Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors from the Letrozole and Vorozole Templates

DOI: 10.1002/cmdc.201100145

Aromatase and Dual Aromatase-Steroid Sulfatase

Inhibitors from the Letrozole and Vorozole Templates

Paul M. Wood,^[a]L. W. Lawrence Woo,^[a]Mark P. Thomas,^[a]Mary F. Mahon,^[b]Atul Purohit,^[c]

and Barry V. L. Potter*^[a]

Concurrent inhibition of aromatase and steroid sulfatase (STS)

may provide a more effective treatment for hormone-depen-

dent breast cancer than monotherapy against individual en-

zymes, and several dual aromatase–sulfatase inhibitors (DASIs)

have been reported. Three aromatase inhibitors with sub-

nanomolar potency, better than the benchmark agent letro-

zole, were designed. To further explore the DASI concept, a

new series of letrozole-derived sulfamates and a vorozole-

based sulfamate were designed and biologically evaluated in

JEG-3 cells to reveal structure–activity relationships. Amongst

achiral and racemic compounds, 2-bromo-4-(2-(4-cyanophen-

yl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phenyl sulfamate is the most

potent DASI (aromatase: IC₅₀=0.87 nm; STS: IC₅₀=593 nm).

The enantiomers of the phenolic precursor to this compound

were separated by chiral HPLC and their absolute configura-

tion determined by X-ray crystallography. Following conversion

to their corresponding sulfamates, the S-(+)-enantiomer was

found to inhibit aromatase and sulfatase most potently (aro-

matase: IC₅₀=0.52 nm; STS: IC₅₀=280 nm). The docking of

each enantiomer and other ligands into the aromatase and sul-

fatase active sites was also investigated.

Introduction

The growth and development of the most common form of

breast malignancies, hormone-dependent breast cancer

(HDBC), is promoted by the presence of oestrogenic steroids.

Currently, the most widely used therapies for the treatment of

this disease focus on blocking the action of these steroids,

either by the use of selective, oestrogen receptor modulators,

such as tamoxifen, or by inhibiting their biosynthesis through

inhibition of the aromatase enzyme complex. Third-generation

aromatase inhibitors (AIs), currently finding widespread appli-

cation in the clinic, comprise the nonsteroidal compounds

anastrozole and letrozole, and the steroidal exemestane.^[1–3]Al-

though these compounds were initially used in patients for

whom tamoxifen therapy had failed, data from a number of

clinical trials suggest that AIs provide a more effective first-line

therapy against HDBC as a result of their superior efficacy and

toxicology profile.^[4–6]Of these AIs, there is evidence to suggest

that letrozole is superior to anastrozole in suppressing oestro-

gen levels in breast tissue and plasma in patients with postme-

nopausal breast cancer.^[7]It is unclear how this difference will

translate to the clinic, however, the Femara versus Anastrozole

Clinical Evaluation (FACE) trial should help to determine wheth-

er any differences in efficacy exist between these two AIs.^[8]

A promising new therapy for the treatment of HDBC has

arisen from the development of inhibitors of steroid sulfatase

(STS).^[9]This enzyme is believed to be virtually ubiquitous

[a] Dr. P. M. Wood, Dr. L. W. L. Woo, Dr. M. P. Thomas, Prof. B. V. L. Potter

Medicinal Chemistry, Department of Pharmacy and Pharmacology

University of Bath, Claverton Down, Bath BA2 7AY (UK)

Fax: (+44)1225-386-114

E-mail: b.v.l.potter@bath.ac.uk

[b] Dr. M. F. Mahon

X-Ray Crystallographic Suite, Department of Chemistry

University of Bath, Claverton Down, Bath, BA2 7AY (UK)

[c] Dr. A. Purohit

Division of Diabetes, Endocrinology & Metabolism

Imperial College London, Hammersmith Hospital, London, W12 0NN (UK)

Supporting information for this article is available on the WWW under

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B. V. L. Potter et al.

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throughout the body and is responsible for the conversion of

alkyl and aryl steroid sulfates to their unconjugated and bio-

logically active forms. Primarily, STS catalyses the conversion of

oestrone sulfate, a biologically inactive steroid found at high

levels in the plasma of postmenopausal women, to oestrone.

In breast cancer tissue, it has been shown that ten times more

oestrone originates from oestrone sulfate than from androste-

nedione.^[10]In addition, STS controls the formation of dehy-

droepiandrosterone (DHEA) from DHEA-sulfate (DHEA-S). DHEA

can be subsequently converted to androst-5-ene-3b,17b-diol,

an androgen with oestrogenic properties capable of stimulat-

ing the growth of breast cancer cells in vitro^[11]and inducing

mammary tumours in vivo.^[12]The pharmacophore for irreversi-

ble STS inhibition has been identified as a substituted phenol

sulfamate ester, and a number of steroidal (e.g., oestrone-3-O-

sulfamate, also known as EMATE) and nonsteroidal inhibitors

(e.g., Irosustat, also known as STX64, BN83495) have been de-

veloped.^[9,13–14]Irosustat, discovered by our group, has been

evaluated in a phase 1 clinical trial for the treatment of post-

menopausal patients with metastatic breast cancer and has

shown promising results.^[15]

on anastrozole.^[25]In a complementary approach, we also re-

ported a series of DASIs obtained following introduction of the

pharmacophore for aromatase inhibition into a biphenyl tem-

plate primarily designed for STS inhibition (e.g, 6).^[26]

In preliminary work on the design of a prototype letrozole-

based DASI, it was hoped that dual aromatase and sulfatase in-

hibition could be achieved by replacing both para-cyano

groups present in letrozole with sulfamate groups, whilst re-

taining both the triazole and the diphenylmethane moieties

necessary for potent aromatase inhibition.^[19]A lead com-

pound, bis-sulfamate 1 exhibited IC₅₀values of 3044 nm for ar-

omatase and >10 mm for STS when evaluated in JEG-3 cells.

Further iterations improved inhibition of both aromatase and

sulfatase,^[20]and the most potent AI identified was (ꢀ)-2 in

which only one of the para-cyano groups is replaced with a

sulfamate group. Compound 2 inhibited aromatase and STS

with IC₅₀values of 3 nm and 2600 nm, respectively. The enan-

tiomers of 41, the phenolic precursor of 2, were separated by

chiral HPLC and converted into their corresponding sulfa-

mates;^[27]the R-configuration provided the most potent aroma-

tase inhibitor (R: 3.2 nm; S: 14.3 nm), whilst the S-configuration

proved to be the best STS inhibitor (S: 553 nm; R: 4633 nm).^[20]

Here, we report the further investigation of the structure–ac-

tivity relationships (SAR) of letrozole-derived DASIs by evaluat-

ing the effect on inhibitory activity of increasing linker length

between the triazole and the STS pharmacophore, and replac-

ing the para-cyano-substituted ring with a para-chloro-substi-

tuted ring. The enantiomers of one compound were separated

and their absolute configuration was determined by X-ray crys-

tallography. We also report the synthesis and in vitro inhibitory

activities of the first dual inhibitor derived from the third gen-

eration AI, vorozole.

The advantages of a single chemical agent with the ability

to interact with multiple biological targets have been recently

highlighted.^[16–18]A possible application of this concept for the

treatment of HDBC would be the combination of the pharma-

cophores for both aromatase and STS inhibition into a single

molecular entity. One approach to achieve this would be inser-

tion of the pharmacophore for STS inhibition into an estab-

lished AI, whilst maintaining the features necessary for aroma-

tase inhibition. We previously reported three series of dual aro-

matase–sulfatase inhibitors (DASIs) based on different AIs: ex-

amples include, compounds 1 and 2 based on letrozole,^[19–20]

compound 3 based on YM511 (4),^[21–24]and compound 5 based

Results and Discussion

Chemistry

All of the novel sulfamates and phenols described in this

paper were prepared according to Schemes 1–4. The final

compounds and intermediates were characterised by standard

analytical methods and, in addition, the purity of the com-

pounds tested in vitro was evaluated using HPLC. The refer-

ence AI, letrozole, was prepared by the alkylation of 1,2,4-tria-

zole with a-bromomethyl-p-tolunitrile followed by reaction

with 4-fluorobenzonitrile according to the procedure described

by Bowman et al.^[19,28]The reference STS inhibitor, STX64, was

synthesised according to the method of Woo et al.^[29]

The synthetic route for the sulfamates 11, 14 and 18 is

shown in Scheme 1. Both sulfamates 11 and 18 were prepared

from 3-bromo-4-(triisopropylsilyloxy)benzaldehyde (7)^[20]and

sulfamate 14 was prepared from (4-(bromomethyl)phenoxy)trii-

sopropylsilane (12).^[30]For 11, treatment of aldehyde 7 with 4-

chlorophenylmagnesium bromide followed by deprotection of

the phenol afforded 9. Displacement of the hydroxy group in

9 was achieved with 1,2,4-triazole in refluxing toluene to afford

10, which was converted to sulfamate 11 by reaction with an

excess of sulfamoyl chloride in N,N-dimethylacetamide (DMA)

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Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

furnish 22. Sulfamate 29 was

prepared in a similar manner

using bromide 26, which was

itself prepared from methyl 2-(3-

bromo-4-hydroxyphenyl)acetate

23.^[23]Following triisopropylsilyl

(TIPS) protection of the phenolic

hydroxy group in 23, ester 24

was reduced with lithium boro-

hydride and the resulting benzyl

alcohol was converted to benzyl

bromide 26, and the synthesis of

29 was completed using the

steps described above. The

bottom part of Scheme 2 de-

scribes the route for the synthe-

sis of sulfamates 35 and 40. Sul-

famate 35 was synthesised from

30, which was prepared as de-

scribed by Avery et al.^[33]From

alcohol 30, Dess–Martin oxida-

Scheme 1. Reagents and conditions: a) 4-ClPhMgBr, THF, RT; b) TBAF, THF, RT; c) 1,2,4-Triazole, p-TsOH, toluene,

reflux; d) H₂NSO₂Cl, DMA, RT; e) NaBH₄, EtOH/H₂O, RT; f) SOCl₂, CH₂Cl₂, RT; g) nBuLi, 4-((1H-1,2,4-triazol-1-yl)methyl)-

benzonitrile, THF, ꢁ788C!RT.

according to the conditions described by Okada et al.^[31]For

the synthesis of sulfamate 14, it was envisaged that alkylation

of n-butyllithium-deprotonated^[32]4-((1H-1,2,4-triazol-1-yl)me-

thyl)benzonitrile^[19]with (4-(chloromethyl)phenoxy)triisopropyl-

silane would provide a route to 13; this reaction failed to pro-

vide the desired product. However, when the alkylating agent

was switched to the more reactive 12, the desired product

could be obtained. Deprotection of the phenol was achieved

tion gave aldehyde 31, which was reacted with 4-chlorophe-

nylmagnesium bromide to give 32. The alcohol was converted

to chloride 33 with thionyl chloride, and this was reacted with

1,2,4-triazole in acetone with concomitant loss of the TIPS pro-

tecting group to give 34. Finally, sulfamoylation as described

above furnished 35. Sulfamate 40 was prepared analogously

from 23 following TIPS protection of the phenol and lithium

borohydride reduction of ester 24.

using

tetra-n-butylammonium

fluoride and the product could

be used without further purifica-

tion. Phenol 13 was subsequent-

ly converted to sulfamate 14

using the conditions described

above. Starting from aldehyde 7,

reduction with sodium borohy-

dride and conversion of the re-

sulting benzyl alcohol to the

chloride gave compound 16.

This is a more reactive alkylating

agent than its nonbrominated

counterpart, and it was success-

fully used as the alkylating agent

for the synthesis of sulfamate 18

according to the route described

above.

The synthesis of sulfamates

22, 29, 35 and 40 is detailed in

Scheme 2. Compound 22 was

obtained in three steps from 19,

which was prepared according

to Avery et al.^[33]Reaction of 19

with 4-((1H-1,2,4-triazol-1-yl)me-

thyl)benzonitrile as described

above was followed by depro-

Scheme 2. Reagents and conditions: a) Triisopropylsilyl chloride, imidazole, DMF, RT; b) LiBH₄, B(OMe)₃, Et₂O, RT;

c) Br₂, PPh₃, imidazole, Et₂O/CH₃CN, RT; d) nBuLi, 4-((1H-1,2,4-triazol-1-yl)methyl)benzonitrile, THF, ꢁ788C!RT;

e) TBAF, THF, RT; f) H₂NSO₂Cl, DMA, RT; g) Dess–Martin periodinane, CH₂Cl₂, RT; h) 4-ClPhMgBr, THF, RT; i) SOCl₂/

tection and sulfamoylation to DMF, CH₂Cl₂, RT; j) 1,2,4-Triazole, K₂CO₃, KI, acetone, 558C.

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N,N-Dimethysulfamate 42 was successfully prepared by heat-

ing a mixture of 1-[(4-cyanophenyl)(3-bromo-4-hydroxyphenyl)-

methyl]-1H-[1,2,4]triazole^[20]and N,N-dimethylsulfamoyl chlo-

ride in N,N-diisopropylethylamine (DIPEA) (Scheme 3).

chloride and quickly reacted with 1,2,4-triazole in the presence

of potassium carbonate to give 49. Deprotection of the phenol

was achieved by catalytic hydrogenation with palladium on

carbon to give 50, and the formation of the corresponding sul-

famate was achieved using the conditions described above to

furnish 51.

Inhibition of aromatase and steroid sulfatase activity by sul-

famoylated compounds

The in vitro inhibition of aromatase and STS activity by each

sulfamate was measured in a preparation of an intact monolay-

er of JEG-3 cells. The results are reported as either IC₅₀values

or as a percentage of inhibition at 10 mm, and are compared to

the reference AI letrozole^[19]and the reference STS inhibitor

STX64^[21](Table 1). The biological activities of both 2 and 52

have been reported previously.^[20]

Scheme 3. Reagents and conditions: a) N,N-Dimethylsulfamoyl chloride,

DIPEA, reflux.

Vorozole-derived sulfamate 51 was prepared from benzoic

acid 43, which was synthesised as described by Dener et al.^[34]

from 3-methoxy-4-nitrobenzoic acid (Scheme 4). Formation of

the benzotriazole ring was achieved by treatment of 43 with a

mixture of sodium nitrite and hydrochloric acid in water. Sub-

sequent formation of methyl ester 45 and lithium borohydride

reduction gave compound 46. This approach to the synthesis

of the benzotriazole ring ensures that the methyl group is

placed on the correct nitrogen atom in the triazole ring. A

more concise route to 45 was explored via the alkylation of

methyl-1H-benzotriazole-5-carboxylate with methyl iodide in

the presence of potassium carbonate but this gave a mixture

of three regioisomers from which it was difficult to separate

the individual benzotriazol-1-yl isomers. The oxidation of alco-

hol 46 with potassium permanganate in dichloromethane^[35]

gave aldehyde 47 in moderate yields. However, excellent yields

of the aldehyde could be obtained by oxidation with the tri-

chloroisocyanuric acid/catalytic 2,2,6,6-tetramethylpiperidi-

nooxy (TEMPO) system reported by Giacomelli et al.^[36]Alde-

hyde 47 was subsequently reacted with the Grignard reagent

generated from 4-benzyloxybromobenzene to give alcohol 48.

This was converted to the corresponding chloride with thionyl

Table 1. In vitro inhibition of the aromatase and STS activity in JEG-3 cells

by letrozole, STX64, 52, 2 and the sulfamates described herein.

Arom

50

STS

50

Compd

R¹

R²

n

IC

[nm]

IC [nm]

Letrozole

STX64

2

11

14

18

22

29

35

40

42

51

52

–

CN

Cl

CN

Cl

–

CN

–

0

1

2

1

–

0

0.89ꢀ0.13^[a]

–

1.5ꢀ0.3^[b]

Br

H

Br

H

Br

H

Br

–

3.0ꢀ0.1^[c]

2.5ꢀ0.7

2.8ꢀ0.2

0.87ꢀ0.21

0.22ꢀ0.01

0.12ꢀ0.02

39ꢀ10

2600ꢀ100^[c]

1400ꢀ234

3517ꢀ625

593ꢀ6

>10000

2233ꢀ666

180ꢀ26

>10000

21.5%^[c,d]

1.4ꢀ1.1

23.04ꢀ2.92

29ꢀ10

–

H

13ꢀ4^[c]

[a] Data taken from Wood et al.^[19][b] Data taken from Woo et al.^[21][c] Data

taken from Wood et al.^[20][d] Percent inhibition at 10 mm. Mean IC₅₀values ꢀ

SD were determined from incubations carried out in triplicate in a minimum

of two separate experiments.

All of the sulfamates tested in this series are potent inhibi-

tors of aromatase with IC₅₀values ꢂ39 nm and, in addition,

some compounds also exhibit moderate-to-potent STS inhibi-

Arom

50

tion. In this assay, two compounds, 22 and 29 (IC

=0.22

and 0.12 nm, respectively) are more potent than the reference

Arom

50

AI letrozole (IC

=0.89 nm).

Scheme 4. Reagents and conditions: a) NaNO₂, 6n HCl, H₂O, 08C!RT;

b) SOCl₂, MeOH, reflux; c) LiAlH₄, THF, RT; d) Trichloroisocyanuric acid,

TEMPO, CH₂Cl₂, 08C, e) 4-Benzyloxybromobenzene, Mg, THF, RT; f) SOCl₂,

CH₂Cl₂, RT; g) 1,2,4-Triazole, KI, K₂CO₃, acetone, 608C; h) 10% Pd/C, THF/

MeOH, RT; i) H₂NSO₂Cl, DMA, RT.

Several compounds in this study contain a para-chloro-sub-

stituted phenyl ring rather than the para-cyano-substituted

ring found in letrozole and letrozole-based DASI 2. This substi-

tution is present in compounds capable of potent aromatase

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Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

inhibition. For instance, this moiety is present in the potent AI

ed counterparts. This trend is exhibited in both the para-

Arom

50

Arom

=0.9 nm) and para-

50

(ꢀ)-vorozole (IC

=2.59 nm),^[37]and furthermore, a derivative

cyano- (14: IC

chloro-substituted series (35: IC

=2.8 nm vs 18: IC

50

Arom

50

of letrozole with both para-cyano groups replaced by para-

chloro groups has been reported to inhibit aromatase activity

with an EC₅₀value of 8.8 nm in a rat ovarian microsome

assay.^[38]For this series, comparison of the activities of the

para-cyano-substituted compounds with their para-chloro-sub-

stituted counterparts reveals that, for the two sets of com-

=39 nm vs 40: IC

=

50

1.4 nm). This and previous results suggest that the higher aro-

matase inhibition can be attributed to the increased lipophilici-

ty conferred by the halogen.^[20]Similarly, we previously discov-

ered that the presence of a halogen group ortho to the sulfa-

mate increases STS inhibitory activity, and this trend holds true

STS

50

pounds 2/11 and 18/40, dual inhibitory activity is retained fol-

in this series for both pairs of compounds 14/18 (14: IC

=

Arom

50

STS

lowing the switch in substitution (e.g., 2: IC

=3.0 nm,

3517 nm vs 18: IC =593 nm) and 36/40. The increase in STS

50

STS

Arom

50

STS

IC =2600 nm, vs 11: IC

=2.5 nm, IC =1400 nm). Howev-

inhibitory activity is reasoned to be caused by a lowering of

the pK_aof the phenol, enhancing its leaving group ability. Pre-

sumably, the deleterious effect on activity caused by an in-

crease in linker length for compounds 22 and 29 is too large

for any halogen-induced increase in inhibition to be observed.

The N,N-dimethylsulfamate-containing compound 42 is a

weaker AI than its corresponding demethylated counterpart 2,

despite the increase in lipophilicity conferred by dimethylation,

which normally benefits aromatase inhibition. The weak STS in-

hibition exhibited by 42 in vitro is anticipated based on our

previous work on N,N-dimethylated sulfamates.^[13,42]For exam-

ple, despite the poor inhibitory activity against STS^[43]exhibited

by the N,N-dimethylated derivative of STX64 in vitro, this com-

pound has been shown to almost completely inhibit mouse

liver and skin STS activities 24 h after oral administration.^[44]

This suggests that N,N-demethylation may occur in vivo to pro-

vide a compound capable of STS inhibition, and it might also

be the case that, although 42 is inactive in vitro, it may act as

a prodrug of 2 in vivo. Further work is required to explore the

potential in vivo conversion of compound 42 to 2.

50

er, for compounds 14 and 35, replacement of the para-cyano

group with a para-chloro group maintains STS inhibitory activi-

Arom

50

ty but is detrimental to aromatase activity (14: IC

=2.8 nm,

STS

Arom

50

STS

IC =3517 nm, vs 35: IC

=39 nm, IC =2233 nm). The im-

50

portance of the positioning of a hydrogen-bond acceptor (e.g.,

CN, NO₂) in the molecule relative to the triazole/imidazole ring

for potent aromatase inhibition has been extensively discussed

in the literature.^[39–40]Interestingly, in this series, replacement of

the cyano group with the weaker hydrogen-bond accepting

chloro substituent maintains good aromatase inhibitory activi-

ty, possibly due to a complex interaction between the hydro-

gen-bond donor in the active site, the halide and the p system

of the connecting aromatic ring.^[41]

The effect played by the linker between the aromatase and

STS pharmacophores on dual inhibitory activities is illustrated

by three series of compounds: 1) 52, 14 and 22; 2) 2, 18 and

29; 3) 11 and 40. In each series, lengthening the linker (n) re-

sults in an increase in aromatase inhibition; this is illustrated

Arom

50

by comparing compounds 2 (n=0; IC

=3.0 nm), 18 (n=1;

Arom

50

Arom

50

IC

=0.87 nm), and 29 (n=2; IC

=0.12 nm). This corre-

Compound 51 is the first reported example of a sulfamate-

containing vorozole derivative. Vorozole is a third generation,

aromatase-selective AI, that entered phase 3 clinical trials, but

further development was discontinued when no improvement

in median survival was obtained compared to megestrol ace-

tate.^[45]Nonetheless, we explored the feasibility of designing a

DASI that is structurally related to vorozole. Incorporation of

the STS inhibitory pharmacophore into the molecule was ach-

ieved by replacement of the para-chloro substituent attached

to the phenyl ring in vorozole with a sulfamate group. Com-

pound 51 exhibits good inhibitory activity against aromatase,

although it is a weaker AI than the corresponding letrozole de-

rivative 52 and, like 52, also exhibits poor STS inhibition. How-

ever, based on previous observations, the introduction of ap-

propriate substitutions onto the sulfamate-bearing ring in 51

would be expected to improve inhibitory activity against both

enzymes, indicating the feasibility of a DASI based on the voro-

zole template.

lates with the small increase in aromatase inhibition observed

in the aw-diarylalkyltriazole series of compounds with inhibito-

ry activities for 4-(3-(4-fluorophenyl)-1-(1H-1,2,4-triazol-1-yl)pro-

pyl)benzonitrile and linker extended 4-(4-(4-fluorophenyl)-1-

(1H-1,2,4-triazol-1-yl)butyl)benzonitrile reported as 0.19 mm and

0.12 mm, respectively.^[32]These finding are also in agreement

with those in a YM511-derived DASI series, with activities for 4-

(((4-cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)phenyl sul-

famate and 4-(2-((4-cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)e-

thyl)phenyl sulfamate being 100 nm and 2.1 nm, respectively.^[23]

A small increase in linker length is beneficial for enhanced STS

STS

inhibition (2: IC =2600 nm, vs 18: IC =593 nm), but further

50

extension of the linker has a detrimental effect on inhibitory

STS

50

activity (29: IC >10000 nm). Extending the linker length was

shown to be detrimental to STS activity in the YM511-derived

DASI series, with a decrease in activity from 227 nm for 4-(((4-

cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)phenyl sulfa-

mate to >10000 nm for 4-(2-((4-cyanophenyl)(4H-1,2,4-triazol-

4-yl)amino)ethyl)phenyl sulfamate, which has one extra methyl-

ene unit in the linker. This decrease in STS inhibition could be

due to an increase in flexibility in the molecule as the linker is

extended, resulting in less favourable binding of the com-

pound in the active site.

Inhibition of aromatase activity by parent phenols

The aromatase inhibition for the phenols described in this

paper is tabulated in Table 2. The loss of the sulfamate group

following irreversible inactivation of STS by a sulfamate-based

DASI will result in the formation of the corresponding phenol.

The quantity of phenol produced by this mechanism is limited

in principle once all the STS activity has been inactivated.^[46]

As in our previous investigation into letrozole- and YM511-

derived DASIs, derivatives containing a halogen positioned

ortho to the sulfamate are better AIs than their nonhalogenat-

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B. V. L. Potter et al.

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ditions similar to those we reported previously for the separa-

tion of phenol 43, the enantiomers of phenol 17 were separat-

ed on a Chiralpak AD-H analytical column with methanol as

the mobile phase (see Experimental Section for further details).

The first enantiomer eluted from the column with a retention

time of 3.80 min (17a), whereas the second enantiomer eluted

with a retention time of 8.2 min (17b) giving greater peak sep-

aration than that previously obtained for 43. This separation

was subsequently scaled-up and successfully performed on a

Chiralpak AD-H semi-prep column to separate 700 mg of the

racemate with injections of 1.5–2.0 mL of a 20 mgmL^ꢁ1metha-

nol solution of 17. Conversion of 17a and 17b into their corre-

sponding sulfamates was achieved with excess sulfamoyl chlo-

ride in DMA. We previously reported that the sulfamoylation

step proceeds without loss of enantiomeric purity in the prep-

aration of the enantiomers of 2, 2a and 2b.^[20]The optical rota-

tion for each enantiomer of the phenol and corresponding sul-

famate was measured (data given in the Experimental Section).

Previously, in the absence of suitable crystals of 2a,b and

41a,b for X-ray analysis, the absolute configuration of each

enantiomer had to be established using vibrational and elec-

tronic circular dichroism in conjunction with time-dependent

density functional theory calculations of their predicted prop-

erties. Fortuitously, crystals suitable for X-ray analysis could be

obtained from ethyl acetate solutions of both 17a and 17b,

and the absolute configuration of each enantiomer was deter-

mined from the X-ray crystal structure of 17a.^[48]The crystal

structure obtained for 17a is shown in Figure 1, allowing the

unambiguous elucidation of the absolute configuration of 17a

as R-(ꢁ). Figure 1 further illustrates the sheets that stack along

the b axis in the gross structure as a consequence of intermo-

lecular hydrogen bonding between the phenolic hydrogen

(H1) and N2 of a proximate triazole in the crystal: [H1ꢁN2,

1.94 ꢁ; O1···N2, 2.744 ꢁ, O1ꢁH1···N2, 174.88]. The second Cꢁ

H···O type interaction arises between H6 in one molecule and a

triazole nitrogen (N3) from a lattice neighbour: [H6ꢁN3, 2.34 ꢁ;

C6···N3, 3.29 ꢁ; C6ꢁH6···N3, 172.68].

Table 2. In vitro inhibition of the aromatase activity in JEG-3 cells by le-

trozole and the phenols described herein.

Arom

50

Compd

R¹

R²

n

IC

[nm]

Letrozole

10

13

17

21

28

34

39

50

–

Cl

–

0

1

2

1

–

0.89ꢀ0.13^[a]

2.3ꢀ0.8

Br

H

Br

H

Br

H

Br

–

CN

Cl

2.9ꢀ0.3

0.21ꢀ0.02

0.16ꢀ0.02

0.02ꢀ0.01

80ꢀ5

Cl

–

1.8ꢀ0.5

3.8ꢀ1.0

[a] Data taken from Wood et al.^[19]Mean IC₅₀values ꢀSD are shown, de-

termined from incubations in triplicate from two independent experi-

ments.

However, degradation of the sulfamate following prolonged

circulation in plasma might provide an additional route for the

formation of the phenol. As these phenols still contain the

pharmacophore for aromatase inhibition they have the poten-

tial to act as AIs in their own right.

The most potent phenol in this series against aromatase is

Arom

50

28 (IC

=0.02 nm) and three compounds, 17, 21 and 28

Arom

(IC

=0.21, 0.16, 0.02 nm, respectively) are more potent than

the reference AI letrozole (IC

50

Arom

=0.89 nm) in this assay. With

50

the exception of 34, the phenols are either equipotent or

slightly better inhibitors of aromatase compared to their corre-

sponding sulfamates.

In common with the trends observed for their sulfamoylated

counterparts, positioning a halogen ortho to the phenol results

in an increase in aromatase inhibitory activity, as seen for ex-

Arom

50

ample in compounds 13 and 17 (IC

=2.9 nm vs 0.21 nm, re-

spectively), and lengthening the linker is also beneficial for aro-

Inhibitory activities of chiral sulfamates and their parent

phenols

matase inhibition, as seen for example in compounds 13 and

Arom

50

21 (IC

=2.9 nm vs 0.16 nm, respectively).

The difference in aromatase and STS inhibition exhibited by

each enantiomer of 18 was evaluated following separation of

the enantiomers of phenolic precursor 17 by chiral HPLC and

conversion to their corresponding sulfamates. For comparison,

the aromatase and STS inhibitory activities of each enantiomer

of 18 and the aromatase inhibitory activities of the enantio-

mers of 17 are shown in Table 3 along with those previously

obtained for the enantiomers of 2 and 41. Previous studies

have suggested that there is often a large difference in aroma-

tase inhibition observed between the enantiomers of chiral

AIs. For vorozole,^[37]there is a 32-fold difference in activity, with

the S-configuration being the most active (S-(+): IC₅₀=1.38 nm

vs R-(ꢁ): IC₅₀=44.2 nm) and exhibiting a similar inhibition to

the racemate (R,S-(ꢀ): IC₅₀=2.59 nm). There is a larger 210-fold

difference in aromatase inhibitory activity between the enan-

tiomers of fadrozole hydrochloride^[47]with the S-enantiomer

Chiral HPLC and absolute structure determination

In order to enrich the SAR for letrozole-derived DASIs with

their target proteins and to allow comparison with the inhibi-

tory activities of the enantiomers of 2, the activities of each

enantiomer of 18, one of the most promising DASIs in this cur-

rent series, were determined. To avoid any complications aris-

ing from decomposition of the sulfamate during separation,

resolution by chiral HPLC was performed with 17, the parent

phenol of the sulfamate, an approach previously used in the

preparation of the enantiomers of 2.^[20]

The literature contains a number of reports on the resolu-

tion of AIs by chiral HPLC with a particular focus on imidazole-

containing compounds: for example, fadrozole hydrochloride,

which was separated with a Chiralcel OD column.^[47]Using con-

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Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

STS

tween the enantiomers (2a: IC =553 nm vs 2b:

50

STS

50

IC =4633 nm).

In the current study, there is a more significant 60-

fold difference in aromatase activity between the two

Arom

50

enantiomers of 18 (18a: IC

=30.83 vs 18b:

Arom

IC

=0.52 nm) but a smaller difference (approxi-

mately fourfold) in STS inhibitory activity (18a: IC

1150 vs 18b: IC =280 nm). This increase in the dif-

50

STS

50

=

STS

50

ference of aromatase inhibition exhibited by each

enantiomer could be a result of the increase in asym-

metry of the molecule following extension of the

linker length. Comparison of the aromatase inhibitory

activity between the enantiomers of 2 and 18 reveals

that in each case the most potent AI is the dextroro-

tatory enantiomer and that despite possessing differ-

ent absolute configuration, the same three-dimen-

sional relationship between the triazole ring and the

para-cyanophenyl ring is present in the two most

potent enantiomers, R-(+)-2b and S-(+)-18b. The

spatial disposition of the heterocyle and para-cyano-

substituted ring in 2b and 18b resembles that pres-

ent in the most potent enantiomers of the chrome-

none-based AI series,^[49]suggesting that this is the

most favourable orientation of these groups for

potent aromatase inhibition. For STS inhibition, there

is a switch in the most potent enantiomer from the

levorotatory for 2a to the dex-

Figure 1. a) X-ray crystal structure of 17a (CCDC deposition code: 806541); ellipsoids are

represented at 30% probability. b) Portion of extended structure present in 17a illustrat-

ing the network of intermolecular hydrogen bonding.

Table 3. In vitro inhibition of the aromatase and STS activity (nm) in JEG-3 cells by chiral sulfamates and aro-

matase inhibitory activity of their parent phenols.

trorotatory for 18b, and the

reason for this is currently un-

clear.

For the parent phenols, both

17a and 17b are more potent

AIs than their corresponding sul-

famates, which is in accordance

with the trend previously de-

scribed. There is a 36-fold differ-

ence in aromatase inhibition for

the two enantiomers, with the

best AI being S-(+)-17b. Signifi-

cantly, the best aromatase inhibi-

tory activity is obtained with the

S-(+)-enantiomer of both the

phenol and sulfamate providing

further confirmation that this is

the optimal three-dimensional

relationship between the triazole

ring and the para-cyanophenyl

ring for potent inhibition.

Compd

Letrozole

STX64

AR: 0.89ꢀ0.13^[a]

–

STS: 1.5ꢀ0.3^[b]

S-(ꢁ)-41a:

AR: 3.4ꢀ0.6^[c]

R-(+)-41b:

AR: 0.6ꢀ0.1^[c]

S-(ꢁ)-2a:

AR: 14.3ꢀ2^[c]

R-(+)-2b:

AR: 3.2ꢀ0.3^[c]

STS: 553ꢀ50^[c]

STS: 4633ꢀ551^[c]

R-(ꢁ)-17a:

AR: 4.0ꢀ0.06

S-(+)-17b:

AR: 0.11ꢀ0.01

R-(ꢁ)-18a:

AR: 30.83ꢀ2.75

STS: 1150ꢀ50

S-(+)-18b:

AR: 0.52ꢀ0.10

STS: 280ꢀ20

[a] Data taken from Wood et al.^[19][b] Data taken from Woo et al.^[21][c] Data taken from Wood et al.^[21]Mean IC₅₀

values ꢀSD were determined from incubations carried out in triplicate in a minimum of two separate experi-

ments.

Molecular modelling

In order to examine the possible

proving to be the most active (S: IC₅₀=3.2 nm vs R: IC₅₀=

680 nm). Our previous study on the enantiomers of 2 revealed

a more modest fourfold difference in aromatase inhibition for

interaction of 18a and 18b with amino acid residues within

the active site, these molecules were docked into the human

aromatase crystal structure (PDB: 3EQM)^[50]along with the nat-

ural substrate of the enzyme, androstenedione. For the first

time, we also report the result of the docking of letrozole into

Arom

50

Arom

=3.2 nm),

50

the enantiomers (2a: IC

=14.3 nm vs 2b: IC

whilst for STS inhibition there was a ninefold difference be-

ChemMedChem 2011, 6, 1423 – 1438

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www.chemmedchem.org

1429

B. V. L. Potter et al.

MED

the active site of aromatase. We previously verified^[24]the suita-

bility of the crystal structure for use in docking studies by re-

moving the cocrystallised androstenedione from the substrate

binding site and using the docking program GOLD^[51]to dock

the steroid back in. The results of this experiment indicated

that the best pose of the docked androstenedione overlays

the crystal structure very well.

form a hydrogen-bond interaction with Met374. Hydrogen-

bond interactions with either a benzonitrile moiety or another

group placed at a suitable position relative to the triazole/imi-

dazole ring are known to be important for potent aromatase

inhibitory activity in nonsteroidal AIs.^[39–40]The other benzoni-

trile group present in letrozole is able to interact with Ser478

(2.31 ꢁ).

The results of the docking of letrozole and androstenedione

into the aromatase active site are shown in Figure 2. The 17-

keto oxygen atom in androstenedione is able to form a hydro-

gen bond with the backbone amide of Met374 (2.75 ꢁ), and

this interaction is mimicked by one of the benzonitrile groups

present in letrozole (bond distance=3.11 ꢁ). Additionally, for

the AI YM511, we recently reported^[24]that the predicted dock-

ing conformation of this compound places the benzonitrile

group in a similar position in the active site, and this is able to

The docking of 18a and 18b into the aromatase active site

is shown in Figure 2. Both 18a and 18b overlay androstene-

dione with their sulfamates positioned close to the 17-keto

group in androstenedione. In a manner similar to letrozole, the

benzonitrile groups of both 18a and 18b are able to form hy-

drogen-bond interactions with Ser478 (3.54 ꢁ and 2.27 ꢁ, re-

spectively), and for 18b, there is an additional interaction with

His480. There is no obvious structural explanation for the dif-

ference in aromatase inhibitory activity observed for the two

enantiomers. To date, no human aromatase crystal structure

complexed with a nonsteroidal AI has been reported. It might

be more relevant and informative to dock 18a and 18b into

such a crystal structure when it becomes available in the

future.

The docking of STX64, 18a and 18b (two poses) into the

crystal structure of STS (PDB: 1P49^[52]) is shown in Figure 3.

Compounds 18a and 18b dock in a mode that places the sul-

famate group in a satisfactory position for interaction with the

gem-diol form of formylglycine residue 75; this is the first step

in the interaction thought to be necessary for irreversible inac-

tivation of the enzyme. In addition, the sulfamate group is pre-

dicted to be able to form favourable interactions with His290

and Thr165 in the active site and for 18b with Lys368. Like

STX64, the remainder of the skeleton of 18a and 18b occupies

the hydrophobic tunnel leading to the active site. The benzoni-

trile groups of both 18a and 18b can form an interaction with

Arg98, and for 18a and one pose of 18b, interaction of their

triazole ring with Gly100 is possible, whilst for the other pose

of 18b, interaction of its triazole ring with His485 may occur.

Conclusions

A range of DASIs structurally similar to the potent clinical AI le-

trozole and one compound similar to vorozole were synthes-

ised and evaluated for aromatase and sulfatase inhibitory activ-

ity in JEG-3 cells. In order to realise molecules capable of dual

inhibition, the known pharmacophore for STS inhibition (a

phenol sulfamate ester) and the pharmacophore for aromatase

inhibition (an N-containing heterocyclic ring) were incorporat-

ed into a single molecule.

For racemic compounds, the most potent AI identified is 29

Arom

50

STS

50

(IC

=0.12 nm), while the most potent inhibitor of STS is 40

(IC =180 nm). Consideration of the developing SAR for these

derivatives reveals that extending the linker between the aro-

matase and STS pharmacophores is beneficial for aromatase in-

hibition, but this is balanced by the detrimental effects on STS

inhibition resulting from extension of the linker beyond two

carbon atoms. As anticipated, the addition of a halogen in the

ortho position to the sulfamate group results in an increase in

both aromatase and STS inhibitory activity. Compounds capa-

Figure 2. a) The docking of androstenedione and letrozole into the human

aromatase crystal structure. Androstendione (cyan) is in the dark green pro-

tein and letrozole (pink) is in the light green protein. b) The docking of 18a

and 18b into the aromatase active site. 18a (cyan) is in the light green pro-

tein and 18b (pink) is in the dark green protein. In both figures the haeme

group is in purple with the iron represented by the orange sphere and pos-

sible hydrogen bonds are shown by dotted lines.

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ChemMedChem 2011, 6, 1423 – 1438

Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

Figure 3. a) The docking of STX64 (cyan) and 18a (buff) into the crystal structure of human STS. The Ca²⁺ion is depicted as a yellow sphere and FG75 is the

gem-diol form of formylglycine residue 75. b) The docking of 18b (two poses: buff and cyan).

ble of potent aromatase and STS inhibition can be obtained

following exchanging the para-cyano group for a para-chloro

substituent, suggesting that this group is able to replicate in-

teractions within the enzyme active site. Compound 51, the

first sulfamate-containing vorozole derivative, exhibits good in-

hibitory activity against aromatase meriting further investiga-

tion. Even with an IC₅₀value in the micromolar range for STS

inhibition, based on established precedent, it is likely that this

compound will be effective in vivo on both enzymes.

the sulfamate group is predicted to occupy this same area of

space with their benzonitrile group able to form hydrogen

bonds with Ser478. The molecular modelling study suggests

no obvious structural explanation for the difference in aroma-

tase inhibitory activity of 18a and 18b. For STS, both 18a and

18b dock in a similar orientation to that of STX64.

These results further demonstrate the feasibility of designing

a DASI based on the letrozole or vorozole templates and pro-

vide a basis for continuing pre-clinical development of such

compounds for the treatment of HDBC using a multitargeted

strategy.

The enantiomers of 17, the phenolic precursor of 18, one of

the most potent dual inhibitors in the current study, were sep-

arated by chiral HPLC and the absolute configuration of one

enantiomer was unambiguously established using X-ray crys-

tallography. Following conversion to the corresponding sulfa-

mate and biological evaluation, it was established that there is

a 60-fold difference in aromatase inhibition, with S-(+)-18b

being the most potent. This enantiomer has the same spatial

disposition of the heterocycle and para-cyano-substituted ring

as that found in R-(+)-2b, the most potent enantiomer discov-

ered in our previous study. For STS inhibition, there is a four-

fold difference in inhibition with the most potent enantiomer

also being S-(+)-18b.

Experimental Section

In vitro aromatase and sulfatase assays: Biological assays were

performed essentially as described previously.^[22]The extent of in

vitro inhibition of aromatase and sulfatase activities was assessed

using intact monolayers of JEG-3 human choriocarcinoma cells,

which were chosen because these cells constitutively express both

enzymes maximally. Aromatase activity was measured using [1b-

³H]androstenedione (30 Cimmol^ꢁ1, PerkinElmer Life Sciences, Wal-

tham, MA, USA) over a 1 h period. Sulfatase activity was measured

using [6,7-³H]E1S (50 Cimmol^ꢁ1, PerkinElmer Life Sciences) over a

1 h period. Each compound was tested in replicate incubations

(n=3) using a minimum of six concentrations of the inhibitor (0.1–

10000 nm range). Mean IC₅₀values ꢀSD from two independent

experiments are shown.

Molecular modelling studies indicate that both YM511 and

letrozole dock into the human aromatase crystal structure with

a benzonitrile group occupying a similar area of space to the

17-keto oxygen atom of androstenedione. For 18a and 18b,

ChemMedChem 2011, 6, 1423 – 1438

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1431

B. V. L. Potter et al.

MED

Molecular modelling: Models of androstenedione, letrozole,

STX64, 18a and 18b were built and minimised using the Schroe-

dinger software running under Maestro version 9.0. The GOLD

docking program (version 5.0)^[51]was used to dock the models into

the aromatase crystal structure (PDB: 3EQM).^[50]The binding site

was defined as a 10 ꢁ sphere around the androstenedione that is

present in the crystal structure. A distance constraint of 2.30 ꢁ was

applied between the ligating triazole nitrogen atom of the ligand

to the haeme iron atom. The ligands were then docked to the

rigid enzyme a total of 25 times each and scored using the GOLD-

Score fitness function. To remove strain from the docked poses,

the systems were put through an energy minimisation procedure

using the Impact module of the Schroedinger software.

cal ionisation (APCI) or electrospray ionisation (ESI). A Waters “Sym-

metry” C18 column (packing: 3.5 mm, 4.6ꢂ100 mm) and gradient

elution were used (MeCN/H₂O, 5:95 at 0.5 mLmin^ꢁ1!95:5 at

1 mLmin^ꢁ1over 10 min). HPLC was undertaken using a Waters 717

machine with an autosampler and PDA detector. The column used

was either a Waters “Symmetry” C18 (packing: 3.5 mm, 4.6ꢂ

150 mm) or a Waters “Sunfire” C18 (packing: 3.5 mm, 4.6ꢂ150 mm)

with an isocratic mobile phase consisting of CH₃CN/H₂O (as indicat-

ed) with a flow rate of 1 mLmin^ꢁ1. Analytical chiral HPLC was per-

formed on a Chiralpak AD-H column (250ꢂ4.6 mm, 5 mm) with

MeOH as the mobile phase, a flow rate of 1.2 mLmin^ꢁ1and a PDA

detector. Semi-preparative HPLC was performed with a Waters

2525 binary gradient module and a Chiralpak AD-H (250ꢂ20 mm)

semi-prep column with MeOH as the mobile phase at a flow rate

of 10 mLmin^ꢁ1, injecting 1.5–2.0 mL of a 20 mgmL^ꢁ1solution and

a run time of 25 min.

The crystal structure of human placental oestrone/DHEA sulfatase

(PDB: 1P49^[52]) was used for building the gem-diol form of steroid

sulfatase (STS). 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 the Schroedinger software. The resulting struc-

ture was then minimised with the backbone atoms fixed to allow

the gem-diol and surrounding side chain atoms to adopt low

energy confirmations. GOLD was used to dock the ligands 25

times each into the rigid protein. The docked poses were scored

using the GOLDScore fitness function.

Method A: Condensation of carbinols with triazole: Substrate,

1,2,4-triazole and para-toluenesulfonic acid (p-TsOH), dissolved/sus-

pended in toluene were heated at reflux with a Dean–Stark separa-

tor for 24 h. The reaction mixture was allowed to cool, and the sol-

vent was removed in vacuo.

Method B: Hydrogenation: Pd/C (10%) was added to a solution

of substrate in THF/MeOH (1:1). The solution was stirred overnight

under an H₂atmosphere (maintained using a balloon). Excess H₂

was removed, and the reaction mixture was filtered through Celite,

washing with THF and MeOH, and then the solvent was removed

in vacuo.

Crystallographic data: CCDC 806541 (17a) contains the supple-

mentary crystallographic data for this paper. These data can be ob-

tained free of charge from The Cambridge Crystallographic Data

Centre via http://www.ccdc.cam.ac.uk.

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-dimethylfor-

mamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofuran

(THF) were purchased from Aldrich. Sulfamoyl chloride was pre-

pared by an adaptation of the method of Appel and Berger^[53]and

was stored under N₂as a solution in toluene as described by Woo

et al.^[54]

Method C: Sulfamoylation: A solution of sulfamoyl chloride

(H₂NSO₂Cl) in toluene was concentrated in vacuo at 308C to furnish

a yellow oil, which solidified upon cooling in an ice bath. DMA and

substrate were subsequently added and the mixture was allowed

to warm to RT and stirred overnight. The reaction mixture was

poured into H₂O and extracted with EtOAc (2ꢂ). The organic layers

were combined, washed with H₂O (4ꢂ) and brine, dried (MgSO₄),

filtered, and the solvent was removed in vacuo.

Method D: Removal of the TIPS protecting group: Tetra-n-buty-

lammonium fluoride (TBAF; 1m in THF) was added to a solution of

substrate in THF. After stirring for 15 min, H₂O was added, and the

solution was treated with AcOH (3m) until colourless. The product

was extracted with EtOAc, and the combined organic layers were

washed with satd aq NaHCO₃and brine, then dried (MgSO₄), fil-

tered and concentrated in vacuo.

Thin layer chromatography (TLC) was performed on pre-coated

aluminium plates (Merck, silica gel 60 F₂₅₄). Product spots were vi-

sualised either by UV irradiation at 254 nm or by staining with

either alkaline KMnO₄solution or 5% w/v dodecamolybdophos-

phoric acid in EtOH, followed by heating. Flash column chromatog-

raphy was performed using gradient elution on either pre-packed

columns (Isolute) on a Flashmaster II system (Biotage) or on a Tele-

dyne ISCO CombiFlash R_fautomated flash chromatography system

(3-Bromo-4-(triisopropylsilyloxy)phenyl)(4-chlorophenyl)metha-

nol (8): 4-ClPhMgBr (1m in Et₂O, 17.0 mL) was added dropwise to a

solution of 7^[20](2.00 g, 5.60 mmol) in THF (20 mL). After stirring for

2 h, the reaction was quenched by addition of H₂O (50 mL) and

1m aq HCl (50 mL). EtOAc (75 mL) was added, the layers were sep-

arated, and the aqueous layer was further extracted with EtOAc

(75 mL). The combined organic layers were washed with H₂O

(150 mL), satd aq NaHCO₃(150 mL) and brine (150 mL), then dried

(MgSO₄), filtered and concentrated in vacuo. Purification using a

Flashmaster II (EtOAc/hexane) gave 8 as a colourless oil (2.20 g,

84%): ¹H NMR (270 MHz, CDCl₃): d=1.08–1.38 (21H, m, 6CH₃,

3CH), 2.33 (1H, d, J=3.5 Hz, OH), 5.69 (1H, d, J=3.5 Hz, CH), 6.82

(1H, d, J=8.4 Hz, ArH), 7.07 (1H, dd, J=8.4, 2.0 Hz, ArH), 7.22–7.33

(4H, m, ArH), 7.48 ppm (1H, d, J=2.0 Hz, ArH); ¹³C NMR (100 MHz,

[D₆]DMSO): d=12.9 (3CH), 17.9 (6CH₃), 74.6 (CH), 115.1 (C), 119.4

(CH), 126.5 (CH), 127.8 (2CH), 128.6 (2CH), 131.6 (CH), 133.4 (C),

137.1 (C), 141.8 (C), 152.5 ppm (C); LC/MS (APCIꢁ): t_R=3.11 min, m/

z (%): 469.0 (1) [MꢁH]^ꢁ, 310.8 (100).

1

with RediSep R_fdisposable flash columns. H and ¹³C NMR spectra

were recorded on either a Jeol Delta 270 MHz or a Varian Mercury

VX 400 MHz spectrometer. Chemical shifts (d) are reported in parts

per million (ppm) 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 Ser-

vice Centre, University of Bath (UK). Fast atom bombardment (FAB)

mass spectra were measured using m-nitrobenzyl alcohol as the

matrix. Elemental analyses were performed by the Microanalysis

Service, University of Bath (UK). Melting points (mp) were deter-

mined 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

using 5 cm cells.

LC/MS was performed using a Waters 2790 machine with a ZQ Mi-

croMass spectrometer and photodiode array (PDA) detector. The

ionisation technique used was either atmospheric pressure chemi-

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ChemMedChem 2011, 6, 1423 – 1438

Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

2-Bromo-4-((4-chlorophenyl)(hydroxy)methyl)phenol

(9):

As

128.2 (2CH), 129.9 (2CH), 132.5 (2CH), 144.1 (CH), 145.0 (C), 151.7

(CH), 156.0 ppm (C); LC/MS (ESI+): t_R=1.31 min, m/z (%): 291.1

(100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for C₁₇H₁₅N₄O:

291.1240, found: 291.1234; Anal. calcd for C₁₇H₁₄N₄O: C 70.33, H

4.86, N 19.30, found: C 70.30, H 4.88, N 19.20.

method D using TBAF (9.4 mL), 8 (2.15 g, 4.59 mmol) and THF

(20 mL). Purification using a Flashmaster II (EtOAc/hexane) gave 9

as

a

cream solid (1.32 g, 92%): mp: 125.5–1288C; ¹H NMR

(270 MHz, [D₆]DMSO): d=5.61 (1H, d, J=4.2 Hz, CH), 5.94 (1H, d,

J=4.2 Hz, OH), 6.87 (1H, d, J=8.2 Hz, ArH), 7.11 (1H, dd, J=8.2,

2.0 Hz, ArH), 7.34–7.39 (4H, m, ArH), 7.42 (1H, d, J=2.0 Hz, ArH),

10.15 ppm (1H, s, OH); ¹³C NMR (68 MHz, [D₆]DMSO): d=73.0 (CH),

109.4 (C), 116.6 (CH), 127.3 (CH), 128.5 (2CH), 128.6 (2CH), 131.1

(CH), 131.8 (C), 138.2 (C), 145.2 (C), 153.5 ppm (C); LC/MS (APCIꢁ):

t_R=0.89 min, m/z (%): 311.0 (80) [MꢁH]^ꢁ; HRMS-ESI: m/z [MꢁH]^ꢁ

calcd for C₁₃H₉BrClO₂: 310.9480, found: 310.9469.

4-(2-(4-Cyanophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phenyl sulfa-

mate (14): As method C using ClSO₂NH₂(0.57m, 6.8 mL), DMA

(1 mL) and a solution of 13 (0.19 g, 0.66 mmol) in DMA (1 mL). Pu-

rification using a Combiflash R_f(CHCl₃/acetone) gave 14 as a white

foam (0.19 g, 88%): ¹H NMR (270 MHz, [D₆]DMSO): d=3.49 (1H,

dd, J=14.0, 5.5 Hz, CHH), 3.68 (1H, dd, J=14.0, 9.9 Hz, CHH), 6.08

(1H, dd, J=14.0, 9.9 Hz, CH), 7.12 (2H, AA’BB’, ArH), 7.25 (2H,

AA’BB’, ArH), 7.64 (2H, AA’BB’, ArH), 7.86 (2H, AA’BB’, ArH), 7.96

(2H, s, NH₂), 8.02 (1H, s, NCHN), 8.62 ppm (1H, s, NCHN); ¹³C NMR

(100 MHz, [D₆]DMSO): d=39.1 (CH₂), 63.1 (CH), 111.0 (C), 118.5 (C),

121.8 (2CH), 128.2 (2CH), 130.2 (2CH), 132.6 (2CH), 135.3 (C), 144.1

(C), 144.8 (CH), 148.9 (C), 151.9 ppm (CH); LC/MS (ESI+): t_R=

1.27 min, m/z (%): 370.1 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺

calcd for C₁₇H₁₆N₅O₃S: 370.0968, found: 370.0958.

2-Bromo-4-((4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl)phenol

(10): As method A using

9 (0.80 g, 2.55 mmol), 1,2,4-triazole

(1.32 g, 19.1 mmol), p-TsOH (0.11 g) and toluene (200 mL). The resi-

due was dissolved in EtOAc/H₂O (1:1, 300 mL), and the organic

layer was washed with H₂O (3ꢂ100 mL) and brine (150 mL), then

dried (MgSO₄), filtered and concetrated in vacuo. Purification using

a Flashmaster II (EtOAc/hexane) gave 10 as an oil (0.87 g, 96%);

white crystals were obtained by slow evaporation of the solvent

a

concentrated EtOAc solution: mp: 95–988C; ¹H NMR

(3-Bromo-4-(triisopropylsilyloxy)phenyl)methanol (15): A solution

of NaBH₄(0.19 g, 5.00 mmol) in H₂O (2 mL) was added dropwise to

a solution of 7 (0.70 g, 1.96 mmol) in EtOH (6 mL). After stirring for

15 min, the reaction was quenched by cautious addition of aq HCl

(3m, 15 mL), EtOAc (15 mL) and H₂O (15 mL). The layers were sepa-

rated, and the aqueous was extracted with EtOAc (30 mL). The

combined organic layers were washed with H₂O (2ꢂ50 mL) and

satd aq NaHCO₃(50 mL), then dried (MgSO₄), filtered and concen-

trated in vacuo. Purification using a Flashmaster II (EtOAc/hexane)

gave 15 as a colourless oil (0.57 g, 81%): ¹H NMR (270 MHz,

CDCl₃): d=1.10–1.37 (21H, m, 6CH₃, 3CH), 1.58 (1H, t, J=5.7 Hz,

OH), 4.57 (1H, d, J=5.7 Hz, CH₂), 6.85 (1H, d, J=8.2 Hz, ArH), 7.13

(1H, dd, J=8.2, 2.2 Hz, ArH), 7.52 ppm (1H, d, J=2.2 Hz, ArH);

¹³C NMR (100 MHz, CDCl₃): d=12.9 (3CH), 18.0 (6CH₃), 64.4 (CH₂),

115.0 (C), 119.5 (CH), 127.1 (CH), 132.2 (CH), 134.6 (C), 152.5 ppm

(C); LC/MS (ESI+): t_R=2.02 min, m/z (%): 341.2 (100) [MꢁH₂O]⁺.

from

(270 MHz, [D₆]DMSO): d=6.94 (1H, d, J=8.4 Hz, ArH), 7.03 (1H, s,

ArH), 7.08 (1H, dd, J=8.4, 2.2 Hz, ArH), 7.18 (2H, AA’BB’, ArH), 7.37

(1H, d, J=2.2 Hz, ArH), 7.44 (2H, AA’BB’, ArH), 8.08 (1H, s, NCHN),

8.60 (1H, s, NCHN), 10.51 ppm (1H, s, OH); ¹³C NMR (100 MHz,

[D₆]DMSO): d=63.6 (CH), 109.2 (C), 116.4 (CH), 128.7 (2CH), 128.8

(CH), 129.6 (2CH), 130.6 (C), 132.6 (CH), 132.7 (C), 138.2 (C), 144.5

(CH), 152.1 (CH), 154.1 ppm (C); LC/MS (ESIꢁ): t_R=0.85 min, m/z

(%): 362.1 (95) [MꢁH]^ꢁ; HRMS-ESI: m/z [MꢁH]^ꢁcalcd for

C₁₅H₁₀BrClN₃O: 361.9690, found: 361.9700.

2-Bromo-4-((4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl)phenyl

sulfamate (11): As method C using ClSO₂NH₂(0.6m, 3.0 mL), DMA

(3 mL) and 10 (0.15 g, 0.42 mmol). Purification using a Flashmaster

1

II (CH₂Cl₂/acetone) gave 11 as a white solid (0.15 g, 82%): H NMR

(270 MHz, [D₆]DMSO): d=7.20 (1H, s, CH), 7.26 (2H, AA’BB’, ArH),

7.35 (1H, dd, J=8.4, 2.2 Hz, ArH), 7.44–7.53 (3H, m, ArH), 7.61 (1H,

d, J=2.2 Hz, ArH), 8.12 (1H, s, NCHN), 8.32 (2H, s, NH₂), 8.67 ppm

(1H, s, NCHN); ¹³C NMR (68 MHz, [D₆]DMSO): d=63.9 (CH), 116.6

(C), 124.2 (CH), 129.4 (C), 129.5 (2CH), 130.4 (CH), 130.5 (2CH), 133.6

(C), 137.9 (C), 139.0 (C), 145.4 (CH), 147.6 (C), 152.9 ppm (CH); LC/

MS (ESIꢁ): t_R=0.89 min, m/z (%): 443.2 (20) [MꢁH]^ꢁ; HRMS-ESI: m/z

[MꢁH]^ꢁcalcd for C₁₅H₁₁BrClN₄O₃S: 440.9429, found: 440.9421.

(2-Bromo-4-(chloromethyl)phenoxy)triisopropylsilane (16): SOCl₂

(0.98 g, 7.10 mmol) was added dropwise to a solution of 15

(0.51 g, 1.42 mmol) in CH₂Cl₂(8 mL). After 1 h, the solvent was re-

moved in vacuo, and the residue was redissolved in CH₂Cl₂(15 mL)

and evaporated; this process was repeated twice more to give 16

as a white solid (0.54 g, 100%): ¹H NMR (270 MHz, CDCl₃): d=

1.10–1.38 (21H, m, 6CH₃, 3CH), 4.49 (2H, s, CH₂), 6.83 (1H, d, J=

8.4 Hz, ArH), 7.15 (1H, dd, J=8.4, 2.5 Hz, ArH), 7.53 ppm (1H, d, J=

2.5 Hz, ArH); ¹³C NMR (68 MHz, CDCl₃): d=13.0 (3CH), 18.0 (6CH₃),

45.4 (CH₂), 115.1 (C), 119.6 (CH), 128.7 (CH), 131.2 (C), 133.8 (CH),

153.2 ppm (C); LC/MS (ESI+): t_R=3.30 min, m/z (%): 341.2 (40)

[MꢁCl]⁺, 236 (90), 185 (100).

4-(2-(4-Hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)ethyl)benzonitrile

(13): nBuLi (1.95m, 3.00 mL) was added dropwise to a cooled

(ꢁ788C) solution of 4-((1H-1,2,4-triazol-1-yl)methyl)benzonitrile^[19]

(0.95 g, 5.16 mmol) in THF (50 mL). After 30 min, a solution of 12^[30]

(2.30 g, 6.71 mmol) in THF (25 mL) was added, and the mixture

was stirred for 30 min. The reaction mixture was allowed to warm

to RT and stirred for 4 h. The reaction was quenched by cautious

addition of satd aq NH₄Cl. The layers were separated and the or-

ganic was dried (MgSO₄), filtered and concentrated in vacuo. The

crude intermediate was purified using a CombiFlash R_f(EtOAc/PE)

and used without further purification. Deprotection was achieved

using method D with TBAF (2.5 mL) and a solution of the inter-

mediate in THF (10 mL). Purification using a Combiflash R_f(EtOAc/

PE) gave 13 as a white solid (0.35 g, 23%): mp: 221.5–223.58C;

¹H NMR (270 MHz, [D₆]DMSO): d=3.32 (1H, dd, J=14.0, 5.8 Hz,

CHH), 3.51 (1H, dd, J=14.0, 9.6 Hz, CHH), 5.93 (1H, dd, J=9.6,

5.8 Hz, CH), 6.58 (2H, AA’BB’, ArH), 6.92 (2H, AA’BB’, ArH), 7.61 (2H,

AA’BB’, ArH), 7.83 (2H, AA’BB’, ArH), 8.00 (1H, s, NCHN), 8.56 (1H, s,

NCHN), 9.24 ppm (1H, s, OH); ¹³C NMR (100 MHz, [D₆]DMSO): d=

39.3 (CH₂), 63.7 (CH), 110.8 (C), 115.1 (2CH), 118.6 (C), 126.9 (C),

4-(2-(3-Bromo-4-hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)ethyl)-

benzonitrile (17): nBuLi (2.12m, 3.80 mL) was added dropwise to a

cooled (ꢁ788C) solution of 4-((1H-1,2,4-triazol-1-yl)methyl)benzoni-

trile^[19](1.68 g, 9.13 mmol) in THF (50 mL). After 30 min, a solution

of 16 (3.79 g, 10.0 mmol) in THF (10 mL) was added, and the mix-

ture was stirred for 1 h. The reaction mixture was allowed to warm

to RT and stirred for 2 h. The reaction was quenched by cautious

addition of satd aq NH₄Cl. The layers were separated, and the or-

ganic layer was dried (MgSO₄), filtered and concentrated in vacuo.

The crude intermediate was purified using a Flashmaster II (EtOAc/

hexane) and used without further purification. Deprotection was

achieved using method D with TBAF (7.6 mL) and a solution of the

intermediate in THF (30 mL). Purification using a Flashmaster II

(EtOAc/PE) gave 17 as a white solid (2.04 g, 81%): mp: 186–

ChemMedChem 2011, 6, 1423 – 1438

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1433

B. V. L. Potter et al.

MED

188.58C; ¹H NMR (270 MHz, [D₆]DMSO): d=3.30–3.39 (1H, m,

CHH), 3.51 (1H, dd, J=13.9, 9.9 Hz, CHH), 5.98 (1H, dd, J=9.9,

5.7 Hz, CH), 6.74 (1H, d, J=8.2 Hz, ArH), 6.88 (1H, dd, J=8.2,

2.0 Hz, ArH), 7.35 (1H, J=2.0 Hz, ArH), 7.62 (2H, AA’BB’, ArH), 7.84

(2H, AA’BB’, ArH), 8.02 (1H, s, NCHN), 8.59 (1H, s, NCHN),

10.07 ppm (1H, br s, OH); ¹³C NMR (100 MHz, [D₆]DMSO): d=39.1

(CH₂), 63.8 (CH), 109.5 (C), 111.3 (C), 116.5 (CH), 119.0 (C), 128.7

(2CH), 129.4 (C), 129.7 (CH), 133.0 (2CH), 133.6 (CH), 144.6 (CH),

145.3 (C), 152.3 (CH), 153.2 ppm (C); LC/MS (ESI+): t_R=1.39 min, m/

z (%): 369.0 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for

C₁₇H₁₄BrN₄O: 369.0346, found: 369.0334.

(3 mL) and 21 (0.14 g, 0.46 mmol). Purification using a CombiFlash

R_f(CH₂Cl₂/acetone) gave 22 as a white foam (0.16 g, 91%): H NMR

1

(270 MHz, [D₆]DMSO): d=2.42–2.78 (4H, m, CH₂CH₂), 5.71–5.75

(1H, m, CH), 7.18 (2H, AA’BB’, ArH), 7.25 (2H, AA’BB’, ArH), 7.57

(2H, AA’BB’, ArH), 7.85 (2H, AA’BB’, ArH), 7.94 (2H, br s, NH₂), 8.08

(1H, s, NCHN), 8.79 ppm (1H, s, NCHN); ¹³C NMR (100 MHz,

[D₆]DMSO): d=31.1 (CH₂), 35.4 (CH₂), 61.8 (CH), 110.9 (C), 118.5 (C),

122.1 (2CH), 127.9 (2CH), 129.5 (2CH), 132.7 (2CH), 138.8 (C), 144.2

(CH), 145.2 (C), 148.5 (C), 152.0 ppm (CH); LC/MS (ESI+): t_R=

1.26 min, m/z (%): 384.3 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺

calcd for C₁₈H₁₈N₅O₃S: 384.1108, found: 384.1125.

2-Bromo-4-(2-(4-cyanophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)-

phenyl sulfamate (18): As method C using ClSO₂NH₂(0.6m,

4.0 mL), DMA (2 mL) and 17 (0.15 g, 0.41 mmol). Purification using

a Flashmaster II (CH₂Cl₂/acetone) gave 18 as a white solid (0.16 g,

88%): ¹H NMR (270 MHz, [D₆]DMSO): d=3.49 (1H, dd, J=14.0,

5.5 Hz, CHH), 3.67 (1H, dd, J=14.0, 10.2 Hz, CHH), 6.12 (1H, dd, J=

10.2, 5.5 Hz, CH), 7.21 (1H, dd, J=8.6, 1.9 Hz, ArH), 7.32 (1H, d, J=

8.6 Hz, ArH), 7.62–7.68 (3H, m, ArH), 7.86 (2H, AA’BB’, ArH), 8.04

(1H, s, NCHN), 8.23 (2H, s, NH₂), 8.64 ppm (1H, s, NCHN); ¹³C NMR

(100 MHz, [D₆]DMSO): d=38.6 (CH₂), 62.8 (CH), 111.0 (C), 115.5 (C),

118.4 (C), 122.9 (CH), 128.2 (2CH), 129.5 (C), 132.7 (2CH), 133.9 (CH),

137.1 (C), 144.3 (CH), 144.7 (C), 146.1 (C), 152.0 ppm (CH); LC/MS

(ESIꢁ): t_R=0.78 min, m/z (%): 446.1 (100) [MꢁH]^ꢁ; HRMS-ESI: m/z

[M+H]⁺calcd for C₁₇H₁₅BrN₅O₃S: 448.0073, found: 448.0074.

Methyl 2-(3-bromo-4-(triisopropylsilyloxy)phenyl)acetate (24):

Imidazole (8.77 g, 0.13 mol) and triisopropylsilyl chloride (TIPSCl;

14.32 g, 74.2 mmol) were sequentially added to a pale yellow solu-

tion of 23^[23](15.80 g, 64.5 mmol) in DMF (50 mL). The reaction

mixture was stirred overnight, then poured into H₂O (100 mL) and

extracted with EtOAc (3ꢂ50 mL). The combined organic extracts

were washed with H₂O (3ꢂ60 mL) and brine (60 mL), then dried

(MgSO₄), filtered and concentrated in vacuo. Purification using a

CombiFlash R_f(EtOAc/PE) gave 24 as a colourless oil (22.77 g,

88%): ¹H NMR (270 MHz, CDCl₃): d=1.07–1.37 (21H, m, 6CH₃,

3CH), 3.51 (2H, s, CH₂), 3.68 (3H, s, CH₃), 6.81 (1H, d, J=8.3 Hz,

ArH), 7.04 (1H, dd, J=8.3, 2.2 Hz, ArH), 7.42 ppm (1H, d, J=2.2 Hz,

ArH); ¹³C NMR (100 MHz, CDCl₃): d=12.9 (3CH), 18.0 (6CH₃), 39.8

(CH₂), 52.1 (CH₃), 114.9 (C), 119.4 (CH), 129.5 (C), 129.0 (CH), 134.0

(CH), 152.1 (C), 171.8 ppm (C).

4-(1-(1H-1,2,4-Triazol-1-yl)-3-(4-(triisopropylsilyloxy)phenyl)pro-

pyl)benzonitrile (20): nBuLi (1.75m, 0.55 mL) was added dropwise

to a cooled (ꢁ788C) solution of 4-((1H-1,2,4-triazol-1-yl)methyl)ben-

zonitrile^[19](0.16 g, 0.87 mmol) in THF (4 mL). After 30 min, a solu-

tion of 19^[33](0.40 g, 1.12 mmol) in THF (1 mL) was added, and the

mixture was stirred for 2 h. The reaction mixture was allowed to

warm to RT and stirred for 1 h. The reaction was quenched by cau-

tious addition of satd aq NH₄Cl. The layers were separated, and the

organic layer was dried (MgSO₄), filtered and concentrated in

vacuo. Purification using a Flashmaster II (EtOAc/hexane) gave 20

as a colourless oil (0.22 g, 55%): ¹H NMR (270 MHz, CDCl₃): d=

1.04–1.32 (21H, m, 6CH₃, 3CH), 2.30–2.84 (4H, m, CH₂CH₂), 5.23

(1H, dd, J=9.4, 4.4 Hz, CH), 6.80 (2H, AA’BB’, ArH), 6.91 (2H,

AA’BB’, ArH), 7.37 (2H, AA’BB’, ArH), 7.62 (2H, AA’BB’, ArH), 8.02

(1H, s, NCHN), 8.05 ppm (1H, s, NCHN); ¹³C NMR (100 MHz, CDCl₃):

d=12.6 (3CH), 17.9 (6CH₃), 31.2 (CH₂), 36.5 (CH₂), 62.4 (CH), 112.4

(C), 118.2 (C), 120.2 (2CH), 127.6 (2CH), 129.2 (2CH), 131.7 (C), 132.7

(2CH), 143.1 (CH), 144.3(C), 152.5 (CH), 154.7 ppm (C); LC/MS

(ESI+): t_R=5.11 min, m/z (%): 461.2 (80) [M+H]⁺, 263.0 (100);

HRMS-ESI: m/z [M+H]⁺calcd for C₂₇H₃₇N₄OSi: 461.2731, found:

461.2711.

2-(3-Bromo-4-(triisopropylsilyloxy)phenyl)ethanol (25): LiBH₄

(3.04 g, 0.14 mol) and B(OMe)₃(0.57 g, 5.84 mmol) were sequential-

ly added to a stirred solution of 24 (22.10 g, 55.1 mmol) in Et₂O

(150 mL). The reaction mixture was stirred for 48 h and then dilut-

ed with Et₂O (150 mL) and quenched by cautious addition of H₂O

(75 mL) and HCl (3m, 75 mL). The layers were separated, and the

organic layer was washed with brine (150 mL), then dried (MgSO₄),

filtered and concentrated in vacuo. Purification using a CombiFlash

R_f(EtOAc/PE) gave 25 as a colourless oil (18.72 g, 91%): ¹H NMR

(270 MHz, CDCl₃): d=1.09–1.39 (22H, m, 3CH, 6CH_3,OH), 2.76 (2H,

t, J=6.3 Hz, CH₂), 3.80 (2H, q, J=6.3 Hz, CH₂OH), 6.80 (1H, d, J=

8.3 Hz, ArH), 6.98 (1H, dd, J=8.3, 2.2 Hz, ArH), 7.37 ppm (1H, d, J=

2.2 Hz, ArH); ¹³C NMR (100 MHz, CDCl₃): d=12.9 (3CH), 18.0 (6CH₃),

37.9 (CH₂), 63.5 (CH₂), 115.0 (C), 119.5 (CH), 128.7 (CH), 132.1 (C),

133.7 (CH), 151.5 ppm (C); LC/MS (ESI+): t_R=6.14 min, m/z (%):

373.2 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for

C₁₇H₃₀BrO₂Si: 373.1193, found: 373.1181.

(2-Bromo-4-(2-bromoethyl)phenoxy)triisopropylsilane (26): PPh₃

(5.44 g, 20.7 mmol), imidazole (1.41 g, 20.7 mmol) and Br₂(3.22 g,

20.1 mmol) were sequentially added to a solution of 25 (2.50 g,

6.70 mmol) in Et₂O/CH₃CN (55 mL:19 mL). After stirring for 3 h, the

reaction mixture was filtered, washing with Et₂O. The organic

layers were combined and washed with brine, then dried (MgSO₄),

filtered and concentrated in vacuo. Purification using a CombiFlash

R_f(EtOAc/PE) gave 26 as a colourless oil (2.58 g, 88%): ¹H NMR

(270 MHz, CDCl₃): d=1.08–1.37 (21H, m, 3CH, 6CH₃), 3.04 (2H, t,

J=7.7 Hz, CH₂), 3.50 (2H, t, J=7.7 Hz, CH₂), 6.81 (1H, d, J=8.3 Hz,

ArH), 6.96 (1H, dd, J=8.3, 2.2 Hz, ArH), 7.35 ppm (1H, d, J=2.2 Hz,

ArH).

4-(3-(4-Hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)propyl)benzoni-

trile (21): As method D using TBAF (0.42 mL), 20 (0.18 g,

0.39 mmol) and THF (3 mL). Purification using a Flashmaster II

(EtOAc/hexane) gave 21 as a white foam (0.11 g, 95%): ¹H NMR

(270 MHz, CDCl₃): d=2.26–2.82 (4H, m, CH₂CH₂), 5.27 (1H, dd, J=

9.6, 5.2 Hz, CH), 6.75 (2H, AA’BB’, ArH), 6.87 (2H, AA’BB’, ArH), 7.38

(2H, AA’BB’, ArH), 7.59 (2H, AA’BB’, ArH), 8.04 (1H, s, NCHN),

8.13 ppm (1H, s, NCHN); ¹³C NMR (100 MHz, [D₆]DMSO): d=30.8

(CH₂), 35.9 (CH₂), 61.7 (CH), 110.8 (C), 115.2 (2CH), 118.5 (C), 127.9

(2CH), 129.1 (2CH), 130.3 (C), 132.7 (2CH), 144.1 (CH), 145.3 (C),

151.9 (CH), 155.6 ppm (C); LC/MS (ESI+): t_R=3.54 min, m/z (%):

305.2 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for C₁₈H₁₇N₄O:

305.1397, found: 305.1389.

4-(3-(3-Bromo-4-(triisopropylsilyloxy)phenyl)-1-(1H-1,2,4-triazol-

1-yl)propyl)benzonitrile (27): nBuLi (1.75m, 2.63 mL) was added

dropwise to a cooled (ꢁ788C) solution of 4-((1H-1,2,4-triazol-1-yl)-

methyl)benzonitrile^[19](0.77 g, 4.18 mmol) in THF (25 mL). After

30 min, a solution of 26 (2.20 g, 5.05 mmol) in THF (5 mL) was

added, and the mixture was stirred for 2 h. The reaction mixture

4-(3-(4-Cyanophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl)phenyl sulfa-

mate (22): As method C using ClSO₂NH₂(0.5m, 5.0 mL), DMA

1434

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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 1423 – 1438

Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

was allowed to warm to RT and stirred for 1 h. The reaction was

quenched by cautious addition of satd aq NH₄Cl. The layers were

separated, and the organic was dried (MgSO₄), filtered and concen-

trated in vacuo. Purification using a CombiFlash R_f(EtOAc/PE) gave

27 as a yellow oil (0.91 g, 40%): ¹H NMR (270 MHz, CDCl₃): d=

1.08–1.38 (21H, m, 6CH₃, 3CH), 2.30–2.53 (3H, m, 3CHH), 2.72–2.82

(1H, m, CHH), 5.26 (1H, dd, J=9.6, 5.2 Hz, CH), 6.79–6.86 (2H, m,

ArH), 7.24–7.26 (1H, m, ArH), 7.39 (2H, AA’BB’, ArH), 7.62 (2H,

AA’BB’, ArH), 8.02 (1H, s, NCHN), 8.08 ppm (1H, s, NCHN); ¹³C NMR

(100 MHz, CDCl₃): d=12.9 (3CH), 17.9 (6CH₃), 30.9 (CH₂), 36.4 (CH₂),

62.5 (CH), 112.5 (C), 115.2 (C), 118.2 (C), 119.7 (CH), 127.6 (2CH),

128.1 (CH), 132.7 (2CH), 133.0 (CH), 133.1 (C), 143.0 (CH), 144.1 (C),

151.6 (C), 152.5 ppm (CH); LC/MS (ESI+): t_R=6.62 min, m/z (%):

539.5 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for

C₂₇H₃₆BrN₄OSi: 539.1836, found: 539.1816.

tion was quenched by addition of H₂O (10 mL) and 2m HCl

(10 mL). The product was extracted with Et₂O (2ꢂ30 mL), and the

combined organic layers were washed with brine (40 mL), then

dried (MgSO₄), filtered and concentrated in vacuo. Purification

using a CombiFlash R_f(EtOAc/PE) gave 32 as a white solid (2.09 g,

1

90%): mp: 71–738C; H NMR (270 MHz, CDCl₃): d=1.06–1.32 (21H,

m, 3CH, 6CH₃), 1.99 (1H, d, J=3.0 Hz, OH), 2.86–2.93 (2H, m, CH₂),

4.76–4.86 (1H, m, CH), 6.79 (2H, AA’BB’, ArH), 6.95 (2H, AA’BB’,

ArH), 7.14–7.32 ppm (4H, m, ArH); ¹³C NMR (100 MHz, CDCl₃): d=

12.6 (3CH), 17.9 (6CH₃), 45.3 (CH₂), 74.7 (CH), 120.0 (2CH), 127.3

(2CH), 128.4 (2CH), 129.6 (C), 130.4 (2CH), 133.1 (C), 142.1 (C),

154.9 ppm (C); HRMS-ESI: m/z [M+Na]⁺calcd for C₂₃H₃₂BrClNaO₂Si:

505.0936, found: 505.0920.

(4-(2-Chloro-2-(4-chlorophenyl)ethyl)phenoxy)triisopropylsilane

(33): SOCl₂(1.90 g, 1.01 mmol) and DMF (5 drops) were added to a

solution of 32 (1.62 g, 4.00 mmol) in CH₂Cl₂(40 mL). After 1 h, the

solvent was removed in vacuo, the residue was redissolved in

CH₂Cl₂, and the process was repeated. Purification using a Combi-

4-(3-(3-Bromo-4-hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)propyl)-

benzonitrile (28): As method D using TBAF (1.72 mL), 27 (0.83 g,

1.54 mmol) and THF (10 mL). Purification using a CombiFlash R_f

a

foam (0.46 g, 79%): ¹H NMR

1

(CH₂Cl₂/acetone) gave 28 as

Flash R_f(EtOAc/PE) gave 33 as a yellow oil (1.55 g, 92%): H NMR

(270 MHz, CDCl₃): d=2.20–2.80 (4H, m, CH₂CH₂), 5.66 (1H, dd, J=

9.6, 5.8 Hz, CH), 6.84 (1H, d, J=8.3 Hz, ArH), 6.94 (1H, dd, J=8.3,

1.9 Hz, ArH), 7.26 (1H, d, J=1.9 Hz, ArH), 7.54 (2H, AA’BB’, ArH),

7.83 (2H, AA’BB’, ArH), 8.06 (1H, s, NCHN), 8.77 (1H, s, NCHN),

10.06 ppm (1H, br s, OH); ¹³C NMR (100 MHz, [D₆]DMSO): d=35.5

(CH₂), 38.9 (CH₂), 61.7 (CH), 109.1 (C), 110.8 (C), 116.3 (CH), 118.5 (C),

127.9 (2CH), 128.5 (CH), 132.3 (CH), 132.4 (C), 132.7 (2CH), 144.1

(CH), 145.2 (C), 151.9 (CH), 152.3 ppm (C); LC/MS (ESI+): t_R=

1.35 min, m/z (%): 383.1 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺

calcd for C₁₈H₁₆BrN₄O: 383.0502, found: 383.0497.

(270 MHz, CDCl₃): d=1.07–1.31 (21H, m, 3CH, 6CH₃), 3.18 (1H, dd,

J=13.8, 7.7 Hz, CHH), 3.34 (1H, dd, J=13.8, 6.9 Hz, CHH), 4.94 (1H,

t, J=6.6 Hz, CH), 6.75 (2H, AA’BB’, ArH), 6.86 (2H, AA’BB’, ArH),

7.15–7.29 ppm (4H, m, ArH); ¹³C NMR (68 MHz, CDCl₃): d=12.7

(3CH), 18.0 (6CH₃), 46.1 (CH₂), 63.4 (CH), 120.0 (2CH), 128.6 (2CH),

128.7 (2CH), 129.5 (C), 130.5 (2CH), 134.0 (C), 139.6 (C), 155.1 ppm

(C).

4-(2-(4-Chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phenol (34):

1,2,4-Triazole (0.63 g, 9.13 mmol), K₂CO₃(0.60 g, 4.35 mmol) and KI

(0.060 g, 0.36 mmol) were sequentially added to a solution of 33

(1.54 g, 3.64 mmol) in acetone (70 mL). The reaction mixture was

heated at 558C and monitored by TLC with additional portions of

triazole added when required. After 4 d, the reaction mixture was

allowed to cool, and the solvent was removed in vacuo. The resi-

due was redissolved in H₂O/EtOAc (1:1, 160 mL), the layers were

separated, and the aqueous layer extracted with EtOAc (80 mL).

The organic layers were combined, washed with brine (100 mL),

then dried (MgSO₄), filtered and concentrated in vacuo. Purification

using a CombiFlash R_f(PE/EtOAc) gave 34 as a white crystalline

2-Bromo-4-(3-(4-cyanophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl)-

phenyl sulfamate (29): As method C using ClSO₂NH₂(0.57m,

4.0 mL), DMA (2.5 mL) and 28 (0.16 g, 0.42 mmol). Purification

using a CombiFlash R_f(CH₂Cl₂/acetone) gave 29 as a white solid

1

(0.13 g, 67%): H NMR (270 MHz, [D₆]DMSO): d=2.36–2.72 (4H, m,

2CH₂), 5.74 (1H, dd, J=9.2, 5.2 Hz, CH), 7.24 (1H, dd, J=8.3, 2.2 Hz,

ArH), 7.38 (1H, d, J=8.3 Hz, ArH), 7.53–7.59 (3H, m, ArH), 7.85 (2H,

AA’BB’, ArH), 8.06 (1H, s, NCHN), 8.21 (2H, br s, NH₂), 8.79 ppm

(1H, s, NCHN); ¹³C NMR (100 MHz, [D₆]DMSO): d=30.8 (CH₂), 35.1

(CH₂), 61.9 (CH), 110.9 (C), 115.7 (C), 118.5 (C), 123.1 (CH), 128.0

(2CH), 128.8 (CH), 132.7 (2CH), 133.1 (CH), 140.7 (C), 144.1 (C), 145.1

(CH), 145.7 (C), 151.9 ppm (CH); LC/MS (ESI+): t_R=1.30 min, m/z

(%): 462.0 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for

C₁₈H₁₇BrN₅O₃S: 462.0230, found: 462.0216.

1

solid (0.53 g, 49%): mp: 99–1018C; H NMR (270 MHz, [D₆]DMSO):

d=3.29 (1H, dd, J=14.0, 5.8 Hz, CHH), 3.49 (1H, dd, J=14.0,

9.6 Hz, CHH), 5.83 (1H, dd, J=9.6, 5.8 Hz, CH), 6.58 (2H, AA’BB’,

ArH), 6.91 (2H, AA’BB’, ArH), 7.39–7.50 (4H, m, ArH), 7.98 (1H, s,

NCHN), 8.55 (1H, s, NCHN), 9.24 ppm (1H, s, OH); ¹³C NMR

(100 MHz, [D₆]DMSO): d=39.5 (CH₂), 63.5 (CH), 115.0 (2CH), 127.2

(C), 128.5 (2CH), 129.1 (2CH), 129.9 (2CH), 132.6 (C), 138.8 (C), 143.8

(CH), 151.5 (CH), 155.9 ppm (C); LC/MS (ESI+): t_R=1.54 min, m/z

(%): 300.2 (100) [M+H]⁺, 231.1 (70); HRMS-ESI: m/z [M+H]⁺calcd

for C₁₆H₁₅ClN₃O: 300.0898, found: 300.0890; Anal. calcd for

C₁₆H₁₄ClN₃O: C 64.11, H 4.71, N 14.02, found: C 64.20, H 4.72, N

14.00.

2-(4-(Triisopropylsilyloxy)phenyl)acetaldehyde (31): A solution of

30^[33](0.25 g, 0.85 mmol) in CH₂Cl₂(1 mL) was added to a suspen-

sion of Dess–Martin reagent (0.40 g, 0.94 mmol) in CH₂Cl₂(5 mL).

After stirring for 1 h, Et₂O (5 mL), satd aq NaHCO₃(5 mL) and satd

aq Na₂S₂O₃(5 mL) were added and stirring continued for 10 min.

The layers were separated, and the aqueous layer was extracted

with Et₂O (10 mL). The combined organic layers were washed with

satd aq NaHCO₃(20 mL), H₂O (20 mL) and brine (20 mL), then dried

(MgSO₄), filtered and concentrated in vacuo. Purification using a

CombiFlash R_f(EtOAc/PE) gave 31 as a yellow oil (0.20 g, 81%):

¹H NMR (270 MHz, CDCl₃): d=1.05–1.31 (21H, m, 3CH, 6CH₃), 3.59

(2H, d, J=2.2 Hz, CH₂), 6.86 (2H, AA’BB’, ArH), 7.05 (2H, AA’BB’,

ArH), 9.70 ppm (1H, t, J=2.5 Hz, CHO); ¹³C NMR (100 MHz, CDCl₃):

d=12.7 (3CH), 18.0 (6CH₃), 49.9 (CH₂), 120.5 (2CH), 124.1 (C), 130.7

(2CH), 155.6 (C), 199.9 ppm (CH).

4-(2-(4-Chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phenyl sulfa-

mate (35): As method C using ClSO₂NH₂(0.57m, 6.0 mL), DMA

(2.5 mL) and 34 (0.20 g, 0.67 mmol). Purification using a Combi-

Flash R_f(CH₂Cl₂/acetone) gave 35 as a white foam (0.22 g, 88%):

¹H NMR (270 MHz, [D₆]DMSO): d=3.45 (1H, dd, J=14.0, 5.8 Hz,

CHH), 3.66 (1H, dd, J=14.0, 9.9 Hz, CHH), 5.97 (1H, dd, J=9.9,

5.8 Hz, CH), 7.11 (2H, AA’BB’, ArH), 7.24 (2H, AA’BB’, ArH), 7.41–7.52

(4H, m, ArH), 7.94 (2H, s, NH₂), 7.99 (1H, s, NCHN), 8.59 ppm (1H, s,

NCHN); ¹³C NMR (100 MHz, [D₆]DMSO): d=40.2 (CH₂), 62.9 (CH₂),

121.8 (2CH), 128.6 (2CH), 129.1 (2CH), 130.2 (2CH), 132.7 (C), 135.6

(C), 138.6 (C), 143.8 (CH), 148.8 (C), 151.7 ppm (CH); LC/MS (ESI+):

1-(4-Chlorophenyl)-2-(4-(triisopropylsilyloxy)phenyl)ethanol (32):

4-ClPhMgBr (1m in Et₂O, 8.25 mL) was added to a solution of 31

(1.20 g, 4.10 mmol) in Et₂O (20 mL). After stirring for 2 h, the reac-

ChemMedChem 2011, 6, 1423 – 1438

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org

1435

B. V. L. Potter et al.

MED

t_R=1.42 min, m/z (%): 379.0 (100) [M+H]⁺; HRMS-ESI: m/z [M+

CHH), 3.49 (1H, dd, J=13.8, 10.0 Hz, CHH), 5.86 (1H, dd, J=9.9,

5.8 Hz, CHH), 6.73 (1H, d, J=8.3 Hz, ArH), 6.87 (1H, dd, J=8.3,

1.9 Hz, ArH), 7.32 (1H, d, J=1.9 Hz, ArH), 7.39–7.50 (4H, m, ArH),

7.98 (1H, s, NCHN), 8.55 (1H, s, NCHN), 10.06 ppm (1H, s, OH);

¹³C NMR (100 MHz, [D₆]DMSO): d=38.9 (CH₂), 63.1 (CH), 109.0 (C),

116.0 (CH), 128.5 (2CH), 129.1 (2CH), 129.2 (CH), 129.3 (CH), 132.7

(C), 133.1 (CH), 138.6 (C), 143.9 (CH), 151.6 (CH), 152.6 ppm (C); LC/

MS (ESI+): t_R=1.70 min, m/z (%): 378.0 (100) [M+H]⁺; HRMS-ESI:

m/z [M+H]⁺calcd for C₁₆H₁₄BrClN₃O: 378.0003, found: 377.9993.

H]⁺calcd for C₁₆H₁₆ClN₄O₃S: 379.0626, found: 379.0612.

2-(3-Bromo-4-(triisopropylsilyloxy)phenyl)acetaldehyde (36):

A

solution of 25 (4.00 g, 10.7 mmol) in CH₂Cl₂(6 mL) was added to a

suspension of Dess–Martin reagent (5.46 g, 12.9 mmol) in CH₂Cl₂

(60 mL). After stirring for 1 h, Et₂O (100 mL), satd aq NaHCO₃

(100 mL) and satd aq Na₂S₂O₃(100 mL) were added and stirring

continued for 10 min. The layers were separated, and the aqueous

layer was extracted with Et₂O (100 mL). The combined organic

layers were washed with satd aq NaHCO₃(20 mL), H₂O (20 mL) and

brine (20 mL), then dried (MgSO₄), filtered and concentrated in

vacuo. Purification using a CombiFlash R_f(EtOAc/PE) gave 36 as a

colourless oil (2.91 g, 73%); ¹H NMR (270 MHz, CDCl₃): d=1.08–

1.37 (21H, m, 3CH, 6CH₃), 3.57 (2H, d, J=2.2 Hz, CH₂), 6.86 (1H, d,

J=8.5 Hz, ArH), 6.97 (1H, dd, J=8.3, 2.2 Hz, ArH), 7.37 (1H, d, J=

2.2 Hz, ArH), 9.70 ppm (1H, t, J=2.2 Hz, CHO); ¹³C NMR (100 MHz,

CDCl₃): d=13.0 (3CH), 18.1 (6CH₃), 49.3 (CH₂), 115.4 (C), 119.9 (CH),

125.4 (C), 129.5 (CH), 134.5 (CH), 152.5 (C), 199.1 ppm (CH).

2-Bromo-4-(2-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)-

phenyl sulfamate (40): As method C using ClSO₂NH₂(0.57m,

4.5 mL), DMA (1.5 mL) and a solution of 39 (0.15 g, 0.40 mmol) in

DMA (1 mL). Purification using a CombiFlash R_f(CH₂Cl₂/acetone)

gave 40 as a white foam (0.13 g, 71%): ¹H NMR (270 MHz,

[D₆]DMSO): d=3.45 (1H, dd, J=14.0, 5.8 Hz, CHH), 3.65 (1H, dd,

J=14.0, 10.2 Hz, CHH), 6.01 (1H, dd, J=10.2, 5.8 Hz, CH), 7.20 (1H,

dd, J=8.5, 1.4 Hz, ArH), 7.31 (1H, d, J=8.5 Hz, ArH), 7.42–7.52 (4H,

m, ArH), 8.01 (1H, s, NCHN), 8.59 (1H, s, NCHN), 8.24 (2H, s, NH₂),

8.61 ppm (1H, s, NCHN); ¹³C NMR (100 MHz, [D₆]DMSO): d=38.8

(CH₂), 62.5 (CH₂), 115.4 (C), 122.8 (CH), 128.6 (2CH), 129.1 (2CH),

129.5 (CH), 132.8 (C), 133.9 (CH), 137.3 (C), 138.4 (C), 143.9 (CH),

146.1 (C), 151.7 ppm (CH); LC/MS (ESI+): t_R=1.42 min, m/z (%):

457.1 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺calcd for

C₁₆H₁₅BrClN₄O₃S: 456.9731, found: 456.9714.

2-(3-Bromo-4-(triisopropylsilyloxy)phenyl)-1-(4-chlorophenyl)e-

thanol (37): 4-ClPhMgBr (1m in Et₂O, 15.5 mL) was added to a so-

lution of 36 (2.85 g, 7.68 mmol) in Et₂O (60 mL). After stirring for

2 h, the reaction was quenched by addition of H₂O (30 mL) and

2m HCl (30 mL). The product was extracted with Et₂O (2ꢂ75 mL),

and the combined organic layers were washed with brine

(100 mL), then dried (MgSO₄), filtered and concentrated in vacuo.

Purification using a CombiFlash R_f(EtOAc/PE) gave 37 as white

solid (2.66 g, 72%): mp: 75–788C; ¹H NMR (270 MHz, CDCl₃): d=

1.06–1.36 (21H, m, 3CH, 6CH₃), 1.96 (1H, d, J=3.0 Hz, OH), 2.86

(2H, d, J=6.9 Hz, CH₂), 4.82 (1H, td, J=6.9, 3.0 Hz, CH), 6.77 (1H, d,

J=8.3 Hz, ArH), 6.86 (1H, dd, J=8.3, 2.2 Hz, ArH), 7.18–7.35 ppm

(5H, m, ArH); ¹³C NMR (100 MHz, CDCl₃): d=12.9 (3CH), 18.0

(6CH₃), 44.8 (CH₂), 74.6 (CH), 115.0 (C), 119.5 (CH), 127.3 (2CH),

128.5 (2CH), 129.3 (CH), 131.1 (C), 133.3 (C), 134.1 (CH), 141.9 (C),

151.8 ppm (C); LC/MS (ESI+): t_R=2.54 min, m/z (%): 427.3 (100)

[M+Na]⁺; HRMS-ESI: m/z [M+Na]⁺calcd for C₂₃H₃₃ClNaO₂Si:

427.1831, found: 427.1828.

2-Bromo-4-((4-cyanophenyl)(1H-1,2,4-triazol-1-yl)methyl)phenyl

dimethylsulfamate (42): N,N-Dimethylsulfamoyl chloride (0.60 g,

4.16 mmol) was added to a suspension of 41 (0.25 g, 0.70 mmol) in

N,N-diisopropylethylamine (DIPEA; 5 mL). After heating at reflux for

1 h, the reaction mixture was allowed to cool, diluted with H₂O

and EtOAc, and then poured into 3m HCl (100 mL). The layers

were separated, and the organic layer was washed with H₂O

(50 mL) and satd aq NaHCO₃(50 mL), then dried (MgSO₄), filtered

and concentrated in vacuo. Purification using a Flashmaster II

system (hexane/EtOAc) gave 42 as a light brown oil (0.11 g, 34%):

¹H NMR (270 MHz, CDCl₃): d=3.08 (6H, s, 2CH₃), 6.71 (1H, s, CH),

7.13 (1H, dd, J=8.4, 2.3 Hz, ArH), 7.22–7.24 (2H, m, ArH), 7.41 (1H,

d, J=2.3 Hz, ArH), 7.53 (1H, d, J=8.4 Hz, ArH), 7.69 (2H, AA’BB’,

ArH), 8.04 (1H, s, NCHN), 8.05 ppm (1H, s, NCHN); ¹³C NMR

(100 MHz, [D₆]DMSO): d=38.5 (2CH₃), 63.4 (CH), 111.2 (C), 115.6

(C), 118.5 (C), 123.5 (CH), 128.9 (2CH), 129.4 (CH), 132.8 (2CH), 133.5

(CH), 138.2 (C), 143.6 (C), 145.1 (CH), 146.6 (C), 152.5 ppm (CH); LC/

MS (ESIꢁ): t_R=4.23 min, m/z (%): 462.3 (100) [MꢁH]^ꢁ; HRMS-FAB:

m/z [M+H]⁺calcd for C₁₈H₁₇BrN₅O₃S: 462.0230, found: 462.0220.

(2-Bromo-4-(2-chloro-2-(4-chlorophenyl)ethyl)phenoxy)triisopro-

pylsilane (38): SOCl₂(2.44 g, 20.5 mmol) and DMF (5 drops) were

added to a solution of 37 (2.48 g, 5.12 mmol) in CH₂Cl₂(50 mL).

After stirring for 2 h, the solvent was removed in vacuo, the resi-

due was redissolved in CH₂Cl₂, and the process was repeated. Pu-

rification using a CombiFlash R_f(EtOAc/PE) gave 38 as a colourless

1

oil (2.11 g, 82%); H NMR (270 MHz, CDCl₃): d=1.06–1.35 (21H, m,

3CH, 6CH₃), 3.13 (1H, dd, J=14.0, 7.2 Hz, CHH), 3.26 (1H, dd, J=

14.0, 7.2 Hz, CHH), 4.91 (1H, t, J=7.2 Hz, CH), 6.73–6.75 (2H, m,

ArH), 7.18–7.32 ppm (5H, m, ArH); ¹³C NMR (68 MHz, CDCl₃): d=

13.0 (3CH), 18.1 (6CH₃), 45.5 (CH₂), 63.0 (CH), 112.5 (C), 119.5 (2CH),

128.6 (2CH), 128.7 (CH), 128.8 (C), 129.4 (CH), 130.8 (C), 134.2 (CH),

139.3 (C), 152.0 ppm (C).

1-Methyl-1H-benzo[d][1,2,3]triazole-6-carboxylic acid (44): NaNO₂

(0.16 g, 2.32 mmol) dissolved in a small amount of H₂O was added

to a mixture of 43^[34](0.26 g, 1.57 mmol) and 6n HCl (2 mL) at 08C.

After 5 min, the reaction was allowed to warm to RT and stirred for

3 h. H₂O (20 mL) and EtOAc (20 mL) were added, the layers were

separated, and the aqueous layer was extracted with EtOAc

(20 mL). The combined organic layers were dried (MgSO₄), filtered

and concentrated in vacuo. Compound 44 was obtained as a

brown solid and used without further purification (0.23 g, 82%):

¹H NMR (270 MHz, [D₆]DMSO): d=4.39 ppm (3H, s, CH₃), 7.92–7.97

(1H, m, ArH and CH), 8.15–8.09 (1H, m, ArH), 8.50 (1H, s, ArH); LC/

MS (ESI+): t_R=0.77 min, m/z (%): 175.5 (100) [MꢁH]^ꢁ.

2-Bromo-4-(2-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)phe-

nol (39): 1,2,4-Triazole (1.41 g, 20.43 mmol), K₂CO₃(0.68 g,

4.93 mmol) and KI (0.067 g, 0.40 mmol) were sequentially added to

a solution of 38 (2.04 g, 4.08 mmol) in acetone (80 mL). The reac-

tion mixture was heated at 558C for 48 h then allowed to cool,

and the solvent was removed in vacuo. The residue was redis-

solved in H₂O/EtOAc (1:1, 160 mL), the layers were separated, and

the aqueous layer extracted with EtOAc (80 mL). The combined or-

ganic layers were washed with brine (100 mL), then dried (MgSO₄),

filtered and concentrated in vacuo. Purification using a CombiFlash

R_f(PE/EtOAc) and then (CH₂Cl₂/acetone) gave 39 as a white foam

Methyl 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxylate (45):

SOCl₂(0.62 g, 4.52 mmol) added to a suspension of 44 (0.20 g,

1.13 mmol) in MeOH (15 mL), and the resulting mixture was

heated at reflux overnight. The mixture was allowed to cool, and

the solvent was removed in vacuo. Purification using a Flashmaster

1

(0.25 g, 16%): H NMR (270 MHz, [D₆]DMSO): d=3.27–3.34 (1H, m,

II (EtOAc/hexane) gave 45 as a cream solid (0.18 g, 83%): H NMR

1436

www.chemmedchem.org

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 1423 – 1438

Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors

(270 MHz, [D₆]DMSO): d=3.93 (3H, s, OCH₃), 4.40 (3H, s, NCH₃),

7.92–7.98 (1H, m, ArH), 8.13–8.18 (1H, m, ArH), 8.55 ppm (1H, s,

ArH); LC/MS (ESI+): t_R=0.87 min, m/z (%): 191.9 (100) [M+H]⁺,

159.7 (25).

(MgSO₄), filtered and concentrated in vacuo. Purification using a

Flashmaster II (EtOAc/hexane) gave 49 as a yellow solid (0.061 g,

76%): ¹H NMR (400 MHz, [D₆]DMSO): d=4.25 (3H, s, CH₃), 5.09

(2H, s, CH₂), 7.02 (2H, AA’BB’, ArH), 7.20 (1H, s, CH), 7.22–7.45 (8H,

m, ArH), 7.65 (1H, s, ArH), 8.02 (1H, d, J=9.0 Hz, ArH), 8.09 (1H, s,

NCHN), 8.61 ppm (1H, s, NCHN); ¹³C NMR (100 MHz, [D₆]DMSO):

d=34.2 (CH₃), 65.1 (CH), 69.3 (CH₂), 109.6 (CH), 114.9 (2CH), 119.3

(CH), 124.3 (CH), 127.8 (2CH), 127.9 (CH), 128.5 (2CH), 129.6 (2CH),

130.8 (C), 133.3 (C), 136.9 (C), 138.8 (C), 144.5 (CH), 144.6 (C), 152.0

(CH), 158.2 ppm (C); LC/MS (ESI+): t_R=1.62 min, m/z (%): 395.6

(100) [M]⁺; HRMS-ESI: m/z [M+H]⁺calcd for C₂₃H₂₁N₆O: 397.1771,

found: 397.1756.

(1-Methyl-1H-benzo[d][1,2,3]triazole-6-yl)methanol (46): A solu-

tion of 45 (1.88 g, 9.84 mmol) in THF (25 mL) was added to a sus-

pension of LiAlH₄(0.75 g, 19.7 mmol) in THF (60 mL). After stirring

for 20 min, the reaction was quenched by addition of EtOAc

(10 mL), H₂O (10 mL) and 3m HCl (50 mL). The product was extract-

ed with EtOAc (2ꢂ75 mL), and the combined organics were

washed with satd aq NaHCO₃(2ꢂ100 mL), then dried (MgSO₄), fil-

tered and concentrated in vacuo. Purification using a Flashmaster II

(EtOAc/hexane) gave 46 as pale yellow crystals (1.07 g, 67%): mp:

4-((1-Methyl-1H-benzo[d][1,2,3]triazole-6-yl)(1H-1,2,4-triazol-1-

yl)methyl)phenol (50): As method B using Pd/C (30 mg), 49

(0.28 g, 0.71 mmol) and THF/MeOH (1:1, 20 mL). Purification using

a Flashmaster II (CH₂Cl₂/MeOH) gave 50 as a white solid (0.21 g,

97%): ¹H NMR (270 MHz, [D₆]DMSO): d=4.25 (3H, s, CH₃), 6.75

(2H, AA’BB’, ArH), 7.09 (2H, AA’BB’, ArH), 7.16 (1H, s, CH), 7.27 (1H,

d, J=8.5 Hz, ArH), 7.61 (1H, s, ArH), 8.02 (1H, d, J=8.5 Hz, ArH),

8.08 (1H, s, NCHN), 8.56 (1H, s, NCHN), 9.63 ppm (1H, s, OH);

¹³C NMR (100 MHz, [D₆]DMSO): d=34.2 (CH₃), 65.4 (CH), 109.5

(CH), 115.4 (2CH), 119.2 (CH), 124.3 (CH), 128.8 (C), 129.6 (2CH),

133.3 (C), 139.0 (C), 144.3 (CH), 144.6 (C), 152.0 (CH), 157.3 ppm (C);

LC/MS (ESIꢁ): t_R=1.24 min, m/z (%): 305.2 (100) [MꢁH]^ꢁ; HRMS-

ESI: m/z [M+H]⁺calcd for C₁₆H₁₅N₆O: 307.1302, found: 307.1296.

1

70–728C; H NMR (270 MHz, CDCl₃): d=4.11 (3H, s, CH₃), 4.82 (2H,

s, CH₂), 4.87 (1H, br s, OH), 7.21 (1H, d, J=8.8 Hz, ArH), 7.46 (1H, s,

ArH), 7.76 ppm (1H, d, J=8.8 Hz, ArH); LC/MS (ESI+): t_R=0.88 min,

m/z (%): 163.9 (100) [M+H]⁺.

1-Methyl-1H-benzo[d][1,2,3]triazole-6-carbaldehyde (47): Tri-

chloroisocyanuric acid (0.81 g, 3.49 mmol) was added to a solution

of 46 (0.54 g, 3.31 mmol) in CH₂Cl₂(10 mL). The solution was

cooled to 08C, TEMPO (0.006 g, 0.038 mmol) was added, the reac-

tion mixture was stirred for 10 min and then filtered through

Celite. The filtrate was washed with 20% aq Na₂CO₃, 1n HCl and

brine, then dried (MgSO₄), filtered and concentrated in vacuo to

give 47 as a white solid used without further purification (0.50 g,

94%): mp: 148.5–1518C; ¹H NMR (270 MHz, [D₆]DMSO): d=4.42

(3H, s, CH₃), 7.89 (1H, dd, J=8.5, 1.4 Hz, ArH), 8.21 (1H, d, J=

8.5 Hz, ArH), 8.56 (1H, d, J=1.4 Hz, ArH), 10.19 ppm (1H, s, CHO);

LC/MS (ESI+): t_R=0.90 min, m/z (%): 161.9 (100) [M+H]⁺.

4-((1-Methyl-1H-benzo[d][1,2,3]triazole-6-yl)(1H-1,2,4-triazol-1-

yl)methyl)phenyl sulfamate (51): As method C using ClSO₂NH₂

(0.45m, 5.0 mL), DMA (2.5 mL) and 50 (0.11 g, 0.36 mmol). Purifica-

tion using a Flashmaster II (CH₂Cl₂/acetone) gave 51 as a white

1

solid (0.12 g, 84%): H NMR (270 MHz, [D₆]DMSO): d=4.27 (3H, s,

CH₃), 7.26–7.39 (6H, m, ArH and CH), 7.72 (1H, s, ArH), 8.00 (2H, s,

NH₂), 8.05 (1H, d, J=8.8 Hz, ArH), 8.13 (1H, s, NCHN), 8.66 ppm

(1H, s, NCHN); ¹³C NMR (100 MHz, [D₆]DMSO): d=34.3 (CH₃), 64.9

(CH), 110.0 (CH), 119.4 (CH), 122.5 (2CH), 124.4 (CH), 129.7 (2CH),

133.4 (C), 137.0 (C), 138.1 (C), 144.7 (C, CH), 149.8 (C), 152.2 ppm

(CH); LC/MS (ESIꢁ): t_R=1.23 min, m/z (%): 384.4 (100) [MꢁH]^ꢁ;

HRMS-ESI: m/z [M+H]⁺calcd for C₁₆H₁₆N₇O₃S: 386.1030, found:

386.1013.

(4-(Benzyloxy)phenyl)(1-methyl-1H-benzo[d][1,2,3-triazole-6-yl)-

methanol (48): A solution of 4-benzyloxybromobenzene (1.35 g,

5.13 mmol) in THF (1 mL) was added in small portions to a suspen-

sion of Mg (0.11 g, 4.58 mmol) in THF (0.5 mL) containing a crystal

of I₂. Heating was used when required until the preparation of the

Grignard reagent was complete. This reagent (4 equiv) were added

to a suspension of 47 (0.050 g, 0.31 mmol) in THF (1 mL). After stir-

ring overnight, H₂O (5 mL) and HCl (1m, 5 mL) were added, and

the product was extracted with EtOAc (2ꢂ20 mL). The combined

organic layers were washed with satd aq NaHCO₃(20 mL) and

brine (20 mL), then dried (MgSO₄), filtered and concentrated in

vacuo. Purification using a Flashmaster II (EtOAc/hexane) gave 48

as a white crystalline solid (0.095 g, 89%): mp: 131.5–133.58C;

¹H NMR (270 MHz, [D₆]DMSO): d=4.30 (3H, s, CH₃), 5.06 (2H, s,

CH₂), 5.85 (1H, d, J=3.9 Hz, CH), 6.06 (1H, d, J=3.9 Hz, OH), 6.93

(2H, m, AA’BB’, ArH), 7.25–7.45 (8H, m, ArH), 7.87–7.94 ppm (2H,

m, ArH); ¹³C NMR (100 MHz, [D₆]DMSO): d=34.1 (CH₃), 69.1 (CH₂),

73.8 (CH), 106.8 (CH), 114.5 (2CH), 118.6 (CH), 123.2 (CH), 127.6

(2CH), 127.7 (2CH), 127.8 (CH), 128.4 (2CH), 133.4 (C), 137.2 (C),

137.7 (C), 144.3 (C), 145.6 (C), 157.3 ppm (C); LC/MS (ESI+): t_R=

1.64 min, m/z (%): 346.6 (100) [M+H]⁺; HRMS-ESI: m/z [M+H]⁺

calcd for C₂₁H₂₀N₃O₂: 346.1550, found: 346.1534.

(R)-4-(2-(3-Bromo-4-hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)e-

thyl)benzonitrile (17a): Separation of 17 on a Chiralpak AD-H

(250ꢂ20 mm) semi-prep column as described in the text gave

17a: t₁=7.92 min; ½aꢃ_D²⁰=ꢁ18.78 (c=0.89 in EtOH).

(S)-4-(2-(3-Bromo-4-hydroxyphenyl)-1-(1H-1,2,4-triazol-1-yl)e-

thyl)benzonitrile (17b): Separation of 17 on a Chiralpak AD-H

(250ꢂ20 mm) semi-prep column as described in the text gave

17b: t₂=15.96 min; ½aꢃ²_D⁰=+25.18 (c=0.90 in EtOH).

(R)-2-Bromo-4-(2-(4-cyanophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)-

phenyl sulfamate (18a): Prepared from 17a as described for (ꢀ)-

18: ½aꢃ²_D⁰=ꢁ15.98 (c=4.1 in EtOH).

(S)-2-Bromo-4-(2-(4-cyanophenyl)-2-(1H-1,2,4-triazol-1-yl)ethyl)-

phenyl sulfamate (18b): Prepared from 17b as described for (ꢀ)-

18: ½aꢃ²_D⁰=+16.58 (c=3.1 in EtOH).

6-((4-(Benzyloxy)phenyl)(1H-1,2,4-triazol-1-yl)methyl)-1-methyl-

1H-benzo[d][1,2,3]triazole (49): SOCl₂(0.1 mL) was added to a so-

lution of 48 (0.090 g, 0.26 mmol) in CH₂Cl₂(3 mL). After stirring for

2 h, the solvent was removed in vacuo and the residue was dis-

solved in acetone (5 mL). 1,2,4-Triazole (0.036 g, 0.52 mmol), KI

(0.002 g, 0.012 mmol) and K₂CO₃(0.11 g, 0.80 mmol) were added,

and the reaction mixture was stirred at 608C overnight. H₂O

(10 mL) was added, and the product was extracted with EtOAc (2ꢂ

10 mL). The combined organic layers were washed with H₂O

(20 mL), 1m aq NaOH (20 mL) and brine (20 mL), then dried

Acknowledgements

This work was supported by Sterix Ltd, a member of the Ipsen

group, and by the Wellcome Trust, VIP funding and Programme

Grant 082837. We thank Alison C. Smith for technical assistance.

ChemMedChem 2011, 6, 1423 – 1438

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org

1437

B. V. L. Potter et al.

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Keywords: aromatase · breast cancer · dual inhibitors

endocrine therapy · sulfatase

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Published online on May 23, 2011

1438

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ChemMedChem 2011, 6, 1423 – 1438

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Aromatase and Dual Aromatase-Steroid Sulfatase Inhibitors from the Letrozole and Vorozole Templates

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