Identification of quinoline-chalcone hybrids as potential antiulcer agents

European Journal of Medicinal Chemistry 89 (2015) 638e653

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Identification of quinoline-chalcone hybrids as potential antiulcer agents* Koneni V. Sashidhara a, *, Srinivasa Rao Avula a, 1, Vaibhav Mishra b, 1, Gopal Reddy Palnati a, L. Ravithej Singh a, Neetu Singh b, Yashpal S. Chhonker c, Priyanka Swami d, R.S. Bhatta c, Gautam palit b a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India b Pharmacology Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India c Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India d Medicinal Chemistry Division, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229 010, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 February 2014 Received in revised form 22 October 2014 Accepted 23 October 2014 Available online 24 October 2014

Antiulcer activity of novel quinoline-chalcone hybrids (13e37) was investigated. Among them, eight compounds (14, 16, 17, 23, 29, 31, 32 and 35) were found to be active in various ulcer models in Sprague eDawley (SD) rats. To understand the mechanism of action of these hybrids, the effects of the compounds on antisecretory and cytoprotective activities were studied. All these active hybrids improved the depleted levels of mucin and consequently inhibited the formation of erosions in a pyloric ligated ulcer model. In addition, they also significantly increased the gastric PGE2 content in an aspirin induced ulcer model. The additional experiments including the in vitro metabolic stability and in vivo pharmacokinetics led to the identification of compound 17 as an orally active and safe candidate that is worthy of further investigation to be developed as an antiulcer agent. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Quinoline Chalcone Antiulcer activity Pharmacokinetic studies

1. Introduction Peptic ulcer disease (PUD) is a gastrointestinal disease which effects a large population of the world. The gastric mucosal damage and the development of lesions is a complex and multifactorial process, occurring from an imbalance between gastroprotective (mucin, prostaglandin, bicarbonate, nitric oxide, blood supply, etc.) and aggressive factors (acid, pepsin) present in the gastric mucosa [1]. In addition, Helicobacter pylori infection, frequent use of nonsteroidal anti-inflammatory drugs and stress-induced mucosal damage are the risk factors of getting PUD [2]. It is also well established that the reactive oxygen species play an crucial role in the pathogenesis of peptic ulcer, as the free radicals liberated from accumulated neutrophils and monocytes can cause oxidative

*

Part XXXIII in the series, “Advances in drug design and discovery”. * Corresponding author. E-mail addresses: [email protected], [email protected] (K.V. Sashidhara). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ejmech.2014.10.068 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

damage to gastric mucosal cells by protein oxidation and lipid peroxidation [3,4]. To date, antiulcer drugs mainly focus on dwindling the gastric acid secretion and/or strengthening the mucosal defence system. The antisecretory drugs such as histamine H2 receptor antagonists (H2RAs) (Ranitidine), irreversible proton pump inhibitors (PPIs) (Omeprazole) and antacids which treat PUD are based on the principle of either reducing or neutralizing the gastric acid [5]. However, H2RAs and PPIs induce rapid tolerance during therapy and rebound hyper secretion following drug withdrawal, which leads to high ulcer relapse rate. Furthermore, the long term treatment with antisecretory agents can cause serious side effects, such as osteoporosis [6], hypergastrinemia, development of carcinoids in gastric mucosa [7e9] and increased risk of bacterial infection [10]. Sucralfate, a gastric cytoprotective drug, is clinically available, but the usefulness of this drug has been obscure in treating ulcerations caused by NSAID's [11]. Misoprostol, (analogue of prostaglandin E1) can be used to prevent NSAID's (Non steroidal antiinflammatory drugs) associated ulcers, but its application is limited by abnormal side effects [12]. These clinical

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limitations actuate the discovery of more effective and safer antiulcer agents. Quinoline derivatives are versatile biodynamic agents both from synthetic as well as natural origin [13]. Rebamipide (2-(4chlorobenzoylamino)-3-[2-(1H)-quinolinon-4-yl]propionic acid) (1, Fig. 1) is an quinoline derived compound acting as efficient anti gastric ulcer agent, the protective effect of rebamipide is not only because of stimulating endogenous prostaglandin in gastric mucosa, but also inhibiting oxygen derived free radicals production [14]. While, the quinoline derivative 4-(arylamino)quinoline (2, Fig. 1) inhibited the gastric (Hþ/Kþ)-ATPase, the enzyme responsible for the secretion of acid into the gastric lumen [15,16]. Consequently, several research groups have synthesized quinoline based derivatives (including AU-461 (3, Fig. 1) and AS-2646) as potential antiulcer agents [17]. In addition, literature survey revealed that chalcones and its derivatives are largely associated

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with various biological properties [18]. Sofalcone (4, Fig. 1) is an oral gastrointestinal medication used in the treatment of gastritis and peptic ulcer disease [19]. While, Isoliquiritigenin (5, Fig. 1), is the active constituent of Glycyrrhiza uralensis (liquorice) [20]. These studies suggested that compounds containing these privileged scaffolds (quinoline and chalcone) may have significant therapeutic potential for the treatment of ulcer. In the quest for novel drug prototypes, the hybrid approach is a promising one and involves the combination of pharmacophoric moieties of different bioactive substances to produce a hybrid with improved efficacy and reduced undesired side effects. Thus, in continuation of our program targeting the discovery of small molecules and our laboratory experiences on molecular hybridization concept [21], a series of novel quinoline and chalcone hybrids were synthesized (our prototype in Fig. 1) and evaluated for their antiulcer activity in several experimental animal ulcer models.

Fig. 1. Chemical structures of quinoline and chalcones with potent antiulcer activity.

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2. Chemistry The synthesis of intermediate (7e12) and target (13e37) compounds is outlined in Scheme 1. Synthesis of the compounds 7 and 8 was achieved by the nucleophilic substitution of 4, 7-

dichloroquinoline with 1, 2-diaminoethane/1, 3-diaminopropane. The resulting derivatives 7 and 8 were condensed with imidazole2-carboxaldehyde in the presence of ethanol as solvent at room temperature, cleanly afforded compounds 9/10. Furthermore, compounds 9/10 was treated with 4-flouro benzaldehyde under

Reagents and conditions: (i) Ethylenediamine /1,3-diaminopropane, reflux, 8-10 h; (ii) Imidazole-2carboxaldehyde, ethanol, rt, 1-2 h; (iii) p-flouro benzaldehyde, DMF, K2CO3, 110-120 ºC, 6-8 h; (iv) Substituted ketones, 10% KOH-MeOH, rt, 1-2 days Scheme 1. Synthesis of substituted quinoline-chalcone hybrids.

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reflux condition, furnished compounds 11/12 in quantitative yields [22]. Finally, the target quinoline-chalcone hybrids (13e37) were synthesized by a base catalyzed ClaiseneSchmidt condensation on compounds 11/12 with substituted aryl methyl ketones at room temperature. The detailed reaction conditions are illustrated in Scheme 1. All the new synthesized hybrid compounds were confirmed by 1H NMR, 13C NMR, IR spectroscopy and mass spectrometry (see supporting information). The purity of the tested compounds was found to be >95% by HPLC analysis.

3. Results and discussion 3.1. Effect of hybrids (13e37) against cold restraint induced gastric ulcer (CRU) model in rats In our preliminary study, we have screened all these hybrids (13e37) at graded doses (12.5, 25 and 50 mg/kg, p.o.) in cold restraint ulcer model. The effect of omeprazole, a substituted benzimidazole (reference drug) was also investigated for comparison. CRU is a well-accepted model for the induction of gastric ulcers, in which cold and restraint stress alters the regulation of acid secretion, and it increases the acidity of gastric juice through vagal activation [23]. Stress related animal experiments appear to be a very good mimic of the human condition and have allowed studies into pathogenic mechanisms as well as useful therapeutic intervention. A preliminary study for dose fixation was conducted and 25 mg/kg was found to be the optimum dose that can give the highest protection. The screening results are summarized in Table 1. Among the synthesized hybrids 14, 16, 17, 23, 25, 27, 29, 31, 32 and

Table 1 Effect of hybrid compounds (13e37) and reference drug omeprazole on the ulcer index (UI) against cold restraint induced gastric ulcer in rats. Statistical analysis was done by One-Way ANOVA followed by Dunnett's Multiple Comparison Test. *P < 0.05 and **P < 0.01 in comparison to control. n ¼ 6 in each group. CRU Model Comp. no

Control 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Omeprazole (10 mg/kg, p.o)

Ulcer index (UI)

Mean % Protection ulcer score index (PI)

(12.5 mg/ kg, p.o.)

(25 mg/ kg, p.o.)

(50 mg/ kg, p.o.)

(25 mg/kg, (25 mg/kg, p.o.) p.o.)

12 12 9 12 12 9 12 12 12 12 12 9 12 12 12 12 12 12 12 9 9 12 12 9 12 12

12 9 6 12 5.7 6 12 12 12 12 12 3 9 6 9 6 9 6 9 6 6 12 12 6 9 9 2.7

12 9 4.5 12 9 4.5 12 12 12 12 12 3 9 12 9 9 12 6 9 6 6 12 12 6 9 9

20 15 10 20 9.6 10 20 20 20 20 20 05 15 10 15 10 15 10 15 10 10 20 20 10 15 15 4.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.44 0 2.51 25 3.65 50* 1.60 0 2.50 52* 3.05 50* 2.70 0 1.11 0 2.09 0 2.31 0 2.54 0 4.91 75** 2.50 25 2.51 50* 3.50 25 4.66 50* 3.01 25 3.46 50* 2.50 25 3.66 50* 3.00 50* 2.51 0 1.50 0 1.44 50* 2.00 25 2.66 25 2.50 77.5**

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35 exhibited considerable protection in a dose dependent manner. Thus, the significant protection imparted by these compounds in this model reflected the possibility of its involvement in the regulatory mechanism of gastric acid secretion. This interesting finding in CRU model intrigued us to further explore their effects on other gastric ulcer models in rats. 3.2. Effect of active hybrids against alcohol induced gastric ulcer model in rats Based on the primary screening, we chose ten active hybrids (14, 16, 17, 23, 25, 27, 29, 31, 32 and 35) for further pharmacological and biochemical evaluation. The ethanol induced acute gastric mucosal injury model is considered to be one of the important and widely used experimental model for ulcer disease. Ethanol causes the gastric damage by altering protective factors, including decreasing the mucus production and blood circulation within the mucosa [24]. In this alcohol induced gastric lesion model, the hybrids (14, 16, 17, 23, 29, 31, 32 and 35) showed 81.60, 75.03, 84.96, 81.03, 83.82, 59.83, 75.27 and 57.56 percent protection index compared with the ulcer control group respectively (Table 2). The concept of gastric cytoprotection signifies protection against mucosal injury by a mechanism other than inhibition of acid secretion. Interestingly, these hybrids (14, 16, 17, 23, 29, 31, 32 and 35) showed significant protection in ethanol induced gastric lesions in rats, to a greater degree than the reference drug sucralfate (SUC) which showed 64.56% protection (P < 0.01). Sucralfate is an oral cytoprotective drug, primarily indicated for the treatment of active duodenal ulcers. It binds to the mucosa, thus creating a physical barrier that impairs diffusion of hydrochloric acid in the gastrointestinal tract and prevents degradation of mucus by acid. Our active compounds appear to augment the gastric mucosal defence indicating their latent cytoprotective potential. Among all the active hybrid compounds, the compound 17 showed remarkable antiulcer activity (84.96% protection, P < 0.001). The administration of ethanol induced a significant macroscopic damage which was evidenced by presence of ulceration hemorrhagic. Fig. 2 shows the photographs of control, compound 17 treated and reference drug sucralfate treated against alcohol induced gastric ulcer model in rats. It is important to mention that the individual pharmacophores (quinoline and chalcone) did not show significant activity (see the supporting information) demonstrating the importance of the hybrids, which were found to be more potent than their individual pharmacophores.

Table 2 Effect of hybrid compounds (14, 16, 17, 23, 25, 27, 29, 31, 32 and 35) and reference drug sucralfate against alcohol induced gastric ulcer in rats. Data expressed as mean % protection ± S.E.M. Statistical analysis was done by One-Way ANOVA followed by Dunnett's Multiple Comparison Test. *P < 0.05, **P < 0.01 and ***P < 0.001 in comparison to control. n ¼ 6 in each group. Alcohol model (25 mg/kg, p.o.) Comp. no

Total haemorrhagic length % Protection index (PI)

Control 14 16 17 23 25 27 29 31 32 35 Sucralfate (500 mg/kg, p.o)

64.68 11.92 16.17 9.96 12.28 29.93 30.59 10.48 25.98 15.99 27.45 22.92

00.00 81.60*** 75.03** 84.96*** 81.03*** 53.80* 52.79* 83.82*** 59.83* 75.27** 57.56* 64.56**

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Fig. 2. Representative photographs of ethanol induced gastric lesions and protection with compound 17 and sucralfate.

3.3. Effect of active hybrids (14, 16, 17, 23, 29, 31, 32 and 35) in pylorus-ligated rat model To understand the biological basis of antiulcerogenic effect of these active hybrids and to determine whether they have an antisecretory effect in vivo, the test compounds were given intraduodenally to pylorous-ligated (PL) rats [25]. The effects of the hybrids on PL induced gastric ulcer and secretion was examined and compared with the reference drug omeprazole. The compounds 14, 16,17, 23, 29, 31, 32 and 35 showed 6, 6, 9, 6, 6, 9, 9 and 9 ulcer indexes (UI) compared with the ulcer control group respectively (Table 3). Gastric acid plays a central role in ulcer induction in the pyloric ligation model [26]. It is well known that pylorus ligature causes gastric hyper secretion. Moreover, neutrophil migration in the mucosa following pylorus ligature suggests that these cells might be involved in gastric mucosal injury, possibly by the release of free radicals that cause damage to the cell membrane [25]. To find out the effect of these active hybrid compounds (14, 16, 17, 23, 29, 31, 32 and 35) with the gastric secretions, we estimated the free/total acid and mucin content of the accumulated gastric juice in pyloric ligated rats. As evident from Table 4, the treatment with hybrids did not significantly reduce the secretion volume and total acid secretion. However, reference drug omeprazole showed significant lowering of free acidity and total acidity offering 39.72% and 48.17% inhibition respectively over the control group. However, the compounds 14, 16, 17, 23, 29, 31, 32 and 35 significantly up regulated the gastric mucin contents by 35.08%, 33.06%, 43.49%, 30.91%, 34.23%, 27.08%, 30.33% and 24.97% respectively in comparison with the ulcer control group, while omeprazole showed 22.73% increase. Thus, the major mechanism of action of these hybrids appears to be due to their effects on promotion of mucosal protection by endogenous Table 3 Effect of hybrid compounds (14, 16, 17, 23, 29, 31, 32 and 35) and reference drug omeprazole on the ulcer index (UI) against pyloric ligation induced gastric ulcer in rats. Data expressed as ulcer index and % protection. Statistical analysis was done by One-Way ANOVA followed by Dunnett's Multiple Comparison Test. *P < 0.05 and **P < 0.01 in comparison to control. n ¼ 6 in each group. Pyloric ligation model (25 mg/kg, p.o.) Comp. no

Mean ulcer score

Control 14 16 17 23 29 31 32 35 Omeprazole (10 mg/kg, p.o.)

20 10 10 15 10 10 15 15 15 6.6

± ± ± ± ± ± ± ± ± ±

0.90 3.65 1.60 2.46 3.05 2.70 1.11 2.09 2.31 1.89

Ulcer index (UI)

% Protection index (PI)

12 6 6 9 6 6 9 9 9 3.96

0 50* 50* 25 50* 50* 25 25 25 67**

factors (like prostaglandin (PGE2) and inhibition of oxidative stress). This data further complemented our hypothesis that antiulcerogenic effect of these compounds is due to its cytoprotective influences on the gastric mucosa of rats. 3.4. Effect of active hybrids (14, 16, 17, 23, 29, 31, 32 and 35) on aspirin induced gastric ulcers in rats The cytoprotective nature of the hybrids was evident with the increase in mucin content in pyloric ligation model and protection against ethanol induced ulcer model to a greater extent than the reference drugs. In order to provide more compelling evidence for the gastroprotective effect of hybrids, its antiulcer effect against NSAIDs induced ulcer model like aspirin was explored. NSAID are considered another well established and common cause of peptic ulcers in humans. Studies revealed that NSAIDs induces ulcer due to suppression of prostaglandin (PGE2) biosynthesis and disruption of the gastric mucosal barrier [27]. Interestingly, these compounds showed 6, 6, 3, 6, 3, 6, 6 and 9 ulcer index (UI) over the ulcer control group, whereas the reference drug omeprazole showed only 7.5 ulcer index (Table 5) in an aspirin induced gastric ulcer model. All the hybrids (14, 16, 17, 23, 29, 31, 32 and 35) significantly decreased the ulcer incidence, which further reinforces the cytoprotective nature of these compounds, which might be due to increased levels of prostaglandins. Also, the gastroprotective potential of these hybrids in NSAID-induced ulcer model indicate that they could be co administered with NSAID with the aim of minimizing the side effect on gastric mucosa. 3.5. Effect of hybrids (14, 16, 17, 23, 29, 31, 32 and 35) on PGE2 level Since PGE2 play a central role in gastric epithelial defence/repair, we next examined the effect of these compounds on gastric PGE2 Table 4 Effect of active hybrid compounds 14, 16, 17, 23, 29, 31, 32, 35 and Omeprazole (10 mg/kg) on free acidity, total acidity and mucin contents in the pyloric ligation model (n ¼ 6 in each group). Treatment

Free acid mequiv/ Total acid mequiv/ Mucin mg/mL mL mL

Control 14 16 17 23 29 31 32 35 Omeprazole (10 mg/ kg)

47.5 42.60 45.86 44.75 45.20 41.00 45.31 46.85 44.02 28.63

± ± ± ± ± ± ± ± ± ±

4.0 14.89 10.41 9.89 6.29 11.73 7.68 8.05 11.78 1.284**

112.50 99.50 107.00 105.00 106.75 102.25 110.48 109.67 108.31 58.30

± ± ± ± ± ± ± ± ± ±

3.48 13.35 9.27 4.00 12.38 7.47 6.01 4.18 7.25 1.309**

3780.00 5823.00 5647.21 6690.00 5471.50 5747.50 5183.85 5426.05 5038.42 4892.50

± ± ± ± ± ± ± ± ± ±

13.76 8.22* 11.83* 14.83** 11.14* 9.23* 14.93* 18.71* 5.95* 9.918*

*Statistically significant at *P < 0.05 and **P < 0.01 in comparison to control. n ¼ 6 in each group.

K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 89 (2015) 638e653 Table 5 Effect of hybrid compounds (14, 16, 17, 23, 29, 31, 32 and 35) and reference drug omeprazole on the ulcer index (UI) against aspirin induced gastric ulcer in rats. Data expressed as ulcer index and % protection. Statistical analysis was done by One-Way ANOVA followed by Dunnett's Multiple Comparison Test. *P < 0.05 and **P < 0.01 in comparison to control. n ¼ 6 in each group. Aspirin Model (25 mg/kg, p.o.) Comp. no

Mean ulcer score

Control 14 16 17 23 29 31 32 35 Omeprazole (10 mg/kg, p.o.)

20 10 10 5 10 5 10 10 15 12.5

± ± ± ± ± ± ± ± ± ±

1.50 4.65 3.655 4.46 2.50 2.70 2.51 3.66 2.46 2.50

Ulcer index (UI)

% Protection index (PI)

12 6 6 3 6 3 6 6 9 7.5

0 50* 50* 75** 50* 75** 50* 50* 25 37.5

level in aspirin induced gastric ulcer model. We found that PGE2 generation in the ulcer control group was 2756.0 ± 61.87 pg/mg tissue protein. While, the PGE2 value of compounds 14, 16, 17, 23, 29, 31, 32, 35 and misoprostol (prostaglandin analogue) treated group was found to be 3936 ± 64.47 (P < 0.05), 3857 ± 71.67 (P < 0.05), 4224 ± 66.62 (P < 0.01), 3823 ± 57.87 (P < 0.05), 3744 ± 87.31 (P < 0.05), 3439 ± 103.0 (P < 0.05), 3683 ± 170.8 (P < 0.05), 3197 ± 181.30 (P < 0.05) and 4293 ± 47.8 (P < 0.01) respectively (Fig. 3). Results presented in Fig. 3 reveal that the hybrids significantly modulate the PGE2 levels in the gastric homogenate. PGE2 have been shown to be beneficial for gastric ulcer healing wherein it acts as important mediator in gastrointestinal mucosal defence [28]. It is a potent vasodilator which can induce ulcer healing by inhibiting the gastric acid secretion (via action on prostaglandin E (PGE) receptor EP1 and EP3 subtypes or prostacyclin IP receptors) and enhancing the mucosal defence (by stimulating mucus and bicarbonate in the stomach) [29]. Furthermore, the increased gastric PGE2 level reduces the permeability of the epithelium which leads to the reduction of acid back diffusion and induced gastric healing by downregulating the release of a number of inflammatory mediators [30]. Therefore, significant increase in gastric PGE2 levels by the hybrids contributed to the protection of

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gastric mucosa in different ulcer models. In terms of structure activity relationship (SAR) of hybrids in general, the variation in the increasing gastric PGE2 level of these hybrid compounds seems to be modulated by the length of the aliphatic side chain linker and the substitutions on the chalcone ring. In the case of two carbon chain linker (n ¼ 0), we observed compounds (17, 23) with electron releasing groups on R (aryl part) exhibiting potential increasing the gastric PGE2 level than the compounds having heterocyclic ring (32) of chalcone part. Whereas, in the case of three-carbon side chain linker (n ¼ 1), compounds with electron releasing groups (14, 16) displayed effective increasing gastric PGE2 level than compounds with electron withdrawing groups (29, 31) on the chalcone ring. However, the replacement aryl part with the heterocyclic ring (35), did not increase the gastric PGE2 level when compared with other active compounds. Interestingly, compound 17 significantly increased the gastric PGE2 levels and the results were comparable with the reference drug misoprostol (prostaglandin analogue). 3.6. Effect of hybrids (14, 16, 17, 23, 29, 31, 32 and 35) on Hþ KþATPase activity in vitro Though all the above investigations confirmed and reinforced the cytoprotective pathway of ulcer protection, it was necessary to evaluate them for their in vitro antisecretory mechanism of action. Thus, the effects of the hybrids on Hþ Kþ-ATPase (proton pump) activities were examined. As expected (Fig. 4) the hybrids 14, 16, 17, 23, 29, 31, 32 and 35 (10e100 mg/mL) exhibited modest activity in comparison with the control, indicating the lack of antisecretory effects. While omeprazole (an irreversible inhibitor of gastric Hþ Kþ-ATPase) (10e50 mg/mL), significantly reduced the Hþ Kþ-ATPase activity with an IC50 value of 30.24 mg/mL. Gastric Hþ Kþ-ATPase is an enzyme unique for the gastric mucosa that is well known to play a crucial role in the final process of gastric acid secretion. The experimental results obtained with gastric microsomes isolated from rat stomach clearly demonstrated that the mode of antiulcer action of these hybrids is via defensive mechanism (cytoprotective) rather than antisecretory activity. 3.7. Effect of lead hybrid 17 on antioxidant's assays [31] The exquisite and consistent potency of compound 17 in different experimental ulcer models prompted us to choose it for further investigation. It was reported that oxygen free radicals were

Fig. 3. Effect of hybrid compounds (14, 16, 17, 23, 29, 31, 32 and 35) and misoprostol on gastric PGE2 level in comparison to ulcer control group. *Statistically significant at P < 0.05 and **P < 0.01 in comparison to control. n ¼ 6 in each group.

Fig. 4. Effect of active hybrid compounds (14, 16, 17, 23, 29, 31, 32 and 35) and reference drug omeprazole on Hþ Kþ-ATPase activity in the rat gastric microsomes. Dots and lines are mean ± S.E.M. of experiments performed in triplicates (n ¼ 3).

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Table 6 In vitro antioxidant activity of lead hybrid compound 17 on DPPH antioxidant (% inhibition). *Values are expressed as percentage mean of 3 replicates. Concentration of compound 17 (mg/mL) and IC50 values

Scavenging or percentage inhibitory activity of compound 17 in DPPH

10 20 40 60 80 100 IC50 of compound 17 IC50 of ascorbic acid

9.39 ± 2.94 22.06 ± 4.03 38.85 ± 8.52 43.41 ± 6.48 64.84 ± 3.09 70.27 ± 6.75 64.47 ± 3.09 mg/mL 42.5 ± 5.27 mg/mL

Table 7 Effect of lead hybrid 17 and reference ascorbic acid on SOD activity. *Values are expressed as percentage mean of 3 replicates. Treatment

SOD activity in U/mg protein

Control group Compound 17 Ascorbic acid

0.082 ± 0.78 0.179 ± 0.81 0.196 ± 0.69

carried to find out its effect on free radicals and reactive oxygen species. Our results revealed that compound 17 had the property to scavenge the free radicals and reactive oxygen species in a dose dependent manner in DPPH (1,1-diphenyl-2-picryl-hydrazyl) and Superoxide dismutase (SOD) assays. The lead compound 17 exhibited significant inhibition of DPPH as shown in Table 6. It exhibited an IC50 value of 64.47 ± 3.09 mg/mL whereas, the positive control ascorbic acid showed 42.5 ± 5.27 mg/mL in the DPPH inhibition assays. Superoxide dismutase (SOD) was assayed according to Misra and Fridevich based on the inhibition of epinephrine autooxidation by the enzyme. In the control group, the SOD activity was 0.082 ± 0.78 (U/mg protein). However, after the treatment of active hybrid 17, SOD activity was increased to 0.179 ± 0.81 (U/mg protein), while the reference ascorbic acid group exhibited 0.196 ± 0.69 (U/mg protein) (Table 7). The potent antioxidant activity exhibited by the lead hybrid 17 has beneficial implications to play a role in the relief of the oxidative stress and long-term chronic complications that are associated with ulcer pathogenesis.

3.8. Initial toxicity studies on lead hybrid 17 In order to evaluate the toxicity of the lead hybrid compound 17, the MTT assay of compound 17 was performed on gastric cells. The relative number of viable cells/well was determined by formation of blue formazan colour product by the mitochondrial dehydrogenase activity in viable cells. The treatment with the lead hybrid compound 17 at concentrations of 5, 10, 50 and 100 mM showed no toxicity on gastric cells (Fig. 5).

3.9. In vitro metabolism and pharmacokinetic studies of lead 17

Fig. 5. MTT assay of the lead hybrid compound 17.

involved in the development of the ulceration in gastric mucosa. Antioxidants can reduce the oxidative stress and consequently, diminish the progress of stress related diseases like gastric ulceration. Thus, the antioxidant study of active compound 17 was

3.9.1. Optimization of bioanalytical HPLC-PDA method The above positive result prompted us to initiate the metabolism and pharmacokinetic studies on 17. Reversed phase HPLCPDA method was employed for separation and quantitation of lead hybrid compound 17 in rat plasma. A systematic approach was followed and each parameter like stationary phase, mobile phase composition, flow rate etc was carefully optimized for developing a sensitive and reliable HPLC-PDA method. Representative overlay chromatograms of blank plasma and blank plasma fortified with compound 17 at lmax 393 are shown in Fig. 6. This method will therefore be highly useful for future detailed pharmacokinetics and toxicokinetics studies of compound 17 with desired precision and accuracy.

Fig. 6. Representative HPLC-PDA overlay chromatograms of (A) AS blank and AS 2 mg/mL compound 17 spiked in acetonitrile (B) CS blank rat plasma and CS 2 mg/mL compound 17 spiked in rat plasma.

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Fig. 7. A graphical presentation SGF and SIF stabilities of lead compound 17.

3.9.2. Stability of lead 17 The lead compound 17 was stable in both simulated gastric fluid (SGF) and intestinal fluid (SIF) (Fig. 7). This behaviour of compound 17 was favourable for its oral administration and supports its efficacy for antiulcer activity.

Fig. 9. Representative HPLC-PDA overlay chromatograms of (A) Incubated compound 17 with NADPH at 60 min, metabolites (M1, M2 and M3) and (B) compound 17 Incubated without NADPH at 393 nm.

identification of metabolites (in vitro/in vivo), its efficacy and pharmacokinetic evaluation. 3.9.3. In vitro metabolism with rat liver microsomes Metabolism is a major contributor of drug clearance and it directly influences the systemic drug exposure. If metabolites are observed into systemic circulation, they will distribute in tissues and may have pharmacological and/or toxicological effects. The compound 17 metabolic stability at 10 mg/mL was evaluated in pooled rat liver microsomes (RLM). Testosterone was employed as control compound to evaluate the functionality of RLM. The determined intrinsic clearance of these probe substances was comparable to previously reported values. As shown in data presented in Fig. 8, compound 17 was rapidly degraded in RLM, with only 44.5% of the original amount left after 30 min incubation and 80.0% was metabolized after an hour. After incubation of compound 17 with NADPH, three prominent peaks (M1, M2 and M3) were detectable at 2.3, 2.6 and 3.9 min. The M1, M2 and M3 were observed at 393 nm (lmax of compound 17). These peaks were not observed in the control samples (Fig. 9). The calculated in vitro halflife for compound 17 was 30.5 ± 2.7 min. The microsomal intrinsic clearance (CLint, in vitro) of compound 17 was 0.036 mL/min/mg microsomal protein. The predicted in vitro hepatic intrinsic clearance CLint was 64.8 mL/min * Kg of body weight. Compound 17 was stable in simulated gastric and intestinal fluid and it was mainly metabolized by enzymes involved in major metabolizing cellular organelles such as hepatocytes or enterocyte. It was observed that compound 17 shows high in vitro intrinsic clearance with three metabolites (M1, M2 and M3). Detection of M1, M2 and M3 will be a gateway for prospective metabolic studies involving structural

Fig. 8. Time-dependent metabolic depletion of compound 17 in rat liver microsomes (RLM). Metabolic elimination profiles (% turnover or amount remaining vs. incubation time) for: (A) with NADPH and (B) without NADPH. Data are shown as mean ± S.D (n ¼ 3).

3.9.4. In vivo pharmacokinetics Pharmacokinetic studies play an important role in drug development, starting with drug discovery, lead optimization, pharmacology and safety evaluation. The pharmacokinetic and pharmacodynamic data may be helpful in developing dosing regimens and dose escalation strategies. The pharmacokinetic analysis was processed by non-compartmental model using Win Nonlin software Ver 5.1 (Pharsight Corporation, Mountain view, USA). The linear trapezoidal method with linear interpolation was used to calculate pharmacokinetic parameters. The mean plasma concentrationetime profiles of compound 17 after oral administration are shown in Fig. 10 and the main pharmacokinetic parameters are summarized in Table 8. The mean peak concentration (Cmax) 0.46 ± 0.32 mg/mL was achieved at 1.5 ± 0.71 h after oral administration, indicating rapid absorption. The plasma concentration of compound 17 after oral administration decreased with the terminal half-life (t1/2) of 7.67 ± 0.26 h. The volume of distribution and clearance were found to be 49.36 ± 0.20 L/hr/kg and 4.46 ± 0.33 L/h/kg, respectively. After oral administration, the presence of multiple plasma peaks phenomena might be suggestive of absorption of compound 17 from distinct regions of the alimentary tract. Generally, the mechanisms proposed to explain multiple peak phenomenon include biphasic dissolution, site specific absorption and entero-hepatic recycling, as well as other physiological phenomena. In summary, together with our pharmacological finding, this study could provide useful clues

Fig. 10. Pharmacokinetic profile of compound 17 after 25 mg/kg oral administration (n ¼ 3).

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K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 89 (2015) 638e653 Table 8 Pharmacokinetic parameters of compound 17 after 25 mg/kg oral administration (n ¼ 3). Parameters

Estimates (mean ± SD) Oral

Cmax (mg/mL) AUC0e∞(h mg/mL) Tmax (h) Vd/F (L/kg) Cl/F (L/h/kg) t1/2 (h) MRT (h)

0.46 5.61 1.5 49.36 4.46 7.67 12.13

± ± ± ± ± ± ±

0.32 0.43 0.71 0.20 0.33 0.26 0.39

Abbreviation: Cmax: Maximum concentration, Tmax: Time of Maximum concentration, AUC: area under the curve from 0 to ∞ h, Vd/F: uncorrected volume of distribution, Cl/F: uncorrected clearance, t1/2: terminal half life, MRT: mean residence time.

and guidance, such as dosage regimen and application strategy, for developing compound 17 as a promising candidate for the treatment of ulcer. The achieved pharmacokinetics results may be useful for further study of compound 17.

4. Conclusion In conclusion, the present study provide convincing evidence that the newly synthesized quinoline-chalcone hybrids display pronounced gastroprotective activity, as evidenced by the significant inhibition of the formation of ulcers induced by different agents. The modes of antiulcer action of the active hybrids appear to be due to their gastroprotective effect rather than antisecretory activity. The cytoprotection is mainly due to secretion of mucus and the production of prostaglandins. Additional studies on lead 17 revealed its antioxidant aspects, while the preliminary toxicity and pharmacokinetics data confirmed and reinforced the efficacy of the compound 17 as a new class of orally active and safe candidate molecule. Recent reports of serious side effects and frequent ulcer relapse by currently marketed drugs has generated considerable interest in the development of new molecules with improved biological profile. This circumstance heightens the impact of our discovery of lead 17 which will be further evaluated for its clinical utility in gastric ulcer therapy.

5. Experimental section 5.1. General procedure for the synthesis of compounds 7&8 A mixture of 4, 7-dichloroquinoline (1 equiv) and 1, 2diaminoethane/1, 3-diaminopropane (2 equiv) in ethanol/neat condition was heated for 8e10 h at 110/130  C. After completion of the reaction (monitored by TLC) excess solvent was removed under vacuum. The reaction mixture was then poured into ice cold water; the precipitate was filtered and purified by washing with ethyl acetate to furnish compounds 7 and 8 in excellent yields.

5.2. General procedure for the synthesis of compounds 9&10 To a solution of 7/8 (1.0 equiv) in ethanol, imidazole-2carboxaldehyde (1.0 equiv) was added at room temperature under stirring condition. After completion of starting material (monitored by TLC), the reaction mixture was poured into ice cold water, the formed precipitate was filtered and dried under vacuum. Finally, the required Schiff base 9/10 was obtained in good yield.

5.2.1. (E)-N1-((1H-imidazol-2-yl)methylene)-N2-(7chloroquinolin-4-yl)ethane-1,2-diamine (9) White solid, yield: 92%; mp 192e193  C; 1H NMR (DMSO-d6, 300 MHz): d 12.61 (bs, 1H), 8.41 (d, J ¼ 4.0 Hz, 1H), 8.24 (d, J ¼ 6.7 Hz, 1H), 8.20 (bs, 1H), 7.78 (d, J ¼ 1.5 Hz, 1H), 7.45e7.42 (m, 1H), 7.34 (t, J ¼ 3.9 Hz, 1H), 7.20 (bs, 1H), 7.05 (bs, 1H), 6.58 (d, J ¼ 4.0 Hz, 1H), 3.88 (t, J ¼ 4.6 Hz, 2H), 3.60e3.56 (m, 2H); ESI-MS (m/z): 300 (MþH)þ. 5.2.2. (E)-N1-((1H-imidazol-2-yl)methylene)-N3-(7chloroquinolin-4-yl)propane-1,3-diamine (10) White solid, yield: 83%; mp 182e183  C; 1H NMR (DMSO-d6, 300 MHz): d 12.70 (bs, 1H), 8.39 (d, J ¼ 4.0 Hz, 1H), 8.28 (d, J ¼ 6.7 Hz, 1H), 8.24 (s, 1H), 7.78 (d, J ¼ 1.4 Hz, 1H), 7.45 (d, J ¼ 1.3 Hz, 1H), 7.34 (bs, 1H), 7.14 (bs, 2H), 6.49 (d, J ¼ 4.0 Hz, 1H), 3.70 (t, J ¼ 4.8 Hz, 2H), 3.38e3.34 (m, 2H), 2.05e1.99 (m, 2H); ESI-MS (m/z): 314 (MþH)þ. 5.3. General procedure for the synthesis of compounds 11&12 To a solution of 9/10 (1.0 equiv) in DMF, 4-fluoro benzaldehyde (1.0 equiv) followed by potassium carbonate (4.0 equiv) was added. The reaction mixture was heated at 110e120  C for 6e8 h. After completion of reaction (monitored by TLC), reaction mixture was cooled and extracted with EtOAc, resulting solution was concentrated under vacuum and purified by column chromatography. Finally, the required compound 11/12 was obtained in good yield. 5.3.1. 4-(2-(7-chloroquinolin-4-ylamino)ethylamino)benzaldehyde (11) White solid, yield: 85%; mp 184e185  C; 1H NMR (DMSO-d6, 300 MHz): d 9.69 (s, 1H), 8.61 (d, J ¼ 5.0 Hz, 1H), 8.46 (d, J ¼ 9.0 Hz, 1H), 7.96 (d, J ¼ 2.0 Hz, 1H), 7.70 (d, J ¼ 8.6 Hz, 2H), 7.61e7.57 (m, 1H), 7.06 (d, J ¼ 5.1 Hz, 1H), 6.80 (d, J ¼ 8.6 Hz, 3H), 6.58 (s, 1H) 4.32e4.23 (m, 1H), 4.05e4.00 (m, 1H), 3.91e3.86 (m, 1H), 3.77e3.69 (m, 1H); 13C NMR (DMSO-d6, 75 MHz): d 181.7, 152.9, 152.3, 150.5, 149.5, 146.1, 145.0, 133.8, 127.9, 124.5, 124.5, 117.9, 99.1, 58.2, 41.0, 29.5; ESI-MS (m/z): 326 (MþH)þ. 5.3.2. 4-(3-(7-chloroquinolin-4-ylamino)propylamino) benzaldehyde (12) White solid, yield: 85%; mp 192e193  C; 1H NMR (DMSO-d6, 300 MHz): d 9.59 (s, 1H), 8.38 (d, J ¼ 5.4 Hz, 1H), 8.29 (d, J ¼ 9.0 Hz, 1H), 7.78 (d, J ¼ 2.0 Hz, 1H), 7.59 (d, J ¼ 8.5 Hz, 2H), 7.47e7.40 (m, 2H), 6.91 (t, J ¼ 5.0 Hz, 1H), 6.68 (d, J ¼ 8.5 Hz, 2H), 6.51 (d, J ¼ 5.4 Hz, 1H), 3.38 (bs, 2H), 3.30e3.23 (m, 2H), 2.01e1.94 (m, 2H); 13 C NMR (DMSO-d6, 75 MHz): d 189.9, 154.5, 151.2, 151.1, 148.3, 134.3, 132.3, 126.9, 125.3, 124.8, 117.7, 111.7, 99.1, 40.8, 40.7, 27.5; ESIMS (m/z): 340 (MþH)þ. 5.4. General procedure for the synthesis of (E)-3-(4-(2-(7chloroquinolin-4-ylamino)ethylamino)phenyl)-1-(4methoxyphenyl)prop-2-en-1-one (17) To a solution of 11 (325 mg, 1 mmol) in 10% methanolic KOH, 4methoxy acetophenone (150 mg, 1 mmol) was added, the reaction mixture was stirred at room temperature until reaction was completed (monitored by TLC). After completion of reaction (36 h), it was neutralized by dil. HCl and extracted with chloroform, organic solvent concentrated under vacuum further purified by column chromatography. Finally, the required compound (17) was obtained in 71% yields as yellow colour solid. The remaining compounds (13e37) were prepared in a similar way by the procedure described above.

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5.4.1. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-phenylprop-2-en-1-one (13) Reddish yellow solid, yield: 81%; mp 195e197  C; IR (KBr, cm1): 3449, 3019, 2912, 1645, 1217, 765; 1H NMR (DMSO-d6, 300 MHz): d 8.42 (d, J ¼ 5.4 Hz, 1H), 8.25 (d, J ¼ 9.1 Hz, 1H), 8.08 (d, J ¼ 7.1 Hz, 2H), 7.80 (d, J ¼ 2.0 Hz, 1H), 7.68e7.60 (m, 4H), 7.56e7.54 (m, 2H), 7.51e7.46 (m, 2H), 7.43 (s, 1H), 6.68 (d, J ¼ 8.6 Hz, 2H), 6.60e6.59 (m, 1H), 6.55 (d, J ¼ 5.4 Hz, 1H), 3.51e3.34 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 188.7, 151.7, 151.2, 150.2, 148.8, 145.4, 138.4, 133.6, 132.4, 131.1, 128.6, 128.1, 127.3, 124.2, 124.0, 122.2, 117.4, 115.6, 111.9, 98.7, 41.6, 40.8; ESI-MS (m/z): 428 (MþH)þ; HRMS (m/z): calcd for C26H22ClN3O (MþH)þ: 428.1530, Found: 428.1534.

5.4.6. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (18) Yellow solid, yield: 78%; mp 215e216  C; IR (KBr, cm1): 3435, 2954, 2900, 1585, 1161, 1030, 762; 1H NMR (DMSO-d6, 300 MHz): d 8.39 (d, J ¼ 5.1 Hz, 1H), 8.29 (d, J ¼ 9.0 Hz, 1H), 8.10 (d, J ¼ 8.7 Hz, 2H), 7.79 (d, J ¼ 2.0 Hz, 1H), 7.64e7.53 (m, 4H), 7.48e7.41 (m, 2H), 7.05 (d, J ¼ 8.8 Hz, 2H), 6.64 (d, J ¼ 8.6 Hz, 2H), 6.55e6.49 (m, 2H), 3.85 (s, 3H), 3.38 (bs, 2H), 3.25e3.17 (m, 2H), 1.99e1.91 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 186.9, 162.6, 151.5, 151.2, 150.2, 148.6, 144.5, 133.5, 131.1, 130.8, 130.4, 127.1, 124.1, 122.1, 117.4, 115.4, 113.8, 111.8, 98.7, 55.4, 40.3, 40.0, 27.2; ESI-MS (m/z): 472 (MþH)þ; HRMS (m/z):calcd for C28H26ClN3O2 (MþH)þ: 472.1792, Found: 472.1758.

5.4.2. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-phenylprop-2-en-1-one (14) Yellow solid, yield: 71%; mp 178e179  C; IR (KBr, cm1): 3400, 3015, 2951, 1638, 1220, 770; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.5 Hz, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.08 (d, J ¼ 7.0 Hz, 2H), 7.78 (d, J ¼ 2.1 Hz, 1H), 7.67e7.51 (m, 7H), 7.47e7.43 (m, 1H), 7.33 (d, J ¼ 5.0 Hz, 1H), 6.64 (d, J ¼ 8.6 Hz, 2H), 6.56e6.50 (m, 2H), 3.37 (bs, 2H), 3.29e3.24 (m, 2H), 1.99e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 188.7, 152.0, 151.6, 150.1, 149.1, 145.5, 138.4, 133.4, 132.4, 131.1, 128.6, 128.1, 127.4, 124.1, 122.0, 117.5, 115.5, 111.9, 98.8, 40.3, 40.2, 27.3; ESI-MS (m/z): 442 (MþH)þ; HRMS (m/z): calcd for C27H24ClN3O (MþH)þ: 442.1686, Found: 442.1688.

5.4.7. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (19) Yellow solid, yield: 68%; mp 169e171  C; IR (KBr, cm1): 3431, 3020, 2901,1582, 1217, 1072, 756; 1H NMR (DMSO-d6, 300 MHz): d 8.41 (d, J ¼ 5.3 Hz, 1H), 8.25 (d, J ¼ 9.0 Hz, 1H), 7.84e7.79 (m, 2H), 7.65e7.58 (m, 5H), 7.49e7.45 (m, 1H), 7.39 (bs, 1H), 7.07 (d, J ¼ 8.4 Hz, 1H), 6.67 (d, J ¼ 8.4 Hz, 2H), 6.59e6.53 (m, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 3.50e3.44 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 186.9, 152.6, 151.8, 150.9, 150.0, 149.0, 148.6, 144.3, 133.4, 131.2, 130.9, 127.4, 124.1, 124.0, 122.6, 122.4, 117.4, 115.6, 111.9, 110.8, 110.7, 98.6, 55.6, 55.5, 41.5, 40.8; ESI-MS (m/z): 488 (MþH)þ; HRMS (m/z): calcd for C28H26ClN3O3 (MþH)þ: 488.1741, Found: 488.1713.

5.4.3. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-p-tolylprop-2-en-1-one (15) Yellow solid, yield: 79%; mp 210e212  C; IR (KBr, cm1): 3530, 3027, 2934, 1640, 1215, 1035, 781; 1H NMR (DMSO-d6, 300 MHz): d 8.40 (d, J ¼ 5.4 Hz, 1H), 8.31 (d, J ¼ 9.0 Hz, 1H), 8.00 (d, J ¼ 8.0 Hz, 2H), 7.79 (d, J ¼ 2.1 Hz, 1H), 7.66e7.61 (m, 3H), 7.56 (d, J ¼ 15.4 Hz, 1H), 7.47e7.43 (m, 1H), 7.34 (d, J ¼ 8.0 Hz, 2H), 6.67 (d, J ¼ 8.6 Hz, 2H), 6.53 (d, J ¼ 5.4 Hz, 1H), 3.50e3.49 (m, 4H), 2.38 (s, 3H); 13C NMR (DMSO-d6, 75 MHz): d 188.2, 151.9, 151.2, 150.1, 149.1, 145.0, 142.8, 135.8, 133.5, 131.0, 129.2, 128.3, 127.4, 124.3, 124.1, 122.2, 117.5, 115.6, 111.9, 98.7, 41.5, 40.8, 21.7; ESI-MS (m/z): 442 (MþH)þ; HRMS (m/z): calcd for C27H24ClN3O (MþH)þ: 442.1686, Found: 442.1708. 5.4.4. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-p-tolylprop-2-en-1-one (16) Yellow solid, yield: 85%; mp 244e245  C; IR (KBr, cm1): 3569, 3001, 2930, 1647, 1218, 1037, 772; 1H NMR (DMSO-d6, 300 MHz): d 8.52 (s, 1H), 8.38e8.32 (m, 2H), 7.99 (d, J ¼ 8.0 Hz, 2H), 7.77 (d, J ¼ 1.9 Hz, 1H), 7.65e7.57 (m, 3H), 7.52e7.42 (m, 2H), 7.34 (d, J ¼ 8.0 Hz, 2H), 6.64 (d, J ¼ 8.6 Hz, 3H), 6.50 (d, J ¼ 5.4 Hz, 1H), 3.42 (bs, 2H), 3.24e3.22 (m, 2H), 2.38 (s, 3H), 1.97e1.93 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 188.6, 166.4, 152.3, 151.9, 150.6, 149.5, 145.6, 143.1, 136.3, 133.8, 131.4, 129.6, 128.7, 124.7, 124.4, 122.3, 118.0, 112.3, 99.1, 40.7, 40.4, 27.5, 21.5; ESI-MS (m/z): 456 (MþH)þ; HRMS (m/z): calcd for C28H26ClN3O (MþH)þ: 456.1843, Found: 456.1828. 5.4.5. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (17) Yellow solid, yield: 71%; mp 175e176  C; IR (KBr, cm1): 3579, 3024, 2913, 1582, 1222, 1026, 742; 1H NMR (DMSO-d6, 300 MHz): d 8.29 (d, J ¼ 8.9 Hz, 1H), 8.20 (d, J ¼ 5.5 Hz, 1H), 8.10 (d, J ¼ 8.7 Hz, 2H), 7.65e7.54 (m, 5H), 7.31e7.28 (m, 1H), 7.05 (d, J ¼ 8.7 Hz, 2H), 6.67 (d, J ¼ 8.5 Hz, 2H), 6.31 (d, J ¼ 5.5 Hz, 1H), 3.85 (s, 3H), 3.41 (bs, 4H); 13C NMR (DMSO-d6, 75 MHz): d 187.0, 162.7, 152.0, 151.3, 151.2, 149.6, 144.5, 132.8, 131.2, 130.8, 130.4, 126.6, 124.7, 122.5, 122.1, 119.5, 115.3, 113.8, 111.9, 98.0, 55.5, 43.3, 41.8; ESI-MS (m/z): 458 (MþH)þ; HRMS (m/z): calcd for C27H24ClN3O2 (MþH)þ: 458.1635, Found: 458.1582.

5.4.8. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(3,4-dimethoxyphenyl)prop-2-en-1-one (20) Yellow solid, yield: 73%; mp 173e175  C; IR (KBr, cm1): 3472, 3064, 2907,1592, 1282, 1082, 734; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.2 Hz, 1H), 8.28 (d, J ¼ 9.0 Hz, 1H), 7.83e7.77 (m, 2H), 7.63e7.58 (m, 5H), 7.47e7.43 (m, 1H), 7.34 (bs, 1H), 7.07 (d, J ¼ 8.5 Hz, 1H), 6.64 (d, J ¼ 8.4 Hz, 2H), 6.51e6.48 (m, 2H),3.85 (s, 3H), 3.85 (s, 3H), 3.38e3.36 (m, 2H), 3.28e3.23 (m, 2H), 1.98e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 186.9, 152.6, 151.8, 151.3, 150.0, 149.0, 148.6, 144.4, 133.3, 131.3, 130.8, 127.4, 124.0, 124.0, 122.6, 122.1, 117.4, 115.3, 111.8, 110.8, 110.3, 98.7, 55.6, 55.5, 40.3, 40.2, 27.3; ESI-MS (m/z): 502 (MþH)þ; HRMS (m/z): calcd for C29H28ClN3O3 (MþH)þ: 502.1897, Found: 502.1878. 5.4.9. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (21) Reddish yellow solid, yield: 79%; mp 178e180  C; IR (KBr, cm1): 3541, 3060, 2976, 1591, 1286, 1065, 789; 1H NMR (DMSO-d6, 300 MHz): d 8.41 (d, J ¼ 4.8 Hz, 1H), 8.25 (d, J ¼ 8.9 Hz, 1H), 7.80 (s, 1H), 7.68e7.62 (m, 4H), 7.48e7.37 (m, 4H), 6.68 (d, J ¼ 8.1 Hz, 2H), 6.60 (s, 1H), 6.54 (d, J ¼ 5.3 Hz, 1H), 3.89 (s, 6H), 3.75 (s, 3H), 3.44 (bs, 4H); 13C NMR (DMSO-d6, 75 MHz): d 187.3, 152.8, 151.7, 151.1, 150.1, 148.8, 145.1, 141.4, 133.8, 133.5, 131.1, 130.7, 127.3, 124.1, 124.0, 122.3, 121.79, 117.4, 115.5, 111.8, 105.8, 98.6, 60.1, 56.1, 41.6, 40.8; ESIMS (m/z): 518 (MþH)þ; HRMS (m/z): calcd for C29H28ClN3O4 (MþH)þ: 518.1847, Found: 518.1822. 5.4.10. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (22) Yellow solid, yield: 73%; mp 170e171  C; IR (KBr, cm1): 3521, 3024, 2985, 1601, 1267, 1046, 709; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.2 Hz, 1H), 8.28 (d, J ¼ 9.0 Hz, 1H), 7.77 (s, 1H), 7.66e7.60 (m, 4H), 7.45 (d, J ¼ 7.0 Hz, 1H), 7.37e7.34 (m, 3H), 6.64 (d, J ¼ 8.5 Hz, 2H), 6.54e6.49 (m, 2H), 3.89 (s, 6H), 3.74 (s, 3H), 3.39e3.37 (m, 2H), 3.27e3.3.23 (m, 2H), 1.99e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 187.4, 152.9, 152.7, 151.9, 151.5, 150.1, 149.1, 145.3, 141.5, 133.9, 133.4, 131.2, 127.5, 124.1, 124.1, 122.1, 117.5, 115.3, 111.8, 105.8, 98.8, 60.2, 56.2, 40.4, 40.3, 27.3; ESI-MS (m/z):

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532 (MþH)þ; HRMS (m/z): calcd for C30H30ClN3O4 (MþH)þ: 532.2003, Found: 532.1989. 5.4.11. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-(2-(7-chloroquinolin4-ylamino)ethylamino)phenyl)prop-2-en-1-one (23) Yellow solid, yield: 74%; mp 185e186  C; IR (KBr, cm1): 3501, 3069, 2961, 1656, 1232, 765; 1H NMR (DMSO-d6, 300 MHz): d 8.39 (d, J ¼ 5.3 Hz, 1H), 8.34 (d, J ¼ 8.6 Hz, 1H), 7.79e7.78 (m, 2H), 7.64e7.53 (m, 6H), 7.47e7.43 (m, 1H), 7.05 (d, J ¼ 8.1 Hz, 1H), 6.75 (bs, 1H), 6.67 (d, J ¼ 8.5 Hz, 2H), 6.53 (d, J ¼ 5.3 Hz, 1H), 6.13 (d, J ¼ 5.1 Hz, 2H), 3.41 (bs, 4H); 13C NMR (DMSO-d6, 75 MHz): d 186.5, 151.9, 151.0, 150.9, 150.0, 149.0, 147.8, 144.7, 133.4, 133.0, 130.9, 127.5, 124.3, 124.1, 124.0, 122.4, 117.4, 115.5, 111.8, 107.9, 107.7, 101.8, 98.7, 41.5, 40.8; ESI-MS (m/z): 472 (MþH)þ; HRMS (m/z): calcd for C27H22ClN3O3 (MþH)þ: 472.1428, Found: 472.1352. 5.4.12. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-(3-(7-chloroquinolin4-ylamino)propylamino)phenyl)prop-2-en-1-one (24) Yellow solid, yield: 72%; mp 240e241  C; IR (KBr, cm1): 3501, 3027, 2922, 1576, 1218, 1048, 768; 1H NMR (DMSO-d6, 300 MHz): d 8.38e8.33 (m, 2H), 7.79e7.77 (m, 2H), 7.62e7.57 (m, 5H), 7.51e7.42 (m, 2H), 7.05 (d, J ¼ 8.1 Hz, 1H), 6.63 (d, J ¼ 8.4 Hz, 3H), 6.50 (d, J ¼ 5.3 Hz, 1H), 6.13 (d, J ¼ 4.8 Hz, 2H), 3.41 (bs, 2H), 3.24e3.22 (m, 2H), 1.97e1.93 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 186.5, 165.6, 152.8, 151.9, 151.4, 150.9, 150.0, 149.1, 147.8, 144.9, 133.3, 133.1, 131.0, 127.4, 124.3, 123.9, 122.0, 117.5, 115.2, 111.7, 108.0, 107.7, 98.6, 40.3, 40.0, 27.0; ESI-MS (m/z): 486 (MþH)þ; HRMS (m/z): calcd for C28H24ClN3O3 (MþH)þ: 486.1584, Found: 486.1548. 5.4.13. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(4-fluorophenyl) prop-2-en-1-one (25) Reddish yellow solid, yield: 70%; mp 248e249  C; IR (KBr, cm1): 3512, 3015, 2894, 1632, 1234, 760; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.4 Hz, 1H), 8.29 (d, J ¼ 9.0 Hz, 1H), 8.20e8.15 (m, 2H), 7.78 (d, J ¼ 2.1 Hz, 1H), 7.68e7.61 (m, 3H), 7.57 (d, J ¼ 15.3 Hz, 1H), 7.48e7.32 (m, 4H), 6.64 (d, J ¼ 8.6 Hz, 2H), 6.57 (t, J ¼ 5.4 Hz, 1H), 6.52 (d, J ¼ 5.4 Hz, 1H), 3.41 (bs, 2H), 3.25e3.23 (m, 2H), 1.98e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 187.1, 151.6, 151.5, 150.3, 148.6, 145.7, 135.1, 133.6, 131.1, 131.1, 130.9, 127.1, 124.1, 121.9, 117.4, 115.7, 115.4, 115.1, 111.8, 98.7, 40.3, 40.2, 27.2; ESIMS (m/z): 460 (MþH)þ; HRMS (m/z):calcd for C27H23ClFN3O (MþH)þ: 460.1592, Found: 460.1560.

148.2, 146.0, 137.4, 137.1, 133.8, 131.3, 130.1, 128.7, 126.8, 124.3, 121.9, 117.3, 114.9, 111.9, 98.7, 40.3, 40.0, 27.2; ESI-MS (m/z): 476 (MþH)þ; HRMS (m/z):calcd for C27H23Cl2N3O (MþH)þ: 476.1230, Found: 476.1261. 5.4.16. (E)-1-(4-bromophenyl)-3-(4-(2-(7-chloroquinolin-4ylamino)ethylamino)phenyl)prop-2-en-1-one (28) Reddish yellow solid, yield: 69%; mp 170e171  C; IR (KBr, cm1): 3435, 3024, 2923, 1585, 1504, 1243, 1076, 782; 1H NMR (DMSO-d6, 300 MHz): d 8.39 (d, J ¼ 5.3 Hz, 1H), 8.34 (d, J ¼ 9.2 Hz, 1H), 8.03 (d, J ¼ 8.4 Hz, 2H), 7.78e7.72 (m, 3H), 7.68e7.60 (m, 4H), 7.55 (d, J ¼ 15.3 Hz, 1H), 7.46e7.43 (m, 1H), 6.85 (bs, 1H), 6.67 (d, J ¼ 8.5 Hz, 2H), 6.53 (d, J ¼ 5.4 Hz, 1H), 3.41 (bs, 4H); 13C NMR (DMSO-d6, 75 MHz): d 187.6, 151.9, 151.5, 150.1, 149.1, 146.0, 137.4, 133.4, 131.6, 131.3, 130.2, 127.4, 126.4, 124.3, 124.1, 122.0, 117.5, 115.0, 111.9, 98.6, 41.4, 40.8; ESI-MS (m/z): 506 (MþH)þ; HRMS (m/z):calcd for:C26H21ClBrN3O (MþH)þ: 506.0635, Found: 506.0616. 5.4.17. (E)-1-(4-bromophenyl)-3-(4-(3-(7-chloroquinolin-4ylamino)propylamino)phenyl)prop-2-en-1-one (29) Yellow solid, yield: 67%; mp 178e179  C; IR (KBr, cm1): 3556, 3043, 2908, 1581, 1171, 1032, 734; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.4 Hz, 1H), 8.29 (d, J ¼ 8.2 Hz, 1H), 8.49 (d, J ¼ 8.0 Hz, 2H), 7.79e7.76 (m, 3H), 7.69e7.61 (m, 3H), 7.54 (d, J ¼ 15.3 Hz, 1H), 7.48e7.45 (m, 2H), 6.65e6.59 (m, 3H), 6.52 (d, J ¼ 5.5 Hz, 1H), 3.42 (bs, 2H), 3.27e3.21 (m, 2H), 1.98e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 187.5, 151.6, 150.8, 150.6, 147.6, 146.0, 137.4, 134.0, 131.6, 131.2, 130.2, 126.3, 124.4, 121.8, 117.2, 114.9, 111.8, 98.7, 40.3, 40.0, 27.1; ESI-MS (m/z): 520 (MþH)þ; HRMS (m/z):calcd for C27H23ClBrN3O (MþH)þ:520.0791, Found: 520.0793. 5.4.18. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (30) Orange solid, yield: 71%; mp 170e171  C; IR (KBr, cm1): 3502, 3028, 2899, 1597, 1223, 1086, 768; 1H NMR (DMSO-d6, 300 MHz): d 8.42e8.36 (m, 3H), 8.25 (d, J ¼ 9.0 Hz, 1H), 7.98 (d, J ¼ 7.6 Hz, 1H), 7.81e7.68 (m, 6H), 7.49e7.54 (m, 1H), 7.41 (bs, 1H), 6.70e6.67 (m, 3H), 6.54 (d, J ¼ 5.4 Hz, 1H), 3.48e3.45 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 187.1, 151.7, 151.5, 150.0, 148.9, 146.5, 139.1, 133.4, 132.0, 131.4, 129.8, 129.7, 129.2, 128.6, 127.3, 124.4, 124.1, 124.0, 122.1, 117.4, 114.9, 111.8, 98.6, 41.5, 40.7; ESI-MS (m/z): 496 (MþH)þ; HRMS (m/ z):calcd for C27H21ClF3N3O (MþH)þ: 496.1403, Found: 496.1398.

5.4.14. (E)-1-(4-chlorophenyl)-3-(4-(2-(7-chloroquinolin-4ylamino)ethylamino)phenyl)prop-2-en-1-one (26) Yellow solid, yield: 72%; mp 202e203  C; IR (KBr, cm1): 3399, 3020, 2934,1665, 1219, 1086, 776; 1H NMR (DMSO-d6, 300 MHz): d 8.41 (d, J ¼ 5.4 Hz, 1H), 8.25 (d, J ¼ 9.0 Hz, 1H), 8.11 (d, J ¼ 8.5 Hz, 2H), 7.80 (d, J ¼ 2.1 Hz, 1H), 7.70e7.54 (m, 6H), 7.48e7.41 (m, 2H), 6.69e6.63 (m, 3H), 6.53 (d, J ¼ 5.4 Hz, 1H), 3.49e3.46 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 187.4, 151.7, 151.3, 150.1, 148.8, 145.9, 137.3, 137.0, 133.9, 131.2, 130.0, 128.7, 127.3, 124.2, 124.0, 122.1, 117.4, 115.1, 111.9, 98.7, 41.6, 40.8; ESI-MS (m/z): 462 (MþH)þ; HRMS (m/ z):calcd for C26H21Cl2N3O (MþH)þ: 462.1140, Found: 462.1149.

5.4.19. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (31) Orange solid, yield: 77%; mp 167e169  C; IR (KBr, cm1): 3505, 3009, 2935, 1589, 1289, 1076, 709; 1H NMR (DMSO-d6, 300 MHz): d 8.41e8.36 (m, 3H), 8.28 (d, J ¼ 8.9 Hz, 1H), 7.98 (d, J ¼ 7.3 Hz, 1H), 7.81e7.76 (m, 2H), 7.69e7.61 (m, 4H), 7.45 (d, J ¼ 8.8 Hz, 1H), 7.35 (s, 1H), 6.66e6.63 (m, 3H), 6.50 (d, J ¼ 5.1 Hz, 1H), 3.39e3.37 (m, 2H), 3.26e3.24 (m, 2H), 1.99e1.97 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 187.1, 151.8, 150.1, 149.0, 146.7, 139.1, 133.4, 132.1, 131.5, 129.9, 129.7, 129.3, 128.7, 127.4, 124.5, 124.1, 124.0, 121.8, 117.5, 114.6, 111.8, 98.7, 40.2, 40.2, 27.2; ESI-MS (m/z): 510 (MþH)þ; HRMS (m/ z):calcd for C28H23ClF3N3O (MþH)þ: 510.1560, Found: 510.1552.

5.4.15. (E)-1-(4-chlorophenyl)-3-(4-(3-(7-chloroquinolin-4ylamino)propylamino) phenyl)prop-2-en-1-one (27) Reddish yellow, yield: 69%; mp 212e213  C; IR (KBr, cm1): 3486, 3027, 2931, 1645, 1218, 1085, 768; 1H NMR (DMSO-d6, 300 MHz): d 8.39 (d, J ¼ 5.2 Hz, 1H), 8.30 (d, J ¼ 9.0 Hz, 1H), 8.10 (d, J ¼ 8.49 Hz, 2H), 7.79 (d, J ¼ 1.9 Hz, 1H), 7.69e7.58 (m, 6H), 7.52e7.45 (m, 2H), 6.64 (d, J ¼ 8.5 Hz, 2H), 6.58 (t, J ¼ 5.3 Hz, 1H), 6.53 (d, J ¼ 5.5 Hz, 1H), 3.40 (bs, 2H), 3.26e3.24 (m, 2H), 1.99e1.95 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 187.4, 151.7, 151.2, 150.5,

5.4.20. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(furan-2-yl)prop-2-en-1-one (32) Orange solid, yield: 64%; mp 157e159  C; IR (KBr, cm1): 3498, 3098, 2912, 1598, 1245, 1087, 754; 1H NMR (DMSO-d6, 300 MHz): d 8.41 (d, J ¼ 5.1 Hz, 1H), 8.24 (d, J ¼ 9.0 Hz, 1H), 7.99 (bs, 1H), 7.79 (d, J ¼ 1.7 Hz, 1H), 7.65e7.58 (m, 4H), 7.49e7.45 (m, 1H), 7.38e7.33 (m, 2H), 6.74e6.73 (m, 1H), 6.67 (d, J ¼ 8.5 Hz, 2H), 6.63e6.61 (m, 1H), 6.53 (d, J ¼ 5.4 Hz, 1H), 3.48e3.44 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 176.7, 153.4, 151.7, 151.2, 150.1, 148.8, 147.3, 144.0, 133.5,

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130.9, 127.3, 124.1, 124.0, 122.0, 117.7, 117.4, 115.5, 112.4, 111.9, 98.7, 41.5, 40.8; ESI-MS (m/z): 418 (MþH)þ; HRMS (m/z): calcd for C24H20ClN3O2 (MþH)þ: 418.1322, Found: 418.1315. 5.4.21. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(furan-2-yl)prop-2-en-1-one (33) Yellow solid, yield: 62%; mp 165e167  C; IR (KBr, cm1): 3499, 2998, 2901, 1567, 1235, 1091, 784; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.4 Hz, 1H), 8.28 (d, J ¼ 9.0 Hz, 1H), 7.99 (d, J ¼ 1.0 Hz, 1H), 7.78 (d, J ¼ 2.1 Hz, 1H), 7.64e7.62 (m, 1H), 7.59 (s, 2H), 7.56 (s, 1H), 7.47e7.43 (m, 1H), 7.37e7.31 (m, 2H), 6.74 (dd, J ¼ 1.6 Hz, J ¼ 3.4 Hz, 1H), 6.64 (d, J ¼ 8.6 Hz, 2H), 6.57e6.53 (m, 1H), 6.50 (d, J ¼ 5.4 Hz, 1H), 3.41e3.37 (m, 2H), 3.27e3.21 (m, 2H), 1.98e1.91 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 176.7, 153.5, 151.9, 151.6, 150.1, 149.1, 147.4, 144.2, 133.4, 131.0, 127.5, 124.1, 124.1, 121.8, 117.7, 117.5, 115.3, 112.5, 111.9, 98.8, 40.2, 40.1, 27.3; ESI-MS (m/z): 432 (MþH)þ; HRMS (m/z): calcd for C25H22ClN3O2 (MþH)þ: 432.1479, Found: 432.1449.

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75 MHz): d 187.6, 152.6, 151.7, 150.4, 150.1, 149.3, 148.8, 146.2, 141.4, 135.5, 133.5, 133.4, 131.3, 127.3, 124.1, 123.7, 121.7, 117.4, 115.1, 111.8, 98.7, 60.1, 56.1, 40.3, 40.0, 27.2; ESI-MS (m/z): 443 (MþH)þ; HRMS (m/z): calcd for: C26H23ClN4O (MþH)þ:443.1639, Found: 443.1629. 6. Biological assays 6.1. Materials and animals All the chemicals used in the study were obtained from M/s. Sigma Chemicals without further purification. Experimental protocols were agreed by the Institutional Ethical and Usage Committee of Central Drug Research Institute (CDRI), Lucknow, India, following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Adult SpragueeDawley rats, procured from National Laboratory Animal Centre, CDRI, were used for these studies. 6.2. Treatment schedule

5.4.22. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(thiophen-2-yl)prop-2-en-1-one (34) Yellow solid, yield: 71%; mp 173e175  C; IR (KBr, cm1): 3484, 3042, 2909, 1598, 1243, 1065, 767; 1H NMR (DMSO-d6, 300 MHz): d 8.41 (d, J ¼ 4.5 Hz, 1H), 8.26e8.19 (m, 2H), 7.97 (d, J ¼ 4.8 Hz, 1H), 7.79 (d, J ¼ 1.5 Hz, 1H), 7.65e7.60 (m, 3H), 7.54e7.45 (m, 2H), 7.39 (bs, 1H), 7.28e7.26 (m, 1H), 6.68 (d, J ¼ 8.4 Hz, 2H), 6.63e6.61 (m, 1H), 6.54 (d, J ¼ 5.3 Hz, 1H), 3.50e3.44 (m, 4H); 13C NMR (DMSO-d6, 75 MHz): d 181.2, 151.8, 151.2, 150.0, 149.0, 146.3, 144.4, 134.2, 133.4, 132.1, 131.0, 128.6, 127.4, 124.1, 124.0, 122.0, 117.4, 115.4, 111.9, 98.7, 41.5, 40.8; ESI-MS (m/z): 434 (MþH)þ; HRMS (m/z): calcd for: C24H20ClN3OS (MþH)þ: 434.1094, Found: 434.1085. 5.4.23. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(thiophen-2-yl)prop-2-en-1-one (35) Reddish yellow solid, yield: 73%; mp 170e171  C; IR (KBr, cm1): 3561, 3087, 2887, 1589, 1224, 1087, 783; 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, J ¼ 5.2 Hz, 1H), 8.28 (d, J ¼ 9.0 Hz, 1H), 8.19 (d, J ¼ 3.3 Hz, 1H), 7.96 (d, J ¼ 4.7 Hz, 1H), 7.78 (d, J ¼ 1.8 Hz, 1H), 7.64e7.60 (m, 3H), 7.53 (s, 1H), 7.48e7.43 (m, 1H), 7.34 (s, 1H), 7.28e7.25 (m, 1H), 6.64 (d, J ¼ 8.4 Hz, 2H), 6.57e6.54 (m, 1H), 6.50 (d, J ¼ 5.4 Hz, 1H), 3.39 (bs, 2H), 3.25e3.23 (m, 2H), 1.98e1.94 (m, 2H); 13C NMR (DMSO-d6, 75 MHz): d 181.2, 151.8, 151.5, 150.0, 149.0, 146.3, 144.5, 134.1, 133.3, 132.1, 131.0, 128.6, 127.4, 124.0, 124.0, 121.7, 117.4, 115.2, 111.8, 98.7, 40.2, 40.1, 27.2; ESI-MS (m/z): 448 (MþH)þ; HRMS (m/z): calcd for C25H22ClN3OS (MþH)þ: 448.1250, Found: 448.1232. 5.4.24. (E)-3-(4-(2-(7-chloroquinolin-4-ylamino)ethylamino) phenyl)-1-(pyridin-3-yl)prop-2-en-1-one (36) Reddish yellow solid, yield: 63%; mp 181e183  C; IR (KBr, cm1): 3561, 3034, 2924, 1602, 1224, 1091, 751; 1H NMR (DMSO-d6, 300 MHz): d 9.26 (s, 1H), 8.78 (s 1H), 8.42 (s, 2H), 8.25 (d, J ¼ 8.9 Hz, 1H), 7.79 (s, 1H), 7.72e7.55 (m, 5H), 7.49e7.44 (m, 2H), 6.69e6.67 (m, 3H), 6.55 (d, J ¼ 4.8 Hz, 1H), 3.48 (s, 4H); ESI-MS (m/z): 429 (MþH)þ; HRMS (m/z): calcd for: C25H21ClN4O (MþH)þ: 429.1482, Found: 429.1011. 5.4.25. (E)-3-(4-(3-(7-chloroquinolin-4-ylamino)propylamino) phenyl)-1-(pyridin-3-yl)prop-2-en-1-one (37) Reddish yellow solid, yield: 66%; mp 179e181  C; IR (KBr, cm1): 3534, 3018, 2877,1598, 1267, 1081, 778; 1H NMR (DMSO-d6, 300 MHz): d 9.25 (s, 1H), 8.77 (d, J ¼ 3.3 Hz, 1H), 8.38 (s, 2H), 8.28 (d, J ¼ 8.9 Hz, 1H), 7.78 (s, 1H), 7.72e7.56 (m, 5H), 7.45 (d, J ¼ 8.8 Hz, 1H), 7.37 (s, 1H), 6.66e6.63 (m, 3H), 6.51 (d, J ¼ 5.0 Hz, 1H), 3.35 (bs, 2H), 3.26e3.24 (m, 2H), 1.98e1.94 (m, 2H); 13C NMR (DMSO-d6,

Quinoline-chalcone hybrid compounds (13e37) and reference antiulcer drugs omeprazole (Omz) (Sigma Chemicals, USA) sucralfate (SUC) (Menarini pharmaceutical, India) and misoprostol (Sigma Chemicals, USA) were prepared in 1% sodium carboxymethylcellulose (CMC) suspension and administered orally, 45 min prior exposure of ulcerogens to the animals. All animals were deprived of food for 16 h before ulcerogens exposure and were divided into three groups each group comprising of six animals (n ¼ 6). Group 1 (Control): Control group of animals were treated with vehicle 1% CMC. Group 2 (Treated with hybrid compounds): Graded doses of hybrid compounds were tested against the cold restraint ulcer (CRU) model to identify the effective dose and selected for further studies in other ulcer models. Group 3 (Reference drug): Experimental group was treated with reference antiulcer drug such as omeprazole (Omz) (10 mg/kg, p.o.) in cold restraint (CRU), pyloric ligation (PL), aspirin (ASA) and another reference drug sucralfate (SUC) (500 mg/kg, p.o.) in the alcohol (AL) induced gastric ulcer model. 6.3. Antiulcer studies 6.3.1. Cold restraint induced gastric ulcer (CRU) Ulcer was induced by cold and restraint stress after 45 min of treatment with graded doses of hybrid compounds (13e37) (12.5, 25 and 50 mg/kg, p.o.) and reference drug omeprazole (10 mg/kg, p.o.) in rats. Animals were immobilized in restraint cages and kept at 4  C in an environmental chamber for 2 h [32]. Animals were then sacrificed and their stomachs were observed under magnascope for ulcers and scored. 6.3.2. Alcohol induced gastric ulcers (AL) Gastric ulcer was induced in rats by administering absolute alcohol (1 mL/200 g) [32]. The active hybrid compounds (14, 16, 17, 23, 25, 27, 29, 31, 32 and 35) (25 mg/kg, p.o.) and reference drug sucralfate (500 mg/kg, p.o.) were administered 45 min before alcohol treatment. After 1 h, the animals were sacrificed and stomachs were excised to observe the gastric lesions. 6.3.3. Aspirin induced ulcer model (ASA) Gastric lesions were induced with Non steroidal antiinflammatory drugs (NSAIDs) like Aspirin (150 mg/kg) administered to rats after 45 mins of treatment with active hybrid compounds 14, 16, 17, 23, 29, 31, 32 and 35 (25 mg/kg body weight) and reference

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drug omeprazole. Animals were sacrificed after 5 h and their stomachs were taken out and cut along the lesser curvature [32]. 6.3.4. Pyloric ligation induced ulcer model (PL) Under chloral hydrate anesthesia (300 mg/kg, i.p.) pyloric ligation was done [25]. After 45 min pre-treatment with compounds 14, 16, 17, 23, 29, 31, 32 and 35 (25 mg/kg body weight) and reference drug omeprazole (10 mg/kg, p.o.), pyloric end of the stomach was ligated and abdomen was closed by suturing. After 4 h of surgery, rats were sacrificed and the stomach was dissected out and the accumulated gastric juice was collected. Ulcers were also scored after examining the dissected stomach under magnascope. 6.3.5. Gastric secretion study In collected gastric juice from the stomach of pyloric ligated rats free and total acidity were measured by titrating against 0.01 N NaOH, using phenolphthalein as indicator and expressed in terms of mequiv/mL. Mucin level in gastric juice was quantified with a fluorometric assay and expressed as mg of mucin/mL of gastric juice [32]. 6.3.6. Direct fluorometric assay Gastric juice was delipidated by the following protocol described by Wessel et al. [33] before the fluorometric assay of mucin level estimation. Mucin level was quantified as described by Crowther et al. [34]. In brief, 50 mL of gastric juice was diluted with 1:l (v/v) in Phosphate Buffered Saline buffer (PBS) and methanol 400 mL was added. Centrifuged at (9000 g for 1 min), then 200 mL of chloroform and 300 mL of distilled water were added, thoroughly mixed and centrifuged once more. The upper phase was discarded and 300 mL of methanol was added. After a further centrifugation (9000 g for 2 min), lipid-free proteins were recovered in the pellet. For the fluorometric mucin determination, the pellet was resuspended in 200 mL of PBS, alkaline reagent 250 mL (1 mL 0.15 N NaOH and 200 mL of 0.6 M 2-cyanoacetamide) was added and the mixture was incubated at 100  C for 30 min. Afterwards, 2 mL of 0.6 M borate buffer (pH 8) was added and the fluorescence was measured by varion fluorimeter at 383 nm (excitation 336 nm). 6.3.7. In vitro assay of Hþ Kþ-ATPase activity [32] Gastric microsomes were isolated from normal fasted rat stomach. Hþ Kþ-ATPase activity was analysed in gastric microsomes by measuring the inorganic phosphate released after hydrolysis of ATP. For the enzyme assay, 50 mg of gastric microsomes were added to an assay buffer (pH ¼ 7.2) containing (in mM) 150 KCl, 10 PIPES, 1 MgSO4, 5 Mg ATP, 1 EGTA and 0.1 ouabain, 10 mg/mL valinomycin and 2.5 mg/mL oligomycin. Further, the microsomes were incubated with or without different concentrations of compounds as well as reference drug omeprazole for 30 min at 37  C after which the reaction was stopped by adding 10% ice-cold trichloroacetic acid. The inorganic phosphate release was determined from the resulting supernatant spectrophotometrically at 310 nm wavelength and expressed as mM/h/mg protein. 6.3.8. PGE2 estimation PGE2 was determined in mucosal tissue samples obtained from control, treatment and reference drug groups. Briefly, mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH ¼ 7.4) containing 1 mM EDTA and 10 mM indomethacin. The homogenate was centrifuged (10,000 rpm, 10 min, 4  C), and the supernatant was processed for PGE2 estimation using the Biotrak enzyme immunosorbent assay kit (Amersham Biosciences, Piscataway, NJ), following the manufacturer's instructions. Results were expressed in pg/mg PGE2 protein.

6.3.9. Measurement of ulcer index Ulcers formed in the stomach of cold restraint ulcer, aspirin and pyloric ligated rats were scored according to the arbitrary scoring system and graded as following: (i) Shedding of epithelium ¼ 10; (ii) Petechial and frank hemorrhages ¼ 20; (iii) one or two ulcers ¼ 30; (iv) more than two ulcers ¼ 40; and (v) Perforated ulcers ¼ 50. In the alcohol model the length of the lesions were measured using Biovis image analyzer software (Expert Vision Lab Private Ltd., Mumbai, India) and summated to give a total lesion score [32]. Ulcer score is measured in ulcer model and area of ulcer base is measured by the help of Biovis image analysis software in the ulcer model, which is considered as the ulcer index. Ulcer index is calculated from scorings described as follows:

UI ¼ Us þ Up  101 where Us ¼ mean severity of ulcer score; Up ¼ percentage of animals with ulcer incidence. Percentage protection index in case of antiulcer studies is calculated as follows:

¼ ðC  TÞ=C  100 where C ¼ ulcer index in the control group; T ¼ ulcer index in the treated group. 6.3.10. Antioxidant assays 6.3.10.1. Scavenging/inhibitory activity coefficient. The scavenging or percentage inhibitory activity of lead hybrid compound 17 in each assay was calculated from:

% Inhibition ¼ ðA0  A1 Þ  100=A0 A0 ¼ absorbance of the control (without hybrid compound 17), and A1 ¼ absorbance of the treated (with hybrid compound 17). The IC50 value of the active hybrid compound 17 was extrapolated from the reference linear regression curve. 6.3.11. Free radical scavenging activity 6.3.11.1. DPPH method [32]. The antioxidant activity of the lead hybrid compound 17 was assessed on the basis of the radical scavenging effect of the stable DPPH free radical. Compound 17 or reference (10e100 mg/mL) was added to 200 mL of DPPH in methanol solution (100 M) in a 96-well microtitre plate (Tarsons Product (P) Ltd., India). After incubation at 37  C for 30 min, the absorbance of each solution was determined at 490 nm using the ELISA micro plate reader (Bio Rad Laboratories Inc., California, USA and Model 550). IC50 value is the concentration of the sample required to scavenge 50% DPPH free radical. 6.3.11.2. Superoxide anion (SOD) scavenging activity assay. Superoxide dismutase activity was measured based on its ability to inhibit the autoxidation of epinephrine to adrenochrome at alkaline pH [33]. The absorbance of reaction mixture was followed for 4 min at 480 nm in a spectrophotometer (Model 1201, Shimadzu). Enzymatic activity was expressed as U/mg protein at 30  C. The amount of enzyme that caused 50 percent inhibition of epinephrine auto-oxidation was defined as one unit (U). 6.3.12. Preparation of isolated cells from the rat stomach Gastric cell isolation was performed as described by Berglindh [32] with some modifications. Media of the following compositions were used.

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Medium A (mmol/L) NaH2PO4 (0.5), Na2HPO4 (1.0), NaHCO3 (20.0), NaCl (70.0), KCl (5.0), glucose (11.0), EDTA (2.0), HEPES (50.0), and BSA (2%). Medium B was of same composition as A, but it was EDTA free and contained CaCl2 (1.0), MgCl2 (1.5), and BSA (1%). Medium C differed from B in having 0.1% BSA. Hanks' balanced salt solution (HBSS) with 10 mM glucose was used for final suspension of the isolated cells. After anaesthetization, the rats were given in situ perfusion through the heart with normal saline. Stomach was removed and washed with normal saline, mucosa was minced. The minced mucosa was incubated in 5 mL of buffer A and was digested with 1 mg/mL protease with continuous stirring at 37  C. After 30 min, the supernatant was collected into a fresh tube and the undigested mucosa was digested with fresh enzyme solution (0.5 mg/mL) and incubated for 15 min. It was then centrifuged at 600 g for 5 min and the pellet was resuspended in 5 mL buffer B along with 0.5 mg/mL collagenase. Another digestion for 30 min with continuous stirring was given and later it was centrifuged at 600 g for 10 min and the supernatant was collected. The pellet was resuspended in buffer C and was washed twice with buffer C at 600 g for 10 min. The cells were then separated from the final cell suspension by densitygradient centrifugation through the linear optiprep gradient. 6.3.13. MTT assay The metabolic activity within the cells was evaluated using the MTT assay kit using protocol described by manufacturer. The relative number of viable cells/well was determined by formation of blue formazan colour product by the mitochondrial dehydrogenase activity in viable cells [35]. In breif, treated and untreated cells were seeded in a 96-well plate at a density of 2  105 cells per well. 5 mL of MTT (5 mg/mL) was added into each well, and then incubated for 4 h at 37  C in the dark. After incubation, the formazon produced within the cells appeared as dark crystals at the bottom of the wells. After incubation, 100 mL of crystal dissolving solution was added in each well and incubated for 45 min to allow the complete dissolution of the formazan crystals produced. Optical density was measured in an automatic micro plate reader at a test wavelength of 530 nm and reference wavelength of 690 nm to negate the effect of cell debris. The data are presented as the percentage above control (untreated samples). 6.3.14. Bioanalytical HPLC-PDA method for compound 17 The Waters HPLC system, Milford USA consisted of a binary pump (model Waters 515 HPLC pump), auto sampler (model 717 plus Auto sampler) and PDA detector (Waters 2996). Data collection and analysis were performed using Empower pro 2 software. The compound 17 resolution and better peak shape were achieved on a Symmetry-Shield C18 (5 mm, 4.6  150 mm) column, protected with a C18 guard column. The Clopidogrel was used as internal standard (IS). A 20 mL aliquot of each sample was injected into the HPLC system. The system was analysed in isocratic mode with a mobile phase consisting of methanol: 0.5% aqueous formic acid (52: 48, v/v) at a flow rate of 1.0 mL/min. The absorption wavelength was set to 393 nm and 240 nm for compound 17 and IS, respectively. The retention time of compound 17 and IS were 6.5 and 9.8 min, respectively. Calibration standards were prepared by spiking working standard solution into 300 mL rat plasma to obtain a concentration range of 0.062e16 mg/mL. Quality control (QC) samples (0.125, 2 and 16 mg/mL) were prepared in a similar manner with appropriate working stock solution. A Solid phase extraction (SPE) method was followed for extraction of compound 17 and IS from rat plasma. The SPE was carried out using 1 cc, C-18 (DSC-18, Supelco) cartridge. Cartridges were conditioned with 2 mL methanol and followed by

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2 mL aqueous 2% v/v formic acid. Plasma samples (300 mL) were diluted to 0.7 mL with 0.05% aqueous formic acid and then loaded into the cartridges. Then cartridges were washed with 2 mL of aqueous 0.1% formic acid. Analytes were eluted with 2 mL of methanol. The eluents were collected in glass tubes and evaporated to dryness under nitrogen in water bath set at 40  C. The dry residues were finally reconstituted in 100 mL methanol 20 mL supernatant injected to HPLC. The method was validated to meet the acceptance criteria of FDA guidance for the bioanalytical method validation [36]. In vivo pharmacokinetics study was performed in male SpragueeDawley rats (n ¼ 3, weight range 250.0e300.0 g), were obtained from Laboratory Animal Division, CDRI, Lucknow, India. The compound 17 was administered oral at a dose of 25 mg/ kg in 0.5% aqueous carboxy methyl cellulose suspension. Blood samples were collected from the retro orbital pluxes into microcentrifuge tubes containing heparin (20 IU/mL) as anticoagulant at 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 7.0, 8.0, 10.0, 12.0, 18.0, 24.0 and 30.0 h post dosing. Plasma was harvested by centrifuging the blood samples at 2000  g for 5 min and stored at 70 ± 10  C until analysis. All experiments were carried out as per the guidelines of CPCSEA. 6.3.15. Stability in simulated gastrointestinal fluids Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the formulation described in the 26th United Stated pharmacopeia. The compound 17 was incubated in simulated gastric fluid (0.2 g NaCl, 3.2 g pepsin, 7 mL HCl and H2O to 1000 mL, pH 1.2) or simulated intestinal fluid (6.805 g KH2PO4, 0.896 g NaOH, 10.0 g pancreatin and H2O to 1000 mL, pH 6.86) at 37  C at a final concentration of 10 mg/mL for stability study. Samples were removed at 0, 5, 15, 30, 45, 60 and 90 min. The concentration of compound 17 was determined by HPLC. 6.3.16. In vitro metabolism with rat liver microsomes The in vitro drug metabolism studies are used to understand the in vivo pharmacokinetic profile of novel chemical entities, has recently become an area of scientific interest. Thus, in vitro metabolism data is used in a prospective manner to choose those compounds for further development that are expected to possess commercially acceptable pharmacokinetic properties (e.g., half-life permitting dose regimens, low oral clearance to reduce the dose, etc.). The in vitro metabolism studies were preformed in Sprague Dawley (SD) rat liver microsomes (RLM) [37]. The incubation was performed as described and all analyses were performed in triplicate. The metabolism of compound 17 in rat liver microsomes was performed in a microcentrifuge tube at concentrations of 10 mg/mL. The rat liver microsomal suspensions (20 mg protein/mL) were diluted up to 1.0 mg/mL in 50 mM tris buffer, pH 7.4. Microsomal suspensions were preincubated with nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM) for 5 min at 37  C prior fortification with analyte. Immediately, after fortification of analyte into the microsomal suspension containing NADPH, the sampling point for t ¼ 0 was taken, and further sampling points were taken at 5, 10, 20, 30, 45, 60 and 90 min 100 mL of samples were taken and mixed with 200 mL of ice cold methanol containing IS (5 mg/mL) in a 1.5 mL centrifuge tube. Samples were centrifuged at approximately 12,000 g for 15 min and 160 mL of the supernatant was transferred into an injection vial for HPLC-PDA analysis. The result of metabolic stability was expressed as the percentage of drug remaining. The test compound disappearance shows MichaeliseMenten kinetics. All depletion data were fitted with the monoexponential decay model, including a 1/y weighing. The in vitro half life (T1/2, min) intrinsic Clearance (CLi) and hepatic clearance were calculated as

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described previously [38]. The intrinsic clearance was then scaled to predict in vivo hepatic intrinsic clearance. The scaling factor was estimated using a microsomal recovery value of 45 mg microsomal protein/g liver and a rat liver weight of 40 g/kg of body weight. Scaling factor (milligrams of protein per gram of liver * gram of liver per kg body weight) was estimated to be 1800 for rat. In vivo intrinsic hepatic clearance in liver was estimated by multiplying in vitro Clint and scaling factor. 6.3.17. Statistical analysis All values shown in the figures and tables represent the means ± S.E.M. IC50 values with 95% confidence limits were estimated using Maximum Likelihood Iterative Procedure [39]. Statistical analysis was performed with Prism version 5.0 software using one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison Test. P < 0.05 was considered to be statistically significant. 6.3.18. Abbreviations PGE2, prostaglandin; S.E.M, standard error mean; IC50, half maximal inhibitory concentration; Hþ Kþ-ATPase, hydrogen potassium adenosine triphosphatase; EGTA, ethylene glycol-bis(2aminoethylether)-N,N,N0 ,N0 -tetraacetic acid; COX, cyclooxygenase; NSAIDs, non steroidal antiinflammatory drugs; TLC, thin layer chromatography; TMS, tetramethylsilane; ESIMS, electrospray ionization mass spectra; HRMS, high resolution mass spectra; HPLC, high-performance liquid chromatography; Omz, omeprazole; PL, pyloric ligation; ASA, aspirin; AL, alcohol; CRU, cold restraint ulcer; SUS, sucralfate; H2RAs, histamine receptor antagonists; PPIs, proton pump inhibitors; CMC, carboxymethylcellulose; AS, aqueous standard; CS, calibration standard. Acknowledgements The authors are grateful to the Director, CDRI, Lucknow, India for constant encouragement in drug development program, S.P. Singh for technical support, SAIF for NMR, IR, and Mass spectral data. A.S.R., V.M., G.R.P. and L.R.S are thankful to CSIR, New Delhi, India for financial support and P.S. is thankful to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India. This is CSIR-CDRI communication number 8826. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.10.068. References [1] W.A. Hoogerwerf, P.J. Pasricha, Pharmacotherapy of gastric acidity, peptic ulcers, and gastroesophageal reflux disease, in: The Pharmacological Basis of Therapeutics, McGraw Hill Publishing Co, New York, 2006. [2] P. Malfertheiner, F.K.L. Chan, K.E.L. McColl, Peptic ulcer disease, Lancet 374 (2009) 1449e1461. [3] S. Bindu, C. Pal, S. Dey, M. Goyal, A. Alam, M.S. Iqbal, S. Dutta, S. Sarkar, R. Kumar, P. Maity, U. Bandyopadhyay, Translocation of Heme Oxygenase-1 to Mitochondria is a novel cytoprotective mechanism against non-steroidal antiinflammatory drug-induced mitochondrial oxidative stress, apoptosis, and gastric mucosal injury, J. Biol. Chem. 286 (2011) 39387e39402. [4] C. Pal, S. Bindu, S. Dey, A. Alam, M. Goyal, M.S. Iqbal, P. Maity, S.S. Adhikari, U. Bandyopadhyay, Gallic acid prevents nonsteroidal anti-inflammatory druginduced gastropathy in rat by blocking oxidative stress and apoptosis, Free Radic. Bio Med. 49 (2010) 258e267. [5] J.S. Ahn, C.S. Eom, C.Y. Jeon, S.M. Park, Acid suppressive drugs and gastric cancer: a meta-analysis of observational studies, World J. Gastroenterol. 19 (2013) 2560e2568. [6] C.S. Eom, S.M. Park, S.K. Myung, J.M. Yun, J.S. Ahn, Use of acid-suppressive drugs and risk of fracture: a meta-analysis of observational studies, Ann. Fam. Med. 9 (2011) 257e267.

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