N
3
N
3
O
HO
TsO
AcO
AcO
H
H
H
H
15
16
17
14
Herein we report results from biological tests of the following synthesized steroid derivatives: hydrazones of androst-
1,4-dien-3,17-dione (1, 2), 3β-azido-5α-androstan-17-one (3), 3α-hydroxy-5α-pregnan-20-one (4, 5), 3α-hydroxy-5α-pregn-
9(11),16-dien-20-one (6), 3α-hydroxy-5α-androst-9(11)-en-17-one (7), and 3β-hydroxy-5α-pregn-16-en-20-one (8) [4] in
addition to a ketosteroid (9), an amine (10) and azides (11-14).
The antituberculosis activity of 1-14 was studied in a series of dilutions in Middlebrook 7H9 medium using
M. tuberculosis H37Rv as the test culture. Isonicotinoylhydrazones 3 and 4, thiosemicarbazone 6, azides 11 and 12, and
aminosteroid 10 exhibited high activity toward M. tuberculosis H37Rv in in vitro tests at all studied concentrations (5, 10, 25,
50, and 100 μg/mL). Compounds 5 and 9, 7, and 8 and 13 were active at concentrations of 10, 25, and 50 μg/mL, respectively.
Compound 14 exhibited activity only at a concentration of 100 μg/mL whereas hydrazones 1 and 2 were inactive against
M. tuberculosis H37Rv even at 100 μg/mL. The antituberculosis activity of steroidal azides has not been reported. This active
class of compounds is reported for the first time. The biological tests provide a good impetus for more extensive study of the
aforementioned steroidal derivatives and continuation of the search for new highly effective compounds in this series.
EXPERIMENTAL
Melting points were determined on a Gallenkamp block. IR spectra in KBr disks were recorded on a Magna-IR
1
Spectrometer 550. NMR spectra were obtained on a Bruker AC 500 instrument (operating frequency 500 MHz for H).
Chemical shifts of protons are given on the δ scale with TMS internal standard and CDCl solvent. Mass spectra were recorded
3
in a MAT-112 GC—MS (ionizing electron energy 70 eV, ionization chamber temperature 180°C, direct sample introduction
into the source). Elemental analyses was performed on a Perkin—Elmer CHN 2004 instrument and agreed with those
calculated. The course of reactions and purity of products were monitored by TLC on plates (60GF-254, Merck) using
benzene:acetone (15:1). Spots were developed by spraying with phosphomolybdic acid solution (10%) in ethanol with
subsequent heating.
17α-Azido-5α-androstan-3β-ol (17). A mixture of 17α-azido-3β-acetoxy-5α-androstane (16, 5 g, 13.9 mmol) and
KOH (0.7 g, 17.5 mmol) in methanol (40 mL) was refluxed for 10 min, cooled, and poured into water (100 mL). The product
(4.3 g) was separated and crystallized from benzene:hexane (1:4) to afford 17 (4.2 g, 96%), mp 136-138°C. IR spectrum (KBr,
ν, cm−1): 3300-3500 (OH), 2105 (N ). PMR spectrum (500 MHz, CDCl , δ, ppm, J/Hz): 0.78 (3H, s, CH -18), 0.85 (3H, s,
3
3
3
+
CH -19), 3.53 (1H, d, J = 6.5, H-17β), 3.59 (1H, m, H-3α). Mass spectrum (EI, 70 eV, m/z, I, %): 289 (100) [M - N ] .
3
2
17α-Azido-3β-(4-methylsulfonyloxy)-5α-androstane (14). A solution of 17 (3 g, 9.44 mmol) in freshly distilled
pyridine (30 mL) at 0°C was treated with p-toluenesulfonylchloride (4.45 g, 23.35 mmol), held at 20°C for 24 h, poured into
icewater (200 mL), and filtered. The product (4.13 g) was separated and crystallized from benzene:hexane (1:2) to afford 14
(4.09 g, 92%), mp 153-155°C. IR spectrum (KBr, ν, cm−1): 2105 (N ). PMR spectrum (500 MHz, CDCl , δ, ppm, J/Hz): 0.71
3
3
(3H, s, CH -18), 0.77 (3H, s, CH -19), 2.44 (3H, s, CH -Ar), 3.47 (1H, d, J = 6.5, H-17β), 4.41 (1H, m, H-3α), 7.32 (1H, d,
3
3
3
+
J = 8.5, H-Ar), 7.78 (1H, d, J = 8.5, H-Ar). Mass spectrum (EI, 70 eV, m/z, I, %): 471 (100) [M] .
The antituberculosis activitywas determined toward M. tuberculosis H37Rv. Middlebrook 7H9 medium was prepared
in bottlesandsterilizedbyautoclaving. Growth additiveADC (argininedecarboxylase) containing fraction V ofbovine albumin,
dextrose, and catalase was added to each bottle under sterile conditions. A sterile suspension of each tested compound
(10 mg/mL) was prepared in the appropriate solvent. Each compound was tested at concentrations of 5-100 μg/mL that was
5
added to the bottles with Middlebrook 7H9 medium and a suspension of M. tuberculosis culture (10 cells/mL). The medium
was stored for at least four weeks at 37°C in a moist chamber. The reference preparations were ciprofloxacin (5 μg/mL),
streptomycin (7.5 μg/mL), or pyrazinamide (7.5 μg/mL). Medium and growth additive were used as separate controls.
619
Merlani
