2 and 5-LOX inhibitors: Synthesis, biological evaluation and docking study

Bioorganic Chemistry 72 (2017) 102–115

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New hybrid molecules combining benzothiophene or benzofuran with rhodanine as dual COX-1/2 and 5-LOX inhibitors: Synthesis, biological evaluation and docking study Mostafa M.M. El-Miligy a,⇑, Aly A. Hazzaa a, Hanan El-Messmary b, Rasha A. Nassra c, Soad A.M. El-Hawash a a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt Faculty of Pharmacy, Omar-Almukhtar University, Libya c Department of Medical Biochemistry, Faculty of Medicine, Alexandria University, Alexandria, Egypt b

a r t i c l e

i n f o

Article history: Received 21 February 2017 Revised 21 March 2017 Accepted 23 March 2017 Available online 31 March 2017 Keywords: Benzothiophene Benzofuran Rhodanine COX/LOX inhibition

a b s t r a c t New molecular hybrids combining benzothiophene or its bioisostere benzofuran with rhodanine were synthesized as potential dual COX-2/5-LOX inhibitors. The benzothiophene or benzofuran scaffold was linked at position -2 with rhodanine which was further linked to various anti-inflammatory pharmacophores so as to investigate the effect of such molecular variation on the anti-inflammatory activity. The target compounds were evaluated for their in vitro COX/LOX inhibitory activity. The results revealed that, compound 5h exhibited significant COX-2 inhibition higher than celecoxib. Furthermore, compounds 5a, 5f and 5i showed COX-2 inhibitory activity comparable to celecoxib. Compound 5h showed selectivity index SI = 5.1 which was near to that of celecoxib (SI = 6.7). Compound 5h displayed LOX inhibitory activity twice than that of meclofenamate sodium. Moreover, compounds 5a, 5e and 5f showed significant LOX inhibitory activity higher than that of meclofenamate sodium. Compound 5h was screened for its in vivo anti-inflammatory activity using formalin-induced paw edema and gastric ulcerogenic activity tests. The results revealed that, it showed in vivo decrease in formalin-induced paw edema volume higher than celecoxib. It also displayed gastrointestinal safety profile as celecoxib. The biological results were also consistent with the docking studies at the active sites of the target enzymes COX-2 and 5-LOX. Also, compound 5h showed physicochemical, ADMET, and drug-like properties within those considered adequate for a drug candidate. Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction Traditional nonselective NSAIDs inhibit both COX enzymes. This broad inhibitory profile accounts for their anti-inflammatory activity as well as their pronounced side effects [1–5]. Selective COX-2 inhibition would be effective for treatment of inflammation without gastric and renal toxicity. Hence, a number of selective COX2 inhibitors such as celecoxib, rofecoxib and valdecoxib (coxibs) have been developed and were approved for clinical use. However, the long-term use of coxibs has been reported to cause serious cardiovascular side effects and were withdrawn from the market due to these limitations [6–9]. In addition, the inhibition of COX-1/ COX-2-mediated metabolism of arachidonic acid can result in an increased formation of leukotrienes (LTs) via the lipoxygenase ⇑ Corresponding author. E-mail address: [email protected] (M.M.M. El-Miligy). http://dx.doi.org/10.1016/j.bioorg.2017.03.012 0045-2068/Ó 2017 Elsevier Inc. All rights reserved.

(LOX) pathway [10]. In particular, 5-LOX has been associated with several undesirable physiological effects. LTs are known to be involved in the progression of inflammation, osteoarthritis, and asthma [11–13]. Consequently, combined 5-LOX/COX-inhibition could provide anti-inflammatory and analgesic effects with the advantage of reduced adverse effects [14]. Literature survey revealed that, the 2-substituted benzothiophene derivative Zileuton, is a potent and selective 5-lipoxygenase inhibitor that has been approved for the prevention and treatment of chronic asthma [15]. In addition, a series of 3-hydroxybenzo[b]thiophene-2carboxylic acid derivatives I has been reported as dual 5-LOX/ COX-inhibitors [16]. Moreover, hybridization between 1,3,4oxadiazole and benzothiophene in compound II was reported to have direct 5-LOX inhibitory activity [17]. In addition, various 5-arylidenethiazolidinone derivatives III have been reported to possess significant anti-inflammatory activity [18]. Also, 5-arylidene rhodanine derivatives IV showed significant

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anti-inflammatory activity [19]. The enhancement of antiinflammatory activity might be due to the addition of rhodanine to 1,3-diaryl pyrazole anti-inflammatory pharmacophore. Furthermore, many 5-arylidenethiazolidinone derivatives were reported to have dual COX/LOX inhibitory activities; for example, Darbufelone is a well-known selective COX-2/5-LOX inhibitor [20,21]. In addition, 1,3,4-oxadiazole-2-thione and 1,3, 4-thiadiazole-2-thione derivatives V were reported as dual COX/5-LOX inhibitors [22]. Replacement of thione group by carbonyl abolished their activity which confirmed the importance of thione group in 5-LOX inhibition [23]. Furthermore, 3benzylidene-indolin-2-one derivative VI displayed excellent inhibitory activity against COX-2 and 5-LOX [24]. Moreover, pyrazole derivatives like celecoxib displayed significant inhibitory activity against COX-2 and 5-LOX [25]. Also, 1,3-diphenylpyrazole derivative VII showed anti-inflammatory and analgesic responses higher than indomethacin and celecoxib [26]. The dual COX/5-LOX inhibitor Tepoxalin comprised the diarylpyrazole scaffold of selective

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COX-2 inhibition beside hydroxamic acid to chelate the nonheme iron atom of 5-LOX [27] (Fig. 1). Tempted by the aforementioned findings, it was thought of interest to construct some new hybrid molecules that comprise rhodanine moiety linked to benzothiophene or its bioisostere benzofuran through two atoms spacer and attached at position-5 of rhodanine with various arylidene moieties which were proved to enhance COX-2/5-LOX inhibition such as 1,3-diarylpyrazoles, indolin-2-one and different substituted phenyl derivatives in order to investigate the effect of such molecular variation on the anticipated anti-inflammatory efficacy (Fig. 2). The target compounds were evaluated for their in vitro COX-1, COX-2 and LOX inhibition and their in vivo anti-inflammatory activity and ulcerogenic liability in addition to histopathological examination to confirm the degree of inflammatory reaction in the gastric layers of treated rats’ stomachs. Furthermore, docking simulation and drug likeness studies were performed to study the structural features required for COX-2 and 5-LOX inhibitory properties of the new derivatives.

Fig. 1. Structures of reported COX and 5-LOX inhibitors.

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Fig. 2. Design of new dual COX/LOX inhibitors.

2. Results and discussion 2.1. Chemistry The synthetic pathway for the target compounds are illustrated in Schemes 1 and 2. Scheme 1 describes the synthesis of methyl 3-chlorobenzo[b]thiophene-2-carboxylate, 2 from reaction of cinnamic acid, 1 with thionyl chloride in the presence of catalytic amount of pyridine following the reported reaction conditions [28,29]. Its treatment with hydrazine hydrate afforded the corresponding acid hydrazide, 3 [30,31], which was reacted with bis(car boxymethyl)trithiocarbonate to yield rhodanine derivative 4. Its I. R. spectrum was characterized by absorption bands corresponding to NH at 3347 cm
1, C@O stretching at 1757and 1677 cm
1 and C@S at 1242 cm
1. 1H NMR spectrum revealed a deuteriumexchangeable signal assigned for one NH proton at d 11.57 ppm

and appearance of thiazolidine C5AH protons d 4.44 ppm. Condensation of rhodanine derivative, 4 with aromatic aldehydes or isatin in the presence of catalytic amount of piperdine yielded the corresponding arylidene derivatives 5a-i. I.R. Spectra of compounds 5a-i were characterized by absorption bands assigned to NH at 3418– 3190 cm
1, C@O stretching at 1748–1711, 1690–1656 cm
1 and C@S at 1263–1250 cm
1. 1H NMR spectra of compounds 5a-i showed a deuterium-exchangeable signal integrated for one NH proton at d 11.81–11.33 ppm and appearance of signal assigned to C@CH proton at d 8.02–7.69 ppm. The spectra lacked the signal assigned of thiazolidin C5AH protons. 13C NMR spectrum of compound 5h was characterized by appearance of signal corresponding to C@OANH at d 158.85 ppm, C@O, thiazolidine at d 163.11 ppm and C@S at d 189.65 ppm. EIMS for 5c showed [M+ + 4] at m/z 656, [M+ + 2] at m/z 654, molecular ion peak [M+] at m/z 652 and the base peak at m/z 195.

Scheme 1. Synthesis of the target benzothiophene derivatives. Reagents: (i) SOCl2/Pyridine; (ii) Methanol:Benzene (1:1); (iii) NH2NH2H2O, EtOH; (iv) Bis(carboxymethyl) trithiocarbonate, H2O (v) Different aldehydes or Isatin, Piperidine, Benzene.

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Scheme 2. Synthesis of the target benzofuran derivatives. Reagents: (i) ethyl chloroacetate, K2CO3; (ii) NH2NH2H2O; (iii) Bis(carboxymethyl)trithiocarbonate, H2O (iv) 3-(4substitutedphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde, Piperidine, Benzene.

Scheme 2 outlines the synthesis of the target benzofuran derivatives. It started by reacting salicylaldehyde with ethyl chloroacetate in the presence of anhydrous potassium carbonate to yield ethyl benzofuran-2-carboxylate, 7 [32]. Its I.R. spectrum showed stretching absorption bands corresponding to carbonyl group at 1727 and CAOAC at 1257 and 1095 cm
1. Treatment of 7 with hydrazine hydrate in ethanol gave the target hydrazide 8 [33,34]. Its I.R. spectrum revealed distinctive absorption bands of NH and NH2 groups at 3319 and 3173 cm
1. The hydrazide was further reacted with bis (carboxymethyl)trithiocarbonate to produce rhodanine derivative 9. I.R. spectrum showed absorption band corresponding to NH at 3292 cm
1, C@O at 1750and 1684 cm
1 and C@S at 1250 cm
1. 1H NMR spectrum elicited a deuterium exchangeable signal assigned for one NH proton at d 11.89 ppm and thiazolidin C5AH proton at d 4.55 ppm. It is worth mentioning that, it has been observed a decrease in the NMR signal assigned for thiazolidin C5 proton due to tautomeric effect. In some cases, this active methylene undergoes complete deuteriumexchange and its signal was completely disappeared as reported for related compounds [35]. Condensation of 9 with 1,3diarylpyrazole carbaldehydes in the presence of catalytic amount of piperidine afforded the target 5-arylidene-rhodanine derivatives 10a-c. Their I.R. spectra possessed absorption bands corresponding to NH at 3349–3314 cm
1, C@O at 1743–1704and 1704– 1684 cm
1 and C@S at 1257–1247 cm
1. 1H NMR spectra of compounds 10a-c revealed a deuterium-exchangeable signal for one NH proton at d 12.22–12.11 ppm, appearance of signal of C@CH proton at d 7.88–7.81 ppm and disappearance of thiazolidin C5AH protons. 13C NMR spectrum of compound 10b was characterized by signal for C@OANH carbon at d 155.00 ppm, C@O, thiazolidine at d 173.01 ppm and C@S at d 189.83 ppm. EIMS for 10c showed [M+ + 2] at m/z 602, molecular ion peak [M+] at m/z 600 and base peak at m/z 145. 2.2. Biological evaluation 2.2.1. In vitro COX-1 and COX-2 inhibitory assay Compounds 4, 5a-i, 9 and 10a-c were tested for COX-1 and COX-2 inhibition at three concentrations (25, 50 and 100 lM) to determine the concentration produced 50% inhibition of COX-1 and COX-2 enzymes (IC50 values) and their selectivity indices (SI = IC50 COX-1/IC50 COX-2) using celecoxib as reference drug. The results recorded in Table 1 indicated that compound 5h displayedsignificantCOX-2inhibitory activity (IC50 = 0.67 lM respec-

tively) higher than that of celecoxib. In addition, compounds 5a, 5f and 5i showed COX-2 inhibitory activity comparable to that of celecoxib. Moreover, compounds 4, 5b-e, 5g, 9 and 10a-c were found to be slightly less potent than celecoxib. In terms of selectivity index (SI), compound 5h showed SI = 5.1 near to that of celecoxib (SI = 6.7). 2.2.2. In vitro lipoxygenase inhibitory assay Compounds 4, 5a-i, 9 and 10a-c were also tested for 5lipoxygenase inhibition at three concentrations (25, 50 and 100 lM) to determine the concentration produced 50% inhibition of 5-lipoxygenase enzyme (IC50 values) using meclofenamate sodium as a reference drug. The results reported in Table 1 indicated that compound 5h displayed LOX inhibitory activity (IC50 = 2.33 lM) twice than that of the standard meclofenamate sodium (IC50 = 5.64 lM). In addition, compounds 5a, 5e and 5f showed significant LOX inhibitory activity (IC50 = 3.97, 4.97 and 3.74 lM respectively) higher than that of meclofenamate sodium, while compounds 4, 5b-d, 5g, 5i, 9 and 10a-c were found to be slightly less potent 5-lipoxygenase inhibitors than meclofenamate sodium. Additionally, compound 5h exhibited significant dual COX/LOX inhibitory activities higher than the reference drugs. 2.2.3. In vivo assay 2.2.3.1. Formalin-induced paw edema test. The results listed in Table 2 represent the mean changes in paw volume mL ± SD of animals pretreated with the reference drug and test compound 5h after 0 and 4 h from the induction of inflammation, together with the percent inhibition of induced rat paw edema by the test compounds (percent anti-inflammatory activity). The antiinflammatory activities of the test compounds relative to those of the reference drugs (percent relative potency) were also calculated. Statistical differences of control and test groups were carried out using the F test (ANOVA), and pairwise comparison using Post Hoc Test (Tukey). The screening results revealed that, the tested compound 5h showed significant anti-inflammatory activity (% edema reduction = 93.26) higher than that of celecoxib (86.5% edema reduction). 2.2.3.2. Gastric ulcerogenic activity. Gross observation of the isolated rat stomachs showed a normal stomach texture for the tested compound 5h as the reference celecoxib. Further histopathological examination was performed to confirm the degree of inflammatory reaction in the gastric layers of the treated rats’ stomachs. The results revealed that compound 5h showed gastrointestinal safety

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Table 1 In vitro COX-1, COX-2 and LOX enzymes inhibitory activities, IC50 values and selectivity indices (SI) of the tested compounds:

a b c

Comp. ID

IC50(lM)a COX-1

IC50(lM) COX-2

SIb (COX-1/COX-2)

IC50(lM) LOX

4 5a 5b 5c 5d 5e 5f 5g 5h 5i 9 10a 10b 10c Celecoxib Meclofenamate sodium

8.7 5.1 7.6 12.4 10.6 6.3 4.9 10.4 3.4 5.9 6.8 12.4 8.7 13.4 7.6 NDc

2.43 1.49 2.61 4.88 3.12 1.89 1.23 4.22 0.67 1.56 1.83 4.81 2.74 5.32 1.14 NDc

3.6 3.4 2.9 2.5 3.4 3.3 4.0 2.5 5.1 3.8 3.7 2.6 3.2 2.5 6.7 NDc

6.87 3.97 8.71 9.51 8.74 4.97 3.74 6.91 2.33 6.87 5.92 9.21 6.91 10.21 6.87 5.64

IC50 value is the compound concentration required to produce 50% inhibition of COX-1 or COX-2 or LOX for means of three determinations. SI: ratio (IC50 COX-1/ IC50 COX-2). ND: not determined.

Table 2 In vivo anti-inflammatory activities of selected compounds in formalin-induced rat paw edema bioassay. Volume of edema in mL

Vehicle control (DMSO)

1 2 3 4 5 6

0

4h

Absolute difference

% Inhibition

0.5 0.4 0.5 0.5 0.4 0.6

1.8 1.9 2 2.1 1.8 2.2

1.3 1.5 1.5 1.6 1.4 1.6

– – – – – –

1.48 ± 0.12

–

0.2 0.2 0.2 0.3 0.1 0.2

86.5 86.5 86.5 79.8 93.3 86.5

0.20* ± 0.06

86.52 ± 4.26

0.1 0.1 0.1 0.1 0.1 0.1

93.3 93.3 93.3 93.3 93.3 93.3

107.7 107.7 107.7 107.7 107.7 107.7

0.10* ± 0.0

93.26a ± 0.0

107.70a ± 0.0

Mean ± SD Reference standard (Celecoxib)

1 2 3 4 5 6

0.6 0.7 0.6 0.5 0.6 0.6

0.8 0.9 0.8 0.8 0.7 0.8

Mean ± SD 5h

Mean ± SD

1 2 3 4 5 6

0.4 0.5 0.4 0.4 0.5 0.4

0.5 0.6 0.5 0.5 0.6 0.5

% Relative potency (4 h) Celecoxib

100.0 100.0 100.0 100.0 100.0 100.0

Values are expressed as mean ± SD, and SEM and comparison between the three groups using F test (ANOVA), and pairwise comparison using Post Hoc Test (Tukey). * Statistically significant difference in comparison with control group. a Statistically significant difference in comparison with reference group (Celecoxib).

profile as the reference celecoxib in the population of the fasted rats at a single oral dose of 60 mg/kg. 2.3. Structure activity correlation Careful inspection of structures of the tested compounds revealed that benzothiophene derivatives were more potent than benzofuran analogues. Compounds 4 and 5a-i exhibited moderate COX-1, COX-2 and LOX inhibition except compound 5h which showed remarkable inhibitory potency against the three enzymes higher than the reference drugs (celecoxib and meclofenamate sodium). The increase in activity might be due to the presence of 3,4-dimethoxyphenyl moiety. On the other hand, condensation with 1,3-diphenylpyrazole carbaldehydes increased selectivity for

COX-2 than COX-1. Additionally, the presence of indolin-2-one in compound 5i increased the inhibitory potency and selectivity to COX-2 and LOX while decreased the inhibitory activity toward COX-1. 2.4. Molecular modeling 2.4.1. In silico prediction of physicochemical properties and pharmacokinetic profile Molecular property prediction is becoming an useful tool in the generation of molecules with the correct parameters to be useful drug candidates. Drug design and lead optimization benefits from the ability to predict physical properties such as lipophilicity and solubility, as well as molecular properties such as topological polar

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surface area (TPSA) and number of H-bond donors and acceptors to build activity prediction tool which predicts drug likeness [36]. In the present investigation, the most biologically active compound 5h and references celecoxib and meclofenamate sodium were subjected to molecular properties and bioactivity prediction by Molinspiration online property calculation toolkit [37], drug-likeness and solubility parameter calculation by Mol-Soft software [38], ADME profiling by PreADMET [39] calculator and toxicity-risk assessment by Osiris property explorer [40] to filter and their overall potential to qualify for a drug was analyzed, also comparing them to some current anti-inflammatory drugs. Physicochemical properties and drug likeness for the most biologically active compound 5h and references celecoxib and meclofenamate sodium were calculated. Hydrogen-bonding capacity has been identified as an important parameter for describing drug permeability [41]. The tested compound under investigation was predicted to possess considerable ranges for H-bond donors (0–2) and H-bond acceptors (5–6). Also, for good membrane permeability logP value should be 5. The tested compound had log P value 3.28. Moreover, we evaluated the compliance of the compound to the Lipinski’s ‘rule of five’. The results indicated that tested compound obeyed Lipinski’s rule of five. Number of rotatable bonds is important for conformational changes and ultimately for the binding to receptors or channels. Tested compound possessed 5 rotatable bonds and therefore, fulfilling the criterion and exhibiting moderate to high conformational flexibility. Molecular polar surface area (TPSA) is a very useful parameter for the prediction of drug transport properties. Compounds with TPSA > 140 are predicted to have low oral bioavailability. The results predicted that all the tested compound demonstrated acceptable TPSA value, 67.88. In addition, TPSA was used to calculate the percentage of absorption (% ABS) and tested compound displayed the % ABS of 85.58%, which indicated their good bioavailability by oral administration. Furthermore, the investigated compounds were found to fulfill the requirements of solubility of >0.0001 mg/L and could be considered as drug candidates for oral absorption. The tested compound was predicted to have drug-likeness scores with positive values from 0.35 compared to
1.03 and 0.58 for the reference drugs celecoxib and meclofenamate sodium, respectively. Pharmacokinetic properties for studied and standard compounds were calculated using PreADMET software. The in silico prediction of human intestinal absorption, HIA, of a drug involves various methods such as percent human intestinal absorption, HIA, Caco2 and MDCK cell models and BBB (Cbrain/Cblood) for the estimation of oral drug absorption and blood-brain barrier penetrations respectively. The tested compound was predicted to possess extremely high HIA values (97.66%) indicating very well-absorbed compounds. Furthermore, BBB penetration capability calculation of the tested compound revealed that compound 5h could have medium BBB penetration (0.54 respectively). The tested compound was predicted to possess medium cell permeability in the Caco-2 cell model with value from 43.79 compared to 0.49 and 19.66 for the reference drugs celecoxib and meclofenamate sodium, respectively. In addition, compound 5h was predicted to have low cell permeability in the MDCK cell model with value 6.46 compared to 45.04 and 105.41 for the reference drugs celecoxib and meclofenamate sodium, respectively. On the other hand, the tested compound was predicted to be strongly-bound to plasma proteins (91.92). In addition, it was found to be non-inhibitors of CYP2D6 enzyme and thus may be metabolized and excreted successfully. Toxicity risks (mutagenicity, tumorigenicity, irritation, reproductive effects) of the tested compound were calculated by the methodology developed by Osiris. It locates fragments in a molecule which indicates a potential toxicity risk. The results predicted that compound 5h has low toxicity profile.

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2.4.2. Anti-inflammatory docking study The molecular docking studies were performed using the Molecular Operating Environment (MOE 2008.10) software [42]. The three dimensional structures and conformations of the enzymes were acquired from the Protein Data Bank (PDB) Web site [43]. Compound 5h that displayed significant dual COX-2/LOX inhibitory activity was docked into the active sites of the targeted enzymes COX-2 and 5-LOX. The docking study results revealed that compound 5h displayed good binding affinities to the target enzymes. The binding affinity to COX-2 showed score =
12.47 kcal/mol which was relatively similar to celecoxib (
13.53 kcal/mol). In addition, the binding affinity to 5-LOX (Score =
11.79 kcal/mol) was comparable to meclofenamic acid (
10.94 kcal/mol) (Tables 3 and 4 and Figs. 3–6). It is worth mentioning that, benzothiophene and phenyl moieties imparted lipophilic properties to 5h which increased its fitting to COX-2 active site through hydrophobic interactions (Table 3 and Figs. 3 and 4). On the other hand, the two methoxy groups, amide and rhodanine imparted polar properties to 5h which increased its fitting to 5LOX active site through polar and hydrogen bond interactions (Table 4 and Figs. 5 and 6). 2.4.2.1. Docking into COX-2 active site. The docking results showed that, celecoxib interacted mainly by hydrophobic interaction using its phenyl and trifluromethyl moieties. Similarly, compound 5h showed hydrophobic interaction using its benzothiophene and 3,4-dimethoxyphenyl moieties with most of the amino acids included in the interaction with celecoxib. In addition, 5h elicited polar and hydrogen bonding interaction to COX-2 receptor using its rhodanine and amide groups (Figs. 3 and 4). 2.4.2.2. Docking to 5-LOX active site. It is worth mentioning that, both meclofenamic acid and 5h interacted with the same amino acids in hydrophobic interactions and most of the amino acids in polar interactions. In addition, meclofenamic acid showed one hydrogen bond interaction and 5h revealed two hydrogen bond interactions. Moreover, 5h interacted mainly by polar interactions using its 3,4-dimethoxyphenyl, amide and rhodanine moieties. 3. Conclusion The present investigation described the design and synthesis of new molecular hybrids combining benzothiophene or its bioisostere benzofuran with rhodanine as potential dual COX-2/5-LOX inhibitors. Furthermore, the rhodanine was linked at position 5 with various anti-inflammatory pharmacophores such as 1,3diarylpyrazoles, indolin-2-one, 3,4-dimethoxyphenyl and 4hydrox-3-methoxyphenyl moieties so as to investigate the effect of such molecular variation on the anti-inflammatory activity. The target compounds were evaluated for their in vitro COX/LOX inhibitory activity. Compound 5h exhibited significant COX-2 inhibition higher than the reference celecoxib. Furthermore, compounds 5a, 5f and 5i showed COX-2 inhibitory activity comparable to that of celecoxib. Moreover, compounds 4, 5b-e, 5g, 9 and 10a-c were found to be slightly less potent than celecoxib. Compounds 5h showed selectivity index SI = 5.1 near to that of celecoxib, SI = 6.7. On the other hand, compound 5h displayed LOX inhibitory activity twice than that of the standard meclofenamate sodium. Moreover, compounds 5a, 5e and 5f showed significant LOX inhibitory activity higher than that of meclofenamate sodium. Compound 5h that showed potent dual COX-2/LOX inhibitory activity higher than the reference drugs was also screened for its in vivo anti-inflammatory activity using Formalin-induced paw

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Table 3 Docking results of the active compound in COX-2 active site.

*

Compound number

Score in kcal/mol

Hydrogen bond interaction amino acids

Hydrophobic interaction amino acids

Polar interaction amino acids

Celecoxib (Fig. 3)


13.53

Three hydrogen bonds between nitrogen of sulfonamide group and Ser339, Leu338 and Gln178

Trp373, Leu517, Leu345, Val509, Ala502, Phe504, Val335

5h (Fig. 4)


12.47

One hydrogen bond between oxygen of 4-oxo rhodanine and Tyr341*

Leu517, Ala513, Trp373, Phe504, Val335, Leu338, lle98, Trp35, Leu78, Val102 and Phe343

Ser516, Gly512, Arg449, Tyr341, Tyr371, His75 and Arg106 Ser516, Tyr101, Arg106, Tyr371, Tyr334 and Ser339

Amino acids interacted with the reference celecoxib were marked in bold format.

Table 4 Docking results of the active compound in 5-LOX active site.

*

Compound number

Score in kcal/mol

Hydrogen bond interaction amino acids

Hydrophobic interaction amino acids

Polar interaction amino acids

Meclofenamic acid (Fig. 5) 5h (Fig. 6)


10.94

One hydrogen bond between hydroxyl group and Asn554.


11.79

Two hydrogen bonds between the oxygen of amide and Gln557* and between 4-methoxy oxygen and Arg666

Phe556, Phe610, Leu607 and Val604 Leu607, Val604, Phe610 and Phe556

Tyr558, Gln557, Gln363 and His367 Ser608, Tyr558, Asn554 and Gln363

Amino acids interacted with the reference meclofenamic acid were marked in bold format.

edema test and Gastric ulcerogenic activity test. The results revealed that, it showed significant decrease in Formalin-induced paw edema higher than that of celecoxib and gastrointestinal safety profile as celecoxib. This was also consistent with the docking studies in the active sites of the target enzymes (COX-2 and 5-LOX). On the other hand, compound 5h revealed physicochemical, ADMET, and drug-like properties within those considered adequate for a drug candidate. Accordingly, compound 5h could be considered as structural lead that is entitled for further modification and investigation for the development of a new class of dual COX-2/5-LOX inhibitors.

4. Experimental 4.1. Chemistry Melting points were determined in open glass capillaries using an electrothermal capillary tube melting point apparatus or a Griffin melting point apparatus and were uncorrected. Infrared spectra (IR) were recorded, using KBr discs, by a Perkin-Elmer 1430 Infrared spectrophotometer in the Central Laboratory, Faculty of Pharmacy, Alexandria University. Nuclear magnetic resonance (1H NMR and 13C NMR) was determined using Mercury 300 MHz spectrophotometer, Faculty of Science, Cairo University. 1H spectra were run at 300 MHz and 13C spectra were run at 75.46 MHz in deuterated dimethylsulfoxide (DMSO-d6) as a solvent. The data were reported as chemical shifts or d values (ppm) relative to tetramethylsilane (TMS) as internal standard. Signals were indicated by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. Electron impact mass spectra (EIMS) were run on a gas chromatograph/mass spectrophotometer Shimadzu GCMS/QP-2010 plus (70 eV) at the faculty of Science, Cairo University. Relative intensity % corresponding to the most characteristic fragments was recorded. Elemental microanalyses were performed at the microanalytical unit, Faculty of Science, Al-Azhar University. Reaction progress was monitored by thinlayer chromatography (TLC) on silica gel sheets (60 GF254, Merck). The spots were visualized by exposure to iodine vapor or UV-lamp at k 254 nm for few seconds. Compounds 2 [28,29], 3 [30,44], 7 [34] and 8 [34] were prepared according to reported procedures.

4.1.1. 3-Chloro-N-(4-oxo-2-thioxothiazolidin-3-yl)benzo[b]thiophene2-carboxamide, 4 To a stirred suspension of 3-chloro-benzo[b]thiophene-2carbohydrazide, 3 (0.45 g, 2 mmol) in water (20 mL), bis(carboxy methyl)trithiocarbonate (0.49 g, 2.2 mmol) was added. The reaction mixture was heated under reflux with stirring for 15 h and then left to attain room temperature. The obtained precipitate was filtered, washed with water, air-dried and crystallized from isopropyl alcohol. The product was obtained as fine buff powder. Yield 0.54 g, 79%; m.p. 238–240 °C; IR (KBr, cm
1): 3347 (NH), 1757, 1677 (C@O), 1242 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 4.44 (s, 2H, CH2-thiazolidin), 7.62–7.70 (m, 2H, benzothiophene C5,6AH), 7.98 (d, J = 8.1 Hz, 1H, benzothiophene-C4AH), 8.19 (d, J = 7.5 Hz, 1H, benzothiophene C7AH), 11.57 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C12H7ClN2O2S3 (342.83): C, 42.04; H, 2.06; N, 8.17; S, 28.05. Found: C, 42.13; H, 2.03; N, 8.23; S, 28.21. 4.1.2. General procedure for the synthesis of N-(5-Substituted-4-oxo2-thioxothiazolidin-3-yl)-3-chlorobenzo[b]thiophene-2carboxamides, 5a-i A mixture of 3-chloro-N-(4-oxo-2-thioxothiazolidin-3-yl)benzo [b]thiophene-2-carboxamide (4) (0.17 g, 0.5 mmol), the appropriate aldehyde or isatin (0.5 mmol) and piperdine (2 drops) in benzene (6 ml) was heated under reflux for 2–3 h. The resulting precipitate was filtered, washed with benzene, air-dried and crystallized from dioxane/water (8:2). 4.1.2.1. 3-Chloro-N-{5-[(1,3-diphenyl-1H-pyrazol-4-yl)methylene]-4oxo-2-thioxothiazolidin-3-yl}benzo[b]thiophene-2-carboxamide, 5a. The product was obtained as orange powder. Yield 0.09 g, 32%; m.p. 238–240 °C; IR (KBr, cm
1): 3224 (NH), 1711, 1677 (C@O), 1263 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.47–7.71 (m, 10H, C3-phenyl C2,6AH, C3-phenyl C3,5AH, N1-phenyl C4AH, benzothiophene C5,6AH, C3-phenyl C4AH, N1-phenyl C3,5AH), 7.72 (s, 1H,vinyl-CH), 7.92–7.98 (m, 2H, N1-phenyl C2,6AH), 8.00 (dd, J = 6.5, 3 Hz, 1H,benzothiophene- C4AH), 8.21 (dd, J = 6, 3.5 Hz, 1H, benzothiophene C7AH), 8.96 (s, 1H, pyrazol C5AH), 11.77 (br s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C28H17ClN4O2S3 (573.10): C, 58.68; H, 2.99; N, 9.78; S, 16.78. Found: C, 58.82; H, 3.01; N, 9.86; S, 16.84.

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Trp 373

Ser 516

Gly 512

Tyr

Arg

371

Leu

499

517

Tyr 341

Ala Val

513

335

Ser 339

O N

Leu

N

S

338

F N

O

F

Gln

F

178

Ala

Val

502

Phe 504

Leu

509

His 75

345

Arg 106

Fig. 3. Mode of binding of celecoxib inside COX-2 active site.

4.1.2.2. 3-Chloro-N-(5-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol4-yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl)benzo[b]thiophene2-carboxamide, 5b. The product was obtained as yellow powder. Yield 0.12 g, 41%; m.p. 273–275 °C; IR (KBr, cm
1): 3355 (NH), 1743, 1687 (C@O), 1253 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 3.85 (s, 3H, OCH3), 7.15 (d, J = 9 Hz, 2H, 4-methoxyphenyl C3,5AH), 7.41–7.48 (m, 1H, phenyl C4AH), 7.55–7.68 (m, 6H, 4methoxyphenyl C2,6AH, benzothiophene C5,6AH, phenyl C3,5AH), 7.71 (s, 1H, vinyl CH), 7.98 (m, 1H, benzothiophene C4AH), 8.07

(d, J = 8.7 Hz, 2H, phenyl C2,6AH), 8.21 (m, 1H, benzothiophene C7AH), 8.92 (s, 1H, pyrazol C5AH), 11.76 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C29H19ClN4O3S3 (603.13): C, 57.75; H, 3.18; N, 9.29; S, 15.95. Found: C, 57.89; H, 3.19; N, 9.34; S, 16.07. 4.1.2.3. N-(5-{[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl)-3-chlorobenzo[b]thiophene-2carboxamide, 5c. The product was obtained as yellow powder.

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Leu 517

Phe 343

Ser Val Leu

516

Tyr

101

Arg

102

373

513

O

Trp 85

Trp

Ala

106

78

O

S

S O S

N H Ile

N Tyr

Cl

98

371

O Ser

Leu

339

338

Tyr

Val 335

Phe 504

341

Tyr 334

Fig. 4. Mode of binding of 5h inside COX-2 active site.

Yield 0.19 g, 59%; m.p. 278–280 °C; IR (KBr, cm
1): 3286 (NH), 1748, 1690 (C@O), 1263 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.43–7.48 (m, 1H, phenyl C4AH), 7.57–7.62 (m, 2H, phenyl C3,5AH), 7.64–7.69 (m, 5H, benzothiophene C5,6AH, 4bromophenyl C3,5AH, vinyl CH), 7.80 (d, J = 8.4 Hz, 2H, phenyl C2,6AH), 7.97–8.01 (m, 1H, benzothiophene C4AH), 8.08 (d, J = 8.7 Hz, 2H, 4-bromophenyl C2,6AH), 8.19–8.22 (m, 1H, benzothiophene C7AH), 9.01 (s, 1H, pyrazol C5AH), 11.78 (s, 1H, NH, D2O exchangeable); EIMS m/z (% relative abundance): 656 (0.34) [M++4], 654 (1.89) [M++2], 652 (70.00) [M+], 197 (35.00), 195 (100.00); Elemental analysis Calcd for C28H16BrClN4O2S3 (652.00): C, 51.58; H, 2.47; N, 8.59; S, 14.75. Found: C, 51.76; H, 2.51; N, 8.67; S, 14.88. 4.1.2.4. N-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-3-chlorobenzo[b]thiophene-2-carboxamide, 5d. The product was obtained as fine yellow powder. Yield 0.11 g, 52%; m.p. 250–252 °C; IR

(KBr, cm
1): 3418 (NH), 1728, 1681 (C@O), 1257 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.57–7.63 (m, 3H, benzothiophene C5,6AH, phenyl C4AH), 7.64–7.69 (m, 2H phenyl C3,5AH), 7.70– 7.75 (m, 2H, phenyl C2,6AH), 7.99 (dd, J = 7.5, 3 Hz, 1H, benzothiophene C4AH), 8.01 (s, 1H, benzyliden-CH), 8.20 (dd, J = 7.5, 3 Hz, 1H, benzothiophene C7AH), 11.79 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C19H11ClN2O2S3 (430.94): C, 52.96; H, 2.57; N, 6.50; S, 22.32. Found: C, 53.17; H, 2.63; N, 6.57; S, 22.38. 4.1.2.5. 3-Chloro-N-[5-(4-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]benzo[b]thiophene-2-carboxamide, 5e. The product was obtained as fine yellow powder. Yield 0.10 g, 43%; m.p. 248– 250 °C; IR (KBr, cm
1): 3190 (NH), 1735, 1671 (C@O), 1263 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 3.87 (s, 3H, OCH3), 7.17 (d, J = 7.2 Hz, 1H, phenyl C3,5AH), 7.72–7.62 (m, 4H, phenyl C2,6AH, benzothiophene C5,6AH), 7.97 (s, 1H, benzyliden CH), 8.00 (d, J = 7.8 Hz, 1H, benzothiophene-C4AH), 8.20 (d, J = 7.2 Hz,

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Tyr

A558

Val A604

Phe A555

Cl

Asn

OH

N H

A554

O Cl

Phe Gln

A557

Leu

A610

A607

His A367

Gln

A363

Fig. 5. Mode of binding of meclofenamic acid inside 5-LOX active site.

1H, benzothiophene C7AH), 11.74 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C20H13ClN2O3S3 (460.97): C, 52.11; H, 2.84; N, 6.08; S, 20.86. Found: C, 52.23; H, 2.89; N, 6.17; S, 20.97.

C7AH), 11.81 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C19H10Cl2N2O2S3 (465.38): C, 49.04; H, 2.17; N, 6.02; S, 20.67. Found: C, 49.11; H, 2.14; N, 6.13; S, 20.84.

4.1.2.6. 3-Chloro-N-[5-(4-chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]benzo[b]thiophene-2-carboxamide, 5f. The product was obtained as yellow crystal. Yield 0.10 g, 43%; m.p. 250–252 °C; IR (KBr, cm
1): 3204 (NH), 1738, 1656 (C@O), 1257(C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.64–7.70 (m, 4H, benzothiophene C5,6AH, phenyl C3,5AH), 7.75 (d, J = 8.5 Hz, 2H, phenyl C2,6AH), 7. 99 (dd, J = 6.5, 3 Hz, 1H, benzothiophene-C4AH), 8.02

4.1.2.7. 3-Chloro-N-[5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2thioxothiazolidin-3-yl]benzo [b]thiophene-2-carboxamide, 5g. The product was obtained as golden yellow powder. Yield 0.09 g, 39%; m.p. 213–215 °C; IR (KBr, cm
1): 3317 (OAH), 3135 (NH), 1727, 1661 (C@O), 1250 (C@S); 1H NMR (DMSO-d6, 300MHz, dppm): 3.31 (br, s, 1H, OH, D2O exchangeable), 3.87 (s, 3H,

(s, 1H, benzylidene-CH), 8.22 (dd, J = 6.5, 3 Hz, 1H, benzothiophene

OCH3), 7.00 (d, J = 8.1 Hz, 1H, phenyl C5AH), 7.20 (dd, J = 8.1, 3 Hz, 1H, phenyl C6AH), 7.27 (d, J = 3 Hz, 1H, phenyl C2AH),

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Val

Ser

A604

A608

Leu A607

O O N Arg

H N

S

S

A666

O

O

S

Phe Tyr

A610

Cl

Phe A555

Gln

Gln

A363

A557

A558

Asn A554

Fig. 6. Mode of binding of 5h inside 5-LOX active site.

7.57–7.65

(m,

2H,

benzothiophene

C5,6AH),

7.83

(s,

1H,

benzylidene-CH), 7.89–7.96 (m, 1H, benzothiophene-C4AH), 8.08–8.15 (m, 1H, benzothiophene C7AH), 11.46 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C20H13ClN2O4S3 (476.96): C, 50.36; H, 2.75; N, 5.87; S, 20.17. Found: C, 50.57; H, 2.76; N, 5.96; S, 20.32.

4.1.2.8. 3-Chloro-N-[5-(3,4-dimethoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]benzo [b]thiophene-2-carboxamide, 5h. The product was obtained as golden yellow powder. Yield 0.10 g, 42%; m.p. 243–245 °C; IR (KBr, cm
1): 3373 (NH), 1732, 1675 (C@O), 1256 1

(C@S); H NMR (DMSO-d6, 300MHz, dppm): 3.87 (s, 6H, 2OCH3), 7.19 (d, J = 8.4 Hz, 1H, phenyl C5AH), 7.30 (s, 1H, phenyl C2AH), 7.34 (d, J = 8.4 Hz, 1H, phenyl C6AH), 7.63–7.71 (m, 2H, benzothiophene C5,6AH), 7.95 (s, 1H, benzylidene-CH), 8.00 (d, J = 8.1 Hz, 1H, benzothiophene-C4AH), 8.20 (d, J = 6.5 Hz, 1H, benzothiophene C7AH), 11.75 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 75 MHz, dppm): 55.60, 55.78 (2OCH3), 112.23 (phenyl C2),

113.82 (phenyl C5), 115.76 (thiazolidine C5), 122.02 (phenyl C6), 122.68 (benzothiophene C7), 125.89 (benzothiophene C5), 126.20 (benzothiophene C6), 127.12 (benzothiophene C4), 127.29 (phenyl C1), 128.22 (benzothiophene C3), 135.56 (benzothiophene C3a), 135.80 (benzothiophene C7a), 136.32 (benzothiophene C2), 137.22 (benzylidene-CH), 149.11, 151.85 (phenyl C3,4), 158.85 (C@OANH), 163.11 (C@O, thiazolidine), 189.65 (C@S); Elemental analysis Calcd for C21H15ClN2O4S3 (490.99): C, 51.37; H, 3.08; N, 5.71; S, 19.59. Found: C, 51.45; H, 3.06; N, 5.78; S, 19.67. 4.1.2.9. 3-Chloro-N-[4-oxo-5-(2-oxoindolin-3-ylidene)-2-thioxothiazolidin-3-yl]benzo[b]thiophene-2-carboxamide, 5i. The product was obtained as red powder. Yield 0.20 g, 87%; m.p. > 300 °C; IR (KBr, cm
1): 3345, 3190 (NH), 1730, 1683 (C@O), 1258 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 6.99 (m, 1H, indolin C5AH), 7.10 (m, 1H, indolin C7AH), 7.39–7.47 (m, 1H, indolin C6AH), 7.55– 7.71 (m, 3H, benzothiophene C4,5,6AH), 7.95–8.01 (m, 1H, benzothiophene C7AH), 8.15–8.22 (m, 1H, indolin C4AH), 11.33 (s, 1H, amide NH, D2O exchangeable), 14.00 (br, s, 1H, indolin NH, D2O

M.M.M. El-Miligy et al. / Bioorganic Chemistry 72 (2017) 102–115

exchangeable); Elemental analysis Calcd for C20H10ClN3O3S3 (471.95): C, 50.90; H, 2.14; N, 8.90; S, 20.38. Found: C, 51.13; H, 2.17; N, 8.97; S, 20.51. 4.1.3. N-(4-Oxo-2-thioxothiazolidin-3-yl)benzofuran-2-carboxamide, 9 A mixture of benzofuran-2-carbohydrazide, 8 (0.35 g, 2 mmol) and bis (carboxymethyl)trithiocarbonate (0.49 g, 2.2 mmol) in water (20 mL) was heated under reflux with stirring for 12 h and then left to attain room temperature. The obtained precipitate was filtered, washed with water, air-dried and crystallized from ethanol. The product was obtained as fine buff powder. Yield 0.47 g, 81%; m.p. 194–196 °C; IR (KBr, cm
1): 3292 (NH), 1750, 1684 (C@O), 1250 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 4.55 (s, 2H, CH2-thiazolidin, D2O exchangeable), 7.37–7.42 (m, 1H, benzofuran C5AH), 7.52–7.58 (m, 1H, benzofuran C6AH), 7.74 (d, J = 8.4 Hz, 1H, benzofuran C4AH), 7.83 (s, 1H, benzofuran C3AH), 7.85 (d, J = 8.4 Hz, 1H, benzofuran C7AH), 11.89 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C12H8N2O3S2 (292.33): C, 49.31; H, 2.76; N, 9.58; S, 21.93. Found: C, 49.44; H, 2.81; N, 9.62; S, 22.08. 4.1.4. General procedure for the synthesis of N-(5-{[3-(4-Substitutedphenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-4-oxo-2-thioxothiazolidin3-yl)benzofuran-2-carboxamides, 10a-c A mixture of N-(4-oxo-2-thioxothiazolidin-3-yl)benzofuran-2carboxamide, 9, (0.15 g, 0.5 mmol), the appropriate aldehyde (0.5 mmol) and piperdine (2 drops) in benzene (6 ml) was heated under reflux for 1 h. The resulting precipitate was filtered, washed with benzene, air-dried and crystallized from dioxane/water (8: 2). 4.1.4.1. N-{5-[(1,3-Diphenyl-1H-pyrazol-4-yl)methylene]-4-oxo-2thioxothiazolidin-3-yl}benzofuran2-carboxamide, 10a. The product was obtained as yellow powder. Yield 0.13 g, 48%; m.p. 285– 287 °C; IR (KBr, cm
1): 3331 (NH), 1704, 1684 (C@O), 1247 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.35–7.47 (m, 3H, benzofuran C5,6AH, N1-phenyl C4AH), 7.53–7.61 (m, 5H, N1-phenyl C3.5AH, C3-phenyl C3,4,5AH), 7.68–7.70 (m, 3H, benzofuran C3AH, C3-phenyl C2,6AH), 7.76 (d, J = 8.4 Hz, 1H, benzofuran C4AH), 7.86–7.88 (m, 2H, benzofuran C7AH, vinyl CH), 8.09 (d, J = 8.4 Hz, 2H, N1-phenyl C2,6AH) 8.94 (s, 1H, pyrazol C5AH), 12.12 (s, 1H, NH, D2O exchangeable); Elemental analysis Calcd for C28H18N4O3S2 (522.60): C, 64.35; H, 3.47; N, 10.72; S, 12.27. Found: C, 64.52; H, 3.45; N, 10.84; S, 12.41. 4.1.4.2. N-(5-{[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl] methylene}-4-oxo-2-thioxothiazolidin-3-yl)benzofuran-2-carboxamide, 10b. The product was obtained as yellow powder. Yield 0.17 g, 61%; m.p. 234–237 °C; IR (KBr, cm
1): 3314 (NH), 1743, 1701 (C@O), 1251 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 3.84 (s, 3H, OCH3), 7.15 (d, J = 8.4 Hz, 2H, 4-methoxyphenyl C3,5AH), 7.36–7.46 (m, 2H, benzofuran C5,6AH), 7.54–7.64 (m, 5H, 4-methoxyphenyl C2,6AH, phenyl C3,4,5AH), 7.69 (s, 1H, benzofuran C3AH), 7.76 (d, J = 8.4 Hz, 1H, benzofuran C4AH), 7.86–7.88 (m, 2H, benzofuran C7AH, vinyl CH), 8.08 (d, J = 8.4 Hz, 2H, phenyl C2,6AH), 8.91(s, 1H, pyrazol C5AH), 12.11 (s, 1H, NH, D2O exchangeable); 13 NMR (DMSO-d6, 75 MHz, dppm): 55.28 (CH3), 112.03 (benzofuran C3), 112.70 (benzofuran C7), 114.49 (pyrazol C4), 115.15 (4methoxyphenyl C3,5), 117.19 (phenyl C2,6), 119.50 (benzofuran C4), 123.22 (thiazolidine C5), 123.31 (benzofuran C5), 124.17 (benzofuran C6), 125.81 (4-methoxyphenyl C1), 126.56 (phenyl C4), 127.63 (benzofuran C3a), 129.08 (4-methoxyphenyl C2,6), 129.57 (phenyl C3,5), 130.17 (pyrazol C5), 138.64 (phenyl C1), 145.74 (vinyl

113

CH), 145.78 (benzofuran C2), 154.19 (pyrazol C3), 154.22 (benzofuran C7a) 155.00 (C@OANH), 160.61 (4-methoxyphenyl C4), 173.01 (C@O, thiazolidine), 189.83 (C@S); Elemental analysis Calcd for C29H20N4O4S2 (552.62): C, 63.03; H, 3.65; N, 10.14; S, 11.60. Found: C, 63.17; H, 3.69; N, 10.27; S, 11.68. 4.1.4.3. N-(5-{[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl]methylene}-4-oxo-2-thioxothiazolidin-3-yl)benzofuran-2-carboxamide, 10c. The product was obtained as yellow powder. Yield 0.22 g, 71%; m. p. 237–240 °C; IR (KBr, cm
1): 3349 (NH), 1737, 1704 (C@O), 1257 (C@S); 1H NMR (DMSO-d6, 300 MHz, dppm): 7.36–7.48 (m, 2H, benzofuran C5,6AH), 7.55–7.62 (m, 3H, phenyl C3,4,5AH), 7.65– 7.68 (m, 3H, 4bromophenyl C3,5AH, benzofuran C3AH), 7.75–7.81 (m, 3H, benzofuran C4,7AH, vinyl CH), 7.87 (d, J = 7.2 Hz, 2H, phenyl C2,6AH), 8.08 (d, J = 8.4 Hz, 2H, 4-bromophenyl C2,6AH), 8.96 (s, 1H, pyrazol C5AH), 12.12 (s, 1H, NH, D2O exchangeable); EIMS m/z (% relative abundance): 602 (21.65) [M++2], 600 (18.01) [M+], 275 (49.01), 145 (100.00), 89 (63.94); Elemental analysis Calcd for C28H17BrN4O3S2 (601.49): C, 55.91; H, 2.85; N, 9.31; S, 10.66. Found: C, 56.09; H, 2.82; N, 9.44; S, 10.74. 4.2. Biological evaluation 4.2.1. In vitro COX-1 and COX-2 inhibitory assay All the newly synthesized compounds were screened for their ability to inhibit COX-1 and COX-2 enzymes. This was carried out using Cayman colorimetric COX (ovine) inhibitor screening assay kit (Catalog No. 760111) supplied by Cayman chemicals, Ann Arbor, MI, USA, according to reported method [45]. 4.2.2. In vitro lipoxygenase (LOX) inhibitory assay All the newly synthesized compounds were also evaluated in vitro for their ability to inhibit lipoxygenase enzyme. This was carried out using Abnova lipoxygenase inhibitor screening assay kit (Catalog No. (KA1329). Inhibitors were dissolved in DMSO and were added to the assay in a final volume of 10 ll before initiating with substrate. Three concentrations were prepared (25, 50 and 100 lM) and the concentration that produced 50% enzyme inhibition was determined according to manufacturer instructions [46]. 4.2.3. In vivo assay 4.2.3.1. Animals. Adult Male Wistar rats weighing 150–250 g were used (procured from Experimental Animal Centre in Alexandria University). All animals accessed food and water ad libitum and were housed in 12 h dark/light cycle in a controlled condition at 23–25 °C. They were allowed to acclimatize for 1 week prior to experimentation. Animals were transferred to the experiment room 2 h before the experiment. Procedures involving animals and their care were conducted with the Guide for the Care and Use of Laboratory Animals published by US National Institute of Health (NIH publication No. 83-23, revised 1996) and following the ethical guidelines of Alexandria University on laboratory animals. In all tests, adequate considerations were used to reduce discomfort or pain of animals. 4.2.3.2. Compounds. Celecoxib powder (Sigma-Aldrich), formalin 5% made from formaldehyde 37% and saline (Merck, Germany) were used. The novel compounds were synthesized based on the previously described methods. 4.2.3.3. Formalin-induced paw edema test. Compound 5h was evaluated for its in vivo anti-inflammatory activity applying the formalin-induced paw edema screening protocol [47,48]. Celecoxib (20 mg/kg [49]) was used as a reference drug. Animals were

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divided into three groups each of six rats. Rats that were given the vehicle (DMSO) served as control (group I), group II treated with celecoxib served as reference and groups from III to VII treated with test compounds 5h. A solution of freshly prepared formalin 5% was used as a phlogistic agent. A mark was made on the lateral malleolus of the rats’ paws delineating the injection sites. The initial volume of paw was measured by means of digital calibrated Vernier caliper. Then, the novel test compounds (20 mg/kg body weight), celecoxib (20 mg/kg body weight), or DMSO, as the control solution, were administered orally. After 45 min, 40 lL formalin was injected subcutaneously into the sub plantar tissue of the right hind paw of all groups under light ether anesthesia. An equal volume of saline was injected into the left hind paw and used as internal control for the degree of inflammation in the right hind paw. The volume of paw was measured in different treatment groups, 4 h following the formalin injection. The increase in paw volume was calculated by subtracting the volumes before and 4 h after the injection of formalin. Edema was expressed as an increase in the volume of paw, and the percentage of edema inhibition (or percent protection against inflammation) for each rat and each group was calculated according to the following equation:

% Inhibition ¼

ðVt
VoÞcontrol
ðVt
VoÞtest compound  100 ðVt
VoÞcontrol

where Vt is the mean volume of edema at specific time interval (4 h) and Vo is the mean volume of edema at zero time interval. Relative potency of the tested compounds was expressed as % inhibition of edema for the tested compounds relative to % inhibition of edema for the reference drugs at 4 h from the induction of inflammation. % Relative potency ¼

%inhibition of edema for the test compoundð4 hoursÞ %inhibition of edema for the referencesð4 hoursÞ  100

4.3. Molecular modeling 4.3.1. In silico prediction of physicochemical properties and pharmacokinetic profile In the present investigation, the most biologically active compound 5h was subjected to molecular properties and bioactivity prediction by Molinspiration online property calculation toolkit [37], drug-likeness and solubility parameter calculation by MolSoft software [38], ADME profiling by PreADMET [39] calculator and toxicity-risk assessment by Osiris property explorer [40] to filter and analyze their overall potential to qualify for a drug, and also comparing them to some current anti-inflammatory drugs. 4.3.2. Anti-inflammatory docking study The molecular docking studies were performed using the Molecular Operating Environment (MOE 2008.10) software [42]. The three dimensional structures and conformations of the enzymes were acquired from the Protein Data Bank (PDB) Web site [43]. The target compounds were drawn in MOE using the builder module, and collected in a database. The database was prepared using the option ‘‘Protonate 3D” to add hydrogens, calculate partial charges and minimize energy. In addition, the proteins were prepared by deleting the repeating chains, water molecules and any surfactants. Hydrogens were also added to the atoms of the receptor and the partial charges were calculated. The compounds’ database was then docked into the pocket of each protein using the MOE dock. MOE was also used to calculate the best score between the ligands and the enzymes’ binding sites. The resulted database contained the score between the ligands’ conformers and the enzymes’ binding sites in kcal/mol. The obtained poses that showed the best ligand-enzyme interactions were selected. Besides, the target compounds that displayed significant dual in vitro COX-2/LOX inhibitory activity and the reference drugs (celecoxib and meclofenamic acid) were docked to the active sites of Mus musculus COX-2 (PDB: 3LN1) and Homo sapiens 5-LOX (PDB: 3V99) to explore their mode of binding to the receptors and compare that with the mode of binding of target compounds. Acknowledgment

4.2.3.4. Gastric ulcerogenic activity. Compounds 5h was evaluated for its acute gastric ulcerogenic effect in adult male Wistar rats [50]. Rats (200–250 g) were divided into three groups of six rats each and were fasted for 12 h prior to test compound administration. Water was given ad libitum. The vehicle (DMSO) was given to control group, and other groups received celecoxib or the test compound orally at a dose of 60 mg/kg body weight (three times the previously used dose) [51]. Six hours after the treatment they were deep ether anesthetizedand sacrificed and their stomachs were removed and opened through greater curvature, washed under running water and fixed in saline solution. Gross examination was performed for any evidence of hyperemia, hemorrhage, definite hemorrhagic erosion or ulcer. The degree of ulcerogenicity was determined by viewing the gastric epithelial ulceration using a 5 magnifying lens and rated by ulcer score. Ulcer score was used to grade the incidence and severity of the lesions such as (1) (2) (3) (4) (5)

Shedding of epithelium—10 Petechial and frank hemorrhages—20 One or more ulcers—30 More than two ulcers—40 Perforated ulcers—50.

In addition, histopathological examination was also performed to confirm the degree of inflammatory reaction in the gastric layers of the treated rats’ stomachs.

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