Design, Synthesis, and Structure-Activity Relationships of 1,2,3-Triazole Benzenesulfonamides as New Selective Leucine-Zipper and Sterile-α Motif Kinase (ZAK) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00664

ZAK is a new promising target for discovery of drugs with activity against antihypertrophic cardiomyopathy (HCM). A series of 1,2,3-triazole benzenesulfonamides were designed and synthesized as selective ZAK inhibitors. One of these compounds, 6p binds tightly to ZAK protein (K_d = 8.0 nM) and potently suppresses the kinase function of ZAK with single-digit nM (IC₅₀ = 4.0 nM) and exhibits excellent selectivity in a KINOMEscan screening platform against a panel of 403 wild-type kinases. This compound dose dependently blocks p38/GATA-4 and JNK/c-Jun signaling and demonstrates promising in vivo anti-HCM efficacy upon oral administration in a spontaneous hypertensive rat (SHR) model. Compound 6p may serve as a lead compound for new anti-HCM drug discovery.

Full text of DOI:10.1021/acs.jmedchem.9b00664

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Article
Design, Synthesis and Structure-Activity Relationships
of 1,2,3-Triazole Benzenesulfonamides as new Selective
Leucine-Zipper and Sterile-# Motif Kinase (ZAK) Inhibitors
Jianzhang Yang, Marthandam Asokan Shibu, Lulu Kong, Jinfeng Luo, Farheen BadrealamKhan,
Yanhui Huang, Zheng-Chao Tu, Cai-Hong Yun, Chih-Yang Huang, Ke Ding, and Xiaoyun Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00664 • Publication Date (Web): 04 Jun 2019
Downloaded from http://pubs.acs.org on June 4, 2019
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Design, Synthesis and Structure-Activity
Relationships of 1,2,3-Triazole
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Benzenesulfonamides as new Selective Leucine-
Zipper and Sterile-α Motif Kinase (ZAK) Inhibitors
Jianzhang Yang,†# Marthandam Asokan Shibu,‡# Lulu Kong,§# Jinfeng Luo, Farheen
ǁ
‡
ǁ
ǁ
§*
‡,⊥, *
BadrealamKhan, Yanhui Huang, Zheng-Chao Tu, Cai-Hong Yun, Chih-Yang Huang,
Ke
†
*
†*
Ding, Xiaoyun Lu
†International Cooperative Laboratory of Traditional Chinese Medicine Modernization and
Innovative Drug Development of Chinese Ministry of Education (MOE), College of Pharmacy,
Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
‡Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan
§Department of Biochemistry and Biophysics, Institute of Systems Biomedicine and Beijing Key
Laboratory of Tumor Systems Biology, School of Basic Medical Sciences, Peking University
Health Science Center, Beijing 100191, China
ǁGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan
Avenue, Guangzhou 510530, China
⊥Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan
College of Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi
University, Hualien 970, Taiwan
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KEYWORDS: 1,2,3-triazole benzenesulfonamides, bioisoteric, ZAK, hypertrophic
cardiomyopathy
ABSTRACT: ZAK is a new promising target for discovery of drugs with activity against anti-
hypertrophic cardiomyopathy (HCM). A series of 1,2,3-triazole benzenesulfonamides were
designed and synthesized as selective ZAK inhibitors. One of these compounds, 6p binds tightly
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to ZAK protein (K = 8.0 nM), and potently suppresses the kinase function of ZAK with single
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digit nM (IC = 4.0 nM), and exhibits excellent selectivity in a KINOMEscan screening platform
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0
against a panel of 403 wild-type kinases. This compound dose-dependently blocks p38/GATA-4
and JNK/c-Jun signaling and demonstrates promising in vivo anti-HCM efficacy upon oral
administration in a spontaneous hypertensive rat (SHR) model. Compound 6p may serve as a lead
compound for new anti-HCM drug discovery.
INTRODUCTION
Hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiovascular
disorders affecting 0.2% global populations. HCM has been implied as one of the key factors
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,2
causing sudden cardiac death and end-stage heart failure and stroke etc. Typical characteristics
of HCM include left ventricular hypertrophy (LVH) at a pathological level, myocyte disarray on
cellular level and overexpression of brain natriuretic peptide (BNP), atrial natriuretic peptide
3
,4
(
ANP), and β-myosin heavy chain (MHC) on molecular level. Although a variety of genes have
been reported to be involved in HCM symptoms, the molecular phenotype pathways remain
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,6
unclear and the best available therapy for HCM is inadequate. Current drug treatments for HCM
include primary β-receptor blockers and calcium-channel antagonists. However, the effects of
these drugs are unable to control the cardiac disorders and there is no cure for HCM.7
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The leucine-zipper and sterile-α motif kinase (ZAK) is a member of mixed-lineage kinase
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(
MLK) family, and widely expresses in heart, skeletal muscle, lung and pancreas. Collective
evidence suggested that dysregulation of ZAK is heavily involved in the development of
myocardial disease, e.g. cardiac hypertrophy and myocardial fibrosis.9-10 For instance, in H9c2
cardio myoblast cells, overexpression of ZAK leads to induction of characteristic hypertrophic
features, including an increase in cell size, expression of ANF and BNP, and actin fiber
organization.10-12 ZAK-dominant negative (DN) cardiomyoblast cells are obviously resistant to
hypertrophic feature transition induced by doxycycline, and the expression of ANF and BNP is
also significantly down-regulated in ZAK-DN cells.11,12 These results collectively suggest that that
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ZAK might be a novel promising target for anti-HCM drug discovery. Recently, we reported the
first “proof of concept” study that pharmacological inhibition of ZAK by a small molecule
inhibitor demonstrated a therapeutic impact on cardiac hypertrophy in a SHR model.17
Several kinase inhibitors, such as vemurafenib (1, N-(3-(5-(4-chlorophenyl)-1H-pyrrolo[2,3-
13
b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonami- de), PLX-4720 (2, N-[3-[(5-
chloro-1H-pyrrolo[2,3-b]pyridine-3-yl)carbonyl]-2,4-difluoro-phenyl]-1-propanesulfonamide),13
sorafenib (3, 4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]-amino]carbonyl]amino]phenoxy]-N-
methyl-2-pyridinecarboxamide)14 and nilotinib (4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-
(
trifluoromethyl)-phenyl)-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)benzamide),15 have been
reported to bind and suppress the function of ZAK. However, none of them was discovered with
ZAK as the primary target.16 Recently, a series of N-(3-((1H-pyrazolo[3,4-b]pyridin-5-
yl)ethynyl)benzenesulfonamides were designed and synthesized by our group as a first class of
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selective ZAK inhibitors demonstrating promising anti-HCM therapeutic efficacy. In this paper,
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we report our continuous research into the design and synthesis of 1,2,3-triazole
benzenesulfonamides as new selective ZAK inhibitors with in vivo efficacy.
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Vemurafenib (1)
PLX-4720 (2)
Sorafenib ( )
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Figure 1. Reported non-selective ZAK inhibitors.
CHEMISTRY
Syntheses of the designed molecules are described in Schemes 1-4. Briefly, the commercially
available difluorophenyl compound (7) was nitrated and coupled with the carboxylic acid (9) to
yield the key intermediate (10). Reduction of 10 with Fe/HCl, followed by sulfonylation led to
intermediate 11. Cyclization of 11 with ammonium acetate, followed by deprotection with TFA
produced the 1,3-imidazole 5a (Scheme 1).
Scheme 1. Synthesis of a 1,3-imidazole 5a
PMB
N
N
N
PMB
OH
N
N
F
O
F
F
O
O
F
F
O
N
O N
2
Br
O
Br
9
O
NO2
a
b
O
O
F
7
8
10
F
PMB
O O
N
H
N
N
N
S
Cl
O
F
F
H
N
N
N
HN
N
H
O
F
c,d
S
Cl
N
e,f
O
O
O
O
O
5a
11
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Reagents and conditions: (a) conc. HNO , conc. H SO , rt, 2 h, 90%; (b) (i) Cs CO , dry EtOH,
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2
4
2
3
2
h; (ii) DMF, overnight 52%; (c) Fe powder, conc. HCl (aq), EtOH, H O, 70 °C, 2 h, 91%; (d) m-
2
chlorobenzenesulfonyl chloride, pyridine, dry DCM, rt, overnight, 79%; (e) ammonium acetate,
xylene, 160 °C, reflux, 82%; (f) TFA, reflux overnight, 90%.
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Synthesis of a 1,3,4-oxadiazole compound (5b) is outlined in Scheme 2. The difluoro compound
(12) was sulfonylated and hydrolyzed, then condensed with an acylhydrazine (14) to give the key
intermediate 15. Cyclization of 15 with CCl , Ph P and Et N, followed by deprotection with TFA
4
3
3
led to the 1,3,4-oxadiazole (5b).
Scheme 2. Synthesis of a1,3,4-oxadiazole compound 5b
PMB
N
N
N
F
NHNH2
F
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O
H COOC
3
NH2
14
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Cl
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15
Reagents and conditions: (a) m-chlorobenzenesulfonyl chloride, pyridine, dry DCM, rt,
overnight, 84%; (b) NaOH, EtOH, rt, 3 h, 61%; (c) HATU, DIEA, DCM, rt, 4 h, 92%; (d) Ph P,
3
CCl , Et N, DMF, rt, overnight, 59%; (e) TFA, reflux, overnight, 61%.
4
3
Synthesis of the 1,2,4-oxadiazole compound (5c) is described in Scheme 3. Sulfonylation of the
starting material (16) gave intermediate 17, which reacted with hydroxylamine hydrochloride to
produce (Z)-hydroxylcarboximidamide (18) in 96% yield. Condensation of 18 with 9 produced the
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intermediate 19, which was cyclized with tetrabutylammonium fluoride and deprotected to
produce the 1,2,4-oxadiazole 5c.
Scheme 3. Synthesis of a 1, 2, 4-oxadiazole compound 5c
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H
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Cl
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N
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F
O
19
Reagents and conditions: (a) m-chlorobenzenesulfonyl chloride, pyridine, dry DCM, rt,
overnight, 71%; (b) hydroxylamine hydrochloride, NaHCO , EtOH, reflux, 3 h, 96%; (c) HATU,
3
DIEA, DCM, rt, 4 h, 60%; (d) tetrabutylammonium fluoride, toluene, 110 °C, reflux, 1.5 h, 82%;
(e) TFA, reflux, overnight, 82%.
The 1,2,3-triazole derivatives were synthesized by following a protocol outlined in Scheme 4.
Compounds 20a-20e were diazotized with sodium nitrite to form diazonium salts, which were
subsequently converted into azides (21a-21e). A copper-catalyzed click reaction between alkynes
(22a-22c) and azides (20a-20e) generated the triazoles (23a-23e), which are key intermediates.
Sulfonylation of 23a-23e with substituted benzenesulfonyl chlorides followed by deprotection
with TFA led to good yields of the desired compounds 5b, 6a-6f and 6h-6v. Amidation of 23a
with acyl chlorides (24a and 24b) followed by deprotection provided compounds 6g and 6h. In
addition, coupling of 23a with 1-chloro-3-isocyanobenzene (25), followed by deprotection gave
compound 6i.
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Scheme 4. Synthesis of compounds 5d, 6a-6f and 6h-6v.
F
O
R2
N
H N
2
NO2
N
N
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Cl
HN
N
n
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O
N
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Cl
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n
a
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24a,n=0; 24b,n=1
N
N
R3
R3
R2
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O
O
N3
R3
NO2
PMB
Cl
R4
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S
S
N
N
NH2
N
N
N
R4
O
22a
N
HN
N
O
O
H
R2
R2
N
N
N
c,d
21a-21e
b
23a-23e
5d,6c-f
O
O
6j-r
H N
2
N
Cl
NCO
X
f,d
b
22b,22c
25
F
F
O
N
H N
2
N
NH2
N
N
N
N
H
NH
Cl
N
N
3f,23g
F
HN
N
X
F
N
2
c
6
i
O
F
O
S
N
H
O
N
Cl
H N
2
N
N
X
F
N
6a:X=CH
6b:X=N
Reagents and conditions: (a) (i) TFA, 0°C, NaNO , 30 min; (ii) NaN , 0 °C, 2 h, 84-96%; (b) (i)
2
3
sodium ascorbate, CuSO ·5H O, tert-BuOH, H O, 95 °C, reflux, 3 h; (ii) Fe, conc. HCl (aq), 70
4
2
2
°C, 2 h, 50-62% (two steps); (c) benzenesulfonyl chloride, DCM, pyridine, rt, overnight, 42%-
7%; (d) TFA, reflux, overnight, 55-86%; (e) Et N, dry THF, 64%; (f) rt, THF, 82%.
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RESULTS AND DISCUSSION
We
previously
reported
the
N-(3-((1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)
benzenesulfonamides (e.g. compound 4, Figure 2) as selective ZAK inhibitors, which exhibited
strong inhibitory activity against ZAK and displayed extraordinary target specificity in a
1
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KINOMEscan screening. X-ray crystallographic determination revealed that the N-(3-((1H-
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pyrazolo[3,4-b]pyridine-5-yl)ethynyl) benzenesulfonamide scaffold fits well into the active pocket
of ZAK, and the alkynyl moiety served as a linking group for the pyrazolo[3,4-b]pyridine and the
difluorobenzene (Figure 2). In our continuous efforts to identify new selective ZAK inhibitors,
isosteric replacement of the alkynyl moiety with a conformationally restricted and metabolically
stable heterocyclic substituent was first explored (Figure 2). The kinase inhibitory activity against
ZAK was determined using a previously described protocol.17 Inhibitory activity against B-
RafV600E of these compounds was also assessed to monitor their target selectivity, because of the
high sequence identity between ZAK and B-Raf V600E kinase. 17
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Ala85
N N
N N
O
O N
N
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H
N
N
N
N
His158
O
H
Structure-based
optimization
R3
N
Isosteres
F
F
H
H
N
Het
O
R4 R5
N
O
N
S
Cl
N
N N
N
O
R1
S
Cl
pcket
H
O
R2
HN
F
F
N
Gly153
O
4
5
6
Figure 2. Optimization of 1,2,3-triazole benzenesulfonamides based on the lead compound (4).
The results showed that heterocyclic linkers had different impact on the ZAK inhibitory
potency. For example, the 1,3,4-oxadiazole substituted analogue 5b and the 1, 2, 4-oxadiazole
substituted compound 5c were 4.0-fold and 2.0-fold less potent than the original compound 4,
respectively, while the introduction of 1,3-imidazole linker (5a) caused a 66.9-fold potency loss
(Table 1). Encouragingly, the 1,2,3-triazole derivative 5d exhibited a greatly improved ZAK
inhibitory activity with an IC of 1.9 nM. However, this compound also displayed relatively strong
5
0
inhibition against B-Raf (IC = 198 nM). Preliminary computational investigation suggested that
50
5
d bound to the ATP pocket of ZAK and B-RafV600E in a similar mode to that of compound 4
Figure 3). The 1H-pyrazolo[3,4-b]pyridine moiety could form two hydrogen bonds (HBs) with
(
Ala85 and catch a π−π interaction with Tyr84, while the 1,2,3-triazole group had no direct
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interaction with the protein, but functioned as a linking group. Two additional HBs were also made
between sulfonamide group and residues His158 and Gly153. The m-chlorophenyl was directed
towards a hydrophobic pocket formed by Leu57, Phe68 and Phe157, which was significantly
different from the binding mode of 5d with B-RafV600E where an steric hindrance with Leu505 was
observed. Using 5d as a new starting point, further medicinal chemistry optimization was
conducted with the aim of improving the inhibitory potency and target specificity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. The in vitro inhibitory activities of compounds 5a-5d against ZAK and B-Raf V600E
.
F
H
Het
O
N
N
S
Cl
O
F
HN
N
O
Cpds
4
Het
Kinase inhibition (IC nM)
50
ZAK a
B-RafV600E b
>1000
-
14.7  3.8
983  235
N
5
a
>1000
N
H
N N
O
5
b
63.3  20.1
29.0  3.7
1.9  0.1
>1000
O N
N
5c
>1000
N
N
5
d
198  24.3
N
ZAK and B-Raf V600E inhibition was performed using an ADP-GloTM a and FRET-based Z'-Lyte kinase assay b,
repectively. The data are means from three independent experiments.
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2
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2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
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5
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1
2
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1
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1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Computationally docking studies of 5d with ZAK (PDB: 5X5O) (A) and B-RafV600E
(PDB: 3OG7) (B). The key residues of ZAK kinase are shown in a blue and gray stick
representation. Compound 5d is shown in a blue and orange stick structure. HBs to key amino
acids are indicated by yellow dashed lines.
Further computational investigation suggested that π−π stacking interactions with Tyr84 and
Trp531 were critical for the 1H-pyrazolo[3,4-b]pyridine scaffold of 5d to adopt a suitable
orientation to form HBs with the hinge residues of ZAK protein (Figure 3). Substitution of the 1H-
pyrazolo[3,4-b]pyridine moiety (5d) with a 2-pyridinyl (6a) and a 2-pyrimidinyl (6b) group indeed
caused a 24-fold and 4-fold potency decrease against ZAK, respectively, while totally abolished
the B-RafV600E inhibition (Table 2). Contribution of the difluoro substitution in 5d to ZAK
inhibition was also evaluated. Removal of the di-fluoro substituents resulted a 13-fold potency loss
(
6c), but the monofluoro-substituted compounds 6d and 6e retained strong ZAK inhibition (IC =
50
5.7 and 3.0 nM). However, the dichloroderivative 6f exhibited a significantly decreased potency
with an IC of 413.3 nM.
5
0
Modification of the sulfonamide part (R ) was also conducted (Table 2) and it was shown that
4
sulfonamide moiety in 5d was crucial for the compound to demonstrate strong ZAK inhibition.
Replacement of the sulfonamide group with an amide (6g), methylene amide (6h) or urea group
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(
6i) significantly decreased the ZAK inhibitory potency. Compound 6g, 6h and 6i exhibited IC50
values of 54.3, 85.7 and 53 nM, respectively (Table 2).
Table 2. The in vitro inhibitory activities of compounds 6a-6i against ZAK and B-Raf V600E
.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R3
R4
Cl
N
N
R1
R2
N
Cpds
R1
R2
R3
R4
Kinase inhibition (IC nM)
50
ZAKa
B-RafV600E b
H
O
5
6
d
a
N
N
F
F
F
F
1.90.1
19824.3
N
S
N
H O
O
H2N
H2N
N
O
46.2±9.9
>1000
S
N
H O
N
O
6
6
b
c
F
F
8.9±2.3
>1000
>1000
S
N
N
H O
H
N
O
N
H
H
24.7±0.5
S
N
N
H O
O
H
N
O
6
6
6
6
6
d
e
f
N
N
N
N
N
H
F
F
H
Cl
F
5.7±0.9
3.0±0.5
334±141nM
795±8.5
S
N
N
H O
O
H
N
O
S
N
N
H O
O
H
N
O
Cl
F
413.3±75.8
54.3±8.7
436±91.4
11.4±2.3
S
N
N
H O
O
H
N
O
g
h
N
N
H
O
H
N
O
F
F
85.7±25.1
83.1±31.3
N
N
H
O
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
O
6
i
N
F
F
53.0±7.0
51.6±2.8
N
N
N
N
H
H
O
ZAK and B-Raf V600E inhibition was performed using an ADP-GloTM a and FRET-based Z'-Lyte kinase assay b,
repectively. The data are means from three independent experiments.
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
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8
9
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1
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1
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9
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1
2
3
4
5
6
7
8
9
0
Our previous investigation revealed that ZAK possesses a larger binding pocket comparing with
that of B-RafV600E due to the outwardly shifting αC-helix.17 It was thus hypothesized that
introduction of a hydrophobic substituent to the tail phenyl group might improve the inhibitory
17
activity and selectivity of ZAK. Accordingly, extensive modification on R position was
5
conducted. The results showed that removal of the chlorine substituent (6j) indeed caused a 2-fold
potency loss with respect to ZAK, while its inhibitory potency against B-RafV600E was obviously
improved (IC50 = 39 nM), which was consistent to our prediction. The investigation also
demonstrated that the m-chloro substituent could be replaced by a variety of groups such as bromo
(6k), cyano (6l), methoxyl (6m) and methyl (6n) without affecting the ZAK inhibitory potency.
Inhibitors 6k, 6l, 6m and 6n exhibited IC values of 2.1, 3.3, 2.4 and 4.4 nM, respectively (Table
5
0
3
).
Co-crystal structure of 6k with ZAK determination validated our proposed binding mode of the
molecules (Figure 4). Consistent to our prediction, the 1H-pyrazolo[3,4-b]pyridine moiety made
two HBs with Ala85, and captured an additional HB and a π−π interaction with Tyr84. The 1,2,3-
triazole forms a HB network with Lys45 and Asp151 mediated by H O. The sulfonamide
2
participated in a single HB interaction with Gly153 because of the rotation of His158. Similar to
our previous observation,17 the m-bromophenyl moiety could induce an obviously outward
extension of αC helix and caused a rotation of Leu57 to avoid steric hindrance with 6k (Figure 4).
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
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6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Co-crystal structure of ZAK and 6k (PDB: 6JUT). Compound 6k is shown in purple
stick structure. The key residues of ZAK kinase are shown in gray and cyan sticks. Hydrogen
bonds to key amino acids are indicated by yellow dashed lines.
Based on the structural information, it was further hypothesized that a large hydrophobic group
might be accommodated at the R position to achieve strong and selective ZAK inhibition. Indeed,
5
m-isopropyl phenyl (6o), m-biphenyl (6p), 2-naphthyl (6q) and 1-naphthyl (6r) analogues retained
strong ZAK inhibitory activities with IC50 values of 6.9, 4.0, 4.5 and 4.2 nM, respectively.
Significatly, the m-biphenyl-substituted molecule (6p) totally abolished the inhibition against B-
RafV600E, becoming one of the most potent and selective ZAK inhibitors in this series.
Table 3. In vitro inhibitory activities of compounds 6j-6r against ZAK and B-Raf V600E
.
F
O
N
S
O
R5
N
N
N
HN
N
H
F
N
O
Cpds
R5
Kinase inhibition (IC nM)
50
ZAKa
B-RafV600E b
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
5
d
1.90.1
19824.3
6
j
3.8±1.8
39±11.6
Br
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6k
2.1±0.3
272.3±68.9
470.3±165.6
272±35.8
453±98
CN
6
l
3.3±0.5
O
6
m
2.3±0.4
6
n
4.4±1.1
6.9±1.2
4.0±1.0
6
o
190±73.9
>1000
6
p
6q
6r
4.5±0.6
4.2±1.4
856±75.2
125.7±18.3
ZAK and B-Raf V600E inhibition was performed using an ADP-GloTM a and FRET-based Z'-Lyte kinase assay b,
repectively. The data are means from three independent experiments.
The ZAK inhibition by 6p was validated by determining its binding affinity with an active
1
8
site-dependent competition binding assay (DiscoveRx, San Diego, CA). Compound 6p bound
tightly to the ATP-binding site of ZAK (K = 8.0 nM), validating its strong kinase inhibition
d
against ZAK. The target selectivity of 6p was also evaluated by using a KINOMEscan study
against 468 kinases at 1.0 μM (Figure 5A). The results revealed that 6p displayed an excellent
1
8
target selectivity profile (see Supporting information, Table S1). Several kinases, e.g. abelson
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murine leukemia viral oncogene homolog 1 (ABL1), B lymphoid tyrosine kinase (BLK), FMS-
like tyrosine kinase 3 (FLT3 ITD, F691L), lymphocyte-specific protein tyrosine kinase (LCK),
LIM domain kinase 1 (LIMK1), LIM domain kinase 2 (LIMK2) and receptor-interacting
serine/threonine-protein kinase 2 (RIPK2) were identified as the major “off targets” (inhibition >
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
0%, ctrl% < 10) (Figure 5B). Further evaluation reveled that 6p demonstrated 2-66- fold target
selectivity over these “off targets” (Figure 5B). Kinase activity determination also showed that 6p
inhibited ABL1 with an IC of 156 nM, which was approximate 30 folds less potent than that of
5
0
ZAK kinase (Figure 5B).
Figure 5. KINOMEscan profiling of 6p. (A) KINOMEscan profiling of 6p at a concentration of
.0 M against 468 kinases. (B) The K determination of compound 6p against the potential off
1
d
targets.
Collective studies have suggested that activation of ZAKα signaling is critical for cardiac
hypertrophy.19,20 The inhibitory effects of 6p on the activation of ZAKα were therefore
investigated in H9c2 cells (Figure 6A). The results showed that 6p dose-dependently inhibited the
phosphorylated ZAKα, but had no detectable impact on the ZAKα expression level. Consequently,
an in vitro AngII induced H9c2 cardio myoblast cell model was utilized to evaluate the therapeutic
effect of 6p on hypertrophy. The results showed that AngII obviously elevated the expression of
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2
2
2
2
2
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3
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5
6
BNP and ZAKα downstream MAPK kinases, e.g. p38 and JNK. Whereas, compound 6p potently
suppressed phosphorylation of p38, JNK and dose-dependently decreased the expression of BNP
in H9c2 cardio myoblast cells. The results demonstrated the blockage of 6p on the myocardial
hypertrophy process induced by the AngII (Figure 6B).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
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1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. (A) Effects of p-ZAKα and ZAKα inhibition by 6p in H9c2 cells. (B) Effects of ZAKα
downstream signaling inhibition by 6p in AngII induced H9c2 cells. The data means from three
independent experiments
The potential therapeutic effect of 6p was further validated in a cell-based Tet-on ZAKα system
2
1
(
Figures 7 and 8). It was shown that doxycycline (Dox) significantly induced Z hypertrophic
effects in H9c2 cells with increased cell surface area and elongation of actin filaments as
determined by F-actin staining (Figures 7A and 7B), but hypertrophic phenomena could be
potently suppressed by a ZAK inhibitor 6p in a dose-dependent manner (Figure 7A and 7B). The
results also showed that Dox obviously induced ZAKα expression in a Tet-on ZAKα system,
leading to the elevation of p-JNK and p-p38 (Figures 8A and 8B) and hypertrophic transcription
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factors p-GATA-4 and p-c-Jun (Figures 8C and 8D). Whereas, the activation of these key factors
could be evidently suppressed by compound 6p (Figures 8C and 8D). The immunofluorescence
staining further showed that ZAKα overexpression obviously elevated the nuclear translocation of
p-GATA-4, while the corresponding processes were potently suppressed by 6p. These results
further validated that compound 6p could effectively suppress hypertrophy induced by ZAKα
overexpression by blocking p38/GATA-4 and JNK/c-Jun signaling.9
1
1
1
1
1
1
1
1
1
1
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5
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1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Effects of 6p suppressing the hypertrophic effects of ZAKα in a Tet-on ZAKα system.
A) Cells were cultured in serum-free medium for 12 h, and treated with Dox (2 µg/mL) and 6p
(2.5, 5 and 10 g/mL) for 24 h, then stained with phalloidin-rhodamine to measure the cell size.
DAPI staining was used to mark nuclei. B) The statistical results were shown from three
independent experiment (***p<0.001, compared with control group ##p<0.01, ###p<0.001
compared with Dox group)
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0
1
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0
Figure 8. Effects of 6p suppressing the p-JNK and p-p38 and downregulating the levels of p-
GATA-4 (A and B) and p-c-Jun (C and D). The statistical results were shown from three
independent experiment (**p<0.01, ***p<0.001 compared with control group; # p<0.05,
##p<0.01, ###p<0.001 compared with Dox group)
Figure 9. Immunofluorescence staining for p-GATA4. The cells were incubated at 4 ℃ overnight
with primary antibody (1:400 diluted with 1% BSA in PBS), then washed with PBS and incubated
in diluted secondary antibody and incubated in the dark at 37 °C for 1 h. The cells were
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subsequently counterstained by 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain. The arrows
in the Tet-on-Zakα+Dox group indicate nuclear localisation of p-GATA-4.
The in vivo pharmacokinetic (PK) profiles of 6p were also determined in Sprague-Dawley (SD)
rats (Table 4). It was shown that compound 6p displays encouraging PK profiles with an oral AUC
1
1
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.
value of 71790.9 ug/L h, a half-life value (T ) of 1.7 hr and an oral bioavailability of 65.3%.
1
/2
These data provided a foundation for the follow-up in vivo oral dosing regimen.
Table 4. Pharmacokinetic profiles of compound 6p in rats.
parameters
po (10mg/kg)
71790.9
71572.6
1.7
iv (2.5 mg/kg)
27866.1
27424.4
1.6
AUC(0-∞) ug/L*h
AUC(0-t) ug/L*h
T (h)
1/2
Cmax (ug/L)
CL(L/h/kg)
BA (%)
16372.2
nd
34151.6
91.9
65.3
nd. Not determined.
The spontaneous hypertensive rats (SHR) model was utilized to investigate the potential in vivo
anti-HCM effects of 6p with the normotensive Wistar-Kyoto (WKY) rat as a control. The results
showed that the SHR performed greater left ventricular masses than normotensive WKY rats,
indicating the symptoms of hypertrophy (Table 4). Compound 6p was orally administrated at 0,
2
.5 and 10 mg/kg once daily for 8 weeks. The whole heart (WH) and the left ventricle (LV) were
collected for biological determination after the experimental rats were sacrificed. The results
demonstrated that compound 6p suppressed the WH and LV mass elevation and rescued the
hypertrophic symptoms in a dose-dependent manner (Table 5). Briefly, the average LVW value
was 0.89 g for the vehicle control SHRs, while the corresponding values were decreased to 0.82
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and 0.78 g, respectively, after treatment with 2.5 mg/kg and 10 mg/kg of 6p. Western blot analysis
showed that the hypertrophy-related signaling mediators and markers such as p-JNK, p-c-Jun, p-
GATA-4, p-P38 and ANP were considerably higher in SHR than that in the normotensive WKY
control rats (Figures 10A and 10B). Consistent to the observation in cell-based models, compound
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p suppressed the phosphorylation of ZAK downstream signaling JNK, p38, c-Jun, p-GATA-4
and protein levels of hypertrophic markers BNP and ANP in a dose-dependent manner.
Hematoxylin and eosin (H&E) staining investigation also showed normal alignment of
cardiomyocytes in the normotensive WKY control tissues, while much more interstitial spaces in
the SHRs heart tissues (Figure 10C). Whereas, the myocardial cell disarrangement was
significantly relieved by 6p to normal phenomena (Figure 10C). The studies further supported the
oral therapeutic effects of 6p on cardiac hypertrophy in SHRs by suppression of ZAK downstream
p38/GATA-4 and JNK/c-Jun signaling.
Table 5. Compound 6p dose-dependently inhibits myocardial hypertrophy in vivo.
Weight (g)
Whole
WHW)
Left
LVW)
WKY
SHR
6p-2.5 mg/kg
6p-10 mg/kg
heart 1.08±0.009 1.23±0.006###
1.18±0.008
1.13±0.005**
(
ventricular 0.70±0.007 0.89±0.005###
0.82±0.007
0.78±0.007***
(
Values are Mean ± SD; Statistical significance: ### P≤ 0.001 vs WKY; **P ≤ 0.01; ***P ≤ 0.001 vs SHR group
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Figure 10. (A) Effects of 6p suppressing the phosphorylation of ZAK downstream signaling of
JNK, p38, c-Jun and GATA-4, and the expression of hypertrophic marker proteins (BNP and ANP)
in vivo. (B) The statistical results from three independent experiments are shown. (C) H&E
#
staining investigation of the heart tissues by treatment of 6p. Statistical significance: P ≤ 0.05
#
#
and P ≤ 0.01 compared to WKY Control; *P ≤ 0.05 and **P ≤ 0.01 compared to SHR
CONCLUSION
A series of 1,2,3-triazole benzenesulfonamides were designed and optimized as novel selective
inhibitors of ZAK. One of the most potent compounds, 6p bound tightly to ZAK with a K of 8.0
d
nM, and potently inhibited ZAK activity with an IC of 4.0 nM, while it was significantly less
5
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potent on majority of the evaluated kinases at 1.0 μM in KINOMEscan profiling. The compound
significantly suppressed the ZAK overexpression induced hypertrophy both in vitro and in vivo by
blocking p38/GATA-4 and JNK/c-Jun signaling, and displayed promising anti-HCM effect in the
SHR model at the low dose (10mg/kg). Compound 6p may be utilized as a promising lead
compound for further development.
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EXPERIMENTAL SECTION
General Methods for Chemistry. All commercially available reagents and solvents were used
as received. All reactions were monitored by thin layer chromatography (TLC). Compounds were
detected by ultraviolet light (UV) absorption at either 254 or 365 nm. Column chromatography
1
was used with silica gel 300 mesh. H NMR, spectra were performed with Bruker AV-400
13
spectrometer, C NMR spectra were performed on Bruker AV-400 spectrometer at 101 MHz,
internal reference was either TMS or deuterated NMR solvent. The low resolution mass was
performed on an Agilent 1200 HPLC-MSD mass spectrometer. High resolution mass spectral
analysis was performed on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass
spectrometer. Purity of all final compounds were confirmed to be >95% by HPLC analysis with
the Agilent 1260 system. The analytical columns were YMC-Triart C reversed-phase column, 5
18
μm, 4.6×250 mm, flow rate 1.0 mL/min.
General Procedure A for Sulfonylation:
Sulfonyl chlorides (1.2 equiv) and pyridine (1.5 equiv) were gradually added dropwise to a
solution of anilines (1.0 equiv) in anhydrous DCM (20 mL), then stirred at r.t. overnight. The
mixture was concentrated in vacuo and purified by column chromatography (25-50% EtOAc in
petroleum ether) to produce the desired compounds in 42%-97% yields.
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General Procedure B for PMB deprotection:
Benzenesulfonamides (0.15 mmol) were refluxed with trifluoroacetic acid (5 mL) overnight,
then cooled to r.t. and concentrated in vacuo. The mixture was quenched with H O, neutralized to
2
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pH 7 with saturated NaHCO solution and extracted with EtOAc three times. The combined
3
organic phases were washed with brine, dried over anhydrous sodium sulfate and evaporated. The
crude material was further purified by flash column chromatography eluting with 1.25-2.5%
MeOH in DCM to produce the desired compounds. (Yield: 55%-86%).
3
-Chloro-N-(2,4-difluoro-3-(5-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-imidazol-
2
-yl)phenyl)benzenesulfonamide (5a)
Compound 11 (100 mg, 0.15 mmol) and ammonium acetate (59 mg, 7.6 mmol) were dissolved
in xylene (25 mL), then stirred at 160 °C overnight (TLC: EtOAc: petroleum ether = 1: 1, Rf~0.4).
The mixture was concentrated in vacuo and purified by column chromatography (40% EtOAc in
petroleum ether) to afford PMB protection 1,3-imidazole compound as a yellow solid (80 mg,
1
yield 82%). H NMR (400 MHz, DMSO-d ) δ 12.99 (s, 1H), 10.36 (s, 1H), 9.14 (d, J = 2.0 Hz,
6
1
7
H), 8.59 (d, J = 2.0 Hz, 1H), 7.76 (p, J= 3.5 Hz, 2H), 7.70-7.67 (m, 1H), 7.62 (t, J = 8.2 Hz, 1H),
.53 (s, 1H), 7.24-7.20 (m, 2H), 7.16 (d, J = 8.8 Hz, 1H), 6.90-6.86 (m, 2H), 5.47 (s, 2H), 4.03 (d,
J = 4.0 Hz, 3H), 3.71 (s, 3H).
The above product was treated according to the general procedure B to afford 5a as a white
1
solid. (Yield: 90%; TLC: EtOAc: petroleum ether = 1: 1, Rf~0.1). H NMR (400 MHz, DMSO-
d6) δ 12.98 (s, 1H), 12.77 (s, 1H), 10.40 (s, 1H), 9.12 (s, 1H), 8.60 (s, 1H), 7.75 (d, J = 10.2 Hz,
2
H), 7.69 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.27-7.07 (m, 2H), 4.05 (s,
1
3
3
H). C NMR (101 MHz, DMSO-d ) δ 159.06, 156.53, 155.59, 152.87, 152.16, 148.30, 144.88,
6
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42.03, 134.29, 133.48, 131.82, 129.35, 126.58, 125.77, 125.34, 121.17, 119.82, 119.05, 113.14,
12.19 (d, J= 25.5 Hz, 1C), 103.49, 56.25. HPLC analysis: MeOH: H O (50: 50), 15.32 min;
2
+
purity: 97.4%. HRMS (ESI) for C H ClF N O S [M+H] , calcd: 517.0656; found: 517.0648.
2
2
15
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3
-Chloro-N-(2,4-difluoro-3-(5-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1,3,4-
oxadiazol -2-yl)phenyl)benzenesulfonamide (5b)
A mixture of compound 15 (1.4 g, 2.17 mmol), triphenyl-phosphine (682 mg, 2.6 mmol), CCl4
(400 mg, 2.6 mmol) and DIPEA (420 mg, 3.3 mmol) in DMF (50 mL) was stirred at r.t. under
argon for overnight (monitored by TLC: EtOAc: petroleum ether = 1: 1, Rf~0.7). The mixture was
quenched with H O and extracted with EtOAc three times. The organic phases were washed with
2
brine, dried over anhydrous Na SO and evaporated. The residue was purified by flash column
2
4
chromatography (30% EtOAc in petroleum ether) to obtain PMB protection 1,3,4-oxadiazol
1
compound as a yellow solid (800 mg, yield 59%). H NMR (400 MHz, DMSO-d ) δ 10.82 (s, 1H),
6
9.16 (d, J = 2.0 Hz, 1H), 8.61 (d, J = 2.1 Hz, 1H), 7.82 (t, J = 1.9 Hz, 1H), 7.77 (dd, J = 8.0, 2.1
Hz, 1H), 7.73-7.69 (m, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.53 (d, J = 5.7 Hz, 1H), 7.41 (d, J = 9.5 Hz,
1
H), 7.28-7.21 (m, 2H), 6.92-6.85 (m, 2H), 5.51 (s, 2H), 4.06 (s, 3H), 3.71 (s, 3H).
The above product was treated by the general procedure B to afford 5b as white solid (Yield:
1
6
1
2
1%; TLC: EtOAc: petroleum ether = 1: 1, Rf~0.2). H NMR (400 MHz, DMSO-d ) δ 13.17 (s,
6
H), 10.66 (s, 1H), 9.10 (d, J = 2.0 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 7.79 (dd, J = 7.4, 1.4 Hz,
H), 7.70 (dt, J = 7.8, 1.6 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.57 (td, J = 8.9, 5.6 Hz,1H), 7.46-
1
3
7.39 (m, 1H), 4.08 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 164.10, 159.19, 156.61, 156.05,
6
1
1
55.97, 152.91, 148.38, 141.75, 134.42, 133.68, 132.39 (d, J = 10.1 Hz, 1C), 131.97, 128.50,
26.60, 125.84, 122.03 (d, J = 13.0 Hz, 1C), 113.72 (d, J = 22.3 Hz, 1C), 111.79, 103.68, 103.25
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(
d, J = 17.5 Hz, 1C), 56.43. HPLC analysis: MeOH: H O (50: 50), 20.21 min; purity:100%. HRMS
2
+
(ESI) for C H ClF N O S [M+H] , calcd: 519.0448; found: 519.0448.
2
1
13
2
6
4
3-Chloro-N-(2,4-difluoro-3-(5-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1,2,4-
1
1
1
1
1
1
1
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1
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oxadiazol-3-yl)phenyl)benzenesulfonamide (5c)
Compound 19 (100 mg, 0.15 mmol) and tetra-butylammonium fluoride (1.0 M solution in THF,
0
.18 mL) were added in toluene and the mixture was refluxed at 110 °C for 1.5 h monitored by
TLC (EtOAc: petroleum ether = 1: 5, Rf~0.3). The mixture was concentrated in vacuo, and
purified by column chromatography (20% EtOAc in petroleum ether) to obtain PMB protection
1
1
,2,4-oxadiazol compound as a yellow solid (80 mg, yield 82%). H NMR (400 MHz, DMSO-d )
6
δ 10.61 (s, 1H), 9.23 (t, J = 1.8 Hz, 1H), 8.83 (t, J = 1.8 Hz, 1H), 7.80-7.75 (m, 2H), 7.71-7.67 (m,
H), 7.64 (dd, J = 8.2, 1.6 Hz, 1H), 7.54 (d, J = 6.8 Hz, 1H), 7.38 (t, J = 8.9 Hz, 1H), 7.28-7.22
m, 2H), 6.89 (dd, J = 8.6, 1.7 Hz, 2H), 5.52 (s, 2H), 4.06 (d, J = 1.5 Hz, 3H), 3.72 (d, J = 1.5 Hz,
3H).
1
(
The above product was treated by the general procedure B to afford 5c as a white solid. (Yield:
1
8
1
2
9
1
2%; EtOAc: petroleum ether = 1: 3, TLC Rf~0.2). H NMR (400 MHz, DMSO-d ) δ 13.25 (s,
6
H), 10.62 (s, 1H), 9.17 (d, J = 2.1 Hz, 1H), 8.80 (d, J = 2.1 Hz, 1H), 7.77 (dt, J = 4.1, 1.8 Hz,
H), 7.71-7.66 (m, 1H), 7.63 (dd, J = 10.1, 6.2 Hz, 1H), 7.53 (td, J = 8.8, 5.6 Hz, 1H), 7.37 (t, J =
1
3
.1 Hz, 1H), 4.08 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 175.33, 160.80, 159.44, 156.90,
6
56.30, 153.17, 149.53, 141.89, 134.39, 133.64, 131.93, 131.78 (d, J = 11.1 Hz, 1C), 130.38,
126.54, 125.76, 121.91 (d, J = 14.1 Hz, 1C), 113.27 (d, J = 20.2 Hz, 1C), 111.94, 105.72 (t, J =
1
7.2 Hz, 1C), 103.87, 56.50. HPLC analysis: MeOH: H O (50: 50), 26.99 min; purity: 100%.
2
+
HRMS (ESI) for C H ClF N O S[M+H] , calcd: 519.0448; found: 519.0443.
2
1
13
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-Chloro-N-(2,4-difluoro-3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-
triazol-1-yl)phenyl)benzenesulfonamide (5d)
5d was synthesized from 23a (120 mg, 0.26 mmol) with general procedure A to produce 4-
chloro-N-(2,4-difluoro-3-(4-(3-methoxy-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-b]-pyridinyl)-
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H-1,2,3-triazol-1-yl)phenyl)benzenesulfo namideas a yellow solid (135 mg, yield 82%; TLC:
1
EtOAc/petroleum ether=1:3, Rf~0.3). H NMR (400 MHz, DMSO-d ) δ 10.75 (s, 1H), 9.12 (d, J
6
=
2.0 Hz, 1H), 9.11 (s, 1H), 8.58 (d, J = 2.0 Hz, 1H), 7.83-7.77 (m, 2H), 7.74-7.70 (m, 1H), 7.66
(
d, J = 7.8 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.26-7.21 (m, 2H), 6.90-6.86 (m, 2H), 5.48 (s, 2H),
+
4
.04 (s, 3H), 3.71 (s, 3H). HRMS (ESI) for C H ClF N O S [M+H] , calcd: 638.1183; found:
29 22
2
7
4
638.1184.
The above product was treated by the general procedure B to afford 5d as a white solid. (Yield:
1
7
6%; TLC: EtOAc: petroleum ether = 1:3, Rf~0.1). H NMR (400 MHz, DMSO-d ) δ 12.76 (s,
6
1H), 10.75 (s, 1H), 9.09 (s, 1H), 9.06 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 7.82-7.76 (m,
13
2
H), 7.72 (d, J = 7.9 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.56-7.46 (m, 2H), 4.05 (s, 3H). C NMR
(
101 MHz, DMSO-d ) δ 155.44, 152.91, 152.44, 149.97, 148.18, 145.32, 142.17, 134.41, 133.63,
6
1
1
31.96, 129.62 (d, J = 8.4 Hz, 1C), 126.58, 125.84, 125.76, 124.38, 122.79 (d, J = 11.1 Hz, 1C),
18.41, 115.45 (t, J = 14.8 Hz, 1C), 113.11 (d, J = 20.4 Hz, 1C), 103.79, 56.17. HPLC analysis:
+
MeOH: H O (50: 50), 9.61 min, 97.8% purity. HRMS (ESI) for C H ClF N O S [M+H] , calcd:
2
21 14
2
7
3
5
18.0608; found: 518.0601.
N-(3-(4-(6-Aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)-2,4-difluorophenyl)-3-chloro-
benzenesulfonamide (6a)
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Compound 6a was synthesized from 23f (60 mg, 0.21 mmol) with the general procedure A as a
1
white solid (44 mg, yield 50%; TLC: EtOAc: petroleum ether = 1: 1, Rf~0.5). H NMR (400 MHz,
DMSO-d ) δ 10.79 (s, 1H), 8.80 (s, 1H), 8.49-8.40 (m, 1H), 7.87 (dd, J = 8.6, 2.4 Hz, 1H), 7.78
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
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0
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0
(
d, J = 8.6 Hz, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.53-7.42 (m, 2H), 6.56 (d,
1
3
J = 8.6 Hz, 1H), 6.29 (s, 2H). C NMR (101 MHz, DMSO-d ) δ 160.19, 154.85, 152.36, 149.82,
6
1
45.64 (d, J = 8.3 Hz, 1C), 142.84, 134.93, 134.29, 133.25, 131.83, 128.73, (d, J = 9.1 Hz, 1C),
26.54, 125.70, 124.12 (d, J = 11.1Hz, 1C), 122.67, 115.51 (t, J = 14.1 Hz, 1C), 114.47, 112.85
1
(
d, J = 20.2 Hz, 1C), 108.51. HPLC analysis: MeOH: H O (55: 45), 3.65 min, 97.6% purity. HRMS
2
+
(
ESI) for chemical formula: C H ClF N O S[M+H] , calcd: 463.0550; found: 463.0540.
19 13
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6
2
N-(3-(4-(2-Aminopyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-2,4-difluorophenyl)-3-chloro-
benzenesulfonamide (6b)
Compound 6b was synthesized from 23g (70 mg, 0.24 mmol) with the general procedure A as
1
a white solid (69 mg, yield 62%; TLC: MeOH: DCM = 1: 15, Rf~0.5). H NMR (400 MHz,
DMSO-d ) δ 10.75 (s, 1H), 8.89 (s, 1H), 8.75 (s, 2H), 7.79 (dd, J = 7.9, 1.4 Hz, 2H), 7.71 (dt, J =
6
1
3
7
.9, 1.5 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.55-7.44 (m, 2H), 7.03 (s, 2H). C NMR (101 MHz,
DMSO-d ) δ 163.58, 155.78 (s, 2C), 153.20, 152.60, 150.04, 143.21, 141.74, 134.45, 133.81,
6
1
32.02, 129.97 (d, J = 9.4 Hz, 1C), 126.59, 125.80, 123.16, 122.16 (d, J = 11.7 Hz, 1C), 115.47 (t,
J = 14.7 Hz, 1C), 113.18 (t, J = 10.5 Hz, 1C). HPLC analysis: MeOH: H O (50: 50), 8.36 min;
2
+
purity: 100%. HRMS (ESI) for chemical formula: C H ClF N O S [M+H] , calcd: 464.0503;
18 12
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7
2
found: 464.0505.
3
-Chloro-N-(3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)-
phenyl)benzenesulfonamide(6c)
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Journal of Medicinal Chemistry
Page 28 of 55
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Compound 6c was synthesized from 23b by a procedure similar to that of 5d (Yield: 59% (two
1
steps); TLC: EtOAc: petroleum ether = 1: 2, Rf~0.2). H NMR (400 MHz, DMSO-d ) δ 12.75 (s,
6
1H), 10.86 (s, 1H), 9.36 (s, 1H), 9.09 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 7.86 (t, J = 1.9
Hz, 1H), 7.79 (ddt, J = 3.8, 2.7, 1.3 Hz, 2H), 7.74 (ddd, J = 8.1, 2.2, 1.1 Hz, 1H), 7.65-7.58 (m,
0
1
2
3
4
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7
8
9
0
1
2
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7
8
9
0
1
2
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8
9
0
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2
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8
9
0
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5
6
7
8
9
0
1
3
2
1
1
H), 7.54 (t, J = 8.0 Hz, 1H), 4.06 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 155.43, 152.38,
6
48.11, 145.95, 141.39, 139.25, 137.61, 134.46, 133.74, 132.01, 131.53, 126.72, 125.92, 125.65,
20.33, 119.86, 118.84, 116.06, 111.70, 103.80, 56.18. HPLC analysis: MeOH: H O (70: 30), 6.47
2
+
min, 98.1% purity. HRMS (ESI) for C H ClN O S [M+H] , calcd: 482.0797; found: 482.0793.
2
1
16
7
3
3
-Chloro-N-(4-fluoro-3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-
1-yl)phenyl)benzenesulfonamide (6d)
Compound 6d was synthesized from 23c by a procedure similar to that of 5d. (Yield: 71% (two
1
steps); TLC: EtOAc: petroleum ether = 1: 2, Rf~0.1). H NMR (400 MHz, DMSO-d ) δ 12.75 (s,
6
1H), 10.82 (s, 1H), 9.17 (d, J = 2.3 Hz, 1H), 9.11 (d, J = 2.0 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H),
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.83 (t, J = 1.9 Hz, 1H), 7.77 (t, J = 1.6 Hz, 1H), 7.76-7.73 (m, 1H), 7.67 (dd, J = 6.5, 2.7 Hz, 1H),
.63 (t, J = 7.9 Hz, 1H), 7.56 (dd, J = 10.4, 9.0 Hz, 1H), 7.30 (ddd, J = 9.0, 4.0, 2.7 Hz, 1H), 4.05
1
3
(
s, 3H). C NMR (101 MHz, DMSO-d ) δ 155.45, 152.41, 151.83, 149.36, 148.21, 145.73,
6
1
1
41.18, 134.89 (d, J = 2.8 Hz, 1C), 134.51, 133.81,132.07, 126.70, 125.92 (d, J = 4.9 Hz, 1C),
25.27 (d, J = 11.9 Hz, 1C), 123.09 (d, J = 7.7 Hz, 1C), 122.80 (d, J = 5.8 Hz, 1C), 118.91, 118.62
(
d, J = 7.9 Hz, 1C), 117.32, 103.79, 56.18. HPLC analysis: MeOH: H O (85: 15), 5.41 min; purity:
2
+
95.9%. HRMS (ESI) for C H ClFN O S [M+H] , calcd: 500.0702; found: 500.0700.
2
1
15
7
3
3
-Chloro-N-(2-fluoro-3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-triazol-
1
-yl)phenyl)benzenesulfonamide (6e)
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Compound 6e was synthesized from 23d with by a procedure similar to that of 5d. (Yield: 69%
1
(
two steps); TLC: EtOAc: petroleum ether = 1: 3, Rf~0.1). H NMR (400 MHz, DMSO-d ) δ 12.75
6
(s, 1H), 10.81 (s, 1H), 9.11 (s, 1H), 9.10 (t, J = 1.5 Hz, 1H), 8.59 (s, 1H), 7.85-7.81 (m, 1H), 7.76
1
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5
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0
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2
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0
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13
(
d, J = 6.3 Hz, 2H), 7.65 (q, J = 8.2 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 4.05 (s, 3H). C NMR (101
MHz, DMSO-d ) δ 155.42, 152.36, 149.58, 148.19, 147.05, 145.45, 142.13, 134.40, 133.67,
6
1
31.99, 126.99, 126.58, 125.90, 125.81 (s, 2C), 125.76, 123.25, 123.04, 118.65, 103.77, 56.18.
HPLC analysis: MeOH: H O (50: 50), 16.52 min; purity: 97.0%. HRMS (ESI) for
2
+
C H ClFN O S [M+H] , calcd: 500.0702; found: 500.0702 .
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1
15
7
3
3
-Chloro-N-(2,6-dichloro-3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-
triazol-1-yl)phenyl)benzenesulfonamide (6f)
Compound 6f was synthesized from 23e by a procedure similar to that of 5d (Yield: 49%, (two
1
steps); TLC: EtOAc: petroleum ether = 1: 1, Rf~0.1). H NMR (400 MHz, DMSO-d ) δ 12.77 (s,
6
1H), 10.79 (s, 1H), 9.07 (d, J = 2.0 Hz, 1H), 9.05 (s, 1H), 8.55 (d, J = 2.0 Hz, 1H), 7.84 (d, J = 1.9
Hz, 1H), 7.80 (dd, J = 8.7, 2.8 Hz, 2H), 7.75 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.55 (d,
1
3
J = 8.9 Hz, 1H), 4.05 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 155.46, 152.33, 148.07, 145.28,
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1
1
9
42.23, 134.61, 134.52, 133.79, 133.64, 132.03, 130.34, 130.13, 129.52, 129.45, 126.6, 125.79,
25.69, 123.74, 118.50, 103.79, 56.22. HPLC analysis: MeOH: H O (70: 30), 9.68 min; purity:
2
+
8.4%. HRMS (ESI) for C H Cl N O S [M+H] , calcd: 550.0017; found: 550.0016.
2
1
14
3
7
3
3
-Chloro-N-(2,4-difluoro-3-(4-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-1,2,3-
triazol-1-yl)phenyl)benzamide (6g)
Compound 23a (70 mg, 0.15 mmol) was dissolved in anhydrous THF (10 mL). To the solution
was gradually dropwise added m-chlorobenzoyl chloride (32 mg, 0.18 mmol) and triethylamine
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