A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: Design and synthesis of benzylpyrazole acylsulfonamides

Article abstract of DOI:10.1016/j.bmc.2011.12.008

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.

Full text of DOI:10.1016/j.bmc.2011.12.008

Bioorganic & Medicinal Chemistry 20 (2012) 714–733
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A new class of non-thiazolidinedione, non-carboxylic-acid-based highly
selective peroxisome proliferator-activated receptor (PPAR)
Design and synthesis of benzylpyrazole acylsulfonamides
c agonists:
Kentaro Rikimaru a, , , Takeshi Wakabayashi a, , Hidenori Abe a, , Hiroshi Imoto a, , Tsuyoshi Maekawa a,
,
⇑
Osamu Ujikawa a, , Katsuhito Murase b, Takanori Matsuo a,à, Mitsuharu Matsumoto a,à, Chisako Nomura a,à
,
Hiroko Tsuge a,à, Naoto Arimura a,à, Kazutoshi Kawakami a,§, Junichi Sakamoto a,§, Miyuki Funami a,§
Clifford D. Mol c, Gyorgy P. Snell c, Kenneth A. Bragstad c, Bi-Ching Sang c, Douglas R. Dougan c,
Toshimasa Tanaka a,§, Nozomi Katayama a,à, Yoshiaki Horiguchi a, ,||, Yu Momose a,–
,
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
b Takeda Pharmaceuticals International Inc., One Takeda Parkway, Deerfield, IL 60015, USA
c Takeda San Diego Inc., 10410 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe the design, synthesis, and structure–activity relationships of novel benzylpyrazole
acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-
Received 3 October 2011
Revised 3 December 2011
Accepted 5 December 2011
Available online 13 December 2011
activated receptor (PPAR)
c agonists. Docking model analysis of in-house weak agonist 2 bound to the
reported PPAR ligand binding domain suggested that modification of the carboxylic acid of 2 would help
c
strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this
study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of
carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl
group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent
agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we
have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfona-
mide 3d.
Keywords:
PPARc agonist
Diabetes mellitus
SBDD
Acylsulfonamide
Isosteric replacement
Ó 2011 Published by Elsevier Ltd.
1. Introduction
countries, and the number of diabetics is expected to reach 380 mil-
lion by 2025.2 Therefore, there is an urgent need to develop new and
Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder
characterized by chronic hyperglycemia resulting from insulin resis-
tance and impaired insulin secretion, leading to abnormal metabo-
lism of not only glucose but also lipids and amino acids. Long-term
microvascular and macrovascular complications such as neuropa-
thy, retinopathy, nephropathy, myocardial infarction, stroke, and
lower limb amputation, which develop as the disease progresses,
gradually decrease quality of life of diabetic patients.1 Although
several drug treatments are currently available, the prevalence of
T2DM is still increasing rapidly in both developed and developing
safe antidiabetic agents capable of lowering hemoglobin A1c (HbA1c
)
levels while improving the lipid profile of patients.3
Peroxisome proliferator-activated receptors (PPARs) are ligand-
activated transcription factors belonging to the nuclear receptor
superfamily. Three PPAR subtypes, namely, PPARa, PPARc, and
PPARd (also known as PPARb), have been cloned and characterized.
Their effects are exerted through ligand-dependent transcription of
a constellation of genes encoding proteins that regulate nutrient
metabolism, energy homeostasis, and cell differentiation. PPARc,
the most extensively investigated subtype, is predominantly ex-
pressed in adipose tissue, and to a lesser extent, in the intestine,
mammary gland, endothelium, liver, skeletal muscle, and in other
tissues throughout the body, and plays a pivotal role in adipogen-
esis, glucose and lipid homeostasis, insulin sensitivity, inhibition of
inflammatory responses, cell proliferation, and promotion of ter-
minal differentiation.4
⇑
Corresponding author. Tel.: +81 6 6300 6063; fax: +81 6 6300 6306.
E-mail address: Rikimaru_Kentarou@takeda.co.jp (K. Rikimaru).
Medicinal Chemistry Research Laboratories.
Pharmacology Research Laboratories.
Discovery Research Center.
Strategic Research Planning Department.
à
§
–
The introduction of thiazolidinediones (TZDs), represented by
||
Present address: Umicore Precious Metals Japan Company Ltd, 2-3 Kita-aoyama
troglitazone (NOSCALÒ or REZULINÒ), pioglitazone hydrochloride
1-chome, Minato-ku, Tokyo 107-0061, Japan.
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.12.008

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
715
(ACTOSÒ) and rosiglitazone maleate (AVANDIAÒ), as insulin sensi-
1FM6)9 were used for the docking of compounds 1 and 2. The com-
pounds were docked into the PPAR -LBD using the GOLD program
tizers, and the discovery that TZDs are high-affinity PPAR
c
li-
c
gands,5 has led to extensive research in the area of antidiabetic
drug discovery and development.6
(ver. 2.0, the Cambridge Crystallographic Data Centre, UK). Docking
positions with a high score were corrected manually to avoid small
steric bumps, minimized energetically using MMFF94s force field
in MOE program (Molecular Operating Environment, Chemical
Computing Group Inc., Canada), and plausible binding modes were
obtained.
In the last two decades, efforts have been directed toward iden-
tifying novel classes of PPAR ligands as second-generation insulin
sensitizers by using several approaches including PPAR
agonists, PPAR /d dual agonists, and PPAR
/d pan agonists.6a
Numerous non-TZD PPAR ligands belonging to different chemical
a/c dual
c
a/c
c
The docking models of 1 and 2 superposed with the co-crystal
structure with rosiglitazone (PDB code: 1FM6) are shown in Figure
classes have been reported so far; however, most of these ligands
are carboxylic acids. Thus, in this study, we investigated a new
2. Rosiglitazone binds to the Y-shaped cavity of the PPARc-LBD in a
class of non-TZD and non-carboxylic-acid-based PPAR
In this article, we report the design, synthesis, and biological
evaluation of benzylpyrazole acylsulfonamides as a novel class of
c
agonists.
U-shaped motif and the TZD head group interacts with Tyr473 of
the AF-2 helix. Conversely, as expected, the docking study indicates
that compound 1 fully occupies all three arms of the Y-shaped cav-
ity including the ‘ligand entrance’ pocket that is not occupied by
rosiglitazone. The phenylacetic acid moiety of 1 effectively occu-
pies ‘the TZD pocket’ (which accommodates the TZD group of ros-
iglitazone); further, similar to the TZD group of rosiglitazone, the
carboxyl group forms hydrogen bonds with Tyr473 as well as
Ser289 and His323. This model suggests that there is little space
for accommodating an additional group in the TZD pocket, which
is consistent with the fact that further modification of the carbox-
ylic acid of 1 remarkably decreases the potency.
non-TZD, non-carboxylic-acid-based selective PPAR
the optimization study, we identified potent, highly selective,
and orally active PPAR agonists, namely, 25c, 30b, and 30c, which
c agonists. In
c
exhibited significant antidiabetic effects in diabetic rodent models.
X-ray crystallographic analysis revealed that compound 3d had a
unique binding mode, especially of the acidic functionality, which
was different from that observed for compound 1 in the docking
model. Furthermore, we discuss the key elements of PPAR
tivity of these compounds.
c selec-
In the docking model of compound 2, the pyrazole ring slightly
shifts to the right as compared to that in 1; nevertheless, the bind-
ing position of the benzylpyrazole moiety of 2 is very similar to
that of 1. The carboxylic acid of 2 does not fully occupy the TZD
pocket or form a hydrogen bond with Tyr473, which is consistent
with weak agonism. The binding mode of 2 implies that the ben-
zylpyrazole substructure, not the acidic group, serves as the anchor
since the former occupies the two binding pockets of the Y-shaped
cavity through strong hydrophobic interactions. Based on this
observation, we assumed that modification of the carboxylic acid
of 2 could strengthen the interaction with the TZD pocket and af-
ford potent non-carboxylic-acid-based agonists.
Therefore, we opted to pursue the benzylpyrazole chemotype
starting with compound 2 to identify a new class of non-TZD and
non-carboxylic-acid-based PPARc agonists.
To date, heterocyclic moieties such as TZDs, oxazolidinediones,
and tetrazoles have been widely incorporated into PPAR ligands
as isosteric replacements of the carboxylic acid.6 In contrast, acyclic
isosteres of carboxylic acids have drawn little attention and have
2. Lead generation
Several X-ray co-crystal structures of PPARc ligand-binding do-
main (LBD) in complex with various ligands have been solved and
the protein-ligand interactions have been clarified at atomic reso-
lution. These studies suggest that the PPARc-LBD comprises 13 a-
helices and a small 4-stranded b-sheet. The helices 3, 7, and 10
form the Y-shaped ligand-binding site that is located in the bottom
half of the overall structure.7 The X-ray co-crystal analysis of rosig-
litazone with PPARc-LBD indicated that the former binds to the
LBD in a U-shaped motif and the TZD head group interacts with
Tyr473 located in activation function 2 (AF-2) helix. In the co-crys-
tal structure of rosiglitazone, the tail pyridine ring occupies 1 pock-
et of the Y-shaped cavity comprising hydrophobic amino acids
such as Ile281, Met348, Ile341 but it does not use the second pock-
et comprising amino acids such as Phe226, Leu228, Met329.7a
We previously identified benzylpyrazole phenylacetic acid 1 as
a potent PPARc agonist with a half-maximal effective concentra-
tion (EC50) value of 2.3 nM.8 However, its synthetic precursor, (1-
benzylpyrazol-5-yl)propanoic acid derivative 2 proved to be a
weak PPARc agonist with an EC50 value of 500 nM (Fig. 1). We ob-
tained weak or virtually inactive compounds with our initial efforts
to identify non-carboxylic-acid-based agonists using compound 1-
related phenylacetic acids (data not shown). On the basis of these
results, we speculated that the difference in the transactivation
activities resulted from how the compounds occupied all the 3
arms of the Y-shaped cavity. To confirm our assumption, we per-
formed a docking study of compounds 1 and 2. Previously reported
PPARc-LBD structures in complex with rosiglitazone (PDB code:
Cl
Cl
N
Cl
Cl
N
MeO
O
CO2H
N
N
Me
Me
Me
Me
CO2H
O
O
Figure 2. Docking models of the phenylacetic acid 1 (shown in cyan) and the
propionic acid 2 (shown in purple) bound to the co-crystal structure of PPARc-LBD
[PDB code: 1FM6] with rosiglitazone (shown in peach). Nitrogen, oxygen, sulfur,
and chlorine atoms are colored in blue, red, yellow, and light green, respectively.
Ser289, His323, and Tyr473 interacting with the TZD of rosiglitazone or the
carboxylic acid of 1 are highlighted.
1: EC50 = 2.3 nM
2
: EC50 = 500 nM
Figure 1. Chemical structures and in vitro PPAR
benzylpyrazole-based PPAR agonists 1 and 2.
c
transactivation activities of
c

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K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
been rarely reported.10 In this study, we focused on an acylsulfona-
mide group as a ring-opened analog of TZD.11 Similar to the interac-
tions with rosiglitazone, we expected that one or two oxygen atoms
of a flexible acylsulfonamide group might interact with the key
amino acid residues in the TZD pocket (Fig. 2). Furthermore, prepa-
ration of acylsulfonamide from the corresponding carboxylic acid is
easy and the terminal substituent is expected to contribute to in-
crease in binding energy. Indeed, preliminary introduction of alkyl-
sulfonamides into 2 gave encouraging results. While the potency of
methylsulfonamide 3a was comparable to that of the parent car-
boxylic acid 2, the potency of propylsulfonamide 3b and pentyl ana-
log 3c was about threefold and 30-fold more potent, respectively.
Subsequently, we conducted optimization studies of the benzylpy-
razole acylsulfonamides using 3c as the starting compound.
regioselectivity. The resulting adduct was treated with sodium
methoxide with heating, which resulted in the removal of the
N-methoxycarbonyl group and subsequent cyclization afforded
3-hydroxypyrazole 12. The 3-hydroxy group was protected by a
methoxymethyl (MOM) group, and the resulting ester was con-
verted to
a,b-unsaturated ester 14 by following steps similar to
those described in Scheme 1. Catalytic hydrogenation of 14 and
subsequent treatment with hydrochloric acid gave 3-hydroxypy-
razole 16, which was O-alkylated to provide 3-alkoxypyrazoles
17a–d. Alkaline hydrolysis and subsequent condensation with
1-pentanesulfonamide afforded the acylsulfonamides 18a–d.
The synthetic scheme of 3-alkylpyrazole acylsulfonamides is
shown in Scheme 3. Claisen condensation of ketones 19a–c with
diethyl oxalate afforded the corresponding a,c-diketoesters, which
were reacted with hydrazine to obtain the corresponding
3-alkylpyrazoles 20a–c. The following sequence of reactions con-
sisting of the introduction of 1-benzyl group, reduction of the ester
group, Swern oxidation, Horner–Wadsworth–Emmons reaction,
reduction of the spacer double bond, and construction of acylsulf-
onamide were performed according to the standard procedure as
mentioned above to give acylsulfonamides 24a–d. Finally, inter-
mediary esters 22a,c were hydrolyzed and subsequently coupled
with 1-pentanesulfonamide to provide the corresponding prope-
noylsulfonamides 25a,c.
Benzylpyrazole propenoylsulfonamides with a 3-alkoxy group
on the pyrazole ring were prepared as shown in Scheme 4. Pyrazole
4 was successively alkylated with MOM chloride and benzyl chlo-
ride to provide ester 26 according to the standard procedure. Alde-
hyde 27, which was prepared from 26 via a 3-step protocol, that is,
hydrolysis, Weinreb amide formation, and reduction with DIBAL-H,
was subjected to Horner–Wadsworth–Emmons reaction to yield
3. Chemistry
Benzylpyrazole acylsulfonamides are generally synthesized by
the route shown in Scheme 1. Cyclization of dimethyl acetylenedi-
carboxylate (DMAD) with hydrazine proceeded smoothly to afford
hydroxypyrazole 4, which was alkylated successively with 2-iodo-
propane and benzyl bromide to produce ester 6. Both alkylations
occurred at the desired positions with moderate regioselectivity,
and regioisomers of 6 were easily separated by silica gel chroma-
tography. The chemical structure of 6 was determined by two-
dimensional nuclear magnetic resonance (2D NMR) spectroscopy
(data not shown). Reduction of ester 6 with diisobutylaluminum
hydride (DIBAL-H) followed by Swern oxidation gave aldehyde 7.
Horner–Wadsworth–Emmons reaction with compound 7 and tri-
ethyl phosphonoacetate afforded the corresponding
a,b-unsatu-
rated ester. Subsequent catalytic hydrogenation resulted in
removal of the benzyl group and concomitant saturation of the
double bond to afford ester 8. N-benzylation of 8 and subsequent
saponification yielded carboxylic acids, which were condensed
with sulfonamides to produce the corresponding acylsulfonamides
3c–j. The regioselectivity of the benzylation of 8 was moderate
similarly to that of 6, and the regioisomers of the benzylated prod-
ucts were also separated by silica gel chromatography.
The synthesis of the 1-benzylpyrazoles with different 3-alkoxy
groups was performed according to Scheme 2. Reaction of methyl
carbazate with 2,4-dichlorobenzyl chloride (10) gave N,N0-disubsti-
tuted hydrazine derivative 11. Michael addition of 11 to DMAD
took place at the nitrogen adjacent to the benzyl group with high
a,b-unsaturated ester 28. The analogous ester 14 prepared in
Scheme 2 and compound 28 were used for further transformation,
wherein 14 and 28 were converted to the acylsulfonamides 30a–d
by a procedure similar to that described in Scheme 2.
The spacer length between the pyrazole ring and acylsulfona-
mide group was modified according to Scheme 5. The synthesis
was initiated by O-butylation of 3-hydroxypyrazole 12 to give es-
ter 31, which was hydrolyzed to produce carboxylic acid 33a. Ester
31 was also reduced with DIBAL-H and the resulting alcohol was
reacted with acetone cyanohydrin under Mitsunobu conditions to
give cyanide 32a,12 which was converted to acetic acid 33b by
saponification. Similarly, butanoic acid 33c was obtained from
MeO2C
CO2Me
O
H
N
H
R2
N
N
CO2Me
a
c
e
N
N
N
OEt
Me
Me
Me
Me
R1O
O
O
H2N NH2
4: R1 = H
: R1 = isopropyl
8
: R2 = CO2Me
: R2 = CHO
b
6
7
5
d
X
Cl
N
X
Cl
N
: X = Cl, R3 = (CH2)4Me
3c
3d
3
: X = CF3, R = (CH2)4Me
O
O
3e: X = Cl, R3 = (CH2)3Me
3f: X = Cl, R3 = (CH2)5Me
O
O
R3
g
f
S
N
OEt
N
N
H
3g: X = Cl, R3 = (CH2)2CHMe2
Me
Me
Me
Me
: X = Cl, R3 = cyclohexyl
: X = Cl, R3 = Ph
3h
3i
O
O
9a: X = Cl
9b
3j: X = Cl, R3 = CH2Ph
: X = CF3
Scheme 1. Synthesis of 3c–j. Reagents and conditions: (a) AcOH, toluene, rt; (b) 2-iodopropane, K2CO3, DMF, rt; (c) PhCH2Br, K2CO3, DMF, rt; (d) (i) DIBAL-H, THF, rt; (ii)
(COCl)2, DMSO, CH2Cl2, ꢀ78 °C, then Et3N, rt; (e) (i) (EtO)2P(O)CH2CO2Et, NaH, THF, DMF, 0 °C–rt; (ii) HCO2H, Pd/C, EtOH, reflux; (f) 2,4-dichlorobenzyl chloride, NaH, DMF, rt
for 9a; 2-chloro-4-trifluoromethylbenzyl chloride, NaH, DMF, rt for 9b; (g) (i) 1 M NaOH, THF, EtOH, 50 °C; (ii) CDI, THF, reflux, then RSO2NH2, DBU, rt.

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
717
Cl
Cl
N
Cl
Cl
N
Cl
O
e
b
d
Y
R2
N
N
OEt
Cl
10
R1O
MOMO
1
: R = H, R2 = CO2Me
14
: Y = Cl
12
a
c
13: R1 = MOM, R2 = CHO
11: Y = NHNHCO2Me
Cl
Cl
Cl
Cl
Cl
Cl
O
O
O
O
O
g
h
N
N
N
S
N
OEt
N
OEt
N
N
H
Me
R1O
R1O
R1O
1
: R1 = MOM
16: R1 = H
: R = Bu
: R1 = Bu
18a
15
17a
f
17b: R1 = MeO(CH2)2
17c: R1 = PhCH2
18b: R1 = MeO(CH2)2
18c: R1 = PhCH2
17d: R1 = pyridin-2-ylmethyl
18d: R1 = pyridin-2-ylmethyl
Scheme 2. Synthesis of 18a–d. Reagents and conditions: (a) methyl carbazate, K2CO3, DMF, 80 °C; (b) MeO2CC„CCO2Me, MeOH, reflux, then NaOMe, reflux; (c) (i) MOMCl,
K2CO3, THF, DMF, rt; (ii) DIBAL-H, THF, 0 °C–rt; (iii) (COCl)2, DMSO, CH2Cl2, ꢀ78 °C, then Et3N, rt; (d) (EtO)2P(O)CH2CO2Et, NaH, THF, DMF, 0 °C–rt; (e) H2, Pd/C, THF, rt; (f) HCl,
EtOH, reflux; (g) ROH, Bu3P, ADDP, THF, rt for 17a–c; 2-(chloromethyl)pyridine hydrochloride, K2CO3, DMF, rt for 17d; (h) (i) 1 M NaOH, THF, EtOH, 50 °C; (ii) CDI, THF, reflux,
then 1-pentanesulfonamide, DBU, rt for 18a–c; CDI, DMF, rt, then 1-pentanesulfonamide, DBU, 100 °C for 18d.
X
Cl
N
X
Cl
N
O
H
N
O
a
b
c
CO2Et
CO2Et
N
N
N
OEt
R
Me
R
R
R
19a
19b
19c
20a
20b
20c
: R = isopropyl
: R = cyclopropyl
: R = Ph
: R = isopropyl
: R = cyclopropyl
: R = Ph
21a: R = isopropyl, X = Cl
21b
22a: R = isopropyl, X = Cl
22b
: R = isopropyl, X = CF3
: R = isopropyl, X = CF3
22c: R = cyclopropyl, X = CF3
22d
21c: R = cyclopropyl, X = CF3
21d
: R = Ph, X = Cl
: R = Ph, X = Cl
e
X
Cl
N
X
Cl
O
O
O
O
d
e
N
N
S
N
OEt
N
CH3
H
R
R
23a: R = isopropyl, X = Cl
23b
24a (single), 25a (double): R = isopropyl, X = Cl
24b
: R = isopropyl, X = CF3
23c: R = cyclopropyl, X = CF3
23d
(single): R = isopropyl, X = CF3
24c (single), 25c (double): R = cyclopropyl, X = CF3
24d
: R = Ph, X = Cl
(single): R = Ph, X = Cl
Scheme 3. Synthesis of 24a–d and 25a,c. Reagents and conditions: (a) (i) (CO2Et)2, t-BuOK, THF, rt; (ii) H2NNH2ꢁH2O, AcOH, THF, reflux; (b) 2,4-dichlorobenzyl chloride,
K2CO3, DMF, rt for 21a,d; 2-chloro-4-trifluoromethylbenzyl chloride, K2CO3, DMF, rt for 21b,c; (c) (i) DIBAL-H, THF, rt; (ii) (COCl)2, DMSO, CH2Cl2, ꢀ78 °C, then Et3N, rt; (iii)
(EtO)2P(O)CH2CO2Et, NaH, THF, DMF, rt; (d) H2, Pd/C, THF, rt; (e) (i) 1 M NaOH, THF, EtOH, 50 °C; (ii) CDI, THF, reflux, then 1-pentanesulfonamide, DBU, rt for 24a,b,d, 25a; CDI,
DMF, rt, then 1-pentanesulfonamide, DBU, 100 °C for 24c,25c.
propionic acid ester 17a via cyanide 32b. Furthermore, butanoic
acid 33c was homologated in a similar manner as that described
for 32a,b to afford pentanoic acid 33d. Condensation of the carbox-
ylic acids 33a–d with 1-pentanesulfonamide afforded the acylsulf-
onamides 34a–d.
stably expressed in Chinese hamster ovary (CHO) cells and their
metabolic stability in human liver microsomes. We also evaluated
the transactivation activities of these compounds against the other
2 PPAR subtypes, namely, human PPARa and PPARd, which were
transiently expressed in COS-1 cells. Transactivation activity was
assessed by a luciferase reporter gene assay and the activities were
reported as EC50 value. (R)-5-(3-{4-[(2-Furan-2-yl-5-methyl-1,3-
oxazol-4-yl)methoxy]-3-methoxyphenyl}propyl)-1,3-oxazolidine-
2,4-dione,13 ({3-[(5-methyl-2-phenyl-1,3-oxazol-4-yl)methoxy]be
nzyl}sulfanyl) acetic acid,14 and carbacyclin15 were used as the ref-
4. Results and discussion
4.1. Human PPARs transactivation activity and metabolic
stability
erence PPARc, a, and d agonists, respectively. Metabolic stability
was assessed by measuring clearance rate of the parent compound
after treatment with human liver microsomes (see Section 6).
We evaluated the transactivation activity of the newly synthe-
sized benzylpyrazole-based PPARc ligands against human PPARc

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K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
F3C
Cl
N
X
Cl
N
O
O
d
a
c
4
N
R
N
OEt
MOMO
26: R = OMe
27
MOMO
14: X = Cl
28: X = CF3
b
: R = H
X
Cl
N
X
Cl
O
O
O
O
R2
e
N
N
S
N
OEt
N
H
R1O
R1O
: X = Cl, R1 = isoproyl, R2 = (CH2)4Me
1
30a
30b
29a
: X = Cl, R = isopropyl
: X = CF3, R1 = isopropyl
29b
: X = CF3, R1 = isopropyl, R2 = (CH2)4Me
30c: X = CF3, R1 = isopropyl, R2 = (CH2)2CHMe2
30d: X = CF3, R1 = MeO(CH2)2, R2 = (CH2)2CHMe2
29c: X = CF3, R1 = MeO(CH2)2
Scheme 4. Synthesis of 30a–d. Reagents and conditions: (a) (i) MOMCl, K2CO3, DMF, 0 °C–rt; (ii) 2-chloro-4-trifluoromethylbenzyl chloride, K2CO3, DMF, rt; (b) (i) 1 M NaOH,
THF, MeOH, 50 °C; (ii) MeONHMeꢁHCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), HOBt, DMF, rt; (iii) DIBAL-H, THF, 0 °C; (c) (EtO)2P(O)CH2-
CO2Et, NaH, THF, DMF, 0 °C–rt; (d) (i) concd. HCl, EtOH, reflux; (ii) 2-iodopropane, K2CO3, DMF, rt for 29a,b; 2-methoxyethanol, Bu3P, ADDP, THF, rt for 29c; (e) (i) 1 M NaOH,
THF, EtOH, 50 °C; (ii) CDI, DMF, rt, then 1-pentanesulfonamide, DBU, 100 °C for 30a,b; CDI, DMF, rt, then 3-methylbutane-1-sulfonamide, DBU, 100 °C for 30c,d.
Cl
Cl
N
Cl
Cl
N
b
CO2Me
(CH2)nCO2H
a
N
N
12
BuO
BuO
31
33a
: n = 0
33b: n = 1
33c: n = 3
d
c
e
33d
: n = 4
f
Cl
Cl
N
Cl
Cl
c
(CH2)nCN
17a
N
H
N
Me
N
N
(CH2)n
S
BuO
O
O
O
32a
: n = 1
BuO
32b: n = 3
34a
: n = 0
34b: n = 1
34c: n = 3
34d
: n = 4
Scheme 5. Synthesis of 34a–d. Reagents and conditions: (a) BuOH, PBu3, ADDP, THF, rt; (b) 1 M NaOH, THF, EtOH, 50 °C; (c) (i) DIBAL-H, THF, 0 °C; (ii) acetone cyanohydrin,
Bu3P, ADDP, THF, rt; (d) 4 M NaOH, EtOH, reflux; (e) (i) BH3ꢁTHF, THF, rt; (ii) acetone cyanohydrin, Bu3P, ADDP, THF, rt; (iii) 4 M NaOH, EtOH, reflux; (f) CDI, THF, reflux, then
1-pentanesulfonamide, DBU, rt.
Despite showing potent PPAR
c
agonism, by oral administration
on reduction of molecular lipophilicity, increment in molecular
rigidity, and/or protection of the metabolically labile sites.
As shown in Figure 3, alkyl substituents of sulfonamide group
the lead compound 3c exhibited weak glucose-lowering effects in
diabetic rodent models. The preliminary pharmacokinetic data of
3c were obtained by cassette dosing in rats. After oral administra-
tion (1 mg/kg), compound 3c showed insufficient pharmacokinetic
profile (CLtotal = 794 mL/h/kg, AUC0–8 h = 495.3 ng h/mL, F = 37.6%),
presumably due to poor metabolic stability in hepatic microsomes
exhibited positive effects on PPARc activity. Thus, we studied the
SAR by further examining the terminal side chain on the sulfonyl
group with 1-(2,4-dichlorobenzyl)-3-isopropoxypyrazole deriva-
tive 3c; the results are presented in Table 1. Both shortening (3e)
and elongation (3f) of the alkyl chain decreased the activity, while
branching at the terminal of the alkyl chain (3g) maintained the
activity. Incorporation of large substituents such as cyclohexyl
(3h), phenyl (3i), or benzyl (3j) groups resulted in more than six-
fold drop in potency. Nevertheless, all these compounds, except
(metabolic clearance h/r = 195/128 lL/min/mg). Additionally, incu-
bation of 3c with hepatic microsomes indicated that the isopro-
poxy group and the pentyl side chain were metabolically labile
in both humans and rats, which supported the aforementioned re-
sult (data not shown). Thus, we aimed to improve metabolic stabil-
ity and study the structure–activity relationship (SAR) of the
chemotype. Our strategy to improve metabolic stability was based
3j, served as selective PPARc agonists (EC50 >10000 nM for PPARa
and PPARd) similar to 3c. With regard to metabolic stability, no

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
719
Table 2
In vitro PPAR
18a–d, and 24a,d
Cl
Cl
N
c transactivation activities and metabolic clearance of compounds 3c,
EC50
O
O
O
R
450 nM
160 nM
16 nM
3a: R = Me
Cl
Cl
N
S
N
N
H
3b: R = (CH)2Me
3c: R = (CH2)4Me
Me
Me
O
O
O
O
S
N
N
Me
H
Figure 3. In vitro PPAR
agonists 3a–c.
c transactivation activities of benzylpyrazole-based PPARc
R
Compound
R
PPARc transactivation Metabolic clearance
EC50 (nM)a
(l
L/min/mg)b
Table 1
3c
Isopropoxy
Butoxy
MeO(CH2)2O
Benzyloxy
2-Pyridylmethoxy 120
Isopropyl
Phenyl
16
7.5
31
14
192
—
In vitro PPARc transactivation activities and metabolic clearance of compounds 3c,e–j
c
18a
18b
18c
18dd
24a
24d
144
100
224
124
111
Cl
Cl
O
O
O
R
13
11
N
S
N
N
H
a
Me
Me
EC50 value means the effective concentration for 50% response of a given
compound’s intrinsic maximum response.
O
b
In vitro metabolic clearance was determined based on the disappearance of the
parent compound after incubation with human liver microsomes for 20 min.
c
Compound
R
PPAR
c
transactivation
Metabolic clearance
(l
L/min/mg)b
Not determined.
EC50 (nM)a
d
Hydrochloric acid salt.
3e
3c
3f
3g
3h
3i
Butyl
Pentyl
Hexyl
Isoamyl
Cyclohexyl
Phenyl
40
16
21
17
150
110
260
165
192
c
—
—
—
—
Considering the difference in the activities of compounds 1 and 2,
the distance of the acidic function from the lipophilic template
could affect PPARc potency. This assumption prompted us to next
investigate effects of the alkylene spacer length on agonism with
c
c
c
3j
Benzyl
107
1-benzyl-3-butoxypyrazole analog 18a. Shortening of the ethylene
a
EC50 value means the effective concentration for 50% response of a given
spacer of 18a (34a,b) proved to be detrimental to PPAR
vation activity. Similarly, elongation of the spacer (34c,d) reduced
the activity, but the agonists still retained 10ꢀ8
potency
c transacti-
compound’s intrinsic maximum response.
b
In vitro metabolic clearance was determined based on the disappearance of the
M
parent compound after incubation with human liver microsomes for 20 min.
c
Not determined.
(Table 3). Accordingly, we pursued optimization of this series of
compounds with a 2-carbon spacer.
In medicinal chemistry, empirical observation suggests that less
flexible molecules tend to have improved metabolic stability de-
spite high lipophilicity.16 Therefore, we next examined the effect
of incorporation of a double bond into the spacer and replacement
of the para substituent of the benzyl group and 3-substituent on
the pyrazole ring; the results are presented in Table 4. Replace-
ment of the chlorine atoms of 3c and 24a with a trifluoromethyl
group (3d, 24b) gave essentially equipotent activity. Interestingly,
substitution of a cyclopropyl group (24c) for the isopropyl group of
24b retained 10ꢀ8 M order potency with moderately improved
metabolic stability. It is noteworthy that remarkable improvement
in metabolic stability was generally achieved through the
improvement was observed after this modification consistent with
their high lipophilicity. The result suggests that pentyl and isoamyl
groups are optimal for potent PPARc agonism and spatial limita-
tion may exist for the binding pocket that accommodates this part
of the molecule. Consequently, pentyl and isoamyl groups were se-
lected as the tail alkyl chains for further SAR study.
Next, we examined the effects of the 3-substituent on the pyr-
azole ring on PPARs transactivation activity; the results are sum-
marized in Table 2. Overall, a variety of lipophilic substituents
like butoxy (18a) benzyloxy (18c), isopropyl (24a) and phenyl
(24d) groups were tolerated at this position. These results are con-
sistent with the docking study that the binding pocket interacting
with this part of the ligand is relatively large and lipophilic. In
addition, the methoxyethoxy group (18b) was also acceptable;
however, improvement in the metabolic stability was minimal
with these structural changes. Benzyloxy group (18c) retained
the activity as compared with 3c, whereas substitution of a pyri-
dine ring (18d) for the benzene ring of 18c decreased the activity.
Table 3
In vitro PPARc transactivation activities of compounds 18a and 34a–d
Cl
Cl
O
O
O
N
As for PPAR
ited EC50 values of >10000 nM. Among these compounds, com-
pound 18a exhibited slightly higher PPAR activity (data not
a and d transactivation activities, all compounds exhib-
S
N
(CH2)n
N
H
Me
BuO
a
shown). According to the SAR results, isopropoxy, methoxyethoxy,
isopropyl, and their related groups were investigated as the 3-sub-
stituent of the pyrazole in the subsequent optimization study (see
Table 4).
The lead compound 3c possesses unique molecular architecture,
a lipophilic benzylpyrazole template linked by an ethylene spacer to
an acylsulfonamide bearing a tail pentyl group. Unlike most of the
Compound
n
PPARc transactivation
EC50 (nM)a
34a
34b
18a
34c
34d
0
1
2
3
4
Inactive
>10000
7.5
67
29
a
non-carboxylic-acid-based PPARc agonists, the acidic acylsulfona-
EC50 value means the effective concentration for 50% response of a given
compound’s intrinsic maximum response.
mide moiety of 3c is located in the center of the molecule.

720
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
Table 4
In vitro PPAR
c
transactivation activities and metabolic clearance of compounds 3c,d, 24a–c, 25a,c, and 30a–d
X
Cl
X
Cl
O
O
O
O
R2
O
O
R2
N
N
S
S
N
N
H
N
N
H
R1
R1
3c 3d 24a-c
25a c 30a-d
, ,
,
,
Compound
X
R1
R2
PPAR
c
transactivation
Metabolic clearance (l
L/min/mg)b
EC50 (nM)a
3c
24a
3d
Cl
Cl
Isopropoxy
Isopropyl
Isopropoxy
Isopropyl
Cyclopropyl
Isopropoxy
Isopropoxy
Isopropoxy
MeO(CH2)2O
Isopropyl
Pentyl
Pentyl
Pentyl
Pentyl
Pentyl
Pentyl
Pentyl
Isoamyl
Isoamyl
Pentyl
Pentyl
16
13
10
8.4
13
20
9.3
8.3
9.0
8.2
12
192
124
163
136
78
29
26
32
34
CF3
CF3
CF3
Cl
CF3
CF3
CF3
Cl
24b
24c
30a
30b
30c
30d
25a
25c
99
54
CF3
Cyclopropyl
a
EC50 value means the effective concentration for 50% response of a given compound’s intrinsic maximum response.
In vitro metabolic clearance was determined based on the disappearance of the parent compound after incubation with human liver microsomes for 20 min.
b
incorporation of a double bond into the spacer. For example,
propenoylsulfonamide 30a showed 6.6-fold lower metabolic clear-
ance than the corresponding propanoylsulfonamide 3c while
indicating that they are highly selective PPAR
in Table 5, these agonists robustly inhibited the specific binding
of [3H]-AD-5061 to human PPAR
with IC50 values ranging from
c ligands. As shown
c
maintaining the selective PPAR
c
activity. As in the case of com-
12 to 23 nM, which were similar to the EC50 values of their func-
tional activities. The results suggest that the transactivation activ-
ity of the compounds is attributed to direct binding to the receptor,
implying that the affinity of these compounds can be increased by
pounds with the ethylene spacer (3c, 24a vs 3d, 24b), replacement
of the chlorine atom of 30a with a trifluoromethyl group (30b,c) in-
duced marginal increase in the potency. In contrast to the potency
of the propanoylsulfonamide series compounds (3c vs 18b shown
in Table 2), the high potency was preserved by substitution of a
methoxyethoxy group (30d) for the isopropoxy group of com-
pound 30c. Moreover, 3-alkylpyrazole series such as isopropyl
(25a) and cyclopropyl (25c) derivatives showed about 10 nM po-
tency with comparatively limited improvement in metabolic sta-
bility. Overall, the compounds 30b, 30c, and 30d exhibited
occupying all 3 arms of the Y-shaped cavity of the PPARc-LBD. In-
deed, the effective occupancy of the Y-shaped cavity by these ago-
nists was confirmed in an X-ray co-crystallographic study to be
discussed later. These results clearly show that the agonists effec-
tively bind to the PPARc-LBD and result in a potent activation of
PPARc.
favorable metabolic stability and potent PPAR
c
agonism with
4.3. In vivo antidiabetic effects in rodent models of T2DM
EC50 values of 9.3, 8.3, and 9.0 nM, respectively. In addition, com-
pound 25c showed acceptable metabolic stability and potent activ-
ity with EC50 value of 12 nM. Greater than 500-fold subtype
We next evaluated metabolic stability and pharmacokinetic
profile in rats as well as the in vivo antidiabetic efficacy of the ben-
zylpyrazole acylsulfonamides in a genetically obese rodent model
of T2DM, fed male Wistar fatty rats.17b The selected compounds
25c, 30b, and 30c with high metabolic stability were orally admin-
istered to male Wistar fatty rats for 7 days. As shown in Table 6,
these agonists significantly reduced plasma glucose (PG) levels
with ED25 values ranging from 0.12 to 0.42 mg/kg and triglyceride
(TG) levels with ED25 values ranging from 0.04 to 0.55 mg/kg in a
selectivity was observed as all compounds showed PPAR
a and d
transactivation activities with EC50 values of >10000 nM; however,
2-chloro-4-trifluoromethylbenzyl derivative 30c and 30d with iso-
amyl side chain exhibited a slightly high PPARa maximum re-
sponse (48% and 47% of control at 10 M, respectively). These
l
results are consistent with the fact that the metabolic stability of
this class of compounds depends on their flexibility rather than
their lipophilicity.
In the view of the results of the SAR study and the structural
diversity, we selected 3d, 25c, 30b, and 30c as potent and highly
Table 5
selective PPAR
evaluations.
c agonists for further in vitro and in vivo
PPARs transactivation activities and PPARc binding affinities of compounds 3d, 25c,
30b, 30c
Compound
PPARs transactivation EC50 (nM)a
PPARc binding
4.2. PPARc binding activity
PPAR
c
PPARa
PPARd
IC50 (nM)b
3d
10
12
9.3
8.3
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
21
12
23
15
To confirm that the representative agonists 3d, 25c, 30b, and
30c activate PPAR through direct binding to the receptor, we per-
25c
30b
30c
c
formed a competition binding assay using GST-hPPAR
c and the
radioligand [3H]-AD-5061 according to a modified protocol of our
previously established method.17a With regard to the PPAR sub-
type selectivity of the above mentioned compounds, they did not
a
EC50 value means the effective concentration for 50% response of a given
compound’s intrinsic maximum response.
b
IC50 value means the concentration of a given compound required for 50%
inhibition of the specific binding of [3H]-AD-5061 to human PPAR
c.
exhibit significant activities against PPARa and PPARd at 10 lM,

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
721
Table 6
PPAR transactivation activities, plasma glucose (PG)- and triglyceride (TG)-lowering effects in Wistar fatty rats, and metabolic clearance by treatment with rat microsomes of
compounds 25c, 30b, and 30c
c
b
Compound
PPAR
c
transactivation EC50 (nM)a
Wistar fatty rats
Rat metabolic clearance (
l
L/min/mg)e
AUC0-8h (ng h/mL)f
PG ED25 (mg/kg)c
TG ED25 (mg/kg)d
25c
30b
30c
12
9.3
8.3
0.42
0.12
0.21
0.55
0.04
0.09
116
33
51
325.6
2487.7
2888.1
a
EC50 value means the effective concentration for 50% response of a given compound’s intrinsic maximum response.
Test compounds were administered by oral gavage to male Wistar fatty rats for 7 days.
b
c,d
Effective dose for 25% reduction of plasma glucose (PG) and triglyceride (TG), respectively.
e
EC50 value means the effective concentration for 50% response of a given compound’s intrinsic maximum response. Rat liver microsome was used instead of human liver
microsome.
f
Rat cassette dosing at 1 m/kg, po. Average of AUC0–8 h values in three rats.
dose-dependent manner. The improved metabolic stability of 30b
and 30c is considered to be reflected in the higher plasma exposure
(AUC0–8 h). Furthermore, a good correlation between the in vivo
potency of the compounds in Wistar fatty rats and their plasma
exposure in rats was observed, indicating that potent antidiabetic
efficacy of 30b, 30c is mainly attributed to not only potent transac-
tivation activity but also improved pharmacokinetic profile. These
results clearly demonstrated that these agonists exhibited signifi-
cant antidiabetic effects.
moiety forms the following 3 hydrogen bonds: with Tyr327 via
the carbonyl oxygen, with His449 via one sulfonyl oxygen, and
with Gln286 via the other sulfonyl oxygen. In the other conforma-
tion, it forms two hydrogen bonds with Tyr327 and His 449.
The result of superposition with the co-crystal structure of the
acylsulfonamide 3d and docking model of the phenylacetic acid 1
to the PPARc-LBD is shown in Figure 4B. The main difference be-
tween the two binding modes is the interaction of the acidic
groups with the key amino acid residues in the TZD pocket. The
carboxyl group of 1 directly interacts with Tyr473 through hydro-
gen bonding. However, the acylsulfonamide moiety of 3d does not
interact with Tyr473, but it interacts with Tyr327, His449, and/or
Gln286, as mentioned above. The other difference between the
two LBD structures is the conformation of the phenyl side chain
of Phe363 located in helix 3. The phenyl side chain in the docking
model of 1 covers the TZD pocket from above, whereas the side
chain of Phe363 in the co-crystal structure of 3d adopts a
‘flipped-out’ conformation, opening up an additional cleft-like lipo-
philic binding space that vertically extends from the middle of the
TZD pocket. Thus, Phe363 is considered as a ‘gate-keeper’ residue
since it is located in the entrance region of the newly generated
cleft18; the additional binding region can accommodate the pent-
ylsulfonyl group. We speculate that the acylsulfonamide deriva-
tives that appropriately occupy all three arms of the Y-shaped
cavity as well as the new binding cleft adjacent to the TZD pocket
by flipping the ‘gate-keeper’ Phe363 residue could compensate for
decreased binding energy derived from no interaction with Tyr473,
4.4. Docking studies based on X-ray co-crystallographic
analyses of PPAR ligands
To gain a structural insight into the molecular basis underlying
the interaction of acylsulfonamide-based PPAR
lyzed the co-crystal structure of human PPAR
with the compound 3d. The binding mode of 3d to the PPAR
c
c
agonists, we ana-
-LBD in complex
c
-
LBD obtained by the co-crystallographic analysis is shown in Figure
4A. The binding position of the benzylpyrazole moiety of 3d was
found to be similar to that observed in the docking model of propi-
onic acid 2 to the co-crystal structure of the PPARc-LBD [PDB code:
1FM6] (see Fig. 2). The 2-nitrogen atom of the pyrazole ring inter-
acted with the backbone amide oxygen of Leu340 through a water
molecule-mediated hydrogen bond. Both the hydrophobic and
hydrogen-bonding interactions contribute to effective occupancy
of the two arms of the Y-shaped cavity. The pentylsulfonylamino
group adopts two conformations, which are similar except for their
terminal pentyl group. In one conformation, the acylsulfonamide
leading to potent PPARc agonism.
Figure 4. (A) Co-crystal structure of the acylsulfonamide-based agonist 3d (shown in gray) with PPARc-LBD. Residues Gln286, Tyr327, Leu340, and His449 that interact with
3d are highlighted in orange. Hydrogen bonds are shown as yellow dotted lines. Tyr473 is also shown for reference. The water molecule interacting with Leu340 and the
pyrazole nitrogen is depicted as a red sphere. Key hydrogen bonds are shown as yellow dotted lines. (B) Superposition of the docking model of the carboxylic-acid-based
agonist 1 (shown in light purple) bound to the PPARc-LBD [PDB code: 1FM6] and the co-crystal structure of 3d (shown in white). Tyr473 interacting with the carboxyl group
of compound 1 is highlighted in light purple. The conformational change of Phe363 is marked by a red circle. The hydrogen bond between Tyr473 and the carboxylic acid of 1
is shown as a yellow dotted line.

722
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
of Phe363 creates the new cleft-like binding space to accommodate
the terminal pentyl group, which could compensate for the de-
creased binding energy. Furthermore, the acylsulfonamide group
of 3d forms hydrogen bonds with the amino acid residues in TZD
pocket, namely, Gln286, Try327, and His449. Among these interac-
tions, it is speculated that hydrogen bonding with Tyr327 is the
key interaction that might contribute to the high subtype selectiv-
ity. These results suggest that the benzylpyrazole acylsulfonamides
are a new class of potent and highly selective PPARc agonists that
do not possess TZD structure or a carboxylic acid group.
6. Experimental section
6.1. Chemistry
6.1.1. General methods
Melting points (mp) were determined on a Büch melting point
B-545 or a Yanagimoto micro melting point apparatus and were
uncorrected. The proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Bruker AVANCE 300 (300 MHz) spec-
trometer or a Varian MERCURY plus 300 (300 MHz) spectrometer.
Chemical shifts are given in d values (ppm) using tetramethylsilane
as the internal standard. All J values are given in hertz. The follow-
ing abbreviations are used: s, singlet; d, doublet; t, triplet; q, quar-
tet; m, multiplet; dd, doublets of doublet; br s, broad singlet.
Elemental analyses (C, H, N) were within 0.3% of theoretical val-
ues and were determined in Takeda Analytical Research Laborato-
ries (Osaka, Japan). Liquid Chromatography–Mass Spectrometry
(LC–MS) analysis was performed on a Shiseido CAPCELL PACK C-
18 UG120 S-3 column in a Waters Alliance 2795 or an Agilent
1100 System equipped with a Waters 2487 absorbance detector
and a Micromass ZQ2000 mass spectrometer, eluted with water
containing 0.05% TFA with a linear gradient of 10%–100% of MeCN
containing 0.04% TFA. Mass spectra were recorded using electro-
spray ionization (ESI) in positive ion mode. Flash chromatography
was performed with Merck silica gel 60 (0.063–0.200 mm) or Fuji
Figure 5. Superposition of the co-crystal structure of the benzylpyrazole acylsulf-
onamide 3d bound to PPAR
to PPAR -LBD (shown in orange) [PDB code: 1I7G], and PPARd agonist GW2331
bound to PPARd-LBD (shown in yellow) [PDB code: 1Y0S]. Residues Gln286, Tyr327,
Leu340, and His449 in PPAR that interact with 3d are highlighted in white. The
corresponding residues in PPAR and d are highlighted in orange and yellow,
c-LBD (shown in white), PPARa/c agonist AZ242 bound
a
c
a
respectively. The key replacement is marked by a red circle.
One of the most attractive features of the benzylpyrazole acyl-
sulfonamides is high selectivity for PPARc over the PPARa and d
subtypes. To examine the high selectivity profile of these com-
pounds, the co-crystal structure of 3d was superposed with the
complex of PPARa-LBD with PPARa/d dual agonist AZ242 (PDB
code: 1I7G)19 and PPARd-LBD with PPARd agonist GW2331 (PDB
code: 1Y0S). 20 As mentioned above, the key interactions of 3d with
PPAR
Gln286, Leu340, Tyr327, and His449. As shown in Figure 5,
Leu340, Gln286, and His449 are conserved both in PPAR
(Gln277, Leu331, and His440) and PPARd (Gln286, Leu340, and
His449).21 However, Tyr327 in PPAR
is replaced with Phe318
and Phe327 in PPAR and PPARd, respectively. Although the pre-
c-LBD are through direct or indirect hydrogen bonds with
a
c
Silysia Chemical Purif-Pack SI (60
lM) or Varian Bond Elut Si
a
(40 M). High-performance liquid chromatography (HPLC) was
l
cise reason for the high selectivity remains unclear, but we specu-
late that the lack of interaction corresponding to the hydrogen
bond with Tyr327 might influence the selectivity.
performed with Nomura Chemical Co., Ltd. Develosil ODS-UG10
using a Waters HPLC module equipped with a 2487 2-channel
UV/VIS detector. Reaction progress was determined by thin layer
chromatography (TLC) analysis on Merck silica gel 60 F254 plates.
Visualization was with UV light (254 nm) or iodine. Yields are of
purified compounds and were not optimized. Reagents and sol-
vents were obtained from commercial sources and used without
further purification. The following abbreviations are used: AcOH,
acetic acid; ADDP, 1,10-(azodicarbonyl)dipiperidine; CDI, N,N-car-
bonyldiimidazole; DMA, N,N-dimethylacetamide; DMF, N,N-
dimethylformamide; DBU, 1,8-diazabicyclo[5.4.0]-7-undecene;
DCM, dichloromethane; DIBAL-H, diisobutylaluminium hydride;
DMAP, 4-dimethylaminopyridine; DMSO, dimethylsulfoxide; EDCI,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
EtOAc, ethyl acetate; EtOH, ethanol; Et2O, diethyl ether; IPE, diiso-
propyl ether; HOBt, 1-hydroxybenzotriazole; MeOH, methanol;
TEA, triethylamine; THF, tetrahydrofuran.
5. Conclusion
Herein, we described the design, synthesis, and in vitro and
in vivo activities of novel benzylpyrazole acylsulfonamides as
non-carboxylic-acid-based selective PPARc agonists. Our strategy
was based on the assumption that incorporation of an isosteric
substitute of the carboxylic acid of the weak agonist 2, which en-
hances the interaction with the TZD pocket, would afford non-car-
boxylic-acid-based PPARc agonists. The lead compound 3c was
identified by incorporating acylsulfonamide, a ring-opening analog
of TZD, into compound 2. The optimization study revealed the fol-
lowing SAR findings: (i) incorporating an acylsulfonamide with a
linear alkyl side chain, 2-carbon spacer, and 3-substituent on the
pyrazole ring are important for potent PPARc agonism, (ii) substi-
tution of a trifluoromethyl group for the para-chlorine atom of the
benzyl moiety tends to enhance in vitro potency, (iii) incorporation
of a double bond into the ethylene spacer significantly improves
6.1.1.1. Methyl 3-hydroxy-1H-pyrazole-5-carboxylate (4). To a
solution of hydrazine monohydrate (44.8 g, 894 mmol) in toluene
(300 mL) was added AcOH (180 mL) followed by addition of acety-
lenedicarboxylic acid dimethyl ester (100 mL, 813 mmol). After
being stirred at room temperature for 2 h, the mixture was poured
into ice-cooled water. The resulting precipitate was collected,
washed with water and hexane, and dried to give 4 (92.2 g, 80%)
as a white solid. 1H NMR (DMSO-d6): d 3.77 (s, 3H), 5.96 (br s,
1H), 10.04 (br s, 1H), 12.79 (br s, 1H).
metabolic stability. The PPARc agonists 30b, and 30c with suffi-
cient plasma exposure and potent antidiabetic efficacy were iden-
tified. The docking study indicated that the carboxylic-acid-based
agonist 1 directly interacts with Tyr473. However, X-ray co-crys-
tallographic analysis indicates that the acylsulfonamide-based
agonist 3d effectively occupies the Y-shaped cavity of the PPAR
c-
LBD without interacting with Tyr473. The conformational change

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
723
6.1.1.2. Methyl 3-isopropoxy-1H-pyrazole-5-carboxylate (5). To
a mixture of 4 (92.2 g, 649 mmol), K2CO3 (135 g, 974 mmol), and
DMF (500 mL) was added 2-iodopropane (132 g, 779 mmol). After
being stirred at room temperature for 15 h, the mixture was
poured into water and extracted with EtOAc. The extract was
washed with water and brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (hexane/EtOAc, 100:1 to 1:1) to give 5 (105 g,
88%) as a colorless oil. 1H NMR (CDCl3): d 1.36 (d, J = 6.0 Hz, 6H),
3.91 (s, 3H), 4.67–4.83 (m, 1H), 6.20 (s, 1H), 10.12 (br s, 1H).
(CDCl3): d 1.30 (t, J = 7.2 Hz, 3H), 1.34 (d, J = 6.3 Hz, 6H), 4.21 (q,
J = 7.2 Hz, 2H), 4.65–4.77 (m, 1H), 5.27 (s, 2H), 5.96 (s, 1H), 6.23
(d, J = 15.8 Hz, 1H), 7.10–7.15 (m, 2H), 7.20–7.33 (m, 3H), 7.45 (d,
J = 15.8 Hz, 1H). To
a solution of the obtained intermediate
(83.9 g, 268 mmol) in EtOH (700 mL) was added 10% Pd/C (100 g)
followed by formic acid (700 mL). The mixture was heated under
reflux for 15 h. After being cooled, the catalyst was removed by fil-
tration and the filtrate was concentrated in vacuo. The residue was
partitioned between EtOAc and water. The organic layer was
washed with sat. NaHCO3 and brine, dried over MgSO4, and con-
centrated under reduced pressure to give a pale yellow oil. The
oil was dissolved in EtOH (800 mL) and subjected to the same
operations with 10% Pd/C (180 g) and formic acid (800 mL). After
being cooled, the catalyst was removed by filtration and the filtrate
was concentrated in vacuo. The residue was partitioned between
EtOAc and water. The organic layer was washed with sat. NaHCO3
and brine, dried over MgSO4, and concentrated under reduced
pressure to give 8 (49.2 g, 81%) as a colorless oil. 1H NMR (CDCl3):
d 1.26 (t, J = 7.2 Hz, 3H), 1.33 (d, J = 6.3 Hz, 6H), 2.58–2.65 (m, 2H),
2.82–2.90 (m, 2H), 4.15 (q, J = 7.2 Hz, 2H), 4.64–4.76 (m, 1H), 5.46
(s, 1H).
6.1.1.3. Methyl 1-benzyl-3-isopropoxy-1H-pyrazole-5-carboxyl-
ate (6). To a mixture of 5 (105 g, 573 mmol), K2CO3 (119 g,
860 mmol), and DMF (400 mL) was added benzyl bromide (117 g,
687 mmol). After being stirred at room temperature for 15 h, the
mixture was poured into water and extracted with EtOAc. The ex-
tract was washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified
by silica gel chromatography (hexane/EtOAc, 100:1 to 4:1) to give
6 (130 g, 83%) as a pale yellow oil. 1H NMR (CDCl3): d 1.34 (d,
J = 6.3 Hz, 6H), 3.82 (s, 3H), 4.67–4.77 (m, 1H), 5.60 (s, 2H), 6.21
(s, 1H), 7.21–7.39 (m, 5H).
6.1.1.6. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-isopropoxy-1H-pyra-
zol-5-yl]propanoate (9a). To a solution of 8 (10.0 g, 44.2 mmol) in
DMF (150 mL) was added 60% NaH (1.93 g, 48.3 mmol). After being
stirred at room temperature for 10 min, 2,4-dichlorobenzyl chlo-
ride (13.0 g, 66.3 mmol) was added. After being stirred at room
temperature for 15 h, the mixture was poured into sat. NH4Cl
and extracted with EtOAc. The extract was washed with water
and brine, dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by silica gel chromatography (hexane/EtOAc, 50:1
to 6:1) to give 9a (11.3 g, 66%) as a pale yellow oil. 1H NMR (CDCl3):
d 1.24 (t, J = 7.2 Hz, 3H), 1.32 (d, J = 6.3 Hz, 6H), 2.51–2.61 (m, 2H),
2.71–2.81 (m, 2H), 4.12 (q, J = 7.2 Hz, 2H), 4.64–4.77 (m, 1H), 5.18
(s, 2H), 5.52 (s, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1, 8.4 Hz,
1H), 7.38 (d, J = 2.1 Hz, 1H).
6.1.1.4.
1-Benzyl-3-isopropoxy-1H-pyrazole-5-carbaldehyde
(7). To an ice-cooled solution of 6 (120 g, 438 mmol) in THF
(1200 mL) was added 1.5 M DIBAL-H in toluene (729 mL,
1090 mmol). The solution was allowed to warm to room tempera-
ture, and stirred for 2 h. The reaction was quenched by successive
addition of MeOH and 10% Rochelle salt, and the mixture was stir-
red vigorously until the white slurry was dissolved. The solution
was extracted with Et2O and the extract was washed with brine.
The solution was dried over MgSO4 and concentrated under re-
duced pressure. The crude products were purified by silica gel
chromatography (hexane-EtOAc, 19:1 to 1:1) to give (1-benzyl-3-
isopropoxy-1H-pyrazol-5-yl)methanol (77.7 g, 72%) as a colorless
oil. 1H NMR (CDCl3): d 1.33 (d, J = 6.0 Hz, 6H), 1.70 (t, J = 6.0 Hz,
1H), 4.47 (d, J = 6.0 Hz, 2H), 4.64–4.74 (m, 1H), 5.22 (s, 2H), 5.64
(s, 1H), 7.09–7.16 (m, 2H), 7.20–7.37 (m, 3H). To a solution of oxa-
lyl chloride (80.0 g, 630 mmol) in DCM (400 mL) at ꢀ78 °C was
added DMSO (73.8 g, 945 mmol). After being stirred for 10 min, a
solution of the obtained intermediate (77.7 g, 315 mmol) in DCM
(250 mL) was added. After being stirred for 1 h, TEA (159 g,
1.58 mol) was added. The mixture was allowed to warm to room
temperature, and the stirring was continued for 1 h. The mixture
was concentrated in vacuo and the residue was diluted with IPE.
The insoluble materials were removed by filtration and the filtrate
was concentrated under reduced pressure. The residue was puri-
fied by silica gel chromatography (hexane/EtOAc, 19:1 to 4:1) to
give 7 (76.8 g, 99%) as a brown oil. 1H NMR (CDCl3): d 1.35 (d,
J = 6.0 Hz, 6H), 4.69–4.81 (m, 1H), 5.55 (s, 2H), 6.24 (s, 1H), 7.08–
7.15 (m, 5H), 9.70 (s, 1H).
6.1.1.7. Ethyl 3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-iso-
propoxy-1H-pyrazol-5-yl}propanoate (9b). Compound 9b was
prepared in 64% yield by a method similar to that described for
9a. 1H NMR (CDCl3): d 1.19–1.28 (t, J = 7.1 Hz, 3H), 1.33 (d,
J = 6.3 Hz, 6H), 2.58 (t, J = 7.4 Hz, 2H), 2.77 (t, J = 7.4 Hz, 2H), 4.12
(q, J = 7.1 Hz, 2H), 4.65–4.76 (m, 1H), 5.26 (s, 2H), 5.55 (s, 1H),
6.70 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.64 (s, 1H).
6.1.1.8. 3-[1-(2,4-Dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-
yl]-N-(pentylsulfonyl)propanamide (3c). A mixture of 9a
(4.00 g, 12.1 mmol), 1 M NaOH (18 mL, 18.0 mmol), THF (20 mL),
and MeOH (20 mL) was heated at 50 °C with stirring for 2 h. After
being cooled, the mixture was acidified with 1 M HCl (19 mL),
diluted with brine, and extracted with EtOAc. The extract was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue was recrystallized from EtOAc/hexane to give
3-[1-(2,4-dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-yl]propa-
noic ac id (2) (3.54 g, 82%) as colorless crystals: mp 114–115 °C. 1H
NMR (CDCl3): d 1.33 (d, J = 6.2 Hz, 6H), 2.57–2.84 (m, 4H), 4.64–
4.77 (m, 1H), 5.19 (s, 2H), 5.54 (s, 1H), 6.56 (d, J = 8.1 Hz, 1H),
7.14 (dd, J = 2.1, 8.1 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H). Anal. Calcd
for C16H18Cl2N2O3: C, 53.79; H, 5.08; N, 7.84. Found: C, 53.97; H,
5.12; N, 7.89. A mixture of 2 (500 mg, 1.40 mmol), CDI (341 mg,
2.10 mmol), and THF (10 mL) was heated under reflux with stirring
for 1 h. After being cooled, 1-pentanesulfonamide (254 mg,
1.68 mmol) and DBU (320 mg, 2.10 mmol) were successively
added. After being stirred at room temperature for 15 h, the mix-
ture was concentrated in vacuo, acidified with 1 M HCl, and ex-
tracted with EtOAc. The extract was washed with brine, dried
6.1.1.5. Ethyl
3-(3-isopropoxy-1H-pyrazol-5-yl)propanoate
(8). To an ice-cooled solution of ethyl diethylphosphonoacetate
(91.4 g, 408 mmol) in DMF (300 mL) was added 60% NaH (16.3 g,
408 mmol). The mixture was allowed to warm to room tempera-
ture, and stirred for 30 min. After the mixture was cooed to 0 °C,
a solution of 7 (76.8 g, 314 mmol) in THF (250 mL) was added.
After being stirred for 1 h, the reaction was allowed to warm to
room temperature, and the stirring was continued for 30 min.
The mixture was concentrated in vacuo and partitioned between
EtOAc and water. The organic layer was washed with water and
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (hexane/
EtOAc, 80:1 to 1:1) to give ethyl (E)-3-(1-benzyl-3-isopropoxy-
1H-pyrazol-5-yl)propenoate (83.9 g, 85%) as a brown oil. 1H NMR

724
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
over MgSO4, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (hexane/EtOAc, 19:1 to
1:1) and successively recrystallized from EtOAc/hexane to give 3c
(450 mg, 66%) as colorless crystals: mp 126–129 °C. 1H NMR
(CDCl3): d 0.91 (t, J = 7.2 Hz, 3H), 1.24–1.48 (m, 10H), 1.74–1.86
(m, 2H), 2.54–2.62 (m, 2H), 2.77–2.85 (m, 2H), 3.34–3.41 (m,
2H), 4.63–4.76 (m, 1H), 5.18 (s, 2H), 5.50 (s, 1H), 6.57 (d,
J = 8.1 Hz, 1H), 7.15 (dd, J = 2.1, 8.1 Hz, 1H), 7.39 (d, J = 2.1 Hz,
1H), 8.00 (br s, 1H). Anal. Calcd for C21H29Cl2N3O4S: C, 51.43; H,
5.96; N, 8.57. Found: C, 51.32; H, 5.92; N, 8.69.
for 3c. Mp 137–138 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.09–
1.77 (m, 12H), 1.85–1.97 (m, 2H), 2.05–2.16 (m, 2H), 2.53–2.64
(m, 2H), 2.78–2.86 (m, 2H), 3.37–3.50 (m, 1H), 4.63–4.76 (m,
1H), 5.19 (s, 2H), 5.51 (s, 1H), 6.59 (d, J = 8.3 Hz, 1H), 7.16 (dd,
J = 2.1, 8.3 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.90 (br s, 1H). Anal.
Calcd for C22H29Cl2N3O4S: C, 52.59; H, 5.82; N, 8.36. Found: C,
52.53; H, 5.73; N, 8.41.
6.1.1.14. 3-[1-(2,4-Dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-
yl]-N-(phenylsulfonyl)propanamide (3i). Compound 3i was pre-
pared in 77% yield by a method similar to that described for 3c.
Mp 184–185 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.30 (d,
J = 6.0 Hz, 6H), 2.47–2.54 (m, 2H), 2.67–2.75 (m, 2H), 4.58–4.70
(m, 1H), 5.08 (s, 2H), 5.36 (s, 1H), 6.48 (d, J = 8.3 Hz, 1H), 7.10
(dd, J = 2.1, 8.3 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.51–7.59 (m, 2H),
7.62–7.69 (m, 1H), 7.97–8.04 (m, 2H). Anal. Calcd for
6.1.1.9. 3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-isopropoxy-
1H-pyrazol-5-yl}-N-(pentylsulfonyl)propanamide (3d). 3-{1-[2-
Chloro-4-(trifluoromethyl)benzyl]-3-isopropoxy-1H-pyrazol-5-yl}
propanoic acid was prepared in 77% yield by a method similar to
that described for 2. Mp 121–122 °C (EtOAc/hexane). 1H NMR
(CDCl3): d 1.33 (d, J = 6.0 Hz, 6H), 2.62–2.71 (m, 2H), 2.77 (t,
J = 7.1 Hz, 2H), 4.63–4.79 (m, 1H), 5.27 (s, 2H), 5.57 (s, 1H), 6.71
(d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.63 (s, 1H). Anal. Calcd
for C17H18ClF3N2O3: C, 52.25; H, 4.64; N, 7.17. Found: C, 52.31; H,
4.72; N, 7.15. Compound 3d was prepared in 6% yield from the cor-
responding carboxylic acid by a method similar to that described for
3c. Mp 147–149 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.90 (t,
J = 7.1 Hz, 3H), 1.32 (d, J = 6.2 Hz, 6H), 1.34–1.49 (m, 4H), 1.72–
1.86 (m, 2H), 2.62 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.3 Hz, 2H), 3.31–
3.45 (m, 2H), 4.60–4.82 (m, 1H), 5.27 (s, 2H), 5.53 (s, 1H), 6.72 (d,
J = 8.1 Hz, 1H), 7.44 (d, J = 0.9, 8.1 Hz, 1H), 7.64 (d, J = 0.9 Hz, 1H).
Anal. Calcd for C22H29ClF3N3O4S: C, 50.43; H, 5.58; N, 8.02. Found:
C, 50.48; H, 5.60; N, 7.93.
C22H23Cl2N3O4S: C, 53.23; H, 4.67; N, 8.46. Found: C, 53.13; H,
4.58; N, 8.56.
6.1.1.15. N-(Benzylsulfonyl)-3-[1-(2,4-dichlorobenzyl)-3-isopro-
poxy-1H-pyrazol-5-yl]propanamide (3j). Compound 3j was pre-
pared in 87% yield by a method similar to that described for 3c.
Mp 183–184 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.32 (d,
J = 6.0 Hz, 6H), 2.41–2.47 (m, 2H), 2.73–2.80 (m, 2H), 4.58 (s, 2H),
4.64–4.77 (m, 1H), 5.18 (s, 2H), 5.47 (s, 1H), 6.56 (d, J = 8.4 Hz,
1H), 7.11 (dd, J = 2.0, 8.4 Hz, 1H), 7.20–7.27 (m, 2H), 7.30–7.40
(m, 4H). Anal. Calcd for C23H25Cl2N3O4S: C, 54.12; H, 4.94; N,
8.23. Found: C, 54.20; H, 4.92; N, 8.30.
6.1.1.16. Methyl 2-(2,4-dichlorobenzyl)hydrazinecarboxylate
(11). A mixture of 2,4-dichlorobenzyl chloride 10 (108 g,
555 mmol), methyl carbazate (75.0 g, 833 mmol), K2CO3 (84.4 g,
611 mmol), and DMF (600 mL) was heated at 80 °C with stirring
for 4 h. After being cooled, the mixture was poured into water
and extracted with EtOAc. The extract was washed with water
and brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(hexane/EtOAc, 9:1 to 1:1) to give 11 (76.7 g, 55%) as a colorless
oil. 1H NMR (CDCl3): d 3.72 (s, 3H), 4.10 (d, J = 5.7 Hz, 2H), 4.33
(br s, 1H), 6.19 (br s, 1H), 7.23 (dd, J = 2.1, 8.4 Hz, 1H), 7.35 (d,
J = 8.4 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H).
6.1.1.10. N-(Butylsulfonyl)-3-[1-(2,4-dichlorobenzyl)-3-isopro-
poxy-1H-pyrazol-5-yl]propanamide (3e). Compound 3e was pre-
pared in 71% yield by a method similar to that described for 3c. Mp
133–134 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.95 (t, J = 7.3 Hz,
3H), 1.32 (d, J = 6.2 Hz, 6H), 1.40–1.55 (m, 2H), 1.70–1.83 (m,
2H), 2.54–2.62 (m, 2H), 2.78–2.87 (m, 2H), 3.35–3.43 (m, 2H),
4.64–4.77 (m, 1H), 5.19 (s, 2H), 5.51 (s, 1H), 6.60 (d, J = 8.3 Hz,
1H), 7.16 (dd, J = 2.1, 8.3 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.80 (br
s, 1H). Anal. Calcd for C20H27Cl2N3O4S: C, 50.42; H, 5.71; N, 8.82.
Found: C, 50.42; H, 5.64; N, 8.84.
6.1.1.11. 3-[1-(2,4-Dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-
yl]-N-(hexylsulfonyl)propanamide (3f)
pared in 48% yield by a method similar to that described for 3c.
Mp 103–106 °C (EtOAc/hexane). 1H NMR (CDCl3):
0.89 (t,
J = 6.9 Hz, 3H), 1.24–1.48 (m, 12H), 1.70–1.86 (m, 2H), 2.53–2.63
(m, 2H), 2.76–2.86 (m, 2H), 3.33–3.43 (m, 2H), 4.63–4.76 (m,
1H), 5.18 (s, 2H), 5.51 (s, 1H), 6.59 (d, J = 8.4 Hz, 1H), 7.15 (dd,
J = 2.1, 8.4 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 8.30 (br s, 1H). Anal.
Calcd for C22H31Cl2N3O4S: C, 52.38; H, 6.19; N, 8.33. Found: C,
52.35; H, 6.19; N, 8.25.
.
Compound 3f was pre-
6.1.1.17. Methyl
1-(2,4-dichlorobenzyl)-3-hydroxy-1H-pyra-
zole-5-carboxylate (12). To a solution of 11 (76.7 g, 308 mmol)
in MeOH (500 mL) was added dimethyl acetylenedicarboxylate
(43.8 g, 308 mmol). The mixture was heated under reflux with stir-
ring for 2 h. After being cooled, NaOMe (19.1 g, 354 mmol) was
added to the mixture. The mixture was heated under reflux with
stirring for 1 h. After being cooled, the mixture was poured into
1 M HCl. The precipitate was collected and dried to give 12
(61.0 g, 66%) as a dark yellow solid. 1H NMR (DMSO-d6): d 3.77
(s, 3H), 5.58 (s, 2H), 6.16 (s, 1H), 6.73 (d, J = 8.4 Hz, 1H), 7.38 (dd,
J = 2.1, 8.4 Hz, 1H), 7.65 (d, J = 2.1 Hz, 1H), 10.33 (br s, 1H).
d
6.1.1.12. 3-[1-(2,4-Dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-
yl]-N-[(3-methylbutyl)sulfonyl]propanamide (3g). Compound
3g was prepared in 73% yield by a method similar to that described
for 3c. Mp 144 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.92 (d,
J = 6.0 Hz, 6H), 1.32 (d, J = 6.3 Hz, 6H), 1.66–1.77 (m, 3H), 2.54–
2.63 (m, 2H), 2.76–2.86 (m, 2H), 3.35–3.44 (m, 2H), 4.64–4.77
(m, 1H), 5.19 (s, 2H), 5.51 (s, 1H), 6.59 (d, J = 8.4 Hz, 1H), 7.15
(dd, J = 2.1, 8.4 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.95 (br s, 1H). Anal.
Calcd for C21H29Cl2N3O4S: C, 51.43; H, 5.96; N, 8.57. Found: C,
51.29; H, 5.79; N, 8.41.
6.1.1.18. 1-(2,4-Dichlorobenzyl)-3-(methoxymethoxy)-1H-pyra-
zole-5-carbaldehyde (13). To an ice-cooed mixture of 12 (42.0 g,
139 mmol) and K2CO3 (28.8 g, 208 mmol) in DMF (350 mL) was
added a solution of chloromethyl methyl ether (16.7 g, 208 mmol)
in THF (100 mL). The mixture was allowed to warm to room tem-
perature and stirred for 15 h. The mixture was poured into water
and extracted with EtOAc. The extract was washed with water
and brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography
(hexane/EtOAc, 100:1 to 8:1) to give methyl 1-(2,4-dichloroben-
6.1.1.13.
N-(Cyclohexylsulfonyl)-3-[1-(2,4-dichlorobenzyl)-3-
isopropoxy-1H-pyrazol-5-yl]propanamide (3h). Compound 3h
was prepared in 55% yield by a method similar to that described
zyl)-3-(methoxymethoxy)-1H-pyrazole-5-carboxylate
(34.4 g,
72%) as a pale orange oil. 1H NMR (CDCl3): d 3.51 (s, 3H), 3.82 (s,

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
725
3H), 5.22 (s, 2H), 5.70 (s, 2H), 6.43 (s, 1H), 6.56 (d, J = 8.4 Hz, 1H),
7.11 (dd, J = 2.1, 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H). [1-(2,4-Dichlo-
2H), 3.43 (s, 3H), 3.67–3.79 (m, 2H), 4.12 (q, J = 7.2 Hz, 2H), 4.24–
4.30 (m, 2H), 5.18 (s, 2H), 5.59 (s, 1H), 6.51 (d, J = 8.4 Hz, 1H),
7.14 (dd, J = 2.1, 8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H).
robenzyl)-3-(methoxymethoxy)-1H-pyrazol-5-yl]methanol
was
prepared from the obtained methyl ester in 84% yield by a similar
method that described for (1-benzyl-3-isopropoxy-1H-pyrazol-5-
yl)methanol (see Section 6.1.1.4.). 1H NMR (CDCl3): d 1.98 (t,
J = 5.9 Hz, 1H), 3.51 (s, 3H), 4.53 (d, J = 5.9 Hz, 2H), 5.19 (s, 2H),
5.29 (s, 2H), 5.83 (s, 1H), 6.68 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1,
8.1 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H). Compound 13 was prepared
from the obtained alcohol in 99% yield by a method similar to that
described for 7. 1H NMR (CDCl3): d 3.53 (s, 3H), 5.25 (s, 2H), 5.67 (s,
2H), 6.48 (s, 1H), 6.66 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 2.1, 8.4 Hz,
1H), 7.39 (d, J = 2.1 Hz, 1H), 9.76 (s, 1H).
6.1.1.24. Ethyl 3-[3-(benzyloxy)-1-(2,4-dichlorobenzyl)-1H-pyr-
azol-5-yl]propanoate (17c). Compound 17c was prepared in 40%
yield by a method similar to that described for 17a. 1H NMR
(CDCl3): d 1.23 (t, J = 7.2 Hz, 3H), 2.52–2.62 (m, 2H), 2.72–2.82
(m, 2H), 4.12 (q, J = 7.2 Hz, 2H), 5.17 (s, 2H), 5.21 (s, 2H), 5.58 (s,
1H), 6.52 (d, J = 8.1 Hz, 1H), 7.13 (dd, J = 2.1, 8.1 Hz, 1H), 7.30–
7.47 (m, 6H).
6.1.1.25. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-(pyridin-2-ylmeth-
oxy)-1H-pyrazol-5-yl]propanoate (17d). To
a mixture of 16
6.1.1.19. Ethyl (E)-3-[1-(2,4-dichlorobenzyl)-3-(methoxyme-
thoxy)-1H-pyrazol-5-yl]propenoate (14). Compound 14 was
prepared in 90% yield by a method similar to that described for
ethyl (E)-3-(1-benzyl-3-isopropoxy-1H-pyrazol-5-yl)propenoate
(see Section 6.1.1.5.). Mp 96–97 °C (EtOAc/hexane). 1H NMR
(CDCl3): d 1.31 (t, J = 7.2 Hz, 3H), 3.52 (s, 3H), 4.23 (q, J = 7.2 Hz,
2H), 5.22 (s, 2H), 5.35 (s, 2H), 6.15 (s, 1H), 6.30 (d, J = 15.6 Hz,
1H), 6.71 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.1, 8.4 Hz, 1H), 7.40 (d,
J = 2.1 Hz, 1H), 7.41 (d, J = 15.6 Hz, 1H).
(1.00 g, 2.91 mmol) and K2CO3 (1.01 g, 7.28 mmol) in DMF
(20 mL) was added 2-(chloromethyl)pyridine hydrochloride
(573 mg, 3.49 mmol). After being stirred at room temperature for
15 h, the mixture was poured into water and extracted with EtOAc.
The extract was washed with water and brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel chromatography (hexane/EtOAc, 19:1 to 2:1) to
give 17d (1.00 g, 79%) as a colorless oil. 1H NMR (CDCl3): d 1.24
(t, J = 7.2 Hz, 3H), 2.53–2.63 (m, 2H), 2.73–2.83 (m, 2H), 4.12 (q,
J = 7.2 Hz, 2H), 5.19 (s, 2H), 5.29 (s, 2H), 5.63 (s, 1H), 6.54 (d,
J = 8.4 Hz, 1H), 7.12 (dd, J = 2.1, 8.4 Hz, 1H), 7.18–7.24 (m, 1H),
7.38 (d, J = 2.1 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.66–7.73 (m, 1H),
8.57–8.62 (m, 1H).
6.1.1.20 Ethyl 3-[1-(2,4-dichlorobenzyl)-3-(methoxymethoxy)-
1H-pyrazol-5-yl]propanoate (15). A mixture of 14 (19.0 g,
49.3 mmol), 5% Pd/C (2.10 g), and THF (200 mL) was hydrogenated
under atmospheric pressure at room temperature for 6 h. The cat-
alyst was removed by filtration and the filtrate was concentrated in
vacuo to give 15 (19.1 g, quant.) as a pale yellow oil. 1H NMR
(CDCl3): d 1.25 (t, J = 7.2 Hz, 3H), 2.53–2.62 (m, 2H), 2.73–2.82
(m, 2H), 3.51 (s, 3H), 4.13 (q, J = 7.2 Hz, 2H), 5.20 (s, 2H), 5.21 (s,
2H), 5.66 (s, 1H), 6.60 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.1, 8.4 Hz,
1H), 7.38 (d, J = 2.1 Hz, 1H).
6.1.1.26. 3-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)propanamide
(18a). 3-[3-Butoxy-1-(2,4-
dichlorobenzyl)-1H-pyrazol-5-yl]propanoic acid was prepared in
88% yield by a method similar to that described for 2. Mp 123–
124 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.95 (t, J = 7.3 Hz, 3H),
1.37–1.53 (m, 2H), 1.64–1.78 (m, 2H), 2.58–2.67 (m, 2H), 2.70–
2.82 (m, 2H), 4.09 (t, J = 6.6 Hz, 2H), 5.19 (s, 2H), 5.56 (s, 1H),
6.55 (d, J = 8.1 Hz, 1H), 7.14 (dd, J = 2.1, 8.1 Hz, 1H), 7.38 (d,
J = 2.1 Hz, 1H). Compound 18a was prepared from the correspond-
ing carboxylic acid in 66% yield by a method similar to that de-
scribed for 3c. Mp 104–105 °C (EtOAc/hexane). 1H NMR (CDCl3):
d 0.85–1.00 (m, 6H), 1.24–1.87 (m, 10H), 2.53–2.63 (m, 2H),
2.75–2.86 (m, 2H), 3.33–3.43 (m, 2H), 4.08 (t, J = 6.6 Hz, 2H), 5.19
(s, 2H), 5.53 (s, 1H), 6.56 (d, J = 8.5 Hz, 1H), 7.15 (dd, J = 2.2,
8.5 Hz, 1H), 7.39 (d, J = 2.2 Hz, 1H), 8.17 (br s, 1H). Anal. Calcd for
6.1.1.21. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-hydroxy-1H-pyrazol-
5-yl]propanoate (16). A mixture of 15 (19.1 g, 49.3 mmol), concd.
HCl (0.4 mL), and EtOH (200 mL) was heated under reflux with stir-
ring for 6 h. After being cooled, the mixture was concentrated in
vacuo and the residue was diluted with IPE/hexane/EtOAc. The
resultant precipitate was collected and dried to give 16 (16.2 g,
96%) as a white solid. 1H NMR (CDCl3): d 1.24 (t, J = 7.2 Hz, 3H),
2.51–2.63 (m, 2H), 2.68–2.80 (m, 2H), 4.12 (q, J = 7.2 Hz, 2H),
5.18 (s, 2H), 5.49 (s, 1H), 5.60 (br s, 1H), 6.67 (d, J = 8.1 Hz, 1H),
7.18 (dd, J = 2.1, 8.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H).
C22H31Cl2N3O4S: C, 52.38; H, 6.19; N, 8.33. Found: C, 52.22; H,
6.17; N, 8.36.
6.1.1.22. Ethyl 3-[3-butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-
5-yl]propanoate (17a). To a solution of 16 (1.88 g, 5.48 mmol), 1-
butanol (609 mg, 8.22 mmol), and tributylphosphine (2.23 g,
11.0 mmol) in THF (110 mL) was added ADDP (2.78 g, 11.0 mmol)
portionwise. After being stirred at room temperature for 15 h, the
mixture was concentrated in vacuo and the residue was diluted
with IPE. The insoluble material was removed by filtration and
the filtrate was concentrated under reduced pressure. The residue
was purified by silica gel chromatography (hexane/EtOAc, 19:1 to
17:3) to give 17a (2.07 g, 95%) as a colorless oil. 1H NMR (CDCl3):
d 0.95 (t, J = 7.3 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H), 1.38–1.54 (m,
2H), 1.66–1.78 (m, 2H), 2.52–2.64 (m, 2H), 2.72–2.82 (m, 2H),
4.09 (t, J = 6.7 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 5.19 (s, 2H), 5.54
(s, 1H), 6.54 (d, J = 8.3 Hz, 1H), 7.15 (dd, J = 2.1, 8.3 Hz, 1H), 7.38
(d, J = 2.1 Hz, 1H).
6.1.1.27.
3-[1-(2,4-Dichlorobenzyl)-3-(2-methoxyethoxy)-1H-
pyrazol-5-yl]-N-(pentylsulfonyl)propanamide (18b). 3-[1-(2,4-
Dichlorobenzyl)-3-(2-methoxyethoxy)-1H-pyrazol-5-yl]propanoic
acid was prepared in 94% yield by a method similar to that de-
scribed for 2. Mp 111–113 °C (EtOAc/hexane). 1H NMR (CDCl3): d
2.60–2.67 (m, 2H), 2.73–2.80 (m, 2H), 3.43 (s, 3H), 3.68–3.74 (m,
2H), 4.25–4.30 (m, 2H), 5.19 (s, 2H), 5.61 (s, 1H), 6.52 (d,
J = 8.4 Hz, 1H), 7.13 (dd, J = 2.1, 8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz,
1H). Anal. Calcd for C16H18Cl2N2O4: C, 51.49; H, 4.86; N, 7.51.
Found: C, 51.23; H, 5.02; N, 7.36. Compound 18b was prepared
from the corresponding carboxylic acid in 30% yield by a method
similar to that described for 3c. Mp 108–112 °C (EtOAc/hexane).
1H NMR (CDCl3): d 0.90 (t, J = 6.9 Hz, 3H), 1.23–1.48 (m, 4H),
1.65–1.86 (m, 2H), 2.52–2.63 (m, 2H), 2.74–2.85 (m, 2H), 3.32–
3.41 (m, 2H), 3.42 (s, 3H), 3.65–3.75 (m, 2H), 4.23–4.30 (m, 2H),
5.18 (s, 2H), 5.55 (s, 1H), 6.54 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1,
8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 8.35 (br s, 1H). Anal. Calcd for
6.1.1.23. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-(2-methoxyethoxy)-
1H-pyrazol-5-yl]propanoate (17b). Compound 17b was prepared
in 73% yield by a method similar to that described for 17a. 1H NMR
(CDCl3): d 1.24 (t, J = 7.2 Hz, 3H), 2.52–2.62 (m, 2H), 2.70–2.80 (m,
C21H29Cl2N3O5S: C, 49.80; H, 5.77; N, 8.30. Found: C, 49.69; H,
5.72; N, 8.24.

726
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
6.1.1.28. 3-[3-(Benzyloxy)-1-(2,4-dichlorobenzyl)-1H-pyrazol-
5-yl]-N-(pentylsulfonyl)propanamide (18c). 3-[3-(Benzyloxy)-
6.1.1.31. Ethyl 3-cyclopropyl-1H-pyrazole-5-carboxylate (20b).
Compound 20b was prepared in 60% yield (2 steps) by a method sim-
ilar to that described for 20a. Mp 99–100 °C (IPE). 1H NMR (CDCl3): d
0.68–0.82 (m, 2H), 0.96–1.06 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H), 1.88–
2.04 (m, 1H), 4.36 (q, J = 7.2 Hz, 2H), 6.45 (s, 1H), 11.30 (br s, 1H).
1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]propanoic
acid
was
prepared in 88% yield by a method similar to that described for
2. Mp 98–101 °C (EtOAc/hexane). 1H NMR (CDCl3): d 2.60–2.67
(m, 2H), 2.72–2.80 (m, 2H), 5.17 (s, 2H), 5.20 (s, 2H), 5.60 (s, 1H),
6.52 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 2.1, 8.4 Hz, 1H), 7.25–7.45
(m, 6H). Anal. Calcd for C20H18Cl2N2O3: C, 59.27; H, 4.48; N, 6.91.
Found: C, 59.05; H, 4.35; N, 6.75. Compound 18c was prepared
from the corresponding carboxylic acid in 56% yield by a method
similar to that described for 3c. Mp 140 °C (EtOAc/hexane). 1H
NMR (CDCl3): d 0.89 (3H, t, J = 7.1 Hz), 1.27–1.46 (m, 4H), 1.72–
1.85 (m, 2H), 2.52–2.63 (m, 2H), 2.76–2.86 (m, 2H), 3.33–3.43
(m, 2H), 5.17 (s, 2H), 5.20 (s, 2H), 5.57 (s, 1H), 6.55 (d, J = 8.1 Hz,
1H), 7.13 (dd, J = 2.1, 8.1 Hz, 1H), 7.28–7.45 (m, 6H), 8.00 (br s,
1H). Anal. Calcd for C25H29Cl2N3O4S: C, 55.76; H, 5.43; N, 7.80.
Found: C, 55.58; H, 5.39; N, 7.73.
6.1.1.32. Ethyl 3-phenyl-1H-pyrazole-5-carboxylate (20c). Com-
pound 20c was prepared in 81% yield (2 steps) by a method similar
to that described for 20a. 1H NMR (CDCl3): d 1.36 (t, J = 7.2 Hz, 3H),
4.36 (q, J = 7.2 Hz, 2H), 7.10 (s, 1H), 7.30–7.46 (m, 3H), 7.71–7.80
(m, 2H), 11.86 (br s, 1H).
6.1.1.33. Ethyl 1-(2,4-dichlorobenzyl)-3-isopropyl-1H-pyrazole-
5-carboxylate (21a). Compound 21a was prepared in 54% yield by
a method similar to that described for 6. 1H NMR (CDCl3): d 1.29 (d,
J = 7.2 Hz, 9H), 1.30 (t, J = 7.2 Hz, 3H), 2.95–3.09 (m, 1H), 4.26 (q,
J = 7.2 Hz, 2H), 5.77 (s, 2H), 6.34 (d, J = 8.4 Hz, 1H), 6.78 (s, 1H),
7.09 (dd, J = 2.1, 8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H).
6.1.1.29.
3-[1-(2,4-Dichlorobenzyl)-3-(pyridin-2-ylmethoxy)-
hydrochlo-
1H-pyrazol-5-yl]-N-(pentylsulfonyl)propanamide
6.1.1.34. Ethyl 1-[2-chloro-4-(trifluoromethyl)benzyl]-3-isopro-
pyl-1H-pyrazole-5-carboxylate (21b). Compound 21b was pre-
pared in 42% yield by a method similar to that described for 6.
1H NMR (CDCl3): d 1.29 (t, J = 7.1 Hz, 3H), 1.30 (d, J = 7.0 Hz, 6H),
2.91–3.10 (m, 1H), 4.26 (q, J = 7.1 Hz, 2H), 5.85 (s, 2H), 6.48 (d,
J = 8.1 Hz, 1H), 6.80 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.64 (s, 1H).
ride (18d). A mixture was 17d (1.00 g, 2.30 mmol), 1 M NaOH
(5.0 mL, 5.00 mmol), THF (10 mL), and EtOH (10 mL) was heated
at 50 °C with stirring for 1 h. After being cooled, the mixture was
concentrated in vacuo and the residue was diluted with water.
The insoluble product was collected by filtration and dried to give
3-[1-(2,4-dichlorobenzyl)-3-(pyridin-2-ylmethoxy)-1H-pyrazol-5-
yl]propanoic acid (760 mg, 75%) as a white solid. 1H NMR (DMSO-
d6): d 2.46–2.56 (m, 2H), 2.67–2.77 (m, 2H), 5.15 (s, 2H), 5.20 (s,
2H), 5.72 (s, 1H), 6.62 (d, J = 8.4 Hz, 1H), 7.27–7.34 (m, 1H), 7.35
(dd, J = 2.1, 8.4 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 2.1 Hz,
1H), 7.77–7.84 (m, 1H), 8.51–8.56 (m, 1H), 12.30 (br s, 1H). To a
solution of the obtained carboxylic acid (760 mg, 1.87 mmol) in
DMF (15 mL) was added CDI (455 mg, 2.81 mmol). After being stir-
red at room temperature for 1 h, 1-pentanesulfonamide (297 mg,
1.96 mmol) and DBU (428 mg, 2.81 mmol) were successively
added. The mixture was heated at 100 °C with stirring for 15 h.
After being cooled, the mixture was concentrated in vacuo and
the residue was diluted with 1 M HCl/EtOAc (10/1), which was stir-
red at room temperature for 2 h. The insoluble product was col-
lected by filtration and dried to give 18d (500 mg, 46%) as a
white solid: mp 167–169 °C. 1H NMR (DMSO-d6): d 0.81 (t,
J = 7.2 Hz, 3H), 1.17–1.38 (m, 4H), 1.52–1.67 (m, 2H), 2.56–2.67
(m, 2H), 2.72–2.83 (m, 2H), 3.26–3.38 (m, 2H), 5.20 (s, 2H), 5.28
(s, 2H), 5.71 (s, 1H), 6.63 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 2.1,
8.4 Hz, 1H), 7.56–7.74 (m, 3H), 8.08–8.19 (m, 1H), 8.67 (d,
J = 4.8 Hz, 1H), 11.71 (br s, 1H). Anal. Calcd for
6.1.1.35. Ethyl 1-[2-chloro-4-(trifluoromethyl)benzyl]-3-cyclo-
propyl-1H-pyrazole-5-carboxylate (21c). Compound 21c was
prepared in 56% yield by a method similar to that described for
6. 1H NMR (CDCl3): d 0.72–0.80 (m, 2H), 0.92–1.02 (m, 2H), 1.29
(t, J = 7.2 Hz, 3H), 1.90–2.02 (m, 1H), 4.25 (q, J = 7.2 Hz, 2H), 5.82
(s, 2H), 6.53 (d, J = 8.1 Hz, 1H), 6.62 (s, 1H), 7.38 (d, J = 8.1 Hz,
1H), 7.64 (s, 1H).
6.1.1.36. Ethyl 1-(2,4-dichlorobenzyl)-3-phenyl-1H-pyrazole-5-
carboxylate (21d). Compound 21d was prepared in 78% yield by a
method similar to that described for 6. Mp 104–107 °C (EtOAc/hex-
ane). 1H NMR (CDCl3): d 1.33 (t, J = 7.2 Hz, 3H), 4.31 (q, J = 7.2 Hz,
2H), 5.88 (s, 2H), 6.52 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 2.1, 8.5 Hz,
1H), 7.38–7.47 (m, 4H), 7.80–7.87 (m, 2H). Anal. Calcd for
C
19H16Cl2N2O2ꢁ0.1H2O: C, 60.52; H, 4.33; N, 7.43. Found: C,
60.41; H, 4.22; N, 7.44.
6.1.1.37. Ethyl (E)-3-[1-(2,4-dichlorobenzyl)-3-isopropyl-1H-
pyrazol-5-yl]propenoate
(22a). [1-(2,4-Dichlorobenzyl)-3-iso-
propyl-1H-pyrazol-5-yl]methanol was prepared in 99% yield by
a method similar to that described for (1-benzyl-3-isopropoxy-
1H-pyrazol-5-yl)methanol (see experimental procedure of 7).
1H NMR (CDCl3): d 1.27 (d, J = 6.9 Hz, 6H), 1.63 (t, J = 5.6 Hz,
1H), 2.92–3.06 (m, 1H), 4.57 (d, J = 5.6 Hz, 2H), 5.49 (s, 2H),
6.14 (s, 1H), 6.59 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 1.2, 8.0 Hz,
1H), 7.62 (d, J = 1.2 Hz, 1H). 1-(2,4-Dichlorobenzyl)-3-isopropyl-
1H-pyrazole-5-carbaldehyde was prepared from the correspond-
ing alcohol in quantitative yield by a method similar to that de-
scribed for 7. 1H NMR (CDCl3): d 1.30 (d, J = 7.0 Hz, 6H), 2.98–
3.12 (m, 1H), 5.75 (s, 2H), 6.43 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H),
7.10 (dd, J = 2.1, 8.4 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 9.80 (s,
1H). Compound 22a was prepared in 99% yield by a method
similar to that described for ethyl (E)-3-(1-benzyl-3-isopro-
poxy-1H-pyrazol-5-yl)propenoate (see Section 6.1.1.5.). 1H NMR
C
24H28Cl2N4O4SꢁHClꢁH2O: C, 48.53; H, 5.25; N, 9.47. Found: C,
48.80; H, 5.25; N, 9.47.
6.1.1.30. Ethyl 3-isopropyl-1H-pyrazole-5-carboxylate (20a). To
a mixture of t-BuOK (92.0 g, 697 mmol) and THF (500 mL) was
added a mixture of 3-methyl-2-butanone (50.0 g, 581 mmol) and
diethyl oxalate (85.8 g, 587 mmol) dropwise over 1 h. The mixture
was stirred at room temperature for 15 h to form ethyl 5-methyl-
2,4-dioxohexanoate. Thereafter, AcOH (80 mL, 1.39 mol) and
hydrazine hydrate (32.0 g, 639 mmol) were successively added.
The mixture was heated under reflux with stirring for 2 h. After
being cooled, the mixture was concentrated in vacuo and the res-
idue was partitioned between EtOAc and water. The organic solu-
tion was washed with sat. NaHCO3 and brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel chromatography (hexane/EtOAc, 9:1 to 1:1) to
give 20a (57.7 g, 55%) as a brown oil. 1H NMR (CDCl3): d 1.30 (d,
J = 6.9 Hz, 6H), 1.39 (t, J = 7.2 Hz, 3H), 2.95–3.12 (m, 1H), 4.38 (q,
J = 7.2 Hz, 2H), 6.64 (s, 1H), 10.30 (br s, 1H).
(CDCl3):
d 1.28 (d, J = 6.9 Hz, 6H), 1.30 (t, J = 7.2 Hz, 3H),
2.92–3.03 (m, 1H), 4.22 (q, J = 7.2 Hz, 2H), 5.44 (s, 2H), 6.30 (d,
J = 15.9 Hz, 1H), 6.48 (s, 1H), 6.49 (d, J = 8.4 Hz, 1H), 7.13 (dd,
J = 2.1, 8.4 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 7.41 (d, J = 15.9 Hz,
1H).

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
727
6.1.1.38. Ethyl (E)-3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
isopropyl-1H-pyrazol-5-yl}propenoate (22b). {1-[2-Chloro-4-
J = 15.9 Hz, 1H), 6.67 (d, J = 8.5 Hz, 1H), 6.95 (s, 1H), 7.13 (dd,
J = 2.1, 8.5 Hz, 1H), 7.30–7.46 (m, 4H), 7.48 (d, J = 15.9 Hz, 1H),
7.77–7.84 (m, 2H). Anal. Calcd for C21H18Cl2N2O2: C, 62.85; H,
4.52; N, 6.98. Found: C, 62.79; H, 4.64; N, 6.77.
(trifluoromethyl)benzyl]-3-isopropyl-1H-pyrazol-5-yl}methanol
was prepared in 99% yield by a method similar to that described for
(1-benzyl-3-isopropoxy-1H-pyrazol-5-yl)methanol (see Section
6.1.1.4.). 1H NMR (CDCl3): d 1.29 (d, J = 6.9 Hz, 6H), 1.63 (t,
J = 5.6 Hz, 1H), 2.92–3.06 (m, 1H), 4.57 (d, J = 5.6 Hz, 1H), 5.49 (s,
2H), 6.14 (s, 1H), 6.59 (d, J = 8.0 Hz, 1H), 7.39 (dd, J = 1.2, 8.0 Hz,
6.1.1.41. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-isopropyl-1H-pyra-
zol-5-yl]propanoate (23a). Compound 23a was prepared in 89%
yield by a method similar to that described for 15. 1H NMR (CDCl3):
d 1.24 (t, J = 7.2 Hz, 3H), 1.26 (d, J = 6.9 Hz, 6H), 2.55–2.63 (m, 2H),
2.74–2.82 (m, 2H), 2.90–3.04 (m, 1H), 4.12 (q, J = 7.2 Hz, 2H), 5.33
(s, 2H), 5.94 (s, 1H), 6.42 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 2.1, 8.4 Hz,
1H), 7.37 (d, J = 2.1 Hz, 1H).
1H),
7.62
(d,
J = 1.2 Hz,
1H).
1-[2-Chloro-4-(trifluoro-
methyl)benzyl]-3-isopropyl-1H-pyrazole-5-carbaldehyde was pre-
pared from the corresponding alcohol in 99% yield by a method
similar to that described for 7. 1H NMR (CDCl3):
d 1.31(d,
J = 7.2 Hz, 6H), 2.98–3.13 (m, 1H), 5.82 (s, 2H), 6.52 (d, J = 8.1 Hz,
1H), 6.83 (s, 1H), 7.36 (d, J = 1.2, 8.1 Hz, 1H), 7.65 (d, J = 1.2 Hz,
1H), 9.79 (s, 1H). Compound 22b was prepared from the corre-
sponding aldehyde in 29% yield by a method similar to that de-
scribed for ethyl (E)-3-(1-benzyl-3-isopropoxy-1H-pyrazol-5-
yl)propenoate (see Section 6.1.1.5.). 1H NMR (CDCl3): d 1.28 (d,
J = 6.9 Hz, 6H), 1.30 (t, J = 7.2 Hz, 3H), 2.93–3.07 (m, 1H), 4.21 (q,
J = 7.2 Hz, 2H), 5.51 (s, 2H), 6.31 (d, J = 15.8 Hz, 1H), 6.50 (s, 1H),
6.60 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 15.8 Hz, 1H), 7.40 (d, J = 7.8 Hz,
1H), 7.65 (s, 1H).
6.1.1.42. Ethyl 3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-iso-
propyl-1H-pyrazol-5-yl}propanoate (23b). Compound 23b was
prepared in 84% yield by a method similar to that described for
15. 1H NMR (CDCl3): d 1.23 (t, J = 7.2 Hz, 3H), 1.26 (d, J = 7.2 Hz,
6H), 2.57–2.64 (m, 2H), 2.74–2.81 (m, 2H), 2.89–3.02 (m, 1H),
4.11 (q, J = 7.2 Hz, 2H), 5.38 (s, 2H), 5.96 (s, 1H), 6.51 (d,
J = 8.1 Hz, 1H), 7.40 (dd, J = 1.2, 8.1 Hz, 1H), 7.63 (d, J = 1.2 Hz, 1H).
6.1.1.43. Ethyl
3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
cyclopropyl-1H-pyrazol-5-yl}propanoate (23c). Compound 23c
was prepared by a method similar to that described for 15 and
obtained in 72% yield (calculated from 1H NMR) along with an
inseparable byproduct, ethyl 3-{1-[2-chloro-4-(trifluoromethyl)
benzyl]-3-propyl-1H-pyrazol-5-yl}propanoate. 1H NMR (CDCl3): d
0.67–0.74 (m, 2H), 0.87–0.97 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H),
1.85–1.97 (m, 1H), 2.53–2.63 (m, 2H), 2.72–2.82 (m, 2H), 4.11 (q,
J = 7.2 Hz, 2H), 5.36 (s, 2H), 5.79 (s, 1H), 6.60 (d, J = 8.1 Hz, 1H),
7.41 (d, J = 8.1 Hz, 1H), 7.64 (s, 1H).
6.1.1.39. Ethyl (E)-3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
cyclopropyl-1H-pyrazol-5-yl}propenoate (22c). {1-[2-Chloro-4-
(trifluoromethyl)benzyl]-3-cyclopropyl-1H-pyrazol-5-yl}methanol
was prepared in 84% yield by a method similar to that described for
(1-benzyl-3-isopropoxy-1H-pyrazol-5-yl)methanol (see Section
6.1.1.4.). 1H NMR (CDCl3): d 0.68–0.75 (m, 2H), 0.88–0.98 (m,
2H), 1.63 (t, J = 5.7 Hz, 1H), 1.88–1.99 (m, 1H), 4.55 (d, J = 5.7 Hz,
2H), 5.45 (s, 2H), 5.96 (s, 1H), 6.66 (d, J = 8.1 Hz, 1H), 7.41 (d,
J = 8.1 Hz, 1H), 7.63 (s, 1H). 1-[2-Chloro-4-(trifluoromethyl) ben-
zyl]-3-cyclopropyl-1H-pyrazole-5-carbaldehyde was prepared
from the corresponding alcohol in 84% yield by a method similar
to that described for 7. Mp 94–96 °C (EtOAc/hexane). 1H NMR
(CDCl3): d 0.75–0.83 (m, 2H), 0.95–1.05 (m, 2H), 1.93–2.06 (m,
1H), 5.80 (s, 2H), 6.58 (d, J = 8.1 Hz, 1H), 6.67 (s, 1H), 7.38 (d,
J = 8.1 Hz, 1H), 7.65 (s, 1H), 9.77 (s, 1H). Compound 22c was pre-
pared from the corresponding aldehyde in 97% yield by a method
similar to that described for ethyl (E)-3-(1-benzyl-3-isopropoxy-
1H-pyrazol-5-yl)propenoate (see Section 6.1.1.5.). 1H NMR (CDCl3):
d 0.72–0.80 (m, 2H), 0.92–1.01 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H),
1.88–2.00 (m, 1H), 4.22 (q, J = 7.2 Hz, 2H), 5.49 (s, 2H), 6.26 (s,
1H), 6.29 (d, J = 15.8 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 7.38 (d,
J = 15.8 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.66 (s, 1H).
6.1.1.44. Ethyl 3-[1-(2,4-dichlorobenzyl)-3-phenyl-1H-pyrazol-
5-yl]propanoate (23d). Compound 23d was prepared in 77% yield
by a method similar to that described for 15. Mp 91–93 °C (EtOAc/
hexane). 1H NMR (CDCl3): d 1.25 (t, J = 7.2 Hz, 3H), 2.61–2.70 (m,
2H), 2.81–2.90 (m, 2H), 4.13 (q, J = 7.2 Hz, 2H), 5.41 (s, 2H), 6.43
(s, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 2.1, 8.4 Hz, 1H),
7.25–7.42 (m, 4H), 7.72–7.81 (m, 2H). Anal. Calcd for
C
21H20Cl2N2O2ꢁ0.1H2O: C, 62.26; H, 5.03; N, 6.92. Found: C,
62.19; H, 4.96; N, 6.85.
6.1.1.453-[1-(2,4-Dichlorobenzyl)-3-isopropyl-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)propanamide
(24a). 3-[1-(2,4-Dichloroben-
zyl)-3-isopropyl-1H-pyrazol-5-yl]propanoic acid was prepared in
74% yield by a method similar to that described for 2. Mp 105–
108 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.25 (d, J = 6.9 Hz, 6H),
2.63–2.68 (m, 2H), 5.31 (s, 2H), 5.96 (s, 1H), 6.39 (d, J = 8.3 Hz,
1H), 7.13 (dd, J = 2.2, 8.3 Hz, 1H), 7.39 (d, J = 2.2 Hz, 1H). Anal. Calcd
for C16H18Cl2N2O2: C, 56.32; H, 5.32; N, 8.21. Found: C, 56.22; H,
5.35; N, 8.20. Compound 24a was prepared from the corresponding
carboxylic acid in 67% yield by a method similar to that described
for 3c. Mp 115–117 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.90 (t,
J = 7.2 Hz, 3H), 1.25 (d, J = 6.9 Hz, 6H), 1.28–1.47 (m, 4H), 1.72–1.84
(m, 2H), 2.55–2.63 (m, 2H), 2.78–2.86 (m, 2H), 2.88–3.02 (m, 1H),
3.34–3.42 (m, 2H), 5.30 (s, 2H), 5.93 (s, 1H), 6.40 (d, J = 8.4 Hz, 1H),
7.12 (dd, J = 1.8, 8.4 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 8.20 (br s, 1H).
Anal. Calcd for C21H29Cl2N3O3S: C, 53.16; H, 6.16; N, 8.86. Found: C,
53.15; H, 6.17; N, 8.78.
6.1.1.40. Ethyl (E)-3-[1-(2,4-dichlorobenzyl)-3-phenyl-1H-pyra-
zol-5-yl]propenoate (22d). [1-(2,4-Dichlorobenzyl)-3-phenyl-1H-
pyrazol-5-yl]methanol was prepared in 92% yield by a method
similar to that described for (1-benzyl-3-isopropoxy-1H-pyrazol-
5-yl)methanol (see Section 6.1.1.4.). Mp 140–141 °C (EtOAc/hex-
ane). 1H NMR (CDCl3): d 1.95 (t, J = 5.3 Hz, 1H), 4.60 (d, J = 5.3 Hz,
2H), 5.49 (s, 2H), 6.58 (s, 1H), 6.63 (d, J = 8.3 Hz, 1H), 7.11 (dd,
J = 2.1, 8.3 Hz, 1H), 7.27–7.45 (m, 4H), 7.78 (dd, J = 1.5, 8.4 Hz,
2H).
1-(2,4-Dichlorobenzyl)-3-phenyl-1H-pyrazole-5-carbalde-
hyde was prepared from the corresponding alcohol in 67% yield
by a method similar to that described for 7. Mp 136 °C (EtOAc/hex-
ane). 1H NMR (CDCl3): d 5.85 (s, 2H), 6.61 (dd, J = 2.1, 8.4 Hz, 1H),
7.11 (dd, J = 2.1, 8.4 Hz, 1H), 7.27 (s, 1H), 7.32–7.49 (m, 4H),
7.80–7.87 (m, 2H), 9.89 (s, 1H). Anal. Calcd for C17H12Cl2N2O: C,
61.65; H, 3.65; N, 8.46. Found: C, 61.39; H, 3.63; N, 8.42. Com-
pound 22d was prepared from the corresponding aldehyde in
95% yield by a method similar to that described for ethyl (E)-3-
(1-benzyl-3-isopropoxy-1H-pyrazol-5-yl)propenoate (see Section
6.1.1.5.). mp 123–124 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.32
(d, J = 7.2 Hz, 3H), 4.24 (q, J = 7.2 Hz, 2H), 5.55 (s, 2H), 6.39 (d,
6.1.1.46.
3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-isopro-
pyl-1H-pyrazol-5-yl}-N-(pentylsulfonyl)propanamide (24b). 3-
{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-isopropyl-1H-pyrazol-
5-yl}propanoic acid was prepared in 68% yield by a method similar
to that described for 2. Mp 118 °C (EtOAc/hexane). 1H NMR
(CDCl3): d 1.25 (t, J = 7.2 Hz, 6H), 2.51–2.53 (m, 2H), 2.54–2.60

728
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
(m, 2H), 2.90–3.03 (m, 1H), 5.40 (s, 2H), 5.98 (s, 1H), 6.53 (d,
J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.63 (s, 1H). Anal. Calcd for
J = 15.7 Hz, 1H), 6.51 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 7.14 (d,
J = 2.2 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.49 (d, J = 15.7 Hz, 1H).
Compound 25a was prepared from the corresponding carboxylic
acid in 24% yield by a method similar to that described for 3c.
Mp 150 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.89 (t, J = 7.2 Hz,
3H), 1.28 (d, J = 7.0 Hz, 6H), 1.29–1.46 (m, 4H), 1.76–1.88 (m,
2H), 2.93–3.05 (m, 1H), 3.41–3.50 (m, 2H), 5.46 (s, 2H), 6.28 (d,
J = 15.3 Hz, 1H), 6.50 (d, J = 8.3 Hz, 1H), 6.51 (s, 1H), 7.13 (dd,
J = 2.1, 8.3 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.56 (d, J = 15.3 Hz,
1H), 8.12 (br s, 1H). Anal. Calcd for C21H27Cl2N3O3S: C, 53.39; H,
5.76; N, 8.89. Found: C, 53.14; H, 5.70; N, 8.87.
C17H18ClF3N2O2: C, 54.48; H, 4.84; N, 7.47. Found: C, 54.43; H,
4.85; N, 7.38. Compound 24b was prepared from the correspond-
ing carboxylic acid in 38% yield by a method similar to that de-
scribed for 3c. Mp 144–145 °C (EtOAc/hexane). 1H NMR (CDCl3):
d 0.90 (t, J = 7.1 Hz, 3H), 1.26 (d, J = 7.0 Hz, 6H), 1.28–1.47 (m,
4H), 1.72–1.85 (m, 2H), 2.58–2.68 (m, 2H), 2.78–2.87 (m, 2H),
2.89–3.03 (m, 1H), 3.33–3.43 (m, 2H), 5.39 (s, 2H), 5.96 (s, 1H),
6.55 (d, J = 8.1 Hz, 1H), 7.42 (dd, J = 1.0, 8.1 Hz, 1H), 7.65 (d,
J = 1.0 Hz, 1H), 8.10 (br s, 1H). Anal. Calcd for C22H29ClF3N3O3S: C,
52.02; H, 5.75; N, 8.27. Found: C, 52.03; H, 5.76; N, 8.18.
6.1.1.50. (E)-3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-cyclo-
propyl-1H-pyrazol-5-yl}-N-(pentylsulfonyl)propenamide
(25c). (E)-3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-cyclopro-
pyl-1H-pyrazol-5-yl}propenoic acid was prepared in 86% yield by a
method similar to that described for 2. Mp 191–192 °C (EtOAc/hex-
ane). 1H NMR (CDCl3): d 0.71–0.80 (m, 2H), 0.91–1.03 (m, 2H),
1.88–2.02 (m, 1H), 5.50 (s, 2H), 6.27 (d, J = 15.8 Hz, 1H), 6.37 (s,
1H), 6.69 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.45 (d,
J = 15.8 Hz, 1H), 7.66 (s, 1H). Anal. Calcd for C17H14ClF3N2O2: C,
55.07; H, 3.81; N, 7.56. Found: C, 55.05; H, 3.84; N, 7.61. To a solu-
tion of the obtained carboxylic acid (501 mg, 1.35 mmol) in DMF
(5 mL) was added CDI (242 mg, 1.49 mmol). After being stirred at
room temperature for 1 h, 1-pentylsulfonamide (225 mg,
1.49 mmol) and DBU (0.223 mL, 1.49 mmol) were successively
added. The mixture was heated at 100 °C with stirring for 12 h.
After being cooled, the mixture was acidified (pH 4) with 1 M
HCl, diluted with water, and extracted with EtOAc. The extract
was washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by silica gel chroma-
tography (hexane/EtOAc, 20:1 to 2:1) and successively recrystal-
lized from EtOAc/hexane to give 25c (313 mg, 46%) as colorless
crystals: mp 139–141 °C. 1H NMR (CDCl3): d 0.66–0.81 (m, 2H),
0.89 (t, J = 7.1 Hz, 3H), 0.93–1.05 (m, 2H), 1.19–1.50 (m, 4H),
1.71–1.88 (m, 2H), 1.88–2.03 (m, 1H), 3.25–3.58 (m, 2H), 5.50 (s,
2H), 6.28 (d, J = 15.3 Hz, 1H), 6.38 (s, 1H), 6.67 (d, J = 8.1 Hz, 1H),
7.42 (dd, J = 0.9, 8.1 Hz, 1H), 7.53 (d, J = 15.5 Hz, 1H), 7.66 (d,
J = 0.9 Hz, 1H), 8.22 (br s, 1H). Anal. Calcd for C22H25ClF3N3O3S: C,
52.43; H, 5.00; N, 8.34. Found: C, 52.38; H, 4.92; N, 8.37. ESI MS
m/z 504.0 [C22H25ClF3N3O3S+H]+.
6.1.1.47. 3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-cyclopro-
pyl-1H-pyrazol-5-yl}-N-(pentylsulfonyl)propanamide (24c). 3-
{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-cyclopropyl-1H-pyra-
zol-5-yl}propanoic acid was prepared by a method similar to that
described for 2 and obtained in 22% yield along with the corre-
sponding 3-propyl-1H-pyrazole derivative (see Section 6.1.1.43.).
1H NMR (CDCl3): d 0.66–0.74 (m, 2H), 0.87–0.97 (m, 2H), 1.85–
1.98 (m, 1H), 2.60–2.67 (m, 2H), 2.72–2.79 (m, 2H), 5.36 (s, 2H),
5.80 (s, 1H), 6.59 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 0.9, 8.1 Hz, 1H),
7.63 (d, J = 0.9 Hz, 1H). To a solution of the obtained carboxylic acid
(450 mg, 1.21 mmol) in DMF (5 mL) was added CDI (216 mg,
1.33 mmol). After being stirred at room temperature for 1 h, 1-pen-
tylsulfonamide (201 mg, 1.33 mmol) and DBU (0.199 mL,
1.33 mmol) were successively added. The mixture was heated at
100 °C with stirring for 12 h. After being cooled, the mixture was
acidified (pH 4) with 1 M HCl, diluted with water, and extracted
with EtOAc. The extract was washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel chromatography (hexane/EtOAc, 20:1 to 2:1) to
give a mixture of 24c and the corresponding 3-propyl-1H-pyrazole
derivative. The mixture was separated using reversed-phase high-
performance liquid chromatography (HPLC), eluted with water
containing 0.01% TFA with a linear gradient of 0% to 40% of MeCN.
Recrystallization from EtOAc/hexane gave 24c (261 mg, 43%) as
colorless crystals: mp 152–154 °C (EtOAc/hexane). 1H NMR
(CDCl3): d 0.57–0.76 (m, 2H), 0.81–0.98 (m, 5H), 1.19–1.47 (m,
4H), 1.70–1.84 (m, 2H), 1.84–1.96 (m, 1H), 2.60 (t, J = 7.4 Hz, 2H),
2.81 (t, J = 7.3 Hz, 2H), 3.22–3.52 (m, 2H), 5.36 (s, 2H), 5.79 (s,
1H), 6.61 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 0.9, 8.2 Hz, 1H), 7.65 (d,
J = 0.9 Hz, 1H), 8.07 (br s, 1H). ESI MS m/z 506.1
[C22H27ClF3N3O3S+H]+.
6.1.1.51.
Methyl
1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
(methoxymethoxy)-1H-pyrazole-5-carboxylate (26). To an ice-
cooled mixture of 4 (68.6 g, 483 mmol), K2CO3 (100 g, 727 mmol),
and DMF (350 mL) was added chloromethyl methyl ether (46.7 g,
580 mmol). The mixture was allowed to warm to room tempera-
ture and stirred for 15 h. The mixture was poured into water and
extracted with EtOAc. The aqueous layer was further extracted
with chloroform. The combined organic extracts were concen-
trated in vacuo and the residual solid was dissolved in chloroform.
The solution was washed with water and brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel chromatography (hexane/EtOAc, 24:1 to 2:1)
and successively recrystallized from EtOAc/hexane to give methyl
3-(methoxymethoxy)-1H-pyrazole-5-carboxylate (23.4 g, 26%) as
colorless crystals: mp 54–55 °C. 1H NMR (CDCl3): d 3.52 (s, 3H),
3.91 (s, 3H), 5.26 (s, 2H), 6.35 (s, 1H), 10.22 (br s, 1H). Compound
26 was prepared in 71% yield by a method similar to that described
for 6. 1H NMR (CDCl3): d 3.53 (s, 3H), 3.83 (s, 3H), 5.24 (s, 2H), 5.79
(s, 2H), 6.46 (s, 1H), 6.70 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 0.9, 8.1 Hz,
1H), 7.64 (d, J = 0.9 Hz, 1H).
6.1.1.48. 3-[1-(2,4-Dichlorobenzyl)-3-phenyl-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)propanamide
(24d). 3-[1-(2,4-Dichloroben-
zyl)-3-phenyl-1H-pyrazol-5-yl]propanoic acid was prepared in
88% yield by a method similar to that described for 2. Mp 160–
161 °C (EtOAc/hexane). 1H NMR (CDCl3): d 2.67–2.75 (m, 2H),
2.81–2.89 (m, 2H), 5.42 (s, 2H), 6.45 (s, 1H), 6.57 (d, J = 8.7 Hz,
1H), 7.12 (dd, J = 2.1, 8.7 Hz, 1H), 7.26–7.43 (m, 4H), 7.75–7.82
(m, 2H). Compound 24d was prepared in 11% yield by a method
similar to that described for 3c. Mp 145–147 °C (EtOAc/hexane).
1H NMR (CDCl3): d 0.85 (t, J = 7.2 Hz, 3H), 1.18–1.40 (m, 4H),
1.68–1.82 (m, 2H), 2.57–2.67 (m, 2H), 2.83–2.93 (m, 2H), 3.30–
3.42 (m, 2H), 5.41 (s, 2H), 6.42 (s, 1H), 6.59 (d, J = 8.1 Hz, 1H),
7.12 (dd, J = 2.1, 8.1 Hz, 1H), 7.23–7.42 (m, 4H), 7.72–7.80 (m,
2H), 8.17 (br s, 1H).
6.1.1.49. (E)-3-[1-(2,4-Dichlorobenzyl)-3-isopropyl-1H-pyrazol-
5-yl]-N-(pentylsulfonyl)propenamide
(25a). 3-[1-(2,4-dichlo-
robenzyl)-3-isopropyl-1H-pyrazol-5-yl]propenoic acid was pre-
pared in 75% yield by a method similar to that described for 2.
Mp 174–175 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.28 (d,
J = 6.9 Hz, 6H), 2.93–3.08 (m, 1H), 5.46 (s, 2H), 6.29 (d,
6.1.1.52. 1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-(methoxy-
methoxy)-1H-pyrazole-5-carbaldehyde (27). 1-[2-Chloro-4-(tri-
fluoromethyl)benzyl]-3-(methoxymethoxy)-1H-pyrazole-5-car-

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
729
boxylic acid was prepared in 86% yield by a method similar to that
described for 2. Mp 133–134 °C. 1H NMR (CDCl3): d 3.52 (s, 3H),
5.24 (s, 2H), 5.76 (s, 2H), 6.56 (s, 1H), 6.71 (d, J = 8.1 Hz, 1H), 7.40
(d, J = 8.1 Hz, 1H), 7.64 (s, 1H), 8.60 (br s, 1H). Anal. Calcd for
6.84 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 15.8 Hz, 1H), 7.44 (d, J = 8.1 Hz,
1H), 7.67 (s, 1H). Compound 29b was prepared from the corre-
sponding hydroxypyrazole in 90% yield by a method similar to that
described for 17d. 1H NMR (CDCl3): d 1.30 (t, J = 7.2 Hz, 3H), 1.34
(d, J = 6.3 Hz, 6H), 4.22 (q, J = 7.2 Hz, 2H), 4.67–4.81 (m, 1H), 5.40
(s, 2H), 6.03 (s, 1H), 6.29 (d, J = 15.6 Hz, 1H), 6.78 (d, J = 8.1 Hz,
1H), 7.38 (d, J = 15.6 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.65 (s, 1H).
C14H12ClF3N2O4: C, 46.11; H, 3.32; N, 7.68. Found: C, 46.11; H,
3.27; N, 7.66. To a stirred mixture of N,O-dimethylhydroxylamine
(9.01 g, 92.4 mmol), TEA (9.35 g, 92.4 mmol), and DMF (300 mL)
were added the obtained carboxylic acid (28.1 g, 77.0 mmol), EDCI
(17.7 g, 92.4 mmol), and HOBt (14.2 g, 92.4 mmol). After being stir-
red at room temperature for 15 h, the mixture was concentrated in
vacuo and partitioned between EtOAc and water. The organic layer
was washed with water and brine, dried over MgSO4, and concen-
trated under reduced pressure. The residue was diluted with EtOAc
and the insoluble product was removed by filtration. The filtrate
was concentrated in vacuo. The residue was purified by silica gel
chromatography (hexane/EtOAc, 19:1 to 1:1) to give 1-[2-chloro-
4-(trifluoromethyl)benzyl]-N-methoxy-3-(methoxymethoxy)-N-
methyl-1H-pyrazole-5-carboxamide (29.1 g, 93%) as a colorless oil.
1H NMR (CDCl3): d 3.29 (s, 3H), 3.53 (s, 3H), 3.67 (s, 3H), 5.25 (s,
2H), 5.70 (s, 2H), 6.41 (s, 1H), 6.75 (d, J = 8.1 Hz, 1H), 7.40 (d,
J = 8.1 Hz, 1H), 7.62 (s, 1H). To an ice-cooled solution of the ob-
tained carboxamide (29.1 g, 71.4 mmol) in THF (500 mL) at
ꢀ78 °C was added 1.5 M DIBAL-H in toluene (71 mL, 107 mmol).
The mixture was stirred at 0 °C for 1 h. To the solution was further
added 1.5 M DIBAL-H in toluene (10 mL, 6.67 mmol). After being
stirred at the same temperature for 0.5 h, the reaction was
quenched by successive addition of MeOH and 10% Rochelle. The
mixture was extracted with Et2O and the extract was washed with
brine. The solution was dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel chromatography (hex-
ane/EtOAc, 19:1 to 2:1) to give 27 (21.2 g, 85%) as a colorless oil. 1H
NMR (CDCl3): d 3.53 (s, 3H), 5.26 (s, 2H), 5.75 (s, 2H), 6.51 (s, 1H),
6.75 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.65 (s, 1H), 9.76
(s, 1H).
6.1.1.56. Ethyl (E)-3-[1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
(2-methoxyethoxy)-1H-pyrazol-5-yl]propenoate
(29c). Com-
pound 29c was prepared in quantitative yield by a method similar
to that described for 17a. Mp 68–69 °C (IPE/hexane). 1H NMR
(CDCl3): d 1.30 (t, J = 7.2 Hz, 3H), 3.44 (s, 3H), 3.63–3.79 (m, 2H),
4.22 (q, J = 7.2 Hz, 2H), 4.28–4.35 (m, 2H), 5.40 (s, 2 H), 6.09 (s,
1H), 6.30 (d, J = 15.7 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 7.37 (d,
J = 15.7 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.66 (s, 1H). Anal. Calcd
for C19H20ClF3N2O4: C, 52.72; H, 4.66; N, 6.47. Found: C, 52.65; H,
4.73; N, 6.39.
6.1.1.57. (E)-3-[1-(2,4-Dichlorobenzyl)-3-isopropoxy-1H-pyra-
zol-5-yl]-N-(pentylsulfonyl)propenamide (30a). (E)-3-[1-(2,4-
Dichlorobenzyl)-3-isopropoxy-1H-pyrazol-5-yl]propenoic
acid
was prepared in 90% yield by a method similar to that described
for 2. Mp 170–171 °C (EtOAc/hexane). 1H NMR (CDCl3): d 1.34 (d,
J = 6.0 Hz, 6H), 4.66–4.79 (m, 1H), 5.34 (s, 2H), 6.04 (s, 1H), 6.26
(d, J = 15.6 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 2.1,
8.4 Hz, 1H), 7.40 (d, J = 2.1 Hz, 1H), 7.48 (d, J = 15.6 Hz, 1H). Com-
pound 30a was prepared from the corresponding carboxylic acid
in 69% yield by a method similar to that described for 25c. Mp
132–133 °C. 1H NMR (CDCl3): d 0.90 (t, J = 7.2 Hz, 3H), 1.22–1.47
(m, 10H), 1.76–1.89 (m, 2H), 3.42–3.50 (m, 2H), 4.67–4.80 (m,
1H), 5.34 (s, 2H), 6.03 (s, 1H), 6.25 (d, J = 15.4 Hz, 1H), 6.67 (d,
J = 8.5 Hz, 1H), 7.16 (dd, J = 2.1, 8.5 Hz, 1H), 7.40 (d, J = 2.1 Hz,
1H), 7.54 (d, J = 15.4 Hz, 1H), 8.05 (br s, 1H). Anal. Calcd for
C21H27Cl2N3O4S: C, 51.64; H, 5.57; N, 8.60. Found: C, 51.69; H,
6.1.1.53. Ethyl (E)-3-[1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
(methoxymethoxy)-1H-pyrazol-5-yl]pro- penoate (28). Com-
pound 28 was prepared in 60% yield by a method similar to that
described for ethyl (E)-3-(1-benzyl-3-isopropoxy-1H-pyrazol-5-
yl)propenoate (see Section 6.1.1.5.). 1H NMR (CDCl3): d 1.30 (t,
J = 7.2 Hz, 3H), 3.53 (s, 3H), 4.23 (q, J = 7.2 Hz, 2H), 5.23 (s, 2H),
5.43 (s, 2H), 6.18 (s, 1H), 6.32 (d, J = 15.5 Hz, 1H), 6.83 (d,
J = 8.1 Hz, 1H), 7.39 (d, J = 15.5 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H),
7.66 (s, 1H).
5.60; N, 8.59.
6.1.1.58.
(E)-3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-iso-
propoxy-1H-pyrazol-5-yl}-N-(pentylsulfonyl)propenamide
(30b). (E)-3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-isopro-
poxy-1H-pyrazol-5-yl}propenoic acid was prepared in 95% yield
by a method similar to that described for 2. Mp 170–171 °C
(EtOAc/hexane). 1H NMR (CDCl3): d 1.35 (d, J = 6.3 Hz, 6H), 4.67–
4.80 (m, 1H), 5.41 (s, 2H), 6.07 (s, 1H), 6.28 (d, J = 15.8 Hz, 1H),
6.81 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.46 (d, J = 15.8 Hz,
1H), 7.66 (s, 1H). Anal. Calcd for C17H16ClF3N2O3: C, 52.52; H,
4.15; N, 7.21. Found: C, 52.54; H, 4.09; N, 7.10. Compound 30b
was prepared from the corresponding carboxylic acid in 31% yield
by a method similar to that described for 25c. Mp 139–140 °C
(EtOAc/hexane). 1H NMR (CDCl3): d 0.89 (t, J = 7.4 Hz, 3H), 1.23–
1.47 (m, 10H), 1.76–1.89 (m, 2H), 3.40–3.50 (m, 2H), 4.69–4.82
(m, 1H), 5.42 (s, 2H), 6.06 (s, 1H), 6.28 (d, J = 15.3 Hz, 1H), 6.79
(d, J = 8.1 Hz, 1H), 7.43 (dd, J = 0.9, 8.1 Hz, 1H), 7.53 (d,
J = 15.3 Hz, 1H), 7.66 (d, J = 0.9 Hz, 1H), 8.05 (br s, 1H). Anal. Calcd
for C22H27ClF3N3O4Sꢁ0.3H2O: C, 50.10; H, 5.27; N, 7.97. Found: C,
50.06; H, 5.11; N, 7.79.
6.1.1.54. Ethyl (E)-3-[1-(2,4-dichlorobenzyl)-3-isopropoxy-1H-
pyrazol-5-yl]propenoate (29a). Ethyl (E)-3-[1-(2,4-dichloroben-
zyl)-3-hydroxy-1H-pyrazol-5-yl]propenoate was prepared in 95%
yield by a method similar to that described for 16. 1H NMR (CDCl3):
d 1.31 (t, J = 7.2 Hz, 3H), 4.23 (q, J = 7.2 Hz, 2H), 5.28 (s, 2H), 5.95 (s,
1H), 6.32 (d, J = 15.6 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 7.17 (dd,
J = 2.1, 8.4 Hz, 1H), 7.38 (d, J = 15.6 Hz, 1H), 7.41 (d, J = 2.1 Hz,
1H), 10.75 (br s, 1H). Compound 29a was prepared from the corre-
sponding hydroxypyrazole in 87% yield by a method similar to that
described for 17d. 1H NMR (CDCl3): d 1.31 (t, J = 7.1 Hz, 3H), 1.34
(d, J = 6.2 Hz, 6H), 4.23 (q, J = 7.1 Hz, 2H), 4.66–4.79 (m, 1H), 5.33
(s, 2H), 6.00 (s, 1H), 6.27 (d, J = 15.9 Hz, 1H), 6.66 (d, J = 8.3 Hz,
1H), 7.15 (dd, J = 2.1, 8.3 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.40 (d,
J = 15.9 Hz, 1H).
6.1.1.59.
(E)-3-{1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-iso-
propoxy-1H-pyrazol-5-yl}-N-[(3-methylbutyl)sulfonyl]propena-
mide (30c). Compound 30c was prepared in 41% yield by a
method similar to that described for 25c. Mp 138–140 °C (EtOAc/
hexane). 1H NMR (CDCl3): d 0.92 (d, J = 6.6 Hz, 6H), 1.34 (d,
J = 6.0 Hz, 6H), 1.64–1.78 (m, 3H), 3.40–3.53 (m, 2H), 4.69–4.82
(m, 1H), 5.42 (s, 2H), 6.06 (s, 1H), 6.27 (d, J = 15.2 Hz, 1H), 6.69
(d, J = 8.1 Hz, 1H), 7.43 (dd, J = 0.9, 8.1 Hz, 1H), 7.53 (d,
J = 15.2 Hz, 1H), 7.66 (d, J = 0.9 Hz, 1H), 7.98 (br s, 1H). Anal. Calcd
6.1.1.55. Ethyl (E)-3-{1-[2-chloro-4-(trifluoromethyl)benzyl]-3-
isopropoxy-1H-pyrazol-5-yl}propenoate (29b). Ethyl (E)-3-{1-
[2-chloro-4-(trifluoromethyl)benzyl]-3-hydroxy-1H-pyrazol-5-yl}
propenoate was prepared in 87% yield by a method similar to that
described for 16. 1H NMR (CDCl3): d 1.30 (t, J = 7.2 Hz, 3H), 4.23 (q,
J = 7.2 Hz, 2H), 5.36 (s, 2H), 5.98 (s, 1H), 6.34 (d, J = 15.8 Hz, 1H),

730
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
for C22H27ClF3N3O4S: C, 50.62; H, 5.21; N, 8.05. Found: C, 50.81; H,
5.42; N, 8.19.
5.58 (s, 1H), 6.60 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.1, 8.4 Hz, 1H),
7.39 (d, J = 2.1 Hz, 1H).
6.1.1.64.
3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazole-5-car-
6.1.1.60.
methoxyethoxy)-1H-pyrazol-5-yl]-N-[(3-me thylbutyl)sulfonyl]
propenamide (30d). (E)-3-[1-[2-Chloro-4-(trifluoromethyl)
benzyl]-3-(2-methoxyethoxy)-1H-pyrazol-5-yl]propenoic acid
(E)-3-[1-[2-Chloro-4-(trifluoromethyl)benzyl]-3-(2-
boxylic acid (33a). Compound 33a was prepared from 31 in 68%
yield by a method similar to that described for 2. Mp 154–155 °C
(EtOAc/hexane). 1H NMR (CDCl3): d 0.96 (t, J = 7.4 Hz, 3H), 1.40–
1.53 (m, 2H), 1.69–1.79 (m, 2H), 4.12 (t, J = 6.6 Hz, 2H), 5.66 (s,
2H), 6.29 (s, 1H), 6.55 (d, J = 8.5 Hz, 1H), 7.12 (dd, J = 2.1, 8.5 Hz,
1H), 7.38 (d, J = 2.1 Hz, 1H). Anal. Calcd for C15H16Cl2N2O3: C,
52.49; H, 4.70; N, 8.16. Found: C, 52.49; H, 4.71; N, 8.22.
was prepared in 70% yield by a method similar to that described
for 2. Mp 169–172 °C (THF/hexane). 1H NMR (CDCl3): d 3.44 (s,
3H), 3.66–3.81 (m, 2H), 4.22–4.40 (m, 2H), 5.41 (s, 2H), 6.13 (s,
1H), 6.29 (d, J = 15.8 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 7.36–7.52
(m, 2H), 7.66 (d, J = 0.9 Hz, 1H). Anal. Calcd for C17H16ClF3N2O4:
C, 50.44; H, 3.98; N, 6.92. Found: C, 50.38; H, 3.89; N, 6.85. Com-
pound 30d was prepared from the corresponding carboxylic acid
in 54% yield by a method similar to that described for 25c. Mp
114–116 °C (MeOH/water). 1H NMR (CDCl3): d 0.91 (d, J = 6.4 Hz,
6H), 1.63–1.77 (m, 3H), 3.44 (s, 3H), 3.47 (dd, J = 2.2, 5.9 Hz, 2H),
3.60–3.84 (m, 2H), 4.20–4.42 (m, 2H), 5.40 (s, 2H), 6.13 (s, 1H),
6.30 (d, J = 15.3 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 7.41 (dd, J = 1.0,
8.1 Hz, 1H), 7.51 (d, J = 15.3 Hz, 1H), 7.66 (d, J = 1.0 Hz, 1H), 8.56
(br s, 1H). Anal. Calcd for C22H27ClF3N3O5S: C, 49.12; H, 5.06; N,
7.81. Found: C, 49.02; H, 4.94; N, 7.73.
6.1.1.65. [3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]ace-
tic acid (33b). The mixture of crude 32a (2.03 g), EtOH (25 mL),
and 4 M NaOH (13 mL, 52.0 mmol) was heated under reflux with
stirring for 1 h. After being cooled, the mixture was acidified with
1 M HCl, concentrated in vacuo to remove organic solvents, and ex-
tracted with EtOAc. The extract was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
recrystallized from EtOAc/hexane to give 33b (890 mg, 48% for 2
steps) as colorless crystals: mp 105–108 °C. 1H NMR (CDCl3): d
0.95 (t, J = 7.3 Hz, 3H), 1.38–1.58 (m, 2H), 1.65–1.86 (m, 2H), 3.56
(s, 2H), 4.10 (t, J = 6.6 Hz, 2H), 5.21 (s, 2H), 5.74 (s, 1H), 6.66 (d,
J = 8.4 Hz, 1H), 7.15 (dd, J = 2.1, 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz,
1H). Anal. Calcd for C16H18Cl2N2O3: C, 53.79; H, 5.08; N, 7.84.
Found: C, 53.78; H, 5.05; N, 7.88.
6.1.1.61. Methyl 3-butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazole-
5-carboxylate (31). Compound 31 was prepared in 83% yield by a
method similar to that described for 17a. 1H NMR (CDCl3): d 0.96 (t,
J = 7.2 Hz, 3H), 1.40–1.53 (m, 2H), 1.69–1.79 (m, 2H), 3.81 (s, 3H),
4.12 (t, J = 6.6 Hz, 2H), 5.67 (s, 2H), 6.29 (s, 1H), 6.53 (d,
J = 8.7 Hz, 1H), 7.11 (dd, J = 2.1, 8.1 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H).
6.1.1.66.
4-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-
yl]butanoic acid (33c). Compound 33c was prepared from 32b
in 82% yield by a method similar to that described for 33b. Mp
79–81 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.96 (t, J = 7.2 Hz,
3H), 1.39–1.54 (m, 2H), 1.67–1.80 (m, 2H), 1.82–1.94 (m, 2H),
2.33–2.42 (m, 2H), 2.46–2.55 (m, 2H), 4.09 (t, J = 6.6 Hz, 2H), 5.17
(s, 2H), 5.57 (s, 1H), 6.56 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1,
8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H). Anal. Calcd for C18H22Cl2N2O3:
C, 56.11; H, 5.76; N, 7.27. Found: C, 56.35; H, 5.77; N, 7.22.
6.1.1.62. [3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]ace-
tonitrile (32a). [3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-
yl]methanol was prepared from 31 in 88% yield by a method sim-
ilar to that described for (1-benzyl-3-isopropoxy-1H-pyrazol-5-
yl)methanol (see Section 6.1.1.4.). 1H NMR (CDCl3): d 0.95 (t,
J = 7.2 Hz, 3H), 1.33–1.53 (m, 2H), 1.64–1.78 (m, 2H), 4.05–4.17
(m, 2H), 4.53 (d, J = 5.7 Hz, 2H), 5.29 (s, 2H), 5.72 (s, 1H), 6.62 (d,
J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1, 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz,
1H). To a solution of the obtained alcohol (1.71 g, 5.19 mmol), ace-
tone cyanohydrin (0.7 mL, 7.66 mmol), and tributylphosphine
(2.4 mL, 9.61 mmol) in THF (40 mL) was added ADDP (2.40 g,
9.51 mmol). After being stirred at room temperature for 30 min,
the mixture was concentrated in vacuo and the residue was diluted
with IPE. The insoluble product was removed by filtration and the
filtrate was concentrated under reduced pressure. The residue was
subjected to silica gel chromatography (hexane to hexane/EtOAc
3:1) to give crude 32a (2.03 g) as a yellow oil, which was used
for the next step without further purification. 1H NMR (CDCl3): d
0.96 (t, J = 7.3 Hz, 3H), 1.37–1.54 (m, 2H), 1.65–1.83 (m, 2H), 3.60
(s, 2H), 4.05–4.18 (m, 2H), 5.20 (s, 2H), 5.83 (s, 1H), 6.72 (d,
J = 8.4 Hz, 1H), 7.20 (dd, J = 2.1, 8.4 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H).
6.1.1.67.
5-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-
yl]pentanoic acid (33d). To a 1 M BH3ꢁTHF in THF (8.0 mL,
8.0 mmol) was added 33c (300 mg, 0.779 mmol) portionwise. After
being stirred at room temperature for 4 h, the mixture was poured
into 1 M HCl and extracted with EtOAc. The extract was washed
with sat. NaHCO3 and brine, dried over MgSO4, and concentrated
in vacuo. The residue was purified by silica gel chromatography
(hexane/EtOAc, 19:1 to 7:3) to give 4-[3-butoxy-1-(2,4-dichlorob-
enzyl)-1H-pyrazol-5-yl]butan-1-ol (150 mg, 52%) as a colorless oil.
1H NMR (CDCl3): d 0.95 (t, J = 7.2 Hz, 3H), 1.37–1.96 (m, 8H), 2.39–
2.52 (m, 2H), 3.54–3.62 (m, 2H), 4.10 (t, J = 6.9 Hz, 2H), 5.16 (s, 2H),
5.56 (s, 1H), 6.58 (d, J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1, 8.4 Hz, 1H),
7.37 (d, J = 2.1 Hz, 1H). 5-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-
pyrazol-5-yl]pentanenitrile was prepared from the obtained alco-
hol in 68% yield by a method similar to that described for 32a.
1H NMR (CDCl3): d 0.96 (t, J = 7.2 Hz, 3H), 1.39–1.54 (m, 2H),
1.68–1.80 (m, 2H), 1.84–1.97 (m, 2H), 2.34–2.42 (m, 2H), 2.58–
2.67 (m, 2H), 4.10 (t, J = 6.6 Hz, 2H), 5.17 (s, 2H), 5.58 (s, 1H),
6.60 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.1, 8.4 Hz, 1H), 7.39 (d,
J = 2.1 Hz, 1H). Compound 33d was prepared in 93% yield by a
method similar to that described for 33b. Mp 79–81 °C (EtOAc/hex-
ane). 1H NMR (CDCl3): d 0.95 (t, J = 7.4 Hz, 3H), 1.35–1.85 (m, 8H),
2.32 (t, J = 6.9 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 4.10 (q, J = 6.7 Hz,
2H), 5.15 (s, 2H), 5.55 (s, 1H), 6.56 (d, J = 8.5 Hz, 1H), 7.14 (dd,
J = 2.1, 8.5 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H).
6.1.1.63.
4-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-
yl]butanenitrile (32b). 3-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-
pyrazol-5-yl]propan-1-ol was prepared from 17a in 84% yield by
a method similar to that described for (1-benzyl-3-isopropoxy-
1H-pyrazol-5-yl)methanol (see Section 7). 6.1.1.4.). 1H NMR
(CDCl3): d 0.95 (t, J = 7.2 Hz, 3H), 1.35–1.86 (m, 7H), 2.45–2.59
(m, 2H), 3.55–3.70 (m, 2H), 4.02–4.14 (m, 2H), 5.17 (s, 2H), 5.56
(s, 1H), 6.55 (d, J = 8.1 Hz, 1H), 7.13 (dd, J = 2.1, 8.1 Hz, 1H), 7.37
(d, J = 2.1 Hz, 1H). Compound 32b was prepared from the corre-
sponding alcohol in 84% yield by a method similar to that de-
scribed for 32a. 1H NMR (CDCl3): d 0.96 (t, J = 7.2 Hz, 3H), 1.39–
1.54 (m, 2H), 1.68–1.80 (m, 2H), 1.84–1.97 (m, 2H), 2.34–2.42
(m, 2H), 2.58–2.67 (m, 2H), 4.10 (t, J = 6.6 Hz, 2H), 5.17 (s, 2H),
6.1.1.68. 3-Butoxy-1-(2,4-dichlorobenzyl)-N-(pentylsulfonyl)-
1H-pyrazole-5-carboxamide (34a). Compound 34a was prepared
from 33a in 4% yield by a method similar to that described for 3c.

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
731
Mp 139–141 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.82–1.03 (m,
6H), 1.22–1.53 (m, 6H), 1.66–2.01 (m, 4H), 3.36–3.65 (m, 2H),
4.13 (t, J = 6.6 Hz, 2H), 5.65 (s, 2H), 6.15 (s, 1H), 6.61 (d,
J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1, 8.4 Hz, 1H), 7.38 (d, J = 2.1 Hz,
1H), 8.38 (s, 1H). Anal. Calcd for C20H27Cl2N3O4S: C, 50.42; H,
5.71; N, 8.82. Found: C, 50.51; H, 5.84; N, 8.90.
the supernatant was diluted and centrifuged again. The compound
concentrations in the supernatant were measured by LC/MS/MS.
6.1.4. Biology
Establishment of a stable transformed cell expressing human
PPARc1, human RXR
a, and PPAR responsive luciferase repoter
gene. The full length human PPAR
c1, full length human RXRa,
6.1.1.69. 2-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)acetamide (34b). Compound 34b was pre-
pared from 33b in 25% yield by a method similar to that described
for 3c. 1H NMR (CDCl3): d 0.81–1.08 (m, 6H), 1.23–1.90 (m, 10H),
3.25–3.36 (m, 2H), 3.60 (s, 2H), 4.11 (t, J = 6.6 Hz, 2H), 5.20 (s,
2H), 5.74 (s, 1H), 6.75 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 2.1, 8.4 Hz,
1H), 7.39 (d, J = 2.1 Hz, 1H).
and PPAR responsive luciferase reporter gene were stably ex-
pressed in CHO-K1 cells. Transfection of mammalian expression
plasmid, pVGRXR2-hPPARc-zeo, and reporter plasmid, pGL3-
(PPREx4)-Tk-neo, into the cells was performed by electroporation
using a Gene Pulser (Bio-Rad, Japan). G418 (Life Technologies
Inc., U.S.A.) and Zeocin (Invitrogen, U.S.A.) resistant clones were se-
lected and examined for their ability to introduce liciferase expres-
sion in the presence of pioglitazone. Finally, we selected the clone,
6.1.1.70. 4-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)butanamide (34c). Compound 34c was pre-
pared from 33c in 54% yield by a method similar to that described
for 3c. Mp 121–122 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.85–
0.99 (m, 6H), 1.27–1.54 (m, 6H), 1.66–1.85 (m, 4H), 1.86–2.02
(m, 2H), 2.34 (t, J = 7.1 Hz, 2H), 2.51 (t, J = 7.5 Hz, 2H), 3.34–3.45
(m, 2H), 4.08 (t, J = 6.6 Hz, 2H), 5.15 (s, 2H), 5.55 (s, 1H), 6.56 (d,
J = 8.4 Hz, 1H), 7.14 (dd, J = 2.1, 8.4 Hz, 1H), 7.37 (d, J = 2.1 Hz,
1H), 7.93 (br s, 1H). Anal. Calcd for C23H33Cl2N3O4S: C, 53.28; H,
6.42; N, 8.10. Found: C, 53.34; H, 6.45; N, 8.11.
PPARc:RXRa:4ERPP/CHO-K1 No. 10, which expressed high levels
of luciferase activity by pioglitazone.
6.1.5. Transactivation assay of hPPARc1
PPAR
c
:RXR
a
:PPREꢂ4/CHO-K1 cells were used for transactiva-
tion assays of hPPAR
c
1. These cells were seeded into an OPAQUE
PLATE (white 96 well half area plate, COSTAR, U.S.A.) at the density
of 1 ꢂ 104 cells/well, and cultured in 5% CO2 at 37 °C overnight.
After washing the OPAQUE PLATE with PBS, 45
medium containing 0.1% fatty acid free-BSA and 5
l
l
L of HAM F12
L of test com-
pounds were added to the plate, which was then cultured in 5%
CO2 at 37 °C for 1 day. After removing the medium, 20 L of PICA-
l
6.1.1.71. 5-[3-Butoxy-1-(2,4-dichlorobenzyl)-1H-pyrazol-5-yl]-
N-(pentylsulfonyl)pentanamide (34d). Compound 34d was pre-
pared from 33d in 50% yield by a method similar to that described
for 3c. Mp 91–93 °C (EtOAc/hexane). 1H NMR (CDCl3): d 0.84–1.01
(m, 6H), 1.23–1.90 (m, 14H), 2.29 (t, J = 7.1 Hz, 2H), 2.45 (t,
J = 7.2 Hz, 2H), 3.34–3.46 (m, 2H), 4.09 (t, J = 6.6 Hz, 2H), 5.15 (s,
2H), 5.55 (s, 1H), 6.58 (d, J = 8.3 Hz, 1H), 7.15 (dd, J = 2.1, 8.3 Hz,
1H), 7.38 (d, J = 2.1 Hz, 1H), 7.90 (br s, 1H). Anal. Calcd for
GENE-LT7.5 (Wako Pure Chemical Ind., Ltd, Japan), which was di-
luted to half with HANK’S BALANCED SALT SOLUTION, was added
to each well. After stirring, luciferase activities were determined
in microplate-based luminescence reader (Perkinnelmer, U.S.A.)
Transient co-transfection assay of hPPAR
a and hPPARd. COS-1
cells were seeded at 5 ꢂ 106 cells in 150 cm2 tissue culture flask,
and cultured in 5% CO2 at 37 °C overnight. Transfections were per-
formed with LipofectAMINE (Life Technologies, Inc., U.S.A.) accord-
ing to the instructions of manufacturer. Briefly, the transfection
C24H35Cl2N3O4S: C, 54.13; H, 6.62; N, 7.89. Found: C, 54.10; H,
6.54; N, 7.90.
mixture contained 125
agent, 2.5 g of each expression plasmid pMCMVneo-hPPAR
(pMCMVneo-hPPARd) and pMCMVneo-hRXR , 5 g of reporter
plasmid pGL3-PPREꢂ4-tk-luc-neo, and 5 g of pRL-tk (Promega,
lL of LipofectAMINE, 100 lL of plus re-
l
a
6.1.2. Metabolic stability assay
a
l
Human or rat hepatic microsomes were purchased from Xeno-
tech, LLC (Lenexa, KS). An incubation mixture with a final volume
of 0.1 mL consisted of microsomal protein in 50 mmol/L phosphate
l
U.S.A.). Cells were incubated in 25 mL of transfection mixture for
3 h in 5% CO2 at 37 °C. After adding 25 mL of DMEM medium (Life
Technologies, Inc., U.S.A.) containing 0.1% fatty acid free-BSA, the
cells were then incubated for 1 day in 5% CO2 at 37 °C. After trans-
fection, cells were detached by treating with trypsin-EDTA (Life
Technologies INC., U.S.A.) centrifuged and then suspended in
DMEM medium containing 0.1% fatty acid free-BSA. The suspended
cells were added into an OPAQUE PLATE (white 96 well half area
buffer (pH 7.4) and 1 lmol/L test compound. The concentration of
hepatic microsomal protein was 0.2 mg/mL. An NADPH-generating
system containing 50 mmol/L MgCl2, 50 mmol/L glucose-6-phos-
phate, 5 mmol/L beta-NADP+ and 15 unit/mL glucose-6-phosphate
dehydrogenase was prepared and added to the incubation mixture
with a 10% volume of the reaction mixture. After the addition of
the NADPH-generating system, the mixture was incubated at
37 °C for 0 and 20 min. The reaction was terminated by the addi-
tion of equivalent volume of MeCN to that of the reaction mixture.
All incubations were made in duplicate. The amounts of test com-
pound in the reaction mixtures were measured by HPLC analysis
which was performed by Nemoto Science Co., Ltd (Tokyo, Japan).
Metabolic stability was assessed as disappearance rate of the par-
ent compound. Disappearance rate is represented as a percentage
of the amount of the parent compound in the reaction mixture
for 20 min relative to that in the reaction mixture for 0 min taken
as 100%.
plate, COSTAR, U.S.A.) at the density of 5 ꢂ 103 cells/well in 45
lL
of DMEM medium containing 0.1% fatty acid free-BSA and 5 lL of
test compounds and then cultured in 5% CO2 at 37 °C for 2 days.
After removing the medium, 20 L of PICAGENE-LT7.5 (Wako Pure
l
Chemical Ind., Ltd, Japan), which was diluted to half with HANK’S
BALANCED SALT SOLUTION, was added to each well. After stirring,
luciferase activities were determined in microplate-based lumines-
cence reader (Perkinnelmer, U.S.A.).
6.1.6. Evaluation of plasma glucose- and triglyceride-lowering
activities in Wistar fatty rats
Male Wistar fatty rats were obtained from Takeda Rabics
(Japan) and were used in these experiments. Throughout the study,
they were housed in metal cages and fed a commercial diet CE-2
(Clea, Japan) and water ad libitum. At the beginning of experiment,
blood samples were withdrawn from tail vein with heparin as anti-
coagulant, and initial body weight was measured. Plasma samples
were obtained from blood samples by centrifugation. Plasma levels
6.1.3. Pharmacokinetic analyses in rat cassette
Dosing test compounds were administered as a cassette dosing
to nonfasted rats. After oral and intravenous administration, blood
samples were collected. The blood samples were centrifuged to ob-
tain the plasma fraction. The plasma samples were deproteinized
with MeCN containing an internal standard. After centrifugation,

732
K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
Table 7
of glucose and triglyceride were measured enzymatically using
Autoanalyzer 7080 (Hitachi, Japan). Rats were divided based on
body weight, plasma glucose and triglyceride (n = 5/group). Wistar
fatty rats were orally administered test compounds suspended in
0.5% methylcellurose solution once daily for 7 days. The next day
of final administration, blood samples were withdrawn from tail
vein, and body weight was measured. After obtaining of plasma
samples by centrifugation of blood samples, we measured plasma
glucose and triglyceride. The change (%) from the initial levels of
plasma glucose and triglyceride after 7-day treatment was calcu-
lated in each group. The degree of the lowering effect in each group
was estimated from the relative ratio of the change (%) in each
group to the change in the control group. Then, the ED25 for the
glucose- and triglyceride-lowering effects of test compounds were
calculated from the dose-response curves of the relative ratios gen-
erated by logistic regression using the PCP systems.
X-ray data collection and refinement statistics
3d
Crystal
Space group
Unit cell dimensions
P43212
a = 65.5 Å
b = 65.5 Å
c = 154.2 Å
1
Molecules/asymmetric unit
Data collection
Resolution [Å] (outer shell)
Observations (unique)
Redundancy
50ꢀ1.90 (1.97ꢀ1.90)
279699 (26985)
10.4
98.3 (94.1)%
33.3 (5.1)
Completeness overall (outer shell)
I/
r (I) overall (outer shell)
Rsymm overall (outer shell)
0.059 (0.416)
Refinement
Resolution [Å]
Reflections used
R-factor
20–1.9
25478
0.187
0.219
0.006
1.10
6.1.7. Docking study
Rfree
The atomic coordinates of the crystal structures (1FM6 and
1PRG) were obtained from the web site of RCSB protein data bank
(http://www.rcsb.org/pdb/home/home.do). Both protein struc-
tures were added hydrogens and then only hydrogens were ener-
getically optimized in MOE (ver. 2000.02, Chemical Computing
RMS bonds [Å]
RMS angles [o]
Average B-value [Å2]
24.8
Group Inc, Canada.). Each compound was docked into the PPARc-
Acknowledgments
LBD using the GOLD program (ver. 2.0, the Cambridge Crystallo-
graphic Data Centre, UK) with the default parameter set. After
some manual adjustment to remove large steric hindrances, dock-
ing poses showing high score (Gold score) were subjected to en-
ergy minimization using MMFF94s force field in MOE. During the
energy minimization procedure firstly whole protein structure
was fixed and secondly that within 3.5 Å from the each ligand
was relaxed.
We would like to thank Robert Wynands for his help in prepar-
ing the manuscript and the staff of the Advanced Light Source,
which is supported by the Director, Office of Science, Office of Basic
Energy Science of the U.S. Department of Energy, under Contract
No. DE-AC02-05CH11231.
References and notes
6.1.8. X-ray co-crystallography study. Protein expression and
purification
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Crystals of the PPARc-3d complex were obtained by pre-incu-
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25
program suite.
The crystallographic refinement statistics are
presented in Table 7.

K. Rikimaru et al. / Bioorg. Med. Chem. 20 (2012) 714–733
733
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