Synthesis, Biological Evaluation and Docking Study of New Pyrimidine Compounds as Anticancer Agents

Article abstract of DOI:10.1055/a-1306-0202

Objectives The microtubule is composed of αβ tubulin heterodimers and is an attractive target for the design of anticancer drugs. Over the years, various compounds have been developed and their effect on tubulin polymerization has been studied. Despite a great efforts to make an effective drug, no drug has been introduced which inhibit Colchicine binding site. Methods In the current work a series of pyrimidine derivatives were designed and synthesized. Furthermore their cytotoxic activities were evaluated and molecular docking studies were performed. Twelve compounds of pyrimidine were synthesized in 3 different groups. In the first group, 4,6-diaryl pyrimidine was connected to the third aryl group via thio-methylene spacer. In the second group, this linker was substituted by sulfoxide-methylene moiety and in the third group sulfone-methylene group was used as spacer. Results The cytotoxic activity of these compounds were evaluated against 3 different cancerous cell lines (HT-29, MCF-7, T47D) as well as normal cell line (NIH3T3). Compounds in group 2 showed the best cytotoxicity and compound 7d showed the most potent cytotoxic activity against all cell lines. Molecular modelling studies revealed that compound 7d could strongly bind to the colchicine binding site of tubulin. Conclusion Altogether, with respect to obtained results, it is attractive and beneficial to further investigation on pyrimidine scaffold as antimitotic agents.

Full text of DOI:10.1055/a-1306-0202

Published online: 2020-12-07
Thieme
Original Article
Synthesis, Biological Evaluation and Docking Study of New
Pyrimidine Compounds as Anticancer Agents
Authors
Shahin Boumi1, Jafar Moghimirad1, Seyed Nasser Ostad2, Massoud Amanlou1, Shohreh Tavajohi2, Mohsen Amini1
ABSTrAcT
Affiliations
1
Department of Medicinal Chemistry, Faculty of Phar-
macy, Drug Design and Development Research Center,
The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
Objectives The microtubule is composed of αβ tubulin het-
erodimers and is an attractive target for the design of antican-
cer drugs. Over the years, various compounds have been de-
veloped and their effect on tubulin polymerization has been
studied. Despite a great efforts to make an effective drug, no
drug has been introduced which inhibit Colchicine binding site.
Methods In the current work a series of pyrimidine derivatives
were designed and synthesized. Furthermore their cytotoxic
activities were evaluated and molecular docking studies were
performed. Twelve compounds of pyrimidine were synthesized
in 3 different groups. In the first group, 4,6-diaryl pyrimidine
was connected to the third aryl group via thio-methylene
spacer. In the second group, this linker was substituted by
sulfoxide-methylene moiety and in the third group sulfone-
methylene group was used as spacer.
2
Department of Toxicology and Pharmacology, Faculty of
Pharmacy and Poisoning Research Center, Tehran
University of Medical Sciences, Tehran, Iran
Key words
synthesis, pyrimidine, docking, cytotoxic, anticancer, MTT
assay
received
accepted
published online 2020
09.08.2020
01.11.2020
Results The cytotoxic activity of these compounds were eval-
uated against 3 different cancerous cell lines (HT-29, MCF-7,
T47D) as well as normal cell line (NIH3T3). Compounds in group
2 showed the best cytotoxicity and compound 7d showed the
most potent cytotoxic activity against all cell lines. Molecular
modelling studies revealed that compound 7d could strongly
bind to the colchicine binding site of tubulin.
Bibliography
Drug Res
DOI 10.1055/a-1306-0202
ISSN 2194-9379
© 2020. Thieme. All rights reserved.
Georg Thieme Verlag KG, Rüdigerstraße 14,
70469 Stuttgart, Germany
Conclusion Altogether, with respect to obtained results, it is
attractive and beneficial to further investigation on pyrimidine
scaffold as antimitotic agents.
Correspondence
Mohsen Amini
Department of Medicinal Chemistry, Faculty of Pharmacy
Tehran University of Medical Sciences, Tehran,
14176 Tehran
Iran (the Islamic Republic of)
Tel:. + 98 21 66959055
moamini@tums.ac.ir
Introduction
shape, cell division and intracellular transport have been referred
to tubulins [3] and hence, they have been recognized as an impor-
tant target in cancer treatment. Tubulin interacting drugs are clas-
sified into two main groups: microtubule stabilizing (e.g. taxanes)
and microtubule destabilizing compounds (e.g. colchicine). Com-
bretastatin A-4 (▶Fig. 1a) is a lead compound that interacts with
colchicine binding site [4]. Most of colchicine binding site agents
has simple chemical structure, high potency and they have ability
to overcome p-glycoprotein, a known agent that caused to form
drug resistance in cancerous cells. Until know, there is not any FDA
One of the main reasons of death worldwide has been attributed
to cancer. Because of aging and other reasons in most countries,
number of people suffering from cancer is estimated to increase.
While enormous efforts, cancer treatment confronts with more
complex situation due to resistance to chemotherapy and exten-
sive side effects of anti-cancer agents [1]. Therefore, design and
synthesis of more potent, selective, and safe new anticancer agents
have been targeted for many research teams [2]. Microtubules play
crucial roles in different functions of cells. Cell signalling and its
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

Thieme
Original Article
▶Fig. 1 Structure of combretastatin A-4 (a), celecoxib (b), diaryl-heterocyclic derivatives (c), triaryl-heterocyclic derivatives (d), and
3-(alkylthio)-5,6-aryl-1,2,4-triazine deivatives (e).
approved drugs which interact with colchicine binding site hence,
it is too important to synthesize CA-4 analogues as active mole-
cules [5]. According to the structure activity relationship (SAR) of
CA-4 analogues, the cis-configuration between ring A and ring B is
important in biological activity. Celecoxib (▶Fig.1b) and other
COX-2 selective inhibitors belong to cis-diaryl heterocyclic struc-
tures. These drugs were synthesized as low gastrointestinal toxic-
ity anti-inflammatory drugs. Soon after that, COX-2 inhibitors
proved to be effective as chemotherapeutic agents [6]. The mech-
anism of their cytotoxic activity has been referred to inhibition of
COX-2-overexpressed in various cancers [7]. There are a lot of stud-
ies that researchers have fixed ring A and ring B in cis-configuration
with different heterocyclic rings (▶Fig. 1c) such as pyrrole, thia-
zole, pyrazole, oxazole, imidazole, quinazoline, and benzofuran [8–
14] to mimic the rigid form of CA-4. Furthermore, there are also
reports about potent anti-cancer activity of some tri-aryl hetero-
cyclic compounds [15] in different cancerous cell lines (▶Fig. 1d).
Recently, a series of 3-(alkylthio)-5,6-diaryl-1,2,4-triazines
(▶Fig. 1e) were prepared and evaluated for their cytotoxic activi-
ties in vitro against different human cancer cell lines. Some of them
showed remarkable cytotoxic activity in cancerous cell line study
[13]. In the current work we developed the structure of D-ana-
logues (▶Fig. 1d) to new di-aryl pyrimidine scaffold and evaluated
their anticancer activity. Docking simulation was used to find the
best conformation of ligands in colchicine binding site. The inves-
tigated ligands occupy the colchicine binding site.
robenzaldehydes (2) in methanol (10 mL) with constant stirring.
The reaction mixture was stirred for 3-5 hours at room tempera-
ture. After completion of the reaction (monitored by the TLC), the
reaction mixture was diluted with ice cold water and filtered. The
solid was washed and crystallized from ethanol.
General procedure for the preparation of
dihydropyrimidine-2-thiones (4)
A mixture of chalcone (1mmol), thiourea (1mmol) and potassium
hydroxide (1.5 mmol) in ethanol (10mL) was refluxed with stirring
at 90 °C for 8 hours. The solid residue was collected, washed with
water to give intermediate 4.
General procedure for the synthesis of pyrimidine-2(1H)-
thiones (5)
The crude of 4 (1 mmol) was dissolved in acetone and DMF (1:1;
5mL) and DDQ (1.5mmol) was added slowly at 10 °C. The reaction
was monitored by TLC, the solid residue was collected and washed
with water. The product was crystallized from ethanol.
General procedure for the synthesis of 2-(benzylthio)-
pyrimidine derivatives (6)
Compound 5 (1 mmol) was dissolved in dry DMF (3mL), then NaH
(3 mmol) was added in reaction, after 5 minutes appropriate ben-
zyl halides (2 m mol) was added drop wise to the mixture at room
temperature. The mixture was stirred for 3 h, upon completion
(checked by TLC), the reaction mixture was poured into cold water
(5 mL). The precipitated solid was separated by filtration, washed
with cold water and was dried. The crude product was purified by
column chromatography on silica gel using ethyl acetate: petrole-
um ether (1:5) as eluent to give compound 6.
Experimental
1H NMR and 13C NMR spectra were recorded with a Varian FT-500,
using TMS as an internal standard. Coupling constant (J) values are
presented in hertz (Hz) and spin multiples are given as s (singlet),
d (double), t (triple), and m (multiple). Melting points were deter-
mined with a Kofler hot stage apparatus and are uncorrected. In-
frared spectra were acquired on a Nicolet Magna 550-FT spectrom-
eter. IR spectra of solid were recorded in KBr disk and the absorp-
tion band was given in wave numbers in cm− 1. Mass spectra were
obtained with an Agilent Technology (HP) mass spectrometer op-
erating at an ionization potential of 70 eV.
2-(Benzylthio)-4-(4-chlorophenyl)-6-phenylpyrimidine (6a)
Yield 80 %, m.p. 122-125 °C, IR (KBr, cm-1) 489, 692, 760, 836,
1008, 1070, 1091, 1185, 1235, 1286, 1309, 1364, 1408, 1450,
1491, 1514, 1572, 1740; 1H NMR (CDCl3) δ: 4.58 (s, 2H, CH2), 7.20-
7.30 (m, 3H), 7.40-7.55 (m, 3H), 7.74 (s, 1H, H-pyrimidine), 8.06-
8.15 (m, 8H); 13C NMR (CDCl3) δ: 35.68, 108.11, 127.31, 127.45,
128.67, 128.69, 128.73, 129.08, 129.29, 131.28, 135.36, 136.78,
137.40, 138.03, 163.77, 165.19, 172.30; Mass, m/z (%): 388 (100),
355 (32), 311 (18), 266 (25), 231 (34), 91 (50).
Chemistry
General procedure for the preparation of chalcone (3)
An aqueous solution of NaOH (3mL, 60 %) was added dropwise to
a solution of acetophenones 1 (1 mmol) and 1 mmol of 4-chlo-
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

2-[(4-Chlorobenzyl)thio]-4-(4-chlorophenyl)-6-
phenylpyrimidine (6b)
166.35, 172.81. Mass, m/z (%):406 (3), 404 (10), 368 (8), 265 (10),
163 (9), 129 (15), 91 (100), 57 (15).
Yield 85%, m.p. 110-112 °C IR (KBr, cm-1) 486, 502, 556, 634, 686,
725, 757, 778, 834, 1015, 1071, 1092, 1193, 1235, 1283, 1311,
1364, 1406, 1491, 1515, 1575; 1H NMR (CDCl3) δ: 4.51 (s, 2H, CH2),
7.25 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.5 Hz,
2H), 7.50-7.60 (m, 3H), 7.71(s, 1H, H-pyrimidine), 8.02 (d, J= 8.5
Hz, 2H), 8.05-8.15 (m, 2H); 13C NMR (CDCl3) δ: 34.92, 108.21,
127.39, 128.66, 128.74, 129.07, 129.29, 130.37, 131.31, 133.04,
135.28, 136.71, 136.75, 137.42, 163.80, 165.22, 171.90; Mass,
m/z (%): 422 (100), 389 (18), 354 (18), 311 (8), 266 (50), 231 (55),
125 (50), 89 (20).
2-[(4-Chlorobenzyl)sulfinyl]-4-(4-chlorophenyl)-6-
phenylpyrimidine (7b)
Yield 40%, m.p. 148-150°C, IR (KBr, cm-1), 450, 490, 633, 658, 699,
763, 779, 839, 983, 1016, 1025, 1049, 1092, 1198, 1234, 1284,
1369, 1414, 1446, 1496, 1586; 1H NMR (CDCl3) δ: 4.38 (d, J = 13
Hz, 1H, CH2), 4.49 (d, J = 13 Hz, 1H, CH2), 7.18 (d, J = 8.1 Hz, 2H),
7.27 (d, J= 8.1 Hz, 2H), 7.55 (d, J= 8.0 Hz, 2H), 7.58-7.65 (m, 3H),
8.10 (s, 1H, H-pyrimidine), 8.14 (d, J= 8.0 Hz, 2H), 8.19 (d, J = 7.35
Hz, 2H); 13C NMR (CDCl3) δ: 59.49, 112.45, 127.69, 128.62, 128.93,
128.98, 129.34, 129.56, 131.61, 132.14, 134.1, 134.67, 135.53,
138.38, 165.05, 166.38, 172.72; Mass, m/z (%): 439 (20), 297 (10),
265 (22), 163 (20), 125 (100), 77 (25).
4-(4-Chlorophenyl)-2-[(4-fluorobenzyl)thio]-6-
phenylpyrimidine (6c)
Yield 85%, m.p. 114-116 °C, IR (KBr, cm-1) 476, 522, 634, 684, 755,
776, 834, 1012, 1069, 1093, 1240, 1282, 1311, 1363, 1408, 1490,
1518, 1575; 1H NMR (CDCl3) δ: 4.53 (s, 2H, CH2), 6.99 (t, J=8.7 Hz,
2H, H3,5-Ph-F), 7.35-7.60 (m, 7H), 7.72 (s, 1H, H-pyrimidine), 8.05
(d, J=8.6 Hz, 2H, H2,6-Ph-Cl), 8.08-8.15 (m, 2H), ; 13C NMR (CDCl3)
δ: 34.84, 108.07, 115.45 (d, JCF = 21.28 Hz, C3,5-Ph-F), 127.35,
128.62, 127.03, 129.23, 130.56 (d, JCF = 7.95 Hz, C2,6-Ph-F), 133.79,
133.81, 135.23, 136.67, 137.35, 162.05 (JCF = 244.24 Hz, C4-Ph-F),
163.69, 165.11, 172.00; Mass, m/z (%): 406 (90), 373 (40), 315 (45),
284 (40), 249 (42), 222 (20), 91 (100), 71 (50), 55 (60).
4-(4-Chlorophenyl)-2-[(4-fluorobenzyl)sulfinyl]-6-
phenylpyrimidine (7c)
Yield 45%, m.p. 151-153°C, IR (KBr cm-1), 488, 517, 593, 637, 663,
691, 716, 764, 838, 1010, 1090, 1136, 1243, 1319, 1366, 1424,
1496, 1580; 1H NMR (CDCl3) δ: 4.33 (d, J=13.1 Hz, 1H, CH2), 4.46
(d, J=13.1 Hz, 1H, CH2), 6.94 (t, J=8.5 Hz, 2H, H3,5-Ph-F), 7.19 (dd,
J=8.5 & 5.4 Hz, 2H, H3,5-Ph-Cl), 7.55-7.62 (m, 3H, Ph), 8.07 (s, 1H,
H-pyrimidine), 8.15 (d, J=8.5 Hz, 2H, H2,6-Ph-Cl), 8.18 (d, J=7.4 Hz,
2H, H2,6-Ph). 13C NMR (CDCl3) δ: 59.36, 112.36, 115.83 (d, JCF =25.0
Hz, C3,5-Ph-F), 125.97, 125.99, 127.68, 128.96, 129.32, 129.55,
131.98, 132.02 (d, JCF =7.5 Hz, C2,6-Ph-F), 132.13, 134.12, 135.55,
138.38, 164.09 (d, JCF =247.5 Hz, C4-Ph-F), 165.01, 166.35, 172.81;
Mass, m/z (%): 422 (8), 265 (8), 163 (6), 129 (8), 109 (100), 77 (8).
4-(4-Chlorophenyl)-2-[(4-methylbenzyl)thio]-6-
phenylpyrimidine (6d)
Yield 95 %, m.p. 91-93 °C, IR (KBr, cm-1) 477, 526, 632, 688, 725,
758, 781, 833, 1010, 1090, 1152, 1187, 1232, 1253, 1286, 1310,
1367, 1411, 1461, 1512, 1573; 1H NMR (CDCl3) δ: 4.33 (s, 3H, CH3),
4.55 (s, 2H, CH2), 7.12 (d, J=7.9 Hz, 2H), 7.39 (d, J=7.9 Hz, 2H), 7.40-
7.55 (m, 5H), 7.73 (s, 1H, H-pyrimidine), 8.08 (d, J=8.3 Hz, 2H), 8.10-
8.15 (m, 2H); 13C NMR (CDCl3) δ: 21.25, 34.45, 108.07, 127.47
128.74, 129.00, 129.07, 129.29, 129.35, 131.25, 134.86, 135.40,
136.82, 136.97, 137.37, 163.70, 165.57, 172.44; Mass, m/z (%): 402
(100), 369 (20), 293 (15), 262 (40), 247 (42), 109 (33).
4-(4-Chlorophenyl)-2-[(4-methylbenzyl)sulfinyl]-6-
phenylpyrimidine (7d)
Yield 35%, 156-158°C, IR (KBr, cm-1), 455, 487, 564, 633, 687, 759,
781, 819, 847, 883, 1019, 1055, 1068, 1088, 1184, 1242, 1283,
1312, 1368, 1420, 1445, 1502, 1496, 1575, 1900; 1H NMR (CDCl3)
δ: 2.29 (s, 3H, CH3), 4.37 (d, J= 13.0 Hz, 1H, CH2), 4.49 (d, J= 13.0
Hz, 1H, CH2), 7.09 (d, J= 8.0 Hz, 2H, H3,5- tolyl), 7.13 (d, J= 8.0 Hz,
2H, H2,6-tolyl), 7.54 (d, J = 8.5 Hz, 2H, H3,5-Ph-Cl), 7.58-7.63 (m,
3H), 8.09 (s, 1H, H-pyrimidine), 8.14 (d, J= 8.5 Hz, 2H, H2,6-Ph-Cl),
8.19 (d, J= 7.6 Hz, 2H, H2,6-Ph); 13C NMR (CDCl3) δ: 21.26, 60.40,
112.36, 127.04, 127.73, 120.02, 129.27, 129.49, 130.21, 132.02,
134.28, 135.70, 138.25, 138.33, 164.93, 166.28, 173.12: Mass,
m/z ( %): 418 (6), 297 (25), 265 (9), 105 (100), 77 (15).
General procedure for the synthesis of compound 7
To a flask containing compound 6 (1 mmol) and dichloromethane
(10 mL) was added drop wise 1.5mmol of mCPBA in ice bath con-
dition. Upon completion the reaction (checked by TLC), the mix-
ture was washed with NaHCO3 solution (3 × 10 mL), dried over
Na2SO4. The solvent was removed under reduced pressure and
compound 7 was crystallized from diethyl ether.
General procedure for the synthesis of compound 8
To a round bottom flask containing 1 mmol of compound 6 was
added THF: water (20mL, 1:1) was added 2mmol of Oxone at room
temperature for 4h. After completion the reaction (checked by TLC)
the product was extracted with ethylacetate (twice, 30 mL), and
organic phase was separated, dried over sodium sulphate. The sol-
vent was removed under reduced pressure and the product was re-
crystallized from Ethanol to give compounds 8.
2-( Benzylsulfinyl)-4-(4-chlorophenyl)-6-phenylpyrimidine
(7a)
Yield 55%, m.p. 163-165°C, IR (KBr, cm-1) 447, 493, 557, 631, 690,
757, 783, 831, 884, 982, 1006, 1053, 1073, 1088, 1245, 1284,
1312, 1366, 1400, 1425, 1448, 1501, 1584, 1799, 1928; 1H NMR
(CDCl3) δ: 4.41 (d, J = 12.9, 1H, CH2), 4.53 (d, J = 12.9, 1H, CH2),
7.20-7.35 (m, 5H), 7.58 (d, J = 8.5 Hz, 2H, H3,5-Ph-Cl), 7.60-7.64
(m, 3H), 8.09 (s, 1H, H-pyrimidine), 8.19 (d, J=8.5 Hz, 2H, H2,6-Ph-
Cl), 8.21(d, J = 6.7 Hz, 2H, H2,6-Ph); 13C NMR (CDCl3) δ: 59.36,
112.36, 115.83, 125.97, 125.99, 127.68, 128.96, 29.33, 129.55,
131.98, 132.05, 132.13, 134.13, 135.56, 138.38, 163.99, 165.02,
2-(Benzylsulfonyl)-4-(4-chlorophenyl)-6-phenylpyrimidine
(8a)
Yield 80%, m.p. 235-237° C, IR (KBr, cm-1) 489, 514, 533, 561, 640,
693, 763, 780, 838, 878, 984, 1012, 1070, 1090, 1123, 1158, 1199,
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

Thieme
Original Article
▶Fig. 2 Synthetic pathway for preparation of the target compounds
1252, 1311, 1366, 1398, 1427, 1455, 1497, 1583 ; 1H NMR (CDCl3)
δ: 4.91 (s, 2H, CH2), 7.25-7.35 (m, 3H), 7.40-7.50 (m, 2H), 7.50-
7.60 (m, 5H), 8.14 (d, J= 8.0 Hz, 2H), 8.18-8.24 (m, 3H). 13C NMR
(CDCl3) δ: 57.6, 113.92, 127.15, 127.76, 128.90, 128.98, 129.04,
129.39, 129.63, 131.48, 132.42, 133.63, 135.06, 138.70, 165.33,
166.00, 166.71; Mass, m/z ( %): 422 (8), 420 (25), 355 (85), 265
(20), 163 (20), 129 (25), 91 (100), 65 (20).
3H, H 2,6-Ph & H-pyrimidine). 13C NMR (CDCl3) δ: 56.88, 114.05,
125.72, 127.76, 129.04, 129.18, 129.47, 129.71, 132.53, 132.78,
133.58, 135.02, 135.32, 138.85, 165.47, 165. 88, 166.81; Mass,
m/z (%): 458 (3), 456 (14), 454 (25), 389 (100), 355 (15), 265 (25),
163 (20), 125 (90), 77 (19).
4-(4-Chlorophenyl)-2-[(4-fluorobenzyl)sulfonyl]-6-
phenylpyrimidine (8c)
Yield 75%, m.p. 197-199°C, IR (KBr, cm-1) 490, 518, 609, 638, 692,
730, 759, 784, 816, 982, 1009, 1091, 1133, 1244, 1309, 1369,
1426, 1455, 1507, 1580; 1H NMR (CDCl3): δ: 4.92 (s, 2H, CH2), 6.99
(d, J= 8.6 Hz, H3,5-Ph-F), 7.42 (dd, J= 8.5 & J= 5.4 Hz, 2H, H2,6-Ph-
F), 7.53 (d, J= 8.6 Hz, 2H, H3,5-Ph-Cl), 7.56-7.70 (m, 3H), 8.15 (d,
J= 8.6 Hz, 2H, H2,6- Ph-Cl), 8.19-8.24 (m, 3H, H2,6-Ph & H-pyrimi-
2-[(4-Chlorobenzyl)sulfonyl]-4-(4-chlorophenyl)-6-
phenylpyrimidine (8b)
Yield 70%, m.p. 208-210°C, IR (KBr, cm-1) 487, 516, 543, 594, 636,
663, 688, 715, 762, 837, 1011, 1092, 1135, 1244, 1317, 1365,
1424, 1497, 1580; 1H NMR (CDCl3) δ: 4.88 (s, 2H, CH2), 7.25-7.38
(m, 3H), 7.42-7.60 (m, 4H), 8.15 (d, J= 8.6 Hz, 2H), 8.18-8.22 ( m,
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

▶Table 1 In vitro cytotoxic activities of compounds 6–8(a–d).
compounds
r
n
cytotoxicity (Ic50, μm)
HT-29
McF-7
T47D
NIH-3T3
15.1 ± 2.5
> 100
6a
6b
6c
H
0
0
0
0
1
1
1
1
2
2
2
2
> 100
> 100
> 100
Cl
F
> 100
78.9 ± 4.2
28.2 ± 2.5
> 100
> 100
> 100
23.2 ± 4.1
43.1 ± 4.8
15.1 ± 2.1
1.6 ± 0.3
10.7 ± 1.8
10.7 ± 1.9
85.2 ± 5.5
> 100
51.4 ± 5.4
96.0 ± 7.5
16.2 ± 1.7
3.5 ± 0.5
19 ± 3.2
6d
7a
7b
7c
Me
H
> 100
45.2 ± 3.1
> 100
2.3 ± 0.3
15.5 ± 1.7
1.8 ± 0.2
1.5 ± 0.2
75.0 ± 6.7
59.1 ± 3.5
45.2 ± 4.45
> 100
Cl
F
79.6 ± 4.5
60.0 ± 3.4
> 100
7d
8a
8b
8c
Me
H
16 ± 2.5
82.1 ± 5.7
51.1 ± 4.7
36.3 ± 4.4
48.3 ± 5.6
1.5 ± 0.2
Cl
F
53.2 ± 3.2
> 100
24.2 ± 3.2
> 100
8d
PTX
Me
45.4 ± 2.54
7.1 ± 1.15
0.2 ± 0.03
40.2 ± 2.1
dine). 13C NMR (CDCl3) δ: 56.70, 116.10 (d, JCF = 21.7 Hz, C3,5-Ph-
Cytotoxic studies
F), 122.92, 127.68, 127.83, 128.94, 129.12, 129.44, 129.71 (d,
Cells were seeded in 96- well plates at a density of 1000 cell per well
and incubated for 24 h for cell attaching. Attached cultured cells
were treated with different concentrations of synthesized com-
pounds at a range of 0.01-100 µM and were incubated for 48 h. At
the end of incubation period we added Phosphate Buffered Saline
(PBS) for cell washing and then cells were incubated with MTT so-
lution (5mg/ml) for 4h to allow the change of MTT to formazan salt.
After removing the medium we added DMSO (100µL) to the wells
for dissolving the formazan salt. Each plate absorbance was ana-
lysed by anthous 2020 plate reader at 540nm. This test was repeat-
ed three times and the IC50 values were determined by a nonlin-
ear regression analysis [16].
J
J
CF = 7.5 Hz, C2,6-Ph-F), 132.51, 133.59, 135.02, 138.8, 163.64 (d,
CF = 247.05 Hz, C4-Ph-F), 165.43, 165.93, 166.77; Mass, m/z ( %):
438 (30), 402 (20), 373 (70), 297 (25), 265 (20), 165 (23), 149 (25),
129 (35), 109 (70), 85 (60), 69 (100).
4-(4-Chlorophenyl)-2-[(4-methylbenzyl)sulfonyl]-6-
phenylpyrimidine (8d)
Yield 65%, m.p. 178-180°C, IR (KBr, cm-1) 488, 517, 593, 637, 663,
691, 716, 764, 838, 1010, 1090, 1136, 1243, 1319, 1366, 1424,
1496, 1580; 1H NMR (CDCl3) δ: 2.28 (s, 3H, Me), 4.88 (s, 2H, CH2),
7.10 (d, J=7.9 Hz, 2H, H3,5-tolyl), 7.32 (d, J=7.9 Hz, 2H, H2,6-tolyl),
7.51 (d, J=8.5 Hz, 2H, H3,5-Ph-Cl), 7.58-7.65 (m, 3H), 8.15 (d, J=8.5
Hz, 2H, H2,6-Ph-Cl), 8.17 (s, 1H, H-pyrimidine), 8.20 (d, J= 6.8 Hz,
2H, H2,6-Ph). 13C NMR (CDCl3) δ: 21.30, 57.33, 113.88, 123.95,
127.76, 129.04, 129.38, 129.64, 131.34, 132.39, 133.67, 135.09,
138.67, 138.95, 165.26, 166.07, 166.66; Mass, m/z (%): 434 (16),
369 (80), 265 (15), 163 (15), 129 (20), 105 (100), 77 (23).
Results and Discussion
Design
As the provided background in introduction part, some of
3-(alkylthio)-5,6-diaryl-1,2,4-triazine derivatives [13] showed re-
markable cytotoxic activity against different human cancerous cell
lines (▶Fig. 1d). In the current work, the central heterocyclic core
of triazine derivative was changed to pyrimidine ring. Furthermore,
in triazine case two aryl groups connected to central core has been
constructed as 1,2- position. In case of pyrimidine, the station of
two aryl groups was changed as 1,3-position. This displacement of
aryl groups causes to terminate the activity of 6-series on the HT-29
cancerous cell line. However in case of MCF-7 and T47D cell lines,
the cytotoxic activity was observed for one compound, 6c. Subse-
Biological studies
Cell culture
HT29, MCF-7, T47D and NIH-3T3 (mouse embryonic fibroblast) cell
lines were purchased from the Pasteur Institute (Tehran, Iran). The
cells were cultured in RPMI-1640 medium (Sigma Aldrich, USA) sup-
plemented with penicillin (100 U/ml), streptomycin (100 µg/ml)
and 10% heat-inactivated fetal bovine serum (Gibco, USA), at 37°C
in an incubator with 5 % CO2.
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

Thieme
Original Article
▶Fig. 3 Docked pose of 7d overlapped with colchicine in colchicine binding site
between different benzyl halides and thione 5 were carried in dry
DMF using sodium hydride as the base. Mono oxidation of sulfur
atom with mCPBA in CH2Cl2 provided the box series 7, S-oxide deri-
vatization. The S-dioxide derivatives, compounds 8, were prepared
using oxone as oxidizing reagent. The structure for compounds se-
ries 6, 7 and 8 were confirmed with spectroscopic methods. In 1H
NMR spectra of S-Oxide series, the CH2 group showed a diastereo-
topic pattern at around 4.5 ppm and for S-dioxide derivatives the
CH2 group appeared at around 4.9 ppm as a simple singlet.
Biological study
Cytotoxicity evaluation by MTT assay
The cytotoxic effects of compounds were evaluated by MTT assay
against human colon adenocarcinoma (HT-29), two breast cancer
cell lines (MCF-7, T47D), as well as a mouse embryonic fibroblast
cell line, NIH-3T3. Paclitaxel was used as positive control. ▶Table 1
illustrates the ability of inhibiting cell growth for all the synthesized
compounds. In the first series, compounds 6, all of them showed
no activity on HT-29 cell line however, 6b and 6c inhibited the cell
growth in breast cancer cell, MCF-7 with IC50 79 and 28µM respec-
tively. Furthermore, on the other breast cancer cell line, T47D, 6c
showed good activity with IC50 23µM. for 6c, comparison between
cytotoxic activity in normal cell (NIH-3T3) and T47D demonstrates
almost two fold selectivity ratios in cancerous cell line that indi-
cates to the benefit of this property. The same results could be ob-
served for 6d on the T47D in aspect of selectivity however, the po-
tency for 6d was less than 6c. In the second box (7a-d) the potency
for all cell lies is better than 6-series remarkably that shows the ox-
idation of sulphur atom in thio benzyl moiety has positive effect for
increasing the cytotoxic activity. In this series the most cytotoxic
activity of compounds against HT-29 and MCF-7 was observed for
▶Fig. 4 3D binding mode of 7d in colchicine binding site
quently, after oxidation of thio-aryl moiety to related sulfinyl de-
rivatives the activity increased significantly on the breast cancer
cell lines.
Chemistry
All compounds were synthesized as shown in ▶Fig. 2. At first, chal-
cone was synthesized through the Claisen-Schmidt procedure using
equimolar amounts of acetophenone and 4-chlorobenzaldehyde.
Intermediate 4 was prepared from the reaction of chalcone and
thiourea in alkaline media as the reported procedure [16]. In the
next step the oxidation of heterocyclic core for preparation of py-
rimidine-2-(1H) thione was carried out using DDQ in acetone and
DMF at 0 °C [14]. For synthesis of compounds 6a-d, the reaction
Boumi S et al. Pyrimidine Derivatives as Anticancer … Drug Res | © 2020. Thieme. All rights reserved.

Funding
7a and 7c respectively. However all of S-Oxide related derivatives
show significant activity against MCF-7 and T47D, attention to the
potency of 7a, 7c and 7d with considering their selectivity index
indicates that these compounds could be candidate to design new
related derivatives with more potency and safety. In the third box,
8a and 8c were completely inactive on HT-29 cell line however 8b
and 8d showed moderate activity against this cell line also on the
breast cancerous cell lines, among of compounds showed moder-
ate activity. Finally 8c as the best compound in this box showed a
cytotoxic activity better than reference drug, paclitaxel (PTX) on
the T47D with better selectivity index.
This research was supported by a grant from Tehran University of
Medical Sciences (GN: 9321302002).
Conflict of Interest
The authors declare that they have no conflict of interest.
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