Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Design, synthesis and bioactivities of Celecoxib analogues or derivatives

http://dx.doi.org/10.1016/j.bmc.2017.07.0380968-0896/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: huangdoctor226@163.com (G. Huang).

Shiyang Zhou, Shanbin Yang, Gangliang Huang ⇑aCollege of Chemistry, Chongqing Normal University, Chongqing 401331, ChinabUniversity Bioactive Substance Engineering Research Center in Chongqing, Chongqing Normal University, Chongqing 401331, China

a r t i c l e i n f o

Article history:Received 28 June 2017Revised 19 July 2017Accepted 19 July 2017Available online 20 July 2017

Keywords:Celecoxib analogues or derivativesDesignSynthesisBiological activities

a b s t r a c t

A series of Celecoxib analogues or derivatives were designed and synthesized, and their biological activ-ities were studied. The results of inhibitory activity in vitro proved that compounds 1a, 1h, 1i, 1l and 1phad better inhibitory effect on COX-2, and the selectivity was higher. Among them, the inhibitory activityof compound 1h to COX-2 was IC50 = 0.049 lmol/L and SI >1000. Moreover, the experimental results ofanti-inflammatory activity in vivo showed that they had good anti-inflammatory activity and could inhi-bit the release of PGE-2. Therefore, these compounds have better druggability.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Cycloxygenase (COX) is found in the endoplasmic reticulum ofmammalian cells and has high biological activity. In 1990s, studieshave shown that there are two subtypes of cyclooxygenase, namelyCOX-1 and COX-2. COX-1 is a constituent enzyme that is present inmost tissues, generally, COX-1 levels are relatively stable.1–3 Inaddition, maintaining a certain level of COX-1 can promote thesynthesis of prostaglandins (PG) in the gastrointestinal tract to reg-ulate the physiological activities of normal tissue cells, protect thegastrointestinal mucosa, regulate renal blood flow as well as pro-mote platelet aggregation and other important role. COX-2 is aninducible enzyme that is very low in the body under normal phys-iological conditions and is expressed mainly in inflammatory cells.When COX-2 is stimulated by inflammatory substances, such asmitogen (LPS and SPA), inflammatory cytokines (TNF, IL-1, andPAF), endotoxin and other inflammatory substances, it can beinduced and expressed at high levels. For example, the levels ofPGE-1, PGE-2 and PGI-2 are increased, which promote the inflam-mation and tissue damage, resulting in swelling, edema, pain, feverand other symptoms.4–6

COX-1 and COX-2 are 71 kDa membrane-bound proteins, bothof which are basically the same length and contain about 600amino acid molecules.7–9 X-ray diffraction crystal structure analy-sis showed that COX-1 (Fig. 1) and COX-2 (Fig. 2) are in the form ofhomodimers. The crystal structures of their is very similar in shape.As a hairpin, there is a long hydrophobic channel in the entire

molecular space structure, this allows the arachidonic acid to enterdirectly from the membrane, which is converted to synthesizeprostaglandin. The amino acids on the other side of hydrophobicchannel are different. The 434 and 523 bits of COX-1 are isoleucineresidues, and the two sites of COX-2 are valine residues. Becausethe molecular weight of valine residue is less than that of isoleu-cine residue, the structure is small, the larger binding space canbe left, and the drug molecule can be covalently bonded.10–12 Inaddition, COX-1 is a histidine residue at position 513, and COX-2is an arginine residue, which makes the end of COX-2 be more flex-ible than COX-1, allowing it to interact with larger drug molecules.The differences in spatial structure provide the possibility for thedesign of selective inhibitors to COX-2.

The present study suggests that nonsteroidal anti-inflammatorydrugs (NSAIDs) are effective in the treatment of inflammation byinhibiting COX-2, COX-1, and other adverse effects.13–15 Therefore,selective inhibition of COX-2 can be achieve to effectively treatinflammation while avoiding or reducing the adverse effectscaused by inhibition of COX-1. Since 1990s, a number of selectiveCOX-2 inhibitors have been used in clinical settings, such as Cele-coxib, Rofecoxib, Valdecoxib, and Percoxib (Fig. 3). The presentselective COX-2 inhibitors have the selectivity for up to hundredsof times, and inhibit the synthesis of physiological prostaglandins.At present, COX-2 inhibitors have been used as the nonsteroidalanti-inflammatory drugs for the first, and are clinically used inarthritis and other diseases. In recent years, clinical studies haveshown that COX-2 inhibitors have effects on the antitumor andbrain protection.15,16

Celecoxib is a highly selective COX-2 inhibitor, which is rapidlyabsorbed by oral administration and the bioavailability is about

Fig. 1. Crystallographic structure of COX-1.

Fig. 2. Crystallographic structure of COX-2.

Fig. 3. The structures of sele

4888 S. Zhou et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893

99%. After absorption, it is widely distributed in all tissues of thebody, and is oxidized and metabolized in the liver. The methylgroup in the benzene ring is hydroxylated and carboxylated, whichis finally combined with glucuronic acid and excreted in urine.17,18

The anti-inflammatory activity of drug is good, which has lessadverse reactions to the stomach. In 1999, Celecoxib was approvedby the FDA for the treatment of rheumatoid arthritis andosteoarthritis-induced pain. The inhibitory effect of Celecoxib onCOX-2 was 400 times that of COX-1, and the selectivity wasgood.19,20 Celecoxib was used as a lead compound, the structuresof six-membered rings, five-membered heterocycles, and sub-stituents on five-membered heterocycles were transformed byusing the principle of bioisosterism (Fig. 4 and Scheme 1).

2. Results and discussion

2.1. Design and synthesis of Celecoxib analogues or derivatives

The design of novel selective COX-2 inhibitor was based onCelecoxib as the lead compound, and its structure was modifiedby the bioisosterism principle (Fig. 4). In terms of drug structure,these compounds had the same spatial structure as Celecoxib, sothe possibility of drug formation was relatively large. In the mod-ification, the basic frame structure of Celecoxib was retained, andthe selective inhibition of COX-2 essential pharmacophore (sul-phamoyl group) was also retained. In addition, the five-memberedheterocycle was an important binding site of COX-2. It was neces-sary to modify the five-membered heterocycle to make it morestable. In the design process, N, O, S and other bioisosteres wereselected to replace the C-4 on the original-five membered ring,and the C-2 on the five-membered ring was replaced by N atom.Because these atoms were easily linked to COX-2 in the form ofcovalent bonds, theoretically, the inhibitory activity should be bet-ter. Moreover, -CH3 and -CCl3 were introduced as the substituentgroups on five-membered ring, because these substituents had acertain spatial structure, and the two subtypes of COX had a hollow

ctive COX-2 inhibitors.

N

N

SO2NH2H3C

CF3

X2

N

N

X1

SO2NH2H3C

R1

Celecoxib Target compound

Fig. 4. Design of Celecoxib analogues or derivatives.

X2

N

N

X1

SO2NH2H3C

R1

R1 CH

X1

CH

OCH3

OCH3N X2H3C CHO N X2H3C CH

OH

X1 CHCH

R1 OCH3

OCH3

N X2H3C CH

Cl

X1 CHCH

R1 OCH3

OCH3

H2N SO2NH2

N X2H3C CH

HN

X1 CHCHO

R1

SO2NH2

CaCl2 / HCl

SOCl2 CH2Cl2

DMF 1)Toluene Na2CO32)H3O+

EtOH

2a-2b 3a-3r

4a-4r

5a-5r 1a-1r

R1:-CH3,-CF3,-CCl3X1:N,O,SX2:C,N

Scheme 1. The synthetic route of Celecoxib analogues or derivatives.

S. Zhou et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893 4889

structure, which was in order to study which group was easier toaccess the interior of COX. In addition, the methyl-linked benzenering was modified by using N instead of C, which was mainly toimprove the binding ability of target molecule. In the synthesisprocess, a simple synthetic route was selected (Scheme 1). The syn-thesized products were analyzed by 1H NMR, 13C NMR, MS, andelemental analysis, respectively. In the intermediates 5a-5r, thechemical shift of hydrogen on the secondary amine disappeared,indicating that the target products 1a-1r had been produced. Thesynthesis of compounds 3a-3r was carried out by the addition ofaldehyde groups to form hydroxyl groups. The synthesis of com-pounds 4a-4r was a substitution of the hydroxyl group with chlo-rine atom, using dichloromethane as the reaction solvent, N, N-dimethylformamide (DMF) as a catalyst, dropping thionyl chloride,and then reacting under refluxing to synthesize. The compounds5a-5r were synthesized to adopt intermolecular condensation,and the compounds 1a-1r were synthesis by intramolecular con-densation. The synthetic route was simple, the reaction conditionswere mild, and the yields of target products were high, which werefrom 72.6% to 85.6%.

2.2. The biological activities

2.2.1. The inhibitory activity to COX in vitroBefore studying the inhibitory activity of target compounds

in vitro, we first studied the physical and chemical properties ofCelecoxib analogues or derivatives, namely the lipid/water parti-tion coefficient (logP) and solubility (pKa), which might affect theiractivity (Table 1). As could be seen from Table 1, these compoundshad good fat-soluble, and the logP was from 1.04 to 3.50. Thehigher the partition coefficient of lipid and water was, the betterthe absorption of drugs was, and the activity was increased. Inaddition, from the experimental results, it could also be seen thatthe compounds could be digested and absorbed by the small intes-tine, and the pKa was from 7 to 8. The difference in the pKa of drugaffected the effective time because the compounds needed to bedigested and absorbed by the small intestine, and the effectivetime would be slightly delayed. After the completion of logP andpKa studies, we focused on the selective inhibition of COXin vitro. In the experiment, COX-1 and COX-2 were used as the inhi-bitory targets, and the inhibitory activity was measured by semi-

Table 1The inhibitory activity in vitro and some physico-chemical properties.

Compounds R1 X1 X2 logP pKa IC50 (lmol/L) SI COX-1/COX-2

COX-1 COX-2

1a -CH3 N C 1.91 7.26 53.69 ± 0.18 0.103 ± 0.06 5211b -CH3 N N 1.44 7.11 90.65 ± 1.23 16.348 ± 1.43 5.61c -CH3 O C 1.40 7.09 95.22 ± 0.07 17.369 ± 0.58 5.51d -CH3 O N 1.04 7.01 98.55 ± 0.29 20.126 ± 1.37 4.91e -CH3 S C 2.12 7.39 56.86 ± 1.07 1.532 ± 1.04 37.11f -CH3 S N 1.75 7.20 76.87 ± 2.59 16.436 ± 2.62 4.81g -CF3 N C 2.54 7.58 70.34 ± 1.38 1.806 ± 0.82 38.91h -CF3 N N 2.09 7.34 49.24 ± 0.98 0.049 ± 0.09 1004.91i -CF3 O C 2.05 7.33 50.22 ± 4.76 0.053 ± 0.55 947.51j -CF3 O N 1.68 7.18 55.60 ± 0.57 1.270 ± 1.03 43.81k -CF3 S C 2.77 7.62 80.50 ± 3.05 8.079 ± 0.07 10.01l -CF3 S N 2.38 7.50 52.36 ± 2.16 0.0793 ± 0.06 660.31m -CCl3 N C 3.27 7.89 90.33 ± 0.75 12.080 ± 1.38 7.51n -CCl3 N N 2.85 7.68 85.96 ± 5.02 14.590 ± 2.77 5.91o -CCl3 O C 2.78 7.63 83.06 ± 0.68 13.685 ± 0.73 6.11p -CCl3 O N 2.41 7.55 53.39 ± 3.75 0.099 ± 1.00 539.31q -CCl3 S C 3.50 8.00 98.18 ± 1.83 12.673 ± 2.62 7.71r -CCl3 S N 3.13 7.76 88.59 ± 0.09 11.568 ± 1.56 7.7Celecoxib – – – 30.21 ± 2.74 0.071 ± 0.05 425

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inhibitory concentration (IC50). Finally, the selectivity (SI) was cal-culated. The results showed that the compounds 1a, 1h, 1i and 1phad high inhibitory activity and selectivity to COX-2.

2.2.2. The anti-inflammatory activity in vivoIn the course of the bioactivity study, compounds 1a, 1h, 1i, 1l

and 1p with high inhibitory activity and selective inhibition ofCOX-2 were selected and tested in vivo (Table 2). The dose was20 mg/kg, and the anti-inflammatory activity was observed at dif-ferent time. Celecoxib was used as the positive reference sub-stance. The experimental results showed that these fourcompounds had fast effective time and good anti-inflammatoryactivity. The expression of PGE-2 in vivo was also observed after2 h of administration (Fig. 5). It could be seen that the expressionof PGE-2 in vivo was relatively low, and their anti-inflammatoryeffect was better.

Fig. 5. The release of PGE-2.

3. Conclusion

In general, we reported the design and synthesis of a class ofselective COX-2 inhibitors, which was simple, efficient, and hadhigh yield. All the newly synthesized compounds had good inhibi-tory activity and high selectivity for inhibiting COX-2. Amongthem, the selective inhibition experiments in vitro showed thatcompounds 1a, 1h, 1i, 1l and 1p had good inhibitory activity andhigh selectivity, the experiments in vivo also showed that com-pounds 1a, 1h, 1i, 1l and 1p had good anti-inflammatory activity,and their inhibition to PGE -2 expression was also very good.

Table 2The inhibitory activity in vivo.

Compounds Inhibition (%, 20 mg/kg)

0 min 20 min 40 m

1a 0 38.56 59.031h 0 46.22 65.791i 0 44.58 64.891l 0 42.50 63.081p 0 40.58 60.86Celecoxib 0 45.33 68.36

4. Experimental

4.1. Synthesis of compounds 3a-3r

1-Methyl-4-pyridinecarboxaldehyde 2a (0.20 mol) and 1,1-dimethoxy-2-propylamine (0.10 mol) were placed in a 250 mLround bottom flask. anhydrous calcium chloride (0.01 mol) andtoluene (100 mL) were added. The mixture was refluxed for 4 h,and a small amount of hydrogen chloride gas was introducedduring the reaction process. After completion of the reaction, the

in 60 min 90 min 120 min

83.09 90.34 91.5190.36 100 10087.49 99.60 10086.09 97.30 98.0385.89 95.38 97.8689.06 100 100

S. Zhou et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893 4891

filtrate was filtered while it was hot. The filtrate was collected,cooled, allowed to stand for 24 h, and then filtered and dried invacuo to give the crude product of compound 3a. The crude 3a pro-duct was recrystallized in toluene, filtered and dried in vacuo togive pure product as a white crystal. General experimental methodwas used for the synthesis of compounds 3b-3r.

4.2. Synthesis of compounds 4a-4r

0.10 mol of compound 3a was placed in a 250 mL round bottomflask, followed by 100 mL of solvent methylene chloride and 5drops of N,N-dimethylformamide (DMF). Under constant pressureconditions, 29 mL of thionyl chloride was dropped with a droppingfunnel. After completion of the dropwise addition, the reaction wasrefluxed for 6 h. After completion of the reflux, the methylene chlo-ride and the excess amount of thionyl chloride were removedunder normal pressure. The mixture was dried in vacuo to givethe crude product of compound 4a. The crude product of 4a wasrecrystallized in benzene, filtered and dried in vacuo to give pureproduct 4a as a white crystal. General experimental method wasused for the synthesis of compounds 4b-4r.

4.3. Synthesis of compounds 5a-5r

0.10 mol of compound 4a, 0.05 mol of anhydrous sodium car-bonate, and 0.10 mol of p-aminobenzenesulfonamide were placedin a 250 mL round bottom flask. 100 mL of toluene was added asa solvent. Under magnetic stirring, the reaction was heated andrefluxed for 8 h. The reaction mixture was immediately filtered.And then 100 mL 2 mol/L dilute sulfuric acid solution was addedto the filtrate, the solution appeared stratification phenomenon.When the heated reflux occurred, the white solid would appearconstantly in the aqueous phase. When the reflux was completed,it was cooled and remained stationary. The organic phase of upperlayer was removed first, toluene was filtered, the filter residue wascollected and dried, and the crude product 5a was obtained, whichwas recrystallized in absolute ethanol, filtered and dried to give theproduct as a white crystal. General experimental method was usedfor the synthesis of compounds 5b-5r.

4.4. A general method for all titled analogues or derivatives 1a-1r

0.10 mol of compound 5a was placed in a 250 mL round bottomflask, and 100 mL of absolute ethanol was added as solvent. Themixture was heated and refluxed for 4 h. During the refluxing pro-cess, a little sodium ethoxide solid was added. After completion ofthe reflux, the filtrate was collected by filtration. The filtrate wascooled and allowed to stand. The precipitate was completely fil-tered and dried to give a crude product of 1a, which was recrystal-lized in absolute ethanol, filtered and dried to give a pure productas white crystal. General experimental method was used for thesynthesis of compounds 1b-1r.

4-(4-methyl-2-(1-methyl-1,4-dihydropyridin-4-yl)-1H-imidazol-1-yl)benzenesulfonamide (1a): yield 76.3%; m.p. 145–147 �C; 1HNMR (300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H), 7.80 (m, 2H, Ph-H),7.23 (s, 2H, -NH2), 7.20 (s, 1H, Im-H), 5.73 (m, 2H, Py-H), 4.43(m, 1H, Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H, -CH3), 2.23 (s, 3H,-CH3); 13C NMR (75 MHz, CDCl3) d: 149.7, 143.2, 141.1, 139.2,129.3, 128.2, 122.4, 112.0, 110.0, 43.2, 34.2, 14.3; HR-ESI-MS m/z:Calcd. for C16H18N4O2S {[M+H]+} 330.4061, found 330.1154; Anal.Calcd. for C16H18N4O2S: C, 58.16; H, 5.49; N, 16.96; S, 9.70, found:C, 58.17; H, 5.48; N, 16.97; S, 9.69%.

4-(4-methyl-2-(4-methylpyrazin-1(4H)-yl)-1H-imidazol-1-yl)benzenesulfonamide (1b): yield 78.3%; m.p. 147-149 �C; 1H NMR(300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H), 7.80 (m, 2H, Ph-H), 7.23(s, 2H, -NH2), 7.20 (s, 1H, Im-H), 5.43 (s, 4H, Py-H), 3.85 (s, 3H, -

CH3), 2.23 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 152.8,143.2, 141.4, 139.3, 128.2, 122.4, 110.3, 103.7, 42.9, 14.0; HR-ESI-MS m/z: Calcd. for C15H17N5O2S {[M+H]+} 333.3941, found333.1102; Anal. Calcd. for C15H17N5O2S: C, 54.37; H, 5.17; N,21.13; S, 9.67; found: C, 54.38; H, 5.17; N, 21.12; S, 9.68%.

4-(5-methyl-2-(1-methyl-1,4-dihydropyridin-4-yl)oxazol-3(2H)-yl)benzene sulfonamide (1c): yield 82.1%; m.p. 153–155 �C;1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89 (s, 2H, -NH2), 5.73 (s, 2H, Py-H), 4.99 (s, 1H, OX-H), 4.69(m, 1H, OX-H), 4.42 (s, 2H, Py-H), 3.85 (s, 3H, -CH3), 1.99 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 147.6, 131.5, 130.7, 130.0,113.8, 107.0, 95.2, 94.6, 43.2, 19.3; HR-ESI-MS m/z: Calcd. for C16-H19N3O3S {[M+H]+} 333.4058, found 333.1145; Anal. Calcd. ForC16H19N3O3S: C, 57.64; H, 5.74; N, 12.60; S, 9.62; found: C, 57.65;H, 5.75; N, 12.60; S, 9.60%.

4-(5-methyl-2-(4-methylpyrazin-1(4H)-yl)oxazol-3(2H)-yl)benzenesulfonamide (1d): yield 83.5%; m.p. 156–157 �C; 1H NMR(300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89(s, 2H, -NH2), 5.76 (s, 1H, OX-H), 5.43 (s, 4H, Py-H), 4.99 (m, 1H,OX-H), 3.85 (s, 3H, -CH3), 1.99 (s, 3H, -CH3); 13C NMR (75 MHz,CDCl3) d: 147.6, 132.1, 130.0, 129.3, 120.8, 113.8, 103.7, 93.4,42.9, 19.0; HR-ESI-MS m/z: Calcd. for C15H18N4O3S {[M+H]+}334.3941, found 334.1101; Anal. Calcd. for C15H18N4O3S: C,53.88; H, 5.43; N, 16.76; S, 9.59; found: C, 53.89; H, 5.42; N,16.76; S, 9.58%.

4-(5-methyl-2-(1-methyl-1,4-dihydropyridin-4-yl)thiazol-3(2H)-yl)benzenesulfonamide (1e): yield 76.8%; m.p. 161–163 �C;1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89 (s, 2H, -NH2), 6.03 (s, 1H, Th-H), 5.73 (s, 2H, Py-H), 4.42(s, 2H, Py-H), 3.85 (s, 3H, -CH3), 3.73 (s, 1H, Th-H), 3.37 (s, 1H,Py-H), 2.03 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 147.6,130.7, 130.0, 129.3, 121.7, 114.8, 113.8, 107.0, 67.5, 43.2, 39.4,23.9; HR-ESI-MS m/z: Calcd. for C16H19N3O2S2 {[M+H]+}349.4671, found 349.0918; Anal. Calcd. for C16H19N3O2S2: C,54.99; H, 5.48; N, 12.02; S, 18.35; found: C, 54.99; H, 5.49; N,12.01; S, 18.34%.

4-(5-methyl-2-(4-methylpyrazin-1(4H)-yl)thiazol-3(2H)-yl)benzenesulfonamide (1f): yield 77.2%; m.p. 165–167 �C; 1H NMR(300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89(s, 2H, -NH2), 6.03 (s, 1H, Th-H), 5.43 (s, 4H, Py-H), 4.80 (s, 1H,Th-H), 3.85 (s, 3H, -CH3), 3.37 (s, 1H, Py-H), 2.03 (s, 3H, -CH3);13C NMR (75 MHz, CDCl3) d: 147.6, 130.0, 129.3, 121.7, 114.8,113.8, 103.7, 90.9, 42.9, 23.6; HR-ESI-MS m/z: Calcd. for C15H18N4-O2S2 {[M+H]+} 350.4551, found 350.0870; Anal. Calcd. For C15H18-N4O2S2: C, 51.41; H, 5.18; N, 15.99; S, 18.30; found: C, 51.40; H,5.18; N, 15.98; S, 18.31%.

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-4-(trifluoromethyl)-1H-imidazol-1-yl)benzenesulfonamide (1g): yield 77.2%; m.p.148–150 �C; 1H NMR (300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H),7.80 (m, 2H, Ph-H), 7.59 (s, 1H, Im-H), 7.23 (s, 2H, -NH2), 5.73(m, 2H, Py-H), 4.42 (m, 1H, Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H,-CH3); 13C NMR (75 MHz, CDCl3) d: 149.7, 143.2, 141.4, 134.1,129.3, 128.2, 122.4, 121.9, 112.0, 110.0, 43.2, 34.2; HR-ESI-MS m/z: Calcd. For C16H15F3N4O2S {[M+H]+} 384.3771, found 384.0867;Anal. Calcd. For C16H15F3N4O2S: C, 50.00; H, 3.93; F, 14.83; N,14.58; S, 8.34; found: C, 50.01; H, 3.94; F, 14.82; N, 14.59; S, 8.33%.

4-(2-(4-methylpyrazin-1(4H)-yl)-4-(trifluoromethyl)-1H-imi-dazol-1-yl)benzenesulfonamide (1h): yield 78.5%; m.p. 155-157 �C; 1H NMR (300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H), 7.80 (m,2H, Ph-H), 7.59 (s, 1H, Im-H), 7.23 (s, 2H, -NH2), 5.43 (m, 2H, Py-H), 4.43 (m, 1H, Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H,- CH3); 13CNMR (75 MHz, CDCl3) d: 152.8, 143.2, 141.4, 134.2, 128.2, 122.4,121.6, 110.3, 103.7, 42.9; HR-ESI-MSm/z: Calcd. For C15H14F3N5O2S{[M+H]+} 385.3650, found 385.0821; Anal. Calcd. For C15H14F3N5-O2S: C, 46.75; H, 3.66; F, 14.79; N, 18.17; S, 8.32; found: C,46.76; H, 3.67; F, 14.79; N, 18.18; S, 8.30%.

4892 S. Zhou et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-5-(trifluoromethyl)oxazol-3(2H)-yl)benzenesulfonamide (1i): yield 82,4%; m.p. 163–165 �C; 1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m,2H, Ph-H), 6.89 (s, 2H, -NH2), 5.82 (s, 1H, Ox-H), 5.73 (m, 2H, Py-H), 4.69 (s, 1H, Ox-H), 4.42 (m, 1H, Py-H), 3.40 (m, 1H, Py-H),3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 147.6, 131.5,130.7, 130.0, 123.5, 113.8, 107.0, 95.2, 94.6, 43.2, 38.1; HR-ESI-MS m/z: Calcd. for C16H16F3N3O3S {[M+H]+} 387.3771, found387.0863; Anal. Calcd. For C16H16F3N3O3S: C, 49.61; H, 4.16; F,14.71; N, 10.85; S, 8.28; found: C, 49.62; H, 4.15; F, 14.72; N,10.85; S, 8.29%.

4-(2-(4-methylpyrazin-1(4H)-yl)-5-(trifluoromethyl)oxazol-3(2H)-yl)benzenesulfonamide (1j): yield 85.6%; m.p. 166–168 �C; 1HNMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H),6.89 (s, 2H, -NH2), 5.82 (s, 1H, Ox-H), 5.76 (s, 1H, Ox-H), 5.43 (s,4H, Py-H), 3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 147.6,132.1, 130.0, 129.3, 123.2, 120.8, 113.8, 103.7, 93.4, 42.9; HR-ESI-MS m/z: Calcd. for C15H15F3N4O3S {[M+H]+} 388.3651, found388.0815; Anal. Calcd. for C15H15F3N4O3S: C, 46.39; H, 3.89; F,14.68; N, 14.43; S, 8.26; found: C, 46.41; H, 3.89; F, 14.66; N,14.42; S, 8.27%.

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-5-(trifluoromethyl)thiazol-3(2H)-yl)benzenesulfonamide (1k): yield 75.5%; m.p. 170–172 �C; 1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m,2H, Ph-H), 6.89 (s, 2H, -NH2), 6.86 (m, 1H, Th-H), 5.73 (m, 2H,Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H, -CH3), 3.73 (m, 1H, Th-H),3.37 (m, 1H Py-H); 13C NMR (75 MHz, CDCl3) d: 147.6, 130.7,130.0, 129.3, 127.1, 121.7, 114.8, 113.8, 107.0, 67.5, 43.2, 39.4;HR-ESI-MS m/z: Calcd. for C16H16F3N3O2S2 {[M+H]+} 403.4380,found 403.0635; Anal. Calcd. for C15H15F3N4O3S: C, 47.63; H,4.00; F, 14.13; N, 10.42; S, 15.89; found: C, 47.65; H, 4.01; F,14.12; N, 10.41; S, 15.87%.

4-(2-(4-methylpyrazin-1(4H)-yl)-5-(trifluoromethyl)thiazol-3(2H)-yl)benzenesulfonamide (1l): yield 78.7%; m.p. 175–177 �C; 1HNMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H),6.89 (s, 2H, -NH2), 6.86 (m, 1H, Th-H), 5.43 (s, 4H, Py-H), 4.80 (m,1H, Th-H), 3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 147.6,130.0, 129.3, 126.8, 121.7, 114.8, 113.8, 103.7, 90.9, 42.9; HR-ESI-MS m/z: Calcd. for C15H15F3N4O2S2 {[M+H]+} 404.4260, found404.0588; Anal. Calcd. for C15H15F3N4O2S2: C, 44.55; H, 3.74; F,14.09; N, 13.85; S, 15.85; found: C, 44.56; H, 3.76; F, 14.07; N,13.87; S, 15.84%.

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-4-(trichloromethyl)-1H-imidazol-1-yl)benzenesulfonamide (1m): yield 78.5%; m.p.153–155 �C; 1H NMR (300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H),7.80 (m, 2H, Ph-H), 7.23 (s, 2H, -NH2), 7.20 (m, 1H, Im-H), 5.73(m, 2H, Py-H), 4.43 (m, 1H, Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H,-CH3); 13C NMR (75 MHz, CDCl3) d: 149.7, 143.2, 141.4, 129.3,128.2, 124.6, 122.4, 112.0, 110.0, 99.3, 43.2, 34.2; HR-ESI-MS m/z:Calcd. for C16H15Cl3N4O2S {[M+H]+} 433.7321, found 432.9980;Anal. Calcd. for C16H15Cl3N4O2S: C, 44.31; H, 3.49; Cl, 24.52; N,12.92; S, 7.39; found: C, 44.32; H, 3.49; Cl, 24.51; N, 12.92; S, 7.38%.

4-(2-(4-methylpyrazin-1(4H)-yl)-4-(trichloromethyl)-1H-imi-dazol-1-yl)benzenesulfonamide (1n): yield 79.9%; m.p. 160–162 �C; 1H NMR (300 MHz, CDCl3) d: 7.86 (m, 2H, Ph-H), 7.80 (m,2H, Ph-H), 7.59 (m, 1H, Im-H), 7.23 (s, 2H, -NH2), 5.43 (s, 4H, Py-H), 3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d: 152.8, 143.2,141.4, 128.7, 128.2, 122.4, 110.3, 103.7, 99.0, 42.9; HR-ESI-MS m/z: Calcd. for C15H14Cl3N5O2S {[M+H]+} 434.7201, found 433.9935;Anal. Calcd. for C15H14Cl3N5O2S: C, 41.44; H, 3.25; Cl, 24.46; N,16.11; S, 7.37; found: C, 41.45; H, 3.26; Cl, 24.45; N, 16.10; S, 7.38%.

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-5-(trichloromethyl)oxazol-3(2H)-yl)benzenesulfonamide (1o): yield 79.9%; m.p. 166–167 �C; 1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m,2H, Ph-H), 6.89 (s, 2H, -NH2), 5.73 (m, 2H, Py-H), 5.26 (s, 1H, Ox-H), 4.69 (s, 1H, Ox-H), 4.42 (m, 2H, Py-H). 3.85 (s, 3H, -CH3), 3.40

(m, 1H, Py-H); 13C NMR (75 MHz, CDCl3) d: 147.6, 139.1, 130.7,130.0, 113.8, 107.0, 96.1, 94.4, 93.1, 43.2; HR-ESI-MS m/z: Calcd.for C16H16Cl3N3O3S {[M+H]+} 436.7322, found 435.9979; Anal.Calcd. for C16H16Cl3N3O3S: C, 44.00; H, 3.69; Cl, 24.35; N, 9.62; S,7.34; found: C, 44.01; H, 3.69; Cl, 24.34; N, 9.61; S, 7.35%.

4-(2-(4-methylpyrazin-1(4H)-yl)-5-(trichloromethyl)oxazol-3(2H)-yl)benzenesulfonamide (1p): yield 82.5%; m.p. 170–172 �C;1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89 (s, 2H, -NH2), 5.76 (s, 1H, Ox-H), 5.43 (s, 4H, Py-H), 5.26(s, 1H, Ox-H), 3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d:147.6, 139.7, 130.0, 129.3, 119.3, 113.8, 103.7, 95.8, 92.6, 42.9;HR-ESI-MS m/z: Calcd. for C15H15Cl3N4O3S {[M+H]+} 437.7200,found 436.9933; Anal. Calcd. for C15H15Cl3N4O3S: C, 41.16; H,3.45; Cl, 24.30; N, 12.80; S, 7.32; found: C, 41.15; H, 3.46; Cl,24.30; N, 12.80; S, 7.31%.

4-(2-(1-methyl-1,4-dihydropyridin-4-yl)-5-(trichloromethyl)thiazol-3(2H)-yl)benzenesulfonamide (1q): yield 72.6%; m.p. 176–178 �C; 1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m,2H, Ph-H), 6.89 (s, 2H, -NH2), 6.30 (s, 1H, Th-H), 5.73 (m, 2H, Py-H), 4.42 (m, 2H, Py-H), 3.85 (s, 3H, -CH3), 3.73 (s, 1H, Th-H), 3.37(m, 2H, Py-H); 13C NMR (75 MHz, CDCl3) d: 147.6, 130.7, 130.0,129.3, 129.2, 114.0, 113.8, 107.0, 97.4, 66.0, 43.2; HR-ESI-MS m/z:Calcd. for C16H16Cl3N3O2S2 {[M+H]+} 452.7932, found 451.9753;Anal. Calcd. for C16H16Cl3N3O2S2: C, 42.44; H, 3.56; Cl, 23.49; N,9.28; S, 14.16; found: C, 42.43; H, 3.57; Cl, 23.49; N, 9.27; S, 14.15%.

4-(2-(4-methylpyrazin-1(4H)-yl)-5-(trichloromethyl)thiazol-3(2H)-yl)benzenesulfonamide (1r): yield 73.8%; m.p. 180–182 �C;1H NMR (300 MHz, CDCl3) d: 7.60 (m, 2H, Ph-H), 6.92 (m, 2H, Ph-H), 6.89 (s, 2H, -NH2), 6.30 (s, 1H, Th-H), 5.43 (s, 4H, Py-H), 4.80(s, 1H, Th-H), 3.85 (s, 3H, -CH3); 13C NMR (75 MHz, CDCl3) d:147.6, 130.0, 129,3, 129.2, 114.0, 113.8, 103.7, 97.1, 89.4, 42.9;HR-ESI-MS m/z: Calcd. for C15H15Cl3N4O2S2 {[M+H]+} 453.7811,found 452.9703; Anal. Calcd. for C15H15Cl3N4O2S2: C, 39.70; H,3.33; Cl, 23.44; N, 12.35; S, 14.13; found: C, 39.72; H, 3.31; Cl,23.45; N, 12.35; S, 14.14%.

4.5. Biological activities

4.5.1. Preparation of peritoneal macrophages from ratsAdult healthy male rat was weighed about 200 g, and its

abdominal cavity was injected with 0.5% gelatin (5mL). After 5 h,the rats were violently knocked on the head to death. The rats werewashed with cold Hanks solution for three times, and the washingsolution was collected by centrifugation tube (10 mL). The cen-trifuge tube was placed in a centrifuge, centrifuged at 2000 r/minfor 5 min, and washed with RPM1-1640 medium for three times.The number of peritoneal macrophages in the sample was adjustedto 1.0 � 106 CUF/mL, and the cells were inoculated into 48-wellculture plates with 0.2 mL in per well. Incubated at 37 �C with 5%CO2 for 24 h, and the culture was complete and washed withoutadherent cells.

4.5.2. Screening of the inhibitory activity to COX-1The peritoneal macrophages were treated with 0.5% FCS RPM1-

1640 medium for 24 h. The Celecoxib analogues or derivativeswere diluted in a proportion and formulated at different concen-trations, and then 10 lL of the compound solution was added toeach well as a positive control. Cultured at 37 �C with 5% CO2 for30 min, the substrate arachidonic acid was added and the culturewas continued for 30 min. The radioactivity of 125I-6-Keto-PGF1awas measured according to the reagent kit. When the radioactivitywas half, it was the inhibitory concentration IC50 to COX-1 for theassayed sample.

S. Zhou et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4887–4893 4893

4.5.3. Screening of the inhibitory activity to COX-2The peritoneal macrophages were treated with 0.5% FCS RPM1-

1640 medium for 24 h, and then the culture was continued with10% FCS RPM1-1640 medium for 1 h. The supernatant wasremoved and rinsed with PBS solution. Then, 0.5mg/mL LPS-10%FCS RPMI-1640 culture medium (200 lL) was added. After incu-bated for 8 h, the assayed sample was diluted in a predeterminedratio and formulated at different concentrations. Then, 10 lL ofthe assayed sample solution was added to each well as a positivecontrol. Cultured at 37 �C and 5% CO2 for 30 min, the substratearachidonic acid was added and continued for 30 min. The super-natant was used to determine the radioactivity of PGE-2. Whenthe radioactivity was half, it was the inhibitory concentrationIC50 to COX-2 for the assayed sample.

Acknowledgements

The Project Sponsored by the Scientific Research Foundation forthe Returned Overseas Chinese Scholars, State Education Ministry.The work was also supported by Chongqing Key Research Project ofBasic Science & Frontier Technology (No. cstc2017jcyjBX0012), Sci-entific and Technological Research Program of Chongqing Munici-pal Education Commission (Grant No. KJ1400523), FoundationProject of Chongqing Normal University (No. 14XYY020), Chongq-ing General Research Project of Basic Science & Frontier Technol-ogy (No. cstc2015jcyjA10054), Chongqing Normal UniversityPostgraduate’s Research and Innovation Project (No. YKC17004),Open Foundation Project of Engineering Research Center for Bioac-tive Substances (No. AS201614), and Chongqing University Stu-dents’ Training Project of Innovation & Undertaking (No.201610637076), China.

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