Discovery of a highly potent, selective and novel CDK9 inhibitor as an anticancer drug candidate

Article abstract of DOI:10.1016/j.bmcl.2017.06.041

A series of novel hybrid structure derivatives, containing both LEE011 and Cabozantinib pharmacophore, were designed, synthesized and evaluated. Surprisingly, a compound 4d was discovered that highly exhibited effective and selective activity of CDK9 inhi

Full text of DOI:10.1016/j.bmcl.2017.06.041

Accepted Manuscript
Discovery of a highly potent, selective and novel CDK9 inhibitor as an anti-
cancer drug candidate
Yongtao Li, Qingxiang Guo, Chao Zhang, Zhi Huang, Tianqi Wang, Xin Wang,
Xiang Wang, Guangwei Xu, Yanhua Liu, Shengyong Yang, Yan Fan, Rong
Xiang
PII:
S0960-894X(17)30643-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.041
BMCL 25075
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 March 2017
12 June 2017
14 June 2017
Please cite this article as: Li, Y., Guo, Q., Zhang, C., Huang, Z., Wang, T., Wang, X., Wang, X., Xu, G., Liu, Y.,
Yang, S., Fan, Y., Xiang, R., Discovery of a highly potent, selective and novel CDK9 inhibitor as an anticancer
drug candidate, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.06.041
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Discovery of a highly potent, selective and novel
CDK9 inhibitor as an anticancer drug candidate
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Yongtao Li⊥a,b, Qingxiang Guo⊥a,b, Chao Zhang a, Zhi Huang a, Tianqi Wang a, Xin
Wang a, Xiang Wang a, Guangwei Xu a, Yanhua Liu a, Shengyong Yang e, Yan Fana,c,*
and Rong Xianga,d,
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a School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China
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b Tianjin International S & T Cooperation Base, 94 Weijin Road, Tianjin 300071,
China
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c International Collaborative Laboratory of Biomedicine of the Ministry of Education,
94 Weijin Road, Tianjin 300071, China
d
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2011 Project Collaborative Innovation Center for Biotherapy of Ministry of
Education, 94 Weijin Road, Tianjin 300071, China
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e Medical Oncology, Cancer Center, State Key Laboratory of Biotherapy, West China
Hospital, Sichuan University, Chengdu, China
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*
Corresponding authors:
*
Yan Fan: Tel: +86-22-2350-9482. Fax:
+86-22-2350-9482. E-mail: yanfan@nankai.edu.cn; *Rong Xiang: Tel:
+86-22-2350-9482. Fax: +86-22-2350-9482. E-mail: rxiang@nankai.edu.cn.
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Author Contributions:⊥Yongtao Li and Qingxiang Guo are contributed equally to
this work.
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Abstract
A series of novel hybrid structure derivatives, containing both LEE011 and
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Cabozantinib pharmacophore, were designed, synthesized and evaluated.
Surprisingly, a compound 4d was discovered that highly exhibited effective and
selective activity of CDK9 inhibition with IC50 = 12 nM. It effectively induced
apoptosis in breast and lung cancer cell lines at nanomolar level. Molecular docking of
4d to ATP binding site of CDK9 kinase demonstrated a new hydrogen bonding
between F atom of 4-(3-fluorobenzyloxy) group and ASN116 residue, compared with
the positive control, LEE011. The compound 4d could block the cell cycle both in
G0/G1 and G2/M phase to prevent the proliferation and differentiation of cancer cells.
Mice bared-breast cancer treated with compound 4d showed significant suppression
of cancer with low toxicity. Taken together, this novel compound 4d could be a
promising drug candidate for clinical application.
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Keywords: CDK9, LEE011, Inhibitor, Cancer

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Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse received the 2001 Nobel
Prize in Physiology or Medicine for their complete description of how cells regulate
proliferation and the central role of Cyclin-dependent kinases (CDKs).1 The CDKs
have two major functional groups based on their roles that mediate cell cycle
(subtypes 1–4 and 6) and regulate transcription control (subtypes 5, 7-9).2
Cdk4–CyclinD complexes regulate the G0/G1 transition and the early phases of G1
through phosphorylation of the Rb. CDK9 is a component of the multiprotein
complex TAK/P-TEFb and a key regulatory kinase of mRNA polymerase II (PII) and
a well-validated target for treating cancers.3 Inhibition of CDK9 associated with
blocking RNA synthesis by inhibitors can lead to a reduction in the levels of
short-lived pro-survival proteins and the induction of apoptosis, which provides an
effective approach to the control of tumor growth.4-5
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Multiple generations of drugs targeting CDK have been designed and synthesized
through binding in the ATP-binding cleft of CDK enzymes. The first generation of
CDK-directed drugs entered clinical trials in the 1990s and were found to be
nonspecific, pan-CDK, cytotoxic and ineffective. Second-generation CDK-directed
drugs have toxicities which limit their clinical utility. Third-generation CDK-directed
drugs have low toxicity, potent and selective CDK4 inhibition6-8. LEE011
(Novartis/Astex) is a bioavailable, selective inhibitor targeting both CDK4 and CDK6
that prevents Rb phosphorylation, resulting in cell cycle G0/G1 phase arrest.9-10 It
received FDA breakthrough therapy designation as first-line treatment for
HR+/HER2- advanced breast cancer in 2016.7 But in clinical trials, LEE011 needed to
combine with letrozole for the treatment of ER+/HER2− metastatic breast cancer,11-13
which indicated a lack of sensitivity to CDK4 monotherapy. Therefore, we reasoned
that development of single drug with multiple biological targets based on CDK4 to
improve efficacy in cancer treatment.

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Figure 1. Schematic showing design for merged CDK-VEGFR pharmacophore.
The vascular endothelial growth factor (VEGF) is a primary regulator of
physiological new vessel formation. Disruption of VEGF signaling has been
demonstrated to retard angiogenesis and inhibit tumor growth14. It has been reported
that VEGFR-2 inhibitors possess synergistic antitumor effects in rational
combinations with anticancer drugs15-16. Cabozantinib is a highly potent small
molecule inhibitor against VEGFR-2 (IC50 = 0.035 nM).17-18 In our study, a series of
novel dual-action compounds was designed and synthesized to target CDK4 and
VEGFR2. In vitro kinase profiling and cell-based screening together with in vivo
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−activity relationship (SAR) studies finally led to the discovery of
assays and structure
compound 4d, which was a hybrid structure containing both pyrrolo[2,3-d]
pyrimidines-2-amine of LEE011 and
N-(4-fluorophenyl)-N-phenylcyclopropane-1,1-dicarboxamide moiety of cabozantinib.
Fortuitously, it was found that the compound 4d has a high potent inhibition of CDK9
(IC50 = 12 nM). This compound showed potent anti-tumor activities both in vitro and
in vivo, and low toxicity. In this paper, we report the synthesis and biological
evaluation of the resulting novel CDK9 compounds (Figure 1).

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Scheme 1. Synthesis of Compounds 4a-u.
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5
6
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Reagents and conditions: (a) SOCl2, Et3N, THF, then aniline or substituted aniline; (b)
p-phenylenediamine,
EDCI,
HOBt,
DIEA,
rt;
(c)
2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide(5), Pd(AcO)2,
BINAP, Cs2CO3, 100 °C; (d) 6, EDCI, HOBt, DIEA; (e) p-Phenylenediamine, Pd(AcO)2, BINAP,
Cs2CO3, 100 °C.
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Scheme 1 outlines the routes used to synthesize compounds 4a–u. Briefly,
compound 2a-u was synthesized by cyclopropane-1,1-dicarboxylic acid condensating
with aniline or substituted aniline.19 The condensation of p-phenylenediamine with
−f
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carboxylic acids 2a-u afforded 3a
and 3i-u under condensing agent
1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) in the
presence of 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA).20
The condensation of carboxylic acids 2g-h with compound 6, which was obtained
from compound 5 that was coupled to the p-Phenylenediamine using the
Buchwald–Hartwig reaction, afforded 4g-h. A palladium catalyzed cross-coupling
−f
reaction of compounds 3a and 3i-u with compound 5 provided compounds 4a
−f
and 4i-u, respectively.21

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Scheme 2. Synthesis of Compounds 10-12.
Reagents and conditions: (a) Pd(AcO)2, BINAP, Cs2CO3, 100 °C; (b) Fe, NH4Cl, EtOH/H2O, reflux; (c) SOCl2,
then 3-fluoro-4-nitroaniline; (d) EDCI, HOBt, DIEA.
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Synthetic routes for compounds 10-12 are outlined in Scheme 2. A palladium
catalyzed cross-coupling reaction of 2-Amino-5-nitropyridine or compounds 9 with
compound 5 provided compounds 7 or 10, respectively. Compound 7 was treated with
ammonium chloride and iron powder for reduction of nitro to obtain compound 8. The
intermediate aniline 9 was obtained from the nitro, which was prepared by
condensation reaction of 3-fluoro-4-nitroaniline with 2d.19 The condensation of
carboxylic acids 2d with 8 or LEE011 afforded 11 or 12 under condensing agent
EDCI in the presence of HOBt and DIEA, respectively.
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Table 1. Structure and inhibitory activity of 4a-u against CDK4/@ 1 µM and
VEGFER2/@ 1 µMa
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CDK4/@ 1 µM
VEGFR2/@ 1 µM
Compound
R
（Inhibition%）
（Inhibition%）
4a
H
82
8
4b
4c
4d
4e
4f
2-F
3-F
75
61
102
44
65
72
48
66
62
26
32
66
77
94
95
53
84
76
77
24
-2
7
IC >10 µM
4-F
50
3-Cl
15
5
4-Cl
4g
4h
4i
3-Br
6
4-Br
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5
3-CH3
4-CH3
3-CF3
4-CF3
3-OCH3
4-OCH3
3-OH
4j
9
4k
4l
-10
1
4m
4n
4o
4p
4q
4r
4s
4t
-2
19
24
36
29
21
-7
2
4-OH
3-CN
4-CN
3-COOCH3
4-COOCH3
3-CF3, 4-Cl
4u
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4
5
aIC50 values and inhibition values were determined using KinaseProfiler by Eurofins. The data represent the mean
values of two independent experiments.

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Table 2. Structure and inhibitory activity of compound 10-12 against CDK4/@1µM
and VEGFER2 /@1µMa
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CDK4/@ 1 µM
VEGFR2/@ 1 µM
Compound
R
（Inhibition%）
（Inhibition%）
87
85
97
33
-3
-8
10
11
12
a
4
5
IC50 values and inhibition values were determined using KinaseProfiler by Eurofins. The data represent the mean
values of two independent experiments.
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The enzyme inhibitory activities are summarized in Table 1 and Table 2. Analogue
4d displayed a better enzymatic potency than the 2-fluoro analogue 4b and the
3-fluoro analogue 4c, suggesting that 4-fluoro substitution is superior to 2-fluoro and
3-fluoro substitution. Analogue 4a, 10 and 11 displayed similar enzymatic potency,
suggesting that pyridine ring could be replaced by benzene ring and fluoro-substitued
benzene. Compound 4o and 4p have stronger inhibition targeting CDK4 than other
analogs except compound 12, most likely because R groups of compound 4o and 4p
are hydrophilic groups. Compound 12 shows stronger inhibition targeting CDK4 than
other analogs, suggesting that piperazine ring is important for CDK4 enzymatic
activity. Compound 4k, 4l and 4u, which possess a trifluoromethyl group, showed
very low binding affinity. Collectively, among all the compounds synthesized, 4d, 4o,
4p and 12 are the promising compounds targeting CDK4. Unfortunately, these
compounds show low inhibition effect targeting VEGFR2.
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Table 3. In vitro growth inhibitory activities of compound 4d against human cancer
cell lines.
IC50 (nM)
T47D
MCF7
H460
H1299
A549
>10000
Cell lines
>10000
2889
338
>10000
5669
359
7637
1919
221
6227
4448
358
LEE011
Cabozantinib
4d
4205
456
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In the meanwhile, we detected the antiviability activity of these compounds against
a panel of human cancer cell lines using standard CCK8 assay. Cancer cell lines
inhibition profiling assays with a fixed concentration of 1 µM and 10 µM were first
carried out. The data is presented in Table S1. The compounds 4d and 4p that showed
a high inhibitory rate at 1 µM and 10 µM, were consistent with the kinase result of
CDK4. Compared with 4p, compound 4d demonstrated strong inhibition in T47D
(breast cancer cell line) and A549 and H1299 (lung cancer cell lines). Negligible
activity was observed for other selected cell lines, HeLa and SiHa (cervical cancer),
OVCAR5(ovarian cancer), which indicated that 4d has a considerable selectivity.
Then we confirmed the inhibition on tumor cells of compound 4d as shown in Table 3.
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4d potently inhibited the proliferation of the breast cancer cell lines T47D and MCF7,
and lung cancer cell lines, H460, H1299 and A549, with half-maximal inhibitory
concentration (IC50) values of 358 nM, 338 nM, 359 nM, 221 nM and 456 nM,
respectively. The CDK4 selective inhibitor LEE011 was not potent in these cells,
indicating a lack of sensitivity to CDK4 monotherapy. This result demonstrated that
compound 4d and 4p maybe have other target kinase.
Table 4. Inhibition of compound 4d and 4p against CDK kinases at 1 µM. a
Inhibition%/@ 1 µM
Compound
CDK1
/cyclinB(h)
73
CDK2
/cyclinA(h)
29
CDK2
/cyclinE(h)
85
CDK3
/cyclinE(h)
65
CDK5
/p25(h)
86
CDK5
/p35(h)
81
CDK6
CDK7
CDK9
CDK12
/cyclinK(h)
24
CDK18
/cyclinD3(h)
/cyclinH/MAT1(h)
/cyclin T1(h)
/cyclinY(h)
4d
4p
71
86
7
98
26
6
0
90
90
88
89
94
88
-9
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a
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IC50 values and inhibition values were determined using KinaseProfiler by Eurofins. The data represent the mean
values of two independent experiments.

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Table 5. Inhibition of compound 4d and LEE011 against CDK9 and CDK4a
CDK4 IC50 (nM)
CDK9 IC50 (nM)
Compound
13
197
12
LEE011
4d
142
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aIC50 values were determined using KinaseProfiler by Eurofins. The data represent the mean values of two
independent experiments.
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We next evaluated the kinase inhibitory activities against CDK kinases of
compound 4d and 4p with a fixed concentration of 1µM through the Eurofins kinase
profiling. The results are provided in Table 4 indicated 4d have comparable potency
and selectivity with 4p. Both the preliminary study on cell and kinase inhibition
declared 4d is the most promising candidate for the treatment of cancer. In-depth
studies including in vitro and in vivo anti-tumor studies were then carried out on 4d.
CDK4 and CDK9 further IC50 were examined and the results are shown in Table 5. As
indicated in the data, we found that compound 4d has exhibited highly potent
inhibition of CDK9 and selectivity to other CDKs. 4d showed high inhibitory activity
against CDK9 (IC50 = 12 nM) and low inhibitory activity against CDK4 (IC50 = 142
nM). While, LEE011 showed high inhibitory activity against CDK4 with IC50 valueof
13 nM rather than CDK9 with IC50 value of 197 nM. Through a complicated process
of structural synthesis and optimization, we finally discover a new compound, 4d, has
potent effect on CDK9 and selectivity for other CDKs and tumor cells in vitro.
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Figure 2. The docking of compound 4d to the active site of CDK9 (PDB:4bcg), using Discovery
Studio 3.1(Accelrys Inc., San Diego, CA, USA): LEE011 (green), and compound 4d (brown). The
H-bonds are shown by dashed red lines.
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Molecular docking of 4d to ATP binding site of CDK9 kinase was performed by

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using Discovery Studio 3.1(Accelrys Inc., San Diego, CA, USA). Here CDK9 in
complex with cyclin T and a 2-amino-4-heteroaryl-pyrimidine inhibitor crystal
structure (PDB:4bcg)22 was selected as the binding model. The docking results
showed compound 4d and LEE011 were in similar binding mode as shown in Figure
2. LEE011 (green) and 4d (brown), both bound in the ATP binding pocket of the
kinases, form two hydrogen bonds with conserved CYS106 and one hydrogen bond
with conserved ASP167. While the F atom of 4-(3-fluorobenzyloxy) group of
compound 4d was directly involved in hydrogen bonding with ASN116 residue.
These changes should be the reason of their different inhibitory activity on CDK9. In
an attempt to find the CDK4 inhibitory difference between the compound 4d and
LEE011, we docked compound 4d and LEE011 into the CDK4 protein in Figure 3.
Compared with the LEE011, the hydrogen bonding interaction between
dimethylcarbamoyl group and ASP-158 in 4d was too weak, which lead to the
significant selectivity of compound 4d for CDK9 over CDK4.
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Figure 3. The docking of compound 4d to the active site of CDK4 (PDB: 2W96), using Discovery
Studio 3.1(Accelrys Inc., San Diego, CA, USA): LEE011 (green), and compound 4d (brown). The
H-bonds are shown by dashed lines.
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We investigated the effect of compound 4d treatment on breast cancer cell line
(T47D) cell cycle progression (Figure 3). Cells were treated with compound 4d with
varying concentrations (0.1 µM, 0.5 µM and 1 µM), LEE011 and vehicle (DMSO) for
24h, and subjected to flow cytometry analysis to determine the distribution of cells in
various phases of the cell cycle. LEE011 treatment for 24h led to a majority of cells in
G0/G1 phase, which is associated with CDK4 inhibition. Compared to the

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vehicle-treated control, cells treated with compound 4d have been demonstrated an
increase of G2 and G0/G1 phase, with smaller percentages in S phase. Compound 4d
blocked the cells in G2 and G0/G1 phase, which resulted in decreased S-phase
populations. The increase of the G2 phase by compound 4d was indicative of cells
undergoing apoptosis, which may be related to the excellent cellular CDK9 inhibitory.
The result in the appearance of G0/G1 population confirmed that compound 4d had
multi-intracellular targets activity on CDK4.
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Figure 4. Compound 4d induces cell apoptosis of T47D cell line in vitro. (A) Flow cytometry and
(B) quantitative analysis of apoptotic cells. Cells were incubated with the indicated concentrations
of compound 4d or LEE011 for 48h and were stained with FITC-Annexin V/PI, followed by flow
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cytometry analysis. Data are expressed as means SD of the percentages of apoptotic cells from
three independent experiments, P < 0.05.
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Breast cancer cells (T47D) were treated with compound 4d, vehicle (DMSO) and
LEE011 as positive control for 48h and analyzed by annexin V/PI staining. As shown
in Figure 4, compound 4d induced cell apoptosis in a dose-dependent manner. An
increase in dose led to increase in annexin V and PI positive cells, indicating
apoptotic events. Compared with the LEE011 and vehicle condition, compound 4d
was significantly more active in apoptosis induction as the concentration increasing.
This study illustrated that compound 4d could induce cell death via apoptosis. In
addition, migration of 4d-treated T47D cells, suggested by transwell migration assay
(Fig. 5a and b), was significantly decreased compared to the untreated group.
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Figure 5. (A) Representative images of transwell migration assay for compound 4d-treated T47D
cells and (B) statistically analyzed. Cells were incubated in the upper transwell chambers and the
number of migrated cells was counted after 16 h incubation using microscope in five random
fields per chamber.
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The in vivo antitumor efficacy of 4d was assessed using 4T1 xenograft models. 4T1

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cells were orthotopically implanted into the mammary fat pads of 6−8 weeks old
female BALB/c mice. Once the tumors reached an average volume of 100 mm3, 4d
was administered daily via IP injection. They received different doses of compound
4d for 21 consecutive days. As shown in Figure 6A, IP injection dosing of 4d at 50,
100 and 150 mg/kg/day inhibited tumor growth in a dose-dependent manner in
models. During the treatment period, no weight loss (Figure 6B) was observed in all
the treatment groups. Our data clearly demonstrated that 4d had potent antitumor
activity against the growth of tumors in vivo.
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Figure 6. In vivo antitumor efficacy of 4d against 4T1 (A) tumor xenograft models and (B)
average body weights for treated mice. Animals were treated with solvent control, compound 4d at
doses of 150, 100 and 50 mg/kg/day through IP injection. Points indicate mean tumor volumes
(mm3) or mean body weights (g); bars indicate SD.
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In summary, through a hybrid-design approach based upon two privileged
pharmacophores,
namely
pyrrolo[2,3-d]
pyrimidines-2-amine
we
and
N-(4-fluorophenyl)-N-phenylcyclopropane-1,1-dicarboxamide,
finally
successfully discovered a novel inhibitor, compound 4d. Compound 4d is a novel
CDK9 inhibitor, which potently inhibited CDK9 with IC50 values of 12 nM. 4d
showed good selectivity in CDKs kinase profiling assay against CDK kinases and cell
proliferation inhibition. In vitro cellar assays, 4d showed potent activities against
human breast cancer cell lines, T47D and MCF7, human lung cancer cell lines, A549,
H1299 and H460. In vivo, compound 4d exhibited a considerable ability to inhibit
tumor and a low body weight loss of mice. These results confirmed that 4d showed
good antitumor efficacy and could be used as a novel CDK9 inhibitor to be further

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researched on the therapy of solid tumors. More works will be done to characterize
the therapeutic relevance of compound 4d.
Acknowledgments
This work was supported by the Natural Science Foundation of Tianjin (17JCQNJC13500) and
National key scientific research project (2013CB967201) and the National Natural Science
Foundation of China (81470354) and Research Found of the Doctoral Program of Higher
Education of China (No. 20100031120044).
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A series of novel hybrid structure derivatives, containing both LEE011 and
Cabozantinib pharmacophore, were designed, synthesized and evaluated.
Surprisingly, a compound 4d was discovered that highly exhibited effective and
selective activity of CDK9 inhibition with IC50 = 12nM. It effectively induced
apoptosis in breast and lung cancer cell lines at nanomolar level. The compound 4d
could block the cell cycle both in G0/G1 and G2/M phase to prevent the proliferation
and differentiation of cancer cells. Mice bared-breast cancer treated with compound
4d showed significant suppression of cancer with low toxicity.
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