Synthesis, spectroscopy characterization and

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Synthesis, spectroscopy characterization and biological activities of some novel 1-(3-(N,N-dimethylamino)-1-(5-substituted thiophene-2-yl) propylidene semicarbazone Mannich base derivatives ⁎

Chandravadivelu Gopi , Magharla Dasaratha Dhanaraju Research Lab, GIET School of Pharmacy, Rajahmundry, Andhra Pradesh 533296, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Thiophene Mannich base Condensation reaction Anti-inflammatory agent Anti-diabetic agent

The main aim of this work was to synthesise a novel 1-(3-(N,N-dimethylamino)-1-(5-substituted thiophene-2-yl) propylidene semicarbazone Mannich base derivatives and examine the anti-diabetic and anti-inflammatory activities using alloxan-induced diabetic and carrageenan-induced paw oedema methods. These analogues were prepared by performing a condensation reaction between 1-(thiophen 2-yl) ethanone, formaldehyde, N,N-dimethyl amine hydrochloride and semicarbazide. The prepared analogues were characterised by FT-IR, 1H NMR, 13 C NMR, mass spectroscopy and elemental analysis. The result reveals that most of the compounds were significantly reduced in the blood glucose level and inflammation of paw volume of experimental animals as compared to the standard drugs.

1. Introduction The Mannich base is prepared from the condensation reaction of aromatic ketone, aldehyde and primary or secondary amine in alcohol. It deals with an amino alkylation of an acidic proton placed next to a carbonyl functional group of the aromatic ketone by formaldehyde and a primary or secondary amine. The reaction is named as a Mannich reaction after Carl Mannich, who first discovered it in 1912. The literature study reveals that the Mannich base can be modified into their derivative by simple chemical reactions and they are the key intermediate for the preparation of various bioactive molecules. They are responsible for different pharmacological activities, such as anti-inflammatory (Prasanna and Saleel, 2015), anticancer (Bhupendra et al., 2016), antipyretic (Kumar et al., 2016), antibacterial (Bogdanov et al., 2016), antifungal (Idhayadhulla et al., 2014), anticonvulsant (Rybka et al., 2016), anthelmintic (Joshi et al., 2004), antitubercular (Lahbib et al., 2013), analgesic (Datar and Limaye, 2015), anti-HIV (Sah et al., 2014), anti-malarial (Francisca et al., 2004), antipsychotic (Shaw et al., 2010), antiviral (Roman, 2015) agent etc. Thiophene and their derivatives have been widely distributed in many of the naturally occurring compounds and are employed in different health hazards. They are responsible for varying biological activity such as anti-inflammatory (Molvi et al., 2008), antipyretic (Gouda et al., 2016), anti-hypotensive (Desai et al., 2014), anti-convulsant (Deep et al., 2016), anti-viral (Fathima et al., 2011), antitumour

⁎

(Pulipati et al., 2016), fungicidal (Wang et al., 2015), herbicidal (Benachenhou et al., 2013), anti-microbial (Rani and Mohamad, 2014) and plant-growth regulator (Baert et al., 2016). In the present study, aforesaid benefits of a Mannich base and thiophene heterocyclic nucleus promoted us to prepare a series of novel 4-(dimethylamino)-1(thiophen-2-yl) butan-1-one derivatives holding both pharmacophore and evaluate for anti-diabetic, anti-inflammatory activities. These analogues were prepared from appropriate raw materials and structures were elucidated by FT-IR, 1H NMR, 13C NMR, mass spectroscopy and Xray crystallography studies. The experimental result suggested that most of the test compounds are possessing admirable anti-diabetic and anti-inflammatory activities. The potency of new series of 1-(3-(N,Ndimethylamino)-1-(5-substituted thiophene-2-yl)propylidene semicarbazone Mannich base derivatives [4a–f] based on the modification of side chain groups in the thiophene nucleus. Accordingly, in vitro antiinflammatory and anti-diabetic activities of these derivatives as a thiophene analogue [4a–f] in which (i) bromo, nitro, chloro, sulphonic acid substitution at 5 position of thiophene ring are important for antiinflammatory and anti-diabetic activities, were maintained in compounds (4f, 4a, 4e, 4b); (ii) whereas, CH3 and C2H5 group at 5 position of thiophene ring (4c and 4d) would be favourable for the mentioned activity; (iii) semicarbazone Mannich base group at 2 position of thiophene ring is expected to offer better anti-inflammatory and anti-diabetic activity (Fig. 1).

Corresponding author at: GIET School of Pharmacy, NH-16, Rajahmundry, Andhra Pradesh 533 296, India. E-mail address: [email protected] (C. Gopi).

https://doi.org/10.1016/j.bjbas.2018.02.004 Received 12 December 2017; Received in revised form 13 February 2018; Accepted 19 February 2018 2314-8535/ © 2018 Beni-Suef University. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Gopi, C., Beni-Suef University Journal of Basic and Applied Sciences (2018), https://doi.org/10.1016/j.bjbas.2018.02.004


C. Gopi, M.D. Dhanaraju

this work was an excellent condition at the time of usage. FT-IR spectroscopy was employed for functional group determination of synthesised compounds between the ranges of 4000 cm−1 and 400 cm−1. 1H NMR Brucker spectra (500 MHz) was used to count the number of protons present in the compounds from chemical shift (δ) using CDCl3 as a solvent. The Mass spectra were recorded by SHIMADZU mass spectrometer and the Perkin Elmer 2400 CHN analyzer was used to determine the atomic percentage of the atoms present in the synthesised compounds. 3. Experimental part Fig. 1. Chemical structure of 2,5 di-substituted novel Mannich base derivatives.

3.1. Preparation of 1-(thiophen 2yl) ethanone [1]

2. Materials and method

A mixture of thiophene (0.1 mol, 8.4 g) and acetic anhydride (0.1 mol, 10.2 ml) was taken in a round bottom flask fitted with a reflux condenser. Phosphoric acid (25 ml) was added to the above mixture from the dropping funnel with caution. The contents were stirred

The reagent and solvents were procured from Alrich, Ranbaxy and Sigma companies and all are in analytical grades. The apparatus used in

Fig. 2. A schematic representation of Mannich base derivatives formation.

2



magnetically for 10 min and refluxed for 4 h. The mixture was cooled to room temperature and the oil layer was removed by washing with 10% sodium carbonate solution. The separated 1-(thiophen 2yl) ethanone was filtered, washed with a small portion of cold water, dried and crystallized from hot ethanol (Gopi et al., 2016).

Table 1 Name and structure of synthesised compounds. Compound name

Structure of synthesised compounds

4a

3.2. Preparation of 1-(5-substituted thiophen-2-yl) ethanone [2a–f] 1-(3-(N,N-Dimethylamino)-1-(5-nitrothiophene-2yl) propylidene)semicarbazone

A mixture of 1-(thiophen 2yl) ethanone (0.1 mol, 12.6 g) and equal molar quantity of different electrophilic reagent was placed in a round bottom flask of 500 ml capacity. The content was stirred and heated (80–120 °C) in a sand bath for an appropriate time. Progression of the reaction was monitored by TLC using a solvent system of n-hexane and ethyl acetate (3:2) as a mobile phase. The resulting solution was diluted with water (100 ml). The separated 1-(5-substituted thiophen-2-yl) ethanone was filtered, washed with a small portion of cold water, dried and crystallized from hot ethyl acetate. In each reaction, the electrophile (E+) from electrophilic reagent accommodates the C5 position of thiophene and formed different 1-(5-substituted thiophene-2-yl) ethanone derivatives (Salman et al., 2015).

4b

1-(3-(N,N-Dimethylamino)-1-(thiophen-2yl)propylidene) semicarbazone-5-sulphonic acid 4c

1-(3-(N,N-Dimethylamino)-1-(5-methylthiophene-2yl) propylidene)semicarbazone 4d

3.3. General preparation of 1-(3-(N,N-dimethylamino)-1-(5-substituted thiophene-2-yl)propane-1-one Mannich bases [3a–f]

1-(3-(N,N-Dimethylamino)-1-(5-ethylthiophene-2yl) propylidene)semicarbazone 4e

A mixture of 1-(5-substituted thiophen-2-yl) ethanone, formaldehyde (0.1 mol, 3 g) and N,N-dimethylamine hydrochloride (0.1 mol, 8.1 g) was dissolved in a 30 ml of ethanol. The resulting solution was stirred, kept gently at reflux bath for 4 h and allowed to stand for overnight. The separated product was filtered, washed well with cold water, crystallized from hot acetone (Aeluri et al., 2015).

1-(1-(5-Chlorothiophene-2yl)-3-(N,N-dimethylamino) propylidene)semicarbazone 4f

1-(1-(5-Bromothiophene-2yl)-3-(N,N-dimethylamino) propylidene)semicarbazone

3.4. Preparation of 1-(3-(N,N-dimethylamino)-1-(5-substituted thiophene2-yl)propylidene semicarbazone Mannich base derivatives [4a–f] 3-(N,N-dimethylamino)-1-(5-substituted thiophene-2yl)propane-1one derivative (0.01 mol) in a warm ethanol and the solution of semicarbazide (0.01 mol, 0.75 g) in 50 ml of water were mixed slowly with the glass stirrer for 5 h at room temperature (Singhal et al., 2014). The precipitated product was filtered at the pump, washed with water, dried and crystallized from ethanol (Fig. 2 & Table 1). The physical and spectral data are listed below.

137 (100), 112 (21), 99 (27), 28 (24); Anal. Calcd for C10H16N4O4S2, (320) predicted: C, 37.46; H, 5.06; O, 19.99; S, 20.05 and N, 17.44; Found: C, 37.26; H, 5.13 and N, 17.02. 3.4.3. 1-(3-(N,N-Dimethylamino)-1-(5-methylthiophene-2yl)propylidene) semicarbazone: 4c Yield: 60%; Melting point: 118–123 °C; Color: White; IR (νmax/cm−1): 3042 (Ar-H str), 1531 (Ar-C str), 1665 (C]O); 1H NMR (CDCl3, δ, ppm): 1.59 (t, 2H, J = 8.2 Hz, CH2-H), 2.26 (s, CH3-H), 2.37 (s, 6H, N(CH3)2-H), 2.83 (t, 2H, J = 9.1 Hz, CH2-H), 6.12 (s, 2H, NH2H), δ 6.56 (d, 1H, J = 12.4 Hz, Ar-H), 7.17 (s, 1H, NH-H), 7.43 (d, 1H, J = 11.8 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 13.2 (CeCH3), 24.9 (C-6), 46.3 (C-8,9), 56.1 (C-7), 122.4 (C-1), 124.9 (C-2,3), 140.9 (C-4), 157.5 (C-5), 159.4 (C-10); Ms (m/z,%): 254 (26) [M+], 224 (18), 196 (60), 123 (38), 82 (100), 40 (26); Anal. Calcd for C11H18N4OS, (254) predicted: C, 51.92; H, 7.14; O, 6.27; S, 12.63 and N, 22.04; Found: C, 37.26; H, 5.13 and N, 17.02.

3.4.1. 1-(3-(N,N-Dimethylamino)-1-(5-nitrothiophene-2yl)propylidene) semicarbazone: 4a Yield: 82%; Melting point: 123–127 °C; Color: Yellow; IR (νmax/cm−1): 3016 (Ar-H str), 1510 (Ar-C str), 1589 & 1322 (NO2), 1657 (C]O); 1H NMR (CDCl3, δ, ppm): 1.43 (t, 2H, J = 7.1 Hz, CH2H), 2.32 (s, 6H, N(CH3)2-H), 2.62 (t, 2H, J = 11.5 Hz, CH2-H), 6.02 (s, 2H, NH2-H), 6.73 (d, 1H, J = 12.4 Hz, Ar-H), 7.09 (s, 1H, NH-H), 7.40 (d, 1H, J = 15.4 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 24.8 (C-6), 46.9 (C-8,9), 56.2 (C-7), 127.2 (C-2,3), 132.8 (C-1), 150.6 (C-4), 155.1 (C-5), 157.8 (C-10); Ms (m/z, %): 285 (22) [M+], 255 (38), 239 (27), 157 (54), 99 (100), 58 (38), 46 (26); Anal. Calcd for C10H15N5O3S, (285) predicted: C, 42.01; H, 5.39; O, 16.86; S, 11.23 and N, 24.51; Found: C, 42.53; H, 5.16 and N, 24.08.

3.4.4. 1-(3-(N,N-Dimethylamino)-1-(5-ethylthiophene-2yl)propylidene) semicarbazone: 4d Yield: 69%; Melting point: 120–124 °C; Color: White; IR (νmax/cm−1): 3022 (Ar-H str), 1516 (Ar-C str), 1650 (C]O); 1H NMR (CDCl3, δ, ppm): 1.22 (t, 3H, J = 6.8 Hz, CH3-H), 1.68 (t, 2H, J = 7.3 Hz, CH2-H), 2.29 (s, 6H, N(CH3)2-H), 2.62 (t, 2H, J = 12.7 Hz, CH2-H), 2.99 (q, 2H, J = 13.2 Hz, CH2-H), 6.08 (s, 2H, NH2-H), 6.56 (d, 1H, J = 15.2 Hz, Ar-H), 7.12 (s, 1H, NH-H), 7.48 (d, 1H, J = 15.8 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 16.6 (C-CH3), 22.9 (C-CH2), 25.2 (C-6), 46.5 (C-8,9), 56.4 (C-7), 122.5 (C-1), 124.7 (C-3), 126.2 (C2),137.5 (C-4), 154.6 (C-5), 156.2 (C-10); Ms (m/z, %): 268 (19) [M+], 224 (29), 210 (15), 196 (38), 123 (100), 98 (37), 73 (58); Anal. Calcd for C12H20N4OS, (268) predicted: C, 53.68; H, 7.53; O, 5.98; S, 11.96

3.4.2. 1-(3-(N,N-Dimethylamino)-1-(thiophen-2yl)propylidene) semicarbazone-5-sulphonic acid: 4b Yield: 74%; Melting point: 129–134 °C; Color: Fuchsine red; IR (νmax/cm−1): 3082 (Ar-H str), 1534 (Ar-C str), 1312 & 1154 (SO3H), 1652 (C]O); 1H NMR (CDCl3, δ, ppm): 1.84 (t, 2H, J = 7.8 Hz, CH2H), 2.30 (s, 6H, N(CH3)2-H), 2.69 (t, 2H, J = 8.4 Hz, CH2-H), 6.07 (s, 2H, NH2-H), 6.67 (d, 1H, J = 15.8 Hz, Ar-H), 7.05 (s, 1H, NH-H), 7.72 (d, 1H, J = 12.7 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 25.2 (C-6), 47.1 (C-8,9), 56.5 (C-7), 124.8 (C-1), 126.5 (C-4), 128.2 (C-2,3), 155.8 (C-5), 158.1 (C-10); Ms (m/z, %): 320 (21) [M+], 290 (38), 261 (25), 3



and N, 20.85; Found: C, 53.42; H, 7.06 and N, 20.61.

The synthesised Mannich derivatives were given daily to group 4–9 containing animals with the dose of 10 mg/kg body weight for 7 days. The animal tail vein was used to collect the blood from the experimental animals and blood glucose level was monitored by AccuSure Blood Glucose apparatus.

3.4.5. 1-(1-(5-Chlorothiophene-2yl)-3-(N,N-dimethylamino)propylidene) semicarbazone: 4e Yield: 83%; Melting point: 134–138 °C; Color: Cream yellow; IR (νmax/cm−1): 3014 (Ar-H str), 1539 (Ar-C str), 1641 (C]O), Cl (758); 1 H NMR (CDCl3, δ, ppm): 1.80 (t, 2H, J = 4.8 Hz, CH2-H), 2.29 (s, 6H, N(CH3)2-H), 2.81 (t, 2H, J = 5.2 Hz, CH2-H), 6.06 (s, 2H, NH2-H), 6.88 (d, 1H, J = 10.7 Hz, Ar-H), 7.14 (s, 1H, NH-H), 7.45 (d, 1H, J = 15.8 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 23.2 (C-6), 45.4 (C8,9), 54.7 (C-7), 114.6 (C-4), 125.6 (C-1), 128.4 (C-2), 130.3 (C-3), 155.8 (C-5), 157.2 (C-10); Ms (m/z, %): 274 (46) [M+], 276 (14) [M++2], 239 (71), 201 (18), 157 (51), 99 (100), 44 (39); Anal. Calcd for C10H15ClN4OS, (274) predicted: C, 43.75; H, 5.46; O, 5.87; S, 11.71; N, 20.42; Cl, 12.79; Found: C, 43.19; H, 5.20 and N, 20.82.

3.6.3. Anti-inflammatory activity The newly synthesised Chalcone derivatives were screened for antiinflammatory activity using carrageenan induced paw oedema method (Maddila et al., 2016). To test the anti-inflammatory activity, Wistar rats of either sex with an average weight of 175–200 g were selected, grouped (standard, test and control) and placed into different cages. Make a mark on both the hand paw just beyond tibiotarsal junction, so that every time the paw is dipped in the mercury column up to the fixed mark to ensure constant paw volume. Note the initial paw volume of each rat (control, test and standard) by plethysmometer. The group 1 was administered with 0.1% CMC (control), indomethacin and test compound (10 mg/kg) were also given simultaneously to standard and test group of animals. These compounds were given orally with the aid of an oral catheter. After 30 min, inject 0.1 ml of 1% (w/v) carrageenan in the plantar region of the left paw and right paw served as a reference for comparison. The volume of paw oedema was measured at 1, 2, 3 and 4 h by using plethysmometer.

3.4.6. 1-(1-(5-Bromothiophene-2yl)-3-(N,N-dimethylamino)propylidene) semicarbazone: 4f Yield: 85%; Melting point: 126–131 °C; Color: Slight brown; IR (νmax/cm−1): 3135 (Ar-H str), 1537 (Ar-C str), 1682 (C]O), Br (646); 1 H NMR (CDCl3, δ, ppm): 1.56 (t, 2H, J = 4.2 Hz, CH2-H), 2.27 (s, 6H, N(CH3)2-H), 2.87 (t, 2H, J = 5.3 Hz, CH2-H), 6.04 (s, 2H, NH2-H), 6.53 (d, 1H, J = 13.8 Hz, Ar-H), 7.19 (s, 1H, NH-H), 7.58 (d, 1H, J = 14.7 Hz, Ar-H); 13C NMR (DMSO‑d6, δ, ppm): 24.5 (C-6), 46.1 (C8,9), 55.3 (C-7), 124.7 (C-1), 125.4 (C-2), 127.8 (C-3), 130.4 (C-4), 157.1 (C-5), 159.2 (C-10); Ms (m/z, %): 318 (23) [M+], 320 (21) [M++2], 239 (37), 195 (29), 157 (100), 99 (42), 41 (29); Anal. Calcd for C10H15BrN4OS, (318) predicted: C, 37.60; H, 4.76; O, 5.04; S, 10.25; N, 17.30; Br, 25.05; Found: C, 37.71; H, 4.58 and N, 17.38.

Percentage inhibition = (1−Ct/Co) × 100

• Co = Oedema in control group • Co = Oedema in tested group 3.6.4. Ulcer effect Most active compounds (4f and 4a) from anti-inflammatory activity were utilised to perform the ulcer effect by using the magnifying lens. Albino rats of either sex with an average weight of 175–200 g were divided into four groups. Each group maintained with 6 animals. The group 1 was treated with 0.1% carboxymethylcellulose and group 2, 3 & 4 were administered to test compound, indomethacin (10 mg/kg, p.o) respectively. Animals were sacrificed under anaesthesia after 6 h of dosing of 0.1% CMC, test and standard drugs. The stomach of the experimental animal was removed and opened the greater curvature. The inner mucosal surface was observed using the magnifying lens and scoring was done as follows. Zero for normal colouration of the stomach, 0.5 for red colouration, 1 for spot ulcer, 2 for > 3 but < 5 mm, 3 for ulcer > 5, 4 for many ulcers more than 3 mm, and 5 for a perforated ulcer. The mean ulcer score for each animal was calculated as ulcer index.

3.5. X-ray crystallography X-ray crystallography is a tool used to investigate the three-dimensional picture of the atomic and molecular structure of a crystal by using X-ray light, which has wavelengths of 1 Å (10−8 cm). The beam of X-ray hits a crystal and causes the diffraction of light in particular directions, it has fed into the computer and find out the position of every atom in the crystallized molecule. 3.6. Pharmacological activity 3.6.1. Acute oral toxicity study An acute toxicity study was performed as per OECD guideline 423 in healthy male and female mice. Animals were grouped and administered with the graded dose of (5 mg/kg–5000 mg/kg) synthesised compounds in a standard room condition (22 °C). Each group maintained with 3 animals (Kifayatullah et al., 2015). All animals were observed for mortality and behavioural changes such as irritability, restlessness, fearfulness and alertness during 14 days and special care should be given for first 4 h. Experimental protocol has been approved by the institutional animal ethics committee at GIET School of pharmacy, Rajahmundry, India (GSP/IAEC/2016/03/01).

4. Result 4.1. X-ray crystallography determination and refinement Diffraction data of the compound 4a were collected from 7325 reflections using X calibur CCD diffractometer equipped with area detector and a graphite monochromator (λ = 0.72683) at 296 K. The dimension of the crystal employed for data collection was 0.28 × 0.28 × 0.28 mm at 30% probability of selected bond angle and bond length. The refinement was carried out by full-matrix least squares using SHELXL 97. The different parameters, conditions and data collections of refinement process of compound 4a were furnished in Table 2. The ORTEP view of the crystal bond angle and bond length was shown in Fig. 3 and Table 3.

3.6.2. Anti-diabetic activity The newly synthesised Mannich base derivatives were screened for anti-diabetic activity in alloxan-induced diabetic rats (Kumar et al., 2011). Wistar rats of either sex with an average weight of 150–200 g were selected, grouped (standard, test and control) and placed into different cages and maintained 6 animals for each. All the animals were fed with standard food pellet and drinking water. Group 1 animals were treated with saline and considered as a normal control. Remaining groups of animals were injected with alloxan monohydrate 120 mg/kg in sterile saline intraperitoneally to attain an acute diabetic condition. Group 2 animals were treated daily with 1 ml of 0.1% carboxymethylcellulose as consider as a disease control. Glibenclamide was administered to group 3 animals had been considered as the standard.

4.2. Acute toxicity study An acute toxicity study was performed as per OECD guidelines 423 in healthy female albino mice. After administration of the synthesised compound, animals were observed for 14 days and special care should 4



Table 2 Crystal data and structural refinement of compound 4a.

Table 4 Acute toxicity study result of synthesised compounds.

Identification code

Compound 4a

Experimental animal

No. animal used

Dose

Alive

Death

Empirical formula Formula weight Crystal system Crystal size (mm) Temperature (K) Space group Wave length (Å) Volume (Å3) Absorption coefficient (mm−1) F(0 0 0) Z Calculated density (Mg/m3) Theta range for data collection Index range

C10H15N5O3S 285 Monoclinic 0.28 × 0.28 × 0.28 296 P 21/n 0.72683 1027.52 (8) 0.073 739 3 1.027 2.27°–25.31° −9 ≤ h ≤ 10 −14 ≤ k ≤ 12 −19 ≤ l ≤ 19 7325 2159 10371/0/539 Full-matrix least-squares on F2 1.162 R1 = 0.0518, ωR2 = 0.1427 R1 = 0.0119, ωR2 = 0.1712 0.0062 (18) 0.317/−0.254

Mice

03

5 mg/kg 50 mg/kg 300 mg/kg 200 mg/kg 100 mg/kg

3 3 0 0 2

0 0 3 3 1

Measured reflections Independent/observed reflections Data/restraints/parameters Refinement method Goodness-of-fit on F2 Final R indices [I > 2σ(I)] R indices [all data] Extinction coefficient Largest diff. Peak and hole (e Å3)

were shown excellent anti-diabetic activity and reduced the blood glucose extensively as similar to that of standard drug. The result of the anti-diabetic activity of the synthesised compounds was depicted in Table 5.

4.4. Anti-inflammatory activity The paw volume of the foot was measured in the test, standard and control group of rats at the interval of 1, 2, 3, and 4 h by using plethysmometer. The result of anti-inflammatory activity reveals that the entire test compounds (4a–f) have been identified as an excellent antiinflammatory agent and extensively reduced the inflammation of paw volume of experimental animals. The result of anti-inflammatory activity of test compounds was summarised in Tables 6 and 7.

4.5. Ulcer effect Selected compounds (4f and 4a) were slightly damaged the inner mucosal surface of the stomach of experimental animals as like a standard drug. The result of ulcer activity reveals that the tested compounds offered varying degrees of an ulcer effect had been compared against control and the standard group of rats were furnished in Table 8 & Fig. 4. Fig. 3. ORTEP view of compound 4a at 30% probability level.

5. Discussion Table 3 Bond distances and bond angle of 4a compound. Bond distances (Å) C01A–S01A C04A–S01A C01A–N01A N01A–O01A N01A–O02A C04A–C05A C05A–C06A C06A–C07A C07A–N02A N02A–C08A N02A–C09A C05A–N03A N03A–N04A N04A–C10A C10A–O03A C10A–N05A

5.1. Chemistry

Bond angle (°) 1.754(3) 1.832(6) 1.306(5) 1.215(5) 1.126(3) 1.486(7) 1.502(3) 1.462(5) 1.362(3) 1.335(5) 1.286(7) 1.332(3) 1.324(2) 1.305(3) 1.251(2) 1.318(5)

C01A–S01A–C04A C01A–N01A–O01A C01A–N01A–O02A C04A–C05A–N03A C07A–N02A–C08A C07A–N02A–C09A N02A–C08A–C09A C05A–N03A–N04A N03A–N04A–C10A C10A–N05A–O03A

The route of synthesis of the prepared compound was depicted in the synthetic scheme outlines the preparation part of the synthesised analogues. The synthesised 1-(3-(N,N dimethylamino)-1-(5-substituted thiophene-2-yl)propylidene semicarbazone Mannich base derivatives were prepared by a condensation reaction between 1-(5-substituted thiophen-2-yl) ethanone, formaldehyde, N,N-dimethyl amine hydrochloride and semicarbazide. The compounds were shown peaks due to the presence of the different functional group in FT-IR spectrum, a strong peak in the region of 1589, 1322, 1312, 1154, 758, 646, 1657, 1652, 1665, 1650, 1641 and 1682 could be attributed to the nitro, sulphonic acid, chloro, bromo and carbonyl group respectively. The aryl ring was raised stretching peak in between 3135 and 3016 cm−1 and 1541–1510 cm−1. The number of protons present in the synthesised compounds was identified by 1H NMR spectroscopy from the chemical shift. The spectra showed a singlet at δ 1.13–2.30 ppm corresponding to a methyl proton (CH3-H), a singlet at δ 2.21–2.43 ppm corresponding to a secondary amine proton (N(CH3)2-H), a triplet at δ 1.37–2.95 ppm corresponding to methylene proton, a singlet at δ 5.96–6.17 ppm corresponding to an amine proton (NH2-H), a singlet at δ 7.01–7.25 ppm corresponding to NH-H proton, a doublet at δ 6.44–7.81 ppm corresponding to an aromatic proton (Ar-H). The synthesised compound molecular mass was recorded by SHIMADZU mass spectrometer. Finally, the three-dimensional structure of compound 4a was determined by using X-ray diffractometer.

91.7(12) 123.5(13) 119.7(15) 117.4(13) 124.3(15) 128.4(13) 121.4(13) 120.3(17) 116.4(13) 123.2(15)

be given for first 4 h. The result of acute toxicity of the synthesised compounds was depicted in Table 4.

4.3. Anti-diabetic activity The blood glucose was measured in the alloxan-induced diabetic rats at the interval of 0, 3, 5, 7th days by using the AccuSure Blood Glucose apparatus. On the whole, some of the synthesised compounds 5



Table 5 Anti-diabetic activity of synthesised compounds in alloxan-induced diabetic Wister rat. Treatments (mg/kg b.w p.o)

Blood glucose levels (mg/dl) 0-day

Normal Diseased Control Glibenclamide 4a 4b 4c 4d 4e 4f **

102.8 264.5 238.5 241.4 247.5 252.9 256.3 244.8 239.5

3-day ± ± ± ± ± ± ± ± ±

2.15 1.06 4.45* 2.56** 2.03* 1.71 2.69** 2.57* 3.02

102.1 286.7 215.3 230.2 264.6 270.3 269.2 231.9 226.4

5-day ± ± ± ± ± ± ± ± ±

1.69 1.43 1.43** 1.40* 2.05** 2.21** 2.02* 1.15 1.61*

103.0 307.4 156.0 163.9 239.6 253.1 281.4 166.5 158.5

7-day ± ± ± ± ± ± ± ± ±

2.26 1.436 1.08** 1.21* 2.48* 1.72* 2.58 1.26** 1.32**

101.5 373.8 135.4 144.5 254.5 274.1 287.6 154.9 138.7

± ± ± ± ± ± ± ± ±

1.41 1.778 1.26** 0.89** 1.24* 1.74 0.72* 1.87* 0.88*

P < 0.05, *P < 0.01 as compared to blank and standard respectively. Statistical analysis-One way ANOVA.

Table 6 Anti-inflammatory activity of synthesised compound. Compounds

Increases in paw volume 1h

4a 4b 4c 4d 4e 4f Control Indomethacin **

2h

0.29 0.31 0.36 0.34 0.32 0.28 0.38 0.24

± ± ± ± ± ± ± ±

*

0.44 0.46 0.54 0.51 0.49 0.41 0.71 0.35

0.015 0.012** 0.013* 0.025** 0.014* 0.012** 0.015 0.018

3h ± ± ± ± ± ± ± ±

**

0.033 0.025* 0.026** 0.037 * 0.042* 0.040** 0.037 0.014

0.42 0.44 0.53 0.50 0.47 0.41 0.74 0.37

4h ± ± ± ± ± ± ± ±

**

0.032 0.037* 0.028** 0.342** 0.031* 0.036** 0.052 0.046

0.43 0.46 0.55 0.52 0.49 0.42 0.74 0.38

± ± ± ± ± ± ± ±

0.015* 0.030** 0.021* 0.050** 0.037** 0.022* 0.024 0.017

P < 0.05, *P < 0.01as compared to blank and standard respectively. Statistical analysis-One way ANOVA.

Table 7 Percentage inhibition of paw oedema of synthesised compounds. Compounds

4a 4b 4c 4d 4e 4f Control Indomethacin

Percentage activity 1h

2h

3h

4h

23.68 18.42 05.26 10.52 15.78 26.31 – 36.84

38.02 35.21 23.94 28.16 30.98 42.25 – 50.70

43.24 40.54 28.37 32.43 36.48 44.59 – 51.35

41.89 37.83 25.67 29.72 33.78 43.24 – 48.64

Table 8 Mean ulcer index value of selected compounds.

Fig. 4. Graphical displays of stomach ulcer index of synthesised compounds against indomethacin and control groups.

Treatment

No of animals treated

Mean ulcer index (mm)

Control Compound 4a Compound 4f Indomethacin

6 6 6 6

0.33 8.25 5.59 3.42

± ± ± ±

Table 9 Structural activity relationship of synthesised compounds [4a–f] related to anti-diabetic activity.

0.14 0.28 0.56 0.17

5.2. Acute toxicity study Newly synthesised Mannich base derivatives were administered in a single dose of 5–300 mg/kg by using the stomach tube after suspended in 0.1% CMC. Mortality and behavioural changes were not observed with the dose of 5–50 mg/kg body weight. The animals died between the doses of 100–300 mg/kg body weight. Through OECD guidelines 423, LD5o of Mannich base derivatives was found to be 100 mg/kg. 1/ 10th of the LD5o value of synthesised compounds have been fixed as an oral dose for animals during the activity evaluation.

Compound

Anti-diabetic activity

4f (Br), 4a (NO2) and 4e (Cl) 4b (SO3H) 4c (CH3), 4d (C2H5)

Significant Interesting Least

Fig. 5. Structural activity relationship of synthesised compounds [4a–f].

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divergence in the anti-diabetic and anti-inflammatory activities due to the commitment of different electron withdrawing/donating groups on thiophene heterocyclic nucleus. Compounds bearing with electron withdrawing groups were shown highly significant activities as compared to standard drugs.

Table 10 Structural activity relationship of synthesised compounds [4a–f] related to anti-inflammatory activity. Compound

Anti-inflammatory activity

4f (Br), 4a (NO2) and 4b (SO3H) 4e (Cl) 4d (C2H5), 4c (CH3)

Significant Interesting Least

Acknowledgments All authors of this manuscript wish to express the gratitude to GIET School of Pharmacy, Rajahmundry, Andhra Pradesh, India, for providing research facilities and also thankful to Dr N. Murugeshan, Indian Institute of Technology, Chennai, Tamil Nadu, India, for providing a spectral data of synthesised compounds time to time.

5.3. Anti-diabetic activity The anti-diabetic activity of Mannich base derivatives (4a–f) was investigated in alloxan-induced diabetic rats using AccuSure Blood Glucose apparatus. The results revealed that all the synthesised compounds were shown a significant reduction in the serum glucose as compared with the control group of animals and exhibited remarkable anti-diabetic activity (Table 5). Compounds bearing with an electron withdrawing moiety on thiophene ring were found to be an excellent anti-diabetic activity. Therefore, compounds with bromo, nitro, chloro substitution (4f, 4a, 4e) were playing a crucial role in anti-diabetic activity. Moreover, a compound composed of with sulphonic acid moiety (4b) showed an interesting activity. Whereas, compounds with an electron donating group on thiophene ring abolishes the anti-diabetic activity. Therefore, compounds substituted with CH3 and C2H5 moiety (4c and 4d) would be un-favourable for anti-diabetic activity. On the whole, compound 4f has shown highest activity as compared to rest of the synthesised compound and the level of activity as similar to that of standard drugs. Here, Glibenclamide was used as a standard drug for the determination of anti-diabetic activity of synthesised compounds. The sequence of in vitro anti-inflammatory activity was found to be 4f > 4a > 4e > 4b > 4c > 4d (Table 9 & Fig. 5).

Declaration of interest The authors of this manuscript report that there are no conflicts of interest relevant to this research work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bjbas.2018.02.004. References Aeluri, R., Alla, M., Polepalli, S., Jain, N., 2015. Synthesis and anti-proliferative activity of imidazo[1,2-a]pyrimidine Mannich bases. Eur. J. Med. Chem. 100, 18–23. Baert, F., Cabanetos, C., Allain, M., Silvestre, V., Leriche, P., Blanchard, P., 2016. Thieno [2,3-b] indole-based small push-pull chromophores: synthesis, structure, and electronic properties. Org. Lett. 18, 1582–1585. Benachenhou, F., Mesli, A., Guilard, R., 2013. Synthesis of Schiff bases by aromatic amine condensation with 3,3′-bithiophenes-2,2′ and 4,4′-dicarbaldehydes. Arab. J. Chem. 6, 313–317. Bhupendra, M., Rahul, P.V., Soo, K.Y., Rafi, N., Enkhtaivan, G., Hwan, K.D., 2016. Synthesis of mannich base derivatives of berberine and evaluation of their anticancer and antioxidant effects. J. Chem. Res. 40, 63–125. Bogdanov, A.V., Vazykhova, A.M., Khasiyatullina, N.R., Krivolapov, D.B., Dobrynin, A.B., Voloshina, A.D., Mironov, V.F., 2016. New N-Mannich bases obtained from isatin and piperazine derivatives: the synthesis and evaluation of antimicrobial activity. Chem. Heterocycl. Compd. 52, 25–30. Datar, P.A., Limaye, S.A., 2015. Design and synthesis of mannich bases as benzimidazole derivatives as analgesic agents. Antiinflamm. Antiallergy Agents Med. Chem. 14, 35–46. Deep, A., Narasimhan, B., Aggarwal, S., Kaushik, D., Sharma, A.K., 2016. Thiophene scaffold as prospective central nervous system agent: a review. Cent. Nerv. Syst. Agents Med. Chem. 16, 58–64. Desai, N.C., Dodiya, A.M., Rajpara, K.M., Rupala, Y.M., 2014. Synthesis and antimicrobial screening of 1,3,4-oxadiazole and clubbed thiophene derivatives. J. Saudi Chem. Soc. 18, 255–261. Fathima, N., Ziaulla, M., Banu, A., Panchamukhi, S.I., Khazi, I.A., Begum, N.S., 2011. Synthesis, spectroscopy and X-ray crystal structure of 9-methyl-3-thiophen-2-Ylthieno [3,2-e][1,2,4] triazolo [4,3-c] pyrimidine-8-carboxylic acid ethyl ester. Am. J. Anal. Chem. 2, 371–375. Francisca, L., Rita, C., Jose, G.O., Peter, H.N., Michael, H.B., Jim, I., Catarina, C.M., Joana, B., Rui, M., 2004. Amidomethylation of amodiaquine: antimalarial N-Mannich base derivatives. Tetrahedron Lett. 45, 7663–7666. Gomez-Rivera, A., Aguilar-Mariscal, H., Romero-Ceronio, N., Roa-de la Fuente, L.F., Lobato-Gracia, C.E., 2013. Synthesis and anti-inflammatory activity of three nitro chalcones. Bioorg. Med. Chem. Lett. 23, 5519–5522. Gopi, C., Sastry, V.D., Dhanaraju, M.D., 2016. Synthesis and spectroscopic characterisation of novel bioactive molecule of 3-(2-substituted)-1H-indol-3-yl)-1-(thiophen-2yl) prop-2-en-1-one chalcone derivatives. Beni-Suef Univ. J. Appl. Sci. 5, 236–243. Gouda, M.A., Eldien, H.F., Girges, M.M., Berghot, M., 2016. Synthesis and antitumor evaluation of thiophene based azo dyes incorporating pyrazolone moiety. J. Saudi Chem. Soc. 20, 151–157. Idhayadhulla, A., Kumar, R.S., Nasser, A.J.A., Selvin, J., Manilal, A., 2014. Synthesis of some Mannich base derivatives and their antimicrobial activity study. Arab. J. Chem. 7, 994–999. Joshi, S., Khosla, N., Tiwari, P., 2004. In vitro study of some medicinally important Mannich bases derived from anti-tubercular agent. Bioorg. Med. Chem. 12, 571–576. Kifayatullah, M., Mustafa, M.S., Sengupta, P., Sarker, M.M.R., Das, A., Das, S.A., 2015. Evaluation of the acute and sub-acute toxicity of the ethanolic extract of Pericampylus glaucus (Lam.) Merr. In BALB/c mice. J. Acute Dis. 4, 309–345. Kumar, A.Y., Nandakumar, K., Handral, M., Talwar, S., Dhayabaran, D., 2011. Hypoglycaemic and anti-diabetic activity of stem bark extracts Erythrina indica in normal and alloxan-induces diabetic rats. Saudi Pharm. J. 19, 35–42. Kumar, R.S., Arif, I.A., Ahamed, A., Idhayadhulla, A., 2016. Anti-inflammatory and

5.4. Anti-inflammatory activity The anti-inflammatory activity of Mannich base derivatives (4a–f) was investigated by using carrageenan-induced paw oedema method using plethysmography (Gomez-Rivera et al., 2013). The results revealed that all the compounds exhibited the varying degree of antiinflammatory activity and are listed in Tables 6 and 7. Compounds bearing with an electron withdrawing moiety on thiophene ring were found to be an excellent anti-inflammatory activity. Therefore bromo, nitro, sulphonic acid substituted compounds 4f, 4a, 4b played a crucial role in the anti-inflammatory activity. Moreover, chloro moiety substituted compounds (4e) showed an interesting activity. Whereas, compounds with an electron donating groups on thiophene ring abolishes the anti-inflammatory activity. Hence, compounds substituted with C2H5 and CH3 moiety (4d and 4c) offered minimum anti-inflammatory activity. On the whole, compound 4f had shown highest anti-inflammatory activity as compared to rest of the synthesised compound and the level activity as similar to that of standard drugs. Indomethacin was used as a standard drug for the determination of anti-inflammatory activity. The sequence of in vitro anti-inflammatory activity of the synthesised compound was found to be 4f > 4a > 4b > 4e > 4d > 4c (Table 10). 6. Conclusion This study stated that easy way of synthesis of novel 1-(3-(N,N dimethylamino)-1-(5-substituted thiophene-2-yl)propylidene semicarbazone Mannich base derivatives and their pharmacological activities. The entirely synthesised compounds were characterised by FT-IR, 1 H NMR, 13C NMR, mass spectroscopy and estimated their pharmacological activities using alloxan-induced diabetic & carrageenan-induced paw oedema methods. The structural activity relationship of these compounds demonstrated that most of the test compounds were shown 7



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