Meiosis activating sterols derived from diosgenin

Article abstract of DOI:10.1016/S0960-894X(01)00840-X

Continuing research based on the meiosis activation properties of the endogenous sterol FF-MAS is reported. The synthesis and SAR of 16- and 26-substituted sterols are described, utilising diosgenin as starting material. Selected sterols were tested for their ability to induce oocyte maturation in hypoxanthine arrested mouse oocytes in vitro.

Full text of DOI:10.1016/S0960-894X(01)00840-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 715–717
Meiosis Activating Sterols Derived from Diosgenin
Anthony Murray,* Christian Grøndahl, Jan L. Ottesen and Peter Faarup
˚
Novo Nordisk A/S, Novo Nordisk Park, 2760 Maløv, Denmark
Received 5 November 2001; accepted 12 December 2001
Abstract—Continuing research based on the meiosis activation properties of the endogenous sterol FF-MAS is reported. The
synthesis and SAR of 16- and 26-substituted sterols are described, utilising diosgenin as starting material. Selected sterols were
tested for their ability to induce oocyte maturation in hypoxanthine arrested mouse oocytes in vitro. # 2002 Elsevier Science Ltd.
All rights reserved.
4,4-Dimethyl-cholest-8,14,24-trien-3b-ol (FF-MAS) is a
naturally occurring sterol isolated from human folli-
cular fluid. It is an upstream intermediate in the choles-
terol biosynthetic pathway, initially formed via
enzymatic 14a-demethylation of lanosterol, and has
been reported to have a positive meiosis inducing effect
on immature mouse oocytes cultured in vitro.1,2 More
recently, FF-MAS has also been shown to promote
meiosis in other species such as rat3 and human.4
of FF-MAS, which lacks Á14 unsaturation was isolated
from bull testicular tissue and given the acronym T-
MAS, was also found to be a potent meiosis inducer.
Based on follow up work on these original findings we
recently reported a novel synthesis of FF-MAS,5 and
this letter describes further research relating to other
sterols which have an influence on the regulation of
meiosis.
The synthetic route reported here (Scheme 1) utilised
diosgenin as the starting material, allowing access to
sterols containing substituents at the 16 and 26 position.
Diosgenin was converted to 26-hydroxy cholesterol (1)
using the established literature method,6 and this diol
could be selectively protected in the side chain with
TBSCl. Introduction of the gem dimethyl group at the 4
position was done via Oppenauer oxidation of the 3-
hydroxy group, dimethylation at C4 with potassium
tert-butoxide and methyl iodide and finally lithium alu-
minium hydride reduction of the 3-keto group to give
alcohol 2, which underwent silyl deprotection to give
the corresponding diol 3.
It has been shown in vitro that FF-MAS can induce
meiotic maturation in hypoxanthine arrested denuded
mouse oocytes and restart the meiotic process, which
results in germinal vesicle breakdown (GVB)/polar
body formation (PB) of the oocyte as the initial steps.
The mode of action of FF-MAS exposure to immature
oocytes and their effect on GVB induction is not yet
known, however, in the original article by Byskov et
al.,1 a number of sterols were isolated and their effect on
meiosis determined. It was demonstrated that whilst
FF-MAS was active, both lanosterol and cholesterol
were inactive in this assay. In addition, a close analogue
Alternatively, silyl protection of the 3b-alcohol of 2
afforded compound 4, which underwent subsequent
allylic bromination and elimination7 to provide the Á5,7
intermediate 5. The two TBS groups in 5 could be cleaved
using HF to provide the Á5,7 diol 6. Similarly, reaction
of 5 with HCl in ethanol at reflux isomerised the Á5,7
diene system to the desired Á8,14 diene with con-
cominant deprotection of both TBS groups to generate
the 26-hydroxylated compound 8. In general for Á5,7 to
Á8,14 isomerisation reactions, traces of unwanted diene
isomers were conveniently removed by recrystallisation.
*Corresponding author. Tel.: +45-4443-4668; fax: +45-4443-4547;
e-mail: amur@novonordisk.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00840-X

716
A. Murray et al. / Bioorg. Med. Chem. Lett. 12 (2002) 715–717
Scheme 1. Reagents and conditions: (a) (i) TBSCl, imidazole, DMF (65%); (ii) Al(OiPr)3, N-methyl morpholine (91%); (iii) KOtBu, MeI, tBuOH
(76%); (iv) LiAlH4, THF (93%); (b) TBAF, THF (64%); (c) TBSCl, imidazole, DMF (98%); (d) (i) dimethyl bromohydantoin, hexane/benzene;
(ii) quinaldine/o-xylene (90% over two steps); (e) HCl, EtOH, benzene (49%); (f) TsCl, py (71%); (g) NaI, DMF (80%); (h) DBU, CHCl3 (44%);
(j) TBAF, THF (75%); (k) HF (72%); (m) TsCl, py (89%); (n) (i) as for (f), (g), (h) (38% over three steps); (ii) HCl, EtOH, benzene (70%); (p) LiCl,
DMF (89%); (q) HCl, EtOH, benzene (36%).
It was also possible to prepare the Á8,14,25 regioisomer
of FF-MAS by regioselective modification of the 26-
hydroxy group in 8. Initially, since the 26-tosyloxy
intermediate 9 did not react in the base induced elim-
ination step, it was necessary to prepare the corre-
sponding 26-iodo derivative 10, which on treatment
with DBU converted smoothly to compound 11. Com-
pound 5 could also be regioselectively deprotected in the
26-position using TBAF at room temperature to afford
compound 7, and in our hands it was observed that for
sterols bearing the 4,4-dimethyl functionality, the 3-
OTBS group was very robust to this deprotection pro-
tocol. This allowed for conversion of the 26-hydroxy
group in 7 to tosylate 12, which in turn was treated with
lithium chloride in DMF to give the corresponding
chloro compound 13. Exposure of this compound to the
acid isomerisation conditions reported above afforded
the Á8,14 compound 14.
under acidic conditions underwent silyl deprotection to
diol 19. Silyl protection of compound 18 at the 3-
hydroxy group followed by introduction of the Á5,7
diene system afforded the protected diol 20. In this case
complete deprotection of the two silyl ethers, and in
particular the 16-OTBS group was not possible using
either TBAF or hydrogen fluoride, however exposure to
DIBAL in refluxing hexane afforded the desired diol 21.
It should be noted that it was not possible to access the
corresponding 16-hydroxy-Á8,14 diene, most likely due
to the instability of the allylic alcohol moiety in this
compound. Based on the initial biological activity of
sterols containing the 16-hydroxy group (see Table 1),
compound 23 was also prepared from 17 via the tosylate
Table 1. Effect of selected sterols on Germinal Vesicle Breakdown9
Compd
GVB relative (%)a
FF-MAS
100
Tosylate 12 could also be transformed to the corre-
sponding Á25 alkene in a similar manner to that descri-
bed for compound 11. In this case, HCl induced
isomerisation also gave addition across the Á25 double
bond to afford the 25-chloro compound 15 which was
isolated as the major product (ꢀ70% yield, traces of
diene impurities could not be completely removed).
19
11
21
6
23
14
10
3
113 (Æ4)
106 (Æ11)
90 (Æ35)
89 (Æ4)
87 (Æ9)
87 (Æ3)
85 (Æ8)
71 (Æ26)
62 (Æ9)
43 (Æ15)
na
15
16
1
16-Hydroxy containing sterols were prepared from
sterols 16 and 17 (Scheme 2), which were again con-
veniently derived from diosgenin.8 The 4,4-dimethyl
group was introduced in a similar fashion to that
described in Scheme 1, providing compound 18, which
8
na
aValues are means of 2–3 experiments, standard deviation is given in
parentheses. na, not active.

A. Murray et al. / Bioorg. Med. Chem. Lett. 12 (2002) 715–717
717
Scheme 2. Reagents and conditions: (a) (i) Al(OiPr)3, N-methyl morpholine (90%); (ii) KOtBu, MeI, tBuOH (75%); (iii) LiAlH4, THF (99%);
(b) HCl, EtOH (60%); (c) (i) TBSCl, imidazole, DMF (93%); (ii) dimethyl bromohydantoin, hexane/benzene; (iii) quinaldine/o-xylene (62% over
two steps); (d) DIBAL-H, THF (46%); (e) LiAlH4 (83%); (f) Super-H, THF (55%).
22, which could be reduced to the desired alcohol on
treatment with lithium triethylborohydride.
In conclusion, we have described the synthesis and pre-
liminary SAR of a series of new sterols which have a
stimulating effect on mouse oocyte maturation in vitro.
Lipophilic substituents on the 25 and 26 positions of the
sterol side chain are generally tolerated and it appears
that in the 16-hydroxy series the Á8 or Á8,14 double
bond moiety is not essential for activity. Compounds of
this type provide further knowledge and understanding
to an area which has potential for the treatment of
infertility, and further research in this area will be
reported in due course.
Selected sterols were tested in a cell based assay for their
ability to induce meiosis in immature mouse oocytes by
measuring the rate of Germinal Vesicle Breakdown
(GVB) compared to FF-MAS. From Table 1 it can be
seen that for the Á8,14 diene compounds 10 and 14,
added iodo or chloro functionality can be tolerated at
the 26 position, the 26-hydroxy compound 8, however,
showed no meiosis inducing effect. The 25-chloro com-
pound 15 also shows moderate activity, and it appears
that lipophilic groups are tolerated at these positions.
Compound 11, the Á8,14,25 regioisomer of FF-MAS,
also showed a similar activity to FF-MAS, and it should
be noted that the saturated side chain analogue of FF-
MAS (not shown) also has a relative GVB value of
around 70%.
References and Notes
1. Byskov, A. G.; Andersen, C. Y.; Nordholm, L.; Thøgersen,
H.; Guoliang, X.; Wassman, O.; Andersen, J. V.; Guddal, E.;
Roed, T. Nature 1995, 374, 559.
2. Grøndahl, C.; Ottesen, J. L.; Lessl, M.; Faarup, P.; Mur-
ray, A.; Grønvald, F. C.; Hegele-Hartung, C.; Ahnfelt-Rønne,
I. Biol. Reprod. 1998, 58, 1297.
The 26-hydroxy cholesterol (1) has poor meiosis pro-
moting activity, however it is interesting to note that the
activity in this series increases with the introduction of
the gem dimethyl group at position 4 (3) and again after
further unsaturation at Á7 (6). In the case of the 16-
hydroxylated sterols we found that moderate activity
could be seen even when only the Á5 double bond was
present (16). In a similar fashion to the 26-hydroxy ser-
ies, increased activity was observed by introduction of
methyl groups at 4 and to a slightly lesser extent further
unsaturation at Á7, and in fact compound 19 in this
assay had a comparable activity to that of FF-MAS. It
appears that the 16-hydroxy group is essential for this
series of compounds as verified by retention of activity
of 16-hydroxy-cholest-5-ene (23). There are still many
open questions relating to the mechanism of FF-MAS
and related sterols as meiosis regulators, however, the in
vitro activity of these compounds gives valuable infor-
mation towards improving our understanding of the
structural constraints required for activity within this
family of compounds. A recent article by Boer et al. has
also discussed the connection between electrostatic
potential and activity of a series of sterols related to FF-
MAS.10
3. Hegele-Hartung, C.; Grutzner, M.; Lessl, M.; Grøndahl,
C.; Ottesen, J. L.; Brannstrom, M. Biol. Reprod. 2001, 64, 418.
4. (a) Grøndahl, C.; Hansen, T. H.; Marky-Nielsen, K.; Otte-
sen, J. L.; Hyttel, P. Hum. Reprod. 2000, 15, 3. (b) Cavilla,
J. L.; Kennedy, C. R.; Baltsen, M.; Klentzeris, L. D.; Byskov,
A. G.; Hartshorne, G. M. Hum. Reprod. 2001, 16, 547.
5. Murray, A.; Grønvald, F. C.; Nielsen, J. K.; Faarup, P.
Bioorg. Med. Chem. Lett. 2000, 10, 1067.
6. Arunachalam, T.; MacKoul, P. J.; Green, N. M.; Caspi, E.
J. Org. Chem. 1981, 46, 2966.
7. Prelle, A.; Winterfeldt, E. Heterocycles 1989, 28, 333.
8. Kim, H. S.; Oh, S. H.; Kim, D. I.; Kim, I. C.; Cho, K. H.;
Park, Y. B. Bioorg. Med. Chem. 1995, 3, 367.
9. Oocytes were obtained from immature female mice weigh-
ing 13–16 g, placed in culture medium and cultured with 10
mM sterol for 24 h at 37 ꢁC according to the procedure descri-
bed in ref 2. At the end of the culture period the oocytes which
had undergone germinal vesicle breakdown (GVB) and polar
body formation (PB) were counted using a stereomicroscope.
% GVB relative was defined as [(number of GVB+number of
PB/total number of oocytes)Â100] indexed to FF-MAS which
was defined as 100.
10. Boer, D. R.; Kooijman, H.; van der Louw, J.; Groen, M.;
Kelder, J.; Kroon, J. Bioorg. Med. Chem. 2001, 9, 2653.