Synthesis and antiproliferative activity in vitro of new 2-aminobenzimidazole derivatives. Reaction of 2-arylideneaminobenzimidazole with selected nitriles containing active methylene group

Article abstract of DOI:10.2478/s11532-014-0533-3

A series of pyrimido[1,2-a]benzimidazole and a-cyanocinnamic acid derivatives have been synthesized in the reactions of Schiff bases 2-7 with selected nitriles containing an active methylene group: malononitrile 8-12, cyanoacetamide 13-16, benzyl cyanide 17-21, benzoylacetonitrile 22-24, cyanoacetate methyl ester 25-28 and benzylacetamide 29. The structures 8-29 were confirmed by the results of elementary analysis and their IR, 1H-, 13C-NMR and MS spectra. The products 8-29 of various chemical structure pyrimido[1,2-a] benzimidazole 8-12, 14-16, 17-21, 23-24, 26 and a-cyanocinnamic acid derivatives 13, 22, 25, 27, 28 were obtained, which are of interest for biological studies or which can be substrates for further synthesis. The selected compounds 10, 13, 14, 17, 19, 21, 23-25 and 28 were screened for their antiproliferative activity in vitro against neoplastic and normal cell lines. The most active two compounds were: 2-(o-bromophenylene)-3-cyano-4-phenyl-1,2-dihydropyrimido[1,2-a] benzimidazole (24) and 3-cyano-4-phenyl-2-(2,4-dimethoxyphenyl)-1,2- dihydropyrimido[1,2-a]benzimidazole (23). However, similarly like cisplatin used as the control, they showed no selectivity towards cancer cells, by inhibiting proliferation of normal mouse fibroblasts in similar manner. Versita Sp. z o.o.

Full text of DOI:10.2478/s11532-014-0533-3

Cent. Eur. J. Chem. • 12(10) • 2014 • 1047-1055
DOI: 10.2478/s11532-014-0533-3
Central European Journal of Chemistry
Synthesis and antiproliferative activity in vitro of
new 2-aminobenzimidazole derivatives. Reaction
of 2-arylideneaminobenzimidazole with selected
nitriles containing active methylene group
Research Article
Anna Nowicka1*, Hanna Liszkiewicz1, Wanda P. Nawrocka1,
Joanna Wietrzyk2, Katarzyna Kempińska2, Andrzej Dryś3
1Department of Drug Technology, Wroclaw Medical University,
50-556 Wroclaw, Poland
2“NeoLek” Laboratory of Experimental Anticancer Therapy,
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Science, 53-114 Wroclaw, Poland
3Department of Physical Chemistry, Wroclaw Medical University,
50-556 Wroclaw, Poland
Received 25 September 2013; Accepted 22 January 2014
Abstract:
A series of pyrimido[1,2-a]benzimidazole and α-cyanocinnamic acid derivatives have been synthesized in the reactions of Schiff bases
2-7 with selected nitriles containing an active methylene group: malononitrile 8-12, cyanoacetamide 13-16, benzyl cyanide 17-21,
benzoylacetonitrile 22-24, cyanoacetate methyl ester 25-28 and benzylacetamide 29. The structures 8-29 were confirmed by the results
1
of elementary analysis and their IR, H-, 13C-NMR and MS spectra. The products 8-29 of various chemical structure pyrimido[1,2-a]
benzimidazole 8-12, 14-16, 17-21, 23-24, 26 and α-cyanocinnamic acid derivatives 13, 22, 25, 27, 28 were obtained, which are
of interest for biological studies or which can be substrates for further synthesis. The selected compounds 10, 13, 14, 17, 19, 21,
23-25 and 28 were screened for their antiproliferative activity in vitro against neoplastic and normal cell lines. The most active two
compounds were: 2-(o-bromophenylene)-3-cyano-4-phenyl-1,2-dihydropyrimido[1,2-a]benzimidazole (24) and 3-cyano-4-phenyl-2-
(2,4-dimethoxyphenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole (23). However, similarly like cisplatin used as the control, they showed
no selectivity towards cancer cells, by inhibiting proliferation of normal mouse fibroblasts in similar manner.
Keywords: 2-arylideneaminobenzimidazole • Pyrimido[1,2-a]benzimidazole • α-cyanocinnamic acid derivatives • Structure • Antiproliferative
activity in vitro
© Versita Sp. z o.o.
neurodegeneration and in the future they may be used
in a treatment of Alzheimer’s disease or Parkinson’s
1. Introduction
There are c.a. 50 derivatives of 2-aminobenzimidazole
registered in the world as drugs with anticancer, antiviral,
antifungal, anthelmintic and antihistamine properties
[1].
Basedonareviewofthechemicalliterature,derivatives
of 2-aminobenzimidazole showed multipharmacological
effects such as hypotensive effect [2], anti-inflammatory
effects [3], analgesic [4] or antiaggregatory [5]
activity. Some chemical compounds, which contain
in their structure 2-aminobenzimidazole motif inhibit
disease [6]. Recently, a lot of literature has revealed that
2-amino-1H-benzimidazole derivatives could effectively
inhibiting the growth of various microorganisms, which
suggest that 2-aminobenzimidazole compounds should
have large potential as a new type of antibacterial [7],
antifungal [8] or antiviral [9] agents.
Some of 2-amino-1H-benzimidazole derivatives
possess nonmedical use eg. anticorrosive or antifungal
agents. They are also common ligands in coordination
chemistry.
* E-mail: anna.nowicka@umed.wroc.pl
1047

Synthesis and antiproliferative activity in vitro of new 2-aminobenzimidazole
derivatives. Reaction of 2-arylideneaminobenzimidazole
with selected nitriles containing active methylene group
During the past 20 years the biological activity of added. The solution was refluxed ca 8 h (or 16 h for
2-amino-1H-benzimidazole has been studied [10,11]. compound 26). After cooling the precipitate was filtered,
In our last review, we presented selected methods of washed with diethyl ether and crystalized.
synthesis for 2-amino-1H-benzimidazoles [12], drugs,
4-Amino-3-cyano-2-(2,4-dimethoxyphenyl)-1,2-
containing in their structures 2-aminobenzimidazole dihydro[1,2-a]benzimidazole (8)
compound [13], and new derivatives, which possess
biological activity and their mechanism of action [14].
Yield 1.56 g (45%), white precipitate, crystallized
from ethanol, mp 206-208oC. IR (KBr): υmax/cm-1 = 3420,
2-Amino-1H-benzmidazoles, because of presence in 3340 (NH2), 3010 (CH), 2960 (CH3), 2200 (CN), 1640
1
theirstructuresguanidinegroup, areuniversalsubstrates (NH), 1610 (C=N), 830, 760 (CH, aromat). H NMR
for the synthesis of biological active substituted or (DMSO-d6): δ[ppm] = 3.67 and 3.71 (2s, 6H), 5.21 (s,
polycyclic derivatives.
1H), 6.55 (s, 1H), 6.70 (s, 2H), 6.98 (m, 3H), 7.06 (lt,
In our recently published papers, we have described J ≈ 7.7 Hz, 1H), 7.20 and 7.61 (2d, J = 7.6 Hz, 2H),
the synthesis and antyproliferative activity in vitro of 8.17 (s, 1H). MS (70 eV): m/z (%) = 348 (M++1.9), 347
pyrimido[1,2-a]benzimidazole derivatives using selected (M+, 43), 346 (M+-1, 20), 316 (17), 210 (15), 144 (3), 133
2-arylideneaminobenzimidazoles
and
compounds (100), 118 (4), 106 (5), 91 (3), 77 (3), 65 (2). Anal. calcd.
containing an active methylene group eg. 1,3-diketones for C16H17N5O2 (347.28): C, 65.70; H, 4.93; N, 20.16%.
[15] or selected β-ketoesters [16].
Found: C, 65.96; H, 5.01; N, 19.98%.
The aim of this work was to synthesize the new
4-Amino-3-cyano-2-(3,4-dimethoxyphenyl)-1,2-
derivatives of 2-amino-1H-benzimidazole using Schiff dihydropyrimido[1,2-a]benzimidazole (9)
bases and selected nitriles containing an active
methylene group.
Yield 1.98 g (57%), white solid, crystallized from
dioxane, mp 215-217C°. IR (KBr): υmax/cm-1= 3440,
Selected compounds of different chemical structures, 3340 (NH2), 3010 (CH), 2960 (CH3), 2220 (CN),
1
were examined for their antiproliferative activity in vitro 1640 (NH), 840. 760 (CH, aromat). H NMR (DMSO-
against the cells of human cancer cell lines and normal d6): δ[ppm] = 3.94 (s, 6H), 5.16 (s, 1H), 6.78 (d, J =
mouse fibroblasts.
8.4 Hz, 1H), 6.82 (s, 2H), 6.90 (d, J = 8.4 Hz, 1H), 6.98
(m, 2H), 7.10 (lt, J ≈ 6, 6 Hz, 1H), 7.21 and 7.68 (2d,
J = 7.8 Hz, 2H), 8.52 (s, 1H). 13C NMR (DMSO-d6):
δ[ppm] = 52.92, 55.41, 55.44, 62.02, 110.29, 111.71,
112.31, 115.99, 117.94, 119.16, 119.84, 123.25, 129.24,
134.90, 143.53, 148.47, 148.69, 149.15, 151.77. Anal.
2. Experimental procedure
2.1. Chemistry
Melting points were measured with a Boethius melting calcd. for C19H17N5O2 (347.37): C, 65.69; H, 4.93; N,
point apparatus. The new products were analyzed using 20.16%. Found: C, 65.52; H, 5.05; N, 19.95%.
a Perkin Elmer 2400 analyzer. IR spectra (in KBr) were
4-Amino-3-cyano-2-(p-chlorophenylene)-1,2-
recorded with an IR 75 spectrophotometer, H NMR dihydropyrimido[1,2-a]benzimidazole (10)
1
spectra–onaBrukerAVANCEDRX300MHzor500MHz
Yield 1.80 g (58%) white solid, crystallized from
using DMSO-d6 as an internal standard. Mass spectra dioxane, mp 224-225oC. IR (KBr): υmax/cm-1= 3430,
were determined on a GCMS-LK 82091 spectrometer 3330 (NH2), 2900 (CH), 2200 (CN), 1670 (C=N, C=C),
at the ionization energy 15 or 70 eV. The course of 1640 (NH), 820, 740 (CH aromat). 1H NMR (DMSO-d6):
reaction and the purity of products were checked by δ[ppm] = 5.27 (s, 1H), 6.87 (s, 2H), 7.01 and 7.12 (2lt,
TLC (Kiesselgel G. Merck) in diethyl ether:ethanol = 5:1 J ≈ 7.8 Hz, 2H), 7.23 (m, 1H), 7.31 and 7.43 (2d, J =
as eluent.
8.5 Hz, 2H), 7.63 (m, 3H), 8.63 (s, 1H). 13C NMR
The syntheses of 2-aminobenzimidazole (1) and (DMSO-d6): δ[ppm] = 52.62, 61.52, 112.51, 116.19,
Schiff bases 2-7 have been presented in our previous 118.19, 119.11, 120.03, 123.48, 127.76 (2C), 129.29,
article [17].
131.58 (2C), 141.83, 143.58, 149.30, 151.65. MS
(70 eV): m/z % = 323 (M+2, 16), 322 (M+1, 15) 321
(M+, 51), 320 M-1, 18), 254 (11), 210 (77), 118 (11),
2.1.1. General procedure for the preparation 8-29
Reaction of Schiff bases 2-7 with selected nitriles 151 (12), 133 (100), 90 (11), 76 (3), 65 (2). Anal. calcd.
containing active methylene group: malononitrile, for C17H12N5Cl (321.77): C, 63.46; H, 3.76; N, 21.77%.
cyanoacetamide, benzyl cyanide, benzoylacetonitrile, Found: C, 63.53; H, 3.60; N, 21.70%.
cyanoacetate methyl ester and benzylacetamide. To a
solution of appropriate Schiff bases 2-7 (0.01 mol) in dihydropyrimido[1,2-a]benzimidzole (11)
ethanol (30 mL) (or methanol (30 mL) for compound 28)
Yield 1.28 g (35%) white solid, crystallized from
containing triethylamine (0.3 mL) selected nitriles were ethanol, mp 228-229oC. IR (KBr): υmax/cm-1= 3440, 3300
4-Amino-2-(p-bromophenylene)-3-cyano-1,2-
1048

A. Nowicka et al.
(NH2+NH), 2850 (CH), 2190 (CN), 1650 (NH), 1595 2H), 7.31 (s, 2H), 7.43 (m, 1H), 7.59 (m, 4H), 7.81 (m,
1
(NH sec), 830, 740 (CH aromat). H NMR (DMSO-d6): 1H), 8.53 (s, 1H). 13C NMR (DMSO-d6): δ[ppm] = 58.46,
δ[ppm] = 5.24 (s, 1H), 6.87 (s, 2H), 7.01 and 7.12 (2lt, J 61.23, 110.60, 115.28, 115.86, 118.63, 121.36, 128.70,
≈ 8.1 Hz, 2H), 7.23 (m, 3H), 7.59 (m, 3H), 8.62 (s, 1H). 129.31 (2C), 130.48 (2C), 131.60, 133.93, 134.47,
13C NMR (DMSO-d6): δ[ppm] = 52.68, 64.44, 112.51, 152.93, 163.51. Anal. calcd. for C17H14N5O Cl (339.78):
116.20, 120.04, 121.06, 123.47, 128.32 (2 C), 129.29, C, 60.009; H, 4.15; N, 20.61%. Found: C, 60.15; H,
130.75, 131.68 (2 C), 142.25, 143.57, 149.30, 151.63. 4.32; N, 21.00%.
Anal. calcd. for C17H12N5Br (366.22): C, 55.76; H, 3.30;
N, 19.12%. Found: C, 55.59; H, 3.30; N, 19.18%.
4-Amino-2-(o-bromophenylene)-3-cyano-1,2-
dihydropyrimido[1,2-a]benzimidazole (12)
4-Amino-2-(p-bromophenylene)-3-carbamoyl-
1,2-dihydropyrimido[1,2-a]benzimidazole (16)
Yield 2.23 g (58%) white solid crystallized from
ethanol, mp 228-230oC. IR (KBr): υmax/cm-1= 3500, 3400
Yield 1.73 g ( 47%) white solid crystallized from (broad, NH2 and CONH2), 2850 (CH), 1690 (CO), 1630
1
ethanol, mp 216-219oC. IR (KBr): υmax/cm-1= 3440, 3300 (NH), 835, 740 (CH, aromat). H NMR (DMSO-d6):
(NH2+ NH), 2860 (CH), 2200 (CN), 1650 (NH), 760 (CH, δ[ppm] = 5.25 (s, 1H), 6.08 (s, 2H), 6.68 and 6.97 (2lt,
1
aromat). H NMR (DMSO-d6): δ[ppm] = 5.63 (s, 1H), J = 7.3 Hz, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.32 (br, 2H),
6.87 (s, 2H), 7.03 and 7.13 (2lt, J ≈ 7.1 Hz, 2H), 7.33 7.46 (m, 1H), 7.67 (m, 3H), 7.84 (m, 1H), 8.58 (s, 1H).
(m, 4H), 7.66 (m, 2H), 8.50 (s, 1H). 13C NMR (DMSO- 13C NMR (DMSO-d6): δ[ppm] = 58.52, 61.28, 110.59,
d6): δ[ppm] = 53.21, 61.12, 112.42, 116.09, 118.35, 116.94, 118.61, 121.37, 123.16, 125.93, 130.38 (2C),
119,99, 121.72, 123.38, 128.52, 128.75, 129.26, 130.08, 131.15, 131.61 (2C), 134.33, 149.39, 152.87, 163.49.
132.93, 140.75, 143.52, 149.42, 151.61. Anal. calcd. Anal. calcd. for C17H14N5OBr (384.24): C, 53.14; H, 3.67;
for C17H12N5Br (366.22): C, 55.76; H, 3.30; N, 19.12%. N, 18.23 %. Found: C, 52.85; H, 3.84; N, 18.00.
Found: C, 55.82; H, 3.31; N, 19.28%.
2-(2,4-Dimethoxyphenyl)-4-imino--3-phenyl-
1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole (17)
Yield 2.44 g (62%) white solid, crystallized from
α-Cyano-2,4-dimethoxycinnamicamide (13)
Yield 1.54 g (66%) yellow solid crystallized from
ethanol, mp 203-206oC. IR (KBr): υmax/cm-1= 3540, dioxane, mp 207-210oC. IR (KBr): υmax/cm-1= 3300 (NH),
3380 (NH2), 3100 (=CH), 2950 (CH3), 2830 (OCH3), 3080 (CH), 2950 (CH3),2840 (OCH3), 1660 (C=C), C=N),
2210 (CN), 1680 (CONH2), 1610 (C=C), 1270 (Ar O-C), 1340 (NH), 830 and 740 (CH, aromat). 1H NMR (DMSO-
1
860 (CH, aromat). H NMR (DMSO-d6): δ[ppm] = 3.85 d6): δ[ppm] = 3.72 and 3.92 (2s, 6H), 4.37 and 4.94 (2d,
and 3.88 (2s, 6H), 6.68 (s, 1H), 6.71 (m, 1H), 7.62 and J = 5.6 Hz, 2H), 6.48 (d, J = 7.6 Hz, 1H), 6.65 (s, 1H),
7.70 (2s, 2H), 8.07 (d. J = 8 Hz, 1H), 8.34 (s, 1H). 13C 6.93 (m, 2H), 7.24 (m, 7H), 8.07 (d, J = 7.8 Hz, 1H),
NMR (DMSO-d6): δ[ppm] = 55.74, 56.07, 98.32, 102.12, 8.23 (s, 1H), 9.34 (s, 1H). 13C NMR (DMSO-d6): δ[ppm]
106.80, 113.24, 117.34, 129.58, 144.73, 160.47, 163.04, = 51.38, 53.50, 55.77, 66.28, 98.84, 104.62, 112.13,
164.60.Anal. calcd. for C12H12N2O3 (232.24): C, 62.06; H, 115.30, 119.54, 121.21, 123.29, 126.06, 127.09 (2C),
5.21; N, 12.06%. Found: C, 62.30; H, 5.40; N, 12.17%.
4-Amino-3-carbamoyl-2-(4-methoxyphenylene)- 156.97, 157.72, 160.14. Anal. calcd. for C24H22N4O2
1,2-dihydro[1,2-a]benzimidazole (14) (398.46): C, 73.34; H, 5.57; N, 14.06%. Found: C, 72.98;
127.36, 128.77 (2C), 130.80, 139.13, 143.96, 152.94,
Yield 2.79 g (83%) white solid crystallized twice from H, 5.75; N, 14.21%.
1
dioxane, mp 337-339oC. H NMR (DMSO-d6): δ[ppm]
4-Imino-2-(4-methoxyphenylene)-3-phenyl-
= 3.35 (s, 3H), 5.27 (s, 1H), 6.50 (s, 2H), 6.97 (d, J = 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole (18)
8.7 Hz, 2H), 7.04 (br, 2H), 7.19 (lt, J ≈ 8.5 Hz, 1H), 7.39
Yield 2.6 g (74%) white solid, crystallized from
(m, 2H), 7.54 (m, 2H), 7.68 (m, 1H), 8.53 (s, 1H).13C ethanol, mp 212-215oC. IR (KBr): υmax/cm-1= 3340 (NH),
NMR (DMSO-d6): δ[ppm] = 55.09, 56.23, 60.63, 110.17, 3080 (CH), 2950 (CH3), 1660 (C=C), 1350 (NH), 840,
1
114.39 (2C), 118.99, 120.79, 122.17, 128.25 (2C), 760 (CH aromat). H NMR (DMSO-d6): δ[ppm] = 3.70
128.46 (2C), 130.64, 131.86, 145.51, 159.40. Anal. (s, 3H), 4.44 and 4.94 (2d, J = 5.5 Hz, 2H), 6.15 (s, 1H),
calcd. for C18H17N5O2 (335.35): C, 64.47; H, 5.11; N, 6.86 (d, J = 8.7 Hz, 2H), 6.97 and 7.11 (lt, J ≈ 7.5 Hz,
20.88%. Found: C, 64.81; H, 5.1; N, 20.55%.
2H), 7.27 (m, 7H), 8.10 (d, J = 8.10 Hz, 1H), 8.45 (s,
1
4-Amino-3-carbamoyl-2-(p-chlorophenylene)- 1H), 8.87 (s, 1H). H NMR (CDCl3): δ[ppm] = 1.85 (br,
pyrimido[1,2-a]benzimidazole (15)
1H), 3.74 (s, 3H), 4.02 and 4.88 (2d, J = 5.8 Hz, 2H),
Yield 1.98 g (58%) white solid crystallized from 6.33 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 8.7 Hz, 2H), 7.07
dioxane, mp 231-232oC. IR (KBr): υmax/cm-1= 3500, (m, 5H), 7.76 (m, 4H), 8.03 (s, 1H), 8.22 (d, J = 6.9 Hz,
3400, (NH2 and CONH2), 2830, 1690 (CO), 1635 (NH), 1H). 1H NMR (CDCl3 + D2O): δ[ppm] = 3.78 (s, 3H), 4.16
840, 740 (CH, aromat). 1H NMR (DMSO-d6): δ[ppm] = = and 4.94 (2d, J = 5.5 Hz, 2H), 6.43 (d, J = 7.8 Hz, 1H),
5.28 (s, 1H), 6.10 (s, 2H), 6.68 and 7.19 (2lt ≈ 7.7 Hz, 6.77 (d, J = 8.7 Hz, 2H), 7.07 (m, 5H), 7.28 (m, 4H),
1049

Synthesis and antiproliferative activity in vitro of new 2-aminobenzimidazole
derivatives. Reaction of 2-arylideneaminobenzimidazole
with selected nitriles containing active methylene group
8.20 (d, J = 6.9 Hz, 1H). 13C NMR (DMSO-d6): δ[ppm]
= 53.28, 55.02, 57.53, 113.81, 114.18, 115.34, 119.57,
α-Benzoyl-2,4-dimethoxycinnamic nitrile (22)
Yield 2.49 g (85%), yellow solid, recrystallized from
123.30, 127.55, 127.72 (2C), 128.29 (2C), 128.89 (2C), ethanol, mp 148-150oC. IR (KBr): υmax/cm-1= 3000 (CH,
130.98 (2C), 132.67, 137.29, 143.82, 152.64, 158.63, C=C), 2970 (OCH3) 2220 (CN), 1670 (C=CO), 1625
1
158.81. Anal. calcd. for C23H20N4O (368.43): C, 74.98; H, (C=C), 1265 (C-O-C), 835, 760 (CH, aromat). H NMR
5.47; N, 15.21%. Found: C, 75.36; H, 5.47; N, 15.17%.
2-(p-Chlorophenylene)-4-imino-3-phenyl-1,2,3,4- 1H), 6.79 (m, 1H), 7.56 and 7.67 (2d, J = 7.5 Hz, 2H),
tetrahydropyrimido[1,2-a]benzimidazole (19) 7.55 (m, 3 H), 8.32 (m, 2H). MS (70 eV): m/z [%] = 294
(DMSO-d6): δ[ppm] = 3.84 and 3.89 (2s, 6H), 6.70 (s,
Yield 2.23 g (60%) white solid, crystallized from (M+1, 14), 293 (M+, 74), 278 (24), 263 (20), 262 (M+ -
n-butanol, mp 207-208oC. IR (KBr): υmax/cm-1= 3300 OCH3, 99), 106 (7), 105 (100), 77 (58), 51 (10), 39 (1).
(=NH), 2940 (CH), 1625 (NH), 820, 740 (CH, aromat). Anal. calcd. for C18H15NO3 (293.32): C, 73.69; H, 5.16;
1H NMR (DMSO-d6): δ[ppm] = 4.45 and 5.03 (2d, J = N, 4.78%. Found: C, 74.53; H, 5.30; N, 4.90%.
6.0 Hz, 2H), 6.10 (s, 1H), 6.92 and 7.04 (2lt, J ≈ 7.5 Hz,
2H), 7.34 (m, 9H), 8.08 (d, J = 7.5 Hz, 1H), 8.53 (s, 1H), dihydropyrimido[1,2-a]benzimidazole (23)
8.85 (s, 1H). Yield 1.47 g (53%) white solid crystallized from
3-Cyano-4-phenyl-2-(2,4-dimethoxyphenyl)-1,2-
13C NMR (DMSO-d6): δ[ppm] = 53.01, 57.44, ethanol, mp 270-273oC. IR (KBr): υmax/cm-1= 3420 (NH),
114.20, 115.45, 119.73, 123.37, 127.71, 128.43 (2C), 2940 (CH3), 2840 (ArOCH3), 2210 (CN), 1650 (Ph-C=C),
128.60 (2C), 128.75 (2C), 129.75, 130.97 (2C), 132.24, 1625 (C=C), 1570 (NH), 846, 780, 740 (CH, aromat).
136.76, 139.74, 143.71, 152.49. 158.64. Anal. calcd. 1H NMR (500 MHz, DMSO-d6): δ[ppm] = 3.72 and 3.75
for C22H17N4Cl (374.87): C, 70.87; H, 4.60; N, 15.03%. (2s, 6H), 6.54 (m, 2H), 6.75 (s, 1H), 6.95 (m, 2H), 7.05
Found: C, 70.98, H, 4.49; N, 14.87%.
2-(p-Bromophenylene)-4-imino-3-phenyl-1,2,3,4- 3H), 7.84 (m, 2H), 11.36 (s, 1H) – exchangeable with
tetrahydropyrimido[1,2-a]benzimidazole (20)
D2O. 13C NMR (500 MHz, DMSO-d6): δ[ppm] = 55.24,
(m, 1H), 7.32 and 7.43 (2d, J = 7.8 Hz, 2H), 7.54 (m,
Yield 2.59 g ( 62%), white solid crystallized from 55.77, 99.12, 105.17, 109.63, 116.26, 119.14, 120.51,
ethanol, mp 204-206oC. IR (KBr): υmax/cm-1= 3300 128.36 and 128.59 (6C), 130.16, 130.57, 137.00, 137.15,
(=NH), 2950 (CH), 3630 (NH), 820, 740 (CH, aromat). 146.01, 158.56, 161.08. Anal. calcd. for C25H20N4O2
1H NMR (DMSO-d6): δ[ppm] = 4.46 and 5.04 (2d, (408.46): C, 73.51; H, 4.94; N, 13.72%. Found: C, 73.93;
J = 6.0 Hz, 2H), 6.98 and 7.12 (2 lt, J ≈ 7.4 Hz, 2H), H, 4.72; N, 14.05%.
7.22 (d, J = 7.8 Hz, 1H), 7.32 (m, 7H), 7.52 (d, J =
2-(o-Bromophenylene)-3-cyano-4-phenyl-1,2-
8.4 Hz, 2H), 8.13 (d, J = 7.8 Hz, 1H), 8.53 (s, 1H), 8.85 dihydropyrimido[1,2-a]benzimidazole (24)
(s, 1H). 13C NMR (DMSO-d6): δ[ppm] = 52.97, 57.50,
Yield 2.15 g (50%), white solid, crystallized from
114.19, 115.46, 119.72, 120.81, 123.36, 127.71, 128.40 ethanol, mp 248-250oC. IR (KBr): υmax/cm-1= 3420 (NH),
(2C), 128.75 (2C), 128.94 (2C), 130.98 (2C), 131.34, 2940 (CH), 2210 (CN), 1650 (C=C), 1570 (NH), 840, 760
1
136.76, 140.18, 143.73, 152.48, 159.63. Anal. calcd. (CH, aromat). H NMR (DMSO-d6): δ[ppm] = 6.74 (d,
for C22H17N4Br (417.31): C, 63.32; H, 4.11; N, 13.43%. J = 7.8 Hz, 1H), 6.83 (s, 1H), 6.97 and 7.10 (2m, 2H),
Found: C, 63.79; H, 3.97; N, 13.52%.
2-(o-Bromophenylene)-4-imino-3-phenyl-1,2,3,4- (m, 4H), 7.67 (m, 4H), 11.67 (s, 1H). 13C NMR (DMSO-
tetrahydropyrimido[1,2-a]benzimidazole (21) d6): δ[ppm] = 57.79, 109.43, 110.43, 116.39, 118.36,
7.34 (lt, J ≈ 7.6 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.52
Yield 1.84 g (44%), white solid crystallized from 121.02, 121.55, 122.31, 127.20, 128.32 and 128.55
ethanol, mp 200-203oC. IR (KBr): υmax/cm-1= 3300 (6C), 130.71, 130.87, 131.03, 132.80, 143.70, 145.49,
(=NH), 2950 (CH), 1630 (NH), 820, 740 (CH, aromat). 152.47, 156.83. Anal. calcd. for C23H15N4Br (427.30): C,
1H NMR (DMSO-d6): δ[ppm] = 4.49 and 5.12 (2d, J = 64.65; H, 3.54; N, 13.21%. Found: C, 64.35; H, 3.48; N,
5.6 Hz, 2H), 7.01 (lt, J = 7.6 Hz, 1H), 7.16 (m, 3H), 13.21%.
7.26 (m, 3H), 7.35 (m, 4H), 7.70 (dd, J = 1.1 Hz and J =
7.8 Hz, 1H), 8.08 (d, J = 7.5 Hz, 1H), 8.51 (s, 1H), 9.46
α-Cyano-2,3-dimethoxycinnamic ethyl ester (25)
Yield 1.50 g (61%), yellow solid, crystallized from
(s, 1H). 13C NMR (DMSO-d6): δ[ppm] = 50.96, 57.79, ethanol, mp 131-132oC. IR (KBr): υmax/cm-1= 3030 (CH),
114.10, 119.84, 122.02, 123.45, 127.07, 127.16 (2C), 2860 (ArOCH3), 2220 (CN), 1730 (C=C), 1660 (C=C),
1
127.76, 128.36, 128.92 (2C), 129.93, 130.81, 133.34, 840 (CH, aromat). H NMR (DMSO-d6): δ[ppm] = 1.28
138.08, 139.72, 140.21, 143.82, 152.45, 156.83. Anal. (t, J = 7.1 Hz, 3H), 3.89 and 3.91 (2s, 6H), 4.28 (q, J =
calcd. for C22H17N4Br (417.31): C, 63.32; H, 4.11; N, 7.1 Hz, 2H), 6.73 (m, 2H), 8.19 (d, J = 8.9 Hz, 1H), 8.49
13.43%. Found: C, 63.75; H, 4.17; N, 13.91%.
(s, 1H). MS (70 eV): m/z [%] = 262 (M+1, 15), 261 (M+,
1050

A. Nowicka et al.
100), 233 (7), 216 (36), 189 (32), 161 (8), 145 (2), 77 = 42.12, 55.44, 55.76, 102.40, 111.81, 112.35, 117.08,
(58), 102 (5), 75 (2). Anal. calcd. for C14H15NO4 (261.28): 124.45, 125.47, 126.86, 127.35 (2C), 128.26 (2C),
C, 64.36; H, 5.79; N, 5.36%. Found: C, 64.27; H, 5.61; 139.00, 148.64, 150.70, 152.52, 161.40. Anal. calcd.
N, 5.44%.
3-Cyano-2-(2,3-dimethoxyphenyl)-1,2,3,4- Found: C, 70.63; H, 5.87; N, 8.67%.
tetrahydropyrimido[1,2-a]benzimidazol-4-one (26)
for C19H18N2O3 (322.36): C, 70.79; H, 5.63; N, 8.69%.
Yield 1.12 g (35%) white solid crystallized from 2.2. Biology
ethanol, mp 206-208oC. IR (KBr): υmax/cm-1= 3340 (NH- 2.2.1. Anti-proliferative assay in vitro
sec), 3080 (CH, aromat), 2840 (CH3), 2240 (CN), 1680 Anti-proliferative tests were performed on human cancer
(C=O), 1640 (NH, sec), 830 and 740 (CH, aromat). cell lines: A549 (lung), MCF-7 (breast), leukemia HL-60
1H NMR (DMSO-d6): δ[ppm] = 3.87 and 3.89 (2s, 6H), and mouse embryonic fibroblast BALB/3T3 according
3.98 and 4.33 (2d, J = 6.0 Hz, 2H), 7.09 (m, 1H), 7.28 to standard procedure [22]. All cell lines were obtained
(m, 2H), 8.15 (d, J = 8.9, 1H), 8.25 (m, 2H), 8.40 (s, from American Type Culture Collection (Rockville,
1H). MS (70 eV): m/z [%] = 349 (M+1, 27), 348 (M+, 63), Maryland, U.S.A.) and have been maintained in
321 (37), 293 (41), 233 (61), 156 (38), 133 (100), 121 culture or frozen in thaw Cell Culture Collection
(17), 105 (23), 97 (7), 77 (6), 65 (4), 39 (4). Anal. calcd. of the Institute of Immunology and Experimental
for C19H16N4O3 (348.36): C, 65.51; H, 4.63; N, 16.08%. Therapy, Polish Academy of Sciences (IIET,PAS,
Found: C, 65.97; H, 4.61; N, 15.94%.
Wrocław, Poland). The A549 cells were cultured in a
α-Cyano-3,4-dimethoxycinnamic ethyl ester (27)
mixture of Opti-MEM and RPMI 1640 medium (1:1,
Yield 1.20 g (46%) yellow precipitate, crystallized both from Gibco, Scotland, UK) supplemented with
with ethanol, mp 65-67oC. IR (KBr): υmax/cm-1= 3020 2 mM L-glutamine and 5% fetal bovine serum. MCF-
(CH), 2860 (ArOCH3), 2200 (CN), 1720 (CO), 1660 7 cells were cultured in Eagle medium (IIET, Wroclaw,
1
(C=C), 840 (CH, aromat). H NMR (DMSO-d6): δ[ppm] Poland), supplemented with 2 mM L-glutamine and
= 1.31 (t, J = 7.2 Hz, 3H), 3.81 and 3.88 (2s, 6H), 4.30 1.0 mM sodium pyruvate, 10% fetal bovine serum
(q, J = 7.2 Hz, 2H), 7.16 (d, J = 8.40 Hz, 1H), 7.73 (m, and with 0.8 mg L-1 of insulin (Sigma-Aldrich Chemie
2H), 8.28 (s, 1H). 13C NMR (DMSO-d6): δ[ppm] = 55.65, GmbH, Steinheim, Germany). HL-60 cells were
61.97, 78.68, 79.35, 98.45, 111.80, 112.89, 116.29, cultured in RPMI 1640 medium (Gibco, Scotland, UK)
123.89, 126.77, 148.69, 153.49, 154.72, 162.34. Anal. with 2 mM L-glutamine, adjusted to contain 1.5 g L-1
calcd. for C14H15NO4 (261.27): C, 64.36; H, 5.79; N, sodium bicarbonate and 1.0 mM sodium pyruvate, 10%
5.36%. Found: C, 64.01; H, 5.93; N, 5.02%.
fetal bovine serum (Sigma-Aldrich Chemie GmbH,
α-Cyano-3,4-dimethoxycinnamic methyl ester Steinheim, Germany). Mouse fibroblasts BALB/3T3
(28)
were maintained in Dulbecco medium (DMEM, Gibco,
Yield 1.38 g (56%), yellow solid crystallized from Scotland, UK) supplemented with 2 mM L-glutamine
methanol, mp 118-120oC. IR (KBr): υmax/cm-1= 3020 and 10% fetal bovine serum (Sigma-Aldrich Chemie
(CH), 2850 (ArOCH3), 2200 (CN), 1720 (CO), 1660 GmbH, Steinheim, Germany). All culture media
(C=C), 840 (CH, aromat). H NMR (DMSO-d6): δ[ppm] were supplemented with 100 units L-1 penicillin, and
1
= 3.80 (s, 3H), 3.84 and 3.86 (2s, 6H), 7.13 (d, J = 100 µg L-1 streptomycin (Polfa Tarchomin S.A., Warsaw,
8.7 Hz, 1H), 7.73 (m, 2H), 8.28 (s, 1H). 13C NMR (DMSO- Poland). Cell lines were grown at 37°C with 5% CO2
d6): δ[ppm] = 53.07, 55.44, 55.87, 98.04, 111.84, 112.83, humidified atmosphere. The anti-proliferative effect
116.35, 123.94, 126.89, 138.68, 153.55, 154.86, 162.88. of the tested compounds was examined after 72 h
Anal. calcd. for C13H13NO4 (247.25): C, 63.15; H, 5.30; exposure of the cultured cells to varying concentrations
N, 5.67%. Found: C, 62.87; H, 5.33; N, 5.35%.
α-Cyano-3,4-dimethoxycinnamic
benzylamide (29)
of the tested compound (total plate incubation time
acid time: 96 h), using the sulforhodamine B (SRB) assay
for adherent cells (A549, BALB/3T3 and MCF-7) and
Yield 1.82 g (57 %), yellow precipitate crystallized MTT assay for leukemia cells (HL-60). The results
from ethanol, mp 156-159oC. IR (KBr): υmax/cm-1= was shown as an IC50 value (inhibitory concentration
3450(NH), 3010 (CH), 2220 (CN), 1650 (CO), 1620 50% - a concentration in μg L-1 of tested agent which
1
(C+C), 820, 760 (CH, aromat). H NMR (DMSO-d6): inhibits proliferation of 50% of cancer cells population).
δ[ppm] = 3.81 and 3.86 (2s, 6H), 4.43 (d, J = 6.0 Hz, Each compound was tested at every concentration in
2H), 7.15 (d, J = 8.4 Hz, 1H), 7.29 (m, 5H), 7.60 (dd, triplicate in a single experiment, which was repeated
J = 2.1 Hz, J = 8.6 Hz, 1H), 7.68 (m, 1H), 8.15 (s, 1H), 3 times.The activity of tested compounds was compared
9.81 (t, J = 6.0 Hz, 1H). 13C NMR (DMSO-d6): δ[ppm] to the activity of cisplatin, used as a reference agent.
1051

Synthesis and antiproliferative activity in vitro of new 2-aminobenzimidazole
derivatives. Reaction of 2-arylideneaminobenzimidazole
with selected nitriles containing active methylene group
benzimidazole derivatives 17-21 were obtained,
instead of expected 4-amino-1,2-dihydropyrimido[1,2-a]
benzimidazole structures 17A-21A. IR spectra of
3. Results and discussion
3.1. Chemistry
Schiff bases 2-7 were obtained earlier [17] from obtained compounds contain, among other absorption
2-amino-1H-benzimidazole (1) with substituted aromatic bands, one band ν=3300-3340 cm-1 characteristic for
aldehydes: 2,4-dimethoxy, 3,4-dimethoxy-, 4-methoxy-, =NH group. 1H NMR spectra (CDCl3) of the product 18
4-chloro-. 4-bromo- and 2-bromobenzaldehyde. contain one proton signal at δ=1.85 ppm corresponding
2-Arylideneaminobenzimidazoles 2-7 and selected to =NH group in position 4, one three proton singlet:
nitriles containing an active methylene group: the signal at δ=3.70 ppm corresponding to protons of
1
malononitrile, cyanoacetamide, benzyl cyanide, –OCH3. In addition, the H NMR spectra displayed two
benzoylacetonitrile, cyanoacetate methyl ester and doublets at δ=4.02 and 4.88 ppm (J=5.8 Hz), which
benzylacetamide have been the substrates for the were assigned to –CH-CH- protons in position 2 and
synthesis presented in Schemes 1 and 2. In earlier 3 pyrimidine ring. Additionally, one singlet signal at
articles [15,16,18-20] it has been suggested that the δ=8.03 ppm, from –NH-CH-CH- proton was observed in
compounds containing an active methylene group the 1H NMR spectra . The signals corresponding to =NH
add to the azomethine double bond (-N=CH-) to give group and –NH-CH-CH- proton exchanged with D2O.
an unstable adduct. There were two possible paths of
α-Benzoyl-2,4-dimethoxycinnamic
nitrile
(22)
reaction: addition-elimination or addition-cyclization. was obtained in the reaction of Schiff base 2 with
The elimination of the amine molecule to yield cinnamic benzoylacetonitrile. 3-Cyano-2-(2,4-dimethoxyphenyl)-
acid derivatives [18,19] or the cyclization of unstable 4-phenyl-1,2-dihydropyrimido[1,2-a]benzimidazole
adduct gives pyrimido[1,2-a]benzimidazole derivatives (23) was produced in synthesis of compound 22 with
[15,16,20]. Using 1.5-fold excess nitriles/Schiff base 2-amino-1H-benzimidazole (1) in boiling diglime.
allowed avoiding an addition of the nitriles molecule to
In next synthesis 2,4-dimethoxy- (2) and 3,4-dime
2 molecules of Schiff base. The reactions were carried thoxybenzylideneaminobenzimidazole (3) in reaction
out in boiling ethanol or methanol, containing a few with cyanoacetate methyl ester in refluxing ethanol for 4
drops triethylamine and single products were obtained.
hours gave α-cyano-2,4-dimethoxycinnamic ethyl ester
The results of elemental analysis and MS spectra (25) and α-cyano-3,4-dimethoxycinnamic ethyl ester
indicated that the formation of pyrimido[1,2-a] (27). The compounds were obtained from an unstable
benzimidazole derivatives 8-12 involved cyclization of adduct, after elimination of 2-amino-1H-benzimidazole
unstableadductsofSchiffbase2-3, 5-7andmalononitrile. (1) molecule and additionally transesterification. The
IR spectra of the obtained compounds 8-12 contain, same reaction of Schiff base 2 with cyanoacetate methyl
among other absorption bands, those within the ester carried out in boiling methanol provide to α-cyano-
range of two sharp bands ν=3440-3300 cm-1 and ca 2,4-dimethoxycinnamic methyl ester (28). Heating for 8
ν=2200 cm-1 characteristic for the NH2 and CN groups.
1H NMR spectra of the products 8-12 contain e.g. one
two protons singlet: the signals at δ = 6.70-6.87 ppm
corresponding to protons of -NH2 group at position 4.
hours Schiff base 2 with cyanoacetate methyl ester in
boiling ethanol gave 3-cyano-2-(2,4-dimethoxyphenyl)-
1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-4-one
(26).
1
Additionally,
these
spectra
for
1,2-
In addition, the H NMR spectra displayed two singlet
dihydropyrimido[1,2-a]benzimidazole derivatives, reveal
the presence of –NH-CH- protons signals as two sharp
one-proton singlets. The possibility of the formation
of two derivative structures: 1H-pyrimido[1,2-a]
benzimidazoleor10H-pyrimido[1,2-a]benzimidazolehas
been considered. The presence of 1H-pyrimido[1,2-a]
benzimidazole structures may originate in fast exchange
of NH protons and the absence of CH protons splitting.
The number of signals for the aromatic protons
signals at δ = 8.17-8.63 ppm and δ = 5.16-5.63 ppm,
which were assigned to the -NH and -CH protons,
respectively.
In the next synthesis, the reaction of
2-arylideneaminobenzimidazole
3-6
with
cyanoacetamide produced products 13,14-16 of
different structures. Compound 13 was produced from
unstable adduct, after the elimination of 2-amino-
1H-benzimidazole (1) molecule. 4-Amino-2-arylo-3-
carbamoyl-1,2-dihydropyrimido[1,2-a]benzimidazole
1
in the H NMR spectra of compounds 8-29 is in good
derivatives 14-16 were obtained from an unstable agreement with their structures.
adduct after the cyclization (vide Experimental).
Ab initio geometry optimization calculations for three
In the reaction of Schiff bases 2,4-7 with benzoyl pairs 1H and 10H of possible products 10 and 10a, 17
cyanide, new 4-imino-1,2,3,4-tetrahydropyrimido[1,2-a] and 17a, 24 and 24a was performed within the density
1052

A. Nowicka et al.
Table 1. Comparison of calculated energies of selected compounds.
Compound
Total energy
(Hartree)
ΔE
(Hartree)
ΔE
(kJ mol-1)
10
-1388.9829027
-1388.9748138
-1182.7152963
-1182.7040707
-3676.1827237
-3676.1747843
R1=CN, R2=NH2
-0.008089
-21.24
-29.47
-20.84
10a
17
R1=Ph, R2=NH
-0.011226
-0.007939
17a
24
R1=CN, R2=Ph
24a
Table 2. The antiproliferative activity of selected compounds.
3.2. Biology
The antiproliferative activity in vitro of compounds
10,13,15,17,19,21,23-25 and 28 were examined
against human cell lines: lung A549 and breast
MCF-7 cancer, HL-60 leukemia and normal mouse
fibroblast cell line BALB/3T3. The most sensitive to
their antiproliferative activity was the cells of human
leukemia HL-60 with the highest IC50 obtained
Compound /
cell line
IC50 [µg mL-1] mean ± SD
HL-60
MCF-7
A549
BALB/3T3
10
54.1 ± 38.6
NA
NA
NA
NA
61.7 ± 14.3
8.2 ± 5.5
2.2 ± 1.2
2.7 ± 0.7
NA
13
29.2 ± 9.3 48.5 ± 9.4
1.9 ± 1.6
15
6.7 ± 1.6 27.6 ± 3.4
for
dihydropyrimido[1,2-a]benzimidazole
3-cyano-4-phenyl-2-(2,4-dimethoxyphenyl)-1,2-
dihydropyrimido[1,2-a]benzimidazole (23) (0.09
2-(o-bromophenylene)-3-cyano-4-phenyl-1,2-
17
8.4 ± 7.6 28.8 ± 4.1 23.0 ±7.1
2.0 ± 0.6 31.2 ± 8.0 24.8 ±4.5
(24) and
19
21
12.4 ± 11.6 72.2 ± 13.5
0.3 ± 0.1 0.5 ± 0.1 2.6 ± 3.4
0.09 ± 0.07 0.3 ± 0.1 0.4 ± 0.3
NA
and 0.3 µg mL-1, respectively). The cells of A549
human lung cancer was the most resistant to the
compounds tested, however the IC50 value counted
for two most active compounds was still very low
(2.6 µg mL-1 for 23 and 0.4 µg mL-1 for 24) towards this
cells.The4-amino-3-carbamoyl-2-(p-chlorophenylene)-
pyrimido[1,2-a]benzimidazole (15) and 2-aryl-3-phenyl-
4-imino-pyrimido[1,2-a]benzimidazole derivatives 17,
19 and 21 revealed moderate antiproliferative activity
23
11.5 ± 7.5
0.4 ± 0.4
NA
24
25
33.2 ± 8.0 82.0 ± 17.1
NA
NA
28
35.4 ± 10.8
0.3 ± 0.1
NA
NA
cisplatin
5.3 ± 0.3 3.3 ± 0.3
3.1 ± 0.7
NA – compound not active in the range of concentrations applied
functional theory (DFT). The computational study was against HL-60 and very low against A549 and MCF-7
performed using Gaussian 03 program [21]. Calculated cells. The weakest activity revealed 4-amino-3-cyano-
energies for optimized structures are presented in 2-(p-chlorophenylene)-1,2-dihydropyrimido[1,2-a]
Table 1. Energy differences ΔE suggested that 10, 17 benzimidazole (10), and α-cyanocinnamic acid
and 24 were more probable products than corresponding derivatives 13, 25 and 28 (Table 2). Unfortunately, the
structures 10a, 17a and 24a respectively.
compounds tested, similarly like control drug cisplatin,
1053

Synthesis and antiproliferative activity in vitro of new 2-aminobenzimidazole
derivatives. Reaction of 2-arylideneaminobenzimidazole
with selected nitriles containing active methylene group
Ar
R
CN
8
CONH2
Ph
17
COPh
22, 23
2,4-C6H3(OCH3)2
3,4-C6H3(OCH3)2
4-C6H4OCH3
4- C6H4Cl
2
3
4
5
6
7
9
13
14
15
16
18
19
20
21
10
11
12
4- C6H4Br
2- C6H4Br
24
Scheme 1. Synthesis of pyrimido[1,2-a]benzimidazole and α-cyanocinnamic acid derivatives.
Scheme 2. Reaction of 2-arylideneaminobenzimidazoles with with cyanoacetate methyl ester.
1054

A. Nowicka et al.
inhibited proliferation of normal mouse fibroblasts spectroscopic and elemental analysis techniques.
BALB/3T3 in similar range of concentrations like Selected synthesized compounds 10, 13, 14, 17, 19, 21,
observed for neoplastic cells.
23-25 and 28 were screened for their antiproliferative
activity in vitro. Two: 2-(o-bromophenylene)-3-cyano-
4-phenyl-1,2-dihydropyrimido[1,2-a]benzimidazole
(24) and 3-cyano-4-phenyl-2-(2,4-dimethoxyphenyl)-
1,2-dihydropyrimido[1,2-a]benzimidazole (23) were
4. Conclusions
Twenty-two new compounds 9-29 of various found to possess very high activity towards leukaemia,
chemical structure: pyrimido[1,2-a]benzimidazole breast and lung cancer cells. Further studies will be
or α-cyanocinnamic acid derivatives were obtained concentrated on finding compounds with sustained
from the syntheses described here. Their structures antiproliferative activity but much more selective
were confirmed by IR, 1H NMR, 13C NMR, mass towards cancer cells.
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