Chiral aromatase and dual aromatase-steroid sulfatase inhibitors from the letrozole template: Synthesis, absolute configuration, and in vitro activity

Article abstract of DOI:10.1021/jm800168s

To explore aromatase inhibition and to broaden the structural diversity of dual aromatase-sulfatase inhibitors (DASIs), we introduced the steroid sulfatase (STS) inhibitory pharmacophore to letrozole. Letrozole derivatives were prepared bearing bis-sulfamates or mono-sulfamates with or without adjacent substituents. The most potent of the achiral and racemic aromatase inhibitor was 40 (IC₅₀ = 3.0 nM). Its phenolic precursor 39 was separated by chiral HPLC, and the absolute configuration of each enantiomer was determined using vibrational and electronic circular dichroism in tandem with calculations of the predicted spectra. Of the two enantiomers, (R)-phenol (39a) was the most potent aromatase inhibitor (IC₅₀ = 0.6 nM, comparable to letrozole), whereas the (S)-sulfamate, (40b) inhibited STS most potently (IC₅₀ = 553 nM). These results suggest that a new structural class of DASI for potential treatment of hormone-dependent breast cancer has been identified, and this is the first report of STS inhibition by an enantiopure nonsteroidal compound.

Full text of DOI:10.1021/jm800168s

4226
J. Med. Chem. 2008, 51, 4226–4238
Chiral Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors from the Letrozole
Template: Synthesis, Absolute Configuration, and In Vitro Activity†
Paul M. Wood,‡ L. W. Lawrence Woo,‡ Jean-Robert Labrosse,‡ Melanie N. Trusselle,‡,† Sergio Abbate,§ Giovanna Longhi,§
Ettore Castiglioni,§,| France Lebon,§ 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, United Kingdom, Dipartimento di Scienze Biomediche e Biotecnologie, UniVersita` di Brescia, Viale Europa 11, 25133 Brescia, Italy,
JASCO Corporation, 2967-5 Ishikawa-cho, Hachioji-shi, Tokyo, Japan, Endocrinology and Metabolic Medicine and Sterix Ltd., Imperial
College London, Faculty of Medicine, St. Mary’s Hospital, London, W2 1NY, United Kingdom
ReceiVed February 18, 2008
To explore aromatase inhibition and to broaden the structural diversity of dual aromatase-sulfatase inhibitors
(DASIs), we introduced the steroid sulfatase (STS) inhibitory pharmacophore to letrozole. Letrozole derivatives
were prepared bearing bis-sulfamates or mono-sulfamates with or without adjacent substituents. The most
potent of the achiral and racemic aromatase inhibitor was 40 (IC50 ) 3.0 nM). Its phenolic precursor 39 was
separated by chiral HPLC, and the absolute configuration of each enantiomer was determined using vibrational
and electronic circular dichroism in tandem with calculations of the predicted spectra. Of the two enantiomers,
(R)-phenol (39a) was the most potent aromatase inhibitor (IC50 ) 0.6 nM, comparable to letrozole), whereas
the (S)-sulfamate, (40b) inhibited STS most potently (IC50 ) 553 nM). These results suggest that a new
structural class of DASI for potential treatment of hormone-dependent breast cancer has been identified,
and this is the first report of STS inhibition by an enantiopure nonsteroidal compound.
Introduction
tane is a mechanism-based or suicide inhibitor, which is
metabolized to a reactive species that irreversibly binds to the
enzyme active site causing enzyme inactivation. In contrast,
letrozole and anastrozole are reversible inhibitors, whose
mechanism of action involves coordination to the heme iron of
aromatase by one of the N atoms contained within the triazole
moiety.
The position of tamoxifen as the standard adjuvant treatment
for hormone-dependent breast cancer (HDBC)a in postmeno-
pausal women is now being challenged by the third generation
of aromatase inhibitors (AIs), which comprises the nonsteroidal
compounds anastrozole and letrozole and the steroidal exemes-
tane (Figure 1).1–4 This modification of the treatment paradigm
has arisen from the results of a number of large randomized
neoadjuvant and adjuvant clinical trials in which the AIs have
been shown to exhibit an improved efficacy compared to
tamoxifen.5–8 Of the third generation AIs, letrozole is 2-5 times
more potent than both anastrozole and exemestane in noncellular
systems and 10-20 times more potent in cellular systems.9 At
present it is unclear whether these differences in potency will
be meaningful in the clinic; however, the Femara versus
Anastrozole clinical evaluation (FACE) trial is seeking to
address this issue in the adjuvant setting.10
While the potential application of AIs has broadened, a new
strategy for estrogen deprivation has arisen from the develop-
ment of inhibitors of the enzyme steroid sulfatase (STS). STS
is responsible for the hydrolysis of alkyl and aryl steroid sulfates
to their unconjugated and biologically active forms. In breast
cancer tissue, it has been shown that 10 times more estrone
originates from estrone sulfate than from androstenedione.11 In
addition, STS controls the formation of dehydroepiandrosterone
(DHEA) from DHEA-sulfate (DHEA-S). DHEA can be sub-
sequently converted to androst-5-ene-3ꢀ,17ꢀ-diol, an androgen
with estrogenic properties capable of stimulating the growth of
breast cancer cells in vitro12 and supports the growth of
carcinogen induced mammary tumors in rodents.13 A number
of steroidal and nonsteroidal irreversible STS inhibitors have
been developed,14–16 and the nonsteroidal compound STX64
(BN83495, Figure 1)17–19 has been evaluated in a phase I clinical
trial for the treatment of patients with advanced breast cancer.20
In this trial, stable disease was observed in 5 out of 8 evaluable
patients receiving a treatment protocol consisting of either a 5
or a 20 mg daily dose of STX64.
The development of a dual aromatase-sulfatase inhibitor (DASI)
is an attractive concept as this single agent should inhibit estrogen
synthesis by both the aromatase and STS pathway and additionally
inhibit the formation of DHEA from DHEA-S. The synthesis and
biological activity of the first series of DASIs based on the known
AI YM511 (4-((4-bromobenzyl)(4H-1,2,4-triazol-4-yl)amino)ben-
zonitrile) have been previously reported by us (1, Figure 2).21,22
Furthermore, we have recently reported on the synthesis and in
vitro and in vivo inhibitory activity of novel compounds based
upon the letrozole and anastrozole templates.23,24 In addition to
Whereas tamoxifen antagonizes the binding of estrogens to
the estrogen receptor in a breast tumor, AIs inhibit the
cytochrome P450 enzyme aromatase, which is responsible for
the final step in the conversion of androgens to estrogens,
resulting in a reduction in circulating estrogen levels. Exemes-
†
This paper is dedicated to Dr. Melanie Trusselle (1973-2008).
* To whom correspondence should be addressed. Phone: +44 1225
386639. Fax: +44 1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
‡
Department of Pharmacy and Pharmacology and Sterix Ltd., University
of Bath.
§
Dipartimento di Scienze Biomediche e Biotecnologie, Universita` di
Brescia.
|
JASCO Corporation, Tokyo.
Endocrinology and Metabolic Medicine and Sterix Ltd., Imperial
College London.
a Abbreviations: AC, Absolute configuration; AIs, aromatase inhibitors;
DASI, dual aromatase-sulfatase inhibitor; DFT, density functional theory;
DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate;
ECD, electronic circular dichroism; FACE, femara versus anastrozole
clinical evaluation; HDBC, hormone-dependent breast cancer; STS, steroid
sulfatase; TDDFT, time dependent density functional theory; VCD,
vibrational circular dichroism.
10.1021/jm800168s CCC: $40.75
2008 American Chemical Society
Published on Web 07/01/2008

Chiral Aromatase and DASIs from the Letrozole Template
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4227
Figure 1. Third generation aromatase inhibitors anastrozole, letrozole, and exemestane and steroid sulfatase inhibitor STX64.
Figure 2. A YM511-based DASI 1, 4,4′-benzophenone bis-sulfamate 2 and prototype DASI 3.
Scheme 1. Synthesis of 7a
a Reagents and conditions: (a) NaNO2, conc H2SO4, 85% H3PO4, 0 °C; (b) NaBH4, EtOH/H2O, r.t.; (c) 1,2,4-triazole, p-toluenesulfonic acid (p-TSA),
toluene, reflux; (d) H2NSO2Cl, DMA, r.t.
our nonsteroidal DASIs, there has been interest in a steroidal DASI,
with Numazawa et al.25 reporting the conversion of a series of
steroidal AIs26 to give a range of STS inhibitors based on estrogen-
3-sulfamates. The sulfamates were good to potent STS inhibitors
(best IC50 STS ) 15 nM) but were found to be weak AIs (best IC50
AROM ) 41.8 µM).
followed by reaction with 4-fluorobenzonitrile according to the
procedure described by Bowman et al.23,29 The reference STS
inhibitor STX64 was synthesized according to the method of
Woo et al.17
All of the novel sulfamates and phenols described in this
paper were prepared according to the schemes 1–4. The final
compounds and intermediates were characterized by standard
analytical methods and, in addition to this, the purity of the
compounds tested in vitro was evaluated using HPLC and they
were >99% pure.
The first two compounds (3 and 7) synthesized differ from
letrozole by having no cyano groups but instead sulfamate
groups at either the para or meta positions on the aromatic ring.
We previously reported the synthesis of 3 from the commercially
available 4,4′-dihydroxybenzophenone.23 The synthesis of 7
commences with the formation of 3,3′-dihydroxybenzophenone
4 from 3,3′-diaminobenzophenone according to the method of
Wittig via diazotization followed by hydroxy-dediazoniation
(Scheme 1).30 Hejaz et al.27 previously reported the synthesis
of the starting 3,3′-dihydroxybenzophenone, albeit via a longer
route, from the nitration of benzophenone followed by reduction,
diazotization, and hydroxy-dediazoniation reactions. The reduc-
tion of ketone 4 with NaBH4 gave 5, from which the hydroxyl
group could be displaced with 1,2,4-triazole in refluxing toluene
to provide 6. This bis-phenol was converted to 3,3′-bis-sulfamate
7 with an excess of sulfamoyl chloride in N,N-dimethylaceta-
mide (DMA) according to the conditions described by Okada
et al.31
In the design of the first letrozole-based DASI, dual aromatase
and sulfatase inhibition was achieved by replacing both of the
para-cyano groups present in letrozole with sulfamates while
retaining both the triazole and the diphenylmethane moieties
necessary for aromatase inhibition. Evidence supporting the
design of an STS inhibitor based on this template was provided
by the benzophenone-based series of STS inhibitors. In this
series of compounds, a bis-sulfamate was found to be a more
potent STS inhibitor than its corresponding mono-sulfamate,27,28
with IC50 values of 0.19 and 5.1 µM for 4,4′-benzophenone bis-
sulfamate (2, Figure 2) and its 4-mono-sulfamate congener
against recombinant human STS.28 This design process resulted
in the synthesis of the prototype DASI, bis-sulfamate 3, which
exhibited IC50 values of 3044 nM for aromatase and >10 µM
for STS in JEG-3 cells. However, at a single oral dose of 10
mg/kg, 3 inhibited aromatase and rat liver STS by 60% and
88% respectively 24 h after administration.23
The objective of this current work was to investigate the
structure-activity relationship (SAR) of letrozole derived
DASIs. We report the effects on activity of relocating the
sulfamate groups to the meta-positions and the synthesis of the
first chiral DASI candidates, in which one of the para-cyano
groups of letrozole was replaced by a sulfamate group, as well
as the assignment of absolute configuration using chiroptical
techniques.
A variety of different reagents were investigated for the
synthesis of dibromo derivative 10 from the corresponding bis-
phenol (8, Scheme 2). Treatment of 8 with bromine in either
acetic acid or carbon tetrachloride gave complex mixtures
comprising of tetrabromo-, tribromo-, dibromo-, and mono-
bromo- compounds with the tetrabromo- derivative predominat-
ing. The reaction of 8 with bromine in tetrahydrofuran (THF)
Results and Discussion
Chemistry. The reference AI, letrozole, was prepared by the
alkylation of 1,2,4-triazole with R-bromomethyl-p-tolunitrile

4228 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Scheme 2. Synthesis of 10a
Wood et al.
a Reagents and conditions: (a) C6H5CH2N(CH3)3Br3, DCM/MeOH, -78 °C to r.t.; (b) H2NSO2Cl, DMA, r.t.
Scheme 3. Synthetic Routes to 16 and 22a
a Reagents and conditions: (a) NaH, BnBr, DMF, r.t.; (b) n-BuLi, 3-benzyloxy-4-methoxybenzaldehyde with 12 or 4-benzyloxy-3-methoxybenzaldehyde
with 18, THF, -78 °C to r.t.; (c) 1,2,4-triazole, p-TSA, toluene, reflux; (d) H2, Pd/C (10%), THF/MeOH, r.t.; (e) H2NSO2Cl, DMA, r.t.
Scheme 4. Synthetic Routes to 28, 34, and 40a
a Reagents and conditions: (a) 4-cyanobenzoyl chloride, AlCl3, r.t.; (b) pyridine hydrochloride, 220 °C with 24 and 30 or BBr3, DCM, r.t. with 36; (c)
NaBH4, EtOH/H2O, r.t.; (d) 1,2,4-triazole, p-TSA, toluene, reflux; (e) H2NSO2Cl, DMA, r.t.; (f) triisopropylsilyl chloride, imidazole, DMF, r.t.; (g) (4-
cyanophenyl)magnesium chloride, THF, 0 °C to r.t.; (h) TBAF, THF, r.t.
and sodium bicarbonate at -78 °C produced a mixture of
starting material and the monobromo derivative. More promising
results were obtained when either benzyl trimethylammonium
tribromide or tetrabutylammonium tribromide was used, with
the reagent of choice being the former. At -78 °C in di-
chloromethane (DCM)/MeOH, this reagent gave 9 in good yield
(72%). Subsequent sulfamoylation of the bis-phenol in the usual
manner furnished the desired dibromo-bis-sulfamate 10.
The syntheses of the two methoxy group-containing sulfa-
mates 16 and 22 are outlined in Scheme 3. For 16, the synthesis
commences with the preparation of 5-bromo-2-methoxyphenol
11 from 5-bromo-2-methoxy-benzaldehyde by Baeyer-Villiger
oxidation followed by hydrolysis according to the method of
Van der Mey et al.32 The resulting phenol was subsequently
benzyl protected to give 12 from which the methoxyphenyl-
lithium salt was generated in situ and reacted with 3-benzyloxy-
4-methoxybenzaldehyde to give benzhydrol 13. From this
intermediate, the preparation of 16 was achieved using the
methodology described above via reaction with triazole, depro-
tection and sulfamoylation. Compound 22 was synthesized
analogously from the commercially available 4-bromoguaiacol
17 and 4-benzyloxy-3-methoxybenzaldehyde (Scheme 3).
The initial synthetic pathways for the three mono-sulfamates
28, 34, and 40 are shown in Scheme 4. For the preparation of
28, the first step is the synthesis of 24 from anisole 23 and
4-cyanobenzoyl chloride according to the method of Leigh et
al.33 Both chloro- and bromo-substituted benzophenones 30 and
36 were prepared in an analogous fashion from 2-chloroanisole
29 and 2-bromoanisole 35 (a small amount of 24 was also
obtained from this reaction), respectively. The demethylation

Chiral Aromatase and DASIs from the Letrozole Template
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4229
modified route provides a faster and cleaner method for the
synthesis of 40 and could also be applied for the synthesis of
28 and 34.
Chiral HPLC. The three monosulfamates in this series were
initially evaluated as racemic mixtures; to explore the full SAR
of interactions with target proteins, it later became desirable to
ascertain the individual activities of each enantiomer of the most
potent compound. To avoid any possible complications arising
from the potential decomposition of the sulfamate, the resolution
was undertaken with the parent phenol, which could later be
converted to the sulfamate. Attempts to separate the enantiomers
of 39 by the preparation of diastereomeric esters and separation
by either crystallization or column chromatography failed, and
efforts were subsequently focused on separation by chiral HPLC.
The literature contains reports of a number of studies on the
resolution of AIs by chiral HPLC with particular focus on
imidazole containing derivatives. Fadrozole hydrochloride was
separated by Furet et al.35 using a Chiralcel OD column with a
hexane/2-propanol mixture as the mobile phase, and the absolute
configuration of the most active enantiomer was established by
an X-ray crystal structure analysis of its tartrate salt. A number
of 3-methyl-6-[1-(imidazo-1-yl)-1-phenylmethyl] benzothiaz-
olinones and 3-methyl-6-[1-(imidazo-1-yl)-1-phenylmethyl] ben-
zoxazolinones were separated by Danel et al.36 using a Chiralpak
AD and a Chiralcel OD-H column, with mobile phases
consisting of hexane/ethanol mixtures. The absolute configu-
ration of these enantiomers was determined by X-ray crystal
structure analysis. A series of seven triazole and imidazole
containing AIs has been separated on a number of cellulose-
and amylose-based chiral stationary phases by Aboul-Enein et
al.,37 with optimum resolution obtained with a Chiralpak AD-
RH column.
Initially, the separation of the enantiomers of 39 was
investigated with a range of amylose and cellulose packed
columns, and the best separations obtained from this initial
screen were with the Chiralpak AD-H (R ) 1.55, Rs ) 3.2)
and the Chiralpak AS-H (R ) 1.3, Rs ) 1.5) columns with
EtOH/MeOH (50:50) as the mobile phase. These conditions
were further optimized to give the best separation with a
Chiralpak AD-H column and MeOH as the mobile phase (R )
1.98, Rs ) 5.11). As shown in Figure 3, the first enantiomer
was eluted from the column with a retention time of 4.76 min
(39a), whereas the second enantiomer eluted with a retention
time of 6.23 min (39b).
The configurational stability of a series of enantiopure
imidazole containing AIs has been investigated by Danel et al.38
It was reported that the enantiomeric purity of these compounds
decreased following removal of the solvent on a rotary evapora-
tor (bath temperature 40-50 °C), although this was attributed
to the presence of basic impurities in the HPLC solvent. As a
result of this observation, the configurational stability of 39a
was investigated by heating a solution of the compound in
MeOH at 40 °C overnight. Under these conditions, the enan-
tiomeric purity of 39a was found39 to decrease to 95.5%. As a
precaution, solutions containing the separated individual enan-
tiomers were stored in the refrigerator and evaporation of
solvents was undertaken at room temperature. No racemization
was detected during this study.
Figure 3. Top trace: Separation of racemic 39 with a Chiralpak AD-H
column (250 mm × 4.6 mm, 5 µm). Mobile phase: MeOH; flow rate:
1.0 mL/min; detection UV: 230 nM; temperature: 25 °C; t1 4.76 min
(39a), t2 6.23 min (39b). Middle trace: HPLC trace of 40a (A, tr 12.69
min) obtained with a Chiralpak AD-H column (250 mm × 4.6 mm, 5
µm). Mobile phase: 85% MeOH/H2O; flow rate: 0.8 mL/min; PDA
detector; temperature: 25 °C. Bottom trace: HPLC trace of 40b (B, tr
10.96 min) details as for 40a.
of 24 and 30 to their corresponding phenols 25 and 31 was
achieved by heating at 220 °C with pyridine hydrochloride. This
method was unsuitable for the demethylation of 36, as it
produced a mixture of the three phenols 25, 31, and 37, which
could not be easily separated. Demethylation of 36 was achieved
by treatment with BBr3 in DCM which gave 37 as the sole
phenolic product, albeit at the expense of extended reaction
times. The three benzophenones 25, 31, and 37 were success-
fully reduced with NaBH4 to give secondary alcohols 26, 32,
and 38, respectively, which were converted to sulfamates 28,
34, and 40 using the methodology described above.
The problematic demethylation step encountered in the
synthesis of 40, as well as the larger quantity required for various
studies, was the driving force for the development of a
modification to the synthesis, and this is outlined in Scheme 4.
The commercially available 3-bromo-4-hydroxybenzaldehyde
was triisopropylsilyl protected and reacted with (4-cyanophe-
nyl)magnesium chloride34 (generated by halogen metal exchange
between 4-iodobenzonitrile and isopropylmagnesium chloride)
to give 43, which was subsequently deprotected with tetra-n-
butylammonium fluoride (TBAF) to give 38 as above. This
Suitable quantities of phenols 39a and 39b for sulfamoylation
were obtained following successful optimization of the separa-
tion on a Chiralpak AD-H (250 mm × 20 mm) semiprep
column. The individual enantiomers obtained from the above
separation were converted to their corresponding sulfamates
using the previously described conditions of ClSO2NH2 in

4230 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Wood et al.
DMA. To confirm that this step had not resulted in a loss of
enantiomeric purity, a method was developed for the chiral
HPLC separation of 40 with a Chiralpak AD-H analytical
column and 85% MeOH/15% water as the mobile phase. The
order of elution for the two enantiomers was found to switch
following sulfamoylation, i.e., phenol 39a with tr ) 4.76 min
corresponds to the slower eluting sulfamate with tr ) 12.69 min
(40a) and phenol 39b with tr ) 6.23 min corresponds to the
sulfamate with tr ) 10.96 min (40b). Although the resolution
obtained for the separation of 40 was not as good as that
obtained for 39, this was satisfactory for analytical purposes.
The HPLC traces of 40a and 40b are shown in Figure 3, and
both show that there was no significant loss of enantiomeric
purity during the sulfamoylation step.
in Figure 4. For the measured spectra, a good mirror-image
condition was obtained for the two enantiomers. However, to
eliminate baseline drift, the half-difference 39a-39b is reported
for 39a and the half-difference 39b-39a is reported for 39b.
The R configuration of 39 was arbitrarily chosen for the
calculations of both the VCD and the ECD spectrum. After a
full conformational search at PM3 level, subsequent B3LYP/
6-3G* optimization in vacuo was performed using Gaussian
03,48 and this gave eight conformers with energy values
remarkably lower than all the others. The conformations were
verified to be real energy minima, and the free energy value of
each conformation was calculated in order to evaluate the
relative population of each conformation at room temperature
using Boltzmann statistics. The calculated spectrum40 was
obtained from the weighted average of each of the eight
conformations (data not shown and will be published elsewhere).
The calculated VCD spectrum for the R configuration corre-
sponds well to the VCD spectra obtained for 39a. Therefore,
VCD permits the unambiguous assignment of the absolute
configuration of 39a as R-(+) and 39b as S-(-).
The optical rotation for each enantiomer of the phenol and
corresponding sulfamate was measured in an ethanolic solution.
For the phenols, the rotation of 39a is +84.3° (c ) 3.9) and for
39b, it is -79.1° (c ) 3.6) and for the sulfamates, 40a is +34.2°
(c ) 0.97) and 40b is -30.7° (c ) 0.95).
Determination of Absolute Configuration. Following isola-
tion of the separated enantiomers of 39 by semiprep chiral
HPLC, it was essential to determine the absolution configuration
(AC) of each enantiomer 39a and 39b. Crystals suitable for
X-ray crystal structure analysis could be obtained from racemic
39 following slow evaporation of the solvent from an EtOAc
solution. However, when this was attempted with either of the
single enantiomers, the product obtained was an oil. In an
attempt to obtain a crystalline compound suitable for X-ray
crystallography, a range of derivatives were prepared (including
N,N-diphenylcarbamates and 1-naphthoates), but these com-
pounds were also all obtained as oils. Extensive efforts to solve
these problems ended in failure, and this necessitated the use
of an alternative technique for the determination of AC.
The measurement of the chiroptical properties of a molecule
in tandem with quantum mechanical calculations provides a
powerful method for the determination of AC. The principle
behind this is the determination of the vibrational circular
dichroism (VCD)40–42 and/or the electronic circular dichroism
(ECD)43,44 of an enantiomer experimentally and the comparison
of these spectra with those predicted by density functional theory
(DFT) calculations for one AC. As these are solution phase
techniques, this approach has found particular application for
the determination of AC for compounds, which are precluded
from X-ray crystallography. The popularity of these techniques
has increased in recent years, and in one example, VCD has
been used to correct the AC of a molecule previously assigned
by X-ray crystallography.45 A combination of DFT calculations
with VCD,40 and TDDFT (time dependent DFT) calculations
with ECD and optical rotation measurements, has been reported
in a study by Stephens et al.46 to determine the AC of a chiral
oxadiazol-3-one, which exhibits myocardial calcium entry
channel blocking activity. All three chiroptical techniques
assigned identical ACs which were R-(+) and S-(-). Recently,
and more relevantly to this work, Cavalli et al.47 have reported
the design of a new series of nonsteroidal AIs and the
determination of AC by ECD and TDDFT calculations.
Although ACs can be determined by the study of a single
chiroptical property alone, agreement between two or more
techniques clearly produces a more definitive assignment of AC.
As these techniques have proved to be reliable, the AC of 39a
and 39b was studied using VCD and ECD in conjunction with
DFT and TDDFT calculations.
The ECD measurement for each enantiomer of 39 along with
that simulated by TDDFT calculations is shown in Figure 5.
The spectra were obtained from methanolic solutions at
concentrations of 10-4 M or 10-3 M and the spectra are solvent
subtracted. The ECD spectrum of the enantiomer of 39 with
the R configuration was calculated with a TDDFT approach; at
the same functional and basis set level, rotational strength in
the velocity representation have been calculated and averaged
over the same conformations and populations employed for
VCD.
The ECD spectrum is quite rich in bands and is reproduced
quite well by the simulated spectrum despite the fact that the
level of calculus is not very high and only pure electronic
transitions in vacuo have been considered. In this case, the data
obtained from the ECD study are sufficient to determine the
AC and once again there is good correlation between the ECD
spectra for 39a and that calculated for the R configuration,
confirming the results obtained from the VCD study.
As mentioned previously, the sulfamoylation step proceeded
without loss of enantiomeric purity, allowing the AC of 40a
and 40b to be assigned as R-(+) and S-(-) respectively.
Biological Results and Discussion
Sulfamates. The in vitro inhibition of aromatase and STS
activity by each sulfamate was measured in a preparation of an
intact monolayer of JEG-3 cells. The results are reported either
as IC50 values or as a percentage of aromatase/STS inhibition
at 10 µM (Table 1). The biological activity of 4,4′-bis-sulfamate
3 was reported previously.23
The relocation of the two sulfamate groups from the para-
positions in 3 to the meta- positions in 7 gives a 30-fold increase
in aromatase inhibition (IC50
) 3044 vs 99 nM). This
AROM
resembles the phenomenon previously observed in the YM511-
based DASI series22 and strongly suggests that a sulfamate group
positioned at the meta-position of the phenyl ring of a DASI is
better tolerated by the aromatase enzyme than a corresponding
para-sulfamate. Both 3 and 7 are weak STS inhibitors, and no
clear difference in their inhibitory activities can be obtained
from the results obtained here. This is in contrast to the finding
of the benzophenone-based series of STS inhibitors where the
difference in IC50 values for 4,4′-benzophenone bis-sulfamate
(2, Figure 2) and its 3,3′-bis-sulfamate congener against
recombinant human steroid sulfatase is much more prominent
at 0.19 and 3.2 µM, respectively.28 These results clearly
The experimental mid-IR VCD for 39a and 39b and the
calculated mid-IR VCD for the R configuration of 39 are shown

Chiral Aromatase and DASIs from the Letrozole Template
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4231
Figure 4. VCD spectra. Bottom trace: experimental spectrum of 39a (0.256 M CDCl3). Middle trace: calculated spectrum for R configuration
(weighted average taking into account populations obtained by calculated Gibbs free energies). Upper trace: experimental spectrum of 39b (0.23
M CDCl3). Calculated frequencies have been shifted by 30 cm-1
.
Table 1. In Vitro Inhibition of the Aromatase and STS Activity in
JEG-3 Cells by Novel Sulfamates
aromatase
IC50 (nM)
STS
IC50(nM)
R1
R2
R3
R4
letrozole
STX64
3
0.89 ( 0.13
300 ( 42
3044 ( 48
99 ( 0.01
43 ( 3.2
1.5 ( 0.3
H
X
Br
X
OMe
H
X
H
X
X
H
X
H
X
Br
X
>10 000
7
31.4% @10 µM
476 ( 36
40% @10 µM 10.9% @10 µM
10
16
22
28
34
40
OMe OMe
Figure 5. ECD spectra. Dark-blue trace: experimental spectrum of
39a. Light-blue trace: experimental spectrum of compound 39b. Black
trace: calculated spectrum for R configuration (average over eight
conformers with calculated population). The spectra were recorded on
10-4 M solutions in the range 190-240 nm, and on 10-3 M solutions
in the range 240-340 nm.
X
X
X
X
X
X
X
OMe 33% @10 µM 11.9% @10 µM
CN
CN
CN
CN
CN
H
H
H
H
H
13 ( 4
21.5% @10 µM
920 ( 20
2600 ( 100
4633 ( 551
553 ( 50
Cl
Br
12 ( 3.4
3.0 ( 0.1
3.2 ( 0.3
14.3 ( 2.1
40a R-(+) Br
40b S-(-) Br
demonstrate the important role played by the carbonyl group
in 2 in increasing the STS inhibitory activity compared to the
two triazole derivatives 3 and 7. The carbonyl group increases
the leaving group potential of the corresponding phenol, thus
increasing the “sulfamoylation potential” of the aryl sulfamate
and facilitating the irreversible inactivation of STS by protein
sulfamoylation. This effect is expected to be strongest, with the
carbonyl group para- to the sulfamate group explaining why 2
is a much stronger STS inhibitor than the 3,3′-bis-sulfamate
derivative. For 3 and 7, the absence of a similar electron-
withdrawing group positioned para- to the sulfamate group
contributes toward the weak STS inhibitory activities observed
for these two compounds. However, based on past observations,
the introduction of appropriate substituents onto the aromatic
ring to increase the “sulfamoylation potential” should increase
the potency of the DASIs in this series against STS.
in vitro. This prompted us to investigate if introduction of
methoxy groups to 7 would further improve the potency of
aromatase inhibition. Hence, 16 was prepared but, as shown in
Table 1, the ability of this compound to inhibit aromatase in
JEG-3 cells was substantially reduced, giving an IC50 value of
>10 µM. The exchange in position of the methoxy and
sulfamate groups in 22 failed to improve the aromatase
inhibitory activity as demonstrated by the comparable IC50 value
of 16 to 22. Both bis-methoxy derivatives 16 and 22 showed
very weak inhibitions of STS in vitro (Table 1), suggesting that
the electron-donating effect of the methoxy groups is deleterious
to the STS inhibitory activity of these derivatives.
There is an increase in both aromatase and STS inhibition in
the derivatives with a halogen ortho to the sulfamate group.
Comparing 3 with 10 reveals a significant increase in potency
against both enzymes, from 3044 to 43 nM for aromatase and
from >10000 to 476 nM for STS. This mirrors the results
obtained from our previous SAR studies on YM511-type
Results published by Jones et al.49 demonstrated that a diaryl
imidazolemethane substituted with bis-methoxy groups at the
para-position of the phenyl rings is 10-fold more active than
the unsubstituted parent compound in the inhibition of aromatase

4232 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Wood et al.
DASIs.21,22 The increase in aromatase inhibition is reasoned to
be due to an increase in lipophilicity of the compound, which
results in tighter binding of the halogenated derivative to the
active site. The increase in STS inhibition can be attributed to
a lowering of the pKa of the phenol, enhancing its leaving group
ability. This beneficial electronic effect on STS inhibition has
consistently been observed in the development of steroidal STS
inhibitors.50,51
Table 2. In Vitro Inhibition of the Aromatase Activity in JEG-3 Cells
by Novel Phenols
R1
R2
R3
R4
aromatase IC50 (nM)
The introduction of two meta-bromo groups to give 10
improves the potency of aromatase inhibition and in addition
gave the most potent STS inhibitor in this study. However, the
best aromatase inhibition is obtained from a small series of
monosulfamates in which more of the features of letrozole were
retained. This series of compounds contains a chiral center, and
these derivatives were initially synthesized and evaluated as
racemic mixtures. Derivative 28 has an IC50 value against
aromatase in JEG-3 cells of 13 nM, making it some 250-fold
more potent than bis-sulfamate 3 and three times more potent
than 10. Clearly, this increase in potency against aromatase can
be attributed to the presence of a para-cyanophenyl moiety in
the molecule, which contributes significantly to the binding of
the inhibitor to the enzyme active site possibly in a similar
manner to letrozole. The importance of the positioning of an
H-bond accepting group relative to the triazole/imidazole ring
has been extensively discussed in the literature,52,53 and it has
been postulated by Furet et al.35 that in (S)-fadrozole the CN
group acts as an H-bond acceptor in a similar manner to the
steroidal 17-carbonyl group. Compared with 28, meta-chloro
derivative 34 is surprisingly not a stronger AI given the role
that a halogen plays in the improvement of aromatase inhibition
for this class of compound. However, the bromo derivative 40
exhibits a substantial improvement in aromatase inhibitory
activity compared to its chloro congener 34 (IC50 AROM ) 3.0
vs 12 nM). This is a trend reflected in our finding that the meta-
chloro derivative of 1, a YM511-based DASI (Figure 2, R )
Cl), is 3-fold weaker as an AI than its bromo-congener (1, Figure
2, R ) Br).21 This increase in inhibitory activity may be caused
by the increase in lipophilicity for the halogenated derivatives,
which results in enhanced binding to the active site of the
enzyme. In the current series, the meta-bromo derivative 40 is
a weaker STS inhibitor than 34 (IC50 STS ) 920 nM vs 2600
nM). This effect could possibly be caused by the increased steric
bulk of the bromine atom, which results in interference of the
binding of this derivative in the enzyme active site.
letrozole
6
8
9
15
21
27
33
0.89 ( 0.13
2457 ( 132
178
OH
H
Br
OH
OMe
H
Cl
Br
Br
Br
H
H
OH
H
Br
OH
OMe
H
H
H
H
H
OH
OH
OMe
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OH
CN
CN
CN
CN
CN
10 ( 2.7
169 ( 50
215 ( 16
7.2 ( 1.1
5.5 ( 3.7
1.8 ( 0.3
0.6 ( 0.1
3.4 ( 0.6
39
39a R-(+)
39b S-(-)
similar in potency to the enantiomer with R configuration (40a),
a finding similar to that observed for vorozole. While for STS
inhibition, the activity of the racemate roughly lies halfway
between the activity of each enantiomer, the activity of 40b
(IC50
) 553 nM) against STS is similar to that of 10 (IC50
STS
STS ) 476 nM), the most potent STS inhibitor in this study.
Interestingly, compounds 40a and 40b comprise the first
enantiopure, sulfamate-based, nonsteroidal STS inhibitors to be
evaluated. Here, for the first time, we have demonstrated that
the enantiomers of a chiral sulfamate-containing compound
inhibit STS to a different extent. This 9-fold difference in
potency observed for 40a and 40b warrants further investigation.
Phenols. Displayed in Table 2 are the IC50s for aromatase
inhibition obtained for the novel phenols described in this paper,
with values ranging from 0.6 to 2457 nM. The loss of the
sulfamate group following irreversible inactivation of STS by
a sulfamate-based DASI or for some sulfamates, through
degradation on prolonged circulation in plasma, will result in
the formation of the corresponding phenol. As these phenols
still contain the pharmacophore for aromatase inhibition, they
have the potential to act as AIs in their own right.
With the exception of 6, all of the phenols tested in this series
are better AIs than their corresponding sulfamates. This trend,
which was previously observed in the YM511 series, suggests
that the sulfamate group is too large for optimum binding in
the aromatase active site. As previously observed, the presence
of a halogen ortho to the sulfamate group results in an increase
in aromatase inhibition and this trend is also seen with the
corresponding phenols. This is particularly illustrated by the
difference in aromatase inhibition between 27 (IC50 AROM ) 7.2
nM) and 39 (IC50 AROM ) 1.8 nM) and is also seen for 8 (IC50
AROM ) 178 nM) and 9 (IC50 AROM ) 10 nM). Introduction of
methoxy groups to 8 to give 21 failed to give the expected
beneficial effects in aromatase inhibition (IC50 AROM ) 178 nM
To determine the difference in aromatase and STS inhibition
for each enantiomer of 40, the preceding phenols were separated
by chiral HPLC (details given above) and converted to their
corresponding sulfamates. Previous studies have suggested that
there is often a large difference in aromatase inhibition between
the enantiomers of chiral AIs. For fadrozole hydrochloride,35
there is a 210-fold difference in aromatase inhibitory activity
between enantiomers, with the S enantiomer proving to be the
most active (S-form IC50 3.2 nM vs R-form IC50 680 nM).
Moreover, for vorozole,54 there is a 32-fold difference in activity,
with the R configuration being the most active (R-(+) IC50 1.38
nM, S-(-) IC50 44.2 nM) and exhibiting a similar inhibition to
the racemate (RS-(() IC50 2.59 nM). Of the two enantiomers
in the current study, 40a (R-(+)) is more potent against
aromatase while 40b (S-(-)) is more potent against sulfatase.
The more modest 4-fold difference in aromatase inhibition
vs 215 nM), although 15 is a better AI than 6 (IC50 AROM
169 nM vs 2457 nM).
)
The best AI among the achiral and racemic compounds is
phenol 39 (IC50 ) 1.8 nM), while the corresponding
AROM
sulfamate, 40 (IC50 AROM ) 3.0 nM), is the best AI in the same
category for the sulfamate series. However, of the two enanti-
omers of 39, the R configuration (39a) is not only nearly 6-fold
more active than the S configuration (39b), but is also the most
potent AI discovered herein, with an IC50 of 0.6 nM, comparable
to that of letrozole. The same trend in activity is observed with
observed for 40a (IC50 AROM ) 3.2 nM) and 40b (IC50 AROM
)
14.3 nM) could be due to the presence of the sulfamate groups,
resulting in these compounds not being optimized for aromatase
inhibition. For the inhibition of aromatase, the racemate (40) is

Chiral Aromatase and DASIs from the Letrozole Template
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4233
the corresponding sulfamates where the compound with the R
configuration is the more active, in this case by around 4-fold
(Table 1).
and their AC was determined using VCD and ECD in conjunc-
tion with TDDFT calculations of their predicted properties. This
allowed the AC of 39a to be assigned as R and that of 39b to
be assigned as S. Each of the enantiomers was converted to its
corresponding sulfamate, 40a and 40b, respectively, without loss
of enantiopurity. Of the two enantiomers, 40a is the better AI
and 40b is the better STS inhibitor, and in this assay, 39a (IC50
Molecular Modeling. A molecular modeling study was
instigated in an attempt to investigate the difference in inhibitory
activities between 39a and 39b for aromatase inhibition and
between 40a and 40b for both aromatase and sulfatase inhibition.
Each of the four compounds and letrozole were individually
docked into the human aromatase homology model55 using
GOLD.56 Compound 39a was found to dock in two modes,
which differed by rotation of the bromine substituted aromatic
ring by ∼180°, and each of these modes resembled the fittest
solution of letrozole (rmsd over equivalent atoms of the fittest
39a and letrozole solutions ) 0.22 Å). There was only one
docking mode for 39b in which the two phenyl rings were
transposed compared to 39a. The docking studies suggest
different binding modes for 39a and 39b, with the important
benzonitrile group positioned in different positions in the active
site. Compound 40a docked into aromatase in the same mode
as 39a and the docking of 40b was similar to that of 39b with
the sulfamate group positioned on the opposite side of the active
site to 40a. These results suggest that there is adequate room
for the sulfamoylated compounds to bind to the active site in
the same mode as the equivalent phenols.
AROM ) 0.6 nM) is similar in potency to letrozole (IC50 AROM
)
0.89 nM). Compounds 40a and 40b are the first enantiopure
nonsteroidal STS inhibitors to undergo biological evaluation.
The phenolic precursors of the sulfamates still possess the
pharmacophore for aromatase inhibition and will be formed in
vivo from the sulfamate ester, following either compound
degradation or inactivation of STS. In general, the phenols are
better inhibitors than their respective sulfamates and there is a
good correlation between the activity of the phenols and their
corresponding sulfamates.
These results illustrate the feasibility of designing a letrozole-
based DASI and provide a basis for development of compounds
with potential for the treatment of HDBC. Furthermore, this
work demonstrates the growing utility of chiroptical techniques
for determination of absolute configuration.
Experimental Section
From the docking of 40a and 40b into the crystal structure
of STS,57 it was observed that compound 40a docked in two
modes, both of which placed the sulfamate group satisfactorily.
Compound 40b had one major binding mode, which places the
groups in similar positions to those observed with 40a. Both
enantiomers docked into the same region of the active site as
that occupied by STX64 with their sulfamate groups directed
toward the Ca2+ ion and the catalytically important formylglcine
residue. Given the well-established difficulties in correlating in
silico binding models using scoring functions with real biological
activity,58 we have not attempted a detailed rationalization of
our rather limited set of compounds.
General Methods for Synthesis. All chemicals were purchased
from either Aldrich Chemical Co. (Gillingham, UK) or Alfa Aesar
(Heysham, UK). All organic solvents of AR grade were supplied
by Fisher Scientific (Loughborough, UK). Anhydrous N,N-dim-
ethylformamide (DMF), N,N-dimethylacetamide (DMA), and tet-
rahydrofuran (THF) were purchased from Aldrich. Sulfamoyl
chloride was prepared by an adaptation of the method of Appel
and Berger59 and was stored as a solution under N2 in toluene as
described by Woo et al.60
Thin layer chromatography (TLC) was performed on precoated
aluminum plates (Merck, silica gel 60 F254). Product spots were
visualized either by UV irradiation at 254 nm or by staining with
either alkaline potassium permanganate solution or 5% w/v dode-
camolybdophosphoric acid in ethanol, followed by heating. Flash
column chromatography was performed on silica gel (Davisil silica
60A) or using prepacked columns (Isolute) and gradient elution
(solvents indicated in text) on the Flashmaster II system (Biotage).
1H and 13C NMR spectra were recorded with either a Jeol Delta
270 MHz or a Varian Mercury VX 400 MHz spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) relative
to tetramethylsilane (TMS) as an internal standard. Coupling
constants, J, are recorded to the nearest 0.1 Hz. Mass spectra were
recorded at the Mass Spectrometry Service Center, University of
Bath. FAB mass spectra were carried out using m-nitrobenzyl
alcohol as the matrix. Elemental analyses were performed by the
Microanalysis Service, University of Bath. Melting points were
determined using either a Stuart Scientific SMP3 or a Stanford
Research Systems Optimelt MPA100 and are uncorrected. Optical
rotations were measured with a machine supplied by Optical
Activity Ltd. with 5 cm cells.
HPLC. LC/MS was performed using a Waters 2790 machine
with a ZQ MicroMass spectrometer and PDA detector. The
ionization technique used was either APCI or ES (as indicated in
the text). A Waters “Symmetry” C18 (packing: 3.5 µm), 4.6 mm
× 100 mm column and gradient elution (flow rate) 5:95 MeCN/
H2O (0.5 mL/min) to 95:5 MeCN/H2O (1 mL/min) over 10 min
were used. HPLC was undertaken using a Waters 717 machine with
Autosampler and PDA detector. The column used was a Waters
“Symmetry” C18 (packing: 3.5 µm), 4.6 mm × 150 mm with an
isocratic mobile phase consisting of MeOH/H2O (as indicated) with
a flow rate of 1.4 mL/min. Analytical chiral HPLC was performed
with a Chiralpak AD-H column (250 mm × 4.6 mm, 5 µm) with
methanol as the mobile phase, a flow rate of 1.0 mL/min, and a
PDA detector. Semipreparative HPLC was performed with a Waters
Conclusions
A number of sulfamate containing compounds structurally
related to letrozole and their precursor phenols were synthesized
and tested for aromatase inhibition and dual inhibition of
aromatase and STS in JEG-3 cells. The aromatase inhibition of
their corresponding phenolic precursors was also determined.
To achieve dual inhibition, both the pharmacophore for aro-
matase inhibition (a N-containing heterocyclic ring) and that
for STS inhibition (a phenol sulfamate ester) were incorporated
into a single molecule.
Comparison of the bis-sulfamates tested in this work reveals
that switching the position of the sulfamate groups from the
para- to the meta-positions gave better aromatase inhibition but
no improvement in STS inhibition; however, the best inhibition
of aromatase and STS in this subseries is achieved with bis-
bromo bis-sulfamate 10 (IC50 STS ) 476 nM), which is the best
STS inhibitor discovered in the letrozole-based DASI series.
The two methoxy-containing derivatives are both weak inhibitors
of both aromatase and STS. Better aromatase inhibition is
obtained from the monosulfamates, which contain more of the
features of letrozole, and the inhibition of both enzymes could
be further enhanced by the presence of a halogen ortho to the
sulfamate. The best AI of the achiral and racemic sulfamoylated
compounds is 40 with an IC50 value of 3 nM. Compound 39,
the corresponding phenolic precursor to 40, is the best AI of
the phenolic achiral and racemic compounds (IC50 AROM ) 1.8
nM). The enantiomers of 39 were separated by chiral HPLC

4234 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Wood et al.
2525 binary gradient module and a Chiralpak AD-H (250 mm ×
20 mm) semiprep column with MeOH as the mobile phase at a
flow rate of 18 mL/min, injecting 1.5-2.0 mL of a 10 mg/mL
solution and a run time of 7 min.
VCD and ECD Measurements. VCD measurements were
conducted with a JASCO FVS4000 apparatus equipped with a MCT
detector, using a BaF2 cell of 50 µm path length and 4 cm-1
resolution. Measurements were taken on CDCl3 solutions. ECD
spectra were obtained at room temperature on a JASCO 500
spectropolarimeter, flushed with dry nitrogen using 0.1 cm and 1
cm quartz cuvettes adopting the following conditions: 100 nm/min
scanning rate, 2 nm resolution and 32 scans. Samples were dissolved
in methanol at 10-4 M or 10-3 M concentrations, and spectra were
solvent subtracted.
General Method A: Condensation of Carbinols with Triazole.
The substrate, 1,2,4-triazole and para-toluenesulfonic acid (p-TSA),
dissolved/suspended in toluene were heated at reflux with a
Dean-Stark separator for 24 h. The reaction mixture was allowed
to cool, and the solvent was removed in vacuo.
General Method B: Hydrogenation. Pd/C (10%) was added
to a solution of the substrate in THF/MeOH. The solution was
stirred under an atmosphere of H2 (provided by addition from a
balloon) overnight. The excess H2 was removed and the reaction
mixture was filtered through celite with additional THF and MeOH,
then the solvent was removed in vacuo.
DMSO-d6): 8.64 (1H, s, NCHN), 8.11 (1H, s, NCHN), 8.04 (4H,
br s, 2 × NH2), 7.49 (2H, t, J ) 7.9, ArH), 7.36-7.16 (7H, m,
ArH and CH). LRMS (FAB+) 426.0 ([M + H]+, 100%), 357.0
(50), 85.1 (50). HRMS (FAB+) found 426.0540, C15H16N5O6S2
requires 426.0542.
1-[Bis-(3-bromo-4-hydroxyphenyl)methyl]-1H-[1,2,4]triazole
(9). A solution of benzyltrimethylammonium tribromide (1.46 g,
3.74 mmol) in CH2Cl2/MeOH 1:1 (10 mL) was added dropwise
over 45 min to a cooled (-78 °C) solution of 1-[bis-(4-hy
droxyphenyl)methyl]-1H-[1,2,4]triazole23 8 (0.50 g, 1.87 mmol)
in CH2Cl2/MeOH 1:1 (40 mL). The mixture was allowed to
warm to 0 °C and stirred for 7 h and then stirred at room
temperature overnight. The solvent was removed in vacuo, and
the residue was dissolved in H2O and EtOAc. The aqueous layer
was extracted with EtOAc, and the combined organic layers were
washed with brine, dried (Na2SO4), and the solvent was removed
in vacuo. The crude product was purified by flash column
chromatography (hexane/EtOAc 2:1) to give the title compound
as a white solid (0.57 g, 72%, mp 198-205 °C). A small sample
was purified by semipreparative HPLC (MeOH/H2O, 60:40) tR )
5.5 min. δH (400 MHz, DMSO-d6): 10.45 (2H, br s, 2 × OH),
8.56 (1H, s, NCHN), 8.06 (1H, s, NCHN), 7.30 (2H, d, J ) 2.1,
ArH), 7.03 (2H, dd, J ) 8.6, 2.1, ArH), 6.93 (2H, d, J ) 8.6,
ArH), 6.90 (1H, s CH). LCMS (APCI+) 425.9 ([M + H]+, 4%),
356.9 (100). Anal. (C15H11N3O2Br2) C, H, N.
General Method C: Sulfamoylation. A solution of sulfamoyl
chloride (H2NSO2Cl) in toluene (0.7 M) was concentrated in vacuo
at 30 °C to furnish a yellow oil, which solidified upon cooling in
an ice bath. DMA and the substrate were subsequently added, and
the mixture was allowed to warm to room temperature and stirred
overnight.
1-[Bis-(3-bromo-4-sulfamoyloxyphenyl)methyl]-1H-[1,2,4]triazole
(10). As general method C using ClSO2NH2 (2.6 mL), DMA (2.5
mL), and 9 (0.18 g, 0.41 mmol). The resulting solution was added
to brine (20 mL) and this was extracted with EtOAc (3 × 20 mL).
The organic layers were combined, washed with H2O (3 × 40 mL),
dried (MgSO4), and the solvent removed in vacuo. The title
compound was obtained as a white solid (0.21 g, 88%, mp 98-102
°C). δH (270 MHz, DMSO-d6): 8.69 (1H, s, NCHN), 8.32 (4H, br
s, 2 × NH2), 8.15 (1H, s, NCHN), 7.66 (2H, d, J ) 2.2, ArH),
7.55 (2H, d, J ) 8.4, ArH), 7.38 (2H, dd, J ) 8.4, 2.2, ArH), 7.21
(1H, s CH). LRMS (FAB+) 584.0 ([M + H]+, 10%), 515.0 (10),
391.2 (100). HRMS (FAB+) found 581.8759, C15H14N5O6S2Br2
requires 581.8752.
Bis-(3-benzyloxy-4-methoxyphenyl)methanol (13). n-BuLi (1.6
M, 5.2 mL) was added slowly to a cooled (-78 °C) solution of 12
(2.10 g, 7.2 mmol) in THF (28 mL). After stirring for 1 h, a solution
of 3-benzyloxy-4-methoxybenzaldehyde (1.74 g, 7.2 mmol) in THF
(16 mL) was added dropwise, and the reaction mixture was stirred
for 1.5 h. The reaction mixture was allowed to warm to room
temperature, and after stirring for 2 h, it was quenched by the
addition of H2O (100 mL). The product was extracted with EtOAc
(3 × 100 mL) and the combined organics were dried (MgSO4) and
the solvent was removed in vacuo. The crude product was purified
by precipitation from EtOAc/hexane to give the title compound
(1.70 g, 52%, mp 103-105 °C) as a white solid. δH (270 MHz,
CDCl3): 7.42-7.21 (10H, m, ArH), 6.89-6.79 (6H, m, ArH), 5.63
(1H, d, J ) 3.5, CH), 5.06 (4H, s, 2 × CH2), 3.86 (6H, s, 2 ×
CH3), 2.02 (1H, d, J ) 3.5, OH). LRMS (FAB+) 456.1 (M+, 45%),
439.1 (85), 349.2 (10), 243.1 (20), 91.1 (100).HRMS (FAB+) found
456.1944, C29H28O5 requires 456.1937.
Bis-(3-hydroxyphenyl)methanol (5). A solution of sodium
borohydride (NaBH4) (0.19 g, 5.14 mmol) in H2O (5 mL) was added
to a solution of 3,3′-dihydroxybenzophenone30 4 (1.00 g, 4.67
mmol) in EtOH (15 mL). After stirring for 1 h, the reaction mixture
was added to a mixture of ice-water (40 mL) and conc HCl (2.5
mL), and the product was extracted with EtOAc (2 × 60 mL). The
combined organic layers were subsequently washed with NaOH
(3M, 2 × 50 mL). Following acidification of the combined basic
washes, the product was extracted with EtOAc (2 × 100 mL). The
combined EtOAc extracts were dried (MgSO4), and the solvent was
removed in vacuo. The crude product was purified by recrystalli-
zation from H2O to give the title compound as a brown crystalline
solid (0.86 g, 85%, mp 141-142 °C). δH (270 MHz, DMSO-d6):
9.27 (2H, br s, ArOH), 7.05 (2H, t, J ) 8.2, ArH), 6.84-6.72 (4H,
m, ArH), 6.63-6.54 (2H, m, ArH), 5.72 (1H, br s, CHOH), 5.50
(1H, s, CHOH). LCMS (ES-) 215.0 ([M - H]-, 30%), 196.9 (10),
168.8 (15), 120.7 (100). HRMS (FAB+) found 216.0785, C13H12O3
requires 216.0786.
1-[Bis-(3-hydroxyphenyl)methyl]-1H-[1,2,4]triazole (6). As
general method A using 5 (0.75 g, 3.47 mmol), 1,2,4-triazole (0.48
g, 6.94 mmol), p-TSA (125 mg), and toluene (300 mL). The
resulting residue was dissolved in EtOAc (75 mL), and the organic
layer was washed with H2O (3 × 75 mL), dried (MgSO4), and the
solvent was removed in vacuo. The crude product was purified by
flash column chromatography (EtOAc:hexane 3:1) to give the title
compound (0.82 g, 88%) as a pale-yellow oil, which formed a foam
under high vacuum. δH (270 MHz, DMSO-d6): 9.50 (2H, br s,
ArOH), 8.55 (1H, s, NCHN), 8.06 (1H, s, NCHN), 7.16 (2H, t, J
) 7.9, ArH), 6.89 (1H, s, CH), 6.75-6.68 (2H, m, ArH), 6.67-6.60
(4H, m, ArH). LRMS (FAB+) 268.3 ([M + H]+, 100%), 199.2
(85). HRMS (FAB+) found 268.1091, C15H14N3O2 requires 268.1086.
1-[Bis-(3-sulfamoyloxyphenyl)methyl]-1H-[1,2,4]triazole (7).
As general method C using ClSO2NH2 (8.8 mL), DMA (7 mL),
and 6 (0.30 g, 1.12 mmol). The resulting yellow solution was added
to brine (30 mL), and this was extracted with EtOAc (3 × 50 mL).
The organic layers were combined, washed with H2O (3 × 50 mL),
dried (MgSO4), and the solvent was removed in vacuo. The crude
product was purified by flash column chromatography (CHCl3:
acetone 1:1) to give the title compound (0.42 g, 88%) as a pale-
yellow oil, which formed a foam under high vacuum. δH (270 MHz,
1-[Bis-(3-benzyloxy-4-methoxyphenyl)methyl]-1H-[1,2,4]triazole
(14). As general method A using 13 (1.60 g, 3.50 mmol), 1,2,4-
triazole (0.48 g, 6.95 mmol), p-TSA (160 mg), and toluene (230
mL). The resulting residue was dissolved in EtOAc (100 mL), and
the organic layer was washed with H2O (3 × 100 mL), dried
(MgSO4), and the solvent was removed in vacuo. The crude product
was purified using Flashmaster II (EtOAc/hexane) to give the title
compound (1.51 g, 84%, mp 96-99 °C) as a yellow oil, which
solidified after standing. δH (400 MHz, CDCl3): 7.95 (1H, s, NCHN),
7.67 (1H, s, NCHN), 7.34-7.24 (10H, m, ArH), 6.81 (2H, d, J ) 8.4,
ArH), 6.58-6.48 (5H, m, ArH), 5.00 (4H, s, 2 × CH2), 3.84 (6H, s,
2 × CH3). LRMS (FAB+) 507.2 (M+, 20%), 439.2 (100), 349.2 (15),
257.2 (15), 91.1 (35). HRMS (FAB+) found 507.2159, C31H29N3O4
requires 507.2158. Anal. (C31H29N3O4) C, H, N.

Chiral Aromatase and DASIs from the Letrozole Template
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4235
1-[Bis-(3-hydroxy-4-methoxyphenyl)methyl]-1H-[1,2,4]triazole
(15). As general method B using Pd/C (80 mg), 14 (1.65 g, 3.25
mmol), and THF/MeOH (1:1, 200 mL). The crude product was
purified using Flashmaster II (EtOAc/hexane) to give the title
compound (0.86 g, 81%, mp 217.5-219 °C) as a white solid. δH
(270 MHz, DMSO-d6): 9.08 (2H, s, 2 × OH), 8.47 (1H, s, NCHN),
8.03 (1H, s, NCHN), 6.88 (2H, d, J ) 8.4, ArH), 6.76 (1H, s, CH),
6.64 (2H, s, ArH), 6.57 (2H, d, J ) 8.4, ArH), 3.74 (6H, s, 2 ×
CH3). LCMS (ES-) 326.2 ([M - H]-, 100%), 255.9 (70), 240.4
(60), 211.4 (70). HRMS (FAB+) found 327.1226, C17H17N3O4
requires 327.1219. Anal. (C17H17N3O4) C, H, N.
1-[Bis-(3-sulfamoyloxy-4-methoxyphenyl)methyl]-1H-[1,2,4]tri-
azole (16). As general method C using ClSO2NH2 (5.8 mL), DMA
(6 mL), and 15 (0.30 g, 0.92 mmol). The resulting mixture was
poured onto brine (50 mL), and this was extracted with EtOAc (3
× 50 mL). The organic layers were combined, washed with H2O
(2 × 50 mL) and brine (2 × 50 mL), dried (MgSO4), and the solvent
was removed in vacuo. The crude product was purified using
Flashmaster II (EtOAc/hexane) to give the title compound (0.40 g,
90%) as a white foam. δH (270 MHz, DMSO-d6): 8.56 (1H, s,
NCHN), 8.07 (1H, s, NCHN), 7.93 (4H, br s, 2 × NH2), 7.26-7.07
(6H, m, ArH), 7.00 (1H, s, CH), 3.80 (6H, s, 2 × CH3). LRMS
(FAB+) 486.1 ([M + H]+, 20%), 417.0 (100), 338.1 (14), 257.1
(6). HRMS (FAB+) found 485.0659, C17H19N5O8S2 requires
485.0675.
1-[Bis-(4-hydroxy-3-methoxyphenyl)methyl]-1H-[1,2,4]triazole
(21). As general method B using Pd/C (55 mg), 20 (1.70 g, 3.35
mmol), and THF/MeOH (1:1, 200 mL). The crude product was
purified using Flashmaster II (EtOAc/Hexane) to give the title
compound (0.73 g, 67%, mp 159 °C dec) as a cream solid. δH (270
MHz, DMSO-d6): 9.12 (2H, s, 2 × OH), 8.44 (1H, s, NCHN), 8.02
(1H, s, NCHN), 6.82-6.70 (5H, m, ArH), 6.59 (2H, dd, J ) 8.2,
2.0, ArH), 3.68 (6H, s, 2 × CH3). LCMS (ES-) 326.3 ([M - H]-,
50%), 256.5 (50), 240.4 (100), 211.2 (20). HRMS (FAB+) found
327.1218, C17H17N3O4 requires 327.1219. Anal. (C17H17N3O4) C,
H, N.
1-[Bis-(4-sulfamoyloxy-3-methoxyphenyl)methyl]-1H-[1,2,4]tri-
azole (22). As general method C using ClSO2NH2 (5.8 mL), DMA
(6 mL), and 21 (0.30 g, 0.92 mmol). The resulting reaction mixture
was poured onto brine (50 mL), and this was extracted with EtOAc
(3 × 50 mL). The organic layers were combined, washed with H2O
(2 × 50 mL) and brine (2 × 50 mL), dried (MgSO4), and the solvent
was removed in vacuo. The crude product was purified using
Flashmaster II (EtOAc/hexane) to give the title compound (0.41 g,
93%) as a white foam. δH (270 MHz, DMSO-d6): 8.60 (1H, s,
NCHN), 8.11 (1H, s, NCHN), 7.95 (4H, br s, 2 × NH2), 7.31 (2H,
d, J ) 8.4, ArH), 7.13 (2H, d, J ) 2.0, ArH), 7.07 (1H, s, CH),
6.90 (2H, dd, J ) 8.4, 2.0, ArH), 3.75 (6H, s, 2 × CH3). LRMS
(FAB+) 485.9 [M + H]+, 20%), 416.9 (35), 338.0 (10). HRMS
(FAB+) found 485.0683, C17H19N5O8S2 requires 485.0675.
(R,S)-1-[(4-Cyanophenyl)(4-hydroxyphenyl)methyl]-1H-
[1,2,4]triazole (27). As general method A using 26 (0.20 g, 0.89
mmol), 1,2,4-triazole (0.13 g, 1.88 mmol), p-TSA (50 mg), and
toluene (70 mL). The resulting residue was dissolved in EtOAc
(20 mL) and the organic layer was washed with H2O (3 × 40 mL)
and brine (40 mL), then dried (MgSO4), and the solvent was
removed in vacuo. The crude product was purified using Flash-
master II (EtOAc/hexane) to give the title compound (0.21 g, 81%)
as a white solid. δH (270 MHz, DMSO-d6): 9.64 (1H, br s, OH),
8.56 (1H, s, NCHN), 8.08 (1H, s, NCHN), 7.84 (2H, AA′BB′, ArH),
7.32 (2H, AA′BB′, ArH), 7.10 (2H, AA′BB′, ArH), 7.08 (1H, s
CH), 6.76 (2H, AA′BB′, ArH); LRMS (FAB+) 277.1 ([M + H]+,
55%), 208.1 (100). HRMS (FAB+) found 277.1095, C16H13N4O
requires 277.1089. Anal. (C16H12N4O) C, H, N.
product was purified using Flashmaster II (EtOAc/hexane) to give
the title compound (0.42 g, 81%) as a white powder. δH (270 MHz,
DMSO-d6): 8.68 (1H, s, NCHN), 8.08 (1H, s, NCHN), 8.05 (2H,
br s, NH2), 7.88 (2H, AA′BB′, ArH), 7.44-7.28 (7H, m, 6 × ArH,
CH). LCMS (ES+) 356.0 ([M + H]+, 10%), 286.8 (100), 207.7
(90), 157.6 (30), 140.6 (35). HRMS (FAB+) found 356.0806,
C16H14N5O3S requires 356.0817.
1-[(4-Cyanophenyl)(3-chloro-4-hydroxyphenyl)methyl]-1H-
[1,2,4]triazole (33). As general method A using 32 (dissolved in
min vol EtOAc) (1.53 g, 5.88 mmol), 1,2,4-triazole (4.00 g, 0.058
mol), p-TSA (0.30 g), and toluene (350 mL). The resulting residue
was dissolved in EtOAc (70 mL), and the organic layer was washed
with H2O (4 × 70 mL) and brine (70 mL), then dried (MgSO4),
and the solvent was removed in vacuo. The crude product was
purified using Flashmaster II (EtOAc/hexane) to give the title
compound (1.24 g, 68%, mp 166.5-168 °C) as a white solid; an
analytical sample was obtained by recrystallization from EtOAc.
δH (400 MHz, DMSO-d6): 10.50 (1H, br s, OH), 8.60 (1H, s,
NCHN), 8.12 (1H, s, NCHN), 7.57 (2H, AA′BB′, ArH), 7.35 (2H,
AA′BB′, ArH), 7.29 (1H, d, J ) 2.1, ArH), 7.16 (1H, s CH), 7.10
(1H, dd, J ) 8.8, 2.1, ArH), 6.99 (1H, d, J ) 8.8, ArH). LCMS
(APCI-) 309.1 ([M - H]-, 60%), 241.0 (100). HRMS (FAB+)
found 311.0709, C16H12N4OCl requires 311.0700.
1-[(4-Cyanophenyl)(3-chloro-4-sulfamoyloxyphenyl)methyl]-
1H-[1,2,4]triazole (34). As general method C using ClSO2NH2 (4.8
mL), DMA (4 mL), and 33 (0.25 g, 0.81 mmol). The resulting
reaction mixture was poured onto brine (20 mL), and this was
extracted with EtOAc (3 × 20 mL). The organic layers were
combined, washed with H2O (3 × 30 mL) and brine (30 mL), dried
(MgSO4), and the solvent was removed in vacuo. The crude product
was purified using Flashmaster II (EtOAc/hexane) to give the title
compound (0.25 g, 80%) as a white solid. δH (270 MHz, DMSO-
d6): 8.71 (1H, s, NCHN), 8.33 (2H, br s, NH2), 8.15 (1H, s, NCHN),
7.89 (2H, AA′BB′, ArH), 7.53 (1H, d, J ) 2.2, ArH), 7.52 (1H, d,
J ) 8.4, ArH), 7.41 (2H, AA′BB′, ArH), 7.35 (1H, dd, J ) 8.4,
2.2, ArH), 7.31 (1H, s, CH). LCMS (APCI+) 390.2 ([M + H]+,
100%), 311.2 (20), 242.1 (20), 241.1 (60). HRMS (FAB+) found,
390.0436 C16H13N5O3SCl requires 390.0428.
4-Cyano-3′-bromo-4′-methoxybenzophenone (36). Aluminum
chloride (7.08 g, 53.1 mmol) was added in small portions over 15
min to a solution of 4-cyanobenzoyl chloride (8.00 g, 48.3 mmol)
in o-bromoanisole (40 mL) After stirring for 24 h, the reaction
mixture was poured onto ice-water (120 mL) and stirred for 1 h.
EtOAc (100 mL) and 3 M NaOH (400 mL) were added and the
layers were separated; the aqueous was further extracted with EtOAc
(2 × 150 mL). The organic layers were combined, washed with
brine (2 × 200 mL), and dried (MgSO4). The solvent was removed
in vacuo to give a dark-brown oil to which hexane (200 mL) was
added. The white precipitate was collected and was purified by
recrystallization from EtOH and then further purified (to remove
debrominated material) using Flashmaster II (EtOAc/hexane) to give
the title compound (4.49 g, 29%, mp 172.5-174 °C) as a white
crystalline solid. δH (270 MHz, DMSO-d6): 8.03 (2H, AA′BB′,
ArH), 7.95 (1H, d, J ) 1.9, ArH), 7.83 (2H, AA′BB′, ArH), 7.76
(1H, dd, J ) 8.5, 2.2, ArH), 7.27 (1H, d, J ) 8.5, ArH), 3.98 (3H,
s, CH3). LCMS (APCI+) 316.1 (M+, 80%), 214.0 (45), 198.2 (100).
HRMS (FAB+) found 315.9982, C15H11NO2Br requires 315.9973.
4-Cyano-3′-bromo-4′-hydroxybenzophenone (37). BBr3 (1 M
solution in DCM, 52 mL) was slowly added to a cooled (0 °C)
solution of 36 (3.30 g, 10.4 mmol) in DCM (40 mL). The resulting
solution was allowed to warm to room temperature and was stirred
for 6 days. The reaction mixture was poured onto ice-water (200
mL) and extracted with DCM (2 × 200 mL), the combined organics
were washed with H2O (250 mL) and NaHCO3 (250 mL) and were
extracted with NaOH (3 M, 3 × 150 mL). The combined basic
extracts were acidified with conc HCl and extracted with DCM (2
× 200 mL), the combined extracts were washed with H2O (2 ×
100 mL), dried, (MgSO4) and the solvent was removed in vacuo.
The title compound (2.00 g, 64%, mp 216 °C (dec)) was obtained
as a cream solid. δH (270 MHz, DMSO-d6): 11.50 (1H, br s, OH),
8.01 (2H, AA′BB′, ArH), 7.88 (1H, d, J ) 1.9, ArH) 7.81 (2H,
1-[(4-Cyanophenyl)(4-sulfamoyloxyphenyl)methyl]-1H-
[1,2,4]triazole (28). As general method C using ClSO2NH2 (5.8
mL), DMA (6 mL), and 27 (0.30 g, 0.92 mmol). The resulting
reaction mixture was poured onto brine (50 mL), and this was
extracted with EtOAc (3 × 50 mL). The organic layers were
combined, washed with H2O (2 × 50 mL) and brine (2 × 50 mL),
dried (MgSO4), and the solvent was removed in vacuo. The crude

4236 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Wood et al.
AA′BB′, ArH)), 7.62 (1H, dd, J ) 8.5, 1.9, ArH), 7.08 (1H, d, J
) 8.5, ArH). LCMS (APCI-) 302.1 (M-, 50%), 222.0 (100), 212.0
(25). HRMS (FAB+) found 301.9823, C14H9NO2Br requires
301.9817.
aromatase enzyme, which is based on the crystal structure of the
human CYP2C962 metabolic enzyme as described by Favia et al.,55
was constructed using SYBYL7.3. Hydrogen atoms were built onto
the model and all atoms except hydrogens were fixed in aggregates.
Hydrogen atom positions were then optimized to convergence using
the Tripos force field with Gasteiger-Hu¨ckel charges. The GOLD
docking program V3.1.1 was used to dock the models to the
aromatase model. The aromatase active site was defined as a 12 Å
sphere around the heme group iron atom. A distance constraint
(minimum ) 2.00 Å, maximum ) 2.30 Å) was applied between
the ligating triazole nitrogen atom of the ligand to the heme iron
atom. The coordination number of the iron atom was defined as 6.
The ligands were then docked to the enzyme a total of 25 times
each using the GOLDScore fitness function.
The 1P49.pdb crystal structure of human placental estrone/
DHEA sulfatase was used for the building of the gem-diol form
of STS.57 This involved a point mutation of the ALS75 residue
in the crystal structure to the gem-diol form of the structure
using editing tools within SYBYL 7.3. The resulting structure
was then minimized with the backbone atoms fixed to allow the
gem-diol and surrounding side chain atoms to adopt low energy
confirmations. Minimisations were undertaken using SYBYL 7.3
applying the AMBER7 99 force field with Gasteiger-Hu¨ckel
charges as implemented within SYBYL 7.3. To mimic the
sulfamate group of E1S, all sulfamate based compounds were
docked into the active site with their sulfamate group in its
monoanionic form (i.e., -OSO2NH-).
(4-Cyanophenyl)(3-bromo-4-hydroxyphenyl)methanol (38). A
solution of NaBH4 (0.53 g, 13.9 mmol) in H2O (8 mL) was added
dropwise to a solution of 37 (2.00 g, 7.75 mmol) in EtOH (20 mL).
After stirring overnight, the reaction mixture was added to aq HCl
(3 M, 40 mL) and the product was extracted with DCM (3 × 30
mL). The combined organic layers were washed with brine (60
mL), dried (MgSO4), and the solvent was removed in vacuo. The
crude product was purified using Flashmaster II (EtOAc/hexane)
to give the title compound (1.67 g, 83%) as an oil which solidified
on standing to give a pink solid. δH (270 MHz, DMSO-d6): 10.17
(1H, br s, OH) 7.76 (2H, AA′BB′, ArH), 7.54 (2H, AA′BB′, ArH),
7.44 (1H, d, J ) 2.0, ArH), 7.13 (1H, dd, J ) 8.4, 2.0, ArH), 6.87
(1H, d, J ) 8.4, ArH), 6.08 (1H, d, J ) 3.9, OH), 5.69 (1H, d, J
) 3.9, CH). LCMS (APCI-) 302.2 ([M - 1]-, 80%), 284.2 (100).
HRMS (FAB+) found 303.9968, C14H11BrNO2 requires 303.9968.
1-[(4-Cyanophenyl)(3-bromo-4-hydroxyphenyl)methyl]-1H-
[1,2,4]triazole (39). As general method A using 38 (dissolved in
min vol EtOAc) (1.32 g, 4.34 mmol), 1,2,4-triazole (3.00 g, 43.4
mmol), p-TSA (0.26 g), and toluene (350 mL). The resulting residue
was dissolved in EtOAc (70 mL) and the organic layer was washed
with H2O (4 × 70 mL) and brine (70 mL), then dried (MgSO4),
and the solvent was removed in vacuo. The crude product was
purified using Flashmaster II (EtOAc/hexane) to give the title
compound (0.94 g, 61%, mp 171.5-173.5 °C) as a white solid. δH
(270 MHz, DMSO-d6): 10.54 (1H, br s, OH), 8.62 (1H, s, NCHN),
8.09 (1H, s, NCHN), 7.84 (2H, AA′BB′, ArH), 7.41 (1H, d, J )
2.0, ArH), 7.32 (2H, AA′BB′, ArH), 7.13-7.07 (2H, m, ArH and
CH), 6.94 (1H, d, J ) 8.4, ArH). LCMS (APCI+) 354.8 ([M +
H]+, 100%), 287.8 (20), 213.9 (40), 112.9 (100). HRMS (FAB+)
found 355.0180, C16H12N4OBr requires 355.0190. Anal.
(C16H11BrN4O) C, H, N.
In Vitro and in Vivo Aromatase and Sulfatase Assays. Bio-
logical activities were performed essentially as described previ-
ously.22 The extent of in vitro inhibition of aromatase and sulfatase
activities was assessed using intact monolayers of JEG-3 cells.
Aromatase activity was measured using [1ꢀ-3H] androstenedione
(30 Ci/mmol, Perkin-Elmer Life Sciences, MA) over a 3 h period.
Sulfatase activity was measured using [6,7-3H] E1S (50 Ci/mmol,
Perkin-Elmer Life Sciences) over a 3 h period.
1-[(4-Cyanophenyl)(3-bromo-4-sulfamoyloxyphenyl)methyl]-
1H-[1,2,4]triazole (40). As general method C using ClSO2NH2 (4.8
mL), DMA (4 mL), and 39 (0.40 g, 1.13 mmol). The resulting
reaction mixture was poured onto H2O (25 mL), and this was
extracted with EtOAc (2 × 30 mL). The organic layers were
combined, washed with H2O (4 × 30 mL) and brine (30 mL), dried
(MgSO4), and the solvent was removed in vacuo. The crude product
was purified using Flashmaster II (EtOAc/hexane) to give the title
compound (0.46 g, 88%) as a white solid. δH (270 MHz, DMSO-
d6): 8.69 (1H, s, NCHN), 8.30 (2H, br s, NH2), 8.12 (1H, s, NCHN),
7.86 (2H, AA’BB’, ArH), 7.65 (1H, d, J ) 2.2, ArH), 7.50 (1H, d,
J ) 8.2, ArH), 7.42-7.33 (3H, m, ArH), 7.29 (1H, s, CH). LCMS
(APCI-) 433.9 ([M - H]-, 100%), 354.9 (50), 284.8 (60), 211.8
(40). HRMS (FAB+) found, 433.9915 C16H13N5O3SBr requires
433.9922. HPLC (MeOH/H2O, 80:20) tR) 1.75 min (purity: 99%).
(R)-1-[(4-Cyanophenyl)(3-bromo-4-hydroxyphenyl)methyl]-
1H-[1,2,4]triazole (39a). This was obtained following separation
of 39 on a Chiralpak AD-H (250 × 20 mm) semiprep column as
Acknowledgment. This work was supported by Sterix Ltd.,
a member of the Ipsen group. We thank A. C. Smith for
technical assistance and Dr. Brian Freer of Chiral Technologies
Europe for assistance with the chiral HPLC.
Supporting Information Available: Synthetic procedures for
12, 18-20, 25, 26, 30-32, 42, 43; 13C NMR data for 5-7, 13-16,
21, 22, 27, 28, 33, 34, 36-40; Microanalysis data for 9, 14-15,
21, 26-27, 39 and HPLC data for 6-7, 10, 16, 22, 28, 33-34, 40.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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