Dysfunction of the androgen receptor (AR) signalling axis plays a pivotal role in the development and progression of prostate cancer (PCa). Steroidal and non-steroidal AR antagonists can significantly improve the survival of PCa patients by blocking the action of the endogenous ligand through binding to the hormone receptor and preventing its activation. Herein, we report two synthetic strategies, each utilizing the advantages of microwave irradiation, to modify the A-ring of natural androgen 5α-dihydrotestosterone (DHT) with pyridine scaffolds. Treatment of DHT with appropriate Mannich salts led to 1,5-diketones, which were then converted with hydroxylamine to A-ring-fused 6'-substituted pyridines. To extend the compound library with 4',6'-disubstituted analogues, 2-arylidene derivatives of DHT were subjected to ring closure reactions according to the Kröhnke's pyridine synthesis. The crystal structure of a monosubstituted pyridine product was determined by single crystal X-ray diffraction. AR transcriptional activity in a reporter cell line was investigated for all novel A-ring-fused pyridines and a number of previously synthesized DHT-based quinolines were included to the biological study to obtain information about the structure-activity relationship. It was shown that several A-ring-fused quinolines acted as AR antagonists, in comparison with the dual or agonist character of the majority of A-ring-fused pyridines. Derivative 1d (A-ring-fused 6'-methoxyquinoline) was studied in detail and showed to be a low-micromolar AR antagonist (IC50 = 10.5 µM), and it suppressed the viability and proliferation of AR-positive PCa cell lines. Moreover, the candidate compound blocked the AR downstream signalling, induced moderate cell-cycle arrest and showed to bind recombinant AR and to target AR in cells. The binding mode and crucial interactions were described using molecular modelling.

• Klíčová slova
• Androgen receptor, Crystal structure, Dihydrotestosterone, Pyridines, Transcriptional activity,
• MeSH
• androgenní receptory metabolismus   MeSH
• antagonisté androgenních receptorů farmakologie   MeSH
• dihydrotestosteron * farmakologie   metabolismus   MeSH
• lidé MeSH
• mikrovlny MeSH
• nádorové buněčné linie MeSH
• nádory prostaty * farmakoterapie   metabolismus   MeSH
• pyridiny farmakologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• androgenní receptory MeSH
• antagonisté androgenních receptorů MeSH
• dihydrotestosteron * MeSH
• pyridiny MeSH

High expression of the androgen receptor (AR) and the disruption of its regulation are strongly responsible for the development of prostate cancer (PCa). Therapeutically relevant non-steroidal or steroidal antiandrogens are able to block the AR effect by eliminating AR-mediated signalling. Herein we report the synthesis of novel steroidal pyrazoles derived from the natural sex hormone 5α-dihydrotestosterone (DHT). 2-Ethylidene or 2-(hetero)arylidene derivatives of DHT obtained by regioselective Claisen-Schmidt condensation with acetaldehyde or (hetero)aromatic aldehydes in alkaline ethanol were reacted with monosubstituted hydrazines to give A-ring-fused 1,5-disubstituted pyrazoles as main or exclusive products, depending on the reaction conditions applied. Spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the primarily formed pyrazolines resulted in the desired products in moderate to good yields, while 17-oxidation also occurred by using the Jones reagent as a strong oxidant. Transcriptional activity of the AR in a reporter cell line was examined for all novel compounds, and several previously synthesized similar DHT-based pyrazoles with differently substituted heteroring were also included to obtain information about the structure-activity relationship. Two specific regioisomeric groups of derivatives significantly diminished the transcriptional activity of the AR in reporter cell line in 10 μM concentration, and displayed reasonable antiproliferative activity in AR-positive PCa cell lines. Lead compound (3d) was found to be a potent AR antagonist (IC50 = 1.18 μM), it generally suppressed AR signalling in time and dose dependent manner, moreover, it also led to a sharp decrease in wt-AR protein level probably caused by proteasomal degradation. We confirmed the antiproliferative activity of 3d in AR-positive PCa cell lines (with GI50 in low micromolar ranges), and its cellular, biochemical and in silico binding in AR ligand-binding domain. Moreover, compound 3d was shown to be potent even ex vivo in patient-derived tissues, which highlights the therapeutic potential of A-ring-fused pyrazoles.

• Klíčová slova
• A-ring-fused pyrazoles, Androgen receptor, Prostate cancer, Protein degradation, Structure-activity relationship, Transcriptional activity,
• MeSH
• androgenní receptory metabolismus   MeSH
• dihydrotestosteron * farmakologie   metabolismus   MeSH
• down regulace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty * farmakoterapie   metabolismus   MeSH
• pyrazoly MeSH
• steroidy terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• androgenní receptory MeSH
• dihydrotestosteron * MeSH
• pyrazoly MeSH
• steroidy MeSH

One of the main directions of steroid research is the preparation of modified derivatives in which, in addition to changes in physicochemical properties, receptor binding is significantly altered, thus a bioactivity different from that of the parent compound predominates. In the frame of this work, 2-arylidene derivatives were first synthesized by regioselective modification of the A-ring of natural sex hormone, 5α-dihydrotestosterone (DHT). After Claisen-Schmidt condensations of DHT with (hetero)aromatic aldehydes in alkaline EtOH, heterocyclizations of the α,β-enones were performed with 3-amino-1,2,4-triazole, 3-aminopyrazole and 3-amino-5-methylpyrazole in the presence of t-BuOK in DMF to afford 7'-epimeric mixtures of A-ring-fused azolo-dihydropyrimidines, respectively. Depending on the electronic demand of the substituents of the arylidene moiety, spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the heteroring led to triazolo[1,5-a]pyrimidines and pyrazolo[1,5-a]pyrimidines in good yields, while, using the Jones reagent as a strong oxidant, 17-oxidation also occurred. The crystal structures of an arylidene and a triazolopyrimidine product have been determined by single crystal X-ray diffraction and both were found to crystallize in the monoclinic crystal system at P21 space group. Most derivatives were found to diminish the transcriptional activity of androgen receptor (AR) in reporter cell line. The candidate compound (17β-hydroxy-2-(4-chloro)benzylidene-5α-androstan-3-one, 2f) showed to suppress androgen-mediated AR transactivation in a dose-dependent manner. We confirmed the cellular interaction of 2f with AR, described the binding in AR-binding cavity by the flexible docking and showed the ability of the compound to suppress the expression of AR-regulated genes in two prostate cancer cell lines.

• Klíčová slova
• 2-arylidene-dihydrotestosterone, Androgen receptor, Azolo[1,5-a]pyrimidines, Crystal structures, Flexible docking, Heterocyclization, Transcriptional activity,
• MeSH
• androgenní receptory chemie   metabolismus   MeSH
• antagonisté androgenních receptorů chemická syntéza   farmakologie   MeSH
• dihydrotestosteron chemie   MeSH
• lidé MeSH
• molekulární konformace MeSH
• nádorové buňky kultivované MeSH
• nádory prostaty farmakoterapie   metabolismus   patologie   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• androgenní receptory MeSH
• antagonisté androgenních receptorů MeSH
• AR protein, human MeSH Prohlížeč
• dihydrotestosteron MeSH