Synthesis of D‑Ring Annulated Pyridosteroids from β‑Formyl Enamides and Their Biological Evaluations Geetmani Singh Nongthombam, † Kasmika Borah, ‡ Thingreila Muinao, ‡ Yumnam Silla, ‡ Mintu Pal, ‡ Hari Prasanna Deka Boruah, ‡ and Romesh Chandra Boruah* ,† † Chemical Science and Technology Division, CSIR-North East Institute of Science & Technology, Jorhat, Assam, India 785006 ‡ Biological Science and Technology Division, CSIR-North East Institute of Science & Technology, Jorhat, Assam, India 785006 * S Supporting Information ABSTRACT: Herein, we report the synthesis of a novel class of substituted androst[17,16-b]pyridines (pyridosteroids) from the reaction of β-formyl enamides with alkynes in high yields. The optimized reaction protocol was extended to acyclic and cyclic β-formyl enamides to afford nonsteroidal pyridines. Cell survival assay of all compounds were carried against prostate cancer PC-3 cells wherein 3-hydroxy-5-en- 2′,3′-dicarbethoxy-androst[17,16-b]pyridine showed the high- est cytotoxic activity. Phase contrast microscopy and flow cytometry studies exhibited marked morphological features characteristic of apoptosis in 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine and abiraterone treated PC-3 cells. The treatment of 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine induces G 2 /M phase cell cycle arrest in prostate cancer PC-3 cells. Enhancement of apoptotic inductions of PC-3 cells by 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16- b]pyridine and abiraterone through the activation of caspases-6, -7, and -8 pathways were supported by qRT-PCR. In silico study of the compound 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine showed stable and promising interaction with the key caspase proteins. Our studies revealed that the pyridosteroid 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16- b]pyridine, bearing pyridine-2,3-dicarbethoxy pharmacophore, facilitated initiation of caspase-8 and activates downstream effectors caspase-6 and caspase-7 and thereby triggering apoptosis of PC-3 cancer cells. KEYWORDS: β-formyl enamide; azasteroid, pyridine, alkyne, SRB assay; molecular dynamic simulation; qRT-PCR, apoptosis ■ INTRODUCTION The pyridine substructure is one of the most ubiquitous heterocyclic motifs in organic and bioorganic chemistry because of its widespread distribution in medicinally important natural products, pharmaceuticals, and bioactive molecules. 1-4 A great deal of attention has been directed toward development of new synthetic strategies for these biologically important substituted pyridines. 5-8 Notable examples for substituted pyridine syn- thesis involve cycloadditions, 9 cycloisomerization, 10 ketoxime ester coupling with alkenylboronic acids, 11 triflic anhydride/2- chloropyridine, 12 alkyne coupling with unsaturated imines, 13 multicomponent reaction, 14 transition-metal-catalyzed reac- tions of substituted ketoxime, 15 and Vilsmeier-Haack-based cyclocondensation reaction of conjugated ketoxime. 16 Enamides constitute the building block of many biologically active compounds and attract considerable attention as prochiral substrates in asymmetric synthesis of amino acids. 17,18 Few metal-catalyzed coupling reactions of enamides with alkynes 19 have been reported for synthesis of pyridine through amide activation reaction. 20 Enamides are also utilized for the preparation of pyridosteroids by the reaction of Vilsmeier reagent. 21 Most of the reactions involve the electron deficient carbonyl of N-acetyl group via intermolecular cycloaddition reactions. 22 On the other hand, β-formyl enamides have emerged as interesting synthons for their potential diversity in heterocyclic synthesis. 23,24 The research effort at our laboratory afforded β- formyl enamide, which was further utilized as an important reagent for one-pot preparation of pyridines. 25 Despite the cross coupling reaction of enamide with alkyne via amide activation for synthesis of substituted pyridines as reported, 26 the scope of β-formyl enamide for such an approach is yet to be explored. Anticancer drug discovery has emerged as a major clinical challenge primarily due to the existence of multidrug resistance, poor absorption of compounds, and several side effects. 27 Among the established drugs, glucocorticoids have shown considerable clinical significance in targeting various cancer cells. 28 Continued efforts have been made for the development of steroid-based novel therapeutic agents for prostate cancer. 29-33 The steroidal heterocycles received particular interests because of their important biological significance. 34 Received: September 17, 2018 Revised: December 19, 2018 Published: December 21, 2018 Research Article pubs.acs.org/acscombsci Cite This: ACS Comb. Sci. 2019, 21, 11-27 © 2018 American Chemical Society 11 DOI: 10.1021/acscombsci.8b00140 ACS Comb. Sci. 2019, 21, 11-27 Downloaded via CSIR-NORTH EAST INST SCI & TECHLGY on January 16, 2019 at 07:05:18 (UTC). 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