Interaction of New Sulfaphenazole Derivatives with Human Liver Cytochrome P450 2Cs: Structural Determinants Required for Selective Recognition by CYP 2C9 and for Inhibition of Human CYP 2Cs

Archives of Biochemistry and Biophysics Vol. 394, No. 2, October 15, pp. 189 –200, 2001 doi:10.1006/abbi.2001.2511, available online at http://www.idealibrary.com on

Interaction of New Sulfaphenazole Derivatives with Human Liver Cytochrome P450 2Cs: Structural Determinants Required for Selective Recognition by CYP 2C9 and for Inhibition of Human CYP 2Cs Nguyeˆt-Thanh Ha-Duong, Cristina Marques-Soares, Sylvie Dijols, Marie-Agne`s Sari, Patrick M. Dansette, and Daniel Mansuy 1 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite´ Paris V, 45 Rue des Saints-Pe`res, 75270 Paris Cedex 06, France

Received March 20, 2001, and in revised form June 14, 2001; published online September 26, 2001

A series of new derivatives of sulfaphenazole (SPA), in which the NH 2 and phenyl substituents of SPA are replaced by various groups or in which the sulfonamide function of SPA is N-alkylated, were synthesized in order to further explore CYP 2C9 active site and to determine the structural factors explaining the selectivity of SPA for CYP 2C9 within the human P450 2C subfamily. Compounds in which the NH 2 group of SPA was replaced with R 1 ⴝ CH 3, Br, CH ⴝ CH 2, CH 2CH ⴝ CH 2, and CH 2CH 2OH exhibited a high affinity for CYP 2C9, as shown by the dissociation constant of their CYP 2C9 complexes, K s, which was determined by difference visible spectroscopy (K s between 0.1 and 0.4 ␮M) and their constant of CYP 2C9 inhibition (K i between 0.3 and 0.6 ␮M). This indicates that the CYP 2C9-iron(III)-NH 2R bond previously described to exist in the CYP 2C9 –SPA complex does not play a key role in the high affinity of SPA for CYP 2C9. Compounds in which the phenyl group of SPA was replaced with various aryl or alkyl R 2 substituents only exhibited a high affinity for CYP 2C9 if R 2 is a freely rotating and sufficiently electron-rich aryl substituent. Finally, compounds resulting from a N-alkylation of the SPA sulfonamide function (R 3 ⴝ CH 3, C 2H 5, or C 3H 7) did not retain the selective inhibitory properties of SPA toward CYP 2C9. However, they are reasonably good inhibitors of CYP 2C8 and CYP 2C18 (IC 50 ⬃ 20 ␮M). These data allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site. They also provide us with 1

To whom correspondence and reprint requests should be addressed. Fax: 33-1-42 86 83 87. E-mail: Daniel.Mansuy@ biomedicale.univ-paris5.fr. 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

clues towards new selective inhibitors of CYP 2C8 and CYP 2C18. © 2001 Academic Press Key Words: yeast-expressed P450 2Cs; drug metabolism; tienilic acid; active site topology; ⌸–⌸ interactions.

Cytochromes P450 2 constitute a superfamily of hemoproteins that plays an important role in the metabolism of a large variety of xenobiotics and endogenous compounds (1). In order to interpret or to predict various problems that may occur with some drugs in relation to genetic polymorphism and drug– drug interaction, it is crucial to determine which human liver cytochrome P450 is involved in the metabolism of a given drug. This requires simultaneous approaches using hepatocytes, recombinant enzymes, and human liver microsomes in the presence of specific inhibitors of the various human P450s. Cytochromes P450 of the 3A and 2C subfamilies are the major isoforms present in human liver (1). From the four members of the 2C subfamily, CYP 2C8, 2C9, 2C18, and 2C19, CYP 2C9 is the protein expressed at the highest level in human liver (2, 3). It is involved in the metabolism of many drugs (4), such as diclofenac (5), tolbutamide (6), and (S)-warfarin (7). A model showing the structural factors that are required for strong interaction between CYP 2C9 and its substrates and inhibitors has been 2 Abbreviations used: CI, chemical ionization; CYP, cytochrome P450; Et 2O, diethylether; DMF, dimethylformamide; EtOAc, ethylacetate; EtOH, ethanol; MS, mass spectrum; SPA, sulfaphenazole; TA, tienilic acid.

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described on the basis of biochemical, UV-visible and 1 H NMR results (8 –12). Moreover, several 3-D models based on homology molecular modeling have been proposed for CYP 2C9 (13–15). Sulfaphenazole (SPA) is a well-known inhibitor of CYP 2C9 (1, 16). In vitro studies using liver microsomes (6, 17) and recombinant cytochromes P450 (18, 19) have shown that SPA is a selective, competitive inhibitor of CYP 2C9. A previous study on the effects of SPA on CYP 2C9 has led us to propose that the high affinity of SPA for this enzyme (K i ⬃ 0.3 ␮M) would be based on three main interactions (19). The first one comes from the coordination of the NH 2 function of SPA to CYP 2C9 iron. The second one would be an ionic bond between the negative charge of the deprotonated sulfonamide function of SPA with a putative cationic residue of the protein. Finally, the third one would correspond to an hydrophobic interaction between the phenyl substituent of the SPA pyrazole ring with aminoacid residues of CYP 2C9. However, the relative importance of these interactions in the inhibitory effects of SPA toward CYP 2C9, and the structural factors explaining the selectivity of SPA for CYP 2C9 within the human CYP 2C subfamily remain to be determined. For that purpose and in order to further explore the CYP 2C9 active site, a series of new derivatives of SPA have been synthesized and compared as inhibitors of recombinant CYP 2C8, 2C9, 2C18, and 2C19. The mode of interaction of the compounds exhibiting the highest affinities for CYP 2C9 was also studied by UV-visible difference spectroscopy. The corresponding results allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site. They also provide us with clues towards new selective inhibitors of CYP 2C8 and CYP 2C18. MATERIALS AND METHODS

Chemicals All chemicals used were of the highest quality commercially available. Tienilic acid (TA) was provided by Anphar-Rolland (ChillyMazarin, France). 3-[2,3-dichloro-4-(2-thenoyl)phenoxy]propane-1-ol and 2-[2,3-dichloro-4-(2-thenoyl)phenoxy]ethanol were prepared by previously described procedures (9, 20). Sulfaphenazole was purchased from Sigma.

Physical Measurements UV-visible spectra were recorded on Kontron Uvikon 860 and 820 spectrophotometers equipped with a diffusion sphere. 1H NMR spectra were recorded at 27°C on a Bruker ARX-250 instrument; chemical shifts are reported downfield from (CH 3) 4Si and coupling constants are in Hz. The abbreviations s, d, t, q, m, bs, and dd are used for singlet, doublet, triplet, quadruplet, multiplet, broad singlet, and doublet of doublet, respectively. Mass spectra (MS) were performed with chemical ionization (CI) using NH 3 on a Nermag R1010 apparatus. Elemental analyses were carried out at Centre regional de Microanalyse (Paris).

Synthesis of Sulfaphenazole Derivatives N-(2-Phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 1. Benzenesulfonyl chloride (0.13 ml) was added to a solution of 2-phenyl-2Hpyrazol-3-ylamine (21) (143 mg, 0.9 mmol) in anhydrous pyridine (1 ml). After 5 min at room temperature, the reaction mixture was heated for 1 h at 95°C. The solvent was evaporated and the residue suspended in 1 N HCl and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4, evaporated, and the residue was purified by column chromatography (SiO 2, CH 2Cl 2–Et 2O, 4 then 6%). After crystallization from Et 2O-cyclohexane, 100 mg of 1 were obtained (37% yield); mp 112–113°C; 1H NMR (CDCl 3) ␦ 7.68 (d, 2H, J ⫽ 7.3), 7.58 (t, 1H, J ⫽ 7.3), 7.53 (d, 1H, J ⫽ 1.9), 7.43 (t, 2H, J ⫽ 7.3), 7.35 (m, 3H), 7.08 (m, 2H), 6.45 (bs, 1H, NH), 6.24 (d, 1H, J ⫽ 1.9). Anal. calcd. for C 15H 13N 3O 2S: C, 60.19; H, 4.38; N, 14.04. Found : C, 60.24; H, 4.34; N, 14.15. 4-Methyl-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 2. p-Toluenesulfonyl chloride was added to a solution of 2-phenyl2H-pyrazol-3-yl-amine (21) in anhydrous pyridine. After 1 h at 95°C, the solvent was evaporated and the residue was dissolved in EtOAc, washed with 0.1 N HCl, and dried over MgSO 4. Compound 2 was purified by column chromatography (SiO 2, CH 2Cl 2–Et 2O 5%) and obtained in 43% yield; mp 142°C (lit. (22): 142–143°C); 1H NMR (CDCl 3) ␦ 2.4 (s, 3H, CH 3), 6.21 (d, 1H, J ⫽ 1.9), 6.53 (bs, 1H, NH), 7.12 (m, 2H, mPh), 7.20 (d, 2H, J ⫽ 8.2), 7.35 (m, 3H, o, pPh), 7.51 (d, 1H, J ⫽ 1.9), 7.55 (d, 2H, J ⫽ 8.2). MS (CI, NH 3) m/z ⫽ 314 ([M ⫹ H] ⫹, 100%), 160 (74%). Anal. calcd. for C 16H 15N 3O 2S: C, 61.32; H, 4.82; N, 13.41. Found: C, 61.36; H, 4.91; N, 13.48. 4-Bromo-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 3. 4Bromobenzenesulfonyl chloride (350 mg, 1.37 mmol) was added to a solution of 2-phenyl-2H-pyrazol-3-yl-amine (200 mg, 1.26 mmol) in 2 ml of anhydrous pyridine. After 5 min at room temperature, the reaction mixture was heated for 1 h at 95°C. The solvent was evaporated and the residue dissolved in 1 N HCl and extracted with CH 2Cl 2. The organic phase was dried over Mg SO 4, evaporated and the residue was purified by column chromatography (SiO 2, CH 2Cl 2– Et 2O 3%). This led to 290 mg of 3. Recrystallization from CH 2Cl 2 gave compound 3 as pale yellow crystals (61% yield); mp 185–186°C; 1 H NMR (CDCl 3) ␦ 7.55 (d, 1H, J ⫽ 2), 7.53-7.43 (4H, PhBr), 7.37 (m, 3H, o, pPh), 7.11 (m, 2H, mPh), 6.55 (bs, 1H, NH), 6.27 (d, 1H, J ⫽ 2). Anal. calcd. for C 15H 12BrN 3O 2S: C, 47.63; H, 3.20; N, 11.11. Found: C, 47.73; H, 3.22; N, 11.18. N-(2-Phenyl-2H-pyrazol-3-yl)-4-vinylbenzenesulfonamide, 4. To a solution of KOH (35 mg) in EtOH (1.65 ml) was added 102 mg of compound 6. The solution was heated under reflux for 1 h with traces of hydroquinone. Then the solution was cooled, acidified with 1 M HCl (1.35 ml), extracted with diethyl ether, and dried over MgSO 4. After filtration of a red impurity, compound 4 was recrystallized from diethyl ether and obtained as white crystals (40% yield); mp 147°C (decomposition); 1H NMR (CDCl 3) ␦ 5.45 (d, 1H, J cis ⫽ 10.9), 5.87 (d, 1H, J trans ⫽ 17.6), 6.25 (d, 1H, J ⫽ 1.8), 6.49 (bs, 1H, NH), 6.71 (dd, 1H, J cis ⫽ 10.9, J trans ⫽ 17.6), 7.10 (m, 2H, mPh), 7.34 (m, 3H, o, pPh), 7.40 (d, 2H, J ⫽ 8.4), 7.53 (d, 1H, J ⫽ 1.8), 7.60 (d, 2H, J ⫽ 8.4). Anal. calcd. for C 17H 15N 3O 2S: C, 62.75; H, 4.65; N, 12.91. Found: C, 62.67; H, 4.54; N, 13.00. 4-Allyl-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 5. The day before tetrakis(triphenylphosphine)palladium was prepared as previously described (23). Then 210 mg (0.56 mmol) of compound 3 and 36 mg of tetrakis(triphenylphosphine)palladium(0) were introduced in a dry schlenck with septum and magnetic stirring. All the experiments were performed under argon. Five milliliters of anhydrous and deoxygenated DMF and 0.19 ml of allyltributyltin were introduced successively with a syringe. The solution was heated at 110°C for 7.5 h. The mixture was filtered and DMF was evaporated. After purification by column chromatography (SiO 2, CH 2Cl 2–Et 2O 5%) 145 mg of 5 were obtained (77% yield). Recrystallization from ether led to 102 mg of compound 5 as pale yellow crystals; mp

INTERACTION OF SULFAPHENAZOLE DERIVATIVES WITH CYP 2Cs 115–116°C; 1H NMR (CDCl 3) ␦ 7.58 (d, 2H, J ⫽ 8.3), 7.53 (d, 1H, J ⫽ 1.9), 7.35 (m, 3H, o, pPh), 7.23 (d, 2H, ⫽8.3), 7.07 (m, 2H, mPh), 6.47 (bs, 1H, NH), 6.25 (d, 1H, J ⫽ 1.9), 5.91 (m, 1H, H a, J ⫽ 6.7, J ab ⫽ 10.1, J ab⬘ ⫽ 16.9), 5.14 (m, 1H, H b, J ba ⫽ 10.1, J bb⬘ ⫽ 1.6), 5.08 (m, 1H, H b⬘, J b⬘a ⫽ 16.9, J bb⬘ ⫽ 1.6), 3.43 (d, 2H, CH 2, J ⫽ 6.7). Anal. calcd. for C 18H 17N 3O 2S: C, 63.70; H, 5.05; N, 12.38. Found: C, 63.69; H, 5.03; N, 12.41. 4-(2-Bromoethyl)-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 6. 0.74 ml (5.4 mmol) of (2-bromoethyl)-benzene was added dropwise to chlorosulfonic acid (2 ml, 0.03 mol). After 1 h at 22°C, the mixture was poured on ice, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4 and evaporated. The two isomers (para and ortho) of (2-bromoethyl)-benzenesulfonylchloride (1.4 g) were separeted by column chromatography (SiO 2, cyclohexane-CH 2Cl 2 70:30 and then 50:50). This allowed one to obtain 220 mg (14%) of ortho-isomer and 710 mg (46%) of the expected paraisomer; 1H NMR (CDCl 3) ␦ 3.28 (t, 2H, J ⫽ 7), 3.60 (t, 2H, J ⫽ 7), 7.46 (d, 2H, J ⫽ 8.4), 7.98 (d, 2H, J ⫽ 8.4); IR (cm ⫺1): 1370, 1170. To an ice cold solution of the latter compound, 4-(2-bromoethyl)-benzenesulfonyl chloride, (350 mg, 1.23 mmol) in CHCl 3 (6 ml) were added 2-phenyl2H-pyrazol-3-ylamine (200 mg, 1.26 mmol) and pyridine (0.1 ml, 1.24 mmol). The mixture was stirred for 45 min at 0°C. After 60 h at room temperature, the solution was washed with 0.1 M HCl, extracted with CH 2Cl 2, dried over MgSO 4, and concentrated. The residue was purified by column chromatography (SiO 2, CH 2Cl 2-acetone 93:7). Recrystallization from diethyl ether gave compound 6 in 30% yield; mp 127°C (decomposition); 1H NMR (CDCl 3) ␦ 3.22 (t, 2H, J ⫽ 7), 3.58 (t, 2H, J ⫽ 7), 6.26 (d, 1H, J ⫽ 1.8), 6.48 (bs, 1H, NH), 7.09 (m, 2H, mPh), 7.28 (d, 2H, J ⫽ 8.4), 7.36 (m, 3H, o, pPh), 7.54 (d, 1H, J ⫽ 1.8), 7.63 (d, 2H, J ⫽ 8.4). Anal. calcd. for C 17H 16BrN 3O 2S: C, 50.25; H, 3.97; N, 10.34. Found: C, 50.28; H, 3.99; N, 10.32. 4-(2-Hydroxyethyl)-N-(2-phenyl-2H-pyrazol-3yl)-benzenesulfonamide, 7. Phenyl-2-ethyl acetate (0.74 ml, 4.6 mmol) was added dropwise to chlorosulfonic acid (1.5 ml) at 0°C. The mixture was stirred for 1h at room temperature, poured on ice, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4 and evaporated. The arylsulfonyl chloride was purified by column chromatography (SiO 2, CH 2Cl 2) and obtained in 44% yield. Then 530 mg (2 mmol) of this compound was dissolved in 10 ml of dry CH 2Cl 2, and 0.165 ml of pyridine and 330 mg (2.07 mmol) of 2-phenyl-2H-pyrazol-3-yl-amine were added successively to the CH 2Cl 2 mixture. After 65 h at room temperature, the reaction mixture was washed with 1 N HCl. The organic phase was dried over MgSO 4. Chromatography on silica gel (CH 2Cl 2–Et 2O 8%) gave 100 mg of pure 7 acetate; 1H NMR (CDCl 3) ␦ 2.00 (s, 3H, CH 3CO), 2.99 (t, 2H, J ⫽ 6.8), 4.29 (t, 2H, J ⫽ 6.8), 6.24 (d, 1H, J ⫽ 1.9), 6.43 (bs, 1H, NH), 7.09 (m, 2H, mPh), 7.27 (d, 2H, J ⫽ 8.4), 7.36 (m, 3H, o, pPh), 7.53 (d, 1H, J ⫽ 1.9), 7.61 (d, 2H, J ⫽ 8.4). It also led to 220 mg of product contaminated by 10% of 2-phenyl-2H-pyrazol-3-yl-amine, which was eliminated as described later. This contaminated compound (220 mg) was hydrolyzed in a solution of 49 mg NaOH in 10 ml H 2O at room temperature. The aqueous phase was washed with CH 2Cl 2 in order to eliminate the impurities, acidified with 1 N HCl, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. After evaporation, the residue was purified by column chromatography (SiO 2, CH 2Cl 2–EtOAc 5050) leading to 175 mg of compound 7; 1H NMR (CDCl 3) ␦ 1.40 (s, 1H, OH), 2.91 (t, 2H, J ⫽ 6.4), 3.88 (t, 2H, J ⫽ 6.4), 6.25 (d, 1H, J ⫽ 2), 6.52 (sl, 1H, NH), 7.09 (m, 2H, mPh), 7.29 (d, 2H, J ⫽ 8.4), 7.35 (m, 3H, o, pPh), 7.53 (d, 1H, J ⫽ 2), 7.62 (d, 2H, J ⫽ 8.4). 4-Amino-N-[2-(3,4-dichlorophenyl)-2H-pyrazol-3-yl]-benzenesulfonamide, 8, and 4-amino-N-(2-ethyl-2H-pyrazol-3-yl)-benzenesulfonamide, 9, were prepared by previously described procedures (19). 4-Amino-N-(2-cyclohexyl-2H-pyrazol-3-yl)-benzenesulfonamide, 10. 2-Cyclohexyl-2H-pyrazol-3-yl-amine was prepared as previously described (24). N-Acetylsulfanilyl chloride (310 mg, 1.33 mmol) was added to a solution of 2-cyclohexyl-2H-pyrazol-3-ylamine (200

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mg, 1.21 mmol) in 2 ml of anhydrous pyridine. After 1 h at 95°C, the solvent was evaporated, and the residue was dissolved in EtOAc. The organic phase was washed with 0.1 N HCl, and dried over MgSO 4. The corresponding product (N-acetyl-10) was recrystallized from EtOAc (54% yield); 1H NMR (CDCl 3–CD 3OD) ␦ 7.65 (m, 4H), 7.26 (d, 1H, J ⫽ 1.8), 5.59 (d, 1H, J ⫽ 1.8), 4.21 (m, 1H, CHN), 2.12 (s, CH 3), 1.80 –1.63 (m, 7H), 1.33–1.17 (m, 3H). This product was boiled in 6 ml 2 N NaOH for 2 h. The solution was filtered and put to pH 5 with 1 N HCl. The solid was filtered and purified by column chromatography (SiO 2, CH 2Cl 2-acetone 90-10). Recrystallization from Et 2O gave 100 mg of crystals of 10 (47% yield); mp 177–178°C; 1H NMR (CD 3SOCD 3) ␦ 9.73 (bs, 1H, NH), 7.31 (d, 2H, J ⫽ 8.7), 7.26 (d, 1H, J ⫽ 1.8), 6.57 (d, 2H, J ⫽ 8.7), 6.00 (bs, 2H, NH 2), 5.65 (d, 1H, J ⫽ 1.8), 4.00 (m, 1H, CHN), 1.74 –1.51 (m, 7H), 1.31–1.06 (m, 3H). Anal. calcd. for C 15H 20N 4O 2S: C, 56.23; H, 6.29; N, 17.49. Found: C, 56.05; H, 6.22; N, 17.58. 4-Amino-N-[2-(2,3,5,6-tetrafluoro-phenyl)-2H-pyrazol-3-yl]-benzenesulfonamide, 11. The synthetic strategy was already described for sulfaphenazole (21). Acrylonitrile (1.25 ml, 19 mmol) and 2,3,5,6tetrafluorophenylhydrazine (3 g, 16.7 mmol) in 20 ml EtOH were heated under reflux for 18 h. The solvent was then evaporated under vacuum and the residue recrystallized from MeOH to yield 2.52 g 3-[N⬘(2,3,5,6-tetrafluorophenyl)-hydrazino]-propionitrile; 1H NMR (CDCl 3) ␦ 7.22 (bd, 1H, NH), 7.07 (m, 1H), 5.12 (bq, 1H, NH), 2.99 (q, 2H, J ⫽ 6.4), 2.60 (t, 2H, J ⫽ 6.4). A solution of this product (2.3 g, 9.87 mmol) in 18 ml of 2 N H 2SO 4 was stirred 17 h at room temperature with 4 g of anhydrous Fe 2(SO 4) 3. The mixture was extracted with Et 2O. After drying over MgSO 4 and evaporation of the solvent, the residue was suspended in CH 2Cl 2/cyclohexane 30%. The solution was filtered and the filtrate concentrated. The residue was heated under reflux in 7 ml 1 N NaOH for 1 h. The mixture was cooled, extracted with CH 2Cl 2 and dried over MgSO 4. After purification by column chromatography (SiO 2, CH 2Cl 2), 260 mg of 2-(2,3,5,6-tetrafluorophenyl)-2H-pyrazol-3-yl-amine was obtained; 1H NMR (CDCl 3) ␦ 7.55 (s, 1H), 7.20 (m, 1H), 5.70 (s, 1H), 3.64 (bs, 2H, NH 2). 259 mg (1.12 mmol) of 2-(2,3,5,6-tetrafluorophenyl)-2H-pyrazol-3-ylamine were dissolved in 2.5 ml of anhydrous pyridine. 262 mg (1.12 mmol) of N-acetylsulfanilyl chloride were added. After 5 min at room temperature, the mixture was heated at 95°C for 2 h. Pyridine was evaporated, the residue was dissolved in CH 2Cl 2 with traces of MeOH, and washed with 1 N HCl. The aqueous phase was extracted with CH 2Cl 2. The organic phase was dried over MgSO 4 and evaporated. After purification by column chromatography (SiO 2, CH 2Cl 2– MeOH 5%) 140 mg of an impure product were obtained. They were heated under reflux in 2 ml 2 N NaOH for 4 h. The mixture was neutralized with 1 N HCl, extracted with CH 2Cl 2 and dried over MgSO 4. The product was purified on preparative TLC plates (SiO 2, 1 mm, CH 2Cl 2–MeOH 5%) leading to 67 mg of 11. 1H NMR (CDCl 3) ␦ 7.67 (s, 1H), 7.36 (d, 2H, J ⫽ 7.5), 7.19 –7.06 (m, 1H), 6.72 (bs, 1H, NH), 6.52 (d, 2H, J ⫽ 7.5), 6.16 (s, 1H), 4.20 (bs, 2H, NH 2). Anal. calcd. for C 15H 10F 4N 4O 2S, 0.5 H 2O: C, 45.57; H, 2.80; N, 14.17. Found: C, 45.82; H, 2.63; N, 14.30. MS (CI, NH 3): m/z ⫽ 387 ([M ⫹ H] ⫹, 84%); 404 ([M ⫹ NH 4] ⫹, 6%); 369 (7%); 232 (65%); 212 (100%). 4-Methyl-N-[2-(3,4-dichloro-phenyl)-2H-pyrazol-3-yl]-benzenesulfonamide, 13. Para-toluenesulfonyl chloride (300 mg, 1.57 mmol) was added to a solution of 2-(3,4-dichlorophenyl)-2H-pyrazol-3-ylamine (340 mg, 1.49 mmol) in 3 ml of anhydrous pyridine at room temperature. After 5 min, the reaction mixture was heated at 95°C for 75 min. The solvent was then evaporated and the residue was dissolved in 1 N NaOH. The aqueous phase was washed with CH 2Cl 2, acidified with 1 N HCl and extracted with CH 2Cl 2. The product was purified by column chromatography (SiO 2, CH 2Cl 2–Et 2O 5%) and crystallized from Et 2O– cyclohexane; yield 35%; mp 93– 95°C; 1H NMR (CDCl 3) ␦ 7.56 (d, 1H, J ⫽ 1.9), 7.53 (d, 2H, J ⫽ 8.3), 7.42 (d, 1H, J ⫽ 8.3), 7.26 –7.15 (m, 4H), 6.33 (bs, 1H, NH), 6.20 (d, 1H, J ⫽ 1.9), 2.42 (s, 3H, CH 3). Anal. calcd. for C 16H 13Cl 2N 3O 2S: C, 50.27; H, 3.43; N, 10.99. Found: C, 50.20; H, 3.65; N, 10.96.

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4-Amino-N-(2-naphthalen-1-yl-2H-pyrazol-3-yl)-benzenesulfonamide, 12. To a suspension of ␣-naphthylamine (1.42 g, 9.93 mmol) in 6 M aqueous hydrochloric acid (6 ml) at 0°C was added dropwise a solution of sodium nitrite (760 mg) in water (2.2 ml), and the reddish mixture was stirred for 15 min. A solution of stannous chloride (7 g, 31 mmol) in concentrated hydrochloric acid (12 ml) was added dropwise to this mixture during 1 h. After stirring for 1 h at 0°C, the mixture was poured into 10 M NaOH (32 ml) at 0°C. The brown gray solid was filtered, suspended in water (15 ml) and extracted with diethyl ether (5 ⫻ 30 ml). Extracts were dried over anhydrous sodium sulfate. The crude product was recrystallized from diethyl ether to yield 1.12 g of ␣-naphthylhydrazine (71%); mp 116 –117°C (lit. (25): 116 –117°C ). ␣-Naphtylhydrazine (1.1 g) and acrylonitrile (0.52 ml) were heated under reflux for 15 h in 10 ml EtOH, and the solvent was then evaporated under vacuum. The products were separated by column chromatography (SiO 2, CH 2Cl 2). 200 mg of 3-(naphthalen-1-ylazo)-propionitrile were obtained (Rf 0.67) and 710 mg of 3-(N⬘-naphthalen-1-yl-hydrazino)-propionitrile (Rf 0.37) mixed with 15% of the azo derivative; 1H NMR (CDCl 3) ␦ 7.85–7.72 (m, 2H), 7.47–7.36 (m, 4H), 7.13 (d, 1H), 3.26 (t, 2H, CH 2NH), 2.64 (t, 2H, CH 2CN). A solution of the hydrazino-propionitrile (710 mg) in 6 ml 2 N H 2SO 4 was stirred 17 h at room temperature with 1.33 g of anhydrous Fe(SO 4) 3. The product was extracted with Et 2O and purified by column chromatography (SiO 2, CH 2Cl 2). Finally, this led to 810 mg of the azo derivative (global yield 55%); 1H NMR (CDCl 3) ␦ 8.72 (d, 1H, J ⫽ 8.2), 7.98 –7.88 (m, 2H), 7.66 –7.45 (m, 4H), 4.53 (t, 2H, J ⫽ 6.7), 3.01 (t, 2H, J ⫽ 6.7). This compound was heated at 70°C in 12 ml 1 N NaOH for 1.5 h. The mixture was cooled, diluted with water and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. 620 mg of 2-naphthalen1-yl-2H-pyrazol-3-ylamine were thus obtained after column chromatography (SiO 2, CH 2Cl 2–Et 2O 8%) (76% yield). 1H NMR (CDCl 3) ␦ 7.95–7.90 (m, 2H), 7.56 –7.48 (m, 6H), 5.69 (d, 1H, J ⫽ 1.9), 3.57 (bs, 2H, NH 2). N-Acetylsulfanilyl chloride (325 mg, 1.39 mmol) was added to a solution of 2-naphthalen-1-yl-2H-pyrazol-3-yl-amine (290 mg, 1.39 mmol) in 3 ml of pyridine at room temperature. After 5 min, the reaction mixture was heated at 95°C for 1 h. The solvent was evaporated, the residue was dissolved in CH 2Cl 2/MeOH, washed with 1 N HCl, and extracted with dichloromethane. The organic phase was dried over MgSO 4. The product was purified by column chromatography (SiO 2, CH 2Cl 2–MeOH, 5 then 10%), and 139 mg was obtained; mp 228 –229°C; 1H NMR (CD 3COCD 3) ␦ 9.46 and 8.89 (2bs, 2H, 2NH), 7.98 (t, 2H), 7.65 (d, 2H), 7.62 (d, 1H), 7.53 (d, 2H), 7.49 (m, 2H), 7.37 (t, 1H), 7.18 (d, 1H), 7.07 (d, 1H), 6.27 (d, 1H, J ⫽ 1.8), 2.14 (s, 3H, CH 3). Anal. calcd. for C 21H 18N 4O 3S, 0.5 H 2O: C, 60.71; H, 4.61; N, 13.49; Found: C, 60.64; H, 4.68; N, 13.75. MS (CI, NH 3): m/z ⫽ 407 ([M ⫹ H] ⫹, 9%); 210 (100%). This compound (129 mg, 0.32 mmol) was heated under reflux in 3 ml of 2 N NaOH for 2 h. The solution was diluted, neutralized with 1 M HCl, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. The product was purified by crystallization from CH 2Cl 2– cyclohexane and 90 mg of 12 was obtained; mp 192–194°C; 1H NMR (CDCl 3) ␦ 7.86 (m, 2H), 7.59 (s, 1H), 7.52–7.24 (m, 5H), 6.99 (m, 2H), 6.65 (bs, 1H, NH), 6.44 (d, 2H, J ⫽ 8.4), 6.37 (s, 1H), 4.28 (bs, 2H, NH 2). Anal. calcd. for C 19H 16N 4O 2S: C, 62.62; H, 4.43; N, 15.37. Found: C, 62.46; H, 4.43; N, 15.43. 4-Methyl-N-[2(4-methoxy-phenyl)-2H-pyrazol-3-yl]-benzenesulfonamide, 14. 4-Methoxyphenylhydrazine, hydrochloride (1.74 g, 10 mmol), acrylonitrile (0.75 ml, 11.4 mmol) and sodium carbonate (530 mg, 5 mmol) were heated under reflux for 9 h in 20 ml EtOH and kept at room temperature overnight. Salts were filtered and the solvent was then evaporated under vacuum. The products were separated by column chromatography (SiO 2, CH 2Cl 2–Et 2O 2%), and 450 mg of 3-(4-methoxyphenylazo)-propionitrile was obtained as a red oil, (24% yield); 1H NMR (CDCl 3) ␦ 7.69 (d, 2H, J ⫽ 9), 6.94 (d, 2H, J ⫽ 9), 4.28 (t, 2H, J ⫽ 6.9), 2.90 (t, 2H, J ⫽ 6.9). IR (cm ⫺1): 2250, 1600, 1585, 1250. This compound was heated at 85°C in 9 ml 1 N NaOH for

0.5 h. The mixture was cooled, diluted with water, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. This led to 2-(4-methoxyphenyl)-2H-pyrazol-3-yl-amine, which was purified by column chromatography (SiO 2, CH 2Cl 2–EtOAc, 10 then 15%) giving 370 mg of auburn solid (82% yield); mp 84 – 85°C; 1H NMR (CDCl 3) ␦ 7.43 (d, 2H, J ⫽ 8.8), 7.38 (s, 1H), 6.97 (d, 2H, J ⫽ 8.8), 5.59 (s, 1H), 3.83 (s, 3H, CH 3), 3.71 (bs, 2H, NH 2). 4-Toluenesulfonyl chloride (200 mg, 1.05 mmol) was added to a solution of 2-(4-methoxyphenyl)-2Hpyrazol-3-yl-amine (200 mg, 1.06 mmol) in 2 ml of pyridine at room temperature. After 5 min, the reaction mixture was heated at 95°C for 1 h and kept at room temperature overnight. The solvent was evaporated, the residue dissolved in 1 N NaOH, washed with dichloromethane, acidified with 1 N HCl, and extracted with dichloromethane. The organic phase was dried over MgSO 4. The product was purified by column chromatography (SiO 2, CH 2Cl 2–EtOAc 15%) and by recrystallization from Et 2O-cyclohexane. 200 mg of white solid 14 were obtained; mp 158 –159°C; 1H NMR (CDCl 3) ␦ 7.57 (d, 2H, J ⫽ 8.3), 7.48 (d, 1H, J ⫽ 2), 7.23 (d, 2H, J ⫽ 8.3), 6.98 (d, 2H, J ⫽ 9), 6.84 (d, 2H, J ⫽ 9), 6.39 (bs, 1H, NH), 6.20 (d, 1H, J ⫽ 2), 3.82 (s, 3H, CH 3), 2.41 (s, 3H, CH 3). Anal. calcd. for C 17H 17N 3O 3S: C, 59.46; H, 4.99; N, 12.24. Found: C, 59.27; H, 5.00; N, 12.12. 4-Methyl-N-[2-(2,6-dichlorophenyl)-2H-pyrazol-3-yl]-benzenesulfonamide, 15. 2,6-Dichlorophenylhydrazine (300 mg, 1.69 mmol) and acrylonitrile (three additions of 0.11 ml at t ⫽ 0, 8, and 16 h) were heated in 3 ml DMF at 40°C for 90 h. The solution was evaporated under vacuum and the residue was purified by column chromatography (SiO 2, CH 2Cl 2–CH 3CN 4%). Pale yellow crystals of 3-[N⬘-(2,6-dichorophenyl)-hydrazino]-propionitrile were obtained in a 87% yield; mp 63– 64°C; IR (cm ⫺1) 3340, 3360, 2240; 1H NMR (CDCl 3) ␦ 2.54 (t, 2H, J ⫽ 6.8), 2.99 (q, 2H, J ⫽ 6.8), 4.48 (bq, 1H, NH), 5.71 (bs, 1H, NH), 6.91 (t, 1H, J ⫽ 8.1), 7.26 (d, 2H, J ⫽ 8.1). This product (320 mg, 1.39 mmol) and 2.5 ml 2 N H 2SO 4 were stirred for 17 h at room temperature with 560 mg anhydrous Fe 2(SO 4) 3. The mixture was extracted with CH 2Cl 2, dried over MgSO 4, evaporated, and the brown oil was purified by column chromatography (SiO 2, CH 2Cl 2) leading to 146 mg of a yellow oil (46% yield); 1H NMR (CDCl 3) ␦ 2.98 (t, 2H, J ⫽ 6.8), 4.60 (t, 2H, J ⫽ 6.8), 7.17 (t, 1H, J ⫽ 8.1), 7.36 (d, 2H, J ⫽ 8.1). This product, 3-(2,6-dichlorophenylazo)propionitrile, was heated at 80°C for 40 min in 2.5 ml 1 N NaOH. The mixture was cooled, diluted with water, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4, evaporated and the residue was purified by column chromatography (SiO 2, CH 2Cl 2-EtOAc 5%). This led to 125 mg of 2-(2,6-dichlorophenyl)-2H-pyrazol-3-ylamine in a 86% yield; mp 106 –107°C; 1H NMR (CDCl 3) ␦ 3.52 (bs, 2H, NH 2), 5.67 (d, 1H, J ⫽ 1.8), 7.53 (d, 1H, J ⫽ 1.8), 7.48 –7.31 (3H). p-Toluenesulfonyl chloride (110 mg, 0.58 mmol) was added to a solution of 2-(2,6-dichlorophenyl)-2H-pyrazol-3-yl-amine (120 mg, 0.53 mmol) in 1.2 ml of anhydrous pyridine at room temperature. After 5 min, the reaction mixture was heated at 95°C for 2 h. The solvent was then evaporated and the residue was dissolved in 1 N NaOH. The aqueous phase was washed with CH 2Cl 2, made acidic with 1 N HCl and extracted with CH 2Cl 2. This organic phase was dried over MgSO 4. Product 15 was purified by column chromatography (SiO 2, CH 2Cl 2–EtOAc 10%) and obtained in a 48% yield; mp 173–174°C; 1H NMR (CDCl 3) ␦ 2.40 (s, 3H, CH 3), 6.23 (d, 1H, J ⫽ 2), 6.30 (s, 1H, NH), 7.41–7.30 (3H), 7.21 (d, 2H, J ⫽ 8), 7.61 (m, 3H). Anal. Calcd. for C 16H 13Cl 2N 3O 2S: C, 50.27; H, 3.43; N, 10.99. Found: C, 50.42; H, 3.38; N, 10.74. 4-Methyl-N-(2-benzyl-2H-pyrazol-3-yl)-benzenesulfonamide, 16. 2-Benzyl-2H-pyrazol-3-ylamine was prepared as previously described (24). 4-Toluenesulfonyl chloride (100 mg, 0.52 mmol) was added to a solution of 2-benzyl-2H-pyrazol-3-ylamine (90 mg, 0.52 mmol) in 1 ml of pyridine at room temperature. After 5 min, the reaction mixture was heated at 95°C for 1 h. The solvent was evaporated and the residue was dissolved in 1 N NaOH, washed with CH 2Cl 2, acidified with 1 N HCl, and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. The product was purified by

INTERACTION OF SULFAPHENAZOLE DERIVATIVES WITH CYP 2Cs column chromatography (SiO 2, CH 2Cl 2-acetone 10%) and by two recrystallizations from Et 2O-cyclohexane. 36 mg of 16 were obtained; mp 141–142°C; 1H NMR (CDCl 3) ␦ 7.62 (d, 2H, J ⫽ 8.3), 7.37 (d, 1H, J ⫽ 1.9), 7.29 –7.25 (m, 5H), 7.10 (m, 2H), 6.00 (bs, 1H, NH), 5.74 (d, 1H, J ⫽ 1.9), 5.22 (s, 2H, CH 2), 2.43 (s, 3H, CH 3). Anal. calcd. for C 17H 17N 3O 2S: C, 62.37; H, 5.23; N, 12.83. Found: C, 62.23; H, 5.21; N, 12.79. 4-Amino-N-methyl-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 17. 4-Acetylamino-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide was prepared as previously described (19). Methyl iodide (0.05 ml, 0.80 mmol) was added to a solution of 4-acetylaminoN-[(2-phenyl)-2H-pyrazol-3-yl)-benzenesulfonamide (165 mg, 0.48 mmol) in 1.5 ml of anhydrous DMF containing 60 mg of Na 2CO 3. After 1 h at 80°C, DMF was evaporated and the residue was dissolved in CH 2Cl 2. The organic phase was washed with water and dried over MgSO 4. After purification by column chromatography (SiO 2, CH 2Cl 2–MeOH 5%), 145 mg of product was obtained in a 84% yield; 1H NMR (CDCl 3) ␦ 2.21 (s, 3H, CH 3CO), 3.03 (s, 3H, CH 3), 5.89 (d, 1H, J ⫽ 2), 7.48 –7.39 (m, 3H, o, pPh), 7.59 –7.55 (m, 4H), 7.64 (s, 4H). The corresponding compound was heated under reflux in 3 ml 3 N NaOH and 3 ml EtOH for 2 h. EtOH was evaporated, the solution was diluted with water and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. Product 17 was purified by crystallization from CH 2Cl 2–Et 2O; mp 210 –211°C. 1H NMR (CDCl 3) ␦ 7.61– 7.56 (m, 3H), 7.47–7.30 (m, 5H), 6.63 (d, 2H, J ⫽ 8.7), 5.94 (d, 1H, J ⫽ 1.9), 4.17 (bs, 2H, NH 2), 3.01 (s, 3H, CH 3). Anal. calcd. for C 16H 16N 4O 2S: C, 58.52; H, 4.91; N, 17.06. Found: C, 58.63; H, 5.08; N, 17.13. 4-Amino-N-ethyl-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 18. Ethyl bromide (0.07 ml, 0.94 mmol) was added to a solution of 4-acetylamino-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide (150 mg, 0.42 mmol) in 1.5 ml anhydrous DMF containing 100 mg of Na 2CO 3. After one night at room temperature, the reaction was not finished, and then 0.17 ml of ethyl bromide in 3 ml DMF was added and the mixture was stirred for 24 h. The solvent was evaporated and the residue was dissolved in CH 2Cl 2. The organic phase was washed with water and dried over MgSO 4. After purification by column chromatography (SiO 2, CH 2Cl 2–EtOAc 60-40), 108 mg of product were obtained in a 67% yield; 1H NMR (CDCl 3) 7.66 –7.59 (m, 8H), 7.47–7.32 (m, 3H, o, pPh), 5.89 (d, 1H, J ⫽ 2), 3.37 (q, 2H, J ⫽ 7.2), 2.21 (s, 3H, J ⫽ 7.2), 0.89 (t, 3H, J ⫽ 7.2). The corresponding compound, N-acetyl-18, (108 mg, 0.28 mmol) was heated under reflux in 2.2 ml 3 N NaOH and 4.5 mL EtOH for 2 h. EtOH was evaporated, and the solution was diluted with water and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. Product 18 was recrystallized from CH 2Cl 2– cyclohexane in quantitative yield; mp 209 –210°C; 1H NMR (CDCl 3) ␦ 7.50 –7.32 (m, 5H), 7.66 (d, 2H, J ⫽ 8.7), 7.60 (d, 1H, J ⫽ 2), 6.64 (d, 2H, J ⫽ 8.7), 5.93 (d, 1H, J ⫽ 2), 4.15 (bs, 2H, NH 2), 3.35 (q, 2H, J ⫽ 7.2), 0.88 (t, 3H, J ⫽ 7.2). Anal. calcd. for C 17H 18N 4O 2S: C, 59.63; H, 5.30; N, 16.36. Found: C, 59.47; H, 5.45; N, 16.30. 4-Amino-N-propyl-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide, 19. Propyl iodide (0.1 mL, 1.02 mmol) was added to a solution of 4-acetylamino-N-(2-phenyl-2H-pyrazol-3-yl)-benzenesulfonamide (165 mg, 0.46 mmol) in 2 ml of anhydrous DMF containing 100 mg of Na 2CO 3. After 8 h at 80°C and 1 night at 60°C, DMF was evaporated, and the residue was dissolved in CH 2Cl 2. The organic phase was washed with water and dried over MgSO 4. After purification by column chromatography (SiO 2, CH 2Cl 2–EtOAc 60-40) 150 mg of product were obtained in a 81% yield; 1H NMR (CDCl 3) ␦ 0.64 (t, 3H, J ⫽ 7.4), 1.23 (m, 2H), 2.21 (s, 3H), 3.22 (t, 2H, J ⫽ 7.4), 5.86 (d, 1H, J ⫽ 1.9), 7.46 –7.36 (m, 3H, o, pPh), 7.67–7.58 (m, 8H). This product (N-acetyl-19) (115 mg, 0.29 mmol) was heated under reflux in 2.4 ml 3 N NaOH and 3 ml EtOH for 2 h. EtOH was evaporated, and the solution was diluted with water and extracted with CH 2Cl 2. The organic phase was dried over MgSO 4. Product 19 (83 mg) was obtained after recrystallization from CH 2Cl 2–Et 2O; mp 169 –170°C;

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1 H NMR (CDCl 3) ␦ 7.67 (d, 2H, J ⫽ 7.6), 7.59 (d, 1H, J ⫽ 2), 7.50 (d, 2H, J ⫽ 8.7), 7.47–7.35 (m, 3H), 6.65 (d, 2H, J ⫽ 8.7), 5.91 (d, 1H, J ⫽ 2), 4.16 (bs, 2H, NH 2), 3.21 (t, 2H, J ⫽ 7.4), 1.21 (m, 2H), 0.63 (t, 3H, J ⫽ 7.4). Anal. calcd. for C 18H 20N 4O 2S, 1/8H 2O: C, 60.27; H, 5.69; N, 15.62. Found: C, 60.25; H, 5.70; N, 15.64.

Yeast Transformation, Cell Culture, and Preparation of the Yeast Microsomal Fraction The expression system used for human liver P450s was based on a yeast strain W(R) fur 1 previously described (26), in which yeast cytochrome P450 reductase was overexpressed. Transformation by a pYeDP60 vector containing one of the human liver CYP 2C8, 2C9, 2C18 and 2C19 cDNAs (27–30) was then performed according to a general method of construction of yeast strain W(R) fur 1 expressing various human liver cytochrome P450s (31, 32). Yeast culture and microsomes preparation were performed by using previously described techniques (33). Microsomes were homogenized in 50 mM Tris buffer (pH 7.4), containing 1 mM EDTA and 20% glycerol (v/v), aliquoted, frozen under liquid N 2, and stored at ⫺80°C until use. P450 contents of yeast microsomes were 40, 90, 40, and 20 pmol P450 per mg protein for CYP 2C8, 2C9, 2C18, and 2C19, respectively. Microsomal P450 content was determined according to the method of Omura and Sato (34). The protein content in microsomal suspensions was determined by the Lowry procedure (35) using bovine serum albumin as the standard.

Study of Substrate Binding to Yeast-Expressed CYP 2C9 by Difference Visible Spectroscopy Difference visible spectra produced by sulfaphenazole derivatives were recorded at room temperature with a Kontron 820 spectrophotometer. Yeast microsomes were suspended in 50 mM Tris, 1 mM EDTA, pH 7.4, to obtain a P450 concentration of 0.13 ␮M. The solution was equally divided between two 500 ␮l quartz cuvettes (1-cm path length) and a baseline was recorded. Aliquots of solutions containing the studied compound either in Tris buffer or in DMSO (total volume 3–10 ␮l) were added to the sample cuvette, the same volume of solvent being added to the reference cuvette. The difference spectra were recorded between 380 and 520 nm (36).

Enzyme Activities Assay 5-Hydroxylation of 2-aroylthiophenes. Quantitation of 5-hydroxy2-aroylthiophenes was based on a spectrophotometric method (37) adapted to yeast microsomes expressing CYP 2C9 in the case of tienilic acid (38). Incubations for metabolic activity with yeast microsomes were carried out at 28°C, using glass tubes in a shaking bath. The incubations mixtures contained the yeast microsomal suspension, providing 0.2, 0.2, 0.1, and 0.075 ␮M P450 for CYP 2C8, 2C9, 2C18, and 2C19, respectively, the substrate, and a NADPHgenerating system (1 mM NADP ⫹, 10 mM glucose 6-phosphate, and 2 units of glucose 6-phosphate dehydrogenase/ml) diluted in 0.1 M Tris buffer, pH 7.4, containing 1 mM EDTA and 8% glycerol (final concentrations). Activity assays were routinely initiated (t 0 ⫽ 0 min) by incorporation of the NADPH-generating system into the incubation mixture after 3 min of separate preincubation at 28°C for temperature equilibration. At t 0 and regularly after, aliquots (140 ␮l) were taken and the reaction was quickly stopped by treatment with 70 ␮l of a cold CH 3CN/CH 3COOH (10:1) mixture. As previously reported (20), the 2-aroylthiophene substrates used for measuring CYP 2C8 and CYP 2C9 dependent activities were 3-[2,3-dichloro-4-(2-thenoyl)phenoxy]ethanol and tienilic acid respectively. In the case of CYP 2C18 and CYP 2C19, 3-[2,3-dichloro4-(2-thenoyl)phenoxy]propane-1-ol was used as substrate (20). IC 50s of sulfaphenazole derivatives were determined after addition of variable concentrations of these derivatives to incubates containing 250,

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6, 15, and 100 ␮M substrate of CYP 2C8, 2C9, 2C18, and 2C19, respectively. These concentrations correspond to the Km values of the hydroxylation of these substrates (20). The studied compound dissolved in CH 3OH or DMSO was added to the incubation mixture (final solvent concentration: 1%). The residual activity was plotted versus the inhibitor concentration. IC 50s were estimated by curve fit analyses. Inhibition studies of CYP 2C9 were performed at various concentrations of inhibitors (1–500 ␮M) and five concentrations of TA in the range 10 –100 ␮M. The inhibitor and the subtrate were added simultaneously to the incubation mixture. K i values were derived from analyses of Lineweaver–Burk plots corresponding to the various enzymatic activities in the presence of increasing concentrations of inhibitors.

RESULTS

Synthesis of a Series of Sulfaphenazole Derivatives in Order to Further Explore the CYP 2C9 Active Site In order to determine the relative importance of the three main interactions previously proposed (19) for explaining the high affinity of SPA for CYP 2C9, a series of SPA derivatives have been synthesized. In the first group of compounds (Fig. 1A), the NH 2 group of SPA that binds to CYP 2C9 iron was replaced by various R 1 substituents. Their synthesis generally involved the reaction of 2-phenyl-2H-pyrazol-3-yl-amine with the most appropriate para-substituted benzenesulfonyl chloride, and further transformation of this para-substituent into R 1 , if necessary. In the second group of compounds (Fig. 1B), the phenyl substituent of SPA, that was proposed to give a strong hydrophobic interaction with CYP 2C9 aminoacid residues, was replaced by various R 2 aryl or alkyl groups. Their synthesis was performed in two steps as shown in Fig. 1B. Synthesis of the derivatives of the third group (Fig. 1C), in which the NH 2 and phenyl substituents of SPA were respectively replaced by a methyl and R 2 (aryl or alkyl) group, was based on the reaction of para-methyl benzenesufonyl chloride with the appropriate 2-substituted-2H-pyrazol-3-ylamine (Fig. 1C). Finally, the compounds of the fourth group (Fig. 1D) were obtained by N-alkylation of the SO 2NH function of SPA. Contrary to SPA, they are not anionic at physiological pH. The detailed synthesis of all these compounds as well as that of their 2-substituted 2H-pyrazol-3-yl-amines precursors are described under Materials and Methods. The structure of each compound was completely established from 1H NMR and mass spectrometry and elemental analysis. Only six of these compounds, 1, 2, 8, 9, 16, and 17, have been previously reported in the literature (22, 39, 19, 19, 39, and 40, respectively). Importance of the R 1 Substituent on the Interaction of SPA Derivatives with CYP 2C9 As previously reported (19), addition of SPA to microsomes from yeast expressing human liver CYP 2C9

led to the appearance of a typical type II difference visible spectrum characterized by a peak at 425 nm and a through at 390 nm. This spectrum is due to the formation of a CYP 2C9 iron-NH 2Ar (SPA) complex, with a spectral dissociation constant, K s, of 0.4 ␮M. As expected, compounds 1–7, that no longer contain a NH 2 function, did not lead to any type II interaction with CYP 2C9 (Table I). However, most of them, 2, 3, 5, and 6 (for which R 1 ⫽ CH 3, Br, allyl and CH 2CH 2Br) bound with a high affinity to CYP 2C9, as they produced a type I difference spectrum characterized by a peak at 385 nm and a trough at 420 nm (Table I). It is well established that such a spectrum is due to the loss of the H 2O ligand of the iron caused by the binding of the substrate to the protein in close proximity of the heme (36). The K s values corresponding to the binding of 2, 3, 5, and 6 to CYP 2C9 (between 0.25 and 0.5 ␮M) are very similar to that of SPA. Interestingly, compound 7, that involves a CH 2CH 2OH group, led to a difference spectrum characterized by a peak at 415 nm and a trough at 390 nm, which is called reverse type I and is due to the binding of H 2O or any similar weak oxygencontaining ligand to pentacoordinate high-spin P450 Fe(III) (36). Two other compounds, 1 and 4, also led to reverse type I difference spectra. It is noteworthy that SPA and all compounds 2–7 bind with a very high affinity to CYP 2C9 (K s values between 0.12 and 0.5 ␮M), whatever their mode of binding (type I, II, or reverse type I). Compound 1 for which R 1 ⫽ H exhibits a lower affinity, with a K s value of 1.8 ⫾ 0.5 ␮M. Kinetic studies about the inhibitory effects of compounds 1–7 toward CYP 2C9 showed that they act as competitive inhibitors of this enzyme (data not shown), as SPA itself (19). Table I shows that compounds 2, 3, 4, 5, and 7 act as good inhibitors of CYP 2C9 with K i values between 0.3 and 0.6 ␮M that are very close to the K i previously reported for SPA (19). For most compounds, the K i values are almost identical to the K s values. Only two compounds, 1 and 6, exhibited markedly different K i and K s values. Anyway, compound 1, which showed the highest K s value, was also the worst inhibitor of CYP 2C9. The almost identical K s and K i values found for SPA and for many of the studied compounds such as 2, 3, 4, 5, and 7, which do not contain a NH 2 function, show that the iron-NH 2Ar bond is not a major determinant of the high affinity of SPA for CYP 2C9. Importance of the R 2 Substituent on the Interaction of SPA Derivatives with CYP 2C9 The results of Table I have shown us that SPA and 2 have almost identical affinities and inhibitory potencies towards CYP 2C9, even though they differently bind in the active site (type II and type I interaction, respectively). Therefore, in order to study the relative

INTERACTION OF SULFAPHENAZOLE DERIVATIVES WITH CYP 2Cs

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FIG. 1. Formula and schematic routes of synthesis of the derivatives of sulfaphenazole used in this study. A–D correspond to the four different classes of compounds that have been synthesized. These figures only give a simplified, general view of the synthesis methods used. Detailed syntheses of all compounds are described under Materials and Methods.

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Effects of Various SPA Derivatives on the Visible Spectrum and Tienilic Acid 5-Hydroxylation Activity of Microsomes from Yeast-Expressing CYP 2C9

Compound

R1

Sulfaphenazole 1 2 3 4 5 6 7

ONH 2 OH OCH 3 OBr OCHACH 2 OCH 2OCHACH 2 OCH 2OCH 2OBr OCH 2OCH 2OOH

Difference spectrum a (␭ min ⫺ ␭ max (nm) type d) 390–425 390–415 420–385 420–385 386–418 420–385 420–385 390–415

K s b (␮M) II RI I I RI I I RI

0.4 ⫾ 0.1 1.8 ⫾ 0.5 0.25 ⫾ 0.1 0.38 ⫾ 0.1 0.12 ⫾ 0.05 0.5 ⫾ 0.2 0.34 ⫾ 0.1 0.13 ⫾ 0.07

K i c (␮M) 0.3 5.8 0.3 0.6 0.5 0.5 2.6 0.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.1 0.5 0.1 0.1 0.2 0.2 0.2 0.2

Positions of the through (␭ min) and peak (␭ max) of the difference visible spectrum appearing after addition of each compound to a suspension of microsomes from yeast expressing CYP 2C9 (0.2 ␮M) in 0.1 M Tris, 0.1 mM EDTA, pH 7.4. b Spectral dissociation constants (K s) were obtained by nonlinear regression analysis (KaleidaGraph 3.0). c K i values for the inhibitory effects of each compound toward 5-hydroxylation of TA were determined as described under Materials and Methods. d I, II, and RI mean type I, II, and reverse type I difference spectra. Values are mean ⫾ SD from three to five experiments. a

importance of the pyrazole substituent (R 2 ) on the binding of SPA to CYP 2C9, two classes of molecules have been synthesized. They are derived either from SPA or from 2 by replacement of their N-Ph group by various alkyl and aryl substituents. The presence of a N-alkyl substituent, as in 9, 10, and 16, led to a dramatic decrease of the inhibitory effects towards CYP 2C9 (IC 50 ⬎ 100 ␮M) (Table II). In that regard, the most spectacular result was obtained with compound 10 (R 2 ⫽ cyclohexyl), which exhibits a K i value (170 ⫾ 30 ␮M; data not shown) almost three orders of magnitude higher than SPA. A possible explanation for these very different behaviours of 10 and SPA, despite their similar size and hydrophobicity, would be that the interaction between the N-phenyl group of SPA and CYP 2C9 involves a ␲-stacking interaction with aromatic residues of the protein. This would be in agreement with the results of Table II, which show that all compounds involving a freely rotating N-aryl substituent, such as SPA, 8, 2, 13, and 14, are good inhibitors of CYP 2C9 with IC 50 values about 0.5 ␮M. Compounds 12 and 15, for which the rotation of the N-aryl ring is restricted by the presence of bulky substituents in the vicinity of the N-C bond are bad inhibitors of CYP 2C9. The lack of inhibitory effects of compound 11 (R 2 ⫽ 2,3,5,6-tetrafluorophenyl) (IC 50 ⬎ 100 ␮M) would suggest that the ␲–␲ interaction between the N-aryl group of the SPA derivatives and an aryl group from the protein is weaker with very much electron-deficient N-aryl substituents. The requirement of a freely rotating N-aryl R 2 substituent for efficient recognition by CYP 2C9, that was deduced from inhibition studies, was also indicated by spectral studies performed on representative examples of compounds of Table II. Thus, good CYP 2C9 inhibi-

tors such as SPA, 8 and 2 lead to type II (SPA and 8) and type I (2) difference spectra with very low K s values of 0.4, 0.1, and 0.25 ␮M, respectively (Table I for SPA and 2, and Ref. 19 for 8). Conversely, bad inhibitors of CYP 2C9 such as compounds 10 and 15 (IC 50 values ⬎ 100 ␮M, Table II) led to type II and reverse type I spectra, respectively, with K s values two to three orders of magnitude higher (120 ⫾ 30 and 1000 ⫾ 300 ␮M, respectively; data not shown).

TABLE II

Inhibition of CYP 2C9 by SPA Derivatives Bearing Various R 2 Substituents on the Pyrazole Ring (see Figs. 1B and 1C) Compound R 1 ⫽ NH 2 Sulfaphenazole 8 9 10 11 12 Compound R 1 ⫽ CH 3 2 13 14 15 16

R2

IC 50 a (␮M)

Phenyl 3,4-Dichlorophenyl Ethyl Cyclohexyl 2,3,5,6-Tetrafluorophenyl ␣-Naphthyl

0.6 0.3 ⬎100 b ⬎100 b ⬎100 b 40

R2

IC 50 a (␮M)

Phenyl 3,4-Dichlorophenyl 4-Methoxyphenyl 2,6-Dichlorophenyl Benzyl

0.6 0.6 0.8 ⬎100 b ⬎100 b

a IC 50 values were measured by increasing concentrations of potential inhibitors added to the assay incubation mixture including yeast microsomes expressing CYP 2C9 (0.2 ␮M P450) in 0.1 M Tris buffer, pH 7.4, 6 ␮M TA, and a NADPH-generating system. b Activity higher than 50% of control activity at 100 ␮M. Values are mean from three experiments.

197

INTERACTION OF SULFAPHENAZOLE DERIVATIVES WITH CYP 2Cs TABLE III

Comparison of the Inhibitory Effects of SPA Derivatives Bearing R 3 Substituents (see Fig. 1D) toward Recombinant CYP 2C8, 2C9, 2C18, and 2C19 Expressed in Yeast IC 50 a (␮M) Compound

R3

CYP 2C8

CYP 2C9

CYP 2C18

CYP 2C19

17 18 19

OCH 3 OCH 2OCH 3 OCH 2OCH 2OCH 3

50 13 8

100 90 55

15 7 8

200 225 250

a IC 50 values were measured from increasing concentrations of potential inhibitors added to the assay incubation mixture including yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH 7.4, substrate, and a NADPH-generating system. The experimental details are described under Materials and Methods. Values are mean from three experiments.

Importance of the R 3 Substituent on the Interaction of SPA Derivatives with CYP 2C9 Compounds 17, 18, and 19 bearing a N-alkyl substituent on the SO 2NH function of SPA were prepared (Fig. 1D) in order to evaluate the importance of the presence of a negative charge able to interact with a putative cationic amino acid residue of CYP 2C9 (19) in the inhibitory effects of SPA. Table III clearly shows that the absence of this negative charge in 17, 18, and 19 leads to a dramatic decrease of the inhibitory properties of the corresponding compounds towards CYP 2C9, with an increase of the IC 50 value from 0.6 ␮M to 55, 90, and 100 ␮M. Accordingly, the absence of this negative charge in compound 19 also leads to a dramatic increase of the K s value determined for its interaction with CYP 2C9 from difference visible spectroscopy (K s ⫽ 27 ⫾ 10 ␮M to be compared with 0.4 ␮M for SPA) (data not shown). Comparison of the Inhibitory Effects of the SPA Derivatives Toward CYP 2C9, 2C8, 2C18, and 2C19 The inhibitory effects of the various SPA derivatives toward the other members of the CYP 2C subfamily in human liver, CYP 2C8, 2C18, and 2C19, were compared to those previously found toward CYP 2C9. This study was done by using microsomes of the W(R) fur 1 yeast expressing each of these CYP 2Cs and typical substrates at concentrations equal to K m (see Materials and Methods). Table III shows that compounds 17, 18, and 19 are bad inhibitors not only for CYP 2C9, as mentioned above, but also for CYP 2C19. However, interestingly, compounds 18 and 19 were found to inhibit recombinant CYP 2C8 and CYP 2C18 with IC 50 values between 8 and 13 ␮M. The inhibitory potency of these N-alkylSPA derivatives towards CYP 2C8 increases when increasing the chain length of the alkyl group (IC 50 ⫽ 50 ␮M for R 3 ⫽ CH 3 to IC 50 ⫽ 8 ␮M for R 3 ⫽ CH 2CH 2CH 3). Table IV compares the inhibitory effects of SPA and compounds 2–7 bearing various R 1 substituents (Fig.

1A). The SPA derivatives bearing relatively small R 1 substituents with a one-atom chain (SPA, 2 and 3) appear to be very selective toward CYP 2C9, as the ratio between the IC 50 values for CYP 2C8, 2C18, or 2C19 and the IC 50 value for CYP 2C9 (all IC 50 values were measured for substrate concentrations equal to K M) was always higher than 100 (except in one case, inhibition of CYP 2C18 by 3, for which IC 50 for 2C18/ IC 50 for 2C9 ⫽ 70). Upon increasing the length of the R 1 chain, one keeps strong inhibitory effects toward CYP 2C9 (IC 50 between 0.6 and 4 ␮M) and weak effects on CYP 2C18 and 2C19 (IC 50 ⬎ 100 ␮M, except for 6 and CYP 2C18 for which IC 50 ⫽ 80 ␮M). However significant inhibitory effects towards CYP 2C8 begin to appear. The IC 50 value toward CYP 2C8 even reaches 10 ␮M in the case of 5 that bears an allyl R 1 group, and the IC 50 for 2C8/IC 50 for 2C9 ratio decreases down to 10. Finally, compounds 8 –16 for which R 1 ⫽ NH 2 or CH 3, R 3 ⫽ H, and R 2 ⫽ various alkyl and aryl groups (Figs. 1B and 1C, Table II) failed to inhibit the activities catalyzed by CYP 2C8, 2C18, and 2C19 (IC 50 ⬎ 100 ␮M, data not shown). DISCUSSION

Structural Determinants Required for Efficient Recognition by CYP 2C9 Nineteen derivatives of the classical CYP 2C9 inhibitor, SPA, bearing various R 1 , R 2 and R 3 substituents (Fig. 1) have been synthesized in order to further explore the CYP 2C9 active site and to determine the interactions with the protein that are responsible for the high affinity of SPA toward CYP 2C9. The dramatic decrease of the inhibitory effects toward CYP 2C9 observed after N-alkylation of the SPA sulfonamide function (compounds 17, 18, and 19, Table III) clearly shows that the presence of a SO 2NH or SO 2N ⫺ (due to deprotonation at physiological pHs) function is required for efficient recognition by CYP 2C9. This should correspond to the ionic interaction

198

HA-DUONG ET AL. TABLE IV

Comparison of the Inhibitory Effects of SPA Derivatives Bearing Various R 1 Substituents toward Recombinant CYP 2C8, 2C9, 2C18, and 2C19 Expressed in Yeast IC 50 a (␮M) Compound

R1

CYP 2C8

CYP 2C9

CYP 2C18

CYP 2C19

Sulfaphenazole 2 3 4 5 6 7

ONH 2 OCH 3 OBr OCHACH 2 OCH 2OCHACH 2 OCH 2OCH 2OBr OCH 2OCH 2OOH

130 220 200 50 10 80 110

0.6 0.6 1.2 1 1 4 0.6

60 170 80 125 150 80 ⬎200 b

⬎500 b 100 200 500 450 ⬎500 b 300

a

IC 50 values were measured from increasing concentrations of potential inhibitors added to the assay incubation mixture including yeast microsomes expressing CYP 2C in 0.1 M Tris buffer, pH 7.4, substrate, and a NADPH-generating system. The experimental details are described under Materials and Methods. b CYP 2C activity higher than 50% of control activity, at the indicated inhibitor concentration. Values are mean from three experiments.

between such a negative charge and a putative cationic amino acid residue of the protein that has been proposed in several models of CYP 2C9-substrate (or inhibitor) complexes (9 –12, 15). This could also correspond to an hydrogen bond between the SO 2NH function of CYP 2C9 inhibitors and an hydrogen bond acceptor from CYP 2C9 (8, 11–13). Because of the pK A of the SPA sulfonamide function (⬃6.2) (41), this second possibility seems less likely; however, it may be particularly important for neutral CYP 2C9 substrates. The results of Table II show that the second structural factor that appears to be crucial for an efficient recognition of SPA derivatives by CYP 2C9 is the presence of a reasonably electron-rich and freely rotating N-aryl substituent on the pyrazole ring. In previous models proposed for CYP 2C9-substrate (or inhibitor) complexes, the requirement of a strong hydrophobic interaction between CYP 2C9 and a hydrophobic zone of the substrates has been very often mentioned (8 –15, 19). Recent studies using site directed mutants of CYP 2C9 have shown that Phe114 plays an important role in recognition of substrates and SPA by CYP 2C9 (42). Mutation of this residue into leucine increased the K i of SPA from 0.32 to 60 ␮M. This has led the authors to propose that a ⌸–⌸ interaction between the phenyl rings of SPA and CYP 2C9 Phe114 is involved in SPA recognition. The results of Table II are easily explained on the basis of this interaction. They show that the presence of a freely rotating aryl R 2 substituent, which may well establish such a ⌸–⌸ interaction, is required for selective recognition by CYP 2C9. Moreover, recent X-ray structure data on a genetically modified P450 of the 2C family, CYP 2C5, clearly showed that Phe114 is well located in the distal region of the heme, in the substrate binding site (43). The comparison of the effects of two compounds, SPA (R 2 ⫽ Ph) and 10 (R 2 ⫽

cyclohexyl), which exhibit very similar size and hydrophobicity, is particularly illustrative. Both compounds bind to CYP 2C9 iron via their NH 2 function, as shown by the appearance of type II difference spectra. However, the affinity of 10 for CYP 2C9 is two to three orders of magnitude lower than that of SPA, as shown by the K s (120 instead of 0.4 ␮M) and K i values (170 instead of 0.3 ␮M) of these two compounds. An analysis of this kind of interactions between aromatic rings in proteins has shown that the distance between the ring centers should be between 4.5 and 7.0 Å and the dihedral angle between 50 and 90° (44). Such geometric constraints could explain why compounds 12 and 15, which contain N-aryl substituents with restricted free rotation because of bulky substituents in the vicinity of the N-C bond, are much weaker inhibitors of CYP 2C9 than SPA, 8, 13, and 14. The weak inhibitory effects of compound 11, for which R 2 ⫽ 2,3,5,6-tetrafluorophenyl, could be due to low electron density of its R 2 substituent, when compared to SPA, 8, 13, and 14, which would disfavor ␲–␲ interaction with CYP 2C9 Phe114. All SPA derivatives whose structure permits the two interactions discussed above—i.e., the ionic and ␲–␲ interactions—are good inhibitors of CYP 2C9. This is true for compounds 2–7, 8, 13, and 14, which exhibit IC 50 values between 0.3 and 4 ␮M (Tables II and IV). Their high affinity for CYP 2C9 is confirmed by their dissociation constants, K s, which have been determined by visible spectroscopy and that vary between 0.1 and 2 ␮M (Table I). Conversely, SPA derivatives whose structure does not favor the two interactions discussed above, such as 10, 15 (Table II), and 19 (Table III), lead to much higher K s values (120 ⫾ 30, 1000 ⫾ 300 and 27 ⫾ 10 ␮M), in agreement with their high IC 50 values (⬎55 ␮M).

INTERACTION OF SULFAPHENAZOLE DERIVATIVES WITH CYP 2Cs

199

leading generally to type I spectra (2, 3, 5, and 6) and sometimes to reverse type I spectra (1 and 4). The type I spectra observed with 2, 3, 5, and 6 are easy to understand, as it is generally admitted that binding of hydrophobic residues close to P450 iron leads to a dissociation of the iron-OH 2 bond and formation of high-spin pentacoordinate iron(III). Naturally, the model of CYP 2C9-inhibitor complexes shown in Fig.2, which assumes that all the SPA derivatives bind in the same manner to CYP 2C9, remains to be established. The low affinity observed for N-alkylated compounds and for SPA derivatives involving an alkyl R 2 substituent could also be interpreted by changes of the inhibitor conformation. However, a relatively great number of molecules with quite different R 1 , R 2 , and R 3 substituents have been compared in this study, and it is noteworthy that most results can be easily explained on the basis of the model of Fig. 2. Moreover, this model is in agreement with previously published site-directed mutagenesis data (42). FIG. 2. Schematic view of the binding mode of SPA derivatives in the active site of CYP 2C9. This figure illustrates that SPA derivatives leading to the ionic and ␲–␲ interactions with residues of the CYP 2C9 active site are positioning their R 1 substituent above the heme close to the iron. Conformation of SPA is in agreement with previous reports (47). The type of difference spectra observed and the possible corresponding iron-ligand bond are also indicated.

Interestingly, the high affinity of the SPA derivatives 2– 8 toward CYP 2C9 was observed whatever their type of spectral interaction with CYP 2C9. Thus, SPA (R 1 ⫽ NH 2), 2 (R 1 ⫽ CH 3), and 7 (R 1 ⫽ CH 2CH 2OH), that lead to type II, type I or reverse type I difference spectra respectively, all exhibit K s values between 0.13 and 0.4 ␮M. This result shows that the iron-nitrogenous ligand bond observed with SPA is not a key factor explaining the high affinity of this compound for CYP 2C9. This result is also a good evidence that the binding of these inhibitors in the CYP 2C9 active site leads to a positioning of their R 1 substituent above the heme in close proximity to the iron (Fig. 2). In that situation, the type of difference spectrum observed will depend on the nature of R 1 . If R 1 is a potential good iron ligand such as NH 2, it will bind to the iron leading to a type II difference spectrum as in the case of SPA itself. If R 1 may act as a weak oxygencontaining ligand, it will bind to high-spin CYP 2C9 Fe(III) possibly leading to a reverse type I spectrum. This seems to occur for compound 7 the R 1 ⫽ CH 2CH 2OH substituent of which is well located to establish an iron-oxygen bond. Finally, in the absence of any potential iron ligand in R 1 , as in 1, 2, 3, 4, 5, and 6, binding to the protein close to iron will modify the position of the spin state equilibrium of CYP 2C9 iron,

Selectivity of the SPA Derivatives as Inhibitors of Human CYP 2Cs None of the 20 compounds that have been compared as inhibitors of human CYP 2Cs in this study was able to inhibit CYP 2C19 at a reasonable level—i.e., their IC 50 values for this isozyme were greater than 100 ␮M. As mentioned above, all compounds that are able to give the two major interactions with CYP 2C9 shown in Fig. 2, are good inhibitors of this P450 (Tables I and II). However, only those having a relatively small R 1 substituent are selective toward CYP 2C9 (Table IV). Some of those which bear more extended substituents (R 1 ⫽ vinyl, allyl, or CH 2CH 2Br) begin to be relatively good inhibitors of CYP 2C8 (IC 50 between 10 and 80 ␮M). N-Alkylation of the sulfonamide function of SPA leads not only to a dramatic decrease of the inhibitory effects toward CYP 2C9, but also to the appearance of marked inhibitory effects towards CYP 2C8 and CYP 2C18, with IC 50 values between 7 and 50 ␮M, whereas CYP 2C19 is not touched (Table III). The high affinity of 19 for CYP 2C8, which was suggested by its IC 50 value of 8 ␮M (Table III), was further confirmed by a study of the interaction of 19 with CYP 2C8 by difference visible spectroscopy. The appearance of a type II difference spectrum (␭ max ⫽ 425 nm and ␭ min ⫽ 395 nm) should result from the binding of the NH 2 group of 19 to CYP 2C8-Fe(III); it occurs with a K s value of 4 ⫾ 1 ␮M (data not shown). Thus, the aforementioned results that concern a first series of 20 derivatives of SPA allowed us to find three new potent and selective inhibitors of CYP 2C9, compounds 2, 3, and 7, as well as molecules, 18 and 19,

200

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