Structure-activity relationships of C-17 cyano-substituted estratrienes as anticancer agents

Article abstract of DOI:10.1021/jm701319c

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17β-cyanomethylestra-1,3,5(10)-trien-3-ol (14), but not the related 2-ethyl derivative 7, and the related 3-O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI₅₀ 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

Full text of DOI:10.1021/jm701319c

J. Med. Chem. 2008, 51, 1295–1308
1295
Structure–Activity Relationships of C-17 Cyano-Substituted Estratrienes as Anticancer Agents†
Mathew P. Leese,‡ Fabrice L. Jourdan,‡ Keira Gaukroger,‡ Mary F. Mahon,§ Simon P. Newman,| Paul A. Foster,|
Chloe Stengel,| Sandra Regis-Lydi, Eric Ferrandis, Anna Di Fiore,# Giuseppina De Simone,# Claudiu T. Supuran,3
Atul Purohit,| Michael J. Reed,| and Barry V. L. Potter*,‡
Medicinal Chemistry, Department of Pharmacy and Pharmacology & Sterix Ltd., and Department of Chemistry, UniVersity of Bath,
Bath BA2 7AY, U.K., Endocrinology and Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College London,
St. Mary’s Hospital, London W2 1NY, U.K., IPSEN System Biology, 91966 Les Ulis, France, Istituto di Biostrutture e Bioimmagini-CNR,
Naples, Italy, and Laboratorio di Chimica Bioinorganica, UniVersità degli Studi di Firenze, Florence, Italy
ReceiVed October 19, 2007
The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as
anticancer agents are discussed. 2-Methoxy-17ꢀ-cyanomethylestra-1,3,5(10)-trien-3-ol (14), but not the related
2-ethyl derivative 7, and the related 3-O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7
GI50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals
that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced
activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a
microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase
and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was
investigated by protein crystallography. The potential of these multimechanism anticancer agents was
confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-
MB-231 human breast cancer xenograft model.
Introduction
The discovery of angiogenesis inhibitors, which block the
formation of new blood vessels required by tumors to support
their growth,1 offered a novel therapeutic approach to the
treatment of cancer and stimulated extensive research in the
area.2 Although a number of potent angiogenesis inhibitors have
been discovered, their application as single agent cancer
therapies has not shown the hoped for clinical benefits. However,
use in combination with conventional cytotoxic agents has
produced more encouraging results. This approach, illustrated
by the use of bevacizumab,3 a monoclonal antibody against
vascular endothelial growth factor (VEGF), in combination with
chemotherapy has been shown to improve the survival of
patients with colorectal,4 lung, and breast cancer.5 The success
of this treatment strategy highlights the potential that exists for
drugs that can exert an antiangiogenic effect in concert with a
second antitumor activity.
In previous studies, we detailed the discovery of a number
of sulfamoylated 2-substituted estratriene derivatives6–9 that, like
the endogenous estrogen metabolite 2-methoxyestradiol (1) (2-
MeOE2), exhibit antiproliferative activity against cancer cells
and also display an antiangiogenic effect.10–13 Although notable
mechanistic differences between the 2-substituted estratriene-
Figure 1. Structures of 2-methoxyestradiol 1, 2-MeOE2MATE 2, and
2-MeOE2bisMATE 3.
3-O-sulfamates 2 and 3 (Figure 1) and 1 have been reported
and are discussed in depth elsewhere,11,14,15 in essence the
antitumor effects of both compound series appear to stem from
their ability to disrupt normal microtubule dynamics, which
arrests the cell cycle and leads the rapidly dividing cancer cells
to undergo apoptosis.11 The estratriene sulfamate series is,
however, differentiated from the corresponding substituted
estradiol derivatives by the enhanced antitumor activity and high
oral bioavailability,16 which they display and, additionally, by
their ability to inhibit both steroid sulfatase (STSa, irreversibly)17,18
and carbonic anhydrase (CA, reversibly).9,19 STS is a clinical
target for the treatment of hormone dependent cancer,20,21 while
the carbonic anhydrase IX isoform, which is highly expressed
in a number of tumors, has also been proposed as a target for
cancer therapy.22 It should be noted that the activities of these
2-substituted estratriene derivatives are independent of the
estrogen receptor, toward which they exhibit negligible affinity.
They are thought to bind to the colchicine binding site of
R-tubulin.9
†
The X-ray crystal structure of 15 has been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication CCDC
658208. Copies of the data can be obtained free of charge on application
to CCDC, 12 Union Rd., Cambridge CB2 1 EZ, U.K. (e-mail: deposit@
ccdc.cam.ac.uk). Protein crystal data: accession code 3BET.pdb.
* To whom correspondence should be addressed. Tel: +44 1225 386639.
Fax: +44 1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
‡
Department of Pharmacy and Pharmacology & Sterix Ltd., University
of Bath.
a Abbreviations: EMATE, estrone-3-O-sulfamate; E2MATE, estradiol-
3-O-sulfamate; E2bisMATE, estradiol-3,17-O,O-bis-sulfamate; STS, Steroid
sulfatase; CA, carbonic anhydrase; hCAII, human carbonic anhydrase II;
ER, estrogen receptor; E2S, estradiol-3-O-sulfate; HDBC, hormone-
dependent breast cancer; HIF, hypoxia inducible factor; DMA, N,N-
dimethylacetamide.
§
Department of Chemistry, University of Bath.
Imperial College London.
|
IPSEN System Biology.
Istituto di Biostrutture e Bioimmagini-CNR.
Università degli Studi di Firenze.
#
3
10.1021/jm701319c CCC: $40.75
2008 American Chemical Society
Published on Web 02/09/2008

1296 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Leese et al.
SAR studies showed that both substitution at C-2 of the
steroidal A-ring and an unsubstituted sulfamate group at C-3
are requisite for high antiproliferative activity.6 Optimal activity
was conferred by a C-2 ethyl, methylsulfanyl, or methoxy
substituent. Although a number of 2-substituted estradiol
derivatives displayed antiproliferative and antiangiogenic
activity,23–25 these nonsulfamoylated molecules are typically
much less potent, as illustrated by the mean activities of 1 and
2-methoxyestradiol-3-O-sulfamate (2) (2-MeOE2MATE) across
the NCI 60 cell line panel (MGM values of 1.3 µM and 110
nM, respectively).6 This finding is especially significant in light
of the extensive evaluation of 1 as an anticancer agent in
numerous academic studies and ongoing phase II clinical trials.
It would seem that the advantages conferred by the incorporation
of the 3-O-sulfamate group are manifold, in that this group
affords both enhanced antiproliferative activity and resistance
to inactivating conjugation of the C-3 hydroxyl group, to which
the corresponding estradiol derivatives are subject. Furthermore,
estrogen 3-O-sulfamates are also highly active reversible inhibi-
tors of carbonic anhydrase II (CAII), an enzyme that is highly
expressed in red blood cells, through a coordination of the
monoanionic form of the sulfamate moiety to the zinc atom in
the enzyme active site.26 This interaction is believed to underlie
the high oral bioavailability observed for estradiol-3-O-sulfamate
(E2MATE) (which is atypical of estrogen derivatives), wherein
reversible uptake by red blood cells and interaction with carbonic
anhydrase II results in avoidance of first pass liver metabolism,27
and it is plausible to suggest that such a mechanism could well
operate advantageously for 2-substituted derivatives of E2MATE.
Figure 2. Computational overlay of minimal energy conformations
of 17-O-sulfamoyl 3 and 17-cyanomethyl 15 functionalized estra-
1,3,5[10]-triene derivatives.
catalytic zinc of the enzyme active site in a manner analogous
to that observed for the 3-O-sulfamate group of E2MATE,9
although interaction also of the 3-O-sulfamate in vivo is not
necessarily excluded. Thus, it appears that, in addition to the
properties discussed above, the 17-O-sulfamate of 3 may also
enhance oral bioavailability by allowing a reversible uptake into
red blood cells and through interaction with hCAII affords a
mechanism for avoiding first pass liver metabolism.
Encouraged by the high activity and excellent pharmacoki-
netic profile displayed by the compounds such as 3, we explored
whether further D-ring modification could afford still greater
enhancement in activity. To this end, a series of C-17-carbamate
derivatives was synthesized, allowing us to establish that the
carbamate function could, in certain cases, successfully function
as a bioisostere for the C-17-sulfamate group.30 We were thus
also drawn to examine further C-17 functional groups that could
serve as hydrogen bond acceptors in the hope of discovering
even more active compounds and herein report the discovery
of 17-cyano-substituted estra-1,3,5(10)-trienes as compounds
that exhibit potent antiproliferative and antiangiogenic activity,
despite only possessing a single sulfamate group.6
To date, SAR knowledge of these compounds outside the
A-ring is limited to variation of the oxidation level of, and/or
substitution at, the C-17-position of the estratriene core. 17-
Hydroxy, 17-keto, and 17-oximino derivatives at C-17 display
similar potency, though the 17-deoxy- and 17R-benzyl deriva-
tives exhibit greatly reduced antiproliferative activity.8 An
improvement in in vitro antiproliferative activity was achieved
by installation of a second O-sulfamate group at C-17, and
significantly, this enhanced activity was seen to translate into
enhanced in vivo antitumor effects. The C-17 sulfamate group
of the 2-substituted estradiol-3,17-O,O-bis-sulfamates thus not
only provides for greater activity, by presumably exploiting
electrostatic interactions at the site of action unavailable to the
smaller oxygen functions of the first generation compounds but
also serves to block inactivating metabolism and conjugation
reactions to which they (especially the 17-hydroxyl derivatives)
are subject. Compound 1 has been shown to be rapidly
metabolized to inactive 2-methoxyestrone (2-MeOE1) by 17ꢀ-
hydroxysteroid dehydrogenase type II (17ꢀ-HSD type II), while
the 3,17-O,O-bis-sulfamate derivatives are not subject to 17ꢀ-
HSD type II metabolism.15 The combination of inactivating
metabolism with rapid conjugative inactivation through reaction
of the hydroxyl groups, to which 1 is subject, emphasizes the
need for molecules with improved pharmacokinetic profiles as
well as potency, as amply illustrated by a recent clinical trial
where a dose escalation of up to 6 g/day of 1 was used in an
attempt to achieve satisfactory plasma concentrations.28,29
Pharmacokinetic studies in rats showed that 2-methoxyestradiol-
3,17-O,O-bis-sulfamate (3) (2-MeOE2bisMATE), however,
displayed 85% bioavailability on oral dosing and was still
detectable after 24 h, while identically dosed 1 was undetectable
after just 5 min.16 X-ray crystallography was used to examine
the interaction of 2-MeOE2bisMATE with human carbonic
anhydrase II (hCAII) and proved, unexpectedly, that the 17-O-
sulfamate group of this compound could also interact with the
Chemistry
Earlier SAR studies demonstrated that a 3-O-sulfamate group
is critical for highly potent activity, but that 2-methoxyestradiol-
17-O-sulfamate is essentially inactive.6,9 We were, therefore,
drawn to examine whether the 17-O-sulfamate group of the
2-substituted estradiol-3,17-O,O-bis-sulfamates could be re-
placed with a small hydrogen bond-accepting group tethered
to C-17, reasoning that a sterically smaller functional group
might provide enhanced in vitro antiproliferative activity. In
order to mimic the distance between the proposed hydrogen
bond acceptor and C-17 of the sulfamate group, a two atom
linker would be required between C-17 and the H-bond acceptor.
We thus decided to install a cyanomethyl group at C-17 since
the alkyl linker would afford a reasonable degree of flexibility,
and the cyano group should offer sterically unhindered H-
bonding interactions through the terminal nitrogen atom. This
is clearly illustrated by computational overlay of bis-sulfamate
3 and nitrile compound 15 (vide infra) presented in Figure 2.
The close spatial proximity of the nitrile nitrogen and 17-O-
sulfamate NH2 would suggest that they could function analo-
gously as H-bond acceptors, albeit with differing electronic and
steric properties, which might allow the nitrile to interact more
strongly with those residues (Asn349 and Val315) around the
colchicine binding site of tubulin identified by docking studies
as potential H-bond donors to the 17-O-sulfamate group of 3.9

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1297
Scheme 1. Synthesis of 17-Cyanomethyl and
Scheme 2. Synthesis of 17-(1,1-Dicyano)methyl
17-Cyanomethylene Estra-1,3,5(10)-triene Derivativesa
Estra-1,3,5(10)-triene Derivativesa
a Reagents and conditions: (i) CH2(CN)2, ꢀ-alanine, PhMe; (ii) TBAF,
THF; (iii) H2NSO2Cl, DMA; (iv) NaBH4, THF/MeOH.
the alkyl substituent C-17 and confirms that the integrity of the
chiral centers of the estra-1,3,5(10)-triene core are retained
throughout the synthesis. Intermolecular hydrogen bonding
between the sulfamate NH2 group and both the nitrile of
crystallographically adjacent 15 and acetone (the crystallization
solvent) molecules is observed. The X-ray crystal structure of
2-methoxyestrone-3-O-sulfamate showed no such intermolecular
interaction, with the sulfamate appearing instead to hydrogen
bond to the adjacent methoxy group in an intramolecular
fashion.6 The appearance of intra or intermolecular hydrogen
bonding may simply derive from the choice of crystallization
solvent.
a Reagents and conditions: (i) (EtO)2POCH2CN, NaH, THF; (ii) H2, Pd/
C, THF/EtOH; (iii) TBAF, THF; (iv) H2NSO2Cl, DMA.
A Horner-Wadsworth-Emmons reaction between TBS-
protected 2-ethylestrone 4 and diethylcyanomethyl phosphonate
was thus carried out in refluxing THF to give the cyanometh-
ylene derivative 5 in an excellent 94% yield as a mixture of its
E- and Z-isomers (Scheme 1). Hydrogenation of the double bond
delivered the 17ꢀ-alkane 6, which was then desilylated with
TBAF in THF to deliver phenol 7. Sulfamoylation of 7 was
then achieved by reaction with a solution of sulfamoyl chloride
in DMA following the method of Okada and co-workers31 to
give sulfamate 8. Desilylation of alkene 5 was also carried out
to deliver the phenol as a mixture of the geometric isomers from
which the E-alkene 9 could be isolated by chromatography.
Sulfamoylation of 9 was then carried out to deliver sulfamate
10. The analogous series of reactions was performed in the
2-methoxy series with 11 being transformed into the respective
phenol (14 and 16) and sulfamate (15 and 17) derivatives.
An X-ray crystal structure of 15 was obtained and is presented
in Figure 3. This structure confirms the ꢀ-stereochemistry of
To assess whether the presence of a second cyano group
would be beneficial, the (dicyano)methylene and (dicyano)-
methyl derivatives of 2-ethylestrone were synthesized (Scheme
2). Condensation of 4 with malononitrile in the presence
ꢀ-alanine was thus carried out. Although this reaction proved
relatively slow, requiring the addition of extra aliquots of
malononitrile after 24 and 48 h, the yield of 18, however (91%),
proved excellent. Sequential desilylation of 18 and sulfamoyl-
ation afforded phenol 19 and sulfamate 20, respectively.
Reduction of the ∆-17 double bond of estratetraene 18 was
achieved with sodium borohydride in THF/MeOH to give 21,
Figure 3. X-ray crystal structure of 15 displaying the intermolecular hydrogen bonding through the sulfamate NH to the nitrile of the neighboring
15 and also to the carbonyl oxygen in the solvent of crystallization (acetone). Ellipsoids are represented at 30% probability. Primed labeled atoms
were generated via the –1 + x, y, 1 + z symmetry operation.

1298 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Scheme 3. Synthesis of 1-(Cyano)ethyl Side Chains at C-17a
Leese et al.
Scheme 4. Synthesis of 2-Ethyl-3-O-sulfamoyl-17ꢀ-prop-2-ynyl
Estra-1,3,5(10)-trienea
a Reagents and conditions: (i) DIBALH, PhMe/CH2Cl2 0 °C to rt; (ii)
(MeO)2POC(N2)COMe, MeOH, K2CO3; (iii) H2NSO2Cl, DMA.
a Reagents and conditions: (i) LDA, THF, MeI, -78 °C to rt; (ii) EtCN,
LDA, THF, -78 °C; (iii) MsOH, Et3N, DCM, or Burgess reagent, DMF;
(iv) H2, Pd/C, THF/EtOH; (v) H2NSO2Cl, DMA.
Scheme 5. Synthesis of 17-Cyano Estra-1,3,5(10)-triene
Derivativesa
which was then converted to phenol 22 and sulfamate 23 as
described above.
In order to study further steric effects around C-17, we were
drawn to methylate 6 at C-20. After some investigation, it was
determined that deprotonation of the C-20 methylene group
could be achieved with 2.2 equivalents of LDA in THF at -78
°C with methyl iodide quench delivering C-20 monomethyl
compound 24 in good yield (Scheme 3). Attempts to introduce
a gem-dimethyl group in a one pot procedure directly from 6
or by lithiation and electrophilic quench of 24, however, proved
abortive, and we thus decided to inspect an alternate approach
to compounds with alkyl substituted side chains. Reaction of
benzyl protected 2-ethylestrone 25 with the lithio-anion of
propionitrile afforded the 17R-alkylated estradiol derivative 26
in reasonable 76% yield, which could then be dehydrated with
methanesulfonic acid/triethylamine to give a 95:5 mixture of
the ∆-17 and ∆-16 dehydration products with the former, 27,
being isolated by column chromatography as a single isomer
(Z-stereochemistry as assigned by NOE experiments) in 85%
yield. Dehydration of 26 with Burgess reagent32 delivered
spectroscopically identical material. Concurrent debenzylation
and double bond hydrogenation delivered 17ꢀ-(1-cyano)ethyl
phenol (28), which was then sulfamoylated to give 29. Reaction
of the lithio-anion of isobutyronitrile with benzyl estrone was
performed to test the approach to C-20 gem-dimethyl target
compounds, and though this reaction proved successful, dehy-
dration of the product alcohol proved intractable.
a Reagents and conditions: (i) KCN, AcOH, MeOH; (ii) SOCl2, pyridine,
reflux; (iii) H2, Pd/C, THF/MeOH; (iv) NaHCO3, MeOH/acetone; (v)
H2NSO2Cl, DMA.
35, which could then be deprotected and sulfamoylated to give
40 or hydrogenated to give the 17ꢀ-cyano derivative 36, which
was then converted to phenol 37 and sulfamate 38 derivatives,
as previously described.
To confirm whether the activities observed for the nitrile
series (vide infra) were attributable to their H-bond acceptor
capabilities, the isosteric 17ꢀ-prop-2-ynyl compound 32 was
synthesized (Scheme 4). DIBALH reduction of the nitrile group
of 6 was thus carried out to give the aldehyde 30 in reasonable
61% yield, which could then be reacted with Ohira’s reagent33
to afford the 3-hydroxy alkyne 31, as the phenolic TBS group
proved unstable to the conditions of the reaction. The desired
sulfamate 32 was then formed by reaction with sulfamoyl
chloride in DMA.
To assess the effects of direct linkage of the nitrile group to
C-17 the cyanohydrin 34 was formed by reaction of 2-ethyl-
3-O-acetyl estrone 33 with potassium cyanide over 5 days at
room temperature (Scheme 5). Dehydration of the cyanohydrin
with thionyl chloride in refluxing pyridine gave the ∆-16 product
Results and Discussion
As a first stage predictor of antitumor activity, compounds
were assayed for their ability to inhibit the proliferation of a
panel of cancer cell lines in vitro. All compounds were screened
against DU-145 cells, a human androgen receptor negative
(AR-) prostate cancer cell line. The majority of compounds
were additionally screened for their ability to inhibit the
proliferation of MCF-7 and MDA MB-231 cells (human breast
cancer estrogen receptor positive (ER+) and estrogen receptor
negative (ER-), respectively). The results obtained from this
in vitro screening are presented in Table 1. The first striking
result in the table is the activity of the 2-methoxy-17-cyanom-
ethyl estra-1,3,5(10)-trien-3-ol (14), which is 2.5-fold more
potent against the proliferation of DU-145 cells and ca. 8-fold

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1299
Table 1. Antiproliferative Activities of Estratriene Derivatives against
respectively, presumably reflecting the inability of the rigid 17-
substituent of these compounds to attain optimal geometry at
the site of action.
Human Cancer Cells in Vitroa
MDA
DU-145
MCF-7
MB-231
The activities of the remaining compounds in the series serve
to characterize the spatial and electronic requirements for high
activity. Introduction of a second cyano substituent on the C-17
tethered alkyl group resulted in a slight reduction in antipro-
liferative activity in the case of both phenol 22 and sulfamate
23, albeit 23 is of a similar potency to the corresponding bis-
sulfamate 42. Introduction of a second H-bond acceptor group
thus appears only to hinder the key interaction between the C-17
functionality and the site of action. Introduction of a methyl-
substituent on the nitrile substituted carbon 29 caused a reduction
in activity similar to that effected by the additional nitrile of
23, which would suggest that the increased steric bulk around
the hydrogen bond acceptor interferes with attainment of the
optimal conformation at the site of action. The rigid dicyano-
alkene substitution gave a significant decrease in activity in the
phenol series 19, although sulfamate 20 was essentially equi-
potent with the corresponding alkane 23. As we had anticipated,
alkyne 32 wherein the hydrogen bond acceptor nitrogen of 8 is
replaced with with a CH group proved 30-fold less active than
the nitrile compound, thus strongly supporting the postulate that
the hydrogen bond acceptor group is essential for high anti-
proliferative activity. By extension, this supports our conclusions
on the function of the C-17 sulfamate, principally as a hydrogen
bond acceptor, in the bis-sulfamate series. Shortening of the
linker group between C-17 and the hydrogen bond acceptor was
also found to be deleterious to antiproliferative activity with a
6-fold reduction in activity observed for the 17ꢀ-nitrile deriva-
tive 38 relative to the corresponding 17-cyanomethyl compound
8. The ∆-16 unsaturated derivatives proved to be essentially
inactive as both phenol 39 and sulfamate 40.
Thus, it can be seen that C-17 cyanomethyl substitution of
2-substituted estra[1,3,5]-triene-3-O-sulfamates affords a series
of molecules with enhanced in vitro antiproliferative activity
relative to the C-17 sulfamate series. An enhancement in activity
is also evident in the nonsulfamoylated series with 7 and 14
found to be more potent than the corresponding estradiol
derivatives. The 3-O-sulfamates 8 and 15 are the most potent
2-substituted estra[1,3,5]-triene-3-O-sulfamates discovered to
date, with 15 proving to be up to 33-fold more active than 1 in
vitro. Substitution, or deletion, of the CH2 linker between C-17
and the nitrile group results in reduced activity, while replace-
ment of the hydrogen bond-accepting nitrile group of 8 with
an isosteric ethynyl group (compound 32) results in a 30-fold
reduction in activity and a more than 40-fold reduction for the
unsulfamoylated pair 7 and 31. On the evidence of these results,
it appears that the C-17 cyanomethyl substituent most likely
affords enhanced antiproliferative activity by functioning as a
sterically unhindered hydrogen bond acceptor group, even in
the simple 2-methoxyestradiol analogue series, which is posi-
tioned at the appropriate distance from C-17 in the biological
site of action.
No R1
R2
R3
17ꢀ-OH
(µM)
(µM)
(µM)
1
2
3
MeO H
MeO SO2NH2 17ꢀ-OH
MeO SO2NH2 17ꢀ-OSO2NH2
1.22
0.39
0.34
10.3
0.21
2.35
0.36
0.25
10.5
0.07
0.3
0.94
0.170
0.28
8.0
0.21
0.117
0.071
41 Et
42 Et
14 MeO H
15 MeO SO2 NH 17ꢀ -CH2 CN
16 MeO H
H
17ꢀ-OH
17ꢀ-OSO2 NH2
17ꢀ-CH2CN
SO2NH
2
0.485
0.062
0.07
28
2
(E) dCHCN
17 MeO SO2NH2 (E) dCHCN
0.24
3.24
0.06
>100
0.48
25.9
0.21
3.9
7
8
9
Et
Et
Et
H
17ꢀ-CH2CN
SO2NH2 17ꢀ-CH2CN
(E) dCHCN
SO2NH2 (E) dCHCN
:C(CN)2
SO2NH2 :C(CN)2
2.5
0.054
0.141
H
10 Et
19 Et
20 Et
22 Et
23 Et
29 Et
30 Et
31 Et
32 Et
37 Et
38 Et
39 Et
40 Et
H
>100
0.267
2.99
0.159
>100
9.9
20.0
0.289
2.62
0.249
7.50
10.3
H
17ꢀ-CH(CN)2
SO2NH2 17ꢀ-CH(CN)2
0.33
29.1
0.32
H
17ꢀ-CHMe
SO2NH2 17ꢀ-CHMe
H
17ꢀ-CH2CCH >100
SO2NH2 17ꢀ-CH2CCH
1.69
n/a
<0.5
H
17ꢀ-CN
>100
0.324
>100
>100
>100
0.342
57.3
SO2NH2 17ꢀ-CN
H
∆-16,17-CN
SO2NH2 ∆-16,17-CN
75.4
a Data for compounds 1–3, 41, and 42 are taken from the literature.9,30
GI50 figures are the mean values obtained from experiments performed in
triplicate; s.e.m. ) (7%.
more potent against the proliferation of both breast cancer cell
lines than 1. To the best of our knowledge, this is the first time
substitution at C-17 alone has provided such an enhancement
in antiproliferative activity with respect to the corresponding
17-hydroxy compound, although derivatives of 1 bearing
unsaturation in the D-ring, notably the 14-dehydro derivative,
have been reported to have significantly enhanced effects on
cancer cell proliferation.34 In comparison, the corresponding 17-
O-sulfamate derivative of 1 proved less active than 1 with a
GI50 of >10 µM.9 Interestingly, the corresponding 2-ethyl
substituted phenol 7 though 5-10-fold less active than 14, was
found to be 4-fold more active than 2-ethylestradiol 41 against
DU-145 cells, thus mirroring the beneficial effects of the C-17
cyanomethyl group on antiproliferative activity in the phenol
series, an effect that could potentially be used to provide for
further enhanced activity if used in conjunction with C-2
substituents, which are reported to deliver enhanced activity in
the estradiol series. The 17-(E)-cyanomethylene functionalized
phenols 9 and 16, in contrast, displayed no significant activity,
a result that suggests that rotation around the C-17-C-20 bond
is required to allow either optimal electrostatic interaction or
minimize disfavorable steric effects.
The 3-O-sulfamate derivatives bearing the 17-cyanomethyl
group in both the 2-methoxy 15 and 2-ethyl 8 series proved to
be exceptionally potent being, in DU-145 cells, 5.5- and 3.5-
fold more active than the 2-methoxy- and 2-ethyl-E2bisMATE
compounds (3 and 42, respectively), which they were designed
to mimic. The effects of 15 and 8 against breast cancer cell
proliferation were similarly profound with 50% growth inhibi-
tion obtained in the low nanomolar range. As observed in the
phenol series, the 3-O-sulfamoylated 2-methoxy 17 and 2-ethyl
10 compounds bearing a C-17 cyanomethylene group were less
potent than the corresponding cyanomethyl compounds, with
activities against MCF-7 proliferation reduced by 4- and 8-fold,
A number of the novel 17-cyanomethyl derivatives were
screened for antiproliferative activity by the NCI in their 60-
cell line assay (Table 2).35,36 Unfortunately, the most active
compounds in the series, 8 and 15, were not selected by the
NCI for evaluation, though the relative activities of compounds
23 and 29 compare favorably with both 1 and 3, for which
comparative data are included. Compounds 23 and 29 caused
>50% growth inhibition in the majority of cell lines at
concentrations below 10 nM, with mean growth inhibition
(MGM) of 30 and 22 nM, respectively (an MGM of 10 nM is

1300 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Leese et al.
Table 2. Antiproliferative Activity of Selected Compounds in the NCI 60-cell Line Panela
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12-C
prostate
DU-145
breast
MDA-MB-435
MGM
(µM)
1
3
23
29
37
38
40
0.7
0.051
0.47
0.045
<0.01
<0.01
>100
0.724
4.07
0.32
0.036
<0.01
<0.01
>100
4.16
0.36
<0.01
<0.01
<0.01
>100
0.588
0.21
<0.01
<0.01
NA
0.95
0.126
<0.01
<0.01
>100
6.76
1.8
0.083
<0.01
NA
0.08
<0.01
<0.01
<0.01
>100
3.16
1.3
0.087
0.03
0.022
>100
3.16
>100
0.512
2.63
>100
>100
14.7
3.55
2.69
2.51
0.524
4.07
4.47
2.95
3.63
a Results are GI50 values in micromolar. Data for 125 and 39 are taken from the literature.
Figure 4. Activity of 8 in an in vitro model of angiogenesis. (a) Plot of quantified tubule length per well in control, VEGF stimulated and 8 treated
wells at 20 and 40 nM. Treated wells are stimulated by 2 ng/mL VEGF. (b) Images of the control, stimulated, and treated wells in this assay.
maximal in this case because of the range of concentrations
(10 nM to 100 µM) tested and would be achieved only if 50%
growth inhibition was observed in all 60 cell lines at 10 nM).
The 17-cyanomethyl compounds were thus seen to show slightly
greater activity than 3 and are, notably, 40-fold more active
than 1 across the NCI panel. In comparison, sulfamates 40 and
38, whose cyano-groups are directly linked to C-17, showed
greatly reduced activity, while 37, the nonsulfamoylated ana-
logue of 38, showed no activity. These NCI results confirm the
activity observed in our preliminary screens and, furthermore,
demonstrate the effects of these compounds against a wide range
of cancer phenotypes.
To assess potential antiangiogenic activity, the compounds
were assayed for the ability to inhibit the proliferation of human
umbilical vein endothelial cells (HUVECs). The IC50 values
obtained for 1, 3, and 15 against HUVEC proliferation were
523, 44, and 30 nM, respectively.37 It can thus be seen that the
sulfamate derivatives express their in vitro antiangiogenic effects
at far lower concentrations than 1. Compound 8 was also assayed
for antiangiogenic activity in an in vitro model of angiogenesis,
wherein endothelial cells cocultured in a matrix of human dermal
fibroblasts are used to assess antiangiogenic potential (Figure
4). When stimulated with VEGF, the endothelial cells proliferate
and migrate through the matrix to form tubule-like structures
(see Figure 4b) and the extent of tubule formation can be
quantified as described elsewhere.12 As can be seen in Figure
4a, treatment with 20 and 40 nM concentrations of 8 almost
completely inhibits the formation of tubule-like structures.
Quantification carried out by calculating total pixel length
(Figure 4a) reflects the lack of tubule formation, which can be
clearly seen in the images of the treated and untreated wells
(Figure 4b). We have recently shown that the in vitro antian-
giogenic effects of the sulfamoylated 2-substituted estratriene
compounds translate well into in vivo activity with compounds
including 15 showing impressive activity upon oral administra-
tion in mouse Matrigel plug assay for angiogenic activity.13
We have previously established that the antiproliferative and
antiangiogenic activities of the 2-substituted estratriene-3-O-
sulfamates are derived from their ability to disrupt normal
microtubule dynamics11 and wished to confirm that the 17-
cyanomethyl series was acting, as we anticipated, at this target.
We thus inspected the effects of 8 on the taxol stimulated
polymerization of tubulin (10 µM Taxol and 20 µM 8). As can
be seen in Figure 5, 8 causes a substantial inhibition of both
the rate and extent of microtubule formation stimulated by
Taxol, thus supporting the postulate that these compounds, as
in previously studied 2-substituted estra-1,3,5(10)-triene-3-O-
sulfamates and the related estradiol derivatives, act as micro-

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1301
2-substitution on CA inhibitory effects are known, as estrone-
3-O-sulfamate (IC50 42nM) is 8-fold more active than 2-MeOE-
MATE,19 and o-bromination of even a simple aryl monosulf-
amate reduces CAII binding from 27 to 137 nM.39 This reflects,
presumably, simple steric constraints close to the sulfamate
binding region of the CAII active site. However, we had not
previously observed that a 17-substituent, which we would
expect to occupy the large lipophilic pocket above the enzyme
active site, could have such an effect on inhibitory activity. We
were thus drawn to further explore the interaction of 15 with
hCAII to see if we could elucidate the origins of the effect of
the C-17 substituent.
Figure 5. Compound 8 disrupts microtubule dynamics. The ability of
compound 8 to inhibit paclitaxel-stimulated tubulin stabilization was
assessed. In vitro tubulin assembly was measured by turbidity at 350
nm. Tubulin was preincubated with or without compound 8 (10 µM)
for 5 min at 37 °C. Tubulin assembly was then stimulated by adding
paclitaxel (10 µM). The change in the absorbance was continuously
monitored at 350 nm for 15 min at 37 °C.
As can been seen in Figure 6b, the sulfamate NH of 15
coordinates to the catalytic zinc ion (NsZn2+ ) 2.08 Å) of the
enzyme active site displacing the hydroxide ion present in the
active site of the uninhibited enzyme. The zinc ion is also
coordinated to three histidine residues (His94, His96, and
His119) with an overall tetrahedral geometry. Two hydrogen
bonds between the sulfamate and Thr 199 through their
respective NH and O groups (see Figure 6b) are also apparent,
with the steroid nucleus lying in the hydrophobic part of the
active site pocket. The presence of the 2-substituent has
previously proved to be detrimental to CAII inhibitory activity,
and it would thus appear that groups at the 2-position render
the interaction between the sulfamate group and the zinc atom
less favorable through steric hindrance with the proximal
residues of the active site. It is not, however, readily apparent
from this structure why 15 should be 4-fold less active than
either 2-MeOEMATE or 3 as an inhibitor of CAII. The electron
density around C-17 is ill defined in this structure, presumably
reflecting the rotational freedom of the cyanomethyl group and
in turn the absence of substantial energetically beneficial or
detrimental interactions with the protein at this site. The structure
of 3/hCAII did contrast to that of 15 in two respects, namely,
the unexpected coordination of the C-17 sulfamate to the
catalytic zinc and the presence of a second binding site for the
ligand, not observed in the 15/hCAII structure. Given the small
difference in inhibitory activity of the respective 3- and 17-O-
sulfamate derivatives of 1 (376 and 526 nM), we believe that
the bis-sulfamate derivative can bind through both of its
sulfamate groups and that the crystal structure only reflected
one of these modes. The presence of a second binding site (ca.
60% occupancy) may, however, be more relevant since it could
be possible that the nitrile substituted compound 15 has lower,
or negligible affinity, for this second site, thus resulting in a
lower overall affinity between the ligand and enzyme and
consequently a reduced inhibitory activity. Given the large size
of the pocket above the enzyme active site, it may be possible
that occupation of both of the ligand binding sites identified in
the 3/hCAII structure is required for high activity. Even with a
protein crystal structure, it is not possible to draw a definitive
conclusion on the origin of the reduced inhibitory activity of
15 against hCAII relative to other 2-substituted estratriene
sulfamate derivatives.
tubule disruptors. No further investigation on the interaction of
these novel ligands with tubulin has yet been made, although it
is notable that these ligands, like the previously studied broadly
similar 2-substituted estra-1,3,5(10)-triene-3-O-sulfamates, which
have been observed to bind tubulin in a competitive manner to
radio-labeled colchicine, destabilize the polymerization of
tubulin.11,37
Having determined that the 17-cyanomethyl compounds act
against cancer cell proliferation in vitro, we assayed the
compounds for activity against both STS and carbonic anhy-
drase. It was thought unlikely that the 17ꢀ-cyanomethyl
substituent would have a profound effect on STS inhibitory
activity since the key pharmacophore for inhibition of this
enzyme is the phenolic sulfamate group, with activity most
strongly influenced by the steric and electronic environment
around C-3. Compound 15 was evaluated for its ability to inhibit
the STS mediated conversion of tritiated E1S to E1 in placental
microsomes.38 An IC50 of 130 nM was obtained, thus confirming
that 17-cyanomethyl substitution has little detrimental effect on
STS inhibitory activity relative to the analogous 17-hydroxy
compound, 2-MeOE2MATE (2) (16 nM), or the 17-O-sulfamate
compound, 2-MeOE2bisMATE (3) (39 nM). The 2-ethyl
compound 8 was found to be less active as an STS inhibitor
with an IC50 of 1.4 ( 0.4 µM, reflecting the lower activity
generally observed for 2-ethyl compounds as inhibitors seen in
previous studies.6,9 It is thus evident that in addition to their
other anticancer activities, molecules such as 15, by functioning
as inhibitors of STS, could act against hormone dependent
cancers by lowering the levels of circulatory estrogen that
stimulate their growth.
The ability of estrogen-3-O-sulfamates to reversibly inhibit
carbonic anhydrase is well established, with the key interaction
being the coordination of the monoanionic form of the sulfamate
to the zinc ion of the active site. This reversible interaction of
phenolic sulfamates with carbonic anhydrase II could potentially
allow an estratriene sulfamate derivative the advantage of
sequestration by red blood cells in vivo and consequent
avoidance of first pass liver metabolism.27 Compound 15 was
thus assayed for inhibitory activity against CAII and was found
to have an IC50 value of 1.5 µM against this enzyme. The
activity of 15 is thus approximately 4-fold lower than the activity
of a number of 2-substituted estratriene-3-O-sulfamate deriva-
tives (e.g., 2-methoxyestrone-3-O-sulfamate (2-MeOEMATE),
IC50 of 376 nM and 3, IC50 of 379 nM) and also 3-fold less
In order to evaluate this class of compounds for in vivo
antitumor activity, it was decided to test 2-methoxy-17-
cyanomethyl estratriene-3-O-sulfamate (15) and the correspond-
ing 3-hydroxy compound 14 for their ability to inhibit the growth
of MDA-MB-231 xenografts in female nude athymic mice (n
) 6). The compounds were dosed orally once daily over a period
of 28 days as a solution in THF/PG (10%/90%) at doses of 40
and 80 mg/kg (15) and 120 mg/kg (14). As can be seen in Figure
7, promising inhibition of tumor growth was achieved with both
compounds. The higher 80 mg/kg dose of sulfamate 15 proved
active
than
2-methoxyestradiol-17-O-sulfamate
(2-
MeOE2–17MATE, IC50 of 526 nM), which possesses a less
acidic alkyl sulfamate group. The detrimental effects of

1302 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Leese et al.
Figure 7. Effects of daily oral administration of 14 and 15 on the
growth of MDA-MB-231 xenografts in athymic female nude mice.
the same model, caused tumor regression, although it is 4-fold
less potent than 15 in vitro. The difference in in vitro and in
vivo activities observed for 15 and 3 may derive from a number
of factors. The formulation used for this study has not been
rigorously investigated, and we have no information on the oral
bioavailability achieved for 15. It may also be that the nitrile
group of 15 is subject to rapid metabolism, a factor that could
lead to inactivation or accelerated clearance. In contrast,
pharmacokinetic studies have shown that 3 displays high oral
bioavailability in rodents (>85%) and does not undergo
significant metabolism. As noted above, the relative inhibitory
activity of 15 for carbonic anhydrase is 4-fold less than that
observed for 3, and this could possibly result in increased
exposure to metabolism for 15 relative to the bis-sulfamate. It
is thus clear that, although promising in vivo activity has been
observed for 15, pharmacokinetic studies would be highly
advantageous to establish the in vivo fate of the compound and
could direct the development of an enhanced formulation or
further structural modification to block potential metabolism.
Nevertheless, the structural modification intrinsic to 15 is clearly
worthy of further investigation, particularly that for 14, given
the problems already noted with 2-MeOE2.
Conclusions
In conclusion, we have discovered that a number of 17-
cyanomethyl functionalized 2-substituted estra-1,3,5(10)-triene
derivatives display high activity against the proliferation of
cancer cells in vitro. Enhanced activity is observed in both the
3-hydroxy and 3-O-sulfamoyl series of compounds, although
as observed in previous studies, the sulfamoylated compounds
display the most potent antiproliferative effects. SAR studies
defined that the C-17 cyanomethyl group most likely functions
as a hydrogen bond acceptor and that substitution of the
methylene linker between the nitrile and C-17, presumably due
to steric factors, is deleterious to activity. Direct linkage of the
nitrile group to C-17 and replacement of the nitrile group or
replacement an isosteric ethynyl group caused a great reduction
in activity. Antiproliferative activity against a wide range of
hormone dependent and independent cancer cells was observed,
and it appears that this activity, as with previously studied
2-substituted estra-1,3,5(10)-triene-3-O-sulfamates, stems from
their ability to interfere with the dynamics of tubulin polym-
erization. The sulfamate compounds showed activity against the
proliferation of HUVECs and in an in vitro model of angio-
genesis. Furthermore, the antiangiogenic profiles of these
compounds in vivo has recently been established.13 In addition,
Figure 6. (a) Ribbon diagram of the 15-hCAII complex obtained by
X-ray crystallography. The inhibitor molecule, metal coordinating
residues His94, His96, and His119, and the zinc ion are represented in
ball and stick format. (b) Detailed schematic representation of 15 within
the hCAII active site.
more effective than the 40 mg/kg dose, with growth inhibitions
of 66% and 34% being achieved, respectively. Dosing with the
phenol 14, albeit at the higher 120 mg/kg dose, caused a 44%
inhibition of tumor growth. No toxic effects were observed at
these dose levels (data not shown). A dose dependent inhibition
of tumor growth was thus observed with 15 treatment, while
significant activity was observed with 14 treatment. The relative
in vivo activities were found to correlate with their relative in
vitro activities. It is notable that 15 proved less active than the
corresponding bis-sulfamate, 2-MeOE2bisMATE 3 which, in

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1303
ments were taken weekly using electronic callipers. Tumor volume
(V), in mm3, was determined using the following equation: length
× width2/2 (l × w2/2).
activity of the sulfamoylated molecules against two tumor
relevant enzymes, namely, steroid sulfatase (a clinical target
for the treatment of HDBC) and carbonic anhydrase was
observed. The interaction of 15 with carbonic anhydrase was
studied by protein crystallography revealing, as expected, co-
ordination of the 3-O-sulfamate group to the catalytic zinc of
the hCAII active site. Confirmation of the potential of these
molecules for development as anticancer agents was demon-
strated by the significant growth inhibition achieved on treatment
of human breast cancer xenografts in mice with two of the novel
compounds being orally bioavailable. Further development of
antitumor agents based on structural modification of 2-MeOE2
is presently underway.
Protein Crystallography. The hCA II-15 complex was ob-
tained by adding a 5-molar excess of the inhibitor to a 10 mg/mL
protein solution in 100 mM Tris-HCl at pH 8.5. Crystals of the
complex were obtained by the hanging drop vapor diffusion
technique at 20 °C. The drops were prepared by mixing 2 µL of
the complex solution and 2 µL of the precipitant solution containing
2.8 M (NH4)2SO4, 300 mM sodium chloride in 100 mM Tris-HCl
(pH 8.4), and 1 mM dithiothreitol to improve the crystal quality.
A complete data set was collected at 1.85 Å resolution from a single
crystal of about 0.2 mm × 0.3 mm × 0.3 mm. Data collection was
carried out at Synchrotron source Elettra in Trieste, using a Mar
CCD detector. The crystal belonged to the P21 space group with
unit cell parameters a ) 42.17 Å, b ) 41.49 Å, c ) 71.58 Å, and
ꢀ ) 104.07°. Diffracted intensities were processed using the HKL
Experimental Procedures
crystallographic data reduction package (Denzo/Scalepack).42
A
Materials and Methods. Chemistry. All chemicals were either
purchased from Aldrich Chemical Co. (Gillingham, U.K.), Fluka
(Gillingham, U.K.) or Lancaster Synthesis (Morecambe, U.K.).
Organic solvents of A.R. grade were supplied by Fisher Scientific
(Loughborough, U.K.) and used as supplied. Anhydrous N,N-
dimethylformamide (DMF) and N,N-dimethylacetamide (DMA)
were purchased from Aldrich and stored under positive pressure
of N2 after use. THF was distilled from sodium with benzophenone
indicator. Sulfamoyl chloride was prepared by an adaptation of the
method of Appel and Berger40 and was stored in the refrigerator
under positive pressure of N2 as a solution in toluene as described
by Woo et al.41 An appropriate volume of this solution was freshly
concentrated in vacuo immediately before use. Estrone was
purchased from Sequoia Research Products (Oxford, U.K.). Reac-
tions were carried out at room temperature (rt) unless otherwise
stated. Flash column chromatography was performed on silica gel
(MatrexC60).
total of 59499 reflections was measured and reduced to 20038
unique reflections. Crystal parameters and data processing statistics
are summarized in tabular form in the Supporting Information. The
structure of the complex was analyzed by difference Fourier
techniques, using the PDB file 1CA243 as a starting model for
refinement. Water molecules were removed from the starting model
prior to structure factor and phase calculations. The inspection of
the Fourier maps, calculated with 3Fo - 2Fc and Fo - Fc
coefficients, revealed prominent electron density features in the
active site region. After an initial refinement limited to the enzyme
structure, a model for the inhibitor was easily built and introduced
into the atomic coordinates set for further refinement, which
proceeded to convergence with continuous map inspections and
model updates. The refinement was carried out with the program
CNS,44 and model building and map inspections were performed
using the O program.45 The final model contains 2068 protein
atoms, 28 inhibitor atoms, and 292 water molecules, and has Rfactor
and Rfree values of 17.0 and 20.3%, respectively. The correctness
of stereochemistry was finally checked using PROCHECK.46 The
statistics for refinement are summarized in the Supporting Informa-
tion. Coordinates and structure factors have been deposited in the
Brookhaven Protein Data Bank (accession code 3BET.pdb).
Crystal Data for 15. Empirical formula: C24H31N2O5S, M )
459.57, T ) 150(2) K, λ ) 0.71073 Å, monoclinic, space group P21,
a ) 8.2860(2) Å, b ) 13.6050(4) Å, c ) 11.2540(1) Å, ꢀ )
107.430(1)°, U ) 1210.42(6) Å3, Z ) 2, Dc ) 1.261 gcm-3, µ )
0.170 mm-1; F(000) ) 490, crystal size 0.35 × 0.35 × 0.30 mm,
unique reflections ) 7011 [R(int) ) 0.0759], observed I >2σ(I) )
5870, data/restraints/parameters: 7011/3/303, R1 ) 0.0470 wR2 )
0.1085 (obs. Data), R1 ) 0.0636, wR2 - 0.1162 (all data), max. peak/
hole: 0.493 and -0.293 eÅ-3, absolute structure parameter ) 0.00(6).
Data were collected on a Nonius kappaCCD diffractometer.
2-Ethyl-3-O-(t-butyldimethylsilyl)estrone (4). A solution of
2-ethylestrone6 (2.0 g, 6.7 mmol) and imidazole (1.14 g, 16.8 mmol)
in DMF (20 mL) was treated with t-butyl-dimethylsilyl chloride
(1.11 g, 7.38 mmol) and stirred for 14 h. Ethyl acetate (50 mL)
and water (100 mL) were then introduced and the organic layer
separated. After washing with water (3 × 50 mL), and brine (50
mL), the organic layer was dried and evaporated to give a colorless
oil, which solidified on standing. Crystallization from ethyl acetate/
hexane gave a white powder (1.98 g, 72%) with mp 95–97 °C,
which showed δH 0.22 (6H, s), 0.91 (3H, s), 1.01 (9H, s), 1.16
(3H, t, J 7.4), 1.32–2.50 (13H, m), 2.56 (2H, q, J 7.4), 2.78–2.88
(2H, m), 6.49 (1H, s) and 7.05 (1H, s); HRMS [ES+] m/z found
413.2851; C26H41O2Si (M+ + H) requires 413.2870.
1H NMR and 13C NMR spectra were recorded with a Varian
Mercury VX400 NMR spectrometer at 400 and 100 MHz,
respectively, and chemical shifts are reported in parts per million
(ppm, δ) relative to tetramethylsilane (TMS) as an internal standard.
Mass spectra were recorded at the Mass Spectrometry Service
Center, University of Bath, U.K. or at the EPSRC National Mass
Spectrometry service, Swansea. FAB-MS was carried out using
m-nitrobenzyl alcohol (NBA) as the matrix. Elemental analyses
were performed by Microanalysis Service, University of Bath.
Melting points were determined using a Reichert-Jung Thermo
Galen Kofler block and are uncorrected.
Biology. The effects of compounds on MCF-7, DU-145 and
MDA MB-231 cell growth were determined using a microtiter plate
assay as described previously.6,30 STS inhibition was assayed by
the method of Purohit et al.,38 and carbonic anhydrase inhibition
was carried out as described elsewhere.19 The effects of compound
8 on in vitro polymerization of purified bovine brain tubulin
(Cytoskeleton, Denver, CO) were measured by turbidometry using
a previously validated assay.11,37 Tubulin (1 mg/mL) in MES buffer
(0.1 M MES at pH 6.5, 0.5 mM MgCl2, 1 M monosodium
glutamate, and 1 mM GTP) was preincubated with or without test
compounds (10 µM, 1% v/v ethanol) for 5 min at 37 °C. Tubulin
assembly was then stimulated by adding paclitaxel (10 µM). The
change in the absorbance was continuously monitored at 350 nm
for 15 min at 37 °C. Because the amount of tubulin polymerized is
directly proportional to the area under the curve (AUC), it was
used to determine the extent of tubulin polymerization by different
test compounds. The AUC of paclitaxel alone, with a maximal
extent of polymerization, was set to 100% polymerization.
In Vivo Efficacy Studies. Female MF-1 nu/nu mice from Harlan
(Bicester, Oxon, U.K.) were injected subcutaneously (s.c.) in the
flank with 2 × 106 MDA-MB-231 cells, which resulted in a single
tumor per animal. All experiments were carried out under conditions
that complied with institutional guidelines. Daily oral administration
of 15 (40 and 80 mg/kg) and 14 (120 mg/kg) was initiated when
the tumors reached approximately 100–150 mm3 in volume. Dosing
was performed for 28 days. Animal weights and tumor measure-
2-Ethyl-3-O-(t-butyldimethylsilyl)-17-cyanomethylene Estra-
1,3,5(10)-triene (5). Sodium hydride (302 mg, 7.6 mmol) was
washed with three aliquots of hexane (1 mL each), dried under
nitrogen, then treated with THF (10 mL), and then diethyl
(cyanomethyl)phosphonate (1.17 mL, 7.3 mmol), at which point
vigorous gas evolution was observed. The resultant solution was
then treated with a solution of 4 (1.82 g, 4.4 mmol) in THF (20
mL) and brought to reflux for 18 h. The reaction mixture was then

1304 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Leese et al.
cooled to rt, diluted with ethyl acetate (25 mL), and poured onto
water (10 mL); the aqueous layer was separated and the organic
layer washed with water (3 × 25 mL) and brine (25 mL), dried,
and evaporated. The crude product was purified by column
chromatography (3% ethyl acetate/hexane) to give the desired
product 5 as a mixture of isomers. White foam (1.81 g, 4.16 mmol,
94%). δH (selected) 0.21 (6H, s), 0.87 and 0.98 (3H, 2 × s), 0.99
(9H, s), 1.15 (3H, t, J 7.4), 2.55 (2H, q, J 7.4),:CH at 5.12 (app t,
J 2.0) and 5.04 (app t, J 2.4), 6.47 (1H, s) and 7.03, 7.04 (1H, 2 ×
s); MS [FAB+] m/z 435.3 (M+, 100%); HRMS [FAB+] m/z found
435.2961, C28H41ONSi requires 435.2957.
2-Ethyl-3-O-(t-butyldimethylsilyl)-17ꢀ-cyanomethyl Estra-
1,3,5(10)-triene (6). Compound 5 (1.80 g, 4.13 mmol) was
dissolved in THF (5 mL) and ethanol (50 mL) then treated with
Pd/C (250 mg, 10%). The degassed solution was then stirred under
a hydrogen atmosphere for 16 h before filtering through a pad of
celite. Evaporation gave the desired product 6 (1.58 g, 87%) as a
white foam. δH 0.21 (6H, s), 0.66 (3H, s), 0.99 (9H, s), 1.16 (3H,
t, J 7.4), 1.22–2.42 (16H, m), 2.55 (2H, q, J 7.4), 2.76–2.84 (2H,
m), 6.47 (1H, s) and 7.03 (1H, s); MS [FAB+] m/z 437.4 (M+,
100%); HRMS [FAB+] m/z found 437.3121; C28H43OSiN requires
437.3114. Anal. (C28H43NO2Si) C, H, N.
2-Ethyl-17ꢀ-cyanomethyl Estra-1,3,5(10)-trien-3-ol (7). A
solution of 6 (1.18 g, 2.70 mmol) in THF (12 mL) was treated
with TBAF (4 mL, 1 M in THF). After 1 h, the reaction was diluted
with ethyl acetate (25 mL) and then washed with water (3 × 30
mL), brine (30 mL), dried, and evaporated to give a yellowy solid.
The crude product was suspended in hot hexane, and the desired
product 7, a white solid with mp 180–182 °C, was collected by
filtration (770 mg, 88%). δH 0.66 (3H, s), 1.15–2.45 (19H, m
including 1.21 (3H, t, J 7.6)), 2.58 (2H, q, J 7.6), 2.75–2.85 (2H,
m), 4.46–4.56 (1H, br), 6.49 (1H, s) and 7.03 (1H, s); MS [FAB+]
m/z 323.2 (M+, 100%); HRMS [FAB+] m/z found 323.2252;
C22H29ON requires 323.2249.
was purified by chromatography (9:1 hexane/EtOAc) to give a white
powder (1.5 g, 73%) with mp 162–64 °C, which showed δH 0.15
(6H, s), 0.92 (3H, s), 0.99 (9H, s), 1.38–2.54 (13H, m), 2.75–2.83
(2H, m), 3.77 (3H, s), 6.56 (1H, s), 6.76 (1H, s); MS [ES-] m/z
413.3 (70%, M+ - H); HRMS [ES+] m/z found 415.2660;
C25H39O3Si (M+ + H) requires 415.2663.
2-Methoxy-3-O-(t-butyldimethylsilyl)-17-cyanomethylene
Estra-1,3,5(10)-triene (12). Sodium hydride (204 mg, 5.1 mmol)
in THF (10 mL) was reacted with diethyl (cyanomethyl)phospho-
nate (776 µL, 4.8 mmol) and 2-methoxy-3-O-t-butyldimethylsilyl
estrone (1.82 g, 4.4 mmol) in THF (10 mL) as described for the
synthesis of 5. The crude product was purified by column
chromatography (4% ethyl acetate/hexane) to give the desired
product 12 (a 3:2 mixture of E- and Z-isomers) as a white solid
(1.04 g, 79%). δH (selected) 0.13 (6H, s), 0.98 (9H, s), 0.99, 0.88
(3H, 2 × s), 3.77 (3H, s), 5.12, 5.04 (1H, 2 × m, :CH, both
isomers), 6.54 (1H, s) and 6.75, 6.74 (1H, 2 × s); MS [FAB+] m/z
438.1 [(M + H)+, 50%] and 380.1 (100%); HRMS [FAB+] m/z
found 437.2731; C27H39NO2Si requires 437.2750.
2-Methoxy-3-O-(t-butyldimethylsilyl)-17ꢀ-cyanomethyl Estra-
1,3,5(10)-triene (13). A solution of 12 (0.98 g, 2.24 mmol) in THF
(15 mL) and ethanol (100 mL) was reacted with Pd/C (200 mg,
5%) and hydrogen over 48 h as described for the synthesis of 6.
The desired product 13 was isolated as a white solid (0.92 g, 94%)
with mp 149–151 °C. δH 0.14 (6H, s), 0.68 (3H, s), 0.98 (9H, s),
1.24–2.42 (16H, m), 2.70–2.76 (2H, m), 3.76 (3H, s), 6.53 (1H, s)
and 6.75 (1H, s). Anal. (C27H41NO2Si) C, H, N.
2-Methoxy-17ꢀ-cyanomethyl Estra-1,3,5(10)-trien-3-ol (14).
A solution of 13 (0.92 g, 2.09 mmol) in THF (20 mL) was reacted
with TBAF (3 mL, 1 M in THF) as described for the synthesis of
7. The desired phenol 14 was crystallized from ethyl acetate/hexane
to give colorless crystals (600 mg,88%) with mp 172–174 °C. δH
0.68 (3H, s), 1.24–2.42 (16H, m), 2.72–2.80 (2H, m), 3.85 (3H, s),
5.42 (1H, s), 6.63 (1H, s) and 6.77 (1H, s); MS [ES-] m/z 309
((M+ -H)-, 100%). Anal. (C21H27NO2) C, H, N.
2-Ethyl-3-O-sulfamoyl-17ꢀ-cyanomethyl
Estra-1,3,5(10)-
triene (8). An ice cold solution of sulfamoyl chloride (4.76 mmol)
in DMA (5 mL) was added to 7 (770 mg, 2.38 mmol). After 14 h
of stirring, ethyl acetate (25 mL) was added, and the solution was
washed with water (3 × 20 mL) and brine (20 mL), dried (Na2SO4),
and evaporated. The crude product was purified by column
chromatography (10% acetone in chloroform) to give 8 as a white
crystalline solid with mp 175–177 °C (700 mg, 73%). δH 0.67 (3H,
s), 1.21 (3H, t, J 7.4), 1.25–2.42 (16H, m) 2.68 (2H, q, J 7.4),
2.80–2.88 (2H, m), 4.91 (2H, br), 7.07 (1H, s) and 7.17 (1H, s);
MS [FAB+] m/z 402.0 (M+, 100%); HRMS [FAB+] m/z found
402.1975; C22H30O3N2S requires 402.1977. Anal. (C22H30N2O3S).
E-2-Ethyl-17-cyanomethylene Estra-1,3,5(10)-trien-3-ol (9).
A solution of 5 (540 mg, 1.24 mmol) in THF (10 mL) was reacted
with TBAF (3 mL, 3 mmol) as described for the synthesis of 7.
Column chromatography (20% ethyl acetate/hexane) afforded the
desired phenol 9 as a pale yellow oil (284 mg, 0.882 mmol, 71%).
Crystallization from ethyl acetate/hexane gave white crystals with
mp 168–170 °C. δH 0.88 (3 H), 1.22 (3 H, t, J 7.4), 1.25–2.45 (11
H, m), 2.59 (2 H, q, J 7.4), 2.63–2.84 (4 H, m), 4.53 (1 H, s), 5.04
(1 H, t, J 2.3), 6.49 (1 H, s) and 7.02 (1 H, s); MS [FAB+] m/z
321.3 (M+, 100%); HRMS [FAB+] m/z found 321.2088; C22H27ON
requires 321.2093. Anal. (C21H25NO2) C, H, N.
E-2-Ethyl-3-O-sulfamoyl-17-cyanomethylene Estra-1,3,5(10)-
triene (10). Compound 9 (110 mg, 0.34 mmol) was reacted with
sulfamoyl chloride (2 equiv) in DMA (1.5 mL) as described for
the synthesis of 8. Column chromatography (10% acetone in
chloroform) gave the desired product as a white foam (86 mg, 63%).
δH 0.87 (3H, s), 1.21 (3H, t, J 7.4), 1.23–1.63 (6H, m), 1.90–1.99
(3H, m), 2.20–2.29 (1H, m), 2.39–2.46 (1H, m), 2.58–2.82 (4H,
m, including 2.69 (2H, q, J 7.4)), 2.83–2.89 (2H, m), 5.00 (2H,
br), 5.05 (1H, t, J 2.6), 7.09 (1H, s) and 7.18 (1H, s); MS [ES-]
m/z 399.5 (M+ - H, 100%). Anal. (C21H28N2O4S) C, H, N.
2-Methoxy-3-O-(t-butyldimethylsilyl)estrone (11). 2-Meth-
oxyestrone6 (1.5 g, 5 mmol) and imidazole (408 mg, 6 mmol) in
DMF (20 mL) was reacted with t-butyl-dimethylsilyl chloride (829
mg) as described for the synthesis of 4. The resultant crude oil
2-Methoxy-3-O-sulfamoyl-17ꢀ-cyanomethyl Estra-1,3,5(10)-
triene (15). Compound 14 (570 mg, 1.75 mmol) was reacted with
sulfamoyl chloride and DMA as described for the synthesis of 8.
Column chromatography (chloroform/aceone 9:1) followed by
recrystallization from acetone/hexane afforded the sulfamate as a
white crystalline solid (15) (590 mg, 83%) with mp 183–185 °C.
δH 0.69 (3H, s), 1.20–2.50 (16H, m, alkyl H), 2.74–2.84 (2H, m),
3.87 (3H, s), 5.06 (2H, s), 6.92 (1H, s) and 7.04 (1H, s); MS [FAB+]
m/z 404.1 (M+, 100%); HRMS [FAB+] m/z found 404.1767;
C21H28O4N2S requires 404.1770. Anal. (C21H28O4N2S) C, H, N.
E-2-Methoxy-17-cyanomethylene
Estra-1,3,5(10)-trien-3-ol
(16). A solution of 12 (400 mg, 0.95 mmol) in THF (10 mL) was
reacted with TBAF (3 mL) as described for the synthesis of 7. Column
chromatography (10% ethyl acetate in hexane) afforded two fractions,
the first being a mixture of the two isomers as a white powder (91
mg, 30%) and the second E-isomer 16 as a white solid with mp
183–185 °C (115 mg, 0.36 mmol, 37%). δH (CDCl3) 0.89 (3H, s),
1.25–2.39 (11H, m), 2.56–2.82 (4H, m), 3.86 (3H, s), 5.05 (1 H, t, J
2.7), 5.43 (1H, s), 6.65 (1H, s), 6.77 (1H, s); MS (FAB+) m/z 323.1
(M+, 100%); HRMS [ES+] m/z found 324.1961; C21H26O2N (M++H)
requires 324.1958. Anal. (C22H27NO) C, H, N.
2-Methoxy-3-O-sulfamoyl-17-cyanomethylene Estra-1,3,5(10)-
triene (17). Compound 16 (40 mg, 0.124 mmol) was treated with
sulfamoyl chloride (1.5 mmol) in DMA (1.5 mL) as described for
the synthesis of 8. Flash column chromatography (hexane/ethyl
acetate 4:1) afforded the desired sulfamate 17 as white needles with
mp 202–204 °C (36 mg, 72%). δH (d6-acetone) 0.95 (3H, s),
1.25–2.66 (12H, m), 2.74–2.84 (3H, m), 3.84 (3H, s), 5.27 (1H, t,
J 2.3), 6.90 (2H, s), 7.02 (1H, s), 7.04 (1H, s); MS [FAB+] m/z
402.0 (M+, 100%); HRMS [FAB+] m/z found 402.1611;
C21H26O4N2S requires 402.1613. Anal. (C22H28N2O3S) C, H, N.
2-Ethyl-3-O-(t-butyldimethylsilyl)-17-(1,1-dicyano)methylene Es-
tra-1,3,5(10)-triene (18). A solution of 4 (830 mg, 2 mmol),
malononitrile (0.38 mL, 10 mmol), and ꢀ-alanine (535 mg, 6
mmol) in toluene (150 mL) and acetic acid (30 mL) was refluxed
for 3 days with extra aliquots of malononitrile (0.13 mL, 2 mmol)

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1305
added after 24 and 48 h. The solution was then cooled to rt, the
solvents evaporated, and the residual solid stirred with water
(50 mL). The mixture was then extracted with ethyl acetate (2
× 100 mL), and the resultant organic layer was washed with
water and brine, then dried, and evaporated. Column chroma-
tography (hexane/ethyl acetate 5:1) gave the desired alkene 18
as a white powder with mp 168–169 °C (840 mg, 91%). δH 0.21
(6H, s), 1.00 (9H, s), 1.06 (3H, s), 1.15 (3H, t, J 7.4), 1.40–2.26
(9H, m), 2.45–3.05 (m, 7H), 6.40 (1H, s) and 7.02 (1H, s); MS
[ES-] m/z 459.4 (M+ - H)-, 100%); HRMS [ES+] m/z found
461.2985; C29H41N2OSi (M++H) requires 461.2983.
further 0.5 h, methyl iodide (212 mg, 1.5mmol) was added. The
mixture was stirred for 4 h at -78 °C and 12 h at rt before the
addition of ethyl acetate (100 mL) and water (30 mL). The organic
layer was then separated, washed with water and brine, dried, and
evaporated. The crude yellow oil was purified by column chroma-
tography (hexane/ethyl acetate 20:1) to give the desired product
24 as a colorless oil (176 mg, 78%) as a 1:1 mixture of
diastereoisomers. δH (selected) 0.23 (6H, s), 0.76 and 0.78 (3H, 2
× s), 1.01 (9H, s), 1.17 (2H, t, J 7.4), 1.25–1.96 (13H, m), 2.05–2.50
(3H, m), 2.57 (2H, q, J 7.4), 2.66–2.88 (4H, m), 6.48 and 6.50
(1H, 2 × s), 7.03 and 7.06 (1H, 2 × s); HRMS [ES+] m/z found
452.3349; C29H46OSi (M+ + H) requires 452.3347.
2-Ethyl-3-O-hydroxy-17-(1,1-dicyano)methylene
Estra-
2-Ethyl-3-O-benzyl Estrone (25). A mixture of 2-ethyl estrone6
(5.6 g, 18.8 mmol) and potassium carbonate (5.19 g, 37.5 mmol)
in DMF (100 mL) was treated with benzyl chloride (2.81 mL) and
then heated to 120 °C for 4 h. The reaction was then cooled to rt
prior to the addition of sufficient water to dissolve the potassium
carbonate then precipitate the product. The resultant beige powder
was removed by filtration then resuspended in hot methanol to
remove colored impurities. The product was then collected by
filtration to give 25 as a white powder (6.1 g, 84%) with mp
169–171 °C, which showed 0.90 (3H, s), 1.20 (3H, t, J 7.4),
1.34–2.56 (13H, m), 2.66 2H, q, J 7.4), 2.83–2.92 (2H, m), 5.04
(2H, s), 6.64 (1H, s), 7.10 (1H, s) and 7.28–7.45 (5H, m); HRMS
[ES+] m/z found 389.2466; C27H33O2 (M+ + H) requires 389.2475.
Anal. (C27H32O2) C, H.
1,3,5(10)-triene (19). A solution of 18 (230 mg, 0.5 mmol) in THF
(50 mL) was cooled to 0 °C and treated with TBAF (0.6 mmol,
0.6 mL) in a dropwise manner. After 2 h at 0 °C, the reaction was
allowed to come to rt and then treated with water (10 mL) and
ethyl acetate (80 mL). The resultant organic layer was separated,
washed with water and brine, then dried, and evaporated. The
residual solid was purified by column chromatography (hexane/
ethyl acetate 5:1 to 2:1) to give phenol 19 as a white solid with mp
269–270 °C (125 mg, 72%). δH 1.06 (3H, s), 1.21 (3H, t, J 7.5),
1.39–2.52 (10H, m), 2.61 (2H, q, J 7.5), 2.63–3.02 (5H, m), 4.52
(1H, s), 6.50 (1H, s) and 7.02 (1H, s); HPLC: 98.9%, MS [APCI-]
m/z 345.4 ((M+ - H)-, 100%).
2-Ethyl-3-O-sulfamoyl-17-(1,1-dicyano)methylene
Estra-
1,3,5(10)-triene (20). Compound 19 (69 mg, 0.2 mmol) was reacted
with sulfamoyl chloride (0.6 mmol) in DMA (2 mL) as described
for the synthesis of 8. Column chromatography (hexane/ethyl acetate
5:1 to 3:1) gave the desired sulfamate 20 as a white powder with
mp 227–228 °C (62 mg, 73%). δH 1.06 (s, 3H), 1.20 (3H, t, J 7.4),
1.44–2.55 (10H, m), 2.69 (2H, q, J 7.4), 2.75–3.04 (5H, m), 5.03
(2H, s), 7.09 (1H, s) and 7.16 (1H, s); HPLC 100%. Anal.
(C23H27N3O3S) C, H, N.
2-Ethyl-3-O-t-butyldimethylsilyl-17ꢀ-(1,1-dicyano)methyl
Estra-1,3,5(10)-triene (21). A solution of 18 (460 mg, 1 mmol) in
methanol (50 mL) and THF (5 mL) was cooled to -10 °C, then
treated with NaBH4 (76 mg, 2 mmol) in a portionwise manner.
After 4 h of stirring, water (50 mL) was added, and then sufficient
ammonium chloride was added to acidify the solution. The mixture
was then extracted with ethyl acetate (2 × 60 mL) and the combined
organics washed with water and brine, dried, and evaporated.
Column chromatography (hexane/ethyl acetate 5:1) gave the desired
product 21 as a white powder with mp 161–162 °C (380 mg, 83%).
δH 0.22 (6H, s). 0.80 (3H, s), 1.00 (9H, s), 1.16 (3H, t, J 7.5),
1.33–2.40 (14H, m), 2.55 (2H, q, J 7.5), 2.78 (2H, m), 3.56 (1H,
d, J 9.9), 6.47 (1H, s) and 7.03 (1H, s); MS [ES-] m/z 461.3 ((M+
- H)-, 100%); HRMS [ES+] m/z found 463.3152; C29H43N2OSi
(M++H) requires 463.3139.
2-Ethyl-17ꢀ-(1,1-dicyano)methyl Estra-1,3,5(10)-trien-3-ol
(22). A solution of 21 (230 mg, 0.5 mmol) in THF (50 mL) was
reacted with TBAF (0.6 mmol, 0.6 mL) as described for the
synthesis of 6. Column chromatography (hexane/ethyl acetate 5:1
to 2:1) gave 22 as a white solid with mp 247–248 °C (124 mg,
71%). δH 0.80 (3H, s), 1.21 (3H, t, J 7.4), 1.30–2.41(14H, m), 2.59
(2H, q, J 7.4), 2.81 (2H, m), 3.56 (1H, d, J 9.9), 4.51 (1H, br),
6.49 (1H, s) and 7.02 (1H, s); MS [APCI-] m/z 347.4 (M+ - H)-,
HRMS [ES+] m/z found 349.2264; C23H29N2O (M+ + H) requires
349.2274. Anal. (C23H28N2O) C, H, N.
2-Ethyl-3-O-sulfamoyl-17ꢀ-dicyanomethyl Estra-1,3,5(10)
-triene (23). A solution of 22 (0.2 mmol) was reacted with
sulfamoyl chloride (0.6 mmol) in DMA (2 mL) as described for
the synthesis of 8. Column chromatography (hexane/ethyl acetate
5:1 to 3:1) gave the desired sulfamate 23 (170 mg, 80%) as a white
powder with mp 169–170 °C. δH 0.80 (3H, s), 1.21 (3H, t, J 7.4),
1.32–2.38 (14H, m), 2.68 (2H, q, J 7.4), 2.83 (2H, m), 3.57 (1H,
d, J 9.9), 4.91 (2H, br), 7.08 (1H, s) and 7.16 (1H, s); MS [FAB-]
m/z 347.4 ((M+ - H)-, 100%). Anal. (C23H29N3O3S) C, H, N.
2-Ethyl-3-O-(t-butyldimethylsilyl)-17ꢀ-(1-cyanoethyl)
2-Ethyl-3-O-benzyl-17r-hydroxy-17ꢀ-(1-cyanoethyl) Estra-
1,3,5(10)-triene (26). A -78 °C solution of propionitrile (50 mmol)
in dry THF (50 mL) was treated with LDA (50 mmol) in a dropwise
manner, and then stirred for 1 h prior to the addition of 25 (8 mmol)
in THF (20 mL) over 3 h. The reaction was then stirred for an
additional 3 h prior to ammonium chloride quench. The mixture
was then extracted into ethyl acetate and the organic layer washed
with water and brine, dried, and evaporated. The crude oil was
purified by column chromatography (hexane/ethyl acetate gradient
10:1 to 4:1) to give the desired alcohol 26 (2.70 g, 76%) as a white
powder with mp 75–77 °C, which showed δH 0.97 (3H, s), 1.20
(3H, t, J 7.6), 1.25–2.49 (16H, m), 2.66 (2H, q, J 7.6), 2.77–2.91
(3H, m), 5.04 (2H, s), 6.62 (1H, s), 7.09 (1H, s) and 7.28–7.46
(5H, m). MS [ES+] m/z 466.36 (M+ + Na)+, 100%).
2-Ethyl-3-O-benzyl-17-(1-cyano)ethylene
Estra-1,3,5(10)-
triene (27). Methane sulfonic acid (0.1 mL, 1.4 mmol) was added
dropwise to a solution of 26 (500 mg, 1.1 mmol) in triethylamine
(5 mL) and DCM (50 mL), and then stirred at 0 °C for 4 h. The
reaction mixture was poured onto ice/water (20 mL), and the
organics were extracted with ethyl acetate (2 × 50 mL). The organic
layer was washed with water then brine, dried, and evaporated to
give a crude oil containing a mixture of the two possible dehydration
products (∆17:∆16 ratio ca. 95:5 as measured by NMR). Column
chromatography (hexane/ethyl acetate 10:1) gave the desired ∆-17
alkene 27 (400 mg, 85%) as a white powder with mp 63–64 °C,
which showed δH 0.95 (3H, s), 1.21 (3H, t, J 7.3), 1.30–1.71 (6H,
m), 1.84 (3H, s), 1.85–2.51 (6H, m), 2.64 (2H, q, J 7.3), 2.75–2.90
(3H, m), 5.04 (2H, s), 6.63 (1H, s), 7.10 (1H, s) and 7.28–7.48
(5H, m); MS [ES-] m/z 424.35 ((M+ - H)-, 100%); HRMS [ES+]
m/z found 426.2788, C30H37NO (M++H) requires 426.2791.
Alternatively, a solution of 26 (220 mg, 0.5 mmol) and Burgess
reagent (358 mg, 1.5 mmol) in DMF (5 mL) was stirred at rt for 1
day. After the addition of water, the organics were extracted with
ethyl acetate, the organic layer washed with water and brine, dried,
and evaporated. The resultant yellow oil (230 mg) was purified by
chromatography (hexane/EtOAc 20:1 to 15:1) to give 27 as a white
powder (165 mg, 78%), which was spectroscopically identical to
that described above.
2-Ethyl-3-hydroxy-17ꢀ-(1-cyano)ethyl Estra-1,3,5(10)-triene
(28). A solution of 27 (430 mg, 1mmol) in methanol (45 mL) and
THF (5 mL) was treated with 5% Pd/C (50 mg) and then was stirred
under an atmosphere of H2. After standard workup column
chromatography (hexane/ethyl acetate 10:1 to 1:1), recrystallization
(hexane/ethyl acetate 5:1) gave the desired phenol 28 as a white
powder with mp 235–236 °C (225 mg, 67%). δH 0.76 (3H, s), 1.20
Estra-1,3,5(10)-triene (24). A stirred -78 °C solution of 6 (220
mg, 0.5 mmol) in THF (50 mL) was treated with LDA (1.2 mL,
1.2 mmol, 1 M in THF) in a dropwise manner, and then after a

1306 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Leese et al.
(3H, t, J 7.4), 1.24–1.69 (13H, m), 1.71–2.48 (4H, m), 2.57 (2H,
q, J 7.3), 2.71 (1H, m), 2.78 (2H, m), 4.47 (1H, br), 6.49 (1H, s)
and 7.03 (1H, s); MS [ES-] m/z 336.3 ((M+ - H)-, 100%). Anal.
(C23H31NO) C, H, N.
2-Ethyl 3-O-acyl-17-hydroxy-17-cyano Estra-1,3,5(10)-
triene (34). A solution of 33 (1.7 g, 5 mmol) and KCN (3.26 g,
50mmol) in methanol (20 mL) and acetic acid (5 mL) was stirred
at room temperature for 5 days. Ice–water (50 mL) was then added
to the mixture, and the resulting precipitate was separated by
filtration. The residue was washed with copious amounts of water
and then dissolved in ethyl acetate (100 mL). The resultant solution
was then washed with water and brine, dried, and evaporated to
give 34 as a white solid with mp 147–148 °C (1.6 g, 87%). δH
0.85 (3H, s), 1.16 (3H, t, J 7.4), 1.33–2.06 (11H, m), 2.30 (3H, s),
2.25–2.58 (4H,m), 2.70 (2H, q, J 7.4), 2.82 (2H, m), 6.71 (1H, s)
and 7.15 (1H, s); HRMS [FAB+] m/z found 368.2229; C23H30NO3
(M+ + H) requires 368.2220. Anal. (C23H29NO3) C, H, N.
2-Ethyl-3-O-acyl-17-cyano Estra-1,3,5(10),16-tetraene (35). A
solution of 34 (1.47 g, 4 mmol) in dry pyridine (10 mL) was treated
with SOCl2 (1.46 mL, 20 mmol) in a dropwise manner. The solution
was brought to reflux for 1 h, then cooled to 0 °C before bringing
to pH 1 with HCl (5 M aqueous). After extraction into ethyl acetate
(3 × 30 mL), the combined organic layers were washed with water
and brine, then dried and evaporated to give a dark brown oil.
Column chromatography (hexane/ethyl acetate 8:1 to 6:1) gave 35
(500 mg, 36%) as a white powder with mp 176–177 °C, which
showed δH 0.94 (3H, s), 1.16 (3H, t, J 7.4), 1.36–2.26 (9H, m),
2.30 (3H, s), 2.39–2.52 (4H, m), 2.86 (2H, m), 6.65 (1H, dd, J 3.5
and 2.0), 6.72 (1H, s) and 7.14 (1H, s); HRMS [FAB+] m/z found
350.2115; C23H28NO2 requires 350.2115. Anal. (C23H28NO2.
0.25H2O) C, H, N.
2-Ethyl-3-O-sulfamoyl-17ꢀ-(1-cyano)ethyl Estra-1,3,5(10)-
triene (29). Compound 28 (100 mg, 0.3mmol) was reacted with a
solution of sulfamoyl chloride (0.8 mmol) in DMA (2 mL) as
described for the synthesis of 8. Column chromatography (hexane/
ethyl acetate 5:1 to 2:1), then recrystallization (hexane/ethyl acetate
5:1) gave the desired sulfamate 29 as a white powder with mp
171–172 °C (86 mg, 69%). δH 0.76 (3H, s), 1.20 (3H, t, J 7.4),
1.22–1.64 (11H, m), 1.75–2.35 (6H, m), 2.66 (2H, q, J 7.3), 2.68
(1H, m), 2.84 (2H, m), 4.92 (2H, br), 7.07 (1H, s) and 7.16 (1H,s);
MS [ES-] m/z 415.4 ((M+ - H)-, 100%). Anal. (C23H32N2O3S)
C, H, N.
2-Ethyl-3-O-t-butyldimethylsilyl-17ꢀ-(2′-oxoethyl)
Estra-
1,3,5(10)-triene (30). A 0 °C solution of 6 (550 mg, 1.26 mmol)
in dichloromethane (7 mL) and toluene (15 mL) was treated with
DIBALH (1.26 mL, 1.26 mmol, THF solution) and then allowed
to warm to room temperature. Further aliquots of DIBALH (1 and
0.5 mL) were added (after 1 h and 1.5 h) until TLC analysis showed
that no starting material remained. A solution of Rochelle’s salt
(20 mL, sat.) was then added, and the reaction was stirred for a
further 0.5 h before extracting with ethyl acetate (3 × 30 mL).
The combined organic layers were then washed with water and
brine, then dried, and evaporated. The resulting oil was purified
by column chromatography (hexane/ ethyl acetate to 19:1) to afford
the desired aldehyde 30 as a white powder with mp 78–80 °C (340
mg, 61%). δH 0.25 (6H, s), 0.66 (3H, s), 1.02 (9H, s), 1.19 (3H, t,
J 7.4), 1.23–2.56 (16H, m), 2.58 (2H, q, J 7.4), 2.74–2.88 (2H,
m), 6.50 (1H, s), 7.07 (1H, s) and 9.81 (1H, t, J 2.2). MS [APCI+]
m/z 441.4 (100%, (M+ + H)).
2-Ethyl-3-O-acyl-17ꢀ-cyano Estra-1,3,5(10)-triene (36). A
solution of 35 (350 mg, 1 mmol) in THF (5 mL) and methanol (30
mL) was reacted with 5% Pd/C (50 mg) under an atmosphere of
hydrogen for 24 h as described for the synthesis of 6. Column
chromatography (hexane/ethyl acetate 8:1 to 3:1) gave 36 as a white
powder with mp 195–196 °C (330 mg, 94%). δH 0.99 (3H, s), 1.21
(3H, t, J 7.4), 1.22–2.32 (12H, m), 2.35 (3H, s), 2.38–2.47 (2H,
m), 2.53 (2H, q, J 7.4), 2.88 (2H, m), 6.76 (1H, s) and 7.19 (1H,
s); MS [APCI-] m/z 350.11 (M+ - H)-.
2-Ethyl-17ꢀ-prop-2-ynyl Estra-1,3,5(10)-trien-3-ol (31). A
mixture of (1-diazo-2-oxo-propyl)-phosphonic acid dimethyl ester33
(0.19 g, 1.0 mmol) and dry K2CO3 (138 mg, 1.0 mmol) in anhydrous
methanol (2.5 mL) was stirred under nitrogen and cooled to 0 °C,
then treated with a solution of 30 (130 mg, 0.3mmol) in DCM (1
mL). The mixture was stirred for 24 h at room temperature after
which water (10 mL) and DCM (50 mL) were added to the solution.
The organic layer was washed with water and brine, dried, and
evaporated. The resulting oil was purified by flash chromatography
(gradient hexane/ethyl acetate 40:1 to 10:1) to give the desired
alkyne 31 as a colorless oil (60 mg, 62%), which showed δH 0.63
(3H, s), 1.21 (3H, t, J 7.3), 1.20–2.31 (17H, m), 2.57 (2H, q, J
7.3), 2.78 (2H, m), 4.58 (1H, s), 6.45 (1H, s) and 7.04 (1H, s);
HRMS [FAB+] m/z found 322.2288; C23H30O requires 322.2296.
NB: A 7% yield of the TBDMS protected alkyne product was also
isolated.
2-Ethyl-17ꢀ-cyano Estra-1,3,5(10)-trien-3-ol (37). A solution
of 36 (280 mg, 0.8 mmol) in acetone (10 mL) and methanol (10
mL) was treated with NaHCO (0.34 g, 4 mmol) then stirred for
3
24 h. Solids were then removed by filtration and the filtrate
evaporated. The resulting solid was treated with 2 M aqueous HCl
to pH 1, and the solution was extracted with ethyl acetate (3 × 25
mL). The combined organics were then washed with water and
brine, then dried, and evaporated. The resulting solid was purified
by chromatography (hexane/ethyl acetate 8:1 to 6:1) and then
recrystallized from hexane/ethyl acetate (4:1) to give 37 as a white
powder with mp 284–285 °C (210 mg, 85%). δH 0.95 (3H, s), 1.21
(3H, t, J 7.4), 1.30–2.42 (14H, m, 2.58 (2H, J 7.4), 2.78 (2H, m),
4.50 (1H, s), 6.49 (1H, s) and 7.03 (1H, s); MS [APCI-] m/z 308.29
(M+ - H)-. Anal. (C21H27NO) C, H, N.
2-Ethyl-3-O-sulfamoyl-17ꢀ-cyano Estra-1,3,5(10)-triene (38). Com-
pound 37 (125 mg, 0.4 mmol) was reacted with sulfamoyl chloride
(0.8 mmol) in DMA (2 mL) as described for the synthesis of 8.
Column chromatography (hexane/ethyl acetate 5:1) followed by
crystallization (hexane/ethyl acetate 5:1) gave the desired sulfamate
38 as a white powder (135 mg, 86%) with mp 193–194 °C, which
showed δH 1.00 (3H, s), 1.25 (3H, t, J 7.4), 1.28–2.42 (14H, m),
2.73 (2H, q, J 7.4), 2.88 (2H, m), 4.99 (2H, s), 7.13 (1H, s) and
7.23 (1H, s); MS [APCI-] m/z 387.31 (M+ - H)-. Anal.
(C21H28N2O3S) C, H, N.
2-Ethyl-17-cyano Estra-1,3,5(10),16-tetraen-3-ol (39). A solu-
tion of 35 (210 mg, 0.6 mmol) in acetone (10 mL) and methanol
(10 mL) was reacted with NaHCO3 (150 mg, 1.8 mmol) as
described for the synthesis of 37. Column chromatography (hexane/
ethylacetate 8:1 to 6:1) followed by recrystallization (hexane/ethyl
acetate 6:1) gave the desired phenol 39 as a white powder (165
mg, 90%) with mp 213–214 °C, which showed δH 0.88 (3H, s),
1.16 (3H, t, J 7.5), 1.31–2.41 (11H, m), 2.53 (2H, q, J 7.5),
2.70–2.84 (2H, m), 4.48 (1H, s), 6.44 (1H, s), 6.60 (1H, dd, J 3.3
and 1.8) and 6.97 (1H, s); MS [APCI-] m/z 306.21 (M+ - H)-.
Anal. (C21H25NO) C, H, N.
2-Ethyl-3-O-sulfamoyl-17ꢀ-prop-2-ynyl
Estra-1,3,5(10)-
triene (32). Compound 31 (50 mg, 0.16 mmol) was reacted with
sulfamoyl chloride (0.3 mmol) in DMA (1 mL) as described for
the synthesis of 8. Column chromatography (hexane/ethyl acetate
10:1 to 7:1) gave the desired sulfamate 32 as a colorless oil (40
mg, 62%), which showed δH 0.61 (3H, s), 1.18 (3H, t, J 7.3),
1.20–2.30 (17H, m), 2.66 (2H, q, J 7.3), 2.79 (2H, m), 4.97 (2H,
br), 7.03 (1H, s) and 7.15 (1H, s); HRMS [FAB-] m/z found
400.1937; C23H30NO3S requires 400.1946.
2-Ethyl-3-O-acyl Estrone (33). A solution of 2-ethyl estrone6
(0.89 g, 3 mmol) in DCM (20 mL) and TEA (1 mL, 7.2 mmol)
was stirred and cooled to 0 °C. Acetyl chloride (0.29 mL, 4 mmol)
was added in a dropwise manner, and the mixture was stirred at 0
°C for an hour. After the addition of DCM (20 mL) and water (20
mL), the organic layer was separated, washed with water and brine,
dried (MgSO4), filtered, and concentrated. The crude white powder
was purified by flash chromatography (hexane/EtOAc 20/1 to 10:
1) to afford the desired acetate 33 (880 mg, 88%) as a white powder
with mp 138–139 °C. δH 1H NMR (CDCl3, 270 MHz): 0.89 (3H,
s, CH3), 1.16 (3H, t, J 7.4 Hz), 1.38–1.68 (6H, m), 1.92–2.25 (5H,
m), 2.30 (3H, s), 2.38–2.54 (4H, m), 2.83–2.88 (2H, m), 6.72 (1H,
s) and 7.15 (1H, s); HRMS [FAB+] m/z 341.2098; C22H29O3
requires 341.2111. Anal. (C22H28O2) C, H.

C-17 Cyano-Substituted Estratrienes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1307
(14) Wood, L.; Leese, M. P.; Mouzakiti, A.; Purohit, A.; Potter, B. V. L.;
Reed, M. J.; Packham, G. 2-MeOE2bisMATE induces caspase-
dependent apoptosis in CAL51 breast cancer cells and overcomes
resistance to TRAIL via cooperative activation of caspases. Apoptosis
2004, 9, 323–332.
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M. F. C.; Leese, M. P.; Potter, B. V. L.; Reed, M. J.; Purohit, A. The
role of 17ꢀ-hydroxysteroid dehydrogenases in modulating the activity
of 2-methoxyestradiol in breast cancer. Cancer Res. 2006, 66, 324–
330.
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Parish, D.; Leese, M. P.; Smith, A. C.; Potter, B. V. L.; Reed, M. J.
Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-meth-
oxyoestradiol-bis-sulphamate in ViVo in rodents. Br. J. Cancer 2004,
90, 932–937.
(17) Reed, M. J.; Purohit, A.; Newman, S. P.; Potter, B. V. L. Steroid
sulfatase: Molecular biology, regulation and inhibition. Endocr. ReV.
2005, 26, 171–202.
2-Ethyl-3-O-sulfamoyl-17-cyano Estra-1,3,5(10),16-tetraene
(40). Compound 39 (75 mg, 0.25 mmol) was reacted with sulfamoyl
chloride (0.5 mmol) in DMA (1 mL) as described for the synthesis
of 8. Column chromatography (hexane/ethyl acetate 5:1) followed
by crystallization (hexane/ethyl acetate 5:1) gave the desired
sulfamate 40 as a white powder (70 mg, 73%) with mp 212–213
°C, which showed δH 0.99 (3H,s), 1.26 (3H, t, J 7.5), 1.41–2.54
(11H, m), 2.74 (2H, q, J 7.5), 2.92 (2H, m), 5.03 (2H, s), 6.71
(1H, dd, J 3.3 and 1.9), 7.13 (1H, s) and 7.22 (1H, s); MS [APCI-]
m/z 385.23 (M - H)-; HRMS [FAB+] m/z found 386.1653;
C21H23O3N2S requires 386.1664. Anal. (C21H26N2O3S) C, H, N.
Acknowledgment. This work was supported by Sterix Ltd.,
a member of the IPSEN Group. We thank Sincrotrone Trieste
CNR/Elettra for the opportunity to collect data at the Crystal-
lographic Beamline, Ms. Alison Smith and Dr. James Robinson
for technical support, and the NCI DTP program for providing
in vitro screening resources.
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Supporting Information Available: Table of elemental analy-
ses, 13C NMR data and crystallographic data for the hCA II/15
complex. This material is available free of charge via the Internet
at http://pubs.acs.org.
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