Synthesis of substituted 15ß-alkoxy estrone derivatives and their cofactor-dependent inhibitory effect on 17ß-HSD1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a key enzyme in the biosynthesis of 17ß-estradiol. Novel estrone-based compounds bearing various 15ß-oxa-linked substituents and hydroxy, methoxy, benzyloxy, and sulfamate groups in position C3 as potential 17ß-HSD1 inhibitors have been synthesized. In addition, in vitro inhibitory potentials measured in the presence of excess amount of NADPH or NADH were investigated. We observed substantial inhibitory potentials for several derivatives (IC50 < 1 µM) and increased binding affinities compared to unsubstituted core molecules. Binding and inhibition were found to be cofactor-dependent for some of the compounds and we propose structural explanations for this phenomenon. Our results may contribute to the development of new 17ß-HSD1 inhibitors, potential drug candidates for antiestrogen therapy of hormone-dependent gynecological cancers.

Synthesis and structure-activity relationships of 2- and/or 4-halogenated 13ß- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis.

Ring A halogenated 13α-, 13ß-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure-activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17ß-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Synthesis of novel 13α-estrone derivatives by Sonogashira coupling as potential 17ß-HSD1 inhibitors.

Novel 13α-estrone derivatives were synthesized by Sonogashira coupling. Transformations of 2- or 4-iodo regioisomers of 13α-estrone and its 3-methyl ether were carried out under different conditions in a microwave reactor. The 2-iodo isomers were reacted with para-substituted phenylacetylenes using Pd(PPh3)4 as catalyst and CuI as a cocatalyst. Coupling reactions of 4-iodo derivatives could be achieved by changing the catalyst to Pd(PPh3)2Cl2. The product phenethynyl derivatives were partially or fully saturated. Compounds bearing a phenolic OH group furnished benzofurans under the conditions used for the partial saturation. The inhibitory effects of the compounds on human placental 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated by an in vitro radiosubstrate incubation method. Certain 3-hydroxy-2-phenethynyl or -phenethyl derivatives proved to be potent 17ß-HSD1 inhibitors, displaying submicromolar IC50 values.

Comparative investigation of the in vitro inhibitory potencies of 13-epimeric estrones and D-secoestrones towards 17ß-hydroxysteroid dehydrogenase type 1.

The inhibitory effects of 13-epimeric estrones, D-secooxime and D-secoalcohol estrone compounds on human placental 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated. The transformation of estrone to 17ß-estradiol was studied by an in vitro radiosubstrate incubation method. 13α-Estrone inhibited the enzyme activity effectively with an IC50 value of 1.2 µM, which indicates that enzyme affinity is similar to that of the natural estrone substrate. The 13ß derivatives and the compounds bearing a 3-hydroxy group generally exerted stronger inhibition than the 13α and 3-ether counterparts. The 3-hydroxy-13ß-D-secoalcohol and the 3-hydroxy-13α-D-secooxime displayed an outstanding cofactor dependence, i.e. more efficient inhibition in the presence of NADH than NADPH. The 3-hydroxy-13ß-D-secooxime has an IC50 value of 0.070 µM and is one of the most effective 17ß-HSD1 inhibitors reported to date in the literature.

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone-Nucleoside Bioconjugates.

2'-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne-azide click reaction (CuAAC). For the introduction of the azido group the 5'-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3'-hydroxy groups of the nucleosides were protected by acetyl groups and the 5'-hydroxy groups were modified by the tosyl-azide exchange method. The commonly used conditions for click reaction between the protected-5'-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17ß-hydroxysteroid dehydrogenase 1 (17ß-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 µM). The thymidine conjugate displayed considerable 17ß-HSD1 inhibitory activity (IC50 = 19 µM).

Heterocyclic androstane and estrane d-ring modified steroids: Microwave-assisted synthesis, steroid-converting enzyme inhibition, apoptosis induction, and effects on genes encoding estrogen inactivating enzymes.

d-ring-fused and d-homo lactone compounds in estratriene and androstane series were synthesized using microwave-assisted reaction conditions. Microwave-irradiated synthesis methods were convenient and effective, and provided high yields with short reaction times. Their inhibition of C17,20-lyase and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities were studied in in vitro enzyme assays. d-ring-fused triazolyl estrone analog 24 showed potent inhibition of NADH-complexed 17ß-HSD1, with a binding affinity similar to that of the substrate estrone; its inhibition against NADPH-complexed 17ß-HSD1 was markedly weaker. Compound 24 also significantly and selectively reduced proliferation of cancer cell lines of gynecological origin. This estrane triazole changed the cell cycle and induced apoptosis of HeLa, SiHa, and MDA-MB-231 cancer cells, measured by both increased subG1 fraction of cells and activation of caspase-independent signaling pathways. A third mode of anti-estrogenic action of 24 saw increased mRNA expression of the SULT1E1 gene in HeLa cells; in contrast, its 3-benzyloxy analog 23 increased mRNA expression of the HSD17B2 gene, thus showing pronounced pro-drug anti-estrogenic activity. Estradiol-derived d-ring triazole compound 24 thus acts at the enzyme, gene expression and cellular levels to decrease the production of active estrogen hormones, demonstrating its pharmacological potential.

Steroidal ferrocenes as potential enzyme inhibitors of the estrogen biosynthesis.

The potential inhibitory effect of diverse triazolyl-ferrocene steroids on key enzymes of the estrogen biosynthesis was investigated. Test compounds were synthesized via copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes using our efficient methodology published previously. Inhibition of human aromatase, steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities was investigated with in vitro radiosubstrate incubations. Some of the test compounds were found to be potent inhibitors of the STS. A compound bearing ferrocenyl side chain on the C-2 displayed a reversible inhibition, whereas C-16 and C-17 derivatives displayed competitive irreversible binding mechanism toward the enzyme. 17α-Triazolyl-ferrocene derivatives of 17ß-estradiol exerted outstanding inhibitory effect and experiments demonstrated a key role of the ferrocenyl moiety in the enhanced binding affinity. Submicromolar IC50 and Ki parameters enroll these compounds to the group of the most effective STS inhibitors published so far. STS inhibitory potential of the steroidal ferrocenes may lead to the development of novel compounds able to suppress in situ biosynthesis of 17ß-estradiol in target tissues.

Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase.

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17ß-azidoandrost-5-en-3ß-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a-k and 9a-k). For the preparation of 5'-iodo-1',2',3'-triazoles (8m-n and 9m-n), an improved method was developed, directly from steroidal azides and terminal alkynes, in reaction mediated by CuI and ICl as iodinating agents. Acetolysis and subsequent hydrolysis of 8n and 9n yielded 5'-hydroxy-1',2',3'-triazoles 8o and 9o. The inhibitory effect of 8a-o, 9a-o, 3, and 5 on rat testicular C17,20-lyase was investigated by means of an in vitro radioincubation technique. The results revealed that the C-17 epimers of steroidal triazoles influence the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series (8a-o), whereas in the C-17 azide pair the 17ß compound (5b) was more potent.

Synthesis of A-ring halogenated 13α-estrone derivatives as potential 17ß-HSD1 inhibitors.

13α-Estrone and its 3-methyl or benzyl ether were halogenated in ring A with N-bromo- or N-iodosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin as electrophile triggers. The chemo- and regioselectivities of the reactions depended greatly on the nature of the substituent on C-3. Bromination of the ethers led to 2- and 4-regioisomers. Bis-halogenation occurred only in the case of the phenolic derivative. Iodination and bromination resulted in similar products, except that the 3-benzyl ether could not be iodinated under the applied conditions. The potential inhibitory action of the new halogenated 13α-estrones on human 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Some compounds proved to be effective inhibitors, with IC50 values in the submicromolar range.