Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) cause peptic lesions in the gastrointestinal mucosa by inhibiting the cyclooxygenase-1 (COX-1) enzyme. Selective COX-2 inhibition causes decreased side effects over current NSAIDs. Therefore, the studies about selective inhibition of COX-2 enzyme are very important for new drug development. The design, synthesis and biological activity evaluation of novel derivatives bearing thiazolylhydrazine-methyl sulfonyl moiety as selective COX-2 inhibitors were aimed in this paper. The structures of synthesized compounds were assigned using different spectroscopic techniques such as 1H NMR,13C NMR and HRMS. In addition, the estimation of ADME parameters for all compounds was carried out using in silico process. The evaluation of in vitro COX-1/COX-2 enzyme inhibition was applied according to the fluorometric method. According to the enzyme inhibition results, synthesized compounds showed the selectivity against COX-2 enzyme inhibition as expected. Compounds 3a, 3e, 3f, 3g, 3i and 3j demonstrated signiicant COX-2 inhibition potencies. Among them, compound 3a was found to be the most effective derivative with an IC50 value of 0.140 ± 0.006 mM. Moreover, it was seen that compound 3a displayed a more potent inhibition proile at least 12-fold than nimesulide (IC50 = 1.684 ± 0.079 mM), while it showed inhibitory activity at a similar rate ofcelecoxib (IC50 = 0.132 ± 0.005 mM). Molecular modelling studies aided in the understanding of the interaction modes between this compound and COX-2 enzyme. It was found Infected total joint prosthetics that compound 3a had a signiicant binding property. In addition, the selectivity of obtained derivatives on COX-2 enzyme could be explained and discussed by molecular docking studies.
1. Introduction
Steroid drugs, which are highly effective for chronic inflammatory conditions, prevent the synthesis of leukotrienes and PGs via the phospholipase A2 pathway. Prostaglandins are known to be biosynthesized from arachidonic acid by the action of cyclooxygenase enzymes (COXs) present in two isoforms (constitutive (COX-1) and inducible form (COX-2)). The mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) includes inhibition of cyclooxygenases (COX-1 and COX-2) and lipoxygenase (5-LOX), which act in the prostaglandin (PG) synthesis. However, side effects of this heterogeneous class click here of compounds on gastrointestinal (GI) and kidney function are their main problem. COX-1, which is mainly involved in the regulation of gastric mucosal blood flow, renal function and vasomotor contraction, is a structural protein found in the blood vessels, kidneys, stomach and other tissues, whereas COX-2, expressed in the nuclear membrane, is an inducible enzyme that is almost undetectable in normal physiological state. Although COX-1 is a structural form that maintains renal function and regulates gastrointestinal cytoprotection, it catalyzes the formation of other isozyme (COX-2) (PG1) and is in charge for fever, pain and other inflammatory symptoms. NSAIDs, large class of therapeutic compounds, are
often used for pain relief, arthritis and rheumatism in everyday of life with variable beneit/risk proile. However, long-term NSAID use is associated with side effects such as
gastrointestinal (GI) ulcers, cardiovascular toxicity, hepatic and renal toxicity, bleeding and platelet disorder, aplastic anemia, and reduced bone healing. It is an urgent need to use new and safe anti-inflammatory drugs for common chronic inflammatory disorders such as rheumatoid arthritis. Unrelated and nonspeciic side effects of conventional NSAIDs result from inhibition of the physiologically important cyclooxygenase-1 (COX-1) enzyme. Accordingly, the use of COX2 selective inhibitors has been seen as a more useful approach in reducing these negative effects. Among new selective COX-2 inhibitors (coxibs), rofecoxib was withdrawn from the market in 2004 due to cerebrovascular risk and cardiac toxicity caused by decreased prostacyclin (PGI2) at the standard dose. Due to these facts, treatment with NSAIDs is limited to side effects. However, beneits and risks of both conventional NSAIDs and selective COX-2 inhibitors should be evaluated together, and COX-2 inhibitors should be suggested for the patients having digestive system problems. Thus, there is still a need for new selective COX-2 inhibitors to increase the number of alternative agents in the treatment of inflammation and pain [1e16].
Human COX-1 and COX-2 enzymes, which have very similar active site architecture, are structurally 60% similar, with 70% amino acid homology. Although they are similar enzymes, the active site of COX-2 enzyme is 20% bigger than COX-1 and slightly different. Compared to Ile523, His513 and Ile434 found in COX-1, the amino acids of COX-2, Val523, Arg513 and Val434, form a larger polar binding pocket speciic to this isoenzyme [17e19]. When most COX-2 selective inhibitors are examined, two aryl groups attached to adjacent atoms of a central ring, which can be homocyclic or heterocyclic, are remarkable, and one of these aryl groups is replaced by a methyl sulfonyl (SO2CH3) or an amino sulfonyl (SO2NH2) group in the para position. Methyl sulfonyl group, a more lipophilic group with low acidic properties compared with acetic or propionic group, increases the selective COX-2 inhibition by facilitating the interaction of the molecule with the polar side pockets [13,18,20e25].In this study, we aim to present the new thiazole derivatives including methyl sulfonyl moiety as selective COX-2 inhibitors. For this purpose, para-methylsulfonylphenyl substructure on the central thiazole ring was used to provide lipophilicity and to form interactions with the polar side pocket of COX-2 enzyme.
2. Result and discussion
2.1. Chemistry
Compounds in this study (3ael) were gained by using a ring closure reaction between equimolar amounts of 2-bromo-1-(4(methylsulfonyl)phenyl)ethan-1-one and 2-substituehydrazine
-1carbothioamide. The synthesis pathway of compounds was summarized in Scheme 1.Methyl sulfonyl group of the inal compounds were recorded as singlet peak at 3.23 or 3.24 ppm in the 1H NMR and 44.0 or 44.1 ppm in the 13C NMR. Thiazole CH and hydrazine NH protons were less affected by the change of the electronic environment with respect to the imine CH proton, so they varied in a more limited range. Thiazole CH was observed as singlet between 7.51 ppm and 7.63 ppm while NH proton was over 12.02 ppm. Imine CH were seen as singlet between 7.91 ppm and 8.37 ppm. Sulfonyl phenyl protons were recorded as two doublets at 7.9 ppm and 8.1 ppm. All other protons and carbons were observed at expected values. And MS results were also compatible with theoretical m/z.
2.2. COX inhibition assay
All the gained thiazolylhydrazine-methyl sulfonyl derivatives were evaluated for their ability to inhibit COX-1 and COX-2 enzymes using fluorometric inhibitor screening kits (Biovision, Switzerland) according to the manufacturer’s instructions [26,27]. The enzyme activity protocol was applied in 2 steps according to the inhibition percentages and concentrations of the compounds. The synthesized compounds and reference agents were used at their concentrations of 10-3 and 10-4 M for the irst stage of the assay. The results of this step are given in Table 1. The selection of the compounds to pass to the second step of inhibition protocol was made based on analyzing these results which showed more than 50% inhibitory activity at 10-4 M concentration. The selected compounds and reference agents were prepared in their further concentrations by serial dilutions (ranging from 10-5 M to 10-9 M) for second step. Therefore, the half maximal inhibitory concentration (IC50) values of the selected compounds and reference inhibitors could be calculated and these results are given in Table 2.
As seen in Table 1, many of the test compounds showed more than 50% inhibitory activity at 10-3 M concentration for both enzymes. But similar eficacy was not the case for the inhibition at 10-4 M concentration. In this concentration, none of the compounds displayed remarkable activity against COX-1 enzyme. On the other hand, compounds 3a, 3e, 3f, 3g, 3i and 3j demonstrated more than 50% inhibitory activity on COX-2 enzyme. So, these compounds were selected for second step of inhibition assay and their IC50 values were determined as seen in Table 2.Generally, it can be said that all synthesized compounds showed selective inhibitory potency against COX-2 enzyme. In this context, selectivity indexes (SI) were expressed as IC50(COX-1)/IC50(COX-2). It was observed that synthesized compounds had selective inhibitory activity on COX-2 enzyme with SI ranging from >10 to >714.286.Among the selected derivatives, compound 3a was found to be the most active agent with an IC50value of 0.140 ± 0.006 mM. When this value compared to that of reference drugs, it was seen that compound 3a displayed a more potent inhibition proile at least 12fold than nimesulide (IC50 = 1.684 ± 0.079 mM), while showed inhibitory activity at a similar rate with celecoxib (IC50= 0.132 ± 0.005 mM). Besides, compound 3a was determined to have the highest SI of >714.286. Moreover, it was detected that except for compound 3a, compounds 3e, 3f, 3g, 3i and 3j were determined to be the other active derivatives against COX-2 enzyme. Compounds 3e, 3f, 3g, 3i and 3j executed more potent inhibitory activity than nimesulide (IC50= 1.684 ± 0.079 mM) with IC50 values of 0.213 ± 0.008 mM, 0.175 ± 0.007 mM, 0.184 ± 0.008 mM, 0.239 ± 0.009 mM and 0.195 ± 0.008 mM, respectively.
2.3. Prediction of ADME parameters
Absorption, distribution, metabolism and elimination (ADME) properties are very important in current drug discovery and development. In addition to good eficacy, the success of drug candidate is also determined by good ADME proiles. Although in vitro ADME screens are available, in silico prediction of ADME properties proves to be valuable in terms of abstaining of the high cost and time consumption in current drug discovery. In silico methods estimates ADME properties using statistical approaches [28,29], molecular descriptors [30] and experimental data to model complex biological processes (e.g. partition coeficient, aqueous solubility, brain/blood partition coeficient, central nervous system activity, apparent Caco2 and MDCK cell permeability, total solvent-accessible volume, Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms, human oral absorption percent, drug likeness score, namely Lipinski’s rule of ive and Jorgensen’s rule of three) [31e34]. In this study, the prediction of ADME parameters of the synthesized compounds were carried out with the help of QikProp 4.8 software [35] by computational method. The predicted parameters and their recommended values are presented in Table 3.
Scheme 1. Synthesis way of the compounds 3a-3l.
QikProp 4.8 software also allows to estimate the drug-likeness as per Lipinski’s “Rule of Five” and Jorgensen’s “Rule of Three” [36e39]. In accordance with the rules of three and ive, the gained compounds (3a-3l) were in full compliance with the parameters set, since they did not cause any violation. Moreover, in Table 3 it was shown that all the physicochemical parameters were found to be within the acceptable range for drug candidacy. Among these parameters, polar surface area (PSA), which is an important measure of drug-likeness, is an index to show the likelihood of transporting a molecule through cell membranes-allows for the prediction of human intestinal absorption and blood-brain barrier penetration among others. The synthesized compounds showed medium to high PSA feature range from 79.613 to 116.022. Therefore, it was understood that the inal compounds had a good ability to transport through cell membranes.
Additionally, it was seen from Table 3 that the investigated compounds displayed signiicant cell permeability in Caco-2 and MDCK cell lines which are very essential for drug absorption and bioavailability.Based on Laboratory Supplies and Consumables the indings of the ADME parameter trials, the synthesized compounds were found to be drug-like having obeyed the “Rule of Five” and “Rule of Three”, displayed good physicochemical properties.
2.4. Molecular docking
As mentioned in the COX enzymes inhibitory activity assay, compound 3a was found to be the most active derivative in the series against COX-2 enzyme. Therefore, docking experiments were carried out to determine its inhibitory potential as in silico. Owing to the docking studies, further information into the binding mode of compound 3a and assessment of the impact of structural modiications on the inhibitory activity against COX-2 enzyme could be researched. Studies were performed by using the X-ray crystal structure of COX-2 (COX-2 PDB Code: 3LN1) [40] retrieved from Protein Data Bank database (www.pdb.org). Firstly, the docking procedure was validated by executing the protocol with celecoxib. Then, compound 3a was subjected to the same docking procedure. The rendered docking poses of celecoxib and compound 3a are presented in Fig. 1-5.
When analyzed the structures of the COX isoenzymes, it was seen that COX-2 enzyme had a 20% wider structure than COX-1 and has structural changes different from COX-1. The principal difference was the speciic side pocket of the hydrophilic structure contained in the COX-2 enzyme. This speciic side pocket consisted of Leu338, Ser339, Arg499, Phe504 and Val509 amino acid residues, and especially selective inhibitors that carried selective groups such as sulfone (-SO2), sulfonamide (-SO2NH2) or methyl sulfonyl (-SO2CH3) could bind to this area. This situation mentioned could be seen in the docking pose of selective COX-2 inhibitor celecoxib (Fig. 1). The amino group of sulfonamide established two hydrogen bonds with carbonyls of Leu338 and Ser339. The other two hydrogen bonds formations were observed between the oxygen atoms of sulfonamide and amino groups of Arg499 and Phe504. In addition, the pyrazole ring in celecoxib formed a cation-π interaction with the amino of Arg106.
Fig. 2 presents the superimposition pose of celecoxib and compound 3a, which was determined to be the most active agent, in the active site of COX-2 enzyme. According to this igure, it was understood that compound 3a bounded to the active region in a very similar position with celecoxib.
The 3D interacting mode of compound 3a is given in Fig. 3. The phenyl ring near to the sulfonamide was in interaction with the phenyl of Tyr341 by a π-π interaction. Moreover, there was a formation of hydrogen bond between the nitrogen atom of thiazole ring and the carbonyl of Val335. Furthermore, it was seen that the hydrazine moiety was very essential for polar interaction. Because, it was detected that the imine nitrogen formed a hydrogen bond with the hydroxyl of Ser516. Similarly, the methyl sulfonyl moiety showed the same interactions with the sulfonamide ofcelecoxib.
Fig. 1. The 3D interacting mode ofcelecoxib in the active region of COX-2 enzyme (PDB Code: 3LN1). Celecoxib is shown in a tube pattern and dark blue colored. (For interpretation of the references to color in this igure legend, the reader is referred to the Web version of this article.
Fig. 3. The 3D interacting mode of compound 3a in the active region of COX-2 enzyme (PDB Code: 3LN1). Compound 3a is shown in a tube pattern and dark green colored. (For interpretation of the references to color in this igure legend, the reader is referred to the Web version of this article.
The oxygen atoms of methyl sulfonyl formed two hydrogen bonds with the amino groups of Arg499 and Phe504. These interactions especially with Arg499 and Phe504 amino acids were very important in terms of speciic side pocket placement and COX-2 selectivity. These indings were an evidence of the selectivity on COX-2 enzyme of the synthesized compounds.The main difference of the gained compounds was the substituents at the phenyl ring near to hydrazine moiety. Compound 3a had a hydroxyl at the meta position of phenyl ring. The contributions of this substituent and its position to enzyme inhibitory activity could be explained owing to molecular docking studies. According to Fig. 3, the hydroxyl of compound 3a established a hydrogen bond with the carbonyl of Met508. Therefore, it can be understood that the substituents especially at the meta position of the phenyl ring contribute positively to enzyme activity and this mined to be the most active derivative. Moreover, this result supported the inding that compounds 3e, 3f, 3g, 3i and 3j, which had the substituents at the especially meta and other positions, displayed a more potent inhibition proiles than compounds 3b, 3c, 3d and 3h, which did not carry any substituents at the meta position. Furthermore, it was thought that compounds 3k and 3l could not display any additional interaction belonging to the substituents at the phenyl ring because of the steric obstacle created by the trisubstitution (namely at the orto, meta and para positions). So, these compounds could not show remarkable inhibitory potency likewise the compounds mentioned above because of this situation.
Fig. 2. The superimposition pose of celecoxiband compound 3a in the enzyme active site (PDB Code: 3LN1).
The docking experiments were detailed using Glide [41] according to the Per-Residue Interaction panel to examine the role of the van der Waals and electrostatic interactions in binding to the enzyme active site. Figs. 4 and 5 present the van der Waals and electrostatic interactions of compound 3a. This compound displayed beneicial interaction of van der Waals with amino acids Gln178, Tyr341, Val335, Leu338, Ser339, Phe367, Leu370, Arg499, Ile503, Phe504, Met508, Val509, Gly512, Ala513, Ser516 and Leu517; shown in red and pink (Fig. 4), as described in the Glide user guide. Likewise, successful electrostatic contributions of compound 3a with amino acids Hid75, Arg106, Gln178, Leu338, Ser339, Tyr371, Trp373, Arg499, Ile503, Met508, Val509, Glu510 and Gly512 were observed (Fig. 5).
Fig. 4. The van der Waals interaction between compound 3a and active site of COX-2 (PDB Code: 3LN1). The active ligand has several advantageous (pink and red) van der Waals interactions. (For interpretation of the references to color in this igure legend, the reader is referred to the Web version of this article.
Fig. 5. The electrostatic interaction between compound 3a and active site of COX-2 (PDB Code: 3LN1). The active ligand has several advantageous (blue, pink, and red) electrostatic interactions. (For interpretation of the references to color in this igure legend, the reader is referred to the Web version of this article.
3. Conclusion
In the current investigation, a diverse series of thiazolylhydrazine-methyl sulfonyl derivatives were designed, synthesized and evaluated for their ability to inhibit COX enzyme by an in vitro fluorometric method. The enzyme inhibitory activity results proved that all synthesized compounds demonstrated the selectivity against COX-2 enzyme and especially compounds 3a, 3e, 3f, 3g, 3i and 3j showed remarkable inhibition potencies. Moreover, the results of in vitro enzyme inhibition assay highlighted that compound 3a had the highest COX-2 inhibitory activity (IC50=0.140 ± 0.006 μM). This inding indicated that this compound had a more potent inhibition proile at least 12-fold than nimesulide (IC50= 1.684 ± 0.079 μM), while showed inhibitory activity at a similar rate with celecoxib (IC50= 0.132 ± 0.005 μM). Furthermore, detailed docking results revealed the reason of selectivity of the synthesized compounds against COX-2 enzyme. According to molecular docking experiments, compound 3a bounded in a similar mode to celecoxib with COX-2 enzyme. Overall, all synthesized compounds could serve as promising candidates due to their good ADME parameters and drug-likeness properties. Based on the indings in this study, it was hypothesized that thiazolylhydrazinemethyl sulfonyl derivatives can serve as a new approach to improve the new selective COX-2 inhibitor candidates. Further development of new powerful anti-inflammatory agents can be carried out in light of these results.
4. Experimental
4.1. Chemistry
All the chemicals used in the synthesis were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA). A MP90 digital melting point apparatus (Mettler Toledo, OH, USA) was used to determine the melting points of the resulting compounds and was presented uncorrected. 1H NMR and13C NMR spectra were recorded by a Bruker 300 MHz and 75 MHz digital FTNMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in DMSO-d6, respectively. In the NMR spectra, splitting patterns were determined and recognized as follows: s: singlet, d: doublet, t: triplet, dd: double doublet, and m: multiplet. Coupling constants (J) were reported in units of Hertz (Hz). Mass spectra were recorded on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan) by means of the ESI method. Silica gel 60 F254 with thin-layer chromatography (Merck KGaA, Darmstadt, Germany) was used to check the purity of compounds.
4.1.1. General procedure for the synthesis of the compounds
4.1.1.1. Synthesis of 2-bromo-1-(4-(methylsulfonyl)phenyl)ethan-1one ( 1). Bromine (0.019 mol, 1 mL) in acetic acid (10 mL) was added to a solution of substituted 1-(4-(methylsulfonyl)phenyl) ethan-1-one (0.016 mol, 3.168 gr) and 3e5 drops of hydrogen bromide in acetic acid (10 mL) at 0 。C. The reaction was processed under magnetic stirring for 20 h. After completion of the reaction the mixture was poured in an ice-bath, precipitated product was iltered, dried, and recrystallized from EtOH.
4.1.1.2. Synthesis of 2-substituehydrazine-1-carbothioamide (2a-2l). Substituted benzaldehyde (0.004 mol) was dissolved in ethanol (10 mL) and an equimolar quantity of a thiosemicarbazide was added to the reaction mixture. The mixture was refluxed for 3he6h. After completion of the reaction the mixture was cooled in an icebath, precipitated product was iltered, dried, and recrystallized from EtOH [42e44].
4.1.1.3. General procedures of target compounds (3a-3l). The compounds 2ae2l (1 mmol) and 2-bromo-1-(4-(methylsulfonyl) phenyl)ethan-1-one (1.2 mmol) were refluxed in ethanol (20 mL) until all of the starting compounds disappeared, as determined by TLC. The mixture was cooled in an ice-bath, precipitated product was iltered, dried, and recrystallized from EtOH [42e44].
4.1.1.3.1. 3-((2-(4-(4-(methylsulfonyl)phenyl)thiazol-2-yl)hydrazineylidene)methyl)phenol (3a). M.P: 202.9e205.3 。C. 1H NMR (300 MHz, DMSO-d6): δ = 3.24 (3H, s, eCH3), 6.79 (1H, dd, J= 8.0 Hze1.8 Hz, phenol CH), 7.05 (1H, d, J= 7.7 Hz, phenol CH), 7.10 (1H, br. s, phenol CH), 7.23 (1H,t, J= 7.8 Hz, phenol CH), 7.63 (1H, s, thiazoleCH), 7.94e7.97 (3H, m, disubs. phenyl CH, N]CH), 8.10 (2H, d, J= 8.5 Hz, disubs. phenyl CH), 9.60 (1H, br. s, OH), 12.22 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ = 44.1,107.7,112.5,117.3,118.5, 126.5, 128.0, 130.4, 136.0, 139.6, 139.7, 142.3, 149.3, 158.1, 169.0. HRMS (m/z): [MþH]þ calcd for C17H15N3O3S2: 374.0628; found: 374.0615.
4.1.1.3.2. 4-((2-(4-(4-(methylsulfonyl)phenyl)thiazol-2-yl)hydrazineylidene)methyl)phenol (3b). M.P: 255.3e257.9 。C. 1H NMR (300 MHz, DMSO-d6): δ = 3.23 (3H, s, eCH3), 6.83 (2H,d, J= 8.6 Hz, phenol CH), 7.50 (2H, d, J= 8.6 Hz, phenol CH), 7.51 (1H, s, thiazole CH), 7.93e7.96 (3H, m, disubs. phenyl CH, N]CH), 8.10 (2H, d, J= 8.5 Hz, disubs. phenyl CH), 9.86 (1H, br. s, OH),12.03 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ = 44.1, 107.3, 116.2, 125.8, 126.5, 128.0, 128.5, 139.6, 139.7, 142.6, 149.3, 159.3, 169.2. HRMS (m/z): [MþH]þ calcd for C17H15N3O3S2: 374.0628; found: 374.0615.
4.1.1.3.3. 2-(2-(2-methoxybenzylidene)hydrazineyl)-4-(4-(methylsulfonyl)phenyl)thiazole (3c). M.P: 225.9e226.8 。C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.86 (3H, s, eOCH3), 7.02 (1H, t, J¼ 7.5 Hz, methoxyphenyl CH), 7.09 (1H, d, J¼ 8.3 Hz, methoxyphenyl CH), 7.38 (1H, td,J¼ 7.9 Hze1.7 Hz,methoxyphenyl CH), 7.62 (1H, s, thiazole CH), 7.79 (1H, dd, J¼ 7.7 Hze1.6 Hz, methoxyphenyl CH), 7.95 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 8.10 (2H,d,J¼ 8.5 Hz, disubs. phenyl CH), 8.37 (1H, s, N]CH),12.24 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 56.1, 107.6, 112.3, 121.2,122.7,125.3,126.5,128.0,131.3,137.6,139.6,139.7,149.3,157.5, 169.0. HRMS (m/z): [MþH]þ calcd for C18H17N3O3S2: 388.0784; found: 388.0769.
4.1.1.3.4. 4-((2-(4-(4-(methylsulfonyl)phenyl)thiazol-2-yl)hydrazineylidene)methyl)benzene-1,3-diol (3d). M.P: 280.2e281.9。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.23 (3H, s, eCH3), 6.33e6.35 (2H, m, dihydroxyphenyl CH), 7.42e7.45 (1H, m, dihydroxyphenyl CH), 7.57 (1H, s, thiazole CH), 7.95 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 8.10 (2H, d, J¼ 8.4 Hz, disubs. phenyl CH), 8.22 (1H, s, N]CH), 9.80 (1H, s, OH), 10.08 (1H, s, OH), 12.02 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 102.9, 106.9, 108.4, 112.0, 126.5, 128.0, 128.6, 139.6, 139.7, 141.7, 149.4, 158.1, 160.6, 168.8. HRMS (m/z): [MþH]þ calcd for C17H15N3O4S2: 390.0577; found: 390.0574.
4.1.1.3.5. 4-Methoxy-2-((2-(4-(4-(methylsulfonyl)phenyl)thiazol2-yl)hydrazineylidene)methyl)phenol (3e). M.P: 248.2e250.1。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.72 (3H, s, eOCH3), 6.84e6.85 (2H, m, methoxyphenol CH), 7.18 (1H, s, methoxyphenol CH), 7.62 (1H, s, thiazoleCH), 7.96 (2H,d,J¼ 8.6 Hz, disubs. phenyl CH), 8.11 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 8.31 (1H, s, N]CH), 9.65 (1H, s, OH),12.26 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 55.8,107.5,110.0,117.6,117.7,120.9,126.5,128.0, 139.6, 139.7, 139.8, 149.4, 150.6, 152.7, 168.8. HRMS (m/z): [MþH]þ calcd for C18H17N3O4S2: 404.0733; found: 404.0715.
4.1.1.3.6. 2-Methoxy-5-((2-(4-(4-(methylsulfonyl)phenyl)thiazol2-yl)hydrazineylidene)methyl)phenol (3f). M.P: 247.0e249.6。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.80 (3H, s, eOCH3), 6.94e7.02 (2H, m, methoxyphenol CH), 7.19 (1H, s, methoxyphenol CH), 7.60 (1H, s, thiazole CH), 7.91 (1H, s, N]CH), 7.96 (2H, d, J¼ 8.6 Hz, disubs. phenyl CH), 8.10 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 9.26 (1H, s, OH), 12.07 (1H, s, NH)$13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 56.1, 107.4, 112.1, 112.4, 120.0, 126.5, 127.6, 128.0, 139.6, 139.7, 142.2, 147.3, 149.3, 149.8, 169.1. HRMS (m/ z): [MþH]þ calcd for C18H17N3O4S2: 404.0733; found: 404.0713.
4.1.1.3.7. 2-(2-(2,3-dimethoxybenzylidene)hydrazineyl)-4-(4(methylsulfonyl)phenyl)thiazole (3g). M.P: 263.6e265.9。C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.78 (3H, s, eOCH3), 3.83 (3H, s, eOCH3), 7.07 (1H, dd, J¼ 8.2 Hze1.7 Hz, dimethoxyphenyl CH), 7.12 (1H, t, J¼ 7.9 Hz, dimethoxyphenyl CH), 7.40 (1H, dd, J¼ 7.7 Hze1.6 Hz, dimethoxyphenyl CH), 7.63 (1H, s, thiazole CH), 7.96 (2H,d,J¼ 8.6 Hz, disubs. phenyl CH), 8.11 (2H,d,J¼ 8.5 Hz, disubs. phenyl CH), 8.31 (1H, s, N]CH), 12.27 (1H, s, NH)$13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 56.2, 61.6, 107.8, 114.0, 117.0, 124.9, 126.5, 128.0, 128.2, 137.7, 139.6, 139.7, 147.7, 149.4, 153.2, 168.9. HRMS (m/z): [MþH]þ calcd for C19H19N3O4S2: 418.0890; found: 418.0874.
4.1.1.3.8. 2-(2-(2,4-dimethoxybenzylidene)hydrazineyl)-4-(4(methylsulfonyl)phenyl)thiazole (3h). M.P: 209.8e211.7。C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.23 (3H, s, eCH3), 3.81 (3H, s, eOCH3), 3.85 (3H, s, eOCH3), 6.62e6.64 (2H, m, dimethoxyphenyl CH), 7.59 (1H, s, thiazole CH), 7.70e7.73 (1H, m, dihydroxyphenyl CH), 7.94 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 8.10 (2H, d, J¼ 8.4 Hz, disubs. phenyl CH), 8.28 (1H, s, N]CH), 12.06 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 55.9, 56.2, 98.7, 107.0, 107.3, 115.6, 126.5, 128.0, 137.8, 137.9, 139.6, 139.7, 149.3, 158.9, 162.4, 169.1. HRMS (m/z): [MþH]þ calcd for C19H19N3O4S2: 418.0890; found: 418.0870.
4.1.1.3.9. 2-(2-(2,5-dimethoxybenzylidene)hydrazineyl)-4-(4(methylsulfonyl)phenyl)thiazole (3i). M.P: 233.2e235.7。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.76 (3H, s, eOCH3), 3.80 (3H, s, eOCH3), 6.97 (1H, dd, J¼ 9.0 Hze3.0 Hz, dimethoxyphenyl CH), 7.03 (1H,d,J¼ 9.0 Hz, dimethoxyphenyl CH), 7.31 (1H, d, J¼ 3.0 Hz, dimethoxyphenyl CH), 7.63 (1H, s, thiazole CH), 7.95 (2H, d, J¼ 8.6 Hz, disubs. phenyl CH), 8.10 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 8.33 (1H, s, N]CH), 12.26 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 55.8, 56.6, 98.7, 107.7, 109.7, 113.7, 116.7, 123.4, 126.5, 128.0, 137.3, 139.6, 139.7, 149.4, 152.3, 153.7, 169.0. HRMS (m/z): [MþH]þ calcd for C19H19N3O4S2: 418.0890; found: 418.0877.
4.1.1.3.10. 2-(2-(3,4-dimethoxybenzylidene)hydrazineyl)-4-(4(methylsulfonyl)phenyl)thiazole (3j). M.P: 240.2e242.2。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.80 (3H, s, eOCH3), 3.82 (3H, s, eOCH3), 7.01 (1H, d, J¼ 8.4 Hz, dimethoxyphenyl CH), 7.18 (1H, dd,J¼ 8.4 Hze1.7 Hz, dimethoxyphenyl CH), 7.28 (1H,d,J¼ 1.7 Hz, dimethoxyphenyl CH), 7.61 (1H, s, thiazole CH), 7.95 (2H, d, J¼ 8.6 Hz, disubs. phenyl CH), 7.98 (1H, s, N]CH), 8.10 (2H, d, J¼ 8.6 Hz, disubs. phenyl CH), 12.15 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.1, 55.8, 56.0,107.4,108.8,112.1,120.8,126.5,127.5, 128.0, 139.6, 139.7, 142.3, 149.3, 149.5, 150.6, 169.1. HRMS (m/z): [MþH]þ calcd for C19H19N3O4S2: 418.0890; found: 418.0875.
4.1.1.3.11. 2,6-Dimethoxy-4-((2-(4-(4-(methylsulfonyl)phenyl) thiazol-2-yl)hydrazineylidene)methyl)phenol (3k). M.P: 255.7e258.0。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.82 (6H, s, eOCH3), 6.95 (2H, s, dimethoxyphenol CH), 7.60 (1H, s, thiazole CH), 7.94e7.96 (3H, m, disubs. phenyl CH, N]CH), 8.10 (2H,d,J¼ 8.5 Hz, disubs. phenyl CH),12.14 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.0, 56.4, 104.4, 107.4, 125.1, 126.5, 127.8, 137.8, 139.6, 139.7, 142.8, 148.6, 149.3, 169.1. HRMS (m/z): [MþH]þ calcd for C19H19N3O5S2: 434.0839; found: 434.0822.
4.1.1.3.12. 4-(4-(methyls ulf onyl)phenyl)-2-(2-(3,4,5 trimethoxybenzylidene)hydrazineyl)thiazole (3l). M.P: 244.5e245.9。 C. 1H NMR (300 MHz, DMSO-d6): δ ¼ 3.24 (3H, s, eCH3), 3.69 (3H, s, eOCH3), 3.83 (6H, s, eOCH3), 6.99 (2H, s, trimethoxyphenyl CH), 7.63 (1H, s, thiazoleCH), 7.96 (2H,d,J¼ 8.6 Hz, disubs. phenyl CH), 7.98 (1H, s, N]CH), 8.11 (2H, d, J¼ 8.5 Hz, disubs. phenyl CH), 12.29 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δ ¼ 44.0, 56.3, 60.6, 104.0, 107.7, 126.5, 128.0, 130.3, 139.1, 139.6, 139.7, 142.0, 149.3, 153.6, 169.0. HRMS (m/z): [MþH]þ calcd for C20H21N3O5S2: 448.0995; found: 448.00973.
4.2. In vitro COX-1 and COX-2 inhibition assay
The in vitro inhibitory potency of the compounds against COX-1/ COX-2 was measured by using fluorometric COX-1 and COX-2 inhibitor screening kits (Biovision, Switzerland) according to the manufacturer’s instructions [26,27]. The assay is based on the fluorometric detection of prostaglandin G2, the intermediate product generated by the COX enzyme.Each kit comprises COX-1/COX-2 enzymes, COX assay buffer, COX probe (in DMSO), COX cofactor (in DMSO), arachidonic acid, NaOH and selective inhibitors ibuprofen, celecoxib and nimesulide for COX-1 and COX-2 enzymes.
The components of kits were prepared as follow to apply in the inhibition assay. COX-1/COX-2 enzyme solutions were prepared by adding 110 μL of ddH2O to the lyophilized powder in the kit. COX assay buffer (398 μL) and COX Cofactor (2 μL) were mixed to prepare diluted COX cofactor. Dilute arachidonic acid/NaOH solution was prepared by adding 5 μL arachidonic acid to 5 μL of NaOH, then diluted with 90 μL ddH2O. Then, the prepared solutions were put together to gain the reaction mixture (80 μL) for a well. This reaction mixture consisted of followings: COX assay buffer (76 μL), COX probe (1 μL), diluted COX cofactor (2 μL) and COX-1/COX-2 enzyme solution (1 mL) and was added into a 96-well white opaque plate. The test compounds (10 mL) were added to above solution. The assay mixture was incubated at 25 。C for 5e10 min.10 mL of diluted arachidonic acid/NaOH solution was added into each well to stop the reaction after incubation. Fluorescence (Ex/Em = 535/587 nm) of the samples were kinetically measured by BioTek-Synergy H1 multimode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) for 5 min intervals. Blank, control and all concentrations of inhibitors were analyzed in quadruplicate. The percentage of inhibition results were displayed as the mean ± standard deviation (SD). Moreover, the IC50 values were calculated with the help of GraphPad ‘PRISM’ software (version 5.0) by using a dose-response curve achieved by plotting the percentage inhibition versus the log concentration. Selectivity index (SI) was calculated as IC50(COX 1)/IC50(COX-2).
4.3. Prediction of ADME parameters
In order to predict the pharmacokinetic proiles of synthesized compounds 3a-3l, QikProp 4.8 software [35] was used and their physicochemical parameters were calculated via in silico method.
4.4. Molecular docking studies
Molecular docking studies were carried out using a structurebased protocol to reveal the binding mechanisms of compound 3a to the active site of the COX-2 enzyme. For this purpose, the crystal structure of COX-2 crystallized with celecoxib (PDB ID: 3LN1) [40] was extracted from the Protein Data Bank database (www.pdb.org).The ligands’ conigurations were designed using the Schro(€)dinger Maestro [45] tool and submitted to the Schro(€)dinger Suite 2016 Up-date 2 Protein Preparation Wizard method. The ligands were processed using LigPrep 3.8 [46] to correctly detect the atom groups as well as the protonation conditions at a pH of 7.4 ± 1.0. Bond orders were assigned, and hydrogen atoms were added to the structures. Glide 7.1 was used to construct the grid generation [41]. Flexible docking runs were performed in single-precision docking mode (SP).
