INTRODUCTION:

Tuberculosis (TB) caused by Mycobacterium tuberculosis, is one of the oldest documented infectious disease. Theresurgence of TB is due to HIV infection as well as Multiple Drug- Resistant Tuberculosis (MDR-TB) resulting from inefficient management¹. As per WHO Almost half of a million individuals worldwide in 2019, rifampicin-resistant TB (RR-TB) developed, 78% of which had MDR-TB. The three countries that accounted for the greater share of the global burden were India (27%), China (14%), and the Russian Federation (8%)².

Benzimidazolenucleus withholds number of biological activities such as antihistaminic³, antiulcer⁴, antitubercular⁵, antioxidant⁶, antiparasitic^7,8, anti-inflammatory⁹, analgesic¹⁰, antimicrobial¹¹, antiprotozoal¹², antihypertensive¹³ and anticancer activities¹⁴. The imidazole group possesses significant pharmacokinetic profile that influences the ability of the drug to reach the target and exhibit promising activity against resistant TB by inhibiting the biosynthesis of lipids¹⁵. On the other hand, oxo-thiazolidine derivatives have found their potential pharmaceutical applications in different kinds of disease such as antimicrobial^5,16, antitubercular¹⁷, antifungal, antioxidant activities and anti-HIV activities¹⁸.

After an extensive literature search, it was observed that till date, enough effort has not been made to combine these moieties as a single molecular scaffold and design new candidates that may be a novel, potent, selective and less toxic antitubercular agent. Prompted by these findings, we designed and synthesis a new series of N-[(4-oxo-2-substituted aryl-1,3-thiazolidine)-acetamidyl]-5-nitrobenzimidazoles derivatives. The synthesis was undertaken two ways: conventional and microwave method. Microwave-assisted synthesis has been explored by a number of scientists for various organic synthesis involving heteroatoms and their comparisons have made to see their advantage over conventional one pot synthesis^19-23. A comparison between the two methods has been made in this study.

MATERIALS AND METHODS:

The chemicals used for the synthesis of Benzimidazole derivatives were procured from SD fine chemicals. The synthesized compounds were characterized by mp, IR, NMR and MASS spectral analysis. TLC was performed to monitor the reactions and determine the purity of the products. Further, the compounds were purified by recrystallisation using suitable solvents. The melting points of the compounds were determined using Veego VMP-1 Apparatus expressed in ^°C and are uncorrected. The IR spectra of the compounds were recorded on FT-IR spectrometer (Perkin Elmer Infrared-283) using KBr pellet technique. ¹H NMR spectra were recorded on AV-III (400 MHz FT-NMR) using DMSO-d₆ as solvent and TMS as an internal standard. Microwave-assisted synthesis was performed using scientific microwave system CATA R from Catalyst Systems (Pune, India).

METHODOLOGY:

Synthesis of novel benzimidazole derivatives:

A series of twelve novel N-(4-alkyl-4-oxo-1,3-thiazolidin-3-yl)-2-(5-nitro-1H-benzimidazole-1-yl) acetamide benzimidazole derivatives were designed and synthesized using scheme in fig 1.

Code	Ar	Code	Ar
b	2-OH C₆H₅	h	3-NO₂ C₆H₅
c	- C₆H₅	I	4-N(CH₃)₂ C₆H₅

d	4-Cl C₆H₅	J	2-Cl C₆H₅
e	2-Br C₆H₅	k	2-OCH₃ C₆H₅
f	4-OCH₃ C₆H₅	L	3-Br C₆H₅

Fig 1: Scheme for the synthesis of substituted oxo-thiazole-benzimidazole derivatives

Two approaches: Conventional and Microwave-assisted synthesis were undertaken for these compounds. The methodologies adopted for their synthesis using both the approaches and their characterization in given below.

Synthesis of Ethyl (5-nitro-1H-benzimidazole-1-yl) acetate (1):

Conventional method:

A mixture of 5-nitrobenzimidazole (30g, 0.18mol) and ethyl chloroacetate (22.55g, 0.18mol) in acetone (100ml) was stirred for about 48 hr. The solvent was removed in vacuo and the residue thus obtained was purified over the column of silica gel and recrystallized from acetone to furnish compound 1. Yield 78%; m.p. 260-264 ⁰C; FTIR (KBr, cm^-1): 3046 (C-H, Ar-H), 1740 (C=O of ester),1601 (N=CH-, cyclic), 1530,1335 (ArNO₂), 1430 (N-CH₂); H¹ NMR (δ ppm, DMSO-d₆): δ 7.90 (s, 1H, N=CH-, cyclic), 7.26-7.60 (m,3H, Ar-H), 4.25 (q, 2H, J =7 Hz, COOCH₂CH₃), 2.51 (s, 2H, N-CH₂), 1.25 (t, 3H, J = 7 Hz, COOCH₂CH₃); C¹³ NMR (δ ppm, DMSO-d₆): 168.9, 142.0, 135.0,110.91, 61.1, 33.3, 14.5.

Microwave method:

A mixture of 5-nitrobenzimidazole (30g, 0.18mol) and ethyl chloroacetate (22.55g, 0.18mol) was taken in 250 ml beaker, mixed well and irradiated in microwave oven 565W for 45 min. The completion of the reaction was monitored by TLC. After completion of the reaction, the beaker was removed from the oven. The mixture was cooled and the product was recrystallized from acetone to furnish Ethyl (5-nitro-1H-benzimidazole-1-yl) acetate (Compound 1). Yield 81%; m.p. 260-264⁰C Spectral and analytical data were found to similar as reported for the conventional method.

Synthesis of 2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide (2):

Conventional method:

A mixture of compound Ethyl (5-nitro-1H-indol-1-yl) acetate (27g, 0.11mol) and hydrazine hydrate (5.43g, 0.11 mol) in dioxane (90ml) was stirred for about 48 hr. The solvent was removed in vacuo and the residue thus obtained was purified over the column of silica gel and recrystallized from acetone to furnish compound 2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide (Compound 2). Yield 80%; m.p. 210- 211⁰C; FTIR (KBr, cm^-1): 3252, 3378 (-NHNH2), 3045(C-H, Ar-H), 1678 (C=O, amide), 1600 (N=CH-, cyclic), 1527,1333 (ArNO₂), 1427 (N-CH₂); H¹ NMR (δ ppm, DMSO-d₆): δ 8.20 (s, 1H, CONH), 7.89 (s, 1H, N]CHe, cyclic), 7.25-7.61 (m, 3H, Ar-H), 4.45 (s, 2H, NH2), 2.49 (s, 2H, NeCH2); C¹³ NMR (δ ppm, DMSO-d₆): 168.0, 142.2, 135.2, 111.9, 122 , 33.5.

Microwave method:

A mixture of compound Ethyl(5-nitro-1H-indol-1-yl) acetate (27g, 0.11mol) and hydrazine hydrate (5.43g, 0.11 mol) was taken in 250ml beaker, mixed well and irradiated in microwave oven 800 for 4 min. The completion of the reaction was monitored by TLC. After completion of reaction, the beaker was removed from the oven. The mixture was cooled and product was recrystallized from acetone to furnish 2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide (Compound 2). Yield 87%; m.p. 210- 211⁰C Spectral and analytical data were found to similar as reported for conventional method.

Synthesis of N'-[(1Z)-alkyllidene]-2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide (3a-l):

Conventional method:

A mixture of the compound 2-(5-nitro-1H-indol-1-yl) acetohydrazide (1.8g, 0.008mol) and different aldehydes (0.008 mol) in dioxane (30ml) was stirred for about 24 hr. The solvent was removed in vacuum and the residue thus obtained was purified over the column of silica gel and recrystallized from chloroform to furnish N'-[(1Z)-alkyllidene]-2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide (3a-l).

Microwave method:

A mixture of the compound 2-(5-nitro-1H-indol-1-yl) acetohydrazide (1.8g, 0.008mol) and different aldehydes (0.008mol) were taken in 250ml beaker, mixed well and irradiated in microwave oven 800W for 8 min. The completion of the reaction was monitored by TLC. After completion of the reaction, the beaker was removed from the oven. The mixture was cooled and product was recrystallized from chloroform to furnish N'-[(1Z)-alkyllidene]-2-(5-nitro-1H-benzimidazole-1-yl) acetohydrazide. Other compounds were prepared similarly by treating with various aromatic aldehydes (3a-l)

N’-ethylidene-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3a)

m.p. 200-202⁰C; FTIR (KBr, cm^-1): 3043 (C-H, Ar-H), 1674(C=O, amide), 1635 (N=CH-, acyclic), 1604 (N=CH-, cyclic),1524,1329 (ArNO₂), 1428 (N-CH₂).);¹ H NMR (δ ppm, DMSO-d₆): δ 8.21 (s, 1H, CONH), 8.70 (s, 1H, N=CH-, acyclic), 7.86 (s, 1H, N=CH-,cyclic), 7.27-7.63 (m, 8H, Ar-H), 2.52 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):168.1, 143.2 ,142.3, 135.6 , 112.1-122.2 , 33.4.

N’-(2-hydroxybenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide(3b)

m.p. 262-264 ⁰C; FTIR (KBr, cm^-1): 3246 (Ar-OH), 3047 (C-H,Ar-H), 1672 (C=O, amide), 1637 (N=CH-, acyclic), 1602(N=CH-, cyclic), 1520, 1324 (ArNO₂), 1431 (N-CH₂); ¹H NMR (δ ppm, DMSO- d₆): δ 11.95 (s, 1H, Ar-OH), 8.71 (s, 1H, N=CH-,acyclic), 8.22 (s, 1H, CONH), 7.84 (s, 1H, N=CH-, cyclic), 7.23-7.59(m, 7H, Ar-H), 2.50 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 167.9 , 165.1 , 143.1,142.4 , 135.4 , 112.3,122.6 , 33.6.

N’-benzylidene-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3c)

m.p. 252-253 ⁰C; FTIR (KBr, cm^-1): 3043 (C-H,Ar-H), 1670 (C=O, amide), 1630 (N=CH-, acyclic), 1612(N=CH-, cyclic), 1520, 1324 (ArNO₂), 1431 (N-CH₂); ¹H NMR (δ ppm, DMSO- d₆): δ 8.71 (s, 1H, N=CH-,acyclic), 8.22 (s, 1H, CONH), 7.84 (s, 1H, N=CH-, cyclic), 7.23-7.59(m, 7H, Ar-H), 2.50 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 167.9 ,165.1 ,143.1,142.4 ,135.4 , 112.3,122.6,33.6.

N’-(4-chlorobenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3d)

m.p. 156-158 ⁰C; FTIR (KBr, cm^-1): 3041 (C-H, Ar-H), 1671 (C=O, amide), 1636 (N=CH-, acyclic), 1603 (N=CH-, cyclic), 1528,1325 (ArNO₂), 1434 (N-CH₂), 757 (Ar-Cl); ¹H NMR (δ ppm, DMSO- d₆): δ 8.72 (s, 1H, N=CH-, acyclic), 8.16 (s, 1H, CONH), 7.85 (s,1H, N=CH-, cyclic), 7.21-7.57 (m, 7H, Ar-H), 2.55 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 167.5 , 143.3,142.5, 135.3 , 134.0, 112.0,122.1, 33.7.

N’-(2-bromobenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3e)

m.p. 190-192 ⁰C; FTIR (KBr, cm^-1): 3040 (C-H, Ar-H), 1666 (C=O, amide), 1638 (N=CH-, acyclic), 1607 (N=CH-, cyclic),1526,1323 (ArNO₂), 1436 (N-CH₂), 618 (Ar-Br); ¹H NMR (δ ppm, DMSO- d₆): δ 8.69 (s, 1H, N=CH-, acyclic), 8.14 (s, 1H, CONH), 7.88 (s,1H, N=CH-, cyclic), 7.22-7.58 (m, 7H, AreH), 2.53 (s, 2H, NeCH2); C¹³ NMR (δ ppm, DMSO-d₆): 167.8, 143.6 , 141.9 , 135.1 , 118.2, 111.7,122.4 , 33.9.

N’-(4-methoxybenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3f)

m.p. 224-225 ⁰C; FTIR (KBr, cm^-1): 3053 (C-H, Ar-H), 2828 (Ar-OCH₃),1665 (C=O, amide),1643 (N=CH-, acyclic),1613 (N=CH-, cyclic), 1532,1335 (ArNO₂), 1439 (N-CH₂); ¹H NMR (δ ppm, DMSO- d₆): δ 8.64 (s, 1H, N=CH-, acyclic), 8.17 (s, 1H, CONH), 7.95 (s,1H, N=CH-, cyclic), 7.29-7.66 (m, 7H, Ar-H), 3.95 (s, 3H, OCH3), 2.57 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 168.6, 143.8 , 143.1 , 136.3 ,110.4,123.0 , 55.5, 34.2.

N’-(2-nitrobenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3g)

m.p. 210-212 ⁰C; FTIR (KBr, cm^-1): 3048 (C-H, Ar-H),1662 (C=O, amide), 1642 (N=CH-, acyclic), 1609 (N=CH-, cyclic),1525,1328 (ArNO₂), 1429 (N-CH₂); ¹H NMR (δ ppm, DMSO- d₆): δ8.68 (s, 1H, N=CH-, acyclic), 8.13 (s, 1H, CONH), 7.93 (s, 1H, N=CH-,cyclic), 7.28-7.62 (m, 7H, Ar-H), 2.60 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 168.5 , 143.5 ,141.6,135.7, 111.2,122.5,34.1.

N’-(3-nitrobenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3h)

m.p. 214-215 ⁰C; FTIR (KBr, cm^-1): 3050 (C-H, Ar-H), 1664 (C=O, amide), 1641 (N=CH-, acyclic), 1608 (N=CH-, cyclic),1529,1332 (ArNO2), 1435 (N-CH₂), ¹H NMR (δ ppm, DMSO- d₆): δ 8.66 (s, 1H, N=CH-, acyclic), 8.12 (s, 1H, CONH), 7.92 (s, 1H, N=CH-, cyclic),7.31-7.65 (m, 7H, Ar-H), 2.59 (s, 2H, N-CH2); C¹³ NMR (δ ppm, DMSO-d₆): 168.3, 143.7, 141.8, 135.9, 111.3, 122.7,34.3.

N’-(4-dimethylaminobenzylidene)-2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3i)

m.p. 205-206 ⁰C; FTIR (KBr, cm^-1): 3054 (C-H, Ar-H), 2910, 2851 (N-CH₃), 1668 (C=O, amide), 1647 (N=CH-, acyclic),1612 (N=CH-, cyclic), 1536,1336 (ArNO₂), 1438 (N-CH₂); ¹H NMR (δ ppm, DMSO- d₆): δ 8.63 (s, 1H, N=CH-, acyclic), 8.23 (s, 1H,CONH), 7.98 (s, 1H, N=CH-, cyclic), 7.34-7.64 (m, 7H, Ar-H), 3.12 (s, 6H, 2 CH₃), 2.63 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 168.7, 144.1, 143.0, 136.4, 110.3,123.1 , 34.7, 23.5.

N’-(2-chlorobenzylidene) -2-(5-nitro-1H-benzo[d]imidazole-1-yl) acetohydrazide (3j)

m.p. 152-155 ⁰C; FTIR (KBr, cm^-1): 3042 (C-H, Ar-H), 1669 (C=O, amide), 1639 (N=CH-, acyclic), 1605 (N=CH-, cyclic), 1523,1327 (ArNO₂), 1432 (N-CH₂), 754 (Ar-Cl); ¹H NMR (δ ppm, DMSO- d₆): δ 8.73 (s, 1H, N=CH-, acyclic), 8.18 (s, 1H, CONH), 7.87 (s,1H, N=CH-, cyclic), 7.20-7.55 (m, 7H, Ar-H), 2.54 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 167.7, 143.0 ,142.7, 135.8, 134.3, 112.8,122.3, 33.8.

N’-(2-methoxybenzylidene)-2-(5-nitro-1H-benzo[d]imidazole1- yl) acetohydrazide (3k)

m.p. 220-222 ⁰C; FTIR (KBr, cm^-1): 3049 (C-H, Ar-H),2825(Ar-OCH3),1663 (C=O, amide),1644 (N=CH-, acyclic),1610 (N=CH-, cyclic), 1533,1331 (ArNO₂), 1437 (N-CH₂), ¹H NMR (δ ppm, DMSO- d₆): δ 8.67 (s, 1H, N=CH-, acyclic), 8.15 (s, 1H, CONH), 7.94 (s,1H, N=CH-, cyclic), 7.30e7.67 (m, 7H, Ar-H), 3.96 (s, 3H, OCH₃), 2.58 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 144.0 , 142.6, 136.1 ,110.5,122.9 , 55.8 , 34.5.

N’-(3-bromobenzylidene) -2-(5-nitro-1H-benzo[d]imidazole-1- yl) acetohydrazide (3l)

m.p. 188-190⁰C; FTIR (KBr, cm^-1): 3044 (C-H, Ar-H), 1661 (C=O, amide), 1640 (N=CH-, acyclic), 1606 (N=CH-, cyclic), 1522, 1326 (ArNO₂), 1433 (N-CH₂), 620 (Ar-Br). ¹H NMR (δ ppm, DMSO- d₆): δ 8.65 (s, 1H, N=CH-, acyclic), 8.11 (s, 1H, CONH), 7.91 (s,1H, N=CH-, cyclic), 7.24-7.56 (m, 7H, Ar-H), 2.56 (s, 2H, N-CH₂); C¹³ NMR (o ppm, DMSO-d₆): 168.2, 143.4, 141.7, 135.5, 118.4, 111.5-122.8, 34.0.

Synthesis of N-(2-methyl-4-oxo-1,3-thiazolidin-3-yl)-2-(5-1H-benzimidazole-1-yl) acetamide (4a):

Conventional method:

A mixture of the compound 3a (1.5g, 0.005mol) and thioglycolic acid (0.43g, 0.005mol) with a pinch of anhydrous ZnCl₂ in acetone (25ml) was stirred for about 24 hr. The solvent was removed in vacuo and the residue thus obtained was purified over column of silica gel and recrystallized from chloroform to furnish compound 4a. Yield 75%; m.p. 220-221⁰C; FTIR (KBr, cm^-1): 3052 (C-H, Ar-H), 2980 (N-CH-S), 1712 (C=O, cyclic), 1670 (C=O, amide), 1614 (N=CH-, cyclic), 1538, 1339 (ArNO₂), 1441 (N-CH₂), 690 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.25 (s, 1H, CONH), 7.99 (s, 1H, N=CH-, cyclic), 7.36-7.70 (m, 8H, Ar-H), 4.10 (s, 1H, N=CH-), 3.40 (s, 2H, CH₂, cyclic), 2.62 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 171.0 , 168.9 , 141.3, 136.6 , 110.1-123.4 , 44.2 , 40.3 , 34.9.

Microwave Method:

A mixture of the compound 3a (1.5g, 0.005mol) and thioglycolic acid (0.43g, 0.005mol) with a pinch of anhydrous ZnCl₂ was taken in 250ml beaker, mixed well and irradiated in microwave oven 800W for 2 min. The completion of the reaction was monitored by TLC. After completion of reaction, the beaker was removed from the oven. The mixture was cooled and the product was recrystallized from chloroform to furnish compound 4a. Yield 80%; m.p. 220-221⁰C Spectral and analytical data were found to similar as reported for the conventional method.Other compounds (4b-l) were prepared similarly by using (3b-l) respectively

N-[2-(2-hydroxyphenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl)acetamide (4b)

m.p. 222-223 ⁰C; FTIR (KBr, cm^-1): 3243 (Ar-OH), 3056 (C-H, Ar-H), 2981 (N-CH-S), 1714 (C=O, cyclic), 1673 (C=O, amide),1616 (N=CH-, cyclic), 1535,1342 (ArNO₂), 1443 (N-CH₂), 691(C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 11.97 (s, 1H, Ar-OH), 8.26 (s,1H, CONH), 7.97 (s, 1H, N=CH-, cyclic), 7.33-7.72 (m, 7H, Ar-H),4.12 (s, 1H, N=CH-), 3.41 (s, 2H, CH₂, cyclic), 2.64 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 171.2 , 169.0, 164.9, 141.0 , 136.2 , 110.2-123.6 , 44.4, 40.6 , 34.8.

2-(5-nitro-1H-benzimidazol-1-yl)-N-(4-oxo-2-phenyl-1,3-thiazolidin-3-yl)acetamide (4c)

m.p. 240-241 ⁰C; FTIR (KBr, cm^-1): 3056 (C-H, Ar-H), 2981 (N-CH-S), 1714 (C=O, cyclic), 1673 (C=O, amide),1616 (N=CH-, cyclic), 1535,1342 (ArNO₂), 1443 (N-CH₂), 691(C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.26 (s,1H, CONH), 7.97 (s, 1H, N=CH-, cyclic), 7.33-7.72 (m, 7H, Ar-H),4.12 (s, 1H, N=CH-), 3.41 (s, 2H, CH₂, cyclic), 2.64 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 171.2 , 169.0, 164.9, 141.0 , 136.2 , 110.2-123.6 , 44.4, 40.6 , 34.8.

N-[2-(4-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl)acetamide (4d)

m.p. 229-230 ⁰C; FTIR (KBr, cm^-1): 3059 (C-H, Ar-H), 2985 (N-CH-S), 1711 (C=O, cyclic), 1677 (C=O, amide), 1615 (N=CH-, cyclic), 1540,1340 (ArNO₂), 1442 (N-CH₂), 725 (Ar-Cl), 695(C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.27 (s, 1H, CONH), 8.04 (s,1H, N=CH-, cyclic), 7.37-7.71 (m,7H, Ar-H), 4.15 (s, 1H, N=CH-),3.47 (s, 2H, CH₂, cyclic), 2.69 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 171.3, 169.3, 141.1,136.7, 134.6, 110.8-123.5, 44.3 , 41.2, 35.0.

N-[2-(2-bromophenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl) acetamide (4e)

m.p. 250-251 ⁰C; FTIR (KBr, cm^-1): 3062 (C-H, Ar-H), 2983 (N-CH-S), 1713 (C=O, cyclic), 1676 (C=O, amide), 1619 (N=CH-, cyclic), 1542,1344 (ArNO₂), 1444 (N-CH₂), 694 (C-S-C), 621(Ar-Br); ¹H NMR (δ ppm, DMSO- d₆): δ 8.29 (s, 1H, CONH), 8.02 (s,1H, N]CHe, cyclic), 7.35-7.74 (m, 7H, Ar-H), 4.13 (s, 1H,N=CH-), 3.71 (s, 2H, N-CH2), 3.43 (s, 2H, CH2, cyclic), 2.66 (s, 2H,N-CH2); C¹³ NMR (δ ppm, DMSO-d₆):171.5 , 169.2,141.4 ,136.8 , 118.5, 111.0-123.7 , 44.5 , 41.4 , 35.2 .

N-[2-(4-methoxyphenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl) acetamide (4f)

m.p. 210-211 ⁰C; FTIR (KBr, cm^-1): 3057 (C-H, Ar-H), 2978 (N-CH-S), 2827 (Ar-OCH₃), 1718 (C=O, cyclic), 1682 (C=O,amide), 1623 (N=CH-, cyclic), 1546,1352 (Ar-NO₂), 1449(N-CH₂), 697 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.33( s,1H, CONH),8.07 (s, 1H, N=CH-, cyclic), 7.41-7.79 (m, 7H, Ar-H), 4.21 (s, 1H, N=CH-), 3.93 (s, 3H, OCH₃), 3.36 (s, 2H, CH₂, cyclic), 2.74 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 172.0, 169.9, 142.1,137.4, 112.2-124.6, 55.4, 44.9, 41.6, 35.6.

2-(5-nitro-1H-benzimidazol-1-yl)-N-[2-(2-nitrophenyl)-4-oxo-1,3-thiazolidin-3-yl]acetamide (4g)

m.p. 236-237 ⁰C; FTIR (KBr, cm^-1): 3064 (C-H, Ar-H), 2976(N-CH-S), 1715 (C=O, cyclic), 1679 (C=O, amide), 1620 (N=CH-, cyclic), 1544,1347 (Ar-NO2),1448 (N-CH2), 693 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.30 (s, 1H, CONH), 8.03 (s, 1H, N=CH-,cyclic), 7.42-7.73 (m, 7H, Ar-H), 4.14 (s, 1H, N=CH-), 3.37 (s, 2H,CH₂, cyclic), 2.70 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):171.6, 169.4 , 140.0 , 136.9 , 111.6-123.9 , 45.0 , 41.3 , 35.3.

2-(5-nitro-1H-benzimidazol-1-yl)-N-[2-(3-nitrophenyl)-4-oxo-1,3-thiazolidin-3-yl]acetamide (4h)

m.p. 245-246 ⁰C; FTIR (KBr, cm^-1): 3063 (C-H, Ar-H), 2977(N-CH-S), 1717 (C=O, cyclic), 1681 (C=O, amide), 1617 (N=CH-, cyclic), 1545,1346 (Ar-NO2), 1447 (N-CH2), 696 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.32 (s, 1H, CONH), 8.06 (s, 1H, N=CH-, cyclic),7.40-7.77 (m, 7H, Ar-H), 4.18 (s, 1H, N=CH), 3.35 (s, 2H,CH₂, cyclic), 2.68 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):171.9 , 169.6, 146.3 , 140.8 ,137.2 , 111.4-124.4 , 44.8, 41.5, 35.5.

2-(5-nitro-1H-benzimidazol-1-yl)-N-[2-(4-dimethylaminophenyl)-4-oxo-1,3-thiazolidin-3-yl] acetamide (4i)

m.p. 225-226 ⁰C; FTIR (KBr, cm^-1): 3066 (C-H, Ar-H), 2986(N-CH-S), 2912, 2850 (N-CH₃), 1719 (C=O, cyclic), 1683 (C=O, amide), 1626 (N=CH-, cyclic), 1549,1351 (Ar-NO₂), 1452(N-CH₂), 700 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.34 (s, 1H,CONH), 8.11 (s, 1H, N=CH-, cyclic), 7.44e7.80 (m, 7H, Ar-H), 3.34(s, 2H, CH₂, cyclic) 4.16 (s, 1H, N=CH-), 3.14 (s, 6H, 2 CH₃),2.73 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):172.1, 170.0,145.5 , 143.8 ,137.3, 112.5-124.1, 45.3, 41.7, 35.7, 23.9.

N-[2-(2-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl) acetamide (4j)

m.p. 248-249 ⁰C; FTIR (KBr, cm^-1): 3060 (C-H, Ar-H), 2982 (N-CH-S), 1710 (C=O, cyclic), 1675 (C=O, amide), 1618 (N=CH-, cyclic), 1537,1341 (ArNO₂), 1445 (N-CH₂), 727 (Ar-Cl), 692(C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.24 (s, 1H, CONH), 8.01 (s,1H, N=CH-, cyclic), 7.38e7.69 (m, 7H, Ar-H), 4.11 (s, 1H, N=CH-),3.42 (s, 2H, CH₂, cyclic), 2.65 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):171.1 , 169.1, 141.2 ,136.5, 134.5, 110.6-123.3, 44.1, 41.0, 34.6.

N-[2-(2-methoxyphenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl) acetamide (4k)

m.p. 207-208 ⁰C; FTIR (KBr, cm^-1): 3058 (C-H, Ar-H), 2975(N-CH-S), 2826 (Ar-OCH₃), 1720 (C=O, cyclic), 1685 (C=O,amide), 1621 (N]CHe, cyclic), 1548,1349 (Ar-NO₂), 1450 (N-CH₂),

698 (C-S-C); ¹H NMR (δ ppm, DMSO- d₆): δ 8.31 (s, 1H, CONH), 8.08 (s, 1H, N]CH-cyclic), 7.43e7.75 (m, 7H, Ar-H), 3.38 (s, 2H, CH₂, cyclic) 4.19 (s, 1H, eNeCHe), 3.92 (s, 3H, OeCH3), 2.72 (s, 2H,N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆):171.7 , 169.8, 141.5 , 137.0 ,111.8-124.2 , 55.6 , 44.7 , 41.8, 35.4.

N-[2-(3-bromophenyl)-4-oxo-1,3-thiazolidin-3-yl]-2-(5-nitro-1H-benzimidazol-1-yl) acetamide (4l)

m.p. 254-255⁰C; FTIR (KBr, cm^-1):3065 (C-H, Ar-H),: 2979(N-CH-S), 1716 (C=O, cyclic), 1680 (C=O, amide), 1622 (N=CH-, cyclic), 1541,1343 (ArNO₂), 1446 (N-CH₂), 689 (C-S-C), 622(Ar-Br); ¹H NMR (δ ppm, DMSO- d₆): δ 8.28 (s, 1H, CONH), 8.05 (s,1H, N=CH-, cyclic), 7.39-7.76 (m, 7H, Ar-H), 4.17 (s, 1H, N=CH-), 3.39 (s, 2H, CH₂, cyclic), 2.67 (s, 2H, N-CH₂); C¹³ NMR (δ ppm, DMSO-d₆): 171.4, 169.5, 143.9, 137.1, 118.8, 111.1-124.0, 44.6,41.1 , 35.1.

Antitubercular activity:

The antimycobacterial activity of compounds was assessed against M. tuberculosis using the microplate Alamar Blue assay (MABA) [24]. This methodology is non-toxic procedure which uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. In this method, 200µl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate to minimize evaporation of medium in the test wells during incubation. 96 wells plate received 100µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on the plate. The final drug concentrations tested were 100 to 0.2µg/ml.

The plates were sealed with parafilm and incubated at 37ºC for five days. After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs. A blue colour in the well was interpreted as no bacterial growth, and pink colour was scored as growth. The MIC was defined as the lowest drug concentration which prevented the colour change from blue to pink. Mycobacteria tuberculosis (Vaccine strain, H37 RV strain): ATCC No- 27294 was used for this study. Standard for Anti-Tb activity was considered taking Pyrazinamide- 3.125µg/ml Ciprofloxacin-3.125µg/ml and Streptomycin- 6.25µg/ml.

RESULTS AND DISCUSSION:

Twelve new N-(4-alkyl-4-oxo-1,3-thiazolidin-3-yl)-2-(5-nitro-1H-benzimidazole-1-yl) acetamide benzimidazole derivatives were synthesized (fig1) using conventional one pot synthesis and microwave assisted synthesis. The compounds were characterized using melting point, IR, H¹-NMR and C^13-NMR spectroscopy. The details of which are given under methodology section. The data reporting the percentage yield and time taken to complete the reaction is given in table 1.

Table 1: Comparison of conventional and microwave synthesis of 3a-l and 4a-l

Compd	Conventional		Microwave (800W)
Compd	Yield (%)	Time in hrs	Yield (%)	Time in min
3a	80	24	86	4
3b	67	48	77	4
3c	65	24	74	6
3d	70	24	76	8
3e	66	24	70	4
3f	78	24	82	4
3g	80	48	87	6
3h	77	24	84	8
3i	68	48	77	3.5
3j	69	24	80	8
3k	61	24	70	6
3l	71	24	76	4
4a	75	24	80	3.5
4b	70	24	76	2
4c	66	24	77	2
4d	74	24	81	2
4e	73	24	80	2
4f	65	48	77	2
4g	60	24	71	2
4h	71	48	78	2
4i	70	24	80	2
4j	74	24	82	2
4k	70	24	85	2
4l	66	24	79	2

The reactions under microwave irradiation were completed within 4-8 min whereas similar reactions under conventional way by reflux took 24-48 h. The yield was also better in the reactions carried out using microwave-assisted synthesis. The table I, clearly indicates the advantages of using green chemistry(microwave assisted synthesis) in terms of not only saving the environment but it is also cost and time effective.

Anti tubercular activity:

The synthesized compounds were tested for their antimycobacterial activity of compounds was assessed against M. tuberculosis (H37 RV strain; ATCC 2729) using the microplate Alamar Blue assay (MABA). The results of the assay in terms of MIC (μg/ml) are given in table 2 and figure 2. Pyrazinamide, streptomycin and ciprofloxacin were used as standards for comparison.

Table 2: In vitro antitubercular activity of the compounds using MABA

S. No	Compounds	*MIC(µg/ml) M. tuberculosis* H37RV**
1.	4a	1.6
2.	4b	1.6
3.	4c	6.25
4.	4d	3.125
5.	4e	3.125
6.	4f	3.125
7.	4g	1.6
8.	4h	12.5
9.	4i	6.25
10.	4j	3.125
11.	4k	3.125
12.	4l	3.125
13.	Pyrazinamide	3.125
14.	Ciprofloxacin	3.125
15.	Streptomycin	6.25

Figure 2: Antitubercular activity of synthesized compounds in comparison with the standards

Compounds 4a, 4b, 4g have shown better activity than the standard drugs with MIC values 1.6 μg/ml and all other compounds also exhibited good activity with MIC values between 3.125‐6.25μg/ml, which is either equivalent to or better than the standards used.

CONCLUSIONS:

We were able to standardize the microwave assisted synthesis of N-[(4-oxo-2-substituted aryl-1,3-thiazolidine)-acetamidyl]-5-nitrobenzimidazoles derivatives to get quicker reactions and better yields, in comparison to conventional method of reflux. This led to saving of time, effort and cost. The in vitro anti-tubercular studies on these 12 new derivatives gave promising results. The compounds 4a and 4b have shown best MIC value against M. tuberculosis by MABA, which is better than the standard drugs used for the assay. This series bears a potential to give good anti TB agent, more modifications can be explored.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENT:

We are thankful to Rajiv Gandhi University of Health Sciences, Karnataka, India, for providing us with the financial assistance to carry work this research work.

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24. PermatasariDesy Ayu Irma, Ritmaleni, NuryastutiTitik. N-(Chlorobenzyl) Formamide as an Antituberculosis Agent from Multicomponent Reaction Synthesis. Research Journal of Pharmacy and Technology. 2021; 14(6):3253-1I:10.52711/0974-360X.2021.00566.

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Received on 24.05.2022 Modified on 18.07.2022

Research J. Science and Tech. 2022; 14(4):199-207.

DOI: 10.52711/2349-2988.2022.00033