Bioorganic & Medicinal Chemistry 22 (2014) 6655–6664

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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Rational design of novel CYP2A6 inhibitors Niina Tani a,⇑, Risto O. Juvonen a, Hannu Raunio a, Muluneh Fashe a, Jukka Leppänen a, Bin Zhao b, Rachel F. Tyndale b, Minna Rahnasto-Rilla a,⇑ a b

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, POB 1627, 70211 Kuopio, Finland Departments of Pharmacology and Toxicology and Psychiatry, University of Toronto, Campbell Family Mental Health Research Institute, M5S 1A8 Toronto, Ontario, Canada

a r t i c l e

i n f o

Article history: Received 18 June 2014 Accepted 2 October 2014 Available online 13 October 2014 Keywords: Cytochrome P450 (CYP) CYP2A6 Nicotine Inhibition Smoking reduction therapy

a b s t r a c t Inhibition of CYP2A6-mediated nicotine metabolism can reduce cigarette smoking. We sought potent and selective CYP2A6 inhibitors to be used as leads for drugs useful in smoking reduction therapy, by evaluating CYP2A6 inhibitory effect of novel formyl, alkyl amine or carbonitrile substituted aromatic core structures. The most potent CYP2A6 inhibitors were thienopyridine-2-carbaldehyde, benzothienophene-3-ylmethanamine, benzofuran-5-carbaldehyde and indole-5-carbaldehyde, with IC50 values below 0.5 lM for coumarin 7-hydroxylation. Nicotine oxidation was effectively inhibited in vitro by two alkyl amine compounds and benzofuran-5-carbonitrile. Some of these molecules could serve as potential lead molecules when designing CYP2A6 inhibitory drugs for smoking reduction therapy. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Tobacco smoking increases the risk for numerous types of cancer, as well as cardiovascular and respiratory diseases, duodenal and gastric ulcers, osteoporosis and diabetes, resulting in a significantly shorter life expectancy in smokers relative to non-smokers.1,2 Most smokers would like to quit smoking and many attempt to do so, but they often fail to quit, and/or resume smoking after some time.1 The primary addictive agent in cigarettes and smokeless tobacco (chewing tobacco, snuff, nicotine gum) is nicotine. Nicotine is a bicyclic alkaloid with pyridine and pyrrolidine rings. It binds to nicotine acetylcholine receptors in the brain evoking psychoactive effects, including pleasure and feelings of relaxation. Smoking also helps to control mood and concentration.3 Nicotine metabolism is two-step reaction, where nicotine is first oxidized in a rate-limiting step to nicotine-D50 (10 )-iminium ion, mainly by the cytochrome P450 (CYP) 2A6 enzyme, and then to cotinine by cytosolic aldehyde oxidase.4 The CYP2A6 gene is polymorphic, and several loss-of-function alleles lead to inactivity or reduced activity of the enzyme (www.cypalleles.ki.se/cyp2a6.htm). Genetic variation in this enzyme alters many smoking behaviors. For example, individuals with reduced or loss of function variants associated with 50% ⇑ Corresponding authors. Tel.: +358 40 355 2011; fax: +358 17 162424 (N.T.); tel.: +358 40 355 3786; fax: +358 17 162424 (M.R.-R.). E-mail addresses: Niina.Tani@uef.fi (N. Tani), Minna.Rahnasto@uef.fi (M. Rahnasto-Rilla). http://dx.doi.org/10.1016/j.bmc.2014.10.001 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

CYP2A6 activity in vivo are more likely to be non-smokers than their counterparts with normal CYP2A6 activity;5 smoking cessation trials have also demonstrated that smokers who have slower CYP2A6 activity have better cessation rates.6,7 It has been suggested that inhibition of CYP2A6 activity, that is, phenocopying the reduced activity genotype, could help smokers to both reduce how much they smoke and help them quit smoking. Pilot studies with nonspecific CYP2A6 inhibitors have yielded promising results.8–11 Most compounds that inhibit CYP enzymes are reversible competitive inhibitors. In this case the interaction between the inhibitor and enzyme occurs via non-covalent bonds, and the inhibition is transient. Mechanism based irreversible CYP inhibition causes longer duration enzyme inactivation. Here stable covalent bonds are formed between the inhibitor and the enzyme active site, and the recovery of enzyme activity requires de novo synthesis of enzyme. Irreversible inhibitors contain specific substructures that are transformed to reactive electrophilic functional groups in the enzyme active site, reacting with nucleophilic amino acid residues or protoporphyrin IX (heme). Numerous compounds with CYP2A6 inhibiting properties have been discovered.12–16 We have used several different chemical scaffolds to obtain compounds with more potent and selective inhibitory properties. Thus far, we have obtained these compounds from commercial sources, including extensive chemical libraries (e.g. Maybridge). We have shown previously that the benzothiophene core with acceptor atoms evoked potent CYP2A6 inhibition.17,18

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Figure 1. Refining of the novel compounds. Hydrogen bonding and p–p-stacking sites are illustrated as light blue and light green half-moon shapes, respectively. The main interacting amino acid residues (Phe107, Phe118, Asn297) and the heme moiety are illustrated as spheres. The reference compound: R = NH2, X = S. Main scaffolds: R = NH2, NHCH3, N(CH3)2, CH2O, X = S, O, NH.

The main aims of this study were to expand the chemical spectrum of known CYP2A6 inhibitors, and to clarify the optimal steric and electrostatic interactions occurring between the inhibitor and the enzyme active site. A further goal was to discover irreversible inhibitors of CYP2A6. We used the previously identified benzothiophene, benzofuran and benzoindole cores and used molecular modeling to detect optimal interactions with the enzyme active site (Fig. 1). Novel thienopyridine derivatives were synthesized based on this information. Two molecules showed the desired properties and will be taken to further development as lead compounds. 2. Materials and methods 2.1. Chemicals 5-Chloro-1-benzothiophen-3-yl)methylamine hydrochloride (compound 3), N-(1-benzothien-2-ylmethyl)-N-methylamine hydrochloride (4), 5-bromo-1-benzothiophen-3-yl)methylamine (5), 1-benzothiophene-5-carbaldehyde (6), N-methyl-1-(thieno[2,3-b]pyridin-2-ylmethyl)amine (10), 1-benzothiophene-2carbaldehyde (11), 5-methyl-1-benzothiophene-2-carbaldehyde (12), 5-bromobenzo[b]thiophene-3-carbaldehyde (13), thieno [3,2-c]pyridine-3-carbothioamide (15), thieno[2,3-b]pyridine-2carbaldehyde (16), benzo[b]thiophene-7-carbaldehyde (17), 1-benzothiophen-2-ylmethanol (18), 1-benzothiophen-3-ylmethanol (19), 5-chloro-1-benzothiophene-3-carbonitrile (20), 2-(5-chloro-

benzo[b]thiophen-3-yl)acetonitrile (21), 5-chloro-3-methylbenzo[b]thiophene (22), 1-benzothiophene-3-carbonyl chloride (23), 1-benzofuran-5-carbaldehyde (24), benzofuran-5-carbonitrile (25) 1-benzofuran-2-carbaldehyde (26), 2-benzofuran-carbonitrile (27) 5-bromo-1-benzofuran-2-carbaldehyde (28), Benzo[b]furan2-carboxaldehyde (29), (5-bromobenzo[b]furan-2-yl)methylamine (30), 2-(benzo[b]thiophen-3-yl)acetaldehyde (31), 2-bromo-1-benzofuran (32), were all purchased from Maybridge, Great Britain. 1Hindole-5-carbaldehyde (33), 1-methyl-1H-indole-5-carbaldehyde (34), 1H-indole-6-carbaldehyde (35), 5-bromo-1H-indole (36), 1methyl-1H-indole-6-carbaldehyde (37), 1H-indole-2-carbaldehyde (38), 4-bromo-1H-indole-3-carbaldehyde (39), 2-methyl-1H-indol5-amine (40), 6-bromo-1H-indole-3-carbaldehyde (41), 1H-indole3-carbaldehyde (42), 4-ethylbenzonitrile (43) and p-tolunitrile (44) were all purchased from Sigma Aldrich, USA. Thieno[3,2-c]pyridine-2-carbaldehyde (1), thieno[2,3-c]pyridine-2-carbaldehyde (2), thieno[3,2-c]pyridin-2-ylmethanamine (7), N,N-dimethyl-1-(thieno[3,2-c]pyridin-2-yl)methanamine (8), N-methyl-1-(thieno[3,2-c]pyridin-2-yl)methanamine (9) and thieno[2,3-c]pyridin-2-ylmethanamine (14) were synthesized in-house (see Scheme 1). The synthesis reactions were monitored by thinlayer chromatography using aluminum sheets coated with silica gel 60 F245 (0.24 mm) with UV and ninhydrin visualization. Purifications by flash chromatography were performed either on a Combiflash Companion Instrument with RediSep Columns (Teledyne ISCO, Lincoln, CA, USA) or by normal column chromatography with silica gel 60 (0.063–0.200 mm mesh). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance AV 500 spectrometer (Bruker Biospin, Fallanden, Switzerland) operating at 500.13 MHz using the solvent peak as an internal standard. Furthermore, the products were characterized by mass spectrometry with a Finnigan LCQ quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an electrospray ionization source, and the purity was determined by elemental analysis (C, H, N) with a ThermoQuest CE Instruments EA 1110-CHNS-O elemental analyzer (CE Instruments, Milan, Italy). All compounds tested in the assays were characterized by combustion analysis to be of at least 95% of purity unless otherwise stated. Thieno[3,2-c]pyridine-2-carbaldehyde (1). To the cooled to 78 °C mixture of thieno[3,2-c]pyridine (1.3 g, 9.62 mmol) in dry tetrahydrofuran (40 ml), n-butyllithium (1.6 M in hexane, 7.51 ml) was added dropwise. Dimethylformamide (0.88 g, 12 mmol) was added to the solution after 20 min stirring and the mixture was allowed to warm up to room temperature and was treated with saturated ammonium chloride solution. The mixture was extracted with diethyl ether and the organic phase was dried with sodium sulfate. The product was purified by column chromatography using 1:1 petroleum ether/ethyl acetate as an eluent to give 1.2 g (76%) of a yellowish solid. 1H NMR CDCl3: d 7.83 (d, 1H), 8.13 (s, 1H), 8.57 (d, 1H), 9.25 (s, 1H), 10.14 (1H, s). ESI-MS: 164.07 (M+1). Anal. Calcd for C8H5NOS: C, 58.88; H, 3.09; N, 8.58; found C, 58.92; H, 3.23; N, 8.29.

R1 Y Z

a S

N

O b or c Y d,e

Y Z

S 1 Y=N, Z=CH 2 Y=CH, Z=N

H

Z

S

R2

H

7 Y=N, Z=CH, R1, R2 =CH3 8 Y=N, Z=CH, RI=CH3, R2=H 9 Y=N, Z=CH, R1, R2 =H 10 Y=CH, Z=N, R1, R2=H

Scheme 1. Synthesis of 1,2: (a) n-BuLi, DMF; 7: (b) Me2NH2, NaCNBH3, AcONa, MeOH; 8: (c) MeNH2, NaBH4 EtOH; 9, 10: (d) NH2OH*HCl, EtOH/Pyridine (e) Zn, AcOH/MeOH.

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Thieno[2,3-c]pyridine-2-carbaldehyde (2). Thieno[2,3-c]pyridine (330 mg, 2,44 mmol) was reacted and purified similar method to (1) to give 200 mg (50%) of a yellowish solid. 1H NMR CDCl3: d 7.79 (d, 1H), 8.03 (s, 1H), 8.58 (d, 1H), 9.23 (s, 1H), 10.18 (1H, s). ESI-MS: 164.07 (M+1). Anal. Calcd for C8H5NOS: C, 58.88; H, 3.09; N, 8.58; found C, 58.82; H, 3.14; N, 8.28. N,N-Dimethyl-1-(thieno[3,2-c]pyridin-2-yl)methanamine (8). Thieno[3,2-c]pyridine-2-carbaldehyde (73 mg, 0.45 mmol), sodium cyanoborohydride (31 mg, 0.49 mmol), dimethylamine hydrochloride (77 mg, 0.94 mmol) and sodium acetate (55 mg, 0.67 mmol) were dissolved in methanol (8 ml) and stirred for 24 h. Ethyl acetate (20 ml) was added and the mixture was acidified with 6 N HCl and washed twice with saturated NaHCO3 solution. Organic phase was dried and evaporated and the product was purified by column chromatography using dichloromethane/MeOH 20–100% to yield 70 mg (59%) of a white solid. 1H NMR CD3OD: d 2.3 4 (s, 6H), 4.91 (s, 2H), 7.43 (d, 1H), 7.95 (dd, 1H), 8.34 (m, 1H), 8.97 (s, 1H). ESI-MS: 193.06 (M+1). Anal. Calcd for C10H12N2S*2HCl: C, 45.29; H, 5.32; N, 10.56; found C, 46.95; H, 4.40; N, 8.50. N-Methyl-1-(thieno[2,3-c]pyridin-2-yl)methanamine, (9) Thieno[2,3-c]pyridine-2-carbaldehyde (43 mg, 0.26 mmol) and methylamine 8 M in EtOH (65 ll, 0.52 mmol) were dissolved in ethanol (8 ml) and stirred for 18 h in sealed vial. Sodium borohydride (11 mg, 0.31 mmol) was added to the vial and the stirring was continued for 2 h. Ethyl acetate (20 ml) was added and the mixture was washed twice with saturated NaHCO3 solution. The organic phase was dried, evaporated and the product was purified by column chromatography using dichloromethane/MeOH from 0% to 20% to yield 41 mg (63%) of a white solid. 1H NMR CD3OD: d 2.5 4 (s, 3H), 4.11 (s, 2H), 7.20 (s, 1H), 7.57 (d, 1H), 8.48 (d, 1H), 9.07 (s, 1H). ESI-MS: 179.06 (M+1). Anal. Calcd for C9H10N2S*2HCl: C, 42.42; H, 4.79; N, 10.99; found C, 42.78; H, 4.44; N, 10.48. Thieno[3,2-c]pyridin-2-ylmethanamine (7). Thieno[3,2-c]pyridine-2-carbaldehyde (50 mg, 0.31 mmol) and hydroxylamine hydrochloride (32 mg, 0.46 mmol) were dissolved in ethanol (4 ml) and pyridine (1 ml) reacted at 80 °C for 20 min. The mixture was evaporated to dryness and the solid was dissolved in MeOH (10 ml) and AcOH (10 ml) and reacted with zinc powder (98 mg) for 1 h. The mixture was filtered and evaporated to dryness. The product was purified by column chromatography using dichloromethane/MeOH 20% and was washed with hexane to yield 55 mg (82%) of a white solid. 1H NMR CD3OD: d 1.9 4 (s, 3H, AcOH), 4.31 (s, 2H), 7.55 (s, 1H), 7.97 (d, 1H), 8.36 (d, 1H), 9.00 (s, 1H). ESI-MS: 164.98 (M+1). Anal. Calcd for C8H8N2S1.3*AcOH: C, 52.55; H, 5.49; N, 11.56; found C, 52.52; H, 5.12; N, 11.59. Thieno[2,3-c]pyridin-2-ylmethanamine (14), thieno[2,3-c]pyridine-2-carbaldehyde (80 mg, 0.49 mmol) was treated similar method as (7) to give 90 mg (65%) of a white solid. 1H NMR CD3OD: d 1.84 (s, 3H, AcOH), 4.21 (s, 2H), 7.37 (s, 1H), 7.69 (d, 1H), 8.30 (d, 1H), 8.97 (s, 1H). ESI-MS: 165.07 (M+1). Anal. Calcd for C8H8N2S*AcOH: C, 53.55; H, 5.39; N, 12.49; found C, 53.33; H, 5.31; N, 12.34.

IC50-values: No.1-44

The characterization by 1H NMR, mass spectrometry (MS), and high-performance liquid chromatography (HPLC) methods confirmed that the purities were greater than 95%. ()-Nicotine hydrogen tartrate and ()-cotinine were purchased from Sigma-Aldrich (St. Louis, MO). The internal standard 5-methylcotinine was custom-made by Toronto Research Chemicals (Toronto, ON). Tris–hydrochloric acid (MP Biomedicals LLC, USA), Tris (MP Biomedicals LLC, USA), magnesium chloride hexahydrate (Riedel-de HaenÒ, Germany), coumarin (Sigma Aldrich, USA), 7-hydroxycoumarin (Aldrich), trichloroacetic acid (TCA) (Sigma– Aldrich), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) (Darmstadt, Germany), dimethyl sulfoxide (DMSO) (J.T. Baker). 2.2. Biological materials Human liver samples were obtained from patients undergoing surgery to remove hepatic tumors.19 The use of surplus tissue was approved by the Ethics Committee of the University of Kuopio. Liver samples were frozen in liquid nitrogen and stored at 70 °C. Only tumor-free tissue was used for the experiments. Baculovirusinsect cell-expressed human CYP2A6 was purchased from BD Biosciences Discovery Labware (Bedford, MA, USA). 2.3. Determination of IC50 and Ki values The IC50 values for inhibition of coumarin 7-hydroxylation were determined as described previously.20 The Ki values were measured or calculated (Ki = IC50/(1 + S/Km)) for the most potent CYP2A6 inhibitors. The concentrations of the inhibitors used in this experiment were the same as those for the IC50 assay. The reaction was initiated with the addition of NADPH (final concentration 0.33 mM) and the reaction mixture was preincubated for 10 min at 37 °C and stopped with the addition of 10% TCA. The fluorescence of the product 7-hydroxycoumarin was measured immediately after addition of 140 lL of 1.6 M glycine-NaOH at excitation and emission wavelengths of 355 nm 450 nm, respectively. 2.4. Determination of inhibition type The IC50 shift assay detects both reversible and time-dependent inhibitors. The effect of preincubation on the IC50 values of all the compounds was determined (Fig. 2). The reaction pool (50 mM Tris–HCl, 5 mM MgCl2, 0.33 mM NADPH and 1 pmol cDNA expressed CYP2A6 enzyme), was preincubated (without or with the NADPH-generating system) for 0–60 min at 37 °C. After preincubation, coumarin (final concentration 10 lM) was added and the mixture was further incubated for 10 min with cofactors at 37 °C. The reactions were stopped with 60 ll of 10% TCA. The remaining enzyme activity in the presence of inhibitor was measured in a Victor2 fluorometer (Perkin Elmer) immediately after adding 140 lL of 1.6 M (pH 10.4) glycine buffer.

Preincubaon test II: irreversible inacvaon Preincubaon test I: IC50 shi

IC50 Shi: No. 11, 13, 16, 24, 25, 38

Catalyc reacon study

Figure 2. Scheme of the tests to elucidate mechanism of inhibition.

Inacvaon: No. 16, 24, 25, 38

Slow-binding No.11-13

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For determining an irreversible mode of inhibition, five concentrations of the test compounds (plus a vehicle control) were preincubated for various times (including 0 min) with 5–20 pmol cDNA-expressed CYP2A6 and NADPH (Fig. 2). The reaction mixture was diluted 10–40 fold with 50 mM Tris–HCl buffer (pH 7.4), 5 mM MgCl2, 10 lM coumarin, and 0.3 mM NADPH, and the incubation was continued for 10 min at 37 °C. The reaction was terminated and measured as described for the IC50 assay. From the data generated, two kinetic constants, the maximum inactivation rate constant (Kinact) and the inhibitor concentration that produced half maximal rate of inactivation (Ki) were determined using linear regression of double reciprocal of values of observed inactivation rate constant (Kobs) against [I] where Ki is the negative reciprocal value of x-intercept and Kinact is the reciprocal of the value of y-intercept. Kobs values were determined from the initial slope of the linear regression of the natural log (ln) of enzyme activity remaining against preincubation time. 2.5. Nicotine oxidation Nicotine metabolizing activity was analyzed as previously described21 where drug concentrations are expressed as the free base. For expressed CYP2A6 (containing P450 reductase and cytochrome b5) (BD Biosciences, Mississauga, Canada), the linear conditions of nicotine metabolism were obtained under assay conditions of 10 pmol CYP enzyme and 10 pmol cytochrome b5/ml with an incubation time of 15 min. All incubation mixtures contained 1 mM NADPH and 50 ll cytosol in 50 mM Tris–HCl buffer, pH 7.4 and were performed at 37 °C in a final volume of 0.5 ml. The reactions were stopped with a final concentration of 4% v/v Na2CO3. After incubation, 5-methylcotinine (70 lg) was added as the internal standard and the samples were prepared and analyzed for nicotine and metabolites by HPLC system as described previously.22 The limits of quantification were 5 ng/ml for nicotine, 12.5 ng/ml for cotinine and 10 ng/ml for 30 -hydroxycotinine. 2.6. Data analysis The data were analyzed using GraphPad Prism software v5.01. Enzyme inactivation kinetic values (IC50, Ki, Km, Vmax) were determined with the GraphPad Prism software using nonlinear modes and various enzyme kinetics equations. For the estimation of IC50 values, the fluorescence was measured at different concentrations of the inhibitors. Control incubations (blanks) were carried out without the substrate, enzyme, or inhibitor. The blank values were deducted from all groups and the percentage of remaining activity was calculated at each inhibitor concentration taking control group values as 100% enzyme activity.

A

2.7. Modeling The crystal structure of CYP2A623 was obtained from the Protein Data Bank (PDB) database (pdb: 2FDW). The structure and the test compounds were further refined as described previously.18 All compounds were docked to the active site of this refined CYP2A6 structure CYP2A6 using the Sybyl X 1.224 with SurflexDock module.25 The docking procedure was allowed to produce up to 30 different docking poses for each inhibitor. The poses were then ranked based on CScore (consensus score)26 ranking values and visual inspection. 3. Results 3.1. Selection of compounds Selection of novel inhibitor molecules was initiated by docking the previously identified inhibitors, including benzothiophene derivatives17,18 to the active site of CYP2A6. The main interactions between the inhibitors and the active site residues were hydrogen bonding with Asn297 and p–p stacking with phenylalanines 107 and 118 (Fig. 1). To identify potential irreversible inhibitors of CYP2A6, compounds with known inactivating substructures were screened. These substructures were expected to form covalent interactions and thus inactivate the enzyme. Figure 3A illustrates the docking pose of the reference compound benzo[b]thiophen2-ylmethanamine18 that has otherwise an optimal structure but lacks a proper acceptor atom and thus did not form a hydrogen bond with Asn297. Many of the purchased novel compounds did not form desired hydrogen bonding interactions. To identify potent inhibitors with a properly placed acceptor atom, compounds 1, 2, 7, 8, 9, and 14 were synthesized (Table 1). To optimize the position of the acceptor atom, the initial benzothiophene core was replaced with a thienopyridine with formyl or alkyl amine substitutions at position two, and a nitrogen atom at position five or six. The docking poses showed that the nitrogen atom of the thienopyridine with formyl substitution formed a hydrogen bond with residue Asn297 (Fig. 3B). The alkyl amine substituent in most of the thienopyridine derivatives was coordinated towards heme although for most compounds Asn297 was too far for hydrogen bonding interaction (Fig. 3C). 3.2. Inhibition of coumarin 7-hydroxylation Basic coumarin 7-hydroxylation inhibition kinetic parameters were determined for 44 compounds (Table 1). The IC50 values varied from 0.3 lM to more than 100 lM. The core structure as well as the type and position of substituents affected the IC50 value. The most potent inhibitors (IC50 60.5 lM) were the formyl derivatives of thiophene, benzofuran and indole, for example, compounds 1, 2,

B PHE 107

C PHE 107

PHE 118

PHE 107

PHE 118

PHE 118

ASN 297 2.3 Å

HEME

HEME

3-4 Å

ASN 297 HEME

Figure 3. Main interactions between CYP2A6 inhibitors and residues at the enzyme active site. Light blue sphere with dotted line, hydrogen bond interaction; light green sphere, p–p-stacking interaction; grey sphere, heme interaction. (A) Reference compound; (B) Compound 8; C, compound 1. The images are 2-dimensional projections of the 3-dimensional docking poses; the distances are not in correct scale. The average distance between the nitrogen atom and heme Fe was 2.3 Å for 1 and 3–4 Å for 8.

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N. Tani et al. / Bioorg. Med. Chem. 22 (2014) 6655–6664 Table 1 Structures of compounds and IC50 values for coumarin 7-hydroxylation No.

Structure

IC50a (lM)

Effect of preincubationb

Ki (lM)

0.10 ± 0.04

No effect

0.01

0.30 ± 0.03

No effect

0.07

0.40 ± 0.03

No effect

0.06

0.40 ± 0.1

No effect

0.09

0.50 ± 0.06

No effect

ND

0.70 ± 0.1

No effect

0.2

0.90 ± 0.4

No effect

ND

1.0 ± 0.6

No effect

ND

1.4 ± 0.2

No effect

0.6

1.5 ± 0.3

No effect

ND

2.2 ± 0.8

No effect

0.8

3.2 ± 1.5

0.8;

ND

3.4 ± 0.4

No effect

ND

5.0

0.5;

ND

5.2

No effect

ND

6.6 ± 1.6

No effect

ND

NH2

Reference compound S

Benzothiophenes and thienopyridines O

N *

1

S O *

2

N

S NH2

Cl

3 S

NH

4 S

NH2 Br

5 S O

6 S

NH2

N *

7

S

8*

N

N

S

HN

N

9* S

NH

10 N

S

O

11 S O

12 S O Br

13 S

NH2

14* N

S

H2N S

15

N

S

(continued on next page)

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Table 1 (continued) No.

Structure

IC50a (lM)

Effect of preincubationb

Ki (lM)

8.0 ± 1.4

0.4 ± 0.05;

ND

11 ± 1.1

No effect

ND

18 ± 1.6

No effect

ND

37 ± 4.2

No effect

ND

41 ± 17

No effect

ND

51 ± 15

No effect

ND

88 ± 10

No effect

ND

270

ND

ND

0.40 ± 0.09

0.05;

0.07

1.6 ± 0.3

0.6;

0.1

17 ± 25

ND

ND

52 ± 13

ND

ND

72 ± 33

ND

ND

Rational design of novel CYP2A6 inhibitors.

Inhibition of CYP2A6-mediated nicotine metabolism can reduce cigarette smoking. We sought potent and selective CYP2A6 inhibitors to be used as leads f...
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