Discovery and SARs of 5-Chloro- N4-phenyl- N2-(pyridin-2-yl)pyrimidine-2,4-diamine Derivatives as Oral Available and Dual CDK 6 and 9 Inhibitors with Potent Antitumor Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b02121

Cyclin-dependent kinases (CDKs) are promising therapeutic targets for cancer therapy. Herein, we describe our efforts toward the discovery of a series of 5-chloro-N⁴-phenyl-N²-(pyridin-2-yl)pyrimidine-2,4-diamine derivatives as dual CDK6 and 9 inhibitors. Intensive structural modifications lead to the identification of compound 66 as the most active dual CDK6/9 inhibitor with balancing potency against these two targets and good selectivity over CDK2. Further biological studies revealed that compound 66 was directly bound to CDK6/9, resulting in suppression of their downstream signaling pathway and inhibition of cell proliferation by blocking cell cycle progression and inducing cellular apoptosis. More importantly, compound 66 significantly inhibited tumor growth in a xenograft mouse model with no obvious toxicity, indicating the promising therapeutic potential of CDK6/9 dual inhibitors for cancer treatment. Therefore, the above results are of great importance in the development of dual CDK6/9 inhibitors for cancer therapy.

Full text of DOI:10.1021/acs.jmedchem.9b02121

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Article
Discovery and SARs of 5-chloro-N4-phenyl-N2-(pyridin-2-
yl)pyrimidine-2,4-diamine derivatives as oral available and
dual CDK 6 and 9 inhibitors with potent anti-tumor activity
yang wang, Xing Chen, Yaoyao Yan, Xiaochen Zhu, Mingming Liu, and XinHua Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02121 • Publication Date (Web): 04 Mar 2020
Downloaded from pubs.acs.org on March 5, 2020
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Discovery and SARs of 5-chloro-N -phenyl-N -(pyridin-2-yl)
pyrimidine-2,4-diamine derivatives as oral available and dual CDK 6
and 9 inhibitors with potent anti-tumor activity
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Yang Wang , Xing Chen , Yaoyao Yan , Xiaochen Zhu , Mingming Liu* and
,1
Xinhua Liu*
1. School of Pharmacy, Anhui Medical University, Hefei 230032, China
2.
Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235025, China
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Abstract
The cyclin-dependent kinases (CDKs) are promising therapeutic targets for
cancer therapy. Herein, we describe our efforts toward the discovery of a series of 5-
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chloro-N -phenyl-N -(pyridin-2-yl)pyrimidine-2,4-diamine derivatives as dual CDK6
and 9 inhibitors. Intensive structural modifications lead to the identification of
compound 66 as the most active dual CDK6/9 inhibitor with balancing potency
against these two targets and good selectivity over CDK2. Further biological studies
revealed that compound 66 directly bound to CDK6/9, resulting in suppression of
their down-stream signaling pathway and inhibition of cell proliferation by blocking
cell cycle progression and inducing cellular apoptosis. More importantly, compound
6
6 significantly inhibited tumor growth in an xenograft mouse model with no obvious
toxicity, indicating the promising therapeutic potential of CDK6/9 dual inhibitors for
cancer treatment. Therefore, the above results are of great importance in the
development of dual CDK6/9 inhibitors for cancer therapy.
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Graphic Abstract
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1
. Introduction
Cyclin-dependent kinases (CDKs) belong to a class of serine/threonine protein
kinases that act synergistically with specific cyclins that control cell cycle progression,
transcription, DNA repair, epigenetic regulation and other important processes
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involved in proliferation, differentiation, and apoptosis . Deregulation of cell cycle
progression is one of hallmarks of cancer, and inappropriate activation and/or
abnormal expression of CDKs were observed in a majority of human cancers. There is
considerable evidence showing that CDK4/6 and cyclin D are hyper-activated or
amplified in a wide spectrum of tumors, including breast carcinoma, melanomas,
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, 5
squamous cell carcinomas, and leukemias . Deregulation of the CDK4/6-cyclin D
pathway promotes cell cycle progression by sequentially phosphorylating the
retinoblastoma protein and facilitating the G1-S phase transition. Therefore, inhibition
of CDKs’ activity by synthetic small molecular inhibitors have been emerged as a
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logical approach in the development of novel cancer therapies .
A wide range of small-molecule CDKs inhibitors have been synthesized and
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-9
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studied
(Figure 1). Among them, Palbociclib (Pfizer), the highly selective
CDK4/6 inhibitor, has been approved for the treatment of ER positive/HER2 negative
breast cancer as the first-line medicine in February 2015, and it is currently prescribed
as a combination therapy together with anti-estrogen agents. Followed closely,
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Ribocilib (Novartis) and Abemaciclib (Eli Lilly) as CDK4/6 inhibitors have also
been approved by the FDA for breast cancer therapy. Although these have achieved
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good response , drug resistance often occurs after a short period of medication and
severe adverse reactions such as aneutropenia, anemia, fatigue and diarrhea constantly
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4, 15
appeared
. Increasing studies have indicated that resistance of tumor cells to
CDK4/6 inhibitors is not only due to the mutations at the binding site, resulting in loss
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of inhibitors’ binding affinity, but also response for mutations at down-stream factors
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such as Rb-E2F signaling pathway.
Therefore, development of resistance is
normally unavoidable for CDK4/6 inhibitors, and this is also true for other therapeutic
agents targeting one single target. Discovery of multi-targeting small molecule has
been emerged as a promising strategy to overcome resistance to signal-targeting
therapeutics.
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Figure 1. Structures of representative selective CDK inhibitors.
With the elucidation of the mechanism of cycle-dependent kinases and the
increasing number of co-crystal structures, the research of multi-target inhibitors has
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continued to expand
. Among them, co-targeting cycle-related kinases and non-
cycle-related kinases have achieved encouraging results, including simultaneously
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targeting CDK1/5 , CDK2/5 , CDK1/2/5 , CDK2/9 , CDK1/2/9 , and CDK4/9 .
These studies tend to achieve better results than single-target inhibitors. By analyzing
the characteristics of these and earlier discovered inhibitors, the researchers have
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gained more information for further development such as potent inhibitors of CDK1/2
often lead to increased toxicity, and some CDK2 inhibitors will act on unexpected
targets such as CDK5 and GSK3, which will increase the complexity of inhibitors
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development and adverse drugability
. Recently, Del Re et al. reported that CDK9
over expression in patients with metastatic breast cancer is related to the clinical
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resistance of CDK4/6 inhibitors and displays reduced sensitivities to anti-oestrogen
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therapies . Lee et al. also reported that CDK9 and CDK6 affect critical processes
involved in tumorigenesis, maintaining immaturity, proliferation, and potential
resistance pathways to currently available therapies in AML. Inhibiting CDK9 is now
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2
a novel approach to treat AML in target therapy combinations . Collectively, CDK9
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has been widely recognized as a potential target for antitumor , and its over
expression has been firmly confirmed to contribute the development of resistance to
CDK4/6 inhibitors. Therefore, we reasoned that small-molecule inhibitors
simultaneously targeting both CDK6 and 9 would not only result in
synergistic/additive anticancer activity, but also circumvent the resistance against
CDK4/6 inhibitors. Aligned with this need, we attempted to design and synthesize
selective CDK6/9 dual inhibitors, which can inhibit the cell cycle process and
transcription activity at the same time, so as to improve the efficacy, reduce the side
effects and delay the appearance of drug resistance.
2
. Results and Discussions
2
.1 Design
In order to discover potent dual CDK6 and 9 inhibitors, we screened our in-
house compounds library by enzymatic activity assay. From the screen, a pyrimidine
diamine derivative (compound 8) was identified as a preliminary hit with moderate
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inhibitory effects on CDK6 and 9 but more effective potency on CDK2. Cell
proliferation assay showed that compound 8 could suppress cell growth in a variety of
tumor cell lines with IC ranging from 38.6 to 56.2 μM. Starting from compound 8,
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with the aim to improve and balance potency on CDK6/9 and to elevate selectivity
over CDK2, a small molecule library containing 59 compounds were designed and
synthesized based on receptor structural-based drug design and in-depth SARs studies.
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4
Based on the co-crystal complex structures of CDK6-Palbociclib (5L2I)
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(
Figure 2A) and CDK9-A86 (6GZH) (Figure 2B), the binding mode compound 8 to
CDK6 and 9 were established by ZDOCK module in Discovery Studio 2017 R2.
From the docking models, key interactions between compound 8 and CDK6 (Figure
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C) or CDK9 (Figure 2D) were observed similar with Palbociclib or A86, including
CDK6
two hydrogen-bonds between 2-amino pyrimidine and key residues (Val101
CDK9
/Cys106
) located at the hinge region of CDK6 or 9. The o-methyl phenyl
substituent of compound 8, similar with the cyclopentyl of Palbociclib and the
cyclopropyl of A89, occupied the hydrophobic pockets formed by Val27, Ile19 and
Asn150 of CDK6 (Figure 2E), or by Val33, Ile25 and Phe30 of CDK9 (Figure 2F).
However, when compared with Palbociclib, compound 8 didn’t interact with
hydrophilic region of CDK6, in which hydrophilic residues Asp163, Lys43 formed
hydrogen bonds with acetyl of Palbociclib. CDK9 also possesses hydrophilic region
formed by Asp167 and Lys48. However, to our knowledge, few CDK9 inhibitor has
been found to occupy this region. In addition, the piperazine ring of Palbociclib was
located at solvent exposure region, but was identified to be critical for the inhibition
of CDK4/6 (Figure 2).
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Figure 2. (A) The interaction regions between CDK6 inhibitors represented by
Palbociclib and CDK6 (PDB: 5L2I); (B) CDK9 inhibitors represented by A86 and
CDK9 (PDB: 6GZH); Docking model of compound 8 and CDK6 (C) or CDK9 (D);
2D histogram of docking model of compound 8 and CDK6 (E) or CDK9 (F) to
indicate the active and potential sites of modifications of compound 8.
Based on the information observed from docking model, modifications and SAR
studies on hit 8 were carried out in a step-by-step manner. Firstly, the pyridine group
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(
region B) was replaced by phenyl, benzyl, phenylethyl or other heterocycles,
resulting in compound 16 with improved activity (step 1), which indicates essential
role of pyridine for potency. To simulate the piperazine ring of Palbociclib, we tried to
introduce various aliphatic heterocyclic structure at the end of pyridine ring in
compound 16, leading to discover N-methylpiperazine derivatives 23 with much
improvement on activity (step 2). Next, the substituents and length between phenyl
and pyrimidine (region A) was modified, but no compound achieved improvement
activity unfortunately. Any change in the distance between the pyrimidine and phenyl
is not conducive. So we identified the type of structure represented by compound 34
(step 3). To simulate cyclopentyl of Palbociclib occupying the hydrophobic pocket of
CDK6, we examined position and volume of hydrophobic substituents on the phenyl
ring. However, introduction of bulk hydrophobic groups can only increase the
inhibitory effect on CDK6, but diminish the effect on CDK9, probably due to small
size hydrophobic pocket of CDK9. Therefore, considering the balancing effects
between two targets, acetyl amino was introduced to interact with key residue located
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CDK6
CDK9
CDK6
CDK9
in hydrophilic region, including Lys43
resulting in the discovery of compound 66 with almost 100-fold (CDK6) and 170-fold
CDK9) increasing potency than that of hit 8 (step 4). More importantly, compared
/ Lys48
and Asp163
/ Asp167
,
(
with Palbociclib and Abemaciclib, compound 66 exhibited balancing potency against
CDK6 and 9 with almost equal IC values, and good selectively over CDK2 (Figure
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Figure 3. Step by step structural optimization on hit compound 8.
2.2 Synthesis
As depicted in Scheme 1, depending on the reactivity of the 2-Cl and the 4-Cl on
the pyrimidine ring, intermediates 3a~3ai were obtained by the reaction of compound
1
2
,4,5 trichloropyrimidine (1) with different amine R -NH (compounds 2a~2ai) in the
2
presence of TBAI and triethylamine. 1-bromo-4-nitrobenzene (4a) or 5-bromo-2-
nitropyridine (4b) is dissolved in DMSO and reacted with heterocyclic amine
(
compounds 5a~5f) at 90 °C in the presence of potassium carbonate and TBAI to give
nitro compounds (6p~6z). Hydrogenation of the nitro compounds (6p~6z) affords the
amine (compounds 7p~7z). Finally, the targeted compound 8-33 and 34-67 were
obtained by Buchwald–Hartwig reaction between compounds 3ai and 7a-7z as well as
between 3a-3ai and 7p, catalyzed by Pd (dba) and phosphorous ligands (BINAP or
2
3
XANTPHOS).
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a
Scheme 1. Synthesis of compounds 8~67
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Reagents and conditions: (a) 2,4,5-trichloropyrimidine, Substituted aniline or
substituted benzylamine or substituted phenethylamine, TBAI, Triethylamine,
DMSO, r.t. (b) 5-bromo-2-nitropyridine or 1-bromo-4-nitrobenzene, azaheterocyclic
amine, potassium carbonate, DMSO, 70 °C. (c) Pd/C 10%, H2. (d) compounds
3a~3ai,7a~7z, Pd (dba) , BINAP, NaOtBu, dioxane, 110 °C, Ar atmosphere. (e)
2
3
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compounds 3a~3ai,7a~7z, Pd (dba) , XANTPHOS, Cs CO , dioxane, 110 °C, Ar
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2
3
2
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atmosphere.
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5
5
5
5
6
2.3 SAR Studies.
Biological evaluation of CDK2, 6 and 9 inhibitory activities were conducted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TM
using ADP-Glo
kinase assay. The anti-proliferative activities of targeted
compounds were also determined in several human cancer cell lines, including MCF-
7, MDA-MB-231 and Jurkat. Meanwhile, the inhibition on normal human hepatocytes
L02 growth was measured in order to assess the selectivity for malignant over normal
cells. Palbociclib, NVP-2 and Abemaciclib were served as the positive controls.
In order to examine the effect of pyridine ring of hit 8 on inhibitory activities,
pyridine was replacing with aliphatic ring, benzene and furan, resulting in synthesis of
compounds 9~15. As listed in Table 1, these synthesized compounds exhibited much
weaker inhibitory activities against both CDK6 and 9 than hit 8. It is clear that
pyridine ring is critical for inhibitory activity on both enzyme and cell proliferation.
Based on this finding, pyridine was retained in the following structural modification.
Table 1. SAR at Position 2 of the Pyrimidine Scaffold.
a
b
IC (μM)
GI (μM)
50
1
50
No.
R
CDK6
CDK9
CDK2
MDA-MB-231
41.19±4.21
Jurkat
MCF-7
L-02
8
9
6.65±0.11
3.43±0.93
0.030±0.001
38.60±2.86
56.52±1.28
22.87±0.29
＞10
＞10
＞10
＞100
＞100
＞100
＞100
10
＞10
＞10
＞10
＞10
＞10
＞10
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
11
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Journal of Medicinal Chemistry
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
12
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
1
1
1
3
4
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Palbociclib
NVP-2
0.005±0.000 1.207±0.190
＞10
0.002±0.000
8.75±0.84
3.46±0.17
＞100
< 0.16
＞100
< 0.16
＞100
＞100
43.36±2.80
4.01±0.23
28.33±1.65
c
Abemaciclib
0.010±0.001 0.065±0.020 0.273±0.014
3.45±0.20
4.63±0.51
N.D.
a
Inhibition of cell-free kinases activities.
Inhibition of cell proliferation.
Not determined.
b
c
Next, as listed in Table 2, compounds 16~22 were synthesized to explore the
effects of N position, distance between the pyridine and pyrimidine, and substitution
of the pyridine ring on inhibitory activities. Extension of the distance between the
pyridine and pyrimidine significant decreased the inhibitory activities on both enzyme
and cell proliferation (compounds 20~22). Introduction of substituents including Cl
and methoxyl group (17 and 19) failed to increase activities. Comparing potencies
among 2-, 3- and 4-pyridyl derivatives (compounds 16, 8 and 18), we found that 2-
pyridyl derivative 16 possessed increased potency against CDK 6, but decreased
inhibition against CDK2. Although CDK9 inhibition of compound 16 was slightly
decreased, considering the improvement of activities on cell proliferative inhibition,
2-pyridyl substitution was fixed in the following modification.
Table 2. SAR at Position 2 of the Pyrimidine Scaffold.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
IC (μM)
GI (μM)
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
No.
R
CDK6
CDK9
CDK2
MDA-MB-231
Jurkat
MCF-7
L-02
16
2.76±0.14
8.11±0.15
0.643±0.016
11.24±2.06
52.43±9.78
68.09±0.03
20.07±2.57
31.37±0.26
＞100
6.15±0.34
41.53±3.61
＞100
17
18
＞10
＞10
＞10
﹥10
＞10
39.59±2.10
6.23±0.42
＞100
98.49±2.34
19
＞10
0.908±0.062 0.074±0.005
42.58±0.42
9.38±1.05
＞100
18.36±0.51
2
2
0
1
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞10
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
＞100
22
a
Inhibition of cell-free kinases activities.
Inhibition of cell proliferation.
b
Previous studies have highlighted the critical role of piperazine groups for CDK6
inhibitory activities of Palbociclib and Abemaciclib. Considering the structural
similarity between Palbociclib and compound 16, we tried to introduce an aliphatic
heterocyclic ring onto the pyridine of compound 16 in order to improve CDK6
inhibitory activity. By introducing N-methylpiperazine, N-ethylpiperazine, piperazine,
morpholine and piperidine, we obtained compounds 23~27. For comparison, we also
replaced the pyridine with phenyl ring to obtain corresponding diphenylprimidine
diamine analogs 28~33. In general, majority of the derivatives with aliphatic
heterocyclic ring substituents (compounds 23, 25~26, and 28~31) exhibited
significantly increasing effects on both CDK6/9 and cell proliferative inhibition.
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Journal of Medicinal Chemistry
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7
8
9
However, phenyl substituted pyridine analogs showed higher cytotoxicity against L02
normal cells and lost the selectivity for CDK6/9 over CDK2, indicating that pyridine
might be critical for the selectivity. Although piperazine and morpholine-substituted
pyridyl pyrimidine analogs (25~26) possessed potent inhibitory activities on CDK6/9,
cytotoxicity of these two compounds were also obvious. In particular, N-
methylpiperazin-substituted pyridine analogue (23) exhibited highest potency on
CDK6 inhibition, good selectivity for CDK6/9 over CDK2, and moderate effects on
cell proliferation, however, its inhibitory activities were almost 10-fold difference
between CDK6 and 9. Therefore we next kept N-methylpiperazin-substituted pyridine
unchanged and focused on balancing the potency on CDK6 and 9 inhibitions in the
following modification.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. SAR at Position 4 of the Benzene or pyridine ring Scaffold.
a
b
IC (μM)
GI (μM)
50
No
.
2
50
R
X
CDK6
CDK9
CDK2
MDA-MB-231
7.47±1.85
Jurkat
MCF-7
L-02
23
N
0.016±0.001 0.152±0.009
＞10
8.08±1.88
5.21±0.13
5.98±0.09
24
N
N
N
N
C
＞10
＞10
9.18±0.57
2.06±0.08
＞10
﹥100
5.06±0.20
5.98±0.36
﹥100
﹥100
7.87±0.24
14.09±1.46
﹥100
﹥100
﹥100
2
2
5
6
0.042±0.006 0.114±0.007
0.046±0.008 0.051±0.022
3.41±0.48
6.63±0.56
21.36±1.20
20.62±0.77
1.85±0.06
1.99±0.11
42.38±2.44
6.51±0.74
27
＞10
＞10
4.84±0.02
1.19±0.77
2
2
3
8
9
0
0.143±0.022 0.434±0.006
7.02±0.02
13.69±1.09
C
C
0.114±0.003 0.751±0.174 0.718±0.032
0.116±0.005 0.176±0.013 0.192±0.022
3.04±0.08
3.26±0.07
2.06±0.07
5.12±0.13
3.16±0.58
2.25±0.19
0.97±0.27
1.75±0.09
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
31
C
C
1.69±0.08
1.12±0.14
2.68±0.23
3.69±0.23
11.30±1.00
8.36±0.27
﹥100
﹥100
5.37±0.35
3
3
2
3
＞10
﹥10
19.87±1.13
6.39±0.10
﹥100
5.69±0.42
1.24±0.02
C
0.536±0.008
0.88±0.13
1.59±0.31
11.26±1.20
4.29±0.88
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Inhibition of cell-free kinases activities.
Inhibition of cell proliferation.
b
Docking models of hit 8 with CDK6/9 revealed that 2-methyl phenyl occupied
hydrophobic pockets formed by Val27, Ile19 and Asn150 of CDK6 as well as by
Val33, Ile25 and Phe30 of CDK9, respectively. Based on the similarity between
hydrophobic pockets of CDK6 and 9, we believed that modification of 2-methyl
phenyl might be critical for the balancing inhibitory potency on these two kinases.
Therefore, we first examined the effects of distance between phenyl ring and
pyrimidine on the inhibitory activity. As listed in Table 4, compared with compound
23, deletion of methyl from phenyl ring (compound 34) or replacement of 2-methyl
phenyl with substituted or non-substituted benzyl (compounds 35~41) or phenethyl
(
compounds 42~45) dramatically diminished the inhibitory effects on both enzymes’
activities and cell proliferation, especially on CDK9, possible due to the hydrophobic
pockets of CDK9 was much smaller than that of CDK6. Next, the favorite position
and size of substituents on phenyl ring for occupancy the hydrophobic pockets of
CDK6 and 9 were screened, resulting in the synthesis of methyl (compounds 47-48),
ethyl (compound 49), i-propyl (compounds 50-52), t-butyl (compound 53), trimethyl
(
compound 54) and methoxyl (compounds 55-57) substituted derivatives. Biological
evaluation indicated that the inhibitory activities of these targeted compounds were
decreased more or less, when compared with compound 23. Especially para-bulk
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Journal of Medicinal Chemistry
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5
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7
8
9
groups substituted derivatives (compounds 49, 50 and 53) lost the potency on CDK9.
Next, we introduced F (compounds 58-60), trifluoromethyl (compounds 61 and 64) or
trifluoromethoxyl (compounds 62-63) as hydrophobic groups to suit the hydrophobic
pockets. Unfortunately, these derivatives containing F didn’t exhibit enough potency,
especially on CDK9. Among them, 2-F substituted phenyl derivative (compound 60)
showed effective cell proliferative inhibition and good selectively for malignant cells
over normal cells, however its inhibitory effects on CDK6/9 was much weaker than
that of compound 23, probably representing alternative molecular mechanism.
Moreover, acetylamino group was also introduced to para-(65), meta-(66) or ortho-
(67) position of phenyl in order to form additional hydrogen bonds with key residues
located at hydrophilic region of CDK6/9. Biological evaluation showed that,
compared with compound 23, although CDK6 inhibition was slightly decreased, the
potency on CDK9 inhibition achieved nearly 4-fold elevation, leading to good balance
between inhibition of CDK6 and 9 with nearly equal IC as well as high selectivity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
for inhibition of CDK6/9 over CDK2. More importantly, compound 66 exhibited
considerable effects against cancer cells with IC values ranging from 4.01 to 8.08
50
µM, but much lower cytotoxicity against L02 normal cells with IC value more than
5
0
1
00 µM, representing excellent selectivity for malignant over normal cells.
To investigate the selectivity among other CDKs, we tested inhibition ratios of
compounds 66 against CDK3, 5 and 11 at 1 μM. As listed in Table S1, The results
showed that the inhibition ratios (at 1μM) of compound 66 against CDK3, 5 and 11
were less than 6.6%, indicating good selectivity of CDK6/9 (100%).
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. SAR at Position 4 of the Pyrimidine Scaffold.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
IC (μM)
GI (μM)
50
50
2
No.
R
CDK6
CDK9
CDK2
MDA-MB-231
37.41±6.70
Jurkat
MCF-7
L-02
34
0.113±0.017
9.85±0.18
＞10
>100
51.78±1.71
17.29±0.72
28.44±0.29
7.99±0.15
3
3
5
6
＞10
﹥10
﹥10
＞10
＞10
24.84±1.36
19.08±2.61
>100
20.26±0.20
26.21±0.44
8.93±0.14
7.96±1.92
3
3
3
7
8
9
1.04±0.06
＞10
﹥10
＞10
＞10
＞10
＞10
＞10
18.58±2.13
>100
12.67±0.35
>100
38.10±1.32
19.26±0.19
17.32±0.32
>100
39.73±2.83
12.87±0.29
0.640±0.098
14.88±2.70
12.86±3.40
4
4
0
1
1.60±0.22
3.29±0.18
＞10
＞10
20.33±5.41
29.28±7.61
73.39±7.43
10.84±0.98
19.46±0.22
>100
16.16±0.47
25.47±1.13
＞10
＞10
4
4
2
3
＞10
＞10
＞10
＞10
＞10
＞10
>100
>100
>100
11.76±0.46
>100
14.87±2.07
>100
28.50±1.73
44
3.71±0.11
＞10
＞10
>100
14.28±0.56
8.73±0.36
9.67±1.29
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Journal of Medicinal Chemistry
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5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
5
＞10
＞10
＞10
>100
>100
38.41±0.57
>100
4
4
6
7
0.279±0.021
0.088±0.020
＞10
＞10
＞10
11.03±12.51
4.03±0.24
>100
21.33±2.19
11.41±0.54
45.70±2.36
13.92±0.07
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.45±0.24
30.88±2.39
4
4
8
9
0.080±0.009 0.431±0.088
＞10
＞10
5.27±1.08
5.56±0.70
15.29±4.50
11.11±4.53
7.16±0.10
8.23±0.40
6.08±0.12
0.281±0.049
0.126±0.004
6.87±0.17
9.17±0.18
12.08±1.02
5
5
0
1
＞10
＞10
＞10
7.02±0.18
5.59±0.03
4.09±0.14
7.53±1.05
12.25±0.19
5.14±0.08
1.88±0.01
8.64±0.40
4.36±0.37
13.65±1.12
12.50±0.97
0.452±0.187 0.654±0.022
c
52
0.033±0.001
2.65±0.89
N.D.
c
5
5
3
4
0.735±0.078
8.87±0.03
6.82±0.47
＞10
＞10
16.16±1.15
21.03±0.82
N.D.
12.60±0.30
9.47±0.40
6.07±1.53
﹥10
7.60±0.82
20.49±0.61
5
5
5
5
6
7
0.606±0.040
0.103±0.008
6.00±0.89
1.38±0.36
6.20±0.14
﹥10
6.11±0.17
0.701±0.04
＞10
8.02±2.42
8.51±4.61
11.16±2.19
15.83±4.28
8.45±0.15
7.54±0.16
12.12±0.24
3.15±0.61
3.47±0.08
8.77±0.01
c
c
N.D.
N.D.
c
c
c
58
＞10
﹥10
＞10
N.D.
N.D.
27.26±1.40
N.D.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
9
＞10
﹥10
﹥10
1.12±0.07
15.45±1.81
6.67±0.71
7.53±1.58
24.41±0.53
6.08±0.05
38.14±0.49
11.41±0.38
60
4.18±0.20
0.169±0.012
5.83±0.41
46.12±1.18
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
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6
7
8
9
0
c
c
c
6
6
6
6
1
2
3
4
1.79±0.21
1.88±0.13
﹥10
﹥10
＞10
＞10
N.D.
N.D.
N.D.
c
c
c
c
N.D.
N.D.
N.D.
N.D.
c
c
0.112±0.011
0.378±0.033
3.10±0.49
＞10
＞10
N.D.
23.32±2.60
9.37±2.00
4.37±0.20
7.96±1.29
3.07±0.09
0.733±0.022
9.13±0.51
13.82±0.57
c
6
6
6
5
6
7
0.633±0.155 0.934±0.393
0.041±0.005 0.040±0.014
3.42±0.35
9.21±0.52
4.51±0.08
31.39±3.52
N.D.
31.37±0.26
4.01±0.09
43.08±0.19
10.20±2.23
>100
＞10
8.08±0.52
8.53±1.87
＞10
4.01±0.27
5.90±0.96
5.22±0.06
a
b
c
Inhibition of cell-free kinases activities.
Inhibition of cell proliferation.
Not determined.
2.4 Cell cycle analysis.
In order to detect effect of compound 66 on cell cycle distribution, MDA-MB-
2
31 cells were stained with PI probes and subjected to flow cytometry analysis. As
shown in Figure 4, treatment of MDA-MB-231 cells with lower concentrations (4 or
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μM) of compound 66 induced G0/G1 cell cycle arrests (71.85% and 77.51%,
respectively) which is typical for CDK6 inhibitors, when compared with the control
(57.65%). However, with the increasing concentration (12 μM), the effect of
compound 66 on cell cycle distribution was converted to G2/M arrest (34.27%) when
compared with the control (15.90%), which is characteristic of CDK9 inhibitors.
Meanwhile, the percentages of cells distributed at S phase were decreased from 26.45%
to 18.97%, 14.36% and 15.23%, respectively. These data indicated that, different
from that of Palbociclib, compound 66 may exert dual CDK6 and CDK9 inhibitory
potency.
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9
0
Figure 4. Effects of compound 66 on cell cycle distribution. The experiment was
performed independent three times and representative results are presented.
2
.5 Reactive Oxygen Species (ROS) Assay
Previous studies have reported that inhibition of CDK6 can affect cellular
glycolysis, thereby resulting in loss of NADPH, increase of reactive oxygen species
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36
(
ROS), and subsequent induction of cell death. Therefore, to detect the effects of
compound 66 on cellular ROS, MDA-MB-231 cells were stained with DCFH-DA dye
and analyzed by flow cytometry after treatment with indicated concentrations of
compound 66. Results from flow cytometry analysis showed that compound 66
increased the level of intracellular ROS in a dose-dependent manner (Figure 5).
0
1
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Figure 5. ROS of MDA-MB-231 cells treated with compound 66 at different
concentrations after 48 h. The results are given in the form of superposition graph.
The experiment was performed independent three times and representative results are
presented.
2
.6 Apoptosis analysis.
Induction cellular apoptosis were one of the main characters of anti-tumor agents.
To determine the capacity of compound 66 inducing cell apoptosis, MDA-MB-231
cells were double stained by Annexin V-FITC and PI assay and subjected to flow
cytometry analysis after treatment with indicated concentrations. As shown in Figure
+
-
6, the total proportion of apoptotic cells, including Annexin V /PI (the right lower
+
+
quadrant, representing early apoptosis) and Annexin V /PI (the right upper quadrant,
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representing late apoptosis and necrosis) cells does-dependently increased from 5.23%
to 42.85%. Especially, treatment with compound 66 at 12 μM dramatically induced
apoptosis with the apoptotic ratio of 42.85%, which was much higher than that of
Palbociclib (8.69% at 20 μM), indicating different mechanism of compound 66 from
typical CDK4/6 inhibitor. These results revealed that compound 66 could induce
cellular apoptosis at relative high condensations through an unique cellular
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7
mechanism , probably due to the dual inhibition of CDK6 and 9.
Figure 6. Effects of compound 66 on cellular apoptosis. Apoptosis of MDA-MB-231
cells treated with compound 66 at different concentrations after 48 h. Cells in Q1-UR
and Q1-LR are considered to be apoptotic. The experiment was performed
independent three times and representative results are presented.
2
.7 Cellular Thermal Shift Assay (CETSA)
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CETSA is based on ligand-induced thermal stabilization of target proteins . This
experiment is likely to validate drug binding for a series of vital clinical targets, off-
target effects or drug resistance in cancer cell lines. To avoid the endogenous
cofactors such as ATP disturbance to the experiment, we used purified recombinant
proteins in this assay. As shown in Figure 7, both CDK6 and 9 were stabilized in the
presence of compound 66. Especially at 57 °C, a majority of both CDK6 and 9 were
stabilized to avoid degradation in the presence of compound 66. In the contrast, at the
same temperature, target protein was almost totally degradation in the absence of
compound 66. The results indicated that compound 66 directly binded to CDK6 and 9.
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8
9
0
Figure 7. CETSA assay to detection the thermal stability of recombinant CDK6 and
CDK9 proteins treated with compound 66.
2
.8 Microscale Thermophoresis Assay (MST)
In order to qualified the binding affinity (K value) between compound 66 and
d
CDK6 or 9, GFP-fused CDK6 or 9 recombinant protein as well as their corresponding
cyclin D3 or T1 were expressed in HEK293T cells and purified by affinity
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chromatography. A couple of CDK6 or 9 as well as their corresponding Cyclin D3 or
T1 was treated with indicated concentrations of compound 66 and the binding affinity
3
9
was then analyzed by microscale thermophoresis assay. As shown in Figure 8,
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0
results from MST assay showed that compound 66 exhibited high affinity to both
CDK6 and 9, with K value of 71 nM and 30 nM, respectively, which agrees well
d
with the results of enzymatic inhibition assay.
Figure 8. MST assay to determination the affinity of compound 66 to CDK6 and
CDK9 proteins. The experiment was performed independent three times and
representative results are presented.
2
.9 Effects of compound 66 on CDK6/9 signaling pathway.
To further clarify effects of compound 66 on signaling pathway of CDK6 and 9,
Western blot analysis was used to detect the expression of CDK6/9 and their down-
stream related proteins, after treatment with different concentrations. As shown in
Figure 9, treatment with compound 66 didn’t affect the expression of CDK6 or 9 but
significantly decreased CDK6 downstream targets p-Rb and E2F1, as well as CDK9
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downstream effectors cyclin T1 and p-Pol II CTD (S2) expression, indicating the
inhibition effects of compound 66 on CDK6 and 9 signaling pathway.
Since previous studies have indicated that CDK9 regulates transcriptional
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elongation of important genes involved in cell proliferation and survival, such as MYC
40, 41
and MCL-1.
Thus, in our studies, the effects of compound 66 on c-Myc and Mcl-1
mRNA levels were analyzed by qualitative real-time PCR (qRT-PCR). As shown in
Figure 9, treatment with various concentrations of compound 66 significantly
decreased mRNA levels of both c-Myc and Mcl-1, which are well-known oncogenes
with the functions for promoting proliferation and suppressing cellular apoptosis.
Altogether, the results from western blotting and qRT-PCR clearly indicated that
compound 66 acted as a dual CDK6/9 inhibitor and blocked both CDK6 and 9
signaling pathway.
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Figure 9. Compound 66 inhibits the function of CDK6 and CDK9 and significantly
down-regulated the mRNA expression of c-MYC and Mcl-1. (A) Immunoblot
analysis of the indicated proteins in MDA-MB-231 cells treated with compound 66,
β-actin used as loading control. The experiment was performed independent three
times and representative results are presented. Phosphorylation level of Rb and RNA
polymerase II CTD are presented as Means ± SEM. Significant difference is shown as
*
p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANVOA. (B) qRT-RCR analysis of
the mRNA expression of oncogene c-MYC and Mcl-1. The experiment was
performed independent three times and presented as Means±SEM. Significant
difference is shown as *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANVOA.
2.10 Aqueous solubility, lipid-water partition coefficient, Caco-2 permeability
determination and metabolic stability assay in hepatocyctes.
In order to initially evaluated the drug ability of compound 66 before in vivo
experiments. We tested the aqueous solubility, lipid-water partition coefficient and
liver microsome stability of this compound. The thermodynamic equilibrium
solubility of compound 66 in water and normal saline were determined as 0.340 and
0.214 mg/mL, respectively. However, the solubility of the hydrochloride of
compound 66 was greatly increased to 7.70 and 3.96 mg/mL. This is very beneficial
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for oral administration. The experimental LogP of compound 66 was found to be 1.23
which follows the Lipinski Rule of Five. In Caco-2 permeability experiments,
compound 66 exhibited moderate permeability and no obvious efflux phenomena with
-6
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the apparent permeability coefficient (P ) value of 1.7×10 cm/s.
app
We further evaluated the in vitro metabolic stability of compound 66 through
both human and mouse hepatocyte metabolism. As listed in Table 5, in general,
compound 66 showed acceptable stability against metabolism of both human and
mouse hepatocyte. It was more stable in human hepatocyte metabolism than mouse’s,
with half-life (t ) of 91.2 and 31.2 min, respectively. In addition, we also detect the
1
/2
effects of compound 66 on the activity of CYPs. The results showed that compound
6 exhibited non-significant effects on 1A2, 2C9, 3C19 or 2D6 isoforms of CYP with
IC values more than 50 μM, and weak inhibition on 3A4 isoform with IC values of
6
5
0
50
31.4 μM, indicating low potential on drug interactions.
Table 5. Metabolic Stability in Hepatocyctes.
Human
Mouse
Compd
CL
CL
(μL/min/million
cells)
Remaining
@120 min (%)
Remaining
@120 min (%)
t1/2 (min)
(μL/min/millio
n cells)
t1/2 (min)
66
91.9
7.5
40.4
39.2
17.7
12.0
2
.11 In vivo PK study
On the basis of biochemical and cellular activities, compound 66 was further
evaluated for in vivo pharmacokinetic (PK) properties via i.v. and p.o. route. As
summarized in Table 6, compound 66 showed moderate PK properties. After iv
administration of 2 mg/kg compound 66, the area under the concentration−time curve
(
AUC0−∞) was about 640.8 μg/L·h, and the half-life (t ) was 1.76 h. In addition, via
1/2
the oral administration (10 mg/kg), compound 66 showed a area under the
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concentration−time curve (AUC0−∞) of 925.7 μg/L×h, a half-life (t ) of 3.24 h and a
1/2
maximum plasma concentration (Cmax) of 100.6 μg/L. The bioavailability (F%) was
about 28.9%, indicating orally available property of compound 66.
Table 6. In vivo PK properties of compound 66
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0
C
μg/L)
max
AUC0−∞
Dose/routes
t
1/2 (h)
1.76
3.24
T
max (h)
CL (L/h/kg) F (%)
(
(μg/L×h)
2
mg/kg(i.v.)
/
994.7
640.8
3.13
10.8
/
1
0 mg/kg(p.o.)
4.0
100.6
925.7
28.9
2
.12 Safety and in vivo anticancer activity.
To evaluate the safety of compound 66, ICR mice were orally administered with
single administration of this compound at 500 mg/kg. No significant weight loss and
toxic reaction were observed for 7 days. Next, the in vivo anti-tumor activities of
compound 66 was further evaluated in a human breast cancer xenograft mouse model.
Approved clinical agent Abemaciclib was selected as positive control. As shown in
Figure 10A~C, tumor growth was dose-dependently inhibited by oral administration
of compound 66, and treatment with 90 mg/kg compound displayed almost equivalent
potency with 45 mg/kg Abemaciclib. No significant weight loss was observed in
animals treated with compound 66. (Figure 10D). The pathological examination
showed that this compound induced significant apoptosis in tumor tissue, which is
presented as deep staining nucleoplasm, increasing intercellular space and shrinkage
of cytoplasm (Figure 10E). Overall, results from in vivo studies indicated the potent
anti-tumor and good safety of this compound.
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