egyptian pharmaceutical journal

Vol 11 No 2 December 2012

EGYPTIAN PHARMACEUTICAL JOURNAL Editorial board Editor-in-Chief Aida El-Azzouny Egypt [email protected] +202 33371362/33371433 +2 0100 52 54 161 +202 33370931/3601877 Complete professional affiliations: Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC), Dokki-Cairo, 12622-Egypt Specialization: Medicinal and Pharmaceutical Chemistry

Deputy-Editors Abdel-Hamid Zaki Abdel-Hamid Amer Egypt [email protected]. +201002020747 Complete professional affiliation: Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC), Dokki-Cairo, 12622-Egypt Specialization: Applied Biochemistry

Mohamed Ahmed Abdel-Naby Egypt +202 24708049 +201149921388 +202 3370931/3601877 [email protected] Complete professional affiliations: Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC), Dokki-Cairo, 12622-Egypt Specialization: Professor of Biochemistry

Editorial Assistants Hassan Abdel Zaher Mohamed Mohamed AMER Egypt [email protected] 00201227341899 Complete professional affiliations: National Research Centre Center of Excellence for Advanced Sciences, Dept of Natural and Microbial Products Chemistry Division of Pharmaceutical and Drug Industries Dokki, El-behoos Street Cairo, Egypt Tel: +201227341899 Specialization: Associate professor of bioorganic Chemistry Mohammad H. A. Ibrahim Egypt [email protected] +201150935326 Complete professional affiliations: Chemistry of Natural and Microbial Products Dept., Pharmaceutical & Drugs Industries Research Division, National Research Centre, Al-Bohoos st., Dokki, 12622 Cairo, Egypt Specialization: Microbial Biotechnology, Fermentation Technology, Bioplastics Mona E. Aboutabl Egypt [email protected] +2011155 330 72 Complete professional affiliations: Researcher of Pharmacology, Room # 374, Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, El-bohous St., Dokki, Cairo, 12311 Egypt Specialization: Pharmacology and Toxicology


EGYPTIAN PHARMACEUTICAL JOURNAL Table of contents Original articles 67

Safety evaluation of needle-like hydroxyapatite nanoparticles in female rats Azza I. Hafez, Fatma Hafez, Maaly Khedr, Omayma Ibrahim, Rania Sabry and Mossad A. Abdel-Wahhab

73

Comparative evaluation of in-vitro cytotoxicity, antiviral and antioxidant activities of different soyasapogenols from soybean saponin Hala A. Amin, Hanem M. Awad and Atef G. Hanna

80

Synthesis, antioxidant, and antimicrobial activities of new 2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro1H-pyrazole-4-carbonitriles, (1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanones, and (1,3,4-oxadiazol-2-yl)-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitriles: QSAR and molecular docking analysis Fatma A. Bassyouni, Hanaa A. Tawfik, Ahmed R. Hamed, Maha M. Soltan, Mahmoud ElHefnawi, Ahmed A. ElRashedy, Maysa E. Moharam and Mohamed Abdel Rehim

93

Characterization and purification of the crude Trematosphaeria mangrovei laccase enzyme Atalla M. Mabrouk, Zeinab H. Kheiralla, Eman R. Hamed, Amani A. Youssry and Abeer A. Abd El Aty

99

Phycochemistry of some Sargassum spp. and their cytotoxic and antimicrobial activities Azza A. Matloub and Nagwa E. Awad

109

Regioselective addition of alkyl phosphites on 6-(aryliminomethyl)-furobenzopyran-5-one derivatives Nabila M. Ibrahim, Asmaa A. Magd-El-Din, Amira S. Abd El-All, Eman F. Al-Amrousi and Hisham Abdallah A. Yosef

116

Synthesis of certain new fused pyranopyrazole and pyranoimidazole incorporated into 8-hydroxyquinoline through a sulfonyl bridge at position 5 with evaluation of their in-vitro antimicrobial and antiviral activities Emad M. Kassem, Eslam R. El-Sawy, Howaida I. Abd-Alla, Adel H. Mandour, Dina Abdel-Mogeed and Mounir M. El-Safty

124

In-vitro bioassays on the metabolites of the fungus Emericella nidulans isolated from the Egyptian Red Sea algae Usama W. Hawas, Lamia T.A. El-Kassem, Eman F. Ahmed and Mahmoud Emam

129

Synthesis of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindolin-2-one derivatives: part I Fatma A. Bassyouni, Amira S. Abdel All, Wafaa M. Haggag, Madiha Mahmoud, Mamoun M.A. Sarhan and Mohamed Abdel-Rehim

136

Antimicrobial, anti-inflammatory, and antinociceptive activities of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindoline-2-one derivatives and a study of their computational chemistry: part II Fatma A. Bassyouni, Amira S. Abdel All, Wafaa M. Haggag, Madiha Mahmoud, Mamoun M.A. Sarhan and Mohamed Abdel-Rehim

144

The efficacy of Silybum marianum (L.) Gaertn. (Silymarin) in the treatment of physiological neonatal jaundice: a randomized, double-blind, placebo-controlled, clinical trial Lamyaa M. Kassem, Mohamed E.A. Abdelrahim and Hassan F. Naguib


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Original article 67

Safety evaluation of needle-like hydroxyapatite nanoparticles in female rats Azza I. Hafeza, Fatma Hafezc, Maaly Khedra, Omayma Ibrahimc, Rania Sabrya and Mossad A. Abdel-Wahhabb Departments of aChemical Engineering & Pilot-Plant, b Food Toxicology & Contaminants, National Research Center and cDepartment of Physical Chemistry, Faculty of Science (Girls), Al-Azhar University, Cairo, Egypt Correspondence to Azza I. Hafez, PhD, Department of Chemical Engineering & Pilot-Plant, National Research Center, Tahrir Street, 12622 Cairo, Egypt Tel: + 20 2 33371211; fax: + 20 2 33370931; e-mail: [email protected] Received 8 January 2012 Accepted 24 June 2012 Egyptian Pharmaceutical Journal 2012, 11:67–72

Objective The present study was designed to evaluate the safety of synthesized needle-like hydroxyapatite (HAp) nanoparticles ranging from 3 to 7 nm in diameter and from 27 to 46 nm in length when administered in female rats orally or subcutaneously at different concentrations. Methods Animals in different treatment groups were maintained on their respective diets as follows: group 1, untreated control; group 2, treated orally with HAp (300 mg/kg body weight) for 3 weeks; group 3, treated orally with a low dose of HAp (150 mg/kg body weight) for 3 weeks; and group 4, implanted subcutaneously with HAp (600 mg/kg body weight) once and left for 5 weeks. At the end of the experimentation period, blood samples were collected from all animals for biochemical analysis (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, uric acid, urea, and creatinine). After sacrifice, histopathological examination of the liver and kidney was carried out. Results and conclusion The biochemical results showed an increase in alanine aminotransferase and aspartate aminotransferase in the groups treated orally and those treated subcutaneously. There was an increase in alkaline phosphatase only in the group receiving the high oral dose; however, animals treated with the low dose or those treated subcutaneously were comparable with the control group. All the rats showed normal kidney function because of normal levels of creatinine, urea, and uric acid. The histopathological results indicated that the liver and kidney of all rats treated with HAp (oral or subcutaneously) had a normal structure. The previous results confirmed the safety of the synthesized nanoneedle HAp when administered orally or subcutaneously at the suggested dose. Keywords: hydroxyapatite nanoparticles, kidney, liver, rats, safety evaluation Egypt Pharm J 11:67–72 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Nanotechnology is known to be a revolutionary manufacturing technology of the 21st century involving multidisciplinary research issues that rely on the understanding and control of substances at the nanoscale length of around 1–100 nm. It has been established that nanotechnology offers a unique approach to overcome the limitations of many conventional materials. From nanomedicine to nanofabrics, this promising technology has encompassed almost all fields of human life. Hydroxyapatite (HAp) Ca10(PO4)6(OH)2 is one of the most important materials affected by nanotechnology. One of the main reasons for the intense focus on HAp nanoparticles is because of its structural and compositional similarity to the mineralized matrix of natural bone, enamel, and dentin [1–4]. Synthetic HAp nanoparticles are increasingly being used in medical applications as a bioresorbable carrier material

for controlled drug delivery in the treatment of diseases such as cancer [5], osteoporosis [6], osteomyelitis [7], and diabetes. Also, HAp nanoparticles have excellent biocompatibility with soft tissues such as skin, muscle, and gums, making them an ideal candidate for orthopedic and dental implants or as components of implants. It has been used widely in the repair of hard tissues, and common uses including bone repair, bone augmentation, as well as coating of implants or as fillers in bone or teeth [8,9]. Furthermore, it has been found to have an obvious inhibitory function on the growth of many kinds of tumor cells, and its nanoparticle exerts a stronger antitumor effect than macromolecule microparticles [10]. Because of the previously mentioned benefits of HAp nanoparticles, the current study was carried out to evaluate the safety of the prepared needle-like HAp nanoparticles when administered in Sprague–Dawley female rats orally or subcutaneously (s.c) at different concentrations.

1687-4315 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre

DOI: 10.7123/01.EPJ.0000418505.66044.c8

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Egyptian Pharmaceutical Journal

Subjects and methods

Results and discussion

Chemicals

During this study, no animal died in any of the treatment groups and all animals appeared healthy during the entire treatment period. No significant changes were observed in the body weight of the animals (Fig. 1).

Experimental animals

One-month-old Sprague–Dawley female rats (100–120 g, purchased from the animal house colony, Giza, Egypt) were maintained on a standard lab diet (protein: 160.4; fat: 36.3; fiber: 41 g/kg, and metabolizable energy 12.08 MJ) obtained from Meladco Feed Co. (Aubor City, Cairo, Egypt). Animals were housed in a room free from any source of chemical contamination, artificially illuminated and thermally controlled, at the Animal House Lab (National Research Centre, Dokki, Cairo, Egypt). After an acclimatization period of 1 week, the animals were divided into four groups (seven rats/group) and housed in filter-top polycarbonate cages. All animals were received humane care in compliance with the guidelines of the Animal Care and Use Committee of the National Research Center.

Experimental design

Needle-like HAp nanoparticles ranging from 3 to 7 nm in diameter and from 27 to 46 nm in length were synthesized according to the technique of Sabry [11]. Animals in different treatment groups were maintained on their respective diets as follows: group 1, untreated control; group 2, treated orally with HAp (300 mg/kg body weight) for 3 weeks; group 3, treated orally with a low dose of HAp (150 mg/kg body weight) for 3 weeks; and group 4, implanted s.c. with HAp (600 mg/kg body weight) once and left for 5 weeks. Body weight was recorded weekly during the experimental period. At the end of the experimentation period, blood samples were collected from all animals from the retro-orbital venous plexus for the determination of ALT and AST [12], ALP [13], uric acid [14], urea [15], and creatinine [16]. After the collection of blood samples, all animals were sacrificed and samples of the liver and kidney from all animals from different treatment groups were excised and fixed in 10% neutral formalin, followed by dehydration in ascending grades of alcohol, clearing in xylene, and embedding in paraffin wax. Liver and kidney sections (5 mm thickness) were stained with hematoxylin and eosin for histological examination [17]. The histopathological study was carried out in a clinical pathology private lab.

The biochemical results indicated that animals treated with HAp showed an increase in liver function parameters (Table 1). ALT showed a significant increase in the groups treated orally at the two tested doses or those treated s.c. with HAp (Fig. 2a). This increase was more pronounced in the group treated with the high dose and reached 92.4%, whereas it reached 60.19 and 29.9% in the group received the low oral dose and the group treated s.c. with Hap, respectively. AST showed a significant increase (Fig. 2b) in the orally treated groups. This increase reached 48.9% in the group that received the high dose and reached 20.4 and 23.46% in the group that received the low dose and the s.c. treated group, respectively. ALP also showed the same trend of increase (Fig. 2c) only in the group that received the high oral dose, reaching 20.8%. However, animals treated with the low dose or those treated s.c. with HAp were comparable with the control group. The current results showed that HAp did not exert any significant effects on kidney function tests (Table 2). No significant differences were found between the control group and the groups treated orally or s.c. with HAp. Uric acid tended to increase insignificantly in the group treated orally with the high dose and the group treated s.c. with HAp (Fig. 3a). The same trend was observed in Figure 1

g

Kits of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, uric acid, and creatinine were purchased from Biodiagnostic Co. (Cairo, Egypt).

184 182 180 178 176 174 172 170 168 166 164 Control

All data were analyzed statistically using the General Linear Model Procedure of the Statistical Analysis System [18]. The significance of the differences among the treatment groups was determined using the Waller– Duncan k-ratio [19]. Significance was determined on the basis of P less than 0.05.

Low dose

Sc group

(150 mg/kg b.w)

(600 mg/kg b.w)

Effect of oral or subcutaneous (s.c.) treatment with hydroxyapatite on body weight in rats.

Table 1 Effect of oral or subcutaneous treatment with hydroxyapatite on liver function tests in rats Parameters Groups

Statistical analysis

High dose (300 mg/kg b.w)

Control High dose Low dose Subcutaneous group

ALT (IU/l)

AST (IU/l) a

26.4 ± 0.82 50.8 ± 2.49b 42.29 ± 1.27c 34.29 ± 2.41d

ALP (IU/l) a

56.7 ± 1.45 84.4 ± 1.72b 68.29 ± 1.54c 70.0 ± 2.0d

45.97 ± 0.88a 55.54 ± 1.26b 48.52 ± 2.34a 47.39 ± 3.19a

Within each column, means with different letters are significantly different (Po0.05). ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Safety evaluation of needle-like hydroxyapatite Hafez et al. 69

Figure 2

Figure 3

(a) 1.8

50

1.6

40

1.4 1.2

30

mg/dl

IU/L

(a) 60

20 10

1 0.8 0.6

0

0.4

Control

High dose

Low dose

Sc group

(300 mg/kg b.w)

(150 mg/kg b.w)

(600 mg/kg b.w)

0.2 0

(b) 100 90 80 70 60 50 40 30 20 10 0

Control

Low dose

Sc group

(150 mg/kg b.w)

(600 mg/kg b.w)

mg/dl

IU/L

(b) 45

Control

High dose

Low dose

Sc group

(300 mg/kg b.w)

(150 mg/kg b.w)

(600 mg/kg b.w)

40 35 30 25 20 15 10 5 0 Control

(c) 60 50

High dose

Low dose

Sc group

(300 mg/kg b.w)

(150 mg/kg b.w)

(600 mg/kg b.w)

(c) 0.4

40 30

mg/dl

IU/L

High dose (300 mg/kg b.w)

20 10 0 Control

High dose

Low dose

Sc group

(300 mg/kg b.w)

(150 mg/kg b.w)

(600 mg/kg b.w)

Effect of oral or subcutaneous (s.c.) treatment with hydroxyapatite on: (a) alanine aminotransferase, (b) aspartate aminotransferase, and (c) alkaline phosphatase activity in rats.

Table 2 Effect of oral or subcutaneous treatment with hydroxyapatite on kidney function tests in rats Parameters

Groups Control High dose Low dose Subcutaneous group

Uric acid (mg/dl)

Urea (mg/dl)

Creatinine (mg/dl)

1.41 ± 0.07a 1.54 ± 0.09a 1.40 ± 0.09a 1.50 ± 0.07a

38.30 ± 2.16a 40.41 ± 1.74a 37.76 ± 1.98a 34.99 ± 0.84b

0.30 ± 0.03a 0.25 ± 0.007a 0.25 ± 0.01a 0.33 ± 0.02a

Within each column, means with different letters are significantly different (Po0.05).

urea (Fig. 3b). No significant differences were observed in the creatinine level between animals treated orally with the high and the low dose, although these groups were decreased insignificantly compared with the control group and the s.c. treated group (Fig. 3c). The microscopic examination of the liver tissues in the control group showed normal central vein and hepatocyte architecture (Fig. 4a). The liver of animals treated with the high dose of HAp nanoparticles showed a normal histological structure of the central vein and surrounding

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Control

High dose

Low dose

Sc group

(300 mg/kg b.w)

(150 mg/kg b.w)

(600 mg/kg b.w)

Effect of oral or subcutaneous (s.c.) treatment with nano-hydroxyapatite on: (a) uric acid (b) urea, and (c) creatinine level in rats.

hepatocytes (Fig. 4b). Rats treated with the low dose had a normal structure of hepatocytes, portal vein, and bile ducts (Fig. 4c). The same group showed a normal structure of hepatocytes and the central vein (Fig. 4d). Histopathological investigation of the liver of rats treated s.c. with HAp nanoparticles showed a normal structure of hepatocytes as shown in Fig. 4e. The biochemical results were in good agreement with the histopathological studies of kidney tissue. The microscopic examination of the kidney tissue of the control group showed normal appearance of parenchyma, glomeruli with mesangial cells, normal Bowman’s space and capillaries, normal tubules with normal lining cells, and normal interstitium (Fig. 5a). No differences were found between the control group and those treated orally (high and low dose) or s.c. with HAp nanoparticles during the histological examination of kidney tissues as shown in Fig. 5b–d. The results indicated that the synthesized HAp nanoparticles exerted no toxic effects on the kidney as indicated by kidney function tests (uric acid, urea, and creatinine) and histological examination. However, it exerted a significant effect on the liver function ALT and AST; this shows that the metabolism of HAp nanoparticles

70 Egyptian Pharmaceutical Journal

Figure 4

(a)

(b) (H&E X 200)

(H&E X 100)

(d)

(c) (H&E X 400)

(H&E X 200)

(e) (H&E X 200) A photomicrograph in the liver of (a) the control group showing normal central vein and hepatocyte architecture; (b) rats treated orally with a high dose of nano-hydroxyapatite (Hap) (300 mg/kg body weight) showed a normal histological structure of the central vein and surrounding hepatocytes; (c, d) rats treated orally with a low dose of nano-HAp (150 mg/kg body weight) showed a normal structure of hepatocytes, portal vein, and bile ducts; (e) rats treated subcutaneously with nano-HAp (600 mg/kg body weight) showed a normal structure of hepatocytes. H&E, 100.

Safety evaluation of needle-like hydroxyapatite Hafez et al.

71

Figure 5

A photomicrograph in rats’ kidney of: (a) the control group; (b) rats treated orally with a high dose of nano-hydroxyapatite (Hap) (300 mg/kg body weight); (c) rats treated orally with a low dose of nano-HAp (150 mg/kg body weight); and (d) rats treated subcutaneously with nano-HAp (600 mg/kg body weight) showing the normal appearance of parenchyma, glomeruli (G) with mesangial cells, normal Bowman’s space and capillaries, normal tubules (T) with normal lining cells, and normal interstitium. H&E, 100.

was mainly through the liver as reported by Hou et al. [20]. These results were in agreement with those of Abdel Gawad et al. [21], who reported that the animals injected with 300 and 600 mg/kg body weight showed a significant increase in serum AST and reverted to almost the normal level after 48 h. It is well documented that HAp particles are converted into Ca2 + and PO34 – ions by a natural metabolic process and eliminated over a period of 6 weeks [22,23]. Moreover, Xie [24] has reported that the maximum concentration of intravenous nano-HAp was detected in the liver and spleen at 1 h after administration and decreased significantly after 72 h, which explains the increase in liver function in the treated rats. However, all the animals had normal ALP levels, indicating that bone metabolism was not disturbed with nano-HAp. The ALP level is known to be indicative of hepatobiliary disease [25,26] or a mild hepatocellular injury [27].

Taken together, the current results showed that HAp is safe when administered orally or s.c. in Sprague–Dawley female rats at different concentrations (150, 300, and 600 mg/kg body weight). Similar to the current observation, Hu et al. [10] reported that HAp is safe and could induce inhibition of implanted hepatic VX2 tumor growth in rabbits and cell p53/c-Myc protein expression. Moreover, Abdel Gawad et al. [21] have reported that the liver and kidney in animals treated with HAp showed a normal structure and HAp could restore most of the normal structure of liver and kidney after treatment with lead nitrate.

Conclusion The current study showed that the synthesized HAp nanoparticles ranging from 3 to 7 nm in diameter and from range 27 to 46 nm in length was safe when


administered orally or s.c. in Sprague–Dawley female rats at different concentrations (150, 300, and 600 mg/kg body weight). The biochemical results showed an increase in liver function, whereas kidney function was normal as shown by biochemical results as in the control group. The histopathological examination indicated that liver and kidney tissues of all rats treated with HAp nanoparticles (orally or s.c.) showed a normal structure compared with the control group.

Acknowledgements The authors are indebted to the National Research Center for financial support.

Conflicts of interest There are no conflicts of interest.

9 Li L, Pan H, Tao J, Xu X, Mao C, Gu X, Tang R. Repair of enamel by using hydroxyapatite nanoparticles as the building blocks. J Mater Chem 2008; 18:4079–4084. 10 Hu J, Liu ZS, Tang SL, He YM. Effect of hydroxyapatite nanoparticles on the growth and p53/c-Myc protein expression of implanted hepatic VX 2 tumor in rabbits by intravenous injection. World J Gastroenterol 2007; 13:2798–2802. 11 Sabry R. 2012. Preparation of hydroxyapatite nanoparticles by using emulsion liquid membrane [PhD Thesis]. Egypt: Al-Azhar University. 12 Reitman S, Frankel S. Colorimetric method for aspartate and alanine tranferases. Am J Clin Pathol 1957; 28:56–63. 13 Roy AV. Rapid method for determining alkaline phosphatase activity in serum with thymolphthalein monophosphate. Clin Chem 1970; 16:431–436. 14 Haisman P, Muller BR. Glossary of clinical chemistry terms. London: Butterworth-Heinemann; 1974. 15 Fawcett JK, Scotte JE. A rapid and precise method for the determination of urea. J Clin Pathol 1960; 13:156–159. 16 Bartels H, Bo¨hmer M, Heierli C. Serum creatinine determination without protein precipitation. Clin Chim Acta 1972; 37:193–197. 17 Drury RAV, Wallington EA. Carltons histological techniques. 5th ed. New York: Oxford University Press; 1980. p. 206SY. 18 SAS Institute Inc., SAS user’s guide: statistics. Cary, NC: SAS Institute Inc.; 1982. 19 Waller RA, Duncan DB. A Bayes rule for the symmetric multiple comparison problems. J Am Stat Ass 1969; 64:1484–1503.

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20 Hou CH, Hou SM, Hsueh YS, Lin J, Wu HC, Lin FH. The in vivo performance of biomagnetic hydroxyapatite nanoparticles in cancer hyperthermia therapy. Biomaterials 2009; 30:3956–3960. 21 Abdel Gawad EI, Awwad SA. In-vivo and in-vitro prediction of the efficiency of nano-synthesized material in removal of lead nitrate toxicity. J Am Sc 2011; 7:105–119. 22 Chinol M, Vallabhajosula S, Goldsmith SJ, Klein MJ, Deutsch KF, Chinen LK, et al. Chemistry and biological behavior of samarium-153 and rhenium-186labeled hydroxyapatite particles: potential radiopharmaceuticals for radiation synovectomy. J Nucl Med 1993; 34:1536–1542. 23 Unni PR, Chaudhari PR, Venkatesh M, Ramamoorthy N, Pillai MR. Preparation and bioevaluation of 166Ho labelled hydroxyapatite (HA) particles for radiosynovectomy. Nucl Med Biol 2002; 29:199–209. 24 Xie JSG. Tissue distribution of intravenously administrated hydroxyapatite nanoparticles labeled with 125I. Nanoelectronics Conference (INEC), 3rd International, Hong Kong, China, January 3-8, 2010. pp. 1415–1416. 25 Abdel-Wahhab MA, Omara EA, Abdel-Galil MM, Hassan NS, Nada SA, Saeed A, el-Sayed MM. Zizyphus spina-christi extract protects against aflatoxin B1-initiated hepatic carcinogenicity. Afr J Tradit Complement Altern Med 2007; 4:248–256. 26 Abdel Wahhab MA, Hassan NS, El Kady AA, Khadrawy YA, El Nekeety AA, Mohamed SR, et al. Red ginseng extract protects against aflatoxin B1 and fumonisins-induced hepatic pre-cancerous lesions in rats. Food Chem Toxicol 2010; 48:733–742. 27 Sharma A, Sharma V, Kansal L. Amelioration of lead-induced hepatotoxicity by Allium sativum extracts in Swiss albino mice. Libyan J Med 2010; 5:1–10.

Original article 73

Comparative evaluation of in-vitro cytotoxicity, antiviral and antioxidant activities of different soyasapogenols from soybean saponin Hala A. Amina, Hanem M. Awadb and Atef G. Hannac Departments of aChemistry of Natural and Microbial Products, bTanning Materials and Leather Technology and cChemistry of Natural Compounds, National Research Center, Cairo, Egypt Correspondence to Hala A. Amin, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, 12622 Cairo, Egypt Tel: + 20 2 33464472; fax: + 20 2 37622603; e-mail: [email protected] Revised 15 February 2012 Accepted 7 June 2012 Egyptian Pharmaceutical Journal 2012,11:73–79

Objectives The aim of this study was to evaluate comparative and structure–activity relationships of in-vitro cytotoxicity, antiviral and antioxidant activities of soyasapogenols A, B, D and F (SSA, SSB, SSD and SSF) together against the total soyasaponin extract (TSSE) itself. Methods The cytotoxicity of soyasapogenols and TSSE against human colon carcinoma cell line (HCT-116), liver carcinoma cell line (Hep-G2), human breast carcinoma cell line (MCF-7) and normal human melanocytes (HFB-4) cell lines was assessed using sulforhodamine B assay. Their antiviral activities were investigated against Rift Valley fever virus (RVFV), hepatitis C virus model (vesicular stomatitis virus, VSV), and hepatitis A virus (HAV). The antioxidant activity of soyasapogenols and TSSE was assessed using a stable DPPH free radical. Results and conclusion The results obtained showed that both TSSE and soyasapogenols have a potent cytotoxic effect on Hep-G2, HCT-116, MCF-7 and HBF-4 cell lines in a concentrationdependent manner. SSA and SSF showed the highest cytotoxic activities against tested cell lines. Analysis of the three-dimensional structure of the measured soyasapogenols indicated that if the b-hydroxyl group at C-21 or C-22 was aligned with the plane of the molecule, a marked increase in the cytotoxic activity of the soyasapogenol was produced. Their antiviral activities against RVFV, VSV and HAV showed significant inhibition activities compared with both TSSE and interferon. SSB showed the best activity against RVFV and HAV, whereas SSA was the best inhibitor against VSV. It was concluded that the hydroxylation at C-21 as well as the presence of a double bond in ring D might enhance anti-VSV activity, whereas they may not be essential for anti-RVFV and anti-HAV activities. On the other hand, the tested soyasapogenols and TSSE did not show good antioxidant activities. Keywords: antioxidant, antiviral activity, cytotoxicity, soyasapogenols, soyasaponins Egypt Pharm J 11:73–79 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Soyasaponins are major phytochemical compounds present in legume seeds [1], soybeans and soy products [2]. The basic structure of soyasaponins is an oleanene-type triterpenoid aglycone to which one or more polysaccharide chains are attached, resulting in the amphiphilic nature of the molecules. They are divided into three groups, on the basis of the structure of the aglycone moiety, A, B and E saponins [3]. Soyasapogenols are the aglycone moieties of soyasaponins. They can be obtained by acid or alkali hydrolysis of soyasaponins or by enzymatic hydrolysis using microorganisms with soyasaponinhydrolyzing activity [4–6].The current consensus is that soyasapogenols A, B, and E (SSA, SSB and SSE) are true aglycones, whereas C, D and F (SSC, SSD and SSF) are artifacts produced during the hydrolysis process [7].

Soyasaponins have been reported to have several healthbeneficial activities including hepatoprotective [8], antiviral [9], anticarcinogenic [10], antioxidant [11] and anti-inflammatory activities [12]. Recent studies have shown that a total soyasaponin extract (TSSE) can inhibit the growth of hepatocarcinoma (Hep-G2) cells [4], colon adenocarcinoma cells (HCT-15) [13] and cervical tumor (Hela) cells [14] by inducing programmed cell death, either apoptosis, or microautophagy [15]. Soyasapogenols have been shown to be more effective than their glycosides in the suppression of 2-acetoxyacetylaminofluorene (2-AAAF)-induced genotoxicity in Chinese hamster ovary cells [16]. Both SSA and SSB have shown almost complete suppression of HT-29 colon cancer cell growth. Moreover, soyasaponins might be an important dietary chemopreventive agent against colon cancer after alternation by microflora [17]. Both SSA-containing and


DOI: 10.7123/01.EPJ.0000421669.78647.e9


SSB-containing extracts have also been reported to be capable of inducing apoptosis. SSA extract-treated Hep-G2 cells were reported to induce 47 ± 3.5% of the cells to undergo apoptosis, whereas SSB extract induced 15 ± 4.2% of cells to undergo apoptosis after 72 h of treatment [4]. In addition, SSB (10 mmol/l) was growth inhibitory to MDA-MB-231 human breast cancer cells in vitro [18]. SSA, SSB, SSE and soyasaponin I, a major constituent of group B saponins, completely inhibited HIV-induced cytopathic effects 6 days after infection at a concentration greater than 0.25 mg/ml, but exerted no direct effect on HIV reverse transcriptase activity [19]. TSSE showed a significant inhibitory effect on the replication of HSV-1 and CoxB3 [20]. In a structure–activity relationship study, the activity of SSA was less than 1/20 of that of SSB and the hydroxylation at C-21 seemed to reduce anti-HSV-1 activity [21]. Soyasaponin II was found to inhibit the replication of the human cytomegalovirus and influenza virus. This action was not because of the inhibition of virus penetration and protein synthesis, but may because of a virucidal effect [22]. The effect of TSSE from soybean on acute alcohol-induced hepatotoxicity in mice has been investigated. Mice treated with TSSE showed a better profile of the antioxidant system with normal superoxide dismutase, glutathione S-transferase, and glutathione peroxidase activities, which were associated with the increase in hepatic glutathione levels relative to the acute alcohol-treated group [23]. However, TSSE and its five main constituent saponins had a much weaker in-vitro inhibitory effect on lipid peroxidation induced by NADPH in mouse liver microsomes than a-tocopherol [11]. All of these reports have led to our interest in a comparative evaluation of in-vitro cytotoxicity, antiviral and antioxidant activities of SSA, SSB, SSD and SSF isolated from a hydrolyzed soybean saponin extract against TSSE itself and to discuss their structure–activity relationships.

Materials and methods Materials

Soybean saponin (50%) was purchased from Organic Technologies Co. Ltd (Coshocton, Ohio, USA). SSA, SSB, SSD and SSF were isolated from a hydrolyzed soybean saponin extract [6]. Sulforhodamine B (SRB), Roswell Park Memorial Institute (RPMI) 1640 medium, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) were purchased from Sigma-Aldrich Co. (St Louis, Missouri, USA). Fetal bovine serum (FBS), 199 E-Hepes buffer medium and fetal calf serum (FCS) were purchased from Gibco (Paysley, UK). Recombinant human interferon a2a (rh-IFN a2a) was obtained from Galaxo Smithkline (Milan, Italy). Dimethyl sulfoxide (DMSO) and methanol were of HPLC grade, and all other reagents and chemicals were of analytical reagent grade. To determine the structure–activity relationships, the three-dimensional (3D) structure of the measured compounds was

created using VEGA ZZ software (Drug Design Laboratory, University of Milan, Milan, Italy), and energy minimization was carried out by AMMP calculation provided by the same software. Cell culture

Four human cell lines, HCT-116 (colon carcinoma cell line), Hep-G2 (liver carcinoma cell line), MCF-7 (human breast carcinoma cell line) and HFB-4 (normal human melanocytes) were purchased from the American Type Culture Collection (Rockville, Maryland, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were grown at 371C in a humidified atmosphere of 5% CO2. Cytotoxic activity (sulforhodamine B assay)

Human cancer cell lines were grown in RPMI-1640 medium (371C, 5% CO2) to assess the growth inhibition by a colorimetric assay, which estimates the cell number indirectly by staining total cellular protein with SRB dye [24]. Logarithmically growing cells were seeded at a density of 104 cells/well into 96-well plates and allowed to adhere for 24 h at 371C. Then, the supernatant was replaced by 100 ml culture medium supplemented with each tested compound in DMSO at specified concentrations and incubated at 371C for 48 h. The final concentration of DMSO in the solution in each well was 0.5%. Treatment with DMSO only was always used as a control. At the end of the treatment, the supernatant from each well was discarded and cells were fixed by layering 100 ml ice-cold 15% trichloroacetic acid on top of the growth medium. They were then incubated at 41C for 1 h. The plates were then washed five times with cold water, the excess water was drained off, and the plates were air dried. SRB stain [100 ml; 0.4 (w/v) in 1% acetic acid] was added to each well and left in contact with the cells for 1 h. Subsequently, the cells were washed with 1% acetic acid and rinsed four times. The plates were dried, and 1 ml of 10 mmol/l Tris base was added to each well to dissolve the dye. The plates were shaken gently for 20 min on a gyratory shaker, and the absorbance (OD) of each well was read on a spectrophotometer at 540 nm. Cell survival was measured as the percentage of absorbance compared with the control. DPPH radical-scavenging assay

The antioxidant activity of soyasapogenols (SSA, SSB, SSD, and SSF), TSSE and standards (ascorbic acid and rutin) was assessed on the basis of the radical-scavenging effect of a stable DPPH free radical [25]. A volume of 10 ml of each tested compound or standard (from 0.0 to 100 mg/ml) was added to 90 ml of a 100 mmol/l methanolic solution of DPPH in a 96-well microtiter plate (SigmaAldrich Co.). After incubation in the dark at 371C for 30 min, the decrease in the absorbance of each solution was measured at 520 nm using an ELISA micro plate reader (Model 550; Bio-Rad Laboratories Inc., Hercules, California, USA). The absorbance of the blank sample containing the same amount of DMSO and DPPH solution was also prepared and measured. All experiments

Comparative evaluation of soyasapogenols biological activities Amin et al.

were carried out in triplicate. The scavenging potential was compared with a solvent control (0% radical scavenging) and ascorbic acid. Radical-scavenging activity was calculated using the following formula: % Reduction of absorbance¼½ðABAAÞ/AB100; where AB is the absorbance of the blank sample and AA is the absorbance of the tested compound (t = 30 min) [26]. Antiviral activity Cells and viruses

Vero clone CCL-81 was obtained from the Cell Culture Department, VACSERA (Cairo, Egypt). Cells were grown in 199 E-Hepes buffer growth medium supplemented with 10% inactivated FCS, 5 mmol/l Hepes buffer, and antibiotics (100 U of penicillin/ml and 100 g of streptomycin/ml) at 371C and incubated in a 5% CO2 atmosphere. Vesicular stomatitis virus (hepatitis C virus model, VSV, Indiana strain), Rift Valley fever virus (RVFV, Menya/sheep/258) and hepatitis A virus (HAV, a local isolate) were kindly supplied by Applied Research Sector, VACSERA. The infectivity titer of the viruses was determined according to the reported method of Specter et al. [27]. The viruses were 10-fold serially diluted and each dilution was dispensed as 100 ml/well onto precultured Vero cells. Noninfected wells were considered as a negative control. Plates were incubated at 371C. Seven days after infection, the 50% cell culture infective dose end point (CCID50) was determined. Cytotoxicity assay

The investigated compounds were dissolved in DMSO and diluted with sterile culture medium at specified concentrations. The cytotoxicity assay of each compound compared with sterile rh-IFN a2a was carried out according to previous reports [28,29], and a negative cell control was included. Plates were incubated at 371C for 24 h. Cell culture-treated plates were examined microscopically using an inverted microscope for the detection of cellular changes or cytotoxicity. The medium was discarded and plates were washed using phosphate-buffer saline (pH 7). Cell viability was evaluated using a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to determine the safe concentration range for each compound. Viability percentage was determined as follows: Viability % ¼ðODcontrol ODtreated Þ/ODcontrol Þ100;

75


All experiments were conducted in triplicate (n = 3). All the values were represented as mean ± SD. Significant differences between the means of parameters as well as IC50 values were determined by probit analysis using the SPSS software program (SPSS Inc., Chicago, Illinois, USA).

Results and discussion In-vitro cytotoxic activity

Four soyasapogenols (SSA, SSB, SSD, and SSF; Fig. 1), were examined in-vitro for their cytotoxic activities against three human cancer cell lines (HCT-116, Hep-G2 and MCF-7) and one normal human cell line (HFB-4) using SRB assay. Their activities were compared with cytotoxicity of TSSE and doxorubicin, a positive control. The compounds examined were produced in our previous work by acid or enzymatic hydrolysis of the crude soybean saponin extract [6]. Results show that all tested soyasapogenols together with TSSE showed dose-dependent cytotoxic activities against four tested human cell lines (Figs 2 and 3). Cytotoxic activities, reflected by their IC50 values, against HCT-116 and Hep-G2 were in the following order: SSF4SSA4 doxorubicin4SSB4SSD4TSSE, whereas, against MCF-7, they were in the following order: SSA = doxorubicin4SSF4SSB4SSD4TSSE. However, cytotoxic activities against HFB-4 were in the following order: SSF = SSA4doxorubicin4SSB4SSD4TSSE. The obtained IC50 values of SSA, SSB, and TSSE (3.89, 15.8, and 37.5 mg/ml, respectively) against Hep-G2 after 48 h were much lower than those reported by Zhang and Popovich [4] (50, 130, and 600 mg/ml, respectively) after 72 h. In terms of the cytotoxic activity against MCF-7, both

Figure 1

12

C A

3

B

13

OH

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E

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22

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D

OH

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HO 24

CH2OH

CH2OH

23

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Antiviral activity

The antiviral activity of tested compounds and interferon against RVFV, VSV, and HAV was determined, where nontoxic concentrations of each compound and rh-IFN (10 IU/0.1 ml) as a positive control were used for the treatment of precultured Vero cells for 24 h. A negative cell control plate was included for viral control titration. Viruses were 10-fold serially diluted in 199 E-Hepes buffer (10–1–10–8). Antiviral activity was determined by evaluating each virus mean titer in treated and nontreated cells. The difference between both titers indicates the antiviral activity [28].

OMe

OH

HO

HO

CH2OH

CH2OH SSF

SSD

Structure of the investigated soyasapogenols. SSA, soyasapogenol A; SSB, soyasapogenol B; SSD, soyasapogenol D; SSF, soyasapogenol F.


Figure 2

(a)

(b) 1.0 SAA SSB SSD SSF

0.8

Surviving fraction (Au)


1.0

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Soy A Soy B Soy D Soy F

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Cytotoxic activities of soyasapogenols (SSA, SSB, SSD and SSF) against (a) human liver carcinoma cell line (Hep-G2); (b) human breast carcinoma cell line (MCF-7); (c) human colon carcinoma cell line (HCT-116); (d) normal human melanocytes (HFB-4) using SRB assay. Values are mean ± SD of three separate experiments, each in triplicate.

Analysis of the results in Table 1 comparing the structure of the measured soyasapogenols (Fig. 1) showed that the hydroxyl groups at C-21 and C-22 play a major role in the activity of the measured compounds in addition to the double bond between C-12 and C-13 as well as C-13 and C-18. SSF, which has a b-hydroxyl group at C-22, has a good activity as compared with the positive control, doxorubicin. This activity decreased markedly on just replacing the b-hydroxyl group by a b-methoxyl group in SSD. However, this did not explain the decrease in the

Figure 3

1.0


SSA and SSB had potent cytotoxic effects after 48 h on MCF-7 cells with IC50 values of 2.97 mg/ml (6.27 mmol/l) and 11.4 mg/ml (24.89 mmol/l), respectively (Table 1). In contrast, Rowlands et al. [18] reported that SSA stimulated the proliferation of estrogen-sensitive cells MCF-7 2.5-fold; however, SSB exerted no significant effect on MCF-7 cells at all concentrations up to 10 mmol/l after 72 h [18]. It is worth noting that there are no previously reported data on the cytotoxic activity of TSSE against MCF-7; however, TSSE showed an IC50 value of 39.3 mg/ml after 48 h in this study.

Hep-G2 MCF-7 HCT-116 HFB-4

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Cytotoxic activities of total soyasaponin extract against human liver carcinoma cell line (Hep-G2), human breast carcinoma cell line (MCF-7), human colon carcinoma cell line (HCT-116), and normal human melanocytes (HFB-4) using the SRB assay. Values are mean ± SD of three separate experiments, each in triplicate.

Comparative evaluation of soyasapogenols biological activities Amin et al. 77

Table 1 Cytotoxicity of soyasapogenols (SSA, SSB, SSD and SSF) and TSSE against HCT-116, Hep-G2, MCF-7 and HFB-4 cell lines as measured by 50% cell toxicity (IC50) using SRB assay

Figure 4

IC50 (mg/ml) Compounds SSA SSB SSD SSF TSSE Doxorubicin (positive control)

HCT-116

Hep-G2

MCF-7

HFB-4

3.12 8.76 11.8 3.0 36.2 3.73

3.89 15.8 30.7 3.12 37.5 4.0

2.97 11.4 21.4 6.0 39.3 2.97

5.1 12.6 16.5 5.1 45.6 8.0

HCT-116, human colon carcinoma cell line; Hep-G2, liver carcinoma cell line; HFB-4, normal human melanocytes; MCF-7, human breast carcinoma cell line; SRB, sulforhodamine B, TSSE, total soyasaponin extract.

Moreover, all soyasapogenols showed very good cytotoxic activities against all cell lines compared with TSSE itself. These results are in agreement with previously reported data [3,30,31]. Gurfinkel and Rao [17] have reported that there was a relationship between structure and bioactivities, with SSA and SSB generally being more bioactive compared with their glycosides [17]. There is some evidence, as with many other saponins, that the bioactivity of soyasaponins increases as sugar moieties are eliminated from the saponin structure, thereby reducing the polarity [29]. Generally, the SSA-containing extract was found to show the greatest propensity to affect the cell cycle compared with the SSB-containing extract compared with a fractionated extract or a total saponin mixture [4,30]. To the best of our knowledge, there are no previously reported data on the cytotoxicity of SSD and SSF. However, there are some reports on the cytotoxicity and hepatoprotective effects of soyasaponins and SSA and SSB.

The energetically optimized three-dimensional structure of the measured soyasapogenols. Hydrogen atoms were deleted after energy minimization to clarify the plane of the compounds and the hydroxyl groups.

Figure 5

100 90 80 % inhibition of DPPH

activity of SSB, which also has a b-hydroxyl group at C-22. The energetically optimized 3D structure of the measured compounds (Fig. 4) shows that both SSA and SSB have rings A, B, C, and D in the same plane and because of the presence of a double bond between C-12 and C-13, ring E adopts a position perpendicular to the molecular plane. Consequently, the b-hydroxyl group at C-21 is aligned with the molecular plane, whereas that at C-22 is perpendicular to it (Fig. 4). SSF, which has a double bond between C-13 and C-18, has rings A, B, C, D and E in the same plane; consequently, the b-hydroxyl group at C-22 is aligned with the molecular plane (Fig. 4). In conclusion, if the b-hydroxyl group at C-21 or C-22 was aligned with the plane of the molecule, a marked increase in the activity of the soyasapogenol was produced. These may be responsible for the good activity of both SSA and SSF compared with the other soyasapogenols and the positive control.

70 60 50 40 30 20 10 0

In-vitro antioxidant activity

The antioxidant activity of four soyasapogenols and TSSE was evaluated using the DPPH radical-scavenging method. Results presented in Fig. 5 show that all soyasapogenols and TSSE did not show appreciable

SSA

SSB

SSD

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TSSE

Vit C

Rutin

Antioxidant activities of soyasapogenols (SSA, SSB, SSD and SSF) and total soyasaponin extract (TSSE) using the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical-scavenging assay. Values are mean ± SD of three separate experiments, each in triplicate.


Figure 6

Antiviral activity of soyasapogenols; SSA (25 mg/ml), SSB (25 mg/ml), SSD (25 mg/ml), SSF (12.5 mg/ml) and total soyasaponin extract (TSSE) (100 mg/ml) against three viruses: Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV) and hepatitis A virus (HAV). Recombinant human interferon a2a (rh-IFN a2a, 10 IU/0.1 ml) was used as a positive control. Values are mean ± SD of three separate experiments, each in triplicate.

scavenging activity compared with the standards (ascorbic acid and rutin), reflected by their DPPH inhibition percentage at a concentration of 100 mmol/l. The DPPH inhibition was in the following order: ascorbic acid4 rutin4SSF4SSB4TSSE4SSA4SSD, where their DPPH inhibition percentages were: 92.35489.62440.72433.194 29.9427.3243.09 (respectively). To the best of our knowledge, there are no previously reported data on the direct antioxidant activity of all tested compounds.

In-vitro antiviral activity

Researchers believe that saponins can stimulate the immune system, ward off microbial and fungal infections, protect against viruses and even act as a spermicide [31]. Therefore, the antiviral activity of the four soyasapogenols and TSSE was evaluated against three viruses (RVFV, VSV and HAV) using the highest nontoxic concentration for each compound. Figure 6 shows that all tested soyasapogenols had a significant antiviral activity against the three viruses, reflected by their high inhibition percentage of the log virus titer count. Their activities against RVFV and HAV viruses were in the following order: rh-IFN4SSB4SSA4SSF4SSD4TSSE. However, those against VSV were in the following order: rh-IFN4 SSA4SSD4SSF4SSB4TSSE. Although the concentration of TSSE (100 mg/ml) used was greater than those used for SSB, SSA, SSD and SSF (25, 25, 25 and 12.5 mg/ml, respectively), it showed no activity against the VSV virus and an elevation in the HAV virus count (2.5%). Consequently, sugar moieties attached at the C-3 position and/or at the C-22 position of the aglycone seemed to eliminate or reduce its antiviral activity. The chemical structure of the tested compounds also shows that the hydroxyl group and the double bond control the activity of the compounds. SSB showed the highest activity against both RVFV and HAV viruses. Analysis of the 3D structure (Fig. 4) shows that the presence of a b-hydroxyl group at C-22 in a position perpendicular to the plane of the molecule enhances the antiviral activity of the compound. However, SSA showed

the maximum activity against the VSV virus. As SSA is a hydroxylated derivative of SSB at C-21, it might enhance the anti-VSV activity, whereas it may not be essential for the anti-RVFV and anti-HAV activities. In addition, SSD and SSF antiviral activities were comparable against the tested viruses. They showed better anti-VSV activities compared with SSB, indicating that the double bond in ring D may play a role in their anti-VSV activities. To the best of our knowledge, there are no previous reports on the antiviral activities of the tested soyasapogenols or TSSE against those three viruses (RVFV, HAV, and VSV).

Conclusion Among the tested soyasapogenols, SSA and SSF showed the best therapeutic values against Hep-G2, HCT-116 and MCF-7 cell lines. Analysis of the 3D structure of these compounds indicated that if the b-hydroxyl group at C-21 or C-22 was aligned with the plane of the molecule, a marked increase in the cytotoxic activity of the soyasapogenol was produced. In terms of their antiviral activity against RVFV, VSV and HAV viruses, all soyasapogenols showed significant inhibition activities compared with TSSE itself. These results indicate that the hydroxylation at C-21 as well as the presence of a double bond in ring D instead of ring C might enhance anti-VSV activity, whereas it may not be essential for anti-RVFV and anti-HAV activities. However, the tested soyasapogenols and TSSE did not show appreciable antioxidant activity. These comparative data suggested that the investigated soyasapogenols could be candidate therapeutic agents as anticancer and antiviral agents. However, further studies may be required to examine the mode of action of each compound.

Acknowledgements Conflicts of interest There are no conflicts of interest.

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Original article

Synthesis, antioxidant, and antimicrobial activities of new 2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2, 3-dihydro-1H-pyrazole-4-carbonitriles, (1,3,4-oxadiazol-2-yl)-1Hbenzo[d]imidazol-5-yl)(phenyl)methanones, and (1,3,4-oxadiazol2-yl)-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitriles: QSAR and molecular docking analysis Fatma A. Bassyounia,b, Hanaa A. Tawfika, Ahmed R. Hamedb,c, Maha M. Soltanb,c, Mahmoud ElHefnawid, Ahmed A. ElRashedyd, Maysa E. Moharame and Mohamed Abdel Rehimf a Department of Chemistry of Natural and Microbial Products, bPharmaceutical Research Group, Center of Excellence for Advanced Sciences, cDepartment of Chemistry of Medicinal Plants, dBiomedical Informatics and Chemo Informatics Group, Department of Informatics and Systems, Centre of Excellence for Advanced Sciences, eDepartment of Microbial Chemistry, National Research Centre, Dokki, Cairo, Egypt and fDepartment of Analytical Chemistry, Stockholm University, Stockholm, Sweden

Correspondence to Fatma A. Bassyouni, PhD, Department Chemistry of Natural and Microbial Products and Pharmaceutical Research Group, Center of Excellence for Advanced Sciences, National Research Centre, 12311 Cairo, Egypt Tel: + 02 1118596967; fax: + 0202 33370931; e-mail: [email protected] Received 19 February 2012 Accepted 6 June 2012 Egyptian Pharmaceutical Journal 2012,11:80–92

Objectives A new series of 2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro-1Hpyrazole-4-carbonitrile (6a,b), (1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl) methanone (9–11), and (1,3,4-oxadiazol-2-yl)-1,5-dihydro-[1,2,4]triazolo[1,5-a] pyridine-8-carbonitrile (14–16) derivatives were synthesized and evaluated for their antioxidant and antimicrobial activities; in addition, their quantitative structure–activity relationships and molecular docking were investigated. Methods The target compounds 6a,b were synthesized by the following method: reaction of 5,6-dimethyl-1H-benzoimidazole-2-carbohydrazide (2) with 4-(dimethyl amino)benzaldehyde or anthracene-9-carbaldehyde yielded Schiff’s bases 3a,b, which were reacted with ethyl cyanoacetate to yield 1H-pyrazole-4-carbonitriles 4a,b; N-methylation of 4a,b afforded 5a,b, which reacted with 4-aminoantipyrine to give 6a,b. In addition, 5-benzoyl-1H-benzo[d]imidazole-2-carbohydrazide (8) or 8-cyano-6isocyano-5-oxo-7-phenyl-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-2-carbohydrazide (13) reacted with different carboxylic acids such as crotonic acid, 3,4-diaminobenzoic acid, and 6-hydroxy-4-methoxybenzofuran-5-carboxylic acid to form compounds 9–11 and 14–16, respectively. The synthesized compounds were evaluated for their antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, and the diffusion plate method for antimicrobial activity. Results and conclusion Among other tested compounds, compounds 15, 11, and 10 possessed the highest antioxidant activity, whereas compounds 4a, 5b, 6b, 10, and 11 displayed high activity against Staphylococcus aureus, Salmonella typhimurium, and Candida albicans. The quantitative structure–activity relationships of the studied compounds 4a, 4b, 5b, 6b, 10, 11, 14, 15, and 16 indicated a high correlation (r2 = 0.82) between the predicted and actual activities as obtained from molecular descriptors and the inhibitory activity of this set of tested molecules measured as antioxidant activity. Moreover, the three-dimensional (3D) pharmacophore was generated, and docking of the most active antibacterial compound 4a against the dihydropteroate synthase enzyme gave comparable scores for hydrogen bond interaction (– 13.5 kcal/mol) and binding mode to the reference antibiotic sulfamethoxazole (– 13.00 kcal/mol). Keywords: antibacterial, antioxidant, benzimidazoles, molecular docking, quantitative structure–activity relationships, triazoles Egypt Pharm J 11:80–92 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Benzimidazoles have a wide range of biological activities such as anticancerous and fungicidal activities; they

also serve as antioxidants and central nervous system depressants [1–6]. A large number of benzotriazole derivatives are currently used in clinical applications [7–9]. In addition, they are of great value as intermediates


DOI: 10.7123/01.EPJ.0000422113.69898.e0

Synthesis, antioxidant, antimicrobial effects Bassyouni et al.

and final products in organic synthesis [10]. Fused heterocyclic compounds containing 1,2,4-triazoles have biological potency such as central nervous system depressant [11], antifungal [12], antiviral, and antibacterial activities [13]. Therefore, triazole derivatives have consistently attracted scientific and practical interest because of their widely varying chemical properties, synthetic versatility, and pharmacological activities [14,15]. Herein, we aim to prepare a new series of 2(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3dihydro-1H-pyrazole-4-carbonitrile, (1,3,4-oxadiazol-2yl-1H-benzo[d]imidazol-5-yl)(phenyl) methanone, and (1,3,4-oxadiazol-2-yl)-1,5-dihydro-[1,2,4]triazolo [1,5-a] pyridine-8-carbonitrile derivatives and evaluate them for their antioxidant and antibacterial activities. In addition, the quantitative structure–activity relationships (QSAR) of this series will be investigated.

Experimental Chemistry

All melting points were determined using the Electro thermal capillary (Stuart, SMP10, UK) melting point apparatus and were uncorrected. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were measured in DMSO-d6 using a JEOL-500 spectrometer (Japan) with Me4Si as an internal standard. Mass spectra were obtained using a Finigan gas chromatography–mass spectrometry (USA) at 70 eV. The IR spectra (4000–400 cm – 1) were recorded using KBr pellets in a Jasco FT/IR 300 E Fourier transform infrared spectrophotometer (USA) and in the 500–100 cm – 1 region using polyethylene-sandwiched nujol mulls on a Perkin Elmer FT-IR 1650 spectrophotometer (Norwalk, USA). Elemental analyses were carried out at the Micro analytical Laboratory of the National Research Centre, Cairo, Egypt. Silica gel thin-layer chromatography cards were purchased from Merck (Darmstadt, Germany) (silica gel precoated aluminum cards with fluorescent indicator at 254 nm). Visualization was performed by illumination with a UV light source. Compounds 1, 7, and 12 were prepared according to the reported literature [16]. 5,6-Dimethyl-ethyl-2-benzimidazole carboxylate (1)

Yield 9.1 g (84%), melting point (MP) 180–1821C. 1 H NMR d:1.30 (3H, t, CH3), 2.15 (6H, s, 2CH3), 4.10–4.30 (2H, q, CH2), 10.70 (1H, s, NH benzimidazole), 7.22–7.46 (2H, m, Ar-H). IR (KBr; cm–1): 3350 (NH), 1730 (C = O), 1640 (C = N). MS: m/z = 218 [M + ]. Anal. calcd for C12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found: C, 66.09; H, 6.40; N, 12.80. Synthesis of 5,6-dimethyl-1,3-benzimidazole-2-carboxyhydrazide (2)

5,6-Dimethyl-ethyl-2-benzimidazole carboxylate (1; 0.025 mol, 5.40 g) was dissolved in absolute ethanol (30 ml). The hydrazine hydrate (0.050 mol, 1 ml) was added dropwise under stirring and refluxed for 6 h. The reaction mixture was cooled to room temperature and poured into ice-cold water.

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The solid formed was filtered, washed with water, dried, and then recrystallized from absolute ethanol to yield 4.15 g (80%), MP 230–2321C. 1H NMR d: 2.15 (6H, s, 2CH3), 5.56 (2H, br, NH2, D2O– exchangeable), 9.56 (1H, s, NH), 10.70 (1H, s, NH benzimidazole), 7.22–7.46 (2H, m, Ar-H). IR (KBr; cm–1): 3340–3350 (NH), 3290 (NH2), 1725 (C = O), 1640 (C = N). MS: m/z = 208 [M + ]. Anal. calcd for C10H12N4O: C, 58.81; H, 5.92; N, 27.43. Found: C, 58.86; H, 5.87; N; 27.48. General procedure for the synthesis of N0 -(4-(dimethylamino) benzylidene)-5,6-dimethyl-1H-benzo[d]imidazole-2carbohydrazide (3a) and N0 -(anthracen-9-ylmethylene)-5, 6-dimethyl-1H-benzo[d]imidazole-2-carbohydrazide (3b).

To a solution of 5,6-dimethyl-1,3-benzimidazole-2-carboxyhydrazide (2; 1 mmol, 0.208 g) in absolute ethanol (30 ml), the appropriate aromatic aldehydes, namely 4-dimethyl amino benzaldehyde (1 mmol, 0.133 g) and anthracene-9-carbaldehyde (1 mmol, 0.206 g), were added with a few drops of piperidine and refluxed under stirring for 7 h. After cooling, the product was poured into crushed ice; the formed solid was filtered, washed with water, and recrystallized from absolute ethanol to give 3a and 3b. N0 -(4-(Dimethylamino)benzylidene)-5,6-dimethyl-1H-benzo [d]imidazole-2-carbohydrazide (3a)

Yield 0.22 g (86%), MP 166–1681C. 1H NMR d: 2.15 (6H, s, 2CH3), 3.17 (6H, s, N–CH3), 6.88–7.10 (4H, m, Ar-H), 7.22–7.46 (2H, m, Ar-H), 8.10 (1H, s, N = CH), 9.60 (1H, s, NH), 10.70 (1H, s, NH benzimidazole). C13 NMR: 19.80 (CH3), 40.50 (N–CH3), 110.30, 115.50, 123.50, 125.30, 130.50, 135.90 (Ar-CH), 145.80 (C = N). IR (KBr; cm–1): 3350–3360 (NH), 1720 (C = O), 1640 (C = N). Anal. calcd for C19H21N5O: C, 68.04; H, 6.31; N, 20.88. Found: C, 68.09; H, 6.38; N, 20.84. N0 -(Anthracen-9-ylmethylene)-5,6-dimethyl-1H-benzo[d] imidazole-2-carbohydrazide (3b)

Yield 0.32 g (84%), MP 190–1921C. 1H NMR d: 2.15 (6H, s, 2CH3), 7.20–7.46 (2H, m, Ar-H), 7.50–7.95 (9H, m, Ar-H), 8.10 (1H, s, N = CH), 9.60 (1H, s, NH), 10.70 (1H, s, NH benzimidazole), C13 NMR: 19.80 (CH3), 115.50, 110.30, 123.50, 125.30, 128.20, 130.50, 131.80, 135.90 (Ar-CH), 145.80 (N = CH), 142.30 (C = N), 155.50 (C = O). IR (KBr; cm–1): 3350–3365 (NH), 1720 (C = O), 1640 (C = N). Anal. calcd for C25H20N4O: C, 76.51; H, 5.14; N, 14.28. Found: C, 76.56; H, 5.18; N, 14.33. General procedure for the synthesis of 2-(5,6-dimethyl1H-benzo[d]imidazole-2-carbonyl)-5-(4-(dimethylamino) phenyl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (4a) and 5-(anthracen-9-yl)-2-(5,6-dimethyl-1H-benzo[d]imidazole-2carbonyl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (4b)

Compound 3a (1 mmol, 0.335 g) or 3b (1 mmol, 0.392 g) was dissolved in dry benzene (25 ml) and ethyl cyanoacetate (1 mmol, 0.15 ml), and a few drops of TEA were added. The reaction mixture was heated under reflux with stirring for 5 h. It was cooled to room temperature and poured into ice-cold water. The precipitate was filtered off and purified by crystallization from chloroform to form compounds 4a or 4b.


2-(5,6-Dimethyl-1H-benzo[d]imidazole-2-carbonyl)-5-(4(dimethylamino)phenyl)-3-oxo-2,3-dihydro-1H-pyrazole4-carbonitrile (4a)

Yield 0.3 g (84%), MP 242–2441C. 1H NMR d:2.15 (6H, s, 2CH3), 6.85–7.10 (4H, m, Ar-H), 7.20–7.46 (2H, m, Ar-H), 10.70 (1H, s, NH benzimidazole), 11.20 (1H, s, NH pyrazole). C13 NMR: 19.80 (CH3), 40.50 N(CH3)2, 115.90 (CN), 110.50, 123.50, 125.30, 130.50, 131.80, 135.90 (Ar-CH), 142.30 (C = N), 150.50 (C = O), 165.20 (C = O). IR (KBr; cm–1): 3370–3380 (NH), 1710– 1720 (C = O), 1640 (C = N), 2150 (CN). MS: m/z = 401[M + + 1]. Anal. calcd for C22H20N6O2: C, 65.99; H, 5.03; N, 20.99. Found: C, 65.95; H, 5.07; N, 20.96. MS: m/z = 401[M + + 1]. 5-(Anthracen-9-yl)-2-(5,6-dimethyl-1H-benzo[d]imidazole-2carbonyl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (4b)

Yield 0.35 g (80%), MP 210–2121C. 1H NMR d: 2.15 (6H, s, 2CH3), 3.17–3.30 [6H, s, N–(CH3)2], 7.20–7.46 (2H, m, Ar-H), 7.50–7.95 (9H, m, Ar-H), 10.70 (1H, s, NH), 11.20 (1H, s, NH pyrazole). C13 NMR: 19.80 (CH3), 32.40 (CH3), 115.90 (CN), 110.50, 123.50, 125.30, 130.50, 131.80, 135.90 (Ar-CH), 142.30 (C = N), 150.50 (C = O), 165.20 (C = O). IR (KBr; cm–1): 3354–3360 (NH), 1 710–1720 (C = O), 1640 (C = N), 2165 (CN). MS: m/z = 458 [M + + 1]. Anal. calcd for C28H19N5O2: C, 73.51; H, 4.19; N, 15.31. Found: C, 73.56; H, 4.24; N, 15.36. General procedure for the synthesis of 5-(4-(dimethylamino) phenyl)-3-oxo-2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (5a) and 5-(anthracen-9-yl)-3-oxo-2-(1,5,6-trimethyl-1H-benzo[d] imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (5b)

Method A: A solution of methyl iodide (2 ml) in n-hexane (2 ml; 1/1, v/v) was added to the reaction mixture of compound 4a (1 mmol, 0.40 g) or 4b (1 mmol, 0.45 g) and sodium hydride/DMF (0.1 g sodium hydride/1.0 ml DMF) and stirred at room temperature. The reaction was stopped by the careful addition of a few drops of water followed by 20 ml of water under stirring at room temperature for 8 h. The product was extracted with 30 ml of n-hexane, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under vacuum to afford 5a or 5b in yields up to 78–80%, respectively. Method B: A mixture of compound 4a (1 mmol, 0.40 g) or 4b (1 mmol, 0.45 g), anhydrous K2CO3 (0.01 mol, 1.0 g), and dimethyl carbonate (DMC; 0.03 mol, 2.5 ml) in DMF (10 ml) was refluxed for 3 h. The reaction mixture was cooled to room temperature, following which ice water was added; the precipitated solid was filtered, dried, and crystallized from ethanol to afford 5a or 5b in yields up to 85–88%, respectively. 5-(4-(Dimethylamino)phenyl)-3-oxo-2-(1,5,6-trimethyl-1Hbenzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4carbonitrile (5a)

Yield 0.36 g (88%) by method B, MP 234–2361C. 1 H NMR d: 2.15 (6H, s, 2CH3), 4.00 (3H, s, CH3),

6.88–7.10 (4H, m, Ar-H), 7.20–7.46 (2H, m, Ar-H), 10.70 (1H, s, NH), 11.20 (1H, s, NH pyrazole). IR (KBr; cm–1): 3320 (NH), 1710–1720 (C = O), 1640 (C = N). MS: m/z = 413 [M + – 1]. Anal. calcd for C23H22N6O2: C, 66.65; H, 5.35; N, 20.28. Found: C, 66.70; H, 5.40; N; 20.25. MS: m/z = 414 [M + ]. 5-(Anthracen-9-yl)-3-oxo-2-(1,5,6-trimethyl-1H-benzo[d] imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (5b)

Yield 0.40 g (85%) by method B, MP 205–2071C. 1H NMR d: 2.15 (6H, s, 2CH3), 4.00 (3H, s, CH3), 7.20–7.46 (2H, m, Ar-H), 7.50–7.95 (9H, m, Ar-H), 10.70 (1H, s, NH), 11.20 (1H, s, NH pyrazole). IR (KBr; cm–1): 3345 (NH), 1710–1720 (C = O), 1640 (C = N). MS: m/z = 471 [M + ]. Anal. calcd for C29H21N5O2: C, 73.87; H, 4.49; N, 14.85. Found: C, 73.84; H, 4.55; N, 14.90. General procedure for the synthesis of 3-(1,5-dimethyl-3oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)-5-(4(dimethylamino)phenyl)-2-(1,5,6-trimethyl-1H-benzo[d] imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (6a) and 5-(anthracen-9-yl)-3-(1,5-dimethyl-3-oxo-2-phenyl2,3-dihydro-1H-pyrazol-4-ylimino)-2-(1,5,6-trimethyl-1Hbenzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4carbonitrile (6b)

Compound 5a (1 mmol, 0.41 g) or 5b (1 mmol, 0.47 g) was dissolved in absolute ethanol (25 ml), followed by the addition of 4-aminoantipyrine (1 mmol, 0.20 g) in the presence of acetic acid (2 ml). The reaction mixture was heated under reflux for 8 h. After cooling, the solvent was evaporated under vacuum and the precipitated product was crystallized from ethanol to afford 6a or 6b. 3-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol4-ylimino)-5-(4-(dimethylamino)phenyl)-2-(1,5,6-trimethyl1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole4-carbonitrile (6a)

Yield 0.5 g (85%), MP 168–1701C. 1H NMR d: 2.10 (6H, s, 2CH3), 4.00 (3H, s, CH3), 6.85–7.10 (4H, m, Ar-H), 7.20–7.46 (2H, m, Ar-H), 10.70 (1H, s, NH). IR (KBr; cm–1): 3358 (NH), 1640–1645 (C = N), 1690, 1685 (C = O), 2100 (CN). MS: m/z = 598 [M + – 1]. Anal. calcd for C34H33N9O2: C, 68.10; H, 5.55; N, 21.02. Found: C, 68.02; H, 5.50; N, 20.96. 5-(Anthracen-9-yl)-3-(1,5-dimethyl-3-oxo-2-phenyl-2,3dihydro-1H-pyrazol-4-ylimino)-2-(1,5,6-trimethyl-1H-benzo[d] imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (6b)

Yield 0.54 g (83%) MP 177–1791C. 1H NMR d: 2.10 (6H, s, 2CH3), 4.00 (3H, s, CH3), 7.20–7.46 (2H, m, Ar-H), 7.50–7.95 (9H, m, Ar-H), 10.90 (1H, s, NH). IR (KBr; cm–1): 3369 (NH), 1640–1665 (C = N), 1690, 1680 (C = O), 2120 (CN). Anal. calcd for. C40H32N8O2: C, 73.15; H, 4.91; N, 17.06. Found: C, 73.11; H, 4.87; N, 16.96. Synthesis of compounds 7 and 12

Compounds 7 and 12 were synthesized using the same procedure as that described for the synthesis of


compound 1 and were obtained in 77 and 74% yields, respectively. Ethyl 5-benzoyl-1H-benzo[d]imidazole-2-carboxylate (7): Yield (77%), MP 158–160 1C. 1H NMR d: 2.25 (3H, s, CH3), 3.80 (2H, q, CH2), 7.4 0–7.60 (2H, m, Ar-H), 7.80–7.90 (4H, m, Ar-H), 11.80 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 1700 (C = O), 1660 (C = N). Anal. calcd for C16H14N2O3: C, 68.00, H, 6.90, N, 9.90. Found: C, 68.15; H, 6.82; N, 9.81. Synthesis of compounds 8 and 13

Compounds 8 and 13 were synthesized by adopting the general procedure used for the preparation of compound 2 and were obtained in 75 and 78% yields, respectively. 5-Benzoyl-1H-benzo[d]imidazole-2-carbohydrazide (8): Yield (75%), MP 182–1841C. 1H NMR d: 5.60 (2H, s, NH2), 7.4 0–7.60 (2H, m, Ar-H), 7.80–7.90 (4H, m, Ar-H), 11.80 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 3250 (NH2), 1700 (C = O), 1660 (C = N). Anal. calcd for C15H12N4O: C, 68.18; H, 5.30; N, 10.60. Found: C, 68.1; H, 5.23; N, 10.61. General procedure for the synthesis of compounds 9–11

To a solution of compound 8 (3 mmol, 0.792 g) in dry DMF (5 ml), POCl3 was added dropwise (6 mmol, 1 ml), followed by 3 mmol of each acid, namely crotonic acid (3 mmol, 0.30 g), 3,4-diaminobenzoic acid (3 mmol, 0.75 g), and 6-hydroxy-4-methoxybenzofuran-5-carboxylic acid (3 mmol, 0.63 g). The reaction mixture was stirred at room temperature for 15 min, thereafter at 80–901C for 5 h. After cooling, the reaction mixture was poured into crushed ice and neutralized by NaHCO3 (20%). The precipitated product 9, 10, or 11 was filtered, washed with ice water, dried, and crystallized from ethanol. Phenyl(2-(5-(prop-1-enyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)methanone (9): Yield 0.25 g (75%), MP 223–2251C. 1H NMR d: 2.20 (3H, d, CH3), 5.80 (1H, d, CH), 5.95 (1H, m, CH), 7.4 0–7.60 (2H, m, Ar-H), 7.80–7.90 (4H, m, Ar-H), 11.80 (1H, s, NH). IR (KBr) cm–1: 3320 (NH), 1700 (C = O), 1640–1645 (C = N). MS: m/z = 330 [M + ]. Anal. calcd for C19H14N4O2: C, 69.08; H, 4.27; N, 16.96. Found: C, 69.12; H, 4.20; N, 16.90. (2-(5-(3,4-Diaminophenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(Phenyl) methanone (10): Yield 0.308 g (78%), MP 188–190 1C. 1H NMR d: 5.60 (2H, m, NH2), 6.80–7.10 (3H, m, Ar-H), 7.40–7.60 (2H, m, Ar-H), 7.80–7.90 (4H, m, Ar-H), 11.80 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 1700 (C = O), 1640–1650 (C = N). MS: m/z = 396 [M + ]. Anal. calcd. for C22H16N6O2: C, 66.66; H, 4.07; N, 21.20. Found: C, 66.71; H, 21.25; N, 8.12. (2-(5-(6-Hydroxy-4-methoxybenzofuran-5-yl)-1,3,4-oxadiazol2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (11): Yield 0.346 g (77%), MP 198–2001C. 1H NMR d: 2.35 (3H, s, CH3), 3.60 (3H, s, OCH3), 6.40–6.60 (2H, m, CH furan), 6.70 (1H, m, CH furan), 6.90 (1H, m, CH aromatic), 7.40–7.60 (4H, m, Ar-H), 7.80–7.90 (2H, m, Ar-H), 11.80

83

(1H, s, NH). IR (KBr; cm–1): 3320 (NH), 1700 (C = O), 1640–1655 (C = N). Anal. calcd for C25H16N4O5: C, 66.30; H, 3.54; N, 12.39. Found: C, 66.39; H, 3.59; N, 12.45. Ethyl 8-cyano-6-isocyano-5-oxo-7-phenyl-1,5-dihydro-[1,2,4] triazolo[1,5-a]pyridine-2-carboxylate (12): Yield 0.215 g (74%), MP 236–2381C. 1H NMR d: 2.20 (3H, s, CH3), 3.80 (2H, q, CH2), 7.30–7.75 (4H, m, Ar-H), 10.90 (1H, s, NH). IR (KBr; cm–1): 3350 (NH), 1720 (C = O), 2120–2127 (CN). Anal. calcd f or C16H11N5O3: C, 63.90; H, 3.78; N, 24.05. Found: C, 63.82; H, 3.70; N, 24.10. 8-Cyano-6-isocyano-5-oxo-7-phenyl-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-2-carbohydrazide (13): Yield 0.25 g(78%), MP193–1951C. 1H NMR d: 5.80 (1H, m, CH), 5.90 (1H, d, CH), 6.50 (2H, s, NH2), 7.30–7.75 (4H, m, Ar-H), 9.80 (1H, s, NH), 10.90 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 3210 (NH2), 1710 (C = O), 2150–2155 (CN). Anal. calcd for C14H9N7O2: C, 52.33; H, 2.80; N, 34.89. Found: C, 52.28; H, 2.72; N, 34.82.

General procedure for the synthesis of compounds 14–16

To a solution of compound 13 (1 mmol, 0.321 g) in dry DMF, POCl3 (5 ml) was added dropwise (3 mmol, 0.50 ml), followed by the addition of 1 mmol of each of the following carboxylic acids: crotonic acid, 3,4-diaminobenzoic acid, or 6-hydroxy-4-methoxybenzofuran-5carboxylic acid. The reaction mixture was stirred at room temperature for 15 min and then at 80–901C for 6 h. After cooling, the reaction mixture was poured into crushed ice and neutralized by NaHCO3 (20%); the precipitated products 14, 15, or 16 were filtered, washed with ice water, dried and, crystallized from methanol. 6-Isocyano-5-oxo-7-phenyl-2-(5-(prop-1-enyl)-1,3,4-oxadiazol2-yl)-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (14): Yield 0.30 g (82%), MP 239–2411C. 1H NMR d: 2.28 (3H, d, CH3), 5.80 (1H, m, CH), 5.90 (1H, d, CH), 7.30–7.75 (4H, m, Ar-H), 10.90 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 1710 (C = O), 2150–2156 (CN). MS: m/z = 369 [M + ]. Anal. calcd for C19H11N7O2: C, 61.79; H, 3.00; N, 26.55. Found: C, 61.84; H, 3.06; N, 26.59. 2-(5-(3,4-Diaminophenyl)-1,3,4-oxadiazol-2-yl)-6-isocyano-5oxo-7-phenyl-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitril (15): Yield 0.34 g (80%), MP 220–2221C. 1H NMR d: 5.60 (2H, m, NH2), 6.90–7.10 (3H, m, Ar-H), 7.30–7.75 (5H, m, Ar-H), 10.90 (1H, s, NH). IR (KBr; cm–1): 3320 (NH), 1710 (C = O), 2140–2150 (CN). Anal. calcd for C22H13N9O2: C, 60.69; H, 3.01; N, 28.95. Found: C, 60.75; H, 3.06; N, 29.05. 1,5-Dihydro-2-(5-(6-hydroxy-4-methoxybenzofuran-5-yl)-1,3,4oxadiazol-2-yl)-6-isocyano-5-oxo-7-phenyl-[1,2,4]triazolo[1,5-a] pyridine-8-carbonitrile (16): Yield 0.40 g (81%), MP 230–2321C. 1H NMR d: 2.35 (3H, s, CH3), 3.60 (3H, s, OCH3), 6.40–6.60 (2H, m, CH furan), 6.70 (1H, m, CH furan), 6.90 (1H, m, CH aromatic), 7.30–7.75 (5H, m, Ar-H), 10.90 (1H, s, NH). IR (KBr; cm – 1): 3320 (NH), 1710 (C = O), 2150–2156 (CN). Anal. calcd forC25H13N7O5: C, 61.09; H, 2.64; N, 19.96. Found: C, 61.15; H, 2.70; N, 20.09.

84


amounts of acetic acid to give 3-(1,5-dimethyl-3-oxo-2phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)-5-(4-(dimethylamino)phenyl)-2-(1,5,6-trimethyl-1H-benzo[d]imidazole2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (6a) and -5-(anthracen-9-yl)-3-(1,5-dimethyl-3-oxo-2-phenyl2,3-dihydro-1H-pyrazol-4-ylimino)-2-(1,5,6-trimethyl-1Hbenzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4carbonitrile (6b), respectively (Scheme 2).

Results and discussion Compound 5,6-dimethyl-1H-benzo[d]imidazole-2-carbohydrazide (2) was prepared by the condensation reaction of 5,6-dimethyl-ethyl-2-benzimidazole carboxylate (1) with hydrazine hydrate in the presence of absolute ethanol. Schiff’s bases N0 -(4-(dimethylamino)benzylidene)5,6-dimethyl-1H-benzo[d]imidazole-2-carbohydrazide (3a) and N0 -(anthracen-9-ylmethylene)-5,6-dimethyl-1H-benzo [d]imidazole-2-carbohydrazide (3b) were obtained by the reaction of 2 with 4-(dimethylamino) benzaldehyde and anthracene-9-carbaldehyde in ethanol, respectively, in the presence of a few drops of piperidine. The target compounds 2-(5,6-dimethyl-1H-benzo[d]imidazole-2-carbonyl)-5-(4-(dimethylamino)phenyl)-3-oxo-2,3-dihydro-1 H-pyrazole-4-carbonitrile (4a) and 5-(anthracen-9-yl)-2(5,6-dimethyl-1H-benzo[d]imidazole-2-carbonyl)-3-oxo-2,3dihydro-1H-pyrazole-4-carbonitrile (4b) were synthesized by the reaction of 3a and 3b with ethyl cyanoacetate in ethanol in the presence of triethylamine, respectively. Methylation of 4a and 4b was achieved by their reaction with methyl iodide or DMC that yielded 5-(4-(dimethylamino)phenyl)-3-oxo-2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (5a) and 5-(anthracen-9-yl)-3-oxo-2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (5b), respectively (Scheme 1). Their structures were established on the basis of elemental analysis and spectral data.

In Scheme 3, ethyl 5-benzoyl-1H-benzo[d]imidazole-2carboxylate (7) and 5-benzoyl-1H-benzo[d]imidazole2-carbohydrazide (8) were prepared according to the method used for the synthesis of compounds 1 and 2. Compounds 9–11 were formed by intermolecular cyclization of the hydrazide derivative 8 through a condensation reaction with crotonic acid, 3,4-diaminobenzoic acid, and 6-hydroxy-4-methoxybenzofuran-5-carboxylic acid in the presence of POCl3 and DMF that yielded the corresponding substituted oxadiazol derivatives identified as phenyl(2-(5-(prop-1-enyl)-1,3,4-oxadiazol-2-yl)-1H-benzo [d]imidazol-5-yl)methanone (9), 2-(5-(3,4-diaminophenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (10), and (2-(5-(6-hydroxy-4-methoxybenzofuran-5-yl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5yl)(phenyl)methanone (11), respectively. The structures of compounds 9–11 were confirmed on the basis of their elemental analysis and spectral data (cf. Experimental data).

Furthermore, compounds 6a or 6b were synthesized by the following reaction: 5a and 5b reacted with 4-aminoantypyrine in ethanol and in the presence of catalytic

Moreover, the condensation reaction of compound 12 with hydrazine hydrate yielded 8-cyano-6-isocyano-5-oxo7-phenyl-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-2-car-

Scheme 1 H C

N

H C

N H

COOC H

i

1

H C

N

H C

N H

CONHNH

ii

2 O H N

H C

N

H C

N H

CONHN

iii

CHAr

4a,b

HC

CH

O

O

N

N N

CN

iv

HN

5a,b

HC

CN

HN Ar

3a,b

HC

N

HC

O N

Ar

HC

Ar : a =

N

;

b=

HC

Synthesis of compounds 1–5a,b. Condition and reagents: (i) NH2NH2 H2O/ethanol/reflux, (ii) ArCHO/ethanol/piperidine/reflux, (iii) ethyl cyanoacetate/ethanol/TEA/reflux, (iv) method A: MeI/NaH/DMF/RT, method B: DMC/K2CO3/DMF/reflux.


85

Scheme 2 CH3 H3 C

N

CH3

H3 C

O

N N

H3 C

N

+

O

N

H2 N

HN O Ar

C N

5a,b

i

CH3

H3 C

N

N

N

H3 C

CH3

O H3 C N

N

N

HN Ar

C N

O

6a,b

H3 C

Ar : a =

N

;

b=

H3 C

Synthesis of compounds 6a,b. Condition and reagents: (i) ethanol/acetic acid/reflux.

bohydrazide (13), which was reacted with crotonic acid, 3,4-diaminobenzoic acid, or 6-hydroxy-4-methoxybenzofuran-5-carboxylic acid in the presence of POCl3 in DMF to produce the corresponding derivatives 6-isocyano-5oxo-7-phenyl-2-(5-(prop-1-enyl)-1,3,4-oxadiazol-2-yl)-1,5dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (14), 2-(5-(3,4-diaminophenyl)-1,3,4-oxadiazol-2-yl)-6-isocyano5-oxo-7-phenyl-1,5-dihydro-[1,2,4]triazolo[1,5-a]pyridine-8carbonitrile (15), or 1,5-dihydro-2-(5-(6-hydroxy-4-methoxybenzofuran-5-yl)-1,3,4-oxadiazol-2-yl)-6-isocyano-5-oxo7-phenyl-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (16), respectively (Scheme 3). The structure of compounds 14–16 was assigned on the basis of their elemental analyses and spectral data. Biological screening Antioxidant activity

2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) scavenging assay Compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16 were prepared in DMSO as 10 stocks from each of the tested concentrations (0, 10 – 3, 10 – 4, 10 – 5, 10 – 6 and 10 – 7 mol/l) and briefly sonicated when necessary in an ultrasonic water bath. The compounds were submitted for testing to determine the effective concentration of the compound producing 50% scavenging of the DPPH (EC50). Two reference radical scavengers, quercetin, and

gallic acid were tested in the assay as positive controls. The method used in the present study was based on previously published methods in literature. The compound stock solutions (15 ml/well) were pipetted in duplicates into 96-well plates. The assay was started with the addition of DPPH reagent (0.004% w/v) in methanol (135 ml/well). Appropriate negative controls were simultaneously run using methanol as a correction for the optical density of colored compounds at 540 nm. The plate was immediately shaken for 30 s and incubated in the dark for 30 min at room temperature. The remaining DPPH was measured in a microplate reader (BMG Fluostar Optima, Ortenberg, Germany) at 540 nm. The percentage of antioxidant activity (%AA) was calculated using the following equation: % Antioxidant activity ð % AAÞ¼ 100

½OD540 nm ðblankÞ OD540 nm ðsampleÞ : OD540nm ðblankÞ


Regression analysis was used to determine the EC50 values for each compound using the concentration–%AA relationship. All data were represented as the mean value of the duplicate absorbance measurement. Table 1 illustrates the antioxidant effects of the tested compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16,

86


Scheme 3 N

H N

a

N

O

N

O

H N N

COOC2 H5

H N

i

CONHNH2

ii

9

O

O

7

N NH2

O

N

N

O

N

H N

b

NH2

10

8

H N

c

N

N

N

OH

O

O H3CO

11

a

O CN

N N

O

N H

CN

N N CN

CN COOC2 H5

N H

i

N N CN

N H

CONHNH2

ii

b

14

O CN

NH2

N N

O NH2

N H

CN

12

N

N

CN

O

O

O

N

N

15

13

c NC

O

H3CO

O N

N

O

N H

N

N

OH

CN

16

a: CH3CH=CHCOOH, b: (NH)2C6H3COOH, OCH3 COOH

C:

O

OH

Synthesis of compounds 7–11 and 12–16. Condition and reagents: (i) NH2NH2.H2O/ethanol/reflux, (ii) POCl3/DMF/ (a) crotonic acid, (b) 3,4diaminobenzoic acid, (c) 6-hydroxy-4-methoxybenzofuran-5-carboxylic acid.

represented as EC50 values. The compounds showed antioxidant activities against the DPPH radical in the following order of higher activity (lower EC50): 1541141041641444b45a46b45b44a. As shown in Table 1, compounds 2-(5-(3,4-diaminophenyl)-1,3,4-oxadiazol-2-yl)-6-isocyano-5-oxo-7-phenyl-1,5dihydro-[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (15), (2-(5-(6-hydroxy-4-methoxybenzofuran-5-yl)-1,3,4-oxadiazol2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (11), and 2-(5-(3,4-diaminophenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (10) showed the highest anti-

oxidant activity compared with other tested compounds and their activities were comparable with the activities of the reference antioxidants quercetin and gallic acid. Antimicrobial activity

The antibacterial and antifungal activities of the tested compounds 4a, 4b, 5b, 6a, 6b, 10, 11, 14, 15, and 16 were estimated using the diffusion plate method. A sterilized filter paper disc saturated with a measured quantity (25 ml) of the sample (1 mg/ml) was placed on a plate (9 cm in diameter) containing a solid bacterial

Synthesis, antioxidant, antimicrobial effects Bassyouni et al. 87

medium (nutrient agar) or a fungal medium (potato dextrose agar) that had been seeded with the spore suspension of the test organism. After incubation of the bacterial culture at 371C for 24 h (in the case of fungi, 251C for 72 h), the diameter of the clear zone of inhibition surrounding the sample is taken as a measure of the inhibitory power of the sample against the particular test organism (% inhibition = sample inhibition zone (cm)/plate diameter 100). All measurements were taken with methanol as a solvent, which has zero inhibitory activity [22–26].

1H-pyrazole-4-carbonitrile (6b) exhibited high antimicrobial activity against S. aureus, reaching an 18 mm clear zone; other compounds showed variable antibacterial activity with different extents. With respect to C. albicans, compounds 2-(5-(3,4-diaminophenyl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (10) and (2-(5-(6-hydroxy-4-methoxybenzofuran5-yl)-1,3,4-oxadiazol-2-yl)-1H-benzo[d]imidazol-5-yl) (phenyl)methanone (11) exhibited significantly high activity against the species.

The antimicrobial activity of the tested compounds 4a, 4b, 5b, 6a, 6b, 10, 11, 14, 15, and 16 was examined with gram-positive bacteria Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus, gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium, and the fungus Candida albicans. The obtained results were compared with that of the reference antibiotic amoxicillin that was purchased from Pfizer (Cairo, Egypt).

Antioxidant quantitative structure–activity relationships study

The flexagen algorithm develops predictive pharmacophore models with both activity and structural data of the training set molecules [27]. To guarantee the construction of robust models, it is crucial to generate a representative training set with sufficient coverage of both biological and chemical spaces occupied by the original data set. Therefore, diversity sampling of the original data set by considering both activity and structural information is the premise for rational selection of training sets. Furthermore, simultaneous inclusion of several similar compounds in the training sets should be avoided, as it may only provide redundant information and bias the resulting model toward those similar structures. Other important guidelines are in the literature [28]. Pharmacophoric hypotheses are important tools in drug design and discovery as they provide excellent insights into ligand macromolecule recognition. However, their predictive value as three-dimensional (3D)-QSAR models is limited by steric clashes and bioactivity enhancing or reducing auxiliary groups. This point, combined with the fact that pharmacophore modeling of antioxidant activity furnished numerous binding hypotheses of comparable success criteria, prompted us to use classical QSAR analysis to search for the best combination of pharmacophore(s) and 2D descriptors capable of explaining bioactivity variation. Furthermore, 3D QSAR modeling was implemented in the current case as grounds of competition to select the best pharmacophore(s) that could explain bioactivity variation across the whole training list.

Table 2 shows that compounds 2-(5,6-dimethyl-1H-benzo [d]imidazole-2-carbonyl)-5-(4-(dimethylamino)phenyl)3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (4a) and (2(5-(6-hydroxy-4-methoxybenzofuran-5-yl)-1,3,4-oxadiazol-2yl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (11) were the most active compounds against S. typhimurium, whereas compounds 5-(anthracen-9-yl)-3-oxo-2-(1,5,6-trimethyl-1Hbenzo[d]imidazole-2-carbonyl)-2,3-dihydro-1H-pyrazole-4carbonitrile (5b) and 5-(anthracen-9-yl)-3-(1,5-dimethyl-3oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)-2-(1,5,6-trimethyl-1H-benzo[d]imidazole-2-carbonyl)-2,3-dihydro-

Table 1 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity (cell-free system) of compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16 represented as EC50 values Compounds

DPPH EC50 (mmol/l) 1135.0 186.1 227.9 412.7 361.8 32.9 27.8 133.8 23.3 90.4 8.9 7.1

4a 4b 5a 5b 6b 10 11 14 15 16 Quercetin Gallic acid

All computational work was carried out using the molecular operating environment (MOE) program (Chemical Computing Group Inc., Quebec, Canada; 2008). All compounds were drawn using Chem (Cambridge Soft Corporation, Cambridge, Massachusetts, USA). Draw 11,

DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; EC50, effective concentration of the compound producing 50% scavenging of the DPPH.

Table 2 The antibacterial and antifungal activities of the tested compounds 4a, 4b, 5b, 6a, 6b, 10, 11, 14, 15, and 16 Inhibition zone diameter (mm/mg sample) Microorganism Bacillus cereus Bacillus subtilis Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Candida albicans Salmonella typhimurium

Gram-stain reaction

4a

4b

5b

6a

6b

10

11

14

15

16

Amoxicillin

Positive Positive Negative Negative Positive Fungi Negative

10 – 7 10 10 – 40

7 – 12 9 10 – –

9 10 8 11 18 – –

7 7 8 11 10 – –

10 9 – 13 18 – 11

8 8 – 9 9 17 10

8 7 – 11 9 14 16

8 9 – 11 10 – 10

– – 10 – – 12 –

10 8 – 10 10 10 11

22 25 22 30 16 25 20

88


Figure 1

Correlation plot showing a linear relationship between the actual and predicted activities of compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16.

and energy was minimized by force-field MMFF94 optimization with a gradient of 0.0001 for determining the low-energy conformations with the most favorable (lowest energy) geometry. The purpose of a QSAR descriptor is to calculate properties of molecules that serve as numerical descriptions or to characterize molecules that are correlated with activity. The descriptors computed using MOE could be classified into three classes: 2D descriptors based on the atom and connection information of the molecule, internal 3D descriptors (i3D) based on the 3D information on each molecule and variant to rotation and translation of the conformation, and external descriptors (x3D) based on the 3D information, fitting the antioxidant activity of the dependent variable pKi to that of the independent variables, namely the molecular descriptors.

molecular properties’ protocol implemented in the QSAR module. The PCA method was then applied using the ‘calculate principal components’ protocol in the ‘library analysis’ module to extract three principal components. The program initially generated more than 400 descriptors for each compound and it is predicted that some of the descriptors are highly correlated. Therefore, the PCA method was applied to reduce the dimensionality of the descriptor space and alleviate the correlations [29]. Basically, the PCA method is a mathematical procedure that converts multiple sets of possibly correlated variables into a few orthogonal ‘principal components’ that are usually linear combinations of the correlated variables, each corresponding to an axis in multiple-dimensional space, as represented by the following equation: v X Cij xj ; ð1Þ PCi ¼ j¼1

Partial least squares

Partial least squares analysis was used to derive linear equations from the resulting matrices. Leave one out cross-validation was used to select the number of principal components and to calculate the cross-validated statistics. Regression analysis modules of statistical analysis tool were used to build the 3D QSAR models. Regression analysis was carried out using the pKi activity (antioxidant activity) as the dependent variable and the calculated descriptor as the predicted variable. As shown in the Blow correlation plot, a linear relationship exists between the actual and predicted activities; the correlation coefficient is 0.817603 (Fig. 1). Principal component analysis

The 2D molecular properties of 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16 were estimated using the ‘calculate

where PCi is the principle component, Cij is the coefficient of the variable xj, and is the number of variables. For clear graphical representation of the molecular diversity, we extracted three principal components that account for the variation in the descriptor space and plotted the molecules as discrete spots in a 3D coordinated system (Fig. 2). Pharmacophore generation

The pharmacophore generation protocol used a flex align algorithm [30]. The features of the hydrogen bond acceptor, hydrogen bond donor, hydrophilic or hydrophobic aromatic center, and the aromatic ring were predefined using a stochastic search conformation, and the parameter of ‘maximum excluded volumes’ (MEV)

Synthesis, antioxidant, antimicrobial effects Bassyouni et al. 89

Figure 2

The three-dimensional scatter plot is a visual representation of the molecules as described by the three selected principal components (PCA1, PCA2, and PCA3). Each point corresponds to a molecule and is colored according to the molecule’s pKi value.

Figure 3

The three-dimensional quantitative structure–activity relationships pharmacophore generated from compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16 showed a hydrophobic center (green), two aromatic planar ring centers (orange), and a hydrogen bond acceptor (blue).

had a value of 5. The fundamental approach for pharmacophore elucidation described herein is to exhaustively search for all pharmacophore queries that induce good overlay of most of the active molecules

(Fig. 3). Thus, the plausibility of the pharmacophore is measured by overlay of actives, and the relationship with activity is measured by classification accuracy and HipHop/HypoGen methods described in attempt to

90


Figure 4

The ligand interaction and the binding mode of the native ligand sulfamethaxazole (O8D) showed one H-bond donor with HOH 333 with a distance of 2.76 (black color); it bonded with one H-bond acceptor with SER 222 at a distance of 2.9 (blue color) and one H-bond acceptor with HOH 289 (black color) depicted as hatched line. It gave a score of – 13.0424.

relate common feature geometries with activity or complexity [31].

potency profile for the antioxidant activity of the tested compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16.

Sensitivity and specificity

The sensitivity and specificity of the models should be established during validation to assess the propensity of the QSAR models for correct qualitative prediction of the dependent variable [32]. Sensitivity can be calculated as follows: TPFN Sensitivity ð % Þ¼100 : ð2Þ TP Specificity can be calculated as follows: TPFP Specificity ð % Þ¼100 : TP

ð3Þ

TP – FN represents the number of corrected predictions, TP represents the number of true positives, and TN represents the number of true negatives. To confirm the probability of identifying true selective molecules, sensitivity and specificity tests were performed on the observed versus predicted selectivity values for the dataset. The observed values for sensitivity and specificity were 90 and 93%, respectively. These results were in accordance with those expected from the earlier examination of probability of predicting the

Antibacterial molecular docking study

Molecular docking was performed and analyzed with the MOE program. Docking calculations were carried out using standard default variables for the MOE program. The binding affinity was evaluated from the binding free energies (S-score, kcal/mol), hydrogen bonds, and root mean square deviation values. Compound 4a docked into the same groove of the binding site of the native cocrystallize ligand. Scoring in MOE software was performed using the London dG scoring function and enhanced using two different refinement methods; the force-field and grid-min poses were updated to ensure that refined poses satisfy the specified conformations. Rotatable bonds were allowed; the best 10 poses were retained and analyzed for the binding pose’s best score. Energy was minimized through force-field MMFF94 optimization with a gradient of 0.0001 for determining low-energy conformations with the most favorable (lowest energy) geometry. The antibiotic sulfamethaxazole was used as a reference for the docking study. It inhibits the dihydropteroate synthase enzyme, a key enzyme in the folate pathway.


91

Figure 5

The ligand interaction and the binding mode of the compound 4a. It binds with one H-bond the acceptor with HOH 333 at a distance of 0.96. It gave a score of – 13.5451 kcal/mol greater than that of the cocrystallized ligand.

Figure 6

Superposition of compound 4a (blue color). The cocrystallized ligand sulfamethaxazole (O8D; gray color) showed binding with HOH 333 as a cocrystallized ligand. Compound Ligand 4a

Score

H-bond involved

– 13.0424 HOH 333, SER 222, HOH 289 – 13.5451 HOH 333


Compound 4a was investigated for its binding affinity with the dihydropteroate synthase receptor (pdb 3TZF) [33] for the purpose of lead optimization and to study the interaction between compound 4a and the dihydropteroate synthase receptor (Figs 4–6).

8 Boido A, Vazzana I, Mattioli F, Sparatore F. Antiinflammatory and antinociceptive activities of some benzotriazolylalkanoic acids. Farmaco 2003; 58:33–44. 9 Biagi G, Calderone V, Giorgi I, Livi O, Scartoni V, Baragatti B, Martinotti E. Some structural changes on triazolyl-benzotriazoles and triazolyl-benzimidazolones as potential potassium channel activators. III. Farmaco 2001; 56:841–849. 10 Katritzky AR, Lan X, Yang JZ, Denisko OV. Properties and synthetic utility of n-substituted benzotriazoles. Chem Rev 1998; 98:409–548.

Conclusion QSAR between the molecular structure and the inhibitory antioxidant activity of the synthesized compounds 4a, 4b, 5a, 5b, 6b, 10, 11, 14, 15, and 16 were studied. Compounds 15, 11, and 10 displayed the highest antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. The antimicrobial activity showed that compound 4a was the most active against S. typhimurium and its activity exceeded the activity of the reference antibiotic amoxicillin. Compounds 5b and 6b exhibited high antimicrobial activity against S. aureus, whereas compounds 10 and 11 showed significantly high activities against C. albicans. The molecular modeling study showed that compound 4a gave score of – 13.5451 kcal/mol, which was greater than the score of the cocrystallized ligand sulfamethoxazole (– 13.0 kcal/mol).

Acknowledgements The authors thank the National Research Center, Cairo, Egypt, and the Science and Technology Development fund (STDF Grant No.: 1169) for financial support.


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11 Parmar SS, Gupta AK, Singh HH, Gupta TK. Benzimidazolyl-1,2,4(H)triazoles as central nervous system depressants. J Med Chem 1972; 15:999–1000. 12 Hwang LC, Tu CH, Wang JH, Lee GH. Synthesis and molecular structure of 6-amino-3-benzylmercapto-1,2,4-triazolo[3,4-f][1,2,4]triazin-8(7H)-one. Molecules 2006; 11:169–176. 13 Hiremath SP, Ullagaddi A, Shivaramayya K, Purohit MG. Amino acid derivatives, VI: synthesis, antiviral, and antimicrobial evaluation of amino acid esters bearing a 1,2,3-triazolo[4,5-d]pyrimidinedione side chain. Indian J Heterocycl Chem 1999; 3:145–148. 14 Shivarama Holla B, Veerendra B, Shivananda MK, Poojary B. Synthesis characterization and anticancer activity studies on some Mannich bases derived from 1,2,4-triazoles. Eur J Med Chem 2003; 38:759–767. 15 Kritsanida M, Mouroutsou A, Marakos P, Pouli N, Papakonstantinou-Garoufalias S, Pannecouque C, et al. Synthesis and antiviral activity evaluation of some new 6-substituted 3-(1-adamantyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles. Farmaco 2002; 57:253–257. 16 John BW. The chemistry of the benzimidazoles. Chem Rev 1951; 84: 397–541. 17 Klein ATJ, Holschbach M. Labelling of the solvent DMSO as side reaction of methylations with n.c.a. [11C]CH 3I. Appl Radiat Isot 2001; 55:309–313. 18 Ouk S, Thie´baud S, Borredon E, Chabaud B. N-methylation of nitrogencontaining heterocycles with dimethyl carbonate. Syn Commun 2005; 35:3021–3026. 19 Braca A, De Tommasi N, Di Bari L, Pizza C, Politi M, Morelli I. Antioxidant principles from Bauhinia tarapotensis. J Nat Prod 2001; 64:892–895. 20 Nara K, Miyoshi T, Honma T, Koga H. Antioxidative activity of bound-form phenolics in potato peel. Biosci Biotechn Biochem 2006; 70:1489–1491. 21 Hamed A. In ‘‘Investigation of multiple cytoprotective actions of some individual phytochemicals and plant extracts’’. Chapter 7: Development of cellfree and intracellular screening systems for testing antioxidant properties of plant extracts. PhD Thesis (Biomedical Sciences), the University of Nottingham, United Kingdom; 2009. 22 Pelczar MJ, Chan EC, Kruz NR. Microbiology. 5th ed. New Delhi: Tata McGraw-Hill Publishing Company Ltd.; 2006. 23 Grayer RJ, Harborne JB. A survey of antifungal compounds from higher plants, 1982–1993. Phytochemistry 1994; 37:19–42. 24 Irobi ON, Moo-Young M, Anderson WA, Daramola SO. Antimicrobial activity of bark extracts of Bridelia ferruginea (Euphorbiaceae). J Ethnopharmacol 1994; 43:185–190. 25 Jawetz E, Melnick JL, Adelberg EA. Review of Medical Microbiology. Los Atos, California: Lang Medical Publication; 1974. 26 Muanza DN, Kim BW, Euler KL, Williams L. Antibacterial and antifungal activities of nine medicinal plants from Zaire. Int J Pharmacogn 1994; 32:337–345. 27 Fang C, Xiao Z, Guo Z. Generation and validation of the first predictive pharmacophore model for cyclin-dependent kinase 9 inhibitors. J Mol Graphics Modell 2011; 29:800–808. 28 Labute P, Williams C, Feher M, Sourial E, Schmidt JM. Flexible alignment of small molecules. J Med Chem 2001; 44:1483–1490. 29 Kurogi Y, Gu¨ner OF. Pharmacophore modeling and three-dimensional database searching for drug design using catalyst. Curr Med Chem 2001; 8:1035–1055. 30 Leach AR. Molecular Modeling: principles and applications. 2nd ed. New York: Prentice Hall Pearson Ed. Ltd.; 2001. 31 Guner OF. Pharmacophore perception, development and use in drug design. La Jolla, CA: International University Line; 2000. 32 Walker JD, Carlsen L, Jaworska J. Improving opportunities for regulatory acceptance of QSARs: the importance of model domain, uncertainty, validity and predictability. QSAR and Combinatorial Science 2003; 22:346–350. 33 Yun M-K, Wu Y, Li Z, Zhao Y, Waddell MB, Ferreira AM, et al. Catalysis and sulfa drug resistance in dihydropteroate synthase. Science 2012; 335:1110–1114.

Original article 93

Characterization and purification of the crude Trematosphaeria mangrovei laccase enzyme

Atalla M. Mabrouka, Zeinab H. Kheirallab, Eman R. Hameda, Amani A. Youssryb and Abeer A. Abd El Atya a Department of Chemistry of Natural and Microbial Products, National Research Centre and bDepartment of Botany, Faculty of Girls for Arts, Science and Education, Ain Shams University, Cairo, Egypt

Correspondence to Eman R. Hamed, PhD, Department of Chemistry of Natural and Microbial Products, National Research Centre, 12311 Dokki, Cairo, Egypt Tel: + 20 2 33464472; fax: + 20 2 37622603; e-mail: [email protected] Received 4 April 2012 Accepted 2 September 2012 Egyptian Pharmaceutical Journal 2012, 11:93–98

Objectives The aim of this work was to study the purification and characterization of the crude extracellular laccase produced by the marine-derived fungus Trematosphaeria mangrovei. Methods The general properties of the crude laccase enzyme produced by T. mangrovei were investigated. These include the effect of temperature, pH, thermal and pH stabilities, and enzyme and substrate concentrations on the laccase activity. Partial purification of the T. mangrovei laccase enzyme was carried out by fractional precipitation with ammonium sulphate, ethanol and acetone. Further purification was carried out on a Sephadex G-100 column. Results and conclusion The results obtained showed that the crude enzyme reached its maximal activity at 351C, pH 4.5, at an enzyme concentration of 5.429 mg protein/reaction mixture and at a substrate concentration of 40 mmol/l 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). The enzyme was stable for 60 min at 351C and retained about 80–90% of its activity after treatment for 60 min from 40 to 501C. The enzyme showed maximum stability (100%) at pH 4.5 and 91.6% at pH 4.0 after 60 min. Fractional precipitation of the fungal extracellular T. mangrovei laccase enzyme with different methods showed that the enzyme fraction precipitated at 60% acetone was the most favourable enzyme fraction; it showed 4.84 purification fold. Laccase obtained from the 50–60% acetone fraction was purified by Sephadex G-100. The final preparation thus obtained reached 31.47-fold that of the culture filtrate (1466.49 U/mg protein) and showed a single band on native polyacrylamide gel electrophoresis. Keywords: characterization, laccase, lignin-degrading enzymes, ligninolytic enzymes, marine-derived fungi, purification, Trematosphaeria mangrovei Egypt Pharm J 11:93–98 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Laccase (E.C.1.10.3.2, p-benzenediol : oxygen oxidoreductase) is a copper-protein belonging to a small group of enzymes denominated blue oxidase. It is an oxidoreductase that can catalyse the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water [1]. Moreover, in the presence of primary substrates that act as electron transfer mediators, such as 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) or 1-hydroxybenzotriazole, the substrate range can be extended to nonphenolic compounds [2]. Laccase or laccase-like activity has been found in higher plants, some insects and a few bacteria [3]. However, most known laccases are from fungi, especially from the white rot fungi. Finally, although most laccases have been characterized from white rot basidiomycetes, there are other groups of fungi (marine-derived fungi) that produce laccases but they have been studied to a much lesser extent [4].

Fungal laccases are involved in various processes in nature including the biodegradation of lignin [5] and their application in the detoxification of various aquatic and terrestrial pollutants and in the treatment of industrial wastewater has been suggested [6]. Laccase production by the filamentous marine-derived fungus Trematosphaeria mangrovei has been described in a previous study [7]. The characterization and purification of the crude laccase enzyme obtained from the optimized fermentation medium is described in this work.

Materials and methods Chemicals

ABTS diammonium salt was obtained from MP Bio (LCN) (USA). Sephadex G-100 was purchased from Fluka Company (Germany). The standard of the laccase


DOI: 10.7123/01.EPJ.0000419801.40087.2a

94


enzyme from Trametes versicolor was purchased from Sigma Chemical Company. The fungal strain and culture condition

The filamentous marine-derived fungus T. mangrovei used in this study was isolated from decayed wood samples collected from Abou Keer (Alexandria, Egypt), and identified in the National Research Centre, Chemistry of Natural and Microbial Products Department (Microbial Culture Collection Unit) according to Kohlmeyer and Kohlmeyer [8]. The final optimized medium composition was found to be as follows (g/l): 16 sucrose, 2 peptone, 1 yeast extract, 50% sea water and addition of copper sulphate (2.5 mmol/l) on the sixth day of incubation with a 20-day incubation period. The optimum initial pH was 6 during incubation at a temperature of 251C under static conditions. Assay of laccase activity

Laccase (EC 1.10.3.2) activity was measured using the method described by Bourbonnais et al. [9] on the basis of the oxidation of the substrate ABTS. The rate of ABTS oxidation was determined spectrophotometrically at 420 nm. The reaction mixture contained 600 ml sodium acetate buffer (0.1 M, pH 5.0 at 271C), 300 ml ABTS (5 mmol/l), 300 ml culture filtrate and 1400 ml distilled water. The mixture was then incubated for 2 min at 301C and the absorbance was measured immediately at 1-min intervals. One unit of laccase activity was defined as the activity of an enzyme that catalyses the conversion of 1 mol of ABTS per minute.

pH stability of the crude laccase

In the present experiment, the crude enzyme solution was subjected to different pH values using acetate buffer pH 3.5–5.5 and phosphate buffer pH 6.0–7.0 at 0.1 mol/l for each buffer for different periods at 351C. The residual activity was assayed after each incubation period. Effect of enzyme concentration

The reaction was carried out under standard conditions with varying amounts of crude enzyme (0.181–9.047 mg protein/reaction mixture), and then the enzyme activity was determined. Effect of substrate concentration

The effect of substrate concentration on the crude enzyme activity was studied using different concentrations of ABTS ranging from 1 to 100 mmol/l in the reaction mixture. The reaction was carried out at pH 4.5 at 351C and incubated for 2 min. Controls were prepared using the same substrate concentrations and dead enzyme. Partial purification of laccase enzyme

Fractional precipitation of the enzyme preparation was achieved with ammonium sulphate, ethanol or acetone. In all cases, an ice-salt bath was used and the precipitant was added to the cold culture filtrate until the required concentration was achieved. After isolating the precipitated fraction by centrifugation in a refrigerated centrifuge, the supernatant was subjected to further precipitation and the process was repeated. Each fraction was suspended in distilled water and dialysed against distilled water in a refrigerator [11].

Determination of total proteins

The protein content of the culture filtrate was estimated according to the method of Lowry et al. [10]. General properties of the crude laccase enzyme Effect of the temperature of the reaction

In the present experiment, identical reaction mixtures were incubated at different temperatures (15–601C) for 2 min at pH 5.

Purification of the laccase enzyme

The enzyme fractions obtained from acetone 60% concentration were collected, lyophilized and applied to a column (65 2.0 cm) packed with 50 ml volume of Sephadex G-100 [11]. The column was eluted with 0.1 mol/l acetate buffer (pH 5.0). Five milliliter fractions were collected at a flow rate of 15 ml/h. The protein content and laccase activity were determined.

Effect of the pH value of the reaction

This experiment was conducted to determine the optimum pH value at which the enzyme showed its maximal activity. The reaction mixtures of different pH values (3.5–7.0) were prepared using 0.1 mol/l acetate buffer (pH 3.5–5.5) and 0.1 mol/l phosphate buffer (pH 6.0–7.0).

Native polyacrylamide gel electrophoresis

It was carried out according to Smith [12].

Results

Thermal stability of the crude laccase

General properties of the crude laccase enzyme preparation from Trematosphaeria mangrovei

The effect of temperature on enzyme stability was studied by preheating the crude enzyme in 0.1 mol/l acetate buffer (pH 4.5) at different temperatures (35–701C) for different time intervals (15–60 min). The activity was then measured at 351C. Controls were carried out using the enzyme solutions without preheating and its activity was taken as 100%.

In this series of experiments, the general properties of the crude laccase enzyme produced by T. mangrovei grown on the optimized culture medium were determined; these included the effects of temperature and pH of the reaction mixture, thermal and pH stability as well as the effects of enzyme and substrate concentrations on laccase activity.

Trematosphaeria mangrovei laccase enzyme Mabrouk et al.

95

Effect of temperature of the reaction mixture

Thermal stability of the crude laccase

Data presented in Fig. 1 show that the enzyme reached its maximal activity at 351C, followed by 401C; deviation of temperatures beyond this range had adverse effects on enzyme activity. At 35 and 401C, the laccase activity increased 11.38 and 3.29%, respectively, compared with the control 100% at 301C.

The results presented in Fig. 3 show that the stability of the enzyme activity depended on the temperature and the time of heating. At 351C, the enzyme was stable for 60 min However, the enzyme retained about 80–90% of its activity after treatment for 60 min at 401C up to 501C. At 55 and 601C, the enzyme began to lose its activity partially. More adverse effect was observed on heating at 65 and 701C, with a loss of B77.89 and 84.31%, respectively.

Effect of the pH value of the reaction mixture

From Fig. 2, it can be seen that the activity of the enzyme was increased in the acidic range until the maximum was achieved at pH 4.5, and then decreased markedly with increasing ionic strength of the buffer to alkaline. At pH 4.5, the laccase activity increased 62% as compared with the control 100% at pH 5.0; higher alkalinity led to a decrease in the activity until the enzyme became inactive at pH 6.5–7.0.

pH stability of the crude laccase

The results presented in Fig. 4 show that at pH 4.5, the enzyme activity had the highest stability after 60 min of exposure. Furthermore, at pH 4, the enzyme retained about 91.60% of its activity after 60 min of exposure.

Figure 3

Figure 1

120

120

40ºC

45ºC

50ºC

55ºC

60ºC

65ºC

70ºC

100

Relative activity (%).

100


35ºC

80

60

40

80

60

40

20 20

0 0 15

20

25

30

35

40

45

50

55

0

60

15

30

Temperature (ºC)

Effect of temperature of the reaction mixture on crude laccase enzyme activity.

45

60

Time (min) Thermal stability of the crude laccase enzyme from Trematosphaeria mangrovei at pH 4.5.

Figure 4 Figure 2 120 pH3.5

pH4

pH4.5

pH5

pH5.5

pH6

180 100


160


140 120 100 80 60

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40 20

0 0

0 3.5

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4.5

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5.5

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6.5

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Time (min).

pH. Effect of pH of the reaction mixture on crude laccase enzyme activity.

pH stability of the crude laccase enzyme from Trematosphaeria mangrovei at 351C.

96


At pH 3.5 and 5.0, the enzyme lost about 28.55 and 22.33% of its activity after 60 min, respectively. The results also indicated that pH 5.5 had an adverse effect on the enzyme activity; about 36.94% of the enzyme activity was lost after 60 min, whereas it lost 94.54% of its activity after 60 min of exposure at pH 6. Effect of enzyme concentration on crude enzyme activity

The results presented in Fig. 5 show that the enzyme concentration had a major effect on the enzyme activity. The activity of enzyme was increased directly with protein increase. The maximum of enzyme activity was obtained by the enzyme concentration from 0.181 to 5.429 mg per reaction mixture. At the enzyme concentration of 5.429 mg protein, the laccase activity increased 130.74% compared with the control experiment, which was 100% at 0.543 mg protein. However, on further increasing the enzyme concentration, the activity remained almost constant.

Fractional precipitation and partial purification of the Trematosphaeria mangrovei laccase enzyme

Partial purification of the T. mangrovei laccase enzyme was carried out by fractional precipitation with ammonium sulphate, ethanol and acetone. A total of 21 fractions were obtained, including seven with ammonium sulphate, seven with ethanol and seven with acetone.

200

150

Gel filtration on a Sephadex G-100 column

250


The data presented in Fig. 6 indicate that the substrate concentration 40 mmol/l ABTS was the optimum for the laccase enzyme; the enzyme activity increased 73.85% as compared with the control. The increase in enzyme activity was parallel to the increase in the substrate concentration until it reached a maximum at 40 mmol/l ABTS.

The laccase activities recovered by precipitations were 0.34, 2.75 and 2.82%, respectively. Of all the fractions obtained by three precipitants, the fraction obtained by precipitation at the 50–60% acetone concentration showed the highest specific laccase activity (225.68 U/mg protein) and good enzyme activity (8734.79 U). In addition, the highest specific activity (225.68 U/mg protein) of this fraction reached 4.84-fold of the culture filtrate; therefore, this fraction (38.71 mg protein) was further purified by Sephadex G-100 column chromatography.

Figure 5

Laccase enzyme fractions obtained by 50–60% concentration of acetone were collected, lyophilized and subjected to further purification on Sephadex G-100 (Fluka Company) column. Elution was performed with 0.1 mol/l acetate buffer (pH 5.0). The results are presented graphically in Fig. 7.

100

50

0 0.181 0.362 0.543 0.723 0.904 1.085 1.266 1.447 1.628 1.809 1.99 2.171 2.714 3.618 5.428 6.333 7.237 9.047

Enzyme conc. (mg protein/reaction mixture) Effect of different enzyme concentrations on crude enzyme activity.

Figure 6

The column yielded two protein components: the first was the minor component (8.86 mg protein) covered by fractions 6–10 and had the highest recovered activity (8440.443 U). The second was the major component (26.79 mg protein) covered by fractions 11–20 and had weak enzyme activity, indicating that this protein component was not related to laccase. The activity of laccase enzyme covered by fractions 6–14 represented about 96.91% of the applied activity. The fraction number 8 of the laccase component was the most active and showed 6.5-fold purification.

200 180 160


Effect of substrate concentration on crude enzyme activity

The purification scheme of the T. mangrovei laccase enzyme showed that the fraction number 8 had the highest specific activity (1466.49 U/mg protein) and the highest purification, 31.47-fold, compared with the culture filtrate (Table 1).

140 120 100 80 60

The purity of laccase enzyme fraction 8 was determined by native polyacrylamide gel electrophoresis.

40 20 0

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Substrate conc. (mM). Effect of substrate concentrations on crude enzyme activity.

Discussion After optimization of the chemical composition of the production medium, the general properties of the crude

Trematosphaeria mangrovei laccase enzyme Mabrouk et al. 97

laccase enzyme were determined. These included the effects of temperature and pH of the reaction, thermal and pH stability and also the effects of enzyme and substrate concentrations. The enzyme showed an optimum temperature of 351C and it was stable at 401C up to 501C, at which the enzyme retained about 90–80% of its activity. It retained about 78.14% of its initial activity after 60 min of incubation at 551C. The enzyme began to lose large amounts of its activity on heating at 65 and 701C. This result is similar to that reported by Sadhasivam et al. [11], Figure 7 3500

10

Enzyme activity (U/f) Protein content (mg/f) 9 3000

6

5

1500 4

Protein content (mg/f)

Enzyme activity (U/f)

7

2000

3

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2 500 1

0

The crude enzyme showed its maximum activity at pH 4.5 in 0.1 mol/l sodium acetate buffer, which was similar to the result of Sadhasivam et al. [11], who reported that the maximum laccase activity of T. harzianum WL1 was observed at 4.5 pH when ABTS was used as a substrate, and at pH values higher than 4.5, the enzyme activity decreased gradually and was completely inactivated at higher alkaline pH. This phenomenon can be attributed to the difference in the redox potential between a reducing substrate and the type 1 copper in the active site of the enzyme and the inhibition of type 3 copper by hydroxide ion at a higher pH [14]. On studying the pH stability of the T. mangrovei laccase enzyme at 351C, the results showed that the enzyme was more stable at pH values of 4.0 and 4.5 [15]. It was found that the Chalara (syn. Thielaviopsis) paradoxa CH32 laccase enzyme was stable in a pH range from 4.0 to 9.0, but it was inactive at pH 3.0.

8

2500

who found that the Trichoderma harzianum WL1 laccase enzyme was active in a temperature range from 30 to 501C, with the maximum activity at 351C, and the T. harzianum laccase enzyme retained 70% of its initial activity after 1 h of incubation at 551C. However, Cambria et al. [13] found that the laccase from Rigidoporus lignosus had the maximum activity at 401C.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Fraction number Gel filtration on a Sephadex G-100 column.

The results indicated that the optimum enzyme and substrate concentrations in the reaction mixture were 5.429 mg protein and 40 mmol/l ABTS, respectively. Partial purification of the fungal extracellular laccase enzyme was achieved by fractional precipitation with ammonium sulphate, acetone and ethanol. From the 21 fractions obtained, the enzyme fraction precipitated at 60% acetone was the most active (2.82% recovered activity, 225.68 specific activity) and had the highest purification: 4.84-fold. Therefore, it was the most favourable enzyme fraction and was used in future work. These results are higher than those reported by Sadhasivam et al. [11] for the T. harzianum WL1 laccase enzyme (1.30 specific activity and 1.51-fold purification). Further purification of the partially purified extracellular laccase enzyme (the fraction precipitated at 60% acetone concentration) was achieved when it was loaded onto a Sephadex G-100 column. When the laccase enzyme was covered by fractions 6–14, its activity represented about 96.91% of the applied activity. The fraction number 8 of the laccase component was the most active and showed 6.5-fold purification compared with the loaded sample (60% acetone) and 31.47-fold purification compared with the culture filtrate.

Table 1 Purification scheme of the Trematosphaeria mangrovei laccase enzyme Steps of purification

Total activity (U/fraction)

Protein content (mg/fraction)

Specific activity (U/mg/fraction)

Yield (%)

Purification fold

Culture filtrate (crude enzyme) Precipitation with 60% acetone (partial purified enzyme) Sephadex G-100 column chromatography

309 814 9945.23 2997.49

6648.70 44.09 2.04

46.60 225.58 1466.49

100 3.21 0.97

1 4.84 31.47

98


This result is similar to that reported by Sadhasivam et al. [11] for the T. harzianum WL1 laccase enzyme (30.6fold purification).

Conclusion Fungal laccases are involved in various processes in nature including the biodegradation of lignin, detoxification of various aquatic and terrestrial pollutants and treatment of industrial wastewater. The crude enzyme reached its maximal activity at 351C, pH 4.5, at an enzyme concentration of 5.429 mg protein/reaction mixture and at a substrate concentration of 40 mmol/l ABTS. The enzyme was stable for 60 min at 351C and retained about 80–90% of its activity after treatment for 60 min from 40 to 501C. The enzyme showed maximum stability of 100% at pH 4.5 and 91.6% at pH 4.0 after 60 min. Fractional precipitation of the fungal extracellular T. mangrovei laccase enzyme by different methods showed that the enzyme fraction precipitated at 60% acetone was the most favourable enzyme fraction, showing 4.84-fold purification. Laccase obtained from the 50–60% acetone fraction was purified by Sephadex G-100. The final preparation thus obtained reached 31.47-fold that of the culture filtrate (1466.49 U/mg protein) and showed a single band on native polyacrylamide gel electrophoresis. Thus, T. mangrovei is an excellent producer of laccase, especially for use in biotechnological processes.


References 1 Durań N, Rosa MA, D’Annibale A, Gianfreda L. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb Technol 2002; 31:907–931. 2 Minussi RC, Pastore GM, Durań N. Laccase induction in fungi and laccase/ N-OH mediator systems applied in paper mill effluent. Bioresour Technol 2007; 98:158–164. 3 Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paanen A, Koivula A, Rouvinen J. Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nat Struct Biol 2002; 9:601–605. 4 Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev 2006; 30:215–242. 5 Eggert C, Temp U, Eriksson KE. Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus. FEBS Lett 1997; 407: 89–92. 6 Mai C, Schormann W, Milstein O, Huttermann A. Enhanced stability of laccase in the presence of phenolic compounds. Appl Microbiol Biotechnol 2000; 54:510–514. 7 Atalla MM, Zeinab HK, Eman RH, Amani AY, Abeer AA. Screening of some marine-derived fungal isolates for lignin degrading enzymes (LDEs) production. Agric and Biol J North Am 2010; 1:591–599. 8 Kohlmeyer J, Kohlmeyer BV. Illustrated key to the filamentous higher marine fungi. Botanica Marina 1991; 34:1–61. 9 Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl Environ Microbiol 1995; 61:1876–1880. 10 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275. 11 Sadhasivam S, Savitha S, Swaminathan K, Lin FH. Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem 2008; 43: 736–742. 12 Smith I. Acrylamide gel disc electrophoresis. Electrophoretic techniques. New York: Academic Press; 1969. pp. 365–515. 13 Cambria M, Cambria A, Ragusa S, Rizzarelli E. Production, purification, and properties of an extracellular laccase from Rigidoporus lignosus. Protein Expr Purif 2000; 18:141–147. 14 Xu F. Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J Biol Chem 1997; 272:924–928. 15 Robles A, Lucas R, Martıńez-Canãmero M, Ben Omar N, Pe´rez R, Ga´lvez A. Characterisation of laccase activity produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32. Enzyme Microb Technol 2002; 31:516–522.

Original article 99

Phycochemistry of some Sargassum spp. and their cytotoxic and antimicrobial activities Azza A. Matloub and Nagwa E. Awad Pharmacognosy Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Cairo, Egypt Correspondence to Azza A. Matloub, Pharmacognosy Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, El-Bohouth St., 12311 Dokki, Cairo, Egypt Tel: + 20 1001405293; fax: + 20 233370931; e-mail: [email protected] Received 12 March 2012 Accepted 30 August 2012 Egyptian Pharmaceutical Journal 2012, 11:99–108

Purpose A comparative study on the chemical composition as well as cytotoxic and antimicrobial activities of the brown algae Sargassum asperifolium, Sargassum dentifolium, and Sargassum linifolium (family: Sargassaceae) from the Red Sea, Hurghada, Egypt, is carried out. Methods The volatile constituents obtained by hydrodistillation as well as the isolated unsaponifiable matter and the fatty acids were analyzed using the gas chromatography/mass spectrometry technique. Antitumorigenic activities of the crude extracts of the three algae have been evaluated in vitro on different human cell lines. Furthermore, the antimicrobial activities of the volatile constituents, successive extractives, unsaponifiable matter, and fatty acids have been tested on 11 different microorganisms. Results The analysis of the volatile fraction led to the identification of sexual pheromones, terpenes, phenolic compounds, free fatty acids, and esters. The most abundant sterols of unsaponifiable matter were fucosterol and cholesterol in all algae. Palmitic acid was found in all investigated algae as a major fatty acid. Biological screening proved that the tested algae have various cytotoxic and antimicrobial activities. Conclusion S. asperifolium, S. dentifolium, and S. linifolium are rich in cytotoxic and antimicrobial bioactive metabolites. Keywords: antimicrobial activity, cytotoxic activity, Sargassum asperifolium, Sargassum dentifolium, Sargassum linifolium Egypt Pharm J 11:99–108 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Marine natural resources are a treasury of a large group of structurally unique secondary metabolites useful to medicine, which have yielded a large number of drug candidates [1]. The anticarcinogenic properties of brown seaweeds are well known in some cultures such as traditional Chinese medicine [2] and in ancient Ayurvedic texts [3]. In addition, they are mentioned in the Ebers Papyrus of the ancient Egyptians, who used seaweed to treat breast cancer [4]. There are numerous reports on compounds that have been derived from Sargassum spp. with a broad range of biological activities. Patra and colleagues, 2007, reported that the methanol extract of Sargassum spp. showed strong antioxidant activity and had antimicrobial activity against Gram-positive and Gram-negative bacteria [5]. Further, the methanol extract of Sargassum swartzii had chronic and acute anti-inflammatory effects [6], whereas the methanol extract of Sargassum henslowianum and Sargassum siliquastrum and the ethanol extract of Sargassum

dentifolium acted as antidotes against the hepatotoxicity induced by carbon tetrachloride [7,8]. Other studies have reported that the hot water extract of Sargassum horneri is the most potent anticoagulant and has a high activated partial thromboplastin time [9], and that polysaccharides isolated from Sargassum trichophyllum show antiviral activity against herpes simplex virus type 2 [10]. Tang et al. [11] isolated several sterols from Sargassum carpophyllum that showed various cytotoxic activities against several cancer cell lines. Moreover, farnesylacetones isolated from S. siliquastrum showed a moderate vasodilatation effect on the basilar arteries [12]. Chandraraj et al. [13] reported that the ethyl acetate fraction of Sargassum ilicifolium stimulated in-vitro chemotatic, phagocytic, and intracellular killing of human neutrophil immunostimulants and showed prominent immunostimulator activity in vivo. S. siliquastrum acts as a food preservative that reduces the microbial count of bread and increases the time of storage [14]. The hydroalcoholic extracts of Sargassum asperifolium, Sargassum dentifolium, and Sargassum linifolium have shown


DOI: 10.7123/01.EPJ.0000419800.62958.79


various insecticidal and antiviral activities in vitro on isolated cell lines of Spodoptera littoralis (Sl52 cells) and Spodoptera frugiperda (Sf9 cells) with or without inoculation of nucleopolyhedrovirus and in vivo on S. littoralis nucleopolyhedrovirus replication [15]. Furthermore, Aboutabl et al. [16] have reported that the different extracts of S. dentifolium, collected from the Mediterranean coast of Egypt, showed potential insecticidal activity against S. littoralis at different stages of the life cycle. Numerous substances such as 24-vinylcholest-4-ene24-ol-3-one, saringosterone, saringosterol, and a hydroazulene diterpene dictyone were isolated from S. asperifolium [17]. Abdel-Fattah et al. [18] isolated sargassan (a sulfated heteropolysaccharide) from S. linifolium and Aboutabl et al. [16] isolated diisooctyl phthalate from S. dentifolium. Other constituents such as pheromones [19], phlorotannins [20], polyphenols, benzoquinone, hydroquinones with a diterpenoid side chain, cyclopentenones, bisnorditerpene derivatives [21], and phthalic acid derivatives [22,23] have been isolated from different Sargassum spp. The current literature and the lack of the data and information on the composition of the volatile matter and other active constituents of S. asperifolium, S. dentifolium, and S. linifolium led us to isolate and identify their volatile constituents and lipoidal matter. During our search for active cancer chemoprotective agents in these marine algae, we also evaluated the volatile constituents, successive extracts, and unsaponifiable and saponifiable matter as antimicrobial agents.

Materials and methods Thallus material

The three brown algae S. asperifolium (Hering and G. Martens ex J. Agardh), S. dentifolium (Agardh), and S. linifolium (C. Agardh) (family: Sargassaceae) were collected at about 2–4 ft under the water surface on the Red Sea coasts in Hurghada, Egypt, during May 2007 and authenticated by Prof. S.A. Shaalan, Faculty of Science, Alexandria University.

Preparation of crude extract

In total, 100 g of the air-dried powdered thallus from each collected sample was extracted successfully with 70% methanol. Each extract was filtered and evaporated under vacuum.

Isolation of the volatile constituents

Pure and fresh homogenized algae (1 kg) were hydrodistilled in a modified Likens–Nickerson apparatus [24] using n-pentane (AR grade). The n-pentane layer was evaporated under pressure to yield a pale-yellow oil.

Isolation of lipoidal matter

Each petroleum ether residue was saponified using 0.5 N alcoholic KOH. The unsaponifiable matter was extracted with ether, washed with water, dried over anhydrous sodium sulfate, evaporated to dryness, weighed, and analyzed by gas chromatography/mass spectrometry (GC/MS). The fatty acids were liberated by acidification of the saponifiable matter and then extracted with ether and dried in vacuo. The fatty acids obtained were methylated (MeOH, 4–5% dry H2SO4) to yield the methyl ester derivatives and then analyzed by GC/MS.

Gas chromatography/mass spectrometry analysis

GC/MS analysis was carried out using a Finnigan SSQ 7000 (ThermoFinnigan, San Jose, California, USA) GC/MS spectrophotometer equipped with library software Wiley 138 and NBS 75 under the following conditions: DB-5fused silica capillary column, 30 m in length, 0.32 mm ID, and with a film thickness of 0.25 mm; carrier gas, helium at a flow rate of 10 ml/min; temperature programmed to 60–2601C at a rate of 41C/min (volatile constituents), 70–2901C at a rate of 51C/min (unsaponifiable matter), 60–2201C at a rate of 41C/min (fatty acid methyl ester derivatives), chart speed: 0.5 cm/min, ionization voltage 70 eV, and detector: flame ionization detector. The identification of the constituents was carried out depending on the fragmentation of the spectra obtained and by comparing them with those of available authentic standards such as an alkane standard mixture, hexadecanol, palmitic acid, geranylgeraniol, a-copanene, longifolene, aromadendrene, D-limonene, caryophyllene, germacrene D, phytol, b-ionone, cholesterol, campesterol, stigmasterol, b-sitosterol, fucosterol, and ergosterol (Sigma-Aldrich Chemie GmbH, Germany). In addition, brassicasterol, 22-dehydrocholesterol, fucostenone, clerosterol, and avensterol have been isolated previously and identified by our research group at the Pharmacognosy Department, NRC, Egypt, or by published data [25–28], and a library database [Wiley (Wiley Institute, USA) and NIST (National Institute of Technology, USA)]. Quantitative determination was carried out on the basis of peak area measurements of the GC chromatograms.

Antitumor activity Cells Preparation of successive extracts

In total, 100 g of the air-dried powdered thallus from each collected sample was extracted exhaustively with petroleum ether (40–601C), ether, chloroform, ethyl acetate, and methanol in a Soxhlet apparatus, followed by maceration in water. Each extract was filtered, evaporated under vacuum, and weighed.

Authentic culture, H460 (lung carcinoma human cell line), Hela (cervix carcinoma human cell line), HepG2 (liver carcinoma human cell line), Mcf7 (breast carcinoma human cell line), Molt4 (leukemia carcinoma human cell line), and U251 (brain carcinoma human cell line) were obtained from the American Type Culture Collection, USA.

Phycochemistry and biological activities of Sargassum spp. Matloub and Awad 101

Culture media

The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% antibiotic– antimycotic mixture (10 000 U/ml K-penicillin, 10 000 mg/ml streptomycin sulfate, and 25 mg/ml amphotericin B), and 1% L-glutamine (all purchased from Lonza, Braine-l’Alleud, Belgium). Assay method for cytotoxic activity

The cytotoxicity against H460, Hela, HepG2, Mcf7, Molt4, and U251 was determined at the National Cancer Institute, according to the method used by Skehan et al. [29]. Adriamycin (Doxorubicin; Pharmacia, Stockholm, Sweden) 10 mg vials were used as the reference drug. The cell lines were plated in 96-multiwell plates (104 cells/well) for 24 h before treatment with the tested samples to allow the attachment of cells to the wall of the plate. Then, a 50 ml aliquot of serial dilutions of the crude extract (1.0, 2.5, 5, and 10 mg/ml) was added and the plates were incubated for 48 h at 371C in a humidified incubator containing 5% CO2 in air. Triplicate wells were prepared for each individual dose. Cells were fixed, washed, and stained with sulforhodamine B stain (Sigma, USA). Excess stain was washed with acetic acid and the attached stain was recovered with Tris EDTA buffer (Sigma, USA). The color intensity was measured in an ELISA reader spectrophotometer (Tecan Group Ltd.Sunrise, Mannedorf, Switzerland). Microbiological activity

The antimicrobial activity of the volatile constituents, successive extracts, saponifiable matter, and fatty acids of algae examined was determined against that of several microbes using the antibiotic assay method [30]. Pure strains of bacteria, yeasts, and fungi were kindly provided by the Microbial Genetics Department, National Research Center, Egypt. The bacterial strains used were Bacillus cereus (Gram positive, G + ), Bacillus subtillis (G + ), Staphylococcus aureus (G + ), Escherichia coli (Gram negative, G – ), Pseudomonas aeruginosa (G – ), and Pseudomonas fluorescens (G – ). The yeast strains were Saccharomyces carles and Saccharomyces cerevisiae, whereas the fungi were Aspergillus flavus, Aspergillus niger, and Diplodia oryzea. The bacteria were cultured on Lauria–Bertani Medium [31], whereas the yeast strains were cultivated on Yeast Extract Peptone Medium [32]. The fungi were cultured on Potato-Dextrose Agar growth medium [33]. The oils, successive extracts, unsaponifiable matter, and fatty acids were sterilized by filtration through a bacterial membrane filter (0.45 mm, 2.5 mm diameter; Millipore, Billerica, Massachusetts, USA). A concentration of 100 mg/ disc was used. The discs (6 mm diameter), after being air dried, were firmly applied to the surface of inoculated agar plates. The diameters of inhibition zones were measured per applied disc after incubation at 371C for 24 h with the bacteria strains, whereas those containing yeast and fungi were incubated at 301C for 48–72 h. Amoxycillin (Medical Union Pharmaceuticals Co., Ismailia, Egypt) as an antibacterial agent (100 mg/disc) and canesten (Alexandria

Co., Alexandria, Egypt) as an antifungal agent (100 mg/ disc) were used as reference drugs. Statistical analysis of data

All values were expressed as means, with three replicates for each treatment. Data were subjected to a paired sample t-test using SPSS (version 17.0; SPSS Inc., Chicago, Illinois, USA). P less than 0.05 was considered as significant.

Results and discussion The yields of volatile oils of fresh algae S. asperifolium, S. dentifolium, and S. linifolium were 0.038, 0.041, and 0.043% (w/w), respectively. Fifty-seven, 53, and 54 compounds were identified, which represent 93.93, 92.36, and 89.43% of the total volatile compounds released from S. asperifolium, S. dentifolium, and S. linifolium; respectively. Table 1 shows that the volatile constituents of the algae are composed of alcohol (15.74, 16.44, and 15.76 %), aldehyde (– , 0.25 and 0.09%), esters (27.98, 29.05, and 10.08%), free acids (6.17, 9.43, and 1.85%), halogenates (0.33, 0.22, and 0.25%), C11 hydrocarbon pheromones (2.34, 5.89, and 24.38%), sesquiterpenes (1.31, 3.44, and 0.72%), hydrocarbons (28.28, 22.95, and 26.36%), ketones (11.43, 2.75, and 9.72%), and miscellaneous compounds (0.35, 1.94, and 0.22%), respectively. Dictyopterene D0 , which is an odoriferous C11 hydrocarbon, was a major constituent in S. linifolium (20.26%) and was also identified in the oil of S. asperifolium and S. dentifolium. Another C11 hydrocarbon pheromone dictyopterene C was detected in S. asperifolium; dictyopterene A was detected in S. dentifolium and S. linifolium. These hydrocarbons have been detected here for the first time in S. asperifolium, S. dentifolium, and S. linifolium. However, ectocarpene and dictyotene have been detected previously in S. asperifolium [34]. Characteristic aroma dictyopterenes have been identified as constituents of brown algae with male gameteattracting activity [19]. Bis-2-ethylhexyl phthalate was identified as the principal constituent in S. asperifolium and S. dentifolium (24.25 and 25.28%, respectively), and this was also found in the Sargassum wightii [22], S. dentifolium [16], and Sargassum spp. [23]. The biosynthesis of di-(2-ethylhexyl) phthalate by red alga Bangia atropurpurea has been described by Chen [35]. Furthermore, di-(2-ethylhexyl) phthalate showed antimicrobial activity against various microorganisms [36], antileukemic and antimutagenic [14]. In addition, dibutyl phthalate has been detected in some edible brown algae such as Undaria pinnatifida and Laminaria japonica as a natural product [37]. Furthermore, sesquiterpenoid compounds a-copaene, b-bourbonene, longifolene, g-elemene, aromdenderene, and muurola-4(14),5-diene have been detected for the first time in Sargassum spp. under study. These compounds were detected in Dictyopteris spp. [38]. b-Ionone was detected


Table 1 Chemical composition of the volatile constituents of brown algae Sargassum asperifolium, Sargassum dentifolium, and Sargassum linifolium Relative %

Compounds Acid Propionic acid Tetradecanoic acid Palmitic acid Alcohol Cis-9-octadecen-1-ol 4-Nonylphenol 1-Hexadecanol 9-cis-Octadecanol Phytol Geranylgeraniol Aldehyde 2,6-Di-t-butyl-4-hydroxybenzaldehyde Esters Isobutyl phthalate Dibutyl phthalate Methyl eicosa-5,8,11,14,17-pentaenoate Dioctyl adiptate Bis(ethyl hexyl) phthalate Halogenates 2,2-Dicholoro-3-methylbutane Iodo-2-methylundecane 1-Chlorooctadecane Hydrocarbons Nonane 2,6-Dimethylheptane Decane 1-Undecene Undecane Dictyopterene D Dictyopterene C Dictyopterene A 6-[(1E)Butenyl]-cyclohepato-1,4-diene Dodecane Tridecane Decahydro-1,4-ethanonaphthalene a-Copaene b-Bourbonene Tetradecane Longifolene g-Elemene Aromodendrene Muurola-4(14)5-diene (cis) Pentadecane Cuparene Hexadecene Hexadecane 8-Heptadecene 1-Heptadecene Heptadecane 4,9-Di-nor-propyldodecane 1-Octadecene Octadecane Eicosane Totarene Heneicosane 1-Docosene Docosane Dictyone Tricosane Tetracosane Pentacosane Hexacosane Heptacosane Octacosane Ketones 3-Methyl, 2-hexanone 3-Methyl-2-heptanone 2,6,6-Trimethyl-2,4-cycloheptadien-1-one b-Ionone 6,10,14-Trimethyl,2-pentadecanone

Base peak

Molecular weight

59 73 43

74 228 256

45, 41, 43, 58, 75 60, 43, 58, 85, 129 41, 57, 55, 73

41 135 43 41 71 41

268 220 242 268 296 288

43, 55, 57, 83, 69, 97 57, 121, 43, 71 55, 41, 83, 97, 69, 57 43, 55, 57, 69 43, 41, 57, 55, 123 159, 105, 69, 91

219

234

57, 41, 220, 91

149 149 79 129 149

278 278 316 370 390

41, 57, 223, 150 41, 55, 150, 223 91, 67, 105, 41, 119 57, 43, 55, 41, 71 167, 279, 57, 43, 71

43 57 57

140 262 288

41, 42, 69, 105 43, 71, 41, 55, 185 41, 43, 55, 69, 71, 85

43 43 43 41 57 91 79 79 79 57 57 67 105 81 43 91 41 41 161 57 133 43 57 55 43 57 57 43 43 43 41 43 41 43 159 57 57 57 57 57 57

128 128 142 154 156 148 150 150 148 170 184 164 204 204 198 204 204 204 204 212 202 224 226 238 238 240 254 252 254 282 272 296 308 310 304 324 338 352 366 380 394

57, 41, 42, 85, 71 57, 41, 42, 71, 57 57, 41, 71, 85, 55 43, 55, 56, 69 43, 41, 71, 85 79, 91, 105, 41, 77, 119, 66 91, 77, 41, 93, 65, 51, 66 93, 91, 77, 41, 67, 66, 55 91, 41, 66, 119, 55 43, 71, 41, 85 43, 71, 85, 55 82, 41, 80, 54, 121, 55, 93 119, 161, 91, 41, 93 79, 80, 123, 41 57, 71, 41, 85, 55 41, 161, 105, 79, 93 121, 93, 91, 67, 105 91, 79, 105, 67, 93 41, 105, 91, 119, 133 43, 41, 85, 71, 55 132, 145, 41, 91 41, 55, 69, 83, 97 43, 71, 41, 55, 85 41, 43, 69, 82 41, 55, 57, 97, 83 43, 71, 41, 85 43, 41, 71, 55, 85 55, 57, 41, 69, 97 41, 57, 55, 71, 85 57, 41, 71, 55, 85 43, 55, 81, 175 57, 41, 71, 55 55, 43, 83, 97, 69 57, 43, 71, 55, 85 43, 71, 286, 107, 145 43, 41, 71, 55, 85 43, 71, 85, 55, 41 71, 43, 85, 55, 113 43, 71, 85, 55, 69 43, 71, 85, 41, 55 43, 71, 85, 41, 55

43 43 107 177 43

114 128 150 192 268

42, 41, 55, 57 42, 41, 57, 58 91, 108, 41, 77, 53, 55 43, 121, 105, 135, 77 58, 71, 57, 109, 124

Main fragments

Sargassum asperifolium

Sargassum dentifolium

Sargassum linifolium

6.17 – 0.91 5.26 15.74 1.93 – 5.77 4.65 0.71 2.68 – – 27.98 0.95 1.30 1.23 0.25 24.25 0.33 – – 0.33 31.93 1.37 0.31 1.14 – 1.14 2.02 0.32 – – 0.85 0.26 – 0.19 0.36 0.76 – 0.44 0.40 0.92 1.56 – 0.35 1.65 – – 3.50 – 0.33 3.00 0.21 0.45 0.70 – 0.65 8.13 – 0.20 0.50 – 0.22 – 11.43 8.38 0.19 0.51 1.16 1.19

9.43 5.66 1.31 2.46 16.44 1.43 – 11.90 – 1.17 1.94 0.25 0.25 29.05 0.32 1.13 2.02 0.30 25.28 0.22 – 0.22 – 32.28 – – 0.23 0.05 0.56 4.63 – 1.02 0.24 0.62 – 0.24 0.25 0.25 0.60 0.63 – 1.18 0.44 2.38 0.69 – 1.37 0.34 0.57 4.41 0.30 0.10 3.08 0.59 0.29 0.51 0.11 0.35 5.14 – 0.25 0.35 0.20 0.31 – 2.75 – – 0.37 0.85 1.53

1.85 – – 1.85 15.76 2.26 0.35 10.69 0.10 1.43 0.93 0.09 0.09 10.08 0.57 0.72 1.07 – 7.72 0.25 0.11 0.14 – 51.46 0.69 0.17 0.92 0.65 0.65 20.26 – 3.18 0.94 0.52 0.19 1.91 0.35 0.10 0.58 – – – 0.27 2.75 – 0.12 0.99 0.56 1.59 6.44 – 0.42 2.50 1.11 0.07 1.06 0.25 0.57 – 0.61 0.29 0.23 0.22 0.18 0.12 9.72 5.37 0.17 0.32 1.39 2.47


Table 1 (continued) Relative %

Compounds Miscellaneous 4,5-Dithiaoctane Anethole Butylated hydroxytoluene Total

Base peak

Molecular weight

43 148 205

150 148 220

Main fragments




0.35 0.35 – – 93.93

1.94 0.12 1.61 0.21 92.36

0.22 0.22 – – 89.43

41, 108, 57, 71, 113 147, 79, 55, 117 206, 57, 41, 177, 145

Table 2 Chemical composition of the unsaponifiable fraction of the brown algae Sargassum asperifolium, Sargassum dentifolium, and Sargassum linifolium Relative %

Compounds Alcohol 2-Butanol 1-Hexadecanol 9-Heptadecanol Octadecanol Geranylgeraniol Phytol 1-Eicosanol 1-Docosanol 1-Tricosanol 1-Tetracosanol 1-Hexacosanol Aldehyde 2-Nor-heptylundec-2-enal Octadecanal Esters Dibutyl phthalate Undecyl laurate Nor-butyl benzyl phthalate Di-2-ethylhexyl phthalate Nor-docosyl acetate Di-cyclohexyl phthalate Hydrocarbons 4,4-Dimethyl-1-pentene 3-Ethyltridecane Pentadecane 6,9-Dimethyltetradecane 1-Heptadecene Heptadecane Octadecane 5-Methyloctadecane 2,6,10,14-Tetramethylpentadecane 2-Phenyltridecane 8-Nor-hexylpentadecane 2-Nor-butyl-8-nor-hexylbicyclo(4,4,0) decane Dictyone Cyclodocosane Nor-tricosane 2-Methyltricosane Nor-pentacosane Nor-heptacosane Nor-octacosane Nonacosane Squalene Ketone 1,4-Benzoquinone Hydroquinone 2,6-Di-butyl-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one 1,4-Naphthaquinone 6,10,14-trimethylpentadecane-2-one Benzophenone Phenol 2,6-Di-t-butyl-4-bromomethyl phenol 2,6-Di-t-butyl-4-methyl phenol 2,6-Di-t-butyl-4-formyl phenol

Base peak

Molecular weight

Main fragments

45 43 43 43 41 71 43 43 43 43 43

74 242 256 270 288 296 298 326 340 354 382

43, 59, 44, 41 41, 55, 57, 97, 69 57, 71, 41, 58 41, 57, 55, 111, 69, 97 159, 105, 69, 91 43, 123, 41, 57, 81 41, 55, 57, 69 55, 57, 41, 71, 97 55, 57, 41, 69, 83 57, 55, 41, 83, 69 57, 55, 41, 83, 69

43 85

266 268

55, 41, 69, 95 41, 57, 157, 139

149 43 149 149 43 149

278 354 312 390 368 368

41, 150, 57, 55, 104 57, 41, 55, 201, 69 91, 41, 56, 206, 104 167, 279, 57, 43 83, 57, 97, 61, 69 167, 55, 41, 150

57 43 57 43 43 57 43 43 57 105 43 41 159 55 57 43 43 57 57 57 69

98 212 212 226 238 240 254 268 268 260 296 278 304 308 324 338 352 380 394 408 410

41, 55, 43, 83 57, 71, 85, 41, 55 43, 41, 85, 71, 55 41, 71, 85, 55 41, 55, 57, 97, 83 43, 71, 41, 85 41, 57, 55, 71, 85 57, 85, 71, 84, 41 43, 71, 41, 85, 69 91, 43, 41, 106, 104 57, 71, 41, 85, 55 95, 55, 81, 67, 43 43, 71, 286, 107, 145 57, 43, 41, 69, 83 43, 41, 71, 55, 85 57, 41, 55, 85, 97 57, 41, 69, 97, 83 43, 71, 55, 41, 85, 99 43, 71, 85, 55, 41, 99 43, 71, 55, 41, 85, 99 81, 41, 95, 136, 55

54 110 165 158 43 105

108 110 236 158 268 182

53, 52, 82, 50, 81 81, 53, 109, 55 180, 41, 57, 43 104, 76, 102, 130 58, 41, 57, 71, 55 77, 51, 106, 181

161 205 219

218 220 234

203, 175, 163, 176 206, 91, 57, 41 57, 220, 41, 191, 55




7.01 0.14 – – 0.24 – 5.48 0.11 – 0.92 0.12 – 0.27 0.03 0.24 3.58 0.42 – 0.70 0.80 – 1.66 15.87 0.27 0.03 – 0.03 0.02 – 0.05 0.03 – 0.02 0.06 0.13 – – 0.20 – 0.17 0.38 0.73 0.62 13.13 0.43 0.02 0.04 0.05 0.03 0.27 0.02 2.03 – 2.03 –

14.87 10.95 0.09 0.04 – 0.16 3.08 0.12 – 0.34 – 0.09 0.11 0.11 – 14.01 0.67 0.05 – 11.12 1.09 1.08 4.98 – 0.08 – 0.09 – 0.01 0.22 0.16 – – – 0.53 1.43 0.41 – – 0.24 – – – 1.81 3.21 0.12 2.27 0.19 – 0.63 – 19.64 0.07 18.52 1.05

7.15 0.36 – – – – 5.67 0.22 0.24 0.66 – – – – – 10.46 2.61 – – 6.07 – 1.78 5.48 – – 0.09 – – 1.02 0.22 – 0.14 – – – – – 0.38 0.28 0.17 – – – 3.18 5.91 0.13 3.44 0.77 – 1.57 – 18.45 0.17 17.72 0.37

104


Table 2 (continued) Relative %

Compounds 2,6-Di-t-butyl-4-methoxy phenol 1,2-Bis-(3,5-di-t-butyl-4-hydroxyphenyl) ethane Sterols 3-Hydroxy androst-2-en-17-one Pregna-4,16-diene-3,20-dione Dehydro-22-cholesterol Cholesterol Brassicasterol Ergosta-5,7,22-trien-3b-ol D5 Ergosterol Campesterol Stigmasterol Clerosterol b-Sitosterol Fucosterol 24-Isoethylidene cholest-5-en-3b ol 24-Ethylcholesta-5,24(25) dienol Fucostenone Miscellaneous 2,2-Diethoxy ethanamine Dihydroactinidiolide Phenanthrene 4-Hydroxyoctadec-9-enolide 2-Ethylhexyldiphenyl phosphate Total

Base peak

Molecular weight

221 219

236 438

57, 41, 91, 222 43, 55, 57, 69, 220

91 312 384 386 398 396 400 55 43 412 91 314 314 314 312

288 312 384 386 398 396 400 398 412 412 414 412 412 412 410

105, 255, 41, 79, 55 43, 136, 160, 159, 297 69, 300, 255, 133 275, 301, 368, 107 380, 300, 271, 255 381, 288, 271, 255 385, 382, 367, 315 69, 81, 383, 253 55, 57, 41, 69, 314 394, 314, 271, 371 91, 55, 43, 95, 108 55, 43, 69, 83, 281 55, 43, 69, 83, 95 299, 281, 271, 296 313, 297, 124, 231

47 111 249 41 251

103 180 264 280 362

75, 60, 89, 45 137, 109, 41, 67, 95 43, 55, 57, 121 55, 67, 81, 54 43, 41, 55, 94, 250

in all algae under investigation, and it has antibacterial and antifungal activity [39]. Two aliphatic chains diterpenes, phytol and geranylgeraniol, were identified in the tested algae, which had bactericidal activity against S. aureus. These diterpenes exerted both growth-inhibitory and growth-accelerating effects depending on their concentration [40]. The yields of unsaponifiable matter of S. asperifolium, S. dentifolium, and S. linifolium were 0.22, 0.22, and 0.61% (w/w), respectively. Fifty-seven, 44, and 40 compounds were identified, which represent 82.17, 79.74, and 80.44% of the total unsaponifiable matter of S. asperifolium, S. dentifolium, and S. linifolium, respectively. Table 2 shows that the unsaponifiable matter of S. asperifolium, S. dentifolium, and S. linifolium is composed of sterols (51.27, 22.23, and 32.35%), which represent the mean fraction of unsaponifiable matter, alcohol (7.01, 14.87, and 7.15%), aldehydes (0.27, 0.11%, and –), esters (3.58, 14.01, and 10.46%), hydrocarbons (15.87, 4.98, and 5.48%), ketones (0.43, 3.21, and 5.91%), phenols ( 2.03, 19.64, and 18.45%), and miscellaneous compounds (1.71, 0.69, and 0.64%). Fucosterol was detected as a major sterol in the algae tested as other Sargassum spp. showed cytotoxic activity against various carcinoma human cell lines [11,41]. In addition, it showed antifungal activity against Curvularia lunata, Stachybotrys atra, and Microsporum canis. These results were obtained for the first time in this work for unsaponifiable matter from Sargassum spp. The percentages of fatty acids of the brown algae S. asperifolium, S. dentifolium, and S. linifolium were 0.09, 0.041, and 0.043% (w/w), respectively. Table 3 shows that the saturated fatty acids represent the main fraction

Main fragments




– – 51.27 – 0.30 1.99 12.21 – – – 0.28 – 2.63 0.88 29.35 – – 3.63 1.71 0.02 0.08 0.04 1.05 0.52 82.17

– – 22.23 1.93 1.86 1.16 5.92 0.65 1.12 1.71 – 0.20 – – 7.13 – 0.55 – 0.69 0.18 0.46 – 0.05 – 79.74

0.08 0.11 32.35 5.33 0.81 1.17 10.05 0.91 – 0.53 – 2.92 1.05 0.12 7.34 0.34 0.58 1.20 0.64 – 0.64 – – – 80.44

(56.12, 65.39, and 56.42%, respectively) of fatty acid, and palmitic acid was found in all Sargassums spp. under study as a major fatty acid. Furthermore, oleic acid represents the main unsaturated fatty acid of S. dentifolium and S. linifolium. However, 9,12-octadecadienoic acid represents the major unsaturated fatty acid of S. asperifolium.

Cytotoxic activity

The cytotoxic activity of crude extracts of Sargassum spp. under study against human cells H460, Hela, HepG2, MCF7, Molt4, and U251 cultured in vitro was examined. The percentages of inhibition and relative inhibition related to the reference drug doxorubicin are shown in Tables 4 and 5 and illustrated in Figs 1–3. The crude extract of S. linifolium has significantly promising in-vitro cytotoxic activity against HepG2 and Molt4, with an effective dose (ED50) of 5.97 and 2.28 mg/ml, respectively, compared with the control, and at concentrations of 5 and 10 mg/ml, they showed good cytotoxic activity against HepG2, which was comparable to that of the reference drug doxorubicin. Whereas the crude extract of S. dentifolium showed significant cytotoxic activity against HepG2 with an ED50 of 11.03 mg/ml, H460 and MCF7 related to the control test and at concentrations of 5 and 10 mg/ml showed good cytotoxic activity against H460 in comparison with doxorubicin. Furthermore, the crude extract of S. asperifolium at a concentration of 1 mg/ml showed high cytotoxic activity against H460, whereas it showed good cytotoxic activity at concentrations of 1 and 2.5 mg/ml against U251 and H460, respectively, when compared with doxorubicin as a reference drug.


Table 3 Methyl ester of the fatty acid composition of the brown algae Sargassum asperifolium, Sargassum dentifolium, and Sargassum linifolium % of total fatty acid derivatives

Compounds Saturated fatty acids Methyl laurate Methyl myristate Methyl pentadecanoate Dimethyl azelate Methyl palmitate Methyl heptadecanoate Methyl stearate Methyl docosanoate Methyl tricosanoate Methyl tetracosanoate Unsaturated Fatty Acids Methyl palmitoleate Methyl oleate Methyl 9,12-octadecadienoate Methyl 12,15-octadecadienoate Methyl 9-cis,12-cis,15-cis-octadecatrienoate Methyl eicosa-5,8,11,14,17-pentaenoate Methyl heptadec-trans-10-en-8-ynoate Methyl eicosa-11-yn- trans-13-enoate Methyl 4,7,10,13,16,19-docosahexaenoate Hydroxylated fatty acids Methyl 3-hydroxyoctadecanoate

Base peak

Molecular weight

74 74 74 55 74 74 74 74 74 74

214 242 256 216 270 284 298 354 368 382

87, 43, 41, 55, 143, 171, 183 87, 43, 41, 55, 143, 199, 213 87, 43, 41, 55, 75, 143, 213 74, 83, 43, 59, 152, 41 74, 43, 41, 55, 75, 143, 227 87, 43, 57, 75, 143, 241, 199 87, 43, 55, 75, 143, 255, 199 256, 43, 129, 87, 213, 185 87, 43, 75, 57, 55, 143 87, 75, 55, 43, 41, 69, 143

55 55 41 67 79 79 79 79 79

268 296 294 294 292 316 278 320 342

74, 69, 41, 87, 96, 236, 194 74, 41, 69, 43, 264, 87, 222 67, 81, 95, 55, 79, 109 81, 82, 95, 55, 109, 123 67, 41, 93, 55, 107, 150, 194 91, 67, 105, 41, 119, 147, 201 41, 67, 93, 91, 108, 121 80, 150, 67, 93, 77, 55 91, 67, 41, 55, 77, 105

43

314

103, 41, 57, 55, 82, 83, 79, 229

Main fragments




56.12 – 9.71 – – 20.72 – 18.61 4.38 – 2.70 43.40 1.20 5.51 18.73 9.21 7.15 – 1.60 – – 0.48 0.48

65.39 0.73 3.85 2.19 9.80 18.21 1.50 10.75 11.12 3.44 3.80 34.61 1.05 11.96 10.07 – 6.70 1.92 2.91 – – – –

56.42 – 7.78 4.02 – 40.16 – 4.46 – – – 43.58 – 25.26 6.95 – – – 2.49 5.97 2.91 – –

Table 4 Cytotoxic activity of the crude extract of the brown algae Sargassum asperifolium (S1), Sargassum dentifolium (S2), and Sargassum linifolium (S3) against different cultured human cell lines Sample Human cell line H460

Hela

HepG2

MCF7

Molt

U251

% of inhibition ± SEM

Concentration (mg/ml)

1

2.5

5

10

ED50

S1 S2 S3 Dox S1 S2 S3 Dox S1 S2 S3 Dox S1 S2 S3 Dox S1 S2 S3 Dox S1 S2 S3 Dox

28.00 ± 0.18*,** – 50.00 ± 0.15*,** – 130.0 ± 0.8*,** 24.40 ± 0.05* – 30.13 ± 0.04*,** – 63.31 ± 0.03*,** – 69.93 ± 0.11*,** 22.6 ± 0.08* – 16.67 ± 0.11*,** 14.07 ± 0.02** 13.15 ± 0.06*,** 64.90 ± 0.11* – 3.23 ± 0.03** 13.19 ± 0.03*,** 6.00 ± 0.09** 31.80 ± 0.11 – 41.2 ± 0.18* – 9.54 ± 0.34 67.40 ± 0.22* NT 39.12 ± 0.03*,** 14.84 ± 0.08*,** 16.00 ± 0.12*,** 67.1 ± 0.20*

24.00 ± 0.15*,** 12.00 ± 0.16*,** – 36.00 ± 0.05** 36.50 ± 0.10* – 45.60 ± 0.13*,** – 34.62 ± 0.09*,** – 39.74 ± 0.09*,** 37.6 ± 0.09 – 0.55 ± 0.06** 27.4 ± 0.05*,** 25 ± 0.07*,** 90.7 ± 0.11* 5.85 ± 0.07** 22.77 ± 0.07*,** – 56.59 ± 0.10*,** 48.50 ± 0.013* – 75.12 ± 0.11* – 43.77 ± 0.47* 53.78 ± 0.18* NT 29.55 ± 0.07*,** 25.82 ± 0.07*,** – 0.75 ± 0.12** 85.20 ± 0.05*

0 ± 0.12** 30.00 ± 0.26*,** 10.00 ± 0.24** 51.60 ± 0.09* – 23.14 ± 0.09*,** – 4.06 ± 0.04** – 44.48 ± 0.07*,** 79.7 ± 0.02* 15.75 ± 0.06** 31.29 ± 0.03*,** 46.11 ± 0.06*,** 86.90 ± 0.15* 4.23 ± 0.07** 11.32 ± 0.15*,** – 77.73 ± 0.10*,** 80.80 ± 0.03* – 54.77 ± 0.20 – 62.31 ± 0.02* 25.13 ± 0.18* NT 32.12 ± 0.01*,** 14.14 ± 0.05*,** – 3.94 ± 0.20** 85.80 ± 0.05*

– 2.00 ± 0.03** 34.00 ± 0.12*,** 28.00 ± 0.22*,** 59.90 ± 0.12* – 12.72 ± 0.04** – 40.61 ± 0.12*,** – 32.50 ± 0.10*,** 80.90 ± 0.01* 37.59 ± 0.05*,** 45.72 ± 0.06*,** 65.56 ± 0.06*,** 95.00 ± 0.10* 10.45 ± 0.07*,** 35.33 ± 0.03*,** 3.00 ± 0.04** 82.80 ± 0.02* – 57.62 ± 0.29* – 65.32 ± 0.58* 44.73 ± 0.07* NT 7.05 ± 0.05*,** 12.18 ± 0.04*,** – 17.76 ± 0.12*,** 92.80 ± 0.05*

– 410 410 4.77 – – – 3.64 410 11.03 5.97 0.80 410 410 – 2.97 – – 2.28 – – – – o0

Sargassum asperifolium (S1), Sargassum dentifolium (S2), and Sargassum linifolium (S3). Each value represents the mean of percentage of inhibition cells of three replicates ± SEM. Dox, doxorubicin; ED, effective dose; NT, not tested. *Significantly different from the control value at Po0.005 according to a paired sample t-test. **Significantly different from the reference drug doxorubicin value at Po0.005 according to a paired sample t-test.

It is noteworthy that the authors have isolated many bioactive cytotoxic constituents such as diterpenes and polysaccharides from different marine algae [42–44]. Khanavi et al. [41] found that fucosterol, the most

abundant phytosterol in the brown algae, is responsible for the cytotoxic effect against a breast carcinoma cell line [inhibitory concentration (IC50) 27.94 mg/ml] and a colon carcinoma cell line (IC50 70.41 mg/ml).

106


Table 5 Relative inhibition of growth of different human cell lines related to doxorubicin Relative inhibition to doxorubicin (%) H460 Concentration (mg/ml) 1 2.5 5 10

S1

Hela

S2

S3

114.75 – – 65.75 32.87 – – 58.14 19.37 – 56.76 46.74

HepG2

S1 S2 S3 – – – –

– – – –

– – – –

S1

S2

– – 18.12 39.56

21.67 30.20 36.00 48.12

MCF7 S3

S1

S2

Molt4 S3

20.26 – 52.76 24 27.56 10.08 39.25 – 53.06 5.25 14.05 – 69.01 11.42 38.61 3.68

U251

S1 S2 S3 NT NT NT NT

NT NT NT NT

S1

S2

S3

NT 58.30 22.11 23.84 NT 34.68 30.30 – NT 37.43 16.48 – NT 7.59 13.13 –

Sargassum asperifolium (S1), Sargassum dentifolium (S2), and Sargassum linifolium (S3). The activity was evaluated according to the inhibition growth related to doxorubicin. Activity >75%, high; 75–50%, good; 50–25%, normal; and < 25%, weak activity. –, no cytotoxic activity; NT, not tested.

Figure 1

Figure 3

60

H460

40

Hela

Hela 50

HepG2

20

1

2.5

5

Molt4

10

U251

−40

0 % of Inhibition

% of Inhibition

−20

HepG2 MCF7

MCF7 0

H460

100

Molt4 1

2.5

5

−50

10

U251

−100

−60

−150

−80 −100

−200

Conc. μg/ml

Cytotoxic activity of a crude extract of Sargassum asperifolium on different human cell lines.

Conc. μg/ml

Cytotoxic activity of a crude extract of Sargassum linifolium on different human cell lines.

% of Inhibition

Figure 2 80

H460

60

Hela

40

HepG2

20

MCF7 Molt4

0 −20

1

2.5

5

10

U251

−40 −60 −80

−100

Conc. μg/ml

compared with canesten as a reference drug. Ether, ethyl acetate, and methanol fractions of S. linifolium were found to have potent antimicrobial activities against D. oryzea when compared with standard canesten. It has been reported that the antimicrobial activity of some algal species is because of the presence of a mixture of fatty acids such as capric, lauric, linoleic, myristic, oleic, palmitic, and stearic acid [45]. It is clear from the present study that these fractions can be utilized as good natural antimicrobial agents in the pharmaceutical industry.

Cytotoxic activity of a crude extract of Sargassum dentifolium on different human cell lines.

Conclusion The antimicrobial activity

The antimicrobial activities of the volatile constituents, successive extracts, unsaponifiable fractions, and fatty acids fractions of Sargassum spp. under study are summarized in Table 6. The different fractions of S. asperifolium showed significant antimicrobial activity against B. cereus compared with amoxycillin as a reference drug. However, the various fractions of S. dentifolium showed pronounced antimicrobial activity against S. carles

The volatile constituents as well as unsaponifiable matter and fatty acids isolated and identified from S. asperifolium, S. dentifolium, and S. linifolium, collected from the Red Sea coasts in Hurghada, for the first time comprise alcohol, aldehydes, esters, free acids, halogenates, C11 hydrocarbon pheromones, sesquiterpenes, hydrocarbons, and ketones. The different extracts of these three algae have various antimicrobial and cytotoxic activities and can act as promising natural sources of these bioactive products.

S1

Algae species (100 mg/disc)

8 ± 0.0a 10 ± 0.57* 9 ± 0.57* 8 ± 0.0a 10 ± 0.0a 24 ± 0.0

10 ± 0.0a – 12 ± 0.57a 8 ± 0.0a 15 ± 0.57* 10 ± 0.0 9 ± 0.57* 9 ± 0.57* – – – 22 ± 0.0

– 9 ± 0.0a – – – 10 ± 0.0a 10 ± 0.57* 10 ± 0.0a – – – 9 ± 0.0a – – 8 ± 0.57* 8 ± 0.0a 12 ± 0.0a – 9 ± 0.0a – 8 ± 0.0a

–

8 ± 0.0a

– 9 ± 0.0a 8 ± 0.0a – 8 ± 0.57* 12 ± 0.57* 11 ± 0.0a 10 ± 0.0* 10 ± 0.0a – 10 ± 0.0a – – 9 ± 0.57* 9 ± 0.0a – 12 ± 0.0a 8 ± 0.0a – – 8 ± 0.0a

Staphylococcus aureus

Bacillus subtillis

– – 20 ± 0.57* 8 ± 0.0a 14 ± 0.57* 14 ± 0.57* 10 ± 0.57 12 ± 1.15 12 ± 0.0a – – 10 ± 0.57 – – 11 ± 0.0a – – 9 ± 0.57 – – 13 ± 0.57*

18 ± 0.57*

Bacillus cereus

10 ± 0.57* 11 ± 0.57* – – – 16 ± 0.0

13 ± 0.57* – 9 ± 0.0a 12 ± 0.0a – 12 ± 0.57* 10 ± 0.0a 9 ± 0.0a 8 ± 0.0a – – 10 ± 0.57* – – 8 ± 0.57* – – 11 ± 0.0a – 9 ± 0.0a 12 ± 0.0a

11 ± 0.57 *

Escherichia coli

8 ± 0.0a 8 ± 0.0a 8 ± 0.57 9 ± 0.57 9 ± 0.0a 9 ± 0.57

8 ± 0.57 – 8 ± 0.0a 8 ± 0.0a 8 ± 0.57 8 ± 0.57 – – – – – 8 ± 0.57 9 ± 0.57 8 ± 0.57 – 9 ± 0.57 9 ± 0.57 8 ± 0.0a 8 ± 0.57 9 ± 0.0a 8 ± 0.57

a

–

Pseudomonas aeruginosa

Each value represents the mean of inhibition zones (mm) of three replicates ± SEM. The correlation and t could not be computed because the standard error of the difference is zero. *Significantly different from the reference drug at Po0.05 according to a paired sample t-test.

S2 S3 Petroleum ether S1 S2 S3 Ether S1 S2 S3 Chloroform S1 S2 S3 Ethyl acetate S1 S2 S3 Methanol S1 S2 S3 Water extract S1 S2 S3 Unsaponifiable S1 matter S2 S3 Fatty acid S1 S2 S3 Reference drug Amoxycillin Canesten

Volatile constituent

Fractions

– – 9 ± 0.57* 9 ± 0.0a 9 ± 0.0a 26 ± 0.0

– 9 ± 0.0a – – – 10 ± 0.57* – – 8 ± 0.57* 8 ± 0.0a 10 ± 0.0a 10 ± 0.0a 9 ± 0.57* 9 ± 0.57* – – 12 ± 0.0a – – 10 ± 0.57* –

–

Pseudomonas fluorescens

11 ± 0.0

12 ± 0.57

20 ± 0.57

– 11 ± 0.57* 9 ± 0.57* – –

– – – – – – – – – – – – – – – – – 9 ± 0.0* – – 9 ± 1.15*

9 ± 0.0a 10 ± 0.0a 8 ± 0.57* – – 10 ± 0.57 12 ± 0.0a 11 ± 0.0a 8 ± 0.57* – 9 ± 0.57 – – – 8 ± 0.57* – 9 ± 0.0a 9 ± 0.0a – 9 ± 0.57 12 ± 0.57 – 10 ± 0.57 8 ± 0.57* 8 ± 0.0a 9 ± 0.0a

–

Aspergillus flavus

9 ± 0.57

Saccharomyces cerevisiae

12 ± 0.57a 8 ± 0.57 9 ± 0.0* 12 ± 0.0 –

14 ± 0.0 – – 12 ± 0.0 – 9 ± 0.0* 14 ± 0.57a 9 ± 0.57 – 11 ± 0.57 – 9 ± 0.0* 9 ± 0.0* – – 9 ± 0.57* – – 8 ± 1.15 – 9 ± 0.57*

–

Saccharomyces carles

Mean of inhibition zones (mm ± SEM)

9 ± 0.0

– – – – –

– – – – 8 ± 0.0a 8 ± 0.57 – 8 ± 0.0a – – – – – – – 8 ± 0.57 8 ± 0.0a – – 8 ± 0.0a –

–

Aspergillus niger

14 ± 0.0

8 ± 0.0a 9 ± 0.57* 14 ± 0.57 9 ± 0.57* 11 ± 0.0a

9 ± 0.0* 8 ± 0.0* 9 ± 0.57* 10 ± 0.57* 8 ± 0.57* 8 ± 1.15* – 14 ± 0.0a 8 ± 0.0a – – 9 ± 0.0a 9 ± 0.57* 14 ± 0.57 9 ± 0.0a 8 ± 0.0a 16 ± 0.0a – – 8 ± 0.0a 10 ± 0.57*

–

Deplodia oryzea

Table 6 Inhibitory response of the different fractions of the brown algae Sargassum asperifolium (S1), Sargassum dentifolium (S2), and Sargassum linifolium (S3) on the tested microbes in comparison with standard antibacterial and antifungal substances


108



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25 Mass Spectrometry Data Centre. Eight peaks index of mass spectra. 2nd ed. Aldermaston, Reading: AWRE; 1974. pp. 1–1190. 26 Jennings W, Shibamoto T. Qualitative analysis of flavor and fragrance volatiles by glass capillary gas chromatography. New York: Academic Press; 1980. pp. 1–472. 27 Adams RP. Identification of essential oils by ion trap mass spectroscopy. San Diego: Academic Press; 1989. pp. 1–302. 28 Adams RP. Identification of essential oil components by gas chromatography/mass spectroscopy. Carol Stream, IL, USA: Allured Publishing Corp.; 1995. pp. 1–459. 29 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990; 82:1107–1112. 30 Gnanamanickam SS, Mansfield JW. Selective toxicity of wyerone and other phytoalexins to gram-positive bacteria. Phytochemistry 1981; 20:997–1000. 31 Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli physiology in Luria– Bertani broth. J Bacteriol 2007; 189:8746–8749. 32 Dillon JR, Nasim A, Nestmann ER. Recombinant DNA Methodology. New York: John Wiley Sons Inc.; 1985. p. 127. 33 Subba Rao NS. Soil micro-organism and plant growth. Oxford: Mohan Primlani Publisher; 1977. p. 252. 34 Elsayed OH, Bolan W, Omer EA, Mohamed FM. Volatiles and pheromones of brown algae Sargassum latifolium and Sargassum asperifolium. Bull NRC Egypt 1997; 22:215–220. 35 Chen CY. Biosynthesis of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) from red alga – Bangia atropurpurea. Water Res 2004; 38:1014–1018. 36 Lyutskanova D, Ivanova V, Stoilova-Disheva M, Kolarova M, Aleksieva K, Peltekova V. Isolation and characterization of a psychrotolerant Streptomyces strain from permafrost soil in Spitsbergen, producing phthalic acid ester. Biotechnol Biotechnol Equipment 2009; 23:1220–1224. 37 Namikoshi M, Fujiwara T, Nishikawa T, Ukai K. Natural abundance 14C content of dibutyl phthalate (DBP) from three marine algae. Marine Drugs 2006; 4:290–297. 38 Hattab ME, Culioli G, Piovetti L, Chitour SE, Valls R. Comparison of various extraction methods for identification and determination of volatile metabolites from the brown alga Dictyopteris membranacea. J Chromatogr A 2007; 1143:1–7. 39 Demirel Z, Yilmaz-Koz FF, Karabay-Yavasoglu UN, Ozdemir G, Sukatar A. Antimicrobial and antioxidant activity of brown algae from the Aegean Sea. [Russian Source]. J Serb Chem Soc 2009; 74:619–628. 40 Inoue Y, Hada T, Shiraishi A, Hirose K, Hamashima H, Kobayashi S. Biphasic effects of geranylgeraniol, teprenone, and phytol on the growth of Staphylococcus aureus. Antimicrob Agents Chemother 2005; 49:1770–1774. 41 Khanavi M, Gheidarloo R, Sadati N, Shams Ardekani MR, Bagher Nabavi SM, Tavajohi S, Ostad SN. Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line. Pharmacogn Mag 2012; 8:60–64. 42 Awad NE, Ibrahim NA, Matloub AA. Phycochemical and cytotoxic activity of some marine algae. Planta Med 2009; 75:877–1095. 43 Awad NE, Selim MA, Metawe HM, Matloub AA. Cytotoxic xenicane diterpenes from the brown alga Padina pavonia (L.) Gaill. Phytother Res 2008; 22:1610–1613. 44 Awad NE, Motawe HM, Selim MA, Matloub AA. Antitumourigenic polysaccharides isolated from the brown algae, Padina pavonia (L.) Gaill. and Hydroclathrus clathratus (C. Agardh) Howe. Med Aromat Plant Sci Biotechnol 2009; 3:6–11. 45 Kanias GD, Skaltsa H, Tsitsa E, Loukis A, Bitis J. Study of the correlation between trace elements, sterols and fatty acids in brown algae from the Saronikos gulf of Greece. Fresenius J Anal Chem 1992; 344: 334–339.

Original article 109

Regioselective addition of alkyl phosphites on 6-(aryliminomethyl)-furobenzopyran-5-one derivatives Nabila M. Ibrahima, Asmaa A. Magd-El-Dinb, Amira S. Abd El-Allb, Eman F. Al-Amrousia and Hisham Abdallah A. Yosefa a Department of Organometallic and Organometalloid Chemistry, Division of Organic Chemistry and b Department of Natural and Microbial Products Chemistry, Division of Pharmaceutical and Drug Industries Research, National Research Centre, Dokki, Cairo, Egypt

Correspondence to Hisham Abdallah A. Yosef, Department of Organometallic and Organometalloid Chemistry, National Research Centre, Dokki, Cairo, 12622, Egypt Tel: + 20 33 371 615; fax: + 20 233 370 931; e-mail: [email protected] Received 1 March 2012 Accepted 13 May 2012 Egyptian Pharmaceutical Journal 2012,11:109–115

Aim Trialkyl phosphites 2a,b attack 6-(aryliminomethyl)furobenzopyran-5-ones 1a–e regiospecifically at the carbon–carbon double bond of the g-pyrone ring to yield new 1,2addition phosphonate products for which structures 3a–e have been respectively assigned. Methods The alkyl phosphites 2a,b attacked the monoanils 1a–e at the azomethine carbon of the C = N bond to yield corresponding phosphonate adducts 5a–e when reactions were carried out in the presence of a controlled amount of acetic acid. Phosphonates 5a–e could also be obtained by the reaction of dialkyl phosphites 4a,b with anils 1a–e. Structures of the new phosphonates 3a–e were elucidated by elemental analyses as well as spectroscopic methods. The 1H and 13C nuclear magnetic resonance and infrared measurements were helpful tools in confirming the structures of the new products. Results and conclusion The insecticidal activities of phosphonates 3a–e and their respective regioisomers 5a–e against adult Aphis gossypii (Glover), which infest cotton crops, were determined. The structure–activity relationship has been discussed. Keywords: 6-(aryliminomethyl)furobenzopyran-5-ones, 13C nuclear magnetic resonance, insecticidal activities, phosphorylation, regioselectivity, spectroscopic evidences, structural elucidation Egypt Pharm J 11:109–115 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction It is evident that compounds of phosphorus play vital roles in the living process. Our interest in the field of organophosphorus chemistry led us to study the preparation of organophosphorus compounds because of their increasing importance in industry and biology [1–3]. Aldehydes [4–6], ketones [7,8], aldimines [9–11] and ketimines [12–14] are suitable substrates for the preparation of members of this class of compounds. Recently, we have reported on the reaction of monoarylimines 1a–e, derived from 4methoxy-5-oxo-5H-furo[2,3-g]benzopyran-6-carboxaldehyde and 4,9-dimethoxy-5-oxo-5H-furo[2,3-g]benzopyran-6-carboxaldehyde, with dialkyl phosphites 4a,b [1]. Now, with our growing interest in the field of organophosphorus chemistry of arylimines derived from carbonyl compounds [9,12–14], we have studied the behaviour of trialkyl phosphites 2a,b towards the monoarylimines 1a–e under different reaction conditions.

were prepared according to the given procedures. Melting points were recorded on an electrothermal melting point apparatus and were uncorrected. The infrared spectra were obtained from KBr disks using a Bru ¨ker Vector 22 infrared spectrophotometer (Germany) and/or a JASCO FT/IR300E fourier transform infrared spectrophotometer (Japan) and reported in cm – 1. 1H-nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury VX-300 spectrometer (Japan) (at 300 MHz) and/or a JEOL JNMEX 270 FT spectrometer (Japan) (at 270 MHz). Proton chemical shifts (d) are reported in ppm downfield from tetramethylsilane. 13C NMR spectra were recorded on a JEOL JNM-EX 270 FT (at 68 MHz) and/or a JOEL 500 AS (at 125 MHz) spectrometer. Mass spectra were recorded on a Finnigan SSQ 7000 spectrometer (Japan) and/or a Shimadzu GC MS-Q 1000 EX spectrometer at 70 eV (Electron Impact, Japan). Microanalyses were carried out at the Microanalytical Unit, Cairo University (Cairo, Egypt).


Experimental

Solvents were purified and dried according to usual procedures. Trialkyl phosphites are commercially available from Aldrich Chem. Co. (New Jersey, USA) and were freshly distilled immediately before use. The starting compounds 1a [1], 1b [1], 1c [15], 1d [1] and 1e [1]

Reactions of 1a–e with trialkyl phosphites 2a,b General procedure

A mixture of the appropriate anil 1a–e (0.005 mol) and trialkyl phosphite (TAP) 2a (or 2b; 0.01 mol) was heated in the absence of a solvent at 1001C until the starting anil


DOI: 10.7123/01.EPJ.0000421666.94096.f5


could no longer be detected (TLC) [TLC silica gel aluminum sheet (type 60 F254, Merk, Damstadt, Germany)]. After removal of the volatile materials in vacuo, the residual substance was washed with cold diethyl ether and then recrystallized from petroleum ether at 60–801C to yield the respective phosphonates 3a–e in yellow crystalline forms. Diisopropyl 6-((4-fluorophenylimino)methyl)-4-methoxy-5-oxo6,7-dihydro-5H-furo[3,2-g]chromen-7-ylphosphonate (3a, C25H27FNO7P)

Yield 81%, melting point (MP) 128–1301C; IR (KBr, cm – 1): 3096, 3064 (C–H aromatic), 2982 (C–H aliphatic, asymmetric), 2936 (C–H aliphatic, symmetric), 1713 (exocyclic C = O of a saturated 4-pyranone ring), 1647 (C = N), 1611, 1552 (C = C), 1247 (P = O), 1135 (C – O) and 1015 (P – O – C). 1H NMR (DMSO-d6): 1.00, 1.05, 1.10, 1.14 (12, O–CH–(CH3)2, 4d, JHH = 6.0 Hz), 4.03 (1H, CH–CH–CH = N, dd, JHH = 8.0 Hz, JHP = 10.8 Hz), 4.08 (3H, –OCH3, s), 4.51 (2H, O–CH– CH3, dsp, JHH = 6.0 Hz, JHP = 10.8 Hz), 5.43 (1H, O– CH–P, dd, JHH = 8.0 Hz, JHP = 18.0 Hz), 6.90 (1H, O – CH – CH, furan ring d, JHH = 7.17 (2H, aromatic meta to F atom, d, AB system, JHH = 9 Hz); 7.26 (2H, aromatics ortho to F atom, d, AB system, JHH = 9 Hz), 7.40 (1H, aromatic, s), 7.82 (1H, CH = N, d, 3JHH = 10.8 Hz), 7.91 (1H, O–CH, furan, d, JHH = 2.0 Hz). 13

C NMR (DMSO-d6): 21.02 (P–O–CH–CH3), 23.67 (P– C–CH–CH = N, 2JCP = 28.0 Hz), 60.84 (O–CH3), 70.71 (P–O–CH–CH3, d, 2JCP = 25.0 Hz), 74.45 (O–CH–P, d, 1 JCP = 146.4 Hz), 94.88 (C–H aromatic), 98.37 (quaternary aromatic carbon), 106.05 (O–CH–CH, furan), 112.25 (quaternary aromatic carbon), 116.80 (carbons ortho to fluorine atom, d,2JCF = 22.62 Hz), 118.36 (carbons meta to fluorine atom, d, 3JCF = 8.3 Hz), 137.26 (C = N–C), 143.48 (O–CH furan), 145.27 (CH = N), 154.92 (C– OCH3), 157.81, 158.56 (quaternary aromatic carbons), 171.97 (C–F) and 181.25 (C = O). MS m/z (%): 503 [M] + (3%). Anal. calcd (%) for C25H27FNO7P (503.46): C, 59.64; H, 5.41; F, 3.77; N, 2.78; P, 6.15. Found (%): C 59.76; H, 5.28; 2.61; P, 5.22. Diisopropyl 6-((4-chlorophenylimino)methyl)-4-methoxy-5oxo-6,7-dihydro-5H-furo[3,2-g]chromen-7-ylphosphonate (3b, C25H27ClNO7P)

Yield 80%; MP 118–1201C. IR (KBr, cm – 1): 3110, 3066 (C– H aromatic), 2980 (C–H aliphatic, asymmetric), 2934 (C–H aliphatic, symmetric), 1717 (exocyclic C = O of a saturated 4-pyranone ring), 1645 (C = N), 1601, 1553 (C = C); 1246 (P = O); 1129 (C – O), and 983 (C – Cl aromatic). 1H NMR (DMSO-d6): 0.966, 1.04, 1.11, 1.18 (6H, O–CH(CH3)2, 4d, JHH = 8.1 Hz), 4.03 (1H, CH–CH–CH = N, d, JHH = 10.8 Hz), 4.06 (3H, OCH3, s), 5.41 (1H, P–CH–CH, dd, JHH = 10.8 Hz, JHP = 15.5 Hz), 6.87 (1H, O–CH–CH furan, d, JHH = 3 Hz), 7.12 (2H, aromatic meta to chlorine, AB system, d, JHH = 8.1 Hz), 7.25 (2H, aromatics ortho to chlorine, AB system, d, JHH = 8.1 Hz), 7.41 (1H, s, aromatic), 7.80 (1H, –CH = N, d, JHH = 10.8 Hz), 7.88 (1H, O–CH furan, d, JHH = 3.0 Hz). MS m/z (%): [M] + at 519 and 521 (2.7% and 0.83%). Anal. calcd (%) for C25H27ClNO7P

(519.92): C, 57.75; H, 5.23; Cl, 6.82; N, 2.69; P, 5.96. Found (%): C, 57. 86; H, 5.02; Cl, 6.57; N, 2.61; P, 6.12. Diisopropyl 6-((4-bromophenylimino)methyl)-4-methoxy-5oxo-6,7-dihydro-5H-furo[3,2-g]chromen-7-ylphosphonate (3c, C25H27BrNO7P)

Yield 83%, MP 150–1521C. IR (KBr, cm – 1): 3066 (C–H, aromatic), 2979 (C–H aliphatic, asymmetric), 2933 (C–H aliphatic, symmetric), 1717 (exocyclic C = O of a saturated 4-pyranone ring), 1645 (C = N), 1601, 1553 (C = C), 1246 (P = O) and 1127 (P–O–C). 1

H NMR (DMSO-d6): 0.09, 1.04, 1.11, 1.17 (12H, CH– (CH3)2, four doublets, each of 1,3JHH = 5.4 Hz), 4.04 (1H, CH–CH–CH = N, d, JHH = 10.8 Hz), 4.06 (3H, O–CH3), 4.48 (2H, O–CH–(CH3)2, m), 5.41 (1H, O–CH–CH, dsp, JHH = 10.8 Hz, JHP = 19.3 Hz), 6.87 (1H, O–CH–CH, furan ring, d, JHH = 2 Hz), 7.15 (2H, aromatic meta to bromine, AB system, d, JHH = 8.1 Hz), 7.33 (2H, aromatic para to bromine, AB system, d, JHH = 8.1 Hz), 7.56 (1H, aromatic, s), 7.82 (1H, CH = N, d, JHH = 10.8 Hz),7.88 (1H, O–CH, furan, d, JHH = 2.0 Hz). MS m/z (%): [M] + at 564 and 566 (2.3 and 2.4%). Anal. calcd (%) for C25H27BrNO7P (564.37): C, 53.21; H, 4.82; Br, 14.16; N, 2.48; P, 5.49. Found (%): C, 53.58; H, 4.57; Br, 14.00; N, 2.24; P, 5.62.

Diisopropyl 6-((4-fluorophenylimino)methyl)-4,9-dimethoxy-5oxo-6,7-dihydro-5H-furo[3,2-g]chromen-7-ylphosphonate (3d, C26H29FNO8P)

Yield 79%; MP 98–1001C. IR (KBr, cm – 1): 3135, 3072 (C–H aromatic), 2983, 2935 (C–H aliphatic), 1714 (exocyclic C = O of a saturated 4-pyranone ring), 1644 (C = N), 1605, 1553 (C = C aromatic), 1240 (P = O) and 1197 (P–O– C). 1H NMR (DMSO-d6): 1.05, 1.11, 1.15, 1.16 (12H, CH(CH3), four doublets each with JHH = 6.0 Hz), 3.86 (1H, CH–CH–CH = N, dd, 3JHH = 6.8 Hz, 3JHH = 8.3 Hz), 3.88 (3H, OCH3,s), 3.92 (3H,OCH3,s), 4.47 (2H, CH(CH3)2,dsp, JHH = 6.3 Hz, JHP = 11.5 Hz), 5.46 (1H, O–CH–P, dd, 3JHH = 6.8 Hz, 2JHP = 15 Hz), 7.07 (1H, O– CH–CH, furan ring, d, JHH = 2.0 Hz), 7.21 (2H, aromatics meta to fluorine, AB system, 3d, JHH = 8.1 Hz), 7.36 (2H, aromatics ortho to fluorine, AB system, d, 3JHH = 8.1 Hz). 7.77 (1H, CH–CH = N, d, JHH = 9.3 Hz), 7.88 (1H, O–CH, furan ring, d, JHH = 2.0 Hz). 13C NMR (DMSO-d6): 21.56 (O–CH–(CH3)2), 24.25 (CH–CH = N), d, 2JCP = 14.31), 61.42 (OCH3), 61.75 (OCH3), 71.41 (O–CH–(CH3)2, d, 2 JCP = 7.2 Hz), 74.41 (O–CH–P, d, 1JCP = 156.3 Hz), 98.79 (quaternary aromatic carbon), 105.99 (CH–CH furan), 113.79 (quaternary fused aromatic carbon), 116.92 (two carbons ortho to fluorine, d, 2JCF = 13.6 Hz), 118.52 (two carbons meta to fluorine, d, 3JCF = 5.4 Hz), 129.91 (C = N– C), 143.97 (O–CH furan), 145.86 (CH = N), 148.99, 149.49, 150.84, 159.02 (quaternary aromatic carbons), 172.67 (C–F), 181.25 (C = O). MS m/z (%): 533 (5%). Anal. Calcd. (%) for C26H29FNO8P (533.49): C, 58.54; H, 5.48; F, 3.56; N, 2.63; P, 5.81. Found (%): C, 58.28; H, 5.20; F, 3.31; N, 2.59; P, 5.95. Diethyl 6-((4-chlorophenylimino)methyl)-4,9-dimethoxy-5oxo-6,7-dihydro-5H-furo[3,2-g]chromen-7-ylphosphonate (3e, C24H25ClNO8P)

Yield 86%; MP 198–2001C. IR (KBr, cm – 1): 3131, 3083 (C – H aromatic); 2975 (C – H aliphatic, asymmetric);

Regioselective addition of phosphites Ibrahim et al. 111

2930 (C – H aliphatic, symmetric); 1715 (exocyclic C = O of a saturated 4-pyranone ring); 1645 (C = N), 1600, 1552 (C = C); 1246 (P = O); 1129 (P–O–C); 984 (C – Cl aromatic). 1H NMR (DMSO-d6): 1.08 (6H, CH2–CH3, t, JHH = 7.1 Hz), 3.92 (3H, OCH3, s), 3.71 (1H, CH– CH = N, dd, 3JHH = 6.7 Hz, 3JHH = 8.3 Hz) 3.97 (3H, OCH3, s), 3.93–4.00 (4H, OCH2CH3, m), 5.58 (1H, O– CH–P, dd, 3JHH = 10.0 Hz, 2JHP = 15.0 Hz), 7.15 (1H, O– C–CH, furan, d, 3JHH = 3.2 Hz), 7.40–7.45 (4H, aromatic, m), 7.85 (1H, CH = N, d, 3JHH = 10.2 Hz), 7.96 (1H, O– CH furan, d, JHH = 3.2 Hz). 13C NMR (DMSO-d6): 16.18 (OCH2–CH3, d, 3JCP = 5.4 Hz), 38.72 (P–CH–CH, d, 2JCP = 20.9 Hz), 60.92 (OCH3), 61.34 (OCH3), 62.28 (P–O–CH2–CH3, d, 2JCP = 16.3 Hz), 74.20 (O–CH–P, d, 1 JCP = 162.0 Hz), 98.95 (quaternary aromatic carbon), 105.34 (O–CH–CH, furan), 113.13 (quaternary aromatic carbon), 117.61 (carbons meta to chlorine), 127.25 (carbons ortho to chlorine), 129.39, 129.54 (quaternary aromatic carbons), 139.17 (C = N–C), 142.47 (O–CH, furan), 145.51 (CH = N), 148.36, 149.10, 150.39 (quaternary aromatic carbons) and 180.79 (C = O). MS: m/z (%): 521and 523 (3 and 0.92%). Anal. calcd (%) for C24H25ClNO8P (521.89): C, 55.23; H, 4.83; Cl, 6.79; N, 2.68; O, 24.53; P, 5.93. Found (%): C, 55.01; H, 4.69; Cl, 6.51; N, 2.50; P, 6.19. Reactions of 1a–e with trialkyl phosphites 2a,b in the presence of acetic acid General procedure

A mixture of the appropriate anil 1a–e (0.005 mol), trialkyl phosphite 2a (or 2b; 0.01 mol) and a few drops of acetic acid was heated in the absence of a solvent at 1001C until the starting anil could no longer be detected (TLC). After removal of the volatile materials in vacuo, the residual substance was washed with cold diethyl ether and then recrystallized from ethanol to yield the respective phosphonates as yellow crystalline products, which were identified to be the corresponding phosphonates 5a–e (MP, mixed MP, comparative TLC and comparative IR) [1]. Insecticidal evaluation

Toxicity of the tested compounds was studied under laboratory conditions using the slide-dip method [16] at the Central Agricultural Laboratory of Pesticides, Cairo, Egypt. Concentrations were prepared by dissolving each compound (0.05 g) in 5 ml of acetone and then diluting it with water in a ratio of 2 : 3 (this dilution did not cause any mortality in insects). Aphis gossypii was transferred onto a slide using a fine brush and then adults were affixed to a double-face scotch tape and fixed tightly onto the slide on their dorsal side. The slides were then dipped into the toxicant solution for 10 s and the excess toxicant was blotted off with filter paper. Mortality counts were carried out 2 h after treatment. A mortality of 50% for each compound was determined from the corresponding average mortality percentage that was corrected, if necessary, using Abbott’s formula [17].

Results and discussion Chemistry

On performing the reaction of monoanils 1a–e with trialkyl phophites 2a,b in the absence of a solvent at

100 1C, yellow crystalline products were obtained. They were assigned the phosphonate structures 3a–e (Scheme 1). However, phosphonates 3a–e were found to be isomeric (microanalyses and mass spectrometry) but not identical (comparative MPs, TLC, IR and 1H NMR spectra) to phosphonates 5a–e [1] that were obtained from the reaction of anils 1a–e with dialkyl phosphites 4a,b (Scheme 2). In contrast, the reaction of anils 1a–e with phosphites 2a,b, in the presence of a few drops of acetic acid, was found to yield phosphonates 5a–e [1] (MP, mixed MP, TLC and IR, Scheme 2). Thus, it is clear that anils 1a–e behave differently towards alkyl phosphites depending on the nature of the reagent and/or the reaction conditions. The spectroscopic measurements, particularly decoupled and distortionless enhancement by polarization transfer 13 C NMR experiments, IR and 1H NMR, have provided valuable evidence in confirming the structure of phosphonates 3a–e. The assigned structure 3a is established by analytical and spectral data:

(1) The IR spectrum (KBr) showed intense bands at 1247, 1135 and 1015 cm – 1, corresponding to P = O, C–O and P–O–C alkyl stretching vibrations [18,19], respectively. Absorption bands of the carbonyl group appeared at 1650 cm – 1 in anil 1a and at 1647 cm – 1 in Scheme 1

X CH3O 3 4

O 2

1

O H 5

O

N

+ (RÒ)3P

6 7

2a , R`= C 2 H5 b , R`= i -C 3H7

R 1a, R = H, X = F b , R = H, X = Cl c, R = H, X = Br d, R = OCH3, X = F e, R = OCH3, X = Cl

100 o C without solvent X

CH3 O 3 4

O 2

1

R

O H H 5

N

6

H OR` O P O OR` 7

3a, R = H, X = F, R`= i -C3 H7 b, R = H, X = Cl, R`= i -C3 H7 c, R = H, X = Br, R`= i -C 3H7 d, R = OCH 3 , X = F, R`= i -C 3H7 e, R = OCH 3 , X = Cl, R`= C 2H5 Preparation of phosphonate compounds (3a-e).


Scheme 2 X CH3O

O H N O

O

O

R

(RÒ)2PH

1a, R = H, X = F b, R = H, X = Cl c, R = H, X = Br d, R = OCH3 , X = F e, R = OCH3 , X = Cl

..

(RÒ) 2 P(OH)

(RÒ)3P/H+ 2a, R`=C2H5 b, R`=C3H7-i

4a, R`=C2H5 b, R`=C3H7-i

X CH3 O

H N P OR` O OR`

O H

O

O R

5a, R = H, X = F, R`= C 3 H7 -i b, R = H, X = Cl, R`= C3 H7 -i c, R = H, X = Br, R`= C 3 H7 -i d, R = OCH 3 , X = F, R`= C 3 H7 -i e, R = OCH3 , X = Cl, R`= C 2 H5

Preparation of phosphonate compounds (5a-e).

Scheme 3 +.

F CH 3 O

O X O p. _ i-C 3 H7 O i-C 3 H7 O

O

H Hy

C N H OC3 H 7 -i O P OC3 H 7 -i O x

O

CH 3 O

H

O H

H

+

C N O

+

O

.

- C 7 H5 FN

F CH 3 O

Y

a, (M +), C 25 H27 FNO 7 P, m/z 503 (3%)

O

O

H

b, C 19 H13 FNO 4, m/z 338 (100%)

H OC3 H7 -i P OC3 H7 -i O

d, C18 H22 O7 P, m/z 381 (1%)

- CH 2 =CH-CH 3

- CH 2 =O +

F O H H

CH 3 O

H C N O

O

O

+

O

H

c, C18 H11 FNO 3, m/z 308 (2%)

O

H OC3 H7 -i P OH O

e, C 15 H16 O7 P, m/z 339 (20%)

MS fragmentation of 3a.

phosphonate 5a [1], whereas in phosphonate 3a, it appeared at 1713 cm – 1, a value that is typical for exocyclic C = O of a saturated six-membered ring [19]. Moreover, the spectrum of 3a indicated the presence of C = N absorption at 1647 cm – 1 (which appeared in the IR spectrum of 1a at 1616 cm – 1) [1] and the absence of any absorption band because of the NH group, which generally appears in the region of

3350–3300 cm – 1 [18,19] (which appeared in the spectrum of 5a at 3303 cm – 1) [1]. (2) The 1H NMR spectrum of 3a (DMSO-d6, d ppm) showed four doublets at 1.00, 1.05, 1.10 and 1.14 ppm, each with JHH = 6.0 Hz, because of protons of the four methyl groups of the two isopropoxy groups attached to phosphorus. Apparently, the asymmetry of the molecule because of the presence of a stereocenter would render the four methyl groups diastereotopic, and hence anisochronous, thus resulting in the observed splitting pattern [20,21]. Moreover, the isopropoxy-CH protons attached to phosphorus (2H) appeared as a doublet of septet at 4.51 ppm (with 3JHH = 6.0 Hz and 3JPH = 10.8 Hz) because of coupling with the methyl groups and the phosphorus atom, respectively. Signals were also recorded at 4.03 ppm (1H, CH–CH–CH = N, dd, 1,3 JHH = 8.0 Hz, 1,3JHH = 10.8 Hz) and 5.43 ppm (1H, O – CH – P, dd, 1,3JHH = 8.0 Hz and 1,2JHP = 18.0 Hz) [22,23]. Moreover, the doublet (1,3JHH = 10.8 Hz) that appeared at d of 7.82 ppm could be attributed to the azomethine CH = N proton. The spectrum also lacked absorption due to D2O-exchangeable protons (NH or OH). (3) The 13C NMR spectrum of 3a showed signals at 181.25 and 145.27 ppm because of carbon atoms of C = O and CH = N groups, whose signals appeared at 180.48 and 144.48 ppm, respectively, in the spectrum of 1a. The signals present at 111.31 and 154.17 ppm in the 13C NMR spectrum of 1a because of carbon-6 and carbon-7, respectively, disappeared in the spectrum of phosphonate 3a. Instead, two doublets centred at 23.67 ppm (2JCP = 28.0 Hz) and 74.45 ppm (1JCP = 146.4 Hz) appeared in the spectrum of 3a, assignable to carbon-6 and carbon-7, respectively. The last doublet was in the region of ter-carbon– oxygen chemical shifts and its large J-value was typical for spin–spin coupling between directly bonded carbon and phosphorus atoms [24,25]. Meanwhile, in phosphonate 5a, the signal because of the carbon attached to phosphorus appeared at d of 42.22 ppm (1JCP = 147.0 Hz) [1], cf. Experimental. (4) In the mass spectrum of 3a, the molecular ion peak (M + ; the cation radical a) appeared at m/z 503 (3%), showing its relative instability under electron bombardment. Cleavage of [M] + at axis x produced the base peak cation b at m/z 338 (100%), which released a neutral CH2O molecule from the methoxy group to yield cation c at m/z 308 (2%). Radical (cation a) [M] + was also cleaved at axis y to yield cation d at m/z 381 (1%), which in turn lost a neutral 1-propene molecule to yield cation e at m/z 339 (20% Scheme 3). Thus, in their reactions with anils 1a–e, phosphites 3a and 3b undergo a Michael-type addition reaction [26,27] in which the phosphite–phosphorus atom regiospecifically attacks carbon-7 of the C = C bond in the g-pyrone ring to yield 1,2-addition phosphonate products 3a–e exclusively (Scheme 4). The negative charge, formed on carbon-6 in phosphonium betaine intermediate 6, is


resonance stabilized through conjugation with the carbonyl and/or imine groups. This conjugation reduces the activation energy for addition. Thereafter, the betaine 6 undergoes solvation to yield intermediate 7 with pentacovalent phosphorus similar to that in many phosphobetaine structures [28]. Collapse of 7 through the release of an alcohol molecule yielded the respective phosphonates 3a–e. However, when the reactions were performed in acetic acid, trialkyl phosphites 2a,b were hydrolysed first to the corresponding dialkyl analogous 4a,b, which in turn reacted with anils 1a–e to yield phosphonates 5a–e. Apparently, the regiochemistry of the nucleophilic addition of alkyl phosphites 2 and 4 to anils 1a–e may be

Scheme 4

X CH 3 O

O

H -

N H OR` O +P OR` OR`

1a-e O R

H2O

Insecticidal activity

6 X CH 3 O 3a-e

(RÒ)3P

O H -

N H OR` O P OR` OR` +O H H

- RÒH O R

7

H+

(RÒ)2P-OH RÒH

1a-e

CH 3 O

O

RÒ O RÒ O +P C N H O

attributed to the steric and electronic nature of both reactants. Thus, in terms of the hard–soft acid–base principle [29,30], alkyl phosphites are considered to be soft bases. However, trialkyl phosphites are softer bases than dialkyl phosphites because of the tautomerism shown by the latter (Scheme 2). However, because of the + R effect exerted by the adjacent oxygen atom, carbon-7 may be relatively less positively polarized (softer acid) than the azomethine carbon of the C = N bond. Therefore, it may be energetically more favourable for alkyl phosphites 2a,b (softer base) with their relatively bulky size to add to carbon-7 (softer acid), rather than to the azomethine carbon, to avoid the steric crowding [31] exerted by the substituted phenyl group on nitrogen in the transition states, leading to intermediates 6 and 7. However, phosphites 4 (harder base), with their relatively smaller size, tend to add preferentially at the more electrophilic azomethine carbon atom (harder acid) of the polarized C = N bond to yield 5. In this case, delocalization of the negative charge that developed on nitrogen over the substituted phenyl group stabilized the intermediate transition state.

H X

R 8

5a-e 5a, R = H, X = F, R`= C 3 H7 -i b, R = H, X = Cl, R`= C3 H7 -i c, R = H, X = Br, R`= C 3 H7 -i d, R = OCH 3 , X = F, R`= C 3 H7 -i e, R = OCH 3 , X = Cl, R`= C 2 H5

Pesticidal activities are associated with a variety of organophosphorus compounds [32,33], which may become less effective as a result of development of crossresistance in pests. Therefore, continuous efforts are being made towards developing a new generation of these pesticides. This led us to study the effect of phosphonates 3a–e and 5a–e on A. gossypii (Glover) using actillic (O-[2-(diethylamino0–6methyl-4-pyridiminyl]O,O-dimethylphosphorothioate) as a standard reference (cf. Experimental). This sucking aphid pest infests cotton crops worldwide. It affects cotton yield by direct feeding and fibre quality by excreting honeydew, which supports the growth of harmful microorganisms. The results for the insecticidal activity of phosphonates 5a–e have been reported [1]. However, as the biological and pharmaceutical activities of many organic compounds [34,35], including phosphonate derivatives [36–38], strongly depend on the position of a given group in a certain class of compounds, it will be interesting to report the insecticidal activity for phosphonates 3a–e and compare it with the activity of their regioisomers 5a–e (Table 1). From the results in Table 1, it is evident that: (1) The LC50 values indicated that all of the tested compounds showed insecticidal activity against adult A. gossypii (Glover).

Mechanism of preparation phosphonates compounds (5a-e).

Table 1 Effect of treatment with phosphonates 3a–e and 5a–e on adults of Aphis gossypii (Glover) Compound Actilicd (pirimiphosmethyl) 3a 3b 3c 3d 3e a

LCa50 (ppm)

TIb (%)

Compound

LCa,c 50 (ppm)

TIb,c (%)

1240.67 1489.71 2816.69 1567.55 5377.57 14714.36

100 83.28 44.05 79.15 23.07 8.43

5a 5b 5c 5d 5e

2520.48 4912.93 1907.59 3906.69 6303.25

49.22 25.25 65.03 31.75 19.68

LC50: lethal concentration that killed 50%. TI: toxicity index = LC50 of the most effective compound 100/LC50 of the tested compound. Values for phosphonates 5a–e have been reported previously in reference [1]. d The standard pesticide actillic: O-[2-(diethylamino0-6-methyl-4-pyridiminyl]O,O-dimethyl phosphorothioate. b c


(2) Among phosphonates 3 and 5, compound 3a was found to be the most effective, with a toxicity index (TI) value of 83.28% (LC50 = 1489.71), when compared with the reference insecticide actillic (TI = 100%, LC50 = 1240.67). Compound 3e was found to be the least effective (TI = 8.43%, LC50 = 14714.36). The order of decreasing activity was as follows: 3a43c45c45a43b45d45b43d45e43e. (3) Among phosphonates 3, the order of decreasing activity was 3a43c43b43d43e, whereas for phosphonates 5, the order was 5c45a45d45b45e. (4) For the phosphonates that were derived from compound 1a (R = H), 3a–c, in which the phosphorus atom is linked to carbon-7, were found to be more active than their respective regioisomers 5a–c, in which the phosphorus atom is linked to the azomethine carbon. However, for the derivatives of 1b (R = OCH3), phosphonates 5d and 5e (phosphorus–azomethine carbon bond) were found to be more active than phosphonates 3d and 3e (phosphorus–carbon-7 bond), respectively. (5) Phosphonates 3e and 5e that have the diethylphosphoryl moiety (R0 = C2H5) are less effective than the other derivatives that have the diisopropylphosphoryl moiety (R0 = i-C3H7).

from 6-(aryliminomethyl)-furobenzopyran-5-ones. Phosphorus, sulfur and silicon and the related elements 2010; 185:2277–2285. 2 Hafez TS, Essawy SA, Al-Amrousi EF, Mahran MRH. Reactions of furobenzopyran-6-carboxaldehydes with alkyl phosphites and ylidenetriphenyl phosphoranes (Wittig reagents). Egypt J Chem 2007;Special Issue (M. Sidky): 45–58. 3 Savignac P, Iogra B. Modern phosphorus chemistry. Boca Raton: CRC Press, Fl; 2003. 4 El Kaı¨m L, Gaultier L, Grimaud L, Dos Santos A. Formation of new phosphates from aldehydes by a DBU-catalysed phospha-brook rearrangement in a polar solvent. Synlett 2005; 15:2335–2336. 5 Deron A, Milewska M, Barycki J, Sawka-Dobrowolska W, Gancarz R. NMR studies of the bishydroxy bisphosphonate synthesis from o-phthalic aldehyde and diethyl phosphate. Heteroatom Chem 2002; 13:157–164. 6 Plazuk D, Zakrzewski J, Rybarczyk-Pirek A. Diastereoselective addition of dimethyl phosphite to 3,30 ,4,40 -tetramethyl-1,10 -diphosphaferrocene-2-carbox aldehyde. J Organometal Chem 2006; 691:3098–3102. 7 Mahran MR, Khidre MD, Abdou WM. The reaction of isatin, 5-methylisatin and their monoximes with alkyl phosphites, triphenylphosphine and phosphorus ylides. Phosphorus, sulfur and silicon 1995; 101:17–27. 8 Borowitz IJ, Anschel M, Readio PD. Reactions of fluorenones and tetraphenyl cyclopentadienones with tricovalent phosphines and phosphites. J Org Chem 1971; 36:553–560. 9 Khidre MD, Abou-Yousef HM, Mahran MRH. On the reaction of 3-(aryliminomethyl)chromones with alkyl phosphites and methylenetriphenylphosphoranes (Wittig reagents). Phosphorus, sulfur and silicon and related elements 1998; 140:147–157. 10 Nazarski RB, Lewkowski JA, Skowron´ski R. Rationalization of the stereochemistry of an addition of dialkyl phosphites to certain chiral aldimines: the experimental and theoretical approach. Heteroatom Chem 2002; 13:120–125. 11 Yuan C, Li S, Li C, Chen S, Huang W, Wang G, Pan C, Zhang Y. New strategy for the synthesis of functionalized phosphonic acids. Pure Appl Chem 1996; 68:907–912. 12 Sidky MM, Mahran MR, Abdou WA, Hafez TS. Reaction of alkyl phosphites with mono-anils of benzil and o-naphthoquinone. Egypt J Chem 1984; 27:809–816.

Conclusion The present study clearly shows that anils 1a–e behave as ambident electrophiles, as they could be attacked by the phosphite reagents 2 and/or 4 either on carbon-7 or the azomethine carbon, yielding a variety of phosphonate regioisomers. The regioselectivity of attack depends on the steric and electronic nature of both the reagents and the substrates as well as the reaction conditions. Thus, although trialkyl phosphites 2 regiospecifically attack carbon-7 in anils 1a–e to yield phosphonates 3a–e exclusively, the dialkyl phosphites 4 attack the azomethine carbon regiospecifically to yield phosphonates 5a–e [1]. However, the reaction of 2 with 1 in the presence of a few drops of acetic acid yielded phosphonates 5. The new phosphonates 3a–e which is derived from furochromones belong to many biologically active centers which can be used as drugs [39,40]. They also belong to the pharmacologically interesting a-aminophosphonates [41–43]. Insecticidal activity tests have confirmed that the phosphonate adduct 3a shows marked potency against adult A. gossypii (Glover), which infest cotton crops. Moreover, there is a marked difference in activity between the respective regioisomers, which confirms the structure–activity relationship principle.


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116 Original article

Synthesis of certain new fused pyranopyrazole and pyranoimidazole incorporated into 8-hydroxyquinoline through a sulfonyl bridge at position 5 with evaluation of their in-vitro antimicrobial and antiviral activities Emad M. Kassema, Eslam R. El-Sawyb, Howaida I. Abd-Allab, Adel H. Mandourb, Dina Abdel-Mogeeda and Mounir M. El-Saftyc Departments of aTherapeutic Chemistry, bChemistry of Natural Compounds, National Research Centre, Dokki, Giza, Egypt and cCentral Laboratory for Evaluation of Veterinary Biologics, Abbassia, Cairo, Egypt Correspondence to Eslam R. El-Sawy, Department of Natural Compounds Chemistry, National Research Centre, 12311 Dokki, Giza, Egypt Tel: + 20238339394; fax: + 2033370931; e-mail: [email protected] Received 14 June 2012 Accepted 9 September 2012 Egyptian Pharmaceutical Journal 2012, 11:116–123

Background and objectives Heterocyclic systems with a quinoline nucleus display a wide spectrum of biological activities such as antimicrobial and antiviral activities. The aim of the present study was the synthesis of new fused pyranopyrazoles, 5a-e and 6a-e, and pyranoimidazoles, 10a-e and 11a-e, incorporated to 8-hydroxyquinoline through a sulfonyl bridge at position 5 and evaluation of their antimicrobial and antiviral activities. Methods The synthesis of the titled quinoline derivatives was achieved through cyclization of 8-hydroxyquinoline-5-sulfonyl chloride (1) with 20 -acetyl-2-cyanoacetohydrazide, 2-cyanoacetic acid hydrazide, and 3-amino-5-pyrazolone to afford 2, 3, and 4, respectively. Moreover, reaction of 1 with glycine gives 7, which on heterocyclization with ammonium thiocyanate yielded the 2-thioxoimidazolidin-2-one derivative 8. Cyclocondensation reaction of 3, 4, 8, and 9 with different arylidene malononitriles afforded fused systems, 5a-e, 6a-e, 10a-e, and 11a-e, respectively. The synthesized compounds were evaluated for their in-vitro antimicrobial activity using the disc diffusion method. In addition, they were evaluated for their in-vitro antiviral activity against avian paramyxovirus type 1 (APMV-1) and laryngotracheitis virus (LTV). Results and conclusion In-vitro antimicrobial activity of the newly synthesized compounds included an inhibitory effect toward the growth of Escherichia coli and Pseudomonas aeruginosa (Gram-negative bacteria). Furthermore, of the six selected compounds (2, 3, 4, 7, 8 and 9) tested for their antiviral activity, compounds 2, 3, and 4 at a concentration range of 3–4 mg/ml showed marked viral inhibitory activity for APMV-1 of 5000 tissue culture infected dose fifty (TCID50) and LTV of 500 TCID50 in Vero cell cultures on the basis of their cytopathic effect. Chicken embryo experiments show that compounds 2, 3, and 4 possess high antiviral activity in vitro, with inhibitory concentration fifty (IC50) ranging from 3 to 4 mg/egg against avian APMV-1 and LTV and toxic concentration fifty (CC50) ranging from 200 to 300 mg/egg. Keywords: antimicrobial, antiviral activities, 8-hydroxyquinoline-5-sulfonyl chloride, pyrano[2,3-c] pyrazole, pyrano[2,3-d]imidazole Egypt Pharm J 11:116–123 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Heterocyclic systems with a quinoline nucleus represent privileged moieties in medicinal chemistry and are ubiquitous substructures associated with biologically active natural products. Quinoline derivatives have been shown to display a wide spectrum of biological activities such as antibacterial [1–3], antifungal [4,5], antiparasitic [6], and antiviral activities [7,8]. Because of their wide range of biological activities, quinoline compounds have been considered to be good starting materials for the search of novel antimicrobial and antiviral agents. Accordingly, the aim of the present work was the synthesis of new fused pyranopyrazoles, 5a-e

and 6a-e, and pyranoimidazoles, 10a-e and 11a-e, incorporated into 8-hydroxyquinoline through a sulfonyl bridge at position 5. Moreover, the study includes testing of the target compounds for their expected antimicrobial and antiviral activities.

Subject and methods Chemistry

Melting points were determined in open capillary tubes, on an Electrothermal 9100 digital melting point apparatus (Bu ¨chi, Mount Holly, New Jersey, USA), and were reported


DOI: 10.7123/01.EPJ.0000421482.33940.0b

Biologically active new quinoline derivatives Kassem et al. 117

uncorrected. Elemental analyses were carried out using the Perkin-Elmer 2400 analyzer (Norwalk, Connecticut, USA) and the results were found to be within ± 0.4% of the theoretical values (Table 1). Infrared (IR) spectra were Scheme 1

recorded on a Perkin-Elmer 1600 Fourier transform infrared spectroscope against KBr discs. 1H NMR spectra were measured on a JEOL 270 MHz spectrometer (JEOL, Tokyo, Japan) in dimethyl sulfoxide-d6 and chemical shifts were recorded in d ppm relative to tetramethylsilane as an internal standard. Mass spectra (EI) were measured at 70 eV using a JEOL-JMS-AX500 mass spectrometer (JEOL). 8-Hydroxyquinoline-5-sulfonyl chloride [9], 2-cyanoacetic acid hydrazide [10], 3-amino-5-pyrazolone [11], 20 -acetyl2-cyanoacetohydrazide [12], and arylidene malononitrile [13] were prepared as reported. 1-Acetyl-5-amino-4-[(8-hydroxyquinoline-5-yl)sulfonyl]-1,2-dihydro-pyrazol-3-one (2)

A mixture of 8-hydroxyquinoline-5-sulfonyl chloride (1; 2.4 g, 0.01 mol) and 20 -acetyl-2-cyanoacetohydrazide (1.3 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3 h. After cooling, the precipitate formed was filtered off, washed with water, air dried, and recrystallized from aqueous ethanol (Scheme 1, Table 1). 5-Amino-1-[(8-hydroxyquinoline-5-yl)sulfonyl]-1,2-dihydropyrazol-3-one (3)

A mixture of 8-hydroxyquinoline-5-sulfonyl chloride (1; 2.4 g, 0.01 mol) and 2-cyanoacetic acid hydrazide (0.99 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 2 h. The hot solid that formed was filtered off, washed with water, air dried, and recrystallized from absolute ethanol (Scheme 1, Table 1). 3-[(8-Hydroxyquinoline-5-yl)sulfonamido]-1,2-dihydropyrazol5(4H) one (4)

A mixture of 8-hydroxyquinoline-5-sulfonyl chloride (1; 2.4 g, 0.01 mol) and 3-amino-5-pyrazolone (0.99 g,

Synthesis of compounds 2, 3, 4, 5a-e, and 6a-e.

Table 1 Physical and analytical properties of the newly synthesized compounds Analysis (%; calculated/found) Compd number 2 3 4 5a 5b 5c 5d 5e 6a 6b 6c 6d 6e 7 8 9 10a 10b 10c 10d 10e 11a 11b 11c 11d 11e

Formula (Mw)

MP (oC)

Yield (%)

C

H

N

C14H12N4O5S (348.33) C12H10N4O4S (306.30) C12H10N4O4S (306.30) C22H16N6O4S (460.47) C22H15ClN6O4S (494.91) C22H15N7O6S (505.46) C23H18N6O5S (490.49) C24H21N7O4S (503.53) C22H14N6O4S (458.45) C22H13ClN6O4S (492.89) C22H13 N7O6S (503.45) C23H16 N6O5S (488.48) C22H19N7O4S (501.52) C11H10N2O5S (282.27) C12H9N3O4S2 (323.35) C12H9N3O5S (307.28) C22H13N5O4S2 (475.50) C22H12ClN5O4S2(509.99) C22H12N6O6S2 (520.50) C23H15N5O5S2 (505.53) C24H18N6O4S2 (518.08) C22H13N5O5S (459.43) C22H12ClN5O5S (493.88) C22H12N6O7S (504.43) C23H15N5O6S (489.46) C24H18N6O5S (502.50)

222–4 222–4 312–4 218–220 253–5 263–5 310–2 166–8 76–8 289–291 205–7 199–200 164–6 215–7 205–7 290–2 271–3 243–5 221–3 305–7 176–8 207–9 199–201 350 dec. 145–7 279–281

60 66 85 20 18 30 22 20 22 20 18 30 34 90 70 60 18 30 22 24 20 33 20 18 25 20

48.27/48.33 47.05/47.22 47.05/47.21 57.38/57.54 53.39/53.21 52.28/52.01 56.32/56.43 57.25/57.44 57.64/57.87 53.60/53.88 52.84/52.91 56.55/56.76 57.48/57.65 46.80/46.91 44.58/44.78 46.90/47.01 55.57/55.50 51.82/52.00 50.77/50.89 54.65/54.77 55.59/55.61 57.51/57.68 53.50/53.77 52.38/52.49 56.44/56.23 57.36/57.43

3.44/3.21 3.26/3.34 3.26/3.45 3.50/3.67 3.05/2.99 2.99/3.03 3.70/3.87 4.20/4.35 3.27/3.47 2.63/2.44 2.58/2.76 3.30/3.55 3.79/3.66 3.54/3.66 2.78/3.00 2.93/3.00 2.76/2.88 2.37/2.55 2.32/2.55 2.99/3.02 3.50/3.52 2.85/3.00 2.45/2.65 2.40/2.65 3.09/2.99 3.61/3.77

16.09/15.99 18.30/18.55 18.30/18.55 18.25/18.44 16.98/17.01 19.40/19.60 17.13/17.33 19.47/19.44 18.34/18.11 17.05/17.21 19.48/19.55 17.20/17.44 19.56/19.77 9.92/10.01 13.00/13.22 13.68/13.77 17.73/17.88 13.73/13.82 16.15/16.32 13.85/14.00 16.21/16.42 15.24/15.28 14.16/14.33 16.66/16.82 14.13/14.22 16.72/16.99

dec., decomposition; MP, melting point; Mw, molecular weight.


Scheme 2

off, washed with water, air dried and recrystallized from absolute ethanol (Scheme 1, Table 1). 2-(2-(8-Hydroxyquinoline-5-yl)sulfonamido)acetic acid (7)

A suspension of 8-hydroxyquinoline-5-sulfonyl chloride (1; 0.24 g, 0.001 mol) and glycine (0.07 g, 0.001 mol) in a saturated solution of potassium carbonate (5 ml, 1.1 mol/l) was stirred and heated at 501C for 10 min and then at 1001C for 30 min. After cooling, the reaction mixture was neutralized with diluted hydrochloric acid (1 : 1). The precipitate formed was filtered off and recrystallized from dioxane (Scheme 2, Table 1). 1-[(8-Hydroxyquinoline-5-yl)sulfonyl]-2-thioxoimidazolidin-4one (8)

A suspension of 2-(2-(8-hydroxyquinoline-5-yl)sulfonamido)acetic acid (7; 3.38 g, 0.012 mol), acetic anhydride (6.3 g, 0.067 mol), anhydrous pyridine (15 ml), and ammonium thiocyanate (1.2 g, 0.015 mol) was heated at 1101C for 1 h. The volatiles were removed in vacuo and the residue was suspended in water (100 ml) and stirred for 1 h. The solid formed was filtered off, air dried, and recrystallized from benzene petroleum ether (60–801C; Scheme 2, Table 1). 1-[(8-Hydroxyquinoline-5-yl)sulfonyl]imidazolidin-2,4-dione (9)

A suspension of 1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2thioxo-imidazolidin-4-one (8) (1.77 g, 0.0055 mol), chloroacetic acid (10 g, 0.1 mol), and water (3 ml) was heated at 1201C for 12 h on a sand bath. The reaction mixture was then diluted with water (50 ml) and set aside in a refrigerator at 51C. The solid formed was filtered off, air dried, and recrystallized from benzene petroleum ether (60–801C; Scheme 2, Table 1).

Synthesis of compounds 7, 8, 9, 10a-e and 11a-e.

General procedure for the synthesis of 5-amino-7-aryl-1,2dihydro-1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2thioxopyrano[3,2-d]imidazole-6-carbonitriles (10a-e)

0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3 h. After cooling, the precipitate formed was filtered off, washed with water, air dried, and recrystallized from aqueous ethanol (Scheme 1, Table 1).

A solution of the appropriate arylidene malononitriles (0.01 mol) and 1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2thioxoimidazolidin-4-one (8; 3.23 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3–6 h. After cooling, the precipitate formed was filtered off, air dried, and recrystallized from absolute ethanol (Scheme 2, Table 1).

General procedure for the synthesis of 4-aryl-3,6-diamino-2,4dihydro-2-[(8-hydroxyquinoline-5-yl)sulfonyl]pyrano[2,3-c] pyrazole-5-carbonitriles (5a-e)

A solution of the appropriate arylidene malononitriles (0.01 mol) and compound 3 (3.06 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3–6 h. After cooling, the precipitate formed was filtered off, washed with water, air dried, and recrystallized from absolute ethanol (Scheme 1, Table 1). General procedure for the synthesis of 6-amino-4-aryl3-[(8-hydroxyquinoline-5-yl)sulfonamido]pyrano[2,3-c]pyrazole-5carbonitriles (6a-e)

A solution of the appropriate arylidene malononitriles (0.01 mol) and compound 4 (3.06 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3–6 h. After cooling, the precipitate formed was filtered

General procedure for the synthesis of 5-amino-7-aryl-1,2dihydro-1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2-oxopyrano[3,2-d]imidazole-6-carbonitriles (11a-e)

A solution of the appropriate arylidene malononitriles (0.01 mol) and 1-[(8-hydroxyquinoline-5-yl)sulfonyl]imidazolidin-2,4-dione (9; 3.07 g, 0.01 mol) in dioxane (20 ml) containing triethylamine (1 ml) was refluxed for 3–6 h. After cooling, the precipitate formed was filtered off, air dried, and recrystallized from absolute ethanol (Scheme 2, Table 1). Biological assay Antimicrobial evaluation

The antimicrobial activities of the test compounds 2, 3, 4, 5a-e, 6a-e, 8, 9, 10a-e, and 11a-e against a variety of pathogenic microorganisms such as Escherichia coli,


Pseudomonas aeruginosa (Gram-negative bacteria), Staphylococcus aureus, Bacillus cereus (Gram-positive bacteria), and one strain of fungi (Candida albicans) were determined in vitro using the disc diffusion method [14]. They were isolated from clinical samples and identified to the species level according to different API 20E systems (Analytab Products Inc., New York, USA). The antimicrobial activities of the tested compounds were estimated by placing presterilized filter paper discs (6 mm in diameter) impregnated with different doses of the tested compounds (100, 50, and 25 mg/disc) on Nutrient and MacConky agar media for bacteria and on Sabouraud dextrose agar for the fungus. Dimethyl formamide was used as a solvent for impregnation. The inhibition zones of the tested compounds were measured after 24–48 h of incubation at 371C for bacteria and after 5 days of incubation at 281C for fungi. Cefotaxime [a standardized 30 mg cefotaxime disc (BBL, Lot 104026; assayed content of 30 mg/disc) was used in the disc diffusion test; Hoechst-Roussel Pharmaceuticals Inc., Somerville, New Jersey, USA] and Pipracillin (Pipracillin, 100 mg/disc; Bristol-Myers Squibb, Giza, Egypt) were used as reference drugs for bacteria, whereas nystatin (30 U/disc; Bristol-Myers Squibb; European unit = 0.04 mg/disc) was used as the reference drug for the fungus (C. albicans). Antiviral evaluation Viruses

Live avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV) were obtained from the Strain Bank of Central Laboratory for Evaluation of Veterinary Biologics, Cairo, Egypt. Cell line

Vero (normal, African green monkey kidney) cell culture was obtained from Veterinary Vaccines and Serum Research Institute, Cairo, Egypt. Cells were cultured in sterile growth medium (RPMI-1640; Sigma-Aldrich, Germany) supplemented with 10% of heat activated new born calf serum (Sigma-Aldrich Chemie GmeH, Taufkirchen, Germany) and antibiotics (1000 IU/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml amphotericinB; Gibco, Rockville, Maryland, USA). The cells were maintained at 371C in a humidified atmosphere with 5% CO2 and were subcultured twice a week. The virus was propagated in Vero cells and the infective titer of the stock solution was 10 – 7tissue culture infected dose fifty (TCID50) per ml (50% tissue culture infective dose). Viruses were adapted on Vero cells throughout seven successive passages, by which the viruses showed a distant cytopathic effect (degeneration and floatation of the infected cells) on the third day after infection. Specific-pathogen-free egg

Specific-pathogen-free (SPF) embryonated chicken eggs were obtained from Nile SPF Farm, Koam Oshiem, Fayoum, Egypt. In-vitro cytotoxicity screening

Cytotoxicity of the tested compounds was determined using the 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetra-

zolium bromide (MTT) assay [15]. The subconfluent cell cultures were trypsinized and collected. The cells at a concentration of 3 103 cells/ml in 100 ml RPM1-1640 culture medium were incubated for 3 h at 371C in a 5% CO2 incubator. The seed cells were incubated in the 96well microplates (3 103 cells/well) at 371C in a 5% CO2 incubator for 24 h. After 24 h, when the cells became confluent, the supernatant was flicked off and added previously diluted with media of 100 ml of different concentrations of test compounds in microplates and kept for incubation at 371C in a 5% CO2 incubator for 72 h. The cells were periodically checked for granularity, shrinkage, and swelling. After 72 h, the sample solution in the wells was flicked off and 100 ml of MTT (0.5 mg/ml) was added to each well. The plates were gently shaken and incubated for 4 h at 371C in a 5% CO2 incubator. The purple crystals that developed were dissolved in 100 ml dimethyl sulfoxide and absorbance was measured using an ELISA microplate reader (Bio-Rad Laboratories, Hercules, California, USA) at a wavelength of 570 nm. In-vitro antiviral assay

Different nontoxic concentrations of test compounds, that is lower than the CTC50 (concentration required to reduce viability by 50%), were checked for antiviral property using the cytopathic effect assay against a challenge dose of 10 TCID50. Cells were seeded in 96well microtitre plates at a population of 10 000 cells/well and incubated at 371C in a 5% CO2 atmosphere for a period of 48 h. The plates were washed with fresh RPMI1640 medium and then with maintenance medium containing the virus (10 TCID50); thereafter, they were incubated at 371C for 90 min for adsorption of the virus. After this, the cultures were treated with different dilutions of the test compounds in fresh maintenance medium and incubated at 371C for 5 days. Observations were made every 24 h and cytopathic effects were recorded. Anti-APMV-1 and anti-LTV activities were determined by the inhibition of the cytopathic effect compared with control – that is the protection offered by the test samples to the cells scored [16]. In Vero cell cultures

These assays were performed in nine 24-well tissue culture plates according to the procedure described by Cox et al. [17]. Confluent monolayer’s of Vero cells were infected with 5000 TCID50/0.2 ml/well of APMV-1 or 500 TCID50 of LTV and incubated for 2 h (for virus adsorption); thereafter, inoculum was decanted, followed by addition of different 10-fold concentrations of each test sample separately (from 3 to 5 mg/ml/well of each concentration). Virus infectivity and cytotoxicity of each test compound were controlled. Test plates were incubated at 371C in a 5% CO2 incubator for 3 days. Cytotoxicity concentration fifty (CC50) of each test compound was defined as the concentration of compounds that induced any deviation in the morphology from that of the normal control cells in 50% of Vero cell monolayers. Antiviral inhibitory concentration fifty (IC50) of test compounds was defined as the concentration of compounds that fully inhibited the cytopathic effect of viruses (100 TCID) in 50% of


monolayers. In addition, the therapeutic index of samples was expressed as CC50/IC50 [18]. In embryonated chicken eggs

Groups of 9–11-day-old SPF embryonated chicken eggs were inoculated with 500 embryo infective dose fifty (EID50) per 0.2 ml per egg of APMV-1 or 50 EID50 of LTV, immediately followed by injection of different concentrations of each compound (from 2 to 500 mg/ 0.2 ml/egg) separately. The virus infectivity control and test sample toxicity control were inoculated through the chorioallantoic cavity. Test eggs were incubated for 3–4 days at 371C and 80% humidity. The CC50, IC50, and therapeutic index values were determined as mentioned before. APMV-1 infectivity in embryonated chicken eggs was detected by haemagglutinating activity of the allantoic fluids of the inoculated eggs, as measured by a microtechnique of the haemagglutination test [19], whereas LTV infectivity was determined on the basis of distension of the abdominal region, mottled necrotic or hemorrhagic liver, and mortality scores in embryos. CC50 and IC50 were calculated by the reported method [18].

Results and discussions Chemistry

The reaction routes for the synthesis of the title compounds are described in Schemes 1 and 2. Condensation of 8-hydroxyquinoline-5-sulfonyl chloride (1) with 20 acetyl-2-cyanoacetohydrazide in refluxing dioxane in the presence of triethylamine led to the formation of 1-acetyl5-amino-4-[(8-hydroxyquinoline-5-yl)sulfonyl]-1,2-dihydropyrazol-3-one (2), Scheme 1. The reaction may be preceded by reaction of the chlorine atom of 1 with the active methylene group of 20 -acetyl-2-cyanoacetohydrazide, followed by intramolecular cyclization to give 2. The structure of 2 was confirmed by its correct elemental analysis, Table 1 as well as its IR, 1H NMR, and MS spectra (Table 2). 1H NMR of 2 revealed two singlet signals at 10.45 and 8.01 ppm for the OH and NH group, respectively, multiple signals at 7.20–7.88 ppm for five aromatic protons, and two singlet signals at 4.66 and 2.99 ppm for the amino (NH2) and acetyl (COCH3) group, respectively (Table 2). In contrast, reaction of 1 with the amino group of 2cyanoacetic acid hydrazide and its cyclic form 3-amino5-pyrazolone in refluxing dioxane in the presence of triethylamine gave 5-amino-1-[(8-hydroxyquinoline-5yl)sulfonyl]-1,2-dihydropyrazol-3-one (3) and 3-[(8-hydroxyquinoline-5-yl)sulfonamido]-1,2-dihydropyrazol-5(4H) one (4) in 66 and 85% yield, respectively (Scheme 1). The characteristic features of 3 are the absence of the absorption bands for the Cl atom in the IR spectrum and the presence of absorption bands at 3209, 3163, 1686, 1385 and 1188/cm for NH2, NH, C = O, and SO2, respectively. The 1H NMR spectrum of 3 revealed signals at 10.55 (s, 1H, OH), 9.52 (s, 1H, NH), 9.11 and 8.82 (2d, 2H, H-2, and H-4 quinoline), 7.81 and 6.90 (2d, 2H, H-6 and H-7 quinoline), 7.51 (m, 1H, H-3 quinoline), 5.62 (s, 2H, NH2), and 4.44 ppm (s, 1H, CH-pyrazole; Table 2).

Cyclocondensation reaction of compounds 3 and 4 with some arylidene malononitriles such as benzylidenemalononitrile, p-chlorobenzylidenemalononitrile, p-nitrobenzylidenemalononitrile, p-methoxybenzylidenemalononitrile, and p-(N,N-dimethylamino) benzylidenemalononitrile in refluxing dioxane in the presence of triethylamine as a catalyst led to the formation of fused systems 4-aryl-3,6diamino-2,4-dihydro-2-[(8-hydroxyquinoline-5-yl)sulfonyl]pyrano [2,3-c]pyrazole-5-carbonitriles (5a-e) and 6-amino4-aryl-3-[(8-hydroxyquinoline-5-yl)sulfonamido]pyrano[2, 3-c]pyrazole-5-carbonitriles (6a-e), respectively, in 18–34% yields (Scheme 1; Table 1). Moreover, reaction of 8-hydroxyquinoline-5-sulfonyl chloride (1) with glycine in the presence of saturated potassium carbonate solution led to the formation of 2-(2-(8-hydroxyquinoline-5-yl)sulfonamido)acetic acid (7; Scheme 2). Heterocyclization of the latter compound through its reaction with ammonium thiocyanate in acetic anhydride in the presence of anhydrous pyridine gave 1-[(8hydroxyquinoline-5-yl)sulfonyl]-2-thioxoimidazolidin-4-one (8; Scheme 2). The IR spectrum of 8 showed absorption bands at 1240/cm for C = S besides those for the sulfonamido group at 1371 and 1136/cm. Its 1H NMR spectrum revealed a singlet signal at 8.76 ppm for NH and at 4.24 ppm for CH2 of the imidazole moiety besides other signals that were located at those positions (Table 2). Acid hydrolysis of compound 8 using aqueous monochloroacetic acid yielded the corresponding imidazolidin-2,4dione derivative 9 (Scheme 2). The IR spectrum of 9 showed the absence of the absorption bands of C = S and the presence of absorption bands at 1705 and 1715/cm for C = O groups (Table 2). In a manner similar to that used to obtain compounds 5a-e and 6a-e, 1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2thioxoimidazolidin-4-one (8) and 1-[(8-hydroxyquinoline5-yl)sulfonyl]- imidazolidin-2,4-one (9) were condensed with the previous arylidine malononitriles to yield the fused systems 5-amino-7-aryl-1,2-dihydro-1-[(8-hydroxyquinoline5-yl)sulfonyl]-2-thioxopyrano[3,2-d]imidazole-6-carbonitriles (10a-e) and 5-amino-7-aryl-1,2-dihydro-1-[(8-hydroxyquinoline-5-yl)sulfonyl]-2-oxo-pyrano[3,2-d]imidazole-6-carbonitriles (11a-e), respectively, in 18–33 yields (Scheme 2; Table 1). The 1H NMR spectra of compounds 10a,c,e and 11a,c,e lack the presence of the CH2 proton of imidazole and revealed new singlet signals for NH2 at 8.99, 9.15, 6.76, 8.87, 8.81 and 8.57 ppm, respectively (Table 2). Antimicrobial activity

All the newly synthesized compounds were tested for their antimicrobial activity against a variety of pathogenic microorganisms, E. coli, P. aeruginosa (Gram-negative bacteria), S. aureus, B. cereus (Gram-positive bacteria), and one strain of fungi (Candida albicans), at different doses of the tested compounds (100, 50, and 25 mg/disc) (Table 3). The results showed that compounds 3, 4, 5c, 8, and 9 were the most active of all test compounds with growth inhibition of 28, 27, 22, 22, and 20 mm, respectively, at 100 mg/disc against E. coli when compared with the reference drug cefatoxime (32 mm) at 30 mg/disc.


Table 2 Spectral characterization of the newly synthesized compounds Compound number 2 3

4 5a 5b

5c 5d 5e 6a 6b

6c 6d 6e 7 8 9 10a 10b

10c 10d 10e 11a 11b

IR (nmax,/cm) 3420 (OH), 3200 and 3106 3163 (NH and NH2), 1702 and 1656 (C = O), 1618 (C = N), 1577 (C = C) 3335 (OH), 3209 and 3163 (NH and NH2), 1686 (C = O), 1651 (C = N), 1606 (C = C), 1385 and 1188 (SO2N) 3325 (OH), 3218 and 3164 ((NH and NH2), 1687 (C = O), 1651 (C = N), 1601 (C = C), 1368 and 1163 (SO2N) 3358 (OH), 3200 and 3103 (NH and NH2), 2230 (CN), 1641 (C = N), 1597 (C = C), 1340 and 1131 (SO2N) 3421 (OH), 3200 and 3102 (NH and NH2), 2223 (CN), 1599 (C = N), 1565 (C = C), 1370 and 1126 (SO2N)

1

H NMR (d, ppm)

Mass (m/z, %)

10.45 (s, 1H, OH), 8.01 (s, 1H, NH), 7.20–7.88 (m, 5H, Ar-H), 4.66 (s, 2H, NH2), 2.99 (3H, s, COCH3) 10.55 (s, 1H, OH), 9.52 (s, 1H, NH), 8.82 and 9.11 (2d, 2H, H-2 and H-4 quinoline), 6.90 and 7.81 (2d, 2H, H-6 and H-7 quinoline), 7.51 (m, 1H, H-3 quinoline) 5.62 (s, 2H, NH2), 4.44 ppm (s, 1H, CH-pyrazole) 10.56 (s, 1H, OH), 9.52 (s, 1H, NH), 7.51–8.65 (m, 5H, Ar-H quinoline) 5.66 (s, 1H, SO2NH), 4.24 ppm (s, 2H, CH2-pyrazole) 10.56 (s, 1H, OH), 9.51 (s, 2H, NH2), 8.42 (s, 1H, H-pyrane), 7.66–8.12 (m, 10H, Ar-H), 4.24 (s, 2H, NH2)

348 (M + , 10), 291 (3), 209 (30), 57 (100)

3400 (OH), 3200 and 3103 (NH and NH2), 2230 (CN), 1641 (C = N), 1597 (C = C), 1346 and 1132 (SO2N) 3400 (OH), 3218 and 3100(NH and NH2), 2200 (CN), 1620 (C = N), 1587 (C = C), 1346 and 1132 (SO2N), 1009 (C–O–C) 3445 (OH), 3212 and 3135 (NH and NH2), 2219 (CN), 1601 (C = N), 1529 (C = C), 1369 and 1131 (SO2N) 4200 (OH), 3320 and 3200 (NH and NH2), 2215 (CN), 1620 (C = N), 1365 and 1131 (SO2N), 1009 (C–O–C) 3350 (OH), 3318 and 3191 (NH and NH2), 2219 (CN), 1618 (C = N), 1365 and 1136 (SO2N), 1019 (C–O–C), 740 (Cl)

9.91 (s, 1H, OH), 8.91 (s, 2H, NH2), 8.47 (s, 1H, H-pyrane), 7.67–8.19 (m, 9H, Ar-H), 5.66 (s, 2H, NH2)

3419 (OH), 3354 and 3255 (NH and NH2), 2223 (CN), 1664 (C = N), 1584 (C = C), 1348 and 1178 (SO2N), 1040 (C–O–C) 3228 (OH), 3198 and 3105 (NH and NH2), 2219 (CN), 1645 (C = N), 1579 (C = C), 1348 and 1137 (SO2N), 1009 (C–O–C) 3423 (OH), 3370 and 3250 (NH and NH2), 2206 (CN), 1612 (C = N), 1565 (C = C), 1358 and 1174 (SO2N), 1031 (C–O–C) 3403 (OH), 3178 (NH), 1740 (C = O), 1651 (C = N), 1620 (C = C), 1339 and 1127 (SO2N) 3353 (OH), 3138 (NH), 1721 (C = O), 1674 (C = N), 1528 (C = C), 1240 (C = S), 1371 and 1136 (SO2N) 3424 (OH), 3164 (NH), 1705 and 1715 (C = O), 1631 (C = N), 1543 (C = C), 1345 and 1154 (SO2N) 4210 (OH), 3220 and 3108 (NH2), 2219 (CN), 1620 (C = N), 1578 (C = C), 1245 (C = S), 1375 and 1168 (SO2N), 1009 (C–O–C) 3390 (OH), 3321 and 3218 (NH2), 2219 (CN), 1620 (C = N), 1558 (C = C), 1245 (C = S), 1336 and 1168 (SO2N), 1009 (C–O–C), 740 (Cl) 4350 (OH), 3310 and 3200 (NH2), 2219 (CN), 1620 (C = N), 1555 (C = C), 1245 (C = S), 1335 and 1173 (SO2N), 1009 (C–O–C) 3330 (OH), 3218 and 3108 (NH2), 2219 (CN), 1620 (C = N), 1568 (C = C), 1245 (C = S), 1375 and 1167 (SO2N), 1009 (C–O–C) 3350 (OH), 3300 and 3208 (NH2), 2219 (CN), 1620 (C = N), 1567 (C = C), 1245 (C = S), 1375 and 1167 (SO2N), 1009 (C–O–C) 3355 (OH), 3320 and 3218 (NH2), 2219 (CN), 1678 (C = O), 1620 (C = N), 1567 (C = C), 1375 and 1156 (SO2N), 1009 (C–O–C) 4320 (OH), 3352 and 3210 (NH2), 2219 (CN), 1645 (C = O), 1620 (C = N), 1567 (C = C), 1375 and 1167 (SO2N), 1009 (C–O–C), 740 (Cl)

11.24 (s, 1H, OH), 9.91 (s, 1H, NH), 8.87 (s, 2H, NH2), 7.11–8.37 (m, 9H, Ar-H)

11.44 (s, 1H, OH), 8.01 (s, 1H, H-pyrane), 7.88 (s, 2H, NH2), 6.67–7.61 (m, 9H, Ar-quinoline), 4.14 (s, 2H, NH2), 2.99 (s, 6H, 2CH3) 10.56 (s, 1H, OH), 8.91 (s, 1H, NH), 8.56 (s, 2H, NH2), 7.44–8.32 (m, 5H, Ar-H quinoline), 7.03–7.37 (m, 5H, Ar-H)

306 (M + , 46), 205 (100), 99(50), 89 (45), 77 (30)

494 (M + , 10), 496 (M + + 2, 3), 383 (20), 205 (30), 111 (70), 89 (100), 77 (30)

490 (M + , 1), 33 (40), 355 (100), 205 (50), 87 (46)

492 (M + , 12), 494 (M + + 2, 2), 396 (100), 330 (30), 206 (45), 111 (40), 87 (50)

488 (M + , 40), 412 (30), 385 (5), 340 (100), 205 (16), 77 (50) 10.88 (s, 1H, OH), 9.01 (s, 1H, NH), 8.56 (s, 2H, NH2), 7.01–8.34 (m, 9H, Ar-H), 3.11 and 3.34 (2s, 6H, CH3) 10.51 and 9.90 (2s, 2H, 2OH), 7.30–7.67 (m, 5H, Ar-H quinoline), 6.66 (s, 1H, NH), 4.24 (s, 2H, CH2) 10.51 (s, 1H, OH), 8.76 (s, 1H, NH), 7.31–7.87 (m, 5H, Ar-H quinoline), 4.24 (s, 2H, CH2) 10.51 (s, 1H, OH), 9.99 (s, 1H, NH), 7.10–7.67 (m, 5H, Ar-H quinoline), 4.44 (s, 2H, CH2) 10.52 (s, 1H, OH), 8.99 (s, 2H, NH2), 7.10–7.37 (m, 5H, Ar-H), 7.31–8.37 (m, 5H, Ar-H quinoline)

509 (M + , 30), 511 (M + + 2, 10), 482 (24), 399 (30), 205 (43), 77 (70), 65 (100)

10.37 (s, 1H, OH), 9.15 (s, 2H, NH2), 7.10–8.37 (m, 9H, Ar-H) 505 (M + , 10), 451 (20), 383 (40), 329 (20), 205 (50), 87 (100), 77 (70) 10.51 (s, 1H, OH), 6.76 (s, 2H, NH2), 7.10–8.37 (m, 9H, Ar-H), 2.99 and 3.04 (2s, 6H, 2CH3) 10.57 (s, 1H, OH), 8.87 (s, 2H, NH2), 7.01–7.23 (m, 5H, Ar-H), 7.30–8.37 (m, 5H, Ar-H quinoline)

493 (M + , 10), 495 (1), 383 (100), 358 (30), 330 (45), 205 (10), 111 (12)


Table 2 (continued) Compound number 11c 11d 11e

1

IR (nmax,/cm) 3340 (OH), 3222 and 3108 (NH2), 2219 (CN), 1670 (C = O), 1620 (C = N), 1567 (C = C), 1375 and 1167 (SO2N), 1009 (C–O–C) 4340 (OH), 3320 and 3208 (NH2), 2219 (CN), 1678 (C = O), 1620 (C = N), 1567 (C = C), 1375 and 1167 (SO2N), 1009 (C–O–C) 3380 (OH), 3320 and 3211 (NH2), 2219 (CN), 1670 (C = O), 1620 (C = N), 1567 (C = C), 1375 and 1167 (SO2N), 1009 (C–O–C)

H NMR (d, ppm)

Mass (m/z, %)

9.99 (s, 1H, OH), 8.81 (s, 2H, NH2), 7.01–8.47 (m, 9H, Ar-H) 489 (M + , 20), 382 (10), 330 (34), 206 (20), 77 (70), 65 (100) 10.51 (s, 1H, OH), 8.57 (s, 2H, NH2), 7.01–8.27 (m, 9H, Ar-H), 2.66 and 3.04 (2s, 6H, 2CH3)

IR, infrared; NMR, nuclear magnetic resonance.

Table 3 Antimicrobial activity of the newly synthesized compounds Inhibition zone (mm) E. coli

P. aeruginosa

S. aureus

B. cereus

C albicans

Compounds (mg/disc) Compound number

100

50

25

100

50

25

100

50

25

100

50

25

100

50

25

2 3 4 5a 5b 5c 5d 5e 6a 6b 6c 6d 6e 8 9 10a 10b 10c 10d 10e 11a 11b 11c 11d 11e Cefatoxime (30 mg/disc) Piperacillin (100 mg/disc) Nystatin (30 U/disc)

17 28 27 19 19 22 17 17 17 17 17 17 17 22 20 18 17 17 17 17 12 12 12 12 12 32 – –

14 18 18 14 12 16 12 10 9 8 8 8 8 14 14 12 10 10 10 10 9 9 9 9 9 22 – –

– 12 13 – – – – – – – – – – 9 9 9 8 8 – – – – – – – 17 – –

18 19 20 16 16 16 14 14 12 12 12 12 12 18 18 16 16 16 14 14 10 10 10 10 10 22 20 –

14 14 14 10 10 12 9 8 8 8 8 8 8 12 12 10 10 10 – – – – – – – 18 15 –

– 10 10 – – – – – – – – – – 9 9 – – – – – – – – – – 12 10 –

14 17 17 12 12 12 12 10 10 12 12 12 12 14 14 14 14 14 12 12 12 12 12 12 12 31 27 –

9 10 12 – – – – – – – – – – 8 9 8 8 8 8 8 8 8 8 8 8 26 18 –

– – – – – – – – – – – – – – – – – – – – – – – – – 17 10 –

12 14 12 12 10 10 10 10 9 10 12 12 12 14 14 14 14 14 14 14 12 12 12 12 12 26 20 –

– 9 – – – – – – – – – – – 8 8 8 8 8 8 8 – – – – – 20 15 –

– – – – – – – – – – – – – – – – – – – – – – – – – 14 10 –

12 12 12 10 10 10 10 10 10 10 10 10 10 12 12 9 9 9 9 9 9 9 9 9 9 – – 40

– – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – –

In addition, they showed growth inhibition of 18, 18, 16, 14, and 14 mm, respectively, at 50 mg/disc against E. coli when compared with the reference drug cefatoxime (22 mm) at 30 mg/disc. In contrast, compounds 3, 4, 8, and 9 were found to be the most active of all the test compounds with growth inhibition of 19, 20, 18, and 18 mm, respectively, at 100 mg/disc against P. aeruginosa when compared with the reference drugs cefatoxime (22 mm) at 30 mg/disc and piperacillin (20 mm) at 100 mg/ disc. The rest of the tested compounds were inactive against all microorganisms tested.

effect inhibitory assay. The results revealed that compounds 2 and 3 as well as 4 were completely inhibited by 5000 TCID50 of APMV-1 and 500 TCID50 of LTV infectivity at concentrations of 3, 4, 3 mg/ml, respectively (Table 4). Substantial therapeutic indices of 66, 75, and 66 were recorded. A cytotoxicity assay indicated that CC50 of 2, 3, and 4 were greater than 200, 300, and 200 mg/ml, respectively (Table 4). These results proved that the three compounds possessed antiviral activity in Vero cells with the absence of apparent cytotoxicity. In chicken embryos

Antiviral activity In Vero cell cultures

Six selected compounds were tested for their antiviral activity against avian paramyxovirus type1 (APMV-1) and laryngotracheitis virus (LTV) using the virus cytotoxicity

Studies on the activity of the six selected compounds (2, 3, 4, 7, 8, and 9), as determined by haemagglutinating activity in allantoic fluids and LTV infectivity criterion in embryos, showed that 4, 3, and 4 mg/0.2 ml/egg of compounds 3, 4, and 2 were fully reduced by the


Table 4 Cytotoxic effect of test compounds on normal Vero cell lines IC50

CC50 Compound number APMV-1 2 3 4 7 8 9

4500 4500 4400 4300 4200 4200

LTV 4500 4500 4400 4300 4200 4200

TI

APMV-1 LTV APMV-1 LTV r5 r4 r3 r4 r3 r3

P. aeruginosa (Gram-negative bacteria) and exhibit marked viral inhibitory activity against APMV-1 and LTV.

r5 r4 r4 r4 r3 r3

100 125 100 75 66 66

100 100 100 75 66 66

Acknowledgements The authors thank Ibrahim Hassan Mohamed Tolba, Department of Agriculture Botany, Faculty of Agriculture, Al-Azher University, Cairo, Egypt, for carrying out the antimicrobial activity screening. The authors are also grateful to the Micro analytical Center, Cairo University, Egypt, for providing permission to carry out elemental analyses and IR, 1H NMR, and mass spectrometry.

Avian paramyxovirus type 1 (APMV-1) = 5000 TCID50; laryngotracheitis virus (LTV) = 500 TCID50; CC50 (mg/ml): toxic concentration fifty; IC50 (mg/ml): inhibiting concentration fifty; TCID50,tissue culture infected dose fifty; TI: therapeutic index.

Conflicts of interest

Table 5 Cytotoxic effect of test compounds in embryonated chicken specific-pathogen-free eggs

References

IC50

CC50 Compound number APMV-1 2 3 4 7 8 9

4400 4400 4400 4300 4200 4200

LTV 4400 4400 4400 4300 4200 4200

TI

APMV-1 LTV APMV-1 LTV r5 r4 r4 r4 r3 r4

r4 r4 r4 r3 r4 r4

80 100 100 75 66 50

100 100 100 66 50 50

Avian paramyxovirus type 1 (APMV-1) = 500 EID50; laryngotracheitis virus (LTV) = 50 EID50; CC50 (mg/ml), toxic concentration fifty; EID50, embryo infective dose fifty; IC50 (mg/ml), inhibiting concentration fifty; TI, therapeutic index.

infectivities of 500 EID50 of APMV-1 and 50 EID50 of LTV, respectively (Table 5). The toxicity assays of compounds 3, 4, and 2 in chicken embryos at concentrations of 300, 200, and 200 mg/egg showed 100% survival of the inoculated eggs on the fifth day after inoculation. Thus, the recorded therapeutic indices of the three compounds were 75, 66, and 50, respectively, in the case of APMV-1 and 66, 50, and 50, respectively, in the case of LTV. In conclusion, chicken embryo experiments showed that compounds 3, 4, and 2 had high antiviral activities in vitro, with IC50 ranging from 3 to 4 mg/egg against avian APMV-1 and LTV and toxic CC50 ranging from 200 to 300 mg/egg. The results showed that a concentration range of 3–4 mg/ml of compounds 2, 3, and 4 showed marked viral inhibitory activity for APMV-1 of 5000 TCID50 and LTV of 500 TCID50 in Vero cell cultures on the basis of their cytopathic effect. Chicken embryo experiments show that compounds 2, 3, and 4 had high antiviral activity in vitro, with IC50 ranging from 3 to 4 mg/egg against avian APMV-1 and LTV and toxic CC50 ranging from 200 to 300 mg/egg.

Conclusion A series of 5-substituted sulfonyl-8-hydroxyquinoline derivatives have been prepared. 8-Hydroxyquinolines that were incorporated into rings of pyrazole 2, 3, and 4 and imidazole 8 and 9 through a sulfonyl bridge at position 5 showed inhibition growth towards E. coli and

There are no conflicts of interest.

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124 Original article

In-vitro bioassays on the metabolites of the fungus Emericella nidulans isolated from the Egyptian Red Sea algae Usama W. Hawasa, Lamia T.A. El-Kassemb, Eman F. Ahmedc and Mahmoud Emama Departments of aPhytochemistry and Plant Systematic, b Pharmacognosy and cNatural and Microbial Products Chemistry, National Research Centre, Dokki, Cairo, Egypt Correspondence to Usama W. Hawas, Phytochemistry and Plant Systematic Department, National Research Centre, Dokki, 12311 Cairo, Egypt Tel: + 20 233 54974; fax: + 20 233 70931; e-mail: [email protected] Received 3 June 2012 Accepted 12 September 2012 Egyptian Pharmaceutical Journal 2012,11:124–128

Aim There are a number of theories on which organisms provide the most interesting bioactive metabolites. In this study, we discuss the biochemical activities of the marinederived endophyte Emericella nidulans, isolated from the Egyptian Red Sea algae. Methods The fungus E. nidulans was isolated as an endophyte from the Egyptian Red Sea brown alga Turbinaria elatensis. The fungus was identified by a morphological method and 18S rDNA sequence comparison. Chemical constituents were isolated using chromatographic techniques. Results and conclusion Cultivation of this fungus in Czapek’s peptone media led to the isolation of five known metabolites: sterigmatocystin (1), emericellin (2), cordycepin (3), ergosterol peroxide (4), and myristic acid (5) from the ethyl acetate extract of the culture broth. The structures were elucidated on the basis of NMR spectroscopic analysis and mass spectrometry. The ethyl acetate extract and the isolated compounds were tested for antimicrobial properties, activity against cancer cell lines, and inhibition of the hepatitis C virus protease. Keywords: anti-hepatitis C virus protease, brown algae, Emericella nidulans, Turbinaria elatensis Egypt Pharm J 11:124–128 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction


Marine-derived microbes, fungi in particular, have long been recognized as a potential source of structurally novel and biologically potent metabolites [1–3]. The fungal genus Emericella is one of the sexual states of Aspergillus [4]. Several species of this genus are saprobes, whereas others are either pathogenic or endophytic on living plants [5,6]. Emericella spp. have been reported as a source of a remarkable diversity of secondary metabolites with interesting biological properties, including antitumor indole alkaloids and quinones [7,8], neuritogenic and antimicrobial polyketides [9], cytotoxic sesterterpenes [10], aflatoxins, and sterigmatocystin [11], as well as xanthones and cyclic depsipeptides with antimicrobial, immunostimulatory, and calmodulin inhibition activities [12–15].

General experimental

Within the scope of our program aiming at the isolation of bioactive natural products from marine endophytic fungi, we have isolated and identified Emericella nidulance from the inner tissue of the brown algae Turbinaria elatensis collected from the Egyptian Red Sea. The culture broth extract of the fungus was subjected to detailed chemical analysis as well as in-vitro bioassays for estimation of antimicrobial, anticancer, and antiviral properties.

Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and silica gel (60–120 mesh; Qualigens, Mumbai, India) were used for column chromatography. Czapek’s agar and potato dextrose broth were procured from Lab M (Lancashire, UK). Flash chromatography was carried out on silica gel (230–400 mesh). Thin-layer chromatography (TLC) was performed on Polygram SIL G/UV254 (Macherey-Nagel & Co., Du ¨ren, Germany). A mixture of methanol and methylene chloride (3 : 2 and 1 : 1, v/v) was used as a mobile phase for TLC analysis. Compounds were visualized as intense dark, blue, and yellow colored spots on TLC under ultraviolet (UV) light. Most of the colored spots changed after spraying with anisaldehyde/sulfuric acid followed by heating at 1201C. UV/vis spectra were recorded on a Shimadzu model UV-240 spectrometer (Shimadzu, Tokyo, Japan). NMR spectra were measured on a Varian Inova 500 spectrometer (International Equipment Trading Ltd, Vernon Hills, Illinois, USA) (1H, 500 MHz; 13C, 125.7 MHz). Electrospray ionization mass spectra were recorded on a Finnigan LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, California, USA). High-resolution mass spectra were recorded by


DOI: 10.7123/01.EPJ.0000421517.80892.ed

Bioassays of Emericella nidulans metabolites Hawas et al. 125

electrospray ionization mass spectrometry on an Apex IV 7 T Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Bremen, Germany). Enzymes and chemicals

Sensolyte 520 HCV protease assay kit Fluorimetric (Lot #AK71145-1020), HCV NS3/4A protease, and Hepatitis Virus C NS3 protease inhibitor 2 (cat #25346) were purchased from AnaSpec Inc. (San Jose, California, USA). Becton Dickinson Falcon Microtest 384-well 120 ml black assay plates, nonsterile, no lid, were purchased from Becton Dickinson Inc. (Tokyo, Japan). Fungal isolation and culture conditions

The brown algae T. elatensis was collected from the Egyptian Red Sea site at a depth of 3–6 m from the coast of Rass Mohamed (South Sina, Egypt) in March 2010. The sample was selected solely on the basis of a clean and healthy exterior and brought to the laboratory in ice. In the laboratory, the specimens were washed with sterile water and processed immediately. The sample was identified by the Coral Reef Ecology and Biology group, National Institute of Oceanography and Fisheries, Suez, Egypt. The fungus E. nidulans was isolated as an endophyte using Czapek’s agar containing (g/l) glucose (30), peptone (10), yeast (2), NaNO3 (3), KH2PO4 (0.5), KCl (0.5), KH2PO4 (0.5), and agar (30) at 281C. The pH of 50% seawater supplemented with penicillin benzyl sodium salt (0.02) was adjusted to 7.5 to avoid any bacterial growth. After 6–7 days, sand brown, velvety colonies were observed. The strain was identified as E. nidulans from the morphological features of its conidiophores and a voucher specimen of the fungus was deposited at the Microbiology Department, Assiut University, Egypt. Stock cultures of the fungus were used to inoculate 500 ml of liquid medium in an Erlenmeyer flask (NovaTech International, Houston, Texas, USA) (3 l) containing biomalt broth in 50% seawater. It was then cultured at 35 ± 21C on a rotary shaker at 200 rpm. The flask was incubated for 72 h and used as first stage inoculum. The same medium (10 l) was made in 75 Erlenmayer flasks (1 l) and inoculated with 5% of first stage inoculum. The flasks were incubated statically for 15 days at 351C.

the GenJET sequencing kit (Signa Gen Laboratories, Gaithersburg, Maryland, USA). The DNA fragment of the ITS regions was amplified by PCR with the pair of primers ITS1 (50 -TGCCAGCMGCCGCGGTA-30 ) and ITS4 (50 -GACGGGCGGTGTGTRCAA-30 ), and the PCR products were assayed using the method of Kumeda and Asao [17]. DNA sequencing was carried out at Sequencer Scientific City (Borg El-Arab, Egypt). Extraction and isolation of metabolites

Fifteen-day-old fermentation broth (10 l) was separated from the fungal mat. The liquid medium and fungal mycelia were extracted with ethyl acetate. The resultant extract was dried using a Rotavapour (Heidolph, Schwabach, Germany) with a heating water bath (r401C), after which defatting was carried out using n-hexane solvent. The crude extract was applied to a silica gel column using n-hexane as the starting nonpolar eluent and by gradually increasing the polarity using ethyl acetate as a polar solvent in the eluent mixture (5, 10%, until 100% ethyl acetate), followed by 20 and 50% methanol/ethyl acetate regarding to the TLC of the crude extract as reference during the fractionation. The combined semifractions are further purified on a Sephadex LH-20 column with MeOH, MeOH/CH2Cl2 (1 : 1 and 2 : 3), and MeOH/CH2Cl2/n-hexane (2 : 2 : 1) to yield pure compounds (1–5). Sterigmatocystin (1)

Pale yellow crystals (26 mg); 1H NMR (CDCl3, 600 MHz): d 13.24 (1H, s, 7-OH), 7.51 (1H, t, J = 8.4 Hz, H-9), 6.83 (1H, d, J = 7.2 Hz, H-4), 6.82 (1H, d, J = 8.4 Hz, H-8), 6.76 (1H d, J = 8.4 Hz, H-10), 6.51 (1H, t, J = 2.5 Hz, H1), 6.44 (1H, s, H-5), 5.45 (1H, t, J = 2.5 Hz, H-2), 4.81 (1H, dd, J = 7.2 and 2.4 Hz, H-3), 3.98 (3H, s, 6-OCH3); (CDCl3, 150 MHz): d 181.4 (C-11), 164.6 (C-12a), 163.2 (C-10a), 162.5 (C-6), 154.9 (C-7), 154.0 (C-5a), 145.4 (C-1), 135.7 (C-9), 113.3 (C-4), 111.2 (C-10), 108.9 (C-7a), 106.5 (C-12b), 105.9 (C-11a), 105.8 (C-8), 102.5 (C-2), 90.2 (C-5), 56.8 (6-OCH3), 48.2 (C-3); (+) ESIMS: m/z 325 [M + H] + , 347 [M + Na] + , 671 [2M + Na] + , and 995 [3M + Na] + . Emericellin (2)

Identification of the endophytic isolate

The endophytic fungus was isolated from brown algae (T. elatensis), grown on Czapek’s peptone agar at 281C for 7 days, and morphologically characterized as E. nidulans. The mycelium was scraped directly from the surface of the agar culture (6 days old) and weighed. Nucleic acid was extracted and purified using the GenElute DNA isolation kit for genomic DNA (Sigma-Aldrich, St Louis, Missouri, USA) by the Chomczynski method [16]. For identification and differentiation, the internal transcript spacer regions (ITS1 and ITS4) and the intervening 5.8S rRNA regions were amplified and sequenced by electrophoretic sequencing on a 3130 genetic analyzer (Fermentas Company, Glen Burnie, Maryland, USA; Taq polymerase, deoxynucleotide triphosphates) using

White crystals (25 mg); 1H NMR (CDCl3, 500 MHz): d 12.56 (1H, s, 1-OH), 7.46 (1H, d, J = 8.5 Hz, H-3), 7.34 (1H, s, H-5), 6.78 (1H, d, J = 8.5 Hz, H-2), 5.64 (1H, m, H-20 ), 5.31 (1H, m, H-20 ), 5.11 (2H, s, 10 -CH2), 4.46 (2H, d, J = 7.0 Hz, 11-CH2), 3.51 (2H, d, J = 7.0 Hz, 10 CH2), 2.46 (3H, s, 6-CH3), 1.82 (3H, s, 40 -CH3), 1.80 (3H, s, 50 -CH3), 1.76 (3H, s, 40 -CH3), 1.73 (3H, s, 50 CH3); 13C NMR (CDCl3, 500 MHz): d 184.6 (C-9), 160.1 (C-1), 153.9 (C-7), 152.9 (C-4a), 152.6 (C-10a), 142.6 (C-6), 138.6 (C-8), 136.9 (C-3), 134.3 (C-30 ), 133.3 (C-30 ), 121.7 (C-20 ), 119.8 (C-20 ), 119.6 (C-5), 118.9 (C4), 118.1 (C-8a), 110 (C-2), 109.1 (C-9a), 72.3 (C-10 ), 57.2 (C-11), 27.4 (C-10 ), 25.8 (C-40 ), 25.7 (C-40 ), 18.2 (C-50 ), 17.9 (C-50 ), 17.6 (6-CH3); ESI-MS: m/z 409 [M + H] + and 839 [2M + Na] + .


Table 1 Anticancer results of the secondary metabolites of Emericella nidulans Solid tumors Normal cells Samples Ethyl acetate extract Sterigmatocystin (1) Cordycepin (3) Ergosterol peroxide (4)

Leukemia

Colon cancer

CFU-GM

L1210

CCRF-CEM

250 250 150 –

– – 100 0

200 100 100 100

HCT-116 0 – 0 100

Colon 38

Lung cancer H-125

Liver cancer HEP-G2

250 50 200 –

100 – 100 100

550 300 400 50

–, samples not tested against examined cell lines.

Table 2 Anti-hepatitis C virus NS3/4A protease activity for Emericella nidulans metabolites Sample

(C-25), 12.9 (C-18); (+) ESI-MS: m/z 451 [M + Na] + , 879 [2M + Na] + and 1308 [3M + Na + H] + .

HCV protease inhibitory activity IC50 (mg/ml)

Ethyl acetate extract Sterigmatocystin (1) Emericellin (2) Cordycepin (3) Ergosterol peroxide (4) Myristic acid (5). HCV-I2

30.0 ± 2.2 48.5 ± 4.2 50.0 ± 3.8 24.5 ± 2.3 47.0 ± 3.4 51.0 ± 2.6 1.5 ± 0.5

Results are represented as means ± SD (n = 3). HCV-I2: hepatitis virus C NS3/4A; protease inhibitor 2 (positive control for HCV protease).

Myristic acid; tetradecanoic acid (5)

White powder (7 mg); 1H NMR (CDCl3, 500 MHz): d 2.42 (2H, m, 2-CH2), 1.51 (2H, m, 13-CH2), 1.28 (20H, br, 3-CH2 to 12- CH2), 0.85 (3H, t, J = 2.5 Hz, 14-CH3); 13 C NMR (CDCl3, 125.4 MHz): d 179.9 (1-COOH), 34.1 (C-2), 31.9 (C-12), 29.7 (C-6/8), 29.6 (C-9), 29.4 (C-5/ 7), 29.2 (C-10), 29.1 (C-4), 24.7 (C-3), 22.6 (C-3), 14.1 (14-CH3); (+) ESI-MS: m/z 227 [M – H] – , 454 [M – H] – and 479 [2M + Na] + . Assay for hepatitis C virus protease inhibitory activity

Cordycepin; 9-cordyceposidoadenosine; 30 -deoxyadenosine; adenine cordyceposide (3)

White powder (23 mg); 1H NMR (DMSO-d6, 500 MHz): d 8.31 (1H, s, H-8), 8.12 (1H, s, H-2), 7.25 (2H, s, 6-NH2), 5.81 (1H, d, J = 2.5 Hz, H-10 ), 5.64 (1H, d, J = 4.0 Hz, 20 -OH), 4.51 (1H, m, H-20 ), 4.29 (1H, m, H-40 ), 3.63/3.45 (2H, m, 50 -CH2), 2.20/1.87 (2H, m, 30 -CH2); 13C NMR (DMSO-d6, 125.4 MHz): d 156.0 (C-4), 152.3 (C-2), 148.8 (C-6), 138.9 (C-8), 118.9 (C-5), 90.7 (C-10 ), 80.6(C-40 ), 74.6 (C-20 ), 62.5 (C-50 ), 34.0 (C-30 ); (+) ESI-MS: m/z 252 [M + H] + , 274 [M + Na] + and 525 [2M + Na] + ; (+) HRESI-MS m/z 252.10912 [M + H] + (calc for C10H14N5O3).

Ergosterol peroxide; 5,8-epidioxy-5a,8a-ergosta-6,22Edien-3b-ol (4)

In total, 2 ml of a compound solution (DMSO as solvent) were dispensed in each well of a 384-well microplate; thereafter, 8 ml of recombinant HCV protease (0.5 mg/ml) was added to the well containing the sample and the plate was briefly agitated. Finally, 10 ml of the freshly prepared substrate [Ac-Asp-Glu-Dap (QXLTM520)-GluGlu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2; 100 dilution of a DMSO stock solution] was added with sequential rotational shaking. The reaction mixture was incubated for 30 min at 371C. Fluorometric analyses were carried out on an automated Tecan GENios plate reader (Tecan Group Ltd, Ma¨nnedorf, Switzerland) with an excitation wavelength of 485 nm and emission wavelength 530 nm. Each test compound was analyzed in triplicates. The HCV protease percentage inhibition was calculated using the following equation: % Inhibition¼ðFsubstrate Ftest compound Þ100/Fsubstrate ;

1

Colorless needles (17 mg); H NMR (CDCl3, 500 MHz): d 6.54 (1H, d, J = 8 Hz, H-6), 6.27 (1H, d, J = 8, H-7), 5.26 (1H, dd, J = 15 and 6.7 Hz, H-22), 5.18 (1H, dd, J = 15 and 6.7 Hz, H-23), 3.98 (1H, m, H-3), 2.19 (1H, dd, J = 6.7 and 4.5 Hz, H-20), 1.92 (1H, m, H-24), 1.82–1.21 (17H, m, remaining methines and methylenes), 1.12 (3H, d, J = 6.5 Hz, 21-CH3), 0.87 (3H, d, J = 6.6 Hz, 25-CH3), 0.84 (3H, s, 19-CH3), 0.84 (3H, d, J = 6.6 Hz, 27-CH3), 0.82 (3H, d, J = 6.6 Hz, 28-CH3), 0.82 (3H, s, 18-CH3); 13C NMR (CDCl3, 500 MHz): d 135.9 (C-6), 135.7 (C-22), 132.8 (C-23), 131.4 (C-7), 82.3 (C-8), 79.7 (C-5), 66.7 (C-3), 52.1 (C-14), 51.6 (C-9), 44.7 (C-13), 42.8 (C-24), 39.7 (C-20), 39.4 (C-12), 37.0 (C-4/10), 34.7 (C-1), 33.1 (C-26), 30.1 (C-2), 28.6 (C-16), 23.4 (C-15), 20.9 (C-21), 20.6 (C-11), 19.9 (C-27), 19.6 (C-28), 18.1 (C-19), 17.5

where Fsubstrate is the fluorescence value of the substrate and enzyme without the test compound, Ftest compound is the fluorescence value of the test compound dissolved in DMSO.

Results and discussion The culture of E. nidulans grown in a common liquid medium, namely Czapek’s peptone broth, was extracted with ethyl acetate, and the resultant extract was subjected to chemical screening based on TLC analyses under staining with anisaldehyde/sulfuric acid reagent. Five known compounds were separated from the organic extract by Sephadex LH-20 and silica gel column chromatography and identified as sterigmatocystin (1), emericellin (2),

Bioassays of Emericella nidulans metabolites Hawas et al. 127

OH

O

1

OH

O

NH2

11

6

3

5a

10a

4a

O

4

7a

8

3'' 2 5''

10a

O

1'

11

N

4

N

5

11a 7

1''

5

12

10

7

1 7

O

12a

N

5

N

9

1

12b

O

O

8a

9a

4

4''

HO

6

1'

4'

3'

OH

O

O 5'

OH

4'

1

2 25

3

27

24 26 21

28 20

18

17

11

19

1

14 10

8

3

HO

O

O

5

O

O

4

cordycepin (3), ergosterol peroxide (4), and myristic acid (5). The structures were elucidated by mass spectrometry and one-dimensional (1D) and 2D NMR spectroscopy. In-vitro bioassays Antimicrobial

Antimicrobial activity of the ethyl acetate extract and isolated compounds was evaluated against different bacteria and fungi. The extract showed only moderate activity against Bacillus megaterium (Gram-positive bacteria), whereas the isolated compounds were inactive against all tested pathogens.

5

A zone of 0 implied that there was no inhibition. A zone of less than 250 implied that there was minimal activity. From the bioassay data (Table 1), it can be seen that the ethyl acetate extract of E. nidulance exhibited selective anticancer activity against the solid tumor of liver cancer cells (HEP-G2) compared with leukemia (CCRF-CEM) and normal cells (CFU-GM) at a concentration of 30 mg/disc. Cordycepin (3) showed potent activity against the same cells with an inhibition zone difference of 250 between liver cancer cells (HEP-G2) and normal cells (CFU-GM) when 3 mg of the pure compound was applied to the filter disc, whereas sterigmatocystin (1) and ergosterol peroxide (4) showed mild to weak activity in comparison with the activity of the extract.

Anticancer activity

The extract of the culture broth of the fungus E. nidulans and the isolated compounds (1), (3), and (4) were screened for their in-vitro anticancer activity against two leukemias (murine L1210 and human CCRF-CEM), four solid tumors (murine colon 38, human colon HCT-116, human lung H-125, human liver HEP-G2), as well as human normal cells (CFU-GM) using the disc diffusion assay [18]. The samples were initially prepared in DMSO and then applied to the filter disc. After 7 days of incubation with the examined cells, the cells that had survived had grown into colonies, and the zones of inhibition of colony formation were assessed.

Hepatitis C virus NS3/4A protease inhibitory action

The ethyl acetate extract along with compounds (1–5) isolated from E. nidulans were tested for their inhibitory activity against HCV protease using HCV NS3 protease inhibitor 2 as a positive control [19,20]. The ethyl acetate extract showed potent activity against HCV NS3/4A protease with an inhibitory concentration fifty (IC50) of 30.0 mg/ml (Table 2). Cordycepin (3) exhibited potent inhibition with an IC50 of 24.5 mg/ml, whereas compounds (1), (2), (4), and (5) showed a mild inhibitory effect with IC50 values of 48.5, 50.0, 47.0, 51.0 mg/ml, respectively.


However, it is noteworthy that this is the first report on the anti-HCV protease activity of adenosine, which warrants further investigation of other members of this widely distributed class of compounds.

Conclusion The present study reveals that the ethyl acetate extract of the culture broth of endophytic E. nidulans possesses promising anti-HCV protease activity and selective anticancer activity against liver cancer cell lines, which may be attributed to the presence of adenosine class compounds.

7 Kralj A, Kehraus S, Krick A, Eguereva E, Kelter G, Maurer M, et al. Arugosins G and H: prenylated polyketides from the marine-derived fungus Emericella nidulans var. acristata. J Nat Prod 2006; 69:995–1000. 8 Wang W, Zhu T, Tao H, Lu Z, Fang Y, Gu Q, Zhu W. Two new cytotoxic quinone type compounds from the halotolerant fungus Aspergillus variecolor. J Antibiot 2007; 60:603–607. 9 Nozawa K, Udagawa S-I, Nakajima S, Kawai K-I. Studies on fungal products. XIV. Emestrin B, a new epitrithiodioxopiperazine, from Emericella striata. Chem Pharm Bull 1987; 35:3460–3463. 10 Wei H, Itoh T, Kinoshita M, Nakai Y, Kurotaki M, Kobayashi M. Cytotoxic sesterterpenes, 6-epi-ophiobolin G and 6-epi-ophiobolin N, from marine derived fungus Emericella variecolor GF10. Tetrahedron 2004; 60: 6015–6019. 11 Frisvad JC, Samson RA. Emericella venezuelensis, a new species with stellate ascospores producing sterigmatocystin and aflatoxin B1. Syst Appl Microbiol 2004; 27:672–680. 12 Figueroa M, Gonza´lez M.d.C., Rodrı´guez-Sotres R, Sosa-Peinado A, Gonza´lez-Andrade M, Cerda-Garcıá-Rojas CM, Mata R. Calmodulin inhibitors from the fungus Emericella sp. Bioorg Med Chem 2009; 17:2167–2174.

Acknowledgements The authors thank Professor Frederick Valeriote, USA, for the cell line test. This work was supported by a Basic and Applied Research Grant from the Egyptian Science and Technological Development Fund (STDF, Grant No. 990).


13 Fujimoto H, Asai T, Kim Y-P, Ishibashi M. Nine constituents including six xanthone-related compounds isolated from two ascomycetes, Gelasinospora santi-florii and Emericella quadrilineata, found in a screening study focused on immunomodulatory activity. Chem Pharm Bull 2006; 54:550–553. 14 Pornpakakul S, Liangsakul J, Ngamrojanavanich N, Roengsumran S, Sihanonth P, Piapukiew J, et al. Cytotoxic activity of four xanthones from Emericella variecolor, an endophytic fungus isolated from Croton oblongifolius. Arch Pharm Res 2006; 29:140–144. 15 Blunt JW, Copp BR, Munro MHG, Northcote PT, Prinsep MR. Marine natural products. Nat Prod Rep 2011; 28:196–268. 16 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156–159.

References 1 Faulkner DJ. Marine natural products. Nat Prod Rep 2000; 17:7–55. 2 Bugni TS, Ireland CM. Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat Prod Rep 2004; 21:143–163. 3 Saleem M, Ali MS, Hussain S, Jabbar A, Ashraf M, Lee YS. Marine natural products of fungal origin. Nat Prod Rep 2007; 24:1142–1152. 4 Geiser DM. Sexual structures in Aspergillus: morphology, importance and genomics. Med Mycol 2009; 47 (Suppl 1):S21–S26. 5 Berbee ML. The phylogeny of plant and animal pathogens in the Ascomycota. Physiol Mol Plant Pathol 2001; 59:165–187. 6 Thongkantha S, Lumyong S, McKenzie EHC, Hyde KD. Fungal saprobes and pathogens occurring on tissues of Dracaena lourieri and Pandanus spp. in Thailand. Fungal Divers 2008; 30:149–169.

17 Kumeda Y, Asao T. Single-strand conformation polymorphism analysis of PCRamplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi. Appl Environ Microbiol 1996; 62:2947–2952. 18 Valeriote F, Grieshaber CK, Media J, Pietraszkewicz H, Hoffmann J, Pan M, McLaughlin S. Discovery and development of anticancer agents from plants. J Exp Ther Oncol 2002; 2:228–236. 19 Love RA, Parge HE, Wickersham JA, Hostomsky Z, Habuka N, Moomaw EW, et al. The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell 1996; 87: 331–342. 20 Wei Y, Ma C-M, Hattori M. Synthesis of dammarane-type triterpene derivatives and their ability to inhibit HIV and HCV proteases. Bioorg Med Chem 2009; 17:3003–3010.

Original article 129

Synthesis of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindolin-2-one derivatives: part I Fatma A. Bassyounia,b, Amira S. Abdel Alla, Wafaa M. Haggagc, Madiha Mahmoude, Mamoun M.A. Sarhand and Mohamed Abdel-Rehimf a Department of Chemistry of Natural and Microbial Products, bPharmaceutical Research Group, c Department of Plant Pathology and Safe of Agriculture group, Center of Excellence for Advanced Sciences, National Research Center Dokki, dAtomic and Molecular Physics Unit, Department of Nuclear Physics. Atomic Energy Authority, Cairo, eDepartment of Pharmacology, Theodor Bilharz Research Institute, Giza, Egypt and fDepartment of Analytical Chemistry, Stockholm University, Stockholm, Sweden

Correspondence to Fatma A. Bassyouni, PhD, Department Chemistry of Natural and Microbial Products, Center of Excellence for Advanced Sciences, National Research Centre, 12622, Cairo, Egypt Tel: + 20 211 185 96967; fax: + 20 233 370 931; e-mail: [email protected] Received 15 April 2012 Accepted 26 August 2012 Egyptian Pharmaceutical Journal 2012,11:129–135

Objective The aim of part I is the synthesis of different series of 1H-1,2,4-triazol-3yl)phenylimino)(methylbenzyl)-5-nitroindolin-2-ones, 1H-pyrazole-1carbonyl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-ones, 3-(4-(1,3,4-oxadiazin6-one)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-ones, 1,3,4-oxadiazol-2yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-ones, and 4-(-1-(p-methylbenzyl)5-nitro-2-oxoindolin-3-ylideneamino) sugar hydrazone derivatives (4–13) through the reaction of 4-[1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylidineamino]benzohydrazide (3) with different reagents to be evaluated biologically. Materials and methods Derivatives of (1H-1,2,4-triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2one and (1H-pyrazole-1-carbonyl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (4–6) were prepared by the reaction of 4-[(1-(p-methylbenzyl)-5-nitro2-oxoindolin-3-ylideneamino)] benzohydrazide (3) with benzyl, benzoyl isothiocyanate, or acetyl acetone to form 1H-1,2,4-triazole and 1H-pyrazole-5-nitroindolin-2-one derivatives. The reaction of 3 with ethyl bromoacetate, ethyl acetoacetate, or acetyl chloride afforded 1,3,4 oxadiazin-6-one, 3-methyl-5-oxo-4,5-dihydro-1H-pyrazole, or 1,3,4-oxadiazole-5-nitroindolin-2-one derivatives (7–9), respectively. Sugar hydrazone5-nitroindolin-2-ones (10–13) were archived by the reaction of 3 with D-glucose, D-mannose, D-arabinose, and D-ribose using both conventional and green chemistry. Results and conclusion Conventional and microwave methods used for the synthesis of various triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindolin-2-one derivatives were applied for the synthesis of compounds 4–13. These methods were simple and gave good yields of the target compounds in short reaction times. Keywords: indoline-2,3-dione, oxadiazine, oxadiazoles, pyrazoles, sugar hydrazones, triazoles Egypt Pharm J 11:129–135 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction

Experimental

Several heterocyclic compounds are widely found in nature and are essential for life. Indoline-2,3-dione is an endogenous compound with pharmacological importance [1]. Many indoline-2,3-dione derivatives are known to possess antimicrobial, anti-inflammatory, analgesic, antiviral, antifungal, antitubercular, and antidepressant activities [2–7]. Moreover, the Schiff ’s bases [8–11] and the hydrazides [12–13] of indoline-2,3-dione derivatives have been reported and evaluated for different pharmacological activities such as antibacterial, anticonvulsant, antiprotozoal, and antitubercular. Also, 1,2,4 triazoles, pyrazoles, 1,3,4 oxadiazoles derivatives [14–15] and their sugar hydrazones [16–17] possess several biological effects. The objective of the present study is the synthesis of several derivatives of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindolin-2-one derivatives from 4-(1-(p-methylbenzyl)-5-nitro2-oxoindolin-3-ylideneamino)benzohydrazide using the conventional and microwave-assisted technique (part I).

Chemistry


Melting points were determined in open capillary tubes on an Electro thermal digital melting point apparatus (Stuart, SMP10, UK) and were uncorrected. IR spectra were recorded on a Jasco FT/IR Fourier transform infrared spectrophotometer (USA) using KBr disks. 1H NMR spectra were determined on a JOEL 270 MHz spectrometer (Japan) in DMSO-d6 using TMS as the internal reference. Mass spectra were recorded on mass spectrometer JOEL at 70 eV. Elemental analyses were performed at the Microanalytical Lab, National Research Center, and the results were found to be in agreement ( ± 0.4%) within the calculated values. The microwave oven used was LG 900 W (LG group, Seoul, South Korea). Purity of the synthesized compounds was checked by thin-layer chromatography (TLC) silica-gel alumina sheet-Merck 60F254 precoated sheets (Darmstadt, Germany). 5-Nitroindoline-2,3-dione was purchased from Sigma-Aldrich (North Teutonia Avenue, Milwaukee, USA). DOI: 10.7123/01.EPJ.0000422115.68888.f2

130


General procedures for the synthesis of ethyl 4-(5-nitro-2oxoindolin-3-ylideneamino) benzoate (1)

Method A: An equimolar amount of 5-nitroindoline-2,3dione (0.03 mol, 5.76 g) and ethyl-4-amino benzoate (0.03 mol, 3.78 g) was dissolved in absolute ethanol (20 ml) containing glacial acetic acid (2 ml). The reaction mixture was heated under reflux at 1201C for 8 h and then kept at room temperature overnight. The solvent was evaporated under vacuum, and the resulting solid was recrystallized from absolute ethanol to afford compound 1 of yield 2.50 g (75%). Method B: A solution of 5-nitroindoline-2,3-dione (0.01 mol, 1.92 g) and ethyl-4-amino benzoate (0.01 mol, 1.26 g) was dissolved in a mixture of water (10 ml) and absolute ethyl alcohol (10 ml) (1 : 1) and refluxed under stirring for 12 h. After cooling, the formed crystalline product was collected by filtration and dried to give 2.50 g (82%) of compound 1. Method C: A mixture of 5-nitroindoline-2,3-dione (0.01 mol, 1.92 g) and ethyl-4-amino benzoate (0.01 mol,1.26 g) in polyethylene glycol-600 (PEG-600) (10 ml) was refluxed at 1001C for 2 h. The reaction mixture was cooled and poured onto ice-cold water. The separated solid was filtered off, washed with cold water, and recrystallized from methanol to give product 1 in good yield (2.88 g, 85%). Method D (microwave): A mixture of 5-nitroindoline-2,3dione (0.01 mol, 1.92 g) and ethyl-4-amino benzoate (0.01 mol, 1.26 g) in the presence of sodium acetate (0.01 mol, 0.82 g) was taken in an open 125 ml Erlenmeyer flask. After thorough mixing, the reaction was irradiated in an automated microwave oven (LG 900 W and temperature 1001C) for 3 min. The oven was turned off after 2 min of heating to avoid evaporation of the reagent, and the progress of the reaction was monitored by TLC. After completion of the reaction, the flask was cooled, and the product was extracted from the flask. Hot ethanol was added. The solvent was then evaporated under vacuum and the product was filtered and recrystallized from methanol to give the compound. Yield 2.70 g (80%); mp 211–2131C; IR cm – 1 (KBr): 3395 (NH), 1705 (C = O), 1730 (C = O ester), 1653 (C = N); 1 H NMR (DMSO-d6, ppm): 2.10 (t, 3H, COOCH2CH3), 4.55–4.75 (q, 2H, COOCH2CH3), 6.85–7.20 (m, 4H, ArH), 7.30–7.50 (m, 3H, Ar-H); MS: m/z: 339 [M + ]; Anal. Calcd. (%) for C17H13N3O5 : C: 60.18, H: 3.86, N: 12.38; found: C: 60.12, H: 3.80, N: 12.32. Synthesis of ethyl 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino)] benzoate (2)

A mixture of compound 1 (0.02 mol, 6.78 g) and 4methylbenzyl chloride (0.03 mol, 3.4 ml) in dry pyridine (5 ml) was heated under reflux for 6 h. After completion of the reaction, the reaction mixture was cooled and then washed with diluted hydrochloric acid (1 : 1) and then with water (50 ml). The formed precipitate was filtered and crystallized from methanol to give compound 2, which was used directly for the preparation of compounds 3–13. Yield 3.28 g (74%); mp 190–192; IR cm – 1 (KBr):

3395(NH), 1705 (C = O), 1735 (C = O ester), 1648 (C = N); 1H NMR (DMSO-d6, ppm): 1.8 (s, 3H, CH3) 2.15 (t, 3H, –COOCH2CH3), 4.20 (s, 2H, CH2-benzyl), 4.50–4.70 (q, 2H, –COOCH2CH3), 6.75–7.00 (m, 4H, ArH), 7.20–7.40 (m, 4H, Ar-H), 7.50–7.65 (m, 3H-Ar-H); MS: m/z = 443 [M + ]; Anal. Calcd. (%) for C25H21N3O5: C: 67.71, H: 4.77, N: 9.48; found: C: 67.69, H: 4.70, N: 9.42. Synthesis of 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3ylideneamino)] benzohydrazide (3)

Compound 2 (2 mmol, 0.90 ) was dissolved in absolute ethyl alcohol (30 ml), and hydrazine hydrate (2 mmol, 1 ml) was added dropwise with stirring at room temperature. Thereafter, the reaction mixture was heated under reflux with stirring for 8 h. The reaction was monitored by TLC and kept in a refrigerator for 4 h. The separated solid was filtered and washed with water and then with a small amount of absolute ethanol (15 ml). The product was dried and recrystallized with absolute ethanol to give compound 3. Yield 3.2 g (76%); mp 270–2721C; IR cm – 1 (KBr): 3380 (NH), 3217 (NH2), 1705 (C = O), 1665 (C = O amide), 1644 (C = N); 1H NMR (DMSOd6, ppm): 2.10 (s, 3H, CH3), 4.30 (s, 2H, CH2-benzyl), 5.63 (s, 2H, NH2), 6.86–7.10 (m, 4H, Ar-H), 7.15–7.40 (m, 4H, Ar-H), 7.50–7.65 (m, 3H, Ar-H), 9.86 (s,1H, NH); MS: m/z: 430 [M + + 1]; Anal. Calcd. (%) for C23H19N5O4: C: 64.33, H: 4.46, N: 16.31; found: C: 64.30, H: 4.42, N:16.25. Synthesis of 3-(4-(4-benzyl-5-thioxo-4,5-dihydro-1H-1,2,4triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2one (4) and 3-(4-(4-benzoyl-5-thioxo-4,5-dihydro-1H-1,2,4triazol-3-yl)phenylimino)-1-(4-methylbenzyl)-5-nitroindolin-2one (5)

Method A: A mixture of compound 3 (1 mmol, 0.43 g), benzyl isothiocyanate (1 mmol, 0.15 ml), or benzoyl isothiocyanate (1 mmol, 0.2 ml) in absolute ethanol (10 ml) and potassium hydroxide solution (10 ml, 10% KOH in absolute ethanol) was taken. The reaction mixture was refluxed under stirring for 8 h, and the progress of the reaction was monitored by TLC. After cooling, the separated product was filtered and acidified with diluted hydrochloric acid (10% HCl). The precipitate formed was collected by filtration and recrystallized from methanol to give compounds 4 and 5 with yields 75 and 78%, respectively. Method B: A mixture of compound 3 (1 mmol, 0.43 g), benzyl isothiocyanate (2 mmol, 0.30 ml) or benzoyl isothiocyanate (2 mmol, 0.4 ml), p-toluenesulfonic acid (1 mmol, 0.17 g), and ammonium acetate (1 mmol, 0.7 g) was mixed together with a pestle and mortar at room temperature. The reaction mixture was solidified. The contents were quenched by adding water and absolute ethanol (5 : 5 ml) with stirring at room temperature for 30 min and then for 3 h at 501C. The reaction was monitored by TLC. The formed precipitate was washed with 5% NaHCO3 (10 ml) and then with water (25 ml) and dried. The resulting solid product was recrystallized

Synthesis of 5-nitroindolin-2-ones Bassyouni et al. 131

from methanol to give pure products 4 and 5 (yields 80 and 84%, respectively). Method C: A mixture of compound 3 (1 mmol, 0.43 g), benzyl isothiocyanate (2 mmol, 0.30 ml) or benzoyl isothiocyanate (2 mmol, 0.40 ml), and polyethylene glycol-600 (PEG-600) (10 ml) was refluxed with stirring at 1001C for 2.30–3 h. The reaction was monitored by TLC. After completion of the reaction, the mixture was poured onto cold water. The precipitated substance was filtered and washed with ice-cold water and then recrystallized with methanol to give products 4 and 5 in excellent yields of 90 and 92%, respectively. Method D: An open 125 ml Erlenmeyer flask containing compound 3 (1 mmol, 0.43 g) and benzyl isothiocyanate (1 mmol, 0.15 ml) or benzoyl isothiocyanate (1 mmol, 0.2 ml) in the presence of a solution of potassium hydroxide (10 ml, 10% KOH in water) was irradiated in an automated microwave oven for 2.30–3.30 min (LG 900 W at 1001C). The reaction was monitored by TLC. The oven was turned off after 2–3 min of heating to avoid evaporation of the reagents. After completion of the reaction the flask was cooled and the product was extracted from the flask by scratching. The product was then acidified by diluted hydrochloric acid and washed with water. The precipitate was collected by filtration and recrystallized from absolute ethanol to give products 4 and 5 in yields of 80 and 82%, respectively. 3-(4-(4-Benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (4)

Yield 0.42 g (75%); mp 253–2551C; IR cm – 1 (KBr): 3399 (NH), 1705 (C = O), 1658 (C = N), 1050 (C = S); 1H NMR (DMSO-d6, ppm): 2.10 (s, 3H, CH3), 3.75 (s, 2H, CH2-benzyl), 4.20 (s, 2H, benzyl-CH2), 6.70–6.90 (m, 5H, Ar-H), 7.02–7.26 (m, 4H, Ar-H), 7.30–7.50 (m, 4H, Ar-H), 7.60–7.78 (m, 3H, Ar-H), 11.34 (s,1H, NH); MS: m/z: 559 [M + – 1], Anal. Calcd. (%) for C31H24N6O3S: C: 66.41, H: 4.31, N: 14.99, S: 5.72; found: C: 66.48, H: 4.25, N: 14.92, S: 5.80. 3-(4-(4-Benzoyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (5)

Yield 0.45 g (78%); mp 280–2821C; IR cm – 1 (KBr): 3390 (NH), 1705, 1715 (2C = O), 1654 (C = N), 1065 (C = S); 1H NMR (270 MHz, DMSO-d6, ppm): 2.00 (s, 3H, CH3), 4.10 (s, 2H, CH2-benzyl), 6.75–6.99 (m, 5H, Ar-H), 7.10–7.30 (m, 4H, Ar-H), 7.40–7.60 (m, 4H, ArH), 7.65–7.80 (m, 3H, Ar-H), 11.34 (s,1H, NH); MS: m/z: 575 [M + + 1]; Anal. Calcd. (%) for C31H22N6O4S: C: 64.80, H: 3.86; N: 14.63, S: 5.58; found: C: 64.76, H: 3.80, N: 4.55, S: 5.55. Synthesis of 3-(4-(3,5-dimethyl-1H-pyrazole-1carbonyl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin2-one (6)

A cold mixture of acetyl acetone (1 mmol, 0.1 ml) and anhydrous sodium acetate (0.01 mol, 0.1 g) in absolute ethanol (10 ml) was added dropwise with stirring for 10 min to a solution of compound 3 (1 mmol, 0.43 g) in ethanol (10 ml) and a few drops of TEA. The reaction

mixture was stirred for 5 h at room temperature and the progress of the reaction was monitored by TLC. The mixture was left at room temperature overnight. The solvent was evaporated under vacuum and the residue obtained was washed with water (50 ml), filtered, dried, and recrystallized from absolute ethanol to form compound 6. Yield 0.33 g (68%), mp 272–2741C; IR cm – 1 (KBr): 1705,1710 (2C = O), 1655 (C = N); 1H NMR (DMSOd6,ppm): 2.10 (s, 3H, CH3), 2.30 (s, 3H, CH3, 2.40 (s, 3H, CH3), 4.20 (s, 2H, CH2-benzyl), 6.10 (s, 1H, CH pyrazole), 6.80–7.00 (m, 4H, Ar-H), 7.20–7.50 (m, 4H, Ar-H), 7.60–7.75 (m, 3H, Ar-H), MS: m/z: 493 [M + ]; Anal. Calcd. (%) for C28H23N5O4: C: 68.14, H: 4.70, N: 14.19; found: C: 68.10, H: 4.68, N: 14.15. Synthesis of 3-(4-(1,3,4-oxadiazin-6-one)phenylimino)-1-(pmethylbenzyl)-5-nitroindolin-2-one (7)

To a solution of compound 3 (1 mmol, 0.43 g) and DMF (10 ml), ethyl bromoacetate (2 mmol, 0.33 ml) was added dropwise with stirring for 30 min. This was followed by addition of anhydrous sodium acetate (2 mmol, 0.139 g). The reaction mixture was heated under reflux at 1001C for 8 h with stirring, and the reaction was monitored by TLC. The reaction mixture was cooled to room temperature and water (50 ml) was added. The product was extracted with ethyl acetate, concentrated under vacuum, washed with water (100 ml), and recrystallized from absolute ethanol to give compound 7. Yield 0.23 g (65%); mp 260–2621C; IR cm – 1 (KBr): 3388 (NH), 1705 (C = O), 1740 (C = O ester), 1650–1656 (C = N); 1H NMR (DMSO-d6, ppm): 2.10 (s, 3H, CH3), 4.20 (s, 2H, CH2-benzyl), 4.40 (s, 2H, CH2), 6.75–7.00 (m,4H, ArH), 7.22–7.45 (m, 4H, Ar-H), 7.55–7.75 (m, 3H, Ar-H), 9.60 (s,1H, NH); Anal. Calcd. (%) for C25H19N5O5: C: 63.96, H: 4.08, N: 14.92; found: C:63.91, H:4.12, N:14.96. Synthesis of 3-(4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1carbonyl) phenylimino)-1-(p-methylbenzyl)-5-nitroindolin2-one (8)

A mixture of compound 3 (1 mmol, 0.43 g) and ethyl acetoacetate (2 mmol, 0.26 ml) was heated under reflux without solvent at 1001C for 15 min with stirring. The mixture was then heated under reflux in DMF (10 ml) containing a few drops of triethylamine for 6 h. The reaction was monitored by TLC. After cooling, the precipitate formed was collected by filtration and recrystallized from absolute ethanol to give compound 8. Yield 0.31 g (64%); mp 272–2741C; IR cm – 1 (KBr): 1705,1720(2C = O),1690 (C = O), 1645–1655 (C = N); 1H NMR (DMSO-d6,ppm): 1.90 (s, 3H, CH3), 1.90 (s, 3H, CH3) 2.10 (s, 2H, CH2), 4.20 (s, 2H, CH2-benzyl), 6.72–7.00 (m, 4H, Ar-H), 7.20–7.35 (m, 4H, Ar-H), 7.40–7.60 (m, 3H, Ar-H), 9.30; Anal. Calcd. (%) for C27H21N5O5: C: 65.45, H: 4.72, N: 14.13; found: C: 65.41, H: 4.68, N: 14.09. Synthesis of 3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylimino)1-(p-methylbenzyl)-5-nitroindolin-2-one (9)

A mixture of compound 3 (1 mmol, 0.43 g), acetyl chloride (2 mmol, 0.20 ml) in DMF (10 ml), and a few drops of TEA was stirred at room temperature for 8 h. The reaction was monitored by TLC. After completion of


the reaction, the reaction mixture was cooled and then poured onto crushed ice. The solid obtained was filtered and recrystallized from methanol to give compound 9. Yield 0.29 g (66%); mp 213–2151C; IR cm – 1 (KBr): 1705(C = O), 1640–1650 (C = N); 1H NMR (DMSO-d6, ppm): 2.10 (s, 3H, CH3), 2.35 (s, J = 6 Hz, 3H, CH3), 4.20 (s, 2H, CH2-benzyl), 6.80–7.00 (m, 4H, Ar-H), 7.20–7.45 (m, 4H, Ar-H), 7.50–7.70 (m, 3H, Ar-H); MS: m/z: 453 [M + ]; Anal. Calcd. (%) for C25H19N5O4: C: 66.22, H: 4.22, N: 15.44; found: C: 66.25, H: 4.25, N: 15.48. General procedure for the synthesis of compounds 6–9 using polyethylene glycol (PEG-600)

A mixture of compound 3 (0.1 mmol, 0.43 g), acetyl acetone, ethyl bromoacetate, ethyl acetoacetate, and acetyl chloride (0.1 mmol) and polyethylene glycol (PEG-600) (10 ml) in the presence of TEA was heated at 1001C for 2–3 h. The reaction monitored by TLC. The reaction mixture was cooled and poured onto cold water. The precipitated solid was filtered and washed with cold water and then crystallized from methanol to give compounds 6–9 in good yields of 80–82%. General procedures for synthesis of compounds 6–9 using a microwave

Into an open round flask were added compound 3 (1 mmol, 0.43 g) and acetyl acetone, ethyl bromoacetate, ethyl acetoacetate, or acetyl chloride (1 mmol) in the presence of p-toluenesulfonic acid (2 mmol, 1.4 g), and DMF (5 ml) absorbed over acidic alumina (1 g). The reaction was mixed at room temperature and then irradiated in an automated microwave oven (900 W LG at 1001C) for 2–3 min. The oven was turned off after 1–2 min of heating to avoid evaporation of the reagents. The reaction was monitored by TLC, and after completion of the reaction the flask was cooled. The product was extracted from the flask by scratching; the solid substance separated was washed with water (30 ml), filtered, and recrystallized from methanol and then weighted to yield compounds 6–9 (78–80%, respectively). General procedure for the synthesis of N0 -ethylidene 4(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) sugar hydrazone derivatives (10–13) D -Glucose

(1 mmol, 0.20 g), D-mannose (1 mmol, 0.20 g), (1 mmol, 0.15 g), or D-ribose (1 mmol, 0.15 g) mixed in water (1 ml) was added to a solution of compound 3 (1 mmol, 0.43 g) in ethanol (20 ml) and acetic acid (0.5 ml). The reaction mixture was stirred at room temperature for 1 h, and then refluxed with stirring at 501C for 5–6 h. Water (0.5 ml) was added after 3 h. The reaction was monitored by TLC. After cooling, the mixture was evaporated under vacuum, and the resulting mixture was washed with absolute ethanol to afford the respective hydrazone derivatives 10–13 in yields of 70–74%. D -arabinose

D-Glucose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (10): Yield 0.44 g (74%), mp

175–1771C; IR cm – 1 (KBr): 3420–3445(OH), 1705 (C = O), 1664 (C = O amide), 1652 (C = N), 1635(N = CH); 1 H NMR (MeOD-d4, ppm): 2.10 (s, 3H, CH3), 3.70–3.80 (m, 2H, CH2-OH), 3.90–4.10 (m, 5H, OH, exchangeable D2O), 4.20 (s, 2H, CH2-benzyl), 4.30–4.50 (m, 2H, H-2, H-1a), 4.60 (dd,1H, J = 12.1, 6.7 Hz, H-1b), 4.70 (1H, dd, J = 7.5, 2.3 Hz, H-3), 4.80 (dd,1H, J = 3.8, 2.5 Hz, H4), 5.00 (dd, 1H, J = 7.8, 3.6 Hz, H-5), 5.10 (d,1H, J = 7.6 Hz, H-6), 6.80–7.10 (m, 4H, Ar-H), 7.30–7.50 (m, 4H, Ar-H), 7.60–7.75 (m, 3H, Ar-H), 8.10 (d, 1H, N = CH), 9.30 (s,1H, NH); Anal. Calcd. (%) for C29H29N5O9: C: 58.88, H, 4.94, N, 11.84; found: C: 58.83, H: 4.90, N: 11.80. D-Mannose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (11): Yield 0. 41 g(70%), mp 183–185oC; IR cm – 1 (KBr): 3425–3440 (OH), 1715(C = O), 1665 (C = O amide) 1650 (C = N), 1630 (N = CH),); 1H NMR (MeOD-d4, ppm): 2.10 (s, 3H, CH3), 3.60–3.70 (m, 2H, CH2-OH), 3.90–4.10 (m, 5H, OH, exchangeable D2O), 4.25 (s, 2H, CH2-benzyl), 4.40 (d, 1H, m, J = 3.5Hz, H-6) 4.60 (1H, dd, J = 8.3, 3.2 Hz, H-5), 4.70 (1H, d, J = 8.2 Hz, H-4), 4.96 (1H, d, J = 7.7 Hz, H-3), 5.20 (1H, m, H-2), 5.40 (d, 1H, H1a), 3.56 (1H, dd, J = 11.2, 3.6 Hz, H-1b), 6.90–7.20 (m, 4H, Ar-H), 7.30–7.50 (m, 4H, Ar-H), 7.60–7.80 (m, 3H, Ar-H), 8.20 (d, 1H, N = CH), 9.40 (s,1H, NH); MS: m/z: 590 [M + – 1]; Anal. Calcd. (%) for C29H29N5O9: C: 58.88, H: 4.94, N: 11.84; found: C: 58.83, H: 4.90, N: 11.80. D-Arabinose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-yli-

deneamino) hydrazone derivative (12): Yield 0.41 g (73%), mp 180–1821C; IR cm – 1 (KBr): 3425–3445 (OH), 1715 (C = O), 1660 (C = O amide); 1649 (C = N); 1630 (N = CH).1H NMR (DMSO-d6, ppm): 2.10 (s, 3H, CH3), 3.60–3.70 (2H, m,H-3, H-1a), 3.80 (1H, dd, J = 7.3, 4.5 Hz, H-2), 4.00 (1H, dd, J = 12.3, 7.3 Hz, H-1b), 4.20 (s, 2H, CH2 -benzyl), 4.40 (1H, Br, H-4), 4.60 (1H, d, J = 10.3 Hz, H-5), 4.80–5.10 (1H, t, J = 6.4 Hz, CH2OH), 5.20–5.40 (m, 5H, OH, exchangeable D2O), 6.80–7.15 (m, 4H, Ar-H), 7.20–7.50 (m, 4H, Ar-H), 7.60–7.80 (m, Hz, 3H, Ar-H), 8.15 (d, 1H, N = CH), 9.25 (s,1H, NH); MS (relative intensity): m/z: 560 [M + + 1]; Anal. Calcd. (%) for C28H27 N5O8: C: 59.89, H: 4.85, N: 12.47; found: C: 59.83, H: 4.80, N, 12.42. D-Ribose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (13): Yield 0.39 g (71%), mp 179–1811C; IR cm – 1 (KBr): 3425–3440 (OH), 1715 (C = O), 1665 (C = O amide); 1650 (C = N); 1635 (N = CH) 1H NMR (DMSO-d6, ppm): 2.10 (s, 3H, CH3), 3.70–3.80 (m, 2H, CH2-OH), 3.90–4.10 (m, 4H, OH, exchangeable D2O), 4.25 (s, 2H, CH2 -benzyl), 4.40 (1H, dd, J = 11.2, 3.5 Hz, H-1a), 4.60–4.70 (2H, m, H-3, H-1b); 4.80 (1H, d, J = 3.2 Hz, H-5), 5.18 (1H, dd, J = 8.7, 3.3 Hz, H-4), 5.44 (1H, dd, J = 9.3, 5.6 Hz, H2),6.80–7.10 (m, 4H, Ar-H), 7.20–7.40 (m, 4H, Ar-H), 7.50–7.70 (m, 3H, Ar-H), 8.35 (d, 1H, N = CH), 9.35 (s,1H, NH); Anal. Calcd. (%) for C28H27 N5O8: C: 59.89, H: 4.85, N: 12.47; found: C: 59.83, H: 4.80, N: 12.42.


Scheme 1

Results and discussion Indole and related compounds have shown diverse biological activities. Furthermore, some of their derivatives have also been reported to exhibit significant biological activities. Considerable interest has arisen in the design and synthesis of oxoindoline derivatives containing oxadiazole, pyrazole, triazole, and sugar hydrazone to explore their pharmacological activities such as antibacterial and anti-inflammatory activities. Compound 1 was prepared by the reaction of 5-nitro indoline-2,3-dione with ethyl-4-amino benzoate to give the corresponding ethyl(-5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1), which reacted with p-methyl benzyl chloride to form ethyl 4-(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino)benzoate (2). The hydrazinolysis of compound 2 with hydrazine hydrate gave the corresponding 4-(1-(pmethylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) benzohydrazide (3) (Scheme 1). On the basis of elemental analyses and spectral data, structures of compounds 1, 2, and 3 were confirmed. The mass spectrum of compound 1 exhibited the molecular ion peak (M + ) at m/z 339 and the mass spectrum of compound 2 showed the molecular ion

C H OOC

N ON O N H C H OOC

H NHNOC

1

N

N

ON

ON

NH NH .H O

O

O N N

HC HC

2

3

Synthesis of 5-nitroindoline-2-one derivatives 1–3.

Scheme 2 H2NHNOC

N O2N O N

H3C

3

iii

i ii

N

N

O2N C

O

O

O2N

CH3

N

N O

N N CH3

H3C

NH

N

N

S

N O2N

N O

6

N

O

NH

N

H3C

4

S

H3C

5

Synthesis of 1H-1,2,4-traizole and 1H-pyrazole-5-nitroindoline-2-one derivatives 4–6. Conditions and reagents: (i) benzyl isothiocyanate/10% KOH/ reflux, (ii) benzoyl isothiocyanate/10% KOH/reflux, (iii) acetyl acetone/anhydrous sodium acetate/RT.

134


peak at m/z 443 (M + ). The IR spectrum of compound 3 was characterized by absorption bands C = N at 1644 cm – 1, C = O at 1705 cm – 1, NH at 3380 cm – 1, and NH2 at 3217 cm – 1.

Scheme 3 N ON

O

The reaction of compound 3 with benzyl isothiocyanate or benzoyl isothiocyanate in the presence of an aqueous solution of 10% KOH provided the corresponding 3-(4-(4benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenylimino)-1-(4-methylbenzyl)-5-nitroindolin-2-one (4) and 3-(4-(4-benzoyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (5). The IR spectrum of compound 5 exhibited two absorption bands for C = O stretching at 1705 and 1715 cm – 1 and for C = S at 1065 cm – 1.

O C

C

O

i N

N

N H

7 HC N ON

ii

3

C

O

O

N

N

N CH

O

8 HC

In addition, condensation reaction of 4-(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) benzohydrazide (3) with acetyl acetone afforded the corresponding -3-(4(3,5-dimethyl-1H-pyrazole-1-carbonyl)phenylimino)-1-(pmethylbenzyl)-5-nitroindolin-2-one (6) (Scheme 2). The IR spectrum of compound 6 displayed two absorption bands at 1705 and 1710 cm – 1 for C = O.

N

iii

ON N O

N

N

O CH

9 HC

Moreover, compound 3 was reacted with ethyl bromoacetate to afford the corresponding 3-(4-(1,3,4-oxadiazin6-one)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2one (7). The IR spectrum of compound 7 showed absorption bands at 3388 cm – 1 for NH, 1740 cm – 1 and 1705 cm – 1 for C = O ester, and carbonyl groups, respectively, and 1656–1650 cm – 1 for C = N. In a similar manner, compound 3 converted into 3-(4-(3methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-carbonyl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (8) by reacting with ethyl acetoacetate to form compound 8. Compound 8 showed absorption bands at around 1705, 1720, 1690, and 1645–1655 cm – 1 regions, resulting from the C = O and C = N functions. In contrast, treatment of compound 3 with acetyl chloride led to the formation of 3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylimino)-1-(pmethylbenzyl)-5-nitroindolin-2-one (9) (Scheme 3). The latter compound showed C = O absorption band at 1705 cm – 1 and C = N at 1640–1650 cm – 1. Green chemistry is an approach for the synthesis of most of the synthesized compounds 1–9 using polyethylene glycol (PEG-600), which is a very effective, efficient, and more environmentally friendly solvent (cf. Materials and methods section). In recent years, microwave-assisted synthesis has become a powerful synthetic tool for rapid synthesis of a variety of beneficial organic intermediates and biologically active compounds. Therefore, we applied microwave conditions to synthesize ethyl-5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1), 3-(4-(4-benzyl-5-thioxo-4,5-dihydro-1H-1,2,4triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (4), 3-(4-(4-benzoyl-5-thioxo-4,5-dihydro-1H1,2,4-triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (5), and compounds 6–9. Synthesis of the sugar hydrazone derivatives 10–13 was accomplished by reaction of target 3 with monosaccharides, namely, D-glucose, D-mannose, D-arabinose, or D -ribose, in the presence of water and a few drops of

Synthesis of 1,3,4 oxadiazin-6-one, 1H-pyrazole, and 1,3,4-oxadiazole5-nitroindolin-2-one derivatives 7–9. Conditions and reagents: (i) ethyl bromoacetate/anhydrous sodium acetate/DMF/reflux, (ii) ethyl acetoacetate/DMF/reflux, (iii) acetyl chloride/DMF/RT.

Scheme 4

CONHN=CH

N

O2N

(CHOH)n O N

CH2OH

n=3or n= 4

H3C

10, D- glucose 11, D- mannose 12, D- arabinose 13, D- ribose

Synthesis of sugar hydrazone-5-nitroindolin-2-one derivatives 10–13. Conditions and reagents: D-glucose, D-mannose, D-arabinose, and D-ribose/water/acetic acid.

acetic acid to give the respective sugar hydrazones 10–13 (Scheme 4). The IR spectra gave strong absorption bands at 3420–3445 cm – 1 characterized by the presence of CHOH function groups and an absorption band at 1705 cm – 1 for the C = O group in addition to N = CH at 1630–1635 cm – 1. 4-(1-(p-Methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) benzohydrazide (3) was attached to a series of open-chain monosaccharides linked to the nitrogen atom of the benzohydrazide group to give sugar hydrazone-5-nitroindolin-2-one derivatives.


Effects of microwave power and reaction time

Microwave heating is a powerful technique for promoting a variety of chemical reactions. The reaction time has an impact on the microwave-assisted method for the synthesis of ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino)benzoate (1) using sodium acetate, 3-(4-(4-benzyl-5thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenylimino)-1(p-methylbenzyl)-5-nitroindolin-2-one derivatives, and 3(4-(4-benzoyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (4 and 5) in the presence of aqueous KOH. In addition, 3-(4(3,5-dimethyl-1H-pyrazole-1-carbonyl)phenylimino)-1-(pmethylbenzyl)-5-nitroindolin-2-one (6), 3-(4-(1,3,4-oxadiazin-6-one)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (7), 3-(4-(3-methyl-5-oxo-4,5-dihydro-1Hpyrazole-1-carbonyl)phenylimino)-1-(4-methylbenzyl)-5nitroindolin-2-one (8), and 3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin2-one (9) in the presence of p-toluenesulfonic acid are also used. When a microwave was used for irradiation, the reaction time was markedly reduced from several hours (in conventional heating) to a few minutes under solvent-free conditions; other advantages of using a microwave are low cost, high yield, and simplicity in processing and handling [18].

Conclusion A series of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindolin-2-one derivatives were synthesized from 4-(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) benzohydrazide in good yields using conventional and microwave-assisted techniques.

Acknowledgements The authors are grateful to National Research Center, Cairo, Egypt for providing generous financial support and Science for NRC project no: 8040204.


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Original article

Antimicrobial, anti-inflammatory, and antinociceptive activities of triazole, pyrazole, oxadiazine, oxadiazole, and sugar hydrazone-5-nitroindoline-2-one derivatives and a study of their computational chemistry: part II Fatma A. Bassyounia,b, Amira S. Abdel Alla, Wafaa M. Haggagc, Madiha Mahmoudd, Mamoun M.A. Sarhane and Mohamed Abdel-Rehimf a Department of Chemistry of Natural and Microbial Products, bPharmaceutical Research Group, c Department Plant Pathology and Departments of Safe of Agriculture, Pharmaceutical Research Group, Center of Excellence for Advanced Sciences, National Research Centre, Dokki, dDepartment of Pharmacology, Theodor Bilharz Research Institute, Giza, eDepartment of Nuclear Physics, Atomic and Molecular Physics Unit, Atomic Energy Authority, Cairo, Egypt and fDepartment of Analytical Chemistry, Stockholm University, Stockholm, Sweden

Correspondence to Fatma A. Bassyouni, PhD, Department of Chemistry of Natural and Microbial Products, Center of Excellence for Advanced Sciences, National Research Centre, Dokki, 12311 Cairo, Egypt Tel: + 20 211 185 96967; fax: + 20 233 370 931; e-mail: [email protected] Received 15 April 2012 Accepted 9 September 2012 Egyptian Pharmaceutical Journal 2012, 11:136–143

Objective The aim of this study (part II) is to evaluate the antibacterial, anti-inflammatory, and antinociceptive activities of a series of 1H-1,2,4-triazol-3-yl)phenylimino) (methylbenzyl)-5-nitroindolin-2-ones, 1H-pyrazole-1-carbonyl)phenylimino)-1(p-methylbenzyl)-5-nitroindolin-2-ones, 3-(4-(1,3,4-oxadizine-6-one)phenylimino)-1(p-methylbenzyl)-5-nitroindolin-2-ones, 1,3,4-oxadiazol-2-yl)phenylimino)1-(p-methylbenzyl)-5-nitroindolin-2-ones and 4-(-1-(p-methylbenzyl)-5-nitro-2oxoindolin-3-ylideneamino) sugar hydrazone derivatives (1–13) and, in addition, to investigate their computational chemistry. Methods The synthesized compounds in (part I) 1–9 were evaluated for their antibacterial and antifungal activities using different strains of Gram-positive bacteria (Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa), yeast (Candida albicans), and four mold fungi (Fusarium solani, Aspergillus niger, Colletotrichum gloeosporioides, and Phomopsis obscurans). The anti-inflammatory and antinociceptive activities of compounds 1–13 were evaluated using a hot-plate test, acetic acid-induced writhing in mice, formalin-induced nociception, a tail immersion test, and carrageenan-induced hind paw edema. For computational chemistry, a semiempirical MNDO method (Modified Neglect of Differential Overlap is a semi-empirical method for the quantum calculation of molecular electronic structure in computational chemistry) associated with HyperChem professional 7.5 programs was adapted. Results and conclusion Compounds 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino)] benzohydrazide (3) and 3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin2-one (9) showed the highest antibacterial and antifungal activities compared with clotrimazole and sulfamethoxazole as reference drugs. In contrast, compounds ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1), 3-(4-(3-methyl-5-oxo-4,5-dihydro1H-pyrazole-1-carbonyl) phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (8), D-glucose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (10), and D-arabinose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino) hydrazone derivative (12) showed significantly high anti-inflammatory and antinociceptive activities when compared with indomethacin and morphine as reference drugs. From the computational chemistry compounds, ethyl 4-(5-nitro-2oxoindolin-3-ylideneamino) benzoate (1), ethyl 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino)] benzoate (2), and 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino)] benzohydrazide (3) yielded the lowest values of total energy and heat of formation, and had higher stability than other molecules. Keywords: antibacterial and antifungal, anti-inflammatory, antinociceptive, computational chemistry, oxadiazole, pyrazoles, sugar hydrazones, triazoles Egypt Pharm J 11:136–143 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction A large number of indoline-2,3-one have powerful antibacterial, antifungal, anti-inflammatory activities [1–3].

The importance of oxadiazole, pyrazole, and triazole derivatives as chemotherapeutic agents is well established and associated with potent biological activities such as antimicribial and anti-inflammatory activities [4–6].


DOI: 10.7123/01.EPJ.0000422114.91245.38

Biological effects of 5-nitroindoline-2-ones Bassyouni et al. 137

Table 1 In-vitro antimicrobial activity expressed as diameter of the growth-inhibitory zone of the tested compounds 2–13 Activity expressed in mm of inhibition zone diameter

Compounds 2 3 4 5 6 7 8 9 10 11 12 13 Clotrimazole Sulfamethoxazole

Bacillus subtilis

Candida albicans


Phomopsis obscurans

Colletotrichum gloeosporioides

Fusarium solani

Aspergillus niger

6.87 10.7 1.45 ND ND ND ND 6.65 0.67 ND ND ND – 3.87

6.67 9.45 1.78 ND ND ND ND 5.45 0.23 ND ND ND – 2.80

7.45 8.86 1.55 ND ND ND ND 6.76 0.54 ND ND ND – 4.98

6.56 9.76 ND ND ND ND ND 4.65 ND ND ND ND 3.76 –

10.6 13.9 1.56 ND ND ND ND 8.56 ND ND ND ND 2.76 –

7.67 11.7 1.54 ND ND ND ND 6.45 0.78 ND ND ND 3.67 –

11.7 15.7 1.87 ND ND ND ND 9.65 1.67 ND ND ND 2.91 –

ND, not detected.

Table 2 In vitro, antifungal activity of the tested compounds 3 and 9 against the strain of Aspergillus niger (zone of inhibition in mm) Compounds Control (medium) 3 9 Clotrimazole

Concentration (mg/ml)

Inhibition zones (mm) ± SE

Spores count/ml

Myceliadry weight (mg) ± SE

Cell concentration (OD610 nm)

0.0 0.007 0.07 0.1 0.007 0.07 0.1 0.1

0.0 7.0 ± 1.4a 16.0 ± 1.4 26.5 ± 2.1 4.5 ± 1.7 12.0 ± 0.0 15.5 ± 2.3 2.54

542.9 67.92 32.8 0.87 94.7 42.7 2.76 231.8

28.9 ± 1.6 14.7 ± 1.0 6.80 ± 1.4 0.27 ± 1.1 17.8 ± 1.0 9.65 ± 0.3 1.56 ± 0.4 11.8

2.570 0.7665 0.347 0.0321 3.213 0.956 0.254 1.45

OD, optical density.

In view of the biological activity, and as a continuation of our research work on the synthesis of 1,2,4 triazole, 1-H pyrazole, 1,3,4 oxadiazin, 1,3,4 oxadiazole, and sugar hydrazone -5-nitro-2-oxoindolin derivatives (part I), it was of interest to evaluate their effects as antibacterial, antifungal, anti-inflammatory, and antinociceptive agents in addition to studying their computational chemistry.

Subjects and methods Antibacterial and antifungal assays

The antibacterial activities of the tested compounds 1–9 were determined using a cup plate method [7].The invitro antibacterial method was carried out using one bacterium (Bacillus subtilis), and four fungal strains (Fusarium solani, Aspergillus niger, Colletotrichum gloeosporioides, and Phomopsis obscurans) and yeast (Candida albicans) were used for the antibacterial assay. Compounds 3 and 9 were assayed for their antifungal activity against A. niger; potato dextrose agar was used in this study. The agar cup (8 mm) diffusion method was utilized in this study [8]. A volume of 200 ml of each compound was dispensed into wells, bored in agar plates, freshly seeded with the tested microorganisms under aseptic conditions. The diameter of the clear zone was recorded after 3 days at 281C. Clotrimazole and sulfamethoxazole were used as the standard antifungal and antibacterial agents as reference drugs, respectively. The inoculated plates were placed in an incubator at 301C for 3

days. One milliliter of the product configuration was dispensed into the first series of sterile test tubes immediately before the test procedure was initiated. Starting with the second sterile test tube using broth medium appropriate for each microorganism, 1 : 2 dilutions developed for the product configuration to achieve a dilution series of 1 : 1, 1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32, 1 : 64, 1 : 128, 1 : 256, 1 : 512, and 1 : 024. A volume of 1.0 ml was left after removing 1.0 ml from the last test tube. A volume of 1.0 ml inoculum of each challenge suspension was introduced into each tube in the series for the product configuration, the dilution containing B5 106 CFUml of challenge microorganism. A positive control tube containing only the appropriate broth and inoculums was prepared for each of the challenge microorganisms. Antifungal assay against Aspergillus niger

The antagonistic activities of compounds 3 and 9 against A. niger were assayed in potato dextrose broth medium. Erlenmeyer flasks containing 100 ml of potato dextrose broth medium containing 0.007, 0.07, and 0.1 mg/ml were inoculated with 2 103 spores of A. niger organism and incubated for 4 days on a rotary shaker (120 rpm) at 281C. The growth of the tested yeast strains was estimated by measuring the optical density at 610 nm. Antibacterial and antifungal activities

The results of antimicrobial activity were tabulated as inhibition zone diameter in millimeter from the pure compounds 2–13 as shown in Table 1. The tested

138


Table 3 Minimum inhibitory concentrations (lg/ml) of the tested compounds 3 and 9 against various microorganisms MIC (expressed as product dilution)

Compounds 3 9 (Clotrimazole) (Sulfamethoxazole)

Bacillus subtilis

Candida albicans


Phomopsis obscurans

Colletotrichum gloeosporioides

Fusarium solani

Aspergillus niger

1 : 32 1 : 16 – 1:8

1 : 64 1 : 32 – 1 : 16

1 : 32 1 : 16 – 1:8

1 : 64 1 : 66 1:8 –

1 : 32 1 : 32 1 : 16 –

1:8 1:8 1:8 –

1 : 64 1 : 66 1:8 –

MIC, minimum inhibitory concentration.

compounds showed high to moderate antibacterial activity against both gram-positive and gram-negative strains of bacteria and yeast at a concentration of 0.007 mg/ml. Compounds 3 and 9 showed the highest activity. It was also observed that compounds 3 and 9 showed activities against Bacillus subtilis, with an inhibition zone of diameter 10.7 and 6.65 mm, respectively. The antibacterial activity observed in this study was concentration dependent. The antifungal activity of the synthesized compounds was studied for the four pathogenic fungi at a concentration of 0.007 mg/ml. Some of the tested compounds showed weak to strong antifungal activity against all three strains of fungi. It was also observed that compounds 3 and 9 showed the highest activity (Table 2). This was observed strongly against A. niger, where an inhibition zone of diameter 15.7 and 9.65 mm, respectively, was observed. The antagonistic activity of compounds 3 and 9 against A. niger was assayed using three concentrations (0.007, 0.07, and 0.1 mg/ml). Ethyl-4(1-benzyl-5-nitro-2-oxoindalin-3-ylideneamino) benzohydrazide (3) was the most active compound that was antifungal. The growth of A. niger was strongly inhibited in the presence of compounds 3 than 9, expressed as spores/ml, biomasses, growth rate per hour, and concentration. Minimal inhibitory concentration measurement

The bacteriostatic activity of the active compounds 3 and 9 was then evaluated using the two-fold serial dilution technique [9,10]. Two-fold serial dilutions of the test compounds and reference drugs solutions were prepared using the proper nutrient broth. The final concentration of the solutions varied between 500 and 7.81 mg/ml, with the concentration of DMF not exceeding 2.5%. Each 0.10 ml from the tested compounds in DMF was mixed with 1, 2, and 3 ml of sterilized distilled water and 0.10 ml from each diluted samples was added to the test tubes. The tubes were then inoculated with the test organisms, grown in a suitable broth at 371C for 24 h for bacteria and 48 h for fungi (about 1 106 cells/ml); each 5 ml received 0.10 ml of the above inoculum and were incubated at 371C for 48 h. The minimum inhibitory concentration for compounds 3 and 9 was significantly higher than those of compound 9 as shown in Table 3. Anti-inflammatory activity and antinociceptive activities Animals

Male Swiss albino CD-1 mice (6–8 weeks old) were obtained from the Schistosome Biology Supply Center, Theodor Bilharz Research Institute (Giza, Egypt), and

were housed under suitable laboratory conditions throughout the period of investigation. Animals were fed standard pellet chow (El-Nasr Chemical Company, Cairo, Egypt) and allowed free access to tap water. Drugs and dosages

Morphine sulfate was administered intraperitoneally at a dose of 10 mg/kg (El-Nasr Pharmaceutical Co.). Indomethacin was administered orally at a dose of 20 mg/kg (El-Kahira Pharmaceutical Co.). Aspirin was administered orally at a dose of 100 mg/kg (Alexandria Pharmaceutical Co., Cairo, Egypt). All tested compounds were suspended in 2% cremophore-El (Sigma Chemical Co., St. Louis, Missouri, USA) and administered orally at a dose of 200 mg/kg. This dose was chosen after determination of the LD50 of compounds 1–13 according to Litchfield and Wilcoxon [11]. Anti-inflammatory activity Carrageenan-induced hind paw edema

A carrageenan-induced hind paw edema model was used for the determination of anti-inflammatory activity [12] 60 min after the oral administration of vehicle, indomethacin, and compounds 1–4 and 6–13, and each mouse was injected with a freshly prepared (0.5 mg/25 ml) suspension of carrageenan in physiological saline (154 nmol/l NaCl) into subplantar tissue of the right hind paw. As a control, 25 ml saline solutions were injected into the left hind paw. Paw edema was measured every 1 h after the induction of inflammation. The difference in footpad thickness was measured using a plethysymometer 7150 (Ugo Basile, Como, Italy). The mean values of the treated groups were compared with the mean values of the control group and analyzed using statistical methods. Indomethacin (20 mg/kg) was used as a reference drug. Antinociceptive activity Acetic acid-induced writhing test in mice

Acetic acid (0.6% v/v, 10 ml/kg) was injected into the peritoneal cavities of mice, which were placed in a large glass cylinder, and the intensity of nociceptive behavior was quantified by counting the total number of writhes that occurred between 0 and 20 min after the stimulus injection, as described earlier [13]. Treatments with vehicle, indomethacin for compounds 1–4 and 6–13, were administered 1 h before acetic acid injection (n = 6/group). Morphine sulfate was administered intraperitoneally


30 min before the test. The writhing response consists of a contraction of the abdominal muscle together with a stretching of the hind limbs. The antinociceptive activity was expressed as writhing scores over a period of 20 min.

Table 4 Antinociceptive activity of the tested compounds 1–4 and 6–13 compared with the reference drug (aspirin) using the acetic acid-induced writhing test

Hot-plate test

Compounds

The hot-plate test was used to measure the response of latencies according to the method described previously by Eddy and Leimback [14], with minor modifications. In this experiment, the hot plate (Ugo Basile; Model-DS37) was maintained at 55 ± 0.21C. The reaction time was noted by observing either the licking of the hind paws or the jumping movements before and after drug administration. The cut-off time was 20 s and morphine sulfate 10 mg/kg (El-Nasr Pharmaceutical Co.) was administered intraperitoneally and used as a reference drug [15]. Formalin-induced nociception

A formalin solution (5% in 0.9% saline; 20 ml/paw) was injected into the hind paw plantar surface (intraperitoneally), and the animals were individually placed in transparent observation chambers, as described previously [16]. Oral treatments with vehicle, indomethacin, and compounds 1–4 and 6–13 were administered 1 h before formalin injection. Morphine sulfate was administrated (intraperitoneally) 30 min before the test. The time spent in licking the injected paw was recorded and expressed as the total licking time in the early phase (0–5 min) and the late phase (20–30 min) after formalin injection. Tail immersion test

The lower two-thirds of the tail were immersed in a beaker containing water maintained at 50 ± 0.51C [17]. The time (s) until the tail was withdrawn from the water was defined as the reaction time. The reaction time was measured at 0, 30, 60, and 120 min after the oral administration of vehicle, compounds 1–4 and 6–13 and morphine (n = 6/group), with the reaction time of 0 min being the start of the test. The mice were exposed to hot water for no longer than 20 s to avoid tissue injury. Statistical analysis

The data obtained were analyzed using the Graph Pad software program Version 4.0 (Inc-La Jolla, California, USA) and expressed as mean ± SE. Statistically significant differences between groups were calculated using an analysis of variance, followed by the Newman–Keuls test. P-values less than 0.05 were considered as significant.

Results and discussion Anti-inflammatory activity

No morbidity or mortality was recorded for any of the tested compounds 1–13; LD50 was found to be 3000 mg/kg body weight as carrageenan-induced hind paw edema model was used for the determination of antiinflammatory activity. After 1 h, the best inhibition was observed after the administration of compound ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1) (53.92%) compared with the indomethacin reference drug

Dose of drug

Control (acetic acid 0.7%/ saline) 1 2 3 4 6 7 8 9 10 11 12 13 Aspirin

Writhing number (count/20 min) (mean ± SE) %Inhibition %Potency

0.01 ml/g

67.00 ± 1.69

–

–

200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 200 mg/kg 100 mg/kg

20.83 ± 0.83* 40.50 ± 3.82* 32.33 ± 2.26* 33.50 ± 5.16* 28.67 ± 3.15* 26.83 ± 2.30* 22.67 ± 2.23* 28.83 ± 2.55* 23.33 ± 4.88* 44.67 ± 3.94* 22.00 ± 1.00* 26.83 ± 4.66* 16.17 ± 1.40*

68.91 39.55 51.74 50.00 57.20 59.95 66.16 56.97 65.18 33.34 67.16 59.96 75.86

90.83 52.13 68.21 65.91 75.41 79.02 87.21 76.08 85.91 43.93 90.16 79.03 100

*Significant difference from the control group at Po0.05. Table 5 Antinociceptive activity of the tested compounds 1–4 and 6–13 compared with reference drug (morphine) using a hot-plate test Compounds Normal control 1 2 3 4 6 7 8 9 10 11 12 13 Morphine

Dose of drug Reaction time (s) (mg/kg) Mean ± SE %Increase %Potency Saline 200 200 200 200 200 200 200 200 200 200 200 200 10

11.00 ± 1.63

–

–

18.50 ± 0.67* 13.83 ± 0.87 14.83 ± 1.58 15.00 ± 1.00 16.17 ± 1.51* 16.17 ± 1.14* 17.00 ± 0.68* 15.67 ± 1.87 16.67 ± 1.09* 13.50 ± 2.29 16.67 ± 2.30* 16.00 ± 1.37* 20.67 ± 1.74*

68.18 25.73 34.18 36.36 47.00 47.00 54.54 42.45 51.54 22.72 51.54 45.45 87.90

77.55 39.60 39.60 41.36 53.46 53.46 62.04 48.29 58.63 25.85 58.63 44.77 100

*Significant difference from the control group at Po0.05.

(50.66%), followed by compounds 3-(4-(3-methyl-5-oxo-4, 5-dihydro-1H-pyrazole-1-carbonyl) phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (8), D-glucose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (10), D-arabinose-4-(-1-(p-methylbenzyl)-5-nitro2-oxoindolin-3-ylideneamino) hydrazone derivative (12) (about 35–37%), and then compounds 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino)] benzohydrazide (3), 3-(4-(4-benzyl-5-thioxo-4,5-dihydro-1H-1,2, 4-triazol-3-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (4), 3-(4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (6), 3-(4-(1,3,4-oxadiazin-6-one)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2-one (7), 3-(4-(5-methyl-1,3,4-oxadiazol-2-yl)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin2-one (9), D-ribose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (13) (22–27%), whereas ethyl 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino)]benzoate (2) and D-mannose-4-(-1(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino) hydrazone derivative (11) showed weak effects (12.5,

– 46.91 12.5 22.75 22.23 27.07 25.75 37.00 23.50 35.24 17.24 37.91 26.28 50.66

23.50 ± 2.03

20.60 ± 2.62

20.74 ± 2.00

19.45 ± 1.82

19.80 ± 3.17

16.80 ± 2.57*

20.40 ± 1.90

17.27 ± 2.02*

22.07 ± 2.70

16.56 ± 3.73*

19.66 ± 2.14

13.16 ± 1.44*

%Inhibition

26.67 ± 3.01 14.16 ± 2.18*

Mean ± SE

*Significant difference from the control group.

Control Saline 1 200 2 200 3 200 4 200 6 200 7 200 8 200 9 200 10 200 11 200 12 200 13 200 Indomethacine 20

Compounds/dose (mg/kg)

%Edema at 1 h

100

51.89

74.83

34.05

69.58

46.41

73.06

50.85

53.44

43.89

44.93

23.46

– 92.59

%Potency

16.75 ± 1.70*

26.90 ± 3.09*

23.08 ± 1.64*

32.32 ± 3.78

21.74 ± 3.26*

27.63 ± 3.17*

22.14 ± 2.41*

25.12 ± 3.10*

27.10 ± 0.84*

28.18 ± 2.31*

26.81 ± 2.56*

30.65 ± 2.34

37.92 ± 2.40 17.68 ± 1.67*

Mean ± SE

55.83

100

52.05

70.10

39.13 29.06

26.45

76.43

48.61

74.54

60.46

51.11

46.01

52.48

34.34

– 95.61

%Potency

14.76

42.66

27.14

41.61

33.76

28.53

25.69

29.30

19.17

– 53.38

%Inhibition

%Edema at 2 h

19.55 ± 1.77*

30.57 ± 3.24*

27.32 ± 1.95*

38.09 ± 6.85

22.57 ± 5.05*

29.69 ± 4.96*

24.45 ± 2.18*

30.02 ± 4.63*

27.72 ± 2.53*

27.84 ± 4.36*

27.58 ± 0.89*

36.12 ± 4.76

47.69 ± 2.68 20.75 ± 1.66*

Mean ± SE

59.01

35.89

42.71

20.13

52.67

37.74

48.73

37.05

41.87

41.62

42.17

24.26

– 56.49

%Inhibition

%Edema at 3 h

100

60.84

72.39

34.12

89.27

63.97

82.59

62.79

70.97

70.54

71.46

41.12

– 95.74

%Potency

14.00 ± 1.63*

20.63 ± 3.30*

18.07 ± 3.40*

25.50 ± 1.71*

17.80 ± 4.59*

21.51 ± 3.07*

19.65 ± 2.03*

23.24 ± 3.47*

23.56 ± 2.36*

19.99 ± 4.76*

20.67 ± 3.11*

31.01 ± 5.64*

41.58 ± 2.80 15.49 ± 2.58*

Mean ± SE

66.33

50.38

56.54

38.67

57.19

48.26

52.74

44.12

43.33

51.92

50.28

25.42

– 62.74

%Inhibition

%Edema at 4 h

100

75.96

85.24

58.30

86.22

72.77

79.51

66.50

65.34

78.28

75.82

38.32

– 94.59

%Potency

Table 6 Anti-inflammatory activities of tested compounds 1–4 and 6–13 compared with the reference drug (indomethacin) using a carrageenan-induced hind paw edema model (n = 6)



Figure 1

17.24%). The highest effect of the anti-inflammatory activity of the tested compounds was observed after 4 h (Table 4). The percentage potency of the anti-inflammatory activity was measured and it was found that compound ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1) had the highest potency (492%) compared with the reference drug indomethacin. Antinociceptive activity

The total number of writhings produced 20 min after the intraperitoneal injection of acetic acid control was about 67.00 ± 1.69. Reduction in the number of writings was found for ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1) (68.91%), then compound 13474649, followed by 344, whereas the least reduction was shown by compound 2 (33%) and 11 (39%). In the hot-plate test (Table 6), compound ethyl 4-(5-nitro2-oxoindolin-3-ylideneamino) benzoate (1) showed increase in the latency time as compared with the control group (Po0.05). The % analgesia of 1 was found to be 77.55% when compared with the reference drug (morphine).

Effect of the synthesized compounds 1–13 administered orally (200 mg/kg) on licking induced by formalin in mice compared with indomethacin (20 mg/kg) or morphine (10 mg/kg) before formalin. The total time spent licking the hind paw was measured (a) in the early phase (0–5 min) and (b) the late phase (20–30 min) after an intraplanter injection of formalin. Each column represents the mean for six mice in each group.

Figure 2

In the tail immersions test shown in Fig. 1, an increase in the tail-flick response latency time was recorded for compound 1 (91.57%), followed by 84124 104749464134443 as compared with the control group (Po0.05), either after 60 or 120 min from drug administration. The least antinociceptive effect was recorded for ethyl 4-[(1-(p-methylbenzyl)-5-nitro-2oxoindolin-3-ylideneamino)] benzoate (2) (29%) and D -mannose-4-(-1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino) hydrazone derivative (11) (27%) when compared with the reference drug (morphine). Figure 2 shows that compound 1 had the best antinociceptive activity compared with the control group in both the early and the late phase (Po0.05). Computational chemistry study

Effect of the synthesized compounds 1–13 administered orally (200 mg/kg) on licking induced by formalin in mice compared with morphine (morph) (10 mg/kg) before tail immersion at 501C. The time of tail withdrawal (s) was measured after (a) 0 and 30 min and after (b) 60 and 120 min. Each column represents the mean for six mice in each group.

HyperChem professional 7.5 programs [18] procedure was used and compared with the experimental data. The results were investigated through regression and correlation analysis, after optimization of geometries, to calculate the thermochemical values for the synthesized compounds 1–9 (part I) and their bond lengths using the semiempirical molecular orbital procedure MNDO [19] (Table 7). Use of the semiempirical method for these types of calculations provides considerable insights into the structure and reactivity of such molecules [20,21]. Different transition structures and reactive pathways were obtained in Fig. 3. From Table 7, we found that compound ethyl 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzoate (1), 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino)] benzoate (2) and 3 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin-3-ylideneamino)] benzohydrazide (3) had the lowest values of total energy and heat of formation respectively, which means that they have higher stability than the other molecules 4, 5, 6, 7, 8, and 9, with higher values of heat of formation. Compound 4-[(1-(p-methylbenzyl)-5-nitro-2-oxoindolin3-ylideneamino)] benzohydrazide (3) is the most stable of the other tested compounds.


Figure 3

Geometry optimization of the studied molecules using the MNDO method (with bond length values).

Table 7 Total energy, dipole moment, and heat of formation for tested compounds 1–9 after optimization using the MNDO method Compounds

Total energy (kcal/mol) Dipole moment Heat of formation (kcal/mol)

1

2

3

4

5

6

7

8

9

– 107 042 1.834 –40

– 133 588 4.633 –48

– 129 503 5.912 –11

– 155 987 8.577 103

– 163 332 9.184 148

– 148 034 5.513 127

– 149 220 6.895 95

– 157457 5.491 45

– 153 125 5.887 121

Conclusion A series of aromatic heterocyclic compounds containing 1H1,2,4-triazol-3-yl)phenylimino)(methylbenzyl)-5-nitroindolin-2-ones, 1H-pyrazole-1-carbonyl)phenylimino)-1-(p-

methylbenzyl)-5-nitroindolin-2-ones, 3-(4-(1,3,4-oxadiazin6-one)phenylimino)-1-(p-methylbenzyl)-5-nitroindolin-2ones, 1,3,4-oxadiazol-2-yl)phenylimino)-1-(p-methylbenzyl)5-nitroindolin-2-ones, 4-(-1-(p-methylbenzyl)-5-nitro-2-ox-


oindolin-3-ylideneamino) sugar hydrazone derivatives (part1) were evaluated for their antibacterial, antifungal, anti-inflammatory, and antinociceptive activities. It was found that compounds 3 and 9 were the most active in terms of antibacterial and antifungal activities, with paramethyl substitution at the benzyl ring and para-hydrazide moiety or the oxadiazol-2-yl ring, in addition to the nitro substitution in the 5-position of oxoindoline-2-one, enhancing the antibacterial and antifungal properties. Compounds 1, 8, 10, and 12 showed significant antiinflammatory and antinociceptive activities compared with the reference drugs. Using the semiempirical molecular orbital procedure MNDO, compounds 1, 2, and 3 were found to be the most stable compounds.

Acknowledgements The authors are very grateful to the National Research Center, Cairo, Egypt, for the generous financial support.


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144

Original article

The efficacy of Silybum marianum (L.) Gaertn. (Silymarin) in the treatment of physiological neonatal jaundice: a randomized, double-blind, placebo-controlled, clinical trial Lamyaa M. Kassema, Mohamed E.A. Abdelrahima and Hassan F. Naguibb a Department of Clinical Pharmacy, Faculty of Pharmacy and bDepartment of Pediatrics, Faculty of Medicine, Beni-Suef University, Egypt

Correspondence to Lamyaa M. Kassem, Department of Clinical Pharmacy, Faculty of Pharmacy, Beni-Suef University, 82524 Beni Suef, Egypt Tel: +002 010 0482 2858; fax: +002 023 567 6109; e-mail: [email protected] Received 9 May 2012 Accepted 27 August 2012 Egyptian Pharmaceutical Journal 2012, 11:144–149

Back ground and aim of work Unconjugated hyperbilirubinemia (UCB) is one of the most common conditions in neonates. Conventional treatments are phototherapy and exchange transfusion. Phototherapy is safe and effective, but it has several disadvantages, which indicates the need to develop alternative pharmacological treatment strategies. These alternative treatment strategies should be less invasive and at least as effective and safe as phototherapy. The present study was designed to investigate the effects of Silybum marianum (silymarin) on the duration of phototherapy, which is known to have antioxidant, anti-inflammatory, hepatic-protective, and regenerative properties, including enhancing glucuronidation activities. Patients and methods A randomized double-blind clinical trial was conducted on 170 full-term healthy neonates with UCB divided into two well-matched groups. Of the 170 neonates, 85 received 3.75 mg/kg of silymarin orally, twice daily, in addition to phototherapy, and 85 received placebo and phototherapy. Total serum bilirubin was measured every 24 h, and alanine aminotransferase (SGPT) and alanine transaminase (SGOT) levels were measured before and after therapy in both groups. Results The mean duration of phototherapy was found to be significantly reduced from 5.3 ± 0.82 days in the control group to 4.2 ± 0.76 days in the silymarin-treated group (P = 0.001). SGPT and SGOT levels were significantly normalized (P = 0.001). Conclusion Silymarin at a dose of 3.75 mg/kg twice daily along with phototherapy was more effective than phototherapy alone in treating full-term healthy neonates with UCB. Keywords: bilirubin, neonatal jaundice, phototherapy, silymarin Egypt Pharm J 11:144–149 & 2012 Division of Pharmaceutical and Drug Industries Research, National Research Centre 1687-4315

Introduction Neonatal hyperbilirubinemia is the most common clinical symptom in neonatal medicine; it is usually physiological but only rarely is it associated with bilirubin neurotoxicity or with significant underlying disease. It reflects accumulation of a yellow–orange pigment of bilirubin in the skin, sclera, and other mucous tissues of the neonate. The serum bilirubin level is increased because of imbalance between the production and elimination of bilirubin [1]. When the breakdown of erythrocytes and heme-containing protein is accelerated, the liver is unable to function adequately to metabolize the extra load of bilirubin produced [2]. It was shown in the study by Tazawa et al. [3] that 31% of breast-fed infants with jaundice had at least one item of abnormal liver function that may suggest mild hepatic dysfunction, decreasing bilirubin elimination. Newborns appear jaundiced when the serum bilirubin level is greater than 7 mg/dl [4]. Significant elevation of serum bilirubin levels can result

in brain damage, known as kernicterus, which is a life-long neurologic sequelae and may lead to death [4]. Treating indirect hyperbilirubinemia at the appropriate time is of high importance in neonates. The intensity and invasiveness of therapy are determined by many factors such as gestational age, relative health of the neonate, total serum bilirubin (TSB), and etiology of jaundice. Phototherapy and exchange transfusion are two main interventions that are used to decrease TSB. Phototherapy has several disadvantages. Most notably, short-term phototherapy does not always decrease plasma UCB to nontoxic levels in neonates, whereas long-term phototherapy, such as that needed for patients with Crigler–Najjar disease, becomes less effective with age and has a profound impact on social life. Under conditions of very severe unconjugated hyperbilirubinemia (UCB) or hyperbilirubinemia with an insufficient response to phototherapy, a ‘rescue’ treatment consists of exchange transfusion in which the hyperbilirubinemic blood is removed and is replaced with nonjaundiced blood. Exchange transfusion,


DOI: 10.7123/01.EPJ.0000421667.70605.8a

Silymarin in treatment of neonatal jaundice Kassem et al. 145

however, has considerable morbidity, especially in sick preterm newborns; mortality has also been reported [5,6]. The potential neurotoxicity of UCB and the disadvantages of the present treatments have prompted the investigation into and development of alternative pharmacological treatment strategies for UCB. These alternative treatment strategies should be less invasive and at least as effective and safe as phototherapy. Pharmacological agents used in the management of hyperbilirubinemia can accelerate bilirubin clearance through the normal metabolic pathways, inhibit the enterohepatic circulation of bilirubin, or interfere with bilirubin formation either by blocking the degradation of heme or by inhibiting hemolysis [5,6]. Metalloporphyrin [7], D-penicillamine [8], phenobarbital, and clofibrate [8] are pharmacological agents that can be used in the management of hyperbilirubinemia. Herbal therapy, including silymarin, has recently received special attention as a mode of complementary therapy. Silymarin is a flavonoid complex that is extracted from seeds of milk thistle (family: Asteraceae/Compositae) [9]. This has been approved by FDA as a herbal medicine and has been indicated as a dietary supplement. It has been widely used in traditional European medicine as a liver tonic for almost 2000 years[10]. The main component of the silymarin complex is silybin [11]. The extracts are still widely used to protect the liver against toxins and to control chronic liver diseases, hepatic viruses, fibroses, and jaundice. Recent experimental and clinical studies have suggested that milk thistle extracts also have anticancer, antidiabetic, cardioprotective, and antihypercholesterolemic effects and induce the flow of breast milk [9,12]. Milk thistle extracts are known to be safe and well tolerated. Toxic or adverse effects, observed in the reviewed clinical trials, seem to be minimal [9,13]. Attempts to decrease the risk of hyperbilirubinemia should be directed at the early establishment of effective lactation and at adequate caloric intake [14]. No clinical trials examining the effect of silymarin in the treatment of neonatal jaundice have been completed in neonates. However, it is used safely in the treatment of neonatal lupus erythematosus with cholestatic hepatitis [15]. The aim of the present study was to investigate the efficacy of silymarin as an adjunct therapy that decreases the duration of phototherapy for treatment of neonatal jaundice.

enrolled into this study and randomly assigned to one of two study groups. All infants were consecutively studied by one blinded investigator after informed parental consent had been obtained. The study group received phototherapy and silymarin [n = 85 (40 girls)], and the control group received phototherapy and placebo [n = 85 (33 girls)]. Inclusion criteria: (1) Patients who fulfilled the criteria of the 2004 American Academy of Pediatrics guidelines for the treatment of hyperbilirubinemia using phototherapy [16]. (2) Healthy neonates with UCB, nonhemolytic jaundice, and who did not require an urgent exchange transfusion. (3) Healthy near-term and full-term newborns with a gestational age of 38–42 weeks, having jaundice at the age of 1–10 days. (4) Those with negative results for the direct coombs test. Exclusion criteria: (1) Newborns with birth weight less than 2500 g. (2) Prior or current use of phenobarbitone by the mother or the child [17]. (3) Initial indication of double or triple phototherapy. (4) Newborns subjected to blood transfusions. (5) Newborns with congenital defects, hereditary disease of erythrocytes, or autoimmune diseases with intense hemolysis. (6) Newborns with conjugated hyperbilirubinemia or with any disease other than jaundice (severe sepsis, pneumonia, respiratory distress, anemia, etc.). (7) Newborns with ABO or Rh incompatibility. (8) Newborns with decreased levels of glucose-6phosphate dehydrogenase. After the initial selection, the neonates were excluded from the research if any of the following criteria were met: (1) Spectral irradiance below 4.0 mW/cm/nm was registered for any of the measurements for phototherapy calibrations. [18] (2) The modality of phototherapy was changed to double or triple. (3) Determination of TSB was technically or clinically impossible. (4) Death occurred during the period of phototherapy.

Patients and methods A blind, randomized, placebo-controlled clinical trial was conducted at Doctor Abdu Al-Naser Badawy’s Neonatal Intensive Care Clinical Center in Sohag, Egypt. Approval from the local ethical committee was obtained for the study protocol and all patients were subjected to through history taking and clinical examination before enrollment. A total of 170 (73 girls) healthy, full-term neonates were

The inclusion criteria for starting phototherapy according to American Academy of Pediatrics and to stop phototherapy according to internal guidelines depended on TSB, age, and gestational age of the neonate. All the criteria of inclusion and discharge were the same for both control and silymarin-treated groups. No infant was excluded from either group during this study.

146


Laboratory tests

The laboratory technician was blinded to the patient group.

Table 1 Basic clinical data and risk factors for jaundice in the two study groups Silymarin-treated group

Variables

TSB was measured on admission, after 12 h of admission, and then every 24 h until discharge. Aspartate aminotransferase (SGOT) and Alanine aminotransferase (SGPT) were measured in all neonates both before and after the study. A dose of 3.75 mg/kg of silymarin syrup was administered orally, twice daily, to the infants in the silymarin-treated group within 12 h of admission. Laboratory tests including determination of complete blood count, total and direct serum bilirubin levels, reticulocyte count, maternal and neonatal blood groups, and glucose-6-phosphate dehydrogenase level; direct Coombs agglutination test and peripheral blood smear were also performed and recorded routinely before initiating therapy in all jaundiced infants in both groups. Total and direct serum bilirubin levels were measured daily until phototherapy was discontinued. Phototherapy was initiated immediately on admission for both patients and controls until TSB decreased to a safe level according to the internal guidelines, which depended on the infant’s gestational and postnatal age. A nurse who was not involved in drug administration recorded the duration of phototherapy. Each phototherapy unit consisted of eight special white fluorescent tubes labeled TL 52/20w (Philips, Eindhoven, the Netherlands) adjusted 20 cm above the infant. During the study, all neonates underwent careful physical observation for any symptoms such as vomiting, loose stools, skin rashes, and hyperthermia. Laboratory tests were conducted 48 h and 1 week after discharge and included complete blood count and TSB for detection of rebound hyperbilirubinemia. Lamps of phototherapy units were changed regularly after 15:00 h of usage to maintain irradiance in the photoeffective range. TSB measurements were taken on the basis of spectrophotometric principles using Bilimeter3 (Pfaff Medical GmbH, Germany). Direct bilirubin measurement was taken using Autoanalyser Random Access (Selectra E; Vital Scientific, the Netherlands). The equipments were standardized periodically. All data were analyzed using SPSS V15.0 (SPSS Inc., Chicago, Illinois, USA). Statistical analysis of the data was carried out using Student’s t-test for between group comparisons and the paired t-test for within group comparisons. P-values less than 0.05 were considered significant for all checked results.

Age [mean (SD), days]

(2–7), 3.69 (2.488)* (38–42), 38.94 (4.2)**

Gestational age [mean (SD), weeks] Sex [N (%)] Male 45 (53) Female 40 (47) Mode of delivery [N (%)] Normal 55 (65) Cesarean section 30 (35) Consanguinity [N (%)] Yes 62 (73) No 23 (27) Feeding [N (%)] Formula 37 (44) Mixed 48 (56) Hyperbilirubinemia in previous siblings [N (%)] No previous sibling 10 (12) Present 47 (52) Absent 28 (36)

Control group (2–8), 3.54 (2.58)* (38–42), 39.01, (2.8)** 52, (61) 33, (39) 56, (66) 29, (44) 60 (71) 25 (29) 33 (39) 52 (41) 7 (8) 56 (66) 29 (26)

*P-value = 0.695 (insignificant). **P-value = 0.904.

Table 2 Main result of the study in both control and silymarin-treated groups Variables

Minimum Maximum

TSB Control 8.79 Silymarin-treated 9.43 Duration of phototherapy Control 84 Silymarin-treated 64 SGOT before Control 23 Silymarin-treated 17 SGOT after Control 34 Silymarin-treated 63 SGPT before Control 10 Silymarin-treated 10 SGPT after Control 15 Silymarin-treated 12

24.38 24.35 168 132 91 89 89 123

Mean ± SD

P-value

15.38 ± 3.61 15.26 ± 3.80

0.837

127.47 ± 19.61 o0.001* 100.66 ± 18.30 50.67 ± 14.55 48.35 ± 17.04

0.342

58.88 ± 14.68 o0.001* 94.84 ± 14.26

31 35

19.51 ± 4.84 19.80 ± 5.84

0.721

35 43

24.80 ± 4.53 35.15 ± 5.23

o0.001*

SGOT, alanine transaminase; SGPT, alanine aminotransferase; TSB, total serum bilirubin. *Significant at 0.001 level.

The mean duration of phototherapy was significantly lower in the control group compared with the silymarintreated group (Po0.01). Both SGPT and SGOT were significantly increased (Po0.01) in the silymarin-treated group at the end of therapy, where as it was increased insignificantly at the end of therapy in the control group.

Results A total of 170 neonates (73 girls) completed the study. Table 1 shows the characteristics and clinical data of the control and silymarin-treated groups collected before therapy. Table 2 shows the mean ± SD and statistical significance of both the control and silymarin-treated groups; no significant differences in age, gestational age, mean TSB, SGPT, and SGOT at the time of admission of neonates were noted between the groups.

As shown in Fig. 1, the reduction rate (the amount removed per unit time) of total and indirect plasma bilirubin levels was significantly higher in the silymarintreated group compared with the control group. Asterisks at 60 and 84 h of life in Fig. 1 are signs of significance. The difference in mean TSB between the two groups became significant (Po0.05) on day 3 of therapy. Table 3 shows a comparison between the number and percentage


of symptoms recorded during therapy in both groups. During the study, two cases of rebound hyperbilirubinemia were recorded in the control group.

Discussion Jaundice is the most common condition that requires medical attention in newborns. Conventional treatment for jaundice includes phototherapy and exchange transfusion in severe cases. These therapeutic modalities have various and serious adverse effects. Development of intensified phototherapy units and the use of drugs have contributed significantly to a reduction in the need for exchange transfusion, which is associated with a high risk of morbidity and mortality. Efficacy of phototherapy needs a lot of precautions to justify the required minimal effective dose. Hence, numerous newborns continue to be subjected to subtherapeutic doses of phototherapy, which may lead to neurological sequelae that may not be detected in childhood [18,19]. Several pharmacological drugs are used to treat neonatal jaundice [8]. The belief that natural medicines are safer compared with synthetic drugs has gained popularity in recent years and has led to tremendous growth in phytopharmaceutical usage [20]. Physiological jaundice in neonates may be because the liver is unable to function adequately, and hence there is a need to support liver function [2]. Silymarin is a natural herbal supplement that supports liver activity with evidence of a wide margin of safety. It has several Figure 1

Mean total serum bilirubin (mg/dl) measured every 24 h throughout the duration of therapy in the two groups. **Highly significant P-value.

mechanisms of action that may contribute to reduction of serum bilirubin. No study has been published previously on the use of silymarin in the treatment of neonatal jaundice. Silymarin was used in the treatment of a neonatal lupus erythmatosus with cholestatic hepatitis [15]. It has several mechanisms of action, one or more of which can reduce the TSB level. It can enhance glucuronidation [21–23], inhibit reabsorption of bilirubin by enterohepatic circulation through its mild laxative effect [12,24,25], and stimulate ribosomal RNA polymerase and subsequent protein synthesis and thus enhance hepatocyte regeneration, which may drive the liver to function adequately to metabolize bilirubin. It has an antioxidant effect that may resemble the adaptive role of physiological neonatal jaundice in scavenging reactive oxygen species. It also has the ability to regulate membrane permeability [21], thus increasing membrane stability and decreasing excess heme metabolism by stabilizing RBCs. Orally administered syrups with enhanced bioavailability was used in the study, and thus we were able to avoid contamination of herbal medicines by heavy metals, microbial toxins, and other contaminants. Silymarin increased the incidence of loose stools with phototherapy, which may have a beneficial effect in lowering hyperbilirubinemia. In the present study, there was increased incidence of jaundice and increased duration of therapy in breast-fed infants and increased incidence of hyperbilirubinemia in previous siblings. Hence, breastfeeding and hyperbilirubinemia in previous siblings might be considered risk factors for neonatal jaundice. There was no correlation between sex or blood group of the neonate and appearance of jaundice or duration of therapy. The duration of phototherapy and hospitalization was significantly shorter in infants treated with silymarin in addition to phototherapy in comparison with that in those treated with only phototherapy. As shown in Fig. 1, TSB was significantly decreased on day 3 of silymarin therapy. No important side effects were identified during the short-term follow-up. Statistics demonstrated that the duration of phototherapy was significantly reduced from 5.3 ± 0.82 days in the control group to 4.2 ± 0.76 days in the silymarin-treated group (P = 0.001). SGPT and SGOT liver function tests were used in previous studies to indicate the safety of certain drugs with regard to the

Table 3 Number and percentage of symptoms that appeared during the duration of therapy in the control and silymarin-treated groups N (%) Other symptoms Number of neonates who showed no other symptoms during study Skin rash (from mild to severe) Vomiting Hyperthermia *Significant at 0.01 level.

Silymarin-treated group 24.0 7.0 21.0 8.0

(28.2) (8.2) (24.7) (9.4)

Control group 16.0 36.0 46.0 12.0

(18.8) (42.4) (54.1) (14.1)

P-value 0.205 0.001* 0.001* 0.475

148


liver of the neonate, such as the safety of paracetamol on neonatal liver [26]; in addition, these tests were used in the follow-up of cholestasis in neonates [27,28] and to evaluate the efficiency and health of the liver [29]. In the silymarin-treated group, the initial values of SGPT and SGOT before therapy were either lower than the normal range or at the lower limit. At the end of therapy, the mean SGPT and SGOT values were found to have increased significantly to higher values within the normal range. In the control group there was no significant increase in mean SGPT and SGOT values. This may indicate better activity of the liver, which means that silymarin can normalize SGPT and SGOT [30], which was concluded after the statistical analysis and on the basis of the significant increase in SGPT and SGOT values in the silymarin-treated group. In addition, there was little significant Pearson’s correlation between SGPT and duration of therapy (r = 0.23, P-value = 0.032) and weak highly significant Pearson’s correlation between SGOT and duration of therapy (r = 0.43, P-value = 0.001) in the silymarin-treated group. This correlation was not found in the control group. No serious side effect was observed during the duration of therapy with silymarin. Similar to phenobarbital, silymarin also enhances bilirubin conjugation and excretion [21–23] and is a better herbal drug with a wide margin of safety. Phenobarbital has a long half-life [31], and many factors can affect the clearance of phenobarbital during the neonatal period [32]. In the study by Heiman and Gladlk, phenobarbital half-life was significantly longer in neonates (118.6 ± 16.1 h) [33]. This means that its half-life may reach more than 2 days, whereas the clearance half-life of silymarin is 6–8 h [21,34]. Phenobarbital also causes drowsiness in neonates and may slow down the oxidation of bilirubin in the brain, leading to more severe bilirubin toxicity [8]. Silymarin reduced and restored the phenobarbitone-induced sleeping time [35]. Table 3 shows that silymarin significantly reduced the incidence of skin rash as a side effect of phototherapy and also significantly decreased the incidence of vomiting in neonates.

Conclusion Silymarin at a dose of 3.75 mg/kg, twice daily, along with phototherapy is more effective than phototherapy alone in treating full-term healthy neonates with UCB. Further studies are required to fully understand silymarin’s role in the treatment of neonatal jaundice, its most effective dose, and the possibility of it being used as a mode of prophylactic therapy and for managing pathological neonatal jaundice.

Acknowledgements The authors thank Professor Hassan F. Naguib for his keen supervision, judicious guidance, and unlimited support. They thank Dr Mohamed Emam Abdelrahim for his kind help, fruitful directions, and constructive criticism during supervision. They are deeply grateful to the Neonatal

Intensive Care Clinical Center of Dr Abd Alnaser Badawy for providing the financial support and facilities necessary for carrying out this study.


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