Synthesis and biological profile of substituted benzimidazoles.

BACKGROUND: A series of benzimidazole derivatives was developed and its chemical scaffolds were authenticated by NMR, IR, elemental analyses and physicochemical properties. The synthesized compounds were screened for their antimicrobial and antiproliferative activities. RESULTS AND DISCUSSION: The synthesized benzimidazole compounds were evaluated for their antimicrobial activity using the tube dilution method and were found to exhibit good antimicrobial potential against selected Gram negative and positive bacterial and fungal species. The compounds were also assessed for their anticancer activity exhibited using the SRB assay and were found to elicit antiproliferative activity against MCF7 breast cancer cell line, which was comparable to the standard drug.
CONCLUSION: Antimicrobial screening results indicated that compounds 1, 2 and 19 to be promising antimicrobial agents against selected microbial species and comparable to standard drugs which included norfloxacin and fluconazole. The anticancer screening results revealed that compounds, 12, 21, 22 and 29 to show the highest activity against MCF7 and their IC50 values were more potent than 5-fluorouracil.

Background

The emergence of antibiotic-resistant microorganisms such as fluoroquinolone-resistant Escherichia coli, Streptococcus pneumonia, carbapenem-resistant Klebsiella pneumonia, vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus is becoming a serious health issue worldwide. There is a critical need to develop new chemotherapeutic agents with different mechanism of action [1].

Cancer is a deadly disease prevalent in both the developing as well as the developed countries. In spite of significant improvements in recognition and treatment of cancer, the incidence of certain types of malignancy is still on the rise. Current treatments such as cytotoxic chemotherapy and radiotherapy yielded only transient therapeutic aids that are accompanied by severe adverse effects. This is due to their toxic effects against normal growing cells. Concerted effort is, therefore, required to eliminate or at least reduce these incidences significantly [2].

Recent findings suggest that substituted benzimidazole derivatives possess potential chemotherapeutic activity with reduced toxic effects. Antibacterial activity of substituted benzimidazole derivatives can be explained by their competition with purines, an integral part of bacterial strain, resulting in inhibition of bacterial nucleic acids and proteins synthesis [3]. Compounds containing benzimidazole moiety such as thiabendazole, parbendazole, mebendazole, albendazole, cambendazole and flubendazole had also been reported for their antihelminthic activity. Similarly, the proton pump inhibitors, omeprazole, lansoprazole, rabeprazole, pantoprazole, had been reported for their use in the management of acid related disorders. In fact, benzimidazole derivatives had found their applications as antioxidant [4], antimicrobial [5], antihelmintic [6], anticancer [7], antiviral [8], antiallergic [9], antiarthritic [10] and anti-mycobacterial agents [11].

In light of above, the present study was undertaken to synthesise and evaluate the antimicrobial and anticancer potentials of substituted benzimidazole derivatives.

Results and discussion

Chemistry

Target compounds (1–30) were synthesized by following procedure outlined in Scheme 1. The physicochemical data of the target compounds are presented in Table 1. The synthesized compounds were evaluated on the basis of spectral analysis: IR, NMR and Mass and elemental analyses which were in full agreement with their proposed molecular structures. The formation of Schiff bases is confirmed by the presence of N=CH str., at around 1560 cm−1 in the IR spectra of synthesized compounds (1–11). Asym str., at around 1550 cm−1 indicated the presence of aromatic nitro group in 5, 7, 13, 14, 16, 21-28 compounds. The presence of C–O–C str., of aralkyl showed methoxy group in 3, 9, 11, 13, 17–20, 27 compounds. The C–H str., at 1727 cm−1 confirmed the aliphatic aldehyde group in 10, 14 and 15 compounds. Furthermore, the appearance of C=O str., at 1660 cm−1 and the absence of NH str., of imidazole at 3400 cm−1 confirmed the synthesis of methanone derivatives (12–30). The multiplet corresponds to 6.697–7.823 δ ppm confirmed the presence of aromatic protons of aryl nucleus and benzimidazole. The appearance of singlet at around 9.580 δ ppm confirmed the Schiff bases (N=CH–). The singlet peak at 3.426 δ ppm indicated the presence of dimethyl group in compounds, 1, 6, 22, 29 and 30. The doublet peak observed at 1.273–1.276 δ ppm which confirmed the presence of aliphatic methyl group in the synthesized compounds, 8, 12 and 15. The multiplet showed at 1.243–2.496 δ ppm confirmed the presence of CH2 chain of palmitoyl group in the structure of compounds (20, 28 and 29). Further confirmation was made on the basis of 13C-NMR and MS spectral analyses. The results of C, H, N analysis are within limits of ± 0.3%.

Scheme 1

Synthesis of benzimidazole derivatives (1–30). Reaction condition: Step i: 2-Aminobenzimidazole, substituted aldehyde, ethanol, glacial acetic acid, reflux for 4–5 h (RT), Step ii: Schiff’s base, different acylchlorides, dimethylformamide, triethylamine, stir for 24 h (RT)

Table 1

Physicochemical characteristic of the synthesized compounds

Comp.	Molecular structures with stereochemistry	M. formula and CHN analyses	M. wt.	Rf value	% Yield	M. Pt. (°C)
1	(E)-N-((E)-3-(4-(Dimethylamino)phenyl) allylidene)-1H-benzo[d]imidazol-2-amine	C18H18N4: Anal calcd: C, 74.46; H, 6.25; N, 19.30; Found: C, 74.43; H, 6.27; N, 19.33	290.40	0.76a	76	228–230
2	(E)-1-(((1H-Benzo[d]imidazol-2-yl)imino)methyl)naphthalen-2-ol	C18H13N3O: Anal calcd: C, 75.25; H, 4.56; N, 14.63; Found: C, 75.27; H, 4.59; N, 14.60	287.34	0.79a	74	255–257
3	(E)-N-(3,4-Dimethoxybenzylidene)-1H-benzo[d]imidazol-2-amine	C16H15N3O2: Anal calcd: C, 68.31; H, 5.37; N, 14.94; Found: C, 68.34; H, 5.35; N, 14.97	281.34	0.77a	78	225–227
4	(E)-4-(((1H-Benzo[d]imidazol-2-yl)imino)methyl)phenol	C14H11N3O: Anal calcd: C, 70.87; H, 4.67; Cl, 17.71; Found: C, 70.88; H, 4.65; Cl, 17.73	237.28	0.75a	67	220–222
5	(E)-N-(4-Nitrobenzylidene)-1H-benzo[d]imidazol-2-amine	C14H10N4O2: Anal calcd: C, 63.15; H, 3.79; N, 21.04; Found: C, 63.13; H, 3.77; N, 21.07	266.28	0.72a	72	236–238
6	(E)-N-(4-(Dimethylamino)benzylidene)-1H-benzo[d]imidazol-2-amine	C16H16N4: Anal calcd: C, 72.70; H, 6.10; N, 21.20; Found: C, 72.73; H, 6.12; N, 21.22	264.36	0.79a	82	238–240
7	(E)-N-(3-Nitrobenzylidene)-1H-benzo[d]imidazol-2-amine	C14H10N4O2: Anal calcd: C, 63.15; H, 3.79; N, 21.04; Found: C, 63.18; H, 3.77; N, 21.05	266.28	0.76a	76	190–192
8	(E)-N-Ethylidene-1H-benzo [d]imidazol-2-amine	C9H9N3: Anal calcd: C, 67.90; H, 5.70; N, 26.40; Found: C, 67.88; H, 5.72; N, 26.42	159.21	0.72a	80	172–175
9	(E)-N-(4-Methoxybenzylidene)-1H-benzo[d]imidazol-2-amine	C15H13N3O: Anal calcd: C, 71.70; H, 5.21; N, 16.72; Found: C, 71.73; H, 5.22; N, 16.74	251.31	0.71a	75	198–200
10	(E)-5-((1H-Benzo[d]imidazol-2-yl)imino)pentanal	C12H13N3O: Anal calcd: C, 66.96; H, 6.09; N, 19.52; Found: C, 66.94; H, 6.11; N, 19.55	215.28	0.75a	78	265–267
11	(E)-N-(3,4,5-Trimethoxybenzylidene)-1H-benzo[d]imidazol-2-amine	C17H17N3O3: Anal calcd: C, 65.58; H, 5.50; N, 13.50; Found: C, 65.61; H, 5.53; N, 13.52	311.37	0.72a	84	242–245
12	(E)-1-(2-(Ethylideneamino)-1H-benzo[d]imidazol-1-yl)ethanone	C11H11N3O: Anal calcd: C, 65.66; H, 5.51; N, 20.88; Found: C, 65.65; H, 5.54; N, 20.86	201.22	0.63b	74	262–265
13	(E)-(3,5-Dinitrophenyl)(2-((4-methoxy-benzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C22H15N5O6: Anal calcd: C, 59.33; H, 3.39; N, 15.72; Found: C, 59.35; H, 3.42; N, 15.75	445.38	0.58b	68	243–245
14	(E)-5-((1-(3,5-Dinitrobenzoyl)-1H-benzo[d]imidazol-2-yl)imino)pentanal	C14H11N3O2: Anal calcd: C, 55.75; H, 3.69; N, 17.11; Found: C, 55.78; H, 3.71; N, 17.14	253.26	0.66b	65	162–164
15	(E)-5-((1-Acetyl-1H-benzo[d]imidazol-2-yl)imino)pentanal	C14H15N3O2: Anal calcd: C, 65.35; H, 5.88; N, 16.33; Found: C, 65.37; H, 5.90; N, 16.36	257.29	0.62b	72	226–228
16	(E)-(3,5-Dinitrophenyl)(2-((4-hydroxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C21H13N5O6: Anal calcd: C, 58.47; H, 3.04; N, 16.24; Found: C, 58.49; H, 3.05; N, 16.25	431.36	0.64b	70	175–177
17	(E)-(2-((2,4-Dimethoxybenzylidene)amino) -1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone	C27H21N3O3: Anal calcd: C, 74.47; H, 4.86; N, 9.65; Found: C, 74.49; H, 4.88; N, 9.68	435.47	0.54b	67	120–122
18	(E)-Naphthalen-2-yl(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C28H23N3O4: Anal calcd: C, 72.24; H, 4.98; N, 9.03; Found: C, 72.27; H, 4.95; N, 9.05	465.5	0.65b	75	210–212
19	(E)-(3,5-Dinitrophenyl)(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C24H19N5O8: Anal calcd: C, 57.03; H, 3.79; N, 13.86; Found: C, 57.07; H, 3.76; N, 13.88	505.44	0.66b	66	141–143
20	(E)-1-(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one	C33H47N3O4: Anal calcd: C, 72.10; H, 8.62; N, 7.64; Found: C, 72.11; H, 8.65; N, 7.67	549.74	0.62b	78	136–138
21	(E)-(3-Nitrophenyl)(2-((4-(pyridin-2-yl)benzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C26H17N5O3: Anal calcd: C, 69.79; H, 3.83; N, 15.65; Found: C, 69.77; H, 3.86; N, 15.68	447.44	0.57b	67	142–144
22	(2-((E)-((E)-3-(4-(Dimethylamino)phenyl) allylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone	C25H21N5O3: Anal calcd: C, 68.33; H, 4.82; N, 15.94; Found: C, 68.37; H, 4.80; N, 15.97	439.47	0.59b	82	126–128
23	(E)-(2-((4-(Dimethylamino)benzylidene) amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone	C23H19N5O3: Anal calcd: C, 66.82; H, 4.63; N, 16.94; Found: C, 66.83; H, 4.66; N, 16.97	413.43	0.63b	76	131–133
24	(E)-(2-(((2-hydroxynaphthalen-1-yl)-methylene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone	C25H16N4O4: Anal calcd: C, 68.80; H, 3.70; N, 12.84; Found: C, 68.83; H, 3.72; N, 12.87	436.42	0.64b	65	134–136
25	(E)-(2-((4-Nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl) methanone	C21H13N5O5: Anal calcd: C, 60.72; H, 3.15; N, 16.86; Found: C, 60.75; H, 3.17; N, 16.89	415.36	0.66b	74	119–121
26	(E)-Naphthalen-2-yl(2-((4-nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone	C25H16N4O3: Anal calcd: C, 71.42; H, 3.84; N, 13.33; Found: C, 71.43; H, 3.87; N, 13.37	420.42	0.52b	62	176–178
27	(E)-(2-((3,4-Dimethoxybenzylidene)amino) -1H-benzo[d]imidazol-1-yl)(3-nitro phenyl)methanone	C23H18N4O5: Anal calcd: C, 64.18; H, 4.22; N, 13.02; Found: C, 64.21; H, 4.25; N, 13.04	430.41	0.63b	65	235–237
28	(E)-1-(2-((4-Nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one	C30H40N4O3: Anal calcd: C, 71.40; H, 7.99; N, 11.10; Found: C, 71.39; H, 7.97; N, 11.12	504.66	0.59b	66	126–128
29	(E)-1-(2-((4-(Dimethylamino)benzylidene) amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one	C34H48N4O: Anal calcd: C, 77.23; H, 9.15; N, 10.60; Found: C, 77.21; H, 9.16; N, 10.63	528.77	0.64b	68	131–133
30	(E)-(2-((4-(Dimethylamino)benzylidene) amino)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone	C27H22N4O: Anal calcd: C, 77.49; H, 5.30; N, 13.39; Found: C, 77.51; H, 5.32; N, 13.42	418.49	0.62b	76	192–195

TLC mobile phase: a Ethyl acetate: Methanol (7:3); b Chloroform: Methanol (8:2)

Anticancer activity

The synthesized benzimidazole derivatives were screened for their anticancer activity against MCF7 (ATCC HTB-22), an oestrogen receptor positive human breast adeno-carcinoma cell line. Anticancer screening results (Table 2) indicated that compound 22 (IC50 = 0.9 µM) was found to be the most potent when compared to the standard drug, 5-fluorouracil (IC50 = 35.4 µM). Other compounds which included 12, 21 and 29 also exhibited more potent antiproliferative results (IC50 = 7.0, 5.4 and 5.5 µM, respectively) when compared to the standard drug. These compounds may be used as drug leads for discovery of new anticancer agents.

Table 2

Anticancer screening results of synthesized compounds

Comp.	MCF-7 cell line	Comp.	MCF-7 cell line
Anticancer screening (IC50 = µM)
1	31.0	16	231.8
2	41.8	17	39.0
3	170.6	18	161.1
4	101.1	19	15.8
5	67.6	20	23.6
6	> 378.3	21	5.4
7	112.7	22	00.9
8	> 628.1	23	50.8
9	> 310.4	24	61.9
10	157.9	25	18.8
11	> 321.2	26	176.0
12	7.0	27	123.1
13	19.1	28	31.7
14	> 394.9	29	5.5
15	11.7	30	138.6
5-Fluorouracil	35.4	5-Fluorouracil	35.4

Antimicrobial activity

Antimicrobial activity results (Table 3) indicated that the compounds possessed good antimicrobial activity against the tested bacterial and fungal strains. Compound 1 showed good antibacterial activity against E. coli (MIC = 5.4 µM) and B. subtilis (MIC = 10.7 µM), whereas compound 19 was found to be more potent against S. aureus (MIC= 12.4 µM). The reference drug, norfloxacin, yielded MIC of 4.7 µM against the tested microorganisms. The antifungal activity results indicated that compound 2 showed good activity against C. albicans (MIC = 5.4 µM. Compound 19, on the other hand, was the most potent antifungal agent against A. niger (MIC = 3.1 µM) in comparison to fluconazole (MIC = 5.0 µM), the reference drug. Thus, compound 19 may serve as a potential lead compound for the design of novel antifungal agents.

Table 3

Antimicrobial activity of synthesized compounds

Comp.	Antimicrobial screening (MIC = µM)
	Bacterial species			Fungal species
	E. coli	B. subtilis	S. aureus	C. albicans	A. niger
1	05.4	10.7	43	10.7	21.5
2	43.5	21.8	43.5	5.4	10.9
3	22.2	22.2	44.4	11.1	22.2
4	06.6	13.1	52.7	13.1	26.3
5	46.9	23.5	46.9	23.5	23.5
6	47.3	23.6	47.3	47.3	47.3
7	23.5	23.5	46.9	93.9	23.5
8	39.3	39.3	78.5	78.5	78.5
9	24.9	24.9	49.7	24.9	49.7
10	14.5	29	58.1	29	58.1
11	10.0	20.1	40.1	20.1	20.1
12	248.5	248.5	15.5	62.1	31.1
13	56.1	28.1	14	14	7
14	197.4	197.4	24.7	98.7	24.7
15	194.3	194.3	24.3	48.6	24.3
16	58.0	29	14.5	14.5	7.2
17	28.7	28.7	14.4	28.7	7.2
18	26.9	13.4	13.4	26.9	6.7
19	49.5	12.4	12.4	6.2	3.1
20	45.5	11.4	22.7	22.7	11.4
21	27.9	14	14	55.9	7
22	56.9	28.4	14.2	28.4	7.1
23	60.5	30.2	15.1	30.2	7.5
24	28.6	28.6	14.3	28.6	7.1
25	60.2	30.1	15	30.1	7.5
26	29.7	29.7	14.9	59.5	7.4
27	58.1	29	14.5	29	7.2
28	49.5	24.8	24.8	24.8	12.4
29	47.3	11.8	23.6	23.6	11.8
30	29.9	29.9	14.9	59.7	7.5
DMSO	NA	NA	NA	NA	NA
Std. drugs	4.7a	4.7a	4.7a	5.1b	5.1b

NA no activity, DMSO dimethyl sulphoxide

Std. drugs: a Norfloxacin, b Fluconazole

Structure activity relationship

The following structure activity relationship may be drawn from the antimicrobial and anticancer activities of the benzimidazole derivatives (Fig. 1):

Fig. 1

Structural requirements for antimicrobial and anticancer activity of synthesized benzimidazole derivatives

It has been noticed that the antibacterial activity of Schiff bases against E. coli enhanced due to the presence of vinyl group between benzimidazole amine and N-benzylidene moiety and the substitution of electron releasing group at phenyl nucleus as in the compound 1 and the same moiety improved anticancer activity of methanone derivatives as in compound 22.

The electron donating group placed at phenyl ring attached to N-alylidene/arylidene moiety along with presence of electron withdrawing group on phenyl ring attached to methanone moiety improved antibacterial and antifungal activity of synthesized benzimidazole derivatives against bacterial and fungal strains as in compound 19.

Experimental

Materials and methods

All the laboratory reagents were procured from Sigma Aldrich and were used without any purification. Melting points were determined on Sonar melting point apparatus in an open capillary tube and are uncorrected. Purity of the compound was ascertained by commercialized (E-Merck Kieselgel 60 F254) TLC plates. The Infrared spectrum was recorded in KBr discs on a Shimadzu-FTIR 8400S spectrometer (νmax in cm−1). Proton and

13C NMR spectra of the synthesized compounds were recorded on Bruker Advance-II 400 NMR spectrometer with DMSO as a solvent and the chemical shift data were expressed as delta values related to tetramethylsilane. Mass spectra were recorded using Waters, Q-TOF micromass spectrometer.

Procedure for the title compounds (1–11)

2-Aminobenzimidazole (0.01 mol) was refluxed with different substituted aromatic aldehyde (0.01 mol) in ethanol (20 ml) for 4–5 h (RT) in presence of glacial acetic acid (few drops). Then the reaction mixture was allowed to cool at RT and the precipitated compound was filtered and dried [12].

Synthesis of 2-(alkyl/arylideneamino)-1H-benzo[d]imidazol-1-yl-alkyl/aryl-methanones (12–30)

Compound of Schiff’s bases (1–11) (0.005 mol) were stirred at RT with different acylchlorides (0.005 mol) in dimethylformamide for 24 h with the addition of small amount of triethylamine. The resulting reaction mixture was precipitated using ice cold water and the crude product was filtered through a vacuum pump, washed with cold water, dried and recrystallized using rectified spirit [13].

Spectral data of synthesized compounds

(E)-N-((E)-3-(4-(Dimethylamino)phenyl)allylidene)-1H-benzo[d]imidazol-2-amine (1)

IR (KBr cm−1): 1550 (N=CH str.), 3475 (N–H str.), 1431 (Ar., C=C str.), 1253 (C–N str.), 1300 (–N(CH3)2 str); 1H NMR (DMSO): 9.557–9.575 (d, 1H, N=CH), 6.646–6.714 (d, 1H, –CH=CH), 6.416–7.614 (m, 8H, ArH), 3.426 (s, 6H, (CH3)2); 13C NMR (DMSO): 40, 115, 119, 123, 127, 135, 138, 148, 159, 162; MS: m/z = 291.12 (M+ +1).

(E)-1-(((1H-Benzo[d]imidazol-2-yl)imino)methyl)naphthalen-2-ol (2)

IR (KBr cm−1): 3066 (N–H str., of imidazole), 3012 (C–H aromatic ring str.), 1442 (Ar., C=C str.), 1550 (N=CH str.), 1253 (C–N str.), 3518 (O–H str.); 1H NMR (DMSO): 10.295 (s, 1H, N=CH), 7.096–8.108 (m, 10H, ArH), 4.481 (s, 1H, OH), 10.809 (s,1H, NH of imidazole); 13C NMR (DMSO): 115, 118, 123, 127, 128, 129, 132, 135, 138, 159, 162; MS: m/z = 288.39 (M+ +1).

(E)-N-(3,4-Dimethoxybenzylidene)-1H-benzo[d]imidazol-2-amine (3)

IR (KBr cm−1): 3410 (N–H str.), 3058 (Ar., C–H str.), 1542 (C=C str.), 1610 (N=CH str.), 2827 (Ar., OCH3 str.); 1H NMR (DMSO): 9.487 (s, 1H, N=CH), 6.982–7.849 (m, 7H, ArH), 10.452 (s, 1H, NH of imidazole) 3.502 (s, 6H, (OCH3)2); 13C NMR (DMSO): 56, 115, 123, 127, 138, 152, 159, 162; MS: m/z = 282.14 (M+ +1).

(E)-4-(((1H-Benzo[d]imidazol-2-yl)imino)methyl)phenol (4)

IR (KBr cm−1): 3440 (N–H str.), 3063 (Ar., C–H str.), 1537 (C=C str.), 1613 (N=CH str.), 3452 (O–H str.); 1H NMR (DMSO): 9.582 (s, 1H, N=CH), 7.106–8.367 (m, 8H, ArH), 10.809 (s, 1H, NH of imidazole); 13C NMR (DMSO): 115, 117, 123, 126, 129, 131, 138, 159, 162; MS: m/z = 238.17 (M+ +1).

(E)-N-(4-Nitrobenzylidene)-1H-benzo[d]imidazol-2-amine (5)

IR (KBr cm−1): 3240 (N–H str., of imidazole ring), 2974 (C–H aromatic ring str.), 1465 (Ar., C=C str.), 1550 (N=CH str.), 1548 (Ar–C–NO2, asym str.); 1H NMR (DMSO): 9.550 (s, 1H, N=CH), 7.103–8.105 (m, 4H, ArH), 8.116–8.376 (d, 4H, Ar-NO2), 12.73 (s, 1H, NH of imidazole); 13C NMR (DMSO): 115, 120, 123, 130, 138, 149, 159, 162; MS: m/z = 267.26 (M+ +1).

(E)-N-(4-(Dimethylamino)benzylidene)-1H-benzo[d]imidazol-2-amine (6)

IR (KBr cm−1): 1550 (N=CH str.), 3374 (N–H str.), 1462 (Ar., C=C str.), 1298 (C–N str. –N(CH3)2); 1H NMR (DMSO): 9.206 (s, 1H, N=CH), 6.697–7.823 (m, 8H, ArH), 3.043 (s, 6H, (CH3)2), 12.42 (s, 1H, NH of imidazole); 13C NMR (DMSO): 40, 115, 123, 138, 159, 162; MS: m/z = 265.35 (M+ +1).

(E)-N-(3-Nitrobenzylidene)-1H-benzo[d]imidazol-2-amine (7)

IR (KBr cm−1): 3428 (N–H str.), 3068 (Ar., C–H str.), 1531 (C=C str.), 1618 (C=N str.), 1547 (Ar-NO2 str.); 1H NMR (DMSO): 9.515 (s, 1H, N=CH), 7.213–8.378 (m, 8H, ArH), 10.23 (s, 1H, NH of imidazole); 13C NMR (DMSO): 115,123, 127, 135, 138, 150, 159, 162; MS: m/z = 267.28 (M+ +1).

(E)-N-Ethylidene-1H-benzo[d]imidazol-2-amine (8)

IR (KBr cm−1): 3267 (N–H str., of imidazole ring), 2924 (C–H aromatic ring str.), 1465 (Ar., C=C str.), 1550 (N=CH str.), 2877 (R-CH3, sym str.); 1H NMR (DMSO): 8.654 (s, 1H, N=CH), 6.897–7.143 (m, 4H, ArH), 1.243 (s, 3H, CH3); 13C NMR: 22, 111, 120, 154, 175. MS: m/z = 160.28 (M+ +1).

(E)-N-(4-Methoxybenzylidene)-1H-benzo[d]imidazol-2-amine (9)

IR (KBr cm−1): 3340 (N–H str., of imidazole ring), 2970 (C–H aromatic ring str.), 1496 (Ar., C=C str.), 1566 (N=CH str.), 1257 (C–O–C str.); 1H NMR (DMSO): 9.383 (s, 1H, N=CH), 7.027–7.960 (m, 8H, ArH), 3.523 (s, 3H, OCH3), 12.497 (s, 1H, NH of imidazole); 13C NMR (DMSO): 57, 114, 115, 123, 126, 130, 138, 162; MS: m/z = 252.28 (M+ +1).

(E)-5-((1H-Benzo[d]imidazol-2-yl)imino)pentanal (10)

IR (KBr cm−1): 3426 (N–H str.), 3054 (Ar., C-H str.), 1562 (C=C str.), 1623 (N=CH str.), 2773 (Aliphatic C–H str.), 1724 (Aliphatic aldehyde C=O str.); 1H NMR (DMSO): 8.454 (t, 1H, N=CH), 6.856–7.143 (m, 4H, ArH), 1.243–2.567 (m, 6H, CH2), 9.700 (t, 1H, CH = O); 13C NMR (DMSO): 18, 28, 44, 115, 123, 138, 160, 162, 202. MS: m/z = 216.26 (M+ +1).

(E)-N-(3,4,5-Trimethoxybenzylidene)-1H-benzo[d]imidazol-2-amine (11)

IR: 3429 (N–H str.), 3064 (Ar., C–H str.), 1577 (C=C str.), 1606 (N=CH str.), 2835 (Ar., O–CH3 str.); 1H NMR (DMSO): 9.476 (s, 1H, N=CH), 6.962–7.859 (m, 6H, ArH), 10.462 (s, 1H, NH of imidazole) 3.382 (s, 9H, (OCH3)3); 13C NMR (DMSO): 56, 106, 115, 123, 127, 138, 141, 152, 159, 162; MS: m/z = 312.14 (M+ +1).

(E)-1-(2-(Ethylideneamino)-1H-benzo[d]imidazol-1-yl)ethanone (12)

IR (KBr cm−1): 1661 (C=O str.), 2919 (C–H aromatic str.), 1575 (N=CH str.), 2849 (CH str. (sym), R-CH3); 1H NMR (DMSO): 7.305–7.627 (m, 4H, Ar–H), 7.233 (s, 1H, N=CH), 1.273–1.276 (d, 3H, CH3), 2.856 (s, 3H, CH3); 13C NMR (DMSO): 16, 24, 115, 123, 129, 138, 141, 162, 168; MS: m/z = 201 (M+ +1).

(E)-(3,5-Dinitrophenyl)(2-((4-methoxy-benzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone (13)

IR (KBr cm−1): 1710 (C=O str.), 2924 (C–H aromatic str.), 1537 (N=CH str.), 1545 (Ar-NO2 str.), 1110 (C–O–C str., OCH3); 1H NMR (DMSO): 6.785–7.943 (m, 8H, ArH), 8.632 (s, 1H, N=CH), 2.984 (s, 3H, (OCH3), 8.912–9.063 (m, 3H, Ar(NO2)2); 13C NMR (DMSO): 56, 115, 123, 125, 130, 150, 163, 168; MS: m/z = 445 (M+ +1).

(E)-5-((1-(3,5-Dinitrobenzoyl)-1H-benzo[d]imidazol-2-yl)imino)pentanal (14)

IR (KBr cm−1): 1701 (C=O str.), 3122 (C–H aromatic str.), 1627 (N=CH str.), 1543 (Ar., NO2 str.), 1727 (Aliphatic aldehyde C=O str.); 1H NMR (DMSO): 6.875-7.946 (m, 4H, ArH), 8.632 (s, 1H, N=CH), 8.912–9.063 (m, 3H, Ar(NO2)2), 9.254–9.678 (m, 1H, CHO); 13C NMR (DMSO): 19, 28, 44, 115, 123, 125, 130, 150, 163, 168; MS: m/z = 409 (M+ +1).

(E)-5-((1-Acetyl-1H-benzo[d]imidazol-2-yl)imino)pentanal (15)

IR (KBr cm−1): 1695 (C=O str.), 3050 (C–H aromatic str.), 1606 (N=CH str.), 1535 (C-NO2 str.), 2860 (C–H sym. str., R-CH3), 1728 (Aliphatic aldehyde C=O str.); 1H NMR (DMSO): 7.875–8.246 (m, 4H, ArH), 7.632 (s, 1H, N=CH), 9.254–9.678 (m, 1H, CHO), 2.856 (s, 3H, CH3); 13C NMR (DMSO): 19, 24, 28, 44, 115, 123, 138, 142, 163, 168, 202; MS: m/z = 257 (M+ +1).

(E)-(3,5-Dinitrophenyl)(2-((4-hydroxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone (16)

IR (KBr cm−1): 1685 (C=O str.), 3094 (C–H aromatic str.), 1630 (N=CH str.), 1544 (C–NO2 str.), 3465 (O–H str); 1H NMR (DMSO): 6.885–7.632 (m, 8H, ArH), 8.654 (s, 1H, N=CH), 8.912–9.063 (m, 3H, Ar (NO2)2); 13C NMR (DMSO): 115, 123, 125, 130, 132, 138, 150, 160, 168; MS: m/z = 431 (M+ +1).

(E)-(2-((2,4-Dimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (17)

IR (KBr cm−1): 1695 (C=O str.), 2919 (C–H aromatic str.), 1634 (N=CH str.), 2850 (Ar., OCH3 str.); 1646 (naph. ring str.); 1H NMR (DMSO): 6.844–8.213 (m, 14H, ArH), 8.612 (s, 1H, N=CH), 2.804 (s, 6H, (OCH3)2); 13C NMR (DMSO): 56, 101, 107, 109, 115, 123, 127, 129, 132, 138, 142, 160, 168; MS: m/z = 435 (M+ +1).

(E)-Naphthalen-2-yl(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone (18)

IR (KBr cm−1): 1691 (C=O str.), 1548 (N=CH str.), 1140 (C–O–C str., OCH3); 795 (C–H out of plane bending, naphthalene ring); 1H NMR (DMSO): 8.590 (s, 1H, N=CH), 4.194 (s, 9H, (OCH3)3), 6.971–8.070 (m,11H, Ar–H); 13C NMR (DMSO): 57, 107, 115, 123, 124, 127, 128, 131, 139, 142, 151, 160, 168; MS: m/z = 465 (M+ +1).

(E)-(3,5-Dinitrophenyl)(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone (19)

IR (KBr cm−1): 1681(C=O str.), 1539 (N=CH str.), 2850 (CH3 sym. str., R-OCH3); 1345 (C–NO2 str.); 1H NMR (DMSO): 8.947 (s, 1H, N=CH), 3.955 (s, 9H, (OCH3)3), 9.860–9.865 (m, 3H, Ar-(NO)2), 7.948–7.951 (d, 2H, Ar–H), 7.343–7.366 (m, 2H, Ar–H); 13C NMR (DMSO): 57, 106, 115, 125, 128, 129, 131, 139, 142, 147, 151, 168; MS: m/z = 505 (M+ +1).

(E)-1-(2-((3,4,5-trimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one (20)

IR (KBr cm−1): 1685 (C=O str.), 3061 (C–H aromatic str.), 1623 (N=CH str.), 2843 (Ar., O–CH3 str.), 1266 (Palmitoyl group str.); 1H NMR (DMSO): 7.283–7.286 (m, 6H, ArH), 1.278–2.386 (m, 28H, CH2 of palmitoyl), 0.884–0.903 (t, 3H, CH3), 3.264 (s, 9H, (OCH3)3); 13C NMR (DMSO): 14, 23, 26, 30, 32, 56, 106, 115, 123, 128, 130, 139, 142, 160, 170; MS: m/z = 549 (M+ +1).

(E)-(3-Nitrophenyl)(2-((4-(pyridin-2-yl)benzylidene)amino)-1H-benzo[d]imidazol-1-yl) methanone (21)

IR (KBr cm−1): 1682 (C=O str.), 2922 (C–H aromatic str.), 1525 (N=CH str.), 1557 (C=C and C=N str. of pyridine ring), 1543 (C–NO2 str.); 1H NMR (DMSO): 10.019 (s, 1H, N=CH), 7.305–8.658 (m, 15H, ArH), 8.662 (s, 1H, Ar–NO2); 13C NMR (DMSO): 115, 123, 126, 129, 130, 132, 142, 150, 155, 160, 168; MS: m/z = 447 (M+ +1).

(2-((E)-((E)-3-(4-(Dimethylamino)phenyl)allylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone (22)

IR (KBr cm−1): 1723(C=O str.), 2920 (C–H aromatic str.), 1530 (N=CH str.), 1549 (Ar-NO2 str.), 1349 (C–N str., of ter. arylamine); 1H NMR (DMSO): 8.390–8.410 (d, 1H, N=CH), 6.731–6.740 (d, 1H, –CH = CH), 6.250–8.355 (m, 11H, ArH), 8.919 (s, 1H, Ar-NO2) 3.559 (s, 6H, (CH3)2); 13C NMR (DMSO): 40, 115, 123, 127, 131, 138, 149, 164, 168; MS: m/z = 439 (M+ +1).

(E)-(2-((4-(Dimethylamino)benzylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl) methanone (23)

IR (KBr cm−1): 1719 (C=O str.), 3085 (C–H aromatic str.), 1615 (N=CH str.), 1545 (Ar-NO2 str.), 1514 (C–N str.); 1H NMR (DMSO): 7.169–8.987 (m, 12H, ArH), 9.568 (s, 1H, N=CH), 2.909 (s, 6H (CH3)2); 13C NMR (DMSO): 40, 115, 123, 127, 131, 138, 149, 160, 168; MS: m/z = 413 (M+ +1).

(E)-(2-(((2-hydroxynaphthalen-1-yl)-methylene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone (24)

IR (KBr cm−1): 1696 (C=O str.), 2924 (C–H aromatic str.), 1553 (N=CH str.), 1546 (Ar-NO2 str.), 752 (O–H bending (out of plane)); 1H NMR (DMSO): 6.748–8.632 (m, 14H, ArH), 9.652 (s, 1H, N=CH); 13C NMR (DMSO):115, 118, 123, 125, 127, 128, 131, 138, 149, 160, 168; MS: m/z = 436 (M+ +1).

(E)-(2-((4-Nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitrophenyl)methanone (25)

IR (KBr cm−1): 1704 (C=O str.), 3107 (C–H aromatic str.), 1617 (N=CH str.), 1549 (Ar., NO2 str.); 1H NMR (DMSO): 7.206–8.689 (m, 12H, ArH), 9.672 (s, 1H, N=CH); 13C NMR (DMSO): 115, 121, 123, 125, 127, 131, 136, 139, 151, 160, 168; MS: m/z = 415 (M+ +1).

(E)-Naphthalen-2-yl(2-((4-nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)methanone (26)

IR (KBr cm−1): 1686 (C=O str.), 3056 (C–H aromatic str.), 1600 (N=CH str.), 1545 (Ar., NO2 str.), 1592 (Naphthalene ring str.); 1H NMR (DMSO): 6.865–7.954 (m, 11H, ArH), 8.765 (s, 1H, N=CH), 8.923–8.967 (m, 4H, Ar(NO2); 13C NMR (DMSO): 115, 123, 124, 128, 129, 131, 135, 136, 139, 142, 149, 160, 168; MS: m/z = 420 (M+ +1).

(E)-(2-((3,4-Dimethoxybenzylidene)amino)-1H-benzo[d]imidazol-1-yl)(3-nitro phenyl)methanone (27)

IR (KBr cm−1): 1684 (C=O str.), 1611 (N=CH str.), 2875 (Ar., O–CH3 str.); 1543 (Ar., NO2 str.); 1H NMR (DMSO): 7.463–8.932 (m, 11H, ArH), 8.185 (s, 1H, N=CH), 2.904 (s, 6H, (OCH3)2); 13C NMR (DMSO): 56, 115, 123, 125, 127, 136, 139, 142, 147, 150, 152, 160, 168; MS: m/z = 430 (M+ +1).

(E)-1-(2-((4-Nitrobenzylidene)amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one (28)

IR (KBr cm−1): 1685 (C=O str.), 2954 (C–H aromatic str.), 1618 (N=CH str.), 1271 (Palmitoyl group. str.), 1547 (Ar-NO2str.); 1H NMR (DMSO): 7.624–8.163 (m, 8H, ArH), 8.672 (s, 1H, N=CH), 1.243–2.496 (m, 28H, CH2 of palmitoyl), 0.845–0.878 (t, 3H, CH3); 13C NMR (DMSO): 14, 23, 26, 30, 32, 56, 106, 115, 120, 123, 125, 128, 131, 135, 136, 139, 142, 149, 160, 170; MS: m/z = 504 (M+ +1).

(E)-1-(2-((4-(Dimethylamino)benzylidene)amino)-1H-benzo[d]imidazol-1-yl)hexadecan-1-one (29)

IR (KBr cm−1): 2927 (C–H, aromatic str.), 2813 (C–H str. aliphatic), 1659 (C=O str.), 1594 (N=CH str.), 1303 (C–N str.), 1278 (palmitoyl group str.); 1H NMR (DMSO): 7.878–7.901 (d, 1H, N=CH), 6.606–6.622 (d, 1H, –CH = CH), 6.661–7.519 (m, 8H, ArH), 3.773 (s, 6H, (CH3)2), 1.252–2.368 (m, 28H, CH2 of palmitoyl), 0.861–0.894 (t, 3H, CH3); 13C NMR (DMSO): 14, 23, 26, 30, 32, 56, 106, 115, 120, 123, 125, 128, 130, 139, 142, 149, 164, 170; MS: m/z = 528 (M+ +1).

(E)-(2-((4-(Dimethylamino)benzylidene)amino)-1H-benzo[d]imidazol-1-yl)(naphthalen-2-yl)methanone (30)

IR (KBr cm−1): 1683 (C=O str.), 3054 (C–H aromatic str.), 1608 (N=CH str.), 1521 (Ar. NO2 str.), 1448 (C–N str.), 778 (C–H out of plane bending, naphthalene ring); 1H NMR (DMSO): 6.668–7.985 (m, 15H, ArH), 9.584 (s, 1H, N=CH), 2.909 (s, 6H (CH3)2); 13C NMR (DMSO): 40, 115, 123, 124, 128, 130, 139, 142, 160, 168; MS: m/z = 418 (M+ +1).

Biological evaluation

In vitro antimicrobial assay

Tube dilution method [15] was used to determine the antimicrobial activity of synthesized compounds against Gram-positive bacteria: Staphylococcus aureus (MTCC-3160); Bacillus subtilis (MTCC-441), the Gram-negative bacterium Escherichia coli (MTCC-443) and fungal species: Candida albicans (MTCC-227) and Aspergillus niger (MTCC-281). Dilutions were made for test and standard compounds in appropriate double strength nutrient broth—I.P. (bacteria) or Sabouraud dextrose broth—I.P. (fungi) [16]. The test and standard compounds were incubated at 37 °C for 24 h (bacteria), at 25 °C for 7 days (A. niger) and at 37 °C for 48 h (C. albicans) and the minimum inhibitory concentration (MIC) was recorded in µg/mL.

In vitro anticancer assay

The in vitro anticancer activity of the developed compounds was performed by the Sulforhodamine B (SRB) assay as described by Skehan et al. [14]. The optimal MCF-7 cell count was seeded on flat-bottom well plates and allowed to attach overnight. The compounds (20 μL) were added in quadruplicates and incubated for 72 h (both drug-free control and treated cells). Cells in each well were fixed with 200 μL of 10% cold trichloroacetic acid. After incubation for 30 min, the individual wells were rinsed with water, allowed to stain in 100 µL 0.4% SRB [Sigma-Aldrich, St Louis, Missouri, USA] (w/v; in 1% acetic acid) for 15 min. The air-dried plates were placed on a plate shaker and bound SRB was solubilised in 100 µL 10 mM Tris base solution. Absorbance was measured using a spectrophotometer at 570 nm and a dose–response curve was plotted from which the IC50 value of each compound against each cell type was determined.

Conclusion

In conclusion, a series of 1,2-disubstituted benzimidazole derivatives were synthesized and assessed for in vitro antimicrobial and anticancer activities against five representative microbial species and cancer cell line. Antimicrobial activity results indicated that the synthesized compound 1 has promising activity towards Gram negative bacteria E. coli. None of the compound showed more potent activity against Gram positive bacteria B. subtilis and S. aureus when compared to reference drug norfloxacin. Moreover, compounds 2 and 19 showed interesting results against fungal strains C. albicans and A. niger and comparable to fluconazole. The results from anticancer activity indicated that compounds 12, 21, 22 and 29 showed promising activity against MCF7. These active compounds may be taken as lead compounds for discovery of novel antimicrobial and anticancer agents in future.