Synthesis and Antiviral Activity of Modified 5α-Steroids.

Hydrazones are a class of organic compounds possessing various biological activities [1, 2].

Steroid oximes, thiosemicarbazones, semicarbazones, and hydrazones have attracted interest because of their high pharmacological activity, e.g., antibacterial, antiviral, antitumor, etc. [3-6].

Transformation into ester derivatives is a common method of structural modification of steroids. Addition of an ester into the steroid core affects the biological activity. The cytotoxicity of several steroid esters synthesized via esterification of a hydroxyl group was shown to be significantly less than that of their precursors [7].

Previously, we reported that several epiandrosterone hydrazones modified by phenylacetic acid chloride exhibited antiviral activity [8].

In continuation of research on the synthesis of new biologically active steroids, several new (2–7) and previously known hydrazones (8 and 9) [9, 10] were prepared from 5α-pregnenolone acetate 1 and tested for antiviral activity. Compound 2 was synthesized from steroid 1; hydrazones 3–6, from ketone 10 via reaction with hydrazides or hydrazines (hydrazides of m-nitrobenzoic, m-bromobenzoic, and salicylic acids and 2,4-dinitrophenyl- and p-nitrophenylhydrazine) in EtOH in the presence of a catalytic amount of AcOH. Oxime 7 was obtained from 3β-(1-adamantoate)-5α-androstan-17-one; this ketone and starting 10, via esterification of epiandrosterone 11 by the literature method [8].

The structures of the synthesized steroids 2–7 were confirmed using IR, 1H NMR, 13C NMR, and mass spectra. IR spectra of 2, 3, 6, and 7 contained absorption bands for ester C=O in the range 1750–1668 cm–1; for NHCO (hydrazones 2 and 3), at 1667 and 1665 cm–1; for stretching vibrations of C=N and aromatic C=C double bonds (steroids 2, 3, and 6), at 1640–1602 and 1562–1560 cm–1, respectively; for hydrazone NH of 6 and OH and C=N of oxime 7, at 3373, 3278, and 1658 cm–1, respectively. IR spectra of nitro derivatives 2 and 6 also had characteristic Ar–NO2 stretching vibrations at 1500 and 1506 and 1374 and 1375 cm–1, respectively.

The 1H NMR spectra of 2–7 exhibited singlets for CH3-18 and CH3-19 methyls at δ 0.95–0.85 and 1.05–0.89 ppm, respectively. The aromatic protons of 2–6 had chemical shifts (CSs) in the range 9.11–6.85 ppm. Singlets for the methylene protons of the phenylacetoxy groups of hydrazones 3–6 were observed at δ 3.51; the resonances of NH groups of steroids 5 and 6, at 10.76 and 9.30; the NHCO protons of 2–4, in the range δ 9.00–8.35 ppm. Multiplets of the 3α protons from the 3β esters of steroids 2–7 showed CSs at δ 4.65–4.59 ppm. A broad singlet from the C=N–OH proton of oxime 7 appeared at δ 7.43 ppm. Resonances of other protons agreed with the proposed structures. 13C NMR spectra of hydrazones 4 and 5 had C-3 resonances at δ 74.0 ppm; aromatic C atoms, in the range δ 154.9–116.4; of C=N bonds, δ 171.2–161.9; of O–C=O groups, at δ 171.2 and 172.3 ppm, respectively. The resonance of the NH–CO C atom of 4 had a CS of δ 158.8 ppm.

Mass spectra confirmed the empirical formulas of 2–7.

Screening for specific antiviral activity of 6 was performed by the National Institute of Allergy and Infectious Diseases at the University of Utah (USA) using the following virus strains: Polio virus (Vero 76 cell culture, strain Type 3, WM-3); SARS-corona virus (Vero 76 cell culture, strain Urbani); Rift Valley fever virus (Vero 76 cell culture, strain MP-12); Tacaribe virus (Vero cell culture, strain TRVL-11573); Venezuelan equine encephalitis virus, Respiratory syncytial virus, Influenza A virus H1N1, Dengue virus (Vero cell culture, MA-104, MDCK; Vero 76; strains TC-83, A-2, California 07/2009, Type 2, New Guinea C, respectively); and Cytomegalovirus (strains Davis and AD-169) and varicella-zoster virus (strains OKA and 07-1); of compounds 2–5 and 7–9, at Rega Institute for Medical Research, Belgium. As it turned out, only hydrazone 6 exhibited moderate antiviral activity against Polio virus. All other compounds did not possess any significant activity against these strains.

Steroids 2–6 and 8 were prepared by the literature method [11]; oxime 7, as before [8]; androst-4-en-3,17-dione bis-thiosemicarbazone 9, by the literature method [10].

3β-Acetoxy-5α-pregn-16-en-20-one -Nitrobenzoylhydrazone (2). Yield 72%, mp 186–188°C. IR (KBr, ν, cm–1): 3425 (NH), 1750 (C=O), 1667 (NH-CO), 1640 (C=N), 1561 (arom. ring), 1500 and 1374 (Ar-NO2). 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.89 (3H, s, CH3-18), 1.05 (3H, s, CH3-19), 1.96 (3H, s, CH3-21), 2.07 (3H, s, OCOCH3), 4.59 (1H, m, H-3), 6.19 (1H, s, H-16), 7.61 (1H, t, J = 8.2, H-5′), 8.10 (1H, d, J = 7.1, H-6′), 8.35 (1H, d, J = 7.3, H-4′), 8.64 (1H, s, H-2′), 9.00 (1H, br.s, NHCO). LC-MS, m/z 522 [M + H]+. C30H39N3O5. MW 521.

3β-Phenylacetoxy-5α-androstan-17-one -Bromobenzoylhydrazone (3). Yield 81%, mp 159–161°C. IR (KBr, ν, cm–1): 3412 (NH), 1729 (C=O), 1665 (NH-CO), 1602 (C=N), 1562 (arom. ring). 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.85 (3H, s, CH3-18), 0.95 (3H, s, CH3-19), 2.18–2.25 (2H, m, H-16), 3.51 (2H, s, CH2C6H5), 4.61 (1H, m, H-3), 7.24–7.34 (5H, m, C6H5), 7.52–8.25 (4H, ArH), 8.35 (1H, br.s, NHCO). LC-MS, m/z 606 [M + H]+. C34H41BrN2O3. MW 605.

3β-Phenylacetoxy-5α-androstan-17-one Salicyloylhydrazone (4). Yield 67%, mp 254–256°C. 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.95 (3H, s, CH3-18), 0.98 (3H, s, CH3-19), 2.40–2.51 (2H, m, H-16), 3.51 (2H, s, CH2C6H5), 4.61 (1H, m, H-3), 6.85–7.41 (9H, ArH), 8.65 (1H, br.s, NH-CO). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.2, 16.9, 20.7, 23.4, 25.2, 27.4, 28.3, 31.4, 33.8, 33.9, 34.9, 35.7, 36.7, 41.8, 44.6, 45.3, 53.3, 54.4, 74.0 (C-3), 117.2 (C-1′′), 117.6 (C-3′′), 118.6 (C-5′′), 125.0 (C-6′′), 126.9 (C-4′), 128.5 (C-3′, 5′), 129.2 (C-2′, 6′), 134.3 (C-4′′), 134.5 (C-1′), 154.9 (C-2′′), 158.8 (NHCO), 161.9 (C=N), 171.2 (O–C=O). LC-MS, m/z 543 [M + H]+. C34H42N2O4. MW 542.

3β-Phenylacetoxy-5α-androstan-17-one 2,4-Dinitrophenylhydrazone (5). Yield 76%, mp 198–200°C. 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.87 (3H, s, CH3-18), 0.95 (3H, s, CH3-19), 2.50–2.65 (2H, m, H-16), 3.51 (2H, s, CH2C6H5), 4.61 (1H, m, H-3), 7.26–7.34 (5H, m, C6H5), 7.93 (1H, d, J = 9.5, ArH), 8.28 (1H, dd, J = 9.5, 2.5, ArH), 9.11 (1H, d, J = 2.5, ArH), 10.76 (1H, s, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 12.3, 17.2, 20.7, 23.5, 26.4, 27.4, 28.3, 31.4, 33.9, 34.1, 35.0, 35.7, 36.7, 41.8, 44.7, 45.3, 53.5, 54.4, 74.0 (C-3), 116.4 (C-6′′), 123.6 (C-3′′), 126.9 (C-4′), 128.5 (C-3′, 5′), 128.8 (C-2′′), 129.2 (C-2′, 6′), 129.9 (C-5′′), 134.3 (C-1′), 137.5 (C-4′′), 145.4 (C-1′′), 171.2 (C=N), 172.3 (O-C=O). LC-MS, m/z 589 [M + H]+. C33H40N4O6. MW 588.

3β-Phenylacetoxy-5α-androstan-17-one -Nitrophenylhydrazone (6). Yield 83%, mp 221–223°C. IR (KBr, ν, cm–1): 3373 (NH), 1668 (C=O), 1615 (C=N), 1560 (arom. ring), 1506 and 1375 (Ar-NO2). 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.85 (3H, s, CH3-18), 0.89 (3H, s, CH3-19), 2.38–2.50 (2H, m, H-16), 3.51 (2H, s, CH2C6H5), 4.61 (1H, m, H-3), 7.10 (2H, d, J = 9.0, ArH), 7.18–7.28 (5H, m, C6H5), 8.00 (2H, d, J = 9.1, ArH), 9.30 (1H, s, NH), LC-MS, m/z 544 [M + H]+. C33H41N3O4. MW 543.

17-Hydroximino-3β-(1-adamantoate)-5α-androstane (7). Yield 87%, mp 245–247°C. IR (KBr, ν, cm–1): 3278 (OH), 2924, 2851 (CH-Ad), 1731 (C=O), 1658 (C=N). 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.87 (3H, s, CH3-18), 0.89 (3H, s, CH3-19), 1.74 (3H, s, CH-Ad), 1.88 (10H, s, CH2-Ad), 1.99 (2H, s, CH2-Ad), 2.40–2.55 (2H, m, H-16), 4.65 (1H, m, H-3), 7.43 (1H, br.s, C=N-OH). LC-MS, m/z 468 [M + H]+. C30H45NO3. MW 467.