Discovery of novel intracellular receptor modulating drugs

ft. Steroid Biochem. Molec. Biol. Vol. 56, Nos 1-6, pp. 61--66, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760(95)00224-3 0960-0760/96 $15.00 + 0.00

Pergamon

Discovery of Novel Intracellular Receptor Modulating Drugs Todd K. Jones,* Charles Pathirana, Mark E. G o l d m a n , t Lawrence G. H a m a n n , Luc J. Farmer, Teodoro Ianiro, Michael G. Johnson, Steven L. Bender,: Dale E. Mais and Robert B. Stein* Ligand Pharmaceuticals, Inc., 9393 Towne Centre Drive, San Diego, CA 92121, U.S.A. U t i l i z i n g the c o - t r a n s f e c t i o n assay as a guide to d e t e r m i n i n g s t r u c t u r e activity r e l a t i o n s h i p s , we have b e e n p u r s u i n g the ddiscovery o f n o n - s t e r o i d a l h P R m o d u l a t o r s . S m a l l m o l e c u l e , n o n - s t e r o i d a l lead s t r u c t u r e s have b e e n identified. O p t i m i z a t i o n o f these s t r u c t u r e s has y i e l d e d m o r e p o t e n t h P R m o d u l a t o r s . I m p r o v e d c r o s s - r e a c t i v i t y profiles with o t h e r i n t r a c e l l u l a r r e c e p t o r s are a feature o f these c o m p o u n d s o w i n g to their n o n - s t e r o i d a l nature.

ft. Steroid Biochem. Molec. Biol., Vol. 56, Nos 1-6, pp. 61-66, 1996

INTRODUCTION

agonists and antagonists of the human progesterone receptor (hPR). Utilizing cloned cDNAs for the individual IRs, powerful cell-culture-based assays have been created [11] to assess the abilities of small organic molecules to act as agonists or antagonists of any one of the hormone-activated IRs. In these assays, termed cotransfection assays, the c D N A for the human IR drug target of interest is transfected into a mammalian cell line lacking appreciable levels of IRs of its own; also transfected with the IR c D N A is a hormone-responsive reporter plasmid. T h e reporter plasmid contains c D N A for a readily detectable protein (e.g., firefly luciferase or chloramphenicol acetyl transferase) under control of a promoter containing the H R E cognate to the transfected IR c D N A (e.g. a progesterone response element is used when the PR c D N A is used). Co-transfection assays can be miniaturized to run in 96-well microtitre plate format and robotized to provide excellent reproducibility and high throughput. In this form, co-transfection assays can contribute strongly to the drug discovery process. Co-transfection assays can be used to profile an existing drug to determine the extent to which the compound crossreacts with other IRs in addition to the intended target, providing useful insights into the basis for its therapeutic actions and side effects. This can delineate objectives for creation of improved compounds acting through clinically proven mechanisms, i.e. demonstrably effective IR targets. In this light, the evaluation of the PR antagonists RU486 [12] and ZK98,299 [13]

T h e cloning of the glucocorticoid receptor in 1985 [1] facilitated the rapid identification of related Intracellular Receptors (IRs) mediating the actions of the other steroid hormones (Fig.l), including progestins [2,3], estrogens [4], androgens [5-7] and mineralocorticoids [8]. Identification of the IR superfamily [ 1, 9] and consequent elucidation of the molecular basis for non-peptidyl hormone action are among the major advances in biological sciences over the last decade. More than fifty members of the IR superfamily have been identified to date [1]. For over twenty of these IRs, known non-peptidyl hormones trigger latent biochemical activities resulting in their activation as transcription factors regulating specific sets of target genes containing appropriate H o r m o n e Response Elements (I-IREs) in their promoters. Increased understanding of hormone action following molecular characterization of IRs has brought new opportunities and new tools to create novel and potentially better medicines that mimic or block hormone action [10]. This report describes the use of ,;ome of these molecular drug discovery approaches to identify novel non-steroid Proceedings of the 12th International Symposium of The ffournal of Steroid Biochemistry & Molecular Biology, Berlin, Germany, 21-24 May 1995. *Correspondence to Todd K. Jones or Robert B. Stein. afPresent address: Signal Pharmaceuticals, Inc., 5555 Oberlin Drive, San Diego, CA 92121, U.S.A. ~Present address: Agouron Pharmaceuticals, Inc., 10350 N. Torrey Pines Road, Suite 100, La Jolla, CA 92037, U.S.A. 61

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Todd K. Jones et al.

(Fig.2) highlights the need for PR pharmacophores with less cross-reactivity with the most closely related IRs: glucocorticoid receptor (GR), androgen receptor (AR) and mineralocorticoid receptor (MR). Co-transfection assays can also be used to carry out high throughput screening (HTS) of structurally diverse collections of chemicals (compound libraries) to allow the identification of novel pharmacophores to serve as chemical leads for drug discovery. Further, co-transfection assays can be used to quantitatively assay synthesized analogues of a lead compound to facilitate the elucidation of structure activity relationships and to guide compound optimization for potency and for desired functional effect on gene expression, i.e. for acting as a hormone agonist to stimulate transcription or as a hormone antagonist to inhibit transcription. The reproductive and endocrine actions of progesterone are mediated by PR-A and PR-B, two closely related members of the IR protein superfamily [14]. Additional ("non-genomic") pharmacological activities of progesterone and related endogenous metabolites are mediated by interactions with an unrelated protein receptor in the plasma membranes of neurons, associated with the receptor for the inhibitory amino acid neurotransmitter gamma amino butyric acid (GABA) [15]. Medically, there is increasing interest in postmenopausal Hormone Replacement Therapy (HRT) [16] in which progestins are added to Estrogen Replacement Therapy (ERT) in order to counter-poise the 5- to 20-fold uterine cancer risk attributable to unopposed estrogen action [17, 18]. Because of the susceptibility of progesterone to hepatic oxidations, relatively large oral doses of progesterone are required to achieve systemic concentrations sufficient to give endocrine activity suitable for cancer therapy or for H R T [19]. An undesirable consequence of oral administration of the massive doses of progesterone required to achieve systemic PR activation is the flooding of the body with oxidized progesterone metabolites. This is felt to be the cause of one of the main impediments to the use of oral progesterone for H R T : the side effect of

somnolence, apparently due to the production of neuroactive sedating progesterone metabolites [20] interactive with the GABA receptor as the progesterone traverses the liver after oral absorption. T he discovery of non-steroidal PR pharmacophores such as we describe offers the opportunity to pursue from a novel structural starting point widely divergent structure activity relationships governing the interactions of compounds with PR-A and PR-B and the GABAreceptor progesterone binding site. These novel pharmacophores therefore should facilitate the separation of the endocrine and neural pharmacology of natural progesterone and provide an attractive approach to "dialing out" the side effect of somnolence seen with oral progesterone. RESULTS As part of our effort to identify new pharmacophores for IRs, we screened natural product extracts and defined chemical collections with the hPR-B cotransfection assay. T he hPR-B1 co-transfection and hormone binding assays have previously been described [21]. CV-1 cells were transiently transfected with hPR-B1 and appropriate reporters, then incubated with an ECs0 concentration of progesterone along with the test sample. Antagonists blocked the progesterone-stimulated chemiluminescence activity, whereas agonists enhance the activity. Organic extracts from Cymopolia barbara possessed agonist activity. Further purification of the extracts, followed by structure elucidation and assay of the two individual components revealed that a pair of diastereomers of cyclocymopol monomethyl ether were responsible for the observed activity. Details of the isolation [22, 23], and analogue preparation [24, 25] of the cyclocymopols have been reported. Antifungal activity was previously reported for these compounds. T he major component (LG100127) of the 4:1 mixture showed antagonist activity and the minor component (LG100128) exhibited agonist activity (Fig.2) in the hPR-B1 co-transfection assay. LG100127 and LG100128 also competitively displaced Mo

MemO Me Me

0"

v

v

~

Progesterone (natural l igand)

Promegestone (R 5020)

Fig. 1. Steroidal progesterone receptor agonists.

Discovery of Novel IR Modulating Drugs Me I

63

Me I

RU486 (ICso = 0.16 nM; Ki = 0.6)

ZK98,299 (ICso = 0.9 nM; Ki = 18)

OMe

OMe

B

LG100127 (IC5o = 549 nM; Ki = 490)

r

~

B

r

LG 100128 (ECso = 35 nM; Ki = 343)

Fig. 2. Steroidal PR antagonists and the cyclocymopol monomethyl ethers. ICs0sand ECs0sare data from the hPR-B1 co-transfection assay. The K~s are derived from competitive binding measurements of the test compound and [3H]progesterone from baculovirus expressed hPR-A.

[3H]progesterone from baculovirus-expressed hPR-A (Ki = 490 and 343 nM, respectively). A series of analogues of LG100127 was prepared, through both semi-synthesis and total synthesis. Removal of the aliphatic bromine from LG100127 generated an enantiomerically pure compound, 1 (Fig.3), that showed equivalent antagonist activity in the co-transfection assay. Since debromination of LG100128 also provided an antagonist (data not shown), further work centered around aliphatic desbromo compounds (2-6). On exposure to acid, the LG100127 cyclizes to form a cymobarbatol structure, (Fig.4). In an attempt to circumvent this chemical inactivation, the phenol was acylated and suitable aromatic replacements were sought through total synthesis. Cyclocymopol analogues are synthetically accessible as shown (Fig.5). Dimedone or another suitable cyclic 1,3-diketone was treated with ethanol in the presence of acid under dehydrating conditions to form a vinylogous ester. Reduction followed by hydrolysis provided a cyclohexenone ready for alkylation. Formation of the enolate under kinetic conditions followed by treatment with the appropriate benzyl bromide yielded the carbon skeleton of the cyclocymopols. The naturally occurring phenyl substituent is available through literature methods [26]. Hydrogenation

followed by methylenation and deprotection provided cyclocymopol analogues. Simplification of the aromatic group was realized by replacement with a 4-nitrophenyl substituent as exemplified by 4 (Fig.3). For comparative purposes, aliphatic modifications (2, 3) were examined with the naturally occurring aromatic portion. These studies included compounds with an added double bond in the cyclohexane ring as well as additional alkyl groups around the cyclohexane. Cyclopropanation of the exomethylene substituent was performed (6) to circumvent the cymobarbitol cyclization. The 4-aromatic substituent appeared to be important for maintaining activity. In general, these structures showed no agonist or antagonist cross-reactivity with the glucocorticoid receptor, mineralocorticoid receptor or estrogen receptor (Table 1). Activity with the androgen receptor, as measured with the hAR co-transfection assay, was in some cases comparable with that measured with hPR. CONCLUSION Non-steroidal PR pharmacophores have a number of potential advantages over steroidal PR pharmacophores. From a pharmaceutical standpoint, nonsteroidal PR modulators may be easier and more

64

Todd K. Jones et al.

OMe

OMe

I

,,'"

Br

Me.,~O

0

(ICso = 741 nM; Ki = 218 nM)

(ICso = 710 nM; Ki = 60 nM)

OMe

(ICso = 3567 nM; Ki = 31 nM)

(ICso = 1050 nM; Ki = 54 nM) OMe

OaN~r~,1

~ M e

MeyO 0

(ICso = 260 nM; Ki = 243 nM)

(ICso = 160 nM; Ki = 103 nM)

Fig. 3. Synthetic analogues of cyclocymopol monomethyl ether. ICs0s are data from the hPR-B1 co-transfection assay. The K~s are derived from competitive binding measurements of the test compound and [~H]progesterone from baculovirus expressed hPR-A.

economical to synthesize than steroids. T h e y may show less cross-reactivity a m o n g the various steroidactivated IRs. T h e y are likely to be less encumbered by chemical patent entanglements than are steroids. N o n steroids are also less likely to be metabolized to I R cross-reactive compounds by endogenous steroid metabolizing enzymes.

Me

MeO ~

v

~

M ee~ M ~-

Fig. 4. Representative cymobarbitol structure.

T h e cymopols described in this article resulted from this use of the co-transfection assay for H T S using hPR-B1. Novel pharmacophores such as the cyclocymopols may serve as lead compounds for directed medicinal chemistry to produce optimized clinical development candidates. T h e cyclocymopols are a novel class of structures for pharmaceutical use. T h e y are readily prepared f r o m inexpensive starting materials in 5-10 chemical steps. T h e better analogues have potencies in the hPR-B1 co-transfection assay of 160-500 n M with binding Kis to h P R - A of 30-50 nM. T h e s e structures have no cross-reactivity with h G R , h M R or h E R and have manageable antagonist crossreactivity with hAR. T h e identification of the cyclocymopols as a lead for non-steroidal progesterone receptor modulators demonstrates the utility of the co-transfection assay for

0 0"

D i s c o v e r y of N o v e l I R M o d u l a t i n g D r u g s

EtOH,TsOH

65

1. LAH

92%

2. H2SO4 98%

Me

Me

LDA,BnBr 88%

OMe

OMe

TBSO

e

e

TBSO

Me

Me

Me3SiCH2 li 100% OMe

Br

~ ,SiMes

HO

"I'BSO

OMe

B n ~ ~ M e

i

e

e

OH

Fig. 5. S c h e m e for preparing synthetic cyclocymopol m o n o m e t h y l e t h e r analogues.

detecting new pharmacophores for IRs. Further, this assay has shown great utility for rapidly providing the data essential for studying structure activity relationships used for improving the biochemical profiles of a n a l o g u e s . S i n c e t h e c o - t r a n s f e c t i o n a s s a y is c e l l - b a s e d , additional data are provided concerning compound s t a b i l i t y , t o x i c i t y a n d a b i l i t y t o e n t e r a cell a n d a c t i v a t e the receptor of interest.

Table 1. Cross reacti'vities o f the steroidal and nonsteroidal P R antagonists Ligand

hAR (nM)

hER (nM)

hGR (nM)

hMR (nM)

RU486 ZK98,299 LG100127 1 4

5 269 449 377 250

> 1000 > 1000 > 10,000 > 10,000 > 10,000

0.8 27 > 10,000 > 10,000 > 10,000

> 1000 > 1000 ND 1500 > 10,000

Values are potencies (nM) in the co-transfection assay with the appropriate receptor and agonist (dihydrotestosterone (hAR), estradiol (hER), dexamethasone (hGR) and aldosterone (hMR)). N D = not determined.

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19. Freeman E. W., Purdy R. H., Coutifaris C., Rickels K. and Paul S. M.: Anxiolytic metabolites of progesterone: correlation with mood and performance measures following oral progesterone administration to healthy female volunteers. Neuroendocrinology 58 (1993) 478-484. 20. Arafat E. S., Hargrove J. T., Maxson W. S., Desiderio D. M., Wentz A. C. and Andersen R. N.: Sedative and hypnotic effects of oral administration of micronized progesterone may be mediated through its metabolites. Am. ft. Obstet. Gynecol. 159 (1988) 1203-1209. 21. Pathirana C., Stein R. B., Berger T. S., Fenical W., Ianiro T., Mais D. E., Torres A. and Goldman M. E.: Nonsteroidal human progesterone receptor modulators from the marine alga Cymopolia barbara. Mol. Pharm. 47 (1995) 630-635. 22. Hogberg H.-E., Thomson R. H. and King T.: The cymopols, a group of phenylated bromohydroquinones from the green calcereous alga Cymopolia barbara, ft. Chem. Soc. Perkin Trans. I (1976) 1696-1701. 23. McConnell O. J., Hughes P. A. and Targett N. M.: Diastereomers of cyclocymopol and cyclocymopol monomethyl ether from Cymopolia barbata. Phytochemistry 21 (1982) 2139-2141. 24. Hamann L. G., Farmer L. J., Johnson M. G., Goldman M. E., Mais D. E., Davtian A., Bender S. L. and Jones T. K.: Synthesis and biological activity of novel nonsteroidal progesterone receptor antagonists. In Steroid Receptors and Antihormones (Edited by D. Henderson, D. Philibert, A. K. Roy and G. Teutsch). The New York Academy of Sciences, New York, Vol. 761 (1995) pp. 383-387. 25. Hamann L. G., Farmer L. J., Johnson M. G., Bender S. L., Mals D. E., Wang M.-W., Crombie D., Goldman M. E. and Jones T. K.: Synthesis and biological activity of novel nonsteroidal progesterone receptor antagonists based on cyclocymopol monomethyl ether, submitted. 26. Clavel J.-M., Guillamel J., Demerseman P. and Royer R.: Recherches sur le benzofuranne--LVI. Syntheses dans la serie de l'Euparone par acetylation de benzofurannes acetyles et methoxyles sur homocycle. J. Heterocyclic Chem. 14 (1977) 219-224.