Design and synthesis of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazol-7-yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine as a highly potent and selective cyclin-dependent kinases 4 and 6 inhibitors and the discovery of structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2017.12.068

Cyclin-dependent kinases 4/6 play an important role in regulation of cell cycle, and overexpress in a variety of cancers. Up to now, new CDK inhibitors still need to be developed due to its poor selectivity. Herein we report a novel series of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazole-7-yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine anologues as potent CDK 4/6 inhibitors based on LY2835219 (Abemaciclib). Compound 10d, which exhibits approximate potency on CDK4/6 (IC₅₀ = 7.4/0.9 nM), has both good pharmacokinetic characters and high selectivity on CDK1 compared with LY2835219. Overall, compound 10d could be a promising candidate and a good starting point as anticancer drugs.

Full text of DOI:10.1016/j.bmcl.2017.12.068

Accepted Manuscript
Design and synthesis of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazol-7-
yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine as a highly
potent and selective Cyclin-Dependent Kinases 4 and 6 Inhibitors and the dis-
covery of structure-activity relationships
Yan Wang, Wen-Jian Liu, Lei Yin, Heng Li, Zhen-Hua Chen, Dian-Xi Zhu,
Xiu-Qing Song, Zhen-Zhen Cheng, Peng Song, Zhan Wang, Zhi-Gang Li
PII:
S0960-894X(17)31242-8
https://doi.org/10.1016/j.bmcl.2017.12.068
BMCL 25524
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 November 2017
22 December 2017
29 December 2017
Please cite this article as: Wang, Y., Liu, W-J., Yin, L., Li, H., Chen, Z-H., Zhu, D-X., Song, X-Q., Cheng, Z-Z.,
Song, P., Wang, Z., Li, Z-G., Design and synthesis of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazol-7-yl)-N-
(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine as a highly potent and selective Cyclin-Dependent
Kinases 4 and 6 Inhibitors and the discovery of structure-activity relationships, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.068
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Design and synthesis of 4-(2,3-dihydro-1H-
benzo[d]pyrrolo[1,2-a]imidazol-7-yl)-N-(5-
(piperazin-1-ylmethyl)pyridine-2-
Leave this area blank for abstract info.
yl)pyrimidin-2-amine-as a highly potent and
selective Cyclin-Dependent Kinases 4 and 6
Inhibitors and the discovery of structure-
activity relationships
Yan Wanga, b , Wen-Jian Liub , Lei Yinb , Heng Lib , Zhen-Hua Chenb , Dian-Xi Zhub , Xiu-Qing Songa , Zhen-
Zhen Chenb , Peng Songa , Zhan Wanga , * and Zhi-Gang Lic , *

Bioorganic & Medicinal Chemistry Letters
Design and synthesis of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-a]imidazol-7-yl)-
N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine as a highly potent
and selective Cyclin-Dependent Kinases 4 and 6 Inhibitors and the discovery of
structure-activity relationships
Yan Wanga b , Wen-Jian Liub , Lei Yinb , Heng Lib , Zhen-Hua Chenb , Dian-Xi Zhub , Xiu-Qing Songa ,
Zhen-Zhen Chengb , Peng Songa , Zhan Wanga,  and Zhi-Gang Lic, 
a Beijing Key Laboratory for Green Catalysis and Separation, Department of Chemistry and Chemical Engineering Beijing University of Technology, Beijing
100124, P.R China
b Gan&lee Pharmaceuticals R&D, No.8 jingsheng north 3rd street, majuqiao town, tongzhou, Beijing 101102, P.R. China
c Beijing Handian Pharmaceutical Co. Ltd. Kuntai international building, chaoyang, Beijing 100020, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Cyclin-dependent kinases 4/6 play an important role in regulation of cell cycle, and overexpress
in a variety of cancers. Up to now, new CDK inhibitors still need to be developed due to its poor
Revised
selectivity. Herein we report a novel series of 4-(2,3-dihydro-1H-benzo[d]pyrrolo[1,2-
Accepted
Available online
a]imidazole-7-yl)-N-(5-(piperazin-1-ylmethyl)pyridine-2-yl)pyrimidin-2-amine anologues as
potent CDK 4/6 inhibitors based on LY2835219 (Abemaciclib). Compound 10d, which exhibits
approximate potency on CDK4/6 (IC50=7.4/0.9 nM), has both good pharmacokinetic characters
and high selectivity on CDK1 compared with LY2835219. Overall, Compound 10d could be a
promising candidate and a good starting point as anticancer drugs.
Keywords:
CDK4/6 inhibitors
CDK1
Abemaciclib
potent
2017 Elsevier Ltd. All rights reserved.
selective
anticancer
Cell cycle is a highly regulated process that leads to the
transition from quiescence or cytokinesis to cell proliferation
through its checkpoints ensures genome stability.1 More
importantly, cyclins and cyclin-dependent kinases (Cdks) play an
important role in regulation of cell cycle.2 In particular, D-type
cyclins are overexpressed in tumor cells,3 associated with Cdk4/6
to activate retinoblastoma protein (pRb) phosphorylation activity,
which results in the release of E2F transcription factor and the
activation of genes required for G1 phase to S phase transition.4,5
Previous studies have shown that the CDK4/6-Rb-E2F pathway
is disrupted in 90% of cancers.6−9.
inhibitor of CDK4/6 may have a wider therapeutic window than
pan-CDK inhibitors in cancer.
Kinase inhibitors can be classified into two types based on
their modes of action: ATP-competitive inhibitors (I) and non-
competitive inhibitors (II). Type I inhibitors bind to the ATP
binding site through the formation of hydrogen bonds with the
kinase “hinge” residues and hydrophobic interactions in
surrounding the region occupied by the adenine ring of ATP. It is
important to note that the recently reported CDK4/6 inhibitors
(LY2835219, Palbociclib and Ribociclib) are all ATP
competitive inhibitors.10,16,17
CDK4/6 are critical regulators of cell cycle progression.10
Surprisingly, genetic studies display that CDK4/6 are dispensable
for the cell cycle.11,12 Ablation of CDK4 kinase activity leads to
complete tumor growth inhibition in CDK4/cyclin D1-dependent
tumors.13,14 Furthermore, genetic knock out experiments
involving CDK4/6 in fibroblast cells are well tolerated due to
In the past few years, several small-molecule CDK inhibitors
have been advanced to clinical trials and even approved for
marketing (Palbociclib). More recently, another CDK 4/6
inhibitor, i.e. Ribociclib, has been used for the treatment of
metastatic HR-positive, HER2-negative breast cancer combined
with aromatase. LY2835219 is also a selective oral CDK4/6
inhibitor approved by the FDA recently (Fig. 1). In assessment,
compensation by CDK1.15 Thus, it is suggested that a selective
———
 Corresponding author. Tel.: +8610-67396186; fax: +8610-67396186; e-mail: wangzhan3401@163.com.
+8610-56903383; fax: +8610-56903383; e-mail: lizhigang@handian.com

LY2835219 could selectively inhibit CDK4 and CDK6 with half
maximal inhibitory concentration values of 2 and 10 nM,10
respectively. However, its selectivity towards CDK1 is not good
enough compared with Palbociclib and Ribociclib, which may
bring more side effects and toxicities.18 Therefore, we performed
scaffold modification and structure–activity relationship (SAR)
investigations of LY2835219 and its analogues to discover novel
selective CDK4/6 inhibitors with drug-like properties.
To preserve the activity and selectivity, the 1-ethylpiperazine
ring with other scaffolds were modified, i.e. substituted
piperazine or piperidine, which allowed for synthetic flexibility
Fig. 2. 2D model of LY2835219 bound to the active site of
CDK6
Fig. 1. Structure of Palbociclib, Ribociclib and LY2835219
preclinical and also were the correct size for this position in
structural modification. The pyridine ring and the imidazole were
retained in our compounds, we attempted to add small groups to
investigate the SAR surrounding the pyridine ring unit, and other
naphthenic rings were incorporated into imidazole to investigate
the inhibitory activities and selectivity, which we concluded this
position might affect the molecule’s selectivity for CDK1.21, 22 In
all, a series of compounds were designed, synthesized for CDK
inhibitory activities evaluation.
According to the 2D interaction (Fig. 2),19 some of these
residues are critical structures for the CDK inhibition and
selectivity. First, the introduction of the pyridine could enhance
the CDK inhibition activity and selectivity over other kinases via
interactions with the sidechain of hinge residue His100. Second,
hydrogen bond between the ligands and the side chain of Lys43
also seems to contribute to a potent and selective CDK4/6 profile.
Finally, the positively-charged piperazine ring of LY2835219 is
stabilized by lying against a solvent-exposed ridge consisting of
Asp104 and Thr107.20 In CDK1/2/3/5, the residue analogous to
CDK6-Thr107 is a lysine, which should lead to electrostatic
repulsion between the piperazine and lysine, and thereby lower
the CDK1/2/3/5 potency.
The synthetic routes of the target compounds (10a-10r, 11a,
11b and 12a-12c) were outlined in Scheme 1. The key
intermediate 5a-5j were prepared from the commercially
available 4-bromo-2-fluoroaniline (1a-1b). First, the starting
material were treated with pyrrolidin-2-one to provide 2a-2j,
which was cyclized with Cs2CO3 and DMA at 180°C to obtain the
benzimidazole analogues (3a-3j).
Scheme 1. Synthetic route for the target compounds. Reactions and conditions: (a) Toluene, pyrrolidin-2-one, TEA, POCl3, reflux 3 h;
(b) DMA, Cs2CO3, 180 °C,5 h; (c) Dioxane, Bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, 100 °C, 6 h, 24.9%, three steps; (d)
Dioxane/H2O, pyrimidine, Pd(PPh3)2Cl2, Na2CO3,100 °C, 3 h, 64%; (e) DCM, NaHB(OAc)3, piperazine, RT, 5 h, 92%; (f) Toluene, 2-
(Dicyclohexylphosphino)biphenyl, Pd2(dba)3, LiHMDS, 80 °C, 12 h, 61%; (g) Dioxane, Pd2(dba)3, XantPhos, Cs2CO3, 120 °C, 1 h,
microwave, 12.4%; (h) DCM, TFA, RT, 3 h, 42%; (i) DMF, K2CO3, 80 °C,5 h, 14%.

Second, compound 3a-3j was converted to the corresponding
heteroarylboronate ester (4a-4j) which underwent palladium
catalyzed Suzuki reaction with 2,4-dichloropyrimidine to yield
5a-5j, Compound 8a-8f was prepared from substituted 6-
bromonicotinaldehyde reductive amination with piperazine,
catalyzed with Pd2(dba)3 and LiHMDS to give 9a-9f. The target
compounds 10a-10r was obtained by utilizing Buchward
coupling reaction of 5a-5j and 9a-9f. 10h, 10i and 10r were
deprotected to afford 11a-11c, then docking other group to give
target compounds 12a-12c.
substituent methyl was drawn to the R3 (11b, 10j, 10k, 10q), the
activities dropped largely, probably due to steric hindrance. R
substituted compounds did not modulate CDK4/6 inhibitory
activity significantly, however, CDK1’s activity was deeply
exchanged, which need further experiments to explain this
phenomenon. The group A was substituted by bulkier aliphatic
rings, like cyclopentyl, cyclohexyl, cycloheptyl (10a, 10b, 10c),
they had obvious effect on the CDK4/6 activity, when the group
A
substituted by methylcyclopentyl, ethylcyclopentyl or
dimethylcyclopentyl analogues, and they had minor effect on the
CDK4/6 activity, because they could tightly fit into the cavity.
On the basis of Docking studies,19 this effect did not derive from
nonspecific lipophilicity but requires the specific steric demand
available, In turn, unavailable spatial position also affected
hydrogen bonding between the ligands and the side chain of
Lys43(Fig 3).
The novel LY2835219 analogue inhibitors (10a-10r, 11a, 11b,
12a-12c) showed good activities and achieved the initial goals.
the structure-activity relationship was summarized in Table 1.
When R1 or R2 was substituted with hydrogen（10f and 10g）,
they had slight impact to the CDK4/6 activities, however, it
showed high inhibition to the CDK1. When an additional
Table 1. The structures and enzyme activity of compounds 10a-10r, 11a, 11b, 12a-12c.
IC50
CDK1/D3IC50
(nM)
IC50(CDK4)/
IC50(CDK6)
IC50 (nM)
Compd.
LY2835219
10a
R1
F
R2
F
R3
H
R
Et
Et
A
-
56.0
1.7/7.8
F
F
H
1460.0
18/ 244.1
F
F
F
F
H
H
Et
Et
1535.0
1550.0
6.8/45.6
10b
10c
147.3/247.8
F
F
F
H
F
F
F
H
F
F
H
H
H
H
H
Et
Me
Et
2670.0
353.9
610.0
733.0
1365.0
7.4/0.9
0.8/4.0
10d
10e
10f
10g
11a
1.0/17.4
1.72/15.8
0.6/3.7
Et
H
F
F
F
F
Me
H
H
2333.0
1610.0
18.9/18.7
5.7/11.2
11b
12a
2-fluoroethyl

F
F
F
F
Me
Me
Et
1914.0
2268.0
16.0/46.0
10j
Me
25.0/271.0
10k
F
F
F
F
H
H
Cyclopropyl
Et
1874.0
1920.0
9.7/19.0
12b
10l
15.8/392.0
F
F
F
F
H
H
Et
Et
1880.0
2075.0
49.7/339.0
20.0/36.0
10m
10n
F
F
F
F
F
F
F
F
F
F
F
F
F
H
H
H
Et
Me
Et
2189.0
2988.0
2950.0
3072.0
5050.0
5562.0
3363.0
27.0/28.0
5.7/275.0
39.0/178.0
0.6/12.7
10o
10p
10q
12c
10s
10t
Me
H
i-Pr
Me
Et
H
10.0/2.4
H
14.0/22.0
17.6/752.0
H
Et
10u
Fig. 3. Binding of compound 10c and 10d in CDK6 catalytic domain.
The above discussions have been validated by a docking study
of compound 10c and 10d into the catalytic domain of
LY2835219 (PDB code: 5L2S), and the result is shown in Figure
3. 19 We found the dimethylcyclopentyl group of compound 10d
tightly fit into the catalytic domain which contributed to
hydrogen bond between the nitrogen atoms of imidazole and the
NH of Lys43. However, bulkier cycloheptyl of compound 10c is
tortuous in the cavity which results in the loss of the binding
affinity to the kinase target. i.e, the losing of hydrogen bond with
Lys43.

On the basis of low activity of CDK1 and high activity of
CDK4/6, we chose 10d, 11b, 10n, 10o, 12c, 10s, 10t to test cell
activity (Table 2). Only 10d exhibited good potency among these
compounds. In addition, the hERG inhibitory of 10d was also
tested (Table 3), and compound 10d showed low heart toxicity.
Table 2. The cell activity of compounds 10d, 11b, 10n, 10o, 12c, 10s, 10t.
Compd.
MDB-MA-231
IC50 (nM)
LY2835219
10d
11b
10n
10o
12c
10s
10t
191
232
2126
5560
2108
1159
2630
2527
Table 3. The properties of hERG channel of 10d and LY2835219.
Amitriptyline
Compd.
LY2835219
10d
6.38
(Positive Control)
3.29
10.90
IC50 (μM)/hERG
Table 4. Pharmacokinetic parameters of rat (5mg/kg)
code
LY2835219
Cmax (ng/mL) AUC (ng/Ml*h)
T1/2 (h)
MRT (h)
CL/F (ml/min/kg)
24.20±2.35
Vz/F (ml/kg)
8533.0±1580.0
8751.0±1657.0
312.0±33.0
343.0±67.0
3275.0±731.0
3746.0±697.0
4.07±2.31 7.97±1.17
4.62±0.20 7.58±0.15
21.90±3.67
10d
In summary, a novel series of substituted of LY2835219
derivatives were developed by changing A (Table 1) regions
with the combination of potent activity on CDK4/6 and
selectivity on CDK1, Compound 10d also demonstrated good
pharmacokinetic effects and druglike properties suitable for
development (Table 4). Thus, the compound 10d was
worthwhile for deeper research as anticancer drugs. Further
biological evaluation of these compounds is currently underway
in our laboratory.
Acknowledgments
We gratefully acknowledge financial support from Gan&lee
Pharmaceuticals R&D fund.
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