Biomedical Research

Abstract

A series of 2`-mercapto-3`-cyano-4`-(aryl)-4-pregneno-[3,2-e]pyridine-20-ones (3a-h) and 1`- thiocarbamoyl -2`-amino-4`-(aryl)-4-pregneno-[3,2-e]pyridine-20-one derivatives (4a-h) were synthesized using progesterone 1 "17-acetyl-1,7,8,10,11,12,13,15,16,17-decahydro-10,13-dimethyl-2Hcyclopenta[ a]phenanthren-3(6H,9H,14H)-one" as starting material. The synthesized compounds were evaluated for 5α-reductase inhibitors and anti-prostate cancer activities compared to Anastrozole® as positive control. Some of the tested compounds exhibited better activities than Anastrozole®. Compounds 3a-h and 4a-h showed potent 5α-reductase inhibitors and anti-prostate cancer activities than Anastrozole®.

Keywords

Progesterone, pregneno[3,2-e]pyridinones, 5α-reductase inhibitor, anti-prostate cancer.

Introduction

Pyridine derivatives were reported to possess anticonvulsant [1,2], cardiotonic [3], antihypertensive [4], b-adrenergic blocking activity [5]. In the previous work, we found that certain substituted steroidal derivatives were reported to be associated with anti-arrhythmic [6], reductase inhibitor [7], antiandrogenic [8,9], anti-inflammatory [10], and anti-alzheimer [11,12] activities.

Some of heterocyclic compounds containing nitrogen atom were evaluated and reported as anticancer [13], antiparkinsonian [14], anti-inflammatory agents [15] and antimicrobial [16]. In addition, several of heterocyclic candidates exhibited analgesic [17], anticonvulsant, anti-inflammatory and antimicrobial [18,19], antitumor [20], antipyretics [21], and anti-arrhythmic [22] activities.

Also the synthesis and pharmacological activities of steroidal derivatives exhibited androgen receptor antagonists [23], cytotoxic activities [24], anti-proliferative activities in a human androgen-responsive prostate cancer cell line [25]. In view of these observation, we report synthesis of 4`-(aryl)-4- pregneno[3,2-e]pyridinone derivatives and evaluated as 5α- reductase inhibitors and anti-prostate cancer agents.

Material and Methods

Chemistry

All melting points are uncorrected and were measured using an electrothermal capillary melting point apparatus. The IR spectra were recorded on a Shimadzu FT-IR 8101 PC infrared spectrophotometer. The ¹H-NMR spectra were determined with Bruker AM-200 MHz spectrometer. The chemical shifts are expressed on the δ (ppm) scale using TMS as the standard reference. Mass spectra were recorded on Finnigan SSQ operating at 70 ev. Elemental analysis determined on a Perkin Elmer 240 (microanalysis), Microanalysis Center, Cairo University, Cairo, Egypt.

Synthesis of 2`-mercapto-3`-cyano-4`-(aryl)-4- pregneno-[3,2-e]pyridine-20-ones (3a-h) and 1`- thiocarbamoyl -2`-amino-4`-(aryl)-4-pregneno-[3,2- e]pyridine-20-ones (4a-h)

General procedure: To a solution of progesterone 1 (0.314 g, 1 mmol), thiocyanoacetamide (0.100 g, 1.2 mmol), and appropriate aromatic aldehydes, namely, benzaldehyde, 4- bromo-, 2-chloro-, 4-chloro-, 4-flouro-, 2-nitro-, 4-nitro- or 4- methoxybenzaldehyde (2) (1 mmol) in n-butanol (10 mL), ammonium acetate (0.608 g, 8 mmol) was added. The reaction mixture was refluxed for 8 hours with stirring, and evaporated to dryness. The formed residue dissolved 10% hydrochloric acid (100 mL), washed with dichloromethane several time. The aqueous solution was alkalinized with 10% sodium bicarbonate, the resulted precipitate product was filtered off, dried and resorting to flash chromatography technique to separate compounds (3a-h) and (4a-h) by using eluent (benzene : MeOH; 9 : 1, v/v).

2`-Mercapto-3`-cyano-4`-(phenyl)-4-pregneno[3,2- e]pyridine-20-one (3a). Yield 32%, m.p. 255-257°C, [α]²⁵ D= + 79 (c 1, CHCl₃); IR (KBr): 3650 (SH), 3055 (CH-Ar), 2900 (CH-aliph), 2150 (CN), 1723 (C=O), 1630 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.86 (s, 3H, CH₃), 0.99-1.00 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.26-1.35 (m, 4H, 2CH₂), 1.42-1.54 (m, 4H, 2CH₂), 1.62-1.66 (m, 6H, 3CH₂), 1.72-1.85 (m, ¹H, CH), 2.17 (s, 3H, COCH₃), 2.35-2.40 (m, ¹H, CH), 2.55 (m, ¹H, CH), 6.10 (s, ¹H, SH exchangeable with D₂O), 6.24 (s, ¹H, CH), 7.22-7.56 (m, 5H, Ar-H); MS (EI): m/z 482 (54%) [M⁺]. Anal. C₃₁H₃₄N₂OS (582.68): Calcd. C, 77.14; H, 7.10; N, 5.80; S, 6.64; found C, 77.10; H, 7.01; N, 5.71; S, 6.55.

2`-Mercapto-3`-cyano-4`-(4-bromophenyl)-4-pregneno[3,2- e]pyridine-20-one (3b). Yield 21%, m.p. 267-269°C, [α]²⁵ D = + 57 (c 1, CHCl₃); IR (KBr): 3651 (SH), 3055 (CH-Ar), 2910 (CH-aliph), 2150 (CN), 1725 (C=O), 1630 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.89 (s, 3H, CH₃), 0.98-1.02 (m, ¹H, CH), 1.22 (s, 3H, CH₃), 1.28-1.36 (m, 4H, 2CH₂), 1.41-1.56 (m, 4H, 2CH₂), 1.61-1.65 (m, 6H, 3CH₂), 1.72-1.84 (m, ¹H, CH), 2.17 (s, 3H, COCH₃), 2.32-2.42 (m, ¹H, CH), 2.55 (m, ¹H, CH), 6.12 (s, ¹H, SH exchangeable with D₂O), 6.22 (s, ¹H, CH), 7.18-7.60 (m, 4H, Ar-H); MS (EI): m/z 561 (45%) [M⁺]. Anal. C₃₁H₃₃BrN₂OS (561.58): Calcd. C, 66.30; H, 5.92; N, 4.99; S, 5.71; found C, 66.15; H, 5.84; N, 4.90; S, 5.60.

2`-Mercapto-3`-cyano-4`-(2-chlorophenyl)-4-pregneno[3,2- e]pyridine-20-one (3c). Yield 22%, m.p. 284-285°C, [α]²⁵ D = + 75 (c 1, CHCl₃); IR (KBr): 3650 (SH), 3058 (CH-Ar), 2900 (CH-aliph), 2158 (CN), 1723 (C=O), 1637 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.90 (s, 3H, CH₃), 0.97-1.05 (m, ¹H, CH), 1.21 (s, 3H, CH₃), 1.27-1.35 (m, 4H, 2CH₂), 1.42-1.57 (m, 4H, 2CH₂), 1.60-1.64 (m, 6H, 3CH₂), 1.70-1.85 (m, ¹H, CH), 2.16 (s, 3H, COCH₃), 2.34-2.43 (m, ¹H, CH), 2.56 (m, ¹H, CH), 6.15 (s, ¹H, SH exchangeable with D₂O), 6.34 (s, ¹H, CH), 7.18-7.60 (m, 4H, Ar-H); MS (EI): m/z 517 (65%) [M⁺]. Anal. C₃₁H₃₃ClN₂OS (517.12): Calcd. C, 72.00; H, 6.43; Cl, 6.86; N, 5.42; S, 6.20; found C, 71.88; H, 6.32; Cl, 6.80; N, 5.33; S, 6.08.

2`-Mercapto-3`-cyano-4`-(4-chlorophenyl)-4-pregneno[3,2- e]pyridine-20-one (3d). Yield 36%, m.p. 269-271°C, [α]²⁵ D = + 91(c 1, CHCl₃); IR (KBr): 3651 (SH), 3056 (CH-Ar), 2904 (CH-aliph), 2155 (CN), 1722 (C=O), 1632 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.87 (s, 3H, CH₃), 0.96-1.00 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.27-1.36 (m, 4H, 2CH₂), 1.40-1.57 (m, 4H, 2CH₂), 1.62-1.66 (m, 6H, 3CH₂), 1.73-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.30-2.41 (m, ¹H, CH), 2.57 (m, ¹H, CH), 6.24 (s, ¹H, CH), 6.27 (s, ¹H, SH exchangeable with D₂O), 7.14-7.64 (m, 4H, Ar-H); MS (EI): m/z 517 (16%) [M⁺]. Anal. C₃₁H₃₃ClN₂OS (517.12): Calcd. C, 72.00; H, 6.43; Cl, 6.86; N, 5.42; S, 6.20; found C, 71.90; H, 6.32; Cl, 6.79; N, 5.34; S, 6.17.

2`-Mercapto-3`-cyano-4`-(4-fluorophenyl)-4-pregneno[3,2- e]pyridine-20-one (3e). Yield 22%, m.p. 253-255°C, [α]²⁵ D = + 22.5(c 1, CHCl₃); IR (KBr): 3650 (SH), 3056 (CH-Ar), 2909 (CH-aliph), 2159 (CN), 1723 (C=O), 1630 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.86 (s, 3H, CH₃), 0.97-1.04 (m, ¹H, CH), 1.17 (s, 3H, CH₃), 1.26-1.35 (m, 4H, 2CH₂), 1.38-1.48 (m, 4H, 2CH₂), 1.60-1.64 (m, 6H, 3CH₂), 1.70-1.84 (m, ¹H, CH), 2.19 (s, 3H, COCH₃), 2.31-2.40 (m, ¹H, CH), 2.56 (m, ¹H, CH), 6.10 (s, ¹H, SH exchangeable with D₂O), 6.26 (s, ¹H, CH), 7.14-7.58 (m, 4H, Ar-H); MS (EI): m/z 500 (18%) [M⁺]. Anal. C₃₁H₃₃FN₂OS (500.67): Calcd. C, 74.37; H, 6.64; N, 5.60; S, 6.40; found C, 74.28; H, 6.55; N, 5.50; S, 6.30.

2`-Mercapto-3`-cyano-4`-(2-nitrophenyl)-4-pregneno[3,2- e]pyridine-20-one (3f). Yield 31%, m.p. 265-267°C, [α]²⁵ D = + 88 (c 1, CHCl₃); IR (KBr): 3651 (SH), 3054 (CH-Ar), 2933 (CH-aliph), 2145 (CN), 1734 (C=O), 1641 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.85 (s, 3H, CH₃), 0.98-1.05 (m, ¹H, CH), 1.20 (s, 3H, CH₃), 1.27-1.35 (m, 4H, 2CH₂), 1.40-1.55 (m, 4H, 2CH₂), 1.61-1.66 (m, 6H, 3CH₂), 1.71-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.32-2.42 (m, ¹H, CH), 2.55 (m, ¹H, CH), 6.25 (s, ¹H, CH), 6.30 (s, ¹H, SH exchangeable with D₂O), 7.30-7.96 (m, 4H, Ar-H); MS (EI): m/z 528 (32%) [M⁺]. Anal. C₃₁H₃₃N₃O₃S (527.68): Calcd. C, 70.56; H, 6.30; N, 7.96; S, 6.08; found C, 70.50; H, 6.22; N, 7.88; S, 6.00.

2`-Mercapto-3`-cyano-4`-(4-nitrophenyl)-4-pregneno[3,2- e]pyridine-20-one (3g). Yield 31%, m.p. 278-280°C, [α]²⁵ D = + 34(c 1, CHCl₃); IR (KBr): 3651 (SH), 3058 (CH-Ar), 2914 (CH-aliph), 2158 (CN), 1722 (C=O), 1632 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.88 (s, 3H, CH₃), 0.99-1.04 (m, ¹H, CH), 1.19 (s, 3H, CH₃), 1.25-1.33 (m, 4H, 2CH₂), 1.38-1.54 (m, 4H, 2CH₂), 1.60-1.67 (m, 6H, 3CH₂), 1.70-1.86 (m, ¹H, CH), 2.17 (s, 3H, COCH₃), 2.30-2.40 (m, ¹H, CH), 2.58 (m, ¹H, CH), 6.22 (s, ¹H, CH), 6.27 (s, ¹H, SH exchangeable with D₂O), 7.28-7.98 (m, 4H, Ar-H); MS (EI): m/z 527 (52%) [M⁺]. Anal. C₃₁H₃₃N₃O₃S (527.68): Calcd. C, 70.56; H, 6.30; N, 7.96; S, 6.08; found C, 70.48; H, 6.13; N, 7.87; S, 6.01.

2`-Mercapto-3`-cyano-4`-(4-methoxyphenyl)-4- pregneno[3,2-e]pyridine-20-one (3h). Yield 28%, m.p. 258-260°C, [α]²⁵ D = + 46 (c 1, CHCl₃); IR (KBr): 3625 (SH), 3055 (CH-Ar), 2922 (CH-aliph), 2148 (CN), 1734 (C=O), 1628 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.87 (s, 3H, CH₃), 0.96-1.00 (m, ¹H, CH), 1.19 (s, 3H, CH₃), 1.26-1.36 (m, 4H, 2CH₂), 1.41-1.57 (m, 4H, 2CH₂), 1.61-1.65 (m, 6H, 3CH₂), 1.72-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.31-2.41 (m, ¹H, CH), 2.57 (m, ¹H, CH), 3.75 (s, 3H, OCH₃), 6.10 (s, ¹H, SH exchangeable with D₂O), 6.24 (s, ¹H, CH), 7.15-7.57 (m, 4H, Ar-H); MS (EI): m/z 512 (24%) [M⁺]. Anal. C₃₂H₃₆N₂O₂S (512.71): Calcd. C, 74.96; H, 7.08; N, 5.46; S, 6.25; found C, 74.87; H, 7.00; N, 5.33; S, 6.18.

1`-Thiocarbamoyl-2`-amino-4`-(phenyl)-4-pregneno[3,2- e]pyridine-20-one (4a). Yield 28%, m.p. 232-234°C, [α]²⁵ D = + 46 (c 1, CHCl₃); IR (KBr): 3505 (NH₂), 3021 (CH-Ar), 2950 (CH-aliph), 1730 (C=O), 1628 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.86 (s, 3H, CH₃), 0.97-1.01 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.26-1.34 (m, 4H, 2CH₂), 1.42-1.55 (m, 4H, 2CH₂), 1.60-1.65 (m, 6H, 3CH₂), 1.72-1.87 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.36-2.41 (m, ¹H, CH), 2.54 (m, ¹H, CH), 4.29 (s, ¹H, NH exchangeable with D₂O), 6.23 (s, ¹H, CH), 7.21-7.54 (m, 5H, Ar-H), 8.21 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 500 (32%) [M⁺]. Anal. C₃₁H₃₇N₃OS (499.71): Calcd. C, 74.51; H, 7.46; N, 8.41; S, 6.42; found C, 74.42; H, 7.34; N, 8.32; S, 6.33.

1`-Thiocarbamoyl-2`-amino-4`-(4-bromophenyl)-4- pregneno[3,2-e]pyridine-20-one (4b). Yield 25%, m.p. 256-258°C, [α]²⁵ D = + 123 (c 1, CHCl₃); IR (KBr): 3509 (NH₂), 3021 (CH-Ar), 2959 (CH-aliph), 1730 (C=O), 1627 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.88 (s, 3H, CH₃), 0.98-1.00 (m, ¹H, CH), 1.16 (s, 3H, CH₃), 1.24-1.32 (m, 4H, 2CH₂), 1.41-1.55 (m, 4H, 2CH₂), 1.61-1.66 (m, 6H, 3CH₂), 1.70-1.85 (m, ¹H, CH), 2.16 (s, 3H, COCH₃), 2.35-2.40 (m, ¹H, CH), 2.55 (m, ¹H, CH), 4.30 (s, ¹H, NH exchangeable with D₂O), 6.34 (s, ¹H, CH), 7.24-7.56 (m, 4H, Ar-H), 8.18 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 578 (42%) [M⁺]. Anal. C₃₁H₃₆BrN₃OS (578.61): Calcd. C, 64.35; H, 6.27; N, 7.26; S, 5.54; found C, 64.22; H, 6.20; N, 7.20; S, 5.46.

1`-Thiocarbamoyl-2`-amino-4`-(2-chlorophenyl)-4- pregneno[3,2-e]pyridine-20-one (4c). Yield 29%, m.p. 288-290°C, [α]²⁵ D = + 44 (c 1, CHCl₃); IR (KBr): 3518 (NH₂), 3021 (CH-Ar), 2967 (CH-aliph), 1745 (C=O), 1631 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.89 (s, 3H, CH₃), 0.98-1.05 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.25-1.34 (m, 4H, 2CH₂), 1.40-1.54 (m, 4H, 2CH₂), 1.62-1.65 (m, 6H, 3CH₂), 1.71-1.86 (m, ¹H, CH), 2.17 (s, 3H, COCH₃), 2.32-2.38 (m, ¹H, CH), 2.53 (m, ¹H, CH), 4.67 (s, 2H, NH₂ exchangeable with D₂O), 6.34 (s, ¹H, CH), 7.18-7.565(m, 4H, Ar-H), 8.16 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 534 (12%) [M⁺]. Anal. C₃₁H₃₆ClN₃OS (534.16): Calcd. C, 69.70; H, 6.79; Cl, 6.64; N, 7.87; S, 6.00; found C, 69.59; H, 6.71; Cl, 6.54; N, 7.82; S, 5.89.

1`-Thiocarbamoyl-2`-amino-4`-(4-chlorophenyl)-4- pregneno[3,2-e]pyridine-20-one (4d). Yield 17%, m.p. 345-247°C, [α]²⁵ D = + 78 (c 1, CHCl₃); IR (KBr): 3522 NH₂), 3034 (CH-Ar), 2980 (CH-aliph), 1740 (C=O), 1631 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.87 (s, 3H, CH₃), 0.96-1.00 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.27-1.36 (m, 4H, 2CH₂), 1.40-1.57 (m, 4H, 2CH₂), 1.62-1.66 (m, 6H, 3CH₂), 1.73-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.36-2.41 (m, ¹H, CH), 2.57 (m, ¹H, CH), 4.80 (s, 2H, NH₂ exchangeable with D₂O), 6.24 (s, ¹H, CH), 7.28-7.60 (m, 4H, Ar-H), 8.36 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 534 (52%) [M⁺]. Anal. C₃₁H₃₆ClN₃OS (534.16): Calcd. C, 69.70; H, 6.79; Cl, 6.64; N, 7.87; S, 6.00; found C, 69.58; H, 6.70; Cl, 6.55; N, 7.80; S, 5.88.

1`-Thiocarbamoyl-2`-amino-4`-(4-fluorophenyl)-4- pregneno[3,2-e]pyridine-20-one (4e). Yield 19%, m.p. 276-278°C, [α]²⁵ D = + 69 (c 1, CHCl₃); IR (KBr): 3524 (NH₂), 3030 (CH-Ar), 2977 (CH-aliph), 1738 (C=O), 1631 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.88 (s, 3H, CH₃), 0.97-1.04 (m, ¹H, CH), 1.17 (s, 3H, CH₃), 1.26-1.35 (m, 4H, 2CH₂), 1.38-1.48 (m, 4H, 2CH₂), 1.60-1.64 (m, 6H, 3CH₂), 1.70-1.84 (m, ¹H, CH), 2.19 (s, 3H, COCH₃), 2.34-2.40 (m, ¹H, CH), 2.56 (m, ¹H, CH), 4.93 (s, 2H, NH₂ exchangeable with D₂O), 6.26 (s, ¹H, CH), 7.15-7.62 (m, 4H, Ar-H), 8.35 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 517 (24%) [M⁺]. Anal. C₃₁H₃₆FN₃OS (517.7): Calcd. C, 71.92; H, 7.01; N, 8.12; S, 6.19; found C, 71.80; H, 6.96; N, 8.00; S, 6.10.

1`-Thiocarbamoyl-2`-amino-4`-(2-nitrophenyl)-4- pregneno[3,2-e]pyridine-20-one (4f). Yield 31%, m.p. 236-238°C, [α]²⁵ D = + 150 (c 1, CHCl₃); IR (KBr): 3509 (NH₂), 3021 (CH-Ar), 2949 (CH-aliph), 1730 (C=O), 1634 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.91 (s, 3H, CH₃), 0.98-1.05 (m, ¹H, CH), 1.20 (s, 3H, CH₃), 1.27-1.35 (m, 4H, 2CH₂), 1.40-1.55 (m, 4H, 2CH₂), 1.61-1.66 (m, 6H, 3CH₂), 1.71-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.36-2.42 (m, ¹H, CH), 2.55 (m, ¹H, CH), 4.70 (s, 2H, NH₂ exchangeable with D₂O), 6.25 (s, ¹H, CH), 7.29-7.92 (m, 4H, Ar-H), 8.32 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 544 (100%) [M⁺]. Anal. C₃₁H₃₆N₄O₃S (544.71): Calcd. C, 68.35; H, 6.66; N, 10.29; S, 5.89; found C, 68.13; H, 6.58; N, 10.18; S, 5.79.

1`-Thiocarbamoyl-2`-amino-4`-(4-nitrophenyl)-4- pregneno[3,2-e]pyridine-20-one (4g). Yield 26%, m.p. 358-360°C, [α]²⁵ D = + 179(c 1, CHCl₃); IR (KBr): 3520 (NH₂), 3033 (CH-Ar), 2956 (CH-aliph), 1742 (C=O), 1646 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.89 (s, 3H, CH₃), 0.99-1.03 (m, ¹H, CH), 1.18 (s, 3H, CH₃), 1.28-1.33 (m, 4H, 2CH₂), 1.39-1.55 (m, 4H, 2CH₂), 1.60-1.65 (m, 6H, 3CH₂), 1.70-1.85 (m, ¹H, CH), 2.17 (s, 3H, COCH₃), 2.35-2.40 (m, ¹H, CH), 2.58 (m, ¹H, CH), 4.68 (s, 2H, NH₂ exchangeable with D₂O), 6.22 (s, ¹H, CH), 7.32-7.95 (m, 4H, Ar-H), 8.45 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 544 (100%) [M⁺]. Anal. C₃₁H₃₆N₄O₃S (544.71): Calcd. C, 68.35; H, 6.66; N, 10.29; S, 5.89; found C, 68.27; H, 6.60; N, 10.20; S, 5.80.

1`-Thiocarbamoyl-2`-amino-4`-(4-methoxyphenyl)-4- pregneno[3,2-e]pyridine-20-one (4h). Yield 18%, m.p. 291-293°C, [α]²⁵ D = + 100 (c 1, CHCl₃); IR (KBr): 3515 (NH₂), 3031 (CH-Ar), 2960 (CH-aliph), 1740 (C=O), 1630 (C=C) cm^-1. ¹H NMR (CDCl₃): δ 0.87 (s, 3H, CH₃), 0.94-1.00 (m, ¹H, CH), 1.21 (s, 3H, CH₃), 1.26-1.36 (m, 4H, 2CH₂), 1.41-1.57 (m, 4H, 2CH₂), 1.61-1.65 (m, 6H, 3CH₂), 1.72-1.85 (m, ¹H, CH), 2.18 (s, 3H, COCH₃), 2.33-2.41 (m, ¹H, CH), 2.57 (m, ¹H, CH), 3.72 (s, 3H, OCH₃), 4.48 (s, ¹H, NH exchangeable with D₂O), 6.24 (s, ¹H, CH), 7.25-7.55 (m, 4H, Ar-H), 8.27 (s, 2H, NH₂ exchangeable with D₂O); MS (EI): m/z 529 (40%) [M⁺]. Anal. C₃₂H₃₉N₃O₂S (529.74): Calcd. C, 72.55; H, 7.42; N, 7.93; S, 6.05; found C, 72.44; H, 7.35; N, 7.86; S, 6.00.

5α-Reductase inhibitors

Treatment of animals: Animals were obtained from the animal house colony of the NRC Cairo Egypt. All animals were allowed free access to water and were kept on a constant standard diet. 19 Groups, each of 12 male Sprague-Dawley rats in the postnatal third days, were treated subcutaneously with the 5α-reductase inhibitor (tested compound or reference standard).

The tested compounds were suspended in 5% Tween 80 in water. The vehicle was used for both standard and negative control group, beginning on the postnatal third day until the age of seven weeks. 18 Groups were used to test the activities, of which one was used as the positive control for anastrozole and another served as the negative control group. After scarifying, blood was withdrawn for testosterone and dihydrotestosterone (DHT) determination [26]. Moreover, intraprostatic concentrations of testosterone and DHT were determined [27]. The biological experiments were performed according to the official standards.

Anti-prostate cancer screening anti-androgenic bioassay in human prostate cancer cells

Human prostate cancer LNCaP and PC-3 cells were maintained in RPMI medium and Dulbecco's minimum essential medium (DMEM), respectively. Both media were supplemented with penicillin (25 units/mL), streptomycin (25 μg/mL), and 10% fetal calf serum. For the androgen receptor transactivation assay, an androgen-dependent reporter gene transcription test was employed as the primary screening for potential antiandrogen identification. This assay was first performed in LNCaP cells, which express a clinically relevant mutant AR. Once anti-androgenic activity was detected in the LNCaP AR transactivation assay, compounds were re-examined for their potential activity against wild type AR. Wild type AR transactivation assay was performed in PC-3 host cells, which lack an endogenous, functional AR.

The method and conditions of cell and gene transfection have been described previously. In brief, cells were plated in 24-well tissue culture dishes for 24 (PC-3 cells) or 48 (LNCaP cells) h prior to transfection. Subsequently, LNCaP cells were transfected with a reporter gene, MMTV-luciferase, which contains MMTV-LTR promoter and androgen receptor binding element, and PRL-SV40, which served as an internal control for transfection efficiency. PC-3 cells were transfected with a wild type AR expression plasmid, pSG5AR, in addition to the above-mentioned MMTV-luciferase reporter gene and PRL-SV40 internal control. SuperFect (Qiagen, Chatsworth, CA) was employed as the transfection reagent following manufacturer's recommendations.

At the end of a five-hour transfection, the medium was changed to DMEM or RPMI supplemented with 10% charcoal dextran-stripped, i.e., androgen-depleted, serum. After 24 h, the cells were treated with 1 nM of DHT and/or test compounds at the designated concentration for another 24 h. The cells were harvested for luciferase activity assay using Dual Luciferase Assay System (Promega, Madison, WI). The derived data were expressed as relative luciferase activity normalized to the internal luciferase control. Cells cultured in medium containing DHT (androgen), as a control, induced a marked reporter gene expression. Test compounds capable of significantly suppressing this DHT-induced reporter gene expression were identified as potential antiandrogens [27,28].

Results and Discussion

Chemistry

A series of 2`-mercapto-3`-cyano-4`-(aryl)-4-pregneno-[3,2- e]pyridine-20-one (3a-h) and 1`-thiocarbamoyl -2`-amino-4`- (aryl)-4-pregneno-[3,2-e]pyridine-20-one derivatives (3a-h) were synthesized using progesterone (1) as starting materials. Treatment of 1 with thiocyanoa-cetamide and appropriate aromatic aldehydes, namely, benzaldehyde, 4-bromo-, 2- chloro-, 4-chloro-, 4-flouro-, 2-nitro-, 4-nitro- or 4- methoxybenzaldehyde (2) in the presence of ammonium acetate in n-butanol afforded the corresponding 4- pregneno[3,2-e]pyridine-20-one derivatives 3a-h and 4a-h as a mixture which was chromatographically separated (Figure 1).

Figure 1. Synthetic route for compounds 3a-h and 4a-h.

Biological activities

The authors synthesized many steroidal derivatives having 5α- reductase inhibitor activities [29,30]. It is worth to mention that in most of these derivatives the ring D of the steroid part was fused mainly with heterocyclic ring system [31,32]. The newly synthesized derivatives here belongs to a large extent to the aforementioned derivatives for their 5α-reductase inhibitors activities but these new derivatives containing pregnane nucleus where the heterocyclic fused ring system was onto ring A so it was bioassayed for their 5α-reductase inhibitors activities. So here the authors study the effects of the pregnane nucleus and fusion of heterocyclic ring system on the 5α- reductase inhibitors activities.

5α-Reductase inhibitors

All the tested compounds showed potent 5α-reductase inhibitors activities. The descending order of 5α-Reductase inhibitor activities was as follow: 3g (ED₅₀: 0.28 μM), 3f (ED₅₀: 0.29 μM), 3a (ED₅₀: 0.30 μM), 3e (ED₅₀: 0.32 μM), 3c (ED₅₀: 0.33 μM), 3d (ED₅₀: 0.35 μM), 3b (ED₅₀: 0.36 μM), 3h (ED₅₀: 0.38 μM), 4g (ED₅₀: 0.39 μM), 4f (ED₅₀: 0.40 μM), 4a (ED₅₀: 0.41 μM), 4e (ED₅₀: 0.42 μM), 4c (ED₅₀: 0.43 μM), 4d (ED₅₀: 0.45 μM), 4b (ED₅₀: 0.46 μM), 4h (ED₅₀: 0.47 μM), Anastrozole^® (ED₅₀: 1.09 μM). It was worth to mention that all tested compounds were more potent than Anastrozole^® (Table 1).

Table 1. 5α-Reductase inhibitors activities and anti-proliferate activity against prostate cancer cell lines.

Anti-proliferate activity against prostate cancer cell lines

All the tested compounds were screened as anti-tumor activities in two prostate cell lines namely, LNCaP and PC-3.

Regarding the in vitro anti prostate cancer activities against prostate cancer cell lines LNCaP the descending order of activities were: 3g (IC₅₀: 2.34 μM), 3f (IC₅₀: 2.45 μM), 3a (IC₅₀: 2.56 μM), 3e (IC₅₀: 2.76 μM), 3c (IC₅₀: 2.77 μM), 3d (IC₅₀: 2.87 μM), 3b (IC₅₀: 2.90 μM), 3h (IC₅₀: 2.91 μM), 4f (IC₅₀: 3.01 μM), 4a (IC₅₀: 3.10 μM), 4e (IC₅₀: 3.29 μM), 4c (IC₅₀: 3.39 μM), 4d (IC₅₀: 3.47 μM), 4b (IC₅₀: 3.56 μM), 4g (IC₅₀: 2.95 μM), 4h (IC₅₀: 3.76 μM), Anastrozole^® (IC₅₀: 11.23 μM). It was worth to mentioned that all tested compounds were more potent than Anastrozole^® (Table 1).

Regarding the in vitro anti prostate cancer activities against prostate cancer cell lines PC3 the descending order of activities were: 3g (IC₅₀: 7.45 μM), 3f (IC₅₀: 7.67 μM), 3a (IC₅₀: 9.54 μM), 3e (IC₅₀: 9.78 μM), 3c (IC₅₀: 9.9 μM), 3d (IC₅₀: 10.45 μM), 3b (IC₅₀: 10.56 μM), 3h (IC₅₀: 10.67 μM), 4g (IC₅₀: 10.78 μM), 4f (IC₅₀: 11.23 μM), 4a (IC₅₀: 11.56 μM), 4e (IC₅₀: 11.89 μM), 4h (IC₅₀: 14.21 μM), 4c (IC₅₀: 12.54 μM), 4d (IC₅₀: 12.78 μM), 4b (IC₅₀: 13.89 μM), Anastrozole^® (IC₅₀: 34.24 μM). It was worth to mention that all tested compounds were more potent than Anastrozole^® (Table 1).

Structural activity relationship

Careful examination of the relationship between chemical structure of the newly synthesized derivatives and their biological activities as 5α-reductase inhibitors, anti PC3 and anti LNCaP carcinoma activities to lead the following structural activities relationship.

1. The fusion of the pyridine heterocyclic ring system onto ring A of the steroidal scaffold is essential for 5α-reductase inhibitors, anti PC3 and anti LNCaP carcinoma activities.

2. The thiol and cyano groups provid more 5α-reductase inhibitors, anti PC3 and anti LNCaP carcinoma activities than the amino and thioamide ones due to the higher lipophilic characters of thiol and cyano groups.

3. Regarding the substitutes on the aromatic moiety the descending order of activity was 4-NO₂, 2-NO₂, H, 4-F, 2- Cl, 4-Cl, 4-Br, 4-OCH₃.

Acknowledgement

The project was financially supported by King Saud University, Vice Deanship of Research Chairs.

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