Pharmacokinetic profile of PBRM in rodents, a first selective covalent inhibitor of 17ß-HSD1 for breast cancer and endometriosis treatments.

The development of a covalent inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a promising approach for the treatment of hormone-dependent breast cancer and endometriosis. After reporting the steroid derivative PBRM as a first potent covalent inhibitor of 17ß-HSD1 without estrogenic activity, we are now interested in studying its pharmaceutical behavior. The metabolism study in a human liver microsomal preparation showed a gradual transformation of PBRM into PBRM-O, an oxidized ketonic form of PBRM at position C17. Interestingly, PBRM-O also inhibits 17ß-HSD1 and is not estrogenic in estrogen-sensitive T-47D cells. However, when PBRM was injected subcutaneously (sc) in mice, a very small proportion of PBRM-O was measured in a 24 h-time course experiment. A pharmacokinetic study in mice revealed suitable values for half-life (T1/2 = 3.4 h), clearance (CL = 2088 mL/h kg), distribution volume (Vz = 10.3 L/kg) and absolute bioavailability (F = 65%) when PBRM was injected sc at 14.7 mg/kg. A good F value of 33% was also obtained when PBRM was given orally. A tritiated version of PBRM, 3H-PBRM, was synthesized and used for an in vivo biodistribution study that showed its gradual accumulation in various mouse tissues (peak at 6 h) followed by elimination until complete disappearance after 72 h. Elimination was found to occur in feces (93%) and urine (7%) as revealed by a mass balance experiment. PBRM was also evaluated for its toxicity in mice and it was found to be very well tolerated after weekly sc administration (30-405 mg/kg for 8 weeks) or by po administration (300-900 mg/kg for 4 weeks). Overall, these experiments represent important steps in the preclinical characterization of the pharmaceutical behavior of PBRM, as well as for its translation to clinical trials.

Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17ß-hydroxysteroid dehydrogenase type 1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16ß,17ß)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17ß-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off-targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki=368nM) and the enzyme inactivation rate (kinact=0.087min-1) values for PBRM were determined with purified 17ß-HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3-5days) is required to recover 17ß-HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17ß-HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non-estrogenic and steroidal covalent irreversible inhibitor of 17ß-HSD1 warrant its further development as a potential drug candidate for estrogen-dependent disorders.

Impact of structural modifications at positions 13, 16 and 17 of 16ß-(m-carbamoylbenzyl)-estradiol on 17ß-hydroxysteroid dehydrogenase type 1 inhibition and estrogenic activity.

The chemical synthesis of four stereoisomers (compounds 5a-d) of 16ß-(m-carbamoylbenzyl)-estradiol, a potent reversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1), and two intermediates (compounds 3a and b) was performed. Assignment of all nuclear magnetic resonance signals confirmed the stereochemistry at positions 13, 16 and 17. Nuclear overhauser effects showed clear correlations supporting a C-ring chair conformation for 5a and b and a C-ring boat conformation for 5c and d. These compounds were tested as 17ß-HSD1 inhibitors and to assess their proliferative activity on estrogen-sensitive breast cancer cells (T-47D) and androgen-sensitive prostate cancer cells (LAPC-4). Steroid derivative 5a showed the best inhibitory activity for the transformation of estrone to estradiol (95, 82 and 27%, at 10, 1 and 0.1µM, respectively), but like the other isomers 5c and d, it was found to be estrogenic. The intermediate 3a, however, was weakly estrogenic at 1µM, not at all at 0.1µM, and showed an interesting inhibitory potency on 17ß-HSD1 (90, 59 and 22%, at 10, 1 and 0.1µM, respectively). As expected, no compound showed an androgenic activity. The binding modes for compounds 3a and b, 5a-d and CC-156 were evaluated from molecular modeling. While the non-polar interactions were conserved for all the inhibitors in their binding to 17ß-HSD1, differences in polar interactions and in binding conformational energies correlated to the inhibitory potencies.

Identification of fused 16ß,17ß-oxazinone-estradiol derivatives as a new family of non-estrogenic 17ß-hydroxysteroid dehydrogenase type 1 inhibitors.

A new family of cyclic carbamate-estradiol derivatives has been designed to remove the intrinsic estrogenic activity of a parent acyclic compound reported as a potent inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). The synthesis of two series of fused 16ß,17ß-oxazinone-estradiol derivatives, saturated compounds 7a-d and unsaturated compounds 10a-d, led to the identification of 10b, a 17ß-HSD1 inhibitor (IC50 = 1.4 µM) without estrogenic activity in estrogen-sensitive T-47D cells. Interestingly, this compound was found selective over 17ß-HSD2 and 17ß-HSD12. A computational analysis of inhibitors into 17ß-HSD1 by molecular docking also revealed interesting structure-activity relationships that could be helpful in the design of new generation of 16ß,17ß-oxazinone-estradiol analogs.

Identification of a first enzymatic activator of a 17ß-hydroxysteroid dehydrogenase.

Small molecule activators that directly modulate the activity of an enzyme are uncommon entities, and such activators had never yet been identified for any 17ß-hydroxysteroid dehydrogenase (17ß-HSD). We hereby report the fortuitous discovery of a steroid derivative that caused an up to 3-fold increase in the activity of 17ß-HSD12. The stimulation of estrone to estradiol conversion has been characterized in intact and homogenized stably transfected HEK-293 cells and has also been observed in T47D breast cancer cells. Structure-activity relationships closely linked to the nature of the substituent on the [1,3]oxazinan-2-one ring of an estradiol derivative emerged from this study and may help in the identification of a previously unsuspected endogenous activation of 17ß-HSD12. This activator will therefore be a useful tool to study this relatively unknown enzyme as well as the possible activation of other 17ß-HSD family members.

Discovery of a non-estrogenic irreversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 from 3-substituted-16ß-(m-carbamoylbenzyl)-estradiol derivatives.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is thought to play a pivotal role in the progression of estrogen-sensitive breast cancer by transforming estrone (E1) into estradiol (E2). We designed three successive series of E2-derivatives at position C3 of the potent inhibitor 16ß-(m-carbamoylbenzyl)-E2 to remove its unwanted estrogenic activity. We report the chemical synthesis and characterization of 20 new E2-derivatives, their evaluation as 17ß-HSD1 inhibitors, and their proliferative (estrogenic) activity on estrogen-sensitive cells. The structure-activity relationship study provided a new potent and steroidal nonestrogenic inhibitor of 17ß-HSD1 named 3-{[(16ß,17ß)-3-(2-bromoethyl)-17-hydroxyestra-1(10),2,4-trien-16-yl]methyl}benzamide (23b). In fact, this compound inhibited the transformation of E1 into E2 by 17ß-HSD1 in T-47D cells (IC50 = 83 nM), did not inhibit 17ß-HSD2, 17ß-HSD7, 17ß-HSD12, and CYP3A4, and did not stimulate the proliferation of estrogen-sensitive MCF-7 cells. We also discussed the results of kinetic and molecular modeling (docking) experiments, suggesting that compound 23b is a competitive and irreversible inhibitor of 17ß-HSD1.

Facile photoinduced charge separation through a cyanoacetylide bridge in a heterobimetallic Fe(II)-Re(I) complex.

Photoinduced Fe-to-bpy charge transfer in [{Cp(dppe)Fe}(mu-C[triple bond]CC[triple bond]N){Re(CO)(3)(bpy)}]PF(6) has been observed by ps-TRIR spectroscopy, supported by UV-Vis/IR spectroelectrochemistry and DFT calculations.

On the dynamics of the H+ +D2(v=0,j=0)-->HD+D + reaction: a comparison between theory and experiment.

The H+ +D2(v=0,j=0)-->HD+D + reaction has been theoretically investigated by means of a time independent exact quantum mechanical approach, a quantum wave packet calculation within an adiabatic centrifugal sudden approximation, a statistical quantum model, and a quasiclassical trajectory calculation. Besides reaction probabilities as a function of collision energy at different values of the total angular momentum, J, special emphasis has been made at two specific collision energies, 0.1 and 0.524 eV. The occurrence of distinctive dynamical behavior at these two energies is analyzed in some detail. An extensive comparison with previous experimental measurements on the Rydberg H atom with D2 molecules has been carried out at the higher collision energy. In particular, the present theoretical results have been employed to perform simulations of the experimental kinetic energy spectra.