T.G. Lobastova et al. / Steroids 74 (2009) 233–237
235
affording the analytically pure dibenzoate IV as white long needles.
s, t-BuSi + C(18)H3), 1.04 (3H, s, C(19)H3), 3.58 (1H, tdd, J = 10.5, 4.6
and 6.0 Hz, H(3␣)), 4.57 (1H, ddd, J = 1.6, 3.5 and 5.3 Hz, H(7)), 5.63
(1H, dd, J = 1.4 and 5.3 Hz, H(6)); MS m/z (%) 421 (0.6, M-Me+), 400
(5, M-HCl+), 379/381 (53/19, M-Bu+), 343 (33, M-Bu–HCl+), 269
(60, M-HCl–BMSO+), 227 (38, 269-CH2CO+), 75 (100, Me2SiOH+).
Yield: 229 mg (84% from 7␣-OH-DHEA), mp 196 ◦C, [␣]D −85.5◦
27
(ca. 0.33, EtOH); IR (KBr) 1744 (C O), 1716 (O CAr), 1600, 1584 (Ph),
1272 (C–O), 712 (Ph); 1H NMR (CDCl3) ı 0.93 (3H, s, C(18)H3), 1.15
(3H, s, C(19)H3), 4.90 (1H, m, H(3␣)), 5.39 (1H, t, J = 4.5 Hz, H(7)),
5.82 (1H, d, J = 5.0 Hz, H(5)), 7.42 and 7.46 (each 2H, t, J = 7.4 Hz, m-
HBz), 7.55 and 7.58 (each 1H, t, J = 7.4 Hz, p-HBz), 8.00 and 8.02 (each
2H, d, J = 7.1 Hz, o-HBz); MS m/z (%) 390 (7, M-BzOH+), 268 (3, M-
2BzOH+), 253 (1, M-2BzOH–Me+), 105 (100, PhCO+), 77 (24, Ph+).
C25H41ClO2Si: calcd. C, 68.69; H, 9.45; Cl, 8.11; Si, 6.42%. Found: C,
68.79; H, 9.66; Cl, 8.02; Si, 6.61%. The crude product was stable dur-
ing storage in a refrigerator for 3–5 weeks, as a solid or as a solution
in hexane, CDCl3 or CH2Cl2. It rapidly reacted with methanol and
ethanol with HCl elimination. Analogously, the chloride VI spotted
on the start line of silicagel TLC plate easily converted into the cor-
responding alcohol V. Thus, for the reliable TLC analysis, the plate
was developed immediately after drying of the spotted solution of
chloride VI.
C33H36O5: calcd. C, 77.32; H, 7.08%. Found: C, 77.25, 76.97; H, 7.54,
7.37%.
2.5.3. d-Androsta-2,4,6-trien-17-one (III)
Solution of dibenzoate IV (15 mg) in freshly distilled PhNMe2
(0.37 mL, bp 194 ◦C) was gently refluxed for 8 h. TLC analysis (PhMe,
triple development) showed ∼50% conversion of starting IV (Rf
0.07) into a product with Rf 0.26 (triene III), and negligible traces
of a product with intermediate polarity (Rf 0.19) (analogous 3,7-
dibenzoates fully converted for 7.5–8 h). The dark reaction mixture
was poured into 2.5 M HCl (3 mL), extracted with Et2O, and the
extract was dried and evaporated to dryness. Chromatography (sil-
ica gel, Et2O) of the residual yellow oil (12 mg) isolated triene III
as colorless crystals. Yield: 4 mg, (50% from IV); mp 120–125 ◦C; UV
(EtOH) ꢁmax 296, 307, 320 nm (peak height ratio 85:100:65, respec-
tively) [13]; 1H NMR (CDCl3) ı 0.93 (3H, s, C(18)H3), 0.97 (3H, s,
C(19)H3), 5.63–5.76 (3H, m, H(3 + 4 + 6)), 5.94 (1H, ddd, J = 3.9, 5.2
and 9.5 Hz, H(2)), 6.05 (1H, dd, J = 2.5, 9.9 Hz, H(7)).
2.8. d-3ˇ-Hydroxyandrosta-5,7-dien-17-one (7-dehydro-DHEA)
An aliquot (3.4 mL) of 1 M THF solution of n-Bu4NF·3H2O was
concentrated in vacuum at 80 ◦C/0.5 Torr until constant weight was
reached (1–2 h). The residue, a light yellow crystalline mass of
the corresponding monohydrate (950 mg, 3.4 mmol), was dissolved
in absolute THF (4 mL) and mixed with the solution of chloride
VI (350 mg, 0.80 mol) in THF (4.5 mL). The mixture was kept
closed for 2 h at 25 ◦C, and then for 20 h at +5 ◦C before diluting
it with t-BuOMe (100 mL). The resultant solution was washed with
H2O (2× 50 mL), dried and evaporated to dryness during 1 min at
100 ◦C/3 Torr. The residue constituted of the analytically pure 7-
dehydro-DHEA, a white crystalline powder, with a yield of: 210 mg
(91.6% from VI), mp 156–160 ◦C; Rf 0.37 (CH2Cl2–Et2O, 19:1, dou-
ble development); UV (EtOH) ꢁmax 272, 282, 293.5 nm (peak height
ratio 96:100:55, respectively) [7]; 1H NMR (CDCl3) ı 0.82 (3H, s,
C(18)H3), 0.97 (3H, s, C(19)H3), 3.67 (1H, m, H(3␣)), 5.62 (2H, m,
H(6) + H(7)); MS m/z (%) 286 (62, M+), 271 (1.7, M-Me+), 268 (1.3,
M-H2O+), 253 (66, M-Me–H2O+), 227 (66), 211 (12), 197 (12), 185
(14), 183 (16), 171 (22), 169 (17),159 (17), 157 (29), 155 (20), 143
(100), 128 (40), 119 (30), 115 (31), 105 (42), 91 (67), 77 (46), 55 (77).
2.6.
d-3ˇ-tert-Butyldimethylsilyloxy-7˛-hydroxyandrost-5-en-17-one
(V)
A clear solution of 7␣-OH-DHEA (3.04 g, 10.0 mmol) and t-
BuMe2SiCl (2.26 g, 15 mmol) in anhydrous pyridine (4.8 l, 60 mmol)
and CH2Cl2 (9 mL) was kept closed for 24 h at 25 ◦C. Water and
CH2Cl2 were added, and the organic layer was dried and evaporated
to dryness. The white crystalline residue afforded the analytically
pure silyl ether V as thin needles (3.50 g, 95% from 7␣-OH-DHEA)
after recrystallization from toluene (100 mL), mp 163-165 ◦C, [␣]D
3. Results and discussion
24
−46.0◦ (c 0.90, EtOH), Rf 0.54 (Et2O); IR (KBr) 3460 (OH), 1740
(C O), 1252 (SiMe2), 1096 (Si–O), 840 (SiMe2); 1H NMR (CDCl3)
ı 0.06 (6H, s, Me2Si), 0.88 (3H, s, C(18)H3), 0.89 (9H, s, t-BuSi), 1.01
(3H, s, C(19)H3), 3.53 (1H, tt, J = 5.2 and 10.4 Hz, H(3␣)), 3.97 (1H,
br.q, J = 5.4 Hz, H(7)), 5.61 (1H, dd, J = 1.3 and 5.2 Hz, H(6)); MS m/z
(%) 403 (1.5, M-Me+), 361 (33, M-Bu+), 343 (8, M-Bu–H2O+), 269
(23, M-H2O–BMSO+), 227 (26, 269-CH2CO+), 75 (100, Me2SiOH+).
C25H42O3Si: calcd. C, 71.72; H, 10.11; Si, 6.71%. Found: C, 71.53, 71.76;
H, 10.54, 10.25; Si, 6.40%.
3.1. Bioconversion of DHEA to 7˛-OH-DHEA by G. zeae VKM
F-2600
Recently, we screened the ability of 481 fungal strains to carry
out hydroxylation of DHEA. A few of the most active strains, includ-
ing G. zeae VKM F-2600, were capable of introducing the hydroxyl
hydroxylation in position 15␣, thus forming 7␣,15␣-diOH-DHEA as
a by-product.
2.7.
d-3ˇ-tert-Butyldimethylsilyloxy-7˛-chloroandrost-5-en-17-one
(VI)
The mode of formation of an aqueous suspension of DHEA
affected its conversion by G. zeae VKM F-2600 (Fig. 1). When DHEA
was added as an alcohol or a DMF solution, 7␣-OH-DHEA formation
increased as compared with the control, in which DHEA was added
as a powder. A highest 7␣-OH-DHEA level (2.75 g/L) was reached at
the use of 4% DMF (v/v). The accumulation of 7␣,15␣-diOH-DHEA
decreased in the presence of DMF and did not exceed 0.8 g/L, thus
evidencing the DMF-mediated suppression of 15␣-hydroxylation
of 7␣-OH-DHEA.
The maximum level of 7␣-OH-DHEA (3 g/L) was achieved at
28 h with the medium supplement with mCD (Fig. 1). At the same
time, the double hydroxylation seemed to be suppressed by mCD:
the yield of 7␣,15␣-diOH-DHEA did not exceed 15% (0.6–0.7 g/L)
while it reached 1–1.5 g/L in the absence of mCD. The effect can be
attributed to the solubilizing action of mCD towards the hydropho-
bic substrate. Besides, the higher product accumulation can be
Solution of the silyl ether V (400 mg, 0.96 mmol) in CH2Cl2
(8 mL) was mixed at 25 ◦C with a solution of freshly distilled SOCl2
(0.14 mL, 1.9 mmol) and pyridine (0.77 mL, 9.6 mmol) in CH2Cl2
(4 mL), and stirred for 30 min. Then, the mixture was diluted with
CH2Cl2, rapidly filtered with a suction through a small column with
Al2O3 (10 g) and washed out with additional CH2Cl2. The combined
filtrate and washings were evaporated until dry, the residue was
suspended in hexane (60 mL), filtered through a small plug of cot-
ton wool and evaporated again. The residue was crude chloride VI
recovered as long white needles. Yield: 390 mg (93% from V). This
crude material was used for the next step. An analytical sample was
prepared by recrystallization from 1 mL of hexane: mp 156–158 ◦C,
Rf 0.68 (Et2O); 1H NMR (CDCl3) ı 0.07 (6H, s, Me2Si), 0.89 (12H,