Synthesis and biological evaluation of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as potent CDK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.08.035

As serine/threonine kinase, the cyclin dependent kinase 2 (CDK2) is a promising target for various diseases such as cerebral hypoxia, cancer, and neurodegenerative diseases. Here we reported the structure-based synthesis and biological evaluation of novel

Full text of DOI:10.1016/j.bmcl.2018.08.035

Accepted Manuscript
Synthesis and biological evaluation of novel 5,6-dihydropyrimido[4,5-f]quina-
zoline derivatives as potent CDK2 inhibitors
Xiaoxia Hu, Hui Zhao, Youzhi Wang, Zhan Liu, Bainian Feng, Chunlei Tang
PII:
S0960-894X(18)30710-8
https://doi.org/10.1016/j.bmcl.2018.08.035
BMCL 26015
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
11 July 2018
19 August 2018
27 August 2018
Please cite this article as: Hu, X., Zhao, H., Wang, Y., Liu, Z., Feng, B., Tang, C., Synthesis and biological evaluation
of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as potent CDK2 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.08.035
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Synthesis and biological evaluation of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as potent CDK2
inhibitors
Xiaoxia Hua, Hui Zhaoa, Youzhi Wangb, Zhan Liua, Bainian Fenga, Chunlei Tang a,*
aSchool of Pharmaceutical Science, Jiangnan University, Wuxi, China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
China
*Correspondence: Chunlei Tang, School of Pharmaceutical Science, Jiangnan University, Wuxi, China
E-mail address: tangcl@jiangnan.edu.cn
Abstract
As serine/threonine kinase, the cyclin dependent kinase 2 (CDK2) is a promising target for various diseases such as cerebral
hypoxia, cancer, and neurodegenerative diseases. Here we reported the structure-based synthesis and biological evaluation
of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as CDK2 inhibitors, which exhibited potent CDK2 inhibitory
activities, as well as anticancer activities in low concentration against two human cancer cell lines (MCF-7 and HCT116).
In particular, compounds 11a and 11f (IC50 values of 0.11 and 0.09 μM for CDK2, respectively) have demonstrated
significantly inhibitory potency against CDK2 and have showed great inhibitory activities against MCF-7 and HCT116 cell
lines.
Keywords: cyclin dependent kinase, anticancer, design and discovery, molecular docking
Cells will progress only through four phases of the cell cycle to divide and replicate, namely, G1, S phase, G2, and M phase
[1-2]. Since unrestrained growth is one of the hallmarks of cancer, it is common to disrupt the cell-cycle regulation in
malignant cells [3]. Although targeting the pathways that regulate the cell cycle has drawn great attention in oncology for
many years [4], the more recent development of selective and potent inhibitors of specific CDKs leads to burgeoning interest
in this field.
CDK2 is a member of the CDK family which plays a central role in regulating cell cycle progression. CDK2 is frequently
over-expressed in human tumors, and needed for the proliferation of tumor cell [5-6]. The CDK2 inhibitors such as
[7-10]
Dinaciclib, Milciclib, BMS-387032, PHA-793887
have been studied extensively in clinical research and it has been
demonstrated that cell cycle arrest would cause cell apoptosis. Meanwhile, ongoing clinical trials have produced promising
data on the potential efficacy of CDK2 inhibitors [11]
.
[12-13]
Most of the reported CDK2 inhibitors
interact with Leu83 residue at ATP-site. The tricyclic scaffold of
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline was previously identified as an inhibitor of other cell cycle kinases like Aur-A,
PLK1 and CDK2 [14-16]. The pyrimidine portion of the three-membered ring scaffold could form a pair of important
donor-acceptor-donor hydrogen bonds with the CDK2 hinge region (Leu83), which was essential. Our team referred to the
4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline scaffold [16] to retain the structure of pyrimidine and then altered the portion of
pyrazolo
ring.
Through
the
previous
screening,
we
discovered
the
compound
8-(methylthio)-5,6-dihydropyrimido[4,5-f]quinazolin-2-amine (a) which displayed weak inhibition activity (IC50=4.72 μM)
against CDK2. As a further development of the fused quinazoline ring systems, we identified a novel series of
5,6-dihydropyrimido[4,5-f]quinazoline as CDK2 inhibitors (Fig. 1). We report the synthetic process and the
structure-activity relationship that led from the initial hit (a) to identification of a potent CDK2 inhibitor.
1

Figure 1. The structure of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline and 5,6-dihydropyrimido[4,5-f]quinazoline scaffold
According to the related literatures [17], The synthetic approach to this class of compounds (scheme 1) started from
commercially available 1,3-cyclohexanedione. Condensation with N,N-dimethylformamide dimethyl acetal gave rise to
compound 2. The compound 2 was condensed with guanidine derivatives in DMF to afford the pyrimidine derivatives
3a-3c, which were converted into the corresponding 5,6-dihydropyrimido[4,5-f]quinazoline analogues 4a-4c and 5a-5b.
Hydrolysis of carboxylic ester 3c gave the corresponding acid 6, which were submitted to the coupling with amines to
provide amides 7a-7c. Then, 7a-7c reacted with N,N-dimethylformamide dimethyl acetal and guanidine hydrochloride to
afford the fused
5,6-dihydropyrimido[4,5-f]quinazoline derivatives 8a-8c. The
synthetic route to
5,6-dihydropyrimido[4,5-f]quinazoline analogues is shown in Scheme 2. Compound 2 reacted with S-methylisothiourea
sulfate to obtain 9 under 80 °C. Pyrimidine ring compounds 10a-10f were obtained by cyclization of compound 9 and
aniline guanidine. Compound 10e were submitted to the coupling with amines to provide amides 11a-11h.
2

Scheme 1. Synthesis of compounds 8a-8c. Reagents and conditions: (i) N,N-dimethylformamide dimethyl acetal, r.t., 8 h,
90%; (ii) phenylguanidine derivatives, Na2CO3, 1,4-dioxane, reflux, 80-90%; (iii) N,N-dimethylformamide dimethyl acetal,
DMF, 110 °C, 4 h; guanidine hydrochloride, Na2CO3, DMF, 110 °C, 50-70%; (iv) N,N-dimethylformamide dimethyl acetal,
DMF, 110 °C, 4 h; phenylguanidine derivatives, Na2CO3, DMF, 110 °C, 60-70%; (v)1.5M NaOH in 95% EtOH, 80 °C, 4 h,
85%; (vi)R3NH2, HOBT, EDC, DMF, r.t., 8 h, 60-80%; (vii) N,N-dimethylformamide dimethyl acetal, DMF, 110 °C, 4 h;
guanidine hydrochloride, Na2CO3, DMF, 110 °C, 40-60%.
Scheme 2. Synthesis of compounds 11a-11h. Reagents and condition: (i) S-methyllisothiourea sulfate, Na2CO3, 1,4-dioxane,
reflux, 8 h, 90%; (ii) N, N-dimethylformamide dimethyl acetal, DMF, 110 °C, 4 h; phenylguanidine derivatives, Na2CO3,
DMF, 110 °C, 60-80%. (iii) R5NH2, HOBT, EDC, DMF, r.t., 8 h, 50-80%.
All synthesized compounds were evaluated for inhibitory activity against CDK2 and anti-proliferative activity against
human cell lines MCF-7 and HCT116 taking BMS-387032 as a positive control. Table 1 summarized the structure-activity
relationship (SAR) of derivatives modified at the 2-position and 8-position of the hit compound a. Firstly, we examined the
substituent effect of 8-position of 5,6-dihydropyrimido[4,5-f]quinazoline scaffold. All the compounds, with the exception of
4c, 8b and 8c, showed an increased activity with respect to the initial compound a (IC50=4.72 μM) against CDK2.
Subsequently work continued on introducing different substitution at para position of benzene ring of compound 4a, but
there was no significant improvement on inhibitory activity. As is showed in the results, the amino carboxylate compounds
8a-8c demonstrated the reduced inhibitory potency for CDK2, when compared with compound 4a.
According to the molecular modeling studies reported in the literature [18], we analyzed the structure in detail, the
introduction of phenylamine at C-8 resulted in hydrogen bonds forming with Leu83. Likewise, the replacement at C-2 with
phenylamine showed the same effect. Then, we defined R2 as phenyl and X as sulfur atom, we found that compound 10a
(IC50=0.41 μM) showed good inhibitory activity to CDK2. Next, we systematically analyzed substituent effects on
4-position of benzyl ring with various substituent, including hydrogen, methyl amino and so on (Table 1). The introduction
of amino and amide on the 4-position of phenyl resulted a significant improvement in activity against CDK2. The
compound 10d (IC50=0.16 μM) exhibited stronger inhibitory activity against CDK2. The halogen compounds (10b-10c) and
ester compounds (10e-10f) showed weaker inhibitory activities compared to 10e. Compound 11a (IC50=0.11 μM) exhibited
much better inhibitory potencies against CDK2 kinases as compared to 10d (IC50=0.16 μM).
Based on the overall anticancer activity parameter, mean IC50, the potencies of the tested compounds could be generally
ranked in the following order: 2-substituted phenylamine compounds (11a-11h) >2-substituted phenylamine compounds
(10a-10f) > 8-substituted phenylamine compounds (4a-4c, 5a-5b and 8a-8c). The results indicated that the phenylamine at
3

the C-2 position derivatives 10a-10f and 11a-11h were again relatively more potent than the phenylamine at the C-8
position derivatives 4a-4c, 5a-5b and 8a-8c. The compounds (4a-4c, 5a-5b and 8a-8c) showed weak inhibitory activity
against CDK2 and anti-proliferative activity against MCF-7 and HCT116 as well. When we defined the C-2 position as
phenylamine and the C-8 position as methyl sulfide, compounds (10a-10f and 11a-11h) performed better. Furthermore,
compound 11f with the N1,N1,N2-trimethylethane-1,2-diamine moiety displayed potent inhibitory activity (IC50=0.09 μM)
and anti-proliferative activity (MCF-7, IC50=1.1 ± 0.1 μM; HCT116, IC50= 1.4 ± 0.2 μM) of all compounds.
Table 1
Results for inhibition of CDK2 and anti-proliferative activity against MCF-7 and HCT116 by compounds 4a-4c, 5a-5b,
8a-8c, 10a-10f, 11a-11h.
ID
4a
4b
4c
X
N
N
N
R1
R2
H
H
H
IC50a(μM)
CDK2/Cyclin A2
1.70
IC50b ± SD (μM)
MCF-7
HCT116
27.6 ± 1.1
25.3 ± 2.4
1.85
NA
>30
>30
>30
>30
20.6 ± 0.5
23.5 ± 0.6
>30
18.1 ± 3.7
26.3 ± 0.7
>30
5a
5b
8a
N
N
N
1.54
2.03
3.2
H
H
H
5.9
>30
>30
>30
>30
8b
8c
N
N
NA
15.6 ± 0.4
8.4 ± 0.3
7.0 ± 0.7
12.7 ± 1.0
7.9 ± 0.4
8.3 ± 0.2
10a
10b
10c
S
S
S
Me
Me
Me
0.41
0.35
0.38
4

1.4 ± 0.3
1.6 ± 0.2
10d
10e
S
S
Me
Me
0.16
0.98
17.8 ± 0.6
12.8 ± 0.9
1.25
0.11
0.53
1.26
0.19
0.16
0.09
0.18
3.7
25.4 ± 0.2
1.3 ± 0.2
3.7 ± 0.4
6.5 ± 1.7
2.1 ± 0.5
1.5 ± 0.3
1.1 ± 0.1
2.5 ± 0.6
19.4 ± 0.4
1.0 ±0.3
14.3 ± 0.4
0.9 ± 0.2
5.3 ± 0.5
9.1 ± 0.2
3.2 ± 1.4
1.3 ± 0.1
1.4 ± 0.2
3.8 ± 0.5
17.2 ± 0.7
0.7 ±0.2
10f
11a
S
S
S
S
S
S
S
S
S
Me
Me
Me
Me
Me
Me
Me
Me
Me
11b
11c
11d
11e
11f
11g
11h
0.052
BMS-387032
a Values are means of at least two or more experiments. Standard deviations were <10% of the mean. NA means data not
available. b Values are means of at least three or more experiments.
Kinases selectivity of compound 11f was further evaluated with other CDK kinase and was summarized in Table 2.
Compound 11f displayed better inhibitory activity against CDK2 and CDK5 (IC50 values of 0.09 μM and 0.68 μM) than
CDK1 and CDK7 (IC50 values of 1.25 μM and 3.60 μM). Compound 11f showed weak inhibitory activity against CDK4
and CDK6 (IC50>5 μM). Thus far it could be identified as a potential and selective CDK2 inhibitor.
Table 2
Selectivity profile of compound 11f
CDKs
CDK1
CDK2
0.09
CDK4
>5
CDK5
0.68
CDK6
>5
CDK7
3.60
IC50a(μM) 1.25
a Values are means of at least two or more experiments. Standard deviations were <10% of the mean.
5

To better understand the mechanism of inhibition, molecular docking was used to analyze the putative binding mode of the
designed compound with CDK2 [19]. The crystal structure of CDK2 in complex with an ATP-competitive inhibitor (PDB
code 2BPM) was selected for the docking studies. As depicted in docking mode of compound 10d with CDK2 (Fig. 2), the
pyrimidine core of 10d formed a pair of donor-acceptor-donor hydrogen bonds with the CDK2 hinge region (Leu83). And
the amino forms a hydrogen bond to the main-chain of Asp86.
Figure 2. Compound 10d bound to CDK2. Hydrogen bonds are shown as green dashed lines, residues of CDK2 and are
shown with carbon and nitrogen colored green and blue, respectively.
Conclusions
In summary, we have designed and synthesized a series of novel 5,6-dihydropyrimido[4,5-f]quinazoline derivatives as
CDK2 inhibitors. The biochemical activity of all compounds against CDK2/A2 kinase and in vitro cell proliferation
cytotoxicity on human cancer cell lines MCF-7 and HCT116 were reported in table 1 and table 2. The brief SAR study
demonstrated that the phenylamine at the C-2 position had better inhibitory potency in comparison with the phenylamine at
the C-8 position derivatives. Similarly, amide derivatives had better inhibitory potency than amino carboxylate derivatives
for CDK2 kinase. Derivatives 11a and 11f were proved to be the best compounds of the series, showing anti-proliferative
activity in the low micro molar range on the MCF-7 and HCT116 cell lines, remarkable activity on CDK2 kinase (IC50
values of 0.11 and 0.09 μM for CDK2, respectively). Studies to explore the mechanism of these compounds are needed and
now in progress. The evolution of these series of compounds will be reported in due course.
Acknowledgements
This work was supported by the National First-class Discipline Program of Food Science and Technology
(JUFSTR20180101), the National Natural Science Foundation of China (Grant Nos. 21305051) and the Jiangsu prospective
joint research project (BY2015019-25).
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Graphical Abstract
Highlights
•
New 5,6-dihydropyrimido[4,5-f]quinazoline derivatives were designed and
synthesized
•
•
•
CDK2 inhibitory activity was evaluated
Anti-proliferative activity was in vitro evaluated against MCF-7 and HCT116 cells
Molecular docking in the active site of CDK2 enzyme
8