18-substituted progesterone derivatives as inhibitors of aldosterone biosynthesis

J. steroid Biochem.Vol. 33, No. 1, pp. 119-124,1989 Printed in Great Britain. All rights reserved

0022-4731/89$3.00+ 0.00 Copyright 0 1989MaxwellPergamon Macmillan plc

l&SUBSTITUTED PROGESTERONE INHIBITORS OF ALDOSTERONE

DERIVATIVES AS BIOSYNTHESIS

ANTOINETTEVIGER,* SUZY COUSTAL, SERGEPERARD, ANNIE PIFFETEAU and AND& MARQIJET Laboratoire de Chimie Organique Biologique, UA CNRS 493, Universitk Paris VI, 4, place Jussieu, 15252 Paris Cedex 05. France (Received 30 July 1988; receivedfor publication3 February 1989)

Summary-The synthesis of new progesterone derivatives substituted at the 18 methyl group is described. These compounds are designed as 18-monooxygenase, cytochrome P-450-dependent potential k, inhibitors. Preliminary results on the in vitro biological investigation of these modified progesterones

are presented.

INTRODUCTION

Aldosterone is a potent mineralocorticoid which regulates the electrolyte balance of body fluids by promoting potassium elimination and sodium retention [l]. An aldosterone overproduction leads to oedematous diseases and hypertension. These disorders are currently treated by aldosterone antagonists at the receptor level, belonging to the spironolactone series [2]. However, their clinical use is limited by side effects attributed to their antiandrogenic and progestational properties (31. With a view to developing more specifically targeted compounds, we investigated the specific inhibition of aldosterone biosynthesis by mechanism based (&) inhibitors [4]. Aldosterone is biosynthesized in mammalian adrenals [S] according to Scheme 1. In the final stages, progesterone is sequentially hydroxylated on carbons 21, 11 and 18 by cytochrome P-450-dependent monooxygenases. 1l/I and 21 hydroxylases have been isolated and fully characterized [6,7]. It has been shown by in vitro experiments [8] that cytochrome P-450 1l/3 catalyses 1l/I as well as 18 hydroxylations. However it is not yet established whether it is the only enzyme responsible for the in vivo 18 hydroxylation or if another cytochrome is involved. Inhibition of cytochrome P-450 monooxygenases is well documented [9]. Three classes of inhibitors, giving intermediates which either covalently bind to the protein, or to the prosthetic heme group, or coordinate quasiirreversibly with the iron atom have been developed. This approach has been widely used for inhibiting the biosynthesis of various steroidal hormones [lo, 111. However, very few studies have been devoted to the inhibition of 18-hydroxylase: the Merrell Dow research group reported acetylenic compounds for the *To whom correspondence

should be addressed.

inhibition of aldosterone production, without biochemical data [12, 131. We have already published results obtained with several progesterones modified at C-18 which are representative of the above mentioned three classes of inhibitors [14]. This paper describes the synthesis of other progesterone derivatives and their biological investigation. EXPERIMENTAL

Melting points were determined on a Kofler apparatus and were uncorrected. “C and [‘H]NMR spectra were recorded either on a Jeol FX 9oQ or on a Bruker AC200 spectrometer in CDCl,. Chemical shifts are reported as values (ppm) relative to TMS. IR spectra were recorded on a Philips SP3-100. Optical rotations were measured with a Perkin Elmer 241 polarimeter. Materials

[1,2,6,7-‘Hlaldosterone (75-105 Ci/mmol) was purchased from the Radio-chemical Centre Amersham (England). Unlabelled aldosterone, pregnenolone and progesterone were from Roussel-Uclaf Laboratories (Romainville, France). Buffers

Buffer A was composed of 20mM Tris-HCl, 250 mM sucrose, pH 7.4. Buffer B contained 20 mM Tris-HCl, KC1 (4.17 mM), MgCl, (12.3 mM), CaCl, (5.6 mM), NaCl (88.3 mM), pH 7.4. BufIer C was a 1: 1 mixture of buffers A and B. Inhibition test

Sprague-Dawley rats were decapitated and adrenal glands were immediately removed and homogenized in buffer A (1 gland in 0.5 ml) at 4°C using a teflon

119

ANTOINETTE VIOERet al.

120

PROGESTERONE

ALOOSTERONE

Scheme 1.

Potter homogeniser. After centrifugation (20 min at 9OOg) the supernatant was dialyzed against buffer A for 2 h at 4°C. The incubation mixture consisted of dialysate (700~1), buffer B (700~1), NADP+ (0.48 mg), isocitrate (2.6 mg), progesterone (0 or 4Opg) and inhibitor. The steroids were dissolved in ethanol, then diluted with buffer C in order to introduce the minimal amount of ethanol (N 2 11). The blank consisted of the same mixture except that inhibitor was omitted. Incubations were performed for 1 h at 37°C under aerobic conditions and steroids were extracted by using SEP-PAK C,, Cartridges (25 ml of ethanol were used for elution). The amount of aldosterone biosynthesized in each assay was then determined using a conventional RIA method, with highly specific antibodies raised against aldosterone [ 151. The stability of compounds 1,2 and 4 was assayed omitting the adrenal homogenate under otherwise identical conditions. After 1 h at 37°C the mixture was extracted with methylene chloride and the purity of the compounds was assessed by TLC on silica gel (Merck Kieselgel 6OF-254, 0.25 mm; hexan+ethyl acetate 1: 1).

ll-methane

sulfonate pregn-4-ene

3,20-dione 1

Methane sulfonic chloride (0.1 mmole) was added to 25 mg (0.08 mmole) of 1I-hydroxypregn4-ene 3,20-dione [16] as an equilibrium mixture 9&Y dissolved in 3 ml of dry pyridine at 0°C and stirred for 16 h at room temperature. The crude mixture was

then poured into cold water, extracted in the usual way and purified by chromatography on silica gel (hexane-ethyl acetate 1: 1) yielding 1 mg of pure 1 (2%). Mass spectrum (C.I./NH$): m/z 409 (MH)+. [‘I-IjNMR: 1.20 (3H, s, Me 19), 2.20 (3H, s, Me 21), 2.95 (3H, s, CHS-SO*-), 3.75 and 4.3 (2H, AB system, J = 9 Hz, CH,-18), 5.75 (lH, s, H-4). 3,3-ethylenedioxy-I8-iodo-20-tetrahydropyranyloxy pregn-5-ene 11

Dihydropyran (DHP) (1.2 ml) and pyridinium ptoluenesulfonate (PPTS) (23 mg) were added to 427 mg of iodo compound 10 (as a mixture of 20a and 20/I isomers) prepared according to Kalvoda et a!.[ 171 and dissolved in dry methylene chloride (5 ml). After 4 h at room temperature the mixture was diluted with ether, washed with saturated brine, dried and concentrated under vacuum, yielding 385 mg of crude 11. Id-chloro-20-hydroxy

pregn-4-ene3-one

14a

Two mmol(556 mg) of tetrabutylammonium chloride (TBACl) were dried under vacuum (0.05 mm) at 50°C for 24 h and 57 mg of 11, dissolved in 3.5 ml of dry HMPA were added. The mixture was stirred at 90°C for 6 h, diluted with CH,Q (100 ml) washed with water and concentrated in oacuo (0.1 mm). Silica gel chromatography (cyclohexanwthyl acetate 3 : 1) yielded 38 mg (81%) of a mixture of (20a and 208 isomers) 12a. This mixture (35 mg, 0.07 mmole) was dissolved in ethanol (4 ml), 4mg of pyridinium ptoluene sulfonate were added and the mixture was

18-Substitute pro~terone stirred at room temperature for 24 h. Ethanol was then evaporated, methylene chloride was added and work-up was achieved in the usual way affording 25 mg of 14a. [‘H]NMR: 1.1 (3H, d, Me 21), 1.2 (3H, s, Me 19), 3.5 and 3.7 (2H, AB system, J= 13 Hz, CH,-18), 4.0 (IH, m, H-20), 5.7 (lH, s, H-4). [“CINMR: 45 (CH,CI-18). 18-chloro-pregn-4-ene-3,20-dione

3

Pyridinium dichromate (PDC) (250 mg) was added to 25 mg of i4a dissolved in 2 ml of dry methylene chloride and the mixture was stirred for 24 h at room temperature. Ethyl acetate (100 ml) was then added, the precipitate was filtered on celite and the organic solution washed to neutrality. The crude product was purified by TLC (cyclohexane-ethyl acetate 1: 1) yielding 8 mg (32%) of pure 3, mp: 135-136°C. Mass s~tr~ (C.I./NH,+): m/z 349, 351 (MH)‘. [‘H]NMR: 1.2 (3H, s, Me 19), 2.25 (3H, s, Me 21), 3.45 and 3.6 (2H, AB system, J = 13 Hz, CH,-18), 5.7 (lH, s, H-4). [cc]::= + 88” (c = 0.077, CHCl,). 18-methylthio-ZO-hydroxy pregn-4-ene-3-one

146

The iodide 11 (195 mg) and 300 mg of sodium thiomethoxide were dissolved in dry HMPA (8 ml) and heated overnight at 100°C under argon. HMPA was then removed by vacuum distillation, the residue redissolved in ethyl acetate and work-up achieved as usual. A catalytic amount of p-toluene sulfonic acid was added to the crude reaction product dissolved in ethanol (10 ml) and the mixture was allowed to react for 90min at 60°C. After conventional work-up the residue was purified by flash chromatography on silica gel (cyclohexane-ethyl acetate 2.5 : 1) affording 54mg (43%) of X4b, mp: 145-146°C. [‘HINMR: 1.13 (d, 3H, J=6Hz, Me 211, 1.18 (s, 3H, Me 19), 2.19 (s, 3H, SMe), 3.77 (m, IH, H-20), 5.71 (s, lH, H-4). Id-methylthio-pregn-I-ene-3,20-dione

7

Dry DMSO (337~1) was added to a solution of oxalyl chloride (183 ~1) in 4.5 ml dry dichloro-

Scheme 2. 38 33/1-l

a:

121

methane at -60°C. After stirring the mixture for 30 min, 61 mg of 14b in 5 ml of methylenechloride were added, followed after 40 min stirring at - 60°C by 1 ml of triethylamine. The mixture was atlowed to react at room temperature for 1 h. After dilution with water and conventional work-up, the residue was chromatographed on silica gel (cyclohexane-ethyl acetate 4: 1) yielding 31 mg of pure 7 (51%), mp: 155-158°C. Mass spectrum (C.I./NH:): m/z 361 (MH+), 378 (M +NH$). [‘HJNMR: 1.18 (s, 3H, Me 19), 1.92 (s, 3H, SMe), 2.25 (s, 3H, Me 21), 5.70 (s, lH, H-4). Anal (C,H,,O$) C,H. [aj2Dt= -l-209” (c = 0.185, CHCI,). RESULTS AND DISCUSSION

A. Synthesis

The synthesis of the new compo~ds 1,3 and 7 is described. Preparation of 2 and 5 was achieved according to Kalvoda et ul.[ 17,191. We have reported elsewhere the synthesis of 4 [20], 6 and 8 [21]. The mesylate 1 was prepared from an equilibrium mixture consisting of 18-hydroxyprogesterone !Y and its more stable hemiacetal9 (Scheme 2). However the ~spla~ment of the equilib~um could not be achieved and the reaction proceeded with a very poor yield. We expected that the iodide 10 could be a convenient starting material to prepare C-18 modified progesterones. First attempts to achieve a direct nucleophilic displa~ment of the iodine failed due to the formation of the cyclic ether 13 (Scheme 3) and we had to protect the hydroxy group at C-20. Under the reactions conditions which are suitable to prepare the tert-butyldimethylsilyl ether, only 13 was again formed. Protection was achieved with dihydropyran under acidic catalysis. Several reactions were tried to displace the iodine at C-18 in compound 11. We checked that the substitution by NaN, can be achieved in HMPA, conditions already described by Choay et a[.[221 on a

I&Cl, pyridine.

d

I

X

A@+ OTHP

0

\

0

c

3X=CI I

X=SMe

12a -

X=CI

l&s

X-Cl

la

X=SMe

I

Scheme 3. a: DHP, PFTS; b: + SiCl, DMAP; c: KF, 18-crown-6, DMSO or TBAF, HMPA; d: TBACl,

I

HMPA or NaSMe, HMPA; e: PPTS, EtOH; f: PDC, CH,CI,; f’: DMSO, (COCI),

I?

r

-‘o

MO 0

\ 4@+ c 0

0

-10

Scheme 4.

18Substituted progesterone related compound. However, treatment of 11 with tetrabutylammonium fluoride in HMPA or with KF/l%crownd in CH,CN (or DMSO) afforded again as main product the cyclic ether 13, obviously formed by neighboring group participation of the lone pair of the oxygen at C-20 (Scheme 4). But tetrabutylammonium chloride and sodium thiomethoxide in HMPA led to the expected products 12a and 12b,respectively (Scheme 3). Cleavage of the tetrahydropyranyl ether followed by Swem oxidation [28] yielded the desired C-18 modified progesterones 3 and 7.

Table

I. Aldosterone biosynthesis inhibition induced by compounds 13 % Inhibition of aldosterone biosynthesis (concentration)

Compound tested

0% (10pM)

1

0 1

B. Inhibition tests Aldosterone biosynthesis was studied using rat adrenal homogenates after addition of progesterone as substrate. Incubation was performed at 37°C for 1 h in air. The amount of aldosterone was evaluated by a RIA [ 151.Inhibition by compounds l-g (Table 1) was estimated by measuring the amount of aldosterone biosynthesized with or without inhibitor, all the other parameters being constant. Results obtained with compound 4, already described [14], are given for comparison. At this stage of our work, using crude adrenal homogenates offers several advantages for testing aldosterone biosynthesis inhibition. First it is easier to handle than the labile purified l&hydroxylase [23]. Furthermore, it contains the different enzymes involved in the last biosynthetic steps, allowing the use of progesterone derivatives rather than corticosterone. However, this preparation is inappropriate for obtaining convincing kinetic data due to the inevitable presence of endogenous precursors, which cannot be removed even after prolonged dialysis. Consequently the results obtained in this study are qualitative and do not disclose the nature of the inhibition which may be only competitive. Nevertheless within the series of compounds tested under the same conditions the inhibition tests are comparative and may be interpreted as follows. 1 and 2 do not inhibit aldosterone biosynthesis even at high concentration (10 PM), but 3 is slightly active. These results suggest that introduction of a bulky group at C-18 precludes binding and (or) reaction at the active site of this enzyme. Work is in progress to synthesize the 18-fluoro derivative which might be a valuable inhibitor. Compound 7 was prepared since previous studies with aromatase [24,25] have shown that thiomethylethers were good competitive inhibitors of the cytochrome P-450 dependent monooxygenase. We also synthesized compound 8 [20] for checking the ability of other thioethers to inhibit the 18-monooxygenase. The thiomethyl derivative 7, although more potent, is still a poor inhibitor. 19-Mercapto compounds were described as k,, inhibitors of aromatase [26] or P450s~~ [27]. We did not attempt to synthesize the corresponding 18-mercapto progesterone, since in that case formation of a stable hemithioketal is expected.

123

dP d?@ 0

0% (10fiM)

0

0

2

Cl

0

40% (IOpM)

0

0

3

0

0

4

A?@ /

100% (0.8 PM) 75% (0.2 PM) 30% (0.08j~M)

0 20% (IOJIM) 0 A&+

/

5

0

d? 0

0

...-

0% (10pM)

6

45% (IOjtM)

7

0% (IOpM)

8

Unsaturated compounds are designed to react after oxidation with an heminic N-atom [9]. We have already reported that derivative 4 is a very potent

124

ANTOINET~VIGERer al.

inhibitor [14]. Compound 5 had also to be tested to check the influence of the 18 side chain length. It is a poor inhibitor. Compound 6 (unexpectedly obtained [21]) does not inhibit aldosterone biosynthesis. The synthesis of other potential inhibitors of the 18-monooxygenase, as well as the determination of inhibition characteristics is underway.

Acknowledgements-We thank the CNRS (AIP No. 06931), the MRES (DBcision No 87CO484) and the RousselUCLAF Company for financial support. Dr A. Carayon (CHU Pitie Salpttritre) is gratefully thanked for the generous gift of antialdosterone antibodies.

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