No abstract

Retinoids are natural and synthetic derivatives of vitamin A that have great promise for cancer therapy and chemoprevention. Of the retinoids developed so far, 4-(N-hydroxyphenyl)retinamide (4-HPR or fenretinide) appears to have the best therapeutic potential in vitro and in vivo and is currently being tested in clinical trials for cancer prevention and therapy. To develop other potentially potent antitumor agents, we synthesized 85 retinoid derivatives. In an initial screening of these synthetic retinoids using the HCT116 colon cancer cell line, we found that 4-amino-2-(butyrylamino)phenyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-2,4,6,8-nonatetraenoate (ABPN or CBG41) induced the greatest growth inhibition, with an IC 50 value of 0.6 M. Subsequent studies in other cancer cell lines indicated that ABPN was much more growth-inhibitory than all-trans retinoic acid or 4-HPR. Compared to 4-HPR, ABPN induced 5.5-to 70.0-fold more growth inhibition in most cancer cells, with the exception of gynecologic cancer cells. In these cells, the antiproliferative effect was only 1.5-to 2.8-fold more than 4-HPR. We examined the molecular mechanism underlying the difference in growth inhibition between 4-HPR and ABPN. DAPI staining, DNA fragmentation, FACS and Western blotting analyses suggest that ABPN induced apoptosis by activating caspase-3 and -8, which may result in increased PARP cleavage. Unlike 4-HPR, ABPN activated all 3 RAR isotypes to an extent similar to AtRA. In addition, ABPN significantly inhibited AP-1 transcriptional activity and thus greatly suppressed the expression of the matrix metalloproteinase -1, -2 and -3 genes, which are involved in tumor invasion. These results suggest that ABPN may be a promising retinoid derivative offering not only enhanced cytotoxicity, but also increased inhibition of tumor invasiveness.

Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against cancer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation, which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that the N-acylated derivatives (2A-5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARbeta and RARgamma. In contrast, the O-acylated retinoate derivatives (1B-5B) activated all three RAR isotypes without specificity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon cancer cells, and the IC(50) of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was 1.67 microM and 0.65 microM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 microM), a prototype N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt derivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the relationship between the structure and function of retinoate and retinamide derivatives.

Retinoids act as potential chemopreventive and chemotherapeutic agents by inhibiting uncontrolled cell growth, inducing apoptosis, and promoting the differentiation of cancer cells.1-3) Natural retinoids, such as all-transretinoic acid (AtRA) and its isomer 9-cis RA, are physiological metabolites. RA-bound receptors, RAR and RXR, associate with cis-acting RA response elements (RARE) and stimulate the transcription of the responsive genes. 4) In addition to receptor activation, RA interferes with the transactivation function of activation protein-1 (AP-1), which is mostly composed of c-Jun and c-Fos. The RA-dependent interference of AP-1 may inhibit the expression of the matrix metalloproteinases collagenase (MMP-1) and stromelysin (MMP-3), the expression of the cytokines IL-1 and TGF-b, and the expression of the HPV-16/18 oncogenes E6 and E7. These functions make RA a promising therapeutic agent for the treatment of tumor invasion, inflammation, and skin aging. 5-7)However, RA is frequently teratogenic and causes severe side effects in tissues of the skin, blood vessels, liver, nerves, and bone when large doses are used clinically. 8) Thus, the toxicity associated with natural retinoids limits their clinical use. This has led to the development of novel RA derivatives that are currently being tested to evaluate their clinical effectiveness. 9) One of the newly developed RA derivatives, 4-hydroxyphenyl retinamide (4-HPR), has demonstrated increased anticancer activity associated with a favorable toxicity profile.10) It has been reported that 4-HPR is cytotoxic against a wide variety of cancer cell lines, including ovarian cancer cells in vitro, 11,12) and is effective in reducing the sizes of breast, prostate, and ovarian cancer tumors in animal models.13-16) Currently, 4-HPR is in clinical trials for the treatment of ovarian, breast, bladder, prostate, and lung cancers. [17][18][19][20][21] However, the plasma levels in patients receiving 200 mg of 4-HPR daily are under 1 mM, which is far less than the effective concentration (usually 10 mM) required to induce apoptosis in vitro.22) The higher doses of 4-HPR that appear to be required may cause skin irritation and tissue injury. De-rivatives with less irritating side effects and better clinical efficacy are needed. Recently, several retinamide derivatives were identified that were effective in inhibiting growth and inducing the apoptosis of several human cancer cells. 23,24) These derivatives bear hydroxyl, carboxy, or methoxy substitutions on the terminal phenylamine ring. Of the derivatives developed, 3-HPR showed the most active growth inhibition in the four bladder cancer cell lines, 23) and 2-HPR was the most effective in some head and neck cancer and lung cancer cell lines. 24)In our studies, we synthesized a series of fatty acid derivatives of 4-HPR by the addition of acetate (compound 1), propionate (2), pyruvate (3), butyrate (4), or stearate (5) to the 4-hydroxylphenyl moiety of 4-HPR. In our initial proliferation assays, we identified compound 3, in w...

Fenretinide, 4-(N-hydroxyphenyl) retinamide (4-HPR), has demonstrated anticancer activity associated with a favorable toxicity profile and is now being investigated in several clinical trials. However, its plasma levels in patients have been far lower than the effective concentration required to induce apoptosis (usually 10 M). This result has led to the synthesis of derivatives with better efficacy. Sodium butyrate's potential as an anticancer agent prompted us to synthesize a butanoate derivative of 4-HPR, 5-hydroxyphenyl butanoate retinamide (5-HPBR) and compare it to the parent compound for antitumor potential in vitro. The cytotoxicity of 5-HPBR was 2-to 6-fold greater than that of 4-HPR against cancer cell lines derived from various tissues. In premalignant bronchial cells (BEAS2B), 5-HPBR exhibited about a 10-fold stronger cytotoxicity than did 4-HPR. Normal CHANG liver cells were unaffected by either 4-HPR or 5-HPBR. Subsequent assays using DNA fragmentation, DAPI staining, FACS and Western blotting suggested that the potent inhibitory effect of 5-HPBR is mediated by apoptosis; the exact mechanism appears to differ among cancer cell types. In transcription assays with COS-1 cells, 5-HPBR selectively activated RAR␤ and RAR␥ but was a weaker ligand for all 3 subtypes of RAR than either all-trans retinoic acid or 4-HPR. Overall, these data suggest that 4-BHPR may be a promising retinoid with enhanced antitumor activity and reduced toxicity.