4-Aryl-1,2,3-triazole: A novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo

Article abstract of DOI:10.1021/jm050408c

Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.

Full text of DOI:10.1021/jm050408c

5
644
J. Med. Chem. 2005, 48, 5644-5647
4
-Aryl-1,2,3-triazole: A Novel Template
for a Reversible Methionine
Aminopeptidase 2 Inhibitor, Optimized
To Inhibit Angiogenesis in Vivo
Lara S. Kallander,* Qing Lu, Wenfang Chen,‡
#
Thaddeus Tomaszek, Guang Yang, David Tew,
Thomas D. Meek, Glenn A. Hofmann,
|
Christina K. Schulz-Pritchard, Ward W. Smith,
§
⊥
Cheryl A. Janson, M. Dominic Ryan,
X
Figure 1. Structure of fumagillin (1), TNP-470 (2), and the
hMetAP2 screening hits 3, 4, and 5.
Gui-Feng Zhang, Kyung O. Johanson,
∇
Robert B. Kirkpatrick, Thau F. Ho, Paul W. Fisher,
b
4
Michael R. Mattern, Randall K. Johnson,
Scheme 1a
†
]
Michael J. Hansbury, James D. Winkler,
£
Keith W. Ward, Daniel F. Veber, and
Scott K. Thompson
GlaxoSmithKline Pharmaceuticals,
King of Prussia, Pennsylvania 19406
Received April 29, 2005
a
Reagents, conditions, and yields: (a) (MeO)2P(O)CN2C(O)CH3,
K2CO3, MeOH, 71-99%; (b) TMSCCH, cat. PdCl2, PPh3, CuI, Et3N,
then MeOH, KOH, 63%; (c) TMSN3, PhMe, reflux, then H2O, 57-
Abstract: Inhibitors of human methionine aminopeptidase
type 2 (hMetAP2) are of interest as potential treatments for
cancer. A new class of small molecule reversible inhibitors of
hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-
triazoles. Compound 24, a potent inhibitor of cobalt-activated
hMetAP2, also inhibits human and mouse endothelial cell
growth. Using a mouse matrigel model, this reversible hMetAP2
inhibitor was also shown to inhibit angiogenesis in vivo.
2%.
a pita-bread shaped fold.14 TNP-470,15 a synthetic
analogue of fumagillin, is a hMetAP2 inhibitor that has
advanced to clinical trials1
6,17
(Figure 1, 2). This ir-
reversible inhibitor is limited by its short half-life in
serum and dose-limiting neurotoxicity. Reversible in-
hibitors are generally favored due to decreased likeli-
hood of toxicity or immunogenic responses. Concurrent
with our studies, reversible inhibitors based on fuma-
Inhibition of angiogenesis is considered a desirable
pathway for stopping tumor growth and metastasis
primarily because of the low potential for toxicity or
1
resistance, as well as the promise of treating a broad
gillin,18 bestatin, or a 1,2,4-triazole
19
13,20-22
template
2
spectrum of tumor types. Fumagillin (Figure 1, 1), a
have been reported. In this report, we describe the
discovery and optimization of 4-aryl-1,2,3-triazoles as
reversible inhibitors of hMetAP2.
natural product that exerts its antiangiogenic effects3
through the inhibition of endothelial cell proliferation,
was discovered to be an irreversible inhibitor of me-
4
,5
The 4-aryl-1,2,3-triazoles were prepared as described
in Scheme 1. Our synthetic route made use of the
availability of diversely substituted benzaldehydes,
which were readily converted to terminal alkynes under
mild conditions using a modified Corey-Fuchs homolo-
gation.23 This method could also be applied to pyridyl-
aldehydes, or alternatively, the ethynylpyridines were
prepared from the corresponding bromopyridines through
a Sonogashira cross-coupling reaction followed by basic
hydrolysis of the trimethylsilyl group.24 Cycloaddition
with trimethylsilyl azide then formed the desired tria-
zole derivatives after aqueous hydrolysis of the tri-
thionine aminopeptidase 2 (MetAP2) by covalently
6-8
binding to His231. There is evidence that the MetAP2
4
,9,10
inhibition by fumagillin (and analogues)
is respon-
sible for the observed antiangiogenic activity. Additional
validation for MetAP2 as a target for cancer therapy
was published more recently; a reversible bestatin-based
MetAP2 inhibitor was shown to inhibit angiogenesis and
1
1
suppress tumor growth.
Human MetAP2 is one of two enzymes that catalyze
the removal of N-terminal methionine residues from
intracellular proteins. The enzyme is characterized by
12
a bimetallic center activated by either cobalt or
1
3
25
manganese, which is supported by residues that form
methylsilyl group.
Our efforts began by screening our compound collec-
tion to identify inhibitors of cobalt-activated hMetAP2.
Several potent inhibitors were discovered (Ki,app < 100
nM) from a novel structural class, the 1,2,3-triazoles
(Figure 1, 3, 4, and 5). These compounds were surpris-
ingly potent for very small molecules (MW < 160). A
full kinetic analysis revealed that compound 3 is a
reversible, competitive inhibitor with a Ki of 4.2 nM, IC50
*
To whom correspondence should be addressed. Phone: (610)-270-
4
814. Fax: (610)-270-4490. E-mail: lara.s.kallander@gsk.com.
‡
Current address: Finnegan Henderson et al., Palo Alto, CA.
#
Current address: Tanabe Research Lab. USA, San Diego, CA.
|
Current address: Argonne National Laboratory, Argonne, IL.
§
Current address: Shamrock Structures, Woodridge, IL
⊥
Current address: Millennium Pharmaceuticals, Cambridge, MA.
X
Current address: Applied Molecular Evolution, San Diego, CA.
3
Current address: Centocor Inc., Radnor PA.
b
Current address: Progenra Inc., Malvern, PA.
4
Current address: Independent consultant, Santa Fe, NM.
)
10 nM (fumagillin IC50 ) 1.2 nM in our cobalt-
†
Current address: Incyte Corporation, Wilmington, DE.
]
activated hMetAP2 assay). Compound 3 was found to
Current address: Array Biopharma Inc., Boulder, CO.
£
Current address: Independent consultant, Ambler, PA.
be very selective for MetAP2 over other metallopro-
1
0.1021/jm050408c CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/06/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5645
Table 1. hMetAP2 Inhibition Activity of 4-Aryl-1,2,3-triazolesa
compd
Ar
Ki,app (nM)
3
4
5
8
9
4-Me-phenyl
phenyl
15
70
as shown in Figure 1
2-Me-phenyl
2-OMe-phenyl
2-Ph-phenyl
4-Et-phenyl
4-n-Pr-phenyl
4-OH-phenyl
4-NH2-phenyl
4-Cl-phenyl
4-Br-phenyl
4-I-phenyl
52
112
150
>1000
30
>1000
>1000
337
6
10
11
12
13
14
15
16
17
18
19
20
3
1
18
300
280
29
16
4
1
13
41
Figure 2. X-ray crystal structure of compound 18 (grey and
blue) bound to hMetAP2 (turquoise). Cobalts are given in red.
The atomic coordinates and structure factors (code 2ADU) have
been deposited in the Protein Data Bank. (http://www.rcsb.org/).
3-Me-phenyl
3-OH-phenyl
3-NH2-phenyl
21
22
3-(PhCH NH)phenyl
2
2
6
This figure was generated using the program MolMol.
3-I-phenyl
2
2
2
2
2
2
2
3
3
3
4
5
6
7
8
9
0
1
3-Br-phenyl
3,4-di-Br-phenyl
2-benzofuran
2-pyridyl
3-pyridyl
4-pyridyl
6-Me-2-pyridyl
5-Me-2-pyridyl
4-Me-2-pyridyl
3-Me-2-pyridyl
260
>1000
6
Figure 3. hMetAP2 inhibitiory activity of pyrazoles.
3
8
52
teases such as MetAP1 (IC50 ) 7100 nM) and matrix
metalloprotease-9 (IC50 > 10 000 nM). Working within
this chemical class, we wanted to improve the inhibitory
potency and gain a structural understanding of the
interaction of these compounds with the enzyme active
site.
32
a
Ki,app values were determined using the Dixon method assum-
ing competitive inhibition for compounds of the same structural
series.
aromatic ring relative to the triazole ring is important
for binding; the coplanar orientation observed in the
X-ray crystal structure may be preferred because it
avoids steric crowding between the aromatic ring and
His231.
A methyl group in the 4-position of a 4-phenyl-1,2,3-
triazole enhances the enzyme inhibition 5-fold over the
unsubstituted ring (3, 4); however, compounds with
chain lengths longer than ethyl were significantly less
potent. A reduction in inhibition was also observed by
introducing a hydroxy or an amino group in the same
position (13, 14). Analysis of the crystal structure
indicates that Tyr444 may be responsible for the size
limitations and hydrophobic nature of this area of the
binding pocket. Significant improvements in potency via
Concurrent with lead optimization, the X-ray cocrys-
tal structure of compound 18 bound in the active site of
hMetAP2 was solved at 1.9 Å resolution (Figure 2). This
structure revealed that there is a key binding interac-
tion between triazole nitrogens N-1 and N-2 and the
active site cobalt atoms, similar to that noted in the
crystal structure of a 1,2,4-triazole bound to cobalt-
2
0
activated bacterial MetAP. This tight coordination
interatomic distance 2.0 Å) probably accounts for much
(
of the strong binding of these small molecules. The third
triazole nitrogen appears not to be directly involved in
binding to the metals but engages in a H-bond to
His339.
To further explore whether N-1 and N-2 of the triazole
contributed to the binding affinity of the compounds,
pyrazole analogues of 3 were prepared. Replacing N-3
with carbon (Figure 3, 6) led to a modest reduction in
potency, suggesting a utility for 4-arylpyrazoles in the
inhibition of metalloproteases such as MetAP2. How-
ever, replacing N-1 with carbon (7) significantly reduced
the MetAP2 potency. These results provide further
evidence that N-1 and N-2 in the heteroaryl ring are
responsible for maintaining direct coordination with the
active site metals and are thus important for inhibition.
The potent inhibitory activity of the tricyclic com-
pound 5 suggested that there may be room in the active
site for additional substitution at the 2-position of a
4
-substitution were achieved through the introduction
of halogens (15-17).
The SAR for substitution at the 3-position of the aryl
ring was generally similar to the corresponding 4-posi-
tion, i.e., compounds with small hydrophobic groups
demonstrated better inhibition than compounds bearing
polar substituents such as hydroxy and amino. How-
ever, larger substituents were accommodated at the
3-position, as might be predicted based on the available
space observed in the crystal structure. For example
compound 21, with a benzylamino substituent, shows
good inhibitory potency. Again, the greatest enhance-
ments in inhibition were obtained by introducing halo-
gens, especially bromine, at the 3-position (23).
4
-phenyl-1,2,3-triazole (Figure 1). Instead, the potency
was shown to decrease as the 2-substituent increased
To take advantage of potency enhancements resulting
from the introduction of halogens at the 3- and 4-posi-
tion of the phenyl ring, a 3,4-dibromophenyl compound
in size from methyl to phenyl (compounds 8-10, Table
1
). This appears to indicate that the orientation of the

5
646 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Letters
Table 2. Enzymatic and Cellular Inhibition by
-Aryl-1,2,3-triazoles
4
enzyme Ki,app (µM)
Mn2+
cellular IC50 (µM)a
compd Co2
+
HUVEC
MS-1
3
1
1
1
2
2
3
3
0.015 4.4
0.006 11
0.003 11
0.001 0% inhibition at 20 µM
0.001 16
0.013 26% inhibition at 20 µM
0.003 15
0.008 14
30
5
20
20
20
20
2
30
3
8
5
6
7
4
5
0
1
6
20
2
20
2
>30
a
IC50 was that concentration of compound that reduced cell
viability to 50% of control (untreated) viability, as determined from
plots of concentration vs percent viability. HUVEC ) human
umbilical vein endothelial cell. These compounds did not inhibit
nonendothelial cell proliferation (human foreskin fibroblast IC50
>
30 µM).
Figure 4. Matrigel plug angiogenesis model. Matrigel plugs
were injected subcutaneously in mice, excised after 6 days, and
analyzed for hemoglobin content as an index of vascularization.
bFGF promotes the vascularization of Matrigel implants in
mice as compared to unstimulated control. Oral dosing of
compound 24 was performed t.i.d at 50 mg/kg or 100 mg/kg
and both where shown to inhibit vascularization. Bars ) SEM,
n ) 8, p ) 0.037 (50 mg/kg), 0.029 (100 mg/kg).
was prepared. There is adequate space for both bromine
substituents to fit into the active site; compound 24 is
an inhibitor of 1 nM potency, a 70-fold improvement
over compound 4.
To further explore the active site, analogues were
prepared where the phenyl ring was replaced with a
heteroaryl ring at the 4-position of the triazole. The
active site is reasonably accommodating of large rings
such as the benzofuran (25). Having a pyridine nitrogen
oriented at the 2-position (26) also gave a slight en-
hancement in potency relative to the phenyl compound
larly oral exposure. Compound 24 demonstrates low
clearance and ∼70% oral bioavailability in the rat.
Furthermore, following oral administration in the mouse
(53 mg/kg), 24 displays systemic exposure at or above
the 2 µM level required to maintain potency (MS-1 IC50)
for about 8 h postdose (DNAUC ) 61 min‚kg/L; half-
life ∼1 h).
4
, while the 3- and 4-pyridyl analogues (27, 28) were
substantially less potent. A pyridyl nitrogen at the
-position would be within 3.4 Å of His339 and may
2
benefit from an additional hydrogen bond. A hydropho-
bic methyl substituent on the 2-pyridyl ring improved
potency particularly when introduced at the 5-position
A mouse matrigel model was selected to test the effect
a 4-aryl-1,2,3-triazole MetAP2 inhibitor would have on
angiogenesis.27 In this study, mice were injected sub-
cutaneously with growth factor (bFGF) enriched matri-
gel matrix. Oral (tid) dosing of inhibitor 24 was main-
tained for 6 days, after which the matrix was excised
and a quantitative analysis of vascularization was
determined as a direct measure of angiogenesis. Shown
in Figure 4, compound 24 inhibited angiogenesis, 83%
at 50 mg/kg and 90% at 100 mg/kg. This animal model
demonstrates that compound 24, a 4-aryl-1,2,3-trizaole,
is a potent inhibitor of angiogenesis in vivo.
(30).
Several of the most potent inhibitors of cobalt-
activated hMetAP2 were tested as inhibitors of endo-
thelial cell proliferation, both human (HUVEC) and
mouse (MS-1, Table 2). For reference, compound 3 was
tested and was found to inhibit cell proliferation in the
micromolar range, less potent than would be expected
based on the enzyme inhibition data. This is possibly
due to low cellular penetration or some other cell specific
phenomenon. Another alternative is that manganese,
instead of cobalt, is the physiologically relevant metal
In conclusion, the 4-aryl-1,2,3-triazoles were discov-
ered to be a unique template for the inhibition of the
metalloprotease MetAP2. The N-1 and N-2 nitrogens of
the triazole structure actively participate in binding to
the enzyme active site metals and are key elements for
inhibition. Considering their small size, the 4-aryl-1,2,3-
triazoles may provide a promising template for the
design of new inhibitors of other metalloproteases.
Additionally, compound 24 inhibits angiogenesis in a
mouse model. The 4-aryl-1,2,3-triazoles are a novel and
interesting chemical class which could be further opti-
mized for the treatment of cancer.
1
3
for hMetAP2, so we also tested our inhibitors against
the manganese-activated form of the enzyme (Table 2).
The potency order of magnitude is more similar between
the manganese-activated enzyme and the inhibition of
HUVEC proliferation for several compounds (3, 15, and
16). However, some compounds are micromolar inhibi-
tors of HUVEC proliferation and are not as potent
against the manganese form of the enzyme (17, 24, 25,
and 30). On the basis of these results, we cannot
conclude which metalloenzyme is physiologically rel-
evant; however, we were able to use the cobalt-activated
form of the enzyme to progress our program toward the
identification of a tool compound for in vivo studies.
Compound 24 was identified as a low-micromolar
inhibitor of cellular proliferation (HUVEC and MS-1)
based on its strong potency against the cobalt-activated
enzyme.
Acknowledgment. We thank Dr. Yie-Hwa Chang
and St. Louis University as the source of the intellectual
property for the type 2 human methionine aminopep-
tidase, Prof. Jon Clardy and Dr. Cristina Nonato for
guidance in crystallizing hMetAP2, and Dr. Martha S.
Head for Figure 2.
One advantage of the 4-aryl-1,2,3-triazole series is the
favorable in vivo pharmacokinetic properties, particu-
Supporting Information Available: Method of enzyme
inhibition, cellular inhibition, pharmacokinetic analysis, matri-

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5647
gel angiogenesis model, crystal structure determination, and
refinement statistics, and synthetic procedures and charac-
terization for 3-32 are given. This material is available free
of charge via the Internet at http://pubs.acs.org.
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JM050408C