Discovery of potent liver-selective stearoyl-CoA desaturase-1 (SCD1) inhibitors, thiazole-4-acetic acid derivatives, for the treatment of diabetes, hepatic steatosis, and obesity

Article abstract of DOI:10.1016/j.ejmech.2018.09.003

SCD1 is a rate-limiting enzyme in the conversion of saturated fatty acids to monounsaturated fatty acids. SCD1 inhibitors have potential effects on obesity, diabetes, acne, and cancer, but the adverse effects associated with SCD1 inhibition in the skin and eyelids are impediments to clinical development. To avoid mechanism-based adverse effects, we explored the compounds that selectively inhibit SCD1 in the liver in an ex vivo assay. Starting from a systemically active lead compound, we focused on the physicochemical properties tPSA and cLogP to minimize exposure in the off-target tissues. This effort led to the discovery of thiazole-4-acetic acid analog 48 as a potent and liver-selective SCD1 inhibitor. Compound 48 exhibited significant effects in rodent models of diabetes, hepatic steatosis, and obesity, with sufficient safety margins in a rat toxicology study with repeated dosing.

Full text of DOI:10.1016/j.ejmech.2018.09.003

European Journal of Medicinal Chemistry 158 (2018) 832e852
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of potent liver-selective stearoyl-CoA desaturase-1 (SCD1)
inhibitors, thiazole-4-acetic acid derivatives, for the treatment of
diabetes, hepatic steatosis, and obesity
Tetsuya Iida*, Minoru Ubukata, Ikuo Mitani, Yuichi Nakagawa, Katsuya Maeda,
Hiroto Imai, Yosuke Ogoshi, Takahiro Hotta, Shohei Sakata, Ryuhei Sano, Hisayo Morinaga,
Tamotsu Negoro, Shinichi Oshida, Masahiro Tanaka, Takashi Inaba
Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka, 569-1125, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
SCD1 is a rate-limiting enzyme in the conversion of saturated fatty acids to monounsaturated fatty acids.
SCD1 inhibitors have potential effects on obesity, diabetes, acne, and cancer, but the adverse effects
associated with SCD1 inhibition in the skin and eyelids are impediments to clinical development. To
avoid mechanism-based adverse effects, we explored the compounds that selectively inhibit SCD1 in the
liver in an ex vivo assay. Starting from a systemically active lead compound, we focused on the physi-
cochemical properties tPSA and cLogP to minimize exposure in the off-target tissues. This effort led to the
discovery of thiazole-4-acetic acid analog 48 as a potent and liver-selective SCD1 inhibitor. Compound 48
exhibited significant effects in rodent models of diabetes, hepatic steatosis, and obesity, with sufficient
safety margins in a rat toxicology study with repeated dosing.
Received 18 June 2018
Received in revised form
10 August 2018
Accepted 3 September 2018
Keywords:
Stearoyl-CoA desaturase 1
Liver-selective SCD1 inhibitor
Thiazole-4-acetic acid derivative
Physicochemical properties
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
Mice with a targeted disruption in the SCD1 isoform (SCD1ꢀ/ꢀ
)
are resistant to diet-induced accumulation of hepatic TGs and fat in
their adipose tissues throughout their bodies, concomitantly pre-
venting obesity due to the increased energy expenditure. The mice
also exhibited improved insulin sensitivity because of the reduced
adipose tissue mass [8]. From these findings, SCD1 inhibitor was
considered to be a novel potential agent for the treatment of
obesity and type-2 diabetes [9]. However, the adverse events in the
eye and skin, such as eye abnormalities characterized by squinting
and alopecia, were observed in mice with systemic SCD1 knockout
[10,11]. Systemically distributed SCD1 inhibitors have also been
reported to have mechanism-based adverse effects in eye and skin,
as well as beneficial metabolic effects [12].
Although SCD1 is expressed ubiquitously, it is predominant in
lipogenic tissues, especially hepatocytes and adipocytes [13]. SCD1
antisense oligonucleotide (ASO) treatment protects mice from high
fat diet-induced weight gain and improves insulin resistance
without any mechanism-based adverse effects because most ASO is
distributed to the liver [14]. In addition, liver-specific SCD1
knockout mice are resistant to high carbohydrate diet-induced
obesity and hepatic steatosis, without adverse effects [15]. There-
fore, SCD1 inhibitors selectively distributed in the liver are hy-
pothesized to circumvent mechanism-based adverse effects, and
Stearoyl-CoA desaturase (SCD) is a lipogenic enzyme involved in
the de novo lipogenesis of monounsaturated fatty acids (MUFAs)
and catalyzes insertion of the cis double bond at the delta-9 posi-
tion (between carbons 9 and 10) of saturated fatty acids (SFAs) [1].
There are four isoforms of SCD (SCD1e4) in the mouse and two
isoforms in the human (hSCD1 and hSCD5) [2e4]. Human SCD1
shows a high degree of homology with mouse SCD1 [5]. The
products of SCD1, such as oleic acid and palmitoleic acid, are
incorporated into the lipids, including phospholipids, triglycerides
(TGs), cholesteryl esters, and wax esters. Maintaining the optimal
balance of MUFA and SFA composition in lipids is important
because an imbalance in the ratio affects membrane fluidity and
lipoprotein metabolism [6]. In an epidemiological survey, the
serum SCD1 activities were significantly higher in men who
developed metabolic syndrome than those who did not [7]. SCD1
affects the ratio of SFAs to MUFAs, though dietary MUFAs also have
an effect.
* Corresponding author.
E-mail address: tetsuya.iida@jt.com (T. Iida).
https://doi.org/10.1016/j.ejmech.2018.09.003
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
833
much effort has been made to discover liver-selective SCD1 in-
hibitors [16,17]. In particular, Merck disclosed MK-8245 as a potent
and liver-selective SCD1 inhibitor, proceeding to clinical trials
(phase IIa). They screened their compounds using a cell-based
assay and identified MK-8245 as a substrate of liver-specific
organic anion transporting polypeptides [18]. We took an ex vivo
assay approach, measuring the activity of all SCD isozymes in both
the liver and off-target tissues after oral administration of com-
pounds [19]. This method could be expected to detect the liver-
selective distribution of compounds for various reasons. First,
based on a high-throughput screening (HTS) hit, we developed a
potent but systemically distributed lead compound (4) and hy-
pothesized that modifying its physicochemical properties could
change the distribution pattern (Fig. 1). Lead optimization focused
on the topological polar surface area (tPSA) and calculated loga-
rithm of the partition coefficient (cLogP), leading to the discovery of
highly potent, orally bioavailable thiazole-4-acetic acid analogs.
The 48 was demonstrated to be liver-selective SCD1 inhibitor [20].
Here, we report the generation of potent and liver-selective small
molecule inhibitors of SCD1 and the discovery of 48.
thereafter. Therefore, we assessed whether 4 inhibits SCD1 enzyme
in off-target tissues 6 h and 24 h after oral dosing (0.1e10 mg/kg) in
mice. In the ex vivo assay, compound 4 dose-dependently inhibited
SCD1 in both the liver and eyelids at 6 h, and sustained inhibition of
SCD1 was observed in the eyelids at 24 h at a dose of 10 mg/kg
(Fig. 2, Supporting Information Table S1). Thus, the PK profiles of
the lead compound had to be changed to the desired profile. The
screening flow shown in Fig. 3 was set to guide the lead optimi-
zation. All compounds with IC50 < 20 nM were advanced to the
ex vivo experiments. First, we screened SCD1 activities in the liver
and eyelids of mice 6 h after oral administration at a dose of 1 mg/
kg. Second, select compounds from the first step were investigated
in the ex vivo assay at higher doses (10 and 100 mg/kg) 24 h after
dosing.
2.2. Lead optimization
Physicochemical properties are one of the factors affecting the
PK profiles [22]. We postulated that altering the physicochemical
properties of lead compound 4 may result in different compound
exposure between the liver and eyelids. Our lead optimization
approach based on the hit-to-lead study is shown in Fig. 4. Com-
pounds having different physicochemical properties were designed
for each part of the molecule, synthesized, and tested with the
SCD1 enzyme assay. The tPSA and cLogP were employed as the
index physicochemical properties of the compounds [23]. Initially,
we examined the substituents on the left-hand phenyl ring. As
shown in Table 1, hydrophobic groups, including CF3 (6) and n-
propyl (9) on the ortho-position and Cl group on the meta-position
(7), were well tolerated. However, removing the ortho substituent
(5) or Cl group at the para-position (8) decreased the potency. Polar
substituents, such as COOH (10) and CONMe2 (11), were not
2. Results and discussion
2.1. HTS hit to lead identification
We performed HTS with our small molecule compound library
looking for inhibitors of human recombinant SCD1. The HTS com-
pound library and synthesized compounds were screened in a
biochemical assay measuring the production of tritiated water from
the desaturation of radio-labeled stearoyl-CoA. We identified a
novel 4-aminopyridine-based SCD1 inhibitor (1) with a moderate
IC50 of 1400 nM (Fig. 1). The selectivity of other lipogenic enzymes
and structural features amenable to the accelerated synthesis of
derivatives made 1 an attractive starting point. Our first hit-to-lead
exploration was performed on the pyridine moiety. Screening a
small molecule library of various heterocyclic amide analogs
revealed that a 2-amino-4-methyl thiazole of 2 (IC50 ¼ 150 nM) was
a preferred replacement for the 4-aminopyridine. A further in-
crease in potency was obtained by replacing an oxygen atom in the
ether linker group with a nitrogen atom. The IC50 value shifted
approximately 16-times in amine analog 3 from ether 2 (Fig. 1). The
lead identification chemistry culminated in compound
4
(IC50 ¼ 6.4 nM), which had good ligand binding efficiency (IC50
/
number of non-hydrogen atoms, LE ¼ 0.5) and selectivity from
other lipogenic enzymes [21]. As the pharmacokinetic (PK) data in
mice indicated that 4 had moderate plasma exposure (10 mg/kg, po,
AUC0einf ¼ 2.4
mM h) with a resulting oral bioavailability (%F) of 15%,
we examined the in vivo activity of 4 in mice. Anti-diabetic effects
and reduced diet-induced body weight gains were observed in db/
db mice at a dose of 10 mg/kg (data not shown). However, adverse
effects, such as squinting and alopecia, were observed on day 9 and
Fig. 2. Effects of 4 on liver and eyelid SCD activities in mice. Compound 4 suspended in
vehicle (0.5% methylcellulose aqueous solution) was orally administered to C57BL/6 J
mice. Six or 24 h after administration, liver and eyelid samples were collected and
homogenized to measure tissue SCD activity. Results are expressed as percentage of
control for remaining SCD activity.
Fig. 1. Hit-to-lead identification and in vitro profiles of lead compound 4.
aRecombinant human SCD1. bMouse liver microsome SCD. cAcetyl-CoA acetyltransferase. dD-5 Fatty acid desaturase. eLigand efficiency.

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T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
Table 2
Effects of replacing the central phenyl ring with heterocyclic rings.
Compound
R1
A
IC50 (nM)a
6.4
cLogPb
3.8
tPSAc
51
4
Me
12
13
Me
Me
46
3.0
2.6
65
61
Fig. 3. Screening flow for identifying liver-selective SCD1 inhibitor.
*SCD1 enzyme activity was measured using recombinant human SCD1.
**Compounds were orally administered to mice and then the liver and eyelid collected
6 h (1 mg/kg) or 24 h (10 and 100 mg/kg) after dosing. Tissue homogenates were ob-
tained and SCD activity measured. Results are expressed as percentage of control for
remaining SCD activity.
310
14
15
CF3
CF3
30
4.1
4.5
49
53
9.7
a
Recombinant human SCD1 enzyme.
Calculated logarithm of the partition coefficient.
Topological polar surface area.
b
c
Table 3
Exploring the thiazole moiety with polar substituents.
Fig. 4. Design strategies to modify the physicochemical properties of compound 4.
Table 1
Exploring the left-hand phenyl ring with substituents.
Compound R1
R2
R3
IC50 (nM)a cLogPb tPSAc
4
Me
Me
Me
Me
CO2H
CH2CO2H
H
H
H
H
H
H
H
H
6.4
>10,000
14
5.1
6.3
2700
3.8
4.2
5.2
8.1
3.8
3.7
2.6
3.5
4.0
4.4
3.2
3.4
3.8
4.4
3.5
4.0
3.2
3.4
3.8
3.0
3.0
51
96
96
95
95
94
74
75
75
95
94
94
74
73
75
117
99
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
CF3 CH2CO2H
CF3 (CH2)2CO2H
CF3 (CH2)3CO2H
CF3 CH2OH
CF3 (CH2)2OH
CF3 (CH2)3OH
CF3
CF3
CF3
CF3
CF3
CF3
Compound
R1
IC50 (nM)a
cLogPb
tPSAc
5
6
3
7
8
9
10
11
H
2-CF3
2-Cl
3-Cl
4-Cl
2-n-Pr
2-CO2H
2-CONMe2
95
4.1
9.2
18
760
8.9
3600
2700
3.3
4.7
4.3
4.3
4.3
4.9
4.1
2.3
54
50
52
54
54
51
92
73
H
H
H
H
H
H
H
CO2H
CH2CO2H
(CH2)2CO2H 570
58
CH2OH
2.8
3.8
260
22
(CH2)2OH
(CH2)3OH
CO2H
a
Recombinant human SCD1 enzyme.
Calculated logarithm of the partition coefficient.
Topological polar surface area.
b
c
CF3 CH2OH
CF3 (CH2)3OH
(CH2)2OH
31
a
Recombinant human SCD1 enzyme.
Calculated logarithm of the partition coefficient.
Topological polar surface area.
b
c
allowed. Next, we replaced the central phenyl ring with heterocy-
clic rings. Unfortunately, the pyridines (12, 13) and piperidine (14)
were less potent, whereas thiophene ring 15 was equipotent to
phenyl ring 4 (Table 2). Finally, we tested polar functional groups at
the thiazole moiety. As shown in Table 3, introduction of a COOH
group at position 4 on the thiazole ring (16) resulted in a loss of
activity. However, the loss in potency was recovered by inserting a
methylene unit between the COOH group and thiazole ring (17). A
higher potency was obtained with a trifluoromethyl group on the
left-hand phenyl ring (18). Extending the methylene chain to
e(CH2)2COOH (19) maintained the single digit nanomolar potency,
but further extension to e(CH2)3COOH (20) decreased the potency
by more than 400-fold. Alcohols with different methylene lengths
(21e23) had single digit nanomolar activities. Introduction of these
polar substituents at position 5 on the thiazole ring resulted in a
structure-activity relationship (SAR) different from that of sub-
stituents at position 4. Single digit nanomolar activity was found for
compounds having COOH (24), eCH2OH (27), and e(CH2)2OH (28),
whereas other carboxylic acids with a methylene chain (25, 26) and
an alcohol with a longer methylene chain (29) decreased the po-
tency. Combination of the alcohols and COOH at positions 4 and 5
(30, 31) resulted in slightly decreased potency. All compounds with
an IC50 < 20 nM were tested in the ex vivo experiments 6 h after

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
835
dosing with 1 mg/kg, but only compound 21 demonstrated the first
sign of liver selectivity (SCD activity in the liver 20% and in the
eyelid 73%). This compound had a higher tPSA value of 74 and lower
cLogP value of 3.2 compared to lead compound 4 (tPSA ¼ 51,
cLogP ¼ 3.8). In light of this result, further investigation focused on
the combination of polar functional groups, which had highly
potent activities in the SCD1 enzyme assay at position 4 on the
thiazole ring and hydrophobic groups at the ortho-position on the
left-hand phenyl ring (Table 4). A benzene ring and thiophene ring
were selected for the central part. This combination resulted in
compounds with tPSA values of 73e98, and cLogP values of
2.6e6.3. The compounds exhibited potent SCD1 inhibitory activ-
ities (IC50 < 20 nM) and advanced to the ex vivo assay in mice.
Eleven compounds (21, 34, 40, 43, 45, 46, 48, 49, 50, 51, and 52) met
the criteria for the ex vivo assay 6 h after dosing with 1 mg/kg (liver
SCD activity < 30%, eyelid SCD activity > 70%). To further examine
the tissue selectivity and duration of SCD1 inhibitory activity in
these 11 compounds, we performed a higher dose ex vivo assay 24 h
after dosing mice with 10 and 100 mg/kg (Fig. 5, Supporting In-
formation Table S2). Among all tested compounds, five thiophene
analogs (46, 48, 49, 50, and 52) significantly inhibited SCD1 in the
liver at 10 mg/kg. However, central-part phenyl analogs (21, 34, 40,
and 43) had largely diminished activities in the liver at 10 mg/kg.
Among five thiophene analogs, 48 and 49, which bear an acetic acid
moiety at position 4 on the thiazole ring and alkyl chain (n-butyl
and n-hexyl, respectively) on the left-hand phenyl ring, achieved
liver selectivity and efficacy in the liver (SCD activity in the
liver < 20% at 10 mg/kg and in the eyelid > 50% at 100 mg/kg).
However, when the alkyl chain length on the left-hand phenyl ring
was extended to n-octyl (50), liver selectivity markedly decreased
in the higher dose ex vivo assay (SCD activity 11% in the liver at
10 mg/kg and 8% in the eyelid at 100 mg/kg).
We then analyzed the higher dose ex vivo assay results against
the physicochemical properties cLogP and tPSA. The correlation
plots indicated that compounds in the ranges of 3.9 ꢁ cLogP ꢁ5.0
and 90 ꢁ tPSA ꢁ98 (43, 48, 49, 51, and 52) were highly selective for
the liver (Fig. 6A and B). Conversely, systemically distributed
compound 4 (tPSA ¼ 51, cLogP ¼ 3.8) exhibited strong SCD1 inhib-
itory activity in the eyelid 24 h after dosing mice with 10 mg/kg, and
it lost its SCD1 inhibitory activity in the liver (Fig. 5, Supporting
Information Table S2). Other compounds (21, 34, 40, 45, 46, and 50)
outside the ranges of 3.9 ꢁ cLogP ꢁ 5.0 and 90 ꢁ tPSA ꢁ 98 also had
no liver selectivity. These results indicate that altering the physi-
cochemical properties of lead compound 4 led to a difference in
compound exposure between the liver and eyelid. In addition to
physicochemical properties, liver selectivity can be caused by the
functionality of the compounds. Hepatocellular compound con-
centrations can be affected by multiple factors, such as passive
diffusion of the compounds across membranes, uptake/efflux
transporters, and metabolism in the hepatocyte [24]. Hepatic up-
take of acidic compounds with relatively large molecular weights
(>400 Da) through liver-specific OATPs (1B1, 1B3) is known [25]. All
of the liver selective compounds identified in this study are car-
boxylic acids; however, compound 50, which is also a carboxylic
acid, was not liver selective. Thus, the liver/eyelid tissue selectivity
of our compounds may be due, at least in part, by the incorporation
of a carboxylic acid substituent into the chemical structure.
little difference in exposure between mice and rats. Compound 48
was highly permeable in the Caco-2 permeability assay and had
good solubility in PBS buffer (pH 7.2). Against hERG channels, 48
had no significant measurable activity (14.7% inhibition at 10
mM).
In addition, 48 had an IC50 > 50 M against the six CYP 450 isoforms
m
tested (3A4, 2C9, 2D6, 1A2, 2A6, and 2C19).
2.3. Pharmacological effects of compound 48
We assessed anti-diabetic and anti-obesity effects in mice fed a
high fat diet (HFD). Compound 48 was administered orally as a
dietary mixture for 43 days. As shown in Fig. 7, 48 improved glucose
tolerance in a dose-dependent manner and had a significant effect
at a dose of 10 mg/kg. As shown in Fig. 8, a mild dose-dependent
decrease in body weight was measured (6% body weight reduc-
tion at 10 mg/kg vs. vehicle-treated mice on day 43).
Compound 48 exhibited liver-selective SCD1 inhibition not only
in mice, but also in rats. As shown in Fig. 9, dose-dependent SCD1
inhibitory activity was observed in epididymal adipose tissue and
the liver 6 h after oral administration, whereas the inhibitory ac-
tivity in the eyelid was milder. Confirming the selectivity, we
measured the concentration of compound 48 in select tissues 6 h
after dosing rats with 10 mg/kg of the compound
(plasma ¼ 0.552
mM, liver ¼ 1.002
mM, and eyelid ¼ 0.160
mM;
n ¼ 3). The liver to eyelid ratio was 6.3:1, which indicates a liver/
eyelid selective tissue distribution profile. To assess the efficacy of
hepatic TG content, 48 was administered orally to rats fed a high
sucrose very low fat (HSVLF) diet for 8 days. As shown in Fig.10, rats
fed a HSVLF diet had hepatic TG content of approximately 80 mg/g
tissue and compound 48 significantly attenuated hepatic TG accu-
mulation in a dose-dependent manner. Similar results have been
reported in liver-specific SCD1 knockout (LKO) mice [15]. In this
report, the LKO mice fed a high-carbohydrate diet were protected
from liver TG accumulation and hepatic steatosis observed in the
control mice fed a high-carbohydrate diet. Therefore, compound 48
could be effective for hepatic steatosis.
2.4. Preclinical toxicology study
To evaluate adverse effects, compound 48 was orally adminis-
tered to male rats once daily at 100 mg/kg and 1000 mg/kg for 2
weeks. As noted above, systemically distributed compound 4 had
adverse effects in the eyelid at therapeutic doses in db/db mice. In
contrast, liver-selective SCD1 inhibitor 48 did not have significant
adverse events during the treatment period at 100 mg/kg, and only
very slight loss of fur in one out of five rats from day 9 and there-
after at a dose of 1000 mg/kg. Comparing plasma exposure at the
no-observed adverse effect level (NOAEL) of 100 mg/kg (179 mM h)
with the plasma exposure at therapeutic doses, 48 had a favorable
safety margin (10e81-fold, AUC levels). This result was consistent
with our ex vivo assay, which demonstrated different exposures
between the liver and off-target tissues, such as the eyelid and skin.
3. Chemistry
The synthesis of HTS hit compound 1 and its analog 2 is outlined
in Scheme 1. Commercially available methyl 4-(bromomethyl)
benzoate (53; CAS 2417-72-3) reacted with 2-chlorophenol in the
presence of K2CO3 to provide 54, followed by alkaline hydrolysis of
ester 54 to give 55. Carboxylic acid 55 reacted with the corre-
sponding amine using EDC$HCl in pyridine-mediated amide
coupling to give 1 and 2. The synthesis of 3e11 to explore the
substituents on the left-hand phenyl ring is outlined in Scheme 2.
Coupling commercially available 56 (CAS 1642-81-5) with 2-
amino-4-methylthiazole (65; CAS 1603-91-4) in the presence of
We chose compound 48 for further investigation of biological
activities. Compound 48 maintained single digit nanomolar po-
tency in rats, and there were no differences in SCD enzyme inhib-
itory activities in rodents (mouse liver microsome SCD
IC50 ¼ 5.4 nM, rat liver microsome SCD IC50 ¼ 6.5 nM). Prior to the
pharmacological studies, the ADME profiles of the compound were
explored. As summarized in Table 5, compound 48 was orally
absorbed moderately in both rats and mice. There was relatively

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T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
Table 4
In vitro and ex vivo assays in mice 6 h after dosing with thiazole-2-carboxyamide derivatives.
Compound
R1
A
R2
IC50 (nM)a
Ex vivob
cLogPc
tPSAd
1 mg/kg 6 h
% of control
Liver
24
Eyelid
41
4
Me
Me
6.4
3.8
4.3
3.2
4.2
6.4
3.0
5.3
5.2
14
3.8
3.2
2.8
3.9
3.4
3.0
4.1
4.5
3.8
2.6
3.5
3.1
4.2
5.2
6.3
4.6
4.0
4.2
2.9
3.1
4.9
3.2
3.9
5.0
6.0
4.3
3.9
51
74
75
73
75
77
75
75
75
97
95
97
95
96
95
96
95
93
76
77
77
98
98
98
98
98
96
21
32
33
22
34
35
36
23
17
18
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
CF3
CH2OH
20
12
10
33
29
61
26
64
NT
98
72
76
31
15
19
74
18
9
73
Cl
CH2OH
37
n-Bu
CF3
CH2OH
29
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)3OH
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2OH
136
74
Cl
n-Bu
iso-Pen
CF3
94
63
75
Me
NT
107
132
74
CF3
5.1
13
Cl
n-Bu
n-Hex
n-Oct
iso-Pen
Ph
5.5
3.0
4.5
4.8
8.7
5.5
5.1
3.9
4.5
9.0
8.8
4.8
17
119
127
68
187
76
Bn
CF3
33
CF3
(CH2)2OH
(CH2)2OH
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
10
12
51
13
12
7
171
104
124
97
n-Hex
CF3
n-Bu
n-Hex
n-Oct
iso-Pen
Bn
88
77
6.7
7.5
19
22
160
128
a
Recombinant human SCD1 enzyme.
b
Compounds (1 mg/kg) were orally administered to mice and then the liver and eyelid collected 6 h later. Tissue homogenates were obtained and SCD activity measured.
Results are expressed as percentage of control for remaining SCD activity.
c
Calculated logarithm of the partition coefficient.
Topological polar surface area.
d

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
837
Compounds 12 and 13, which have a pyridine ring in the central
part, were prepared according to Scheme 3. The reaction of
commercially available carboxylic acids 59a (CAS 1154575-21-9)
and 59b (CAS 1154323-05-3) with borane-THF at appropriate
temperatures provided corresponding alcohols 60a and 60b. The
primary alcohols were converted to 4-chloromethylpyridines 61a
and 61b, and alkylation with 2-methylaniline in the presence of KI
provided compounds 12 and 13.
Piperidinecarboxamide 14 was obtained as outlined in Scheme
4. 2-amino-4-methylthiazole (65) was converted to imidazole
amide 66. Subsequently, a coupling reaction was carried out with 4-
substituted piperidine hydrochloride (64) synthesized by the
palladium-assisted coupling of 62 (CAS 144222-22-0) with 2-
bromobenzotrifluoride, followed by treatment with HCl.
Fig. 5. Higher dose ex vivo assay in mice. Compounds 4, 21, 34, 40, 43, 45, 46, and
48e52 (10 or 100 mg/kg) suspended in vehicle (0.5% methylcellulose aqueous solution)
were orally administered to C57BL/6 J mice. Twenty-four hours after administration,
liver and eyelid samples were collected and homogenized to measure tissue SCD ac-
tivity. The results are expressed as percentage of control for remaining SCD activity.
*Dose was 10 mg/kg.
The general synthesis of phenyl carboxamide analogs 16e43 to
explore the polar functional groups at the thiazole moiety is
described in Schemes 5 and 6. As shown in Scheme 5, benzyl bro-
mide 53 was reacted with the corresponding 2-alkylaniline analogs
in the presence of NaI to yield the corresponding N-alkylated
compounds (67e75), which were subsequently hydrolyzed to the
corresponding carboxylic acids (76e84). Compounds 27e29 were
synthesized by coupling of carboxylic acid 77 with the corre-
sponding amines. Compounds 16e20, 24e26, 30, and 37e43 were
synthesized by amide coupling, followed by hydrolysis of corre-
sponding carboxylic esters 88e92, 100e102, 103, and 93e99. Hy-
droxyl compounds 22, 23, and 34e36 were synthesized by sodium
borohydride reduction of corresponding carboxylic esters 90, 91,
and 93e95. Alkaline hydrolysis of acetates 85, 86, and 87 produced
hydroxyl compounds 21, 32, and 33.
The synthesis of 4,5-disubstituted thiazole 31 was performed as
described in Scheme 6. Commercially available diester 104 (CAS
6317-49-3) was converted to 2-amino-4,5-disubstitued thiazole
106 in two steps: bromination with bromine in CHCl3, followed by
cyclization with thiourea. Amide coupling between 106 and car-
boxylic acid 77, followed by ester reduction with lithium borohy-
dride, resulted in the formation of 31.
The general synthesis of thiophene carboxamide analogs 15 and
44e52 is described in Scheme 7. Imines 109e114 were prepared by
reaction of commercially available 5-formyl-2-thiophenecarboxylic
acid (108; CAS 4565-31-5) with corresponding 2-alkyl anilines
under Dean-Stark conditions. Subsequent reduction with sodium
borohydride yielded amines 115e120. Compound 115 was then
transformed into amide derivative 15 via EDC$HCl coupling in
pyridine with 2-amino-4-methylthiazole (65). Amide coupling
between 115e120 and ethyl (2-amino-4-thiazolyl) acetate, fol-
lowed by alkaline hydrolysis of esters 124e129, afforded carboxylic
acids 47, 51, 52, and 130e132. Compounds 48e50 were obtained as
a sodium salt by treatment of 130e132 with aqueous NaOH in
EtOH. Analogously, compounds 44e46 were prepared from the
coupling of carboxylic acid 115 or 117 with the corresponding
amine in the presence of EDC$HCl and DMAP, followed by hydro-
lysis of the ester 121 or deprotection of the TBS ethers 122 and 123.
Fig. 6. Plot of higher dose ex vivo assay results against physicochemical properties.
A. Liver SCD activity in higher dose ex vivo assays (y-axis, mice, 10 mg/kg, 24 h after
dosing, Fig. 5) and the cLogP of the compounds (x-axis). Points are color-coded by tPSA
(blue, brown, green) and labeled with compound identifiers. B. Eyelid SCD activity in
higher dose ex vivo assays (y-axis, mice, 100 mg/kg, 24 h after dosing, Fig. 5) and the
cLogP of the compounds (x-axis). Points are color-coded by tPSA (blue, brown, green)
and labeled with compound identifiers. *Dose was 10 mg/kg.
4. Conclusion
HATU yielded 4-(chloromethyl)phenyl carboxamide (57), followed
by nucleophilic substitution of the benzylchloride with corre-
sponding 2-alkylaniline analogs in the presence of KI, provided
3e9. Carboxylic acid analog 10 was obtained from the basic hy-
drolysis reaction of ester intermediate 58. Compound 10 was then
transformed into dimethylamide derivative 11 via PyBOP coupling
with dimethylamine.
In conclusion, novel thiazole-4-acetic acid analog 48 was iden-
tified as a highly potent, orally active, and liver-selective SCD1 in-
hibitor in ex vivo assay screening starting from systemically
distributed lead compound 4. Lead optimization focused on the
physicochemical properties tPSA and cLogP, which was effective for
achieving tissue selectivity. Exposure in eyelids was minimized by
introducing a carboxylic acid group on the thiazole ring and

838
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
Table 5
PK data for 48.
species
po
iv
BAg (%)
Dose (mg/kg)
Cltoth (L/h/kg)
c
e
f
Dose (mg/kg)
Cmax
(
m
M)
tmaxd (h)
t1/2 (h)
AUC0-inf (mM$h)
Rata
10
10
8.0
1.7
0.5
1.0
4.7
5.2
16.5
10.2
16
28
1
1
0.21
0.61
Mouseb
Metabolic stability in liver S9 (remaining %, 1 h)
Caco2
Solubility in PBS buffer (pH 7.2)
(mM)
Papp (cm/sec$10ꢀ6
)
Human
Mouse
90
Rat
87
91
43.60
82
a
Based on a per os (po) dose of 10 mg/kg and intravenous (iv) dose of 1 mg/kg in two male Sprague Dawley (SD) rats.
Based on a per os (po) dose of 10 mg/kg and intravenous (iv) dose of 1 mg/kg in two male C57BL/6 J mice.
Maximum observed concentration of the compound in plasma.
Time of maximum observed concentration of the compound in plasma.
Apparent half-life of the terminal phase of elimination of the compound from plasma.
Area under the concentration-time curve from the time of dosing to infinity.
Bioavailability.
b
c
d
e
f
g
h
Total body clearance of compound.
Fig. 9. Effects of 48 on the liver, epididymal adipose tissue, and eyelid SCD activities in
Sprague Dawley rats fed a high sucrose (HS) diet. Samples were collected and ho-
mogenized 6 h after administration. Results are expressed as percentage of control for
remaining SCD activity. Data are presented as the mean standard deviation (n ¼ 3).
Fig. 7. Effects of 48 on glucose tolerance in C57BL/6 J mice fed a high fat diet (HFD).
Compound 48 was administered with food admixture for 43 days. On day 22 of
administration, a glucose tolerance (1 g/kg) test was performed with the mice fasted
overnight. Area under the curve of plasma glucose level (AUC glucose) was calculated
from plasma glucose levels 0, 0.5, 1, and 2 h after glucose loading. Data are presented as
the mean standard deviation (n ¼ 5e8). #p < 0.05 vs. Normal group (Student's t-
test), **p < 0.01 vs. HFD group (Dunnett's test).
Fig. 10. Effects of 48 on liver triglyceride (TG) contents in Sprague Dawley rats fed a
high sucrose very low fat (HSVLF) diet. Samples were collected after 8 days of treat-
ment. Data are presented as the mean standard deviation (n ¼ 6). ##p < 0.01 vs.
Normal group (Student's t-test), **p < 0.01 vs. HSVLF group (Dunnett's test).
Fig. 8. Effects of 48 on body weight in C57BL/6 J mice fed a high fat diet for 43 days.
Compound 48 was administered with food admixture for 43 days. Data are presented
as the mean standard deviation (n ¼ 5e8). ##p < 0.01 vs. Normal group (Student's t-
test), *p < 0.05 vs. Ctrl group (Dunnett's test).
5. Experimental section
5.1. General
hydrophobic groups on the left-side phenyl ring with a central
thiophene ring. Compound 48 ameliorated diet-induced obesity
and hepatic TG accumulation, and improved glucose tolerance in
diabetic mouse and rat models in the absence of adverse skin or eye
effects. Based on a preliminary toxicology study, compound 48
could have a sufficient therapeutic window for chronic treatment.
All reagents and solvents obtained from commercial sources
were used without further purification or drying. TLC was per-
formed using silica gel 60 F254 plates purchased from Merck. Silica
gel chromatography was performed using either silica gel (Kanto

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
839
Scheme 1. Synthesis of phenyl carboxamide 1 and 2.
Scheme 2. Synthesis of phenyl carboxamide 3e11.
Scheme 3. Synthesis of picolinamide 12 and nicotinamide 13.
Chemical, 40e100
m
m) with standard techniques or prepacked
(ESIþ) were recorded on a ThermoQuest LCQ mass spectrometer.
All compounds that appear in SAR tables in the manuscript were
determined to be ꢂ 90% pure by NMR. The purity of compounds
SNAP Ultra C18 and the Biotage Isora One flash purification system.
Analytic HPLC was performed on a SHIMAZU Prominence instru-
ment. Melting points were determined using a Yanagimoto micro
melting point apparatus or BüCHI B-540 melting point instrument
and were uncorrected. 1H NMR was recorded on a JEOL JNM-
A300W, Bruker AMX-300, or JEOL JNM-AL400 spectrometer in
the indicated solvent. Chemical shifts are reported in parts per
48e50 was determined by HPLC (4.6 ꢃ 150 mm, 5
mm SHISEIDO
CAPCELL PAK C18 UG120 column; mobile phase A: 0.1% formic acid
in water; mobile phase B: 0.1% formic acid in MeCN; gradient: 50% B
to 90% B from 0 to 17 min, 90% B from 17 to 20 min, 90% B to 50% B
from 20 to 30 min; flow rate: 0.5 ml/min; detection wavelength:
250 nm).
million (ppm) relative to internal standard tetramethylsilane in d-
units, and coupling constants (J-values) are given in hertz (Hz).
Elemental analysis was performed with an Elementar Analy-
sensysteme GmbH vario EL III Element Analyzer. Mass spectra
The following section comprises the synthetic methods and
analytical data for all compounds reported in this publication.

840
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
Scheme 4. Synthesis of piperidinecarboxamide 14.
5.1.1. 4-((2-Chlorophenoxy)methyl)eN-(pyridin-4-yl)benzamide
(1)
temperature overnight, water was poured into the reaction and the
mixture extracted with EtOAc. The organic layer was washed with
brine and dried over anhydrous MgSO4. After filtration and con-
centration, the residue was purified by preparative TLC (CHCl3/
MeOH ¼ 9/1) to afford the title compound 4-((2-chlorophenoxy)
methyl)eN-(4-methylthiazol-2-yl)benzamide (2; 30 mg, 44%
yield). MS ESI m/e: 357 (M þ H), 359 (M ꢀ H); 1H NMR (DMSO‑d6,
Step 1. Mixture of methy 4-(bromomethyl)benzoate (53; 2.3 g,
10 mmol), K2CO3 (4.1 g, 30 mmol), and 2-chlorophenol (1.2 ml,
12 mmol) in DMSO (23 ml) was stirred at 65 ꢄC for 3 h under argon
atmosphere. After cooling to room temperature, water and EtOAc
were added and the water layer extracted twice with EtOAc. The
combined organic layer was washed with brine and dried over
anhydrous MgSO4. Filtration and concentration afforded the title
compound methyl 4-((2-chlorophenoxy)methyl)benzoate (54;
400 MHz)
d: 2.31 (d, J ¼ 0.9 Hz, 3H), 5.32 (s, 2H), 6.83 (s, 1H), 6.98
(td, J ¼ 7.6, 1.5 Hz, 1H), 7.24 (dd, J ¼ 8.3, 1.4 Hz, 1H), 7.31 (d,
J ¼ 17.3 Hz, 1H), 7.46 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.62 (d, J ¼ 8.3 Hz, 2H),
8.11 (d, J ¼ 8.6 Hz, 2H), 12.56 (br s, 1H); Anal. Calcd for
2.57 g, 98% yield). 1H NMR (DMSO‑d6)
d: 3.86 (s, 3H), 5.32 (s, 2H),
6.96e7.00 (m, 1H), 7.22 (dd, J ¼ 8.3, 1.4 Hz, 2H), 7.30 (dd, J ¼ 8.6,
7.0 Hz, 2H), 7.46 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.61 (d, J ¼ 8.3 Hz, 1H), 8.00
(dd, J ¼ 6.7, 1.9 Hz, 1H).
C18H15ClN2O2S: C, 60.25; H, 4.21; N, 7.81. Found: C, 60.19; H, 4.20; N,
7.79.
Step 2. To a solution of 54 (2.57 g, 9.78 mmol) in MeOH (20 ml)
and THF (6 ml) was added 4 N NaOH (3.8 ml, 15 mmol) at room
temperature. The reaction mixture was heated to reflux for 3 h. After
cooling to 0 ꢄC, water, followed by 2 N HCl, was added portion-wise
and the reaction mixture allowed to stir at room temperature. The
slurry was filtered and the filter cake washed with water, followed by
drying in a vacuum to afford 4-((2-chlorophenoxy)methyl)benzoic
5.1.3. N-(4-methyl-thiazol-2-yl)-4-(o-tolylamino-methyl)-
benzamide (4)
Step 1. To a solution of 4-(chloromethyl)benzoic acid (56; 1.71 g,
10 mmol) in DMF (17 ml) was added DIPEA (2.6 ml, 15 mmol), fol-
lowed by 2-amino-4-methylthiazole (65; 1.14 g, 10 mmol) at room
temperature. HATU (4.56 g, 12 mmol) was then added and the re-
action mixture stirred at room temperature overnight. To the re-
action was added saturated NaHCO3 (10 ml), followed by extraction
with EtOAc twice. The combined organic layer was washed with
brine, dried over anhydrous MgSO4, filtered, and concentrated. The
insoluble material was precipitated from the evaporated residue
and filtered off. The filter cake was washed with EtOAc. The filtrate
was concentrated and purified by reverse phase flash chromatog-
raphy (MeCN/H2O) to afford 57 (1.3 g, 47%). MS ESI m/e: 267
acid (55; 2.43 g, 95%) as a white solid. 1H NMR (DMSO‑d6)
d: 5.31 (s,
2H), 6.98 (td, J ¼ 7.5, 1.2 Hz, 1H), 7.22 (dd, J ¼ 8.3, 1.2 Hz, 1H),
7.28e7.32 (m,1H), 7.46 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.59 (d, J ¼ 8.3 Hz, 2H),
7.98 (d, J ¼ 8.3 Hz, 2H), 12.99 (br s, 1H).
Step 3. To a solution of 55 (50 mg, 0.19 mmol) in pyridine (1 ml)
was added EDC$HCl (44 mg, 0.23 mmol), followed by 4-
aminopyridine (18 mg, 0.19 mmol) at room temperature. The re-
action mixture was stirred at room temperature overnight. Water
was added to the reaction, and extraction with EtOAc performed
twice. The combined organic layer was washed with brine and
dried over anhydrous MgSO4. After filtration and concentration, the
residue was purified by preparative TLC (CHCl3/MeOH ¼ 9/1) to
afford the title compound 4-((2-chlorophenoxy)methyl)eN-(pyr-
idin-4-yl)benzamide (1; 8 mg, 12% yield). MS ESI m/e: 339 (M þ H),
(M þ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.31 (s, 3H), 4.84 (s, 2H),
6.83 (s, 1H), 7.59 (d, J ¼ 8.1 Hz, 2H), 8.08 (d, J ¼ 10.0 Hz, 2H), 12.60
(br s, 1H).
Step 2. To a solution of 57 (50 mg, 0.187 mmol) and KI (31 mg,
0.187 mmol) in DMF (0.5 ml) was added 2-methylaniline (0.06 ml,
0.562 mmol). The reaction mixture was stirred at 60 ꢄC for 2 h,
cooled to room temperature, and water (1 ml) added, followed by
saturated NaHCO3 (pH 8). The water layer was extracted with EtOAc
and separated. The organic layer was washed with saturated
NaHCO3, followed by brine, then dried over Na2SO4. After filtration
and removal of the solvent under reduced pressure, the residue was
purified by flash chromatography (CHCl3/EtOAc ¼ 9/1) to afford N-
(4-methyl-thiazol-2-yl)-4-(o-tolylamino-methyl)-benzamide (4;
45 mg, 67%) as an amorphous solid. MS ESI m/e: 338 (M þ H), 336
337 (M ꢀ H); 1H NMR (DMSO‑d6)
: 5.34 (s, 2H), 6.98 (td, J ¼ 7.6,
d
1.5 Hz, 1H), 7.24 (dd, J ¼ 8.3, 1.6 Hz, 1H), 7.29e7.33 (m, 1H), 7.46 (dd,
J ¼ 7.9, 1.4 Hz, 1H), 7.64 (d, J ¼ 8.3 Hz, 2H), 7.80 (d, J ¼ 6.5 Hz, 2H),
7.99 (d, J ¼ 8.3 Hz, 2H), 8.49 (dd, J ¼ 5.0, 1.5 Hz, 2H), 10.64 (br s, 1H);
Anal. Calcd for C19H15ClN2O2$0.5H2O: C, 65.62; H, 4.64; N, 8.05.
Found: C, 65.87; H, 4.49; N, 7.93.
5.1.2. 4-((2-Chlorophenoxy)methyl)eN-(4-methylthiazol-2-yl)
benzamide (2)
To a solution of 4-((2-chlorophenoxy)methyl)benzoic acid (55;
50 mg, 0.19 mmol) and 2-amino-4-methylthiazole (65; 22 mg,
0.19 mmol) in pyridine (0.5 ml) was added EDC$HCl (44 mg,
0.23 mmol) at room temperature. After stirring at room
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.18 (s, 3H), 2.30 (d,
J ¼ 0.9 Hz, 3H), 4.44 (d, J ¼ 6.0 Hz, 2H), 5.72 (t, J ¼ 6.0 Hz, 1H), 6.32
(d, J ¼ 7.5 Hz, 1H), 6.47 (td, J ¼ 7.3, 1.0 Hz, 1H), 6.80 (q, J ¼ 1.1 Hz, 1H),
6.86e6.90 (m, 1H), 6.95e7.00 (m, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 8.03
(d, J ¼ 8.4 Hz, 2H), 12.46 (br s, 1H); Anal. Calcd for
C19H19N3OS$0.25H2O: C, 66.74; H, 5.75; N,12.29. Found: C, 66.83; H,

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
841
Scheme 5. Synthesis of phenyl carboxamide 16e30, 32e43.

842
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
Scheme 6. Synthesis of phenyl carboxamide 31.
Scheme 7. Synthesis of thiophene carboxamide 15, 44e52.

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
843
5.72; N, 12.10; mp 157e159 ꢄC.
ESI m/e: 366 (M þ H), 364 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d:
0.98 (t, J ¼ 7.4 Hz, 3H), 1.61 (td, J ¼ 15.0, 7.4 Hz, 2H), 2.30 (s, 3H),
2.48e2.55 (m, 2H), 4.43 (d, J ¼ 6.0 Hz, 2H), 5.85 (t, J ¼ 5.9 Hz, 1H),
6.30 (d, J ¼ 8.1 Hz, 1H), 6.48 (dd, J ¼ 7.3, 6.4 Hz, 1H), 6.81 (s, 1H), 6.86
(t, J ¼ 6.9 Hz,1H), 6.94 (dd, J ¼ 7.3,1.5 Hz,1H), 7.47 (d, J ¼ 8.3 Hz, 2H),
8.02 (d, J ¼ 8.3 Hz, 2H), 12.46 (br s, 1H).; Anal. Calcd for C21H23N3OS:
C, 69.01; H, 6.34; N, 11.50. Found: C, 69.12; H, 6.31; N, 11.47.
5.1.4. 4-[(2-Chloro-phenylamino)-methyl]eN-(4-methyl-thiazol-2-
yl)-benzamide (3)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and 2-chloroaniline. MS
ESI m/e: 358 (M þ H), 356 (M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d:
2.30 (d, J ¼ 0.8 Hz, 3H), 4.49 (d, J ¼ 6.4 Hz, 2H), 6.24 (t, J ¼ 6.0 Hz,
1H), 6.48e6.58 (m, 2H), 6.80 (br s, 1H), 6.97e7.05 (m, 1H), 7.25 (dd,
J ¼ 7.9, 1.5 Hz, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.03 (d, J ¼ 8.3 Hz, 2H),
12.46 (br s, 1H); Anal. Calcd for C18H16ClN3OS: C, 60.41; H, 4.51; N,
11.74. Found: C, 60.33; H, 4.56; N, 11.54.
5.1.10. 2-((4-((5-Methylthiazol-2-yl)carbamoyl)benzyl)amino)
benzoic acid (10)
Step 1. To a solution of 57 (150 mg, 0.562 mmol) and KI (93 mg,
0.562 mmol) in DMF (1.5 ml) was added 2-aminobenzoic acid
methyl ester (0.22 ml, 1.69 mmol). The reaction mixture was stirred
at 60 ꢄC for 2 h, cooled to room temperature, and water (3 ml)
added, followed by saturated NaHCO3 (pH 8). The water layer was
extracted with EtOAc and separated. The organic layer was washed
with saturated NaHCO3, followed by brine, and then dried over
MgSO4. After filtration and removal of the solvent under reduced
pressure, the residue was purified by reverse phase flash chroma-
tography (MeCN/H2O) to afford methyl 2-((4-((4-methylthiazol-2-
yl)carbamoyl)benzyl)amino)benzoate (58; 89 mg, 42%) as an
amorphous solid. MS ESI m/e: 382 (M þ H), 380 (M ꢀ H); 1H NMR
5.1.5. N-(5-methylthiazol-2-yl)-4-((phenylamino)methyl)
benzamide (5)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and aniline. MS ESI m/e:
324 (M þ H), 322 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.52 (s,
1H), 4.35 (d, J ¼ 6.2 Hz, 2H), 6.33 (t, J ¼ 6.2 Hz, 1H), 6.49e6.57 (m,
3H), 6.81 (s, 1H), 7.04 (td, J ¼ 6.9, 1.6 Hz, 2H), 7.49 (d, J ¼ 8.3 Hz, 2H),
8.04 (d, J ¼ 8.3 Hz, 2H), 12.47 (br s, 1H); Anal. Calcd for C18H17N3OS:
C, 66.85; H, 5.30; N, 12.99. Found: C, 66.80; H, 5.41; N, 13.02.
(DMSO‑d6, 400 MHz)
d
: 2.30 (s, 3H), 3.83 (s, 3H), 4.59 (d, J ¼ 6.0 Hz,
2H), 6.58e6.62 (m, 1H), 6.66 (d, J ¼ 8.3 Hz, 1H), 6.81 (s, 1H),
7.30e7.34 (m, 1H), 7.48 (d, J ¼ 8.1 Hz, 2H), 7.82 (dd, J ¼ 7.9, 1.6 Hz,
1H), 8.05 (d, J ¼ 8.3 Hz, 2H), 8.21 (t, J ¼ 6.0 Hz, 1H), 12.51 (br s, 1H).
Step 2. To a solution of 58 (73 mg, 0.19 mmol) in THF (0.35 ml)
5.1.6. N-(4-methyl-thiazol-2-yl)-4-[(2-trifluoromethyl-
phenylamino)-methyl]-benzamide (6)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and 2-(trifluoromethyl)
aniline. MS ESI m/e: 392 (M þ H), 390 (M ꢀ H); 1H NMR (DMSO‑d6,
and MeOH (0.35 ml) was added 4 N NaOH (144 ml, 0.57 mmol) at
room temperature. The reaction mixture was stirred for 3 h at 60 ꢄC.
After cooling to 0 ꢄC, water (5 ml), followed by 2 N HCl (0.2 ml), was
added portion-wise and the reaction mixture allowed to stir at
room temperature. The slurry was filtered and the filter cake
washed with water (4 ꢃ 30 ml), followed by drying in a vacuum to
afford 2-((4-((5-methylthiazol-2-yl)carbamoyl)benzyl)amino)ben-
zoic acid (10; 50 mg, 71%) as a white solid. MS ESI m/e: 368 (M þ H);
300 MHz)
d
: 2.30 (s, 3H), 4.54 (d, J ¼ 5.7 Hz, 2H), 6.38 (t, J ¼ 6.2 Hz,
1H), 6.58e6.68 (m, 2H), 6.80 (br s, 1H), 7.28 (t, J ¼ 7.3 Hz, 1H),
7.39e7.50 (m, 3H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.47 (br s, 1H); Anal.
Calcd for C19H16F3N3OS: C, 58.30; H, 4.12; N, 10.74. Found: C, 58.31;
H, 3.97; N, 10.69; mp 159e161 ꢄC.
1H NMR (DMSO‑d6, 400 MHz)
d: 2.30 (s, 2H), 4.58 (s, 1H), 6.55e6.59
(m, 1H), 6.62 (d, J ¼ 8.3 Hz, 1H), 6.81 (s, 1H), 7.26e7.31 (m, 1H), 7.48
(d, J ¼ 8.3 Hz, 1H), 7.81 (dd, J ¼ 7.9, 1.6 Hz, 1H), 8.05 (d, J ¼ 8.3 Hz,
1H), 12.51 (br s, 1H); Anal. Calcd for C19H17N3O3S: C, 62.11; H, 4.66;
N, 11.44. Found: C, 62.14; H, 4.71; N, 11.39.
5.1.7. 4-(((3-Chlorophenyl)amino)methyl)eN-(5-methylthiazol-2-
yl)benzamide (7)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and 3-chloroaniline. MS
5.1.11. N,N-dimethyl-2-((4-((5-methylthiazol-2-yl)carbamoyl)
benzyl)amino)benzamide (11)
ESI m/e: 358 (M þ H), 356 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d:
2.32 (s, 3H), 4.37 (d, J ¼ 6.0 Hz, 2H), 6.52 (dd, J ¼ 8.0, 2.0 Hz, 2H),
6.57 (t, J ¼ 2.1 Hz, 1H), 6.69 (t, J ¼ 6.1 Hz, 1H), 6.81 (br s, 1H), 7.04 (t,
J ¼ 8.0 Hz, 1H), 7.48 (d, J ¼ 8.3 Hz, 2H), 8.05 (d, J ¼ 8.3 Hz, 2H), 12.49
(br s, 1H).; Anal. Calcd for C18H16ClN3OS: C, 60.41; H, 4.51; N, 11.74.
Found: C, 60.44; H, 4.57; N, 11.62.
To a solution of 10 (50 mg, 0.135 mmol) in DMF (0.5 ml) was
added a 2 M THF solution of dimethylamine (0.2 ml, 0.405 mmol),
followed by PyBOP (77 mg, 0.149 mmol) at room temperature. The
mixture was stirred at room temperature overnight under argon
atmosphere. Water was added to the reaction mixture and the
water layer extracted with EtOAc twice. The combined organic layer
was washed with brine and dried over anhydrous MgSO4. After
filtration and removal of the solvent under reduced pressure, the
residue was purified by flash chromatography (hexane/EtOAc ¼ 4/1
to 2/1) to afford N,N-dimethyl-2-((4-((5-methylthiazol-2-yl)car-
bamoyl)benzyl)amino)benzamide (11; 39 mg, 73%) as an amor-
phous solid. MS ESI m/e: 395 (M þ H), 393 (M ꢀ H); 1H NMR
5.1.8. 4-(((4-Chlorophenyl)amino)methyl)eN-(5-methylthiazol-2-
yl)benzamide (8)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and 4-chloroaniline. MS
ESI m/e: 358 (M þ H), 356 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d:
2.30 (s, 3H), 4.35 (d, J ¼ 6.2 Hz, 2H), 6.55e6.58 (m, 3H), 6.81 (s, 1H),
7.06 (dt, J ¼ 9.7, 2.7 Hz, 2H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.04 (d,
J ¼ 8.3 Hz, 2H), 12.48 (br s, 1H); Anal. Calcd for C18H16ClN3OS: C,
60.41; H, 4.51; N, 11.74. Found: C, 60.28; H, 4.67; N, 11.62.
(DMSO‑d6, 400 MHz)
d
: 2.32 (s, 3H), 2.96 (s, 6H), 4.43 (d, J ¼ 6.0 Hz,
2H), 6.04 (t, J ¼ 6.1 Hz, 1H), 6.49 (d, J ¼ 8.1 Hz, 1H), 6.58 (td, J ¼ 7.4,
0.9 Hz, 1H), 6.81 (s, 1H), 7.04 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.06e7.10 (m,
1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.03 (d, J ¼ 8.6 Hz, 2H), 12.47 (br s, 1H);
Anal. Calcd for C21H22N4O2S: C, 63.94; H, 5.62; N, 14.20. Found: C,
63.55; H, 5.59; N, 14.04; mp 244e246 ꢄC.
5.1.9. N-(5-methylthiazol-2-yl)-4-(((2-propylphenyl)amino)
methyl)benzamide (9)
This compound was prepared according to the synthetic pro-
tocols described for compound 4, starting from 4-(chloromethyl)
eN-(4-methylthiazol-2-yl)benzamide (57) and 2-propylaniline. MS
5.1.12. N-(4-methylthiazol-2-yl)-5-((o-tolylamino)methyl)
picolinamide (12)
Step 1. To
a stirred mixture of 6-((4-methylthiazol-2-yl)

844
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
carbamoyl)nicotinic acid (59a; 200 mg, 0.76 mmol) in THF (2 ml)
was added borane tetrahydrofuran complex solution (1.0 M) in THF
(1.5 ml, 1.5 mmol) via syringe at room temperature. The mixture
was stirred at 55 ꢄC for 1 h under argon atmosphere. The reaction
was then cooled to 0 ꢄC, followed by the careful addition of water
and then K2CO3 (260 mg). The reaction mixture was stirred at room
temperature for 30 min and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated. To the residue isopropyl ether was added and the
slurry filtered. The filter cake was dried in a vacuum and purified by
reverse phase preparative TLC (THF/EtOH/H2O ¼ 1/1/1) to afford
the compound 5-(hydroxymethyl)eN-(4-methylthiazol-2-yl)pico-
linamide (60a; 90 mg, 48%) as a solid. 1H NMR (DMSO‑d6, 400 MHz)
gel column chromatography (hexane/EtOAc ¼ 100/0e4/1) to give
tert-butyl 4-(((2-(trifluoromethyl)phenyl)amino)methyl)piperi-
dine-1-carboxylate (63; 0.64 g, 59%) as a pale yellow oil. 1H NMR
(CDCl3, 400 MHz) : 1.31e1.11 (m, 3H), 1.46 (s, 9H), 1.85e1.71 (m,
3H), 2.70 (t, J ¼ 12.4 Hz, 2H), 3.08 (t, J ¼ 6.0 Hz, 2H), 4.14 (s, 2H), 4.39
(s, 1H), 6.74e6.67 (m, 2H), 7.36 (t, J ¼ 7.8 Hz, 1H), 7.43 (d, J ¼ 7.9 Hz,
1H).
d
Step 2. To a solution of 63 (0.64 g, 1.79 mmol) in dioxane (1 ml)
and MeOH (0.5 ml) was added 4 N HCl/dioxane solution (4 ml).
After stirring at room temperature for 12 h, the solvent was
removed under reduced pressure. The residue was purified by
trituration with ethyl acetate to give N-(piperidin-4-ylmethyl)-2-
(trifluoromethyl)aniline hydrochloride (64; 0.51 g, 97%) as a solid.
d
: 2.38 (s, 3H), 4.70 (d, J ¼ 4.2 Hz, 2H), 5.59 (s, 1H), 7.25 (s, 1H), 8.09
1H NMR (DMSO‑d6, 400 MHz)
d: 1.42e1.28 (m, 2H), 1.86e1.72 (m,
(d, J ¼ 7.9 Hz, 1H), 8.24 (d, J ¼ 7.9 Hz, 1H), 8.76 (s, 1H), 12.03 (br s,
2H), 1.97e1.86 (m, 1H), 2.88e2.72 (m, 2H), 3.15e3.06 (m, 2H),
3.30e3.20 (m, 2H), 4.99 (s, 1H), 5.58 (s, 1H), 6.66 (t, J ¼ 7.5 Hz, 1H),
6.84 (d, J ¼ 8.6 Hz, 1H), 7.43e7.33 (m, 2H), 8.62 (s, 1H).
1H).
Step 2. To a mixture of 60a (90 mg, 0.361 mmol) in CHCl3 (2 ml)
was added thionyl chloride (0.067 ml, 1.08 mmol) at room tem-
perature. The reaction mixture was stirred at 50 ꢄC for 10 min and
then catalyst DMF added. After the reaction mixture was stirred for
1 h at 50 ꢄC, it was cooled to room temperature. The solvent was
removed under reduced pressure and used directly in the next step
without further purification.
Step 3. To
a
solution of 1,10-carbonyldiimidazole (0.50 g,
3.08 mmol) in CHCl3 (4 ml) was added a solution of 2-amino-4-
methylthiazole (65; 0.39 g, 3.42 mmol) in CHCl3 (1 ml). After stir-
ring at room temperature for 3 h, precipitated solids were collected
to give N-(4-methylthiazol-2-yl)-1H-imidazole-1-carboxamide
(66; 0.57 g, 89%). 1H NMR (DMSO‑d6, 400 MHz)
d: 13.18 (s, 1H),
Step 3. To a solution of 5-(chloromethyl)eN-(4-methylthiazol-2-
yl)picolinamide (61a; 0.361 mmol, crude) and 2-methylaniline
(0.154 ml, 1.44 mmol) in THF (5 ml) was added KI (60 mg,
0.361 mmol). After stirring for 1 h at 60 ꢄC under argon atmosphere,
the reaction was cooled to room temperature, water poured into it,
and the mixture extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated. The residue was purified by preparative TLC (CHCl3/
EtOAc ¼ 2/1), hexane/Et2O (20/1) added, and the slurry filtered. The
filter cake was dried in a vacuum to afford the title compound N-(4-
8.26 (s, 1H), 7.63 (s, 1H), 7.02 (s, 1H), 6.72 (s, 1H), 2.23 (s, 3H).
Step 4. To a solution of 64 (62 mg, 0.211 mmol) and N-(4-
methylthiazol-2-yl)-1H-imidazole-1-carboxamide (66; 40 mg,
0.192 mmol) in CHCl3 (10 ml) was added TEA (53 ml, 0.384 mmol).
After stirring for 24 h at room temperature, the mixture was puri-
fied by silica gel column chromatography (hexane/EtOAc ¼ 100/
0e2/3) to give N-(4-methylthiazol-2-yl)-4-(((2-(trifluoromethyl)
phenyl)amino)methyl)piperidine-1-carboxamide (14; 75 mg, 99%)
as an oil. MS ESI m/e: 399 (M þ H), 397 (M ꢀ H); 1H NMR (DMSO‑d6,
400 MHz)
d
: 1.01e1.14 (m, 2H), 1.69 (d, J ¼ 11.2 Hz, 2H), 1.79e1.92
methylthiazol-2-yl)-5-((o-tolylamino)methyl)picolinamide
(12;
(m, 1H), 2.19 (s, 3H), 2.76 (t, J ¼ 11.7 Hz, 2H), 3.09 (t, J ¼ 6.4 Hz, 2H),
4.19 (d, J ¼ 12.1 Hz, 2H), 5.45 (t, J ¼ 5.6 Hz, 1H), 6.51 (s, 1H), 6.65 (t,
J ¼ 7.4 Hz,1H), 6.84 (d, J ¼ 8.6 Hz,1H), 7.36e7.42 (m, 2H), 10.83 (br s,
1H); Anal. Calcd for C18H21F3N4OS$0.25H2O: C, 53.65; H, 5.38; N,
13.90. Found: C, 53.88; H, 5.50; N, 13.63.
15 mg, 12%) as a pale yellow solid: MS ESI m/e: 339 (M þ H), 337
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.18 (s, 3H), 2.30 (s, 3H),
4.52 (d, J ¼ 4.4 Hz, 2H), 5.79 (br s,1H), 6.39 (d, J ¼ 8.2 Hz,1H), 6.49 (t,
J ¼ 7.3 Hz, 1H), 6.89e6.91 (m, 2H), 6.98 (d, J ¼ 7.3 Hz, 1H), 8.01 (dd,
J ¼ 7.9, 2.0 Hz, 1H), 8.11 (d, J ¼ 7.9 Hz, 1H), 8.73 (s, 1H), 11.76 (s, 1H);
Anal. Calcd for C18H18N4OS: C, 63.88; H, 5.36; N, 16.55. Found: C,
63.86; H, 5.29; N, 16.48; mp 117e120 ꢄC.
5.1.15. N-(4-hydroxymethyl-thiazol-2-yl)-4-[(2-trifluoromethyl-
phenylamino)-methyl]-benzamide (21)
Step 1. To a solution of methyl 4-(bromomethyl)benzoate (53;
25 g, 0.109 mol) in DMSO (250 ml) was added 2-(trifluoromethyl)
aniline (40.6 ml, 0.327 mol) and NaI (16.3 g, 0.109 mol). After stir-
ring at 60 ꢄC for 3 h, the mixture was cooled to room temperature.
Water (1 L) and EtOAc were added to the reaction and separated.
The organic layer was washed with 5% sodium thiosulfate, followed
by brine, and dried over MgSO4, filtered, and the solvent removed
under reduced pressure. The residue was purified by flash chro-
matography (hexane/EtOAc ¼ 1/1) to afford methyl 4-(((2-(tri-
fluoromethyl)phenyl)amino)methyl)benzoate (68; 19.34 g, 57%). 1H
5.1.13. N-(4-methylthiazol-2-yl)-6-((o-tolylamino)methyl)
nicotinamide (13)
This compound was prepared according to the synthetic pro-
tocols described for compound 12, starting from 6-(chloromethyl)-
3-pyridinecarboxylic acid (59b). MS ESI m/e: 339 (M þ H), 337
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.20 (s, 3H), 2.31 (s, 3H),
4.51 (d, J ¼ 6.0 Hz, 2H), 5.82 (t, J ¼ 5.8 Hz,1H), 6.30 (d, J ¼ 7.9 Hz,1H),
6.50 (t, J ¼ 7.3 Hz, 1H), 6.84 (s, 1H), 6.90 (t, J ¼ 7.6 Hz, 1H), 7.00 (d,
J ¼ 7.1 Hz, 1H), 7.46 (d, J ¼ 8.2 Hz, 1H), 8.33 (d, J ¼ 8.3 Hz, 1H), 9.17 (s,
1H), 12.70 (s, 1H); Anal. Calcd for C18H18N4OS: C, 63.88; H, 5.36; N,
16.55. Found: C, 63.77; H, 5.39; N, 16.17; mp 167e168 ꢄC.
NMR (DMSO‑d6, 400 MHz)
d
: 3.83 (s, 3H), 4.55 (d, J ¼ 6.0 Hz, 2H),
6.41 (t, J ¼ 6.0 Hz, 1H), 6.56 (d, J ¼ 8.6 Hz, 1H), 6.65 (t, J ¼ 7.5 Hz, 1H),
7.27 (t, J ¼ 7.7 Hz, 1H), 7.42 (d, J ¼ 7.8 Hz, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H),
7.93 (d, J ¼ 8.3 Hz, 2H).
5.1.14. N-(4-methylthiazol-2-yl)-4-(((2-(trifluoromethyl)phenyl)
amino)methyl)piperidine-1-carboxamide (14)
Step 2. To a solution of methyl ester 68 (19.34 g, 65.5 mmol) in
MeOH (48 ml) and THF (48 ml) was added 4 N LiOH (25 ml,
98 mmol). The reaction mixture was heated to reflux for 4 h. After
the solvent was removed under reduced pressure and the mixture
acidified with 2 N HCl, it was extracted with EtOAc. The organic
layer was washed with brine and dried over MgSO4. After filtration
and removal of the solvent under reduced pressure, hexane and
EtOAc were added to the resulting residue and the slurry filtered.
The filter cake was washed with hexane and dried in a vacuum to
afford 4-(((2-(trifluoromethyl)phenyl)amino)methyl)benzoic acid
Step 1. To a mixture of 2-bromobenzotrifluoride (0.41 ml,
3 mmol), tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (62;
0.77 g, 3.6 mmol), sodium tert-butoxide (0.41 g, 4.2 mmol), and 2-
dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
(0.14 g,
0.3 mmol) in toluene (10 ml) was added tris(dibenzylideneacetone)
dipalladium(0) (0.14 g, 0.15 mmol). The mixture was stirred at 80 ꢄC
for 16 h under argon atmosphere. After cooling to room tempera-
ture, the mixture was passed through a celite pad. The solvent was
removed under reduced pressure and the residue purified by silica

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
845
(77; 18.94 g, 98%) as a white solid. 1H NMR (DMSO‑d6, 400 MHz)
d:
EDC$HCl (95 mg, 0.500 mmol) at 0 ꢄC. The reaction mixture was
stirred at room temperature overnight under N2 atmosphere. Wa-
ter and EtOAc were added to the reaction and separated. The
organic layer was washed with brine, dried over MgSO4, filtered,
and concentrated. The residue was purified by flash chromatog-
raphy (hexane/EtOAc ¼ 4/1) to afford 89 (71 mg, 42%) as an oil. 1H
4.52 (d, J ¼ 5.8 Hz, 2H), 6.39 (t, J ¼ 5.8 Hz, 1H), 6.56 (d, J ¼ 8.3 Hz,
1H), 6.64 (t, J ¼ 7.5 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz,
3H), 7.89 (d, J ¼ 8.1 Hz, 2H), 12.84 (br s, 1H).
Step 3. To a solution of 77 (200 mg, 0.68 mmol), (2-amino-1,3-
thiazol-4-yl)methyl acetate hydrochloride (213 mg, 1.02 mmol),
and DIPEA (0.30 ml, 1.7 mmol) in DMF (2 ml) was added PyBOP
(451 mg, 1.02 mmol). The reaction mixture was heated to 65 ꢄC for
9 h. After cooling to room temperature, water and EtOAc were
added and separated. The organic layer was washed with brine,
dried over MgSO4, filtered, and concentrated. The residue was pu-
rified by flash chromatography (hexane/EtOAc ¼ 4/1) to afford (2-
(4-(((2-(trifluoromethyl)phenyl)amino)methyl)benzamido)thia-
zol-4-yl)methyl acetate (85; 26.9 mg, 66%) as a solid. MS ESI m/e:
NMR (DMSO‑d6, 400 MHz)
d: 1.19 (t, J ¼ 7.2 Hz, 3H), 2.18 (s, 3H), 3.73
(s, 2H), 4.09 (q, J ¼ 7.1 Hz, 2H), 4.43 (d, J ¼ 6.0 Hz, 2H), 5.73 (t,
J ¼ 6.0 Hz, 1H), 6.31 (d, J ¼ 7.9 Hz, 1H), 6.47 (t, J ¼ 6.9 Hz, 1H), 6.87 (t,
J ¼ 7.6 Hz, 1H), 6.97 (d, J ¼ 6.9 Hz, 1H), 7.04 (s, 1H), 7.49 (d, J ¼ 8.1 Hz,
2H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.58 (br s, 1H).
Step 2. To a solution of 89 (73 mg, 0.178 mmol) in EtOH (0.7 ml)
was added 4 N NaOH (0.13 ml, 0.534 mmol) at 0 ꢄC. The reaction
mixture was stirred at room temperature for 30 min. After cooling
to 0 ꢄC, 10% citric acid was added portion-wise and the reaction
mixture was allowed to stir at room temperature. The slurry was
filtered and the filter cake washed with water and dried in a vac-
uum to afford {2-[4-(o-tolylamino-methyl)-benzoylamino]-thia-
zol-4-yl}-acetic acid (17; 67 mg, 99%) as a white solid. MS ESI m/e:
450 (M þ H), 448 (M ꢀ H). 1H NMR (DMSO‑d6, 400 MHz)
d: 2.06 (s,
3H), 4.54 (d, J ¼ 6.0 Hz, 2H), 5.06 (s, 2H), 6.42 (t, J ¼ 6.0 Hz, 1H), 6.59
(d, J ¼ 8.3 Hz, 1H), 6.65 (t, J ¼ 7.5 Hz, 1H), 7.25 (s, 1H), 7.28 (t,
J ¼ 7.7 Hz, 1H), 7.41e7.43 (m, 1H), 7.47 (d, J ¼ 8.1 Hz, 2H), 8.05 (d,
J ¼ 8.3 Hz, 2H), 12.66 (br s, 1H).
Step 4. K2CO3 (27 mg, 0.2 mmol) was added to a solution of 85
(176 mg, 0.39 mmol) in water (0.24 ml) and MeOH (2.6 ml). The
reaction mixture was stirred at room temperature overnight, and
then heated at 65 ꢄC for 1 h under N2 atmosphere. After adding
water to the reaction mixture, the slurry was filtered. The filter cake
was washed with water and dried in a vacuum to afford N-(4-
hydroxymethyl-thiazol-2-yl)-4-[(2-trifluoromethyl-phenylamino)-
methyl]-benzamide (21; 55.9 mg, 35%) as a white solid. MS ESI m/e:
382 (M þ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.18 (s, 3H), 3.64 (s,
2H), 4.44 (d, J ¼ 4.9 Hz, 2H), 5.72e5.74 (br m, 1H), 6.32 (d, J ¼ 8.2 Hz,
1H), 6.47 (t, J ¼ 7.3 Hz, 1H), 6.88 (t, J ¼ 7.7 Hz, 1H), 6.97 (d, J ¼ 7.1 Hz,
1H), 7.01 (s, 1H), 7.49 (d, J ¼ 8.2 Hz, 2H), 8.04 (d, J ¼ 8.4 Hz, 2H),
12.48 (br s, 1H); Anal. Calcd for C20H19N3O3S$0.25H2O: C, 62.24; H,
5.09; N, 10.89. Found: C, 62.39; H, 5.05; N, 10.76.
5.1.19. 2-[4-(o-Tolylamino-methyl)-benzoylamino]-thiazole-4-
carboxylic acid (16)
408 (M þ H), 406 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 4.50 (d,
J ¼ 5.8 Hz, 2H), 4.54 (d, J ¼ 6.0 Hz, 2H), 5.23 (t, J ¼ 5.7 Hz,1H), 6.41 (t,
J ¼ 6.0 Hz, 1H), 6.58 (d, J ¼ 8.3 Hz, 1H), 6.65 (t, J ¼ 7.5 Hz, 1H), 6.98 (s,
1H), 7.28 (t, J ¼ 7.8 Hz, 1H), 7.42 (d, J ¼ 6.5 Hz, 1H), 7.47 (d, J ¼ 8.3 Hz,
2H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.53 (br s, 1H); Anal. Calcd for
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 76 and ethyl 2-
aminothiazole-4-carboxylate. MS ESI m/e: 368 (M þ H); 1H NMR
(DMSO‑d6, 400 MHz)
d
: 2.19 (s, 3H), 4.45 (s, 2H), 6.35 (d, J ¼ 7.9 Hz,
C
19H16F3N3O2S: C, 56.01; H, 3.96; N, 10.31. Found: C, 56.28; H, 3.90;
1H), 6.49 (t, J ¼ 7.2 Hz, 1H), 6.89 (t, J ¼ 7.7 Hz, 1H), 6.98 (d, J ¼ 7.1 Hz,
1H), 7.51 (d, J ¼ 8.4 Hz, 2H), 8.03 (s, 1H), 8.07 (d, J ¼ 8.4 Hz, 2H),
12.90 (s,1H); Anal. Calcd for C19H17N3O3S: C, 62.11; H, 4.66; N,11.44.
Found: C, 61.97; H, 4.76; N, 11.47.
N, 10.07.
5.1.16. 4-[(2-Chloro-phenylamino)-methyl]eN-(4-hydroxymethyl-
thiazol-2-yl)-benzamide (32)
This compound was prepared according to the synthetic pro-
tocols described for compound 21, starting from methyl 4-(bro-
momethyl)benzoate (53) and 2-chloroaniline. MS ESI m/e: 374
5.1.20. (2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazol-4-yl)-acetic acid (18)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 77 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 436 (M þ H), 434
(M þ H), 372 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 4.49e4.51
(m, 4H), 5.22 (t, J ¼ 5.8 Hz,1H), 6.26 (t, J ¼ 6.3 Hz,1H), 6.50e6.57 (m,
2H), 6.97 (s, 1H), 6.99e7.03 (m, 1H), 7.24e7.27 (m, 1H), 7.47 (d,
J ¼ 8.3 Hz, 2H), 8.04 (d, J ¼ 8.3 Hz, 2H), 12.52 (br s, 1H); Anal. Calcd
for C18H16ClN3O2S: C, 57.83; H, 4.31; N, 11.24. Found: C, 57.72; H,
4.11; N, 11.20.
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 3.60 (s, 2H), 4.54 (d,
J ¼ 6.0 Hz, 2H), 6.40 (t, J ¼ 5.7 Hz,1H), 6.59 (d, J ¼ 8.2 Hz,1H), 6.65 (t,
J ¼ 7.5 Hz, 1H), 6.97 (s, 1H), 7.28 (t, J ¼ 7.7 Hz, 1H), 7.42e7.46 (m, 3H),
8.06 (d, J ¼ 8.2 Hz, 2H); Anal. Calcd for C20H16F3N3O3S: C, 55.17; H,
3.70; N, 9.65. Found: C, 55.33; H, 3.87; N, 9.72.
5.1.17. 4-[(2-Butyl-phenylamino)-methyl]eN-(4-hydroxymethyl-
thiazol-2-yl)-benzamide (33)
This compound was prepared according to the synthetic pro-
tocols described for compound 21, starting from methyl 4-(bro-
momethyl)benzoate (53) and 2-butylaniline. MS ESI m/e: 396
5.1.21. 3-(2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazol-4-yl)-propionic acid (19)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 77 and methyl 3-
(2-amino-1,3-thiazol-4-yl)propanoate hydrobromide. MS ESI m/e:
(M þ H), 394 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 0.83 (t,
J ¼ 6.8 Hz, 6H), 1.40e1.47 (m, 8H), 3.01e3.03 (m, 1H), 3.54 (s, 2H),
4.48 (d, J ¼ 5.7 Hz, 2H), 6.08 (t, J ¼ 6.0 Hz, 1H), 6.45 (d, J ¼ 7.9 Hz,
1H), 6.53 (t, J ¼ 7.5 Hz, 1H), 6.92 (s, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 7.27
(d, J ¼ 7.5 Hz, 1H), 7.44 (d, J ¼ 7.9 Hz, 2H), 8.06 (d, J ¼ 7.9 Hz, 2H);
Anal. Calcd for C22H25N3O2S: C, 66.81; H, 6.37; N, 10.62. Found: C,
66.74; H, 6.34; N, 10.59; mp 181e182 ꢄC.
450 (M þ H), 448 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.61 (t,
J ¼ 7.5 Hz, 2H), 2.86 (t, J ¼ 7.5 Hz, 2H), 4.53 (d, J ¼ 6.0 Hz, 2H), 6.39 (t,
J ¼ 6.0 Hz, 1H), 6.58 (d, J ¼ 8.6 Hz, 1H), 6.64 (t, J ¼ 7.5 Hz, 1H), 6.84 (s,
1H), 7.27 (t, J ¼ 7.8 Hz, 1H), 7.40e7.43 (m, 1H), 7.45 (d, J ¼ 8.3 Hz,
2H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.18 (br s, 1H), 12.50 (br s, 1H); Anal.
Calcd for C21H18F3N3O3S$0.5H2O: C, 55.02; H, 4.18; N, 9.17. Found: C,
54.95; H, 4.15; N, 9.01.
5.1.18. {2-[4-(o-Tolylamino-methyl)-benzoylamino]-thiazol-4-yl}-
acetic acid (17)
Step 1. To a solution of (2-amino-4-thiazolyl)acetic acid ethyl
ester (77 mg, 0.413 mmol) and 4-((o-tolylamino)methyl)benzoic
acid (76; 100 mg, 0.413 mmol) in pyridine (1 ml) was added
5.1.22. 4-(2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazol-4-yl)-butyric acid (20)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 77 and ethyl 4-(2-

846
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
amino-1,3-thiazol-4-yl)butanoate. MS ESI m/e: 464 (M þ H), 462
3.61 (s, 2H), 4.43 (d, J ¼ 5.3 Hz, 2H), 5.77 (t, J ¼ 5.8 Hz, 1H), 6.32 (d,
J ¼ 7.7 Hz, 1H), 6.49 (t, J ¼ 7.5 Hz, 1H), 6.84e6.88 (m, 1H), 6.94 (dd,
J ¼ 7.2, 1.4 Hz, 1H), 6.98 (s, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.04 (d,
J ¼ 8.3 Hz, 2H); Anal. Calcd for C24H27N3O3S: C, 65.88; H, 6.22; N,
9.60. Found: C, 65.58; H, 6.19; N, 9.40.
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 1.83e1.90 (m, 2H), 2.24 (t,
J ¼ 7.3 Hz, 2H), 2.64 (t, J ¼ 7.7 Hz, 2H), 4.53 (d, J ¼ 5.8 Hz, 2H), 6.39 (t,
J ¼ 6.0 Hz, 1H), 6.58 (d, J ¼ 8.3 Hz, 1H), 6.64 (t, J ¼ 7.5 Hz, 1H), 6.83 (s,
1H), 7.27 (t, J ¼ 7.7 Hz, 1H), 7.40e7.43 (m, 1H), 7.45 (d, J ¼ 8.3 Hz,
2H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.10 (s, 1H), 12.47 (s, 1H); Anal. Calcd
for C22H20F3N3O3S: C, 57.01; H, 4.35; N, 9.07. Found: C, 57.20; H,
4.22; N, 8.76.
5.1.28. {2-[4-(Biphenyl-2-ylaminomethyl)-benzoylamino]-thiazol-
4-yl}-acetic acid (42)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 83 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 444 (M þ H), 442
5.1.23. (2-{4-[(2-Chloro-phenylamino)-methyl]-benzoylamino}-
thiazol-4-yl)-acetic acid (37)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 78 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 402 (M þ H), 400
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 3.63 (s, 2H), 4.39 (d,
J ¼ 5.8 Hz, 2H), 5.44 (t, J ¼ 5.9 Hz,1H), 6.46 (d, J ¼ 8.3 Hz,1H), 6.64 (t,
J ¼ 7.3 Hz, 1H), 6.98e7.00 (m, 2H), 7.03e7.07 (m, 1H), 7.36e7.41 (m,
1H), 7.46e7.52 (m, 6H), 8.04 (d, J ¼ 8.3 Hz, 2H), 12.49 (br s, 1H);
Anal. Calcd for C25H21N3O3S: C, 67.70; H, 4.77; N, 9.47. Found: C,
67.56; H, 4.80; N, 9.36.
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 3.64 (s, 2H), 4.49 (d,
J ¼ 6.2 Hz, 2H), 6.26 (t, J ¼ 6.3 Hz, 1H), 6.50e6.57 (m, 2H), 6.99e7.04
(m, 2H), 7.25 (dd, J ¼ 7.8, 1.4 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 8.04 (d,
J ¼ 8.4 Hz, 2H), 12.52 (br s, 1H); Anal. Calcd for C19H16ClN3O3S: C,
56.79; H, 4.01; N, 10.46. Found: C, 56.80; H, 4.12; N, 10.24.
5.1.29. (2-{4-[(2-Benzyl-phenylamino)-methyl]-benzoylamino}-
thiazol-4-yl)-acetic acid (43)
5.1.24. (2-{4-[(2-Butyl-phenylamino)-methyl]-benzoylamino}-
thiazol-4-yl)-acetic acid (38)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 84 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 458 (M þ H), 456
This compound was prepared according to the synthetic protocols
described for compound 17, starting from 79 and (2-amino-4-
thiazolyl)acetic acid ethyl ester. MS ESI m/e: 424 (M þ H), 422
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 3.64 (s, 2H), 3.94 (s, 2H),
4.41 (d, J ¼ 5.7 Hz, 2H), 5.76 (t, J ¼ 6.0 Hz,1H), 6.38 (d, J ¼ 7.9 Hz,1H),
6.51 (t, J ¼ 7.2 Hz, 1H), 6.86e6.96 (m, 2H), 7.01 (s, 1H), 7.20e7.36 (m,
7H), 7.99 (d, J ¼ 8.3 Hz, 2H), 12.47 (br s, 2H); Anal. Calcd for
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
: 0.94 (t, J ¼ 7.4 Hz, 3H),
d
1.35e1.42 (m, 2H), 1.54e1.61 (m, 2H), 2.55 (t, J ¼ 7.7 Hz, 2H), 3.58 (s,
2H), 4.43 (d, J ¼ 5.7 Hz, 2H), 5.81 (t, J ¼ 6.0 Hz, 1H), 6.31 (d, J ¼ 7.9 Hz,
1H), 6.47e6.49 (m,1H), 6.86 (t, J ¼ 7.4 Hz,1H), 6.93e6.95 (m, 2H), 7.46
(d, J ¼ 8.2 Hz,2H), 8.06(d, J ¼ 7.9 Hz,2H);Anal.CalcdforC23H25N3O3S:
C, 65.23; H, 5.95; N, 9.92. Found: C, 65.46; H, 5.86; N, 9.84.
C26H23N3O3S$0.25H2O: C, 67.59; H, 5.13; N, 9.09. Found: C, 67.77; H,
5.02; N, 9.13.
5.1.30. 4-Hydroxymethyl-2-{4-[(2-trifluoromethyl-phenylamino)-
methyl]-benzoylamino}-thiazole-5-carboxylic acid (30)
5.1.25. (2-{4-[(2-Hexyl-phenylamino)-methyl]-benzoylamino}-
thiazol-4-yl)-acetic acid (39)
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 77 and methyl 2-
amino-4-(hydroxymethyl)-1,3-thiazole-5-carboxylate. MS ESI m/
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 80 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 452 (M þ H), 450
e: 452 (M þ H), 450 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 4.53
(d, J ¼ 6.2 Hz, 2H), 4.57 (s, 2H), 6.38 (t, J ¼ 6.1 Hz, 1H), 6.59 (d,
J ¼ 8.6 Hz, 1H), 6.65 (t, J ¼ 7.7 Hz, 1H), 7.27 (t, J ¼ 7.8 Hz, 1H),
7.39e7.48 (m, 3H), 8.02 (d, J ¼ 8.4 Hz, 2H); Anal. Calcd for
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
: 0.89 (t, J ¼ 7.1 Hz, 3H),
d
1.34 (dt, J ¼ 17.4, 7.0 Hz, 6H), 1.57 (dd, J ¼ 15.0, 7.3 Hz, 2H),
2.49e2.56 (m, 2H), 4.43 (d, J ¼ 6.0 Hz, 2H), 5.81 (t, J ¼ 5.8 Hz, 1H),
6.31 (d, J ¼ 7.9 Hz, 1H), 6.48 (t, J ¼ 6.8 Hz, 1H), 6.86 (t, J ¼ 7.8 Hz, 1H),
6.94 (d, J ¼ 7.4 Hz, 1H), 7.01 (s, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.03 (d,
J ¼ 8.3 Hz, 2H), 12.56 (br s, 1H); Anal. Calcd for C25H29N3O3S: C,
66.49; H, 6.47; N, 9.30. Found: C, 66.49; H, 6.36; N, 9.23.
C20H16F3N3O4S$0.25H2O: C, 52.69; H, 3.65; N, 9.22. Found: C, 52.67;
H, 3.66; N, 8.89.
5.1.31. N-[4-(2-hydroxy-ethyl)-thiazol-2-yl]-4-[(2-trifluoromethyl-
phenylamino)-methyl]-benzamide (22)
To a solution of ethyl 2-(2-(4-(((2-(trifluoromethyl)phenyl)
5.1.26. (2-{4-[(2-Octyl-phenylamino)-methyl]-benzoylamino}-
thiazol-4-yl)-acetic acid (40)
amino)methyl)benzamido)thiazol-4-yl)acetate
(90;
100 mg,
0.222 mmol) in t-BuOH (0.9 ml) and THF (0.9 ml) was added so-
dium borohydride (21 mg, 0.555 mmol) under argon atmosphere,
followed by MeOH (0.2 ml) drop-wise while heating to reflux. The
reaction mixture was heated to reflux for 4 h. After cooling to room
temperature, the reaction mixture was quenched with water. The
water layer was extracted with EtOAc and separated. The organic
layer was washed with brine, dried over MgSO4, filtered, and the
solvent removed under reduced pressure. After adding hexane/
EtOAc (1/1) to the residue, the slurry was filtered. The filter cake
was washed with hexane and dried in a vacuum to afford N-[4-(2-
hydroxy-ethyl)-thiazol-2-yl]-4-[(2-trifluoromethyl-phenylamino)-
methyl]-benzamide (22; 4.1 mg, 4%) as a white solid. MS ESI m/e:
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 81 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 480 (M þ H), 478
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
: 0.86 (t, J ¼ 6.8 Hz, 3H),
d
1.28e1.33 (m, 8H), 1.55e1.59 (m, 2H), 2.51e2.56 (m, 2H), 3.64 (s,
2H), 4.43 (d, J ¼ 5.7 Hz, 2H), 5.79 (t, J ¼ 6.2 Hz, 1H), 6.32 (d,
J ¼ 8.2 Hz,1H), 6.46e6.50 (m, 1H), 6.84e6.87 (m,1H), 6.93e6.95 (m,
1H), 7.01 (s, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 8.03 (d, J ¼ 8.4 Hz, 2H);
Anal. Calcd for C27H33N3O3S: C, 67.61; H, 6.93; N, 8.76. Found: C,
67.72; H, 6.76; N, 8.64.
5.1.27. [2-(4-{[2-(3-Methyl-butyl)-phenylamino]-methyl}-
benzoylamino)-thiazol-4-yl]-acetic acid (41)
422 (M þ H), 420 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.79 (t,
J ¼ 7.1 Hz, 2H), 3.70 (dd, J ¼ 12.3, 7.0 Hz, 2H), 4.54 (d, J ¼ 6.0 Hz, 2H),
4.65 (t, J ¼ 5.3 Hz, 1H), 6.41 (t, J ¼ 6.1 Hz, 1H), 6.59 (d, J ¼ 8.6 Hz, 1H),
6.65 (t, J ¼ 7.5 Hz, 1H), 6.86 (s, 1H), 7.28 (t, J ¼ 7.7 Hz, 1H), 7.42 (d,
J ¼ 7.7 Hz, 1H), 7.46 (d, J ¼ 8.1 Hz, 2H), 8.03 (d, J ¼ 8.1 Hz, 2H), 12.50
(br s, 1H); Anal. Calcd for C20H18F3N3O2S: C, 57.00; H, 4.31; N, 9.97.
Found: C, 57.04; H, 4.40; N, 9.69.
This compound was prepared according to the synthetic pro-
tocols described for compound 17, starting from 82 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 438 (M þ H), 436
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
: 0.96 (d, J ¼ 6.5 Hz, 6H),
d
1.49 (dd, J ¼ 15.2, 7.3 Hz, 2H), 1.58e1.68 (m, 1H), 2.49e2.57 (m, 2H),

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
847
5.1.32. N-[4-(3-hydroxy-propyl)-thiazol-2-yl]-4-[(2-
2H), 12.45 (br s, 1H).
trifluoromethyl-phenylamino)-methyl]-benzamide (23)
Step 2. To a solution of 101 (70 mg, 0.156 mmol) in MeOH
(0.7 ml) was added 2 N NaOH (0.234 ml, 0.467 mmol) at 0 ꢄC and
stirred at room temperature for 30 min 2 N HCl (0.234 ml) was
added portion-wise. After adding water, the reaction mixture was
allowed to stir at room temperature. The slurry was filtered and the
filter cake washed by water and dried in a vacuum to afford (2-{4-
[(2-trifluoromethyl-phenylamino)-methyl]-benzoylamino}-thia-
zol-5-yl)-acetic acid (25; 63 mg, 93%) as a solid. MS ESI m/e: 436
This compound was prepared according to the synthetic pro-
tocols described for compound 22, starting from 91. MS ESI m/e:
436 (M þ H), 434 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d:
1.75e1.82 (m, 2H), 2.66 (t, J ¼ 7.5 Hz, 2H), 3.44 (dd, J ¼ 11.7, 6.4 Hz,
2H), 4.47 (t, J ¼ 5.2 Hz, 1H), 4.54 (d, J ¼ 6.3 Hz, 2H), 6.41 (t, J ¼ 6.0 Hz,
1H), 6.59 (d, J ¼ 8.3 Hz, 1H), 6.65 (t, J ¼ 7.4 Hz, 1H), 6.82 (s, 1H), 7.28
(t, J ¼ 7.2 Hz,1H), 7.42 (d, J ¼ 6.7 Hz, 1H), 7.46 (d, J ¼ 8.3 Hz, 2H), 8.03
(d, J ¼ 8.3 Hz, 2H), 12.50 (br s, 1H); Anal. Calcd for
(M þ H), 434 (M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 3.78 (s, 2H),
C
21H20F3N3O2S$0.25H2O: C, 57.33; H, 4.70; N, 9.55. Found: C, 57.29;
4.54 (d, J ¼ 6.0 Hz, 2H), 6.39 (t, J ¼ 6.0 Hz, 1H), 6.59 (d, J ¼ 8.7 Hz,
1H), 6.65 (t, J ¼ 7.5 Hz, 1H), 7.26 (t, J ¼ 7.9 Hz, 1H), 7.31 (s, 1H), 7.42
(d, J ¼ 7.9 Hz, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.02 (d, J ¼ 8.3 Hz, 2H);
Anal. Calcd for C20H16F3N3O3S: C, 55.17; H, 3.70; N, 9.65. Found: C,
54.86; H, 3.79; N, 9.49.
H, 4.66; N, 9.54.
5.1.33. 4-[(2-Chloro-phenylamino)-methyl]eN-[4-(2-hydroxy-
ethyl)-thiazol-2-yl]-benzamide (34)
This compound was prepared according to the synthetic pro-
tocols described for compound 22, starting from 93. MS ESI m/e:
5.1.37. 2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazole-5-carboxylic acid (24)
388 (M þ H), 386 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 2.79 (t,
J ¼ 7.1 Hz, 2H), 3.68e3.72 (br m, 2H), 4.49 (d, J ¼ 6.2 Hz, 2H), 4.63 (br
s, 1H), 6.24 (t, J ¼ 6.2 Hz, 1H), 6.50e6.57 (m, 2H), 6.84 (s, 1H),
6.99e7.04 (m, 1H), 7.24e7.27 (m, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 8.04
(d, J ¼ 8.2 Hz, 2H), 12.48 (br s, 1H); Anal. Calcd for C19H18ClN3O2S: C,
58.83; H, 4.68; N, 10.83. Found: C, 58.47; H, 4.79; N, 10.45.
This compound was prepared according to the synthetic pro-
tocols described for compound 25, starting from 77 and ethyl 2-
aminothiazole-5-carboxylate. MS ESI m/e: 422 (M þ H), 420
(M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
: 4.52 (d, J ¼ 5.6 Hz, 2H),
d
6.37 (t, J ¼ 7.5 Hz, 1H), 6.58e6.68 (m, 2H), 7.28 (t, J ¼ 12.1 Hz, 2H),
7.42e7.40 (m, 3H), 7.48 (s, 1H), 8.02 (d, J ¼ 8.4 Hz, 2H); Anal. Calcd
for C19H14F3N3O3S$0.5H2O: C, 53.58; H, 3.43; N, 9.87. Found: C,
53.20; H, 3.71; N, 9.55.
5.1.34. 4-[(2-Butyl-phenylamino)-methyl]eN-[4-(2-hydroxy-
ethyl)-thiazol-2-yl]-benzamide (35)
This compound was prepared according to the synthetic pro-
tocols described for compound 22, starting from 94. MS ESI m/e:
5.1.38. 3-(2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazol-5-yl)-propionic acid (26)
410 (M þ H), 408 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 0.94 (t,
J ¼ 7.3 Hz, 3H), 1.35e1.44 (m, 2H), 1.54e1.61 (m, 2H), 2.53e2.57 (m,
2H), 2.79 (t, J ¼ 7.0 Hz, 2H), 3.67e3.72 (m, 2H), 4.43 (d, J ¼ 5.6 Hz,
2H), 4.62e4.65 (m, 1H), 5.80e5.83 (m, 1H), 6.31 (d, J ¼ 7.9 Hz, 1H),
6.48 (t, J ¼ 7.0 Hz, 1H), 6.84e6.88 (m, 2H), 6.93e6.95 (m, 1H), 7.47
(d, J ¼ 8.3 Hz, 2H), 8.03 (d, J ¼ 8.3 Hz, 2H), 12.47 (br s, 1H); Anal.
Calcd for C23H27N3O2S: C, 67.45; H, 6.65; N, 10.26. Found: C, 67.40;
H, 6.50; N, 10.24.
This compound was prepared according to the synthetic pro-
tocols described for compound 25, starting from 77 and methyl 3-
(2-aminothiazol-5-yl)propanoate. MS ESI m/e: 450 (M þ H), 448
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
: 2.57 (t, J ¼ 7.2 Hz, 2H),
d
2.98 (t, J ¼ 7.2 Hz, 2H), 4.54 (d, J ¼ 5.7 Hz, 2H), 6.39 (t, J ¼ 5.8 Hz, 1H),
6.55e6.70 (m, 2H), 7.23e7.32 (m, 2H), 7.38e7.51 (m, 3H), 8.01 (d,
J ¼ 7.9 Hz, 2H), 12.32 (s, 2H); Anal. Calcd for C21H18F3N3O3S: C,
56.12; H, 4.04; N, 9.35. Found: C, 56.19; H, 3.84; N, 9.36.
5.1.35. N-[4-(2-hydroxy-ethyl)-thiazol-2-yl]-4-{[2-(3-methyl-
butyl)-phenylamino]-methyl}-benzamide (36)
5.1.39. N-[5-(2-hydroxy-ethyl)-thiazol-2-yl]-4-[(2-trifluoromethyl-
phenylamino)-methyl]-benzamide (28)
This compound was prepared according to the synthetic pro-
tocols described for compound 22, starting from 95. MS ESI m/e:
To a solution of 77 (80 mg, 0.271 mmol), 2-(2-aminothiazol-5-
yl)ethan-1-ol (43 mg, 0.298 mmol), and DIPEA (0.057 ml,
0.325 mmol) in DMF (0.8 ml) was added PyBOP (155 mg,
0.298 mmol). The reaction mixture was stirred at room tempera-
ture overnight. Water and EtOAc were added to the reaction and
separated. The organic layer was washed with saturated NaHCO3,
followed by brine, and dried over Na2SO4, filtered, and concen-
trated. The residue was purified by preparative TLC (CHCl3/
MeOH ¼ 10/1) to afford N-[5-(2-hydroxy-ethyl)-thiazol-2-yl]-4-
424 (M þ H), 422 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d: 0.96 (d,
J ¼ 6.7 Hz, 6H), 1.49 (dd, J ¼ 15.5, 7.0 Hz, 2H), 1.60e1.67 (m, 1H), 2.54
(t, J ¼ 8.2 Hz, 2H), 2.79 (t, J ¼ 7.0 Hz, 2H), 3.70 (q, J ¼ 6.5 Hz, 2H), 4.43
(d, J ¼ 5.3 Hz, 2H), 4.64 (t, J ¼ 5.3 Hz,1H), 5.77 (t, J ¼ 5.9 Hz, 1H), 6.32
(d, J ¼ 7.9 Hz, 1H), 6.49 (t, J ¼ 7.3 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 2H), 6.95
(d, J ¼ 7.2 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz, 2H), 8.03 (d, J ¼ 8.1 Hz, 2H),
12.48 (br s, 1H); Anal. Calcd for C24H29N3O2S$0.25H2O: C, 67.34; H,
6.95; N, 9.82. Found: C, 67.35; H, 6.83; N, 9.71.
[(2-trifluoromethyl-phenylamino)-methyl]-benzamide
(28;
5.1.36. (2-{4-[(2-Trifluoromethyl-phenylamino)-methyl]-
benzoylamino}-thiazol-5-yl)-acetic acid (25)
105 mg, 46%) as a solid. MS ESI m/e: 422 (M þ H), 420 (M ꢀ H); 1H
NMR (DMSO‑d6, 300 MHz)
d
: 2.88 (t, J ¼ 6.4 Hz, 2H), 3.60 (q,
Step 1. To a solution of 77 (80 mg, 0.271 mmol), methyl 2-(2-
aminothiazol-5-yl)acetate hydrobromide (82 mg, 0.324 mmol)
and DIPEA (0.123 ml, 0.706 mmol) in DMF (0.8 ml) was added
PyBOP (169 mg, 0.325 mmol). The reaction mixture was stirred at
65 ꢄC for 2 h. After cooling to room temperature, water and EtOAc
were added and separated. The organic layer was washed with
brine, dried over MgSO4, filtered, and concentrated. The residue
was purified by preparative TLC (CHCl3/MeOH ¼ 12/1) to afford
methyl 2-(2-(4-(((2-(trifluoromethyl)phenyl)amino)methyl)ben-
zamido)thiazol-5-yl)acetate (101; 98 mg, 80%) as a solid. 1H NMR
J ¼ 6.0 Hz, 2H), 4.54 (d, J ¼ 6.0 Hz, 2H), 4.85 (t, J ¼ 5.3 Hz,1H), 6.39 (t,
J ¼ 6.0 Hz, 1H), 6.55e6.69 (m, 2H), 7.23e7.31 (m, 2H), 7.38e7.50 (m,
3H), 8.02 (d, J ¼ 8.3 Hz, 2H), 12.33 (s, 1H); Anal. Calcd for
C20H18F3N3O2S: C, 57.00; H, 4.31; N, 9.97. Found: C, 56.72; H, 4.22;
N, 9.75.
5.1.40. N-(5-hydroxymethyl-thiazol-2-yl)-4-[(2-trifluoromethyl-
phenylamino)-methyl]-benzamide (27)
This compound was prepared according to the synthetic pro-
tocols described for compound 28, starting from 77 and (2-
aminothiazol-5-yl)methanol. MS ESI m/e: 408 (M þ H), 406
(DMSO‑d6, 300 MHz)
d
: 3.66 (s, 3H), 3.93 (s, 2H), 4.54 (d, J ¼ 6.0 Hz,
2H), 6.40 (t, J ¼ 5.8 Hz, 1H), 6.58e6.67 (m, 2H), 7.27 (t, J ¼ 7.5 Hz,
(M ꢀ H). 1H NMR (DMSO‑d6, 400 MHz)
: 4.54 (d, J ¼ 6.0 Hz, 2H),
d
1H), 7.35 (s, 1H), 7.45 (dd, J ¼ 14.7, 7.9 Hz, 3H), 8.02 (d, J ¼ 7.9 Hz,
4.61 (d, J ¼ 5.6 Hz, 2H), 5.39 (t, J ¼ 5.7 Hz, 1H), 6.41 (t, J ¼ 6.0 Hz, 1H),

848
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
6.58 (d, J ¼ 8.3 Hz, 1H), 6.65 (t, J ¼ 7.5 Hz, 1H), 7.27 (t, J ¼ 7.9 Hz, 1H),
7.36 (s, 1H), 7.42 (d, J ¼ 6.7 Hz, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 8.03 (d,
J ¼ 8.3 Hz, 2H), 12.41 (br s, 1H); Anal. Calcd for C19H16F3N3O2S: C,
56.01; H, 3.96; N, 10.31. Found: C, 56.01; H, 4.03; N, 10.13.
After adding EtOAc and hexane, the slurry was filtered and the filter
cake washed with EtOAc and hexane before drying in a vacuum to
afford N-[5-(2-hydroxy-ethyl)-4-(3-hydroxy-propyl)-thiazol-2-yl]-
4-[(2-trifluoromethyl-phenylamino)-methyl]-benzamide
26 mg, 25%) as a white solid. MS ESI m/e: 480 (M þ H), 478 (M ꢀ H);
1H NMR (DMSO‑d6, 400 MHz)
: 1.69e1.76 (m, 2H), 2.59 (t,
(31;
5.1.41. N-[5-(3-hydroxy-propyl)-thiazol-2-yl]-4-[(2-
d
trifluoromethyl-phenylamino)-methyl]-benzamide (29)
J ¼ 7.4 Hz, 2H), 2.81 (t, J ¼ 6.6 Hz, 2H), 3.37e3.42 (m, 2H), 3.55 (q,
J ¼ 6.3 Hz, 2H), 4.44 (t, J ¼ 5.2 Hz,1H), 4.53 (d, J ¼ 5.8 Hz, 2H), 4.83 (t,
J ¼ 5.3 Hz, 1H), 6.39 (t, J ¼ 5.9 Hz, 1H), 6.59 (d, J ¼ 8.6 Hz, 1H), 6.65 (t,
J ¼ 7.5 Hz, 1H), 7.27 (t, J ¼ 7.8 Hz, 1H), 7.41e7.46 (m, 3H), 8.02 (d,
J ¼ 8.1 Hz, 2H), 12.31 (br s, 1H); Anal. Calcd for C23H24F3N3O3S$H2O:
C, 55.52; H, 5.27; N, 8.45. Found: C, 55.76; H, 5.03; N, 8.13.
This compound was prepared according to the synthetic pro-
tocols described for compound 28, starting from 77 and 3-(2-
aminothiazol-5-yl)propan-1-ol. MS ESI m/e: 436 (M þ H), 434
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 1.68e1.81 (m, 2H), 2.79 (t,
J ¼ 7.5 Hz, 2H), 3.45 (q, J ¼ 5.9 Hz, 2H), 4.48e4.58 (m, 3H), 6.39 (t,
J ¼ 6.2 Hz, 1H), 6.55e6.69 (m, 2H), 7.23 (s, 1H), 7.27 (t, J ¼ 7.7 Hz,
1H), 7.42 (d, J ¼ 7.9 Hz, 1H), 7.46 (d, J ¼ 8.3 Hz, 2H), 8.01 (d,
5.1.43. N-(4-methylthiazol-2-yl)-5-(((2-(trifluoromethyl)phenyl)
amino)methyl)thiophene-2-carboxamide (15)
J ¼ 7.9 Hz,
2H),
12.33
(s,
1H);
Anal.
Calcd
for
C
21H20F3N3O2S$0.25H2O: C, 57.33; H, 4.70; N, 9.55. Found: C, 57.14;
Step 1. A solution of commercially available 5-formylthiophene-
2-carboxylic acid (108; 1 g, 6.40 mmol) and 2-(trifluoromethyl)an-
iline (0.8 ml, 6.40 mmol) in AcOH (5 ml) and DME (5 ml) was stirred
at room temperature for 1 h under argon atmosphere. The reaction
mixture was cooled to 0 ꢄC before adding sodium borohydride
(315 mg, 8.32 mmol) portion-wise over 10 min. The reaction
mixture was then allowed to stir at room temperature overnight.
After cooling to 0 ꢄC, water was added drop-wise over 10 min and
the reaction mixture allowed to stir at room temperature for
30 min. The slurry was filtered and the filter cake washed with
water and dried in a vacuum to afford 5-(((2-(trifluoromethyl)
phenyl)amino)methyl)thiophene-2-carboxylic acid (115; 1.58 g,
H, 4.70; N, 9.41.
5.1.42. N-[5-(2-hydroxy-ethyl)-4-(3-hydroxy-propyl)-thiazol-2-yl]-
4-[(2-trifluoromethyl-phenylamino)-methyl]-benzamide (31)
Step 1. A solution of 104 (5 ml, 23.5 mmol) in CHCl3 (32 ml) was
cooled to 0 ꢄC. A solution of Br2 (1.2 ml, 23.5 mmol) in CHCl3 (5 ml)
was added drop-wise over 2 min and the reaction mixture allowed
to stir at room temperature for 30 min CHCl3 and 10% Na2S2O3 were
added and separated. The organic layer was washed with 10%
Na2S2O3, followed by brine, and dried over MgSO4, filtered, and
concentrated to afford crude diethyl 3-bromo-4-oxoheptanedioate
(105) as a pale yellow oil, which was used directly in the next step
without further purification.
82%) as a solid. 1H NMR (DMSO‑d6, 400 MHz)
d
: 4.66 (d, J ¼ 5.8 Hz,
2H), 6.39 (t, J ¼ 6.0 Hz, 1H), 6.70 (t, J ¼ 7.5 Hz, 1H), 6.77 (d, J ¼ 8.6 Hz,
1H), 7.09 (d, J ¼ 3.7 Hz, 1H), 7.34 (t, J ¼ 7.6 Hz, 1H), 7.43 (dd, J ¼ 7.9,
1.2 Hz, 1H), 7.57 (d, J ¼ 3.7 Hz, 1H), 12.89 (br s, 1H).
Step 2. To a solution of 105 (23.5 mmol theoretical yield) in EtOH
(70 ml) was added thiourea (1.97 g, 25.9 mmol) and the reaction
mixture heated to 100 ꢄC for 30 min under argon atmosphere. After
cooling to room temperature, saturated NaHCO3 and EtOAc were
added and separated. The combined organic layer was washed with
brine, dried over Na2SO4, filtered, and the solvent removed under
reduced pressure. The residue was purified by flash chromatog-
raphy (hexane/EtOAc ¼ 5/3) to afford ethyl 3-(2-amino-5-(2-
ethoxy-2-oxoethyl)thiazol-4-yl)propanoate (106; 4.69 g, 70%) as a
Step 2. To a solution of 115 (50 mg, 0.166 mmol) and 2-amino-4-
methylthiazole (65; 19 mg, 0.166 mmol) in pyridine (1 ml) was
added EDC$HCl (38 mg, 0.200 mmol) at room temperature. After
stirring at room temperature overnight, water was poured into the
reaction and the mixture extracted with EtOAc. The organic layer
was washed with brine and dried over anhydrous MgSO4. After
filtration and concentration, the residue was purified by prepara-
tive TLC (hexane/EtOAc ¼ 1/1) to afford the title compound N-(4-
methylthiazol-2-yl)-5-(((2-(trifluoromethyl)phenyl)amino)
methyl)thiophene-2-carboxamide (15; 59 mg, 90% yield). MS ESI
pale yellow oil. 1H NMR (CDCl3, 400 MHz)
d: 1.22e1.29 (m, 6H), 2.63
(t, J ¼ 6.1 Hz, 2H), 2.77 (t, J ¼ 7.3 Hz, 2H), 3.63 (s, 2H), 4.08e4.19 (m,
4H), 4.85 (s, 2H).
Step 3. To a solution of 106 (516 mg, 1.8 mmol), 4-(((2-(tri-
fluoromethyl)phenyl)amino)methyl)benzoic acid (77; 532 mg,
1.8 mmol), and DIPEA (0.32 ml, 1.8 mmol) in DMF (5 ml) was added
PyBOP (937 mg, 1.8 mmol). The reaction mixture was heated to
65 ꢄC overnight. After cooling to room temperature, water and
EtOAc were added and separated. The organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated. After
adding EtOAc and IPE, the slurry was filtered and the filter cake
washed with IPE and dried in a vacuum to afford ethyl 3-(5-(2-
ethoxy-2-oxoethyl)-2-(4-(((2-(trifluoromethyl)phenyl)amino)
methyl)benzamido)thiazol-4-yl)propanoate (107; 897 mg, 88%) as
m/e: 398 (M þ H), 396 (M ꢀ H); 1H NMR (DMSO‑d6, 400 MHz)
d:
2.28 (s, 3H), 4.67 (d, J ¼ 6.0 Hz, 2H), 6.42 (t, J ¼ 6.0 Hz, 1H), 6.70 (t,
J ¼ 7.5 Hz, 1H), 6.78 (d, J ¼ 8.3 Hz, 2H), 7.13 (d, J ¼ 3.7 Hz, 1H), 7.34 (t,
J ¼ 7.7 Hz,1H), 7.43 (t, J ¼ 3.9 Hz, 1H), 8.04 (br s, 1H),12.54 (br s, 1H);
Anal. Calcd for C17H14F3N3OS2: C, 51.38; H, 3.55; N, 10.57. Found: C,
51.27; H, 3.64; N, 10.24.
5.1.44. Sodium 2-(2-(5-(((2-butylphenyl)amino)methyl)thiophene-
2-carboxamido)thiazol-4-yl)acetate (48)
Step 1. A solution of 2-butylbenzenamine (101 ml, 640.4 mmol)
in toluene (100 ml) was added drop-wise to a mixture of 5-
formylthiophene-2-carboxylic acid (108; 100 g, 640.4 mmol) in
toluene (600 ml) and the resulting mixture heated to reflux for 3 h.
After cooling to 90 ꢄC, n-heptane (500 ml) was added and the
resulting solution seeded with small crystals of the desired product
at 68 ꢄC. After cooling to room temperature, the precipitated solid
was filtered and washed with n-heptane (1 L). The filter cake was
dried in a vacuum to afford (E)-5-(((2-butylphenyl)imino)methyl)
thiophene-2-carboxylic acid (110; 171.5 g, 93%) as a pale yellow
a white solid. 1H NMR (CDCl3, 400 MHz)
d: 1.21e1.30 (m, 6H), 2.68
(t, J ¼ 7.4 Hz, 2H), 2.91 (t, J ¼ 7.6 Hz, 2H), 3.76 (s, 2H), 4.12 (q,
J ¼ 7.1 Hz, 2H), 4.19 (q, J ¼ 7.1 Hz, 2H), 4.54 (d, J ¼ 5.5 Hz, 2H), 4.92
(br s, 1H), 6.59 (d, J ¼ 8.4 Hz, 1H), 6.75 (t, J ¼ 7.6 Hz, 1H), 7.26e7.31
(m, 1H), 7.47e7.51 (m, 3H), 7.93 (t, J ¼ 4.2 Hz, 2H).
Step 4. To a solution of 107 (123 mg, 0.219 mmol) in THF (1.2 ml)
was added LiBH4 (16 mg, 0.735 mmol). The reaction mixture was
stirred at room temperature for 4 h under argon atmosphere.
Additional LiBH4 (17 mg, 0.781 mmol) was added and heated to
65 ꢄC for 1 h. After cooling to room temperature, dilute HCl was
added and the reaction stirred at room temperature overnight.
EtOAc and water were added and separated. The organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated.
solid. 1H NMR (CDCl3, 400 MHz)
d
: 0.93 (t, J ¼ 7.3 Hz, 3H), 1.33e1.42
(m, 2H), 1.58 (tt, J ¼ 8.6, 3.6 Hz, 2H), 2.80 (t, J ¼ 7.8 Hz, 2H),
6.99e7.02 (m,1H), 7.18e7.27 (m, 3H), 7.47 (d, J ¼ 3.9 Hz,1H), 7.90 (d,
J ¼ 3.9 Hz, 1H), 8.53 (s, 1H).
Step 2. A solution of 110 (171.3 g, 596.1 mmol) in DME (580 ml)

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
849
was cooled to 0 ꢄC, and sodium borohydride (16.9 g, 447.1 mmol)
was added portion-wise over 30 min with DME (105 ml). The re-
action mixture was allowed to stir at room temperature for 2 h.
After cooling to 0 ꢄC, AcOH (136.5 ml, 2.384 mol) was added drop-
wise over 20 min, and the reaction mixture was allowed to stir
for 40 min. Subsequently, water (260 ml) was added drop-wise over
15 min and the reaction mixture allowed to stir at room tempera-
ture for 15 min. Next, MeOH (377 ml) was added drop-wise over
10 min and the reaction mixture allowed to stir at room tempera-
ture for 5 min, followed by the drop-wise addition of water
(500 ml) over 20 min. The resulting solution was seeded with small
crystals of the desired product at room temperature, and the
mixture was allowed to stir at room temperature for 20 min.
Additional water (350 ml) was added drop-wise over 45 min, and
the mixture was allowed to stir at room temperature for 30 min.
After 690 ml of water was added drop-wise over 60 min, the
mixture was allowed to stir at room temperature overnight. The
precipitated solid was filtered and washed with water (1.8 L). The
filter cake was dried in a vacuum to give 5-(((2-butylphenyl)amino)
methyl)thiophene-2-carboxylic acid (116; 166.1 g, 96%) as a yellow
2H), 3.62 (s, 2H), 4.55 (d, J ¼ 5.7 Hz, 2H), 5.86 (t, J ¼ 6.1 Hz, 1H),
6.48e6.55 (m, 2H), 6.90e6.98 (m, 3H), 7.12 (d, J ¼ 4.0 Hz, 1H), 8.07
(d, J ¼ 4.0 Hz, 1H), 12.54 (br s, 1H).
Step 5. A solution of 4 N NaOH (6.1 ml, 24.50 mmol) was added
drop-wise to a stirred suspension of 130 (10 g, 23.30 mmol) in EtOH
(350 ml) and water (150 ml) over 30 min while heating to 80 ꢄC,
and the reaction mixture was allowed to stir at room temperature
overnight. The slurry was filtered and the filter cake washed with
EtOH (1 L) and dried in a vacuum to afford sodium 2-(2-(5-(((2-
butylphenyl)amino)methyl)thiophene-2-carboxamido)thiazol-4-
yl)acetate (48; 8.18 g, 78%) as a white solid. MS ESI m/e: 452
(M þ H), 450 (M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 0.93 (t,
J ¼ 7.3 Hz, 3H), 1.30e1.45 (m, 2H), 1.48e1.61 (m, 2H), 2.47e2.55 (m,
2H), 3.36 (s, 2H), 4.53 (d, J ¼ 5.7 Hz, 2H), 5.83 (t, J ¼ 5.7 Hz, 1H),
6.46e6.56 (m, 2H), 6.72 (s, 1H), 6.87e6.98 (m, 2H), 7.03 (d,
J ¼ 3.8 Hz, 1H), 8.59 (s, 1H); Anal. Calcd for C21H22N3O3S2$Na: C,
55.86; H, 4.91; N, 9.31. Found: C, 55.79; H, 4.83; N, 9.24; Purity by
HPLC 99.9%; mp 243e249 ꢄC.
5.1.45. [2-({5-[(2-Trifluoromethyl-phenylamino)-methyl]-
solid. 1H NMR (DMSO‑d6, 400 MHz)
d
: 0.92 (t, J ¼ 7.3 Hz, 3H),
thiophene-2-carbonyl}-amino)-thiazol-4-yl]-acetic acid (47)
This compound was prepared according to the synthetic pro-
tocols described for compound 48, starting from 115 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 442 (M þ H), 440
1.32e1.41 (m, 2H), 1.50e1.58 (m, 2H), 2.48e2.52 (m, 2H), 4.54 (s,
2H), 5.83 (br s, 1H), 6.47e6.54 (m, 2H), 6.90e6.95 (m, 2H), 7.08 (d,
J ¼ 3.7 Hz, 1H), 7.57 (d, J ¼ 3.5 Hz, 1H), 12.83 (br s, 1H).
Step 3. To a solution of ethyl (2-amino-4-thiazolyl)acetate
(106.2 g, 570.1 mmol) and 116 (150.0 g, 518.3 mmol) in CHCl3
(1500 ml) was added EDC$HCl (148.6 g, 777.5 mmol), followed by
DMAP (31.7 g, 259.2 mmol) with CHCl3 (250 ml) at 0 ꢄC. The reac-
tion mixture was stirred for 30 min under N2 atmosphere at 0 ꢄC,
and then at room temperature for 4 h. The solvent was removed
under reduced pressure. To the resulting residue was added 750 ml
of EtOAc and the solvent removed under reduced pressure. EtOAc
(750 ml) and additional water (300 ml) were then added and the
solvent removed under reduced pressure. EtOAc (900 ml), water
(300 ml), and 5% KHSO4 (600 ml) were added to the resultant res-
idue and the layers separated. After additional n-hexane (600 ml)
was added, the organic portion was washed with 5% KHSO4, brine,
5% NaHCO3, and brine, and dried over MgSO4. Silica gel (45 g) and
activated charcoal (4 g) were added and the mixture stirred at room
temperature for 1 h. The mixture was filtered through a celite pad
and the filtrate concentrated to about half its initial volume. After
adding EtOAc (59 ml) and EtOH (75 ml), the mixture was warmed to
80 ꢄC and IPA (2100 ml) added carefully. The resulting solution was
cooled to 45 ꢄC and small crystals of the desired product seeded
before stirring at room temperature overnight. The precipitated
solid was filtered and washed with IPE (300 ml), followed by IPE/
EtOH (50/1; 765 ml). The filter cake was dried in a vacuum to afford
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 3.62 (s, 2H), 4.67 (d,
J ¼ 6.0 Hz, 2H), 6.40 (t, J ¼ 6.0 Hz, 1H), 6.70 (t, J ¼ 7.3 Hz, 1H), 6.78 (d,
J ¼ 8.7 Hz, 1H), 6.98 (s, 1H), 7.13 (d, J ¼ 3.8 Hz, 1H), 7.34 (t, J ¼ 7.7 Hz,
1H), 7.43 (d, J ¼ 7.9 Hz, 1H), 8.06 (d, J ¼ 4.1 Hz, 1H), 12.48 (br s, 2H);
Anal. Calcd for C18H14F3N3O3S2: C, 48.97; H, 3.20; N, 9.52. Found: C,
49.23; H, 3.49; N, 9.45.
5.1.46. Sodium [2-({5-[(2-hexyl-phenylamino)-methyl]-thiophene-
2-carbonyl}-amino)-thiazol-4-yl]-acetate (49)
This compound was prepared according to the synthetic pro-
tocols described for compound 48, starting from 117 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 480 (M þ H), 456
(M ꢀ Na); 1H NMR (DMSO‑d6, 300 MHz)
: 0.88 (t, J ¼ 7.0 Hz, 3H),
d
1.23e1.42 (m, 6H), 1.49e1.62 (m, 2H), 2.46e2.54 (m, 2H), 3.34 (s,
2H), 4.53 (d, J ¼ 5.7 Hz, 2H), 5.80 (t, J ¼ 5.7 Hz, 1H), 6.47e6.56 (m,
2H), 6.68 (s, 1H), 6.88e6.97 (m, 2H), 7.02 (d, J ¼ 3.8 Hz, 1H), 8.41 (s,
1H); Anal. Calcd for C23H26N3O3S2$Na: C, 57.60; H, 5.46; N, 8.76.
Found: C, 57.42; H, 5.42; N, 8.68; Purity by HPLC 99.6%; mp
236e242 ꢄC.
5.1.47. Sodium [2-({5-[(2-octyl-phenylamino)-methyl]-thiophene-
2-carbonyl}-amino)-thiazol-4-yl]-acetate (50)
This compound was prepared according to the synthetic pro-
tocols described for compound 48, starting from 118 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 508 (M þ H), 506
ethyl
carboxamido)thiazol-4-yl)acetate (125; 182.1 g, 77%) as a pale yel-
low solid. 1H NMR (DMSO‑d6, 400 MHz)
: 0.93 (t, J ¼ 7.3 Hz, 3H),
2-(2-(5-(((2-butylphenyl)amino)methyl)thiophene-2-
d
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
: 0.85 (t, J ¼ 7.0 Hz, 3H),
d
1.18 (t, J ¼ 7.1 Hz, 3H), 1.33e1.42 (m, 2H), 1.51e1.59 (m, 2H),
2.49e2.53 (m, 2H), 3.71 (s, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 4.55 (d,
J ¼ 6.0 Hz, 2H), 5.86 (t, J ¼ 5.8 Hz, 1H), 6.48e6.55 (m, 2H), 6.90e6.96
(m, 2H), 7.01 (s,1H), 7.12 (d, J ¼ 3.5 Hz,1H), 8.07 (s,1H),12.62 (s,1H).
Step 4. To a solution of 125 (35 g, 76.5 mmol) in THF (175 ml) and
MeOH (350 ml) was added 2 N NaOH (115 ml, 229 mmol) at 0 ꢄC.
The reaction mixture was stirred at room temperature for 3 h. After
cooling to 0 ꢄC, water (700 ml), followed by 2 N HCl (115 ml), was
added portion-wise and the reaction mixture allowed to stir at
room temperature. After adding water (700 ml), the slurry was
filtered and the filter cake washed with water (4 ꢃ 300 ml) and
1.26e1.36 (m, 10H), 1.51e1.59 (m, 2H), 2.47e2.49 (m, 2H), 3.32 (s,
2H), 4.52 (d, J ¼ 5.6 Hz, 2H), 5.79 (t, J ¼ 6.1 Hz, 1H), 6.50e6.53 (m,
2H), 6.65 (s, 1H), 6.90e6.94 (m, 2H), 7.01 (d, J ¼ 3.7 Hz, 1H), 8.24 (br
s, 1H); Anal. Calcd for C25H30N3O3S2$Na: C, 59.15; H, 5.96; N, 8.28.
Found: C, 59.09; H, 5.88; N, 8.20; Purity by HPLC 99.8%; mp
226e230 ꢄC.
5.1.48. {2-[(5-{[2-(3-Methyl-butyl)-phenylamino]-methyl}-
thiophene-2-carbonyl)-amino]-thiazol-4-yl}-acetic acid (51)
This compound was prepared according to the synthetic pro-
tocols described for compound 48, starting from 119 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 444 (M þ H), 442
dried in
methyl)thiophene-2-carboxamido)thiazol-4-yl)acetic acid (130;
32.3 g, 98%) as a white solid. 1H NMR (DMSO‑d6, 400 MHz)
: 0.93 (t,
J ¼ 7.4 Hz, 3H), 1.33e1.42 (m, 2H), 1.51e1.59 (m, 2H), 2.48e2.54 (m,
a vacuum to afford 2-(2-(5-(((2-butylphenyl)amino)
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
: 0.95 (d, J ¼ 6.4 Hz, 6H),
d
d
1.40e1.52 (m, 2H), 1.54e1.69 (m, 1H), 2.47e2.55 (m, 2H), 3.62 (s,
2H), 4.56 (d, J ¼ 5.7 Hz, 2H), 5.81 (t, J ¼ 5.7 Hz, 1H), 6.46e6.57 (m,

850
T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
2H), 6.88e7.00 (m, 3H), 7.13 (d, J ¼ 3.8 Hz, 1H), 8.07 (d, J ¼ 3.8 Hz,
1H), 12.49 (br s, 2H); Anal. Calcd for C22H25N3O3S2: C, 59.57; H,
5.68; N, 9.47. Found: C, 59.61; H, 5.77; N, 9.51; mp 218e223 ꢄC.
and concentrated. The residue was purified by preparative TLC
(hexane/EtOAc ¼ 3/1) to afford N-(4-(2-((tert-butyldimethylsilyl)
oxy)ethyl)thiazol-2-yl)-5-(((2-(trifluoromethyl)phenyl)amino)
methyl)thiophene-2-carboxamide (122; 110 mg, 87%) as a solid. 1H
5.1.49. [2-({5-[(2-Benzyl-phenylamino)-methyl]-thiophene-2-
carbonyl}-amino)-thiazol-4-yl]-acetic acid (52)
NMR (CDCl3, 300 MHz) d: 0.00 (s, 6H), 0.86 (s, 9H), 2.84 (t,
J ¼ 6.4 Hz, 2H), 3.88 (t, J ¼ 6.6 Hz, 2H), 4.65 (d, J ¼ 5.7 Hz, 2H), 4.94
(br s, 1H), 6.64 (s, 1H), 6.72 (d, J ¼ 8.3 Hz, 1H), 6.79 (t, J ¼ 7.5 Hz, 1H),
7.05 (d, J ¼ 3.8 Hz, 1H), 7.34 (t, J ¼ 7.7 Hz, 1H), 7.48 (d, J ¼ 7.5 Hz, 1H),
7.56 (d, J ¼ 3.8 Hz, 1H).
This compound was prepared according to the synthetic pro-
tocols described for compound 48, starting from 120 and (2-amino-
4-thiazolyl)acetic acid ethyl ester. MS ESI m/e: 464 (M þ H), 462
(M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d
: 3.62 (s, 2H), 3.89 (s, 2H),
Step 2. To a solution of 122 (110 mg, 0.203 mmol) in THF
(0.55 ml) was added 1 M THF solution of TBAF (0.609 ml,
0.609 mmol) at room temperature. The reaction mixture was stir-
red at 50 ꢄC for 1 h under N2 atmosphere. EtOAc and saturated
NaHCO3 were added to the reaction mixture and the layers sepa-
rated. The organic layer was washed with brine and dried over
Na2SO4. The mixture was filtered and concentrated. The residue
was purified by preparative TLC (CHCl3/MeOH ¼ 9/1). After adding
hexane and EtOAc to the resulting residue, the slurry was filtered.
The filter cake was washed with hexane and dried in a vacuum to
afford 5-[(2-trifluoromethyl-phenylamino)-methyl]-thiophene-2-
carboxylic acid [4-(2-hydroxy-ethyl)-thiazol-2-yl]-amide (45;
82 mg, 94%) as a solid. MS ESI m/e: 428 (M þ H), 426 (M ꢀ H); 1H
4.55 (d, J ¼ 5.7 Hz, 2H), 5.90 (t, J ¼ 5.8 Hz, 1H), 6.51e6.59 (m, 2H),
6.88 (d, J ¼ 6.4 Hz, 1H), 6.93e7.01 (m, 2H), 7.05 (d, J ¼ 3.8 Hz, 1H),
7.16e7.33 (m, 5H), 8.05 (d, J ¼ 3.8 Hz, 1H), 12.51 (br s, 2H); Anal.
Calcd for C24H21N3O3S2: C, 62.18; H, 4.57; N, 9.06. Found: C, 62.23;
H, 4.39; N, 9.28; mp 188e191 ꢄC.
5.1.50. 5-[(2-Trifluoromethyl-phenylamino)-methyl]-thiophene-2-
carboxylic acid (4-hydroxymethyl-thiazol-2-yl)-amide (44)
Step 1. To a solution of 5-(((2-(trifluoromethyl)phenyl)amino)
methyl)thiophene-2-carboxylic acid (115; 70 mg, 0.232 mmol) and
(2-amino-1,3-thiazol-4-yl)methyl acetate hydrochloride (58 mg,
0.280 mmol) in CHCl3 (1.4 ml) was added EDC$HCl (53 mg,
0.280 mmol), followed by DMAP (65 mg, 0.530 mmol) at room
temperature. The reaction mixture was stirred for 3 h under N2
atmosphere. EtOAc and saturated NaHCO3 were added to the re-
action mixture and the layers separated. The organic layer was
washed with brine and dried over Na2SO4. The mixture was filtered
and concentrated. The residue was purified by preparative TLC
(hexane/EtOAc ¼ 1/1) to afford (2-(5-(((2-(trifluoromethyl)phenyl)
amino)methyl)thiophene-2-carboxamido)thiazol-4-yl)methyl ace-
NMR (DMSO‑d6, 300 MHz)
d
: 2.77 (t, J ¼ 6.8 Hz, 2H), 3.62e3.75 (m,
2H), 4.56e4.71 (m, 3H), 6.40 (t, J ¼ 5.7 Hz, 1H), 6.70 (t, J ¼ 7.5 Hz,
1H), 6.74e6.85 (m, 2H), 7.12 (d, J ¼ 3.8 Hz, 1H), 7.34 (t, J ¼ 7.7 Hz,
1H), 7.43 (d, J ¼ 7.5 Hz, 1H), 8.04 (s, 1H), 12.55 (s, 1H); Anal. Calcd for
C
18H16F3N3O2S2: C, 50.58; H, 3.77; N, 9.83. Found: C, 50.55; H, 3.50;
N, 9.77; mp 182e184 ꢄC.
5.1.52. 5-[(2-Hexyl-phenylamino)-methyl]-thiophene-2-carboxylic
acid [4-(2-hydroxy-ethyl)-thiazol-2-yl]-amide (46)
tate (121; 90 mg, 85%) as a solid. 1H NMR (CDCl3, 300 MHz)
d: 2.11
(s, 3H), 4.66 (d, J ¼ 6.0 Hz, 2H), 4.95 (br s, 1H), 5.08 (s, 2H), 6.71 (d,
J ¼ 8.3 Hz, 1H), 6.80 (t, J ¼ 7.7 Hz, 1H), 6.95 (s, 1H), 7.06 (d, J ¼ 3.8 Hz,
1H), 7.34 (t, J ¼ 7.7 Hz, 1H), 7.49 (d, J ¼ 7.5 Hz, 1H), 7.57 (d, J ¼ 4.1 Hz,
1H), 9.47 (br s, 1H).
Step 2. To a solution of 121 (90 mg, 0.198 mmol) in THF (0.45 ml)
and MeOH (0.9 ml) was added K2CO3 (55 mg, 0.396 mmol) at room
temperature. The reaction mixture was stirred for 1 h under N2
atmosphere. EtOAc and saturated NaHCO3 were added to the re-
action mixture and the layers separated. The organic layer was
washed with brine and dried over Na2SO4. The mixture was filtered
and concentrated. After adding hexane and EtOAc to the resulting
residue, the slurry was filtered. The filter cake was washed with
hexane and dried in a vacuum to afford 5-[(2-trifluoromethyl-
This compound was prepared according to the synthetic pro-
tocols described for compound 45, starting from 117 and 4-(2-
((tert-butyldimethylsilyl)oxy)ethyl)thiazol-2-amine. MS ESI m/e:
444 (M þ H), 442 (M ꢀ H); 1H NMR (DMSO‑d6, 300 MHz)
d: 0.88 (t,
J ¼ 5.7 Hz, 3H), 1.20e1.43 (m, 6H), 1.48e1.62 (m, 2H), 2.46e2.54 (m,
2H), 2.77 (t, J ¼ 6.8 Hz, 2H), 3.69 (dd, J ¼ 11.7, 6.8 Hz, 2H), 4.55 (d,
J ¼ 5.7 Hz, 2H), 4.62 (t, J ¼ 4.9 Hz, 1H), 5.85 (t, J ¼ 5.8 Hz, 1H),
6.46e6.57 (m, 2H), 6.82 (s, 1H), 6.87e6.98 (m, 2H), 7.11 (d,
J ¼ 3.8 Hz, 1H), 8.05 (s, 1H), 12.53 (s, 1H); Anal. Calcd for
C
23H29N3O2S2$0.25H2O: C, 61.65; H, 6.64; N, 9.38. Found: C, 61.80;
H, 6.37; N, 9.29; mp 108e111 ꢄC.
5.2. Inhibitory effects on SCD1 enzyme activity
phenylamino)-methyl]-thiophene-2-carboxylic
acid
(4-
hydroxymethyl-thiazol-2-yl)-amide (44; 70 mg, 86%) as a solid.
SCD1 enzyme activity was evaluated by measuring the pro-
duction of [3H] H2O from [9, 10-3H] stearoyl-CoA catalyzed by SCD1
enzyme. The microsomal fraction (recombinant human SCD;
rhSCD1) of cells over-expressing human SCD1 via a baculovirus
expression system was used as an enzyme source. The rhSCD1
MS ESI m/e: 414 (M þ H), 412 (M ꢀ H); 1H NMR (DMSO‑d6,
300 MHz)
d
: 4.49 (d, J ¼ 5.7 Hz, 2H), 4.67 (d, J ¼ 6.0 Hz, 2H), 5.20 (t,
J ¼ 5.7 Hz, 1H), 6.41 (t, J ¼ 6.2 Hz, 1H), 6.70 (t, J ¼ 7.5 Hz, 1H), 6.78 (d,
J ¼ 8.3 Hz, 1H), 6.94 (s, 1H), 7.13 (d, J ¼ 3.8 Hz, 1H), 7.34 (t, J ¼ 7.5 Hz,
1H), 7.43 (d, J ¼ 7.5 Hz, 1H), 8.05 (d, J ¼ 3.0 Hz, 1H), 12.59 (s, 1H);
Anal. Calcd for C17H14F3N3O2S2: C, 49.39; H, 3.41; N, 10.16. Found: C,
49.40; H, 3.08; N, 10.17; mp 183e190 ꢄC.
(0.5
and the compound solution dissolved in DMSO were added to 96-
well plates with 80 l of the reaction buffer containing 100 mmol/L
Tris-HCl (pH 7.4), 2 mmol/L NADH, 6 mmol/L MgCl2, 125 mmol/L
sucrose, 0.005% (w/v) BSA, and 0.25 g cytochrome b5. Thirty mi-
nutes after incubation at room temperature, the reaction was
terminated by adding 100 of stopping buffer containing
mg), 0.12 m mCi, Perkin Elmer),
mol/L [9, 10-3H] stearoyl-CoA (1.4
m
5.1.51. 5-[(2-Trifluoromethyl-phenylamino)-methyl]-thiophene-2-
carboxylic acid [4-(2-hydroxy-ethyl)-thiazol-2-yl]-amide (45)
Step 1. To a solution of 5-(((2-(trifluoromethyl)phenyl)amino)
methyl)thiophene-2-carboxylic acid (115; 70 mg, 0.232 mmol) and
4-(2-((tert-butyldimethylsilyl)oxy)ethyl)thiazol-2-amine (72 mg,
0.280 mmol) in CHCl3 (1.4 ml) was added EDC$HCl (53 mg,
0.280 mmol), followed by DMAP (31 mg, 0.254 mmol) at room
temperature. The reaction mixture was stirred for 2 h under N2
atmosphere. EtOAc and saturated NaHCO3 were added to the re-
action mixture and the layers separated. The organic layer was
washed with brine and dried over Na2SO4. The mixture was filtered
m
ml
100 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA, and 62.5 mg/ml
activated charcoal. [3H] H2O (liquid phase) was separated by
filtration (Unifilter, GE Healthcare) from the charcoal-adsorbed [9,
10-3H] stearoyl-CoA and scintillation cocktail (MicroScint-40, Per-
kin Elmer) added. The radioactivity of the mixture was measured by
a scintillation counter (TopCount, Perkin Elmer). Control values
were measured by evaluating the radioactivity of a well containing
DMSO instead of the compound solution. Background values were

T. Iida et al. / European Journal of Medicinal Chemistry 158 (2018) 832e852
851
measured by evaluating the radioactivity of a well to which stop-
ping buffer was added before the reaction. The remaining SCD1
activity (%) was calculated for each well using the following
formula:
Research Diets, Inc.). Compound 48 was administered orally at 1 or
10 mg/kg. Six hours after administration, liver, epididymal adipose
tissue, and eyelid samples were collected and homogenized to
measure tissue SCD activity as described above.
SCD1 activity (%) ¼ (Compound e Background)/(Control e
5.6. Effects of 48 on the liver triglyceride contents in rats fed a high
sucrose very low fat diet
Background)
The 50% inhibitory concentration (IC50) values were calculated
from two measurements of the remaining SCD1 activity crossing
50%.
SD rats were obtained from Charles River Laboratories (Yoko-
hama, Japan) and fed a HSVLF diet (D08030601, Research Diets,
Inc.). Compound 48 was administered orally at 1 or 10 mg/kg once
daily for 8 days and liver slices collected to measure the TG contents
of the liver on day 8 of administration. Methanol (0.4 ml) was added
to the liver slices before homogenization using one zirconia ball and
a mixer mill. The homogenates were combined with 0.4 ml meth-
anol and 0.8 ml chloroform, mixed, and centrifuged at 10,000 ꢃ g
for 5 min. A total of 0.2 ml of supernatant was collected and dried
5.3. Effects on SCD1 activity in the liver and eyelid
C57BL/6 J mice were obtained from Charles River Laboratories
(Japan). Compounds were orally administered to the mice (male, 6
weeks of age). Six or 24 h after administration, the liver or eyelids
were dissected from the mice and added to 9-fold (liver) or 4-fold
(eyelid) homogenizing buffer (100 mmol/L Tris-HCl (pH 7.4),
0.25 mol/L sucrose), respectively. The tissues were sufficiently ho-
mogenized using one zirconia ball (YTZ® Ball, Nikkato Corp.) and a
mixer mill (MM300, Retsch Co., Ltd.), followed by centrifugation at
10,000 ꢃ g at 4 ꢄC for 5 min to obtain the S9 fraction. The SCD1
with N2 gas. The residues were resolved in 200 ml of 2-propanol and
TG levels measured using an automatic analyzer (Hitachi7170S,
Tokyo, Japan).
5.7. Safety studies
reaction was initiated by adding 40
solution containing 100 mmol/L Tris-HCl (pH 7.4), 4 mmol/L NADH,
12 mmol/L MgCl2, 0.5 g cytochrome b5, and 12
mol/L [14C]
ml of S9 fraction to the substrate
In order to investigate any toxicity after repeated dosing for 2
weeks in rats, 48 suspended in vehicle (0.5% methylcellulose
aqueous solution) was administered orally once daily to 6-week-
old SD rats (5 males per group) at 100 and 1000 mg/kg. The animals
in the control group (5 males) were given the vehicle. During the
dosing period, clinical observations and measurements were made
of body weight and food consumption. Ophthalmoscopy was con-
ducted in week 2. At the end of the dosing period, necropsy, he-
matology, clinical chemistry, organ weight measurements, and
histopathological examination were performed. The plasma con-
centrations of 48 were determined 24 h after dosing on days 1, 5,
and 10.
m
m
stearoyl-CoA. The mixtures were incubated at room temperature
for 10 min (liver) or 30 min (eyelid). The reaction was terminated by
adding 1 ml of HCl in methanol (Wako Pure Chemical Industries).
Two milliliters of hexane was added and the mixtures incubated at
100 ꢄC for 1 h to saponify the lipids. The mixtures were centrifuged
at 1000 ꢃ g for 10 min and the upper phases (hexane phases)
collected. Hexane was evaporated by N2 gas and the residues
resolved in 100 ml of hexane and separated on a 10% AgNO3-
impregnated TLC plate with hexane/diethyl ether (9:1). The TLC
plate was exposed to an imaging plate (BAS-MS2040, Fuji film
Corporation, Japan) to visualize and quantify [14C]-incorporated
stearic acid and oleic acid using an imaging analyzer (BAS2500,
Fujifilm Corporation, Japan).
Acknowledgments
The authors thank Dr. Atsushi Sakai, Toshihiro Kiguchi, Noboru
Nagahashi, Masakazu Terashita, and Satoshi Watanabe for sup-
porting this work. We sincerely appreciate Dr. Hiromasa Hashimoto
for reviewing the manuscript. We also thank Dr. Jun-ichi Haruta for
helpful discussion.
5.4. Effects of 48 in mice fed a high fat diet
C57BL/6 J mice were obtained from Charles River Laboratories
(Yokohama, Japan) and individually housed under controlled
temperature (23 3 ꢄC), humidity (55 15%), and lighting (12 h
light/dark cycle with lights on at 8:00 a.m.). The mice were pro-
vided a HFD (D12330, Research Diets, Inc., USA) and water ad
libitum. After being fed the HFD for a month, the mice were divided
into three groups: a control group and 3 and 10 mg/kg 48. The mice
in the 48-treated groups were fed a HFD containing appropriate
amounts of 48 (0.0040% at 3 mg/kg/day, 0.0133% at 10 mg/kg/day)
to achieve a daily dose of approximately 3 or 10 mg/kg for 43 days
from 10 to 16 weeks of age. A normal chow diet (CRF-1; Oriental
Yeast, Osaka, Japan) was provided for the control group. Body
weight was measured every 3 or 4 days during the experimental
period. On day 22 of administration, a glucose tolerance test (1 g/
kg) was performed to the mice fasted overnight. The area under the
curve of plasma glucose level (AUC glucose) was calculated from
plasma glucose levels 0, 0.5, 1, and 2 h after glucose loading.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.09.003.
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ABBREVIATIONS USED
SCD: stearoyl-CoA desaturase
MUFA: monounsaturated fatty acid
SFA: saturated fatty acid
TG: triglyceride
ASO: antisense oligonucleotide
HTS: high-throughput screening
PK: pharmacokinetic
SAR: structure-activity relationship
cLogP: calculated logarithm of the partition coefficient
tPSA: topological polar surface area
LE: ligand efficiency
HFD: high fat diet
HSVLF: high sucrose very low fat
SD: Sprague Dawley
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HS: high sucrose
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NOAEL: no-observed adverse effect level
hERG: human ether a-go-go-related gene
CYP: cytochrome P450
155e175.
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desaturase inhibitors, Chem. Phys. Lipids 197 (2016) 3e12.
NT: not tested
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Y.K. Ramtohul, K. Skorey, D. Sorensen, W. Sturkenboom, A. Styhler,
D.M. Waddleton, H. Wang, S. Wong, L. Xu, L. Zhang, Development of a liver-
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TLC: thin-layer chromatography
CDI: 1,10-carbonyldiimidazole
TBS: tert-butyldimethylsilyl
EDC·HCl: 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride
HOBt: 1-hydroxybenzotriazole monohydrate
PyBOP:
hexafluorophosphate
TEA: triethylamine
1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium
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DMAP: 4-dimethylaminopyridine
DMSO: dimethyl sulfoxide
DMF: dimethylformamide
DME: 1,2-dimethoxyethane
AcOH: acetic acid
MeOH: methanol
EtOH: ethanol
CHCl3: chloroform
Pd2(dba)3: tris(dibenzylideneacetone)dipalladium(0)
X-Phos: 2-dicyclohexylphosphino-20,40,60-triisopropylbiphenyl
rt: room temperature
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