Design, synthesis, and structure-activity relationships of 1-ethylpyrazole-3-carboxamide compounds as novel hypoxia-inducible factor (HIF)-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.02.038

Hypoxia-inducible factor (HIF)-1 is well known as a promising target for cancer chemotherapy. By screening an in-house chemical library using a hypoxia-responsive luciferase reporter gene assay, we identified CLB-016 (1) containing 1-ethylpyrazole-3-carbo

Full text of DOI:10.1016/j.bmc.2015.02.038

Bioorganic & Medicinal Chemistry 23 (2015) 1776–1787
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure–activity relationships of
1-ethylpyrazole-3-carboxamide compounds as novel
hypoxia-inducible factor (HIF)-1 inhibitors
Yorinobu Yasuda a, Takeaki Arakawa a, Yumi Nawata a, Sayaka Shimada a, Shinya Oishi b, Nobutaka Fujii b,
Shinichi Nishimura a, Akira Hattori a, Hideaki Kakeya a,
⇑
a Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences,
Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Department of Bioorganic Medicinal Chemistry & Chemogenomics, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences,
Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hypoxia-inducible factor (HIF)-1 is well known as a promising target for cancer chemotherapy. By
screening an in-house chemical library using a hypoxia-responsive luciferase reporter gene assay, we
Received 27 December 2014
Revised 14 February 2015
Accepted 17 February 2015
Available online 25 February 2015
identified CLB-016 (1) containing 1-ethylpyrazole-3-carboxamide as a HIF-1 inhibitor (IC50 = 19.1
In a subsequent extensive structure-activity relationship (SAR) study, we developed compound 11Ae
with an IC50 value of 8.1 M against HIF-1-driven luciferase activity. Compounds 1 and 11Ae were shown
lM).
l
to significantly suppress the HIF-1-mediated hypoxia response, including carbonic anhydrase IX (CAIX)
gene expression and migration of human sarcoma HT1080 cells. These results revealed 1-ethylpyra-
zole-3-carboxamide as a novel scaffold to develop promising anti-cancer drugs targeting the HIF-1 sig-
naling pathway.
Keywords:
Hypoxia-inducible factor (HIF)
Cancer
Chemotherapy
Structure–activity relationship
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
transcription through the promotion of hydroxylation at Asn-803
of HIF-1a by factor-inhibiting-HIF (FIH), which, in turn, disrupts
Tumor cells survive in harsh conditions, such as hypoxia and
exposure to poor nutrition by changing their metabolic pathway.1
Hypoxia-inducible factor (HIF)-1, a basic helix–loop–helix tran-
scriptional factor, promotes the transcription of a number of genes
involved in cellular adaptive responses to low availability of oxy-
gen and nutrients, such as glucose transporter-1 (GLUT), vascular
endothelial growth factor (VEGF) and carbonic anhydrase IX
(CAIX).2 Therefore, it has been proposed that HIF-1 is a promising
molecular target for cancer chemotherapy.
the association of its C-terminal transactivation domain with
p300/CBP, a critical co-factor of HIF-1.5 The inhibition of HIF-1
activity by YC-1 suppresses tumor metastasis and decreases the
incidence of post-irradiation tumor recurrence.6
To generate new inhibitors against HIF-1, we screened an in-
house chemical library using a hypoxia-responsive luciferase
reporter gene assay. We here report CLB-016 (1) with 1-ethyl pyra-
zole-3-carboxamide as a potent inhibitor. Through an extensive
structure–activity relationship (SAR) study of CLB-016 (1), we have
successfully developed more potent HIF-1 inhibitors.
To date, dozens of compounds have been reported to suppress
the HIF-1 signaling pathway (Fig. 1).3,4 For example, YC-1, the
most-widely used HIF-1 inhibitor, suppresses HIF-1-mediated
2. Results and discussion
2.1. Discovery of CLB-016 (1)
Abbreviations: HIF-1, hypoxia-inducible factor-1; CA, carbonic anhydrase; FIH,
factor-inhibiting-HIF; SAR, structure–activity relationship; SEM, 2-(trimethylsi-
lyl)ethoxymethyl; HRE, hypoxia-response element; VEGF, vascular endothelial growth
factor; TBAI, tetrabutylammonium iodide; HATU, 1-(bis(dimethylamino)methylene)-
1H-1,2,3-triazolo(4,5-b)pyridinium 3-oxide hexafluorophosphate.
To identify novel HIF-1 inhibitors, we screened an in-house
chemical library using a hypoxia-response element (HRE)-driven
luciferase assay system. Human fibrosarcoma HT1080 cells with
stably-integrated x5HRE-luciferase reporter were treated with
⇑
Corresponding author. Tel.: +81 75 753 4524; fax: +81 75 753 4591.
E-mail address: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya).
the tested compounds at 100 lM under hypoxic conditions (1%
http://dx.doi.org/10.1016/j.bmc.2015.02.038
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1777
region 3
OH
region 4
O
N
N
O
N
N
N
N
H
O
H2N
N
O
region 2
region 1
CLB-016 (1)
YC-1
O
O
O O
S
O
O
O
N
O
N
H
O
OH
CAY10585
KCN-1
N
N
O
O
H
N
N
O
N
N
O
H
O S
S
O
O
O
O
O
O
H
N
N
N
N
N
N
H
O
Echinomycin
Figure 1. Chemical structures of CLB-016 (1) and representative HIF-1 inhibitors.
O2) for 24 h. Among approximately 6000 compounds, CLB-016 (1,
Fig. 1) significantly decreased hypoxia-induced luciferase activity
(89.8% inhibition compared with vehicle control). Interestingly,
CLB-016 (1) exhibited a novel chemotype among various HIF-1
inhibitors,3,4 which promoted us to undertake a SAR study of this
compound.
hydrogenation in the presence of a catalytic amount of 10% Pd/C
to produce amines 4A and 4B, respectively. The structures of
the isomers (4A, 4B) were determined by NOESY analysis: a
NOESY correlation was observed between H-5 and ethyl protons
in 4A.
Next, dimethyl pyrazole (6), the right segment, was reacted
with various substituted benzyl halides, and heteroarylmethyl
halides (5a–5c) in the presence of KOtBu to yield the correspond-
ing alkyl 4-substituted aryl or heteroaryl-2-carboxylates (7a–7e).
The ester compounds 7a–7e were hydrolyzed with NaOH to gener-
ate carboxylic acids (8a–8e). These carboxylic acids were con-
densed with aminopyrazole 4A or 4B to yield the corresponding
amides (9Aa–9Ae, 9Ba). Hydrolysis of the ester group with NaOH
gave the corresponding acids, followed by their conversion into
the amide compounds (1, 11Aaa–11Ae, 11Ba).
Compound 18 lacking an ethyl group in CLB-016 (1) was syn-
thesized in a similar fashion to CLB-016 (1) (Scheme 2). 4-Nitro-
1H-pyrazole-3-carboxylic acid (2) was first converted to the
methyl ester 12, followed by protection with 2-(trimethylsi-
lyl)ethoxymethyl (SEM) group to afford 13, which could be
separated from the 1,5-isomer by silica gel flash chromatography.
The nitro compound 13 was reduced and conjugated with the mid-
dle-right part 8a. The obtained methyl ester 15 was hydrolyzed,
converted to carboxamide 17, and finally deprotected to yield the
target compound 18.
2.2. Design and synthesis of CLB-016 (1) and its derivatives
We planned to synthesize analogues by conjugation of 4-
amino-pyrazole (left segment), pyrazole (right segment), and the
remaining middle segment (Fig. 1). The furan ring in the middle
segment was replaced by other aromatic rings (region 1). Next,
substitutions of the left-pyrazole ring were investigated (regions
2 and 3). Then, the size and lipophilicity of the right-pyrazole ring
were examined (region 4).
The synthetic route of 1-ethylpyrazole-3-carboxamide is shown
in Scheme 1. The left segments were first synthesized.
Commercially available 4-nitro-1H-pyrazole-3-carboxylic acid (2)
was alkylated with excess ethyl iodide in the presence of potassi-
um carbonate in DMF. After flash chromatography on silica gel,
two regioisomers, ethyl 1-ethyl-4-nitro-1H-pyrazole-3-carboxy-
late (3A, 1,3-isomer) and ethyl 1-ethyl-4-nitro-1H-pyrazole-5-car-
boxylate (3B, 1,5-isomer) were obtained in 34.4% and 20.7% yield,
respectively. This regioselectivity might be due to a steric hin-
drance of both the ethyl group and the carboxyl group at pyrazole
3-position. The nitro group of 3A and 3B was reduced by
We designed the other compound 23 to test the importance of
the carboxamide in the left segment. Synthesis was commenced

1778
Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
H
N
N
N
N
N
N
N
N
O
a
b
N
N
O
O
NH2
HO
NO2
NO2
O
O
NO2
NH2
O
O
O
O
2
3A
3B
4A
4B
R2
2
6a
: R =H
2
6d
: R =Cl
O
O
R1
6e: R2=Br
R1
O
R2
HN
R2
N
N
N
O
N
N
O
X
HO
c
d
Ar
e
Ar
Ar
1
1
2
2
5a
7a
8a
: Ar=furan, R =H
: Ar=furan, R =Et, X=Cl
: Ar=furan, R =Et, R =H
5b: Ar=thiophene, R1=Me, X=Br
5c: Ar=phenylene, R1=Me, X=Cl
7b: Ar=thiophene, R1=Me, R2=H
8b: Ar=thiophene, R2=H
7c: Ar=phenylene, R1=Me, R2=H
8c: Ar=phenylene, R2=H
1
2
2
7d
7e
8d
8e
: Ar=furan, R =Et, R =Cl
: Ar=furan, R =Cl
1
2
2
: Ar=furan, R =Et, R =Br
: Ar=furan, R =Br
N
N
O
N
N
N
O
O
N
O
f
R2
R2
g
R2
N
N
N
N
R3
N
N
H
N
N
H
O
HO
H
N
N
Ar
Ar
O
R4
Ar
O
2
2
2
3
4
9Aa
10Aa
1
: Ar=furan, R =H
: Ar=furan, R =H
: Ar=furan, R =H, R =H, R =H
9Ab: Ar=thiophene, R2=H
10Ab: Ar=thiophene, R2=H
11Aaa: Ar=furan, R2=H, R3=Me, R4=H
9Ac: Ar=phenylene, R2=H
10Ac: Ar=phenylene, R2=H
11Aab: Ar=furan, R2=H, R3=Me, R4=Me
2
2
2
3
4
9Ad
9Ae
10Ad
10Ae
11Ab
11Ac
11Ad:
: Ar=furan, R =Cl
: Ar=furan, R =Cl
: Ar=thiophene, R =H, R =H, R =H
: Ar=phenylene, R =H, R =H, R =H
2
2
2
3
4
: Ar=furan, R =Br
: Ar=furan, R =Br
2
3
4
Ar=furan, R =Cl, R =H, R =H
11Ae: Ar=furan, R2=Br, R3=H, R4=H
N
N
O
N
N
N
O
O
N
O
N
N
N
N
N
N
N
N
H
O
O
N
H
HO
O
H
O
H2N
O
O
9Ba
10Ba
11Ba
Scheme 1. Synthesis of CLB-016 (1) and its derivatives. Reagents and conditions: (a) ethyl iodide, K2CO3, DMF, rt; (b) Pd/C, H2, EtOH, rt; (c) 6a/6d/6e, KOtBu, TBAI, THF, rt; (d)
5 N NaOH aq, EtOH, rt; (e) 4A/4B, HATU, Hunig’s base, DMF, MW 50 °C; (f) 5 N NaOH aq, EtOH, rt; (g) R3R4NH, HATU, Hunig’s base, DMF, rt or MW 50 °C.
by alkylation of the methyl ester 12 with tert-butyl 2-bromoethyl-
carbamate (19) to obtain methyl 1-(2-(tert-butoxycarbony-
lamino)ethyl)-4-nitro-1H-pyrazole-5-carboxylate 20 (Scheme 2). As
in the case of alkylation from 2 to 3, the desired 1,5-isomer 20 was
separated from the 1,3-isomer by silica gel flash chromatography.
The Boc group in 20 was removed in an acidic condition, and the result-
ing primary amine in an HCl salt form was reacted with the methyl
ester to give the cyclic amide 21 in a basic condition. The nitro group
in 21 was reduced by hydrogenation to give an amine 22. Finally, con-
densation of 22 with 8a furnished the target compound 23.
decrease in the HIF-1 inhibitory activity (IC50: >100 and 72.0 lM,
respectively). This could be attributed to the difference in the bond
angle of bisection among furan (C–O–C), thiophene (C–S–C), and
1,3-disubstituted benzene (C1–C2–C3). Thus, the species of the
central ring determined the overall molecular conformation, which
consequently affected the HIF-1 inhibitory activity.7,8 It is alterna-
tively conceivable that a heteroatom in the central ring plays an
important role in its interaction with target molecule(s).
We could modulate potency by changing the substitution pat-
tern of the carboxamide group. Non-methylated amide 1 and
mono-methylated amide 11Aaa exhibited equipotent HIF-1
inhibitory activity, while dimethylated amide 11Aab exhibited
decreased potency both in the HIF-1 inhibitory activity and in
the anti-proliferative activity against HT1080 cells. Since the 1H
NMR signals of the two carboxamide protons in compound 1 were
not equivalent (dH 6.74 and 5.62 ppm), there may exist a five-
membered ring formed by intramolecular hydrogen bonding
between the carboxamide proton and the pyrazole nitrogen. This
five-membered ring can fix the direction of the carbonyl oxygen
atom, which might be prerequisite for its activity. In contrast, com-
pound 11Aab cannot fix the conformation of carboxamide, as this
compound has no proton atom on the nitrogen. We also
2.3. Biological activities
HIF-1 inhibitory activity of the synthesized analogues was
examined in HT1080 cells stably-expressing x5HRE-luciferase
reporter. In our system, YC-1, a HIF-1 inhibitor, exhibited marginal
inhibitory activity with an IC50 value of 48.4
inhibited proliferation of cells cultured under hypoxia with an
IC50 value of 30.9 M. In contrast, CLB-016 (1) (IC50 = 19.1 M)
lM. This compound
l
l
inhibited the HIF-1 signaling pathway in a dose-dependent manner
(Fig. 2A). As shown in Table 1, replacement of the furan ring of 1
with thiophene (11Ab) or benzene (11Ac) caused a substantial

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1779
SEM
SEM
N
SEM
N
H
N
N
N
h
N
O
j
i
k
O
N
O
N
O
2
N
N
N
H
O
O
O
NO2
O
O
NH2
NO2
O
12
13
14
15
SEM
SEM
H
N
N
l
N
m
n
N
N
N
O
O
O
N
N
N
N
N
N
N
H
N
H
N
H
O
O
O
H2N
HO
H2N
O
O
O
16
17
18
Boc
Br
N
H
H
N
19
N
N
N
H
N
N
N
O
o
N
N
O
p
q
Boc
HN
HN
NO2
NH2
O
O
NO2
NO2
O
O
22
21
20
12
N
N
O
r
N
N
N
O
HN
H
O
23
Scheme 2. Synthesis of 18 and 23. Reagents and conditions: (h) Acetyl chloride, MeOH, rt; (i) SEM-Cl, NaH, THF, rt; (j) H2, Pd/C, 40 °C; (k) 8a, HATU, Hunig’s base, DMF, MW
50 °C; (l) 5 N NaOH aq, EtOH, rt; (m) NH4Cl, HATU, Hunig’s base, DMF, MW 50 °C; (n) 2 M HCl/EtOH, MW 80 °C; (o) 19, K2CO3, TBAI, DMF, rt; (p) (i) 2 M HCl/EtOH, 50 °C; (ii)
Hunig’s base, toluene, 100 °C; (q) Pd/C, H2, EtOH, 40 °C; (r) 8a, HATU, Hunig’s base, DMF, MW 50 °C.
investigated the effect of the alkyl chain in the left pyrazole ring.
The ethyl group in CLB-016 (1) was indispensable for the HIF-1
inhibitory activity, because compound 18 with no alkylation
showed only marginal inhibition compared with CLB-016 (1). In
addition, the effect of compound 11Ba, a regioisomer of 1, on both
the reporter activity and proliferation of HT1080 cells was found to
be negligible. Thus, it is suggested that both formation of the intra-
molecular hydrogen bond and proper substitution of an ethyl
group in the pyrazole ring are indispensable for the potent HIF-1
inhibitory activity. This was also supported by the invalidity of
compound 23 with 6,7-dihydropyrazolopyrazinone containing a
six-membered cyclic amide.
We evaluated the inhibitory activities of CLB-016 (1) and its
derivatives toward HIF-1 transcriptional activity by measuring
the endogenous mRNA levels of CAIX gene, a HIF-1 target gene.
As shown in Figure 2B, the CAIX gene in HT1080 cells was actively
transcribed under hypoxia (153-fold over normoxia). Quantitative
RT-PCR analysis demonstrated that compound 1 significantly sup-
pressed the hypoxia-induced CAIX expression with comparable
dose-dependency to its HRE-luc inhibition. In contrast, CLB-016
(1) did not affect the mRNA levels of HIF-1a and -1b up to
100 M, suggesting that the inhibition of hypoxia-induced
l
responses, such as elevation of luciferase activity and CAIX mRNA
expression, by the compound was not due to general inhibition
of transcription. As shown in Figure 2C, CLB-016 derivatives
(11Ad and 11Ae, but not 11Ba) decreased the hypoxia-induced
CAIX mRNA levels with good correlation to their HRE-reporter inhi-
bition. Finally, these active compounds including 1, 11Ad and
11Ae, were found to significantly suppress the motility of
HT1080 cells (Fig. 3), which is known to be facilitated by several
HIF-1-regulated genes including VEGF and CAIX.10 Taken together,
it is conceivable that a series of 1-ethylpyrazole-3-carboxamide
compounds are able to modulate cellular adaptive responses to
hypoxia by specifically inhibiting the HIF-1 signaling pathway.
We next investigated the effect of the size and lipophilicity of the
right pyrazole ring on the HIF-1 inhibitory activity. As shown in
Table 2, the importance of the dimethyl substitution was demon-
strated by compound 24, which had no methyl group and exhibited
marginal HIF-1 inhibitory activity (IC50 = 81.8
compound 25 with dimethylisoxazole lost its inhibitory activity
(IC50 >100 M), suggesting the importance of the lipophilic surfaces
lM). Furthermore,
l
of the molecule for its activity. In fact, introduction of a halogen atom
at position 4 of dimethyl pyrazole furnished much more potent inhi-
bitors, for example, 11Ad (Cl, HRE-Luc IC50 = 8.7
lM) and 11Ae (Br,
HRE-Luc IC50 = 8.1 M). Alternatively, it is also plausible that an
l
3. Conclusions
increase in the lipophilicity of the molecules contributed to the
elevation of their biological activities by increasing the permeability
of the plasma membrane9: each ALogP for 1, 11Ad, and 11Ae is 1.18,
1.84, and 1.93, respectively.
In the current study, we identified CLB-016 (1) and developed
its derivatives containing 1-ethylpyrazole-3-carboxamide as

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Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
HIF-1 inhibitors. SAR studies revealed that: (1) 1-ethylpyrazole-3-
carboxamide moiety (regions 2 and 3) required both protons on
the carboxamide group and proper substitution of an ethyl group
for HIF-1 inhibitory activity; (2) investigation of the central ring
(region 1) suggested the importance of the bond angle of the bisec-
tion among the hetero-aromatic ring for activity; and (3) introduc-
tion of a halogen atom on the right pyrazole ring (region 4)
improved activity. Because CLB-016 derivatives possess drug-like
physicochemical properties (molecular weight of 100–350, LogP
value of 1–3),11 1-ethylpyrazole-3-carboxamide compounds are
promising lead compounds from which to develop novel anti-can-
cer drugs targeting the HIF-1 signaling pathway. Although we have
not identified the target molecule(s) of the compounds, we demon-
strated that CLB-016 (1) and its derivatives should affect the HIF-1-
dependent pathway. Detailed studies of the mechanism of action
of 1-ethylpyrazole-3-carboxamide compounds on HIF-1 signaling
are required, and are currently being undertaken in our laboratory.
4. Experimental section
4.1. Chemistry
Reactions in microwave reactors were performed by single-
node heating in an Initiator (Biotage). Flash column chromatogra-
phy was performed manually or by using a Biotage SP-1 Flash.
High-resolution mass spectra were measured on LC-IT-TOF MS
(Shimadzu) mass spectrometers. The 1H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were recorded on JEOL AL-400 and JEOL
ECS-400 with tetramethylsilane as an internal standard.
Abbreviations are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad. Melting points were measured
by BÜCHI Melting Point B-545 (BÜCHI Labortechnik AG). IR spectra
were measured on
a SHIMADZU FTIR-8400S spectrometer.
Compounds 25 and YC-1 were purchased from Princeton
BioMolecular Research, Inc. and Santa Cruz Biotechnology, Inc.,
respectively.
4.1.1. Ethyl 1-ethyl-4-nitro-1H-pyrazole-3-carboxylate (3A) and
ethyl 1-ethyl-4-nitro-1H-pyrazole-5-carboxylate (3B)
4-Nitro-1H-pyrazole-3-carboxylic acid (1) (300 mg, 1.91 mmol)
was added to a stirred solution of ethyl iodide (324 ll, 4.01 mmol)
and K2CO3 (660 mg, 4.77 mmol) in DMF (9 ml). The reaction mix-
ture was stirred at rt for 3 h, and then the mixture was filtrated
and concentrated. The residue was suspended in H2O and
extracted with EtOAc. The organic layer was washed with H2O
and brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(hexane/EtOAc) to give 3A (140 mg, 0.656 mmol, 34.4%) as a pale
brown oil and 3B (84 mg, 0.395 mmol, 20.7%) as a pale brown oil.
Compound 3A: 1H NMR (CDCl3) d 8.16 (s, 1H), 4.47 (q, J = 7.2 Hz,
2H), 4.26 (q, J = 7.3 Hz, 2H), 1.57 (t, J = 7.3 Hz, 3H), 1.41 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3) d 160.3, 138.8, 134.1, 129.1,
62.5, 48.9, 14.9, 14.0; HRMS (ESI) m/z: [M+Na]+ calcd for
C8H11N3O4Na: 236.0642, found: 236.0643.
Compound 3B: 1H NMR (CDCl3) d 8.03 (s, 1H), 4.50 (q, J = 7.2 Hz,
2H), 4.31 (q, J = 7.3 Hz, 2H), 1.50 (t, J = 7.3 Hz, 3H), 1.42 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3) d 158.6, 135.2, 134.4, 131.2,
63.4, 47.7, 15.3, 13.8; HRMS (ESI) m/z: [M+Na]+ calcd for
C8H11N3O4Na: 236.0642, found: 236.0639.
Figure 2. Inhibition of HIF-1-mediated transcription by CLB-016 (1) and its
derivatives. (A) Dose-dependent inhibition of hypoxia-induced luciferase activity
by CLB-016 (1). x5HRE/HT1080 cells were incubated with the compound for 24 h
under hypoxia (1% O2). The luciferase activity of CLB-016 (1)-treated cells relative to
that of DMSO-treated cells are shown. (B) Effects of CLB-016 (1) on the expression
of the human CAIX, HIF-1a, and HIF-1b genes. Cells were treated with the compound
for 4 h. mRNA levels were quantified by real-time PCR, normalized to 18S rRNA
levels, and presented as relative values against that of DMSO-treated cells. (C)
Effects of CLB-016 (1) and its derivatives against the mRNA levels of CAIX. Cells were
treated with compounds at 100 lM for 16 h. mRNA levels were measured by
quantitative PCR analysis as in (B), and the values relative to that under normoxic
4.1.2. Ethyl 4-amino-1-ethyl-1H-pyrazole-3-carboxylate (4A)
and Ethyl 4-amino-1-ethyl-1H-pyrazole-5-carboxylate (4B)
After purging with N2 gas, to a solution of compound 3A
(140 mg, 0.657 mmol) in MeOH (1.3 ml) was added 10% Pd/C
conditions are shown. Data represent the mean standard error (n = 3).

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1781
Table 1
Effects of CLB-016 derivatives (in regions 1, 2 and 3) on HIF-1 transcriptional activity and HT1080 cell proliferation
O
R1
N
N
N
H
Ar
a
a
Compd
R1
Ar
HRE Luc IC50
M)
HT1080 IC50
M)
ALogPb
(
l
(l
N
O
S
N
1 (CLB-016)
19.1 2.8
15.1 1.8
1.18
H2N
O
11Ab
11Ac
>100
>100
1.74
1.71
72.0 7.3
50.2 2.9
N
O
N
11Aaa
11Aab
19.4 6.2
18.1 0.8
1.38
1.59
H
N
O
N
N
>100
75.0 10.9
N
O
H
N
N
18
71.6 1.1
66.6 2.9
0.62
1.04
H2N
O
N
N
11Ba
>100
>100
H2N
HN
O
N
N
23
>100
>100
0.72
4.08
O
YC-1
48.4 2.3
30.9 4.3
a
Data represent the mean standard error (n = 3).
ALogP was calculated using Pipeline pilot (Accelrys, Inc.).
b
(6.99 mg, 6.57
l
mol). The reaction mixture was purged with H2 gas
4.1.3. Synthesis of pyrazoles 7a–e
4.1.3.1. Ethyl 5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxylate (7a). Ethyl 5-(chloromethyl)furan-2-carboxylate
and stirred at rt for 22 h. The mixture was filtrated through celite
and concentrated. The residue was subjected to column chro-
matography using amino-functionalized silica gel (EtOAc/MeOH)
to furnish the carboxylate 4A (92.0 mg, 76.8%) as a colorless oil.
The isomer 4B was synthesized in a similar way (66.0 mg, 90.7%).
Compound 4A: 1H NMR (CDCl3) d 6.99 (1H, s), 4.41 (2H, q,
J = 7.2 Hz), 4.13 (2H, q, J = 7.3 Hz), 4.07 (2H, br s), 1.46 (3H, t,
J = 7.2 Hz), 1.41 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3) d 163.7,
135.0, 129.6, 115.8, 60.4, 48.1, 15.4, 14.5; HRMS (ESI) m/z:
[M+Na]+ calcd for C8H13N3O2Na: 206.0900, found: 206.0904.
Compound 4B: a colorless oil; 1H NMR (CDCl3) d 7.09 (1H, s),
4.45 (2H, q, J = 7.2 Hz), 4.39 (2H, q, J = 7.2 Hz), 4.10 (2H, br s),
1.41 (3H, t, J = 7.2 Hz), 1.36 (3H, t, J = 7.2 Hz); 13C NMR (CDCl3) d
160.4, 137.2, 126.9, 116.3, 60.4, 47.5, 15.7, 14.4; HRMS (ESI) m/z:
[M+H]+ calcd for C8H14N3O2: 184.1081, found: 184.1083.
(5a, 2.0 g, 10.60 mmol) was added to a solution of 3,5-dimethyl-
1H-pyrazole (6a) (1.019 g, 10.60 mmol), KOtBu (1.547 g,
13.79 mmol) and TBAI (0.392 g, 1.060 mmol) in THF (53 ml) at
0 °C. The mixture was allowed to warm up to rt and was stirred
at rt for 24 h. To the reaction mixture was added satd NH4Cl aq,
and then the mixture was extracted with EtOAc. The organic layers
were combined, washed with H2O and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was subjected to silica
gel column chromatography (hexane/EtOAc), which yielded the
pyrazole 7a (1.25 g, 47.5%) as a brown oil. Pyrazoles 7b–e were
synthesized in a similar way.
Compound 7a: yield, 47.5%; a brown oil; 1H NMR (CDCl3) d 7.07
(d, J = 3.4 Hz, 1H), 6.18 (d, J = 3.7 Hz, 1H), 5.84 (s, 1H), 5.21 (s, 2H),

1782
Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
Table 2
4.1.3.3. Methyl 3-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zoate (7c). After extraction, washing and drying, evaporation of
the solvent gave a residue that was used without further purifica-
tion in the next step.
Effects of CLB-016 derivatives (in region 4) on HIF-1 transcriptional activity and
HT1080 cell proliferation
N
N
O
4.1.3.4. Ethyl 5-((4-chloro-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxylate (7d). Yield, 55.4%, a pale yellow solid; 1H
NMR (CDCl3) d 7.08 (d, J = 3.4 Hz, 1H), 6.25–6.22 (m, 1H), 5.21 (s,
2H), 4.34 (q, J = 7.1 Hz, 2H), 2.28 (s, 3H), 2.20 (s, 3H), 1.36 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3) d 158.6, 153.7, 145.4, 144.7, 136.1,
118.6, 110.2, 61.1, 46.8, 29.7, 14.3, 11.2, 9.4; HRMS (ESI) m/z:
[M+Na]+ calcd for C13H15ClN2O3Na: 305.0663, found: 305.0666.
N
H
O
H2N
R2
O
a
a
Compd
R2
HRE Luc IC50
M)
HT1080 IC50
M)
ALogPb
(
l
(l
N
4.1.3.5. Ethyl 5-((4-bromo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxylate (7e). Yield, 41.5%, a pale yellow powder; 1H
NMR (CDCl3) d 7.08 (d, J = 3.4 Hz, 1H), 6.24 (d, J = 3.7 Hz, 1H), 5.24
(s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 2.20 (s, 3H), 1.36 (t,
J = 7.1 Hz, 3H); 13C NMR (CDCl3) d 158.5, 153.8, 146.9, 144.7, 137.7,
118.6, 110.2, 95.0, 61.1, 47.0, 14.3, 12.3, 10.4; HRMS (ESI) m/z:
[M+Na]+ calcd for C13H15BrN2O3Na: 349.0158, found: 349.0149.
1 (CLB-016)
19.1 2.8
15.1 1.8
1.18
N
N
N
24
25
81.8 6.9
>100
75.1 0.2
>100
0.75
1.17
N
O
4.1.4. Synthesis of carboxylic acids 8a–e
4.1.4.1. 5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-car-
boxylic acid (8a). A solution of 5 N NaOH (7.25 ml, 36.2 mmol)
was added to a solution of compound 7a (3.0 g, 12.08 mmol) in
EtOH (60.4 ml), which was stirred at rt for 24 h. The aqueous phase
was acidified to a pH of approx. 3 with 6 N HCl on ice. The majority
of the solvent was removed on a rotary evaporator. After the
precipitate was filtrated and washed with H2O, the carboxylic
acid 8a was obtained as a pale yellow powder (1.5 g, 6.81 mmol,
56.4%). Carboxylic acids 8b–e were synthesized in a similar
way.
Cl
N
N
11Ad
8.7 0.6
7.4 0.2
1.84
Br
N
N
11Ae
YC-1
8.1 0.2
7.0 0.3
1.93
4.08
48.4 2.3
30.9 4.3
a
Data represent the mean standard error (n = 3).
ALogP was calculated using Pipeline pilot (Accelrys, Inc.).
b
Compound 8a: yield, 56.4%; a pale yellow powder; 1H NMR
(DMSO-d6) d 13.07 (s, 1H), 7.14 (d, J = 3.5 Hz, 1H), 6.46 (d,
J = 2.9 Hz, 1H), 5.84 (s, 1H), 5.25 (s, 2H), 2.26 (s, 3H), 2.07 (s, 3H);
13C NMR (DMSO-d6) d 159.1, 154.7, 146.3, 144.4, 139.0, 118.5,
110.3, 105.2, 45.0, 13.2, 10.5; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
4.34 (q, J = 7.2 Hz, 2H), 2.28 (s, 3H), 2.21 (s, 3H), 1.36 (t, J = 7.1 Hz,
3H); 13C NMR (CDCl3) d 158.6, 154.7, 148.3, 144.4, 139.5, 118.7,
109.7, 105.8, 60.9, 46.0, 14.3, 13.5, 11.0; HRMS (ESI) m/z:
[M+Na]+ calcd for C13H16N2O3Na: 271.1053, found: 271.1046.
C11H11N2O3: 219.0775, found: 219.0769.
4.1.4.2. 5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)thiophene-2-
carboxylic acid (8b). Yield, 46.4%; a pale yellow powder; 1H
NMR (DMSO-d6): d 13.0 (s, 1H),7.56 (d, J = 3.6 Hz, 1H), 7.01 (d,
J = 3.6 Hz, 1H), 5.84 (s, 1H), 5.38 (s, 2H), 2.21 (s, 3H), 2.09 (s, 3H);
13C NMR (DMSO-d6) d 162.7, 147.6, 146.6, 138.7, 133.9, 132.9,
126.6, 105.3, 46.8, 13.3, 10.5; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
4.1.3.2. Methyl 5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)thio-
phene-2-carboxylate (7b). Yield, 42.7%; a white solid; 1H NMR
(CDCl3) d 7.63 (d, J = 3.9 Hz, 1H), 6.86–6.84 (m, 1H), 5.84 (s, 1H),
5.33 (s, 2H), 3.84 (s, 3H), 2.23 (s, 3H), 2.23 (s, 3H); 13C NMR
(CDCl3) d 162.4, 148.3, 147.4, 138.9, 133.5, 133.0, 125.8, 106.0,
52.1, 47.8, 13.5, 11.0; HRMS (ESI) m/z: [M+H]+ calcd for
C11H11N2O2S: 235.0546, found: 235.0539.
C12H15N2O2S: 251.0849, found: 251.0845.
Figure 3. Inhibition of HT1080 cell motility by CLB-016 (1) and its derivatives. Each compound (100
lM) was added to scratched monolayers. Representative images of
HT1080 cell migration after 48 h of incubation are shown (Scale bar, 500 m). Double-headed arrows indicate the scratched wound gaps.
l

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1783
4.1.4.3. 3-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)benzoic acid
(8c).
Yield, 73.0% (from 2 steps); a white powder; 1H NMR
164.2, 163.8, 148.0, 139.2, 138.5, 133.8, 130.2, 130.0, 129.3,
126.0, 125.6, 125.5, 121.1, 105.8, 61.4, 52.2, 48.5, 15.4, 14.4, 13.5,
11.1. HRMS (ESI) m/z: [M+Na]+ calcd for C21H25N5O3Na:
418.1849, found: 418.1841.
(DMSO-d6) d 13.00 (s, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.68 (s, 1H),
7.46 (dd, J = 8.1, 7.5 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 5.87 (s, 1H),
5.26 (s, 2H), 2.15 (s, 3H), 2.10 (s, 3H); 13C NMR (DMSO-d6) d
167.1, 146.2, 138.8, 138.5, 131.3, 131.0, 128.8, 128.2, 127.6,
105.1, 51.1, 13.3, 10.6; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
4.1.5.4. Ethyl 4-(5-((4-chloro-3,5-dimethyl-1H-pyrazol-1-yl)-
methyl)furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylate
(9Ad). Yield, 71.1%; a white powder; 1H NMR (CDCl3) d 9.94 (s,
1H), 8.28 (s, 1H), 7.13 (d, J = 3.7 Hz, 1H), 6.37 (d, J = 3.4 Hz, 1H),
5.25 (s, 2H), 4.50 (q, J = 7.2 Hz, 2H), 4.25 (q, J = 7.4 Hz, 2H), 2.40 (s,
3H), 2.21 (s, 3H), 1.53 (t, J = 7.3 Hz, 3H), 1.47 (t, J = 7.2 Hz, 3H); 13C
NMR (CDCl3) d 163.9, 154.9, 152.3, 147.2, 145.5, 136.0, 130.1,
124.7, 121.0, 116.1, 110.9, 108.5, 61.4, 48.5, 46.7, 15.4, 14.5, 11.4,
9.5; HRMS (ESI) m/z: [M+Na]+ calcd for C19H22N5O4ClNa: 442.1252,
found: 442.1266.
C13H13N2O2: 229.0982, found: 229.0974.
4.1.4.4. 5-((4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)methyl)fu-
ran-2-carboxylic acid (8d).
Yield, 79.8%; a white powder;
1H NMR (DMSO-d6) d 13.09 (s, 1H), 7.15 (d, J = 3.4 Hz, 1H), 6.54
(d, J = 3.4 Hz, 1H), 5.33 (s, 2H), 2.26 (s, 3H), 2.08 (s, 3H); 13C NMR
(DMSO-d6) d 159.1, 153.8, 144.7, 143.5, 135.7, 118.5, 110.8,
106.5, 46.1, 11.0, 9.0; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
C11H10ClN2O3: 253.0385, found: 253.0378.
4.1.5.5. Ethyl 4-(5-((4-bromo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylate
(9Ae). Yield, 67.9%; a white powder; 1H NMR (CDCl3) d 9.94 (1H,
s), 8.28 (1H, s), 7.13 (1H, d, J = 3.4 Hz), 6.37 (1H, d, J = 3.4 Hz), 5.27
(2H, s), 4.50 (2H, q, J = 7.1 Hz), 4.25 (2H, q, J = 7.3 Hz), 2.42 (3H, s),
2.22 (3H, s), 1.53 (3H, t, J = 7.3 Hz), 1.47 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3) d 163.9, 154.9, 152.2, 147.2, 147.0, 137.8, 130.1, 124.7,
121.0, 116.1, 110.9, 94.9, 61.4, 48.5, 46.8, 15.4, 14.5, 12.3, 10.4;
HRMS (ESI) m/z: [M+Na]+ calcd for C19H22N5O4BrNa: 486.0747,
found: 486.0742.
4.1.4.5. 5-((4-Bromo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)fu-
ran-2-carboxylic acid (8e).
Yield, 92.7%; a white powder;
1H NMR (DMSO-d6) d 13.12 (s, 1H), 7.15 (d, J = 3.4 Hz, 1H), 6.54
(d, J = 3.4 Hz, 1H), 5.35 (s, 2H), 2.28 (s, 3H), 2.08 (s, 3H); 13C NMR
(DMSO-d6) d 159.1, 153.8, 145.0, 144.7, 137.4, 118.5, 110.8, 93.4,
46.2, 11.9, 9.9; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for C11H10BrN2O3:
296.9880, found: 296.9880.
4.1.5. Synthesis of carboxylates 9Aa–e and 9Ba
4.1.5.1. Ethyl 4-(5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)furan-
2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylate (9Aa). To a
solution of compound 4A (150 mg, 0.819 mmol) and compound 8a
4.1.5.6. Ethyl 4-(5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-5-carboxylate
(180 mg, 0.819 mmol) in DMF (1637
(374 mg, 0.982 mmol) and Hunig’s base (214
l
l) were added HATU
(9Ba).
Purified by column chromatography over silica gel
ll, 1.228 mmol). The
(hexane/EtOAc). Yield, 42.5%; a white powder; 1H NMR (CDCl3) d
9.67 (s, 1H), 8.37 (s, 1H), 7.14 (d, J = 3.6 Hz, 1H), 6.28 (d,
J = 3.2 Hz, 1H), 5.85 (s, 1H), 5.23 (s, 2H), 4.54 (q, J = 6.8 Hz, 2H),
4.50 (q, J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.22 (s, 3H), 1.50 (t,
J = 7.2 Hz, 3H), 1.43 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) d 159.9,
155.1, 152.9, 148.3, 147.2, 139.4, 130.3, 127.6, 118.4, 116.3,
110.7, 106.0, 61.5, 48.1, 45.9, 15.8, 14.4, 13.5, 11.1; HRMS
(ESI) m/z: [M+Na]+ calcd for C19H23N5O4Na: 408.1642, found:
408.1649.
mixture was stirred at 50 °C for 5 min in a microwave instrument.
The reaction mixture was diluted with H2O and stirred for 0.5 h.
The precipitate was filtrated to give the carboxylic acid 9Aa
(221 mg, 0.57 mmol, 70.0%) as a white powder. Carboxylates 9Ab–e
and 9Ba were synthesized in a similar way.
Compound 9Aa: yield, 70.0%; a white cotton; 1H NMR (CDCl3) d
9.90 (1H, s), 8.29 (1H, s), 7.13 (d, J = 3.4 Hz, 1H), 6.31 (d, J = 3.4 Hz,
1H), 5.86 (s, 1H), 5.25 (s, 2H), 4.49 (q, J = 7.1 Hz, 2H), 4.25 (q,
J = 7.3 Hz, 2H), 2.39 (s, 3H), 2.22 (s, 3H), 1.53 (t, J = 7.3 Hz, 3H),
1.47 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3) d 163.8, 155.1, 153.2,
148.4, 147.0, 139.6, 130.1, 124.7, 121.1, 116.2, 110.5, 105.9, 61.3,
48.5, 45.8, 15.4, 14.5, 13.5, 11.1; HRMS (ESI) m/z: [M+Na]+ calcd
for C19H23N5O4Na: 408.1642, found: 408.1646.
4.1.6. Synthesis of carboxylic acids 10Aa–e and 10Ba
4.1.6.1. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-car-
boxamido)-1-ethyl-1H-pyrazole-3-carboxylic acid (10Aa). 5 N
NaOH aq (311 ll, 1.557 mmol) was added to a solution of compound
9Aa (200 mg, 0.519 mmol) in EtOH (5 ml), and then the mixture
was stirred at rt for 9 h. The reaction mixture was adjusted to a
pH of approx. 3 with 5 N HCl aq on ice. The majority of the solvent
was removed on a rotary evaporator. After the precipitate was
filtrated and washed with H2O, the carboxylic acid 10Aa was obtained
as a white powder (169 mg, 0.47 mmol, 91.1%). Carboxylic acids
10Ab–e and 10Ba were synthesized in a similar way.
4.1.5.2. Ethyl 4-(5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)thio-
phene-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylate
(9Ab).
Purified by column chromatography over silica gel
(hexane/EtOAc). Yield, 74.7%; a white powder; 1H NMR (CDCl3) d
9.74 (s, 1H), 8.27 (s, 1H), 7.49 (d, J = 4.1 Hz, 1H), 6.88 (d,
J = 4.1 Hz, 1H), 5.85 (s, 1H), 5.35 (s, 2H), 4.47 (q, J = 7.2 Hz, 2H),
4.24 (q, J = 7.3 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.52 (t,
J = 7.2 Hz, 3H), 1.45 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3) d 164.1,
158.6, 148.4, 146.1, 139.0, 137.9, 129.8, 128.6, 126.2, 125.2,
121.0, 106.1, 61.4, 48.5, 47.8, 15.4, 14.5, 13.5, 11.1. HRMS (ESI)
m/z: [M+Na]+ calcd for C19H23N5O3SNa: 424.1414, found:
424.1416.
Compound 10Aa: yield, 91.1%; a white powder; 1H NMR
(DMSO-d6) d 13.37 (br s, 1H), 9.86 (s, 1H), 8.35 (s, 1H), 7.19 (d,
J = 3.4 Hz, 1H), 6.58 (d, J = 3.7 Hz, 1H), 5.84 (s, 1H), 5.28 (s, 2H),
4.22 (q, J = 7.2 Hz, 2H), 2.39 (s, 3H), 2.06 (s, 3H), 1.39 (t,
J = 7.3 Hz, 3H); 13C NMR (DMSO-d6) d 165.0, 153.8, 153.8, 146.5,
146.3, 139.2, 130.2, 123.3, 121.3, 116.1, 111.1, 105.1, 47.4, 44.7,
15.2, 13.3, 10.6; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for C17H18N5O4:
356.1364, found: 356.1369.
4.1.5.3. Ethyl 4-(3-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)ben-
zamido)-1-ethyl-1H-pyrazole-3-carboxylate (9Ac).
Purified
by column chromatography over silica gel (hexane/EtOAc). Yield,
93.0%; a pale yellow oil; 1H NMR (CDCl3) d 9.98 (s, 1H), 8.37 (s,
1H), 7.81 (d, J = 7.8 Hz, 1H), 7.69 (s, 1H), 7.44 (dd, J = 7.8, 7.8 Hz,
1H), 7.22 (d, J = 7.6 Hz, 1H), 5.88 (s, 1H), 5.30 (s, 2H), 4.49 (q,
J = 7.1 Hz, 2H), 4.26 (q, J = 7.3 Hz, 2H), 2.26 (s, 3H), 2.18 (s, 3H),
1.54 (t, J = 7.3 Hz, 3H), 1.47 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3) d
4.1.6.2. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)thiophene-
2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylic acid (10Ab). Yield,
93.7%; a white powder; 1H NMR (DMSO-d6) d 13.30 (br s, 1H), 9.84 (s, 1H),
8.26 (s, 1H), 7.55 (d, J = 4.1 Hz, 1H), 7.07 (d, J = 4.1 Hz, 1H), 5.84 (s, 1H), 5.40
(s, 2H), 4.20 (q, J = 7.3 Hz, 2H), 2.21 (s, 3H), 2.09 (s, 3H), 1.37 (t, J = 7.2 Hz,
3H); 13C NMR (DMSO-d6) d 164.7, 157.6, 146.5, 146.3, 138.6, 137.4, 130.9,

1784
Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
128.5, 126.8, 123.4, 121.7, 105.2, 47.3, 46.7, 15.2, 13.2, 10.5; HRMS (ESI) m/
z: [MꢀH]ꢀ calcd for C17H18N5O3S: 372.1136, found: 372.1132.
10.39 (s, 1H), 8.23 (s, 1H), 7.09 (d, J = 3.4 Hz, 1H), 6.82–6.75 (m, 1H),
6.21 (d, J = 3.4 Hz, 1H), 5.85 (s, 1H), 5.25 (s, 2H), 4.15 (q, J = 7.3 Hz,
2H), 3.01 (d, J = 5.1 Hz, 3H), 2.37 (s, 3H), 2.22 (s, 3H), 1.50 (t, J = 7.3 Hz,
3H); 13C NMR (CDCl3) d 164.3, 155.2, 153.4, 148.3, 147.1, 139.7, 132.3,
122.9, 121.3, 115.8, 110.2, 105.8, 48.0, 46.0, 25.4, 15.3, 13.5, 11.1;
HRMS (ESI) m/z: [M+H]+ calcd for C18H23N6O3: 371.1826, found:
371.1812.
4.1.6.3. 4-(3-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)benzami-
do)-1-ethyl-1H-pyrazole-3-carboxylic acid (10Ac). After the
reaction mixture was adjusted to a pH of approx. 3, evaporation
of the solvent gave a white powder that was used without further
purification to use in the next step.
4.1.7.2.
carboxamido)-1-ethyl-N,N-dimethyl-1H-pyrazole-3-carboxam-
ide (11Aab). The reaction mixture was stirred for 5 h at rt.
Yield, 30.2%; a white powder; mp 185.8–186.3 °C; IR (cmꢀ1
3319, 3002, 2955, 1661, 1618, 1603, 1576, 1551, 1533, 1425,
1367, 1213, 1191, 1163, 1136, 1084, 1016, 966; 1H NMR (CDCl3)
d 10.84 (s, 1H), 8.27 (s, 1H), 7.08 (d, J = 3.5 Hz, 1H), 6.25 (d,
J = 3.5 Hz, 1H), 5.84 (s, 1H), 5.24 (s, 2H), 4.17 (q, J = 7.3 Hz, 2H),
3.59 (s, 3H), 3.14 (s, 3H), 2.39 (s, 3H), 2.21 (s, 3H), 1.51 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3) d 164.5, 155.1, 153.1, 148.3,
147.4, 139.8, 133.4, 124.9, 120.5, 115.5, 110.2, 105.8, 47.9, 45.9,
39.0, 36.5, 15.2, 13.5, 11.1; HRMS (ESI) m/z: [M+H]+ calcd for
4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
4.1.6.4. 4-(5-((4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylic acid
(10Ad).
13.37 (br s, 1H), 9.86 (s, 1H), 8.35 (s, 1H), 7.20 (d, J = 3.4 Hz, 1H),
6.66 (d, J = 3.4 Hz, 1H), 5.37 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H), 2.40
(s, 3H), 2.08 (s, 3H), 1.39 (t, J = 7.3 Hz, 3H); 13C NMR (DMSO-d6) d
165.0, 153.8, 152.9, 146.5, 143.6, 135.9, 130.2, 123.3, 121.4,
116.1, 111.6, 106.5, 47.4, 45.8, 15.2, 11.0, 9.1; HRMS (ESI) m/z:
[MꢀH]ꢀ calcd for C17H17N5O4Cl: 390.0974, found: 390.0972.
Yield, 95.4%; a white powder; 1H NMR (DMSO-d6) d
)
4.1.6.5. 4-(5-((4-Bromo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxylic acid
C19H25N6O3: 385.1982, found: 385.1975.
(10Ae).
Yield, 96.8%; a white powder; 1H NMR (DMSO-d6) d
13.38 (br s, 1H), 9.87 (s, 1H), 8.35 (s, 1H), 7.20 (d, J = 3.7 Hz, 1H),
6.66 (d, J = 3.7 Hz, 1H), 5.39 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H), 2.41
(s, 3H), 2.08 (s, 3H), 1.39 (t, J = 7.3 Hz, 3H); 13C NMR (DMSO-d6) d
165.0, 153.8, 152.9, 146.5, 145.2, 137.6, 130.2, 123.3, 121.4,
116.1, 111.6, 93.4, 47.4, 45.9, 15.2, 11.9, 10.0; HRMS (ESI) m/z:
[MꢀH]ꢀ calcd for C17H17BrN5O4: 434.0469, found: 434.0462.
4.1.7.3. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)thiophene-
2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxamide (11Ab). The
reaction mixture was stirred for 13 h at rt. Recrystallized from EtOH.
Yield, 64.4%; a white cotton; mp 230.0–230.2 °C; IR (cmꢀ1) 3522,
3402, 3346, 3009, 2959, 1666, 1583, 1545, 1487, 1420, 1375, 1223,
1213, 1171, 1132, 1022, 960, 901; 1H NMR (CDCl3) d 10.18 (s, 1H),
8.21 (s, 1H), 7.48 (d, J = 3.9 Hz, 1H), 6.87 (d, J = 3.7 Hz, 1H), 6.68 (br s,
1H), 5.84 (s, 1H), 5.34 (s, 3H), 4.16 (q, J = 7.3 Hz, 2H), 2.24 (s, 3H),
2.23 (s, 3H), 1.51 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) d 165.9, 158.7,
148.3, 145.9, 139.0, 138.1, 131.3, 128.5, 126.1, 124.0, 121.3, 106.0,
48.1, 47.8, 15.3, 13.5, 11.1; HRMS (ESI) m/z: [M+H]+ calcd for
4.1.6.6.
4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxamido)-1-ethyl-1H-pyrazole-5-carboxylic acid (10Ba). Yield,
89.7%; a pale yellow powder; 1H NMR (DMSO-d6) d 9.75 (s, 1H), 8.12
(s, 1H), 7.20 (d, J = 3.5 Hz, 1H), 6.58 (d, J = 3.5 Hz, 1H), 5.84 (s, 1H),
5.27 (s, 2H), 4.48 (q, J = 7.2 Hz, 2H), 2.36 (s, 3H), 2.06 (s, 3H), 1.33 (t,
J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) d 161.1, 154.2, 153.8, 146.4, 146.2,
139.1, 129.3, 126.2, 119.4, 116.2, 111.0, 105.1, 47.0, 44.6, 15.5, 13.2,
10.6; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for C17H18N5O4: 356.1364, found:
356.1359.
C17H21N6O2S: 373.1441, found: 373.1441.
4.1.7.4. 4-(3-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)benzami-
do)-1-ethyl-1H-pyrazole-3-carboxamide (11Ac). Purified by
column chromatography over silica gel (hexane/EtOAc). Yield,
19.0% (from 2 steps); a white powder; mp 152.6–153.5 °C; IR
(cmꢀ1) 3522, 3402, 3354, 3018, 1666, 1583, 1539, 1420, 1375,
1329, 1219, 1211; 1H NMR (CDCl3) d 10.41 (s, 1H), 8.31 (d,
J = 1.2 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.42 (t,
J = 7.5 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 6.72 (br s, 1H), 5.87 (s,
1H), 5.41 (br s, 1H), 5.29 (s, 2H), 4.18 (q, J = 7.3 Hz, 2H), 2.25 (s,
3H), 2.17 (s, 3H), 1.52 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3) d 165.9,
163.9, 147.9, 139.3, 138.4, 133.9, 131.6, 130.2, 129.3, 126.1,
125.7, 124.3, 121.4, 105.8, 52.3, 48.1, 15.3, 13.5, 11.2; HRMS (ESI)
m/z: [M+Na]+ calcd for C19H22N6O2Na: 389.1696, found: 389.1681.
4.1.7. Synthesis of carboxamides 1, 11Aa–b, 11Ab–e and 11Ba
To a solution of compound 10Aa (90 mg, 0.252 mmol) and
ammonium chloride (67.4 mg, 1.259 mmol) in DMF (1.5 ml) were
added HATU (115 mg, 0.302 mmol) and Hunig’s base (286 ll,
1.637 mmol). The mixture was stirred for 5 min at 50 °C in a micro-
wave instrument. The reaction mixture was diluted with H2O and
stirred for 0.5 h. The precipitate was filtrated, and then purified by
column chromatography over amino-functionalized silica gel (hex-
ane/EtOAc) to give the carboxamide 1 (76 mg, 0.21 mmol, 84.7%) as
a white powder. Carboxamides 11Aa–b, 11Ab–e and 11Ba were
synthesized in a similar way.
4.1.7.5. 4-(5-((4-Chloro-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxamide
Compound 1: yield, 84.7%; a white powder; mp 209.7–210.2 °C;
IR (cmꢀ1) 3522, 3404, 3346, 3009, 1668, 1605, 1583, 1553, 1420,
1375, 1331, 1236, 1221, 1213, 1138, 1016, 964; 1H NMR (CDCl3)
d 10.27 (s, 1H), 8.25 (s, 1H), 7.11 (d, J = 3.4 Hz, 1H), 6.74 (br s,
1H), 6.29 (d, J = 3.7 Hz, 1H), 5.85 (s, 1H), 5.62 (br s, 1H), 5.24 (s,
2H), 4.16 (q, J = 7.3 Hz, 2H), 2.39 (s, 3H), 2.21 (s, 3H), 1.51 (t,
J = 7.3 Hz, 3H); 13C NMR (CDCl3) d 165.7, 155.2, 153.2, 148.3,
147.1, 139.7, 131.6, 123.4, 121.4, 116.0, 110.5, 105.8, 48.1, 45.8,
15.3, 13.5, 11.1; HRMS (ESI) m/z: [M+H]+ calcd for C17H21N6O3:
357.1669, found: 357.1670.
(11Ad).
Yield, 71.9%; a white powder; mp 220.1–220.8 °C; IR
(cmꢀ1) 3522, 3404, 3344, 2997, 1668, 1605, 1583, 1553, 1477,
1421, 1377, 1331, 1223, 1209, 1140, 1088, 1016, 962; 1H NMR
(CDCl3) d 10.29 (s, 1H), 8.24 (s, 1H), 7.11 (d, J = 3.7 Hz, 1H), 6.70
(br s, 1H), 6.33 (d, J = 3.4 Hz, 1H), 5.44 (br s, 1H), 5.23 (s, 2H),
4.17 (q, J = 7.3 Hz, 2H), 2.40 (s, 3H), 2.20 (s, 3H), 1.51 (t,
J = 7.3 Hz, 3H); 13C NMR (CDCl3) d 165.6, 155.0, 152.4, 147.3,
145.4, 136.0, 131.6, 123.4, 121.4, 115.9, 110.9, 108.5, 48.1, 46.8,
15.3, 11.4, 9.5; HRMS (ESI) m/z: [M+H]+ calcd for C17H20ClN6O3:
391.1280, found: 391.1282.
4.1.7.1. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-carbox-
amido)-1-ethyl-N-methyl-1H-pyrazole-3-carboxamide (11Aaa). The
reaction mixture was stirred for 5 h at rt. Yield, 24.5%; a white powder;
mp 178.9ꢀ179.0 °C; IR (cmꢀ1) 3431, 3335, 3009, 1655, 1584, 1554,
1437, 1373, 1223, 1209, 1173, 1146, 1016, 962; 1H NMR (CDCl3) d
4.1.7.6. 4-(5-((4-Bromo-3,5-dimethyl-1H-pyrazol-1-yl)methyl)-
furan-2-carboxamido)-1-ethyl-1H-pyrazole-3-carboxamide
(11Ae).
Yield, 81.6%; a white powder; mp 229.8–230.2 °C; IR

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1785
(cmꢀ1) 3522, 3404, 3344, 3009, 1668, 1605, 1585, 1553, 1477,
1421, 1377, 1331, 1221, 1209, 1140, 1072, 1016, 964; 1H NMR
(CDCl3) d 10.30 (s, 1H), 8.24 (s, 1H), 7.11 (d, J = 3.4 Hz, 1H), 6.72
(br s, 1H), 6.34 (d, J = 3.7 Hz, 1H), 5.49 (br s, 1H), 5.26 (s, 2H),
4.17 (q, J = 7.4 Hz, 2H), 2.41 (s, 3H), 2.21 (s, 3H), 1.51 (t,
J = 7.3 Hz, 3H); 13C NMR (CDCl3) d 165.6, 155.0, 152.3, 147.3,
146.9, 137.8, 131.6, 123.4, 121.4, 115.9, 110.9, 94.9, 48.1, 46.9,
15.3, 12.3, 10.5; HRMS (ESI) m/z: [M+H]+ calcd for C17H20BrN6O3:
435.0775, found: 435.0783.
EtOH (15 ml). The reaction mixture was purged with H2 gas and
stirred for 1 h at 40 °C. The mixture was filtrated and concentrated.
The residue was subjected to column chromatography over amino-
functionalized silica gel (EtOAc/MeOH) to give the compound 14
(580.9 mg, 2.14 mmol, 70.9%) as a colorless oil.
1H NMR (CDCl3) d 7.16 (1H, s), 5.37 (2H, s), 4.12 (2H, br s), 3.94
(3H, s), 3.55–3.51 (2H, m), 0.92–0.88 (2H, m), ꢀ0.02 (9H, s); 13C
NMR (CDCl3) d 164.1, 135.6, 130.3, 115.8, 81.6, 67.0, 51.7, 17.9,
ꢀ1.4; HRMS (ESI) m/z: [M+Na]+ calcd for C11H21N3O3SiNa:
294.1244, found: 294.1235.
4.1.7.7.
4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxamido)-1-ethyl-1H-pyrazole-5-carboxamide (11Ba). The
reaction mixture was stirred at rt for 5 h. Yield, 47.4%; a white pow-
der; mp 189.9–190.5 °C; IR (cmꢀ1) 3468, 3404, 3292, 2999, 1676,
1604, 1585, 1551, 1474, 1379, 1221, 1209, 1148, 1090, 1016, 970;
1H NMR (CDCl3) d 9.09 (s, 1H), 7.83 (d, J = 1.2 Hz, 1H), 7.13 (dd,
J = 3.5, 1.2 Hz, 1H), 6.42 (br s, 2H), 6.33 (d, J = 3.5 Hz, 1H), 5.85 (s,
1H), 5.20 (s, 2H), 4.45 (q, J = 7.3 Hz, 2H), 2.31 (s, 3H), 2.20 (s, 3H),
1.48 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3) d 161.6, 157.2, 152.9, 148.5,
146.9, 139.3, 133.4, 127.4, 121.3, 116.8, 110.7, 106.0, 47.3, 45.6,
15.6, 13.4, 11.1; HRMS (ESI) m/z: [M+H]+ calcd for C17H21N6O3:
357.1669, found: 357.1665.
4.1.11. Methyl 4-(5-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)
furan-2-carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-pyrazole-3-carboxylate (15)
To a solution of compound 14 (0.58 g, 2.137 mmol) and com-
pound 8a (0.471 g, 2.137 mmol) in DMF (10 ml) were added
HATU (0.97 g, 2.56 mmol) and Hunig’s Base (0.560 ml, 3.21 mmol).
The mixture was stirred for 5 min at 50 °C, 3 times in a microwave
instrument. The reaction mixture was diluted with H2O and satd
NaHCO3 aq and then, the mixture was extracted with EtOAc. The
organic layer was washed with H2O and brine, and dried over
Na2SO4, and evaporated. The residue was subjected to silica gel col-
umn chromatography (hexane/EtOAc) to give the compound 15
(493 mg, 1.04 mmol, 48.7%) as a white powder.
4.1.8. Methyl 4-nitro-1H-pyrazole-3-carboxylate (12)
Acetyl chloride (6.34 ml, 89 mmol) was added to the mixture of
4-nitro-1H-pyrazole-3-carboxylic acid (2, 7 g, 44.6 mmol) in MeOH
(223 ml) at 0 °C. The mixture was stirred at rt for 16 h. After con-
centrated in vacuo, the residue was azeotroped with MeOH two
times. The residue was diluted with MeOH and EtOAc, and the mix-
ture was adjusted to a pH of approx. 9 with satd NaHCO3 aq. The
mixture was extracted with EtOAc. The organic layer was washed
with H2O and brine, dried over Na2SO4, and concentrated to give
the compound 12 (5.545 g, 32.4 mmol, 72.7%) as a white powder.
1H NMR (DMSO-d6) d 14.39 (br s, 1H), 8.99 (s, 1H), 3.89 (s, 3H);
13C NMR (DMSO-d6) d 161.1, 138.1, 133.2, 130.9, 52.8; HRMS (ESI)
m/z: [MꢀH]ꢀ calcd for C5H4N3O4: 170.0207, found: 170.0200.
1H NMR (CDCl3) d 9.86 (s, 1H), 8.46 (s, 1H), 7.14 (d, J = 3.7 Hz,
1H), 6.33 (d, J = 3.7 Hz, 1H), 5.86 (s, 1H), 5.48 (s, 2H), 5.26 (s, 2H),
4.02 (s, 3H), 3.59–3.55 (m, 2H), 2.39 (s, 3H), 2.23 (s, 3H), 0.94–
0.90 (m, 2H), ꢀ0.03 (s, 9H); 13C NMR (CDCl3) d 164.1, 155.1,
153.2, 148.4, 146.9, 139.6, 130.7, 125.2, 121.6, 116.3, 110.6,
105.9, 81.8, 67.3, 52.2, 45.8, 17.8, 13.5, 11.1, ꢀ1.5; HRMS (ESI) m/
z: [M+Na]+ calcd for C22H31N5O5SiNa: 496.1986, found: 496.1989.
4.1.12. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyra-
zole-3-carboxylic acid (16)
An aqueous solution of 5 N NaOH (624 ll, 3.12 mmol) was
added to a solution of compound 15 (493 mg, 1.04 mmol) in
EtOH (5 ml), and then the mixture was stirred at rt for 2 h. The
pH of the reaction mixture was adjusted to approx. 3 with 6 N
HCl on ice. The majority of solvent was removed on a rotary
evaporator. After the precipitate was filtrated, the solid was
washed with H2O to give the compound 16 (455 mg, 0.990 mmol,
95.2%) as a white powder.
4.1.9. Methyl 4-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
pyrazole-3-carboxylate (13)
To a solution of methyl 4-nitro-1H-pyrazole-3-carboxylate (12,
3.0 g, 17.53 mmol) in THF (88 ml) was added NaH (1.052 g,
26.3 mmol) at 0 °C in an Ar atomosphere. After being stirred for
10 min, SEM-Cl (3.73 ml, 21.04 mmol) was added to the reaction
at 0 °C and stirred for another 20 min at rt. The reaction was
quenched with H2O and extracted with EtOAc. The organic layer
was washed with brine and dried over Na2SO4. After filtration
and concentration, the residue was subjected to silica gel column
chromatography to give the compound 13 (2.83 g, 9.39 mmol,
53.6%) as a colorless oil and methyl 4-nitro-1-((2-(trimethylsi-
1H NMR (DMSO-d6) d 9.90 (s, 1H), 8.45 (s, 1H), 7.21 (d, J = 3.4 Hz,
1H), 6.59 (d, J = 3.4 Hz, 1H), 5.84 (s, 1H), 5.49 (s, 2H), 5.28 (s, 2H),
3.57–3.54 (m, 2H), 2.38 (s, 3H), 2.06 (s, 3H), 0.86–0.82 (m, 2H),
ꢀ0.05 (s, 9H); 13C NMR (DMSO-d6) d 171.7, 165.0, 154.0, 153.8,
146.5, 146.2, 139.3, 123.5, 122.2, 116.2, 111.1, 105.1, 80.5, 66.1,
44.7, 17.1, 13.3, 10.7, ꢀ1.4; HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
C21H28N5O5Si: 458.1865, found: 458.1869.
lyl)ethoxy)methyl)-1H-pyrazole-5-carboxylate
130
(1.60 g,
5.31 mmol, 30.3%) as a colorless oil.
Compound 13: 1H NMR (CDCl3) d 8.32 (s, 1H), 5.48 (s, 2H), 4.00
(s, 3H), 3.66–3.62 (m, 2H), 0.97–0.93 (m, 2H), 0.00 (s, 9H); 13C NMR
(CDCl3) d 160.3, 138.4, 135.2, 129.7, 82.1, 68.4, 53.2, 17.8, ꢀ1.5;
HRMS (ESI) m/z: [M+Na]+ calcd for C11H19N3O5SiNa: 324.0986,
found: 324.0982.
4.1.13. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
pyrazole-3-carboxamide (17)
To a solution of compound 16 (90 mg, 0.196 mmol) and ammo-
nium chloride (52.4 mg, 0.979 mmol) in DMF (1 ml) were added
HATU (89 mg, 0.235 mmol) and Hunig’s base (0.222 ml,
1.273 mmol). The mixture was stirred for 5 min at 50 °C in a micro-
wave instrument. The reaction mixture was diluted with H2O. The
precipitate was filtrated and washed with H2O to give the com-
pound 17 (75 mg, 0.16 mmol, 83.5%) as a white powder.
1H NMR (CDCl3) d 10.27 (s, 1H), 8.41 (s, 1H), 7.12 (d, J = 3.4 Hz,
1H), 6.76 (br s, 1H), 6.30 (d, J = 3.4 Hz, 1H), 5.85 (s, 1H), 5.47 (br s,
1H), 5.40 (s, 2H), 5.24 (s, 2H), 3.59–3.54 (m, 2H), 2.40 (d, J = 0.7 Hz,
3H), 2.21 (s, 3H), 0.95–0.91 (m, 2H), ꢀ0.01 (s, 9H); 13C NMR (CDCl3)
d 165.4, 155.2, 153.3, 148.3, 147.0, 139.7, 132.8, 123.9, 122.2,
Compound 130: 1H NMR (CDCl3) d 8.06 (1H, s), 5.61 (2H, s), 4.03
(3H, s), 3.59–3.54 (2H, m), 0.92–0.86 (2H, m), ꢀ0.02 (9H, s); 13C
NMR (CDCl3) d 158.6, 135.3, 135.2, 131.4, 80.8, 67.9, 53.9, 17.6,
ꢀ1.5; HRMS (ESI) m/z: [M+Na]+ calcd for C11H19N3O5SiNa:
324.0986, found: 324.0986.
4.1.10. Methyl 4-amino-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-pyrazole-3-carboxylate (14)
After purging with N2 gas, 10% Pd/C (32 mg, 0.030 mmol) was
added to the solution of compound 13 (913 mg, 3.02 mmol) in

1786
Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
116.1, 110.5, 105.8, 81.2, 67.2, 45.8, 17.7, 13.5, 11.1, ꢀ1.4; HRMS
(ESI) m/z: [M+H]+ calcd for C21H31N6O4Si: 459.2170, found:
459.2151.
4.1.17. 3-Amino-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one
(22)
After being purged with N2 gas, 10% (w/w) Pd/C (3.80 mg,
3.57
0.357 mmol) in EtOH (2 ml). The reaction mixture was purged with
2 gas and stirred at 40 °C for 1.5 h. The mixture was filtrated and
lmol) was added to a solution of compound 21 (65 mg,
4.1.14. 4-(5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)furan-2-
carboxamido)-1H-pyrazole-3-carboxamide (18)
H
To a suspension of compound 17 (40 mg, 0.087 mmol) in EtOH
(174 ll) was added 2 M HCl/EtOH (436 ll, 0.872 mmol). The mix-
concentrated. The residue was subjected to column chromatogra-
phy over amino-functionalized silica gel (EtOAc/MeOH) to give
the compound 22 (46 mg, 0.298 mmol, 83.5%) as a white powder.
1H NMR (DMSO-d6) d 7.75 (1H, s), 6.99 (1H, s), 4.67 (2H, s),
4.10–4.05 (2H, m), 3.54–3.48 (2H, m); 13C NMR (DMSO-d6) d
160.9, 134.9, 126.9, 117.1, 45.8, 39.4; HRMS (ESI) m/z: [M+Na]+ cal-
cd for C6H8N4ONa: 175.0596, found: 175.0589.
ture was stirred at 80 °C for 1.5 h in a microwave instrument.
After the white precipitate was filtrated, the solid was washed with
EtOH. The resultant pellet was suspended in satd NaHCO3 aq, and
stirred for 0.5 h. After the white precipitate was filtrated, the solid
was washed with H2O. The residue was subjected to column chro-
matography over amino-functionalized silica gel (CH2Cl2/MeOH) to
give the compound 18 (15 mg, 0.05 mmol, 52.4%) as a white
powder.
4.1.18. 5-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)-N-(4-oxo-
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-3-yl)furan-2-
carboxamide (23)
Mp 234.6–235.1 °C; IR (cmꢀ1) 3676, 3522, 3447, 3404, 3344,
3007, 1670, 1605, 1583, 1555, 1448, 1420, 1375, 1340, 1300,
1236, 1103, 1016, 968, 943, 881; 1H NMR (DMSO-d6) d 13.26 (s,
1H), 10.49 (s, 1H), 8.24 (s, 1H), 7.81 (s, 1H), 7.58 (s, 1H), 7.14 (d,
J = 3.4 Hz, 1H), 6.57 (d, J = 3.7 Hz, 1H), 5.84 (s, 1H), 5.28 (s, 2H),
2.41 (s, 3H), 2.06 (s, 3H); 13C NMR (DMSO-d6) d 165.7, 153.7,
153.5, 146.5, 146.4, 139.2, 132.6, 121.7, 119.8, 115.6, 110.9,
105.0, 44.6, 13.2, 10.6; HRMS (ESI) m/z: [M+H]+ calcd for
HATU (135 mg, 0.355 mmol) and Hunig’s base (77 ll,
0.444 mmol) were added to a solution of compound 22 (45 mg,
0.296 mmol) and compound 8a (65.1 mg, 0.296 mmol) in DMF
(1.5 ml) at rt. The mixture was stirred at 50 °C for 5 min in a micro-
wave instrument. The mixture was diluted in H2O and satd
NaHCO3 aq, and extracted with EtOAc. The organic layer was
washed with H2O and brine, and dried over Na2SO4, and concen-
trated in vacuo. The residue was subjected to a column chromatog-
raphy over silica gel (hexane/EtOAc) to give the compound 23
(44 mg, 0.123 mmol, 41.6%) as a yellow powder.
C
15H17N6O3: 329.1356, found: 329.1362.
4.1.15. Methyl 1-(((tert-butoxycarbonyl)amino)methyl)-4-
nitro-1H-pyrazole-5-carboxylate (20)
Mp 288.4–289.6 °C; IR (cmꢀ1) 3418, 3254, 2997, 1668, 1610,
1551, 1491, 1429, 1379, 1354, 1335, 1304, 1223, 1209, 1142,
1055, 1016, 970; 1H NMR (DMSO-d6) d 9.56 (s, 1H), 8.46 (s, 1H),
8.04 (s, 1H), 7.20 (d, J = 3.7 Hz, 1H), 6.58 (d, J = 3.7 Hz, 1H), 5.83
(s, 1H), 5.28 (s, 2H), 4.32–4.25 (m, 2H), 3.68–3.61 (m, 2H), 2.39
(s, 3H), 2.06 (s, 3H). 13C NMR (DMSO-d6) d 160.0, 154.1, 153.8,
146.4, 146.1, 139.2, 129.4, 123.0, 120.4, 116.2, 111.0, 105.0, 45.6,
44.6, 13.2, 10.5; HRMS (ESI) m/z: [M+H]+ calcd for C17H19N6O3:
355.1513, found: 355.1501.
To a solution of compound 12 (1.333 g, 7.79 mmol), K2CO3
(1.615 g, 11.68 mmol) and TBAI (0.288 g, 0.779 mmol) in DMF
(39 ml) was added tert-butyl (2-bromoethyl)carbamate (19)
(1.92 g, 8.57 mmol). The mixture was stirred at rt for 2 h, and then
filtrated on Celite. After concentration, the pellet was suspended in
EtOAc and H2O, and extracted with EtOAc. The organic layer was
washed with H2O and brine, dried over Na2SO4, and concentrated
in vacuo. The residue was subjected to column chromatography
over silica gel (hexane/EtOAc) to yield compound 20 (271 mg,
11.1%, 0.863 mmol) as a white powder and methyl 1-(((tert-
butoxycarbonyl)amino)methyl)-4-nitro-1H-pyrazole-3-carboxy-
late 200 (28.6%, 701 mg, 2.228 mmol) as a white powder.
4.1.19. 4-(5-((1H-Pyrazol-1-yl)methyl)furan-2-carboxamido)-1-
ethyl-1H-pyrazole-3-carboxamide (24)
Compound 24 was prepared according to the synthetic proce-
dure in a similar way of CLB-016 (1) starting from a commercially
available 5-((1H-pyrazol-1-yl)methyl)furan-2-carboxylic acid.
Yield: 32.8% in 3 steps as a white powder; mp 199.5–200.3 °C;
IR (cmꢀ1) 3522, 3402, 3344, 3009, 1668, 1607, 1585, 1555, 1420,
1375, 1330, 1217, 1209, 1135, 1088, 1016, 962; 1H NMR (CDCl3)
d 10.33 (s, 1H), 8.24 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.55 (d,
J = 1.7 Hz, 1H), 7.12 (d, J = 3.4 Hz, 1H), 6.74 (br s, 1H), 6.40 (d,
J = 3.4 Hz, 1H), 6.32 (dd, J = 2.1, 2.1 Hz, 1H), 5.58 (s, 1H), 5.40 (s,
2H), 4.17 (q, J = 7.3 Hz, 2H), 1.51 (t, J = 7.3 Hz, 3H). 13C NMR
(CDCl3) d 165.7, 155.1, 152.2, 147.4, 140.0, 131.6, 129.6, 123.4,
121.4, 115.8, 111.1, 106.5, 48.7, 48.1, 15.3; HRMS (ESI) m/z:
[M+H]+ calcd for C15H17N6O3: 329.1356, found: 329.1348.
Compound 20: 1H NMR (CDCl3) d 8.06 (1H, s), 4.85–4.77 (1H,
m), 4.40 (2H, t, J = 5.5 Hz), 4.02 (3H, s), 3.60 (2H, q, J = 5.5 Hz),
1.42 (9H, s); 13C NMR (CDCl3) d 158.8, 155.7, 135.8, 134.7, 131.8,
80.0, 53.9, 52.0, 40.2, 28.3; HRMS (ESI) m/z: [M+Na]+ calcd for
C
12H18N4O6Na: 337.1118, found: 337.1114.
Compound 200: 1H NMR (CDCl3) d 8.18 (1H, s), 4.91–4.81 (1H,
m), 4.39–4.30 (2H, m), 3.99 (3H, s), 3.64–3.56 (2H, m), 1.43 (9H,
s); 13C NMR (CDCl3) d 160.5, 155.9, 138.8, 134.2, 131.0, 80.4,
53.2, 53.2, 40.1, 28.3; HRMS (ESI) m/z: [M+Na]+ calcd for
C12H18N4O6Na: 337.1118, found: 337.1113.
4.1.16. 3-Nitro-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one
(21)
2 M HCl/EtOH (3.6 ml, 7.22 mmol) was added to a solution of
compound 20 (227 mg, 0.722 mmol) in EtOH (1.5 ml) at rt. The
mixture was stirred at 50 °C for 1 h. The precipitate was collected
by filtration as a white powder, and subjected to the next reaction.
The mixture of the white powder and Et3N (0.150 ml, 1.077 mmol)
in toluene (5 ml) was stirred at 100 °C for 1 h. After cooling to room
temperature, H2O was added to the reaction mixture. The pre-
cipitate was filtered, and dried in vacuo to give the compound 21
(60 mg, 0.329 mmol, 45.6%) as a white powder.
4.2. Biology
4.2.1. Cell-based HRE-driven luciferase reporter assay
x5HRE/HT1080 cells, stable transformant cell lines with a
5xHRE/pGL3/VEGF/E1b reporter plasmid,12 were cultured in
Dulbecco’s modified Eagle’s medium containing 10% fetal bovine
serum at 37 °C in a humidified 5% CO2 incubator. For reporter
assay, cells (1.0 ꢁ 104) were seeded in 96-well plates (in 100
ll)
and pre-incubated for 24 h. After 1 h from the addition of chemical
compounds, the cells were incubated under hypoxia (1% O2) for
24 h. The cells were washed with ice-cold phosphate-buffered sal-
1H NMR (DMSO-d6) d 8.71 (1H, br s), 8.35 (1H, s), 4.41–4.38 (2H,
m), 3.67–3.61 (2H, m); 13C NMR (DMSO-d6) d 154.6, 136.3, 134.0,
129.4, 47.3, 38.4; HRMS (ESI) m/z: [M+Na]+ calcd for
C6H6N4O3Na: 205.0332, found: 205.0333.
ine (PBS) 3 times, followed by lysis in 30
Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA and 0.1% Triton X-100).
ll of lysis buffer (20 mM

Y. Yasuda et al. / Bioorg. Med. Chem. 23 (2015) 1776–1787
1787
Twenty-microliter of the cellular lysate was employed for the
luciferin-luciferase assay (50 mM Tris pH 8.0, 5 mM MgSO4,
0.125 mM D-luciferin, 0.5 mM ATP, and 0.5 mM CoA).
Epiluminescence was measured using an EnVision luminometer
(Perkin Elmer). Each compound was tested in triplicate. To calcu-
late the relative luciferase activity, the luminescence units were
normalized with total protein content in each well.
24 h incubation under hypoxia. Images of scratch-wound were
acquired using phase contrast microscopy.
Acknowledgements
We thank Drs. H. Osada (RIKEN) and M. Hiraoka (Kyoto
University) for providing x5HRE/HT1080 cells and a 5xHRE/pGL3/
VEGF/E1b reporter plasmid, respectively. We are also grateful to
Mrs. M. Morioka and E. Moriyoshi (Kyoto University) for support-
ing the HIF-1 inhibitor screening. This work was supported in part
by research grants from the Japan Society for the Promotion of
Science (JSPS), the Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT), the Ministry of Health, Labour
and Welfare of Japan (MHLW).
4.2.2. Cell proliferation assay
HT1080 cells (1.0 ꢁ 103 cells/well) were seeded in a 96-well
plate (in 100 ll) and cultured under hypoxia (1% O2). After 72 h
from the addition of test compounds, cell growth was evaluated
by using the WST-8 cell counting kit (Dojindo) according to
manufacturer’s instructions. Absorbance was measured at
450 nm in a microplate reader (Bio-rad).
Supplementary data
4.2.3. RNA extraction and quantitative real-time PCR
TotalRNAofHT1080cells wasisolatedusingTotalRNAExtraction
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.02.038.
kit (Viogene). cDNA was reverse transcribed from 0.5
lg of the total
RNA using ReverTra Ace (TOYOBO) using a random primer. For the
a
quantitative real-time PCR, DNA amplification was carried out using
the Fast Start Universal SYBR Green Master (ROX) (Roche). Primers
used for PCR were as follows: human CA-IX, 50-ACCCTC
TCTGACACCCTGTG-30 (sense) and 50-GGCTGGCTTCTCACATTCTC-30
References and notes
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a,
50-TACTAGCTTTGCAGAATGCTC-30
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4.2.4. Wound-healing assay
HT1080 cells were plated at 7.5 ꢁ 104 cells per well in a 6-well
plate. When the cells were confluent, a monolayer of the cells was
scratched using a sterile 200 ll pipette tip. After replacing the
medium with fresh medium to remove cell debris, vehicle or the
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test compound was added to the cells, followed by an additional