Novel progestogenic androgens for male contraception: design, synthesis, and activity of C7 α-substituted testosterone†.

Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates.

In vitro metabolism and stability of the actinide chelating agent 3,4,3-LI(1,2-HOPO).

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.

The stereoselective sulfate conjugation of 4'-methoxyfenoterol stereoisomers by sulfotransferase enzymes.

The presystemic sulfate conjugation of the stereoisomers of 4'-methoxyfenoterol, (R,R')-MF, (S,S')-MF, (R,S')-MF, and (S,R')-MF, was investigated using commercially available human intestinal S9 fractions, a mixture of sulfotransferase (SULT) enzymes. The results indicate that the sulfation was stereospecific and that an S-configuration at the ß-OH carbon of the MF molecule enhanced the maximal formation rates with (S,R')-MF (S,S')-MF (R,S')-MF ≈ (R,R')-MF, and competition studies demonstrated that (S,R')-MF is an effective inhibitor of (R,R')-MF sulfation (IC(50) = 60 µM). In addition, the results from a cDNA-expressed human SULT isoform screen indicated that SULT1A1, SULT1A3, and SULT1E1 can mediate the sulfation of all four MF stereoisomers. Previously published molecular models of SULT1A3 and SULT1A1 were used in docking simulations of the MF stereoisomers using Molegro Virtual Docker. The models of the MF-SULT1A3 and MF-SULT1A1 complexes indicate that each of the two chiral centers of MF molecule plays a role in the observed relative stabilities. The observed stereoselectivity is the result of multiple hydrogen bonding interactions and induced conformational changes within the substrate-enzyme complex. In conclusion, the results suggest that a formulation developed from a mixture of (R,R')-MF and (S,R')-MF may increase the oral bioavailability of (R,R')-MF.

Effect of resveratrol on 17beta-estradiol sulfation by human hepatic and jejunal S9 and recombinant sulfotransferase 1E1.

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4'-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17beta-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3-20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 microM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated K(i) values of 1.1 microM (liver), 0.6 microM (jejunum), and 2.3 microM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms.

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.