Dual aromatase-sulfatase inhibitors based on the anastrozole template: Synthesis, in vitro SAR, molecular modelling and in vivo activity

Article abstract of DOI:10.1039/b707768h

The synthesis and biological evaluation of a series of novel Dual Aromatase-Sulfatase Inhibitors (DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished via selective radical bromination and substitution reactions to furnish a series of inhibitory aromatase pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed S_N2, Heck and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC₅₀ 3.5 nM), comparable with that of Anastrozole (IC₅₀ 1.5 nM) whereas, only moderate activity against steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition. Compound 11 was modelled into the active site of a homology model of human aromatase and the X-ray crystal structure of steroid sulfatase. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b707768h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Dual aromatase–sulfatase inhibitors based on the anastrozole template:
synthesis, in vitro SAR, molecular modelling and in vivo activity†
Toby Jackson,a L. W. Lawrence Woo,a Melanie N. Trusselle,a Surinder K. Chander,b Atul Purohit,b
Michael J. Reedb and Barry V. L. Potter*a
Received 23rd May 2007, Accepted 18th July 2007
First published as an Advance Article on the web 7th August 2007
DOI: 10.1039/b707768h
The synthesis and biological evaluation of a series of novel Dual Aromatase–Sulfatase Inhibitors
(DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase
enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of
hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase
inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme
inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent
irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished
via selective radical bromination and substitution reactions to furnish a series of aromatase inhibitory
pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed SN2, Heck
and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via
sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC50
3.5 nM), comparable with that of Anastrozole (IC50 1.5 nM) whereas, only moderate activity against
steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition.
Compound 11 was modelled into the active site of a homology model of human aromatase and the
X-ray crystal structure of steroid sulfatase.
inhibition of the enzymes involved in this process, e.g. aromatase
Introduction
and steroid sulfatase (STS). The inhibition of aromatase, which
In 2003 there were 36 509 new cases of breast cancer diagnosed in
converts androgens to oestrogens in the final step of steroid
biosynthesis (see Fig. 1), has been the focus of extensive research2,3
,
England. This represented 32% of all cancers in women and was
the most common cause of female cancer related death. However,
despite an increase in incidence, mortality rates are declining as a
result of earlier detection and improved treatment.1 Four in five
cases of breast cancer are diagnosed in women over the age of 50
with the postmenopausal 50–64 age group the most at risk. In the
majority of these cases breast tumours are found to be hormone-
dependent, with oestrogens playing a key role in the growth and
development of the disease. This has led to the development of
endocrine therapies to remove the influence of oestrogen on breast
cancer cells. Currently, the preferred strategy to tackle hormone-
dependent breast cancer (HDBC) involves blocking oestrogens at
the receptor level by using selective oestrogen receptor modula-
tors (SERMs) such as (Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-
N,N-dimethyl-ethanamine (Tamoxifen). Alternative approaches
concentrate on restricting the availability of oestrogens by inter-
fering with the steroid biosynthetic pathway, achievable via the
which, in particular, has resulted in two significant clinical non-
steroidal agents; 2-[3-(cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-
ylmethyl-phenyl]-2-methyl-propionitrile (Anastrozole) and 4-[(4-
cyanophenyl)–(1,2,4-triazol-1-yl)methyl]benzonitrile (Letrozole)
(Fig. 2), that show improved efficacies and superior toxicity
profiles when compared to Tamoxifen.4,5
Most oestrogens that orginate from the aromatase pathway are
stored in the body as steroid sulfates. It is now widely recognised
that this store provides an important source of oestrogens in
tumours when oestrone sulfate (E1S) is hydrolysed to oestrone
(E1) by STS (see Fig. 1).6–12 Considerable progress has been
made in developing steroidal and non-steroidal STS inhibitors
containing a phenol sulfamate ester pharmacophore.13,14 This
moiety effects highly potent irreversible STS inhibition. Indeed,
STX64 (now BN83495) (Fig. 2) has become the first STS inhibitor
to enter clinical trial and has shown highly promising Phase 1
results.15
We have previously reported and validated the concept of dual
aromatase–sulfatase inhibitors (DASIs).16–18 The rationale to this
hypothesis is that concomitant inhibition of aromatase and STS
should achieve a more effective oestrogen ablation than that
affected by singular inhibition of either enzyme. Whereas this
could be achieved by co-administrating two stand-alone drugs, or,
by administrating a fixed-dose bi-component drug (one tablet-two
agents), an alternative strategy is to design a dual-pharmacophore
single agent to act against both aromatase and STS. Recently,
aMedicinal Chemistry, Department of Pharmacy and Pharmacology and
Sterix Ltd, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: b.v.l.potter@bath.ac.uk; Fax: +44 1225 826114; Tel: +44 1225
386693
bEndocrinology and Metabolic Medicine and Sterix Ltd, Imperial College
London, Faculty of Medicine, St. Mary’s Hospital, London, W2 1NY, UK
† Electronic supplementary information (ESI) available: Synthesis and
spectroscopic data for Anastrozole and preceding intermediates following
two different routes, and experimental data for compounds 1, 2, 3(a–c),
4(a–c), 5(a–c) and 20–23. See DOI: 10.1039/b707768h
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Fig. 1 The contribution of the aromatase and sulfatase pathways to oestrogen synthesis from androstenedione and oestrone sulfate in HDBC. 17b-HSDs
are associated dehydrogenase/reductase enzymes.
compounds. The in vitro biological activities of these compounds
were evaluated in a JEG-3 cells assay from which extensive SAR
information is derived. The leading compound from this series,
compound 11 was studied in vivo alongside synthesised reference
compounds. This compound was also docked into a homology
model of the human aromatase enzyme20 and into the crystal
structure of STS21 in silico in order to understand how it might
interact with the individual enzymes.
Results and discussion
Chemistry
The synthesis of Anastrozole has been documented only in the
patent literature.22 We adapted two routes for the synthesis of
Fig. 2 Leading aromatase inhibitors (Anastrozole and Letrozole) and
STS inhibitor (STX64).
Anastrozole and report for the first time full spectroscopic and
analytical data (see ESI†). In contrast to Anastrozole the targeted
Morphy and Rankovic have reasoned that such a multiple-
ligand strategy, as opposed to developing a multicomponent drug
or drug cocktail, may provide significant pharmaceutical and
development advantages.19 Initial design of DASIs led to the
successful incorporation of the phenol sulfamate ester moiety
into the scaffold of potent aromatase inhibitors YM511,16 and
Letrozole.17 These first examples of DASIs were shown to exhibit
potent dual inhibition in vivo, introducing STS inhibition while
preserving a high degree of the aromatase inhibition inherent in
the original scaffold.
Adopting a similar strategy, we report here a new class of DASIs
that is structurally based upon the Anastrozole template. Whilst
retaining the haem-ligating triazole moiety key to the reversible
inhibition of aromatase, the removal of one of the two dimethyl-
propionitrile side-arms of Anastrozole allows the incorporation of
a phenol sulfamate ester group via linkers into the hybrid inhibitor.
The use of ether, thioether and alkene linking units, as well as
variation of the remaining dimethylpropionitrile side-arm to form
cycloalkanecarbonitriles, generates a structurally diverse set of
DASI compounds are synthetically more demanding, largely
owing to a loss of symmetry with three different substituents
around the central aromatic ring as opposed to two. Whereas
the preparation of Anastrozole involves the simultaneous con-
struction of two dimethylpropionitrile side-arms, the DASI motif
requires just one such side-arm with the second position left free
to allow for the incorporation of the irreversible STS inhibiting
pharmacophore. The synthetic strategy adopted for the synthesis
of these Anastrozole-based DASIs (see Scheme 1) involved
modification of one of the two routes to Anastrozole (see method
B, reference 22 and ESI†). This was achieved by starting with the
mono-bromination (instead of the di-bromination required for
Anastrozole) of methyl 3,5-dimethylbenzoate, using the reaction
conditions described by Kikuchi et al.,23 to give the benzyl bromide
1. Reaction of 1 with potassium cyanide gave the phenylacetoni-
trile 2 followed by gem-dimethylation to complete the carbonitrile
side-arm in 3(a). Variation of the carbonitrile side-arm was
possible by using 1,3-dibromopropane and 1,2-dibromoethane as
alkylating species to form the cyclic side-arms in 3(b) and 3(c)
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Scheme 1 Reagents and conditions: (i) NaHSO3, NaBrO3, H2O–EtOAc, 85%; (ii) KCN, TBAB, H2O–CH2Cl2, D, 75%; (iii) NaH, alkyl halide, THF,
35–78%; (iv) NaHSO3, NaBrO3, H2O–EtOAc, 75–96%; (v) 1,2,4-triazole, K2CO3, KI, acetone, D, 43–59%.
respectively.24 Incorporation of the haem-ligating heteroaryl unit
was achieved via bromination of the remaining tolyl functionality
of 3(a–c) to give 4(a–c) and then substitution with 1,2,4-triazole
to complete the Anastrozole derivatives 5(a–c) (Scheme 1).
Once these Anastrozole derivatives were complete the next
task was to introduce the STS inhibiting pharmacophore in
the form of phenol sulfamates (Scheme 2). Initially, the methyl
ester groups in 5(a–c) were reduced using sodium borohydride
in PEG400, a system which is inert towards nitrogen-containing
functional groups.25 Of the resulting benzyl alcohols, 6(a) was
converted to the unstable benzyl chloride 7, which was used with-
out purification or characterisation, and subsequently coupled
with 4-hydroxythiophenol to form the thioether compound 8.
Sulfamoylation26 of this compound afforded the first example of
this DASI series, 11. Derivatives of 11 were prepared by a modified
strategy (Scheme 2). Thioether intermediates were prepared by
an alternative transformation directly from the benzyl alcohols,
6(a–c), using (cyanomethyl)trimethylphosphonium iodide27 and
appropriately substituted thiophenols,28 to form the mesylated
compounds 9(a–e) and 9(f). Deprotection of the mesylated phenols
Scheme 2 Reagents and conditions: (i) NaBH4, PEG400, 80 ◦C, 64–78%; (ii) SOCl2, pyridine, DCM; (iii) 4-hydroxythiophenol, K2CO3, DMF, 26%;
(iv) (CH3)3P+CH2CN I−, DIPEA, EtCN, thiophenol or thiophenol mesylate, 30–92%; (v) 2 M NaOH(aq), THF, 68–99%; (vi) ClSO2NH2, DMA, 83–96%.
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Scheme 3 Reagents and conditions: (i) DEAD, triphenylphosphine polystyrene, 4-(benzyloxy)phenol, 82%; (ii) 5% Pd/C, THF–MeOH, 76%;
(iii) ClSO2NH2, DMA, 68%.
provided the free phenols 10(a–e). Incorporation of the phenol
sulfamate ester moiety was completed via direct application of
sulfamoyl chloride to the phenols (10(a–e)) to furnish the DASI
compounds 12–16.
In addition to the thioether linker between the Anastrozole
fragment and the phenol sulfamate ester fragment in 11, alter-
native linking groups were also introduced. An alkylether linked
DASI was prepared via a Mitsunobu reaction between 6(a) and 4-
(benzyloxy)phenol to form 17 (Scheme 3). Debenzylation followed
to give the free phenol 18 which was successfully converted to the
sulfamate 19.
An alkene linked DASI, compound 26, was also prepared
(Scheme 4). Starting with 5-bromo-m-xylene, mono-bromination
was undertaken to form the benzyl bromide 20. Substitution
with potassium cyanide gave 21 and the Anastrozole side-arm
was completed via dimethylation to give 22. Further bromination
of the remaining tolyl group yielded 23 and the Anastrozole
derivative 24 was completed via substitution with 1,2,4-triazole.
A Heck reaction between this fragment and 4-vinylphenol gave
the phenolic precursor 25 which was sulfamoylated to furnish
26. Proton NMR confirms that 26 exists as one geometrical
isomer with the coupling constant between the signals for the vinyl
protons (1H NMR dH 7.35 and 1H NMR dH 7.38) being consistent
with that expected for a trans stilbene (JH–H = 16.6 Hz).
In relation to compound 11, two reference compounds were
prepared to assess the role of each pharmacophore on both target
enzymes. The synthesis of 9(f), which does not contain a phenol
sulfamate ester and therefore would not be expected to exhibit any
significant STS inhibitory activity, has been previously described
(see Scheme 2). The preparation of a control compound that
does not contain a triazole moiety and hence would be expected
to have relatively insignificant aromatase inhibition was also
undertaken (see Scheme 5). To this end, commercially available
2-(3,5-dimethylphenyl)acetonitrile was dimethylated to give the
gem-dimethyl compound 27 and then mono-brominated to yield
the benzyl bromide 28. Reaction with 4-hydroxythiophenol in
the presence of base furnished the phenol 29 which was then
sulfamoylated to give compound 30.
Inhibition of aromatase and sulfatase in vitro
The in vitro inhibitory activities of compounds 9(f), 11–16, 19, 26
and 30 against aromatase and STS in a JEG-3 cells preparation
are shown in Table 1. Anastrozole, synthesised as described,22
and STX64,29 established potent aromatase and STS inhibitors
respectively, are included in the same assay for reference.
All the compounds evaluated (except compound 30) show
potent inhibition of aromatase with IC50 values ranging from
Scheme 4 Reagents and conditions: (i) KCN, TBAB, H2O–DCM, D, 74%; (ii) NaH, CH3I (2 eq.), THF, 83%; (iii) NaHSO3, NaBrO3, H2O–EtOAc, 55%;
(iv) 1,2,4-triazole, K2CO3, KI, acetone, D, 64%; (v) Pd(OAc)2, PPh3, NEt3, 4-vinylphenol, THF, D, 71%; (vi) ClSO2NH2, DMA, 36%.
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Scheme 5 Reagents and conditions: (i) ((CH3)3Si)3NLi, −78 ◦C, CH3I (2 eq.), THF, 77%; (ii) NBS, Bz2O2, CCl4, D, 66%; (iii) 4-hydroxythiophenol,
K2CO3, DMF, 85%; (iv) ClSO2NH2, DMA, 74%.
Table 1 In vitro inhibition of aromatase and STS activity in JEG-3 cells by anastrozole, STX64 and compounds 9(f), 11–16, 19, 26 and 30
Aromatase IC50 or
% inhibition @ 1 lM
STS IC50 or % inhibition
@ 10 lM
Compound
R1 (X = OSO2NH2)
R2
R3
Anastrozole
STX64
9(f)
—
—
—
—
CH3
—
—
CH3
1.5 0.5 nM
300 42 nM
2.3 0.09 nM
<10%
1.5 0.3 nM
—
11
CH3
CH3
CH3
CH3
CH3
CH3
3.5 1.5 nM
10.7 0.8%
12
13
14
15
16
4.2 0.10 nM
5.0 0.43 nM
20.7 4.2 nM
97.1 0.3%
3000 200 nM
73.6 0.1%
21.4 7.9%
75.3 0.6%
<10%
–CH2CH2CH2–
–CH2CH2CH2–
–CH2CH2–
3.4 0.28 nM
19
26
30
CH3
CH3
—
CH3
CH3
—
5.5 0.2 nM
25.5 2.7 nM
<10%
<10%
21.5 4.7%
38.5 2%
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3.5–25.5 nM. The best aromatase inhibitor is the non-
sulfamoylated compound 9(f) (IC50 2.3 nM), although the leading
DASI 11 (3.5 nM) is similarly potent. Given the IC50 for
Anastrozole is 1.5 nM the introduction of the phenol sulfa-
mate ester STS inhibiting pharmacophore at the expense of a
dimethylpropionitrile side-arm is well tolerated in this series of
DASI compounds. With the exception of compounds 14 and 26,
all the other DASI compounds maintain aromatase inhibition
within an order of magnitude of that of Anastrozole. The lack
of a triazole group in compound 30 results in a complete loss
of aromatase inhibitory activity. This confirms that the nitrogen-
containing heterocycle triazole is crucial for potent aromatase
inhibition in these non-steroidal inhibitors. Although our previous
DASI work16 has shown the introduction of a halogen ortho to the
sulfamate group to be beneficial to aromatase inhibition, possibly
due to steric and/or lipophilic factors, no significant effect is
observed between the unsubstituted compound 11, the chloro
compound 12 (IC50 4.2 nM) and the bromo compound 13 (IC50
5.0 nM). Converting the dimethylpropionitrile side-arm in 11 to
a cyclopropanecarbonitrile group in 16 (IC50 3.4 nM) results in
no significant change in activity. However, the introduction of a
cyclobutanecarbonitrile side-arm in 14 (IC50 20.7 nM) causes a six-
fold reduction in potency against aromatase, presumably due to
unfavourable steric interactions affecting the binding motif of the
molecule in the aromatase active site. Replacement of the thioether
in 11 with an ether linker in compound 19 (IC50 5.5 nM) results in
a minimal effect on activity despite the decrease in lipophilicity the
introduction of an oxygen atom imparts. However, replacement of
the thioether linker with a trans alkene group is clearly deleterious
for aromatase inhibition, as borne out by the seven-fold difference
in activity between 11 (IC50 3.5 nM) and 26 (25.5 nM). It seems
reasonable to suggest that the alkene linker of 26 restricts the
conformational flexibility of the molecule between the two phenyl
rings which, in turn, reduces the binding affinity of 26 to the
aromatase active site.
Fig. 3 Docking of Anastrozole (pink) and 11 (green) into the human
aromatase homology model of Favia et al.20 using the GOLD docking
program version 3.1.1.30 The haem iron atom is represented by a purple
ball and active site amino acid residues are indicated.
˚
in compound 11 appearing to be in the closest proximity (3.0 A)
to this residue. The phenol sulfamate ester group of 11 is in close
proximity to His475, His480 and Trp224 and there is a potential
for stabilizing interactions of this group with one or more of these
amino acids.
From the set of compounds tested there are no examples
of strong STS inhibitors in vitro. Of the best examples the
ortho chlorinated derivative 12 has an IC50 of 3000 nM which
is significantly less potent than STX64 (1.5 nM). Some trends are
apparent, however. The presence of the triazole group introduces
increased possibility of steric constraints for effective binding
within the STS active site. Indeed, the comparative activities of
30, and its triazole substituted analogue 11 (38.5% versus 10.7%
inhibition at 10 lM) appear to bear this out. Secondly, as antici-
pated, and in line with previous observations,16 the introduction of
a halogen atom ortho to the sulfamate group increases the ability
of the resulting compounds to inhibit STS (cf. 12 and 13 with 11
and also 14 versus 15). The electron-withdrawing effect produced
by the halogen atom improves the leaving group ability of the
corresponding phenol which, inter alia, facilitates the inactivation
of STS through sulfamoylation. Finally, trans-stilbenes are known
to structurally mimic oestrogens31 and hence, their sulfamate ester
derivatives may mimic oestrogen sulfamates. However, although
oestrone 3-O-sulfamate is a potent STS inhibitor, the stilbene
diethylstilboestrol monosulfamate is relatively much less active.32
Similarly, 26, a congener of diethylstilboestrol monosulfamate, is
also a weak inhibitor of STS in vitro.
Compounds 11, together with the STS inhibitor STX64, were
docked into the crystal structure of STS.21 As illustrated in Fig. 4,
the DASI compound occupies the same region of the active site as
STX64. The sulfamate groups lie in close proximity to the Ca2+ ion
(purple ball) and the gem-diol form of the formylglycine residue in
the catalytic cavity. However, it is likely that, compared to STX64,
compound 11 with a larger more flexible motif, particularly
taking into account the protruding triazolylmethyl moiety and
the thioether linker, does not interact as efficiently with the amino
acid residues in the hydrophobic tunnel approaching the active
A molecular modelling study was performed to identify possible
ligand–enzyme interactions by which Anastrozole and compound
11 might exert inhibitory effects on aromatase. Both were docked
into the human aromatase homology model20 using GOLD.30 The
second highest scoring modes of both inhibitors demonstrate
a satisfactory representation of triazole ligation to the haem
and are illustrated in Fig. 3 with Anastrozole depicted in pink,
compound 11 in green and the haem iron atom as a purple ball.
The substructures common to both ligands, the central phenyl
ring, the triazolylmethyl moiety and one dimethylpropionitrile
group, occupy the same space in the enzyme active site. Potential
hydrogen bonding interactions are observed between the main
chain nitrogen atom of Leu479 and the cyano component of
the dimethylpropionitrile groups in both inhibitors. A number of
lipophilic residues lining the enzyme active site (Val370, Leu477,
Val373, Phe134, Ile305) form contacts with the common aromatic
ring and the nitrile groups. The Ser478 side chain, which has
been implicated in the binding of the non-steroidal inhibitor
Vorozole,20 is in close proximity (4.6 A) to the cyano group
˚
of the dimethylpropionitrile side arm of 11. With induced fit
these groups may form a hydrogen bond in the enzyme active
site. It is interesting to note there is the possibility for both the
dimethylpropionitrile groups in Anastrozole to form hydrogen
˚
˚
bonds with Ser478 (5.1 A and 3.0 A) with the group replaced
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inhibitor and/or delivering the active species to the target enzyme
in the in vivo setting. Whereas, and as expected, Anastrozole,
without a phenol sulfamate ester moiety, was completely inef-
fective in suppressing STS activity, complete inhibition (100%) of
aromatase, shown as a measure of plasma E2 levels, was achieved
at 10 mg kg−1 p.o. The DASI compound 11 showed only 70%
inhibition against aromatase, which is a low figure in contrast to
Anastrozole when taking into consideration the similarity of the
in vitro IC50 values for these two compounds. It is possible that
other factors such as DMPK issues could be responsible for this
result. Compound 30, however, in agreement with the in vitro
results (Table 1), showed no significant inhibition against
aromatase due to the absence of a haem-ligating triazole group.
Conclusions
Fig. 4 Docking of 11 (green) and STX64 (purple) into the active site of
STS. The Ca2+ ion is depicted as a purple ball. Hydrated FGly75 is the
crucial mechanistic residue for desulfation.
A series of novel dual enzyme inhibitors has been synthesised,
based upon the clinically potent aromatase inhibitor Anastrozole.
These compounds include not only a haem-ligating triazole group
important for aromatase inhibition, but also a phenol sulfamate
ester group to act against STS activity. Evaluation in vitro
shows these compounds to be highly potent towards aromatase
with inhibitory activities close to the Anastrozole benchmark
(IC50 = 1.5 nM). In contrast, the level of STS inhibition in vitro
is not as potent across the series of compounds tested when
compared to the non-steroidal STS inhibitor STX64. However,
when compound 11 was tested in vivo, both the plasma E2 levels
and liver STS activity were inhibited potently (STS: 83% and
Aromatase: 70%) confirming that 11 is indeed a DASI. Analysis
of the structure activity relationship for these compounds suggests
that the incorporation of the second pharmacophore in the DASI
compounds results in a slight impediment to the activity against
both enzymes when compared to the (pharmacophores working
as) independent entities. However, any decrease in inhibitory
activity against one enzyme is expected to be largely compensated
by the dual inhibition provided by these DASIs, which may
strengthen the overall effectiveness of this type of endocrine
therapy. Optimisation of this new structural class of DASI is still
required to maximise the potencies against both aromatase and
STS, but this series is clearly worthy of further development. With
site. The modes shown represent the binding event before any
irreversible interaction takes place.
Inhibition of aromatase and sulfatase in vivo
Compounds 11, 30 and Anastrozole were studied in vivo (Fig. 5).
Female Wistar rats pre-treated with 200 IU/0.1 mL s.c. of PMSG
(pregnant mares’ serum gonadotropin) were treated 3 days later
with individual agent at a single 10 mg kg−1 p.o. dose. Plasma
oestradiol (E2) levels and liver STS activity were determined
3 hours after dosing.
At 10 mg kg−1 p.o. 11 and 30 were found to be highly active
against STS, with near complete inhibition observed for 30 (98.5%)
and 83% inhibition evident for 11. These are encouraging results
given that these compounds were observed to be only weak
STS inhibitors in vitro (Table 1). However, this phenomenon
is not unprecedented and the inhibitory activity difference of
phenol sulfamate esters in vitro and in vivo has been previously
observed.16,17 Although the explanation for such a phenomenon
is yet to be elucidated, other DMPK (Drug Metabolism and
Pharmacokinetics) factors could be involved in transforming the
Fig. 5 % Inhibition of aromatase and STS activities in 7 Wistar rats. Results are expressed as the % of PMSG stimulated E2 levels or % of liver STS
activity in untreated rats (means se, n = 3). NS, not significant. * Figures above bars represent % inhibition. ** 2 rats gave >80% inhibition, 1 rat
gave ∼50%.
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the DASI concept validated, the development of a novel effective
treatment for HDBC is a realistic possibility.
glycol (10 : 90) as a single dose. Blood and liver samples were
obtained 3 h after drug administration. Plasma concentrations
of oestradiol were measured using a radio-immunoassay kit
(Diagnostic Products Corporation, CA) to monitor the extent
of aromatase inhibition. Liver STS activity was determined to
assess the extent of STS inhibition. Results are expressed as the
percentage of PMSG stimulated oestradiol levels for aromatase
activity or percentage of activity in untreated animals for STS
activity (means se, n = 3).
Experimental
Chemistry
Unless otherwise stated HPLC grade solvents were used and
commercial reagents and starting materials were used without
further purification. Nuclear magnetic resonance spectra were
recorded on either a Jeol Delta 270 MHz or Varian Mercury
Molecular modelling
1
VX 400 MHz spectrometer. H NMR spectra were recorded at
i) Homology model of the human aromatase enzyme. Models
of Anastrozole and 11 were built in the SYBYL7.135 molecular
modelling program. To obtain low energy conformations of these
models energy minimization was performed to convergence using
the MMFF94s force field with MMFF94 charges.
270 MHz or 400 MHz with shifts reported in parts per million
(ppm, d) relative to residual chloroform (dH = 7.26 ppm) or residual
DMSO (dH = 2.50 ppm). Coupling constants, J, are reported in
Hertz. 13C NMR spectra were recorded at either 67.9 MHz or
100.6 MHz with the central peak of chloroform (dC = 77.16 ppm)
or DMSO (dC = 39.52 ppm) as internal standard. Low resolution
mass spectra were obtained from a Micromass platform LCZ
(APCI+). FAB high-resolution mass spectra were determined by
the EPSRC mass spectrometry centre (Swansea, UK). HPLC
analyses were performed on a Waters 996 PDA detector using
A homology model of the human aromatase enzyme, which is
based on the crystal structure of the human CYP2C9 metabolic
enzyme,36 as described by Favia et al.20 (PDB accession code
1TQA) was read into using SYBYL7.1. Hydrogen atoms were
built onto the model and all atoms except hydrogens were fixed
in aggregates. Hydrogen atom positions were then optimized to
convergence using the Tripos force field with Gasteiger–Hu¨ckel
charges.
R
a Symmetryꢀ C18 column (4.6 × 150 mm) eluting with MeCN–
H2O at 1.0 mL min−1. Elemental analyses were performed by the
Microanalysis Service, University of Bath. Melting points were
determined using a Stanford Research Systems Optimelt MPA100
automated melting point system and are uncorrected. Thin layer
chromatography was undertaken using Kieselgel 60 F254 plates
(Merck). Flash column chromatography was performed on silica
gel (Sorbsil/Matrex C60) or using Argonaut pre-packed columns
with FlashMaster II. Sulfamoyl chloride was prepared by an
adaptation of the method of Appel and Berger,33 and was stored as
a solution in toluene as described by Woo et al.34 The experimental
data for 1, 2, 3(a–c), 4(a–c), 5(a–c), 20–23 are available as ESI.†
The GOLD30 docking program v3.1.1 was used to dock
the Anastrozole and 11 models to the aromatase model. The
˚
aromatase active site was defined as a 12 A sphere around
the haem group iron atom. A distance constraint (minimum =
˚
˚
2.00 A, maximum = 2.30 A) was applied between the ligating
triazole nitrogen atom of the ligand to the haem iron atom. The
coordination number of the iron atom was defined as 6. The
ligands were then docked to the enzyme a total of 25 times each
using the GOLDScore fitness function.
ii) Crystal structure of steroid sulfatase. The 1P49.pdb crys-
tal structure of Human Placental Oestrone/DHEA Sulfatase was
used for the building of the gem-diol form of STS.21 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
SYBYL7.1. The resulting structure was then minimised with the
backbone atoms fixed to allow the gem-diol and surrounding side
chain atoms to adopt low energy confirmations. Minimisations
were undertaken using SYBYL7.1 applying the AMBER7 99
forcefield with Gasteiger–Hu¨ckel charges as implemented within
SYBYL7.1. In order to mimic the sulfamate group of E1S, all
sulfamate based compounds are docked into the active site with
their sulfamate group in its mono-anionic form (i.e. –OSO2NH−).37
Biology
The extent of in vitro inhibition of aromatase and STS activities
was assessed using intact monolayers of JEG-3 cells. Cells were
seeded into 24-well culture plates and maintained in MEM
(Flow Laboratories, Irvine, UK) containing supplements and
used when 80% confluent. To determine STS activity, cells were
incubated for 1 h with [6,7–3H]E1S (5 pmol, 7 × 105 dpm,
60 Ci/mmol; Perkin Elmer LS, Wellesley, MA) in the presence
or absence of (0.001–10 000 nmol L−1) inhibitor. The product E1
was separated from E1S by a toluene partition using [4–14C]E1 to
monitor procedural losses, and the radioactivity was measured by
scintillation spectrometry. Similarly, for aromatase activity, [1b-
3H]androstenedione (5 pmol, 30 Ci/mmol, Perkin Elmer LS, MA)
was incubated with JEG-3 cells for 1 h in the presence or absence
of inhibitor. The product, 3H2O, was separated from the substrates
using dextran-coated charcoal at 4 ◦C for 2 h and remaining
radioactivity measured by scintillation spectrometry. Each IC50
value represents the mean SE of triplicate measurements.
The in vivo inhibition of aromatase and STS activity by
11 and STX64 was assessed in female Wistar rats. Animals
received a single subcutaneous injection of pregnant mare’s
serum gonadotropin (PMSG, 200 IU, Sigma). Three days later
drugs (10 mg kg−1) were administered orally in THF–propylene
Syntheses
2-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethylphenyl)-2-methyl-
propionitrile 6(a). A mixture of compound 5(a) (0.500 g,
1.76 mmol) and polyethylene glycol 400 (6.0 g) was heated to
80 ◦C with stirring until a clear solution had formed. NaBH4
(0.200 g, 5.28 mmol) was added carefully resulting in evolution of
gas. The reaction mixture was stirred vigorously at 80 ◦C for 16 h.
An extremely viscous residue formed that gradually dissolved in
CH2Cl2 (50 mL) with warming (30 ◦C). This solution was washed
with 1 M HCl(aq) (10 mL) and then carefully neutralised with
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NaHSO3 (aq). Washed with distilled H2O (50 mL × 4) and brine
(50 mL) and dried (MgSO4). Solvent was removed in vacuo to leave
a yellow viscous oil. Column chromatography (EtOAc) elu◦ted
6(a) as a white crystalline solid (0.351 g, 78%), mp 114–116 C.
1H NMR dH (270 MHz, CDCl3) 1.70 (6H, s, ArC(CH3)2CN),
2.19–2.28 (1H, t, J = 5.5 Hz, ArCH2OH), 4.69–4.71 (2H, d, J =
5.5 Hz, ArCH2OH), 5.35 (2H, s, ArCH2N), 7.17 (1H, s, ArH),
7.29 (1H, s, ArH), 7.44 (1H, s, ArH), 7.95 (1H, s, C2H2N3) and
8.09 (1H, s, C2H2N3); m/z (APCI+) 257 ((M + H)+, 100%), 188
(88); HRMS (ES+) 257.1392. C14H17N4O requires 257.1397.
Methanesulfonic acid 2-chloro-4-[3-(cyanodimethylmethyl)-5-
[1,2,4]triazol-1-ylmethylbenzylsulfanyl]phenyl ester 9(a). (Cyano-
methyl)trimethylphosphonium iodide (0.114 g, 0.470 mmol) was
added to a mixture of 6(a) (0.100 g, 0.390 mmol), methanesulfonic
acid-2-chloro-4-mercaptophenyl ester (0.136 g, 0.570 mmol),
diisopropylethylamine (88.0 lL, 0.510 mmol) and propionitrile
(1.0 mL) under inert conditions. The mixture was then set to stir at
92 ◦C. After 17 h the reaction was allowed to cool. CH2Cl2 (20 mL)
and distilled H2O (20 mL) were added and the aqueous layer
separated and extracted with CH2Cl2 (20 mL × 2). The organic
fractions were combined and washed with brine (20 mL), dried
(MgSO4) and solvent removed in vacuo to leave yellow residues.
Column chromatography (EtOAc) eluted 9(a) as a yellow viscous
1-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethylphenyl)cyclobu-
tanecarbonitrile 6(b). Compound 6(b) was prepared from 5(b)
using similar conditions to those described for the synthesis of
compound 6(a). Column chromatography (EtOAc) eluted 6(b) as
1
oil (0.114 g, 61%). H NMR dH (270 MHz, CDCl3) 1.60 (6H, s,
ArC(CH3)2CN), 3.19 (3H, s, ArOSO2CH3), 4.01 (2H, s, ArCH2S),
5.26 (2H, s, ArCH2N), 6.96 (1H, s, ArH), 7.07–7.12 (1H, dd, J =
2.3 & 6.3 Hz, ArH), 7.19–7.26 (4H, m, ArH), 7.92 (1H, s, C2H2N3)
and 8.04 (1H, s, C2H2N3); m/z (APCI+) 479 ((37ClM + H)+, 45%),
477 ((35ClM + H)+, 100).
1
a colourless viscous oil (0.238 g, 64%). H NMR dH (270 MHz,
CDCl3) 2.08–2.15 (1H, m, CH2), 2.20–2.23 (1H, t, J = 5.8 Hz,
ArCH2OH), 2.46–2.55 (1H, m, CH2), 2.61–2.68 (2H, m, CH2),
2.84–2.89 (2H, m, CH2), 4.76–4.77 (2H, d, J = 5.8 Hz, ArCH2OH),
5.41 (2H, s, ArCH2N), 7.25 (1H, s, ArH), 7.28 (1H, s, ArH), 7.45
(1H, s, ArH), 8.02 (1H, s, C2H2N3), and 8.15 (1H, s, C2H2N3); m/z
(APCI+) 269 ((M + H)+, 100%).
Methanesulfonic acid 2-bromo-4-[3-(cyanodimethylmethyl)-5-
[1,2,4]triazol-1-ylmethylbenzylsulfanyl]phenyl ester 9(b). Com-
pound 9(b) was prepared from 6(a) and 2-bromo-4-mercapto-
phenyl methanesulfonate using similar conditions to those
described for the synthesis of compound 9(a). Column chromato-
graphy (EtOAc) eluted 9(b) as a yellow viscous oil (0.152 g, 75%).
1H NMR dH (270 MHz, CDCl3) 1.62 (6H, s, ArC(CH3)2CN),
3.25 (3H, s, ArOSO2CH3), 4.04 (2H, s, ArCH2SAr), 5.31 (2H, s,
ArCH2N), 7.00 (1H, s, ArH), 7.16–7.31 (4H, m, ArH), 7.45–7.46
(1H, d, J = 2.2 Hz, ArH), 7.97 (1H, s, C2H2N3) and 8.10 (1H, s,
C2H2N3); m/z (APCI+) 523 ((81BrM + H)+, 100%), 521 ((79BrM +
H)+, 85).
1-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethyl-phenyl)-cyclo-
propanecarbonitrile 6(c). Compound 6(c) was prepared from
5(c) using similar conditions to those described for the synthesis
of compound 6(a). Chromatography (EtOAc) eluted 6(c) as a
1
colourless viscous oil (0.274 g, 78%). H NMR dH (270 MHz,
CDCl3) 1.36–1.40 (2H, dd, J = 2.5 & 5.3 Hz, CH2), 1.70–1.75
(2H, dd, J = 2.5 & 5.3 Hz, CH2), 1.89–1.94 (1H, t, J = 5.8 Hz,
ArCH2OH), 4.66–4.68(2H, d, J = 5.8 Hz, ArCH2OH), 5.31 (2H, s,
ArCH2N), 7.12 (1H, s, ArH), 7.14 (1H, s, ArH), 7.24 (1H, s, ArH),
7.95 (1H, s, C2H2N3), and 8.08 (1H, s, C2H2N3); m/z (APCI+) 255
((M + H)+, 100%); HRMS (ES+) 255.1237. C14H15N4O requires
255.1240
Methanesulfonic acid 4-[3-(1-cyanocyclobutyl)-5-[1,2,4]triazol-
1-ylmethylbenzylsulfanyl]phenyl ester 9(c). Compound 9(c) was
prepared from 6(b) and methanesulfonic acid 4-mercaptophenyl
ester using similar conditions to those described for the synthesis of
compound 9(a). Column chromatography (EtOAc) eluted 9(c) as a
yellow viscous oil (0.151 g, 92%). 1H NMR dH (270 MHz, CDCl3)
1.99–2.03 (1H, m, CH2), 2.32–2.58 (3H, m, CH2), 2.71–2.85 (2H,
m, CH2), 3.15 (3H, s, ArOSO2CH3), 4.06 (2H, s, ArCH2S), 5.29
(2H, s, ArCH2N), 6.97 (1H, s, ArH), 7.08–7.35 (6H, m, ArH), 7.97
(1H, s, C2H2N3) and 8.08 (1H, s, C2H2N3); m/z (APCI+) 455 ((M +
H)+, 100%).
2-[3-(4-Hydroxyphenylsulfanylmethyl)-5-[1,2,4]triazol-1-ylmethyl-
phenyl]-2-methylpropionitrile 8. To a solution of 6(a) (0.322 mg,
1.26 mmol) in anhydrous CH2Cl2 (15 mL) at 0 ◦C under inert
conditions was added anhydrous pyridine (0.15 mL, 1.88 mmol)
followed by SOCl2 (0.14 mL, 1.88 mmol). The reaction mixture
was stirred at room temperature for 2 h and then refluxed for
1 h. After cooling, solvent was removed in vacuo to obtain 7 as
a brown oil. Compound 7 was dissolved in anhydrous DMF
(5 mL) at room temperature and under inert conditions and
treated with K2CO3 (1.74 g, 12.6 mmol) followed by a solution
of 4-hydroxythiophenol (0.196 g, 1.51 mmol) in anhydrous DMF
(0.5 mL). The resulting dark yellow suspension was heated at
50 ◦C for 18 h and then diluted with EtOAc (40 mL). The organic
layer was washed with brine (50 mL), dried (MgSO4), filtered and
solvent removed in vacuo to give dark brown residues. Column
chromatography (EtOAc) eluted 8 as a light brown viscous oil
(0.090 g, 20%). 1H NMR dH (400 MHz, CDCl3) 1.66 (6H, s,
ArC(CH3)2CN), 3.87 (2H, s, ArCH2S), 5.25 (2H, s, ArCH2N),
6.68 (2H, m, ArH), 6.74 (1H, s, ArH), 7.04 (2H, m, ArH), 7.19
(1H, m, ArH), 7.24 (1H, m, ArH), 7.95 (1H, s, C2H2N3) and
7.99 (1H, s, C2H2N3) and 8.70 (1H, br s, OH); m/z (ES+) 365
((M + H)+, 100%), 296 (7), 269 (8); HRMS (FAB+) 365.1441
C20H21N4OS requires 365.1436.
4-({3-[(1H-1,2,4-triazol-1-yl)methyl]-5-(1-cyanocyclobutyl)ben-
zyl}sulfanyl)-2-chlorophenyl methanesulfonate 9(d). Compound
9(d) was prepared from 6(b) and methanesulfonic acid-2-chloro-
4-mercaptophenyl ester using similar conditions to those described
for the synthesis of compound 9(a). Column chromatography
1
(EtOAc) eluted 9(d) as a yellow viscous oil (0.054 g, 30%). H
NMR dH (270 MHz, CDCl3) 1.96–2.08 (1H, m, CH2), 2.31–2.58
(3H, m, CH2), 2.70–2.83 (2H, m, CH2), 3.24 (3H, s, ArOSO2CH3),
4.05 (2H, s, ArCH2S), 5.31 (2H, s, ArCH2N), 7.00 (1H, s, ArH),
7.08–7.32 (5H, m, ArH), 7.96 (1H, s, C2H2N3) and 8.10 (1H, s,
C2H2N3); m/z (APCI+) 489 ((M + H)+, 100%).
Methanesulfonic acid 4-[3-(1-cyanocyclopropyl)-5-[1,2,4]triazol-
1-ylmethylbenzylsulfanyl]phenyl ester 9(e). Compound 9(e) was
prepared from 6(c) and methanesulfonic acid 4-mercaptophenyl
ester using similar conditions to those described for the synthesis
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of compound 9(a). Column chromatography (EtOAc) eluted 9(e)
as a yellow viscous oil (0.134 g, 77%). H NMR dH (270 MHz,
m, CH2), 2.31–2.58 (3H, m, CH2), 2.70–2.82 (2H, m, CH2), 3.86
(2H, s, ArCH2S), 5.23 (2H, s, ArCH2N), 6.66–6.72 (3H, m, ArH),
7.01–7.08 (2H, dd, J = 2.2 & 6.7, ArH), 7.15 (2H, s, ArH), 7.92
(1H, s, C2H2N3), 7.99 (1H, s, C2H2N3) and 8.18 (1H, br s, ArOH);
m/z (APCI+) 377 ((M + H)+, 100%).
1
CDCl3) 1.29–1.34 (2H, dd, J = 5.0 & 7.9 Hz, CH2), 1.69–1.74
(2H, dd, J = 5.0 & 7.9 Hz, CH2), 3.16 (3H, s, ArOSO2CH3), 4.01
(2H, s, ArCH2S), 5.27 (2H, s, ArCH2N), 6.94 (1H, s, ArH), 7.05
(1H, s, ArH), 7.08 (1H, s, ArH), 7.14–7.28 (4H, dd, J = 8.6 &
27.9 Hz, ArH), 7.96 (1H, s, C2H2N3) and 8.07 (1H, s, C2H2N3);
m/z (APCI+) 441 ((M + H)+, 100%).
1-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-[(3-chloro-4-hydroxyphenyl-
thio)methyl]phenyl}cyclobutanecarbonitrile 10(d). Compound
10(d) was prepared from 9(d) using similar conditions to those
described for the synthesis of compound 10(a) and was obtained
as a light yellow viscous oil (0.039 g, 99%). 1H NMR dH
(270 MHz, CDCl3) 1.92–2.12 (1H, m, CH2), 2.31–2.62 (3H, m,
CH2), 2.65–2.88 (2H, m, CH2), 3.89 (2H, s, ArCH2SAr), 5.28
(2H, s, ArCH2N), 6.81–7.28 (6H, m, ArH), 7.99 (1H, s, C2H2N3),
7.99 (1H, s, C2H2N3) and 8.06 (1H, br s, ArOH); m/z (APCI+)
411 ((M + H)+, 100%).
2-Methyl-2-(3-phenylsulfanylmethyl-5-[1,2,4]triazol-1-ylmethyl-
phenyl)propionitrile 9(f). Compound 9(f) was prepared from 6(a)
and thiophenol using similar conditions to those described for the
synthesis of compound 9(a). Column chromatography (EtOAc)
eluted 9(f) as a yellow oil (0.047 g, 68%). 1H NMR dH (270 MHz,
CDCl3) 1.61 (6H, s, ArC(CH3)2CN), 4.04 (2H, s, ArCH2SAr),
5.30 (2H, s, ArCH2N), 7.05 (1H, s, ArH), 7.18–7.25 (7H, m, ArH),
7.97 (1H, s, C2H2N3) and 8.01 (1H, s, C2H2N3); 13C NMR dC
(100.6 MHz, CDCl3) 29.1 (CH3), 37.1 (C), 39.2 (CH2), 53.3 (CH2),
123.6 (CH), 124.1 (C), 125.9 (CH), 127.2 (CH), 127.9 (CH), 129.1
(CH), 131.1 (CH), 135.0 (C), 135.8 (C), 139.9 (C), 142.6 (C), 143.2
(CH) and 152.4 (CH); m/z (APCI+) 349 ((M + H)+, 100%); HRMS
(ES+) 349.1481.C20H21N4S requires 349.1481.
1-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-[(4-hydroxyphenylthio)-
methyl]phenyl}cyclopropanecarbonitrile 10(e). Compound 10(e)
was prepared from 9(e) using similar conditions to those described
for the synthesis of compound 10(a). Column chromatography
(CH2Cl2–acetone 80 : 20) eluted 10(e) as a white solid (0.076 g,
72%), mp 135–137 ◦C. 1H NMR dH (270 MHz, CDCl3) 1.31–1.35
(2H, dd, J = 5.2 & 8.0 Hz, CH2), 1.68–1.73 (2H, dd, J = 5.2
& 8.0 Hz, CH2), 3.83 (2H, s, ArCH2S), 5.21 (2H, s, ArCH2N),
6.65–6.70 (3H, m, ArH), 6.99–7.04 (3H, m, ArH), 7.11 (1H, s,
ArH), 7.91 (1H, s, C2H2N3), 7.99 (1H, s, C2H2N3) and 8.12 (1H, s,
ArOH); m/z (APCI+) 363 ((M + H)+, 100%).
2-[3-(3-Chloro-4-hydroxyphenylsulfanylmethyl)-5-[1,2,4]triazol-
1-ylmethylphenyl]-2-methylpropionitrile 10(a). Compound 9(a)
(0.100 g, 0.210 mmol) was dissolved in THF (2.5 mL) and MeOH
(1.5 mL) to which 2 M NaOH(aq) (0.52 mL) was added. The mixture
was set to stir at room temperature for 1 h. THF was removed
in vacuo and the residues taken up in EtOAc (20 mL) and washed
with 2 M KHSO4 (aq) (20 mL), distilled H2O (20 mL × 2) and
brine (20 mL). The organic layer was then dried over MgSO4
and solvent removed in vacuo to leave a colourless viscous oil.
Column chromatography (CH2Cl2–acetone 80 : 20) eluted 10(a)
as a colourless viscous oil that crystallised on st◦anding to a white
Sulfamic acid 4-[3-(cyanodimethylmethyl)-5-[1,2,4]triazol-1-
ylmethylbenzylsulfanyl]phenyl ester 11. Sulfamoyl chloride in
toluene (1.94 mL, 1.17 mmol) was transferred to a reaction vessel
and the solvent removed under vacuum at 30 ◦C. On cooling a
white solid formed to which was added a solution of 8 (0.085 g,
0.233 mmol) in N,N -dimethylacetamide (1.5 mL) at 0 ◦C to form
a colourless solution. The reaction mixture was left to stir at
room temperature under inert conditions for 20 h. The reaction
mixture was then poured into EtOAc (30 mL) and washed with
distilled H2O (30 mL × 4) and brine (30 mL). Dried (MgSO4)
and solvent removed in vacuo to leave off white residues. Column
chromatography (CH2Cl2–acetone 80 : 20) eluted 11 as a colourless
1
crystalline solid (0.071 g, 84%), mp 131–132 C. H NMR dH
(400 MHz, DMSO-d6) 1.59 (6H, s, ArC(CH3)2CN), 4.10 (2H, s,
ArCH2SAr), 5.43 (2H, s, ArCH2N), 6.85–6.87 (1H, d, J = 8.6 Hz,
ArH), 7.07–7.10 (1H, dd, J = 2.3 & 8.6 Hz, ArH), 7.14 (1H, s,
ArH), 7.20 (1H, s, ArH), 7.26 (1H, s, ArH), 7.34 (1H, s, ArH), 8.00
(1H, s, C2H2N3), 8.66 (1H, s, C2H2N3) and 10.37 (1H, s, ArOH);
m/z (APCI+) 401 ((37ClM + H)+, 30%), 399 ((35ClM + H)+, 100).
1
viscous oil (0.060 g, 58%). H NMR dH (400 MHz, CDCl3) 1.73
2-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-[(3-bromo-4-hydroxyphenyl-
thio)methyl]phenyl}-2-methylpropanenitrile 10(b). Compound
10(b) was prepared from 9(b) using similar conditions to those
described for the synthesis of compound 10(a). Column chro-
matography (CH2Cl2–acetone 80 : 20) eluted 10(b) as a colourless
(6H, s, ArC(CH3)2CN), 4.04 (2H, s, ArCH2SAr), 5.25 (2H, s,
CH2N), 6.58 (1H, s, ArH), 6.87 (2H, br s, ArOSO2NH2), 7.15
(4H, s, ArH), 7.23 (1H, s, ArH), 7.34 (1H, m, ArH), 7.57 (1H, s,
C2H2N3) and 7.85 (1H, s, C2H2N3); 13C NMR dC (100.6 MHz,
DMSO-d6) 28.2 (CH3), 36.5 (C), 36.8 (CH2), 51.8 (CH2), 122.8
(C), 123.6 (CH), 124.4 (CH), 125.4 (CH), 127.5 (CH), 130.1 (CH),
133.8 (C), 137.3 (C), 138.8 (C), 142.0 (C), 144.4 (CH), 148.5 (C)
and 151.9 (CH); m/z (ES+) 444 ((M + H)+, 100%); HRMS (FAB+)
444.1170. C20H22N5O3S2 requires 444.1164.
1
viscous oil (0.087 g, 68%). H NMR dH (270 MHz, CDCl3) 1.63
(6H, s, ArC(CH3)2CN), 3.90 (2H, s, ArCH2SAr), 5.29 (2H, s,
ArCH2N), 6.75 (1H, br s, ArOH), 6.83–6.87 (2H, m, ArH),
7.06–7.09 (1H, dd, J = 2.2 & 8.4 Hz, ArH), 7.14 (1H, s, ArH),
7.24 (1H, s, ArH), 7.30–7.31 (1H, d, J = 2.2 Hz, ArH), 7.99
(1H, s, C2H2N3) and 8.04 (1H, s, C2H2N3); m/z (APCI+) 445
((81BrM + H)+, 89%), 443 ((79BrM + H)+, 100).
Sulfamic acid 2-chloro-4-[3-(cyanodimethylmethyl)-5-[1,2,4]-
triazol-1-ylmethylbenzylsulfanyl]phenyl ester 12. Compound 12
was prepared from 10(a) using similar conditions to those
described for the synthesis of compound 11. Column chromato-
graphy (CH2Cl2–acetone 80 : 20) eluted 12 as a white solid
(0.051 g, 85%), mp 120–124 ◦C. 1H NMR dH (270 MHz, DMSO-
d6) 1.62 (6H, s, ArC(CH3)2CN), 4.32 (2H, s, ArCH2S), 5.44 (2H, s,
ArCH2N), 7.21 (1H, s, ArH), 7.34–7.39 (3H, m, ArH), 7.45 (1H, s,
ArH), 7.53–7.54 (1H, d, J = 1.5 Hz, ArH), 7.99 (1H, s, C2H2N3),
1-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-[(4-hydroxyphenylthio)-
methyl]phenyl}cyclobutanecarbonitrile 10(c). Compound 10(c)
was prepared from 9(c) using similar conditions to those described
for the synthesis of compound 10(a). Column chromatography
(CH2Cl2–acetone 80 : 20) eluted 10(c) as a colourless viscous oil
1
(0.091 g, 82%). H NMR dH (270 MHz, CDCl3) 1.93–2.03 (1H,
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Org. Biomol. Chem., 2007, 5, 2940–2952 | 2949
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8.27 (2H, br s, ArOSO2NH2) and 8.66 (1H, s, C2H2N3); 13C
NMR dC (100.6 MHz, DMSO-d6) 28.3 (CH3), 36.2 (CH2), 36.5
(C), 51.8 (CH2), 123.7 (CH), 124.1 (CH), 124.3 (C), 125.5 (CH),
127.0 (CH), 127.5 (CH), 128.3 (C), 129.6 (CH), 135.5 (C), 137.4
(C), 138.5 (C), 142.0 (C), 144.2 (C), 144.4 (CH) and 151.9 (CH);
m/z (APCI−) 478 ((37ClM − H)−, 18%), 476 ((35ClM − H)−, 40),
399 (40), 397 ((35ClM − C2H2N3)−, 100). HRMS (ES+) 478.0773.
C20H21ClN5O3S2 requires 478.0769.
Sulfamic acid 4-[3-(1-cyanocyclopropyl)-5-[1,2,4]triazol-1-
ylmethylbenzylsulfanyl]phenyl ester 16. Compound 16 was pre-
pared from 10(e) using similar conditions to those described for
the synthesis of compound 11. Column chromatography (CH2Cl2–
acetone 80 : 20) eluted 16 as a colourless viscous oil (0.055 g,
1
73%). H NMR dH (270 MHz, CDCl3) 1.39–1.44 (2H, dd, J =
5.0 & 7.9 Hz, CH2), 1.74–1.79 (2H, dd, J = 5.0 & 7.9 Hz, CH2),
4.01 (2H, s, ArCH2SAr), 5.21 (2H, s, ArCH2N), 6.55 (1H, s, ArH),
6.88 (2H, br s, ArOSO2NH2), 7.03 (1H, s, ArH), 7.11–7.21 (5H, m,
ArH), 7.54 (1H, s, C2H2N3) and 7.85 (1H, s, C2H2N3); 13C NMR dC
(67.9 MHz, CDCl3) 13.7 (CH2), 18.7 (C), 38.3 (CH2), 53.2 (CH2),
122.2 (C), 123.4 (CH), 123.9 (CH), 126.5 (CH), 126.6 (CH), 132.4
(CH), 132.9 (C), 135.6 (C), 137.6 (C), 140.1 (C), 142.9 (CH), 149.3
(C) and 151.1 (CH); m/z (APCI+) 442 ((M + H)+, 100%), 363 (35);
HRMS (ES+) 442.1002. C20H20N5O3S2 requires 442.1003.
Sulfamic acid 2-bromo-4-[3-(cyanodimethylmethyl)-5-[1,2,4]-
triazol-1-ylmethylbenzylsulfanyl]phenyl ester 13. Compound 13
was prepared from 10(b) using similar conditions to those
described for the synthesis of compound 11. Column chromato-
graphy (CH2Cl2–acetone 80 : 20) eluted 13 as a white solid (0.082 g,
96%), mp 111–115 ◦C. 1H NMR dH (270 MHz, CDCl3) 1.63 (6H, s,
ArC(CH3)2CN), 4.06 (2H, s, ArCH2SAr), 5.28 (2H, s, ArCH2N),
6.54 (1H, s, ArH), 6.97–7.01 (1H, dd, J = 2.2 & 8.6 Hz, ArH),
7.09 (2H, br s, ArOSO2NH2), 7.25–7.31 (2H, m, ArH), 7.35 (1H, s,
ArH), 7.40–7.41 (1H, d, J = 2.2 Hz, ArH), 7.63 (1H, s, C2H2N3)
and 7.87 (1H, s, C2H2N3); 13C NMR dC (100.6 MHz, DMSO-d6)
28.7 (CH3), 36.3 (C), 36.5 (CH2), 51.8 (CH2), 116.5 (C), 123.6
(C), 123.7 (CH), 124.3 (CH), 125.4 (CH), 127.5 (CH), 129.0 (CH),
132.6 (CH), 135.6 (C), 137.4 (C), 138.5 (C), 142.0 (C), 144.4 (CH),
145.6 (C) and 151.9 (CH); m/z (APCI+) 524 ((81BrM + H)+, 100%),
522 ((79BrM + H)+, 80). HRMS (ES+) 522.0267. C20H21BrN5O3S2
requires 522.0264.
2-(3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-{[4-(benzyloxy)phenoxy]-
methyl}phenyl)-2-methylpropanenitrile 17. Under inert con-
ditions compound 6(a) (0.150 g, 0.586 mmol) and 4-(benzyloxy)-
phenol (0.098 g, 0.488 mmol) were dissolved in anhydrous
CH2Cl2 (3 mL) to form a clear solution to which was added
triphenylphosphine polystyrene (0.586 g, 0.586 mmol) and the
mixture cooled to 0 ◦C. DEAD (92.3 lL, 0.586 mmol) was added
drop-wise and the reaction allowed to warm to room temperature
and left to stir for 16 h. The reaction mixture was then filtered to
remove the polystyrene which was washed with CH2Cl2 (25 mL ×
6). These washings were combined with the filtrate and washed
with distilled H2O (25 mL × 2) and brine (25 mL) then dried
(MgSO4) and solvent removed in vacuo. Column chromatography
(EtOAc) eluted 17 as a light yellow solid (0.175 g, 82%), mp
Sulfamic acid 4-[3-(1-cyanocyclobutyl)-5-[1,2,4]triazol-1-
ylmethylbenzylsulfanyl]phenyl ester 14. Compound 14 was
prepared from 10(c) using similar conditions to those described
for the synthesis of compound 11. Column chromatography
(CH2Cl2–acetone 80 : 20) eluted 14 as a colourless viscous oil
◦
1
119.3–120.5 C. H NMR dH (270 MHz, CDCl3) 1.70 (6H, s,
ArC(CH3)2CN), 4.98 (2H, s, ArCH2O), 5.01 (2H, s, ArCH2O),
5.37 (2H, s, ArCH2N), 6.84–6.92 (4H, m, ArH), 7.29–7.49 (8H,
m, ArH), 7.99 (1H, s, C2H2N3) and 8.10 (1H, s, C2H2N3); m/z
(APCI+) 439 ((M + H)+, 100%).
1
(0.058 g, 55%). H NMR dH (270 MHz, CDCl3) 2.03–2.10 (1H,
m, CH2), 2.42–2.61 (3H, m, CH2), 2.77–2.82 (2H, m, CH2), 4.03
(2H, s, ArCH2SAr), 5.27 (2H, s, ArCH2N), 6.59 (1H, s, ArH), 6.88
(2H, br s, ArOSO2NH2), 7.11–7.33 (6H, m, ArH), 7.59 (1H, s,
C2H2N3) and 7.92 (1H, s, C2H2N3); 13C NMR dC (67.9 MHz,
CDCl3) 17.2 (C), 34.6 (CH2), 38.2 (CH2), 40.0 (CH2), 53.2 (CH2),
116.0 (C), 123.4 (CH), 124.0 (CH), 126.5 (CH), 126.8 (CH), 132.5
(CH), 132.7 (C), 135.6 (C), 140.1 (C), 141.2 (C), 143.9 (CH), 149.3
(C) and 151.2 (CH); m/z (APCI+) 456 ((M + H)+, 100%), 377
(20); HRMS (ES+) 456.1159. C21H22N5O3S2 requires 456.1159.
2-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-[(4-hydroxyphenoxy)-
methyl]phenyl}-2-methylpropanenitrile
18. Compound
17
(0.100 g, 0.228 mmol) was dissolved in THF (2.5 mL) and
MeOH (2.5 mL) to form a clear solution to which was added 5%
Pd/C (0.010 g) to form a black suspension on vigorous stirring.
The reaction vessel was degassed and the mixture set to stir
under a hydrogen atmosphere for 16 h. The reaction mixture
was filtered through Celite which was subsequently washed
with THF (30 mL × 2). Solvent was removed in vacuo to leave
brown residues. Column chromatography (EtOAc) eluted 18 as
Sulfamic acid 2-chloro-4-[3-(1-cyanocyclobutyl)-5-[1,2,4]triazol-
1-ylmethylbenzylsulfanyl]phenyl ester 15. Compound 15 was pre-
pared from 10(d) using similar conditions to those described for
the synthesis of compound 11. Column chromatography (CH2Cl2–
acetone 80 : 20) eluted 15 as a white amorphous solid (0.037 g,
1
a colourless viscous oil (0.060 g, 76%). H NMR dH (270 MHz,
CDCl3) 1.70 (6H, s, ArC(CH3)2CN), 4.97 (2H, s, ArCH2O), 5.17
(1H, s, ArOH), 5.37 (2H, s, ArCH2N), 6.73–6.82 (4H, m, ArH),
7.22 (1H, s, ArH), 7.33 (1H, s, ArH), 7.49 (1H, s, ArH), 7.99
(1H, s, C2H2N2) and 8.10 (1H, s, C2H2N2); m/z (APCI+) 349
((M + H)+, 100%).
1
77%). H NMR dH (270 MHz, CDCl3) 2.04–2.13 (1H, m, CH2),
2.44–2.67 (3H, m, CH2), 2.80–2.87 (2H, m, CH2), 4.06 (2H, s,
ArCH2SAr), 5.28 (2H, s, ArCH2N), 6.56 (1H, s, ArH), 6.89–6.97
(1H, dd, J = 2.2 & 8.6 Hz, ArH), 7.10 (2H, br s, ArOSO2NH2),
7.23–7.30 (4H, m, ArH), 7.62 (1H, s, C2H2N3) and 7.86 (1H, s,
C2H2N3); 13C NMR dC (67.9 MHz, DMSO-d6) 17.3 (C), 34.0
(CH2), 34.4 (CH2), 36.8 (CH2), 52.2 (CH2), 124.6 (CH), 124.7
(CH), 126.4 (CH), 127.5 (C), 128.2 (CH), 128.8 (CH), 130.2 (CH),
136.0 (C), 138.1 (C), 139.2 (C), 140.8 (C), 144.7 (C), 144.9 (CH)
and 152.4 (CH) (one overlapping resonance); m/z (APCI+) 492
((37ClM + H)+, 40%), 490 ((35ClM + H)+, 100); HRMS (ES+)
490.0768. C21H21ClN5O3S requires 490.0769.
Sulfamic acid 4-[3-(cyanodimethylmethyl)-5-[1,2,4]triazol-1-
ylmethylbenzyloxy]phenyl ester 19. Compound 19 was prepared
from 18 (0.057 g, 0.164 mmol) using similar conditions to
those described for the synthesis of compound 11. Column
chromatography (CH2Cl2–acetone◦80 : 20) eluted 19 as a white
solid (0.049 g, 69%), mp 158–160 C (found: C, 56.3; H, 5.2; N,
16.0. C20H21N5O4S requires C, 56.2; H, 5.0; N, 16.4%). 1H NMR
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dH (270 MHz, DMSO-d6) 1.68 (6H, s, ArC(CH3)2CN), 5.11 (2H, s,
ArCH2OAr), 5.48 (2H, s, ArCH2N), 7.05–7.21 (4H, dd, J = 8.8 &
53.2 Hz, AAꢀBBꢀ), 7.30 (1H, s, ArH), 7.46 (1H, s, ArH), 7.57 (1H, s,
ArH), 7.91 (2H, br s, ArOSO2NH2), 8.00 (1H, s, C2H2N3) and 8.69
(1H, s, C2H2N3); 13C NMR dC (100.6 MHz, DMSO-d6) 28.3 (CH3),
36.6 (C), 51.8 (CH2), 69.3 (CH2), 115.6 (CH), 123.4 (CH), 124.2
(CH), 124.4 (CH), 126.5 (CH), 137.4 (C), 138.2 (C), 142.2 (C),
143.8 (C), 144.4 (CH), 151.9 (CH) and 156.5 (C) (one overlapping
resonance); m/z (APCI+) 428 ((M + H)+, 100%); HRMS (ES+)
428.1385. C20H22N5O4S requires 428.1387.
bis(trimethylsilyl)lithium amide (1.0 M in THF, 38.0 mL,
37.9 mmol). After stirring for 60 min at −75 ◦C, iodomethane
(2.36 mL, 37.9 mmol) was introduced drop wise and the resulting
light orange mixture was allowed to warm to room temperature
and stir for an additional 4 h. Solvent was removed in vacuo to
give a dark brown viscous oil This was diluted in EtOAc (100 mL),
washed with 1 M Na2S2O3 (aq) (100 mL × 2), brine (100 mL) and
dried (MgSO4), filtered and solvent removed in vacuo to give a
brown liquid. Column chromatography (EtOAc–hexane, 20 : 80
to 35 : 65) eluted 27 as a clear light brown liquid (2.30 g, 77%).
1H NMR dH (270 MHz, CDCl3) 1.65 (6H, s, ArC(CH3)2CN), 2.29
(6H, s, ArCH3), 6.97 (1H, s, ArH) and 7.11 (2H, s, ArH); m/z
(FAB+) 173 (M+, 100%).
2-(3-Bromo-5-[1,2,4]-triazole-1-ylmethylphenyl)-2-methylpropio-
nitrile 24. Compound 24 was prepared from 23 (3.20 g,
10.09 mmol) using similar conditions to those described for the
synthesis of compound 5(a). Column chromatography (EtOAc)
eluted 23 as a clear viscous oil that crystallised on standing
to give a colourless crystalline solid (1.97 g, 6.46 mmol, 64%),
mp 71–72 ◦C. 1H NMR dH (270 MHz, CDCl3) 1.68 (6H, s,
ArC(CH3)2CN), 5.33 (2H, s, ArCH2N), 7.40–7.41 (2H, t, J =
1.7 Hz, ArH), 7.54–7.55 (1H, t, J = 1.7 Hz, ArH), 7.99 (1H, s,
C2H2N3) and 8.12 (1H, s, C2H2N3); m/z (EI+) 307 ((81BrM + H)+,
100%), 305 ((79BrM + H)+, 99).
2-(3-Bromomethyl-5-methylphenyl)-2-methylpropionitrile 28.
To a solution of 27 (2.25 g, 12.98 mmol) in CCl4 (50 mL) was
added N-bromosuccinimide (2.33 g, 13.0 mmol) and benzoyl
peroxide (0.126 g, 0.520 mmol). The light yellow suspension was
then refluxed under inert conditions for 18 h. After cooling to
room temperature, the suspension was filtered and the filter cake
collected was washed with CHCl3 (30 mL × 5). The combined
filtrates were evaporated in vacuo to give a yellow liquid. Column
chromatography (EtOAc–hexane, 10 : 90 to 20 : 80) eluted 28 as
1
2-{3-[(1H-1,2,4-Triazol-1-yl)methyl]-5-(4-hydroxystyryl)phenyl}-
2-methylpropanenitrile 25. Under inert conditions compound
24 (0.100 g, 0.328 mmol), 4-vinylphenol [10% wt in propylene
glycol] (0.787 g, 0.655 mmol), NEt3 (91.0 lL, 0.655 mmol)
and PPh3 (0.029 g, 0.112 mmol) were dissolved in anhydrous
THF (1.5 mL) and the resulting clear solution was thoroughly
degassed. Pd(OAc)2 (0.012 g, 0.056 mmol) was added and the
reaction mixture set to reflux for 22 h with constant stirring.
After the reaction had been allowed to cool THF was removed
in vacuo and the residues dissolved in CH2Cl2 (20 mL), washed
with 2 M KHSO4 (aq) (20 mL), distilled H2O (20 mL × 3) and brine
(20 mL). The organic portion was dried (MgSO4) and solvent
removed in vacuo. Column chromatography (EtOAc) eluted 25 as
a colourless viscous oil (0.080 g, 71%) which was used without
further purification. m/z (APCI+) 345 ((M + H)+, 100%).
a light yellow oil (2.15 g, 66%). H NMR dH (400 MHz, CDCl3)
1.67 (6H, s, ArC(CH3)2CN), 2.33 (3H, s, ArCH3), 4.70 (2H, s,
ArCH2Br), 7.24 (1H, s, ArH), 7.28 (1H, s, ArH) and 7.39 (1H,
d, J = 1.9 Hz, ArH); m/z (FAB+) 252 ((79BrM + H)+, 33%), 225
(50), 172 (100); HRMS (FAB+) 254.0381. C12H1581BrN requires
254.0367.
2-[3-(4-Hydroxyphenylsulfanylmethyl)-5-methylphenyl]-2-methyl-
propionitrile 29. A solution of 28 (1.5 g, 5.95 mmol) in anhydrous
DMF (30 mL) at room temperature and under inert conditions
was treated with K2CO3 (8.25 g, 59.5 mmol) followed by a solution
of 4-hydroxythiophenol (0.940 g, 7.14 mmol) in anhydrous DMF
(0.5 mL). The resulting dark yellow suspension was stirred at
room temperature for 30 min and then acidified with 1 M HCl(aq)
.
After diluting with EtOAc (100 mL), the organic layer was washed
with distilled H2O (50 mL × 3), brine (50 mL), dried (MgSO4),
filtered and solvent removed in vacuo to give yellow residues.
Column chromatography (EtOAc–hexane 20 : 80 to 50 : 50) eluted
Sulfamic acid 4-{2-[3-(cyanodimethylmethyl)-5-[1,2,4]triazol-1-
ylmethylphenyl]vinyl}phenyl ester 26. Compound 26 was pre-
pared from 25 using similar conditions to those described for the
synthesis of compound 11. Column chromatography (CH2Cl2–
acetone 80 : 20) eluted 26 as a white solid (0.035 g, 36%), mp
151–152 ◦C. 1H NMR dH (400 MHz, DMSO-d6) 1.73 (6H, s,
ArC(CH3)2)CN), 5.50 (2H, s, ArCH2N), 7.30–7.33 (3H, m, ArH
& vinyl), 7.35–7.38 (1H, d, J = 16.6 Hz, trans vinyl), 7.42 (1H, s,
ArH), 7.51 (1H, s, ArH), 7.71 (1H, s, ArH), 7.72–7.74 (2H, d, J =
8.4 Hz, ArH), 8.04 (1H, s, C2H2N3), 8.06 (2H, br s, ArOSO2NH2)
and 8.73 (1H, s, C2H2N3); 13C NMR dC (100.6 MHz, DMSO-d6)
28.3 (CH3), 36.6 (C), 51.9 (CH2), 122.6 (CH), 123.0 (CH), 124.2
(CH), 124.5 (C), 125.4 (CH), 127.9 (CH), 128.2 (CH), 128.5 (CH),
135.2 (C), 137.5 (C), 138.0 (C), 142.5 (C), 144.4 (CH), 149.7 (C)
and 151.9 (CH); m/z (APCI+) 424 ((M + H)+, 100%), 345 (27).
HRMS (ES+) 424.1435. C21H22N5O3S requires 424.1438.
◦
29 as a white solid (1.50 g, 85%), mp 77–78 C (Found: C, 72.5;
H, 6.4; N, 4.5. C18H19NOS requires C, 72.7; H, 6.4; N, 4.7%). 1H
NMR dH (400 MHz, CDCl3) 1.63 (6H, s, ArC(CH3)2CN), 2.33
(3H, s, ArCH3), 3.91 (2H, s, ArCH2SAr), 5.49 (1H, br s, ArOH),
6.73 (2H, m, ArH), 6.85 (1H, s, ArH), 6.99 (1H, s, ArH), 7.12
(1H, s, ArH) and 7.17 (2H, m, ArH).
Sulfamic acid 4-[3-(cyanodimethylmethyl)-5-methylbenzyl-
sulfanyl]phenyl ester 30. Compound 30 was prepared from 29
using similar conditions to those described for the synthesis
of compound 11. Column chromatography (EtOAc) eluted 30
as a pale yellow viscous oil that on standing ◦crystallised to a
1
pale yellow solid (0.282 g, 74%), mp 107–110 C. H NMR dH
(270 MHz, CDCl3) 1.61 (6H, s, ArC(CH3)2CN), 2.35 (3H, s,
ArCH3), 4.00 (2H, s, ArCH2SAr), 5.27 (2H, br s, OSO2NH2),
6.80 (1H, s, ArH), 7.01 (1H, s, ArH), 7.08 (1H, s, ArH), 7.22
(2H, m, ArH) and 7.32 (2H, m, ArH); 13C NMR dC (100.6 MHz,
DMSO-d6) 21.0 (CH3), 28.3 (CH3), 36.5 (C), 36.9 (CH2), 122.8
2-(3,5-Dimethylphenyl)-2-methylpropionitrile 27. To a solution
of 3,3-dimethylphenylacetonitrile (2.50 g, 17.2 mmol) in an-
hydrous THF under inert conditions at −75 ◦C was added
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(CH), 122.9 (CH), 124.6 (C), 124.7 (CH), 128.9 (CH), 130.0 (CH),
134.1 (C), 138.0 (C), 138.4 (C), 141.5 (C) and 148.4 (C); m/z (ES+)
377 ((M + H)+, 100%); HRMS (ES+) 377.0972. C18H21N2O3S2
requires 377.0988.
18 L. W. L. Woo, C. Bubert, O. B. Sutcliffe, A. Smith, S. K. Chander,
M. F. Mahon, A. Purohit, M. J. Reed and B. V. L. Potter, J. Med.
Chem., 2007, 50, 3540.
19 R. Morphy and Z. Rankovic, J. Med. Chem., 2005, 48, 6523.
20 A. D. Favia, A. Cavalli, M. Masetti, A. Carotti and M. Recanatini,
Proteins: Struct., Funct., Bioinf., 2006, 62, 1074–1087.
21 F. G. Hernandez-Guzman, T. Higashiyama, W. Pangborn, Y. Osawa
and D. Ghosh, J. Biol. Chem., 2003, 278, 22989.
Acknowledgements
22 (a) P. N. Edwards and M. S. Large, U.S. Pat. 4,935,437, 1990; (b) P. N.
Edwards and M. S. Large, U.S. Pat. RE36617, 2000. Two predominant
methods exist for the synthesis of Anastrozole. The first route (A)
employs 3,5-bis(bromomethyl)toluene as a starting material. Reaction
with potassium cyanide followed by alkylation with iodomethane neatly
completes the two dimethylpropionitrile side-arms. Radical bromina-
tion of the tolyl group furnishes a benzyl bromide that is reacted
with sodium triazole to finish the synthesis in four steps. The second
route (B) begins with the bromination with 3,5-dimethylbenzoate. The
two dimethylpropionitrile side-arms are constructed via reaction with
potassium cyanide followed by gem-dimethylation with methyl iodide.
The ester functionality is then reduced and the corresponding alcohol
converted to a benzyl chloride intermediate before the synthesis is
completed by substitution with sodium triazole. For the synthesis
and spectroscopic data for Anastrozole and preceding intermediates
following route (A) and route (B) see ESI†.
This work was supported by Sterix Ltd which is part of the Ipsen
Group. We thank Dr C. Sharland for the synthesis of Anastrozole
and Ms A. C. Smith for technical support.
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