Application research of 16-dehydropregnenolone acetate

Introduction

16-Dehydropregnenolone acetate, also known as 3β-(acetyloxy) pregna-5,16-dien-20-one, is used for chemical synthesis of steroid hormones. It can be a potential raw material for steroid bioconversion. 16-Dehydropregnenolone acetate is produced by chemical degradation of solasodine and diosgenin obtained from plant sources. Banerjeeet al.  reported the microbial conversion of 16-dehydropregnenolone acetate to 17-ketosteroids, by mixed culture of Pseudomonas diminuta MTCC 3361 and Commamonas acidovorans MTCC 3362. Conversion of 16-dehydropregnenolone acetate could be a  potential alternative route for production of 4-androstene-3,17-dione (AD),which is currently dependent mainly on soy sterol bioconversion. Solasodine can be directly converted to AD but lower bioconversion yield is a concern. Moreover, availability of solasodine is limited. Microbial conversions offer a single step route to important steroidal intermediates under mild conditions of temperature and pressure.[1]

Bioconversion of 16-dehydropregnenolone Acetate

Delftia acidovorans MTCC 3363 was found to convert 16-dehydropregnenolone acetate (16-DPA) exclusively to 4-androstene-3, 17-dione (AD). Addition of 9α-hydroxylase inhibitors was not required for preventing the accumulation of byproducts. The effect of pH, temperature, substrate concentration, surfactants and carrier solvents on this bioconversion has been studied. 16-DPA was maximally converted in buffered medium at pH 7.0, at temperature 30°C and 0.5 mg ml-1 substrate concentration. Detergent addition and temperature above 35°C had deleterious effect on bioconversion. Dioxan was found to be the best carrier solvent for biotransformation of 16-dehydropregnenolone acetate to AD.[1]

The reaction of 16-dehydropregnenolone acetate with 2-aminobenzimidazole

Wojtkielewicz et al. commenced with this project by investigation into the reaction of 16-dehydropregnenolone acetate (1) and 2-aminobenzimidazole (2) under various conditions. The reaction may produce two different types of pyrimidobenzimidazoles depending on which nitrogen atom (N₃ or NH₂ group) of 2-aminobenzimidazole attacks first the steroid and which carbon atom (C16 or C20) is attacked.Among two alternative pathways leading to cyclic products shown in Scheme 1, the pathway A seems to be preferred over B, as this course of reaction allows to interact nucleophiles and electrophiles of the same properties with each other (hard ones, the NH₂ group and carbonyl group, and soft ones, the imidazole N3 atom and β-carbon atom of the conjugated system, in pairs). Also previous literature reports confirm this hypothesis.[2]

The initial experiment was carried out with 2 equivalents of 2-aminobenzimidazole in presence of DIPEA as a base in DMF,according to Xue protocol. After heating for 16 h at 140℃ a mixture of two products was obtained, pyrimidobenzimidazole 3 and an unexpected ketone 4, in 51% and 17% yield, respectively(Scheme 2).

As 2-aminobenzimidazole possesses two nucleophilic nitrogen atoms(NH₂ and N₃) the condensation may lead to two isomeric pyrimidines. However, the aromatic product 3 was formed selectively,by imine formation followed by conjugated addition of N3 of benzimidazole to C16 of steroid, cyclization, autoxidation, and aromatization. The major product 3 was accompanied by the D-homoketone 4, produced by the competitive α-ketol type rearrangement of the hydroperoxide intermediate, and the aza-Michael adduct.The choice of solvent and base strongly affected the condensation process. Employing polar aprotic solvents and secondary or tertiary amine pyrimidine 3 was obtained in high yield.[2]

Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives

Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-reductase enzyme. This androgen is more active than T, since it has a higher affinity for the androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases, including prostate cancer, could result. The aim of this study was to synthesize several 16-dehydropregnenolone acetate derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines. The results from this study showed that with the exception of steroids 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-LU-1 cell line. These results also indicated that the triazole derivatives, which have a hydroxyl or acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a alicyclic ester group at C-3 do not show biological activity.[3]

References

[1] Awadhiya P, Banerjee T, Patil S. Bioconversion of 16-dehydropregnenolone Acetate to Exclusively 4-androstene-3,17-dione by Delftia acidovorans MTCC 3363. Pol J Microbiol. 2017;66(3):321-326. doi:10.5604/01.3001.0010.4858

[2] Wojtkielewicz A, Uścinowicz P, Siergiejczyk L, Kiełczewska U, Ratkiewicz A, Morzycki JW. A study on the reaction of 16-dehydropregnenolone acetate with 2-aminobenzimidazole. Steroids. 2017;117:71-76. doi:10.1016/j.steroids.2016.09.003

[3] Silva-Ortiz AV, Bratoeff E, Ramírez-Apan T, et al. Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1. Bioorg Med Chem. 2015;23(24):7535-7542. doi:10.1016/j.bmc.2015.10.047

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