Development of sulfonamide compounds as potent methionine aminopeptidase type II inhibitors with antiproliferative properties

Article abstract of DOI:10.1016/j.bmcl.2006.03.085

We have screened molecules for inhibition of MetAP2 as a novel approach toward antiangiogenesis and anticancer therapy using affinity selection/mass spectrometry (ASMS) employing MetAP2 loaded with Mn²⁺ as the active site metal. After a series

Full text of DOI:10.1016/j.bmcl.2006.03.085

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3574–3577
Development of sulfonamide compounds as potent methionine
aminopeptidase type II inhibitors with antiproliferative properties
Megumi Kawai,* Nwe Y. BaMaung, Steve D. Fidanze, Scott A. Erickson,
Jason S. Tedrow, William J. Sanders, Anil Vasudevan, Chang Park, Charles Hutchins,
Kenneth M. Comess, Douglas Kalvin, Jieyi Wang, Qian Zhang, Pingping Lou,
Lora Tucker-Garcia, Jennifer Bouska, Randy L. Bell, Richard Lesniewski,
Jack Henkin and George S. Sheppard
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 13 January 2006; revised 22 March 2006; accepted 23 March 2006
Available online 2 May 2006
Abstract—We have screened molecules for inhibition of MetAP2 as a novel approach toward antiangiogenesis and anticancer ther-
apy using affinity selection/mass spectrometry (ASMS) employing MetAP2 loaded with Mn2+ as the active site metal. After a series
of anthranilic acid sulfonamides with micromolar affinities was identified, chemistry efforts were initiated. The micromolar hits were
quickly improved to potent nanomolar inhibitors by chemical modifications guided by insights from X-ray crystallography.
Ó 2006 Elsevier Ltd. All rights reserved.
Methionine aminopeptidases (MetAPs) play an impor-
tant role in removing the initiator methionine residue
from nascent polypeptide chains and are believed to be
one of the essential enzymes involved in protein matura-
tion. There are three known mammalian methionine
aminopeptidases, MetAP1, MetAP2, and the recently
reported MetAP1D.1,2 The latter is closely related to
MetAP1 and is located in the mitochondria. The well-
studied types 1 and 2 remove methionine from the N-
terminus of proteins during protein synthesis, but ap-
pear to play different roles in cell function.3 Eukaryotic
cells contain two types, (MetAP1 and MetAP2), while
prokaryotes have only MetAP1. MetAP2 appears to
play a critical role in cell proliferation and tumor
growth, and is expressed at higher concentrations in tu-
mors as compared to normal cells.4 Many efforts have
been focused on the inhibition of MetAP2 since the en-
zyme was identified as an endogenous target for the anti-
angiogensis/antitumor agent TNP-470, a well-studied
semi-synthetic product under clinical development.5
Recently, it has been reported that an irreversible
MetAP2 inhibitor potently inhibits the proliferation of
human fibroblast-like synoviocytes (HFLS-RA),
suggesting the possibility of usage for the treatment of
rheumatoid arthritis.6
Since the finding of clinical implication of an inhibition
of MetAP2 as an anti-tumor agent, several classes of
compounds have been reported to possess inhibitory
activities against MetAP2.
Various synthetic analogues of the natural products
fumagillin, ovacillin, and bengamide have been evaluat-
ed as MetAP2 inhibitors.7 We have also previously
examined bestatin analogues as MetAP2 inhibitors,
and revealed that a reversible inhibitor effectively sup-
presses tumor growth and inhibits angiogenesis in
animal models.8 After establishing that MetAP2 inhibi-
tors are promising anti-cancer agents, we shifted our
focus to search for orally active reversible inhibitors.
In this study, we have used an affinity selection/mass
spectrometry (ASMS) screening method9 to identify
molecules that bind to human MetAP2 containing
Mn2+ in the enzyme active site.10 Activity was confirmed
by evaluation of enzyme inhibitory activity and inhibi-
tion of cell proliferation using assay described
previously.8b
Keywords: MetAP2; Sulfonamide; Antiproliferative.
*
From several classes of small molecule inhibitors
possessing less than 10 lM inhibitory activities against
Corresponding author. Tel.: +1 847 937 5939; fax: +1 847 937
8378; e-mail: megumi.kawai@abbott.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.085

M. Kawai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3574–3577
3575
MetAP2, anthranilic acid sulfonamide 1 (IC50 = 9 lM
Br
Br
(b)
(d)
(a)
for MetAP2) was chosen as a starting point to develop
a structure–activity relationship study. We envisioned
that a sulfonamide is an attractive lead, showing prom-
ising pharmacokinetics (data not shown) as well as a
high synthetic feasibility.
(I)
H2N
O
H2N
O
COOH
Br
COOMe
R
(c)
R
O
O
O
O
S
N
S
S
N
N
H
COOMe
H
H
COOMe
COOH
Based on the X-ray structures shown in Figure 1, we felt
the active site would be able to accommodate substitut-
ed anthranilic acids with substituents at 5 and 6
positions. We also observed that the sulfonamide back-
bone is angled and fits tightly to allow both aromatic
rings to reside in a hydrophobic environment. Chloro
substitution at the para-position on the sulfonyl phenyl
shows a relatively tight binding due to a narrow hydro-
phobic region of the enzyme.
O
O
(f)
O
NH2
(e)
OH
NH2
NH
(II)
(g)
O
OH
H
R
N
S
O O
Scheme 1. Reagents and conditions: (a) HCl in MeOH; (b) ArSO2Cl,
Py–CH2Cl2; (c) R ZnBr, CuI, PdCl2(dppf), THF, lwave, 150 °C,
10 min; (d) LiOH, dioxane–water, lwave, 160 °C, 10 min; (e) diethyl
ketomalonate, AcOH, 120 °C, 3 h; (f) 30% H2O2, 1 N NaOH; (g)
TMS–Cl, ArSO2Cl, Py–CH2Cl2.
Compounds 1–5 were prepared from commercially
available anthranilic acids and well-established chemis-
try. Compounds 6–8 were synthesized by the route illus-
trated in Scheme 1 (I). Commercially available 2-amino-
5-bromo benzoic acid was methylated, followed by ben-
zenesulfonylation to yield the 5-bromo methyl ester.
Organometallic couplings, followed by the saponifica-
tion of the ester under microwave conditions, provided
the final products.
(a) (b)
(c)
O
O
R
S
O2N
HBr.H2N
N
O
H
Br
O
Br O
Compounds 9 and 10 were synthesized by the procedure
illustrated in Scheme 1 (II). 2-Aminonaphthalene was
reacted with diethyl ketomalonate in acetic acid to form
an isatin derivative.12 Ring opening was carried out by
treatment with 30% hydrogen peroxide in 1 N sodium
hydroxide to yield 2-aminonaphthalene-1-carboxylic
acid.13 Sulfonamide formation was carried out with
in situ protection of the carboxyl group as its TMS ester,
followed by reaction with appropriate arylsulfonyl
chloride.
O
O
(f) (g)
(d) (e)
R
O
O
S
N
R
S
N
H
O
O
H
Br
O
O
(h)
R
S
N
H
O
OH
Scheme 2. Reagents and conditions: (a) Fe, NH4Cl, EtOH–H2O (4:1);
(b) Br2, CHCl3–DMF; (c) ArSO2Cl, Py–CH2Cl2; (d) 3 M MeMgBr,
THF–Et2O; (e) p-TosOH toluene; (f) PdCl2(dppf)-CH2Cl2, CO, Et3N,
MeOH (g) Pd/C, H2, EtOAc; (h) LiOH, dioxane–water, lwave, 160 °C,
10 min.
Sulfonamides 12 and 14 were prepared by the route
shown in Scheme 2.14 Commercially available 7-ni-
tro-1-tetralone was reduced, followed by bromination
to yield its hydrobromic acid salt. The resulting amine
was reacted with the aryl sulfonyl chloride to generate
sulfonamides, and the ketone was reacted with
MeMgBr to insert a methyl group. Carbonylation
was carried out in methanol to yield methoxycarbonyl
group insertion, followed by hydrogenation, and the
ester was then removed. The enantiomeric mixture
was separated by a chiral column to obtain compound
14 (S-configuration). Compound 12 was synthesized
from the same route except omitting the hydrogena-
tion step.
Tetrahydro naphthalene derivatives 11 and 13 were pre-
pared from the route shown in Scheme 3. A substituted
naphthalene derivative was hydrogenated to obtain the
tetrahydronaphthalene derivative, followed by treat-
ment with trifluoromethane sulfonic acid anhydride to
activate the hydroxyl group. After formation of a Schiff
base, it was reacted with hydrochloric acid to obtain the
amine. Final analogues were then prepared according to
the method described in Scheme 1. Compounds 11 and
13 can also be made by hydrogenation of 9 and 10
(PtO2, H2, and HOAc).
H331
E364
MN
H231
E459
D251
Enzyme inhibition assay results ([E] IC50) as well as pro-
liferation inhibition data on human microvascular endo-
thelial cells (HMVEC, IC50) are shown in Table 1. Some
analogues were examined in the enzymatic assay to
Figure 1. Stereo view of MetAP2 complex of compound 1 from an
X-ray crystal structure of an inhibitor–enzyme complex.11 Mn2+ ions
are shown as pink spheres.

3576
M. Kawai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3574–3577
Table 2. % protein binding of some MetAP2 inhibitors
(a) (b)
(c)
(d) (e)
Ph
Compound
Rat plasma binding
HO
Ph
TfO
N
COOMe
COOMe
COOMe
3
4
98.1% 0.2
99.7% 0.1
99.8% 0.1
99.8% 0.1
99.2% 0.2
6
9
10
(f)
O
O
O
O
R
S
R
S
N
N
H
H
COOMe
O
OH
Scheme 3. Reagents and conditions: (a) Pd/C, H2, MeOH–H2O; (b)
Tf2O, Py–CH2Cl2; (c) Pd(OAc)2, xantphos, Cs2CO3, HN@C(Ph)2,
dioxane; (d) HCl, THF–H2O; (e) ArSO2Cl, Py–CH2Cl2; (f) lwave,
LiOH, dioxane–water, 160 °C, 10 min.
site of the enzyme. Interestingly, even weak inhibitors,
such as 3–5, showed relatively potent inhibition of
HMVEC proliferation, which might be due to off-target
effects. On the other hand, compounds 9–11 were potent
inhibitors of MetAP2 with good selectivity against Me-
tAP1 (e.g., 9: MetAP1 IC50 = 47lM) and other amino-
peptidases (e.g., 9, Pfu-MetAP, AP, AP-1, and AP-M;
IC50 > 100 lM), and displayed moderate activity in the
cellular proliferation (HMVEC) assay.
Table 1. Inhibition of human methionine aminopeptidase type-2
a
b
Compound
[E] IC50 (lM)
[E] IC50 (lM)
IC50 (lM)
1
2
9.1
3.9
>100
>100
3
2
3
11
1
0.3
0.1
0.4
10
Compounds 11–14 possess excellent inhibitory activity.
Among them, an S-configuration on the compound 14
was preferred over the R-configuration in every assay
tested (data not shown).
4
5
0.35
0.09
1.1
>100
>100
6
7
60
8
10
100
0.5
0.5
0.4
0.15
0.18
0.13
In light of the propensity of serum albumin to bind
hydrophobic anionic compounds, we examined the ex-
tent of protein binding of selected MetAP2 inhibitors,
as shown in Table 2. Rat plasma (pH 7.4) was spiked
with compounds to a concentration of 10 lM. The plas-
ma sample was centrifuged and the sample was
fractionated, extracted and unbound compound was
measured in the supernatant by LC methods. These
compounds are found to be highly protein bound.
9
0.019
0.015
0.009
0.02
0.01
0.027
3.8
10
11
12
13
14
3.3
1.9
2.6
9.2
a Enzyme inhibition in the presence of 40 mg/mL of human serum
albumin.8b
b HMVEC cell line growth inhibition.8b
determine the effect of 40 mg/mL of human serum albu-
min. As was expected from the X-ray data, relatively
larger hydrophobic groups were accepted at the active
In conclusion, we have shown that the modification of
anthranilic acid sulfonamide 1 has led to inhibitors of
human MetAP2 with good to potent activities against
Br
O
O
O
O
O
O
S
S
N
S
N
N
H
H
COOH
Br
H
COOH
COOH
COOH
Cl
COOH
OH
1
6
11
S
N
N
O
O
O
O
O
O
S
S
S
N
N
N
H
H
H
COOH
F
O
7
12
2
O
O
O
O
O
O
S
S
S
N
H
N
N
H
H
COOH
COOH
Cl
F
OH
OH
O
O
3
8
13
O
O
O
S
O
O
O
S
N
S
N
H
N
H
H
COOH
COOH
F
4
9
14
O
O
O
S
O
S
N
H
N
H
COOH
F
5
10

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compounds also display high affinity to serum albumin.
Therefore, our efforts shifted to identifying molecules
having less protein binding, while maintaining potent
biological activity.
Acknowledgments
We thank Professor Lester Mitscher (University of
Kansas) for providing useful suggestions. We also thank
Hugh Nellans, Dan Hoffman, and Anthony Borre of
Abbott Laboratories for rat plasma protein binding
measurements.
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