New aromatic substituted pyrazoles as selective inhibitors of human adipocyte fatty acid-binding protein

Article abstract of DOI:10.1016/j.bmcl.2011.03.063

a-FABP is indespensible in inflammation and may serve as a new potential drug target for inflammation related diseases. We have successfully designed and synthesized a series of aromatic substituted pyrazoles as new human a-FABP inhibitors. The compounds

Full text of DOI:10.1016/j.bmcl.2011.03.063

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2949–2952
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New aromatic substituted pyrazoles as selective inhibitors of human
adipocyte fatty acid-binding protein
Xiujie Liu a,b, Xiaoli Huang a,b, Wanhua Lin a,b, Dongye Wang a, Yanyan Diao c, Honglin Li c, Xiaoyan Hui d,
a,
Yu Wang a,d, Aimin Xu a,d, Donghai Wu a, , Ding Ke
⇑
⇑
a Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan Avenue,
Guangzhou Science Park, Guangzhou 510530, PR China
b Graduate School of Chinese Academy of Sciences, PR China
c Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
d Department of Medicine and Pharmacology, University of Hong Kong, Pok Fu Lam Road, Hong Kong 999077, China
a r t i c l e i n f o
a b s t r a c t
Article history:
a-FABP is indespensible in inflammation and may serve as a new potential drug target for inflammation
related diseases. We have successfully designed and synthesized a series of aromatic substituted pyra-
zoles as new human a-FABP inhibitors. The compounds strongly bound to the hydrophobic binding
pocket of a-FABP, while showed significantly lower binding affinities to the closely related homologue
protein h-FABP. The most potent and selective compound 5g bound to a-FABP with an apparent Ki value
Received 8 December 2010
Revised 11 March 2011
Accepted 16 March 2011
Available online 22 March 2011
below 1.0 nM, while did not inhibit h-FABP at 50 lM and thus represents one of the most potent and
Keywords:
a-FABP inhibitor
Aromatic substituted pyrazoles
Inflammatory response
Inflammation related diseases
selective a-FABP inhibitors to date. The strong binding capacity of these inhibitors was further validated
by their effective blockade of inflammatory responses as determined by the production of pro-inflamma-
tory cytokines upon LPS stimulation. Compound 5g may serve as a lead compound for developing new
effective therapeutic agent for prevention and treatment of atherosclerosis, type 2 diabetes and other
inflammatory and metabolic related diseases.
Ó 2011 Elsevier Ltd. All rights reserved.
Fatty acid-binding proteins (FABPs) are a class of low molecular
weight cytoplasmic lipid chaperones that bind with hydrophobic
lipids inside the cells.1 Studies have suggested that FABPs coordi-
nate intracellular lipid storage and trafficking, and cholesterol
and phospholipids metabolism as well.1 At least 9 FABPs have been
identified which exhibit distinct expression profiles (FABP1–9, or
liver (l-), intestinal (i-), heart (h-), adipocyte (a-), epidermal (e-),
ileal (il-), brain (b-), myelin (m-) and testis (t-) FABPs).1 The FABPs
share highly similar three-dimensional structures, although their
primary sequences show significant diversity (15–70% sequence
identity).2
Adipocyte FABP (a-FABP), also known as ap2 or FABP4, is abun-
dantly expressed in adipocytes and macrophages.3 a-FABP defi-
cient mice are significantly protected from the development of
insulin resistance in both genetic and high-fat diet induced ob-
ese.4–8 Molecular studies demonstrated that a-FABP physically
associates with adipocyte hormone sensitive lipase (HSL), Jak2
and nuclear hormone receptor PPAR and thereby acts as a lipid
sensor in adipocytes.9–13 Furthermore, recent studies revealed that
macrophage is a critical site of its action where a-FABP plays a key
role in mediating inflammatory responses.5,8,14 Additionally, a-
FABP knockout mice are resistant to several other inflammation re-
lated disorders, including allergic airway inflammation11 and
experimental autoimmune encephalomyelitis (EAE)/multiple scle-
rosis (MS).12 These evidence collectively support the notion that a-
FABP is indespensible in inflammation and it may represent a new
potential drug target for inflammation related diseases. Actually, it
has been recently reported that inhibition of a-FABP by specific
inhibitors suppressed inflammatory responses in macrophage
cells.9,10,14 More importantly, oral administration of the selective
a-FABP inhibitor BMS309403, mitigated the onset of insulin resis-
tance and atherosclerosis in mice, which are two main pathologies
by chronic inflammation.15 Several other small molecular a-FABP
inhibitors have also been discovered with promising potential
(Fig. 1).16–22
In this Letter, we report the design, synthesis and biological
evaluation of a series of aromatic substituted pyrazoles (5) as
new human a-FABP inhibitors.
The designed compounds were synthesized by condensation–
cyclization of chalone derivatives 6 with phenylhydrazines 7 as
the key step (Scheme 1). Briefly, condensation of chalone deriva-
tives 6 with phenylhydrazine 7 under acidic conditions yielded
tri-aromatic substituted 4,5-dihydro-1H-pyrazoles which were
further converted to pyrazoles 8 by oxidization. Intermediates 9
were obtained by Suzuki coupling of pyrazoles 8 with boronic acid
⇑
Corresponding authors. Tel.: +86 20 32015276; fax: +86 20 32015299 (D.K.).
E-mail address: ding_ke@gibh.ac.cn (D. Ke).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.063

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X. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2949–2952
Table 1
HO
NH
The binding affinities of the designed compounds against a-FABP and h-FABPa
O
Compds
R1 R2
R3
Ar
Binding affinity (Ki,
h-FABP a-FABP
lM)
O
S
CF3
CONH2
N
N
BMS309403
BMS309403
3.47 0.18
NBb
0.015 0.008
NBb
S
O
N
CO2H
HO
N
S
ester
5a
O
O
H
Cl
Cl
H
H
H
H
H
H
Ph
Ph
2-
Furanyl
2-
Thienyl
2-
Thienyl
2-
5.24 3.70
17.00 1.83 0.013 0.008
0.032 0.004
5b
5c
2
1, HTS01037
3
4.13 1.03
4.24 0.46
2.27 0.92
3.72 1.52
>50
0.007 0.004
0.005 0.002
<0.001
R1
5d
5e
5f
Cl
H
H
H
H
Cl Me
Cl Et
HO2C
O
Ar
N
0.006 0.002
<0.001
N
N
HO2C
R2
N
Thienyl
O
5g
Cl Me Me 2-
Thienyl
R3
a
The binding affinity with a-FABP or h-PABP was determined by an in vitro 1,8-
4, BMS309403
ANS based fluorescence displacement assay.23 The proteins were prepared and
purified based on the published procedure.24 The data were reported as the means
of at least five independent experiments. The apparent Ki values were calculated
using an equation reported by Sulsky.20 The determined Kd values of 1,8-ANS for a-
5
Figure 1. Chemical structures of some important reported a-FABP inhibitors and
the designed aromatic substituted pyrazoles 5.
FABP and h-FABP were 0.25 and 0.60 lM, respectively.
b
NB, no obvious binding at 10 lM.
R1
Ar
NHNH2
N
N
O
carboxyl group formed direct hydrogen bonds with Arg126 and
Tyr128 in a-FABP. It is also likely to form an extra interaction with
Arg106 through a water bridge. The phenyl rings (A, B, C, D)
achieved the hydrophobic interactions with Ile104, Val115,
Met40 and Pro38. Additionally, the phenyl rings A and C could
Ar
a, b
c, d
Br
+
Br
R1
6
7
8
form
p–p stacking interactions with aromatic residues of His93
and Phe57. Further analysis of the binding mode of compound 5a
with a-FABP indicated that an extra small hydrophobic space
formed by the residues Met20, Val23 and Val25 in a-FABP was
available. A tiny hydrophobic group might be introduced to the
R1-position of the compound 5a to capture the interaction with
this pocket. Therefore, the p-Cl substituted compound 5b was de-
signed and synthesized to display a binding affinity to a-FABP with
R1
R1
A
(B)
Ar
Ar
N
N
HO2C
R2
N
N
e, f
O
HO
R3
a
Ki value of 13 nM, which was almost equally potent to
D
C
BMS309403 (Table 1).
Our modeling also indicated that the 5-phenyl group (phenyl
ring B) could be replaced with a smaller aromatic group to achieve
better binding mode with the less opened pocket surrounded by
residues Ala33, Ala36, Pro38 and Ser55 (Supplementary data).
Compounds 5c and 5d, which possess a 5- furanyl or 2-thienyl
group respectively, indeed had a higher binding affinity than that
of compound 5b. Specifically, compound 5d bound to a-FABP with
a Ki value of 5.0 nM, which is three times more potent than com-
pound 5b or BMS309403 in a parallel comparison (Table 1).
Interestingly, compound 5e, which was derived from the meth-
5
9
Scheme 1. Synthesis of the designed aromatic substituted pyrazoles. Reagents and
conditions: (a) CH3CO2H, C2H5OH, reflux, 80–85%; (b) MnO2, toluene, separator, 75–
80%; (c) 3-methoxyphenylboronic acid (10), Pd(P(Ph)3)4, dioxane, reflux, 70–95%;
(d) BBr3, CH2Cl2, 75–85%; (e) K2CO3, DMF, rt or 50–60 °C, 60–85%; (f) NaOH, H2O,
85–95%.
10 and subsequent demethylation. O-Alkylation of compounds 9
with 2-bromoacetatic acid esters followed by hydrolysis of the cor-
responding esters provided the designed compounds 5a–5g.
The binding affinity of the compounds with a-FABP was deter-
mined by an in vitro 1-anilinonapthalene 8-sulfonic acid (1,8-
ANS) based fluorescence displacement assay.23 BMS309403 or
BMS309403 ethyl ester was respectively utilized as the positive
or negative control to validate the screening assay. Under the
screening conditions, BMS309403 displayed a Ki value of 15.0 nM
binding to a-FABP, while its ethyl ester did not show obvious bind-
ylation of carboxyl a-carbon atom of 5d, displayed the best binding
affinity with a-FABP with the Ki value below 1 nM. This might be
due to the fact that the methyl group would further strengthen
the hydrophobic effects and van der Waals (VDW) interactions
with Val115 and Cys117 (Fig. 2C). Furthermore, the introduction
of a methyl group also caused a decrease in flexibility and degree
of freedom for the carboxyl side chain to reduce the entropy effect
of the conformation. The constraints in the orientation were con-
ducive to the formation of salt bridge between the carboxyl group
and the nitrogen atoms of Arg126, while oxygen atom on the side
chains was fitted in right place to participate in the formation of
hydrogen bonds with the receptor via a water bridge (Fig. 2C).
The binding affinity to a-FABP was barely affected by the introduc-
tion of an extra methyl group at this position (compound 5g)
(Fig. 2D and Table 1). However, when a slightly bigger ethyl group
ing to the protein at 10 lM, which were consistent to the previous
report (Table 1).20 The results from the affinity binding assay
showed that 5a bound to a-FABP protein with an apparent Ki value
of 32 nM which was twice less potent than BMS309403 (Table 1).
The predicted binding pose of compound 5a with a-FABP had a
similar orientation to that of BMS309403 (Fig. 2A and B).25,26 The

X. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2949–2952
2951
Figure 2. Co-crystal structure of a-FABP–BMS309403 complex (A) and the binding modes of the compounds 5a (B), 5e (C) and 5g (D) with a-FABP. a-FABP is colored yellow.
Key residues of the binding site are shown as solid lines and the hydrogen bonds are labeled as dash lines. The water molecules are represented with red balls. All the figures
are prepared using Pymol 0.99 (http://www.pymol.org).
was introduced (5f), the binding affinity decreased by >6-fold. It is
possible that the hydrophobic groove formed by Val115 and
Cys117 is too small to accommodate an ethyl group in a proper
orientation.
Nine FABPs have been identified to date and they share almost
identical three-dimensional structures. In order to examine the
specificity of these inhibitors, the binding affinities of these com-
pounds against the human heart type FABP, that is, h-FABP (also
ctrl
ctrl
LPS
LPS
MCP-1
TNFa
LPS + BMS309403
LPS + 5e
LPS + BMS309403
LPS + 5e
140
120
100
80
LPS + 5g
LPS + 5g
8
7
*
6
5
*
*
**
**
**
**
*
4
3
2
1
0
60
**
**
**
**
**
**
40
20
0
1µM
5µM
20µM
1µM
5µM
20µM
Figure 3. Compounds 5e and 5g potently inhibited the pro-inflammatory cytokine production in dose dependent manners. RAW264.7 cells were pretreated with
BMS309403, 5e and 5g under indicated concentrations or DMSO for 1 h, followed by LPS stimulation. Cytokine production was determined by quantitative PCR. (A) MCP-1. (B)
TNFa.
⁄p <0.1, ⁄⁄p <0.05.

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X. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2949–2952
known as FABP3) were also evaluated by the fluorescence binding
assay. Although h-FABP and a-FABP have a sequence identity of
64.4% and share a highly conserved functional domain, our data
showed that almost all of the designed inhibitors and
BMS309403 exhibited remarkable selectivity to a-FABP over h-
FABP (Table 1). For instance, compound 5d selectively bound to
a-FABP with a Ki value of 5.0 nM, while its binding apparent Ki va-
talent program of Chinese Academy of Sciences, and National Nat-
ural Science Foundation (Grant #30811120429, 3097063 and
90813033) for their financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.063.
lue against h-FABP was about 4.2 lM. This might be due to the rel-
atively smaller hydrophobic groove in h-FABP as compared to a-
FABP. The increase in the number of three-dimensional conforma-
tions compromised the binding affinity since it causes spatial col-
lision with some larger residues (such as Tyr115, Tyr117 and
Phe16), and hinders the formation of hydrogen bonds between
key carboxyl pharmacophore with residues Phe16 and Arg126 on
h-FABP as well (Supplementary data). Noticeably, when the car-
boxyl was substituted with dimethyl groups (compound 5g), the
biochemical activity against h-FABP was almost completely abol-
ished due to the unavoidable spatial collision caused by the rigidity
of the side chains.
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FABP inhibitors. The compounds strongly bound to the hydropho-
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binding affinities to the closely related homologue protein h-FABP.
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Acknowledgments
We thank National Basic Research Program of China (Grant
#2010CB529706, 2010CB9455000 and 2009CB940904), the 100-