Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.10.003

FGF receptors (FGFRs) are tyrosine kinases that are overexpressed in diverse tumors by genetic alterations such as gene amplifications, somatic mutations and translocations. Owing to this characteristic, FGFRs are attractive targets for cancer treatment. It has been demonstrated that most multi-targeted, ATP competitive tyrosine kinase inhibitors are active against FGFRs as well as other kinases. The design of new and more selective inhibitors of FGFRs, which might be reduced off-target and side effects, is a difficult yet significant challenge. The results of the current investigation, show that novel Ponatinib analogues are highly active as FGFR inhibitors and that they possess reduced kinase insert domain receptor (KDR) activities. Observations made in a structure and activity relationship (SAR) investigation led to the development of a promising, orally available lead compound 4, which displays a 50–100 fold in vitro selectivity for inhibition of FGFR1-3 over KDR. In addition, biological evaluation of compound 4 showed that it displays significant antitumor activities in FGFR1-amplificated H1581 and FGFR2-amplificated SNU-16 xenograft models.

Full text of DOI:10.1016/j.ejmech.2016.10.003

Accepted Manuscript
Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs
inhibitors
Dr Yang Liu, Xia Peng, Xiaocong Guan, Dong Lu, Yong Xi, Shiyu Jin, Hui Chen, Limin
Zeng, Jing Ai, Meiyu Geng, Youhong Hu
PII:
S0223-5234(16)30865-0
10.1016/j.ejmech.2016.10.003
EJMECH 8968
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2016
Revised Date: 1 October 2016
Accepted Date: 1 October 2016
Please cite this article as: D.Y. Liu, X. Peng, X. Guan, D. Lu, Y. Xi, S. Jin, H. Chen, L. Zeng, J. Ai,
M. Geng, Y. Hu, Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs
inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.003.
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ACCEPTED MANUSCRIPT
Title:
Discovery of Novel Ponatinib Analogues for Reducing KDR Activity as Potent FGFRs Inhibitors
1

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Graphical abstract
A series of novel Ponatinib analogues were synthesized. Observations made in a structure and
activity relationship (SAR) investigation led to compound 4 as a predominant FGFR inhibitor.
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Highlights
‹
‹
‹
‹
‹
Novel Ponatinib analogues as selective FGFRs inhibitors were synthesized.
The SAR of these novel Ponatinib analogues were investigated.
The target selectivity for FGFR1 over KDR was improved by structural optimization.
Lead compound with both high potent enzymatic and cellular activity was achieved.
The evaluation of the lead compound 4 in vivo was reported.
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Title:
Discovery of Novel Ponatinib Analogues for Reducing KDR Activity as Potent FGFRs Inhibitors
Dr. Yang Liu,†, § Xia Peng,‡, § Xiaocong Guan,†, § Dong Lu,† Yong Xi,‡ Shiyu Jin,† Hui Chen,‡
Limin Zeng,† Jing Ai,*, ‡ Meiyu Geng,*, ‡ and Youhong Hu*, †
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, 555 ZuChongZhi Road, Shanghai, 201203, China.
‡Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 ZuChongZhi Road, Shanghai,
201203, China
*Corresponding author: E-mail: yhhu@simm.ac.cn, mygeng@simm.ac.cn, jai@simm.ac.cn.
§Y. L., X. P. and X. G. contributed equally to this work
Abstract:
FGF receptors (FGFRs) are tyrosine kinases that are overexpressed in diverse tumors by genetic
alterations such as gene amplifications, somatic mutations and translocations. Owing to this
characteristic, FGFRs are attractive targets for cancer treatment. It has been demonstrated that
most multi-targeted, ATP competitive tyrosine kinase inhibitors are active against FGFRs as well
as other kinases. The design of new and more selective inhibitors of FGFRs, which might be
reduced off-target and side effects, is a difficult yet significant challenge. The results of the current
investigation, show that novel Ponatinib analogues are highly active as FGFR inhibitors and that
they possess reduced kinase insert domain receptor (KDR) activities. Observations made in a
structure and activity relationship (SAR) investigation led to the development of a promising,
orally available lead compound 4, which displays a 50-100 fold in vitro selectivity for inhibition
of FGFR1-3 over KDR. In addition, biological evaluation of compound 4 showed that it displays
significant antitumor activities in FGFR1-amplificated H1581 and FGFR2-amplificated SNU-16
xenograft models.
Key words:
FGFR / KDR/ inhibitor/ anticancer/ Ponatinib
1. Introduction
The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway
plays a fundamental role in many physiological processes, including embryogenesis, adult tissue
homeostasis and wound healing [1-3]. Substantial evidence has been accumulated that the aberrant
FGFR signaling is activated in diverse tumor types by genetic alterations including genetic
amplifications, somatic mutations and translocations [4-6]. Moreover, the results of studies using
preclinical models and patient biomarker validation have demonstrated the oncogenic potential of
these aberrations in driving tumor growth, promoting tumor metastasis as well as conferring
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resistance to anticancer therapies [7-11]. Consequently, the targeted inhibition of FGFRs is an
attractive modality for cancer treatment. However, most multi-targeted, ATP competitive tyrosine
kinase inhibitors are active against FGFRs as well as other kinases. The lack of kinase selectivity
of these inhibitors has the potential of causing significant side effects [12-14]. For example, their
activity against VEGF receptor 2 (VEGFR2, KDR) is the likely source of grade 3/4 hypertension
induction and dose-limiting toxicity of these inhibitors [15,16]. The discovery of substances that
selectively inhibit kinases is a difficult yet significant challenge [17-22].
Ponatinib (AP24534, Figure 1) is a multi-targeted tyrosine kinase inhibitor that exhibits potent
inhibitory activity against many tyrosine kinases including Abl, Src, PDGFRα, FGFR1 and KDR
with respective IC50 values of 0.37 nM, 2.5 nM, 2.6 nM, 1.2 nM and 3.7 nM [23,24]. As can be
seen by viewing the information in Figure 1, changes in the nature of the heterocyclic ring system
attached to the Ponatinib core structure alter the specificities of binding to various kinases. This
feature has been used advantageously to uncover selective Bcr-Abl inhibitors, which do not suffer
from drug resistance [25-29], as well as selective DDR1 [30] and Src inhibitors [31] (Figure 1). In
our group, compound 1 as an analogue of ponatinib was identified for a potent FGFR1 inhibitor
with an IC50 value of 1.2 nM and a weaker activity against KDR with an IC50 of 79.1 nM at the
enzymatic level. While the activity of 1 against FGFR1 translocated KG1 cells is only modest
(IC50 = 175.5 nM). A following structure activity relationship (SAR) investigation based on
compound 1 led to the development of a promising, orally available substance 4 which displays a
50-100 fold in vitro selectivity for inhibition of FGFR1-3 over VEGFR1-2. In addition, biological
evaluation of this substance 4 showed that it displays significant antitumor activities in
FGFR1-amplificated H1581 and FGFR2-amplificated SNU-16 xenograft models.
Figure 1
2. Results and Discussion
2.1 Chemistry
The designed compounds for SAR investigation focused on the modification of various
six-membered aryl ring for A while retaining the isoquinoline group and changing the B ring
component while retaining the methyl-substituted phenyl ring (Scheme 1). The general pathway
employed to prepare these targets is depicted in Scheme 1. The routes begin with transformation
of the appropriate halogen-substituted aryl acid 5 to the corresponding acyl chloride, which is then
reacted with the piperazine containing aniline derivative. This process forms amide 6, which is
then coupled with the appropriate TMS-substituted alkyne to generate the target compound.
Scheme 1
2.2 SAR study and lead generation
The initial FGFR1 enzyme inhibition of the compounds that focused on the modification of
various six-membered aryl ring A was tested, and then the potent inhibitors were further
investigated against the FGFR1-translocated KG1 cell and KDR profiles (Table 1). As can be seen
by viewing the data listed in Table 1, removal of the methyl group on the phenyl A-ring or
replacement of it by a fluorine group (2a and 2b, respectively) lead to an improved cellular
activity but a diminished FGFR1/KDR selectivity. In contrast, replacing the methyl group by a
chlorine substituent (2c) dramatically enhances the cellular activity while maintaining enzyme
selectivity. In addition, introduction of electron donating or withdrawing group (2d and 2e) and a
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bulky group (2f) in place of the methyl group on the A-ring phenyl moiety cause a decrease in
FGFR1 inhibitory activity. Moreover, replacement of the phenyl ring by variously positioned
pyridine groups (2g-2k) do not bring about improvement of cellular activity or selectivity. These
observations indicate that the substituents on the phenyl A-ring have a significant effect on
enzymatic activity and selectivity of the Ponatinib analogues.
Table 1
Meanwhile, the enzyme and cell level inhibitory properties of Ponatinib analogues containing
the methyl-substituted phenyl A-ring and diverse N-heterocycles as the B-ring unit (Table 2) were
also explored. The results show that 3a-3b with N-heteroindole display an observable selectivity
for FGFR1 over KDR. After opened the indole ring to the different N-substituted pyridines, 3c-3g
also exhibit the promised selectivity. The methoxyl substituted compound 3h decrease the activity
of FGFR1 slightly. Significantly, 3b and 3f have excellent selectivity and inhibitory activities for
KG1 cells.
The results of this effort show that 2c, which contains a chloro-phenyl A-ring, and 3f, which
contains a morpholine substituted pyridine group as the B-ring, have ideal properties. This finding
led to the design and synthesis of derivative 4 (see Table 2), which exhibits potent inhibitory
activity against FGFR1 (IC50 = 0.5 nM), weak KDR inhibitory activity (IC50 = 48.7 nM) and a
potent KG1 cellular activity ((IC50 = 7.4 nM) as expected.
Table 2
The kinase inhibition profiles and selected anti-proliferation activities in related cell lines of
the newly designed inhibitor 4 were furtherly evaluated and contrasted with Ponatinib (Table 3).
The results show that 4 has a significantly higher potency against FGFRs and displays a greater
inhibitory selectivity for KDR and VEGFR1 than does Ponatinib. Paralleling these enzyme level
activities, 4 exhibits anti-proliferation properties against FGFRs and KDR translocated cell lines.
Interestingly, 4 inhibits RET and the RET-V804M gatekeeper mutant kinase and its driven cell
growth to the same degrees that Ponatinib does [32]. In addition, the fact that 4 does not inhibit
EGFR, its use as an anticancer drug could be associated with less adverse side effects. It is likely
that 4 will have inhibitory activities against FGFR1-3 and reduced inhibitory activities against Src
and ABL.
Table 3
The cellular kinase-targeting activity of 4 in representative FGFR1 translocated KG1 cells and
FGFR2 amplified SUN16 cells was evaluated. The results show that 4 inhibits FGFR
phosphorylation as well as the phosphorylation of key downstream Erk, and PLCγ proteins in a
dose-dependent manner (Figure 2A and B). These findings demonstrate that 4 could be used to
block the FGFR signaling pathway.
Figure 2
A preliminary study was carried out to assess the pharmacokinetic properties of 4 in mice. For
this purpose, the plasma concentrations of 4, following oral administration at a dose of 10 mg/kg,
were determined (Figure 3). The results reveal that compound 4 can be absorbed and reach a high
plasma concentration (AUC0-24h of 11.38 ug*h/mL). This property stimulated a study of the in vivo
effects of 4 on the two representative tumor xenograft models including a FGFR1-amplificated
H1581 human lung cancer model and a FGFR2-amplificated SNU-16 human gastric carcinoma
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model. As shown in Figure 4, when administered orally at a dosing regimen of 20 mg/kg once
daily, 4 displays significant antitumor activity in both tumor xenografts.
Figure 3
Figure 4
3. Conclusions
As described above, the SAR study based on compound 1 led to the identification of
compound 4 as an ideal inhibitor. An enzyme level investigation showed that 4 is a more potent
and selective FGFRs tyrosine kinase inhibitor than is Ponatinib. In addition, the use of 4 as a drug
could be associated with a lower level of adverse side effects caused by VEGFR and EGFR
inhibition. Finally, the results of further studies show that 4 has favorable pharmacokinetic
properties in mice and better antitumor activity than does Ponatinib in a FGFR2-driven SNU-16
xenograft model. The combined observations indicate that 4 is an excellent starting point for
optimization studies targeted at the development of clinical candidates for treatment of patients
with FGFRs driven cancer.
4. Experimental Section
4.1 General Information
Unless otherwise noted, all reagents and solvents employed were purchased commercially and
used as received. All reactions involving air- or moisture-sensitive reagents were performed under
a N2 atmosphere. All reactions were conducted in microwave vials or flasks containing
Teflon-coated magnetic stirrer. Microwave irradiation experiments were performed in a
CEM-Discover® LabMate mono-mode microwave apparatus equipped with an IntelliVentTM
pressure control system and a vertically-focused IR temperature sensor. Reactions were monitored
by the thin layer chromatography (TLC) on precoated TLC glass plates (silica gel GF254,
0.2±0.03mm thickness) or by Agilent 1200 series LC-MS system (100mm x 3mm 3.5µm column
+ column; 5µL injection; 3-98% MeOH/H2O + 0.1% HCOOH gradient over 6 min; 0.7 mL/min
flow; ESI; positive ion mode; UV detection at 254 nM). TLC glass plates were developed in a
covered chamber and were visualized with ultraviolet light using a 254 nm fluorescent indicator.
Low-resolution mass spectral (MS) data were gathered on the LC-MS system. Column
chromatography was carried out on silica gel (200-300 mesh). All 1H NMR and 13C NMR spectra
were measured in CDCl3, CD3OD or DMSO-d6 with TMS as the internal standard on Varian
Mercury NMR spectrometers. Chemical shifts were expressed in parts per million (ppm, δ units)
and coupling constants were given in Hz. Data for NMR spectra were reported as follows:
chemical shift (δ ppm), multiplicity (s = singlet, brs = broad singlet, d = doublet, t = triplet, q =
quartet, dd = doublet of doublets, m = multiplet), coupling constant (Hz), integration.
4.2 Procedure for Preparation of Novel Ponatinib Analogues
4.2.1. The Preparation of 3-(isoquinolin-4-ylethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (1).
Step 1: To a solution of 3-iodo-4-methylbenzoic acid 5-1 (262 mg, 1.0 mmol) in dry DCM (10
mL) at 0 oC was added oxalyl chloride (0.13 ml, 1.5 mmol) and 3 drops of dry DMF. The resulting
reaction mixture was stirred at room temperature for 3h, and then the solvent was removed under
reduced pressure. The crude product was used directly for the next step without further
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purification. Step 2: To a solution of 3-iodo-4-methylbenzoyl chloride obtained above in dry DCM
(10 mL) at 0 oC was added Et3N (0.28 ml, 2.0 mmol) and 4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) aniline (328 mg, 1.2 mmol). The mixture was stirred at room
temperature for 5h, and then the solvent was removed under reduced pressure. The residue was
purified by using column chromatography to afford the corresponding product 6-1 (439 mg, 2
steps yield: 85%). Step 3: To a solution of 6-1 (260 mg, 0.5 mmol) in dry MeCN (2 mL) was
added Pd(PPh3)4 (30 mg, 0.025 mmol), CuI (10 mg, 0.05 mmol), Et3N (0.28 ml, 2.0 mmol), CsF
(152 mg, 1.0 mmol) and 4-((trimethylsilyl) ethynyl) isoquinoline 7-1 (225 mg, 1.0 mmol) at room
temperature under nitrogen atmosphere. After stirring for 5 min, the mixture was irradiated for 15
min at 90 °C (monitored by TLC). The mixture was extracted with ethyl acetate (10 mL×3). The
combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered
and concentrated to give the crude product, which was further purified by column chromatography
to afford compound 1 as a light yellow solid (176 mg, yield 65%): mp 128~130 ℃. 1H NMR (400
MHz, CDCl3) δ 9.51 (s, 1H), 9.14 (s, 1H), 8.68 (s, 1H), 8.23 (d, J = 5.0 Hz, 2H), 8.10 (s, 1H), 8.02
(d, J = 8.3 Hz, 1H), 7.93 (t, J = 9.1 Hz, 2H), 7.74 (t, J = 7.5 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.53
(d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 3.62 (s, 2H), 3.00 (s, 4H), 2.74 (s, 4H), 2.65 (s, 3H),
2.57 (s, 3H).13C NMR (151 MHz, CDCl3) δ 165.59, 152.01, 146.16, 144.30, 137.96, 135.32,
131.99, 131.48, 131.39, 131.27, 129.97, 129.18 (q, J = 30.5 Hz), 128.10, 128.05, 127.99, 127.73,
124.93, 124.04 (q, J = 273.31 Hz), 123.81, 122.96, 118.48 (d, J = 5.8 Hz), 115.94, 94.68, 89.18,
57.38, 54.07, 49.97, 43.85, 21.05.19F NMR (471 MHz, CDCl3) δ -59.08. HRMS [M]+ Calculated
for C32H29F3N4O 542.2293, found 542.2295.
4.2.2.
The
Preparation
of
3-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (2a).
The procedures applied to the synthesis of 1 were used with 3-iodobenzoic acid 5-2a (62 mg,
0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline, (82 mg, 0.30 mmol)
and 4-((trimethylsilyl) ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2a as a white solid
1
(87 mg, yield: 66%): mp 118~120 ℃. H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 9.18 (s, 1H),
8.73 (s, 1H), 8.29 (s, 1H), 8.26 (s, 1H), 8.07 (s, 1H), 8.02 (d, J = 8.1 Hz, 2H), 7.98 (d, J = 8.2 Hz,
1H), 7.82 – 7.72 (m, 2H), 7.66 (t, J = 7.5 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.8 Hz,
1H), 3.66 (s, 2H), 2.95 (s, 4H), 2.75 (s, 4H), 2.62 (s, 3H).13C NMR (126 MHz, CDCl3) δ 164.95,
151.71, 145.83, 137.17, 134.97, 134.39, 134.27, 131.38, 131.02, 130.98, 130.24, 128.87 (q, J =
30.24 Hz), 128.52, 127.65, 127.53, 127.43, 127.25, 124.49, 123.52 (q, J = 274.4 Hz), 123.25,
122.87, 117.96 (d, J = 5.8 Hz), 115.16, 95.14, 85.08, 56.99, 53.72, 49.79, 43.61.19F NMR (471
MHz, CDCl3) δ -59.18. HRMS [M]+ Calculated for C31H27F3N4O 528.2137, found 528.2132.
4.2.3. The Preparation of 4-fluoro-3-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (2b).
The procedures applied to the synthesis of 1 were used with 4-fluoro-3-iodobenzoic acid 5-2b
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline 8 (82 mg,
0.30 mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2b as
1
a white solid (83 mg, yield: 61%): mp 118~120 ℃. H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H),
9.20 (s, 1H), 8.76 (s, 1H), 8.30 (d, J = 8.0 Hz, 2H), 8.02 (dd, J = 18.5, 10.6 Hz, 4H), 7.81 (t, J =
7.6 Hz, 1H), 7.71 – 7.59 (m, 2H), 7.25 (t, J = 8.6 Hz, 1H), 3.66 (s, 2H), 2.88 (s, 3H), 2.72 (s, 4H),
2.58 (s, 3H).13C NMR (126 MHz, CDCl3) δ 164.08 (d, J =259.56 Hz), 163.98, 152.03, 145.87,
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137.00, 134.94, 132.43, 131.73, 131.19, 131.00, 130.25 (d, J = 3.2 Hz), 129.84 (d, J = 8.9 Hz),
128.85 (q, J = 30.7 Hz), 127.75, 127.53, 127.24, 124.49, 123.53 (q, J = 274.1 Hz), 123.25, 117.90
(d, J = 5.9 Hz), 115.66 (d, J = 21.6 Hz), 114.91, 111.48 (d, J = 16.7 Hz), 90.14, 88.56, 57.05,
53.89, 50.24, 43.92. 19F NMR (471 MHz, CDCl3) δ -59.29, -103.84. HRMS [M]+ Calculated for
C31H26F4N4O 546.2043, found 546.2041.
4.2.4. The Preparation of 4-chloro-3-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (2c).
The procedures applied to the synthesis of 1 were used with 4-chloro-3-iodobenzoic acid 5-2c
(70 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2c as a
white solid (97 mg, yield: 69%): mp 132~134 ℃. 1H NMR (400 MHz, CDCl3) δ 9.41 (s, 1H), 9.19
(s, 1H), 8.76 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.08 (s, 1H), 8.04 (d, J =
8.5 Hz, 1H), 8.01 – 7.95 (m, 2H), 7.80 (t, J = 7.6 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.60 (d, J = 8.4
Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 3.67 (s, 2H), 2.95 (s, 4H), 2.75 (s, 4H), 2.63 (s, 3H).13C NMR
(126 MHz, CDCl3) δ 164.09, 152.01, 146.06, 139.09, 137.11, 134.98, 132.49, 131.87, 131.46,
131.16, 131.01, 129.29, 128.86 (q, J = 30.24 Hz), 128.42, 127.72, 127.49, 127.25, 124.62,123.73
(q, J = 275.94 Hz), 123.27, 122.68, 117.98 (d, J = 5.9 Hz), 114.93, 92.03, 90.23, 57.00, 53.74,
49.82, 43.63.19F NMR (471 MHz, CDCl3) δ -59.16. HRMS [M]+ Calculated for C31H26ClF3N4O
562.1747, found 562.1751.
4.2.5. The Preparation of 3-(isoquinolin-4-ylethynyl)-4-methoxy-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (2d).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methoxybenzoic acid
5-2d (70 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg,
0.30 mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2d as
an off-white solid (70 mg, yield: 50%): mp 112~114 ℃. 1H NMR (400 MHz, CDCl3) δ 9.16 (d, J
= 12.6 Hz, 1H), 9.13 (s, 1H), 8.72 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 1.9 Hz, 1H), 8.01
– 7.91 (m, 4H), 7.71 (dd, J = 14.0, 7.5 Hz, 2H), 7.61 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H),
3.98 (s, 3H), 3.62 (s, 2H), 2.55 (s, 8H), 2.34 (s, 3H).13C NMR (126 MHz, CDCl3) δ 165.12,
162.75, 151.91, 145.93, 137.27, 135.57, 133.08, 132.29, 131.30, 130.33, 129.11 (q, J = 30.5 Hz),
128.00, 127.86, 127.75, 126.74, 125.18, 124.11 (q, J = 272.16 Hz), 123.56, 123.02, 117.95 (d, J =
5.9 Hz), 116.12, 112.18, 110.66, 92.36, 89.33, 57.75, 56.19, 55.01, 52.53, 45.64.19F NMR (471
MHz, CDCl3) δ -59.30. HRMS [M]+ Calculated for C32H29F3N4O2 558.2243, found 558.2241.
4.2.6.
The
Preparation
of
3-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl)-4-(trifluoromethyl) benzamide (2e).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-(trifluoromethyl) benzoic
acid 5-2e (79 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82
mg, 0.30 mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2e
as a white solid (67 mg, yield: 45%): mp 106~108 ℃. 1H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H),
9.15 (s, 1H), 8.73 (s, 1H), 8.34 (s, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.04 (dd, J = 13.3, 6.1 Hz, 3H),
7.95 (d, J = 8.2 Hz, 1H), 7.76 (dd, J = 14.6, 8.0 Hz, 2H), 7.69 (d, J = 8.4 Hz, 1H), 7.64 (t, J = 7.5
Hz, 1H), 3.64 (s, 2H), 2.65 (d, J = 22.1 Hz, 8H), 2.44 (s, 3H).13C NMR (126 MHz, CDCl3) δ
164.33, 152.64, 146.68, 137.71, 137.05, 135.45, 133.81 (q, J = 30.9 Hz), 133.24, 132.97, 131.72,
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131.42, 129.28 (q, J = 30.5 Hz), 128.27, 127.94, 127.72, 126.56 (d, J = 4.8 Hz), 124.83, 124.01 (q,
J = 274.2 Hz), 123.77, 123.11 (q, J = 273.8 Hz), 118.29 (d, J = 5.9 Hz), 115.30, 91.50, 91.23,
57.63, 54.76, 51.78, 45.11.19F NMR (471 MHz, CDCl3) δ -59.33, -62.33. HRMS [M]+ Calculated
for C32H26F6N4O 596.2011, found 596.2016.
4.2.7. The Preparation of 4-isopropyl-3-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) benzamide (2f).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-isopropylbenzoic acid
5-2f (73 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg,
0.30 mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2f as
1
an off-white solid (61 mg, yield: 43%): mp 112~114 ℃. H NMR (400 MHz, CDCl3) δ 9.20 (s,
1H), 9.00 (s, 1H), 8.75 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 1.4 Hz, 1H), 8.07 – 7.95 (m,
4H), 7.79 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.2 Hz,
1H), 3.74 – 3.60 (m, 3H), 2.85 (s, 4H), 2.71 (s, 4H), 2.55 (s, 3H), 1.39 (s, 3H), 1.37 (s, 3H).13C
NMR (126 MHz, CDCl3) δ 166.02, 155.36, 152.83, 147.06, 138.26, 136.15, 132.86, 132.69,
132.22, 132.13, 130.09 (q, J = 30.6 Hz), 128.94, 128.79, 128.74, 128.52, 126.42, 125.63, 124.74
(q, J = 274.2 Hz), 124.27, 122.90, 118.92 (d, J = 5.8 Hz), 116.66, 95.09, 89.83, 58.29, 55.10,
51.56, 45.18, 32.76, 30.40, 23.65.19F NMR (471 MHz, CDCl3) δ -59.25. HRMS [M]+ Calculated
for C34H33F3N4O 570.2606, found 570.2608.
4.2.8.
The
Preparation
of
4-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) picolinamide (2g).
The procedures applied to the synthesis of 1 were used with 4-bromopicolinic acid 5-2g (51
mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2g as a
1
brown solid (47 mg, yield: 36%): mp 115~117 ℃. H NMR (400 MHz, CDCl3) δ 10.11 (s, 1H),
9.27 (s, 1H), 8.82 (s, 1H), 8.67 (d, J = 5.0 Hz, 1H), 8.48 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.09 –
7.99 (m, 3H), 7.87 (t, J = 7.6 Hz, 1H), 7.71 (dd, J = 15.5, 6.2 Hz, 3H), 3.71 (s, 2H), 2.85 (s, 4H),
2.75 (s, 4H), 2.57 (s, 3H).13C NMR (126 MHz, CDCl3) δ 161.17, 152.75, 149.08, 147.78, 146.75,
136.25, 134.91, 132.58, 131.96, 131.22 (d, J = 8.2 Hz), 129.12 (q, J = 30.7 Hz), 127.82, 127.74,
127.69, 127.24, 124.24, 124.07, 123.52 (q, J = 274.3 Hz), 122.15, 116.94 (d, J = 6.0 Hz), 113.98,
92.56, 90.25, 57.16, 54.00, 50.61, 44.18.19F NMR (471 MHz, CDCl3) δ -59.12. HRMS [M]+
Calculated for C30H26F3N5O 529.2089, found 529.2096.
4.2.9.
The
Preparation
of
6-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) picolinamide (2h).
The procedures applied to the synthesis of 1 were used with 6-bromopicolinic acid 5-2h (51
mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2h as a
1
light brown solid (52 mg, yield: 39%): mp 118~120 ℃. H NMR (400 MHz, CDCl3) δ 10.11 (s,
1H), 9.32 (s, 1H), 8.93 (s, 1H), 8.41 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.13 – 8.00 (m,
4H), 7.91 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 8.5 Hz,
1H), 3.77 (s, 2H), 3.03 (s, 4H), 2.86 (s, 4H), 2.70 (s, 3H).13C NMR (151 MHz, CDCl3) δ 161.60,
153.18, 149.80, 147.40, 141.64, 138.27, 136.92, 135.57, 130.45, 127.99(q, J = 30.6 Hz), 128.31,
128.25, 127.79, 124.88, 123.77 (q, J = 273.32 Hz), 122.87, 122.11, 117.84 (d, J = 5.7 Hz), 114.51,
10

ACCEPTED MANUSCRIPT
94.56, 85.73, 57.64, 54.22, 50.26, 44.14.19F NMR (471 MHz, CDCl3) δ -59.35. HRMS [M]+
Calculated for C30H26F3N5O 529.2089, found 529.2091.
4.2.10.
The
Preparation
of
5-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) nicotinamide (2i).
The procedures applied to the synthesis of 1 were used with 5-bromonicotinic acid 5-2i (51 mg,
0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30 mmol)
and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2i as a brown solid
(54 mg, yield: 41%): mp 106~108 ℃. 1H NMR (400 MHz, CDCl3) δ 9.75 (s, 1H), 9.23 (d, J = 1.9
Hz, 1H), 9.20 (s, 1H), 8.93 (d, J = 1.7 Hz, 1H), 8.74 (s, 1H), 8.56 (d, J = 1.9 Hz, 1H), 8.26 (d, J =
8.3 Hz, 1H), 8.10 (s, 1H), 8.05 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.82 (t, J = 7.6 Hz,
1H), 7.69 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 3.66 (s, 2H), 2.87 (s, 4H), 2.70 (s, 4H),
2.57 (s, 3H).13C NMR (126 MHz, CDCl3) δ 163.21, 153.83, 152.19, 147.27, 146.10, 137.63,
136.80, 134.84, 132.07, 131.21, 130.98, 129.46, 128.83 (q, J = 30.8 Hz), 127.81, 127.64, 127.22,
124.31, 123.51 (q, J = 274.3 Hz), 123.38, 119.60, 118.01 (d, J = 5.8 Hz), 114.51, 91.68, 88.49,
57.05, 53.91, 50.37, 43.97.19F NMR (471 MHz, CDCl3) δ -59.23. HRMS [M]+ Calculated for
C30H26F3N5O 529.2089, found 529.2088.
4.2.11.
The
Preparation
of
2-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) isonicotinamide (2j).
The procedures applied to the synthesis of 1 were used with 2-bromoisonicotinic acid 5-2j (51
mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-((trimethylsilyl)ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain 2j as a
1
brown solid (46 mg, yield: 35%): mp 116~118 ℃. H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H),
9.17 (s, 1H), 8.76 (d, J = 5.1 Hz, 1H), 8.72 (s, 1H), 8.27 (d, J = 8.3 Hz, 1H), 8.20 (s, 1H), 8.06 (s,
1H), 8.02 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.86 (dd, J = 5.1, 1.3 Hz, 1H), 7.76 (t, J =
7.4 Hz, 1H), 7.69 – 7.61 (m, 2H), 3.63 (s, 2H), 2.71 (s, 4H), 2.62 (s, 4H), 2.45 (s, 3H).13C NMR
(126 MHz, CDCl3) δ 163.19, 152.28, 150.45, 146.37, 143.17, 141.80, 136.47, 134.97, 132.69,
131.27, 130.94, 127.85, 127.56, 127.17, 123.49 (q, J = 274.1 Hz), 123.36, 120.57, 117.91 (d, J =
6.0 Hz), 114.32, 94.60, 92.56 – 91.55 (m), 85.06, 54.12, 51.02, 44.42.19F NMR (471 MHz, CDCl3)
δ -59.29. HRMS [M]+ Calculated for C30H26F3N5O 529.2089, found 529.2093.
4.2.12.
The
Preparation
of
2-(isoquinolin-4-ylethynyl)-N-(4-((4-methylpiperazin-1-yl)
methyl)-3-(trifluoromethyl) phenyl) pyrimidine-4-carboxamide (2k).
The procedures applied to the synthesis of 1 were used with 2-chloropyrimidine-4-carboxylic
acid 5-2k (51 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82
mg, 0.30 mmol) and 4-((trimethylsilyl) ethynyl) isoquinoline 7-1 (112 mg, 0.50 mmol) to obtain
1
2k as a dark brown solid (20 mg, yield: 15%): mp 102~104 ℃. H NMR (400 MHz, CDCl3) δ
9.96 (s, 1H), 9.35 (s, 1H), 9.14 (d, J = 5.0 Hz, 1H), 8.99 (s, 1H), 8.46 (d, J = 8.5 Hz, 1H), 8.21 (d,
J = 5.0 Hz, 1H), 8.07 (dd, J = 18.4, 8.3 Hz, 3H), 7.91 (t, J = 7.6 Hz, 1H), 7.76 (t, J = 8.0 Hz, 2H),
3.74 (s, 2H), 2.82 (s, 4H), 2.75 (s, 4H), 2.56 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 160.22,
160.03, 156.56, 153.77, 151.96, 148.14, 135.98, 135.76, 133.51, 131.89, 131.72, 128.45, 128.26,
127.75, 124.97, 123.09, 117.88 (d, J = 5.9 Hz), 117.26, 113.81, 94.15, 84.66, 57.69, 54.60, 51.33,
44.85.19F NMR (471 MHz, CDCl3) δ -59.43. HRMS [M]+ Calculated for C29H25F3N6O 530.2042,
found 530.2045.
11

ACCEPTED MANUSCRIPT
4.2.13.
The
Preparation
of
3-((1H-pyrrolo[2,3-c]
pyridin-4-yl)
ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) phenyl) benzamide
(3a).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-((trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-c] pyridine 7-3a (107 mg, 0.50 mmol) to
1
obtain 3a as a white solid (62 mg, yield: 47%): mp 160~162 ℃. H NMR (400 MHz,
CD3OD_SPE) δ 8.72 (s, 1H), 8.28 (s, 1H), 8.17 (s, 2H), 7.97 (d, J = 8.3 Hz, 1H), 7.88 (dd, J = 8.0,
1.8 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 3.0 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 6.77 (d, J
= 3.0 Hz, 1H), 3.72 (s, 2H), 3.09 (s, 4H), 2.73 (s, 3H), 2.70 (s, 4H), 2.65 (s, 3H).13C NMR (126
MHz, MeOD) δ 165.89, 143.58, 139.08, 137.64, 133.50, 132.54, 131.62, 131.25, 130.75, 130.44,
130.14, 129.15, 128.22 (q, J = 30.3 Hz), 127.04, 124.91, 123.83 (q, J = 273.2 Hz), 123.24, 122.66,
117.40 (d, J = 6.0 Hz), 99.96, 91.14, 89.43, 56.51, 53.41, 49.94, 42.41, 19.25.19F NMR (471 MHz,
MeOD) δ -60.29. HRMS [M]+ Calculated for C30H28F3N5O 531.5832, found 531.5837.
4.2.14.
The
Preparation
of
4-methyl-3-((1-methyl-1H-pyrrolo[2,3-c]
pyridin-4-yl)
ethynyl)-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) phenyl) benzamide (3b).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 1-methyl-4-((trimethylsilyl)ethynyl)-1H-pyrrolo[2,3-c] pyridine 7-3b (114 mg, 0.50
1
mmol) to obtain 3b as a white solid (56 mg, yield: 41%): mp 117~119 ℃. H NMR (400 MHz,
CDCl3) δ 8.84 (s, 1H), 8.68 (s, 1H), 8.46 (s, 1H), 8.09 (s, 1H), 7.97 (d, J = 5.7 Hz, 2H), 7.83 (d, J
= 8.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 3.4 Hz, 1H), 6.68 (d,
J = 2.9 Hz, 1H), 3.91 (s, 3H), 3.66 (s, 2H), 3.12 (s, 4H), 2.63 (s, 3H), 2.59 (s, 4H), 2.39 (s, 3H).13C
NMR (126 MHz, CDCl3) δ 165.40, 144.33, 141.49, 137.16, 134.28, 133.51, 133.11, 133.01,
132.16, 132.02, 131.41, 130.39, 130.08, 129.26 (q, J = 30.5 Hz), 127.50, 123.53, 123.43, 117.85
(d, J = 6.0 Hz), 111.28, 100.47, 92.00, 90.65, 57.75, 54.80, 52.31, 45.38, 21.02.19F NMR (471
MHz, CDCl3) δ -59.33. HRMS [M]+ Calculated for C31H30F3N5O 545.2402, found 545.2405.
4.2.15.
The
Preparation
of
3-((5-(isopropylamino)
pyridin-3-yl)
ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) phenyl) benzamide
(3c).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and N-isopropyl-5-((trimethylsilyl)ethynyl) pyridin-3-amine 7-3c (116 mg, 0.50 mmol) to
1
obtain 3c as a brown solid (70 mg, yield: 51%): mp 121~123 ℃. H NMR (400 MHz,
CD3OD_SPE) δ 8.18 (d, J = 1.9 Hz, 1H), 8.12 (d, J = 1.7 Hz, 1H), 7.98 (d, J = 6.8 Hz, 1H), 7.89
(dd, J = 8.0, 1.8 Hz, 2H), 7.78 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.12 (s, 1H), 3.74 (s,
2H), 3.66 (dt, J = 12.7, 6.3 Hz, 1H), 3.09 (s, 4H), 2.73 (s, 7H), 2.60 (s, 3H), 1.24 (t, J = 6.0 Hz,
6H).13C NMR (126 MHz, CDCl3) δ 166.11, 145.21, 143.67, 140.97, 138.18, 136.42, 132.95,
132.70, 132.19, 131.43, 130.75, 130.02 (q, J = 30.6 Hz), 128.36, 124.74 (q, J = 274.0 Hz), 124.27,
123.82, 121.24, 120.72, 118.86 (d, J = 5.9 Hz), 92.41, 90.07, 58.30, 55.16, 51.75, 45.32, 44.74,
23.39, 21.56.19F NMR (471 MHz, CDCl3) δ -59.27. HRMS [M]+ Calculated for C31H34F3N5O
549.2715, found 549.2712.
12

ACCEPTED MANUSCRIPT
4.2.16.
The
Preparation
of
3-((5-(diethylamino)
pyridin-3-yl)
ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) phenyl) benzamide
(3d).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and N, N-diethyl-5-((trimethylsilyl)ethynyl) pyridin-3-amine 7-3d (123 mg, 0.50 mmol) to
1
obtain 3d as a brown solid (66 mg, yield: 47%): mp 108~110 ℃. H NMR (400 MHz, CDCl3) δ
9.05 (s, 1H), 8.08 (s, 1H), 8.04 (s, 1H), 7.99 (d, J = 9.1 Hz, 3H), 7.86 (d, J = 7.9 Hz, 1H), 7.60 (d,
J = 8.2 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.03 (s, 1H), 3.69 (s, 2H), 3.37 (q, J = 7.1 Hz, 4H), 3.04
(d, J = 40.2 Hz, 4H), 2.79 (s, 4H), 2.65 (s, 3H), 2.56 (s, 3H), 1.18 (t, J = 7.1 Hz, 6H).13C NMR
(126 MHz, CDCl3) δ 164.97, 144.01, 142.54, 138.04, 137.26, 133.13, 131.46, 131.17, 131.05,
130.35, 129.53, 128.91 (q, J = 30.7 Hz), 127.17, 123.52 (q, J = 274.2 Hz), 123.12, 122.67, 119.46,
119.39, 117.82 (d, J = 5.9 Hz), 91.46, 88.62, 57.01, 53.67, 49.68, 43.64, 43.55, 20.38, 11.83. 19F
NMR (471 MHz, CDCl3) δ -59.23. HRMS [M]+ Calculated for C32H36F3N5O 563.2872, found
563.2877.
4.2.17. The Preparation of 4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl)
phenyl)-3-((5-(pyrrolidin-1-yl) pyridin-3-yl) ethynyl) benzamide (3e).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 3-(pyrrolidin-1-yl)-5-((trimethylsilyl)ethynyl) pyridine 7-3e (122 mg, 0.50 mmol) to
1
obtain 3e as a brown solid (60 mg, yield: 43%): mp 137~139 ℃. H NMR (400 MHz, CDCl3) δ
9.10 (s, 1H), 8.07 (d, J = 1.8 Hz, 1H), 8.06 (d, J = 1.4 Hz, 1H), 8.01 (d, J = 1.9 Hz, 1H), 7.98 (d, J
= 8.4 Hz, 1H), 7.89 – 7.84 (m, 2H), 7.59 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 6.90 (d, J =
2.6 Hz, 1H), 3.69 (s, 2H), 3.29 (t, J = 6.5 Hz, 4H), 3.03 (s, 4H), 2.81 (d, J = 4.3 Hz, 4H), 2.68 (s,
3H), 2.55 (s, 3H), 2.06 – 2.02 (m, 4H).13C NMR (126 MHz, CDCl3) δ 166.28, 145.08, 143.75,
139.45, 138.44, 134.26, 132.72, 132.62, 132.14, 131.55, 130.66, 129.96 (q, J = 30.6 Hz), 128.46,
124.76 (q, J = 274.4 Hz), 124.37, 123.78, 120.50, 118.98 (d, J = 6.0 Hz), 92.65, 89.88, 58.24,
55.05, 51.40, 47.99, 45.09, 26.06, 21.56.19F NMR (471 MHz, CDCl3) δ -59.20. HRMS [M]+
Calculated for C32H34F3N5O 561.2715, found 561.2713.
4.2.18. The Preparation of 4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl)
phenyl)-3-((5-morpholinopyridin-3-yl) ethynyl) benzamide (3f).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-(5-((trimethylsilyl)ethynyl) pyridin-3-yl) morpholine 7-3f (130 mg, 0.50 mmol) to
obtain 3f as a light yellow solid (79 mg, yield: 55%): mp 128~130 ℃. 1H NMR (400 MHz, DMSO)
δ 10.66 (s, 1H), 8.37 (d, J = 2.8 Hz, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.22 (d, J = 1.4 Hz, 1H), 8.21 (d,
J = 1.5 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.99 – 7.95 (m, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.52 (d, J
= 8.2 Hz, 2H), 3.79 – 3.73 (m, 4H), 3.63 (s, 2H), 3.39 (s, 4H), 3.26 – 3.22 (m, 4H), 2.89 (s, 4H),
2.56 (s, 3H), 2.54 (s, 3H).13C NMR (126 MHz, CDCl3) δ 166.12, 147.02, 145.15, 143.48, 138.41,
137.98, 132.70, 132.50, 132.17, 131.75, 130.69, 129.94 (q, J = 30.5 Hz), 128.55, 124.75 (q, J =
274.1 Hz), 124.65, 124.39, 123.57, 120.73, 118.99 (d, J = 5.5 Hz), 91.88, 90.66, 67.20, 58.19,
54.96, 51.17, 48.84, 44.91, 21.57.19F NMR (471 MHz, CDCl3) δ -59.15. HRMS [M]+ Calculated
13

ACCEPTED MANUSCRIPT
for C32H34F3N5O2 577.2665, found 577.2661.
4.2.19. The Preparation of 4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl)
phenyl)-3-((5-(4-methylpiperazin-1-yl) pyridin-3-yl) ethynyl) benzamide (3g).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 1-methyl-4-(5-((trimethylsilyl) ethynyl) pyridin-3-yl) piperazine 7-3g (137 mg, 0.50
1
mmol) to obtain 3g as a dark brown solid (90 mg, yield: 61%): mp 109~111 ℃. H NMR (400
MHz, CD3OD_SPE) δ 8.25 (d, J = 2.8 Hz, 1H), 8.15 (dd, J = 5.8, 1.8 Hz, 2H), 8.10 (d, J = 1.9 Hz,
1H), 7.96 (dd, J = 8.4, 2.0 Hz, 1H), 7.87 (dd, J = 8.0, 1.9 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.51
(dd, J = 2.7, 1.6 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 3.71 (s, 2H), 3.44 – 3.33 (m, 4H), 3.06 (s, 4H),
2.84 – 2.76 (m, 4H), 2.71 (s, 6H), 2.57 (s, 3H), 2.49 (s, 3H).13C NMR (126 MHz, MeOD) δ
166.89, 147.16, 145.02, 142.04, 138.84, 137.36, 132.83, 132.50, 131.95, 131.67, 130.38, 129.40
(q, J = 30.2 Hz), 128.48, 125.14, 125.03 (q, J = 273.4 Hz), 124.39, 123.27, 121.08, 118.54 (d, J =
6.0 Hz), 91.13, 90.42, 57.74, 54.61, 51.20, 47.40, 44.92, 43.66, 20.30.19F NMR (471 MHz, MeOD)
δ -60.24. HRMS [M]+ Calculated for C33H37F3N6O 590.2981, found 590.2983.
4.2.20.
The
Preparation
of
3-((5-methoxypyridin-3-yl)
ethynyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) phenyl) benzamide
(3h).
The procedures applied to the synthesis of 1 were used with 3-iodo-4-methylbenzoic acid 5-1
(66 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 3-methoxy-5-((trimethylsilyl)ethynyl) pyridine 7-3h (102 mg, 0.50 mmol) to obtain 3h
as a white solid (51 mg, yield: 39%): mp 106~108 ℃. 1H NMR (400 MHz, CDCl3) δ 8.96 (s, 1H),
8.35 (d, J = 1.5 Hz, 1H), 8.25 (d, J = 2.8 Hz, 1H), 8.07 (d, J = 1.6 Hz, 1H), 7.97 (d, J = 10.2 Hz,
2H), 7.86 (dd, J = 8.0, 1.7 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.30 (dd, J
= 2.7, 1.6 Hz, 1H), 3.87 (s, 3H), 3.66 (s, 2H), 2.91 (s, 4H), 2.73 (s, 4H), 2.59 (s, 3H), 2.55 (s,
3H).13C NMR (126 MHz, CDCl3) δ 166.17, 155.84, 145.16, 144.93, 138.41, 138.10, 132.63,
132.48, 132.06, 131.82, 130.64, 129.82 (q, J = 30.5 Hz), 128.68, 124.76 (q, J = 274.3 Hz), 124.51,
123.39, 123.03, 121.12, 119.05 (d, J = 5.6 Hz), 91.27, 91.07, 58.13, 56.38, 54.93, 51.15, 44.88,
21.51.19F NMR (471 MHz, CDCl3) δ -59.12. HRMS [M]+ Calculated for C29H29F3N4O2 522.2243,
found 522.2239.
4.2.21. The Preparation of 4-chloro-N-(4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl)
phenyl)-3-((5-morpholinopyridin-3-yl) ethynyl) benzamide (4).
The procedures applied to the synthesis of 1 were used with 4-chloro-3-iodobenzoic acid 5-2c
(70 mg, 0.25 mmol), 4-((4-methylpiperazin-1-yl) methyl)-3-(trifluoromethyl) aniline (82 mg, 0.30
mmol) and 4-(5-((trimethylsilyl)ethynyl) pyridin-3-yl) morpholine 7-3f (130 mg, 0.50 mmol) to
obtain 4 as a white solid (87 mg, yield: 58%): mp 132~134 ℃. 1H NMR (400 MHz, CDCl3) δ 9.34
(s, 1H), 8.28 (s, 1H), 8.20 (d, J = 2.7 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 7.99 (d, J = 9.5 Hz, 2H),
7.92 (dd, J = 8.4, 1.8 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 3.90
– 3.83 (m, 4H), 3.66 (s, 2H), 3.23 – 3.16 (m, 4H), 2.85 (s, 4H), 2.70 (s, 4H), 2.56 (s, 3H).13C
NMR (126 MHz, CDCl3) δ 164.08, 145.81, 142.34, 139.04, 137.10, 136.90, 132.50, 131.89,
131.83, 130.98, 129.25, 128.79 (q, 30.7 Hz), 128.36, 123.58, 123.52 (q, J = 274.2 Hz), 123.25,
122.46, 119.02, 117.83 (d, J = 6.0 Hz), 91.99, 87.37, 65.99, 57.06, 50.42, 47.58, 44.07.19F NMR
14

ACCEPTED MANUSCRIPT
(471 MHz, CDCl3) δ -59.16. HRMS [M]+ Calculated for C31H31ClF3N5O2 597.2118, found
597.2119.
4.3 ELISA Kinase Assay
The effects of indicated compound on the activities of receptor tyrosine kinases were
determined using enzyme-linked immunosorbent assays (ELISAs) with purified recombinant
proteins. Briefly, 20 µg/mL poly (Glu,Tyr) 4:1 (Sigma, St. Louis, MO, USA) was pre-coated in
96-well plates as a substrate. A 50-µL aliquot of 10 µmol/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 µL of various concentrations of indicated
compound diluted in 1% DMSO (v/v) (Sigma) 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
or commercial tyrosine kinase proteins diluted in 49 µL of kinase reaction buffer. After incubation
for 60 min at 37 °C, the plate was washed three times with phosphate-buffered saline (PBS)
containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody (100 µL; 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 µL 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-µL aliquot of a solution 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 terminated
by the addition of 50 µL of 2 mol/L H2SO4 as the color changed, and the plate was analyzed using
a multi-well spectrophotometer (SpectraMAX 190, Molecular Devices, and Sunnyvale, CA, USA)
at 490 nm. The inhibition rate (%) was calculated using the following equation: [1 − (A490/A490
control)] × 100%. The IC50 values were calculated from the inhibition curves in two separate
experiments.
4.4 Western Blot Analysis
KG1 and SNU16 cells were treated with indicated concentrations of compounds for 2 h at
37°C followed by lysed in 1×SDS sample buffer. Cell lysates were subsequently resolved by
SDS–PAGE and transferred to nitrocellulose membranes. The membranes were probed with the
appropriate primary antibodies [phospho-FGFR1, phospho-FGFR2, FGFR, phospho-PLCγ, PLCγ,
phospho-ERK1/2, ERK1/2 (all from Cell Signaling Technology, Beverly, MA, USA), GAPDH
(Epitomics, Burlingame, CA, USA)], and then with horseradish peroxidase-conjugated anti-rabbit
or antimouse IgG. The immunoreactive proteins were detected using an enhanced
chemiluminescence detection reagent (Thermo Fisher Scientific, Rockford, IL, USA) and images
were captured with ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, Buckinghamshire,
UK.).
4.5 Cell Proliferation Assay
Cells were seeded in 96-well tissue culture plates. On the next day, cells were exposed to
various concentrations of compounds and further cultured for 72 h. Finally, cell proliferation was
determined using sulforhodamine B (SRB) assay or Cell Counting Kit(CCK-8) assay. IC50 values
were calculated by concentration–response curve fitting using a Soft Max pro-based
four-parameter method.
4.6 In vivo Antitumor Activity Assay
15

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Male nude mice (4–6 weeks old) were housed and maintained under specific-pathogen free
conditions. Animal procedures were performed according to institutional ethical guidelines of
animal care. The cells at a density of 5×106 in 200 µL were injected s.c. into the right flank of
nude mice and then allowed to grow to 700–800 mm3, defined as a well-developed tumor. After
that, the well-developed tumors were cut into 1 mm3 fragments and transplanted s.c. into the right
flank of nude mice using a trocar. When the mean tumor volume reached 120–150 mm3, the mice
were randomly assigned into vehicle and treatment groups (n = 6 in treated group, n = 12 in
vehicle group). Vehicle groups were given vehicle alone, and treatment groups received the
compound as indicated doses orally once daily for 10 days in NCI-H1581 model and 18 days in
SNU16 model. The sizes of the tumors were measured twice per week using microcaliper. The
tumor volume (V) was calculated as follows: V = [length (mm) ×width2 (mm2)]/2. The individual
relative tumor volume (RTV) was calculated as follows: RTV = Vt/V0, where Vt is the volume on
each day, and V0 represents the volume at the beginning of the treatment. RTV was shown on
indicated days as mean ± SD indicated for groups of mice. Percent (%) inhibition values （TGI）
were measured on the final day of study for drug-treated compared with vehicle-treated mice and
were calculated as 100% × (1 – ((treatedfinal day – treatedday 0)/(controlfinal day –controlday 0))).
5. Statistical Analysis
Data of in vitro and in vivo efficacy are presented as the mean ± SD, and significance was
determined by Student’s t-test. Differences were considered statistically significant at ***P<0.001,
**P<0.01.
Acknowledgments
We are grateful for financial support from the National Natural Science Foundation of China
(Grant No. 81225022, 81473243, 81321092); The National Science & Technology Major Project
"Key New Drug Creation and Manufacturing Program" of China (Grant No.
2012ZX09301001-007). SA-SIBS Scholarship Program is also gratefully acknowledged.
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Table, Figure and Scheme Captions
Table 1. FGFR1/KDR enzymatic and FGFR1 translocated KG1 cellular activities of the designed
Ponatinib derivatives 1-2.
Table 2. FGFR1/KDR enzymatic and FGFR1 translocated KG1 cellular activities of the designed
Ponatinib derivatives 3-4.
Table3. Kinase selectivity profiles and anti-proliferative activity for the related cell lines displayed
by 4 and Ponatinib.
Figure 1. Ponatinib analogues with different biological activities and the strategy for discovery of
novel selective FGFRs inhibitors.
Figure 2. Compound 4 suppresses FGFR phosphorylation and downstream signaling in KG1
Cells (A) and SNU16 cells (B). Cells were treated with indicated concentrations of 4 for 2 hr and
analyzed by using immunoblotting.
Figure 3. Plasma concentration vs time plots after oral administration of 10 mg/kg of compound
4. Plasma concentration was monitored at 0.5, 1, 2, 3, 5, 7, 9, 12 and 24 h after dosing.
Figure 4. Compound 4 potently inhibits FGFR-dependent tumor growth through effects on tumor
cell growth as well as through antiangiogenic mechanisms. Inhibitory activity of 4 on tumor
growth in NCI-H1581 (A) and SNU16 (B) xenografts. 4 was administrated to the tumor-bearing
mice once a day for 10 or 18 d. Mean relative tumor volume ± SE on the final day is shown (n = 6
per group).
Scheme 1. Synthetic route of designed compounds 1-4.
20

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IC50 (nM)
Compound A ring
KDR (nM)
FGFR1
KG1 (Cell)
1
1.2± 0.4 175.4±49.5 79.1±3.0
2a
1.1±0.5
67.6 ±11.5
2.5 ± 0.5
5.5 ± 1.6
91.4 ±8.1
NTb
2b
1.3 ±0.5 27.8 ±10.3
5.2± 0.7 88.2± 21.9
26.5±7.8 > 500
F
2c
2d
2e
> 100
> 100
> 500
NTb
NTb
2f
NTb
2g
2h
2i
8.1±2.6
> 100
> 500
NTb
NTb
NTb
1.6±0.3
2.2±0.4
>500
2.6±0.2
2j
427.1 ±21.4 6.5 ± 0.5
NTb
2k
11.2±1.6 > 500
aIC50 values are given as the mean±SD (nM) from two separate
experiments. bNot tested.
Table 1. FGFR1/KDR enzymatic and FGFR1 translocated KG1 cellular activities of the designed
Ponatinib derivatives 1-2.a
21

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IC50 (nM)
Compound B ring
KDR (nM)
FGFR1
KG1 (Cell)
3a
6.7 ± 2.3 489.8±140.8 > 100
3b
3c
3d
3e
3f
1.1 ± 0.2 10.0 ± 5.9
3.6 ± 1.9 84.5 ± 24.3
6.5 ± 2.6 58.3 ± 5.8
63.5±2.6
59.9 ±3.8
104.5±4.7
7.6 ± 1.5 119.2± 18.2 108.1±15.6
3.0 ± 1.8 24.3 ± 7.6
5.7 ± 1.4 21.7 ± 3.7
89.9± 13.4
21.8 ± 4.8
3g
3h
4
12.8±2.2 125.2±45.9
0.5 ± 0.0 7.5 ± 3.6
110.2±27.3
48.7 ± 0.1
aIC50 values are given as the mean±SD (nM) from two separate
experiments. bNot tested.
Table 2. FGFR1/KDR enzymatic and FGFR1 translocated KG1 cellular activities of the designed
Ponatinib derivatives 3-4.a
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IC50 (nM)
Ponatinb
IC50 (nM)
Ponatinib
Kinase
FGFR1
Cell linesb
4
4
KG1
7.5±3.6
17.2±3.8
0.5±0.0 1.2±0.1
H1581
57.0±2.8
194.2±29.5
FGFR2
FGFR3
FGFR4
KDR
0.3±0.1 1.3±0.2
1.0±0.1 9.4±2.3
5.5±0.6 7.1±1.1
SNU16
26.4±0.9
514.4±1.2
131.0±8.1
33.6±2.1
RT112
736.5±55.5
126.8±4.5
BaF3/TEL-FGFR4
BaF3/TEL-KDR
BaF3/CCDC6-RET
48.7±0.
3.7±1.5
1
489.0±42.2 53.7±11.2
RET
1.3±0.3 0.3±0.1
37.2±3.9
39.1±2.6
71.5±2.4
RETV804M 7.5±1.3 1.9±0.3
62.7±6.
BaF3/CCDC6-RET-V804M 33.4±5.4
VEGFR1
20.9±0.8
-
-
-
-
-
-
4
EGFR
>100
>1000
PDGFR-α
PDGFR-β
c-Src
5.6±0.3 2.6±0.4
5.5±0.9 1.2±02
-
-
-
-
-
-
-
-
-
-
-
-
10.8±1.
2.5±0.6
5
23.2±1.
ABL
7.7±0.1
1
aIC50 values are given as the mean ± SD (nM) or estimated values from two separate experiments.
bCell line KG1, H1581, KATOIII, SNU16, RT112 are driven by FGFR1 translocation, FGFR1
amplification, FGFR2 amplification, FGFR2 amplification, FGFR3 amplification, respectively.
BaF3/TEL-FGFR4, BaF3/TEL-KDR, BaF3/CCDC6-RET, BaF3/CCDC6-RET-V804M cells were
stably expressing constitutively active oncogenic version TEL-FGFR4, TEL-KDR, CCDC6-RET,
CCDC6-RET-V804M respectively.
Table 3. Kinase selectivity profiles and selected anti-proliferative activity for the related cell lines
displayed by 4 and Ponatinib.a
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O
CF3
HN
F3C
N
N
N
F3C
N
B
DDR1
inhibitor
N
N
N
O
O
N
H
N
H
CF3
N
C
O
N
A
N
N
Predominant
Src inhibitor
Bcr-AblT3151
N
N
H
H2N
N
inhibitor
N
H2N
N
N
Ponatinib
multi-targeted TKI
N
N
N
N
N
N
CF3
CF3
O
O
N
N
SAR
Study
N
N
O
N
N
N
H
H
Cl
4 Prodominant
FGFRs inhibitor
1 FGFRs inhibitor
Enzyme
Enzyme
FGFR1, IC50 = 1.2 0.4 nM
FGFR1, IC50 = 0.5 0.0 nM
±
±
KDR, IC50 = 48.7 0.1 nM
KDR, IC50 = 79.1 3.0 nM
±
±
Cell KG1, IC50 = 7.5 3.6 nM
Cell KG1, IC50 = 175.4 49.5 nM
±
±
Figure 1. Ponatinib analogues with different biological activities and the strategy for discovery of
novel selective FGFRs inhibitors.
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Figure 2. Compound 4 suppresses FGFR phosphorylation and downstream signaling in KG1
Cells (A) and SNU16 cells (B). Cells were treated with indicated concentrations of 4 for 2 hr and
analyzed by using immunoblotting.
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Figure 3. Plasma concentration vs time plots after oral administration of 10 mg/kg of compound
4. Plasma concentration was monitored at 0.5, 1, 2, 3, 5, 7, 9, 12 and 24 h after dosing.
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Figure 4. Compound 4 potently inhibits FGFR-dependent tumor growth through effects on tumor
cell growth as well as through antiangiogenic mechanisms. Inhibitory activity of 4 on tumor
growth in NCI-H1581 (A) and SNU16 (B) xenografts. 4 was administrated to the tumor-bearing
mice once a day for 10 or 18 d. Mean relative tumor volume ± SE on the final day is shown (n = 6
per group).
.
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Scheme 1. Synthetic route of designed compounds 1-4a.
aReagents and conditions: (i) (a) Oxalyl chloride, DMF (cat.), CH2Cl2, rt, 3 h; (b)
4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline, Et3N, CH2Cl2, rt, 5h; (ii) 7,
Pd(PPh3)4, CuI, Et3N, CsF, MeCN, MW: 90 oC, 15 min.
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