Virtual coupling of pyran protons in the 1H NMR spectra of C- and N-glucuronides [correction]: dependence on substitution and solvent.

We have observed that certain C- and N-glucuronides prepared as intermediates for breast cancer preventives demonstrate non-first order 1H NMR spectra that are not the result of impurities or degradation but are instead due to virtual coupling in the pyran proton network. This virtual coupling shows the expected dependence on solvent and field strength and, more importantly, on the nature of the C-1 substitution. Although the hybridization of the atom bonded to C-1 may play a role, it appears that steric and/or electronic factors, which have the effect of increasing Delta(v)/J for H-3 and H-4, are critical for eliminating the spectral complexity. These observations, which appear to be fairly general, suggest that this phenomenon should be considered when addressing the purity of pharmaceutical agents containing these types of structural units.

Chemotherapeutic evaluation of N-(4-hydroxyphenyl) retinamide-O-glucuronide in the rat mammary tumor model.

The growth inhibitory effects of N-(4- Hydroxyphenyl) retinamide (4-HPR) and its glucuronide derivative, N-(4-Hydroxyphenyl) retinamide-O-glucuronide (4-HPROG) on established DMBA induced rat mammary tumors were compared. The results indicate that the glucuronide analog had a greater antitumor potency than equimolar concentration of the free retinoid. Tumor regression occurred in 75% of the rats fed 2 mmol/Kg diet of 4-HPROG. In a 6-week study, the maximum tolerated dietary dose (MTD) was found to be 3.5 mmol/Kg diet for 4-HPR and 5 mmol/Kg diet in the case of 4-HPROG. The higher potency and lower toxicity of the glucuronide suggests that this conjugate may have an in vivo chemotherapeutic advantage over the parent free retinoid.

Chemopreventive activities of C-glucuronide/glycoside analogs of retinoid-O-glucuronides against breast cancer development and growth.

The O-glucuronide analog of N-(4-hydroxyphenyl)retinamide (4-HPROG) has shown a greater chemopreventive activity than the parent N-(4-hydroxyphenyl)retinamide (4-HPR). However, this compound is relatively unstable. In order to improve stability and efficacy, we have prepared a number of stable C-linked analogs of 4-HPROG (C-phenyl and C-benzyl glucuronosyl, glucosyl, and xylosyl analogs). These analogs are stable toward acid hydrolysis and the glucuronosyl analogs resist the actions of beta-glucuronidase. The analogs were prescreened for their antiproliferative potential in vitro using cultured human MCF-7 breast cancer cells. Selected analogs were then evaluated for their ability to inhibit the development and growth of tumors in the 7,12-dimethylbenzanthracene-induced rat mammary tumor model. Although the stable C-linked analogs bound poorly to the nuclear retinoic acid receptors, many showed more potency than the less stable 4-HPROG in inhibiting tumor incidence and multiplicity in vivo. The glucuronide/glucoside analogs are more potent than the xylosides, and the C-benzyl more effective than the C-phenyl analogs. The higher potency of at least two C-linked analogs (retinamidobenzyl glucuronide and retinamidobenzyl glucose) suggests that these analogs may have a chemopreventive advantage over the parent retinamide and its natural O-glucuronide.

Stereospecific assignments of glycine in proteins by stereospecific deuteration and 15N labeling.

A method is described for stereospecifically assigning the alpha-protons of glycine residues in proteins. The approach involves the stereospecific deuteration and 15N labeling of glycine and subsequent selective incorporation of this residue into the protein. The stereospecific assignments of the glycine alpha-protons are obtained from a comparison of a 3D 15N-resolved TOCSY spectrum of the uniformly 15N-labeled protein with a 2D/3D 15N-edited TOCSY spectrum of the protein, containing the stereospecifically deuterated and 15N-labeled glycine. The approach is demonstrated by stereospecifically assigning the glycine alpha-protons of the FK506 binding protein when bound to the immunosuppressant ascomycin.

In vivo use of N-(4-hydroxyphenyl retinamide)-0-glucuronide as a breast cancer chemopreventive agent.

The inhibitory effects of N-(4-hydroxyphenyl) retinamide and its glucuronide derivative on the development and growth of 7,12-dimethylbenz (a) anthracene - induced rat mammary tumors in vivo were compared. The results indicate that the glucuronide had a greater chemopreventive potency than equimolar concentration of the free retinoid by all the criteria measured, mainly the inhibition of tumor incidence, multiplicity and tumor growth. HPLC analysis of the blood of the rats showed no hydrolysis of the glucuronide during its chronic consumption, indicating that the retinoid glucuronide is probably acting in vivo per se rather than through hydrolysis to the free retinoid. The higher potency and lower toxicity of the glucuronide suggests, for the first time, that the conjugate may have an in vivo chemopreventive advantage over the parent retinamide.