Quinolone antibiotic derivatives as new selective Axl kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.01.065

Axl is a new promising molecular target for antineoplastic therapies. A series of quinolone antibiotic derivatives were designed and synthesized as new selective Axl inhibitors. One of the most promising compound 8i bound tightly to Axl with a K_d value of 1.1 nM, and inhibited its kinase activity with an IC₅₀ value of 26 nM. The compound also significantly inhibited the phosphorylation of Axl and dose dependently inhibited cell invasion and migration in TGF-β1 induced MDA-MD-231 breast cancer cells. In addition, 8i demonstrated reasonable pharmacokinetic properties and exhibited extraordinary target selectivity over 468 kinases except for Flt3 (IC₅₀ = 50 nM)), with a S(10) and S(35) value of 0.022 and 0.42 at 1.0 μM, respectively. Compound 8i may serve as a new valuable lead compound for future anticancer drug discovery.

Full text of DOI:10.1016/j.ejmech.2019.01.065

Accepted Manuscript
Quinolone antibiotic derivatives as new selective Axl kinase inhibitors
Li Tan, Zhang Zhang, Donglin Gao, Shingpan Chan, Jinfeng Luo, Zheng-Chao Tu,
Zhi-Min Zhang, Ke Ding, Xiaomei Ren, Xiaoyun Lu
PII:
S0223-5234(19)30085-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.065
EJMECH 11072
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2018
Revised Date: 27 January 2019
Accepted Date: 27 January 2019
Please cite this article as: L. Tan, Z. Zhang, D. Gao, S. Chan, J. Luo, Z.-C. Tu, Z.-M. Zhang, K. Ding, X.
Ren, X. Lu, Quinolone antibiotic derivatives as new selective Axl kinase inhibitors, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.065.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Quinolone Antibiotic Derivatives as New Selective Axl Kinase
Inhibitors
Li Tana,#, Zhang Zhanga,#, Donglin Gaob,#, Shingpan Chana, Jinfeng Luob, Zheng-Chao Tub,
Zhi-Min Zhanga, Ke Dinga, * Xiaomei Rena,*, Xiaoyun Lu a,*
aInternational Cooperative Laboratory of Traditional Chinese Medicine Modernization and
Innovative Drug Development , Ministry of Education (MOE) of People’s Republic of China and
Guangzhou City Key Laboratory of Precision Chemical Drug Development, School of Pharmacy,
Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
bState Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou 510530, China
OH
N
O
N
F
H
F
N
O
O
O
O
8i
N
Axl IC50= 26 nM
Axl Kd = 1.1 nM
Mer Kd = 25 nM
Tyro3 Kd = 750 nM
N
Compound 8i bound to Axl with a Kd value of 1.1 nM, and inhibited its kinase activity with an
IC50 value of 26 nM. Further, 8i exhibited extraordinary target selectivity over 468 kinases, with a
S(10) and S(35) value of 0.022 and 0.42 at 1.0 µM, and significantly inhibited the phosphorylation
of Axl and dose dependently inhibited cell invasion and migration in TGF-β1 induced
MDA-MD-231 breast cancer cells.

ACCEPTED MANUSCRIPT
Quinolone Antibiotic Derivatives as New Selective Axl Kinase
Inhibitors
Li Tana,#, Zhang Zhanga,#, Donglin Gaob,#, Shingpan Chana, Jinfeng Luob, Zheng-Chao Tub,
Zhi-Min Zhanga, Ke Dinga,* Xiaomei Rena,*, Xiaoyun Lu a,*
a
International Cooperative Laboratory of Traditional Chinese Medicine Modernization and
Innovative Drug Development , Ministry of Education (MOE) of People’s Republic of China and
Guangzhou City Key Laboratory of Precision Chemical Drug Development, School of Pharmacy,
Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
b
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan
Avenue, Guangzhou 510530, China
Corresponding
authors:
Tel:
+86-20-85221523.
E-mail:
ding_ke@gibh.ac.cn;
ren_xiaomei@jnu.edu.cn; luxy2016@jnu.edu.cn.
Abstract
Axl is a new promising molecular target for antineoplastic therapies. A series of quinolone
antibiotic derivatives were designed and synthesized as new selective Axl inhibitors. One of the
most promising compound 8i bound tightly to Axl with a Kd value of 1.1 nM, and inhibited its
kinase activity with an IC50 value of 26 nM. The compound also significantly inhibited the
phosphorylation of Axl and dose dependently inhibited cell invasion and migration in TGF-β1
induced MDA-MD-231 breast cancer cells. In addition, 8i demonstrated reasonable
pharmacokinetic properties and exhibited extraordinary target selectivity over 468 kinases except
for Flt3 (IC50 = 50 nM)), with a S(10) and S(35) value of 0.022 and 0.42 at 1.0 µM, respectively.
Compound 8i may serve as a new valuable lead compound for future anticancer drug discovery.
Keyword: Axl, quinolones, selective inhibitor, breast cancer
1. Introduction
Axl is a tyrosine kinase that belongs to TAM subfamily which consists of three highly
conserved members (i.e., Tyro3, Axl and c-mer proto-oncogene tyrosine kinase (Mer)) [1]. Upon
binding with the endogenous ligand growth arrest-specific 6 (Gas6), Axl activates various signal
transduction cascades (e.g. PI3K/Akt, MEK/ERK and SOCS/STAT etc) to mediate a number of
fundamental cellular processes including cell growth and survival, proliferation, differentiation,

ACCEPTED MANUSCRIPT
cell adhesion and migration, invasion and angiogenesis etc [2]. Over-expression and/or
over-activation of Axl is frequently detected in a variety of human tumors, and closely correlated
to tumor aggressiveness and drug resistance [3-5]. Dysregulation of Axl correlates with poor
prognosis in various types of human cancer. For instance, increasing levels of Axl and Gas6 are
frequently found and Axl is one of most phosphorylated RTKs in over 50% of non-small cell lung
cancer cells (NSCLCs) [6-7]. Recent results also indicated that up-regulation of Axl tends to be
closely correlated to lymph node metastasis and poor survival of NSCLC patients and is one of the
important mechanisms mediated resistance to EGFR-targeted therapies [8]. The pathological role
of Axl in breast cancer has also been evidenced by its prominent role to trigger
epithelial-mesenchymal transition (EMT) and mediate the clinical resistance against lapatinib and
traztuzumab in Her2+ and/or ER+ breast cancer patients [9]. Most recent studies revealed that Axl
is closely associated with the expression of programmed death-ligand 1 (PD-L1) and inhibition of
Axl led to significant reduction of PD-L1 expression under basal conditions, suggesting its
promising potential in cancer immunotherapy [10]. Collectively, Axl becomes a new attractive
molecular target for anti-neoplastic drug discovery [11].
A number of well characterized small molecule kinase inhibitors, e.g. NPS-1034 (1) [12] and
Gilteritinib (2) [13] were reported to exhibit potent Axl inhibition activity (Figure 1), but few of
them was developed by using Axl as the primary target. Several classes of selective Axl kinase
inhibitors were also developed, among which TP0903 (3) [14] and R428 (BGB324, 4) [15]
(Figure 1) have been advanced into different stages of clinical investigation. Particularly, inhibitor
4, which is recognized as first selective Axl inhibitor with an IC50 value of 14.0 nM, was granted
as “orphan-drug” designation for treatment of acute myeloid leukemia (AML) in 2014 [16]. Most
recently, drug 4 also met the first efficacy endpoint in a phase II clinical trial in NSCLC patients
by combination with erlotinib [17]. However, Myers and our previous work independently
demonstrated that inhibitor 4 is actually also target other kinases (e.g. RET, VEGFR2 and Flt3) as
potently as Axl [18-19]. Thus, it is in urgent need to discovery a more selective Axl inhibitor to
explore the biological function of Axl. We have designed and synthesized a series of 4-oxo-1,
4-dihydroquinoline-3-carboxamides as novel selective Axl inhibitors (5, Figure 2) in which a
substituted quinazoline was utilized as a potential hinge region binding moiety and a quinolone
was adapted as a “dual hydrogen bond acceptor (DHBA) group [19]. Further improving the

ACCEPTED MANUSCRIPT
pharmacokinetic properties of 5 led to compound 6 with potent Axl inhibitory activity and ideal
pharmacokinetic property, which indicated that a hydrophilic group as larger as N-methyl
piperazine is tolerant in the DHBA region (Figure 2). This promising result inspired us that
quinolone antibiotics with similar hydrophilic group may be utilized as favourable substitutes for
the DHBA segment of 6. Quinolone represents the pharmacophore of an important class of FDA
approved antibiotics, including ciprofloxacin, enoxacin, sarafloxacin, and marbofloxacin etc [20].
Following James Black’s famous saying “the most fruitful basis of the discovery of a new drug is
to start with an old drug” [21], we introduced clinically used quinolone drugs into compound 6 as
a “DHBA group” and successfully obtained a new class of quinolone-based selective Axl
inhibitors (Figure 2).
Figure 1. Chemical structures of reported Axl inhibitors.
Figure 2. Design of the new potential selective Axl inhibitors

ACCEPTED MANUSCRIPT
2. Chemistry
The designed compounds were readily synthesized via a protocol outlined in Scheme 1.
Briefly, commercially available 4-chloro-6, 7-dimethoxyquinazoline (9) was treated with
4-amino-2-fluorophenol (10) to yield compound 11 by a nucleophilic substitution. Intermediate 11
was then condensed with various FDA approved quinolone antibiotics (7) to afford the final
products 8 in good yields
Scheme 1. Synthesis of compounds 8a-l. Reagents and conditions: (a) Sodium hydride (NaH),
N,N-dimethylformamide
(DMF),
0
°C,
75%;
(b)
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), ethyl
diisopropylamine (DIPEA), DMF, room temperature (rt), overnight, 75-90%.
3. Results and Discussion
Kinase inhibitory activities of the designed compounds against Axl were evaluated via a
well-established fluorescence resonance energy transfer (FRET)-based Z′-Lyte assays [22].
Inhibitor 4 was utilized as a positive control to validate the screening conditions. Under the
experimental conditions, 4 exhibited strong inhibitory potency against Axl with an IC50 value of
8.8 nM, which is similar to the reported data (Table 1) [15].
Table 1. In Vitro Axl Kinase Inhibitory Activities of Inhibitors 8a-l.a
Axl
Axl
Quinolone
antibiotics
Quinolone
antibiotics
Cpds
8a
Cpds
8h
(IC50, µM)
(IC50, µM)
0.020
0.230
0.054
0.026
8b
8i

ACCEPTED MANUSCRIPT
0.068
0.042
0.059
1.246
8c
8j
8d
8k
0.787
0.149
0.024
>10
8e
8f
8l
7
-
0.035
-
-
>10
8g
4
11
6
-
0.0088
0.011
a
Axl kinase inhibition was determined by using an FRET-based Z’-Lyte assay according to the
manufactory’s instructions (Invitrogen, Carsbad, USA). The compounds were incubated with the
kinase reaction mixture for 1.0-1.5 h before measurement. Reported data are means from 3
independent experiments in which the variation is less than 20%.
All of the designed compounds exhibited potent kinase activity against Axl with IC50 values
ranged from 0.020 µM to 1.25 µM (Table 1). It was also shown that the quinolone antibiotics with
mono-substituted piperazine group or free amino or hydroxyl group, such as ciprofloxacin (8a),
enoxacin (8d), sarafloxacin (8g), tosufloxacin (8h), nadifloxacin (8i) and the intermediate of
prulifloxacin (8l), displayed favourable Axl inhibitory activities (IC50 = 0.020-0.054 µM). While
the derivatives of quinolone antibiotics with N-substituted (8b, 8f and 8k) or steric hindrance
piperazine (8e) displayed less potency. We also determined the Axl inhibitory activity of
compound 11 and quinolone antibiotic 7i. The results showed that both 11 and 7i have no
inhibitory activity against Axl kinase, thus confirmed the contribution of quinolone block on Axl
inhibitory activity of designed compounds. Taken into consideration both the kinase inhibitory
potency and structural novelty, we chose 8i to do further evaluation.
The binding affinity of compound 8i with Axl protein was further determined using an
active-site-dependent competition binding assay (conducted by DiscoveRx Corporation, San
Diego, USA) [23]. It was shown that compound 8i tightly bound to Axl with a binding constant
(Kd) value of 1.1 nM. We also profiled the target selectivity of 8i against a panel of 468 kinases

ACCEPTED MANUSCRIPT
(including 403 non-mutated kinases) using the DiscoveRx screening platform at a concentration of
1.0 µM, which is approximately 909 times higher than its Kd value against Axl. The results
revealed that 8i displayed excellent target selectivity with S(1), S(10) and S(35) scores of 0.01,
0.022 and 0.042, respectively (Table S1). The major “off target” hits included colony stimulating
factor-1 receptor (CSF1R), discoidal domain receptor 1 (DDR1), DDR2, FMS-like tyrosine kinase
3 (Flt3), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (Kit), serine/threonine
kinase 10 (LOK) and nerve growth factor receptor A (TrkA) etc (Supporting Information). The
binding affinities (Kd) or kinase inhibitory activities (IC50) of compound 8i against these “off
targets” were further determined by using DiscoveRx’s platform or our in-house kinase assays
(Table 2). It was shown that compound 8i exhibited an approximately 25~1000-fold less potency
against the majority of the “off target” kinases, with the exception of Flt3 and LOK, which
displayed an IC50 value of 50 nM and a Kd value of 8 nM, respectively. The binding affinities of 8i
with the other TAM tyrosine kinase members (Tyro3 and Mer) were also determined. It was
shown that 8i exhibited a 25 fold less potency against Mer with a Kd value of 25 nM, while it was
much less potent to Tyro3 with a Kd value of 750 nM. These results collectively supported that
compound 8i achieved an obviously better target selectivity than the positive control 4 at the same
concentration (Figure 3).
Figure 3. KINOMEscan kinase selectivity profiles of 8i and 4 (R428).
Table 2. Binding affinities or kinase inhibition of compound 8i against a panel of “off target”
kinases.
Kinase
Kd or IC50 (µM)
Kinase
Kd or IC50 (µM)

ACCEPTED MANUSCRIPT
Axl
ABL
0.001a/0.026b
>10b
KIT
LOK
>10b
0.008a
0.025a
>10b
AKT1/2/3
CSF1R
DDR1
DDR2
EGFR
Flt3
>10b
MER
>10b
PDGFRA
PDGFRB
TRKA
TYRO3
ZAK
>10b
0.793b
0.125b
0.750a
>10b
>10b
>10b
0.050b
a
b
The binding affinities (Kd) were determined by using DiscoveRx’s platform. The kinase
inhibitory activities (IC50) were evaluated by using “in-house” kinase assays. Reported data are
means from 3 independent experiments in which the variation is less than 20%.
The Axl inhibitory effect of compound 8i on the activation of Axl and downstream Akt signal
was also validated by determining the phosphorylation level of Axl and Akt in MDA-MB-231
breast cancer cells which expressing a high level of Axl (Figure 4). The results revealed that 8i
dose-dependently inhibited the phosphorylation of Axl (pAxl (Tyr702)) and the downstream Akt
(pAkt(Thr308)) at a low concentration, while the total amount of corresponding proteins remained
unchanged as determined by western blot analysis in MDA-MB-231 cancer cells.
Figure 4. Compound 8i and 4 inhibits activation of Axl and its downstream signaling pathway in
MDA-MB-231 cells. The cells were treated with or without compound 8i or 4 for 4h at the
indicated concentrations, respectively.
It is well documented that Axl is closely involved in the tumor cell EMT transition process. In
breast cancer, Axl was identified as an EMT induced effector to participate in a positive feedback

ACCEPTED MANUSCRIPT
loop that sustains the malignant mesenchymal phenotype of breast tumor cells [24]. As
transforming growth factor beta receptor (TGF-β1) is a widely recognized EMT inducer [25],
therefore, the potential antitumor activity of 8i was further validated by determining the levels of
EMT associated proteins in MDA-MB-231 cells (Figure 5). As expected, TGF-β1 treatment
significantly induced EMT process as determined by the expression level of EMT markers (a
decrease of epithelial cell biomarker E-cadherin and increase of mesenchymal cell marker
N-cadherin). The results also revealed that treatment of 8i dose dependently increases the
expression of epithelial marker E-cadherin and decreases the expression of mesenchymal marker
N-cadherin in MDA-MB-231 cells as determined by western blot analysis, which suggested 8i can
reverse TGF-β1 induced expression of the EMT markers in a dose dependent manner.
It has been demonstrated that EMT mediated Axl up-regulation is essential for breast cancer
invasiveness and metastasis [24]. Thus, encouraged by its promising repressing ability on the
TGF-β1 induced EMT transition, we also determined the suppression activity of 8i on TGF-β1
induced tumor migration by a well-established transwell assay. It was shown that treatment of
compound 8i moderately inhibited the migrating process in MDA-MB-231 cells, suppressing the
TGF-β1 (10 ng/mL) induced cell migration by ~14.9%, and 69.7% at concentrations of 1.0 and
5.0 µM, respectively, compared with the untreated control (Figures 6A, 6B). We further
evaluated the inhibitory effect of 8i on the invasiveness of MDA-MB-231 breast cancer cells by
using a matrigel mediated Boyden chamber assay. It was shown that compound 8i
dose-dependently inhibited the invasiveness of MDA-MB-231 cancer cells. Treatment of 8i at
0.04, 0.2, 1.0 or 5.0 µM for 24 hours inhibited cancer cell invasion by ~17.4%, ~34.4%, ~73.8%
and ~90.3%, respectively (compared to the TGF-β1 (10 ng/mL) treatment; Figures 6A and 6C).
These results collectively suggested the promising potential of compound 8i to serve as a lead
compound for further drug discovery.

ACCEPTED MANUSCRIPT
Figure 5. Compound 8i reverses the expression of the EMT markers induced by TGF-β1 in
MDA-MB-231 cells as determined by western blot analysis.
Figure 6. Compound 8i suppresses migration and invasion of MDA-MB-231 cells. (A)
Compound 8i suppresses migration and invasion induced by TGF-β1 (10 ng/mL) in
MDA-MB-231 cells. (B, C) Quantity analysis of migration (B) and invasion (C). The results are
presented as the mean ± standard deviation, *P<0.05 (compare with TGF-β1 treatment).
Given the promising Axl inhibitory activity in vitro, we further investigated preliminary in vivo
pharmacological properties of 8i in Sprague-Dawley (SD) rats (Table 3). Plasma levels of the
compounds were monitored after a single oral dose of 25 mg/kg or an i.v. dose of 2.5 mg/kg. The
results showed that, 8i exhibited reasonable pharmacokinetic (PK) properties with an area under
concentration−time curve (AUC(0-∞)) value of 25944.7 µg/L*h and a T1/2 value of 5.68 h at an oral
dose of 25 mg/kg. The Cmax (2386.9 µg/L=3.6 µM) occurred at 4.0 h postdose, which was 138
times higher than its IC50 value against Axl kinase.
Table 3. Pharmacokinetic profile of compound 8i in SD rats
8i
oral (25 mg/kg)
25944.7
i.v. (2.5 mg/kg)
20680.6
AUC (0-∞) (µg/L*h)
Cmax (µg/L)
2386.9
4358.2

ACCEPTED MANUSCRIPT
T1/2 (h)
Tmax (h)
CLz (L/h/kg)
BA (%)
5.68
4.0
4.26
-
--
0.12
--
12.5%
Moreover, 8i was well tolerated and there was no animal death or obvious body weight change
and obvious toxicity to liver and kidney after the mice received 200 mg/kg, 400 mg/kg or 800
mg/kg administration of 8i (Tables 4 and Figure 7). These results suggested that, 8i may serve as a
start point for further drug discovery.
Table 4. 8i has no effects on survival and weight of ICR mice
Number
0d
Weight(g)
Dose(mg/Kg)
7d
6
0d
7d
0
6
6
6
6
6
6
6
23.8±0.9
23.5±1.2
23.9±1.2
23.3±0.9
23.7±1.1
23.9±1.2
24.1±1.3
27.9±1.1
27.7±1.5
27.6±1.6
25.8±1.2
26.1±1.3
25.6±1.5
25.2±1.6
50
6
100
200
400
600
800
6
6
6
6
6
Figure 7. HE staining showed that 8i had no obviously toxicity to liver and kidney. ICR mice
were singly treated with vehicle or 8i, then the tissues of liver and kidney were harvested after 7
days for HE staining.
4. Conclusion
In summary, a series of quinolone antibiotic derivatives were designed and synthesized as novel
selective Axl inhibitors. One of the most promising compounds 8i tightly bound with Axl and
suppressed Axl kinase activity with a Kd value of 1.1 nM and an IC50 value of 26 nM, respectively,

ACCEPTED MANUSCRIPT
but it is significantly less potent for a panel of 403 nonmutated kinases evaluated at 1.0 µM (which
is approximately 909 times higher than its Kd value against Axl) with the exception of Flt3. Thus,
to the best of our knowledge, 8i represents one of the most selective Axl inhibitors to date. 8i also
demonstrated reasonable PK properties and potently inhibit the phosphorylation levels of Axl.
Furthermore, 8i dose dependently blocked TGF-β1 induced EMT and suppressed tumor invasion
and migration in MDA-MB-231 breast cancer cells. Its strong Axl binding affinity and excellent
target selectivity make compound 8i not only a promising lead compound for drug discovery but
also a valuable research probe for further biological investigation of its target.
5. Experimental Section
5.1 General methods for chemistry
All reagents and solvents were purchased from commercial sources without further purification.
Flash chromatography was performed using 300 mesh silica gel. All reactions were monitored by
1
TLC using silica gel plates with fluorescence F254 and UV light visualization. H NMR spectra
was recorded on a Bruker AV-400 spectrometer at 400 MHz or a Bruker AV-500 spectrometer at
500 MHz. 13C NMR spectra was recorded on a Bruker AV-500 spectrometer at 125 MHz.
Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in
parts per million (ppm) units relative to an internal standard (TMS). Low resolution ESI-MS were
recorded on an Agilent 1200 HPLC-MSD mass spectrometer and high resolution ESI-MS on an
Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Purity of compounds was
determined by reverse-phase high performance liquid chromatography [HPLC, Dionex Summit
HPLC (Column: Diamonsil C18, 5.0 µm, 4.6 × 250 mm (Dikma Technologies); detector:
PDA-100 photodiode array; injector: ASI-100 autoinjector; pump: p-680A)] to be >95%. A flow
rate of 1.0 mL/min was used with mobile phase of MeOH in H2O with 0.1% modifier (ammonia,
v/v).
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6-ethyl-1,2-dimethyl-7-(4-methylp
iperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (6)
1
Compound 6 was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
DMSO-d6) δ 11.19 (s, 1H), 8.57 (s, 1H), 8.08 (s, 1H), 7.95 (dd, J = 13.3, 2.2 Hz, 1H), 7.59 (s, 1H),
7.49 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.42 (s, 1H), 4.00 (s, 3H), 3.99 (s, 3H), 3.82 (s,
3H), 3.03 (m, 4H), 2.74 (q, J = 7.5 Hz, 2H), 2.66 (s, 3H), 2.54 (m, 4H), 2.27 (s, 3H), 1.27 (t, J =

ACCEPTED MANUSCRIPT
7.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.67, 166.23, 164.58, 156.42, 156.11, 152.71,
152.60, 150.77, 149.45, 140.62, 138.92 (d, J = 10.9 Hz, 1C), 135.14, 134.80 (d, J = 13.0 Hz, 1C),
125.80, 124.80, 121.67, 118.29, 115.98, 109.62, 108.06, 107.28, 106.50, 101.06, 56.68, 56.56,
55.47, 52.10, 46.28, 35.62, 23.09, 19.57, 14.94. HRMS (ESI) for C32H28F2N6O5S [M+H]+,
calcd: 641.2854, found: 641.2882. HPLC analysis: MeOH-H2O (85:15), 10.76 min, 97.64%
purity.
Cyclopropyl-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6-fluoro-4-oxo-7-(pip
erazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (8a)
A mixture of 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (11, 100mg, 1
mmol),7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquino
line-3-carboxylic acid (7a) (150 mg, 1.1 mmol), HATU (180 mg, 1.5 mmol), and DIPEA
(0.16 mL, 3 mmol) in DMF (20 mL) was stirred at rt overnight. Next, water was added to
the mixture, and the precipitates were filtrated. The filter cake was washed with water,
diluted with DCM, and concentrated in vacuo. The resulting residue was purified by flash
chromatography on silica gel to get the product as a white solid. The resulting compound
(150 mg) was dissolved in 10 mL DCM, 5 mL F3CCOOH was added slowly under ice bath, then
stirred at rt overnight. The organic solvent was removed under vacuum, and the resulting residue
was washed by saturated NaOH, then water was added to the residue. The precipitate was filtered
1
and washed with EA and acetone to give the white product (100 mg, 78%). H NMR (500 MHz,
CDCl3) δ 12.50 (s, 1H), 8.89 (s, 1H), 8.62 (s, 1H), 8.05 (d, J = 13.5 Hz, 1H), 8.00 (dd, J1 = 2.0 Hz,
J2 = 12.0 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.34 (m, 2H), 7.26 (s, 1H), 4.07 (s, 3H),
4.06 (s, 3H), 3.52 (m, 1H), 3.28 (t, J = 4.5 Hz, 4H), 3.10 (t, J = 4.5 Hz, 4H), 1.37 (m, 2H), 1.22 (m,
2H). 13C NMR (125 MHz, CDCl3) δ 175.57, 164.87, 163.18, 155.96, 154.26 (d, J = 246.3 Hz, 1C),
153.66 (d, J = 248.5 Hz, 1C), 152.89, 150.29, 149.44, 147.04, 145.69 (d, J = 10.1 Hz, 1C), 138.61,
137.80 (d, J = 9.8 Hz, 1C), 135.32 (d, J = 12.6 Hz, 1C), 123.82, 121.43 (d, J = 7.3 Hz, 1C), 116.09
(d, J = 3.0 Hz, 1C), 112.68 (d, J = 23.3 Hz, 1C), 110.98, 110.31, 109.28 (d, J = 23.0 Hz, 1C),
106.83, 104.67 (d, J = 2.8 Hz, 1C), 101.10, 56.41, 56.39, 51.17, 51.14, 45.97, 35.01, 8.24. HRMS
(ESI) for C33H30F2N6O5 [M+H]+, calcd: 629.2319, found: 629.2306. HPLC analysis: MeOH-H2O
(85:15), 17.78 min, 97.22% purity.

ACCEPTED MANUSCRIPT
1-cyclopropyl-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-7-(4-ethylpiperazin
-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamide (8b)
1
The compound was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
CDCl3) δ 12.50 (s, 1H), 8.89 (s, 1H), 8.63 (s, 1H), 8.06 (d, J = 13.5 Hz, 1H), 8.00 (dd, J1 = 2.5 Hz,
J2 = 12.5 Hz, 1H), 7.58 (s, 1H), 7.46 (dd, J1 = 0.5 Hz, J2 = 8.5 Hz, 1H), 7.36 (m, 1H), 7.33(s, 1H)
7.28 (m, 1H), 4.08 (s, 3H), 4.06 (s, 3H), 3.52 (m, 1H), 3.35 (t, J = 4.5 Hz, 4H), 2.69 (s, 4H), 2.52
(q, J = 7.0 Hz, 2H), 1.36 (m, 2H), 1.21 (m, 2H), 1.15 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 175.55, 164.86, 163.15, 155.94, 154.26 (d, J = 246.1 Hz, 1C), 153.64 (d, J = 248.6 Hz,
1C), 152.92, 150.28, 149.46,147.03, 145.33 (d, J = 10.4 Hz, 1C), 138.59, 137.82 (d, J = 9.9 Hz,
1C), 135.32 (d, J = 13.0 Hz, 1C), 123.80, 121.47 (d, J = 7.3 Hz, 1C),116.07 (d, J = 2.6 Hz, 1C),
112.66 (d, J = 23.1 Hz, 1C), 111.01, 110.32, 109.26 (d, J = 23.3 Hz, 1C), 106.86, 104.72, 104.70,
101.10, 56.40, 56.38, 52.53, 52.31, 50.00, 49.96, 34.99, 11.99, 8.23. HRMS (ESI) for
C35H34F2N6O5 [M+H]+, calcd: 657.2632, found: 657.2625. HPLC analysis: MeOH-H2O
(85:15), 20.53 min, 98.36% purity
.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1-ethyl-6,8-difluoro-7-(3-methylpi
perazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (8c)
A mixture of 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (11, 315 mg, 1.0
mmol),
7-(4-(tert-butoxycarbonyl)-3-methylpiperazin-1-yl)-1-ethyl-6,8-difluoro-4-oxo-1,4-
dihydroquinoline -3-carboxylic acid (7c, 490 mg, 1.0 mmol), HATU (574 mg, 1.5 mmol),
and DIPEA (0.5 mL, 3 mmol) in DMF (20 mL) was stirred at rt overnight. Next, water
was added to the mixture, and the precipitates were filtrated. The filter cake was washed
with water, diluted with DCM, and concentrated in vacuo. The product was then dissolved
in 10 mL DCM, 5 mL F3CCOOH was added slowly under ice bath and stirred at rt overnight. The
organic solvent was removed under vacuum, and the resulting residue was alkailized by saturated
NaOH. The precipitate was filtered and washed with EA and acetone to give the white product
(485 mg, 75%). 1H NMR (400 MHz, d6-DMSO) 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H),
8.93 (s, 1H), 8.56 (s, 1H), 7.99 (dd, J = 12.0, 1.6 Hz, 1H), 7.94 (dd, J = 12.0, 1.6 Hz, 1H), 7.56 (s,
1H), 7.51-7.45 (m, 2H), 7.40 (s, 1H), 4.62-4.57 (m, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.58 (m, 2H),
3.49-3.40 (m, 3H), 3.29-3.17 (m, 2H), 1.48-1.45 (m, 3H), 1.26 (d, J = 6.4 Hz, 3H). 13C NMR (125
MHz, d6-DMSO) δ 173.87, 164.49, 162.76, 158.70, 158.45, 156.43, 154.09 (d, J = 243.4 Hz, 1C),

ACCEPTED MANUSCRIPT
153.85, 152.57, 151.17, 150.76, 149.44, 137.83 (d, J = 10.3 Hz, 1C), 135.26 (d, J = 13.1 Hz, 1 C),
132.29 (d, J = 13.6 Hz, 1C), 127.141 (d, J = 6.9 Hz, 1C), 125.11, 123.73 (d, J = 8.0 Hz, 1C),
116.50 (d, J = 11.9 Hz, 1C), 110.23, 109.59, 108.53 (d, J = 22.9 Hz, 1C), 107.56 (d, J = 22.9 Hz,
1C), 101.02, 56.69, 56.53, 53.93, 53.77, 51.46, 47.40, 43.59, 16.45 (d, J = 4.9 Hz, 1C), 15.83.
HRMS (ESI) for C33H31F3N6O5 [M+H]+, calcd: 649.2333, found: 649.2361. HPLC
analysis: MeCN-H2O (70:30), 6.81 min, 97.43% purity.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-1-ethyl-6-fluoro-4-oxo-7-(piperazi
n-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (8d)
The compound was prepared by following a procedure similar to that of 8a. 1H NMR (500 MHz,
CDCl3) δ 12.51 (s, 1H), 8.79 (s, 1H), 8.63 (s, 1H), 8.13 (d, J = 13.5 Hz, 1H), 7.80 (dd, J1 = 2.0 Hz,
J2 = 12.0 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J = 8.5 Hz, 1 H), 7.33 (s, 1H), 7.28 (m, 1H), 4.41 (q, J =
7.0 Hz, 2H), 4.08 (s, 3H), 4.06 (s, 3H), 3.82 (t, J = 4.5 Hz, 4H), 3.04 (t, J = 4.5 Hz, 4H), 1.82 (s,
1H), 1.51 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 175.65, 164.86, 163.05, 155.95,
154.26 (d, J = 245.9 Hz, 1C), 152.91, 150.37 (d, J = 9.0 Hz, 1C), 150.28, 149.46, 147.36 (d, J =
257.0 Hz, 1C), 146.02, 144.65, 137.80 (d, J = 9.6 Hz, 1C), 135.34 (d, J = 13.1 Hz, 1C), 123.81,
120.66 (d, J = 21.9 Hz, 1C), 116.10 (d, J = 2.9 Hz, 1C), 115.11 (d, J = 3.0 Hz, 1C), 112.20,
110.32, 109.29 (d, J = 23.3 Hz, 1C), 106.86, 101.10, 56.40, 56.38, 48.38, 48.32, 47.44, 46.11,
15.03. HRMS (ESI) for C31H29F2N7O5 [M+H]+, calcd: 618.2271, found: 618.2264. HPLC
analysis: MeOH-H2O (85:15), 16.99 min, 96.81% purity.
5-amino-1-cyclopropyl-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)
-7-((3S,5R)-3,5-dimethylpiperazin-1-yl)-6,8-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxam
ide (8e)
1
The compound was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
d6-DMSO) δ 12.19 (s, 1H), 8.58 (s, 1H), 8.53 (s, 1H), 7.96 (d, J = 12.5 Hz, 1H), 7.55 (s, 1H),
7.44-7.37 (m, 5H), 3.98 (s, 3H), 3.97 (s, 3H), 3.34 (d, J = 12.0 Hz, 2H), 3.10 (s, 2H), 2.92 (t, J =
11.5 Hz, 2H), 1.90 (s, 2H), 1.13-1.07 (m, 10H). 13C NMR (125 MHz, d6-DMSO) δ 178.94,
172.67, 164.49, 162.77, 156.42, 154.03 (d, J = 243.3 Hz, 1C), 152.56, 150.73, 149.67, 149.38,
139.76 (d, J = 230.5 Hz, 1C), 137.85 (d, J = 9.75 Hz, 1C), 136.92 (d, J = 13.3 Hz, 1C), 135.11 (d,
J = 13.1 Hz, 1C), 133.20, 128.35, 124.96, 116.46, 109.58, 109.15, 108.50 (d, J = 23.5 Hz, 1C),
107.28, 107.15, 101.00, 56.66, 56.50, 56.05, 21.59, 17.94, 9.01, 8.95. HRMS (ESI) for

ACCEPTED MANUSCRIPT
C35H34F3N7O5 [M+H]+, calcd: 690.2646, found: 690.2622. HPLC analysis: MeOH-H2O
(90:10), 10.22 min, 95.56% purity.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6,8-difluoro-1-(2-fluoroethyl)-7-(4
-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (8f)
1
The compound was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
d6-DMSO) δ 12.35 (s, 1H), 8.84 (s, 1H), 8.57 (s, 1H), 8.00 (d, J = 12.5 Hz, 1H), 7.88 (d, J = 12.0
Hz, 1H), 7.57 (s, 1H), 7.48 (m, 2H), 7.41 (s, 1H), 4.95-4.86 (m, 4H), 4.00 (s, 3H), 3.99 (s, 3H),
3.33 (s, 4H), 2.45 (s, 4H), 2.23 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 174.61, 164.83, 162.44,
155.97, 155.01 (d, J = 249.0 Hz, 1C), 154.27 (d, J = 246.3 Hz, 1C), 152.88, 150.87, 150.30,
149.47, 145.94 (dd, J1 = 6.4 Hz, J2 = 244.4 Hz, 1C), 137.54 (d, J = 9.5 Hz, 1C), 135.51 (d, J =
13.0 Hz, 1C), 133.82 (t, J = 13.8 Hz, 1C), 126.58 (d, J = 5.2 Hz, 1C), 123.87, 122.87 (d, J = 7.9
Hz, 1C), 116.12 (d, J = 2.8 Hz, 1C), 111.03, 110.30, 109.33 (d, J = 23.1 Hz, 1C), 108.81 (d, J =
22.9 Hz, 1C), 106.86, 101.08, 81.25 (dd, J1 = 5.4 Hz, J2 = 172.3 Hz, 1C), 58.30 (dd, J1 = 14.9 Hz,
J2 = 20.4 Hz, 1C), 56.40, 56.38, 55.52, 50.89(t, J = 4.0 Hz, 2C), 46.34. HRMS (ESI) for
C33H30F4N6O5 [M+H]+, calcd: 667.2287, found: 667.2290. HPLC analysis: MeOH-H2O (85:15),
14.66 min, 99.56% purity.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6-fluoro-1-(4-fluorophenyl)-4-oxo-
7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (8g)
The compound was prepared by following a procedure similar to that of 8a. 1H NMR (500 MHz,
d6-DMSO) δ 12.51 (s, 1H), 8.61 (s, 1H), 8.54 (s, 1H), 7.96 (m, 1H), 7.92 (d, J = 13.0 Hz, 1H),
7.81 (m, 2H), 7.55 (m, 3H), 7.45 (m, 2H), 7.39 (s, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 2.91 (s, 4H),
2.77 (s, 4H). 13C NMR (125 MHz, d6-DMSO) δ 179.96, 169.27, 167.73, 167.65 (d, J = 246.1 Hz,
1C), 161.20, 158.84 (d, J = 243.5 Hz, 1C), 158.03 (d, J = 247.0 Hz, 1C), 157.33 (d, J = 24.4 Hz,
1C), 155.53, 154.21, 152.94 (d, J = 23.1 Hz, 1C), 150.41(d, J = 10.4 Hz, 1C), 143.81, 142.61 (d, J
= 9.8 Hz, 1C), 141.87, 140.00 (d, J = 12.9 Hz, 1C), 135.11, 129.81, 125.13 (d, J = 7.3 Hz, 1C),
122.58 (d, J = 23.4 Hz, 1C), 121.18, 116.58, 115.24, 114.37, 113.28, 112.03, 111.17, 105.79 (d, J
=12.6 Hz, 1C), 61.43, 61.27, 55.73, 50.46. HRMS (ESI) for C36H29F3N6O5 [M+H]+, calcd:
683.2224, found: 683.2226. HPLC analysis: MeOH-H2O (85:15), 13.93 min, 95.12%
purity.

ACCEPTED MANUSCRIPT
7-(3-aminopyrrolidin-1-yl)-1-(2,4-difluorophenyl)-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)
-3-fluorophenyl)-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxamide (8h)
The compound was prepared by following a procedure similar to that of 8a. 1H NMR (500 MHz,
d6-DMSO) δ 12.52 (s, 1H), 8.75 (s, 1H), 8.57 (s, 1H), 8.06 (d, J = 13.0 Hz, 1H), 7.98 (d, J = 12.5
Hz, 1H), 7.85 (m, 1H), 7.59 (m, 2H), 7.47 (d, J = 12.5 Hz, 2 H), 7.41 (s, 1H), 7.36 (t, J = 8.0 Hz,
1H), 4.62 (s, 2H), 4.00 (s, 3H), 3.99 (s, 3H), 3.62 (m, 5H), 2.02 (s, 1H), 1.76 (s, 1H). 13C NMR
(125 MHz, CDCl3) δ 176.04 (d, J = 1.6 Hz, 1C), 164.78, 163.03 (dd, J1 = 10.9 Hz, J2 = 251.1 Hz,
1C), 162.59, 157.83 (dd, J1 = 12.4 Hz, J2 = 253.9 Hz, 1C), 155.96, 154.19 (d, J = 246.1 Hz, 1C),
152.77, 150.29, 149.38, 148.92 (d, J = 12.6 Hz, 1C), 146.39 (d, J = 256.3 Hz, 1C), 146.15, 145.98,
137.62 (d, J = 9.6 Hz, 1C), 135.36 (d, J = 13.3 Hz, 1C), 130.00 (d, J = 10.1 Hz, 1C), 124.72 (dd,
J1 = 4.3 Hz, J2 = 13.3 Hz, 1C), 123.82, 118.45 (d, J = 20.6 Hz, 1C), 116.07 (d, J = 2.6 Hz, 1C),
112.69, 112.41 (d, J = 2.5 Hz, 1C), 112.01 (dd, J1 = 3.4 Hz, J2 = 22.5 Hz, 1C), 110.24, 109.20 (d,
J = 23.0 Hz, 1C), 106.76, 104.94 (t, J = 23.5 Hz, 1C), 101.03, 56.61, 56.57, 56.38, 56.35, 46.92,
30.32. HRMS (ESI) for C35H27F4N7O5 [M+H]+, calcd: 702.2083, found: 702.2074. HPLC
analysis: MeOH-H2O (85:15), 12.42 min, 99.81% purity.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-9-fluoro-8-(4-hydroxypiperidin-1-
yl)-5-methyl-1-oxo-1,5,6,7-tetrahydropyrido[3,2,1-ij]quinoline-2-carboxamide.（8i）
A mixture of 4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluoroaniline (11, 315 mg, 1
mmol), Nadifloxacin (433 mg, 1.2 mmol), HATU (570 mg, 1.5 mmol), and DIEA (0.5
mL, 3 mmol) in DMF (20 mL) was stirred at rt overnight. Next, water was added to the
mixture, and the precipitates was filtrated. The filter cake was washed with water, diluted
with DCM, and concentrated in vacuo. The resulting residue was purified by flash
chromatography on silica gel to get the product as a white solid (460 mg, 70 %). 1H NMR
(500 MHz, d6-DMSO)
δ 12.66 (s, 1H), 8.95 (s, 1H), 8.57 (s, 1H), 8.02 (d, J = 13.0 Hz,
1H), 7.89 (d, = 13.0 Hz, 1H), 7.58 (s, 1H), 7.48 (m, 2H), 7.41 (s, 1H), 4.87 (s, 1H), 4.75
J
(s, 1H), 4.00 (s, 3H), 3.99 (s, 3H), 3.68 (s, 1H), 3.20-3.13 (m, 3H), 2.96-2.92 (m, 2H),
=6.5 Hz, 3H). 13C
2.16-2.06 (m, 2H), 1.90-1.83 (m, 2H), 1.63-1.50 (m, 2H), 1.43 (d,
J
NMR (125 MHz, d6-DMSO) δ 175.04, 164.47, 163.30, 157.47 (d, J = 247.3 Hz, 1C), 156.41,
154.04 (d, J = 243.5 Hz, 1C), 152.51, 150.74, 149.41, 147.12, 142.33 (d, J = 13.6 Hz, 1C), 138.01

ACCEPTED MANUSCRIPT
(d, J = 9.4 Hz, 1C), 135.06 (d, J = 12.9 Hz, 1C), 133.57, 126.65, 124.92, 124.13 (d, J = 8.1 Hz,
1C), 116.22, 109.79, 109.60, 109.56, 108.37 (d, J = 23.1 Hz, 1C), 107.21, 101.03, 57.09, 56.60,
56.46, 49.08, 35.86, 35.50, 25.58, 20.05, 18.94. HRMS (ESI) for C35H33F2N5O6 [M+H]+, calcd:
658.2424, found: 658.2416. HPLC analysis: MeOH-H2O (85:15), 10.63 min, 99.51% purity. MW:
657.66, cLogP: 3.314, tPSA: 125.29.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-9-fluoro-3-methyl-10-(4-methylpi
perazin-1-yl)-7-oxo-3,7-dihydro-2H-[1,3,4]oxadiazino[6,5,4-ij] quinolone-6-carboxamide (8j).
1
The compound was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
d6-DMSO) δ 12.47 (s, 1H), 8.73 (s, 1H), 8.56 (s, 1H), 7.99 (m, 1H), 7.57 (m, 2H), 7.45 (m, 2H),
7.41 (s, 1H), 5.32 (s, 2H), 4.00 (s, 3H), 3.99 (s, 3H), 3.33 (m, 4H), 3.03 (s, 3H),2.45 (s, 4H), 2.24
(s, 3H). 13C NMR (125 MHz, d6-DMSO) δ 174.54, 164.51, 162.78, 156.44, 155.69 (d, J = 244.5
Hz, 1C), 154.08 (d, J = 243.5 Hz, 1C), 152.59, 150.77, 149.46, 144.71, 138.66 (d, J = 7.4 Hz, 1C),
137.88 (d, J = 10.0 Hz, 1C), 135.20 (d, J = 12.8 Hz, 1C), 131.45 (d, J = 14.1 Hz, 1C), 125.07,
123.89, 121.49 (d, J = 8.1 Hz, 1C), 116.39, 109.60, 109.53, 108.48 (d, J = 22.8 Hz, 1C), 107.27,
104.45 (d, J = 23.4 Hz, 1C), 101.02, 82.64, 56.68, 56.52, 55.72, 50.54, 46.47, 43.22. HRMS (ESI)
for C33H31F2N7O6 [M+H]+, calcd: 660.2377, found: 660.2373. HPLC analysis:
MeOH-H2O (85:15), 15.48 min, 99.89% purity.
(S)-N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-9-fluoro-3-methyl-10-(4-methy
lpiperazin-1-yl)-7-oxo-3,7-dihydro-2H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxamide (8k)
1
The compound was prepared by following a procedure similar to that of 8i. H NMR (500 MHz,
d6-DMSO) δ 12.59 (s, 1H), 8.95 (s, 1H), 8.57 (s, 1H), 8.00 (m, 1H), 7.58 (m, 2H), 7.46 (m, 2H),
7.40 (s, 1 H), 4.92 (m, 1H), 4.57 (d, J = 10.5 Hz, 1H), 4.38 (d, J = 9.5 Hz, 1H), 4.00 (s, 3H), 3.99
(s, 3H), 3.33 (m, 4H), 2.63 (s, 4H), 2.37 (s, 3H), 1.47 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz,
d6-DMSO) δ 174.84, 164.52, 163.30, 156.43, 155.78 (d, J = 244.0 Hz, 1C), 154.09 (d, J = 243.5
Hz, 1C), 152.59, 150.76, 149.45, 146.00, 140.79 (d, J = 7.1 Hz, 1C), 138.02 (d, J = 10.1 Hz, 1C),
135.11 (d, J = 12.9 Hz, 1C), 131.39 (d, J = 14.8 Hz, 1C), 125.06, 124.81, 122.07 (d, J = 8.6 Hz,
1C)116.36, 109.84, 109.60, 108.46 (d, J = 22.9 Hz, 1C), 107.27, 103.97 (d, J = 23.3 Hz, 1C),
101.02, 68.64, 56.68, 56.52, 55.39, 54.85, 50.01, 45.80, 18.36. HRMS (ESI) for C34H32F2N6O6
[M+H]+, calcd: 659.2424, found: 659.2425. HPLC analysis: MeOH-H2O (85:15), 15.81

ACCEPTED MANUSCRIPT
min, 98.54% purity.
N-(4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl)-6-fluoro-1-methyl-4-oxo-7-(pipera
zin-1-yl)-1,4-dihydro-[1,3]thiazeto[3,2-a]quinoline-3-carboxamide (8l)
The compound was prepared by following a procedure similar to that of 8a. 1H NMR (400 MHz,
DMSO-d6) δ 12.12 (s, 1H), 8.57 (s, 1H), 7.98-7.91 (m, 1H), 7.82 (d, J = 13.9 Hz, 1H), 7.58 (s,
1H), 7.44 (dd, J = 3.9, 1.6 Hz, 2H), 7.42 (s, 1H), 6.86 (d, J = 7.3 Hz, 1H), 6.36 (q, J = 6.1 Hz, 1H),
4.00 (s, 3H), 3.99 (s, 3H), 3.17 (t, J = 4.6 Hz, 4H), 2.94-2.82 (m, 4H), 2.11 (d, J = 6.1 Hz, 3H). 13C
NMR (125 MHz, CDCl3) δ 174.25, 164.81, 163.82, 162.12, 154.24 (d, J = 245.9 Hz, 1C), 153.62,
152.87, 151.65, 150.267, 149.44, 145.60 (d, J = 10.6 Hz, 1C), 137.38 (d, J = 10.0 Hz, 1C), 135.56,
135.35 (d, J = 13.3 Hz, 1C), 123.79 (d, J = 1.8 Hz, 1C), 120.75 (d, J = 7.4 Hz, 1C), 115.82 (d, J =
3.1 Hz, 1C), 114.05 (d, J = 23.5 Hz, 1C), 110.05 (d, J = 23.5 Hz, 1C), 106.84, 105.23, 101.07,
99.89, 69.37 , 56.37, 56.36, 51.03, 51.00, 45.87, 21.28, 1.00. HRMS (ESI) for C32H28F2N6O5S
[M+H]+, calcd: 647.1883, found: 647.1882. HPLC analysis: MeOH-H2O (80:20), 15.66
min, 96.78% purity.
5.2 Cell Culture and Reagents
MDA-MB-231 cells were purchased from American type culture collection (ATCC).
MDA-MB-231 were maintained in Roswell Park Memorial Institute (RPMI)-1640 supplemented
with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 50 mg/mL streptomycin, and 2 mmol/L
glutamine in a humidified CO2 incubator at 37 °C. All cells were passaged for less than 3 months
before renewal from frozen, early-passage stocks obtained from the indicated sources.
The compound was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a concentration
of 10 mmol/L and stored at -20 °C. Primary antibodies against Axl (8661), phosphor-Axl (Tyr702
,
5724), E-cadherin (3195), N-cadherin (13116), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, 2118) and anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP)-linked
secondary antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA).
Primary antibodies against v-akt murine thymoma viral oncogene homolog (AKT, SC8312),
phosphor-AKT (SC16646R, SC7985R) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA)
5.3 Animals
Male Sprague Dawley mice were obtained from the Animal Center of Jinan University. Animal

ACCEPTED MANUSCRIPT
experiments were performed in accordance with the Guide for the Care and Use of Laboratory
Animals. All animal experimental procedures were approved by the Jinan University Animal
Policy and Welfare Committee.
5.4 In Vitro Enzymatic Activity Assay
Axl and the Z’-Lyte Kinase Assay Kit were purchased from Invitrogen. The experiments were
performed according to the instructions of the manufacturer. The concentrations of kinases were
determined by optimization experiments and the respective concentration was: Axl (PV3971,
Invitrogen) 0.22 µg/µL. First, the compounds were diluted by three-fold from 10-10 M to 1x10-4 M
in DMSO and a 400 µM of compound solution was prepared (4 µL compound dissolved in 96 µL
water). Second, a 100 µM ATP solution in 1.33×Kinase Buffer was prepared. Third, a
kinase/peptide mixture containing 2×kinase and 4 µM Tyr6 peptide (PV4122; Invitrogen) was
prepared right before use. Kinase/peptide mixture was prepared by diluting Z’-LYTE Tyr6 peptide
(PV4122; Invitrogen) and kinase in 1×Kinase Buffer, and 0.2 µM Tyr6 phospho-peptide solution
was made by adding Z’-LYTE Tyr6 phospho-peptide to 1×Kinase Buffer. The final 10 µL
reaction consists of 0.002 ng of Axl, 2 µM Tyr6 peptide in 1×kinase buffer. For each assay, 10 µL
kinase reactions were made at first (including 2.5 µL compound solution, 5 µL Kinase/Peptide
Mixture, and 2.5 µL ATP solution). Mixed the plate thoroughly and incubated for one hour at
room temperature. Then 5 µL development solution was added to each well and the plate was
incubated for 1.0 h at room temperature; the nonphosphopeptides were cleaved at this time. In the
end, 5 µL stop reagent was loaded to stop the reaction. For the control setting, 5 µL
phospho-peptide solution instead of kinase/peptide mixture was used as 100% phosphorylation
control. 2.5 µL 1.33×Kinase Buffer instead of ATP solution was used as 100% inhibition control,
and 2.5 µL 4% DMSO instead of compound solution was used as the 0% inhibitor control. The
plate was measured on an EnVision Multilabel Reader (Perkin-Elmer). Curve fitting and data
presentations were performed using Graph Pad Prism, version 5.0. Every experiment was repeated
at least 2 times.
5.5 Western Blot Analysis
The western blot analysis was carried out by following the protocol described before. Briefly,
after the indicated treatment, cell lysates were collected dissolving cells in 1×SDS sample lysis
buffer (CST recommended). After being sonicated and boiled, the supernatant of cell lysate were

ACCEPTED MANUSCRIPT
used for western blot analysis. Cell lysates were loaded to 8-12% SDS-PAGE and separated by
electrophoresis. Separated proteins were then electrically transferred to a PVDF film. After being
blocked with 1×TBS containing 0.5% Tween-20 and 5% non-fat milk, the film was incubated
with corresponding primary antibody followed by HRP-conjugated secondary antibody. And the
protein lanes were visualized using ECL Western Blotting Detection Kit (Thermo Scientific,
USA).
5.6 Cell Migration and Invasion Assay
Cell migration assays were evaluated in Transwell chambers (Corning Costar). Cell invasion
assays were evaluated in Magrigel invasion chambers (Corning Costar). 0.2~1 x 105 tumor cells
were plated in the top chamber with medium without FBS. RPMI-1640 medium containing 2%
FBS with or without TGF- β1 of 10 ng/ml and test compound (1.25~10 µmol/L) was added to the
bottom chamber. After incubation for 24 hrs at 37 °C, the cells were fixed in 100% methanol and
stained with 0.25% crystal violet; and the cells that had not migrated from the top surface of the
filters were removed with cotton. Migrated cells were quantitated by counting cells in six
randomly selected fields on each filter under a microscope at 200 magnification and graphed as
the mean of three independent experiments.
5.7 Determination of Pharmacokinetic Parameters in Rats
Compound 8i was dissolved in 0.5% CMC-Na solvents. Male Sprague−Dawley rats (190−230 g)
were dosed with the test compounds intravenously (iv) at 2.5 mg/kg and by oral gavage (po) at 25
mg/kg. Blood samples (0.2 mL) were then obtained via external jugular vein puncture at 0.25 h,
0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h time points and collected into heparinized tubes. Samples were
centrifuged for cell removal, and the plasma supernatant was then transferred to a clean vial and
subsequently stored in -80 °C prior to analysis. Test sample concentrations were determined by
LC/MS and pharmacokinetic parameters were calculated using Analyst 1.5.1 software.
5.8 In vivo Acute Toxicity Study of compound 8i
The ICR mice were randomly divided into an experimental group and a control group. Each
group contains 6 mice (male). The experimental group mice were fed with 8i solution once at a
dose of 50, 100, 200, 400, 600 or 800 mg/kg. The control animals were treated with the same
volume of vehicle. After administration, all animals were observed at 30 minutes, 1, 2, 4 and 6
hours, then during the remaining experimental period, for a total of 7 days. All animals were

ACCEPTED MANUSCRIPT
observed at least once per day. The animals’ body weight and survival were monitored every day.
In addition, their histopathology was all measured at the final observation time points.
Acknowledgements
The authors thank the financial support from National Natural Science Foundation of China
(21702074, 21572230, 81425021 and 81673285), Guangdong Natural Science Funds
(2105A030312014), Guangdong Nanyue-Baijie Award, Guangzhou City Key Laboratory of
Precision Chemical Drug Development (201805010007) and Jinan University (21616321).
Abbreviations
Mer, c-mer proto-oncogene tyrosine kinase; CML, chronic myeloid leukemia; Gas6, ligand
growth arrest-specific 6; EMT, epithelial-mesenchymal transition; TNBC, triple negative breast
cancer; EGFR, epidermal growth factor receptor; AML, acute myeloid leukemia; DHBA, dual
hydrogen bond acceptor; NaH, Sodium hydride; DMF, N,N-dimethyl formamide; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo
[4,5-b]pyridinium
3-oxid
hexafluorophosphate; DIPEA, N,N-diisopropylethylamine; rt, room temperature; DCM,
dichloromethane; Et3N, trimethylamine; DMAP, dimethyla-minopyridine; (BOC)2O, Di-tert-butyl
dicarbonate; FRET, fluorescence resonance energy transfer; IC50, the half maximal (50%)
inhibitory concentration of a substance; Kd, binding constant; CSF1R, colony stimulating factor 1
receptor; DDR, discoidin domain receptor; Flt3, FMS-like tyrosine kinase 3; Kit, v-kit
Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; LOK, serine/threonine kinase 10;
Trk, nerve growth factor receptor; TGF-β1, transforming growth factor β1; SD, Sprague-Dawley;
PK, pharmacokinetic; AUC, Area Under The Curve.
References
[1] Liu, E.; Hjelle, B.; Bishop, J. M., Transforming genes in chronic myelogenous leukemia. Proc.
Natl. Acad. Sci. U S A. 85 (1988) 1952-1956.
[2] Varnum, B. C.; Young, C.; Elliott, G.; Garcia, A.; Bartley, T. D.; Fridell, Y. W.; Hunt, R. W.;
Trail, G.; Clogston, C.; Toso, R. J.; Yanagihara, D.; Bennett, L.; Sylber, M.; Merewether, L. A.;
Tseng, A.; Escobar, E.; Liu. E. T.; Yamane, H. K., Axl receptor tyrosine kinase stimulated by the
vitamin K-dependent protein encoded by growth-arrest-specific gene 6. Nature 373 (1995)
623-626.

ACCEPTED MANUSCRIPT
[3] Antony, J.; Huang, R. Y., AXL-Driven EMT State as a Targetable Conduit in Cancer. Cancer
Res. 77 (2017) 3725-3732.
[4] Li, Y.; Ye, X.; Tan, C.; Hongo, J. A.; Zha, J.; Liu, J.; Kallop, D.; Ludlam, M. J.; Pei, L., Axl as
a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis.
Oncogene 28 (2009) 3442-3455.
[5] Postel-Vinay, S.; Ashworth, A., AXL and acquired resistance to EGFR inhibitors. Nat. Genet.
44 (2012) 835-836.
[6] Shinh, Y.-S.; Lai, C.-Y.; Kao, Y.-R.; Shiah, S.-G.; Chu, Y.-W.; Lee, H.-S.; Wu, C.-W.,
Expression of Axl in Lung Adenocarcinoma and Correlation with Tumor Progression. Neoplasia 7
(2005) 1058-1064.
[7] Ishikawa, M.; Sonobe, M.; Nakayama, E.; Kobayashi, M.; Kikuchi, R.; Kitamura, J.; Imamura,
N.; Date, H., Higher expression of receptor tyrosine kinase Axl, and differential expression of its
ligand, Gas6, predict poor survival in lung adenocarcinoma patients. Ann. Surg. Oncol. 20 (2013)
S467-S476.
[8] Bivona, T.; Okimoto, R., AXL receptor tyrosine kinase as a therapeutic target in NSCLC. Lung
Cancer (Auckl) 6 (2015) 27-34.
[9] Liu, L.; Greger, J.; Shi, H.; Liu, Y.; Greshock, J.; Annan, R.; Halsey, W.; Sathe, G. M.; Martin,
A. M.; Gilmer, T. M., Novel mechanism of lapatinib resistance in HER2-positive breast tumor
cells: activation of AXL. Cancer Res. 69 (2009) 6871-6878.
[10] Skinner, H. D.; Giri, U.; Yang, L. P.; Kumar, M.; Liu, Y.; Story, M. D.; Pickering, C. R.;
Byers, L. A.; Williams, M. D.; Wang, J.; Shen, L.; Yoo, S. Y.; Fan, Y. H.; Molkentine, D. P.;
Beadle, B. M.; Meyn, R. E.; Myers, J. N.; Heymach, J. V., Integrative Analysis Identifies a Novel
AXL-PI3 Kinase-PD-L1 Signaling Axis Associated with Radiation Resistance in Head and Neck
Cancer. Clin. Cancer Res. 23 (2017) 2713-2722.
[11] Myers, S. H.; Brunton, V. G.; and Unciti-Broceta, A., AXL Inhibitors in Cancer: A Medicinal
Chemistry Perspective. J. Med. Chem. 59 (2016) 3593-3608.
[12] Shin, J. S.; Hong, S. W.; Moon, J. H.; Kim, J. S.; Jung, K. A.; Kim, S. M.; Lee, D. H.; Kim,
I.; Yoon, S. J.; Lee, C. G.; Choi, E. K.; Lee, J. Y.; Kim, K. P.; Hong, Y. S.; Lee, J. L.; Kim, B.;
Choi, E. K.; Lee, J. S.; Jin, D. H.; Kim, T. W., NPS-1034, a novel MET inhibitor, inhibits the

ACCEPTED MANUSCRIPT
activated MET receptor and its constitutively active mutants. Invest. New Drugs 32 (2014)
389-399.
[13] Ueno, Y.; Kaneko, N.; Saito, R.; Kondoh, Y.; Shimada, I.; Mori, M.; Kuromitsu, S., ASP2215,
a novel FLT3/AXL inhibitor: Preclinical evaluation in combination with cytarabine and
anthracycline in acute myeloid leukemia (AML). 2014 ASCO Annual Meeting.
[14] Mollard, A.; Warner, S. L.; Call, L. T.; Wade, M. L.; Bearss, J. J.; Verma, A.; Sharma, S.;
Vankayalapati, H.; Bearss, D. J., Design, Synthesis and Biological Evaluation of a Series of Novel
Axl Kinase Inhibitors. ACS med. Chem. Lett. 2 (2011) 907-912.
[15] Holland, S. J.; Pan, A.; Franci, C.; Hu, Y.; Chang, B.; Li, W.; Duan, M.; Torneros, A.; Yu, J.;
Heckrodt, T. J.; Zhang, J.; Ding, P.; Apatira, A.; Chua, J.; Brandt, R.; Pine, P.; Goff, D.; Singh, R.;
Payan, D. G.; Hitoshi, Y., R428, a selective small molecule inhibitor of Axl kinase, blocks tumor
spread and prolongs survival in models of metastatic breast cancer. Cancer Res. 70 (2010)
1544-1554.
[16] https://clinicaltrials.gov/ct2/show/NCT02488408
[17] https://clinicaltrials.gov/ct2/show/NCT02424617
[18] Myers, S. H.; Temps, C.; Houston, D. R.; Brunton, V. G.; Unciti-Broceta, A., Development of
Potent Inhibitors of Receptor Tyrosine Kinases by Ligand-Based Drug Design and Target-Biased
Phenotypic Screening. J Med. Chem. 61 (2018) 2104-2110.
[19] Tan, L.; Zhang, Z.; Gao, D.; Luo, J.; Tu, Z. C.; Li, Z.; Peng, L.; Ren, X.; Ding, K.,
4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New Axl Kinase Inhibitors. J. Med.
Chem. 59 (2016) 6807-6825.
[20] Singh, S.; Kaur, G.; Mangla, V.; Gupta, M. K., Quinoline and quinolones: promising scaffolds
for future antimycobacterial agents. J. Enzyme Inhib. Med. Chem. 30 (2015) 492-504.
[21] Liu, K. K.; Sakya, S. M.; O'Donnell, C. J.; Li, J., Synthetic approaches to the 2008 new
drugs. Mini. Rev. Med. Chem. 9 (2009) 1655-1675.
[22] Rodems, S. M.; Hamman, B. D.; Lin, C.; Zhao, J.; Shah, S.; Heidary, D.; Makings, L.; Stack,
J. H.; Pollok, B. A. A FRET-based assay platform for ultra-high density drug screening of protein
kinases and phosphatases. Assay Drug Dev. Technol. 1 (2002) 9−19.
[23] Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.;
Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D.

ACCEPTED MANUSCRIPT
J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.;
Zarrinkar, P. P., A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26 (2008)
127-32.
[24] Gjerdrum, C.; Tiron, C.; Hoiby, T.; Stefansson, I.; Haugen, H.; Sandal, T.; Collett, K.; Li, S.;
McCormack, E.; Gjertsen, B. T.; Micklem, D. R.; Akslen, L. A.; Glackin, C.; Lorens, J. B., Axl is
an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis
and patient survival. Proc. Natl. Acad. Sci. U S A. 107 (2010) 1124-1129.
[25] Wu, J.; Ru, N. Y.; Zhang, Y.; Li, Y.; Wei, D.; Ren, Z.; Huang, X. F.; Chen, Z. N.; Bian, H.,
HAb18G/CD147 promotes epithelial-mesenchymal transition through TGF-beta signaling and is
transcriptionally regulated by Slug. Oncogene 30 (2011) 4410-4427.

ACCEPTED MANUSCRIPT
1. Quinolone 8i bound tightly to Axl with a Kd value of 1.1 nM
2. 8i dose dependently inhibited cell invasion and migration in TGF-β1 induced breast cancer
cells.
3. 8i exhibited extraordinary target selectivity over 468 kinases with a S(10) and S(35) value of
0.022 and 0.42 at 1.0 µM, respectively.