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Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors Ankita Rathorea, Mujeeb-Ur-Rahmana, Anees A. Siddiquia, Abuzer Alib and Mohammad Shahar Yar a,* a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi110062, India; bPhytochemistry Research Laboratory, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi-110062, India Abstract: A new series of 1-{(5-substituted-alkyl/aryl-1,3,4-oxadiazol-2-yl)methyl}-2-(piperidin-1-ylmethyl)-1Hbenzimidazoles (5a-5r) was synthesized and screened for their inhibitory activity against COX (1 and 2). In vivo antiinflammatory activity of potent compounds was done by carrageenan-induced rat paw edema model. In vitro anticancer activity of synthesized compounds was also performed at the National Cancer Institute (NCI) against NCI 60 cell lines panel. Out of the 18 compounds screened, 5h, 5i, 5j and 5l were found to be potent COX-2 inhibitors in the range of IC50 0.06-0.81 M. In vivo anti-inflammatory screening results revealed that the compounds 5h and 5j manifested profound percent protection of 72.8 and 75.0%, respectively. Compound 5f exhibited moderate cytotoxicity with 58.79% growth inhibition against SNB-75 (CNS Cancer) cell lines and moderate activity against COX-2 (IC50 = 8.0 M).
Keywords: Anti-inflammatory, anti-cancer, benzimidazoles, lipid peroxidation, piperidine, ulcerogenicity. 1. INTRODUCTION Inflammation is a multi-step and complex biological reaction involving variety of pro-inflammatory cellular proteins, enzymes and cytokines. Prostaglandins are endogenous substances which are potent mediators of inflammation, also involved in variety of physiological effects. Cyclooxygenase (COX) enzyme catalyzes the conversion of arachidonic acid into prostaglandins through oxidation process in the arachidonic acid metabolism [1, 2]. The COX-1 isozyme is mainly responsible to maintain the physiological functions such as gastroprotection and homeostasis of vascular system, while the COX-2 isozyme is involved in various processes of inflammation. Therefore, selective inhibition of COX-2 is meaningful for the treatment of inflammatory disorders [3, 4]. The non-steroidal anti-inflammatory drugs (NSAIDs) are extensively used in various inflammatory conditions but the gastrointestinal toxicities (ulcerogenicity, GI bleeding etc.) associated with them limit their long term usage [5]. Conversely, the selective COX-2 inhibitors are not clinically preferred due to their cardiovascular side effects [6] which undoubtedly necessitate the discovery of new scaffolds with significant COX-2 inhibitory potential. Stimulation of promitogenic factors, carcinogenesis and tumor angiogenesis leads to upregulation of COX-2 ultimately ending up in significant clinical concerns [7-10]. Hence, the suppressive and preventive effects of COX-2 activity on various types of cancers such as colon, lung, gastric and intestinal cancers have been widely studied [11-13]. The involvement of COX-
*Address correspondence to this author at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi-110062, India; Tel: +91 11 26059681, Ext: 688x5890, +91 9811811033; Fax: +91-11-26059666; E-mail:
[email protected] /15 $58.00+.00
2 in the propagation of cancer and the evaluation of several COX-2 inhibitors for reducing cancer propagation impelled us to investigate the present compounds for their anticancer activities. Recently, the literature has been upgraded with progressive investigations related to the synthesis and biological evaluation of benzimidazole derivatives and have received much more attention because they have emerged as potent cytotoxic and anti-inflammatory agents [14-18]. Therefore it was considered worthy to synthesize and explore several hybrid compounds of benzimidazoles. In light of the aforementioned facts and continuation of our previous work [19], some new heteroaryl linked benzimidazoles (5a-5r) were synthesized, characterized and screened for their in vitro COX inhibitory potential, in vivo anti-inflammatory as well as in vitro anticancer activities. The objective of the work is an endeavor to accomplish the active anticancer and antiinflammatory agent having selectivity towards COX-2 isozyme. 2. RESULTS AND DISCUSSION 2.1. Chemistry The synthetic strategy has been explored to obtain the title compounds (5a-5r) in excellent yields with economical cost, as per the sequence of reactions delineated in (Scheme 1). The initial step of the synthesis involves preparation of 2(chloromethyl)-1H-benzimidazole (1) followed by the synthesis of 2-(piperidin-1-ylmethyl)-1H-benzimidazole (2) by reaction of 2-chloro-1H-benzimidazole and piperidine as reported previously [20-24]. Compound (2) on nucleophilic substitution reaction afforded ethyl [2-(piperidin-1ylmethyl)-1H-benzimidazol-1-yl]acetate (3) which is further treated with hydrazine hydrate to form 2-[2-(piperidin-1© 2015 Bentham Science Publishers
Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors NH2
COOH +
Medicinal Chemistry, 2015, Vol. 11, No. 2 Cl
N
a
Cl
189
N H
NH2 1
b
N
N
N
N c
N
N H
O 2
3
O
d N
N
N N N
4
N
e
O HN
N N
NH2
O R
5a-5r
Reaction and Condition (a) 4N HCl, 4 h, reflux (b) Piperidine/Ethanol, 8 h, reflux (c) Ethyl chloroactate/dry Acetone/K2CO3, reflux, 6 h (d) NH2NH2.H2O/ methanol, reflux, 6 h (e) POCl3, substituted carboxylic acid, reflux, 8-12 h.
Scheme 1. Synthesis of intermediate and target compounds.
ylmethyl)-1H-benzimidazol-1-yl]acetohydrazide (4). 1-{(5Substitutedalkyl/aryl-1,3,4-oxadiazol-2-yl)methyl}-2-(piperidin-1-ylmethyl)-1H-benzimidazoles (5a-5r) were synthesized by the cyclization of 2-[2-(piperidin-1-ylmethyl)-1Hbenzimidazol-1-yl]acetohydrazide (4) with respective carboxylic acids and POCl3. The purity of the compounds was checked by elemental analyses. Both the analytical and spectral data (IR, 1H NMR, 13C NMR and Mass) of the compounds were in full harmony with the proposed structures. The results of elemental analysis (C, H and N) were within ±0.4% of the theoretical values. 2.2. Biological Evaluation 2.2.1. In Vitro COX Inhibition The ability of the synthesized compounds (5a-5r) to inhibit ovine COX-1 and human recombinant COX-2 was determined using an enzyme immunoassay kit. IC50 (M) values were calculated which is the concentration of compound that affords 50% inhibition of COX-2 for average of two determinations (Table 1). The IC50 values of celecoxib for COX-1 and COX-2 were found to be 9.41 and 0.08 M, respectively; signifying the high selectivity of celecoxib to-
wards COX-2 enzyme [COX-2 (SI) 117.52]. The data revealed that the most of the compounds exhibited excellent inhibition against COX-2 (IC50 0.061-0.812 M; Fig. 1) than COX-1 (IC50 > 100 M). In vitro COX (1&2) isozyme inhibition studies further indicated that the compounds (5f-5l) were found to be more potent inhibitors of COX-2 (IC50 = 0.061-8.03 M range) as compared to COX-1 (IC50 = 16.0>100 M range). Compound (5j) emerged as the most potent inhibitor of the series with IC50 = 0.06 M (1.3-fold higher) in comparison to reference drug celecoxib (IC50 = 0.08 M). Compound (5f) containing unsubstituted phenyl ring exhibited moderate COX-2 inhibitory activity (IC50 = 8.0 M). The substitution of functional groups into the aromatic moiety of compounds (5g-5r) displayed that the directional effects were related to the electronic nature of the substituent. Structure-activity relationship studies revealed that the electron withdrawing groups were the prime factor of COX-2 inhibitory potency and selectivity. The relative profile was pF > p-Cl > p-NO2 > o-F > o-NO2 > o-Cl. The para substituted derivatives (having electron withdrawing groups) were identified as more active as compared to the ortho substituted derivatives. The SAR studies revealed p-F derivative
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Table 1. In vitro COX-1 and COX-2 inhibition and COX-2 selectivity index (SI) data.
N
N N N N
O
R (5a-5r) IC50 (M)a Compound No
SIb
Substituted Value (-R) COX-1
COX-2
5a
-CH3
>100
46.1
>2.1
5b
-CH2CH 3
>100
54.9
>1.8
5c
-CH2Cl
>100
38.8
>2.5
5d
-CH2CH 2Cl
>100
30.4
>3.2
5e
-CH2NH2
>100
62.3
>1.6
5f
-C6H5
>100
8.0
>12.5
5g
o-ClC6H4
92.4
5.8
15.9
5h
p-ClC6H4
32.0
0.20
160
5i
o-FC6H4
18.8
0.81
23.2
5j
p-FC6H4
16.0
0.06
266.6
5k
o-NO2C6H4
80.2
3.4
23.5
5l
p-NO2C6H4
44.1
0.7
63.0
5m
o-OHC6H4
>100
32.4
>3.0
5n
p-OHC6H4
>100
20.2
>4.9
5o
o-CH3C6H 4
68.1
9.2
7.4
5p
p-CH3C6H 4
74.7
9.0
8.3
5q
o-OCH3C6H 4
>100
26.4
>3.7
5r
p-OCH3C6H 4
80.6
9.8
8.2
Celecoxib
--
9.4
0.08
117.5
Indomethacin
--
0.20
6.2
0.03
a
IC50 value is the compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2 for means of two determinations using the enzyme immunoassay kit. In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
b
(5j) possess greater COX-2 potency and selectivity. Likewise, p-Cl (5h) and p-NO2 (5l) derivatives were selective and potent COX-2 inhibitors but lesser than p-F. Whereas, o-Cl (5g) derivative was least potent and least selective among compounds containing electron withdrawing groups. The compounds (5m-5r) containing electron releasing substituents were less potent inhibitors than their electron withdrawing counterparts (5g-5l), with moderate COX-2 potency (IC50 = 9.0-32.4 M range) and COX-2 selectivity index and the order was p-CH3 > o-CH3 > p-OCH3 > p-OH > o-OCH3 > o-OH). Compounds (5a-5e) were less potent inhibitors of
COX-2 (IC50 = 30.4-62.3 M range) and the order of selectivity was chloroethyl > chloromethyl > methyl > ethyl > methylamine substituents. Diminished activity of compounds (5a-5e) may be attributed to the absence of phenyl ring linked with oxadiazole moiety, which indicated the significance of aromatic fragment for the activity. 2.2.2. In Vivo Anti-Inflammatory Activity Compounds with good COX-2 inhibitory activity were chosen for in vivo anti-inflammatory activity. The results of
Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors
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90
80
70
% COX-2 inhibition
60
50
40 5j 30
5i
20
5h 5l
10
0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Concentration (μMͿ
Fig. (1). Represents the % inhibition of COX-2 enzyme for potent compounds. Data represents the mean ± SEM of four representative compounds for the inhibition of COX-2. Table 2. Anti-inflammatory activity of potent compounds.
Compound No
Paw Volume (mL) (mean ± SEM) a
(S.I. ) (mean ± SEM) 4h
1.92±0.05 **
2.76±0.07 1.06±0.07
3h
4h
--
--
**
54.1±1.0
c
0.00±00
nM of MDA Formed/ hr/mg of Protein (mean ± SEM)c 3.51±0.28
61.5±2.8
0.30±0.36
**
4.12±0.32**
5f
0.88±0.01
5g
0.90±0.02**
1.12±0.04**
53.1±1.3
59.4±1.6
0.40±0.14**
4.80±0.40**
5h
0.62±0.02**
0.75±0.02**
67.7±1.1
72.8±0.9
0.38±0.20**
4.66±0.26**
5i
0.66±0.03**
0.84±0.04**
65.6±1.0
69.5±1.0
0.32±0.18**
4.26±0.32
5j
0.64±0.02**
0.69±0.03**
66.6±1.1
75.0±0.8
0.46±0.30**
4.74±0.40**
5k
0.80±0.02**
1.00±0.03**
58.3±1.1
63.7±1.2
0.60±0.16**
5.00±0.46**
5l
0.74±0.03**
0.90±0.05**
61.4±1.8
67.3±1.0
0.50±0.36**
4.38±0.38**
5m
1.11±0.05**
1.42±0.06**
42.1±1.6
48.5±1.8
ND
ND
5n
1.03±0.04**
1.37±0.10**
46.3±2.4
50.3±3.6
ND
5o
0.84±0.03
**
**
56.2±2.0
60.1±1.2
5p
0.95±0.02**
1.19±0.05**
50.5±1.4
56.8±2.0
ND
ND
5q
1.09±0.04**
1.30±0.06**
43.2±2.1
52.8±2.3
ND
ND
5r
0.93±0.02
**
1.23±0.04
**
51.5±1.1
55.4±1.7
ND
ND
0.96±0.04
**
1.16±0.04
**
50.0±2.4
57.9±1.9
1.96±0.13
6.98±0.40
Indomethacin a
Ulcerogenic Activity b
3h Control
% Inhibition ± SEM
1.10±0.03
0.26±0.14
ND **
Data were analyzed by ANOVA followed by Dunnett’s multiple comparison test, (n = 6). **P < 0.01, compared to control. b Severity index (S.I.): mean score of each treated group minus the mean score of the control group. c Relative to standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test, (n = 6). **P < 0.01, compared to standard. ND denotes not determined.
4.70±0.40**
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Developmental Therapeutics Program One Dose Mean Graph Panel/Cell Line
Mean Delta Range
Conc: 1.00E-5 Molar
Experiment ID: 1211OS78
Growth Percent
Leukemia HL-60(TB) K-562 RPMI-8226 SR Non-Small Cell Lung Cancer A549/ATCC HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 Colon Cancer COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 CNS Cancer SF-268 SF-539 SNB-19 SNB-75 U251 Melanoma MALME-3M M14 MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 Ovarian Cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 NCI/ADR-RES SK-OV-3 Renal Cancer 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 Prostate Cancer PC-3 DU-145 Breast Cancer MCF7 MDA-MB-231/ATCC HS 578T BT-549 T-47D MDA-MB-468
NSC: D-768275 / 1
Test Date: Nov 13, 2012 Report Date: Dec 10, 2013
Mean Growth Percent - Growth Percent
101.62 94.09 96.26 100.48 96.84 102.70 94.28 105.21 107.23 116.33 99.96 96.40 119.48 97.64 102.90 96.60 108.69 110.54 97.43 103.72 95.53 97.94 41.21 96.48 109.43 103.90 104.99 101.82 112.54 101.87 100.68 94.39 111.98 107.30 104.80 100.97 106.74 110.80 103.48 71.90 96.08 94.27 104.34 98.08 124.14 79.50 88.17 112.32 90.01 83.63 80.74 106.58 108.37 104.82 99.97 58.76 82.93
150
Fig. (2). Single dose mean graph of compound (5f).
100
50
0
-50
-100
-150
Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors
in vivo anti-inflammatory activities of selected compounds (5f-5r) are shown in (Table 2). Out of 13 compounds, 9 compounds possessed potent anti-inflammatory activity and a few of them displayed superior inhibition as compared to reference drug indomethacin. Compound (5j) emerged as the most promising compound of the series with the percent protection of 75.0% whereas compounds (5h), (5i) and (5l) also displayed remarkable protection against inflammation 72.8, 69.5 and 67.3%, respectively as compared to indomethacin (57.9%). Likewise, compounds (5f), (5g), (5k) and (5o) exhibited significant percent protection than indomethacin (61.5, 59.4, 63.7 and 60.1%, respectively). Conversely, remaining derivatives (5m, 5n and 5p-5r) have shown moderate percent protection. SAR studies revealed that introduction of electron withdrawing groups such as fluoro, chloro or nitro (5g-5l) resulted in enhancement in the activity whereas introduction of an electron-donating hydroxyl, methyl or methoxy group (5m-5q) resulted in decrease in the activity. 2.2.3. Acute Ulcerogenicity Study Tested compounds exhibiting significant antiinflammatory activity were further screened for their ulcerogenic potential. The results (Table 2) showed that the tested compounds exhibited a better G.I. safety profile (S.I. values 0.26-0.60) as compared to standard drug indomethacin (S.I. value 1.96±0.13). Most potent compounds (5h) and (5j) possessed a severity index of 0.38±0.20 and 0.46±0.30, respectively; which was very less than the value obtained from the standard drug. These outcomes further suggested that tested compounds may be regarded safer in terms of gastric toxicity than the standard drug indomethacin. 2.2.4. Lipid Peroxidation Study The results of ulcerogenicity were validated by determining the lipid peroxidation profile of the selected compounds and the results are tabulated in (Table 2). All the tested compounds exhibited significant reduction in lipid peroxidation (in the range of 4.12-5.00 nM of MDA formed/hr/mg of protein). Most active anti-inflammatory compounds (5h) and (5j) showed less lipid peroxidation 4.66±0.26 and 4.74±0.40 nM of MDA formed/hr/mg of protein, respectively. It is worthwhile to mention that indomethacin (reference standard) manifested the highest lipid peroxidation, 6.98±0.40 nM of MDA formed/hr/mg of protein, while the control group displayed a lipid peroxidation of 3.51±0.28 nM of MDA formed/hr/mg of protein. 2.2.5. In Vitro Anticancer Activity All the synthesized compounds were submitted to National Cancer Institute (NCI, USA), for anticancer activity. Six compounds were selected according to the NCI protocol and granted NSC codes viz; NSC D-768276/1 (5a), NSC D768275/1 (5f), NSC D-768274/1 (5g), NSC D-768273/1 (5h), NSC D-768272/1 (5q), and D-768271/1 (5r) and screened for in vitro anticancer activity at a single high dose (10 M) in full NCI 60 cell lines panel. Among the tested compounds, (5f) showed the moderate activity at single dose with 58.79% growth inhibition against SNB-75 (CNS Cancer) cell lines (Fig. 2). Compounds (5g), (5h), (5q) and (5r) exhibited 47.60%, 38.15%, 38.57% and 37.96% growth inhibition, respectively against SNB-75 (CNS Cancer) cell
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lines. The compound (5a) did not exhibit significant growth inhibition against any of the cancer cell lines. Furthermore the mean of growth percent for the compounds (5f), (5g), (5h), (5q) and (5r) was found to be 41.21%, 52.40%, 61.85%, 61.43% and 62.04%, respectively. Overall results of the primary in vitro anticancer assay showed that the synthesized 1-{(5-substitutedalkyl/aryl-1,3,4-oxadiazol-2-yl)methyl}-2-(piperidin-1-ylmethyl)-1H-benzimidazoles (5f), (5g), (5h), (5q) and (5r) were moderately active while compound (5a) was inactive on tested cell lines (Table 3). 3. CONCLUSION A new series of heteroaryl linked benzimidazoles was successfully synthesized in good yields. Compound (5j) was found to be most potent (IC50 = 0.06 M), and highly selective [COX-2 (SI) = 266.6] compared to celecoxib [COX-2 (SI) > 117.5] as well as most potent anti-inflammatory agent of the series with the percent protection of 75.0%. This compound was proved to have no gastric toxicity with a better safety profile. Compound (5f) exhibited moderate anticancer activity with 58.79% growth inhibition against SNB-75 (CNS Cancer) cell lines and moderate activity against COX2 (IC50 = 8.0 M). Furthermore, it was inferred that further optimization of these analogues may possibly help in the designing of more active and safer anti-inflammatory as well as anticancer agents. 4. EXPERIMENTAL METHODS 4.1. Chemistry Chemicals used for experimental work were of laboratory grade and purchased from Sigma-Aldrich (India) and E. Merck (Germany) and generally used without further purification. COX assay kit (catalogue no. 560131) was procured from Cayman Chemical Company, Ann Arbor, MI, U.S.A. Melting points were determined by open capillary tubes on an electrical melting point apparatus and are uncorrected. Nuclear Magnetic Resonance (1H NMR and 13C NMR) spectra were taken on Bruker spectrospin DPX-500 MHz in CDCl3. Chemical shift () were expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. The splitting pattern abbreviations are as follows: s, singlet; br, broad singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet. The exchangeable protons (OH and NH) were verified by D2O exchange. Mass spectra recorded on LCMS/MS (Perkin-Elmer and LABINDIA, Applied Biosystem), presented as m/z. IR spectra were recorded on FT-IR (Jasco, Japan, model no. 410) by means of KBr pellets of the compounds. Elemental analyses were carried out using PerkineElmer 240 analyzer. The steps of the chemical reactions and purity of the synthesized products were confirmed on precoated thin layer chromatographic plates (silica gel 60 F254) and toluene/ethyl acetate/formic acid (5:4:1, v/v/v) and benzene/acetone (8:2, v/v) as eluants. The chromatograms were visualized under iodine vapour/UV light. 4.1.1. Ethyl [2-(piperidin-1-ylmethyl)-1H-benzimidazol-1yl]acetate (3) To a solution of 2-((piperidin-1-yl)methyl)-1Hbenzimidazole (0.01 M, 2.15 g) in dry acetone (30 mL), ethyl chloroacetate (0.01 M, 1.06 mL) and anhydrous K2CO 3
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Table 3.
a
Rathore et al.
Sensitivity, NSC: Code, growth percent, delta value, mean growth percent of NCI cancer cell lines treated with synthesized compounds (10 M).
Compound No
NSC: Code
The Most Sensitive Cell Line
Growth % of the Most Sensitive Cell Line
% Growth Inhibition
Range of Growth %
Mean
Delta
Range
Activitya
5a
768276/1
UO-31 (Renal Cancer)
83.72
16.28
83.72115.73
101.50
17.78
32.01
Inactive
5f
768275/1
SNB-75 (CNS Cancer)
41.21
58.79
41.21124.14
99.97
58.76
82.93
Active
5g
768274/1
SNB-75 (CNS Cancer)
52.40
47.60
64.38125.60
101.21
48.81
75.91
Active
5h
768273/1
SNB-75 (CNS Cancer)
61.85
38.15
61.85139.01
102.93
41.08
77.16
Active
5q
768272/1
SNB-75 (CNS Cancer)
61.43
38.57
61.43127.77
103.42
41.99
66.34
Active
5r
768271/1
SNB-75 (CNS Cancer)
62.04
37.96
62.04122.69
102.24
40.20
70.04
Active
Compounds active of that particular cell lines, which showed growth inhibition 32% cell growth reduction following 48-h incubation with test compounds.
(1 g) were added and the reaction mixture was refluxed for 6 h. After completion of the reaction, K2CO3 was removed through filtration. Solvent was evaporated at room temperature. The residue thus obtained was washed with water (which also washes out KCl formed in the reaction) and crystallized from ethanol to give compound 3. Yellowish brown mass, Yield: 78%, Rf : 0.70, M.P. (°C): 210-214. IR (KBr) cm-1: 3018 (Ar-H), 2862 (C-H, CH3), 2854 (C-H, CH2), 1742 (C=O), 1636 (C=N), 1582 (C=C). 1 H NMR (CDCl3): ( ppm) 7.74-7.32 (m, 4H, Ar-H), 4.20 (q, 2H, CH2), 3.96 (s, 2H, CH2), 3.12 (s, 2H, CH2), 2.48 [t, 4H, J = 3.6 Hz, (CH2)2N], 1.68-1.51 (m, 6H, CH2), 1.38 (t, J = 6.3 Hz, 3H, CH3). 13C NMR (CDCl3): ( ppm) 173.70 (C=O, ester), 154.80 (C=N), 135.82-123.80 (Ar-C), 69.40 (CH2CH3), 52.06 (CH2 COO), 50.22 (CH2), 58.88 (CH2)2N, 27.50 (CH2), 16.80 (CH3). ESI-MS (m/z): 302 [M+1] + (63.8%). Anal. Calcd for C17H23N3O2: C(67.75) H(7.69) N(13.94). Found: C(67.69) H(7.66) N(13.97). 4.1.1.1. 2-[2-(Piperidin-1-ylmethyl)-1H-benzimidazol-1-yl] acetohydrazide (4) A solution of compound 3 (0.01 M, 3.01 g) in methanol (60 mL) and 99% hydrazine hydrate (1 mL) was refluxed for 6 h. After the completion of reaction, mixture was cooled and the solid was filtered, washed with water and recrystallized with ethanol to afford the compound 4. Pale yellow powder, Yield: 72%, Rf : 0.62, M.P. (°C): 284-286. IR (KBr) cm-1: 3482 (N-H, NH2), 3446 (N-H, NH), 3030 (Ar-H), 2880 (C-H, CH2), 1724 (C=O hydrazide), 1652 (C=N), 1590 (C=C). 1H NMR (CDCl3): ( ppm) 9.54 (s, 1H, NH, D2O exchangeable), 7.75-7.10 (m, 4H, Ar-H), 4.68 (br, 2H, NH2, D2O exchangeable), 3.91 (s, 2H, CH2), 3.08 (s, 2H, CH2), 2.70 [t, 4H, J = 4.6 Hz, (CH2)2N], 1.68-1.50 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 168.44 (C=O, hydrazide), 155.70 (C=N), 133.90-118.60 (Ar-C), 56.38 (CH2CO), 57.80 (CH2)2N, 50.63 (CH2), 28.68 (CH2). ESI-MS (m/z): 288 [M+1]+ (100%). Anal. Calcd for C15H21N5O: C(62.70) H(7.37) N(24.37). Found: C(62.58) H(7.40) N(24.33).
4.1.1.2. Common method for synthesis of 1-{(5-substitutedalkyl/aryl -1,3,4-oxadiazol-2-yl)methyl}-2-(piperidin-1-ylmethyl)-1H-benzimidazoles (5a-5r) A mixture of compound 4 (0.001 M, 2.87 g) and respective carboxylic acids was refluxed for 8-12 h in presence of 5-6 mL of phosphoryl chloride (POCl3) The reaction mixture was then poured into ice-cold water and neutralized with 20% NaHCO3 solution. The solid thus obtained was filtered, washed with water and recrystallized from ethanol to obtain the title compounds. 4.1.2. 1-[(5-Methyl-1,3,4-oxadiazol-2-yl)methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5a) Yellow amorphous powder, Yield: 70%, Rf : 0.60, M.P. (°C): 210-212. IR (KBr) cm-1 : 3022 (Ar-H), 2912 (C-CH3), 2880 (C-H, CH2), 1630 (C=N), 1560 (C=C), 1120 (C-O-C, asymmetric), 1062 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.64-7.18 (m, 4H, Ar-H), 3.66 (s, 2H, CH2), 2.80 (s, 2H, CH2), 2.30 [t, 4H, J = 3.6 Hz, (CH2)2N], 2.32 (s, 3H, CH3), 1.58-1.40 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.61, 161.90 (2C, oxadiazole), 157.52 (C=N), 132.70118.40 (Ar-C), 54.72 (CH2)2N, 48.44 (CH2), 46.20 (CH2), 24.88 (CH2), 16.90 (CH3). ESI-MS (m/z): 312 [M+1] + (67.5%). Anal. Calcd for C17H21N5O: C(65.57) H(6.80) N(22.49). Found: C(65.82) H(6.77) N(22.42). 4.1.3. 1-[(5-Ethyl-1,3,4-oxadiazol-2-yl)methyl]-2-(piperidin1-yl-methyl)-1H-benzimidazole (5b) Dark brown solid, Yield: 64%, Rf : 0.65, M.P. (°C): 266268. IR (KBr) cm-1: 2988 (Ar-H), 2900 (C-CH3), 2860 (C-H, CH2), 1658 (C=N), 1570 (C=C), 1133 (C-O-C, asymmetric), 1052 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.687.30 (m, 4H, Ar-H), 3.74 (s, 2H, CH2), 3.11 (q, 2H, CH2), 2.86 (s, 2H, CH2), 2.37 [t, 4H, J = 4.0 Hz, (CH2)2N], 1.501.38 (m, 6H, CH2), 1.40 (t, 3H, J = 3.4 Hz, CH3). 13C NMR (CDCl3): ( ppm) 161.80, 159.22 (2C, oxadiazole), 154.90 (C=N), 135.10-123.26 (Ar-C), 52.80 (CH2)2N, 49.80 (CH2), 48.22 (CH2), 25.30 (CH2), 22.44 (CH2CH3), 16.88 (CH3).
Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors
ESI-MS (m/z): 326 [M+1]+ (71.9%). Anal. Calcd for C18H23N5O: C(66.44) H(7.12) N(21.52). Found: C(66.19) H(7.14) N(21.59). 4.1.4. 1-[{5-(Chloromethyl)-1,3,4-oxadiazol-2-yl}methyl]-2(piperidin-1-yl-methyl)-1H-benzimidazole (5c)
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156.70 (C=N), 130.88-119.40 (Ar-C), 56.93 (CH2)2N, 49.91 (CH2), 46.22 (CH2), 28.20 (CH2). ESI-MS (m/z): 374 [M+1] + (92.6%). Anal. Calcd for C22H23N5O: C(70.76) H(6.21) N(18.75). Found: C(71.01) H(6.18) N(18.69). 4.1.8. 1-[{5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl}methyl]2-(piperidin-1-yl-methyl)-1H-benzimidazole (5g)
Yellow amorphous powder, Yield: 58%, Rf : 0.69, M.P. (°C): 222-224. IR (KBr) cm-1: 2990 (Ar-H), 2901, 2858 (CH, CH2), 1630 (C=N), 1549 (C=C), 1144 (C-O-C, asymmetric), 1028 (C-O-C, symmetric), 776 (C-Cl). 1H NMR (CDCl3): ( ppm) 7.70-7.24 (m, 4H, Ar-H), 4.68 (s, 2H, CH2), 4.52 (s, 2H, CH2), 3.80 (s, 2H, CH2), 2.34 [t, 4H, J = 3.5 Hz, (CH2)2N], 1.56-1.39 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 160.10, 159.22 (2C, oxadiazole), 153.90 (C=N), 132.22-119.56 (Ar-C), 53.70 (CH2)2N, 49.48 (CH2), 48.20 (CH2Cl), 46.11 (CH2), 28.88 (CH2). ESI-MS (m/z): 346 [M+1]+ (74.2%). Anal. Calcd for C17H20ClN5O: C(59.04) H(5.83) N(20.25). Found: C(58.84) H(5.85) N(20.32).
Dark brown solid, Yield: 74%, Rf : 0.68, M.P. (°C): 258260. IR (KBr) cm-1: 3029 (Ar-H), 2878 (C-H, CH2), 1660 (C=N), 1574 (C=C), 1130 (C-O-C, asymmetric), 1020 (C-OC, symmetric), 754 (Ar-Cl). 1H NMR (CDCl3): ( ppm) 7.84-7.30 (m, 8H, Ar-H), 3.76 (s, 2H, CH2), 2.79 (s, 2H, CH2), 2.32 [t, 4H, J = 4.4 Hz, (CH2)2N], 1.54-1.38 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 163.20, 159.33 (2C, oxadiazole), 155.68 (C=N), 137.94 (C-Cl), 132.18-120.44 (Ar-C), 56.30 (CH2)2N, 50.22 (CH2), 47.10 (CH2), 29.80 (CH2). ESI-MS (m/z): 408 [M+1]+ (85.3%). Anal. Calcd for C22H22ClN5O: C(64.78) H(5.44) N(17.17). Found: C(64.69) H(5.46) N(17.23).
4.1.5. 1-[{5-(2-Chloroethyl)-1,3,4-oxadiazol-2-yl}methyl]-2(piperidin-1-yl-methyl)-1H-benzimidazole (5d)
4.1.9. 1-[{5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl}methyl]2-(piperidin-1-yl-methyl)-1H-benzimidazole (5h)
Pale yellow powder, Yield: 64%, Rf : 0.70, M.P. (°C): 196-198. IR (KBr) cm-1: 3010 (Ar-H), 2868 (C-H, CH2), 1666 (C=N), 1584 (C=C), 1120 (C-O-C), asymmetric), 1038 (C-O-C, symmetric), 770 (C-Cl). 1H NMR (CDCl3): ( ppm) 7.77-7.30 (m, 4H, Ar-H), 4.60 (t, 2H, CH2), 3.72 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.79 (t, J = 2.8 Hz, 2H, CH2), 2.38 [t, 4H, J = 4.4 Hz, (CH2)2N], 1.52-1.42 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 163.62, 161.28 (2C, oxadiazole), 152.94 (C=N), 134.22-123.65 (Ar-C), 58.80 (CH2)2N, 48.14 (CH2), 46.66 (CH2), 43.40 (CH2Cl), 34.26 (CH2 CH2Cl), 29.80 (CH2). ESI-MS (m/z): 360 [M+1]+ (100%). Anal. Calcd for C18H22ClN5O: C(60.08) H(6.16) N(19.46). Found: C(60.30) H(6.13) N(19.40).
Brown amorphous powder, Yield: 70%, Rf : 0.62, M.P. (°C): 229-231. IR (KBr) cm-1: 2984 (Ar-H), 2864 (C-H, CH2), 1655 (C=N), 1590 (C=C), 1140 (C-O-C, asymmetric), 1026 (C-O-C, symmetric), 760 (Ar-Cl). 1H NMR (CDCl3): ( ppm) 7.78-7.22 (m, 8H, Ar-H), 3.69 (s, 2H, CH2), 2.83 (s, 2H, CH2), 2.36 [t, 4H, J = 5.5 Hz, (CH2)2N], 1.57-1.42 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.22, 161.50 (2C, oxadiazole), 153.88 (C=N), 138.04 (C-Cl), 132.80-120.44 (Ar-C), 58.00 (CH2)2N, 49.82 (CH2), 47.00 (CH2), 26.80 (CH2). ESI-MS (m/z): 408 [M+1]+ (81.0%). Anal. Calcd for C22H22ClN5O: C(64.78) H(5.44) N(17.17). Found: C(64.66) H(5.45) N(17.21).
4.1.6. 1-(5-[{2-(Piperidin-1-yl-methyl)-1H-benzimidazol-1yl}methyl]-1,3,4-oxadiazol-2-yl)methanamine (5e) Reddish brown solid mass, Yield: 72%, Rf : 0.64, M.P. (°C): 218-220. IR (KBr) cm-1 : 3430, 3362 (C-NH2), 3016 (Ar-H), 2880 (C-H, CH2), 1655 (C=N), 1570 (C=C), 1136 (C-O-C, asymmetric), 1020 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.50-7.11 (m, 4H, Ar-H), 4.20 (br, 2H, NH2, D2O exchangeable), 3.90 (s, 2H, CH2), 3.83 (s, 2H, CH2), 2.80 (s, 2H, CH2), 2.34 [t, 4H, J = 3.2 Hz, (CH2)2N], 1.50-1.36 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 163.82, 161.08 (2C, oxadiazole), 155.34 (C=N), 131.98-123.10 (ArC), 59.16 (CH2)2N, 50.26 (CH2), 47.56 (CH2), 42.80 (CH2NH2), 26.96 (CH2). ESI-MS (m/z): 327 [M+1] + (69.8%). Anal. Calcd for C17H22N6O: C(62.56) H(6.79) N(25.75). Found: C(62.79) H(6.76) N(25.66). 4.1.7. 1-[(5-Phenyl-1,3,4-oxadiazol-2-yl)methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5f) Yellow amorphous powder, Yield: 69%, Rf : 0.70, M.P. (°C): 225-227. IR (KBr) cm-1: 3010 (Ar-H), 2850 (C-H, CH2), 1656 (C=N), 1580 (C=C), 1111 (C-O-C, asymmetric), 1030 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.547.22 (m, 9H, Ar-H), 3.70 (s, 2H, CH2), 2.85 (s, 2H, CH2), 2.39 [t, 4H, J = 4.0 Hz, (CH2)2N], 1.57-1.41 (m, 6H, CH2). 13 C NMR (CDCl3): ( ppm) 163.66, 159.10 (2C, oxadiazole),
4.1.10. 1-[{5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-yl}methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5i) Pale yellow mass, Yield: 68%, Rf : 0.61, M.P. (°C): 174176. IR (KBr) cm-1: 3012 (Ar-H), 2884 (C-H, CH2), 1648 (C=N), 1554 (C=C), 1226 (Ar-F), 1152 (C-O-C, asymmetric), 1056 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 8.00-7.40 (m, 8H, Ar-H), 3.76 (s, 2H, CH2), 2.84 (s, 2H, CH2), 2.30 [t, 4H, J = 3.0 Hz, (CH2)2N], 1.49-1.30 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.20, 160.26 (2C, oxadiazole), 157.00 (C=N), 152.20 (C-F), 131.95-118.83 (Ar-C), 56.26 (CH2)2N, 50.20 (CH2), 48.40 (CH2), 27.88 (CH2). ESI-MS (m/z): 392 [M+1]+ (100%). Anal. Calcd for C22H22FN5O: C(67.50) H(5.66) N(17.89). Found: C(67.31) H(5.64) N(17.86). 4.1.11. 1-[{5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl}methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5j) Light brown powder, Yield: 58%, Rf : 0.69, M.P. (°C): 192-194. IR (KBr) cm-1: 2988 (Ar-H), 2910, 2860 (C-H, CH2), 1658 (C=N), 1550 (C=C), 1382 (C-N), 1226 (Ar-F), 1133 (C-O-C, asymmetric), 1052 (C-O-C, symmetric). 1 H NMR (CDCl3): ( ppm) 8.18-7.33 (m, 8H, Ar-H), 3.84 (s, 2H, CH2), 2.88 (s, 2H, CH2), 2.40 [t, 4H, J = 3.8 Hz, (CH2)2N], 1.58-1.39 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.80, 162.96 (2C, oxadiazole), 157.88 (C=N), 154.80 (C-F), 132.21-120.79 (Ar-C), 59.96 (CH2)2N, 50.40
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(CH2), 47.80 (CH2), 25.00 (CH2). ESI-MS (m/z): 392 [M+1] + (100%). Anal. Calcd for C22H22FN5O: C(67.50) H(5.66) N(17.89). Found: C(67.39) H(5.65) N(17.94).
(m/z): 390 [M+1]+ (100%). Anal. Calcd for C22H23N5O2: C(67.85) H(5.95) N(17.98). Found: C(68.69) H(5.92) N(18.04).
4.1.12. 1-[{5-(2-Nitrophenyl)-1,3,4-oxadiazol-2-yl}methyl]2-(piperidin-1-yl-methyl)-1H-benzimidazole (5k)
4.1.16. 2-(Piperidin-1-yl-methyl)-1-[(5-o-tolyl-1,3,4-oxadiazol-2-yl)methyl]-1H-benzimidazole (5o)
Brown powder, Yield: 65%, Rf : 0.73, M.P. (°C): 188190. IR (KBr) cm-1: 3020 (Ar-H), 2870 (C-H, CH2), 1666 (C=N), 1585 (C=C), 1520, 1455 (NO2), 1126 (C-O-C, asymmetric), 1040 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.88-7.30 (m, 8H, Ar-H), 3.75 (s, 2H, CH2), 2.89 (s, 2H, CH2), 2.33 [t, 4H, J = 3.6 Hz, (CH2)2N], 1.52-1.33 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.82, 161.58 (2C, oxadiazole), 157.20 (C=N), 147.36 (C-NO2), 132.00-120.18 (Ar-C), 55.70 (CH2)2N, 49.90 (CH2), 47.70 (CH2), 27.40 (CH2). ESI-MS (m/z): 419 [M+1]+ (76.3%). Anal. Calcd for C22H22N6O3: C(63.15) H(5.30) N(20.08). Found: C(63.38) H(5.28) N(20.01).
Pale yellow flakes, Yield: 70%, Rf : 0.65, M.P. (°C): 161163. IR (KBr) cm-1: 2988 (Ar-H), 2954 (C-H, CH3), 2880 (C-H, CH2), 1676 (C=N), 1569 (C=C), 1120 (C-O-C, asymmetric), 1040 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.11-6.68 (m, 8H, Ar-H), 3.78 (s, 2H, CH2), 2.94 (s, 2H, CH2), 2.40 (s, 3H, CH3), 2.29 [t, 4H, J = 5.0 Hz, (CH2)2N], 1.60-1.48 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 165.22, 163.40 (2C, oxadiazole), 157.40 (C=N), 137.60 (C-CH3), 132.68-121.60 (Ar-C), 59.40 (CH2)2N, 49.80 (CH2), 47.10 (CH2), 27.88 (CH2), 19.30 (CH3). ESIMS (m/z): 388 [M+1]+ (89.4%). Anal. Calcd for C23H25N5O: C(71.29) H(6.50) N(18.07). Found: C(71.48) H(6.52) N(18.02).
4.1.13. 1-[{5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl}methyl]2-(piperidin-1-yl-methyl)-1H-benzimidazole (5l) Pale yellow mass, Yield: 62%, Rf : 0.71, M.P. (°C): 237239. IR (KBr) cm-1: 2980 (Ar-H), 2872 (C-H, CH2), 1668 (C=N), 1584 (C=C), 1518, 1446 (NO2), 1140 (C-O-C, asymmetric), 1036 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.94-7.24 (m, 8H, Ar-H), 3.80 (s, 2H, CH2), 2.86 (s, 2H, CH2), 2.40 [t, 4H, J = 4.0 Hz, (CH2)2N], 1.60-1.44 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 163.82, 160.50 (2C, oxadiazole), 155.22 (C=N), 148.90 (C-NO2), 132.20-121.84 (Ar-C), 59.76 (CH2)2N, 48.00 (CH2), 46.90 (CH2), 27.82 (CH2). ESI-MS (m/z): 419 [M+1]+ (71.7%). Anal. Calcd for C22H22N6O3: C(63.15) H(5.30) N(20.08). Found: C(62.96) H(5.28) N(20.14). 4.1.14. 2-[5-{(2-(Piperidin-1-yl-methyl)-1H-benzimidazol-1yl)methyl}-1,3,4-oxadiazol-2-yl]phenol (5m) Yellowish brown flakes, Yield: 61%, Rf : 0.69, M.P. (°C): 264-266. IR (KBr) cm-1: 3340 (Ar-O-H), 3026 (Ar-H), 2878 (C-H, CH2), 1670 (C=N), 1570 (C=C), 1150 (C-O-C, asymmetric), 1042 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.34-6.90 (m, 8H, Ar-H), 5.14 (s, 1H, OH, D2O exchangeable), 3.90 (s, 2H, CH2), 3.00 (s, 2H, CH2), 2.40 [t, 4H, J = 4.8 Hz, (CH2)2N], 1.62-1.38 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.90, 161.88 (2C, oxadiazole), 158.24 (C-OH), 156.80 (C=N), 131.66-122.80 (Ar-C), 59.70 (CH2)2N, 51.44 (CH2), 48.68 (CH2), 29.80 (CH2). ESI-MS (m/z): 390 [M+1]+ (100%). Anal. Calcd for C22H23N5O2: C(67.85) H(5.95) N(17.98). Found: C(68.02) H(5.93) N(17.92). 4.1.15. 4-[5-{(2-(Piperidin-1-yl-methyl)-1H-benzimidazol-1yl)methyl}-1,3,4-oxadiazol-2-yl]phenol (5n) Dark brown solid, Yield: 66%, Rf : 0.59, M.P. (°C): 140142. IR (KBr) cm-1: 3368 (Ar-O-H), 3006 (Ar-H), 2912 (CH, CH2), 1676 (C=N), 1569 (C=C), 1138 (C-O-C, asymmetric), 1055 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.19-6.82 (m, 8H, Ar-H), 5.10 (s, 1H, OH, D2O exchangeable), 3.78 (s, 2H, CH2), 2.96 (s, 2H, CH2), 2.48 [t, 4H, J = 3.9 Hz, (CH2)2N], 1.70-1.48 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.56, 161.80 (2C, oxadiazole), 159.22 (C-OH), 155.80 (C=N), 132.00-121.66 (Ar-C), 56.90 (CH2)2N, 50.30 (CH2), 48.40 (CH2), 27.40 (CH2). ESI-MS
4.1.17. 2-(Piperidin-1-yl-methyl)-1-[(5-p-tolyl-1,3,4-oxadiazol-2-yl)methyl]-1H-benzimidazole (5p) Dark brown solid, Yield: 68%, Rf : 0.68, M.P. (°C): 206208. IR (KBr) cm-1: 2974 (Ar-H), 2877 (C-H, CH3), 2862 (C-H, CH2), 1659 (C=N), 1542 (C=C), 1136 (C-O-C, asymmetric), 1060 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.23-6.60 (m, 8H, Ar-H), 3.88 (s, 2H, CH2), 2.94 (s, 2H, CH2), 2.39 [t, 4H, J = 4.8 Hz, (CH2)2N], 2.38 (s, 3H, CH3), 1.56-1.38 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.98, 162.36 (2C, oxadiazole), 156.20 (C=N), 140.40 (CCH3), 131.66-120.94 (Ar-C), 58.66 (CH2)2N, 50.42 (CH2), 48.66 (CH2), 28.90 (CH2), 22.90 (CH3). ESI-MS (m/z): 388 [M+1]+ (84.7%). Anal. Calcd for C23H25N5O: C(71.29) H(6.50) N(18.07). Found: C(71.08) H(6.49) N(18.14). 4.1.18. 1-[{5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-yl}methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5q) Brownish yellow mass, Yield: 74%, Rf : 0.72, M.P. (°C): 177-179. IR (KBr) cm-1: 3018 (Ar-H), 2888 (C-H, CH2), 2855 (OCH3), 1672 (C=N), 1580 (C=C), 1154 (C-O-C, asymmetric), 1028 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.20-6.48 (m, 8H, Ar-H), 3.84 (s, 3H, OCH3), 3.74 (s, 2H, CH2), 2.94 (s, 2H, CH2), 2.33 [t, 4H, J = 5.1 Hz, (CH2)2N], 1.57-1.36 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 164.78, 161.90 (2C, oxadiazole), 155.20 (C=N), 156.52 (C-OCH3), 134.88-121.28 (Ar-C), 58.70 (CH2)2N, 54.84 (OCH3), 49.90 (CH2), 47.26 (CH2), 26.48 (CH2). ESIMS (m/z): 404 [M+1]+ (100%). Anal. Calcd for C23H25N5O2: C(68.47) H(6.25) N(17.36). Found: C(68.28) H(6.24) N(17.42). 4.1.19. 1-[{5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl}methyl]-2-(piperidin-1-yl-methyl)-1H-benzimidazole (5r) Dark brown mass, Yield: 66%, Rf : 0.58, M.P. (°C): 213215. IR (KBr) cm-1: 2989 (Ar-H), 2910 (C-H, CH2), 2870 (O-CH3), 1672 (C=N), 1570 (C=C), 1142 (C-O-C, asymmetric), 1042 (C-O-C, symmetric). 1H NMR (CDCl3): ( ppm) 7.38-6.52 (m, 8H, Ar-H), 3.80 (s, 2H, CH2), 2.64 (s, 2H, CH2), 2.96 (s, 3H, OCH3), 2.20 [t, 4H, J = 3.6 Hz, (CH2)2N], 1.63-1.41 (m, 6H, CH2). 13C NMR (CDCl3): ( ppm) 165.20, 162.10 (2C, oxadiazole), 159.50 (C-OCH3), 157.12 (C=N), 131.90-119.60 (Ar-C), 59.20 (CH2)2N, 55.42 (OCH3), 50.20
Synthesis and Evaluation of Benzimidazole Derivatives as Selective COX-2 Inhibitors
(CH2), 48.90 (CH2), 28.88 (CH2). ESI-MS (m/z): 404 [M+1] + (100%). Anal. Calcd for C23H25N5O2: C(68.47) H(6.25) N(17.36). Found: C(68.62) H(6.27) N(17.31). 4.2. Biological Methods 4.2.1. In Vitro Cyclooxygenase (COX) Inhibition Assay All the synthesized compounds (5a-5r) were subjected to inhibit ovine COX-1 and human recombinant COX-2 using an enzyme immunoassay (EIA) kit according to a previously described method [25-28]. COX catalyzes the first step of biosynthesis of arachidonic acid to produce PGH2. PGH2 is reduced with stannous chloride to form PGF2, which is measured by enzyme immunoassay. The assay was performed in duplicate according to the manufacturer’s guidelines. This enzymatic reaction produces a distinct yellow color product that absorbs at 412 nm. The intensity of this color, determined spectrophotometrically, is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the well during the incubation. Percent inhibition was calculated by the comparison of compounds treated to various control incubations. The concentration of the test compounds that affords 50% inhibition of COX-2 (IC50, M) was calculated from the concentration inhibition response curve. 4.2.2. Animal Male Wistar albino rats weighing (150-200 g) were selected to determine anti-inflammatory activity and ulcerogenicity. All animals were assigned into groups of six animals each. Animals were kept at 25-27 °C and 30-70% relative humidity. All experimental protocols were carried out after the approval of Committee for the Purpose of Control and Supervision of Experiments in Animal (CPCSEA) [Institutional Animal Ethics Committee (IAEC) [Reg. No: JH/CAHF/173/CPCSEA]. 4.2.3. In Vivo Anti-Inflammatory Activity In vivo anti-inflammatory activity of potent compounds was determined by the method of Winter et al. [29-31]. The Group I served as control; received 0.5% w/v carboxymethylcellulose (CMC). Group II received standard drug indomethacin, at a dose level of 10 mg.kg-1 and other groups received tested compounds, at a dose level of 50 mg.kg-1 b.w. The hind paw edema was induced in each rat by the subplantar injection of 0.1 ml of carrageenan suspension (1.0% w/v in 0.9% saline) 1 h after the administration of the tested compounds and standard drug orally. The paw volume was measured by a digital plethysmometer (Panlab LE 7500) at 0, 1, 3 and 4 h after the carrageenan injection. The percent inhibition of edema was calculated in the control and treated animals according to the following equation: Percent edema inhibition = {1 - Vt/ Vc} 100 Where, Vc represents the paw volume of control (mean increase in paw volume in absence of tested compound) and Vt represents the mean increase of paw volume after treatment with the tested compound and standard drug.
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32]. The animals were fasted for 24 h before the experiment with free access to water and were treated orally with two equal doses of either indomethacin (10 mg.kg-1 b.w.) or the tested compounds (50 mg.kg-1 b.w.) at 0 and 12 h intervals, except the control group, which received 0.5% w/v CMC. After 17 h of the drug treatment, the rats were sacrificed. The stomach was removed and opened along the greater curvature, washed with distilled water and cleaned gently by dipping in normal saline. The mucosal damage was examined by means of a microscope with a 4 magnifying lens. The mucosal damage in each stomach was assessed according to the following scoring system: [{0.5 for Redness; 1.0 for spot ulcers; 1.5 for hemorrhagic streaks; 2.0 for ulcers more than 3 but less than 5; 3.0 for more than five ulcers}. The mean score of each treated group minus the mean score of control group was regarded as severity index of gastric mucosal damage.] 4.2.5. Lipid Peroxidation Study Lipid peroxidation study was done by the method of Ohkawa et al. [30, 33-34]. After the evaluation of stomach for ulcers, the gastric mucosa of glandular portion was scrapped with the help of two glass slides, weighed (100 mg) and homogenized in 1 mL of a 0.15 M, ice cold potassium chloride (KCl) solution and centrifuged at 3,000 rpm for 10 minutes (R-BC DX REMI centrifuge). 1 mL of suspension was taken from the above tissue homogenate in test tube and 0.5 mL of 30% w/v TCA was added to it, followed by 0.5 mL of 0.8% w/v TBA reagent. The tubes were then covered with aluminium foil and kept in water bath for 30 minutes at 80 °C. After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes and centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was measured in spectrophotometer (UV-1601, SHIMADZU) at 540 nm against blank. Blank consisted of 1 mL distilled water, 0.5 mL of 30% w/v TCA and 0.5 mL of 0.8% w/v TBA. The results were expressed as nM of MDA (malondialdehyde) formed /hr/ mg of protein. 4.2.6. In Vitro Anticancer Activity Primary single high dose (10 M) in vitro assay (SRB assay method). In vitro anticancer activity of the selected compounds was done by NCI-USA at single dose (10 M) against full NCI 60 human cell lines panels in accordance with the standard protocol. The compounds were added at a single concentration (10 M) and the culture was incubated for 48 hours. End point determinations were made with a protein binding dye, sulforhodamine B. The results for each compound were reported as percentage growth (GP %) of the treated cells and compared to the untreated control cells. Only significant results have been reported. Range of growth (%) showed the lowest and the highest growth that was found among different cancer cell lines. For NCI criteria, a compound which reduces the growth of any of the cell line by 32% or less is considered in vitro active [35-36].
4.2.4. Acute Ulcerogenicity Study
CONFLICT OF INTEREST
Potent compounds were subjected to acute ulcerogenecity study by following the method of Cioli et al. [30-
The authors confirm that this article content has no conflict of interest.
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ACKNOWLEDGEMENTS The authors are grateful to Jamia Hamdard for providing infrastructures and other facilities. The author (Ankita Rathore) is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for the award of SRF. SUPPLEMENTARY MATERIALS 1
C NMR spectrum of compound (5h)
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Revised: July 23, 2014
Accepted: July 23, 2014
Corona, P.; Carta, A.; Loriga, M.; Vitale, G.; Paglietti, G. Synthesis and in vitro antitumor activity of new quinoxaline derivatives. Eur. J. Med. Chem., 2009, 44, 1579-91.
Received: March 06, 2014
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