Novel acyl coenzyme A (CoA): Diacylglycerol acyltransferase-1 inhibitors: Synthesis and biological activities of diacylethylenediamine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.01.067

A series of diacylethylenediamine derivatives were synthesized and evaluated for their inhibitory activity against DGAT-1 and pharmacokinetic profile to discover new small molecule DGAT-1 inhibitors. Among the compounds, N-[2-({[1-phenyl-3-(trifluoromethy

Full text of DOI:10.1016/j.bmc.2010.01.067

Bioorganic & Medicinal Chemistry 18 (2010) 2785–2795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel acyl coenzyme A (CoA): Diacylglycerol acyltransferase-1 inhibitors:
Synthesis and biological activities of diacylethylenediamine derivatives
e
e
e
e
Yoshihisa Nakada a, , Thomas D. Aicher , Yvan Le Huerou , Timothy Turner , Scott A. Pratt ,
Stephen S. Gonzales e, Steve A. Boyd e, Hiroshi Miki b, Toshihiro Yamamoto c, Hiroshi Yamaguchi c,
Koki Kato d, Shuji Kitamura a
*
a Medicinal Chemistry Research Laboratories, Takeda Pharmaceutical Company Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
b Discovery Research Center, Takeda Pharmaceutical Company Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
c Pharmacology Research Laboratories, Takeda Pharmaceutical Company Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
d Strategic Research Planning Department, Takeda Pharmaceutical Company Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
e Array Biopharma Inc, 3200 Walnut Street, Boulder, CO 80301, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diacylethylenediamine derivatives were synthesized and evaluated for their inhibitory activity
against DGAT-1 and pharmacokinetic profile to discover new small molecule DGAT-1 inhibitors. Among
the compounds, N-[2-({[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}amino)ethyl]-6-(2,2,2-
trifluoroethoxy)pyridine-3-carboxamide 3x showed potent inhibitory activity and excellent PK profile.
Oral administration of 3x to mice with dietary-induced obesity resulted in reduced body weight gain
and white adipose tissue weight.
Received 25 December 2009
Revised 28 January 2010
Accepted 29 January 2010
Available online 4 February 2010
Keywords:
DGAT-1 inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
Diet-induced obesity
Inhibition of triacylglycerol synthesis
1. Introduction
of the pharmacological features of DGAT-1 deficient mice. We have
also reported the discovery of the potent DGAT-1 inhibitor
The concurrent epidemics of obesity, diabetes and cardiovascu-
lar disease have become a rapidly increasing health problem facing
the world’s industrialized countries. Obesity is one of the main risk
factors in cardiovascular disease. The high unmet need for treat-
ments to control obesity has encouraged the identification of many
new mechanistic targets, one of which is triacylglycerol (TG) syn-
thesis inhibition. One of the key enzymes in TG synthesis is acyl
coenzyme A (CoA): diacylglycerol acyltransferase (DGAT), which
catalyzes the final and only committed step of the TG synthesis
pathway. Two DGAT enzymes, DGAT-1 and DGAT-2, have been
identified, and both are ubiquitously expressed. The highest levels
of expression of these enzymes are found in tissues that are active
in TG synthesis, such as white adipose tissue, small intestine, and
liver.1,2 The phenotype of DGAT-1 deficient mice has already been
described.3–5 These mice are viable, resistant to diet-induced obes-
ity (DIO) and show increased sensitivity to insulin and leptin.
These findings suggest that pharmacological inhibition of DGAT-1
may be a feasible therapeutic strategy for human obesity and type
2 diabetes. Recent studies6,7 have demonstrated that treatment of
rodents with small molecule DGAT-1 inhibitors (I–II) showed some
N-(5-benzyl-4-phenyl-1,3-thiazol-2-yl)-4-(4,5-diethoxy-2-methyl-
phenyl)-4-oxobutanamide (1).8 Administration of 1 to KKAy mice
as a food admixture, exhibited anti-obesity effects in a four-week
study. However, after administration by methylcellulose suspen-
sion 1 did not show sufficient in vivo efficacy due to its poor
pharmacokinetic (PK) profile, which might be caused by poor
aqueous solubility and metabolic stability. Compound 1 was also
suspected of having potential bioactivation-related toxicities as a
consequence of oxidative ring-opening of the thiazole moiety to
yield potentially toxic thioureas.9–11 It also had the potential to
give rise to aromatic amine metabolites, which have the risk of
mutagenicity.12 Therefore we started to research a new type of
compound to address these concerns.
As a modification of compound 1, the reverse amide compound
2, which avoids the risk of giving rise to aromatic amine metabo-
lites, was found to have DGAT-1 inhibitory activity; however it still
exhibited poor solubility and poor metabolic stability (Fig. 1). To
improve the potency of compound 2, and to replace the thiazole
moiety, we first synthesized a lead library 3, focused on the acyl
group (R1CO) and the five-membered heteroaromatic ring bearing
the phenyl group. From this library we discovered compound 3a,
which possessed potent inhibitory activity and improved aqueous
solubility. However 3a still exhibited a poor PK profile that may be
* Corresponding author. Tel.: +81 6 6300 6458; fax: +81 6 6300 6306.
E-mail address: Nakada_Yoshihisa@takeda.co.jp (Y. Nakada).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.067

2786
Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
activity relationships (SAR) and biological properties of this novel
series of diacylethylenediamine derivatives.
2. Chemistry
The target compounds 3a–l, designed for optimization of the
substituted pyrazole moiety, were synthesized as illustrated in
Scheme 1. The key intermediate 5 was prepared from 4-ethoxy-
benzoic acid 4 by condensation with mono-protected ethylenedia-
mine and subsequent removal of the tert-butyl carbamate group.
Compounds 3a–c, f–l and the intermediate 7 were obtained from
5 by a second condensation with pyrazole carboxylic acids 6a–
d,13–15 f–l. Compounds 3d and 3e were prepared from 7 by regio-
selective alkylation with corresponding alkyl bromides. Pyrazole
carboxylic acids 6a, f–l were obtained from pyrazole ester 9 by reg-
ioselective arylation16,17 and subsequent hydrolysis.
The synthesis of target compounds 3m–x, designed for optimi-
zation of the linker and benzoyl moieties, is shown in Scheme 2.
Use of the mono-protected ethylenediamine derivatives 11i–iv
led to compounds 3q–v and the intermediate esters 14m–p.
Hydroxymethyl derivatives 3m–p were obtained by reduction of
the esters 14m–p. The 2-alkoxypyridine derivatives 3w and 3x
were prepared from the 2-chloropyridine derivative 3v by etherifi-
cation with the corresponding alcohols.
Figure 1. Known compounds, general design 3 and novel DGAT-1 inhibitor 3a.
3. Results and discussion
attributed to insufficient metabolic stability (Table 3). To achieve
good in vivo efficacy, we started the optimization of 3a, focusing
on the pyrazole ring substituents, the linker portion and the termi-
nal benzoyl group, with the goal of improving the PK profile while
maintaining good potency. At each position we sought to decrease
or maintain the overall lipophilic nature of the compound, and to
utilize substituents which we believed would alleviate oxidative
metabolism. In this paper, we describe the synthesis, structure–
The compounds 3a–x were evaluated for their inhibitory activ-
ities against human DGAT-1 enzyme and TG synthesis in the C2C12
cell line, their in vitro metabolic stability (human, rat, and mouse
microsomes), and their PK profile in rats via cassette dosing. The
results are summarized in Tables 1–3.
We initially examined the effect of optimizing the substituents
on the pyrazole ring, as shown in Table 1. The reduced activity of
Scheme 1. Modification of the pyrazole moiety. Reagents and conditions: (a) tert-butyl (2-aminoethyl)carbamate, EDCl, HOBt, CH3CN; (b) 4 N HCl in AcOEt; (c) EDCl, HOBt,
Et3N, DMF; (d) HATU, DIEPA, THF; (e) (i) CH(OEt)3, Ac2O; (ii) NH2NH2–H2O, CH3OH; (f) R2-B(OH)2, Cu(OAc)2, pyridine, DMF, air; (g) R2-Br, CuI, (1S,2S)-N1,N2-
dimethylcyclohexane-1,2-diamine, toluene; (h) 2 N NaOH, EtOH; (j) alkyl bromide, K2CO3.

Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
2787
Scheme 2. Modification of Linker fragment and benzyl moiety. Reagents and conditions: EDCl, HOBt, Et3N, DCM; (b) TFA; (c) 10% Pd/C, THF, H2 gas; (d) LiCl, NaBH4, THF–
EtOH; (e) 4 N HCl AcOEt; (f) HATU, DIEPA, THF; (g) ROH, 60% NaH, THF.
compounds 3b and 3c suggested that the existence of a trifluoro-
methyl group and the position of the pyrazole ring nitrogen atoms
are important to give high potency. Replacement of the phenyl ring
by an alkyl or an arylalkyl group exhibited decreased potency and
insufficient metabolic stability (3a vs 3d, e). Incorporating substit-
uents in the para-position (3f and 3g) and in the meta-position (3h)
did not improve metabolic stability versus the parent (3a).
Replacement of the phenyl ring with a heteroaromatic group (to
decrease the overall lipophilicity) afforded the 2-pyridyl derivative
3i (IC50 = 28 nM). This compound exhibited good potency, however
its metabolic stability was not significantly improved. Isomeric
pyridyl derivatives 3j and 3k exhibited drastically decreased
potency. Based on the reasonable potency of 3i, we introduced
polar groups at the 2-position of the phenyl ring. However, the
2-hydroxyphenyl derivative 3l exhibited decreased potency, insuf-
ficient metabolic stability and a poor PK profile. Overall, we be-
lieved the 1-phenyl-3-trifluoromethylpyrrazol-4-carbamoyl group
was an acceptable pyrazole moiety to use for the remaining SAR,
as it provided potent inhibitory activity and moderate metabolic
stability.
Concurrently, we examined substituents on the ethylene linker
(R1 or R2) as shown in Table 2. Our design concept was based on a
belief that the ethylene linker of 3a serves as a mimic of 1,2-diac-
ylglycerol, the substrate of the DGAT-1 enzyme. With this modifi-
cation, we aimed to enhance potency with the insertion of a
hydroxymethyl group. Alternatively, even if the 1,2-diacylglycerol
hypothesis was incorrect, it could lead to an improvement of met-
abolic stability by affording an overall decrease in lipophilicity.
However, the hydroxymethyl derivatives 3m–p showed drastically
reduced potency. Methyl derivatives 3q and 3r also decreased en-
zyme activities, and these results suggested that substitution on
the ethylene linker would be disfavored.
The third concurrent focus of SAR effort was modification of the
benzoyl moiety in 3a to improve the metabolic stability (Table 3).
Moving the ethoxy group from para- to meta-position decreased
potency significantly (3s vs 3a). The 4-pyrazol-1-ylphenyl deriva-
tive 3t showed a slightly decreased potency. These results sug-
gested that an alkoxy substituent at the para-position of the
phenyl ring was desirable. Subsequently, we examined the replace-
ment of the phenyl ring by pyridyl, in an attempt to improve met-
abolic stability by reducing the lipophilicity. In order to compare
the insertion position of the nitrogen atom we synthesized two
types of chloropyridines 3u and 3v. 2-Chloro-5-pyridine derivative
3v showed more potent DGAT-1 inhibition and better in vitro met-
abolic stability than that of the 5-chloro-2-pyridine derivative 3u.
Several of these analogs were evaluated for rat PK, as shown in Ta-
ble 3. Chloropyridyl amide 3v exhibited a great improvement of
the PK profile compared to 3a, with much lower clearance and a
large increase in area under the blood concentration-time curve
(AUCpo) and bioavailability. We desired to replace the potential
metabolically labile halogen, and consequently we introduced alk-
oxy groups at the para-(2-) position. The 2-ethoxy-5-pyridine
derivative 3w (IC50 = 25 nM) exhibited potent DGAT-1 inhibitory
activity. Compound 3w showed improved metabolic stability as
compared to 3a; however it still showed low plasma concentra-
tion. In our further optimization of the substituent at the 2-posi-
tion of the pyridine ring, 2-(2,2,2-trifluoroethoxy) derivative 3x
exhibited potent DGAT-1 inhibitory activity in both enzyme
(IC50 = 22 nM) and cell-based (IC50 = 44 nM) assays. Compound 3x
also showed a significant improvement of the metabolic stability
and >17,000-fold higher AUCpo than that of 3a.
Encouraged by the potency and excellent PK profile of com-
pound 3x, we tested it for efficacy in animal models of obesity.
In one study, C57BL/6 mice on a high-fat diet were dosed with

2788
Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
Table 1
SAR around the pyrazole ring substituent
Compd
Ar
Enzyme IC50 (nM)
Cell IC50 (nM)
Metabolic stabilitya (human/rat/mouse)
Rats cassette dosing PK
AUCpob
BAc
3a
3b
3c
33
650
77
260
9.0 / 6.5 / 7.0
5.0 / 5.8 / 2.5
12 / 12 / 14
0.3
0.1
NT*
NT*
NT*
NT*
2000
1200
3d
3e
3f
220
790
94
650
770
8.7 / 11 / 7.8
5.5 / 18 / 7.5
4.5 / 7.2 / 3.4
ND**
NT*
22
ND**
NT*
3.3
1500
3g
3h
3i
140
44
380
600
7.5 / 8.7 / 14
7.4 / 7.1 / 6.5
14 / 10 / 7.0
NT*
NT*
54
NT*
7.4
28
91
28
8.1
3j
1900
5500
98
3400
>10,000
610
NT*
NT*
0.9
NT*
NT*
1.4
3k
NT*
3l
5.7 / 18 / 0.5
a
b
c
%/min/mg.
Area under the blood concentration-time curve (ng h/mL).
Bioavailability (%).
*
Not tested.
Not detected.
**
compound 3x (3 mg/kg, b.i.d., 0.5% MC suspension) for four weeks.
The treated animals in the study had a significant and sustainable
reduction in body weight gain (ꢀ75%) and white adipose tissue
weight (mesenteric: ꢀ25%, perirenal: ꢀ12%, subcutaneous:
ꢀ2.8%) without affecting total food intake. The details of this study
and other advanced pharmacology experiments with this com-
pound will be reported separately.
benzoyl group of compound 3a, with the goal of improving the
PK profile while maintaining good potency. Replacement of the
4-ethoxybenzoyl group of 3a with
a 2-(2,2,2-trifluoroeth-
oxy)pyrid-5-yl group gave compound 3x, which exhibited both
potent DGAT-1 inhibitory activity and an excellent PK profile
due to good metabolic stability. Compound 3x has a unique
structure, and in contrast to many known DGAT-1 inhibitors,
does not possess carboxylic acid or amino functionalities. Fur-
thermore, 3x exhibited superior efficacy in the reduction of body
weight gain and white adipose tissue weight in DIO mice. These
results suggest that the small molecule DGAT-1 inhibitor 3x
might have potential in the treatment of obesity and metabolic
syndrome.
4. Conclusion
In this study, we designed the lead library 3 from compounds
1 and 2, leading to the discovery of lead compound 3a. We opti-
mized substituents on the pyrazole ring, the linker portion, and

Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
2789
Table 2
Reactions were carried out at room temperature unless other-
wise noted and followed by thin layer chromatography (TLC) on
silica gel 60 F254 precoated TLC plates (E. Merck) or by HPLC using
an octadecyl silica (ODS) column (A-303, 4.6 mm id ꢁ 250 mm,
YMC Co., Ltd). Standard workup procedures were as follows. The
reaction mixture was partitioned between the indicated solvent
and water. Organic extracts were combined and washed in the
indicated order using the following aqueous solutions: water, 5%
aqueous sodium carbonate solution (aqueous NaHCO3), saturated
sodium chloride (NaCl) solution (brine), 1 N aqueous sodium
hydroxide solution (1 N NaOH) and 1 N hydrochloric acid (1 N
HCl). Extracts were dried over anhydrous magnesium sulfate
(MgSO4), filtered, and evaporated in vacuo. Chromatographic sep-
arations were carried out on Silica gel 60 (0.063–0.200 mm, E.
Effect of insertion into ethylene Linker
Compd
R1
R2
Enzyme IC50 (nM)
3a
3m
3n
3o
3p
3q
3r
H
H
H
H
33
S-CH2OH
R-CH2OH
H
H
Me
H
>10,000
>10,000
>10,000
>10,000
5300
S-CH2OH
R-CH2OH
H
Me
2800
Merck) on Purif-Pack (SI 60 lm or NH 60 lm, Fuji Silysia, Ltd) or
ODS (CPO-273L, prepacked column, 22–300 mm, Kusano Kagaku
Kikai Co.) using the indicated eluents. Yields were not maximized.
5. Experimental section
5.1. Chemistry
5.1.1. N-(2-Aminoethyl)-4-ethoxybenzamide hydrochloride (5)
A mixture of 4-ethoxybenzoic acid 4 (3.00 g, 18.0 mmol),
All melting points were determined on a Yanagimoto micro-
melting point apparatus or Stanford Research Systems OptiMelt
automated melting point system and are uncorrected. Proton nu-
clear magnetic resonance (1H NMR) spectra were recorded on a
Varian Mercury-300 (300 MHz), Bruker DPX-300 (300 MHz) or
Bruker-400 (400 MHz) with tetramethylsilane as internal standard.
The following abbreviations are used: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet and br = broad. Coupling con-
stants (J values) are given in hertz (Hz). LC/MS (ESI-MS) analyses
were carried out using a Waters Open-Lynx system. Optical rota-
tions were determined with a JASCO P-1030 digital polarimeter.
Elemental analyses were carried out by Takeda Analytical Labora-
tories Ltd and were within 0.4% of the theoretical values for the
elements indicated unless otherwise noted.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDCI) (5.19 g, 27.0 mmol), tert-butyl (2-aminoethyl)carbamate
(3.08 g, 19.9 mmol) and 1-hydroxybenzotriazole monohydrate
(HOBt) (4.15 g, 27.0 mmol) in acetonitrile (30 ml) was stirred for
4 h. The reaction mixture was concentrated in vacuo. The residue
was diluted with water and extracted with ethyl acetate. The ex-
tracts were washed with saturated aqueous sodium hydrogen car-
bonate solution and brine, dried over anhydrous magnesium
sulfate and concentrated in vacuo. The residue was dissolved in
ethyl acetate (50 ml). To the mixture was added 4 N hydrogen
chloride in ethyl acetate (19.0 ml, 76.2 mmol) and the mixture
was stirred for 24 h. The reaction mixture was concentrated in va-
cuo and the residue was collected by filtration to give 5 (3.12 g,
71%) as a white solid. Compound 5 was used for next step without
Table 3
SAR around benzoyl moiety
Compd
Ar
Enzyme IC50 (nM)
Cell IC50 (nM)
Metabolic stabilitya (human/rat/mouse)
Rats cassette dosing PK testb
Vdss
CLtotal
Cmax
AUCpo
MRTpo
BAh
0.1
c
d
e
f
g
3a
3s
33
77
9.0 / 6.5 / 7.0
14 / 8.3 / 19
1453
5535
0.4
0.3
0.38
*
*
*
*
*
*
3600
5100
NT
NT
NT
NT
NT
NT
3t
79
400
5.9 / 6.5 / 3.5
1346
1838
6.4
25
3.2
4.5
*
*
*
*
*
*
3u
3v
3w
3x
1300
49
1200
370
87
4.8 / 2.6 / 1.8
2.2 / 1.1 / 0
5.1 / 3.4 / 2.5
2.1 / 0.4 / 0
NT
NT
NT
NT
NT
NT
728
399
325
343
1098
95
182
103
800
964
74
3.3
33
7.6
50
25
0.73
4.2
22
44
5292
a
b
c
%/min/mg.
iv: 0.1 mg/0.5 mL/kg, po: 1 mg/5 mL/kg, n = 3.
Distribution volume (mL/kg).
Total clearance (mL/h/kg).
Maximum blood concentration (ng/mL).
Area under the blood concentration-time curve (ng h/mL).
Mean residence time (h).
d
e
f
g
h
Bioavailability (%).

2790
Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
further purification. mp 179–180 °C; 1H NMR (DMSO-d6) d: 1.34
(3H, t, J = 7.0 Hz), 2.97 (2H, t, J = 6.0 Hz), 3.51 (2H, q, J = 5.8 Hz),
4.08 (2H, q, J = 6.8 Hz), 6.94–7.02 (2H, m), 7.84–7.93 (2H, m),
8.11 (2H, br s.), 8.64 (1H, t, J = 5.3 Hz); LC/MS m/z: 209 (MH+); Anal.
Calcd for C11H17N2O2Cl: C, 53.99; H, 7.00; N, 11.45. Found: C,
53.88; H, 7.04; N, 11.41.
5.1.7. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-1-
pyridin-3-yl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide
(3j)
Solid (yield 39%); mp 199–201 °C; 1H NMR (DMSO-d6) d: 1.33
(3H, t, J = 7.0 Hz), 3.41 (4H, m), 4.08 (2H, q, J = 7.0 Hz), 6.97 (2H,
d, J = 9.0 Hz), 7.65 (1H, m), 7.81 (2H, d, J = 9.0 Hz), 8.23 (1H, m),
8.43 (1H, m), 8.51 (1H, m), 8.68 (1H, m), 9.06 (1H, m), 9.13 (1H,
m); LC/MS m/z: 448 (MH+); Anal. Calcd for C21H20N5O3F3: C,
56.37; H, 4.51; N, 15.65. Found: C, 56.26; H, 4.57; N, 15.60.
5.1.2. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-1-
phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3a)
A mixture of 5 (98 mg, 0.40 mmol), EDCI (115 mg, 0.60 mmol), 6a
(102 mg, 0.40 mmol), HOBt (92 mg, 0.60 mmol) and triethylamine
(0.112 ml, 0.80 mmol) in N,N-dimethylformamide (DMF) (5 ml)
was stirred for 24 h. The reaction mixture was diluted with aqueous
NaHCO3 and extracted with ethyl acetate. The extracts were washed
with brine, dried over anhydrous magnesium sulfate and concen-
trated in vacuo. The residue was recrystallized from ethyl acetate–
hexane to give 3a (148 mg, 83%) as a white solid. mp 202–203 °C;
1H NMR (CDCl3) d: 1.42 (3H, t, J = 7.0 Hz), 3.57–3.77 (4H, m), 4.06
(2H, q, J = 6.9 Hz), 6.85–6.98 (2H, m), 7.36–7.45 (1H, m), 7.46–7.61
(3H, m), 7.66–7.74 (2H, m), 7.74–7.89 (3H, m), 8.55 (1H, s); LC/MS
m/z: 447 (MH+); Anal. Calcd for C22H21N4O3F3: C, 59.19; H, 4.74; N,
12.55. Found: C, 59.23; H, 4.78; N, 12.60.
5.1.8. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-1-
pyridin-4-yl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide
(3k)
Solid (yield 55%); mp 228–230 °C; 1H NMR (DMSO-d6) d: 1.33
(3H, t, J = 7.0 Hz), 3.41 (4H, m), 4.08 (2H, q, J = 7.0 Hz), 6.97 (2H,
d, J = 9.0 Hz), 7.82 (2H, d, J = 9.0 Hz), 7.85 (2H, m), 8.43 (1H, s),
8.55 (1H, s), 8.78 (2H, m), 9.26 (1H, s); LC/MS m/z: 448 (MH+); Anal.
Calcd for C21H20N5O3F3: C, 56.37; H, 4.51; N, 15.65. Found: C,
56.39; H, 4.63; N, 15.59.
5.1.9. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-1-(2-
hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide (3l)
The following compounds 3f–l, q–s, 7, 14m–p were prepared
from the corresponding starting materials in a similar manner to
that described for 3a.
White solid (yield 83%); mp 220–223 °C; 1H NMR (DMSO-d6) d:
1.33 (3H, t, J = 7.0 Hz), 3.39 (4H, br s), 4.07 (2H, q, J = 6.8 Hz), 6.91–
7.00 (3H, m), 7.11 (1H, dd, J = 8.3, 1.1 Hz), 7.26–7.35 (1H, m), 7.57
(1H, dd, J = 7.9, 1.5 Hz), 7.77–7.84 (2H, m), 8.43 (1H, br s), 8.52 (1H,
br s), 8.85 (1H, s), 10.73 (1H, br s); LC/MS m/z: 463 (MH+); Anal.
Calcd for C22H21N4O4F3: C, 57.14; H, 4.58; N, 12.12. Found: C,
56.93; H, 4.81; N, 12.04.
5.1.3. 1-(4-Chlorophenyl)-N-(2-{[(4-
ethoxyphenyl)carbonyl]amino}ethyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (3f)
Solid (yield 24%); mp 192–194 °C; 1H NMR (DMSO-d6) d: 1.34
(3H, t, J = 7.0 Hz), 3.40 (4H, m), 4.07 (2H, q, J = 7.0 Hz), 6.97
(2H, m), 7.68 (2H, m), 7.83 (4H, m), 8.43 (1H, m), 8.48 (1H,
m), 9.08 (1H, m); LC/MS m/z: 481 (MH+); Anal. Calcd for
C22H20N4O3ClF3: C, 54.95; H, 4.19; N, 11.65. Found: C, 54.85;
H, 4.03; N, 11.77.
5.1.10. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}propyl)-1-
phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3q)
White solid (yield 44%); mp 191–193 °C; 1H NMR (DMSO-d6) d:
1.18 (3H, d, J = 6.6 Hz), 1.34 (3H, t, J = 7.0 Hz), 3.35–3.41 (2H, m),
4.07 (2H, q, J = 7.0 Hz), 4.15–4.25 (1H, m), 6.94–6.99 (2H, m),
7.45–7.50 (1H, m), 7.57–7.64 (2H, m), 7.78–7.84 (4H, m), 8.12
(1H, d, J = 8.2 Hz), 8.45 (1H, t, J = 5.9 Hz), 9.05 (1H, s); LC/MS m/z:
461 (MH+); Anal. Calcd for C23H23N4O3F3: C, 59.99; H, 5.03; N,
12.17. Found: C, 59.91; H, 5.08; N, 12.12.
5.1.4. 1-(4-Ethoxyphenyl)-N-(2-{[(4-
ethoxyphenyl)carbonyl]amino}ethyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (3g)
White solid (yield 91%); mp 199–200 °C; 1H NMR (DMSO-d6) d:
1.29–1.40 (6H, m), 3.35–3.44 (4H, m), 4.02–4.14 (4H, m), 6.93–7.01
(2H, m), 7.08–7.16 (2H, m), 7.65–7.73 (2H, m), 7.77–7.85 (2H, m),
8.44 (2H, d, J = 2.3 Hz), 8.93 (1H, d, J = 1.1 Hz); LC/MS m/z: 491
(MH+); Anal. Calcd for C24H25N4O4F3: C, 58.77; H, 5.14; N, 11.42.
Found: C, 58.60; H, 5.24; N, 11.39.
5.1.11. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}-1-
methylethyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide (3r)
White solid (yield 42%); mp 174–176 °C; 1H NMR (DMSO-d6) d:
1.17 (3H, t, J = 8.6 Hz), 1.32 (3H, t, J = 7.0 Hz), 3.34–3.45 (2H, m),
4.07 (2H, q, J = 7.0 Hz), 4.15–4.24 (1H, m), 6.94–6.98 (2H, m),
7.45–7.51 (1H, m), 7.57–7.65 (2H, m), 7.78–7.85 (4H, m), 8.17
(1H, d, J = 8.2 Hz), 8.38 (1H, t, J = 5.9 Hz), 9.07 (1H, s); LC/MS m/z:
461 (MH+); Anal. Calcd for C23H23N4O3F3ꢂ0.4H2O: C, 59.07; H,
5.13; N, 11.98. Found: C, 59.43; H, 5.28; N, 11.54.
5.1.5. 1-(3-Ethoxyphenyl)-N-(2-{[(4-
ethoxyphenyl)carbonyl]amino}ethyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (3h)
Solid (yield 32%); mp 174–176 °C; 1H NMR (DMSO-d6) d: 1.35
(6H, m), 3.40 (4H, m), 4.10 (4H, m), 6.96 (2H, m), 7.02 (1H, m),
7.35 (2H, m), 7.49 (1H, t, J = 7.8 Hz), 7.81 (2H, m), 8.44 (2H, m),
9.07 (1H, m); LC/MS m/z: 491 (MH+); Anal. Calcd for
C24H25N4O4F3ꢂ0.1H2O: C, 58.56; H, 5.16; N, 11.38. Found: C,
58.30; H, 5.21; N, 11.42.
5.1.12. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-3-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (7)
Solid (yield 34%); mp 160–162 °C; 1H NMR (CDCl3) d: 1.34 (3H,
t, J = 7.0 Hz), 3.36 (4H, m), 4.07 (2H, q, J = 7.0 Hz), 6.96 (2H, m), 7.80
(2H, m), 8.34 (2H, m), 8.41 (1H, m), 13.74 (1H, br s); Anal. Calcd for
C16H17N4O3: C, 51.89; H, 4.63; N, 15.13. Found: C, 51.71; H, 4.64; N,
15.15.
5.1.6. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-1-pyridin-
2-yl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3i)
White solid (yield 88%); mp 223–224 °C; 1H NMR (DMSO-d6) d:
1.33 (3H, t, J = 7.0 Hz), 3.40 (4H, br s), 4.08 (2H, q, J = 6.8 Hz), 6.94–
7.00 (2H, m), 7.53 (1H, ddd, J = 7.4, 4.9, 0.9 Hz), 7.78–7.85 (2H, m),
7.99 (1H, d, J = 8.3 Hz), 8.07–8.14 (1H, m), 8.44 (1H, br s), 8.56–8.60
(1H, m), 8.63–8.70 (1H, m), 9.38 (1H, d, J = 0.8 Hz); LC/MS m/z: 448
(MH+); Anal. Calcd for C21H20N5O3F3: C, 56.37; H, 4.51; N, 15.65.
Found: C, 56.29; H, 4.60; N, 15.64.
5.1.13. (S)-Methyl N-[(4-ethoxyphenyl)carbonyl]-3-({[1-phenyl-
3-(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}amino)alaninate
(14m)
White solid (yield 63%); mp 77–79 °C; 1H NMR (CDCl3) d:
1.41 (3H, t, J = 7.0 Hz), 3.77 (3H, s), 3.86–4.00 (2H, m), 4.03

Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
2791
(2H, q, J = 7.0 Hz), 4.88 (1H, m), 6.85 (2H, m), 7.25 (1H, m),
7.34–7.44 (3H, m), 7.54–7.62 (3H, m), 7.26 (2H, m), 8.43
(1H, s); LC/MS m/z: 505 (MH+); Anal. Calcd for C24H23N4O5F3ꢂ
0.2H2O: C, 56.74; H, 4.64; N, 11.03. Found: C, 56.55; H, 4.65;
N, 10.98.
J = 8.8 Hz), 7.34 (1H, t, J = 7.3 Hz), 7.52 (2H, t, J = 7.9 Hz), 7.82 (2H, d,
J = 8.8 Hz), 7.89 (2H, d, J = 7.8 Hz), 8.37 (2H, br s), 8.44 (1H, s); LC/MS m/z:
393 (MH+); Anal. Calcd for C22H24N4O3: C, 67.33; H, 6.16; N, 14.28. Found:
C, 67.10; H, 6.22; N, 14.05.
5.1.19. N-(2-{[(3-Ethoxyphenyl)carbonyl]amino}ethyl)-1-
phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3s)
Solid (yield 18%); mp 157–158 °C; 1H NMR (DMSO-d6) d: 1.32
(3H, t, J = 6.9 Hz), 3.42–3.43 (4H, m), 4.03–4.08 (2H, m), 7.05–
7.08 (1H, m), 7.34–7.43 (3H, m), 7.48 (1H, t, J = 7.4 Hz), 7.61 (2H,
t, J = 8.0 Hz), 7.80 (1H, s), 7.82 (1H, s), 8.49 (1H, s), 8.56 (1H, s),
9.06 (1H, s); LC/MS m/z: 447 (MH+); Anal. Calcd for
C22H21N4O3F3ꢂ0.5H2O: C, 58.02; H, 4.87; N, 12.30. Found: C,
57.74; H, 4.74; N, 12.04.
5.1.14. (R)-Methyl N-[(4-ethoxyphenyl)carbonyl]-3-({[1-
phenyl-3-(trifluoromethyl)-1H-pyrazol-4-
yl]carbonyl}amino)alaninate (14n)
White solid (yield 73%); mp 73–75 °C; 1H NMR (DMSO-d6) d:
1.34 (3H, t, J = 7.0 Hz), 3.60–3.70 (1H, m), 3.66 (3H, s), 3.82 (1H,
dt, J = 13.7, 5.8 Hz), 4.09 (2H, q, J = 7.0 Hz), 4.62 (1H, dt, J = 7.4,
5.5 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.48 (1H, tt, J = 7.4, 1.5 Hz), 7.56–
7.63 (2H, m), 7.78–7.88 (4H, m), 8.61 (1H, t, J = 5.9 Hz), 8.67 (1H,
d, J = 7.4 Hz), 9.04 (1H, s); LC/MS m/z: 505 (MH+); Anal. Calcd for
C24H23N4O5F3: C, 57.14; H, 4.60; N, 11.11. Found: C, 56.84; H,
4.64; N, 10.98.
5.1.20. 1-Phenyl-N-[2-({[4-(1H-pyrazol-1-
yl)phenyl]carbonyl}amino)ethyl]-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide hydrochloride (3t)
White solid (yield 65%); mp 235–236 °C; 1H NMR (DMSO-d6) d:
3.39–3.51 (4H, m), 6.59 (1H, dd, J = 2.6, 1.9 Hz), 7.44–7.51 (1H, m),
7.57–7.65 (2H, m), 7.77–7.85 (3H, m), 7.92–8.03 (4H, m), 8.52 (1H,
br s.), 8.61 (1H, d, J = 2.6 Hz), 8.67 (1H, br s.), 9.08 (1H, d, J = 1.1 Hz);
LC/MS m/z: 469 (MH+); Anal. Calcd for C23H19N6O2F3: C, 58.97; H,
4.09; N, 17.94. Found: C, 58.97; H, 4.13; N, 17.89.
5.1.15. (S)-Methyl 3-{[(4-ethoxyphenyl)carbonyl]amino}-N-{[1-
phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}alaninate
(14o)
White solid (yield 84%); mp 73–75 °C; 1H NMR (CDCl3) d: 1.41
(3H, t, J = 7.0 Hz), 3.80 (3H, m), 3.95 (2H, m), 4.04 (2H, q,
J = 7.0 Hz), 4.89 (1H, m), 6.88 (3H, m), 7.40 (1H, m), 7.49 (2H, m),
7.64–7.74 (5H, m), 8.43 (1H, m); LC/MS m/z: 505 (MH+); Anal.
Calcd for C24H23N4O5F3ꢂ0.2H2O: C, 56.74; H, 4.64; N, 11.03. Found:
C, 56.60; H, 4.69; N, 11.06.
5.1.21. 5-Chloro-N-[2-({[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbonyl}amino)ethyl]pyridine-2-carboxamide
(3u)
Solid (yield 15%); mp 166–168 °C; 1H NMR (CDCl3) d: 3.71 (4H,
br s), 6.83 (1H, br s), 7.41 (1H, t, J = 7.2 Hz), 7.51 (2H, t, J = 8.4 Hz),
7.68–7.71 (2H, m), 7.82 (1H, dd, J = 2.4, 8.4 Hz), 8.13 (1H, d,
J = 8.4 Hz), 8.31 (1H, br s), 8.42 (1H, s), 8.52 (1H, d, J = 1.6 Hz);
LC/MS m/z: 438 (MH+); Anal. Calcd for C19H15N5O2ClF3ꢂ0.6H2O: C,
49.64; H, 3.42; N, 15.24. Found: C, 49.59; H, 3.41; N, 15.02.
5.1.16. (R)-Methyl 3-{[(4-ethoxyphenyl)carbonyl]amino}-N-
{[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-
yl]carbonyl}alaninate (14p)
White solid (yield 76%); mp 73–75 °C; 1H NMR (DMSO-d6) d:
1.27 (3H, t, J = 7.0 Hz), 3.51–3.61 (1H, m), 3.60 (3H, s), 3.65–3.75
(1H, m), 4.02 (2H, q, J = 7.0 Hz), 4.61 (1H, dd, J = 13.6, 6.6 Hz),
6.92 (2H, d, J = 9.0 Hz), 7.44 (1H, tt, J = 7.4, 1.6 Hz), 7.53–7.61 (2H,
m), 7.72 (2H, d, J = 9.0 Hz), 7.75–7.80 (2H, m), 8.45 (1H, t,
J = 5.9 Hz), 8.68 (1H, d, J = 7.4 Hz), 9.08 (1H, s); LC/MS m/z: 505
(MH+); Anal. Calcd for C24H23N4O5F3ꢂ0.2H2O: C, 56.74; H, 4.64; N,
11.03. Found: C, 56.60; H, 4.68; N, 10.97.
5.1.22. 6-Chloro-N-[2-({[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbonyl}amino)ethyl]pyridine-3-carboxamide
(3v)
Solid (yield 73%); mp 199–200 °C 1H NMR (DMSO-d6) d: 3.39–
3.49 (4H, m), 7.44–7.52 (1H, m), 7.57–7.68 (3H, m), 7.78–7.84
(2H, m), 8.24 (1H, dd, J = 8.3, 2.3 Hz), 8.48–8.56 (1H, m), 8.82–
8.86 (1H, m), 8.88 (1H, br s.), 9.09 (1H, d, J = 1.1 Hz); LC/MS m/z:
438 (MH+); Anal. Calcd for C19H15N5O2ClF3ꢂ0.2H2O: C, 51.70; H,
3.52; N, 15.87. Found: C, 51.58; H, 3.41; N, 15.86.
5.1.17. N-(2-(4-Ethoxybenzamido)ethyl)-3-methyl-1-phenyl-
1H-pyrazole-4-carboxamide (3b)
A mixture of 5 (200 mg, 0.981 mmol), N,N-diisopropylethyl-
amine (DIPEA) (349 mg, 2.697 mmol), 2-(7-aza-1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
(373 mg, 0.981 mmol) and 3-methyl-1-phenyl-1H-pyrazole-4-car-
boxylic acid 6b (165 mg, 0.817 mmol) in THF (15 ml) was stirred at
room temperature overnight. The reaction mixture was quenched
by adding water (150 ml). The obtained solid were filtered and
washed with water and dried under high vacuum to give 3b
(146 mg, 43%) as a tan solid. mp 158–160 °C; 1H NMR (DMSO-d6)
d: 1.33 (3H, t, J = 6.9 Hz), 2.43 (3H, s), 3.39 (4H, s), 4.05–4.10 (2H,
m), 6.97 (2H, d, J = 8.8 Hz), 7.33 (1H, t, J = 7.4 Hz), 7.52 (2H, t,
J = 7.9 Hz), 7.73 (2H, d, J = 7.8 Hz), 7.82 (2H, d, J = 8.8 Hz), 8.12
(1H, s), 8.44 (1H, s), 8.83 (1H, s); LC/MS m/z: 393 (MH+); Anal. Calcd
for C22H24N4O3ꢂ0.4H2O: C, 64.42; H, 6.04; N, 13.76. Found: C, 64.67;
H, 6.20; N, 13.88.
5.1.23. 1-Cyclohexyl-N-(2-{[(4-ethoxyphenyl)carbonyl]amino}-
ethyl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3d)
To a solution of 7 (0.1 g, 0.27 mmol) in bromocyclohexane
(0.44 g, 2.70 mmol) was added potassium carbonate (0.11 g,
0.81 mmol). The mixture was stirred at 120 °C for 16 h. The mix-
ture was diluted with EtOAc and filtered, and the filtrate was con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (9:0.5:0.5 DCM/EtOAc/MeOH) then recrystallized
from ethyl acetate to give 3d (0.030 g, 25%) as a solid. mp 185–
187 °C; 1H NMR (DMSO-d6) d: 1.23 (1H, m), 1.34 (3H, t,
J = 7.0 Hz), 1.43 (2H, m), 1.65 (2.5H, m), 1.77–1.90 (2.5H, m), 2.05
(2H, m), 3.36 (4H, m), 4.08 (2H, q, J = 7.0 Hz), 4.24 (1H, m), 6.96
(2H, d, J = 8.9 Hz), 7.80 (2H, d, J = 8.9 Hz), 8.31 (1H, s), 8.39 (1H,
s), 8.40 (1H, s); LC/MS m/z: 453 (MH+); Anal. Calcd for
C22H27N4O3F3: C, 58.40; H, 6.01; N, 12.38. Found: C, 58.36; H,
6.08; N, 12.44.
The following compounds 3c, s–v were prepared from the cor-
responding starting materials in a similar manner to that described
for 3b.
5.1.18. N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}ethyl)-4-
methyl-1-phenyl-1H-pyrazole-3-carboxamide (3c)
5.1.24. 1-Benzyl-N-(2-{[(4-ethoxyphenyl)carbonyl]amino}-
ethyl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (3e)
The compound 3e was prepared from compound 7 and benzyl
bromide in a similar manner to that described for 3d. White solid
Solid (yield 15%); mp 195–198 °C; 1H NMR (DMSO-d6) d: 1.33 (3H, t,
J = 6.9 Hz), 2.27 (3H, s), 3.40–3.44 (4H, m), 4.02–4.10 (2H, m), 6.97 (2H, d,

2792
Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
(yield 74%); mp 186 °C; 1H NMR (CDCl3) d: 1.43 (3H, t, J = 7.0 Hz),
3.55–3.65 (4H, m), 4.07 (2H, q, J = 7.2 Hz), 5.30 (2H, s), 6.81–6.98
(4H, m), 7.22–7.31 (2H, m), 7.34–7.42 (3H, m), 7.66–7.73 (2H,
m), 7.84 (1H, s); LC/MS m/z: 461 (MH+); Anal. Calcd for
C23H23N4O3F3: C, 59.99; H, 5.03; N, 12.17. Found: C, 60.09; H,
5.01; N, 12.23.
5.05 mmol), and 9 (0.500 g, 2.40 mmol). (1S,2S)-N1,N2-dimethyl-
cyclohexane-1,2-diamine (0.0683 g, 0.481 mmol) and 2-bromo-
phenol (0.334 ml, 2.88 mmol) were then added along with
toluene (3 ml). The tube was sealed and heated to 110 °C over-
night, then cooled to room temperature. The mixture was filtered
through celite to remove the solid. The filtrate was concentrated
under vacuum and the residue was purified by silica gel column
chromatography (10% Et2O/DCM) to give 10l (0.393 g, 55%) as a
white solid. mp 122–123 °C; 1H NMR (CDCl3) d: 1.40 (3H, t,
J = 7.2 Hz), 4.39 (2H, q, J = 7.2 Hz), 6.99 (1H, ddd, J = 8.3, 7.2,
1.5 Hz), 7.15 (1H, dd, J = 8.3, 1.1 Hz), 7.30 (1H, ddd, J = 8.5, 7.4,
1.5 Hz), 7.43 (1H, dd, J = 8.3, 1.5 Hz), 8.57 (1H, d, J = 0.8 Hz), 9.69
(1H, s); LC/MS m/z: 301 (MH+); Anal. Calcd for C13H11N2O3F3: C,
52.01; H, 3.69; N, 9.33. Found: C, 51.97; H, 3.71; N, 9.33.
The following compounds 10i–k were prepared from the corre-
sponding starting materials in a similar manner to that described
for 10l.
5.1.25. Ethyl 3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (9)
A mixture of ethyl 3,3,3-trifluoro-2-oxo-butanoate 8 (64.8 g,
0.352 mol), triethyl orthoformate (117 ml, 0.703 mol) and acetic
anhydride (100 ml, 1.06 mol) was stirred at 120 °C for 2 h and at
140 °C for 5 h. The reaction mixture was concentrated in vacuo.
The residue was dissolved in methanol (500 ml), hydrazine hydrate
(25.6 ml, 0.528 mol) added, and the reaction heated at reflux for
3 h. The reaction mixture was concentrated in vacuo. The residue
was extracted with ethyl acetate, washed with water and brine,
dried over anhydrous magnesium sulfate and concentrated in va-
cuo. The residue was recrystallized from ethyl acetate–hexane to
give 9 (50 g, 68%) as a white solid. 1H NMR (DMSO-d6) d: 1.28
(4H, t, J = 7.1 Hz), 4.25 (2H, q, J = 7.0 Hz), 8.59 (1H, s), 14.13 (1H,
br s); LC/MS m/z: 209 (MH+).
5.1.31. Ethyl 1-(pyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-
4-carboxylate (10i)
Solid (yield 87%); 1H NMR (CDCl3) d 1.39 (3H, t, J = 7.4 Hz), 4.37
(2H, q, J = 7.0 Hz), 7.34 (1H, m), 7.90 (1H, m), 8.06 (1H, m), 8.48
(1H, m), 9.13 (1H, m).
5.1.26. Ethyl 1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylate (10a)
To a solution of 9 (15.2 g, 73.0 mmol) in DMF were added phen-
ylboronic acid (17.8 g, 146 mmol), copper(II) acetate (19.9 g,
110 mmol) and pyridine (11.8 ml, 146 mmol) and the reaction stir-
red for 24 h in air. The precipitate was removed by filtration with
Celite. The filtrate was concentrated in vacuo. The residue was ex-
tracted with ethyl acetate, washed with saturated aqueous sodium
hydrogen carbonate solution and brine, dried over anhydrous
magnesium sulfate and concentrated in vacuo. The residue was
purified by silica gel column chromatography to give 10a (15.5 g,
75%) as a white solid. 1H NMR (CDCl3) d: 1.39 (3H, t, J = 7.0 Hz),
4.37 (2H, q, J = 7.0 Hz), 7.42 (1H, m), 7.52 (2H, m), 7.72 (2H, m),
8.48 (1H, m).
5.1.32. Ethyl 1-(pyridin-3-yl)-3-(trifluoromethyl)-1H-pyrazole-
4-carboxylate (10j)
Solid (yield 74%); 1H NMR (CDCl3) d: 1.40 (3H, t, J = 7.4 Hz), 4.38
(2H, q, J = 7.4 Hz), 7.49 (1H, m), 8.11 (1H, m), 8.54 (1H, m), 8.69
(1H, m), 9.02 (1H, m).
5.1.33. Ethyl 1-(pyridin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-
4-carboxylate (10k)
Solid (yield 20%); 1H NMR (CDCl3) d: 1.39 (3H, t, J = 7.2 Hz), 4.39
(2H, q, J = 7.2 Hz), 7.68–7.73 (2H, m), 8.63 (1H, d, J = 1.1 Hz), 8.74–
8.81 (2H, m); LC/MS m/z: 286 (MH+).
The following compounds 10f–h were prepared from the corre-
sponding starting materials in a similar manner to that described
for 10a.
5.1.34. 1-Phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic
acid (6a)
To a solution of 10a (15.5 g, 54.5 mmol) in ethanol (150 ml) and
THF (50 ml) was added 2 N NaOH (110 ml, 220 mmol). The mixture
was stirred at 50 °C for 1 h. The reaction mixture was concentrated
in vacuo. The residue was extracted with water and washed with
diethyl ether. The water layer was acidified by 6 N hydrochloric acid
(36.7 ml, 220 mmol) and extracted with ethyl acetate. The extracts
were washed with brine, dried over anhydrous sodium sulfate and
concentrated in vacuo. The obtained solid was collected and
washed with water to give 6a (11.8 g, 84%) as a white solid. mp
182–184 °C; 1H NMR (DMSO-d6) d: 7.47 (1H, t, J = 7.3 Hz), 7.54–
7.61 (2H, m), 7.91–7.98 (2H, m), 9.23 (1H, d, J = 0.9 Hz), 13.23 (1H,
s); LC/MS m/z: 279 (M+Na+); Anal. Calcd for C11H7N2O2F3ꢂ0.2H2O:
C, 50.86; H, 2.87; N, 10.78. Found: C, 50.81; H, 2.57; N, 10.81.
The following compounds 6f–l were prepared from the corre-
sponding starting materials in a similar manner to that described
for 6a.
5.1.27. Ethyl 1-(4-chlorophenyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylate (10f)
Solid (yield 87%); mp 54–55 °C; 1H NMR (CDCl3) d: 1.39 (3H, t,
J = 7.0 Hz), 4.37 (2H, q, J = 7.0 Hz), 7.49 (2H, m), 7.68 (2H, m),
8.47 (1H, m); Anal. Calcd for C13H10N2O2ClF3: C, 49.00; H, 3.16;
N, 8.79. Found: C, 49.05; H, 3.11; N, 8.87.
5.1.28. Ethyl 1-(4-ethoxyphenyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylate (10g)
Solid (yield 82%); mp 102 °C; 1H NMR (CDCl3) d: 1.38 (3H, t,
J = 7.2 Hz), 1.44 (3H, t, J = 7.0 Hz), 4.08 (2H, q, J = 7.2 Hz), 4.36
(2H, q, J = 7.2 Hz), 6.94–7.03 (2H, m), 7.55–7.64 (2H, m), 8.37 (1
H, s); LC/MS m/z: 329 (MH+); Anal. Calcd for C15H15N2O3F3: C,
54.88; H, 4.61; N, 8.53. Found: C, 54.94; H, 4.57; N, 8.66.
5.1.29. Ethyl 1-(3-ethoxyphenyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylate (10h)
5.1.35. 1-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6f)
Solid (yield 72%); 1H NMR (CDCl3) d: 1.38 (3H, t, J = 7.0 Hz), 1.45
(3H, t, J = 7.0 Hz), 4.10 (2H, q, J = 7.0 Hz), 4.36 (2H, q, J = 7.0 Hz),
6.93 (1H, m), 7.23 (1H, m), 7.28 (1H, m), 7.38 (1H, t, J = 8.2 Hz),
8.45 (1H, m).
Solid (yield 29%); mp 192–193 °C; 1H NMR (CDCl3) d: 7.64 (2H,
m), 7.98 (2H, m), 9.26 (1H, s), 13.27 (1H, br s); Anal. Calcd for
C11H6N2O2ClF3ꢂ0.3H2O: C, 44.63; H, 2.25; N, 9.46. Found: C,
44.47; H, 2.22; N, 9.50.
5.1.30. Ethyl 1-(2-hydroxyphenyl)-3-(trifluoromethyl)-1H-
pyrazole-4-carboxylate (10l)
To a 50 mL sealed tube flushed with argon were added copper(I)
iodide (0.0229 g, 0.120 mmol), potassium carbonate (0.697 g,
5.1.36. 1-(4-Ethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6g)
White solid (yield 78%); mp 176–177 °C; 1H NMR (CDCl3) d:
1.35 (3H, t, J = 7.0 Hz), 4.09 (2H, q, J = 7.1 Hz), 7.05–7.12 (2H, m),

Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
2793
7.78–7.86 (2H, m), 9.10 (1H, d, J = 0.8 Hz), 13.15 (1H, br s); LC/MS
m/z: 301 (MH+); Anal. Calcd for C13H11N2O3F3ꢂ0.1H2O: C, 51.38; H,
3.68; N, 9.22. Found: C, 51.13; H, 3.69; N, 8.91.
q, J = 7.0 Hz), 4.70–4.76 (1H, m), 6.89–6.94 (2H, m), 7.70–7.83
(2H, m); LC/MS m/z: 267 (MH+ꢀBoc).
5.1.44. (R)-Methyl N-[(benzyloxy)carbonyl]-3-({[1-phenyl-3-
(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}amino)alaninate
(12n)
5.1.37. 1-(3-Ethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6h)
White solid (yield 90%); 1H NMR (DMSO-d6) d: 3.50–3.70 (2H,
m), 3.64 (3H, s), 4.28–4.35 (1H, m), 5.05 (2H, s), 7.25–7.37 (5H,
m), 7.45–7.51 (1H, m), 7.58–7.63 (2H, m), 7.70–7.75 (1H, m),
7.78–7.83 (2H, m), 8.48 (1H, t, J = 5.9 Hz), 9.03 (1H, s).
Solid (yield 75%); mp 177–178 °C; 1H NMR (CDCl3) d: 1.36 (3H,
t, J = 7.0 Hz), 4.13 (2H, q, J = 7.0 Hz), 7.00 (1H, m), 7.47 (3H, m), 9.27
(1H, m); Anal. Calcd for C13H11N2O3F3: C, 52.01; H, 3.69; N, 9.33.
Found: C, 51.83; H, 3.63; N, 9.34.
5.1.45. (R)-Methyl N-[(benzyloxy)carbonyl]-3-{[(4-ethoxyphenyl)-
carbonyl]amino}alaninate (12p)
5.1.38. 1-(Pyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6i)
1H NMR (DMSO-d6) d: 1.34 (3H, t, J = 7.1 Hz), 3.61–3.54 (2H, m),
3.61 (3H, s), 4.08 (2H, q, J = 7.1 Hz), 4.33–4.26 (1H, m), 5.07–4.98
(2H, m), 7.00–6.95 (2H, m), 7.37–7.29 (5H, m), 7.79–7.71 (3H,
m), 8.41–8.35 (1H, m).
Solid (yield 95%); mp 231–232 °C; 1H NMR (CDCl3) d: 7.55 (1H, m),
7.99 (1H, m), 8.11 (1H, m), 8.58 (1H, m), 9.10 (1H, m), 13.33 (1H, br s);
Anal. Calcd for C10H6N3O2F3: C, 46.70; H, 2.35; N, 16.34. Found: C,
46.46; H, 2.49; N, 16.15.
5.1.46. tert-Butyl (2-{[(4-ethoxyphenyl)carbonyl]amino}-
propyl)carbamate (12q)
5.1.39. 1-(Pyridin-3-yl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6j)
White solid (yield, 93%); 1H NMR (CDCl3) d: 1.23 (3H, d,
J = 6.2 Hz), 1.39 (9H, s), 1.42 (3H, t, J = 7.0 Hz), 3.15–3.23 (1H, m),
3.30–3.40 (1H, m), 4.06 (2H, q, J = 7.0 Hz), 4.14–4.20 (1H, m),
5.04 (1H, t, J = 6.6 Hz), 6.86–6.90 (2H, m), 6.99 (1H, d, J = 6.6 Hz),
7.75–7.80 (2H, m); LC/MS m/z: 223 (MH+ꢀBoc).
Solid (yield 96%); mp 216–217 °C; 1H NMR (CDCl3) d: 7.62 (1H, m),
8.34 (1H, m), 8.66 (1H, m), 9.17 (1H, br s), 9.33 (1H, m); Anal. Calcd for
C10H6N3O2F3: C, 46.70; H, 2.35; N, 16.34. Found: C, 46.68; H, 2.35; N,
16.34.
5.1.47. tert-Butyl [2-({[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbonyl}amino)propyl]carbamate (12r)
White solid (yield 88%); 1H NMR (CDCl3) d: 1.25 (3H, d,
J = 6.6 Hz), 1.40 (9H, s), 3.25–3.30 (2H, m), 4.16–4.24 (1H, m),
4.95–5.01 (1H, m), 6.74–6.79 (1H, m), 7.38–7.43 (1H, m), 7.47–
7.52 (2H, m), 7.66–7.71 (2H, m), 8.39 (1H, s); LC/MS m/z: 313
(MH+ꢀBoc).
5.1.40. 1-(Pyridin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylic acid (6k)
Solid (yield 70%); mp 310–311 °C; 1H NMR (CDCl3) d: 8.01 (2H,
m), 8.75 (2H, m), 9.47 (1H, m); Anal. Calcd for C10H6N3O2F3: C,
46.70; H, 2.35; N, 16.34. Found: C, 46.35; H, 2.34; N, 16.26.
5.1.41. 1-(2-Hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazole-
4-carboxylic acid (6l)
5.1.48. tert-Butyl [2-({[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbonyl}amino)ethyl]carbamate (12s)
White solid (4.1 g, 86%); 1H NMR (CDCl3) d: 1.43 (9H, s), 3.37
(2H, q, J = 5.8 Hz), 3.56 (2H, q, J = 5.4 Hz), 4.98 (1H, s), 6.84 (1H,
s), 7.37–7.44 (1H, m), 7.47–7.54 (2H, m), 7.69 (2H, d, J = 7.9 Hz),
8.41 (1H, s). LC/MS m/z: 421 (MNa+).
White solid (yield 73%); mp 223–224 °C; 1H NMR (DMSO-d6) d:
6.98 (1H, td, J = 7.6, 1.3 Hz), 7.11 (1H, dd, J = 8.3, 1.1 Hz), 7.28–7.37
(1H, m), 7.58 (1H, dd, J = 7.9, 1.9 Hz), 8.78 (1H, d, J = 1.1 Hz), 10.64
(1H, br s.), 13.10 (1H, br s.); LC/MS m/z: 273 (MH+); Anal. Calcd for
C11H7N2O3F3: C, 48.54; H, 2.59; N, 10.29. Found: C, 48.51; H, 2.59;
N, 10.29.
5.1.49. (R)-Methyl 2-amino-3-(1-phenyl-3-(trifluoromethyl)-
1H-pyrazole-4-carboxamido)propanoate (13n)
5.1.42. (S)-Methyl 3-[(tert-butoxycarbonyl)amino]-N-{[1-
phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}alaninate
(12o)
To a solution of 12n (4.50 g, 9.20 mmol) in dry THF (300 mL)
was added 10%wt palladium on charcoal (dry) (1.00 g) under argon
atmosphere. The mixture was shaken under hydrogen (45 psi) for
4 days at room temperature. The palladium was filtered off and
the filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography (DCM/MeOH = 95:5) to give
13n (2.57 g, 84%) as a white solid. 1H NMR (DMSO-d6) d: 3.17
(1H, s), 3.18 (1H, s), 3.40–3.45 (2H, m), 3.62 (3H, s), 4.06–4.12
(1H, m), 7.45–7.50 (1H, m), 7.58–7.63 (2H, m), 7.81–7.85 (2H,
m), 8.37 (1H, t, J = 5.9 Hz), 9.09 (1H, s).
To a mixture of 11i (1.00 g, 3.90 mmol) in DCM (50 mL) were
added successively 6a (1.0 g, 3.90 mmol), EDCI (0.9 g, 4.71 mmol),
HOBtꢂH2O (0.690 g, 5.10 mmol) and triethylamine (0.800 g,
7.90 mmol). The mixture was stirred at room temperature for
12 h and 2 N HCl was added (100 mL). The organic layer was iso-
lated and washed with 10% aqueous K2CO3 and brine. The com-
bined organic layers were dried over MgSO4, concentrated and
purified by silica gel column chromatography (DCM/MeOH 99:1)
to give 12o (1.2 g, 67%) as a white solid. 1H NMR (CDCl3) d: 1.42
(9H, s), 3.64–3.68 (2H, m), 3.80 (3H, s), 4.74–4.82 (1H, m), 4.91–
4.97 (1H, m), 7.32–7.38 (1H, m), 7.39–7.44 (1H, m), 7.48–7.54
(2H, m), 7.69–7.72 (2H, m), 8.44 (1H, br); LC/MS m/z: 357
(MH+ꢀBoc).
5.1.50. (R)-Methyl 3-{[(4-ethoxyphenyl)carbonyl]amino}-
alaninate (13p)
Compound 13p was prepared from 12p in a similar manner to
that described for 13n. 1H NMR (DMSO-d6) d: 1.34 (3H, t,
J = 7.1 Hz), 3.33 (1H, br s), 3.45–3.35 (2H, m), 3.56–3.51 (1H, m),
3.59 (3H, s), 4.08 (2H, q, J = 7.1 Hz), 6.99–6.94 (2H, m), 7.70–7.77
(2H, m), 8.35–8.30 (1H, m).
The following compounds 12m, n, p–s were prepared from the
corresponding starting materials in a similar manner to that de-
scribed for 12o.
5.1.43. (S)-Methyl 3-[(tert-butoxycarbonyl)amino]-N-[(4-
ethoxyphenyl)carbonyl]alaninate (12m)
5.1.51. (S)-Methyl 3-amino-N-{[1-phenyl-3-(trifluoromethyl)-
1H-pyrazol-4-yl]carbonyl}alaninate (13o)
To 12o (1.0 g, 2.2 mmol) was added TFA (10 mL). The mixture
was stirred at room temperature for 1 h and concentrated in vacuo.
White solid (yield 60%); 1H NMR (CDCl3) d: 1.26 (3H, t,
J = 7.0 Hz), 1.42 (9H, s), 3.60–3.70 (2H, m), 3.78 (3H, s), 4.12 (2H,

2794
Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
The residue was dissolved in water (50 mL) and sodium carbonate
was added until alkaline. After stirring 1 h at room temperature,
the aqueous layer was extracted with DCM. The combined organic
layers were dried over MgSO4 and concentrated to give 13o
(780 mg, quant.): 1H NMR (CDCl3) d: 3.17 (1H, dd, J = 13.3,
4.7 Hz), 3.25 (1H, dd, J = 13.3, 3.9 Hz), 3.81 (3H, s), 4.78 (1H, dt,
J = 7.0, 4.3 Hz), 7.17 (1H, br s), 7.39–7.44 (1H, m), 7.48–7.54 (2H,
m), 7.69–7.73 (2H, m), 8.49 (1H, s).
5.1.57. (R)-N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}-3-
hydroxypropyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide (3n)
White solid (yield 85%); mp 209–212 °C; ½a D25
ꢃ
+87.9° (c 0.951,
methanol); 1H NMR (DMSO-d6) d: 1.33 (3H, t, J = 7.0 Hz), 3.39–
3.60 (4H, m), 4.07 (2H, q, J = 7.0 Hz), 4.08–4.16 (1H, m), 4.79 (1H,
t, J = 5.8 Hz), 6.96 (2H, d, J = 9.0 Hz), 7.47 (1H, tt, J = 7.4, 1.5 Hz),
7.56–7.63 (2H, m), 7.77–7.84 (4H, m), 7.97 (1H, d, J = 7.4 Hz),
8.44 (1H, t, J = 5.9 Hz), 9.04 (1H, s); LC/MS m/z: 459 (M+ꢀH2O);
Anal. Calcd for C23H23N4O4F3: C, 57.98; H, 4.87; N, 11.76. Found:
C, 58.11; H, 4.76; N, 11.76.
The following compounds 13m, q, r were prepared from the
corresponding starting materials in a similar manner to that de-
scribed for 13o.
5.1.58. (R)-N-[2-{[(4-Ethoxyphenyl)carbonyl]amino}-1-
(hydroxymethyl)ethyl]-1-phenyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (3p)
5.1.52. (S)-Methyl 3-amino-N-[(4-
ethoxyphenyl)carbonyl]alaninate (13m)
Clear paste (quant.); 1H NMR (400 MHz, DMSO-d6) d: 1.35 (3H,
t, J = 7.0 Hz), 3.19–3.40 (2H, m), 3.69 (3H, s), 4.10 (2H, q, J = 7.0 Hz),
4.74–4.80 (1H, m), 7.00–7.05 (2H, m), 7.85–7.90 (2H, m), 8.01 (2H,
br s), 8.20 (1H, d, J = 8.2 Hz).
White solid (yield 88%); mp 223–225 °C; ½a D25
ꢃ
+88.6° (c 0.957,
methanol); 1H NMR (DMSO-d6) d: 1.33 (3H, t, J = 7.0 Hz), 3.35–
3.57 (4H, m), 4.06 (2H, q, J = 7.0 Hz), 4.10–4.18 (1H, m), 4.83 (1H,
t, J = 5.8 Hz), 6.96 (2H, d, J = 9.0 Hz), 7.48 (1H, tt, J = 7.4, 1.6 Hz),
7.57–7.64 (2H, m), 7.76–7.84 (4H, m), 8.06 (1H, d, J = 8.2 Hz),
8.38 (1H, t, J = 5.9 Hz), 9.11 (1H, s); LC/MS m/z: 477 (MH+), 459
(M+ꢀH2O); Anal. Calcd for C23H23N4O4F3: C, 57.98; H, 4.87; N,
11.76. Found: C, 58.06; H, 4.70; N, 11.84.
5.1.53. N-(2-Amino-1-methylethyl)-4-ethoxybenzamide (13q)
Clear oil (quant.); 1H NMR (CDCl3) d: 1.22 (3H, d, J = 7.0 Hz),
1.39 (3H, t, J = 7.0 Hz), 3.02–3.11 (2H, m), 3.99 (2H, q, J = 7.0 Hz),
4.35–4.43 (1H, m), 6.77–6.82 (2H, m), 7.39 (1H, d, J = 8.6 Hz),
7.64–7.69 (2H, m), 8.03 (2H, br s); LC/MS m/z: 223 (MH+).
5.1.59. N-(2-Aminoethyl)-1-phenyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide hydrochloride (13s)
5.1.54. N-(2-Amino-1-methylethyl)-1-phenyl-3-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (13r)
White solid (quant.); 1H NMR (CDCl3) d: 1.22 (3H, d, J = 6.6 Hz),
3.10–3.20 (2H, m), 4.40–4.50 (1H, m), 6.35 (2H, br s), 7.30–7.43
(3H, m), 7.47 (1H, d, J = 7.8 Hz), 7.58–7.63 (2H, m), 8.67 (1H, s);
LC/MS m/z: 313 (MH+).
To a solution of 12s (4.10 g, 10.3 mmol) in ethyl acetate (40 ml)
was added 4 N hydrogen chloride in ethyl acetate (25.7 ml,
103 mmol) and stirring continued for 24 h. The reaction mixture
was concentrated in vacuo and the residue was collected by filtra-
tion to give 13s (3.40 g, 99%) as a white solid. Compound 13s was
used for next step without further purification. 1H NMR (DMSO-d6)
d: 3.02 (2H, d, J = 5.3 Hz), 3.52 (2H, q, J = 5.8 Hz), 7.48 (1H, t,
J = 7.4 Hz), 7.58–7.65 (2H, m), 7.82–7.88 (2H, m), 8.21 (3H, s),
8.95 (1H, t, J = 5.4 Hz), 9.59 (1H, d, J = 0.9 Hz). LC/MS m/z: 321
(MNa+).
5.1.55. (S)-N-[2-{[(4-Ethoxyphenyl)carbonyl]amino}-1-
(hydroxymethyl)ethyl]-1-phenyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide (3o)
To a solution of 14o (647 mg, 1.30 mmol) in anhydrous THF/
EtOH (2:1, 10 mL) was added lithium chloride (109 mg, 2.60
mmol). Upon dissolution, sodium borohydride (97 mg, 2.60 mmol)
was added and the mixture was stirred at room temperature for
18 h. The mixture was concentrated to dryness, water (50 mL)
was added and the pH was brought to 2–3 by addition of 2 N
HCl. The aqueous layer was extracted with DCM (3 ꢁ 50 mL) and
the combined organic layers were dried over MgSO4. Concentration
5.1.60. N-[2-({[1-Phenyl-3-(trifluoromethyl)-1H-pyrazol-4-
yl]carbonyl}amino)ethyl]-6-(2,2,2-trifluoroethoxy)pyridine-3-
carboxamide (3x)
To a solution of 2,2,2-trifluoroethanol (1.00 g, 10.0 mmol) in dry
THF (8 ml) was added 60% NaH in oil (320 mg, 8.00 mmol) and the
mixture was stirred for 30 min at room temperature. 3v (876 mg,
2.00 mmol) was added and the mixture was stirred at 75 °C for
4 h. The solution was then cooled and concentrated. The residue
was diluted with water and extracted with ethyl acetate. The or-
ganic layer was then dried over anhydrous sodium sulfate and con-
centrated in vacuo. The residue was recrystallized from ethyl
acetate–hexane to give 3x (968 mg, 97%) as a white solid. mp
155–156 °C; 1H NMR (DMSO-d6) d: 3.36–3.49 (4H, m), 5.06 (2H,
q, J = 9.1 Hz), 7.04–7.11 (1H, m), 7.44–7.52 (1H, m), 7.56–7.65
(2H, m), 7.75–7.86 (2H, m), 8.21 (1H, dd, J = 8.7, 2.3 Hz), 8.50
(1H, br s.), 8.63–8.76 (2H, m), 9.07 (1H, d, J = 0.8 Hz); LC/MS m/z:
502 (MH+); Anal. Calcd for C21H17N5O3F6: C, 50.31; H, 3.42; N,
13.97. Found: C, 50.55; H, 3.45; N, 13.86.
yielded 3o (450 mg, 74%) as a white solid. mp 220–222 °C; ½a D25
ꢃ
ꢀ82.4° (c 0.965, methanol); 1H NMR (DMSO-d6) d 1.32 (3H, t,
J = 7.0 Hz), 3.40 (1H, m), 3.45–3.68 (3H, m), 4.07 (2H, q, J =
7.0 Hz), 4.13 (1H, m), 6.96 (2H, m), 7.48 (1H, m), 7.61 (2H, m),
7.78–7.82 (4H, m), 8.08 (1H, d, J = 8.2 Hz), 8.40 (1H, t, J = 5.9 Hz),
9.13 (1H, s); LC/MS m/z: 477 (MH+); Anal. Calcd for C23H23N4
O4F3ꢂ0.2H2O: C, 57.55; H, 4.91; N, 11.67. Found: C, 57.36; H,
4.93; N, 11.69.
The following compounds 3m, n, p were prepared from the cor-
responding starting materials in a similar manner to that described
for 3o.
The compound 3w was prepared from the corresponding start-
ing materials in a similar manner to that described for 3x.
5.1.56. (S)-N-(2-{[(4-Ethoxyphenyl)carbonyl]amino}-3-
hydroxypropyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide (3m)
5.1.61. 6-Ethoxy-N-[2-({[1-phenyl-3-(trifluoromethyl)-1H-
pyrazol-4-yl]carbonyl}amino)ethyl]pyridine-3-carboxamide
(3w)
White solid (yield 63%); mp 211–212 °C; ½a D25
ꢀ88.5° (c 0.986,
ꢃ
methanol); 1H NMR (DMSO-d6) d: 1.33 (3H, t, J = 7.0 Hz), 3.40–
3.60 (4H, m), 4.07 (2H, q, J = 7.0 Hz), 4.13 (1H, m), 4.60 (1H, br s),
6.96 (2H, m), 7.48 (1H, m), 7.61 (2H, m), 7.78–7.82 (4H, m), 8.00
(1H, d, J = 7.4 Hz), 8.48 (1H, t, J = 5.9 Hz), 9.08 (1H, s); LC/MS m/z:
459 (M+ꢀH2O); Anal. Calcd for C23H23N4O4F3ꢂ0.4H2O: C, 57.12; H,
4.96; N, 11.58. Found: C, 56.83; H, 4.94; N, 11.55.
White solid (yield 48%); mp 179–181 °C; 1H NMR (DMSO-d6) d:
1.32 (3H, t, J = 7.0 Hz), 3.42 (4H, br s), 4.33–4.38 (2H, m), 6.85 (1H,
d, J = 8.8 Hz), 7.48 (1H, t, J = 7.3 Hz), 7.61 (2H, t, J = 7.9 Hz), 7.81
(2H, d, J = 7.8 Hz), 8.09–8.12 (1H, m), 8.48 (1H, br s), 8.59 (1H, br
s), 8.64 (1H, s), 9.06 (1H, s); LC/MS m/z: 448 (MH+); Anal. Calcd

Y. Nakada et al. / Bioorg. Med. Chem. 18 (2010) 2785–2795
2795
for C21H20N5O3F3: C, 56.37; H, 4.51; N, 15.65. Found: C, 56.45; H,
4.55; N, 15.67.
with a final volume of 0.1 mL consisted of microsomal protein in
50 mmol/L KH2PO4–K2HPO4 phosphate buffer (pH 7.4) and
1 lmol/L test compound. The concentration of hepatic microsomal
5.2. Biology
protein was 0.2 mg/mL. An NADPH-generating system containing
50 mmol/L MgCl2, 50 mmol/L glucose-6-phosphate, 5 mmol/L
beta-NADP+ and 15 unit/mL glucose-6-phosphate dehydrogenase
was prepared and added to the incubation mixture with a 10% vol-
ume of the reaction mixture. After the addition of the NADPH-gen-
erating system, the mixture was incubated at 37 °C for 0 and
20 min. The reaction was terminated by the addition of acetonitrile
equivalent to the volume of the reaction mixture. All incubations
were made in duplicate. Test compound in the reaction mixture
was measured by HPLC system equipped with a UV detector.
For metabolic stability determinations, chromatograms were ana-
lyzed for parent compound disappearance from the reaction
mixtures.
5.2.1. Assay of hDGAT-1 inhibitory activity in vitro
Human DGAT-1 (hDGAT-1) was expressed in Sf9 insect cells
using a baculovirus expression system. The microsome of the Sf9
cells was obtained as an enzyme source.
The hDGAT-1 reaction mixtures contained 100 mM Tris–HCl
(pH 7.5), 250 mM sucrose, 150 mM MgCl2, 0.01% bovine serum
albumin (BSA fatty acid free), 1% acetone, 5
of Sf9 expressing hDGAT-1 and 25 M 1,2-dioleoyl-sn-glycerol at
lg/mL the microsome
l
the final concentration. For inhibitor testing, serial dilutions of
the compounds were added to the reaction mixture in the final
concentration of 0.1% DMSO. The reaction was initiated by adding
25 l
M [14C]-Oleoyl-CoA at the final concentration. The reactions
were performed in a 96-well plate in a final volume of 100
lL
Acknowledgments
and were terminated after 15 min at 32 °C by addition of 300
lL
of chloroform/methanol (1:2 by volume) mixed solvent. After mix-
ing the solution, 200 L of phosphate buffer saline (PBS) was added
to facilitate phase separation. After centrifugation (3000 rpm,
3 min), 50 L of the chloroform layer was spotted on a thin layer
We thank Dr. K. Kubo for encouragement and helpful advice.
We thank Mr. Y. Nakano for technical assistance.
l
l
chromatography (TLC) plate. Lipids were separated by TLC with a
solvent system of n-hexane/diethyl ether/ethyl acetate/acetic acid
(74:15:15:1 by volume). The radioactivity of [14C]-triglyceride was
counted with BAS-2500 imaging system (Fujifilm) to calculate the
hDGAT-1 activity or inhibition rates of the compounds.
References and notes
1. Cases, S.; Smith, S. J.; Zheng, Y.; Myers, H. M.; Lear, S. R.; Sande, E.; Novak, S.;
Collins, C.; Welch, C. B.; Lusis, A. J.; Erickson, S. K.; Farese, R. V., Jr. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 13018.
2. Cases, S.; Stone, S. J.; Zhou, P.; Yen, E.; Tow, B.; Lardizabal, K. D.; Voelker, T.;
Farese, R. V., Jr. J. Biol. Chem. 2001, 276, 38870.
5.2.2. Assay of intracellular DGAT inhibitory activity in mouse
myoblast C2C12 cell line in vitro
C2C12 cells were cultured with Dulbecco’s Modified Eagle Med-
ium (D-MEM) supplemented with 10% FBS, 50 U/mL of penicillin
3. Smith, S. J.; Cases, S.; Jensen, D. R.; Chen, H. C.; Sande, E.; Tow, B.; Sanan, D. A.;
Raber, J.; Eckel, R. H.; Farese, R. V., Jr. Nat. Genet. 2000, 25, 87.
4. Chen, H. C.; Smith, S. J.; Ladha, Z.; Jensen, D. R.; Ferreira, L. D.; Pulawa, L. K.;
McGuire, J. G.; Pitas, R. E.; Eckel, R. H.; Farese, R. V., Jr. J. Clin. Invest. 2002, 109,
1049.
5. Chen, H. C.; Stone, S. J.; Zhou, P.; Buhman, K. K.; Farese, R. V., Jr. Diabetes 2002,
51, 3189.
and 50 lg/mL of streptomycin for two days as myoblast cells with-
out differentiation to myotube and were treated by trypsine-EDTA,
following were suspended in phosphate buffer saline(PBS). The se-
rial dilutions of compound were added in the C2C12 cells suspen-
sion and incubated at 37 °C for 20 min. Then, [14C]-Oleic acid was
added as a substrate of DGAT reaction. The reactions were per-
6. Zhao, G.; Souers, A. J.; Voorbach, M.; Falls, H. D.; Droz, B.; Brodjian, S.; Lau, Y. Y.;
Iyengar, R. R.; Gao, J.; Judd, A. S.; Wagaw, S. H.; Ravn, M. M.; Engstrom, K. M.;
Lynch, J. K.; Mulhern, M. M.; Freeman, J.; Dayton, B. D.; Wang, X.; Grihalde, N.;
Fry, D.; Beno, D. W. A.; Marsh, K. C.; Su, Z.; Diaz, G. J.; Collins, C. A.; Sham, H.;
Reilly, R. M.; Brune, M. E.; Kym, P. R. J. Med. Chem. 2008, 51, 380.
7. Birch, A. M.; Birtles, S.; Buckett, L. K.; Kemmitt, P. D.; Smith, G. J.; Smith, T. J. D.;
Turnbull, A. V.; Wang, S. J. Y. J. Med. Chem. 2009, 52, 1558.
8. Nakada, Y.; Ogino, M.; Asano, K.; Aoki, K.; Miki, H.; Yamamoto, T.; Kato, K.;
Masago, M.; Tamura, N.; Shimada, M. Chem. Pharm. Bull. 2010, 58, in press.
9. Guengerich, F. P.; MacDonald, J. S. Chem. Res. Toxicol. 2007, 20, 344.
10. Hobbs, D. C. Drug Metab. Dispos. 1977, 5, 75.
11. Onderwater, R. C.; Commandeur, J. N.; Menge, W. M.; Vermeulen, N. P. Chem.
Res. Toxicol. 1999, 12, 396.
12. Schwab, C. E.; Huber, W. W.; Parzefall, W.; Hietsch, G.; Kassie, F.; Schulte, -H.
R.; Knasmuller, S. Crit. Rev. Toxicol. 2000, 30, 1.
13. 3-Methyl-1-phenylpyrazole-4-carboxylic acid 6b was prepared as described in
Abdelilah, B.; Michel, H.-H. Bull. Soc. Chim. Fr. 1988, 3, 540.
14. 4-Methyl-1-phenylpyrazole-3-carboxylic acid 6c was prepared as described in
Martins, M. A. P.; Freitag, R. A.; Rosa, A. da.; Flores, A. F. C.; Zanatte, N.;
Bonacorso, H. G. J. Hetrocycl. Chem. 1999, 36, 217.
formed in a 96-well plate in a final volume of 100
tration of 106 C2C12 cells/mL, 5 M [14C]-Oleic acid, 0.001% BSA
(fatty acid free) and 0.2% ethanol and 0.1% DMSO. The reactions
were terminated after 20 min at 37 °C by addition of 300 L of
chloroform/methanol (1:2 by volume) mixed solvent. After mixing
the solution, 200 L of DW was added to facilitate phase separa-
tion. After centrifugation (2000 rpm, 2 min), 45 L of the chloro-
lL at the concen-
l
l
l
l
form layer was spotted on a thin layer chromatography (TLC)
plate. Lipids were separated by TLC with a solvent system of n-hex-
ane/diethyl ether/ethyl acetate/acetic acid (255:30:15:0.6 by vol-
ume). The radioactivity of [14C]-triglyceride was counted with
BAS-2500 imaging system (Fujifilm) to calculate intracellular
DGAT inhibitory activity of the compounds.
15. 3-(Trifluoromethyl)-1H-pyrazole-4-carboxylic acid 6d was prepared as
described in Kazmierski, W. M.; Aquino, C. J.; Bifulco, N.; Boros, E. E.;
Chauder, B. A.; Chong, P. Y.; Duan, M.; Deanda, Jr. F.; Koble, C. S.; Mclean, E.
W. Patent WO 2004/054974.
16. Compounds 10a, f, g, h: Lam, P. Y. S.; Clarkt, C. G.; Saubernt, S.; Adamst, J.;
Winters, M. P.; Chan, D. M. T.; Combst, A. Tetrahedron Lett. 1998, 39, 2941.
17. Compounds 10i–l: Antilla, J. C.; Baskin, J. M.; Barder, T. E.; Buchwald, S. L. J. Org.
Chem. 2004, 69, 5578.
5.2.3. Metabolic stability assay
Hepatic microsomes from mice, rats and humans were pur-
chased from Xenotech, LLC (Lenexa, KS). An incubation mixture