Synthesis of simplified hybrid inhibitors of type 1 17β-hydroxysteroid dehydrogenase via cross-metathesis and Sonogashira coupling reactions

Article abstract of DOI:10.1021/ol048820u

(Chemical Equation Presented) The inhibitor of type 1 17β- hydroxysteroid dehydrogenase EM-1745 (1) exhibits affinity for both the substrate (estrone or estradiol) and the cofactor (NAD(P)H) binding domains. However, to increase its bioavailability, this compound needs to be simplified. The efficient and convergent synthesis of simplified substrate/cofactor hybrid inhibitors (compounds 2) involving a cross-metathesis and a Sonogashira coupling reaction as key steps is described. Compounds 2a-c were also tested as enzyme inhibitors and compared to EM-1745.

Full text of DOI:10.1021/ol048820u

ORGANIC
LETTERS
2004
Vol. 6, No. 18
3127-3130
Synthesis of Simplified Hybrid Inhibitors
of Type 1 17â-Hydroxysteroid
Dehydrogenase via Cross-Metathesis
and Sonogashira Coupling Reactions
Marie Be´rube´ and Donald Poirier*
Medicinal Chemistry DiVision, Oncology and Molecular Endocrinology Research
Center and UniVersite´ LaVal, Centre Hospitalier UniVersitaire de Que´bec (CHUQ),
PaVillon CHUL, 2705 Laurier BouleVard, Quebec, Qc, G1V 4G2, Canada
donald.poirier@crchul.ulaVal.ca
Received June 21, 2004
ABSTRACT
The inhibitor of type 1 17â-hydroxysteroid dehydrogenase EM-1745 (1) exhibits affinity for both the substrate (estrone or estradiol) and the
cofactor (NAD(P)H) binding domains. However, to increase its bioavailability, this compound needs to be simplified. The efficient and convergent
synthesis of simplified substrate/cofactor hybrid inhibitors (compounds 2) involving a cross-metathesis and a Sonogashira coupling reaction
as key steps is described. Compounds 2a−c were also tested as enzyme inhibitors and compared to EM-1745.
Estrogens are known to be an important factor in the
development of estrogen-dependent breast cancer.1 A comple-
mentary approach to the treatment of this type of cancer with
an antiestrogen2 is to lower the level of estradiol by inhibiting
one of the enzymes involved in its biosynthesis.3 Among
these enzymes, type 1 17â-hydroxysteroid dehydrogenase
(17â-HSD) catalyzes the last step in the biosynthesis of
estradiol.4 During this enzymatic process, the nicotinamide
moiety of the cofactor NADPH transfers a hydride to the
R-face of the steroid carbon-17 to reduce estrone to the most
potent estrogen estradiol.5 Furthermore, the reductive activity
of type 1 17â-HSD is more important in breast tumors than
in normal breast tissue.6
The design of type 1 17â-HSD inhibitors has been
attempted during the past decades, but without achieving
progress significant enough to justify their use in the
treatment of estrogen-dependent breast cancer.7 Recently, a
new class of inhibitors was developed based on the three-
dimensional structures of the apoenzyme8 and of a binary
(1) (a) von Angerer, E. The Estrogen Receptor as a Target for Rational
Drug Design; R.G. Landes Co.: Austin, TX, 1995. (b) Dickson, R. B.;
Lippman, M. E. Endocr. ReV. 1987, 8, 29-43. (c) Poulin, R.; Labrie, F.
Cancer Res. 1986, 46, 4933-4937.
(2) Macgregor, J. I.; Jordan, V. C. Pharmacol. ReV. 1998, 50, 151-
196.
(3) (a) Labrie, F.; Luu-The, V.; Lin, S. X.; Simard, J.; Labrie, C.; El-
Alfy, M.; Pelletier, G.; Be´langer, A. J. Mol. Endocrinol. 2000, 25, 1-16.
(b) Smith, H. J.; Nicholls, P. J.; Simons, C.; Le Lain, R. Exp. Opin. Ther.
Patents 2001, 11, 789-824.
(4) (a) Luu-The, V.; Zhang, Y.; Poirier, D.; Labrie, F. J. Steroid Biochem.
Mol. Biol. 1995, 55, 581-587. (b) Luu-The, V. J. Steroid Biochem. Mol.
Biol. 2001, 76, 143-151.
(5) Breton, R.; Housset, D.; Mazza, C.; Fontecilla-Camps, J. C. Structure
1996, 4, 905-915.
(6) McNeil, J. M.; Reed, M. J.; Beranek, P. A.; Bonney, R. C.; Ghilchik,
M. W.; Robinson, D. J.; James, V. H. T. Int. J. Cancer 1986, 38, 193-
196.
(7) For review on type 1 17â-HSD inhibitors, see: (a) Poirier, D. Curr.
Med. Chem. 2003, 10, 1241-1253. (b) Penning, T. M. Endocr. Relat.
Cancer 1996, 3, 41-56.
10.1021/ol048820u CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/07/2004

complex (the enzyme and estradiol)9 and on results of a
structure/activity relationship (SAR) study.10 This new
category of substrate/cofactor hybrid inhibitor was designed
to interact with both the substrate (estrone or estradiol) and
the cofactor (NAD(P)H) enzyme-binding domains.11 An
eight-methylene spacer between the estradiol and the ad-
enosine moiety of the cofactor gave the best in vitro inhibitor
of type 1 17â-HSD (EM-1745, 1, Figure 1) with an IC50 of
groups of the adenosine mimic would interact with Ser11
and Asp65. Furthermore, it is known that the cofactor
NADPH has a better affinity for the enzyme than NADH.12
The structural difference between these two entities is the
presence of a phosphate group (NADPH) on the ribose. So,
a phosphate derivative, or a carboxylic acid as a bioisostere,13
added on aniline could interact well with the cofactor-binding
domain. Other functionalities (ester, bromide, alcohol) will
also be added to the aniline to extend the SAR study, and
the alkyl spacers (n ) 13-15, m ) 0-2) will be optimized.
We report herein a convergent and efficient route to prepare
inhibitors 2a-c using olefin cross-metathesis and Sonogash-
ira coupling reactions as key steps. Preliminary biological
evaluation is also reported.
Scheme 1. Retrosynthetic Analysis of Compounds 2a-c
Figure 1. Type 1 17â-HSD hybrid inhibitor 1 (EM-1745) showing
main interactions with this enzyme, as well as proposed simplified
hybrid inhibitors 2.
52 nM and a Ki value of 3.0 nM.11a Furthermore, the complex
EM-1745/type 1 17â-HSD was crystallized and X-ray
analysis confirmed that compound 1 interacts with both
binding domains.11a However, this inhibitor needs to be
simplified, especially on the adenosine moiety and on the
ester link, to improve its in vivo stability and activity.
Compounds 2 were proposed as simplified hybrid inhibitors
of type 1 17â-HSD on the basis of main interactions between
1 and the enzyme (Figure 1). It was assumed that estradiol
core of 2 would form strong hydrogen bonds with the amino
acids His221, Ser142, and Tyr155, while the amino and R
The retrosynthetic strategy selected to synthesize inhibitors
2 is outlined in Scheme 1. To limit the number of steps in
the preparation of compounds 2a-c, introduction of molec-
ular diversity (R ) COOMe, COOH, CH2OH) and removal
of protective groups (TBDMS, THP, and BOC) would be
done in the last stages. Because our synthetic plan is based
on a convergent strategy, compound 3 would be obtained
by a Sonogashira coupling reaction between the highly
functionalized aryl iodide 4, generated from 5, and the alkyne
6. A cross-metathesis between the suitably protected allyl-
estradiol 7 and the olefin aldehyde 8 would be used to link
the steroid and the alkyl spacer to give 6. Thus, any alkyl
spacers’ length can be attached to the estradiol derivative 7
by using the appropriate olefin aldehyde.
(8) (a) Ghosh, D.; Pletnev, V. Z.; Zhu, D. W.; Wawrzak, Z.; Duax, W.
L.; Pangborn, W.; Labrie, F.; Lin, S. X. Structure 1995, 3, 503-513. (b)
Lin, S. X.; Zhu, D. W.; Azzi, A.; Campbell, R. L.; Breton, R.; Labrie, F.;
Ghosh, D.; Pletnev, V.; Duax, W. L.; Pangborn, W. J. Endocrinol. 1996,
150, S13-20.
(9) Azzi, A.; Rhese, P. H.; Zhu, D. W.; Campbell, R.; Labrie, F.; Lin,
S. X. Nat. Struct. Biol. 1996, 3, 665-668.
(10) (a) Tremblay, M. R.; Poirier, D. J. Steroid Biochem. Mol. Biol. 1998,
66, 179-191. (b) Tremblay, M. R.; Lin, S. X.; Poirier, D. Steroids 2001,
66, 821-831.
Aniline 4 was prepared from the commercially available
2,6-diiodo-4-nitroaniline (5) (Scheme 2). Diazotization of 5
(11) (a) Qiu, W.; Campbell, R. L.; Gangloff, A.; Dupuis, P.; Boivin, R.
P.; Tremblay, M. R.; Poirier, D.; Lin, S. X. FASEB J. 2002, 16, 1829-
1831. (b) Poirier, D.; Boivin, R. P.; Be´rube´, M.; Lin, S. X. Synth. Commun.
2003, 33, 3183-3192.
(12) Huang, Y. W.; Pineau, I.; Chang, H. J.; Azzi, A.; Bellemare, V.;
Laberge, S.; Lin, S. X. Mol. Endocrinol. 2001, 15, 2010-2020.
(13) Silverman, R. B. The Organic Chemistry of Drug Design and Drug
Action; Academic Press: San Diego, CA, 1992; pp 19-23.
3128
Org. Lett., Vol. 6, No. 18, 2004

>95% yield (NMR analysis of aliquots of the reaction
mixture after quenching with water). This Grignard reagent
was quenched with allyl bromide in the presence of CuCN‚
2LiCl18 to give 12 in excellent yield (98%). Finally, oxidative
cleavage19 of allyl 12 with RuCl3 and NaIO4 followed by
esterification with trimethylsilyldiazomethane20 produced
ester 4.
Scheme 2. Synthesis of Aniline 4
For the attachment of the spacer to the steroid, several
strategies were considered. For example, R-alkylation of
steroidal 17-ketone, using LiHMDS as a base and a bro-
moalkyl chain, could be one way of joining the alkyl spacer
to the TBDMS-estrone. However, this strategy was used
when synthesizing 1 and only a low alkylation yield was
obtained, which is explained by the less reactive 17-ketone
of estrone. In fact, R-alkylation of 17-ketosteroid can only
be obtained in good yield with an activated electrophile such
as allyl bromide21,10 or with an unactivated electrophile when
the ketone is activated.22 This later methodology was not
selected because it is not a convergent strategy and its three-
step sequence would have added too many steps to the
synthesis of alkyne 6.
We developed instead a convergent and improved strategy
in which various alkyne lengths can be obtained in three
steps (Scheme 3). This new approach is based on a cross-
followed by reduction with copper sulfate in refluxing ethanol
afforded 9 as described by Brink et al.14 Nitro compound 9
was smoothly reduced with NaBH4 and SnCl2 to provide
Scheme 3. Synthesis of Alkyne 6
15
aniline 10 in good yield and purity without purification.
Protection of aniline 10 with a BOC group was more difficult
than expected. Classic protective conditions (BOC2O, Et3N
in DCM or DMF, or BOC2O in refluxing THF) did not allow
completion of the reaction even after a long time. However,
a biphasic system (BOC2O, NaHCO3, NaCl, H2O, and
CHCl3)16 gave a complete reaction, affording the protected
aniline 11. Desymmetrization of 11 was accomplished using
a metal halogen exchange methodology. Adapting the
chemistry of Knochel,17 metalation of 11 using iso-propyl-
magnesium chloride was explored as a means for generating
the arylmagnesium. When exactly 2 equiv of metalating
agent were used, we found that only one I-Mg exchange
took place. Thus, treatment of 11 with 2 equiv of iso-
propylmagnesium chloride at -20 °C for 30 min afforded
the formation of the carbamate anion and the I-Mg
exchange, giving the corresponding Grignard reagent in
(14) Brink, G. J. T.; Vis, J. M.; Arends, I. W. C. E.; Sheldon, R. A.
Tetrahedron 2002, 58, 3977-3983.
(15) (a) Satoh, T.; Mitsuo, N.; Nishiki, M.; Inoue, Y.; Ooi, Y. Chem.
Pharm. Bull. 1981, 29, 1443-1445. (b) Halterman, R. L.; McEvoy, M. A.
J. Am. Chem. Soc. 1992, 114, 980-985.
(16) Tarbell, D. S.; Yamamoto, Y.; Pope, B. M. Proc. Natl. Acad. Sci.
U.S.A. 1972, 69, 730-732.
metathesis23 between allyl-estradiol 724 and aldehyde 8,
obtained from oxidation of alcohol 13 with TPAP, which
(17) (a) Boymond, L.; Rottla¨nder, M.; Cahiez, G.; Knochel, P. Angew.
Chem., Int. Ed. 1998, 37, 1701-1703. (b) Abarbri, M.; Thibonnet, J.;
Be´rillon, L.; Dehmel, F.; Rottla¨nder, M.; Knochel, P. J. Org. Chem. 2000,
65, 4618-4634. (c) Rottla¨nder, M.; Boymond, L.; Be´rillon, L.; Lepreˆtre,
A.; Varchi, G.; Avolio, S.; Laaziri, H.; Que´guiner, G.; Ricci, A.; Cahiez,
G.; Knochel, P. Chem. Eur. J. 2000, 6, 767-770. (d) Herrinton, P. M.;
Owen, C. E.; Gage, J. R. Org. Process Res. DeV. 2001, 5, 80-83. (e)
Knochel, P.; Dohle, W.; Gommermann, N.; Kneisel, F. F.; Kopp, F.; Korn,
T.; Sapountzis, I.; Anh Vu, V. Angew. Chem., Int. Ed. 2003, 42, 4302-
4320.
(18) Knochel, P.; Yeh, M. C. P.; Berk, S. C.; Talbert, J. J. Org. Chem.
1988, 53, 2390-2392.
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Org. Lett., Vol. 6, No. 18, 2004
3129

led to the product 14 in an acceptable but not optimized 55%
yield. Hydrogenation of alkene 14 with palladium, followed
by treatment with LDA, trimethylsilyl-diazomethane, and
heat provided alkyne 6 in 59% yield for two steps.25
The next step is the Sonogashira coupling26 between alkyne
6 and aryl iodide 4 (Scheme 4). Under optimized conditions
protective groups in acidic solution and hydrogenation of
the alkyne, ester 2a was successfully obtained. From ester
2a, acid 2b and alcohol 2c were obtained after a hydrolysis
and a hydride reduction treatment, respectively.
Compounds 2a-c were evaluated for their ability to inhibit
the in vitro transformation of estrone into estradiol by type
1 17â-HSD according to an established procedure.10b Pre-
liminary results revealed that 2b (90% inhibition) was a more
potent inhibitor than 2a and 2c (32 and 16% inhibition,
respectively) at 1 µM. When compared to EM-1745 (1), the
inhibitory potency of 2b was approximately the same (90%
versus 95% for 1) at 1 µM and slightly weaker at 0.1 µM
(56% versus 90% for 1). The good inhibition obtained for
2b could be explained by the fact that the carboxylic acid is
a bioisostere of the phosphate of NADPH and probably
interacts with the same amino acids in the cofactor-binding
domain.
Scheme 4. Final Coupling and Molecular Diversities
In conclusion, we developed an efficient strategy for the
preparation of simplified hybrid inhibitors 2a-c in six steps
from known intermediate 7 and with an overall yield of 20%.
Halogen-metal exchange and olefin metathesis and Sono-
gashira coupling reactions are key steps in this synthesis.
This methodology has been applied to the synthesis of
analogues of compounds 2 (spacers of 14 and 15 methylenes,
aryl diversity) with similar overall yields (data not shown).
This strategy could also be used for the preparation of any
bisubstrate inhibitors joined by a methylene spacer. For this
purpose, the steroidal nucleus could be replaced by a phenolic
ring, extending our methodology to other families of
therapeutic agents.
Acknowledgment. We thank the Canadian Institutes of
Health Research (CIHR) for financial support and scholarship
(M.B.). We also thank Ms. Sylvie Me´thot for careful reading
of the manuscript and Drs. Rene´ Maltais and Shankar M.
Singh for helpful suggestions.
(Pd(PPh3)4, CuI, Et3N, benzene),27 product 15 was obtained
in excellent yield (95%). Finally, after cleavage of all three
Supporting Information Available: Experimental pro-
cedure and characterization data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(21) Fevig, T. L.; Katzenellenbogen, J. A. J. Org. Chem. 1987, 52, 247-
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Chem. 1995, 5, 505-523.
OL048820U
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