A simple route toward new clomiphene metabolites

Article abstract of DOI:10.1055/s-0029-1219383

A new clomiphene metabolite, extremely useful for doping analysis, was synthesized in 19% overall yield. The approach involved a Grignard reaction via a N-acylbenzotriazole intermediate to afford a key aromatic ketone and a HWE reaction. Both stereo-isomers were separated and identified. Georg Thieme Verlag Stuttgart ? New York.

Full text of DOI:10.1055/s-0029-1219383

LETTER
753
A Simple Route Toward New Clomiphene Metabolites
A
S
imple Route
T
r
oward N
u
ew
C
lomip
n
hene Metabo
o
lite
s
C. Vitoriano,a Luísa C. R. Carvalho,a Mónica S. Estevão,a Michael H. Sekera,b M. Manuel B. Marques*a
a
REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa,
2829-516 Caparica, Portugal
Fax +351(21)2948550; E-mail: mmbmarques@dq.fct.unl.pt
Laboratório de Análises de Dopagem, Autoridade Antidopagem de Portugal, Av. Prof. Egas Moniz (Estádio Universitário),
1600-190 Lisboa, Portugal
b
Received 25 November 2009
This work is dedicated to Professor Sundaresan Prabhakar, extraordinary teacher and mentor, for his dedication to chemistry.
Due to the anti-estrogenic activity, tamoxifen (1) and re-
lated anti-estrogens induce a decrease in side effects after
abusive consumption of anabolic androgenic agents.
Thus, since 2000, these compounds have been banned in
male athletes by the International Olympic Committee
and by the World Anti-Doping Agency. The presence of
tamoxifen (1) and clomiphene (2) metabolites in urines is
a proof of doping. Indeed, some metabolites have been de-
tected and identified, such as 3 (Figure 1).8
Abstract: A new clomiphene metabolite, extremely useful for dop-
ing analysis, was synthesized in 19% overall yield. The approach
involved a Grignard reaction via a N-acylbenzotriazole intermediate
to afford a key aromatic ketone and a HWE reaction. Both stereo-
isomers were separated and identified.
Key words: clomiphene, Grignard reactions, N-acylbenzotriazole,
olefination, drugs
The nonsteroidal anti-estrogens tamoxifen (1) and clomi-
phene (2, Figure 1) are synthetic triphenylethylene deriv-
atives and have been widely used in the treatment of
estrogen-dependent cancers.1 Moreover, they have been
found to act like as estrogen mimetic in prevention of
bone loss in postmenopausal women.2
N
N
O
O
Cl
N(R2)2
Cl
O
enclomiphene (E)
zuclomiphene (Z)
X
Figure 2 The two geometric isomers of clomiphene (2)
R1
In order to study the molecular action of synthetic nonste-
roidal anti-estrogens protocols have been developed to
address the synthesis of clomiphene (2)9 and its metabo-
lites.9c,10 Concerning the preparation of clomiphene me-
tabolites, some general synthetic approaches to establish
the carbon skeleton have been reported, such as: the use of
organometallic reagents, in particular Grignard reagents;
and the McMurry coupling, also applied to tamoxifen (1)
synthesis,11 which has the disadvantage of generating a
mixture of the hetero-ketone coupling and the undesired
homo-ketone coupling.
tamoxifen (1) R1 = H, R2 = Me, X = Et
clomiphene (2) R1 = H, R2 = Et, X = Cl
4-hydroxy-3-methoxytamoxifen (3) R1 = 4-OH, 3-OMe, R2 = Me, X = E
4-hydroxy-clomiphene (4) R1 = 4-OH, R2 = Et, X = Cl
4-hydroxy-3-methoxyclomiphene (5) R1 = 4-OH, 3-OMe, R2 = Et, X = C
Figure 1 Structures of tamoxifen (1), clomiphene (2) and its meta-
bolites
Tamoxifen (1) is an important estrogen for the treatment,3
and more recently, prevention of breast cancer.4 Clomi-
phene citrate (CC), structurally related to diethylstil-
bestrol, is since the early 1960s, used in the treatment of
women with absent or irregular ovulation by inducting
ovulation.5 In 1961 were reported the first results of CC on
ovulation induction.6 Clomiphene citrate is administrated
as a mixture of two geometric isomers, enclomiphene (E)
and zuclomiphene (Z) (Figure 2) in the ratio 62:38.7
Recently, analysis of human urine, taken after clomiphene
(1) administration, revealed the presence of a metabolite
with m/z = 424, compatible with the structure of 6a/6b.12
However, no authentic sample was available and structur-
al confirmation could not be achieved.
The therapeutic application of the anti-estrogens as well
as the interest in the identification of novel metabolites for
detection in doping analysis has stimulated us to synthe-
size novel clomiphene metabolites. Thus, we now report a
SYNLETT 2010, No. 5, pp 0753–0756
Advanced online publication: 10.02.2010
DOI: 10.1055/s-0029-1219383; Art ID: G36509ST
1
6.
3
.2
0
1
0
© Georg Thieme Verlag Stuttgart · New York

754
B. C. Vitoriano et al.
LETTER
NHEt
Grignard
O
O
Cl
Et2N
O
OPG
HWE
OMe
MeO
7
6
OH
O
MgBr
X
Et2N
+
O
OPG
OMe
8
9
MeO2C
OH
OMe
10
Scheme 1 Retrosynthetic analysis of clomiphene metabolite 6
modified and alternative synthetic approach toward the the authors have used this phosphonates to prepare trisub-
triphenylethylene moiety. stituted vinyl chlorides, via a HWE reaction.
Our strategy for the synthesis of the novel clomiphene A similar strategy was followed to prepare the vinyl chlo-
metabolite 6 is outlined in Scheme 1. The preparation of ride 6. The use of easily accessible and stable N-acylben-
tetrasubstituted olefins13 from ketones has been addressed zotriazoles, as acylating agents,17 has been successfully
by the Horner–Wadsworth–Emmons (HWE) approach,14 applied for the preparation of aromatic ketones via orga-
however, due to ketone lower reactivity and steric bulki- nometallic reactions, such as the Grignard reaction.18 In
ness this route has been barely applied. According to order to avoid the difficulties associated with the use of a
Kumara Swamy et al.15 cyclic phosphonates show en- carboxylic acid or acid chloride, we applied a Grignard
hanced reactivity when compared to acyclic ones,16 and approach reacting a N-acylbenzotriazole derivative 9 with
O
MeO2C
HO2C
1. BnBr, K2CO3, acetone,
reflux overnight (82%)
3. Et3N, THF, r.t. overnight
(90%)
N
N
N
OH
OBn
2. NaOH (5 M), EtOH,
reflux overnight (70%)
N
N
N
OBn
OMe
OMe
11
OMe
10
13
SO2Me
12
MgBr
Br
2 steps
4. THF, 0 °C to r.t. 3 h (75%)
O
OH
14
8
NEt2
O
Et2N
O
OBn
OMe
15
Scheme 2 Preparation of the ketone 15 via a Grignard reaction with a N-acylbenzotriazole intermediate
Synlett 2010, No. 5, 753–756 © Thieme Stuttgart · New York

LETTER
A Simple Route Toward New Clomiphene Metabolites
755
the known Grignard reagent 819 to obtain the ketone 7 via ficient when applied to the ketone 15. Subsequent mono-
a mild, simple, and effective procedure. Thus, our ap- N-dealkylation of isomers 17a/17b using the procedure
proach started with protection of the commercial methyl described by Olofson et al.23a afforded the corresponding
vanillate 10 with the benzyl group, followed by hydrolysis secondary amines 18a/18b as a mixture of both E/Z iso-
to afford the acid 1120 in 57% yield (2 steps, Scheme 2).
First attempts to obtain ketone 15 via Grignard reaction
mers that were next separated by preparative thin-layer
chromatography.
using the corresponding acid chloride of 11 failed. In or- The assignment of the mixture 17a/17b as isomers E and
der to overcome the problems associated with the use of Z was done by NMR. This assignment was confirmed by
the acid chloride, the N-acylbenzotriazole derivative 13 NMR analysis (COSY, NOESY, HMQC, HMBC) of the
was prepared in 90% yield, by treatment of the carboxylic compounds 18a and 18b, after chromatographic separa-
acid 11 with the N-(1-methanesulfonyl)benzotriazole (12, tion. The 1H NMR spectrum of the major isomer (E)-18a
readily prepared from benzotriazole and methanesulfonyl shows the H1 and H2 resonances shifted to upfield (d =
chloride).21 The next step consisted on the Grignard reac- 6.44 and 6.41 ppm, respectively), while in the minor iso-
tion of N-acylbenzotriazole 13 with the known magne- mer (Z)-18b H1 and H2 proton resonances appear at d =
sium reagent 8 [readily prepared from 4-bromophenol 6.93 and 6.61 ppm, respectively. This observation sug-
(14) in 2 steps].19 The reaction proceeded under very mild gests that the ring current effect of ring A moves H1 and
conditions and ketone 15 was isolated in 75% yield, after H2 proton resonances to higher frequency in the E-isomer
chromatographic purification, and the benzotriazole could (Figure 3).
be recovered.
Moreover, the NOE experiment of 18a shows a correla-
In order to avoid a stepwise installation of the trisubstitut- tion between H1 and H2 from ring B with the aromatic
ed vinyl chloride moiety (formation of the double bond protons from ring A. This correlation is not observed in
followed by chlorination), the use of a proper a-chloro- the NOE spectra of 18b (Figure 3).
phosphonate was required, allowing formation of the dou-
Finally, removal of the benzyl group by hydrogenation
ble bond and insertion of a chlorine atom simultaneously.
(H2, 10% Pd/C, AcOH)10a afforded the desired metabolite
Thus, having in hand useful quantities of ketone 15, the
6 in 70% yield.
assembly of the olefinic system was next undertaken via a
The LC-MS analysis of 6 gave a peak at m/z = 424 with
HWE reaction using the chlorophosphonate 16 (prepared
retention time of 7.50 min. Subsequently a urine sample
in 2 steps from the commercially available 5,5-dimethyl-
of clomiphene administration was analyzed under the
same chromatographic conditions. Using multiple reac-
tion monitoring, a peak was observed at retention time of
7.50 min with ion transitions (m/z = 424 – 71 and m/z =
424 – 317), consistent with the data of the authentic sam-
1,3,2-dioxaphosphorinan-2-one)15,22 (Scheme 3). Com-
pound 17 was obtained as a mixture of two isomers 17a
and 17b (E/Z, 60:40 by 1H NMR) in 70% yield. Other pro-
tocols previously described for clomiphene (2) synthesis,
involving the use of acyclic chlorophosphonates,9a were
also tested. However, these conditions proved to be insuf-
O
Et2N
Cl
O
O
EtHN
t-BuLi, THF, –78 °C to r.t.
(70%)
+
Et2N
Cl
Cl
OBn
O
O
OMe
MeO
P
15
O
O
OBn
MeO
17b
17a
16
OBn
1. ClCOCHClMe, toluene,
reflux, 16 h (quant.)
2. chromatographic separation
O
O
EtHN
EtHN
Cl
Cl
hydrogenation (70%)
6a
6b
+
+
MeO
18b
MeO
OBn
OBn
18a
Scheme 3 Synthesis of the metabolites 6a/6b via a HWE reaction using the chlorophosphonate 16
Synlett 2010, No. 5, 753–756 © Thieme Stuttgart · New York

756
B. C. Vitoriano et al.
LETTER
(3) Early Breast Cancer Trialists’ Collaborative Group Lancet
1998, 351, 1451.
(4) Cuzick, J.; Powles, T.; Veronesi, U.; Forbes, J.; Edwards, R.;
Ashley, S.; Boyle, P. Lancet 2003, 361, 296.
(5) Casper, R. F.; Mitwally, M. F. M. J. Clin. Endocrinol.
Metab. 2008, 91, 760.
(6) Greenblatt, R. B.; Barfield, W. E.; Jungck, E. C.; Ray, A. W.
J. Am. Med. Assoc. 1961, 14, 101.
NH
NH
O
O
C
B
A
C
B
Cl
(7) Ernest, S.; Hite, G.; Cantrell, J. S.; Richardson, A. Jr.;
1
1
Cl
Benson, H. D. J. Pharm. Sci. 1976, 65, 148.
(8) Xin, Z.; Kairong, C.; Moutian, W. Recent Adv. Doping Anal.
2004, 12, 421.
MeO
A
MeO
BnO
2
2
BnO
(9) For the synthesis of 2 involving Horner–Emmons-type
reaction, see: (a) Crenshaw, M. D.; Zimmer, H. J. Org.
Chem. 1983, 48, 2782. Stannylcupration of diphenyl-
acetylene, see: (b) Cummins, C. H. Synth. Commun. 1995,
25, 4071. Palladium-catalyzed cross-coupling, see: (c) Al-
Hassan, M. I. Synth. Commun. 1987, 17, 1787 . Grignard
reaction, see: (d) Palopoli, F. P.; Feil, V. J.; Allen, R. E.;
Holtkamp, D. E.; Richardson, A. Jr. J. Med. Chem. 1967, 10,
84. (e) Rao, P. N.; Cessac, J. W. J. Labelled Compd.
Radiopharm. 1985, 22, 245.
NOESY
(E)-isomer
major
(Z)-isomer
minor
18a
18b
Figure 3 The two geometric isomers of the isolated metabolites
18a/18b
ple 6, suggesting that the observed metabolite12 has the
(10) (a) Ruenitz, P. C.; Arrendale, R. F.; Schmidt, W. F.;
Thompson, C. B.; Nanavati, N. T. J. Med. Chem. 1989, 32,
192. (b) Ruenitz, P. C.; Bagley, J. R.; Watts, C. K. W.; Hall,
R. E.; Sutherland, R. L. J. Med. Chem. 1986, 29, 2511.
(11) For recent references, see: (a) Detsi, A.; Koufaki, M.;
Calogeropoulou, T. J. Org. Chem. 2002, 67, 4608. (b) Top,
S.; Kaloun, E. B.; Vessières, A.; Laïos, I.; Leclercq, G.;
Jaouen, G. J. Organomet. Chem. 2002, 643. (c) Jaoun, G.;
Top, S.; Vessières, A.; Pigneon, P.; Leclercq, G.; Laïos, I.
Chem. Commun. 2001, 383.
(12) Sample obtained at the Laboratório de Análises de
Dopagem, Instituto do Desporto de Portugal for the Master
degree of: Vitoriano, B. Master Thesis; New University of
Lisbon: Portugal, 2009.
(13) For preparation of tetrasubstituted olefins, see: Zhou, C.;
Emrich, D. E.; Larock, R. C. Org. Lett. 2003, 5, 1579; and
references cited therein.
(14) (a) Petrova, J.; Coutrot, P.; Drefux, M.; Savignac, P.
Synthesis 1975, 658. (b) Crenshaw, M. D.; Zimmer, H.
J. Org. Chem. 1983, 48, 2782. (c) Gajda, T. Synthesis 1970,
717. (d) Morise, X.; Savignac, P.; Denis, M. J. Chem. Soc.,
Perkin Trans. 1 1996, 2179.
same structure.
In summary, we have prepared new clomiphene metabo-
lites using a mild, simple, and straightforward route. Me-
tabolites 6a and 6b were synthesized in 19% overall yield
from commercially available starting materials.
The key steps consisted on the preparation of the ketone
15 using a N-acylbenzotriazole derivative as acylating
agent and the HWE approach, using a cyclic a-chloro-
phosphonate, to establish the chloro vinyl skeleton. Both
stereoisomers could be separated by chromatography and
their structures assigned by NMR. Furthermore the syn-
thesized compound 6 was used as control for analysis of
the biological sample. This approach represents a simple
and effective strategy to prepare novel and more substitut-
ed clomiphene metabolites useful for doping analysis.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
(15) Muthiah, C.; Kumar, P.; Kumaraswamy, S.;
Kumara Swamy, K. C. Tetrahedron 1998, 54, 14315.
(16) Larsen, R. O.; Aksnes, G. Phosphorus, Sulfur Relat. Elem.
1983, 16, 339.
Acknowledgment
The authors acknowledge Fundação para a Ciência e Tecnologia for
the fellowship to M. S. Estevão SFRH/BD/46234/2008 and Institu-
to do Desporto de Portugal for partial financial support. We would
like to thank Prof. Ricardo B. Ferreira from Instituto Superior de
Agronomia for the support running HPLC analysis. We acknowl-
edge Fundação para a Ciência e Tecnologia (FCT) for the project
REDE/1502/REM/2005 and M.C. Oliveira for providing prelimina-
ry data from the LC-MS at the Instituto Superior Técnico, Portugal.
(17) For a review of N-acylbenzotriazoles as acylating agents,
see: Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005,
1656.
(18) Katritzky, A. R.; Le, K. N. B.; Khelasvili, L.; Mohapatra,
P. P. J. Org. Chem. 2006, 71, 9861.
(19) Short, J. H.; Biermacher, U.; Dunnigan, D. A.; Lambert,
G. F.; Martin, D. L.; Nordeen, C. W.; Wright, H. B. J. Med.
Chem. 1965, 8, 223.
(20) Herbert, R. B.; Jackson, F. B.; Nicolson, I. T. J. Chem. Soc.,
Perkin Trans. 1 1984, 825.
(21) Katritzky, A. R.; Shobana, N.; Pernak, J.; Afridi, A. S.; Fan,
References and Notes
W. Q. Tetrahedron 1992, 48, 7817.
(22) Kumaraswamy, S.; Selvi, R. S.; Kumara Swammy, K. C.
Synthesis 1997, 207.
(23) (a) Olofson, R. A.; Martz, J. T.; Senet, J.-P.; Piteau, M.;
Malfroot, T. J. Org. Chem. 1984, 49, 2081. The same
procedure was applied to tamoxifen, see: (b) da Costa,
G. G.; McDaniel-Hamilton, L. P.; Heflich, R. H.; Marques,
M. M.; Beland, F. A. Carcinogenesis 2001, 22, 1307.
(1) Ruenitz, P. C. Burger’s Medicinal Chemistry and Drug
Discovery, 5th ed., Vol.4; Wiley: New York, 1997, 553–587.
(2) (a) Love, R. R.; Mazess, R. B.; Tormey, D. C.; Barden,
H. S.; Newcomb, P. A.; Jordan, V. C. Breast Cancer Treat.
1988, 12, 297. (b) Young, R. L.; Goldzieher, J. W.; Elkind-
Hirsch, K.; Hickox, P. G.; Chakraborty, P. K. Int. J. Fertil.
1991, 36, 167.
Synlett 2010, No. 5, 753–756 © Thieme Stuttgart · New York

Copyright of Synlett is the property of Georg Thieme Verlag Stuttgart and its content may not
be copied or emailed to multiple sites or posted to a listserv without the copyright holder's
express written permission. However, users may print, download, or email articles for
individual use.