Discovery of Novel Pazopanib-Based HDAC and VEGFR Dual Inhibitors Targeting Cancer Epigenetics and Angiogenesis Simultaneously

Article abstract of DOI:10.1021/acs.jmedchem.8b00384

Herein a novel series of pazopanib hybrids as polypharmacological antitumor agents were developed based on the crosstalk between histone deacetylases (HDACs) and vascular endothelial growth factor (VEGF) pathway. Among them, one ortho-aminoanilide 6d and one hydroxamic acid 13f exhibited considerable total HDACs and VEGFR-2 inhibitory activities. The HDAC inhibitory activities endowed 6d and 13f with potent antiproliferative activities, which was not observed in the approved VEGFR inhibitor pazopanib. Compounds 6d and 13f possessed comparable HDAC isoform selectivity profiles to the clinical class I HDAC inhibitor MS-275 and the approved pan-HDAC inhibitor SAHA, respectively. 6d and 13f also exhibited uncompromised multiple tyrosine kinases inhibitory activities relative to pazopanib. The intracellular dual inhibition to HDAC and VEGFR of 6d and 13f was validated by Western blot analysis. In both HUVECs tube formation assay and rat thoracic aorta rings assay, 6d and 13f showed comparable antiangiogenic potencies to pazopanib. What's more, 6d possessed desirable pharmacokinetic profiles with the oral bioavailability of 72% in SD rats and considerable in vivo antitumor efficacy in a human colorectal adenocarcinoma (HT-29) xenograft model.

Full text of DOI:10.1021/acs.jmedchem.8b00384

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Article
Discovery of Novel Pazopanib-based HDAC and VEGFR Dual Inhibitors
Targeting Cancer Epigenetics and Angiogenesis Simultaneously
Jie Zang, Xuewu Liang, Yongxue Huang, Yuping Jia,
Xiaoyang Li, Wenfang Xu, C. James Chou, and Yingjie Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00384 • Publication Date (Web): 22 May 2018
Downloaded from http://pubs.acs.org on May 22, 2018
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Discovery of Novel Pazopanib-based HDAC and VEGFR Dual Inhibitors Targeting Cancer
Epigenetics and Angiogenesis Simultaneously
†
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‡
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∥
Jie Zang , Xuewu Liang , Yongxue Huang , Yuping Jia , Xiaoyang Li , Wenfang Xu , C. James Chou
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†,*
and Yingjie Zhang
†Department of Medicinal Chemistry, School of Pharmaceutical of Science, Shandong University, Ji’nan, Shandong,
50012, PR China
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‡Weifang Bochuang International Biological Medicinal Institute, Weifang, Shandong, 261061, PR China
§Shandong Academy of Pharmaceutical Sciences, Ji’nan, Shandong, 250101, PR China
∥
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of
South Carolina, Charleston, SC, 29425, United States
ABSTRACT: Herein a novel series of pazopanib hybrids as polypharmacological antitumor agents
were developed based on the crosstalk between histone deacetylases (HDACs) and vascular endothelial
growth factor (VEGF) pathway. Among them, one orthoꢀaminoanilide 6d and one hydroxamic acid 13f
exhibited considerable total HDACs and VEGFRꢀ2 inhibitory activities. The HDAC inhibitory
activities endowed 6d and 13f with potent antiproliferative activities, which was not observed in the
approved VEGFR inhibitor pazopanib. Compounds 6d and 13f possessed comparable HDAC isoform
selectivity profiles to the clinical class I HDAC inhibitor MSꢀ275 and the approved panꢀHDAC
inhibitor SAHA, respectively. 6d and 13f also exhibited uncompromised multiple tyrosine kinases
inhibitory activities relative to pazopanib. The intracellular dual inhibition to HDAC and VEGFR of 6d
and 13f was validated by western blot analysis. In both HUVECs tube formation assay and rat thoracic
aorta rings assay, 6d and 13f showed comparable antiangiogenic potencies to pazopanib. What’s more,
6d possessed desirable pharmacokinetic profiles with the oral bioavailability of 72% in SD rats and
considerable in vivo antitumor efficacy in a human colorectal adenocarcinoma (HTꢀ29) xenograft
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model.
INTRODUCTION
Mechanismꢀbased drugs targeting a specific biological molecule or pathway is one of the major
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breakthroughs in cancer therapy in the past several decades. However, the clinical effectiveness of
such single target drugs is generally transitory, being followed by almostꢀinevitable resistance and
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relapses due to the adaptive nature and heterogeneity of tumor cells and tumor microenvironment.
The limitations of single target drugs can be overcome by multiple target drugs, which not only can
attack multiple hallmark capabilities of cancer simultaneously to get more robust and durable
therapeutic effects, but also can avoid the risks involved in multicomponent drug cocktails, such as
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unpredictable pharmacokinetic profiles, drug−drug interactions and poor patient compliance.
Both genetic and epigenetic aberrations contribute to tumorigenesis and development. Compared
with genetic aberrations, epigenetic aberrations are more druggable due to its reversibility regulated by
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various enzymes.
Among the numerous epigenetic enzymes, histone deacetylases (HDACs) are a
family of important therapeutic targets for cancer, inflammation, neurological disorders and
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infections.
Thus far, four HDAC inhibitors, vorinostat (SAHA), romidepsin (FK228), belinostat
(PXD101) and panobinostat (LBH589) were approved by the US Food and Drug Administration
(FDA), and one HDAC inhibitor chidamide (CS005) was approved by the China Food and Drug
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Administration (CFDA) for the treatment of hematologic cancer (Figure 1) . Despite the efficacy in
hematologic malignancies, HDAC inhibitor and other epigenetic agents have not shown significant
efficacy as monotherapy against solid tumors. Inspiringly, combination therapies offer hope to conquer
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this challenge.
In addition, considering the aboveꢀmentioned advantages compared with
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combination therapy, intense interest is drawn to the discovery of HDAC inhibitorꢀbased multitarget
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antitumor agents.
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Angiogenesis, one key physiology of solid tumors required for tumor growth and metastasis, has
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long been recognized as a target for cancer therapy.
Among the various angiogenic factors, vascular
endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling
pathway is considered as the primary antiangiogenesis target, with over ten monoclonal antibodies and
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small molecular inhibitors approved for the treatment of solid tumors.
Pazopanib (Figure 1), a
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multiple VEGFR inhibitor, has already been approved by the FDA for the treatment of advanced renal
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cell carcinoma and advanced soft tissue sarcoma in 2009 and 2012, respectively. A phase II study
indicated that secondꢀline treatment with pazopanib in relapsed and refractory smallꢀcell lung cancer
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SCLC) was well tolerated and resulted in promising objective responses and disease control.
However, drug resistance and tumor relapse occurred in nearly all patients treated with pazopanib and
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other VEGFR inhibitors.
To overcome the drug resistance and enhance the antitumor efficacy,
combination therapies using pazopanib and diverse HDAC inhibitors were carried out and showed
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encouraging preclinical in vitro and in vivo results.
Notably, the results of a phase I clinical study
supported further evaluation of the combination of pazopanib and HDAC inhibitor SAHA in cancer
patients with mutated TP53, especially in those with metastatic sarcoma or metastatic colorectal
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cancer. Another phase I clinical study of pazopanib plus HDAC inhibitor abexinostat (Figure 1)
showed that epigenetic modification with HDAC inhibition could enhance response and reverse
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resistance to pazopanib in patients with renal cell carcinoma and other solid tumor malignancies. This
study also showed that the clinical benefit was significantly correlated with both peripheral blood
mononuclear cell (PBMC) HDAC2 expression and fold change in PBMC histone H4 acetylation,
supporting HDAC2 as the clinically relevant target to restore and enhance response to VEGFꢀtargeting
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therapy.
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Figure 1. The structures of several representative HDAC inhibitors and one VEGFR inhibitor pazopanib. The
three parts of HDAC inhibitor pharmacophore model are indicated in three different colors, respectively.
Herein, based on the prevalent strategy of multitarget drug and the clinical benefit of HDAC
inhibitor and VEGFR inhibitor combination, a novel series of pazopanibꢀbased HDAC and VEGFR
dual inhibitors were rationally designed and readily synthesized. Comprehensive evaluation identified
compound 6d with simultaneous HDAC/VEGFR inhibitory activity, potent in vitro antiproliferative
and antiangiogenic potency, desirable in vivo pharmacokinetic profiles and antitumor efficacy.
RESULTS AND DISCUSSION
Rational Design of Novel HDAC and VEGFR Dual Inhibitors Derived from Pazopanib. Most
HDAC inhibitors share a general pharmacophore (Figure 1) composed of a zinc binding group that
chelates the catalytic zinc ion, a hydrophobic linker that occupies the tunnel of the active site, and a
surface recognition cap that interacts with the amino acid residues around the entrance of the active
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site. Considering the surface recognition cap can tolerate extraordinarily variable structures, even like
the cyclic depsipeptide in FK228, we believed that incorporation of pazopanib into the surface
recognition cap of HDAC inhibitor pharmacophore could obtain dual inhibitors of HDAC and VEGFR.
To determine which site of pazopanib can be modified without compromising its VEGFR inhibitory
activity, the proposed binding mode of pazopanib in the ATP pocket of VEGFRꢀ2 was analyzed using
our inꢀhouse molecular docking study (Figure 2A). We could see that the indazole moiety of pazopanib
fit well into the inside pocket of VEGFRꢀ2, and the 2ꢀaminopyrimidine moiety formed two important
hydrogen binds with Cys917 in the hinge region. Therefore, modification of these two moieties could
not be well tolerated. Note that the benzenesulfonamide moiety was projected towards the solvent
region, where could be installed with HDAC inhibitor module while minimally interfering the binding
with VEGFR. Based on these analysis, two privileged zinc binding groups (hydroxamic acid and orthoꢀ
aminoanilide) were attached to the solventꢀexposed phenyl group of pazopanib directly or via various
linkers, leading to the structurally diverse pazopanibꢀbased HDAC and VEGFR dual inhibitors (Figure
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Figure 2. A. The proposed binding mode of pazopanib in the ATP pocket of VEGFRꢀ2 (PDB code: 3CJG). The
figure was generated using PyMol (http://www.pymol.org/); B. The design strategy of Pazopanibꢀbased
HDAC and VEGFR dual inhibitors. Note that the delimitation of the surface recognition cap and the linker is
variable dependent on the linker length.
Chemistry. The procedures to synthesize the target compounds 6aꢀ6e were outlined in Scheme 1.
Commercially available 2,3ꢀDimethylꢀ2Hꢀindazolꢀ6ꢀamine (1) as the starting material reacted with 2,
4
ꢀdichloropyrimidine to afford 2, which was treated with CH I to obtain 3. Then compound 3 was
3
treated with 4ꢀaminobenzoic acid, 3ꢀaminobenzoic acid and 5ꢀaminoꢀ2ꢀmethylbenzoic acid to produce
the key intermediates 4aꢀ4c, respectively. Reaction of 4aꢀ4c with SOCl in methanol afforded 5aꢀ5c,
2
which were directly converted into hydroxamic acids 6aꢀ6c by NH OK in dry methanol. In addition,
2
the intermediate 4a and 4b could react with 1,2ꢀdiaminobenzene by TBTUꢀmediated amide formation
to afford orthoꢀaminoanilides 6d and 6e, respectively.
a
Scheme 1. Synthesis of Compounds 6a-6e
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a
Reagents and conditions: (a) 2,4ꢀdichloropyrimidine, NaHCO , EtOH, reflux, 4 h, 90%; (b) CH I,
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Cs CO , DMF, rt, 2 h, 80%; (c) various aminobenzoic acids, isopropanol, conc. HCl, reflux, 4 h, 79ꢀ
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0%; (d) SOCl , CH OH, reflux, 4 h, 93ꢀ95%; (e) NH OH.HCl, KOH, anhydrous CH OH, rt, 2 h, 40ꢀ
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57%; (f) 1,2ꢀdiaminobenzene, TBTU, TEA, anhydrous DMF, ice bath, 30 min, rt, 12 h, 43ꢀ65%;
Target compounds 10a and 10b were synthesized using the procedures described in Scheme 2. The
intermediate 4a and 4b were treated with N, Oꢀdimethylhydroxylamine hydrochloride to give Weinreb
amides 7a and 7b, which were then reduced via LiAlH to afford aldehydes 8a and 8b, respectively.
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The HornerꢀWadsworthꢀEmmons reaction of 8a and 8b with triethyl phosphonoacetate led to 9a and
9b, which were directly converted into hydroxamic acids 10a and 10b by NH OK in dry methanol,
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respectively.
a
Scheme 2. Synthesis of Compounds 10a-10b
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Reagents and conditions: (a) N,Oꢀdimethylhydroxylamine hydrochloride, TBTU, TEA, anhydrous
DMF, ice bath, 30 min, rt, 12 h, 71ꢀ72%; (b) LiAlH , anhydrous THF, ꢀ40 °C, 4 h, 70ꢀ72%; (c) triethyl
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phosphonoacetate, NaH, anhydrous THF, ice bath, 20 min, rt, 12 h, 65ꢀ68%; (d) NH OH.HCl, KOH,
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anhydrous CH OH, rt, 2 h, 60ꢀ64%.
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The procedures to prepare the target compounds 13aꢀ13i and 14aꢀ14g were outlined in scheme 3.
The intermediates 4aꢀ4c were coupled with various methyl aminoalkanoates to get compounds 11aꢀ11i,
which could be transformed to hydroxamic acids 13aꢀ13i, respectively. Besides, hydrolysis of
compounds 11aꢀ11g, followed by condensation with 1,2ꢀdiaminobenzene led to orthoꢀaminoanilide
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4a-14g, respectively.
a
Scheme 3. Synthesis of Compounds 13a-13i and 14a-14g
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0 min, rt, 12 h, 69ꢀ80%; (b) NH OH.HCl, KOH, anhydrous CH OH, rt, 2 h, 51ꢀ61%; (c) 3M NaOH,
2 3
CH OH, rt, 4 h, 81ꢀ90%; (d) 1,2ꢀdiaminobenzene, TBTU, TEA, anhydrous DMF, ice bath, 30 min, rt,
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2 h, 38ꢀ48%.
In Vitro HDAC and VEGFR Inhibition Assay. To minimally alter the structure of pazopanib, two
zinc binding groups, hydroxamic acid and orthoꢀaminoanilide, were directly attached to the paraꢀ or
metaꢀposition of the solventꢀexposed phenyl group of pazopanib, leading to compounds 6aꢀ6e. The
HDAC inhibitory activities of 6aꢀ6e were evaluated by determining the IC50 values against HeLa
nuclear extract, with the hydroxamic acidꢀbased compound SAHA and orthoꢀaminoanilideꢀbased
compound MSꢀ275 as positive controls. Their VEGFR inhibitory activities were preliminarily tested by
determining the VEGFRꢀ2 inhibitory rates at 200 nM with pazopanib as the positive control. Results in
Table 1 showed that all hydroxamic acids (6aꢀ6c) displayed much less potent HDAC inhibitory
activities than the positive control SAHA, probably because the short linkers of 6aꢀ6c could not lead
their hydroxamate groups to the zinc ion. Note the paraꢀsubstituted 6a exhibited better HDAC and
VEGFRꢀ2 inhibitory activities than the metaꢀsubstituted 6b and 6c. For orthoꢀaminoanilide analogs (6d
and 6e), paraꢀsubstituted 6d showed more potent HDAC and VEGFRꢀ2 inhibitory activities than metaꢀ
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substituted compound 6e, which was in accordance with the trend in hydroxamates. Importantly,
compound 6d not only exhibited comparable HDAC inhibitory activity to MSꢀ275, but also exhibited
comparable VEGFRꢀ2 inhibitory activity to pazopanib, validating our compound design strategy that
incorporation of zinc binding group to the solventꢀexposed phenyl group of pazopanib could obtain
HDAC and VEGFR dual inhibitors. To get dual inhibitors with improved HDAC inhibitory activity,
compounds 10a and 10b with Nꢀhydroxycinnamamide fragment were designed and synthesized. Nꢀ
hydroxycinnamamide is a privileged structure used by many potent HDAC inhibitors including the
approved drug LBH589 and PXD101. Enzyme inhibition results (Table 1) showed that compared with
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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
6
a and 6b, 10a and 10b exhibited dramatically increased HDAC activities and uncompromised
VEGFRꢀ2 activities, indicating that introduction of longer linker was benefit to HDAC inhibition and
not detrimental to VEGFR inhibition. However, 10a and 10b were still over 10ꢀfold less potent than
SAHA.
Table 1. Structure and In Vitro Enzymatic Inhibition of 6a-6e and 10a-10b
VEGFRꢀ2
HDAC
Compd
Structures
a
inhibition rate
at 0.2 ꢁM
IC (ꢁM)
5
0
6
a
11.3
>20
99%
77%
6
b
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c
>20
74%
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
6d
4.6
100%
63%
6
e
21.3
1
0a
1.4
3.0
95%
97%
1
0b
b
SAHA
MS275
Pazopanib
ꢀ
ꢀ
ꢀ
0.13
1.8
NDb
ND
b
ND
95%
a
b
Assays were performed in replicate (n≥2); the SD value are <20% of the mean.
Not determined.
To further investigate the effects of linker on HDAC and VEGFR inhibition, hydroxamic acidꢀbased
compounds 13aꢀ13i and orthoꢀaminoanilideꢀbased compounds 14aꢀ14g were designed and synthesized.
Their enzyme inhibition results were shown in Table 2. For paraꢀsubstituted hydroxamic acids 13aꢀ
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9
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
1
3g, their HDAC inhibitory activities first increased and then decreased with the linker length,
culminating in compound 13f (n = 6), which was about 40ꢀfold more potent than SAHA. Such
structureꢀactivity relationship was also found in orthoꢀaminoanilides 14aꢀ14g, among which compound
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
1
4e with the optimal linker length (n = 5) exhibited comparable HDAC inhibitory activity to MSꢀ275.
Similar with the results in Table 1, metaꢀsubstituted hydroxamic acids 13h and 13i also exhibited
inferior HDAC inhibition compared with their paraꢀsubstituted analog 13e. Interestingly, neither the
linker length nor the substituted position had significant influence on VEGFRꢀ2 inhibition of these dual
inhibitors.
Table 2. Structure and In Vitro Enzymatic Inhibition of 13a-13i and 14a-14g
VEGFRꢀ2
HDAC
Compd
Structures
a
inhibition rate
at 0.2 ꢁM
IC (ꢁM)
5
0
>20
95%
99%
99%
94%
1
3a
>20
1
3b
19.8
0.68
1
3c
1
3d
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0
.017
98%
93%
92%
1
3e
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
0.0033
13f
0.025
13g
1
3h
0.18
90%
87%
1
3i
0.82
14a
14b
14c
>20
>20
19.0
98%
97%
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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5
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5
5
5
5
5
5
6
1
4d
15.1
3.0
5.0
8.7
94%
95%
93%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14e
14f
14g
92%
b
SAHA
MS275
Pazopanib
ꢀ
ꢀ
ꢀ
0.13
1.8
NDb
ND
b
ND
95%
a
b
Assays were performed in replicate (n≥2); the SD value are <20% of the mean.
Not determined
In Vitro Antiproliferation Assay. Considering their potent HDAC inhibitory potencies, four
hydroxamic acids (10a, 13e, 13f, 13g) and four orthoꢀaminoanilides (6d, 14e, 14f, 14g) were evaluated
for their antiproliferative activities against seven solid tumor cell lines and four hematological tumor
cell lines. According to the results in Table 3, all the HDAC and VEGFR dual inhibitors showed
moderate to potent activities, while the VEGFR inhibitor pazopanib was not active, demonstrating that
the HDAC inhibitory activities of these dual inhibitors contributed to their antiproliferative capacities.
Generally, our compounds and the two positive controls SAHA and MSꢀ275 were more potent against
the hematological tumor cell lines over the solid tumor cell lines. However, because the main purpose
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of our research was to find dual inhibitors for solid tumor therapy, we focused our attention on
identifying target compounds with potent activities against solid tumor cell lines. Among the tested
solid tumor cell lines, the human colorectal adenocarcinoma cell line HTꢀ29 seemed to be the most
sensitive one to our compounds. It was worth noting that the orthoꢀaminoanilideꢀbased compound 6d
exhibited more potent antiproliferative activity against HTꢀ29 than both SAHA and MSꢀ275. Besides,
though less potent than SAHA in HTꢀ29, the hydroxamic acidꢀbased compound 13f was slightly more
potent than MSꢀ275. The cytotoxicities of 13f and 6d against normal cells were determined using
human umbilical vein endothelial cells (HUVECs). It was revealed that the IC values of 13f and 6d
5
6
7
8
9
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
5
0
against HUVECs were 21.1 ꢁM and 16.0 ꢁM, respectively, much higher than their IC values against
50
tumor cell lines. Note that 13f and 6d showed lower or comparable cytotoxicities against HUVECs
than pazopanib plus HDAC inhibitors (SAHA or MS275), despite their higher cytotoxicities relative to
pazopanib, SAHA or MS275 alone (Table S1 in Supporting Information).
Table 3. In Vitro Growth-Inhibitory Activities of Selected Compounds
a
IC50 (ꢁM)
Compd
solid tumor cell lines
hematological tumor cell lines
HTꢀ29
ACHN
AGS
HeLa
PCꢀ3
MDAꢀMBꢀ231
HTꢀ1080
3.25
>5
KG1
1.02
2.31
1.60
0.93
1.52
0.67
3.15
3.14
2.68
HEL
0.75
2.56
2.80
1.04
0.19
1.52
2.06
1.16
0.49
MOLTꢀ4
0.59
0.60
0.31
0.27
0.36
0.94
0.87
1.15
0.56
K562
1.66
2.89
0.99
1.10
1.52
1.82
4.31
4.15
3.97
1
0a
3e
2.96
3.85
>5
4.93 1.60
2.59
b
b
b
b
b
1
ND
ND
ND
ND
>5
ND
1
3f
3g
SAHA
2.51
>5
4.44 4.36 1.69
2.96
1.97
>5
b
b
b
b
b
1
ND
ND
ND
ND
3.42
>5
ND
1.51
1.07
2.08
>5
4.23 2.06
3.02
3.74
2.54
>5
6
d
>5
ND
ND
>5
1.71 2.38
b
b
b
b
b
b
b
1
4e
4f
4g
ND
ND
ND
ND
>5
>5
>5
ND
ND
b
b
b
b
b
1
ND
ND
ND
ND
1
2.96
3.62
3.07
2.57
>5
MS275
Pazo
3.10
>5
2.40
>5
0.34
>5
>5
>5
>5
>5
2.55
>5
>5
>5
>5
>5
0.44
>5
0.45
>5
3.66
>5
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5
5
5
5
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5
5
6
Pazo represents Pazopanib
a
Assays were performed in replicate (n≥2); the SD value are <20% of the mean.
Not determinded
b
0
1
2
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6
7
8
9
0
1
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9
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9
0
HDAC and Kinase Selectivity Profile. In general, hydroxamic acidꢀbased HDAC inhibitors show
2
+
little discrimination among different Zn ꢀdependent HDACs (class I, class II and class IV), while
orthoꢀaminoanilideꢀbased HDAC inhibitors are selective for class I. Therefore, to profile the selectivity
of these HDAC and VEGFR dual inhibitors, two representative hydroxamic acids 10a and 13f were
tested against two class I isoforms (HDAC2/8) and one class II isoform (HDAC6) with SAHA as
positive control, two representative orthoꢀaminoanilides 6d and 14g were tested against three class I
isoforms (HDAC1/2/3) with MSꢀ275 as positive control. The results in Table 4 indicated that compared
with SAHA, compound 13f possessed comparable HDAC2/6 inhibitory activities, and much better
HDAC8 inhibition. It also revealed that compound 6d and MSꢀ275 exhibited almost equivalent
inhibition towards HDAC1/2/3.
Table 4. HDACs Isoform Selectivity Profile of Representative Compounds
a
IC (ꢁM)
HDAC3
5
0
Compd
HDAC1
HDAC2
5.30
HDAC6
1.03
HDAC8
2.89
b
b
10a
ND
ND
b
b
1
3f
ND
0.38
0.22
0.91
ND
0.072
0.091
1.66
>5
b
b
SAHA
6d
ND
ND
b
b
0.59
0.43
ND
ND
b
b
1
4g
1.46
0.16
1.26
0.40
0.66
0.61
ND
ND
b
b
MS275
ND
ND
a
Assays were performed in replicate (n≥2); the SD value are <20% of the mean.
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6
b
Not determinded
Based on the considerable HDAC inhibitory and antiproliferative activities of 6d and 13f, their
VEGFRꢀ2 IC values were determined and compared with pazopanib (Table 5). Satisfyingly, both 6d
5
0
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
(
IC = 37 nM) and 13f (IC = 46 nM) exhibited uncompromised VEGFRꢀ2 inhibitory activities
50 50
relative to pazopanib (IC = 34 nM), which was in line with our preliminary VEGFRꢀ2 inhibition
50
results shown in Table 1 and Table 2. Moreover, compounds 6d and 13f were evaluated against several
other kinases including VEGFRꢀ1, VEGFRꢀ3, PDGFRβ, FGFR1, CꢀFms and CꢀKit. We chose these
28
tyrosine receptor kinases for the reason that they are all tumorꢀrelated targets inhibited by pazopanib.
Strikingly, 6d and 13f could potently inhibit all the tested kinases with IC values at the same range of
50
pazopanib (Table 5). Note that 6d was more potent than 13f and was even equivalent to pazopanib,
probably because compared with 13f, the structure of 6d was more similar to that of pazopanib. These
results provided solid evidence that rational structural modification of the solventꢀexposed phenyl
group of pazopanib was well tolerated for its kinase inhibition profile.
Table 5. Kinase Selectivity Profile of Representative Compounds
a
IC (ꢁM)
5
0
Compd
VEGFRꢀ2
0.037
VEGFRꢀ1
0.022
VEGFRꢀ3
PDGFRβ
FGFR1
0.192
CꢀFms
0.351
CꢀKit
6
d
0.046
0.088
0.125
1
3f
0.046
0.034
0.035
0.098
0.113
0.046
0.111
0.083
0.409
0.163
0.581
0.274
0.163
0.118
Pazopanib
a
Assays were performed in replicate (n≥2); the SD value are <20% of the mean.
Molecular Docking Study. The proposed binding modes of 6d and 13f in the ATP binding pocket of
VEGFRꢀ2 were analyzed using Sybyl X_2.1. As we expected, the attached HDAC functional groups of
6
d (Figure 3A) and 13f (Figure 3B) were projected towards the solvent region. Comparison of 6d and
3f with pazopanib revealed that their indazole and 2ꢀaminopyrimidine moieties were superimposed
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pretty well. Just like pazopanib, the 2ꢀaminopyrimidine moiety of 6d and 13f could also form two
hydrogen bonds with Cys917 in the hinge region. For pazopanib, another hydrogen bond between the
sulfamide group and Asn921 was observed. In the case of 6d, a similar hydrogen bond was formed
between Asn921 and the amide group. Though 13f could not form hydrogen bond with Asn921, two
extra hydrogen bonds with Glu848 were formed via its hydroxamic acid group. These results
rationalized the potent VEGFRꢀ2 inhibitory activities of 6d and 13f.
0
1
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1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. A. Overlap of the proposed binding modes of compounds 6d (green) and pazopanib (yellow)
in VEGFRꢀ2 (PDB code: 3CJG); B. Overlap of the proposed binding modes of compounds 13f (purple)
and pazopanib (yellow) in VEGFRꢀ2 (PDB code: 3CJG). Yellow dashed lines represent the hydrogen
bonds. Oxygen, nitrogen and polar hydrogen atoms are shown in red, blue and white, respectively. The
figure was generated using PyMol (http://www.pymol.org/).
The binding modes of 6d and 13f in relevant HDAC isoforms proposed by molecular docking study
also supported their considerable HDAC inhibitory activities. Figure 4A showed the overlap of 6d and
MSꢀ275 in the active site of HDAC2. We could see that the Nꢀ(2ꢀaminophenyl)benzamide moieties of
1
3f and MSꢀ275 were well superimposed, forming multiple key interactions with HDAC2, such as
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2+
chelation with Zn , hydrogen bonds with Asp161 and Gly154, and faceꢀtoꢀface πꢀπ stacking with
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
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6
Phe155. The overlaps of 13f and SAHA in HDAC2 (Figure 4B) and HDAC6 (Figure 4C) displayed the
2
+
classical binding mode of aliphatic hydroxamic acids: the hydroxamic acid groups chelate the Zn in a
bidentate manner and form multiple hydrogen bonds, the aliphatic chains occupy the hydrophobic
channel, and the aromatic terminal groups interact with the amino acid residues around the entrance of
the active site.
0
1
2
3
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9
0
1
2
3
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0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. A. Overlap of the proposed binding modes of compounds 6d (green) and MS275 (orange) in
HDAC2 (PDB code: 5IWG). B. Overlap of the proposed binding modes of compounds 13f (purple)
and SAHA (magenta) in HDAC2 (PDB code: 5IWG); C. Overlap of the proposed binding modes of
compounds 13f (purple) and SAHA (magenta) in HDAC6 (PDB code: 5EEI). Yellow dashed lines
indicate the hydrogen bonds or metal interactions. Oxygen, nitrogen and polar hydrogen atoms are
shown in red, blue and white, respectively. The figure was generated using PyMol
(http://www.pymol.org/).
Western Blot Analysis. Western blot analysis was conducted to confirm the intracellular efficacies
of 6d and 13f. Results in Figure 5A showed that compound 6d (1ꢁM) could significantly increase the
intracellular levels of the HDAC1/2/3 substrate acetylꢀhistone H4 (AcꢀHH4), which was comparable, if
not better than that of MSꢀ275 at the same concentration. Neither 6d nor MSꢀ275 had significant
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influence on the levels of the HDAC6 substrate acetylꢀtubulin (AcꢀTub), confirming the class I HDAC
selectivity of these orthoꢀaminoanilides. In contrast, the hydroxamic acid 13f (100 nM) could
simultaneously increase the levels of AcꢀHH4 and AcꢀTub. At the same concentration, the intracellular
efficacies of 13f on AcꢀHH4 and AcꢀTub were inferior to and superior to those of SAHA, respectively,
which were in line with their enzyme inhibition results shown in Table 4 that 13f possessed inferior
HDAC2 inhibitory activity but superior HDAC6 inhibitory activity relative to SAHA. In addition, 100
nM of 6d, 13f and pazopanib could completely inhibit the phosphorylation of VEGFRꢀ2 in HUVECs
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(Figure 5B), verifying their potent VEGFRꢀ2 inhibitory activities in cellular context. However, HDAC
inhibitors SAHA and MSꢀ275 had no influence on phosphorylation of VEGFRꢀ2.
Figure 5. A. HeLa cells were treated with DMSO or compounds for 6 h. The levels of acetylꢀtubulin
(AcꢀTub) and acetylꢀhistone H4 (AcꢀHH4) were determined by immunoblotting. βꢀActin was used as a
loading control. B. HUVEC cells were treated with DMSO or compounds for 2 h, then stimulated with
VEGF (50 ng/ml). The levels of phosphorylated VEGFRꢀ2 (pꢀVEGFRꢀ2) were determined by
immunoblotting. Total VEGFRꢀ2 was used as a loading control. Pazo represents pazopanib.
In Vitro HUVECs Tube Formation Assay. Encouraged by their remarkable VEGFR inhibitory
activities, compounds 6d and 13f were progressed to in vitro HUVECs tube formation assay to evaluate
their antiꢀangiogenesis potencies. To avoid the cytotoxicities of tested compounds, HUVECs were
treated with compounds at the low concentration of 100 nM. Results in Figure 6 showed that 6d and
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3f could inhibit the tube formation as effectively as pazopanib.
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Ctrl
Pazopanib (0.1 ꢁM)
6d (0.1 ꢁM)
13f (0.1 ꢁM )
Figure 6. Representative images of the capillaryꢀlike tubular network of HUVECs treated with DMSO
or compounds.
Ex Vivo Rat Thoracic Aorta Rings (TARs) Assay. Compared with HUVECs tuber formation
model, rat thoracic aorta rings (TARs) assay could better simulate the in vivo angiogenic environment
and condition. Therefore, we performed ex vivo rat TARs assay to further evaluate the antiangiogenic
activity of 6d and 13f. As clearly presented in Figure 7, in the negative control group, a mass of
microvessels sprouted from the thoracic aorta ring. In contrast, compounds 6d and 13f as well as the
positive control pazopanib, dramatically inhibited the microvessel outgrowth.
Ctrl
Pazopanib (5 ꢁM)
6d (5 ꢁM)
13f (5 ꢁM)
Figure 7. Representative images of rat thoracic aorta rings treated with DMSO or compounds.
In Vivo Pharmacokinetic (PK) Profile. Though the in vitro activities of 6d and 13f were
comparable, only 6d was progressed to in vivo pharmacokinetic study because of the potential
40
metabolic liability of hydroxamic acidꢀbased compounds. The hydrochlorate of 6d was administrated
intravenously (IV) at 2 mg/kg or orally (PO) at 10 mg/kg in Sprague−Dawley (SD) rats. The
concentration–time curves and PK parameters of 6d were respectively shown in Figure 8 and Table 6,
which indicated the desirable PK properties of 6d (hydrochlorate). Importantly, 6d (hydrochlorate)
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possessed the high oral bioavailability (F) of 72 %, warranting further evaluation of its oral antitumor
activity.
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Figure 8. The concentration–time curves of 6d in SD rats following IV (2 mg/kg) or PO (10 mg/kg)
administration.
Table 6. PK Parameters of 6d in SD Rats (N = 6)
Dose
mg/kg)
t1/2
(h)
Tmax
(h)
Cmax
(ng/mL)
AUC0ꢀt
(ng*h/mL)
MRT0ꢀt
(h)
Vz
(mL/kg)
CL
(mL/h/kg)
F
(%)
Route
(
2
IV
1.64
2.10
0.08
0.5
2775
3245
2377
8571
1.13
2.54
2061
876
1
0
PO
72
In Vivo Antitumor Activity Evaluation. A HTꢀ29 xenograft model in nude mice was established to
7
evaluate the in vivo antitumor potency of compound 6d (hydrochlorate). HTꢀ29 cells (1 × 10 ) were
subcutaneously implanted in the right flanks of male nude mice (BALB/cꢀnu). When tumor size
3
reached about 100 mm , mice were randomized to six per group and were orally administrated with 6d
(hydrochlorate), SAHA or pazopanib for 25 consecutive days. Tumor growth inhibition (TGI) and
relative increment ratio (T/C) were calculated at the end of treatment to evaluate the antitumor effects
in the aspects of tumor weight and tumor volume, respectively. As shown in Table 7, 6d (hydrochlorate,
5
0 mg/kg/d) exhibited potent oral antitumor activity, which was comparable to pazopanib at the same
dosage (50 mg/kg/d) and SAHA at the higher dosage (100 mg/kg/d). The tumor growth delay induced
by 6d (hydrochlorate) were visualized by the tumor growth curve in Figure 9A and the final tumor
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tissue size in Figure 9B. Moreover, in the mice group treated by 6d (hydrochlorate), no significant body
weight loss and no evident toxic signs in liver and spleen were observed.
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Table 7. In Vivo Antitumor Efficacy in HT-29 Xenograft Model
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a
a
Compd
Administration
50 mg/kg/d, PO
T/C (%)
65
TGI (%)
40
6
d
Pazopanib
SAHA
50 mg/kg/d, PO
100 mg/kg/d, PO
61
66
44
39
a
Compared with the control group, all treated groups showed statistically significant (P < 0.05) T/C and
TGI by Student’s twoꢀtailed t test.
Figure 9. A. Growth curve of implanted HTꢀ29 xenograft in nude mice. B. Picture of dissected HTꢀ29
tumor tissue.
CONCLUSIONS
In summary, a novel series of pazopanib derivatives simultaneously inhibiting HDAC and VEGFR
were rationally designed, synthesized and identified. Among them, one pazopanibꢀbased orthoꢀ
aminoanilide 6d and one pazopanibꢀbased hydroxamic acid 13f exhibited comparable HDACs
inhibitory activities to MSꢀ275 and SAHA respectively, which transformed to their potent
antiproliferative activities. Compounds 6d and 13f also exhibited uncompromised multiple tyrosine
kinases inhibitory activities and antiangiogenic potencies compared to pazopanib. Importantly,
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compound 6d possessed desirable pharmacokinetic profiles with the oral bioavailability of 72 % in SD
rats and considerable in vivo antitumor efficacy in a HTꢀ29 xenograft model. Though 6d showed no
significant advantage over pazopanib in our preliminary in vivo antitumor model, it should be noted
that 6d is of great therapeutic potential for mutant TP53ꢀexpressing tumors due to the clinical benefit in
a phase I trial of pazopanib plus SAHA. Moreover, based on the encouraging results of another phase I
clinical study that combination of pazopanib and HDAC inhibitor resulted in strikingly durable
responses in patients with pazopanibꢀrefractory diseases, further evaluation of 6d in pazopanibꢀ
refractory tumor models is warranted. Currently, related in vitro and in vivo studies are in progress in
our lab.
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EXPERIMENTAL SECTION
Chemistry. The chemical reagents and solvents were purchased from commercial sources and were
1
13
used without further purification. H NMR and C NMR spectra were obtained using a Bruker DRX
spectrometer at 400 and 100 MHz respectively. Chemical shifts were reported in parts per million
1
(
ppm). Multiplicity of H NMR signals was reported as singlet (s), doublet (d), triplet (t), quartert (q)
and multiplet (m). ESIꢀMS data was recorded on an API 4000 spectrometer. High resolution mass
spectra were conducted by Shandong Analysis and Test Center in Ji’nan. Melting points were
determined using open capillary on an uncorrected electrothermal melting points apparatus. HPLC
analysis was carried out on an Agilent 1200 with an ODS HYPERSIL column (4.6 mm×250 mm, 5
ꢁ
m). The flow rate is 1 mL / min. All final compounds achieved a minimum of 95% purity.
N-(2-Chloropyrimidin-4-yl)-2,3-dimethyl-2H-indazol-6-amine (2). To a stirred solution of 1 (5.00
g, 31.0 mmol) and NaHCO (10.4 g, 124 mmol) in EtOH (100 mL) was added 2,4ꢀdichloropyrimidine
3
(13.9 g, 93 mmol) at room temperature. The reaction was stirred at 75 °C for 4 h. The resulting
suspension was filtered, and the residue was washed thoroughly with EtOAc. The filtrate was
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concentrated under vacuum to leave residue. The residue was triturated with EtOAc to afford
5
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9
1
1
1
1
1
1
1
1
1
1
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2
2
2
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2
2
2
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3
3
3
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5
6
1
compound 2, pale yellow solid (7.64 g, 90%). Mp: 215ꢀ216 ℃. H NMR (400 MHz, DMSOꢀd ) δ 10.02
6
(s, 1H, ArNHAr), 8.16 (d, J = 5.9 Hz, 1H, ArH), 7.96 (s, 1H, ArH), 7.63 (d, J = 8.9 Hz, 1H, ArH), 6.98
0
1
2
3
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(
dd, J = 8.9, 1.8 Hz, 1H, ArH), 6.78 (d, J = 5.9 Hz, 1H, ArH), 4.01 (s, 3H, NCH ), 2.58 (s, 3H, ArCH ).
3 3
N-(2-Chloropyrimidin-4-yl)-N,2,3-trimethyl-2H-indazol-6-amine (3). To a solution of compound
2 (1.00 g, 3.65 mmol) in DMF (50 mL) was added cesium carbonate (2.38 g, 7.31 mmol). The reaction
mixture was stirred for 30 min under nitrogen. Iodomethane (0.78 g, 5.48 mmol) was added and the
reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured into ice cold
water and stirred for 30 min. The resulting precipitate was filtered off, washed with water, and airꢀ
dried, the crude was recrystallization by EtOAc to afford the desired compound 3, pale yellow solid
1
(
0.84 g, 80%). Mp: 173ꢀ174 ℃. H NMR (400 MHz, DMSOꢀd ) δ 7.95 (d, J = 6.1 Hz, 1H, ArH), 7.80
6
(
dd, J = 0.8, 8.7 Hz, 1H, ArH), 7.51 (dd, J = 0.8, 1.8 Hz, 1H, ArH), 6.88 (dd, J = 1.8, 8.8 Hz, 1H, ArH),
6.24 (d, J = 6.1 Hz, 1H, ArH), 4.06 (s, 3H, NCH ), 3.42 (s, 3H, NCH ), 2.63 (s, 3H, ArCH ).
3
3
3
4
-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)benzoic acid (4a).
To a solution of 3 (0.50 g, 1.74 mmol) and 4ꢀaminobenzoic acid (0.29 g, 2.09 mmol) in isopropanol (30
mL) was added 2 drops of conc. HCl, and the mixture was heated to reflux with stirring for 4 h. The
mixture was cooled to room temperature and the resulting precipitate was collected via filtration and
1
washed with EtOAc, affording compound 4a, white solid (0.53 g, 79%). Mp: >250 ℃. H NMR (400
MHz, DMSOꢀd ) δ 12.16 (s, 1H, ArCOOH), 9.80 (s, 1H, ArNHAr), 7.91 (d, J = 6.3 Hz, 1H , ArH),
6
7
.79 (d, J = 8.7 Hz, 3H, ArH), 7.74 (d, J = 8.6 Hz, 2H , ArH), 7.49 (d, J = 1.7 Hz, 1H , ArH), 6.91 (dd,
J = 1.8, 8.8 Hz, 1H , ArH), 5.94 (d, J = 6.2 Hz, 1H, ArH), 4.08 (s, 3H, NCH ), 3.51 (s, 3H, NCH ), 2.64
3
3
ꢀ
(
s, 3H, ArCH ). ESIꢀMS m/z: 387.5 [MꢀH] .
3
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Compounds 4b and 4c were prepared from compound 3 in a similar manner as described for
compound 4a.
3-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)benzoic acid (4b).
0
1
2
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1
White solid, 81% yield. Mp: >250 ℃. H NMR (400 MHz, DMSOꢀd ) δ 12.22 (s, 1H, ArCOOH), 9.68
6
(s, 1H, ArNHAr), 8.52 (s, 1H, ArH), 7.88 (dd, J = 2.1, 7.6 Hz, 1H, ArH), 7.84 (d, J = 6.3 Hz, 1H,
ArH), 7.79 (d, J = 8.8 Hz, 1H, ArH), 7.54 (d, J = 7.7 Hz, 1H, ArH), 7.50 (d, J = 1.7 Hz, 1H, ArH), 7.34
(t, J = 7.9 Hz, 1H, ArH), 6.91 (dd, J = 1.8, 8.7 Hz, 1H, ArH), 5.83 (d, J = 6.3 Hz, 1H, ArH), 4.07 (s,
ꢀ
3
H, NCH ), 3.52 (s, 3H, NCH ), 2.64 (s, 3H, ArCH ). ESIꢀMS m/z: 387.6 [MꢀH] .
3
3
3
5-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-2-methylbenzoic
ꢀ
acid (4c). White solid, 90% yield. Mp: 230ꢀ232 ℃. ESIꢀMS m/z: 401.6 [MꢀH] .
Methyl 4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino) benzoate
(
5a). To a solution of compound 4a (0.50 g, 1.29 mmol) in MeOH (50 mL) was added SOCl (0.61 g,
2
5
.16 mmol) dropwise at 0 ℃. The mixture was heated to reflux for 4 h. The solvent was evaporated
under vacuum. The crude product was recrystallized from isopropanol to obtain 5a, white solid (0.49 g,
1
9
5%). Mp: 198ꢀ200 ℃. H NMR (400 MHz, DMSOꢀd ) δ 9.63 (s, 1H, ArNHAr), 7.92 (d, J = 6.0 Hz,
6
1H, ArH), 7.83 (d, J = 8.6 Hz, 2H, ArH), 7.78 (d, J = 8.6 Hz, 1H, ArH), 7.72 (d, J = 8.6 Hz, 2H, ArH),
7
4
.46 (dd, J = 0.8, 1.7 Hz, 1H, ArH), 6.89 (dd, J = 1.8, 8.8 Hz, 1H, ArH), 5.91 (d, J = 6.0 Hz, 1H, ArH),
.07 (s, 3H, NCH ), 3.80 (s, 3H, COOCH ), 3.49 (s, 3H, NCH ), 2.64 (s, 3H, ArCH ). ESIꢀMS m/z:
3
3
3
3
+
403.5 [M+H] .
Compounds 5b and 5c were prepared from compounds 4b and 4c in a similar manner as described
for 5a, respectively.
Methyl
3-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)benzoate
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(
5b). White solid. 93% yield. Mp: 192ꢀ194 ℃. H NMR (400 MHz, DMSOꢀd ) δ 9.77ꢀ9.61 (m, 1H,
6
5
6
7
8
9
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6
ArNHAr), 8.67 (s, 1H, ArH), 7.86 (t, J = 6.4 Hz, 2H, ArH), 7.79 (d, J = 8.7 Hz, 1H, ArH), 7.54 (d, J =
7.7 Hz, 1H, ArH), 7.50 (d, J = 1.7 Hz, 1H, ArH), 7.36 (t, J = 7.9 Hz, 1H, ArH), 6.91 (dd, J = 1.8, 8.7
Hz, 1H, ArH), 5.82 (d, J = 6.3 Hz, 1H, ArH), 4.07 (s, 3H, NCH ), 3.84 (s, 3H, COOCH ), 3.54 (s, 3H,
0
1
2
3
4
5
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0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
3
3
+
NCH ), 2.64 (s, 3H, ArCH ). ESIꢀMS m/z: 403.5 [M+H] .
3
3
Methyl
5-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-2-
+
methylbenzoate (5c). White solid. 94% yield. Mp: 218ꢀ220 ℃. ESIꢀMS m/z: 417.6 [M+H] .
4
-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-N-
hydroxybenzamide (6a). KOH (28.55 g, 509 mmol) and NH OH.HCl (23.84 g, 343 mmol) were
2
dissolved, respectively, in 70 mL and 120 mL MeOH to get solution A and solution B. Then solution A
was added dropwise to solution B. After filtering the precipitate (KCl), a NH OK solution was
2
obtained. Compound 5a (0.20 g, 0.50 mmol) was dissolved in 30 mL NH OK solution and stirred for 2
2
h. After the reaction was complete, it was evaporated under vacuum. The residue was acidified by
addition of 1 M HCl to a pH 5ꢀ6. The resulting precipitate was collected by filtration and dried to
1
afford compound 6a, white solid (0.081 g, 40% yield). Mp: 210ꢀ212 ℃. H NMR (400 MHz, DMSOꢀ
d6) δ 10.99 (s, 1H, OH), 9.49 (s, 1H, ArNHAr), 8.85 (s, 1H, NH), 7.89 (d, J = 6.1 Hz, 1H, ArH), 7.84ꢀ
7
.70 (m, 3H, ArH), 7.59 (d, J = 8.3 Hz, 2H, ArH), 7.46 (s, 1H, ArH), 6.90 (d, J = 8.7 Hz, 1H, ArH),
1
3
5
.86 (d, J = 6.2 Hz, 1H, ArH), 4.07 (s, 3H, NCH ), 3.49 (s, 3H, NCH ), 2.64 (s, 3H, ArCH ). C NMR
3
3
3
(100 MHz, DMSOꢀd ) δ 164.82, 162.88, 159.63, 156.25, 147.45, 144.33, 142.37, 132.64, 127.88,
6
1
24.75, 122.21, 120.19, 119.95, 117.88, 114.42, 97.36, 38.42, 37.85, 9.90. HRMS (APꢀESI) m/z calcd
+
for C H N O [M+H] 404.1757, found 404.1739. Retention time: 14.1 min, eluted with 40%
2
1
21
7
2
methanol/60% water (containing 0.1% trifluoroacetic acid).
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Compounds 6b and 6c were prepared from compounds 5b and 5c in a similar manner as described
for compound 6a, respectively.
3-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-N-
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hydroxybenzamide (6b). White solid. 53% yield. Mp: 200ꢀ202 ℃. H NMR (400 MHz, DMSOꢀd ) δ
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1.09 (s, 1H, OH), 9.32 (s, 1H, ArNHAr), 8.96 (s, 1H, NH), 8.32 (s, 1H, ArH), 7.91ꢀ7.81(m, 2H, ArH),
7.77 (d, J = 8.7 Hz, 1H, ArH), 7.45 (d, J = 1.7 Hz, 1H, ArH), 7.29ꢀ7.17 (m, 2H, ArH), 6.89 (dd, J = 1.8,
.8 Hz, 1H, ArH), 5.77 (d, J = 6.0 Hz, 1H, ArH), 4.06 (s, 3H, NCH ), 3.50 (s, 3H, NCH ), 2.63 (s, 3H,
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ArCH ). C NMR (100 MHz, DMSOꢀd ) δ 164.95, 162.75, 156.87, 151.57, 147.24, 141.57, 140.33,
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33.86, 132.85, 128.91, 122.61, 122.28, 120.43, 120.19, 119.62, 118.87, 114.52, 97.33, 38.98, 37.92,
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.92. HRMS (APꢀESI) m/z calcd for C H N O [M+H] 404.1835, found 404.1812. Retention time:
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8.8 min, eluted with 36% methanol/64% water (containing 0.1% trifluoroacetic acid).
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-((4-((2,3-Dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)-N-hydroxy-2-
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methylbenzamide (6c). White solid. 57% yield. Mp: 130ꢀ132 ℃. H NMR (400 MHz, DMSOꢀd ) δ
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0.76 (s, 1H, OH), 9.21 (s, 1H, ArNHAr), 9.02 (s, 1H, NH), 7.83 (d, J = 6.0 Hz, 1H, ArH), 7.79ꢀ7.74
m, 2H, ArH), 7.71 (dd, J = 2.4, 8.4 Hz, 1H, ArH), 7.44 (d, J = 1.7 Hz, 1H, ArH), 7.04 (d, J = 8.4 Hz,
1H, ArH), 6.88 (dd, J = 1.7, 8.8 Hz, 1H, ArH), 5.75 (d, J = 5.8 Hz, 1H, ArH), 4.06 (s, 3H, NCH ), 3.47
(
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s, 3H, NCH ), 2.63 (s, 3H, ArCH ), 2.23 (s, 3H, ArCH ). C NMR (100 MHz, DMSOꢀd ) δ 166.03,
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62.65, 155.16, 151.56, 146.56, 142.98, 140.13, 135.72, 134.82, 133.61, 132.21, 131.47, 123.39,
122.65, 120.62, 118.89, 114.71, 97.69, 39.45, 37.97, 19.11, 9.94. HRMS (APꢀESI) m/z calcd for
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C H N O [M+H] 418.1991, found 418.1990. Retention time: 14.2 min, eluted with 36%
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methanol/64% water (containing 0.1% trifluoroacetic acid).
N-(2-Aminophenyl)-4-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)
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Journal of Medicinal Chemistry
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benzamide (6d). To a solution of 4a (0.50g, 1.29 mmol) in anhydrous DMF (50 mL) in ice bath, was
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added 2ꢀ(1HꢀBenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium tetrafluoroborate (TBTU, 0.50 g, 1.54
mmol), followed by Et N (0.16 g, 1.54 mmol). 30 min later, oꢀdiaminobenzene (0.17 g, 1.54 mmol)
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was added, 12 h later, the solution was diluted by water and extracted with ethyl acetate. The combined
organic extract was washed with saturated NaHCO and brine, dried over Na SO overnight, and the
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solvent was evaporated under vacuum. The crude product was purified by silica gel column
chromatography (MeOH/CH Cl , 1/50ꢀ1/20) to afford compound 6d (0.40 g, 65% yield), white solid.
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Mp: 138ꢀ140 ℃. H NMR (400 MHz, DMSOꢀd ) δ 9.54 (s, 1H, NH), 9.49 (s, 1H, NH), 7.91 (d, J = 6.0
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Hz, 1H, ArH), 7.87 (d, J = 9.0 Hz, 2H, ArH), 7.83 (d, J = 8.8 Hz, 2H, ArH), 7.80ꢀ7.76 (m, 1H, ArH),
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.47 (d, J = 2.3 Hz, 1H, ArH), 7.17 (dd, J = 1.5, 7.9 Hz, 1H, ArH), 6.96 (td, J = 1.6, 7.6 Hz, 1H, ArH),
.91 (dd, J = 1.8, 8.8 Hz, 1H, ArH), 6.79 (dd, J = 1.5, 8.0 Hz, 1H, ArH), 6.60 (td, J = 1.5, 7.5 Hz, 1H,
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ArH), 5.87 (d, J = 6.0 Hz, 1H, ArH), 4.89 (s, 2H, ArNH ), 4.06 (s, 3H, NCH ), 3.51 (s, 3H, NCH ),
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2.63 (s, 3H, ArCH ). C NMR (100 MHz, DMSOꢀd ) δ 165.39, 162.91, 159.45, 156.02, 147.46,
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44.54, 143.42, 142.33, 132.64, 128.88, 126.97, 126.63, 126.60, 124.33, 122.23, 120.17, 119.99,
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17.79, 116.84, 116.69, 114.46, 97.41, 38.48, 37.85, 9.90. HRMS (APꢀESI) m/z calcd for C H N O
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[M+H] 479.2230, found 479.2257. Retention time: 8.7 min, eluted with 65% methanol/35% water.
N-(2-Aminophenyl)-3-((4-((2,3-dimethyl-2H-indazol-6-yl)(methyl)amino)pyrimidin-2-yl)amino)
benzamide (6e). Compound 6e was prepared from compound 4b in a similar manner as described for
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compound 6d. Offꢀwhite solid. 43% yield. Mp: 230ꢀ232 ℃. H NMR (400 MHz, DMSOꢀd ) δ 9.60 (s,
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1H, NH), 9.37 (s, 1H, NH), 8.49 (s, 1H, ArH), 7.91 (dd, J = 2.2, 8.2 Hz, 1H, ArH), 7.86 (d, J = 6.0 Hz,
1H, ArH), 7.76 (d, J = 8.8 Hz, 1H, ArH), 7.51 (d, J = 7.6 Hz, 1H, ArH), 7.45 (d, J = 1.7 Hz, 1H, ArH),
7.32 (t, J = 7.9 Hz, 1H, ArH), 7.23ꢀ7.16 (m, 1H, ArH), 6.97 (td, J = 1.6, 7.6 Hz, 1H, ArH), 6.89 (dd, J =
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