Synthesis of Furoxan Derivatives of Diclofenac As Potent Anti-Inflammatory Agents with Reduced GI Toxicity

Indian Journal of Chemistry Vol. 55B, August 2016, pp. 989-998

Synthesis of furoxan derivatives of diclofenac as potent anti-inflammatory agents with reduced GI toxicity

Mohd Amir*, Md Wasim Akhter & K Somakala Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110 062, India E-mail: [email protected]

Received 12 February 2016; accepted (revised) 2 May 2016

A new series of NO donating furoxan derivatives of diclofenac have been synthesized by linking diclofenac to selected furoxan moieties and have been investigated for their anti-inflammatory, analgesic, ulcerogenic, lipid peroxidation, hepatotoxicity, histopathological and NO releasing properties. All the hybrid derivatives are endowed with anti- inflammatory activity comparable to diclofenac but unlike this drug they show reduced GI toxicity. The compounds 4-[(2-(2-(2-(2,6-dichlorophenylamino)phenyl)acetamido)ethoxy)carbonyl]-3-methyl furoxan 12 having amide-ester linkage and 4-[(2-(2-(2,6-dichlorophenylamino)phenyl)acetoxy)methyl]-3-methyl-furoxan 13a having ester linkage show greater anti-inflammatory activity comparable to standard drug diclofenac. These compounds 12 and 13a also show higher gastrointestinal protection in comparison to standard drug diclofenac.

Keywords: Diclofenac, nitric oxide donors, furoxan, anti-inflammatory, ulcerogenicity, lipid peroxidation, hepatotoxic effect, histopathological studies

Nonsteroidal anti-inflammatory drugs (NSAIDs) viz. Such NO-donating NSAIDs are emerging as a new ibuprofen, naproxen and diclofenac are commonly class of effective anti-inflammatory compounds employed drugs for the treatment of various chronic having an enhanced safety profile8,9. The rationale anti-inflammatory diseases. Among these, diclofenac behind this drug design is based on the biological has been approved in 120 countries since its proprieties of NO. It has been reported that an introduction 36 years ago and ranked among the top increased generation of endothelial NO or release of 200 drugs with respect to new prescription. NSAIDs NO from a nitric oxide donor drug produces therapy effectively reduces the symptoms of many beneficial effects such as reduction of blood pressure painful arthritic syndromes, but invites adverse and prevention of atherosclerosis10. At nanomolar gastrointestinal (GI) complications ranging from concentrations, NO reversibly activates soluble stomach irritation to life threatening GI ulceration, guanylate cyclase (sGC) by 400 folds, catalyzing the bleeding, and perforation to more serious small-bowel conversion of guanosine triphosphate to cyclic ulceration1-3. It occurs due to the inhibition of guanosine monophosphate (cGMP)11. Elevation of cyclooxygenase (COXs) as well as acidic character of cGMP relaxes smooth muscles in blood vessels, NSAIDs themselves4. In order to reduce GI toxicity inhibits platelet aggregation and blocks the adhesion various COX-2 selective inhibitors viz. celecoxib, of white cells to blood vessels walls12. Besides these rofecoxib have been developed which showed marked cardiovascular effects, NO is also recognized as a anti-inflammatory activity but induce less GI side critical mediator of gastrointestinal mucosal defense. effect in comparison to non selective COX inhibitors NO is able to protect gastric mucosa by a number of such as aspirin, ibuprofen and diclofenac5,6. But mechanisms including promotion of mucus secretion selective COX-2 inhibitors have been withdrawn from and increased mucosal blood flow resulting in the market due to their adverse cardiovascular side enhanced mucosal resistance to ulceration. NO also effects7. Therefore, development of NSAIDs with increases the ability of mucous cells to undertake improved safety profile is still a necessity. healing and repair of the existing ulcers13. Encouraged One interesting innovative approach relies on the by these observations and in continuation of our incorporation of a nitric-oxide (NO) releasing moiety ongoing research program14-17 to discover new and into the structure of established NSAIDs molecules. useful agents for treatment of anti-inflammatory 990 INDIAN J. CHEM., SEC B, AUGUST 2016

disease, we report herein the synthesis and treatment with ethanol amine forms an intermediate pharmacological profile of furoxan derivatives of 2-[2-(2,6-dichlorophenylamino)phenyl)-N-(2-hydroxyethyl] diclofenac as potential NO donor drugs. acetamide 11 which on further treatment with intermediate 6a resulted in the formation of [(2-(2-(2-(2,6- Result and Discussion dichlorophenylamino)phenyl) acetamido)ethoxy)carbonyl]- The reaction sequence leading to the formation of 3-methyl-furoxan 12. The final compounds 4-[(2-(2- the desired heterocyclic compounds are outlined in (2,6-dichlorophenylamino)phenyl)acetoxy)methyl]-3- Scheme I and Scheme II. Compounds 1 (Ref 18), substituted-furoxan 13a-b of the series were prepared by 2 (Ref 19), 3 (Ref 20), 4 (Ref 21), 5a-b (Ref 22) and treating intermediate 7 with 4-(hydroxymethyl)-3- 6a-b (Ref 22) were synthesized according to methods methyl/phenyl furoxan in presence of triethyl amine reported in literature. Intermediate compounds 8 and (Scheme I). The compounds 4-[(2-(2-(2-(2,6- 11 were synthesised according to literature method by dichlorophenylamino)phenyl)acetyl)hydrazono)methyl]- 23 slightly modifying reaction conditions . 3-substituted-furoxan 14a-b were prepared by treating 2-

Substituted furoxans were synthesized from [2-(2,6-dichloro phenylamino)phenyl]acetohydrazide crotonaldehyde and cinnamaldehyde (Figure 1). with 4-formyl-3-methyl/phenyl-furoxan in presence of Compound 9 was synthesized by treating 2-[2-(2,6- concentrated sulphuric acid. The compound 4-[2-(2-(2-(2,6- dichlorophenylamino)phenyl]acetyl chloride 7 with dichlorophenylamino)phenyl)acetyl)hydrazinecarbonyl]- ethylene glycol to form an intermediate 2-hydroxyethyl- 3-substituted-furoxan 15a-b were synthesized by stirring 2-[2-(2,6-dichlorophenylamino)phenyl]acetate which on 2-[2-(2,6-dichlorophenylamino)phenyl]acetohydrazide further treatment with 4-(chlorocarbonyl)-3-methyl and 4-(chlorocarbonyl)-3-substituted-furoxan 6a-b in furoxan 6a resulted in the formation of 4-[(2-(2-(2-(2,6- presence of triethylamine. The compound 4-[5-(2-(2,6- dichlorophenylamino)phenyl)acetoxy)ethoxy)carbonyl]- dichlorophenylamino)benzyl)-1,3,4-oxadiazol-2-yl]-3- 3-methyl-furoxan 9. The ethyl ester of diclofenac 10 on methyl-furoxan 16 was synthesized by refluxing 2-[2-

Scheme I — Synthesis of furoxan derivatives of 9, 12 and 13a-b AMIR et al.: FUROXAN DERIVATIVES OF DICLOFENAC 991

Scheme II — Synthesis of furoxan derivatives 14a-b, 15a-b and 16

guidelines in accordance with the rules laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. All the test compounds and standard drug were administered in the form of solution (0.5% w/v carboxymethyl cellulose as a vehicle) by an oral route. Each group consisted of six animals. All the animals were procured from the CPCSEA and maintained in colony cages at 25 ± 2°C, with relative humidity of 45–55%, under a 12 h light and dark cycle and were fed a standard animal feed. All

Figure 1 — Different substituted furoxans the animals were acclimatized for a week before use. The anti-inflammatory activity of the test compounds (2,6-dichlorophenyl amino)phenyl]acetohydrazide with were compared with the control. The analgesic, 4-carboxy-3-methyl furoxan 5a in phosphorus ulcerogenic and lipid peroxidation activities were oxychloride (Scheme II). The purity of the compounds compared with the standard drug diclofenac. Data were was checked by elemental analyses and spectral data. analysed by student’s t test for n = 6. 1 Both the analytical and spectral data (IR, H and 13C NMR, and mass spectroscopy) of the compounds Measurement of nitric oxide release were in full harmony with the proposed structures. The All the compounds 9, 12, 13a-b, 14a-b, 15a-b and results of elemental analysis (C, H and N) were within ± 0.3% 16 were tested for their NO releasing properties in of the theoretical values. vitro in both phosphate buffer of pH 7.4 and 0.1N HCl of pH 1 by using Griess reagent. The reaction Biological Studies was carried out in the presence of L-cysteine as a The synthesized compounds were evaluated for their source of the SH group. It was found that a reduced anti-inflammatory, analgesic, ulcerogenic and lipid thiol group from endogenous L-cysteine could peroxidation, hepatotoxic and histopathological mediate the release of NO from furoxan derivatives. properties. The Wistar rats and albino mice used in the The amount of NO release from test compounds was present study were housed and kept with the Hamdard measured relative to NO released from standard University Animal Care Unit, which applies the sodium nitrite solution and calculated as amount of 992 INDIAN J. CHEM., SEC B, AUGUST 2016

NO released (mol/mol)24 and listed in Table I. Linker Analgesic activity groups which attach furoxan with diclofenac had The compounds which showed significant anti- significant effect on NO releasing capacity of inflammatory activity (more than 65%) were further compounds. The compound 12 having an amide-ester tested for their analgesic effect at the same oral dose linkage and a methyl group in the furoxan ring and as used for the anti-inflammatory activity. The compound 13a having an ester linkage and a methyl analgesic activity was carried out by tail immersion group in the furoxan ring showed maximum nitric method27 and the results are reported as % analgesia oxide releasing capacity. The compound 14b having (Table III). Analgesic activity of furoxan derivatives amide-Schiff base linkage showed minimum nitric showed 57.15% to 79.05% whereas standard drug oxide releasing capacity. Other compound of the diclofenac showed 74.49% activity. Compound 13a series having an amide-amide linkage 15a-b and having ester linkage with a methyl group at furoxan 1,3,4-oxadiazole spacers 16 showed moderate nitric ring showed maximum activity (79.05%). Other oxide releasing capacity. On the other hand the tested compounds 12, 13b and 15a also showed very good compounds were not able to release NO at pH 1, activity ranging from 65.40% to 74.19%. Compound which suggests that NO donating furoxan derivatives 14a having amide-Schiff base linkage with a methyl of diclofenac are weakly hydrolysed in the gastric group at furoxan ring showed poor activity (57.15%). lumen and this confirmed that the suggested gastro 25 Ulcerogenic activity protective action of NO is mediated systemically The compounds 9, 12, 13a-b and 15a which (Table I). showed significant anti-inflammatory and good Anti-inflammatory activity analgesic activity were further tested for their acute All the synthesized compounds were screened for ulcerogenicity by the method of Cioli et al28 their anti-inflammatory activity by carrageenan induced 26 (Table III). The compounds were tested at an rat paw edema method as described by Winter et al . equimolar oral dose relative to 30 mg/Kg diclofenac. The results revealed that compounds 12, 13a and 15a The tested compounds showed severity index ranging exhibited excellent anti-inflammatory activity (85.97%, from 0.250 ± 0.11 to 0.583 ± 0.08 whereas the 80.44% and 79.16% respectively), at an equimolar oral standard drug diclofenac showed severity index of dose relative to 10 mg/Kg diclofenac which showed 0.666 ± 0.10. Compound 12 having amide-ester 77.41% inhibition after 4 h. Compounds 13b and 15b linkage and a methyl group at furoxan ring showed showed anti-inflammatory activity comparable to minimum ulcerogenicity (severity index 0.250 ± 0.11), standard drug diclofenac i.e. 75.83% and 73.06% respectively. Compound 14a also showed good anti- Table I — The amount of NO released from tested compounds in inflammatory activity whereas compounds 9, 14b and phosphate buffer (pH = 7.4) 16 showed lesser degree of activity. Thus it was Compd Amount of Compd Amount of concluded that furoxan derivatives having amide-ester NO release (mol/mol) NO release (mol/mol) linkage or ester-ester linkage or ester linkage showed 9 0.54 ± 0.033 14b 0.21 ± 0.039 very good activity. These results indicate that the 12 0.65 ± 0.082 15a 0.43 ± 0.043 incorporation of NO donating furoxan moieties to 13a 0.62 ± 0.061 15b 0.31 ± 0.061 diclofenac have not only retained but showed enhanced 13b 0.53 ± 0.026 16 0.35 ± 0.047 anti-inflammatory activity (Table II). 14a 0.40 ± 0.038 − − Table II — Anti-inflammatory activity of furoxan derivatives 9, 12, 13a-b, 14a-b, 15a-b and 16 Compd Anti-inflammatory activity % inhibition# Compd Anti-inflammatory activity % inhibition# After 3 h After 4 h After 3 h After 4 h 9 54.71 ± 0.87 61.77 ± 0.67d 14b 54.89 ± 1.07 58.11 ± 0.96d 12 72.28 ± 0.73 85.97 ± 0.55d 15a 75.46 ± 1.04 79.16 ± 0.59d 13a 78.07 ± 0.71 80.44 ± 0.62c 15b 70.83 ± 0.65 73.06 ± 0.97d 13b 71.01 ± 0.54 75.83 ± 0.53a 16 59.78 ± 0.74 64.02 ± 0.47b 14a 64.49 ± 0.77 67.53 ± 0.84b Diclofenac 73.80 ± 0.72 77.41 ± 0.98 Test compounds and diclofenac were tested at 10 mgkg–1 body weight #Mean ± SEM, n = 6. Significance levels compared to the control: ap < 0.5, bp < 0.05, cp < 0.001, dp < 0.0001 AMIR et al.: FUROXAN DERIVATIVES OF DICLOFENAC 993

whereas compounds 13a and 15a having an ester and derivatives showing lower ulcerogenic activity also an amide-amide linkage also showed lesser showed reduction in lipid peroxidation. The ulcerogenicity (severity index 0.333 ± 0.10 and compound 12 having amide-ester linkage and 0.416 ± 0.15 respectively). Thus, the results indicated compound 13a having ester linkage showed that nitric oxide released from the diclofenac-furoxan maximum reduction 3.58 ± 0.10 and 4.70 ± 0.07 derivatives have a significant effect on ulcerogenic respectively whereas compound 9 having ester-ester activity (Figure 2). The decreased severity index may linkage showed minimum reduction 6.67 ± 0.09 nmol be due to the release of NO that increased mucosal MDA/100 mg tissue. Thus these studies showed that blood flow resulting in enhanced mucosal resistance the synthesized compounds have inhibited the to ulceration and/or an enhanced ability of the NO induction of gastric mucosal lesions and the results donating furoxan derivatives to cross the gastric further suggested that their protective effect might be mucosal layer prior to the subsequent release of NO. related to the inhibition of lipid peroxidation in the gastric mucosa due to release of nitric oxide from Lipid peroxidation activity furoxan derivatives (Table III). All the compounds screened for ulcerogenic activity were also analyzed for lipid peroxidation Hepatotoxic and histopathological activity according to the method of Ohkawa et al29. The lipid Furoxan derivatives 12 and 13a showing potent peroxidation was measured as nanomoles of anti-inflammatory and analgesic activities with malonodialdehyde (MDA/100mg) of gastric mucosa reduced ulcerogenicity and lipid peroxidation were tissue. Diclofenac exhibited high lipid peroxidation further studied for their hepatotoxic effect. 7.15 ± 0.06 whereas the control group showed Assessment of liver function such as serum glutamate 3.46 ± 0.10. It was found that all the furoxan oxaloacetate transaminase (SGOT) and serum

Table III — Analgesic, ulcerogenic and lipid peroxidation activities of synthesized furoxan derivatives 9, 12, 13a, 13b, 14a and 15a Compd Analgesic Activity Ulcerogenic activity nmol MDA content#/100 mg # Pre-treatment/ Post-treatment/ % Inhibition# (Severity index) tissue normal 0 h after 4 h 9 1.55 ± 0.01 2.56 ± 0.02 65.40 ± 0.97b 0.583 ± 0.08 6.67 ± 0.09b 12 1.72 ± 0.01 2.98 ± 0.01 70.71 ± 0.98b 0.250 ± 0.11b 3.58 ± 0.10b 13a 1.69 ± 0.01 2.86 ± 0.02 79.05 ± 1.22 0.333 ± 0.10c 4.70 ± 0.07d 13b 1.47 ± 0.01 2.52 ± 0.02 74.19 ± 1.79 0.500 ± 0.00a 5.97 ± 0.08d 14a 1.55 ± 0.01 2.44 ± 0.01 57.15 ± 0.97e − − 15a 1.75 ± 0.01 3.09 ± 0.01 70.15 ± 1.50a 0.416 ± 0.15 5.72 ± 0.11a Control − − − 0.000 ± 0.00 3.46 ± 0.10 Diclofenac 1.43 ± 0.00 2.50 ± 0.01 74.49 ± 0.96 0.666 ± 0.10 7.15 ± 0.06 Test compounds and diclofenac sodium were tested at 10mgkg–1 body weight. #Mean ± SEM, n = 6. Significance levels compared to the standard drug: ap < 0.5, bp < 0.05, cp < 0.001, dp < 0.0001

Figure 2 — Gastric ulcer which is induced by diclofenac drug was reduced by linking NO releasing furoxan moieties with diclofenac 994 INDIAN J. CHEM., SEC B, AUGUST 2016

Table IV — Effect of compounds 12 and 13a on serum enzymes, total proteins and total albumin Compd Alkaline Phosphatase# SGOT# SGPT# Total Protein (g/100 mL)# Albumin (g/100 mL)# Control 32.50 ± 0.45 149.76 ± 0.81 31.54 ± 1.46 1.69 ± 0.012 1.59 ± 0.017 Diclofenac 42.69 ± 0.32d 159.07 ± 0.79d 46.23 ± 0.77d 1.86 ± 0.027c 1.65 ± 0.020b 12 35.06 ± 0.47b 155.91 ± 1.49b 36.11 ± 1.49a 1.73 ± 0.007b 1.56 ± 0.098 13a 34.29 ± 0.38a 155.16 ± 1.09b 44.84 ± 1.17d 1.78 ± 0.018b 1.64 ± 0.013b Test compounds and diclofenac were tested at 10 mgkg–1 body weight. #Mean ± SEM, n = 6. Significance levels compared to the control: bp < 0.5, bp < 0.05, cp < 0.001, dp < 0.0001

Figure 3 — Histopathological studies of the liver glutamate pyruvate transaminase (SGPT) were show any significant pathological changes in estimated by a reported method30. The alkaline comparison to standard drug diclofenac (Figure 3). phosphatase, total protein and total albumin were No hepatocyte necrosis or degeneration was seen in measured according to reported procedures31,32 as any of the samples. shown in Table IV. Activities of liver enzyme SGOT, SGPT, alkaline phosphatase, total protein and total Experimental Section albumin were less than for the standard drug The melting points were determined in open diclofenac. Histopathological studies were also capillary tubes in a Hicon melting point apparatus and carried out by reported methods33. The are uncorrected. Elemental analysis (C,H,N,S) was histopathological studies of the liver samples do not performed on the CHNS Elimentar (Analysen AMIR et al.: FUROXAN DERIVATIVES OF DICLOFENAC 995

Systime, GmbH) Germany Vario EL III instrument. Synthesis of 4-[(2-(2-(2-(2,6-dichlorophenylamino)- FTIR spectra were recorded as KBr pellets on a Jasco phenyl)acetamido)ethoxy) carbonyl]-3-methyl-furoxan, FT-IR 410 spectrometer and frequency was expressed 12 in cm−1. 1H NMR spectra were recorded on a Ethyl 2-[2-(2,6-dichlorophenylamino)phenyl]acetate Bruker DPX 400 NMR spectrometer. Chemical shifts 10 was added to ethanolamine (20 mmol) in CH2Cl2 (δ) are expressed in ppm relative to tetramethylsilane (30 mL) with stirring. The mixture was refluxed for (TMS); coupling constants (J) are reported in Hertz, 6 h, cooled to RT and the solvent removed by and refer to apparent peak multiplicities, and may not distillation at reduced pressure. Pale brown oil was necessarily be true coupling constants. Mass spectra obtained which became a sticky solid on cooling in were measured on a Jeol SR-102 (FAB) mass the refrigerator. Subsequent grinding of the sticky spectrometer. Unless otherwise specified, all reactions solid with acetone and diethyl ether gave a white solid were carried out in oven-dried glassware, and which was the intermediate 2-[2-(2,6-dichlorophenylamino)- commercially available starting materials were used phenyl)-N-(2-hydroxyethyl]acetamide 11. The compound without further purification. thus obtained was purified by recrystallization from petroleum ether. A solution of 4-(chlorocarbonyl)-3- Synthesis of 4-[(2-(2-(2-(2,6-dichlorophenylamino)- methyl furoxan 6a (10 mmol) in THF (3 mL) was phenyl)acetoxy)ethoxy)carbonyl]-3-methyl furoxan, 9 added drop-wise to a stirred mixture of intermediate Ethylene glycol (0.015 mol) and dry CH2Cl2 (20 11 in dichloromethane (60 mL) in the presence of mL) were slowly mixed together at RT. 2-[2-(2,6- triethylamine (1 mL). The stirring was continued for Dichlorophenylamino)phenyl]acetyl chloride 7 (10 5 h, after which the reaction mixture was poured into mmol) in dry CH2Cl2 (5 mL ) was added drop-wise to water. The organic layer was separated, washed twice this mixture with continuous stirring under ice cold with water dried with anhydrous sodium sulphate and conditions. The stirring was continued for 5 h and distilled under reduced pressure. The residue thus progress of the reaction was monitored by TLC using obtained was purified by silica gel column 30% ethyl acetate-hexane solvent system. After chromatography using EtOAc/hexane as eluent to completion, it was washed twice with water, dried and afford the pure compound. Yield 58%. m.p.80-82°C. distilled under reduced pressure to afford the IR (KBr): 3288 (NH), 1743(C=Oester), 1656 intermediate 2-hydroxyethyl 2-[2-((2,6- −1 1 (C=Oamide), 1592 cm (C=N); H NMR: δ 6.98-7.35 dichlorophenyl)amino)phenyl]acetate 8 which was (m, 8H, 7ArH and 1NH), 6.27 (bs, 1H, CONH), 4.50 used immediately for the next step of the reaction. A (t, 2H, OCH2), 3.70 (s, 2H, CH2CO), 3.62 (t, 2H, solution of 4-(chlorocarbonyl)-3-methyl furoxan 6a NCH ), 2.28 (s, 3H, CH ); 13C NMR: δ 172.10 (10 mmol) in anhydrous THF ( 5 mL) in the presence 2 3 (CCONH), 169.61 (C=O), 148.53, 141.92, 135.76, of triethylamine (0.5 mL) was added drop-wise at RT 128.41, 128.23, 127.96, 127.04, 126.45, 125.65, to a stirred solution of 8 in dry CH2Cl2 (30 mL). The 120.06, 118.10, 110.21, 60.34 (CH2), 38.11 (CH2), stirring was continued for 6 h and the reaction mixture + 35.34 (CH2), 7.8 (CH3); MS: m/z 465 (M ), 467 was poured into ice cold water. The organic layer was + (M +2). Anal. Calcd for C20H17Cl2N3O6: C, 51.63; H, separated, washed twice with water, dried and 3.90; N, 12.04. Found: C, 51.43; H, 3.74; N, 11.92%. distilled under reduced pressure. The residue thus obtained was purified by silica gel column General method for synthesis of 4-[(2-(2-(2,6- chromatography using EtOAc/hexane as eluent to dichlorophenylamino)phenyl)acetoxy) methyl]-3- afford pure compound. Yield 55%. m.p.78-80°C. IR substituted-furoxan, 13a-b −1 (KBr): 3283 (NH), 1758, 1747 (C=Oester), 1614 cm An equimolar mixture of 4-(hydroxymethyl)-3- (C=N); 1H NMR: δ 6.12-7.95 (m, 8H, 7ArH and methyl furoxan/4-(hydroxymethyl)-3-phenyl furoxan 1NH), 4.13 (t, 2H, CH2), 4.22 (t, 2H, CH2), 3.61 (10 mmol) and triethylamine (10 mmol) was 13 (s, 2H, CH2CO), 2.04 (s, 3H, CH3); C NMR: dissolved in dry toulene (20 mL) at 0°C with stirring. δ 171.84 (C=O), 165.32 (C=O), 149.43, 140.42, To this solution 2-[2-(2,6- 135.86, 129.89, 128.31, 127.78, 127.37, 126.80, dichlorophenylamino)phenyl]acetyl chloride 7 was 125.23, 121.37, 119.12, 110.53, 59.38 (CH2), 54.31 added drop-wise with continuous stirring. The + (CH2), 37.89 (CH2), 7.72 (CH3); MS: m/z 466 (M ), reaction mixture was further stirred for 4-5 h and then + 468 (M +2). Anal. Calcd for C20H18Cl2N4O5: C, 51.52; poured into ice cold water. The organic layer was H, 3.67; N, 9.01. Found: C, 51.38; H, 3.53; N, 8.87%. separated, washed with water, dried and distilled 996 INDIAN J. CHEM., SEC B, AUGUST 2016

13 under reduced pressure. The separated solid thus 2.53 (s, 3H, CH3); C NMR: δ 171.52 (CCONH), 146.26, obtained was purified by silica gel column 143.31, 139.32, 135.27, 128.87, 128.34, 127.83, 127.09, chromatography using EtOAc/hexane as eluent to 126.45, 125.72, 121.73, 119.73, 110.11, 36.71 (CH2), + + afford the title compounds. 9.08 (CH3); MS: m/z 420 (M ), 422 (M +2). Anal. Calcd for C18H15Cl2N5O3: C, 51.44; H, 3.60; N, 16.66. Found: 4-[(2-(2-(2,6-Dichlorophenylamino)phenyl)acetoxy)methyl]- C, 51.27; H, 3.42; N, 16.48%. 3-methyl-furoxan, 13a: Yield 65%. m.p.91-93°C. IR (KBr): −1 1 3323 (NH), 1731 (C=Oester), 1586 cm (C=N); H NMR: 4-[(2-(2-(2-(2,6-Dichlorophenylamino)phenyl)acetyl)- δ 7.07-7.52 (m, 7H, 6ArH and 1NH), 6.39 (d, 1H, hydrazono)methyl]-3-phenyl-furoxan, 14b: Yield ArH), 4.58 (s, 2H, OCH2), 3.78 (s, 2H, CH2CO), 2.26 13 54%. m.p.162-64°C. IR (KBr): 3297 (NH), 1656 (s, 3H, CH3); C NMR: δ 173.69 (C=O), 154.41, −1 1 (C=Oamide), 1587 cm (C=N); H NMR: δ 8.31-8.86 143.31, 135.50, 130.89, 130.44, 129.08, 127.97, (m, 6H, 3ArH and one proton each of NH, CONH, 125,13, 124.87, 124.31, 123.12, 109.55, 35.77 (CH2), + N=CH merged with ArH), 7.39-7.60 (m, 9H, ArH) 34.57 (CH2), 7.79 (CH3); MS: m/z 408 (M ), 410 13 δ + 5.62 ( s, 2H, CH2CO); C NMR: 173.50 (CCONH), (M +2). Anal. Calcd for C18H15Cl2N3O4: C, 52.96; H, 147.26, 140.32, 137.13, 134.20, 131.37, 130.65, 3.70; N, 10.29. Found: C, 52.76; H, 3.52; N, 10.11%. 129.81, 128.87, 128.31, 127.56, 127.12, 126.36,

125.83, 124.71, 121.28, 118.11, 109.17, 35.26 (CH2); 4-[(2-(2-(2,6-Dichlorophenylamino)phenyl)acetoxy)methyl]- MS: m/z 482 (M+), 484 (M++2). Anal. Calcd for 3-phenyl-furoxan, 13b: Yield 58%. m.p.58-60°C. IR (KBr): −1 C23H17Cl2N5O3: C, 57.27; H, 3.55; N, 14.52. Found: 3313 (NH), 1712 (C=Oester), 1594 cm (C=N); C, 57.07; H, 3.71; N, 14.28%. 1 δ H NMR: 7.10-7.80 (m, 12H, 11ArH and 1NH), 6.39 (d, 1H, ArH), 4.59 (s, 2H, OCH2), 3.74 (s, 2H, 13 General method for synthesis of 4-[2-(2-(2-(2,6- CH2CO); C NMR: δ 171.24 (C=O), 152.43, 140.25, 135.58, 131.65, 130.89, 130.21, 129.27, 128.54, dichlorophenylamino)phenyl)acetyl) hydrazinecarbonyl]- 128.13, 127.4, 126.85, 125.83, 124.37, 123.63, 3-substituted-furoxan, 15a-b 2-[2-(2,6-Dichlorophenylamino)phenyl] acetohydrazide 123.10, 110.52, 38.26 (CH2), 36.43 (CH2); MS: m/z 470 (M+), 472 (M++2). Anal. Calcd for (10 mmol) was dissolved in dry toluene (20 mL) and triethylamine (10 mmol) was added to it. C23H17Cl2N3O4: C, 58.74; H, 3.64; N, 8.93. Found: C, 58.56; H, 3.59; N, 8.75%. 4-(Chlorocarbonyl)-3-methyl/phenyl-furoxan in dry toluene (5 mL) was added to the solution drop-wise General method for synthesis of 4-[(2-(2-(2-(2,6- with continuous stirring. The reaction mixture was dichlorophenylamino)phenyl)acetyl) hydrazono)methyl]-3- further stirred for 4-5 h at RT and then was poured substituted-furoxan, 14a-b into ice cold water. The organic layer was separated, 2-[2-(2,6-Dichlorophenylamino)phenyl] acetohydrazide washed with water, dried and distilled under reduced (10 mmol) and 4-formyl-3-methyl/phenyl-furoxan pressure. The separated solid thus obtained was (10 mmol) were dissolved in ethanol (25 mL) and heated purified by silica gel column chromatography using until a clear solution was obtained. Few drops of conc. EtOAc/hexane as eluent to afford pure compounds. sulphuric acid were added to the mixture and the solution was refluxed for 6-8 h. Progress of the reaction was 4-[2-(2-(2-(2,6-Dichlorophenylamino)phenyl)acetyl)- monitored by TLC using a mixture of ethyl acetate and n- hydrazinecarbonyl]-3-methyl-furoxan, 15a: Yield hexane (2:3). After completion, reaction mixture was 65%. m.p.102-104°C. IR (KBr): 3239, 3243 (NH), poured into ice cold water. The separated solid thus 1704, 1680 (C=O), 1611 cm−1 (C=N); 1H NMR: obtained was purified by silica gel column chromatography δ 8.68 (s, 1H, CONH), 8.16 (s, 1H, CONH), 6.93-7.45 using EtOAc/hexane as eluent to afford pure compound. (m, 7H, 6ArH and 1NH), 6.53 (d, 1H, ArH), 4.16 ( s, 13 2H, CH2CO), 2.53 (s, 3H, CH3); C NMR: δ 174.52 4-[(2-(2-(2-(2,6-Dichlorophenylamino)phenyl)acetyl)- (CCONH), 171.60 (CCONH), 146.17, 143.21, 139.22, hydrazono)methyl]-3-methyl-furoxan, 14a: Yield 135.29, 128.89, 128.37, 127.81, 127.29, 126.54, 50%. m.p.100-102°C. IR (KBr): 3289 (NH), 1662 121.76, 119.71, 110.17, 36.69 (CH2), 9.23 (CH3); MS: −1 1 + + (C=Oamide), 1589 cm (C=N); H NMR: δ 11.97 (s, 1H, m/z 436 (M ), 438 (M +2). Anal. Calcd for CONH), 8.16 (s, 1H, N=CH), 6.93-7.45 (m, 7H, 6ArH C18H15Cl2N5O4: C, 49.56; H, 3.47; N, 16.05. Found: and 1NH), 6.53 (d, 1H, ArH), 4.16 ( s, 2H, CH2CO), C, 49.37; H, 3.29; N, 15.87%. AMIR et al.: FUROXAN DERIVATIVES OF DICLOFENAC 997

4-[2-(2-(2-(2,6-Dichlorophenylamino)phenyl)acetyl)- ulceration. Thus the use of the hybrid molecules hydrazinecarbonyl]-3-substituted-furoxan, 15b: containing NO releasing furoxan moieties may be a Yield 65%. m.p.110-12°C. IR (KBr): 3248, 3254 useful approach for the development of GI-safe −1 (NH), 1684, 1689 (C=Oamide), 1593 cm (C=N); anti-inflammatory analgesic agents. 1H NMR: δ 10.54 (bs, 1H, CONH), 10.10 (bs, 1H, CONH), 7.18-8.80 (m, 13H, 12ArH and 1NH), 4.92 Acknowledgment 13 (s, 2H, CH2CO); C NMR: δ 174.12 (CCONH), 170.66 The authors are grateful to University Grants (CCONH), 155.55, 149.36, 142.92, 137.86, 135.84, Commission (UGC) New Delhi for financial support 132.32, 131.43, 130.87, 128.92, 128.25, 127.99, [F. No. 36-1-08/2008(SR)]. Authors are also thankful 125.26, 124.37, 122.06, 118.14, 109.25, 38.11 (CH2); to the Central Instrumentation Facility (CIF) Faculty MS: m/z 498 (M+), 500 (M++2). Anal. Calcd for of Pharmacy for spectral analysis of compounds and C23H17Cl2N5O4: C, 55.44; H, 3.44; N, 14.05. Found: to Dr. Ambrish Kumar Tiwari, in-charge animal C, 55.31; H, 3.27; N, 13.85%. house, Jamia Hamdard, for providing animals. The authors are also thankful to Dr. A. Mukharjee, MD, Synthesis of 4-[5-(2-(2,6-dichlorophenylamino)benzyl)- Department of Pathology, All India Institute of 1,3,4-oxadiazol-2-yl]-3-methyl-furoxan, 16 Medical Sciences (AIIMS), New Delhi, for carrying 2-[2-(2,6-Dichlorophenyl amino)phenyl]acetohydrazide out histopathological studies. (10 mmol) was dissolved in phosphorus oxychloride (10 mL) and to it was added 4-carboxy-3-methyl References furoxan 5a (10 mmol). The reaction mixture was 1 Palomer A, Cabre F, Pascual J, Campos J, Trugillo M A, refluxed for 6 h, cooled to RT and poured onto Entrena A, Gallo M A, Garcia L, Macleon D & Epinsoma A, crushed ice. The solution was neutralized with sodium J Med Chem, 45 (2002) 1402. 2 Sorbera L A, Lesson P A, Gastanar J & Gastanar R M, Drug bicarbonate (10%). The solid mass thus separated was Future, 26 (2001) 133. washed with water and dried. It was purified by silica 3 Cash J M & Klippel J H, N Engl J Med, 330 (1994) 1368. gel column chromatography using EtOAc/hexane as 4 El-Nezhway A O, Biuomy A R, Hasan F S, Ismaiel A K & eluent to afford the pure compound. Yield 62%. Omar H A, Bioorg Med Chem, 21 (2013) 1661. 5 Lanza F L, Rack M F, Simon T J, Quan H, Bolognese J A, m.p.188-90°C. IR (KBr): 3293 (NH), 1610 (C=N), −1 1 Hoover M E, Wilson F R & Harper S E, Aliment Pharmacol 1134 cm (C-O-C); H NMR: δ 7.02-7.57 (m, 7H, Therap, 13 (1999) 761. 6ArH and 1NH), 6.47 (d, 1H, ArH), 4.19 (s, 2H, 6 Ray W A, Stein C M, Daugherty J R, Hall K, Arbogast P G CH CO), 2.52 (s, 3H, CH ); 13C NMR: δ 168.15, & Griffin M R, Lancet, 360 (2002) 1071. 2 3 164.32, 147.54, 144.31, 140.16, 139.63, 137.35, 7 Donge J M, Supuran C T & Pratico D, J Med Chem, 48 (2005) 2251. 135.90, 129.38, 127.76, 126.54, 122.30, 121.36, 8 Keeble J E & Moore P K, Br J Pharmacol, 137 (2002) 295. + 112.23, 25.77 (CH2), 11.10 (CH3); MS: m/z 418 (M ), 9 Lolli M L, Cena C, Medana C, Lazzarato L, Morini G, + 420 (M +2). Anal. Calcd for C18H13Cl2N5O3: C, Coruzzi G, Manarini S, Fruttero R & Gasco A, J Med Chem, 51.69; H, 3.13; N, 16.75. Found: C, 51.53; H, 3.23; N, 44 (2001) 3463. 10 Vallance P, Fundam Clin Pharmacol, 17 (2003) 1. 16.56%. 11 Ignarro L J, Int Suppl, 55 (1996) S2. 12 Russwurm M & Koesling D, Mol Cell Biochem, 230 (2002) Conclusions 159. Nine furoxan derivatives of diclofenac were 13 Iwata F & Leung F W, Am J Physol, 268 (1995) G153. synthesized and evaluated for their anti-inflammatory 14 Amir M, Akhter M & Alam O, Monat Chem (2015) DOI 10.1007/s00706-015-1557-x. and nitric oxide releasing properties. Compounds 9, 12, 15 Amir M, Saifullah K & Akhter W, J Enz Inhib Med Chem, 13a, 13b and 15a showing high anti-inflammatory and 26 (2011) 141. analgesic activity were also tested for their ulcerogenic 16 Amir M, Kumar H & Khan S A, Bioorg Med Chem Lett, 18 potential and lipid peroxidation. It was found that (2008) 918. 17 Amir M, Kumar H & Javed S A, Eur J Med Chem, 43 (2008) compounds 12 and 13a showing high anti- 2056. inflammatory activity also exhibited reduced 18 Fruttero R, Ferrarotti B, Serafino A, Stilo A D & Gasco A, J ulcerogenic potential when compared with diclofenac. Heterocycl Chem, 26 (1989) 1345. Furthermore, increased gastro protective activities of 19 Kim G Y, Kim J, Lee S H, Kim H J & Hwang K J, Bull these compounds may be due to the release of NO that Korean Chem Soc, 30 (2009) 459. 20 Collins J C & Hess W W, Org Synth, 6 (1988) 644. promoted mucous secretion and increased mucous 21 Stilo A D, Visentin S, Cena C, Gasco A M, Ermondi G & blood flow resulting in enhanced mucosal resistance to Gasco A, J Med Chem, 41 (1998) 5393. 998 INDIAN J. CHEM., SEC B, AUGUST 2016

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