Highly potent first examples of dual aromatase-steroid sulfatase inhibitors based on a biphenyl template

Article abstract of DOI:10.1021/jm901705h

Single agents against multiple drug targets are of increasing interest. Hormone-dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS). The aromatase inhibitory pharmacophore was thus introduced into a known biphenyl STS inhibitor to give a series of novel dual aromatase-sulfatase inhibitors (DASIs). Several compounds are good aromatase or STS inhibitors and DASI 20 (IC₅₀: aromatase, 2.0 nM; STS, 35 nM) and its chlorinated congener 23 (IC₅₀: aromatase, 0.5 nM; STS, 5.5 nM) are examples that show exceptional dual potency in JEG-3 cells. Both biphenyls share a para-sulfamate-containing ring B and a ring A, which contains a triazol-1-ylmethyl meta to the biphenyl bridge and para to a nitrile. At 1 mg/kg po, 20 and 23 reduced plasma estradiol levels strongly and inhibited liver STS activity potently in vivo. 23 is nonestrogenic and potently inhibits carbonic anhydrase II (IC₅₀ 86 nM). A complex was crystallized and its structure was solved by X-ray crystallography. This class of DASI should encourage further development toward multitargeted therapeutic intervention in HDBC.

Full text of DOI:10.1021/jm901705h

J. Med. Chem. 2010, 53, 2155–2170 2155
DOI: 10.1021/jm901705h
Highly Potent First Examples of Dual Aromatase-Steroid Sulfatase Inhibitors based on a Biphenyl
Template†,‡
§
§
§
§
L. W. Lawrence Woo, Toby Jackson, Aurelien Putey, Gyles Cozier, Philip Leonard, ,# K. Ravi Acharya,
ꢀ
Surinder K. Chander,^ Atul Purohit,^ Michael J. Reed,^ and Barry V. L. Potter*,§
§Medicinal Chemistry, Department of Pharmacy and Pharmacology and Sterix Ltd, University of Bath, Claverton Down, Bath BA2 7AY, U.K.,
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K., and ^Endocrinology and Metabolic
Medicine and Sterix Ltd, Imperial College London, Faculty of Medicine, St. Mary’s Hospital, London W2 1NY, U.K. # Present address: Biofocus
DPI, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, U.K.
Received November 17, 2009
Single agents against multiple drug targets are of increasing interest. Hormone-dependent breast cancer
(HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS).
The aromatase inhibitory pharmacophore was thus introduced into a known biphenyl STS inhibitor to
give a series of novel dual aromatase-sulfatase inhibitors (DASIs). Several compounds are good
aromatase or STS inhibitors and DASI 20 (IC50: aromatase, 2.0 nM; STS, 35 nM) and its chlorinated
congener 23 (IC50: aromatase, 0.5 nM; STS, 5.5 nM) are examples that show exceptional dual potency in
JEG-3 cells. Both biphenyls share a para-sulfamate-containing ring B and a ring A, which contains a
triazol-1-ylmethyl meta to the biphenyl bridge and para to a nitrile. At 1 mg/kg po, 20 and 23 reduced
plasma estradiol levels strongly and inhibited liver STS activity potently in vivo. 23 is nonestrogenic and
potently inhibits carbonic anhydrase II (IC50 86 nM). A complex was crystallized and its structure was
solved by X-ray crystallography. This class of DASI should encourage further development toward
multitargeted therapeutic intervention in HDBC.
Introduction
sulfatase (STS) catalyzes the hydrolysis of substrates such as
estrone sulfate (E1S) to estrone (E1).4 Thus, STS has now
become an attractive new target for therapeutic intervention.
Considerable progress has been made in developing steroidal
and nonsteroidal STS inhibitors containing the aryl sulfamate
ester moiety, which is the pharmacophore for irreversible
inhibition of STS.4,5 One such compound, 1 (STX64 or
BN83495, Figure 1), has become the first STS inhibitor to
enter clinical trial for postmenopausal patients with advanced
HDBC and has shown encouraging results.6,7
Designing hybrid molecules that target multiple biological
targets is an emerging and attractive strategy in drug design
and discovery.8 For our application, because of the therapeu-
tic potential of STS inhibitors and the established clinical
effectiveness of AIs, it has been reasoned that a concomitant
inhibition of aromatase and STS may lead to a more compre-
hensive estrogen deprivation in patients with HDBC. To this
end, we successfully developed three series of single agent dual
aromatase andsulfatase inhibitors(DASIs) that are sulfamate
derivatives of nonsteroidal AIs 4-((4-bromobenzyl)-[1,2,4]-
triazol-4-yl-amino)benzonitrile (e.g., 2, Figure 1),9-11 letro-
zole(e.g., 3, Figure 1),12,13 and anastrozole(e.g., 4, Figure 1).14
The design of these DASIs shares a common strategy that is to
engender the STS inhibitory pharmacophore into an estab-
lished or experimental AI with minimal structural change
incurred to the original scaffold in order to retain and maxi-
mize aromatase inhibition.
The majority of postmenopausal breast tumors are hor-
mone-dependent and estrogen receptor (ER)-positive. A
common therapeutic intervention for this type of cancer is
throughendocrinetherapy. Endocrineagents canactbyeither
diminishing the availability or inhibiting the binding of estro-
gens to ER. Aromatase catalyzes the conversion of androgens
to estrogens in the final step of the biosynthesis of estrogens
and is therefore an attractive therapeutic target for inhibition.
To this end, a large number of steroidal and nonsteroidal
aromatase inhibitors (AIs)a have been developed1-3 and
several of them are now in clinical use showing high efficacy
against hormone-dependent breast cancers (HDBC).
Most estrogens that originate from the aromatase pathway
are stored in the body as steroid sulfates. It is now widely
recognized that this reservoir of estrogen sulfates provides
an important source of estrogens in tumors when steroid
†This article is dedicated to Professor Michael John Reed, B.Sc., M.
Sc., Ph.D., D.Sc., FRCPath (1944-2009).
‡Protein Data Bank accession code: 2wd3.pdb.
*To whom correspondence should be addressed. Phone: þ44 1225
386639. Fax: þ44 1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
a Abbreviations: AIs, aromatase inhibitors; DASI, dual aromatase-
sulfatase inhibitor; DMA, N,N-dimethylacetamide; DMPK, drug meta
bolism and pharmacokinetics; E1, estrone; E2, estradiol; E1S, estrone
sulfate; ER, estrogen receptor; hCAII, human carbonic anhydrase II;
HDBC, hormone-dependent breast cancer; NSAIs, nonsteroidal aro-
matase inhibitors; 2-MeOE2bisMATE, 2-methoxyestradiol-3-O,17β-O-
bissulfamate; PMSG, pregnant mare’s serum gonadotropin; STS,
steroid sulfatase; STX641, 17β-cyanomethyl-2-methoxyestra-1,3,5(10)-
trien-3-O-sulfamate; TBAB, tetrabutylammonium bromide.
With the intention of widening further the structural diversity
of DASIs, a new design strategy was explored that involves
introducing the aromatase inhibitory pharmacophore into a
r
2010 American Chemical Society
Published on Web 02/11/2010
pubs.acs.org/jmc

2156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
Figure 1. Structures of 1 and its N,N-dimethylated prodrug 8, AI 4-((4-bromobenzyl)-[1,2,4]triazol-4-yl-amino)benzonitrile and its DASI
derivative 2, AI letrozole and letrozole-based DASI 3, AI anastrozole and anastrozole-based DASI 4, biphenyl STS inhibitor 5, biphenyl AI 6,
steroidal STS inhibitors 7, and steroid-like STS inhibitor 9. X = OSO2NH2.
template that has been designed primarily for STS inhibition.
While the modification of established STS inhibitors like 1
may be an attractive approach, it was reasoned that the series
of biphenyl STS inhibitors reported by Okada et al.15 repre-
sents a more accessible platform for the incorporation of the
aromatase inhibitory pharmacophore. One example, 5
(Figure 1), which has two electron withdrawing cyano groups
incorporated at the 20- and 40-positions and a sulfamate ester
at the 4-position, was shown to be a highly potent STS
inhibitor.
In a separate development, we also recently exploited the
biphenyl motif and successfully designed a new class of
nonsteroidal aromatase inhibitors (NSAIs).16 A particularly
potent example is 6 (Figure 1), the IC50 value of which against
aromatase in JEG-3 cells was found to be 0.22 nM.
Figure 2. Retrosynthesis of the biphenyl DASIs.
Herein, we report a new class of DASI that is based on a
biphenyl template. The phenyl ring A of these decorated
biphenyls is substituted with a triazol-1-ylmethyl group with
or without a second substituent, whereas their phenyl ring B
bears a sulfamate groupwithorwithout a chlorine atomortho
to it. The in vitro activities of these sulfamoylated compounds,
their phenolic precursors, and other derivatives were studied
in JEG-3 cells. The extent to which rat liver STS activity is
inhibited and plasma estradiol (E2) levels are reduced in vivo
by sulfamates 20 and 23 and their respective parent phenols 19
and 22 was determined. A uterine weight gain assay was
carried out to assess the estrogenicity of 23. Because the in
vivo sequestration of sulfamate esters by carbonic anhydrase
II (hCAII) in red blood cells is thought to enhance the
bioavailability and pharmacokinetics of this emerging struc-
tural class of drug, the ability of a representative biphenyl
derivative 23 to interact with human hCAII was investigated
in vitro and an hCAII/23 complex was investigated using
X-ray crystallography.
Figure 3. Bromobenzyltriazoles.
where functionalization of the biphenyl scaffold is inherited
from already derivatized starting bromobenzyltriazoles and
phenylboronic acid (see Figure 2).
We previously described the syntheses of the bromoben-
zyltriazoles 10, 11, and 13-16 (Figure 3).14,16 Likewise,
starting material 12 herein (Figure 3) was synthesized by
radical bromination of 3-bromo-5-methylbenzonitrile followed
by reacting the resulting 3-bromo-5-(bromomethyl)benzonitrile
with 1H-1,2,4-triazole in the presence of potassium carbonate.
3-Bromo-5-methylbenzonitrile was synthesized in a similar
manner as described by Fisher et al.20 from 2-amino-5-
methylbenzoic acid, although the 3-bromo-5-methylbenzoic
Results and Discussion
Chemistry. A palladium catalyzed Suzuki reaction17-19
was employed in the preparation of the biphenyl compounds

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2157
Scheme 1a
a (i) Pd(OAc)2, K2CO3, TBAB, H2O/EtOH, 13-89%; (ii) ClSO2NH2, DMA, 62-93%; (iii) ClSO2N(CH3)2, NEt3, CH2Cl2, 60-90%. Substituents
are numbered as shown for presentational uniformity; see Experimental Section for correct numbering of substituents.
acid intermediate was converted to the product 3-bromo-5-
methylbenzonitrile in two steps via the 3-bromo-5-methyl-
benzamide intermediate.
letrozole,12 the investigational STS inhibitor 1 (Figure 1),
and the biphenyl STS inhibitor 5 (Figure 1) were studied in
the same assay. Although the STS inhibitory potential of
anastrozole and letrozole was not investigated here, they are
expected to be ineffective against STS and act primarily as
AIs. Compound 1 has an IC50 value of 1.5 nM against STS
but shows relatively insignificant aromatase inhibition. The
biphenyl 5 inhibits STS strongly, albeit being more than 10-
fold less active than 1. As anticipated, there was no signifi-
cant aromatase inhibition shown by 5. Therefore, all these
reference compounds are acting individually either as AIs or
STS inhibitors but not as DASIs in JEG-3 cells.
Compounds 20, 21, 23, 24, and 26 of structural group I
have in common a cyano group on phenyl ring A ortho to the
biphenyl bridge and para to the triazol-1-ylmethyl moiety.
The simplest derivative in this series is 20, which has a
sulfamate group on phenyl ring B at the 40-position. This
compound inhibits aromatase and STS with IC50 values of 2
and 35 nM, respectively. It is interesting to note that the STS
inhibitory potency of 20 is of the same order of magnitude as
5, perhaps reflecting in part the similar pKa values of their
parent phenols (9.32 and 9.08 as calculated by ACD/La-
boratories Software v 8.14 for 19 and 40-hydroxy-2,4-biphe-
nyldicarbonitrile, respectively). The introduction of a
chlorine atom ortho to the sulfamate group and at the 30-
position of 20 renders a marked improvement on the inhibi-
tory activities of the resulting compound 23, the IC50 values
of which against aromatase and STS are 0.5 and 5.5 nM,
respectively. Hence, in JEG-3 cells, 23 is 4-fold and 6-fold
better than 20 in inhibiting aromatase and STS, respectively,
is equipotent to letrozole as an AI and inhibits STS in the
Phenylboronic acid, 4-fluorophenylboronic acid, 4-cya-
nophenylboronic acid, 3-hydroxyphenylboronic acid, 4-hy-
droxyphenylboronic acid, and 3-chloro-4-hydroxyphenyl-
boronic acid are all available commercially. 3-Hydroxy-4-
chlorophenylboronic acid 17 (Scheme 1) was prepared via
lithiation of the commercially available 5-bromo-2-chloro-
phenol and subsequent treatment with triisopropyl borate.
Several series of biphenyl compounds (18-58) were pre-
pared (see Scheme 1). The syntheses of the standalone AIs 18,
27, 41, 46, 51, and 54 (where R = H) were reported
previously,16 and herein we report the syntheses of congeners
34-40. Sulfamoylated biphenyl compounds were prepared
as follows. Corresponding boronic acids and bromobenzyl-
triazoles were coupled under either convection or micro-
wave initiated palladium catalyzed Suzuki cross-coupling
conditions.21 The resulting phenolic biphenyl precursors
(19, 22, 25, 28, 30, 32, 37, 39, 42, 44, 47, 49, 52, 55, and 57)
were then sulfamoylated22 with sulfamoyl chloride23 to
give 20, 23, 26, 29, 31, 33, 38, 40, 43, 45, 48, 50, 53, 56, and 58.
The N,N-dimethylsulfamates 21 and 24 were prepared by reac-
tion of the phenols 19 and 22 with N,N-dimethylsulfamoyl
chloride in dichloromethane in the presence of base.
Inhibition of Aromatase and Steroid Sulfatase in Vitro by
Sulfamoylated Compounds. The in vitro inhibitory activities
of sulfamoylated compounds against aromatase and STS in
JEG-3 cells are shown in Table 1. The various structural
subclasses of inhibitors have been broken down into groups
I-VII. As references, the clinical AIs anastrozole14 and

2158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
Table 1. In Vitro Inhibition of Aromatase and STS Activity in JEG-3 Cells by Anastrozole, Letrozole, 1, 5, and Sulfamoylated Compounds 20, 21, 23,
24, 26, 29, 31, 33, 38, 40, 43, 45, 48, 50, 53, 56, and 58
structural
group
aromatase IC50 (nM)
or % inhibition @ 1 μM
STS IC50 (nM)
or % inhibition @ 10 μM
compd
Ra
-
-
-
-
anastrozole
letrozole
-
-
-
-
1.5 ( 0.5
0.89 ( 0.13
300 ( 42
nd
nd
1
5
1.5 ( 0.3
16.3 ( 2
47 ( 3.7%b
I
20
21
23
24
26
40-OSO2NH2
2.0 ( 0.20
0.50 ( 0.05
0.50 ( 0.01
2.40 ( 1.0
35.0 ( 5.0
>10000
5.50 ( 0.5
>10000
>10000
40-OSO2N(CH3)2
30-Cl, 40-OSO2NH2
30-Cl, 40-OSO2N(CH3)2
30-OSO2NH2, 40-Cl
0.35 ( 0.07
II
29
31
33
30-OSO2NH2
63.4 ( 2.1%
176 ( 25
146 ( 11
>10000
140 ( 10
32.6 ( 6.3
40-OSO2NH2
30-Cl, 40-OSO2NH2
III
IV
V
38
40
40-OSO2NH2
280 ( 10
80 ( 18
>10,000
6333 ( 577
30-Cl, 40-OSO2NH2
43
45
40-OSO2NH2
30-Cl, 40-OSO2NH2
275 ( 6
70 ( 5
5500 ( 300
160 ( 10
48
50
40-OSO2NH2
30-Cl, 40-OSO2NH2
284 ( 26
571 ( 53
28.6 ( 2.0
29 ( 2.0
VI
53
30-Cl, 40-OSO2NH2
1.90 ( 0.06
>10000
VII
56
58
40-OSO2NH2
30-Cl, 40-OSO2NH2
17.5 ( 2.0
0.3 ( 0.1
>10000
68.2 ( 1.6%
a Substituents are numbered as shown for presentational uniformity; see Experimental Section for correct numbering of substituents. b Inhibition @
10 μM, -: not applicable, nd: not determined.
same order of magnitude as 1. On the basis of past ob-
servations,9-11 the higher aromatase inhibition observed for
23 compared to 20 can be attributed to the increase in the
lipophilicity of 23 conferred by its chlorine atom. We have
demonstrated in previous work, within limits, that a general
inverse relationship exists between the STS inhibitory po-
tency of a sulfamate and the pKa value of its corresponding
parent phenol.4,5,24,25 Given that the pKa value of 22 is
7.89 (calculated according to ACD/Laboratories Software
v 8.14), which is significantly lower than that of 19 (9.32), the
increase in STS inhibition observed for 23 compared with 20
could be rationalized by the presence of the chlorine atom
ortho to its sulfamate group. The electron-withdrawing
effect of the halogen increases the leaving group ability of
the parent phenol 22, which in return facilitates the sulfa-
moylation and hence inactivation of the enzyme by the
sulfamate group of 23. While the electron-withdrawing
cyano groups of 5 are both conjugated to its sulfamate group
and are hence conducive to the reduction of the pKa of its
parent phenol (40-hydroxybiphenyl-2,4-dicarbonitrile), the
presence of a chlorine atom at the 30- and a cyano group at
the 2-position of the biphenyl system confers a more marked
reduction in the pKa value of the phenol 22. This factor
may help in part to explain why 23 is a stronger STS inhibitor
than 5. A recent study with Pseudomonas aeruginosa aryl-
sulfatase has shown that the stoichiometry of inactivation by
1 has a value of around 3, with one interpretation being that
the inactivation process of this arylsulfatase could involve
multiple sulfamoylation events.26 Although no similar experi-
ment has yet been carried out with STS, it is possible that the
inhibition of STS elicited by 23 may also involve more than
just one single sulfamoylation event or, perhaps more likely,
proceed by a complex process.
When the positions of the chlorine atom and the sulfamate
group in 23 are transposed, the resulting compound 26 shows
a slight increase in aromatase inhibition but a substantial
reduction in STS inhibition. A similar finding was made in
the series of DASIs, which are derivatives of AI 4-((4-
bromobenzyl)-[1,2,4]triazol-4-yl-amino)benzonitrile.10 It was
observed that substituted or unsubstituted meta-sulfamate

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2159
derivatives are stronger AIs but much weaker STS inhibitors
than their para-sulfamate counterparts.
The STS inhibitory activities of sulfamates in structural
groups II-IV are significantly weaker than those of deriva-
tives from group I. The best STS inhibitor out of these groups
is 33 (IC50 = 32.6 nM) that has the electron-withdrawing
cyano group and the chlorine atom both conjugated to the
sulfamate group.
The N,N-dimethylation of the sulfamate group of 20 to
give 21 improves aromatase inhibition by 4-fold (IC50
for 21 = 0.5 nM, cf. 2 nM for 20). In contrast, the same
dimethylation of the sulfamate group of 23 to give 24 reduces
aromatase inhibition by nearly 5-fold. It is not clear why the
effects of N,N-dimethylation of 20 and 23 are so disparate
despite both 21 and 24 being more lipophilic than their
nonmethylated counterparts. In general, an increase in the
lipophilicity of an inhibitor normally benefits aromatase
inhibition. For STS inhibition, the very weak activities
observed for 21 and 24 are anticipated. It has been shown
that the N-piperidino- and N,N-dibenzyl derivatives of
estrone 3-O-sulfamate (7, Figure 1) are significantly weaker
STS inhibitors than 7 in vitro, although the N-acetyl deriva-
tive, which has one free proton on the sulfamate nitrogen
atom, shows moderate irreversible STS inhibition.27 Simi-
larly, when the sulfamate group of the clinical agent 1
(Figure 1) was N,N-dimethylated, the resulting derivative 8
(Figure 1) was shown to be inactive against STS in vitro.28
These findings clearly suggest that a ligand is to have a fully
unsubstituted sulfamate group, i.e., H2NSO2O-Ar, in order
to maximize active site-directed irreversible STS inhibition in
vitro. However, like 1, 8 at 1 and 10 mg/kg doses inhibits
almost completely mouse liver and skin STS activities 24 h
after oral administration of the compound.29 There is evi-
dence to suggest that 8 is a pro-drug of 1 that is metabolized
in vivo and undergoes N,N-demethylation to give 1 that
subsequently inhibits STS in treated animals. By the same
token, it is entirely possible that 21 and 24, although inactive
as STS inhibitors in vitro, are indeed pro-drugs of 20 and 23,
respectively, and hence can be demethylated and become
active against STS in vivo. More studies are required to
explore this potential property of 21 and 24.
While keeping the triazol-1-ylmethyl moiety meta to the
biphenyl bridge, the effects on biological activities of relocat-
ing the cyano group of 20 and 23 to other positions on phenyl
ring A or removing it entirely were explored. Compounds 29,
31, and 33 (structural group II) have a cyano group ortho to a
triazol-1-ylmethyl moiety but para to the biphenyl bridge,
whereas compounds 38 and 40 (structural group III) have
a cyano group meta to both the biphenyl bridge and the
triazol-1-ylmethyl moiety. Compounds 43 and 45 (structural
group IV) have no cyano group on phenyl ring A apart from
a triazol-1-ylmethyl moiety meta to the biphenyl bridge. As
shown in Table 1, these derivatives are relatively much
weaker AIs. Apart from 33 of group II, the introduction of
a chlorine atom ortho to the sulfamate group significantly
improves the aromatase inhibitory potency of 40 (group III)
and 45 (group IV) over their respective nonchlorinated
counterparts 38 and 43. However, despite 40 and 45 being
the two most active AIs in structural groups II-IV, their
activities are at least 2 orders of magnitude lower than those
of their congeners 20, 23, and 26 in group I. These results
clearly demonstrate the importance of having a triazol-1-
ylmethyl moiety meta to the biphenyl bridge and para to the
cyano group on phenyl ring A for high aromatase inhibitory
potency to be maintained. Any relocation or removal of the
cyano group is detrimental to biological activity. Compound
29, which has a 30-sulfamate, is a much weaker AI than the 40-
sulfamate derivative 31, although the relocation of the
sulfamate group in a similar fashion from the 40-position
of 23 to the 30-position of 26 results in a small beneficial effect.
On keeping the cyano group ortho to the biphenyl bridge
and moving the triazol-1-ylmethyl moiety from the 3- to the
4-position of phenyl ring A where it is meta to the cyano
group and para to the biphenyl bridge, the resulting struc-
tural group V derivatives 48 and 50 remain relatively weak
AIs (IC50 = 284 and 571 nM, respectively). Although both
these two sulfamates have a cyano group ortho to the
biphenyl bridge, the substantial reduction in their inhibitory
activities observed compared to 20 and 23 further demon-
strate the importance of having a para relationship bet-
ween the triazol-1-ylmethyl moiety and the cyano group
for potent aromatase inhibition. Interestingly, the STS in-
hibitory activities of 48 and 50 are comparable to that of 20,
with IC50 values at around 30 nM. However, it is not clear
why in this structural class the chlorine atom of 50 does not
confer as anticipated an additional advantage over its non-
chlorinated congener 48 on STS inhibition. Nonetheless, for
48 and 50 as well as other relatively more active STS
inhibitors identified in this work, i.e., 20, 23, 31, 33, and
45, they all share a common feature, which is the conjugation
of the cyano group and/or the chlorine atom to the sulfamate
group.
To further investigate the para relationship between the
triazol-1-ylmethyl moiety and the cyano group on phenyl
ring A, the two functionalities were placed respectively ortho
to and meta to the biphenyl bridge to give 53 (structural
group VI). As shown in Table 1, 53 (IC50 = 1.9 nM) is
equipotent to 20 (IC50 = 2.0 nM) but some 4-fold weaker
than 23 (IC50 = 0.5 nM) as an AI. In conjunction with the
SAR obtained so far, this finding further confirms the
importance of having a para relationship between the tria-
zol-1-ylmethyl moiety and the cyano group on phenyl ring A
for this structural class of biphenyl-based DASIs. However,
for optimal aromatase inhibition, it requires the heme ligat-
ing triazol-1-ylmethyl moiety to be meta to and the cyano
group ortho to the biphenyl bridge, which is a feature of
those compounds in structural group I. In contrast to their
similar abilities in inhibiting aromatase, compounds 20, 23,
and 53 inhibit STS quite differently. While 20 and 23 are
inhibitors of STS with nanomolar IC50 values, the inhibition
of STS by 53 remains ineffective at micromolar concentra-
tions.
In our recent work, it was observed that DASIs could be
designed by replacing one of the anastrozole 2-methylpro-
panenitrile groups with linkers attached to an aryl sulfamate,
i.e., the STS pharmacophore for irreversible inhibition.14
Herein, we replace one of the side-arms of anastrozole
directly with a phenyl sulfamate to give the biphenyl deriva-
tives 56 and 58 (structural group VII). These two compounds
are related to 20 and 23 structurally except they have no
cyano group ortho to the biphenyl bridge but instead a 2-
methylpropanenitrile group meta to it. As shown in Table 1,
while 56 is a moderate to good AI, its chlorinated congener
58 is exceptionally potent; the IC50 value (0.3 nM) is compar-
able to those of top AIs discovered in this work (i.e., 21, 23,
and 26). On comparing 56 and 58 with 38 and 40 from
structural group III, the replacement of the cyano group with
a 2-methylpropanenitrile group at the 3-position of phenyl

2160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
Table 2. In Vitro Inhibition of Aromatase Activity in JEG-3 Cells by Anastrozole, Letrozole, and Nonsulfamoylated Compounds 18, 19, 22, 25, 27, 28,
30, 32, 34-37, 39, 41, 42, 46, 47, 49, 51, 52, 54, 55, and 57
structural
group
aromatase IC50 (nM)
or % inhibition @ 1 μM
compd
Ra
-
-
anastrozole
letrozole
-
-
1.5 ( 0.5
0.89 ( 0.13
I
18
19
22
25
H
0.20 ( 0.02 b
0.20 ( 0.01
0.15 ( 0.03
0.22 ( 0.01
40-OH
30-Cl, 40-OH
30-OH, 40-Cl
II
III
27
28
30
32
H
68.8 ( 1.4%b,c
14.4 ( 1.4
30-OH
40-OH
30-Cl, 40-OH
76.4 ( 2.4% d
74.0 ( 12
34
35
36
37
39
H
40-F
40-CN
40-OH
30-Cl, 40-OH
29.3 ( 2.4
19 ( 1
52 ( 3
112 ( 10
24 ( 1
IV
V
41
42
44
H
210 ( 30 b
40-OH
30-Cl, 40-OH
79.0 ( 1.9% e
85.3 ( 1.2% f
46
47
49
H
4.6 ( 1.2 b
920 ( 121
380 ( 190
40-OH
30-Cl, 40-OH
VI
51
52
H
1.7 ( 0.8 b
0.800 ( 0.03
30-Cl, 40-OH
VII
54
55
57
H
0.50 ( 0.10 b
6.0 ( 1.0
0.1 ( 0.1
40-OH
30-Cl, 40-OH
a Substituents are numbered as shown for presentational uniformity; see Experimental Section for correct numbering of substituents. b Data from ref
16; -: not applicable. c 23.9 ( 2.9% @ 0.1 μM. d 35.1 ( 6.0% @ 0.1 μM. e 24.6 ( 7.0% @ 0.1 μM. f 36.6 ( 2.2% @ 0.1 μM.
ring A enhances aromatase inhibition significantly. Previous
molecular modeling studies carried out on the nonsulfamoy-
lated congener 54 (R = H, Scheme 1 and Table 2) has
suggested that its 2-methylpropanenitrile group might form
a H-bond with Ser478 of the aromatase active site.16 It is
possible that, inter alia, a similar favorable interaction with
the aromatase enzyme active site also operates in 38 and 40.
Compounds 38 and 40 are very weak inhibitors of STS in
JEG-3 cells. Their lack of in vitro activity against STS can be
attributed to a combination of unfavorable steric and elec-
tronic factors, as postulated for the structurally related
anastrozole-based DASIs.14
sulfamate-based STS inhibitor is expected to be its corre-
sponding phenol, although the quantity released by this
route in vivo is considered restricted once all the STS activity
has been inactivated.30 However, given the chemical stability
issues observed for some aryl sulfamates, it is reasonable to
expect the formation of the parent phenol could in principle
continue as a result of gradual hydrolytic cleavage of the
sulfamate group of any ligand that presents in the plasma
over time. For this reason and also because they possess the
pharmacophore for aromatase inhibition, the parent phenols
of sulfamates and also the new compounds 34-36 were
assayed for aromatase inhibition in JEG-3 cells (Table 2).
Anastrozole and letrozole were used as reference AIs, and the
IC50 values for the unsubstituted derivatives (18, 27, 41, 46,
51, and 54) reported previously16 are included for compa-
rison.
Inhibition of Aromatase in Vitro by Nonsulfamoylated
Compounds. According to the various mechanisms of act-
ion proposed by our group,4,5,23,24 the immediate product
released after the irreversible inactivation of STS by a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2161
Figure 4. Generalized summary of the SAR for aromatase and STS inhibition in JEG-3 cells by biphenyl-based DASIs and their parent
phenols (R = 40-OH; 40-OH, 30-Cl; 40-OSO2NH2; 40-OSO2NH2, 30-Cl).
Table 3. The Effects of 19, 20, 22,and 23, Letrozole (an AI) and 9 (an
STS Inhibitor) on PMSG-Induced Plasma E2 Concentrations
(Aromatase Activity) and Liver STS Activity in the Immature Rat 3 h
after Oral Dosing
As shown in Table 2, the parent phenol of most structural
groups is in general a stronger AI than its corresponding
sulfamate. This observation is expected because a similar
profile was observed for the parent phenols from other
classes of DASIs. Of the phenols tested, 19, 22, and 25
(structural group I: 5-triazolylmethyl and 2-cyano), 52
(group VI: 6-triazolylmethyl and 3-cyano), and 55 and 57
(group VII: 5-triazolylmethyl and 3-(2-methylpropanenit-
rile)) were found to inhibit aromatase most strongly. Their
IC50 values are comparable to or within an order of magni-
tude of those observed for anastrozole and letrozole. Inter-
estingly, although not comparable in terms of the level of
potency, the three most potent groups of AIs within the series
of nonhydroxylated biphenyl-based AIs studied by Jackson
et al.16 also belong to these three structural groups. Hence, it
appears that these biphenyl templates and their substituents
concertedly bind well to the aromatase active site and inter-
act favorably with the heme and certain neighboring amino
acid residues. The fact that the respective parent phenols, 19
(IC50 = 0.2 nM) and 22 (IC50 = 0.15 nM), of the two most
active DASIs 20 and 23 are themselves even more potent AIs
suggest that they should in their own right further inhibit
aromatase, and do so potently, on release either chemically
or after STS inhibition.
Of phenols belonging to the structural groups II-V, the
hitherto unexplored compounds 34-37 and 39 of structural
group III, whose triazol-1-ylmethyl moiety and cyano group
are both meta to the biphenyl bridge, turn out to be the more
potent AIs. However, the phenols from groups I, VI, and VII
phenols are clearly much more potent AIs whose aromatase
IC50 values are between 2 and 3 orders of magnitude lower
than those observed for phenols from structural groups
II-V.
In summary, for inhibition of individual enzymes, com-
pounds primarily from structural groups VI and VII are
good to potent aromatase inhibitors, whereas some examples
from group II, IV, and V show moderate to good STS
inhibition. However, for dual inhibition of aromatase and
STS, the best two DASIs in this work are 20 (IC50Arom =
2.0 nM and IC50STS = 35 nM) and 23 (IC50Arom = 0.5 nM
plasma E2
levels (pg/mL)
aromatase
inhibition (%)b
STS inhibition
(%)
treatmenta
control
PMSG
letrozole
17 ( 3
334 ( 85
49 ( 11
94 ( 33
124 ( 74
297 ( 90
132 ( 22
nd
95 ( 1
0 ( 26
85 ( 3
72 ( 10
63 ( 22
11 ( 27
60 ( 7
nd
0 ( 8
16 ( 6
0 ( 10
10 ( 7
77 ( 12
5 ( 22
92 ( 6
99 ( 0.1
19
20
22
23
9
a Dose: letrozole, 0.1 mg/kg; others, 1 mg/kg. b Results are expressed
as the percentage inhibition of PMSG stimulated E2 levels. The statis-
tical significance for aromatase and STS activities in control and treated
groups (n = 3) was assessed using Student’s t test. nd: not determined.
and IC50STS = 5.5 nM) from structural group I, whose
triazol-1-ylmethyl moiety on phenyl ring A is meta to the
biphenyl bridge and para to the cyano group. Both of their
sulfamate groups are para to the biphenyl bridge on phenyl
ring B, but 23 has an additional chlorine atom ortho to its
sulfamate group. In general, parent phenols of DASIs are
better AIs and those from structural groups I, VI, and VII are
exceptionally potent. A generalized summary of the key SAR
for dual aromatase and STS inhibition by para-sulfamoy-
lated biphenyl-based DASIs and their parent phenols is
shown in Figure 4.
Inhibition of Aromatase and Sulfatase in Vivo. Compounds
19, 20, 22, 23, letrozole, and 9 (Figure 1, a highly potent
steroid-like STS inhibitor31-33) were studied in vivo (Table 3).
Immature female Wistar rats (20 days), pretreated with 200
IU/0.1 mL sc of PMSG (pregnant mare’s serum gon-
adotropin), were treated 3 days later with individual agent
at a 1 mg/kg po dose (letrozole, 0.1 mg/kg po). Plasma E2
levels and liver STS activity were determined 3 h after dosing.
The phenol 19 inhibits aromatase strongly and reduces E2
plasma levels by 72% compared with those of rats pretreated
with PMSG (Table 3). In contrast and unexpectedly, the
phenol 22 failed to reduce plasma E2 levels significantly.

2162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
Table 4. IC50 Values for the Inhibition of hCAII by 7 and 23 from Initial
Rate Experiments Performed in Triplicate
compd
hCAII IC50 (nM)
7
23
56 ( 10
86 ( 8
Interactions with hCAII. Like sulfonamides, sulfamate
esters also reversibly inhibit hCAII. In its monoanionic form,
the sulfamate moiety (OSO2NH-) coordinates to the zinc ion
of the active site. Many aryl sulfamates have been shown to
inhibit hCAII in vitro.28,36-39 Given that 23 represents a new
structural class of highly potent DASI, the potential of 23 to
inhibit hCAII in vitro was assessed alongside 7 (Figure 1) as
reference, which is a known potent hCAII inhibitor.
As shown in Table 4, DASI 23 inhibits hCAII potently
with an IC50 value in the same order of magnitude as 7.
This result demonstrates the potential of 23 to be seque-
strated into hCAII in erythrocytes, which is an important
mechanism for optimizing oral activity, pharmacokinetics,
and protecting against first pass metabolism as shown
with 1.40
Figure 5. The effect on uterine growth in the ovariectomized rat
after an oral dosing of 2 and 23 at 10 mg/kg for 4 days. Mean ( SD
of triplicate measurements. ANOVA: control vs compounds; P >
0.05, nonsignificant.
Given the high aromatase inhibitory potency of 22 in vitro
(IC50 = 0.15 nM, Table 2), it is possible that its weak
aromatase inhibition observed in vivo could be related to a
poor oral bioavailability or drug delivery of 22. In the
absence of a sulfamate group, 19 predictably has negligible
inhibitory effect on liver STS activity (Table 3). Putting aside
the potential DMPK issue postulated for 22, this compound
is not expected to be active against STS for the same reason
because it lacks a sulfamate group. DASIs 20 and 23 inhibit
aromatase strongly, rendering a reduction in E2 plasma
levels by 63% and 60%, respectively, albeit not as potent
as letrozole (85%) at 0.1 mg/kg po. Although the aromatase
IC50 value for 23 is 4-fold lower than that of 20 in JEG-3 cells
(Table 1), 20 and 23 apparently show similar inhibitory
activities in vivo. Both DASIs inhibit liver STS activity
potently but to a different extent with 75% and 92% inhibi-
tion observed for 20 and 23, respectively. In vitro, DASI 23 is
some 6-fold more potent than 20 as an STS inhibitor
(Table 1). In the same assay, 9 inhibits liver STS activity
almost completely. These results clearly demonstrate the
significant dual inhibition of aromatase and STS exhibited
by 20 and 23 in vivo on oral administration.
Estrogenicity. It would be undesirable for an antiendo-
crine agent to possess any endocrinological effect(s) that it is
designed to counteract. Hence, only a nonestrogenic DASI
will be of suitable use in endocrine therapy for HDBC. With
the exception of 18, 25-29, 34, 41, 46, 51, and 54 (Scheme 1),
other compounds studied in this work share a common
4-hydroxybiphenyl template. It has been shown in the litera-
ture that compounds which bear this template and are
structurally similar to E2 can possess estrogenic activity.34,35
Given that the immediate product or metabolite released
upon either STS inhibition or by gradual chemical degrada-
tion of a biphenyl sulfamate is its parent phenol (vide supra),
DASI 23 was therefore selected and tested for estrogenicity
in vivo. Using adult ovariectomized rats, 23 was adminis-
tered in a 10 mg/kg po dose daily for 4 days, after which time
the rats were weighed, sacrificed, and uteri removed for
weighing. As shown in Figure 5, 23 is not estrogenic as there
was no increase in uterine weight observed in treated ani-
mals. Similarly, DASI 2 (Figure 1) was also found to be
nonestrogenic. This result, in conjunction with the report
that the biphenyl STS inhibitor 5 (Figure 1) is also nones-
trogenic in vivo,15 suggests the substituted biphenyl template
adopted in this work for the design of DASIs may not be
intrinsically pro-estrogenic.
Several X-ray crystal structures of sulfamate-based com-
pounds bound to hCAII have been reported recently. These
include steroid sulfatase inhibitors 1,41 7,37 and 3-sulfamoyl-
oxy-N-(30-pyridin-10-ylmethyl)-16,17-seco-estra-1,3,5(10)-tri-
ene-16,17-imide,33 two examples of a DASI,42 2-MeOE2bis-
MATE, a multitargeted antitumor agent,38 and a related
agent STX641.39 With the excellent potency shown by 23
against hCAII, we explored the co-crystallization of 23 with
the enzyme and now report the interaction of this compound
with hCAII using protein crystallography (Figure 6).
Co-crystallization of hCAII with 23 produced crystals of
the enzyme-inhibitor complex in the P21 space group.
During the early stages of refinement, the presence in the
binding site of the active site zinc(II) atom and 23 was
identified by strong positive density in the |Fo| - |Fc| map
˚
corresponding to the sulfamate group of 23 within 2.5 A of
the active site zinc(II) atom. The final model (Figure 6A) was
produced by addition of the inhibitor molecule, Zn(II) atom,
and water molecules, with manual rebuilding and further
refinement using CCP4i45 and Coot,46 giving final Rfree and
Rcryst values of 0.19 and 0.15 respectively.
DASI 23 was refined with an occupancy of 0.8, and there
was almost complete density for each part of the molecule in
the final model (Figure 6B). The interactions involving the
sulfamate group of 23, the active site Zn(II) atom and the
histidine triad (His94, His96, and His119), as well as the
residues at the bottom of the active site funnel, are similar to
those observed in previous structures of the enzyme with
sulfamate containing inhibitors.33,37,41 The biphenyl moiety
of 23 is located coming out of the center of the active site
funnel. Many hCAII inhibitors, including 1,41 7,37 and a
steroid-like STS inhibitor,33 have a significant degree of
interaction with hydrophobic residues lining the funnel. In
contrast, 23 bound more toward the center of the funnel,
only Val121, Phe131, and Leu198 are close enough to the
biphenyl skeleton to form productive hydrophobic interac-
tions (Figure 6B).
With there only being a few of these hydrophobic inter-
actions, the binding of 23 to hCAII could be bolstered
through the triazole ring being orientated toward a small
hydrophilic cleft situated close to the entrance of the active
site funnel which is flanked by Leu60, Asn62, Glu69, and
Glu238 (Figure 6B). There are two possible orientations of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2163
Figure 6. (A) Ribbon diagram showing the overall structure of the hCAII-23 complex. Secondary structure R-helices are shown in blue and β-
sheets in green. The inhibitor molecule, residues His94, His96, and His119 are shown as sticks, and the zinc ion is shown in dark green. (B)
Diagram of the active site region in the hCAII-23 complex. Only residues with interactions to 23 or the active site zinc are shown for clarity.
Glu238 from the symmetry related molecule is shown in yellow. Density for the 2|Fo| - |Fc| map (light blue) contoured at 1.0σ is shown for 23.
(B-1 and B-2) Close up views of the two possible triazole orientations are shown, with only the residues that could hydrogen bond to the triazole
shown for clarity. Black lines link atoms capable of hydrogen bonding and the distances between these atoms are shown. This diagram was
prepared using CCP4 mg43 and POV-Ray.44
˚
the triazole ring in this cleft, where the difference between
these two orientations involves a 180ꢀ rotation of the ring. In
one orientation (Figure 6B-1), the 2-N of the triazole ring
3.13 A distance between the triazole and the symmetry
related Glu238, and therefore may be the predominant
orientation in the crystal structure. It is also worth noting
that in solution there would be no symmetry molecule;
therefore there is only the possibility of one hydrogen bond
from either Asn62 or Glu69 that could anchor the triazole in
this cleft. During the refinement of the structure of hCAII in
complex with 23, density for a second inhibitor molecule was
observed in the 2|Fo| - |Fc| map contoured at 1.0σ. This was
in a similar location to the second inhibitor binding site
observed in the structure of hCAII/2-MeOE2bisMATE
complex.38 Thus, in summary, the potent inhibition of
hCAII observed for 23 can be attributed to the anchoring
of the ligand at both ends to the binding site of the protein,
˚
would be 3.45 A from Asn62, and the 4-N of the triazole
˚
being 3.13 A from the backbone nitrogen of Glu238 from a
symmetry related molecule. In the second orientation
˚
(Figure 6B-2), the 2-N of the triazole ring is 3.54 A from
˚
Glu69, and the 4-N of the triazole is 3.58 A from the side-
chain oxygen of Glu238 from a symmetry related molecule.
These distances are all too long for optimal hydrogen bond-
ing. From the electron density of 23, it is not possible to
conclude which orientation is adopted, or whether it is a
mixture of the two orientations, but the orientation shown in
Figure 6B-1 does have the shorter distances, in particular the

2164 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
bolstered by additional hydrophobic interactions of its bi-
phenyl ring with some neighboring amino acid residues. It
seems likely that this hCAII interaction will also engender
good in vivo properties for this new class of dual inhibitor.
II run Argonaut prepacked columns or on ISCO CombiFlash
Rf automated flash chromatography system run RediSep Rf
disposable flash columns. Nuclear magnetic resonance spectra
were recorded on either a Jeol Delta 270 MHz or Varian
1
Mercury VX 400 MHz spectrometer. H NMR spectra were
recorded at 270 or 400 MHz with shifts reported in parts per
million (ppm, δ) relative to residual chloroform (δH = 7.26 ppm)
or residual DMSO (δH = 2.50 ppm). Coupling constants, J, are
reported in hertz. 13C NMR spectra were recorded at either 67.9
or 100.6 MHz with the central peak of chloroform (δC = 77.16
ppm) or DMSO (δC = 39.52 ppm) as internal standard. The
Conclusions
By introducing the aromatase inhibitory pharmacophore
into the biphenyl STS inhibitor 5, a series of biphenyl-based
DASIs was designed and synthesized. The phenyl ring A of
these biphenyls is substituted with a triazol-1-ylmethyl group
with or without a second substituent, whereas phenyl ring B
bears a sulfamate groupwithorwithout a chlorine atomortho
to it. SAR studies derived from seven structural groups have
shown that while individual good to potent AIs or moderate
to good STS inhibitors are obtained, potent DASIs are
realized primarily from structural group I, where the para-
sulfamate containing phenyl ring B is coupled with phenyl
ring A which contains a triazol-1-ylmethyl moiety meta to the
biphenyl bridge and para to the cyano group. The best two
DASIs in this work are 20 (IC50Arom = 2.0 nM and IC50STS =
35 nM) and its chlorinated congener 23 (IC50Arom = 0.5 nM
and IC50STS = 5.5 nM). As expected, the parent phenols of
sulfamoylated compounds including 20 and 23 are generally
even stronger AIs, with those from structural groups I, VI, and
VII exceptionally potent. The N,N-dimethylsulfamate deriva-
tives of 20 and 23 are potent AIs but inactive as STS inhibitors in
JEG-3 cells. On oral administration of 20 and 23 at 1 mg/kg to
immature female Wistar rats 3 days after pretreating them with
PMSG, both compounds inhibit aromatase strongly 3 h after
dosing, rendering a reduction in E2 plasma levels by 63% and
60%, respectively, albeit not as potent as letrozole (85%) at 0.1
mg/kg po. From the same experiment, 20 and 23 were found to
inhibit liver STS activity by 75% and 92%, while 9, the potent
steroid-like STS inhibitor, inhibits liver STS activity almost
completely. At an oral dose of 10 mg/kg given for 4 days, DASI
23 was found to be nonestrogenic, as shown by the lack of uterine
growth observed in treated ovariectomized rat. Like other
sulfamoylated compounds reported in the literature, 23 inhibits
hCAII potently. The structure of a complex of 23 with hCAII
was solved through X-ray crystallography. In summary, a new
class of biphenyl-based DASIs has been discovered. The two best
compounds 20 and 23 are highly potent against aromatase and
STS in JEG-3 cells and show strong dual inhibitory activity in
vivo on oral administration. Compound 23, in addition, is
nonestrogenic and shows potential for sequestration into hCAII
in erythrocytes, which influences its in vivo behavior. All these
results suggest that biphenyl-based DASIs like 20 and 23 can be
further developed for potential therapeutic use in the treatment
of HDBC and other hormone-dependent malignant diseases.
1
following abbreviations are used to describe resonances in H
NMR and 13C NMR spectra: br, broad; s, singlet; d, doublet;
dd, double doublet; q, quartet; m, multiplet; t, triplet. HPLC
analyses were performed on a Waters Millenium 32 instrument
equipped with a Waters 996 PDA detector. For chromatographic
conditions, refer to Supporting Information. All biologically
tested compounds attained a purity level of 95% or above by
HPLC. LC-MS analysis was performed on a Waters 2790
Alliance linked up with a ZQ MicroMass spectrometer and a
Waters 996 PDA detector. FAB accurate mass spectra were
recorded at the EPSRC National Mass Spectrometry Service
Centre, University of Wales Swansea, Chemistry Department,
using m-nitrobenzyl alcohol as the matrix. Atmospheric pres-
sure chemical ionization (APCI) or electrospray (ES) high
resolution mass spectra were recorded on a Bruker micrOTOF
Focus. Elemental analyses were performed by the Microanalysis
Service, University of Bath. Melting points (mp) were deter-
mined using a Stanford Research Systems Optimelt MPA100
automated melting point system and are uncorrected. Micro-
wave reactions were carried out using a CEM Discover micro-
wave.
Biology. The extent of in vitro inhibition of STS and aroma-
tase activity by compounds was assessed using JEG-3 human
choriocarcinoma cells which were chosen because these cells
constitutively express both enzymes maximally. In our hands,
these enzyme activities remain stable during repeated subcultur-
ing over several months. Cells were seeded into 24-well culture
plates and maintained in MEM (Sigma, Poole, UK) containing
supplements and used when 80% confluent. To determine STS
activity, cells were incubated for 1 h with [6,7-3H]E1S (3 nM, 60
Ci mmol-1; PerkinElmer Life Sciences, Wellesley, MA) in the
presence or absence of (0.001-10000 nmol L-1) inhibitor. The
product E1 was separated from E1S by toluene partition using
[4-14C]E1 to monitor procedural losses, and the radioactivity
was measured by scintillation spectrometry. Similarly, for aro-
matase activity, [1β-3H]androstenedione (2-3 nM, 30 Ci
mmol-1; PerkinElmer Life Sciences, Wellesley, MA) was incu-
bated with JEG-3 cells for 1 h in the presence or absence of
inhibitor. The product, tritiated water (3H2O), was separated
using dextran-coated charcoal at 4-8 ꢀC for 2 h, and remaining
radioactivity was measured by scintillation spectrometry. Each
IC50 value represents the mean ( SE of triplicate measurements.
The in vivo inhibition of aromatase and STS activity by
letrozole, 9, 19, 20, 22, and 23 was assessed in immature female
Wistar rats (20 days) purchased from Charles River, UK.
Animals received a single subcutaneous injection of PMSG,
200 IU, Sigma. Three days later, compounds (1 mg kg-1) and
letrozole (0.1 mg kg-1) were administered orally in THF/
propylene glycol (10:90) as a single dose. Blood and liver
samples were obtained 3 h after drug administration. Plasma
concentrations of E2 were measured using a radio-immuno-
assay kit (Diagnostic Products Corporation, CA) to monitor the
extent of aromatase inhibition. Liver STS activity was deter-
mined by incubating rat liver homogenate with [3H]E1S [7 ꢀ
10-5 dpm, 60 Ci mmol-1, adjusted to 20 μM with nonradioac-
tive E1S (Sigma)] for 1 h. The product was extracted with
toluene, and the radioactivity was measured by liquid scintilla-
tion spectrometry. Results are expressed as the percent inhibi-
tion of PMSG-stimulated E2 levels for aromatase activity or the
Experimental Section
General Methods for Synthesis. Unless otherwise stated,
HPLC grade solvents were used and commercial reagents and
starting materials were used without further purification. Sulfa-
moyl chloride was prepared by an adaptation of the method of
Appel and Berger47 and was stored at 4 ꢀC under positive N2
pressure as a solution in toluene, as described by Woo et al.23
Thin-layer chromatography (TLC) was performed on precoated
plates (Merck TLC aluminum sheets silica gel 60 F254, art. no.
5554). Product(s) and starting materials(s) were detected by
either viewing under UV light or treatment with an ethanolic
solution of phosphomolybdic acid followed by heating. Flash
column chromatography was performed on glass columns
packed with silica gel (Sorbsil/Matrex C60) or on FlashMaster

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2165
percent activity in untreated animals for STS activity (mean (
SE, n = 3-5).
ure maintained at -78 ꢀC. After 15 min of stirring at this
temperature, the dry ice/acetone bath was removed. At 0 ꢀC,
2 M HCl(aq) was added dropwise until the pH of the reaction
mixture was neutral and the reaction left to stir for a further
15 min. THF was removed in vacuo and residues obtained taken
up in EtOAc (50 mL). Distilled H2O (50 mL) was added and the
organic layer separated. The aqueous layer was extracted with
EtOAc (50 mL ꢀ 2) and the organic portions combined, dried
(MgSO4), and solvent removed in vacuo. Addition of CH2Cl2
(100 mL) to the viscous yellow oil obtained resulted in a white
precipitate that was collected via filtration to give 17 as a white
solid (0.88 g, 25%), mp >250 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 7.18-7.32 (m, 3H), 8.06 (s, 2H) and 9.94 (br s, 1H).
LCMS (APCI-) m/z (rel intensity) 173 (25, [37ClM - H]-), 171
(80, [35ClM - H]-), 127 (100 [(35ClM - H) - B(OH)2]-).
5-((1H-1,2,4-Triazol-1-yl)methyl)-40-hydroxybiphenyl-2-car-
bonitrile 19. A 10 mL microwave vial was loaded with 10 (0.150
g, 0.570 mmol), 4-hydroxyphenylboronic acid (0.118 g, 0.855
mmol), K2CO3 (0.198 g, 1.43 mmol), tetrabutylammonium
bromide (0.189 g, 0.570 mmol), distilled H2O (3.5 mL), and
EtOH (1.5 mL). The vial was sealed and loaded (with no prior
degassing) into a CEM Discover microwave. After a run time of
5 min at 120 ꢀC (150 W), the reaction mixture was allowed to
cool and diluted with EtOAc (50 mL). The resulting mixture was
then washed with distilled H2O (25 mL ꢀ 3) and brine (25 mL).
The organic layer separated was dried (MgSO4), filtered, and
solvent removed in vacuo to leave a yellow/brown residue.
Column chromatography (EtOAc) eluted 19 as a white solid
(0.102 g, 65%), mp 211-213 ꢀC. 1H NMR δH (270 MHz,
CDCl3) 5.62 (s, 2H), 6.90 (d, J = 8.6, 2H), 7.32 (d, J = 8.2,
1H), 7.38 (d, J = 8.5, 2H), 7.45 (s, 1H), 7.89 (d, J = 7.9, 1H),
8.02 (s, 1H), 8.17 (s, 1H) and 9.87 (br s, 1H). LCMS (APCIþ) m/z
(rel intensity) 277 (100, [M þ H]þ). HRMS (ESþ) m/z calcd for
C16H13N4O [Mþ H]þ: 277.1084, found 277.1084. Anal. (C16H12-
N4O) C, H, N.
Uterine Weight Gain Assay. Ovariectomized female Wistar
rats (200 g) were purchased from Charles River. Groups of three
rats were treated with 10% THF/90% propylene glycol vehicle
(po), 1 μg E1/rat (sc in 10% ethanol/90% propylene glycol), or
orally with 2 and 23 at 10 mg/kg/d for 4 days. Animals were
sacrificed 24 h after administration of the last dose of compound
and uteri were removed, excised of fat, and weighed. Total body
weights of the rats were also recorded, and the uterine weight
was expressed as % total body weight.
hCAII Studies. To assess the inhibition of hCAII in vitro, an
adaptation of a colorimetric assay previously developed was
used.48 CAII-catalyzed hydrolysis of p-nitrophenyl acetate pro-
duces p-nitrophenol, which has an absorption peak at 384 nm
which allows for colorimetric determination of product and
subsequent calculation of enzyme activity.
Inhibition constants (IC50 values) were measured based on
the method previously described,28 using a 96-well plate spectro-
photometric assay except the initial rates were measured rather
than the absorbance after 20 min. Assays contained hCAII
(Sigma, 180 nM) and 1 mM 4-nitrophenol acetate in 50 mM
Tris-HCl, pH 7.6 in a final volume of 0.20 mL. Initial rates of the
enzyme reaction were monitored for 2 min at 20 ꢀC. Stock
solutions and dilutions of 23 were prepared in ethanol such that
there was a constant 5% ethanol in the assay. At least three
replicates at each concentration were used.
Protein Crystallography. The hanging drop vapor diffusion
method was used for crystallization. Protein (2.5 μL, ∼10 mg/
mL, ∼0.3 mM) containing 0.5 mM inhibitors and 30 mM 2-
mercaptoethanol was mixed with well buffer (2.5 μL; 0.1 M Tris/
HCl, pH 8.0, 1 mM ZnSO4, and 2.49 M ammonium sulfate),
with crystals appearing after 3-4 weeks at 4 ꢀC.
X-ray diffraction data were collected at station PX14.2 at the
Synchrotron Radiation Source (Daresbury, U.K.) under cryo-
genic conditions with a 5 s exposure and a crystal to detector
distance of 168.6 mm, with 100 frames of 1ꢀ oscillation collected.
Before data collection, the crystals were flash-cooled to 100 K in
a cryoprotectant containing the reservoir solution and 33% (v/v)
8 M sodium formate. Data were indexed and reduced with
DENZO and SCALEPACK modules of the HKL suite49 in the
orthorhombic P21 space group. The structure was determined
by the molecular replacement method using 1TTM.pdb41 as the
search model with the program Phaser50 from the CCP4i
program suite.45 Refinement was performed using Refmac551
from the program package CCP4i in addition to manual model
building using the program COOT.46 Clear density for the active
site zinc, and the inhibitor was observed after the first round of
refinement. The topology files for each inhibitor were generated
using the Dundee PRODRG2 Server.52 After alternating
cycles of water addition, refinement and manual rebuilding
50-((1H-1,2,4-Triazol-1-yl)methyl)-20-cyanobiphenyl-4-yl Sul-
famate 20. As general method using H2NSO2Cl (4.63 mL, 1.39
mmol), DMA (1.5 mL) and 19 (0.077 g, 0.279 mmol). The
reaction mixture was poured into EtOAc (30 mL). The organic
layer separated was washed with distilled H2O (30 mL ꢀ 4),
brine (30 mL), dried (MgSO4), and evaporated in vacuo to give
white residues. Column chromatography (CH2Cl2/acetone
80:20) eluted 15 as a white solid (0.074 g, 75%), mp 159-162
ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.60 (s, 2H), 7.41-7.46
(m, 3H), 7.51 (s, 1H), 7.67 (d, J = 8.2, 2H), 7.98 (d, J = 7.9, 1H),
8.03 (s, 1H), 8.17 (br s, 2H) and 8.73 (s, 1H). LCMS (APCI-) m/z
(rel intensity) 354 (100, [M - H]-). HRMS (FABþ) m/z calcd for
C16H14N5O3S (M þ H)þ: 356.0812, found 356.0811.
50-((1H-1,2,4-Triazol-1-yl)methyl)-20-cyanobiphenyl-4-yl Di-
methylsulfamate 21. To a white suspension of 19 (0.075 g,
0.271 mmol) and NEt3 (0.292 mL, 2.71 mmol) in anhydrous
CH2Cl2 (2 mL) at 0 ꢀC under an inert atmosphere was added to a
colorless solution of N,N-dimethylsulfamoyl chloride (0.408
mL, 3.80 mmol) in anhydrous CH2Cl2 (3 mL). The resulting
white suspension was left to stir under an inert atmosphere at
room temperature for 70 h, forming a clear orange/brown
solution. The reaction mixture was diluted in CH2Cl2 (30 mL)
and washed with distilled H2O (30 mL ꢀ 3) and brine (30 mL).
The organic fraction was separated, dried (MgSO4), filtered,
and solvent removed in vacuo to leave yellow residues. Column
chromatography (CH2Cl2/acetone 75:25) eluted 21 as a viscous
yellow oil (0.093 g, 90%). 1H NMR δH (270 MHz, CDCl3) 3.00
(s, 6H), 5.44 (s, 2H), 7.28-7.33 (m, 2H), 7.39 (d, J = 8.7 Hz,
2H), 7.53 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 7.9 Hz, 1H), 8.00 (s,
1H) and 8.18 (s, 1H). LCMS (APCI-) m/z (rel intensity) 382
(100, [M - H]-). HRMS (ESþ) m/z calcd for C18H18N5O3S
[M þ H]þ: 384.1125, found 384.1143.
the final inhibitor complex had a final Rfree = 0.19 and Rcryst
=
0.15. The statistics for refinement are summarized in the Sup-
porting Information. Coordinates and structure factors have
been deposited with the Protein Data Bank (accession code
2wd3.pdb).
General Method of Sulfamoylation. A solution of sulfamoyl
chloride (H2NSO2Cl) in toluene (ca. 0.7 M)23 was concentrated
in vacuo at 30 ꢀC to furnish a yellow oil which solidified upon
cooling in an ice bath. The substrate in anhydrous N,N-di-
methylacetamide (DMA) was subsequently added, and the
reaction mixture was allowed to warm to room temperature
and stirred overnight.
3-Hydroxy-4-chlorophenylboronic Acid 17. With stirring and
under an atmosphere of N2, a solution 5-bromo-2-chlorophenol
(4.25 g, 20.5 mmol) in anhydrous THF (100 mL) was cooled to
-78 ꢀC (dry ice/acetone). After 30 min, n-BuLi (16.4 mL, 40.9
mmol, 2.5 M in hexanes) was added dropwise over 30 min. The
reaction was left to stir for 1 h. Triisopropyl borate (5.65 mL,
24.6 mmol) was added dropwise with the reaction temperat-
5-((1H-1,2,4-Triazol-1-yl)methyl)-30-chloro-40-hydroxybiphe-
nyl-2-carbonitrile 22. The title compound was prepared from
10 and 3-chloro-4-hydroxyphenylboronic acid using similar

2166 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
conditions to those described for the synthesis of 19. Chromato-
graphy (EtOAc) eluted 22 as a white solid (0.218 g, 62%), mp
188-189 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.56 (s, 2H),
7.10 (d, J = 8.4 Hz, 1H), 7.34-7.39 (m, 2H), 7.52 (d, J = 1.2 Hz,
1H), 7.56 (d, J = 2.2 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 8.02 (s,
1H), 8.72 (s, 1H) and 10.69 (br s, 1H). LCMS (APCI-) m/z (rel
intensity) 311 (30, [37ClM - H]-), 309 (100, [35ClM - H]-).
HRMS (ESþ) m/z calcd for C16H12ClN4O [M þ H]þ: 311.0694,
found 311.0684. Anal. (C16H11ClN4O) C, H, N.
similar conditions to those described for the synthesis of 20.
Chromatography (CH2Cl2/acetone 80:20) eluted 29 as a white
solid (0.071 g, 92%), mp 137-138 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.69 (s, 2H), 7.38 (d, J = 7.6 Hz, 1H), 7.59-7.69 (m,
3H), 7.83-7.89 (m, 2H), 8.01-8.03 (m, 2H), 8.09 (s, 2H) and
8.74 (s, 1H). LCMS (APCIþ) m/z (rel intensity) 356 (100, [M þ
H]þ). HRMS (ESþ) m/z calcd for C16H14N5O3S [M þ H]þ:
356.0812, found 356.0811.
3-((1H-1,2,4-Triazol-1-yl)methyl)-40-hydroxybiphenyl-4-car-
bonitrile 30. The title compound was prepared from 11 and 4-
hydroxyphenylboronic acid using similar conditions to those
described for the synthesis of 19. Chromatography (EtOAc)
50-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-20-cyanobiphenyl-
4-yl Sulfamate 23. The title compound was prepared from 22
using similar conditions to those described for the synthesis of
20. Chromatography (CH2Cl2/acetone 80:20) eluted 23 as a
white solid (0.292 g, 93%), mp 133-136 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.59 (s, 2H), 7.41-7.46 (d, J = 8.2 Hz, 1H),
7.59 (s, 1H), 7.65 (s, 2H), 7.84 (s, 1H), 7.99 (d, J = 7.9 Hz, 1H),
8.03 (s, 1H), 8.45 (br s, 2H) and 8.72 (s, 1H). LCMS (APCIþ) m/z
(rel intensity) 392 (43, [37ClM þ H]þ), 390 (100, [35ClM þ H]þ).
HRMS (FABþ) m/z calcd for C16H13ClN5O3S [M þ H]þ:
390.0422, found 390.0421. Anal. (C16H12ClN5O3S): C, H, N.
50-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-20-cyanobiphenyl-
4-yl Dimethylsulfamate 24. The title compound was prepared
from 22 using similar conditions to those described for the
synthesis of 21. Chromatography (CH2Cl2/acetone 75:25)
1
eluted 30 as a white solid (0.082 g, 79%), mp 203-204 ꢀC. H
NMR δH (270 MHz, DMSO-d6) 5.62 (s, 2H), 6.86 (d, J = 8.7
Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.67-7.89 (m, 3H), 7.99 (s,
1H), 8.71 (s, 1H) and 9.83 (br s, 1H). LCMS (APCI-) m/z (rel
intensity) 275 (100, [M þ H]þ). HRMS (ESþ) m/z calcd for
C16H13N4O [M þ H]þ: 277.1084, found 277.1080.
30-((1H-1,2,4-Triazol-1-yl)methyl)-40-cyanobiphenyl-4-yl Sul-
famate 31. The title compound was prepared from 30 using
similar conditions to those described for the synthesis of 20.
Chromatography (CH2Cl2/acetone 80:20) eluted 31 as a white
solid (0.106 g, 82%), mp 179-180 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.67 (s, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.7
Hz, 2H), 7.82-7.86 (s, 2H), 7.98 (d, J = 8.2 Hz, 1H), 8.01 (s,
1H), 8.08 (s, 2H) and 8.72 (s, 1H). LCMS (APCIþ) m/z (rel
intensity) 356 (100, [M þ H]þ). HRMS (ESþ) m/z calcd for
C16H14N5O3S [M þ H]þ: 356.0812, found 356.0813. Anal.
(C16H13N5O3S) C, H, N.
1
eluted 24 as a white solid (0.189 g, 60%), mp 119-120 ꢀC. H
NMR δH (270 MHz, CDCl3) 3.07 (s, 6H), 5.45 (s, 2H),
7.30-7.33 (m, 2H), 7.43 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.58 (d,
J = 2.2 Hz, 1H), 7.62 (d, J = 10.9 Hz, 1H), 7.77 (d, J = 8.7 Hz,
1H), 8.01 (s, 1H) and 8.18 (s, 1H). LCMS (APCI-), m/z 418 (40,
[37ClM - H]-), 416 (100, [35ClM- - H]-). HRMS (ESþ) m/z
calcd for C18H17ClN5O3S [M þ H]þ: 418.0735, found 418.0725.
Anal. (C18H16ClN5O3S) C, H, N.
3-((1H-1,2,4-Triazol-1-yl)methyl)-30-chloro-40-hydroxybiphe-
nyl-4-carbonitrile 32. The title compound was prepared from 11
and 3-chloro-4-hydroxyphenylboronic acid using similar con-
ditions to those described for the synthesis of 19. Chromato-
graphy (EtOAc) eluted 32 as a white solid (0.065 g, 37%), mp
244-247 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.67 (s, 2H),
7.09 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 2.2 Hz, 8.2 Hz, 1H), 7.75
(d, J = 2.2 Hz, 1H), 7.80-7.83 (m, 2H), 7.89-7.93 (m, 2H), 8.04
(s, 1H) and 8.73 (br s, 1H). LCMS (APCIþ) m/z (rel intensity)
313 (35, [37ClM þ H]þ), 311 (100, [35ClM þ H]þ). HRMS
(FABþ) m/z calcd for C16H12ClN4O [M þ H]þ: 311.0694, found
311.0690. Anal. (C16H11ClN4O) C, H, N.
5-((1H-1,2,4-Triazol-1-yl)methyl)-40-chloro-30-hydroxybiphe-
nyl-2-carbonitrile 25. The title compound was prepared from 10
and 17 using similar conditions to those described for the
synthesis of 19. Chromatography (EtOAc) eluted 25 as a white
solid (0.111 g, 54%), mp 228-229 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.58 (s, 2H), 6.96 (dd, J = 2.2 Hz, 8.2 Hz, 1H), 7.11
(d, J = 2.2 Hz, 1H), 7.41 (dd, J = 2.2 Hz, 7.9 Hz, 1H), 7.47 (d,
J = 8.2 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.2 Hz,
1H), 8.03 (s, 1H), 8.72 (s, 1H) and 10.60 (br s, 1H). LCMS
(APCI-) m/z (rel intensity) 311 (25, [37ClM - H]-), 309 (100,
[35ClM - H]-). HRMS (ESþ) m/z calcd for C16H12ClN4O [M þ
H]þ: 311.0694, found 311.0680. Anal. (C16H11ClN4O) C, H, N.
50-((1H-1,2,4-Triazol-1-yl)methyl)-4-chloro-20-cyanobiphenyl-
3-yl Sulfamate 26. The title compound was prepared from 25
using similar conditions to those described for the synthesis of
20. Chromatography (CH2Cl2/acetone 80:20) eluted 26 as a
white solid (0.087 g, 87%), mp 148-150 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.59 (s, 2H), 7.45 (dd, J = 1.5 Hz, 7.9 Hz, 1H),
7.57 (dd, J = 2.2 Hz, 8.4 Hz, 1H), 7.61 (d, J = 1.5 Hz, 1H), 7.66
(d, J = 2.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 7.9 Hz,
1H), 8.03 (s, 1H), 8.37 (br s, 2H) and 8.72 (s, 1H). LCMS
(APCI-) m/z (rel intensity) 390 (20, [37ClM - H]-), 388 (100,
[35ClM - H]-). HRMS (ESþ) m/z calcd for C16H13ClN5O3S
[M þ H]þ: 390.0422, found 390.0421.
30-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-40-cyanobiphenyl-
4-yl Sulfamate 33. The title compound was prepared from 32
using similar conditions to those described for the synthesis of
20. Chromatography (CH2Cl2/acetone 80:20) eluted 33 as a
white solid (0.057 g, 78%), mp 155-158 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.73 (s, 2H), 7.62 (d, J = 8.7 Hz, 1H), 7.78 (d,
J = 8.7 Hz, 1H), 7.89-7.98 (m, 4H), 8.01 (s, 1H), 8.38 (br s, 2H)
and 8.73 (s, 1H). LCMS (APCIþ) m/z (rel intensity) 392
(40, [37ClM þ H]þ), 390 (100, [35ClM þ H]þ). HRMS (ESþ)
m/zcalcd for C16H13ClN5O3S[M þ H]þ: 390.0422, found 390.0426.
5-((1H-1,2,4-Triazol-1-yl)methyl)biphenyl-3-carbonitrile 34.
The title compound was prepared from 12 and phenylboronic
acid using similar conditions to those described for the synthesis
of 19. Chromatography (CHCl3/acetone 90:10) eluted 34 as a
white solid (0.050 g, 77%), mp 109-110 ꢀC. 1H NMR δH (270
MHz, CDCl3) 5.45 (s, 2H), 7.44-7.53 (m, 6H), 7.67 (s, 1H), 7.84
(s, 1H), 8.03 (s, 1H) and 8.19 (s, 1H). LCMS (ES-) m/z (rel
intensity) 261 (100, [M þ H]-). HRMS (ESþ) m/z calcd for
C16H13N4 [M þ H]þ: 261.1135, found 261.1122.
3-((1H-1,2,4-Triazol-1-yl)methyl)-30-hydroxybiphenyl-4-car-
bonitrile 28. The title compound was prepared from 11 and 3-
hydroxyphenylboronic acid using similar conditions to those
described for the synthesis of 19. Chromatography (EtOAc)
1
eluted 28 as a white solid (0.184 g, 89%), mp 183-184 ꢀC. H
5-((1H-1,2,4-Triazol-1-yl)methyl)-40-fluorobiphenyl-3-carbo-
nitrile 35. The title compound was prepared from 12 and 4-
fluorophenylboronic acid using similar conditions to those
described for the synthesis of 19. Chromatography (CHCl3/
acetone 90:10) eluted 35 as a white solid (0.040 g, 50%), mp
134-135 ꢀC. 1H NMR δH (270 MHz, CDCl3) 5.45 (s, 2H),
7.13-7.21 (m, 2H), 7.46-7.51 (m, 3H), 7.62 (s, 1H), 7.79 (s, 1H),
8.03 (s, 1H) and 8.19 (s, 1H). LCMS (ESþ) m/z (rel intensity) 279
(100, [M þ H]þ). HRMS (ESþ) m/z calcd for C16H12FN4 [M þ
H]þ: 279.1041, found 279.1030.
NMR δH (270 MHz, DMSO-d6) 5.68 (s, 2H), 6.85 (dd, J = 2.2
Hz, 8.2 Hz, 1H), 7.01-7.10 (m, 2H), 7.30 (t, J = 8.2 Hz, 1H),
7.68 (s, 1H), 7.78 (dd, J = 2.2 Hz, 8.2 Hz, 1H), 7.95 (d, J = 8.2
Hz, 1H), 8.06 (s, 1H), 8.75 (s, 1H) and 9.73 (br s, 1H). LCMS
(APCIþ) m/z (rel intensity) 277 (100, [M þ H]þ). HRMS (ESþ)
m/z calcd for C16H13N4O [M þ H]þ: 277.1084, found 277.1071.
Anal. (C16H12N4O) C, H, N.
30-((1H-1,2,4-Triazol-1-yl)methyl)-40-cyanobiphenyl-3-yl Sul-
famate 29. The title compound was prepared from 28 using

Article
5-((1H-1,2,4-Triazol-1-yl)methyl)biphenyl-3,40-dicarbonitrile
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2167
(APCIþ) m/z (rel. intensity) 251 (18, [M]þ). HRMS (ESþ) m/z
calcd for C15H14N3O [M þ H]þ: 252.1131, found 252.1144.
Anal. (C15H13N3O) C, H, N.
36. The title compound was prepared from 12 and 4-cyanophe-
nylboronic acid using similar conditions to those described for
the synthesis of 19. Chromatography (CHCl3/acetone 80:20)
30-((1H-1,2,4-Triazol-1-yl)methyl)biphenyl-4-yl Sulfamate 43.
The title compound was prepared from 42 using similar condi-
tions to those described for the synthesis of 20. Column chro-
matography (CH2Cl2/acetone 80:20) eluted 43 (0.055 g, 71%) as
a white solid, mp 149-152 ꢀC. 1H NMR δH (400 MHz, DMSO-
d6) 5.51 (s, 2H), 7.29 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 8.7 Hz,
2H), 7.48 (t, J = 7.5 Hz, 1H), 7.60-7.64 (m, 2H), 7.73 (d, J = 8.7
Hz, 2H), 8.01 (s, 1H), 8.07 (br s, 2H) and 8.73 (s, 1H). LCMS
(ES-) m/z (rel intensity) 329 (100, [M - H]-). HRMS (FABþ)
m/z calcd for C15H14N4O3S [M]þ: 330.0787, found 330.0782.
30-((1H-1,2,4-Triazol-1-yl)methyl)-3-chlorobiphenyl-4-ol 44.
The title compound was prepared from 13 and 3-chloro-4-
hydroxyphenylboronic acid using similar conditions to those
described for the synthesis of 19. Column chromatography
(EtOAc) eluted 44 as a white solid (0.129 g, 72%), mp
153-154 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.54 (s, 2H),
7.05 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.36-7.45 (m,
2H), 7.55 (d, J = 7.4 Hz, 2H), 7.61 (d, J = 2.2 Hz, 1H), 7.99 (s,
1H), 8.70 (s, 1H) and 10.36 (br s, 1H). LCMS (APCI-) m/z (rel
intensity) 286 (30, [37ClM - H]-), 284 (100, [35ClM - H]-).
HRMS (ESþ) m/z calcd for C15H13ClN3O [M þ H]þ: 286.0742,
found 286.0737.
1
eluted 36 as a white solid (0.038 g, 47%), mp 154-155 ꢀC. H
NMR δH (270 MHz, CDCl3) 5.47 (s, 2H), 7.49-7.67 (m, 4H),
7.76-7.79 (m, 2H), 7.84 (t, J = 1.4 Hz, 1H), 8.03 (s, 1H) and 8.21
(s, 1H). LCMS (ESþ) m/z (rel intensity) 286 (100, [M þ H]þ).
HRMS (ESþ) m/z calcd for C17H12N5 [M þ H]þ: 286.1087,
found 286.1075.
5-((1H-1,2,4-Triazol-1-yl)methyl)-40-hydroxybiphenyl-3-car-
bonitrile 37. The title compound was prepared from 12 and 4-
hydroxyphenylboronic acid using similar conditions to those
described for the synthesis of 19. Chromatography (CHCl3/
acetone 80:20) eluted 37 as a white solid (0.086 g, 81%), mp
186-187 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.52 (s, 2H),
6.87 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H),
7.86 (s, 1H), 8.02 (s, 2H), 8.72 (s, 1H) and 9.78 (br s, 1H). LCMS
(ES-) m/z (rel intensity) 277 (100, [M þ H]-). HRMS (ESþ) m/z
calcd for C16H13N4O [M þ H]þ: 277.1084, found 277.1071.
Anal. (C16H12N4O) C, H, N.
30-((1H-1,2,4-Triazol-1-yl)methyl)-50-cyanobiphenyl-4-yl Sul-
famate 38. The title compound was prepared from 37 using
similar conditions to those described for the synthesis of 20.
Chromatography (CH2Cl2/MeOH 96:4) eluted 38 as a white
solid (0.115 g, 89%), mp 166-167 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.56 (s, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.76 (s, 1H),
7.81 (d, J = 8.5 Hz, 2H), 7.96 (s, 1H), 8.02 (s, 1H), 8.09 (br s,
2H), 8.15 (s, 1H) and 8.73 (s, 1H). LCMS (ESþ) m/z (rel
intensity) 356 (100, [M þ H]þ). HRMS (ESþ) m/z calcd for
C16H14N5O3S [M þ H]þ: 356.0812, found 356.0800.
30-((1H-1,2,4-Triazol-1-yl)methyl)-3-chlorobiphenyl-4-yl Sul-
famate 45. The title compound was prepared from 44 using
similar conditions to those described for the synthesis of 20.
Chromatography (CH2Cl2/acetone 80:20) eluted 45 as a white
solid (0.055 g, 62%), mp 144-147 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.49 (s, 2H), 7.29 (d, J = 7.4 Hz, 1H), 7.47 (t, J = 5.9
Hz, 1H), 7.57-7.72 (m, 4H), 7.87 (d, J = 2.0 Hz, 1H), 8.00 (s,
1H), 8.34 (br s, 2H) and 8.72 (s, 1H). LCMS (APCIþ) m/z (rel
intensity) 367 (20, [37ClM þ H]þ), 365 (55, [M þ H]þ), 288 (30,
[37ClM - SO2NH2]þ), 286 (100, [35ClM - SO2NH2]þ). HRMS
(ESþ) m/z calcd for C15H14ClN4O3S [M þ H]þ: 365.0470, found
365.0471.
5-((1H-1,2,4-Triazol-1-yl)methyl)-30-chloro-40-hydroxybiphe-
nyl-3-carbonitrile 39. The title compound was prepared from 12
and 3-chloro-4-hydroxyphenylboronic acid using similar con-
ditions to those described for the synthesis of 19. Chromato-
graphy (CHCl3/acetone 80:20) eluted 39 as a white solid (0.189 g,
80%), mp 208-209 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.52
(s, 2H), 7.06 (d, J = 8.5 Hz, 1H), 7.53 (dd, J = 2.2 and 8.5 Hz, 1H),
7.65 (s, 1H), 7.76 (d, J= 2.2 Hz, 1H), 7.95 (s, 1H), 8.01 (s, 1H), 8.10
(t, J = 1.6 Hz, 1H) and 8.72 (s, 1H). LCMS (ESþ) m/z (rel
intensity) 313 (36, [37ClM þ H]þ), 311 (100, [35ClM þ H]þ).
HRMS (ESþ) m/z calcd for C16H12ClN4O [M þ H]þ: 311.0694,
found 311.0686. Anal. (C16H11ClN4O) C, H, N.
4-((1H-1,2,4-Triazol-1-yl)methyl)-40-hydroxybiphenyl-2-car-
bonitrile 47. The title compound was prepared from 14 and 4-
hydroxyphenylboronic acid using similar conditions to those
described for the synthesis of 19. Chromatography (EtOAc))
1
eluted 47 as a white solid (0.061 g, 60%), mp 168-171 ꢀC. H
NMR δH (270 MHz, DMSO-d6) 5.51 (s, 2H), 6.89 (d, J = 8.7
Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.2 Hz, 1H), 7.62
(dd, J = 2.0 Hz, 8.2 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H), 8.02 (s,
1H), 8.72 (s, 1H) and 9.85 (br s, 1H). LCMS (APCI-) m/z (rel
intensity) 275 (100, [M - H]-). HRMS (FABþ) calcd for
C16H13N4O [M þ H]þ: 277.1084, found 277.1077. Anal.
(C16H12N4O) C, H, N.
30-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-50-cyanobiphenyl-
4-yl Sulfamate 40. The title compound was prepared from 39
using similar conditions to those described for the synthesis of
20. Chromatography (CHCl3/acetone 80:20) eluted 40 as a white
solid (0.120 g, 87%), mp 163-164 ꢀC. 1H NMR δH (270 MHz,
DMSO-d6) 5.55 (s, 2H), 7.61 (d, J = 8.8 Hz, 1H), 7.77-7.80 (m,
2H), 8.00-8.04 (m, 3H), 8.22 (s, 1H), 8.36 (br s, 2H) and 8.72 (s,
1H). LCMS (ESþ) m/z (rel intensity) 392 (38, [37ClM þ H]þ), 390
(100, [35ClM þ H]þ), 313 (18, [37ClM - SO2NH2]þ), 311 (56,
40-((1H-1,2,4-Triazol-1-yl)methyl)-20-cyanobiphenyl-4-yl Sul-
famate 48. The title compound was prepared from 47 using
similar conditions to those described for the synthesis of 20.
Chromatography (CH2Cl2/acetone 80:20) eluted 48 a white
solid (0.051 g, 66%), mp 157-160 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.55 (s, 2H), 7.43 (d, J = 8.6 Hz, 2H),
7.62-7.70 (m, 4H), 7.92 (s, 1H), 8.03 (s, 1H), 8.16 (br s, 2H)
and8.73 (s, 1H);LCMS(APCI-) m/z (rel intensity) 354 (100, [M -
H]-). HRMS (FABþ) calcd for C16H14N5O3S [M þ H]þ:
356.0812, found 356.0759.
[35ClM
-
SO2NH2]þ). HRMS (ESþ) m/z calcd for
C16H13ClN5O3S [M þ H]þ: 390.0422, found 390.0408. Anal.
(C16H12ClN5O3S) C, H. N.
30-((1H-1,2,4-Triazol-1-yl)methyl)biphenyl-4-ol 42. Com-
pound 13 (0.100 g 4.20 mmol), 4-hydroxyphenylboronic acid
(0.087 g, 0.630 mmol), K2CO3 (0.145 g, 1.05 mmol), tetrabutyl-
ammonium bromide (0.139 g, 0.420 mmol), distilled H2O
(7 mL), and EtOH (3 mL) were combined and degassed with
N2 for 30 min. Pd(OAc)2 (3 mol %) was added and the reaction
mixture heated with vigorous stirring at 70 ꢀC for 1 h. Upon
cooling, EtOAc (50 mL) was added and washed with distilled
H2O (50 mL ꢀ 2) and brine (50 mL). The organic portion was
separated and dried (MgSO4), filtered, and solvent removed in
vacuo to leave a yellow/brown residue. Column chromatog-
raphy (EtOAc) eluted 42 as a white solid (0.092 g, 88%), mp
163-164 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.44 (s, 2H),
6.83 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 7.7 Hz, 1H), 7.34-7.51
(m, 5H), 7.92 (s, 1H), 8.68 (s, 1H) and 9.58 (br s, 1H). LCMS
4-((1H-1,2,4-Triazol-1-yl)methyl)-30-chloro-40-hydroxybiphe-
nyl-2-carbonitrile 49. The title compound was prepared from 14
and 3-chloro-4-hydroxyphenylboronic acid using similar con-
ditions to those described for the synthesis of 19. Chromato-
graphy (EtOAc) eluted 49 as a white solid (0.072 g, 41%), mp
195-197 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.51 (s, 2H),
7.09 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 2.2 Hz, 8.7 Hz, 1H),
7.57-7.61 (m, 3H), 7.86 (s, 1H), 8.02 (s, 1H), 8.72 (s, 1H) and
10.68 (br s, 1H). LCMS (APCI-) m/z (rel intensity) 311 (25,
[37ClM - H]-), 309 (100, [35ClM - H]-). HRMS (ESþ) m/z
calcd for C16H12ClN4O [M þ H]þ: 311.0694, found 311.0681.

2168 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Woo et al.
40-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-20-cyanobiphenyl-
4-yl Sulfamate 50. The title compound was prepared from 49
using similar conditions to those described for the synthesis of
20. Chromatography (CH2Cl2/acetone 80:20) eluted 50 as a
white solid (0.071 g, 82%), mp 151-153 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.55 (s, 2H), 7.61-7.68 (m, 4H), 7.87 (t, J =
1.0 Hz, 1H), 7.94 (s, 1H), 8.04 (s, 1H), 8.43 (br s, 2H) and 8.74 (s,
1H). LCMS (APCI-) m/z (rel intensity) 391 (20, [37ClM - H]-),
388 (100, [35ClM - H]-). HRMS (ESþ) m/z calcd for
C16H13ClN5O3S [M þ H]þ: 390.0422, found 390.0418.
synthesis of 20. Chromatography (CH2Cl2/acetone 80:20)
eluted 58 as a white solid (0.097 g, 88%), mp 71-74 ꢀC. H
1
NMR δH (270 MHz, DMSO-d6) 1.74 (s, 6H), 5.53 (s, 2H), 7.53
(s, 1H), 7.58-7.61 (m, 2H), 7.71 (d, J = 2.2 Hz, 1H), 7.73 (s,
1H), 7.94 (d, J = 2.2 Hz, 1H), 8.01 (s, 1H), 8.35 (s, 2H) and 8.74
(s, 1H). LCMS (APCI-) m/z (rel intensity) 432 (15, [37ClM -
H]-), 430 (35, [35ClM - H]-), 353 (32, [37ClM - SO2NH2]-),
351 (100, [35ClM - SO2NH2]-). HRMS (ESþ) m/z calcd for
C19H19ClN5O3S [M þ H]þ: 432.0892, found 432.0889.
6-((1H-1,2,4-Triazol-1-yl)methyl)-30-chloro-40-hydroxybiphe-
nyl-3-carbonitrile 52. The title compound was prepared from 15
and 3-chloro-4-hydroxyphenylboronic acid using similar con-
ditions to those described for the synthesis of 19. Chromato-
graphy (EtOAc) eluted 52 as a white crystalline solid (0.089 g,
46%), mp 243-245 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 5.46
(s, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.15-7.23 (m, 2H), 7.44 (d, J =
2.2 Hz, 1H), 7.75 (d, J = 1.7 Hz, 1H), 7.80 (dd, J = 1.7 Hz, 7.9
Hz, 1H), 7.98 (s, 1H), 8.43 (s, 1H) and 10.51 (br s, 1H). LCMS
(APCI-) m/z (rel intensity) 311 (35, [37ClM - H]-), 309 (100,
[35ClM - H]-). HRMS (ESþ) m/z calcd for C16H12ClN4O [M þ
H]þ: 311.0694, found 311.0689.
Acknowledgment. This work was supported by Sterix Ltd.,
which is part of the Ipsen group. We thank A. C. Smith for
technical support and C. M. Parker for assistance in preparing
the manuscript.
Supporting Information Available: 13C NMR data for 19-26,
28-40, 42, 43, 45, 48-50, 52, 53, and 55-58. Combustion
analysis of 19, 22-25, 28, 31, 32, 37, 39, 40, 42, and 47. HPLC
and purity data of biologically tested compounds without
combustion analysis. Crystallographic statistics of data collec-
tion and refinement for the hCA II-23 complex. This material is
available free of charge via the Internet at http://pubs.acs.org.
20-((1H-1,2,4-Triazol-1-yl)methyl)-3-chloro-50-cyanobiphenyl-
4-yl Sulfamate 53. The title compound was prepared from 52
using similar conditions to those described for the synthesis of
20. Chromatography (CH2Cl2/acetone 80:20) eluted 53 as a
white solid (0.051 g, 82%), mp 139-142 ꢀC. 1H NMR δH (270
MHz, DMSO-d6) 5.47 (s, 2H), 7.26 (d, J = 7.9 Hz, 1H), 7.52 (dd,
J = 2.2 Hz, 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.73 (d, J =
2.2 Hz, 1H), 7.83 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 1.7 Hz, 7.9
Hz, 1H), 7.99 (s, 1H), 8.38 (br s, 2H) and 8.45 (s, 1H). LCMS
(APCI-) m/z (rel intensity) 390 (20, [37ClM - H]-), 388 (60,
[35ClM - H]-), 311 (35 [37ClM - SO2NH2]-), 309 (100, [35ClM -
SO2NH2]-). HRMS (ESþ) m/z calcd for C16H13ClN5O3S [M þ
H]þ: 390.0422, found 390.0405.
2-(5-((1H-1,2,4-Triazol-1-yl)methyl)-40-hydroxybiphenyl-3-yl)-
2-methylpropanenitrile 55. The title compound was prepared
from 16 and 4-hydroxyphenylboronic acid using similar condi-
tions to those described for the synthesis of 19. Chromatography
(EtOAc) eluted 55 as a pale-yellow solid (0.216 g, 89%), mp
66-68 ꢀC. 1H NMR δH (270 MHz, DMSO-d6) 1.71 (s, 6H), 5.49
(s, 2H), 6.86 (d, J = 8.7 Hz, 2H), 7.38 (s, 1H), 7.42 (s, 1H), 7.46
(d, J = 8.7 Hz, 2H), 7.59 (s, 1H), 8.00 (s, 1H), 8.72 (s, 1H) and
9.64 (br s, 1H). LCMS (APCI-) m/z (rel intensity) 317 (100, [M -
H]-). HRMS (ESþ) m/z calcd for C19H19N4O [M þ H]þ:
319.1553, found 319.1550.
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solid (0.111 g, 86%), mp 74-76 ꢀC. H NMR δH (270 MHz,
DMSO-d6) 1.72 (s, 6H), 5.52 (s, 2H), 7.37 (d, J = 8.7 Hz, 2H),
7.49 (s, 1H), 7.67 (s, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.99 (s, 1H),
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Article
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