WO1995033745A2 - Novel compounds useful in modulating gene expression of retinoid responsive genes and/or having anti-ap-1 activity - Google Patents

Abstract

Novel compounds are provided which are useful for the regulation of gene expression by retinoids; these compounds are represented by structural formula (I), wherein the substituents R1 through R5 are as defined herein. Additional compounds are provided which are useful for selectively inhibiting AP-1 or an AP-1 component; one group of such compounds is exemplified by structural formula (II), wherein the substituents R?1, R2, R20, R21, R22 and R23¿ are defined herein. Pharmaceutical compositions are provided as well, as are methods of using the compounds in a variety of contexts.

Description

NOVEL COMPOUNDS USEFUL IN MODULATING GENE

EXPRESSION OF RETINOID RESPONSIVE GENES

AND/OR HAVING ANTI-AP-1 ACTIVITY

Acknowledgement of Government Support

This invention was made with government support under Grants P01 CA 51993, CA 50676, CA 55681 and CA 60988 awarded by the National Cancer Institute and 2RT0109 awarded by the University of California. The U.S. government has certain rights in the invention.

Technical Field

This invention relates generally to the regulation of gene expression by retinoids. More particularly, the invention relates to novel compounds that are useful in modulating gene expression by retinoids, as well as other effects of these retinoids. The compounds are thus also useful for regulating cell differentiation processes and other processes controlled or regulated by retinoids. The invention additionally relates to certain novel compounds useful for selectively inhibiting AP-1 or an AP-1 component. Finally, the invention relates to pharmaceutical compositions and methods for treating mammalian individuals with the novel compounds.

Background

Retinoids, which regulate cell differentiation by modulating gene expression and are able to reverse the preneoplastic transformation of cells, have excellent potential as therapeutic agents for the treatment and prophylaxis of cancer. See, e.g.: A. B. Roberts et al., "Cellular Biology and Biochemistry of Retinoids," in The Retinoids, vol. 2, eds. M. B. Sporn et al., Orlando:

Academic Press, Inc., 1984, at pp. 209-286 (1984); and M. B. Sporn et al., "Biological Methods for Analysis and Assay of Retinoids," also in The Retinoids, vol. 2 (1984). Retinoids, particularly retinoic acid (RA) analogs, have been used in the treatment of leukemia, mycosis fungoides, basal cell carcinoma, and psoriasis and other hyperproliferative diseases of the skin (see R. C. Moon et al. , "Retinoids and Cancer" in The Retinoids, Vol. 2 (1984), supra). However, the systemic side effects of the compounds and their teratogenicity limit their utility. Side effects include, for example, bone remodeling, palmoplantar peeling, dermatitis, alopecia, hepatotoxicity, and systemic toxicity as documented by H.

Mayer et al., Experientia 34: 1105 (1978) and by R. A. Pittsley et al, New Eng. J. Med. 308: 1012 (1983). Recently, retinoids have also been shown to activate several viruses including HIV-1 (Turpin, J., et al., J. Immunol. 148:2539-2546 (1992); Poli, G., et al., Proc. Natl. Acad. Sci. USA 89:2689-2693 (1992); Huan, B., et al. , Proc. Natl. Acad. Sci. USA 89:9059-9063 (1992); Ghazal, P., et al ,

Proc. Natl. Acad. Sci. USA 89:7630-7634 (1992)).

The physiological activities of retinoids are mediated by two types of nuclear receptors, the Retinoic Acid Receptors (RARs), and the Retinoid X Receptors (RXRs). These receptors interact as dimers with specific DNA sequences known as the retinoic acid response elements (RAREs).

The synthesis of particular retinoids has been disclosed. For example, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid was reported by M. I. Dawson et al. in J. Med Chem. 32:1504-1517 (1989), and by B. Shroot et al. in European Patent Publication 210929 A3 (1987), which also discloses analogs. Other analogs were reported by M. I. Dawson et al in J.

Label. Comp. Radiopharm. 31:865-869 (1992), and by M. I. Dawson et al. in J. Label Comp. Radiopharm. 33:633-637 (1993). The synthesis of heterocyclic analogs was reported by J. Eustache et al. in Tetrahedron Lett. 29:4409-4410 (1988), L. Eyrolles et al. in Med. Chem. Res. 2:361-367 (1992), H. Kagechika et al. in J. Med. Chem. 32: 1098-1108 (1989), J. M. Lehmann et al. in Cancer Res. 51:4804-4809 (1991), and B. Shroot et al. in European Patent Publication 292348 A1 (1988). The biological activities of these compounds were reported by M. I. Dawson et al. in J. Med Chem. 32: 1504-1517 (1989), M. I. Dawson et al. in Retinoids: Progress in Research and Clinical Applications, M. A. Livrea and L. Packer, eds., New York: Marcel Dekker, Inc., 1993, pp. 205-221, H. Kagechika et al. in J. Med. Chem. 32: 1098-1108 (1989), and J. M. Lehmann et al. in Cancer Res. 51:4804-4809 (1991). L. Eyrolles et al. in Med. Chem. Res. 2: 361-367 (1992) describe receptor antagonists. G. Graupner et al, Biochem. Biophys. Res. Commun. 179(3): 1554-1561 (1991), describe analogs of

6'-substituted naphthalene-2-carboxylic acid that elicit activation of the RARγ receptor but not of the RARα receptor.

The present invention provides in part a new class of compounds useful for modulating gene expression by receptors in the retinoic acid family, e.g., RARs, RXRs and the like. The invention also provides methods for using these compounds novel compounds for a number of purposes related to regulating and/or eliciting selective gene expression of a receptor in the retinoic acid family.

The invention additionally provides compounds useful for selectively inhibiting AP-1 or an AP-1 component. Differences in the two major mechanisms of retinoid receptor action which have now been identified make it possible to define conformationally restricted retinoids that differ in their receptor transcriptional activation and receptor anti-AP-1 activities. Such retinoids, in particular compounds that have low transcriptional activation activity but strong anti-AP-1 activity, can serve as anti-proliferative agents that do not induce the full retinoid response and could thus be expected to have fewer side effects.

Disclosure of the Invention

Accordingly, it is a primary object of the invention to provide novel compounds useful in a variety of contexts, including modulation of gene expression by a receptor in the retinoic acid family of receptors, and inhibition of AP-1 induced transcription. It is another object of the invention to provide pharmaceutical compositions containing one or more of the novel compounds.

It is a further object of the invention to provide a method of using the novel compounds to modulate gene expression in a mammalian individual.

It is still a further object of the invention to provide other methods related to the use of the compounds in the modulation of gene expression by a receptor in the retinoic acid family of receptors.

It is yet a further object of the invention to provide methods related to the use of certain novel compounds to inhibit AP-1 induced transcription.

Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.

In one aspect, the invention relates to novel compounds having the structural formula (I)

in which the substituents R¹ through R⁵ are defined as follows.

R¹ is selected from the group consisting of lower alkyl, 1-methyl-

1-cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶ where R⁶ is lower alkyl, and when R¹ is ortho to R , R¹ and R² can be linked together to form a 5- or

6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R³ is selected from the group consisting of

in which R⁷ is hydrogen or methyl, k is 0 or 1, and * represents the point of attachment of the R³ substituent to the remainder of the molecule.

R⁴ is selected from the group consisting of: hydrogen; hydroxy; lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- or di-substituted amino, and

combinations thereof; amino; lower alkyl mono- or di-substituted amino; cyano; carboxyl; -(CO)-R⁸ wherein R⁸ is hydroxyl, amino, lower alkyl mono- or di- substituted amino, -OR⁹ where R⁹ is lower alkyl, or lower alkyl substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- and di- substituted amino, and combinations thereof; and -(CH₂)_m-C₆H_(5-p)-(R¹⁰)_p where m is an integer of from 0 to 6, more preferably 0 or 1, p is an integer of from 1 to 5, and R¹⁰ is independently hydrogen, hydroxyl, amino, lower alkyl mono- or di-substituted amino.

R⁵ is selected from the group consisting of: hydrogen; hydroxy; lower alkyl or lower alkoxy substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- and di-substituted amino, and combinations thereof; amino; lower alkyl mono- or di-substituted amino; cyano; carboxyl; -(CO)-R¹¹ where R¹¹ is hydroxyl, lower alkyl substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- or di-substituted amino, and combinations thereof, or wherein R¹¹ may be amino, lower alkyl and/or cycloalkyl mono- or di-substituted amino, or -OR¹⁴ where R¹⁴ is lower alkyl; -(CH₂)_q-C₆H_(5-p)-(R^14a)_r, where q is an integer of from 0 to 6, more preferably 0 or 1, r is an integer of from 1 to 5, and R*^4a is independently hydrogen, hydroxyl, amino, or mono- or di-substituted lower alkyl amino; or lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- and di-substituted amino, and S(O)_tCH₃, where t is 0, 1, or 2; -NHCO(CH₂)_v-R ¹⁵ where v is an integer of from 1 to 6 and R¹⁵ is lower alkyl substituted with from 0 to 6 fluorine substituents;

-NHCO(CH₂)_x-CONH-(CH₂)_y-R¹⁶ where x is an integer of from 1 to 10, y is an integer of from 1 to 6, and R¹⁶ is lower alkyl substituted with from 0 to 6 fluorine substituents; -CH₂S(O)_zR¹ where z is 0, 1, or 2 and R^{1 7} is lower alkyl substituted with from 0 to 6 fluorine substituents; and -C(R¹⁸)=N-OR19, where R¹⁸ is lower alkyl substituted with from 0 to 6 fluorine substituents and R¹⁹ is lower alkyl, with the proviso that when R⁴ is hydrogen, R⁵ is other than hydrogen or -NH₂.

In another aspect, then, the invention relates to novel compounds having the structural formula (II)

wherein the substituents R¹ and R² are as defined above with respect to the compounds of Formula (I), and R²⁰ through R²³ may be defined as follows.

R²⁰ and R²¹ are the same and are selected from O and S;

R²² is selected from the group consisting of hydrogen, lower alkyl and halogen; and R²² is selected from the group consisting of hydrogen, lower alkyl and halogen; and

R²³ is selected from the group consisting of

in which R²⁴ is lower alkyl and ** represents the point of attachment of R²³ to the remainder of the molecule.

In still another aspect, the invention relates to novel compounds having the structural formula (III)

wherein the substituents R¹, R², R²², and R²³ are as defined above and R²⁵ is -O- R²⁶ or -S-R²⁶ wherein R²⁶ is lower alkyl or lower alkyl carbonyl. (IV)

wherein the substituents R¹, R² and R²² are as defined above, R²⁷ is hydroxyl, =O, =CH₂, lower alkyl, or a six-membered cycloheteroalkylene ring

-O(CH₂)₃O- or -S(CH₂)₃S-, and wherein R²⁸ is a moiety of the structure

in which R²⁹ is S, NH or O and * represents the point of attachment of the R²⁸ substituent to the remainder of the molecule.

In a further aspect of the invention, novel compounds having the formula (V) are provided

in which R¹, R² and R²³ are as defined above, R³⁰ is O, S or lower alkylene, and R³¹ and R³ are independently selected from the group consisting of hydrogen, lower alkyl unsubstituted or substituted with halogen, lower alkoxy, halogen, amino, and amino substituted with one or two lower alkyl moieties. In still a further aspect of the invention, novel compounds having the structural formula (VI) are provided

or the 6-cis or 11-cis isomers thereof, and in which R ¹, R², R³¹ and R³² are as defined above.

In yet a further aspect of the invention, novel compounds are provided having the structural formula (VII)

in which R¹, R², R²², R²³, R³¹ and R³² are as defined above.

The compounds of formulae (I) through (VII)

are sometimes referred to herein as the "inventive" compounds.

The invention further encompasses pharmaceutically acceptable esters, amides and salts of the aforementioned compounds, as well as

pharmaceutical compositions containing one or more of the inventive compounds in combination with a pharmaceutically acceptable carrier.

The invention further provides a method for modulating gene expression in a mammalian individual comprising administering to the individual an effective modulating amount of an inventive compound or a pharmaceutical composition thereof.

The invention also provides a method for treating an individual afflicted with a disease caused by malfunction of cell differentiation processes, or other diseases, regulated by retinoids, comprising administering to the individual a therapeutically effective amount of an inventive compound or a pharmaceutical composition thereof.

The invention further provides a method of treating an individual infected with Human Immunodeficiency Virus ( HIV) comprising administering to the individual a therapeutically effective amount of an inventive compound or a pharmaceutical composition thereof. In a related aspect, the invention provides a method of inhibiting the replication of HIV in a subject with a receptor or protein antagonist which binds the -348 to -328 region of the long terminal repeat of HIV corresponding to an RARE of HIV. Methods are also provided for screening the capability of candidate compounds to inhibit HIV replication.

Related to these aspects of the invention, an isolated nucleic acid is provided comprising the -348 to -328 region of the long terminal repeat of HIV-1 and a non-HIV reporter gene, wherein the region includes two half-sites and a spacer region, wherein one or more nucleotides of the spacer region can be substituted by another nucleotide.

The invention also provides a method of selectively inhibiting, in a subject, transcription of a first gene which is activated by AP-1 or an AP-1 component over transcription of a second gene which is activated by a first nuclear receptor comprising administering to the subject an inventive compound. In related aspects, methods are provided for screening candidate compounds for the ability to selectively inhibit AP-1 or AP-1 component transcriptional activation.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention. Brief Description of the Drawings

Fig. 1 shows a retinoid receptor binding site in the HIV-1 LTR. a. Sequence comparison of a putative HIV-1-RARE (SEQ ID NO:1) with other RAREs (SEQ ID NOS: 11-16). Sequences that are closely related to the

AGG/TTCA (SEQ ID NOS:4 and 5) motif are indicated by thick arrows, while a less related sequence is shown by a thin arrow. TREpal is the synthetic RA response element (Windom et al, Mol. Cell. Biol. 12:3380-3389 (1992)); the other RAREs are naturally occurring sequences (see, e.g., Tini, M., et al, Genes & Dev. 7:295-307 (1993)). b. Binding of the retinoid receptors to the

HIV-1-RARE. An oligonucleotide corresponding to the HIV-1-RARE shown in a was synthesized with a Bg/ll adapter sequences at both ends and used in the gel retardation assay with in vitro-synthesized receptor proteins. 2 μl of RXRα was used for the experiment shown in the left panel while 4 μl of RXRα was used for the assay shown in the right panel, c. Receptor binding to mutated

HIV-1-RAREs. Oligonucleotides containing point mutations (indicated by solid triangles) in the first (Ml) (SEQ ID NO: 17) and second (M2) (SEQ ID NO: 18) half site of HIV-1-RARE as shown were synthesized with Bg/II adapter sequences at both ends and used in the gel retardation assay. Binding of receptors to the wild-type HIV-1-RARE was used for comparison (first two lanes). Open triangles indicate non-specific binding of the reticulocyte lysate.

Fig. 2 shows the activation of HIV-1-RAREs by all-trans-RA and 9-m-RA. a. Transcriptional activation of RARs and RXRα on the

HIV-1-RARE. Transient transfection assays were carried out as described (Zhang, X-k., et al., Nature 358:587-591 (1992); Pfahl, M., et al, Methods Enzymol. 153:256-270 (1990)). CV-1 cells were cotransfected with 100 ng

HIV- 1 -RARE reporter plasmid and 25 ng empty pECE expression vector or pECE-RXRα, pECE-RARα, pECE-RARβ, pECE-RARγ, or a combination of the receptor expression vectors as indicated. The HIV-1-RARE reporter construct was obtained by inserting two copies of the oligonucleotides shown in Fig. 1a with additional 5'-GATC (SEQ ID NO:6) overhangs into the Bg/II site of pBL-CAT2 as previously described (Pfahl, M. et al. (1990), supra), b.

Activation of a HIV-1 LTR by all-trans-RA and 9-cis-RA. To construct the HIV-1 LTR-CAT reporter, specific oligonucleotide primers complementary to sequences in the HIV-1 LTR (variant A) (5'-AAAGGGGGGACTGGAAG (SEQ ID NO:7); 5'- TGAAGCACTCAAGGCAAG (SEQ ID NO:8)) were used to amplify HIV-1 LTR from HXB2 genomic DNA (Ratner, L., et al., Aids Res. Hum. Retroviruses 3:57-69 (1987)). The resulting fragment (-464 to +97) was purified and cloned into pBS-CAT (Hoffmann, B., et al., Mol. Endocrinol. 4: 1727-1736 (1990)). The HIV-1 LTR-CAT reporter construct Geft panel) (100 ng) was cotransfected with 50 ng pECE-RARα and/or pECE-RXRα into CV-1 cells. For comparison an experiment where HIV-1 LTR CAT was replaced by pBL-CAT2, a reporter that carried a single HIV-1 -RARE is also shown (right panel). Transfected cells were treated with no hormone (open bars), 10^-7 M all-trans-RA (striped bars) or 10^-7 M 9-cis-RA (filled bars) and 24 hr later assayed for CAT activity. Results of a representative experiment with duplicate measurements are shown. In four independent experiments, induction profiles did not vary significantly, c. Activation of HIV-1-RARE by retinoids is

concentration-dependent. The HIV-1-RARE (variant A) plasmid was

cotransfected into CV-1 cells with 10 ng pECE-RXRα alone Geft panel) or together with 10 ng pECE-RARα (right panel). Cells were grown in the presence of the indicated amount of 9-cis-RA (black columns) or sH-trans-RA (striped columns), d. Activation of HIV-RAREs present in different HIV-1 variants. RARE sequences present in different HIV-1 variants are shown on top (SEQ ID NOS: 1-3). Triangles indicate differences in the sequences. Reporter plasmids of variants B and C were obtained by inserting two copies of the oligonucleotides corresponding to the sequences shown, with Bg/Il adapter sequences, into pBL-CAT2. CV-1 cells were cotransfected with the reporter construct and the receptor expression vectors as described in a.

Fig. 3 shows the repression of retinoid-induced activation of the HIV-1-RARE by COUP-TF. The HIV-1-RARE (variant A) a. or the HIV-1 LTR-CAT b. reporter plasmid (100 ng) were cotransfected together with pECE-RARα and/or pECE-RXRα (25 ng for a and 50 ng for b) into CV-1 cells in the presence or absence of COUP-TFα expression vector as indicated.

Transfected cells were treated with no hormone (open bars), 10^-7 M

all-trans-RA (striped bars) or 10^-7 M 9-cis-RA (filled bars) and 24 hr later assayed for CAT activity. The data shown represent the means of duplicate experiments.

Fig. 4 shows the activity of a retinoid antagonist, a. Repression of all-trans-RA and b. 9-cis-RA activation of the HIV-1-RARE by a retinoid antagonist. CV-1 cells were cotransfected with 100 ng HIV-1-RARE (variant

A) reporter plasmid and pECE-RARα and pECE-RXRα expression vector.

Transfected cells were treated with 10^-7 M, 10^-8 M or 10^-9 M all-trans-RA or 9-cis-RA in the presence or absence of the indicated concentration of the retinoid antagonist, AR-1. The activation obtained with retinoic acid treatment only was considered as 100%. The data points represent the mean ±S.E. of three independent experiments.

Fig. 5 shows transcriptional activation and anti-AP-1 activities of retinoids. a-f. Transient transfection assays were used to determine

transcriptional activation and anti-AP-1 activities of various retinoids. Methods: To measure receptor activation, CV-1 cells were transfected with 50 ng of expression plasmids for the receptors RARα, β, and y and RXRα, 100 ng of the reporter gene (TRE)₂-tk-CAT and 150 ng of β-galactosidase expression plasmid pCH1000. Cells were treated subsequently for 24 h with 10% charcoal-treated DME medium containing various concentrations of the retinoids to be analyzed. To determine anti-AO-1 activity of retinoids, HeLa cells were transiently transfected with 100 ng of -73ColCAT (containing an AP-1 site) reporter gene and 50 ng of expression plasmids for RARα, β, y and RXRα. After transfection, cells were grown in 0.5% charcoal-treated fetal calf serum in the presence or absence of retinoids with or without TPA (100 ng/ml) for 24 h before harvesting. The β- galactosidase expression vector (pCH1000, Pharmacia) was cotransfected to normalize the CAT activity. The assays were carried out in general as described by Yang- Yen, H.F., et al, New Biol. 3:1206-1219 (1991) and Salbert, G., et al., Mol. Endocrinol. 7: 1347-1356 (1993).

Fig. 6 shows that anti-AP-1 selective retinoids show antagonist activity. Transient transfections into CV-1 cells as described in Fig. 5 were used to measure antagonist activity of anti-AP-1 selective retinoids. After transfection cells were grown in medium containing 10^-9 M t-RA alone or t-RA and the indicated amounts of the synthetic retinoids. 100% CAT activity represent the activity measured in the presence of t-RA only.

Fig. 7 shows biological activities of anti-AP-1 selective retinoids. a. F9 cell proliferation. F9 cells were seeded in plastic wells (Costar, 24-well plates) coated with 0.1 % gelatin at different densities as indicated. After adhesion (day 0), cells were treated (day 1) with 10^-7 M or 10^-6 M retinoid. Solvent controls received an identical dilution of ethanol only (0.01 % or 0.001 % final concentration of ethanol). Treatment was repeated 48 hr after the initial treatment (day 3). Cells were analyzed on day 6 with a commercial cell proliferation kit (Promega) based on formation and extraction of a tetrazolium salt. After addition of 150 μl of substrate to 1 ml of medium for 4 h at 37°C, cells were lysed overnight at 37°C by addition of 1 ml of lysis solution.

Absorption at 550 nm was determined and plotted against retinoid concentration at the respective cell densities, b. Morphological changes in F9 cells. Cells were seeded at densities of 5,000, 7,500, or 10,000 cells pere well on

microscopic coverslips coated with 0.1 % gelatin and treated with retinoids as under (a). On day 6, cells were fixed in acetone at -20°C for 5 min, stained with 0.1 % trypan blue (Sigma) in 10% acetic acid/methanol at room temperature for 2 h, and mounted. Photomicrographs were taken through a 25x objective (total magnification 62.5x) at tungsten lamp illumination and recorded on Kodak B&W 400 ASA film. Shown are data (A to G) from treatment with 10^-6 M retinoid. A: all trans-retinoic acid, B: 9-cis-retinoic acid, C: solvent control, D: SR11220, E: SR11327, F: SR11238, G: SR11302. Essentially identical results were seen after treatment with 10^-7 M retinoid. Methods: Cell proliferation was analyzed with a commercial cell proliferation kit (Promega) based on formation and extraction of a tetrazolium salt following the manufacturer's instructions.

Fig. 8 shows growth inhibition of cancer cells by retinoids. a-b: Calu-6 and H661 human lung cancer cells and T-47D human breast cancer cells were seeded at a concentration of 3000 cells per well in 96-well plates (Costar, Cambridge, MA). After 24 h, the cells were treated with various concentrations of the indicated retinoids for 4 days and assayed for their capability to reduce MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to a colored formazan product as an index of cell survival and proliferation. The results are expressed as a percentage of absorbency at 550 nm of MTT-derived formazan developed by cells treated with control solvent.

Fig. 9 shows Differential Display of RNAs from human breast carcinoma T-47 D cells treated with 100 nm t-RA, SR11302 or R10. A 1 μg total RNA was reversed transcribed individually by four sets of primers

T12MN(N=G,A,T,C). One tenth of each sample was amplified by PCR using identical 3 '-primer and a random 5'-primer in presence of ³⁵S-dATP. The final cDNA fragments were resolved on 6% DNA sequencing gel. The solid arrows mark bands which are enhanced or reduced in the presence of the various retinoids. Methods: PCR and Differential Display. T-47D cells were treated with 100nM t-RA or SR11302 or R10 for 72 h. Total RNA was purified by the guanidinium method. RNAs were reverse transcripted using four sets of 3' primer symbolized as T12MG, T12MA, T12MC, and T12MT (T12 stands for poly(dT)12, M id degenerate nucleotide for G, A, and C. Each primer was anchored by G, A, T, C respectively. The cDNAs were then amplified by PCR using same sets of 3' primers and a 5' random primer ABR1 with the sequence 5'-GCGGACACAC-3'. The condition for reverse transcription and PCR were identical to those published by Liang, P., et al., Science 257:967-971 (1992). The products were resolved on a 6% DNA sequence gel. Modes for Carrying Out the Invention

The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.

Before the present compounds, compositions and methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods, specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a novel compound" includes mixtures of compounds, reference to "a pharmaceutical carrier" includes mixtures of two or more carriers, and the like.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkyl groups herein contain from 1 to 12 carbon atoms. The term "lower alkyl" intends an alkyl group of from one to six carbon atoms, preferably from one to four carbon atoms. The term

"cycloalkyl" intends a cyclic alkyl group of from three to eight, preferably five or six carbon atoms.

The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be defined as -OR where R is alkyl as defined above. A "lower alkoxy" group intends an alkoxy group containing from one to six, more preferably from one to four, carbon atoms.

The term "alkylene" as used herein refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for example, methylene (-CH₂-), ethylene

(-CH₂-CH₂-). propylene (-CH₂-CH₂-CH₂-), 2-methylpropylene

[-CH₂-CH(CH₃)-CH₂-], hexylene [-(CH₂)₆-] and the like. "Lower alkylene" refers to an alkylene group of from 1 to 6, more preferably from 1 to 4, carbon atoms. The term "cycloalkylene" as used herein refers to a cyclic alkylene group, typically a 5- or 6-membered ring.

The term "alkene" as used herein intends a mono-unsaturated or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Preferred groups within this class contain 2 to 12 carbon atoms. Asymmetric structures such as (AB)C=C(CD) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol—.

The term "Me" is an abbreviation for methyl. "Ac" is an abbreviation for acetyl.

"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase "optionally substituted lower alkyl" means that the lower alkyl group may or may not be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.

By the term "effective amount" of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired effect, e.g., regulation of gene expression. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art using only routine experimentation.

By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected bicyclic compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

"Eliciting," "modulating" or "regulating" selective gene expression is intended to mean that a compound is capable of acting as an activator or an antagonist of gene expression by a particular receptor, i.e., a receptor in the retinoic acid family.

"AP-1" is composed of cJun homodimers and cJun/cFos heterodimers; the term "AP-1 component" refers to a Jun protein or a Fos protein or portions thereof which either individually or in combination with other components activate transcription through AP-1 responsive nucleotide sequences.

Thus, "AP-1" means any compound having the structure necessary for the binding of AP-1 or AP-1 components to its responsive element.

"Nuclear receptor" means a receptor, such as a retinoid receptor, a glucocorticoid receptor, a vitamin D3 receptor, a thyroid receptor, a

mineralocorticoid receptor, an androgen receptor, a progesterone receptor, an arylhydrocarbon receptor and an estrogen receptor, or portions of these receptors, which retain the function of binding AP-1 or transcriptionally activating fragments of AP-1. The Retinoic Acid Receptor (RAR) includes RARα, RARβ, RARγ, RARε and the related RXR proteins. The thyroid receptor includes erbA- T, TRα-2 and TRα -2 variants. Typically, the nuclear receptor binds to its ligand, e.g. Retinoic Acid Receptor to retinoic acid, prior to binding to AP-1 or an AP-1 component. Thus, the invention provides methods in which

ligand/receptor binding is required. However, circumstances can exist where the receptor directly binds to AP-1 or an AP-1 component. The present discovery also provides a novel composition of matter comprising AP- 1 or an AP- 1 component bound to a nuclear receptor. The binding of the complex to AP- 1 can result in an increased anti-cancer or anti-arthritis effect compared to a ligand known to bind a nuclear receptor. Thus, one can screen for ligands with increased specificity or affinity of the receptor/ligand complex for AP- 1 or AP- 1 components. These ligands can be made by standard organic synthesis and screened using the methods of the invention.

The present invention provides compounds of structural formulae (I), (II), (III), (IV), (V), (VI) and (VII) shown above.

Preferred compounds within the generic structure of Formula (I) include

where R³³ is selected from the group consisting of O, S, C(CH₃)₂, and CH₂, with the proviso that R³³ is other than (CH₃)₂C for formula (lc), and R³⁴ is hydrogen or methyl. Particularly preferred compounds within this group are as shown in structural formula (lb).

When R⁵ is hydrogen or alkyl, R⁴ is preferably selected from the group consisting of

where R¹⁰ is defined above.

When R⁴ is hydrogen or alkyl, R⁵ is preferably selected from the group consisting of

where R¹⁴ is defined above. Examples of preferred structures encompassed by formula (lb) thus include

Examples of specific and particularly preferred compounds within the class of compounds defined by formula (lb) include:

With respect to compounds of formulae (II) through (VII), also included are compounds wherein R¹ and R² are linked together to form the structures VIII, IX, X, XI or XII

wherein R³⁵ is O, S, SO, SO₂, C(CH₃)₂, or CH₂, but is other than C(CH₃)₂ for structure X, and R³⁶ is hydrogen or methyl.

In yet a further embodiment, preferred compounds within generic formulae (II) through (VII), wherein R¹ and R² form structure VIII, include:

the structure (II), wherein R¹⁶ is H, R²² is hydrogen, methyl, or halogen, and R²⁰ and R²¹ are both S;

the structure (III), wherein R²⁵ is OAc, R²² is hydrogen, methyl, or halogen, and R² is hydrogen or lower alkyl;

the structure (IV), wherein R²⁷ is =O, and R²² is hydrogen, methyl, or halogen;

the structure (V), wherein R¹⁶ is H, R³⁰ is CH₂, O, or S, R³¹ is meta or para to the ring carbon attached to R³⁰ , and R³² is hydrogen or is ortho or meta to R^{3 1} , but is only meta to R³¹ when R³¹ is amino, monosubstituted amino, disubstituted amino, or lower alkyl (a further preferred embodiment for structure (V) is where R³⁰ is CH₂, R³¹ is para -N(CH₃)₂HCl, and R³² is hydrogen);

the structure (VI), wherein R³¹ is meta or para to the ring carbon attached to the carbon at position 6 of the alkene chain; and R³² is hydrogen or is ortho or meta to R^{3 1}, but is only meta to R³¹ when R³¹ is amino,

monosubstituted amino, disubstituted amino, or lower alkyl (a further preferred embodiment for structure (VI) is where R³¹ is para-methyl and R³² is hydrogen); and

the structure (VII), wherein R¹⁶ is H, R³¹ is meta or para to the ring carbon attached to the methylene group, R³² is hydrogen or is ortho or meta to R³¹, but is only meta to R³¹ when R³¹ is amino, monosubstituted amino, disubstituted amino, or lower alkyl, and R²² is hydrogen, methyl, or halogen (a further preferred embodiment for structure (VII) is where R³¹, R³², and R²² are all hydrogen).

Specific and preferred compounds within generic formulae (II) through (VII) include:

The invention also encompasses pharmaceutically acceptable nontoxic ester, amide, and salt derivatives of those compounds of formulae G) through (VII) containing a carboxylic acid moiety.

Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base.

Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine,

2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. The reaction is conducted in water, alone or in combination with an inert, water-miscible organic solvent, at a temperature of from about 0°C to about 100°C, preferably at room temperature. The molar ratio of compounds of structural formulae G) through (VII) to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material-a particular preferred embodiment-the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt. When calcium salts are prepared, approximately one-half a molar equivalent of base is used to yield a neutral salt, while for aluminum salts, approximately one-third a molar equivalent of base will be used.

Ester derivatives are typically prepared as precursors to the acid form of the compounds--as illustrated in the examples below--and accordingly may serve as prodrugs. Generally, these derivatives will be lower alkyl esters such as methyl, ethyl, and the like. Amide derivatives -(CO)NH₂, -(CO)NHR and -(CO)NR₂, where R is lower alkyl, may be prepared by reaction of the carboxylic acid-containing compound with ammonia or a substituted amine.

The present invention further provides a method for modulating gene expression in a mammalian individual comprising administering to the individual an effective modulating amount of the inventive compound or a pharmaceutical composition thereof. Typically, the gene expression is inhibited by this method.

The invention also provides a method for treating an individual afflicted with a disease caused by malfunction of cell differentiation processes, or other diseases, regulated by retinoids, comprising administering to the individual a therapeutically effective amount of the inventive compound or a pharmaceutical composition thereof. Examples of such diseases include a neoplastic condition such as cancer.

The present invention also provides a method of treating an individual infected with a viral infection where the virus has a RARE that corresponds to a RARE which is bound by an RAR/RXR heterodimer or a RXR homodimer. As described in the Examples, one such virus is the Human

Immunodeficiency Virus (HIV). The method comprises administering to the individual a therapeutically effective amount of the inventive compound or a pharmaceutical composition thereof.

In another embodiment, the present invention provides a method of inhibiting the replication of HIV in a subject, comprising contacting HIV with a receptor or protein antagonist which binds the -348 to -328 region of the long terminal repeat of HIV corresponding to an RARE of HIV. As described in the

Examples, COUP-TFα and β can be used to bind this region and competitively prevent the binding of the retinoid receptor to this region. This binding can inhibit the viral replication. Other receptors or proteins that bind this region can be screened utilizing the methods set forth in the examples.

In one embodiment, hematopoietic cells or hematopoietic stem cells can be isolated and transfected with a viral vector containing the COUP-TF gene such that the cell expresses COUP-TF. This cell can be reintroduced into the patient and such transfected cells would produce COUP-TF and inhibit HIV that infects these cells or the progeny of these cells. Alternatively, homologous recombination can be utilized to introduce the COUP-TF gene into the cell.

Methods of isolating hematopoietic stem cells have been described, for example, in U.S. Patent No. 5,061,620.

Also provided is a method of screening for a candidate compound for the inhibition of the replication of HIV, comprising administering the compound to a suitable host having a promoter region comprising a retinoic acid response element (RARE) found in positions -348 to -328 of the long terminal repeat from an HIV which is operably linked to a reporter gene and a gene functionally expressing an RXR either alone or with a gene functionally expressing an RAR and determining whether the activation of transcription of the reporter gene is inhibited by the compound, the inhibition of transcription indicating a candidate compound for inhibiting the replication of HIV. In this method, the RARE may be selected from the group consisting of the sequences set forth in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. In addition, the candidate compound may be an analog or derivative of retinoic acid.

The present invention further provides a method of screening for a candidate compound to inhibit the replication of HIV, comprising administering the compound to a suitable host having the promoter region of either COUP-TFα or COUP-TFβ orphan receptor which is operably linked to a reporter gene or to the COUP-TFα or COUP-TFβ protein and detecting the increased expression of the reporter gene, the COUP-TFα or COUP-TFβ, the presence of increased expression indicating the presence of a candidate compound to inhibit the replication of HIV (Tran et al , Mol Cell Biol , 12:4666-4676). The regulatory region of the COUP-TF orphan receptor can be cloned using standard techniques (Sambrook et al, Molecular Cloning, 2d Ed. (1982)).

Also provided is an isolated nucleic acid comprising the -348 to -328 region of the long terminal repeat of HIV-1 and a non-HIV reporter gene, wherein the region includes two half-sites and a spacer region, wherein one or more nucleotides of the spacer region can be substituted by another nucleotide. The reporter gene may be CAT2. The nucleic acid can be synthesized using standard synthesis techniques. This nucleic acid can be placed into a vector and the vector can be placed into a host. Such molecular biology techniques and suitable vectors and hosts are well known (see, e.g., Sambrook et al, supra).

Furthermore, in more preferable embodiments, the nucleic acid is selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

In addition, the present invention provides a method of screening for a candidate compound to inhibit the replication of HIV comprising contacting a nucleic acid comprising an RARE found in positions -348 to -328 of the long terminal repeat from HIV with either a Retinoid X Receptor (RXR), either alone or with a Retinoic Acid Receptor (RAR), with the candidate compound and determining the absence of a complex between the nucleic acid and a receptor, the absence of a complex indicating a candidate compound to inhibit the replication of HIV. One example of such a method is a Gel Retardation Assay as described in the Examples.

The above methods primarily relate to compounds having the structural formula (I), while compounds of formulae (II) through (VII) are primarily useful for inhibiting AP-1 induced transcription.

The invention, then, also provides a method of screening a compound for an ability to selectively inhibit AP-1 or AP-1 component transcriptional activation over transcriptional activation of a gene regulated by a nuclear receptor. By this method, it is meant that compounds, including retinoid derivatives, can be screened, as in the Examples, for relative differences in promoting transcriptional activation between AP-1 activated genes and other nuclear receptor activated genes. For example, the method allows for the selection of those compounds which minimize AP-1 transcriptional activation by assisting a nuclear receptor to bind and inhibit AP-1 or AP-1 components dun and cFos. This selective binding to AP-1 minimizes transcriptional activation mediated by other nuclear receptors. Thus, such a compound would have the selective effect of inhibiting AP-1 activated genes while minimizing

transcriptional activation of other nuclear receptor controlled genes. However, it should be understood that the method provides for the screening of compounds which effect any change in anti-AP-1, or AP-1 component transcriptional activation relative to transcriptional activation of other nuclear receptor controlled genes.

This method of screening involves the steps of: (a.) combining the compound within a first cell containing AP-1 or an AP-1 component, a first nuclear receptor and an AP-1 responsive element linked to a first reporter gene;

(b.) combining the compound within a second cell containing a second nuclear receptor and a receptor specific responsive element linked to a second reporter gene; (c.) detecting the transcriptional activation of the first and second reporter genes; and (d.) selecting the compound which selectively inhibits transcriptional activation of the first gene over transcriptional activation of the second reporter gene, thereby screening the compound for the ability to selectively inhibit AP-1 or AP-1 component transcriptional activation over transcriptional activation of the gene regulated by the nuclear receptor.

The invention also provides a method of screening a compound for an ability to selectively inhibit AP-1 or an AP-1 component transcriptional activation over transcriptional activation of a gene regulated by a nuclear receptor involving the steps of: (a.) combining the compound within a cell containing AP- 1 or an AP-1 component, a nuclear receptor, and AP-1 or AP-1 component responsive element linked to a first reporter gene and a receptor specific responsive element linked to a second reporter gene; (b.) detecting

transcriptional activation of the first and second reporter genes; and (c.) selecting the compound which selectively inhibits transcriptional activation of the first reporter gene over transcriptional activation of the second reporter gene, thereby screening the compound for the ability to selectively inhibit AP-1 or AP-1 component transcriptional activation over transcriptional activation of the gene regulated by the nuclear receptor. Therefore, this method represents a single cell approach to screening for ligands.

In one embodiment of these methods, the AP-1 or AP-1 component responsive element is a collagenase promoter. In another embodiment of these methods, the compound is a retinoid or retinoid derivative. In another

embodiment of these methods, the first and second nuclear receptors are the same receptors, such as retinoid receptors. In another embodiment of these methods, the retinoid receptors are Retinoic Acid Receptors (RARs) and Retinoid X

Receptors (RXRs). In another embodiment of these methods, the receptor contained in the first cell is non-endogenous. In another embodiment of these methods, the receptor contained in the second cell is non-endogenous. In still another embodiment of these methods, the AP-1 component is dun or cFos. In still another embodiment of these methods, the first and second reporter genes are the same genes. In yet another embodiment of these methods, the resultant anti- AP-1 activity is at least 50% at 10^-6 M and the transcription activation activity is less than 20% to 10^-5 M. In another embodiment of these methods, the resultant anti-AP-1 activity is at least 70% at 10^-6 M and the transcription activation activity is less than 15% at 10^-5 M.

The present invention also provides a method of screening a compound for an ability to selectively promote transcription activation of a gene regulated by a nuclear receptor over the ability to inhibit AP-1 or an AP-1 component transcriptional activation. Therefore, this method permits the selection of compounds which do not promote nuclear receptor binding to AP-1 or AP-1 component and thus do not inhibit AP-1 or AP-1 component-mediated transcriptional activation, and which do promote transcription mediated by other nuclear receptors. This method involves the steps of: a. combining the compound within a cell containing AP-1 or an AP-1 component, a nuclear receptor, an AP-1 or AP-1 component responsive element linked to a first reporter gene and a receptor-specific responsive element linked to a second reporter gene; b. detecting transcriptional activation of the first and second reporter genes; and, c. selecting the compound which selectively promotes transcriptional activation of the second reporter gene over the ability to inhibit transcriptional activation of the first reporter gene, thereby screening the compound for the selective ability promote transcriptional activation of the gene regulated by the nuclear receptor over the ability to inhibit AP-1 or AP-1 component transcriptional activation.

In one embodiment of this invention, the AP-1 or AP-1 component responsive element is a collagenase promoter. In another embodiment of this method, the compound is a retinoid. In another embodiment of this method, the first and second nuclear receptors are the same receptors. In another embodiment of this method, the first and second nuclear receptors are retinoid receptors, such as Retinoic Acid Receptors (RARs) and Retinoid X Receptors (RXRs). In another embodiment of this method, the AP-1 component is cJun or cFos. In another embodiment of this method, the resultant anti-AP-1 activity is less than 20% at 10^-6 M and the transcription activation activity is at least 75% at 10^-5 M. Synthetic Methods:

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of synthetic organic chemistry, biological testing, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. The compounds of the invention may be prepared in high yield using relatively simple, straightforward methods as exemplified in the experimental section herein. All starting materials and reagents used are commercially available or may be readily synthesized using conventional techniques. Utility and Administration:

The compounds of the invention defined by structural formula (I), including the pharmacologically acceptable esters, amides or salts thereof, are useful to elicit and/or regulate selective gene expression through receptors of the retinoic acid family and to control cell differentiation processes regulated by retinoids. The compounds can also be specifically used to antagonize

transcriptional activation of Human Immunodeficiency Virus (HIV) and other viruses. Thus, diseases and cell processes which are regulated by retinoic acid, can be treated using the compounds of the invention, as well as viral infections and diseases that are mediated by genes having RAREs to which these receptors bind.

The compounds of formulae (II) through (VII), including the pharmacologically acceptable esters, amides or salts thereof, are useful for many contemplated uses, including the prevention and treatment of various diseases. The compounds permit the identification of therapeutic compounds which can be administered to inhibit AP-1 mediated transcription, while minimizing the side effects of transcription mediated by other nuclear receptors. Since AP-1 is a transcriptional activator of the gene encoding coUagenase, and coll agenase is one of the enzymes known to break down collagen, a component of most connective tissues including bone, the control of AP-1 mediated transcription can be utilized to treat arthritis, for example. Likewise, since AP-1 is comprised of the protooncogene encoded products Jun and Fos, the control of AP-1 mediated

transcription can be utilized to treat those cancers associated with AP-1, a Jun or Fos component or another oncogene that regulates AP-1 activity. Examples of control of AP- 1 mediated cancer include the over expression of AP- 1, the expression of mutated forms of AP-1 and the increased AP-1 activity caused by expression of oncogenes such as H-ras. Other disease states associated with AP-1 stimulation which could be treated by the present invention include tumor formation, asthma, allergies, and skin rashes.

A variety of cancers may be treated with the compounds of formulae (II) through (VII), including those cancers represented by the isolated tumor cell lines found in the "American Type Culture Collection," Catalogue of Cell Lines and Hybridomas, 7th Ed. (1992).

Compounds (II) through (VII) and methods of the present invention are also useful as tools to study the molecular biology of genetic regulation. AP- 1 responsive promoter regions and other nuclear receptor responsive promoters can be operably linked to respective reporter genes to monitor the transcriptional effects of various ligands. These tools permit the discernment of specific ligand, nuclear receptor, AP-1 and DNA interactions which result in negative and positive inducible or repressible transcription. This in turn permits the design and use of particularly desirable ligands. The selective regulatory mechanisms revealed by the invention permit the creation and use of a genetic system for selectively controlling the expression of any desirable gene products.

The compounds of the invention may be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's

Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that may be used in conjunction with the preparation of formulations of the inventive compounds.

The compounds may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like, although oral administration is preferred. The amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the manner of administration and the judgement of the prescribing physician. Generally, however, dosage will approximate that which is typical for the administration of retinoic acid, and will preferably be in the range of about 2 μg/kg/day to 2 mg/kg/day.

Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary compounds such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example see Remington's Pharmaceutical Sciences, referenced above.

For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup.

Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

Parental administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parental administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795.

For topical administration, liquids, suspension, lotions, creams, gels or the like may be used as long as the active compound can be delivered to the surface of the skin.

Experimental

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C and pressure is at or near atmospheric.

(a.) 1,2,3,4-Tetrahydro-1, 1,4,4-tetramethylanthracene (2): To a solution of 2-bromonaphthalene (18.2 g, 87.7 mmol) and 2,5-dichloro-2,5- dimethylhexane (Kagechika et al. , J. Med. Chem. , 31:2182-2192 (1988)) (16.6 g, 90.7 mmol) in 120 mL of CH₂Cl₂ at -5°C was added 2.0 g (15 mmol) of AICI₃ over a 15-min period. The red-brown suspension was allowed to warm to 0° to 5°C, then stirred at this temperature for T h. The reaction mixture was poured into ice-water and extracted with dichloromethane. The organic layer was washed twice with water, then dried (Na₂SO₄), filtered, and concentrated to give a pale-yellow semisolid, which was extracted into 150 mL of hexanes and filtered, and the filtrate was concentrated to give 26.1 g of a yellow gum. To the crude 6-bromo-1 ,2,3,4-tetrahydro-1, 1,4,4-tetramethylanthracene was added 200 mL of ethyl acetate, 25.3 g (0.25 mol) of triethylamine, and 1.5 g of 5% Pd/C. The mixture was hydrogenated at 20°C at atmospheric pressure for 22 h and then filtered. The precipitate was washed with ethyl acetate. The yellow filtrates were washed twice with water, then dried (Na₂SO₄), and concentrated. The resultant oil was extracted into 100 mL of hexane, and filtered. The filtrate was

3,4-tetrahydro-1,1,4,4-tetramethylanthracene (2) as a 95%): m.p. 81.5° to 82.5°C (from toluene); R_f 0.55 (hexane); IR (CHCl₃) 1590, 1290, 1275, 1130, 1100, 1010, 940 cm^-1. Anal. calcd. for C₁₈H₂₂: C, 90.69; H, 9.31; found: C, 90.67; H, 9.60.

(b.) 1-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-1- anthracenyl)-1-ethanone (2): To a suspension of AICI₃ (12.0 g, 90.0 mmol) in 60 mL of 1,2-dichloroethane at 0°C was added a solution of acetyl chloride (7.4 g, 94 mmol) in 25 mL of 1,2-dichloroethane, followed by a solution of

1,2,3,4-tetrahydro-1,1,4,4-tetramethylanthracene (2) (19.5 g, 81.8 mmol) in 50 mL of 1,2-dichloroethane over a 25-min period. The mixture was allowed to warm to room temperature over 22 h. The dark-brown suspension was poured onto ice-2 N HCl (200 mL) and extracted with CH₂Cl₂. The organic phase was washed with water, aq. NaHCO₃, and dilute brine, dried (Na₂SO₄), and concentrated. The yellow solid was crystallized from CH₂Cl₂-hexane to give white crystals (12.33 g, 54%) of the ketone (3): m.p. 154° to 156°C; % 0.47

(50% CH₂Cl₂/hexane); IR (CHCl₃) 2950, 1660, 1560, 1240 cm^-1. Anal, calcd. for C₂₀H₂₄O: C, 85.67; H, 8.63; found: C, 86.02; H, 8.53. Silica gel column chromatography (gradient elution, 30% CH₂Cl₂/hexanes to 100% CH₂CI₂) of the crystallized mother liquor yielded additional 2 (7.16 g; total yield 19.49 g, 85%) and then the isomeric ketone 1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- anthracen-2-yl)-1-ethanone (2.53 g, 11 %): m.p. 133-133.5°C (hexanes); R_f 0.34 (50% CH₂Cl₂/hexane); IR (CHCl₃) 2967, 2932, 2862, 1676, 1627, 1595, 1461, 1361, 1309, 1292, 1257, 1212, 1192, 1138, 1109, 1021, 914, 898, 822, 638 cm^-1; Anal, calcd. for C₂₀H₂₄O: C, 85.67; H, 8.63; found: C, 85.75; H, 8.67. (c.) 1-(3-Bromo-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 1-anthracenyl)-1-ethanone (4): To a solution of TiCl₄ (21 mL, 0.19 mol) in 50 mL of CH₂Cl₂ at 0°C was added over a 15-min period a solution of 1-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-anthracen-1-yl)-1-ethanone (3) (18.06 g, 64.4 mmol) in 65 mL of CH₂CI₂, and then a solution of Br₂ (13.0 g, 81.4 mmol) in

35 mL of CH₂CI₂. The red-purple solution was stirred at 0°C for 15 min, and then at room temperature for 18 h. The mixture was poured onto ice-1 N HCl and extracted with CH₂Cl₂. The organic extract was washed with water, aq. NaHCO₃, and water, dried (Na₂SO₄), and concentrated to give a dark semisolid. Silica gel column chromatography (20 to 30% CH₂CI₂/hexanes) eluted the ketone

4 (11.25 g, 52%) as pale-yellow crystals, m.p. 123° to 124°C; % 0.39 (25% CH₂Cl₂/hexane); IR (CHCl₃) 2950, 1660, 1560, 1460, cm^-1. Anal, calcd. for

C₂₀H₂₃BrO: C, 66.85; H, 6.40; Br, 22.28; found: C, 66.96; H, 6.64; Br, 22.10.

(d.) Ethyl 3-Bromo-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-anthracene-1-carboxylate (5): To a solution of methyl ketone 4 (2.08 g, 5.79 mmol) in 40 mL of ethanol was added at room temperature 40 mL (about 28 mmol) of 5.25% aq. NaOCl. The suspension was heated at reflux for 1.25 h, then treated with an additional 15 mL (about 10 mmol) of 5.25% NaOCl solution and heated at reflux a further 1 h. The pale-yellow solution was decanted, then concentrated to approximately 40 mL, and poured into aq. NaHSO₃. The solution was diluted to 150 mL with water and acidified to pH 3 with 1 N HCl at 0°C. The suspension was filtered and the precipitate was washed repeatedly with water, dried at reduced pressure, and extracted into ether. The extract was filtered, concentrated, refiltered, and again concentrated to give crude 3-bromo-

5,6,7,8-tetrahydro-5,5,8,8-tetramethylanthracene-1-carboxylic acid (2.0 g, 98%) as a pale-yellow glass, R_f 0.0 (10% ethyl acetate/hexane). To the product was added potassium carbonate (1.2 g, 8.7 mmol), dimethylformamide (10 mL), and ethyl iodide (2 mL, 25 mmol). This suspension was stirred at room temperature for 48 h. The mixture was diluted with 100 mL of water and extracted with 20% ethyl acetate/hexanes. The organic phase was washed three times with water, dried (Na₂SO₄), and concentrated. The yellow gum was chromatographed on a silica gel column (20 to 25% CH₂CI₂ /hexanes) to yield 2.10 g (95%) of the ester 5 as a pale-yellow gum. A sample was crystallized from hexane to give white needles, m.p. 113° to 113.5°C; R_f 0.76 (35% CH₂Cl₂/hexanes); IR (CHCl₃) 1705, 1575, 1220, 1160, 1090, 1040, 1010, 885 cm^-1. Anal, calcd. for

C₂₁H₂₅BrO₂: C, 64.79; H, 6.47; Br, 20.52; found: C, 64.91; H, 6.50; Br, 20.44.

(e.) 3-Bromo-1-hydroxymethyl-5,6,7,8-tetrahydro-

5,5,8,8-tetramethyl-anthracene (6): To 13 mL (13 mmol) of 1 M LiA1H₄ in tetrahydrofuran was added at 0°C over a 15-min period a solution of ester 5

(4.18 g, 10.74 mmol) in 15 mL of tetrahydrofuran. The clear solution was stirred for 1 h with warming to room temperature, then cooled to 0°C. The excess reagent was quenched by the dropwise addition of a solution of ethyl acetate in tetrahydrofuran. The mixture was poured into 100 mL of 15% aq. NaOH and extracted three times with Et₂O. The combined organic phases were washed twice with brine, dried (Na₂SO₄), and concentrated to yield a colorless oil. Silica gel column chromatography (15% ethyl acetate/hexanes) gave 6 (3.37 g, 90%) as a colorless, viscous oil. A sample was crystallized (CH₂CI₂/hexanes) to give white crystals, m.p. 82.5 to 83°C; R_f 0.52 (20% ethyl acetate/hexane); IR (CHCl₃) 3620, 1605, 1110, 1075, 1000, 890, 880, 860 cm^-1. Anal, calcd. for C₁₉H₂₃BrO: C, 65.71; H, 6.68; Br, 23.01; found: C, 65.78; H, 6.70; Br, 22.99.

(f.) 4-Carbethoxyphenylboronic Acid (S):

To a suspension of activated Mnθ2 (152 g, 1.75 mol) in 400 mL of ethanol containing 4-formylphenylboronic acid (Snyder et al., J. Am. Chem.

Soc. , 80:835-838 (1958)) (6.00 g, 44.8 mmol) and NaCN (12.0 g, 249 mmol) at 5° to 10°C under argon, was added acetic acid (4.0 mL, 70 mmol). The suspension was allowed to warm to room temperature, stirred for 90 h, and filtered. The precipitate was washed with hot ethanol (4 times 150 mL). The combined filtrates were concentrated, and the semisolid residue was extracted with 40 mL of water and filtered. The filtrate was cooled to 0°C and acidified with 1 N HCl to pH 2 to 3 to give a semisolid yellow precipitate, which solidified on standing at 4°C for 2 h. The solid was filtered, washed repeatedly with water, and dried to give 8 g of crude product, which was extracted with hot ethanol (6 times 20 mL). The combined extracts were filtered and concentrated to 30 mL, before dilution with 60 mL of water, concentration to about 50 mL, and cooling to 4°C for 18 h. The yellow-brown crystals were filtered, washed with water (2 times 20 mL), and dried for 30 min under reduced pressure to yield 4-carboethoxyphenylboronic acid (8) (4.96 g, 57%): m.p. 136° to 138°C; IR (KBr) 3330, 1690, 1560, 1275, 1155, 1225, 1095, 1010, 845 cm^-1. Anal, calcd. for C₉H ₁₁BO₄: C, 55.72; H, 5.72; B, 5.57; found: C, 55.54; H, 5.90; B, 5.66. CI-HRMS (i-C₄H₉) calcd. for C₉H₁₂BO₄(MH⁺): 195.0828; found: 195.0832.

(g.) Ethyl 4-(1-Hydroxymethyl-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl)benzoate (9): To a stirred solution of

3-bromo-1-hydroxy-methyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethylanthracene (6)

(3.15 g, 9.07 mmol) and tetrakis(triphenylphosphine) palladium(0) (0.030 g, 0.026 mmol) in 45 mL of 1,2-dimethoxyethane under argon was added at room temperature a solution of 4-carboethoxyphenylboronic acid (1.80 g, 9.28 mmol) in 14 mL of ethanol, followed by 28.5 mL of saturated aq. NaHCO₃. The pale-yellow suspension was heated at reflux for 1 h. The yellow solution was poured into dilute brine and extracted three times with Et₂O. The organic phase was twice washed with dilute brine, dried (Na₂SO₄), and concentrated. The mixture was chromatographed on a silica gel column (15 to 20% ethyl

acetate/hexanes) to yield 9 (2.93 g, 78%) as white crystals: m.p. 136° to

137.5°C (CH₂Cl₂/hexanes); R_f 0.24 (20% ethyl acetate/hexanes); IR (CHCl₃)

3620, 1710, 1280, 1105, 1015, 845 cm^-1. CI-HRMS (i-C₄H₉) calcd. for

C₂₈H₃₂O₃: 416.2351; found: 416.2344. (h.) 4-(1-Hydroxymethyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-3-anthracenyl)benzoic Acid (10):

A mixture of the ester 9 (0.100 g, 0.24 mmol), 1.5 mL of ethanol, and 0.5 mL of 40% aq. KOH (about 3 mmol) was heated at reflux for 1 h to give a colorless solution, which was then concentrated in an argon stream, and acidified with 1 N H₂SO₄ at 0°C. The white suspension was stirred at room temperature for 20 min, filtered, washed repeatedly with water, dried at reduced pressure, and crystallized from ethanol to give 0.073 g (78%) of 10 as white crystals: m.p. 249° to 250°C; IR (KBr) 3500-2300, 1690, 1607, 1272, 1184, 1112, 1015, 903, 850, 776, 694 cm^-1. Anal, calcd. for C₂₆H₂₈O₃: C, 80.38; H, 7.26; found: C, 80.16; H, 7.31.

(a.) Ethyl 4-[1-(1-Hydroxy-2,2,2-trifluoroethyl)-5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoate (12):

To CF₃COCI (6.0 g, 45 mmol) at -35°C was added 30 mL of 1,2-dichloroethane. .To this solution was added at -40°C a solution of ethyl 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylanthracen-2-yl)benzoate (Dawson et al., J. Med. Chem., 32: 1504-1517 (1989) (11) (2.50 g, 6.47 mmol) in 12 mL of 1,2-dichloroethane, followed by AICI₃ (1.35 g, 10.0 mmol). The red-brown

suspension was warmed to 10°C over a 3-h period, and then to room temperature over 68 h. The dark red-brown suspension was poured into ice-1 N H₂SO₄ and extracted twice with CH₂CI₂. The organic extracts were washed three times with dilute brine, dried (Na₂SO₄), and concentrated to give a dark gum. The mixture was chromatographed on a silica gel column (gradient elution, 2% ethyl acetate/10% CH₂CI₂/hexanes to 10% ethyl acetate/10% CH₂Cl₂/hexanes) to yield 2.73 g of an orange gum containing unreacted 11 and aryl trifluoromethyl ketones: R_f 0.28 (10% ethyl acetate/hexanes) 0.35, 0.41, 0.50, 0.58, 0.65. The mixture was dissolved in 40 mL of ethanol at 40°C, cooled to room temperature, and treated with NaBH₄ (0.35 g, 10 mmol) over a 15-min period. After stirring for 15 min more, the mixture was treated with 20% aq. acetic acid until gas evolution ceased, concentrated, and partitioned between CH₂CI₂ and dilute brine. The organic phase was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The residue was chromatographed on a silica gel column (gradient elution 2% ethyl acetate/10% CH₂Cl₂/hexanes to 10% ethyl acetate/10%

CH₂Cl₂/hexanes) to yield 1.25 g of crude aryl trifluoromethyl carbinol 12 as an orange gum. The crude product was crystallized (ethyl acetate/hexanes) to yield 0.640 g (20%) of 12 as white needles: m.p. 174.5° to 176.5°C; R_f 0.40 (15% ethyl acetate/hexanes); IR (KBr) 3408, 1692, 1608, 1291, 1164, 1123, 1020, 774, 704 cm^-1. Anal, calcd. for C₂₉H₃₁F₃O₃: C, 71.88; H, 6.45; F, 11.76; found: C, 71.76; H, 6.81; F, 12.04. Further silica gel column chromatography (2% ethyl acetate/10% CH₂Cl₂/hexanes) of the crystallization residues gave an additional 0.182 g (6%) of 12 after two crystallizations (ethyl acetate/hexanes) for a total yield of 0.822 g (26%).

(b.) 4-[1-(Hydroxy-2,2,2-trifluoromethyl)- 5,6,7,8-tetrahydro-

5,5,8,8-tetramethyl-3-anthracenyl]benzoic Acid (13): A suspension of ester 12 (0.670 g, 1.38 mmol) in ethanol (6 mL) and 40% aq. KOH (2 mL, about 11 mmol) was heated at 80°C for 1 h, then concentrated under an argon stream, and cooled. The mixture was acidified with 1 N H₂SO₄ at 0°C, and filtered. The white precipitate was washed repeatedly with water, then dried. The crude product was extracted with ethanol and the extract was filtered and concentrated to give 0.63 g of white powder. Crude 13 was crystallized (ethyl acetate/ethanol) to give white needles, m.p. 170°C, then dried at 100°C/0.1 mm for 16 h to yield 0.549 g (87%) of white powder. The powder was extracted into CHCl₃ at reflux and the solution concentrated and cooled to yield 0.465 g (74%) of 13 as a white powder, m.p. 262° to 265°C; IR (KBr) 3500-2300, 3448, 1688, 1609, 1297, 1171, 1126 cm^-1. Anal, calcd. for C₂₇H₂₇F₃O₃: C, 71.04; H, 5.96; F, 12.48; found: C, 70.67; H, 6.19; F, 12.26.

(a.) Ethyl 4-(1-Formyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl)-benzoate (14):

To a solution of alcohol 9 (0.420 g, 1.01 mmol) in 5 mL of CH₂Cl₂ was added pyridinium chlorochromate (0.43 g, 2.00 mmol). The suspension was stirred at room temperature for 3 h. The black suspension was diluted with 30 mL of Et₂O, filtered through a Celite pad, and washed with 20% ethyl acetate/Et₂O. The combined yellow filtrates were concentrated to give a yellow-brown solid, which was chromatographed on a silica gel column (3% ethyl acetate/40% CH₂Cl₂/hexanes) to yield 14 as a white solid (0.398 g, 95%): m.p. 148.5° to 149.5°C (CH₂Cl₂/hexanes); R_f 0.50 (10% ethyl acetate/hexanes); IR (KBr) 2765, 1704, 1686, 1607, 1275, 1214, 1108 cm^-1. Anal, calcd. for

C₂₈H₃₀O₃: C, 8 1.13; H, 7.29; found: C, 80.97; H, 7.35.

(b.) Ethyl 4-[1-(1-Hydroxy-2,2,2-trifluoroethyl)-5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoate (12): To a suspension of aldehyde 14 (0.380 g, 0.917 mmol) in 1.5 mL of dimethylformamide containing Zn dust (0.130 g, 2.0 mmol) under argon was added at room temperature a solution of F₃CI (0.7 g, 3.6 mmol) in dimethylformamide (2 mL). The suspension was ultrasonicated (Kitazume and Ishikawa, Chem. Lett. , pp. 1679- 1680 (1981)) at room temperature in a 175 W ultrasonication bath (Branson, Shelton, CT) for 1.75 h. The yellow suspension was decanted from unreacted Zn onto ice-1 N H₂SO₄. This mixture was twice extracted with ethyl acetate. The pale-yellow extract was washed twice with water, dried (Na₂SO₄), and concentrated. The yellow gum was chromatographed on a silica gel column (gradient elution, 2% ethyl acetate/10% CH₂Cl₂/hexanes to 10% ethyl acetate/ 10% CH₂Cl₂/hexanes) to yield successively unreacted aldehyde 14 (0.306 g, 81 %) as a pale-yellow solid; R_f 0.45 (10% ethyl acetate/hexanes); and crude trifluoromethylcarbinol 12 (0.076 g, 17%) as a yellow gum, R_f 0.22 (10% ethyl acetate/hexanes). To the recovered aldehyde 14 and Zn dust (0.50 g, 7.7 mmol) was added under argon a solution of F₃CI (0.8 g, 4.1 mmol) in 5.5 mL of dimethylformamide. The mixture was ultrasonicated at room temperature for 14 h, then worked up and chromatographed as before to yield 0.20 g (53%) of unreacted aldehyde 14, and an additional 0.085 g (19%) of 12 as a pale-yellow solid. The combined product fractions of 12 were crystallized (ethyl

acetate/hexanes) to give 0.120 g (27%) of 12 as white crystals, m.p. 174° to 176°C, identical with the product 12 described in Example 2. Silica gel chromatography of the crystallization residues of 12, and two further

crystallizations (ethyl acetate/hexanes) yielded a further 0.014 g of 12, for a total yield of 0.134 g (30%).

4-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-1-trifluoroacetyl- 3-anthracenyl)benzoic Acid (15):

To a stirred solution of the trifluoromethylcarbinol 12 (6.0 mg, 0.013 mmol) in 2 mL of acetone was added dropwise over a 3-h period at room temperature 0.21 g (0.25 mmol) of a solution of CrO₃ in aq. H₂SO₄ [Jones reagent prepared from CrO₃ (2.65 g) in water (8.0 g) and concentrated H₂SO₄ (4.2 g)]. The suspension was stirred at room temperature for 24 h, diluted with 10 mL of water, and centrifuged to give a yellow solid. The solid was washed with water (3 times 1 mL) by centrifugation, dried at reduced pressure, and crystallized (MeCN, to -15 °C) to yield 4.0 mg (65%) of the aryl trifluoromethyl ketone 15 as yellow crystals: m.p. 258° to 262°C; IR (KBr) 3500-2300, 1690, 1609, 1295, 1207, 1138, 1094, 1006, 924, 774, 720 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₂₇H₂₆F₃O₃ (MH+): 455.1834; found: 455.1824.

(a.) Ethyl 4-[1-(1-Methoxy-2,2,2-trifluoroethyl)-

5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoate (16) :

To a solution of the trifluoromethylcarbinol 12 (0.075 g, 0.155 mmol) in 0.5 mL of dimethylformamide was added anhydrous K₂CO₃ (0.050 g, 0.36 mmol) and Mel (1 mL, 16 mmol). The suspension was stirred under reflux in a 65°C bath for 76 h. Mel was added periodically to maintain the original volume. The suspension was diluted with 20 mL of water and extracted three times with ethyl acetate. The pale-yellow extract was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The mixture was chromatographed on a silica gel column (4% ethyl acetate/hexane) to give 16 (0.048 g, 62%) as white crystals: m.p. 163° to 164°C; R_f 0.55 (10% ethyl acetate/hexanes); TR (KBr) 1710, 1606, 1275, 1175, 1136, 1112, 1024, 852, 774, 706 cm^-1. Anal, calcd. for C₃₀H₃₃F₃O₃: C, 72.27; H, 6.67; F, 11.43; found, C, 72.40; H, 7.02; F,

11.10. (b.) 4-[1-(1-Methoxy-2,2,2-trifluoroethyl)- 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoic Acid (17): A mixture of ester 16 (0.028 g, 0.056 mmol), ethanol (1 mL), and 40% aq. KOH (0.25 mL, about 1.5 mmol) was heated at 80°C for 1 h, and then concentrated under an argon stream. The residue was cooled to 0°C, acidified with aq. citric acid to pH 2, and stirred at room temperature for 15 min. The white suspension

was filtered, and the precipitate washed with water (6 times 1 mL) and dried under reduced pressure to give 0.026 g (100%) of 17 as white crystals, m.p. 286° to 288°C (ethyl acetate); IR (KBr) 3500-2350, 1688, 1608, 1426, 1296, 1170, 1140, 909, 851, 773, 702, 668 cm^-1. Anal, calcd. for C₂₈ H₂₉F₃O₃: C 71.47; H, 6.21; F, 12.11; found: C, 71.25; H, 6.26; F, 12.07. CI-HRMS (i-C₄H₉) calcd. for C₂₈H₃₀F₃O₃ (MH+): 471.2147; found: 471.2149.

(a.) Ethyl 4-(1-Bromomethyl-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl)benzoate (18) :

To CBr₄ (0.66 g, 1.99 mmol) and Et₂O (2 mL) was added at 0°C a solution of (C₆H₅)₃P (0.52 g, 1.98 mmol) in 6 mL of Et₂O. The mixture was stirred for 15 min before alcohol 9 (0.42 g, 1.01 mmol) in benzene (4 mL) was introduced. The reaction mixture was stirred with warming to room temperature for 18 h. The thick white suspension was diluted with hexane (30 mL) and filtered. The white precipitate was washed three times with 25% Et₂O/hexanes. The combined filtrates were concentrated and the residue was chromatographed on a silica gel column (40% CH₂Cl₂/hexanes) to yield benzylic bromide 18

(0.479 g, 99%) as white crystals: m.p. 159° to 160°C (hexanes); R_f 0.54 (50% CH₂Cl₂/hexanes); IR (KBr) 1701, 1603, 1275, 1213, 1131, 1108, 1021, 907,

852, 771, 704, 562 cm^-1. Anal, calcd. for C₂₈H₃₁BrO₂: C, 70.14; H, 6.52; Br, 16.67; found: C, 70.05; H, 6.57; Br, 16.76.

(b.) Ethyl 4-(1-Benzyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-3-anthracenyl)benzoate (19): To a solution of bromide 18 (0.120 g, 0.25 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.03 g, 0.026 mmol) in

1,2-dimethoxyethane (2 mL) was added under argon a solution of phenylboronic acid (0.050 g, 0.41 mmol) in ethanol (0.25 mL), followed by saturated aq.

NaHCO₃ (0.75 mL, about 0.9 mmol). The suspension was heated at reflux under argon for 2 h. The black reaction mixture was diluted with water and extracted with ethyl acetate. The yellow organic extract was washed twice with water, dried (Na₂SO₄), and concentrated. The mixture was chromatographed twice on a silica gel column (25% CH₂Cl₂/hexanes, then 20% CH₂Cl₂/hexanes) to yield 19 (0.074 g, 62%) as white crystals; m.p. 106.5° to 107°C (hexanes); R_f 0.42 (35% CH₂Cl₂/hexanes); IR (CHCl₃) 1710, 1605, 1270, 1220, 1105, 1010, 895, 840 cm^-1. Anal, calcd. for C₃₄H₃₆O₂: C, 85.67; H, 7.61; found: C,

85.41; H, 7.67.

(c.) 4-(1-Benzyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl)benzoic Acid (20):

A suspension of ester 19 (0.074 g, 0.155 mmol) in ethanol (1.5 mL) and 40% aq. KOH (0.5 mL, about 3 mmol) was heated at reflux for 1 h to give a clear solution and then concentrated under an argon stream. The mixture was then treated at 0°C with 1 N HCl (7 mL). The white suspension was stirred at room temperature for 20 min, filtered, washed repeatedly with water, and dried at reduced pressure. The product was recrystallized from ethanol to yield 20 (0.057 g, 82%) as white crystals: m.p. 239° to 242°C; IR (KBr) 3500-2300,

1688, 1607, 1423, 1290, 1112, 903, 776, 731, 700 cm^-1. Anal, calcd. for

C₃₂H₃₂O₂: C, 85.68; H, 7.19; found: C, 85.45; H, 7.26.

Ethyl 4-[1-(4-Dimethylaminophenyl)methyl- 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoate (21): To a solution of 18 (0.240 g, 0.50 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (0.03 g, 0.026 mmol) in 1,2-dimethoxy ethane (4 mL) was added under argon at room temperature a solution of 4-dimethylaminophenylboronic acid (Staab and

Meissner, Liebigs Ann. Chem. , 753:80-91 (1971) (0.083 g, 0.51 mmol) in ethanol (1.0 mL), and then saturated NaHCO₃ (1.5 mL, about 1.8 mmol). The suspension was heated at reflux for 1 h. Next, the yellow suspension was partitioned between CH₂CI₂ and dilute brine with centrifugation to break up the resulting emulsion. The organic phase was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The resultant orange gum was chromatographed twice on silica gel columns (10% ethyl acetate/hexanes, then 7% ethyl acetate/hexanes) to yield 21 (0.052 g, 20%) as pale-yellow crystals: m.p.142° to 143.5°C (CH₂Cl₂/hexanes); R_f 0.22 (15% ethyl acetate/hexanes); TR (CHCl₃) 1705, 1605, 1510, 1275, 1105, 1015, 940, 900, 845 cm^-1. CI-HRMS (i-C4H9) calcd. for C₃₆H₄₁NO₂: 519.3137; found: 519.3118.

(a.) Ethyl 4-[1-(1-Oxoethyl)-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl]benzoate (22): To a solution of 4 (0.360 g, 1.00 mmol) and tetrakis(triphenyl- phosphine)palladium(0) (0.03 g, 0.026 mmol) in 1,2-dimethoxyethane (6 mL) was added at room temperature under argon a solution of 4-carbethoxyphenylboronic acid (8) (0.213 g, 1.10 mmol) in ethanol (1.5 mL), followed by saturated NaHCO₃ (3 mL, about 3.6 mmol). The mixture was heated at reflux under argon for 1.5 h to give an orange suspension, which was partitioned three times between Et₂O and dilute brine. The organic extract was washed three times with dilute brine, dried (Na₂SO₄), and concentrated. The orange solid was twice chromatographed on a silica gel column (3% ethyl acetate/20% CH₂Cl₂/hexanes, then 2% ethyl acetate/ 10% CH₂Cl₂/hexanes) to yield ester 22 (0.303 g, 71 %) as a white solid: m.p. 130° to 132°C

(CH₂Cl₂/hexanes); R_f 0.38 (3% ethyl acetate/20% CH₂Cl₂/hexanes); IR

(CHCl₃) 1705, 1675, 1605, 1270, 1220, 1105, 1010, 845 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₂₉H₃₃O₃, (MH+): 429.2429; found: 429.2360.

(b.) Ethyl 4-[1-(1-Hydroxyethyl)-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl]benzoate (23): To ketone 22 (0.180 g, 0.42 mmol) suspended in ethanol (5 mL) and tetrahydrofuran (2 mL) was added NaBH₄ (0.04 g, 1.0 mmol). The reaction mixture was stirred at room

temperature for 2 h, diluted with dilute brine, and extracted three times with Et₂O. The organic extract was washed twice with brine, dried (Na₂SO₄), and concentrated to give a colorless glass. Silica gel column chromatography (10% ethyl acetate/hexanes) yielded alcohol 23 (0.162 g, 90%), as a colorless glass. A sample was crystallized from CH₂Cl₂/hexane to give white needles, m.p. 159.5° to 160°C; R_f 0.61 (20% ethyl acetate/hexanes); IR (CHCl₃) 3600, 1700, 1605, 1270, 1100, 1010, 895, 840 cm^-1. Anal, calcd. for C₂₉H₃₄O₃: C, 80.89; H, 7.96; found: C, 80.67; H, 8.00.

(c.) 4-[1-(1-Hydroxyethyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl]benzoic Acid (24):

A suspension of ester 23 (0.107 g, 0.249 mmol) in ethanol (1.5 mL) and 40% aq. KOH (0.5 mL, about 3 mmol) was heated at reflux for 1 h to give a clear solution, which was concentrated in an argon stream and acidified at 0°C with aq. acetic acid. The white suspension was stirred at room temperature for 30 min, and filtered. The precipitate was washed repeatedly with water and dried under reduced pressure. The crude product was recrystallized from ethanol (60 mL), which on concentration gave 24 (0.079 g, 79%) as white crystals: m.p. 264° to 265°C; IR (KBT) 3500-2300, 3423, 1700, 1608, 1284, 1116, 1018, 903, 776 cm^-1. Anal, calcd. for C₂₇H₃₀O₃: C, 80.56; H, 7.51; found: C, 80.52; H, 7.56.

(d.) 4-[1-(1-Oxoethyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl]benzoic Acid (25): To alcohol 24 (0.020 g, 0.050 mmol) suspended in acetone (3 mL) was added at room temperature over a 5-min period Jones reagent (0.175 g, containing about 0.18 mmol CrO₃), prepared from CrO₃ (3.5 g), water (25 mL) and cone. H₂SO₄ (3.1 mL). The mixture was stirred for

10 min more to give a green suspension, diluted with water (20 mL), and centrifuged. The precipitate was washed with water (3 times 1.5 mL) with centrifugation. The white solid was dried at reduced pressure and dissolved in chloroform. After filtration, the filtrate was concentrated to give acid 25 (0.017 g, 85%) as white crystals: m.p. 205° to 206°C; IR (KBr) 3500-2500, 1684 (sh 1680), 1607, 1431, 1317, 1297, 1250, 1158, 923, 853, 774 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₂₇H₂₈O₃: 400.2038; found: 400.2073.

Example 9

(a.) Ethyl 4-(1-Carbethoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl)benzoate (26):

To a solution of 5 (0.390 g, 1.00 mmol) and tetrakis(triphenyl- phosphine)palladium(0) (0.03 g, 0.026 mmol) in 1,2-dimethoxyethane (6 mL) was added a solution of 4-carbethoxyphenylboronic acid (8) (0.213 g, 1.10 mmol) in ethanol (1.5 mL), followed by saturated aq. NaHCO₃ (3 mL, about 3.6 mmol). The mixture was heated at reflux for 1.5 h. The yellow-orange suspension was poured into dilute brine and extracted three times with Et₂O. The organic extract was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The pale-yellow solid was chromatographed twice on silica gel columns (3% ethyl acetate/20% CH₂Cl₂/hexanes, then 1 % to 2% ethyl acetate/ 10%

CH₂Cl₂/hexanes) to yield diester (26) (0.353 g, 77%) as white crystals: m.p. 158° to 160.5°C (CH₂Cl₂/hexanes); R_f (3% ethyl acetate/20% CH₂Cl₂/hexanes) 0.49; TR (kBr) 1705, 1605, 1270, 1160, 1110, 1040, 845 cm^-1. Anal, calcd. for C₃₀H₃₄O₄: C, 78.57; H, 7.47; found: C, 78.57; H, 7.47.

(b.) 4-(1-Carboxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3- anthracenyl)benzoic Acid (22): A suspension of diester 26 (0.250 g, 0.55 mmol) in ethanol (3 mL) and 40% aq. KOH (1.0 mL, about 6 mmol) was heated at reflux for 1 h to give a yellow solution. The solution was concentrated under an argon stream and acidified at 0°C with 1 N H₂SO₄. The precipitate was filtered, washed with water, and dried at reduced pressure to yield a pale-pink solid. The crude dicarboxylic acid was treated with ethanol (6 mL) and 0.2 g of decolorizing charcoal (Bamebey-Cheney, Inc., Columbus, OH) and stirred at 80°C for 2h, filtered while hot, and concentrated. The product was recrystallized from ethanol (5 mL) to give 27 (0.075 g, 34%) as white crystals, m.p. > 280°C; TR (KBr) 3600-2400, 1687, 1609, 1426, 1260, 1182, 1113, 910, 852, 775, 706 cm^-1.

Anal, calcd. for C₂₆H₂₆O₄: C, 77.59; H, 6.51; found: C, 77.34; H, 6.56.

Ethyl 4-(1-Carboxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3- anthracenyI)benzoate (28): To a solution of alcohol 9 (0.113 g, 0.271 mmol) in acetone (20 mL) was added dropwise over a 1.5-h period at room temperature Jones reagent (1.6 g, about 1.7 mmol of CrO₃), prepared from CrO₃ (3.5 g) in water (25 mL), and H₂SO₄ (3.1 mL). After 30 min at room temperature, the suspension was diluted with water (150 mL) and filtered. The yellow precipitate was washed repeatedly with water and dried at reduced pressure. The crude product was recrystallized (MeCN) to give 28 (0.093 g, 79%) as white crystals: m.p. 197° to 198°C; IR (KBr) 3500-2400, 1708, 1682, 1608, 1412, 1274, 1105, 1021, 771 cm^-1. Anal, calcd. for C₂₈H₃₀O₄: C, 78.11; H, 7.02; found: C, 77.88; H, 7.02.

(a.) 7-Bromo-5-(1,1,Dimethylethyl)-1,2,3,4-tetrahydro- 1,1,4,4-tetramethyl-anthracene (22): Reetz's geminal dimethylation reaction (method B) (Reetz and Westermann, J. Org. Chem. , 48:254-255 (1983)) was used. To a solution of 4 (0.32 g, 0.89 mmol) in CH₂Cl₂ (10 mL) under argon, cooled to -45°C, was added TiCl₄ (0.33 mL, 3 mmol) by syringe, followed by dimethylzinc (3 mmol) in CH₂CI₂ (1 mL). The red-brown reaction mixture was allowed to slowly reach room temperature, stirred overnight, then poured into ice (20 g), and extracted with CHCl₃ (2 times 30 mL). The organic layer was washed twice with water (30 mL) and dried (MgSO₄). The filtrate was partially evaporated, and the white precipitate that was formed was removed by filtration. The clear filtrate was then evaporated and the white solid recrystallized from CH₂Cl₂/hexanes to give 29 (0.100 g, 30%) as a white powder; R_f 0.70 (30% CH₂Cl₂/hexanes); m.p. 165° to 166°C; IR (KBr) 2959, 2924, 1582, 1458, 1364, 1249, 1210, 1170, 1112, 1098, 1049, 1018, 951, 898, 871 , 777, 727 cm^-1.

Anal, calcd. for C₂₂H₂₉Br: C, 70.77; H, 7.83; Br, 21.40; found: C, 70.56; H, 7.82; Br, 21.38.

(b.) Ethyl 4-[1-(1, 1-Dimethylethyl)-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl]benzoate (30): To a solution of 29 (0.373 g, 1.00 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (0.060 g, 0.052 mmol) in 6 mL of 1,2-dimethoxyethane was added under argon a solution of

4-carbethoxyphenyl boronic acid 8 (0.21 g, 1.10 mmol) in 1.3 mL of ethanol, followed by 3.0 mL of saturated aq. NaHCO₃. The yellow mixture was heated at reflux under argon for 1 h to give a dark suspension, which was poured into dilute brine and extracted with CH₂CI₂. The organic extract was washed twice with dilute brine, dried (Na₂SO₄), and concentrated to give a yellow gum, which was chromatographed on a silica gel column (30% CH₂Cl₂/hexanes) to yield 30 (0.358 g, 81 %) as white crystals, m.p. 144.5° to 145°C (hexane); Rf 0.53 (50% CH₂Cl₂/hexane): IR (CHCl₃) 1705, 1605, 1270, 1105, 1010, 895 cm^-1. Anal, calcd. for C₃₁H₃₈O₂: C, 84.12; H, 8.65; found: C, 84.16; H, 8.65.

(c.) 4-[1-(1,1-Dimethylethyl)-5,6,7,8-tetrahydro-

5,5,8,8-tetramethyl-3-anthracenyl]benzoic Acid (31): A suspension of ester 30 (0.165 g, 0.373 mmol) in 5 mL of ethanol and 1.0 mL (6 mmol) of 40% aq. KOH was heated at reflux for 1 h. Then the clear solution was concentrated in an argon stream and acidified at 0°C with 10 mL of 1 N HCl. The white suspension was stirred for 2 h and filtered. The precipitate was washed repeatedly with water and dried to give 0.153 g (99%) of 31 as a white powder. Crystallization (ethanol/2-propanol) gave 31 (0.138 g, 89%) as white needles, m.p. > 300°C: IR (KBr) 3600-2300, 3428, 2958, 2670, 1690, 1608, 1289, 1181, 1114, 903, 850, 777 cm^-1. Anal, calcd. for C₂₉H₃₄O₂: C, 84.02; H, 8.27; found: C, 83.86; H, 8.32.

Example 12

(a.) Ethyl 4-(5,6,7,8-Tetrahydro-1,5,5,8,8-pentamethyl- 3-anthracenyl)benzoate (22): To a solution of IS (0.212 g, 0.442 mmol) in 10 mL of ethyl acetate containing diisopropylethylamine (0.50 g, 3.9 mmol) was added 0.10 g of 5% Pd/C. The stirred mixture was hydrogenated at atmospheric

pressure at 21°C for 3 h. The suspension was filtered, and the precipitate was washed with ethyl acetate. The filtrates were washed three times with water, dried (Na₂SO₄), and concentrated. The residue was chromatographed on a silica gel column (25-50% CH₂Cl₂/hexane) to yield 32 (0.122 g, 69%), as a colorless gum; crystallization from CH₂Cl₂/hexanes gave colorless crystals, m.p. 129° to

130°C;R_f 0.50 (5% ethyl acetate/hexane); IR (CHCl₃) 1710, 1610, 1275, 1110, 1015, 900, 845 cm^-1. Anal, calcd. for C₂₈H₃₂O₂: C, 83.96; H, 8.05; found: C, 83.88; H, 8.12.

(b.) 4-(5,6,7,8-Tetrahydro-1,5,5,8,8-pentamethyl-3- anthracenyl)benzoic Acid (33): A mixture of ester 32 (0.074 g, 0.18 mmol), ethanol (1.5 mL), and 40% aq. KOH (0.5 mL, 3 mmol) was heated at reflux for 1 h to give a colorless solution, which was acidified at 0°C with 7 mL of 1 N HCl, then stirred at ambient temperature for 20 min, and filtered. The white precipitate was washed with water (5 times 1.5 mL) and dried, then crystallized from ethanol to give white crystals of 33 (0.061 g, 88%), m.p. 208.5° to

209.5°C; IR (KBr) 3600-2300, 2959, 1690, 1607, 1424, 1292, 900, 775 cm^-1. Anal, calcd. for C₂₆H₂₈O₂: C, 83.83; H, 7.58; found: C, 83.72; H, 7.63.

(a.) N-[(3-Bromo-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 1-anthracenyl)-methyl]pyrrolidine (34): A suspension of 60% NaH in mineral oil (0.048 g, 1.2 mmol) was washed under argon with hexane (2 times 1 mL). The dry NaH was treated with a solution of 6 (0.35 g, 1.01 mmol) in 1.3 mL of dimethylformamide. The suspension was stirred for 10 min at ambient temperature. To this mixture was added a solution of

N,N-methylphenylaminotriphenylphosphonium iodide (Tanigawa et al. ,

Tetrahedron Lett. , pp. 471-472 (1975)) (0.50 g, 1.01 mmol) and pyrrolidine (0.14 g, 1.97 mmol) in 1.3 mL of dimethylformamide. The red suspension was heated at 80 °C for 2 h, and then concentrated under reduced pressure at ambient temperature. The orange-brown solid was treated with 40 mL of 1 N NaOH and extracted three times with Et₂O. The organic extract was washed with brine, dried (Na₂SO₄), and concentrated. The pale-yellow gum was chromatographed on a silica gel column (12% ethyl acetate/hexane) to yield amine 34 (0.208 g, 51 %) as a pale yellow gum; R_f 0.66 (20% ethyl acetate/hexane); IR (film) 2970, 2930, 2780, 1585, 1485, 1460, 1385, 1365, 1345, 1220, 1185, 1110, 990, 885, 780 cm^-1; CI-HRMS (i-C₄H₉) calcd. for C₂₃H₃₀BrN: 399.1562; found: 399.1571.

(b.) Ethyl 4-[1-(1-Pyrrolidinomethyl)-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl]benzoate (35): To a solution of amine 34

(0.200 g, 0.50 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (0.03 g, 0.026 mmol) in 3 mL of 1,2-dimethoxyethane was added under argon a solution of 4-carbethoxyphenylboronic acid 8 (0.107 g, 0.55 mmol) in 0.95 mL of ethanol followed by 1.5 mL of saturated aq. NaHCO₃. The yellow suspension was heated at reflux for 1.25 h. The dark mixture was then partitioned between dilute brine and Et₂O. The ether extract washed twice with brine, dried (Na₂SO₄), and concentrated. Silica gel column chromatography (15% ethyl acetate/hexanes) yielded 35 (0.176 g, 75%), as a pale-yellow gum; R_f 0.50 (20% ethyl

acetate/hexane); TR (CHCl₃) 2780, 1700, 1605, 1270, 1105, 1015, 900, 865, 845 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₃₂H₄₀NO₂ (MH+): 470.3069; found: 470.3060.

(c.) 4-[1-(1-Pyrrolidinomethyl)-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-3-anthracenyl]benzoic Acid Hydrochloride Salt (36): A mixture of 35

(0.142 g, 0.30 mmol) and 0.5 mL (about 3 mmol) of 40% aq. KOH in 1.5 mL of ethanol was heated at reflux for 1 h under argon. The yellow solution was concentrated in an argon stream, acidified with 1.5 N HCl, stirred for 30 min at ambient temperature, and filtered. The precipitate was washed with water, dried, and crystallized from ethanol to give 26 as a white powder (0.127 g, 89%), m.p.

281° to 284°C (decomp.): IR (KBr) 3600-2300, 3425, 1704, 1608, 1215, 1182, 1113, 1016, 906, 777, 693 cm^-1. Anal, calcd. for C₃₀H₃₈ClNO₂: C, 75.37; H, 7.59; Cl, 7.42; N, 2.93; found: C, 75.16; H, 7.64; Cl, 7.32; N, 2.84.

(a.) Ethyl 4-(1-Hydroxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl)benzoate (37): To a solution of aldehyde 14 (0.207 g, 0.50 mmol) in 5 mL of 1 ,2-dichloroethane was added 85% m-chloroperbenzoic acid (0.20 g, 1.0 mmol). The mixture was heated under argon at 85°C for 2.75 h. The orange solution was cooled, treated with a further 0.20 g (1.0 mmol) of 85% m-chloroperbenzoic acid, and heated at 80 °C for 24 h, and then cooled. The pale-brown solution was concentrated and the residue was partitioned between ethyl acetate and 5% aq. NaHCO₃. The organic extract was twice washed with dilute brine, dried (Na₂SO₄) and concentrated to give a dark semisolid, then dissolved in 4 mL of ethanol and treated under argon with 0.75 g (about 1.1 mmol) of 10% aq. KOH. The dark solution was stirred for 1.5 h, then concentrated in an argon stream and acidified at 0°C with 1 N HCl to give a pale-brown precipitate. The suspension was filtered, washed repeatedly with water and dried, then dissolved in CH₂CI₂, and the solution was filtered and concentrated. The crude product was chromatographed on a silica-gel column

(5%-10% ethyl acetate/20% CH₂Cl₂/hexane) to give first unreacted 14 (0.057 g,

25%), R_f 0.67 (10% ethyl acetate/20% CH₂Cl₂/hexane), and then the phenol 37 (0.049 g, 24%) as a yellow powder; R_f 0.43 (10% ethyl acetate/20%

CH₂Cl₂/hexane); TR(KBr) 3386, 2959, 1690, 1604, 1577, 1464, 1414, 1367, 1279, 1184, 1106, 1020, 907, 852, 773 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₂₇H₃₀O₃: 402.2195; found: 402.2220.

(b.) 4-(1-Hydroxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 3-anthracenyl)benzoic Acid (38): A suspension of ester 37 (0.029 g, 0.058

mmol) in 2 mL of acetic acid and 1.0 mL of 5 N H₂SO₄ was heated at reflux under argon for 2.5 h. The yellow-orange suspension was cooled, treated with 0.5 mL of 18 N H₂SO₄, and heated at reflux under argon for 24 h, then cooled. The pale-brown suspension was diluted with 10 mL of water and filtered. The precipitate was washed with water (6 times 3 mL) and dried. The crude acid was dissolved in ethyl acetate, the yellow solution was filtered, concentrated, and purified by reversed-phase HPLC [Waters Novapak C₁₈ 8 mm X 100 mm, 90% MeCN/10% (1 % acetic acid/H₂O), 1.5 mL/min, 260 nm]. The product 38 eluted at t_R 3.4 min to give, after concentration, an off-white powder (0.019 g, 70%), m.p. 265° to 268°C: IR (KBr) 3500-2400, 3417, 2960, 2670, 2544, 1688, 1605, 1575, 1462, 1416, 1365, 1292, 1244, 1182, 1105, 1046, 906, 844, 778 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₂₅H₂₆O₃: 374.1882; found: 374.1867.

(a.) Methyl 8-[(n-Pentylamino)carbonyl]octanoate (40): To a solution of azelaic acid monomethyl ester 39 (4.04 g, 20.0 mmol),

N-hydroxysuccinimide (2.53 g, 22.0 mmol), and 4-dimethylaminopyridine (0.12 g, 0.98 mmol) in 20 mL of tetrahydrofuran was added over 10 min at 0°C 5.3 g (25.6 mmol) of dicyclohexylcarbodiimide. The mixture was stirred at ice-bath temperature for 30 min. To the white suspension was added a solution of n-pentylamine (1.74 g, 20.0 mmol) in 3 mL of tetrahydrofuran and the mixture was stirred for 1.5 h and filtered. The filtrate was concentrated to give a white semisolid, which was washed with hexane, dried, washed with water, and again dried to give a white solid. The crude product was chromatographed on a silica gel column (25% ethyl acetate/CH₂Cl₂) to yield the amide 40 (4.43 g, 82%), as a white powder. A sample was recrystallized (CH₂Cl₂/hexane) to give colorless needles, m.p. 47.5° to 48°C; R_f 0.42 (33% ethyl acetate/CH₂Cl₂); TR (KBτ) 3307, 2930, 1731, 1636, 1543, 1472, 1438, 1420, 1375, 1315, 1236, 1173, 703 cm^-1. Anal, calcd. for C₁₅H₂₉NO₃: C, 66.38; H, 10.77; N, 5.16; found: C, 66.45; H, 10.81; N, 5.22.

(b.) 8-[(n-Pentylamino)carbonyl]octanoic Acid (41): To the ester 40 (4.06 g, 15.0 mmol) in 75 mL of tetrahydrofuran was added 100 mL (200 mmol) of 2 N NaOH, and the mixture was stirred at ambient temperature under argon for 2.5 h. The aqueous phase was separated, the tetrahydrofuran phase was washed with dilute brine, and the combined aqueous phases were acidified with concentrated HCl at 0°C to pH 1-2. The suspension was extracted three times with CHCl₃, and the extract was washed twice with water, dried, and concentrated to give 41 as a white solid (3.20 g, 83%); m.p. 75° to 76°C

(CH₂Cl₂/hexane); IR (KBr) 3400-2400, 3313, 3056, 2932, 1699, 1636, 1540, 1473, 1416, 1315, 1251, 1192, 938, 685 cm^-1. Anal, calcd. for C₁₄H₂₇NO₃: C, 65.33; H, 10.57; N, 5.44; found: C, 65.45; H, 10.59; N, 5.49.

(c.) Ethyl 4-(1-{7-[(n-Pentylamino)carbonyl]heptanecarbox- amido}-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl)benzoate (43): A solution of acid 41 (0.040 g, 0.16 mmol) and triethylamine (32 mL, 0.23 mmol) in 0.3 mL of tetrahydrofuran was treated at 0°C with isobutyl chloroformate (21 μh, 0.16 mmol). This mixture was stirred for 2 h. The thick white suspension was treated at 0°C with a solution of ethyl 4-(1-amino-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-3-anthracenyl)benzoate (Dawson et al, J. Label. Comp.

Radiopharm. , 28:89-98 (1990) (42) (0.024 g, 0.060 mmol) in 0.3 mL of tetrahydrofuran containing triethylamine (10 μl, 0.07 mmol) and then allowed to warm to ambient temperature over a 2.5-h period. The suspension was partitioned between CHCl₃ and water, and the CHCl₃ extract was washed twice with 0.2 N NaOH, then twice with water, dried (Na₂SO₄), and concentrated to give a pink semisolid. Two silica gel column chromatographies (25% ethyl acetate/CH₂Cl₂) gave 43 (0.023 g, 60%) as white needles, m.p. 146° to 147°C

(CH₂Cl₂/pentane); R_f 0.45 (35% ethyl acetate/CH₂Cl₂); IR (CHCl₃) 3450, 1700, 1660, 1605, 1510, 1270, 1100, 1010, 840 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₄,H₅₆N₂O₄: 640.4240; found: 640.4265.

(d.) 4-(1-{7-[(n-Pentylamino)carbonyl]heptanecarboxamido}- 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl)benzoic Acid (44): To a solution of ester 42 (0.020 g, 0.031 mmol) in 2 mL of ethanol was added 0.2 g (0.8 mmol) of 4 N aq. KOH, the mixture was stirred at ambient temperature under argon for 2 h, and then acidified with 1 N HCl. The suspension was concentrated at < 30° C, then diluted with water and extracted three times with CHCl₃. The organic extract was washed twice with water, dried (Na₂SO₄) and concentrated to yield a pale-yellow glass. The crude product was purified by reversed-phase HPLC [Waters Novapak C₁₈, 8 mm X 100 mm; 80% MeOH/10% tetrahydrofuran/10% (1 % aq. acetic acid), 2.0 mL/min, 260 nm; t_R 2.4 min] to yield 42 (0.011 g, 58%), as a pale-yellow glass; TR (KBr) 3500-2400, 3289, 2929, 2626, 1689, 1649, 1607, 1542, 1462, 1408, 1364, 1261, 1181, 1112,

1018, 901, 850, 777, 704 cm^-1. Positive-ion FAB-MS calcd. for C₃₉H₅₃N₂O₄ (MH⁺): 613.4005; found, 613.3982.

(a.) Ethyl 4-(3-Methyl-2-naρhthalenyl)benzoate (46): To Mg turnings (0.53 g, 22 mmol) was added over a 20-min period under argon while heating at 50-55°C a solution of 2-bromo-3-methylnaphthalene (Smith et al, J. Org. Chem., 51:3762-3768 (1986)) (45) (3.98 g, 18.0 mmol) in 28 mL of tetrahydrofuran. The reaction was initiated with Mel (2 times 10 μl) after one-quarter of the solution had been introduced, and then moderated by removal of the heating bath until the addition was complete. Then the suspension was stirred at 50-55°C for an additional 1 h. The cooled supernatant solution was added under argon by syringe to a solution of fused ZnCl₂ (3.0 g, 22 mmol), in 20 mL of tetrahydrofuran with ice-water cooling. The light-brown suspension was stirred at ambient temperature for 30 min, then treated with a solution of ethyl 4-iodobenzoate (6.07 g, 22.0 mmol) in 5 mL of tetrahydrofuran, immediately followed by three 2.5-mL aliquots at 30-min intervals of a dark-brown Ni(0) catalyst solution, prepared by treatment of a mixture of bis(triphenylphosphine)nickel (II) chloride (0.66 g, 1.01 mmol),

triphenylphosphine (0.52 g, 1.98 mmol), and 28 mL of tetrahydrofuran with 2.5 mL (2.5 mmol) of 1 M diisobutylaluminum hydride in cyclohexane. The dark-brown suspension was stirred at ambient temperature for 18 h, and then poured into 1 N HCl and twice extracted with Et₂O. The organic extract was washed twice with brine, dried (Na₂SO₄), and concentrated. The resulting gum was chromatographed three times on silica gel columns (2% ethyl acetate/5% CH₂Cl₂/hexane; 3.5% ethyl acetate/hexane; then 3% ethyl acetate/hexane) to yield ester 46 (1.88 g, 36%), as a colorless gum. Crystallization from hexane gave white crystals, m.p. 73° to 75°C; R_f 0.55 (10% ethyl acetate/hexane); IR

(KBr) 3055, 2980, 2932, 1713, 1607, 1562, 1494, 1465, 1401, 1366, 1275, 1174, 1123, 1099, 1021, 982, 880, 864, 774, 745, 711 cm^-1. Anal, calcd. for C₂₀H₁₈O₂: C, 82.73% 5%; found: C, 82.67%; H, 6.33%.

(b.) Ethy ethyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 2-anthracenyl)benzoate (47): To a solution of 46 (1.45 g, 4.99 mmol) and

2,5-dichloro-2,5-dimethyl-hexane (1.01 g, 5.52 mmol) in 20 mL of CH₂Cl₂ at 0°C was added AICI3 (1.33 g, 10.0 mmol). The yellow-brown solution was stirred for 30 min before the ice-bath was removed, and the mixture was stirred for 3 h more with warming to ambient temperature. The suspension was poured onto ice-2 N HCl and extracted twice with CH₂CI₂. The yellow extract was washed three times with dilute brine, then dried (Na₂SO₄), and concentrated. The residue was chromatographed on a silica gel column (3% ethyl

acetate/hexane) to yield 47 (1.26 g, 63%) as white crystals, m.p. 94° to 95 °C (from MeCN); R_f 0.54 (50% CH₂Cl₂/hexane); TR (KBr) 2961, 2924, 1716, 1608, 1564, 1460, 1385, 1365, 1273, 1176, 1102, 1022, 980, 909, 869, 853, 775, 711 cm^-1. Anal, calcd. for C₂₈H₃₂O₂: C, 83.96; H, 8.05; found: C, 84.05; H, 8.07.

(c.) 4-(3-Methyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-anthracenyl)benzoic Acid (48): A suspension of ester 47 (0.774 g, 1.93 mmol) in 25 mL of ethanol and 5 mL of 40% aq. KOH (about 30 mmol) was stirred at ambient temperature under argon for 18 h, then concentrated and acidified with 1 N HCl. The suspension was filtered, and the precipitate was washed repeatedly with water and dried to give 47 (0.705 g, 98%) as a white solid, m.p. 247.5° to 248.5°C (from ethanol); TR (KBr) 3500-2400, 2959, 2926, 2669, 2541, 1691, 1608, 1563, 1460, 1418, 1363, 1313, 1284, 119, 1107, 1020, 980, 907, 870, 779, 713 cm^-1. Anal, calcd. for C₂₆H₂₈O₂: C, 83.83; H, 7.58; found: C, 83.74; H, 7.63.

(a.) Ethyl 4-(3-Bromomethyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-anthracenyl)benzoate (49): A solution of ester 47 (0.536 g, 1.34 mmol), N-bromosuccinimide (0.286 g, 1.61 mmol), and dibenzoyl peroxide (about 0.01 g) in 5 mL of CCl₄ was heated at reflux for 1.5 h while irradiated from a distance of 10 cm with a 200 W tungsten lamp. The orange suspension was filtered, the precipitate was washed with CCl4, and the filtrate was concentrated. The residue was chromatographed on a silica gel column (20%-50% CH₂Cl₂/hexane) to yield 49 (0.531 g, 83%) as a white solid, m.p. 129° to 130°C (hexanes); R_f 0.47 (50% CH₂Cl₂/hexane); IR (CHCl₃) 1705, 1600, 1455, 1270, 1095, 1010, 980, 900, 860, cm^-1. CI-HRMS (i-C₄H₉); calcd. for C₂₈H₃₂BrO₂ (MH⁺): 479.1586; found: 479.1551.

(b.) Ethyl 4-(3-Benzyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 2-anthracenyl)benzoate (50): To a solution of 49 (0.192 g, 0.40 mmol) and tetrakis(triphenyl-phosphine)palladium(0) (0.030 g, 0.026 mmol) in 3 mL of 1,2-dimethoxyethane was added under argon a solution of phenylboronic acid

(0.075 g, 0.61 mmol) in 0.7 mL of ethanol, followed by 1.5 mL of saturated aq. NaHCO₃. The mixture was heated at reflux for 1.5 h, before the dark suspension was partitioned between CH₂CI₂ and dilute brine. The yellow organic phase was washed with brine, dried (Na₂SO₄), and concentrated. The dark resultant oil was chromatographed on a silica gel column (25-35%

CH₂Cl₂/hexane) to yield 50 (0.063 g, 33%), a pale-yellow gum. Crystallization (hexane) gave a white solid, m.p. 131 ° to 132°C; R_f 0.36 (35%

CH₂Cl₂/hexane); IR (CHCl₃) 1700, 1605, 1265, 1170, 1095, 1015, 980, 905 cm^-1. Anal. calcd. for C₃₄H₃₆O₂: C, 85.67; H, 7.61; found: C, 85.50; H, 7.65.

(c.) 4-(3-Benzyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 2-anthracenyl)benzoic Acid (51): A mixture of ester 50 (0.028 g, 0.059 mmol), 40% aq. KOH (0.3 g, about 1.8 mmol), and 0.6 mL of ethanol was heated at reflux under argon for 0.75 h to give a colorless solution, concentrated in an argon stream, cooled in ice, and acidified with 1 N HCl. The suspension was filtered, washed with water (6 times 1 mL), dried, and crystallized (ethanol) to give white crystals (0.018 g, 68%), m.p. 284° to 286°C: IR (KBr) 3600-2300, 3448, 2959, 1689, 1608, 1459, 1420, 1285, 1178, 1107, 911, 699 cm^-1. Anal, calcd. for C₃₂H₃₂O₂: C, 85.68; H, 7.19; found: C, 85.50; H, 7.25.

(a.) Ethyl 4-[3-(4-Dimethylaminophenyl)methyl-5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl]benzoate (52): To a solution of 47 (0.240 g, 0.50 mmol) and tetrakis(triphenyl- phosphine)palladium(0) (0.030 g, 0.026 mmol) in 3 mL of 1,2-dimethoxyethane was added under argon a solution

of 4-dimethylamino-phenylboronic acid (0.10 g, 0.61 mmol) in 0.7 mL of ethanol, followed by 1.5 mL of saturated aq. NaHCO₃. The mixture was heated at reflux for 1.5 h, and then partitioned between CH₂CI₂ and dilute brine. The yellow CH₂CI₂ extract was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The gum was chromatographed twice on silica gel (25%-100%

CH₂Cl₂/hexane, then 35%-100% CH₂Cl₂/hexane) to yield 52 (0.139 g, 54%), as a pale-yellow gum. Crystallization from ethyl acetate-hexanes gave pale-yellow crystals, m.p. 151° to 152°C; R_f 0.54 (15% ethyl acetate/hexanes); TR (CHCl₃) 1700, 1605, 1565, 1510, 1475, 1455, 1270, 1170, 1095, 1015, 985, 940, 905 cm^-1. CI-HRMS (i-C₄H₉) calcd. for C₃₆H₄₁NO₂: 519.3137; found: 519.3125.

(b.) 4-[3-(4-Dimethylaminophenyl)methyl-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-anthracenyl]benzoic Acid (53): A mixture of the ester 52 (0.105 g, 0.202 mmol), 40% aq. KOH (0.5 mL, about 3 mmol), and ethanol (1.5 mL) was heated at reflux for 0.75 h to give a pale-yellow solution. The solution was concentrated in an argon stream and acidified with 1 N HCl at 0°C. The suspension was filtered, and the white solid was washed repeatedly with water, dried, and crystallized (ethanol) to yield 53 (0.095 g, 89%), as a white powder, m.p. 243° to 253°C (decomp.); IR (KBr) 3600-2300, 3424, 2628, 1708, 1608, 1511, 1461, 1364, 1214, 1176, 1108, 1019, 909, 783, 707 cm^-1. Anal. calcd. for C₃₄H₃₈ClNO₂: C, 77.32; H, 7.25; Cl, 6.71; N, 2.65; found: C, 77.38; H, 7.25; Cl, 6.44; N, 2.61.

(a.) Methyl 4-[(3,4-Dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)- carbonyl]benzoate (56) (Belgian Patent BE 1,000,195): To a suspension of aluminum chloride (1.6 g, 12 mmol) in 1 mL of 1,2-dichloroethane under argon at room temperature was added a solution of 4,4-dimethyl-3,4-dihydro-2H-1- benzopyran (54) (1.5 g, 9.25 mmol) (Dawson, M. I., et al. J. Med. Chem.

27: 1516-1531 (1984)) and 4-carbomethoxybenzoyl chloride (55) (1.79 g, 9 mmol) in 9 mL of 1,2-dichloroethane. The reaction mixture was stirred overnight, poured onto ice-water, and extracted with 40% ethyl acetate/hexane. The combined organic layers were washed with saturated aqueous NaHCO₃ and brine. The solution was dried over anhydrous MgSO₄, filtered, and concentrated to afford a yellow solid (3.24 g). Flash chromatography (80% dichloromethane/ hexane) yielded the desired product (56) as a white powder (1.42 g, 49%): m.p. 129-131 °C; R_f 0.26 (CH₂Cl₂); IR (KBr) 2954, 1725, 1645, 1572, 1278, 1251, 1110 cm^-1; ¹H NMR (CDCI₃) δ 1.36 (s, 6, CH₃), 1.88 (m, 2, OCH₂CH₂), 3.97 (s, 3, CO₂Me), 4.28 (m, 2, OCH₂CH₂), 6.84 (d, J = 8.5 Hz, 1, ArH), 7.53 (dd, J = 2.2, 8.5 Hz, 1, ArH), 7.78 (d, J = 8.5 Hz, 2, ArH), 7.86 (d, J = 2.2

Hz, 1, ArH), 8.14 (d, J = 8.5 Hz, 2, ArH). Anal, calcd. for C₂₀H₂₀O₄: C, 74.06; H, 6.21. Found: C, 73.97; H, 6.22.

(b.) 2-(3,4-Dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)- 2-(4-carbomethoxyphenyl)-1,3-dithiane (57): To a solution of the keto-ester (56) (0.152 g, 0.469 mmol) in dichloromethane (3 mL) at 0°C under argon was added

1,3-propanedithiol (0.061 g, 0.563 mmol), followed by boron trifluoride etherate (0.07 mL, 0.57 mmol). The resulting mixture was stirred at 0°C for 1 h and at ambient temperature overnight. The reaction mixture was quenched by pouring into saturated aqueous Na₂CO₃, and then extracted with 40% ethyl

acetate/hexane. The combined organic layers were dried over anhydrous

MgSO₄, filtered, and concentrated to afford an oil. Flash chromatography (80% dichloromethane/hexane) yielded the desired dithiane (57) as a white solid (0.175 g, 90%): m.p. 103-105°C; R_f 0.2 (50% CH₂Cl₂/hexane); TR (KBr) 2955, 1722, 1490, 1276, 1109 cm^-1; ¹H NMR (CDCI₃) δ 1.28 (s, 6, CH₃), 1.82 (m, 2, OCH₂CH₂), 2.01 (m, 2, SCH₂CH₂CH₂S), 2.76 (m, 4, SCH₂CH₂CH₂S ), 3.92

(s, 3, CO₂Me), 4.18 (m, 2, OCH₂CH₂), 6.17 (d, J = 8.7 Hz, 1, ArH), 7.20 (dd, J = 2.3, 8.7 Hz, 1, ArH), 7.57 (d, J = 2.3 Hz, 1, ArH), 7.84 (d, J = 8.6 Hz, 2, ArH), 8.02 (d, J = 8.6 Hz, 2, ArH). Anal, calcd. for C₂₃H₂₆O₃S₂: C, 66.63; H, 6.32; S, 15.47. Found: C, 66.32; H, 6.28; S, 15.26.

(c.) 2-(3,4-Dihydro-4,4-dimethyl-2H-1-benzopyran-

6-yl)-2-(4-carboxy-phenyl) 1,3-dithiane (58): To a suspension of the dithiane (57) (0.145 g, 0.349 mmol) in 75% aqueous methanol (4 mL) was added one pellet of potassium hydroxide (0.106 g). The reaction mixture was stirred at 70°C for 1 h, during which time the material dissolved. The solution was cooled to room temperature, acidified with 1 N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate/hexane. The combined organic layers were dried over anhydrous MgSO₄, filtered, and concentrated to afford a white solid.

Recrystallization from dichloromethane-hexane afforded the desired acid (58) as a white powder (0.127 g, 90%): m.p. 204-205°C; IR (KBr) 2955, 1722, 1490, 1276, 1109 cm^-1 ; ¹H NMR (CDCI₃) δ 1.29 (s, 6, CΗ₃), 1.83 (m, 2, OCH₂CH₂), 2.02 (m, 2, SCH₂CH₂CH₂S), 2.78 (m, 4, SCH₂CH ₂CH₂S), 4.19 (m, 2, OCH₂CH₂), 6.72 (d, J = 8.7 Hz, 1, ArH), 7.22 (dd, J = 2.4, 8.7 Hz, 1, ArH), 7.59 (d, J = 2.4 Hz, 1, ArH), 7.88 (d, J = 8.7 Hz, 2, ArH), 8.09 (d, J = 8.7 Hz, 2, ArH). HRMS calcd. for C₂₂H₂₅O₃S₂ (M⁺ +H): 401.1245. Found: 401.1261.

(a.) Methyl 4-[1-Oxo-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 2-naphthalenyl)ethyl]benzoate (60): A flask containing anhydrous nickel iodide (2.031 g, 6.5 mmol), naphthalene (0.083 g, 0.65 mmol), and clean oil-free lithium wire (0.105 g, 15.15 mmol) was subjected to three cycles of evacuation and argon purging prior to adding dry glyme (10 mL). The resulting suspension was stirred at room temperature for 14 h and then heated at 70 °C for 4 h to ensure that the reaction of the Ni(TI) species was complete. A solution of acid chloride (55) (1.043 g, 5.25 mmol) and 2-bromomethyl-5,6,7,8-tetrahydro- 5,5,8,8-tetramethylnaphthalene (59) (1.406 g, 5.00 mmol) (Dawson, M. I., et al., J. Med. Chem. 32: 1504-1517 (1989)) in dry glyme (10 mL) was added, and the reaction mixture was heated at reflux for 18 h. After cooling, the reaction mixture was poured into cold 2 N hydrochloric acid (75 mL) and extracted with ether. The combined organic layers were washed with water and brine. The solution was dried over anhydrous MgSO₄, filtered, and concentrated to afford a brown solid (1.97 g). Flash chromatography (50% dichloromethane/hexane), followed by recrystallization from hexane, afforded (60) as a white fluffy solid (0.611 g, 34%): m.p. 110-112°C; R_f 0.29 (benzene); TR (KBr) 1720, 1700, 1280 cm^-1; ¹H NMR (CDCI₃) δ 1.25 (s, 6, CH₃), 1.26 (s, 6, CH₃), 1.66 (s, 4, CH₂CH₂), 3.95 (s, 3, CO₂CH₃), 4.25 (s, 2, CH₂CO), 7.02 (dd, J = 2, 8 Hz, 1, ArH), 7.18 (d, J = 2 Hz, 1, ArH), 7.25 (d, J = 8 Hz, 1, ArH), 8.06 (d, J =

9 Hz, 2, ArH), 8.12 (d, J = 9 Hz, 2, ArH). Anal, calcd. for C₂₄H₂₈O₃: C, 79.09; H, 7.74. Found: C, 79.18; H, 7.67.

(b.) Methyl (Z)-4-[1-Acetoxy-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra- methyl-2-naphthalenyl)ethenyl]benzoate (61): To a solution of keto-ester (60) (0.092 g, 0.25 mmol) in acetic anhydride (5 Ml) under argon at room

temperature was added a catalytic amount of concentrated sulfuric acid (3 drops), and the reaction mixture was heated at 100°C for 14 h. After cooling, the reaction mixture was diluted with water and extracted with ether. The combined organic layers were washed with water, saturated aqueous NaHCO₃, and brine. The solution was dried over anhydrous MgSO₄, filtered, and concentrated to afford a brown solid (0.117 g). Flash chromatography (10% ethyl

acetate/hexane, 50% dichloromethane/hexane) followed by recrystallization from hexane afforded (61) as a white crystalline solid (0.036 g, 35%): m.p.

144-146°C; R_f 0.10 (50% CH₂Cl₂/hexane); TR (KBr) 1754, 1721, 1608, 1284, 1200 cm^-1 ; ¹H NMR (acetone-d₆) δ 1.29 (s, 6, CH₃), 1.31 (s, 6, CH₃), 1.72 (s,

4, CH₂CH₂), 2.41 (s, 3, OCOCH₃), 3.90 (s, 3, CO₂CH₃), 6.96 (s, 1, HC = C), 7.34 (d, J = 8 Hz, 1, ArH), 7.40 (dd, J = 2, 8 Hz, 1, ArH), 7.57 (d, J = 2 Hz, 1, ArH), 7.69 (d, J = 9 Hz, 2, ArH), 8.01 (d, J = 9 Hz, 2, ArH). Anal^. calcd. for C₂₆H₃₀O₄: C, 76.82; H, 7.44. Found: C, 76.48; H, 7.60.

(a.) 6-Cyano-1-naphthoyl Chloride (62): To a solution of

6-cyano-1-naphthoic acid (62) (0.221 g, 1.12 mmol) (Dewar, M. J. S., et al , J. Am. Chem. Soc. 84:3541-3546 (1962)) in thionyl chloride (10 mL) under argon at room temperature was added one drop of DMF, and the reaction mixture was heated at reflux for 14 h. After cooling, the excess thionyl chloride was removed at reduced pressure, and the residue azeotroped twice with dry toluene (10 mL) to give the acid chloride (63) as a pale-yellow solid (0.244 g, 99%): ¹H NMR

(acetone-d₆) δ 7.95 (dd, J = 7, 8 Hz, 1, ArH), 8.03 (dd, J = 2, 9 Hz, 1, ArH), 8.54 (d, J = 8 Hz, 1, ArH), 8.69 (d, J = 2 Hz, 1, ArH), 8.84 (dd, J = 1, 7 Hz, 1, ArH), 8.86 (d, J = 9 Hz, 1, ArH). (b.) 5-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- carbonyl]-2-naphthonitrile (65): To a suspension of aluminum chloride (0.165 g, 1.24 mmol) in 10 mL of 1,2-dichloroethane at room temperature under argon was added a solution of 1,2,3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene (64) (0.223 g, 1.18 mmol) (Kagechika, H., et al., J. Med. Chem. 31:2182-2192 (1988)) and acid chloride (63) (0.243 g, 1.13 mmol) in 10 mL of 1,2-dichloroethane. The resulting solution was then heated at reflux for 16 h. After cooling, the reaction mixture was poured onto a mixture of crushed ice (25 g) and 2 N hydrochloric acid (25 mL) and extracted with dichloromethane. The combined organic layers were washed with water, 1 M NaHCO₃, and brine. The solution was dried over anhydrous MgSO₄, filtered, and concentrated to afford an orange solid (0.285 g). Flash chromatography (5% ethyl acetate/hexane) followed by recrystallization from hexane afforded the desired product (65) as a light-yellow crystalline solid (0.148 g, 36%): m.p. 179-181°C; R_f 0.10 (5% EtOAc/hexane); IR (KBr) 2231, 1657, 1253 cm^-1; ¹H NMR (CDCI₃) δ 1.27 (s, 6, CH₃), 1.31 (s, 6, CH₃), 1.72

(s, 4, CH₂CH₂), 7.37 (d, J = 8 Hz, 1, ArH), 7.49 (dd, J = 2, 8 Hz, 1, ArH), 7.63 (dd, J = 2, 8 Hz, 1, ArH), 7.65 (dd, J = 7, 9 Hz, 1, ArH), 7.74 (dd, J = 1, 7 Hz, 1, ArH), 7.91 (d, J = 2 Hz, 1, ArH), 8.06 (d, J = 8 Hz, 1, ArH), 8.21 (d, J = 9 Hz, 1, ArH), 8.32 (d, J = 2 Hz, 1, ArH). Anal, calcd. for C₂₆H₂₅NO: C, 84.98; H, 6.86; N, 3.81. Found: C, 84.91; H, 6.87; N, 3.60.

(c.) Methyl 5-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl- 2-naphthalenyl)-carbonyl]-2-naphthalenecarboxylate (66): To a suspension of the keto-nitrile (65) (0.136 g, 0.37 mmol) in 3.7 mL of ethanol at room temperature under argon was added 40% aqueous sodium hydroxide (0.37 mL, 3.7 mmol), and the resulting solution was heated at reflux for 14 h. After cooling, the reaction mixture was acidified with 2 N hydrochloric acid and extracted with ether. The combined organic layers were washed with water and brine. The solution was dried over anhydrous MgSO₄, filtered, and concentrated to afford an orange gummy solid (0.135 g). Attempts to isolate the pure acid by

recrystallization failed. Therefore, the crude acid (67) was esterified to aid in purification. To a solution of crude acid (67) (0.084 g, 0.216 mmol) in DMF (2 mL) under argon at room temperature was added potassium carbonate (0.060 g,

0.433 mmol) and methyl iodide (27 μL, 0.432 mmol). This mixture was stirred for 18 h and then diluted with water and extracted with ether. The combined organic layers were washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated to afford a yellow foam (0.076 g). Flash

chromatography (75% dichloro-methane/hexane) followed by recrystallization from ether/hexane afforded (66) as an off-white crystalline solid (0.042 g, 28%): m.p. 138-140°C; R_f 0.36 (CH₂Cl₂); IR (KBr) 2959, 1722, 1657, 1282, 1245, 1201, 779 cm^-1; ¹H NMR (CDCI₃) δ 1.27 (s, 6, CH₃), 1.31 (s, 6, CH₃), 1.72 (s, 4, CH₂CH₂), 4.0 (s, 3, CO₂CH₃), 7.36 (d, J = 8 Hz, 1, ArH), 7.51 (dd, J = 2, 8 Hz, 1, ArH), 7.58 (dd, J = 7, 8 Hz, 1, ArH), 7.69 (dd, J = 2, 7 Hz, 1,

ArH), 7.93 (d, J = 2 Hz, 1, ArH), 8.06 (dd, J = 2, 9 Hz, 1, ArH), 8.11 (d, J = 9 Hz, 1, ArH), 8.15 (d, J= 7 Hz, 1, ArH), 8.68 (d, J = 2 Hz, 1, ArH). Anal. calcd. for C₂₇H₂₈O₃: C, 80.97; H, 7.05. Found: C, 81.33; H, 6.96.

(d.) 5-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- carbonyl]-2-naphthalenecarboxylic Acid (67): To a suspension of the ester (66)

(0.035 g, 0.087 mmol) in 75% aqueous methanol (0.39 mL) was added 0.015 g of potassium hydroxide and the mixture was stirred at 70°C for 5 h, during which time the solid dissolved. The solution was cooled to room temperature, acidified with 1 N hydrochloric acid, and then extracted with 80% ethyl acetate/hexane. The combined organic layers were dried over anhydrous

MgSO₄, filtered, and concentrated to afford a white solid. Recrystallization from benzene/hexane afforded (67) as a white crystalline solid (0.033 g, 98%): m.p. 253-255°C; IR (KBr) 3426, 2961, 1696, 1858, 1290, 1245, 1191, 1065 cm^-1; ¹H NMR (CDCI₃) δ 1.28 (s, 6, CH₃), 1.32 (s, 6, CH₃), 1.72 (s, 4, CH₂CH₂), 7.37 (d, J = 8 Hz, 1, ArH), 7.52 (dd, J = 2, 8 Hz, 1, ArH), 7.62 (dd, J = 7, 8 Hz, 1, ArH), 7.72 (dd, J = 2, 7 Hz, 1, ArH), 7.93 (d, J = 2 Hz, 1, ArH), 8.13 (dd, J = 2, 9 Hz, 1, ArH), 8.17 (d, J = 9 Hz, 1, ArH), 8.19 (d, J = 7 Hz, 1, ArH), 8.78 (d, J = 2 Hz, 1, ArH). Anal. calcd. for C₂₆H₂₆O₃: C, 80.80; H, 6.78. Found: C, 80.66; H, 6.57.

(a.) Ethyl 4-(3-Methylnaphthalen-2-yl)benzoate (69): To Mg turnings (0.53 g, 22 mmol) was added over a 20-min period under argon while heating at 50-55°C a solution of 2-bromo-3-methyl-naphthalene (68) (3.98 g, 18.0 mmol) (Smith, J. G., et al., J. Org. Chem. 51:3762-3768 (1986)) in 28 mL of THF. The reaction was initiated with Mel (2 x 10 μL) after one-quarter of the solution had been introduced, and then moderated by intermittent removal of the

heating bath until the addition was complete. Then, the suspension was stirred at 50-55 °C for an additional hour. The cooled supernatant solution was added under argon by syringe to a solution of fused ZnCl₂ (3.0 g, 22 mmol) in 20 mL of THF with ice-water cooling. The light-brown suspension was stirred at ambient temperature for 30 min, then treated with a solution of ethyl

4-iodobenzoate (6.07 g, 22.0 mmol) in 5 mL of THF, immediately followed by three 2.5-mL aliquots at 30-min intervals of the dark-brown Ni(0) catalyst solution, which was prepared by treatment of a mixture of

bis(triphenylphosphine)nicke(II) chloride (0.66 g, 1.01 mmol),

triphenylphosphine (0.52 g, 1.98 mmol), and THF (28 mL) with 2.5 mL (2.5 mmol) of 1.0 M diisobutylaluminum hydride in cyclohexane. The dark-brown suspension was stirred at ambient temperature for 18 h, then poured into 1 N HCl and twice extracted with Et₂O. The organic extract was washed twice with brine, dried (Na₂SO₄), and concentrated. The resulting gum was

chromatographed three times on silica gel (2% EtOAc/5% CH₂Cl₂/hexane, 3.5%

EtOAc/hexane, 3% EtOAc/hexane) to yield ester (69) as a colorless gum (1.88 g, 36%). Crystallization from hexane gave white crystals: m.p. 73-75°C; R_f (10% EtOAc/hexane) 0.55; IR (KBr) 3055, 2980, 2932, 1713, 1607, 1562, 1494, 1465, 1401, 1366, 1275, 1174, 1123, 1099, 1021, 982, 880, 864, 774, 745, 711 cm^-1; ¹H NMR (CDCI₃) δ 1.41 (t, J = 7 Hz, 3, CH₂CH₃), 2.39 (s, 3,

ArCH₃), 4.42 (q, J = 7 Hz, 2, OCH₂), 7.46 (m, 2, 6,7-Naph-H), 7.46 (d, J = 8 Hz, 2, ArH meta to CO₂Et), 7.69 (s, 1, ArH), 7.72 (s, 1, ArH), 7.80 (m, 2, 5,8-Naph-H), 8.11 (d, J = 8 Hz, 2, ArH ortho to CO₂Et). Anal, calcd. for C₂₀H₁₈O₂: C, 82.73; H, 6.25. Found: C, 82.67; H, 6.33.

(b.) Ethyl 4-(3-Methyl-5,6,7,8-tetrahydro-5,5,8,8 tetramethyl-

2-anthracenyl)benzoate (70): To a solution of (69) (1.45 g, 4.99 mmol) and 2,5-dichloro-2,5-dimethylhexane (1.01 g, 5.52 mmol) (Kagechika, H., et al., J. Med. Chem. 31:2182-2192 (1988)) in 20 mL of CH₂Cl₂ at 0°C was added AICI₃ (1.33 g, 10.0 mmol). The yellow-brown solution was stirred for 30 min before the ice-bath was removed, and the mixture was stirred for 3 h more with warming to room temperature. The suspension was poured onto ice/2 N HCl and extracted twice with CH₂CI₂. The yellow extract was washed three times with dilute brine, then dried (Na₂SO₄) and concentrated. The residue was

chromatographed on a silica gel column (3% EtOAc/hexane) to yield (70) as white crystals (1.26 g, 63%): m.p. 94-95°C (from MeCN); R_f 0.54 (50%

CH₂Cl₂/hexane): IR (KBr) 2961, 2924, 1716, 1608, 1564, 1460, 1385, 1365, 1273, 1176, 1102, 1022, 980, 909, 869, 853, 775, 711 cm^-1 ; ¹H NMR (CDCI₃) δ 1.37 and 1.39 (2s, 12, 5,5,8,8-CH₃), 1.41 (t, J = 7 Hz, 3, CH₂CH₃), 1.76 (s, 4, 6,7-CH₂), 2.34 (s, 3, Ar-CH₃), 4.41 (q, J = 7 Hz, 2, OCH₂), 7.44 (d, J = 8 Hz, 2, ArH meta to CO₂Et), 7.59 (s, 1, ArH), 7.63 (s, 1, ArH), 7.73 (s, 1, ArH), 7.75 (s, 1, ArH), 8.10 (d, J = 8 Hz, 2, ArH ortho to CO₂Et). Anal. calcd. for C₂₈H₃₂O₂: C, 83.96; H, 8.05. Found: C, 84.05; H, 8.07.

(c.) Ethyl 4-(3-Bromomethyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-anthracenyl)benzoate (71): A solution of ester (70) (0.536 g, 1.34 mmol), N-bromosuccinimide (0.286 g, 1.61 mmol), and dibenzoyl peroxide (about 0.01 g) in 5 mL of CCI₄ was heated at reflux for 1.5 h while being irradiated from a distance of 10 cm with a 200-W tungsten lamp. The orange suspension was filtered, the precipitate was washed with CCI₄, and the filtrate was concentrated. The residue was chromatographed on a silica gel column (20%-50% CH₂Cl₂/hexane) to yield (71) as a white solid (0.531 g, 83%): m.p. 129-130°C; R_f 0.47 (50% CH₂Cl₂/hexane); IR (CHCl₃) 1705, 1600, 1455, 1270, 1095, 1010, 980, 900, 860, cm^-1. ¹H NMR (CDCI₃) δ 1.36 and 1.38 (2s,

12, 5,5,8,8-CH₃), 1.43 (t, J = 7 Hz, 3, CH₂CH₃), 1.76 (s, 4, 6,7-CH₂), 4.42 (q, J = 7 Hz, 2, OCH₂), 4.57 (s, 2, CH₂Br), 7.58 (dm, J = 8 Hz, 2, ArH meta to CO₂Et), 7.62 (s, 1, ArH), 7.78 (s, 1, ArH), 7.81 (s, 1, ArH), 7.93 (s, 1, ArH), 8.15 (dm, J = 8 Hz, 2, ArH ortho to CO₂Et). CI-HRMS (i-C₄H₉) calcd. for C₂₈H₃₂BrO₂ (MH⁺): 479.1586. Found: 479.1551.

(d.) Ethyl 4-[3-(4-Dimethylaminophenyl)methyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl]benzoate (72): To a solution of (71)

(0.240 g, 0.50 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.030 g, 0.026 mmol) in 3 mL of 1,2-dimethoxyethane was added under argon a solution of 4-dimethylaminophenylboronic acid (0.10 g, 0.61 mmol) (Staab, H.; Meissner, B. Liebigs Ann. Chem. 753, 80-91, 1971) in 0.7 mL of EtOH, followed by 1.5 mL of saturated aq. NaHCO₃. The mixture was heated at reflux for 1.5 h and then partitioned between CH₂CI₂ and dilute brine. The yellow CH₂CI₂ extract was washed twice with dilute brine, dried (Na₂SO₄), and concentrated. The gum was chromatographed twice on silica gel (25% -100% CH₂Cl₂/hexane, then 35%-100% CH₂Cl₂/hexane) to yield (72) as a pale-yellow gum (0.139 g, 54%). Crystallization from EtOAc/hexanes gave pale-yellow crystals: m.p. 151-152°C;

R_f 0.54 (15% EtOAc/hexanes); IR (CHCl₃) 1700, 1605, 1565, 1510, 1475, 1455, 1270, 1170, 1095, 1015, 985, 940, 905 cm^-1 ; ¹H NMR (acetone-d₆) δ 1.43 (m, 15, 5,5,8,8-CH₃, CH₂CH₃), 1.82 (s, 4, 6,7-CH₂), 2.88 (s, 6,

N(CH₃)₂), 3.98 (s, 2, ArCH₂), 4.42 (q, J = 7 Hz, 2, OCH₂), 6.61 (d, J = 8 Hz, 2, ArH ortho to N(CH₃)₂), 6.82 (d, J = 8 Hz, 2, ArH meta to N(CH₃)₂),

7.48 (d, J = 8 Hz, 2, ArH meta to CO₂Et), 7.67 (s, 2, ArH), 7.87 (s, 1, ArH), 7.92 (s, 1, ArH), 8.07 (d, J = 8 Hz, 2, ArH ortho to CO₂Et). CI-HRMS (i-C4H9) calcd. for C₃₆H₄₁NO₂ (M⁺): 519.3137. Found: 519.3125.

(e.) 4-[3-(4-Dimethylaminophenyl)methyl-5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-anthracenyl]benzoic Acid, Hydrochloride Salt (73): A mixture of the ester (72) (0.105 g,0.202 mmol), 40% aqueous KOH (0.5 mL, about 3 mmol), and EtOH (1.5 mL) was heated at reflux for 0.75 h to give a pale-yellow solution. The solution was concentrated in an argon stream and acidified with 1 N HCl at 0°C. The suspension was filtered, and the white solid was washed repeatedly with water, dried, and crystallized (EtOH) to yield (73) as a white powder (0.095 g, 89%): m.p. 243-253°C (decomp.); IR (KBr)

3600-2300, 3424, 2628, 1708, 1608, 1511, 1461, 1364, 1214, 1176, 1108, 1019,

909, 783, 707 cm^-1; ¹H NMR (CDCI₃) δ 1.39 and 1.42 (2 s, 12, 5,5,8,8-013), 1.79 (s, 4, 6,7-CH₂), 3.12 (s, 6, N(CH₃)₂), 4.11 (s, 2, ArCH₂), 7.01 (broad d, J = 8 Hz, 2, ArH ortho to N (CH₃)₂), 7.30 (dm, J = 8 Hz, 2, ArH meta to N(CH₃)₂), 7.50 (broad d, J = 8 Hz, 2, ArH meta to CO₂H), 7.60 (s, 1, ArH), 7.63 (s, 1, ArH), 7.78 (s, 1, ArH), 7.78 (s, 1, ArH), 8.06 (dm, J = 8 Hz, 2,

ArH ortho to CO₂H); UV (EtOH) λ max 234 nm (e 7.93 x 10⁴). Anal. calcd. for C₃₄H₃₈ClNO₂: C, 77.32; H, 7.25; Cl, 6.71; N, 2.65. Found: C, 77.38; H, 7.25; Cl, 6.44; N, 2.61.

(a.) 3-(4-Methylphenyl)-1-(2,6,6-trimethylcyclohexen- 1-yl)propen-3-ol (75): To 31 mL (31 mmol) of 1.0 M p-tolylmagnesium bromide in Et₂O was added over a period of 10 min at 0°C under argon a solution of 3-(2,6,6-trimethylcyclohexen-1-yl)prop-2-enal (74) (5.00 g, 28.0 mmol) (Van Den Tempel, P. J., et al., Tetrahedron 22:293-299 (1966)) in 15 mL of Et₂O. The yellow suspension was stirred at ice-bath temperature for 15 min, then allowed to warm to ambient temperature over a 1.5-h period. The mixture was poured onto ice and aqueous NH₄Cl and extracted twice with Et₂O. The extract was washed three times with dilute brine, dried (Na₂SO₄), and concentrated to give crude

(75) as a white solid (7.63 g, 101 %), which was used without purification for the next step: R_f (10% EtOAc/hexanes) 0.38 (major), 0.93 (minor). A sample was crystallized (CH₂Cl₂/hexane) to give large white crystals of (75): m.p.

85.5-86°C; R_f (10% EtOAc/hexanes) 0.37; IR (CHCl₃) 3600, 2930, 1605, 1450, 1355, 1165, 1075, 1000, 960 cm^-1; 300 MHz ¹H NMR (CDCI₃) 5 1.00

(s, 6, 16_R,17_R-CH₃), 1.35-1.7 (m, 4, 2_R,3_R-CH₂), 1.64 (s, 3, 18_R-CH₃), 1.82 (m, 1, OH), 1.95 (m, 2, 4_R-CH₂), 2.34 (s, 3, ArCH₃), 5.25 (m, 1, CHOH), 5.64 (dd, J = 16, 8 Hz, 1, 8_R-HC=CH), 6.19 (d, J = 16 Hz, 1, 7_R-HC=CH), 7.17 (d, J = 8 Hz, 2, ArH), 7.30 (d, J = 8 Hz, 2, ArH). Anal. calcd. for C₁₉H₂₆O: C, 84.39; H, 9.69. Found: C, 84.19; H, 9.65.

(b. ) 3-(4-Methylphenyl)-1-(2,6,6-trimethylcyclohexen-1-yl)- propen-3-one (26): A solution of crude (75) (4.60 g, 17.0 mmol) in a mixture of hexane (180 mL) and acetone (20 mL) was treated at room temperature with active MnO₂ (80 g, 0.92 mmol), and the suspension was stirred at ambient temperature under argon for 27 h. The mixture was filtered, the precipitate was washed with CH₂CI₂, and the combined filtrates were refiltered and then concentrated to give a yellow oil. Silica gel chromatography (5-15%

EtOAc/hexanes) yielded the enone (76) as a yellow, viscous oil (3.33 g, 73%): R_f (7% EtOAc/hexanes) 0.52; IR (CHCl₃) 2920, 1650, 1590, 1445, 1305, 1290, 1270, 1255, 1170, 1110, 1005, 970 cm^-1; 300 MHz ¹H NMR (CDCI₃) δ 1.13

(s, 6, 16R,17_R-CH₃), 1.45-1.7 (m, 4, 2_R,3_R-CH₂), 1.85 (s, 3, 18_R-CH₃), 2.10 (t, J = 6 Hz, 2, 4_R-CH₂), 2.43 (s, 3, ArCH₃), 6.91 (d, J = 16 Hz, 1,

8_R-HC=CH), 7.26 (d, J = 8 Hz, 2, ArH meta to C(O)), 7.58 (d, J = 16 Hz, 1, 7_R-HC=CH), 7.87 (d, J = 8 Hz, 2, ArH ortho to C(O)). CI-HRMS (i-C₄H₉) calcd. for C₁₉H₂₅O (MH⁺): 269.1905. Found: 269.1911.

(c.) (2Z,4E)-3-(4-Methylphenyl)-5-(2,6,6-trimethylcyclohexen- 1-yl)-penta-2,4-dienal (78): To a solution of diisopropylamine (4.0 mL, 29 mmol) in Et₂O (50 mL) at 0°C was added under argon 17.5 mL (28.0 mmol) of 1.6 M n-Buli in hexane and the solution was stirred for 10 min. To the solution was added, at 0°C, 35 mL of THF, followed by a solution of

trimethylsilylacetaldehyde t-butylimine (5.05 g, 29.5 mmol) (Corey, E. J., et al., Tetrahedron Lett., 7-10 (1976)) in 14 mL of THF over a 25-min period. The mixture was stirred at ice-bath temperature for 80 min and then cooled in a -78°C bath. To the dark-orange solution was added over a 40-min period a solution of (26) (3.60 g, 13.4 mmol) in 12 mL of THF. The mixture was allowed to warm to -10°C over a 1.5-h period, and this temperature was maintained for an additional 1.5 h. To the stirred mixture was added (CO₂H)₂●(H₂O)₂ (14 g, 110 mmol) and 120 mL of H₂O, and the suspension was stirred under argon with warming to ambient temperature over a 16-h period. The dark orange-red suspension was diluted with 150 mL of H₂O and extracted three times with Et₂O. The red extract was washed twice with dilute brine, dried (MgSO₄), and concentrated to give an orange-brown gum. Flash silica gel chromatography (6%

Et₂O/hexanes) yielded first the (2E,4E)-aldehyde (77) as a yellow gum (0.40 g, 10%): R_f (10% Et₂O/hexane) 0.44; IR (CHCl₃) 2930, 1650, 1610, 1450, 1350, 1145, 1010, 965 cm^-1; 90 MHz ¹H NMR (CDCI₃) δ 1.04 (s, 6, 16_R,17_R-CH₃), 1.4-1.7 (m, 4, 2_R,3_R-CH₂), 1.80 (s, 3, 18_R-CH₃), 2.07 (m, 2, 4_R-CH₂), 2.40 (s, 3, ArCH₃₎, 6.14 (d, J = 8 Hz, 1, 10_R-C =CH), 6.42 (broad d, J = 16 Hz,

1, 7R-HC=CH), 6.92 (d, J = 16 Hz, 1, 8_R-HC=CH), 7.21 (d, J = 8 Hz, 2, ArH), 7.36 (d, J = 8 Hz, 2, ArH), 10.15 (d, J = 8 Hz, 1, CHO); UV (EtOH)λ max 326 nm (ε 1.39 X 10⁴), 266 nm (ε 1.01 x 10⁴). CI-HRMS (i-C₄H₉) calcd. for C₂₁H₂₇O (MH⁺): 295.2062. Found: 295.2052. Continued chromatography provided a 1:1 mixture (by ¹H NMR) of (77) and the (2Z,4E)-isomer (78) (0.94 g, 24%), and then the (2Z,4E)-aldehyde (78) as a yellow solid (2.45 g, 62%): m.p. (hexane) 59-59.5°C; R_f (10% Εt₂O/hexane) 0.41; IR (CHCl₃) 2930, 1650, 1600, 1450, 1355, 1145, 1125, 1015, 970 cm^-1; 90 MHz ¹H NMR (CDCI₃) δ 0.99 (s, 6, 16_R,17_R-CH₃), 1.3-1.7 (m, 4, 2_R,3_R-CH₂), 1.70 (s, 3, 18_R- CH₃), 2.02 (m, 2, 4_R-CH₂), 2.41 (s, 3, ArCH₃), 6.09 (d, J = 8 Hz, 1, 10_R-C =CH), 6.39 (m, 2, 7_R, 8_R-HC=CH), 7.23 (m, 4, ArH), 9.42 (d, J = 8 Hz, 1, CHO); UV (EtOH) λ_max 332 nm (ε 1.72 x 10⁴), 265 nm (ε 1.00 x 10⁴). Anal, calcd. for C₂₁H₂₆O: C, 85.66; H, 8.90. Found: C, 85.52; H, 8.71. The isomer mixture fraction (0.94 g) in Et₂O (20 mL) was treated with I₂ (a few mg), and the solution was allowed to stand at ambient temperature under argon for 1.5 h, and then washed with sodium thiosulfate solution and twice with dilute brine. The yellow solution was concentrated to give a mixture of (77) and (78) (1:4 by

¹H NMR). Flash silica gel chromatography (6-8% Et₂O/hexanes) yielded an additional 0.094 g (2%) of (77), 0.168 g (4%) of a mixture of (77) and (78), and 0.59 g (15%) of (78). The total yield of (78) was 3.04 g (77%).

(d.) Ethyl (2E,4E,6Z,8E)-3-Methyl-7-(4-methylphenyl)-9-(2,6,6- trimethylcyclohexen-1-yl)-2,4,6,8-nonatetraenoate (81): To a solution of triethyl

3-methyl-4-phosphono-2-butenoate (79) as a mixture of E- and Z-isomers) (Pattenden, G., et al., J. Chem. Soc , 1984-1997 (1968)) (2.80 g, 10.6 mmol) in 20 mL of THF was added 6.3 mL (10.1 mmol) of 1.6 M n-BuLi in hexane over a 5-min period under argon at -78°C. The cooling bath was removed after an additional 15 min, and the mixture was allowed to warm to ambient temperature over a 15-min period. The orange-red solution was cooled in a -50°C bath, and a solution of (78) (2.60 g, 8.83 mmol) in 8 mL of THF was introduced over a 15-min period. The mixture was then allowed to warm to ambient temperature over a 20-h period. The orange solution was poured into dilute brine and extracted twice with Et₂O. The yellow extract was washed twice with brine, dried (Na₂SO₄), and concentrated. The viscous yellow oil was twice

chromatographed by flash silica gel chromatography (2-2.5% Et₂O/hexanes) to yield successively the (2Z,4E,6Z,8E)-isomer (80) as a yellow gum (0.261 g, 7%): R_f (5% Et₂O/hexane) 0.51; IR (CHCl₃) 2930, 1690, 1605, 1580, 1450, 1370, 1220, 1160, 1045, 975, 820 cm^-1; ¹H NMR (CDCl₃) δ 0.94 (s, 6,

16_R,17_R-CH₃). 1.29 (t, J = 7 Hz, 3, CH₂CH₃), 1.43 and 1.58 (2 m, 4, 2_R,3_R-CH₂), 1.70 (s, 3, 18_R-CH₃), 1.83 (d, J = 1.2 Hz, 1, 20_R-CH₃), 2.00

(m, 2, 4_R-CH₂), 2.40 (s, 3, ArCH₃), 4.16 (q, J = 7 Hz, 2, OCH₂), 5.58 (s, 1, 14_R-C=CH), 5.92 (d, J = 16 Hz, 1, 7R-HC=CH), 6.32 (d, J = 16 Hz, 1, 8_R-HC=CH), 6.43 (d, J = 11 Hz, 1, 10_R-C=CH), 6.56 (dd, J = 15, 11 Hz, 1, 11_R-HC=CH), 7.11 (d, J = 8 Hz, 2, ArH), 7.20 (d, J = 8 Hz, 2, ArH), 7.80 (d, J = 15 Hz, 1, 12_R-HC=CH); UV (EtOH) λ max 363.5 nm (ε = 3.53 x

10⁴); HPLC (Waters Radialpak Novapak silica 8-mm x 100-mm, 1 %

Et₂O/hexane, 2.0 mL/min, 260 nm) t_R 9.3 min. CI-HRMS (i-C₄H₉) calcd. for C₂₈H₃₇O₂ (MH ⁺ )- 405.2793. Found: 405.2770. Next was eluted a mixture of isomers (0.61 g, 17%): HPLC t_R 7.0 (3%), 7.7 (14%), 9.4 (21 %), 10.8 min (62%); and then the (2E,4E,6Z,8E)-isomer (81) as a yellow gum (2.23 g, 62%):

R_f (5% Εt₂O/hexane) 0.43; IR (CHCl₃) 2950, 1700, 1605, 1580, 1450, 1370, 1360, 1225, 1160, 1045, 975, 880, 825 cm^-1 ; ¹H NMR (CDCI₃) δ 0.94 (s, 6, 16_R,17_R-CH₃), 1.27 (t, J = 7 Hz, 3, CH₂CH₃), 1.42 and 1.58 (2 m, 4,

2_R,3_R-CH₂), 1.70 (s, 3, 18_R-CH₃), 2.00 (m, 2, 4_R-CH₂), 2.12 (s, 3,

20_R-CH₃), 2.41 (s, 3, ArCH₃), 4.15 (q, J = 7 Hz, 2, OCH₂), 5.73 (s, 1,

14_R-C=CH), 5.92 (d, J = 16 Hz, 1, 7_R-HC=CH), 6.30 (d, J = 15 Hz, 1, 12_R-HC=CH), 6.31 (d, J = 16 Hz, 1, 8_R-HC=CH), 6.32 (d, J = 11 Hz, 1, 10_R-C=CH), 6.58 (dd, J = 15, 11 Hz, 1, 11_R-HC=CH), 7.11 (d, J = 8 Hz, 2, ArH), 7.22 (d, J = 8 Hz, 2, ArH); UV (EtOH) λ_max 361 nm (c 4.22 x 10⁴); HPLC t_R 10.7 min. CI-HRMS (i-C₄H₉) calcd. for C₂8H₃₆O₂ (M⁺): 404.2715.

Found: 404.2711. (e.) (2E,4E,6Z,8E)-3-Methyl-7-(4-methylphenyl)-9-(2,6,6- trimethylcyclohexen-1-yl)-24,6,8-nonatetraenoic Acid (82): To a stirred solution of (81) (2.00 g, 4.95 mmol) in 12 mL of EtOH was added under argon 4.0 g (about 24 mmol) of 40% aqueous KOH. The mixture was heated at 70°C for 1 h to give a dark-orange solution that then was concentrated in an argon stream.

The mixture was acidified at 0°C with 25 mL of 10% HOAC, and then the yellow suspension was diluted with 25 mL of H₂O and extracted twice with EtOAc. The extract was washed three times with H₂O, dried (Na₂SO₄), and concentrated to give an orange solid. The crude product was crystallized twice from MeOH (60 mL, then 50 mL) under argon to yield (82) as orange crystals

(1.18 g, 63%), m.p. 168.5-169.5°C. The combined crystallization liquors were concentrated, and the residue was recrystallized three times from MeOH to yield an additional 0.39 g (21 %) of (82). The total yield was 1.57 g (84%): IR

(CHCl₃) 3300-2400, 1675, 1605, 1575, 1440, 1255, 1110, 1015, 970, 825 cm^-1; 400 MHz ¹H NMR (CDCI₃) δ 0.94 (s, 6, 16_R,17_R-CH₃), 1.42 and 1.59 (2 m,

4, 2_R,3_R-CH₂), 1.70 (s, 3, 18_R-CH₃), 2.00 (m, 2, 4_R-CH₂), 2.13 (s, 3, 20_R-CH₃), 2.41 (s, 3, ArCH₃), 5.76 (s, 1, 14_R-C=CH), 5.94 (d, J = 16 Hz, 1, 7_R-HC=CH), 6.32 (d, J = 16 Hz, 1, 8_R-HC=CH), 6.32 (d, J = 15 Hz, 1, 12_R-HC=CH), 6.33 (d, J = 11 Hz, 1, 10_R-C=CH), 6.62 (dd, J = 15, 11 Hz, 1, 11_R-HC=CH), 7.11 (d, J = 8 Hz, 2, ArH), 7.22 (d, J = 8 Hz, 2, ArH); UV

(EtOH) λ_max 346 nm (e 4.64 x 104); HPLC (Waters Radialpak Novapak C₁₈, 8-mm x 100-mm, 90 MeOH/10 H₂O/0.1 HOAc, 2.0 mL/min, 260 nm) t_R 8.8 min (>99%). Anal, calcd. for C₂₆H₃₂O₂: C, 82.93; H, 8.57. Found: C, 82.86; H, 8.48.

(a.) Ethyl (E)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-3-phenylpropen-1-yl]benzoate (84): A mixture of ethyl(E)-4-[2- (5 , 6, 7, 8-tetrahydro-5 ,5 , 8 , 8-tetramethyl-2-naphthalenyl)propen-1-yl]-benzoate (83) (0.141 g, 0.38 mmol) (Loeliger, P., et al., Eur. J. Med. Chem.-Chim. Ther. 15:9-15 (1980)), N-bromosuccinimide (0.080 g, 0.45 mmol), and dibenzoyl peroxide (0.002 g) in 1.5 mL of CCl₄ was heated at reflux for 2 h while being irradiated with a 100-W tungsten lamp from about 10 cm, then cooled. The pale-yellow suspension was filtered, and the filtrate was concentrated to give a yellow gum, which was chromatographed on a silica gel (1: 1 CH₂Cl₂/hexanes) to give 0.165 g of a colorless gum. To a solution of the crude gum in 2.25 mL of DME was added tetrakis(triphenylphosphine)palladium(0) (0.030 g, 0.026 mmol), a solution of phenylboronic acid (0.060 g, 0.49 mmol) in 0.7 mL of EtOH, and

1.2 mL of saturated aq. ΝaHCO₃. The mixture was heated at reflux under argon for 3 h to give an orange suspension. The cooled suspension was partitioned between CH₂CI₂ and dilute brine, and the organic phase was washed with dilute brine, dried (Na₂SO₄), and concentrated. The orange gum was chromatographed on silica gel (30% CH₂Cl₂/hexanes) to yield 0.064 g (40%) of a mixture of (83), (84) and the (Z)-isomer of (84): HPLC (Waters Radialpak silica 8-mm x

100-mm, 1 % EtOAc/hexanes, 1.5 mL/min, 260 nm) t_R 6.9 (9%) (83), 7.5 (28%) (Z-84), 8.8 min (63%) (84); Rf (1:1 CH₂Cl₂/hexanes) 0.56, 0.59. The

(E)-isomer (84) (0.029 g, 18%), a colorless gum, was isolated by HPLC (same conditions): t_R 8.8 min (100%); IR (CHCl3) 2930, 1700, 1600, 1485, 1450, 1360, 1270, 1100, 1010, 875, 825 cm^-1 ; ¹H NMR (CDCI₃) δ 1.20 and 1.25 (2 s, 12, 5,5,8,8-CH₃), 1.38 (t, J = 7 Hz, 3, CH₂CH₃), 1.65 (s, 4, 6,7-CH₂), 4.10 (s, 2, ArCH₂), 4.36 (q, J = 7 Hz, 2, OCH₂), 7.11 (s, 1, C=CH),

7.15-7.30 (m, 7, ArH), 7.41 (m, 3, ArH meta to CO₂Et and 1-NaphH), 8.00 (d, J = 8 Hz, 2, ArH ortho to CO₂Et); UV (EtOH) λ_max 307 nm (6 2.36 x 10⁴). CI-HRMS (i-C₄H₉) calcd. for C₃₂H₃₆O₂: 452.2715. Found: 452.2675. A sample of the (Z)-isomer of (84) was isolated by HPLC for comparison: ¹H NMR (CDCI₃) δ 1.02 and 1.25 (2, 12, 5,5,8,8-Me), 1.35 (t, J = 7 Hz, 2, CH₂CH₃),

1.69 (m, 4, 6,7-CH₂), 3.78 (s, 2, ArCH₂), 4.31 (q, J = 7 Hz, 2, OCH₂) , 6.33 (s, 1, C=CH), 6.85 (m, 2, ArH), 6.95 (d, J = 8 Hz, 2, ArH meta to CO₂Et), 7.15-7.32 (m, 6, ArH), 7.72 (d, J = 8 Hz, 2, ArH ortho to CO₂Et).

(b.) (E)-4-[2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl- 2-naphthalenyl)-3-phenylpropen-1-yl]benzoic Acid (85): A suspension of (84)

(0.019 g, 0.042 mmol) in 0.5 mL of EtOH and 0.2 g (about 1.2 mmol) of 40% aq. KOH was heated at 80°C under argon for 1 h, then concentrated in an argon stream. The residue was treated at 0°C with 3 mL of 1.5 N HCl. The suspension was stirred for 30 min and filtered. The white precipitate was washed repeatedly with H₂O, then dried. Crystallization from EtOH gave white crystals of (85) (0.013 g, 73%): m.p. 204.5-205.5°C; IR (KBr) 3500-2400, 2957, 1688, 1604, 1494, 1420, 1284, 1179, 701 cm^-1; 400 MHz ¹H NMR (CDCI₃) δ 1.21 and 1.26 (2 s, 12, 5,5,8,8-CH₃), 1.66 (s, 4, 6,7-CH₂), 4.12 (s, 2, ArCH₂), 7.13 (s, 1, C=CH), 7.18-7.31 (m, 7, ArH), 7.41 (d, J = 2 Hz, 1, 1-NaphH), 7.44 (dm, J = 8 Hz, 2, ArH meta to CO₂H), 8.03 (dm, J = 8 Hz, 2, ArH ortho to

CO₂H); UV (EtOH) λ_max 299 nm (e = 2.53 x 10⁴); HPLC (Waters Radialpak Nova-pak C₁₈, 8-mm x 100-mm, 40 MeCN/40 MeOH/10 H₂O/0.1 HOAc, 1.4 mL/min, 260 nm) t_R 8.6 min (100%). CI-HRMS (i-C₄H₉) calcd. for

C₃₀H₃₂O₂: 424.2402. Found: 424.2435.

Example 25

Retinoid Antagonist of HIV-1 Promoter

Materials and Methods:

(a.) Retinoids: All-trans-RA was purchased from Sigma Chemical Co. 9-cis-RA was prepared by the method of Sakashita et al. (Sakashita, A., et al. , Blood 81:1009-1016 (1993)). 4-[1-(1-Methoxy-2,2,2-trifluoroethyl)- 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoic acid was prepared by the procedures given in Examples 2 and 5. All compounds gave satisfactory elemental analyses (C, H, F). Retinoid stock solutions (10 mM) were made in a dimethylsulfoxide:ethanol (1:1) mixture and were maintained at -20°C. Further dilutions were made in cell culture medium before use.

(b.) Plasmids: The receptor expression plasmids pECE-RARα,

-RARβ, -RARγ, and -RXRα, as well as pECE-COUP-TF expression plasmids, have been described previously (Zhang, X-k. et al , Nature 358:587-591 (1992); Tran, P., et al., Mol. Cell. Biol 12:4666-4676 (1992)). The HIV-1-RARE reporter constructs (HIV-1-RARE-tk-CAT) were obtained by inserting either one or two copies of the corresponding oligonucleotide sequences shown in Fig.

1a with additional 5'-GATC (SEQ ID NO: 6) overhangs into the Bg/Il site of pBL-CAT2 that carries a thymidine kinase (tk) promoter (Luckow, B. et al., Nucleic Acids Res. 15:5490 (1987)) as previously described (Pfahl, M. et al, Methods Enzymol. 153:256-270 (1990)). The reporters containing two copies of HIV-1 -RARE were used in all transfection experiments except the experiment shown in Fig. 2c. To construct the HIV-1 LTR-CAT reporter, specific oligonucleotide primers complementary to sequences in the HIV-1 LTR (variant A) (5'- AAAGGGGGGACTGGAAG (SEQ ID NO:7);

5'-TGAAGCACTCAAGGCAAG (SEQ ID NO:8)) were used to amplify HIV-1 LTR from HXB2 genomic DNA (Ratner, L. et al., Aids Res. Hum. Retro-viruses 3:57-69 (1987)). The resulting fragment (-464 to +97) was purified and cloned into pBS-CAT (Hoffmann, B. et al., Mol. Endocrinol. 4:1727-1736 (1990)).

Reporter plasmids of HIV-1-RARE variants B and C were obtained by inserting copies of the oligonucleotides shown in Fig. 1c with BglIl adapter sequences into pBL-CAT2. Transfection Assays:

CV-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. A modified calcium phosphate precipitation procedure was used for transient transfection as previously described (Zhang, X-k., et al., Nature 358:587-591 (1992), supra; Lehmann, J.M., et al, Science 258:1944-1946 (1992), supra; Pfahl, M., et al., Methods Enzymol

153:256-270 (1990)). In general, 100 ng of reporter plasmid, 200 ng of β-galactosidase expression vector (pCH110, Pharmacia), and variable amounts of receptor expression vector were mixed with carrier DNA (pBluscript, Stratagene) to 1000 ng of total DNA per well. After the precipitate was removed, retinoids were applied for 24 hours. CAT and β-galactosidase activity were assayed as described (Pfahl, M., et al. (1990), supra). CAT activity was normalized for transfection efficiency by the corresponding β-galactosidase activity.

In vitro Transcription and Translation:

cDNAs of RARα, RXRβ, TRs and COUP-TFs cloned in pBluscript were transcribed by using T7 or T3 RNA polymerase, and the transcripts were translated in the rabbit reticulocyte lysate system (Promega) as previously described (Zhang, X-k., et al. (1992), supra; Lehmann, J.M., et al. (1992), supra; Pfahl, M., et al. (1990), supra). The efficiency of translation was determined by separating the ³⁵S-methionine labeled proteins on a

SDS-polyacrylamide gel, quantitating the amount of incorporated radioactivity, and normalizing it relative to the content of methionine residues in each protein.

Gel Retardation Ass y

Gel retardation assays were performed essentially as previously described (Zhang, X-k., et al and Lehmann, J., et al., referenced in the preceding section). Briefly, in vitro translated receptor proteins were incubated with the ³²P-labeled oligonucleotides in a 20-μl reaction mixture containing 10 mM HEPES, pH 7.9, 50 mM KCl , 1 mM dithiothreitol, 2.5 mM MgCl₂, 10% glycerol, and 1 μg of poly(dl-dC) at 25 °C for 20 minutes. The reaction mixtures were then loaded on a 5% nondenaturing polyacrylamide gel containing 0.5 times TBE (1 times TBE is 89 mM Tris-borate, 89 mM boric acid, and 2 mM EDTA),

9-cw-RA (10^{- 7} M) was incubated with the reticulocyte lysate-containing receptors at room temperature for 30 minutes before performing the experiments described herein. Oligonucleotides corresponding to the HIV-1-RARE (Fig. 1a) and those containing point mutations (indicated by solid triangles) in the first (M1) and second (M2) half-site of HIV-1-RARE (Fig. lc) were synthesized with

Bg/Il adapter sequences at both ends, ³²P-labeled, and used as probes.

Results:

To define the mechanism for retinoid control of HIV-1, the LTR was analyzed for possible RAREs. A region spanning nucleotides -348 to -328 of the HIV-1 LTR was found to contain two consensus (A/GGGTCA (SEQ ID NO:9 and 10)) RARE half sites. In Fig. 1a, this sequence is compared to one synthetic (TREpal) and several natural RAREs. While natural RAREs have so far been found to consist of mostly direct repeats separated by a 1, 2 or 5 bp spacer (Hoffmann, G., et al., Mol. Endocrin. 4:1727-1736 (1990); de The, H., et al., Nature 343:177-180 (1990); Mangelsdorf, D.J., et al., Cell 66:555-561 (1991); Naar, A.M., et al. , Cell 65: 1267-1279 (1991); Rottman, J.N., et al.,

Mol. Cell. Biol. 11:3814-3820 (1991); Umesono, K., et al., Cell 65:1255-1266 (1991); Hussmann, G., et al., Biochem. Biophys. Res. Comm. 187:1558-1564 (1992); Lehmann, J.M., et al , Mol. Cell Biol. 12:2976-2985 (1992) and references therein), the two half-sites of the putative HIV-1-RARE are arranged as a palindrome separated by 9 nucleotides. A possible third half-site was also noted within the spacer (Fig. 1a).

RAREs interact either with RAR/RXR receptor heterodimers or RXR receptor homodimers (Zhang, X-k., et al. (1992), supra; Yu, V.C., et al, Cell 67:1251-1266 (1991); Kliewer, S.A., et al., Nature 355:446-449 (1992); Leid, M., et al., Cell 68:377-395 (1992); Zhang, X-k., et al., Nature 358:587-

591 (1992)). Using a gel shift assay, it was observed that RAR/RXR

heterodimers as well as RXRα homodimers bound effectively to the putative HIV-1-RARE (Fig. 1b). RXRα homodimer binding was seen only in the presence of 9-cis-RA. RARs alone, or RXRα in the absence of 9-cis-RA, did not bind to the putative HIV-1-RARE. The orphan receptors COUP-TFα and

COUP-TFβ, known to function as negative regulators of some RAREs (Tran, P., et al., Mol Cell. Biol. 12:4666-4676 (1992); Kliewer, S.A., et al. Proc. Natl. Acad. Sci. USA 89:1448-1452 (1992); Cooney, A.J., et al , Mol Cell. Biol 12:4153-4163 (1992); Windom, R.L., et al., Mol Cell. Biol. 12:3380-3389 (1992)), also bound very strongly to this sequence (Fig. 1b).

Point mutations in the first (M1) as well as second (M2) half-site of the HIV-1-RARE sequence abolished or strongly reduced binding of RAR/RXR heterodimers and RXR homodimers (Fig. 1c), indicating that each consensus half-site is functionally important. Interestingly, M1 only slightly reduced binding of COUP-TF, whereas M2 did not affect COUP-TF interaction (Fig. 1c). This may indicate that COUP-TF interacts with the first half-site and the putative third half-site, i.e., part of the spacer sequence of this RARE.

When placed into the reporter gene pBL-CAT2 (Luckow, B., et al., Nucleic Acids Res., 15:5490 (1987)), the putative RARE sequence allowed a strong induction of chloramphenicol acetyl transferase (CAT) activity in response to retinoids in CV-1 cells in the presence of expression vectors for the RARs and/or RXR. Very high induction was observed by 9-cis-RA and RXRα alone

(Fig. 2a), showing that this RARE is activated very efficiently by RXRα homodimers, which is consistent with the receptor binding data.

In contrast to the RXRα homodimers, heterodimers could also be activated by all-trans-RA. Comparing the transactivation activities of various heterodimers, weak induction with RARα/RXRα by all-trans-RA and high induction by 9-cis-RA was noted. For RARα/RXRα or RARγ/RXRα heterodimers all-trans-RA was a strong activator. In both cases, however, the higher response was seen with 9-cis-RA (Fig. 2a) when all-trans-RA and 9-cis-RA were compared at concentrations of 10^-7 M. At lower concentrations, all-trans-RA gave a comparable or higher response with the heterodimer (Fig.

2b), consistent with its higher affinity for RARs (Allenby, G., et al., Proc. Natl Acad. Sci. USA 90:30-34 (1993). Together these data show that 9- cis-RA as well as all-trans-RA are potent activators of the HIV-1-RARE and that receptor heterodimers have differential activation profiles with this RARE. When a construct that contained the complete HIV-1 LTR (HIV-1 LTR-CAT) was used, instead of only the RARE, a strong retinoid induction was also observed in the presence of cotransfected RXRα or RARα/RXRα (Fig. 2c). The magnitude of this induction and the receptor response profiles for the LTR to both

all-trans-RA and 9-cis-RA were similar to those observed for a reporter construct carrying a single copy of the HTV-1- RARE.

Because various effects of RA on the replication of HIV-1 have been reported, the RARE sequences of 3 distinct HIV-1 variants were compared.

The RARE sequence was not completely conserved in the different HIV-1 variants. The first half-site in variant B showed a change in position 2, whereas variant C differed in position 3 (Fig. 2d), both of which are digressions from the consensus RARE half-site sequence. Surprisingly, these changes did not significantly change responses to retinoids, but resulted in increased activities of the reporter constructs (Fig. 2d).

Binding data (Fig. 1b) showed that the COUP-TF orphan receptors also interact efficiently with the HIV- 1 -RARE. When COUP-TFα was cotransfected with the retinoid receptor expression vectors, a dramatic repression of the retinoid response was observed (Fig. 3a and b). COUP-TFα effectively repressed RXR homodimer, as well as RAR/RXR heterodimer activity. Very similar results were obtained with COUP-TFβ (not shown). Thus COUP-TF receptors can function as negative regulators of the HIV-1-RARE and prevent retinoid-induced replication of HIV-1.

These results demonstrated that retinoid antagonists provide a way of repressing HIV-1 activation by retinoids in vivo. Structure-activity analyses of retinoids showing low transcriptional activation activity was conducted to establish skeletal features that could be modified to enhance receptor antagonism. Of those compounds thus far designed and synthesized, 4-[1-(1-methoxy-2,2,2- trifluoroethyl)-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-anthracenyl]benzoic acid (SR11335) (AR-1) proved to be the most effective at inhibiting the ability of all-trans-RA to induce HIV-1-RARE (Fig. 4a). Clear inhibition by the antagonist was observed even when all-trans-RA was used at 10^{- 7} M. The antagonist also functioned as a potent inhibitor of 9-cis-RA (Fig. 4b); however, inhibition of the heterodimers was more efficient than inhibition of RXR homodimers. Example 26

Retinoids with Differential Receptor Selectivity in Two Response Mechanisms

The transcriptional activation activity of approximately 50 synthetic retinoids was compared with their anti-AP-1 activity using RARα, β and y and RXRα. For measuring transcriptional activation activity, a standard analysis system was used (Fig. 5a). Briefly, expression vectors for different RAR subtypes or RXRα were cotransfected into CV-1 cells with the reporter gene (TRE)2-tk-CAT. To measure anti-AP-1 activity, a collagenase promoter-CAT construct (Col-CAT) was cotransfected with receptor expression vectors into HeLa cells. As previously shown, the Col-CAT receptor was strongly activated by TPA, but dramatically inhibited by t-RA (Fig. 5a). Inhibition of Col-CAT activity by RARs in the presence of synthetic retinoids was compared to that of t- RA, whereas 9-cis-RA was used as the standard for inhibition by RXRα-specific retinoids.

While t-RA (Fig. 5a) and several synthetic retinoids (not shown) behaved very similarly in transcriptional activation and anti-AP-1 assays, some other retinoids clearly demonstrated distinct receptor selectivities in both assays (Figs. 1b and 1c). For instance R20 (6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-2-naphthalenecarboxylic acid) is an effective transactivator for RARβ and RARβ while it is less effective in inhibiting AP-1 and also shows a preference for RARβ in this assay; R5 ((E)-4-[2-(5,5-dimethyl-5,6,7,8-tetrahydro-

3-naphthalenyl)propenyl]benzoic acid) is RARγ, β-selective in transactivation but RARα, β-selective in the anti-AP-1 assay; SR11217 (4-[2-methyl-1-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl]benzoic acid) is a strong RXRα-selective compound in transactivation but is RARβ-selective in the anti- AP-1 assay (Figs. 5b and 5c).

Overall, these results demonstrated that retinoids can display distinct receptor selectivities in the two different receptor mediated responses. This is in concert with the different mechanisms by which the receptors function in these two pathways. It has been shown that t-RA as well as retinoid antagonists induce conf ational changes when interacting with RARs. This invention indicates that d erent receptor conformations are required for transcriptional activation or anti-AP-1 activity and that conformationally restricted retinoids can induce distinct conformational changes in the receptor.

Example 27

Retinoids with Selective Anti-AP-1 or Transcriptional Activation Activities

To investigate whether retinoid structures could be identified that induce only anti-AP-1 activity with the retinoid receptors, a series of retinoids that were inactive in the transcriptional activation assay were evaluated.

Surprisingly, several of these transcriptionally inactive compounds were potent activators of the RARs or RXRα in the anti-AP-1 assay. The two most potent ones, SR11238 and SR11302 showed the highest anti-AP-1 activity (80% of that seen with t-RA or 9-cis-RA) with RARβ and RARα, respectively (Figs. 5d and

5e). The most potent anti-AP-1 -selective compounds in this series and their receptor specificities are compiled in Table 1 below.

Table 1. Structures and activities of retinoids. Transcriptional activation and anti-AP-1 activity were evaluated as shown in Figure 1. SR11238, [2-(3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-2-(4-carboxyphenyl]-1,3- dithiane; SR11302, (2E,4E,6Z,8E)-3-methyl-7-(4-methylphenyl-9-(2,6,6- trimethylcyclohexen-1-yl)-2,4,6,8-nonatetrenoic acid; SR11220, methyl (Z)-4-[1- acetoxy-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)ethenyl] benzoate; SR11327, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-3-phenylpropenyl]benzoic acid; SR11228, 5-[(5,6,7,8-tetrahydro- 5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]-2-naphthalenecarboxylic acid;

SR11324, 4-[3-(4-dimethylaminophenyl)methyl-5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-anthracenyl]benzoic acid, hydrochloride salt; SR11235, 2-(5,6,7,8- tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-(4-carboxyphenyl)-1,3- oxathiolane.

In addition to those retinoids named in Table 1, SR11105 (R5), (E)-4-[2-(5,5-dimethyl-5,6,7,8-tetrahydro-3-naphthalenyl)-propenyl]benzoic acid;

SR11217, 4-[2-methyl-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)propenyl]benzoic acid; SR3919 (R10), 4-[2-(4,5,6,7-tetrahydro-4,4- dimethyl(benzo[b]thien-2-yl)propenyl]benzoic acid; and SR3957 (R20), 6- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid were evaluated.

Although these retinoids show anti-AP-1 activity with more than one of the receptors, a certain degree of receptor selectivity was usually observed (Figs. 1d and 1e, Table 1). Also analyzed were several of these retinoids with the different receptors in 10 T 1/2 cells, in which TPA also allows induction of the Col-CAT gene. The results obtained in this cell line were essentially the same as the results obtained with HeLa cells (data not shown). Whether retinoids with no anti-AP-1 activity, but potent as transcriptional activators could be found was investigated. On evaluating retinoids that had been shown previously to be transcriptional activators in the anti-AP-1 assay, SR11235 was identified, which induced essentially no (less than 20%) receptor anti-AP-1 activity. Also, as previously shown, SRI 1235 is an

RXRα-selective transcriptional activator (Fig. If and Table 1). Thus retinoids, selective either for anti-AP-1 activity or transcriptional activation, can be defined using the screening assays employed here. Example 28

Anti-AP-1 Selective Retinoids Show Antagonist Activity Retinoids that activate the anti-AP-1 activity of a particular receptor are likely to interact with the ligand-binding pocket of that receptor. If such retinoids are not transcriptional activators, they could be expected to function as antagonists in the transcriptional activation assay. This was indeed the case for several of the anti-AP-1 selective retinoids tested (Fig. 6). For instance SR11302 inhibited effectively t-RA induced transcriptional activation by RARα. SR11302 also inhibited t-RA induced transcriptional activation by RARγ but not by RARβ, consistent with the observation that this retinoid showed strong anti-AP-1 activity with RARα and RAR7 but not with RARβ. A similar correlation between antagonism of transcriptional activation and anti-AP-1 activity was obtained for SR11327 (Fig. 6) and several other compounds (not shown). These data indicate that the same region in the retinoid receptor ligand binding domain is likely responsible for ligand interaction in transcriptional activation as well as for anti-AP-1 activity. However, the anti-AP-1 selective retinoids were only weak antagonists and were only effective antagonists when low

concentrations of RA (10^-9 M) were used and are for instance much poorer inhibitors of t-RA than other antagonists recently described. Thus, the potency of a retinoid in the anti-AP-1 assay cannot be directly correlated to its antagonist activity, pointing to a complex reaction in this former mechanism, where receptor/ AP-1 interaction may stabilize the receptor/ligand interaction. Example 29

Biological Activities of Pathway Selective Retinoids The biological activities of anti-AP-1-selective retinoids were examined in the embryonal carcinoma cell line F9, in which RA is known to induce differentiation and cancer cell lines where retinoids can inhibit growth. As judged from a cell proliferation assay and morphological changes, the anti-

AP-1 -selective retinoids SR11220, SR11238, and SR11302 failed to inhibit the proliferation of F9 cells (Fig. 7a) or to induce their differentiation (Fig. 7b, panels D-G) as observed with t-RA and 9-cis-RA (Fig. 7b, panels A, B). In contrast, these same retinoids were potent inhibitors of the growth of the lung cancer cell lines Calu-6 and H661 and the breast cancer cell line T-47D (Figs. 4a and 4b), as well as several other cancer cell lines (not shown). SR11235, the transcriptional activation selective compound, did not inhibit either one of these cell lines. The anti-AP-1 selective compounds were in general more potent inhibitors of the cancer cell lines than t-RA, especially at lower concentrations. In addition, the activity of the individual compounds varied somewhat from cell line to cell line which could reflect their different receptor selectivities. Thus, the anti-AP-1 activity of SR11220, SR11238 and SR11302 can be correlated with their anti-proliferative activity in lung and breast cancer cell lines as well as several other cancer cell lines (not shown). The observation that these same retinoids do not induce differentiation in F9 cells is consistent with the observation that differentiation is associated with the induction of several t-RA responsive genes via response elements. Example 30

Pifferential Modulation of Gene Expression by t-RA and Anti-AP-1 Selective Retinoids

It has long been known that retinoids can induce complex changes at the level of gene expression. Retinoids with selective anti-AP-1 activity should be expected to alter gene expression in a manner distinct from t-RA but not necessarily completely different. The highly sensitive method of differential display was used to compa effect of t-RA, SR11302, one of the anti-AP-1 selective compounds, and R n RARβ,γ-selective retinoid on the human breast cancer cell line T-47D that is growth inhibited by t-RA as well as SR11302. All three retinoids induced changes in gene expression that showed some overlap but were also clearly distinct (Fig. 9). Not unexpectedly, the induced changes differed much more between SR11302 and t-RA than between t-RA and R10 (Fig. 9). These data thus demonstrate that anti-AP-1 selective retinoids can induce specific patterns of gene expression, that show some overlap with patterns induced by t-RA but are distinct.

The broad range of biological responses to retinoids are mediated by six subtypes of nuclear receptors that are expressed in a developmentally and cell type specific manner. These receptors are all activated by the natural retinoids t-RA and/or 9-cis-RA. Previous research has demonstrated that synthetic retinoids with selective receptor activity can be designed. This invention has found that retinoids which can induce selectively only one of the two major receptor activities-transcriptional activation or anti-AP-1 activity-can also be defined. This is consistent with the notion that distinct mechanisms mediate the two different receptor activities. For transcriptional activation the receptors bind as hetero or homodimers to specific DNA sequences, RAREs, usually found in the promoter regions of RA responsive genes. For the inhibition of AP-1 activity retinoid receptors are not required to interact with specific DNA sequences but appear to inhibit AP-1 via direct protein-protein interaction, as also observed with other nuclear receptors. The two distinct mechanisms could thus be expected to require different receptor configurations induced or stabilized by the ligand. Retinoic acid and with its flexible polyene side chain is apparently able to accommodate both receptor configurations, while the conformationally restricted retinoids identified here favor either the anti-AP-1 configuration of the receptor (SR11238, SR11302 etc.) or the transcriptional activation configuration (of RXRα) (SR11235). That retinoids can stabilize particular receptor conformations has been well documented using limited proteolytic digestions. This invention indicates that indeed different receptor configurations are induced for transcriptional activation and receptor anti-AP-1 activity.

The general observation that selective retinoids have usually lower apparent receptor affinities than the natural non-selective retinoids also applies to this novel series of selective retinoids, and is best explained by the hypotheses that the selective, conformationally restricted retinoids can only interact with a particular configuration of the receptors. Such specific receptor configuration could be stabilized by various factors, including response elements, receptor dimerization and AP-1 components.

Anti-AP-1 selective retinoids have a reduced range of biological activity in that they for instance no longer induce differentiation in F-9 cells.

Importantly, however, these retinoids have maintained the ability to inhibit efficiently proliferation of cancer cell lines and are in fact more potent inhibitors of cell proliferation in these cell lines than t-RA. That the observed inhibition is specific and not due to some "general toxicity" is demonstrated by the specific changes in gene expression. Thus, the instant data show that retinoid anti- proliferative activity can be separated from retinoid differentiation inducing activity, and that retinoids having these separate activities can be identified by using molecular assays. Although the anti-AP-1 selective retinoids are likely to induce only a limited retinoid response, the instant differential display data indicate still a complex pattern of changes in gene expression. These changes can now be analyzed in detail. The selective retinoids will thus be valuable agents for deciphering the complex mechanisms by which retinoids exert their pleiotropic biological roles. The anti-AP-1 selective compounds are in addition of particular interest because of their anti-proliferative activity and their inability to induce transcriptional activation from RAREs. These retinoids are therefore likely to have fewer side effects and could be starting points for a new generation of retinoid therapeutics.

Throughout this application, various publications are referenced. These publications may be referred to for background information not explicitly included herein.

It will be apparent to those skilled in the art that various

modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

We Claim:

1. A compound having the structural formula (I)

wherein:

R¹ is selected from the group consisting of lower alkyl, 1-methyl-

1-cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R³ is selected from the group consisting of

in which R⁷ is hydrogen or methyl, k is 0 or 1, and * represents the point of attachment of the R³ substituent to the remainder of the molecule; R⁴ is selected from the group consisting of: hydrogen; hydroxy; lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl substituted secondary amino, lower alkyl di- substituted tertiary amino, and combinations thereof; amino; lower alkyl substituted secondary amino; lower alkyl di-substituted tertiary amino; cyano; carboxyl; -(CO)-R⁸ wherein R⁸ is hydroxyl, lower alkyl substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl substituted secondary amino, lower alkyl di-substituted tertiary amino, and combinations thereof, amino, lower alkyl substituted secondary amino, lower alkyl di-substituted tertiary amino, or -OR⁹ where R⁹ is lower alkyl; and -(CH₂)_m-C₆H_(5-p)-(R¹⁰)_p where m is an integer of from 0 to 6, more preferably 0 or 1, p is an integer of from 1 to 5, and R¹⁰ is independently hydrogen, hydroxyl, lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl substituted secondary amino, lower alkyl di-substituted tertiary amino, and combinations thereof, amino, lower alkyl substituted secondary amino, and lower alkyl di-substituted tertiary amino;

R⁵ is selected from the group consisting of: hydrogen; hydroxy; lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl substituted secondary amino, lower alkyl or cycloalkyl di-substituted tertiary amino, and combinations thereof; amino; lower alkyl substituted secondary amino; lower alkyl di-substituted tertiary amino; cyano; carboxyl; -(CO)- R¹¹ where R¹¹ is hydroxyl, lower alkyl, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl substituted secondary amino, lower alkyl di-substituted tertiary amino, and combinations thereof, amino, lower alkyl substituted secondary amino, lower alkyl or cycloalkyl di-substituted tertiary amino, or -OR¹⁴ where R¹⁴ is lower alkyl; -(CH₂)_q-C₆H_(5-r)-(R¹⁴)_r, where q is an integer of from 0 to 6, more preferably 0 or 1, r is an integer of from 1 to 5, and R¹⁴ is independently hydrogen, hydroxyl, lower alkyl or lower alkoxy, substituted with from 0 to 6, more preferably from 0 to 4, substituents selected from the group consisting of halogen, hydroxyl, lower alkoxy, amino, lower alkyl mono- or di-substituted amino, and S(O)_tCH₃, where t is 0, 1, or 2; -NHCO(CH₂)_v-R¹⁵ where v is an integer of from 1 to 6 and R¹⁵ is lower alkyl substituted with from 0 to 6 fluorine substituents; -NHCO(CH₂)_x-CONH-(CH₂)_y-R¹⁶ where x is an integer of from 1 to 10, y is an integer of from 1 to 6, and R¹⁶ is lower alkyl substituted with from 0 to 6 fluorine substituents; -CH₂S(O)_zR¹⁷ where z is 0, 1, or 2 and R¹⁷ is lower alkyl substituted with from 0 to 6 fluorine substituents; and

-C(R¹⁸)=N-OR¹⁹, where R¹⁸ is lower alkyl substituted with from 0 to 6 fluorine substituents and R¹⁹ is lower alkyl, with the proviso that when R⁴ is hydrogen, R⁵ cannot simultaneously be hydrogen or -NH₂, or a pharmaceutically acceptable ester, amide or salt of the compound.

2. A compound having the structural formula (II)

wherein: R¹ is selected from the group consisting of lower alkyl, 1-methyl-1- cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R²⁰ and R²¹ are the same and are selected from O and S;

R²² is selected from the group consisting of hydrogen, lower alkyl and halogen; and

R²³ is selected from the group consisting of

in which R²⁴ is lower alkyl and ** represents the point of attachment of R²³ to the remainder of the molecule, or a pharmaceutically acceptable ester, amide or salt of the compound.

3. A compound having the structural formula

wherein:

R¹ is selected from the group consisting of lower alkyl, 1-methyl- 1-cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R , R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R²² is selected from the group consisting of hydrogen, lower alkyl and halogen;

R²³ is selected from the group consisting of

in which R²⁴ is hydrogen or lower alkyl and ** represents the point of attachment of R²³ to the remainder of the molecule; and

R²⁵ is -O-R⁶ or -S-R²⁶ wherein R²⁶ is lower alkyl or lower alkyl carbonyl, or a pharmaceutically acceptable ester, amide or salt of the compound.

4. A compound having the structural formula (IV)

wherein:

R¹ is selected from the group consisting of lower alkyl, 1-methyl- 1-cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R²² is selected from the group consisting of hydrogen, lower alkyl and halogen;

R²⁷ is hydroxyl, =O, =CH₂, lower alkyl, or a six-membered cycloheteroalkylene ring -O(CH₂)₃O- or -S(CH₂)₃S-; and

R²⁸ is a moiety of the structure

in which R²⁹ is S, NH or O and * represents the point of attachment of the R²⁸ substituent to the remainder of the molecule, or a pharmaceutically acceptable ester, amide or salt of the compound.

5. A compound having the structural formula (V)

wherein:

R¹ is selected from the group consisting of lower alkyl, 1 -methyl- 1- cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R²³ is selected from the group consisting of

in which R²⁴ is hydrogen or lower alkyl and ** represents the point of attachment of R²³ to the remainder of the molecule;

R³⁰ is O, S or lower alkylene; and

R³¹ and R³² are independently selected from the group consisting of hydrogen, lower alkyl unsubstituted or substituted with halogen, lower alkoxy, halogen, amino, and amino substituted with one or two lower alkyl moieties, or a pharmaceutically acceptable ester, amide or salt of the compound.

6. A compound having the structural formula (VI)

wherein:

R¹ is selected from the group consisting of lower alkyl, 1-methyl-1- cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl; and

R³¹ and R³² are independently selected from the group consisting of hydrogen, lower alkyl unsubstituted or substituted with halogen, lower alkoxy, halogen, amino, and amino substituted with one or two lower alkyl moieties,

and the 6-cis and 11-cis isomers thereof, or a pharmaceutically acceptable ester, amide or salt of the compound.

7. A compound having the structural formula (VII)

wherein:

R¹ is selected from the group consisting of lower alkyl, 1-methyl-1- cyclohexyl, and adamantyl, and R² is -O-R⁶ or -S-R⁶, where R⁶ is lower alkyl, and wherein when R¹ is ortho to R², R¹ and R² can be linked together to form a 5- or 6-membered cycloalkylene ring, either unsubstituted or substituted with 1 to 6 lower alkyl groups, and optionally containing 1 or 2 ring members selected from the group consisting of O, S, SO, SO₂ and NR where R is hydrogen or lower alkyl;

R²² is selected from the group consisting of hydrogen, lower alkyl and halogen;

R²³ is selected from the group consisting of

in which R²⁴ is hydrogen or lower alkyl and ** represents the point of attachment of R²³ to the remainder of the molecule; and

R³¹ and R³² are independently selected from the group consisting of hydrogen, lower alkyl unsubstituted or substituted with halogen, lower alkoxy, halogen, amino, and amino substituted with one or two lower alkyl moieties, or a pharmaceutically acceptable ester, amide or salt of the compound.

8. A pharmaceutical composition for control of cellular processes regulated by retinoids comprising an effective regulating amount of the compound of claim 1 in combination with a pharmaceutically acceptable carrier.

9. A method for modulating gene expression in a mammalian individual comprising administering to the individual an effective modulating amount of the compound of claim 1 or a pharmaceutical composition thereof.

10. A method for treating an individual afflicted with a disease caused by malfunction of cell differentiation processes, or other diseases, regulated by retinoids, comprising administering to the individual a

therapeutically effective amount of the compound of claim 1 or a pharmaceutical composition thereof.

11. A method of treating an individual infected with Human

Immunodeficiency Virus (HIV) comprising administering to the individual a therapeutically effective amount of the compound of claim 1 or a pharmaceutical composition thereof.

12. A method of inhibiting the replication of HIV in a subject, comprising contacting the HIV with a receptor or protein antagonist which binds the -348 to -328 region of the long terminal repeat of HIV corresponding to an RARE of HIV.

13. A method of screening for a candidate compound to inhibit the replication of HIV, comprising:

a. administering the compound to a suitable host having i) a promoter region comprising a retinoic acid response element (RARE) found in positions -348 to -328 of the long terminal repeat from an HIV which is operably linked to a reporter gene and

ii) a gene functionally expressing an RXR either alone or with a gene functionally expressing an RAR; and

b. determining whether the activation of transcription of the reporter gene is inhibited by the compound, the inhibition of transcription indicating a candidate compound for inhibiting the replication of HIV.

14. A method of screening for a candidate compound to inhibit the replication of HIV, comprising:

a. administering the compound to a suitable host having the promoter region of either COUP-TFα or COUP-TFβ orphan receptor which is operably linked

i) to a reporter gene; or

ii) to the COUP-TFα or COUP-TFβ protein; and b. detecting the increased expression of the reporter gene, the COUP-TFα or COUP-TFβ, the presence of increased expression indicating the presence of a candidate compound to inhibit the replication of HIV.

15. An isolated nucleic acid comprising the -348 to -328 region of the long terminal repeat of HTV-1 and a non-HIV reporter gene, wherein the region includes two half-sites and a spacer region, wherein one or more nucleotides of the spacer region can be substituted by another nucleotide.

16. A method of screening for a candidate compound to inhibit the replication of HIV comprising contacting a. a nucleic acid comprising an RARE found in positions -348 to -328 of the long terminal repeat from HIV with either a Retinoid X Receptor either alone or with a Retinoic Acid Receptor with

b. the candidate compound and determining the absence of a complex between the nucleic acid and a receptor, the absence of a complex indicating a candidate compound to inhibit the replication of HIV.

17. A method of selectively inhibiting in a subject transcription of a first gene which is activated by AP-1 or an AP-1 component over transcription of a second gene which is activated by a first nuclear receptor comprising administering to the subject a compound of any one of claims 2, 3, 4, 5, 6 or 7.

18. A method of screening a compound for an ability to selectively inhibit AP-1 or an AP-1 component transcriptional activation over transcriptional activation of a gene regulated by a nuclear receptor comprising:

a. combining the compound within a first cell containing AP-1 or an AP-1 component, a first nuclear receptor and an AP-1 responsive element linked to a first reporter gene;

b. combining the compound within a second cell containing a second nuclear receptor and a receptor specific responsive element linked to a second reporter gene;

c. detecting the transcriptional activation of the first and

second reporter genes; and

d. selecting the compound which selectively inhibits

transcriptional activation of the first reporter gene over transcriptional activation of the second reporter gene, thereby screening the compound for the ability to selectively inhibit AP-1 or AP-1 component transcriptional activation over transcriptional activation of the gene regulated by the nuclear receptor.

19. A method of screening a compound for an ability to selectively promote transcriptional activation of a gene regulated by a nuclear receptor over the ability to inhibit AP-1 or an AP-1 component transcriptional activation comprising:

a. combining the compound within a cell containing AP-1 or an AP-1 component, a nuclear receptor, an AP-1 or AP-1 component responsive element linked to a first reporter gene and a receptor specific responsive element linked to a second reporter gene;

b. detecting transcriptional activation of the first and second reporter genes; and

c. selecting the compound which selectively promotes

transcriptional activation of the second reporter gene over the ability to inhibit transcriptional activation of the first reporter gene, thereby screening the compound for the selective ability to promote transcriptional activation of the gene regulated by the nuclear receptor over the ability to inhibit AP-1 or AP-1 component transcriptional activation.