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Synthesis and biological evaluation of 2-substituted benzimidazole derivatives
⁎Corresponding author. Tel.: +91 9474530205; fax: +91 3592 246462. gmariappanhpi@yahoo.co.in (Gurusamy Mariappan)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A novel series of 2-substituted benzimidazole derivatives (3a–3j) were synthesized by the reaction of 2-chloro methyl benzimidazole with substituted primary aromatic amines. All the compounds were characterized by UV, IR, 1H NMR, mass spectral data and CHN elemental analysis. The synthesized derivatives were screened for analgesic and anti-inflammatory activities. All the compounds showed significant effect at 100 mg/kg p.o. and the experimental data are statistically significant at p < 0.01 level.
Keywords
Anti-inflammatory
Analgesic
Benzimidazole
1 Introduction
Benzimidazole derivatives have occupied a prominent place in medicinal chemistry because of their significant properties as therapeutics in clinical applications. Benzimidazole is a versatile pharmacophore producing a diverse range of biological activities including anti-inflammatory and analgesic (Bahaa et al., 2006; Kavitha et al., 2010; Khan and Nandan, 1997), anti-ulcer (Bariwal et al., 2008), anti-fungal (Canan, 2003), anti-microbial (Chhonker et al., 2009; Pathak et al., 2010; Reddy et al., 2009), anthelmintic (Theodorides et al., 1976), anti-cancer (Demirayak et al., 2002), anti-asthmatic and anti-diabetic (Vinodkumar et al., 2008), anti-tubercular (Yar et al., 2009), antiprotozoal (Zygmunt et al., 2002), antiviral activities etc. The optimization of benzimidazole derivatives based on their structures has resulted in various potent drugs that are now being currently practiced in the market, like albendazole, omeprazole, mebendazole, etc. Owing to the importance and in continuation of our ongoing project work on benzimidazole derivatives, it was felt worthwhile to synthesize some novel 2-substituted benzimidazole derivatives and screen them for their analgesic and anti-inflammatory activities.
2 Experimental
2.1 Materials and reagents
All the chemicals and reagents were of synthetic grade and commercially procured from S.D. Fine Chem. Ltd. (Mumbai, India). The melting points were determined using open capillary tubes and are uncorrected. The λmax of the compounds was measured by UV–visible spectrophotometer (UV-Pharma Spec 1700, Shimadzu, Kyoto, Japan), IR spectra were recorded on FT-IR8400S, Fourier Transform (Shimadzu) Infrared spectrophotometer using KBr disk method. 1H NMR spectra were recorded on JEOL (JNM-ECS400, 400 MHz) in dimethyl sulfoxide (DMSO-d6) using Tetramethylsilane as an internal standard. The mass spectra were recorded on a Micromass Q-TOF and Shimadzu LC–MS 2010A Mass spectrometer and CHN elemental analysis was performed at Perkin Elmer Autosystem XL analysis.
2.2 General procedure for the preparation of (1H-benzimidazol-2-ylmethyl)-phenyl-amine derivatives
2.2.1 Step-1
A mixture of o-phenylenediamine (0.1 mol) and monochloroacetic acid (0.1 mol) was refluxed for 3 h in 4 N hydrochloric acid (50 mL) on a water bath. The reaction mixture was cooled and basified with ammonium hydroxide solution. The precipitate thus obtained was dried and recrystallized from methanol with activated charcoal treatment. The pure product obtained was a slightly yellow colored crystal whose melting point was 150–152 °C and the yield was 89%.
2.2.2 Step-2
A mixture of 2-chloromethyl benzimidazole (0.01 mol), substituted primary aromatic amine (0.01 mol) and KI (0.01 mol) in 50 mL of ethanol was heated under reflux for 6 h, KOH (0.01 mol in 5 mL of water) was added with continuous stirring for 2 h. Finally the reaction mixture was left aside at room temperature and then poured into crushed ice. The solid product that precipitated was filtered off, recrystallized from ethanol and dried in vacuum desiccators. The synthetic route for the target compounds 3a–3j is shown in Scheme 1.
The synthetic route of the target compounds.
2.2.2.1 (1H-Benzimidazol-2-ylmethyl)-(3,4-dichloro-phenyl)-amine (3a)
Yellow crystal; m.p. 187–192 °C; Yield 81%; UV(ethanol) λmax: 362; IR (KBr): νmax in cm−1: N–H, 3433; C–H(CH2), 3066; C⚌C(Ar), 1600; C⚌N, 1492; C–H, 2891; C–Cl, 744; Ar. Ring Vib., (995, 877, 829, 810), 1H NMR (DMSO-d6, 400 MHz) δ: 2.52(s, 2H, CH2), 4.51(s, 1H, NH aromatic), 6.66(s, 1H, NH benzimidazole), 6.88–7.52 (m, 7H, Ar-H); MS: 291 [M+]; Anal. calcd for C14H11Cl2N3 (292): C 57.55, H 3.79, N 14.38; found C 57.29, H 3.65, N 14.45.
2.2.2.2 (1H-Benoimidazol-2-ylmethyl)-(2,3-dichloro-phenyl)-amine (3b)
Light yellow powder; m.p. 93–96 °C; Yield 56%; UV(ethanol) λmax: 281; IR (KBr): νmax in cm−1: N-H, 3475; C–H(CH2), 3080; C–H(Ar), 2821; C⚌N, 1585; C–Cl, 765; Ar. Ring Vib., (902, 862, 700, 578); 1H NMR (DMSO-d6, 400 MHz) δ: 2.45(s, 2H, CH2), 4.59(s, 1H, NH aromatic), 6.56 (s, 1H, NH benzimidazole), 6.57–7.46(m, 7H, Ar-H); MS: 292 (M++1); Anal. calcd for C14H11Cl2N3 (292): C 57.55, H 3.79, N 14.38; found C 57.34, H 3.73, N 14.33.
2.2.2.3 (1H-Benzimidazol-2-ylmethyl)-(2-nitro-phenyl)-amine (3c)
Orange crystal; m.p. 90–95 °C; yield 55%; UV(ethanol) λmax: 251; IR (KBr): νmax in cm−1: N–H, 3637; C–H(CH2), 3466; C–H(Ar), 2899; C⚌N, 1506; C–NO2, 746; Ar. Ring Vib., (871, 848, 813, 783), 1H NMR (DMSO-d6, 400 MHz) δ: 2.49(s, 2H, CH2), 3.47(s, 1H, NH aromatic), 6.61(s, 1H, NH benzimidazole), 6.69–7.93(m, 8H, Ar-H); MS: 268 [M+]; Anal. calcd for C14H12N4O2 (268): C 62.68, H 4.51, N 20.88; found C 62.56, H 4.58, N 20.73.
2.2.2.4 (1H-Benzimidazol-2-ylmethyl)-(4-nitro-phenyl)-amine (3d)
Brick red crystal; m.p. 140–150 °C; Yield 68%; UV(ethanol) λmax: 382; IR (KBr): νmax in cm−1: N–H, 3481; C–H(CH2), 3363; C–H(Ar), 2914; C⚌N, 1508; C–NO2, 756; Ar. Ring Vib., (840, 632, 534, 489), 1H NMR (DMSO-d6, 400 MHz) δ: 2.50(s, 2H, CH2), 3.41(s, 1H, NH aromatic), 6.60(s, 1H, NH benzimidazole), 6.73–7.99(m, 8H, Ar-H); MS: 268 [M+]; Anal. calcd for C14H12N4O2 (268): C 62.68, H 4.51, N 20.88; found C 62.74, H 4.49, N 20.78.
2.2.2.5 (1H-Benzimidazol-2-ylmethyl)-(3-nitro-phenyl)-amine (3e)
Yellow crystal; m.p. 177–179 °C; Yield 70%; UV(ethanol) λmax: 252; IR (KBr): νmax in cm−1: N–H, 3537; C–H(CH2), 3444; C–H(Ar), 3363; C⚌N, 1585; C–NO2, 738, Ar. Ring Vib., (842, 790, 738, 669); 1H NMR (DMSO-d6, 400 MHz) δ: 2.50(s, 2H, CH2), 3.43(s, 1H, NH aromatic), 7.14(s, H, NH benzimidazole), 7.33–7.45(m, 8H, Ar-H); MS: 268 [M+]; Anal. calcd for C14H12N4O2 (268): C 62.68, H 4.51, N 20.88; found C 62.66, H 4.53, N 20.83.
2.2.2.6 (1H-Benzimidazol-2-ylmethyl)-(4-fluoro-phenyl)-amine (3f)
Light gray crystal; m.p. 161–163 °C; Yield 76%; UV(ethanol) λmax: 281; IR (KBr): νmax in cm−1: N–H, 3456; C–H(CH2), 3358, C–H(Ar), 3059; C⚌N, 1589; C–F, 819; Ar. Ring Vib., (752, 639, 605, 518, 482); 1H NMR (DMSO-d6, 400 MHz) δ: 2.55(s, 2H, CH2), 3.44(s, 1H, NH aromatic), 6.29(s, 1H, NH benzimidazole), 6.30–7.18(m, 8H, Ar-H); MS: 241 [M+]; Anal. calcd for C14H12FN3 (241): C 69.70, H 5.01, N 17.42; found C 69.76, H 5.08, N 17.64.
2.2.2.7 (1H-Benzimidazol-2-ylmethyl)-(2,4,6-tribromo-phenyl)-amine (3g)
White crystal; m.p. 120–123 °C; Yield 68%; UV(ethanol) λmax: 251; IR (KBr): νmax in cm−1: N–H, 3506; C–H(CH2), 3452, C–H(Ar), 3288; C⚌N, 1564; C–Br, 860; Ar. Ring Vib., (844, 732, 707, 673); 1H NMR (DMSO-d6, 400 MHz) δ: 2.08(s, 2H, CH2), 3.38(s, 1H, NH aromatic), 5.44(s, 1H, NH benzimidazole), 7.14–7.80(m, 6H, Ar-H); MS: 460 [M+]; Anal. calcd for C14H10Br3N3 (460): C 36.56, H 2.19, N 9.14; found C 36.44, H 2.22, N 9.34.
2.2.2.8 (1H-Benzimidazol-2-ylmethyl)-(2-chloro-4-nitro-phenyl)-amine (3h)
Dark brown crystal; m.p. 160–161 °C; Yield 65%; UV(ethanol) λmax: 364; IR (KBr): νmax in cm−1: N–H, 3051; C–H(CH2), 2899, C–H(Ar), 2762; C⚌N, 1442; C–NO2, 740; C–Cl, 895, Ar. Ring Vib., (927, 842, 638); 1H NMR (DMSO-d6, 400 MHz) δ: 2.05(s, 2H, CH2), 3.40(s, 1H, NH aromatic), 6.77(s, 1H, NH benzimidazole), 6.79–8.07(m, 7H, Ar-H); MS: 302 [M+]; Anal. calcd for C14H11ClN4O2 (303): C 55.55, H 3.66, N 18.51; found C 55.45, H 3.54, N 18.59.
2.2.2.9 4-[(1H-Benzimidazol-2-ylmethyl)-amino]-benzene sulfonamide (3i)
Pale yellow crystal; m.p. 254–256 °C; Yield 66%; UV(ethanol) λmax: 267; IR (KBr): νmax in cm−1: N–H, 3491; C–H(CH2), 3425; C–H(Ar), 3271; C⚌N, 1597; C–SO2, 1456; Ar. Ring Vib., (1001, 935, 898, 823, 748); 1H NMR (DMSO-d6, 400 MHz) δ: 2.50(s, 2H, CH2), 4.54(s, 1H, NH aromatic), 6.70(s, 1H, NH benzimidazole), 6.7–7.52(m, 8H, Ar-H), 4.55(s, 2H, SO2NH2); MS: 302 [M+]; Anal. calcd for C14H14N4O2S (302): C 55.61, H 4.67, N 18.53; found C 55.44, H 4.31, N 18.70.
2.2.2.10 (1H-Benzimidazol-2-ylmethyl)-(4-iodo-phenyl)-amine (3j)
Colorless powder; m.p.221–223 °C; Yield 72%; UV(ethanol) λmax: 253; IR (KBr): νmax in cm−1: C–I, 806; N–H, 3527; C–H(CH2), 3475; C–H(Ar), 3443; C⚌N, 1589; Ar. Ring Vib., (746, 690, 669, 576, 501); 1H NMR (DMSO-d6, 400 MHz) δ: 2.07(s, 2H, CH2), 4.44(s, 1H, NH aromatic), 6.46(s, 1H, NH benzimidazole), 7.11–7.33(m, 8H, Ar-H); MS: 349 [M+]; Anal. calcd for C14H12IN3 (349): C 48.16, H 3.46, N 12.03, found C 48.97, H 3.37, N 12.29.
2.3 Experimental animals
Adult Swiss albino mice (20–25 g) and albino rats weighing (150–200 g) of either sex were used as experimental animals. All the animals were housed in groups of 4–8 per cage at a temperature of 25 ± 1 °C and a relative humidity of 45–55%. A 12 h dark and 12 h light cycle was followed during the experiments. Animals were allowed free access to food and water ad libitum. During the study period, guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Institutional Animals Ethics Committee (IAEC) were followed for the maintenance of animals.
2.3.1 Acute toxicity studies
The acute toxicity studies were carried out in groups of six Swiss albino mice, weighing 20–25 g which were fasted overnight and treated orally with the test compounds. The dosage was varied from 100–1000 mg/kg body weight orally. All the animal experiments were performed with the approval of Institutional Animal Ethics Committee, Himalayan Pharmacy Institute, East-Sikkim, India.
2.3.2 Analgesic activity by Tail-flick method in mice
The analgesic activity was carried out by Tail-flick (D’Armour and Smith, 1941) method using Swiss albino mice. In this method, heat is used as a source of pain. Overnight fasted healthy and adult male Swiss albino mice weighing between 20 g and 25 g, in a group of six each were taken for the investigation. The animals were kept into a small cage with an opening for the tail at the rear wall. The tail was held gently and a light beam exerting radiant heat was directed to the proximal third of the tail. The tip of the tail of the mice was individually placed on the radiant heat source at constant temperature 55 °C. The cut-off reaction time was fixed at 15 s to avoid tissue damage. The tail flick response was measured at 0 h, 1 h, 2 h, 3 h and 4 h after treatment of test compounds by digital analgesiometer (INCO, Ambala, India). The drug pentazocine (3.9 mg/kg, i.p.) was used as standard drug for comparison and test groups received synthesized benzimidazole derivatives at 100 mg/kg p.o.
2.3.3 Anti-inflammatory activity by Carrageenan-induced rat paws edema method
The anti-inflammatory activity of the test compounds was evaluated by carrageenan induced rat paw edema model of Winter et al. (1962). Rats of either sex were treated with benzimidazole derivatives (100 mg/kg p.o.) and standard drug Diclofenac sodium (100 mg/kg p.o.), one hour prior to the 1% w/v solution injection of 0.1 mL carrageenan into the plantar region of left hind paw. The marking was just made beyond the tibia-tarsal junction of (knee joint) left hind paw in each animal of all groups. Paw volume was measured by Plethysmometer (Model 520, IITC, Life sciences, USA) at 0 h, 1 h, 2 h, 3 h and 4 h after carrageenan injection. The difference between the paw volume at 4th h and 0 h measurement was calculated and taken as edema volume. Percentage inhibition in the paw was calculated by using the formula, percentage inhibition = 100 (1−Vt/Vc), where Vt = mean increase in paw volume of test, and Vc = mean increase in paw volume with the control.
3 Results and discussion
3.1 Synthesis of (1H-benzimidazol-2-ylmethyl)-phenyl-amine derivatives
2-Chloromethyl benzimidazole (2) was synthesized by reacting o-phenylenediamine (1) with monochloroacetic acid in the presence of 4 N hydrochloric acid. Then the comp 2 was reacted with various substituted primary aromatic amine in the presence of KI in ethanol to get the title compounds 3a–3j. From IR spectra, the appearance of peaks at 3400–3500 cm−1 and 3000–3400 cm−1 indicated the presence of NH of benzimidazole and CH2 group attached with benzimidazole, respectively. From NMR spectra, a sharp singlet at 2–2.6 ppm ascertained the presence of CH2 (aliphatic) proton in all the synthesized compounds. The appearance of a sharp singlet at 5.54–7.14 ppm confirmed the presence of NH of benzimidazole in all the compounds. The appearance of multiplet at 6.30–8.0 ppm indicated the presence of aromatic and hetero-aromatic protons. The calculated molecular weight of the compounds was matched with observed m/e value. Hence the compounds synthesized were in conformity with the structure assigned.
3.2 Acute toxicity studies
Acute toxicity and gross behavior studies revealed that the tested compounds in the present investigation were found to be nontoxic up to 1000 mg/kg p.o.
3.3 Analgesic activity
Table 1, revealed that almost all the compounds showed very potent analgesic activity when compared with standard pentazocine. Among the tested compounds 3d, 3g, 3h, 3i, 3j showed profound analgesic activity. The rest of the compounds 3a, 3b, 3c, 3e and 3f showed moderate activity when compared with the control. n = 6 animals in each group. All synthesized compounds tested at a dose of 100 mg/kg p.o. body weight, Std-pentazocine (3.9 mg/kg i.p)., Control-vehicle (0.5% CMC).
Comp. code
Tail withdrawing time in second (Mean ± SEM)
0 h
1 h
2 h
3 h
4 h
Control
1.56 ± 0.16
2.16 ± 0.16
2.33 ± 0.21
2.66 ± 0.21
2.82 ± 0.72
Std
2.16 ± 0.16
8.5 ± 0.34
11.33 ± 0.21⁎⁎
10.16 ± 0.30⁎
10.83 ± 0.30⁎⁎
3a
2.0 ± 0.25
4.66 ± 0.21
3.53 ± 0.33⁎
4.73 ± 0.21⁎
5.66 ± 0.33⁎⁎
3b
2.16 ± 0.16
3.33 ± 0.21
3.5 ± 0.22⁎
3.10 ± 0.22⁎
3.16 ± 0.30⁎
3c
2.0 ± 0.25
2.66 ± 0.10
3.44 ± 0.21⁎
3.33 ± 0.21⁎
3.46 ± 0.33⁎⁎
3d
2.0 ± 0.25
3.0 ± 0.25
4.16 ± 0.33⁎
10.5 ± 0.22⁎⁎
9.83 ± 0.33⁎⁎
3e
1.61 ± 0.30
4.5 ± 0.22
3.23 ± 0.09⁎
4.63 ± 0.21⁎⁎
4.36 ± 0.33⁎⁎
3f
2.16 ± 0.30
3.33 ± 0.21
5.33 ± 0.42⁎⁎
4.33 ± 0.33⁎
4.16 ± 0.30⁎⁎
3g
2.0 ± 0.25
4.33 ± 0.21
3.73 ± 0.30⁎
8.63 ± 0.21⁎⁎
10.03 ± 0.30⁎⁎
3h
2.16 ± 0.16
5.16 ± 0.30
7.0 ± 0.36⁎⁎
8.0 ± 0.30⁎⁎
9.50 ± 0.42⁎⁎
3i
2.0 ± 0.25
6.53 ± 0.21
7.83 ± 0.30⁎
9.73 ± 0.21⁎⁎
9.2.50 ± 0.30⁎⁎
3j
2.16 ± 0.16
10.83 ± 0.30
7.16 ± 0.47⁎⁎
9.83 ± 0.21⁎⁎
8.9 ± 0.30⁎⁎
3.4 Screening of anti-inflammatory activity
From the Table 2, it was found that most of the tested compounds showed significant results in comparison with standard diclofenac sodium. Amongst all the compounds, 3b, 3d, 3f, 3g and 3i showed potent anti-inflammatory activity and the rest of the compounds showed moderate activity. n = 6 animals in each group. All synthesized compounds tested at a dose of 100 mg/kg p.o. body weight, Std-pentazocine (3.9 mg/kg i.p)., Control-vehicle (0.5% CMC).
Comp. code
Mean difference in Paw volume in mL (Mean ± SEM)
% Inhibition
0 h
1 h
2 h
3 h
4 h
4 h
Control
0.14 ± 0.01
0.23 ± 0.01
0.24 ± 0.02
0.25 ± 0.01
0.25 ± 0.01
–
Std
0.14 ± 0.01
0.12 ± 0.01
0.12 ± 0.01⁎
0.10 ± 0.01⁎
0.09 ± 0.01⁎
64
3a
0.15 ± 0.01
0.17 ± 0.01
0.21 ± 0.01
0.23 ± 0.01
0.19 ± 0.01⁎⁎
24
3b
0.12 ± 0.01
0.13 ± 0.01
0.15 ± 0.01
0.14 ± 0.01⁎
0.12 ± 0.01⁎⁎
52
3c
0.13 ± 0.02
0.15 ± 0.01
0.15 ± 0.01
0.2 ± 0.01
0.15 ± 0.01⁎
25
3d
0.14 ± 0.02
0.12 ± 0.02
0.11 ± 0.01⁎
0.11 ± 0.02⁎
0.10 ± 0.01⁎
60
3e
0.12 ± 0.01
0.16 ± 0.02
0.15 ± 0.01
0.14 ± 0.02⁎
0.13 ± 0.02⁎
48
3f
0.14 ± 0.01
0.16 ± 0.01
0.12 ± 0.01⁎
0.13 ± 0.01⁎
0.12 ± 0.02⁎
50
3g
0.15 ± 0.02
0.15 ± 0.01
0.13 ± 0.01⁎
0.13 ± 0.01
0.10 ± 0.01⁎
60
3h
0.14 ± 0.02
0.14 ± 0.01
0.13 ± 0.02⁎
0.12 ± 0.01⁎⁎
0.19 ± 0.02
24
3i
0.14 ± 0.01
0.13 ± 0.01
0.12 ± 0.01⁎
0.10 ± 0.11⁎⁎
0.09 ± 0.02⁎⁎
64
3j
0.23 ± 0.01
0.22 ± 0.02
0.21 ± 0.01⁎
0.18 ± 0.01⁎⁎
0.18 ± 0.01
28
4 Conclusion
In conclusion, we have described a simple protocol for the synthesis of (1H-benzimidazol-2-ylmethyl)-phenyl-amine derivatives with remarkable yields. All the synthesized compounds were screened for their in-vivo analgesic and anti-inflammatory activities and found most of them having significant analgesic and anti-inflammatory activities. The pharmacological activities exhibited by synthesized novel benzimidazole derivatives have confirmed that these compounds may serve the purpose of being accepted as the novel therapeutic agents. Furthermore, an extensive toxicological study of these derivatives are highly recommended to assess the safety and pharmacological efficacy of the compounds studied.
Acknowledgments
The authors are grateful to the Director, Dr. H. P. Chhetri, Himalayan Pharmacy Institute, Majhitar, East Sikkim, for providing laboratory facilities. The authors are also thankful to Professor N. S. Islam, Dept. of Chemical Science, SAIF, Tezpur University, India, for providing spectral data.
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