The synthesis of 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol – Proposed long term metabolite (M4) of oralturinabol

Article abstract of DOI:10.1016/j.steroids.2020.108601

4-Chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol is one of proposed long term metabolites of oralturinabol (anabolic androgenic steroid restricted in sport). The synthesis of 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol was achieved. Isomerisation of configuration of 13-carbon was used for construction of 17β-hydroxymethyl-17α-methyl fragment. The proposed route of synthesis allows to obtain 3β-hydroxy isomer as well.

Full text of DOI:10.1016/j.steroids.2020.108601

Steroids 158 (2020) 108601
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
The synthesis of 4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-
4
,13-diene-3α-ol – Proposed long term metabolite (M4) of oralturinabol
⁎
D. Yu. Shostko, A.I. Liubina, Yu.Yu. Kozyrkov , S.A. Beliaev
National Anti-Doping Laboratory, Lesnoy 31, 223040, Belarus
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dehydrochloromethyltestosterone
Oral turinabol
Long-term metabolite
Total synthesis
4-Chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol is one of proposed long term meta-
bolites of oralturinabol (anabolic androgenic steroid restricted in sport). The synthesis of 4-chloro-17β-hydro-
xymethyl-17α-methyl-18-norandrosta-4,13-diene-3α-ol was achieved. Isomerisation of configuration of 13-
carbon was used for construction of 17β-hydroxymethyl-17α-methyl fragment. The proposed route of synthesis
allows to obtain 3β-hydroxy isomer as well.
1. Introduction
sample is very low [11], thus the way to prove an assigned structure by
synthesis is more preferred [8]. Synthesis of these substances have not
The search for new long-term metabolites of the substances pro-
been reported yet. The aim of the present study was to synthesise and
hibited in sports is one of the fields of anti-doping research [1,2]. To-
gether with a growing sensitivity of analytical instrumentation, it al-
lows to expand a detection window and increase chances of revealing
cases of illicit usage of performance-enhancing drugs [3]. For example,
finding of metabolites containing 17β-hydroxymethyl-17α-methyl
fragment [4] of 17-methylated androgenic-anabolic steroids (AAS) al-
lowed to expand significantly the detection window of such drugs as
metandienone [5], oxandrolone [6], and especially oralturinabol [7]
characterise the structure of proposed for DHCMT long-term metabolite
M4.
2. Experimental
2.1. General information
Commercially available reagents were obtained from Aldrich, Acros
Organics, Fluka or Merck. Anhydrous tetrahydrofuran was dried by
(
dehydrochloromethyltestosterone, DHCMT) 1. In the latter case, it led
to an increase in the number of adverse analytical finding (AAF) more
than 10 times in 2013 [4]. At the same time, discovering of new me-
tabolites makes it necessary to characterise them [8]. Structure as-
signment usually performed on the basis of previous knowledge of
metabolism and MS-spectrometry data [7]. However, an assigned
structure can be inexact and should be proved by compound isolation
and characterization or by comparison with synthesized reference
material [9]. For instance, recent synthesis of the DHCMT metabolite
M3 [10] 2 (Fig. 1), which was discovered by Sobolevsky in 2012 [7],
and its comparison [9] with reference urine showed the difference in
the C-4 and C-5 stereocenters.
The M3 metabolite is used [4] for anti-doping analysis and has the
detection window more than 40 days [7]. The other new DHCMT me-
tabolites M2 4 and M4 3 containing 17β-hydroxymethyl-17α-methyl
fragment are left without attention and their proposed structures are
not proven, although they are at least as valuable as previously known
long-term metabolites [7]. The content of metabolites in a biological
refluxing over LiAlH
on a Bruker AVANCE 500 MHz spectrometer using TMS as an internal
4
and freshly distilled. NMR spectra were obtained
−1
standard. IR spectra (4000–400 cm ) were recorded as KBr pellets on
a Nicolet iS10 IR spectrometer. Melting point was measured in open
capillary with melting point apparatus MPM-HV2 in manual mode.
High-resolution mass measurements were performed on an LTQ
Orbitrap XL, with the external calibration of the instrument, having
accuracy up to 5 ppm. Ionisation was carried out in the positive mode
using the electrospray source at voltage of 4.5 kV. Samples were used as
a solution in methanol or acetonitrile. TLC-analysis was performed with
pre-coated aluminium plates (Silica gel 60 UV254, Macherey–Nagel).
The spots were visualized under 254 nm UV light and/or submerging in
an phosphomolybdic acid solution and heating. Column chromato-
graphy was performed through silica gel (200–300 mesh).
⁎
Corresponding author.
E-mail address: kozyrkovyu@gmail.com (Y.Y. Kozyrkov).
https://doi.org/10.1016/j.steroids.2020.108601
Received 5 December 2019; Received in revised form 29 January 2020; Accepted 5 February 2020
Available online 19 February 2020
0039-128X/ © 2020 Elsevier Inc. All rights reserved.

D.Y. Shostko, et al.
Steroids 158 (2020) 108601
Fig. 1. Structures of oral turinabol (DHCMT) (1) and long-term metabolites proposed for it: M3 (2), M4 (3) and M2 (4) [7].
2
2
.2. Chemical synthesis
2.2.3. 4-Chloro-13α-androst-4-en-3,17-dione (16)
To a solution of 13α-androst-4-en-17-one (15) (700 mg, 2.45 mmol)
.2.1. 3β-Hydroxy-13α-androst-5-en-17-one acetate (14)
A solution of dehydroepiandrosterone 13 (4.16 g, 14.32 mmol) in
2 2
in dry pyridine (7 mL) SO Cl (0.32 mL, 3.92 mmol) was added at 0 °C.
The resulting mixture was stirred at the same temperature for 1 h and
quenched with water (20 mL). The mixture was extracted with EtOAc
(3 × 10 mL), combined organic layers were washed with 5% HCl, sa-
acetic acid (40 mL) and 1,2-phenylenediamine (o-PDA) (2.32
2
g
1.48 mmol) was refluxed for 20 h. The resulting mixture was then
poured into ice water (80 mL) and the beige precipitate formed was
separated by filtration. The precipitate was washed with water and
dried to give crude product (1.5 g). The filtrate was extracted with
3 2 4
turated NaHCO solution and brine and then dried over Na SO .
Solvents were evaporated under reduced pressure and the residue was
chromatographed on silica gel (petroleum ether/ethyl acetate,
88:12–84:16) to afford compound 16 (650 mg) as yellowish crystal.
Additional recrystallization from mixture hexane/ethyl acetate allows
CH
2 × 20 mL) and with saturated NaHCO
Na SO and the solvent evaporated under reduced pressure to give 3 g
2
Cl
2
(2 × 40 mL) and the organic phase washed with water
(
3
solution (3 × 15), dried over
1
2
4
to obtain white crystals of 16 (580 mg, 74%). m.p. 191–193 °C. H NMR
of crude product. The combined solids was chromatographed on silica
3
(500 MHz, CDCl ) δ 3.31–3.23 (m, 1H), 2.63–2.49 (m, 2H), 2.47–2.38
gel (petroleum ether/ethyl acetate 90:10) to give 14 (2.66 g, 56%), 3β-
(m, 1H), 2.29–2.03 (m, 6H), 1.93–1.85 (m, 1H), 1.74 (td, J = 13.6,
Hydroxy-13α-androst-5-en-17-one (0.53 g, 11%) and starting material
5.5 Hz, 1H), 1.63–1.54 (m, 2H), 1.26–1.15 (m, 1H), 1.16–1.06 (m, 1H),
1
13
(
(
0.30 g, 7%). m.p. 143–144 °C. H NMR (500 MHz, CDCl
3
) δ 5.43–5.37
1.09 (s, 3H), 1.06–0.99 (m, 2H), 1.00 (s, 3H), 0.92–0.80 (m, 1H).
NMR (126 MHz, CDCl
50.00, 49.94, 41.51, 37.73, 34.28, 33.99, 33.76, 31.95, 31.85, 29.14,
25.10, 22.86, 21.55, 18.11. HRMS (ESI): m/z calcd for C19 26ClO [M
C
m, 1H), 4.65–4.55 (m, 1H), 2.42–2.22 (m, 4H), 2.25–2.07 (m, 3H),
3
) δ 221.52, 190.73, 164.00, 127.45, 51.83,
2
1
0
.03 (s, 3H), 1.93–1.81 (m, 2H), 1.83–1.75 (m, 1H), 1.69–1.51 (m, 4H),
.22 (td, J = 13.5, 3.6 Hz, 1H), 1.17–1.04 (m, 2H), 1.02–0.93 (m, 1H),
H
2
1
3
+
−1
.99 (s, 3H), 0.92–0.80 (m, 1H), 0.86 (s, 3H). C NMR (126 MHz,
) δ 222.42, 170.65, 139.32, 122.00, 73.89, 51.12, 50.19, 47.99,
7.98, 36.94, 36.73, 34.28, 34.18, 33.16, 31.68, 27.68, 25.25, 23.05,
2.16, 21.54, 19.18. HRMS (ESI): m/z calcd for C21
[M+H]+
+H] : 321.16158; found: 321.16136 (Δ = −0.7 ppm). IR [cm ]:
2953, 1720, 1692, 1581, 1292, 1238, 1188, 1094, 1070, 955, 808, 575.
CDCl
3
3
2
3
1
H
31
O
3
:
2
.2.4. 3β-Hydroxy-4-chloro-13α-androst-4-en-17-one (17)
To solution of 4-chloro-13α-androst-4-en-3,17-dione (16)
470 mg, 1.46 mmol) in dry THF (20 mL) under Ar atmosphere was
−
1
31.22677; found: 331.22729 (Δ = 1.6 ppm). IR [cm ]: 2939, 1735,
a
239, 1026.
(
added a 1 M solution of L-Selectride in THF (1.48 mmol, 1.5 mL)
dropwise at −50 °C. The mixture was stirred at −50 °C for 2 h and
quenched with saturated aqueous NH Cl (1.5 mL) and then was
4
warmed to room temperature. After concentrating under reduced
pressure the mixture was diluted with water and extracted with EtOAc.
Combined organic layers were washed with water and brine and then
2
.2.2. 13α-Androst-4-en-3,17-dione (15)
A solution of 14 (1.29 g, 3.92 mmol) and potassium carbonate
(
1.14 g, 8.23 mmol) in methanol (50 mL) was refluxed for an hour. The
reaction mixture was diluted with deionised water and extracted with
dichloromethane. The pooled extracts were washed with brine, dried
2 4
dried over Na SO . Solvents were evaporated under reduced pressure
over Na
the resulting crude 3β-Hydroxy-13α-methylandrost-5-en-17-one
1.13 g, 100%) was dissolved in dry toluene (70 mL) and cyclohex-
anone (7.7 g, 78.4 mmol) The solution was heated to reflux and after
5 min aluminium isopropoxide (2.0 g, 9.79 mmol) were added, upon
2 4
SO . The solvents were removed under reduced pressure and
and the residue was chromatographed on silica gel (petroleum ether:
ethyl acetate, 86:14) to give allyl alcohol 17 (435 mg, 92%) with ratio
of diastereomers as 9:1. Analytical data were recorded for single isomer
(
1
obtained after recrystallization from MeOH: m.p. 157–160 °C. H NMR
1
(
500 MHz, CDCl
3
) δ 4.16 (ddd, J = 9.4, 6.2, 3.0 Hz, 1H), 2.94 (dt,
which the solution turned yellow. After 2 h the reaction was complete.
It was then washed with water (25 mL), 5% sulfuric acid (120 mL),
saturated NaHCO solution and brine. After drying over Na SO and
3 2 4
evaporating under reduced pressure the mixture was left to crystallize
at 3 °C overnight. The crystals were washed with cold petroleum ether
and dried to give 830 mg of crude product. The filtrate was left to
crystallize at −18 °C overnight and after filtration, washing and drying
additional 230 mg were collected. Combined solids purified via column
chromatography on silica gel (petroleum ether/ethyl acetate 70:30) to
J = 14.1, 3.2 Hz, 1H), 2.48 (d, J = 3.0 Hz, 1H), 2.38 (dd, J = 19.1,
9
1
.0 Hz, 1H), 2.26–2.03 (m, 5H), 1.99–1.90 (m, 1H), 1.90–1.82 (m, 1H),
.81–1.74 (m, 1H), 1.73–1.61 (m, 1H), 1.57–1.47 (m, 2H), 1.35 (td,
J = 13.4, 2.9 Hz, 1H), 1.16 (td, J = 13.4, 3.9 Hz, 1H), 1.04–0.91 (m,
H), 0.97 (s, 3H), 0.95 (s, 3H), 0.90–0.71 (m, 2H). 13C NMR (126 MHz,
CDCl ) δ 222.05, 142.12, 128.51, 69.68, 51.76, 50.26, 50.07, 40.60,
8.02, 33.88, 33.39, 32.55, 32.09, 28.04, 27.28, 25.24, 22.98, 21.54,
9.54. HRMS (ESI): m/z calcd for C19 28ClO
[M+H]+: 323.17723;
2
3
3
1
H
2
−1
afford product 15 (795 mg, 71%) as white solid, m.p. 147–149 °C. 1
NMR (500 MHz, CDCl
2
1
1
NMR (126 MHz, CDCl
5
2
+
found: 323.17745 (Δ = 0.7 ppm). IR [cm ]: 3540, 2949, 2839, 1720,
H
1451, 841, 454.
3
) δ 5.76–5.72 (m, 1H), 2.46–2.28 (m, 5H),
.27–2.04 (m, 5H), 1.90–1.83 (m, 1H), 1.69 (td, J = 13.8, 5.0 Hz, 1H),
.60–1.54 (m, 2H), 1.21 (td, J = 13.5, 3.9 Hz, 1H), 1.16–1.05 (m, 2H),
2.2.5. 3α-Hydroxy-4-chloro-13α-androst-4-en-17-one (18)
.04 (s, 3H), 1.01–0.93 (m, 1H), 1.00 (s, 3H), 0.91–0.81 (m, 1H). 13
C
To a solution of 3β-alcohol 17 (400 mg, 1.24 mmol) and PPh
3
3
) δ 221.70, 199.51, 170.44, 124.07, 51.62,
0.02, 49.99, 38.77, 38.13, 35.56, 33.94, 33.83, 32.93, 32.86, 31.91,
5.18, 22.81, 21.62, 17.70. HRMS (ESI): m/z calcd for C19 [M
H] : 287.20056; found: 287.19977 (Δ = −2.7). IR [cm ]: 2922,
(650 mg, 2.48 mmol) in dry THF (12 mL) under Ar atmosphere neat
formic acid (95 µL, 2.48 mmol) was added at once, followed by solution
of diethyl azodicarboxylate in toluene (1.0 mL, 2.48 mmol, 40%). The
resulted light-yellowish solution was left stirred at room temperature
for 2 h. After concentrating under reduced pressure the reaction
H O
27 2
−1
+
1
735, 1667, 1619, 1447, 1229, 1190, 1095, 871.
2

D.Y. Shostko, et al.
Steroids 158 (2020) 108601
mixture was divided between ethyl ether (10 mL) and aqueous NaHCO
3
3H), 0.91 (s, 9H), 0.88 (s, 3H), 0.14 (s, 3H), 0.10 (s, 3H). 13C NMR
(
5%, 10 mL). The organic phase was washed with water, then metha-
3
(126 MHz, CDCl ) δ 157.40, 142.50, 127.35, 103.04, 70.69, 53.46,
nolic NaOH (1 M, 4 mL) was added and resulting mixture was stirred
for 30 min. The reaction mixture was diluted with water and extracted
with EtOAc. Combined organic layers were washed with water and
brine and then dried over Na
reduced pressure and the residue was chromatographed on silica gel
53.03, 45.94, 40.58, 37.32, 34.50, 32.30, 31.43, 31.39, 29.57, 29.47,
27.09, 26.06(×3), 24.75, 21.89, 18.55, 18.36, −4.18, −4.59. HRMS
26
C H : 435.28445; found:
44ClOSi [M+H]+
(ESI): m/z calcd for
435.28479 (Δ = 0.8 ppm). IR [cm ]: 2950, 2855, 1080, 838, 774.
−1
2
SO
4
. Solvents were evaporated under
(
(
petroleum ether: ethyl acetate, 80:20) to afford 3α-alcohol 18
2.2.8. 3α-[(tert-Butyldimethylsilyl)oxy]-4-chloro-17-spiro[13α-androst-4-
en-17,2′-oxirane] (21, 22)
345 mg, 86%) as white crystals with ratio of diastereomers as 9:1. M.p.
1
1
1
5
3
0
1
3
z
69–171 °C (MeOH). H NMR (500 MHz, CDCl
3
) δ 4.15 (q, J = 2.6, Hz,
NaHCO
were sequentially added to an ice cooled stirring solution of 20
(174 mg, 0.40 mmol) in CH Cl (12 mL). The reaction mixture was
stirred for 1 h at the same temperature and then 15 h at room tem-
perature, diluted with CH Cl (25 mL) and quenched with saturated
aqueous Na (1 mL). The organic layer was separated, washed with
saturated aqueous NaHCO , brine, dried over Na SO and concentrated.
3
(335 mg, 4.0 mmol) and MCPBA (70%, 99 mg, 0.40 mmol)
H), 2.96 (dt, J = 14.5, 3.4 Hz, 1H), 2.44–2.33 (m, 1H), 2.27–2.05 (m,
H), 1.96–1.77 (m, 4H), 1.65 (dt, J = 13.3, 3.8 Hz, 1H), 1.61–1.49 (m,
H), 1.18 (td, J = 13.5, 4.0 Hz, 1H), 1.04–0.94 (m, 2H), 0.97 (s, 3H),
.92–0.74 (m, 2H), 0.89 (s, 3H). C NMR (126 MHz, CDCl
43.05, 127.30, 69.77, 52.10, 50.12, 49.99, 40.58, 37.97, 33.83, 32.26,
2
2
1
3
3
) δ 222.08,
2
2
2 2 3
S O
2.00, 31.35, 27.45, 26.80, 25.17, 23.26, 21.49, 18.29. HRMS (ESI): m/
3
2
4
+
calcd for C19
H
28ClO
2
[M+H] : 323.17723; found: 323.17783
The residue was chromatographed on silica gel preparative plate
(20 × 20 × 0.1 cm) (petroleum ether: ethyl acetate, 96:4; two elua-
tions) to afford compound 21 (21 mg, 12%) as a white solid and 22
−
1
(
1
Δ = 1.8 ppm). IR [cm ]: 3542, 2947, 2881, 1726, 1446, 1274, 1077,
054, 1005, 966, 774, 636.
(
81 mg, 45%) as a white solid. TLC on SiO
2
: (petroleum ether/ethyl
2
.2.6. 3α-[(tert-Butyldimethylsilyl)oxy]-4-chloro-13α-androst-4-en-17-one
acetate 15/1 v/v) R (21): 0.51, R (22): 0.42.
f
f
(
19)
To a solution of 3α-hydroxy-4-chloro-13α-androst-4-en-17-one (18)
345 mg, 1.07 mmol) in DMF (3.5 mL) imidazole (182 mg, 2.67 mmol)
2.2.9. 3α-[(tert-Butyldimethylsilyl)oxy]-4-chloro-17β-hydroxymethyl-17α-
methyl-18-nor-androst-4,13-dien (23)
(
and TBSCl (201 mg, 1.34 mmol) were sequentially added at room
temperature. The reaction mixture was stirred overnight and quenched
with water (15 mL). The mixture was extracted with mixture of EtOAc:
PE (1:1) (3 × 5 mL). The combined extracts were washed with water
2 2
A mixture of dry CH Cl (1.3 mL) and 2,6-lutidine (12 µL,
0.11 mmol) under Ar atmosphere was cooled to −80 °C. Then TMSOTf
(16 µL, 0.09 mmol) was added at that temperature. After five minutes,
2 2
the solution of minor epoxide 21 (20 mg, 0.04 mmol) in CH Cl
(
5 mL), brine (5 mL) and dried over Na
2
SO
4
. Concentration and column
(0.2 µL + 0.2 µL washings) was added dropwise to the chilled reaction
mixture. The reaction mixture is stirred at −80–−70 °C for 1 h and
then quenched by the addition of methanol (1 mL) followed by 2 M HCl
(0.2 mL). The reaction mixture was warmed to room temperature and
stirred for 30 min. The mixture was diluted with water, extracted with
chromatography on silica gel (petroleum ether:ethyl acetate, 97:3 to
1
9
5:4) afforded 19 (440 mg, 94%) as white crystals, m.p. 163–165 °C H
NMR (500 MHz, CDCl ) δ 4.11–4.06 (m, 1H), 2.95 (dt, J = 14.4,
.3 Hz, 1H), 2.42–2.32 (m, 1H), 2.25–1.99 (m, 4H), 1.94–1.81 (m, 2H),
3
3
1
0
.80–1.46 (m, 6H), 1.27–1.12 (m, 1H), 1.06–0.93 (m, 2H), 0.97 (s, 3H),
.92–0.71 (m, 2H), 0.90 (s, 9H), 0.85 (s, 3H), 0.13 (s, 3H), 0.09 (s, 3H).
CH
NaHCO
2
Cl
2
and the combined organic phases were washed with saturated
solution, brine and dried over Na SO . Solvent were evapo-
3
2
4
1
3
C NMR (126 MHz, CDCl
3
) δ 222.20, 141.65, 127.73, 70.47, 51.91,
rated under reduced pressure and the residue was chromatographed on
5
2
0.07, 50.00, 40.37, 38.01, 33.86, 32.20, 32.01, 31.21, 29.24, 26.74,
silica gel (petroleum ether: ethyl acetate, 90:10) to afford 23 (13 mg,
5.92(×3), 25.15, 23.22, 21.51, 18.27, 18.22, −4.32, −4.73. HRMS
65%) as colorless oil. 1H NMR (500 MHz, CDCl
1.9 Hz, 1H), 3.48 (d, J = 10.5 Hz, 1H), 3.31 (d, J = 10.5 Hz, 1H), 3.01
(ddd, J = 13.8, 4.4, 2.0 Hz, 1H), 2.34–2.25 (m, 1H), 2.25–2.12 (m, 2H),
2.03–1.85 (m, 6H), 1.82 (tt, J = 13.8, 3.9 Hz, 1H), 1.74–1.58 (m, 3H),
3
) δ 4.10 (dt, J = 3.8,
+
(
ESI): m/z calcd for
C
25
H
42ClO
2
Si [M+H]
:
437.26371; found:
−
1
4
1
37.26424 (Δ = 1.3 ppm). IR [cm ]: 2948, 2856, 1732, 1470, 1358,
070, 934, 837, 776.
1
.55 (ddd, J = 12.8, 9.4, 5.5 Hz, 1H), 1.32–1.22 (m, 1H), 1.17 (s br,
1H) 1.15–1.02 (m, 2H), 0.98 (s, 3H), 0.94 (s, 3H), 0.92 (s, 9H), 0.13 (s,
3H), 0.10 (s, 3H). 13C NMR (126 MHz, CDCl
2
.2.7. 3α-[(tert-butyldimethylsilyl)oxy]-4-chloro-17-methylene-13α-
androst-4-en (20)
3
) δ 141.94, 140.59,
Diiodomethane (0.42 mL, 5.26 mmol) was added dropwise to a
136.80, 128.17, 70.61, 69.13, 51.93, 51.71, 40.41, 36.75, 34.12, 31.25,
stirred suspension of activated Zn (1.17 g, 17.9 mmol) and PbI
.17 mmol) in THF (5 mL) and the resulting mixture was maintained at
self-reflux during the addition. The reaction mixture was stirred for a
further 30 min at rt before cooling to 0 °C. TiCl (0.22 mL in 1 mL
CH Cl , 1.97 mmol) was added dropwise, and the mixture was allowed
to stir at rt for a further 1 h after the addition. A solution of ketone 19
230 mg, 0.53 mmol) in THF (0.7 mL + 0.7 mL washing) was added
and the resultant mixture was stirred at rt for 20 h. The reaction was
quenched by slow addition of saturated aqueous NH Cl (15 mL) at 0 °C
2
(78 mg,
30.68, 30.55, 29.40, 27.22, 26.04, 23.22, 22.66, 21.89, 18.34, 17.83,
+
0
−4.21, −4.61. HRMS (ESI): m/z calcd for C26
H44ClO
2
Si [M+H]
:
calc: 451.27936; found: 451.28015 (Δ = 1.7 ppm).
4
2
2
2.2.10. 4-Chloro-17β-hydroxymethyl-17α-methyl-18-nor-androst-4,13-
dien-3α-ol (24)
(
To a solution of TBS-ether (23) (13 mg, 0.044 mmol) in THF
2
(0.5 mL) was added TBAF·3H O (18 mg, 0.06 mmol) and stirred for
44 h at room temperature. The mixture was diluted with water (2 mL)
and extracted with EtOAc. Combined organic layers were washed with
brine and dried over Na SO . Solvents were evaporated under reduced
2 4
pressure and the residue was chromatographed on silica gel (petroleum
4
and stirred for 20 min. Resulting suspension was filtered through a pad
of Celite, filter cake was washed with EtOAc (50 mL), then the layers
were separated. The organic phase was washed with water, saturated
aqueous NaHCO
3
, brine, dried over Na
2
SO
4
and concentrated in vacuo.
ether: ethyl acetate, 80:20 to 70:30) to give diol 24 (7 mg, 78%) as
colorless oil. H NMR (500 MHz, Chloroform-d) δ 4.16 (q, J = 2.7 Hz,
1
The residue was chromatographed on silica gel (petroleum ether: ethyl
acetate, 99:1) to afford 20 (152 mg, 66%) as white crystals, m.p.
1H), 3.48 (d, J = 10.5 Hz, 1H), 3.32 (d, J = 10.5 Hz, 1H), 3.01 (ddd,
J = 13.8, 4.4, 2.0 Hz, 1H), 2.37–2.09 (m, 4H), 2.06–1.82 (m, 8H), 1.71
(dt, J = 13.4, 3.6 Hz, 1H), 1.65–1.52 (m, 2H), 1.35–1.23 (m, 1H), 1.18
(s br, 1H), 1.12 (ddd, J = 12.8, 10.7, 2.0 Hz, 1H), 1.08–1.02 (m, 1H),
1
1
30–132 °C. H NMR (500 MHz, CDCl
3
) δ 4.87–4.81 (m, 1H), 4.73–4.67
(
2
(
m, 1H), 4.09 (dt, J = 4.1, 2.2 Hz, 1H), 2.91 (dt, J = 13.9, 3.1 Hz, 1H),
.53–2.34 (m, 2H), 1.93 (ddt, J = 13.5, 10.4, 3.0 Hz, 2H), 1.89–1.78
m, 2H), 1.75 (dt, J = 13.8, 3.5 Hz, 1H), 1.69–1.53 (m, 4H), 1.45 (dq,
1.01 (s, 3H), 0.93 (s, 3H). 13C NMR (126 MHz, CDCl
137.07, 127.74, 69.95, 69.05, 52.30, 51.68, 40.64, 36.68, 34.03, 31.43,
) δ 143.36, 140.24,
3
J = 12.4, 3.3 Hz, 1H), 1.37 (td, J = 13.4, 3.7 Hz, 1H), 1.21 (dd,
J = 9.6, 6.2 Hz, 1H), 1.13–1.04 (m, 1H), 0.96–0.83 (m, 3H), 0.94 (s,
30.67, 30.62, 27.51, 27.28, 23.20, 22.69, 21.89, 17.81. HRMS (ESI): m/
3

D.Y. Shostko, et al.
Steroids 158 (2020) 108601
literature (Scheme 1). One of them is the biotechnological approach
that is based on cytochrome P450 [5,12,13] or fungus [6] oxidation of
17,17-dimethyl fragment 7. In addition, there are two chemical ap-
proaches. First includes isomerization of the C-13 β-methyl group of 17-
ketosteroids 8 to 9 by o-PDA in acetic acid media [14]. After installa-
tion of epoxide function, 10 undergoes ring-opening and cationic mi-
gration of C-13 α-methyl group [10,15,16]. Second is based on Pd-
catalysed C–H oxidation of the 17β-methyl substituent in 12 using di-
recting oxazoline group established at 17α-methyl [17].
For the synthesis of M4, pathway including epimerization of C-13
and cationic rearrangement was chosen. The synthesis was started from
the commercially available dehydroepiandrosterone 13 that at the first
stage was exposed to radical epimerization by o-PDA under refluxing in
acetic acid with formation of 13α-DHEA acetate 14 as the main product
(
Scheme 2). The basic deprotection of acetate with subsequent Oppe-
nauer oxidation leads to enone 15. To incorporate chlorine function in
α-position of 15, it was subjected to SO Cl [18] in pyridine at 0 °C give
16 in 72% yield. With chloroenone in hand, we tried to selectively
reduce conjugated carbonyl group with NaBH , NaBH /CeCl ·7H O,
DIBAL-H, L-Selectride. Only L-Selectride [19] at −30 °C allowed to
reduce selectively 3-carbonyl group in presence of 17-ketone with the
formation of isomers 17 as 9:1. It is known that reduction of 4-en-3-on
fragment by Selectride reagents in steroid substrates without sub-
stituents in A and B ring leads mostly to β-configuration of hydroxyl
group [20–22], whereas reduction of substrates with 2α-, 6α- or 7α-
Scheme 1. Approaches to the construction of the steroidal fragment 5.
2
2
20 2
calcd for C H30ClO
[M+H]+: 337.19288 found: 337.19290
z
3
7
(
(
Δ = 0.04 ppm); calcd for C20
Δ = 0.15 ppm).
H
30 ClO
2
: 339.18993; found: 339.18998
4
4
3
2
3
. Results and discussion
There are only a handful of approaches to form challenging C-17
stereocenter of 17-methylated steroids long-term metabolites 5 in
Scheme 2. Rout of the synthesis of 4-chloro-17β-(hydroxymethyl)-17α-methyl-18-norandrosta-4,13-diene-3α-ol.
4

D.Y. Shostko, et al.
Steroids 158 (2020) 108601
substituents gives α-alcohols [23,24]. At this stage configuration of C-3
was assigned by implication from our previous results [25] as β. Since
the recent research [10] reports that DHCMT metabolite M3 has 3α-
OH, the next step was the inversion of the hydroxyl group configura-
tion. Inversion of C-3 was performed by Mitsunobu reaction [26] of 17
with formic acid followed by treatment with methanolic NaOH solution
to afford compound 18 with the 3α-hydroxyl group in 86% yield that
was subsequently protected as TBS-ether. The next stage was to trans-
form the 17-carbonyl group into some functionality that could be re-
arranged in mild conditions to give proper C–17 stereocenter.
Because of steric hindrance and enolization of C-17 ketone in ster-
oids with cis-C/D rings, such substrate is inactive in Corey-Chaykovsky
reaction [15,16]. Attempts to attach other nucleophiles as bromo-
methyllithium [27] and cyanide failed. When TMSCl was added to
ketone 19 and cyanide in DMSO [28], slight formation of products was
observed, although with low conversion. Therefore, further study was
focused on methylenation of carbonyl group.
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2
f
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1
1
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way as 3α-M4 (24) without inversion stage of C-3.
To summarise, we have performed the first synthesis of two isomers
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(
relative to 3-hydroxy group) 24 and 25 of potential long-term meta-
bolite (M4) of DHCMT. Comparison of this compounds with en-
dogenous metabolite will allow us to determine exact structure of it.
[
[
[
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Appendix A. Supplementary data
1
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