Design and synthesis of 2-phenylpyrimidine coumarin derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2017.08.044

A series of 2-phenylpyrimidine coumarin derivatives with potential telomerase-inhibiting activity was designed and synthesized. All of the compounds were screened for antiproliferative activity against CNE2, KB, and Cal27 cell lines in vitro. The results showed that most of the derivatives had a favorable effect on resisting tumor cell proliferation; compound 13, 3-(4-amino-5-oxo-5H-chromeno[4,3-d]pyrimidin-2-yl)phenyl 4-(dimethylamino)benzenesulfonate, exhibited the best activity. Flow cytometry revealed that compound 13 can inhibit CNE2 proliferation. Telomerase inhibition and in vitro antitumor activity were consistent among the compounds, but compound 13 showed the best telomerase-inhibiting activity and could inhibit telomere extension. Molecular docking results indicated that compound 13 bonded with telomerase reverse transcriptase (TERT) through multiple interactions, including hydrogen bonding and hydrophobic interactions. The results of the study provide further information on 2-phenylpyrimidine coumarins, expanding the types of telomerase inhibitors as the parent structures.

Full text of DOI:10.1016/j.bmcl.2017.08.044

Accepted Manuscript
Design and synthesis of 2-phenylpyrimidine coumarin derivatives as anticancer
agents
Na Lv, Ming Sun, Chen Liu, Jiabin Li
PII:
S0960-894X(17)30846-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.044
BMCL 25238
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
16 July 2017
17 August 2017
19 August 2017
Please cite this article as: Lv, N., Sun, M., Liu, C., Li, J., Design and synthesis of 2-phenylpyrimidine coumarin
derivatives as anticancer agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.08.044
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Design and synthesis of 2-phenylpyrimidine coumarin
derivatives as anticancer agents
Na Lv a,†, Ming Sun a,†, Chen Liu b , and Jiabin Li c,*
a
Department of Stomatology, First Affiliated Hospital of Anhui Medical University, Hefei 230032, China
College of Basic Medicine, Anhui Medical University, Hefei 230032, China
Department of Infectious, First Affiliated Hospital of Anhui Medical University, Hefei 230032, China
b
c
* Correspondence: jbahmu@yeah.net Tel.: +86-551-65161136
† These authors contributed equally to this work.
ABSTRACT A series of 2-phenylpyrimidine coumarin derivatives with potential
telomerase-inhibiting activity was designed and synthesized. All of the compounds were
screened for antiproliferative activity against CNE2, KB, and Cal27 cell lines in vitro. The
results showed that most of the derivatives had a favorable effect on resisting tumor cell
proliferation; compound 13, 3-(4-amino-5-oxo-5H-chromeno[4,3-d]pyrimidin-2-yl)phenyl
4-(dimethylamino)benzenesulfonate, exhibited the best activity. Flow cytometry revealed
that compound 13 can inhibit CNE2 proliferation. Telomerase inhibition and in vitro
antitumor activity were consistent among the compounds, but compound 13 showed the
best telomerase-inhibiting activity and could inhibit telomere extension. Molecular docking
results indicated that compound 13 bonded with telomerase reverse transcriptase (TERT)
through multiple interactions, including hydrogen bonding and hydrophobic interactions.
The results of the study provide further information on 2-phenylpyrimidine coumarins,
expanding the types of telomerase inhibitors as the parent structures.
Keywords: 2-phenylpyrimidine coumarin derivatives; antiproliferative; telomerase
inhibitors

Telomerase, as a ribonucleoprotein enzyme with reverse transcriptase activity, can use its
own RNA as a template to synthesize telomere DNA sequences 1-3. Any abnormalities in
telomerase structure and behavior are closely related to cell senescence, tumorigenesis and
development 4, 5. A large quantity of data confirms that more than 85% of human tumor
cells have high levels of telomerase activity, but most somatic cells and benign tumor cells
lack telomerase activity 6, 7. Therefore, most scholars believe that the proliferation of some
tumor cells can be effectively inhibited through the inhibition of telomerase activity 8, 9
.
Coumarin, namely, benzopyrone, is a substance with an aromatic odor. Coumarin and its
derivatives constitute important organic heterocyclic molecules with diverse physiological
activities. As early as the 1960s, scientists found that coumarin compounds had antitumor
metastasis effects 10. As large quantities of coumarin compounds have been separated and
synthesized, more and more coumarin compounds have been found to have antitumor
effects 11-14 . In recent years, Liu’s group reported on multiple series of coumarin
derivatives, which were found to have favorable telomerase-inhibiting effects and inhibiting
effects on cell proliferation in multiple tumors 15-17. Aromatic sulfonate antitumor drugs are
widely used in the clinic, and aromatic sulfonyl groups are important pharmacophores 18, 19
Based on the above-mentioned studies and for the continuous extension of structural types
of coumarin telomerase inhibitors, our research used 2-phenylpyrimidine coumarin as a
parent nucleus. In addition, aromatic sulfonyl groups were introduced in the substituted
phenyl meta-position, which was expected to increase the antitumor activity and
interactions with telomerase. Then, a series of 2-phenylpyrimidine coumarin derivatives
was obtained (Figure 1), and their antitumor effects and telomerase-inhibiting activities
were evaluated.
.
Figure 1. Synthesis of 3’-sulfonate-substituted 2-phenyl-benzopyranopyrimidine
derivatives. Reagents and conditions: (i) CNCH2COOC2H5, NH4OAc, C2H5OH, reflux, 2 h
and (ii) ClSO2R, DMF, rt, and 5 h (R is shown in table 1).
Figure 1 shows the synthesis route. Parent compound 2 was obtained through the
“one-pot” method, namely, ethyl cyanoacetate, salicylic aldehyde, 3-hydroxybenzaldehyde,
and ammonium acetate were put under reflux for 2 h 20. During the reaction process, large

quantities of yellow solids precipitated, and then parent compound 2 was obtained through
a simple after-treatment. Parent compound 2 can be directly applied in the next step without
further purification, and it reacts with the substituted sulfonyl chloride to obtain the target
product.
To test the antitumor activity of the synthesized compounds, we evaluated the activity of
compounds 3–15 against human nasopharyngeal carcinoma cells (CNE2), human oral
epidermoid carcinoma cells (KB), and human oral squamous cells (Cal27). The results are
summarized in Table 1. Following the interactions between compounds and the cells, the
results revealed that the most examined compounds showed potent activity against the
CNE2 and Cal27 cell lines and moderate activity against the KB cell lines. Compounds 4, 8,
10, and 13 exhibited high activity against the CNE2 cell lines, with IC50 values of
4.24±0.17, 3.44±0.46, 2.34±0.21, and 1.92±0.13 µM, respectively. Compounds 8, 9, 10,
and 13 possessed potent activity against the Cal27 cell lines, with IC50 values of 2.58±0.47,
4.91±0.87, 4.32±0.24, and 1.97±0.51 µM, respectively. Compounds 4, 8, and 13 showed
high activity against the KB cell lines, with IC50 values of 5.62±0.35, 6.74±0.24, and
3.72±0.54 µM, respectively, which are comparable with the positive control doxorubicin
(AMD).
Structure–activity relationship analysis showed that our examined compounds were
divided into three series. Compound 1 has an alkyl moiety and no antiproliferative ability.
Compounds 4–7 have thiophene moieties, while compounds 8–15 have aromatic-ring
moieties; compounds 8–15 are better at resisting tumor cell proliferation being than
compounds 4–7. In the aromatic-ring moiety series of compounds, the compounds
substituted by halogen demonstrated moderate activity. Compound 13, which has
N,N-dimethyl amino benzenesulfonyl moiety, had the best activity; its activity values (IC50)
for the CNE2, KB, and Cal27 cell lines were 1.92±0.13, 3.72±0.54, and 1.97±0.51 µM,
respectively, and the values are comparable with those of potent ADM. Therefore, for this
type of structural moiety, this 2-phenylpyrimidine coumarin derivative structure warrants
further optimization as a potent anticancer agent.

Table 1. In vitro anticancer activity of the synthesized compounds a.
In vitro anticancer activity, IC50 (µM) b
Compound
R
CNE2
>100
KB
Cal27
>100
3
4
–CH3
>100
4.24 ± 0.17
5.62 ± 0.35
10.12 ± 0.78
5
6
14.58 ± 1.98
18.64 ± 3.27
20.86 ± 1.51
36.17 ± 1.41
11.25 ± 0.99
18.34 ± 3.01
38.36 ±
2.98
47.47 ±
3.26
>100
7
8
3.44 ± 0.46
6.74 ± 0.24
2.58 ± 0.47
4.23
±
11.59
4.91
±
±
9
0.18
2.34
±
1.54
7.35
±
0.87
4.32
±
10
0.21
11.07
±
0.16
15.83
±
0.24
18.07
±
11
12
13
14
1.45
16.25
±
1.64
16.99
±
2.21
3.54
±
2.62
1.92
±
1.60
3.72
±
1.33
1.97
±
0.13
0.54
0.51
16.57 ± 2.50
18.62 ± 1.98
9.63 ± 1.04
15
20.64 ± 2.21
2.12 ± 0.56
29.72 ± 2.24
3.04 ± 0.87
17.63 ± 1.85
1.56 ± 0.64
ADM
Negative control 0.1%DMSO, no activity.
a The data represented the mean of three experiments in triplicate and were expressed as means ± SD; only
descriptive statistics were done in the text.
b The IC50 value was defined as the concentration at which 50% survival of cells was observed.

Next, flow cytometry was used to detect the effect of the compounds on inducing
apoptosis in tumor cells. The Annexin V-FITC/PI double-staining method was used to
evaluate the effect of compound 13 on inducing apoptosis of the CNE2 cells (Figure 2).
Figure 2A shows that a blank control was not added to any of the compounds, and only 8.1%
of the cells were in an apoptotic state. After compound 13 was added, and as its
concentration increased, the number of apoptotic cells gradually increased; finally, the
percentage of CNE2 cells in an apoptotic state had escalated to 54.9%. This result
confirmed that compound 13 induced apoptosis in the CNE2 cells.
Figure 2. CNE2 cells were treated with no drug (A) and 0.5 µM (B) and 1 µM (C) of
compound 13 for 4 days and then analyzed with flow cytometry.
Based on the discovered coumarin skeletal structure, the structural types of telomerase
inhibitors were continuously extended. Our research evaluated the telomerase-inhibiting
activities of some of the examined compounds using CNE2 cell extracts with ethidium
bromide as the positive control. Results are shown in Table 2. Compounds 4, 8 13, and 15
show favorable telomerase-inhibiting activities, but compounds 3 and 12 had weak
telomerase-inhibiting activity. These results are consistent with the in vitro antitumor effect
of these compounds, and they indicate that the compounds may resist tumor cell
proliferation by inhibiting telomerase activity.
Table 2. Inhibitory effects of selected compounds against telomerase a.
Compound
IC50 (µM) telomerase b
3
4
58.64 ± 3.42
7.35 ± 0.96

8
3.16 ± 0.67
21.07 ± 2.32
0.82 ± 0.14
6.32 ± 0.56
2.16 ± 0.28
12
13
15
Ethidium bromide
Negative control 0.1%DMSO, no activity.
a The data represented the mean of three experiments in triplicate and were expressed as means ± SD; only
descriptive statistics were done in the text.
b The IC50 value was defined as the concentration at which 50% survival of cells was observed.
Telomere length is jointly regulated by telomerase and the telomere-binding protein.
Telomere length, a biological marker that measures cell senescence and apoptosis, is a good
index to measure the biological characteristics of tumors 6, 21. We conducted additional
research on the length change of restrictive telomere fragments to further study compound
13’s mechanism in inhibiting cell proliferation and inducing cell apoptosis. Telomere
restriction fragment (TRF) experimental results (Figure 3) show that the CNE2 cells’
telomere lengths after treatment with 1 µM of compound 13 for 8 days were shortened by
0.8 kb compared with the blank control group, and telomere shortening was also observed
after treatment with 0.5 µM of compound 13 for 8 days. Telomere dysfunction can activate
p53 to initiate cellular senescence or apoptosis to suppress tumorigenesis. In this study,
senescence induction via compound 13 might be due to telomere-length shortening; this
result is consistent with those previously reported for efficient telomerase inhibitors 6, 21
Hence, compound 13 may inhibit cell proliferation by inhibiting telomere activity and
inhibiting telomere length extension.
.

Figure 3. Effect of compound 13 on telomere length. TRF analysis of CNE2 cells treated
with or without compound 13 for 8 days. Lane 1, with 0 µM compound 13; lane 2, with 0.5
µM compound 13; lane 3, with 1 µM compound 13.
To further examine the mechanism of the telomerase-inhibiting effects of
2-phenylpyrimidine coumarin derivatives and provide an idea for follow-up structural
transformation, compound 13 and telomerase TERT (PDB code: 3DU6) 22, which includes
ASP254 key active regions, were selected for the molecular docking experiment. The
results are shown in Figure 4. In general, compound 13 could adaptively bind to catalytic
subunits of telomerase and replace nucleotides as the active substrate. Multiple interactions,
including hydrogen-bond interactions and hydrophobic interactions, can be seen in the
figure. One hydrogen bond was formed between –NH2 and ILE187 on the
compound-substituted aromatic ring. Two hydrogen bonds were formed between the
substituted amino group on the pyrimidine ring and ASP254. Another two hydrogen bonds
were formed between two Os on the sulfonic group and ASP343. Furthermore, one
pi–cation interaction was formed between the substituted aromatic ring and ASP254. These
results show why compound 13 has favorable enzyme-inhibiting activity.
Figure 4. The binding mode between the active conformation of compound 13 and the
protein TERT (PDB code: 3DU6). Entire view (A) and view of the active site cavity (B).
The compound is rendered in green stick models, and the amino acid residues are rendered
in golden sticks.

In summary, in our quest to find new, efficient compounds with telomerase-inhibiting
activity and to extend the research on coumarin analogs, we designed new
2-phenylpyrimidine coumarin derivatives, followed by their chemical synthesis; we then
performed biological evaluations of them. An in vitro antitumor study showed that these
compounds have strong effects on resisting tumor cell proliferation. Among them,
compound 13 exhibited strong inhibitory activity against the CNE2 cells, and it was
revealed to have the most potent telomerase-inhibiting activity, with an IC50 value of
0.82±0.14 M. Flow cytometric analysis indicated that cell proliferation was also arrested
by compound 13, which was accompanied by a shortening of the telomere length.
Molecular docking indicated that active compound 13 was nicely bound to the catalytic
subunits of telomerase TERT through multiple patterns and could replace nucleotides as
active substrates. The above results certify that 2-phenylpyrimidine coumarin derivatives
have the potential to become lead compounds among antitumor drugs.
Acknowledgments
This work was supported by the key fund project of Anhui Education Department (Grant
No.kj2017A830). We thank LetPub (www.letpub.com) for its linguistic assistance during
the preparation of this manuscript.
A. Supplementary data
Supplementary data associated with this article can be found.
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