Synthesis, crystal structure determination and antiproliferative activity of novel 2-amino-4-aryl-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazoles

Article abstract of DOI:10.1016/j.molstruc.2011.10.054

This manuscript describes the synthesis of novel 2-amino-4-aryl-4,10- dihydro-[1,3,5]triazino[1,2-a]benzimidazoles as hydrochloride salts 4a-n and 5b which were prepared in the reaction of cyclocondensation between 2-guanidinobenzimidazole and versatile h

Full text of DOI:10.1016/j.molstruc.2011.10.054

Journal of Molecular Structure 1007 (2012) 242–251
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, crystal structure determination and antiproliferative activity of novel
2-amino-4-aryl-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazoles
b
a,
Marijana Hranjec a, , Gordana Pavlovic , Grace Karminski-Zamola
⇑
⇑
´
a Faculty of Chemical Engineering and Technology, Department of Organic Chemistry, University of Zagreb, Maruli´cev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
b Faculty of Textile Technology, Department of Applied Chemistry, University of Zagreb, Prilaz Baruna Filipovi´ca 28a, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
This manuscript describes the synthesis of novel 2-amino-4-aryl-4,10-dihydro-[1,3,5]triazino[1,2-
a]benzimidazoles as hydrochloride salts 4a-n and 5b which were prepared in the reaction of cyclocon-
densation between 2-guanidinobenzimidazole and versatile heteroaromatic aldehydes. Structures of all
prepared compounds have been studied by using 1H and 13C NMR, IR and UV/Vis spectroscopy.
The crystal and molecular structure of 4f was determined by X-ray diffraction on single crystals. The
molecule of 2-amino-4-(40-methylphenyl)-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazole hydrochlo-
Received 15 September 2011
Received in revised form 25 October 2011
Accepted 30 October 2011
Available online 7 November 2011
Keywords:
Benzimidazoles
ride 4f (C16H16N5 ꢀClꢁ) exists in the solid state in one of the possible tautomeric forms, being protonated
+
at the one of the nitrogen atoms of the 1,4-dihydrotriazine ring. The molecule is highly delocalized within
the 4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazole moiety with the highest deviation from the plane
for the methine carbon atom and the protonated nitrogen atom of the 1,4-dihydrotriazine ring. The cat-
ions are joined via N–Hꢀ ꢀ ꢀN hydrogen bonds into R22(8) centrosymmetric dimers. Cation dimers are fur-
ther connected with Clꢁ ions via N–Hꢀ ꢀ ꢀCl and C–Hꢀ ꢀ ꢀCl hydrogen bonds into 2D chains spreading
along the b axis. The obtained single-crystal X-ray structure determination unequivocally confirms tau-
tomeric form of the compound present in the solid-state and can represent tantative pattern for other
prepared compounds.
Guanidines
Single-crystal X-ray structure
UV/Vis spectroscopy
Antiproliferative activity in vitro
All prepared compounds were tested on their antiproliferative activity in vitro on several human cancer
cell lines. Compound 4m was the most active one (IC50 ꢂ 20
l
M), while compounds 4d, 4f, 4k, 4l 4m
M.
showed moderate, but non-selective, antiproliferative activity with IC50 25–60
l
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
antiproliferative effects and pronounced selectivity toward tumor
cells in regard to normal cells. We also showed that some
positively charged analogs of benzimidazo[1,2-a]quinolines and
diaza-cyclopenta[c]fluorenes intercalate into ds DNA or RNA,
which might account for their accentuated antiproliferative effect.
On the other hand, guanidine functionalities are found in a large
number of synthetic and naturally occurring compounds such as
sponges, algae or different microorganisms [14]. Their synthesis
has attracted much attention from organic and medicinal chemists
and many synthetic methods were developed [15]. Guanidine group
can be found either as a terminal group of substituents or in the form
of five- or six-membered heterocycles. Their derivatives posses the
diversity of the chemical, physicochemical and biological properties
[16–19]. Guanidines are also efficient ligands for the synthesis of
various organometallic catalysts with a various metal ions [20,21].
Triazino[1,2-a]benzimidazole derivatives which posses a guanidine
group as a part of six-membered ring, have a wide range of biological
effects including antibacterial and antifungal activity and dihydro-
folate reductase (DHFR) inhibitory activities [22,23].
Benzimidazoles represent the key building block for a variety of
numerous medical and biochemical agents possessing different
chemical and pharmacological features. A large number of benzim-
idazoles derivatives play also crucial roles in the functions of a
number of biologically important molecules. In addition, benz-
imidazole derivatives posses diverse biological properties like anti-
cancer [1–3], antiviral [4,5], antibacterial [6,7], antifungal [8],
antihistaminic [9] or anticonvulsant activity [10]. We have recently
reported the synthesis and antiproliferative activity of versatile
heterocyclic benzimidazole derivatives as well as fused benzo-
annulated benzimidazole derivatives related to benzimidazo
[1,2-a]quinolines [11–13]. Thus, our previously published results
reveal that amidino substituted 2-styrylbenzimidazoles and their
cyclic analogs benzimidazo[1,2-a]quinolines showed significant
⇑
Corresponding authors. Tel.: +385 1 4597 245; fax: +385 1 4597 250 (M.
Hranjec), tel.: +385 1 4597 215; fax: +385 1 4597 224 (G. Karminski-Zamola).
E-mail addresses: mhranjec@fkit.hr (M. Hranjec), gzamola@fkit.hr (G. Karminski-
Zamola).
Above mentioned considerations prompted us to explore a
new series of different substituted heteroaromatic 2-amino-
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.10.054

M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
243
4-aryl-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazoles as hydro-
chloride salts. The hydrochloride salts were prepared to improve
their solubility in water. Full details about the synthesis, single
crystal X-ray structure determination and evaluation of antiprolif-
erative activity on the panel of several human tumor cell lines
in vitro are reported herein.
with HCl(g) to gave 0.28 g (83%) of white powder; mp = 212–
213 °C; IR (KBr)
/cmꢁ1: 3467, 3298, 3220, 2964, 1659, 1625; 1H
t
NMR (600 MHz, DMSO-d6): 13.31 (s, 1H, NH), 9.36 (s, 1H, Harom.),
7.95 (d, 2H, J = 8.13 Hz, Harom.), 7.72 (d, 2H, J = 8.19 Hz, Harom.),
7.39 (d, 1H, J = 7.83 Hz, Harom.), 7.26 (t, 1H, J = 7.64 Hz, Harom.),
7.16 (d, 1H, J = 7.92 Hz, Harom.), 7.12 (s, 1H, Harom.), 6.99 (d, 1H,
J = 7.89 Hz, Harom.); 13C NMR (75 MHz, DMSO-d6): 157.0 (s), 151.1
(s), 143.2 (s), 133.8 (d, 2C), 129.8 (s), 128.3 (d, 2C), 128.0 (s),
124.6 (d), 123.6 (d), 118.6 (s), 113.3 (s), 112.3 (d), 110.8 (d), 65.4
(d); Anal. Calcd. for C15H13ClFN5: C, 56.70; H, 4.12; N, 22.04. Found:
C, 56.49; H, 4.08; N, 21.88.
2. Experimental section
2.1. Synthesis
2.1.1. Instrumental methods of detection
2.1.2.3. 2-Amino-4-(40-cyanophenyl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole hydrochloride 4c. Compound 4c was prepared from
2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and 4-cyano-
benzaldehyde 2c (0.22 g, 1.70 mmol) in absolute ethanol (10 ml)
and piperidine (0.15 ml) after refluxing for 4 h and recrystallization
from ethanol to yield 0.31 g (64%) of white powder 3c which was
suspended in absolute ethanol (10 ml) and saturated with HCl(g)
All chemicals were purchased from commercial suppliers. Melt-
ing points were recorded on SMP11 Bibby apparatus. The 1H and
13C NMR spectra were recorded on a Varian Gemini 300 or Varian
Gemini 600 at 300, 600 and 150 and 75 MHz, respectively. NMR
spectra were measured in DMSO-d6 solutions using TMS as an
internal standard. IR spectra were recorded on a Bruker Vertex
70 spectrophotometer with diamond crystal. All compounds were
routinely checked by TLC with Merck silica gel 60F-254 glass
plates. Elemental analysis for carbon, hydrogen and nitrogen were
performed on a Perkin–Elmer 2400 elemental analyzer. Where
analyses are indicated only as symbols of elements, analytical re-
sults obtained are within 0.4% of the theoretical value. All com-
pounds were routinely checked by thin layer chromatography
(TLC) using precoated Merck silica gel 60F-254 plates and the spots
were detected under UV light (254 and 366 nm).
to gave 0.30 g (86%) of white powder; mp >290 °C; IR (KBr)
t/
cmꢁ1 3398, 3304, 3199, 2984, 2225, 1690, 1624; 1H NMR
:
(600 MHz, DMSO-d6): 13.29 (s, 1H, NH), 9.33 (s, 1H, Harom.), 8.01
(brs, 1H, NH+), 7.61 (d, 2H, J = 8.58 Hz, Harom.), 7.38 (t, 1H,
J = 7.78 Hz, Harom.), 7.31 (d, 2H, J = 8.52 Hz, Harom.), 7.13 (t, 2H,
J = 7.68 Hz, Harom.), 7.00 (s, 1H, Harom.), 6.90 (d, 1H, J = 7.98 Hz, Har-
om.); 13C NMR (75 MHz, DMSO-d6): 164.9 (s), 161.8 (s), 157.1 (s),
151.1 (s), 134.8 (s), 134.1 (s), 129.8 (s), 129.7 (d), 129.6 (d), 128.1
(s), 124.4 (d), 123.5 (d), 116.9 (d), 116.5 (d), 112.6 (d), 110.9 (d),
65.6 (d); Anal. Calcd. for C16H13ClN6: C, 59.17; H, 4.03; N, 25.88.
Found: C, 59.05; H, 4.12; N, 26.02.
2.1.2. General method for preparation of compounds 4a-n
Solution of equimolar amounts of 2-guanidinobenzimidazole 1,
corresponding heteroaromatic aldehydes 2a-n and few drops of
piperidine in absolute ethanol, were refluxed for 2–4 h. After reac-
tion mixture was cooled to the room temperature, the crude prod-
uct was filtered off and recrystallized from ethanol to obtain
powder products 3a-n. A stirred suspension of compounds 3a-n
in absolute ethanol (15 ml) was cooled in an ice-salt bath and sat-
urated with HCl(g). After 24 h of stirring small amount of diethyl-
ether was added, resulting product was filtered off and washed
with diethylether to obtain hydrochlorides 4a-n.
2.1.2.4. 2-Amino-4-(40-N,N-dimethylaminophenyl)-4,10-dihydro [1,3,5]-
triazino[1,2-a]benzimidazole hydrochloride 4d. Compound 4d was
prepared from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol)
and 4-N,N-dimethylaminobenzaldehyde 2d (0.26 g, 1.70 mmol) in
absolute ethanol (10 ml) and piperidine (0.15 ml) after refluxing
for 2 h and recrystallization from ethanol to yield 0.33 g (63%) of
yellow powder 3d which was suspended in absolute ethanol
(10 ml) and saturated with HCl(g) to gave 0.32 g (86%) of orange
powder; mp = 263–264 °C; IR (KBr)
t
/cmꢁ1: 3409, 3312, 3199,
2944, 1689, 1641; 1H NMR (300 MHz, DMSO-d6): 13.30 (s, 1H,
NH), 9.33 (s, 1H, Harom.), 8.00 (brs, 1H, NH+), 7.42 (d, 2H,
J = 8.46 Hz, Harom.), 7.36 (d, 1H, J = 7.92 Hz, Harom.), 7.22 (t, 1H,
J = 7.62 Hz, Harom.), 7.11 (t, 1H, J = 7.62 Hz, Harom.), 7.02 (brs, 2H,
2.1.2.1.
2-Amino-4-phenyl-4,10-dihydro[1,3,5]triazino[1,2-a]benz-
imidazole hydrochloride 4a. Compound 4a was prepared from 2-
guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and benzaldehyde
2a (0.18 g, 1.70 mmol) in absolute ethanol (10 ml) and piperidine
(0.15 ml) after refluxing for 2 h and recrystallization from ethanol
to yield 0.31 g (70%) of white powder 3a which was suspended in
absolute ethanol (15 ml) and saturated with HCl(g) to gave 0.32 g
H
arom.), 6.89 (d, 2H, J = 8.42 Hz, Harom.), 5.42 (brs, 3H, NHþ), 2.93
3
(s, 6H, CH3); 13C NMR (75 MHz, DMSO-d6): 156.8 (s), 150.7 (s),
129.3 (s), 128.0 (d, 2C), 127.8 (s), 126.4 (s), 124.8 (d, 2C), 123.8
(d), 122.9 (d), 121.4 (s), 111.6 (d), 110.6 (d), 65.7 (d), 41.6 (q,
2C); Anal. Calcd. for C17H19ClN6: C, 59.56; H, 5.59; N, 24.51. Found:
C, 59.82; H, 5.70; N, 24.61.
(90%) of white powder; mp >290 °C; IR (KBr) t
/cmꢁ1: 3420, 3310,
3209, 2980, 1675, 1635; 1H NMR (600 MHz, DMSO-d6): 13.30
(s, 1H, NH), 9.35 (s, 1H, Harom.), 8.03 (brs, 1H, NH+), 7.52 (brs, 2H,
H
arom.), 7.48–7.44 (m, 3H, Harom.), 7.38 (d, 1H, J = 7.85 Hz, Harom.),
7.23 (t, 1H, J = 7.68 Hz, Harom.), 7.12 (1H, J = 7.80 Hz, Harom.), 6.99
(s, 1H,
arom.), 6.92 (d, 1H, J = 7.98 Hz, Harom.); 13C NMR
2.1.2.5. 2-Amino-4-(N-methylpyrol-2-yl)-4,10-dihydro[1,3,5]triazino
[1,2-a]benzimidazole hydrochloride 4e. Compound 4e was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and N-
methyl-pyrole-2-carboxaldehyde 2e (0.19 g, 1.70 mmol) in abso-
lute ethanol (10 ml) and piperidine (0.15 ml) after refluxing for
3 h and recrystallization from ethanol to yield 0.22 g (50%) of white
powder 3e which was suspended in absolute ethanol (10 ml) and
saturated with HCl(g) to gave 0.20 g (80%) of yellow powder;
H
(150 MHz, DMSO-d6): 157.2 (s), 151.2 (s), 138.4 (d), 130.6 (d),
129.8 (d, 2C), 128.2 (s), 127.3 (d, 2C), 126.4 (s), 124.4 (d), 123.4
(d), 112.2 (d), 110.9 (d), 66.3 (d); Anal. Calcd. for C15H14ClN5: C,
60.10; H, 4.71; N, 23.36. Found: C, 59.96; H, 5.00; N, 23.60.
2.1.2.2. 2-Amino-4-(40-fluorophenyl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole hydrochloride 4b. Compound 4b was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and 4-fluo-
robenzaldehyde 2b (0.21 g, 1.70 mmol) in absolute ethanol
(10 ml) and piperidine (0.15 ml) after refluxing for 3 h and recrys-
tallization from ethanol to yield 0.30 g (63%) of white powder 3b
which was suspended in absolute ethanol (10 ml) and saturated
mp = 289–290 °C; IR (KBr) t
/cmꢁ1: 3395, 3291, 3203, 2963, 1666,
1629; 1H NMR (600 MHz, DMSO-d6): 13.26 (s, 1H, NH), 9.30 (s,
1H, Harom.), 8.04 (brs, 1H, NH+), 7.39 (d, 1H, J = 7.86 Hz, Harom.),
7.25 (t, 1H, J = 7.65 Hz, Harom.), 7.13 (t, 1H, J = 7.68 Hz, Harom.),
7.04 (s, 1H, Harom.), 6.91 (d, 2H, J = 7.98 Hz, Harom.), 6.26 (t, 2H,
J = 3.24 Hz, HPy), 6.04 (t, 2H, J = 3.24 Hz, HPy), 3.56 (s, 3H, CH3);
13C NMR (150 MHz, DMSO-d6): 157.6 (s), 151.6 (s), 129.8 (s),

244
M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
128.5 (s), 126.7 (d), 126.1 (s), 125.9 (s), 124.4 (d), 123.5 (d), 112.2
(d), 111.7 (d), 110.8 (d), 107.4 (d), 60.8 (d), 34.5 (q); Anal. Calcd. for
carboxaldehyde 2i (0.16 g, 1.70 mmol) in absolute ethanol
(10 ml) and piperidine (0.15 ml) after refluxing for 3 h and recrys-
tallization from ethanol to yield 0.25 g (56%) of white powder 3i
which was suspended in absolute ethanol (10 ml) and saturated
with HCl(g) to gave 0.23 g (82%) of white powder; mp >290 °C; IR
C14H15ClN6: C, 55.54; H, 4.99; N, 27.76. Found: C, 55.72; H, 5.12; N,
27.62.
2.1.2.6.
2-Amino-4-(40-methylphenyl)-4,10-dihydro[1,3,5]triazino
(KBr)
(300 MHz, DMSO-d6): 13.42 (s, 1H, NH), 9.64 (d, 1H, J = 1.44 Hz,
arom.), 8.77 (d, 2H, J = 5.97 Hz, HPy), 8.17 (brs, 1H, NH+), 7.71 (d,
t
/cmꢁ1: 3398, 3314, 3202, 2986, 1664, 1650; 1H NMR
[1,2-a]benzimidazole hydrochloride 4f. Compound 4f was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and 4-
methylbenzaldehyde 2f (0.20 g, 1.70 mmol) in absolute ethanol
(10 ml) and piperidine (0.15 ml) after refluxing for 3 h and recrys-
tallization from ethanol to yield 0.34 g (72%) of white powder 3f
which was suspended in absolute ethanol (10 ml) and saturated
with HCl(g) to gave 0.26 g (68%) of white powder; mp >290 °C; IR
H
2H, J = 5.94 Hz, HPy), 7.39 (d, 1H, J = 7.80 Hz, Harom.), 7.26 (t, 1H,
J = 7.24 Hz, Harom.), 7.19–7.09 (m, 2H, Harom.), 7.08 (s, 1H, Harom.);
13C NMR (75 MHz, DMSO-d6): 157.0 (s), 152.8 (s), 151.0 (s), 145.8
(d, 2C), 129.8 (s), 127.9 (s), 124.8 (d), 124.3 (d, 2C), 123.7 (d),
112.5 (d), 110.9 (d), 64.3 (d); Anal. Calcd. for C14H13ClN6: C,
55.91; H, 4.36; N, 27.94. Found: C, 55.72; H, 4.25; N, 27.71.
(KBr)
t
/cmꢁ1: 3412, 3324, 3221, 3004, 1698, 1638; 1H NMR
(600 MHz, DMSO-d6): 13.18 (s, 1H, NH), 9.17 (s, 1H, Harom.), 7.94
(brs, 1H, NH+), 7.38 (d, 2H, J = 8.01 Hz, Harom.), 7.34 (d, 1H,
J = 7.98 Hz, Harom.), 7.24 (d, 2H, J = 7.92 Hz, Harom.), 7.21 (d, 1H,
J = 7.78 Hz, Harom.), 7.18 (d, 1H, J = 7.78 Hz, Harom.), 7.10 (t, 1H,
J = 7.72 Hz, Harom.), 6.89 (s, 1H, Harom.), 6.88 (d, 1H, J = 8.28 Hz,
2.1.2.10. 2-Amino-4-(thiophene-2-yl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole hydrochloride 4j. Compound 4j was prepared from
2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and thiophene-2-
carboxaldehyde 2j (0.19 g, 1.70 mmol) in absolute ethanol (10 ml)
and piperidine (0.15 ml) after refluxing for 3 h and recrystallization
from ethanol to yield 0.42 g (93%) of white powder 3j which was
suspended in absolute ethanol (10 ml) and saturated with HCl(g)
H
arom.), 2.26 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): 157.2 (s),
151.2 (s), 140.3 (s), 135.5 (s), 130.2 (d, 2C), 129.8 (s), 128.2 (s),
127.3 (d, 2C), 124.4 (d), 123.4 (d), 112.2 (d), 111.0 (d), 66.2 (d),
21.3 (q); Anal. Calcd. for C16H16ClN5: C, 61.24; H, 5.14; N, 22.32.
Found: C, 61.36; H, 5.28; N, 22.55.
to gave 0.43 g (90%) of white powder; mp >290 °C; IR (KBr)
t/
cmꢁ1: 3432, 3318, 3226, 2988, 1681, 1642; 1H NMR (300 MHz,
DMSO-d6): 13.24 (s, 1H, NH), 9.45 (s, 1H, Harom.), 8.11 (brs, 1H,
NH+), 7.61 (d, 1H, J = 4.95 Hz, Hthioph), 7.45 (d, 1H, J = 8.15 Hz,
2.1.2.7.
2-Amino-4-(furan-2-yl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole hydrochloride 4g. Compound 4g was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and fur-
ane-2-carboxaldehyde 2g (0.16 g, 1.70 mmol) in absolute ethanol
(10 ml) and piperidine (0.15 ml) after refluxing for 4 h and recrys-
tallization from ethanol to yield 0.24 g (57%) of light yellow pow-
der 3g which was suspended in absolute ethanol (10 ml) and
saturated with HCl(g) to gave 0.25 g (92%) of white powder;
H
H
arom.), 7.38 (d, 1H, J = 5.20 Hz, Hthioph), 7.35 (d, 1H, J = 8.10 Hz,
arom.), 7.26–7.16 (m, 2H, Harom.), 7.05 (t, 1H, J = 4.86 Hz, Harom.);
13C NMR (75 MHz, DMSO-d6): 157.1 (s, 2C), 150.7 (s), 141.8 (s),
129.7 (s), 129.1 (d), 128.8 (d), 127.9 (s), 127.6 (d), 124.6 (d), 123.6
(d), 112.3 (d), 111.1 (d), 61.9 (d); Anal. Calcd. for C13H12ClN5S: C,
51.06; H, 3.96; N, 22.90. Found: C, 50.89; H, 4.17; N, 22.70.
mp >290 °C; IR (KBr)
t
/cmꢁ1: 3411, 3323, 3198, 2956, 1684,
2.1.2.11.
2-Amino-4-(40-chlorophenyl)-4,10-dihydro[1,3,5]triazino
1642; 1H NMR (300 MHz, DMSO-d6):): 13.21 (s, 1H, NH), 9.27 (s,
1H, Harom.), 8.06 (brs, 1H, NH+), 7.67 (d, 1H, J = 1.80 Hz, Harom.),
7.35 (d, 1H, J = 7.36 Hz, Harom.), 7.30–7.18 (m, 2H, Harom.), 7.17 (s,
[1,2-a]benzimidazole hydrochloride 4k. Compound 4k was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and 4-chlo-
robenzaldehyde 2k (0.24 g, 1.70 mmol) in absolute ethanol (10 ml)
and piperidine (0.15 ml) after refluxing for 3 h and recrystallization
from ethanol to yield 0.35 g (70%) of white powder 3k which was
suspended in absolute ethanol (10 ml) and saturated with HCl(g) to
1H,
Harom.), 6.82 (d, 1H, J = 3.15 Hz, Harom.), 6.48 (dd, 1H,
J1 = 3.05 Hz, J2 = 1.78 Hz, Harom.); 13C NMR (75 MHz, DMSO-d6):
157.6 (s), 150.9 (s), 149.5 (s), 145.4 (d), 129.6 (s), 127.9 (s), 126.8
(s), 124.6 (d), 123.6 (d), 112.2 (d), 111.3 (d), 110.7 (d), 59.4 (d);
Anal. Calcd. for C13H12ClN5O: C, 53.89; H, 4.17; N, 24.17. Found:
C, 54.16; H, 4.25; N, 24.32.
gave 0.36 g (91%) of white powder; mp >290 °C; IR (KBr) t :
/cmꢁ1
3433, 3298, 3199, 2976, 1685, 1649; 1H NMR (600 MHz, DMSO-
d6): 13.26 (s, 1H, NH), 9.30 (s, 1H, Harom.), 7.70 (brs, 1H, NH+),
7.52 (s, 4H, Harom.), 7.38 (d, 1H, J = 7.83 Hz, Harom.), 7.25 (t, 1H,
J = 7.38 Hz, Harom.), 7.14 (t, 2H, J = 7.62 Hz, Harom.), 7.01 (s, 1H,
2.1.2.8. 2-Amino-4-(imidazole-2-yl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole dihydrochloride 4h. Compound 4h was prepared
from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and imidaz-
ole-2-carboxaldehyde 2h (0.16 g, 1.70 mmol) in absolute ethanol
(10 ml) and piperidine (0.15 ml) after refluxing for 3 h and recrys-
tallization from ethanol to yield 0.17 g (40%) of white powder 3h
which was suspended in absolute ethanol (10 ml) and saturated
with HCl(g) to gave 0.18 g (80%) of yellow powder; mp >290 °C;
H
arom.), 6.94 (d, 1H, J = 7.86 Hz, Harom.); 13C NMR (75 MHz,
DMSO-d6): 157.1 (s), 157.0 (s), 137.4 (s), 135.2 (s), 129.8 (d, 2C),
129.6 (s), 129.3 (d, 2C), 128.1 (s), 124.5 (d), 123.5 (d), 112.3 (d),
110.9 (d), 65.6 (s); Anal. Calcd. for C15H13Cl2N5: C, 53.91; H, 3.92;
N, 20.96. Found: C, 54.10; H, 4.11; N, 20.69.
2.1.2.12.
2-Amino-4-(20-chloro-50-nitrophenyl)-4,10-dihydro[1,3,5]
IR (KBr)
t
/cmꢁ1: 3413, 3356, 3245, 3029, 1698, 1621; 1H NMR
triazino[1,2-a]benzimidazole hydrochloride 4l. Compound 4l was
prepared from 2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol)
and 2-chloro-5-nitrobenzaldehyde 2l (0.32 g, 1.70 mmol) in abso-
lute ethanol (10 ml) and piperidine (0.15 ml) after refluxing for
3 h and recrystallization from ethanol to yield 0.34 g (59%) of yel-
low powder 3l which was suspended in absolute ethanol (10 ml)
and saturated with HCl(g) to gave 0.30 g (80%) of yellow powder;
(600 MHz, DMSO-d6): 13.20 (s, 1H, NH), 9.31 (s, 1H, Harom.), 8.64
(brs, 1H, Harom.), 8.18 (brs, 1H, NH+), 7.90 (s, 1H, Harom.), 7.36 (d,
1H, J = 7.72 Hz, Harom.), 7.23 (t, 1H, J = 7.28 Hz, Harom.), 7.14 (d,
1H, J = 7.36 Hz,
Harom.), 7.13 (s, 1H, Harom.), 6.95 (d, 1H,
J = 7.65 Hz, Harom.); 13C NMR (75 MHz, DMSO-d6): 157.0 (s), 150.9
(s), 147.7 (d), 130.6 (s), 130.5 (s), 129.8 (s), 128.1 (s), 124.5 (d),
123.5 (d), 120.1 (d), 112.1 (d), 110.5 (d), 59.1 (d); Anal. Calcd. for
mp = 256–258 °C; IR (KBr) t
/cmꢁ1: 3432, 3299, 3205, 2969, 1655,
C12H12ClN7: C, 49.75; H, 4.17; N, 33.84. Found: C, 49.98; H, 4.32;
1639; 1H NMR (600 MHz, DMSO-d6): 13.29 (s, 1H, NH), 8.83 (brs,
1H, NH+), 8.40 (s, 1H, Harom.), 8.29 (dd, 1H, J1 = 8.70 Hz,
J2 = 2.70 Hz, Harom.), 7.85 (d, 1H, J = 8.76 Hz, Harom.), 7.34 (d, 1H,
J = 7.92 Hz, Harom.), 7.20 (brs, 1H, Harom.), 7.13 (t, 1H, J = 7.62 Hz,
N, 33.60.
2.1.2.9.
2-Amino-4-(pyridine-4-yl)-4,10-dihydro[1,3,5]triazino[1,2-
a]benzimidazole hydrochloride 4i. Compound 4i was prepared from
2-guanidinobenzimidazole 1 (0.30 g, 1.70 mmol) and pyridine-2-
H
H
arom.), 6.97 (t, 1H, J = 7.74 Hz, Harom.), 6.72 (d, 1H, J = 7.98 Hz,
arom.); 13C NMR (150 MHz, DMSO-d6): 156.3 (s), 152.0 (s), 147.2

M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
245
(s), 139.1 (s), 136.5 (s), 133.0 (d), 128.9 (s), 126.8 (d), 126.1 (d),
123.7 (d), 122.4 (d), 113.7 (d), 109.9 (d), 65.4 (d); Anal. Calcd. for
(d); Anal. Calcd. for C15H13ClN6: C, 57.60; H, 4.19; N, 26.87. Found:
C, 57.81; H, 4.26; N, 26.70.
C
15H12Cl2N6O2: C, 47.51; H, 3.19; N, 22.16. Found: C, 47.70; H,
2.1.4. Preparation of 2-amino-4-(20-chloro-50-aminophenyl)-4,10-
dihydro[1,3,5]triazino [1,2-a]benzimidazole hydrochloride 5b
3.32; N, 22.25.
2.1.2.13. 2-Amino-4-(40-N,N-diethylamino-20-hydroxyphenyl)-4,10-
dihydro[1,3,5]triazino[1,2-a]benzimidazole hydrochloride 4m. Com-
A stirred suspension of compound 5a (0.05 g, 0.16 mmol) in
absolute ethanol (5 ml) was cooled in an ice-salt bath and satu-
rated with HCl(g). After 24 h of stirring small amount of diethyl-
ether was added, resulting product was filtered off and washed
with diethylether to gave 0.45 g (80%) of yellow powder; mp
pound 4m was prepared from 2-guanidinobenzimidazole
1
(0.30 g, 1.70 mmol) and 4-N,N-diethylamino-2-hydroxybenzalde-
hyde 2m (0.33 g, 1.70 mmol) in absolute ethanol (10 ml) and
piperidine (0.15 ml) after refluxing for 4 h and recrystallization
from ethanol to yield 0.32 g (54%) of light yellow powder 3m
which was suspended in absolute ethanol (10 ml) and saturated
with HCl(g) to gave 0.31 g (88%) of white powder; mp = 209–
264–265 °C; IR (KBr)
t
/cmꢁ1: 3452, 3328, 3253, 3119, 2989,
1652, 1644; 1H NMR (600 MHz, DMSO-d6): 13.30 (s, 1H, NH),
9.33 (s, 1H, Harom.), 8.00 (brs, 1H, NH+), 7.40 (d, 1H, J = 7.92 Hz,
Harom.), 7.31–7.24 (m, 2H, Harom.), 7.14 (d, 1H, J = 7.68 Hz, Harom.),
210 °C; IR (KBr) t
/cmꢁ1: 3395, 3312, 3215, 2965, 1663, 1631; 1H
NMR (600 MHz, DMSO-d6): 13.05 (s, 1H, NH), 8.92 (s, 1H, Harom.),
7.70 (brs, 1H, NH+), 7.34 (d, 2H, J = 7.74 Hz, Harom.), 7.21 (t, 1H,
J = 7.56 Hz, Harom.), 7.12 (t, 2H, J = 7.62 Hz, Harom.), 6.98 (s, 1H,
7.08 (s, 1H, Harom.), 6.91 (brs, 1H, Harom.), 6.85 (d, 1H, J = 7.60 Hz,
H
arom.), 6.75 (d, 1H, J = 8.10 Hz, Harom.), 4.00 (brs, 3H, NHþ); 13C
3
NMR (150 MHz, DMSO-d6): 155.5 (s), 154.0 (s), 149.1 (s), 145.0
(s), 144.1 (s), 137.2 (s), 131.7 (s), 130.6 (d), 121.3 (d), 119.4 (d),
117.1 (s), 116.8 (d), 116.5 (d), 112.8 (d), 108.1 (d), 64.1 (d); Anal.
Calcd. for C15H14Cl2N6: C, 51.59; H, 4.04; N, 24.07. Found: C,
51.80; H, 4.22; N, 24.30.
Harom.), 6.91 (d, 1H, J = 8.14 Hz, Harom.), 6.62 (brs, 1H, Harom.), 3.36
(m, 4H, CH3), 1.01 (t, 6H, J = 6.81 Hz, CH3); 13C NMR (150 MHz,
DMSO-d6): 157.3 (s, 2C), 151.3 (s), 143.0 (s), 129.6 (s), 128.3 (s),
126.0 (s), 124.1 (d), 123.3 (d), 123.2 (d), 119.4 (d), 117.6 (d),
114.6 (d), 111.9 (d), 110.5 (d), 64.3 (d), 48.9 (t), 47.7 (t), 42.7 (q),
41.6 (q); Anal. Calcd. for C19H23ClN6O: C, 58.99; H, 5.99; N, 21.72.
Found: C, 59.20; H, 6.13; N, 21.55.
2.2. Single-crystal X-ray diffraction experiment
Selected crystallographic and refinement data for structure 4f
obtained by the single-crystal X-ray diffraction experiment are re-
ported in Table 1.
Data collection has been performed by applying the CrysAlis
Software system, Version 1.171.33.55 [24]. The Lorentz-polariza-
2.1.2.14. 2-Amino-4-[20-(400-N,N-dimethylaminophenyl)ethylene]-4,
10-dihydro
[1,3,5]triazino[1,2-a]benzimidazole
dihydrochloride
4n. Compound 4n was prepared from 2-guanidinobenzimidazole
1 (0.30 g, 1.70 mmol) and 4-N,N-diethylamino-2-hydroxybenzal-
dehyde 2n (0.26 g, 1.70 mmol) in absolute ethanol (10 ml) and
piperidine (0.15 ml) after refluxing for 3 h and recrystallization
from ethanol to yield 0.45 g (80%) of orange powder 3n which
was suspended in absolute ethanol (10 ml) and saturated with
HCl(g) to gave 0.47 g (87%) of light pink powder; mp = 227–
Table 1
General and crystal data and summary of intensity data collection and structure
refinement for compound 4f.
4f
Formula
C16H16ClN5
229 °C; IR (KBr) t
/cmꢁ1: 3431, 3318, 3224, 2999, 1684, 1645; 1H
Mr
313.79
NMR (300 MHz, DMSO-d6): 13.17 (s, 1H, NH), 9.15 (s, 1H, Harom.),
Crystal system, colour and habit
Space group
Triclinic, colourless, prism
P-1
0.49 ꢃ 0.37 ꢃ 0.29
7.98 (brs, 1H, NH+), 7.48 (d, 2H, J = 8.25 Hz, Harom.), 7.37 (d, 2H,
Crystal dimensions (mm3)
J = 8.28 Hz,
Harom.), 7.30–7.21 (m, 2H, Harom.), 7.04 (d, 1H,
Unit cell parameters
J = 15.51 Hz, Harom.), 7.00 (s, 1H, Harom.), 6.54 (d, 1H, J = 8.40 Hz,
a (Å)
b (Å)
c (Å)
8.6725(4)
9.0395(3)
10.9905(6)
88.008(4)
72.587(5)
80.615(4)
811.02(7)
2
1.285
0.239
328
3–30
ꢁ12 to 11
ꢁ12 to 12
ꢁ15 to 15
x
8462
4724 (0.017)
212/0
H
arom.), 6.33 (dd, 1H, J1 = 15.48 Hz, J2 = 8.30 Hz, Harom.), 4.10 (brs,
2H, NH+), 2.97 (s, 6H, CH3); 13C NMR (150 MHz, DMSO-d6): 157.3
(s, 2C), 150.8 (s), 147.1 (s), 139.2 (s), 134.9 (d, 2C), 129.8 (s),
129.0 (d, 2C), 128.3 (s), 124.3 (s), 124.2 (d), 123.5 (d), 112.1 (d),
111.1 (d), 66.0 (d), 43.5 (q, 2C); Anal. Calcd. for C21H25ClN6: C,
63.55; H, 6.35; N, 21.17. Found: C, 63.80; H, 6.44; N, 21.30.
a
(°)
b (°)
c
(°)
V (Å3)
Z
Dc (g cmꢁ3
)
(mmꢁ1
)
2.1.3. 2Preparation of 2-amino-4-(20-chloro-50-aminophenyl)-3,4-
dihydro[1,3,5]triazino [1,2-a]benzimidazole 5a
l
F(000)
h range for data collection (°)a
h, k, l range
Compound 4l (0.25 g, 7.30 mmol) and solution of SnCl2x2H2O
(1.48 g, 6.57 mmol) in H2O (2 ml) and concentrated HCl (2 ml)
were refluxed for 30 min. After cooling, the reaction mixture was
evaporated under vacuum and dissolved in water (50 ml). The
resulting solution was treated with 20% NaOH to pH 14. The result-
ing product was filtered off and washed with water to gave 0.15 g
Scan type
No. measured reflections
No. independent reflections (Rint.
No. refined parameters/restraints
No. observed reflections, I P 2
g1, g2 in w
)
(66%) of dark yellow powder; mp = 223–224 °C; IR (KBr) t :
/cmꢁ1
r
(I)
3060
3456, 3322, 3268, 3110, 2977, 1658, 1625; 1H NMR (600 MHz,
DMSO-d6): 7.88 (s, 1H, Harom.), 7.25 (d, 1H, J = 8.28 Hz, Harom.),
7.12 (d, 1H, J = 8.28 Hz, Harom.), 6.94 (t, 1H, J = 8.22 Hz, Harom.),
6.88 (s, 1H, Harom.), 6.80 (t, 1H, J = 7.56 Hz, Harom.), 6.61 (d, 1H,
J = 7.68 Hz, Harom.), 6.55 (dd, 1H, J1 = 8.60 Hz, J2 = 2.08 Hz, Harom.),
6.31 (d, 1H, J = 1.98 Hz, Harom.), 6.26 (brs, 2H, NH2), 5.34 (brs, 2H,
NH2); 13C NMR (75 MHz, DMSO-d6): 153.8 (s), 152.9 (s), 148.8
(s), 145.2 (s), 143.3 (s), 135.9 (s), 131.2 (s), 130.1 (d), 122.5 (d),
120.7 (d), 118.2 (s), 116.0 (d), 115.2 (d), 112.2 (d), 109.4 (d), 63.9
0.0689, 0
0.0431, 0.1133
0.0705, 0.1211
0.979
R, wR [I P 2r(I)]
R, wR [all data]
Goodness of fit on F2, S
Min. and max. electron density (e Åꢁ3
)
ꢁ0.19, 0.25
Maximum
D/r
0.001
a
The data collection were collected by
tometer equipped with 4-circle kappa geometry and CCD Sapphire 3 detector and
graphite-monochromated MoK
(296 K).
x
-scans on an Oxford Xcalibur diffrac-
a radiation (k = 0.71073 Å) at ambient temperature

246
M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
the default value, which is preceded by a ‘‘>’’ sign. Each test was
performed in quadruplicate in at least two individual experiments.
tion effect was corrected and the intensity data reduced by the
CrysAlis RED application of the CrysAlis Software system, Version
1.171.33.55 [24]. The diffraction data have been scaled for absorp-
tion effects by the multi-scanning method. Structure was solved by
direct methods and refined on F2 by weighted full-matrix least-
squares. Programs SHELXS97 and SHELXL97 integrated in the
WinGX v. 1.70.01 [25] software system were used to solve and re-
fine structure. All non-hydrogen atoms were refined anisotropi-
cally. Hydrogen atoms belonging to Csp2 and Csp3 (C3 and C16)
carbon atoms were placed in geometrically idealized positions
[Csp2–H 0.93 Å with Uiso(H) = 1.2 Ueq(C); Csp3–H 0.96 Å with
Uiso(H) = 1.5 Ueq(C)] and they were constrained to ride on their par-
ent atoms by using the appropriate SHELXL97 HFIX instructions.
The positions of hydrogen atoms belonging to nitrogen N1, N4
and N5 atoms were determined from difference Fourier syntheses
and their coordinates were refined freely, while isotropic displace-
ment parameters were refined with Uiso(H) = 1.2 Ueq(N). The
molecular geometry calculations and graphics were done using
ORTEP-3 [26,27] integrated in the WinGX software system,
PLATON [28] programme and Mercury [29].
3. Results and discussion
3.1. General aspects of preparation
All newly prepared 2-amino-4-aryl-4,10-dihydro[1,3,5]triazi-
no[1,2-a]benzimidazoles as hydrochloride salts were synthesized
according to the procedure shown in Scheme 1 and Table 2, by
the conventional methods of organic synthesis for the preparation
of similar heterocyclic compounds [23].
In the cyclocondensation reaction between the 2-guanidino-
benzimidazole 1 and versatile heteroaromatic benzaldehydeys
2a-n in absolute ethanol by using piperidine as a catalysts, 2-ami-
no-4-aryl-3,4-dihydro[1,3,5]triazino[1,2-a]benzimidazoles
were
prepared. Hydrochloride salts of 2-amino-4-aryl-4,10-dihy-
dro[1,3,5]triazino[1,2-a]benzimidazoles 4a-n were prepared in
ethanolic solution with HClgas in order to ensure better solubility.
Amino substituted derivative 5a was prepared according to the
experimental procedure shown in Scheme 2, from nitro substituted
derivative 3l by reduction with SnCl2ꢀ2H2O in MeOH and concen-
trated HCl.
All structures of novel prepared compounds 4a-n and 5b were
determined by the NMR analysis based on the analysis of H–H
coupling constants as well as chemical shifts. The formation of
the s-triazine ring was confirmed by the NMR spectral data. Since
prepared compounds could exist in several tautomeric forms,
according to the 1H NMR data, 3,4-dihydro[1,3,5]triazino[1,2-
2.3. Antiproliferative evaluation assay
The experiments were carried out on five human cell lines,
which are derived from four cancer types. The following cell lines
were used: HCT 116 (colon carcinoma), H 460 (lung carcinoma)
and MCF-7 (breast carcinoma). MCF-7, HCT 116 and H 460 cells
were cultured as monolayers and maintained in Dulbecco’s modi-
fied Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 lg/
ml streptomycin in a humidified atmosphere with 5% CO2 at
37 °C. The growth inhibition activity was assessed as described
previously. The panel cell lines were inoculated onto a series of
standard 96-well microtiter plates on day 0, at 1 ꢃ 104–
3 ꢃ 104 cells/ml, depending on the doubling times of a specific cell
H2N
NH
N
Ar
CHO
+
NH
line. Test agents were then added in ten-fold dilutions (10ꢁ8
–
N
H
1
10ꢁ4 M) and incubated for further 72 h. Working dilutions were
freshly prepared on the day of testing. After 72 h of incubation
the cell growth rate was evaluated by performing the MTT assay,
which detects dehydrogenAse activity in viable cells. The absor-
bance (A) was measured on a microplate reader at 570 nm. The
absorbance is directly proportional to the number of living, meta-
bolically active cells. The percentage of growth (PG) of the cell lines
was calculated according to one or the other of the following two
expressions:
2a-n
EtOHabs.
piperidine
Ar
NH
N
If ðmean Atest ꢁ mean AtzeroÞ P 0; then
PG ¼ 100 ꢃ ðmeanAtest ꢁ mean AtzeroÞ=
ðmean Actrl ꢁ mean AtzeroÞ:
N
NH2
N
3a-n
If ðmean Atest ꢁ mean AtzeroÞ < 0; then PG
EtOHabs.
HCl(g)
¼ 100 ꢃ ðmean Atest ꢁ mean AtzeroÞ=Atzero
;
where the mean Atzero is the average of optical density measure-
ments before exposure of cells to the test compound, the mean Atest
is the average of optical density measurements after the desired
period of time and the mean Actrl is the average of optical density
measurements after the desired period of time with no exposure
Ar
NH+Cl-
of cells to the test compound. The results are expressed as IC50
,
N
NH2
which is the concentration necessary for 50% of inhibition. The
IC50 values for each compound are calculated from concentration–
response curves using linear regression analysis by fitting the test
concentrations that give PG values above and below the reference
value (i.e. 50%). If however, for all of the tested concentrations pro-
duce PGs exceeding the respective reference level of effect (e.g. PG
value of 50), then the highest tested concentration is assigned as
N
N
H
4a-n
Scheme 1. Reaction scheme for preparation of 2-amino-4-aryl-4,10 dihy-
dro[1,3,5]triazino[1,2-a]benzimidazole hydrochlorides 4a-n.

M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
247
Table 2
Prepared compounds 3a–3n, 4a–4n and 5a–5b.
Comp.
Ar
Comp.
Ar
3a, 4a
3i, 4i
N
3b, 4b
3c, 4c
3d, 4d
3j, 4j
3k, 4k
3l, 4l
F
S
NC
Cl
(H3C)2N
Cl
O2N
(H3CH2C)2N
3e, 4e
3m, 4m
OH
N
CH3
H3C
3f, 4f
3n, 4n
(H3C)2N
3g, 4g
5a
Cl
O
H2N
3h, 4h
5b
Cl
N
N
-Cl+H3N
H
O2N
Cl
H2N
Cl
NH
N
-Cl+H3N
Cl
SnCl2×2H2O
HCl, MeOH
EtOHabs.
HCl(g)
NH+Cl-
NH2
N
NH
N
N
NH2
N
NH2
N
N
N
N
H
3l
5a
5b
Scheme 2. Reaction scheme for preparation of 2-amino-4-aryl-4,10 dihydro[1,3,5]triazino[1,2-a]benzimidazoles hydrochlorides 5a-b.
a]benzimidazole structures were confirmed. These spectra showed
the presence of NH signal around 8 ppm in 1H NMR as well as sin-
glet of triazine ring at 6.89–7.20 ppm in 1H NMR as it can be
viewed from the 1H NMR spectra of compound 3d (Fig. 1a) and
at 59.1–66.3 ppm in 13C NMR spectra.
carbon and proton chemical shifts of compounds 4a-n and 5b are
given in Table 3.
3.2. Crystal structure description
It is well known that compounds 4a-n could obtain in three
tautomeric forms as 3,4-dihydro, 4,10-dihydo and 1,4-dihy-
dro[1,3,5]triazino[1,2-a]benzimidazoles. By the protonation of
3,4-dihydro[1,3,5]-triazino[1,2-a]benzimidazoles 4a-n with HClgas
4,10-dihydro forms obtained. 1H NMR spectra have shown broad-
ening effect of the NH signal as well as presence of NH singlet of
benzimidazole nuclei at 13.18–13.40 ppm as it can be viewed from
the 1H NMR spectra of compound 4c (Fig. 1b). Some of specific
ORTEP drawing of molecular structure for compound 4f is de-
picted in Fig. 2, while crystal structure is shown in Fig. 3.
The selected molecular geometry is listed in Table 4 and hydro-
gen bonding geometry in Table 5, respectively.
The structure of compound 4f is isostructural with 2-amino-
4-phenyl-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazole hydro-
chloride [30]. The molecule of 2-amino-4-(40-methylphenyl)-4,
10-dihydro[1,3,5]triazino[1,2-a]benzimidazole hydrochloride, 4f,

248
M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
(a)
2.8
2.6
PPM
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
(b)
PPM 13.2
12.8
12.4
12.0
11.6
11.2
10.8
10.4
10.0
9.6
9.2
8.8
8.4
8.0
7.6
7.2
6.8
Fig. 1. 1H NMR spectra of compounds 3d (a) and 4c (b).
C16H16N5 ꢀClꢁ, exists in the solid state in one of the possible tauto-
meric forms being protonated at the N5 atom of the 1,4-dihydrotri-
azine ring (the value of the C3–N5 bond distance of 1.457(2) Å
into R22 (8) centrosymmetric dimers. Dimers further connected with
Clꢁ anions via N–Hꢀ ꢀ ꢀCl and C–Hꢀ ꢀ ꢀCl hydrogen bonds including
dihydrotriazine and imidazole NH proton groups as well as the
remaining amino NH group (Table 5). The Clꢁ anion acts as a quadru-
ple proton acceptor.
+
indicates dominantly
r bond character). The molecule is not planar
containing the sp3 methine C3 atom. Benzimidazole ring is fused
with the dihydrotriazine one containing the C3 atom in a relatively
planar moiety (the atoms N5 and C3 deviate 0.123(1) Å and
0.123(1) Å, respectively, from the dihydrotriazine ring best calcu-
lated plane). The phenyl and dihydrotriazine rings are inclined
mutually by 86.65(8)°. The cations are mutually link via N–Hꢀ ꢀ ꢀN
hydrogen bond between the amino N4 atom acting as proton donor
and the dihydrotriazine N3 atom acting as proton acceptor (Table 5)
3.3. UV/Vis spectroscopy
In order to study the spectroscopic properties of prepared
hydrochloride salts of 2-amino-4-aryl-4,10-dihydro[1,3,5]triazi-
no[1,2-a]benzimidazoles 4a-n and 5b, UV/Vis absorption spectra
were undertaken in ethanol. The UV/Vis spectra of all were

M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
249
Table 3
Characteristic NMR data of compounds 4a–4n and 5b.
4'
3'
5'
2'
Ar
1'
6'
3
4
NH+Cl-
6
5
N
5a
7
NH2
2
N
1
8
10a
10
9a
N
9
H
Comp.
1H NMR (d/ppm)
13C NMR (d/ppm)
NH+-3
NH-10
H-4
C-4
C-10a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
5b
8.03
–
13.30
13.31
13.29
13.30
13.26
13.18
13.21
13.20
13.42
13.24
13.26
13.29
13.05
13.17
13.30
6.99
6.99
7.00
7.02
7.04
6.89
7.17
7.13
7.08
7.22
7.01
7.20
6.98
7.00
6.91
66.3
65.4
65.6
65.7
60.8
66.2
59.4
59.1
64.3
61.9
65.6
65.4
64.3
66.0
64.1
157.2
157.0
161.8
156.8
157.6
157.2
157.6
157.0
157.0
157.1
157.1
156.3
157.3
157.3
155.5
8.01
8.00
8.04
7.94
8.06
8.18
8.17
8.11
7.70
8.83
7.70
7.98
8.00
Fig. 3. Crystal packing of 2-amino-4-(40-methylphenyl)-4,10-dihydro[1,3,5]triazi-
no[1,2-a]benzimidazole hydrochloride, 4f showing assembling of molecules via
N–Hꢀ ꢀ ꢀCl, N–Hꢀ ꢀ ꢀN and C–Hꢀ ꢀ ꢀCl hydrogen bonds.
Table 4
Selected interatomic distances (Å) and valence and torsion
angles (°) for the compound 4f.
4f
Selected bond distances
C2–N4
C2–N5
C1–N3
C2–N3
C3–N5
C3–N2
C1–N2
C1–N1
1.320(2)
1.332(2)
1.321(2)
1.351(2)
1.457(2)
1.459(2)
1.350(2)
1.346(2)
Bond angles
108.0(1)
122.8(1)
105.7(1)
N1–C1–N2
N3–C2–N5
N5–C3–N2
Fig. 2. ORTEP drawing of the molecular structure of 2-amino-4-(40-methylphenyl)-
4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazole hydrochloride, 4f, with the atomic
numbering scheme. The thermal ellipsoids are drawn at the 50% probability level at
296 K. The Clꢁ anion is omitted.
As it might be observed from obtained spectra (Fig. SM1a) most
of aromatic derivatives exhibit one characteristic strong absorption
maxima with one main absorption band at ꢄ300 nm. 4-N,N-
dimethylamino 4d, 2-chloro-5-nitro 4l, 4-N,N-diethylamino-2-hy-
droxy 4m and 2-chloro-5-amino 5b substituted compounds
showed two absorption bands of similar intensity at 301, 300,
302 and 303 nm and at 266, 263, 268 and 252 nm respectively.
4-N,N-(dimethylaminophenyl)ethylene substituted compound 4n
showed hyperchromic shift of absorption maxima at 304 nm in
recorded at the same concentration of 2 ꢃ 10ꢁ5 mol dmꢁ3. Spectra
can be visualized in Fig. SM1 (Supplementary material) and all the
results are summarized in Table 6.

250
M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
Table 5
3.4. Antiproliferative evaluation
Hydrogen bond geometry (Å, °) for compound 4f.
All prepared 10,4-dihydro[1,3,5]triazino[1,2-a]benzimidazoles
4a-n and 5b were tested for their antiproliferative activities
against human malignant tumor cell lines in the Laboratory of
D–Hꢀ ꢀ ꢀA
D–H
Hꢀ ꢀ ꢀA
Dꢀ ꢀ ꢀA
\D–
Symmetry code
–
Hꢀ ꢀ ꢀA
4f
-
C3–H3ꢀ ꢀ ꢀCl1
0.98
2.575
3.517(1) 161
Experimental Therapy, Division of Molecular Medicine, Ruder
N1–H1N1ꢀ ꢀ ꢀCl1 0.86(2) 2.258(17) 3.109(1) 168(2) ꢁx, ꢁy, ꢁz + 2
N5–H1N5ꢀ ꢀ ꢀCl1 0.83(2) 2.335(18) 3.130(1) 162(2) ꢁx, ꢁy + 1, ꢁz + 2
N4–H1N4ꢀ ꢀ ꢀCl1 0.86(2) 2.720(21) 3.379(2) 134(2) x + 1, +y, +z
´
Boškovic Institute, Croatia. The following cell lines were used:
H460 (colon carcinoma), MCF-7 (breast carcinoma) and HCT 116
(colon carcinoma). Most of the tested compounds did not showed
antiproliferative activity while some of compounds showed mod-
erate activity (Table 7). The most active compound is 2-hydroxy-
N4–H2N4ꢀ ꢀ ꢀN3
1.00(2) 1.976(19) 2.971(2) 174(2) ꢁx + 1, ꢁy, ꢁz + 2
4-N,N-diethylamino substituted derivative 4m (IC50 ꢂ 20
which show cytotoxicity at the maximal tested concentration
(100 M). 4-N,N-dimethylamino 4d, 4-methyl 4f, 4-chloro 4k, 2-
chloro-5-nitro 4l and 4-N,N-dimethyl-aminophenyl)ethylene 4m
substituted derivatives showed also moderate antiproliferative
lM),
Table 6
Electronic absorption data of 4a–n and 5b in ethanol (c = 2.0 ꢃ 10ꢁ5 mol dmꢁ3).
l
Comp. kmax
e
ꢃ 103
Comp. kmax
e
ꢃ 103
(nm)
(dm3 molꢁ1 cmꢁ1
)
(nm)
(dm3 molꢁ1 cmꢁ1
)
activity with IC50 25–60 lM. Also, there is no significant selectivity
4a
4b
4c
4d
4e
302
260
301
261
303
261
301
266
301
270
261
302
270
260
301
270
261
20.7
9.8
4i
302
259
303
260
303
260
300
263
302
268
17.95
11.1
18.65
9.85
21.35
11.45
19.3
19.35
21.45
18.8
of the above mentioned compounds between the cell lines. Intro-
duction of heteroaromatic rings caused only decrease of antiprolif-
erative activity.
18.93
13.4
22.75
11.1
21.8
23.7
25.95
11.7
12.35
21.1
8.25
9.45
18.3
8.6
4j
4k
4l
4. Conclusions
4m
In this work we have presented the synthesis of novel 2-amino-
4-aryl-4,10-dihydro-[1,3,5]triazino[1,2-a]benzimidazoles, crystal
structure determination of compound 4f and antiproliferative
activity in vitro of all prepared derivatives. Novel 2-amino-4-aryl-
4,10-dihydro-[1,3,5]triazino[1,2-a]benzimidazoles 4a-n and 5b as
hydrochloride salts were prepared from 2-amino-4-aryl-3,4-dihy-
dro-[1,3,5]triazino[1,2-a]benzimidazoles 3a-n and 5a in ethanolic
solution saturated with HClgas. Compounds 3a-n and 5a were pre-
pared in cyclocondensation reaction between 2-guanidinobenzim-
idazole and versatile heteroaromatic compounds.
4f
4n
5b
332
304
260
303
252
13.45
27.25
8.1
21.3
21.4
4g
8.85
Table 7
GI50 values (in lM).
The molecular and crystal structure of compound 2-amino-4-
(40-methylphenyl)-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazole
hydrochloride, 4f, reveals that one of the nitrogen atoms of 4,10-
dihydro[1,3,5]triazine ring is protonated causing that the Csp3
methine atom and protonated Nsp3 nitrogen atom deviate most
from planarity. The cations are joined via N–Hꢀ ꢀ ꢀN hydrogen bonds
into R22(8) centrosymmetric dimers. Cation dimers are further con-
nected with Clꢁ ions into 2D chains spreading along the b axis. Sin-
gle-crystal X-ray structure determination has been performed for
compound 2-amino-4-(40-methylphenyl)-4,10-dihydro[1,3,5]triaz-
ino[1,2-a]benzimidazole hydrochloride, 4f, in order to establish
correlation between the solid-state molecular structure with IR
spectroscopic data in the context of tautomeric variability of this
class of compounds and further with possible interpretation of bio-
logical activity. Moreover, the obtained structure unequivocally
confirms tautomeric form of the compound present in the solid-
state and can represent tantative pattern for other prepared
compounds.
Compound ID
Cell lines
HCT 116
MCF-7
H460
a
GI50
4a
4b
4c
4d
4e
4f
(lM)
P100
P100
P100
65 36
>100
52 20
>100
>100
>100
>100
34 14
52 39
P100
>100
>100
51 28
>100
38 19
>100
>100
>100
P100
52 45
60 40
>100
>100
>100
72 27
>100
43
4
4g
4h
4i
4j
4k
4l
4m
4n
5a
5b
>100
>100
>100
>100
31 0.05
49 0.5
17
4
22
7
18
3
24 19
P100
P100
23 19
P100
>100
35 15
>100
>100
a
All prepared 10,4-dihydro[1,3,5]triazino[1,2-a]benzimidazoles
4a-n and 5b were tested for their antiproliferative activities and
showed in general no to modest activity. 2-Hydroxy-4-N,N-
diethylamino substituted derivative 4m was the most active com-
GI50; the concentration that causes 50% growth inhibition.
comparison to all other compounds as well as appearance of one
absorption band at 332 nm. 4-fluoro substituted compound 4b
showed hypochromic shift of absorption maxima at 301 nm. UV/
Vis spectra of heteroaromatic derivatives 4e, 4g, 4h, 4i and 4j
(Fig. SM1b) showed that all compounds exhibit one main strong
absorption band at ꢄ300 nm and one lower absorption band at
ꢄ260 nm, while compounds 4e and 4g showed two lower absorp-
tion bands at 270 and 261 nm. 2-N-methylpyrolyl 4e and 5-imid-
azolyl 4h substituted derivatives showed hyperchromic shift of
absorption maxima at 301 and 300 nm.
pound (IC50 ꢂ 20
lM), while compounds 4d, 4f, 4k, 4l 4m showed
also moderate antiproliferative activity with IC50 25–60
lM with
no significant selectivity between the tumor cell lines.
Acknowledgments
This study was supported by Grants 125-0982464-1356 and
09-1191344-2943 by the Ministry of Science, Education and Sports
of the Republic of Croatia. We would also like to thank Dr. M. Kralj

M. Hranjec et al. / Journal of Molecular Structure 1007 (2012) 242–251
251
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