Discovery of a new class of cinnamyl-triazole as potent and selective inhibitors of aromatase (cytochrome P450 19A1)

Article abstract of DOI:10.1016/j.bmcl.2014.07.083

Synthesis of a novel class of natural product inspired cinnamyl-containing 1,4,5-triazole and the potent inhibition of human aromatase (CYP 450 19A1) by select members is described. Structure-activity data generated provides insights into the requirements for potency particularly the inclusion of an aryl bromide or chloride residue as a keto-bioisostere.

Full text of DOI:10.1016/j.bmcl.2014.07.083

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a new class of cinnamyl-triazole as potent and selective
inhibitors of aromatase (cytochrome P450 19A1)
a
b
b
James McNulty a, , Kunal Keskar , Denis J. Crankshaw , Alison C. Holloway
⇑
a Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ont. L8S 4M1, Canada
b Department of Obstetrics and Gynecology, McMaster University, 1280 Main Street West, Hamilton, Ont. L8S 4K1, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a novel class of natural product inspired cinnamyl-containing 1,4,5-triazole and the potent
inhibition of human aromatase (CYP 450 19A1) by select members is described. Structure–activity data
generated provides insights into the requirements for potency particularly the inclusion of an aryl bro-
mide or chloride residue as a keto-bioisostere.
Received 3 June 2014
Revised 9 July 2014
Accepted 11 July 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Triazoles
Huisgen cycloaddition
Vinyl ethers
Aromatase
Anticancer agents
Aromatase inhibitors (AI’s), such as exemestane 1, anastrozole 2
and letrozole 3, have emerged over the last few decades as alterna-
tives to the selective estrogen receptor modulating agent tamoxi-
fen 4, in the treatment of hormone-dependent breast cancer.
Approximately 75% of postmenopausal breast cancer patients have
hormone-dependent (estrogen-dependent) breast cancer,1 now
the leading cancer amongst women.2 Tamoxifen 4 (Fig. 1) served
as the standard anti-estrogen in treating such tumors for many
years.3a–f Tamoxifen and its metabolites function as selective estro-
gen receptor modulators (SERMs).3f Their partial antagonist action
CN
N
O
N
NC
O
N
N
H
N
H
H
N
CN
N
O
CN
1
Exemestane
2 Anastrozole
3
Letrozole
4 Tamoxifen
X
O
O
RO
"aromatase"
H
H
H
H
N
O
Y
HO
N
N
7a. R=Ac, X,Y=H, 2,3-anti
7b. R=Mom, X,Y=H, 2,3-syn
7c. R=H, X,Y=H, 2,3-syn
5 Androstenedione
6 Estrone
Figure 1. Structures of currently used AIs 1–3, and the SERM agent tamoxifen 4. Example of aromatase conversion of 5 to 6 and structure of recently described potent AIs 7
employing aryl halide ketone bioisosteres.
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.07.083
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: McNulty, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.083

2
J. McNulty et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
Br
N
OEt
H
N3
N
O11
OMe
N
Br
CO2Et
Br
OMe
OEt
Br
PR3Br
O
10
9
8
Br
12
Figure 2. Cinnamyl-triazole core structure 8 and retrosynthetic analysis based on the Wittig reaction of alkoxyphosphonium salt 12.
Table 1
Conversion of
equivalents’
in breast tissue prevents estrogen binding and resulting down-
stream effects that include cancer cell proliferation and the activa-
tion of survival and anti-apoptosis pathways.3d,e
a-methoxy phosphonium salts to b-methoxycinnamate ‘alkyne
OMe
OMe
1. THF/ LHMDS
-78 o
AIs are inhibitors of human cytochrome P450 19A1 enzyme
complex (CYP19A1), the rate-limiting enzyme involved in the oxi-
dative decarboxylation of the C19 methyl group in androgens such
as testosterone and androstenedione 5 leading to the estrogens
estradiol and estrone 6, respectively, (Fig. 1).4 Irreversible Type-I
aromatase inhibitors (steroidal) such as exemestane 1 as well as
reversible, nonsteroidal type-II inhibitors exemplified by anastroz-
ole 2 and letrozole 3, are currently approved AIs for the treatment
of metastatic estrogen-dependent breast cancer.1a,5a–e
CO2Et
P(Et)3 Br
C
R
R
2. Ethyl glyoxylate
-78 oC to RT
15
14
Isolated
Alpha-alkoxy P salts
Entry
Beta-alkoxy acrylate
yield(%)
OMe
OMe
CO2Et
1
P(Et)3 Br
80
Despite their clinical success, current AIs are associated with
issues such as osteoporosis, joint pain, reproductive problems
and androgenic side effects. These compounds also partly inhibit
cytochromes 1A1, 1A2, 2D6, 2C8/9 and 3A4, all of which are
involved in the metabolism of xenobiotics, thus increasing the like-
lihood of drug–drug interactions. These factors necessitate the dis-
covery and development of structurally novel, potent and selective
AIs for the next generation treatment of ER positive breast cancer.
Natural products and structural analogues have proven to be
valuable sources in the search for lead compounds as nonsteroidal
AIs. In particular, natural products from the cinnamic and coumaric
acid pathways, including cinnamates, chalcones, flavanones/flav-
ones, isoflavones and stilbenes6 have been shown to be privileged
structures amongst naturally occurring AIs. Work in our own labo-
ratories resulted in the discovery of natural flavones6c that exhib-
ited potent aromatase inhibitory activity as well as a series of
alkaloids and synthetic derivatives that demonstrated selective
activity against aromatase and other cytochrome P450s.7 This
work resulted in the discovery8a of potent triazole-containing AIs
based on a common 5-component pharmacophore 7 (Fig. 1). A
key aspect of the optimization in this work was the inclusion of
aryl bromide residues as carbonyl-mimics at positions correspond-
ing the keto groups on androstenedione.8b The use of aryl halides
as ketone bioisosteres resulted in increased aromatase inhibition
to 20 nM. In continuance of this work, we desired to explore a rap-
idly accessible system capable of mimicking the androstenedione
core and allowing incorporation of aryl halide groups at the critical
positions. Consideration of both natural product and synthetic AI
structures led us to postulate the cinnamyl-triazole core molecule
8 (Fig. 2) as a potential new lead for developing AIs. In this Letter,
we report the synthesis of an initial mini-panel of compounds
based on this cinnamyl-triazole core and discovery of their potent
aromatase activities.
Cl
Cl
14a
14b
15a
OMe
CO2Et
OMe
P(Et)3 Br
2
85
82
15b
Cl OMe
CO2Et
Cl OMe
P(Et)3 Br
3
4
5
14c
15c
OMe
CO2Et
OMe
P(Et)3 Br
80
85
88
H3C
H3C
14d
15d
15e
OMe
P(Et)3 Br
OMe
OMe
CO2Et
O
O
O
O
14e
14f
OMe
P(Et)3 Br
CO2Et
OMe
6
7
Br
Br
15f
15g
15h
OMe
P(Et)3 Br
CO Et 52
2
14g
14h
OMe
P(Et)3 Br
CH3
OMe
CO Et 40
2
8
CH3
The principal method for preparing 1,2,3-triazoles is the cop-
per(I) catalyzed 1,3-dipolar stepwise cycloaddition of an azide onto
an alkyne (Click Reaction).11 The direct synthesis of 1,2,3-triazoles
under metal-free thermal cycloaddition conditions (Huisgen
reaction) is less explored. In addition to alkynes, access to 1,2,3-
triazoles from the cycloaddition of azides onto heteroatom-
substituted alkenes is an alternative route to functionalized
triazoles. Examples of such ‘alkyne equivalents’ include enol
OMe
OMe
OMe
P(Et)3
P(Et)3.HBr
80 oC
Br
R
R
13a-13h
Scheme 1. Synthesis of alkoxyphosphonium salts 14a–14h.
14a-14h
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J. McNulty et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 2
Regiocontrolled thermal Huisgen cycloaddition of benzyl azides onto b-methoxycinnamates leading to potent AIs
CO2Et
N
R2
N
N
OMe
BnN3
CO2Et
Sealed Tube
150 o
C
R1
R1'
15
16
Isolated
Ki (uM)
1.41
pKi
Entry
1
Benzyl azide
Click Product
Beta-alkoxy acrylate
Yield (%)
CO2Et
N
N3
OMe
N
CO2Et
N
5.85
95
16a
15b
CO2Et
N
OMe
N3
N
CO2Et
N
Br
95
96
0.64
0.02
6.20
7.66
2
3
Br
16b
15f
15f
CO2Et
N
N3
OMe
N
Br
CO2Et
CO2Et
CO2Et
N
Br
Br
Br
16c
CO2Et
N
N3
OMe
OMe
N
N
0.71
0.71
6.15
6.15
57
40
4
5
CH3
H3C
16d
15d
15e
CO2Et
N
N3
N
O
O
N
O
O
16e
CO2Et
N
N3
N3
N3
OMe
OMe
OMe
CO2Et Br
N
6
7
40
44
0.03
0.36
7.60
6.45
N
Cl
Br
Cl
Cl
15a
15a
15b
16f
CO2Et
N
N
CO2Et
N
Cl
16g
CO2Et
N
N
Br
CO2Et
0.15
6.84
8
48
N
Br
16h
ethers, enamines, vinylogous amides, vinyl amides, and vinyl sulf-
oxides. The cycloadditions of azides onto functionalized alkenes
typically requires higher temperatures than alkynes, but such
alkenes do possess advantages such easy access from carbonyl
compounds and high regioselectivity in the cycloaddition due to
the polarized nature of the alkene.
dialkylacetal and a trialkylphosphine hydrobromide salt,9a we pre-
pared the series of -methoxybenzyl phosphonium salts 14a–h.
These salts were obtained in high yield (92–98%) without need of
chromatographic purification through simply removing methanol
under high vacuum. We next investigated a-methoxy ylide forma-
tion and olefination of the range of substituted phosphonium salts
with ethyl glyoxylate. Dark red solutions of the ylides were gener-
ated in THF at À78 °C using lithium bistrimethylsilylazide. Efficient
a
Following a recently described method for the synthesis of
a-alkoxy-phosphonium salts (Scheme 1), from the reaction of a
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4
J. McNulty et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
olefination occurred in all cases upon addition of ethyl glyoxylate
and the corresponding methoxycinnamate 15a–h were purified
by silica gel chromatography and isolated in 40–88% yields and
good (E) selectivity. (E/Z ratio ranging between 70:30 and 80:20)
as summarized in Table 1. These b-alkoxycinnamates are relatively
stable to air but are readily hydrolysed to the corresponding b-keto
esters in high yield upon treatment with acids.
References and notes
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Thermal cycloaddition of the series of methoxycinnamates with
benzylic azides occurred smoothly in a sealed tube at 150 °C, lead-
ing to the desired triazoles 16a–h in fair to excellent yield. The cyc-
loadditions were seen to be completely regioselective, leading only
to the 1,4,5 trisubstituted 1,2,3-triazoles. Regiochemical assign-
ments are in accord with earlier examples confirmed via X-ray
crystallographic analysis.10i The well-known Cu-catalyzed azide
alkyne cycloaddition leads to 1,4-triazoles and requires employ-
ment of a terminal alkyne.11 Thus in addition to complete regio-
control, the use of vinyl ethers allows access to functionalized
triazoles that are not available from the conventional click process.
The series of eight novel cinnamyltriazoles 16a–h described in
Table 2 were screened for activity against recombinant human aro-
matase via kinetic monitoring of the conversion of O-dibenzylfluo-
rescein benzyl ester (DBF) substrate to fluorescein by-product.8a
Fluorometric measurement of emission was made at 535 nm after
excitation at 485 nm utilizing ketoconazole as a positive control.6c
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delight, several analogs proved to be potent inhibitors, displaying
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a 100-fold range in potencies ranging from the
lM range down
to 20 nM, Table 2. The most interesting examples proved to be
the dibromo compound 16c and the corresponding bromo-
chloro-analog 16f that exhibited potencies of 20 and 30 nM,
respectively. The positions of halogenation approximate the loca-
tion of the carbonyl groups in natural substrates such as andro-
stenedione. These results add further to the previous postulate of
arylbromide/chloride residues as bioisosteres8b in the develop-
ment of stable analogs as potent AI’s. In addition to potency, the
discovery of this new pharmacophore is significant in view of the
rapid synthetic entry to the series in only four chemical steps fol-
lowing the sequence aldehyde to dimethylacetal 13, to alkoxy-
phosphonium salt 14, to methoxycinnamate 15 and finally
triazole 16. The final thermal Huisgen reaction with benzylic (or
other) azide as the fourth (last) step leading to 16 allows significant
late-stage diversity for further optimization in this new lead series.
In summary, we report a new natural product-inspired hetero-
aromatic 1,4,5-substituted cinnamyltriazole pharmacophore,
examples of which display potent aromatase inhibitory activity.
The core structure can be rapidly prepared in four linear steps from
readily available aldehydes and the synthesis incorporates a regio-
specific, diversity oriented thermal Huisgen reaction as the last
step. The most potent AIs within this new series incorporate aryl
halide residues at positions mimicking the carbonyl substituents
of the natural enzyme substrates. Further development of potent
and selective AIs based on this novel pharmacophore are currently
under investigation.
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Acknowledgments
We thank NSERC and McMaster University for financial support
of this work.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
07.083.
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