This document presents a journal club presentation focusing on the synthesis and anticancer activity of pyrimidine derivatives, particularly compound 4b, which exhibited significant cytotoxic effects against SW480 human colon cancer cells. Compound 4b showed a strong potential for inducing apoptosis and demonstrated the ability to upregulate pro-apoptotic proteins while downregulating anti-apoptotic proteins, suggesting a mitochondria-mediated intrinsic apoptosis pathway. The findings support further exploration of compound 4b as a lead for developing new anticancer therapies.

1. Journal Club Presentation-Round
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As a part of curricular requirement for
M. Pharmacy II year III semester
Presented by
Darshan N U
Register. No. 21MPC101
Department of Pharmaceutical Chemistry
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2. Title, Authors and Affiliations:
Journal Bioorganic chemistry
DOI https://doi.org/10.1016/j.bioorg.2020.104028
Impact factor 2.097 (14 April 2020)
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3. Introduction
Cancer remains the second biggest cause of death after cardiovascular disorders, considering the
great research that has been done in this area. WHO reported Cancer is the biggest disease lots of
people passed away from cancer globally. Across the globe highest number of cancer deaths
occurred in Asia (57.3%), Europe (23.4%) & Africa (7.4%). The class of heterocyclic nitrogenous
compounds known as pyrimidines has many uses in the research and development of anticancer
drugs. Pyrimidine derivatives have attracted great interest in medicinal chemistry because of their
wide range of biological and pharmacological activities. To date, a large array of pyrimidine and
pyrimidine-fused heterocyclic compounds having anticancer activity via multiple different
mechanisms.
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4. A representative pyrimidine derivative Imatinib is the first kinase inhibitor approved for the treatment of
chronic myeloid leukemia(CML). Clinical success of Imatinib has prompted substantial interest in the
further development of novel pyrimidines.
Nilotinib, structurally related to Imatinib, is developed for the treatment of imatinib-resistant chronic
myelogenous leukemia.
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5. Infigratinib, a pan-FGFR(fibroblast growth factor receptor) kinase inhibitor has a manageable safety profile
in phase 2 trial for cholangiocarcinoma treatment.
2,4-Pyrimidine based drugs such as Rociletinib and Osimertinib are presently used for EGFR (epidermal
growth factor receptor) triggered NSCLC (non-small cell lung cancer).
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6. On the other hand, diarylurea scaffold is also of wide interest due to their diverse anticancer properties.
Sorafenib is the first example of kinase inhibitor bearing urea moiety that is used for treatment of advanced
renal cancer. To date many diarylurea derivatives were reported as potential anticancer agents against various
cancer cell lines. Previously, reported some pyrrolopyrimidine derivatives designed by merging substituted
phenylurea moieties. Among them, compounds bearing 3-trifluoromethyl and 4-chloro-3- trifluoromethylphenyl
urea groups showed strong cytotoxic effect through activation of the mitochondrial apoptosis pathway in A549
(isolated from lung tissue) and PC3 (human prostate cancer) cells, respectively.
sorafenib 3-trifluoromethyl pyrrolopyrimidine derivatives
4-chloro-3- trifluoromethylphenyl urea
pyrrolopyrimidine derivatives
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7. Material and methods
Scheme
N
N
H
N
H2
NH2
O
N
N
N
H2
NH2
Cl
6-chloropyrimidine-2,4-diamine
N
N
N
H2 NH
Cl
NH
O
R1
R2
N
N
N
H2 NH
N
NH
O
R1
R2
N
R3
N
N
N
H2 NH
N
NH
O
R1
R2
O
a b
c
1
2
2,6-diaminopyrimidin-4(3H)-one
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8. N
N
N
H2
NH2
Cl
N
N
N
H2
NH2
d
b
N
N
N
H2 NH NH
O
R1
R2
4
6-phenylpyrimidine-2,4-diamine
N-(2-amino-6-phenylpyrimidin-4-yl)-N'-[3-(trifluoromethyl)phenyl]urea
N-(2-amino-6-phenylpyrimidin-4-yl)-N'-[4-chloro-3-(trifluoromethyl)phenyl]urea
1
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9. Synthesis of the target compounds started with the chlorination reaction of 2,4-diamino-6-
hydroxypyrimidine using POCl3. The urea linker at 4-position of pyrimidine ring was formed by the
reaction of 6-chloropyrimidine-2,4-diamine (1) or 6-phenylpyrimidine2,4-diamine (4) with
isocyanate derivatives in the moderate yield. For the preparation of the 6-phenylpyrimidine-2,4-
diamine (4), 6- chloropyrimidine-2,4-diamine underwent a Suzuki coupling reaction with
phenylboronic acid in DME (Dimethyl ether) in the presence of Pd(PPh3)4 and 2 M Na2CO3. Final
compounds (2a-e, 3a-e) were prepared via reaction of the chloropyrimidines (2 and 3) with
appropriate amine derivatives in the mixture of DMF:H2O:IPA (1:1:1) under reflux 48 hours
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10. Synthesis of 6-chloropyrimidine-2,4-diamine (1)
2,6-diaminopyrimidine-4(3-H)-one (2.25 g, 0.017 mol) was refluxed in POCl3 (17 mL) for 3 h. The
reaction mixture was added to ice-water and stirred at 90 °C for 1 h. The pH of the mixture was adjusted
to 7–9 with aqueous ammonia and then it was extracted with EtOAc (ethylacetate). Organic phase was
dried over anhydrous Na2SO4 and evaporated to obtain pure product 1.88 g, yield 73%. MS (ESI) m/z:
144.9 [M]+, 146.8 [M+2]
General procedures for synthesis of compound 2 and 3
6-chloropyrimidine-2,4-diamine (1) in acetonitrile, the phenylisocynate derivatives (1 eq) was added. The
reaction mixture was stirred at room temperature for overnight. After completion of the reaction; the
precipitate formed was filtered, washed with ethanol, and dried to obtain the pure product 2 and 3.
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11. General procedures for synthesis of compound 4a and 4b
6-phenylpyrimidine-2,4-diamine (4) in acetonitrile, the phenylisocynate derivatives (2 eq) was added. The
reaction mixture was stirred at room temperature for overnight. After completion of the reaction; the precipitate
formed was filtered and crystallized by the mixture of ethanol and ethylacetate to obtain the pure product 4a
and 4b
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12. RESULT AND DISSCUSSION
Biological evaluation
Cell proliferation assay
The synthesized compounds (2a-e, 3a-e, 4a-b) were screened for in vitro cytotoxicity against human colon
cancer and human prostate cancer cell lines using MTT assay. The results are given in Table
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13. Compound 4b substituted with the rigid and hydrophobic phenyl ring at the 6-position of pyrimidine ring
showed the best cytotoxic effect against SW480 cell line with the IC50 value of 11.08 µM. Based on the
cytotoxicity profile of the synthesized pyrimidine derivatives, compound 4b can be taken as lead to explore
the effect of the bulky and hydrophobic groups at the 4-position of pyrimidine ring for the design of better
compounds.
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14. Cell cycle analysis
In order to obtain a better understanding of the cytotoxic activity of compound 4b, cell cycle assay was
performed by treating SW480 cells with IC50 concentration of 11.08 µM for 24 h. As shown in Fig.5, the cell
population percent at G2/M phase increased to 45.3% in compound 4b treated group, whereas it was 21.6% in
untreated control group, suggesting that compound 4b induced a significant G2/M phase arrest (p < 0.05).
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15. Apoptosis and possible mechanism
The evaluation of apoptotic effect of compound 4b was performed through Annexin V binding assay (powerful
tool for he quantification of apoptosis). The SW480 cells were treated with compound 4b at IC50 concentration
and the annexin V bound cell population were measured after a 24 h incubation. As shown in Fig. 6, the
percentage of cells in early apoptosis were recorded as to 5.69 ± 0.16% while it was 0.7 ± 0.28% in nontreated
cells. The late apoptotic cell population were also significantly increased to 3.52 ± 0.30% in compound 4b
treated group when compared to control (p < 0.01).
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16. The apoptotic efficiency of compound 4b on SW480 cells were also shown through fluorescein staining (Fig. 7).
The annexin V binding of cells treated with compound 4b at IC50 concentration were observed under a
fluorescence microscope. The annexin V/FITC staining of SW480 cells showed that the cells had undergone
remarkable apoptotic changes by compound 4b treatment.
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17. Western blot analysis were displayed the alteration on expression of apoptosis-related proteins.
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18. Conclusion
Among the synthesized compounds, 4b bearing the rigid and hydrophobic phenyl moiety at the 4-position of
pyrimidine ring showed the most cytotoxic activity against SW480 cell line with IC50 value of 11.08 µM.
Compound 4b also induced apoptosis in SW480 cells by upregulating Bax, Ikb-α and cleaved PARP and
downregulating Bcl-2 expression. Moreover, compound 4b has potent effect on mitochondrial membrane
potential in SW480 cells, indicating compound-4b induced apoptosis occurs through mitochondria-mediated
intrinsic apoptosis pathway. These findings demonstrate that compound 4b could be used as a lead for
designing new anticancer compounds for further studies.
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19. REFERED ARTICLE
Kilic-Kurt Z, Ozmen N, Bakar-Ates F. Synthesis and anticancer activity of
some pyrimidine derivatives with aryl urea moieties as apoptosis-inducing
agents. Bioorganic Chemistry. 2020 Aug 1;101:104028.
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20. THANK YOU
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