ISSN   0974-3618  (Print)                  www.rjptonline.org

            0974-360X (Online)

 

 

RESEARCH ARTICLE

 

Synthesis of New Series of Benzimidazole Acetic Acid Derivatives Bearing Thiophene Moiety for Anti-Tubercular Activity

 

M. S. Palled*, A. R. Bhat, A. Patel

Department of Pharmaceutical Chemistry, K.L.E.S’s College of Pharmacy, KLE University Belagavi, 590 010, Karnataka, India.

*Corresponding Author E-mail: pjpalled@gmail.com

 

ABSTRACT:

Tuberculosis is a disease that has been known from the earliest of recorded history. The development of the multidrug-resistant TB (Mtb) strains, treatment of active TB is more complicated than it used to be. Therefore, there is an urgent need for the development of novel anti-tuberculosis drugs, which will be active against both drug-sensitive and drug-resistant Mtb strains. FtsZ (Filamental temperature-sensitive protein Z), a tubulin homolog, is the most critical protein for bacterial cell division. GTP dependent polymerization of FtsZ forms a dynamic helical structure at the center of the cell called Z-ring. Recruitment of other cell division proteins leads to the contraction of Z-ring, which initiates the cell division. Therefore, novel molecules, which interfere polymeriza­tion or depolymerization of FtsZ can be developed as anti-tuberculosis agents. It has been found that the treat­ment of MTB with albendazole and thiabendazole, known tubulin inhibitors, leads to the cell filamentation, indicative of FtsZ inhibition. Accordingly, we designed and synthesized a library of benzimida­zoles to investigate their microbacterial activities. A number of these compounds demonstrated substantial activity against H37RV strain. We will present the synthesis and biological evaluations of these compounds.

 

KEY WORDS: Thiophene, Anti-tubercular, Antimicrobial, Benzimidazole acetic acid.

 

INTRODUCTION:

TB (Tubercle bacillus) has been one of the leading causes of death among humans. Its easy transmission through air among humans turned TB into a global pandemic1.Tuberculosis, termed the “captain of the men of death” is a major public health problem2. Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergency of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. Novel anti-TB drugs are urgently required3 M. tuberculosis is responsible for most cases of tuberculosis; the reservoir of infection is humans with active tuberculosis4.

 

 

 

 

 

 

 

 

 

 

 

Received on 02.04.2015          Modified on 08.04.2015

Accepted on 15.04.2015         © RJPT All right reserved

Research J. Pharm. and Tech. 8(6): June, 2015; Page 674-678

DOI: 10.5958/0974-360X.2015.00106.7

 

Various derivatives of thiophene5-7 exhibit interesting biological properties like anti-tubercular and antifungal activities hence attempt was made to incorporate benzimidazole acetic acid derivatives bearing thiophene to achieve promising anti-tubercular compounds.

 

Antimicrobial Activity8

The antimicrobial activity of the synthesized compounds was determined by cup-plate method. The antibacterial activity was determined against gram-positive organism Staphylococcus aureus and gram-negative organism Escherichia coli at 50-mcg/ml and 75mcg/ml concentration of sample compounds. Dimethyl Formanide was used as control. The bacteria were subcultured on nutrient agar broth and incubated at 37ᵒC for 18-24 hrs. Standard antibacterial drug Ciprofloxacin was also screened under similar conditions at 50µg/ml and 75 µg/ml concentration for comparison. The antifungal activity was carried out against the fungi Candida albicans and Aspergillus niger at 50 µg/ml and 75 µg/ml concentration of sample compounds. The fungi were subcultured in Sabourod’s dextrose agar medium. The fungal susceptibility testing was done by cup-plate method using Fluconazole (50 µg/ml and 75 µg/ml concentration) as standard. The petridishes were incubated a 370C for18-24 hrs. [Table 3].

 

Anti-Tubercular Evaluation9

The anti-tubercular screening of synthesized compounds was carried out by middle brook 7H9 broth base (M198) medium against H37Rv strain at100 mg/ml, 125 mg/ml and 250 mg/ml. middle brook 7H9 broth base (M198) medium was inoculated with mycobacterium tuberculosis of H37Rv strain. The inoculated medium was incubated for 370C for 6 weeks. At the end of 6 weeks the growth of mycobacterium tuberculosis was read. [Table 4]. Streptomycin (100 mg/ml, 125mg/ml and 250 mg/ml was used as a standard drug.

 

 

                         A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                          

                             A

 

 

 

 

 

 

 

Figure 1: Proposed scheme for synthesis of compounds

 

 

 

 

 

 

 

 

Experimental:

Melting points were determined in open capillary method and are uncorrected. IR spectra were recorded on Thermo Nicolet IR 200 spectrophotometer using KBr disc method. The 1H-NMR spectra were recorded on sophisticated multinuclear FT-NMR Spectrometer model Avance-III (Bruker), using dimethylsulfoxide-d6 as solvent and tetramethylsilane as internal standard.

 

1. General Method for Preparation of Cinnamic Acids:

Aromatic aldehyde (0.02 mole) and malonic acid (0.42 mole) was dissolved in a mixture of dry pyridine (75 ml) and piperidine (1.3 ml) was added. The reaction mixture was heated under reflux for 2 hrs. A rapid evolution of carbon dioxide took place. Cooled, poured into excess of water containing hydrochloric acid (1N) to combine with pyridine. The solid that separated was filtered and recrystallized from hot water10. Yield -91%, m.p. -134-1350C. 

 

2. Synthesis of 2-(2-Phenyl ethenyl)-1H-benzimidazole:

A mixture of aryl cinnamic acid (0.05 mol) and o-Phenylenediamine (0.05 mol) was treated with 4 N Hydrochloric acid and stirred at room temperature for 1hour, until it goes into a solution. The reaction mixture refluxed further for 4-6 hours cooled and neutralized with dilute ammonia. The precipitate that separated was filtered and washed with water and crystallized from methanol to get solid crystal of pure (BZ1) 11 yield - 96%, m.p. –119-1200C.

 

3. Synthesis of 2-(2-Phenyl ethenyl) – 1 carboxy- benzimidazole:

A mixture of 2 (2-Phenyl ethenyl)–1H benzimidazole (0.01 mol) and chloroacetic acid (0.01mol) in 30 ml of dry benzene was refluxed for 7-8 hour using dean stark apparatus. The residue was washed with Sodium bicarbonate solution and the product was washed with water thoroughly and crystallized from alcohol to get solid crystals of pure (BC1) 12Yield- 82%, m.p. – 125-1260C       

 

4. Synthesis of 5-(1-carboxy benzimidazole - 2 - yl)–4-Phenyl dihdrothiophen-3-(2H)-one:

A mixture of 2 (2-Phenyl ethenyl)–1carboxy benzimidazole (0.01 mol) and thioglycollic acid (0.01mol) in 30 ml of dry benzene was refluxed for 7-8 hour using dean stark apparatus. The residue was washed with Sodium bicarbonate solution and the product was washed with water thoroughly and crystallized from alcohol to get solid crystals of pure (BT1) 12-13 Yield- 80%, m.p. –142- 1430C.

 

 

5. Synthesis of (2-substitutedphenyl)-1H-benzimidazole- acetic acid hydrazides:

Hydrazine hydrate (4 ml) and related 1H-benzimidazole acetic acid ethyl ester (1.5 mmol) in ethanol (5 ml) were refluxed for 4 hrs. The reaction mixture was cooled and poured into water. The crude product was filtered off and recrystallised from ethanol to give ethanol to give the desired hydrazide compounds14 yield 85%, m.p.-140-1520C.

 

6. Synthesis of (2-substitutedphenyl)-1H-benzimidazole- N’-(substituted aryl  methylidene) acetic acid hydrazides:

0.0025 mol of (2-substitutedphenyl)-1H-benzimidazole acetic acid hydrazides was refluxed with 0.0025 mol of appropriate aldehyde in 50 ml ethanol (60%) for 5 hrs. The solid separated was washed with ethanol (60%)15 yield 80%, m.p. – 160-1620C.

 

RESULT AND DISCUSSION:

The title compounds were prepared from 2(2-substituted phenyl ethenyl)-1H-benzimidazole by various steps. (Table 1 and 2) The structures of the compounds were confirmed by spectra and analytical studies.

 

Table No. 1: Analytical data of the synthesized compounds

Compound

Mol. Formula

Mol. Wt. (gm)

Melting Point 0C

Yield %

AA

C27H24O3N4S

484

160-162

72

AB

C27H24O4N4S

500

155-157

62

CA

C27H23O4N4SBr

579

180-182

55

CB

C27H24O4N4S

500

189-191

57

DA

C26H20O5N5SBr

594

173-175

61

DB

C27H23O3N4SBr

563

167-169

54

DC

C27H23O4N4SBr

579

164-166

52

 

 

Table No. 2: Elemental analysis of synthesized compounds (calculated)

Compounds

C

H

N

O

S

Br

AA

66.92

4.99

11.56

9.91

6.62

-

AB

64.78

4.83

11.19

12.78

6.41

-

CA

55.96

4.00

9.67

11.04

5.53

13.79

CB

64.78

4.83

11.19

12.78

6.41

-

DA

52.53

3.39

11.78

13.46

5.39

13.44

DB

57.55

4.11

9.94

8.52

5.69

14.18

DC

55.96

4.00

9.67

11.04

5.53

13.79

 

All the compounds synthesized matched with spectral data. All the compounds were screened for antibacterial and antifungal activity from 50µg/ml and 75µg/ml. Compounds AA, DA and DB have shown significant antibacterial activity against E. coli. Compounds AA and AB have shown significant activity against S. aureus, while other compounds showed moderate activity. Ciprofloxacin was used as standard drug. The compounds AA and DB have shown significant antifungal activity against C. albicans, while other compounds show moderate activity. Nystatin was used as standard drug.


Table No. 3: Antibacterial and Antifungal activity of synthesized compounds.

SL. No.

Compound

Zone of inhibition at 75 and 50mg/ml (in mm.)

            E. coli

        S. aureus

         A. niger

       C. albicans

75                   50

75                  50

75             50

75                 50

1

AA

12

11

16

11

10

R

16

15

2

AB

10

5

18

13

12

11

11

10

3

CA

10

8

R

R

R

R

15

10

4

CB

6

5

10

8

R

R

11

10

5

DA

12

8

13

10

R

R

11

9

6

DB

12

11

15

12

R

R

16

14

7

DC

11

9

11

10

R

R

11

10

Standard

Ciprofloxacin

22

21

22

20

-

-

-

-

Standard

Nystatin

-

-

-

-

21

20

21

20

 

Table No. 4: Anti-tubercular activity of the synthesized compounds

Sr. No.

Compounds

10 µg/ml

25µg/ml

50 µg/ml

1

AA

R

R

S

2

AB

S

S

S

3

CA

R

R

S

4

CB

R

R

S

5

DA

S

S

S

6

DB

R

S

S

7

DC

S

S

S

Standard

Streptomycin

S

S

S

R denotes Resistant and S denotes sensitive

 

 

The compounds synthesized were screened mainly for the anti-tubercular activity by using middle brook Media method using H37Rv strain. Compounds BT1, BT3, and BT6 have shown very significant anti-tubercular activity when compared with the standard drug Streptomycin. Remaining compounds have also shown moderate anti-tubercular activity. (Table 4).  All compounds found to be very good anti-tubercular agents and present synthesized compound can definitely as lead compound for future molecular manipulation studies.

 

CONCLUSION:

The present work intended to study the synthesis and evaluation of certain benzimidazoles derivatives and derivatives associated with 4-thiophene for antitubercular, antifungal and antibacterial activities. The detailed review of the literature survey was carried out for the synthesis. The structures of the synthesized compounds were confirmed on the basis of M.P., TLC, IR, 1H NMR and MASS spectra.

 

The compounds synthesized in the present work were screened mainly for the antitubercular activity by using Middle brook 7H9 Broth Base (M198) media method using H37RV strain. It was indeed very much encouraging to note that with some exception most of the compounds have shown better and significant antitubercular activity. Streptomycin was used as the standard drug. The synthesized compounds were also screened for antibacterial activity against S. aureus and E. coli at 75 and 50 µg/ml  and antifungal activity against C. albicans and A. fumigates 75 and 50 µg/ml by disc diffusion method. Ciprofloxacin and Nystatin were used as the standard drug for antibacterial and antifungal activity respectively. With suitable molecular modification of the presently synthesized compounds, it will be possible to find potential lead compounds for better biological activities in future.

 

Analytical data of the Synthesized Compounds:

·         AA: IR(KBr) cm-1: 1750 (-C=O Str.), 1350(-CN Sec.), 1320 (-CN Pri.). 13C NMR: 144.92 (C1 aromatic), 120.17 (C2 aromatic), 130.91 (C3 aromatic), 124.32 (C4 aromatic), 130.91 (C11,C12 benzimidazole), 128.81 (C16 thiophene), 125.07 (C20 thiophene).

·         AB : IR(KBr) cm-1 : 3150 (-NH), 1750 (-C=O Str.), 550 (-Br)

·         CA : IR(KBr) cm-1 : 3300 (-NH), 2800 (-CH2-), 1590 (-C=O), 500 (-Br).

·         CB : IR(KBr) cm-1 : 3190 (-NH), 1650 (-C=O), 1580 (-N=CH-), 700 (-C-S-)

·         DA : IR(KBr) cm-1 : 3190 (-NH), 1680 (-C=O), 1500 (-N=CH-), 1500 (-N=O), 550 (-Br).

·         DB : IR(KBr) cm-1 : 3150 (-NH), 1750 (-C=O), 1550 (-N=CH-), 550 (-Br).

·         DC : IR(KBr) cm-1 : 3150 (-NH), 1700 (-C=O), 1600 (-N=CH-), 1250 (-CH3-), 780 (-Br). 1H NMR (DMSO solvent) d: 2.09 (-CH2), 3.83 (-OCH3), 6.54-7.83 (11H, Ar-H), 8.63 (-NH).

 

 

ACKNOWLEDGEMENT:

The authors sincerely thank to Dr. V P Rasal, Principal, K.L.E.S’s College of Pharmacy, Belgaum, for his kind help and encouragement during our research work.

 
 
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