chapter 4 studies on benzimidazole...

CHAPTER 4

Studies on

Benzimidazole

derivatives

Chapter 4 Studies on benzimidazole derivatives

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Ph.D. Thesis (2013) – Deepkumar S. Joshi, DST-JRF (INSPIRE fellow), MNSC-HNGU, Patan

4. Studies on benzimidazole derivatives

4.1 Introduction and scope of present work

The versatility of benzimidazole and its property of exhibiting high potency

against several microbial strains1 and cancer cell lines2 has drawn attention of many

researchers to concentrate their work around it. Different derivatives of benzimidazole

are found to possess antibacterial, antifungal and molluscicidal activities.3 Benzimidazole

is a class of heterocyclic scaffold exhibiting antiparasitic,4 analgesic,5 anticancer,6

antiprotozoal7,8 and chemotherapeutic properties.9 Some of the benzimidazole

derivatives are utilized as inhibitors of MDA-MB-231 human breast cell proliferation.10

Several benzimidazole derivatives are observed to exhibit anti-inflammatory,11

antihypertensive,12 antidiabetic as well as anti-asthametic properties.13

Drugs available in the market viz. albendazole, mebendazole and bendamustine

are found to possess benzimidazole as its core nucleus. Literature survey also reveals

that benzimidazole ring containing molecules are potent growth inhibitors over a wide

range of bacteria and fungi.14,15 Synthesis and evaluation of differently substituted

benzimidazole analogs resulted in the discovery of some important drugs viz.

omeprazole, lansoprazole, rabeprazole and pantoprazole. Benzimidazole derivatives are

used as CK2 inhibitors against the CK2 proteins responsible for neoplastic growth in

animals.16 Some fused heterocyclic benzimidazole derivatives are used as eukaryotic

topisomerase II inhibitors.17 Variedly substituted benzimidazoles exhibited excellent

activity against a wide variety of viruses including HIV, resulting in development of

several leads possessing benzimidazole nucleus and were found capable of ceasing

adenoviral replication.18

In accordance with earlier work of synthesizing bio-active molecules

possessing benzimidazole nucleus,19 an attempt was made to synthesize and present in

this chapter a series of heterocyclic compounds bearing benzimidazole nucleus as the

core structure. The extraordinary property of benzimidazole as potent anti-cancer6,20

and antimicrobial21-22 has drawn attention to concentrate research around it. Several

other biological properties associated with different benzimidazole derivatives include

antiparasitic,4 analgesic,23 antiprotozoal7 and molluscicidal3 properties. It is also


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observed that benzimidazole derivatives are found to possess anti–HIV,24 anthelmintic,25

antimycobacterial,26 antidiabetic27 and antioxidant properties.28 Several biological

properties exhibited by different benzimidazole scaffolds includes antiallergic,29

analgesic5,30 and anti-hypertensive activity.31 The literature survey helped to understand

the versatility of benzimidazole and its high potency as a therapeutic agent,32 which

directed to synthesize two varied series of benzimidazole derivatives possessing -OCH3

and -OCHF2 substituent.

Benzimidazole analogs bearing electron-withdrawing as well as electron-

donating substituent were synthesized, in order to achieve bioactive molecules with

significant antimicrobial property. The desired compounds were prepared by multi-step

synthesis process. The formation of intermediates and their corresponding derivatives

(III1-13) and (IIIa-j) was confirmed by spectral characterization such as 1H NMR, mass

spectra, IR and elemental analysis. The compounds were screened for their

antimicrobial properties against a broad panel of gram-positive and gram-negative

bacteria as well as fungi. From the SAR study data, it was observed that the derivatives

with electron-withdrawing functional groups were more bio-active than that with

electron-donating functional groups.


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4.1.1 General structure for 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-

N-arylacetamide derivatives (III1-13)

4.1.2 Table-1: List of 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-

arylacetamide derivatives (III1-13)

Compounds Substituent Nomenclature

III1 -3-NO2 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-(3-

nitrophenyl)acetamide



III3 -3-Cl N-(3-chlorophenyl)-2-(5-methoxy-1H-benzo[d]imidazol-2-

ylthio)acetamide

III4 -3-F-4-Cl N-(4-chloro-3-fluorophenyl)-2-(5-methoxy-1H-

benzo[d]imidazol-2-ylthio)acetamide

III5 -H 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-

phenylacetamide

III6 -4-F N-(4-fluorophenyl)-2-(5-methoxy-1H-benzo[d]imidazol-2-

ylthio)acetamide

III7 -2-OCH3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-(2-

methoxyphenyl)acetamide

III8 -3-CH3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-m-

tolylacetamide

III9 -3-OCH3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-(3-

methoxyphenyl)acetamide



III11 2-CH3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-o-

tolylacetamide

III12 4-CH3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-p-

tolylacetamide

III13 3-CF3 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-(3-

(trifluoromethyl)phenyl)acetamide


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4.1.3 List of synthesized derivatives III1-13

4.1.3.1 Derivatives III1-4


5




6




7


4.1.3.4 Derivative III13


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4.1.4 General structure for 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-

2-ylthio)-N-arylacetamide derivatives (IIIa-j)

4.1.5 Table-2: List of 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-

ylthio)-N-arylacetamide derivatives (IIIa-j)

Compounds Substituent Nomenclature

IIIa -3-NO2 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-

N-(3-nitrophenyl)acetamide

IIIb -4-NO2 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-


IIIc -3-Cl N-(3-chlorophenyl)-2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)acetamide

IIId -3-F-4-Cl N-(4-chloro-3-fluorophenyl)-2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)acetamide

IIIe -H 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-N-phenylacetamide

IIIf -4-F 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-N-(4-fluorophenyl)acetamide

IIIg -2-OCH3 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-

N-(2-methoxyphenyl)acetamide

IIIh -3-CH3 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-

N-m-tolylacetamide

IIIi -2-NO2 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-


IIIj -3-CF3 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-

N-(3-(trifluoromethyl)phenyl)acetamide


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4.1.6 List of synthesized derivatives IIIa-j

4.1.6.1 Derivatives IIIa-d


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4.1.6.2 Derivatives IIIe-h


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4.1.6.3 Derivatives IIIi-j


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4.2 Designing the derivatives – Lipinski’s rule of five

4.2.1 Table-3: Screening for drug-likeness of derivatives III1-13

(stage 1 only)

Entry Descriptors for Lipinski’s rule of five No. of violation M.W. Log P No. of

H-bond donor

No. of H-bond acceptor

III1 358 2.955 2 6 0

III2 358 2.955 2 6 0

III3 347 3.619 2 5 0

III4 365 3.762 2 6 0

III5 313 3.015 2 4 0

III6 331 3.158 2 5 0

III7 343 2.858 2 5 0

III8 327 3.529 2 4 0

III9 343 2.858 2 5 0

III10 358 2.955 2 6 0

III11 327 3.529 2 4 0

III12 327 3.529 2 4 0

III13 381 3.893 2 7 0

The data depicted in the table above indicates the presence of H-bond donor and

H-bond acceptor sites in the designed molecules. Only two H-bond donor sites are

found among all the derivatives synthesized III1-13 (indicated by red color). Four

common H-bond acceptor sites were observed in each of the derivatives and other H-

bond acceptor sites varied depending upon the substituent used (-CF3, -NO2, -Br, -Cl, -

F and -OCH3).


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4.2.2 Table-4: Screening for drug-likeness of compounds IIIa-j

(stage 1 only)

Entry Descriptors for Lipinski’s rule of five No. of violation M.W. Log P No. of

H-bond donor

No. of H-bond acceptor

IIIa 394 3.882 2 8 0

IIIb 394 3.882 2 8 0

IIIc 383 4.546 2 7 0

IIId 401 4.689 2 8 0

IIIe 349 3.942 2 6 0

IIIf 367 4.085 2 7 0

IIIg 379 3.782 2 7 0

IIIh 363 4.455 2 6 0

IIIi 394 3.882 2 8 0

IIIj 417 4.82 2 8 0

The H-bond donor sites for all the derivatives IIIa-j remain the same, but the H-

bond acceptor sites were found to vary with the change in the substituent (-R). Six H-

bond acceptor sites were found to be the same in all designed molecules viz. one -O and

two -F atoms in -OCHF2, atoms -O in carbonyl, -S- as a linkage and -N in

benzimidazole ring. H-bond donor sites were found to remain constant through out all

the derivatives IIIa-j.


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4.3 Experimental - Schematic representation and procedure for the

synthesis of compounds III1-13 and IIIa-j

4.3.1 Synthesis of 5-methoxy-1-H-benzo[d]imidazole-2-thiol I (step 1)

Ethanol (40 ml) and potassium hydroxide (0.01 mol, 56.11 gm/mol,

0.56 gm in 2 ml H2O) were taken in a dry round bottom flask. 4-methoxybenzene-1,2-

diamine (0.01 mol, 138.08 gm/mol, 1.38gm) was added to it and stirred well to get a

clear solution. Carbon disulfide (0.02 mol, 76.14 gm/mol, 1.2 ml) was added to the

clear solution obtained above and refluxed for 12 -15 h. The ethanol was distilled off

and then cooled to room temperature. The content was poured into water and acidified

with diluted HCl till the precipitates were separated. The separated solid was washed

with cold water and dried to get the desired product. The completion of the reaction

was monitored on TLC using ethyl acetate: benzene (6:4) as mobile phase. m.p.:258°C;

Yield: 72%.

4.3.2 Synthesis of 5-(difluoromethoxy)-1H-benzo[d]imidazole-2-thiol I’ (step

2)

Ethanol (40 ml) and potassium hydroxide (0.01 mol, 56.11 gm/mol, 0.56 gm in

2 ml H2O) were taken in a dry round bottom flask. 4-(difluoromethoxy)benzene-1,2-

diamine (0.01 mol, 174.14gm/mol, 1.74 gm) was added to it and stirred well to get a

clear solution. Carbon disulfide (0.02 mol, 76.14 gm/mol, 1.2 ml) was added to the

clear solution obtained above and refluxed for 12 -15 h. The ethanol was distilled off

and then cooled to room temperature. The content was poured into water and acidified

with diluted HCl till the precipitates were separated. The separated solid was washed

with cold water and dried to get the desired product. The completion of the reaction


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was monitored on TLC using ethyl acetate: benzene (6:4) as mobile phase. m.p.:241°C;

Yield: 56%.

4.3.3 Synthesis of 2-chloro-N-(aryl)acetamide derivatives II1-13 and IIa-j (step

3)

Various substituted amines (0.01 mol) were added to a solution of DMF (35ml)

containing TEA (3-4 drops). The mixture was stirred for 10 minutes at room

temperature. CAC (0.015 mol, 113gm/mol, 1.19 ml) was added to the above mixture,

maintaining the temperature between 0 to 5°C. The obtained solution was then stirred

at room temperature for 4-6h. The completion of reaction was monitored with TLC

using toluene: acetone (8:2) as mobile phase. The solution was then added onto crushed

ice and the separated precipitates were filtered and dried. The product was crystallized

from methanol.


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4.3.4 Synthesis of 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-

(aryl)acetamide derivatives III1-13 (step 4)

5-methoxy-1H-benzo[d]imidazole-2-thiol I (0.01 mol, 180 gm/mol, 1.8 gm) was

made soluble in acetone. To this well stirred solution different 2-chloro-N-

arylacetamide derivatives II1-13 (0.01 mol) were added to the above solution. K2CO3

(0.02 mol, 138 gm/mol, 2.76 gm) was added to the solution containing mixture of 5-

methoxy-1H-benzo[d]imidazole-2-thiol I and different acetamide derivatives. The mixture

was allowed to stir for 4h at room temperature. The completion of reaction was

monitored using TLC plate with mobile phase ethyl acetate: n-hexane (6:4). The final

products thus obtained were poured into ice cold water and stirred for 30 min. The

precipitates were filtered and washed occasionally. The final products III1-13 obtained

were crystallized from alcohol.


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4.3.5 Synthesis of 2-(5-(difluoromethoxy)-1H-benzo[d]imidazol-2-

ylthio)-N-arylacetamide derivatives IIIa-j (step 5)

5-(difluoromethoxy)-1H-benzo[d]imidazole-2-thiol I’ (0.01 mol, 180 gm/mol,

1.8 gm) was made soluble in acetone. To this well stirred solution different 2-chloro-N-

arylacetamide derivatives IIa-j (0.01 mol) were added to the above solution. K2CO3

(0.02 mol, 138 gm/mol, 2.76 gm) was added to the solution containing mixture of 5-

methoxy-1H-benzo[d]imidazole-2-thiol I and different acetamide derivatives. The mixture

was allowed to stir for 4h at room temperature. The completion of reaction was

monitored using TLC plate with mobile phase ethyl acetate: n-hexane (6:4). The final

products thus obtained were poured into ice cold water and stirred for 30 min. The

precipitates were filtered and washed occasionally. The final products IIIa-j obtained

were crystallized from alcohol.


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4.4 Physical constants for the final derivatives III1-13 and IIIa-j

4.4.1 Table-5 Physical constants data for derivatives III1-13

Derivatives Substituent Molecular

formula

Molecular weight

gm/mol

m.p. °C

% Yield

Elemental Analysis

% Carbon % Hydrogen %Nitrogen

Calcd. Found Calcd. Found Calcd. Found

III1 -3-NO2 C16H14N4O4S 358 118°C 50% 53.62 53.67 3.94 3.98 15.63 15.67

III2 -4-NO2 C16H14N4O4S 358 125°C 59% 53.62 53.66 3.94 3.99 15.63 15.66

III3 -3-Cl C16H14N3O2SCl 347 121°C 63% 55.25 55.29 4.06 4.10 12.08 12.11

III4 -3-F-4-Cl C16H13N3O2SClF 365 202°C 59% 52.53 52.56 3.58 3.62 11.49 11.53

III5 -H C16H15N3O2S 313 118°C 55% 61.32 61.35 4.82 4.85 13.41 13.45

III6 -4-F C16H14N3O2SF 331 158°C 58% 57.99 58.02 4.26 4.30 12.68 12.72

III7 -2-OCH3 C17H17N3O3S 343 156°C 44% 59.46 59.51 4.99 5.04 12.24 12.28

III8 -3-CH3 C17H17N3O2S 327 105°C 54% 62.36 62.41 5.23 5.28 12.83 12.87

III9 -3-OCH3 C17H17N3O3S 343 195°C 62% 59.46 59.50 4.99 5.04 12.24 12.27

III10 -2-NO2 C16H14N4O4S 358 105°C 60% 53.62 53.66 3.94 3.95 15.63 15.66

III11 -2-CH3 C17H17N3O2S 327 156°C 53% 62.36 62.41 5.23 5.28 12.83 12.87

III12 -4-CH3 C17H17N3O2S 327 175°C 76% 62.36 62.41 5.23 5.28 12.83 12.87

III13 -3-CF3 C17H14N3O2SF 381 109°C 66% 53.54 53.58 3.70 3.74 11.02 11.05


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4.4.2 Table-6 Physical constants for derivatives IIIa-j

Derivatives Substituent Molecular

formula

Molecular weight

gm/mol

m.p. °C

% Yield

Elemental Analysis

% Carbon % Hydrogen %Nitrogen

Calcd. Found Calcd. Found Calcd. Found

III a -3-NO2 C16H12F2N4O4S 394 182°C 87% 48.73 48.75 3.07 3.10 14.21 14.25

III b -4-NO2 C16H12F2N4O4S 394 194°C 66% 48.73 48.76 3.07 3.11 14.21 14.25

III c -3-Cl C16H12ClF2N3O2S 383 154°C 89% 50.07 50.11 3.15 3.18 10.95 10.98

III d -3-F-4-Cl C16H11ClF3N3O2S 401 166°C 78% 47.83 47.86 2.76 2.80 10.46 10.49

III e -H C16H13F2N3O2S 349 182°C 56% 55.01 55.05 3.75 3.78 12.03 12.05

III f -4-F C16H12F3N3O2S 367 156°C 85% 52.31 52.34 3.29 3.32 11.44 11.47

III g -2-OCH3 C17H15F2N3O3S 379 135°C 89% 53.82 53.85 3.99 4.03 11.08 11.11

III h -3-CH3 C17H15F2N3O2S 363 194°C 94% 56.19 56.22 4.16 4.20 11.56 11.60

III i -2-NO2 C16H12F2N4O4S 394 195°C 87% 48.73 48.75 3.07 3.10 14.21 14.25

III j -3-CF3 C17H12F5N3O2S 417 158°C 84% 48.92 48.95 2.90 2.93 10.07 10.11


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4.5 Characterization – Spectral data

4.5.1 Spectral status for III1-13 derivatives

4.5.1.1 IR spectra for compound III13

4.5.1.2 table-7: IR spectral data for compound III13

Functional group Frequency

(Cm-1) Functional group

Frequency (Cm-1)

-N-H sec. amine (str.) 3393 -C=O carbonyl group

(str.) 1698

-C-H aromatic ring (str.) 3095 -C=N sec. amine (str.) 1580

-C-H methoxy group (str.) 2921 -C-F (str.) 1161

-CH2 methylene group (str.) 2853 -Nil- -Nil-


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4.5.1.3 1H NMR spectra for compound III13

4.5.1.4 Table-8: 1H NMR spectral data for compound III13

Assignment No. of protons Multiplicity value (ppm)

-NH; -NH (in ring) 1; 1 singlet; singlet 11.21; 12.33

-OCH3 3 Singlet 3.75

-CH2 2 Singlet 4.11

Ar-H 7 Multiplet 6.74-8.03


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4.5.1.5 Mass spectra for compound III13

4.5.1.6 Table-9: Mass spectral data for compounds III13

Peak assignment m/z value

M+1 peak 382.123506


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4.5.2.1 IR spectra for compound IIIj

4.5.2.2 Table-10: IR spectral data for compounds IIIj

Functional group Frequency

(Cm-1) Functional group

Frequency (Cm-1)

-N-H sec. amine (str.) 3403 -C=O carbonyl group

(str.) 1674

-C-H aromatic ring (str.) 3090 -C=N sec. amine (str.) 1581

-C-H –OCHF2 group (str.) 3003 -C-F (-OCHF2) (str.) 1173

-CH2 methylene group (str.) 2840 -Nil- -Nil-


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4.5.2.3 1H NMR spectra of compound IIIj

4.5.2.4 Table-11: 1H NMR data table for compound IIIj

Assignment No. of protons Multiplicity value (ppm)

-OCHF2 1 Singlet 6.93

-NH; -NH 1; 1 singlet; singlet 10.92; 12.35

-CH2 2 singlet 4.17

Ar-H 7 multiplet 6.58-8.03


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4.5.2.5 Mass spectra for compound IIIj

4.5.2.6 Table-12: Mass spectral data for compound IIIj

Peak assignment m/z value

M+1 peak 418


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4.5.3 Spectral interpretation (IR, 1H NMR, Mass)

4.5.3.1 Spectral interpretation for compound III13

If we observe the data for 2-(5-methoxy-1H-benzo[d]imidazol-2-ylthio)-N-(3-

(trifluoromethyl)phenyl)acetamide III13, An absorption peak obtained at 3393 cm-1 has

helped in confirming the presence of -NH in vicinity to the carbonyl group. The

presence of aromatic C-H linkage is confirmed by the peak obtained at 3095 cm-1. A

sharp peak at 2921 cm-1 helped to assign the presence of -C-H bond in -OCH3 group. A

stretching band at 2853 cm-1 concluded the presence of methylene group in the final

motif. The presence of carbonyl (-C=O) in the structure is proved due to the presence

of a sharp peak at 1698 cm-1. The presence of -C=N in the benzimidazole nucleus was

also confirmed by the presence of a sharp absorption band at 1580 cm-1.

Compound III13 when observed under 1H NMR, the different peaks obtained at

desirable values (in ppm) confirmed its formation. A singlet at = 3.75 helped to

prove the presence of three protons of the methoxy group. The presence of methylene

group was indicated by = 4.11 showing a sharp singlet peak. The aromatic protons

present in the phenyl ring were confirmed by the multiplet peak obtained in the range of

= 6.74 to = 8.03. In the structure there are two -NH (secondary amine) present;

the two protons of each secondary amine can be easily differentiated from the shift

observed in the peaks of the corresponding amines. The proton of -NH in the vicinity of

-C=O group was confirmed by value = 11.21 where as the proton of secondary

amine in the ring confirmed its presence by showing a singlet at downfield shift of =

12.33.

From the mass spectral data, it is observed that the molecular ion peak obtained

at 382.1 m/z value confirmed the formation of the desired derivative.


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4.5.3.2 Spectral interpretation for compound IIIj

Let us consider compound IIIj and try to understand its formation from the IR,

1H NMR, 13C NMR and mass analysis. IR spectral data for the compound 2-(5-

(difluoromethoxy)-1H-benzo[d]imidazol-2-ylthio)-N-(3-

(trifluoromethyl)phenyl)acetamide IIIj has shown a stretching vibration at 3403 cm-1

indicating clearly the presence of -NH (secondary amine) in the molecule. The presence

of a band at 3003 cm-1 confirmed the presence of -C-H bond in the -OCHF2

substitution. The -C-H bond in the aromatic ring can be depicted from a sharp band

obtained at 3090 cm-1. A weak absorption band observed at 2840 cm-1 helped to prove

the formation of the -CH2 linkage (methylene group) in the molecule. The presence of -

C=O (carbonyl) group and -C=N was confirmed by a sharp intensified bands at 1674

cm-1 and 1581 cm-1 respectively. Also the presence of fluorine atom in form of -C-F

band (in -OCHF2) was proved by a sharp band at 1173 cm-1.

The chemical shift observed in 1H NMR spectral data added more confirmation

to the formation of final derivatives. In the compounds IIIj, the absorption peak at =

12.35 helped to exhibit the presence of proton of secondary amine group (-NH) in the

benzimidazole nucleus. The proton of -NH (secondary amine) group in between the

methylene and the carbonyl group confirmed its presence with a absorption band at

value 10.92 ppm. The aromatic protons were proved by a broad band between the

values 6.58 to 8.03. A sharp absorption band appearing at = 4.17 confirmed the

presence of -CH2 linkage, as the value appeared corresponded to the two protons of

the methylene group.

A molecular ion peak observed at 318 m/z value confirms the formation of the

compound IIIj.


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4.6 Therapeutic studies – antimicrobial studies

4.6.1 Antimicrobial assay

Broad panels of bacterial and fungal strains were used for testing the

antimicrobial properties of the synthesized molecules III1-13 and IIIa-j. The results

obtained were depicted in the form of Minimum Inhibitory Concentration (MIC) values

for the synthesized derivatives. The samples were tested by standard protocols like

micro dilution/broth titer method. The screening for antimicrobial activity was carried

out by diluting the solution and preparing the sets consecutively from 1000, 500, 250,

125, 62.5, 31.25, 15.62 up to 7.8 μg/ml. Ciprofloxacin was used as a standard drug for

antibacterial activity tests and fluconazole was used as a standard for antifungal activity

tests. The MIC values for standards fluconazole against C. albicans and A. niger were

recorded 125 μg/ml and 62.5 μg/ml respectively. On the other hand, the MIC values

obtained for the standard ciprofloxacin against gram-positive bacteria S. aureus and E.

faecalis were 62.5 μg/ml and 125 μg/ml respectively; similarly the MIC value was

recorded 125 μg/ml when tested against both the gram-negative bacteria E. coli and P.

aeruginosa for the same standard drug ciprofloxacin. The antibacterial and antifungal

details for each compound synthesized are discussed below.


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4.6.1.1 Table-13 Antimicrobial screening of the compounds III1-13

Compounds

Minimum Inhibitory Concentration (MIC) in µg/ml

Gram-positive bacteria Gram-negative bacteria Fungi

S. aureus ATCC 25923

E. faecalis ATCC 29212

E. coli ATCC 25922

P. aeruginosa ATCC 27853

C. albicans ATCC 10231

A. niger ATCC 1015

III1 250 250 125 62.5 250 250

III2 125 250 125 62.5 125 250

III3 125 125 125 62.5 125 250

III4 125 125 62.5 62.5 125 250

III5 125 125 62.5 125 125 125

III6 125 250 62.5 125 125 125

III7 31.25 125 62.5 125 250 125

III8 250 250 125 250 250 250

III9 62.5 125 125 62.5 250 250

III10 62.5 125 62.5 125 250 250

III11 125 125 62.5 125 125 125

III12 250 250 62.5 125 250 250

III13 250 250 62.5 125 250 125

Fluconazole - - - - 125 62.5

Ciprofloxacin 62.5 125 125 125 - -


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4.6.1.2 Table-14: Antimicrobial screening of the compounds IIIa-j

Compounds

Minimum Inhibitory Concentration (MIC) in µg/ml

Gram-positive bacteria Gram-negative bacteria Fungi

S. aureus ATCC 25923

E. faecalis ATCC 29212

E. coli ATCC 25922

P. aeruginosa ATCC 27853

C. albicans ATCC 10231

A. niger ATCC 1015

IIIa 250 250 62.5 125 250 250

IIIb 250 500 250 125 500 500

IIIc 500 500 500 62.5 500 500

IIId 500 500 500 62.5 500 500

IIIe 500 500 500 125 500 500

IIIf 125 250 62.5 125 125 125

IIIg 500 500 500 125 500 500

IIIh 500 500 500 125 500 500

IIIi 62.5 125 62.5 125 250 250

IIIj 250 250 125 125 250 250

Fluconazole - - - - 125 62.5

Ciprofloxacin 62.5 125 125 125 - -


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4.7 Results and discussion

4.7.1 Antibacterial activity for compounds III1-13

Gram-positive bacteria S. aureus (ATCC No. 25923) and E. faecalis (ATCC No.

27853) were introduced for testing the antibacterial potential of the synthesized

molecules III1-13. When tested against S. aureus, it was found that from the complete

series synthesized, compounds III9 (p-OCH3) and III10 (o-NO2) exhibited activity

equivalent to that of the standard ciprofloxacin (62.5 μg/ml). Other derivatives from

the series exhibited higher MIC value than the standard resulting in poor results against

gram-positive bacteria S. aureus. Similarly the synthesized series was tested against

another gram-positive bacterial strain E. faecalis, where it was observed that the

derivatives III3 (m-Cl), III4 (3-F-4-Cl), III5 (H), III9 (p-OCH3), III10 (o-NO2) and

III11 (o-CH3) exhibited equivalent (125 μg/ml ) activity as compared to the standard.

Overall half of the derivatives showed equivalent activity against gram-positive bacteria

S. aureus and E. faecalis when compared with the standard drug ciprofloxacin. The final

derivatives III1-13 were also tested against two gram-negative bacteria E. coli (ATCC No.

25922) and P. aeruginosa (27853) and compared with the same standard ciprofloxacin.

The results of most of the compounds as antibacterial were excellent against both the

gram-negative bacterial strains. The compounds III4 (3-F-4-Cl), III5 (H), III6 (p-F),

III10 (o-NO2), III11 (o-CH3), III12 (p-CH3) and III13 (m-CF3) showed MIC value (62.5

μg/ml) even better than that of the standard drug ciprofloxacin proving excellent

potency as antibacterial. The remaining derivatives from the same series which included

III1 (m-NO2), III2 (p-NO2), III3 (m-Cl), III8 (m-CH3), III9 (p-OCH3) exhibited

activity equivalent to the standard (125 μg/ml). These derivatives when tested against P.

aeruginosa also resulted in very good antibacterial activity. Compounds III1 (m-NO2),

III2 (p-NO2), III3 (m-Cl), III4 (3-F-4-Cl) and III9 (p-OCH3) showed excellent activity

(62.5 μg/ml), even better than ciprofloxacin. There were other compounds exhibiting

equivalent activity (125 μg/ml) as compared to the standard viz. III5 (H), III6 (p-F),

III10 (o-NO2), III11 (o-CH3), III12 (p-CH3) and III13 (m-CF3). Thus from the series of

synthesized derivatives III1-13 more than half of the compounds showed equivalent


32


activity against gram-positive bacteria and all the compounds were excellent or

equivalent when tested against gram-negative bacteria with respect to ciprofloxacin.

4.7.2 Antifungal activity for compounds III1-13

The antifungal tests were carried against two fungal strains C. albicans and A.

niger, where fluconazole was used as a standard drug for comparison and evaluation of

antifungal activity of the synthesized molecules III1-13. The derivatives III2 (p-NO2),

III3 (m-Cl), III4 (3-F-4-Cl), III5 (H), III6 (p-F) and III11 (o-CH3) showed equivalent

activity (125 μg/ml) to that of the standard drug when tested against fungal strain C.

albicans. The rest of the compounds exhibited poor activity as compared to the standard

drug result. A. niger when introduced for antifungal activity test, none of the compounds

exhibited even equivalent activity to that of the standard drug. All the compounds were

seen to exhibit poor activity. Very few compounds among the synthesized derivatives

III1-13 were capable of exhibiting antifungal property.

4.7.3 Antibacterial activity for compounds IIIa-j

The newly synthesized compounds IIIa-j were tested against the broad panel of

gram-positive and gram-negative bacteria. A very few derivatives were found to be

active against the gram-positive bacterial strains S. aureus and E. faecalis. The compounds

IIIi (2-NO2) was found to be exhibiting equivalent activity as that of the standard

ciprofloxacin against S. aureus (62.5 μg/ml) as well as E. faecalis (125 μg/ml). Also

compound IIIf (4-F) was found to exhibit excellent activity (62.5 μg/ml ) against E.

faecalis as compared to the activity of standard drug ciprofloxacin against the same

bacterial strain. The other derivatives were demonstrating poor activity against the

gram-positive bacterial strains. The same synthesized derivatives IIIa-j when tested

against gram-negative bacteria E. coli and P. aeruginosa were found to exhibit much

better activity than that shown against gram-positive strains. The compounds IIIa (3-

NO2) and IIIf (4-F) showed excellent activity (62.5 μg/ml), even better than the

standard (125 μg/ml) against E. coli. The compounds IIIi (2-NO2) and IIIj (m-CF3) also

possessed activity (125 μg/ml) equivalent to that of standard drug ciprofloxacin when

tested against E. coli. The complete series of synthesized motifs were found to exhibit

best results against the gram-negative strain P. aeruginosa as compared to the standard.


33


Compounds IIIc (3-Cl), IIId (3-F-4-Cl) and IIIf (4-F) were found to show excellent

activity (62.5 μg/ml) even better than the standard. The other compounds IIIa (3-

NO2), IIIb (4-NO2), IIIe (H), IIIg (2-OCH3), IIIh (m-CH3), IIIi (2-NO2) and IIIj

(3-CF3) also exhibited activity equivalent (125 μg/ml ) to that of the standard drug

ciprofloxacin.

4.7.4 Antifungal activity for compounds IIIa-j

. The derivatives IIIa-j when tested against the fungal strains C. albicans and A.

niger; it was found that none of the compounds showed good activity even equivalent to

that of the standard drug fluconazole. Overall it was found from the antimicrobial data

that the derivatives synthesized were good antibacterial but none of them could be used

as antifungal due to their poor MIC values as compared to the standard.

4.7.5 SAR study – Structure Activity Relationship (III1-13)

The variation in MIC values of the synthesized derivatives III1-13 and the reason

for such behavior against each bacterial and fungal strain was much understood from the

Structure Activity Relationship study (SAR). It was observed that the use of electron-

withdrawing and electron-donating groups to confer different electronic environments

of the molecules showed a great impact on their biological properties. It is very clear

from the data showed in the table-13, that the derivatives possessing electron-

withdrawing substituent like -NO2, -F, and -Cl exhibit much better activity than the

other derivatives as well as sometimes even better than the standard drugs ciprofloxacin

and fluconazole. Compounds III3 (m-Cl), III4 (3-F-4-Cl), and III10 (o-NO2) possessing

electron-withdrawing functional groups exhibited excellent activity against gram-

positive bacterial strains S. aureus and E. faecalis. One more electron-withdrawing

functional group fluorine was added to the list, as the compound III6 (p-F) and III13 (m-

CF3) also exhibited excellent activity along with the other derivatives possessing electro-

withdrawing groups when tested against the gram-negative bacteria E. coli and P.

aeruginosa. It was found that only the derivatives with electron-withdrawing groups

were present among the few exhibiting equivalent antifungal activity as that of the

standard fluconazole against both the fungal strains A. niger and C. albicans. Henceforth it

can be concluded from the SAR study that the compounds with electron-withdrawing


34


functional groups like -NO2, -F, -CF3 and -Cl exhibited excellent activity even better

than that of the electron-donating substituents. In future, it can be noted that

modifications in the current molecules with more electron-withdrawing substituent can

help to enhance the antimicrobial properties.

4.7.6 SAR study – Structure Activity Relationship (IIIa-j)

The structure activity relationship study helped to analyze the impact of different

functional group in the derivatives towards the resulting biological data against the broad

panel of microorganisms. The variation in the structure of the final derivatives was made

possible by the use of both electron-withdrawing and electron-donating substituents.

The MIC values presented in the table-14 very well stated that the use of electron-

withdrawing substituents like -NO2, -F, -Cl and -CF3 were responsible for the

exhibition of excellent biological activity than the derivatives with electron-donating

groups. Some of the derivatives bearing electron-withdrawing functional groups

demonstrated even lower MIC (μg/ml) value than the standard proving them to be

excellent antibacterial molecules.


35


4.8 Conclusion

From the antimicrobial results and SAR study carried above, it can be concluded

that the derivatives possessing electron with-drawing substituent were found to exhibit

excellent antimicrobial property, both in III1-13 and IIIa-j. On the basis of above results,

attempts are made to optimize the lead structure to obtain more potent antimicrobial

molecules. The derivatives bearing fluoro-substituted benzimidazole (IIIa-j) nucleus

presented here; up to a great extent have proved to be potent antibacterial agents. Also

from the SAR study, it is much clear that the use of electron-withdrawing functional

groups in the final derivatives as substituents has influenced the biological property of

the synthesized motifs IIIa-j. This concept of utilizing electron-withdrawing substituents

will be kept in mind while undertaking other scientific work of the same kind.


36


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