Synthesis and Antimicrobial Activity of Some Novel Pyrrolyl 1, 3, 4-Thiadiazole Derivatives

Introduction

Tuberculosis (TB) is an epidemic disease and its causative organism Mycobacterium tuberculosis is one of the most prolific infectious organisms affecting humans. The 196 countries reporting to WHO in 2008 notified 5.6 million new and relapse cases in 2007, of which 2.6 million (46%) were new smear-positive cases.¹ Furthermore, treatment of TB with human immunodeficiency virus infected patients (HIV) is difficult and results as the leading cause of death among HIV positive patients worldwide. Another factor which contributes to a greater number of deaths is the emergence of multiple drug resistance (MDR).^2-5

Heterocycle bearing thiazoles, sulphur, and nitrogen moiety constitute the core structure of a number of pharmacological and biologically active interesting compounds. The efficiency of azoles as chemotherapeutic agents is well stabilized. Thus the role of nitrogen and sulphur containing heterocyclic compounds which are endowed with unique structure and potent antimicrobial activities need to be over emphasized. The 1, 3, 4-thiadiazole ring system is known to possess several biological activity and its derivatives possess a wide verities of activities such as anti-inflammatory,⁶ analgesic,⁷ antimicrobial,⁸ antitubercular,⁹ and antiviral.¹⁰

The pyrrole ring is a part of many biological compounds such as the enzyme catalyses, the bile pigment bilirubin and the mould pigment prodigiosin; it is also a significant part of macro cyclic porphyrin ring system of chlorophyll and hemin.¹¹ Apart from these properties pyrrole and its derivative possess a number of biological activities such as antiallergic,¹² antibacterial,¹³ antifungal, anti-inflammatory,¹⁴ antitumor,¹⁵ analgesic, anticonvulsant,¹⁶ antitubercular, anticancer, and anti HIV.^17-18

This observation prompted us to undertake the synthesis of some novel compounds involving pyrrole and 1, 3, 4-thiadiazole ring were evaluated for their antibacterial and anti-tubercular properties.

Materials and Methods

Chemistry

Melting points were determined using the Shital-digital programmable melting point apparatus and are uncorrected. FTIR spectra in KBr pellets were recorded on a Bruker FTIR spectrophotometer. The ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE II at 400 and 100/75 MHz, respectively; chemical shifts are expressed in parts per million (ppm) relative to TMS. The abbreviations used to describe the peak patterns are: (b) broad, (s) singlet, (d) doublet, (t) triplet, (q) quartet, and (m) multiplet. Mass spectra (MS) were recorded in a JEOL GCMATE II GC-Mass spectrometer and Schimadzu QP 20105 GC-Mass spectrometer. Analytical thin-layer chromatography (TLC) was performed on precoated TLC sheets of silica gel 60 F254 (Merck, Darmstadt, Germany) visualized by long- and short- wavelength ultraviolet (UV) lamps. Chromatographic purifications were performed on Merck aluminium oxide (70-230 mesh) and Merck silica gel (70-230 mesh).

Procedure for the synthesis of 2-amino-5- (substituted phenyl)-1,3,4-thiadiazoles (3a-e)

A stirring mixture of benzoic acid (50 m mol), thiosemicarbazide (50 m mol) and phosphoryl oxytrichloride (13 mL) was heated at 750 C for 30 min. After cooling down to room temperature, water was added. The reaction mixture was refluxed for 4 h. After cooling, the mixture was basified to pH 8 by adding 50% NaOH solution drop wise with stirring. The precipitate was filtered and recrystalized from ethanol.

Procedure for the synthesis of 4-(1H-pyrrol-1-yl) benzoic acid (4)¹⁹

Compound (4) synthesis was carried out by procedure described by Hallikeri et al. 2,5-Dimethoxy tetrahydrofuran (16 gm, 0.12 mol) was added to p-amino benzoic acid (13.72 gm, 0.1 mol) in glacial acetic acid (100 mL) and mixture was refluxed for 30 min. The reaction mixture was poured in to ice cold water; precipitated solid was filtered and dried. Solid crude product was recrystallized from ethanol and obtained as brown crystals in 62% yield. M.P 286-290⁰ C.

General procedure for synthesis of N- (5-substituted phenyl)-1,3,4-thiadiazole-2-yl)-4- (1H-pyrrol-1-yl) benzamides (5a-e).

Appropriate 2-amino-5-(substituted phenyl) -1,3,4-thiadiazole (0.16 gm, 0.0018 mol) and 4-(1H-pyrrole-1-yl) benzoic acid (0.43 gm, 0.0019 mol) were dissolved in dry DMF, HBTU (N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate) (0.87 gm, 0.0023 mol) and DIEA (N’, N’-Diisopropylethylamine) (0.93 mL, 0.0053 mol) were added and stirred for 5 h at 23⁰ C. The reaction mixture was quenched by brine. The resulting mixture was extracted with ethyl acetate (3 X 50 mL). The ethyl acetate layer washed with 1N HCl then with saturated sodium bicarbonate followed by brine and then solvent was evaporated to dryness. Crude product was recrystallized with chloroform.

N-(5-(4-aminophenyl)-1, 3, 4-thiadiazol-2-yl)-4- (1H-pyrrol-1-yl)benzamide (5a)

(Yield 65%). MP 220-222⁰ C; FTIR (KBr): 3447 (-NH) 2921 (Ar-H), 1680 (C=O), 1601 (C=N); ¹H NMR (400 MHz, CDCl3) δ ppm: 6.33 (t, 2H, pyrrole-C₃, C4-H), 7.15-7.98 (m, 2H-pyrrole-C2, C₅ -H, 8H, bridge phenyl-C₂, C₃, C₅, C6-H, phenyl-C₂, C₃, C₅, C₆-H), 8.62 (s, 2H, -NH₂) 12.81 (s, 1H, amide -NH). MS (EI): m/z = found 361.38 [M+]; calcd. 361.42. Anal. C₁₉H₁₅ N₅OS.

N-(5-(4-nitrophenyl)-1, 3, 4-thiadiazol-2-yl)-4- (1H-pyrrol-1-yl)benzamide (5b)

(Yield 68%). MP 239-242⁰ C; FTIR (KBr): 3476 (-NH) 2927 (Ar-H), 1683 (C=O), 1603 (C=N); ¹ H NMR (400 MHz, CDCl₃) δ ppm: 6.22 (s, 2H, pyrrole-C3, C4-H), 7.21 (s, 2H-pyrrole-C2, C5-H) 7.71-8.12 (m, 8H, bridge phenyl-C2, C3, C5,C6-H, phenyl-C₂, C₃, C₅, C₆-H-H), 12.73 (s, 1H, amide -NH). MS (EI): m/z = found 391.47 [M+]; calcd. 391.41. Anal. C₁₉H₁₃N₅O₃S.

N-(5-(4-chlorophenyl)-1, 3, 4-thiadiazol-2-yl)-4- (1H-pyrrol-1-yl)benzamide (5c)

(Yield 62%). MP 227-229⁰ C; FTIR (KBr): 3378 (-NH) 2920 (Ar-H), 1683 (C=O), 1598 (C=N); MS (EI): m/z = found 380.85 [M+]; calcd. 380.05. Anal. C₁₉H₁₃C_l N₄OS.

N-(5-(4-bromophenyl)-1, 3, 4-thiadiazol-2-yl) -4-(1H-pyrrol-1-yl)benzamide (5d)

(Yield 73%). MP 224-246⁰ C; FTIR (KBr): 3385 (-NH) 2923 (Ar-H), 1689 (C=O), 1613 (C=N); ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.52 (s, 2H, pyrrole-C3, C4-H), 6.77 (s, 2H-pyrrole-C2, C5-H) 7.61-8.12 (m, 8H, bridge phenyl-C2, C3, C5,C6-H, phenyl-C2, C3, C5, C6-H), 12.63 (s, 1H, amide -NH). MS (EI): m/z = found 425.30 [M+]; calcd. 424.00. Anal. C₁₉H₁₃BrN₄OS.

4-(1H-pyrrol-1-yl)-N-(5-(p-tolyl)-1,3,4-thiadiazo l-2-yl)benzamide (5e)

(Yield 66%). MP 246-248⁰ C; FTIR (KBr): 3314 (-NH) 2917 (Ar-H), 1655 (C=O), 1583 (C=N); 1H NMR (400 MHz, CDCl₃) δ ppm: 2.50 (s, 3H, -CH3) 6.33 (t, 2H, pyrrole-C3, C4-H), 7.15-7.98 (m, 2H, pyrrole-C2, C5-H, 8H- bridge phenyl-C2, C3, C5,C6-H, phenyl-C2, C3, 4, C5, C6-H), 12.81 (s, 1H, amide -NH). ¹³C NMR (100 MHz, DMSO-d6) δ (ppm): 14.12 (CH3), 102.34 (pyrrole-C3, C4), 120.13 (pyrrole-C2, C5) 127.49 (methyl phenyl-C2, C6), 129.99 (bridge phenyl-C2, C6), 130.07 (methyl phenyl-C3, C5), 130.48 (bridge phenyl-C3, C5), 131.32 (bridge phenyl-C1), 131.63 (methyl phenyl-C4), 142.81 (bridge phenyl-C4), 158.79 (thiadiazole-C5) 164.33 (C=O), 168.39 (thiadiazole-C2). MS (EI): m/z = found 360.44 [M+]; calcd. 360.10. Anal. C₂₀H₁₆N₄OS.

Antibacterial Activity

The MIC determination of the compounds was carried out simultaneously in comparison with ciprofloxacin, norfloxacin against gram positive (Staphylococcus autreus). Gram negative bacteria (Escherichia coli) by broth micro dilution method.^20-21 Serial dilutions of the test compounds and reference drugs were prepared in Mueller-Hinton broth. Drugs (10 mg) were dissolved in dimethylsulfoxide (DMSO, 1 mL). Further progressive dilutions were done to obtain final concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100 μg/mL. The tubes were inoculated with 105 cfu/mL (colony forming unit/mL) and incubated at 37 °C for 18 h. The MIC was the lowest concentration of the tested compound that yields no visible growth on the plate. To ensure that the solvent had no effect on the bacterial growth, a control was performed with the test medium supplemented with DMSO at the same dilutions as used in the experiments and DMSO had 03 no effect on the microorganisms in the concentrations studied. The MIC values are given in μg/mL. Ciprofloxacin and norfloxacin were used as standard drugs. The preliminary results of antibacterial activities are depicted in Table 1. Compounds showed antibacterial activity between MIC of 100-3.125 μg/mL.

Anti-tubercular Activity

MIC values were determined for the newly synthesized compounds against M. tuberculosis strain H37Rv using the Micro plate Alamar Blue assay (MABA)²² using isoniazid as the standard drug. The 96 wells plate received 100 µL of Middlebrook 7H9 broth and serial dilution of compounds were made directly on the plate with drug concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100 μg/mL. Plates were covered and sealed with parafilm and incubated at 37 °C for 5 days. Then, 25 µl of freshly prepared 1:1 mixture of alamar blue reagent and 10% Tween 80 was added to the plate and incubated for 24 h. A blue color in the well was interpreted as no bacterial growth and pink color was scored as growth. The MIC was defined as the lowest drug concentration, which prevented color change from blue to pink.

Results and Discussion

Chemistry

For the preparation of pyrrolyl thiadiazole derivatives three step procedure was established (Scheme-I). In first step 2-amino-(5-substituted phenyl)-1, 3, 4-thiadiazoles were prepared by stirring appropriate benzoic acid, thiosemicarbazide and phosphorus oxychloride, further mixture was heated 75 °C for 30 min, cooled mixture at room temperature and water was added, then reaction mixture was refluxed for 4h. Further mixture was basified by adding 50% of NaOH solution, precipitate were collected by filtration and recrystalized from ethanol. In second step, 4-pyrrol-1yl-benzoic acid was prepared by refluxing 2, 5-dimethyl tetrahydofuran with para amino benzoic acid in glacial acetic acid for 30 min, reaction mixture poured in ice cold water, precipitate was collected and recrystallized from ethanol. Finally titled compounds were prepared by dissolving appropriate 2-amino-(5-substituted phenyl)-1,3,4-thiadiazole and 4-pyrrol-1yl-benzioc acid in dry DMF, further HBTU and DIEA were added and stirred for 5h. All the newly synthesized compounds were characterized by melting point, Rf value, FTIR, NMR, and MASS spectroscopy. The FTIR spectrum of pyrrolyl thiadiazole derivatives showed characteristic carbonyl (C=O) absorption band between 1655 to 1689 cm^-1 and absence of broad hydroxyl band band around 3400 cm^-1 reveals formation of final compounds. 1H NMR spectra of the compounds displayed presence amide –NH peak in between δ 12.63 to 12.81 ppm and absence of proton peak of hydroxyl group around δ 10 to 11 ppm. In case of 13CNMR spectroscopy carbonyl (C=O) carbon observed around δ 164 ppm. The mass spectra of the compounds displayed base peak at M⁺ and M⁺⁺1 corresponding to their respective molecular weights.

Antibacterial activity

Antibacterial activity of newly synthesized compounds towards gram +ve (Staphylococcus aureus) and gram –ve bacteria (Escherichia coli) by broth micro dilution method and reference ciprofloxacin and norfloxacin were included for comparison under in vitro conditions and the results are shown in Table 1. In this study it reveals that electron donating substituent showed significant activity against gram +ve and gram –ve bacteria.

Antitubercular Activity

Antitubercular activity of newly synthesized compounds pyrrolyl-1, 3, 4-thiadiazole derivatives towards M. tuberculosis H37Rv was determined using Microplate Alamar Blue Assay (MABA) method and the reference ligand Isoniazid was included for comparison under in vitro conditions and the results are shown in Table 2. From this study it reveals that electron withdrawing substituents (Br, Cl) at para position of phenyl ring enhance the antitubrecular activity (MIC 3.2 µg/mL) as compared to other substitutions.

Conclusion

Compounds exhibited anti-bacterial activity (expressed as MIC) in the range of 3.2 to 100 μ g/mL against gram +ve and gram –ve bacteria. Compound 5d showed the most significant activity against both gram +ve (Staphylococcus aureus) and gram –ve bacteria (Escherichia coli). Antitubercular activity at MIC value in the range of 3.125 and 25 μg/mL. Compounds 5c and 5d showed highly significant activity against M. tuberculosis H37Rv. These compounds can be considered for further modification with the molecular modelling and insilico studies to get more potent antitubercular and antibacterial agents.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgement

Authors immensely thank the research support from Indian Council of Medical Research, New Delhi, India (Letter No. BIC/12(13)2014 dated13/02/2017). We also thank Dr. T. M. Aminabhavi, Research Director and Dr. H. V. Dambal, President, S. E. T’s College of Pharmacy, Dharwad for providing the facilities. We thank Dr. K.G. Bhat of Maratha Mandal’s Dental College, Hospital and Research Centre, Belgaum, for providing antibacterial and antitubercular activities. SAIF, Panjab University, Chandigarh, Panjab, India provided some of the NMR and mass spectral data. The authors are grateful to Mr. Manjunath Hiremath for his technical assistance.