Synthesis and biological evaluation of novel imidazopyrimidin-3-amines as anticancer agents

Article abstract of DOI:10.1111/cbdd.12904

Groebke–Blackburn–Bienayme reaction has been utilized for the synthesis of new imidazo[1,2-a]pyrimidine derivatives as novel anticancer agents. The cytotoxic activities of compounds were evaluated against human cancer cell lines including MCF-7, T-47D, an

Full text of DOI:10.1111/cbdd.12904

Received Date : 23-Apr-2016
Revised Date : 08-Oct-2016
Accepted Date : 31-Oct-2016
Article type : Research Article
Synthesis and biological evaluation of novel imidazopyrimidin-3-amines as anticancer agents
1
2
3
4
Mohammad Mahdavi , Shima Dianat , Behnaz Khavari , Setareh Moghimi , Mohammad
5
6
2
3
Abdollahi , Maliheh Safavi , Arash Mouradzadegun , Susan Kabodian-Ardestani , Reyhaneh
7
8
4,7
4†
Sabourian , Saeed Emami , Tahmineh Akbarzadeh , Abbas Shafiee and Alireza Foroumadi
1,4
1 Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Chemistry, Faculty of Sciences, Shahid Chamran University, Ahvaz, Iran
3
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran,
Iran
4
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
5
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran.
 Corresponding author. Tel.: +98-21-66419413; Fax: +98-21-66461178.
E-mail address: aforoumadi@yahoo.com (A Foroumadi).
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12904
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6
Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran
33535-111, Iran
7 Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
8 Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Keywords: imidazo[1,2-a]pyrimidine, anticancer agents, apoptosis, Groebke-Blackburn-
Bienayme reaction, isocyanide
Running title: Synthesis and anticancer activity of imidazopyrimidines
Abstract: Groebke-Blackburn-Bienayme reaction (GBB) has been utilized for the synthesis of
new imidazo[1,2-a]pyrimidine derivatives as novel anticancer agents. The cytotoxic activities of
compounds were evaluated against human cancer cell lines including MCF-7, T-47D and MDA-
MB-231, compared with etoposide as the standard drug. Among the tested compounds, hydroxy-
and/or methoxy-phenyl derivatives (6a-c and 6k) with IC50 values of 6.72-14.36 µM were more potent
than etoposide against all cell lines. The acridine-orange/ethidium bromide double staining and
DNA-fragmentation studies demonstrated that the cytotoxic effect of 3-hydroxy-4-
methoxyphenyl derivative 6c is associated with apoptosis in cancer cells.
The cells constitute the building block of the body, growing, dividing and dying through a
regular mechanism which are necessary for normal human’s life. The uncontrolled cell
proliferation led to severe health consequences, called in the worst case, cancer (1). The life style
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choices (2), environmental factors (3) and family history are responsible risk factors for
mutations (4), resulted in the increased risk of cell malfunction and the subsequent new cancer
cases. Being raised as a worldwide killer (5), many therapeutic strategies have been devised to
date like surgery, chemotherapy, radiotherapy and many others. None of these treatment types
exhibited 100% efficacy.
Programmed cell death or apoptosis normally occurs to maintain cell populations in tissues. Any
abnormalities in this case causes different kinds of diseases. Producing excessive amounts of
anti-apoptotic proteins in cancer cells, regarded as a sign of cancer, interfering in normal cell
cycle and led to tumor progression (6). Caspase-induced activation of apoptosis is one of the
most important treatment methods confronting the vicious cell cycle (7, 8). In this context,
exploring for new chemotherapeutic strategies with the goal of tumor regression by inducing
apoptosis has gained considerable attention (9).
Pyrimidine-fused heterocycles constitute a pivotal framework in the field of anticancer drug
development and discovery (10). In particular, imidazopyrimidines are well-known structures for
their cytotoxic activities. For example, Fig. 1 depicts some 2,3-disubstituted-imidazopyrimidines
(structures A-C) which have been previously reported as cytotoxic agents (11). Pal-Bhadra group
have synthesized imidazopyrimidine-chalcones (structure A) with promising activity (GI50
values = 0.28 to 30.0 μM). A number of imidazo[1,2-a]pyrimidine Mannich bases (structure B)
were synthesized and tested against three different human cancer cell lines. Most of the Mannich
bases displayed GI50 values ranging from 0.01 to 79.4 μM. Furthermore, some
imidazopyrimidine-benzimidazole conjugates were designed by Kamal et al. and evaluated
against the human cervical (Hela), lung (A549), prostate (DU-145) and melanoma (B-16) cancer
cell lines. Among them, the compound with structure C exhibited significant anti-proliferative
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activity against lung cancer cell line A549 (IC50 = 1.48 μM) (11). On the other hand, the 5,7-
diphenyl-triazolo[1,5-a]pyrimidine scaffold (structure D) has been introduced as a potent lead
compound for designing novel anti-cancer agents (12).
In continuation of our ongoing program to design new anticancer agents (13), the prominent
activity of pyrimidine-fused heterocycles (structures A-D) paid our attention on 5,7-
diphenylimidazo[1,2-a]pyrimidine scaffold as a hybrid of structures A-C and D. Thus, we report
here, the synthesis of 2-aryl-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amines 6a-o (Fig. 1) as new
cytotoxic compounds and evaluation of their biological activity as apoptosis-inducing agents.
Methods and Materials
General chemistry
Melting points are uncorrected and were determined with a Kofler hot-stage apparatus (Reichert,
Vienna, Austria). Bruker 500 spectrometers and Nicolet Magna FT-IR 550 spectrophotometer
1
(potassium bromide disks) were used to record H, 13C NMR spectra and IR spectra,
respectively. Agilent Technology (HP) mass spectrometer operating at an ionization potential of
70 eV was utilized to determine mass spectra. The elemental analysis for C, H, N was carried out
with an Elementar Analysen system GmbH VarioEL. All needed chemicals and solvents were
commercially bought and used without further purification.
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Synthesis of 4,6-diphenylpyrimidin-2-amine 5
A solution of chalcone (3, 10 mmol), and guanidine hydrochloride (4, 10 mmol) in 15 mL
ethanol was refluxed for 15 min. To this mixture, NaOH solution (4 mL, 50%) was added
dropwise and the reaction was refluxed for another 6 h. Then, the reaction mixture was
concentrated under the reduced pressure and kept at 0 ºC. The resulting crystals were filtered and
recrystallized from ethanol to give compound 5 as a yellow solid.
General procedure for the synthesis of N-substituted-5,7-diphenyl-imidazo[1,2-
a]pyrimidin-3-amines (6a-o)
To the stirred solution of compound 5 (1 mmol), aldehyde derivatives (1 mmol) and NH4Cl (1
mmol) in toluene (5 mL), isocyanide (1 mmol) was added. The mixture was refluxed for 18 h.
Then, the reaction was cooled and the obtained solid was filtered, washed with water and
recrystallized from petroleum ether/ethyl acetate to give corresponding compounds 6a-o.
4-(3-(Cyclohexylamino)-5,7-diphenylimidazo[1,2-a]pyrimidin-2-yl) phenol (6a)
◦
Yellow solid; yield: 0.33 g (71 %); m.p. 233-236 C. IR (KBr) (νmax, cm−1): 3358, 3059, 2926,
1610. 1H NMR (500 MHz, DMSO-d6): δ = 9.51 (s, OH), 8.28 (d, J = 6.5 Hz, 2H, H-Ph), 8.12 (d,
J = 8.5 Hz, 2H, H2,6-OHC6H4), 7.78-7.80 (m, 2H, H-Ph), 7.50-7.55 (m, 6H, H-Ph), 7.43 (s, 1H,
H6-pyrimidine), 6.81 (d, J = 8.5 Hz, 2H, H3,5-OHC6H4), 3.88 (s, 1H, NH), 2.03 (s, 1H, NCH),
0.59-1.32 (m, 10H, 5CH2, cyclohexyl). 13C NMR (125 MHz, DMSO-d6): δ = 156.9, 153.4, 146.0,
145.2, 140.1, 136.9, 132.8, 130.0, 129.7, 129.2, 128.8, 127.8,126.9, 125.1, 123.0, 115.8, 114.8,
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107.5, 56.5, 31.9, 25.3, 24.0. Anal. Calcd. for C30H28N4O: C, 78.23; H, 6.13; N, 12.16. Found: C,
78.15; H, 6.25; N, 12.31.
N-Cyclohexyl-2-(2-methoxyphenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6b)
◦
Yellow solid; yield: 0.40 g (87 %); m.p. 148–149 C. IR (KBr) (νmax, cm−1): 3303, 2920, 1604,
1483, 1242. 1H NMR (500 MHz, DMSO-d6): δ = 8.29 (d, J = 7.9 Hz, 2H, H-Ph), 7.70-7.77 (m,
3H, H6-MeOC6H4, H-Ph), 7.50-7.58 (m, 6H, H-Ph), 7.49 (s, 1H, H6-pyrimidine), 7.40 (t, J = 7.4
Hz, 1H, H4-MeOC6H4), 7.17 (td, J = 8.4, 1.5 Hz, 1H, H3-MeOC6H4), 7.10 (t, J = 7.5 Hz, 1H, H5-
MeOC6H4), 3.89 (s, 3H, OMe), 3.71 (s, 1H, NH), 1.80 (s, 1H, NCH), 0.28–1.17 (m, 10H, 5CH2,
13
cyclohexyl). C NMR (125 MHz, DMSO-d6): δ = 155.5, 152.4, 146.1, 144.8, 136.9, 135.6,
135.5, 132.0, 131.6, 130.2, 130.0, 129.4, 129.3, 128.9, 127.1, 126.8, 123.5, 121.0, 112.0, 107.8,
55.9, 55.8, 32.0, 25.1, 23.9. MS: m/z (%) = 474 (28) [M+], 443 (100), 345 (23), 268 (57), 192
(17), 77 (65).
5-(3-(Cyclohexylamino)-5,7-diphenylimidazo[1,2-a]pyrimidin-2-yl)-2-methoxyphenol (6c)
Yellow solid; yield: 0.37 g (76 %); m.p. 285-287 ◦C. IR (KBr) (νmax, cm−1): 3369, 1602, 1243. 1H
NMR (500 MHz, DMSO-d6): δ = 8.99 (s, OH), 8.29 (d, J = 6.9 Hz, 2H, H-Ph), 7.79-7.81 (m, 3H,
H2-3-OH-4-MeOC6H3, H-Ph), 7.76 (d, J = 8.4 Hz, 1H, H6-3-OH-4-MeOC6H3), 7.50-7.60 (m, 6H,
H-Ph), 7.46 (s, 1H, H6-pyrimidine), 6.96 (d, J = 8.4 Hz, 1H, H5-3-OH-4-MeOC6H3), 3.80 (s, 3H,
13
OMe), 3.12 (s, 1H, NH), 2.03 (s, 1H, NCH), 0.59-1.30 (m, 10H, 5CH2, cyclohexyl). C NMR
(125 MHz, DMSO-d6): δ = 154.1, 146.4, 145.4, 137.1, 136.7, 136.2 (2C), 132.4, 130.3 (2C),
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129.8, 129.5, 128.9, 127.7, 127.6, 127.0, 126.2, 125.3, 122.7, 107.8, 56.9, 56.8, 32.1, 25.3, 24.3.
Anal. Calcd. for C31H30N4O2: C, 75.89; H, 6.16; N, 11.42. Found: C, 75.71; H, 6.21; N, 11.40.
N-Cyclohexyl-5,7-diphenyl-2-(m-tolyl)imidazo[1,2-a]pyrimidin-3-amine (6d)
◦
Yellow solid; yield: 0.40 g (89 %); m.p. 200-201 C. IR (KBr) (νmax, cm−1): 3312, 2923, 1600,
1
1483. H NMR (500 MHz, DMSO-d6): δ = 8.29 (d, J = 7.8 Hz, 2H, H-Ph), 8.13 (s, 1H, H2-
MeC6H4), 8.10 (d, J = 7.9 Hz, 1H, H6-MeC6H4), 7.80-7.81 (m, 2H, H-Ph), 7.58-7.63 (m, 3H, H-
Ph), 7.51-7.56 (m, 3H, H-Ph), 7.48 (s, 1H, H6-pyrimidine), 7.31 (t, J = 7.6 Hz, 1H, H5-MeC6H4),
7.11 (d, J = 7.4 Hz, 1H, H4-MeC6H4), 3.38 (s, 1H, NH), 2.36 (s, 3H, Me), 2.03 (s, 1H, NCH),
0.55-1.28 (m, 10H, 5CH2, cyclohexyl). 13C NMR (125 MHz, DMSO-d6): δ = 154.0, 146.4,
145.2, 139.2, 137.0, 136.8, 134.1, 132.7, 130.2, 129.8, 129.3, 128.9, 128.2, 128.1, 128.0, 127.8,
126.9, 124.7, 124.4, 107.8, 56.5, 31.9, 25.2, 24.0, 21.2. MS: m/z (%) = 458 (41) [M+], 443 (100),
345 (35), 269 (29), 194 (28), 98 (44), 77 (66). Anal. Calcd. for C31H30N4: C, 81.19; H, 6.59; N,
12.22. Found: C, 81.15; H, 6.71; N, 12.28.
N-Cyclohexyl-2-(2-fluorophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6e)
◦
Light Yellow solid; yield: 0.31 g (68 %); m.p. 134-135 C. IR (KBr) (νmax, cm−1): 3408, 2924,
1606, 1483, 1096.1H NMR (500 MHz, DMSO-d6): δ = 8.30 (d, J = 7.5 Hz, 2H, H-Ph), 7.81 (m,
1H, H6-FC6H4), 7.77 (d, J = 6.9 Hz, 2H, H-Ph), 7.52-7.59 (m, 6H, H-Ph), 7.51 (s, 1H, H6-
pyrimidine), 7.42-7.45 (m, 1H, H4-FC6H4), 7.33 (m, 1H, H3-FC6H4), 7.30 (t, J = 7.7 Hz, 1H, H5-
13
FC6H4), 3.22 (s, 1H, NH), 1.92 (s, 1H, NCH), 0.35-1.20 (m, 10H, 5CH2, cyclohexyl). C NMR
(125 MHz, DMSO-d6): δ = 158.2 (d, JC-F = 224 Hz), 153.7, 146.5, 145.2, 136.7, 134.4, 132.2,
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131.9, 130.2, 129.9, 129.7, 128.9, 127.3, 127.0, 126.3, 124.4, 122.2, 122.1, 115.7, 108.0, 56.0,
31.8, 25.1, 23.8. Anal. Calcd. for C30H27FN4: C, 77.90; H, 5.88; N, 12.11. Found: C, 77.76; H,
5.79; N, 12.18.
N-Cyclohexyl-2-(4-fluorophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6f)
◦
Yellow solid; yield: 0.32 g (69 %); m.p. 220-222 C. IR (KBr) (νmax, cm−1): 3349, 2928, 1740,
1489, 1211.1H NMR (500 MHz, DMSO-d6): δ = 8.29 (d, J = 7.1 Hz, 2H, H–Ph), 8.26 (d, J = 8.0
Hz, 2H, H2,6-FC6H4), 7.82 (d, J = 6.5 Hz, 2H, H-Ph), 7.53-7.62 (m, 6H, H-Ph), 7.49 (s, 1H, H6-
pyrimidine), 7.26 (dd, J = 8.0, 1.5 Hz, 2H, H3,5-FC6H4), 4.02 (s, 1H, NH), 1.99 (s, 1H, NCH),
0.60-1.31 (m, 10H, 5CH2, cyclohexyl). 13C NMR (125 MHz, DMSO-d6): δ = 159.3 (d, JC-F = 221
Hz), 154.3, 146.5, 145.4, 138.6, 136.7, 132.6, 130.7, 130.2, 129.7, 129.6, 129.2, 128.8, 127.8,
126.9, 124.0, 114.8 (d, JC-F = 20 Hz), 107.8, 56.6, 31.9, 25.2, 24.0. Anal. Calcd. for C30H27FN4:
C, 77.90; H, 5.88; N, 12.11. Found: C, 77.83; H, 5.96; N, 12.08.
2-(3-Chlorophenyl)-N-cyclohexyl-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6g)
◦
Deep Yellow solid; yield: 0.41 g (88 %); m.p. 186-188 C. IR (KBr) (νmax, cm−1): 3364, 2922,
1604, 1481, 687.1H NMR (500 MHz, DMSO-d6): δ = 8.30 (d, J = 7.3 Hz, 2H, H-Ph), 8.13 (s,
1H, H2-ClC6H4), 8.11 (d, J = 7.9 Hz, 1H, H6-ClC6H4), 7.80-7.82 (m, 2H, H-Ph), 7.50-7.62 (m,
6H, H-Ph), 7.48 (s, 1H, H6-pyrimidine), 7.30 (t, J = 7.8 Hz, 1H, H5-ClC6H4), 7.11 (d, J = 7.3 Hz,
1H, H4-ClC6H4), 4.03 (s, 1H, NH), 2.49 (s, 1H, NCH), 0.55-1.28 (m, 10H, 5CH2, cyclohexyl).
13C NMR (125 MHz, DMSO-d6): δ = 154.5, 146.6, 145.4, 138.2, 136.7, 133.1, 132.5, 131.9
(2C), 130.3, 129.9 (2C), 129.3, 129.2, 128.9, 128.1, 127.9, 127.0, 124.5, 108.0, 56.7, 31.9, 25.2,
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24.1. Anal. Calcd. for C30H27ClN4: C, 75.22; H, 5.68; N, 11.70. Found: C, 75.30; H, 5.56; N,
11.79.
N-Cyclohexyl-2-(2,4-dichlorophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6h)
◦
Yellow solid; yield: 0.30 g (60 %); m.p. 97–98 C. IR (KBr) (νmax, cm−1): 3393, 2926, 1735,
1481, 695. 1H NMR (500 MHz, DMSO-d6): δ = 8.28 (d, J = 7.1 Hz, 2H, H-Ph), 7.75 (d, J = 8.0
Hz, 2H, H-Ph), 7.72 (d, J = 1.8 Hz, 1H, H3-2,4-Cl2C6H3), 7.67 (d, J = 8.0 Hz, 1H, H6-2,4-
Cl2C6H3), 7.51-7.60 (m, 7H, H–Ph, H5-2,4-Cl2C6H3), 7.50 (s, 1H, H6-pyrimidine), 4.02 (s, 1H,
13
NH), 1.91 (s, 1H, NCH), 0.34-1.17 (m, 10H, 5CH2, cyclohexyl). C NMR (125 MHz, DMSO-
d6): δ = 153.9, 146.5, 144.8, 136.7, 134.0, 133.8, 133.3, 132.4, 132.2, 130.3, 129.7, 129.6, 129.0,
128.9, 127.5, 127.0, 126.1, 108.0, 55.8, 31.6, 25.1, 23.7. Anal. Calcd. for C30H26Cl2N4: C, 70.18;
H, 5.10; N, 10.91. Found: C, 70.24; H, 5.16; N, 10.85.
N-Cyclohexyl-2-(3-nitrophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6i)
◦
Deep Yellow solid; yield: 0.31 g (65 %); m.p. 250-251 C. IR (KBr) (νmax, cm−1): 3377, 2924,
1522, 1345. 1H NMR (500 MHz, CDCl3): δ = 9.42 (d, J = 1.9 Hz, 1H, H2-NO2C6H4), 8.80 (dd, J
= 7.9 Hz, 1H, H4-NO2C6H4), 8.26 (d, J = 7.7 Hz, 2H, H-Ph), 8.13 (d, J = 8.2 Hz, 1H, H6-
NO2C6H4), 7.65-7.66 (m, 5H, H-Ph), 7.59 (t, J = 7.9 Hz, 1H, H5-NO2C6H4), 7.50-7.52 (m, 3H,
H-Ph), 7.22 (s, 1H, H6-pyrimidine), 2.34 (s, 1H, NH), 2.19 (s, 1H, NCH), 0.67-1.41 (m, 10H,
5CH2, cyclohexyl). 13C NMR (125 MHz, CDCl3): δ = 156.2, 148.2, 146.4, 145.8, 136.6, 133.6,
135.8, 133.6, 132.9, 130.58, 130.53, 129.0 (2C), 128.87, 128.84, 128.3, 127.2, 125.2, 122.6,
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122.0, 108.3, 58.2, 32.6, 25.4, 24.6. Anal. Calcd. for C30H27N5O2: C, 73.60; H, 5.56; N, 14.31.
Found: C, 73.51; H, 5.46; N, 14.40.
N-Cyclohexyl-5,7-diphenyl-2-(thiophen-2-yl)imidazo[1,2-a]pyrimidin-3-amine (6j)
◦
Yellow solid; yield: 0.34 g (76 %); m.p.: 232-234 C. IR (KBr) (νmax, cm−1): 3323, 2922, 1604.
1H NMR (500 MHz, DMSO-d6): δ = 8.29 (d, J = 7.6 Hz, 2H, H-Ph), 7.84 (d, J = 3.5 Hz, 1H, H5-
thiophene), 7.80 (d, J = 6.7 Hz, 2H, H-Ph), 7.57-7.61 (m, 3H, H-Ph), 7.52-7.54 (m, 4H, H-Ph,
H3-thiophene), 7.51 (s, 1H, H6-pyrimidine), 7.13 (t, J = 4.0 Hz, 1H, H4-thiophene), 3.50 (s, 1H,
13
NH), 1.92 (s, 1H, NCH), 0.72-1.34 (m, 10H, 5CH2, cyclohexyl). C NMR (125 MHz, DMSO-
d6): δ = 154.1, 146.4, 145.4, 137.1, 136.7, 136.2, 132.3, 130.2, 129.8, 129.4, 128.9, 127.7, 127.5,
126.9, 126.2, 125.2, 122.7, 107.8, 56.9, 32.1, 25.3, 24.3. MS: m/z (%) = 450 (29) [M+], 352
(100), 269 (47), 192 (25), 82 (39), 77 (71). Anal. Calcd. for C28H26N4S: C, 74.63; H, 5.82; N,
12.43. Found: C, 74.51; H, 5.96; N, 12.40.
N-(tert-Butyl)-2-(2-methoxyphenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6k)
◦
Light Yellow solid; yield: 0.37 g (85 %); m.p. 194-196 C. IR (KBr) (νmax, cm−1): 3336, 2956,
1482. 1H NMR (500 MHz, DMSO-d6): δ = 8.31 (d, J = 7.0 Hz, 2H, H-Ph), 7.78 (dd, J = 7.5, 1.5
Hz, 1H, H6-MeOC6H4), 7.72-7.74 (m, 2H, H-Ph), 7.53-7.56 (m, 6H, H-Ph), 7.52 (s, 1H, H6-
pyrimidine), 7.40 (dt, J = 7.2, 1.5 Hz, 1H, H4-MeOC6H4), 7.16 (d, J = 8.5 Hz, 1H, H3-
MeOC6H4), 7.11 (t, J = 7.2 Hz, 1H, H5-MeOC6H4), 3.92 (s, 3H, OMe), 3.65 (s, 1H, NH), 0.30 (s,
13
9H, C(CH3)3). C NMR (125 MHz, DMSO-d6): δ = 155.8, 153.4, 146.9, 145.5, 139.4, 136.8,
132.5, 131.5, 130.5, 130.1, 129.5, 129.4, 128.9, 126.9, 126.8, 126.0, 124.1, 120.9, 111.8, 108.4,
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55.9, 54.9, 28.7. Anal. Calcd. for C29H28N4O: C, 77.65; H, 6.29; N, 12.49. Found: C, 77.58; H,
6.36; N, 12.40.
N-(tert-Butyl)-5,7-diphenyl-2-(m-tolyl)imidazo[1,2-a]pyrimidin-3-amine (6l)
◦
Yellow solid; yield: 0.39 g (90 %); m.p. 273-275 C. IR (KBr) (νmax, cm−1): 3326, 2963, 1596,
1
1484. H NMR (500 MHz, DMSO-d6): δ = 8.32 (d, J = 6.8 Hz, 2H, H-Ph), 8.02 (s, 1H, H2-
MeC6H4), 7.96 (d, J = 7.4 Hz, 1H, H6-MeC6H4), 7.76-7.77 (m, 2H, H-Ph), 7.54-7.59 (m, 6H, H-
Ph), 7.52 (s, 1H, H6-pyrimidine), 7.31 (t, J = 7.6 Hz, 1H, H5-MeC6H4), 7.13 (d, J = 7.0 Hz, 1H,
13
H4-MeC6H4), 3.54 (s, 1H, NH), 2.38 (s, 3H, CH3), 0.38 (s, 9H, C(CH3)3). C NMR (125 MHz,
DMSO-d6): δ = 154.1, 147.0, 145.4, 142.3, 137.3, 134.6, 133.1, 130.4, 130.2, 129.6, 129.0,
128.9, 128.6, 128.2, 127.7, 127.6, 126.9, 125.6, 123.9, 108.3, 55.4, 29.0, 21.0. Anal. Calcd. for
C29H28N4: C, 80.52; H, 6.52; N, 12.95. Found: C, 80.51; H, 6.59; N, 12.84.
N-(tert-Butyl)-2-(4-fluorophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6m)
◦
Light Yellow solid; yield: 0.30 g (69 %); m.p. 291-293 C. IR (KBr) (νmax, cm−1): 3392, 1608,
1482, 1204.1H NMR (500 MHz, DMSO-d6): δ = 8.32 (d, J = 7.8, 2H, H-Ph), 8.21 (m, 2H, H2,6-
FC6H4), 7.79 (m, 2H, H-Ph), 7.56-7.60 (m, 6H, H-Ph), 7.55 (s, 1H, H6-pyrimidine), 7.26-7.28
(m, 2H, H3,5-FC6H4), 3.53 (s, 1H, NH), 0.39 (s, 9H, C(CH3)3). 13C NMR (125 MHz, DMSO-d6):
δ = 162.3 (d, JC-F = 218 Hz), 147.1, 136.5, 132.8, 130.4, 129.6, 128.8, 127.6, 126.9, 114.8, 114.6,
108.4, 55.2, 28.8. Anal. Calcd. for C28H25FN4: C, 77.04; H, 5.77; N, 12.83. Found: C, 77.10; H,
5.81; N, 12.91.
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N-(tert-Butyl)-2-(4-chlorophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6n)
◦
Light Yellow solid; yield: 0.38 g (84 %); m.p. 290-292 C. IR (KBr) (νmax, cm−1): 3390, 1610,
1480, 755. 1H NMR (500 MHz, DMSO-d6): δ = 8.32 (d, J = 7.0 Hz, 2H, H-Ph), 8.21 (d, J = 8.2
Hz, 2H, H2,6-ClC6H4), 7.76-7.78 (m, 2H, H-Ph), 7.57-7.59 (m, 3H, H-Ph), 7.55 (s, 1H, H6-
pyrimidine), 7.52-7.54 (m, 3H, H-Ph), 7.48 (d, J = 8.3 Hz, 2H, H3,5-ClC6H4), 3.55 (s, 1H, NH),
0.38 (s, 9H, C(CH3)3). 13C NMR (125 MHz, DMSO-d6): δ = 154.6, 147.1, 145.6, 140.9, 136.6,
133.7, 132.9, 132.1, 131.2, 130.1, 129.7, 129.3, 128.9, 128.0, 127.7, 127.0, 124.1, 108.5, 55.5,
28.9. Anal. Calcd. for C28H25ClN4: C, 74.24; H, 5.56; N, 12.37; Found: C, 74.30; H, 5.61; N,
12.40.
N-(tert-Butyl)-2-(3-nitrophenyl)-5,7-diphenylimidazo[1,2-a]pyrimidin-3-amine (6o)
◦
Yellow solid; yield: 0.32 g (70 %); m.p. 248-249 C. IR (KBr) (νmax, cm−1): 3392, 1605, 1520,
1
1344. H NMR (500 MHz, DMSO-d6): δ = 9.08 (d, J = 1.7 Hz, 1H, H2-NO2C6H4), 8.66 (d, J =
7.7 Hz, 1H, H4-NO2C6H4), 8.33 (d, J = 7.8 Hz, 2H, H-Ph), 8.17 (d, J = 8.0 Hz, 1H, H6-
NO2C6H4), 7.80-7.82 (m, 2H, H-Ph), 7.74 (t, J = 8.0 Hz, 1H, H5-NO2C6H4), 7.62-7.63 (m, 3H,
H-Ph), 7.58 (s, 1H, H6-pyrimidine), 7.54-7.57 (m, 3H, H-Ph), 3.56 (s, 1H, NH), 0.42 (s, 9H,
C(CH3)3). 13C NMR (125 MHz, DMSO-d6): δ = 155.2, 147.8, 147.4, 145.8, 139.5, 136.5, 136.4,
134.4, 132.8, 130.5, 129.9, 129.6, 128.9, 127.9, 127.1, 124.7, 122.6, 122.5, 122.1, 108.8, 55.6,
29.0. MS: m/z (%) = 463 (27) [M+], 406 (100), 284 (51), 122 (39), 207 (28), 77 (35), 57 (64).
Anal. Calcd. for C28H25N5O2: C, 72.55; H, 5.44; N, 15.11. Found: C, 72.51; H, 5.46; N, 15.20.
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Cell lines and cell culture
The human breast cancer cell lines including MCF-7, MDA-MB-231 and T-47D were received
from Pasture Institute, Tehran (Iran). The cultured cells in RPMI 1640 medium (containing 10%
FBS, 1% L-Glutamine, and Penicillin-Streptomycin) were incubated under humidified
atmosphere with 5% CO2 at 37 ºC.
MTT assay
The stock solutions of compounds were prepared in DMSO. The cells were treated with different
concentrations of the compounds and incubated for 48 h. Etoposide and DMSO were used as
reference drug and control, respectively. The effect of compounds 6a-o on cell viability was
determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
according to the reported method (14). The optical density was read at 492 nm with an ELISA
plate reader (Exert 96, Asys Hitch, Ec Austria).
Acridine orange/ethidium bromide staining method
The cancer cells (MCF-7 and T47D) grown in 12-well plates (50,000 cells/well) were treated
with and without IC50 concentration of compounds 6c and 6k for 24 h. After treatment, cells
were harvested and washed three times with phosphate buffer saline (PBS). Then, the cells were
stained with 100 mL of a mixture of acridine orange and ethidium bromide (1:1, 100 mg/mL)
solutions. The stained cell suspension (10 mL) was placed on a clean microscope slide and
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covered with a coverslip. The cells were immediately analyzed by Axoscope 2 plus fluorescence
microscope (Zeiss, Germany).
DNA-fragmentation assay
DNA fragmentation was measured by a slight modification of the diphenylamine assay described
by Gercel-Taylor (15). The diphenylamine assay is a very useful method for measuring apoptosis
and is based on the notion that extensively fragmented double-stranded DNA can be separated
from apoptotic nuclei into the cytoplasm and constitutes a quantitative measure of soluble DNA
(16).
Briefly, MCF-7 and T-47D cells treated with test compounds for 24 h were harvested and
centrifuged at 2000 RPM, for 10 min (4°C) to obtain a cell pellet. The pellets were resuspended
in ice-cold hypotonic lysis TTE buffer (10 mM tris-base, 1 mM EDTA, 0.2% Triton X-100,
pH=8.0) and samples were allowed to lyse for 30 min at 4°C. Then, the intact chromatin (pellet)
was separated from DNA fragments (supernatant) by centrifugation at 13000 g for 15 min (4°C).
The pellets were then resuspended in TTE buffer. Both the pellets and the supernatant samples
were precipitated with trichloroacetic acid (TCA; 25%). Samples were then centrifuged at 13000
g for 15 min (4°C) and the supernatant was removed. The precipitates were resuspended in 5%
TCA, boiled at 90 ºC for 20 min and centrifuged at 4 ºC to remove proteins. The resulting
supernatants, whether containing whole or fragmented DNA, were left to react with freshly
prepared diphenylamine for 16–20 h at room temperature, and absorbance was measured at 620
nm. DNA fragmentation was expressed as a percentage of fragmented to total DNA by the
formula:
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% DNA fragmentation= [OD of supernatant/(OD of supernatant + OD of pellet)] × 100
Results and Discussion
Chemistry
The reaction pathway toward target compounds 6a-o namely N-substituted-5,7-diphenyl-
imidazo[1,2-a]pyrimidin-3-amines is depicted in Scheme 1. Initially, we prepared chalcone 3
according to the reported method (17), via the reaction of acetophenone 1 and benzaldehyde 2 in
the presence of NaOH in ethanol. In the next step, the cyclization reaction of guanidine
hydrochloride 4 with chalcone 3 provided 4,6-diphenylpyrimidin-2-amine 5 as a yellow powder
o
o
in 75% yield identified according to the literature (m.p. 139 C; m.p. lit. 138-139 C) (18). The
resulting solid was used in the subsequent step after a simple recrystallization from ethanol.
Groebke-Blackburn-Bienayme reaction (GBB) of compound 5 with aromatic aldehydes and
isocyanides afforded N-substituted-5,7-diphenyl-imidazo[1,2-a]pyrimidin-3-amines 6a-o in 60-
90% yields. The reaction was performed in refluxing toluene (19). Accordingly, this condition
was applied for the synthesis of 15 different imidazopyrimidines which were fully characterized
by 1H, 13C-NMR, and IR spectroscopy. The NMR spectra are provided as Appendix 1 file.
Pharmacology
In vitro anticancer screening (MTT assay)
The cytotoxic activities of compounds 6a-o were tested against three different cell lines MCF-7,
T-47D and MDA-MB-231 by using MTT assay (14). The IC50 values were given in Table 1.
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Most of the compounds showed remarkable activity against tested cell lines. Particularly,
compound 6c bearing a methoxyphenolic group showed the most potent activity against MCF-7
and T-47D cells. Its IC50 value against T-47D cell line was 6.72 µM, being 5-fold lower than that
of etoposide as reference drug. The most potent compound against MDA-MB-231 cell line was
compound 6e with IC50 value of 9.90 µM. Besides 6e, compound 6c also showed good activity
against the latter cell line, being more potent than etoposide. The obtained IC50 values revealed
that better results were obtained with hydroxy- and methoxy-phenyl derivatives (compounds 6a-
c and 6k). The IC50s of these compounds were in the range of 6.72-14.36 µM. Their activity was
higher than that of etoposide against all cell lines. These results demonstrated that the presence
of electron-donating groups such as OH and OMe could be favorable for cytotoxic activity. In
contrast, since the compounds such as 6g, 6i, 6n and 6o were less active or inactive derivatives,
it could conclude that the electron-withdrawing groups such as chlorine or nitro diminish the
anti-proliferative activity. Exceptionally, the 2-fluorophenyl derivative 6e exhibited significant
activity against all cell line (IC50s = 8.88-16.97 µM). However, the displacement of fluoro group
to para position resulted in compound 6f with diminished activity. On the other hand, the
activity of 2,4-dichloro derivative 6h was better than that of 3-chloro analog 6g. The comparison
of cyclohexyl and tert-butyl series revealed that the pendent resides (R= cyclohexyl or tert-butyl)
didn't have same effect on different cell lines. For example, while the cyclohexyl derivative 6d
was inactive against MCF-7, its tert-butyl analog 6l showed moderate activity against this cell
line. In contrast, compound 6d was more active than tert-butyl derivative 6l against MDA-MB-
231 and T-47D cells.
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Acridine orange/ethidium bromide double staining test
The morphological assessment of MCF-7 and T-47D cells by acridine orange/ethidium bromide
double staining method was used to indicate the potential of compounds 6c and 6k as apoptotic
inducers. The test cells were treated with the IC50 concentration of compounds 6c and 6k for 24 h
and then stained with acridine orange/ethidium bromide. Using fluorescence microscopy,
uniformly stained green cells can be distinguished as live cells and orange cells are characterized
as apoptotic cells. The observed results demonstrated that the selected compounds can induce
apoptosis in MCF-7 and T-47D cells (Fig. 2 and 3). Also, chromatin condensation and nuclear
fragmentation were occurred following the treatment of cancer cells with compounds 6c and 6k.
DNA-fragmentation assay
DNA fragmentation using diphenylamine assay is a useful tool in measuring apoptosis. The cells
exhibiting the morphological characteristics associated with DNA fragmentation are also referred
to as apoptotic cells (15, 16). The MCF-7 and T-47D cells were treated with selected compounds
6c and 6k for 24 h and subjected to the assay. The obtained results were summarized in Fig. 4 as
mean percentages of DNA fragmentation. Based on the results, the compounds 6c and 6k
induced DNA fragmentation more significantly in the MCF-7 cells compared to the T-47D cells.
Conclusion
In conclusion, we synthesized a series of functionalized imidazo[1,2-a]pyrimidin-3-amines
bearing poly-aromatics and a bulky residue (cyclohexyl or tert-butyl) on the amine, as new
cytotoxic agents. The results of cytotoxicity assay against different tumor cell lines revealed that
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most of the compounds have significant activity against MCF-7, T-47D and MDA-MB-231 cells.
Especially, compounds 6a-c and 6k bearing hydroxy- and/or methoxy group on the 2-phenyl ring
showed more promising results against all cell lines, with IC50 values in the range of 6.72-14.36
µM. Their activities were superior to that of etoposide as reference anticancer agent. In
particular, the 3-hydroxy-4-methoxyphenyl derivative 6c with IC50 value of 6.72 µM against T-
47D cells was 5 times more potent than etoposide. Further biological assays by using acridine
orange/ethidium bromide double staining test and DNA-fragmentation study demonstrated that
the promising compound 6c exhibits its cytotoxic effect partly by the induction of apoptosis in
cancer cells.
Acknowledgment
† In the memory of Professor Abbas Shafiee, a leading Iranian medicinal chemist and the first
corresponding author of this article, who was passed away during the reviewing process. This
study was supported and funded by grants from the Tehran University of Medical Sciences
(TUMS) and Iran National Science Foundation (INSF).
Conflict of interest
The authors declare no conflict of interest
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Table 1: In vitro cytotoxic activities of compounds 6a-o against cancer cell lines.
Ph
N
R
HN
N
N
Ar
Ph
Compound
Ar
R
(IC50, µM)
MCF-7
11.56±0.03
13.65±0.007
10.92±0.04
>100
MDA-MB-231
14.08±0.26
10.47±0.005
10.43±0.18
33.54±0.01
9.90±0.01
>100
T-47D
4-HOC6H4
Cyclohexyl
Cyclohexyl
14.36±0.43
8.42±0.016
6.72±0.81
69.73±1.02
8.88±0.06
21.11±0.02
>100
6a
6b
2-MeOC6H4
3-OH-4-MeOC6H3 Cyclohexyl
6c
3-MeC6H4
2-FC6H4
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
tert-Butyl
-
6d
16.97±0.001
81.43±0.006
>100
6e
4-FC6H4
6f
3-ClC6H4
2,4-diClC6H3
3- O2NC6H4
2-Thienyl
2-MeOC6H4
3-MeC6H4
4-FC6H4
>100
6g
73.59±0.05
>100
34.00±0.004
>100
15.44±0.68
61.23±0.29
27.59±1.48
7.93±0.49
72.53±0.78
9.87±0.35
46.98±0.39
93.98±0.39
36.60±0.71
6h
6i
77.33±0.02
11.41±0.54
27.95±0.27
35.21±0.49
>100
>100
6j
12.64±0.09
42.53±0.40
69.21±1.02
84.11±0.25
>100
6k
6l
6m
6n
4-ClC6H4
3- O2NC6H4
-
93.00±0.08
39.41±0.63
6o
Etoposide
26.01±0.09
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