Structure-guided design, synthesis and in vitro evaluation of a series of pyrazole-based fatty acid binding protein (FABP) 3 ligands

Article abstract of DOI:10.1016/j.bmcl.2013.01.054

We designed a series of pyrazole-based carboxylic acids as candidate ligands of heart fatty acid binding protein (H-FABP, or FABP3), based on a comparison of the X-ray crystallographic structures of adipocyte fatty acid binding protein (FABP4)-selective inhibitor (BMS309403) complex and FABP3-elaidic acid complex. Some of the synthesized compounds exhibited dual FABP3/4 ligand activity, and some exhibited selectivity for FABP3.

Full text of DOI:10.1016/j.bmcl.2013.01.054

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1662–1666
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-guided design, synthesis and in vitro evaluation of a series
of pyrazole-based fatty acid binding protein (FABP) 3 ligands
⇑
Yoko Beniyama, Kenji Matsuno, Hiroyuki Miyachi
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University 1-1-1, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed a series of pyrazole-based carboxylic acids as candidate ligands of heart fatty acid binding
protein (H-FABP, or FABP3), based on a comparison of the X-ray crystallographic structures of adipocyte
fatty acid binding protein (FABP4)–selective inhibitor (BMS309403) complex and FABP3–elaidic acid
complex. Some of the synthesized compounds exhibited dual FABP3/4 ligand activity, and some exhibited
selectivity for FABP3.
Received 22 December 2012
Revised 11 January 2013
Accepted 16 January 2013
Available online 26 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Heart fatty acid binding protein
FABP3
Pyrazole
Structural design
The increasing availability of three-dimensional structures of
proteins and protein–ligand complexes has proved extremely
helpful in allowing medicinal chemists to design ligands with in-
creased potency and selectivity. Here, we adopted this approach
with the aim of obtaining subtype-selective fatty acid-binding pro-
tein (FABP) ligands.
Cytosolic FABPs are a family of low-molecular-weight (about
14–15 kDa) proteins, expressed in a tissue-specific manner, which
bind medium- to long-chain fatty acids as endogenous ligands.1
They are involved in modulating intracellular lipid metabolism,
regulation of gene expression, and intracellular shuttling of fatty
acids.2 Nine FABPs have been identified to date, that is, FABP1–9,
or liver (l-), intestinal (i-), heart (h-), adipocyte (a-), epidermal
(e-), ileal (il-), brain (b-), myelin (m-) and testis (t-) FABPs.3
Although the primary sequences of the FABPs show significant
diversity (15–70% sequence identity), the FABPs exhibit very simi-
lar three-dimensional structures, consisting of 10 antiparallel b-
strands folded into two b-sheets, which form a b-barrel with an
internal ligand-binding cavity (Fig. 2).4 A helix-turn-helix motif
serves to close the main opening to the b-barrel.
macrophage-specific deletion of FABP4 has a protective effect
against atherosclerosis in apolipoprotein E-deficient mice.9 How-
ever, ligands for other subtype-specific FABP ligands have received
relatively little attention. Therefore, we set out to design ligands
for human heart FABP (FABP3), which shows 65% sequence identity
with human FABP4. The function of FABP3 is not completely dis-
closed, but some report indicated that FABP3 is also involved in li-
pid homeostasis, for FABP3 is involved in the uptake of fatty acids
and their subsequent transport towards the mitochondrial b-oxi-
dation systems.10
We focused on pyrazole-based FABP4-selective inhibitors 111
(BMS309403) and 212 as lead compounds (Fig. 1).
Compound 1 is a potent and selective FABP4 inhibitor, and the
X-ray crystallographic structure of FABP4–BMS309403 complex is
available in PDB (pdb: 2nnq: Fig. 2A).11 Pyrazole derivative 2 was
reported to exhibit more potent FABP4-inhibitory activity than
BMS309403. On the other hand, the three-dimensional structure
of FABP3–elaidic acid (a naturally occurring long-chain unsatu-
rated fatty acid) complex is also available (pdb: 1hmr: Fig. 2C).13
As mentioned above, the structural folds of FABP4 and FABP3 are
well conserved, that is, both molecules consist of ten antiparallel
b-strands folded into two b-sheets, which form a b-barrel with
an internal ligand-binding cavity. BMS-309403 and elaidic acid
interact with the same amino acids, which form a hydrophobic
cavity, and their carboxylic acid moieties form hydrogen-bonding
interactions with Arg126 and Tyr128 (FABP4 sequence). The resid-
ual hydrophobic tail part of both molecules lies in the vast hydro-
phobic cavity of each FABP.
Because of the biological activities of FABPs, ligands that may
modulate the functions of FABPs function are of interest. In partic-
ular, many researchers have focused on the creation of FABP4 (adi-
pocyte FABP or aP2) inhibitors with the aim of discovering
candidate drugs for the treatment of diabetes and atherosclerosis
(Fig. 1).5–7 The rationale for this is that disruption of FABP4 in mice
prevents development of diet-induced insulin resistance,8 and
In order to create ligands that preferentially bind to FABP3, we
computationally introduced BMS309403 into the ligand-binding
⇑
Corresponding author.
E-mail address: miyachi@pharm.okayama-u.ac.jp (H. Miyachi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.054

Y. Beniyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1662–1666
1663
S
N
N
N
N
O
O
O
S
COOH
COOH
N
H
Cl
COOH
Me
S
MeOOC
1
2
3
6
O
COOH
H
N
O
S
N
N
N
H2NOC
O
Cl
N
N
N
H
COOH
4
5
Figure 1. Representative synthetic FABP inhibitors. 1–4: FABP4-selective inhibitors. 5: FABP3/4 dual inhibitor. 6: FABP4/5 dual inhibitor.
Figure 2. X-ray crystallographic structures of fatty acid binding protein (FABP)–ligand complexes and superimposed structures of FABP4 and FABP3 complexed with
BMS309403. (A) FABP4–BMS309403 complex (PDB: No. 2NNQ). (B) Superimposed structure of FABP4–BMS309403 complex and FABP3–elaidic acid complex. (C) FABP3–
elaidic acid complex (PDB: No. 1HMR). Amino acids located near (D) the phenoxyacetic acid part, (E) the 3-position of the pyrazole ring, (F) the 4-position of the pyrazole ring,
and (G) the 5-position of the ethyl ring of BMS309403 are shown.
cavity of FABP3 in the Molecular Operating Environment, MOE (we
did not take into account induced fit, for convenience). The results
are depicted in Figure 2D–G.
We noted that the proximal phenyl group of the biphenyloxy-
acetic acid moiety of BMS-309403 interacted with the small
binding cavity of FABP4, formed from Tyr19, Glu72, His93, Glu95,
Ile104, Arg106 and Cys117. However the corresponding amino acid
104 of FABP3 is Leu instead of Ile. The side-chain isobutyl group of
Leu104 appears to have a short contact with the benzene ring of
the biphenyloxyacetic acid moiety of BMS309403. Therefore the

1664
Y. Beniyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1662–1666
rearrange
Introduce substituent
Figure 3. General formula of the present series of FABP3 ligands based on FABP4-selective inhibitor BMS-309403 as a template.
OMe
CH3
OMe
a
b
R1
N
N
N
N
c
OMe
OMe
R2
R2
O
O
R1
9a-u
R1
7
8a-d
10a-u
d
f
e
OH
O
O
N
N
N
N
N
N
n
n
COOH
COOEt (Me)
R2
R2
R2
R1
R1
R1
11a-u
12a-u
13a-u
O
S
O
OH
N
N
N
N
O
CF3
h
COOH
N
N
O
g
11a
15
14
Scheme 1. Synthesis of 1,3,5-trisubstituted pyrazole derivatives. Reagents and conditions: (a) benzaldehyde derivatives, 10% KOH, EtOH, rt, 2 days, 78–96%; (b)
phenylhydrazine HCl derivative (or cycloalkylhydrazine HCl derivative), EtOH, reflux, 20 h, 43–87%; (C) DDQ, benzene, reflux, 16 h, 63–94%; (d) BBr3, dry CH2Cl2, À78 to 0 °C,
7 h, 47–97%; (e) Br-(CH2)n-CO2R (R = Me or Et)), NaH, dry DMF, 0–50 °C, 4 h, 47–91%; (f) 1 mol/L NaOH, EtOH, 50 °C, 4 h, 35–97%; (g) trifluoromethanesulfonic anhydride,
pyridine, dry CH2CL2, 0–20 °C, 4 h, 98%; (h) (1) ethyl 2-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)acetate, Cs2CO3, Pd(OAc)2, BINAP, 1,4-dioxane, reflux,
overnight, 46% (2) 1 mol/L NaOH, MeOH, 50 °C, 3 h, 86%.
presence of this proximal phenyl group attached at the 1-position
nitrogen atom of the pyrazole nucleus might be unfavorable for a
FABP3 selective ligand (Fig. 2D). The phenyl group at the 3-position
of the pyrazole ring of BMS309403 interacted with the small bind-
ing cavity of FABP4, formed from Phe16, Met20, Val25, Ala33,
Phe57, Ala75, Asp76 and Arg78, but the side-chain benzyl group
of the corresponding Phe57 of FABP3 is located far distant
(Fig. 2E). Also, the phenyl group attached at the 4-position of the
pyrazole ring of BMS309403 interacted with the side chain amino
acids of Phe16, Ala33, Ala36 Pro38, Ser55, Phe57 and Arg126 of
FABP4. However the corresponding amino acid 36 of FABP3 is
Thr instead of Ala. As a consequence, the hydrophobic pocket host-
ing the 4-phenyl group of the pyrazole ring of BMS309403 is wider
in the case of FABP3 ( Fig. 2F). These structural differences
prompted us to speculate that the introduction of suitable substi-
tuent(s) at the phenyl groups on the pyrazole ring of BMS309403
might selectively increase the affinity for FABP3. Finally, the ethyl
group at the 5-position of the pyrazole ring of BMS309403 inter-
acted with a small ‘dimple’ composed of the side chain amino acids
of Pro38, Asn39, Met40, Ser53 and Tyr128 of FABP4. But, in the case
of FABP3, the corresponding amino acids 40 and 53 of FABP3 are
Thr and Thr instead of Ser and Met. Notably, Thr53 is located close
to the methylene group of the 5-position ethyl group. Therefore,
the presence of this ethyl group might be unfavorable for FABP3-
selecive ligands (Fig. 2G). Based on these structural considerations,
we focused on the general formula depicted in Figure 3 as a puta-
tively selective ligand structure for FABP3.
The synthetic route to the present series of designed compounds
is shown in Scheme 1. Condensation of chalcone derivatives, pre-
pared from the aldol condensation of 2-methoxyacetophenone
and substituted benzaldehydes with substituted phenylhydrazines
HCl (or cycloalkylhydrazines HCl) under acidic conditions in ethanol
yielded tri-aromatic substituted 4,5-dihydro-1H-pyrazoles 9a–u,
which were aromatized to pyrazoles 10a–u by means of 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidization.14 As pre-
viously reported, when the cyclization reaction was performed
in DMSO as a solvent, cyclization and subsequent aromatization
occurred to form the pyrazole ring,14 but in poor yield, and the prod-
uct was impure. The methoxy group was demethylated with BBr3,
alkylated with ethyl (or methyl) bromo alkanoate, and hydrolyzed
with alkali to afford the desired compounds 13a–u. Biphenyloxy-
acetic acid type compound 15 was also prepared for comparison.
All compounds were tested for binding-inhibitory activity in the
8-anilino-1-naphthalenesulfonic acid (1,8-ANS)-based fluores-

Y. Beniyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1662–1666
1665
cence displacement assay developed by Kane and Bernlohr,15 with
a slight modification (all the test compounds were dissolved in
ethanol instead of DMSO, for DMSO decreased fluorescent inten-
Table 1
FABP-inhibitory activities of the present series of compounds
sity). The results, expressed as IC50 (lM), are summarized in Table
1. BMS309403 was utilized as the positive control to validate the
screening assay. In our assay system, BMS309403 exhibited a
sub-micromolar IC50 value towards FABP4, with more than 50-fold
selectivity over FABP3,15 in agreement with reported data.
Although we did not find a clear-cut structure–activity relation-
ship, some important features were noted. Compound 15, a posi-
tional isomer of BMS309403 lacking the ethyl side chain,
exhibited a somewhat decreased, but still potent, IC50 value against
FABP4, as compared with BMS309403. But, it did not inhibit FABP3
O
N
n
COOH
N
R2
R1
No.
R1
R2
n
IC50
FABP 3
(
l
M)
FABP 4
13a
13b
13c
13d
13e
13f
13g
13h
13i
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-MePh
4-BrPh
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
1
3
4
6
3
3
3
4
4
4
3
3
3
3
3
3
3
3
3
3
3
>10,000
21.0
243
>10,000
4730
224
>10,000
6.13
74.0
31.0
5.41
9.51
3.02
6.39
259
86.0
10.2
5.64
4.32
10.3
13.6
4.27
IA
activity at the highest concentration of 10,000 lM, so its FABP4
selectivity is superior to that of BMS-309403. Alkyl tether length,
connecting the carboxyl group and the phenolic oxygen, strikingly
affected activity towards both FABPs. In the case of FABP3, the ace-
tic acid derivative (13a: n = 1), was ineffective even at the highest
concentration examined. The butyric acid derivative (13b: n = 3),
showed comparable inhibitory activity towards both FABP’s, while
the pentanoic acid derivative (13c: n = 4) exhibited decreased
inhibitory activity as compared with 13b. Therefore, we selected
the tether length of n = 3.
2.18
Ph
>10,000
>10,000
22.3
4-MePh
4-BrPh
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
13j
13k
13l
4-FPh
4-BrPh
4-ClPh
4-iPrPh
4-MeOPh
2-ClPh
2-MeOPh
3-ClPh
Cyclopentyl
Cyclohexyl
Cycloheptyl
2.01
0.733
0.748
0.787
1.65
1.16
505
0.695
6.03
5.26
13m
13n
13o
13p
13q
13r
13s
13t
13u
We next turned our attention to the R1 position. Introduction of
a 4-position substituent at the R1 phenyl group greatly affected
FABP3-inhibitory activity, that is, the 4-methyl and 4-bromo deriv-
atives, 13e and 13f, exhibited reduced FABP3-inhibitory activity,
whereas the unsubstituted phenyl derivative, 13g, retained the
activity. As for FABP4, these compounds exhibited comparable
micromolar-order inhibitory activity. These results indicate that
the introduction of a 4-position substituent did not greatly affect
the FABP4-inhibitory activity, suggesting that there is a difference
between the modes of interaction with FABP3 and FABP4. We
2.50
16.6
23.5
15.5
6.47
1
15
14.7
>10,000
0.221
0.987
Data are the mean of two duplicated independent assay results.
RU
RU
13g
13k
80
80
60
40
20
60
40
20
0
0
-10
-20
-100
-50
0
50
100
150
200
250
s
-100
-50
0
50
100
150
200
250
s
Time
Time
RU
100
RU
13l
13m
100
80
80
60
40
60
40
20
20
0
0
-20
-100
-20
-50
0
50
100
150
200
250
s
-100
-50
0
50
100
150
200
250
s
Time
Time
2.0
1.5
1.0
0.5
0.0
R² = 0.9783
H
ka (1 / Ms) kd (1 / s) KD (µM) IC50 (µM)
F
No.
2.60 x 103
6.43 x 103
5.83 x 103
6.16 x 103
0.0426
0.0338
0.0192
0.0134
16.3
5.26
3.30
2.17
2.01
1.14
13g
13k
13l
Br
Cl
0.749
0.733
13m
0.0
5.0
10.0
15.0
20.0
KD (µM)
Figure 4. Representative Biacore sensorgrams, using FABP3-functionalized CM5 sensors, together with physico-chemical parameters, and the correlation between KD and
IC50 values.

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Y. Beniyama et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1662–1666
selected the unsubstituted phenyl group as the optimum R1
substituent.
Acknowledgments
Then we moved on to the R2 substituent. It is noteworthy that
the effect of a substituent introduced at the 4-position of the ben-
zene ring is small as compared to the case of the R1 substituent.
Compounds 13k–13o exhibited micromolar to sub-micromolar or-
der IC50 values towards FABP3. Change of the position of the sub-
stituent from the 4-position to the 2- or 3-position might be
tolerable in the case of a relatively small chlorine atom. However,
the 2-MeO derivative 13q exhibited decreased FABP3-inhibitory
activity. Similar tendencies were also seen in the case of FABP4,
though the activities were rather weak. As a R2 substituent, an aro-
matic ring is preferable to a cycloalkyl ring, because all three cyclo-
alkyl ring derivatives 13s–13u exhibited decreased inhibitory
activities towards both FABPs. In the present series, compounds
13l, 13m and 13n exhibited selective FABP3-inhibitory activity at
submicromolar concentration.
We thank Drs. Takaaki Miyaji and Asako Kawakami for Biacore
binding analyses.
This work was supported in part by the Drug Discovery for
Intractable Infectious Diseases Project, Graduate School of Medi-
cine, Dentistry and Pharmaceutical Sciences, Okayama University,
the Uehara Memorial Foundation, the Tokyo Biochemical Research
Foundation (TBRF), and the Okayama Foundation for Science and
Technology (OFST).
References and notes
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In order to confirm that the FABP3-inhibitory activities of the
present series of compounds were due to direct binding of the
compounds to FABP3, we performed direct binding assay of repre-
sentative compounds (13g, 13k–13m) based on the principle of
the surface plasmon resonance, using a Biacore X 100 system with
a FABP3-functionalized sensor-chip (Fig. 4). We observed micro-
molar-order KD values, ranging from 2 to 16 lM, which were well
correlated with the IC50 values (R2 = 0.98). These data clearly indi-
cated that the pyrazole compounds directly bind to the binding
pocket of FABP3, and compete with the fluorescent ligand 1,
8-ANS.
In conclusion, we designed and synthesized a series of 1,3,5-tri-
substituted pyrazole derivatives as candidate FABP3 ligands, and
found that 4-(2-(1,5-diphenyl-1H-pyrazol-3-yl)phenoxy)butanoic
acid structure is a good lead structure for FABP3-selective inhibi-
tors. Further structural development and in vitro pharmacological
evaluation of the present series of compounds are under way.
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