Novel dual inhibitors targeting CDK4 and VEGFR2 synergistically suppressed cancer progression and angiogenesis

Article abstract of DOI:10.1016/j.ejmech.2019.07.044

Based on the significantly synergistic effects of CDK4 and VEGFR2 inhibitors on growth of cancer cells, a series of novel multi-kinase inhibitors targeting CDK4 and VEGFR2 were designed, synthesized and evaluated, among which Roxyl-ZV-5J exhibited potent and balanced activities against both CDK4 and VEGFR2 with half-maximal inhibitory concentration at the nanomolar level. It effectively induced breast and cervical cancer cell cycle arrest and cell apoptosis. Roxyl-ZV-5J also inhibited the proliferation, tube formation and VEGFR2 downstream signaling pathways of HUVECs. Oral administration of Roxyl-ZV-5J led to significant tumor regression and anti-angiogenesis without obvious toxicity in SiHa xenograft mouse model. In addition, this compound showed good pharmacokinetics. This study confirmed a new tool for dual CDK-VEGFR2 pathways inhibition achieved with a single molecule, which provided valuable leads for further structural optimization and anti-angiogenesis and anti-tumor mechanism study.

Full text of DOI:10.1016/j.ejmech.2019.07.044

Accepted Manuscript
Novel dual inhibitors targeting CDK4 and VEGFR2 synergistically suppressed cancer
progression and angiogenesis
Zhi Huang, Borui Zhao, Zhongxiang Qin, Yongtao Li, Tianqi Wang, Wei Zhou, Jianyu
Zheng, Shengyong Yang, Yi Shi, Yan Fan, Rong Xiang
PII:
S0223-5234(19)30665-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.044
EJMECH 11541
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2019
Revised Date: 13 July 2019
Accepted Date: 14 July 2019
Please cite this article as: Z. Huang, B. Zhao, Z. Qin, Y. Li, T. Wang, W. Zhou, J. Zheng, S. Yang, Y.
Shi, Y. Fan, R. Xiang, Novel dual inhibitors targeting CDK4 and VEGFR2 synergistically suppressed
cancer progression and angiogenesis, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.07.044.
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ACCEPTED MANUSCRIPT
A novel dual inhibitor, Roxyl-ZV-5J with potent and balanced activities against both
CDK4 and VEGFR2 at the nanomolar level, exhibited improved antitumor and
anti-angiogenesis activities over positive drugs in vitro and in vivo.

ACCEPTED MANUSCRIPT
Novel dual inhibitors targeting CDK4 and VEGFR2
synergistically suppressed cancer progression and angiogenesis
a, b #
a,f #
a
a
a
Zhi Huang
, Borui Zhao , Zhongxiang Qin , Yongtao Li , Tianqi Wang , Wei
a
d
e
a, b
a, b
a,
Zhou , Jianyu Zheng , Shengyong Yang , Yi Shi *, Yan Fan * and Rong Xiang
b, c *
a
Department of Medicinal Chemistry, School of Medicine, Nankai University, 94 Weijin Road,
Tianjin 300071, China
b
2
011 Project Collaborative Innovation Center for Biotherapy of Ministry of Education, 94 Weijin
Road, Tianjin 300071, China
c
State Key Laboratory of Medicinal Chemical Biology, 94 Weijin Road, Tianjin 300071, China
d
State Key Laboratory and Institute of Elemento-Organic Chemistry, Collaborative Innovation
Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China
e
Medical Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China Hospital,
Sichuan University, Chengdu, China
*
Corresponding authors:* Yi Shi, E-mail: yishi@nankai.edu.cn * Yan Fan, E-mail:
yanfan@nankai.edu.cn; Rong Xiang*: E-mail: rxiang@nankai.edu.cn.
#
Author Contributions: Zhi Huang and Borui Zhao are contributed equally to this
work.

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Abstract
Based on the significantly synergistic effects of CDK4 and VEGFR2 inhibitors on
growth of cancer cells, a series of novel multi-kinase inhibitors targeting CDK4 and
VEGFR2 were designed, synthesized and evaluated, among which Roxyl-ZV-5J
exhibited potent and balanced activities against both CDK4 and VEGFR2 with
half-maximal inhibitory concentration at the nanomolar level. It effectively induced
breast and cervical cancer cell cycle arrest and cell apoptosis. Roxyl-ZV-5J also
inhibited the proliferation, tube formation and VEGFR2 downstream signaling
pathways of HUVECs. Oral administration of Roxyl-ZV-5J led to significant tumor
regression and anti-angiogenesis without obvious toxicity in SiHa xenograft mouse
model. In addition, this compound showed good pharmacokinetics. This study
confirmed a new tool for dual CDK-VEGFR2 pathways inhibition achieved with a
single molecule, which provided valuable leads for further structural optimization and
anti-angiogenesis and anti-tumor mechanism study.
Keywords: CDK4, VEGFR2, Inhibitor, Cancer

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1
. Introduction
Cancer is a multigenetic disease involving multiple cross-talks between signaling
networks and often required multiple therapeutic interventions[1]. Kinase inhibitors
are widely employed in clinical oncology as molecularly targeted therapeutics and
lead for drug design. Simultaneous inhibition of multiple mechanisms can yield
superior efficacy, such as synergy effects, as exemplified by multi-kinase inhibitors
[
2]. The utility of these inhibitors simultaneous targeting multiple pathways is critical
for tumor proliferation, angiogenesis and apoptotic regulation[3].
More recently the promising target to control tumor cellular proliferation is
inhibition of cell cycle progression by targeting cyclin-dependent kinases (CDKs)[4,
5
]. Different CDKs forming complexes with cyclins control specific checkpoint of the
cell cycle, which have two major functional groups based on their roles. The subtypes
-4 and 6 of CDKs mainly mediate cell cycle and subtypes 5, 7-9 of CDKs regulate
1
transcription control [6]. Aberrations in CDKs-cyclin pathway have been observed in
various tumors, including those of the breast, colon, liver and brain. The simultaneous
and pharmacological inhibition of these kinases have been proved to provide an
effective approach for cancer compared with the inhibition of a single CDK alone [7,
8
].
Multiple generations of drugs targeting CDK have been designed and synthesized
through binding in the ATP-binding cleft of CDK enzymes. More recently,
third-generation CDK-directed drugs targeting CDK4/6, palbociclib, ribociclib and

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abemaciclib have received significant attention [9-13]. But in clinical trials, these
compounds needed to combine with letrozole or tamoxifen for the treatment of
+
−
ER /HER2 metastatic breast cancer, which indicated a lack of sensitivity to CDK4/6
monotherapy[14, 15]. Therefore, we reasoned that development of single drug with
multiple biological targets based on CDK4/6 to improve efficacy in cancer treatment.
Angiogenesis is required for the growth, survival, and metastasis of most solid
tumors, including breast cancer [16]. Of all angiogenic molecules, vascular
endothelial growth factor (VEGF) is the primary modulator of angiogenesis and
metastasis in human carcinogenesis. Therapies that block VEGF signaling has been
demonstrated to retard angiogenesis and inhibit tumor growth [17, 18]. VEGF
executes its effects by binding to and activating its receptors (VEGFRs). In
endothelial cells, VEGFR2 is the major regulator of VEGF-stimulated cell survival
and vascular permeability during angiogenesis [19]. It has been reported that
VEGFR2 inhibitors possess synergistic anti-tumor effects in rational combinations
with anti-cancer drugs [20, 21]
. Recent research indicated that concurrent of cell cycle
arrest and angiogenesis inhibition showed more effective due to synergy effects [8,
2
2-26].
Cabozantinib is a highly potent small molecule inhibitor against VEGFR2 [27, 28].
In a recent study, we developed a novel hybrid inhibitor based on LEE011 and
cabozantinib structure, which indeed showed a much higher anti-tumor activity than
positive drugs both in vitro and in vivo. Nevertheless, the compound show low
inhibition effect targeting VEGFR2 [29]. In our continued effort to discover novel

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dual-action inhibitors targeting both CDK4 and VEGFR2, a series of compounds were
designed and synthesized based on the remarkably synergistic effects of CDK4
inhibitor and VEGFR2 inhibitor on growth of cancer cell lines (Figure S1). To the
most active compound in enzymatic and cellular assays, an in depth anti-tumor
activities both in vitro and in vivo are evaluated. (Figure 1)
2
2
. Results and discussions
.1 Chemistry
All compounds prepared by routes were outlined in Schemes 1−3. Scheme 1
outlines the routes of compounds 5A-K, 6D, 6F, 6H, 6J, 6K. Briefly, compounds
A-K, 6D, 6F, 6H, 6J and 6K were synthesized from cyclopropane-1,1-dicarboxylic
5
acid condensation with aniline or substituted aniline [29, 30]. The condensation of
p-phenylenediamine or 4-(aminomethyl)aniline dihydrochloride with carboxylic acids
2
A-K afforded 3A-K, 4D, 4F, 4H, 4J and 4K under condensing agent
1
-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) in the
presence of 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA) [29,
3
4
6
1]. A palladium catalyzed cross-coupling reaction of compounds 3A-K, 4D, 4F, 4H,
and 4K with compound
-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imida
J
zole(7) provided compounds 5A-K and 6D, 6F, 6H, 6J, 6K, respectively [29, 32].
We synthesized the key intermediate 13 and the general synthetic routes are
illustrated in Scheme 2. Firstly, commercial compound 4-bromo-2-fluoro-1–

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nitrobenzene (8) reacted with isopropylamine to provide compound 9. Secondly,
compound 9 was reduced to amino group. Compound 11 was obtained by closing the
ring in the presence of conc.HCl and aq NaNO [33]. Finally, compound 11 was
2
converted to the corresponding heteroarylboronate ester (12) which underwent
palladium catalyzed Suzuki reaction with 2, 4-dichloro-5-fluoropyrimidine to yield 13.
Scheme 3 outlines the routes used to synthesize compounds 14A-K. A palladium
catalyzed cross-coupling reaction of compounds 3A-K with compound
6
-(2-chloro-5-fluoropyrimidin-4-yl)-1-isopropyl-1H-benzo[d][1,2,3]triazole
(13)
provided compounds 14A-K, respectively.[29, 32]
2
.2 Enzyme Inhibitory Activity and SAR
Based on the synergistic experimental results with mean CI < 1 across relevant
concentrations of CDK4/6 inhibitors ribociclib or abemaciclib, with VEGFR2
inhibitor cabozantinib in vitro (Figure S1), we initiated our hybrid strategy to merge
two different pharmacophores to link two inhibitors together to obtain compounds 4d,
5
B . Enzymatic-based assays were preliminarily to evaluate the bioactivity of the
synthesized compounds in the SAR studies. As shown in Table 1, substitutions at the
part 1 position substantially impacted the activity. Compared with pharmacophore Y,
pharmacophore X all increased both CDK4 and VEGFR2 inhibitory activities,
revealing that pharmacophore X of abemaciclib are optimal substituent for our dual
inhibitor design. Subsequently, we fixed part 1 as X group and varied the part 3 group
substituent. Compared with 5A, when R position is CF , compounds 5G, 5H
2
3
decreased CDK4 inhibitory activities (31% and 74%, respectively), revealing that the

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electron-withdrawing group may decrease the inhibition activity of CDK4. Other
compounds with CF substituent at R position (5K and 4l) were similar activities
3
2
against CDK4. On the contrary, compounds with the electron-donating group CH3
substituent at R position (5I and 5J) are more active, which indicated that the
2
electron-donating group should be beneficial for enhancing the inhibition activity of
CDK4. We then explored the influence of different substituents on part 2. According
to the structural characteristic of abemaciclib, we introduced a CH group to
2
synthesize 6D, 6F and 6J. Compared with compounds 5D, 5F and 5J, compounds 6D,
6
F and 6J showed parallel potency against CDK4. As expected, compounds 6H and
6
K with electron-withdrawing groups at R position exhibited low binding affinity.
2
Furthermore, we reconstructed the X group to obtain the Z group and synthesized 11
derivatives 14A-K. These compounds were all inactive against either CDK4 or
VEGFR2 at the test concentration (1 µM). It implied that X group is the most suitable
substituent among these three groups of Part 1. It was worth noting that compounds
1
4G, 14H and 14K containing electron-withdrawing group CF exhibited obviously
3
lower inhibition activity against both CDK4 and VEGFR2, compared with
compounds 14I and 14J containing electron-donating group CH . These results were
3
consistent with the above conclusions: the electron-donating group is favorable for the
inhibition activity of CDK4.
Compounds with Y and Z group at part 1 were inactive against VEGFR2 in Table 1,
Therefore, X group is beneficial for inhibition of VEGFR2 activity. From Table 1, we
can see that the derivatives with CF at part 3 position have in-activities in enzymatic
3

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level. These data clearly show substitution of R by CH is the most optimal. We
2
3
identified dual-acting compounds 5A, 5B, 5F, 5I, 5J, 6F and 6J exhibiting better
kinase inhibitory among the derivatives.
The reason was further described that Figure 2A shows a docking model between
5
J and CDK4. It can be seen from the diagram that 5J forms a hydrogen bond
between NH and an oxygen atom in 12-position isoleucine. The substituents of
benzene ring were electron-withdrawing groups, which reduced the density of
electron cloud of nitrogen atom in NH. It made hydrogen atom closer to nitrogen
atom. This increased the distance between NH and an oxygen atom of 12-position
isoleucine, thus lowering the hydrogen bonding and ultimately decreasing the activity.
However, the substituents on benzene ring were electron donating groups, which
increased the electron cloud density of nitrogen atoms in NH. It made hydrogen atom
in NH closer to an oxygen atom of 12-position isoleucine, thus enhancing the
hydrogen bonding and eventually increasing inhibition activity of CDK4.
2
.3 Cellular Assay.
As the aim of this study was to discover dual-action inhibitors targeting both CDK4
and VEGFR2 for cancer treatment, we screened compounds exhibiting better kinase
inhibitory to do the preliminary cell inhibition assays. Compounds 5A, 5B, 5F, 5I, 5J,
6
F and 6J were chosen to test their cellular cytotoxic activity (Figure S2 and S3). We
found that compound 5J had significant cancer cell inhibitory activity. We further
evaluated the effect of compound 5J on proliferation in cervical cancer cell lines

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(SiHa and HeLa), breast cancer cell lines (MDA-MB-231 and T-47D) (Table 2).
Compound 5J potently inhibited the growth of cervical cancer cells and breast cancer
cells. These results demonstrated that Roxyl-ZV-5J performed better inhibitory
ability compared to cabozantinib or abemaciclib. The best tumor cell potency of these
analogues was obtained for compound 5J, which displayed inhibition of CDK4 and
VEGFR2 with IC values of 46 nM and 46 nM, respectively (Figure S4). Therefore,
5
0
as attributed of a truly merged bispecific pharmacophore and the results of anti-tumor
activity in cells, Roxyl-ZV-5J was chosen for further studies both in vitro and in vivo
subsequently.
2
.4 Lipophilicity.
Log P and Log D describe a compound’s lipophilicity, and often correlate with a
number of key biopharmaceutical parameters in drug discovery. We detected the
concentration of Roxyl-ZV-5J in the aqueous and organic phases and calculated Log
P (Log D ) value. The values were measured using the traditional shake flask
7
.4
method. The Log P value for Roxyl-ZV-5J is 2.53 and Log D 7.4 value is 2.51. Our
results indicated that compound Roxyl-ZV-5J has a good lipophilicity.
2
.5 Kinase Selectivity Profiling.
To characterize the kinase inhibitory activities and selectivity, kinase inhibition
profiling assays with a fixed concentration of 1 µM of compound Roxyl-ZV-5J were
carried out against a series of 270 kinases through the Eurofins kinase profiling. The

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results are shown in Table S1. Compound Roxyl-ZV-5J did not show obvious
activity against other kinases, indicated acceptable kinase selectivity.
2
.6 Molecular Modeling of Compound Roxyl-ZV-5J
Molecular docking studies were carried out to investigate the binding modes of
compound Roxyl-ZV-5J in CDK4 and VEGFR2, respectively. Figure 2 illustrated
the predicted binding modes and the detailed protein-inhibitor interactions of
compound Roxyl-ZV-5J with CDK4 and VEGFR2, respectively. For comparison, the
binding mode of Roxyl-ZV-5J was superimposed on that of abemaciclib and
cabozantinib. Our results showed that compound Roxyl-ZV-5J is a potent inhibitor of
CDK4 with an IC of 46 nM. As shown in Figure 2A and 2B, Roxyl-ZV-5J could
5
0
tightly bind to the ATP-binding site of CDK4 in a binding mode. Hydrogen bonds
appear to be formed between the aminopyrimidine and the backbone residue of
VAL96 and ASP97. These hydrogen bonds are critical for binding to CDK4. The
crystal structure of VEGFR2 was taken from the RCSB Protein Data Bank (PDB
code: 2OH4). As shown in Figure 2C and 2D, Roxyl-ZV-5J could tightly bind to the
ATP-binding site of VEGFR2 in a binding mode. In this binding model, hydrogen
bonds between the hinge-region CYS 917 and the fluorine atom of the pyrimidine
ring were observed. Moreover, carbonyl oxygen could form significant hydrogen
bond with the backbone NH of ASP 1044. Other hydrogen bond interaction was
formed between the residue GLU883 and the NH of Roxyl-ZV-5J, which indicated
Roxyl-ZV-5J would be a good inhibitor for VEGFR2.

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2
.7 Roxyl-ZV-5J inhibited the tube formation and VEGFR2 downstream
signaling pathways of HUVECs in vitro.
The compound Roxyl-ZV-5J was next evaluated in a dose response study to
against HUVEC, with cabozantinib, abemaciclib or Vehicle as control groups. Values
of IC50 measured after 72 h were presented in Table 2. The results showed a
significantly higher drug activity of Roxyl-ZV-5J with IC around 40 nM. However,
5
0
cabozantinib or abemaciclib presented IC almost >400 nM. Hence, we selected 20
5
0
nM and 40 nM as research concentrations of HUVECs, then cell viability was
examined after treated with Roxyl-ZV-5J 20 nM, 40 nM, cabozantinib 40 nM,
abemaciclib 40 nM or Vehicle at 0, 12, 24, 36, 48, 60 and 72 h. The results indicated
that Roxyl-ZV-5J effectively inhibited cell viability in dose- and time-dependent
manner compared to other groups (Figure 3A). Although angiogenesis involved
several types of cells, tube formation in endothelial cells was a significant step. After
treated with different concentrations of Roxyl-ZV-5J, cabozantinib or vehicle for 6 h,
HUVECs were observed under a light microscope. It was confirmed that
Roxyl-ZV-5J could effectively attenuate the ability of tube formation of HUVECs
compared to cabozantinib or Vehicle group (Figure 3B). To determine whether
Roxyl-ZV-5J inhibited VEGFR2 and its downstream signaling pathways, we
screened essential kinases involved in VEGFR2 signaling. Western Blot assay showed
that Roxyl-ZV-5J treatment suppressed VEGFR-mediated phosphorylation of
VEGFR2, STAT3, Akt and Src, while phosphorylation of Erk1/2 barely altered
(Figure 3C).

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2
.8 Roxyl-ZV-5J restrained angiogenesis in vivo
We conducted Matrigel plug assay in a mouse model of angiogenesis. As shown in
Figure 4, the subcutaneous new blood vessels of Balb/c mice were significantly
inhibited in Roxyl-ZV-5J groups compared to cabozantinib or vehicle group. The
results revealed that Roxyl-ZV-5J blocked new blood vessel formation in vivo.
2
.9 Roxyl-ZV-5J suppressed the proliferation of cancer cells.
Colony formation assay confirmed that Roxyl-ZV-5J had a robust growth
inhibition on MDA-MB-231, T-47D, SiHa and HeLa cells when comparing to other
treatments (Figure 5A). Moreover, EdU assay demonstrated that the positive ratio of
proliferating cells was decreased after treated with Roxyl-ZV-5J compared to other
groups (Figure 5B).
2
.10 Roxyl-ZV-5J modulated cell cycle arrest in vitro.
We wondered whether the inhibitory of cell proliferation partly result cell cycle
inhibition. We next investigated the effect of Roxyl-ZV-5J treatment on cell cycle
kinetics. As shown in Figure 6, the FACS analysis revealed that abemaciclib
treatment resulted in accumulation of cells in G /G phase, while treatment of
0
1
cabozantinib presented a mildly G arrest. Roxyl-ZV-5J significantly induces G
2
2
arrest, accompanied by decreases in S phase (Figure 6A). As reported, the G2 phase
inhibition was indicative of cells undergoing apoptosis, which attributed to the role for
inhibition of VEGFR2 [34, 35]. Accumulation of cells in G2/M phase is a significant
remark of the apoptotic role of compound Roxyl-ZV-5J in cells. The results

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confirmed that compound Roxyl-ZV-5J had anti-proliferative properties.
2
.11 Roxyl-ZV-5J induced cell apoptosis in vitro.
Subsequently, the effect of Roxyl-ZV-5J on cell apoptosis was conducted using
Annexin V and PI double staining. Treatment with Roxyl-ZV-5J markedly increased
Annexin V positive early-stage and late-stage apoptotic cells, compared with control
groups (Figure 6B). Western Blots assay indicated that Roxyl-ZV-5J decreased the
expression level of p-Rb in dose-dependence. Meanwhile, Roxyl-ZV-5J depressed
the proliferation associated protein Ki67 expression (Figure 6C). Roxyl-ZV-5J also
suppressed the expression level of apoptosis associated proteins Bcl-2 compared to
other groups. These above results showed that the inhibition of cancer cell growth
induced by Roxyl-ZV-5J was associated with cell cycle and apoptosis, confirming the
existence of multi-intracellular targets.
2
. 12. Roxyl-ZV-5J inhibited tumor growth and angiogenesis in vivo.
To better assess whether Roxyl-ZV-5J could efficiently inhibit tumor growth and
angiogenesis in vivo, we constructed xenograft models using Nod/SCID female mice
bearing SiHa cells. Mice were sacrificed at day 21, tumor growth curve demonstrated
that Roxyl-ZV-5J (10 mg/kg, 20 mg/kg, 40 mg/kg) treatments were effective
compared to cabozantinib and abemaciclib 40 mg/kg or vehicle group, especially in
Roxyl-ZV-5J 20 mg/kg and 40 mg/kg group (Figure 7A). The average body weight
of mice was monitored every 3 days after gavage. There were hardly changed in
cabozantinib or abemaciclib group, while in Roxyl-ZV-5J 20 mg/kg and 40 mg/kg

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group, body weight of mice slightly decreased (Figure 7B). An obvious decrease in
tumor weight and volume was also observed at the end-point of the study (Figure 7C
and 7D).As Ki67 and CD31 presented the markers of proliferation and angiogenesis
respectively, IHC staining showed that the expression level of Ki67 and CD31 was
obviously suppressed in Roxyl-ZV-5J groups in dose-dependence (Figure 7E). In
addition, acute general toxicity analysis indicated that Roxyl-ZV-5J did not induce
any toxic effects on mice compared to control group (Figure S5). These results
demonstrated that Roxyl-ZV-5J effectively inhibited tumor growth and angiogenesis
of xenograft tumor mice without obvious toxicity.
2
.13. Pharmacokinetic Characteristics of Roxyl-ZV-5J in Rats.
Pharmacokinetic evaluation in rats following intravenous and oral administration of
Roxyl-ZV-5J was further evaluated (Table 3). Compound Roxyl-ZV-5J showed
good oral bioavailability of 60%. The oral maximum plasma concentration (Cmax)
was 1.47 mg/L. Besides, compound Roxyl-ZV-5J showed oral apparent distribution
volume (Vss) of 5.901 L/kg and a clearance of 1.589 L/h/kg. All of these results
indicated that Roxyl-ZV-5J presented favorable pharmacokinetic properties, which
may be a promising lead compound for the treatment of tumor diseases.
3
. Conclusion
As reported, broad-spectrum inhibitors have exemplified superior efficacy
compared with traditional agents, with increasing use in clinical oncology as
molecular targeted therapeutics. The efficacy of these drugs is often simultaneously

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targeting multiple pathways that are critical for cancer growth, including those that
promote proliferation, angiogenesis, and apoptotic regulation.
In our present work, based on the favorable synergistic effects between CDK4 and
VEGFR2 inhibitors, a series of cyclopropane-1, 1-dicarboxamide derivatives as
CDK4 and VEGFR2 dual inhibitors were designed, synthesized, and evaluated. The
representative compound Roxyl-ZV-5J simultaneously inhibited the oncogenic
kinases CDK4 and VEGFR2, leading to much potent anti-proliferative and
anti-angiogenesis capacities. Roxyl-ZV-5J displayed prominent cytotoxic activity in
various cancer cell lines with IC values in the 16−70 nM range, several times lower
5
0
than that for the reference drugs. Roxyl-ZV-5J induced G2/M phase arrest and
inhibited cancer cell proliferation, as shown in the EdU-incorporation assay. To
understand the mechanism of action of Roxyl-ZV-5J, we identified important
mediators related to the pathways, such as the cell cycle, apoptosis and angiogenesis.
Roxyl-ZV-5J demonstrated effective inhibition of CDK and VEGFR2 signaling
pathways in the relevant in vitro and in vivo models examined. It was much more
potent than abemaciclib in inhibiting phosphorylation of Rb protein and than
cabozantinib in inhibiting angiogenesis. In addition, Roxyl-ZV-5J significantly
inhibited the relative levels of Ki67. We demonstrated that Roxyl-ZV-5J induced
apoptosis in solid tumour cells, which reduced expression of the antiapoptotic protein
BCL-2.In vivo, oral administration of Roxyl-ZV-5J with 20 mg/kg or 40 mg/kg every
day showed significant anti-tumor potency and no obvious toxicity.
In summary, Roxyl-ZV-5J possessed acceptable kinase selectivity and good

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pharmacokinetics. As a mutitarget inhibitor, Roxyl-ZV-5J is noteworthy because the
combined inhibition of transcriptional CDK with cell-cycle regulation and VEGFR2
with angiogenesis regulation has been shown to be advantageous for effective
apoptotic induction in tumor cells. The merit of our research is the development of a
series of CDK4 and VEGFR2 dual inhibitors with excellent anti-tumor and
anti-angiogenesis activity, which validated the biology-oriented multitarget drug
design strategy.
4
. Experimental
4
.1 Chemistry
1
13
19
H NMR (400 MHz), C NMR (101 MHz) and F NMR(376 MHz) spectra were
taken on a Bruker AV-400 MHz spectrometer and chemical shifts were reported in
ppm downfield from internal Me Si. Melting points (mp) were taken in open
4
capillaries on a Mettler MP 50 melting-point system. High-resolution mass spectra
(HRMS) were recorded on a VG ZAB-HS mass spectrometer under electron spray
ionization (ESI). All of the solvents were purified and distilled according to the
standard procedure. The commercially obtained materials were used directly without
further purification unless otherwise noted.
The purity of tested compound was assessed to be ≥ 95% by HPLC analysis on a
Shimadzu Prominence-i LC-2030C 3D system (column, InertSustain C , 4.6 mm ×
1
8
2
50 mm, 5 µm; mobile phase, gradient elution of methanol/H O; low rate, 1.0
2
mL/min; UV wavelength, 190−800 nm; temperature, 40 °C; injection volume, 10 µL).

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4
.1.1. General Procedure for the Preparation of 2A-P. To a stirred solution of
cyclopropane-1,1-dicarboxylic acid (1) (13.01 g, 100 mmol) in isopropyl acetate (150
mL) at 0℃ , thionyl chloride (12.5 g, 105 mmol) was added dropwise. After addition,
the resulting mixture was stirred at room temperature and stirred for 6 hours. The
resulting mixture was then treated with a solution of aniline or substituted aniline (110
mmol) and triethylamine (110 mmol) in isopropyl acetate (40 mL) over 1 hour. After
stirred for 2 hours, the resulting mixture was added ethyl acetate (500 mL). The
solvent was washed by 1 N HCl solution and brine. The organic phase was dried over
MgSO4, evaporated and the residue treated with heptane (200 mL). The product
slurry was stirred for 0.5 h, filtered and dried under vacuum to obtain product.
4
.1.1. General Procedure for the Preparation of 2A-K. To a stirred solution of
cyclopropane-1,1-dicarboxylic acid (1) (13.01 g, 100 mmol) in isopropyl acetate (150
mL) at 0℃ , thionyl chloride (12.5 g, 105 mmol) was added dropwise. After addition,
the resulting mixture was stirred at room temperature and stirred for 6 hours. The
resulting mixture was then treated with a solution of aniline or substituted aniline (110
mmol) and triethylamine (110 mmol) in isopropyl acetate (40 mL) over 1 hour. After
stirred for 2 hours, the resulting mixture was added ethyl acetate (500 mL). The
solvent was washed by 1 N HCl solution and brine. The organic phase was dried over
MgSO4, evaporated and the residue treated with heptane (200 mL). The product
slurry was stirred for 0.5 h, filtered and dried under vacuum to obtain product.
4
.1.1.1. 1-((3-fluorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2A). White
1
solid (76%), H NMR (400 MHz, DMSO-d ) δ 13.12 (s, 1H), 10.73 (s, 1H), 7.61 (dt, J
6

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=
11.3, 2.0 Hz, 1H), 7.41 – 7.27 (m, 2H), 6.88 (ddt, J = 9.1, 6.7, 2.7 Hz, 1H), 1.41 (s,
4
H).
4
.1.1.2.1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2B). White
1
solid (67%), H NMR (400 MHz, DMSO-d ) δ 13.11 (s, 1H), 10.81 – 10.50 (m, 1H),
6
7
.63 (dd, J = 8.9, 5.0 Hz, 2H), 7.13 (tt, J = 8.9, 3.0 Hz, 2H), 1.54 – 1.30 (m, 4H).
4
.1.1.3.1-((3-chlorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2C). White
1
solid (81%), H NMR (400 MHz, DMSO-d ) δ 13.12 (s, 1H), 10.70 (s, 1H), 7.83 (t, J
6
=
1.9 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.32 (td, J = 8.1, 1.5 Hz, 1H), 7.10 (dd, J =
1
3
7
.9, 2.1 Hz, 1H), 1.42 (s, 4H); C NMR (101 MHz, DMSO) δ 173.52, 167.03, 140.20,
1
33.04, 130.33, 123.03, 118.79, 117.69, 28.87, 16.98.
4
.1.1.4.1-((4-chlorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2D). White
1
solid (55%), H NMR (400 MHz, DMSO-d ) δ 13.10 (s, 1H), 10.65 (s, 1H), 7.71 –
6
1
3
7
.54 (m, 2H), 7.49 – 7.23 (m, 2H), 1.41 (s, 4H); C NMR (101 MHz, DMSO) δ
1
73.60, 166.80, 137.75, 128.56, 126.87, 120.85, 28.80, 16.95.
4
.1.1.5.1-((3-bromophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2E). White
1
solid (76%), H NMR (400 MHz, DMSO-d ) δ 13.12 (s, 1H), 10.69 (s, 1H), 7.97 (t, J
6
1
3
=
2.0 Hz, 1H), 7.50 (dt, J = 7.5, 1.9 Hz, 1H), 7.39 – 7.15 (m, 2H), 1.42 (s, 4H); C
NMR (101 MHz, DMSO) δ 174.03, 167.51, 140.83, 131.13, 126.43, 122.14, 122.01,
18.57, 29.38, 17.50.
1
4
.1.1.6.1-((4-bromophenyl)carbamoyl)cyclopropane-1-carboxylic acid (2F). White
1
solid (84%), H NMR (400 MHz, DMSO-d ) δ 13.13 (s, 1H), 10.70 (s, 1H), 7.64 –
6

ACCEPTED MANUSCRIPT
1
3
7
.55 (m, 2H), 7.51 – 7.41 (m, 2H), 1.43 (s, 4H); C NMR (101 MHz, DMSO) δ
1
74.22, 167.32, 138.61, 131.95, 121.73, 115.43, 29.15, 17.70.
4
.1.1.7.1-((3-(trifluoromethyl)phenyl)carbamoyl)cyclopropane-1-carboxylic
acid
1
(
2G). White solid (81%), H NMR (400 MHz, DMSO-d ) δ 13.13 (s, 1H), 10.83 (s,
6
1
7
1
1
H), 8.13 (t, J = 1.9 Hz, 1H), 7.95 – 7.72 (m, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.46 –
1
3
.30 (m, 1H), 1.43 (s, 4H); C NMR (101 MHz, DMSO) δ 173.92, 167.75, 140.04,
30.39, 130.34, 130.08, 129.77, 129.45, 128.60, 125.89, 123.33, 123.18, 120.13,
20.09, 115.89, 115.85, 29.49, 17.34.
4
.1.1.8.1-((4-(trifluoromethyl)phenyl)carbamoyl)cyclopropane-1-carboxylic
acid
1
(
2H). Yellow solid (82%), H NMR (400 MHz, DMSO-d ) δ 12.95 (s, 1H), 10.91 (s,
6
1
3
1
H), 7.82 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 1.44 (s, 4H); C NMR (101
MHz, DMSO) δ 174.04, 167.78, 142.80, 126.48, 126.45, 126.41, 126.13, 124.03,
1
23.71, 123.43, 119.65, 29.44, 17.55.
4
.1.1.9.1-(m-tolylcarbamoyl)cyclopropane-1-carboxylic acid (2I). White solid (75%),
1
H NMR (400 MHz, DMSO-d ) δ 13.14 (s, 1H), 10.58 (s, 1H), 7.52 – 7.31 (m, 2H),
6
7
.17 (t, J = 7.8 Hz, 1H), 6.87 (d, J = 7.4 Hz, 1H), 2.27 (s, 3H), 1.42 (s, 4H).
4
.1.1.10.1-(p-tolylcarbamoyl)cyclopropane-1-carboxylic acid (2J). White solid (79%),
1
H NMR (400 MHz, DMSO-d ) δ 13.11 (s, 1H), 10.54 (s, 1H), 7.60 – 7.36 (m, 2H),
6
7
.20 – 6.99 (m, 2H), 2.25 (s, 3H), 1.42 (s, 4H).
4
.1.1.11.1-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)cyclopropane-1-carboxyli
1
c acid (2K). White solid (67%), H NMR (400 MHz, DMSO-d ) δ 13.13 (s, 1H),
6

ACCEPTED MANUSCRIPT
1
1
1
0.88 (s, 1H), 8.20 (d, J = 2.3 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.71 – 7.43 (m, 1H),
1
3
.42 (s, 4H); C NMR (101 MHz, DMSO) δ 173.24, 167.32, 138.23, 131.95, 131.91,
26.82, 126.51, 124.01, 121.28, 117.97, 117.91, 29.02, 16.75.
4
.1.2. General Procedure for the Preparation of 3A-K.
To a two-necked flask, compound 2A-K (30 mmol), p-Phenylenediamine (60
mmol), EDCI (45 mmol), HOBt (36 mmol), DIEA (60 mmol) and DMF (30 mL)
were charged. The mixture was stirred at room temperature for 12 hours, then
quenched by water. The mixture was extracted by ethyl acetate, and the combined
organic layers were washed by saturated aqueous NaHCO solution, water and brine,
3
dried by anhydrous magnesium sulphate. The solvent was evaporated, and the residue
was purified by silica gel column chromatography.
4
.1.2.1. N-(4-aminophenyl)-N-(3-fluorophenyl)cyclopropane-1,1-dicarboxamide (3A).
1
Brown solid (39%), H NMR (400 MHz, DMSO-d ) δ 10.47 (s, 1H), 9.47 (s, 1H),
6
7
.69-7.58 (m, 1H), 7.40 -7.25 (m, 2H), 7.18 (d, J = 8.5 Hz, 2H), 6.88 (td, J = 7.8, 6.4,
1
3
2
.1 Hz, 1H), 6.50 (d, J = 8.5 Hz, 2H), 4.91 (s, 2H), 1.53 -1.35 (m, 4H); C NMR (101
MHz, DMSO) δ 169.07, 168.25, 162.47 (d, J = 240.8 Hz), 145.82, 141.06 (d, J = 11.2
Hz), 130.55 (d, J = 9.3 Hz), 127.84, 123.22, 116.25, 113.97, 110.33 (d, J = 21.4 Hz),
1
07.39 (d, J = 26.3 Hz), 31.38, 16.02.
4
.1.2.2. N-(4-aminophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (3B).
1
Brown solid (54%), H NMR (400 MHz, DMSO-d ) δ 10.26 (s, 1H), 9.57 (s, 1H),
6
7
.62 (dd, J = 8.9, 5.1 Hz, 2H), 7.20 (d, J = 8.3 Hz, 2H), 7.14 (t, J = 8.8 Hz, 2H), 6.51

ACCEPTED MANUSCRIPT
1
3
(
d, J = 8.4 Hz, 2H), 4.91 (s, 2H), 1.44 (d, J = 4.4 Hz, 4H); C NMR (101 MHz,
DMSO) δ 168.97, 168.33, 158.71 (d, J = 240.0 Hz), 145.80, 135.56, 127.91, 123.11,
1
22.71 (d, J = 7.9 Hz), 115.53 (d, J = 22.1 Hz), 114.03, 31.01, 16.05 .
4
.1.2.3.N-(4-aminophenyl)-N-(3-chlorophenyl)cyclopropane-1,1-dicarboxamide (3C).
1
Brown solid (58%), H NMR (400 MHz, DMSO-d ) δ 10.42 (s, 1H), 9.50 (s, 1H),
6
7
.85 (t, J = 2.1 Hz, 1H), 7.49 (dd, J = 8.1, 1.9 Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.26
-7.16 (m, 2H), 7.11 (dd, J = 7.9, 2.1 Hz, 1H), 6.55-6.44 (m, 2H), 4.91 (s, 2H),
1
3
1
1
.52-1.38 (m, 4H); C NMR (101 MHz, DMSO) δ 169.14, 168.14, 145.80, 140.78,
33.30, 130.63, 127.90, 123.59, 123.18, 120.21, 118.98, 113.99, 31.43, 16.02.
4
.1.2.4. N-(4-aminophenyl)-N-(4-chlorophenyl)cyclopropane-1,1-dicarboxamide (3D).
1
Brown solid (62%), H NMR (400 MHz, DMSO-d ) δ 10.39 (s, 1H), 9.52 (s, 1H),
6
7
.65 (d, J = 8.6 Hz, 2H), 7.39-7.32 (m, 2H), 7.20 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.5
1
3
Hz, 2H), 4.91 (s, 2H), 1.55 -1.38 (m, 4H); C NMR (101 MHz, DMSO) δ 169.01,
1
68.30, 145.80, 138.23, 128.86, 127.88, 127.56, 123.17, 122.27, 114.01, 31.23, 16.08.
4
.1.2.5.N-(4-aminophenyl)-N-(3-bromophenyl)cyclopropane-1,1-dicarboxamide (3E).
1
Brown solid (58%), H NMR (400 MHz, DMSO-d ) δ 10.41 (s, 1H), 9.51 (s, 1H),
6
8
2
1
4
.00 (s, 1H), 7.64 -7.38 (m, 1H), 7.37-7.09 (m, 4H), 6.52 (d, J = 8.2 Hz, 2H), 4.95 (s,
1
3
H), 1.45 (d, J = 8.6 Hz, 4H). C NMR (101 MHz, DMSO) δ 168.66, 167.67, 145.23,
40.41, 130.44, 127.47, 126.02, 122.69, 122.61, 121.31, 118.89, 113.57, 30.90, 15.58.
.1.2.6.N-(4-aminophenyl)-N-(4-bromophenyl)cyclopropane-1,1-dicarboxamide (3F).
1
Brown solid (62%), H NMR (400 MHz, DMSO-d ) δ 10.38 (s, 1H), 9.50 (s, 1H),
6
7
.60 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.50 (d, J

ACCEPTED MANUSCRIPT
1
3
=
8.2 Hz, 2H), 4.92 (s, 2H), 1.44 (d, J = 7.1 Hz, 4H). C NMR (101 MHz, DMSO) δ
1
3
4
68.52, 167.81, 145.31, 138.16, 131.29, 127.38, 122.68, 122.15, 115.12, 113.53,
0.77, 15.59.
.1.2.7.N-(4-aminophenyl)-N-(3-(trifluoromethyl)phenyl)cyclopropane-1,1-dicarboxa
1
mide (3G). Brown solid (78%), H NMR (400 MHz, DMSO-d ) δ 10.50 (s, 1H), 9.73
6
(s, 1H), 8.16 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.53 (t, J = 8.2 Hz, 1H), 7.39 (d, J = 7.7
Hz, 1H), 7.22 (d, J = 8.2 Hz, 2H), 6.52 (d, J = 8.2 Hz, 2H), 4.91 (s, 2H), 1.59 -1.35 (m,
1
3
4
H); C NMR (101 MHz, DMSO) δ 168.94, 167.58, 145.22, 139.96, 129.59, 129.19
(d, J = 31.1 Hz), 127.57, 123.73, 125.65 – 120.14 (m), 122.54, 119.51, 116.42 (d, J =
4
.3 Hz), 113.52, 31.01, 15.49.
4
.1.2.8.N-(4-aminophenyl)-N-(4-(trifluoromethyl)phenyl)cyclopropane-1,1-dicarboxa
1
mide (3H). Brown solid (50%), H NMR (400 MHz, DMSO-d ) δ 10.64 (s, 1H), 9.46
6
(s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H),
1
3
6
.50 (d, J = 8.3 Hz, 2H), 4.93 (s, 2H), 1.45 (d, J = 8.8 Hz, 4H); C NMR (101 MHz,
DMSO) δ 168.78, 167.68, 145.31, 142.45, 127.36, 125.90–125.57 (m), 122.73, 119.91,
13.48, 31.04, 15.59.
1
4
.1.2.9.N-(4-aminophenyl)-N-(m-tolyl)cyclopropane-1,1-dicarboxamide (3I). Brown
1
solid (83%), H NMR (400 MHz, DMSO-d ) δ 10.31 (s, 1H), 9.58 (s, 1H), 7.48 -7.37
6
(m, 2H), 7.20 (dd, J = 8.0, 5.2 Hz, 3H), 6.88 (d, J = 7.5 Hz, 1H), 6.52 (d, J = 8.3 Hz,
1
3
2
H), 4.93 (s, 2H), 2.27 (s, 3H), 1.47 (d, J = 6.4 Hz, 4H); C NMR (101 MHz, DMSO)
δ 168.89, 168.77, 145.84, 139.03, 138.22, 128.87, 127.77, 124.73, 123.23, 121.27,
17.87, 114.04, 30.80, 21.59, 16.24.
1

ACCEPTED MANUSCRIPT
4
.1.2.10.N-(4-aminophenyl)-N-(p-tolyl)cyclopropane-1,1-dicarboxamide (3J). Brown
1
solid (47%), H NMR (400 MHz, DMSO-d ) δ 10.19 (s, 1H), 9.56 (s, 1H), 7.48 (dd, J
6
=
8.1, 5.0 Hz, 2H), 7.21-7.14 (m, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.60 – 6.45 (m, 2H),
1
3
4
1
1
.92 (s, 2H), 2.25 (s, 3H), 1.51-1.40 (m, 4H); C NMR (101 MHz, DMSO) δ 168.79,
68.63, 145.83, 136.59, 132.99, 129.39, 127.78, 123.13, 120.80, 114.00, 30.75, 20.92,
6.16.
4
.1.2.11.N-(4-aminophenyl)-N-(4-chloro-3-(trifluoromethyl)phenyl)cyclopropane-1,1-
1
dicarboxamide (3K). Yellow solid (29%), H NMR (400 MHz, DMSO-d ) δ 10.62 (s,
6
1
8
=
H), 9.54 (s, 1H), 8.26 (d, J = 2.5 Hz, 1H), 7.89 (dd, J = 8.9, 2.5 Hz, 1H), 7.63 (d, J =
.9 Hz, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 8.6 Hz, 2H), 5.26 (s, 2H), 1.44 (d, J
1
3
7.3 Hz, 4H); C NMR (101 MHz, DMSO) δ 169.40, 167.70, 145.70, 138.92,
32.44, 132.22, 128.07, 127.09, 126.78, 125.31, 124.59, 124.47, 123.06, 121.88,
19.41, 114.02, 31.78, 15.90.
1
1
4
.1.3.General Procedure for the Preparation of 4D,4F,4H,4J,4K.
To
a
two-necked
flask,
compound
2D,2F,2H,2J,2K(30
mmol),
4
-(aminomethyl)aniline dihydrochloride (30 mmol), EDCI (45 mmol), HOBt (36
mmol), DIEA (120 mmol) and DMF (30 mL) were charged. The mixture was stirred
at room temperature for 12 hours, then quenched by water. The mixture was extracted
by ethyl acetate, and the combined organic layers were washed by saturated aqueous
NaHCO solution, water and brine, dried by anhydrous magnesium sulphate. The
3
solvent was evaporated, and the residue was purified by silica gel column

ACCEPTED MANUSCRIPT
chromatography.
4
.1.3.1.N-(4-aminobenzyl)-N-(4-chlorophenyl)cyclopropane-1,1-dicarboxamide (4D).
1
Brown solid (70%). H NMR (400 MHz, DMSO-d ) δ 10.79 (s, 1H), 8.27 (d, J = 5.9
6
Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H),
6
4
1
4
.50 (d, J = 7.9 Hz, 2H), 4.96 (s, 2H), 4.15 (d, J = 5.8 Hz, 2H), 1.38 (d, J = 4.6 Hz,
1
3
H). C NMR (101 MHz, DMSO) δ 170.17, 168.34, 147.47, 137.62, 128.45, 128.15,
27.02, 126.08, 121.46, 113.68, 42.35, 29.43, 15.88.
.1.3.2.N-(4-aminobenzyl)-N-(4-bromophenyl)cyclopropane-1,1-dicarboxamide (4F).
1
Brown solid (55%). H NMR (400 MHz, DMSO-d ) δ 10.80 (s, 1H), 8.28 (t, J = 5.8
6
Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 7.9 Hz, 2H),
6
4
1
4
.50 (d, J = 7.9 Hz, 2H), 4.95 (s, 2H), 4.15 (d, J = 5.6 Hz, 2H), 1.38 (d, J = 4.7 Hz,
1
3
H). C NMR (101 MHz, DMSO) δ 170.18, 168.35, 147.49, 138.03, 131.36, 128.16,
26.07, 121.82, 115.05, 113.67, 42.35, 29.44, 15.92.
.1.3.3.N-(4-aminobenzyl)-N-(4-(trifluoromethyl)phenyl)cyclopropane-1,1-dicarboxa
1
mide (4H). Brown solid (70%). H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H), 8.28
6
(d, J = 5.9 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 7.9
Hz, 2H), 6.51 (d, J = 7.9 Hz, 2H), 4.95 (s, 2H), 4.16 (d, J = 5.6 Hz, 2H), 1.41 (d, J =
1
3
3
1
1
4
.2 Hz, 4H). C NMR (101 MHz, DMSO) δ 170.12, 168.71, 147.50, 142.27, 128.16,
26.06, 125.84 (d, J = 3.6 Hz), 124.62 (d, J = 213.2 Hz), 123.12 (d, J = 26.2 Hz),
19.68, 113.66, 42.37, 29.63, 16.02.
.1.3.4.N-(4-aminobenzyl)-N-(p-tolyl)cyclopropane-1,1-dicarboxamide (4J). Brown
1
solid (69%). H NMR (400 MHz, DMSO-d ) δ 10.67 (s, 1H), 8.29 (d, J = 5.9 Hz, 1H),
6

ACCEPTED MANUSCRIPT
7
.46 (dd, J = 8.2, 2.1 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 6.52
(dd, J = 8.2, 2.2 Hz, 2H), 4.97 (s, 2H), 4.16 (d, J = 5.5 Hz, 2H), 2.25 (s, 3H), 1.40 (dd,
1
3
J = 10.4, 2.7 Hz, 4H). C NMR (101 MHz, DMSO) δ 170.56, 168.04, 147.48, 136.05,
1
32.45, 129.00, 128.16, 126.09, 119.92, 113.71, 42.32, 28.94, 20.41, 16.01.
4
.1.3.5.N-(4-aminobenzyl)-N-(4-chloro-3-(trifluoromethyl)phenyl)cyclopropane-1,1-d
1
icarboxamide (4K). Brown solid (80%). H NMR (400 MHz, DMSO-d ) δ 10.94 (s,
6
1
7
2
1
1
4
H), 8.31 (t, J = 5.9 Hz, 1H), 8.25 (d, J = 2.3 Hz, 1H), 7.85 (dd, J = 8.8, 2.3 Hz, 1H),
.63 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 7.9 Hz, 2H), 6.50 (d, J = 7.9 Hz, 2H), 4.95 (s,
1
3
H), 4.15 (d, J = 5.7 Hz, 2H), 1.38 (s, 4H). C NMR (101 MHz, DMSO) δ 169.59,
68.83, 147.49, 138.28, 131.79, 128.15, 126.52 (d, J = 30.6 Hz), 126.09, 124.62,
23.99, 122.73 (d, J = 272.8 Hz), 118.63 (d, J = 5.7 Hz), 113.64, 42.39, 30.03, 15.65.
.1.4.General Procedure for the Preparation of 5A-K, 6D,6F,6H,6J,6K.
To
a
suspension
of
6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-
1
-isopropyl-2-methyl-1H-benzo[d]imidazole (7) (645.48 mg, 2 mmol) in 20 mL
1
,4-dioxane were added compound 3A-K, 4D,4F,4H,4J,4K(2 mmol), Pd(OAc) (11
2
mg, 0.05 mmol), BINAP (62 mg, 0.1 mmol) and Cs CO (978 mg, 3 mmol) and the
2
3
flask was purged with Ar. Then the flask was sealed and the mixture was heated for
2 h at 100℃ . The reaction was cooled to rt, the solvent was removed under reduced
pressure, and the residue was purified by silica gel column chromatography to obtain
1
5
A-K, 6D,6F,6H,6J,6K.
4
.1.4.1.N-(4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)p
yrimidin-2-yl)amino)phenyl)-N-(3-fluorophenyl)cyclopropane-1,1-dicarboxamide(5A

ACCEPTED MANUSCRIPT
1
)
. Light yellow solid, Yield(55%), Mp 227 ℃ . H NMR (400 MHz, DMSO-d ) δ 10.35
6
(s, 1H), 9.89 (s, 1H), 9.74 (s, 1H), 8.59 (dd, J = 3.9, 1.2 Hz, 1H), 8.25 (d, J = 1.3 Hz,
1
H), 7.74 (d, J = 9.0 Hz, 2H), 7.68- 7.61 (m, 2H), 7.57 (d, J = 8.9 Hz, 2H), 7.43-7.29
(m, 2H), 6.94 -6.82 (m, 1H), 4.82 (p, J = 6.9 Hz, 1H), 2.63 (s, 3H), 1.62 (d, J = 6.9 Hz,
1
3
6
H), 1.48 (d, J = 6.8 Hz, 4H). C NMR (101 MHz, DMSO) δ 168.98, 168.26, 162.48
(d, J = 241.3 Hz), 156.79, 154.92, 153.99, 151.59 (d, J = 13.7 Hz), 150.25, 149.15,
148.24 (d, J = 26.8 Hz), 141.15 (d, J = 11.2 Hz), 136.96, 136.77 (d, J = 9.9 Hz),
133.62 (d, J = 16.6 Hz), 133.30, 130.52 (d, J = 8.9 Hz), 127.27 (t, J = 6.2 Hz), 121.47,
119.20, 116.38, 110.36 (d, J = 21.1 Hz), 109.21 (d, J = 6.9 Hz), 107.51 (d, J = 26.2
1
9
Hz), 48.57, 32.04, 21.40, 15.94, 14.99. F NMR (376 MHz, DMSO-d ) δ -107.69(s,
6
1
F), -124.01(s, 1F), -146.01(s, 1F). ESI-HRMS m/z calcd for Chemical Formula:
+
+
C H F N O 600.2329, found 600.2323 [M + H] . HPLC purity 99%.
32
29
3
7
2
4
.1.4.2.N-(4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)p
yrimidin-2-yl)amino)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide(5B).
1
Light yellow solid, Yield(45%), Mp 140 ℃ . H NMR (400 MHz, DMSO-d ) δ 10.12
6
(
s, 1H), 9.98 (s, 1H), 9.73 (s, 1H), 8.64 – 8.55 (m, 1H), 8.25 (d, J = 1.3 Hz, 1H), 7.73
d, J = 9.0 Hz, 2H), 7.68 – 7.61 (m, 3H), 7.57 (d, J = 8.7 Hz, 2H), 7.15 (t, J = 8.9 Hz,
(
2
4
1
1
1
H), 4.83 (p, J = 6.9 Hz, 1H), 2.63 (s, 3H), 1.62 (d, J = 6.8 Hz, 6H), 1.51 – 1.42 (m,
1
3
H). C NMR (101 MHz, DMSO) δ 168.88, 168.34, 158.75 (d, J = 239.8 Hz), 156.81,
54.94, 154.00, 151.59 (d, J = 14.0 Hz), 150.27, 149.16, 148.24 (d, J = 26.2 Hz),
36.92, 136.78 (d, J = 9.8 Hz), 133.62 (d, J = 16.5 Hz), 133.32, 127.27 (t, J = 6.6 Hz),
22.85 (d, J = 7.8 Hz), 121.36, 119.25, 115.50 (d, J = 22.4 Hz), 109.25, 107.42 (dd, J

ACCEPTED MANUSCRIPT
19
=
20.1, 8.1 Hz), 48.57, 31.65, 21.41, 15.96, 15.00. F NMR (376 MHz, DMSO-d ) δ
6
-114.26(s, 1F), -124.04(s, 1F), -145.87(s, 1F).ESI-HRMS m/z calcd for Chemical
+
+
Formula: C H F N O 600.2329, found 600.2322 [M + H] . HPLC purity 100%.
3
2
29
3
7
2
4
.1.4.3.N-(3-chlorophenyl)-N-(4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benz
o[d]imidazol-6-yl)pyrimidin-2-yl)amino)phenyl)cyclopropane-1,1-dicarboxamide
1
(
5C). Light yellow solid, Yield(51%), Mp 155 ℃ . H NMR (400 MHz, DMSO-d ) δ
6
10.29 (s, 1H), 9.90 (s, 1H), 9.73 (s, 1H), 8.60 (d, J = 3.9 Hz, 1H), 8.25 (d, J = 1.3 Hz,
1H), 7.86 (t, J = 2.1 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.65 (dd, J = 11.9, 1.2 Hz, 1H),
7.60 – 7.50 (m, 3H), 7.33 (t, J = 8.1 Hz, 1H), 7.12 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 4.83
(p, J = 6.9 Hz, 1H), 2.63 (s, 3H), 1.62 (d, J = 6.9 Hz, 6H), 1.47 (d, J = 6.0 Hz, 4H).
1
3
C NMR (101 MHz, DMSO) δ 169.02, 168.16, 156.81, 154.94, 154.00, 151.60 (d, J
=
14.4 Hz), 150.29, 149.16, 148.26 (d, J = 27.8 Hz), 140.86, 136.94, 136.79 (d, J =
9
1
2
1
6
.8 Hz), 133.62 (d, J = 16.6 Hz), 133.34, 130.61, 127.31 (d, J = 6.3 Hz), 123.63,
21.44 , 120.33, 119.23, 119.09, 109.26, 107.43 (dd, J = 20.1, 8.2 Hz), 48.58, 32.06,
1
9
1.41, 15.92, 15.01. F NMR (376 MHz, DMSO-d ) δ -123.91 (s, 1F) , -145.72 (s,
6
+
F).ESI-HRMS m/z calcd for Chemical Formula: C H ClF N O 616.2034, found
3
2
29
2
7
2
+
16.2033 [M + H] . HPLC purity 100%.
4
.1.4.4.N-(4-chlorophenyl)-N-(4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benz
o[d]imidazol-6-yl)pyrimidin-2-yl)amino)phenyl)cyclopropane-1,1-dicarboxamide
1
(
5D). Light yellow solid, Yield(57%), Mp 245 ℃ . H NMR (400 MHz, DMSO-d ) δ
6
1
0.22 (s, 1H), 9.90 (s, 1H), 9.73 (s, 1H), 8.61 (d, J = 3.8 Hz, 1H), 8.31 – 8.16 (m, 1H),
.75 – 7.70 (m, 2H), 7.69 – 7.62 (m, 3H), 7.56 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.9 Hz,
7

ACCEPTED MANUSCRIPT
2
H), 4.83 (p, J = 6.9 Hz, 1H), 2.63 (s, 3H), 1.61 (d, J = 6.9 Hz, 6H), 1.46 (d, J = 4.3
13
Hz, 4H). C NMR (101 MHz, DMSO) δ 168.40, 167.78, 156.33, 154.46, 153.50,
51.10 (d, J = 14.1 Hz), 149.83, 148.67, 147.77 (d, J = 28.3 Hz), 136.43, 136.29 (d, J
10.1 Hz), 133.12 (d, J = 16.7 Hz), 132.83, 128.35, 127.11, 126.78 (t, J = 6.6 Hz),
21.90, 120.92, 118.75, 108.76, 106.93 (d, J = 12.0 Hz), 48.08, 31.42, 20.92, 15.43,
1
=
1
1
19
4.52. F NMR (376 MHz, DMSO-d ) δ -124.04 (s, 1F), -145.95(s, 1F). ESI-HRMS
6
+
m/z calcd for Chemical Formula: C H ClF N O 616.2034, found 616.2030 [M +
3
2
29
2
7
2
+
H] . HPLC purity 100%.
4
.1.4.5.N-(3-bromophenyl)-N-(4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benz
o[d]imidazol-6-yl)pyrimidin-2-yl)amino)phenyl)cyclopropane-1,1-dicarboxamide
1
(
5E). Light yellow solid, Yield(43%), Mp 201 ℃ . H NMR (400 MHz, DMSO-d ) δ
6
1
1
7
6
1
0.26 (s, 1H), 9.89 (s, 1H), 9.73 (s, 1H), 8.62 (d, J = 3.9 Hz, 1H), 8.25 (d, J = 1.4 Hz,
H), 8.00 (d, J = 2.3 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.68 – 7.62 (m, 1H), 7.58 –
.54 (m, 3H), 7.31 – 7.19 (m, 2H), 4.83 (p, J = 6.9 Hz, 1H), 2.64 (s, 3H), 1.62 (d, J =
1
3
.8 Hz, 6H), 1.46 (d, J = 5.7 Hz, 4H). C NMR (101 MHz, DMSO) δ 168.50, 167.63,
56.32, 154.46, 153.50, 151.10 (d, J = 14.2 Hz), 149.84, 148.67, 147.78 (d, J = 26.6
Hz), 140.50, 136.42, 136.29 (d, J = 9.8 Hz), 133.12 (d, J = 16.8 Hz), 132.86, 130.44,
1
1
26.78 (t, J = 6.3 Hz), 126.03, 122.70, 121.24, 119.96 (d, J = 195.3 Hz), 118.74,
1
9
08.75, 107.34 – 106.13 (m), 48.08, 31.58, 20.92, 15.40, 14.52. F NMR (376 MHz,
DMSO-d ) δ -123.94(s, 1F), -145.99 (s, 1F). ESI-HRMS m/z calcd for Chemical
6
+
+
Formula: C H BrF N O 660.1529, found 660.1530 [M + H] . HPLC purity 97%.
3
2
29
2
7
2