Synthesis and COX-2 Inhibitory Activity of 4-[(E)-2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethenyl]benzene-1-sulfonamide and Its Analogs.

Some novel 3-phenyl-2-[(E)-2-phenylethenyl]-3,4-dihydroquinazolin-4-one derivatives possessing para-sulfonamides groups on the phenyl ring of the 2-phenylethenyl moiety have been synthesized and their COX-2 inhibitory activity evaluated. The stuctures of the synthesized compounds were confirmed  on the basis of  FT-IR, 1H-NMR, 13C-NMR and mass spectral data. The COX-2 inhibition screening assay revealed that 4-[(E)-2-{3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide had a  maximum COX-2 inhibition (47.1%), at a concentration of 20 μM.

1. Introduction

Compounds containing the 4(3H)-quinazolinone ring system possess various biological activities [1]. Some 2,3-diaryl-4(3H)-quinazolinone derivatives exhibit COX-2 inhibitory and anti-inflammatory activity [2,3,4]. The majority of COX-2 inhibitors are diaryl heterocycles. The presence of para-sulfonamides or para-sulfonylmethanes on one of the aryl rings was found to be essential for optimum COX-2 selectivity and inhibitory potency, while a wide variety of heterocycles, in general a five membered or six membered ring, can be used as the central ring system [5,6]. The results of a molecular docking study showed that 2,3-diaryl-4(3H)-quinazolinones possessing p-benzene-sulfonamide moieties at C-2 and phenyl rings at N-3 were predicted to have potent COX-2 inhibitory activity. The study used SC-558 (Figure 1) as reference ligand [7]. As continuation of our research program, we report herein the synthesis and COX-2 inhibitory activity evaluation of 4-[(E)-2-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethenyl]benzene-1-sulfonamide and its analogs 1a-f (Fig.1).

Figure 1

Chemical structure of SC-558 [5] and general formula of the title compounds 1a-f.

2. Results and Discussion

The title compounds 1a-f were synthesized stepwise by the method summarized in Scheme 1 and Scheme 2. Anthranilic acid (2) was reacted with acetic anhydride (3) at reflux temperature for 1 hour to provide benzoxazinone 4 [8]. Treatment of 4 with corresponding anilines 5a-f in glacial acetic acid under reflux conditions for 6-7 hours gave 2-methyl-3-phenyl-4(3H)-quinazolinones 6a-f [1,8,9]. 4-Formylbenzenesulfonamide (7) was synthesized from sulfanilamide (8). Diazotization of 8 led to diazonium salt 9, which was then neutralized with sodium carbonate and added dropwise into CuCN/KCN (Sandmeyer reaction) to yield benzonitrile 10. Reduction of 10 with Raney nickel alloy in 75% aqueous formic acid gave 4-formylbenzenesulfonamide (7) [10]. Finally, the condensation of 6a-f and 7 in the presence of anhydrous sodium acetate as catalyst and glacial acetic acid as solvent at 90 °C for 4 hours (TLC monitoring) afforded the title compounds 1a-f [11,12].

Scheme 1

Synthesis of the title compounds 1a-f.

Scheme 2

Synthesis of 4-formylbenzenesulfonamide (7).

The IR spectra of compounds 1a-f showed absorption bands at 3,331–3,365 and 3,200–3,265 cm-1 due to the presence of the NH2 group. The bands at 1,330–1,340 and 1,163–1,166 cm-1 correspond to ‑SO2-, while the carbonyl groups of the quinazolinones are observed as strong bands at 1,654–1,691 cm-1. In the 1H-NMR spectra the two protons of the sulfonamide NH2 group appear as a broad singlet at δ 7.40 ppm, while the presence of the two protons of the –(E)-ethenyl chain of the compounds are observed as doublets at 6.43–6.51 ppm (1H, J = 15,0 Hz) and 7.87–7.91 (1H, J = 15,0 Hz), respectively. The structures were further supported by 13C-NMR and HR-ESI-MS of the compounds which showed the complete agreement with the assigned molecular structures.

The results of the COX-2 inhibition screening assay of the compounds (at concentrations of 10, 20 and 50 μM) are listed in Table 1. Compound 1a is inactive as a COX-2 inhibitor, while compounds 1b-f are active. These data indicated that the substituent at the para-position of the 3-phenyl rings is essential for their inhibitory activity. In the present series, 4-[(E)-2-{3-(4-methoxyphenyl)-4-oxo-3-4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide (1c) showed a maximum COX-2 inhibition (47.1%) at a concentration of 20 μM. All our compounds are less potent COX-2 inhibitors than celecoxib (which showed 80.1% inhibition at a concentration of 1 μM), but the COX-2 inhibitory activity of the most active compound is somewhat higher than that of 2-(4-nitrophenyl)-3-(tolyl)-4(3H)-quinazolinone reported earlier (which showed a maximum COX-2 inhibition of 27.72% at a concentration of 22 μM) [4]. The solubility of the compounds in the assay system used is poor, and at concentrations of 50 μM the compounds do not dissolve completely.

Table 1

In vitro COX-2 inhibition data (%) for compounds 1a-f (see Figure 1).

Compd	X	Inhibition ± SD (%)1) at concentration of
		10 μM	20 μM	50 μM
1a	H	ns	ns	ns
1b	CH3	21.2 ±1.4	17.4 ±1.3	30.6 ±2.7
1c	OCH3	39.1 ±0.9	47.1 ±4.3	38.2 ±2.3
1d	Br	29.2 ±1.2	36.3 ±3.6	33.7 ±2.3
1e	Cl	ns	27.2 ±1.8	38.4 ±3.8
1f	COOC2H5	10.8 ±0.5	36.7 ±1.6	33.9 ±3.0
Reference compound		Inhibition ± SD (%)1) at 1 μM concentration
Celecoxib		80.1 ±2.7

Values are the mean (n = 3) and SD (Standard deviation) of the % inhibition acquired using COX inhibition assay kit (Catalog No. 560131, Cayman Chemical Co., Ann Arbor, MI, USA); ns = not significant (%-inhibition <10%).

3. Experimental

3.1. Chemistry. General Procedures

All solvents, chemicals, and reagents were obtained commercially and used without purification. Purity tests of the products was performed by the TLC method on silica gel 60 F254 plates (Merck). Melting points were determined in the capillary tube using melting point apparatus (Stuart Scientific) and are uncorrected. Infrared (IR) spectra were recorded on a FTIR spectrophotometer (8400S, Shimadzu), 1H-NMR and 13C-NMR spectra were recorded on a JEOL JNM 500 spectrometer, using TMS as internal standard, and high resolution mass spectra (HRMS) were measured with a Waters LCT Premier XE (ESI-TOF) system in positive mode.

4-[(E)-2-(4-oxo-3-phenyl-3,4-quinazolin-2-yl)ethenyl]benzene-1-sulfonamide (1a): A mixture of 2-methyl-3-phenyl-4(3H)-quinazolinone (6a; 0.98 g, 5 mmol), 4-formylbenzenesulfonamide (7; 1.02 g, 5.5 mmol) and anhydrous sodium acetate (1 g) were dissolved in glacial acetic acid (6 mL) and refluxed at 100 °C. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was poured onto cold water and filtered off through a Buchner funnel. The solid product was washed with cold water, recrystallized from suitable solvents and dried in vacuum oven at 85 °C for 1 hour to provide 1a as pale yellow crystalline powder (57.9% yield, recrystallized from acetonitril-water, and washed with cold ethanol), m.p. 277-278 °C. IR (KBr), □max, cm-1: 3,362, 3,234 (primary sulfonamide N-H streching), 3,049 (aromatic/alkene C-H streching), 1,672 (C=O lactam), 1,556 (C=C), 1,340, 1,165 (sulfonamide asymmetric and symmetric SO2 streching). 1H-NMR (DMSO-d6), δ/ppm: 8.16 (1H, dd, J = 7.1, 1.9 Hz, 5-Hquinazolinone), 7.88 (1H, td, J = 7.1, 1.9 Hz, 7-Hquinazolinone), 7.87 (1H, d, J = 15.5 Hz, 2-Htrans ethenyl), 7.80 (1H, d, J = 7.8 Hz, 8-Hquinazolinone) 7.74 (2H, d, J = 8.4 Hz, 2”,6”-HAr), 7.53-7.63 (6H, m, overlap of 6-Hquinazolinone, 3”,5”; 2’,6’; and 4’-HAr), 7.48 (2H, d, J = 8.45 Hz, 3’,5’-HAr), 7.39 (2H, s broad, NH2 sulfonamide), and 6.43 (1H, d, J = 15.5 Hz, 1-Htrans ethenyl). 13C-NMR (DMSO-d6, 125 MHz, TMS), δ/ppm: 161.2 (C(=O)-N), C-4quinazolinone, 151.1 (N-C=N-, C-2quinazolinone ), 147.3 (CPhe-N=C, C-9quinazolinone), 144.5 (C-1”Phe-1”-sulfonamide), 138.0, 137.1, 136.8, 134.9, 130.0, 129.7, 129.3, 127.9, 127.3, 126.9, 126.5, 126.3, 122.6, and 120.8 (C aromatic and C ethenyl). HRESIMS (m/z): found 404.1046 ([M+H]+), calculated masses of C22H18N3O3S: 404.1069 (error 5.7 ppm).

4-[(E)-2-{3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide (1b): Compound 1b was prepared as a pale yellow crystalline powder from 2-methyl-3-(4-methylphenyl)-4(3H)-quinazolinone (6b) using the procedure described for 1a (35.8% yield, recrystallized from THF-ethanol, followed by chloroform), m.p. 244-245 °C. IR (KBr), □max, cm-1: 3,329, 3,265 (primary sulfonamide N-H streching), 3,063 (aromatic/alkene C-H streching), 1,691 (C=O lactam), 1,550 (C=C), 1,340, 1,165 (sulfonamide asymmetric and symmetric SO2 streching). 1H-NMR (DMSO-d6) δ/ppm: 8.14 (1H, dd, J = 7.8; 1.3 Hz, 5-Hquinazolinone), 7.90 (1H, td, J = 8.9, 1.9 Hz, 7-Hquinazolinone), 7.89 (1H, d, J = 15.5 Hz, 2-Htrans ethenyl), 7.80 (1H, d, J = 6 Hz, 8-Hquinazolinone), 7.78 (2H, d, J = 8.4 Hz, 2”,6”-HPhe-1”-sulfonamide), 7.56 (1H, td, J = 6.5; 1.3 Hz, 6-Hquinazolinone), 7.53 (2H, d, J = 8.5 Hz, 3”,5”-HPhe-1”-sulfonamide), 7.42 (2H, s, NH2sulfonamide), 7.37 (2H, d, J = 8.45 Hz, 2’,6’-HAr), 7,34 (2H, d, J = 7.8 Hz, 3’,5’-HAr), 6,43 (1H, d, J = 15.5 Hz, 1-Htrans ethenyl), 2,44 (3H, s, CH3-Ar). 13C-NMR (DMSO-d6) δ/ppm: 161.2 ((C(=O)-N), C-4quinazolinone), 151.1 (N-C=N-, C-2quinazolinone), 147.2 (CPhe-N=C, C-9quinazolinone), 144.5 (C-1”Phe-1”-sulfonamide), 138.7, 138.0, 137.1, 136.8, 134.9, 134.8, 130.1, 128.6, 127.9, 127.3, 126.8, 126.3, 122.6, 120.7 (C aromatic and C ethenyl), and 20.4 (CH3-Ar). HRESIMS (m/z): found 418.1237 ([M+H]+), calculated masses of C23H20N3O3S: 418.1225 (error 2.9 ppm).

4-[(E)-2-{3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide (1c): Compound 1c was prepared from 2-methyl-3-(4-methoxyphenyl)-4(3H)-quinazolinone (6c) using the procedure described for 1a as a pale yellow crystalline powder (45.5% yield, recrystallized from ethanol), m.p. 228-229 °C. IR (KBr), □max, cm-1: 3,365, 3,246 (primary sulfonamide N-H streching), 3,080 (aromatic/alkene C-H streching), 1,675 (C=O lactam), 1,555 (C=C), 1,338, 1,163 (sulfonamide asymmetric and symmetric SO2 streching) and 1,250 (Ar-O-Al ether). 1H-NMR (DMSO-d6) δ/ppm: 8.14 (1H, dd, J = 7.8; 1.3 Hz, 5-Hquinazolinone), 7.88 (1H, td, J = 7.0; 1.9 Hz, 7-Hquinazolinone), 7,90 (1H, d, J = 15.6 Hz, 2-Htrans ethenyl), 7.77-7.80 (3H, overlap, d, J = 8.4 Hz, HAr), 7.55 (1H, t, J = 7.9 Hz, 6-Hquinazolinone), 7.58 (2H, d, J = 8.5 Hz (3”,5”-HPhe-1”-sulfonamide), 7,39 (2H, d, J = 6.5 Hz, (2’,6’-HPhe-4’-OMe), 7.34 (2H, d, J = 7.2 Hz, (3’,5’-HPhe-4’-OMe)), 7,42 (2H, s, NH2sulfonamide), 6,51 (1H, d, J = 15.6 Hz, 1-Htrans ethenyl) and 3.86 (3H, s, CH3O-). 13C-NMR (DMSO-d6) δ/ppm: 161.4 ((C(=O)-N), C-4quinazolinone), 159.5 (C-4’Phe-O), 151.5 (N-C=N-, C-2quinazolinone), 147.3 (CPhe-N=C, C-9quinazolinone), 144.5 (C-1”Phe-1”-sulfonamide), 138.0, 136.9, 134.7, 130.0, 129.2, 127.9, 127.3, 126,8, 126.5, 126,3, 122.7, 120.7, 114.8 (C aromatic and C ethenyl), and 55.4 (methoxy). HRESIMS (m/z): found 434.1176 ([M+H]+), calculated masses of C23H20N3O4S: 434.1175 (error 0.2 ppm).

4-[(E)-2-{3-(4-bromophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide (1d): Compound 1d was prepared from 2-methyl-3-(4-bromophenyl)-4(3H)-quinazolinone (6d) using the procedure described for 1a as a pale yellow crystalline powder (41.2% yield, recrystallized from ethanol), m.p. 211-212 °C. IR (KBr), □max, cm-1: 3,335, 3,236 (primary sulfonamide N-H streching), 3,095 (aromatic/alkene C-H streching), 1,683 (C=O lactam), 1,556 (C=C), 1,338, 1,165 (sulfonamide asymmetric and symmetric SO2 streching). 1H-NMR (DMSO-d6) δ/ppm: 8.14 (1H, dd, J = 7.8; 1.3 Hz, 5-Hquinazolinone), 7.88 (1H, td, J = 7.0; 1.3 Hz, 7-Hquinazolinone), 7.93 (1H, d, J = 15.0 Hz, 1H, 2-Htrans ethenyl), 7.78-7.83 (5H, m, overlap, HAr), 7.63 (2H, d, J = 8.5 Hz, 2’,6’-H Phe-4’-Br), 7.57 (1H, t, J = 7.8 Hz, 6-Hquinazolinone), 7.48 (2H, d, J = 7.2 Hz, 3’,5’-H Phe-4’-Br), 7.4 (2H, s, NH2 sulfonamide), and 6.5 (1H, d, J = 15.0 Hz, 1-Htrans ethenyl). 13C-NMR (DMSO-d6) δ/ppm: 161.1 ((C(=O)-N), C-4quinazolinone), 150.9 (N-C=N-, C-2quinazolinone), 147.2 (CPhe-N=C, C-9quinazolinone), 144.6 (C-1”Phe-1”-sulfonamide), 137.9, 137.4, 136.1, 134.9, 132.7, 131.3, 128.1, 127.3, 126.9, 126.3, 120.6, 122.5, 122.4 (C aromatic and C ethenyl). HRESIMS (m/z): found 482.0164 ([M+H]+, 95%) and 484.0157 ([M+H]+, 100%), calculated masses of C22H17N3O3SBr: 482.0174 (error 2.1 ppm).

4-[(E)-2-{3-(4-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide (1e): Compound 1e was prepared from 2-methyl-3-(4-chlorophenyl)-4(3H)-quinazolinone (6e) using the the procedure described for 1a as a pale yellow crystalline powder (49.5% yield, recrystallized from THF-ethanol, followed chloroform), m.p. 248-249 °C. IR (KBr), □max, cm-1: 3340, 3200 (primary sulfonamide N-H streching), 3066 (aromatic/alkene C-H streching), 1672 (C=O lactam), 1552 (C=C), and 1330, 1166 (sulfonamide asymmetric and symmetric SO2 streching). 1H-NMR (DMSO-d6) δ/ppm: 8.14 (1H, dd, J = 7.8; 1.3 Hz, 5-Hquinazolinone), 7.88 (1H, td, J = 7.8; 1.3 Hz, 7-Hquinazolinone), 7.91 (1H, d, J = 15,0 Hz, 2-Htrans ethenyl), 7.8 (1H, t, J = 8 Hz, 6-Hquinazolinone), 7.79 (2H, d, J = 8.4 Hz, 2”,6”-HPhe-1”-sulfonamide), 7.68 (2H, d, J = 8.5 Hz, 3”,5”H-Phe-1”-sulfonamide), 7.63 (2H, d, J = 7.8 Hz, 2’,6’H-Phe-4’-Cl), 7.54 (2H, d, J = 10 Hz, 3’,5’-HPhe-4’-Cl), 7.4 (2H, s, NH2 sulfonamide), 7.57 (1H, d, J = 7.15 Hz, 8-Hquinazolinone), 6.5 (1H, d, J = 15.0 Hz, 1-Htrans ethenyl). 13C-NMR (DMSO-d6) δ/ppm: 161.2 ((C(=O)-N), C-4quinazolinone), 150.9 (N-C=N-, C-2quinazolinone), 147.2 (CPhe-N=C, C-9quinazolinone), 144.6 (C-1”Phe-1”-sulfonamide), 137.9, 137.4, 135.7, 134.9, 133.3, 130.9, 129.7, 128.1, 127.3, 126.9, 126.5, 126.3, 120.7 (C aromatic and C ethenyl). HRESIMS (m/z): found 438.0674 ([M+H]+, 100%), 440,0647 ([M+H]+, 40%), calculated masses of C22H17N3O3SCl: 438,0679 (error 1.1 ppm).

Ethyl 4-{4-okso-2-[(E)-2-(4-sulfamoylphenyl)ethenyl]-3,4-dihydroquinazolin-3-yl}benzoate (1f): Compound 1f was prepared from ethyl 4-(2-methyl-4-oxo-3,4-dihydroquinazolin-3-yl)benzoate (6f) using the procedure described for 1a as a pale yellow crystalline powder (50,2% yield, recrystallized from THF-ethanol, and washed with cold ethanol), m.p. 239-240 °C. IR (KBr), □max, cm-1: 3,331, 3,219 (primary sulfonamide N-H streching), 3,100 (aromatic/alkene C-H streching), 2,960-1,983 (alipatic C-H streching), 1,701 (C=O ester), 1,654 (C=O lactam), 1,556 (C=C), and 1,340, 1,165 (sulfonamide asymmetric and symmetric SO2 streching). 1H-NMR (DMSO-d6) δ/ppm: 8.15 (1H, dd, J = 8.4; 1.3 Hz, 5-Hquinazolinone), 8.17 (2H, d, J = 8.5 Hz, 2’,6’-Phe-1’-COOEt), 7.93 (1H, d, J = 15.5 Hz, 2-Htrans ethenyl), 7.91 (1H, td, J = 7.1; 1.9 Hz, 7-Hquinazolinone), 7.81 (1H, d, J = 7.8 Hz, 8-Hquinazolinone), 7.76 (2H, d, J = 8.5 Hz, 3”,5”-H-Phe-4”-sulfonamide), 7.66 (2H, d, J = 6.5 Hz, 3’,5’-HPhe-1’-COOEt), 7.61 (2H, d, J = 7.8 Hz, H-2”,6”-Phe-4”-sulfonamide ), 7.57 (1H, t, J = 8.5 Hz, 6-Hquinazolinone), 7.39 (2H, s, NH2 sulfonamide), 6.45 (1H, d, J = 15.5 Hz, 1-Htrans ethenyl). 13C-NMR (DMSO-d6) δ/ppm: 165.1 (C=O ester), 161.1 (C(=O)-N), C-4quinazolinone), 150.6 (N-C=N-, C-2quinazolinone), 147.2 (CPhe-N=C, C-9quinazolinone), 144.6 (C-1’-Phe-1’-COOEt), 141.0 (C-4”Phe-4”-sulfonamide), 137.6, 137.5, 134.9, 130.5, 130.4, 129.6, 128.1, 127.3, 127.0, 126.5, 126.2, 120.6 (C aromatic and C ethenyl), 61.1 (CH2-O-) and 14.1 (CH3-C). HRESIMS (m/z): found 476.1278 ([M+H]+), calculated masses of C25H22N3O5S: 476.1280 (error 0.4 ppm).

3.2. In Vitro Cyclooxygenase-2 (COX-2) Inhibition Assays

The ability of the test compounds 1a-f listed in Table 1 to inhibit the ability of COX-2 to catalyze the conversion of arachidonic acid to prostaglandin H2 (PGH2) was determined using a COX inhibitor screening assay kit (catalog No. 560131, Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s instructions. Celecoxib was used as reference compound. The test compounds were dissolved in DMSO and added 20 μL to COX reaction tube to get final concentration mentioned at Table 1.

4. Conclusions

A series of 3-phenyl-2-[(E)-2-phenylethenyl]-3,4-dihydroquinazolin-4-ones possessing para-sulfonamide groups on the phenyl rings of the 2-phenylethenyl moiety and various substituents (X= H, CH3, OCH3, Br, Cl, COOC2H5) at the para-position of the 3-phenyl rings were synthesized and their COX-2 inhibitory activity evaluated. The compounds having substituents at the para-position of the 3-phenyl ring showed COX-2 inhibitory activity. All our compounds are less potent COX-2 inhibitors than celecoxib. 4-[(E)-2-{3-(4-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide showed a maximum COX-2 inhibition (47.1%) at concentration of 20 μM.