sp2-bridged diaryl retinoids: effects of bridge-region substitution on retinoid X receptor (RXR) selectivity.

RXR class selectivity and RXR transcriptional activation activity compared to those for the retinoic acid receptor subtypes were enhanced on the 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylethenyl)be nzoic acid scaffold and its 3-methyl analogue by replacing their 1,1-ethenyl bridge by a 1,1-(2-methylpropenyl) or cyclopropylidenylmethylene group.

4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzoic acid and heterocyclic-bridged analogues are novel retinoic acid receptor subtype and retinoid X receptor alpha agonists.

Aromatic retinoids having a meta-substituted aromatic ring bridge, such as 4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzo ic acid and its 3,5-diaryl-substituted 4,5-dihydroisoxazole analogue, function as retinoid receptor panagonists by activating both retinoic acid and retinoid X receptors to induce gene transcription, and thereby provide novel scaffolds for retinoid drug development. Both classes of these ligand-inducible transcription factors are involved in mediating the inhibitory effects of retinoids on cancer cell growth.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL-60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9-cis-retinoic acid (9-cis-RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9-cis-RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA-MB-468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR-signaling pathways.

Michellamine alkaloids inhibit protein kinase C.

Michellamines A, B, and C have shown antiviral activity against HIV-1 and HIV-2 in cell culture. They act in a complex manner by at least two reported antiviral mechanisms, inhibition of HIV reverse transcriptase and inhibition of HIV-induced cellular fusion. On the basis of their structural similarity to other protein kinase C (PKC) inhibitors, we have investigated another possible mechanism-inhibition of PKC. The michellamines were found to inhibit rat brain PKC with IC50 values in the 15-35 microM range. Michellamine B was a noncompetitive PKC inhibitor with respect to ATP with a Ki value of 4-6 microM, whereas mixed-type inhibition was observed when the peptide concentration was varied. Michellamine B inhibited the kinase domain of PKC similarly. These results indicate that the michellamines bind to the PKC kinase domain and not its regulatory domain. Molecular modeling showed that all three michellamines can bind in the active site cleft of the PKC kinase domain, to block both the ATP and the peptide substrate subsites.

Effects of trans-retinoic acid, 9-cis-retinoic acid, 1alpha,25-(dihydroxy)vitamin D3 and a novel apoptosis-inducing retinoid on breast cancer and endothelial cell growth.

Breast cancer cell growth inhibition was not synergistically enhanced by trans-retinoic acid (RA) or 9-cis-RA plus 1alpha,25-(dihydroxy)vitamin D3 (DHVD). The retinoid/DHVD combinations did lower their 50% effective concentrations for inhibiting retinoid-sensitive MCF-7, but not retinoid-refractory BT-20, breast cancer cell growth. In contrast, the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its analog SR11389 inhibited the growth of both cell lines. Unlike RA, 9-cis-RA and DHVD, AHPN and SR11389 also potently inhibited human umbilical vascular endothelial cell growth. These results on AHPN and SR11389 suggest that angiogenesis of tumor microvasculature should also be an effective therapeutic target for this new compound class.

Effects of receptor class- and subtype-selective retinoids and an apoptosis-inducing retinoid on the adherent growth of the NIH:OVCAR-3 ovarian cancer cell line in culture.

Comparison of the adherent growth inhibition of NIH:OVCAR-3 ovarian cancer cells by retinoid receptor class-selective and subtype-selective compounds with their receptor binding affinities and transcriptional activation activities indicated no correlation for RAR alpha and RAR gamma although both receptors are present. Retinoids that activated RXR alpha inhibited cell growth in the range as all-trans-retinoic acid and 9-cis-retinoic acid. The most potent inhibitor was 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), which has been found to inhibit breast and lung cancer and leukemia cell growth and induce cancer cell apoptosis through a pathway independent of the retinoid receptors.

N-(4-hydroxyphenyl)retinamide (4-HPR)-mediated biological actions involve retinoid receptor-independent pathways in human breast carcinoma.

Retinoid response pathways involve retinoic acid receptors (RARs) and retinoid X receptors. N-(4-hydroxyphenyl) retinamide (4-HPR), a derivative of all-trans-retinoic acid (RA) is currently in clinical trials as a chemopreventive agent for breast cancer. The issue whether 4-HPR mediates its biological actions via classical retinoid receptor pathways remains to be investigated. In this study, we provide several lines of evidence that 4-HPR mediates its biological actions via a novel pathway(s) that does not involve the classical retinoid receptor pathways. For example, 4-HPR was more potent than RA as an antiproliferative agent and inhibited growth of otherwise RA-resistant human breast carcinoma cells. Exposure to 4-HPR resulted in the generation of DNA fragmentation with subsequent cell death in both RA-positive estrogen receptor (ER)-positive as well as RA-refractory ER-negative breast carcinoma cell lines. N-(4-Methoxyphenyl)retinamide (4-MPR), which is the major 4-HPR metabolite in circulation, was biologically inert in this system. 4-HPR and 4-MPR bound poorly to the RAR alpha, beta and gamma in vitro and only minimally activated the retinoic acid receptor element (RARE) and retinoid X receptor response elements (RXREs) in human breast carcinoma cells. Neither 4-HPR nor 4-MPR are metabolized to any of the known conventional retinoids. In addition, 4-HPR or 4-MPR transactivation of RAREs or RXREs transfected into MCF-7 and MDA-MB-231 cells was not noted at 48 h. Nevertheless 4-HPR-mediated cell death was observed at 48 h, further suggesting that neither 4-HPR nor 4-MPR are metabolized to retinoids which activate the RAREs or RXREs in breast carcinoma cells. Furthermore, unlike RA, which exhibited anti-AP1 activity, 4-HPR inhibition of growth did not involve anti-AP1 activity. These results suggest that 4-HPR acts by a unique pathway that is not mediated by retinoid receptors.

Correlation of retinoid binding affinity to retinoic acid receptor alpha with retinoid inhibition of growth of estrogen receptor-positive MCF-7 mammary carcinoma cells.

Both anchorage-dependent growth and anchorage-independent growth of the estrogen receptor-positive mammary carcinoma cell line MCF-7 are inhibited by all-trans-retinoic acid. This cell line has nuclear retinoic acid receptors (RARs) alpha and gamma. The natural retinoids all-trans-retinoic acid and 9-cis-retinoic acid and a series of 12 conformationally restricted retinoids, which showed a range of binding selectivities for these receptors and had either agonist or antagonist activity for gene transcriptional activation by the RARs, were evaluated for their abilities to inhibit anchorage-dependent (adherent) and anchorage-independent (clonal) growth of MCF-7 cells. Correlation analyses were performed to relate growth inhibition by these retinoids with their binding affinity to RAR alpha or RAR gamma. Inhibition of anchorage-dependent growth in culture after 7 days of retinoid treatment correlated with binding to RAR alpha (n = 14; P < or = 0.001) and not to RAR gamma (n = 14; P > 0.1). Both the RAR alpha-selective retinoid agonists and the two RAR antagonists that were evaluated inhibited adherent cell growth. The RAR gamma-selective agonists had very low growth inhibitory activity (< 10%) at concentrations as high as 12.5 microM. These results suggest that RAR alpha is the retinoid receptor involved in the inhibition of adherent cell growth by retinoids and that transcriptional activation by this receptor on a RAR response element does not appear to be required for this process to occur. For this series of retinoids, inhibition of anchorage-independent growth after 21 days of retinoid treatment only correlated (n = 12; P < or = 0.005) with binding affinity to RAR alpha for the retinoid agonists, although the RAR gamma-selective retinoids displayed weak activity. The RAR antagonists were very poor inhibitors of growth. These results suggest that activation of gene transcription by RAR alpha appears to be required for inhibition of anchorage-independent growth by retinoids in this estrogen receptor-positive mammary carcinoma cell line.

Conformational effects on retinoid receptor selectivity. 2. Effects of retinoid bridging group on retinoid X receptor activity and selectivity.

The natural retinoid 9-cis-retinoic acid is an activating ligand for both the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which are members of the retinoid/thyroid hormone/steroid hormone family of nuclear receptor proteins that activate gene transcription through specific response elements. The pharmacophoric groups necessary to confer RXR selectivity were established by evaluating the ability of 21 conformationally restricted retinoids to activate the TREpal retinoic acid receptor response element for gene transcription in the presence of one of the three RAR subtypes or RXR alpha. In contrast to those retinoids selective for the RARs, these RXR-selective retinoids have one less atom in the bridge linking the hydrophobic and carboxylic acid termini of the retinoid skeleton. Therefore, a one-carbon bridge replaces the 19-methyl group and 9E-double bond of 9-cis-retinoic acid and is further functionalized by inclusion in an isopropylidene group, a dioxolane ring, or a cyclopropane ring for optimal RXR alpha activity and selectivity. In addition, the beta-geranylidene and 20-methyl-(11E,13E)-dienoic acid groups of 9-cis-retinoic acid are replaced by a 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl ring and a 4-carboxylphenyl ring, respectively, for optimal activation and selectivity. RXR alpha selectivity is reduced on replacement of the 4-carboxylphenyl group by a 2-carboxyl-5-thienyl group or the 9-cis-retinoic acid methylpentadienoic acid terminus.

Recombinant human retinoic acid receptor beta. Binding of synthetic retinoids and transcriptional activation.

All-trans-retinoic acid mediates cell growth and differentiation by binding to and then activating nuclear retinoid receptor proteins that regulate gene transcription. Recombinant human retinoic acid receptor beta was cloned and expressed in Escherichia coli as a fusion protein rMBP-RAR beta with maltose-binding protein to facilitate purification. After isolation from bacterial lysates, rMBP-RAR beta was used for binding with selected retinoids. Scatchard analysis with [11,12-3H2]all-trans-retinoic acid gave a Kd of 0.34 nM. Competitive binding studies with a series of conformationally restricted aromatic retinoids indicated that the Ki values for binding to rMBP-RAR beta correlated with the logs of the EC50 values for gene transcriptional activation (p < or = 0.05) and with those for the relative activation compared to that of all-trans-retinoic acid (p < or = 0.01). Inspection of binding-activation correlation diagrams indicates candidate structures for improved retinoid agonists or antagonists.

Synthesis and antiinflammatory activity of novel 12 beta-substituted analogues of betamethasone.

A series of 9 alpha-halo-12 beta-hydroxy and 12 beta-acyloxy analogues of betamethasone 17,21-dipropionate were synthesized and tested for topical antiinflammatory potency in the croton oil ear assay. The compounds were assayed for systemic absorption in the contralateral ear assay, in which it was found that 12 beta-hydroxy analogues 9, 13, and 15 were all absorbed but the corresponding 12 beta-esters 11a-e, 14, and 16 were not. On repeated high-dose applications to the mouse ear, there was no evidence of systemic absorption of any 12 beta-propionate ester as gauged by thymus weights (thymic involution) and plasma cortisol levels (adrenal suppression). Results of limited SAR studies showed that topical antiinflammatory activity and systemic absorption were not greatly influenced by the 9 alpha-halogen but were largely dependent on the polarity and size of the 12 substituent. While the optimal compounds 14 and 16 were less topically active than the controls beta- and beclomethasone dipropionate, unlike the controls, they displayed no systemic effects, even after repeated high-dose applications. Surprisingly, propionate 14 was devoid of atrophogenic activity.

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein.

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.

Effect of structural modifications in the C7-C11 region of the retinoid skeleton on biological activity in a series of aromatic retinoids.

A series of conformationally restricted analogues of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl ] benzoic acid--(E)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 - propenyl]benzoic acid, (E)-4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-bu ten- 2-yl]benzoic acid, trans-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) cyclopropyl]benzoic acid, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid, 6-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2- naphthalenecarboxylic acid--were synthesized and screened for retinoid biological activity. Comparison of the conformers of these analogues generated by molecular mechanics calculations with the biological activity profiles of these compounds indicates that geometric constraints required for high biological activity are imposed on the bridge joining the two aromatic ring systems by the retinoid receptor.

Comparison of the inhibitory effects of retinoids on 12-O-tetradecanoylphorbol-13-acetate-promoted tumor formation in CD-1 and Sencar mice.

Linear regression analysis of the dose levels of retinoids required to inhibit the formation of papillomas by 50% in mouse dorsal epidermis topically treated with 7,12-dimethylbenz[a]anthracene (DMBA) (200 nmol) and then promoted 2 weeks later with twice-weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) (8.5 nmol) indicated that there was a good correlation (r = 0.9095, P less than 0.02) between retinoid inhibitory activities in the CD-1 mouse at 20 weeks after the start of promotion and in the Sencar mouse after 12 weeks of promotion. The ID50 values (nmol) determined were as follows: all-trans-retinoic acid (RA) (3.5, CD-1; 17, Sencar); 4-[2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)-1E-propen-1-yl]benzoic acid (0.14, CD-1; 0.71, Sencar); 4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl- 2-naphthalenyl)- 1E-propen-2-yl]benzoic acid (0.54, CD-1; 1.9, Sencar); 6-[1-(4-carboxyphenyl)-1E-propen-2-yl]- 3,4- dihydro-4,4-dimethyl-2H-1-benzothiopyran (3.0 CD-1; 11, Sencar); 7-[1-(4-carboxyphenyl)-1E-propen-2-yl)- 3,4-dihydro- 4,4-dimethyl-2H-1-benzothiopyran (1.5, CD-1; 0.4, Sencar); 2-[1-(4-carboxyphenyl)-1E-propen-2-yl]- 4,5,6,7-tetrahydro-4,4- dimethyl-benzo[b]thiophene (0.30, CD-1; 2.3, Sencar).

Synthesis and biological activity of ketomethylene-containing nonapeptide analogues of snake venom angiotensin converting enzyme inhibitors.

Two ketomethylene-containing nonapeptide analogues were synthesized to determine if ketomethylene analogues of the nonapeptide venom inhibitors of angiotensin converting enzyme (ACE) would have oral ACE inhibition activity. Both ketomethylene-containing nonapeptides 18 and 19 were potent inhibitors of rabbit lung ACE with I50s of 3.4 and 8.0 nM, respectively, compared to 340 nM for their parent nonapeptide and 450 nM for captopril. Peptide 18 was rapidly cleaved by trypsin, but 19 was reasonably stable to all enzyme degradation systems tested with maximum degradation of 50% by pepsin in 3 h. Both 18 and 19 when given iv to normotensive rats were between 3 and 10 times more potent than captopril in inhibiting an angiotensin I induced blood pressure increase. Peptide 19 showed no ACE inhibition activity in unanesthetized normotensive rats when administered orally at doses of 10 or 100 mg/kg. Experiments were conducted to determine whether 19 is adsorbed from the gastrointestinal track following oral administration. These experiments indicated that 19 is adsorbed. It is concluded that the lack of oral activity of 19 is probably due to its rapid excretion, probably into the bile.

Inhibition by retinoids of anthralin-induced mouse epidermal ornithine decarboxylase activity and anthralin-promoted skin tumor formation.

The retinoids all-trans-retinoic acid, 13-cis-retinoic acid, 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]benzoic acid, 6-[1-(4-carboxyphenyl)-1E-propen-2-yl]-3,4-dihydro-4,4-dimethyl-2H -1-benzothiopyran, and 6-(5,6,7,8,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)- 2-naphthalenecarboxylic acid inhibited the induction of ornithine decarboxylase in CD-1 mouse epidermis treated with the weak tumor promoter anthralin (444 nmol). Enzyme activity reached maximal levels 48 h after the application of the promoter. This activity was most effectively inhibited when retinoids were applied to the epidermis 24 h after the promoter. These retinoids also inhibited the appearance of papillomas in mouse epidermis in the two-stage tumorigenesis model using 7,12-dimethylbenz(a)anthracene (200 nmol) as the initiator and anthralin (444 nmol) as the promoter during the 32-week period of promotion. Comparison of the doses of retinoids required to inhibit anthralin-induced ornithine decarboxylase by 50% and those required to inhibit anthralin-induced tumor promotion by 50% demonstrated that these values correlated.

Comparison of the inhibitory effects of retinoids on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activities in CD-1 and Sencar mice.

Linear regression analysis of the dose levels of a series of retinoids required ornithine decarboxylase (ODC) induction by 50% indicated that there was no significant difference in inhibitory activity between CD-1 and Sencar mice (r = 0.9699; P less than 0.001). The ID50 values (nmol) found were as follows: retinoic acid (0.20, CD-1; 0.29, Sencar); 13-cis-retinoic acid (1.4, CD-1; 1.9, Sencar); 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1E-propenyl]benzoic acid (0.04, CD-1; 0.03; Sencar); 2-[1-(4-carboxyphenyl)-1E-propeny-2-yl]-4,5,6,7-tetrahydro-4, 4-dimethylbenzothiophene (0.08, CD-1; 0.14, Sencar); 2-[1-(4-carboxyphenyl)-1E-propen-2-yl]3, 4-dihydro-4,4-dimethyl-2H-1-benzothiopyran (0.56, CD-1; 0.70, Sencar); 6-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-2-naphthalenecarboxylic acid (1.0, CD-1; 0.90, Sencar).

Synthesis and biological activity of pentapeptide analogues of the potent angiotensin converting enzyme inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline.

Two pentapeptide analogues of the ketomethylene-containing angiotensin converting enzyme (ACE) inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline (1) were synthesized and evaluated as ACE inhibitors and antihypertensive agents. Compounds 14 and 15 were very potent ACE inhibitors with I50 values of 7.0 and 3.0 nM, respectively, compared to an I50 value of 70 nM for 1. Neither 14 nor 15 showed significant blood pressure lowering activity in renal hypertensive rats. Investigations conducted on a tritiated analogue of 14 showed that 70% of an oral dose of this compound is absorbed but is rapidly excreted from the blood with a half life of 24 min. Thin-layer chromatography of bile and urine contents in rats given tritiated 14 orally showed that it is excreted in greater than 90% unchanged form. This implies that a ketomethylene linkage can stabilize peptide amide linkages adjacent to it to peptidase degradation.

Synthesis and biological activity of carboxylic acid replacement analogues of the potent angiotensin converting enzyme inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline.

The carboxylic acid group on the proline of 1 was replaced by a phosphoric acid, a hydroxamic acid, and a tetrazole to give compounds 2-4, respectively. Testing of 2-4 as angiotensin converting enzyme (ACE) inhibitors gave I50 values of 100, 1.6, and 22 microM, respectively, compared to 0.07 microM for 1. A hydroxamic acid derivative of the ketomethylene pentapeptide analogue 18 was then synthesized. This compound, 17, had an ACE I50 of 0.011 microM compared to 0.0076 microM for 18. Oral administration of 10 mg/kg of 17 to renal hypertensive rats had no effect on blood pressure or heart rate.

Conformationally restricted retinoids.

A series of conformationally restricted retinoids was synthesized and screened in two assays used to measure the ability of retinoids to control cell differentiation, namely, the reversal of keratinization in tracheal organ culture from vitamin A deficient hamsters and the inhibition of the induction of mouse epidermal ornithine decarboxylase by a tumor promoter. These compounds had bonds corresponding to selected bonds of the E-tetraene chain of retinoic acid (1) held in a planar cisoid conformation by inclusion in an aromatic ring. The meta-substituted analogue 3 of 4-[(E)-2-methyl-4-(2,6,6-trimethylcyclohexenyl)-1,3-butadienyl+ ++]benzoic acid (2) was far less active than 2 in both assays. In contrast, the vinyl homologue of 2 (4) and the 7,8-dihydro and 7,8-methano analogues (5 and 6) had activity comparable to that of 2. Analogues of 4-[(E)-2-(1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-6-naphthyl)propenyl] benzoic acid (7) were also screened. Replacement of the tetrahydronaphthalene ring of 7 by a benzonorbornenyl group (9) significantly reduced activity, as did removal of the vinylic methyl group from 9 (10). Replacement of the propenyl group of 9 by a cyclopropane ring (12) also reduced activity. Replacement of the tetrahydronaphthalene ring of 7 by 4,4-dimethyl-3,4-dihydro-2H-1-benzopyran and -benzothiopyran rings (13 and 14) also decreased activity. Inclusion of the 7,9 double bond system of 1 in an aromatic ring (15 and 16) reduced activity, whereas inclusion of the 5,7 double bond system in an aromatic ring enhanced activity (7 and 19). Inclusion of the 11,13 and 9,11,13 double bond systems in aromatic rings (2 and 18) also reduced activity below that of 1. Retinoic acid, 7, 13, 14, and 19 inhibited papilloma tumor formation in mice. Toxicity testing indicated that 7 was more toxic than 1, 13, 14, and 19, 19 was more toxic than 1, and 13 and 14 were less toxic than 1.