Design, synthesis and biological evaluation of novel 1H-1,2,4-triazole, benzothiazole and indazole-based derivatives as potent FGFR1 inhibitors viafragment-based virtual screening

Article abstract of DOI:10.1080/14756366.2019.1673745

Fibroblast growth-factor receptor (FGFR) is a potential target for cancer therapy. We designed three novel series of FGFR1 inhibitors bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via fragment-based virtual screening. All the newly synthesised compounds were evaluated in vitro for their inhibitory activities against FGFR1. Compound 9d bearing an indazole scaffold was first identified as a hit compound, with excellent kinase inhibitory activity (IC₅₀ = 15.0 nM) and modest anti-proliferative activity (IC₅₀ = 785.8 nM). Through two rounds of optimisation, the indazole derivative 9 u stood out as the most potent FGFR1 inhibitors with the best enzyme inhibitory activity (IC₅₀ = 3.3 nM) and cellular activity (IC₅₀ = 468.2 nM). Moreover, 9 u also exhibited good kinase selectivity. In addition, molecular docking study was performed to investigate the binding mode between target compounds and FGFR1.

Full text of DOI:10.1080/14756366.2019.1673745

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation
of novel 1H-1,2,4-triazole, benzothiazole and
indazole-based derivatives as potent FGFR1
inhibitors viafragment-based virtual screening
Jian Liu, Yu Wen, Lina Gao, Liang Gao, Fengjun He, Jingxian Zhou, Junwei
Wang, Rupeng Dai, Xiaojing Chen, Di Kang & Lihong Hu
To cite this article: Jian Liu, Yu Wen, Lina Gao, Liang Gao, Fengjun He, Jingxian Zhou, Junwei
Wang, Rupeng Dai, Xiaojing Chen, Di Kang & Lihong Hu (2020) Design, synthesis and biological
evaluation of novel 1H-1,2,4-triazole, benzothiazole and indazole-based derivatives as potent
FGFR1 inhibitors viafragment-based virtual screening, Journal of Enzyme Inhibition and Medicinal
Chemistry, 35:1, 72-84, DOI: 10.1080/14756366.2019.1673745
To link to this article: https://doi.org/10.1080/14756366.2019.1673745
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Submit your article to this journal
View Crossmark data
Published online: 04 Nov 2019.
View related articles
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 72–84
https://doi.org/10.1080/14756366.2019.1673745
RESEARCH PAPER
Design, synthesis and biological evaluation of novel 1H-1,2,4-triazole,
benzothiazole and indazole-based derivatives as potent FGFR1 inhibitors
via fragment-based virtual screening
ꢀ
ꢀ
ꢀ
Jian Liu , Yu Wen , Lina Gao , Liang Gao, Fengjun He, Jingxian Zhou, Junwei Wang, Rupeng Dai, Xiaojing Chen,
Di Kang and Lihong Hu
Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization, Stake Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Pharmacy, Nanjing University of Chinese
Medicine, Nanjing, PR China
ABSTRACT
ARTICLE HISTORY
Received 28 December 2018
Accepted 9 September 2019
Fibroblast growth-factor receptor (FGFR) is a potential target for cancer therapy. We designed three novel
series of FGFR1 inhibitors bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via fragment-
based virtual screening. All the newly synthesised compounds were evaluated in vitro for their inhibitory
activities against FGFR1. Compound 9d bearing an indazole scaffold was first identified as a hit
compound, with excellent kinase inhibitory activity (IC50 ¼ 15.0 nM) and modest anti-proliferative activity
(IC50 ¼ 785.8 nM). Through two rounds of optimisation, the indazole derivative 9 u stood out as the
most potent FGFR1 inhibitors with the best enzyme inhibitory activity (IC50 ¼ 3.3 nM) and cellular activity
(IC50 ¼ 468.2 nM). Moreover, 9 u also exhibited good kinase selectivity. In addition, molecular docking
study was performed to investigate the binding mode between target compounds and FGFR1.
KEYWORDS
Anticancer; FGFR1; FGFR1
inhibitor; Fragment-based
virtual screening
GRAPHICAL ABSTRACT
Introduction
inflammation. Targeted inhibition of protein kinases has thereby
become an attractive therapeutic strategy in treatment of rele-
Protein kinases constitute one of the largest protein families in
humans1–5. The kinase enzymes in this family catalyse phosphoryl-
ation of serine, threonine, or tyrosine residues, which regulate the
majority of signal transduction pathways in cell, and thus play an
important role in cell growth, metabolism, differentiation and
apoptosis consequently. Deregulation of protein kinases is impli-
vant diseases6,7
.
Fibroblast growth-factor receptors (FGFRs) form a sub-family
of the receptor tyrosine kinase (RTK) superfamily, which consists
of four highly conserved functional members (FGFR 1–4)8,9. FGFR
signalling is initiated by binding of extracellular FGF ligand
cated in a number of diseases including cancer, diabetes and which leads to receptor dimerisation and cross-phosphorylation
CONTACT Jian Liu
liujian623@njucm.edu.cn; Di Kang
kangdi@njucm.edu.cn; Lihong Hu
lhhu@njucm.edu.cn
Jiangsu Key Laboratory for Functional
Substance of Chinese Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Stake Key Laboratory Cultivation Base for
TCM Quality and Efficacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China.
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
73
of the kinase domains proceed to phosphorylate intracellular further optimisation of the original indazole inhibitors via intro-
substrates such as FRS2, Gab1, PLCc, and STAT1. Subsequent duced halogen atoms, that were often used to improve perme-
downstream signalling is complex and includes activation of the ability through the modulation of modulation of compound’s
PI3K-Akt and the Ras/Raf/Mek/Erk pathways. In normal cells, lipophilicity and halogen-protein interactions.
FGFR plays fundamental roles in many physiologic processes,
Herein, with the fragment-based virtual screening strategy,
including embryogenesis, tissue homeostasis, tissue repairing, we designed three novel series of FGFR1 inhibitors bearing 1H-
wound healing, and inflammation10–12. Therefore, the inhibition 1,2,4-triazole, benzothiazole and indazole scaffold (Figure 2(b)).
of FGFR signalling pathway presents a promising option for can-
cer therapeutics.
In the past few years, different strategies have been pursued
to inhibit abnormal FGFR signalling pathways, among them sev-
eral small-molecule FGFR inhibitors have advanced into clinical
trial (Figure 1)13. While the pan-kinase inhibitors (e.g. dovitinib
(TKI-258)14, nintedanib (BIBF-1120)15, and ponatinib (AP-
24534)16) which were originally adopted for the treatment of can-
cers that harbour aberrant FGFR, severe side effects were
observed due to inhibition of several off-target kinases such as
EGFR, VEGFR, PDGFR, etc. More recently, there has been an
increasing interest in developing selective FGFR inhibitors. Several
selective FGFR inhibitors bear various scaffolds have been devel-
oped into clinical investigation (e.g. NVP-BGJ39817, LY-
287445518, AZD454719, JNJ-4275649320, and Debio-134721).
In our previous studies, a series of indazole derivatives were
discovered as potent FGFR1 inhibitors22. The most promising com-
pound L1 exhibited good enzymatic inhibition against FGFR1 and
modest cellular inhibition (Figure 1). In this study, in order to
improve the cellular inhibition, discover novel scaffold and enrich
the structure-activity relationship, further work has been putting
into modification of indazole scaffold and the hydrophobic sub-
stituents. The goal of this compound modification was to increase
the ligand-receptor interaction, and improve the physico-chemical
property. As a result, we designed the novel FGFR1 inhibitors by
the following two strategies: (a) according to the fragment-based
drug design strategy, we introduced novel flat hetero aromatic Figure 2. (a) Binding model of AZD4547 and NVP-BGJ398 to the FGFR1 kinase
domain (PDB ID: 4V05, 3TTO). (b) The fragment-based virtual screening protocol.
ring which formed key H-bond interaction with hinge region; (b)
Figure 1. Structure of representative FGFR inhibitors.

74
J. LIU ET AL.
The indazole derivative 9 u stood out as the most potent FGFR1 General method for preparation of compounds 9a-9z
(exemplified by 9d)
inhibitor, and it also exhibited good kinase selectivity.
Additionally, the docking studies were carried out to investigate
the receptor-ligand interaction.
The detail experimental procedures of intermediate 2, 3, 4, 6, 7
and
8
are described as previous reference 22 (see in
Supplementary Material).
4–(4-ethylpiperazin-1-yl)-N-(6–(3-methoxyphenyl)-1H-inda-
Materials and methods
zol-3-yl)benzamide (9d) The building block
8
(100.0 mg,
0.2 mmol) was dissolved in dioxane (2 ml), then followed by the
addition of (3-methoxyphenyl)boronic acid (85.2 mg, 0.5 mmol),
Pd(dppf)Cl2 (20 mg), 1 M Cs2CO3 (500 mL). The reaction mixture
was heated at 120 ꢁC. for 1 h, then it cooled to rt. After concen-
trated, the residue was dissolved in EtOAc (50 ml) and washed
with H2O (10 ml ꢂ2), and brine (10 ml ꢂ2), dried over Na2SO4,
concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel DCM-MeOH (10:1) to give the 9d (45.6 mg,
44%). Mp 226.5–230.2 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.80
(brs, 1H), 10.53 (brs, 1H), 7.99 (d, J ¼ 8.9 Hz, 2H), 7.78 (d, J ¼ 8.6 Hz,
1H), 7.68 (s, 1H), 7.35–7.45 (m, 2H), 7.30 (d, J ¼ 7.9 Hz, 1H),
7.24–7.26 (d, J ¼ 2.0 Hz, 1H), 7.03 (d, J ¼ 9.0 Hz, 2H), 6.97
(dd, J ¼ 8.2, 2.0 Hz, 1H), 3.85 (s, 3H), 2.48–2.55 (m, 4H), 2.39
(q, J ¼ 7.1 Hz, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). 13 C NMR (100 MHz,
DMSO-d6) d: 165.5, 160.2, 153.6, 142.6, 142.2, 141.1, 138.9, 130.5,
129.8, 123.1, 123.0, 120.0, 119.8, 117.0, 113.9, 113.6, 113.1, 108.3,
55.6, 52.6, 52.1, 47.4, 12.4. ESI-MS (m/z): [M þ H]þ ¼ 456.7 (Calcd:
456.55). HRMS: calcd for C27H31N5O2 (M þ H)þ 456.2400, found
456.2391. HPLC analysis: MeOH-H2O (80: 20), 6.85 min,
96.8% purity.
Fragment-based virtual screening
Pharmacophore screening
The fragment library was derived from kinase hinge region
directed library provided by enamine (https://enamine.net/index.
php), which contained 11809 fragments. On the basis of the
chemical features of FGFR1 inhibitors, these pharmacophore fea-
tures, hydrogen bond acceptor (HBA), hydrogen bond donor
(HBD), and hydrophobic (HY) were defined as a query for
pharmacophore screening. The database screening was per-
formed using the Ligand Pharmacophore Mapping protocol in
DS 4.0. The fit values were calculated based on the chemical
substructures map the location constraints of the pharmaco-
phoric features and their distance deviation from the feature
centres. Finally, only the compounds showed good fit values in
pharmacophore model could enter the further molecular docking
study.
N-(6–(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-4-((3R,5S)-3,5-
dimethylpiperazin-1-yl)benzamide (9a) White solid: 29.2 mg,
yield 38%; Mp 228.1–232.2 ꢁC. 1H NMR (400 MHz, DMSO-d6) d:
12.81 (brs, 1H), 10.53 (brs, 1H), 7.99 (d, J ¼ 8.3 Hz, 2H), 7.77 (d,
J ¼ 8.0 Hz, 1H), 7.68 (s, 1H), 7.37 (dd, J ¼ 8.6, 1.3 Hz, 1H), 7.04 (d,
J ¼ 9.0 Hz, 2H), 6.85 (d, J ¼ 2.2 Hz, 2H), 6.54 (t, J ¼ 2.1 Hz, 1H), 3.85
(s, 2H), 3.83 (s, 6H), 2.90–3.01 (m, 2H), 2.35–2.41 (m, 2H), 1.12
(s, 3H), 1.10 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) d: 165.7, 161.3,
153.2, 143.3, 142.1, 141.0, 139.0, 129.9, 122.9, 120.0, 117.1, 114.0,
Molecular docking study
Molecular docking of compounds into the three dimensional X-
ray structure of FGFR1 (PDB ID: 4ZSA) was carried out using the
surflex-dock module of the Sybyl-x 2.0 software package.The
three-dimensional structure of compound was constructed using
ChemBio 3 D Ultra software [Chemical Structure Drawing
Standard; Cambridge corporation, USA 2010], then it was ener-
getically minimised by using MMFF94 with 5000 iterations and
minimum RMS gradient 0.05. The protein was prepared by the
Protein preparation wizard of Sybyl-x 2.0. The waters were elimi-
nated from the protein and the polar hydrogen was added.
Receptor grids were generated using Receptor Grid Generation.
The generated binding site was just the ATP-binding pocket of
FGFR1, including several key amino acid residues: Glu562,
Ala564.
108.4, 105.8, 99.9, 55.8, 53.1, 50.6, 18.7. ESI-MS (m/z): [M þ H]þ
¼
486.1 (Calcd: 486.24). ESI-HRMS: calcd for C28H33N5O3 (M þ H)þ
486.2505, found 486.2502. HPLC analysis: MeOH-H2O (80: 20),
7.36 min, 96.0% purity.
4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(6–(3-methoxy-
phenyl)-1H-indazol-3-yl)benzamide (9 b) White solid: 24.8 mg,
yield 30%; Mp 224.8–227.4 ꢁC. 1H NMR (400 MHz, DMSO-d6) d:
12.81 (brs, 1H), 10.52 (brs, 1H), 7.98 (d, J ¼ 8.9 Hz, 2H), 7.78
(d, J ¼ 8.1 Hz, 1H), 7.68 (s, 1H), 7.37–7.44 (m, 2H), 7.30 (d,
J ¼ 7.9 Hz, 1H), 7.26 (s, 1H), 7.03 (d, J ¼ 8.2 Hz, 2H), 6.97 (dd, J ¼ 8.1,
2.2 Hz, 1H), 3.85 (s, 3H), 3.79–3.81 (m, 2H), 2.85–2.93 (m, 2H),
2.28–2.34 (m, 2H), 1.09 (s, 3H), 1.07 (s, 3H). 13 C NMR (100 MHz,
DMSO-d6) d: 165.7, 160.2, 153.1, 142.5, 142.2, 141.0, 139.0, 130.6,
129.9, 123.0, 119.9, 117.0, 114.1, 113.6, 113.1, 108.3, 55.6, 52.8,
50.7, 18.4. ESI-MS (m/z): [M þ H]þ ¼ 456.1 (Calcd: 456.23). ESI-
HRMS: calcd for C27H31N5O2 (M þ H)þ 456.2400, found 456.2390.
HPLC analysis: MeOH-H2O (80: 20), 7.27 min, 95.9% purity.
N-(6–(3-methoxyphenyl)-1H-indazol-3-yl)-4–(4-methylpipera-
zin-1-yl)benzamide (9c) White solid: 40.2 mg, yield 45%; Mp
224.8–227.4 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.82 (brs, 1H),
10.55 (brs, 1H), 8.00 (d, J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 8.8 Hz, 1H), 7.65
(s, 1H), 7.35–7.45 (m, 2H), 7.29 (d, J ¼ 8.0 Hz, 1H), 7.25 (d,
J ¼ 2.0 Hz, 1H), 7.05 (d, J ¼ 8.8 Hz, 2H), 6.95 (dd, J ¼ 8.2, 1.9 Hz, 1H),
3.83 (s, 3H), 2.48–2.55 (m, 4H), 2.39 (q, J ¼ 7.1 Hz, 2H), 1.05 (t,
J ¼ 7.2 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) d: 165.6, 160.2,
153.7, 142.6, 141.1, 139.0, 130.5, 129.8, 123.1, 122.9, 120.0, 119.9,
117.0, 113.9, 113.6, 113.1, 108.3, 56.2, 52.6, 52.1, 46.2. ESI-MS (m/z):
Compound 9d was placed during the molecular docking pro-
cedure. Types of interactions of the docked protein with ligand
were analysed after the end of molecular docking. The detailed
structures and calculation results were shown in Supplementary
Table S1.
Chemistry
Solvents were distilled under the positive pressure of dry argon
before use and dried using standard methods. Chemicals were
obtained from local suppliers and were used without further puri-
fication. All reactions were monitored by thin-layer chromatog-
raphy (silica gel 60 F254 glass plates). NMR spectra were recorded
on Bruker 400 MHz instruments, and the chemical shifts were pre-
sented in terms of parts per million with TMS as the internal refer-
ence. Electron-spray ionisation mass spectra in positive mode
(ESI-MS) data were obtained with a Bruker Esquire 3000þ spec-
trometer. Flash column chromatography was performed on silica
gel (200–300 mesh, Adamas, China).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
75
[M þ H]þ ¼ 442.2 (Calcd: 442.21). ESI-HRMS: calcd for C26H28N5O2 (q, J ¼ 7.3 Hz, 2H), 2.25 (s, 3H), 1.09 (t, J ¼ 7.7 Hz, 3H). 13 C NMR
(M þ H)þ 442.2198, found 442.2190. HPLC analysis: MeOH-H2O (80: (100 MHz, DMSO-d6) d: 165.5, 157.6, 142.4, 141.0, 138.9, 132.9,
20), 7.38 min, 96.5% purity.
129.9, 126.5, 126.1, 122.9, 119.5, 116.4, 114.1, 111.2, 107.3, 56.5,
55.9, 51.8, 46.6, 19.0, 16.6. ESI-MS (m/z): [M þ H]þ ¼ 470.3 (Calcd:
N-(6–(3-ethoxyphenyl)-1H-indazol-3-yl)-4–(4-ethylpiperazin-
1-yl)benzamide (9e) White solid: 22.7 mg, yield 39%; Mp 470.26). ESI-HRMS: calcd for C28H33N5O2 (M þ H)þ 470.2556, found
223.6–226.0 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.80 (brs, 1H),
470.2551. HPLC analysis: MeOH-H2O (80: 20), 7.14 min,
10.54 (brs, 1H), 8.00 (d, J ¼ 8.9 Hz, 2H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.66
96.3% purity.
(s, 1H), 7.35–7.43 (m, 2H), 7.29 (d, J ¼ 7.8 Hz, 1H), 7.24 (s, 1H), 7.03
N-(6–(3,5-bis(trifluoromethyl)phenyl)-1H-indazol-3-yl)-4–(4-
(d, J ¼ 8.9 Hz, 2H), 6.96 (dd, J ¼ 8.1, 1.9 Hz, 1H), 4.13 (q, J ¼ 6.9 Hz, ethylpiperazin-1-yl)benzamide (9j) White solid: 33.6 mg, yield
2H), 3.26–3.40 (m, 4H), 2.51–2.62 (m, 4H), 2.34–2.45 (m, 2H), 1.37 44%; Mp 227.5–213.3 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.95
(t, J ¼ 8.0 Hz, 3H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz, (brs, 1H), 10.59 (brs, 1H), 8.42 (s, 2H), 8.13 (s, 1H), 8.00 (d,
DMSO-d6) d: 165.6, 159.5, 153.6, 142.6, 142.2, 141.1, 139.0, 130.5, J ¼ 8.9 Hz, 2H), 7.94 (s, 1H), 7.87 (d, J ¼ 8.6 Hz, 1H), 7.54 (d,
129.8, 123.1, 123.0, 119.9, 117.0, 114.0, 113.9, 113.6, 108.3, 63.6, J ¼ 8.2 Hz, 1H), 7.04 (d, J ¼ 8.0 Hz, 2H), 3.27–3.38 (m, 4H), 2.44–2.52
52.5, 52.1, 47.4, 15.2, 12.3. ESI-MS (m/z): [M þ H]þ ¼ 470.2 (Calcd: (m, 4H), 2.40 (q, J ¼ 7.1 Hz, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). 13 C NMR
470.25). ESI-HRMS: calcd for C28H33N5O2 (M þ H)þ 470.2556, found (100 MHz, DMSO-d6) d: 165.6, 153.6, 143.7, 141.9, 141.3, 135.7,
470.2550. HPLC analysis: MeOH-H2O (80: 20), 7.42 min, 131.5, 131.2, 129.9, 128.4, 125.0, 123.6, 122.9, 122.8, 119.7, 117.6,
96.9% purity.
113.9, 109.7, 52.6, 52.1, 47.4, 12.4. ESI-MS (m/z): [M þ H]þ ¼ 562.2
(Calcd: 562.20). ESI-HRMS: calcd for C28H27F6N5O (M þ H)þ
562.2042, found 562.2049. HPLC analysis: MeOH-H2O (80: 20),
4–(4-ethylpiperazin-1-yl)-N-(6–(3-isopropoxyphenyl)-1H-
indazol-3-yl)benzamide (9f) White solid: 21.3 mg, yield 39%; Mp
227.9–231.7 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.77 (brs, 1H), 7.08 min, 95.9% purity.
10.52 (brs, 1H), 8.00 (d, J ¼ 8.7 Hz, 2H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.65
N-(6–(3,5-dichlorophenyl)-1H-indazol-3-yl)-4–(4-ethylpipera-
(s, 1H), 7.35–7.41 (m, 2H), 7.27 (d, J ¼ 7.7 Hz, 1H), 7.22 (s, 1H), 7.03 zin-1-yl)benzamide (9k) White solid: 28.9 mg, yield 41%; Mp
(d, J ¼ 8.0 Hz, 2H), 6.92 (dd, J ¼ 8.3, 1.9 Hz, 1H), 4.72–4.78 (m, 1H), 226.4–228.7 ꢁC. 1H NMR (400 MHz, DMSO-d6) d 12.90 (brs, 1H),
3.27–3.38 (m, 4H), 2.40–2.58 (m, 4H), 2.34 (q, J ¼ 7.1 Hz, 2H), 1.32 10.58 (brs, 1H), 7.99 (t, J ¼ 9.4 Hz, 2H), 7.81 (dd, J ¼ 10.2, 5.0 Hz,
(s, 3H), 1.31 (s, 3H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz, 3H), 7.67–7.73 (m, 1H), 7.63 (t, J ¼ 1.7 Hz, 1H), 7.16–7.47 (m, 1H),
DMSO-d6) d: 166.6, 158.4, 153.6, 142.7, 142.2, 141.1, 139.0, 130.6, 7.03 (dd, J ¼ 8.8, 4.5 Hz, 2H), 3.34 (s, 4H), 2.46–2.67 (m, 4H), 2.41 (s,
129.8, 123.1, 119.8, 117.0, 115.0, 114.9, 114.0, 69.7, 52.5, 52.0, 47.3, 2H), 1.06 (t, J ¼ 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) d: 165.5,
22.4, 12.3. ESI-MS (m/z): [M þ H]þ ¼ 484.3 (Calcd: 484.27). ESI- 153.6, 144.6, 141.9, 136.0, 135.1, 129.8, 127.3, 126.4, 124.7, 123.5,
HRMS: calcd for C29H35N5O2 (M þ H)þ 484.2713, found 484.2706. 123.1, 120.2, 119.5, 117.5, 113.9, 113.1, 109.1, 52.5, 52.0, 47.3, 12.3.
HPLC analysis: MeOH-H2O (80: 20), 7.22 min, 97.1% purity.
ESI-MS (m/z): [M þ H]þ ¼ 494.2 (Calcd: 494.15). ESI-HRMS: calcd for
C26H27Cl2N5O (M þ H)þ 494.1514, found 494.1508. HPLC analysis:
N-(6–(3-(sec-butoxy)phenyl)-1H-indazol-3-yl)-4–(4-ethylpi-
perazin-1-yl)benzamide (9 g) White solid: 23.9 mg, yield 47%; Mp MeOH-H2O (80: 20), 7.23 min, 96.5% purity.
225.9–290.0 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.79 (brs, 1H),
N-(6–(2,5-dichlorophenyl)-1H-indazol-3-yl)-4–(4-ethylpipera-
10.54 (brs, 1H), 8.00 (d, J ¼ 8.9 Hz, 2H), 7.78 (d, J ¼ 8.5 Hz, 1H), 7.67 zin-1-yl)benzamide (9 l) White solid: 30.3 mg, yield 39%; Mp
(s, 1H), 7.37–7.42 (m, 2H), 7.29 (d, J ¼ 7.9 Hz, 1H), 7.24–7.26 (m, 231.5–234.6 ꢁC. 1H NMR (400 MHz, DMSO-d6) d 12.93 (brs, 1H),
1H), 7.03 (d, J ¼ 9.0 Hz, 2H), 6.96 (dd, J ¼ 8.1, 1.8 Hz, 1H), 3.86 (s, 10.59 (brs, 1H), 8.01 (d, J ¼ 8.8 Hz, 2H), 7.79 (d, J ¼ 8.5 Hz, 1H), 7.64
1H), 3.84 (s, 1H), 3.26–3.38 (m, 4H), 2.51–2.60 (m, 4H), 2.40–2.46 (d, J ¼ 8.6 Hz, 1H), 7.58 (d, J ¼ 2.5 Hz, 1H), 7.52 (dd, J ¼ 8.3, 2.8 Hz,
(m, 2H), 2.03–2.09 (m, 1H), 1.06 (t, J ¼ 7.1 Hz, 3H), 1.03 (s, 3H), 1.01 2H), 7.09–7.15 (m, 1H), 7.05 (d, J ¼ 8.8 Hz, 2H), 3.36–3.40 (m, 4H),
(s, 3H). 13 C NMR (100 MHz, DMSO-d6) d: 166.6, 159.8, 153.6, 142.6, 2.61–2.68 (m, 4H), 2.46–2.50 (q, J ¼ 7.2 Hz, 2H), 1.10 (t, J ¼ 6.8 Hz,
142.2, 141.1, 139.0, 130.5, 129.8, 123.1, 119.9, 117.0, 114.0, 113.6, 3H). 13 C NMR (100 MHz, DMSO-d6) d: 165.7, 153.4, 142.3, 141.3,
108.3, 74.3, 52.5, 52.0, 47.3, 31.2, 28.3, 19.6, 12.3. ESI-MS (m/z): 141.1, 136.0, 132.4, 131.9, 131.6, 130.9, 128.9, 129.5, 123.2, 122.5,
[M þ H]þ ¼ 498.3 (Calcd: 498.29). ESI-HRMS: calcd for C30H37N5O2 121.6, 117.1, 114.1, 111.2, 52.1, 51.9, 46.8, 11.6. ESI-MS (m/z):
(M þ H)þ 498.2869, found 498.2875. HPLC analysis: MeOH-H2O (80: [M þ H]þ
¼
494.2 (Calcd: 494.15). ESI-HRMS: calcd for
20), 7.30 min, 97.0% purity.
C26H27Cl2N5O (M þ H)þ 494.1514, found 494.1510. HPLC analysis:
methyl 3–(3-(4–(4-ethylpiperazin-1-yl)benzamido)-1H-inda-
zol-6-yl)benzoate (9 h) White solid: 27.5 mg, yield 45%; Mp
MeOH-H2O (80: 20), 7.36 min, 96.7% purity.
N-(6–(2,3-dichlorophenyl)-1H-indazol-3-yl)-4–(4-ethylpipera-
228.7–233.4 ꢁC. 1H NMR (400 MHz, DMSO-d6) d 12.91 (brs, 1H), zin-1-yl)benzamide (9 m) White solid: 22.1 mg, yield 36%; Mp
10.61 (brs, 1H), 8.27 (s, 1H), 7.98–8.06 (m, 4H), 7.84 (d, J ¼ 8.5 Hz, 220.1–222.4 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.89 (brs, 1H),
1H), 7.74 (s, 1H), 7.65–7.69 (m, 1H), 7.42 (d, J ¼ 8.6 Hz, 1H), 7.08 (d, 10.58 (brs, 1H), 8.00 (d, J ¼ 8.4 Hz, 2H), 7.78 (d, J ¼ 8.0 Hz, 1H),
J ¼ 8.5 Hz, 2H), 3.91 (s, 3H), 3.42–3.48 (m, 4H), 2.80–2.90 (m, 4H), 7.70–7.72 (m, 1H), 7.46–7.50 (m, 3H), 7.10 (d, J ¼ 7.8 Hz, 1H), 7.04
2.48–2.52 (q, J ¼ 7.6 Hz, 2H), 1.19 (t, J ¼ 7.1 Hz, 3H). 13 C NMR (d, J ¼ 8.4 Hz, 2H), 3.26–3.40 (m, 4H), 2.51–2.55 (m, 4H), 2.42 (q,
(100 MHz, DMSO-d6) d: 166.7, 165.6, 153.0, 142.2, 141.5, 141.1, J ¼ 7.1 Hz, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). 13 C NMR (100 MHz, DMSO-
137.9, 132.5, 130.9, 130.1, 129.9, 128.6, 128.0, 123.4, 119.6, 117.2, d6) d: 165.6, 153.5, 143.1, 141.3, 141.2, 137.1, 132.8, 130.8, 130.4,
114.3, 108.5, 52.8, 51.6, 46.1, 29.5, 10.8. ESI-MS (m/z): [M þ H]þ
¼
130.2, 129.8, 128.8, 123.0, 122.5, 121.6, 117.0, 114.0, 111.0, 52.4,
484.3 (Calcd: 484.24). ESI-HRMS: calcd for C28H31N5O3 (M þ H)þ 52.0, 47.2, 12.2. ESI-MS (m/z): [M þ H]þ ¼ 494.2 (Calcd: 494.15).
484.2349, found 484.2358. HPLC analysis: MeOH-H2O (80: 20), ESI-HRMS: calcd for C26H27Cl2N5O (M þ H)þ 494.1514, found
7.05 min, 96.5% purity.
494.1509. HPLC analysis: MeOH-H2O (80: 20), 6.88 min,
95.5% purity.
4–(4-ethylpiperazin-1-yl)-N-(6–(3-methoxy-4-methylphenyl)-
1H-indazol-3-yl) benzamide (9i) White solid: 27.7 mg, yield 41%;
N-(6–(3,4-dichlorophenyl)-1H-indazol-3-yl)-4–(4-ethylpipera-
1
Mp 227.3–231.2 ꢁC. H NMR (400 MHz, DMSO-d6) d 12.83 (brs, 1H), zin-1-yl)benzamide (9n) White solid: 21.8 mg, yield 38%; Mp
10.54 (brs, 1H), 8.01 (d, J ¼ 8.7 Hz, 2H), 7.75 (d, J ¼ 8.5 Hz, 1H), 7.60 224.9–228.0 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 12.91 (brs, 1H),
(s, 1H), 7.54 (s, 2H), 7.33 (dd, J ¼ 8.6, 1.1 Hz, 1H), 7.05 (d, J ¼ 9.1 Hz, 10.57 (brs, 1H), 7.99–8.03 (m, 3H), 7.73–7.81 (m, 3H), 7.53–7.60 (m,
3H), 3.84 (s, 3H), 3.33–3.38 (m, 4H), 2.59–2.64 (m, 4H), 2.46 1H), 7.40–7.47 (m, 1H), 7.04 (d, J ¼ 8.4 Hz, 2H), 3.26–3.40 (m, 4H),

76
J. LIU ET AL.
2.51–2.55 (m, 4H), 2.41 (q, J ¼ 7.1 Hz, 2H), 1.06 (t, J ¼ 7.2 Hz, 3H). 141.2, 137.1, 133.3, 129.9, 128.5, 123.6, 122.9, 119.6, 117.5, 113.9,
13 C NMR (100 MHz, DMSO-d6) d: 165.5, 153.6, 142.0, 141.7, 141.2, 110.5, 109.1, 52.5, 52.1, 47.4, 12.3. ESI-MS (m/z): [M þ H]þ ¼ 451.2
136.3, 132.2, 131.5, 130.7, 129.8, 129.4, 127.9, 123.5, 122.9, 119.4, (Calcd: 451.22). ESI-HRMS: calcd for C27H28N6O (M þ H)þ 451.2246,
117.3, 113.9, 108.7, 52.6, 52.1, 47.4, 12.4. ESI-MS (m/z): [M þ H]þ
¼
found 451.2238. HPLC analysis: MeOH-H2O (80: 20), 7.40 min,
494.2 (Calcd: 494.15). ESI-HRMS: calcd for C26H27Cl2N5O (M þ H)þ 96.3% purity.
4–(4-ethylpiperazin-1-yl)-N-(6–(1-methyl-1H-pyrazol-3-yl)-
494.1514, found 494.1514. HPLC analysis: MeOH-H2O (80: 20),
7.05 min, 96.5% purity.
1H-indazol-3-yl) benzamide (9t) White solid: 18.7 mg, yield 35%;
1
4–(4-ethylpiperazin-1-yl)-N-(6–(3-fluoro-5-methoxyphenyl)-
Mp 221.6–224.2 ꢁC. H NMR (400 MHz, DMSO-d6) d 12.67 (brs, 1H),
1H-indazol-3-yl)benzamide (9o) White solid: 22.1 mg, yield 34%; 10.46 (brs, 1H), 8.23 (s, 1H), 7.98 (d, J ¼ 8.8 Hz, 2H), 7.94 (s, 1H),
1
Mp 225.5–227.0 ꢁC. H NMR (400 MHz, DMSO-d6) d: 12.86 (brs, 1H), 7.68 (d, J ¼ 8.5 Hz, 1H), 7.57 (s, 1H), 7.30 (d, J ¼ 8.5 Hz, 1H), 7.02 (d,
10.55 (brs, 1H), 8.00 (d, J ¼ 8.9 Hz, 2H), 7.80 (d, J ¼ 8.5 Hz, 1H), 7.72 J ¼ 8.8 Hz, 2H), 3.89 (s, 3H), 3.37–3.45 (m, 4H), 2.52–2.60 (m, 4H),
(s, 1H), 7.40 (dd, J ¼ 8.6, 1.3 Hz, 1H), 7.12–7.21 (m, 2H), 7.03 (d, 2.41 (q, J ¼ 6.7 Hz, 2H), 1.06 (t, J ¼ 7.2 Hz, 3H). 13 C NMR (100 MHz,
J ¼ 9.0 Hz, 2H), 6.85–6.89 (m, 1H), 3.87 (s, 3H), 3.26–3.41 (m, 4H), DMSO-d6) d: 165.6, 153.6, 142.4, 141.1, 136.8, 131.3, 129.8, 128.7,
2.51–2.57 (m, 4H), 2.39 (q, J ¼ 7.1 Hz, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). 123.0, 122.7, 118.6, 116.1, 113.9, 105.5, 52.5, 52.0, 47.3, 19.0, 12.3.
13 C NMR (100 MHz, DMSO-d6) d: 165.0, 162.2, 161.1, 143.5, 141.5, ESI-MS (m/z): [M þ H]þ ¼ 430.2 (Calcd: 430.24). ESI-HRMS: calcd for
140.7, 137.2, 129.3, 122.8, 122.4, 119.2, 116.8, 113.4, 109.1, 108.1, C24H29N7O (M þ H)þ 430.2355, found 430.2357. HPLC analysis:
106.2, 106.0, 55.7, 52.1, 51.6, 46.9, 11.9. HRMS: calcd for MeOH-H2O (80: 20), 7.11 min, 95.5% purity.
C27H30FN5O2 (M þ H)þ 474.2305, found 474.2296. HPLC analysis:
4–(4-(dimethylamino)piperidin-1-yl)-N-(6–(3-fluoro-5-
methoxyphenyl)-1H-indazol-3-yl)benzamide (9 u) White solid:
14.3 mg, yield 46%; Mp 223.8–227.4 ꢁC. 1H NMR (400 MHz, DMSO-
MeOH-H2O (80: 20), 7.24 min, 96.7% purity.
N-(6–(2-chloro-4-(trifluoromethyl)phenyl)-1H-indazol-3-yl)-
4–(4-ethylpiperazin-1-yl)benzamide (9p) White solid: 19.9 mg, d6) d 12.87 (brs, 1H), 10.54 (brs, 1H), 7.99 (d, J ¼ 8.7 Hz, 2H), 7.79
yield 35%; Mp 227.9–232.0 ꢁC. 1H NMR (400 MHz, DMSO-d6) d (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.40 (d, J ¼ 8.8 Hz, 1H), 7.11–7.22
12.92 (brs, 1H), 10.58 (brs, 1H), 7.97–8.04 (m, 3H), 7.80–7.85 (, 2H), (m, 2H), 7.04 (d, J ¼ 8.8 Hz, 2H), 6.87 (d, J ¼ 10.9 Hz, 1H), 3.98
7.74 (d, J ¼ 8.0 Hz, 1H), 7.55 (s, 1H), 7.14 (dd, J ¼ 8.5, 1.2 Hz, 1H), (d, J ¼ 12.2 Hz, 2H), 3.87 (s, 3H), 2.82–2.90 (m, 2H), 2.59 (s, 1H),
7.03 (d, J ¼ 8.9 Hz, 2H), 3.33–3.40 (m, 4H), 2.48–2.52 (m, 4H), 2.45 2.35 (s, 6H), 1.92 (s, 2H), 1.51 (d, J ¼ 10.8 Hz, 2H). 13 C NMR
(s, 2H), 1.06 (t, J ¼ 7.1 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) d: (100 MHz, DMSO-d6) d: 165.5, 164.8, 162.9, 161.6, 153.2, 143.9,
165.7, 153.5, 144.8, 141.3, 136.0, 133.2, 133.0, 129.9, 127.2, 124.8, 142.0, 141.2, 137.7, 129.9, 123.3, 122.5, 119.6, 117.3, 114.1, 109.6,
123.0, 122.7, 121.4, 117.2, 114.0, 111.2, 52.4, 52.0, 47.1, 12.1. ESI- 108.6, 106.7, 100.9, 62.1, 56.2, 46.9, 41.3, 27.4, 21.6. ESI-MS (m/z):
MS (m/z): [M þ H]þ ¼ 528.1 (Calcd: 528.17). ESI-HRMS: calcd for [M þ H]þ ¼ 488.3 (Calcd: 488.26). HRMS: calcd for C27H30FN5O2
C27H27ClF3N5O (M þ H)þ 528.1778, found 528.1777. HPLC analysis: (M þ H)þ 488.2462, found 488.2459. HPLC analysis: MeOH-H2O (80:
MeOH-H2O (80: 20), 7.18 min, 96.9% purity.
20), 7.22 min, 97.5% purity.
N-(6–(2,6-dimethylphenyl)-1H-indazol-3-yl)-4–(4-ethylpipera-
4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(6–(3-fluoro-5-
zin-1-yl)benzamide (9q) White solid: 30.9 mg, yield 40%; Mp methoxyphenyl)-1H-indazol-3-yl)benzamide (9v) White solid:
235.7–239.2 ꢁC. 1H NMR (400 MHz, DMSO-d6) d 12.75 (brs, 1H), 26.5 mg, yield 38%; Mp 229.6–233.4 ꢁC. 1H NMR (400 MHz, DMSO-
10.56 (brs, 1H), 8.02 (d, J ¼ 8.7 Hz, 2H), 7.77 (d, J ¼ 8.3 Hz, 1H), 7.18 d6) d 12.87 (brs, 1H), 10.56 (brs, 1H), 7.99 (d, J ¼ 8.8 Hz, 2H), 7.79
(d, J ¼ 8.4 Hz, 2H), 7.14 (d, J ¼ 8.2 Hz, 2H), 7.07 (d, J ¼ 8.6 Hz, 2H), (d, J ¼ 8.6 Hz, 1H), 7.72 (s, 1H), 7.40 (d, J ¼ 8.5 Hz, 1H), 7.14–7.18
6.82 (d, J ¼ 8.3 Hz, 1H), 3.37–3.43 (m, 4H), 2.54–2.68 (m, 4H), 2.35 (m, 2H), 7.04 (d, J ¼ 9.0 Hz, 2H), 6.87 (d, J ¼ 10.9 Hz, 1H), 4.47 (brs,
(q, J ¼ 7.2 Hz, 2H), 2.01 (s, 6H), 1.14 (t, J ¼ 7.0 Hz, 3H). 13 C NMR 1H), 3.87 (s, 3H), 3.82 (s, 2H), 2.96 (s, 2H), 2.36 (d, J ¼ 10.7 Hz, 2H),
(100 MHz, DMSO-d6) d: 165.6, 153.0, 142.0, 141.1, 139.1, 135.8, 1.11 (d, J ¼ 6.1 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) d: 165.5,
129.9, 127.8, 127.5, 122.8, 121.5, 116.5, 114.3, 110.2, 51.7, 51.6, 164.8, 162.9, 161.6, 153.2, 143.9, 142.0, 141.1, 137.7, 129.9, 126.7,
46.4, 21.0, 12.2. ESI-MS (m/z): [M þ H]þ ¼ 454.3 (Calcd: 454.26). 123.3, 119.6, 117.3, 113.9, 109.6, 108.6, 106.7, 100.9, 63.3, 56.2,
ESI-HRMS: calcd for C26H28ClN5O (M þ H)þ 454.2607, found 53.3, 50.6, 19.0. ESI-MS (m/z): [M þ H]þ ¼ 474.2 (Calcd: 474.23).
454.2606. HPLC analysis: MeOH-H2O (80: 20), 7.30 min, ESI-HRMS: calcd for C27H30FN5O2 (M þ H)þ 474.2305, found
95.7% purity.
474.2290. HPLC analysis: MeOH-H2O (80: 20), 7.11 min,
95.5% purity.
4–(4-ethylpiperazin-1-yl)-N-(6–(2-(trifluoromethoxy)phenyl)-
1H-indazol-3-yl)benzamide (9r) White solid: 28.2 mg, yield 38%;
N-(6–(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4–(4-
1
Mp 230.9–233.5 ꢁC. H NMR (400 MHz, DMSO-d6) d: 12.87 (brs, 1H), methyl-1,4-diazepan-1-yl)benzamide (9w) White solid: 30.1 mg,
10.57 (brs, 1H), 8.00 (d, J ¼ 8.4 Hz, 2H), 7.79 (d, J ¼ 7.8 Hz, 1H), yield 33%; Mp 230.4–234.6 ꢁC. 1H NMR (400 MHz, DMSO-d6) d
7.62–7.64 (m, 1H), 7.53–7.57 (m, 4H), 7.16 (d, J ¼ 8.0 Hz, 1H), 7.04 12.83 (brs, 1H), 10.44 (brs, 1H), 7.97 (d, J ¼ 8.4 Hz, 2H), 7.79
(d, J ¼ 8.8 Hz, 2H), 3.28–3.43 (m, 4H), 2.47–2.53 (m, 4H), 2.41 (q, (d, J ¼ 8.0 Hz, 1H), 7.71 (s, 1H), 7.39 (d, J ¼ 8.1 Hz, 1H), 7.14–7.18
J ¼ 7.1 Hz, 2H), 1.06 (t, J ¼ 7.2 Hz, 3H). 13 C NMR (100 MHz, DMSO- (m, 2H), 6.85–6.89 (m, 1H), 6.80 (d, J ¼ 7.9 Hz, 2H), 3.87 (s, 7H),
d6) d: 165.6, 153.6, 145.9, 141.6, 141.2, 135.5, 134.7, 132.4, 129.9, 3.63–3.65 (m, 2H), 3.53 (t, J ¼ 6.8 Hz, 2H), 2.70–2.78 (m, 2H),
128.5, 123.0, 122.6, 121.5, 117.0, 113.9, 110.8, 52.5, 52.0, 47.4, 12.3. 2.55–2.62 (m, 2H), 2.48–2.51 (m, 4H), 2.36 (s, 3H). 13 C NMR
ESI-MS (m/z): [M þ H]þ ¼ 510.2 (Calcd: 510.21). ESI-HRMS: calcd for (100 MHz, DMSO-d6) d: 165.6, 164.8, 162.9, 161.6, 151.7, 144.0,
C27H28F3N5O2 (M þ H)þ 510.2117, found 510.2119. HPLC analysis: 142.0, 137.6, 130.16, 123.3, 120.2, 119.6, 117.3, 110.8, 109.6, 108.6,
MeOH-H2O (80: 20), 7.18 min, 96.9% purity.
106.7, 101.1, 57.2, 56.6, 56.2, 48.1, 46.1, 26.8. ESI-MS (m/z):
[M þ H]þ ¼ 474.0(Calcd: 473.22). ESI-HRMS: calcd for C27H30FN5O2
N-(6–(4-cyanophenyl)-1H-indazol-3-yl)-4–(4-ethylpiperazin-
1-yl)benzamide (9 s) White solid: 15.8 mg, yield 38%; Mp (M þ H)þ 474.2305, found 474.2297. HPLC analysis: MeOH-H2O (80:
226.4–230.0 ꢁC. 1H NMR (400 MHz, DMSO-d6) d 12.94 (brs, 1H),
20), 7.15 min, 96.2% purity.
10.52 (brs, 1H), 7.93–8.08 (m, 6H), 7.85 (d, J ¼ 8.5 Hz, 1H), 7.78 (s,
N-(6–(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4-((4-
1H), 7.44 (d, J ¼ 8.6 Hz, 1H), 7.03 (d, J ¼ 8.6 Hz, 2H), 3.28–3.39 (m, methylpiperazin-1-yl)methyl)benzamide (9x) White solid:
4H), 2.51–2.59 (m, 4H), 2.40 (q, J ¼ 6.7 Hz, 2H), 1.06 (t, J ¼ 7.1 Hz, 20.1 mg, yield 44%; Mp 240.3–242.5 ꢁC. 1H NMR (400 MHz, DMSO-
3H). 13 C NMR (100 MHz, DMSO-d6) d: 165.6, 153.7, 145.6, 142.1, d6) d: 13.01 (brs, 1H), 10.84 (brs, 1H), 8.06 (d, J ¼ 8.2 Hz, 2H), 7.80

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
77
(d, J ¼ 8.2 Hz, 1H), 7.74 (s, 1H), 7.46 (d, J ¼ 8.0 Hz, 2H), 7.41 (dd,
J ¼ 8.0, 1.4 Hz, 1H), 7.14–7.18 (m, 2H), 6.85–6.89 (m, 1H), 3.87 (s,
1H), 3.55 (s, 2H), 2.33–2.46 (m, 8H), 2.16 (s, 3H). 13 C NMR
(100 MHz, DMSO-d6) d: 165.9, 164.8, 162.9, 161.6, 144.0, 143.0,
142.0, 140.6, 137.7, 132.9, 130.1, 129.1, 128.4, 123.0, 119.8, 117.1,
109.6, 108.7, 106.7, 100.9, 62.1, 56.2, 55.2, 53.1, 46.2. ESI-MS (m/z):
[M þ H]þ ¼ 474.2 (Calcd: 474.23). ESI-HRMS: calcd for C27H30FN5O2
(M þ H)þ 474.2305, found 474.2298. HPLC analysis: MeOH-H2O (80:
20), 7.11 min, 95.5% purity.
N-(6–(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-4–(4-
methylpiperazin-1-yl)benzamide (9 y) White solid: 17.1 mg, yield
30%; Mp 237.4–241.6 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 13.01
(brs, 1H), 10.84 (brs, 1H), 8.06 (d, J ¼ 8.2 Hz, 2H), 7.80 (d, J ¼ 8.2 Hz,
1H), 7.74 (s, 1H), 7.46 (d, J ¼ 8.0 Hz, 2H), 7.41 (dd, J ¼ 8.0, 1.4 Hz,
1H), 7.14–7.18 (m, 2H), 6.85–6.89 (m, 1H), 3.87 (s, 1H), 3.24–3.39 (s,
4H), 2.44–2.52 (s, 4H), 2.16 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) d:
165.9, 164.7, 163.1, 161.6, 144.0, 143.0, 142.0, 140.7, 137.7, 132.9,
129.1, 128.4, 123.0, 119.8, 117.2, 109.6, 108.7, 106.7, 101.1, 62.1,
56.2, 55.2, 53.1, 46.2. ESI-MS (m/z): [M þ H]þ ¼ 460.2 (Calcd:
460.21). ESI-HRMS: calcd for C27H30FN5O2 (M þ H)þ 460.2149,
found 460.2155. HPLC analysis: MeOH-H2O (80: 20), 7.42 min,
96.4% purity.
N-(6–(3-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-5–(4-
methylpiperazin-1-yl)pyrazine-2-carboxamide (9z) White solid:
19.8 mg, yield 35%; Mp 237.7–240.3 ꢁC. 1H NMR (400 MHz, DMSO-
d6) d: 12.95 (brs, 1H), 10.42 (brs, 1H), 8.80 (s, 1H), 8.44 (s, 1H), 7.94
(d, J ¼ 8.4 Hz, 1H), 7.74 (s, 1H), 7.42 (d, J ¼ 8.3 Hz, 1H), 7.05–7.25
(m, 2H), 6.87 (d, J ¼ 10.9 Hz, 1H), 3.87 (s, 7H), 2.70 (s, 4H), 2.42 (s,
3H). ESI-MS (m/z): [M þ H]þ¼462.2 (Calcd: 462.21). 13 C NMR
(100 MHz, DMSO-d6) d: 164.8, 162.8, 161.6, 155.6, 142.8 142.1,
140.1, 137.8, 132.4, 130.1, 129.5, 123.2, 119.8, 116.7 109.6, 108.6,
106.7, 101.0, 56.2, 54.5, 46.0, 44.2. ESI-MS (m/z): [M þ H]þ ¼ 462.2
(Calcd: 462.20). HRMS: calcd for C24H25FN7O2 (M þ H)þ 462.2054,
found 462.2038. HPLC analysis: MeOH-H2O (80: 20), 7.11 min,
95.5% purity.
4–(4-methylpiperazin-1-yl)-N-(6–(3,4,5-trimethoxyphenyl)-
benzo[d]thiazol-2-yl)benzamide (12c) White solid 50.5 mg, yield
1
47%. Mp 236.4–238.3 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.59 (brs,
1H), 8.17 (s, 1H), 8.07 (d, J ¼ 8.8 Hz, 2H), 7.83 (d, J ¼ 8.4 Hz, 1H),
7.60 (d, J ¼ 8.3 Hz, 1H), 7.24 (t, J ¼ 7.9 Hz, 1H), 7.19 (t, J ¼ 7.5 Hz,
1H), 7.12 (t, J ¼ 6.6 Hz, 1H), 7.04 (d, J ¼ 8.9 Hz, 2H), 3.90 (s, 3H),
2.45 (s, 4H), 2.23 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) d 165.57,
159.85, 154.19, 153.68, 148.40, 137.43, 136.32, 136.26, 132.86,
130.43, 125.67, 120.63, 120.30, 120.08, 113.71, 104.76, 60.54, 56.46,
54.77, 46.94, 46.18. HRMS: calcd for C28H31N4O4S (M þ H)þ
519.1988, found 519.2007. HPLC analysis: MeOH-H2O (85: 15),
7.35 min, 96.8% purity.
N-(6–(2-fluoro-3-methoxyphenyl)benzo[d]thiazol-2-yl)-4–(4-
methylpiperazin-1-yl)benzamide (12d) White solid 64.8 mg, yield
1
52%. Mp 232.5–235.2 ꢁC. H NMR (500MHz, DMSO-d6) d 12.59 (brs,
1H), 8.17 (s, 1H), 8.07 (d, J ¼ 8.8 Hz, 2H), 7.83 (d, J ¼ 8.4Hz, 1H), 7.60
(d, J ¼ 8.3 Hz, 1H), 7.24 (t, J ¼ 7.9 Hz, 1H), 7.19 (t, J ¼ 7.5 Hz, 1H), 7.12
(t, J ¼ 6.6Hz, 1H), 7.04 (d, J ¼ 8.9 Hz, 2H), 3.90 (s, 3H), 2.45 (s, 4H),
2.23 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) d 165.59, 160.39, 154.19,
150.12, 148.69, 148.32, 148.23, 148.17, 132.55, 130.65, 130.44,
129.38, 129.29, 127.56, 127.54, 125.01, 124.97, 122.36, 122.28,
120.49, 120.24, 113.70, 113.24, 56.58, 54.75, 46.92, 46.15. HRMS:
calcd for C26H26FN4O2S (M þ H)þ 477.1682, found 477.1653. HPLC
analysis: MeOH-H2O (85: 15), 7.58 min, 95.5% purity.
General method for preparation of compounds 18a-18c
(exemplified by 18a)
4–(4-methylpiperazin-1-yl)-N-(5-phenethyl-1H-1,2,4-triazol-3-
yl)benzamide (18a) white solid 145.4 mg, yield 32%. Mp
1
218.1–222.5ꢁC. H NMR (500MHz, DMSO-d6) d 13.14 (brs, 1H), 11.59
(brs, 1H), 7.96 (d, J ¼ 7.7 Hz, 2H), 7.24–7.28 (m, 4H), 7.18 (t,
J ¼ 7.1 Hz, 1H), 7.00 (d, J ¼ 8.9 Hz, 2H), 3.31 (s, 4H), 3.00 (t, J ¼ 7.7 Hz,
2H), 2.88 (s, 2H), 2.37–2.48 (m, 4H), 2.22 (s, 3H). 13 C NMR (125MHz,
DMSO-d6) d 165.27, 160.15, 153.93, 130.06, 128.75, 128.71, 126.38,
121.02, 113.77, 111.75, 54.80, 47.09, 46.19, 34.09, 30.25. HRMS: calcd
for C24H31N6O3 (M þ H)þ 391.2168, found 391.2083. HPLC analysis:
MeOH-H2O (85: 15), 9.05min, 97.5% purity.
General method for preparation of compounds 12a-12d
N-(5–(3-methoxyphenethyl)-1H-1,2,4-triazol-3-yl)-4–(4-meth-
ylpiperazin-1-yl) benzamide (18 b) White solid 82.1 mg, yield
(exemplified by 12a)
N-(6–(3-methoxyphenyl)benzo[d]thiazol-2-yl)-4–(4-methylpiper-
azin-1-yl) benzamide (12a) white solid: 73.7 mg, yield 53%. Mp
230.5–233.2 ꢁC. 1H NMR (500 MHz, DMSO-d6) d: 12.57 (brs, 1H),
8.34 (s, 1H), 8.07 (d, J ¼ 8.8 Hz, 2H), 7.81 (d, J ¼ 8.4 Hz, 1H), 7.76 (d,
J ¼ 8.3 Hz, 1H), 7.40 (t, J ¼ 7.9 Hz, 1H), 7.31 (d, J ¼ 7.7 Hz, 1H), 7.28
(s, 1H), 7.03 (d, J ¼ 8.9 Hz, 2H), 6.95 (d, J ¼ 8.0 Hz, 1H), 3.85 (s, 3H),
2.44 (s, 4H), 2.23 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) d 165.57,
160.24, 160.06, 154.19, 148.63, 141.98, 135.97, 132.96, 130.46,
130.43, 125.64, 120.78, 120.29, 120.19, 119.59, 113.71, 113.34,
112.70, 55.62, 54.77, 46.94, 46.18. HRMS: calcd for C26H27N4O2S
(M þ H)þ 459.1776, found 459.1708. HPLC analysis: MeOH-H2O (85:
15), 7.50 min, 97.1% purity.
1
31%. Mp 216.4–219.5 ꢁC. H NMR (500 MHz, DMSO-d6) d 13.09 (brs,
1H), 11.55 (brs, 1H), 7.96 (d, J ¼ 5.2 Hz, 2H), 7.19 (t, J ¼ 7.8 Hz, 1H),
7.00 (d, J ¼ 8.9 Hz, 2H), 6.81 (d, J ¼ 7.6 Hz, 2H), 6.75 (d, J ¼ 8.4 Hz,
1H), 3.72 (s, 3H), 3.31 (s, 4H), 2.97 (t, J ¼ 7.5 Hz, 2H), 2.88 (d,
J ¼ 8.6 Hz, 2H), 2.37–2.47 (m, 4H), 2.23 (s, 3H). 13 C NMR (125 MHz,
DMSO-d6) d 165.29, 159.70, 153.93, 130.05, 129.74, 120.96, 114.42,
113.77, 111.80, 55.33, 54.80, 47.11, 46.21, 34.05. HRMS: calcd for
C23H29N6O2 (M þ H)þ 421.2274, found 421.2215. HPLC analysis:
MeOH-H2O (85: 15), 8.80 min, 97.2% purity.
N-(5–(3,5-dimethoxyphenethyl)-1H-1,2,4-triazol-3-yl)-4–(4-
methylpiperazin-1-yl)benzamide (18c). White solid 77.8 mg, yield
1
31%. Mp 220.9–223.3 ꢁC. H NMR (500 MHz, DMSO-d6) d 13.11 (brs,
N-(6–(3,5-dimethoxyphenyl)benzo[d]thiazol-2-yl)-4–(4-meth-
ylpiperazin-1-yl) benzamide (12 b) White solid 56.7 mg, yield
1H), 11.60 (brs, 1H), 7.96 (s, 2H), 6.94–7.05 (m, 2H), 6.38–6.41 (m,
2H), 6.31 (s, 1H), 3.71 (s, 6H), 3.31 (s, 4H), 2.79–2.97 (m, 4H),
2.40–2.47 (m, 4H), 2.22 (s, 3H). HRMS: calcd for C24H31N6O3
(M þ H)þ 451.2379, found 451.2351. HPLC analysis: MeOH-H2O (85:
15), 8.72 min, 96.9% purity.
1
49%. Mp 234.6–238.2 ꢁC. H NMR (500 MHz, DMSO-d6) d 12.58 (brs,
1H), 8.34 (s, 1H), 8.06 (d, J ¼ 8.3 Hz, 2H), 7.73–7.84 (m, 2H), 7.03 (d,
J ¼ 8.3 Hz, 2H), 6.88 (s, 2H), 6.51 (s, 1H), 3.84 (s, 6H), 2.44 (s, 4H),
2.23 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) d 165.57, 161.33,
160.09, 154.19, 148.72, 142.62, 136.02, 132.89, 130.43, 125.67,
120.69, 120.29, 120.25, 113.70, 105.39, 99.68, 55.77, 54.77, 46.94,
46.17. HRMS: calcd for C27H29N4O3S (M þ H)þ 489.1882, found
489.1852. HPLC analysis: MeOH-H2O (85: 15), 7.70 min,
96.4% purity.
Biological evaluation
Kinase profiling
The kinase profiling of 9 u was conducted using Elisa kinase assay
and the Eurofins Kinase Profiler Selectivity Testing Service.

78
J. LIU ET AL.
Elisa kinase assay. The effects of the indicated compounds on
Accordingly, the inhibitors are commonly involving the follow-
the activities of receptor tyrosine kinases were determined using ing pharmacophore features. (i) The core structure of most inhibi-
enzyme-linked immunosorbent assays (ELISAs) with purified
recombinant proteins. Briefly, 20 lg/mL poly (Glu,Tyr)4:1 (Sigma, St
Louis, MO, USA) was pre-coated in 96-well plates as a substrate. A
50 lL aliquot of 10 lmol/L ATP solution diluted in kinase reaction
buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl2, 0.5 mmol/L
MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L DTT) was added to each
well; 1 lL of different compounds with indicated concentration
diluted in 1% DMSO (v/v) (Sigma, St Louis, MO, USA) were then
added to each reaction well. DMSO (1%, v/v) was used as the
negative control. The kinase reaction was initiated by the addition
of purified tyrosine kinase proteins (FGFR1, FGFR2, FGFR3, FGFR4,
ALK, Bcr-Abl, EPH-A2, Flt-1, RET, c-Src, IGF1R, c-Met, EGFR, ErbB2)
diluted in 49 lL of kinase reaction buffer solution. After incubation
for 60 min at 37 ꢁC, the plate was washed three times with phos-
phate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS).
Anti-phosphotyrosine (PY99) antibody (100 lL; 1:500, diluted in
5 mg/mL BSA T-PBS) was then added. After a 30 min incubation at
37 ꢁC, the plate was washed three times, and 100 lL horseradish
peroxidase-conjugated goat anti-mouse IgG (1:2000, diluted in
5 mg/mL BSA T-PBS) was added. The plate was then incubated at
37 ꢁC for 30 min and washed 3 times. A 100 lL aliquot of a solu-
tion containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in
0.1 mol/l citrate buffer (pH 5.5) was added. The reaction was termi-
nated by the addition of 50 lL of 2 mol/l H2SO4 as the colour
changed, and the plate was analysed using a multi-well spectro-
photometer (SpectraMAX 190, Molecular Devices, Sunnyvale, CA,
USA) at 490 nm. The inhibition rate (%) was calculated using the
following equation: [1-(A490/A490 control)] ꢂ 100%. The IC50 val-
ues were calculated from the inhibition curves in two separate
experiments.
tors consists of a flat hetero aromatic ring system that contains at
least one H-bond acceptor and H-bond donor, then it can form
hydrogen bonds with the key amino acid residues: Glu562 and
Ala564. (ii) Terminal hydrophobic head often interacts with the
hydrophobic region I, which is a phenyl ring with different extra
hydrophobic substitutions. (iii) Another hydrophobic scaffold dir-
ectly linked to the flat hetero aromatic ring system which occupies
the hydrophobic region II. Herein, we initially pay attention to
explore the novel hetero aromatic ring, which was the core struc-
ture of FGFR1 inhibitor, formed the key H-bond interaction with
hinge region, and connected the hydrophobic region I and II.
Furthermore, we superimposed the two FGFR1 crystal struc-
tures of 4V05 and 3TTO, which were complexed with AZD454725
and NVP-BGJ 39817, respectively. The results revealed that
AZD4547 and NVP-BGJ 398 were similar in their binding mode
with the target were similarly, when there was a significant spatial
distance constraint between the pharmacophore features. The
spatial distance of the hydrophobic head and the heterocycle ring
is between 5.9 Å and 6.5 Å (Figure 2(a) and Figure S2). Therefore,
the FGFR1 inhibitors might contain the corresponding pharmaco-
phore features, and these pharmacophore features were also con-
sisted to a space distance constraint.
The fragment library was derived from kinase hinge region
directed library, which contained 11809 scaffolds. Subsequently,
with the built pharmacophore model and molecular docking,
seven hit fragments were obtained through serval round filtration
(Supplementary Table S1 and Figure S4). One of them was inda-
zole scaffold, which was proved to be an efficiency FGFR1 inhibi-
tor scaffold22, which indicated the accuracy of virtual screening
protocol. Then, in view of the difficulties in synthesis, only 1H-
1,2,4-triazole, benzothiazole and indazole scaffold were selected
for further modification (Figure 2(b)). The novel 1H-1,2,4-triazole
and benzothiazole scaffolds were selected for the study of FGFR1
inhibitors research. Furthermore, we also continued to optimise
the original indazole derivatives, improve the physicochemical
property and enrich the structure-activity relationship of indazole
derivatives. Finally, we synthesised several indazole derivatives
through introduced halogen substituents at various positions. As a
result, with the fragment-based virtual screening strategy, novel
derivatives bearing 1H-1,2,4-triazole, benzothiazole and indazole
scaffold were designed, synthesised and evaluated.
Cell proliferation assay. Human gastric cancer cell line SNU-16
was purchased from American Type Culture Collection (ATCC,
Manssas, VA, USA). All the cell lines were routinely maintained in
complete medium according to the suppliers’ recommendations.
Cells were seeded in 96-well tissue culture plates. On the day
when seeding, the cells were exposed to various concentrations
of compounds and further cultured for 72 h at 37 ꢁC. Finally, cell
proliferation was determined using Cell Counting Kit (CCK-8) or
the thiazolyl blue tetrazolium bromide (MTT, from Sigma-Aldrich,
St. Louis, MO, USA) assay. The IC50 values were calculated by con-
centration–response curve fitting using a SoftMax pro-based four-
parameter method.
Chemistry
The preparation of target compounds 9a-t, 12a-d and 18a-c was
described in Schemes 1–3. Compounds 9a-t were synthesised
from starting material 4-fluorobenzoate (1) and 4-bromo-2-fluoro-
benzonitrile (2) through seven steps. The nucleophilic substitution
reactions of 4-fluorobenzoate with various piperazines afforded
the methyl ester 2 intermediate, which then could hydrolysis to
carboxylic acid 3. Subsequently, the carboxylic acid 3 was con-
densed with HATU to provide the activated ester reagent 4.
Meanwhile, the intermediate 5 could be condensed by 4-bromo-
2-fluorobenzonitrile 1 with hydrazine. Then nitrogen atom at the
1-position of 1H-indazol-3-amine scaffold 5 was protected by BOC
group. The key intermediate 8 was obtained by condensation of 4
and 7 in the presence of NaH. Treatment of the intermediate 8
with various substituted-phenylboronic acid under Suzuki coupling
condition26 of Pd(dppf)Cl2 and Cs2CO3 in refluxing dioxane/water
gave target compounds. According to the similar synthetic route,
Results and discussion
Rational drug design
Most selective FGFR1 inhibitors are ATP-competitive inhibitors that
bind to the ATP-binding pocket of FGFR. The ATP binding pocket
of the FGFR1 consists of five regions3,23,24: (i) the adenine region,
(ii) hydrophobic region I, (iii) hydrophobic region II, (iv) the
nucleotide domain, (v) and the phosphate region. The adenine
region is considered as the major binding site in which hetero-
cycle scaffolds are anchored through several H-bonds with a hinge
region of the kinase (Figure 1, red colour). Meanwhile, the hydro-
phobic region I (Figure 1, blue colour) and hydrophobic region II
(Figure 1, pink colour) are the other two important sites, which
plays key factor in binding small molecules by interacting with
lipophilic moieties hydrophobically, and forms van der Waals inter-
actions and H-bonds.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
79
Scheme 1. Synthesis of indazole derivatives. Reagents and conditions: (a) K2CO3, DMSO, 120 ꢁC, 85–90%; (b) NaOH, MeOH, H2O, 80 ꢁC, 93–96%; (c) HATU, K2CO3, DMF,
rt, 62–70%; (d) hydrazine hydrate, n-butanol, 120 ꢁC, 86%; (e) Boc2O, DMAP, THF, 89%; (f) NaH, THF, 60 ꢁC, 52–61%; (g) TFA, CH2Cl2, 0 ꢁC, 70%; (h) R-B(OH)2, Cs2CO3,
Pd(dppf)Cl2, dioxane, 120 ꢁC, 30–48%.
Scheme 2. Synthesis of benzothiazole derivatives. Reagents and conditions: (a) 4, NaH, THF, 60 ꢁC, 51–62%; (b) R-B(OH)2, Cs2CO3, Pd(dppf)Cl2, dioxane,
120 ꢁC, 47–53%.
Scheme 3. Synthesis of 1H-1,2,4-triazole derivatives. Reagents and conditions: (a) propanedioic acid, pyridine, piperidine, 105ꢁC, 77–82%; (b) Pd/C, H2, EtOH, 88–93%;
(c) SO2Cl2, reflux; (d) amino guanidine hydrochloride, 140 ꢁC, 49–53%; (f) 4, NaH, THF, 60 ꢁC, 54–62%; (g) TFA, CH2Cl2, 0 ꢁC, 70%.
compounds 12a-d were synthesised from the starting material 6- Biological evaluation
bromobenzo[d]thiazol-2-amine (10) through two steps.
Biological evaluation for various scaffold derivatives 9a-d, 12a-d,
18a-c
Initially, we designed and synthesised the indazole (9a-d), benzo-
hyde (13) was condensed with propanedioic acid in the presence
The synthesis of the other 1H-1,2,4-triazole derivatives was
shown in Scheme 3. The starting material substituted-benzalde-
of pyridine and piperidine at 105 ꢁC via Aldol condensation, which
the intermediate 14 was obtained. After hydrogenation of 14
released the corresponding aliphatic acid 15, which was con-
nected with the amino guanidine hydrochloride giving triazole
scaffold 16 in 49.1% yield. Then nitrogen atom at the 1-position
of the 1H-1,2,4-triazole heterocycle 16 was protected by BOC
group. The target compounds 18a-c were prepared in similar way
by following condensation and Suzuki-coupling procedures.
thiazole (12a-d) and 1H-1,2,4-triazole (18a-c) derivatives, with the
goal of analysing the effects of different scaffolds on biological
activity (Table 1). All these compounds were evaluated against
FGFR1 enzyme and SNU-16 cell line. The results indicated that
when the heterocycle was substituted by a 1H-1,2,4-triazole scaf-
fold, it would result in almost no enzyme inhibitory activities and
cell anti-proliferative activities. Furthermore, the corresponding
benzothiazole derivatives 12a-d were also revealed poor inhibitory

80
J. LIU ET AL.
Table 1. Structures and activities of various derivatives 9a-d, 12a-d, and 18a-c.
Hydrophobic
group I
FGFR1 kinase SNU-16 cell
No.
heterocycle
R1
IC50(nM)a
IC50(nM) b
30.6
1195.7
9a
9b
9c
20.7
28.2
1045.5
842.8
15.0
785.8
9d
>1000
>1000
>1000
>1000
NAc
NA
NA
NA
12a
12b
12c
12d
>1000
>1000
NA
NA
18a
18b
18c
>1000
0.7
NA
6.1
AZD4547
aThe IC50 values are shown as the average values from two separate experiments.
bSNU-16 is FGFR2-amplified cell type.
c“NA” means IC50 > 5 lM.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
81
Table 2. Structures and activities of indazole derivatives 9e–t.
activity against FGFR1 enzyme (IC50 >1000 nM), thus led to slight
reduction cellular anti-proliferative activities.
Fortunately, compound 9a with an indazole scaffold exhibited
good FGFR1 enzyme inhibitory activity (IC50 ¼ 30.6 nM) and mod-
est cellular inhibition (IC50 ¼ 1195.7 nM). While the 3,5-dimethoxy
phenyl substituent was replaced with the 3-methoxy phenyl
group, the enzyme inhibition of 9 b was relatively lower than 9a.
However it was more potent in cellular assay (9a vs 9 b). Besides,
co-crystal structure analysis revealed that the R1 substituent is at
the solvent region of the target protein, which might be helpful
to improve membrane permeability and enhance cellular poten-
cies. Then the other two derivatives 9c and 9d with N-methyl
piperazine and N-ethyl piperazine substituent at the R1 position,
respectively, were synthesised and evaluated. As a result, the 9d
demonstrated the best enzyme inhibitory (IC50 ¼ 15.0 nM) and
cellular anti-proliferative activities (IC50 ¼ 785.8 nM). The above
results demonstrated that the heterocycle could significantly affect
the enzyme and cell growth inhibitory activity.
FGFR1 kinase
IC50(nM)a
SNU-16 cell
No.
R
IC50(nM)b
13.2
9.8
1790.3
9e
9f
1502.3
NAc
>1000
9g
77.7
>1000
>1000
67.2
757.3
NA
9h
9i
The structure-activity relationship of indazole derivatives 9e-z
The hydrophobic group I located in the hydrophobic cavity of the
active site and formed strong hydrophobic interaction with the
target. To increase the enzyme inhibitory activity, we initially opti-
mised the hydrophobic group I (Table 2). It was indicated that
3-methoxy phenyl (9d, IC50 ¼ 15.0 nM) was substituted by the
3-ethoxy phenyl (9e, IC50 ¼ 13.2 nM), 3-isopropoxy phenyl (9f,
IC50 ¼ 9.8 nM) caused increase of activity. On the contrary, the
compound 9 h with 3-s-butoxy phenyl exhibited low enzymatic
inhibitory activity (IC50 > 1000 nM). Meanwhile, the compound
with 3-COOMe-phenyl (9 h, IC50 ¼ 77.7 nM), 3,5-dichloro-phenyl
(9k, IC50 ¼ 67.2 nM), 2,5-dichloro-phenyl (9 l, IC50 ¼ 50.6 nM), 2,3-
dichloro-phenyl (9 m, IC50 ¼ 44.8 nM), 2,6-methyl-phenyl (9q,
IC50 ¼ 41.7 nM), shown moderate FGFR1 inhibitory activity. On the
other hand, compounds containing a substituent either the elec-
tron-withdrawing or electron-donating group at para-position,
were significant decreasing their activities (9d vs 9i; 9k, 9 l and
9 m vs 9n; 9 m vs 9p). Furthermore, the additional halogen atoms
on phenyl might be helpful to improve physicochemical proper-
ties and enhance the cell anti-proliferative activities. Thus, the
compound 9o (IC50 ¼ 5.5 nM) containing a fluorine atom at the
para-position of phenyl showed better inhibitory activity than
their counterparts lack of these atoms (9d, IC50 ¼ 15.0 nM).
NA
9j
1200.7
780.2
1180.2
NA
9k
9l
50.6
44.8
9m
9n
>1000
9o
9p
9q
9r
9s
9t
5.5
572.2
NA
The active compounds 9e, 9f, 9 h, 9k, 9 l, 9 m, 9o and 9q were
also selected for further evaluation of their cellular potencies
(SNU-16 cell). Although, the FGFR1 enzyme inhibitory activities of
compound 9 h and 9j were higher than that of 9d, while the cell
anti-proliferative activities were similar. Besides, compound 9o
bearing a fluorine atom at meta-position of phenyl resulted in 1.4-
fold enhancement of the cellular inhibition (9o vs 9d), which
exhibited the best enzyme and cellular inhibitory activity of these
indazole compounds 9e-t.
As the above reported, R1 group located at the solvent region
of the target protein, which might be help to improve membrane
permeability and enhance cell anti-proliferative activities. Another
six compounds 9 u-z were designed, prepared and evaluated for
their biological activities (Table 3). The results of inhibition of
FGFR1 assay revealed that (2S, 6 R)-2,6-dimethyl-1-phenylpipera-
zine (9v, IC50 ¼ 13.2 nM), and N-methyl-4-phenylpiperazine (9 y,
IC50 ¼ 10.2 nM) led to slight reduction potency comparing to N-
ethyl-4-phenylpiperazine (9o, IC50 ¼ 5.5 nM), while introducing of
a 1-benzyl-4-methylpiperazine group (9x, IC50 > 1000 nM) demon-
strated poor inhibitory activity against FGFR1 enzyme. Meanwhile,
>1000
41.7
855.7
NA
>1000
>1000
NA
>1000
0.7
NA
6.1
AZD4547
aThe IC50 values are shown as the average values from two separate
experiments.
bSNU-16 is FGFR2-amplified cell type.
c“NA” means IC50 > 5 lM.

82
J. LIU ET AL.
Table 3. Structures and activities of indazole derivatives 9 u–z.
Table 4. Selectivity of 9 u in a panel of kinases.
Tyrosine
kinase
Tyrosine
kinase
IC50 (nM)
IC50 (nM)
FGFR1
FGFR2
FGFR3
FGFR4
ALK
3.3
5.2
12.2
>1000
>1000
>1000
>1000
Flt-1
RET
c-Src
IGF1R
c-Met
EGFR
ErbB2
>100
>100
>100
>1000
>1000
>1000
>1000
Bcr-Abl
EPH-A2
FGFR1 kinase
SNU-16 cell
IC50(nM)b
No.
R1
IC50(nM)a
Molecular docking studies
The docking simulation was performed to elucidate the binding
mode of the various scaffold derivatives 9d, 12a and 18 b, the
potent indazole inhibitor 9e and 9f, the inactive inhibitor 9 g with
FGFR1. These compounds were docked into the ATP-binding
pocket of FGFR1 (4ZSA).
3.3
468.2
437.1
9u
13.2
9v
As a result, the core indazole ring of compound 9d was
entered the adenine region by forming the essential H-bond inter-
action with hinge residue Glu562 and Ala564 (docking score:
8.523), while the N-H of indazole scaffold formed H-bond with
Glu562, and the nitrogen atom of indazole scaffold and N-H
of amide formed another two hydrogen bond with Ala564
(Figure 3(a)). Meanwhile, the 3-methoxy phenyl group occupied
the hydrophobic area I, and hydrophobic interact with the lipo-
philic residue Leu547, Val561 and Phe 642. However, while the
hetero aromatic rings were converted to 1H-1,2,4-triazole or ben-
zothiazole, there was weakly H-bond interaction with Ala564, and
no H-bond interaction with Glu562 (Figure 3(b,c)). Besides, the
hydrophobic group I was deviated from hydrophobic pocket. The
above docking results might explain why the triazole and benzo-
thiazole derivatives lost their biological activities (12a docking
score: 6.380, 18 b docking score: 6.588).
Additionally, the methoxy group of 9d was inserted into a
small hydrophobic cavity of the hydrophobic area I. While this
cavity could contain a certain size of lipophilic group, thus the
ethyloxy substituent of 9e (docking score: 8.983) and the iso-pro-
pyxoy substituent of 9f (docking score: 9.451) were more appro-
priate to this hydrophobic cavity, meanwhile 9e and 9f shown
more potent FGFR1 inhibitory activity than 9d. However, the cav-
ity could not accommodate large volume groups, including the
sec-butoxy group (9 g docking score: 5.668, IC50 > 1000 nM). The
molecular simulation results may further confirm the inhibitory
potency of 9e and 9f against FGFR1, and the 9 g was inactive
(Figure 3(d)).
3.3
>1000
10.2
557.2
NAc
9w
9x
9y
639.1
4.6
0.7
738.5
6.1
9z
AZD4547
aThe IC50 values are shown as the average values from two separate experiments.
bSNU-16 is FGFR2-amplified cell type.
c“NA” means IC50 > 5 lM.
the FGFR1 inhibitory activities of compounds 9 u, 9w and 9z were
enhanced, and the lead compound 9 u was 1.5-fold enhancement.
More importantly, the cellular potencies were improved by intro-
ducing various R1 group (9 u, 9v and 9w vs 9o), and among them
9 u exhibited the good FGFR1 inhibitory activity and cell anti-pro-
liferative activity. Therefore, we selected 9 u as the lead com-
pound for further pharmacological research.
The kinase selectivity profile of compound 9 u
Conclusion
To identify the kinase selectivity of the lead compound 9 u, the
pharmacological experiment was first evaluated by testing it
against our in-house panel of tyrosine kinases. The results were
summarised in Table 4, 9 u effectively inhibited the potent activ-
ities of FGFR1-3 with IC50 values of 3.3 nM, 5.2 nM and 12.2 nM,
respectively. However, 9 u exhibited low enzymatic inhibitory
activity (IC50 > 1000 nM), which indicated that 9 u is a selective
FGFR1-3 inhibitor. The reason was that the sequence of FGFR1-3
was very similar, while the sequence of FGFR4 was quite different.
Therefore, the binding mode of FGFR1-3 inhibitor was different
from FGFR4 inhibitor, and the pharmacophore of FGFR4 inhibitor
usually contained an acrylamide group [27–29]. Furthermore, IC50
values for most of kinases were greater than 1 lM except for Flt-1,
RET and c-Src, which were inhibited at sub-micromolar concentra-
tions of the compound. As a result, compound 9 u exhibited good
kinase selectivity.
In summary, we designed three novel series of FGFR1 inhibitors
bearing indazole, benzothiazole, and 1H-1,2,4-triazole scaffold via
fragment-based virtual screening. Interestingly, 33 new com-
pounds were synthesised and evaluated for their inhibitory activity
against FGFR1. Initially, the indazole derivative 9d was identified
as a promising FGFR1 inhibitor, with the good enzymatic inhib-
ition (IC50 ¼ 15.0 nM) and modest anti-proliferative activity (IC50
¼
785.8 nM). Then, the hit 9d was further optimised, through two
rounds of optimisation, the compound 9 u stood out as the most
potent FGFR1 inhibitors with the best enzyme inhibitory (IC50
¼
3.3 nM) and cellular activity (IC50 ¼ 468.2 nM). Moreover, 9 u also
exhibited good kinase selectivity. Meanwhile, the docking study
was performed to investigate the putative interaction mechanism
with the FGFR1 target. Further studies on the structural optimisa-
tion and biological evaluation of 9 u are currently underway in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
83
Figure 3. The docking mode of compound 9d (a), 12a (b), and 18 b (c). (d) Superposed docking poses of 9e (white), 9f (yellow), and 9 g (green).
our laboratory. Our study would provide a basis for discovering
novel FGFR1 inhibitors.
4. Sharma PS, Sharma R, Tyagi T. Receptor tyrosine kinase
inhibitors as potent weapons in war against cancers. Curr
Pharm Des 2009;15:758–76.
5. Ocana A, Serrano R, Calero R, Pandiella A. Novel tyrosine
kinase inhibitors in the treatment of cancer. Curr Drug
Targets 2009;10:575–6.
6. Huang M, Shen A, Ding J, Geng M. Molecularly targeted can-
cer therapy: some lessons from the past decade. Trends
Pharmacol Sci 2014;35:41–50.
Disclosure statement
No potential conflict of interest was reported by the authors.
7. Clark JD, Flanagan ME, Telliez JB. Discovery and develop-
ment of Janus Kinase (JAK) inhibitors for inflammatory dis-
eases: miniperspective. J Med Chem 2014;57:5023–38.
8. Touat M, Ileana E, Postel-Vinay S, et al. Targeting FGFR sig-
naling in cancer. Clin Cancer Res 2015;21:2684–94.
9. Carter EP, Fearon AE, Grose RP. Careless talk costs lives: fibro-
blast growth factor receptor signalling and the consequences
of pathway malfunction. Trends Cell Biol 2015;25:221–33.
10. Dieci MV, Arnedos M, Andre F, Soria JC. Fibroblast growth
factor receptor inhibitors as a cancer treatment: from a bio-
logic rationale to medical perspectives. Cancer Discovery
2013;3:264–79.
11. Singh D, Chan JM, Zoppoli P, et al. Transforming fusions of
FGFR and TACC genes in human glioblastoma. Science
2012;337:1231–5.
12. Wu YM, Su F, Kalyana-Sundaram S, et al. Identification of tar-
getable FGFR gene fusions in diverse cancers. Cancer
Discovery 2013;3:636–47.
Funding
This work was supported by the National Natural Science
Foundation of China (81703342, 81473110, 81773596), Natural
Science Foundation of Jiangsu Higher Education Institutions
(17KJA360004, 16KJB350003), Natural Science Foundation of
Jiangsu (BK2016105), Postgraduate Research & Practice Innovation
Program of Jiangsu Province (SJCX18_0448, KYCX18_1614).
References
1. Anand P, Kunnumakara A, Sundaram C, et al. Cancer is a
preventable disease that requires major lifestyle changes.
Pharm Res 2008;25:2097–116.
2. Siemann DW, Chaplin DJ, Horsman MR. Vascular-targeting
therapies for treatment of malignant disease. Cancer 2004;
100:2491–9.
3. Traxler P, Furet P. Strategies toward the design of novel and
selective protein tyrosine kinase inhibitors. Pharmacol Ther
1999;82:195–206.
13. Helsten T, Elkin S, Arthur E, et al. The FGFR landscape in
cancer: analysis of 4,853 tumors by next-generation
sequencing. Clin Cancer Res 2016;22:259–67.

84
J. LIU ET AL.
14. Hilberg F, Roth GJ, Krssak M, et al. BIBF 1120: triple angioki-
nase inhibitor with sustained receptor blockade and good
antitumor efficacy. Cancer Res 2008;68:4774–82.
predictor of the sensitivity to CH5183284/Debio 1347, a
novel selective FGFR inhibitor. Mol Cancer Ther 2014;13:
2547–58.
15. Roth GJ, Heckel A, Colbatzky F, et al. Design, synthesis, and 22. Liu J, Peng X, Dai Y, et al. Design, synthesis and biological
evaluation of indolinones as triple angiokinase inhibitors
and the discovery of a highly specific 6-methoxycarbonyl-
evaluation of novel FGFR inhibitors bearing indazole scaf-
fold. Org Biomol Chem 2015;13:7643–54.
substituted indolinone (BIBF 1120). J Med Chem 2009;52: 23. Yosaatmadja Y, Patterson AV, Smaill JB, Squire CJ. The 1.65
4466–80.
Å resolution structure of the complex of AZD4547 with the
kinase domain of FGFR1 displays exquisite molecular recog-
nition. Acta Crystallogr 2015;71:525–33.
16. Gozgit JM, Wong MJ, Moran L, et al. Ponatinib (AP24534), a
multitargeted pan-FGFR inhibitor with activity in multiple
FGFR-amplified or mutated cancer models. Mol Cancer Ther 24. Zhang J, Yang PL, Gray NS. Targeting cancer with small mol-
2012;11:690–9. ecule kinase inhibitors. Nat Rev Cancer 2009;9:28–39.
17. Guagnano V, Furet P, Spanka C, et al. Discovery of 3-(2,6- 25. Tucker JA, Klein T, Breed J, et al. Structural insights into
dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-
ylphenylamino] -pyrimidin-4-yl}-1-methyl-urea (NVP-BGJ398),
a potent and selective inhibitor of the fibroblast growth fac-
FGFR kinase isoform selectivity: diverse binding modes of
AZD4547 and ponatinib in complex with FGFR1 and FGFR4.
Structure 2014;22:1764–74.
tor receptor family of receptor tyrosine kinase. J Med Chem 26. Miyaura N, Suzuki A. Palladium-catalyzed cross-coupling
2011;54:7066–83.
reactions of organoboron compounds. Chem Rev 1995;95:
18. Zhao GS, Li WY, Chen DH, et al. A novel, selective inhibitor
2457–85.
of fibroblast growth factor receptors that shows a potent 27. Ken AB, Timothy DO, Erik V, et al. Discovery of the irrevers-
broad spectrum of antitumor activity in several tumor xeno-
graft models. Mol Cancer Ther 2011;10:2200–10.
19. Gavine PR, Mooney L, Kilgour E, et al. AZD4547: an orally
bioavailable, potent, and selective inhibitor of the fibroblast
ible covalent FGFR inhibitor 8-(3-(4-acryloylpiperazin-1-
yl)propyl)-6-(2,6-dichloro-3,5-dimethoxyphenyl)-2-(methyla-
mino)pyrido[2,3-d]pyrimidin-7(8H)- one (PRN1371) for the
treatment of solid tumors. J Med Chem 2017;60:6516–27.
growth factor receptor tyrosine kinase family. Cancer Res 28. Li X, Guise CP, Taghipouran R, et al. 2-Oxo-3, 4-dihydropyri-
2012;72:2045–56.
20. Perera TPS, Jovcheva E, Mevellec L, et al. Discovery and
pharmacological characterization of JNJ-42756493 (erdafiti-
mido[4, 5-d]pyrimidinyl derivatives as new irreversible pan
fibroblast growth factor receptor (FGFR) inhibitors. Eur J
Med Chem 2017;135:531–43.
nib), a functionally selective smallmolecule FGFR family 29. Lu X, Chen H, Patterson AV, et al. Fibroblast growth factor
inhibitor. Mol Cancer Ther 2017;16:1010–20.
21. Nakanishi Y, Akiyama N, Tsukaguchi T, et al. The fibroblast
receptor 4 (FGFR4) selective inhibitors as hepatocellular car-
cinoma therapy: advances and prospects. J Med Chem 2019;
62:2905–15.
growth factor receptor genetic status as
a potential