A facile synthesis, in vitro antiinflammatory and antioxidant activity of novel benzimidazolylpyrano[2,3-d][1,3]thiazolocarbonitriles

*Corresponding Author:

R. Anisetti
Department of Pharmacy, University College of Technology, Osmania University, Hyderabad-500 007, India
E-mail: ravindernathanisetti@gmail.com

Abstract

The synthesis benzimidazolylpyrano[2,3-d][1,3]thiazolocarbonitriles (5a-j) were achieved by cyclocondensation of arylidene amino-benzo[d]imidazole-2-thiols (3a-j) with mercaptoacetic acid followed by cyclization with 2-(phenylmethylene)malononitrile. Further more, the present study aimed at the evaluation of in vitro antiinflammatory activity and antioxidant activity of synthetic compounds. All tested compounds showed appreciable activity against the standard drugs.

Keywords

Benzimidazolylpyrano[2,3-d][1,3]thiazolocarbonitrile, mercaptoacetic acid, Michael addition, cyclo condensation, antiinflammatory activity, antioxidant activity

Pyrano[2,3-d]thiazoles are biologically interesting compounds with diabetes, obesity, hyperlipidemia, and atherosclerotic diseases [1]. They are also known to show antimicrobial, bactericidal, fungicidal and molluscicidal activities [2,3]. Furthermore, benzimidazole a nitrogen containing heterocyclic provides an interesting building block for the synthesis of various biologically active compounds [4-6]. There are several classic examples of benzimidazole derivatives which possess useful pharmaceutical properties and they are marketed as commercial drugs. Fuberidazole and Benomyl can be used as antifungal agents. Benzimidazole derivatives are also reported to possess analgesic [7], anthelminthic [8], antiinflammatory [9], antiarthritic, and anti HIV activities [10].

Glycosidase inhibitors (GIs) have been isolated from plants and microorganisms. The importance of the glycosidases in the living organisms has been identified by the changing the biochemical processes using glycosidase inhibitors. Glycosidase inhibitors have many potential applications as agrochemicals, viral infection, cancer and genetic disorders [11]. Tetrahydro-3aH-pyrano [3,2,-d] thiazoles are found to be better glycosidase inhibitors (fig. 1) [12].

Fig. 1: Glycosidase inhibitor.
R= OAc, OH. R¹= OAc, OH.R²= CH₂F, CF₃, CH₂-O-CH₃, Ph, Ph-CH₂, NH₂, NH-CH₃, CH₃..

Based on biological activity of pyrano[2,3-d] thiazoles compounds and benzimidazoles, it seemed that introduction of benzimidazole, pyrano[2,3-d] thiazoles rings in a single molecular frame work may enhance the pharmacological activity of these compounds. As a sequel to our research on the design and synthesis of biologically active and pharmacologically important new heterocycles [13-16], it was thought worth while to synthesize the novel title compounds (5a–j) and to have them evaluated for their in vitro antiinflammatory and antioxidant activities.

Materials and Methods

All the melting points were determined on a Cintex melting point apparatus and are uncorrected. Analytical TLC was performed on Merck precoated 60 F254 silica gel plates. Visualization was done by exposing to iodine vapour. IR spectra (KBr pellet) were recorded on a PerkinElmer BX series FT-IR spectrometer. ¹H NMR spectra were recorded on a Varian Gemini 300 MHz spectrometer. ¹³C NMR spectra were recorded on a Bruker 75 MHz spectrometer. Chemical shift values are given in ppm (d) with tetramethylsilane as an internal standard. Mass spectral measurements were carried out by the EI method on a Joel JMC-300 spectrometer at 70 eV. The synthesis of compounds 5a-j was accomplished by synthetic route shown in scheme 1.

Scheme 1. Synthesis of benzimidazolylpyrano[2,3-d][1,3]thiazolo carbonitriles (5a-j). 3, 4, 5: a: R=H, R’=H. b: R=H, R’=NO₂. c: R=H, R’=Cl. d: R=H, R’=OCH₃.e: R=H,R’=N(CH₃)₂. f: R=H, R’=Br. g: R=OH, R’=H. h: R=OH, R’=Br: i: R=OH,R’=OCH₃.j: R=OH,R’=Cl.

Synthesis of (Z)-5-(arylideneamino)-1H-benzo[d] imidazole-2-thiols (3a-j)-general Procedure

A mixture of 5-amino-2-mercaptobenzimidazole 1 (0.01 mol) and aromatic aldehyde 2 (0.01 mol) in ethanol (15 ml) were refluxed for 4 h. The reaction mixture was cooled to room temperature, and poured on to crushed ice. The solid separated was filtered off and purified by recrystallization from ethylacetate to give (Z)-5-(arylidene amino)-1H-benzo[d]imidazole-2-thiols.

5-[(E)-1-Phenylmethylidene]amino-1H-benzo[d] imidazole-2-thiole(3a); Yield: 68%, mp: 116-118o, IR (cm^-1): 3121 (NH). ¹H NMR (300 MHz, CDCl₃): 3.24 (s, 1H, =CH), 4.03 (s, 1H, SH), 7.1-7.34(m, 8H, ArH), 7.93 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 107.4, 114.8, 119.0, 126.8, 130.2, 130.8, 131.2, 133.0, 138.4, 139.2, 142.4, 145.3, 158.0, 166.0. Anal. calcd for C₁₄H₁₁N₃S (MW=254 [M+H]⁺): C, 66.34; H, 4.34; N, 16.54. Found: C, 66.38; H,. 38; N, 16.59. 5-[(E)-1-(4-Nitrophenyl)methylidene]amino-1Hbenzo[ d]imidazole-2-thiole(3b); Yield: 71%, mp: 132-134o, IR(cm^-1): 3210(NH), 1535,1326 (NO₂). ¹H NMR (300 MHz, CDCl₃): 3.33 (s, 1H, =CH), 4.01 (s, 1H, SH), 7.10-7.32 (m, 7H, ArH), 7.98 (s, 1H, NH, D₂Oe changeable).¹³C NMR (75 MHz, CDCl₃): 106.4, 114.2, 115.8, 125.4, 125.5, 126.0, 126.1, 135.4, 142.6, 144.7, 145.1, 153.2, 162.2, 166.2. Anal. Calcd for C₁₄H₁₀N₄O₂S (MW=298[M+H]⁺): C, 56.37; H, 3.38; N, 18.78. Found: C, 56.33; H, 3.34; N, 18.7.

5-[(E)-1-(4-Chlorophenyl)methylidene]amino-1Hbenzo[ d]imidazole-2-thiole (3c); Yield: 69%, mp: 121-123o, IR (cm^-1): 3121 (NH). ¹H NMR (300 MHz, CDCl₃): 4.00 (s, 1H, SH), 4.31 (s, 1H, =CH), 7.00-7.98 (m, 7H, ArH), 8.13 (s, 1H, NH, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 107.8, 114.4, 115.2, 130.9, 131.9, 132.2 ,132.3, 132.4, 133.2, 135.6, 144.3, 145.6, 165.2, 166.1. Anal. Calcd for C₁₄H₁₀ClN₃S (MW=287[M+H]⁺): C, 58.43; H, 3.50; N, 14.60. Found: C, 58.39; H, 3.47; N, 14.57.

5-[(E)-1-(4-Methoxyphenyl)methylidene]amino- 1H-benzo[d]imidazole-2-thiole (3d); Yield: 74%, mp:126-128o, IR (cm^-1): 3245 (NH).¹H NMR (300 MHz, CDCl₃): 3.62(s, 3H, OCH3) 4.08 (s, 1H, SH), 4.19m (s, 1H, =CH), 6.99-7.89 (m, 7H, ArH), 9.00(s, 1H, NH, D₂O exchangeable). ¹³CNMR (75 MHz, CDCl): 53.8, 107.5, 112.4, 116.4, 118.3, 119.2, 128.2, 133.0, 132.4, 135.3, 142.4, 145.3, 160.5, 163.0, 166.4. Anal. Calcd for C₁₅H₁₃N₃OS (MW=283 [M+H]⁺): C, 63.58; H, 4.62; N, 14.83. Found: C, 63.55; H, 4.59; N, 14.80.

5 - ( ( E ) - 1 - [ 4 - ( Dimethylamino ) p h e n y l ] methylideneamino)-1H-benzo[d]imidazole-2-thiole (3e); Yield: 71%, mp: 129-131o, IR (cm^-1): 3115 (NH). ¹H NMR (300 MHz, CDCl₃): 3.00 (s, 6H, N(CH3)2), 3.33 (s, 1H, =CH), 4.00 (s, 1H, SH), 6.93-7.26 (m, 7H, ArH), 8.48 (s, 1H, NH, D₂O exchangeable). ¹³CNMR (75 MHz, CDCl₃): 41.4, 43.1, 109.7, 111.0, 113.9, 114.3, 115.2, 126.0, 127.6, 130.3, 139.2, 142.4, 145.3, 155.4, 162.1, 166.2. Anal.Calcd for C₁₆H₁₆N₄S (MW=296[M+H]⁺): C, 64.84; H, 5.44; N, 18.90. Found: C, 64.81; H, 5.42; N, 18.88.

5-[(E)-1-(4-Bromophenyl)methylidene]amino-1Hbenzo[ d]imidazole-2-thiole(3f); Yield: 70%, mp: 118-120o, IR (cm^-1): 3115 (NH). ¹H NMR (300 MHz, CDCl₃): 4.11 (s, 1H, SH), 4.26 (s, 1H, =CH), 7.13-98 (m, 7H, ArH), 9.31 (s, 1H, NH, D₂O exchangeable).¹³CNMR(75 MHz, CDCl₃):111.2,11 4.5,115.0,127.4,130.2,130.5, 132.7, 132.7, 133.2, 135.2, 138.2, 145.6, 158.0, 166.4. Anal. Calcd for C₁₄H₁₀BrN₃S (MW=332[M+H]⁺): C, 50.62; H, 3.03; N, 12.65. Found: C, 50.66; H, 3.06; N, 12.69.

4-[(2-Sulfanyl-1H-benzo[d]imidazol-5-yl)imino] methylphenol(3g); Yield: 73%, mp: 123-125o, IR (cm^-1): 3210 (NH). ¹H NMR (300 MHz, CDCl₃): 4.00 (s, 1H, SH), 4.21 (s, 1H, =CH),7.04-.89 (m, 7H, ArH), 8.96 (s, 1H, NH, D₂O exchangeable), 9.13 (s, 1H, OH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 111.5, 114.3, 115.4, 119.0, 122.5, 123.4, 133.1, 134.4, 135.2, 142.2, 145.3, 162.0, 163.1, 166.2. Anal. Calcd for C₁₄H₁₁N₃OS (MW =269[M+H]⁺): C, 62.44; H, 4.12; N, 15.60. Found: C, 62.40; H, 4.17; N, 15.58.

5-Bromo-2-[(2-sulfanyl-1H-benzo[d]imidazol-5-yl) imino]methylphenol(3h); Yield: 69%, mp: 136-138o, IR (cm^-1): 3225 (NH). ¹H NMR (300 MHz, CDCl₃): 4.18 (s, 1H, SH), 4.29 (s, 1H, =CH), 7.00-7.79 (m, 6H, ArH), 8.86 (s, 1H, NH, D₂O exchangeable), 9.23 (s, 1H, OH, D₂O exchangeable). ¹³CNMR (75 MHz, CDCl₃): 107.8, 115.0, 117.2, 117.5, 121.5, 122.1, 123.2, 134.2, 135.2, 142.2, 145.6, 162.0, 161.0, 166.2. Anal. Calcd for C₁₄H₁₀BrN₃OS (MW=348[M+H]⁺): C, 48.29; H, 2.89; N, 12.07. Found: C, 48.24; H, 2.93; N, 12.02.

5-Methoxy-2-[(2-sulfanyl-1H-benzo[d]imidazol-5- yl)imino]methylphenol(3i); Yield: 74%, mp:139- 141o, IR(cm^-1): 3205(NH).¹H NMR (300 MHz, CDCl₃): 3.58 (s, 3H, OCH3),4.10 (s, 1H, SH), 4.26 (s, 1H,=CH), 6.99-7.94 (m, 6H, ArH), 8.46 (s, 1H, NH, D₂O exchangeable), 8.73 (s, 1H, OH, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 56.3, 102.1, 109.0, 111.2, 114.3, 119.0, 119.3, 135.3,135.4, 142.1, 145.3, 152.3, 162.1, 165.1, 166.2. Anal. Calcd for C₁₅H₁₃N₃O₂S (MW=299[M+H]⁺): C,60.19; H, 4.38; N, 14.04. Found: C, 60.21; H, 4.41; N, 14.00.

5-Chloro-2-[(2-sulfanyl-1H-benzo[d]imidazol-5-yl) imino]methylphenol(3j); Yield: 75%, mp: 143-145o, IR (cm^-1): 3300 (NH). ¹H NMR(300 MHz, CDCl₃): 4.00(s, 1H, SH), 4.2(s, 1H, =CH), 6.89-7.76(m, 6H, ArH), 8.04 (s, 1H, NH, D₂O exchangeable), 8.61 (s,1H, OH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 111.2, 114.5, 115.0, 116.3, 119.8, 123.2, 134.0, 137.2, 139.8, 143.0, 145.3, 162.0, 164.2, 166.2. Anal. Calcd for C₁₄H₁₀ClN₃OS (MW=303 [M+H]⁺):C,55.36; H, 3.32; N, 13.83.Found: C,55.32; H, 3.36; N, 13.87.

Synthesis of 2-aryl-3-(2-sulfanyl-1H-benzo[d] imidazol-5-yl)-1,3-thiazolan-4-ones (4a-j) general procedure

A solution of (Z)-5-(arylidene amino)-1H-benzo[d] imidazole-2-thiol 3 (0.01 mol), and thioglycollic acid (0.01 mol) in ethanol (15 ml) were heated under reflux for 6 h. The reaction mixture was cool to room temperature and poured into ice-cold water. The precipitate that formed was filtered off, and purified by recrystallization from ethyl acetate to afford the product.

2-Phenyl-3-(2-sulfanyl-1H-benzo[d]imidazol-5-yl)- 1,3-thiazolan-4-one(4a); Yield: 70%, mp: 151-153o, IR(cm^-1): 3310 (NH), 1695 (C=O).1HNMR (300 MHz, CDCl₃): 4.12 (s, 2H, thiazole-CH2), 4.25 (s, 1H, SH), 5.62 (s, 1H, thiazole–CH), 6.89-7.46 (m, 8H, ArH), 9.12 (s, 1H, NH, D₂O exchangeable). ¹³CNMR (75 MHz, CDCl₃): 32.5, 41.2, 66.6, 111.3, 113.2, 116.1, 124.9, 125.5, 128.5, 129.1, 130.2, 130.3, 133.2, 136.3, 137.4, 173.8. Anal. calcd for C₁₆H₁₃N₃OS₂ (MW=327 [M+H]⁺): C, 58.69; H, 4.00; N, 12.83. Found: C, 58.73; H,4.03; N,12.88.

2-(4-Nitrophenyl)-3-(2-sulfanyl-1H-benzo[d]imidazol- 5-yl)-1,3-thiazolan-4-one(4b); Yield: 73%, mp: 171- 173o, IR (cm^-1): 3300 (NH), 1542, 1340 (NO₂), 1715 (C=O).¹H NMR (300 MHz, CDCl₃): 4.10 (s, 2H, thiazole-CH2),4.24 (s, 1H, SH), 5.42 (s, 1H, thiazole– CH), 7.00-7.3 (m, 7H, ArH), 8.63 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 33.5, 65.6, 107.3, 117.3, 125.3, 125.5, 125.6, 126.9, 127.6, 133.4, 136.5, 137.1, 147.3, 148.3, 165.9, 173.3. Anal. calcd for C₁₆H₁₂N₄O₃S₂ (MW=372[M+H]⁺): C, 51.60; H, 3.25; N, 15.04. Found: C, 51.57; H, 3.23; N, 15.01.

2-(4-Chlorophenyl)-3-(2-sulfanyl-1H-benzo[d]imidazol- 5-yl)-1,3-thiazolan-4-one(4c); Yield: 70%, mp: 181- 183o, IR (cm^-1): 3305 (NH), 1710 (C=O). ¹H NMR (300 MHz, CDCl₃): 4.10 (s, 2H, thiazole-CH2), 4.19 (s, 1H, SH), 5.31 (s, 1H, thiazole –CH), 6.94-7.77 (m, 7H, ArH), 8.62 (s, 1H, NH, D₂O exchangeable).13 C NMR (75 MHz, CDCl₃): 34.8, 64.4, 111.5, 113.2,116.2, 125.5, 128.1, 130.5, 130.5, 132.3, 133.2, 135.2, 137.3, 135.5, 137.7, 166.2 Anal.calcd for C₁₆H₁₂ClN₃OS₂ (MW=361[M+H]⁺): C, 53.11; H, 3.34; N, 11.61. Found: C, 53.14; H, 3.32; N, 11.58.

2-(4-Methoxyphenyl)-3-(2-sulfanyl-1H-benzo[d] imidazol-5-yl)-1,3-thiazolan-4-one(4d); Yield: 71%, mp: 178-181o. IR(cm^-1): 3117(NH), 1705 (C=O). 1H NMR(300 MHz, CDCl₃): 3.60 (s, 3H, OCH3).4.00 (s, 2H, thiazole-CH2), 4.26(s, 1H, SH), 5.52 (s, 1H, thiazole–CH), 6.92-7.84 (m, 7H, ArH),8.46 (s, 1H, NH, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 34.5, 56.8, 67.6,111.2,112.5,113.2,116.4,125.5, 127.3, 131.5, 132.3,133.1,133.3,141.1,163.7,170.0,173.2. Anal. calcd for C₁₇H₁₅N₃O₂S₂(MW=357 [M+H]⁺): C, 57.12;H, 4.23 N, 11.76. Found: C, 57.16; H, 4.26; N, 11.73.

2-[4-(Dimethylamino)phenyl]-3-(2-sulfanyl-1Hbenzo[ d]imidazol-5-yl)-1,3-thiazolan-4-one(4e); Yield: 69%, mp:160-162o. IR (cm^-1): 3325 (NH), 1725 (C=O). ¹H NMR (300 MHz, CDCl₃): 4.18 (s, 2H, thiazole-CH2), 4.23 (s, 1H, SH), 5.49 (s, 1H, thiazole–CH), 7.10-7.93 (m, 7H, ArH), 8.24 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 34.5, 41.2, 43.1, 66.8, 111.5, 114.3, 114.4, 117.3, 125.1, 125.5, 129.2, 130.7, 132.3, 133.1, 137.9, 151.5, 166.2, 172.2. Anal. Calcd for C₁₈H₁₈N₄OS₂(MW=370[M+H]⁺): C, 58.36; H, 4.90; N, 15.12. Found: C, 58.34; H, 4.94; N, 15.17.

2-(4-Bromophenyl)-3-(2-sulfanyl-1H-benzo[d]imidazol- 5-yl)-1,3-thiazolan-4-one(4f);Yield:71%,mp: 186-188o. IR (cm^-1): 3310 (NH), 1720(C=O).¹H NMR (300 MHz, CDCl₃): 4.11 (s, 2H, thiazole-CH2), 4.26 (s, 1H, SH), 5.44 (s, 1H, thiazole–CH), 7.00-7.86 (m, 7H, ArH), 8.10 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃):33.5, 65.6, 111.1, 113.2, 123.3, 125.5, 132.1, 132.4, 132.7, 133.2, 133.3, 133.4, 136.2, 141.1, 170.0, 173.2. Anal. calcd for C₁₆H₁₂BrN₃OS₂ (MW=406[M+H]⁺): C, 47.30; H, 2.98; N, 10.34. Found: C, 47.33; H, 2.92; N, 10.30.

2-(2-Hydroxyphenyl)-3-(2-sulfanyl-1H-benzo[d] imidazol-5-yl)-1,3-thiazolan-4-one(4g); Yield: 70%, mp: 189-191o. IR (cm^-1): 3208 (NH), 1709 (C=O). 1H NMR (300 MHz, CDCl₃): 4.12 (s, 2H, thiazole-CH2), 4.4 1(s, 1H, SH), 5.13 (s, 1H, thiazole–CH), 6.93-7.84 (m, 7H, ArH), 9.21 (s, 1H, NH,D₂O exchangeable), 9.46 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 34.8, 60.5, 111.5, 117.2, 117.5, 120.1, 121.2, 125.6, 130.0, 130.3, 133.1, 135.7, 137.2, 155.5, 166.1, 173.3. Anal. Calcd for C₁₆H₁₃N₃O₂S₂ (MW=343[M+H]⁺): C, 55.96; H, 3.82; N, 12.24. Found: C, 55.99; H, 3.86; N, 12.26.

2-(4-Bromo-2-hydroxyphenyl)-3-(2-sulfanyl-1Hbenzo[ d]imidazol-5-yl)-1,3-thiazolan-4-one(4h); Yield: 73%, mp: 175-177 o. IR (cm^-1): 3300 (NH), 1725 (C=O). ¹H NMR(300 MHz, CDCl₃): 4.14(s, 2H, thiazole-CH2), 4.26(s, 1H, SH), 5.26(s, 1H, thiazole –CH), 7.10-7.86 (m, 6H, ArH), 9.00(s, 1H, NH, D₂O exchangeable). ¹³CNMR (75 MHz, CDCl₃): 33.5, 59.4, 111.3, 113.2, 115.1, 121.3, 123.3, 125.3, 126.3, 132.2, 133.2, 135.2, 141.1, 160.0, 166.1, 173.5. Anal. calcd for C₁₆H₁₂BrN₃O₂S₂ (MW=422 [M+H]⁺): C, 45.51; H, 2.86; N, 9.95. Found: C, 45.55; H, 2.89; N,9.99.

2-(2-Hydroxy-4-methoxyphenyl)-3-(2-sulfanyl- 1H-benzo[d]imidazol-5-yl)-1,3-thiazolan-4-one (4i);Yield:71%, mp: 155-157o. IR (KBr cm^-1): 3315(NH), 1745(C=O). 1HNMR(300 MHz, CDCl₃): 3.63(s, 3H, OCH3), 4.00 (s, 2H, thiazole-CH2), 4.18 (s, 1H, SH), 5.20 (s, 1H, thiazole–CH), 7.05-7.93 (m, 6H, ArH), 9.21 (s, 1H, NH, D₂O exchangeable), 10.02(s, 1H, OH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 34.8, 56.9, 58.9, 103.3, 104.4, 111.5, 112.4, 113.2, 125.4, 132.3, 133.1, 133.3, 141.1, 159.2, 158.4, 166.1, 173.0. Anal. calcd for C₁₇H₁₅N₃O₃S₂ (MW=373 [M+H]⁺): C, 54.68; H, 4.05; N, 11.25. Found: C, 54.63; H, 4.01; N, 11.22.

2-(4-Chloro-2-hydroxyphenyl)-3-(2-sulfanyl-1Hbenzo[ d]imidazol-5-yl)-1,3-thiazolan-4-one (4j); Yield: 72%, mp: 156-158o. IR(cm^-1): 3345(NH), 1735(C=O). ¹H NMR (300 MHz, CDCl₃): 4.10 (s, 2H, thiazole- CH2), 4.20(s,1H,SH), 5.31 (s, 1H, thiazole–CH), 7.00-7.36 (m, 6H, ArH), 9.00 (s, 1H, NH, D₂O exchangeable),10.01 (s, 1H, OH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 34.8, 60.4, 111.5, 117.2, 118.2, 118.4, 123.2, 125.6, 133.2, 133.3, 136.3, 137.1, 140.2, 155.2, 166.2, 173.2. Anal. calcd for C₁₆H₁₂ClN₃ O₂ S₂ (MW=377[M+H]⁺): C, 50.86; H, 3.20; N, 11.12. Found: C, 50.89; H, 3.24; N, 11.10.

Synthesis of 5-amino-2,7-diaryl-3-(2-sulfanyl-1Hbenzo[ d]imidazol-5-yl)-3,7-dihydro-2H-pyrano [2,3-d][1,3]thiazole-6-carbonitriles (5a-j), general procedure

To a solution of 2-aryl-3-(2-sulfanyl-1H-benzo[d] imidazol-5-yl)-1,3-thiazolan-4-one 4(0.01 mol) in ethanol (15 ml) 2-(phenyl methylene) malononitrile (0.01 mol) was added. The reaction mixture was heated at 80o for 4 h and then poured into ice-cold water. The separated solid was collected by filtration, dried and purified by recrystallization from ethyl acetate.

(5-Amino-2,7-diphenyl-3-(2-sulfanyl-1H-benzo[d] imidazol-5-yl)-3,7-dihydro-2H-pyrano[2,3-d] [1,3] thiazole-6-carbonitrile (5a); Yield: 73%, mp: 218-220o. IR(cm^-1): 3410(NH₂), 3118 (NH), 2204 (C=N).1H NMR (300 MHz, CDCl₃): 4.01 (s, 1H, SH), 4.26 (s, 1H, CH), 5.56 (s, 1H, CH), 6.86-7.86 (m, 13H, ArH), 8.00 (s, 1H, NH, D₂O exchangeable), 9.10 (s, 2H, NH₂, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 18.7, 23.8, 52.9, 53.4, 85.6, 103.2, 117.3, 118.2, 122.4, 123.3, 125.1, 126.4, 127.2, 129.1, 130.7, 139.4, 141.3, 141.4, 142.1, 143.2, 156.2, 166.1.Anal. Calcd for C₂₆H₁₉N₅OS₂ (MW=481[M+H]⁺): C, 64.84; H, 3.98; N, 14.54. Found: C, 64.80; H, 3.95; N, 14.59.

5-Amino-2-(4-nitrophenyl)-7-phenyl-3-(2-sulfanyl-1Hbenzo[ d]imidazol-5-yl)-3,7-dihydro-2H-pyrano[2,3-d] [1,3]thiazole-6-carbonitrile(5b); Yield: 71%, mp: 231-233o. IR (cm^-1): 3425 (NH₂), 3300 (NH), 2200 (C=N),1538, 1320 (NO₂). ¹H NMR (300 MHz, CDCl₃): 4.16 (s, 1H, SH), 5.15 (s, 1H, CH), 5.36 (s, 1H, CH), 6.93-7.80 (m, 12H, ArH), 8.42 (s, 1H, NH, D₂O exchangeable), 8.82 (s, 2H, NH₂, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 42.5, 53.4, 57.3, 81.2, 103.3, 117.3, 119.0, 120.1, 125.2, 125.6, 126.6, 127.4, 129.4, 129.7, 129.7, 130.2, 130.3, 130.4, 139.4, 141.3, 143.4, 144.2, 148.5, 150.0, 160.1, 170.5. Anal. calcd for C₂₆H₁₈N₆O₃S₂ (MW=526[M+H]⁺): C, 59.30; H, 3.45; N, 15.96.F ound: C, 59.34; H, 3.42; N, 15.94.

5-Amino-2-(4-chlorophenyl)-7-phenyl-3-(2-sulfanyl- 1H-benzo[d]imidazol-5-yl)-3,7-dihydro-2Hpyrano[ 2,3-d][1,3]thiazole-6-carbonitrile(5c); Yield: 70%,mp: 197-199o.IR (cm^-1): 3400 (NH₂), 3200 (NH), 2205 (C=N). ¹H NMR (300 MHz, CDCl₃): 4.26(s, 1H, SH), 5.14(s, 1H, CH), 5.44 (s, 1H, CH), 7.00-7.84 (m, 12H, ArH), 8.26 (s, 1H, NH, D₂O exchangeable), 8.80 (s, 2H, NH₂, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 41.3, 52.4, 58.2, 80.1, 103.4, 117.4, 119.0, 121.1, 127.1, 126.8, 129.3, 127.4, 129.4, 129.4, 130.1, 130.1, 130.2, 130.4, 130.6, 139.4, 141.3, 142.1, 143.4, 144.4, 161.2, 166.6. Anal. calcd for C₂₆H₁₈ClN₅OS₂ (MW=516[M+H]⁺): C, 60.52; H, 3.52; N, 13.57. Found: C, 60.49; H, 3.49; N, 13.54.

5-Amino-2-(4-methoxyphenyl)-7-phenyl-3-(2- sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro-2Hpyrano[ 2,3-d][1,3]thiazole-6-carbonitrile (5d); Yield: 74%, mp: 206-208o. IR (cm^-1): 3410 (NH₂), 3245 (NH), 2200 (C=N). ¹H NMR (300 MHz, CDCl₃): 3.57 (s, 3H, OCH3), 4.08 (s, 1H, SH), 5.21 (s, 1H, CH), 5.34 (s, 1H, CH), 7.05-7.96 (m, 12H, Ar H), 8.00 (s, 1H, NH, D₂O exchangeable), 8.96 (s, 2H, NH₂, D₂O exchangeable).¹³C NMR (75 MHz,CD Cl3): 42.5, 52.2, 54.4, 56.9, 81.8, 103.4, 112.3, 116.3, 117.6, 118.0, 121.1, 126.7, 127.3, 129.0, 129.3, 129.4, 130.3, 130.3 ,130.5, 132.2, 139.5, 141.4, 143.3, 144.1, 160.0, 160.2, 166.2. Anal. calcd for C₂₇H₂₁N₅O₂S₂ (MW=511[M+H]⁺): C, 63.39; H, 4.14; N, 13.69. Found: C, 63.36; H, 4.11; N, 13.64.

5-Amino-2-[4-(dimethylamino)phenyl]-7-phenyl- 3-(2-sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7- dihydro -2H-pyrano[2,3-d][1,3]thiazole-6-carbonitrile (5e); Yield: 69%,mp: 193-195o. IR (cm^-1): 3400 (NH₂), 3210 (NH), 2205 (C=N). ¹H NMR (300 MHz, CDCl₃): 3.12 (s, 6H, N (CH3)2), 4.00 (s, 1H, SH), 5.26 (s, 1H, CH), 5.40 (s, 1H, CH), 7.00-7.85 (m, 12H, ArH), 8.66 (s, 1H, NH,D₂O exchangeable), 9.3 (s, 2H, NH₂, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 39.9, 41.2, 41.5, 55.4, 58.9, 83.0, 103.6, 114.2, 114.9, 117.6, 119.0, 121.4, 128.4, 128.4, 127.4, 129.4, 129.4, 129.3, 130.5, 130.6, 133.5, 139.9, 140.7, 143.6, 142.6, 144.5, 151.4, 161.2, 170.2. Anal. Calcd for C₂₈H₂₄N₆OS₂(MW=524[M+H]⁺): C, 64.10; H, 4.61; N, 16.02. Found: C,64. 15; H, 4.69; N, 16.07.

5-Amino-2-(4-bromophenyl)-7-phenyl-3-(2- sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro- 2H-pyrano[2,3-d][1,3]thiazole-6-carbonitrile (5f); Yield: 70%, mp: 201-203o. IR(cm^-1): 3405 (NH₂), 3200 (NH), 2220 (C=N). ¹H NMR(300 MHz, CDCl₃): 4.14 (s, 1H, SH), 5.18 (s, 1H, CH), 5.36 (s, 1H, CH), 6.86-7.68 (m, 12H, ArH), 8.41(s, 1H, NH, D₂O exchangeable), 9.00 (s, 2H, NH₂, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 42.5, 54.4, 57.9, 83.0, 103.4, 113.6, 119.0, 121.1, 123.4, 125.4, 126.6, 126.6, 127.7, 129.3, 129.7, 133.6, 130.4, 131.9, 132.6, 137.7, 139.9, 144.2, 142.4, 142.9, 161.2, 166.2. Anal. calcd for C₂₆H₁₈BrN₅OS₂ (MW=560[M+H]⁺): C, 55.72; H, 3.24; N, 12.50. Found: C, 55.70; H, 3.28; N, 12.56.

5-Amino-2-(2-hydroxyphenyl)-7-phenyl-3-(2- sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro-2Hpyrano[ 2,3-d][1,3]thiazole-6-carbonitrile(5g);Yield: 73%, mp: 211-213o. IR (cm^-1): 3410 (NH₂),3225 (NH), 2210 (C=N).¹H NMR (300 MHz, CDCl₃): 4.06 (s, 1H, SH), 5.14 (s, 1H, CH), 5.42 (s, 1H, CH), 7.05-7.86 (m, 12H, ArH), 8.66 (s, 1H, NH, D₂O exchangeable), 9.26 (s, 2H, NH₂, D₂O exchangeable), 9.40 (s, 1H, OH, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 42.5, 52.2, 54.9, 81.0, 103.4, 113.6, 113.9, 114.0, 121.1, 122.8, 123.3, 123.7, 125.3, 125.7, 125.7, 126.2, 126.5, 129.0, 130.6, 135.9, 137.7, 143.3, 144.2, 154.7, 157.2, 166.2. Anal. calcd for C₂₆H₁₉N₅O₂S₂ (MW=497[M+H]⁺): C, 62.76; H,3.85; N, 14.07. Found: C, 62.73; H, 3.84; N, 14.09.

5-Amino-2-(4-bromo-2-hydroxyphenyl)-7-phenyl-3- (2-sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro- 2H-pyrano[2,3-d][1,3]thiazole-6-carbonitrile(5h); Yield: 69%, mp: 225-227o. IR (cm^-1): 3405 (NH₂), 3310 (NH), 2200 (C=N).¹H NMR (300 MHz, CDCl₃): 4.10 (s, 1H, SH), 4.26 (s, 1H, CH), 5.26 (s, 1H, CH), 6.93-7.71 (m, 11H, ArH), 8.42 (s, 1H, OH, D₂O exchangeable), 9.01 (s,1H, NH, D₂O exchangeable), 9.25 (s, 2H, NH₂, D₂O exchangeable). ¹³C NMR (75 MHz, CDCl₃): 41.5, 53.2, 54.9, 83.0, 103.4, 113.3, 117.4, 119.0, 121.1, 121.2, 123.7, 123.7, 125.7, 126.2, 126.6, 126.6, 129.3, 129.7, 133.3, 139.4, 141.3, 142.2, 144.2, 159.0, 157.2, 166.2. Anal. calcd for C₂₆H₁₈BrN₅O₂S₂ (MW=576[M+H]⁺): C, 54.17; H, 3.15; N, 12.15. Found: C, 54.18; H, 3.18; N, 12.10.

5-Amino-2-(2-hydroxy-4-methoxyphenyl)-7-phenyl- 3-(2-sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro- 2H-pyrano[2,3-d][1,3]thiazole-6-carbonitrile(5i); Yield: 74%, mp: 215-217o. IR(cm^-1): 3424 (NH₂), 3249 (NH), 2203 (C=N). 1HNMR (300 MHz, CDCl₃): 3.64 (s, 3H, OCH3), 4.10 (s, 1H, SH), 5.25 (s, 1H, CH), 5.26 (s, 1H, CH), 7.05-7.82 (m, 13H, ArH), 8.20 (s, 1H, NH, D₂O exchangeable), 8.64 (s, 2H, NH₂, D₂O exchangeable), 8.92 (s, 1H, OH, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃):41.5, 52.2, 52.2, 53.9, 54.8, 83.0, 103.0, 103.4, 108.9, 112.4, 114.0, 115.1, 117.6, 123.7, 129.3, 129.4, 129.6, 129.7, 130.6, 132.1, 135.9, 141.2, 143.4, 144.2, 153.8, 155.9, 161.2, 170.2. Anal. calcd for C₂₇H₂₁N₅O₃ S₂ (MW=527[M+H]⁺): C, 61.46; H, 4.01; N, 13.27. Found: C, 61.42; H, 4.06; N, 13.22.

5-Amino-2-(4-chloro-2-hydroxyphenyl)-7-phenyl-3-(2- sulfanyl-1H-benzo[d]imidazol-5-yl)-3,7-dihydro-2Hpyrano[ 2,3-d][1,3]thiazole-6-carbonitrile(5j); Yield: 75%, mp: 220-222o. IR (cm^-1): 3410 (NH₂), 3305 (NH), 2205 (C=N). 1HNMR (300 MHz, CDCl₃): 4.25 (s, 1H, SH), 5.05(s, 1H, CH), 5.32 (s, 1H, CH), 7.00-7.63 (m, 11H, ArH), 8.10 (s, 1H, NH, D₂O exchangeable), 8.42 (s, 2H, NH₂, D₂O exchangeable), 8.61 (s, 1H, OH, D₂O exchangeable).¹³C NMR (75 MHz, CDCl₃): 40.5, 50.2, 59.9, 82.0, 103.4, 113.6, 114.7, 115.0, 117.1, 120.9, 123.4, 123.7, 125.7, 125.7, 127.6, 129.3, 130.6, 132.5, 133.3, 137.7, 139.9, 143.4, 144.2, 157.2, 158.2, 166.2. Anal. calcd for C₂₆H₁₈ClN₅O₂S₂ (MW=532[M+H]⁺): C, 58.70; H, 3.41; N, 13.16. Found: C, 58.63; H, 3.44; N, 13.19.

In vitro antiinflammatory activity by human red blood cell membrane stabilization method

Human red blood cell membrane stabilization method (HRBC method) was used for the estimation of antiinflammatory activity in vitro [17]. Blood was collected from healthy volunteers and was mixed with equal volume of sterilized Alsevers solution. This blood solution was centrifuged at 3000 rpm and the packed cells were separated. The packed cells were washed with isosaline solution and a 10% v/v suspension was made with isosaline. This HRBC suspension was used for the estimation of antiinflammatory property. The concentrations of synthetic compounds 100 µg/ml, reference sample and control were separately mixed with 1 ml of phosphate buffer, 2 ml of hyposaline and 0.5 ml of HRBC suspension. All the assay mixtures were incubated at 37° for 30 min and centrifuged at 3000 rpm. The supernatant liquid was decanted and the hemoglobin content was estimated by a spectrophotometer at 560 nm. Content was estimated by a spectrophotometer at 560 nm. The percent hemolysis was estimated by assuming the hemolysis produced in the control as 100%, percent protection =100-(OD sample/OD control)×100. Antiinflammatory activity data of newly synthesized compounds resulted are shown in Table 1.

Table 1: In Vitro anti inflammatory Of Benzimidazolylpyrano[2,3-D][1,3] Thiazolocarbonitriles (5a-J)

Antioxidant assay

For the evaluation of antioxidant activity, we have used a stable-free radical a,a-diphenyl- ß-picrylhydrazyl (DPPH), at the concentration of 0.2 mM in methanol [18]. To 0.1 ml of the test compound (at different concentrations), 1.5 ml of methanol and 0.5 ml of DPPH solution were added, mixed thoroughly and absorbance (OD) was read at 517 nm against the blank. The % reduction of the free radical concentration (OD) with different concentration of test compounds was calculated and was compared with standard ascorbic acid. The results were expressed as IC50values (the concentration of the test required to scavenge 50% free radicals). The antioxidant DPPH free radical scavenging activity of all the synthesized compounds performed using DPPH method results shown in Table 2.

Table 2: Antioxidant Activity Of Benzimidazolylpyrano[2,3-D][1,3] Thiazolocarbonitriles (5a-J)

Results and Discussion

The synthesis of the compounds (3-5) was accomplished by the synthetic route shown in scheme 1. The reaction of 5-amino-2- mercaptobenzimidazole (1), with substituted aldehydes (2) in refluxing ethanol furnished (Z)- 5-(arylideneamino)-1H-benzo[d]imidazole-2-thiols (3) in quantitative yields (68-74%). Cyclocondensation of 3 with mercaptoacetic acid in boiling ethanol afforded benzimidazolyl-1,3-thiazolan-4-ones (4). Compound 4 reacted with 2-(phenylmethylene) malononitrile in boiling ethanol delivered the corresponding benzimidazolylpyrano[2,3-d][1,3] thiazolecarbonitrile (5). The formation benzimidazolyl pyrano[2,3-d][1,3]thiazolecarbonitriles is explained with the plausible mechanism (Scheme 2). The reaction occurs via an initial Michael addition of the endocyclic methylene group in 4 to the activated double bond in 6 to give the intermediate A, which underwent cyclization through addition of oxygen to the nitrile functional group followed by autooxidation [19] to afford the final product 5. The structures of the products (3-5) have been elucidated on the basis of IR, 1HNMR, ¹³CNMR and MS spectral data.

Scheme 2. The mechanistic equations for formation of pyrano[2,3-d] thiazolocarbonitriles.

Compound 3 displayed a characteristic absorption band in the IR spectra around 3121 cm^-1 due to the NH functional group. In the 1HNMR spectra of 3, the absence of NH₂ proton signals, which are present in its precursor 1, establish the formation of Schiff base. The mass spectrum of 3 agrees well with the condensed product formed. Compound 4 displayed a characteristic absorption band in IR spectra around 1695 cm^-1 due to the C=O functional group. In the ¹H NMR spectrum of 4, displayed distinct singlets at d 4.12 and 5.62 due to thiazole-CH2 and thiazole- CH protons of the thiazolone ring, confirming the formation of the thiazole ring.

IR spectra of compounds 5 displayed a characterization absorption band around 3380 and 2204 cm^-1 due to NH₂ and CN functional groups. The absence of C=O functional group absorption band which is present in its precursor 4, clearly confirms the formation of pyran ring. The 1HNMR spectral of 5 exhibited singlet at d 5.56 due to pyran-CH protons, confirming the formation of the pyran ring.

The newly synthesized compounds (5a-j) were tested for in vitro antiinflammatory activity. Compared to the standard, diclofenac sodium, they have shown adequate antiinflammatory activity. In vitro antiinflammatory activity of synthesized compounds is summarized in Table 1. Among all the tested compounds 5c, 5e, 5h and 5j possessing chloro, N,Ndimethylamine, hydroxylbromo and hydroxylchloro groups as substituents on the benzene ring showed potent activity in the compound 5 series. The compound 5a have showed moderate activity because it has no substituent on the benzene ring. While other compounds having weak activity.

The antioxidant or DPPH free radical scavenging activity of all the synthesized compounds (5a-j) performed using DPPH method and the results were found in Table 2. All the synthesized compounds produced a concentration dependant scavenging of free radical, DPPH. The IC50 values of all the compounds (5a-j) were found between 13 and 26 µM, with antioxidant activity. In the series, compounds 5b, 5g and 5h possessing nitro, hydroxyl and hydroxylbromo groups as substitutions on benzene ring showed better activity against DPPH free radicals. This may be due to the increased lipophilicity of molecules because of substitution with electronegative atom such as chloro/bromo at the C2 and C4 positions of the aromatic ring. These results suggest that C2 and C5 substitution increases the antioxidant activity of benzimidazolyl pyrano[2,3-d] [1,3]thiazolocarbonitriles.

In conclusion, a elegant synthesis of benzimidazolyl pyrano[2,3-d][1,3]thiazolo carbonitriles were achieved by using in expensive and commercial available staring material, moreover, fused pyrano ring derivatives are potent pharmacological agent, this study may motivates the researchers concerned in this field to explore the pharmacological activity of the compounds.

Acknowledgements

The authors thank the Director of Central Facilities for Research and Development (CFRD), Osmania University, Hyderabad, India, for the facilities and the Director, Indian Institute of Chemical Technology, Hyderabad, India, for the spectral facility.

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