Arylpiperazines as fatty acid transport protein 1 (FATP1) inhibitors with improved potency and pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2013.02.116

The discovery and optimization of a novel series of FATP1 inhibitors are described. Through the derivatization process, arylpiperazine derivatives 5k and 12a were identified as possessing potent in vitro activity against human and mouse FATP1s as well as

Full text of DOI:10.1016/j.bmcl.2013.02.116

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2560–2565
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylpiperazines as fatty acid transport protein 1 (FATP1) inhibitors
with improved potency and pharmacokinetic properties
Tetsuyoshi Matsufuji a, Mika Ikeda b, Asuka Naito b, Masakazu Hirouchi c, Shoichi Kanda b,
Masanori Izumi b, Jun Harada b, Tsuyoshi Shinozuka a,
⇑
a Lead Discovery & Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and optimization of a novel series of FATP1 inhibitors are described. Through the deriva-
tization process, arylpiperazine derivatives 5k and 12a were identified as possessing potent in vitro activ-
ity against human and mouse FATP1s as well as excellent pharmacokinetic properties. In vivo evaluation
of triglyceride accumulation in the liver, white gastrocnemius muscle and soleus is also described.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 9 January 2013
Revised 21 February 2013
Accepted 28 February 2013
Available online 7 March 2013
Keywords:
Fatty acid transport protein
High-throughput screening
Microsomal stability
Arylpiperazine
Skeletal muscle is one of the most promising target tissues for
the treatment of insulin resistance as it accounts for the largest
proportion of body mass and uptakes glucose in an insulin-depen-
dent manner. However, no drug acting directly on skeletal muscle
is presently available for the treatment of insulin resistance. Fatty
acid transport protein 1 (FATP1) is a transmembrane protein with
acyl-CoA synthase activity, that is, highly expressed in skeletal
muscle.1 FATP1 is reported to play a major role in uptaking and
metabolizing fatty acids through their conversion to the corre-
sponding acyl-CoA in skeletal muscle. In FATP1-deficient mice, less
accumulation of intramuscular fatty acyl-CoA was observed under
a high-fat-diet (HFD)-fed condition, which made them less insulin
resistant.2 Therefore, the inhibition of FATP1 is an attractive thera-
peutic target for the treatment of insulin resistance. In a previous
paper, we reported the potent triazole-based FATP1 inhibitor 1
(Fig. 1) with moderate pharmacokinetic parameters.3 To acquire
potent anti-insulin-resistance activity in vivo, further improve-
ment of pharmacokinetic parameters was required. This led us to
discover a new scaffold with an improved pharmacokinetic profile
for the evaluation of in vivo efficacy. In this Letter, we describe the
structure–activity relationship (SAR) of novel arylpiperazine deriv-
atives as FATP1 inhibitors with improved pharmacokinetic
parameters.
Through the high-throughput screening (HTS) of our corporate
library, phenylpiperazine derivatives 2 and 3 were identified
(Fig. 1). In an in vitro assay, to validate the direct inhibitory activity
against FATP1, cell-free fatty acyl-CoA synthase (ACS) activity was
evaluated with purified human or mouse FATP1 protein, because
the transportation of fatty acid by FATP1 is known to involve ACS
synthesis.4 Although tetrahydrobenzothiazole 2 possessed potent
in vitro activity against human FATP1,
2 showed moderate
in vitro activity against mouse FATP1.5 Furthermore, as the mouse
microsomal stability of 2 was 30%,6 it was also necessary to ad-
dress this issue. On the other hand, imidazopyridine 3 was needed
for the improvement of in vitro activity against both species. Thus,
we initiated the derivatization of 2 and 3 to improve in vitro activ-
ity as well as microsomal stability.
First, the derivatization of compound 2 was commenced be-
cause of the potent in vitro inhibitory activity against human
FATP1. Since the tetrahydrobenzothiazole moiety of compound 2
might cause microsomal instability, the attenuation of lipophilicity
was examined, as described in Table 1. Simple removal of the
methyl group in 2 maintained mouse in vitro activity of 4.0 lM
with reduced activity against human FATP1 (compound 4a). Since
dimethyl derivative 4b showed a further fivefold reduction of hu-
man in vitro activity, while thiazole 4c lost its in vitro potency,
the bicyclic structure in 4a plays a major role for in vitro activity
against human FATP1. Furthermore, partial saturation of the bicy-
clic ring in 4a was also required, because the introduction of the
benzothiazole ring led to the disappearance of mouse in vitro
⇑
Corresponding author.
E-mail address: shinozuka.tsuyoshi.s5@daiichisankyo.co.jp (T. Shinozuka).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.116

T. Matsufuji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2560–2565
2561
showed decreased human and mouse in vitro activity and the 4-
methyl substituent did not improve the activity (compound 5b).
Moreover, replacement with the 3-fluoro substituent resulted in
a complete loss of in vitro potency (compound 5c). Similarly, no
in vitro activity was observed in the benzoyl and cyclohexyl deriv-
atives (5d and 5e). For further enhancement of in vitro activity and
microsomal stability, the replacement of the benzene ring with
heterocycles was also examined. Among pyridine derivatives 5f,
5g and 5h, only 2-pyridyl derivative 5f retained in vitro activity.
Due to the fact that 2-thiazole 5j also possessed moderate activity
as with 2-pyridine 5f, an aromatic nitrogen atom in this position
can be installed. Nevertheless, 2-pyrimidyl derivative 5i lost all
in vitro activity. These results led us to synthesize fluoro-substi-
tuted 2-pyridyl compounds 5k and 5l to find a several-fold in-
crease in in vitro activity against both species. Moreover, the
microsomal stability of 5k was improved to 63%.6 Although the
logD value of tetrahydrobenzooxazole 5l was further reduced to
2.7, the microsomal stability was not improved compared to tetra-
hydrobenzothiazole 5k. To improve the microsomal stability of 5k
by increasing the steric hindrance, N-alkyl derivatives 5m and 5n
were synthesized. Methyl derivative 5m retained in vitro potency
with similar microsomal stability, while ethyl derivative 5n indi-
cated a sharp loss of in vitro activity.
F
F
F
O
O
HN
S
N
N
N
N
1
human IC50 = 0.10 µM
mouse IC50 = 0.091 µM
F
O
N
S
O
N
N
N
N
N
N
N
H
2
3
human IC50 = 0.026 µM
mouse IC50 = 5.1 µM
human IC50 = 3.0 µM
mouse IC50 = Not Determined
Figure 1. Triazole-based FATP1 inhibitor 1 and HTS hits 2 and 3 with FATP1 IC50
values.
activity (compound 4d). Tetrahydrobenzooxazole 4e, an oxazole
analogue of 4a, retained fair human and mouse in vitro activity
(0.18 and 5.4 lM, respectively). Compared to tetrahydrobenzo-
The synthesis of arylpiperazine derivatives listed in Tables 1
and 2 (4a–4g, 5a–5n) is shown in Scheme 1. Compounds 4a–4e
(Table 1) were synthesized in two simple steps. After preparing
thiazoyl or oxazoyl amine 6 in the known manner,7 acylation with
chloroacetyl chloride, followed by SN2 reaction with 4-fluorophen-
ylpiperazine 8 provided compounds 4a–4e. Five- and seven-mem-
bered ring derivatives 4f and 4g were synthesized from carboxylic
acid 9. Starting from 4-fluorophenyl piperazine 8, N-alkylation
thiazole 4a, the five-membered ring derivative 4f lost in vitro
activity against mouse FATP1, and reduced human activity was ob-
served in the seven-membered ring analogue 4g. Thus, the tetra-
hydrobenzothiazole ring seemed to be the most suitable ring
system in this position.
Then, further modifications of compound 4a were performed, as
indicated in Table 2. The 4-fluoro substituent on the phenyl ring is
important for in vitro activity, because phenyl derivative 5a
Table 1
IC50 values of FATP1 inhibitors 2 and 4a–4g4
F
O
N
R
N
H
N
logDb
MSc (%)
30
a
a
Compd
R
Human FATP1 IC50
(
lM)
Mouse FATP1 IC50
5.1
(lM)
S
2
0.026
5.1
N
S
4a
0.077
4.0
4.5
42
N
S
4b
4c
4d
0.43
>1.0
0.49
1.7
3.7
2.8
4.4
56
N
S
>10
>10
NDd
NDd
N
S
N
N
O
4e
4f
0.18
0.49
0.12
5.4
>10
2.0
3.0
4.0
5.0
53
S
NDd
NDd
N
S
4g
N
a
b
c
Inhibition of recombinant human or mouse acyl-CoA synthetase activity of FATP1. The IC50 values represent the average of at least n = 2.
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
Remaining (%) of the test compound after 0.5-h incubation with mouse liver microsome.
Not determined.
d

2562
T. Matsufuji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2560–2565
Table 2
IC50 values of FATP1 inhibitors 4a and 5a–5n4
R2
X
O
N
N
N
N
R1
R1
H
R2
Human FATP1 IC50
(lM)
Mouse FATP1 IC50
4.0
(lM)
logDb
MSc (%)
42
a
a
Compd
X
S
F
4a
0.077
4.5
5a
5b
5c
S
S
S
H
H
H
0.28
0.25
>1.0
9.0
4.3
NDd
NDd
NDd
>10
>10
4.8
NDd
F
O
5d
S
H
>1.0
>10
2.1
NDd
5e
5f
5g
5h
5i
S
S
S
S
S
S
H
H
H
H
H
H
>1.0
0.20
>1.0
>1.0
>1.0
0.52
>10
4.9
3.0
3.9
3.3
2.0
3.7
3.8
NDd
NDd
NDd
NDd
NDd
NDd
N
>10
>10
>10
7.9
N
N
N
N
N
5j
S
F
5k
5l
S
O
S
S
H
0.046
0.10
0.60
1.7
4.1
2.7
3.9
NDd
63
N
N
N
N
F
F
F
H
63
5m
5n
Me
Et
0.041
0.78
0.62
>10
65
NDd
a
b
c
Inhibition of recombinant human or mouse acyl-CoA synthetase activity of FATP1. The IC50 values represent the average of at least n = 2.
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
Remaining (%) of the test compound after 0.5-h incubation with mouse liver microsome.
Not determined.
d
with benzyl bromoacetate, followed by benzyl deprotection
provided carboxylic acid 9, which was condensed with the
corresponding amines to furnish 4f and 4g with good yields. In
the same manner, using the appropriate starting material and re-
agents, compounds 5a–5l (Table 2) were synthesized.8 N-alkyl
amides 5m and 5n were synthesized from N-alkyl thiazoyl amine
11 derived from chlorocyclohexanone 10 and N-alkyl thiourea. In
the same manner, acylation with chloroacetyl chloride, followed
by SN2 reaction with 1-(5-fluoropyridine-2-yl)piperazine hydro-
chloride8 provided N-alkyl amides 5m and 5n, respectively.
Then, the derivatization of imidazopyridine 3 was focused on, as
described in Table 3, because the structural similarity between 2
and 3 allowed us to utilize the structure–activity relationship of
2. As expected, the replacement of the phenyl group in 3 with a flu-
oro-substituted 2-pyridyl group resulted in a dramatic enhance-
ment of in vitro activity against both species compared to hit
compound
IC50 = 0.39
3
(compound 12a; human IC50 = 0.43
l
M, mouse
l
M). Even compared to thiazole 5k, imidazopyridine
12a showed better mouse in vitro activity. Moreover, the elimina-
tion of the methyl group in imidazopyridine 12a led to compound
12b, which showed an enhancement of human and mouse in vitro
activity (0.13 and 0.29 lM, respectively) as well as excellent mouse
microsomal stability of 98%.6 Although several imidazopyridine
ring replacements were examined, only pyrazolopyridine 12e
and benzofuran 12g indicated fair in vitro activity. On the other
hand, tetrahydroimidazopyridines 12h and 12i showed enhanced
in vitro potency, especially 12i, which indicated the best in vitro
activity against mouse FATP1 without an increase in lipophilicity.
Imidazopyridine derivatives and their analogs 12a–12i were
synthesized as illustrated in Scheme 2. Tetrahydroimidazopyridine
15 was synthesized via imidazopyridine 14 with partial hydroge-
nation under acidic conditions. The synthesis of other acetyl

T. Matsufuji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2560–2565
2563
F
R1
R1
R1
a
b
O
X
X
R2
R2
O
N
X
R2
Cl
N
N
NH2
N
H
N
N
N
H
6
7
4a-4e
(X = S or O)
F
F
F
e
c, d
n
S
O
N
N
O
N
N
N
N
HN
N
H
HO
8
9
4f (n = 1)
4g (n = 3)
F
f
a, b
O
S
S
O
N
N
N
R3
N
N
N
H
N
Cl
R3
11
10
5m, 5n
(R3 = Me or Et)
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, Et3N, THF; (b) arylpiperazine, KI, DMF or THF, 80 °C, 2 steps, 14–82%; (c) benzyl bromoacetate, Et3N, KI, THF,
reflux, 97%; (d) H2, Pd/C, EtOH, 86%; (e) thiazoyl amine, WSCÁHCl, HOBtÁH2O, Et3N, CH2Cl2, 71–74%; (f) methyl or ethyl thiourea, EtOH, reflux.
heterocycles is known.9 Finally, after bromination of acetyl hetero-
cycle 16, SN2 reaction with 1-(5-fluoropyridine-2-yl)piperazine
hydrochloride8 yielded compounds 12a–12i.
Compounds 2, 5k and 12a were selected to evaluate pharmaco-
kinetic parameters in mice (Table 4). We believe that FATP1 inhib-
itors should be exposed in blood or skeletal muscle above the IC50
value to show sufficient in vivo efficacy. When hit compound 2 was
orally administered to mice at 10 mg/kg, the Cmax value was 1.8 g/
mL, which was comparable to its IC50 value against mouse FATP1
(2.0 g/mL). Compound 2 showed high plasma protein binding
(99.57%). Therefore, if protein binding was taken into account, only
l
l
0.0077
lg/mL of unbound 2 would be exposed in mouse plasma,
Table 3
IC50 values of FATP1 inhibitors 5k and 12a–12i4
F
O
N
N
N
R
logDb
MSc (%)
63
a
a
Compd
R
Human FATP1 IC50
0.046
(l
M)
Mouse FATP1 IC50
(lM)
S
5k
0.60
4.1
N
N
H
N
12a
12b
12c
0.43
0.13
>1
0.39
0.29
>10
2.8
52
N
N
2.5
98
N
N
NDd
NDd
N
12d
12e
12f
12g
>1
>10
1.1
2.8
2.5
NDd
3.9
NDd
84
N
N
H
0.33
>1
N
N
>10
1.9
NDd
N
H
0.47
76
O
(continued on next page)

2564
T. Matsufuji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2560–2565
Table 3 (continued)
a
a
Compd
R
Human FATP1 IC50
(l
M)
Mouse FATP1 IC50
(lM)
logDb
MSc (%)
55
N
12h
0.19
0.40
2.5
N
N
12i
0.10
0.16
2.1
86
N
a
Inhibition of recombinant human or mouse acyl-CoA synthetase activity of FATP1. The IC50 values represent the average of at least n = 2.
The distribution coefficients (logD) were measured between 1-octanol and phosphate buffered saline (pH 7.4).
Remaining (%) of the test compound after 0.5-h incubation with mouse liver microsome.
Not determined.
b
c
d
excellent Kp soleus value was observed in imidazopyridine 12a,
12a was the most promising compound in this aspect, as well.
Then, to assess the in vivo inhibition of FATP1, we evaluated tis-
sue triglyceride (TG) content in skeletal muscle and liver, as de-
picted in Figure 2, because TG is known as the storage form of
fatty acid. When mice were fed with a high-fat diet (HFD) for
4 weeks, TG content in the liver increased by around threefold
compared to the mice fed with a normal diet. The increase of TG
content was also observed in the white gastrocnemius muscle of
HFD-fed mice compared to the group fed with a normal diet,
whereas TG content in the soleus muscle showed no difference.
When 3, 10, 30 mg/kg of imidazopyridine 12a was orally adminis-
tered to mice fed with a HFD for 4 weeks, TG content of each tissue
did not change at any dose. A similar result was observed with 10,
30, 100 mg/kg administration of 2. When 1, 10, 100 mg/kg of thia-
zole 5k was examined in the same manner for 3 weeks, a reduction
of TG content in each tissue was not observed (data not shown).
In summary, the discovery and optimization of arylpiperazine
derivatives as novel FATP1 inhibitors were described. The replace-
ment of the 4-fluoro phenyl group with a fluoro-substituted 2-pyr-
idyl group increased in vitro activity against human and mouse
FATP1 to identify thiazole 5k. With the improvement of physiolog-
ical properties as well as microsomal stability, 5k showed good
pharmacokinetic parameters. A 2-amino tetrahydrobenzothiazole
moiety can be replaced with an imidazopyridine ring, and imidazo-
pyridine 12a was found to possess reduced plasma protein binding
ability with potent in vitro activity. When 5k was orally adminis-
tered at 10 mg/kg, the Cmax value exceeded the mouse IC50 value
without considering plasma protein binding. Moreover, oral
administration of 12a at 10 mg/kg showed a Cmax value above
the mouse IC50 value even though the free fraction was considered.
When compounds 2, 5k and 12a were orally administered to mice
fed with a HFD, reduction of TG accumulation was not observed in
the liver, white gastrocnemius muscle and soleus. Recently,
O
O
N
c
a, b
N
N
NH2
N
N
14
15
13
F
O
O
O
N
N
d
e
Br
N
Ar
Ar
Ar
16
17
12a-12i
Scheme 2. Reagents and conditions: (a) N,N-dimethylformamide dimethylacetal,
toluene, reflux; (b) bromoacetone, ethanol, reflux, 18% (2 steps); (c) H2 (50 psi), HCl,
EtOH, rt, 59%; (d) bromine, HBr, AcOH, 96–100%; (e) 1-(5-fluoropyridine-2-
yl)piperazine hydrochloride, Et3N, DMF, rt, 24–92%.
which was much lower than its IC50 value. When thiazole 5k was
administered at the same dosage, improved pharmacokinetic
parameters were observed, especially for clearance, which was re-
duced to 5.0 mL/min/kg. The Cmax value of thiazole 5k (5.5
was more than 20 times higher than its IC50 value of 0.22
However, unbound 5k in mouse plasma was only 0.0083
due to its high protein binding ability. The pharmacokinetic
parameters were further improved in imidazopyridine 12a with
better solubility as well as lower plasma protein binding ability
l
l
g/mL)
g/mL.
g/mL
l
compared to that of 5k. The free Cmax value reached 0.61
which is above its in vitro IC50 of 0.14 g/mL, even if the protein
lg/mL,
l
binding was considered. Therefore, imidazopyridine 12a was ex-
pected to show the most potent in vivo efficacy among these
compounds.
The soleus to plasma concentration ratio (Kp soleus) was also
evaluated, as shown in Table 4. Although compound 1 showed a
Kp value of 0.33, Kp soleus of 5k was as low as 0.030. Since an
Table 4
Physical properties and mouse PK parameters of 2, 5k and 12a
b
b
c
d
Compd
In vitro IC50
g/mL)
SolubilityJP1/JP2
g/mL)
PB freea (%)
Tmax (h)
Cmax
g/mL)
Free Cmax
g/mL)
AUCb
(h g/mL)
Vd (L/kg)
Cld
F (%)
Kp
(
l
(
l
(
l
(
l
l
(mL/min/kg)
soleuse
2
5k
12a
2.0
0.22
0.14
540/0.7
700/2.4
>700/350
0.43
0.15
27.5
0.25
0.33
0.33
1.8
5.5
2.2
0.0077
0.0083
0.61
2.3
36
5.5
1.5f
0.57
0.20
46f
5.0
6.4
62
100
21
0.33
0.030
1.1
a
b
c
Unbound fractions (%) in mouse plasma.
Average of three mice dosed at 10 mg/kg po in DMA/PG/(20%HPbCD/saline): 10/10/80.
Free Cmax = PB free (%) Â in vitro IC50
.
d
e
Average of two mice dosed at 1 mg/kg iv in DMA/PG/(20%HPbCD/saline): 10/10/80.
Soleus to plasma concentration ratio. Kp value was determined after 1-week repeated administration of compounds at 10 mg/kg po to mice. The measurement of each
concentration was conducted at 3 h after administration.
f
Dosed at 5 mg/kg iv.

T. Matsufuji et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2560–2565
2565
Figure 2. Inhibition of tissue TG accumulation in vivo (⁄P <0.05 versus HFD control. ⁄⁄P <0.01 versus HFD control).
organic phase was removed. To remove [1À14C] oleic acid completely, the
aqueous phase was washed by heptane again. The aqueous phase was added
Pico-Fluor 40 scintillation cocktail (PerkinElmer) and the radioactivity was
measured by LSC-6100 scintillation counter (Aloka). The radioactivity of FLAG-
tagged luciferase protein instead of FATP1 protein was deducted as
background. The IC50 values represent the average of at least n = 2.
5. The homology between human and mouse FATP1 is calculated to be 89% from NCBI
reference sequence: NP_940982.1 (human FATP1), NP_036107.1 (mouse FATP1).
6. Mouse microsomal stability was evaluated by measuring the remaining (%) of
the test compound after 0.5-h incubation with mouse liver microsome.
7. (a) Chordia, M.; Murphree, L. J.; Macdonald, T. L.; Linden, J.; Olsson, R. A. Bioorg.
Med. Chem. Lett. 2002, 12, 1563; (b) Lang, M.; Seifert, M. H.-J.; Wolf, K. K.;
Aschenbrenner, A.; Baumgartner, R.; Wieber, T.; Trentinaglia, V.; Blisse, M.;
Vitt, D.; Tajima, N.; Yamashita, T.; Noda, H. Bioorg. Med. Chem. Lett. 2011, 21,
5417.
8. For the synthesis of 1-(5-fluoropyridine-2-yl)piperazine hydrochloride, see: Slassi,
A.; Isac, M.; Arora, J.; Brown, D. PCT Int. Appl. 2007, WO 2007087135.
9. (a) Almirante, L.; Mugaini, A.; Toma, D. N.; Gamba, A.; Murmann, W. J. Med.
Chem. 1970, 13, 1048; (b) Hlasta, J. D. Tetrahedron Lett. 1990, 31, 5833; (c)
Desreumaux, P.; Chavatte, P. Tetrahedron Lett. 2007, 63, 419; (d) Bonnet, M.;
Flanagan, J. U.; Hay, M. P.; Chan, D. A.; Lai, E. W.; Nguyen, P.; Giaccia, A. J. Bioorg.
Med. Chem. 2011, 19, 3347; (e) Ottoni, O.; Neder, A.; Dias, V.; Cruz, R.; Aquino, L.
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A.; Wojtczak, A. Heterocycles 2001, 55, 569.
Holloway et al. reported that FATP1 overexpression in mice muscle
did not cause muscular TG accumulation or whole body insulin
resistance.10 Taken together, FATP1 inhibition alone may not have
enough of an impact on fatty acid transport and/or metabolism to
reduce tissue TG contents.
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3. Matsufuji, T.; Ikeda, M.; Naito, A.; Kanda, S.; Izumi, M.; Harada, J.; Takakusa, H.;
Hirouchi, M.; Shinozuka, T. Bioorg. Med. Chem. Lett. 2012, 22, 5067.
4. The inhibition of human or mouse FATP1 was evaluated by cell-free ACS assay:
FLAG-tag fused human or mouse FATP1 protein was incubated with 10 lM
[1-14C] oleic acid and test compound in 100 mM Tris–HCl (pH 7.4), 30 mM
NaCl, 0.5 mM ATP, 5 mM MgCl2, 0.5 mM coenzyme A, 0.05% Triton-X 100,
2 mM dithiothreitol at 37 °C for 20 min. After the reaction, [1-14C] oleoyl-CoA
was extracted by successive additions of fivefold volume of 2-propanol/
heptane/H2SO4 (40:10:1, v/v/v), threefold volume of heptane, and twofold
volume of water. Phases were then separated by centrifugation, and the
10. Holloway, G. P.; Chou, C. J.; Lally, J.; Stellingwerff, T.; Maher, A. C.; Gavrilova, O.;
Haluzik, M.; Alkhateeb, H.; Reitman, M. L.; Bonen, A. Diabetologia 2011, 54,
1457.