Two series of potent retinoid X receptor (RXR)-selective compounds were designed and synthesized based upon recent observation that (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1- propenyl]benzoic acid (TTNBP) binds and transactivates only the retinoic acid receptor (RAR) subtypes whereas (E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid (3-methyl TTNPB) binds and transactivates both the RAR and RXR subfamilies. Addition of functional groups such as methyl, chloro, bromo, or ethyl to the 3 position of the tetrahydronaphthalene moiety of 4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid (5a) and 4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2- naphthyl)ethenyl]benzoic acid (6a) results in compounds which elicit potent and selective activation of the RXR class. Such RXR-selective compounds offer pharmacological tools for elucidating the biological role of the individual retinoid receptors with which they interact. Activation profiles in cotransfection and competitive binding assays as well as molecular modeling calculations demonstrate critical structural determinants that confer selectivity for members of the RXR subfamily. The most potent compound of these series, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]ben zoi c acid (6b), is the first RXR-selective retinoid (designated as LGD1069) to enter clinical trials for cancer indications.

The biological response to progesterone is mediated by two distinct forms of the human progesterone receptor (hPR-A and hPR-B). In most cell contexts, hPR-B functions as a transcriptional activator of progesterone-responsive genes, whereas hPR-A functions as a transcriptional inhibitor of all steroid hormone receptors. We have created mutations within the carboxyl terminus of hPR which differentially effect the transcriptional activity of hPR-B in a cell- and promoter-specific manner. Analogous mutations, when introduced into hPR-A, have no effect on its ability to inhibit the transcriptional activity of other steroid hormone receptors. The observed differences in the structural requirements for hPR-B and hPR-A function suggest that transcriptional activation and repression by PR are mediated by two separate pathways within the cell. In support of this hypothesis, we have shown that hPR-A mediated repression of human estrogen receptor (hER) transcriptional activity is not dependent on hER expression level but depends largely on the absolute expression level of hPR-A. Thus, it appears that hPR-A inhibits hER transcriptional activity as a consequence of a noncompetitive interaction of hPR-A with either distinct cellular targets or different contact sites on the same target. We propose that hPR-A expression facilitates a ligand-dependent cross-talk among sex steroid receptor signaling pathways within the cell. It is likely, therefore, that alterations in the expression level of hPR-A or its cellular target can have profound effects on the physiological or pharmacological responses to sex steroid hormone receptor ligands.

We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Derivatives of pyridinones were found to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevent the spread of HIV-1 infection in cell culture without an appreciable effect on other retroviral or cellular polymerases. 3-{[(4,7-Dimethyl-1,3-benzoxazol-2-yl)methyljamino}-5-ethyl-6methylpyridin-2(LH)-one (L-697,639) and 3-{[(4,7-dichloro-1,3-benzoxazol-2-yl)methylJamino}-5-ethyl--methylpyridin-2(IH)-one (L-697,661) Infection with the human immunodeficiency virus type 1 (HIV-1) causes progressive destruction of the immune system, which ultimately results in AIDS. An essential step in the life cycle of HIV-1 is reverse transcription of the viral RNA genome to produce a double-stranded DNA copy. This process is mediated by the virally encoded reverse transcriptase (RT). Thus, RT is a potential therapeutic target and, indeed, nucleoside analog inhibitors of RT, such as 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI), are clinically effective drugs for treating HIV-1 infection (1, 2). However, their effectiveness is limited by toxicities, which may reflect inhibition of cellular polymerases and/or alteration of nucleoside pools, given that the nucleoside analogs are phosphorylated (in competition with natural nucleosides) to their active form by cellular kinases (3, 4). The emergence of AZT-resistant virus (5) further emphasizes the need to develop selective RT inhibitors that can be used either alone or in combination with nucleoside analogs. The development of specific RT inhibitors is the subject ofthis communication. (L-697,661) were synthesized by alkylation of 3-amino-5-ethyl-6-methylpyridin-2(1H)-one with either N-hydroxymethylphthalimide or the appropriate 2-halomethylbenzoxazole. Requisite aminopyridinone was obtained from condensation of 3-formyl-2-pentanone with nitroacetamide followed by catalytic reduction.Synthesis of ethylene derivative 5-ethyl-6-methyl-3-(2-phthalimidoethyl)pyridin-2(1H)-one (L-693,593) began with the condensation of 3-formyl-2-pentanone and cyanoacetamide to give 3-cyano-5-ethyl-6-methylpyridin-2(1H)-one. Reaction of this cyanopyridinone with POCl3 followed by methanolysis and reduction with diisobutylaluminum hydride led to 5-ethyl-2-methoxy-6-methylpyridine-3-carboxaldehyde. The aldehyde function was treated with trimethylsilyl cyanide, and the resulting cyanohydrin was reduced with lithium aluminum hydride to the amino alcohol. After conversion of the amino group to phthalimide, demethylation ofthe 2-methoxy group and alcohol dehydration was accomplished in one step by heating with pyridine hydrochloride. Catalytic reduction of the olefin formed yielded ethylene analog L-693,593. The structures of all pyridinones synthesized are consistent with their NMR spectra, and all compounds gave an acceptable combustion analysis (within 0.4%).H1V-1 RT Assays. rC-dG.

Two distinct isoforms of the human progesterone receptor (hPR-A and hPR-B) have been identified previously. They differ only in that hPR-B contains an additional 164 amino acids at the amino terminus. Among various species these two forms arise as a result of either alternate initiation of translation from the same mRNA or by transcription from alternate promoters within the same gene. In order to understand the reason for their existence, we studied the transcriptional capacity of these individual receptors and observed that their activity was influenced strongly by cell and promoter context. More surprising was the observation that in promoter and cell contexts where hPR-A was inactive, it acted as a potent trans-dominant repressor of hPR-B-mediated transcription. This event occurred at substoichiometric concentrations of hPR-A and was hormone dependent. Human PR-A was not a general repressor of ligand-mediated transcription, as it had no effect on vitamin D receptor function. Interestingly, hPR-A but not hPR-B was capable of a similar inhibition of glucocorticoid, androgen, and mineralocorticoid receptor-mediated gene transcription. This suggests a specific role for the hPR-A isoform in this regulatory process. The trans-dominant effects of hPR-A were induced also by the antiprogestins ZK112993 and ZK98299 and a DNA binding defective hPR-A mutant, suggesting that the inhibitory function of hPR-A does not require DNA binding. The dual role of hPR-A as an activator or repressor of transcription defines a potential mechanism by which cells can generate dissimilar responses to a single hormone and provides a molecular explanation for the existence of two distinct forms of the hPR.