Synthesis of 2-aryl-4-(benzimidazol-2-yl)-1,2-dihydro[1,2,4]triazino-[4,5-a]benzimida zol-1-one derivatives with preferential cytotoxicity against carcinoma cell lines

Article abstract of DOI:10.1016/j.ejmech.2007.01.008

In this paper we describe the preparation of some derivatives of 1,2,4-triazino[4,5-a]benzimidazol-1-ones (5 and 6), containing additional benzimidazole ring. These compounds were prepared using coupling reactions of diazonium salts with 1,1-bis(1-ethoxyc

Full text of DOI:10.1016/j.ejmech.2007.01.008

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 449e455
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 2-aryl-4-(benzimidazol-2-yl)-1,2-dihydro[1,2,4]triazino-
[4,5-a]benzimidazol-1-one derivatives with preferential
cytotoxicity against carcinoma cell lines
a
a,b
a
Jakub Styskala , Libuse Styskalova , Jan Slouka , Marian Hajduch
b,
*
´
ˇ
´
´
´
´
a
ˇ
Department of Organic Chemistry, Faculty of Science, Palacky´ University, Tr. Svobody 8, 771 46 Olomouc, Czech Republic
b Laboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine,
Palacky´ University and Faculty Hospital in Olomouc, Pusˇkinova 6, 775 20 Olomouc, Czech Republic
Received 14 September 2006; received in revised form 3 January 2007; accepted 8 January 2007
Available online 25 January 2007
Abstract
In this paper we describe the preparation of some derivatives of 1,2,4-triazino[4,5-a]benzimidazol-1-ones (5 and 6), containing additional
benzimidazole ring. These compounds were prepared using coupling reactions of diazonium salts with 1,1-bis(1-ethoxycarbonyl-benzimida-
zol-2-yl)methane (2) to obtain unstable hydrazones 4, which readily undergo cyclization. Interestingly, the selected compounds demonstrated
preferential cytotoxic activities against human carcinoma and glioma cell lines compared with leukemic cells. They showed significant activity
against multidrug-resistant P-glycoprotein expressing cell lines but had less effect on multidrug-resistance protein 1 positive and topoisomerase
IIa negative leukemias.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Condensed 1,2,4-triazine; Benzimidazole; Hydrazones; Topoisomerase; MRP-1; Pgp
1. Introduction
and tumor progression [14e16]. For this reason we have de-
cided to test anticancer activity of those compounds under in
vitro conditions using definitive endpoint cytotoxic assay.
Since a number of benzimidazole derivatives, including
2-aryl-4-(benzimidazol-2-yl)-1,2-dihydro[1,2,4]triazino-[4,5-a]
benzimidazol-1-one [1], have proven biological activities e
inter alia as peptide deformylase inhibitors [2,3], luteinizing
hormone receptor antagonists [4], antitumor agents [5,6], anti-
ulcerative agents [7,8], antiproliferative agents [9] and antimi-
crobial agents [10] e we have re-examined cyclization
reactions that give rise to derivatives of 1,2,4-triazino[4,5-
a]benzimidazole. Our current interest in compounds of this
type increased after a series of the derivatives of the isosteric
condensed 1,2,4-triazino[4,3-a]benzimidazole system which
proved to be effective as selective aldose reductase inhibitors
[11e13]. Recently, several lines of evidence demonstrated
that aldose reductases are directly involved in cancerogenesis
2. Chemistry
The most efficient syntheses of 2-arylderivatives of 1,2,4-tri-
azine include the cyclization reaction of hydrazones, which can
be achieved via coupling reactions of diazonium salts with se-
lected compounds with a reactive CH2 group. The most com-
mon and proven syntheses of this type include the cyclization
of ethyl arylhydrazono-cyanoacetyl carbamates resulting in
1-aryl-6-azauracils [17e19] and the analogous cyclization of
arylhydrazones of ethoxycarbonylated amidines of mesoxalate
acid generating 1-aryl-6-azacytosines [20]. Hydrazones of the
last type include the arylhydrazones that can be generated by
coupling diazonium salts with 2-(1-ethoxycarbonylbenzi-
midazole-2-yl)acetonitrile. However, the arylhydrazones are
barely detectable owing to their very rapid cyclization [21e24].
* Corresponding author.
E-mail addresses: marian.hajduch@fnol.cz, hajduchm@atlas.cz (M. Hajduch).
´
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.01.008

450
J. Sty´skala et al. / European Journal of Medicinal Chemistry 43 (2008) 449e455
O
O
COOC2H5
i
ii
N
N
N
N
N
N
N
N
N
N
N
N
COOC2H5
COOC2H5
H
7
2
8
Scheme 1. Hydrolysis of diester 2 to compound 8. Reagents and conditions: (i) water and pyridine, reflux 1 h; (ii) aqueous ethanolic HCl, reflux 1 h.
In attempts to prepare derivatives of 1,2,4-triazino[4,5-
knowledge of the stability of 1,2,4-triazine ring system with
respect to hydrolytic cleavage [26], we obtained compounds
6ae6d without any difficulty following a short period of
acid hydrolysis of compounds 5ae5e, without any degradation
of 1,2,4-triazine ring system. During long-term refluxing of
compounds 5 and 6 with HCl or in aqueous pyridine, cleavage
of the 1,2,4-triazine ring system occurred, giving rise to hydra-
zones 3ae3e. In the case of NaOH, the hydrolysis and cleav-
age is very fast and compounds 6 are produced from
compounds 5 after just 1 h of refluxing.
a]benzimidazole containing an additional benzimidazole ring,
we used bis(benzimidazol-2-yl)methane (1) [25] as our starting
material, and following its acylation using ethyl chloroformate
in a pyridine medium we obtained the double ethoxycarbony-
lated derivative (2). It is very interesting to note that all our at-
tempts to generate mono-ethoxycarbonyl products were
unsuccessful. Even with the gradual addition of 1 eq of ethyl
chloroformate to compound 1, we could not obtain a mono-
ethoxycarbonylated product, but only a mixture of the double
ethoxycarbonylated derivative 2 and the unreacted starting ma-
terial 1. The mono-ethoxycarbonylated product was not ob-
tained even from selective hydrolysis of compound 2 using
1 mol of NaOH; a mixture of several components was always
obtained. We discovered that under more moderate conditions,
boiling in aqueous pyridine, one of the ester groups was hydro-
lyzed, and in addition the pyrimidine ring formed, giving rise to
ester 7. Following another hydrolysis of compound 7 a con-
densed heterocyclic system, compound 8 is generated; this
compound was previously produced via the reaction of bis(ben-
zimidazol-2-yl)methane with phosgene [27]. It is very proba-
3. Biological experiments: results and discussion
The biological activities of the generated compounds of
interest were examined by comparing their cytoxicity, using
COOC2H5
H
N
N
iii
N
N
1
ble, based on H NMR spectroscopic data that this compound
is present in its 5H-tautomeric form rather than the 6H-tauto-
meric form as indicated earlier [27] (Scheme 1).
N
N
N
N
H
COOC2H5
1
2
iv
iv
The most important point for further synthetic steps was to
ensure the reactivity of the CH2 group in compounds 1 and 2.
We discovered that the coupling reaction of diazonium salts
with these components was very rapid. The diazotization of
aniline and its derivatives and the diazonium salts with the
CH2 group of compound 1 derived from the coupling reaction
of pyridine generate the corresponding hydrazones in good
yields (3ae3e). However, during the analogous reaction of
the diazonium salts with component 2, hydrazones could not
be successfully isolated (4ae4e), as they cyclized immediately
to the corresponding 4-(1-ethoxycarbonylbenzimidazol-2-yl)-
2-aryl-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-ones
(5ae5e). This reaction was expected because of the previous
experimental results [6e9]. Hydrazones 4ae4e could not be
separated even during the ethoxycarbonylation of hydrazones
3ae3e. The reaction of hydrazones (3ae3e) with ethyl chlor-
oformate in pyridine also gave rise to the abovementioned de-
rivatives of 1,2,4-triazino[4,5-a]benzimidazole (5ae5e)
(Scheme 2).
COOC2H5
H
N
N
N
N
N
iii
N
N
H
N
N
H
H
COOC2H5
N
N
N
3
Ar
Ar
4
vi or vii
COOC2H5
H
N
v
N
N
N
N
O
N
N
N
N
N
O
N
N
Ar
Ar
6
5
(a) Ar = phenyl
(b) Ar = 4-methylphenyl
(c) Ar = 4-methoxyphenyl
(d) Ar = 4-chlorophenyl
(e) Ar = 4-nitrophenyl
From the perspective of potential biological activity, we
were mainly interested in compounds with a free NH group
(6ae6e), which have structures that may allow them to inter-
calate with DNA double helices or interact with nucleic acids
through intermolecular hydrogen bonding. Based on previous
Scheme 2. General scheme leading to 2-aryl-4-(benzimidazol-2-yl)-1,2-dihy-
dro[1,2,4]triazino[4,5-a]benzimidazol-1-ones. Reagents and conditions: (iii)
ClCOOC2H5 and pyridine; (iv) arene diazonium salt and pyridine; (v) aqueous
ethanolic HCl, reflux 30 min; (vi) aqueous ethanolic HCl, reflux 48 h; (vii) wa-
ter and pyridine, reflux 24 h.

J. Sty´skala et al. / European Journal of Medicinal Chemistry 43 (2008) 449e455
451
the MTT assay (Table 1) [28,29], towards the drug-sensitive
4. Conclusions
leukemia cell lines CEM (T-lymphoblastic) and K562
(myeloid), and the drug-resistant lines CEM-DNR-bulk
(daunorubicin-resistant, topoisomerase IIa negative, multi-
drug-resistance protein 1 [MRP-1] positive and P-glycopro-
tein [Pgp] negative) and K562-Tax (topoisomerase IIa
positive, MRP-1 negative and Pgp positive) [28]. The effects
of the compounds were also analyzed (in parallel) on model
cell lines of human lung adenocarcinoma (A549), colorectal
carcinoma (HT-29, HCT116 p53wt, HCT116 p53mut),
breast cancer (MCF-7), prostate cancer (PC-3) and glioblas-
toma (U118Mg). The compounds showed preferential activ-
ity against solid tumors compared to leukemia cells, which
is unusual due to the intrinsic chemosensitivity of hemato-
poietic malignant diseases. Interestingly, biological activity
decreased with the level of substitution at position 4. While
compounds with the lipophilic substituents, 4-phenyl, 4-
methylphenyl and 4-methoxyphenyl in compounds 3ae3c,
5ae5c, 6ae6c, were highly active in leukemia cell lines
at low micromolar concentrations, substitutions with 4-
chlorophenyl or 4-nitrophenyl groups substantially decreased
the cytotoxic potency (3de3e, 5de5e, 6de6e). However,
this was not the case in human carcinoma cell lines, where
a higher level of substitution in position 4 demonstrated the
opposite effect and generally increased the cytotoxic activ-
ity. Interestingly, higher activity was also observed in
HCT116 cells expressing a mutated p53 oncosuppressor.
Moreover, majority of the derivatives showed 2- to 10-fold
decrease of cytotoxic activity against MRP-1 positive and
topoisomerase IIa negative CEM-DNR-bulk cells, suggest-
ing that the molecules are transported via MRP-1 and/or tar-
get the topoisomerase IIa gene. The decrease is considered
as significant since it corresponds to reduction of cytotoxic
activity in drug-sensitive versus resistant cell lines or pri-
mary tumor cells using classical anticancer agents [28,29].
However, those observations will require more detailed con-
firmatory studies.
We have successfully prepared derivatives of 1,2,4-tria-
zino[4,5-a]benzimidazol-1-ones (5 and 6), containing an addi-
tional benzimidazole ring. The compounds were prepared using
coupling reactions of diazonium salts with 1,1-bis(1-ethoxycar-
bonylbenzimidazol-2-yl)methane (2) to obtain unstable hydra-
zones 4, which readily undergo cyclization. Analysis of their
biological activity demonstrated that selected compounds pos-
sess preferential cytotoxic activities against human carcinoma
cell lines and showed significant activity in the multidrug-resis-
tant, Pgp expressing cell line K562-Tax, but were inactive in
MRP-1 positive and topoisomerase IIa negative CEM-DNR-
bulk cells. Structureeactivity relationship showed the cytotoxic
activity to be dependent on the substituent at position 4.
5. Experimental
5.1. Chemical synthesis
Melting points were determined on a Boetius stage and are
uncorrected. The IR spectra were recorded in KBr wafers on
an ATI Unicam Genesis FTIR instrument. The NMR spectra
were registered on a Bruker Avance 300 MHz DRX spectrom-
eter; chemical shifts are reported in parts per million, and the
coupling constants J in hertz. Elemental analyses were per-
formed with an EA 1108 Elemental Analyser (Fison Instru-
ments). Mass spectrometric experiments were performed
using an LCQ ion trap mass spectrometer (Finnigan MAT,
San Jose, CA, USA).
5.1.1. 1,1-Bis(1-ethoxycarbonylbenzimidazol-2-yl)-
methane (2)
Bis(benzimidazol-2-yl)methane (1) (10 g; 40.3 mmol) was
dissolved in anhydrous pyridine (500 ml) on boiling and
cooled to 5 ꢀC. To this solution, ethyl chloroformate (9.3 ml;
97.3 mmol) was added dropwise with stirring. After 1 h of
Table 1
Cytotoxic activity of synthesized compounds on human malignant cell lines with varying tissue origins, drug resistance profiles and p53 gene status
Compound
Cytotoxicity (IC50, mM)
CEM
CEM-DNR-bulk
K562
K562-Tax
A549
MCF-7
PC-3
HT-29
HCT116 p53wt
HCT116 p53mut
U118Mg
3a
3b
3c
3d
3e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
13
27.9
87.6
114.5
159.6
116.5
199.5
190.3
69.4
143.8
238.6
235.4
159.2
100.9
42.2
53.7
7.8
3.8
0.6
0.5
2.8
0.7
18
83.2
95.8
215.1
140.4
94.8
35.1
134
248.3
250
250
81.9
95.9
114.4
113.6
17.7
15.2
96.2
38.7
29.7
21.9
8
162.1
194.9
35.7
201
17.6
10.5
66.5
16.8
4.7
160
250
250
250
250
17.7
147.3
54.3
12.4
6.2
26.5
10.9
42.5
58.4
58.6
25.5
31.8
31.8
154.7
12.2
18.5
3.5
96.7
112.1
140.3
18.4
250
218.4
37.5
165.1
145.3
149.8
124.9
13.6
40.8
24.6
242.5
250
11.7
11.4
46.3
42.8
7.7
10.8
44.4
46.3
6.4
29.6
139.5
14
14.1
71
110.9
110.9
223.7
45.6
44.4
4
51.6
21.3
144
7.9
165.5
23.5
18.3
196.4
42.7
20.9
7.5
4.4
11.5
14.7
8.7
1.2
2.2
70.7
38.1
7.8
5.4
8.6
74.8
34.1
10.7
140.5
250
12
61.2
35.4
15.2
6
7.6
3.6
17.9
10
8.7
133.9
209.5
6.9
73.9
250
83.6
102.9
51.2
190.2
166.6
250
88.6
248
2.3
247
250
250
Data represent mean values from 3e5 independent experiments with typical standard deviations within 10e15% of the average.

452
J. Sty´skala et al. / European Journal of Medicinal Chemistry 43 (2008) 449e455
stirring, the reaction mixture was left to stand at 2 ꢀC for 18 h.
Then, it was diluted slowly with water under stirring to a total
volume of 1500 ml. The precipitated solid was collected on
a filter, washed with water, crystallized from ethanol
(250 ml) and dried at 110 ꢀC for 1.5 h. Yield 11.6 g (73.4%,
C22H18N6 (366.4): C 72.11, H 4.95, N 22.93. Found: C
71.97, H 4.83, N 22.89.
5.1.2.3. 1-(4-Methoxyphenylhydrazono)-1,1-bis(benzimidazol-
2-yl)methane (3c). Yield 89.3%, yellow-orange solid, mp
291e293 ꢀC dec. 1H NMR (DMSO-d6): d 3.78 (s, 3H, OCH3),
7.03 (d, 2H, arom, J ¼ 9.0 Hz), 7.20e7.39 (m, 4H, arom),
7.59 (d, 1H, arom, J ¼ 7.5 Hz), 7.67 (d, 2H, arom,
J ¼ 9.0 Hz), 7.81 (d, 1H, arom, J ¼ 7.2 Hz), 7.86e7.94 (m,
2H, arom), 12.68 (bs, 1H, NH), 13.24 (bs, 1H, NH), 14.57 (s,
1H, NH). IR [cmꢁ1]: 3455, 3223, 1562, 1501, 1233, 737. MS
(ESI, m/z) 383.2 [M þ H]þ. Anal. Calcd. for C22H18N6O
(382.4): C 69.10, H 4.74, N 21.98. Found: C 68.99, H 4.62,
N 22.09.
1
colorless crystals), mp 158e160 ꢀC. H NMR (DMSO-d6):
d 1.28 (t, 6H, CH3), 4.42 (q, 4H, CH2), 5.04 (s, 2H, eCH2e),
7.32e7.43 (m, 4H, arom), 7.63 (d, 2H, arom, J ¼ 6.9 Hz),
7.98 (d, 2H, J ¼ 6.9 Hz); 13C NMR (DMSO-d6): d 13.6,
33.9, 64.0, 114.7, 119.3, 124.2, 124.6, 132.5, 141.6, 149.6,
151.5; IR (cmꢁ1): 2992, 1742, 1548, 1454, 1377, 1337,
1189, 766; MS (ESI, m/z): 393.1 [M þ H]þ. Anal. Calcd. for
C21H20N4O4 (392.4): C 64.28, H 5.14, N 14.28. Found: C
64.33, H 5.03, N 14.14.
5.1.2.4. 1-(4-Chlorophenylhydrazono)-1,1-bis(benzimidazol-2-
5.1.2. 1-Arylhydrazono-1,1-bis(benzimidazol-2-yl)methanes
(3ae3e): general procedure
yl)methane (3d). Yield 94.4%, yellow solid, mp 302e304 ꢀC
dec. H NMR (DMSO-d6): d 7.28e7.39 (m, 4H, arom), 7.48
1
A
solution of the corresponding aromatic amine
(d, 2H, arom, J ¼ 8.7 Hz), 7.62 (bs, 1H, arom), 7.75 (d, 2H,
arom, J ¼ 8.7 Hz), 7.81 (d, 1H, arom, J ¼ 7.5 Hz), 7.86e7.98
(m, 2H, arom), 12.80 (bs, 1H, NH), 13.28 (bs, 1H, NH), 14.66
(s, 1H, NH). IR [cmꢁ1]: 3452, 3224, 1538, 1485, 1261, 821.
MS (ESI, m/z) 387.2 [M þ H]þ. Anal. Calcd. for C21H15ClN6
(386.9): C 65.20, H 3.91, N 21.72. Found: C 65.18, H 4.08,
N 21.81.
(2.00 mmol) in a mixture of ice water (5 ml) and HCl (35%;
1.5 ml) was diazotized by sodium nitrite (138 mg;
2.00 mmol) in ice water (4 ml). The mixture was stirred in
an ice bath for 15 min and then added dropwise to the solution
of compound 1 (500 mg; 2.01 mmol) in pyridine (80 ml),
which was boiled and then cooled to 2e5 ꢀC. The mixture
was left to stand at 0e5 ꢀC for 24 h and then slowly diluted
with water to a total volume of 300 ml. The precipitated yel-
low-orange solid was collected by suction, washed with water
and dried. The sample for analysis was obtained by crystalli-
zation from a DMSOeethanol mixture. The purity of these
compounds was determined by TLC on silica gel plates using
toluene as the mobile phase.
5.1.2.5. 1-(4-Nitrophenylhydrazono)-1,1-bis(benzimidazol-2-
yl)methane (3e). Yield 96.7%, orange solid, mp over 360 ꢀC
1
dec. H NMR (DMSO-d6): d 7.33 (t, 2H, arom, J ¼ 8.1 Hz),
7.41 (t, 2H, arom, J ¼ 8.1 Hz), 7.65 (d, 1H, arom, J ¼ 7.5 Hz),
7.90 (d, 2H, arom, J ¼ 9.0 Hz), 7.93e8.00 (m, 3H, arom),
8.31 (d, 2H, arom, J ¼ 9.0 Hz), 13.00 (bs, 1H, NH), 13.38 (bs,
1H, NH), 15.00 (s, 1H, NH). IR [cmꢁ1]: 3420, 3223, 1596,
1562, 1504, 1333, 1265, 1109, 768. MS (ESI, m/z) 398.2
[M þ H]þ. Anal. Calcd. for C21H15N7O2 (397.4): C 63.47, H
3.80, N 24.67. Found: C 63.40, H 3.74, N 24.56.
5.1.2.1. 1-Phenylhydrazono-1,1-bis(benzimidazol-2-yl)methane
(3a). Yield 92.3%, yellow solid, mp 293e295 ꢀC dec. 1H
NMR (DMSO-d6): d 7.07 (t, 1H, arom, J ¼ 8.1 Hz), 7.30 (t,
2H, arom, J ¼ 7.8 Hz), 7.37 (t, 2H, arom, J ¼ 8.1 Hz), 7.45 (t,
2H, arom, J ¼ 7.8 Hz), 7.62 (d, 1H, arom, J ¼ 7.5 Hz), 7.71
(d, 2H, arom, J ¼ 8.1 Hz), 7.84 (d, 1H, arom, J ¼
7.2 Hz), 7.90 (d, 1H, arom, J ¼ 7.8 Hz), 7.93 (d, 1H, arom,
J ¼ 7.5 Hz), 12.70 (bs, 1H, NH), 13.31 (bs, 1H, NH), 14.61 (s,
1H, NH); 13C NMR (DMSO-d6): d 111.4, 113.2, 114.5, 118.3,
118.8, 118.9, 121.7, 122.5, 122.7, 123.2, 124.2, 129.5, 131.9,
134.2, 141.6, 143.0, 143.2, 146.0, 149.9. IR [cmꢁ1]: 3456,
3220, 1598, 1537, 1492, 1262, 750. MS (ESI, m/z) 353.2
[M þ H]þ. Anal. Calcd. for C21H16N6 (352.4): C 71.58, H
4.58, N 23.85. Found: C 71.49, H 4.55, N 23.76.
5.1.3. 2-Aryl-4-(1-ethoxycarbonylbenzimidazol-2-yl)-1,2-
dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-ones (5ae5e):
general procedure
Method A: A solution of the corresponding aromatic amine
(2.00 mmol) in a mixture of ice water (5 ml) and HCl (35%,
1.5 ml) was diazotized by sodium nitrite (138 mg;
2.00 mmol) in ice water (4 ml). The mixture was stirred in
an ice bath for 15 min and then added dropwise to the solution
of compound 2 (784.8 mg; 2.00 mmol) in pyridine (45 ml),
which was cooled to 2e5 ꢀC. The mixture was left to stand
at 0e5 ꢀC for 48 h and then slowly diluted with water to a total
volume of 350 ml. The next day, the precipitated solid was col-
lected by suction, washed with water and dried. The sample for
analysis was obtained by crystallization from ethanol (for com-
pound 5a) ev. ethanolechloroform mixture. The purity of these
compounds was determined by TLC on silica gel plates using
chloroformemethanol mixture (80: 1 v/v) as the mobile phase.
Method B: To a cooled solution (2e5 ꢀC) of the corre-
sponding compound 2 (0.5 mmol) in anhydrous pyridine
(20 ml), ethyl chloroformate (0.12 ml; 1.25 mmol) was added
5.1.2.2. 1-(4-Methylphenylhydrazono)-1,1-bis(benzimidazol-2-
yl)methane (3b). Yield 85.9%, yellow-orange solid, mp
297e299 ꢀC dec. H NMR (DMSO-d6): d 2.33 (s, 3H, CH3),
7.26 (d, 2H, arom, J ¼ 8.4 Hz), 7.29e7.31 (m, 2H, arom),
7.36 (t, 2H, arom, J ¼ 7.8 Hz), 7.62 (d, 2H, arom, J ¼
8.4 Hz), 7.82 (d, 2H, arom, J ¼ 7.2 Hz), 7.87e7.95 (m, 2H,
arom), 12.70 (bs, 1H, NH), 13.27 (bs, 1H, NH), 14.58 (s,
1H, NH). IR [cmꢁ1]: 3462, 3219, 1555, 1537, 1505, 1277,
735. MS (ESI, m/z) 367.2 [M þ H]þ. Anal. Calcd. for
1

J. Sty´skala et al. / European Journal of Medicinal Chemistry 43 (2008) 449e455
453
dropwise with stirring at 2e5 ꢀC. After 1 h of stirring, the
mixture was left to stand at 2 ꢀC for 48 h. Then, it was diluted
slowly with water under stirring to a total volume of 200 ml.
The compound starts to precipitate after standing for some
hours. The precipitated solid was collected on a filter, washed
with water and crystallized.
5.1.3.5. 2-(4-Nitrophenyl)-4-(1-ethoxycarbonylbenzimidazol-2-
yl)-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one (5e).
Yield 72.4% (method A), 65.7 (method B), beige crystals,
mp 138e142 ꢀC dec. 1H NMR (CDCl3): d 1.33 (t, 3H,
CH3), 4.43 (q, 2H, CH2), 7.46e7.59 (m, 2H, arom), 7.64e
7.67 (m, 2H, arom), 7.94e7.98 (m, 2H, arom), 8.06e8.10
(m, 3H, arom), 8.41 (d, 2H, arom, J ¼ 8.4 Hz), 8.54 (t, 1H,
arom, J ¼ 4.5 Hz). IR [cmꢁ1]: 3094, 2984, 1755, 1724,
1523, 1344, 1213, 754. MS (ESI, m/z) 496.2 [M þ H]þ.
Anal. Calcd. for C25H17N7O5 (495.5): C 60.61, H 3.46, N
19.79. Found: C 60.38, H 3.70, N 19.88.
5.1.3.1. 2-Phenyl-4-(1-ethoxycarbonylbenzimidazol-2-yl)-1,2-
dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one (5a). Yield
80.2% (method A), 74.2 (method B), beige crystals, mp
187e189 ꢀC. 1H NMR (DMSO-d6): d 0.99 (t, 3H, CH3),
4.27 (q, 2H, CH2), 7.50e7.68 (m, 7H, arom), 7.77 (d, 2H,
arom, J ¼ 7.8 Hz), 7.92 (d, 1H, arom, J ¼ 7.8 Hz), 7.97 (t,
1H, arom, J ¼ 4.2 Hz), 8.11 (d, 1H, arom, J ¼ 8.1 Hz), 8.43
(t, 1H, arom, J ¼ 4.2 Hz); 13C NMR (DMSO-d6): d 13.3,
64.6, 114.6, 115.0, 120.6, 120.7, 125.1, 126.0, 126.2, 126.6,
126.8, 128.5, 128.9, 129.3, 132.3, 133.6, 139.8, 141.5,
141.9, 143.3, 143.6, 144.4, 148.8. IR [cmꢁ1]: 3070, 2988,
1755, 1719, 1337, 1216, 760. MS (ESI, m/z) 451.2
[M þ H]þ. Anal. Calcd. for C25H18N6O3 (450.5): C 66.66, H
4.03, N 18.66. Found: C 66.63, H 4.11, N 18.50.
5.1.4. 2-Aryl-4-(benzimidazol-2-yl)-1,2-dihydro[1,2,4]-
triazino[4,5-a]benzimidazol-1-ones(6ae6e): general procedure
A solution of compound 5 (0.2 mmol) in ethanol (3 ml) and
hydrochloric acid (35%; 3 ml) was refluxed for 30 min, with
precipitation from the solution. The mixture was diluted
with water (30 ml) then precipitated solid was filtered off
and washed with water. The sample for analysis was obtained
by crystallization from ethanol (about 1 mg per 3 ml ethanol).
The purity of these compounds was checked by TLC on silica
gel plate using a chloroformemethanol mixture (80:2 v/v) as
the mobile phase.
5.1.3.2. 2-(4-Methylphenyl)-4-(1-ethoxycarbonylbenzimidazol-
2-yl)-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one (5b).
Yield 79.4% (method A), 72.3 (method B), beige crystals, mp
1
218e220 ꢀC. H NMR (CDCl3): d 1.25 (t, 3H, CH3), 2.44 (s,
5.1.4.1.
2-Phenyl-4-(benzimidazol-2-yl)-1,2-dihydro[1,2,4]-
3H, CH3), 4.39 (q, 2H, CH2), 7.34 (d, 2H, arom, J ¼ 8.7 Hz),
7.44e7.55 (m, 2H, arom), 7.60 (m, 4H, arom), 7.91e7.99 (m,
2H, arom), 8.07 (d, 1H, arom, J ¼ 7.5 Hz), 7.55 (t, 1H, arom,
J ¼ 4.7 Hz). IR [cmꢁ1]: 3093, 2982, 1762, 1722, 1512, 1333,
1212, 767. MS (ESI, m/z) 465.2 [M þ H]þ. Anal. Calcd. for
C26H20N6O3 (464.5): C 67.23, H 4.34, N 18.09. Found: C
67.22, H 4.24, N 18.15.
triazino[4,5-a]benzimidazol-1-one (6a). Yield 93.8%, pale
yellow needle, mp 331e333 ꢀC. 1H NMR (DMSO-d6):
d 7.26e7.36 (m, 2H, arom), 7.51e7.75 (m, 6H, arom),
7.80e7.83 (m, 3H, arom), 8.17 (d, 1H, arom, J ¼ 7.2 Hz),
8.45 (d, 1H, arom, J ¼ 7.2 Hz), 13.04 (bs, 1H, NH); 13C
NMR (DMSO-d6): d 112.5, 115.1, 119.6, 120.7, 122.2,
123.7, 125.7, 126.4, 126.6, 128.4, 128.7, 129.5, 131.0,
134.4, 140.2, 140.7, 143.4, 143.5, 144.1, 144.6. IR [cmꢁ1]:
3309, 1713, 1366, 1220, 755. MS (ESI, m/z) 379.1
[M þ H]þ. Anal. Calcd. for C22H14N6O (378.4): C 69.83, H
3.73, N 22.21. Found: C 69.78, H 3.66, N 22.16.
5.1.3.3. 2-(4-Methoxyphenyl)-4-(1-ethoxycarbonylbenzimida-
zol-2-yl)-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one
(5c). Yield 81.3% (method A), 70.8 (method B), beige crys-
tals, mp 214e216 ꢀC. 1H NMR (CDCl3): d 1.26 (t, 3H,
CH3), 3.88 (s, 3H, OCH3), 4.39 (q, 2H, CH2), 7.05 (d, 2H,
arom, J ¼ 9.3 Hz), 7.34e7.54 (m, 2H, arom), 7.60e7.66 (m,
4H, arom), 7.90e7.98 (m, 2H, arom), 8.07 (d, 1H, arom,
J ¼ 7.5 Hz), 8.50 (t, 1H, arom, J ¼ 5.0 Hz). IR [cmꢁ1]: 3060,
2980, 1751, 1719, 1510, 1244, 1212, 762. MS (ESI, m/z)
481.1 [M þ H]þ. Anal. Calcd. for C26H20N6O4 (480.5): C
64.99, H 4.20, N 17.49. Found: C 65.07, H 4.31, N 17.33.
5.1.4.2. 2-(4-Methylphenyl)-4-(benzimidazol-2-yl)-1,2-dihydro-
[1,2,4]triazino[4,5-a]benzimidazol-1-one (6b). Yield 92.4%,
1
pale yellow needle, mp 340e342 ꢀC. H NMR (DMSO-d6):
d 2.44 (s, 3H, CH3), 7.25e7.36 (m, 2H, arom), 7.44 (d, 2H,
arom, J ¼ 8.1 Hz), 7.67e7.75 (m, 5H, arom), 7.81 (d, 1H,
arom, J ¼ 8.1 Hz), 8.16 (d, 1H, arom, J ¼ 7.0 Hz), 8.47 (d,
1H, arom, J ¼ 7.0 Hz), 13.03 (bs, 1H, NH). IR [cmꢁ1]:
3282, 1713, 1511, 1368, 1221, 743. MS (ESI, m/z) 393.1
[M þ H]þ. Anal. Calcd. for C23H16N6O (392.4): C 70.40, H
4.11, N 21.42. Found: C 70.34, H 4.19, N 21.36.
5.1.3.4. 2-(4-Chlorophenyl)-4-(1-ethoxycarbonylbenzimidazol-
2-yl)-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one (5d).
Yield 75.4% (method A), 68.1 (method B), beige crystals, mp
1
220e222 ꢀC. H NMR (CDCl3): d 1.29 (t, 3H, CH3), 4.41 (q,
5.1.4.3. 2-(4-Methoxyphenyl)-4-(benzimidazol-2-yl)-1,2-dihydro-
2H, CH2), 7.48e7.55 (m, 4H, arom), 7.63e7.65 (m, 2H,
arom), 7.74 (d, 2H, arom, J ¼ 8.7 Hz), 7.92e7.99 (m, 2H,
arom), 8.08 (d, 1H, arom, J ¼ 7.8 Hz), 8.54 (t, 1H, arom,
J ¼ 4.8 Hz). IR [cmꢁ1]: 3088, 2980, 1759, 1725, 1335, 1207,
768. MS (ESI, m/z) 485.1 [M þ H]þ. Anal. Calcd. for
C25H17ClN6O3 (484.9): C 61.93, H 3.53, N 17.33. Found: C
61.76, H 3.76, N 17.20.
[1,2,4]triazino[4,5-a]benzimidazol-1-one (6c). Yield 91.2%,
1
pale yellow needle, mp 324e326 ꢀC. H NMR (DMSO-d6):
d 3.87 (s, 3H, OCH3), 7.16 (d, 2H, arom, J ¼ 8.7 Hz), 7.29
e7.20 (m, 2H, arom), 7.65e7.78 (m, 6H, arom), 8.15 (d,
1H, arom, J ¼ 7.2 Hz), 8.44 (d, 1H, arom, J ¼ 7.2 Hz), 13.03
(bs, 1H, NH). IR [cmꢁ1]: 3324, 1714, 1510, 1219, 757. MS
(ESI, m/z) 409.1 [M þ H]þ. Anal. Calcd. for C23H16N6O2

454
J. Sty´skala et al. / European Journal of Medicinal Chemistry 43 (2008) 449e455
(408.4): C 67.64, H 3.95, N 20.58. Found: C 67.55, H 4.06,
N 20.73.
275.2 [M þ H]þ. Anal. Calcd. for C16H10N4O (274.3): C 70.07,
H 3.67, N 20.43. Found: C 69.87, H 4.01, N 20.20.
5.1.4.4. 2-(4-Chlorophenyl)-4-(benzimidazol-2-yl)-1,2-dihydro-
5.2. Biological activity
[1,2,4]triazino[4,5-a]benzimidazol-1-one (6d). Yield 96.0%,
1
pale yellow needle, mp 345e347 ꢀC. H NMR (DMSO-d6):
5.2.1. Cell lines
d 7.33e7.36 (m, 2H, arom), 7.68e7.73 (m, 5H, arom),
7.78e7.81 (m, 2H, arom), 7.87 (d, 1H, arom, J ¼ 9.0 Hz),
8.17 (d, 1H, arom, J ¼ 7.0 Hz), 8.44 (d, 1H, arom,
J ¼ 7.0 Hz), 13.04 (bs, 1H, NH). IR [cmꢁ1]: 3337, 1719,
1489, 1219, 739. MS (ESI, m/z) 413.1 [M þ H]þ. Anal. Calcd.
for C22H13ClN6O (412.8): C 64.01, H 3.17, N 20.36. Found: C
64.13, H 3.11, N 20.22.
All cells were purchased from the American Tissue Culture
Collection (ATCC), unless otherwise indicated. The daunorubi-
cin-resistant subline of CEM cells (CEM-DNR-bulk) and pacli-
taxel-resistant subline K562-Tax were selected in our laboratory
by cultivating the original cell lines in increasing concentrations
of daunorubicin or paclitaxel, respectively [28]. The cells were
maintained in Nunc/Corning 80 cm2 plastic tissue culture flasks
and cultured in DMEM/RPMI 1640 cell culture medium, with
5 g/Lglucose, 2 mMglutamine,100 U/mLpenicillin, 100 mg/mL
streptomycin, 10% fetal calf serum, and NaHCO3.
5.1.4.5. 2-(4-Nitrophenyl)-4-(benzimidazol-2-yl)-1,2-dihydro-
[1,2,4]triazino[4,5-a]benzimidazol-1-one (6e). Yield 86.6%,
lemon yellow needle, mp over 360 ꢀC. 1H NMR (DMSO-
d6): d 7.32e7.36 (m, 2H, arom), 7.0e7.73 (m, 2H, arom),
7.79e7.82 (m, 4H, arom), 8.17 (d, 2H, arom, J ¼ 9.0 Hz),
8.49 (d, 2H, arom, J ¼ 9.0 Hz), 13.08 (bs, 1H, NH). IR
[cmꢁ1]: 3314, 1715, 1524, 1353, 1219, 738. MS (ESI, m/z)
424.1 [M þ H]þ. Anal. Calcd. for C22H13N7O3 (423.4): C
62.41, H 3.09, N 23.16. Found: C 62.39, H 2.96, N 23.10.
5.2.2. Cytotoxic MTT assay [29]
Cell suspensions were prepared and diluted according to the
particular cell type and the expected target cell density (2500e
30 000 cells/well based on cell growth characteristics). The cells
were pipetted (80 mL) into 96-well microtiter plates, and cul-
tured in the wells for 24 h at 37 ꢀC in a 5% CO2 atmosphere
for stabilization. Test compounds were then added to the wells,
in 20 mL portions, to the intended concentrations and the incuba-
tions continued for further 72 h at 37 ꢀC, in a 5% CO2 atmo-
sphere at 100% humidity. The time they were added was
defined as time zero, and each concentration was tested in dupli-
cate. At the end of the incubation period, the numbers of viable
cells were assayed using MTT. Aliquots (10 mL) of the MTT
stock solution were pipetted into each well and the cells were in-
cubated for further 1e4 h. After this incubation period the for-
mazan produced was dissolved by adding 100 mL of 10% aq.
SDS (pH ¼ 5.5) to each well, followed by a further incubation
at 37 ꢀC overnight. The optical density (OD) of the mixture in
each well was then measured at 540 nm with a Labsystem
iEMS Reader MF. The tumor cell inhibitory concentration (IC)
of each test compound was calculated using the following equa-
tion: IC ¼ (ODdrug-exposed well/mean ODcontrol wells) ꢂ 100%.
The IC50 value, the drug concentration lethal to 50% of the
tumor cells, was calculated from appropriate doseeresponse
curves.
5.1.5. Ethyl 13-oxo-5H,13H-pyrimido[1,6-a:3,4-a0]bis-
(benzimidazol)-5-carboxylate (7)
A solution of compound 2 (432 mg; 1.1 mmol) in pyridine
(3 ml) and water (1.5 ml) was refluxed for 60 min. The mixture
was diluted with water (15 ml) and the precipitated solid was fil-
tered off, washed with water and dried. The crude product
(330 mg) was dissolved in chloroform (10 ml) and filtered
through Celite. The filtered solution was evaporated under vac-
uum and the residue was crystallized from ethanol. Yield
250 mg (65.6%, colorless crystals), mp 175e178 ꢀC. 1H NMR
(CDCl3): d 1.57 (t, 3H, CH3), 4.60 (q, 2H, CH2), 7.21 (s, 1H,
eCH]), 7.29e7.37 (m, 3H, arom), 7.44 (t, 1H, arom
J ¼ 7.5 Hz), 7.72 (d, 1H, arom, J ¼ 8.1 Hz), 7.96 (t, 1H, arom,
J ¼ 5.2 Hz), 8.39 (d, 1H, arom, J ¼ 8.1 Hz), 8.45 (t, 1H, arom,
J ¼ 6.3 Hz); 13C NMR (CDCl3): d 14.9, 65.5, 81.3, 115.5,
115.7, 115.9, 119.1, 123.3, 125.9, 126.5, 126.8, 128.4, 130.1,
130.4, 141.3, 143.6, 145.6, 149.7, 150.5. IR [cmꢁ1]: 3124,
2968, 1740, 1645, 1550, 1417, 1285, 748. MS (ESI, m/z)
347.1 [M þ H]þ. Anal. Calcd. for C19H14N4O3 (346.3): C
65.89, H 4.07, N 16.18. Found: C 65.54, H 4.18, N 16.01.
Acknowledgments
5.1.6. 5H,13H-Pyrimido[1,6-a:3,4-a0]bis(benzimidazol)-13-
one (8)
The study was supported from the research program of the
Ministry of Education, Youth and Sport of the Czech Republic
(MSM6198959216).
A solution of compound 7 (25.6 mg, 0.074 mmol) in etha-
nol (2 ml) and HCl (35%, 2 ml) was refluxed for 1 h. The pH
of the cooled solution was raised to 10e11 by adding a solu-
tion of NaOH. The precipitated solid was filtered off and
washed with water. Yield 15.8 mg (78.3%, colorless solid),
mp over 360 ꢀC. 1H NMR (DMSO-d6): d 6.16 (s, 1H,
eCH]), 7.28 (t, 2H, arom J ¼ 7.5 Hz), 7.41 (t, 2H, arom
J ¼ 7.8 Hz), 7.47 (d, 2H, arom, J ¼ 7.5 Hz), 8.34 (d, 2H,
arom, J ¼ 8.1 Hz), NeH proton is missing in the spectrum.
IR [cmꢁ1]: 3108, 1714, 1660, 1564, 1251, 779. MS (ESI, m/z)
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