Synthesis of BMS-309403-related compounds, including [14C]BMS- 309403, a radioligand for adipocyte fatty acid binding protein

Article abstract of DOI:10.1248/cpb.60.164

Adipocyte fatty acid binding protein (A-FABP; FABP4), which is predominantly expressed in macrophages and adipose tissue, regulates fatty acid storage and lipolysis, and is also an important mediator of inflammation. Here, we report a synthesis of ¹⁴

Full text of DOI:10.1248/cpb.60.164

—
164
Note
Chem. Pharm. Bull. 60(1) 164 168 (2012)
Vol. 60, No. 1
Synthesis of BMS-309403-Related Compounds, Including [14C]BMS-
309403, a Radioligand for Adipocyte Fatty Acid Binding Protein
Toshihiko Okada,a Makoto Hiromura,b Masato Otsuka,c Shuichi Enomoto,a,b and
,a
Hiroyuki Miyachi*
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University; c Department of
Genomics and Proteomics, Advanced Science Research Center, Okayama University; 1–1–1 Tsushima-Naka, Kita-ku,
b
Okayama 700–8530, Japan: and Laboratory of Multiple Molecular Imaging Research Center for Molecular Imaging
Science, RIKEN Kobe Institute; Kobe 650–0047, Japan.
Received September 14, 2011; accepted October 27, 2011; published online November 2, 2011
Adipocyte fatty acid binding protein (A-FABP; FABP4), which is predominantly expressed in macro-
phages and adipose tissue, regulates fatty acid storage and lipolysis, and is also an important mediator of
14
′
inflammation. Here, we report a synthesis of C-labeled 2-[2 -(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphe-
nyl-3-yloxy]acetic acid (BMS309403), a potent and selective small-molecular FABP4 inhibitor, as a chemical
tool for investigating the roles of FABP4 in inflammatory and metabolic disorders. The structure–activity
relationship of several BMS derivatives for inhibition of FABP4 is also reported.
Key words fatty acid binding protein 4; BMS-309403; radioligand; labelled synthesis; C-14; adipocyte fatty
acid binding protein
—
6)
Fatty acid binding proteins (FABPs) are small-molecular- lipid metabolism and insulin sensitivity,4
recent pharma-
weight hydrophobic proteins containing a large hydrophobic cological and biological findings have indicated a regulatory
—
10)
cavity, into which naturally occurring long-chain fatty acids function of FABP4 in inflammation.7
Because acute and/
and synthetic hydrophobic ligands can be accepted.1) FABPs or chronic inflammation is involved in many diseases, we con-
act as transporters of endogenous fatty acids from the cell sidered that visualization of FABP4 might be a powerful tool
surface to various sites of fatty acid storage and metabolism.2) to investigate not only metabolic disorders, but also inflamma-
Nine isoforms of FABPs are currently known, and each FABP tory disorders.
has a characteristic distribution pattern. Adipocyte FABP (also
Since a labelled FABP4 ligand is expected to be a valu-
known as FABP4, A-FABP, ALBP and aP2), which is the only able tool for FABP studies, we have prepared 14C-labelled
isoform predominantly expressed in macrophages and adipose 2-[2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-yloxy]-
tissue, regulates fatty acid storage and lipolysis in those tis- acetic acid (BMS-309403),11) which is a potent and selective
sues.3) But, in addition to the roles of FABP4 in regulating inhibitor of FABP4. Several derivatives of BMS-309403 were
Chart 1. Synthesis of BMS-309403 Derivatives
*To whom correspondence should be addressed. e-mail: miyachi@pharm.okayama-u.ac.jp
© 2012 The Pharmaceutical Society of Japan

January 2012
165
also prepared, and their structure–activity relationship for in- value, confirming the validity of the assay. We found that the
hibition of FABP4 is also reported. introduction of a side-chain alkyl group at the α-position of
Synthesis of Side-Chain Alkyl Derivatives of BMS- the carboxyl group decreased the activity, and the decrease
309403 The binding pocket of FABP4 is reported to be was greater as the alkyl chain length was increased.
extremely hydrophobic. Therefore, we first examined the ef-
fect of introducing an alkyl side chain at the α-position of the
carboxyl group of BMS-309403.
The three-dimensional structure of the complex of BMS-
309403 with FABP4 indicated that BMS-309403 formed a
2′-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-ol (5a; hairpin-like structure and its acetic acid moiety lay over
R1=H, R2=H), used as a precursor for the present study, the pyrazole moiety.11) One of the methylene protons at the
was prepared according to Sulsky et al., from commercially α-position of the carboxyl group of BMS-309403 was located
available 2-bromophenyhydrazine HCl.11) Compounds 5a was near the side-chain benzyl group of 16Phe of FABP4 (within
treated with either methyl 2-bromopropionate or methyl 2-bro- 3Å), and the other was located near the phenyl group at the
mobutyrate in the presence of potassium carbonate as a base 4-position of the pyrazole ring of BMS-309403 (within 3Å).
to afford ester derivatives 6a and b. Alkaline hydrolysis of the We speculated that the introduction of an alkyl chain at the
ester moiety of 6a,b afforded the desired acids 7a,b (Chart 1). α-position of the carboxyl group changed the conformation of
Synthesis of Fluorinated Derivatives of BMS-309403 BMS-309403, interfering with one or more of these interac-
We also synthesized fluorinated derivatives of BMS-309403 tions. Therefore, alkylation of BMS-309403 does not appear to
(7c,d), in view of the potential applicability of 18F-labeled be a promising avenue, and so we considered radiolabeling of
derivatives for positron emission tomographic (PET) im- BMS-309403 itself.
aging of chronic inflammation. The synthetic routes are
First, we focused on the two benzene rings attached to the
also depicted in Chart 1. 2-Bromophenylhydrazine (1) was pyrazole backbone of BMS-309403. Although the detailed
treated with either 1-(4-fluorophenyl)-2-phenylethanone or structure–activity relationship at the 3- and 4-position ben-
2-(4-fluorophenyl)-1-phenylethanone in the presence of sodium zene rings of BMS-309403 has not been established, we were
acetate in ethanol to afford the condensed derivatives 2b and interested in the introduction of fluorine at the para-positions
c. These compounds were treated with n-propionic anhydride, of both benzene rings, in view of the potential applicability
followed by cyclization and Suzuki coupling reaction to afford of 18F-labeled derivatives for PET imaging. Table 1 summa-
biphenyl-pyrazole derivatives 5b and c. Compounds 5b and rizes the FABP4-inhibitory activity of BMS-309403 and its
c were treated with ethyl 2-bromoacetate in the presence of fluorinated derivatives. It is noteworthy that the introduction
potassium carbonate as a base, and subsequent alkaline hy- of fluorine at the para-position of the 3-phenyl group of BMS-
drolysis of the ester moiety afforded the desired acids 7c and 309403 resulted in retention or improvement of the FABP-4
d (Chart 1).
inhibitory activity, while the introduction of fluorine at the
Structure–Activity
Relationship
of
BMS-309403 para-position of the 4-phenyl group considerably decreased
Derivatives The primary goal of this study is to prepare a the FABP-4 inhibitory activity as compared to that of the par-
FABP-4-selective radio isotope-labeled ligand as a candidate ent BMS-309403. These data might indicate that the binding
probe to image inflammation, especially inflammation-associ- pockets hosting the 3-phenyl group and 4-phenyl group differ
ated macrophages. Table 1 summarizes the FABP4-inhibitory in volume and/or shape.
activity of the prepared alkyl-substituted BMS-309403 deriva-
The X-ray crystal study of BMS-309403 complexed with
tives, in addition to BMS-309403 itself and arachidonic acid, FABP4 indicated that the 3-position phenyl group binds to the
a naturally occurring unsaturated fatty acid, as a positive hydrophobic pocket formed by 16Phe, 19Tyr, 20Met, 25Val,
control. The assay-kit supplier reported that the IC50 value of 57Phe and 76Asp. The distance between the 4-position hydro-
arachidonic acid is 3μM with this kit. We obtained a similar gen atom of the 3-phenyl group of BMS-309403 and the near-
est amino acid (25Val) was estimated to be more than 2.5Å.12)
On the other hand, the 4-position phenyl group binds to the
hydrophobic pocket formed by 33Ala, 34Gly, 36Ala, 38Pro,
Table 1. FABP4-Inhibitory Activity of BMS-309403 Derivatives
55Ser, 57Phe and 126Arg. The distance between the 4-posi-
tion hydrogen atom of the 4-phenyl group of BMS-309403 and
the nearest amino acid (36Ala) was estimated to about 1.7Å.
These results indicated that there may not be enough space to
accommodate a fluorine atom at the 4-position of the 4-phenyl
group of BMS-309403. This might be the reason why the in-
troduction of fluorine at the 4-position decreased the FABP4-
R1
R2
R3
IC50 (μM)
inhibitory activity, even though fluorine is a small atom.
Based on these preliminary results, we decided that appro-
priate labeling positions would the α-position of the carboxyl
group of BMS-309403 for 14C, and the 4-position of the 3-phe-
nyl group of BMS-309403 for 18F.
H
H
H
F
H
H
H
H
F
H
Me
Et
H
(BMS-309403)
0.71
2.35
2.91
0.33
4.67
5.13
Synthesis of [14C]BMS-30940313) [14C]BMS-309403 was
prepared analogously by the reaction of 5 with 2-[14C]ethyl
bromoacetate in the presence of potassium carbonate as a
base. Alkaline hydrolysis, followed by column-chromato-
graphic purification, afforded [14C]BMS-309403 9 in satisfac-
H
H
Arachidonic acid
The inhibitory activities were determined with a Cayman FABP4 assay kit,
according to the supplier s protocol. Arachidonic acid was used as a positive
control.
’

166
Vol. 60, No. 1
tory yield and high radiochemical purity of 94.8%.
1-(2-Bromophenyl)-5-ethyl-3,4-diphenyl-1H-pyrazole,
To a solution of 3 (334mg, 0.793mmol) in dehydrated N,N-
4
dimethylformamide (5mL) at room temperature was added
Conclusion
We synthesized 14C-labeled BMS-309403 from commer- sodium hydride (60% dispersion, 79mg, 3.30mmol) in several
cially available 14C-labeled ethyl bromoacetate in excellent ra- portions over 1min under argon. The reaction mixture was
°
°
diochemical purity and with sufficiently high specific activity. warmed to 38 C and then further heated to 50 C for 16h. The
Several BMS-309403 related compounds were also prepared deep red reaction mixture was cooled and quenched by the ad-
to enable us to select appropriate positions for labeling with dition of water (10mL). The mixture was acidified with aque-
14C and 18F. As BMS309403 is a potent and selective small- ous HCl (1mol/L, 20mL) and then extracted four times with
molecular FABP4 inhibitor, and FABP4 is expressed mainly ether (30mL). The organic extracts were washed with water
to macrophages and inflammatory response was reported to (50mL) and brine (50mL), dried over anhydrous magnesium
—
16)
augment the expression of FABP4,14
the labeled compound sulfate and concentrated in vacuo. The residue was purified
is expected to be useful as a chemical tool for investigating by means of column chromatography (eluant; n-hexane:ethyl
the roles of FABP4 in inflammatory and metabolic disorders. acetate=5/1 v/v) to afford 4 (110mg, 34%) as an orange oil.
1
Studies along this line, including the synthesis of [18F]BMS-
309403, are in progress.
H-NMR (400MHz, CDCl3) δ: 7.76 7.22 (m, 14H), 2.54 (m,
—
2H), 0.91 (t, J=7.8Hz, 3H). MS (FAB) m/z 403, 405 (M+H+).
′
2 -(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-ol,
5
Aqueous NaOH (2mol/L, 1.0mL) was added to a mixture
Experimental
General All reagents were of commercial grade un- of bromophenyl pyrazole (4) (318mg, 0.788mmol), 3-hy-
less otherwise stated. 2-[14C]Ethyl bromoacetate (52mCi/ droxyphenylboronic acid (130mg, 0.946mmol), and Pd2(dba)3
mmol) was purchased from Moravek Biochemicals and (22mg, 0.023mmol) in ethanol (2.0mL). The reaction mixture
Radiochemicals, and was supplied as CH2Cl2 solution was heated to reflux with stirring for 15h under argon, then
(0.5mL). Radioactivity measurements were carried out using cooled to room temperature, quenched with water (50mL) and
a Perkin Elmer liquid scintillation counter Tri-Carb 2800 TRs extracted four times with ethyl acetate (30mL). The organic
and Clera-sol Ias scintillant. Flash column-chromatography extracts were washed with water (50mL) and brine (50mL),
—
was performed using Merck silica gel 0.04 0.063mm. Thin- dried over anhydrous magnesium sulfate, and concentrated
layer chromatography (TLC) was run on Merck Kieselgel in vacuo. The residue was purified by means of column chro-
silica gel 60 F254 glass plates.
matography (eluant; n-hexane:ethyl acetate=5/1 v/v) to give
(E)-1-(2-Bromophenyl)-2-(1,2-diphenylethylidene)hydra- 5 (281mg, 0.675mmol, 86%) as a brown solid. 1H-NMR
—
—
zine, 2 To a stirred solution of 2-bromophenylhydrazine (400MHz, CDCl3) δ: 7.64 6.55 (m, 18H), 2.16 2.05 (m,
hydrochloride (1) (1.00g, 4.47mmol) in ethanol (20mL) were 2H), 0.60 (t, J=7.6Hz, 3H).
′
added sodium acetate trihydrate (877mg, 4.47mmol) and
benzyl phenyl ketone (608mg, 4.47mmol) under argon. The [1,1 -biphenyl]-3-yl)oxy)propanate, 6a To
mixture was heated to reflux for 20h and then cooled and (171mg, 0.411mmol) in anhydrous dehydrated N,N-
Methyl
2-((2 -(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-
′
a
slurry of
5
evaporated. The reaction mixture was poured into saturated dimethylformamide (4mL) was added potassium carbonate
sodium hydrogencarbonate solution (10mL), and extracted (113mg, 0.822mmol), followed by methyl 2-bromopropionate
three times with ethyl acetate (50mL). The combined organic (0.05mL, 0.41mmol). The reaction mixture was stirred at
extracts were washed with water (100mL) and brine (100mL), room temperature for 3h. Additional methyl 2-bromopro-
dried over anhydrous magnesium sulfate, and concentrated in pionate (0.05mL, 0.41mmol) was added and stirring was
vacuo. The residue was purified by means of column chroma- continued for 21h. The reaction mixture was cooled to room
tography (eluant; n-hexane:ethyl acetate=12/1 v/v) to afford 2 temperature, quenched with water (150mL) and extracted
1
(1.21g, 74%) as a white solid. H-NMR (400MHz, CDCl3) δ: four times with ethyl acetate (50mL). The combined organic
—
7.97 6.65 (m, 14H), 4.18 (s, 2H), 3.93 (s, 1H). MS (FAB) m/z extracts were washed once with water (50mL) and once with
365, 367 (M+H)+.
brine (50mL), dried over anhydrous magnesium sulfate, and
(E)-N-(2-Bromophenyl)-N -(1,2-diphenylethylidene)pro- concentrated in vacuo. The residue was purified by means
′
pionohydrazide, 3 To a stirred mixture of sodium hydride of silica gel column chromatography (eluant; n-hexane:ethyl
(60% dispersion, 173mg, 7.20mmol) in N-methylpyrrolidone acetate=3/1, v/v) to give 6a (126mg, 61%) as a yellow oil.
1
—
H-NMR (400MHz, CDCl3) δ: 7.67 6.66 (m, 18H), 4.58
(NMP) (5mL) was added a solution of 2 (1.21g, 3.31mmol) in
—
NMP (5mL) over 5min under argon at room temperature. The (q, J=6.8Hz, 1H), 3.61 (s, 3H), 2.08 2.05 (m, 2H), 1.44 (d,
°
reaction mixture was heated to 60 C for 3h and then cooled J=6.8Hz, 3H), 0.60 (t, J=7.4Hz, 3H).
′ ′
2-((2 -(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-[1,1 -
to room temperature. To this solution was added propionic an-
hydride (0.57mL, 4.62mmol) at a rate appropriate to keep the biphenyl]-3-yl)oxy)propanoic Acid, 7a A solution of 6a
°
reaction temperature below 35 C. The reaction mixture was (121mg, 0.241mmol) in 1,4-dioxane (0.8mL) was treated with
stirred for 3h, then quenched with aqueous HCl (0.5mol/L, sodium hydroxide solution (2mol/L, 0.3mL). The reaction
100mL) and extracted four times with ether (30mL). The mixture was stirred at room temperature for 3h. The reaction
ether extracts were combined, washed with water (100mL) mixture was cooled to room temperature and acidified with
and brine (100mL), dried over anhydrous magnesium sul- aqueous citric acid solution (10%, 5mL). The mixture was
fate, and concentrated in vacuo. The residue was purified by diluted with water (20mL) and stirred vigorously for 30min.
means of column chromatography (eluant; n-hexane:ethyl ac- The precipitate was collected by filtration, washed with wa-
etate=5/1 v/v) to afford 3 (805mg, 58%) as an orange oil. MS ter, and dissolved in ethyl acetate (100mL). The solution was
(FAB) m/z 421, 423 (M+H)+.
dried over anhydrous magnesium sulfate and concentrated

January 2012
167
in vacuo. The resulting residue purified by silica gel column
chromatography (eluant; n-hexane:ethyl acetate=5/1, v/v) to
1
give 7a (111mg, 95%) as a white solid. H-NMR (400MHz,
—
CDCl3) δ: 7.64 6.61 (m, 18H), 4.45 (m, 1H), 2.05 (m, 2H),
1.35 (m, 3H), 0.57 (t, J=6.8Hz, 3H). High resolution (HR)-MS
(FAB, MH+) Calcd for C32H28N2O3 489.2178, Found 489.2165.
′
′
2-((2 -(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)-[1,1 -
biphenyl]-3-yl)oxy)butanoic Acid, 7b This compound
(white solid, 79% yield) was prepared from 6b by means of
1
a procedure similar to that used for 7a. H-NMR (400MHz,
—
—
CDCl3) δ: 7.64 6.64 (m, 18H), 4.32 4.15 (m, 1H), 2.05 (m,
2H), 1.74 (m, 2H), 0.83 (m, 3H), 0.57 (t, J=7.0Hz, 3H). HR-
MS (FAB, (M+H)+) Calcd for C32H28N2O3 503.2335, Found
503.2329.
′
2-((2 -(5-Ethyl-4-(3-fluorophenyl)-3-phenyl-1H-pyrazol-
Fig. 1. X-Ray Crystallographic Structure of BMS-309403 Complexed
with FABP4 (pdb:2NNQ)
′
1-yl)-[1,1 -biphenyl]-3-yl)oxy)acetic Acid, 7c This com-
pound (white amorphous solid) was prepared from 1 by means
of a procedure similar to that used for 7a. H-NMR (400MHz,
CDCl3) δ: 7.66 6.65 (m, 17H), 4.38 (s, 2H), 2.06 2.05 (m,
2H), 0.58 (t, J=7.4Hz, 3H). HR-MS (FAB, (M+H)+) Calcd for
C31H26FN2O3 493.1927, Found 473.1928.
(A) The whole structure of BMS-309403 complexed with FABP4. FABP4 pro-
tein is depicted as ribbon model, and BMS-309403 as boll and stick model. (B)
Expanded view of BMS-309403 (boll and stick model) showing the amino acid side
chains within 4.5A (boll and wire model) from the oxyacetic acid moiety of BMS-
309403. (C) Expanded view of BMS-309403 (cylinder) and the amino acids hosting
the 3-phenyl group of BMS-309403. The 3-phenyl group of BMS-309403 and its
nearest amino acid, Val25, are illustrated as space-filling models. (D) Expanded
view of BMS-309403 (cylinder) and the amino acids hosting the 4-phenyl group
of BMS-309403. The 4-phenyl group of BMS-309403 and its nearest amino acid,
Ala36, are illustrated as space-filling models.
1
—
—
′
2-((2 -(5-Ethyl-4-(4-fluorophenyl)-3-phenyl-1H-pyrazol-
′
1-yl)-[1,1 -biphenyl]-3-yl)oxy)acetic Acid, 7d This com-
pound (white amorphous solid) was prepared from 1 by means
of a procedure similar to that used for 7a. H-NMR (300MHz,
1
—
CDCl3) δ: 7.64 6.65 (m, 17H), 4.40 (s, 2H), 2.05 (m, 2H),
0.58 (t, J=7.5Hz, 3H). HR-MS (FAB, (M+H)+) Calcd for Reagent that exhibits increased fluorescence at 500nm when
C31H26FN2O3 493.1927, Found 493.1949. bound to FABP4. A ligand and/or inhibitor of FABP4 displac-
14
′
2[ C](2 -(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphe- es the Detection Reagent, thereby reducing the fluorescence.
nyl-3-yloxy)acetic Acid, [14C]BMS 309403, 9 To a solu- The assay was performed according to the supplier s protocol.
’
tion of 5 (12.0mg, 30.0μmol), potassium carbonate (4.00mg,
30.0μmol) in dehydrated N,N-dimethylformamide (5mL) was
Acknowledgement This work was supported in part by
added ethyl bromoacetate (3.06μL, 20.0μmol (containing the Targeted Proteins Research Program of the Japan Science
1.06μL 2-[14C]ethyl bromoacetate and 2.00μL ethyl bromoac- and Technology Corporation (JST), the Uehara Memorial
etate: total radioactivity was 6.852MBq)) in a mixed solution Foundation and the Tokyo Biochemical Research Foundation
of CH2Cl2 (0.5mL) and dehydrated N,N-dimethylformamide (TBRF). We are grateful to Dr. Hiroki Kakuta for his help in
°
(4.5mL). The reaction mixture was stirred at 50 C for 27h. FABP4 inhibitory activity assay.
The whole was quenched with water (100mL) and extracted
four times with ethyl acetate (30mL×4). The combined or-
ganic extracts were dried over anhydrous magnesium sulfate,
concentrated in vacuo. To the residue was added 1,4-dioxane
(0.5mL) and 2mol/L sodium hydroxide solution (0.025mL).
References and Notes
—
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—
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—
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168
Vol. 60, No. 1
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