Robert W. Curley

Growth suppression of human breast carcinoma cells in culture by N-(4-hydroxyphenyl)retinamide and its glucuronide and through synergism with glucarate

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (HPR) and its glucuronide derivative on the growth of MCF-7 human breast cancer cells in vitro were compared. The results indicate that the glucuronide had slightly greater potency and much less cytotoxicity than the free retinoid. At a concentration of 10(-6) M, HPR inhibited MCF-7 cell growth by approximately 25%, whereas an equimolar concentration of the glucuronide caused a 40% growth inhibition. Higher concentrations of HPR were highly cytotoxic. At a 10(-5) M concentration of the glucuronide, cell viability was 77%, and 65% of the cells were able to resume growth. On the other hand, at 10(-5) M HPR, cell viability dropped to 49%, and only 15% of the cells were capable of resuming growth. The lower cytotoxicity and higher potency of the retinoid glucuronide compared to the parent retinamide suggest that the conjugate may have a chemotherapeutic advantage over the parent compound. The apparent higher efficacy of HPR in combination with glucarate (GT) compared to the single agents could be due to increased net formation of HPR glucuronide conjugate following conversion of GT to the beta-glucuronidase inhibitor, D-glucaro-1,4-lactone. However, HPLC analysis of the cell metabolites did not show any detectable levels of the retinoid glucuronide upon treatment of MCF-7 cells with HPR and GT.

Effect of dietary soybean and licorice on the male F344 rat: An integrated study of some parameters relevant to cancer chemoprevention

The individual and combined effects of dietary toasted soybean meal (3.13-25%) and dietary licorice root extract (0.38-3.0%) on selected liver and intestinal enzyme levels and on clinical chemistry and histopathological parameters were evaluated on male F344 rats. All parameters were measured one and three months after the 50-day-old rats were started on the diets. By use of newly developed high-performance liquid chromatography-based analytic methods, measurable levels of daidzein (2.67 micrograms/ml) and glycyrrhetinic acid (7.87 micrograms/ml) were detected in the sera of rats on the 25% soybean and 3% licorice diets, respectively. Histopathological evaluations of organs and tissues yielded only nonsignificant strain-related changes. At all dosages, there were no significant soybean- or licorice-related anatomic lesions or hematologic changes. In the clinical biochemistry profile, soybean meal caused moderate but significant dose-dependent decreases in serum cholesterol and increases in alkaline phosphatase, blood urea nitrogen, and phosphorus, which remained within the normal range. Liver glutathione transferase, catalase, and protein kinase C showed significant inductions (up to 50%) in response to increasing doses of soybean meal and licorice extract, with evidence for only marginal interaction between the two additives. Their effects on the intestinal mucosa were not significant. Ornithine decarboxylase levels, an indicator of promotional activity, were unchanged or repressed by the additives. The favorable effects of up to 25% toasted soybean meal and 3% licorice root extract on the levels of the four enzymes, without unfavorable changes in clinical parameters, might account in part for the chemopreventive activities of these additives. These effects would be in addition to direct inhibitory effects of known components in these additives on these or other enzymes or modulation of hormone activity that is not evaluated in this study.

Retinoid chemistry: synthesis and application for metabolic disease.

In this review a discussion of the usual procedures used to synthesize retinoids is followed by an overview of the structure-activity relationships of these molecules. The discussion is then focused on the role and impact of retinoids on metabolic disorders with a particular emphasis on obesity, diabetes, and the metabolic syndrome. In these areas, both natural and synthetic retinoids that are being studied are reviewed and areas where likely future research will occur are suggested. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

A Convenient Synthesis of the Important Retinoid Synthon Ethyl trans-3-formyl-2-butenoate

Abstract The efficient, high-yield synthesis of the important retinoic acid synthon ethyl trans-3-formyl-2-butenoate by Wadsworth-Emmons methodology is described.

Reaction of Glycosyl Halides with Benzyl Grignard Reagents: Unexpected o-Tolyl Alkylation of Tetra-O-acetylglucopyranosyl Bromide and Direct Synthesis of (B-Glycosyl)phenylmethanes

Abstract The synthesis of (β-glycosyl1)phenylmethanes by Grignard alkylation of glycosyl halides is investigated. Reaction of tetra-O-acetylglucopyranosyl bromide with benzylmagnesium chloride gave a good yield of a 3:1 mixture of 2-(β-d-glucopyranosyl)toluene and (β-glucosyl)phenylmethane. The requirement for an equatorial 2-acetoxy group and 6-acetoxymethyl group for the formation of the unexpected o-tolyl rearrangement product is explored by using xylosyl, mannosyl, and 2-deoxyglucosyl halides as substrates for the alkylation. Synthesis of (β-glucosyl)phenylmethane by alkylation of 2,3,4,6-tetra-O-benzylglucosyl bromide with benzylmagnesium chloride is also presented.

Stereoselective route to 15N-labeled-β-deuterated amino acids: synthesis of (2S,3R)-[3-2H,15N]-phenylalanine

Abstract (2 S ,3 R )-[3- 2 H, 15 N]-Phenylalanine hydrochloride was synthesized utilizing 15 N-labeled 8-phenylmenthylhippurate as a chiral glycine equivalent. The key step in the synthesis was the alkylation of the glycine template with ( S )-(+)-benzyl-α-d-mesylate. The doubly labeled alkylation product was obtained in 89% yield with 92% de at the α-carbon and 74% de at the β-carbon. The nature of the electrophile employed in the alkylation step significantly affects the stereochemical outcome at the β-carbon. Hydrolysis of the alkylation product under acidic conditions followed by recrystallization from isopropanol yielded the title compound as the hydrochloride salt. Analysis of the (−)-camphanamide derivative of the final product by NMR spectroscopy and HPLC revealed a 76% de at the α-carbon and a 72% de at the β-carbon. The synthetic strategy described represents a simple yet versatile route to chirally deuterated β-methylene unit containing amino acids.

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-Hydroxyethoxy)retinoic acid

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.

4-oxygenated retinoids: unexpected chemopreventive potential for analogues originally synthesized as affinity labels

Abstract Potential retinoid binding protein affinity labels 3 – 5 were prepared from the retinoic acid metabolite 2 . While unsuccessful as affinity labels, 5 and the serendipitously discovered either 6 show potential as skin cancer chemoproventives, activity not normally associated with 4-oxygenated retinoids.

Affinity chromatographic purification of serum retinol-binding protein using 4-substituted aminoretinoids☆

Retinol-binding protein has been purified from rabbit serum by a new affinity chromatographic phase. Human retinol-binding protein has been shown to bind with vitamin A derivatives and certain other terpenoids. Consequently, this affinity method is based upon the ability of the protein to reversibly bind to beta-ionone and employs a derivatized affinity ligand while preserving the integrity of the beta-ionone molecule via substitution of the allylic 4-position. Purification is relatively simple when compared with other known methods and the yield from serum is similar to other schemes. The protein is obtained in the apo-form and retains the ability of the native protein to bind retinol.

An HPLC–MS/MS method for the separation of α-retinyl esters from retinyl esters

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene.