C?H Acetoxylation-Based Chemical Synthesis of 17 β-Hydroxymethyl-17 α-methyl-18-norandrost-13-ene Steroids

Article abstract of DOI:10.1002/chem.201602957

Palladium-catalyzed C?H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17β-hydroxymethyl-17α-methyl-18-nor-13-ene D-fragment. This C?H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti-doping analysis to control the abuse of this androgenic anabolic steroid.

Full text of DOI:10.1002/chem.201602957

A Journal of
Accepted Article
Title: C-H Acetoxylation Based Chemical Synthesis of 17β-Hydroxymethyl-17α-
methyl-18-norandrost-13-ene Steroids
Authors: Alaksiej L. Hurski; Maryia V. Barysevich; Tatsiana S. Dalidovich; Marharyta
V. Iskryk; Nastassia U. Kolasava; Vladimir N. Zhabinskii; Vladimir A. Khripach
This manuscript has been accepted after peer review and the authors have elected
to post their Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
blished to ensure accuracy of information. The authors are responsible for the content
of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201602957
Link to VoR: http://dx.doi.org/10.1002/chem.201602957
Supported by

Chemistry - A European Journal
10.1002/chem.201602957
COMMUNICATION
C-H Acetoxylation Based Chemical Synthesis of 17β-
Hydroxymethyl-17α-methyl-18-norandrost-13-ene Steroids
[
a]
[a]
[a]
[a]
Alaksiej L. Hurski,* Maryia V. Barysevich, Tatsiana S. Dalidovich, Marharyta V. Iskryk,
[
a]
[a]
[a]
Nastassia U. Kolasava, Vladimir N. Zhabinskii, and Vladimir A. Khripach
Abstract: Palladium-catalyzed C-H acetoxylation has been
proposed as a key transformation in the first chemical synthesis of
steroids bearing a unique 17β-hydroxymethyl-17α-methyl-18-nor-13-
ene D-fragment. This C-H functionalization step was crucial for
inverting the configuration at the quaternary stereocenter of a readily
available synthetic intermediate. The developed approach was
applied to prepare metandienone metabolite needed as a reference
substance in anti-doping analysis to control the abuse of this
androgenic anabolic steroid.
structural elucidation. Inspired by challenging structure of the
[
2a, 4]
compounds 3-6 and analogous metabolites
along with their
importance for anti-doping analysis, we have undertaken studies
on the fully chemical synthesis of the 17β-hydroxymethyl-17α-
methyl-18-norandrost-13-ene steroids.
1
7β-Hydroxymethyl-17α-methyl-18-norandrost-13-enes 20OH-
[
1]
[2]
NorMD (3)
and 4
are human metabolites of the 17-
methylated androgenic anabolic steroids (AAS) metandienone
1) and oxandrolone (2) banned in sports (Scheme 1). The use
(
of 3 and 4 as reference substances in anti-doping analysis
resulted in an enormous increase of adverse analytical findings
of the parent drugs due to the considerable extension of the
[
3]
detection window. The metabolite 3 is detectable for up to 19
days after metandienone (1) abuse whereas the detection period
[
1a]
of its other metabolites is only 4-6 days.
It is expected that
long-term metabolites of dehydrochloromethyltestosterone
[
2a, 4]
[2a]
[4a]
[5]
(
DHCMT)
(including 5a and 5b ), oxymetholone 6 and
[
2a]
metabolites of other frequently abused 17-methylated AAS
also bear analogous D-fragment. But their full structural
elucidation still remains an unresolved issue. These metabolites
cannot be isolated in quantities sufficient for NMR analysis, and
the only way to prove their structures is preparation of synthetic
[
6]
samples for comparison purposes.
Structure of the D-ring in steroids 3-6 is unique due to the
configuration of its quaternary C17 stereocenter which in
combination with an endocyclic C13-C14 double bond makes
this fragment a challenging target for chemical synthesis. This
fact is particularly impressive, since epimeic at C17 D-fragment
[
1c, 7]
[1b, 2a, 8]
7
as well as other 18-nor-13-ene steroids 8-12
bearing non-hydroxylated 17β-methyl group are readily
a
available via a stereospecific Wagner-Meerwein shift of 18-
methyl group in the cationic intermediates 13 (Scheme 1). The
only known routes to steroids 16 have been based on a
biotechnological oxidation of the rearranged intermediates 8
[
1b, 2]
(
Scheme 2).
Application of these methods allowed to
[
1b]
[2b]
prepare metabolites 3 and 4 in quantities sufficient for their
[
a]
Dr. A.L. Hurski, M.V. Barysevich, T.S. Dalidovich, M.V. Iskryk,
N.U. Kolasava, Dr. V.N. Zhabinskii, Prof. Dr. V.A. Khripach
Institute of Bioorganic Chemistry, National Academy of Sciences of
Belarus
Kuprevich str., 5/2, 220141 Minsk, Belarus
E-mail: ahurski@iboch.bas-net.by
Scheme 1. Androgenic anabolic steroids, their metabolites and 17β-methyl-
Supporting information for this article is given via a link at the end of
the document.
18-nor-13-ene steroids.

Chemistry - A European Journal
10.1002/chem.201602957
COMMUNICATION
[
14]
with Ph
3
P and CCl
4
led to oxazoline ring closure.
However,
[
10a]
heating of the oxazoline 22 with iodobenzene diacetate
in the
presence of a catalytic amount of palladium diacetate led to the
destruction of the starting material. We assumed that the
decomposition of 22 could be caused by the reactions of
intermediate palladium-oxazoline complex with a proximate C13-
C14 alkenyl fragment. Another C5-C6 double bond in the
substrate was expected to be stable under the reaction
[
15]
conditions.
Scheme 2. Approaches to the construction of the steroidal fragment 16.
While searching for a suitable starting material, our attention
was focused on availability of steroids with D-fragment 15.
Synthesis of the target steroids 16 in living organisms is based
on steroselective C-H oxidation of 17β-methyl group in 17,17-
[
1-2]
dimethyl intermediate 8 (Scheme 2).
We speculated that
[
9]
chemical C-H activation methods could be applied for oxidation
of 17β-methyl substituent as well. To perform this
functionalization selectively, the appropriate directing group is
necessary. In this respect, steroids with epimeric D-fragment 7
are suitable intermediates for our synthesis as their hydroxyl
group could be transformed to any directing ligand. Finally, our
synthetic plan included two stages (Scheme 2). At first stage,
carbon backbone of the target fragment was thought to be built
through
rearrangement of a readily available epoxide 17 to alcohol 7.
At the second stage of the synthesis we planned to perform C-H
a
Lewis
acid
catalyzed
Wagner-Meerwein
[
7]
[
10]
acetoxylation
of 17β-methyl group and a complete reduction
of functionalities at 17α-substituent. The result of the latter set of
transformations is inversion of configuration at the quaternary
C17 stereocenter of intermediate 7.
Scheme 3. Initial efforts to oxidize the 17β-methyl group.
The synthesis started from androstenolone (18), which reacted
To test this hypothesis, synthesis of steroidal oxazoline 28
bearing a C13-C14 epoxide as the protecting group for double
bond was undertaken staring from 14α-hydoxyandrostenolone
acetate (23). The acetyl group in 23 was replaced by a tert-
butyldimethylsilyl protecting group and the resulting compound
[
11]
with dimethyloxosulfonium methylide to give, after acetylation,
a 2:3 mixture of isomeric spirooxiranes 19 (Scheme 3). The
Wagner-Meerwein rearrangement of the intermediate 19 was
[16]
2
promoted by a catalytic amount of Zn(OTf) at room temperature
to afford alcohol 20 that bears the necessary carbon backbone
with a C13-C14 double bond. To carry out the implied Pd-
catalyzed C–H oxidation, the 17-hydroxymethyl group of
compound 20 had to be converted into an appropriate directing
ligand.
2
4 was treated with dimethyloxosulfonium methylide to afford
the spirooxirane 25 as a single isomer (Scheme 4). Its Wagner-
Meervein rearrangement is expected to proceed via the
formation of a carbocation similar to 14 which undergoes the
nucleophilic attack of the adjacent hydroxyl group to form
oxirane ring.
Existing directing groups can be simply introduced into the
molecules but their reduction to alkane could be problematic as
only amides, oxime derivatives and heterocycles can direct
[17]
This transformation was effected in a yield of
81% using zinc triflate as the catalyst under ambient conditions.
Alternatively, it is possible to synthesize the compound 26 from
commercial androstenolone (18) in four steps and in 44% overall
yield (see SI). Conversion of the hydroxymethyl group of
intermediate 26 to the required directing oxazoline group was
achieved in two efficient synthetic steps. Oxidation of 26 by
Dess-Martin periodinane gave aldehyde 27 that was subjected
to one-pot reaction sequence involving formation of intermediate
sp3
[10]
palladium catalyzed acetoxylation of
C
-H bonds.
Our
[
12]
attention was drawn to the oxazoline group
which can be
smoothly prepared from aldehydes or acids and after the
acetoxylation could be easily converted into aldehydes or
[
13]
alcohols suitable for further deoxygenation.
0 with Jones reagent yielded the acid 21 that was amidated
with 2-amino-2-methylpropanol. Reaction of the resulting amide
The oxidation of
2

Chemistry - A European Journal
10.1002/chem.201602957
COMMUNICATION
[
18]
oxazolidine and its oxidation with NBS to afford oxazoline 28.
the synthesis of compound 22 was not possible as the C13-C14
It should be noted that application of the latter transformation in
alkenyl fragment reacted with NBS.
Scheme 4. Chemical synthesis of the metabolite 3.
Having in hands steroid 28 with oxazoline directing group and a
protected double bond, we moved on to explore the Pd-
Compound 33 possesses the required features of ring D and 3β-
5
hydroxy- -fragment allowing functionalization of this part of the
[
20]
catalyzed C–H acetoxylation. The reaction of 28 with PhI(OAc)
in the presence of catalytic amounts of Pd(OAc) at 100°C
proceeded smoothly over 45 min, providing product 29 in a good
2% yield. The next task to be performed was the conversion of
2
molecule by conventional steroid chemistry methods.
The
2
scope of the developed approach was evaluated through the
synthesis of metandienone metabolite 3. Alcohol 33 was
5
8
oxidized by Dess-Martin reagent to yield the corresponding  -3-
[
21]
the oxazoline heterocycle into a methyl group. It was desirable
to use the synthetic routes that are compatible with the acetyl
protecting group in 29. Quaternization of 29 with methyl triflate
followed by treatment with sodium borohydride and hydrolysis of
ketone, which was isomerized under acidic conditions to give
4
conjugated  -3-ketone 34 in 78% yield. The application of the
Oppenauer method to the oxidation of 33 proved to be less
efficient, resulting in the desired product in only moderate yield
[
13a]
4
1,4
the resulting aminal with oxalic acid
gave the aldehyde 30 in
(58%). Transformation of  -3-ketone to  -3-ketone was
5
6% overall yield. Deoxygenation of the aldehyde carbonyl
performed in 81% yield using benzeneseleninic anhydride as an
[
22]
[20, 23]
group was effected via the dithiane intermediate 31. Zinc triflate
catalyzed thioacetalization was accompanied by loss of the silyl
protecting group. Desulfurization of dithiane 31 under the action
of Raney nickel was unexpectedly accompanied by
deoxygenation providing a mixture of epoxide 32 and the target
oxidant.
Employment of a more common oxidant DDQ
led to the dienone in a yield of only 10%. Removal of the acetate
protecting group at the last step of the synthesis furnished
metabolite 3 whose 1D and 2D NMR spectra were consistent
[1b, c][24]
with the assigned structure.
1
3
13-ene steroid 33. The deprotection of  -double bond by
In conclusion, we have developed the first fully chemical route to
the construction of a key fragment of long-term metabolites of
AAS. These 17β-hydroxymethyl-17α-methyl-18-norandrost-13-
ene steroids are needed as synthetic reference substances for
effective anti-doping programs to ensure fair sport
Raney nickel was desirable, but neither increasing the amount of
the reagent nor lengthening the reaction time could drive the
transformation to completion. Ultimately, epoxide 32 was
successfully transformed into 33 with WCl and n-BuLi in the
6
presence of LiI.
[
19]
[3]
competitions. Palladium catalyzed C-H acetoxylation of 17β-

Chemistry - A European Journal
10.1002/chem.201602957
COMMUNICATION
[
13]
(a) T. G. Gant, A. I. Meyers, Tetrahedron 1994, 50, 2297-2360; (b)
G. S. K. Wong, W. Wu, in Oxazoles: Synthesis, Reactions, and
Spectroscopy, John Wiley & Sons, Inc., 2004, pp. 331-528.
methyl group was successfully used for the inversion of
configuration at
a quaternary stereocenter of the readily
available epimeric synthetic intermediate. Although this
transformation required 2 additional steps for the formation of
the directing oxazoline group and 4 steps were necessary to
reduce the directing group to methyl, the developed approach
allowed the preparation of the challenging compound 3 in an
acceptable 8.5% overall yield.
[14]
H. Vorbrüggen, K. Krolikiewicz, Tetrahedron Lett. 1981, 22, 4471-
4474.
[
15]
For examples of palladium-catalyzed C-H functionalyzation of
substrates with alkenyl fragments, see: (a) M. Wasa, J.-Q. Yu, J.
Am. Chem. Soc. 2008, 130, 14058-14059; (b) S.-Y. Zhang, G. He,
Y. Zhao, K. Wright, W. A. Nack, G. Chen, J. Am. Chem. Soc. 2012,
1
34, 7313-7316; (c) G. Shan, X. Yang, Y. Zong, Y. Rao, Angew.
Chem. 2013, 125, 13851-13855; Angew. Chem. Int. Ed. Engl.
013, 52, 13606-13610; (d) W.-H. Rao, B.-B. Zhan, K. Chen, P.-X.
2
Ling, Z.-Z. Zhang, B.-F. Shi, Org. Lett. 2015, 17, 3552-3555.
A. F. S. André, H. B. MacPhillamy, J. A. Nelson, A. C. Shabica, C.
R. Scholz, J. Am. Chem. Soc. 1952, 74, 5506-5511.
[
[
16]
17]
Acknowledgements
(a) M. Tanabe, D. F. Crowe, J. Org. Chem. 1963, 28, 3197-3199;
(b) V. A. Khripach, V. N. Zhabinskii, A. I. Kotyatkina, G. P. Fando,
A. S. Lyakhov, A. A. Govorova, M. Groen, J. van der Louw, A. de
Groot, Mendeleev Commun. 2001, 144-145.
K. Schwekendiek, F. Glorius, Synthesis 2006, 2006, 2996-3002.
K. B. Sharpless, M. A. Umbreit, M. T. Nieh, T. C. Flood, J. Am.
Chem. Soc. 1972, 94, 6538-6540.
J. Fried, J. A. Edwards, Organic Reactions in Steroid Chemistry,
Van Nostrand Reinhold Comp., New York, 1972.
L. F. Fieser, J. Am. Chem. Soc. 1953, 75, 5421-5422.
D. H. R. Barton, D. J. Lester, S. V. Ley, J. Chem. Soc., Perkin
Trans. 1 1980, 2209-2212.
The financial support of the Belarusian Foundation for Funda-
mental Research (project X16-107) is greatly appreciated. We
thank Dr. Alexander Baranovsky (Institute of Bioorganic
Chemistry) for assistance with NMR spectroscopy, Dr. Aliaksei
Yantsevich (Institute of Bioorganic Chemistry) for high resolution
mass spectrometric analysis.
[18]
19]
[
[20]
[
[
21]
22]
[
[
23]
24]
K. Chen, C. Liu, L. Deng, G. Xu, Steroids 2010, 75, 513-516.
C NMR spectral data of the synthesized compound were in a
Keywords: C-H activation • steroids • rearrangement • oxidation •
13
good agreement with those reported for 20OH-NorMD (3) obtained
protecting groups
[
1b, c]
by biotechnological approach
while small chemical shift
1
differences were observed in the H NMR spectra (see SI).
[
1]
(a) W. Schänzer, H. Geyer, G. Fußholler, N. Halatcheva, M. Kohler,
M.-K. Parr, S. Guddat, A. Thomas, M. Thevis, Rapid Commun.
Mass Spectrom. 2006, 20, 2252-2258; (b) A. Zöllner, M. K. Parr,
C.-A. Drăgan, S. Dräs, N. Schlörer, F. T. Peters, H. H. Maurer, W.
Schänzer, M. Bureik, J. Biol. Chem. 2010 391, 119-127; (c) M. K.
Parr, A. Zöllner, G. Fussholler, G. Opfermann, N. Schlörer, M.
Zorio, M. Bureik, W. Schänzer, Toxicol. Lett. 2012, 213, 381-391.
For detection of 17β-hydroxymethyl-17α-methyl-18-norandrost-13-
ene human metabolites of 17-methylated AAS (oxandrolone,
DHCMT, methyl-1-testosterone and methyltestosteron), and for
formation of steroids with analogous D-ring by biotransfornation of
[
2]
1
7,17-dimethyl-18-norandrost-13-enes, see: (a) M. K. Parr, G.
Fußhöller, M. Gütschow, C. Hess., W. Schänzer, in Recent
advances in doping analysis (18), Sportverlag Strauß, Cologne,
010, 64-73; for biotechnological synthesis of and its full
2
4
characterization, see: (b) Guddat, G. Fußhöller, S. Beuck, A.
Thomas, H. Geyer, A. Rydevik, U. Bondesson, M. Hedeland, A.
Lagojda, W. Schänzer, M. Thevis, Anal. Bioanal. Chem. 2013 405,
8
285-8294.
H. Geyer, W. Schänzer, M. Thevis, Br. J. Sports Med. 2014, 48,
20-826.
[
[
3]
4]
8
For detection of 5a, 5b and further DHCMT metabolites with similar
D-ring, see: (a) T. Sobolevsky, G. Rodchenkov, J. Steroid Biochem.
Mol. Biol. 2012, 128, 121-127; (b) M. Fernández-Álvarez, J. Lu, D.
Cuervo, Y. Xu, J. Muñoz-Guerra, R. Aguilera, in Recent advances
in doping analysis (22), Sportverlag Strauß, Cologne, 2014, 182-
187.
[
[
5]
6]
T. Sobolevsky, G. Rodchenkov, Drug Test. Anal. 2012, 4, 682-691.
W. Abushareeda, A. Fragkaki, A. Vonaparti, Y. Angelis, M. Tsivou,
K. Saad, S. Kraiem, E. Lyris, M. Alsayrafi, C. Georgakopoulos,
Bioanalysis 2014, 6, 881-896.
[
[
7]
8]
(a) D. N. Kirk, M. A. Wilson, J. Chem. Soc. C 1971, 414-424; (b) J.
R. Bull, L. M. Steer, Tetrahedron 1990, 46, 5389-5400.
(a) D. M. P. E. Caspi, Can. J. Chem. 1963, 41, 2294-2298; (b) W.
Schänzer, G. Opfermann, M. Donike, Steroids 1992, 57, 537-550;
(
c) D. G. Loughhead, J. Org. Chem. 1985, 50, 3931-3934; (d) G. R.
Pettit, R. F. Mendonça, J. C. Knight, R. K. Pettit, J. Nat. Prod. 2011,
4, 1922-1930; (e) C.-X. Huang, Y. Shi, J.-R. Lin, R.-H. Jin, W.-S.
7
Tian, Tetrahedron Lett. 2011, 52, 4123-4125; (f) G. Deniau, K. T.
Sillar, D. O’Hagan, J. Fluorine Chem. 2008, 129, 881-887.
(a) J. F. Hartwig, J. Am. Chem. Soc. 2016, 138, 2-24; (b) T.
Newhouse, P. S. Baran, Angew. Chem. 2011, 123, 3422-3435;
Angew. Chem. Int. Ed. 2011, 50, 3362-3374; (c) D. Y. K. Chen, S.
W. Youn, Chem. Eur. J. 2012, 18, 9452-9474; (d) Y. Qiu, S. Gao,
Natural Product Reports 2016, 33, 562-581.
[
[
9]
10]
(a) T. W. Lyons, M. S. Sanford, Chem. Rev. 2010, 110, 1147-
1169; (b) X. Yang, G. Shan, L. Wang, Y. Rao, Tetrahedron Lett.
2016, 57, 819-836.
[
[
11]
12]
G. Drefahl, K. Ponsold, H. Schick, Chem. Ber. 1964, 97, 3529-
535.
R. Giri, J. Liang, J. G. Lei, J. J. Li, D. H. Wang, X. Chen, I. C.
3
Naggar, C. Guo, B. M. Foxman, J. Q. Yu, Angew. Chem. 2005,
117, 7586-7590; Angew. Chem. Int. Ed. Engl. 2005, 44, 7420-7424.

Chemistry - A European Journal
10.1002/chem.201602957
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Alaksiej L. Hurski,* Maryia V.
Barysevich, Tatsiana S. Dalidovich,
Marharyta V. Iskryk, Nastassia U.
Kolasava, Vladimir N. Zhabinskii, and
Vladimir A. Khripach
Page No. – Page No.
Palladium-catalyzed C-H acetoxylation was used in the first chemical synthesis of
C-H Acetoxylation Based Chemical
Synthesis of 17β-Hydroxymethyl-17α-
methyl-18-norandrost-13-ene Steroids
17β-hydroxymethyl-17α-methyl-18-nor-13-ene steroids for inversion of configuration
at quaternary stereocenter of the readily available synthetic intermediate.