4-amino-2-aryl-3-cyano-1,2-dihydropyrimido- [1,2-a]benzimidazoles and their pyrimidine analogs as new anticancer agents

Article abstract of DOI:10.1007/s10593-014-1460-0

A multicomponent condensation between 2-aminobenzimidazole, malononitrile, and an aryl or heteroaryl aldehyde was used for the synthesis of 4-amino-2-aryl-3-cyano-1,2-dihydropyrimido-[1,2-a]benzimidazoles. In addition, a new method of synthesis of the corresponding pyrimido-[2,1-a]benzimidazole derivatives was developed. These syntheses were used to prepare a library of 4-amino-2-aryl-3-cyano-1,2-dihydropyrimido[1,2-a]benzimidazoles and their corresponding pyrimido-[2,1-a]benzimidazole derivatives. This library of compounds was then tested against pancreatic and breast cancer cell lines. A number of compounds were found to possess notable anticancer activity.

Full text of DOI:10.1007/s10593-014-1460-0

Chemistry of Heterocyclic Compounds, Vol. 50, No. 2, May, 2014 (Russian Original Vol. 50, No. 2, February, 2014)
4-AMINO-2-ARYL-3-CYANO-1,2-DIHYDROPYRIMIDO-
[1,2-a]BENZIMIDAZOLES AND THEIR PYRIMIDINE
ANALOGS AS NEW ANTICANCER AGENTS
V. A. Risley1, S. Henry1, M. V. Kosyrikhina2, M. R. Manzanares2, I. Payan2,
2
2
2
1
*
C. D. Downer , C. C. Hellmann , S. Van Slambrouck , and L. V. Frolova
A multicomponent condensation between 2-aminobenzimidazole, malononitrile, and an aryl or
heteroaryl aldehyde was used for the synthesis of 4-amino-2-aryl-3-cyano-1,2-dihydropyrimido-
[1,2-a]benzimidazoles. In addition, a new method of synthesis of the corresponding pyrimido-
[2,1-a]benzimidazole derivatives was developed. These syntheses were used to prepare a library of
4-amino-2-aryl-3-cyano-1,2-dihydropyrimido[1,2-a]benzimidazoles and their corresponding pyrimido-
[2,1-a]benzimidazole derivatives. This library of compounds was then tested against pancreatic and
breast cancer cell lines. A number of compounds were found to possess notable anticancer activity.
Keywords: benzimidazole, 1,2-dihydropyrimidine, 1,2-dihydropyrimido[1,2-a]benzimidazoles, pyrimi-
dine, pyrimido[2,1-a]benzimidazoles, anticancer activity, multicomponent reactions.
Multicomponent reactions (MCRs) are significant and important synthetic tools, which refer to a chemical
reaction where three or more components react to form a single product [1, 2]. MCRs have become popular in
modern synthetic chemistry due to their efficiency and convenience in the construction of multiple new bonds in a
one-pot process and are especially useful for the synthesis of heterocyclic compounds [3].
Heterocycles are found throughout medicinal chemistry, with examples including natural and synthetic
drugs such as quinine, atropine, morphine, diazepam, barbiturates, and antipyrine. Many biologically active
molecules, including those previously mentioned, typically feature five- and six-membered rings containing one
or more nitrogen atoms. Such privileged medicinal scaffolds, benzimidazole and pyrimidine heterocycles being
perfect examples, are frequently used in the development of new drugs. These heterocycles play the roles of key
pharmacophores in many different drugs expressing anticancer and antibacterial activity. It is noteworthy that
the pyrimidine core is a structural constituent of critically important anticancer drugs like fluorouracil, tegafur,
methotrexate, and cytabine [4]. Likewise, compounds based on the benzimidazole core are used in anticancer
therapy alone, or in the mix with other drugs [5-7].
_______
*To whom correspondence should be addressed, e-mail: frolova@nmt.edu.
1New Mexico Institute of Mining and Technology, 801 Leroy Place, Socorro NM 87801, U.S.A.
2School of Science, St. Thomas University, 16401 NW 37th Ave. Miami Gardens, FL 33054, U.S.A.; е-mail:
svanslambrouck@stu.edu.
_________________________________________________________________________________________
Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 209-218, February, 2014. Original
article submitted January 14, 2014.
0009-3122/14/5002-0185©2014 Springer Science+Business Media New York
185

One of the most rapidly developing areas in modern medicinal chemistry is the design of new medicinal
agents using a combination of different pharmacophores in one molecule [4, 8]. Such hybrid agents could offer
several potential advantages over traditional drug combinations, such as improvement of activity against
multidrug resistant cell lines, improvement of pharmacokinetic profile, and synergistic activity between the
pharmacophores. There are two different approaches for the design of hybrid molecules: 1) merging two different
pharmocophoric moieties; 2) linking of two or more pharmacophores together [9]. The first approach is based
on the ability to combine pharmacophoric groups in a new molecular structure and to retain their affinity for the
biological targets. A fusion of benzimidazole and pyrimidine cycles can be a good opportunity for creating
a new scaffold with anticancer properties (Scheme 1).
Scheme 1
Fusion scaffolds based on benzimidazole and pyrimidine
Previously, some compounds based on the 1,2-dihydropyrimido[1,2-a]benzimidazole core have
demonstrated antiproliferative activity [10]. These compounds differ in the mechanism of action based on the
substitution pattern in the dihydropyrimidine ring. Some of them act as topoisomerase I inhibitors [11, 12],
while others act as dual KSP and Aurora-A kinase inhibitors [13]. On the other hand, biological data on the
activity of the pyrimido[1,2-a]benzimidiazole core are scarcely available in the literature, probably due to the
lack of convenient methods of synthesis of core responding derivatives [14]. Interestingly, previous work on
pyridine and dihydropyridine derivatives that inhibit topoisomerase II has shown that pyridine derivatives could
be obtained by intracellular oxidation of dihydropyridine prodrugs [15]. In that case, corresponding pairs of
compounds from both series had the same magnitude of antiproliferative activity. If our title compounds have
similar mechanisms of action, the corresponding 1,2-dihydropyrimidine and pyrimidine derivatives should show
similar degree of activity. For this reason, we decided to investigate systematically the antiproliferative activity
of compound pairs containing 1,2-dihydropyrimido[1,2-a]benzimidazole and pyrimido[1,2-a]benz-imidazole
cores.
The three-component condensation reaction of malononitrile, aromatic aldehyde, and 2-amino-
benzimidazole was used to synthesize a library of 1,2-dihydropyrimido[1,2-a]benzimidazole derivatives. We
tried several different procedures, but only the reaction in water allowed us to obtain all the desired compounds
(Table 1) [16-20]. A slight modification of the procedure (time, ratio of reagents) from [16] allowed us to obtain
the products 1a-k in good yields, especially for aromatic aldehydes possessing six-membered rings. In the cases
of pyrazole 1i and thiophene 1j derivatives, the yields were significantly lower, although it should be mentioned
that we were not able to synthesize compounds bearing heteroaromatic five-membered rings using the other
procedures [17-20].
In principle, two possible heterocycles with the amino group in position 2 (structure A) or 4 (structure
B) can be formed as a result of this reaction (Scheme 2). According to the literature data, the isomer B
186

Scheme 2
N
N
NH
N
NH2
N
N
Ar
H
N
O
H
+
+
A
N
NH2
Ar
N
NH
N
Ar
H2N
N
B
Two theoretically possible isomers of 1,2-dihydropyrimido[1,2-a] benzimidazole
TABLE 1. Multicomponent Synthesis of 1,2-Dihydropyrimido[1,2-a]-
benzimidazole Derivatives 1a-k by the Condensation Reaction of
2-Aminobenzimidazole, Malononitrile, and Aldehyde
Com-
pound
1a
Com-
pound
1g
Ar
Time, h Yield, %
Ar
Time, h
14
Yield, %
61
12
45
12
65
15
42
1b
1h
13
16
54
57
12
14
36
29
1c
1i
1j
1d
12
13
56
60
12
63
1e
1f
1k
187

is the main product of the MCR as confirmed by X-ray crystallography and by NMR spectroscopy in solution
[18, 19]. Very detailed NMR investigation of compound 1k using 1H–13C-COSY and NOESY was made in [20].
Their NMR spectral data supported the existence of isomer B only. Unfortunately, they used a method of
synthesis of compound 1k different from the one presented here. Although the physical and spectral
characteristics of compound 1k obtained by our method and described in [20] are similar, we performed our
own NOESY investigation of this compound. The presence of NOE interactions (2-CH)–H Ar and (H-6)–NH2
and the absence of the interaction (H-6)–H Ar confirm the structure B for compound 1k obtained by our method
and rule out the formation of the structure A.
Synthesis of the corresponding pyrimido[1,2-a]benzimidazole derivatives 2a-j was not straightforward
(Table 2). Known procedures to obtain this type of compounds in a one-pot procedure or in a stepwise
manner gave very low yields or did not work at all for many derivatives [17, 20]. Earlier, we successfully
used DDQ as an oxidation reagent in the synthesis of pyrroles from pyrrolines [21]. Based on our previous
experience, we tried DDQ again as an oxidizing reagent for the synthesis of pyrimido[1,2-a]benzimidazoles
2a-j from the corresponding 1,2-dihydropyrimido[1,2-a]benzimidazole derivatives 1a-j. 1,2-Dihydro-
pyrimido[1,2-a]benzimidazole derivatives were heated at 55°C with 3 equiv. of DDQ for 4 h in acetonitrile.
These reactions worked for compounds with both aromatic and heteroaromatic substituents at position 2, and
the products were obtained with very high yields. The structures of new derivatives were confirmed by NMR
and mass spectroscopy.
TABLE 2. Synthesis of Pyrimido[1,2-a]benzimidazole Derivatives 2a-j
Com-
pound
Com-
pound
Ar
Time, h
4
Yield, %
41
Ar
Time, h Yield, %
5
94
2a
2f
5
5
87
54
4
4
86
64
2b
2c
2g
2h
5
4
73
84
4
4
96
82
2d
2e
2i
2j
188

TABLE 3. Antiproliferative Activity of Compounds 1a-j and 2a-j against
Human Cancer Cell Lines
IC50, *
PANC-1
Compound
Hs578T
MCF-7
>50
37.5 3.5
39.5 0.7
5 1.4
7.5 3.53
9.5 0.7
46.25 2.5
5 0.7
3.2 1.6
10 1.4
8.25 0.353
>50
6.25 2.5
48.5 3.5
18 4.2
21 0
5.5 1.4
19.3 5.1
21.3 1.7
38.5 0.2
42 3.5
10 0
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
>50
47.3 6.4
11.85 2.6
>50
43.5 4.9
>50
>50
>50
>50
>50
>50
7.7 0.7
>50
20.8 5.8
>50
ND
> 50
> 50
>50
>50
ND
>50
>50
>50
2f
1g
2g
1h
2h
1i
ND*2
44.1 7.2
38.75 15.2
>50
> 50
>50
> 50
>50
2i
ND
ND
ND
1j
>50
>50
>50
2j
ND
ND
ND
1k
_______
*Concentration required to reduce the viability of cells by 50% after a 48 h
treatment with the indicated compounds relative to a DMSO control with
standard deviation from two independent experiments, each performed in
four replicates, as determined by the MTT assay.
*2Not determined.
The obtained compounds 1a-j and 2a-j were tested in vitro against human pancreatic and breast cancer
cell lines PANC-1, MCF-7, and Hs578T. Many of these compounds showed micromolar antiproliferative
activity (Table 3) at varying levels, depending on the cancer cell lines and structures of the compounds used.
Activities had a tendency to vary, as in some cases the 1,2-dihydropyrimido[1,2-a]benzimidazole derivatives were
slightly more active than their respective oxidized counterparts, whereas in other cases the 1,2-dihydro-
pyrimido[1,2-a]benzimidazole derivatives were either less active or had similar activity as their oxidized
counterparts.
At the same time, a much stronger variation of antiproliferative activity was observed depending on the
2-aryl substituent. In general, homoaryl derivatives (compounds 1a-f,h, 2a-f,h) demonstrated higher activity
than heteroaryl derivatives (compounds 1g-j, 2g-j). In addition to this, the presence of lipophilic substituents or
hydroxyl groups in the meta position of the 2-phenyl ring generally improved the activity. Studies of SAR and
possible mechanisms of action of synthesized compounds are under way.
In conclusion, we have developed a new method for the synthesis of pyrimido[1,2-a]benzimidazole
derivatives. New 2-aryl- and 2-heteroaryl-1,2-dihydropyrimido[1,2-a]benzimidazoles and their oxidized analogs
were synthesized and tested against several different cancer cell lines. Some pairs demonstrated low micromolar
antiproliferative activity. Possible mechanisms of action will be investigated.
189

EXPERIMENTAL
1H and 13C NMR spectra were recorded on a Bruker Avance III 400 spectrometer (400 and 100 MHz,
respectively) in DMSO-d6, internal standard – TMS. High-resolution ESI+ mass spectra with positive ion-mode
electrospray ionization (ESI+) were recorded at the Mass Spectrometry Facility, University of New Mexico, on
a LCT Premier TOF mass spectrometer (compounds 1b-e, 2b,e) and at the Department of Chemistry and
Biochemistry, Texas State University, on a Waters Synapt G2 LCMS (other compounds). Melting points were
measured on a Mel-Temp Model 1001D apparatus and were uncorrected. All reactions were performed in oven-
dried flasks open to the atmosphere or under nitrogen and monitored by TLC on precoated (250 m) silica gel
60 F254 glass-backed plates (Sorbent Technologies). Visualization was accomplished with UV light. Flash
column chromatography was performed on silica gel (32-63 m, 60 Å pore size). All reagents and solvents were
purchased from commercial sources (Acros Organics and Sigma-Aldrich) and used without purification.
4-Amino-2-(phenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1a). A mixture of
malononitrile (0.0330 g, 0.499 mmol), 2-aminobenzimidazole (0.0666 g, 0.500 mmol), and benzaldehyde
(0.0531 g, 0.500 mmol) was refluxed in water (3 ml) for 12 h. The product was separated by filtration, and the
precipitate was washed with EtOH and diethyl ether. Yield 0.0650 g (45%). White powder. Mp 264-266°C (mp
267-269°C [20]). The 1H NMR spectrum corresponds to the published one [20].
4-Amino-2-(3,5-dibromophenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1b) was
obtained analogously from malononitrile (0.0343 g, 0.519 mmol), 2-amino benzimidazole (0.0660 g, 0.496 mmol),
and 3,5-dibromobenzaldehyde (0.1002 g, 0.380 mmol). Yield 0.1445g (65%). White powder. Mp 246-247°C.
1H NMR spectrum, δ, ppm (J, Hz): 5.35 (1H, s, CH); 6.96 (2H, s, NH2); 7.02 (1H, t, J = 7.2, H Ar); 7.13 (1H, t,
J = 7.2, H Ar); 7.25 (1H, d, J = 7.5, H Ar); 7.50 (2H, s, H Ar); 7.65 (1H, d, J = 7.2, H Ar); 7.79 (1H, s, H Ar);
8.66 (1H, s, NH). 13C NMR spectrum, δ, ppm: 52.0 (C-2); 60.3 (C-3); 112.5 (C-6); 116.2 (C-9); 118.8 (CN);
120.0 (C-7); 122.7 (C-3,5 Ar); 123.5 (C-8); 128.3 (C Ar); 129.2 (C-5a); 132.8 (C Ar); 143.4 (C-9a); 147.4
(C Ar); 149.5 (C-10a); 151.2 (C-4). Found, m/z: 443.9443 [M+H]+. C17H12Br2N5. Calculated, m/z: 443.9459.
4-Amino-2-(3-hydroxyphenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1c) was
obtained analogously from malononitrile (0.0327 g, 0.495 mmol), 2-aminobenzimidazole (0.0664 g, 0.498
mmol), and 3-hydroxybenzaldehyde (0.0659 g, 0.540 mmol). Yield 0.0823 g (54%). White powder. Mp 234-235°C.
1H NMR spectrum, δ, ppm (J, Hz): 5.09 (1H, s, CH); 6.68 (2H, t, J = 7.56, H Ar); 6.78 (2H, s, NH2); 7.00 (1H, t, J
= 7.6, H Ar); 7.12 (2H, s, H Ar); 7.23 (1H, d, J = 7.7, H Ar); 7.63 (1H, d, J = 7.5, H Ar); 8.53 (1H, s, H Ar); 9.46
(1H, s, NH). 13C NMR spectrum, δ, ppm: 53.2 (C-2); 62.0 (C-3); 112.4 (C-6); 112.5 (C Ar), 114.7 (C Ar), 116.0
(C Ar), 116.4 (C-9); 119.2 (CN), 119.8 (C-7); 123.3 (C-8); 129.3 (C-5a), 129.6 (C Ar), 143.6 (C-9a), 144.5
(C Ar), 148.9 (C-4); 151.7 (C-10a); 157.5 (C–OH). Found, m/z: 304.1202 [M+H]+. C17H14N5O. Calculated, m/z:
304.1198.
4-Amino-2-(4-hydroxyphenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1d) was
obtained analogously from malononitrile (0.0336 g, 0.508 mmol), 2-aminobenzimidazole (0.0658 g,
0.494 mmol), and 4-hydroxybenzaldehyde (0.0658 g, 0.516 mmol). Yield 0.0847g (57%). White powder.
Mp 234-235°C. 1H NMR spectrum, δ, ppm (J, Hz): 5.08 (1H, s, CH); 6.71–6.73 (3H, m, NH2, H Ar); 6.99 (1H,
t, J = 7.8, H Ar); 7.06-7.12 (3H, m, H Ar); 7.21 (1H, d, J = 7.7, H Ar); 7.62 (1H, d, J = 8.1, H Ar); 8.43 (1H, s,
13
H Ar); 9.42 (1H, s, NH). C NMR spectrum, δ, ppm: 52.9 (C-2); 62.6 (C-3); 115.0 (C-6); 115.3 (C Ar); 115.9
(C-9); 119.2 (CN); 119.7 (C-7); 123.2 (C-8); 127.3 (C Ar); 129.3 (C-5a); 133.1 (C Ar); 143.6 (C-9a); 148.9
(C-4); 151.7 (C-10a); 157.1 (C Ar). Found, m/z: 304.1202 [M+H]+. C17H14N5O. Calculated, m/z: 304.1198.
4-Amino-2-(3,4,5-trimethoxyphenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1e) was
obtained analogously from malononitrile (0.0331 g, 0.501 mmol), 2-aminobenzimidazole (0.0661 g, 0.496
mmol), and 3,4,5-trimethoxybenzaldehyde (0.0962 g, 0.490 mmol). Yield 0.1887 g (56%). White powder. Mp
215-216°C. 1H NMR spectrum, δ, ppm (J, Hz): 3.70 (9H, s, 3CH3); 5.18 (1H, s, CH); 6.63 (2H, s, H Ar); 6.84 (2H, s,
NH2); 7.00 (1H, t, J = 7.8, H Ar); 7.11 (1H, t, J = 7.6, H Ar); 7.23 (1H, d, J = 7.2, H Ar); 7.64 (1H, d, J = 8.0, H Ar);
13
8.50 (1H, s, NH). C NMR spectrum, δ, ppm: 53.2 (C-2); 55.8 (OCH3); 59.9 (OCH3); 61.7 (C-3); 103.6 (C Ar);
190

112.2 (C-6); 116.0 (C-9); 119.0 (CN); 119.8 (C-7); 123.2 (C-8); 129.2 (C-5a); 137.1 (C Ar); 138.0 (C Ar); 143.5
(C-9a); 149.1 (C-4); 151.6 (C-10a); 152.8 (C Ar). Found, m/z: 378.1560 [M+H]+. C20H20N5O3. Calculated, m/z:
378.1566.
4-Amino-2-(4-fluorophenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1f) was
obtained analogously from malononitrile (0.0332 g, 0.502 mmol), 2-aminobenzimidazole (0.0669 g,
0.502 mmol), and 4-fluorobenzaldehyde (0.0654 g, 0.526 mmol). Yield 0.0919 g (60%). White powder.
Mp 233-235°C (mp 232°C [22]). The 1H NMR spectrum corresponds to the published one [22].
4-Amino-2-(4-pyridinyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1g) was obtained
analogously from malononitrile (0.0334 g, 0.506 mmol), 2-aminobenzimidazole (0.0669 g, 0.502 mmol), and
4-pyridinecarbaldehyde (0.0577 g, 0.539 mmol). Yield 0.0885 g (61%). Tan powder. Mp 232-235°C (mp 236°C
(decomp.) [23]). The 1H NMR spectrum corresponds to the published one [23].
4-Amino-2-(1,3-benzodioxolan-5-yl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1h) was
obtained analogously from malononitrile (0.0331 g, 0.501 mmol), 2-aminobenzimidazole (0.0666 g,
0.500 mmol), and piperonal (0.0752 g, 0.501 mmol). Yield 0.0698 g (42%). Yellow powder. Mp 266-267°C.
1H NMR spectrum, δ, ppm (J, Hz): 5.29 (1H, s, CH); 5.94 (2H, s, CH2); 6.66 (1H, d, J = 7.2, H Ar); 6.79-6.89
(3H, m, NH2, H Ar); 7.01 (2H, t, J = 7.3, H Ar); 7.13 (2H, t, J = 7.7, H Ar); 7.23 (1H, d, J = 7.7, H Ar); 8.44
(1H, s, NH). 13C NMR spectrum, δ, ppm: 49.1 (C-2); 60.7 (C-3); 101.0 (CH2); 108.1 (C Ar); 112.3 (C-6); 116.0
(C-9); 118.8 (CN); 119.6 (C Ar); 119.8 (C-7); 121.8 (C Ar); 123.2 (C-8); 124.2 (C Ar); 129.3 (C-5a); 143.6
(C-9a); 144.2 (C Ar); 147.3 (C Ar); 149.3 (C-4); 151.7 (C-10a). Found, m/z: 332.1145 [M+H]+. C18H12N5O2.
Calculated, m/z: 332.1147.
4-Amino-2-(1-methyl-1H-pyrazol-4-yl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile
(1i) was obtained analogously from malononitrile (0.0333 g, 0.504 mmol), 2-aminobenzimidazole (0.0667 g,
0.501 mmol), and 1-methyl-1H-pyrazole-4-carbaldehyde (0.0552 g, 0.501 mmol). Yield 0.0440 g (30%). White
1
powder. Mp 278-280°C. H NMR spectrum, δ, ppm (J, Hz): 3.71 (3H, s, CH3); 5.08 (1H, s, CH); 6.74 (2H, s,
NH2); 6.95 (1H, t, J = 7.6, H Ar); 7.07 (1H, t, J = 7.6, H Ar); 7.17 (1H, d, J = 7.0, H Ar); 7.24 (1H, s, H Ar);
7.50 (1H, s, H Ar); 7.57 (1H, d, J = 7.0, H Ar); 8.30 (1H, s, NH). 13C NMR spectrum, δ, ppm: 45.9 (C-2); 62.7
(C-3); 112.9 (C-6); 116.5 (C-9); 119.5 (CN); 120.3 (C-7); 123.7 (C-8); 123.9 (C Ar); 128.9 (C Ar); 129.8
(C-5a); 136.9 (C Ar); 144.0 (C-9a); 149.7 (C-4); 152.4 (C-10a). Found, m/z: 292.1314 [M+H]+. C15H14N7.
Calculated, m/z: 292.1311.
4-Amino-2-(4-bromothiophen-2-yl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1j) was
obtained analogously from malononitrile (0.0327 g, 0.495 mmol), 2-aminobenzimidazole (0.0668 g,
0.502 mmol), and 4-bromo-2-thiophenecarbaldehyde (0.0957 g, 0.501 mmol). Yield 0.0499 g (27%). Brown
1
powder. Mp 349-350°C. H NMR spectrum, δ, ppm (J, Hz): 5.47 (1H, s, CH); 6.97-7.01 (5H, m, NH2, H Ar);
7.08 (1H, t, J = 7.6, H Ar); 7.21 (1H, d, J = 7.2, H Ar); 7.60 (1H, d, J = 8.0, H Ar); 8.70 (1H, s, NH). 13C NMR
spectrum, δ, ppm: 49.3 (C-2); 61.7 (C-3); 108.6 (C-Br); 113.0 (C-6); 116.8 (C-9); 119.1 (C Ar); 120.7 (CN);
123.7 (C-7); 124.0 (C-8); 127.3 (C Ar); 129.6 (C-5a); 143.8 (C Ar); 149.8 (C-9a); 150.1 (C-4); 151.5 (C-10a).
Found, m/z: 371.9910 [M+H]+. C15H10BrN5S. Calculated, m/z: 371.9918.
4-Amino-2-(4-chlorophenyl)-1,2-dihydropyrimido[1,2-a]benzimidazole-3-carbonitrile (1k) was
obtained analogously from malononitrile (0.0367 g, 0.50 mmol), 2-aminobenzimidazole (0.0708 g, 0.50 mmol),
and 4-chlorobenzaldehyde (0.0708 g, 0.50 mmol). Yield 0.1080 g (63%). White powder. Mp 239-240°C (mp
238°C [21]). The 1H NMR spectrum corresponds to the published one [20].
4-Amino-2-(phenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2a). Compound 1a (0.0150 g,
0.053 mmol) was treated with DDQ (0.027 g, 0.122 mmol) in acetonitrile (2 ml) under nitrogen atmosphere at
55°C for 4 h. The product was then filtered, and the precipitate was washed with EtOH. Yield 0.0062 g (41%).
1
Yellow powder. Mp >300°C (mp 291-293°C [23]). The H NMR spectrum corresponds to the published
one [24].
4-Amino-2-(3,5-dibromophenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2b) was prepared
analogously from compound 1b (0.0303 g, 0.068 mmol) and DDQ (0.0543 g, 0.239 mmol). Yield 0.0265 g
191

(87%). White powder. Mp >300°C. 1H NMR spectrum, δ, ppm (J, Hz): 7.44 (1H, t, J = 7.5, H Ar); 7.59 (1H, t,
J = 7.0, H Ar); 7.79 (1H, d, J = 7.6, H Ar); 8.06 (2H, s, H Ar); 8.11 (1H, s, H Ar); 8.62 (1H, d, J = 7.2, H Ar).
13C NMR spectrum, δ, ppm: 94.6 (C-3); 115.6 (CN); 116.7 (C-6, C Ar); 122.6 (C-9); 126.1 (C-7); 126.5 (C-8);
127.1 (C-5a); 131.0 (C Ar); 135.7 (C Ar); 141.0 (C-9a); 150.4 (C-10a); 154.8 (C-2); 161.4 (C-4). Found, m/z:
441.9310 [M+H]+. C17H10Br2N5. Calculated, m/z: 441.9303.
4-Amino-2-(3-hydroxyphenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2c) was prepared
analogously from compound 1c (0.1088 g, 0.359 mmol) and DDQ (0.1112 g, 0.490 mmol). Yield 0.0853 g
(78%). Green powder. Mp 262-264°C. 1H NMR spectrum, δ, ppm (J, Hz): 6.96 (1H, d, J = 7.6, H Ar); 7.28-7.40
(5H, m, H Ar, NH2); 7.54 (1H, t, J = 7.6, H Ar); 7.75 (1H, d, J = 7.3, H Ar); 8.58 (1H, d, J = 7.8, H Ar); 8.57
13
(1H, s, H Ar). C NMR spectrum, δ, ppm: 109.4 (C-3); 115.0 (C Ar); 115.4 (C-6); 116.9 (C Ar); 117.5 (CN);
118.0 (C-9); 119.3 (C Ar); 121.5 (C-7,8); 126.0 (C Ar); 126.5 (C Ar); 129.3 (C-5a); 138.4 (C-9a); 150.6
(C-10a); 154.6 (C-OH); 157.1 (C-2); 163.6 (C-4). Found, m/z: 302.1037 [M+H]+. C17H12N5O. Calculated, m/z:
302.1042.
4-Amino-2-(4-hydroxyphenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2d) was prepared
analogously from compound 1d (0.0322 g, 0.106 mmol) and DDQ (0.0541 g, 0.238 mmol). Yield 0.0234 g
(73%). Green powder. Mp >300°C. 1H NMR spectrum, δ, ppm (J, Hz): 7.43-7.49 (5H, m, H Ar, NH2); 7.62 (1H, t,
J = 7.6, H Ar); 7.78 (1H, d, J = 7.9, H Ar); 7.97-8.01 (2H, m, H Ar); 8.64 (1H, d, J = 8.4, H Ar). 13C NMR
spectrum, δ, ppm: 76.8 (C-3); 115.2 (C-6); 115.7 (CN); 116.2 (C Ar); 116.7 (C-9); 122.4 (C-7); 125.8 (C-8); 126.9
(C Ar); 131.2 (C Ar); 131.4 (C-5a); 133.0 (C-9a); 154.4 (C-10a); 162.4 (C-OH); 163.7 (C-2); 164.9 (C-4). Found,
m/z: 302.1045 [M+H]+. C17H12N5O. Calculated, m/z: 302.1042.
4-Amino-2-(3,4,5-trimethoxyphenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2e) was prepared
analogously from compound 1e (0.0303 g, 0.080 mmol) and DDQ (0.0543 g, 0.238 mmol). Yield 0.0265 g (87%).
White powder. Mp >300°C. 1H NMR spectrum, δ, ppm (J, Hz): 3.78 (3H, s, CH3); 3.86 (6H, s, 2CH3); 7.29 (4H, m,
H Ar, NH2); 7.51 (1H, t, J = 7.3, H Ar); 7.66 (1H, t, J = 7.3, H Ar); 7.76 (1H, d, J = 7.2, H Ar); 8.66 (1H, d, J = 7.6,
H Ar). 13C NMR spectrum, δ, ppm: 56.7 (OCH3); 60.8 (OCH3); 71.2 (C-3); 107.2 (C Ar); 116.0 (C-6); 116.4 (CN);
119.2 (C-9); 123.5 (C-7); 123.7 (C-8); 128.1 (C-5a); 131.4 (C Ar); 137.2 (C-9a); 140.8 (C-10a); 153.1(C Ar); 154.9
(C-2); 163.1 (C-4). Found, m/z: 376.1397 [M+H]+. C20H18N5O3. Calculated, m/z: 376.1398.
4-Amino-2-(4-fluorophenyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2f) was prepared
analogously from compound 1f (0.0307 g, 0.101 mmol) and DDQ (0.0542 g, 0.239 mmol). Yield 0.0289 g
1
(94%). White powder. Mp 235-236°C (mp 235-237°C [23]). The H NMR spectrum corresponds to the
published one [24].
4-Amino-2-(4-pyridinyl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2g) was prepared analogously
from compound 1g (0.0332 g, 0.185 mmol) and DDQ (0.0793 g, 0.349 mmol). Yield 0.0320 g (98%). Brown
1
powder. Mp 271-272°C. H NMR spectrum, δ, ppm (J, Hz): 7.41 (1H, t, J = 7.8, H Ar); 7.56 (1H, t, J = 7.4,
H Ar); 7.76 (1H, d, J = 7.8, H Ar); 7.83 (2H, d, J = 5.8, H Ar); 8.60 (1H, d, J = 7.9, H Ar); 8.80 (2H, d, J = 5.2,
H Ar). 13C NMR spectrum, δ, ppm: 102.1 (C-3); 114.2 (C-6); 115.7 (CN); 116.4 (C-8); 117.9 (C-9); 122.9
(C Ar); 123.6 (C-7); 127.3 (C Ar); 129.7 (C-5a); 145.2 (C-9a); 150.0 (C Ar); 151.3 (C-10a); 154.9 (C-2); 162.5
(C-4). Found, m/z: 287.1045 [M+H]+. C16H11N6. Calcu lated, m/z: 287.1045.
4-Amino-2-(1,3-benzodioxolan-5-yl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2h) was prepared
analogously from compound 1h (0.0309 g, 0.064 mmol) and DDQ (0.0522 g, 0.230 mmol). Yield 0.0197 g
1
(64%). Green powder. Mp 235-236°C. H NMR spectrum, δ, ppm (J, Hz): 6.18 (2H, s, CH2); 7.14 (1H, d,
J = 7.8, H Ar); 7.20 (2H, d, J = 7.9, H Ar); 7.60 (2H, t, J = 7.4, H Ar); 7.76 (1H, d, J = 7.8, H Ar); 8.37 (2H, s,
NH2); 8.61 (1H, d, J = 7.7, H Ar). 13C NMR spectrum, δ, ppm: 78.0 (C-3); 102.4 (CH2); 103.4 (C-6); 108.2
(C Ar); 109.2 (C Ar); 109.7 (C Ar); 114.3 (C Ar); 115.1 (CN); 124.3 (C-9a); 126.1 (C Ar); 130.3 (C-5a); 147.2
(C Ar); 148.8 (C-10a); 150.1 (C Ar); 153.5 (C-2); 161.0 (C-4). Found, m/z: 330.0911 [M+H]+. C18H12N5O2.
Calculated, m/z: 330.0911.
4-Amino-2-(1-methyl-1H-pyrazol-4-yl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2i) was prepared
analogously from compound 1i (0.0273 g, 0.094 mmol) and DDQ (0.0547 g, 0.241 mmol). Yield 0.0265 g
192

1
(97%). Olive green powder. Mp >400°C. H NMR spectrum, δ, ppm (J, Hz): 3.97 (3H, s, CH3); 7.48 (1H, t,
J = 7.8, H Ar); 7.62 (1H, t, J = 7.6, H Ar); 7.68 (1H, d, J = 7.8, H Ar); 8.25 (1H, s, H Ar); 8.60–8.61 (2H, m,
H Ar). 13C NMR spectrum, δ, ppm: 71.3 (C-3); 102.0 (C-6); 114.2 (C-6); 115.7 (C Ar); 116.8 (CN); 118.4
(C-9); 119.4 (C-7); 123.1 (C-8); 126.1 (C Ar); 127.8 (C-5a); 129.7 (C Ar); 133.5 (C-9a); 140.0 (C-10a); 151.3
(C-2); 154.8 (C-4). Found, m/z: 290.1157 [M+H]+. C15H12N7. Calculated, m/z: 290.1154.
4-Amino-2-(4-bromothiophen-2-yl)pyrimido[1,2-a]benzimidazole-3-carbonitrile (2j) was prepared
analogously from compound 1j (0.0110 g, 0.030 mmol) and DDQ (0.0205 g, 0.090 mmol). Yield 0.0091 g
1
(84%). Orange powder. Mp 363-364°C. H NMR spectrum, δ, ppm (J, Hz): 7.37 (1H, t, J = 7.8, H Ar); 7.52
(1H, t, J = 7.3, H Ar); 7.69 (1H, d, J = 7.8, H Ar); 8.05 (1H, s, H Ar); 8.07 (1H, s, H Ar); 8.55 (1H, d, J = 7.8,
H Ar). 13C NMR spectrum, δ, ppm: 109.9 (C-3, C-Br); 115.5 (CN); 117.2 (C-6); 122.5 (C-9); 126.9 (C-7, C Ar);
127.0 (C-8, C Ar); 130.7 (C-5a); 131.8 (C-9a); 143.0 (C Ar); 150.1 (C-10a); 154.2 (C-2); 155.0 (C-4). Found,
m/z: 369.9761 [M+H]+. C15H9BrN5S. Calculated, m/z: 369.9762.
Cell Culture. Human pancreatic and breast cancer cell lines PANC-1 (ATCC® CRL-1469TM), MCF-7
(ATCC® HTB-22TM), and Hs578T (ATCC® HTB-126TM) were obtained from the ATCC®. All cell lines were
maintained on a tissue culture plastic substrate in a mixture of Dulbecco's modified Eagle's medium (DMEM)
supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Life Techno-
logies, NY, USA), at 37°C in a humidified atmosphere containing 10% CO2.
Determination of in vitro Cytotoxicity Using the MTT Colorimetric Assay. The antiproliferative
effect of a library of compounds was tested in accordance with Romijn et al. [25]. Briefly, mitochondrial
dehydrogenase activities were measured by MTT reagent (Sigma, MO, USA). The cells were seeded in 96-well
plates at a density of 1.5×104 cells in 100 μl of DMEM medium and initially treated with concentrations of
compounds 1a-k and 2a-j of 50 μM in 100 μl of medium. After 48 h incubation, 100 μl medium was removed
prior to the addition of MTT reagent (5 mg/ml). Subsequently, IC50 values were determined for those
compounds exhibiting strong inhibitory effects at 50 μM, using concentrations ranging from 0.1 to 50 M.
Three or four independent experiments were carried out in which four to eight wells were used for each
concentration to determine the mean optical density, using an Epoch microplate spectrophotometer controlled
with Gen5 Data Analysis software (Biotek, VT, USA).
This work was supported by National Institute of General Medical Sciences (P20GM103451) and
federal and private funding, including STEM-TRAC, USDA, AREA programs, Florida Blue and Domingo
Moreira.
Also the authors wish to thank Prof. Alexander Kornienko, Department of Chemistry and Biochemistry,
Texas State University, and Mass Spectrometry Facility, University of New Mexico for mass spectra.
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