Accepted Manuscript Benzimidazole-Ibuprofen/Mesalamine conjugates: Potential candidates for multifactorial diseases Yogita Bansal, Maninder Kaur, Om Silakari PII:

S0223-5234(14)01017-4

DOI:

10.1016/j.ejmech.2014.10.081

Reference:

EJMECH 7489

To appear in:

European Journal of Medicinal Chemistry

Received Date: 23 May 2014 Revised Date:

22 September 2014

Accepted Date: 29 October 2014

Please cite this article as: Y. Bansal, M. Kaur, O. Silakari, Benzimidazole-Ibuprofen/Mesalamine conjugates: Potential candidates for multifactorial diseases, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.10.081. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Benzimidazole-Ibuprofen/Mesalamine conjugates: Potential candidates for multifactorial diseases Yogita bansal, Maninder Kaur and Om Silakari* Molecular Modelling Lab (MML), Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147002, India.

R

H N

R

N

N

HOOC

O

Benzimidazole (BZ) Immunomodulatory

Ibuprofen

BZ-IB Conjugates

Anti-inflammatory, Ulcerogenic

H N

R

N

2-Aminobenzimidazole (ABZ)

HOOC

NH2

NH2

H N

R

N

HO

HO

Mesalamine

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NH2

ABZ-IB Conjugates

Anti-inflammatory, Immunomodulatory, Less ulcerogenic

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N

R

H N

NH

H N

R

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Graphical abstract

BZ-MES Conjugates

H N

NH2 NH

N O HO

ABZ-MES Conjugates

Conjugates of ibuprofen and mesalamine were designed and synthesized. These exhibited anti-inflammatory and immunostimulatory or immunosuppressive effects. The compounds

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were more selective towards COX-2 enzyme, and therefore less ulcerogenic.

ACCEPTED MANUSCRIPT Benzimidazole-Ibuprofen/Mesalamine conjugates: Potential candidates for multifactorial diseases Yogita Bansal, Maninder Kaur and Om Silakari* Molecular Modelling Lab (MML), Department of Pharmaceutical Sciences and Drug

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*E-mail: [email protected]

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Research, Punjabi University, Patiala - 147002, India.

1

ACCEPTED MANUSCRIPT Abstract Ibuprofen (IB) and mesalamine (MES) are commonly used NSAIDs whereas benzimidazole (BZ)

and

2-aminobenzimidazole

(ABZ)

are

important

pharmacophore

for

immunomodulatory activities. In the present study, IB and MES were coupled with variedly

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substituted BZ or ABZ nucleus to synthesize IB-BZ (2a-2e), IB-ABZ (3a-3e), MES-BZ (4a4e) and MES-ABZ (5a-5e) chimeric conjugates as novel compounds that could elicit both anti-inflammatory and immunomodulatory activities. Each compound retained the anti-

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inflammatory activity of the parent NSAID. The BZ conjugates (2 and 4) were found immunostimulatory whereas the ABZ conjugates (3 and 5) were immunosuppressive. Each

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compound also exhibited good antioxidant activity, which is attributed to the electron rich BZ and ABZ nuclei. Compound 2a, 2e, 3a, 3e and 5b exhibited the most significant antiinflammatory and immunomodulatory activities. Hence, these were evaluated for in vivo acute gastric ulcerogenicity. The compounds were safe to gastric mucosa, probably due to

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masking of the free –COOH group of IB and MES, and/or to the BZ nucleus itself. A benzoyl group at 5-position of BZ and ABZ incurred maximum immunostimulatory activity. In contrast, a –NO2 group incurred the maximum immunosuppressive action. Docking analysis

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revealed the compounds to be more selective towards COX-2 enzyme, which support the

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gastroprotective activity. These results suggest that the compounds can be taken as lead for development of new drugs for the treatment of immune related inflammatory disorders, such as cancer and rheumatoid arthritis.

Keywords:

Benzimidazole,

2-Aminobenzimidazole,

inflammatory, immunomodulatory.

2

Ibuprofen,

Mesalamine,

anti-

ACCEPTED MANUSCRIPT 1. Introduction Multifactorial diseases such as atherosclerosis, Alzheimer’s diseases (AD), metabolic syndrome, rheumatoid arthritis, osteoarthritis, cancer, neurotrauma and multiple sclerosis are highly variable and heterogeneous [1]. Their treatment protocols include the use of drug

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cocktails (independent dosage of the drugs), and combined drugs (fixed combinations of two or more drugs in one dosage form) [2]. But these protocols suffer from the drawbacks of poor patient compliance [3] and possible drug-drug interactions [4]. Therefore, the paradigm has

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shifted to development of multifunctional drug i.e., a single chemical entity having multiple biological activities. Such drugs possess the advantages of having additive or synergistic

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therapeutic responses, and more predictable pharmacokinetics and pharmacodynamic relationships [5,6].

Ibuprofen (IB) is a well known NSAID for the treatment of many inflammatory diseases [7], but its use is accompanied with side effects associated with its non-selectivity

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towards cycloxygenase (COX) enzyme. Mesalamine (MES) is another NSAID like drug used for anti-inflammatory and immunosuppressive actions, especially in ulcerative colitis. It acts through inhibition of IL-1, TNF-α, NF-κB and LOX pathway, and scavenging of free

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radicals. It is not orally bioavilable as such, and hence it is used as derivatives such as

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sulphasalazine, olsalazine and balsalazide that are metabolized in vivo to release MES [7]. On the other hand, imidazole nucleus and guanidine moiety are the common structural components in levamisole, mizoribine, 6-mercaptopurine and methotrexate (Fig. 1) that are clinically

used

immunomodulators.

Imidazole

ring

is

solely

responsible

for

immunostimulatory activity of levamisole [8] whereas guanidine itself is known as a good immunomodulator [9]. Benzimidazole (BZ), the simplest fused imidazole nucleus, is a versatile nucleus as marked by its presence in a wide range of bioactive compounds such as antiparasitic, anticonvulsant, analgesic, antihistaminic, antiulcer, antihypertensive, antiviral, 3

ACCEPTED MANUSCRIPT anticancer, antifungal, anti-inflammatory, antioxidant, immunomodulator and anticoagulant [10]. 2-Aminobenzimidazole (ABZ) is the simplest fused heteroaromatic form of guanidine (Fig. 1), and it is used to develop compounds that exhibit immunosuppressive activity through inhibition of various targets [11-14]. Hence, BZ and ABZ can be exploited as

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important pharmacophore for developing immunomodulatory compounds.

Combination therapy, employing NSAIDs and immunomodulators, is an important therapeutic strategy in treatment of immune system related inflammatory multifactorial

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diseases such as rheumatoid arthritis, ulcerative colitis, multiple sclerosis, osteoarthritis and cancer [15-17]. For instance, the combined use of indomethacin and OK-432 (an

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immunostimulant) reinforces the macrophage-mediated anti-tumour response in advanced cancer cases [18]. Based on these literature reports on importance of NSAIDs, and BZ and ABZ derived immunomodulators in treatment of multifactorial diseases, it is hypothesized that coupling or fusion of pharmacophore from NSAIDs (for anti-inflammatory activity) with

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BZ or ABZ (responsible for immunomodulatory activity) will generate a single hybrid molecule that can exhibit both the activities. Coupling involves connecting two drugs through a stable or metabolisable linker to produce a hybrid drug (HD) whereas fusion involves

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merging or fusing the pharmacophore of two different drugs in one to produce a chimeric

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drug (CD). In comparison to CDs, HDs have higher molecular mass and higher conformational flexibility. These features may be pros in one case and cons in other. Both HDs and CDs are termed as multifunctional compounds (MFCs) that usually augment the potency of both the drugs and/or reduce the dosage [1]. Further, different substituents at 5position of BZ nucleus are known to be responsible for antiulcer, anti-inflammatory and anticancer activities [19-21]. Taking into consideration the importance of IB and MES as potent anti-inflammatory drugs, and of BZ and ABZ nuclei in immunomodulatory drugs, and of substituents at 54

ACCEPTED MANUSCRIPT position of BZ and ABZ for other activities, it is hypothesized that conjugation of BZ or ABZ nucleus, bearing an appropriate functional group at 5-position, with a NSAID (IB and MES) may induce or potentiate immunomodulatory activity in the resultant BZ-NSAID or ABZNSAID conjugate compounds (Fig. 2). These compounds are also expected to be

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gastroprotective or less ulcerogenic due to BZ nucleus, which itself is an important pharmacophore in commonly used antiulcer drugs like omeprazole, pantoprazole, rabeprazole and lansoprazole, and/or due to the masking of free -COOH group of NSAID with BZ.

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Hence, in the present study, IB or MES are fused with BZ or coupled with ABZ nucleus, bearing different substituents at 5-position, to produce different BZ-IB and BZ-MES

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conjugates as chimeric MFCs, and ABZ-IB and ABZ-MES conjugates as hybrid MFCs. These are evaluated for immunomodulatory and anti-inflammatory activities, and for gastric safety through acute ulcerogenic studies. Docking studies of the synthesized conjugate compounds are carried out to investigate the selectivity of the compounds towards COX-1

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and COX-2 enzymes. The experimental findings have supported the proposed hypothesis that fusion of BZ or ABZ with IB and MES not only retains anti-inflammatory activity of the NSAID but also incurs immunomodulatory and antioxidant activities. Moreover, the

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compounds exhibited significantly reduced ulcerogenic potential.

2. Results and Discussion 2.1 Chemistry

Coupling of IB or MES with variedly 4-(un)substituted o-phenylenediamine and 5(un)substituted ABZ yielded the target compounds (2-5) in good yields (Fig. 3). Synthesis of compounds 2 (BZ-IB conjugates) and 3 (BZ-MES conjugates) involved reaction of IB and MES with variedly substituted o-phenylenediamines in the presence of polyphosphoric acid (PPA) using the reaction conditions as reported for 2-susbtituted benzimidazole [22]. 5

ACCEPTED MANUSCRIPT Orthophosphoric acid (OPA) was also used as catalyst but it led to many by-products and longer reaction times. Compounds were characterized through IR, 1H-NMR and mass spectral analyses. In general, IR spectra of compounds 2 and 3 revealed that C=O stretching band (due to –COOH in IB and MES) were absent, which suggested that –COOH group in IB

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and MES was coupled with o-phenylenediamine to form BZ nucleus. Presence of the latter in compounds was supported by appearance of signals due to aromatic and N-H protons of BZ nucleus and absence of signal due to –COOH proton in IB or MES. The intermediates, 5-

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(un)substituted-2-aminobenzimidazole (1a-1e), for synthesis of compounds 4 and 5 were obtained by stirring 4-(un)substituted o-phenylenediamine with cyanogen bromide. While the

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intermediate 1a is a known compound [23], the others (1b-1e) are new compounds. The ABZ conjugates of IB (4, ABZ-IB) and MES (5, ABZ-MES) were synthesized by their coupling with 1a-1e in the presence of dicyclohexyl dicarbodiimide (DCC), which is a high yielding coupling agent for amide/ester formations (Fig. 3). Compounds 4 and 5 were obtained in

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quatitative yields and characterized through comprehensive spectral analysis. A doublet band at 3434 and 3357 cm-1 due to -NH2 group in IR spectra of 1 was converted into a single band at about 3360 cm-1 in IR spectra of 4 and 5, which indicated that -NH2 group in 1 was

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converted into -NH- group in 4 and 5. The IR spectrum of each compound (4 and 5) also

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showed Amide I and Amide II bands at about 1680 and 1575 cm-1, respectively that indicated an amide linkage in the compounds. In addition, the signals due to protons of BZ nucleus in 1 as well as protons of IB or MES were found similarly as in 1H-NMR spectra of 4 or 5, respectively. It suggested that both the coupling moieties (1 and IB or MES) were present in one molecule. The signals due to –CONH and –NH– protons were ascertained through D2O exchange experiments. Structures of all target compounds (2-5) were confirmed by accurate mass spectral data (HRMS), wherein the compounds were detected as [M+H]+ ion at m/z values corresponding to their theoretical accurate masses. 6

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2.2 Pharmacological activity The compounds were evaluated for anti-inflammatory and immunomodulatory activities as well as for gastric safety at a dose of 150 µmol/kg body weight. It was selected on the basis

various benzimidazole derivatives reported in literature [24, 25]. 2.2.1 Anti-inflammatory activity

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of pilot study carried out at various doses, and was found to be almost equal to the dose of

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Anti-inflammatory activity of compounds 2-5, IB and MES was evaluated using carrageenaninduced paw oedema model as reported in literature [26]. The activity was expressed as

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percent inhibition of carrageenan induced paw oedema with respect to control (Table 1). Each compound exhibited the activity in the range of 40 – 65%, which was equal to or higher than that of the parent NSAID. Peak anti-inflammatory response was noted at 60 min. The activity of each compound was attributed to the structural features, which are contributed by

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NSAIDs, in the compound. It suggested that conjugation of IB or MES with BZ as well as ABZ retains or marginally increase their anti-inflammatory activity. Amongst the series, the 5-nitro substituted conjugates (2b-5b) were the least active whereas the 5-benzoyl conjugates

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(2e-5e) were maximally active. These findings are in consonant with the results reported by

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Lo et al [14], which suggest that a bulky lipophillic group at 5-position of BZ may be responsible for favourable binding interactions with targets responsible for inflammation. 2.2.2 Immunomodulatory activity IB is a weak Immunostimulant, and MES is a moderate immunosuppressant [7,27]. Immunomodulatory activity of each compound was evaluated in terms of humoral immunity measured as humoral antibody titre (HAT), and macrophage phagocytic activity (MPA) of reticularendothelial system measured as carbon clearance index (K), taking levamisole as standard drug. The HAT and K values for each compound were significantly different from 7

ACCEPTED MANUSCRIPT the control values (Table 2), which indicated the compounds to be good immunomodulators. Both HAT and K was increased to greater extent by IB-BZ conjugates (2; HAT 19.2 – 61.5% and K 29.4 – >100%) than by IB-ABZ conjuagtes (4; HAT 15.3 – 42.3% and K 23.5 – 76.4%). It indicated that immunostimulatory activity of IB was potentiated to greater extent

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by its conjugation with BZ than with ABZ. On the other hand, conjugation of MES with BZ produced immunostimulatory compounds (3, increase in HAT and K with respect to control) whereas that with ABZ produced immunosuppressive compounds (5, decrease in HAT and K

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with respect to control). It indicated that immunosupressive action of MES is retained or potentiated by its conjugation with ABZ, but it is converted into immunostimulatory action

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upon conjugation with BZ. Further, 5-benzoyl substituted derivatives (2e-4e) caused maximum gain in HAT (2e, +61.5%; 3e, +69.2%; 4e, +42.3% and 5e, -26.9%). On the contrary, 5-nitro substituted derivatives caused minimum gain or maximum decrease in HAT (2b, +19.2%; 3b, +23.0%; 4b, +15.3%; 5b, -50%). The changes in K value shown by each

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compound were similar to that in HAT values (Table 2), which indicated that immunomodulatory activity exhibited by each compound in both the models (K and HAT) is is well agreement .

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2.2.3 Acute ulcerogenicity test

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Compounds 2a, 2e, 3a, 3e and 5b exhibited significant anti-inflammatory and immunomodulatory activities, and hence their gastric safety was evaluated in terms of acute ulcerogenic effect and in vivo oxidative stress (Table 3). The ulcer indexes of these compounds were significantly less than that of indomethacin indicating the compounds to be less ulcer-inducing than indomethacin. This decreased ulcerogenicity of the compounds might be attributed to BZ itself and/or masking of the free –COOH in IB and MES. Safety of the compounds was also estimated through monitoring of oxidative stress induced markers such as reduced glutathione (GSH) levels, catalase (CAT) activity and lipid peroxidation in 8

ACCEPTED MANUSCRIPT terms of thiobarbituric acid reactive substances (TBARS) levels (Table 3). CAT and GSH levels were increased in animals treated with compounds, which suggested the compounds to be good antioxidants and less gastro-toxic. TBARS levels were significantly decreased as

effects against lipid peroxidation-induced gastric damage. 2.3. Structure activity relationship

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compared to indomethacin, which also revealed that these compounds exhibit protective

A subtle increase in anti-inflammatory activity of the target compounds vis-a-vis IB and MES

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was attributed to BZ nucleus. However, it was independent of the linker between BZ and the NSAID. It was also found independent of electronic nature of substituent at 5-position of BZ

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or ABZ. In comparison to 5-unsubstituted derivatives (2a – 5a), nitro substituted derivatives (2b – 5b) decreased the activity whereas methyl and methoxy substituted derivatives (2c – 5c and 2d – 5d) retained or exhibited an insignificantly increased activity. Increased activity of benzoyl group containing compounds (2e – 5e) revealed that presence of bulky lipophillic at

5-position

potentiates

the

activity.

Unlike

anti-inflammatory activity,

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groups

immunomodulatory activity of the compounds was found different for BZ and ABZ derivatives. While BZ potentiates or incurs stimulatory action, ABZ induces either a subtle

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stimulatory or significant inhibitory actions on the immune system. These findings are in

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agreement with the proposed hypothesis, and suggested that BZ and ABZ may be responsible for interaction with immunostimulatory and immunosupressive targets, respectively. A comparison of percent change in HAT, and K vaules by different derivatives has revealed that at 5-position, in comparison to a hydrogen atom, a nitro group, probably, decreases binding interactions with targets responsible for stimulation and/or increases the interactions with those for inhibition of immune system. In contrast, a benzoyl group has marginally increased the immunostimulation, probably, due to H-bonding interactions (C=O being Hbond acceptor) as well as some lipophillic interactions between the bulky phenyl ring and the 9

ACCEPTED MANUSCRIPT targets. The -CH3 and –OCH3 groups have marginally decreased the immunostimulation which suggest that an electron releasing group may not be favorable for stimulant actions on immune system. Based on these results, it is proposed that a small electron withdrawing substituent such as –NO2, -CN or halogen at 5-position of BZ or ABZ nucleus may incur

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immunohibitory activity. In contrast, a large bulky group capable of exhibiting H-bonding as well as lipophilic interactions such as (un)substituted aryl or heteroaryl moiety linked through H-bond acceptor linker such as -C(O)-, -C(S)-, -CO-NH-, -C(O)-O-, -S(O)-, and others at the

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5-position may increase immunostimulatory effects of the compounds. 2.4. In silico docking studies

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NSAIDs interact with COX-1 active site through strong ionic interaction between its free – COOH group and Arg120 residue in the enzyme active site. This interaction is essential for COX-1 inhibition and also responsible for ulcerogenic potential of the NSAIDs [28]. In the present study, molecular docking studies for the target compounds with cycloxygenase

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enzyme (COX-1 and COX-2) were carried out to reveal their COX inhibitory profile, which is an indicative of anti-inflammatory activity as well as gastric safety. It was found that none of the compounds interacted with COX-1 through Arg120 residue, and this finding supported

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the in vivo gastroprotective effect of the compounds. The selected compounds were different

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from the crystal ligand of the selected COX-2 protein (3NT1). It implicated that the active site environment would be different, although even slightly, for the compounds as compared in 3NT1. Therefore, conformational sampling was induced in the active site of COX-2, with respect to the maximally active compound (2e), using induced fit docking (IFD) protocol. In contrast to observation in COX-1 docking analysis, compounds showed significant COX-2 inhibition. The binding interactions displayed by 2a, 2e, 3a, 3e and 5b (the most active compounds from the series) with COX-1 (Fig. 4) and with COX-2 (Fig. 5) are summarised in Table 4. A comparison of binding interactions with COX-1 as well as COX-2 revealed that 10

ACCEPTED MANUSCRIPT the compounds are more selective towards COX-2, and are gastroprotective. 4. Conclusions The pharmacological activity profiles of all compounds conjugate compounds of IB and MES have supported the proposed hypothesis that fusion of BZ or ABZ with IB and MES not only

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retains anti-inflammatory activity of the NSAID but also incurs immunomodulatory activity. Benzoyl group at 5-position of BZ or ABZ nucleus increases the immunostimulatory activity whereas the other substituents decrease it. Compounds 2a, 2e, 3a and 3e, exhibiting

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pronounced immunostimulatory and anti-inflammatory effects, may be of interest in anticancer immunotherapy wherein combination of NSAIDs and immunostimulants has

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already been explored [29]. Compound 5b, showing maximum immunosuppressive response, may be a potential candidate for treatment of multifactorial disorders such as rheumatoid arthritis, osteoarthritis, ulcerative colitis and other autoimmune disorders. The compounds have more displayed selectivity towards COX-2 enzyme in the docking analysis. Their

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binding with COX-1 is not strong due to lack of ionic interaction involving Arg120 residue in the active site. This lack of strong interaction is accounted for the safe gastric profile of the compounds. Further detailed investigations to establish underlying mechanisms involved in

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alteration of immune system by these conjugate compounds will help in delineating the

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therapeutic utility of these molecules in specific immune related disorders. 5. Experimental

IB and MES were procured as generous gift samples from Crystal Pharmaceutical (Ambala, India) and Ranbaxy Research Laboratories (Gurgaon, India), respectively. Drugs were authenticated through melting point determination and IR spectral analyses. The solvents and reagents were dried prior to use, when required, over KOH or anhydrous Na2SO4 or fused CaCl2. All chemicals were obtained from Sigma Aldrich, S.D. fine or Merck chemicals. The chemical reactions were monitored by thin layer chromatography (TLC) using precoated 11

ACCEPTED MANUSCRIPT aluminium plates (Merck, Mumbai, India) visualized in UV chamber at short as well as long wavelengths. Silica gel (100-200 mesh) (Merck, Mumbai, India) was used for purification of the compounds by column chromatography. The melting points were recorded in open sulfuric acid bath and uncorrected. FT-IR spectra were recorded using KBr pellet on FT-IR

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Perkin-Elmer 1710 series. 1H-NMR and 13C-NMR spectra were recorded on Bruker AC 300 NMR spectrophotometer (400 MHz). Chemical shifts were reported in δ values using tetramethylsilane as internal standard with multiplicities (br, broad; s, singlet; d, doublet; t,

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triplet; q, quartet; qv, quintet; sx, sextet; sp, septet; m, multiplet; dd, double doublet) and number of protons in the solvent specified. The coupling constants (J) were expressed in Hz.

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High resolution mass spectral (HRMS) studies were carried out in electrospray ionization (+ESI) mode on a Bruker Daltonics microTOF instrument (Bruker Daltonik GmbH, Bremen, Germany), which was controlled by microTOF control software ver. 2.0. Elementary composition of each compound was confirmed through calculation of molecular formula

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corresponding to accurate mass of its [M+H]+ ion using Elemental Composition Calculator. The calculated molecular formula of [M+H]+ ion of each compound were found to be same as the actual one.

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5.1 Chemistry: General Procedures

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5.1.1 Synthesis of 2-Aminobenzimidazoles intermediates (1a – 1e) 2-Aminobenzimidazole (1a) is a known compound [23], and its synthetic method was employed to synthesize its 5-substituted analogs. The reaction conditions for each analog were optimized to obtain the products in maximum yields. In brief, solutions of cyanogen bromide (0.03 mol), and 4-(un)substituted-o-phenylenediamine (0.02 mol) prepared separately each in 35 mL of aqueous methanol (50% v/v), were mixed in a 250 mL conical flask and stirred at room temperature for 24-48 h. Methanol was distilled in vacuum, the solution was cooled to room temperature and made alkaline with aqueous ammonia. The 12

ACCEPTED MANUSCRIPT product was precipitated and recrystallized from ethanol-water mixture. For synthesis of 2amino-5-methoxy

benzimidazole

(1d),

4-methoxyphenylenediamine

dihydrochloride,

neutralised initially with equimolar quantities of Na2CO3, was used. 2-Aminobenzimidazole (1a).

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Yield 88%; mp 232 ˚C (mpLit 226-230˚C); IR (KBr): 3434, 3357 (N-H str of NH2 group), 3116 (C-H Ar. str), 1636 (C=N str), 1451 (C=C str); 1H-NMR (CDCl3): δ 7.22 (2H, dd, J =

calculated for C7H8N3+: 134.0640, found: 134.0653. 2-Amino-5-nitrobenzimidazole (1b).

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6;2), 6.91 (2H, dd, J = 6;2), 5.44 (2H, br, s), 4.83 (1H, br, s); HRMS (+ESI) m/z: [M+H]+

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Yield 87%; mp 134 ˚C; IR (KBr): 3514, 3456 (N-H str of NH2), 3092 (C-H Ar. str), 1659, 1586, 1504 (skeletal bands), 1470 (N-H scissoring), 876 (N-H wagging); 1H-NMR (CDCl3): 8.07 (1H, br, s), 7.99-7.98 (1H, m), 7.89-7.86 (1H, m), 7.18-7.16 ( 1H, d, J = 6), 6.72 (2H, br, s); HRMS (+ESI) m/z: [M+H]+ calculated for C7H7N4O2+: 179.0491, found: 179.0582.

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2-Amino-5-methylbenzimidazole (1c).

Yield 78%; mp 198 ˚C; IR (KBr): 3415, 3313 (N-H str of NH2), 3130 (C-H Ar. str), 1650, 1562 (skeletal bands), 1464 (N-H scissoring), 851 (N-H wagging); 1H-NMR (CDCl3): 7.84

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(1H, br, s), 7.00-6.98 (1H, m), 6.95 (1H, s), 6.68 (1H, dd, J = 8;1), 6.13 (2H, br, s), 2.35 (3H,

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s); HRMS (+ESI) m/z: [M+H]+ calculated for C8H10N3+: 148.0796, found: 148.0815. 2-Amino-5-methoxybenzimidazole (1d). Yield 65%; mp 168 ˚C; IR (KBr): 3425, 3303 (N-H str of NH2), 3026 (C-H Ar. str), 1642, 1562 (skeletal bands), 1462 (N-H scissoring), 851 (N-H wagging); 1H-NMR (CDCl3): 7.26 (1H, d, J = 8), 6.94 (1H, d, J = 2), 6.77 (2H, br, s), 6.65 (1H, dd, J = 8;2), 3.75 (3H, s); HRMS (+ESI) m/z: [M+H]+ calculated for C8H10N3O+: 164.0746, found: 164.0783. 2-Amino-5-benzoylbenzimidazole (1e). Yield 84%; mp 196 ˚C; IR (KBr): 3415, 3313 (N-H str of NH2), 3130 (C-H Ar. str), 1650, 13

ACCEPTED MANUSCRIPT 1562 (skeletal bands), 1464 (N-H scissoring), 851 (N-H wagging); 1H-NMR (CDCl3): δ 7.697.67 (2H, m), 7.59-7.57 (2H, m), 7.51-7.49 (2H, m), 7.43-7.41 (1H, m), 7.19-7.17 (1H, d, J = 8), 6.54 (2H, br, s); HRMS (+ESI) m/z: [M+H]+ calculated for C14H12N3O+: 238.0902, found:

5.1.2 General procedure for synthesis of compounds 2 and 3

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238.1002.

Compounds 2a-2e and 3a-3e were synthesized through modifications carried out in the method reported in literature [22]. 4-Substituted o-phenylenediamine (1 mol) was refluxed

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with IB and MES (1.5 mol) for compounds 2 and 3, respectively, in the presence of PPA (1g/mmol of the drug) under nitrogen. Reaction for all compounds completed within 30-60

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min. The reaction mixture was poured into cold water and basified with aqueous ammonia. The product was filtered at pump, washed with cold water and recrystallized from hot aqueous ethanol to obtain the product as fine crystals/powder, which was dried in vacuum desiccators. The %yield and melting points were recorded, and structures were elucidated

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through spectral analyses.

2-[α-Methyl-4-(2-methylpropyl)benzyl]benzimidazole (2a). Yield: 72%; mp 184 ˚C; IR (KBr): 3051 (C-H Ar. str), 2954 (C-H Ali. str), 1620 (C=N str), 1591, 1562, 1424 (skeletal

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bands), 1318 (C-H Ali. bend); 1H-NMR (CDCl3): δ 7.52 (2H, d, J = 8), 7.20 (4H, m), 7.14 (2H, d, J = 8), 4.31 (1H, q, J = 7), 2.54 (2H, d, J = 7), 1.83 (4H, m), 0.91 (6H, d, J = 6.6); C-NMR (DMSO-d6): δ 146.1, 141.3, 139.3, 138.6, 130.2, 128.4, 126.5, 119.3, 48.9, 43.5,

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13

34.3, 26.4, 23.8; HRMS (+ESI) m/z: [M+H]+ calculated for C19H22N2+: 279.1861, found: 279.1804.

2-[α-Methyl-4-(2-methylpropyl)benzyl]-5-nitrobenzimidazole (2b). Yield: 52%; mp 283-285 ˚C; IR (KBr): 3094 (C-H Ar. str), 2948 (C-H Ali. str), 1638 (C=N str), 1586, 1506, 1428 (skeletal bands), 1464, 1382 (N=O str.); 1H-NMR (CDCl3): δ 7.97 (1H, d, J = 2), 7.45 (1H, dd, J = 7;2), 7.41 (1H, d, J = 7), 7.19 (2H, d, J = 8), 7.07 (2H, d, J = 8), 4.75 (1H, br, s), 3.60 14

ACCEPTED MANUSCRIPT (1H, q, J = 7), 2.42 (2H, d, J = 7), 1.83 (1H, sp, J = 7), 1.41 (3H, d, J = 7), 0.88 (6H, d, J = 7);

13

C-NMR (DMSO-d6): δ 146.7, 141.5, 139.6, 138.9, 135.8, 131.4, 130.6, 128.7, 125.9,

120.4, 119.7, 48.9, 43.6, 34.5, 26.5, 24.1, 22.3; HRMS (+ESI) m/z: [M+H]+ calculated for C19H22N3O2+: 324.1712, found: 324.1773.

RI PT

2-[α-Methyl-4-(2-methylpropyl)benzyl]-5-methylbenzimidazole (2c). Yield: 78%; mp 156 ˚C; IR (KBr): 3061 (C-H Ar. str), 2914 (C-H Ali. str), 1624 (C=N str), 1572, 1512, 1443 (skeletal bands); 1H-NMR (CDCl3): δ 7.18 (2H, d), 7.07 (2H, d), 6.88 (1H, d, J = 8), 6.77 (1H, s), 6.59

SC

(1H, d, J = 8), 3.64 (1H, q, J = 7), 2.42 (2H, d, J = 7), 2.17 (3H, s), 1.82 (1H, sp, J = 7), 1.40 (3H, d, J = 7), 0.88 (6H, d, J=7); 13C-NMR (DMSO-d6): δ 149.6, 145.8, 147.2, 142.4, 140.7,

M AN U

138.2, 129.8, 128.1, 124.8, 121.7, 117.6, 48.5, 43.3, 33.9, 26.1, 23.8; HRMS (+ESI) m/z: [M+H]+ calculated for C20H25N2+: 293.2018, found: 293.2095.

2-[α-Methyl-4-(2-methylpropyl)benzyl]-5-methoxybenzimidazole (2d). Yield: 67%; mp 172173 ˚C; IR (KBr): 3052 (C-H Ar. str), 2925 (C-H Ali. str), 1632 (C=N str), 1565, 1502, 1423

TE D

(skeletal bands); 1H-NMR (CDCl3): δ 7.24 (3H, m), 7.04 (3H, m), 6.69 (1H, dd, J = 9;2), 3.81 (3H, s), 3.62 (1H, q, J = 7), 2.46 (2H, d, J = 7), 1.87 (1H, sp, J = 7), 1.51 (3H, d, J = 7), 0.90 (6H, d, J = 7);

13

C-NMR (DMSO-d6): δ 160.5, 146.2, 142.3, 141.2, 138.4, 130.5, 131.6,

EP

128.6, 119.6, 110.7, 103.8, 58.2, 48.6, 43.2, 34.4, 26.1, 24.4; HRMS (+ESI) m/z: [M+H]+

AC C

calculated for C20H25N2O+: 309.1967, found: 309.2051. 2-[α-Methyl-4-(2-methylpropyl)benzoyl]-5-benzoylbenzimidazole (2e). Yield: 73%; mp 202203 ˚C; IR (KBr): 3055 (C-H Ar. str), 2914 (C-H Ali. str), 1672 (C=O str), 1647 (C=N str), 1565, 1502, 1423 (skeletal bands); 1H-NMR (CDCl3): δ 7.69-7.67 (2H, m), 7.60-7.56 (2H, m), 7.52-7.48 (2H, m), 7.42 (1H, dd, J = 8;2), 7.19-7.16 (3H, m), 7.06 (2H, d, J = 8), 3.58 (1H, q, J = 7), 2.38 (2H, d, J = 7), 1.78 (1H, sp, J = 7), 1.46 (3H, d, J = 7), 0.86 (6H, d, J = 7);

13

C-NMR (DMSO-d6): δ 193.8, 146.4, 145.9, 141.7, 139.1, 138.2, 137.7, 134.9, 134.1,

132.5, 131.1, 129.7, 128.5, 126.3, 120.8, 118.5, 48.3, 42.8, 33.9, 26.2, 24.2; HRMS (+ESI) 15

ACCEPTED MANUSCRIPT m/z: [M+H]+ calculated for C26H27N2O+: 383.2123, found: 383.2141. 2-[(5-Amino-2-hydroxy)phenyl]benzimidazole (3a). Yield 67%; mp 215 ˚C; IR (KBr): 36003200 (br N-H, O-H str), 3083 (C-H Ar. str), 2851 (C-H Ali. str), 1624 (C=N str); 1H-NMR (DMSO-d6): δ 7.62 (2H, dd, J = 6;3), 7.52 (1H, s), 7.28 (2H, dd, J = 6;3), 6.92 (2H, m), 2.14 13

C-NMR (DMSO-d6): δ 155.6, 148.1, 146.2, 140.6, 129.7, 126.4, 120.5, 119.3,

RI PT

(4H, s);

117.7, 116.4; HRMS (+ESI) m/z: [M+H]+ calculated for C13H12N3O+: 226.0980, found: 226.0956.

SC

2-[(5-Amino-2-hydroxy)phenyl]-5-nitrobenzimidazole (3b). Yield 42%; mp 285-286 ˚C; IR (KBr): 3600-3200 (br N-H, O-H str), 3063 (C-H Ar. str), 2842 (C-H Ali. str), 1614 (C=N str),

M AN U

1457, 1372 (N=O str.); 1H-NMR (DMSO-d6): δ 9.26 (1H, br, s), 8.71 (1H, d, J = 2), 8.22 (1H, dd, J = 8;2), 8.04 (1H, d, J = 8), 7.63 (1H, s), 7.05 (2H, m), 2.18 (2H, br, s);

13

C-NMR

(DMSO-d6): δ 155.2, 150.3, 148.4, 147.8, 146.5, 143.6, 129.9, 123.1, 120.6, 119.1, 117.3, 115.2; HRMS (+ESI) m/z: [M+H]+ calculated for C13H11N4O3+: 271.0831, found: 271.0901. mp 217 ˚C; IR

TE D

2-[(5-Amino-2-hydroxy)phenyl]-5-methylbenzimidazole (3c). Yield 54%;

(KBr): 3600-3200 (br N-H, O-H str), 3071 (C-H Ar. str), 2943 (C-H Ali. str), 1632 (C=N str); 1

H-NMR (DMSO-d6): δ 7.69 (1H, d, J = 7), 7.61 (1H, s), 7.56 (1H, s), 7.14-7.07 (3H, m),

EP

6.09 (1H, br, s), 2.75 (2H, br, s), 2.35 (3H, s); 13C-NMR (DMSO-d6): δ 155.6, 148.1, 146.2, 143.1, 140.1, 136.9, 129.7, 128.3, 120.7, 120.5, 119.2, 117.7, 25.4; HRMS (+ESI) m/z:

AC C

[M+H]+ calculated for C14H14N3O+: 240.1137, found: 240.1164. 2-[(5-Amino-2-hydroxy)phenyl]-5-methoxybenzimidazole (3d). Yield 43%; mp 227-228 ˚C; IR (KBr): 3600-3200 (br N-H, O-H str), 3028 (C-H Ar. str), 2941 (C-H Ali. str), 1624 (C=N str); 1H-NMR (DMSO-d6): δ 7.67-7.60 (2H, m), 7.15-7.11 (3H, m), 6.62 (1H, d, J=7), 5.43 (1H, br, s), 3.73 (3H, s), 2.36 (2H, br, s); 13C-NMR (DMSO-d6): δ 161.4, 155.4, 147.7, 146.1, 143.4, 135.8, 129.4, 122.3, 120.3, 119.1, 117.2, 114.1, 107.2, 62.3; HRMS (+ESI) m/z: [M+H]+ calculated for C14H14N3O2+: 256.1086, found: 256.1114. 16

ACCEPTED MANUSCRIPT 2-[(5-Amino-2-hydroxy)phenyl]-5-benzoylbenzimidazole (3e). Yield 48%; mp 234-235 ˚C; IR (KBr): 3600-3200 (br N-H, O-H str), 3030 (C-H Ar. str), 2911 (C-H Ali. str), 1664 (C=O str), 1630 (C=N str); 1H-NMR (DMSO-d6): δ 9.14 (1H, br, s), 8.09 (1H, s), 7.86 (1H, d, J = 7), 7.78-7.71 (3H, m), 7.63-7.56 (3H, m), 7.27-7.18 (3H, m), 2.93 (2H, br, s);

13

C-NMR

RI PT

(DMSO-d6): δ 195.1, 156.1, 148.5, 147.8, 146.7, 143.7, 143.2, 137.6, 136.7, 135.3, 132.7, 130.2, 129.8, 122.5, 120.9, 120.2, 119.5, 117.5; HRMS (+ESI) m/z: [M+H]+ calculated for C20H16N3O2+: 330.1243, found: 330.1308.

SC

5.1.3 General procedure for synthesis of compounds 4 and 5

DCC (2.06 g, 0.1 mol) was added to a solution of IB or MES (0.1 mol) in dichloromethane

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(10 mL). The contents were stirred for 30 minutes followed by addition of a solution of 1 (0.1 mol) in dichloromethane (10 mL), and pyridine (5 mL). The reaction mixture was stirred at 0 ˚C for the first 2 h followed by stirring at room temperature overnight. The precipitated dicyclohexyurea (DHU) was filtered and the solvent was removed in vacuum. Ethyl acetate

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(10 mL) was added to the residue in order to separate the product from residual DHU. The ethyl acetate solution was filtered, washed with 10% aqueous solution of Na2CO3 followed by distilled water, and then dried over MgSO4 (anhydrous). The solvent was recovered in

EP

vacuum to obtain crude product, which was purified by gradient column chromatography

AC C

using chloroform and methanol as mobile phase to afford the products as amorphous powder. 2-[α-Methyl-4-(2-methylpropyl)phenylacetylamino]benzimidazole (4a). Yield 65%; mp 167 ˚C; IR (KBr): 3361 (N-H str), 3066 (C-H Ar. str), 2922 (C-H Ali. str), 1680 (Amide I), 1576 (Amide II); 1H-NMR (CDCl3): δ 7.38 (2H, dd, J = 7;3), 7.15 (2H, dd, J = 7;3), 6.84-7.12 (4H, m), 6.23 (1H, br, s), 3.94 (1H, q, J = 7), 2.37 (2H, d, J = 7), 1.73 (1H, m), 1.57 (3H, d, J = 7), 0.82 (6H, d, J = 7); 13C-NMR (DMSO-d6): δ 176.4, 144.8, 142.3, 139.6, 134.5, 131.7, 129.6, 125.1, 118.4, 47.5, 43.8, 32.7, 24.8, 18.3; HRMS (+ESI) m/z: [M+H]+ calculated for C20H24N3O+: 322.1919, found: 322.1938. 17

ACCEPTED MANUSCRIPT 2-[α-Methyl-4-(2-methylpropyl)phenylacetylamino]-5-nitrobenzimidazole (4b). Yield 63%; mp 195-196 ˚C; IR (KBr): 3268 (N-H str), 3050 (C-H Ar. str), 2951 (C-H Ali. str), 1644 (Amide I), 1578 (Amide II) ), 1471, 1379 (N=O str.); 1H-NMR (DMSO-d6): δ 8.34 (1H, d, J = 2), 8.14 (1H, dd, J = 8;2), 7.31 (1H, d, J = 8), 7.26-7.17 (2H, m), 7.15-7.08 (2H, m), 6.53

RI PT

(1H, br, s), 5.42 (1H, br, s), 3.72 (1H, q, J = 7), 2.45 (2H, d, J = 7), 1.85 (1H, sp, J = 7), 1.52 (3H, d, J = 7), 0.87 (6H, d, J=7); 13C-NMR (DMSO-d6): δ 175.7, 147.8, 144.7, 143.7, 141.4, 140.2, 134.1, 130.9, 128.7, 120.6, 112.5, 46.3, 43.2, 32.1, 24.3, 17.7; HRMS (+ESI) m/z:

SC

[M+H]+ calculated for C20H23N4O3+: 367.4218, found: 367.4302.

2-[α-Methyl-4-(2-methylpropyl)phenylacetylamino]-5-methylbenzimidazole (4c). Yield 69%;

M AN U

mp 174 ˚C; IR (KBr): 3345 (N-H str), 3052 (C-H Ar. str), 2911 (C-H Ali. str), 1640 (Amide I), 1568 (Amide II); 1H-NMR (DMSO-d6): δ 7.26 (2H, d, J = 8), 7.14 (2H, d, J = 8), 6.91 (1H, d, J = 8), 6.81 (1H, s), 6.64 (1H, d, J = 8), 3.64 (1H, q, J = 7), 2.47 (2H, d, J = 7), 2.28 (3H, s), 1.87 (1H, sp, J = 7), 1.43 (3H, d, J = 7), 0.91 (6H, d, J = 7); 13C-NMR (DMSO-d6): δ

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176.1, 145.2, 141.8, 139.5, 137.7, 134.3, 133.9, 131.4, 129.2, 126.7, 119.4, 118.2, 46.9, 43.5, 32.3, 24.4, 22.1, 17.9; HRMS (+ESI) m/z: [M+H]+ calculated for C21H26N3O+: 326.2076, found: 326.2016.

EP

2-[α-Methyl-4-(2-methylpropyl)phenylacetylamino]-5-methoxybenzimidazole

(4d).

Yield

48%; mp 184 ˚C; IR (KBr): 3315 (N-H str), 3026 (C-H Ar. str), 2841 (C-H Ali. str), 1636

AC C

(Amide I), 1585 (Amide II); 1H-NMR (DMSO-d6): δ 7.30 (3H, m), 7.16-7.11 (3H, m), 6.81 (1H, dd, J = 8;2), 6.72 (1H, br, s), 4.84 (1H, br, s), 3.92 (3H, s), 3.71 (1H, q, J = 7), 2.48 (2H, d, J = 7), 1.83 (1H, sp, J = 7), 1.42 (3H, d, J = 7), 0.86 (6H, d, J = 7); 13C-NMR (DMSO-d6): δ 175.9, 160.3, 145.0, 141.6, 140.4, 134.5, 132.4, 131.2, 129.3, 119.2, 112.5, 104.7, 59.7, 46.8, 43.3, 32.4, 24.1, 17.6; HRMS (+ESI) m/z: [M+H]+ calculated for C21H26N3O2+: 352.2025, found: 352.2125. 2-[α-Methyl-4-(2-methylpropyl)phenylacetylamino]-5-benzoylbenzimidazole 18

(4e).

Yield

ACCEPTED MANUSCRIPT 55%; mp 206-207 ˚C; IR (KBr): 3242 (N-H str), 3094 (C-H Ar. str), 2905 (C-H Ali. str), 1670 (C=O str), 1636 (Amide I), 1564 (Amide II); 1H-NMR (DMSO-d6): δ 7.81-7.78 (2H, m), 7.65-7.52 (4H, m), 7.38 (1H, d, J = 8), 7.27-7.24 (1H, m), 7.20-7.17 (2H, d, , J = 8), 7.097.06 (2H, d, J = 8,2), 6.49 (1H, br, s), 5.14 (1H, br, s), 3.54 (1H, q, J = 7), 2.43 (2H, d, J = 7),

RI PT

1.75 (1H, sp, J = 7), 1.51 (3H, d, J = 7), 0.90 (6H, d, J = 7); 13C-NMR (DMSO-d6): δ 191.4, 176.6, 145.6, 145.3, 141.9, 139.4, 138.9, 134.8, 134.5, 133.9, 132.6, 131.5, 129.8, 129.5, 127.2, 119.6, 117.4, 47.1, 43.7, 32.2, 24.5, 18.1; HRMS (+ESI) m/z: [M+H]+ calculated for

SC

C27H28N3O2+: 426.2182, found: 426.2201.

2-[(5-Amino-2-hydroxy)benzoylamino]benzimidazole (5a). Yield 52%; mp 148-150 ˚C; IR

M AN U

(KBr): 3420-2600 (br N-H, O-H str), 3073 (C-H Ar. str), 1675 (Amide I), 1558 (Amide II), 1634 (C=N str), 1312 (C-O str), 1208 (C-N str); 1H-NMR (DMSO-d6): 10.72 (1H, br, s), 7.73 (3H, m), 7.08 (4H, m), 2.70 (4H, br, s); 13C-NMR (DMSO-d6): δ 171.4, 153.6, 146.5, 144.7, 141.3, 125.4, 123.6, 122.1, 119.7, 118.5, 116.2; HRMS (+ESI) m/z: [M+H]+ calculated for

TE D

C14H13N4O2+: 269.1039, found: 269.1051.

2-[(5-Amino-2-hydroxy)benzoylamino]-5-nitrobenzimidazole (5b). Yield 42%; mp 172-174 ˚C; IR (KBr): 3380-2500 (br N-H, O-H str), 3081 (C-H Ar. str) 1652 (Amide I), 1562 (Amide

EP

II), 1477, 1374 (N=O str.); 1H-NMR (DMSO-d6): δ 10.16 (2H, br, s), 8.59 (1H, s), 8.17 (1H, d, J = 8), 7.92 (1H, d, J = 8), 7.54 (1H, s), 7.13 (2H, m), 2.87 (2H, br, s); 13C-NMR (DMSO-

AC C

d6): δ 171.1, 153.6, 148.6, 147.2, 146.5, 145.6, 142.5, 124.7, 122.3, 121.7, 120.5, 119.4, 117.6, 113.9; HRMS (+ESI) m/z: [M+H]+ calculated for C14H12N5O4+: 314.0889, found: 314.1006.

2-[(5-Amino-2-hydroxy)benzoylamino]-5-methylbenzimidazole (5c). Yield 55%; mp 148 ˚C; IR (KBr): 3400-2600 (br N-H, O-H str), 3073 (C-H Ar. str), 2953 (C-H Ali. str), 1664 (Amide I), 1558 (Amide II); 1H-NMR (DMSO-d6): δ 10.57 (1H, br, s), 8.18 (1H, br, s), 7.78 (2H, m), 7.64 (1H, s), 7.24-7.16 (3H, m), 2.69 (2H, br, s), 2.37 (3H, s); 19

13

C-NMR (DMSO-

ACCEPTED MANUSCRIPT d6): δ 171.7, 154.1, 146.9, 145.2, 141.6, 139.2, 136.2, 127.4, 124.2, 122.7, 120.2, 119.5, 118.3, 117.1, 24.7; HRMS (+ESI) m/z: [M+H]+ calculated for C15H15N4O2+: 283.1195, found: 283.1254. 2-[(5-Amino-2-hydroxy)benzoylamino]-5-methoxybenzimidazole (5d). Yield 51%; mp 158

RI PT

˚C; IR (KBr): 3350-2600 (br N-H, O-H str), 3073 (C-H Ar. str) 1668 (Amide I), 1543 (Amide II); 1H-NMR (DMSO-d6): δ 9.94 (1H, br, s), 7.74-7.69 (2H, m), 7.28-7.21 (3H, m), 6.73 (1H, d, J=7), 5.8 (1H, br, s), 3.83 (3H, s), 2.86 (2H, br, s); 13C-NMR (DMSO-d6): δ 170.6, 153.7,

SC

159.6, 146.2, 144.8, 142.3, 134.6, 123.9, 122.1, 120.7, 119.7, 116.8, 112.2, 105.7, 60.4; HRMS (+ESI) m/z: [M+H]+ calculated for C15H15N4O3+: 299.1144, found: 299.1202.

M AN U

2-[(5-Amino-2-hydroxy)benzoylamino]-5-benzoylbenzimidazole (5e). Yield 43%; mp 183185 ˚C; IR (KBr): 3420-2540 (br N-H, O-H str), 3081 (C-H Ar. str), 1680 (C=O str), 1662 (Amide I), 1548 (Amide II); 1H-NMR (DMSO-d6): δ 10.83 (1H, br, s), 8.17 (1H, s), 7.82 (1H, d, J=7), 7.72-7.65 (3H, m), 7.58-7.51 (3H, m), 7.38 (1H, m), 7.19-7.13 (2H, m), 6.2 (1H, br,

TE D

s), 2.93 (2H, br, s); 13C-NMR (DMSO-d6): δ 192.5, 172.2, 154.7, 147.1, 146.9, 145.7, 141.6, 141.2, 135.1, 134.8, 133.7, 131.5, 127.5, 124.5, 122.4, 120.6, 120.3, 118.7, 117.2; HRMS (+ESI) m/z: [M+H]+ calculated for C21H17N4O3+: 373.1301, found: 373.1354.

EP

5.2 Pharmacological evaluation

AC C

Wistar rats (150-200 g) and albino mice (18-22 g) of either sex were used for evaluation of pharmacological activities. The animals were allowed food and water ad libitum. They were housed in cages at room temperature (about 25 ˚C) with a 12 h light/dark cycle. The animals were randomly allocated into groups at the beginning of the experiments. All test compounds and the reference drugs were administered p.o. or i.p. suspended in 0.5% sodium carboxymethylcellulose solution (SCMC). The experimental protocol was duly approved by Institutional Animal Ethical Committee. Levamisole was obtained as gift sample from Ranbaxy Research Laboratories (Gurgaon, India). 20

ACCEPTED MANUSCRIPT

5.2.1 Anti-inflammatory activity Rat paw oedema model was used to evaluate anti-inflammatory activity of each compound and reference drug (indomethacin) using methodology as reported by Winter et al [26].

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Oedema was induced by injecting a 1.0% carrageenan solution in right hind paw of the rat. The test compounds, and indomethacin was administered i.p. 30 min before carrageenan was injected. SCMC (0.5%) was used as control. Volume of the paw was measured at 0, 30, 60,

SC

120 and 180 minutes after the carrageenan injection. Anti-inflammatory activity was calculated as percent inhibition of carrageenan induced paw oedema using the following

animal) x 100] 5.2.2 Immunomodulatory activity

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formula: % oedema inhibition = 100-[(paw volume in treated animal/paw volume in control

Dosage regimen: Mice were divided in 22 groups with 6 animals in each group. Group I

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(control) was given 0.5% SCMC in water (0.3 mL/mouse, p.o.). Groups II - XXI received test compounds 2a-2e, 3a-3e, 4a-4e and 5a-5e, and Group XXII received levamisole at the dose of 150 µmol/kg p.o.

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Preparation of sheep red blood cells (SRBCs): Fresh blood was collected from sheep

AC C

sacrificed in the local slaughter house. The blood was centrifuged at 4000 rpm for 10 min. Plasma was discarded and pellet was washed with normal saline thrice. SRBCs count was adjusted to about 1×108 cells in 0.1 mL of the suspension for further experiments. Humoral antibody titre (HAT): The immunomodulatory activity was evaluated through HAT using SRBCs in mice. The animals were treated with test compounds and levamisole as per the dosage regimen for seven days. On 7th day, mice from all groups were challenged with SRBCs suspension (0.1 mL) i.p. Blood samples were collected from each animal by retroorbital puncture on 14th day and centrifuged at 2500 rpm for 10 min to separate the serum. 21

ACCEPTED MANUSCRIPT The antibody titre was determined using microtitre plates as reported in literature [30]. Carbon clearence index: The animals were treated with test compounds and levamisol for 5 days by following the dosage regimen. At the end of 5 days, mice were injected carbon ink suspension (Rotring, Zeichentusche ink, Germany) (10 µL/g of body weight) after 48 h via

RI PT

tail vein. Blood samples were withdrawn from retro-orbital vein at 3 and 15 min. A 25 µL of the blood sample was mixed with 0.1% Na2CO3 solution (2 mL) and optical density was measured by turbidometric method using ELISA plate reader (APR4 Microplate reader,

SC

Germany). Carbon clearance index (K) was calculated using the following equation: K= (ln

respectively [31,32]. 5.2.3 Acute ulcerogenecity test

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OD1 - ln OD2)/12, where OD1 and OD2 were the optical densities at 3 and 15 min,

Rats were divided in groups each of 6 animals, and fasted for 20 h before the drug administration. The test compounds (2a, 2e, 3a, 3e and 5b) and standard drug (indomethacin)

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were administered orally as a suspension (150µmol/kg) in 0.5% SCMC (vehicle). The control group received vehicle. Rats were fasted for 2 h, allowed to feed for 2 h, and then fasted for another 20 h. The other two doses were administered on 2nd and 3rd days. On 4th day, rats

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were sacrificed, the stomach removed, opened along with the greater curvature and rinsed

AC C

with 0.9% saline. The gastric mucosa was then examined for the severity of ulceration and mean ulcer score for each treatment group was calculated and reported as ulcer index [33]. 5.2.4 Biochemical estimations for oxidative stress Glandular parts of the excised stomachs were homogenized in ice cold phosphate buffer (pH 7.4) with a tissue homogenizer (Model PT194, Remi Motors, New Delhi, India) for 2 min. The homogenate was centrifuged at 5000 rpm for 10 min. The supernatant was separated and centrifuged at 15000 rpm for 15 min. The clear supernatant was used for the estimation of GSH level [34], CAT level [35] and TBARS level [36]. 22

ACCEPTED MANUSCRIPT

5.3 Statistical analysis Data is expressed as mean±standard deviation (SD)/standard error mean (SEM) of the results. Statistical analysis of the data was performed using analysis of variance (ANOVA) (Sigma

RI PT

stat 3.5) followed by multiple comparison (Tukey’s) test. A value of p< 0.05 was considered statistically significant. 6. Docking studies

SC

Sketching and cleaning of geometry of structures of compounds were performed in Maestro molecular modelling workspace. Energy minimization was done with ‘ligprep’ program of

M AN U

Schrödinger software using OPLS_2005 force field at pH of 7.4. For docking analysis, two protein structures 2OYU (COX-1) and 3NT1 (COX-2) were downloaded, and optimized. The protein optimization protocol included the addition of hydrogen atoms, deletion of water molecules, completion of bond orders, assignment of hydrogen bonds and protein ligand

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complex minimization to RMSD of 0.20Å using OPLS_2005 force field. The active sites of proteins were defined as 10Å around the co-crystal ligand. All compounds were docked using extra precision (XP) docking mode of ‘glide’ program in COX-1 active site whereas IFD was

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performed for docking in COX-2 active site. In first stage of IFD protocol, softened-potential

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docking was performed to generate 20 initial poses, the van der Waal radii were scaled to 0.5, and receptor scaling was set to 0.7. For each of the top poses from the initial docking step, a full cycle of protein refinement was performed that refined residues within 5.0 Å. All side chains underwent conformational search and minimization. The complexes were ranked according to ‘Prime’ energy, and those within 30 kcal/mol of the minimum structure energy were passed for final round of docking and scoring that used Glide XP mode docking protocol. In this way, total 10 different protein environments, ranked on the basis of IFD energy, were generated for the highest active molecule 2e, and subsequently the one with 23

ACCEPTED MANUSCRIPT least IFD energy was utilized for docking analysis of all other molecules using ‘Glide version 5.6’ docking program. 7. Acknowledgments

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The authors are thankful to Crystal Pharmaceuticals (Ambala, India) for providing ibuprofen and indomethacin, and to Ranbaxy Research Laboratories (Gurgaon, India) for providing mesalamine and levamisole as generous gift samples. 8. Conflicts of interest

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There are no conflicts of interest.

[1]

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9. References

Y. Bansal, O. Silakari. Multifunctional compounds: Smart molecules for multifactorial diseases. Eur. J. Med. Chem. 76 (2014) 31-42.

[2]

C.T. Keith, A.A. Borisy, B.R. Stockwell, Multicomponent therapeutics for networked systems. Nat. Rev. Drug Discov. 4 (2005) 71-78.

S.A. Eisen, D.K. Miller, R.S. Woodward, E. Spitznagel, T.R. Przybeck, The effect of

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[3]

prescribed daily dose frequency on patient medical compliance, Arch. Intern. Med. 25

[4]

EP

(1990) 1881-1884.

C.M. Hoh, J. Dankoff, A. Colacone, M. Afilalo, Polypharmacy, adverse drug-related

AC C

events, and potential adverse drug interactions in elderly patients presenting to an emergency department, Ann. Emergency Med. 38 (2001) 666-671. [5]

R. Morphy, C. Kay, Z. Rankovic, From magic bullets to designed multiple ligands. Drug. Discov. Today. 9 (2004) 641-651.

[6]

J.L. Medina-Franco, M.A. Giulianotti, G.S. Welmaker, R.A. Houghten, Shifting from the single to the multitarget paradigm in drug discovery. Drug Discov. Today 18 (2013) 495-501.

24

ACCEPTED MANUSCRIPT [7]

L.J. Roberts, J. D. Morrow, Autacoids; Drug therapy of inflammation, in: J.G. Hardman, L.E. Limbird, (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw Hill, New York, 2001, pp. 687.

[8]

J.M. Redondo, J.A. Lopez-Guerrero, M. Fresno, Potentiation of interleukin-2 activity

[9]

RI PT

by levamisole and imidazole. Immunol. Letts. 14 (1987) 111-116.

T.P. Misko, W.M. Moore, T.P. Kasten, G.A. Nicokols, J.A. Corbett, R.G. Tilton, M.L. Mcdaniel, J.R. Williamson, M.G. Corrie, Selective inhibition of the inducible nitric

SC

oxide synthase by aminoguanidine. Eur. J. Pharm. 233 (1993) 119-122.

[10] Y. Bansal, O. Silakari, The therapeutic journey of benzimidazoles: a review. Bioorg.

M AN U

Med. Chem. 20 (2012) 6208-6236.

[11] P. Hamley, A.C. Tinker, 1,2-Diaminobenzimidazoles: Selective inhibitor of nitric oxide synthase derived from aminoguanidine. Bioorg. Med. Chem. Lett. 5 (1995) 1573-1576. [12] M.E. Hayes, G.A. Wallace, P. Grongsaard, A. Bischoff, D.M. George, W.M. Michael,

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J. McPherson, R.H. Stoffel, D.W. Green, G.P. Roth, Discovery of small molecule benzimidazole antagonists of the chemokine receptor CXCR3. Bioorg. Med. Chem. Letts.18 (2008) 1573-1576.

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[13] G. Zhang, P. Ren, N.S. Gray, T. Sim, X. Wang, Y. Liu, J. Che, W. Dong, S.S.T. Mark,

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L. Sandberg, T.A. Spalding, R. Romeo, M. Iskandar, Z. Wang, H.M. Seidel, D.S. Karanewsky, Y. He, Discovery of pyrimidine benzimidazoles as Src-family selective Lck inhibitors. Part II. Bioorg. Med. Chem. Letts. 19 (2009) 6691-6695. [14] H.Y. Lo, J. Bentzien, R.W. Fleck, S.S. Pullen, H.H. Khine, J.R. Woska, S.Z. Kugler, M.A. Kashem, H. Takahashi, 2-Aminobenzimidazoles as potent ITK antagonists: Trans-stilbene-like moieties targeting the kinase specificity pocket. Bioorg. Med. Chem. Lett. 18 (2008) 6218-6221.

25

ACCEPTED MANUSCRIPT [15] R.I. Spain, M.H. Cameron, D. Bourdette, Recent developments in multiple sclerosis therapeutics, BMC Med. 7 (2009) 74. [16] M.V. Volin, A.E. Koch, Interleukin-18: A mediator of inflammation and angiogenesis in rheumatoid arthritis, J. Interferon Cytokine Res. 31 (2011) 745-751. cancer

therapies.

National

Cancer

Institute.

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[17] Targeted

http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted (Accessed Jan. 10, 2013).

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[18] Y. Ohshika, N. Umesaki, T. Sugawa. Immunomodulating capacity of the monocyte macrophage system in patients with uterine cervical cancer. Nippon Sanka Fujinka

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Gakkai Zasshi, 40, 601-608 (1988).

[19] F. Makovec, A. Giordani, R. Artusi, S. Mandelli, I. Verpiliq, S. Zanzola, L.C. Rovati, Novel anti-inflammatory and analgesic heterocyclic amidines that inhibit nitrogen oxide (NO) production, US Patent Application. US 2010/0120802 A1, May 13, 2010.

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[20] C.G. Neochoritis, T. Zarganes-Tzitzikas, C.A. Tsoleridis, J. Stephanidou-Stephanatou, C.A. Kontogiorgis, D.J. Hadjipavlou-Litina, T. Choli-Papadopoulou, One-pot microwave assisted synthesis under green chemistry conditions, antioxidant screening,

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and cytotoxicity assessments of benzimidazole Schiff bases and pyrimido[1,2-

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a]benzimidazol-3(4H)-ones, Eur. J. Med. Chem. 46 (2011) 297-306. [21] A. Patil, S. Ganguly, S. Surana, A systematic review of benzimidazole derivatives as an antiulcer agent, Rasayan J. Chem. 1 (2008) 447-460. [22] A. Bali, Y. Bansal, M. Sugumaran, J.S. Saggu, P. Balakumar, G. Kaur, G. Bansal, A. Sharma, M. Singh, Design, synthesis, and evaluation of novel substituted benzimidazole compounds as angiotensin II receptor antagonists. Bioorg. Med. Chem. Lett. 15 (2005) 3962-3965.

26

ACCEPTED MANUSCRIPT [23] N.L. Leonard, C.Y. Daviy, B.M. Karl, Sulfonate salts of substituted benzimidazoles. J. Med. Chem. 69 (1947) 2459-2461. [24] K.C.S. Achar, K.M. Hosaman, H.R.S. Reddy, In-vivo analgesic and anti-inflammatory activities of newly synthesized benzimidazole derivatives. Eur. J. Med. Chem. 45

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(2010) 2048-2054.

[25] S.M. Sondhi, S. Rajvanshi, M. Johar, N. Bharti, A. Azam, A.K. Singh, Antiinflammatory, analgesic and antiamoebic activity evaluation of pyrimido[1,6-

SC

a]benzimidazole derivatives synthesized by the reaction of ketoisothiocyanates with mono and diamines. Eur. J. Med. Chem. 37 (2002) 835-843.

M AN U

[26] C.A. Winter, E.A. Risley, G.W. Nuss, Carrageenan-induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs. P. Soc. Exp. Biol. Med. 111 (1962) 544547. [27] J.

Hansbrough,

V.

Peterson,

R.

Zapata-Sirvent,

N.H.

Claman,

Postburn

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immunosuppression in an animal model. II. Restoration of cell mediated immunity by immunomodulating drugs. Surgery. 95 (1984) 290. [28] A.S. Kalgutkar, B.C. Crews, S.W. Rowlinson, A.B. Marnett, K.R. Kozak, R.P.

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Remmel, L.J. Marnett, Biochemically based design of cyclooxygenase-2 (COX-2)

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inhibitors: Facile conversion of nonsteroidal anti-inflammatory drugs to potent and highly selective COX-2 inhibitors. PNAS 97 (2000) 925-930. [29] M. Hussain, A. Javeed, M. Ashraf, N. Al-Zaubai, A. Stewart, M.M. Mukhtar Nonsteroidal anti-inflammatory drugs, tumour immunity and immunotherapy Pharmacol. Res. 66 (2012) 7–18. [30] A. Puri, R. Saxena, K.C. Saxena, J.S. Tandon, Immunostimulant activity of Nyctanthus arbortristis L. J. Ethnopharmacol. 42 (1994) 31–37.

27

ACCEPTED MANUSCRIPT [31] H.I. Luck, R.S. Nussenzweig, Plasmodium chabaodi and Plasmodiom vinckei: Phagocytic activity of mouse reticuloendothelial system. Exp. Parasitol. 25 (1969) 319323. [32] M.G. Jayathirtha, S.H. Mishra, Preliminary immunomodulatory activities of methanol

RI PT

extracts of Eclipta alba and Centella asiatica. Phytomedicine 11 (2004) 361–365.

[33] V. Cioli, S. Putzolu, V. Rossi, P.S. Barcellona, C. Corradino, The role of direct tissue contact in the production of gastrointestinal ulcers by anti-inflammatory drugs in rat.

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Toxicol. Appl. Pharmacol. 50 (1979) 283–289.

[34] G.L. Ellman, Tissue sulfahydryl group. Arch. Biochem. Biophys. 82 (1959) 70-77.

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[35] H. Aebi, Catalase. In: Bergmeyer H (eds) Methods of enzymatic analysis, vol 2. Chemic Academic Press Inc Verlag, New York (1974) pp 673-685. [36] H. Ohkawa, N. Ohishi, K. Yagi, Assay of lipid peroxidation in animal tissues by

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thiobarbituric acid reaction. Anal. Biochem. 95 (1979) 351-358.

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Common structural features of clinically used immunomodulators. Fig. 2. Design of the multifunctional conjugate compounds of ibuprofen and mesalamine.

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Fig. 3. Synthetic scheme for conjugate compounds 2-5. Fig. 4. Docking study of compounds (R)-2a (A), (S)-2a (B), (R)-2e (C), (S)-2e (D), 3a (E), (R)-3e (F) and (S)-5b (G) with COX-1.

Fig. 5. Docking study of compounds (R)-2a (A), (S)-2a (B), (R)-2e (C), (S)-2e (D), 3a (E),

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(R)-3e (F) and (S)-5b (G) with COX-2.

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Table 1: Anti-inflammatory activity of target compounds Compound Paw volume (% oedema inhibition)a 1.13 ± 0.16b,d (57.1) 2a 1.63 ± 0.12b,c,d (38) 2b 1.10 ± 0.08b,c,d (58) 2c 0.92 ± 0.07b,c,d (64.7) 2d 0.83 ± 0.09b,c,d (68.1) 2e 1.19±0.11b,d (54.6) 3a 1.50 ± 0.14b,c (42.9) 3b 1.25 ± 0.11b,d (52.4) 3c 1.16 ± 0.07b,d (55.7) 3d 0.98 ± 0.04 b,c,d (62.6) 3e 1.21 ± 0.04 b,d (53.8) 4a 1.72 ± 0.08b,c,d (34.3) 4b 1.20 ± 0.09b,c,d (54.1) 4c 1.17 ± 0.04b,d (55.3) 4d 1.04 ± 0.12b,c,d (60.1) 4e 1.31 ± 0.12b (50) 5a 1.65 ± 0.14b,c,d (36.9) 5b 1.34 ± 0.11b (48.8) 5c 1.31 ± 0.08b (50) 5d 1.25 ± 0.11b (52.1) 5e 2.63 ± 0.02 Control 1.28 ± 0.04b (53.8) Ibuprofen Mesalamine 1.39 ± 0.12b (50.0) a Percent inhibition of paw volumes of compounds at 60 min. Values are expressed as mean ± SD. bValues are statistically different with respect to control at p 100 3a a,b a,b 6.4±0.17 + 23.0 0.024±0.003 + 41.2 3b 7.4±0.19a,b + 42.3 0.027±0.002 a,b + 58.8 3c a,b a 7.6±0.21 + 46.2 0.029±0.004 + 70.5 3d 8.8±0.22a + 69.2 0.040±0.003 a,b >100 3e a,b a 7.2±0.20 + 38.4 0.030±0.001 + 76.4 4a 6.0±0.18b + 15.3 0.021±0.001 a,b + 23.5 4b a,b a,b 6.4±0.16 + 23.1 0.025±0.002 + 47.1 4c 6.6±0.21a,b + 26.9 0.026±0.003a,b + 52.9 4d a,b a,b 7.4±0.23 + 42.3 0.028±0.002 + 64.7 4e 3.4±0.24a,b - 34.6 0.012±0.002a,b - 29.4 5a a,b a,b 2.6±0.16 50.0 0.010±0.001 - 41.1 5b a,b a,b 3.2±0.20 - 38.4 0.011±0.002 - 35.2 5c - 34.6 0.012±0.002a,b - 29.4 3.4±0.19a,b 5d a,b b 3.8±0.23 - 26.9 0.014±0.001 - 17.6 5e 5.2±0.23 --0.017 ± 0.002 --Control a a + 65.3 0.032±0.006 + 88.2 Levamisole 8.6±0.24 Values are expressed as mean ± SD for HAT and K. aValues are statistically different from the control at p < 0.05. bValues are statistically different with respect to standard drug at p < 0.05.

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Catalase TBARS Glutathione (µmol/mg) (nmol/mg) (µmol/ 100mg) Control 0.33±0.20 22.14±1.19 3.72±0.23 184.10±4.19 a a a Indomethacin 3.17±0.41 7.79±0.38 30.41±1.57 85.20±5.15a 2a 0.92±0.20a,b 18.26±0.26a,b 5.90±0.39b 158.60±16.44a,b 2e 0.70±0.34b 14.12±0.44a,b 11.28±1.12b 131.50±8.25a,b 3a 0.58±0.12b 18.78±0.20a,b 5.47±0.38b 178.18±6.74b 3e 1.10±0.22a,b 16.94±0.33a,b 8.95±0.36b 153.38±7.23a,b 5b 0.40±0.22b 11.87±0.79a,b 11.98±0.64b 127.32±10.36a,b Values are expressed as mean±SD. aValues are statistically different from the control at p < 0.05.bValues are statistically different with respect to indomethacin at p < 0.05.

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H-bond

NH of BZ

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(S)-2a

H-bond

π-π

Benzene ring of BZ

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(R)-2e

π-cation

π-π

Tyr355

π-π

Benzene ring of BZ C6H5CO

H-bond π-π

-NH2 BZ

Ser355 Tyr355

π-π

Tyr385, Trp387 Tyr355

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Phenyl from MES Benzene ring of BZ C=O

H-bond H-bond

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Ser530

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Table 4. Binding interactions of compounds with active sites of COX-1 and COX-2 COX-1 active site Compound COX-2 active site Type of Functional Amino Type of Functional Amino interaction group of acid interaction group of acid compound residue compound residue involved involved involved involved H-bond NH of BZ Met522 π-π Benzene Tyr385 (R)-2a π-cation Phenyl Arg120 ring of BZ from IB

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IB/MES-NSAID conjugates designed and synthesized. All compounds showed anti-inflammatory activity equivalent to the parent NSAID. All compounds also showed mild to good immunomodulatory activity. Docking analysis proved compounds more selective to COX-2 and less ulcerogenic. The compounds may be useful in treatment of multifactorial diseases.

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