Hybrid dual aromatase-steroid sulfatase inhibitors with exquisite picomolar inhibitory activity

Article abstract of DOI:10.1021/ml100273k

Single agents against multiple drug targets are highly topical. Hormone-dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS), and several dual aromatase-sulfatase inhibitors (DASIs) hav

Full text of DOI:10.1021/ml100273k

LETTER
pubs.acs.org/acsmedchemlett
Hybrid Dual Aromatase-Steroid Sulfatase Inhibitors with Exquisite
Picomolar Inhibitory Activity
L. W. Lawrence Woo,† Christian Bubert,† Atul Purohit,‡ and Barry V. L. Potter*,†
†Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY,
United Kingdom
‡Endocrinology and Metabolic Medicine, Imperial College London, Faculty of Medicine, St. Mary’s Hospital, London W2 1NY,
United Kingdom
S Supporting Information
b
ABSTRACT: Single agents against multiple drug targets are highly topical. Hormone-
dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of
aromatase and steroid sulfatase (STS), and several dual aromatase-sulfatase inhibitors
(DASIs) have been recently reported. The best compounds from two leading classes of
DASI, 3 and 9, are low nanomolar inhibitors. In search of a novel class of DASI, core
motifs of two leading classes were combined to give a series of hybrid structures, with
several compounds showing markedly improved dual inhibitory activities in the
picomolar range in JEG-3 cells. Thus, DASIs 14 (IC50: aromatase, 15 pM; STS, 830
pM) and 15 (IC50: aromatase, 18 pM; STS, 130 pM) are the first examples of an
exceptional new class of highly potent dual inhibitor that should encourage further
development toward multitargeted therapeutic intervention in HDBC.
KEYWORDS: Hybrid, dual inhibitors, aromatase, sulfatase, cancer
strogen deprivation has been an effective therapeutic inter-
incorporating the reversible aromatase inhibitory pharmaco-
phore, which is principally a heme-ligating nitrogen-contain-
ing heterocycle, into a known sulfamate-based STS inhibitor
(e.g., 8 and 9, Figure 1).24 On evaluating the lead candidates
from these four structural classes of DASI, all of them showed
in vitro and in vivo dual inhibitory activities with potency
ranging from good to very high.
Given this success in the design of DASIs to date, we explored
further the feasibility of expanding structural diversity but, at the
same time, set a challenge of maintaining or increasing still
further the high potency. One obvious route to take would be to
repeat the two aforementioned strategies for designing a DASI
and apply them to other reported aromatase and STS inhibitors.
However, repetitive application of the same strategies lacks some
novelty and so pursuing a new design approach seemed more
desirable and challenging .
On reviewing computational docking studies of leading DASIs
and other related molecules performed with a homology model
of aromatase,19,23,25 we noticed that the molecules docked in
different orientations with unoccupied space available within the
enzyme site. This observation suggested that further interactions
with available amino acid residues might be exploited in principle
if additional functionality could be built into the docked molecule
in a complementary manner. For STS,19,23 a similar observation
was realized on reviewing docking studies of lead DASIs carried
E
vention for hormone-dependent breast cancer (HDBC).
One established and clinically proven approach involves the
inhibition of the aromatase enzyme,1-3 although the inhibition
of steroid sulfatase (STS) is an emerging new strategy, as demon-
strated by promising results for STX64 (Irosustat, BN83495),4-6
the first STS inhibitor that entered clinical trials. Since both
aromatase and STS are targets for treating HDBC, it has been
reasoned that estrogen deprivation may be more comprehensively
achieved by dual inhibition of both enzymes.
Designing single agents that act against multiple biological
targets is of increasing interest and prominence. In recent years,
an increasing volume of work has been published exemplifying
the successful use of this strategy.7-17 We have pioneered this
approach to augment our strategy directed at the first-in-class
clinical target of STS inhibition and successfully developed three
series of single agent dual aromatase and sulfatase inhibitors
(DASIs) that are sulfamate derivatives of the nonsteroidal
aromatase inhibitor (AI) 4-((4-bromobenzyl)-[1,2,4]-triazol-
4-ylamino)benzonitrile (1) (e.g., 2 and 3),18-20 letrozole 4
(e.g., 5),21,22 and anastrozole 6 (e.g., 7) (Figure 1).23 The design of
these DASIs shares a common principle of engendering the
irreversible STS inhibitory pharmacophore (ie. an aryl sulfa-
mate ester, ArOSO2NH2) into a clinical or experimental AI
with minimal structural change incurred on the original
scaffold in order to retain and maximize aromatase inhibition.
More recently, a different design approach was employed. A
series of biphenyl-based DASIs was developed as a result of
Received: November 17, 2010
Accepted: December 20, 2010
Published: December 29, 2010
r
2010 American Chemical Society
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ACS Medicinal Chemistry Letters
LETTER
Figure 1. Structures of nonsteroidal AIs 1, 4, and 6, and DASIs 2, 3, 5, and 7 - 9.
Figure 2. Derivatives 10-18 of a hybrid structure designed to possess dual inhibition against aromatase and STS by combining core components of the
two leading 4-((4-bromobenzyl)-[1,2,4]-triazol-4-ylamino)benzonitrile-based (I) and biphenyl-based (II) DASIs.
out with the crystal structure of STS. There are pockets and areas
around the docked molecules in regions away from the sulfamate
moiety in the enzyme active site where further interactions could
be engineered.
Based on these observations, we scrutinized our four classes
of DASI and reasoned that introducing an additional moiety
to an existing DASI could be realized by combining the core
components of the two leading 4-((4-bromobenzyl)-[1,2,4]-
triazol-4-ylamino)benzonitrile-based (I) and biphenyl-based
(II) DASIs to form a series of hybrid structures as shown in
Figure 2.
The synthesis of sulfamate-bearing hybrid structures 10, 11,
and 13-17 (see Scheme 1) started with the formation of biphenyl
intermediate (A) by reacting 2-bromo-4-fluorobenzonitrile
with the required boronic acid under Suzuki cross-coupling condi-
tions. Upon performing a nucleophilic aromatic substitution on (A)
with 4H-1,2,4-triazol-4-amine, the resulting secondary amine was
alkylated by an appropriately substituted benzyl halide. As required,
the hybrid intermediate formed was deprotected to give the parent
phenol, which was sulfamoylated to the corresponding sulfamate
under established conditions.26 A different approach was adopted
for the synthesis of 12 and 18 (see Scheme 2). The key difference
was the formation of a secondary amine by nucleophilic aromatic
substitution, followed by conducting a Suzuki cross-coupling reac-
tion on the resulting brominated intermediate.
The extent of in vitro inhibition of STS and aromatase activity
produced by sulfamoylated compounds was assessed using JEG-
3 human choriocarcinoma cells, which were chosen because
these cells constitutively express both enzymes maximally.19,24
In our hands, these enzyme activities remain stable during repeated
subculturing over several months. The results are shown in Table 1.
AI 1 and DASIs 2, 3, 8, and 9, which were tested previously in the
same assay, are included as reference.
The modification of the DASI 8 by replacing the methylene
bridge between the triazolyl group and the biphenyl scaffold with
either a benzylamine or para-bromobenzylamine moiety signifi-
cantly increases the potency of the resulting compounds against
aromatase. Hybrids 10 (IC50: 0.1 nM) and 11 (IC50: 0.05 nM)
are 1 and 2 orders of magnitude, respectively, more potent than
8 (IC50: 2 nM) as AIs. However, the opposite effect is rendered
with regard to STS inhibition. A substantial reduction in potency
against STS is observed for 10 and 11 (both IC50s: 2900 nM, cf.
35 nM for 8). This suggests that the introduction of either a benzyl-
amine or para-bromobenzylamine moiety to 8 results in productive
interactions of 10 and 11 with the active site of aromatase but not
with that of STS. Steric hindrance may be a contributory factor to
the poor STS inhibition observed for 10 and 11.
The introduction of a phenyl ring (either unsubstituted or with a
substituent at the para-position) to the 4-((4-bromobenzyl)-
[1,2,4]-triazol-4-ylamino)benzonitrile-based DASI 2 at the ortho
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Scheme 1. Syntheses of Sulfamates (10, 11 and 13-17): (A) and (B) Are Key Intermediates; (i) [Pd(dba)2]3; (ii) KOtBu/DMSO;
(iii) SOCl2, K2CO3/DMF; (iv) ClSO2NH2/DMA; (v) NaH/DMF; (vi) Pd-C/MeOH/THF; (vii) AcOH/conc HCl (2:1, v/v),
1 h @ 100 °C, X = OSO2NH2
Scheme 2. Syntheses of Sulfamates (12 and 18): (i) KOtBu/DMSO; (ii) NaH/DMF; (iii) [Pd(dba)2]3; (iv) Pd-C/MeOH/THF;
(v) ClSO2NH2/DMA
position to the cyano group renders a prominent increase in
aromatase inhibition of most resulting hybrid compounds
(IC50's: 0.015-0.75 nM vs 0.82 nM of 3 and 0.5 nM of 9).
Likewise, apart from 18 and to a lesser extent with 12, 16, and
17, a significant increase in STS inhibition is observed for
those hybrid compounds. This finding clearly demonstrates
that the formation of hybrid structures has, inter alia, rendered
a positive effect on the binding to the active site of both
aromatase and STS, contributing toward the increased inhi-
bitory activities observed for the molecules.
The best AIs in this series of compounds are 14-16. Their
IC50 values are similar in the low picomolar range (15 pM for 14
and 16, and 18 pM for 15). As anticipated for 14 and 15, because
of past observations in other series of DASIs, the introduction of
a halogen ortho to the sulfamate group, in particular with Cl and
Br, increases aromatase inhibition.18-20,24 This can be attributed
to the increase in lipophilicity effected by halogens on the molecule.
It is not clear why 16 is equally potent against aromatase given the
fact that, unlike 14 and 15, it does not contain a halogen adjacent to
the sulfamate group. The tentative explanation is that the para-
methoxy group of 16 provides a significant contribution to the
binding of the compound to the active site of aromatase. For17 and
18, they are some 2- and 3-fold less active as AI than 12, respectively.
The common feature in 17 and 18 is that they both possess an
electron-withdrawing group (a fluoro atom in 17 and a cyano group
in 18) at the 40-position which conjugates with the cyano group at
the 2-position of the biphenyl scaffold. It is possible that such
conjugation might weaken the ability of the cyano group at the
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LETTER
Table 1. In Vitro Inhibition in JEG-3 Cells of Aromatase and
STS Activity by Sulfamoylated Compounds 10-18, with
Aromatase Inhibitor 1 and DASIs 2, 3, 8, and 9 Included as
Reference
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: þ44 1225 386639. Fax þ44 1225 386114. E-mail:
B.V.L.Potter@bath.ac.uk.
compd
aromatase IC50 (nM)
STS IC50 (nM)
Author Contributions
1
0.5 ( 0.03b
100 ( 7.8b
0.82 ( 0.3b
2.0 ( 0.20c
0.50 ( 0.01c
0.1 ( 0.02
n.d.a
Overall strategy and core structures were designed by L.W.L.W. and
B.V.L.P. in consultation with C.B. C.B. synthesized the compounds,
and A.P. carried out their biological evaluation. The paper was
written by L.W.L.W. and B.V.L.P. in consultation with all authors.
2
277 ( 29b
39 ( 4.2b
35.0 ( 5.0c
5.50 ( 0.5c
2900 ( 180
2900 ( 100
17 ( 2
3
8
9
10
11
12
13
14
15
16
17
18
’ ACKNOWLEDGMENT
0.05 ( 0.01
0.21 ( 0.03
0.15 ( 0.03
0.015 ( 0.005
0.018 ( 0.01
0.015 ( 0.001
0.47 ( 0.18
0.75 ( 0.005
This work was supported by Sterix Ltd., which is part of the
Ipsen group.
2.3 ( 0.9
0.83 ( 0.15
0.13 ( 0.01
22 ( 6
’ ABBREVIATIONS
AI, aromatase inhibitor; DASI, dual aromatase-sulfatase inhibitor;
DMA, N,N-dimethylacetamide; HDBC, hormone-dependent
breast cancer; STS, steroid sulfatase; STX64, 6-oxo-6,7,8,9,10,
11-hexahydrocyclohepta[c]chromen-3-yl sulfamate
55 ( 6
240 ( 60
a n.d.: not determined. b Reference 19. c Reference 24.
2-position to function as a hydrogen bond acceptor, which has been
attributed to be an important factor for strong binding of nonster-
oidal AIs bearing a cyano group similar to that of letrozole.27
As shown in Table 1, 18 is relatively the weakest STS inhibitor
among its congeners (12-17). Since hybrids 12, 16, and 17 have
IC50 values against STS of the same order of magnitude, this
suggests that a fluoro atom or a methoxy group, but not a cyano
group, substituted at the 40-position is better tolerated by STS in
this subgroup of compounds. The strongest STS inhibitors are
those derivatives which have a halogen ortho to their sulfamate
group. This has been a characteristic observed in our previous
work.18-20,21,23,24,28,29 The most potent inhibitor against STS in
the series is the brominated 15, whose IC50 value is 0.13 nM,
which is two orders and one order of magnitude more potent
than the 4-((4-bromobenzyl)-[1,2,4]-triazol-4-ylamino)benzo-
nitrile-based DASI 3 (IC50: 39 nM) and the biphenyl-based
DASI 9 (IC50: 5.5 nM), respectively. This further demonstrates
the productive contribution of the additional phenyl ring to
inhibitory activity and the advantage of hybrid structure.
In conclusion, a novel series of DASIs has been designed by
combining the core components of two leading series of DASIs,
namely 4-((4-bromobenzyl)-[1,2,4]-triazol-4-ylamino)benzo-
nitrile-based and biphenyl-based series. Several resulting derivatives
show a very high level of dual inhibition against aromatase and STS
invitro. The improvement observed compared with the best leading
DASIs is significant and substantial, making this new inhibitor class
worthy of further optimization and arguably the most promising to
date for preclinical studies. This work clearly validates the “hybrid”
strategy and demonstrates that structurally novel and distinctive
DASIs with improved dual inhibitory activities can be designed. It is
now warranted to further develop these “hybrid” compounds and
explore their full potential as therapeutic agents for the treatment of
hormone dependent cancers such as that of the breast.
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