INTRODUCTION:

The benzimidazole nucleus is a useful structural moiety for the development of molecules for various pharmacological activities. Appropriately substituted benzimidazole derivatives have found diverse therapeutic applications such as in antiulcers, antihypertensive, antivirals, antifungals, anticancers, antihistaminics., antimicrobial, anti-inflammatory, anticonvulsant, antidepressant, antioxidant, radioprotective and anti-leishmanial. The benzimidazoles are broad–spectrum group of drug discovered in the 1960 with activity against GI helminthes¹.

The benzimidazoles are an important group of microtubule inhibitors which have found widespread use as broad-spectrum anthelmintics showing high efficacy against a range of nematode, trematode and cestode parasites. It is well established that benzimidazoles bind directly to the β-tubulin of nematodes, interfering with microtubule dynamics and consequently disturbing microtubule-based processes in these helminthes^2,3. Benzimidazole derivatives have been found antidepressant and analgesic activity⁴, inhibitor of Biotin Carboxylase⁵ anti-inflammatory activity⁶, antimicrobial activity^7,8,9, antidepressant activity¹⁰. On that reason the synthesis of new benzimidazoles and the investigation of their antihelminthic activity in order to compare it to the efficacy of Albendazole and to the activity of the most important commercial of pharmacological interest.

MATERIALS AND METHODS:

Melting points were determined by using VEEGO electronic (VMP-D) melting point apparatus and are uncorrected. The IR spectra of the compounds were recorded on a JASCO FT-IR 4100, Japan spectrometerin KBr. The ¹H NMR spectra were obtained on a VARIAN MERCURY YH-300 MHz NMR spectrometer in DMSO-d₆as solvent and TMS as internal standard.

Procedure:

2-(3-nitrophenyl)-1H-benzimidazole (1a):

Equimolar amounts (0.5 mmol) of o-phenylenediamine and 0.5mmol of 3-nitrobenzaldehyde were thoroughly mixed in 2 ml of N,N-dimethylformamide, 0.15 mmol of sodium hydrogen sulfite was added, and the mixture was stirred at 80°C for 40 min until the reaction was complete according to the TLC data. The mixture was cooled to room temperature and added dropwise to 20 ml of water under vigorous stirring. The product separated as a free flowing solid, it was collected by filtration, washed with water, and dried¹¹.

IR [cm^-1, KBr]: 3083 (NH), 2915 (CH), 1588 (C=N), 1518, 1339 (Ar NO₂), 1267 (C-N)

Ethyl [2-(3-nitrophenyl)-1H-benzimidazole-1-Yl] acetate (2a):

A mixture of equimolar alkaline solution (0.5 ml, 4 N NaOH) of 2-(3-nitrophenyl)-1H-benzimidazole (0.01 mol) in 50 ml of methanol and 0.01 mol of ethylchloroacetate in methanol (30 ml) was heated gently on boiling water bath for 0.5 hr. The solid thus obtained on cooling was recrystallized from chloroform to give product.

IR [cm^-1, KBr]: 3062 (CH), 1737 (C=O), 1521, 1348 (Ar NO₂), 1437 (C=N), 1128 (C-N), 1102 (C-O)

Substituted 2-[2-(3-nitrophenyl)-1H-benzimidazole-1-yl] acetamide analogues:

To a solution of ethyl [2-(3-nitrophenyl)-1H-benzimidazole-1-Yl]acetate (0.01 mol) dissolved in dry methanol (50 ml) 99 % primary amines, substituted hydrazine (1 ml) was added and the mixture was refluxed for 4–5 hr. The reaction mixture was cooled and the solid obtained was filtered, washed with small quantity of cold methanol to give product⁵.

1. N-ethyl-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3a)

IR [cm^-1, KBr]: 3044 (NH), 2913 (CH), 1605 (C=O), 1515, 1341 (Ar-NO₂) , 1452 (C=N) 1128 (C-N)

¹H NMR: 11.166 (s, 1H, NH), 7.242-9.010 (m, 8H, Ar), 4.512 (s, 2H, CH₂), 3.385-3.407 (q, 2H, CH₂), 2.495-2.504 (t, 3H, CH₃).

2. N-benzyl-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3b)

IR [cm^-1, KBr]: 3049 (NH), 2905 (CH), 1605 (C=O), 1568, 1348 (Ar-NO₂) , 1428 (C=N) 1090 (C-N)

¹H NMR: 10.025 (s, 1H, NH), 6.902-7.853 (m, 10H, Ar), 3.027 (s, 2H, CH₂), 4.012 (s, 2H, CH₂).

3. N-(naphthalen-2-yl)-2-[2-3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3c)

IR [cm^-1, KBr]: 3154 (NH), 2958 (CH), 1665 (C=O), 1535, 1362 (Ar-NO₂) , 1582 (C=N) 1178 (C-N)

¹H NMR: 10.906 (s, 1H, NH), 7.258-9.286 (m, 16H, Ar), 3.128 (s, 2H, CH₂)

4. 2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl]-N-phenylacetamide (3d)

IR [cm^-1, KBr]: 3055 (NH), 2926 (CH), 1611 (C=O), 1509, 1273 (Ar-NO₂) , 1428 (C=N) 1013 (C-N)

¹H NMR: 11.013 (s, 1H, NH), 7.250-9.010 (m, 13H, Aromatic), 2.800 (s, 2H, CH₂).

5. N-(2-nitrophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3e)

IR [cm^-1, KBr]: 3122 (NH), 2958 (CH), 1681 (C=O), 1528, 1333 (Ar-NO₂) , 1542 (C=N) 1180 (C-N)

¹H NMR: 10.227 (s, 1H, NH), 7.320-8.017 (m, 12H, Ar), 3.242 (s, 2H, CH₂).

6. N-(3-chlorophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3f)

IR [cm^-1, KBr]: 3068 (NH), 2932 (CH), 1655 (C=O), 1518, 1339 (Ar-NO₂) , 1433 (C=N) 1128 (C-N), 747 (C-Cl)

¹H NMR: 10.436 (s, 1H, NH), 7.427-8.148 (m, 12H, Ar), 3.019 (s, 2H, CH₂).

7. N-(3-nitrophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3g)

IR [cm^-1, KBr]: 3156 (NH), 2980 (CH), 1668 (C=O), 1554, 1345 (Ar-NO₂) , 1584 (C=N) 1154 (C-N)

¹H NMR: 10.572 (s, 1H, NH), 7.182-8.923 (m, 12H, Ar), 3.012 (s, 2H, CH₂).

8. N-(4-bromophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3h)

IR [cm^-1, KBr]: 3178 (NH), 2967 (CH), 1678 (C=O), 1557, 1334 (Ar-NO₂) , 1575 (C=N) 1170 (C-N), 667 (C-Br)

¹H NMR: 10.238 (s, 1H, NH), 7.580-8.207 (m, 12H, Ar), 3.549 (s, 2H, CH₂).

9. N-(4-nitrophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3i)

IR [cm^-1, KBr]: 3062 (NH), 2918 (CH), 1771 (C=O), 1588, 1321 (Ar-NO₂) , 1428 (C=N) 1090 (C-N)

¹H NMR: 10.338 (s, 1H, NH), 7.028-8.003 (m, 12H, Ar), 3.129 (s, 2H, CH₂).

10. N-(4-hydroxyphenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetamide (3j)

IR [cm^-1, KBr]: 3318 (OH), 3196 (NH), 2985 (CH), 1676 (C=O), 1516, 1378 (Ar-NO₂) , 1539 (C=N), 1126 (C-N)

¹H NMR: 10.586 (s, 1H, NH), 7.238-8.213 (m, 12H, Ar), 3.028 (s, 1H, OH), 2.728 (s, 2H, CH₂).

11. 2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl]-N'-acetohydrazide (3k)

IR [cm^-1, KBr]: 3452 (NH₂), 3145 (NH), 2962 (CH), 1659 (C=O), 1539, 1343 (Ar-NO₂) , 1552 (C=N), 1183 (C-N)

¹H NMR: 11.203 (s, 1H, NH), 7.210-8.431 (m, 8H, Ar), 4.862 (s, 2H, NH₂) 3.827 (s, 2H, CH₂).

12. 2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl]-N'-phenylacetohydrazide (3l)

IR [cm^-1, KBr]: 3312 (NH), 3168 (NH), 2935 (CH), 1665 (C=O), 1548, 1352 (Ar-NO₂) , 1568 (C=N), 1162 (C-N)

¹H NMR: 11.238 (s, 1H, NH amide), 7.103-8.521 (m, 13H, Ar), 4.147 (s, 1H, NH) 3.802 (s, 2H, CH₂).

13. N'-(4-nitrophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl]- acetohydrazide (3m)

IR [cm^-1, KBr]: 3285 (NH), 3215 (NH), 2987 (CH), 1696 (C=O), 1527, 1346 (Ar-NO₂) , 1558 (C=N), 1142 (C-N)

¹H NMR: 11.272 (s, 1H, NH), 7.012-8.128 (m, 12H, Ar), 4.338 (s, 1H, NH) 3.923 (s, 2H, CH₂).

14. N'-(2,4-dinitrophenyl)-2-[2-(3-nitrophenyl)-1H-benzimidazol-1-yl]-acetohydrazide (3n)

IR [cm^-1, KBr]: 3236 (NH), 3285 (NH), 2994 (CH), 1678 (C=O), 1533, 1324 (Ar-NO₂) , 1548 (C=N), 1135 (C-N)

¹H NMR: 11.427 (s, 1H, NH), 7.578-9.025 (m, 11H, Ar), 4.402 (s, 1H, NH) 3.902 (s, 2H, CH₂).

15. 4-({[2-(3-nitrophenyl)-1H-benzimidazol-1-yl] acetyl} amino) benzoic acid (3o)

IR [cm^-1, KBr]: 3412 (OH), 3215 (NH), 2965 (CH), 1679, 1756 (C=O), 1545, 1334 (Ar-NO₂) , 1532 (C=N), 1245 (C-O), 1183 (C-N)

¹H NMR: 11.282 (s, 1H, NH), 7.624-9.123 (m, 12H, Ar), 9.032 (s, 1H, COOH) 3.435 (s, 2H, CH₂)

Procedure for Antihelminthic activity:

Three groups, of six earthworms each were released in 20 ml of desired formulations prepared in distilled water are as follows: saline, 20 mg/ml of Albendazole (Bandy, Mankind) and compounds 3a-3o (20 mg/ml). Observations were made for the time taken to paralysis and death of individual worms. Paralysis was said to occur when the worm do not revive in normal saline. And death was concluded when the worms lost their motility followed with fading away of their body color^12-17.

RESULTS AND DISCUSSION:

Antihelminthic activity, Benzimidazole derivatives of substituted 2 [2-(3-nitrophenyl)-1H-benzimidazole-1-yl] acetamide 3a-3o were evaluated for their activity against Pheretima posthuma. The antihelminthic activity showed that compounds 3f, 3h, 3i, 3j and 3k good activity against Indian earthworms (Pheretima posthuma) in comparison to albendazole. The docking studies yielded fitness score ranging from -5.380668 to -4.588958. The docking study indicate that compounds bind with β-tubulin by forming hydrophobic interaction with amino acid residues GLU 200B, THR 201B, TYR 202B, LEU 255B, MET 259B, van-der waal interaction with amino acid residues MET 166, THR 168, THR 198, ASP 199, GLU 200, THR 201, LEU 265, PHE 268. From the docking simulation it is found that title analogues have good interaction with β-tubulin.

Table 1: Anthelmintic Activity of Compounds.

Compound	Paralysis Time (min) ±SEM	Death Time (min) ±SEM
Albendazole	17.56 ± 1.73	55.24 ± 1.40
3a	21.43 ± 1.34	63.36 ± 1.54
3b	31.17 ± 1.78	77.21 ± 1.53
3c	34.07 ± 1.10	86.50 ± 1.67
3d	24.31 ± 0.69	63.19 ±1.44
3e	26.08 ± 1.06	63.21 ± 1.50
3f	18.24 ± 1.35	58.56± 1.27
3g	29.04 ± 1.07	72.15 ± 1.44
3h	19.44 ± 1.32	56.59± 1.32
3i	18.58 ± 1.51	55.43 ± 1.33
3j	20.26 ± 1.04	58.43 ± 1.39
3k	19.08 ± 1.43	57.26 ± 0.96
3l	29.16 ± 0.77	63.04 ± 1.37
3m	32.37 ± 0.72	65.49 ± 1.25
3n	38.12 ± 1.55	72.16 ± 1.46
3o	27.24 ± 0.63	68.14 ± 1.08

Control worms were alive up to 24 hours of the experiment.

Figure 2: Hydrophobic interaction of Albendazole with β-tubulin

Figure 3: Hydrophobic interaction of compound 3f with β-tubulin.

Figure 4: Hydrophobic interaction of compound 3h with β-tubulin.

Figure 5: Hydrophobic interaction of compound 3i with β-tubulin.

Figure 6: Hydrophobic interaction of compound 3j with β-tubulin.

Figure 7: Hydrophobic interaction of compound 3k with β-tubulin.

Values are expressed as mean ± SEM, n=6

DOCKING RESULTS:

Table 2: Docking Score of Title Compounds

Sr.no.	Compound	Docking score
1	3a	-5.074641
2	3b	-4.588958
3	3c	-5.045692
4	3d	-4.978864
5	3e	-4.924625
6	3f	-5.380668
7	3g	-4.882526
8	3h	-5.330436
9	3i	-5.263447
10	3j	-5.110342
11	3k	-5.246499
12	3l	-5.080505
13	3m	-4.960719
14	3n	-5.081316
15	3o	-5.088659
16	Albendazole	-5.125293

CONCLUSION:

1. The evaluation of anthelmintic activity reveals that, 3f, 3h, 3i, 3j and 3k, shows good anthelmintic activity and most of these compounds contain electronegative group at first and second position of Benzimidazole moiety.

2. Molecular docking study indicates that the observed anthelmintic activity may be due to inhibition of β-tubulin of the worms.

3. The title compounds are the good anthelmintic agents as per anthelmintic activity and molecular modeling studies.

ACKNOWLEDGEMENT:

The authors are thankful to Department of Chemistry, Savitribai Phule Pune University, for NMR spectra analysis.

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Received on 25.07.2020 Modified on 19.09.2020

Research J. Pharm. and Tech 2021; 14(12):6369-6373.

DOI: 10.52711/0974-360X.2021.01101