مجلة المنصورة للعلوم الصيدلية
MJPS VOL. 27 (2) 2012 1-10
Mansoura Journal of Pharmaceutical Sciences
Synthesis and In Vitro Activity of Novel Non-Nucleoside Derivatives as Anti-HCV Agents Amany S. Mostafa,* Serry A. El Bialy, Waleed A. Bayoumi and Ali M. Abdelal Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt. KEYWORDS Tetrahydropyrimidines, pyrrolo[3,4b]pyridines, non nucleoside anti HCV agents
Abstract: New series of 1,2,3,4-tetrahydropyrimidines (9a-e, 11a-c) and 3,4,6,7tetrahydro-1H-pyrrolo[3,4-b]pyridines (14a-d) were synthesized and investigated for their potential anti-HCV activity, using HCV virus model (Vesicular Stomatitis Virus VSV), compared to interferon. The target derivatives 9a-e were synthesized in moderate to good yield through the acid-catalyzed Biginelli condensation reaction, whereas compounds 11a-c were obtained by esterification of 6bromomethyltetrahydropyrimidine 10 with the appropriate aromatic acid salt. Pyrrolo[3,4-b]pyridines (14a-d) were synthesized via conversion of lactone ring in compound 13 into lactam through condensation with different amines. The structures of the newly synthesized compounds were confirmed by different spectral and analytical techniques. It was found that tetrahydropyrimidine (9b) and tetrahydro1H-pyrrolo[3,4-b]pyridine-2,5-dione (14c) showed the highest activity. Compounds 11c, 11b, 14a and 14d showed moderate activity, while compounds 9d, 11a and 14b showed the least antiviral activity among the series. Received: June 27, 2011 ; Accepted: August 15, 2011
Introduction: Hepatitis C virus (HCV) infection is a serious and predominantly chronic disease, which over time leads to cirrhosis and fibrogenesis.1 It remains a global health threat with about 175 million carriers representing approximately 3% of the worldwide population.2 HCV infection increases the risk of developing serious lifethreatening end-stage liver failure and hepatocellular malignancy, which eventually requires liver transplantation.3,4 Unfortunately, till date HCV has an unmet medical need for therapies that target disease progression. Specifically targeted antiviral therapies against HCV include protease inhibitors, nucleoside inhibitors and non-nucleoside inhibitors.5 Pyrimidine ring was proved to serve as the core structure for several molecules designed to meet antiviral activity. Pyrimidines and annelated pyrimidines attracted interest since 1961, when Nitracin (1) displayed good activity against the viruses of the trachoma group.6,7 Besides, 2amino-4,6-dichloropyrimidine was revealed to be the most active among pyrimidine derivatives on the growth of DNA and RNA viruses.8 HAP-1 (2)9 and Bay 41-4109 (3),10 carrying dihydropyrimidine scaffold, were proved to be highly potent non nucleoside inhibitors of HBV replication with *Corresponding author: e-mail:
[email protected]
in vitro and in vivo antiviral activity. (Figure 1). 6-Substituted pyrimidines, like 1-[(2 hydroxyethyl) methyl]-6-(phenylthio)thymine (HEPT) (4), Emivirine (EMV) (5), N1-ethyl pyrimidinediones (6), 3,4-dihydro2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs) (7),11,12 and S-DABOs (8), were reported as potent and selective inhibitors of HIV reverse transcriptase (RT).13 HCV polymerase and HIV RT were reported to share a common mechanism of action.4 This suggests the possibility that nucleoside or non nucleoside analogs may be designed to fit the RNA polymerase of HCV and the DNA polymerase of HIV RT.14,15 The essential role of polymerase enzyme for viral replication16 and the success obtained in the clinic with inhibitors of HIV RT and HBV DNA polymerase17,18 have prompted many laboratories to search for inhibitors of the HCV polymerase that could be used in the therapeutic intervention against the virus. In this work, rational design for the target compounds was planned through optimization of pyrimidine scaffold that proved to have antiviral activity via structural modification aiming for potential anti HCV activity. In addition, a series of pyrrolo[3,4-d]pyridine-2,5-diones was designed to investigate the effect of isosteric replacement of pyrimidine with pyridine moiety on the 1
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MJPS VOL. 27 (2) 2012 1-10 anti HCV activity. Besides, removal of one nitrogen atom from the pyrimidine moiety might potentially lead to lower polar surface areas (PSA) values of the resulting pyrrolopyridine;19 and as a consequence, might improve permeability, physicochemical properties, pharmacokinetic profiles, and potency relative to the original series.
CH3COONH4 and EAA in a one-pot four-component reaction27 to synthesize dihydro-2(1H)-pyridone (12). The synthesis of γ-lactone fused 1,4-dihydropyridones was also afforded in a one-pot synthesis, through reacting the dihydro-2(1H)-pyridone (12) and Nbromosuccinimide (NBS) as brominating agent in refluxing chloroform. Lactonization could be accounted F
O 2N O MeO
O
O C
NH N H
MeO
N
O R
N
R3
O R1
R Comp. R 4 5 6
R1
R R2
R
O NH
3
N
2: R = H 3: R = F
R X
R
N H
O
1
R
Cl
NH N
2 X R
R1
2
R3
H O(CH2)2OH H H CH3 OCH 2CH 3 H H CH3 CH3 CH3 CN
X S CH2 CO
Comp. 7 8
R1
R
H, Me, Et H, Me, Et, i -Pr
H, 3-Me, 3,5-Me H, 3-Me, 2,6-F2
R2
R3
X
alkyl, cycloalkyl H O alkyl, cycloalkyl H, Me S
Figure 1. Different antiviral agents containing pyrimidine ring
Results and Discussion Chemistry The target compounds 9a-e were synthesized by two different methods to evaluate the resulting yield. The first depends on the classical acid-catalyzed one-pot multicomponent reaction of the appropriate β-keto ester with (un)substituted urea or thiourea and (un)substituted benzaldehyde using concentrated HCl as a catalyst and EtOH as a solvent.20-22 Solvent-free condition was adopted in the second method, according to Yu et al23 and proved to be better yielding. Therefore, compounds 9a-d were prepared by using chloroacetic acid (CAA) as a catalyst in neat condition (scheme 1).
for by the allylic bromination at the methyl group to yield a non isolable bromo-intermediate, followed by nucleophilic cyclization28,29 (scheme 3). The lactone ring in hexahydrofuro[3,4-b]-2(IH)-pyridone 13 was converted into lactam 14a-d by boiling with different amines, using the proper solvent.
The bromination of methyl group was achieved by using stoichiometric amounts of elemental bromine in chloroform, with slow addition to avoid the formation of gem-dibromo derivative, provided the bromomethyldihydropyrimidine derivative 10.24 The carboxylic acid esters 11a-c were obtained by esterification of the 6-bromomethyltetrahydropyrimidine 10 with the appropriate sodium benzoate derivative (scheme 2). Meldrum’s acid25,26 (2,2-dimethyl-1,3-dioxane-4,6dione) was reacted with a mixture of benzaldehyde, 2
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MJPS VOL. 27 (2) 2012 1-10
R1
R1
O
O CHO
EtO R
O
method 1, 2
EtO R
NH 2
HN R2
NH
X
N R2
X
R1
9
x
a b c d e
O CH3 O CH 3 O CH3 O CH 3 S C6 H5
R
R2
H CH 3 CO 2H H OCH 2CO2 H H CO 2C 2H 5 CH3 CO2 C 2H 5 H
9a-e
Scheme 1: Reagents and conditions: (method 1): EtOH, conc.HCl, ref lux, 3h; (method 2): CAA, 90°, 3h
O O
O EtO
NH N
(a)
EtO
EtO
NH N
O
NH
(b)
N O
O
O
Br
9a
O
a b c
R 10
11
R H OH COOH
11a-c
Scheme 2: Reagents and conditions: (a) Br2 , CHCl3 , 5h; (b) 2-R-PhCOONa, DMF, 60°, 12h
CHO O
O O
O (a)
O
O
O (b)
EtO
O
N H
O
EtO BrH2 C
O
N H
O
12
O
O
(c) R
O N H 13
N N H
O
O
14a-d R = H, CH 3, Cl, OCH 3
Scheme 3: Reagents and conditions: (a) HOAc, NH4 OAc, reflux, 6h, 70%; (b) NBS, CHCl3 , reflux,12 h, 55%; (c) (ArNH 2, g.HOAc, reflux, 48 h for comp. 14a-c) or (ArNH2 , dry toluene, ref lux, 36 h f or comp. 14d)
3
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MJPS VOL. 27 (2) 2012 1-10 Biological screening The newly synthesized compounds have been subjected to preliminary biological screening for their potential in vitro anti-HCV using HCV virus model (Vesicular Stomatitis Virus VSV), compared to interferon. Biological screening was performed in the applied research sector of the holding company for biological products, vaccines and drugs (Egy-Vac/ VACSERA). Cells and Viruses African green monkey kidney (Vero) clone CCL-81 was kindly supplied from Cell Culture (CC) Department, VACSERA – Egypt. Cells were grown in 199 E-Hepes buffer (GIBCO-UK) growth medium was supplemented with 10% inactivated fetal calf serum (FCS), 5mM HEPES buffer and antibiotics (100 U of penicillin / mL, 100 gm of streptomycin / mL) at 37 °C and incubated in 5% CO2 atmosphere. Vesicular stomatitis virus (VSV), Indiana strain was kindly supplied by Applied Research Sector, VACSERA-Egypt. Virus infectivity titer was determined according to reported method,30 The VSV was 10 fold serially diluted. Each dilution was dispensed as 100 µL/ well onto pre-culture Vero cells. Non infected wells as negative control were considered. Plates were incubated at 37 °C. 7 Days post infection the 50 % end point (50% cell culture infective dose CCID50) was determined.31 Cytotoxicity assay Tested organic compounds were dissolved in 50% ethanol in the presence of 0.02 µL tween 20 as a surfactant. Test materials were 0.2 µm membrane filtered using Millipore disposable syringe filters (MilliporeUSA). Cytotoxicity assay of synthetic organic compounds compared to sterile r-hIFN-α2a (Galaxo Smithkline-Italy) was performed according to the previous reports,32-35 where 103 µM/mL sterile filtered organic test materials were 13 fold serially diluted in Eagle minimum essential medium (MEM-E)and added to precultured Vero cells. Negative cell culture control was considered.
Plates were incubated at 37 °C for 24 h. Cell culture treated plates were microscopically examined using inverted microscope for detection of cellular changes or cytotoxicity. Test compounds treatment medium was discarded. Plates were washed using PBS. Cell viability was measured using 3-(4,5- dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) method. 36 The cytotoxicity of each compound was expressed as the 50% cytotoxic concentration (CC50), which is the concentration, required for reducing cell viability to 50% of the control. Viability percentage was determined as follows, since OD is the Optical Density: Number of residual living cells = (OD of treated cells/ OD of untreated cells) X number of negative control cells (104 cells/ 0.1 mL). Percentage viability = (number of residual living cells / number of negative control cells) X 100. Cytotoxicity assay results for compounds 9b,d,e, 11a-c and 14a-d are listed in (Table 1). Antiviral assay Antiviral activity of test compounds and interferon against VSV was determined,37 where non toxic concentrations of test chemical compounds were compared to interferon; as a currently used antiviral agent; used as 10 IU/ mL to treat pre-cultured Vero cells for 24 h as 0.1 mL / well. One plate was maintained and left untreated for viral control titration. VSV was 10 fold serially diluted in 199 E-Hepes buffer (10-1 - 10-8). Antiviral activity was determined by subtracting the VSV mean titer in treated and non treated cells. The difference between both titers refers to the antiviral activity.38 Synthetic compounds were tested for antiviral activity and cytotoxicity against HCV virus model (VSV) compared to IFN–α2a (Mena pharm – Egypt, 3 X 106 IU/ mL) according to previously published procedures. 39-41 Evaluation was based on cell treatment with test compounds for 24 h for expression of genes responsible for virus inhibition. Antiviral assay results for compounds 9b,d,e, 11a-c and 14a-d are listed in (Table2).
Table 1: Cytotoxicity assay results for compounds 9b,d,e, 11a-c and 14a-d Test compounds (% Viability) Conc.µM/mL 9b
9d
9e
11a
11b
11c
14a
14b
14c
14d
500
7
7
5
13
10
9
11
14
5
10
250
22
9
9
17
14
14
15
17
7
28
125
25
13
12
21
17
17
22
24
11
34
62.5
68
65
25
39
37
38
39
38
44
51
31.5
99
98
38
88
86
88
89
88
89
82
15
99
99
82
98
99
99
99
98
99
100
7.5
98
99
100
99
99
100
100
101
101
101
3.25
101
102
101
103
102
101
102
99
100
101
1.625
100
101
99
101
101
100
101
100
100
102
0.8125
101
100
98
101
101
99
99
99
101
99
0.406
101
100
100
101
99
100
99
101
100
0.2
101
102
102
99
100
100
102
101
100
101 4 101
0.1
100
99
101
101
101
102
101
102
100
99
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MJPS VOL. 27 (2) 2012 1-10 Table 2: Antiviral assay results for compounds 9b,d,e, 11a-c and 14a-d Test Conc Virus titer Post Safe dilution material µM/ 0.1mL Vero cell treatment 3.125 32 6 9b 50 safe 6.57 9d 3.125 16 6.89 9e 6.25 16 6.57 11a 3.125 32 6.49 11b 3.125 32 6.4 11c 3.125 16 6.5 14a 3.125 16 6.57 14b 6.25 32 5.57 14c 6.25 16 6.5 14d Control 6.89 (0 point virus titer) 10 IU/ mL 0.89 IFN–α2a Experimental Section Melting points were (°C) were recorded using FisherJohn melting point apparatus and are uncorrected. IR spectra were recorded on Mattson 5000 FT-IR spectrometer (υ in cm-1) using KBr disc at Cairo University and at Faculty of Science, Mansoura University. NMR spectra were recorded on a Bruker Avance spectrometer (400 MHz-1H and 100.0 MHz-13C) in DMSO-d6 or CDCl3 at Georgia State University, Atlanta, GA 30303-3083, USA. Chemical shifts in ppm are given as δ values against TMS as the internal standard. MS analyses were performed on JOEL JMS600H spectrometer in Cairo University. Microanalyses were performed by the microanalytical unit, Cairo University. Reactions were monitored by TLC performed on silica gel plates (Merck 60 F250) and using ethyl acetate: petroleum ether (1:1) as eluent. General procedure for the synthesis of ethyl 1-methyl2-oxo (thioxo)-4-phenyl-6-substituted 1,2,3,4tetrahydropyrimidine-5-carboxylate (9a-e) Method 120 To a mixture of the appropriate aromatic aldehyde (1 mmol), the appropriate β-keto ester (1.2 mmol) and (un)substituted urea or thiourea (1.2 mmol) in EtOH (20 mL), conc. HCl (5 drops) was added. The mixture was heated at reflux for 3 h. The solution was allowed to stand at 0 °C for 1 hour, the resulting precipitate was filtered and recrystallized from EtOH to yield the target compounds 9a-e. Method 223 A mixture of the appropriate aromatic aldehyde (25 mmol), the appropriate β-keto ester (27.5 mmol), urea (37.5 mmol) and chloroacetic acid (CAA) (0.24 g, 2.5 mmol) under solvent-free conditions was heated at 90 °C for 3 h. After cooling, the reaction mixture was poured into crushed ice and stirred for 5-10 min. The solid was filtered, washed with ice-cold water and recrystallized from EtOH to afford pure product of compounds 9a-d. Ethyl1,6-dimethyl-2-oxo-4-phenyl-1,2,3,4tetrahydropyrimidine-5-carboxylate (9a)22 White crystals, yield 41% (method 1), 60% (method 2), mp: 174-176 °C (lit.22 175-176 °C).
Virus titer diff. log(10) 0.89 0.32 0 0.32 0.4 0.49 0.39 0.32 1.32 0.39 6
Ethyl6-methyl-2-oxo-4-(4-carboxyphenyl)-1,2,3,4tetrahydropyrimidine-5-carboxylate (9b) White crystals, yield 36% (method 1), 58% (method 2), mp 297-299 °C. 1H-NMR (CDCl3, 400 MHz): 1.06 (t, 3H, CH3), 2.25 (s, 3H, CH3), 3.92 (q, 2H, CH2), 5.22 (s, 1H, CH), 7.35 (d, 2H, Ar-H), 7.79 (s, 1H, N3H), 7.90 (d, 2H, Ar-H), 9.24 (s, 1H, N1H).13C-NMR (DMSO-d6, 100 MHz): 14.5, 18.3, 54.4, 59.8, 99.2, 127.0 (2C), 130.1 (2C), 130.3, 149.3, 150.0, 152.5, 165.7, 167.6. Anal. Calcd for C15H16N2O5 (304.30): C, 59.21; H, 5.30; N, 9.21. Found: C, 59.29; H, 5.21; N, 9.15. 2-(4-(5-(Ethoxycarbonyl)-6-methyl-2-oxo-1,2,3,4tetrahydropyrimidin-4-yl)phenoxy)acetic acid (9c) White crystals, yield 39% (method 1), 61% (method 2), mp 247-249 °C. MS (m/z %): 335 (6.6, M++1), 276 (17.6), 251 (18.3), 226 (36.6), 83 (76), 78 (64.6), 63 (72.2), 44 (100). IR (KBr): υ/cm-1 1508 (C=C Str.), 1637 (C=O amide), 1702 (C=O acid), 1725 (C=O ester), 2940 (O-H Str.), 2977 (C-H Ar.Str.), 3110 (NH), 3239 (NH). Anal. Calcd for C16H18N2O6 (334.32): C, 57.48; H, 5.43; N, 8.38. Found: C, 57.35; H, 5.32; N, 8.41. Ethyl4-(4-(ethoxycarbonyl)phenyl)-1,6-dimethyl-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (9d) White crystals, yield 15% (method 1), 22% (method 2), mp 125-127 °C. 1H-NMR (DMSO-d6, 400 MHz): 1.08 (t, 3H, CH3), 1.31 (t, 3H, CH3), 2.50 (s, 3H, CH3), 3.13 (s, 3H, N-CH3), 4.06 (q, 2H, CH2), 4.31 (q, 2H, CH2), 5.27 (s, 1H, CH), 7.39 (d, 2H, Ar-H), 7.91 (d, 2H, Ar-H), 7.93 (s, 1H, NH).13C-NMR (DMSO-d6, 100 MHz): 13.4, 13.6, 15.5, 29.2, 51.9, 59.0, 60.0, 101.6, 125.7, 125.9, 128.6(2C), 128.8, 148.6, 150.3, 152.4, 164.9, 165.0. MS (m/z %): 346 (100, M+), 304 (25.2), 218 (3.2), 101 (5.7). Anal. Calcd for C18H22N2O5 (346.38): C, 62.42; H, 6.40; N, 8.09. Found: C, 62.60; H, 6.54; N, 8.15. Ethyl4-(4-(ethoxycarbonyl)phenyl)-6-phenyl-2-thioxo1,2,3,4-tetrahydropyrimidine-5-carboxylate (9e) Yellow crystals, yield 39% (method 1), mp 220-222 °C. 1 H-NMR (DMSO-d6, 400 MHz): 0.76 (t, 3H, CH3), 1.33 (t, 3H, CH3), 3.77 (q, 2H, CH2), 4.33 (q, 2H, CH2), 5.40 (s, 1H, CH), 7.33-7.43 (m, 5H, Ar-H), 7.55 (dd, 2H,3JHH 5
MJPS VOL. 27 (2) 2012 1-10
مجلة المنصورة للعلوم الصيدلية
3
Ethyl 6-(2-carboxybenzoyloxy) methyl-1-methyl-2oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5carboxylate (11c)
= 8 Hz, Ar-H), 8.01 (dd, 2H, JHH = 8 Hz, Ar-H), 9.70 (s, 1H, N3H), 10.39 (s, 1H, N1H).13C-NMR (DMSO-d6, 100 MHz): 12.8, 13.6, 53.6, 59.0, 60.2, 101.1, 126.2 (2C), 127.2 (2C), 128.1(2C), 128.6, 129.1(2C), 129.2, 133.4, 145.5, 147.5, 164.3, 164.9, 174.4. MS (m/z %): 411 (100, M++1), 305 (2.2), 218 (1.1), 100 (1.5). Anal. Calcd for C22H22N2O4S (410.49): C, 64.37; H, 5.40; N, 6.82. Found: C, 64.22; H, 5.27; N, 6.77. Ethyl6-(bromomethyl)-1-methyl-2-oxo-4-phenyl1,2,3,4-tetrahydropyrimidine-5-carboxylate (10)24 To a stirred solution of 9a (0.01 mol) in CHCl3 (30 mL) at 4 °C, bromine (1.6 g, 0.01 mol) in CHCl 3 (50 mL) was added dropwise. The reaction mixture was stirred at room temperature for 5 h, and concentrated under reduced pressure. The resulting precipitate was crystallized from EtOH to yield 6bromomethyltetrahydropyrimidine 10: mp 170-171 °C (lit.24 170 °C). General procedure for the synthesis of ethyl 6-((2substituted benzoyloxy) methyl)-1-methyl-2-oxo-4phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (11a-c) To astirred solution of 6-bromomethyl tetrahydropyrimidine 10 (0.35 g, 1 mmol) in DMF (10 mL), sodium benzoate derivative (1 mmol) was added. The reaction mixture was stirred at 60 °C for 12 h, and then allowed to stand at room temperature for 3 h. The reaction mixture was poured into ice-water (50 mL), and the precipitated solid was collected by filtration, washed with water, dried and crystallized from ethanol. Ethyl 6-(benzoyloxymethyl)-1-methyl-2-oxo-4-phenyl1,2,3,4-tetrahydropyrimidine-5-carboxylate (11a) White crystals, yield 71%, mp 146-148 °C. 1H-NMR (CDCl3, 400 MHz): 1.10 (t, 3H, CH3), 3.20 (s, 3H, NCH3), 4.06 (q, 2H, CH2), 5.24 (s, 1H, CH), 5.46 (d, 1H, 2 JHH = 12.8 Hz, CH2), 5.82 (d, 1H, 2JHH = 12.8 Hz, CH2), 7.28-7.36 (m, 5H, Ar-H), 7.54-7.58 (m, 2H, Ar-H), 7.69 (m, 1H, Ar-H), 7.97 (d, 2H, Ar-H), 8.09 (s, 1H, NH).13CNMR (DMSO-d6, 100 MHz): 14.3, 30.3, 53.2, 59.6, 60.9, 107.9, 126.6 (2C), 128.2, 129.1 (2C), 129.4 (2C), 129.5, 129.7 (2C), 134.2, 143.5, 145.6, 153.5, 165.3, 165.6. Anal. Calcd for C22H22N2O5 (394.42): C, 66.99; H, 5.62; N, 7.10. Found: C, 66.81; H, 5.55; N, 7.21. Ethyl 6-((2-hydroxybenzoyloxy) methyl)-1-methyl-2oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5carboxylate (11b) White crystals, yield 68%, mp 103-105 °C. 1H-NMR ( CDCl3, 400 MHz): 1.10 (t, 3H, CH3), 3.20 (s, 3H, NCH3), 4.06 (q, 2H, CH2), 5.24 (s, 1H, CH), 5.48 (d, 1H, 2 JHH = 12.8 Hz, CH2), 5.83 (d, 1H, 2JHH = 12.8 Hz, CH2), 6.94-7.01 (m, 2H, Ar-H), 7.28-7.37 (m, 5H, Ar-H), 7.52 (t, 1H, Ar-H), 7.73 (d, 1H, Ar-H), 8.09 (s, 1H, NH), 10.40 (s, 1H, OH).13C-NMR (DMSO-d6, 100 MHz): 14.3, 30.3, 53.2, 59.8, 60.9, 108.0, 113.7, 118.0, 120, 126.6 (2C), 128.2 (2C), 129.1, 130.6, 136.2, 143.4, 145.4, 153.5, 160.3, 165.2, 168.2. Anal. Calcd for C22H22N2O6 (410.42): C, 64.38; H, 5.40; N, 6.83. Found: C, 64.27; H, 5.36; N, 6.72.
White crystals, yield 74%, mp 109-111 °C. 1H-NMR (DMSO-d6, 400 MHz): 1.14 (t, 3H, CH3), 3.16 (s, 3H, NCH3), 4.09 (q, 2H, CH2), 5.26 (s, 1H, CH), 5.40 (dd, 1H, 2 JHH = 12.4 Hz, CH2), 5.77 (dd, 1H, 2JHH = 12.4 Hz, CH2), 7.26-7.33 (m, 5H, Ar-H), 7.73-7.93 (m, 4H, Ar-H), 8.1 (s, 1H, NH).13C-NMR (DMSO-d6, 100 MHz): 13.6, 29.4, 52.6, 59.5, 60.1, 107.7, 125.9 (2C), 127.4 (2C), 128.3 (2C), 128.6 (2C), 130.7 (2C), 131.8 (2C), 142.8, 144.3, 152.8, 164.5, 165.7. Anal. Calcd for C23H22N2O7 (438.43): C, 63.01; H, 5.06; N, 6.39. Found: C, 63.11; H, 5.13; N, 6.28. 5-Ethoxycarbonyl-6-methyl-4-phenyl-3,4-dihydro2(1H)-pyridone (12)42 A mixture of Meldrum’s acid25,26 (0.144 g, 1 mmol), ethyl acetoacetate (0.130 g, 1 mmol), benzaldehyde (0.106 g, 1 mmol), and ammonium acetate (0.094 g, 1.2 mmol) in acetic acid (8.0 mL) was heated at 118 °C for 6 h. The solvent was evaporated under reduced pressure, and then saturated aqueous NaHCO3 solution and EtOAc were added. The two layers were separated and the aqueous layer was further extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. Recrystallization from 95% ethanol afforded compound 12: m.p. 150-151 °C (lit.42 146-148 °C). 5-Oxo-4-phenyl-l,2,3,4,5,7-hexahydrofuro[3,4-b]2(IH)-pyridone (13)29 A solution of compound 12 (1.3 g, 5 mmol), Nbromosuccinimide (0.89 g, 5 mmol), in 10 mL of dry chloroform was refluxed for 12 h. The reaction mixture was cooled and the precipitated solid was collected by filtration. Recrystallization from ethanol yielded 55% of 13: m.p. 238-239 °C (lit.29 239-240 °C). 4-Phenyl-6-(un)substituted phenyl-3,4,6,7-tetrahydro1H-pyrrolo[3,4-b]pyridine-2,5-dione (14a-d) A mixture of compound 13 (0.49 g, 2.0 mmol) and the appropriate amine (2.2 mmol) in glacial acetic acid (10 mL) was heated at reflux for 48 h. The solvent was evaporated under reduced pressure, and the residue was purified by recrystallization from ethanol. 4,6-Diphenyl-3,4,6,7-tetrahydro-1H-pyrrolo[3,4b]pyridine-2,5-dione (14a) Brown crystals, yield 39%, mp 155-157 °C. 1H-NMR (DMSO-d6, 400 MHz): 2.49 (dd, 1H, H-3b), 3.13 (dd, 1H, H-3a), 4.00 (dd, 1H, H-4), 4.92 (dd, 2H, NCH2), 7.02-7.99 (m, 10H, Ar-H), 10.77 (s, 1H, NH). MS (m/z %): 305 (0.36, M++1), 304 (0.94, M+), 93 (9.7). Anal. Calcd for C19H16N2O2 (304.34): C, 74.98; H, 5.30; N, 9.20. Found: C, 74.80; H, 5.46; N, 9.39.
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مجلة المنصورة للعلوم الصيدلية
MJPS VOL. 27 (2) 2012 1-10 4-Phenyl-6-p-tolyl-3,4,6,7-tetrahydro-1H-pyrrolo[3,4b] pyridine-2,5-dione (14b) Off-white crystals, yield 43%, mp 235-237 °C. 1H-NMR (DMSO-d6, 400 MHz): 2.32 (s, 3H, CH3), 2.56 (dd, 1H, H-3b), 3.14 (dd, 1H, H-3a), 4.06 (dd, 1H, H-4), 4.92 (dd, 2H, NCH2), 6.82-7.62 (m, 9H, Ar-H), 10.77 (s, 1H, NH). MS (m/z %): 319 (2.4, M++1), 318 (5.6, M+), 229 (11.2), 93 (100.0). Anal. Calcd for C20H18N2O2 (318.37): C, 75.45; H, 5.70; N, 8.80. Found: C, 75.58; H, 5.84; N, 8.71. 6-(4-Chlorophenyl)-4-phenyl-3,4,6,7-tetrahydro-1Hpyrrolo[3,4-b]pyridine-2,5-dione (14c) 1
White crystals, yield 42%, mp 165-167 °C. H-NMR (DMSO-d6, 400 MHz): 2.54 (dd, 1H, H-3b), 3.12 (dd, 1H, H-3a), 4.06 (dd, 1H, H-4), 4.95 (dd, 2H, NCH2), 7.19-7.34 (m, 5H, Ar-H), 7.47-7.84 (m, 4H, Ar-H), 10.14 (s, 1H, NH). MS (m/z %): 339 (0.42, M ++1), 338 (0.43, M+), 93 (5.5). Anal. Calcd for C19H15ClN2O2 (338.79): C, 67.36; H, 4.46; N, 8.27. Found: C, 67.27; H, 4.31; N, 8.35. For compound 14d: A mixture of compound 13 (0.49 g, 2.0 mmol) and 4methoxyaniline (0.27 g, 2.2 mmol) in dry toluene (10 mL) was heated at reflux for 36 h. The precipitated solid was collected by filtration, recrystallized from ethanol. 6-(4-Methoxyphenyl)-4-phenyl-3,4,6,7-tetrahydro-1Hpyrrolo[3,4-b]pyridine-2,5-dione (14d) Grey crystals, yield 48%, mp 153-155 °C. 1H-NMR (DMSO-d6, 400 MHz): 2.56 (dd, 1H, H-3b), 3.15 (dd, 1H, H-3a), 3.77 (s, 3H, OCH3), 3.99 (dd, 1H, H-4), 4.91 (dd, 2H, NCH2), 7.01-7.34 (m, 7H, Ar-H), 7.50 (d, 2H, Ar-H), 10.74 (s, 1H, NH). MS (m/z %): 335 (17.3, M++1), 334 (38.7, M+), 229 (21.1), 93 (17.3). Anal. Calcd for C20H18N2O3 (334.37): C, 71.84; H, 5.43; N, 8.38. Found: C, 71.71; H, 5.37; N, 8.27. Conclusion The results of antiviral assay revealed that compounds 14c and 9b showed the highest activity. Compounds 11c, 11b, 14a and 14d showed moderate activity. While compounds 9d, 11a and 14b showed the least antiviral activity among the series of tested compounds. On the other hand, compound 9e showed no activity. It was noted that the antiviral activity of all compounds was low compared to interferone. Acknowledgements The authors are grateful to Prof. David W. Boykin, Dept. of Chemistry, Georgia State University for his generous permission for performing NMR analysis. Authors would like also to provide deepest gratitude to Prof. Dr. Aly Fahmy Mohamed El-Sayed, G.M. of Applied Research Sector, Holding Company for Biological Products, Vaccines and Drugs (Egy-Vac/ VACSERA), for his assistance in performing the antiviral screening in this work
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MJPS VOL. 27 (2) 2012 1-10 12. Petricci E., Mugnaini C., Radi M., Togninelli A., Bernardini C., Manetti F., Parlato M. C., Renzulli M. L., Alongi M., Falciani C., Corelli F. and Botta M.; Towards new methodologies for the synthesis of biologically interesting 6-substituted pyrimidines and 4(3H)-pyrimidinones, ARKIVOC, 2006, vii , 452-478. 13. Mitchell M. L., Son J. C., Guo H., AIm Y., Cho E. J., Wang J., Hayes J., Wang M., Paul A., Lansdon E. B., Chen J. M., Graupe D., Rhodes G., He G. X., Geleziunas R., Xu L. and Kim C. U.; N1-Alkyl pyrimidinediones as non-nucleoside inhibitors of HIV-1 reverse transcriptase, Bioorg. Med. Chem. Lett., 2010, 20, 1589-1592. 14. De Clercq E.; The design of drugs for HIV and HCV, Nat. Rev. Drug Discovery, 2007, 6, 10011018. 15. Petrocchi A., Koch U., Matassa V.G., Pacini B., Stillmock K.A. and Summa V.; From dihydroxypyrimidine carboxylic acids to carboxamide HIV-1 integrase inhibitors: SAR around the amide moiety, Bioorg. Med. Chem. Lett., 2007, 17 (2), 350-353. 16. Kolykhalov A. A., Mihalik K., Feinstone S. M. and Rice C. M.; Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo, J. Virol., 2000, 74(4), 20462051. 17. El Safadi Y., Vivet-Boudou V. and Marquet R.; HIV-1 reverse transcriptase inhibitors, Appl. Microbiol. Biotechnol., 2007, 75 (4), 723-737. 18. Rivkin A.; A review of entecavir in the treatment of chronic hepatitis B infection, Curr. Med. Res. Opin., 2005, 21(11), 1845-1856. 19. Ruebsam F., Tran C. V., Li L., Kim S. H., Xiang A. X., Zhou Y., Blazel J. K., Sun Z., Dragovich P. S., Zhao J., McGuire H. M., Murphy D. E., Tran M. T., Stankovic N., Ellis D. A., Gobbi A., Showalter R. E., Webber S. E., Shah A. M., Tsan M., Patel R. A., LeBrun L. A., Hou H. J., Kamran R., Sergeeva M. V., Bartkowski D. M., Nolan T. G., Norris D. A. and Kirkovsky L.; 5,6-Dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase, Bioorg. Med. Chem.lett., 2009, 19 (2), 451-458. 20. Lewandowske K., Murer P., Svec F. and Fréchet J. M. J.; A combinatorial approach to recognition of chirality: preparation of highly enantioselective aryl-dihydropyrimidine selectors for chiral HPLC, J. Comb. Chem., 1999, 1, 105-112. 21. Kappe C. O., Kumar D. and Varma S. R.; Microwave-assisted high-speed parallel synthesis of 4-aryl-3,4-dihydropyrimidin-2(1H)-ones using a solventless Biginelli condensation protocol, Synthesis, 1999, 10, 1799-1803.
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MJPS VOL. 27 (2) 2012 1-10
تشييد و تقييم الفعالية البيولوجية لمشتقات غير نيوكميوسيدية جديدة كمثبطات لفيروس الكبد الوبائى ج أمانى صالح مصطفى* ،سرى عطا البيمى ،وليد عبدالحكيم بيومى ،عمى ماهر عبدالعال قسم الكيمياء العضوية الصيدلية -كمية الصيدلة -جامعة المنصورة -المنصورة – مصر
تم فى هذا البحث تشييد مشتقات جديدة من 1،2،3،4رباعى هيدروبيريميددين و 3،4،1،2بيرولدو[-2،1ب] بيريددين ختتبدار
فعاليتها كمثبطات محتممه لفيروس الكبدد الوبدائى ج وذلد باسدتتدام فيدروس التهداب الفدم الحويصدمذ كنمدوذج لفيدروس الكبدد الوبدائذ مقارندا لممركبددات الجديددة بدداونترفيرون ،باوضدافة الدى دراسددة تمثيرهدا السددام عمدى التاليداا و قددد تدم اجدراء العديدد مددن
التفاعالت الكيميائيدة لمحصدول عمدى تمد المركبدات الجديددة و التدى تدم تثبدات التراكيدب البنائيدة لهدا باسدتتدام التحميدل الددقي
لعناصدرها اضدافة الدى دراسدة بعضدها باتشدعة تحدت الحمدراء و الدرنين الندووى الموناطيسدى لندواة الهيددروجين ( )4و الكربدون
( ) 42و طيف الكتمةا و قد أظهرت النتائج أن مركبين قدد أظهد ار فاعميدة عاليدة مضدادة لمفيدروس بينمدا اظهدرت أربعدة مركبدات أترى فاعمية متوسطة ،فى حين أبدت ثالثة مركبات فاعمية ضعيفة مضادة
01
لمفيروس.
