Design, synthesis, and target identification of new hypoxia-inducible factor 1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety

Article abstract of DOI:10.1016/j.bmc.2021.116375

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure–activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC₅₀ = 1.2 μM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic β subunit of mitochondrial F_oF₁-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via F_oF₁-ATP synthase suppression.

Full text of DOI:10.1016/j.bmc.2021.116375

Bioorg. Med. Chem. 46 (2021) 116375
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and target identification of new hypoxia-inducible factor
1 (HIF-1) inhibitors containing 1-alkyl-1H-pyrazole-3-carboxamide moiety
Marina Sakaia, Nobuaki Takahashia, Hiroaki Ikedaa, Yutaka Furutanib, Shoko Higuchib,
Takehiro Suzukic, Naoshi Dohmaec, Sayaka Kobayashia, Hiroshi Haradad, Soichi Kojimab,
,
,*
Tomokazu Matsuurab e, Akira Hattoria, Hideaki Kakeyaa
a Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto
University, Kyoto 606-8501, Japan
b Liver Cancer Prevention Research Unit, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan
c Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Saitama 351-0198, Japan
d Laboratory of Cancer Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan
e Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo 105-8471, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program
aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-
5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an
extensive structure–activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037
Hypoxia-inducible factor (HIF)
Cancer
Chemotherapy
Structure–activity relationship
Target identification
(IC50 = 1.2 μM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1)
mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9)
and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional
probes, ATP5B, a catalytic β subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of
KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-
carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase
suppression.
1. Introduction
01759, and CAY105852,3 and the natural products echinomycin and
oligomycin.4,5 For example, YC-1, the most widely used HIF-1 inhibitor,
Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tumor
survival and malignancy.1 The transcriptional activity of HIF-1, a het-
suppresses HIF-1-mediated transcription under hypoxic conditions,
suppresses tumor metastasis and prevents the incidence of post-
irradiation tumor recurrence.6 IACS-01759 and oligomycin inhibit
HIF-1 activity by targeting mitochondrial respiratory complex I and
complex V, respectively.
erodimer comprising HIF-1
α and HIF-1β, depends on HIF-1α, which
degrades in an O2 environment. HIF-1 binds to hypoxia-responsive el-
ements (HRE) to regulate the expression of >800 genes, including car-
bonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF)
genes, leading to diverse phenotypes for adaptation to the tumor
microenvironment; metabolic modulation, angiogenesis, invasion, and
Thus far, we have identified microbial metabolite verucopeptin7 and
synthetic 1-ethylpyrazole-3-carboxamides8 as promising lead molecules
for the inhibition of HIF-1 activity.9 During the ongoing screening pro-
gram aimed at identifying new HIF-1 inhibitors by using a hypoxia-
responsive luciferase reporter gene assay, KUSC-5001 containing the
1-alkyl-1H-pyrazole-3-carboxamide moiety was found as an appropriate
metastasis.1 Therefore, HIF-1
α is one of the most promising targets for
cancer chemotherapy. Inhibitors affecting the HIF-1 signaling pathway
have been identified; they include the synthetic molecules YC-1, IACS-
Abbreviations: HIF-1, hypoxia-inducible factor 1; Boc, t-butoxycarbonyl; CA, carbonic anhydrase; DIPEA, N,N-diisopropylethylamine; SAR, structure-activity
relationship; HRE, hypoxia-response element; VEGF, vascular endothelial growth factor; TBAI, tetrabutylammonium iodide; HATU, 1-(Bis(dimethylamino)methy-
lene)-1H-1,2,3-triazolo(4,5-b)pyridinium 3-oxide hexafluorophosphate.
* Corresponding author.
E-mail address: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya).
https://doi.org/10.1016/j.bmc.2021.116375
Received 25 July 2021; Received in revised form 13 August 2021; Accepted 13 August 2021
Available online 30 August 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
hit molecule from our in-house chemical library. Herein, we report our
finding on the structure–activity relationship (SAR) of KUSC-5001 and
its derivatives and the HIF-1 inhibitory activity (e.g. potency, target
protein, and mode of action) of a more effective HIF-1 inhibitor KUSC-
5037.
carboxylate S1 was reduced by catalytic hydrogenation using 10% Pd/C
to produce amine, which was simultaneously protected by (Boc)2O to
yield the N-Boc protected carbamide S10 in 87% yield. The obtained
carbamide was hydrolyzed with NaOH to generate carboxylic acid S11,
which was condensed with p-bromoaniline to yield the amide S12 in
87% yield over two steps. The N-Boc-protected amide S12 was depro-
tected with 4 M HCl in dioxane to generate the amine hydrochloride
KUSC-5017 in 73% yield, which was acetylated by acetyl chloride to
yield the corresponding analogue KUSC-5025 in 75% yield.
2. Results and discussion
2.1. Design and synthesis of KUSC-5001 and its derivatives
The synthesis route of KUSC-5035 ~ KUSC-5040 is shown in
Scheme 3A. KUSC-5035 ~ KUSC-5040 were synthesized following the
procedure for KUSC-5017 and KUSC-5025, but p-trifluoromethylaniline
was used instead of p-bromoaniline. In addition, KUSC-5035 was acyl-
ated by various acyl chlorides in the final step to yield the corresponding
analogues KUSC-5036 ~ KUSC-5040. The synthesis schemes of KUSC-
5046, KUSC-5049, and KUSC-5056 are shown in Scheme S2.
KUSC-5001 with the 1-alkyl-1H-pyrazole-3-carboxamide moiety
was identified from our in-house chemical library found as a hit mole-
cule for the inhibition of HIF-1 activity. An hypoxia-responsive element
(HRE)-driven luciferase assay using HeLa/HRE/Luc cells revealed that
KUSC-5001 inhibited HIF-1 activity with an IC50 of 18 μM.
We synthesized KUSC-5001 and its derivatives by conjugating the
substituted aniline (left segment), 1-(bromomethyl)-1H-pyrazole-3-
carboxylate (middle segment) and 4-nitro-1H-pyrazole (right segment).
Substitutions of p-bromoaniline (region 1) and 4-nitro-1H-pyrazole
(region 2) were conducted and the 1H-pyrazole ring in the middle
segment was replaced by other aromatic rings (region 3).
Finally, BODIPY-FL NHS ester was activated with DIPEA and
condensed with KUSC-5035 to yield the fluorescent probe KUSC-5054.
Moreover, KUSC-5035 was condensed with 3-(3-(pent-4-yn-1-yl)–3H-
diazirin-3-yl)propanoic acid S15 to produce the bifunctional probe
KUSC-5051 (Schemes 3C and 3D).
The synthesis route of the KUSC-5001 analogues is shown in Scheme
1. First, 1-(bromomethyl)-1H-pyrazole-3-carboxylate was synthesized as
reported.10 Commercially available 4-nitro-1H-pyrazole was reacted
with an excess of 1-(bromomethyl)-3-carboxylate in the presence of
potassium carbonate in DMF to produce the conjugated ester S1 in 96%
yield. The ester was hydrolyzed with NaOH to generate the carboxylic
acid S2 in 61% yield, which was condensed with various aniline ana-
logues to yield the corresponding amides KUSC-5001 ~ KUSC-5008,
KUSC-5010 ~ KUSC-5013, KUSC-5027, and KUSC-5028. The synthesis
route of KUSC-5015 and KUSC-5021 is shown in Scheme S1.
2.2. Structure-activity relationship of KUSC-5001 and its derivatives
The HIF-1 inhibitory activity of the synthetic derivatives was
examined using HeLa cells stably-expressing x5HRE-luciferase re-
porter.11 Camptothecin, a well-known topoisomerase I inhibitor, was
used as a positive control. As mentioned above, KUSC-5001 showed
HIF-1 inhibitory activity with an IC50 of 18 μM. In contrast, KUSC-5015
and KUSC-5021 (Scheme 1B) lacking region 2 and region 3, respec-
tively, did not show any HIF-1 inhibitory activity. Therefore, the three
aromatic groups of regions 1–3 significantly affect the inhibitory activity
of the synthesis derivatives.
KUSC-5014, KUSC-5019, KUSC-5020, and KUSC-5029 were syn-
thesized following the procedure for KUSC-5001, but 4-nitro-1H-pyr-
azole was replaced with 1H-imidazole, 1H-pyrrole, 4-nitro-1H-
imidazole and 3-nitro-1H-pyrrole, respectively (Scheme 2A). The syn-
thesis route of KUSC-5017 and KUSC-5025 is shown in Scheme 2B. The
nitro group of ethyl 1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyrazole-3-
We then evaluated the effect of aromatic substitution in region 1
(Table 1) on the HIF-1 inhibitory activity of the synthetic derivatives.
KUSC-5002, KUSC-5003, and KUSC-5008 exhibited lower HIF-1
inhibitory activity than KUSC-5001, indicating that the p-substitution
Scheme 1. A) Synthesis of KUSC-5001 ~ KUSC-5008, KUSC-5010 ~ KUSC-5013, KUSC-5027, and KUSC-5028. B) Structures of KUSC-5001, KUSC-5015, and
KUSC-5021.
2

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
Scheme 2. A) Synthesis of KUSC-5014, KUSC-5019, KUSC-5020, and KUSC-5029. B) Synthesis of KUSC-5017 and KUSC-5025.
on the phenyl group was the most effective at improving inhibitory
activity. Further derivatives were synthesized and evaluated in accor-
dance with the Topliss Tree.12 The inhibitory activities of KUSC-5004
containing p-CF3 and KUSC-5001 were almost the same, whereas that of
KUSC-5006 containing m-CF3 was lower. Therefore, p-substitution using
electron-withdrawing groups (KUSC-5001, KUSC-5004, KUSC-5005,
and KUSC-5007), not electron-donating groups (KUSC-5010 and KUSC-
5011), improved the HIF-1 inhibitory activity of the synthetic
derivatives.
inhibitory activity, indicating the structural tolerance for these sub-
stituents. Among them, KUSC-5037 exhibited the most effective HIF-1
inhibitory activity with an IC50 of 1.2 μM (Fig. S1), which was compa-
rable to that of KUSC-5056 containing alkylamine moiety. Encouraged
by these results, we designed and synthesized a fluorescent probe KUSC-
5054 containing the BODIPY-FL functional group and a photoaffinity
probe KUSC-5051 containing the diazirine and alkyne functional
groups, which were attached at the C-4 position on the pyrazole ring
(Schemes 3C and 3D).13–15 Finally, we replaced the pyrazole ring in
region 3 of KUSC-5037 with phenyl (KUSC-5046) and furan ring
(KUSC-5049), both of which reduced the inhibitory activity of KUSC-
5037, indicating the significance of the pyrazole ring in region 3.
We next attempted to optimize the 4-nitro-1H-pyrazole moiety in the
region 2 (Table 2). The inhibitory activity of KUSC-5019 containing an
unsubstituted pyrazole ring was ca. 3.5 times less than that of KUSC-
5001. A comparison of the effects of 1H-pyrazole, 1H-imidazole, and
1H-pyrrole (KUSC-5001, KUSC-5020, and KUSC-5029) revealed that
the pyrazole ring (KUSC-5001) was the most effective at enhancing
inhibitory activity. In addition, the nitro group on the pyrazole ring was
replaced by amine (KUSC-5017) and the N-acyl group (KUSC-5025).
Compared with KUSC-5017, the inhibitory activity of KUSC-5025 was
enhanced, which was attributed to the effect of the hydrophobic N-acyl
group on membrane permeability.
2.3. Biological activity of an effective HIF-1 inhibitor KUSC-5037
We evaluated the effectiveness of KUSC-5037 at inhibiting HIF-1
transcriptional activity by measuring the endogenous mRNA levels of
HIF-1 target genes, specifically the carbonic anhydrase 9 (CA9) and
vascular endothelial growth factor (VEGF) genes. Camptothecin was also
used as a positive control. As shown in Fig. 1A, quantitative RT-PCR
analysis revealed that KUSC-5037 suppressed CA9 and VEGF expres-
Based on the optimization results of region 1, as shown in Table 1, we
proceeded to evaluate KUSC-5036 containing p-CF3 on the phenyl group
in region 1 and 4-N-acetyl pyrazole in region 2, respectively, which was
three times more potent than KUSC-5001 in terms of HIF-1 inhibitory
activity. We replaced the acetyl group with a set of acyl groups, namely
propionyl, butyryl, benzyl, and i-propionyl groups (denoted as KUSC-
5037, KUSC-5038, KUSC-5039, and KUSC-5040, respectively)
(Table 3). These four molecules showed almost the same HIF-1
sion with comparable to its HRE-Luc inhibition. Moreover, HIF-1
α
transcription was also suppressed by KUSC-5037 under hypoxic con-
ditions, which in turn significantly suppressed hypoxia-induced CA9 and
VEGF expression. We then examined the effect of KUSC-5037 on the
HIF-1
under hypoxic conditions, the HIF-1
dependent manner, whereas the -tubulin protein level was not affected
α protein level. When the cells were treated with KUSC-5037
α
protein level decreased in a dose-
α
3

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
Scheme 3. A) Synthesis of KUSC-5035 ~ KUSC-5040. B) Structures of KUSC-5046 and KUSC-5049. C) Synthesis of a fluorescence probe KUSC-5054. D) Synthesis of
a bifunctional probe KUSC-5051 for pull-down of binding protein (s) of KUSC-5037.
(Fig. 1B).
binding protein of KUSC-5051 (Fig. 2A), which was strongly supported
by the competition with an excess of KUSC-5037. LC-MS/MS analysis
indicated that the 60 kDa protein was the ATP5B protein (a FoF1-ATP
synthase subunit beta) (Table S1).16 To confirm this, western blotting
was performed using an anti-ATP5B antibody and the 60 kDa protein
was determined as the ATP5B protein, as shown in Fig. 2B. Because FoF1-
ATP synthase is localized in the mitochondria membrane, it is plausible
that KUSC-5037 targets ATP5B in the cells. Furthermore, we tested the
effect of KUSC-5037 on the enzymatic activity of FoF1-ATP synthase
using the Complex V activity assay kit. As shown in Fig. 2C, KUSC-5037
significantly inhibited the FoF1-ATP synthase as oligomycin did, pre-
sumably by binding to ATP5B.
To determine its mode of action, we investigated the subcellular
localization of KUSC-5037. Our SAR study indicated that a few N-acyl
chains, as substituents in region 2, did not significantly affect the HIF-1
inhibitory activity of the synthetic derivatives. Therefore, we designed
and synthesized
a fluorescent probe KUSC-5054 containing the
BODIPY-FL functional group. After treating the HeLa cells with KUSC-
5054, mitochondria and nuclei were stained by MitoTracker Red
CMXRos and Hoechst 33342, respectively. As shown in Fig. 1C, KUSC-
5054 fluorescence merged with the MitoTracker Red CMXRos stains,
indicating that KUSC-5054 was localized in mitochondria.
Next, we attempted to identify the binding protein(s) of KUSC-5037
by using a bifunctional fishing probe KUSC-5051 containing the dia-
zirine and alkyne functional groups. The diazirine and alkyne functional
groups were introduced to facilitate a photoaffinity labeling and pull-
down by click-chemistry, respectively. HeLa cell lysates were fraction-
ated into mitochondria, cytoplasm, and debris. The molecular probe
KUSC-5051 was irradiated at 365 nm in the absence or presence of
KUSC-5037 and then incubated with each fractionated lysate (mito-
chondria, cytoplasm, or debris). Results of the pull-down experiments
suggested that the ca. 60 kDa protein in the mitochondria fraction was a
Finally, we investigated the effect of KUSC-5037 on HeLa cells using
the XF mito-stress test.17 As shown in Fig. 2D, KUSC-5037 inhibited
mitochondrial respiratory complex V with similar potency of a FoF1-ATP
synthase inhibitor oligomycin inhibition.5,18 The oxygen consumption
rate (OCR) decreased after KUSC-5037 (0.1, 1.0 μM) was injected, and
the OCR level immediately recovered after an uncoupled reagent FCCP
was subsequently injected. Furthermore, a final injection of rotenone/
antimycin A reduced the OCR level, suggesting that KUSC-5037 func-
tioned as an inhibitor of the mitochondrial respiratory complex V.
4

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
Table 1
Table 3
Effects of KUSC-5001 derivatives (region 1) on HIF-1 transcriptional activity.
HeLa/HRE/Luc cells were treated with solvent or the test compounds for 1 h
under normoxic conditions (21% O2) and further incubated under hypoxic
conditions (1% O2) for 24 h. Luciferase activity was measured using an EnVision
Effects of KUSC-5036 derivatives on HIF-1 transcriptional activity. HeLa/HRE/
Luc cells were treated with solvent or the test compounds for 1 h under normoxic
conditions (21% O2) and further incubated under hypoxic conditions (1% O2)
for 24 h. Luciferase activity was measured using an EnVision luminometer (n =
3). Representative data from two independent experiments are
luminometer (n = 3). One μM of camptothecin exhibited the 62.3% inhibitory
activity. Representative data from two independent experiments are
shown.
shown.
KUSC-
R
IC50
18
(μM)
KUSC-
R
IC50 (μM)
KUSC-
R
IC50
5.5
(μM)
5001
5008
>100
5036
5002
5003
45
35
5010
5011
21
5037
5038
5039
1.2
1.8
1.4
>100
5004
18
5012
13
5040
5056
6.9
1.2
5005
5006
28
33
5013
5027
>100
35
5007
20
5028
81
Rotenone and antimycin A are inhibitors of mitochondrial respiratory
complex I and complex III, respectively.17,18 In addition, OCR reduction
by KUSC-5037 elevated the intracellular local oxygen concentration,
which likely suppressed the HIF-1α level. Furthermore, we investigated
Table 2
the effect of KUSC-5037 on the OCR level of cells treated with oligo-
mycin as shown in Fig. S2. After the addition of KUSC-5037, the OCR
level recovered by FCCP was significantly reduced in the presence of
oligomycin, suggesting that the mode of action of KUSC-5037 slightly
differs from that of oligomycin. Furthermore, KUSC-5037 exhibited cell
Effects of KUSC-5001 derivatives (region 2) on HIF-1 transcriptional activity.
HeLa/HRE/Luc cells were treated with solvent or the test compounds for 1 h
under normoxic conditions (21% O2) and further incubated under hypoxic
condition (1% O2) for 24 h. Luciferase activity was measured using an EnVision
luminometer (n = 3). Representative data from two independent experiments
growth inhibitory activity with IC50 values of 34.5
μM and 0.6 μM
against human cervical cancer HeLa S3 cells and fibrosarcoma HT1080
cells, respectively, as shown Fig. 2E, while those of oligomycin were 5.3
are shown.
μM and < 0.1
μM (data not shown), respectively.
KUSC-
R
IC50
18
(μM)
3. Conclusion
5001
In the present study, we identified KUSC-5001 containing the 1-
alkyl-1H-pyrazole-3-carboxamide moiety as a potential HIF-1 inhibitor
and developed a more effective derivative KUSC-5037. Our extensive
SAR study revealed that: i) p-substitution of the phenyl group in region 1
using electron-withdrawing groups, not electron-donating groups
improved HIF-1 inhibitory activity; ii) the hydrophobic N-acyl group at
C-4 on the pyrazole ring in the region 3 enhanced inhibitory activity by
facilitating membrane permeability; and iii) the pyrazole ring in the
region 3 played a key role in HIF-1 inhibitory activity. KUSC-5037
5019
5020
66
58
5014
34
5029
5017
5025
27
20
14
blocked the transcription of HIF-1
α gene. Based on the SAR study, a
fluorescent probe KUSC-5054 containing the BODIPY-FL functional
group and a bifunctional probe KUSC-5051 containing the diazirine and
alkyne functional groups were designed and synthesized, both of which
indicated that KUSC-5037 targeted the ATP5B protein, a catalytic β
subunit of FoF1-ATP synthase. Oligomycin also inhibits the HIF-1
signaling pathway via the inhibition of a target protein FoF1-ATP syn-
thase.5 FoF1-ATP synthase plays a key role in the energy production of
cancer cells, which is essential for cell survival.15,19,20 The scaffold of
KUSC-5037 is different from those of the reported natural and synthetic
FoF1-ATP synthase inhibitors.20 Recently, a new synthetic small mole-
cule Gboxin was reported to exhibit antitumor activity especially against
5

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
Fig. 1. Effects of KUSC-5037 on transcriptional activity of HIF-1 and HIF-1
α
protein level.
M, hatched column), or KUSC-5037 (3 μM, black column) for 1 h under normoxic
A) Cells were treated with solvent (blank column), camptothecin (CPT, 0.5
μ
conditions (21% O2), and further incubated under hypoxic conditions (1% O2) for 4 h. RNA was then collected and subjected to qRT-PCR to quantify the mRNA
levels of the indicated genes (n = 2). Representative data from three independent experiments are shown. *P < 0.05 (Student’s t-test).
B) Cells were treated with solvent, camptothecin (CPT, 0.5 μM), or KUSC-5037 (0.3, 1, 3 μM) for 1 h under normoxic conditions (21% O2), and further incubated
under hypoxic conditions (1% O2) for 25 h. The cells were lysed and subjected to western blotting using antibodies against HIF-1
data from three independent experiments are shown.
α and α-tubulin Representative
C) Cells were treated with 1
μM KUSC-5054 for 3 h. MitoTracker Red CMXRos (100 nM) and Hoechst 33342 (10 μM) were then added, and the cells were fixed and
observed using a fluorescence microscope. Scale bar, 10
μ
m. Representative data from three independent experiments are shown.
glioblastoma by inhibiting of FoF1-ATP synthase activity.21 Our experi-
ments on the HeLa cells using the XF mito-stress test also revealed that
KUSC-5037 inhibited mitochondrial respiratory complex V, although its
mode of action was slightly different from that of oligomycin. While the
link between FoF1-ATP synthase activity and HIF-1 activity remains
unclear, KUSC-5037 can be a promising lead compound for the devel-
opment of new antitumor drugs.
4.1.2. Synthesis of KUSC-5001 ~ KUSC-5008, KUSC-5010 ~ KUSC-
5013, KUSC-5027, and KUSC-5028
4.1.2.1. Ethyl 1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyrazole-3-carbox-
ylate (S1). To a stirred solution of 4-nitro-1H-pyrazole (100.2 mg,
0.836 mmol) in DMF (0.49 mL) were added ethyl 1-(bromomethyl)-3-
carboxylate (227.1 mg, 0.974 mmol) and potassium carbonate (244.9
mg, 1.77 mmol) at room temperature. After being stirred for 5.3 h, the
reaction was quenched with water and the layers were separated. The
aqueous layer was extracted with CHCl3, and the combined organic
layers were washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by SiO2 flash column chromatography
(CHCl3/MeOH = 20:1) to afford the carboxylate S1 as a colorless solid
(225.1 mg, 96%).
4. Experimental section
4.1. Chemistry
4.1.1. General
1H and 13C NMR spectra were recorded on a JEOL ECA 500 (500 MHz
for 1H NMR and 125 MHz for 13C NMR), or a JEOL ECZ600 (600 MHz for
1H NMR and 150 MHz for 13C NMR) spectrometer. Chemical shifts are
denoted in δ (ppm) relative to residual solvent peaks as internal standard
(CDCl3, 1H δ 7.26, 13C δ 77.16; CD3OD, 1H δ 3.31, 13C δ 49.00; (CD3)2CO
1H δ 2.05, 13C 29.84; DMSO‑d6 1H δ 2.50, 13C 39.52). ESI-MS and LC-MS
experiments were recorded on a Shimadzu LCMS-IT-TOF. High perfor-
mance liquid chromatography (HPLC) experiments were performed
with SHIMADZU HPLC system equipped with LC-20AD intelligent
pump. All reactions sensitive to air and/or moisture were conducted
under nitrogen atmosphere using dry, freshly distilled solvents, unless
otherwise noted. All reagents were used as supplied unless otherwise
stated. Analytical thin-layer chromatography (TLC) was performed
using E. Merck Silica gel 60 F254 pre-coated plates. Column chroma-
1H NMR (500 MHz, DMSO‑d6): δ1.27 (t, J = 7.0 Hz, 3H), 4.26 (q, J =
7.0 Hz, 2H), 6.59 (s, 2H), 6.81 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 2.0 Hz,
1H), 8.18 (d, J = 3.0 Hz, 1H), 8.35 (s, 1H), 9.21 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 14.17, 60.50, 65.35, 109.16,
131.42, 133.40, 135.61, 136.82, 144.41, 161.29.
HRMS (ESI) m/z: [M + Na]+ calcd for C10H11N5O4Na+ 288.0703;
found, 288.0725.
4.1.2.2. 1-((4-Nitro-1H-pyrazol-1-yl)methyl)-1H-pyrazole-3-carboxylic
acid (S2). To a stirred solution of the carboxylate S1 (53.0 mg, 0.20
mmol) in MeOH (3.0 mL) and water (1.0 mL), was added sodium hy-
droxide (11.2 mg, 0.28 mmol) at room temperature. After being stirred
reflux for 18 h, the mixture was then cooled to room temperature. After
the reaction solution was acidified with conc. HCl at pH 2, CHCl3 was
added and the layers were separated. The aqueous layer was extracted
with CHCl3, and the combined organic layers were dried over Na2SO4,
and concentrated in vacuo to afford the carboxylic acid S2 as a colorless
tography was performed using 40–50
μm Silica Gel 60 N (Kanto
Chemical Co., Inc.).
6

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
Fig. 2. Target identification of KUSC-5037 using a bifunctional probe KUSC-5051 and effects of KUSC-5037 on mitochondrial oxygen consumption rate.
A) Subcellular fractioned HeLa S3 lysates were incubated with KUSC-5051 in the presence or absence of a competitor KUSC-5037. The mixtures were photo-cross-
linked and subjected to click chemistry with biotin-azide followed by NeutrAvidin enrichment. Binding proteins were eluted by boiling in SDS-loading buffer,
subjected to SDS-PAGE, and detected with silver stain followed by tryptic digestion and LC-MS/MS analysis. Arrowhead marks an ATP5B protein band, the binding
protein of KUSC-5051. Representative data from three independent experiments are shown.
B) Binding protein of KUSC-5051 prepared in Fig. 2A was subjected to western blotting using an antibody against ATP5B. Representative data from three independent
experiments are shown.
C) Mitochondrial complex V activity was measured by using a bovine heart mitochondria solution. Absorbance at 340 nm was measured with a spectrophotometer
after the solution was incubated for 2 h in the presence of DMSO (1.0%), oligomycin (12.6 μM), and KUSC-5037 (10, 100 nM) (n = 3). Representative data from
three independent experiments are shown. *P < 0.05 (Student’s t-test).
D) Cells were seeded in Seahorse microplates and OCR was measured. Cells were treated with test substance (oligomycin (0.5
μ
M) or KUSC-5037 (0.1, 1, 10
μM)).
After injections of FCCP (0.5
Representative data from three independent experiments are shown.
μM) and rotenone/antimycin A (AA) (0.5 μM each), OCR was measured using a Seahorse XF analyzer (n = 6, mean ± SEM).
E) HeLa S3 cells (circle) and HT1080 cells (square) were treated with solvent or serial dilutions of KUSC-5037, respectively, and incubated for 72 h. Cell viability was
measured using Cell Counting Kit-8 (n = 3). Representative data from two independent experiments are shown.
7

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
solid (37.8 mg, 61%).
1H), 7.11 (t, J = 8.5 Hz, 2H), 7.81 (t, J = 8.0 Hz, 2H), 8.15 (d, J = 2.5 Hz,
1H), 8.21 (s, 1H), 8.94 (s, 1H), 9.48 (br, 1H).
1H NMR (500 MHz, DMSO‑d6): δ 6.57 (s, 2H), 6.75 (d, J = 2.0 Hz,
1H), 8.15 (d, J = 2.5 Hz, 1H), 8.35 (s, 1H), 9.19 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 65.36, 109.15, 131.41, 133.19,
135.61, 136.79, 145.36, 162.76.
13C NMR (125 MHz, Acetone‑d6): δ 66.94, 108.44, 115.99, 122.63,
131.24, 134.10, 135.86, 137.18, 149.56, 158.88, 160.07, 160.79.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11FN6O3Na+ 353.0769;
found, 353.0774.
HRMS (ESI) m/z: [M + H]+ calcd for C8H8N5O4+ 238.0571; found,
238.0536.
4.1.2.8. 1-((4-Nitro-1H-pyrazol-1-yl)methyl)-N-(3-(trifluoromethyl)
phenyl)-1H-pyrazole-3-carboxamide (KUSC-5006). A colorless solid.
1H NMR (500 MHz, CD3OD): δ 6.57 (s, 2H), 6.90 (d, J = 1.0 Hz, 1H),
7.42 (d, J = 8.5 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz,
1H), 8.04 (s, 1H), 8.16 (s, 1H), 8.20 (s, 1H), 8.92 (br, 1H).
13C NMR (125 MHz, CD3OD): δ 66.96, 108.90, 118.11, 121.69,
124.45, 124.83, 126.60, 130.74, 131.60, 132.15, 134.30, 137.76,
140.28, 149.42, 162.08.
4.1.2.3. N-(4-Bromophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5001). To a stirred solution of the carbox-
ylic acid S2 (10.0 mg, 0.042 mmol) in dichloromethane (0.57 mL) were
added DIPEA (15.0 μL, 0.086 mmol) and HATU (16.0 mg, 0.042 mmol)
at room temperature. After being stirred for 30 min, p-bromoaniline
(8.0 mg, 0.047 mmol) was added, and the mixture was stirred at room
temperature for 22 h. The reaction was then quenched with water and
the layers were separated. The aqueous layer was extracted with CHCl3
and the combined organic layers were washed with 1 M HCl and sat.
NaHCO3 aq., dried over Na2SO4, and concentrated in vacuo. The residue
was purified by preparative TLC (CHCl3/MeOH = 20:1) to afford KUSC-
5001 as a colorless solid (13.4 mg, 81%).
HRMS (ESI) m/z: [M + Na]+ calcd for C15H11F3N6O3Na+ 403.0737;
found, 403.0740.
4.1.2.9. N-(4-Chlorophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5007). A colorless solid.
1H NMR (500 MHz, CDCl3): δ 6.33 (s, 2H), 6.97 (s, 1H), 7.33 (d, J =
8.5 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 2.5 Hz, 1H), 8.12 (s,
1H), 8.43 (s, 1H), 8.58 (br, 1H).
1H NMR (500 MHz, CDCl3): δ 6.38 (s, 2H), 6.96 (m, 1H), 7.47 (dd, J
= 8.8 Hz, 2H), 7.59 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 2.5 Hz, 1H), 8.12 (d,
J = 2.0 Hz, 1H), 8.43 (s, 1H), 8.58 (br, 1H).
13C NMR (150 MHz, CDCl3): δ 66.44, 109.21, 117.15, 121.45,
129.03, 132.22, 132.71, 136.67, 137.18, 137.26, 149.08, 158.78.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11BrN6O3Na+ 412.9968;
found, 412.9983.
13C NMR (125 MHz, CDCl3): δ 66.45, 109.20, 121.16, 129.03,
129.27,129.55 132.70, 136.17, 137.18, 137.28, 149.10, 158.79.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11ClN6O3Na+ 369.0473;
found, 369.0483.
4.1.2.4. N-(2-Bromophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5002). A colorless solid.
1H NMR (600 MHz, Acetone‑d6): δ 6.73 (s, 2H), 6.89 (d, J = 2.5 Hz,
1H), 7.09 (td, J = 6.0, 1.5 Hz, 1H), 7.42 (td, J = 6.5, 1.0 Hz, 1H), 7.67
(dd, J = 3.5, 1.0 Hz, 1H), 8.21 (d, J = 2.0 Hz, 1H), 8.22 (s, 1H),8.48 (dd,
J = 3.5, 1.0 Hz, 1H), 9.05 (s, 1H), 9.38 (br, 1H).
4.1.2.10. 1-((4-Nitro-1H-pyrazol-1-yl)methyl)-N-phenyl-1H-pyrazole-3-
carboxamide (KUSC-5008). A colorless solid.
1H NMR (500 MHz, CDCl3): δ 6.33 (s, 2H), 6.97 (d, J = 2.5 Hz, 1H),
7.15 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 8.0 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H),
7.76 (d, J = 2.5 Hz, 1H), 8.12 (s, 1H), 8.44 (s, 1H), 8.57 (br, 1H).
13C NMR (125 MHz, CDCl3): δ 66.42, 109.12, 119.96, 124.61,
129.05, 129.25, 132.60, 137.12, 137.24, 137.58, 149.35, 158.83.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H12N6O3Na+, 335.0863;
found, 335.0864.
13C NMR (150 MHz, Acetone‑d6): δ 66.94, 108.23, 114.03, 122.30,
126.12, 129.34, 131.62, 133.39, 134.57, 136.89, 137.37, 137.41,
148.98, 159.62.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11BrN6O3Na+ 412.9968;
found, 412.9980.
4.1.2.11. N-(4-Isopropylphenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-
pyrazole-3-carboxamide (KUSC-5010). A colorless solid.
1H NMR (500 MHz, CDCl3): δ 1.25 (d, J = 7.0 Hz, 6H), 2.90 (septet, J
= 7.0 Hz, 1H), 6.32 (s, 2H), 6.96 (d, J = 3.0 Hz, 1H), 7.22 (d, J = 8.5 Hz,
2H), 7.58 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 2.5 Hz, 1H), 8.12 (s, 1H), 8.44
(s, 1H), 8.54 (br, 1H).
4.1.2.5. N-(3-Bromophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5003). A colorless solid.
1H NMR (600 MHz, Acetone‑d6): δ 6.66 (s, 2H), 6.87 (d, J = 2.4 Hz,
1H), 7.27–7.31 (m, 2H), 7.73 (dd, J = 1.8, 9.0 Hz, 1H), 8.16–8.17 (m,
2H), 8.21 (s, 1H), 8.94 (s, 1H), 9.58 (br, 1H).
13C NMR (125 MHz, CDCl3): δ 24.16, 33.76, 66.40, 109.08, 120.10,
127.13, 129.08, 132.54, 135.23, 137.10, 137.20, 145.37, 149.41,
158.76.
13C NMR (150 MHz, Acetone‑d6): δ 66.95, 108.55, 119.44, 122.71,
123.37, 127.32, 131.28, 131.33, 134.21, 137.20, 141.13, 149.28,
160.30.
HRMS (ESI) m/z: [M + Na]+ calcd for C17H18N6O3Na+ 377.1333;
found, 377.1352.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11BrN6O3Na+ 412.9968;
found, 412.9977.
4.1.2.12. N-(4-Methoxyphenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-
pyrazole-3-carboxamide (KUSC-5011). A colorless solid.
1H NMR (500 MHz, Acetone‑d6): δ 3.78 (s, 3H), 6.64 (s, 2H), 6.84 (d,
J = 2.0 Hz, 1H), 6.91 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H), 8.13
(s, 1H), 8.20 (s, 1H), 8.93 (s, 1H), 9.29 (s, 1H).
4.1.2.6. 1-((4-Nitro-1H-pyrazol-1-yl)methyl)-N-(4-(trifluoromethyl)
phenyl)-1H-pyrazole-3-carboxamide (KUSC-5004). A colorless solid.
1H NMR (500 MHz, Acetone‑d6): δ 6.67 (s, 2H), 6.89 (d, J = 3.0 Hz,
1H), 7.69 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 8.18 (d, J = 2.5
Hz), 8.22 (s, 1H), 8.95 (s, 1H) 9.76 (s, 1H).
13C NMR (125 MHz, Acetone‑d6): δ 55.62, 66.90, 108.29, 114.64,
122.37, 131.22, 132.57, 133.99, 137.16, 137.38, 149.85, 157.08,
159.79.
13C NMR (125 MHz, Acetone‑d6): δ 66.97, 108.65, 120.65, 124.36,
125.56, 126.51, 126.82, 126.85, 131.28, 134.29, 137.22, 137.41,
143.09, 149.20, 160.50.
HRMS (ESI) m/z: [M + Na]+ calcd for C15H14N6O4Na+ 365.0969;
found, 365.0990.
HRMS (ESI) m/z: [M + Na]+ calcd for C15H11F3N6O3Na+ 403.0737;
found, 403.0757.
4.1.2.13. N-(3,4-Dichlorophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-
1H-pyrazole-3-carboxamide (KUSC-5012). A colorless solid.
4.1.2.7. N-(4-Fluorophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5005). A colorless solid.
1H NMR (500 MHz, DMSO‑d6): δ 6.60 (s, 2H), 6.89 (d, J = 2.0 Hz,
1H), 7.59 (d, J = 9.0 Hz, 1H), 7.79 (dd, J = 9.0, 2.0 Hz, 1H), 8.15 (d, J =
1H NMR (500 MHz, Acetone‑d6): δ 6.65 (s, 2H), 6.85 (d, J = 2.0 Hz,
8

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
3.0 Hz, 1H), 8.21 (d, J = 3.0 Hz, 1H), 8.39 (s, 1H), 9.17 (s, 1H), 10.42 (s,
1H).
4.1.3.2. N-(4-bromophenyl)-3-(4-nitro-1H-pyrazol-1-yl)propanamide
(KUSC-5021). To a stirred solution of 3-(4-nitro-1H-pyrazol-1-yl)prop-
anoic acid (15.0 mg, 0.0810 mmol) in dichloromethane (1.1 mL) were
13C NMR (125 MHz, DMSO‑d6): δ 65.42, 108.84, 120.41, 121.58,
125.18, 130.51, 130.79, 131.28, 133.71, 135.70, 136.71, 138.74,
147.67, 159.84.
added DIPEA (28.0 μL, 0.161 mmol) and HATU (30.8 mg, 0.0810 mmol)
at room temperature. After being stirred for 30 min, p-bromoaniline
(15.3 mg, 0.047 mmol) was added, and the mixture was stirred at room
temperature for 18.5 h. The reaction was then quenched with water and
the layers were separated. The aqueous layer was extracted with AcOEt
and the combined organic layers were washed with 1 M HCl, sat.
NaHCO3 aq., and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by SiO2 chromatography (CHCl3/MeOH =
20:1), and the purified fraction was further subjected to ODS HPLC on
COSMOSIL 5C18 MS II (φ20 × 250 mm) with H2O/CH3CN (40 : 60) to
afford KUSC-5021 as a colorless solid (20.3 mg, 74%).
HRMS (ESI) m/z: [M + Na]+ calcd for C14H10Cl2N6O3Na+ 403.0084;
found, 403.0092.
4.1.2.14. N-(2-Hydroxyphenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-1H-
pyrazole-3-carboxamide (KUSC-5013). A colorless solid.
1H NMR (500 MHz, DMSO‑d6): δ 6.63 (s, 2H), 6.81 (t, J = 7.5 Hz,
1H), 6.85 (d, J = 2.5 Hz, 1H), 6.92 (m, 2H), 8.17 (d, J = 8.0 Hz, 1H),
8.22 (d, J = 2.0 Hz, 1H), 8.36 (s, 1H), 9.22 (d, J = 7.0 Hz, 2H), 10.19 (s,
1H).
1H NMR (600 MHz, acetone‑d6): δ 3.08 (t, J = 6.0 Hz, 2H), 4.60 (t, J
= 7.2 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 8.10 (s,
1H), 8.64 (s, 1H), 9.43 (br, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 65.28, 107.32, 114.78, 119.22,
119.74, 124.06, 126.02, 131.31, 134.11, 135.66, 136.76, 146.41,
147.84, 158.43.
13C NMR (150 MHz, acetone‑d6): δ 36.72, 49.45, 116.18, 121.88,
131.07, 132.48, 132.65, 136.18, 139.26, 169.03.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H12N6O4Na+ 351.0812;
found, 351.0808.
HRMS (ESI) m/z: calcd for [Mꢀ H]- C12H10BrN4O3 336.9942; found,
336.9963.
4.1.2.15. N-(2,4-Dichlorophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-
1H-pyrazole-3-carboxamide (KUSC-5027). A colorless solid.
1H NMR (500 MHz, DMSO‑d6): δ 6.63 (2H, s), 6.89 (1H, s), 7.47 (1H,
d, J = 8.5 Hz), 7.73 (1H, s), 7.96 (1H, d, J = 8.5 Hz), 8.23 (1H, s), 8.37
(1H, s), 9.21 (1H, s), 9.65 (1H, s).
4.1.4. Synthesis of KUSC-5019, KUSC-5014, KUSC-5020, and KUSC-
5029
13C NMR (125 MHz, DMSO‑d6): δ 65.40, 107.58, 125.89, 127.08,
127.87, 128.90, 129.47, 131.47, 133.55, 134.00, 135.71, 136.76,
147.07, 159.10.
4.1.4.1. Ethyl
1-((1H-pyrazol-1-yl)methyl)-1H-pyrazole-3-carboxylate
(S3). To a stirred solution of ethyl 1-(bromomethyl)-3-carboxylate
(100.0 mg, 0.429 mmol) in CH3CN (5.60 mL) were added 1H-pyrazole
(66.0 mg, 0.969 mmol) and potassium carbonate (130.5 mg, 0.944
mmol). After being stirred at room temperature for 3.5 days, the reaction
was quenched with water and the layers were separated. The aqueous
layer was extracted with ethyl acetate and the combined organic layer
was washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by SiO2 column chromatography (CHCl3/
MeOH = 20:1) to afford the carboxylate S3 as a colorless solid (94.8 mg,
quant.).
HRMS (ESI) m/z: [M + Na]+ calcd for C14H10Cl2N6O3Na+ 403.0084;
found, 403.0106.
4.1.2.16. N-(2,4-Difluorophenyl)-1-((4-nitro-1H-pyrazol-1-yl)methyl)-
1H-pyrazole-3-carboxamide (KUSC-5028). A colorless solid.
1H NMR (500 MHz, DMSO‑d6): δ 6.61 (s, 2H), 6.86 (d, J = 2.5 Hz,
1H), 7.10 (t, J = 3.5 Hz, 1H), 7.36 (t, J = 3.0 Hz, 1H), 7.66 (q, J = 8.5 Hz,
1H), 8.20 (d, J = 2.5 Hz, 1H), 8.36 (s, 1H), 9.18 (s, 1H), 9.77 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 65.38, 104.29, 107.57, 111.19,
121.89, 127.64, 131.35, 133.68, 135.71, 136.71, 147.37, 154.59,
156.53, 159.53.
1H NMR (600 MHz, CDCl3): δ 1.37 (t, J = 7.2 Hz, 3H), 4.39 (q, J =
7.2 Hz, 2H), 6.30 (s, 1H), 6.37 (s, 2H), 6.81 (d, J = 3.0 Hz, 1H), 7.56 (s,
1H), 7.67 (d, J = 1.8 Hz, 1H), 7.70 (d, J = 1.8 Hz, 1H).
13C NMR (150 MHz, CDCl3): δ 14.48, 61.30, 65.85, 107.71, 110.21,
130.20, 131.09, 141.38, 144.93, 162.07.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H10F2N6O3Na+ 371.0675;
found, 371.0683.
HRMS (ESI) m/z: [M + H]+ calcd for C10H13N4O2 221.1034; found,
221.1003.
4.1.3. Synthesis of KUSC-5015 and KUSC-5021
4.1.4.2. 1-((1H-Pyrazol-1-yl)methyl)-1H-pyrazole-3-carboxylic
acid
4.1.3.1. N-(4-bromophnyl)-1-methyl-1H-pyrazole-3-carboxamide (KUSC-
5015). To a stirred solution of 1-methyl-1H-pyrazole-3-carboxylic acid
(30.0 mg, 0.238 mmol) in dichloromethane (3.2 mL) were added DIPEA
(S4). To a stirred solution of the carboxylate S3 (30.0 mg, 0.136 mmol)
in MeOH (2.1 mL) and water (0.7 mL) was added 2 M NaOH (0.10 mL,
0.200 mmol). After being stirred at 48 ◦C for 15 h, the reaction solution
was cooled to room temperature. After the reaction was quenched with
conc. HCl at pH 5, added ethyl acetate and the layers were separated.
The aqueous layer was extracted with ethyl acetate, and the combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo to afford the carboxylic acid S4 as a colorless solid
(9.7 mg, 37%).
(83 μL, 0.477 mmol) and HATU (90.5 mg, 0.238 mmol) at room tem-
perature. After being stirred for 30 min, p-bromoaniline (45.0 mg, 0.262
mmol) was added, and the mixture was stirred at room temperature for
21 h. The reaction was then quenched with water and the layers were
separated. The aqueous layer was extracted with CHCl3 and the com-
bined organic layers were washed with 1 M HCl, sat. NaHCO3 aq., and
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
subjected to ODS HPLC on COSMOSIL 5C18 MS II (φ20 × 250 mm) with
H2O/CH3CN (40 : 60) to afford KUSC-5015 as a colorless solid (34.81
mg, 52%).
1H NMR (500 MHz, MeOH‑d4): δ 6.35 (d, J = 2.0 Hz, 1H), 6.44 (s,
2H), 6.78 (d, J = 2.0 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.90 (d, J = 2.5
Hz, 1H), 7.93 (d, J = 2.5 Hz, 1H).
13C NMR (125 MHz, MeOH‑d4): δ 66.16, 107.99, 110.31, 132.21,
133.22, 142.20, 146.29, 164.93.
1H NMR (600 MHz, CDCl3): 3.97 (s, 3H), 6.88 (d, J = 2.5 Hz, 1H),
7.41 (d, J = 2.5 Hz, 1H), 7.46 (d, J = 9.5 Hz, 2H), 7.61 (d, J = 8.5 Hz,
2H), 8.68 (br, 1H).
HRMS (ESI) m/z: [M + Na]+ calcd for C8H8N4O2Na+ 215.0539;
found, 215.0503.
13C NMR (150 MHz, CDCl3): δ 39.56, 107.40, 1116.50, 121.22,
132.06, 132.38, 137.20, 146.51, 159.84.
HRMS (ESI) m/z: calcd for [M + Na]+ C11H10BrN3ONa+ 303.9879;
found, 303.9828.
4.1.4.3. 1-((1H-pyrazol-1-yl)methyl)-N-(4-bromophenyl)-1H-pyrazole-3-
carboxamide (KUSC-5019). To a stirred solution of the carboxylate S4
(9.72 mg, 0.0506 mmol) in dichloromethane (0.68 mL) were added
9

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
DIPEA (18
μ
L, 0.103 mmol) and HATU (19.43 mg, 0.0505 mmol). After
4.1.4.6. Ethyl
1-((4-nitro-1H-imidazol-1-yl)methyl)-1H-pyrazole-3-
being stirred at room temperature for 30 min, p-bromoaniline (9.60 mg,
0.0558 mmol) was added, and the mixture was stirred at room tem-
perature for 11 h. The reaction was then quenched with water and the
layers were separated. The aqueous layer was extracted with CHCl3, and
the combined organic layers were washed with sat. NaHCO3 aq. and
brine, respectively, dried over Na2SO4, concentrated in vacuo. The res-
idue was purified by SiO2 flash column chromatography (CHCl3/MeOH
= 100:3) to afford KUSC-5019 as a colorless solid (15.9 mg, 14%).
1H NMR (500 MHz, Acetone‑d6): δ 6.31 (t, J = 2.0 Hz, 1H), 6.50 (s,
2H), 6.81 (d, J = 2.5 Hz, 1H), 7.49–51 (m, 3H), 7.82 (d, J = 9.0 Hz, 2H),
7.99 (s, 1H), 8.00 (s, 1H), 9.55 (s, 1H).
carboxylate (S6). To a stirred solution of ethyl 1-(bromomethyl)-pyr-
azole-3-carboxylate (100.0 mg, 0.429 mmol) in DMF (4.3 mL) were
added Cs2CO3 (153.8 mg, 0.472 mmol), NaI (64.3 mg, 0.429 mmol), and
4-nitro-1H-imidazole (72.8 mg, 0.644 mmol). After being stirred at
80 ◦C for 19 h, the reaction was quenched with water and the layers
were separated. The aqueous layer was extracted with ethyl acetate, and
the combined organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by SiO2 flash col-
umn chromatography (CHCl3/MeOH = 20:1) to afford the carboxylate
S6 as a colorless solid (74.8 mg, 66%).
1H NMR (500 MHz, DMSO‑d6): δ 1.27 (t, J = 6.0 Hz, 3H), 4.27 (q, J =
6.0 Hz, 2H), 6.54 (s, 2H), 6.83 (d, J = 1.5 Hz, 1H), 8.15 (s, 1H), 8.23 (d,
J = 2.5 Hz, 1H), 8.59 (s, 1H).
13C NMR (125 MHz, Acetone‑d6): δ 66.17, 107.52, 108.20, 116.38,
122.48, 122.56, 131.08, 132.45, 133.31, 139.10, 141.34, 148.58,
160.43.
13C NMR (125 MHz, DMSO‑d6): δ 14.17, 60.53, 60.90, 109.36,
120.98, 133.09, 137.42, 144.59, 147.39, 161.24.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H12BrN5ONa+ 368.0117;
found, 368.0093.
HRMS (ESI) m/z: [M + Na]+ calcd for C10H11N5O4Na+ 288.0703;
found, 288.0726.
4.1.4.4. Ethyl 1-((1H-imidazol-1-yl)methyl)-1H-pyrazole-3-carboxylate
(S5). To a stirred solution of ethyl 1-(bromomethyl)-3-carboxylate
(50.0 mg, 0.215 mmol) in CH3CN (2.8 mL) were added potassium car-
bonate (65.2 mg, 0.472 mmol) and 1H-imidazole (33.0 mg, 0.485
mmol). After being stirred at room temperature for 3 days, the reaction
was quenched with water and the layers were separated. The aqueous
layer was extracted with ethyl acetate, and the combined organic layers
were washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by SiO2 flash column chromatography (CHCl3/
MeOH = 20:1) to afford the carboxylate S5 as a colorless solid (36.1 mg,
76%).
4.1.4.7. 1-((4-Nitro-1H-imidazol-1-yl)methyl)-1H-pyrazole-3-carboxylic
acid (S7). To a stirred solution of the carboxylate S6 (30.0 mg, 0.113
mmol) in MeOH (1.8 mL) and water (0.6 mL) was added 2 M NaOH
(0.085 mL). After being stirred at 48 ◦C for 10 h, the reaction solution
was cooled to room temperature. After the reaction was quenched with
conc. HCl at pH 5, ethyl acetate was added, and the layers were sepa-
rated. The aqueous layer was extracted with ethyl acetate, and the
combined organic layers were dried over Na2SO4, and concentrated in
vacuo to afford the carboxylic acid S7 as a colorless solid (24.9 mg,
93%).
1H NMR (500 MHz, CDCl3): δ 1.38 (t, J = 7.0 Hz, 3H), 4.39 (q, J =
7.0 Hz, 2H), 6.21 (s, 2H), 6.83 (d, J = 3.0 Hz, 1H), 7.07 (s, 1H), 7.10 (s,
1H), 7.51 (d, J = 2.5 Hz, 1H), 7.73 (s, 1H).
1H NMR (500 MHz, DMSO‑d6): δ 6.51 (s, 2H), 6.77 (d, J = 2.0 Hz,
1H), 8.13 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 2.5 Hz, 1H), 8.56 (d, J = 1.5
Hz, 1H).
13C NMR (125 MHz, CDCl3): δ 14.44, 61.43, 110.58, 118.36, 118.74,
130.25, 131.01, 137.14, 145.38, 161.85.
13C NMR (125 MHz, DMSO‑d6): δ 60.86, 109.23, 120.95, 132.84,
137.37, 145.54, 147.35, 162.68.
HRMS (ESI) m/z: [M + H]+ calcd for C10H13N4O2+, 221.1033; found,
221.1012.
HRMS (ESI) m/z: [M + H]+ calcd for C8H8N5O4 238.0571; found,
238.0548.
4.1.4.5. 1-((1H-imidazol-1-yl)methyl)-N-(4-bromophenyl)-1H-pyrazole-
3-carboxamide (KUSC-5014). To a stirred solution of the carboxylate S5
(36.1 mg, 0.164 mmol) in MeOH (2.4 mL) and water (0.8 mL) was added
2 M NaOH (0.12 mL). After being stirred at 45 ◦C for 20 h, the reaction
mixture was cooled to room temperature. After the reaction solution was
acidified with 1 M HCl at pH 5, ethyl acetate was added, and the layers
were separated. The aqueous layer was extracted with ethyl acetate, and
the combined organic layers were concentrated in vacuo. The residue
was used without further purification.
4.1.4.8. N-(4-bromophenyl)-1-((4-nitro-1H-imidazol-1-yl)methyl)-1H-
pyrazole-3-carboxamide (KUSC-5020). To a stirred solution of the car-
boxylic acid S7 (18.2 mg, 0.0765 mmol) in CH2Cl2 (1.0 mL) were added
DIPEA (27.0 μL, 0.155 mmol) and HATU (29.3 mg, 0.0770 mmol). After
stirred for 30 min, p-bromoaniline (9.60 mg, 0.0558 mmol) was added,
and the mixture was stirred for 12 h. The reaction was then quenched
with water and the layers were separated. The aqueous layer was
extracted with ethyl acetate, and the combined organic layer was
washed with sat. NaHCO3 aq. and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by SiO2 flash chroma-
tography (CHCl3/MeOH = 100:3) to afford KUSC-5020 as a colorless
solid (26.1 mg, 87%).
To a stirred solution of the residue (5.6 mg, 0.0291 mmol) in
dichloromethane (0.39 mL) were added DIPEA (10.2 μL, 0.0586 mmol)
and HATU (11.1 mg, 0.0292 mmol). After being stirred at room tem-
perature for 30 min, p-bromoaniline (5.0 mg, 0.0291 mmol) was added,
and the mixture was stirred at room temperature for 22 h. The reaction
was then quenched with water and the layers were separated. The
aqueous layer was extracted with ethyl acetate and the combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by preparative TLC
(CHCl3/MeOH = 20:1) to afford KUSC-5014 as a colorless solid (5.0 mg,
9% in 2 steps).
1H NMR (500 MHz, DMSO‑d6): δ 6.54 (s, 2H), 6.88 (d, J = 2.5 Hz,
1H), 7.53 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H), 8.16 (d, J = 1.5
Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 8.59 (s, 1H), 10.20 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 60.89, 107.74, 115.52, 120.98,
122.35, 131.46, 133.36, 137.41, 137.89, 147.36, 148.15, 159.59
HRMS (ESI) m/z: [M + Na]+ calcd for C14H11BrN6O3Na+ 412.9968;
found, 412.9965.
1H NMR (600 MHz, DMSO‑d6): δ 6.42 (s, 2H), 6.84 (d, J = 1.8 Hz,
1H), 6.94 (s, 1H) 7.40 (s, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.77 (d, J = 9.0
Hz, 2H), 7.94 (s, 1H), 8.16 (d, J = 1.8 Hz, 1H), 10.23(1H, br).
13C NMR (150 MHz in DMSO‑d6): δ 60.05, 107.60, 115.43, 119.30,
122.32, 129.23, 131.46, 132.83, 137.55, 138.04, 147.57, 159.83.
HRMS (ESI) m/z: [M + Na]+ calcd for C14H12BrN5ONa+ 368.0118;
found, 368.0154.
4.1.4.9. Ethyl 1-((3-nitro-1H-pyrrol-1-yl)methyl)-1H-pyrazole-3-carbox-
ylate (S8). To a stirred solution of 3-nitro-1H-pyrrole (30.0 mg, 0.268
mmol) in DMF (1.5 mL) were added Ethyl 1-(bromomethyl)-pyrazole-3-
carboxylate (68.6 mg, 0.294 mmol) and potassium carbonate (74.0 mg,
0.535 mmol). After being stirred at room temperature for 23 h, the re-
action was quenched with water and the layers were separated. The
aqueous layer was extracted with CHCl3 and the combined organic layer
was washed with brine, dried over Na2SO4, and concentrated in vacuo.
10

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
The residue was purified by SiO2 flash chromatography (CHCl3/MeOH
= 100:3) to afford the carboxylate S8 as a colorless solid (55.7 mg, 79%).
1H NMR (500 MHz, CDCl3): δ 1.38 (t, J = 7.0 Hz, 3H), 4.39 (q, J =
7.0 Hz, 2H), 6.18 (s, 2H), 6.71 (dd, J = 2.0 Hz, 1H), 6.82 (t, J = 2.5 Hz,
1H), 6.85 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 2.5 Hz, 1H), 7.74 (s, 1H).
13C NMR (125 MHz, CDCl3): δ 14.25, 61.34, 64.33, 106.81, 110.55,
121.19, 121.30, 130.69, 138.13, 145.64, 161.63.
110.26, 120.14, 123.41, 130.85, 132.77, 144.87, 152.92, 162.12.
HRMS (ESI) m/z: [M + Na]+ calcd for C15H21N5O4Na+ 358.1486;
found, 358.1487.
4.1.5.2. tert-Butyl (1-((3-((4-bromophenyl)carbamoyl)-1H-pyrazol-1-yl)
methyl)-1H-pyrazol-4-yl)carbamate (S12). To a stirred solution of the
carboxylate S10 (50.0 mg, 0.149 mmol) in MeOH (3.0 mL) and water
(1.0 mL) was added 2 M NaOH (0.45 mL). After being stirred at 45 ◦C for
1 day, the reaction solution was cooled to room temperature. After the
reaction was quenched with conc. HCl at pH 2, added CHCl3 and the
layers were separated. The aqueous layer was extracted with CHCl3, and
the combined organic layers were dried over Na2SO4, and concentrated
in vacuo to afford 1-((4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)
methyl)-1H-pyrazole-3-carboxylic acid (S11) as a colorless solid (51.1
mg).
HRMS (ESI) m/z: [M + Na]+ calcd for C11H12N4O4Na+, 287.0751;
found, 287.0774.
4.1.4.10. 1-((3-Nitro-1H-pyrrol-1-yl)methyl)-1H-pyrazole-3-carboxylic
acid (S9). To a stirred solution of the carboxylate S8 (20.0 mg, 0.0757
mmol) in MeOH (1.20 mL) and water (0.40 mL) was added 2 M NaOH
(57.0 μ
L, 0.114 mmol). After being stirred at 45 ◦C for 13 h, the reaction
solution was cooled to room temperature. After the reaction was
quenched with conc. HCl at pH 3–4, was added ethyl acetate, and the
layers were separated. The aqueous layer was extracted with ethyl ac-
etate, dried over Na2SO4, and concentrated in vacuo to afford the car-
boxylic acid S9 as a colorless solid (17.58 mg, 98%).
To a stirred solution of the carboxylic acid S11 (51.1 mg, 0.149
mmol) in dichloromethane (2.0 mL) were added DIPEA (52.0 μL, 0.299
mmol) and HATU (56.7 mg, 0.164 mmol). After being stirred at room
temperature for 30 min, p-bromoaniline (28.2 mg, 0.164 mmol) was
added, and the mixture was stirred at room temperature for 30 h. The
reaction was then quenched with water and the layers were separated.
The aqueous layer was extracted with CHCl3, and the combined organic
layer was washed with sat. NaHCO3 aq. and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by SiO2 flash col-
umn chromatography (CHCl3/MeOH = 20:1) to afford the carbamate
S12 as a colorless solid (60.0 mg, 87% in 2 steps).
1H NMR (600 MHz, DMSO‑d6): δ 6.39 (s, 2H), 6.71 (d, J = 3.0 Hz,
1H), 6.75 (d, J = 2.0 Hz, 1H), 7.15 (t, J = 2.5 Hz, 1H), 8.17 (d, J = 2.0
Hz, 1H), 8.20 (t, J = 2.0 Hz, 1H).
13C NMR (150 MHz, DMSO‑d6): δ 63.19, 105.48, 109.25, 122.77,
123.01, 132.63, 136.66, 145.26, 162.83.
HRMS (ESI) m/z: [M + Na]+ calcd for C9H8N4O4Na+ 259.0438;
found, 259.0437.
1H NMR (500 MHz, DMSO‑d6): δ 1.43 (s, 9H), 6.42 (s, 2H), 6.82 (d, J
= 2.0 Hz, 1H), 7.38 (s, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 8.5 Hz,
2H), 7.97 (s, 1H), 8.08 (d, J = 2.5 Hz, 1H), 9.27 (s, 1H), 10.28 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 28.10, 65.09, 78.90, 107.53,
115.33, 119.44, 122.32, 123.27, 131.37, 132.81, 138.10, 147.32,
147.43, 152.69, 159.83.
4.1.4.11. N-(4-bromophenyl)-1-((3-nitro-1H-pyrrol-1-yl)methyl)-1H-pyr-
azole-3-carboxamide (KUSC-5029). To a stirred solution of the carbox-
ylic acid S9 (17.58 mg, 0.0744 mmol) in dichloromethane (1.0 mL) were
added DIPEA (26.0
μL, 0.149 mmol) and HATU (28.3 mg, 0.0744
mmol). After being stirred at room temperature for 30 min, p-bromoa-
niline (14.1 mg, 0.0820 mmol) was added and the mixture was stirred at
room temperature for 14 h. The reaction was then quenched with water
and the layers were separated. The aqueous layer was extracted with
CHCl3, and the combined organic layer was washed with sat. NaHCO3
aq. and brine, dried over Na2SO4, and concentrated in vacuo. The res-
idue was purified with SiO2 flash column chromatography (CHCl3/
MeOH = 20:1). The purified fraction was further subjected to ODS HPLC
on COSMOSIL 5C18 MSII (φ20 × 250 mm) with H2O/MeCN (40:60) to
afford KUSC-5029 as a colorless solid (20.2 mg, 69%).
HRMS (ESI) m/z: [M + Na]+ calcd for C19H21BrN6O3Na+ 485.0751;
found, 485.0727.
4.1.5.3. 1-((4-amino-1H-pyrazol-1-yl)methyl)-N-(4-bromophenyl)-1H-
pyrazole-3-carboxamide hydrochloride (KUSC-5017). A solution of the
carbamate S12 (24.1 mg, 0.0522 mmol) in 4 M HCl in 1,4-dioxane (0.52
mL) was stirred at room temperature for 2.7 h and concentrated in vacuo
to afford KUSC-5017 as a colorless solid (15.06 mg, 73%).
1H NMR (500 MHz, DMSO‑d6): δ 6.55 (s, 2H), 6.87 (d, J = 2.5 Hz,
1H), 7.50 (d, J = 8.5 Hz, 2H), 7.65 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 8.17
(d, J = 2.5 Hz, 1H), 8.29 (s, 1H), 10.29 (br, 2H).
1HNMR (500 MHz, Acetone‑d6): δ 6.49 (s, 2H), 6.70 (s, 1H), 6.86 (d,
J = 2.5 Hz, 1H), 7.15 (s, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 8.5
Hz, 2H), 8.06 (s, 1H), 8.15 (s, 1H), 9.57 (s, 1H).
13C NMR (125 MHz, DMSO‑d6): δ 65.05, 107.60, 114.41, 115.38,
122.32, 126.01, 131.38, 133.18, 135.32, 138.08, 147.65, 159.74.
HRMS (ESI) m/z: [Mꢀ HCl + Na]+ calcd for C14H13BrN6ONa+
383.0226; found, 383.0175.
13C NMR (125 MHz in Acetone‑d6): δ 64.71, 106.43, 108.51, 116.52,
122.63, 122.98, 123.06, 132.47, 133.61, 138.46, 138.91, 149.27,
160.23.
HRMS (ESI) m/z: [M + Na]+ calcd for C15H12BrN5O3Na+ 412.0016;
found, 412.0037.
4.1.5.4. 1-((4-acetamido-1H-pyrazol-1-yl)methyl)-N-(4-bromophenyl)-
1H-pyrazole-3-carboxamide (KUSC-5025). To a stirred solution of KUSC-
5017 (15.1 mg, 0.0379 mmol) in dichloromethane (0.38 mL) was added
4.1.5. Synthesis of KUSC-5017 and KUSC-5025
Et3N (13.2
μL, 0.0950 mmol). After being stirred at room temperature
4.1.5.1. Ethyl
1-((4-((tert-butoxycarbonyl)amino)-1H-pyrazol-1-yl)
for 30 min, acetyl chloride (12 μL, 0.169 mmol) was added and the
methyl)-1H-pyrazole-3-carboxylate (S10). To a stirred solution of the
carboxylate S1 (150.0 mg, 0.566 mmol) in MeOH (0.72 mL) and THF
(0.72 mL) were added (Boc)2O (0.13 mL, 0.566 mmol) and 10% Pd/C
(39.5 mg). After being stirred at room temperature for 10 h under H2
atmosphere, the reaction solution was filtered with celite, and the so-
lution was concentrated in vacuo. The residue was purified by SiO2 flash
column chromatography (CHCl3/MeOH = 20:1) to afford the carbox-
ylate S10 as a colorless solid (164.9 mg, 87%).
mixture was stirred at room temperature for 20 h. The reaction was then
quenched with MeOH, added water and AcOEt, and the layers were
separated. The aqueous layer was extracted with ethyl acetate, and the
combined organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified with preparative TLC
(CHCl3/MeOH = 20:1) to afford KUSC-5025 as a colorless solid (11.5
mg, 75%).
1H NMR (600 MHz, MeOH‑d4): δ 2.07 (s, 3H), 6.42 (s, 2H), 6.83 (d, J
= 1.5 Hz, 2H), 7.48 (d, J = 4.0 Hz, 2H), 7.55 (s, 1H), 7.68 (d, J = 4.0 Hz,
2H), 7.93 (d, J = 2.0 Hz, 1H), 8.24 (s, 1H).
1H NMR (500 MHz, CDCl3): δ 1.38 (t, J = 7.0 Hz, 3H), 1.47 (s, 9H),
4.39 (q, J = 7.0 Hz, 2H), 6.27 (s, 2H), 6.38 (s, 1H), 6.79 (d, J = 2.0 Hz,
1H), 7.48 (s, 1H), 7.61 (d, J = 1.5 Hz, 1H), 7.84 (s, 1H).
13C NMR (150 MHz, MeOH‑d4): δ 22.73, 66.49, 108.57, 117.79,
122.78, 123.34, 124.06, 132.82, 133.48, 133.70, 138.77, 148.86,
13C NMR (125 MHz, CDCl3): δ 14.47, 28.39, 61.31, 66.41, 80.81,
11

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
162.10, 170.46.
121.27, 122.75, 124.11, 124.84, 126, 0.76 (q), 127.01 (d), 133.55,
133.79, 143.06, 148.73, 126.27, 174.24.
HRMS (ESI) m/z: [M + Na]+ calcd for C16H15BrN6O2Na+ 425.0332;
found, 425.0314.
HRMS (ESI) m/z: [M + Na]+ calcd for C18H17F3N6O2Na+ 429.1257;
found, 429.1263.
4.1.6. Synthesis of KUSC-5035 ~ KUSC-5040
4.1.6.5. 1-((4-butyramido-1H-pyrazol-1-yl)methyl)-N-(4-(tri-
fluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (KUSC-5038). A color-
less solid.
4.1.6.1. tert-Butyl (1-((3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-1-yl)
methyl)-1H-pyrazol-4-yl)carbamate (S13). To a stirred solution of the
carboxylic acid S11 (24.1 mg, 0.0785 mmol) in dichloromethane (1.1
1H NMR (500 MHz, MeOH‑d4): δ 0.96 (t, J = 8.0 Hz, 3H), 1.68
(sextet, J = 7.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 6.43 (s, 2H), 6.86 (d, J
= 3.0 Hz, 1H), 7.57 (s, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 8.5 Hz,
2H), 7.94 (d, J = 2.5 Hz, 1H), 8.26 (s, 1H).
mL) at room temperature were added DIPEA (27.0 μL, 0.155 mmol) and
HATU (29.9 mg, 0.0786 mmol). After being stirred at room temperature
for 30 min, p-trifluoromethyl aniline (11.0 L, 0.0876 mmol) was added,
μ
and the mixture was stirred for 23 h. The reaction was then quenched
with water and the layers were separated. The aqueous layer was
extracted with CHCl3 and the combined organic layers were washed
with sat. NaHCO3 aq., brine, dried over Na2SO4, and concentrated in
vacuo to afford the crude solid S13. This crude S13 was subjected to the
next reaction without further purification.
13C NMR (125 MHz, MeOH‑d4): δ 13.97, 20.28, 39.01, 66.52,
108.70, 121.28, 122.78, 124.05, 124.66, 126.73 (q), 127.00 (d), 133.55,
133.81, 143.05, 148.74, 162.26, 173.39.
HRMS (ESI) m/z: [M + Na]+ calcd for C19H19F3N6O2Na+ 443.1414;
found, 443.1433.
4.1.6.6. 1-((4-(2-phenylacetamido)-1H-pyrazol-1-yl)methyl)-N-(4-(tri-
fluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (KUSC-5039). A color-
less solid.
4.1.6.2. 1-((4-amino-1H-pyrazol-1-yl)methyl)-N-(4-(trifluoromethyl)
phenyl)-1H-pyrazole-3-carboxamide hydrochloride (KUSC-5035). A solu-
tion of the carbamate S13 (42.2 mg) was dissolved in 4 M HCl in 1,4-
dioxane (0.94 mL) and stirred at room temperature for 7.3 h. After the
reaction mixture was concentrated in vacuo, the residue was subjected
to ODS HPLC on COSMOSIL 5C18 MSII (φ20 × 250 mm) with 0.01% TFA
in H2O/CH3CN (70:30) to afford KUSC-5035 as a colorless solid (22.75
mg, 75% in 2 steps).
1H NMR (600 MHz, DMSO‑d6): δ 3.56 (s, 2H), 6.46 (s, 2H), 6.85 (d, J
= 2.4 Hz, 1H), 7.22–24 (m, 1H), 7.28–7.31 (m, 4H), 7.52 (s, 1H), 7.69
(d, J = 8.4 Hz, 2H), 8.03 (d, J = 7.8 Hz, 2H), 8.09 (d, J = 2.4 Hz, 1H),
8.23 (s, 1H), 10.30 (br, 1H), 10.51 (br, 1H).
13C NMR (150 MHz, DMSO‑d6): δ 42.49, 65.10, 107.73, 120.26,
120.77, 122.58, 123.51 (q), 125.34, 125.89, 126.58, 128.36, 129.04,
131.81, 132.98, 135.95, 142.38, 147.18, 160.18, 167.73.
HRMS (ESI) m/z: [M + Na]+ calcd for C23H19F3N6O2Na+ 491.1414;
found, 491.1423.
1H NMR (500 MHz, MeOH‑d4): δ 6.53 (s, 2H), 6.89 (d, J = 2.5 Hz,
1H), 7.66 (d, J = 8.5 Hz, 2H), 7.68 (s, 2H), 9.34 (d, J = 8.5 Hz, 2H), 7.94
(d, J = 8.5 Hz, 2H), 7.99 (d, J = 2.0 Hz, 1H), 8.19 (s, 1H).
13C NMR (125 MHz, MeOH‑d4): δ 66.69, 108.77, 116.56, 121.27,
124.64, 126.78 (q), 127.06, 127.09, 133.92, 136.48, 142.98, 149.07,
162.09.
4.1.6.7. 1-((4-isobutyramido-1H-pyrazol-1-yl)methyl)-N-(4-(tri-
fluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (KUSC-5040). A color-
less solid.
HRMS (ESI) m/z: [Mꢀ HCl + Na]+ calcd for C15H13F3N6ONa+
373.0995; found, 373.1019.
1H NMR (500 MHz, MeOH‑d4): δ 1.17 (d, J = 7.0 Hz, 6H), 2.56
(septet, J = 7.0 Hz, 1H), 6.44 (s, 2H), 6.86 (d, J = 2.0 Hz, 1H), 7.59 (s,
1H), 7.65 (d, J = 8.5 Hz, 2H), 7.94–96 (m, 3H), 8.25 (s, 1H).
13C NMR (125 MHz, MeOH‑d4): δ 19.85, 36.39, 66.53, 108.70,
121.29, 122.78, 124.15, 124.68, 126.74 (q), 127.02 (d), 133.55, 133.87,
143.07, 148.74, 162.28, 177.43.
4.1.6.3. 1-((4-acetamido-1H-pyrazol-1-yl)methyl)-N-(4-(trifluoromethyl)
phenyl)-1H-pyrazole-3-carboxamide (KUSC-5036). A colorless solid.
1H NMR (500 MHz, MeOH‑d4): δ 2.07 (s, 3H), 6.44 (s, 2H), 6.86 (d, J
= 2.0 Hz, 1H), 7.56 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.94–96 (m, 3H),
8.25 (s, 1H)
HRMS (ESI) m/z: [M + Na]+ calcd for C19H19F3N6O2Na+ 443.1414;
found, 443.1434.
13CNMR (125 MHz, MeOH‑d4): δ 22.72, 66.52, 108.69, 121.29,
122.81, 124.08 (d), 126.83 (t), 127.03 (d), 133.57, 133.74, 143.09,
148.75, 162.29, 170.46.
4.1.7. Synthesis of KUSC-5054 and KUSC-5051
HRMS (ESI) m/z: [M + Na]+ calcd for C17H15F3N6O2Na+ 415.1101;
found, 415.1071.
4.1.7.1. 1-((4-(3-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-
c:2′,1′-f][1,3,2]diazaborinin-2-yl)propanamido)-1H-pyrazol-1-yl)methyl)-
N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (KUSC-5054).
To a stirred solution of KUSC-5035 (6.0 mg, 0.0155 mmol) in DMF
4.1.6.4. 1-((4-propionamido-1H-pyrazol-1-yl)methyl)-N-(4-(tri-
fluoromethyl)phenyl)-1H-pyrazole-3-carboxamide (KUSC-5037). To
a
stirred solution of KUSC-5035 (16.8 mg, 0.0435 mmol) in dichloro-
methane (0.44 mL) at room temperature was added Et3N (7.6 L,
0.0545 mmol). After being stirred at room temperature for 30 min,
propionyl chloride (4.2 L, 0.0481 mmol) was added, and the mixture
(0.13 mL) were added DIPEA (4.5 μL, 0.0258 mmol) and BODIPY FL
μ
NHS ester (5.0 mg, 0.0128 mmol) in DMF (0.13 mL). After being stirred
at room temperature for 22 h, the reaction solution was concentrated in
vacuo. The residue was subjected to ODS HPLC on COSMOSIL 5C18 MSII
(φ20 × 250 mm) with H2O/CH3CN (35:65) to afford KUSC-5054 as a
brown solid (3.63 mg, 45%).
μ
was stirred for 15 h. The reaction was then quenched with water and the
layers were separated. The aqueous layer was extracted with ethyl ac-
etate and the combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified by
preparative TLC (CHCl3/MeOH = 20:1) and the purified fraction was
further subjected to ODS HPLC on COSMOSIL 5C18 MSII (φ20 × 250
mm) H2O/CH3CN (50:50) to afford KUSC-5037 as a colorless solid (8.8
mg, 50%).
1H NMR (500 MHz, MeOH‑d4): δ 2.27 (s, 3H), 2.50 (s, 3H), 2.75 (t, J
= 7.5 Hz, 2H), 3.28 (t, J = 7.5 Hz, 2H), 4.60 (br, 1H), 5.49 (br, 1H), 6.20
(s, 1H), 6.31 (d, J = 4.0 Hz, 1H), 6.44 (s, 2H), 6.86 (d, J = 7.0 Hz, 1H),
6.97 (d, J = 4.0 Hz, 1H), 7.40 (s, 1H), 7.58 (s, 1H), 7.64 (d, J = 8.5 Hz,
2H), 7.94–95 (m, 3H), 8.28 (s, 1H).
13C NMR (125 MHz, MeOH‑d4): δ 11.16, 14.85, 25.41, 36.06, 66.53,
108.70, 117.51, 121.30, 122.92, 123.99, 124.68, 125.78, 126.78 (q),
127.02 (d), 129.55, 133.59, 133.88, 124.87, 136.56, 143.07, 145.90,
148.74, 158.23, 161.45, 162.29, 171.88.
1H NMR (500 MHz, MeOH‑d4): δ 1.18 (t, J = 7.5 Hz, 3H), 2.34 (q, J
= 7.5 Hz, 2H), 6.44 (s, 2H), 6.86 (d, J = 3.0 Hz, 1H), 7.57 (s, 1H), 7.65
(d, J = 3.5 Hz, 1H), 7.94–96 (m, 3H), 8.26 (s, 1H).
13C NMR (125 MHz, MeOH‑d4): δ 10.25, 30.18, 66.51, 108.69,
HRMS (ESI) m/z: [M
+
Na]+ calcd for C29H26BF5N8O2Na+,
12

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
647.2084; found, 647.2071.
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 coenzyme A).
Epiluminescence was measured using an EnVision luminometer (Perkin
Elmer). Luciferase activity was determined in triplicate and showed as a
relative activity normalized with protein concentration.
4.1.7.2. 2-(3-(Pent-4-yn-1-yl)-3H-diazirin-3-yl)ethan-1-cyanide (S15).
To a stirred solution of the iodide S14 (17.38 mg, 0.0663 mmol) in DMF
(0.83 mL) was added KCN (8.7 mg, 0.132 mmol). After being stirred at
70 ◦C for 22 h, the reaction solution was cooled to room temperature.
The reaction solution was diluted with water, extracted with ethyl ac-
etate, washed with brine, dried over Na2SO4, and then concentrated in
vacuo. The crude cyanide S15 was subjected to the next reaction.
4.2.3. RNA extraction and quantitative real-time PCR
Total RNA was isolated from HeLa S3 cells by using Total RNA
Extraction kit (Viogene). The RNA (1.5 µg) was reverse transcribed by
random hexamer (ReverTra Ace with gDNA remover, TOYOBO).
Quantitative real-time PCR analysis was carried out StepOne PCR Real
Time System (Thermo Fisher Scientific) employing with SYBR Select.
Master Mix. Primers used for PCR were as follows: human β -actin, 5′-
CTCCTCCCTGGAGAAGAGCTAC-3′ (sense) and 5′-TGCGGATGTC-
4.1.7.3. 3-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)propanoic acid (S16). To
a solution of the crude cyanide S15 (9.77 mg) was added 10 %NaOH
(1.1 mL). After stirred reflux for 15 h, the reaction solution was cooled to
room temperature, acidified with 1 M HCl at pH 2, extracted with ethyl
acetate, dried over Na2SO4, and concentrated in vacuo. The crude car-
boxylic acid S16 was used without purification.
CACGTCACA-3′ (antisense); human HIF-1
α
, 5′-TACTAGCTTTGCA-
GAATGCTC-3′(sense)
and
5′-GCCTTGTATAGGAGCATTAAC-3′
(antisense); human HIF-1 β, 5′-GGGGGCAGCTTACCTCTAAC-3′ (sense)
and 5′-GGGTAGAGGCACTCGAACTG-3′ (antisense); human CA9, 5′-
ACCCTCTCTGACACCCTGTG-3′ (sense) and 5′-GGCTGGCTTCTCA-
CATTCTC-3′ (antisense); human VEGF, 5′-AATGACGAGGGCCTG-
4.1.7.4. 1-((4-(3-(3-(pent-4-yn-1-yl)-3H-diazirin-3-yl)propanamido)-1H-
pyrazol-1-yl)methyl)-N-(4-(trifluoromethyl)phenyl)-1H-pyrazole-3-carbox-
amide (KUSC-5051). To a stirred solution of the crude carboxylic acid
S16 (10.6 mg, 0.0588 mmol) in dichloromethane (0.79 mL) were added
GAGTGTG-3′
(sense)
and
5′-ATGTGCTGGCCTTGGTGAGGT-3′
(antisense); human 18S rRNA, 5′-GCAATTATTCCCCATGAACG-3′
(sense) and 5′-GGGACTTAATCAACGCAAGC-3′ (antisense). Relative
expression level was calculated by the ΔΔCT method, and normalized
with 18S rRNA level.
DIPEA (41.0 μL, 0.235 mmol) and HATU (22.4 mg, 0.0589 mmol). After
being stirred at room temperature for 30 min, KUSC-5035 (22.75 mg,
0.0598 mmol) was added, and the mixture was stirred at room tem-
perature for 13 h. The reaction was then quenched with water and the
layers were separated. The aqueous layer was extracted with CHCl3, and
the combined organic layers were washed with sat. NaHCO3 aq. and
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
purified by SiO2 flash column chromatography (CHCl3/MeOH = 20:1),
and the purified fraction was further subjected to ODS HPLC on COS-
MOSIL 5C18 MSII (φ20 × 250 mm) with H2O/CH3CN (45:55) to afford
the KUSC-5051 as a brown solid (4.54 mg, 15%).
4.2.4. Subcellular localization analysis of KUSC-5054
HeLa S3 cells grown on cover glass were treated with KUSC-5054 (1
μ
M), MitoTracker Red CMXRos (100 nM) and Hoechst 33342 (10 μM).
Epifluorescence was observed under IX81 fluorescence microscope
(Olympus).
1H NMR (500 MHz, CDCl3): δ 1.32 (pentet, J = 8.0 Hz, 2H), 1.55 (t, J
= 8.5 Hz, 2H), 1.88 (t, J = 7.5 Hz, 2H), 1.93 (t, J = 2.5 Hz, 1H), 2.08 (t, J
= 7.5 Hz, 2H), 2.15 (td, J = 7.0, 2.0 Hz, 2H), 6.25 (s, 2H), 6.92 (d, J =
2.5 Hz, 1H), 7.19 (s, 1H), 7.46 (s, 1H), 7.62 (d, J = 9.0 Hz, 2H), 7.68 (d,
J = 2.0 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 8.27 (s, 1H), 8.82 (s, 1H).
13C NMR (125 MHz in CDCl3): δ 17.82, 22.56, 27.87, 28.28, 30.42,
31.65, 66.10, 69.12, 83.23, 108.55, 119.33, 122.14, 122.99, 125.47,
126.29, 131.89, 132.28, 140.75, 147.60, 159.35, 168.48.
4.2.5. Western blot analysis
MDA-MB-231 cells were treated with KUSC-5037 (0.3, 1, 3 μM) or
camptothecin (0.5
were lysed in PBS(-) containing 0.5% Triton X-100. Whole cell lysate
(20 g) was subjected to SDS-PAGE followed by blotting onto a PVDF
μM) under the hypoxia (1% O2, 25 h) and the cells
μ
membrane (Pall Life Sciences). The membrane was briefly washed with
PBST buffer [PBS(-) containing 0.1% Tween-20] and blocked with
blocking buffer (PBST buffer containing 5% dry non-fat milk or 3%
BSA). The membrane was incubated with the primary antibody in
blocking buffer overnight. After three times wash with PBST, the
membrane was incubated with the secondary antibody for 1 h. Bands
were visualized with nitro-blue tetrazolium chloride/5-bromo-4-chloro-
3′-indolyphosphate p-toluidine salt colorimetric detection system
(Nacalai Tesque).
HRMS (ESI) m/z: [M + Na]+ calcd for C24H23F3N8O2Na+, 535.1788;
found, 535.1737.
4.2. Biology
4.2.1. Reagents and antibodies
4.2.6. Measurement of oxygen consumption rate (OCR)
MitoTracker Red CMXRos and Hoechst 33342 were purchased from
Cell Signaling Technology and Nacalai Tesque, respectively. The anti-
OCR was measured by Seahorse XFe96 Flux Analyzer (Agilent
Technologies), according to the manufacturer’s protocol. Briefly, cells
(80–90% confluency) were equilibrated for 1.5 h in a 37 ◦C incubator
lacking CO2. Oxygen concentration in media was measured at basal
conditions and after sequential addition of compounds (KUSC-5037
bodies used in this study are as follows: anti-human HIF-1
α mouse
monoclonal antibody (54/HIF-1α, BD Transduction Laboratories), anti-
α
-tubulin mouse monoclonal antibody (B-5–1-2, Sigma), anti-human
ATP5B rabbit polyclonal antibody (Proteintech), alkaline phosphatase
or horseradish peroxidase conjugated-secondary antibodies (Promega).
(0.1, 1, and 10
of rotenone (0.5
from at least 6 samples.
μ
M), oligomycin (0.5
μ
M), FCCP (0.5
μM) and a mixture
μ
M) and antimycin A (0.5
μ
M)). OCR was determined
4.2.2. Cell-based HRE-driven luciferase reporter gene assay
HeLa/HRE/Luc cells, stably transfected with a luciferase gene driven
by the hypoxia responsive element, were cultured in Dulbecco’s modi-
fied Eagle’s medium containing 10% fetal bovine serum.8,11 For the
reporter assay, the cells (6.0 × 103) were seeded in 96-well plates (100
µL) and cultured for 48 h. After 48 h incubation, the cells were treated
with test compounds and incubated for 1 h under normoxic conditions
prior to the exposure to hypoxic conditions (1% O2). After further 24 h-
incubation under hypoxic conditions, the cells were washed with PBS(-)
and lysed in 30 µL of lysis buffer (20 mM Tris/HCl pH 7.5, 150 mM NaCl,
1 mM EDTA and 0.1% Triton X-100). Twenty µL of the cell lysate was
4.2.7. In vitro labeling and identification of KUSC-5037/KUSC-5051
binding proteins
HeLa S3 cells were lysed and fractionated by using Mitochondria
Isolation Kit for Cultured Cells (Thermo Fisher Scientific) according to
the manufacturer’s protocol. Debris fraction (1 mg/mL), mitochondrial
fraction (0.8 mg/mL) and cytosol fraction (0.6 mg/mL) were incubated
with DMSO or 300 μM KUSC-5037 in the absence or presence of 30 μM
KUSC-5051 for 1 h at 37 ◦C with agitation, followed by 365 nm UV
irradiation for 30 min. A biotin moiety was conjugated to the covalent
13

M. Sakai et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116375
protein-compound complex by click chemistry at RT for 1 h [200
μ
M
Acknowledgements
biotin azide, 1 mM Tris(2-carboxyethyl)phosphine (TCEP, Cayman),
100
μ
M Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Tokyo
We thank Mr. Haoyu Yang for the preliminary experiments of this
study. This work was financially supported in part by a Research on the
Innovative Development and the Practical Application of New Drugs for
Chemical Industry) and 1 mM CuSO4]. After the removal of excess la-
beling reagents by CHCl3-MeOH precipitation,14 the complexes were
enriched on High Capacity NeutrAvidin agarose (Thermo Fisher Scien-
tific) by overnight incubation in PBS(-) containing 0.1% SDS at 4 ◦C.
After a series of extensive wash of the beads at 4 ◦C with [PBS(-) + 0.02%
SDS/50 mM NH4HCO3/PBS(-)], the assembled proteins were eluted into
Laemmli SDS sample buffer and subjected to SDS-PAGE. The gel was
used in either western blot analysis employing an anti-human ATP5B
antibody or silver stain by using Pierce Silver Stain Kit (Thermo Fisher
Scientific) for the further mass analysis.
Hepatitis
B
Grant [19fk0310112 (H.K.), 20fk0310112 (H.K.),
21fk0310112 (H.K.)] from the Japan Agency for Medical Research and
Development (AMED), Japan., a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan [17H06401 (H.K.) and 19H02840 (H.K.).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116375.
4.2.8. Analysis of KUSC-5037/KUSC-5051 binding proteins by LC-MS/MS
The SDS-PAGE separated band was excised and de-stained. The 60
Da protein band shown in Fig. 2A was digested with trypsin (TPCK-
treated, Worthington Biochemical) for 12 h at 37 ◦C. The digested
mixture was analyzed by nano liquid chromatography-tandem mass
spectrometry (LC-MS/MS) using a Q Exactive HF-X mass spectrometer
(Thermo Fisher Scientific) coupled with Easy-nLC 1200 (Thermo Fisher
Scientific). The peptides were separated on a nano ESI spray column (75
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
14