Discovery of Novel Pyrazolo[3,4- b] Pyridine Derivatives with Dual Activities of Vascular Remodeling Inhibition and Vasodilation for the Treatment of Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.0c01132

Current pulmonary arterial hypertension (PAH) therapeutic strategies mainly focus on vascular relaxation with less emphasis on vascular remodeling, which results in poor prognosis. Hence, dual pathway regulators with vasodilation effect via soluble guanylate cyclase (sGC) stimulation and vascular remodeling regulation effect by AMP-activated protein kinase (AMPK) inhibition provide more advantages and potentialities. Herein, we designed and synthesized a series of novel pyrazolo[3,4-b] pyridine derivatives based on sGC stimulator and AMPK inhibitor scaffolds. In vitro, 2 exhibited moderate vasodilation activity and higher proliferation and migration suppressive effects compared to riociguat. In vivo, 2 significantly decreased right ventricular systolic pressure (RVSP), attenuated pulmonary artery medial thickness (PAMT), and right ventricular hypertrophy (RVH) in hypoxia-induced PAH rat models (i.g.). Given the unique advantages of significant vascular remodeling inhibition and moderate vascular relaxation based on the dual pathway regulation, we proposed 2 as a promising lead for anti-PAH drug discovery.

Full text of DOI:10.1021/acs.jmedchem.0c01132

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Article
Discovery of Novel Pyrazolo[3,4-b] pyridine Derivatives
with Dual Activities of Vascular Remodeling Inhibition and
Vasodilation for the Treatment of Pulmonary Arterial Hypertension
Liqing Hu, Lijun Li, Qi Chang, Songsen Fu, Jia Qin, Zhuo
Chen, Xiaohui Li, Qinglian Liu, Gaoyun Hu, and Qianbin Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01132 • Publication Date (Web): 11 Sep 2020
Downloaded from pubs.acs.org on September 13, 2020
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Discovery of Novel Pyrazolo[3,4-b] pyridine Derivatives with Dual
Activities of Vascular Remodeling Inhibition and Vasodilation for
the Treatment of Pulmonary Arterial Hypertension
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Liqing Hu,1,3 Lijun Li, Qi Chang, Songsen Fu, Jia Qin, Zhuo Chen, Xiaohui
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,1
Li, Qinglian liu, Gaoyun Hu, and Qianbin Li
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Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University,
Changsha 410013, Hunan, China
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Department of Pharmacology, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha
10013, Hunan, China
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Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, VA 23298,
USA
ABSTRACT
Current pulmonary arterial hypertension (PAH) therapeutic strategies mainly focus on vascular
relaxation with less emphasis on vascular remodeling, which results in poor prognosis. Hence, dual
pathway regulators with vasodilation effect via soluble guanylate cyclase (sGC) stimulation and
vascular remodeling regulation effect by AMP-activated protein kinase (AMPK) inhibition will
provide more advantages and potentialities. Herein, we designed and synthesized a series of novel
pyrazolo[3,4-b] pyridine derivatives based on sGC stimulator and AMPK inhibitor scaffolds. In
vitro, 2 exhibited moderate vasodilation activity and higher proliferation and migration suppressive
effects compared to riociguat. In vivo, 2 significantly decreased right ventricular systolic pressure
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(RVSP), attenuated pulmonary artery medial thickness (PAMT) and right ventricular hypertrophy
(RVH) in hypoxia-induced PAH rat models (i.g.). Given the unique advantages of significant
vascular remodeling inhibition and moderate vascular relaxation based on dual pathways regulation,
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we proposed 2 as a promising lead for anti-PAH drug discovery.
INTRODUCTION
Pulmonary arterial hypertension (PAH) is a type of chronic progressive and life-threatening
cardiopulmonary disease with annual mortality about 15% in high-risk patients.1, 2 PAH is
characterized by pulmonary vascular remodeling, elevated pulmonary vascular resistance (PVR)
and increased pulmonary artery pressure (PAP), resulting in right ventricular overload and
hypertrophy, and eventually right heart failure or even death.3, 4
It is widely recognized that advances in PAH pathology contributed enormously to the identification
of several effective therapeutic targets, including endothelin pathway, prostacyclin pathway, and
nitric oxide (NO) pathway.5-9 However, despite considerable progress in conventional therapies or
newly developed targeted therapies, these treatment strategies mainly focus on vasodilation to
reduce blood pressure. As a result, the side effects on systemic hypotension and bottlenecks in
efficiency remain challenging clinical problems, which still lead to poor prognosis.10 The main
reason lies in the lack of efficient treatment strategies targeting pulmonary vascular remodeling.
With the increasing understanding on the pathological biology of PAH, vascular remodeling has
been recognized as the essential pathological characteristic of PAH over the past decade.11, 12 It is
noteworthy that the excessive proliferation and abnormal migration of human pulmonary arterial
smooth muscle cells (HPASMCs) and human lung fibroblasts (HLF1) are pivotal abnormal
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phenotypes of PAH. These abnormalities will thicken the pulmonary vascular wall and ultimately
result in occlusion and fibrosis of vascular.13, 14
In terms of PAH clinical manifestations, high PAP is the primary factor to be dealt with. The heart
overload can be reduced with decreased ventricular pressure, which also results in the improvement
of blood flow and alleviation of hypoxia status. Soluble guanylate cyclase (sGC), the endogenous
receptor of NO, is attracting tremendous enthusiasm in PAH drug discovery. sGC stimulation not
only promotes vasodilation but also slightly inhibits vascular remodeling via increasing the level of
messenger molecule cGMP.15, 16 These pharmacological effects make sGC/cGMP a typical
signaling pathway in the treatment of PAH through regulating a number of downstream targets such
as protein kinases, cyclic nucleotide-gated channels and phosphodiesterases.8, 17 sGC stimulators, a
class of ligands that bind allosterically to the heme-containing sGC, act NO-independently to
stimulate the formation of cGMP.15 Riociguat (Adempas®) (Figure 1), the first approved sGC
stimulator for treating PAH, significantly decreased mean pulmonary artery pressure (mPAP) and
PVR, and improved right ventricular function.18,19 Although riociguat displays many advantages in
attenuating the symptoms of PAH, it is insufficient to block PAH progression due to its limited
inhibition of vascular remodeling. Moreover, the systematic hypotension partially due to high level
of cGMP suggests its limited clinical use. 20, 21
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To overcome the drawbacks of monotherapy by vasodilation, the strategy majorly targeting vascular
remodeling will be a potential addition. In fact, many attempts and efforts to treat PAH by means
of inhibiting vascular remodeling have been investigated. Adenosine monophosphate-activated
protein kinase (AMPK) is a serine-threonine kinase, well known as cellular energy homeostasis
regulator.22 Previous reports have shown that AMPK activation and activity is induced early and
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remains elevated in cancer cells, which is essential for cell growth and proliferation. However,
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AMPK is not required for growth in normal cells with abundance of nutrients. Genetic deletion of
AMPK or attenuation of its activity in tumor cells was sufficient to reduce cell proliferation.
Interestingly, the increased AMPK activation was determined by AMPK phosphorylation at the
residue Thr172, required for the mammalian AMPK activation, and the higher AMPK activity was
assessed by acetyl-CoA carboxylase (ACC) phosphorylation at the residue Ser79.23 Particularly,
AMPK plays a key role in abnormal cell phenotypes of HPASMCs and HLF1 under conditions such
as hypoxia or drug induction.24, 25 The level and activity of phosphorylated AMPK (p-AMPK) in
HPASMCs of PAH and hypoxic mouse PASMCs appeared to be elevated. The over-activated
AMPK promotes cell metabolism by increasing ATP consumption, leading to abnormal cell
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proliferation and migration, and ultimately vascular remodeling. Moreover, compound C (Figure
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), an AMPK inhibitor, prevents hypoxia-induced PAH in vivo. These results suggest that AMPK
pathway may be a significant therapeutic target in the treatment of PAH through inhibiting vascular
remodeling.24, 26
In recent years, it has been reported that monotherapy via vascular remodeling inhibition results in
rapid changes of vascular status. This in turn promotes a vicious circle, leading to the failure of PAH
treatment.27-29 Combination therapy has been widely accepted as simultaneous regulation of
vascular remodeling and vasodilation to produce improved outcomes compared to monotherapy.30
Interestingly, the novel hybrid (CDDO-NO, Figure 1) from bardoxolone methyl (CDDO-Me) and
NO donor exhibited dual actions of vascular remodeling inhibition and pulmonary vascular
relaxation, which was superior to the mono- or combination therapy.31
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Figure 1. Structures of sGC stimulators, AMPK inhibitors and CDDO-NO.
To find novel dual regulators of both high ventricular pressure and vascular remodeling, sGC and
AMPK pathways will be the focus in this study. In modern drug discovery, fragment-based drug
design (FBDD) strategy provides many opportunities for the rational development of drug
candidates. Previously, it was reported that pyrazolo[3,4-b]pyridine derivatives have strong anti-
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proliferation effect. Furthermore, as shown in Figure 1, the pyrimidine and benzyl moieties were
vital for potent sGC stimulation based on extensive structure-activity relationship (SAR) studies.10,
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In addition, the pyrazolo[1,5-a]pyrimidine scaffold in the structure of compound C has been
suggested to bound tightly to a unique elongated binding pocket of AMPK enzyme, while the
piperidine ring resides outside the binding pocket.34 These integrated information led to the
discovery of the lead compound 5-(4,6-diaminopyrimidin-2-yl)-3-methyl-1-phenyl-1,7-dihydro-
6
H-pyrazolo[3,4-b] pyridin-6-one (1, Figure 2). Consistent with our hypothesis, compound 1
exhibited considerable inhibition against both the HPASMCs and HLF1 proliferation compared to
riociguat and compound C.
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Herein, we purposely optimized the structure of 1 to improve its potency using fragment linking
and growing strategies (Figure 2). The subsequent screening based on pre-constricted rat thoracic
aorta rings and cell viability assays resulted in the discovery of 2 with moderate vasodilatory effect
compared with riociguat and significant improved inhibitory activity against HPASMCs and HLF1
proliferation and migration compared to compound C. Compound 2 demonstrated moderate
regulatory effect on sGC/cGMP pathway and considerable inhibition against AMPK signaling
pathway, which resulted in equivalent RVSP control, better RVH and PAMT improvement in vivo.
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Figure 2. Design rationale and optimization of pyrazolo[3,4-b] pyridine derivatives with dual activities of anti-
vascular remodeling and regulating vascular tone.
CHEMISTRY
All targeted compounds were prepared with high yields following synthetic routes outlined in
Schemes 1-5. The key intermediates 10, 14a-i (Scheme 1), and 16, 20a-d (Scheme 2) were
synthesized with initial efforts. As shown in Scheme 1, compound 4 was synthesized through
Vilsmeier-Haack-Arnold reaction with 3 as the starting material, and the chlorine was substituted
by azide to afford 5, followed by the reduction of 5 to acquire the intermediate 6. 7 was the
substitution reaction product through introducing benzyl group on 6 and the subsequent N-alkylation
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product 8 can be obtained via reacting 7 with ethyl cyanoacetate. 3-methyl-6-oxo-1-phenyl-6,7-
dihydro-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (11) was provided by the reaction of compound
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and ethyl cyanoacetate in acetic acid. Actually, we did not directly obtain N-alkylation product
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from compound 11, but only O-alkylation product 12a-i even through different bases such as
potassium carbonate, cesium carbonate and sodium hydride. We speculated that the presence of
phenyl group on N1 of 11 might increase the steric hindrance, resulting in the only formation of O-
alkylation products. Reducing cyano-group is a crucial step to introduce pyrimidine ring.
Compounds 9 or 13a-i were the reaction products of hydroxylamine hydrochloride and 8 or 12a-i.
The key intermediates 10 and 14a-i were from 9 and 13a-i, respectively.36, 37 The synthetic approach
for substituted-malononitrile compounds 16 and 20a-d is outlined in Scheme 2 by referring to
relevant literatures.38, 39
Scheme 1. Synthesis of 3-methyl-1-phenyl-1H-pyrazolo[3,4-b] pyridine-5-carboximidamides
0 and 14a-ia
1
aReagents and conditions: (a) DMF, POCl
, 100 ℃, 2 h; (b) NaN , TBAI, DMSO, rt, 18 h; (c) Fe (power), NH Cl,
3 3 4
2 3 2 3 2 2 3
EtOAc, H O, rt, 22 h; (d) CH CN, K CO , rt, 4 h; (e) NCCH COOEt, AcOH, reflux, 48 h; (f) DMF, K CO , rt, 2 h;
(g) H
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NOH-HCl, DIEA, EtOH, 90 ℃, Ar, 24 h; (h) Zn, AcOH, rt, 8 h.
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Scheme 2. Synthesis of substituted-malononitrile intermediates 16 and 20a-da
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aReagents and conditions: (a) NaNO
, AcOH, H O, AgNO , rt, 12 h; (b) K CO , CH CN, rt, 3 h; (c) NH , MeOH,
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rt, 48 h; (d) POCl , DMF, -5 ~ 0 ℃, 4 h.
As a proof of concept, a series of targeted compounds were designed and synthesized by
introducing the 4,6-diamino-pyrimidine rings so as to increase sGC stimulating activity. The crucial
step for each route was the synthesis of substituted-pyrimidines, such as 21a-e, 25a-d (Scheme 3),
or 1, 2, 31a-f (Scheme 5). After brief investigation, synthetic methodology for preparing this
40
pyrimidine scaffold has been validated. The reaction of 10 or 14a-i with 15 that was commercially
available or the appropriate malononitrile derivatives 20a-d, which were prepared according to
Scheme 2, under microwave irradiation at 130 °C to afford 4,6-diamino-pyrimidines 1, 2, 21a-e, 30
and 31a-f. In addition, compound 23 was the product of the cyclization of 22 and 16 followed by
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the removal of silver chloride using a pyrimidine forming procedure, and subsequent reduction of
the resulting nitroso group with Zn powder furnished the target 24 (Scheme 3). Treatment of the
resulting tri-aminopyrimidine with acyl chloride compounds delivered products 25a-d and 27
(Scheme 4). Target compound 29 was prepared under the reaction of 25b with iodomethane
catalyzed by sodium hydride (Scheme 4).
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Scheme 3. Synthesis of 2-(6-((2-fluorobenzyl)oxy)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]
pyridin-5-yl)pyrimidine-4,6-diamine derivatives 21a-e, 24 and 25a-da
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aReagents and conditions: (a) Microwave (MW), MeOH, 1,4-dioxane, 130 ℃, 2 h; (b) HCl(gas), MeOH, rt, 2 h; (c)
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h to rt 2 h (25a); (f) Pyridine, -5 ~ 0 ℃, 2 h (25b-d).
Scheme 4. Synthesis of Compounds 27, 29 and 30a
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aReagents and conditions: (a) K
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(MW), MeOH, 1,4-dioxane, 130 ℃, 2 h.
Scheme 5. Synthesis of Compounds 1, 2 and 31a-fa
aReagents and conditions: (a) Microwave (MW), MeOH, 1,4-dioxane, 130 ℃, 2 h.
RESULTS AND DISCUSSION
Rational Design of Novel Pyrazolo[3,4-b] pyridines to Improve Anti-proliferation Efficacy and
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Vasodilatory Effect by Introducing Pyrimidine Pharmacophore. Increasing studies in recent
years have demonstrated that the abnormal proliferation and migration of HPASMCs and HLF1 are
the essential features of pulmonary vascular remodeling in the pathogenesis of PAH.16, 42, 43
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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
Considering these basic findings,10, 32 we hypothesized that the introduction of pyrimidine groups
on the basis of pyrazolo[3,4-b]pyridine ring could dramatically increase the effect of stimulating
sGC, which mainly mediates vascular tone. Meanwhile, the designed compounds may also
concomitantly inhibit AMPK to increase the effect of anti-vascular remodeling, which is crucial for
better PAH treatment described above. Therefore, the priority of this study was to investigate the
anti-proliferation effects of compounds against HPASMCs and HLF1 through the cell-based
activity. Riociguat and compound C were applied as the positive control. As our initial effort, we
prepared a novel pyrazolo[3,4-b] pyridine compound (1, IC = 40.6 μM), which had a marked
5
0
improvement against HPASMCs proliferation compared with compounds 11 (IC > 100 μM) and
5
0
1
2a (IC >100 μM). It is clear that introduction of pyrimidine ring replacing the cyano group at the
50
3
-position of the pyridine ring significantly decreased the cell viability of HPASMCs, indicating
that the pyrimidine moiety of 1 is essential for anti-proliferation activity (Table 1). In view of the
considerable anti-proliferation effects of 1 against both HPASMCs and HLF1 compared to
compound C, further structural modification was carried out in order to improve the anti-
proliferation activity. Subsequently, compounds with better anti-proliferation activity were
subjected to evaluating their ability to relax blood vessels based on the consideration of searching
for compounds with moderate vasodilatory effect compared to riociguat.
Table 1. In Vitro SARs of Pyrazolo[3,4-b]pyridines Substituent Groups.
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1
1
1
1
1
1
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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
O
NH2
R2
NH2
N
H N
NH2
N
2
N
N
N
O
N
O
F
N
N
O
N
H
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
O
N
OH
N
N
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
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9
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3
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8
9
0
R1
F
1
29
30
HLF1
HPASMC
Rat aortic ring
Emax
Compd.
R
1
R
2
IC50 (μM)
IC50 (μM)
EC50 (μM)
(1μM, %)
1
--
--
H
N
>100
55.6±1.3
>100
40.6±4.1
59.3±5.3
25.2±1.6
53.0±3.0
29.3±0.8
56.5±5.5
22.4±1.3
34.9±1.8
25.5±1.2
21.7±0.9
24.8±2.0
24.7±1.3
23.2±1.2
>100
nd
nd
2
1a
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
--
2.2±0.1
8.2±0.2
0.79±0.02
12.7±1.1
1.1±0.1
0.96±0.02
0.93±0.01
5.8±0.2
>100
41.3±2.9
28.7±7.4
47.8±5.5
29.8±7.7
46.1±7.3
46.7±9.9
O
21b
2
1c
N
N
N
>100
2
1d
>100
21e
>100
2
4
-NH
2
37.2±0.8
>100
H
N
25a
51.1±10.1
31.4±13.7
15.8±4.1
15.5±9.4
19.7±7.2
32.9±10.4
nd
O
H
N
O
2
5b
>100
O
H
O
2
5c
N
52.8±2.0
63.4±3.1
49.7±0.5
>100
O
H
O
N
25d
>100
O
O
N
2
7
>100
O
29
--
--
H
H
4.9±0.2
nd
3
0
--
>100
3
1a
3-F
67.7±9.8
55.3±2.0
68.4±3.0
>100
0.36±0.02
nd
59.9±13.4
nd
31b
4- F
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31c
3-Me
4-Me
H
H
H
H
H
H
--
41.9±3.5
>100
32.0 ±1.6
89.4±6.5
67.4±5.0
20.4±1.2
17.0±1.0
82.2±0.5
0.99±0.01
nd
48.4±10.0
nd
3
1d
3
1e
52.8±1.9
31.0±2.6
38.5±0.2
>100
10.8±1.2
1.6±0.2
0.29±0.05
nd
20.4±5.2
43.4±11.6
63.2±3.7
nd
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
3
1f
4-CF
2-Me
--
3
2
C
Rioci
guat
--
--
>100
>100
0.18±0.05
93.6±3.9
nd: not determined
In Vitro Structure-Activity Relationships (SARs) of Novel Pyrazolo[3,4-b]pyridine
Derivatives. In view of the fundamental roles of HPASMCs and HLF1 abnormal proliferation in
the pathogenesis of PAH, SAR studies were started to explore their inhibitory activities on cell
viability using CCK-8 assay. Moreover, the aim of SAR studies was to find compounds with good
inhibitory activities against both HPASMCs and HLF1 cell lines. The preliminary results indicated
that the activity of O-alkylation product (21b, IC = 25.2 μM) is stronger than N-alkylation target
5
0
(
30, IC50 >100 μM) against HPASMCs. Hence, we focused on designing and synthesizing O-
alkylation target compounds in a follow-up study. As shown in Table 1, most of the target
compounds exhibited potent anti-proliferation activities, which were more effective than compound
C and riociguat.
The initial investigation on the SARs of the pyrazolo[3,4-b]pyridine derivatives was mainly carried
out by substituting the 5-position of the pyrimidine ring, and the inhibition activities of these
substituted compounds against vascular hyper-proliferation cell lines were also presented in Table
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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
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5
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5
5
5
5
5
5
5
6
1. Compounds with different substituents at the 5-position of the pyrimidine ring displayed a
stronger inhibitory effect on HPASMCs proliferation than HLF1 which also abnormally proliferate
in the pathogenesis of PAH. Compounds 21a (HFL1, IC = 55.6 μM) and 24 (HFL1, IC = 37.2
5
0
50
0
1
2
3
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8
9
0
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2
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9
0
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9
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2
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9
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0
μM) with a hydrogen and an amino group at the 5-position of the pyrimidine ring, respectively,
showed potent anti-proliferation activities, while compounds 21b-e (IC > 100 μM) bearing an
5
0
aliphatic amine groups decreased significantly in potency toward HLF1, suggesting that a steric
hindrance substitution at this position is unfavorable. Based on these results, further SARs were
carried out through changing substituted groups at the benzyl in order to preferentially increase the
inhibitory effect on HLF1, and the results are summarized in Table 1. The classic bioisosterism
replacement of fluorine (21a, HFL1, IC = 55.6 μM; HPASMC, IC = 59.3 μM) with the methyl
5
0
50
group (2, HFL1, IC = 38.5 μM; HPASMC, IC = 17.0 μM) resulted in an increased potency.
5
0
50
Substitutions of the benzyl ring with fluorine atom or methyl group showed a preference at 2-
position over other positions for the anti-proliferation activities, compounds 21a and 2 versus
compounds 31a-b and 31c-d, respectively. The electron withdrawing groups were beneficial to anti-
proliferation activities against HLF1(31f, -CF , IC = 31.0 μM > 31b, -F, IC = 55.3 μM > 31d, -
3
50
50
CH , IC > 100 μM). Taken together, compounds 24, 31f and 2 showed comparable anti-
3
50
proliferation activities against both HPASMCs and HLF1.
Furthermore, we carried out the rat aortic ring assay to evaluate the vasodilation activity of the
potential compounds. As shown in Table 1, the vasodilatory effect of compounds in different
concentrations were measured and the EC50 was calculated. Results showed that the anti-
proliferation activities of the compounds were not in agreement with the vasodilatory activity. We
speculated that the newly designed and synthesized compounds have different effects based on the
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dual target actions. Comparing with riociguat (IC > 100 μM), 25b exhibited more potent anti-
5
0
proliferation activity against HPASMCs (IC = 25.5 μM), with a weak vasodilatory effect (25b,
5
0
EC = 5.8 μM; riociguat, EC = 0.18 μM). These results provide an experimental basis for us to
5
0
50
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
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4
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5
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0
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search for further compounds with better anti-proliferation activities and moderate vasodilatory
effect compared to riociguat. The anti-proliferation activities of 24 (HFL1, IC50 = 37.2 μM;
HPASMC, IC = 22.4 μM), 31f (HFL1, IC = 31.0 μM; HPASMC, IC = 20.3 μM) were roughly
5
0
50
50
equivalent to 2 (HFL1, IC = 38.5 μM; HPASMC, IC = 17.0 μM). However, the vasodilatory
5
0
50
effects of compounds 24 (EC50 = 0.96 μM) and 31f (EC50 = 1.6 μM) displayed an obvious
disadvantage compared to 2 (EC = 0.29 μM) which showed potent anti-proliferation activities and
50
moderate vasodilatory effect. Taken together, compound 2 was selected as the best candidate for
subsequent study.
The Vasodilatory Effect of 2 Through Stimulating sGC. sGC/cGMP signal transduction is
crucial to vascular tone modulation in the pathogenesis of PAH. Pharmacological stimulation of
sGC exerts intracellular effects by increasing the formation of cGMP that can be degraded by
phosphodiesterase 5 (PDE-5). Because 2 was designed through introducing the pharmacophores of
sGC stimulators based on the fragment design strategy, we expected that 2 could up-regulate the
intracellular cGMP levels by activating sGC. Hence, 2 and riociguat were subjected to homogeneous
time-resolved fluorescence (HTRF) cGMP assay to evaluate their effects on cGMP generation in
HPASMCs. Firstly, in the absence of the PDE-5 inhibitor 3-isobutyl-1-methylxanthine (IBMX), 2
and riociguat slightly increased intracellular cGMP levels (about 5% increase at 100 μM, Figure
3A). Moreover, the presence or absence of sGC inhibitor 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-
1
-one (ODQ) had no significant difference on the levels of cGMP (the red and blue lines shown in
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2
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5
5
5
5
5
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6
Figure 3A). This is primarily due to the rapid cGMP degradation by the intracellular PDE-5.
Secondly, with the addition of IBMX, we explored the impact of ODQ on cGMP formation in
HPASMCs. As a result, 2 exhibited distinct ability to produce cGMP and showed a significant dose-
dependent relationship, while the effect was moderate compared to riociguat (about 40% and 50%
increase at 100 μM as the green and black lines shown in Figure 3B, respectively). In addition, the
elevated levels of cGMP were obviously reduced after adding ODQ compared with the groups
without ODQ (green vs red; black vs blue, Figure 3B). Taken together, these findings suggest that
0
1
2
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2
was effective in elevating the intracellular cGMP levels possibly through stimulating sGC.
Figure 3. (A) Dose-dependent curve of compound 2 and riociguat to elevate the cGMP levels in the absence or
presence of 10 μM ODQ in HPASMCs without IBMX, values are the average of three independent experiments. (B)
Dose-dependent curve of compound 2 and riociguat to elevate the cGMP levels in the absence or presence of 10 μM
ODQ in HPASMCs under condition of IBMX (10 μM), values are the average of three independent experiments.
(C) The inhibition curve of compound 2 and riociguat on PDE-5. (D) Concentration-response curve of compound 2
and riociguat relaxing rat thoracic aorta rings. Value represent mean ± REM, n = 3.
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As we noted above, stimulation of sGC promotes the production of cGMP, nevertheless, PDE-5
4
4
inhibition is also an alternative approach to elevate intracellular cGMP levels. However, PDE-5
inhibitors do not stimulate the formation of cGMP, but inhibit its degradation in contrast to sGC
stimulators which have a stronger effect on elevating cGMP levels. Herein, in order to validate
whether the elevated cGMP after 2 treatment was induced via sGC stimulation rather than PDE-5
inhibition, we also tested the inhibitory effect of compound 2 against PDE-5 in this study.45 The
inhibition rate of compound 2 against PDE-5 at 100 nM was only 4.3%, while that of sildenafil at 2
nM was 54.9%. The results indicate that 2 exhibited a significantly weaker inhibitory effect on PDE-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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8
9
0
5
than the positive control sildenafil. In addition, 2 and riociguat showed comparable inhibitory
potency against PDE-5 under different concentrations, as shown in Figure 3C. These data showed
that compound 2 has little inhibitory effect on PDE-5, and its vasodilatory effect caused by cGMP
elevation was achieved through stimulating sGC.
These results supported that compound 2 can noticeably activate sGC signaling pathway to relax
blood vessels. In addition, the ability of 2 to elevate cGMP and lower blood pressure is milder than
that of riociguat at high dosage, as illustrated in Figure 3B and Figure 3D. Furthermore, what
sparked our enthusiasm for this work is that 2 exerted significantly potent anti-proliferation effects
against HPASMCs and HLF1 compared with riociguat. Therefore, we speculated that the anti-
proliferation effects of 2 may mainly through regulating AMPK signaling pathway.
The Vascular Remodeling Inhibition Effect of 2 via Mainly Regulating AMPK. Although the
imbalance between pulmonary artery vasoconstriction and vasodilation contributes to the
pathophysiology of the disease, the remodeling of pulmonary vascular represents the essential
pathologic finding associated with PAH.22, 23 Pulmonary vascular remodeling is characterized by
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the thickening of pulmonary vessel wall. Such thickening is predominantly due to hyper-
proliferation of smooth muscle cells and fibroblasts, such as HPASMCs and HLF1, respectively.46,
47
0
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0
Interestingly, compound 2 significantly inhibited the over-proliferation against both HPASMCs
(
IC = 17.0 μM) and HLF1 (IC = 38.5 μM) induced by transforming growth factor β (TGF-β, 10
50 50
ng/mL) as discussed above. Recent studies reported that AMPK was up-regulated under the status
of hypoxia, which is involved in inducing PASMCs proliferation as well as the formation of
pulmonary vascular remodeling.26, 48 Hence, we also investigated the anti-proliferation activity of 2
under hypoxic condition with compound C and riociguat as the positive controls. Results showed
that 2 has the best IC values of 20.8 μM (HPASMCs, Figure 4A) and 34.9 μM (HLF1, Figure 4B),
5
0
respectively. Accordingly, the dramatic anti-proliferation effects indicate that 2 may display
significant advantages in inhibiting vascular remodeling compared to riociguat. In addition, the
activities of compound C were stronger than that of riociguat against both HPASMCs (Figure 4A)
and HLF1 (Figure 4B), indicating that inhibition of AMPK is more beneficial to inhibit cell
proliferation than activation of sGC. Given the AMPK inhibitor compound C was also taken as a
lead compound, and these data preliminarily support that compound 2 may exhibit anti-proliferation
effects by inhibiting AMPK.
In order to investigate whether compound 2 inhibits AMPK, the inhibitory activity of 2 against
AMPK was tested using HTRF assay. Here we showed that 2 inhibited AMPK activity in a dose-
dependent manner consistent with compound C, while riociguat exerted no effect on AMPK (Figure
4C). Furthermore, 2 showed slightly weaker potency against the AMPK compared to compound C.
According to the X-ray structural analysis of compound C, the pyrrazolo[1,5-a]-pyrimidine ring of
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compound C is fixed in the corresponding region of AMPK and forms a hydrogen bond to Val96,
and the pyrimidine ring forms a hydrogen bond with the water molecule (Figure 4D). However,
docking analysis showed that 2 formed only one hydrogen bond between amino group and Val96
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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0
1
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9
0
3
4
(
Figure 4E). Nevertheless, the computational docking mode led to identification that 2 binds to the
pocket better than compound C, especially the binding of benzyl group to hydrophobic cavity. The
results suggest that the activation of sGC after introducing benzyl moiety has little conflict with
AMPK inhibition. Therefore, the significant anti-proliferation activities of 2 is most likely mediated
by simultaneously regulating both AMPK and sGC pathways.
Figure 4. (A) Dose-dependent curve of 2, riociguat and compound C to inhibit the proliferation of HPASMCs
under hypoxic condition, values are the average of three independent experiments. (B) Dose-dependent curve of 2,
riociguat and compound C to inhibit the proliferation of HLF1 under hypoxic condition, values are the average of
three independent experiments. (C) Dose-dependent curve of 2, riociguat and compound C to inhibit AMPK, values
are the average of three independent experiments. (D) Surface presentation of the compound C binding to the active
site pocket of AMPK (PDB ID: 3aqv). (E) Surface presentation of 2 binding to the active site pocket of AMPK. 2
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and compound C are presented in colors of green and yellow, respectively. Atoms of carbon, nitrogen, and oxygen
of AMPK are presented in colors of white, blue, and red, respectively. Hydrogen bonds are indicated with yellow
dashed lines.
0
1
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As previously mentioned, the abnormal migration of HPASMCs and HLF1 plays a pivotal role in
vascular remodeling. To further explore whether 2 regulates the migration of HPASMCs and HLF1,
scratch assay was used to detect the migratory activity after 2 treatment with the compound C and
riociguat as comparisons. We synchronously investigated the effects of these compounds with
different concentrations and induction conditions against cell lines migration. The effects of
compound 2 to inhibit cells migration are shown in Figure 5. Compared with the control group, cell
migration was significantly increased in the model group (p < 0.01). These results suggest that 2
showed greatest potency among the tested 3 compounds in a dose-dependent manner, which is
consistent with the anti-proliferation activity. In addition, compounds are more sensitive to suppress
HPASMCs migration under the condition of hypoxia (Figure 5A, B), while the suppression of HLF1
migration are more effective in the presence of TGF-β (Figure 5C, D). In a word, 2 remarkably
suppressed cell migration against both HPASMCs and HLF1, which play a key role in pulmonary
vascular remodeling, especially against HPASMCs under hypoxic condition.
These data suggest that 2 exhibited remarkable proliferation and migration suppression effects
mainly mediated by AMPK pathway inhibition. Moreover, both effects can also be enhanced
synergistically through upregulation of cGMP level via moderately stimulating sGC, indicating that
2
will show significant advantages in inhibiting vascular remodeling in vivo.
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Figure 5. Effects of 2 on inhibiting vascular cells migration. Movement of cells into wound was shown for cells
at 0 and 24 h. (A) Effects of 2, riociguat and compound C inhibit HPASMCs migration induced by TGF-β (10
ng/mL). (B) Effects of 2, riociguat and compound C inhibit HPASMCs migration under hypoxic condition (C)
Effects of 2, riociguat and compound C inhibit HLF1 migration induced by TGF-β (10 ng/mL). (D) Effects of 2,
riociguat and compound C inhibit HLF1 migration under hypoxic condition. Data were shown as the mean ± SD
from three independent experiments. (∗) p < 0.05, (∗∗) p < 0.01.
Reasonable Physicochemical Properties to Enable in vivo Studies. To further characterize the
physicochemical properties (Table 2) of compound 2, its water solubility, metabolic stability and
plasma protein binding rate were evaluated. As is well-known that liver microsomes (human and
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rat) are extensively used to perform in vitro drug metabolism assays. Herein, compound 2 was
subjected to metabolic stability evaluation by human and rat liver microsomal experiments in vitro.
The results indicate that 2 was more stable in human liver microsome (t1/2 of 125 min, and Clint of
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4 mL/min/mg, respectively) than that in rat liver microsome (t1/2 of 10 min, and Clint of 248
mL/min/mg, respectively). This difference may be caused by species differences or individual
differences. The plasma protein binding rate of 2 in human (99.8%) and rat (99.3%) was comparable
to the positive control warfarin (98.9% and 99.4%). Compound 2 could be a starting point for a new
strategy in treating PAH although its drug-like properties are limited at the current stage, which
needs further study in the next logic step.
Table 2. Physicochemical Properties of Compound 2
Content
Value
Water solubility (pH: 7.3)
Water solubility (pH: 3.5)
Human liver microsome stability (T )
34 ng/mL
28 μg/mL
125 min
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Rat liver microsome stability (T )
10 min
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Human liver microsome stability (Clint)
Rat liver microsome stability (Clint)
Human plasma binding rate
14 mL/min/mg
248 mL/min/mg
99.8 %
Rat plasma binding rate
99.3 %
In Vivo Notable Therapeutic Effects of 2 in PAH Rat Models. To investigate the
pharmacodynamics of compound 2, a PAH rat model was established using hypoxia method.49, 50
Compound 2 and riociguat at equimolar dose (10 mg/kg) were orally administrated daily for 2 weeks
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under hypoxia after hypoxia-induced PAH rat model has been built in the first 2 weeks. In keeping
with previous studies, RVSP was significantly increased in the hypoxia model group (26.2 mmHg)
compared with the normoxic group (16.2 mmHg, p < 0.01). This result suggested successful PAH
rat models after 4 weeks of exposure to hypoxic condition. After treatment, a significant decrease
of RVSP was measured in the groups treated with 2 (22 mmHg) and riociguat (22.3 mmHg)
compared to that of the hypoxia group (26.2 mmHg) (Figure 6A) at a dose of 10 mg/kg (p < 0.01).
Although the effects of 2 on increasing cGMP in HPASMCs and relaxing rat aortic ring were lower
than that of riociguat, its ability to reduce right ventricular pressure in vivo was comparable to that
of riociguat, suggesting that the vascular remodeling inhibition effect of 2 plays a pivotal role in the
treatment of PAH.
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Figure 6. Effects of 2 and riociguat in hypoxia treated rats. The hypoxia-induced PAH rats were orally treated daily
with 2 and riociguat at a dose of 10 mg/kg for two weeks. (A) Effects of 2 and riociguat on RVSP of the rats. (B)
Effects of 2 and riociguat on RV/ (LV + S) of the rats. (C) Effects of 2 and riociguat on RV/tibial length of the rats.
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(D) Effects of 2 and riociguat on body weight change of the rats. These data are indicated as the mean ± SEM (n =
5
/group). (∗) p < 0.05, (∗∗) p < 0.01.
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The effects of 2 on the RVH are shown in Figure 6. The weight ratio of right ventricle /left ventricle
septum [RV/(LV+S), Figure 6B] was used to indicate RVH and ratio of RV/tibial length (Figure
C) was also measured, the model group [RV/(LV+S) = 0.63] showed significant increase than the
+
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control group (p < 0.01). Notably, oral administration of 2 (10 mg/kg) daily for 14 days dramatically
reduced RV/(LV+S) (0.38), which was superior to riociguat [RV/(LV+S) = 0.44]. Similar trends
were also observed in RV/tibial length and body weight change (Figure 6D).
2
Significantly Ameliorates Pulmonary Vascular Remodeling of PAH Rats. Elevated
pulmonary vascular remodeling presents the hallmarks of PAH, such as increased PAMT and
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aberrant pulmonary vascular fibrosis. In order to investigate the effect of compound 2 on vascular
remodeling, hematoxylin and eosin (H&E) was used to stain the lung tissues. As shown in Figure
7
A and Figure 7B, PAMT was thickened in hypoxia-induced PAH rats in contrast to the control
group, which was remarkably reduced after 2 and riociguat treatment. Moreover, the therapeutic
effect of 2 was more prominent than that of riociguat.
It is well-known that TGF-β plays a key role in the pathogenesis of fibrotic diseases and is also
5
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involved in PAH development. Furthermore, increased TGF-β expression contributes to vascular
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remodeling in the progress of PAH. Accordingly, we performed Western blotting analysis to test
the effects of 2 and riociguat on TGF-β level. Our results indicated that 2 significantly
downregulated hypoxia-induced the over-expression of TGF-β in lung tissues (Figure 7C and Figure
7D). To address whether compound 2 mediates the process of fibrosis via canonical TGF-β signaling
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pathways during hypoxia-induced PAH, we next determined the levels of total Smad2/3 (t-Smad2/3)
and phosphorylated Smad2 (p-Smad2). Consistently, hypoxia significantly induced the levels of p-
Smad2 without statistically altering t-Smad2/3 levels compared with the control group. Treatment
with 2 (10 mg/kg) did not change levels of t-Smad2/3, however, abolished the over-phosphorylation
of Smad2, which agrees with the effect on TGF-β (Figure 7C).
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Figure 7. Effects of 2 and riociguat on attenuating pulmonary vascular remodeling of rats with PAH. (A)
Representative images of pulmonary vascular in the lungs by H&E staining for morphology. Scale bar at the lower
right corner = 50 μm. (B) The PAMT quantifications in the lungs. 2 (10 mg/kg) markedly lowered hypoxia-induced
pulmonary artery medial thickness (n = 5/group). (C) Representative images of TGF-β, Smad2/3 and α-SMA in the
lungs by western blotting. 2 markedly reduced hypoxia-induced over-expression of TGF-β, p-Smad2 and α-SMA in
the lungs. (D) The quantitative ratio of TGF-β to β-actin in the lungs. (E) Quantification of ratio of α-SMA to β-
actin in the lungs. The tests were independently performed for three times. The data are reported as the mean ± SEM
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(n = 3/group). (∗) p < 0.05, (∗∗) p < 0.01.
Studies have shown that α-smooth muscle actin (α-SMA) is the marker of muscularization, and its
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over-expression could mainly contribute to structural changes in PAH. Therefore, the effect of 2
on this marker were evaluated via Western blotting analysis. As expected, 2 significantly attenuated
hypoxia-induced up-expression of α-SMA compared to riociguat (Figure 7C, E).
2 Regulated both sGC and AMPK Signaling Pathways against PAH in Rats. To address
whether 2 inhibits hypoxia-induced PAH via sGC stimulation and AMPK inhibition, we measured
the levels of related proteins downstream of these two pathways. Protein kinases G (PKG) have
been characterized as crucial downstream mediators of sGC/cGMP in the regulation of vascular
tone and remodeling.55, 56 Vasodilator-stimulated phosphoprotein (VASP) is a prime substrate of
PKG, and its activity is commonly assessed by phosphorylation levels of VASP at Ser239 (p-
VASP).57 Recent data showed that downregulation of sGC leads to reduced levels of cGMP and
impaired PKG activity with decreased levels of p-VASP, whereas the levels of PKG1 are increased
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in a compensatory manner to make up for the damaged sGC-cGMP-PKG activity. As shown in
Figure 8A and Figure 8B, 2 observably attenuated the compensatory expression of PKG1 in contrast
to the levels of p-VASP, which in accordance with the effects of elevating cGMP in HPASMCs.
Hence, these results further demonstrated that 2 could protect against the development of hypoxia-
induced PAH via sGC stimulation although at lower level than that of riociguat.
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Figure 8. Effects of 2 on regulating sGC and AMPK dual signaling pathways. (A) Representative images of
Western blotting for PKG1, p-VASP, p-AMPK, t-AMPK, p-ACC and t-ACC in hypoxia-induced PAH rat lungs. 2
significantly decreased hypoxia-induced over-expression of PKG1, p-AMPK and p-ACC in the lungs. (B)
Quantification of ratio of PKG1 to β-actin. (C) Quantification of ratio of p-AMPK to β-actin. (D) Quantification of
ratio of p-ACC to β-actin in the lungs. The tests were independently performed for three times. The data are reported
as the mean ± SEM (n = 3/group). (∗) p < 0.05, (∗∗) p < 0.01.
In addition, it was reported that AMPK is a key player in the hypoxia-induced PAH, which results
in increased phosphorylation of AMPK and ACC, a downstream target and standard reporter for
AMPK activity.23 Thus, we investigated whether compound 2 acts through AMPK pathway
inhibition. Treatment with 2 significantly decreased the phosphorylation levels of AMPK and ACC
without changing total levels of AMPK and ACC in hypoxia-induced PAH rat lungs (Figure 8A, C,
D). These results suggest that 2 could prevent PAH development by inhibiting AMPK signaling
pathway. Taken together, 2 is more efficient in treating PAH through simultaneous regulation of
sGC and AMPK dual pathways, especially through its anti-vascular remodeling profiles.
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In summary, compound 2 significantly inhibited HPASMCs and HLF1 proliferation and migration
mainly by inhibiting AMPK, as well as relaxing vascular vessels via stimulating sGC in vitro.
Moreover, 2 remarkably decreased RVSP, RVH, PAMT and pulmonary vascular remodeling in vivo
even though the moderate vasodilatory effect compared to riociguat, indicating the enormous
advantages of 2 with dual activities of vascular remodeling inhibition and vasodilation. Furthermore,
2 exhibited highlighted anti-vascular remodeling effect and prominently prevented the progression
of tissue fibrosis. These data suggest that with the potent and valuable anti-vascular remodeling
activity, 2 significantly improved the vascular pathological characteristics in PAH compared to the
reference compound riociguat.
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As the essential pathological characteristic of PAH, vascular remodeling has been paid more and
more attention in the field of anti-PAH drug discovery. The abnormal phenotypes in pulmonary
vascular cells under PAH result in vasoconstriction and loss of elasticity of vascular wall, which
will eventually contribute to increased PAP, RVH and right ventricular overload. Clinically, high
PAP is the primary symptom to be dealt with, which is the major strategy for current therapy, i.e.
diuretics, calcium channel blockers, anticoagulants, inhaled NO donors, and targeted therapy
including NO pathway regulators (sGC stimulators or PDE-5 inhibitors), endothelin receptor
antagonists and prostacyclin analogs. However, this kind of “symptom-based” strategy is gradually
running into drawbacks and bottlenecks such as drug resistance, systematic hypotension and
unsatisfactory long-term use due to less consideration of the essential pathological characteristics
of PAH. As a result, agents with dual regulatory activities on vascular remodeling and vasodilation
will show potential advantages in long-term outcomes and achievements of predefined treatment
goals. This will also contribute to the strategy switch from “symptom-based” to “pathology-based”
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in PAH treatment.
CONCLUSION
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In this study, compound 1 was initially designed and synthesized with riociguat and compound C
as the lead compounds based on fragment-guided drug discovery strategy. Subsequently, we
designed, synthesized and evaluated a series of novel pyrazolo[3,4-b] pyridine derivatives with dual
effects of pre-constricted rat thoracic aorta rings and cell viability based on the structure of 1.
Compared with riociguat, 2 displayed moderate vasodilatory effect and significant improved
inhibitory activities against HPASMCs and HLF1 proliferation and migration in vitro. More
importantly, 2 significantly decreased RVSP, myocardial and vascular remodeling (RVH and
PAMT) via moderate regulatory effect on sGC/cGMP pathway and considerable inhibition against
AMPK signaling pathway in vivo. In conclusion, the results in this study demonstrated that agents
characterized by higher anti-vascular remodeling potency and moderate vasodilation effect will
show more advantages in PAH treatment. 2 could be further evaluated as a potential lead for anti-
PAH drug discovery based on “pathology-based” strategy through regulating dual functions of
vascular remodeling and vasodilation instead of mono vasodilation function in “symptom-based”
strategy.
EXPERIMENTAL SECTION
General Chemical Methods. Unless otherwise indicated, all starting materials and reagents were
purchased from standard suppliers (Sigma-Aldrich, Aladdin, Energy Chemical, Shanghai bide) and
used directly without further purification. Melting point was determined on a XT4MP melting
apparatus (Taike Co., LTD). NOVA-2S microwave apparatus (Preekem, Yiyao technology co.,
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