Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmcl.2017.12.066

Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide. Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as anti-breast cancer drugs.

Full text of DOI:10.1016/j.bmcl.2017.12.066

Accepted Manuscript
Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives
as anti-breast cancer agents
Linyi Liu, Zhichao Tang, Chengze Wu, Xinyu Li, Ali Huang, Xiang Lu, Qidong
You, Hua Xiang
PII:
S0960-894X(17)31240-4
https://doi.org/10.1016/j.bmcl.2017.12.066
BMCL 25522
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
21 October 2017
18 December 2017
29 December 2017
Please cite this article as: Liu, L., Tang, Z., Wu, C., Li, X., Huang, A., Lu, X., You, Q., Xiang, H., Synthesis and
biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents, Bioorganic &
Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.066
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Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine
derivatives as anti-breast cancer agents
Linyi Liu a,b,#, Zhichao Tanga,b,#, Chengze Wua,b, Xinyu Lia,b, Ali Huanga,b, Xiang
Lua,b, Qidong Youa,b, Hua Xianga,b,*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
* Corresponding authors. Tel.: +86 025 83271096; Fax: +86 025 83271096(H. Xiang). E-mail
addresses: xianghua@cpu.edu.cn (H. Xiang)
# Same contribution to this paper.
Abstract
Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide.
Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer
patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of
ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic
activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become
resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen
receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that
these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified
compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit
angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as
anti-breast cancer drugs.
Keywords
4,6-diaryl-2-pyrimidinamine derivatives, Anti-breast cancer, SERMs, VEGFR-2
Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide
according to GLOBOCAN 2012.1 Susceptible population of breast cancer have common characteristics, including advanced age,
low parity, delayed age at first delivery, short duration of breastfeeding, overeating, limited exercise and so on. Breast cancer is a
heterogeneous disease and multiple subtypes have been defined. The overexpressed of estrogen receptor α (ERα) which is a
member of the large superfamily of nuclear receptors has been identified as the predominant internal reasons, involving in more
than 70% breast cancer patients, and this type of patients are tagged as ER positive (ER+).2 In this case, the continuous activation
of ERα by estrogens induce the proliferation of tumor cell, so the blocking-up of ER signaling by competitively binding to ER
with anti-estrogens or estrogen deprivation is an effective therapeutic strategy 3. Selective estrogen receptor modulators (SERMs)
which compete with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such
as tamoxifen, raloxifene.4 However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues,
such as endometrial cancer.5 Moreover, up to approximate 50% of patients with ER-positive tumors either initially do not respond
or become resistant to these drugs within 5 years of treatment.6 Thus contribute to a significant obstacle towards ER+ breast

cancer treatment. Hence, there is a great need for the development of new drugs to improve therapeutical effect in the breast
cancer treatment.
The significance of the 2-pyrimidinamine scaffold in medicinal chemistry is widely known. Many of these compounds are
serving as anticancer agent (Figure 1). Here, we noticed that 4,6-diaryl-2-pyrimidinamine derivatives shows good activity in
pharmacological studies.7-10. These compounds have similar toluylene likely to tamoxifen (Figure 2), and the distance of two 4-C
in phenyl is approximate equal to tamoxifen (9.8Å vs 9.3Å, simulation by Chem3D). Moreover, 2-pyrimidinamine scaffold is the
key skeleton of pazopanib and JNJ-1702925911 which are the vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors.
It’s known that VEGFR-2 plays key role in the angiogenesis pathway which participates in the proliferation, metastasis, and
invasion of breast cancer cells.12 And studies identified that VEGFR-2 involve in tamoxifen resistance via the Ras/MAPK
pathway.13,14 The combination of tamoxifen and a low dose of brivanib alaninate, a VEGFR-2 inhibitor, was reported not only to
maximize therapeutic efficacy but also to retard SERM resistant tumour growth.15 Thus urge us to design new molecules which
have potential to anti-breast cancer via the inhibition of ER and VEGFR-2 based on 4,6-diaryl-2-pyrimidinamine scaffold (Figure
2). Studies found that the phenolic groups which exist in 4-hydroxytamoxifen and raloxifene, interact to the binding domain of
ERα, mimicking to estradiol A-ring.4 Meanwhile, the side chains, 4-[2-(dimethylamino)ethoxy]phenyl of tamoxifen or
4-[2-(1-piperidinyl)ethoxy]phenyl of raloxifene are critical for anti-estrogenic activity, and this side chain also exist in
JNJ-17029259 (the Blue Oval, Figure 2). So according to the combination principle of medicinal chemistry, it’s easy to design
new structure based on the 4,6-diaryl-2-pyrimidinamine scaffold (Figure 2).
Figure 1 .Anti-cancer drugs with the 2-pyrimidinamine scaffold
Figure 2 The design of target molecules based on 4,6-diaryl-2-pyrimidinamine scaffold
The synthetic route of target 4,6-diaryl-2-pyrimidinamine derivatives is shown in Scheme 1. The key intermediate,
4,6-bis(4-methoxyphenyl)-3,4-dihydropyrimidin-2(1H)-one (4), was prepared from the commercially available urea,

1-(4-methoxyphenyl)-ethanone, and 4-methoxybenzaldehyde according to Biginelli reaction in high yields. Then, intermediate 4
was dehydrogenize and the hydroxy was replaced by chloro under the reflux of phosphorus oxychloride to give
2-chloro-4,6-diaryl-2-pyrimidine (5). This pyrimidine reacted a nucleophilic replacement with substitutional aniline to give new
compounds III-1A~III-12A. At last, under the catalysis of BBr3, the takeoff of one or two methoxy group of compounds
III-1A~III-12A obtained the end-products III-1B~III-12B and III-1C~III-12C. Finally, we got three series of novel small
compounds. All of the synthesized compounds have been confirmed by NMR and mass spectrometry.
Scheme 1. Reagents and conditions: a) acetonitrile, FeCl3•6H2O, TMSCl, 80°C, 10h; b) POCl3, 110°C, 5h,
c) dry dioxane, Pd(OAc)2, Cs2CO3, BINAP, 100°C, 8h. d) dry dichloromethane, BBr3, 0°C /rt, 4h.
To evaluate the anticancer activity, all synthesized compounds were screened against human breast cancer cell line MCF-7,
human umbilical vein endothelial cell (HUVEC) as well as human endometrial cancer cell line Ishikawa (Table 1). Human Breast
cancer cell line MCF-7 belongs to ER+ cancer cell, and is good target cell for the screen of ER antagonist. Meanwhile, human
umbilical vein endothelial cell (HUVEC) have played a major role as a model system for the study of the regulation of
endothelial cell function and are suitable for the evaluation of antiangiogenesis effect by antiproliferative experiment. Here, most
compounds show low micromole IC50 to MCF-7, and are better than tamoxifen in our antiproliferative experiment. There are no
significant difference between the acylamino side chain and ethyoxyl side chain except in certain amino groups. Certainly,
diethylaminoethoxyl and piperdinoethoxyl are better than the acylamino side chain (III-8 vs III-2, III-10 vs III-4) no matter
whether the methyl of 4,6-phenol in pyrimidine exist or not, but morpholinyl acetamide is better than the another (III-5 vs
III-11). When we removed two methyl, the inhibitory activity of dimethylaminyl or pyrrolidinyl derivatives significant decreased
(III-1C, III-3C, III-7C and III-9C), while other compounds have no difference. Furthermore, studies identified these
compounds have good inhibitory activity to HUVEC and most of them are comparable to sunitinib. However, several double
demethylation derivatives (III-1C, III-5C and III-11C) showed low inhibitory activity. From the results, we can simply
summary the structure-activity relationship. The linker of side chain including the acetamido and ethyoxyl are not the key factor
of activity. When the terminal amino group is diethylamine (III-2), the inhibitory activity significant decreased, this indicated the
terminal may be suitable for the small amino group. Moreover, although many SERMs contain the bisphenol, the demethylation
can’t improve the activity of synthesized compounds in cell level, and several compounds’ activity even declined perhaps
because of physicochemical property.
Table 1 Cancer cells antiproliferative activity (IC50, µM)
MCF-7 HUVEC Ishikawa
MCF-7 HUVEC Ishikawa
Compound
Compound
IC50/μM IC50/μM IC50/μM
IC50/μM IC50/μM IC50/μM
III-1A
III-1B
2.5
3.4
2.8
3.8
3.6
6.3
III-7A
III-7B
3.6
3.6
4.6
4.7
5.1
5.2

III-1C
III-2B
III-2C
III-3A
III-3B
III-3C
III-4B
III-4C
III-5A
III-5B
III-5C
III-6A
III-6B
III-6C
Tamoxifen
39.5
49
60.6
22.0
19.0
2.5
2.3
7.0
10.6
7.4
-
8.0
16.4
17.3
1.9
III-7C
III-8A
19.4
5.0
5.6
5.1
2.6
4.6
57
4.7
2.2
3.5
2.5
2.1
3.1
4.7
5.4
4.5
4.6
7.7
74.9
3.0
2.4
2.5
3.0
1.6
4.3
6.4
4.8
3.9
5.9
4.5
1.9
4.8
5.0
31.9
-
27
III-8B
2.2
III-8C
4.5
5.5
III-9A
19.7
>100
26.4
33.1
19.2
15.6
1.8
8.3
III-9B
1.4
III-9C
13.5
-
III-10A
III-10B
III-10C
III-11B
III-11C
III-12A
III-12B
III-12C
Sunitinib
32.4
17.1
6.1
95.1
-
10.2
47
42.2
60.4
5.7
5.0
4.7
6.8
4.1
4.2
4.2
4.8
5.2
5.7
5.3
5.4
5.2
4.9
19.5
Furthermore, the cell line Ishikawa bears estrogen and progesterone receptors, the cells have been used in numerous basic
research areas. It is known that tamoxifen will stimulate endometrial cell growth and induce endometrial cancer after long term
treatment.16 Here, we evaluated the synthesized compounds to the effect of uterus by the Ishikawa antiproliferative experiment
(Table 1). And the simulation of tamoxifen to Ishikawa had been confirmed. Tamoxifen simulated the cell line Ishikawa’s
proliferation under the concentration of 10µM and weakly inhibited Ishikawa’s proliferation above 40µM in our experiment.
Fortunately, the compounds which we synthesized could significant inhibit the proliferation of Ishikawa cells and the IC50 of
most molecules are under the 10µM. This indicated synthesized compounds will not induce endometrial cancer.
In order to identify the function of synthesized compounds to ER and VEGFR-2, we choose III-3A for further study.
Because progesterone receptor (PgR) is the target gene of estrogen, the expression level of PgR is commonly used to assess
estrogenic or antiestrogenic activity. Here we used real-time polymerase chain reaction (RT-PCR) in the ER positive MCF-7 cells
to evaluate the modulation of progesterone receptor. As shown in Figure 3A, presence of 10 nM estradiol (E2) was able to
remarkably elevate the mRNA expression of PgR gene compared to the vehicle control. While both tamoxifen and III-3A can’t
simulate the mRNA expression of PgR gene without E2. When combination with 10nM E2, tamoxifen under the concentration of
1µM could significant reduce the expression of PgR mRNA compared with the E2 group. Compound III-3A also exhibited
stronger antagonism against the expression of PgR mRNA, although weaker than tamoxifen. This result identified that III-3A
could competitively bind to ER against E2. Moreover, we explored if III-3A could block VEGF signalling pathway by inhibiting
the VEGFR-2 tyrosine kinase phosphorylation. A Western blotting assay for total and phosphorylated (active) VEGFR-2 was
performed (Figure 3B). The positive control group sunitinib could significantly inhibit the phosphorylation of VEGFR-2 in
HUVEC. Despite weaker than sunitinib, Compound III-3A could make p-VEGFR-2 fall to approximate 50% compared to
control group at 1µM. These results identified compound III-3A could antagonize against estrogen and inhibit the
phosphorylation of VEGFR-2.

Figure 3. The effect of III-3A to PR and VEGFR-2. A). The mRNA expression of PR was examined by
Real-time PCR. The data illustrated the increased mRNA expression of PR induced by E2 was reversed by
III-3A in MCF-7 cells. B). III-3A inhibits the phosphorylation of VEGFR-2 in HUVEC cells. Expression of
p-VEGFR-2 and VEGFR-2 in HUVEC cells were examined by western blots. Densitometric analysis was
performed to determine the phosphorylation rate of VEGFR-2. From the chart, the phosphorylation of
VEGFR-2 in HUVEC were inhibited by sunitinib and compound III-3A. Values are mean ±SD (n=3). * P <
0.05, ** P < 0.01, *** P < 0.001 vs. E2 group in A or Control group in B. ### P< 0.001 vs Control group.
Futhermore, compound III-3A was selected to perform chicken chorioallantoic membrane (CAM) assay to investigate their
inhibition of angiogenesis in vivo. Compound III-3A and the positive control sunitinib dissolved in DMSO were placed on
sterile methyl cellulose filter papers at 1µM, 10µM and 20µM with phosphate buffered saline (PBS) as the blank control. Results
are shown in Figure 4. Compared with blank control group, compound III-3A could significantly inhibit angiogenesis and be
similar to sunitinib. Moreover, the inhibitory ability was proportional to the concentration. Overall, compound showed potential
anti-angiogenesis activities in vivo.
Figure 4 Results of CAM assay.
In conclusion, we have designed and synthesized a series of 4,6-diaryl-2-pyrimidinamine derivatives targeting both ER and
VEGFR-2. Biological evaluation showed that most of the synthesized compounds exert stronger inhibitory activity to MCF-7,
HUVEC and Ishikawa cells. Further investigation also showed these compounds were expected to inhibit ER positive breast
cancer through inhibition of ER and VEGFR-2 simultaneously based on the result of III-3A which exhibited stronger antagonism

against the expression of PgR mRNA in MCF-7 and the inhibitory activity to the phosphorylation of VEGFR-2 in HUVEC at
1µM as well as potential anti-angiogenesis effects in vivo. This work may provide a new and potential route to develop effective
drugs for breast cancer and will help us for the next research.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (81373279), the Twelfth
Five-Year Plan Major Project of Candidate Drugs (Ministry of National Science and Technology, 2012ZX09103101048). The
authors are grateful to Dr. Qianming Du and Chao Liu for the help in the biological test.
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1. Approximate 50% of patients with ER-positive tumors either initially do not respond or
become resistant to SERMs.
2. Tamoxifen simulated the cell line Ishikawa’s proliferation under the concentration of 10µM in
our experiment.
3. A series of designed 4,6-diaryl-2-pyrimidinamine derivatives could simultaneously antagonize
ER and inhibit VEGFR-2.