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Available online at www.sciencedirect.com

Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 18–29

Synthesis, biochemical evaluation of a range of potent 4-substitutedphenyl alkyl imidazole-based inhibitors of the enzyme complex

17�-Hydroxylase/17,20-Lyase (P45017�)

Imran Shahid a, Chirag H. Patel a, Sachin Dhanani b, Caroline P. Owen a, Sabbir Ahmed a,∗a Department of Pharmacy, School of Pharmacy and Chemistry, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK

b School of Life Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK

Received 22 May 2007; accepted 19 October 2007

bstract

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitorsf the two components of the cytochrome P-450 enzyme 17�-hydroxylase/17,20-lyase (P45017�), i.e. 17�-hydroxylase (17�-OHase) and 17,20-yase (lyase). The results suggest that the compounds synthesised are potent inhibitors, with 7-phenyl heptyl imidazole (11) (IC50 = 320 nM against7�-OHase and IC50 = 100 nM against lyase); 1-[7-(4-fluorophenyl) heptyl] imidazole (14) (IC50 = 170 nM against 17�-OHase and IC50 = 57 nMgainst lyase); 1-[5-(4-bromophenyl) pentyl] imidazole (19) (IC50 = 500 nM against 17�-OHase and IC50 = 58 nM against lyase) being the mostotent inhibitors within the current study, in comparison to ketoconazole (KTZ) (IC50 = 3.76 �M against 17�-OHase and IC50 = 1.66 �M againstyase). Furthermore, consideration of the inhibitory activity against the two components shows that all of the compounds tested are less potentowards the 17�-OHase in comparison to the lyase component, a desirable property in the development of novel inhibitors of P45017�. From the

odelling of these compounds onto the novel substrate heme complex (SHC) for the overall enzyme complex, the length of the compound, alongith its ability to undergo interaction with the active site corresponding to the C(3) area of the steroidal backbone, are suggested to play a key role

n determining the overall inhibitory activity.2008 Elsevier Ltd. All rights reserved.

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eywords: 17�-hydroxylase/17,20-lyase (P45017�); Synthesis; Imidazole-base

. Introduction

Prostate cancer is the second leading cause of deaths inhe Western World and the majority of prostate cancer haseen found to be androgen-dependent. The enzyme complex7�-hydroxylase/17,20-lyase (P45017�) is a pivotal enzymen the conversion of C21 containing progestins and pregnanesfor example, progesterone and pregnenolone respectively) tohe C19 containing androgen precursor [1] (androstenedionend dehydroepiandrosterone respectively, Fig. 1) and requiresoth NADPH and oxygen in the sequential oxidative steps

2]. P45017� is therefore responsible for the second step inhe steroidal cascade leading to the biosynthesis of the sexormones and glucocorticoids, with the latter two series of

∗ Corresponding author. Tel.: +44 20 8547 2000; fax: +44 20 8547 7562.E-mail address: S.AHMED@KINGSTON.AC.UK (S. Ahmed).

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960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.jsbmb.2007.10.009

bitors; Biochemical evaluation

ompounds being produced directly from the 17�-hydroxy pro-estins/pregnanes.

The enzyme catalyses both the hydroxylation at C(17) of theteroid backbone, via the 17�-hydroxylase (17�-OHase) com-onent, followed by the cleavage of the C(17) and C(20) bond,ia the 17,20-lyase (lyase) component. No specific informations known about the active site of this enzyme as no crystaltructure for it exists, although workers have utilised homol-gy modelling in an attempt to discover detailed informationegarding the active site of this complex enzyme [3]; using thenformation derived from the homology study, it was proposedhat the active site appears to possess two binding sites and thathe hydroxylation and lyase steps are undertaken within eachlobe’. Inhibition of the lyase component has become the focus

f attention in the treatment of hormone-dependent prostateancer through the overall reduction in the biosynthesis of andro-ens. Azole-containing compounds have been shown to inhibit45017� through dative covalent bond formation between the

I. Shahid et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 18–29 19

enzym

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Fig. 1. Action of the overall

zole and the Fe heme within the active site. Here, we report:he synthesis of a range of imidazole-based compounds; theiriochemical evaluation [in comparison to ketoconazole (KTZ)],nd; the rationalisation of their inhibitory activity using theubstrate-heme complex (SHC) approach [4,5].

. Experimental

.1. Methods and materials

Chemicals were purchased from Sigma–Aldrich Companytd. (Poole, Dorset, England), and checked for purity byH and 13CNMR (JEOL 400 and 100 MHz or Brucker 300nd 75.5 MHz respectively) using either CDCl3, or d6-acetones solvent. Infrared spectra were obtained on a PerkinElmerourier Transform-Paragon 1000 IR. High-resolution massesf the synthesised compounds were obtained from the EPSRCass Spectrometry Service Centre at the University of Walesollege Swansea, UK, using a VG ZAB-E instrument. Gashromatography–mass spectrometry was carried out on aewlett 5890 Packard series II GC–MS at a flow rate of.58 mL/min, and a temperature range increasing from 120 to70 ◦C at the rate of 10 ◦C/min. Melting points are uncorrectednd were obtained on a BUCHI 512 or a Gallenkamp Instrument.

lemental analysis was undertaken at the School of Pharmacy,ondon. All non-radioactive steroids and laboratory reagentsere analar grade; �-nicotinamide adenine dinucleotide phos-hate (NADP, mono sodium salt), d-glucose-6-phosphate

s(w(

e complex on progesterone.

mono sodium salt), d-glucose-6-phosphate dehydrogenasesuspension in ammonium sulphate) were obtained fromoche Diagnostics, Lewes, East Sussex and ketocona-ole was obtained from Sigma–Aldrich Company, Poole,orset. [1,2,6,7-3H]Progesterone and 17�-hydroxy[1,2,6,7-

H]progesterone was obtained from Amersham Pharmaciaiotech UK Limited, Buckinghamshire. Optiscint HiSafe wasbtained from PerkinElmer Life and Analytical Sciences, Bea-onsfield, Bucks.

.2. Chemistry

1-(3-Phenylpropyl)-1H-imidazole (1): Imidazole (2 g,9.4 mmol) was added to anhydrous potassium carbonateK2CO3) (1.02 g, 7.34 mmol) and anhydrous tetrahydrofuranTHF) (50 mL). The mixture was stirred at room temperatureor 10 min prior to the addition of 3-phenylpropyl bromide2.92 g, 14.7 mmol). The mixture was then stirred under refluxor 24 h. After filtration, the THF was removed under vacuumo leave a yellow solid which was dissolved in dichloromethaneDCM) (40 mL) and washed with water (3 mL × 50 mL). Therganic layer was then extracted using hydrochloric acid (HCl)2M, 3 mL × 30 mL) followed by water (2 mL × 50 mL). Theombined acid layer was neutralised with solid saturated

odium bicarbonate (NaHCO3) and then extracted into DCM2 mL × 40 mL). The combined DCM layer was washed withater (3 mL × 50 mL), dried over anhydrous magnesium sulfate

MgSO4) and filtered. Removal of DCM under vacuum gave 1

2 mistry

ai(miN1((p6

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3t(twdsdu5e((7OO1C7

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0 I. Shahid et al. / Journal of Steroid Bioche

s a yellow oil (1.53 g, 56%). ν(max)(Film)cm−1: 3387.3 (NCNmidazole), 3031.7 (CH aromatic), 1605.1 (C C aromatic); δH300 MHz, CDCl3): 7.35 (1H, s, NCHN imidazole), 7.13 (5H,, H-Ar), 6.97 (1H, s, NCH imidazole), 6.81 (1H, s, NCH

midazole), 3.85 (2H, t, J = 7 Hz, Ph-CH2), 2.50 (2H, t, J = 7 Hz,CH2), 2.43 (2H, m, Ph-CH2CH2); δC (75 MHz, CDCl3):40.30, (NCN), 130.00 (ImC), 129.38, 128.61, 128.37, 126.31ArC), 118.00 (ImC), 46.11 (Ph-CH2), 32.41 (NCH2), 32.27Ph-CH2CH2); GCMS tR 9.89 min m/z 186 (M+), 82 (baseeak). C12H14N2 requires (C 77.38, H 7.58, N 15.04), found (C8.53, H 6.35, N 12.89 + 1.34 mol H2O).

4-Phenylbutan-1-ol (2): 4-Phenylbutyric acid (1.00 g,.09 mmol) was added drop-wise to a stirred solution of lithiumluminium hydride (LiAlH4) (24 mL, 1 M solution in THF)nd anhydrous THF (30 mL). The mixture was stirred at 3 ◦Cor 15 min and then refluxed for a further 3 h. On cooling,he mixture was added to a beaker containing iced water200 mL) and conc. HCl (50 mL). The solvent was removednder vacuum to leave a clear oil which was dissolved inCM (40 mL) and washed with saturated NaHCO3 solution

3 mL × 50 mL), followed by water (3 mL × 50 mL), dried overnhydrous MgSO4 and filtered. Removal of DCM under vac-um gave 2 as a clear oil (0.49 g, 54%); Rf 0.50 [30/70iethyl ether/petroleum ether (40–60 ◦C)]. ν(max)(Film)cm−1:341.5 (OH), 3031.7 (CH aromatic), 2936.1 (CH aliphatic),605.1 (C C aromatic); δH (400 MHz, CDCl3): 7.28 (5H,, Ph-H), 3.64 (2H, t, J = 6 Hz, Ph-CH2), 2.66 (2H, t,= 8 Hz, OCH2), 1.95 (1H, s, OH), 1.69 (2H, m, Ph-CH2CH2),.61 (2H, m, HOCH2CH2); δC (100 MHz, CDCl3): 142.25,28.31, 128.20, 125.64 (ArC), 62.57 (Ph-CH2), 35.53 (OCH2),2.16 (Ph-CH2CH2), 27.46 (HOCH2CH2); GCMS tR 5.37 minm/z 150 (M+), 104 (base peak).

(4-Bromobutyl)-benzene (3): Phosphorus tribromide (PBr3)1.80 g, 6.66 mmol) was added in a drop-wise manner to atirred solution of compound 2 (1.00 g, 6.66 mmol) and anhy-rous pyridine (5 drops) in anhydrous diethyl ether (50 mL). Theeaction was refluxed for 6 h. The diethyl ether was removednder vacuum to leave an orange oil which was dissolved inCM (40 mL) and washed with saturated NaHCO3 solution

3 mL × 50 mL), followed by water (3 mL × 50 mL), dried overnhydrous MgSO4 and filtered. Removal of DCM under vac-um gave 3 as a pale yellow oil (1.02 g, 72%); Rf 0.89 [10/90iethyl ether/petroleum ether (40–60 ◦C)]. ν(max)(Film)cm−1:031.7 (CH aromatic), 1605.1 (C C aromatic); δH (400 MHz,DCl3): 7.31 (5H, m, Ph-H), 3.44 (2H, t, J = 7 Hz, Ph-CH2), 2.68

2H, t, J = 8 Hz, OCH2), 1.91 (2H, m, Ph-CH2CH2), 1.80 (2H,, BrCH2CH2); δC (100 MHz, CDCl3): 141.75, 128.33, 125.85

ArC), 34.91 (Ph-CH2), 33.64 (OCH2), 32.18 (Ph-CH2CH2),9.81 (BrCH2CH2); GCMS tR 6.88 min, m/z 214 (M+), 91 (baseeak).

1-(4-Phenylbutyl)-1H-imidazole (4): Compound 4 was syn-hesised following the same procedure as for compound 1,xcept that 3 (3.13 g, 14.7 mmol) was added to a stirred solution

f imidazole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g,.34 mmol) in anhydrous THF (50 mL) to give 5 as a yellowil (1.51 g, 51%). ν(max)(Film)cm−1: 3387.3 (NCN imidazole),031.7 (CH aromatic), 1605.1 (C C aromatic); δH (300 MHz,

t(8e

& Molecular Biology 110 (2008) 18–29

DCl3): 7.36 (1H, s, NCHN imidazole), 7.12 (5H, m, Ph-H),.95 (1H, s, NCH imidazole), 6.77 (1H, s, NCH imidazole),.81 (2H, t, J = 7 Hz, Ph-CH2), 2.53 (2H, t, J = 7 Hz, NCH2), 1.702H, m, Ph-CH2CH2), 1.53 (2H, m, NCH2CH2); δC (75 MHz,DCl3): 141.45 (NCN), 136.96 (ImC), 129.22, 128.38, 128.30,25.96 (ArC), 118.75 (ImC), 46.80 (Ph-CH2), 35.19 (NCH2),0.48 (Ph-CH2CH2), 28.17 (NCH2CH2); GCMS tR 10.55 min/z 200 (M+), 91 (base peak). C13H16N2 requires (C 77.96,8.05, N 13.99), found (C 75.66, H 8.17, N 13.42 + 0.34 mol

2O).2-(3-Phenylpropyl)-malonic acid diethyl ester (6): Malonic

cid diethyl ester (0.80 g, 5.02 mmol) was added to anhydrousotassium tert-butoxide (0.56 g, 5.02 mmol) and anhydrous THF50 mL). The mixture was stirred at room temperature for 15 minnd then (3-bromo-propyl)-benzene (1.00 g, 5.02 mmol) wasdded. The mixture was stirred under reflux for a further 14 h.fter filtration, the THF was removed under vacuum to leaveclear oil which was dissolved in DCM (40 mL) and washedith water (3 mL × 50 mL), dried over anhydrous MgSO4 andltered. Removal of DCM under vacuum gave 6 as a clearil (1.28 g, 92%); Rf 0.46 [35/65 diethyl ether/petroleum ether40–60 ◦C)]. ν(max)(Film)cm−1: 3031.7 (CH aromatic), 2982.4CH aliphatic), 1732.2 (C O), 1605.1 (C C aromatic); δH300 MHz, CDCl3): 7.30 (2H, m, Ph-H), 7.18 (3H, m, Ph-H),.20 (4H, m, OCH2), 3.35 (1H, t, J = 7 Hz, OCCH), 2.65 (2H,, J = 7 Hz, Ph-CH2), 1.94 (2H, m, HCCH2), 1.67 (2H, m, Ph-H2CH2), 1.26 (6H, t, J = 7 Hz, CH3); δC (75 MHz, CDCl3):69.42 (C O), 141.71, 128.37, 125.89 (ArC), 61.32 (Ph-CH2),1.90 (OCH2), 35.49 (OCCH), 29.13 (Ph-CH2CH2), 28.36 [Ph-CH2)2CH2], 14.09 (CH3); GCMS tR 9.53 min, m/z 278 (M+),04 (base peak).

5-Phenylpentanoic acid (7): Compound 6 (1.00 g,.59 mmol) was added to a stirred solution of concen-rated HCl (50 mL), water (50 mL) and glacial acetic acid25 mL). The mixture was refluxed for 12 h. After coolingo room temperature, the HCl, water and glacial acetic acidere removed under vacuum to leave a clear oil, which wasissolved in DCM (40 mL) and washed with saturated NaHCO3olution (3 mL × 50 mL), followed by water (3 mL × 50 mL),ried over anhydrous MgSO4 and filtered. Removal of DCMnder vacuum gave 7 as a white solid (0.45 g, 70%) [m.p.8.8–59.4 ◦C (lit. m.p. 58–59 ◦C [6])]; Rf 0.65 [30/70 diethylther/petroleum ether (40–60 ◦C)]. ν(max)(Film)cm−1: 3031.7CH aromatic), 2858.4 (CH aliphatic), 1700.0 (C O), 1605.1C C aromatic); δH (300 MHz, CDCl3): 11.32 (1H, s, COOH),.28 (2H, m, Ph-H), 7.17 (3H, m, Ph-H), 2.64 (2H, m,CCH2), 2.38 (2H, m, Ph-CH2), 1.69 (4H, m, Ph-CH2CH2,CCH2CH2); δC (75 MHz, CDCl3): 180.04 (C O), 141.96,28.34, 128.34, 125.79 (ArC), 35.50 (OCCH2), 33.89 (Ph-H2), 30.73 (OCCH2CH2), 24.25 (Ph-CH2CH2); GCMS tR.08 min, m/z 178 (M+), 91 (base peak).

5-Phenylpentan-1-ol (8): Compound 8 was synthesisedollowing the same procedure as for compound 2, except

hat compound 7 (1.00 g, 5.61 mmol) was added to LiAlH423 mL, 1M solution in THF) and refluxed for 3 h to give

as a clear oil (0.71 g, 77%); Rf 0.56 [20/80 diethylther/petroleum ether (40–60 ◦C)]. ν(max)(Film)cm−1: 3373.7

mistry

((Jm[1((p

fP8d6ν

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pfmi0l2CPPC1δ

I. Shahid et al. / Journal of Steroid Bioche

OH), 2857.8 (CH aliphatic), 1605.1 (C C aromatic); δH400 MHz, CDCl3): 7.25 (5H, m, Ph-H), 3.61 (2H, t,= 7 Hz, Ph-CH2), 2.62 (2H, t, J = 7 Hz, OCH2), 2.31 (1H,, OH), 1.64 (4H, m, Ph-CH2CH2, HOCH2CH2), 1.38

2H, m, Ph-(CH2)2CH2]; δC (100 MHz, CDCl3): 142.48,28.31, 128.21, 125.70, 125.60 (ArC), 62.73 (Ph-CH2), 35.83OCH2), 32.42 (Ph-CH2CH2), 31.21 (HOCH2CH2), 25.31 [Ph-CH2)2CH2]; GCMS tR 6.97 min, m/z 164 (M+), 104 (baseeak).

(5-Bromopentyl)-benzene (9): Compound 9 was synthesisedollowing the same procedure as for compound 3, except thatBr3 (1.65 g, 6.09 mmol) was added to a solution of compound(1.00 g, 6.09 mmol) and anhydrous pyridine (5 drops) in anhy-rous diethyl ether (50 mL) to give 9 as a pale yellow oil (0.92 g,7%); Rf 0.90 [10/90 diethyl ether/petroleum ether (40–60 ◦C)].(max)(Film)cm−1: 2856.4 (CH aliphatic), 1605.1 (C C aro-atic); δH (400 MHz, CDCl3): 7.31 (5H, m, Ph-H), 3.34 (2H,

, J = 7 Hz, Ph-CH2), 2.58 (2H, t, J = 8 Hz, BrCH2), 1.82 (2H,, Ph-CH2CH2), 1.58 (2H, m, BrCH2CH2), 1.41 [2H, m, Ph-

CH2)2CH2]; δC (100 MHz, CDCl3): 142.40, 128.47, 128.42,25.86, (ArC), 35.84 (Ph-CH2), 33.92 (BrCH2), 32.78 (Ph-H2CH2), 30.74 (BrCH2CH2), 27.94 [Ph-(CH2)2CH2]; GCMS

R 7.73 min, m/z 228 (M+), 91 (base peak).1-(5-Phenylpentyl)-1H-imidazole (10): Compound 10 was

ynthesised following the same procedure as for compound 1,xcept that 9 (3.34 g, 14.7 mol) was added to a stirred solutionf imidazole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g,.34 mmol) in anhydrous THF (50 mL) to give 10 as a yellowil (2.84 g, 90%). ν(max)(Film)cm−1: 3387.3 (NCN imidazole),031.7 (CH aromatic), 1605.1 (C C aromatic); δH (300 MHz,DCl3): 7.33 (1H, s, NCHN imidazole), 7.16 (5H, m, Ph-H),.95 (1H, s, NCH imidazole), 6.77 (1H, s, NCH imidazole), 3.782H, t, J = 7 Hz, Ph-CH2), 2.50 (2H, t, J = 7 Hz, NCH2), 1.60 (4H,, Ph-CH2CH2, NCH2CH2), 1.24 [2H, m, Ph-(CH2)2CH2];

C (75 MHz, CDCl3): 142.10 (NCN), 137.00 (ImC), 129.24,28.35, 125.83 (ArC), 118.80 (ImC), 46.90 (Ph-CH2), 35.67NCH2), 30.97 (Ph-CH2CH2), 30.88 (NCH2CH2), 26.11 [Ph-CH2)2CH2]; GCMS tR 11.16 min m/z 214 (M+), 96 (base peak).14H18N2 requires (C 78.46, H 8.47, N 13.07), found (C 76.99,8.28, N 12.69 +0.23 mol H2O).1-(7-Phenyl-heptyl)-1H-imidazole (11): Compound 11 was

ynthesised following the same procedure as for compound, except that 7-phenyl-heptyl bromide (3.75 g, 14.7 mmol)as added to a stirred solution of imidazole (2 g, 29.4 mmol)

nd anhydrous K2CO3 (1.02 g, 7.34 mmol) in anhydrousHF (50 mL) to give 12 as a yellow oil (2.98 g, 84%).(max)(Film)cm−1: 3387.3 (NCN imidazole), 3031.7 (CH aro-atic), 1605.1 (C C aromatic); δH (300 MHz, CDCl3): 7.44

1H, s, NCHN imidazole), 7.21 (5H, m, Ph-H), 7.04 (1H, s,CH imidazole), 6.88 (1H, s, NCH imidazole), 3.88 (2H, t,= 7 Hz, Ph-CH2), 2.59 (2H, t, J = 7 Hz, NCH2), 1.74 (2H,, Ph-CH2CH2), 1.60 (2H, m, NCH2CH2), 1.31 [6H, m, Ph-

CH2)2(CH2)3]; δC (75 MHz, CDCl3): 142.62 (NCN), 137.05

ImC), 129.33, 128.38, 128.27, 125.65, (ArC), 118.77 (ImC),6.99 (Ph-CH2), 35.87 (NCH2), 31.33 (Ph-CH2CH2), 31.04NCH2CH2), 29.02 [Ph-(CH2)2CH2], 28.93 [N(CH2)2CH2],6.46 [Ph-(CH2)3CH2]; GCMS tR 11.49 min m/z 242 (M+), 82

(C3(

& Molecular Biology 110 (2008) 18–29 21

base peak). C16H22N2 requires (C 79.29, H 9.15, N 11.56),ound (C 79.04, H 9.05, N 11.30).

1-[3-(4-Fluorophenyl)-propyl]-1H-imidazole (12): Com-ound 12 was synthesised following the same procedure asor compound 1, except that 3-(4-fluorophenyl)-propyl bro-ide (3.19 g, 14.7 mmol) was added to a stirred solution of

midazole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g,.34 mol) in anhydrous THF (50 mL) to give 12 as a yellowil (2.64 g, 88%). ν(max)(Film)cm−1: 3109.1 (CH aromatic),937.0 (CH aliphatic), 1605.1 (C C aromatic); δH (300 MHz,DCl3): 7.43 (1H, s, NCHN imidazole), 7.08 (2H, d, J = 8 Hz,h-H), 6.99 (1H, s, NCH imidazole), 6.91 (2H, d, J = 8 Hz,h-H), 6.89 (1H, s, NCH imidazole), 3.91 (2H, t, J = 7 Hz, Ph-H2), 2.56 (2H, t, J = 7 Hz, NCH2), 2.10 (2H, m, Ph-CH2CH2);

C (75 MHz, CDCl3): 159.82 (ArC-F), 137.11 (NCN), 135.93ImC), 128.61, 129.66, 126.31 (ArC), 118.00 (ImC), 46.11 (Ph-H2), 32.41 (NCH2), 31.63 (Ph-CH2CH2); GCMS tR 9.89 min/z 204 (M+), 82 (base peak). C12H13F1N2 requires (C 70.57,6.42, N 13.72), found (C 68.48, H 6.25, N 13.90 + 0.34 mol

2O).1-[5-(4-Fluorophenyl)-pentyl]-1H-imidazole (13): Com-

ound 13 was synthesised following the same procedure as forompound 1, except that 4-(4-fluorophenyl)-pentyl bromide3.60 g, 14.7 mmol) was added to a stirred solution of imidazole2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g, 7.34 mmol)n anhydrous THF (50 mL) to give 13 as a yellow oil (3.07 g,0%). ν(max)(Film)cm−1: 3109.1 (CH aromatic), 2935.1 (CHliphatic), 1605.1 (C C aromatic); δH (300 MHz, CDCl3): 7.421H, s, NCHN imidazole), 7.09 (2H, d, J = 8 Hz, Ph-H), 7.031H, s, NCH imidazole), 6.96 (2H, d, J = 8 Hz, Ph-H), 6.861H, s, NCH imidazole), 3.89 (2H, t, J = 7 Hz, Ph-CH2), 2.542H, t, J = 7 Hz, NCH2), 1.77 (2H, m, Ph-CH2CH2), 1.59 (2H,, NCH2CH2), 1.29 [2H, m, Ph-(CH2)2CH2]; δC (75 MHz,DCl3): 159.59 (ArC-F), 137.69 (NCN), 129.67 (ImC),29.32, 115.18, 114.90 (ArC), 118.76 (ImC), 46.90 (Ph-CH2),4.83 (NCH2), 31.01 (Ph-CH2CH2), 30.94 (NCH2CH2), 26.03Ph-(CH2)2CH2]; GCMS tR 11.16 min m/z 232 (M+), 82 (baseeak). C14H17F1N2 requires (C 72.39, H 7.38, N 12.06), foundC 72.09, H 7.39, N 11.98).

1-[7-(4-Fluoro-phenyl)-heptyl]-1H-imidazole (14): Com-ound 14 was synthesised following the same procedure asor compound 1, except that 7-(4-fluoro-phenyl)-heptyl bro-ide (0.30 g, 1.10 mmol) was added to a stirred solution of

midazole (0.30 g, 4.39 mmol) and anhydrous K2CO3 (0.08 g,.55 mmol) in anhydrous THF (50 mL) to give 14 as a dark yel-ow oil (0.25 g, 87%). ν(max)(Film)cm−1: 3109.7 (CH aromatic),930.1 (CH aliphatic), 1605.1 (C C aromatic); δH (400 MHz,DCl3): 7.43 (1H, s, NCHN imidazole), 7.08 (2H, d, J = 8 Hz,h-H), 7.03 (1H, s, NCH imidazole), 6.93 (2H, d, J = 8 Hz,h-H), 6.87 (1H, s, NCH imidazole), 3.91 (2H, t, J = 7 Hz, Ph-H2), 2.56 (2H, t, J = 7 Hz, NCH2), 1.74 (2H, m, Ph-CH2CH2),.55 (2H, m, NCH2CH2), 1.29 [6H, m, Ph-(CH2)2(CH2)3];C (100 MHz CDCl3): 160.01 (ArC-F), 137.11 (NCN), 129.65

ImC), 129.44, 115.02, 114.81 (ArC), 118.72 (ImC), 46.99 (Ph-H2), 35.04 (NCH2), 31.39 (Ph-CH2CH2), 31.09 (NCH2CH2),1.00 [Ph-(CH2)2CH2], 28.96[(N(CH2)2CH2], 26.55 [Ph-CH2)3CH2]; GCMS tR 11.29 min m/z 260 (M+), 109 (base

2 mistry

p(

pf(zi9a7762((Cm

pc((i8a((((mC13[p(

s1wa([3CPH(((C5

pc(

zi9a7762((Cm

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s1aa(7a((s1(23H

2

2

tn

2 I. Shahid et al. / Journal of Steroid Bioche

eak). C16H21F1N2 requires (C 73.81, H 8.13, N 10.76), foundC 73.78, H 8.44, N 10.49).

1-[3-(4-Chlorophenyl)-propyl]-1H-imidazole (15): Com-ound 15 was synthesised following the same procedure asor compound 1, except that 3-(4-chlorophenyl)-propyl bromide3.43 g, 14.7 mmol) was added to a stirred solution of imida-ole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g, 7.34 mol)n anhydrous THF (50 mL) to give 15 as a yellow oil (3.14 g,2%). ν(max)(Film)cm−1: 3112.0 (CH aromatic), 2945.7 (CHliphatic), 1605.1 (C C aromatic); δH (300 MHz, CDCl3):.43 (1H, s, NCHN imidazole), 7.26 (2H, d, J = 8 Hz, Ph-H),.07 (1H, s, NCH imidazole), 7.06 (2H, d, J = 8 Hz, Ph-H),.89 (1H, s, NCH imidazole), 3.91 (2H, t, J = 7 Hz, Ph-CH2),.55 (2H, t, J = 7 Hz, NCH2), 2.07 (2H, m, Ph-CH2CH2); δC75 MHz, CDCl3): 138.71 (ArC-Cl), 137.10 (NCN), 132.07ImC), 129.69, 129.64, 128.71 (ArC), 118.66 (ImC), 45.99 (Ph-H2), 32.19 (NCH2), 31.79 (Ph-CH2CH2); GCMS tR 10.76 min/z 220 (M+), 82 (base peak).1-[5-(4-Chlorophenyl)-pentyl]-1H-imidazole (16): Com-

ound 16 was synthesised following the same procedure as forompound 1, except that 5-(4-chlorophenyl)-pentyl bromide3.84 g, 14.7 mmol) was added to a stirred solution of imidazole2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g, 7.34 mmol)n anhydrous THF (50 mL) to give 16 as a yellow oil (3.37 g,8%). ν(max)(Film)cm−1: 3112.0 (CH aromatic), 2935.0 (CHliphatic), 1605.1 (C C aromatic); δH (400 MHz, CDCl3): 7.421H, s, NCHN imidazole), 7.24 (2H, d, J = 8 Hz, Ph-H), 7.021H, s, NCH imidazole), 7.05 (2H, d, J = 8 Hz, Ph-H), 6.851H, s, NCH imidazole), 3.88 (2H, t, J = 7 Hz, Ph-CH2), 2.552H, t, J = 7 Hz, NCH2), 1.74 (2H, m, Ph-CH2CH2), 1.60 (2H,, NCH2CH2), 1.29 [2H, m, Ph-(CH2)2CH2]; δC (100 MHzDCl3): 140.39 (ArC-Cl), 136.95 (NCN), 131.43 (ImC),29.58, 129.31, 128.34 (ArC), 118.66 (ImC), 46.82 (Ph-CH2),4.91 (NCH2), 30.85 (Ph-CH2CH2), 30.70 (NCH2CH2), 25.97Ph-(CH2)2CH2]; GCMS tR 13.60 min m/z 248 (M+), 82 (baseeak). C14H17Cl1N2 requires (C 67.60, H 6.89, N 11.26), foundC 67.01, H 6.57, N 11.08+0.12 mol H2O).

1-(4-Bromobenzyl)-1H-imidazole (17): Compound 17 wasynthesised following the same procedure as for compound, except that 4-bromobenzyl bromide (3.67 g, 14.7 mmol)as added to a stirred solution of imidazole (2 g, 29.4 mmol)

nd anhydrous K2CO3 (1.02 g, 7.34 mmol) in anhydrous THF50 mL) to give 17 as a pale yellow solid (1.31 g, 36%)m.p. 82.2–83.1 ◦C (lit. m.p. 81–83 ◦C [7])]. ν(max)(Film)cm−1:091.4 (CH aromatic), 1605.1 (C C aromatic); δH (400 MHz,DCl3): 7.53 (1H, s, NCHN imidazole), 7.47 (2H, d, J = 8 Hz,h-H), 7.08 (1H, s, NCH imidazole), 7.00 (2H, d, J = 8 Hz, Ph-), 6.87 (1H, s, NCH imidazole), 5.06 (2H, s, Ph-CH2); δC

100 MHz CDCl3): 137.36 (NCN), 135.14 (ArC-Br), 132.12ImC), 130.00, 128.84, 122.28 (ArC), 119.14 (ImC), 50.12Ph-CH2); GCMS tR 9.41 min m/z 236 (M+), 169 (base peak).10H9Br1N2 requires (C 50.66, H 3.83, N 11.81), found (C0.84, H 3.64, N 11.69).

1-[3-(4-Bromo-phenyl)-propyl]-1H-imidazole (18): Com-ound 18 was synthesised following the same procedure as forompound 1, except that 3-(4-bromophenyl)-propyl bromide4.08 g, 14.7 mmol) was added to a stirred solution of imida-

(71(

& Molecular Biology 110 (2008) 18–29

ole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g, 7.34 mol)n anhydrous THF (50 mL) to give 18 as a yellow oil (3.72 g,1%). ν(max)(Film)cm−1: 3106.5 (CH aromatic), 2937.1 (CHliphatic), 1605.1 (C C aromatic); δH (300 MHz, CDCl3):.45 (1H, s, NCHN imidazole), 7.30 (2H, d, J = 8 Hz, Ph-H),.15 (2H, d, J = 8 Hz, Ph-H), 7.06 (1H, s, NCH imidazole),.90 (1H, s, NCH imidazole), 3.92 (2H, t, J = 7 Hz, Ph-CH2),.60 (2H, t, J = 7 Hz, NCH2), 2.11 (2H, m, Ph-CH2CH2); δC75 MHz, CDCl3): 140.29 (ArC-Br), 137.15 (NCN), 129.54ImC), 128.62, 128.38, 126.33 (ArC), 118.72 (ImC), 46.11 (Ph-H2), 32.44 (NCH2), 32.30 (Ph-CH2CH2); GCMS tR 9.07 min/z 186 (M+, -Br), 82 (base peak).1-[5-(4-Bromo-phenyl)-pentyl]-1H-imidazole (19): Com-

ound 19 was synthesised following the same procedure asor compound 1, except that 5-(4-bromophenyl)-pentyl bro-ide (4.50 g, 14.7 mmol) was added to a stirred solution of

midazole (2 g, 29.4 mmol) and anhydrous K2CO3 (1.02 g,.34 mmol) in anhydrous THF (50 mL) to give 19 as a yel-ow oil (4.03 g, 90%). ν(max)(Film)cm−1: 3112.0 (CH aromatic),934.6 (CH aliphatic), 1605.1 (C C aromatic); δH (400 MHz,DCl3): 7.42 (1H, s, NCHN imidazole), 7.36 (2H, d, J = 8 Hz,h-H), 7.03 (1H, s, NCH imidazole), 6.99 (2H, d, J = 8 Hz,h-H), 6.86 (1H, s, NCH imidazole), 3.88 (2H, t, J = 7 Hz, Ph-H2), 2.52 (2H, t, J = 7 Hz, NCH2), 1.76 (2H, m, Ph-CH2CH2),.58 (2H, m, NCH2CH2), 1.28 [2H, m, Ph-(CH2)2CH2]; δC100 MHz CDCl3): 140.92 (ArC-Br), 136.95 (NCN), 131.32ImC), 130.03, 129.32, 119.46 (ArC), 118.67 (ImC), 46.83 (Ph-H2), 34.99 (NCH2), 30.87 (Ph-CH2CH2), 30.65 (NCH2CH2),5.98 [Ph-(CH2)2CH2]; GCMS tR 12.36 min m/z 292 (M+), 82base peak).

1-(4-Iodo-benzyl)-1H-imidazole (20): Compound 20 wasynthesised following the same procedure as for compound, except that 4-iodobenzyl bromide (4.36 g, 14.7 mmol) wasdded to a stirred solution of imidazole (2 g, 29.4 mmol)nd anhydrous K2CO3 (1.02 g, 7.34 mmol) in anhydrous THF50 mL) to give 20 as a yellow solid (3.85 g, 92%) (m.p. 69-0 ◦C). ν(max)(Film)cm−1: 3110.1 (CH aromatic), 2929.2 (CHliphatic), 1605.1 (C C aromatic); δH (400 MHz, CDCl3): 7.661H, s, NCHN imidazole), 7.52 (2H, d, J = 8 Hz, Ph-H), 7.251H, s, NCH imidazole), 7.08 (2H, d, J = 8 Hz, Ph-H), 6.86 (1H,, NCH imidazole), 5.03 (2H, s, Ph-CH2); δC (100 MHz CDCl3):38.07 (ArC-I), 137.37 (NCN), 135.83 (ImC), 130.06, 129.02ArC), 119.14 (ImC), 50.19 (Ph-CH2); GCMS tR 10.19 min m/z84 (M+), 217 (base peak). C10H9I1N2 requires (C, 42.28, H,.19, N, 9.86), found (C, 43.28, H, 3.26, N, 9.91 + 0.05 mol2O).

.3. Biochemistry

.3.1. 17α-OHase assayAll assays were run in triplicate. Sprague–Dawley rat

esticular microsomal suspension was thawed under cold run-ing water, and vortexed. The final incubation assay mixture

1 mL) consisted of sodium phosphate buffer (50 mM, pH.4, 905 �L), radiolabelled progesterone as substrate (1.5 �M,5 �L), NADPH-generating system (50 �L) and inhibitor10 �M, 20 �L). Tubes were warmed to 37 ◦C for 5 min and

mistry

tcbowfenawtwTcsv

2

tn(7sia(itlatatptw(etwf

2

poidto

2

tb

ac

2

hbghbApaaticgcbt

3

timastXs(iaavetptanaato the alkyl bromide through the use of phosphorus tribromide(PBr3) to give 3-(4-bromophenyl)-propyl bromide.

In the chain elongation step, we did not wish to con-sider the use of −CN due to safety reasons, as such, in

I. Shahid et al. / Journal of Steroid Bioche

he assay initiated by the addition of microsomal enzyme (finaloncentration 0.16 mg/mL, 10 �L). The assay mixture was incu-ated for 15 min. The reaction was quenched by the additionf ether (2 mL), vortexed and placed on ice. The organic layeras then placed into a separate tube. The assay mixture was

urther extracted with ether (2 × 2 mL), and the organic lay-rs combined. The solvent was removed under a stream ofitrogen, and acetone (30 �L) was then added to each tubend the solution spotted onto silica based TLC plates alongith carrier steroids (progesterone, 17�-hydroxyprogesterone,

estosterone and androstenedione, 5 mg/mL). The TLC platesere developed using the mobile phase DCM:ethylacetate (7:3).he separated spots were identified under UV light and each spotut out and placed into scintillation vials. Acetone (1 mL) andcintillation fluid (Optiscint HiSafe) (3 mL) were added to eachial, vortexed and counted for 3 min for 3H.

.3.2. Lyase assayAll assays were run in triplicate. Sprague–Dawley rat

esticular microsomal suspension was thawed under cold run-ing water, and vortexed. The final incubation assay mixture1 mL) consisted of sodium phosphate buffer (50 mM, pH.4, 905 �L), radiolabelled 17�-hydroxyprogesterone as sub-trate (1 �M, 10 �L), NADPH generating system (50 �L) andnhibitor (10 �M, 20 �L). Tubes were warmed to 37 ◦C for 5 minnd the assay initiated by the addition of microsomal enzymefinal concentration 0.23 mg/mL, 15 �L). The assay mixture wasncubated for 30 min. The reaction was quenched by the addi-ion of ether (2 mL), vortexed and placed on ice. The organicayer was then removed and placed into a separate tube. Thessay mixture was further extracted with ether (2 × 2 mL), andhe organic layers combined. The solvent was removed under

stream of nitrogen, and acetone (30 �L) was then addedo each tube and the solution spotted onto silica based TLClates along with carrier steroids (17�-hydroxyprogesterone,estosterone and androstenedione, 5 mg/mL). The TLC platesere developed using the mobile phase DCM:ethylacetate

4:1). The separated spots were identified under UV light andach spot cut out and placed into scintillation vials. Ace-one (1 mL) and scintillation fluid (Optiscint HiSafe) (3 mL)ere added to each vial, vortexed and counted for 3 min

or 3H.

.3.3. IC50 determinationIn determining the IC50 values for the most potent com-

ounds, the inhibitory activity was determined using the methodutlined above, however, for each compound, five or morenhibitor concentrations were used and the inhibitory activityetermined at each concentration (in triplicate); the IC50 washen determined from a graph (using linear regression analysis)f the inhibitory activity versus log [I].

.3.4. Ki determinationEach assay tube contained substrate (of varying final concen-

ration, 15 �L), NADPH-generating system (25 �L), phosphateuffer (445 �L), enzyme (0.10 mg/mL final concentration, 5 �L)

S(

& Molecular Biology 110 (2008) 18–29 23

nd inhibitor (of varying final concentration, 10 �L). The pro-edure was similar to that previously described.

.3.5. Molecular modellingThe general approach for the construction of the SHC

as been described previously [8–11] and has therefore noteen detailed here. In general, however, the structures of pro-esterone, 17�-hydroxyprogesterone, the heme and potentialydrogen bonding groups were all constructed, and then refinedy performing a pre-optimisation calculation in Mechanics usingugmented MM2 as implemented in the molecular modellingrogram CaChe [12]. The atoms of the SHC were then ‘locked’nd the proposed inhibitors attached to the SHC representing thective site of the overall enzyme complex resulting in the produc-ion of the enzyme–inhibitor complex; in the attachment of thenhibitors, interactions at both the heme (that is, the N–Fe dativeovalent bond) and potential hydrogen bonding interactions withroups at the active site were mimicked. The enzyme–inhibitoromplex was minimised and optimised so as to produce the finalinding conformer of the inhibitor within the representation ofhe overall active site of P45017�.

. Results and discussion

In the synthesis of the proposed inhibitors, the azole func-ionality was reacted with a substituted phenyl alkyl haliden the presence of a suitable base (Scheme 1). However, in

ost cases, the phenyl alkyl bromides were not readily avail-ble, and as such, these compounds were synthesised using aeries of reactions to extend the alkyl chain. For example, inhe synthesis of 5-(4-bromophenyl)-pentyl bromide (Scheme 2;= Br), 4-bromocinnamic acid was initially esterified and sub-

equently reduced to the ethyl 3-(4-bromophenyl)-propionateusing hydrogen gas over palladium on activated charcoal),t should be noted that the direct reduction of the cinnamiccid derivatives did not yield any of the appropriate propanoiccid derivative due to solubility problems, whereas the con-ersion of the cinnamic acid derivatives to the correspondingster derivative resulted in no solubility problems. The ester washen reduced to the corresponding alcohol [3-(4-bromophenyl)-ropan-1-ol] using an appropriate reducing agent. In our hands,he use of lithium aluminium hydride (LiAlH4) resulted in theppropriate alcohol in good yield, however, the use of alter-ative reducing agents such as sodium borohydride or dibutylluminium hydride did not result in the synthesis of the targetlcohol in good yield. The resulting alcohol was then converted

cheme 1. Synthesis of potential inhibitors of P45017� (a = K2CO3/THF/�)n = 1–7; X = H, F, Cl, Br or I).

24 I. Shahid et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 18–29

S romie r).

ub[pfapanttiiasemdptbts

cictwidiA[fo(rtmdp

cheme 2. Reactions undertaken in the synthesis of 4-substituted phenyl pentyl b= diethyl malonate/KButO/THF/�; f = CH3COOH/H+/�) (X = H, F, Cl and B

ndertaking the chain extension, 3-(4-bromophenyl)-propylromide was then reacted with diethyl malonate to give 2-3-(4-bromophenyl)-propyl]-malonic acid diethyl ester, usingotassium tertiary butoxide dissolved in anhydrous tetrahydro-uran (THF) under reflux. Following acid hydrolysis (usingqueous hydrochloric acid), the resulting 5-(4-bromophenyl)-entanoic acid was reduced using LiAlH4 to give the appropriatelcohol, namely [5-(4-bromophenyl)-pentan-1-ol]. The bromi-ation of the alcohol (using PBr3) resulted in the synthesis ofhe desired 5-(4-bromophenyl)-pentyl bromide. Conversion ofhe latter compound to the azole derivative involved reflux-ng the resultant alkyl bromide (in anhydrous THF) withmidazole in the presence of anhydrous potassium carbon-te. Attempts to synthesise the phenylbutyl derivatives via theynthesis of 2-[3-(4-bromophenyl)-ethyl]-malonic acid diethylster from the reaction between 2-(4-bromophenyl)-ethyl bro-ide and diethyl malonate using potassium tertiary butoxide

issolved in anhydrous THF, did not result in the target com-ound. Indeed, dehydrohalogenation was observed and attempts

o use weaker bases (for example anhydrous potassium car-onate) did not result in the target compounds, as such,he phenylbutyl and phenylhexyl derivatives were not synthe-ised.

ttpd

de (a = ROH/H+/�; b = H2/Pd/C; c = LiAlH4/THF/�; d = PBr3/diethyl ether/�;

In the synthesis of the iodo derivatives of the longer chainontaining compounds, it was found that the reactions outlinedn Scheme 2 (X = I) could not be utilised due to the lack ofommercially available 4-iodocinnamic acid. As such, alterna-ive routes to ethyl 3-(4-iodophenyl) propionate (or derivatives)ere considered and an alternative route was developed which

nvolved an initial reaction between 4-iodobenzyl bromide andiethyl malonate (step a, Scheme 3) and using this method, 3-(4-odophenyl) propionic acid was obtained in relatively good yield.cid hydrolysis to give the corresponding mono-carboxylic acid

3-(4-iodophenyl)-propionic acid] (via step b, Scheme 3) wasound to occur without any major problems, although the yieldf the target compound was poor. However, the reduction stepvia step c, Scheme 3) to give 3-(4-iodophenyl)-propanol did notesult in the target compound. Indeed, attempts to synthesise thearget compound with numerous reducing agents (lithium alu-inium hydride, sodium borohydride, lithium borohydride or

ibutyl aluminium hydride) resulted in no reaction at all or theroduction of both the de-halogenated derivative together with

he target compound in various ratios – attempts to isolate thewo using flash chromatography failed to resolve the two com-ounds due to their similarity, as such, the synthesis of the 4-iodoerivatives was abandoned – the only compound reported is the

I. Shahid et al. / Journal of Steroid Biochemistry

Smr

4b

damftsthisvi

oa

ppcataKaf(lOtta1a(lt∼19 and ∼29 times respectively more potent against lyase incomparison to KTZ. With regards to selectivity towards the lyasecomponent, of the most potent compounds, 19 appears to pos-

cheme 3. Reactions attempted in the synthesis of the 4-iodophenyl alkyl bro-ide (a = diethyl malonate/KButO/DMF/�; b = CH3COOH/H+/�; c = various

educing agents/THF/�).

-iodobenzyl imidazole which was obtained from the reactionetween 4-iodobenzyl bromide and imidazole.

The synthesised azole compounds were initially screened (theata is not shown here) and IC50 values determined (Table 1)gainst both 17�-OHase and lyase using modified literatureethods [13–15]. The modification involved the use of a dif-

erent mobile phase to that used by previous workers withinhe field in the separation and identification of the radiolabelledubstrate from the radiolabelled products. It should be notedhat the inhibitory acticity for compounds 1, 12, 15, 17 and 18ave been reported previously, however, the compounds werencluded within the assay for consistency between the differenttudies [16–18]. So as to study the mode of inhibition, the Kialues were also determined for a small range of highly potent

nhibitors (Table 2).

Table 1 shows the inhibitory data obtained for the inhibitionf both 17�-OHase and lyase by the synthesised compoundsnd KTZ. Consideration of the inhibitory data for the com-

FD

& Molecular Biology 110 (2008) 18–29 25

ounds shows that compounds 10, 11, 13, 14, 16, 18 and 19ossess potent inhibitory activity in comparison to the standardompound KTZ. For example, compound 16 (IC50 = 570 nMgainst 17�-OHase and IC50 = 86 nM against lyase) was foundo be some ∼7 times more potent than KTZ against 17�-OHasend ∼19 times more potent against lyase in comparison toTZ (IC50 = 3.76 �M against 17�-OHase and IC50 = 1.66 �M

gainst lyase) (Tables 1 and 3). The potent inhibitory activity isurther demonstrated by 4-bromophenyl pentyl imidazole (19)IC50 = 500 nM against 17�-OHase and IC50 = 58 nM againstyase) which is ∼8 times more potent than KTZ against 17�-Hase and ∼29 times more potent against lyase in comparison

o KTZ. The most potent compounds within the series of syn-hesised compounds are 11 (IC50 = 320 nM against 17�-OHasend IC50 = 100 nM against lyase); 14 (IC50 = 170 nM against7�-OHase and IC50 = 57 nM against lyase); 16 (IC50 = 570 nMgainst 17�-OHase and IC50 = 86 nM against lyase), and; 19IC50 = 500 nM against 17�-OHase and IC50 = 58 nM againstyase) which are found to be ∼12, ∼22, ∼7 and ∼8 times respec-ively more potent than KTZ against 17�-OHase and ∼17, ∼29,

ig. 2. (a) Dixon plot to show the mode of inhibition of 16 against lyase; (b)ixon plot to show the mode of inhibition of 16 against 17�-OHase.

26 I. Shahid et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 18–29

Table 1Table to show the IC50 values obtained for a range of imidazole-based compounds against 17�-OHase and lyase (the values are the mean of three determinations,

n = 9; ND = not determined) and the calculated log P values

X n Compound number 17�-OHase IC50 values (�M) Lyase IC50 values (�M) Calculated Log P

H 3 1 30.95 ± 0.68 6.14 ± 1.21 1.959H 4 4 8.65 ± 1.37 2.23 ± 0.38 2.355H 5 10 2.20 ± 0.25 1.31 ± 0.21 2.751H 7 11 0.32 ± 0.05 0.10 ± 0.02 3.544F 3 12 27.81 ± 1.44 1.96 ± 0.01 2.098F 5 13 0.75 ± 0.005 0.10 ± 0.01 2.891F 7 14 0.17 ± 0.001 0.057 ± 0.002 3.683Cl 3 15 5.85 ± 0.19 0.55 ± 0.07 2.477Cl 5 16 0.57 ± 0.03 0.086 ± 0.006 3.269Br 1 17 16.55 ± 0.23 2.85 ± 0.08 2.102Br 3 18 2.95 ± 0.03 0.33 ± 0.02 2.750Br 5 19 0.50 ± 0.04 0.058 ± 0.005 3.543I 1 20 10.06 ± 0.96 1.58 ± 0.17 2.568– – KTZ 3.76 ± 0.01 1.66 ± 0.15 3.178

Table 2Showing the Ki values obtained for the four most potent compounds against both lyase and 17�-OHase; the relative potency in comparison to KTZ and the selectivitybetween lyase and 17�-OHase

Compound Ki values (�M)against 17�-OHase

Potency against17�-OHase w.r.t to KTZ

Ki values (�M)against lyase

Potency against lyasein comparison to KTZ

Selective bindingto lyase

11 0.207 ± 0.011 5.99 0.0553 ± 0.0034 12.03 3.713 0.265 ± 0.013 4.68 0.052 ± 0.002 12.79 5.111K

sacpt

c

Oba

TTt

C

1411111111112K

4 0.0775 ± 0.0025 16.006 0.208 ± 0.006 5.96TZ 1.24 ± 0.01 1

ess the greater selectivity, being over 8 times more selectivegainst lyase compared to 17�-OHase. Furthermore, from theonsideration of the inhibitory data, we observe that those com-

ounds where n = 3 appear to show a greater selectivity withinhe different series of compounds.

Consideration of the mode of inhibition of the most potentompounds show all are competitive inhibitors of both 17�-

acci

able 3able to show relative potency for a small range of synthesised compounds against 17�

han KTZ are highlighted in bold)

ompound number Relative potency (w.r.t KTZ) against 17

0.120.43

1 1.711 11.752 0.143 5.014 22.115 0.646 6.607 0.238 1.279 7.520 0.37TZ 1

0.0215 ± 0.0001 30.93 3.60.040 ± 0.002 16.63 5.20.665 ± 0.015 1 1.9

Hase and lyase [the Dixon plots for compound 16 (Fig. 2a and) and KTZ (Fig. 3a and b) are shown as examples]. Consider-tion of the Ki values (Table 2) show that 11, 13, 14 and 16 are

ll extremely potent inhibitors with potent binding constants inomparison to KTZ and all bind more selectively to the lyaseomponent in comparison to the 17�-OHase component—thiss a highly desirable characteristic of inhibitors of P45017�

-OHase and lyase with respect to (w.r.t) KTZ (compounds that are more potent

�-OHase Relative potency (w.r.t KTZ) against lyase

0.270.741.27

16.600.85

16.6029.12

3.0219.30

0.585.03

28.621.051

I. Shahid et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 18–29 27

se; (b)

sisu

stagidpap

c0aevtmtapi

Fig. 3. (a) Dixon plot to show the mode of inhibition of KTZ against lya

ince the inhibition of the lyase component specifically wouldnhibit the androgen biosynthesis whilst allowing the synthe-is of the corticosteroids and the mineralocorticoids to continuenaffected.

Detailed consideration of the inhibitory activity (Table 1)hows that within the series of compounds considered withinhe current study, a trend is observed in that the length of thelkyl spacer group, between the phenyl moiety and the azoleroup, appears to play an important part in the inhibitory activ-ty of these compounds. That is, we observe that the IC50 valueecreases with an increase in the alkyl spacer group, for exam-

le, compound 1 is found to possess IC50 values of 30.95nd 6.14 �M against 17�-OHase and lyase respectively. Com-ound 11, however, contains four more carbons in the alkyl

tip

Dixon plot to show the mode of inhibition of KTZ against 17�-OHase.

hain (spacer group) and is found to possess IC50 values of.32 and 0.1 �M against 17�-OHase and lyase respectively,n increase in potency of ∼97 and 61 respectively. Consid-ration of the calculated logarithm of the partition coefficientalue (log P) (using Quantum CaChe Project Leader [12]) of thewo respective compounds suggests that the increased potency

ay also be due to the increase in the log P of the respec-ive inhibitors, the calculated log P for 1 and 11 being 1.96nd 3.54 respectively. That the hydrophobicity of the com-ounds may play an important role in determining the overallnhibitory activity is further demonstrated when we consider

he IC50 values for the 4-substituted compounds where theren an increase in potency with an increase in log P. For exam-le, from the consideration of the fluoro derivatives, we observe

2 mistry & Molecular Biology 110 (2008) 18–29

ttpmKttpIt1attsi

lfp2ctiIOac

obwtotapiatbiatpte1t1otebtei

tpitFwsowltsa

R

[5] S. Ahmed, Molecular modelling of inhibitors of 17�-hydroxylase—a

8 I. Shahid et al. / Journal of Steroid Bioche

hat compound 12 (log P = 2.09) is the weakest inhibitor inhe range whilst 14 (log P = 3.68) is the most potent com-ound within the current study and is found to be ∼29 timesore potent against the lyase component when compared toTZ. A similar trend is observed within all the other deriva-

ives, although fewer compounds are considered, for example,he weakest inhibitor within the bromo derivatives is com-ound 17 (n = 1) (IC50 = 16.55 �M against 17�-OHase andC50 = 2.85 �M against lyase and with log P = 2.10) whereashe most potent inhibitor within the same range is compound9 (n = 5) (IC50 = 500 nM against 17�-OHase and IC50 = 58 nMgainst lyase and with log P = 3.54). As such, this would appearo suggest that both the log P and/or length of the compoundogether with the appropriate group to interact with the activeite of the enzyme are all important factors in the design of novelnhibitors of P45017�.

Of the two physicochemical properties, however, we postu-ate that the hydrophobicity of the inhibitor is the more importantactor—our hypothesis is supported by the inhibitory activityossessed by compounds 1, 11 and 20. That is, compounds 1 and0 differ in length as the former compound contains two morearbon spacer units in comparison to compound 20. However,he latter compound possessing a higher hydrophobic character-stic (log P = 2.57), is found to possess greater inhibitory activity.t is interesting to note that the potency of the inhibitors for 17�-Hase within each series increases to a greater extent when the

lkyl chain is extended from n = 3 to n = 5, compared to any otherhain extension. This trend is not observed for lyase.

On consideration of the inhibitory data within Table 1, webserve that within the substituted series of compounds, theromo-derivatives are more potent than the chloro-derivatives,hich are in turn more potent than the fluoro-derivatives and

he non-substituted derivatives. We hypothesise that the trendbserved is due to the ability of the substituted atom being ableo undergo interaction with the H-bonding/polar group at thective site [which normally interacts with the group at the C(3)osition of Preg or P]. As such, the bromine atom is able tonteract with the group(s) at the active site, whereas atoms suchs Cl or F, due to their increased electronegativity, are unableo undergo strong polar–polar interaction, resulting in weakerinding with the active site of the enzyme and thus resultingn weaker enzyme-inhibitor complex and thus poorer inhibitoryctivity. As such, compound 19 is found to possess not onlyhe appropriate alkyl spacer group (alkyl chain consisting of aentyl group) but also a 4-bromo-substituted aromatic ring sys-em allowing this compound to interact favourably within thenzyme active site, resulting in IC50 values of 500 nM against7�-OHase and 58 nM against lyase (Table 1). On modellinghese compounds within the SHC (Fig. 4 shows the modelling of9 onto the SHC), we observe that the increase in chain lengthf the spacer group allows for increased polar–polar interac-ion between the substituted phenyl moiety and the area of thenzyme active site corresponding to the C(3) area of the steroidackbone (without adopting highly unfavourable conformer),

hereby presumably leading to the formation of a more stablenzyme-inhibitor complex and therefore leading to more potentnhibitory activity.

Fig. 4. Binding of compound 19 into the overall SHC for P45017�.

In conclusion, the current study has suggested important fac-ors (length of the alkyl chain between the azole moiety thehenyl ring; log P; and substituent on the phenyl ring of thenhibitor) which may be taken into consideration in the fur-her design of novel and highly potent inhibitors of P45017�.urthermore, from the structure–activity relationship observedithin the series of compounds synthesised within the current

tudy, compounds may be designed which specifically inhibitne (in particular, the lyase component) of the two componentsithin the overall active site of P45017�. The study has also high-

ighted the usefulness of the SHC approach which has allowedhe derivation of a simplified representation of the overall activeite of P45017� and the rationalisation of highly potent inhibitoryctivity observed within the synthesised compounds.

eferences

[1] P.R. Ortiz de Montellano, in: P.R. Ortiz de Montellano (Ed.), CytochromeP-450: Structure Mechanism and Biochemistry, Plenum Press, New Yorkand London, 1986.

[2] P. Robichaud, J.N. Wright, M.J. Akhtar, The involvement of an O2-derivednucleophilic species in acyl-carbon cleavage, catalyzed by cytochrome P-45017�—implications for related P-450 catalyzed fragmentation reactions,Chem. Soc. Chem. Commun. 12 (1994) 1501–1503.

[3] C.A. Laughton, S. Neidle, M.J.J.M. Zvelebil, M.J.E.A. Sternberg, Amolecular-model for the enzyme cytochrome-P45017�, a major target forthe chemotherapy of prostatic-cancer, Biochem. Biophys. Res. Commun.171 (1990) 1160–1167.

[4] S. Ahmed, P.J. Davis, Molecular modelling of inhibitors of aromatase—anovel-approach, Bioorg. Med. Chem. Lett. 5 (1995) 1673–1678.

novel-approach, Bioorg. Med. Chem. Lett. 5 (1995) 2795–2800.[6] A.T. Blomquist, R.E. Stahl, Y.C. Meinwald, B.H. Smith, Many-membered

carbon rings. XIII. Restricted rotation in a simple paracyclophan, J. Org.Chem. 26 (1961) 1687–1691.

mistry

[

[

[

[

[

[

[

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I. Shahid et al. / Journal of Steroid Bioche

[7] Patent; Wellcome Found.; DE 2903653; 1979.[8] S. Ahmed, P.J. Davis, The mechanism of aromatase—a molecular

modelling perspective, Bioorg. Med. Chem. Lett. 5 (1995) 2789–2794.

[9] S. Ahmed, The mechanism of a P-450 enzyme-aromatase; A molecularmodelling perspective for the removal of the C(19) methyl and aromatisa-tion of the steroid A ring, J. Enz. Inhib. 12 (1997) 59–70.

10] S. Ahmed, The use of the substrate–heme complex approach in thedesign, synthesis, biochemical evaluation, and rationalization of theinhibitory activity of a range of azole compounds against cholesterol sidechain cleavage enzyme, Biochem. Biophys. Res. Commun. 275 (2000)75–76.

11] S. Ahmed, A novel molecular modelling study of inhibitors ofthe 17�-hydroxylase component of the enzyme system 17�-hydroxylase/17,20-lyase (P-45017�), Bioorg. Med. Chem. 7 (1999) 1487–1496.

12] Quantum Cache® and Project Leader® are a trademark of Oxford Molec-ular Ltd. (Fujitsu), Oxford Science Park, Oxford, UK.

13] J.S. Li, Y. Li, C. Son, A.M.H. Brodie, Synthesis and evaluation of pregnanederivatives as inhibitors of human testicular 17�-hydroxylase/C-17,C-20-lyase, J. Med. Chem. 39 (1996) 4335–4339.

[

& Molecular Biology 110 (2008) 18–29 29

14] C.P. Owen, P.J. Nicholls, H.J. Smith, R. Whomsley, Inhibition of aromatase(P450Arom) by some 1-(benzofuran-2-ylmethyl)imidazoles, J. Pharm. Phar-macol. 51 (1999) 427–433.

15] S.A. Bahshwan, C.P. Owen, P.J. Nicholls, H.J. Smith, M. Ahmadi,Some 1-[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17�-hydroxylase:17,20-lyase (P45017) and their specificity patterns, J. Pharm.Pharmacol. 50 (1998) 1109–1116.

16] C.P. Owen, S. Dhanani, C.H. Patel, I. Shahid, S. Ahmed, Synthesisand biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex17�-hydroxylase/17,20-lyase (P45017�), Bioorg. Med. Chem. Lett. 16(2006) 4011–4016.

17] C.H. Patel, S. Dhanani, C.P. Owen, S. Ahmed, Synthesis, biochemicalevaluation and rationalisation of the inhibitory activity of a range of4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme com-plex 17�-hydroxylase/17,20-lyase (P45017�), Bioorg. Med. Chem. Lett. 16

(2006) 4752–4757.

18] C.P. Owen, C.H. Patel, S. Dhanani, S. Ahmed, Synthesis and biochemicalevaluation of a range of non-substituted phenyl alkyl triazole-based com-pounds as potential inhibitors of the enzyme complex 17�-hydroxylase/17,20-lyase (P45017�), Lett. Drug Des. Disc. 3 (2006) 761–765.