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

Bioorganic & Medicinal Chemistry 20 (2012) 714–733

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Bioorganic & Medicinal Chemistry

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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,–

,

^aPharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan

^bTakeda Pharmaceuticals International Inc., One Takeda Parkway, Deerﬁeld, IL 60015, USA

^cTakeda 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 2025.²Therefore, 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.¹Although

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 A_1c(HbA_1c

)

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 (NOSCAL^Òor REZULIN^Ò), 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

(ACTOS^Ò) and rosiglitazone maleate (AVANDIA^Ò), as insulin sensi-

1FM6)⁹were 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,⁵has 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.⁶In 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.⁷The 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.⁸However, its synthetic precursor, (1-

benzylpyrazol-5-yl)propanoic acid derivative 2 proved to be a

weak PPARc agonist with an EC₅₀value 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

N

Cl

N

MeO

O

CO₂H

N

Me

CO₂H

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.¹⁰In this study, we focused on an acylsulfona-

mide group as a ring-opened analog of TZD.¹¹Similar 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,¹²which was converted to acetic acid 33b by

saponiﬁcation. Similarly, butanoic acid 33c was obtained from

MeO₂C

CO₂Me

O

H

N

H

R²

N

CO₂Me

a

c

e

N

OEt

Me

R¹O

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

3e: X = Cl, R³= (CH₂)₃Me

3f: X = Cl, R³= (CH₂)₅Me

O

R³

g

f

S

N

OEt

N

H

3g: X = Cl, R³= (CH₂)₂CHMe₂

Me

: X = Cl, R³= cyclohexyl

: X = Cl, R³= Ph

3h

3i

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

N

Cl

N

Cl

O

e

b

d

Y

R²

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

O

g

h

N

S

N

OEt

N

OEt

N

H

Me

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

N

OEt

R

Me

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

22c: R = cyclopropyl, X = CF₃

22d

21c: R = cyclopropyl, X = CF₃

21d

: R = Ph, X = Cl

e

X

Cl

N

X

Cl

O

d

e

N

S

N

OEt

N

CH₃

H

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 EC₅₀value. (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,¹⁴and carbacyclin¹⁵were 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

718

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

F₃C

Cl

N

X

Cl

N

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

R²

e

N

S

N

OEt

N

H

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)CH_2-

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

N

Cl

N

b

CO₂Me

(CH₂)_nCO₂H

a

N

12

BuO

31

33a

: n = 0

33b: n = 1

33c: n = 3

d

c

e

33d

: n = 4

f

Cl

N

Cl

c

(CH₂)_nCN

17a

N

H

N

Me

N

(CH₂)_n

S

BuO

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 (CL_total= 794 mL/h/kg, AUC_{0–8 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

N

c transactivation activities and metabolic clearance of compounds 3c,

EC₅₀

O

R

450 nM

160 nM

16 nM

3a: R = Me

Cl

N

S

N

H

3b: R = (CH)₂Me

3c: R = (CH₂)₄Me

Me

O

S

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

18d^d

24a

24d

144

100

224

124

111

Cl

O

R

13

11

N

S

N

H

a

Me

EC₅₀value 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

3j

Benzyl

107

1-benzyl-3-butoxypyrazole analog 18a. Shortening of the ethylene

a

EC₅₀value 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 10^ꢀ8

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.¹⁶Therefore, 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 10^ꢀ8M 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

O

N

As for PPAR

ited EC₅₀values 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-

EC₅₀value 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

R²

O

R²

N

S

N

H

N

H

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

Isopropoxy

Isopropyl

Isopropoxy

Isopropyl

Cyclopropyl

Isopropoxy

MeO(CH₂)₂O

Isopropyl

Pentyl

Isoamyl

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

Cl

CF₃

Cl

24b

24c

30a

30b

30c

30d

25a

25c

99

54

CF₃

Cyclopropyl

a

EC₅₀value 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 IC₅₀values 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 EC₅₀values 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

EC₅₀values of 9.3, 8.3, and 9.0 nM, respectively. In addition, com-

pound 25c showed acceptable metabolic stability and potent activ-

ity with EC₅₀value 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.^17bThe 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 ED₂₅values ranging from 0.12 to 0.42 mg/kg and triglyceride

(TG) levels with ED₂₅values 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 EC₅₀values 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

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.^17aWith regard to the PPAR sub-

type selectivity of the above mentioned compounds, they did not

a

EC₅₀value means the effective concentration for 50% response of a given

compound’s intrinsic maximum response.

b

IC₅₀value 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

AUC_0-8h(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

EC₅₀value 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

EC₅₀value 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 AUC_{0–8 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

(AUC_{0–8 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

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)¹⁹and PPARd-LBD with PPARd agonist GW2331 (PDB

code: 1Y0S). ²⁰As 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).²¹However, 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 F₂₅₄plates.

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. NaHCO₃and 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 MgSO₄and 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. NaHCO₃and 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 MgSO₄and 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. NaHCO₃and 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 10⁴cells/well, and cultured in 5% CO₂at 37 °C overnight.

After washing the OPAQUE PLATE with PBS, 45

medium containing 0.1% fatty acid free-BSA and 5

l

L of HAM F12

L of test com-

pounds were added to the plate, which was then cultured in 5%

CO₂at 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 10⁶cells in 150 cm²tissue culture ﬂask,

and cultured in 5% CO₂at 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% CO₂at 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% CO₂at 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 10³cells/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% CO₂at 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 ED₂₅for 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)

R_symmoverall (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

R_free

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

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

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Article Doi

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

DOI: 10.1016/j.bmc.2011.12.008

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