Synthesis of Pyrimido[1,2-a]benzimidazol-4(10H)-one Derivatives and Evaluation of their Interactions with DNA

Article abstract of DOI:10.1002/jhet.5570400620

The synthesis of new derivatives of the planar tricyclic pyrimido[1,2-a]benzimidazole system featuring protonable side chains in the 3 and/or 10 positions is described. The reported literature procedures for the preparation of the intermediate 3-ethoxycarbonylpyrimido[1,2-a]benzimidazol-4(10H)2-a]benzimidazol-4(10H)-one 15, starting from 2-aminobenzimidazole 18 and diethyl ethoxymethylenemalonate, were revised. The interaction with DNA, the intrinsic binding constants, and the antiproliferative activity of a number of compounds (1-8, 10, 11) were preliminarly investigated.

Full text of DOI:10.1002/jhet.5570400620

Nov-Dec 2003
1091
Synthesis of Pyrimido[1,2-a]benzimidazol-4(10H)-one Derivatives
and Evaluation of their Interactions with DNA
1
1
1
1
1
A. Da Settimo , G. Primofiore *, F. Da Settimo , A. M. Marini , S. Taliani ,
1
2
S. Salerno and L. Dalla Via
1
Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
2
Dipartimento di Scienze Farmaceutiche, Università di Padova, Via Marzolo 5, 35131 Padova, Italy
Received August 1, 2003
The synthesis of new derivatives of the planar tricyclic pyrimido[1,2-a]benzimidazole system featuring pro-
tonable side chains in the 3 and/or 10 positions is described. The reported literature procedures for the prepa-
ration of the intermediate 3-ethoxycarbonylpyrimido[1,2-a]benzimidazol-4(10H)-one 15, starting from
2-aminobenzimidazole 18 and diethyl ethoxymethylenemalonate, were revised. The interaction with DNA,
the intrinsic binding constants, and the antiproliferative activity of a number of compounds (1-8, 10, 11) were
preliminarly investigated.
J. Heterocyclic Chem., 40, 1091 (2003).
Introduction.
ated by means of an in vitro assay using two human tumor
cell lines (HeLa and HL-60).
The ability to intercalate into DNA constitutes a funda-
mental property for numerous antitumor compounds.
Nowadays, a wide number of intercalating agents have an
established place in clinical therapy for the treatment of
cancer. Among them, the well-known drugs adriamycin
and mitoxantrone represent important examples [1-4].
The molecular requirement for the intercalation process
is the presence of a planar chromophore able to form a
freely reversible complex between base pairs, whose dri-
ving force results from a combination of electrostatic,
hydrogen-bonding, van der Waals and hydrophobic inter-
actions [2,3]. Besides the undoubted role of the intercalat-
ing portion, the presence of appropriate groups or side
chains on the planar moiety often appears to be decisive to
increase the binding capacity to DNA of a molecule, and,
in this connection, we may underline the role of protonable
groups, which are believed to be essential for the binding
of a drug to the negatively-charged nucleic acids.
Chemistry.
The intermediates for the synthesis of compounds 1-6
are represented by the heterocycle 15 and its alkylated
derivatives 16-17 (Figures 1 and 2).
In previous papers [5-7] we described some planar
polycyclic derivatives characterized by different dialky-
laminoalkyl side chains. The results obtained confirmed
the importance of the side chains for the overall cellular
effects of the intercalating nucleus. In particular, the struc-
ture-activity relationships showed that both the length and
the steric hindrance of the side chains are decisive for
cytotoxicity.
With the aim of studying new chemical structures with a
planar chromophore bearing protonable groups, in this
paper we describe the synthesis and the biological evalua-
tion of pyrimido[1,2-a]benzimidazol-4(10H)-one deriva-
tives 1-14, which feature various basic side chains in posi-
tions 3 and/or 10.
Linear flow dichroism experiments allowed us to study
the interaction of compounds 1-8, 10, 11 with DNA and to
establish their mode of binding. Furthermore, fluorimetric
titration was performed to obtain the intrinsic binding con-
stant values (K ). The capacity of the new compounds to
i
exert an antiproliferative activity was preliminary evalu-
Figure 1

1092
A. Da Settimo, G. Primofiore, F. Da Settimo, A. M. Marini, S. Taliani,
S. Salerno and L. Dalla Via
Vol. 40
The pyrimidobenzimidazole derivative 15 was obtained
The synthetic procedure employed to prepare the pyrim-
idobenzimidazoles 7-10, bearing the basic chain in posi-
tion 10, involved condensation of the appropriate 1-
aminoalkyl-2-aminobenzimidazole 22-25 [5,6] with
diethyl ethoxymethylenemalonate by heating at reflux in a
solution of ethanol (Figure 3). For the synthesis of deriva-
tives 11-14, which bear two basic chains in their structure,
compounds 7-10 were heated at reflux in an excess of the
appropriate diamine (Figure 3).
by referring to an experimental procedure described in the
literature [8], which consisted of the condensation of 2-
aminobenzimidazole 18 with diethyl ethoxymethylene-
malonate in refluxing ethanol (Figure 1). The authors
report that from this reaction they directly obtain the 3-
ethoxycarbonylpyrimido[1,2-a]benzimidazol-4(10H)-one
15, which was characterized by analytical and spectral
data [8].
When we repeated this procedure, we obtained a white
solid product which we identified as the imino derivative 21
(Figure 1 and experimental section). The structure of 21 was
unequivocally confirmed by the analytical and spectral data.
1
In particular, the H nmr spectrum of 21 showed two multi-
plets, each integrating for two protons at δ 6.97-7.02 and
7.19-7.23 relative to the pendant benzimidazole nucleus,
distinctly separated from the aromatic envelope of the
pyrimidobenzimidazole system, which exhibited its signals
between δ 7.25 and 8.65 (see experimental section).
We also obtained the same product 21 when the reaction
was carried out following two other reported procedures: a
mixture of 2-aminobenzimidazole 18 and diethyl
ethoxymethylenemalonate was heated at 100° for 5 min-
utes, as described in reference [9], or at 110° for 30 min-
utes, as described in reference [10].
Figure 3
By acid hydrolysis with dilute hydrochloric acid at 50°,
the imino derivative 21 gave compound 15 with a high
yield (Figure 1). The ir and H nmr data shown by our
All products 1-14 were purified by recrystallization
from ethanol and their structures were confirmed by ir, ms,
H nmr and elemental analyses (Tables 1 and 2).
For the biological assays, compounds 1-8, 10, 11 were
converted into their hydrochlorides by treatment with
hydrogen chloride-saturated ethanol, for an easier solubi-
lization in the test media.
1
1
product 15 are in perfect agreement with those reported in
the literature [8-10]. Probably the authors obtain 21 as
their crude product, which in the recrystallization process
undergoes a hydrolysis reaction to compound 15. Actually,
a pure sample of compound 15 was obtained when we
recrystallized product 21 from acetic acid [9,10].
In the reaction of 19-20 [11] with diethyl ethoxymethyl-
enemalonate in refluxing ethanol, the target esters 16 [12]
and 17 [13] were directly obtained (Figure 1).
By heating at reflux in an excess of N,N-dimethylethyl-
enediamine or N,N-diethylethylenediamine for 12 hours,
the ethoxycarbonyl compounds 15-17 were converted to
the corresponding amide derivatives 1, 3, 5, and 2, 4, 6,
respectively (Figure 2).
Biological Results.
The ability of the new derivatives 1-8, 10, 11 to interact
with DNA was investigated by means of both flow linear
dichroism and fluorimetric titration measurements follow-
ing the experimental procedures previously described [7].
For all the considered compounds, a dichroic signal at
wavelengths higher than 260 nm (310-370 nm) appeared,
thus indicating the occurrence of a molecular complex
with the macromolecule [5]. To investigate the geometry
of binding, the calculation of the average orientation angle
α
was performed [14,15] and the resulting values are
L
shown in Table 3. The values of α (80°-83°) obtained for
L
3, 4 and 11 are considered consistent with an intercalative
mode of binding [16], whilst for the others derivatives the
obtained values (64°-77°) indicate a divergence from co-
planarity with DNA base plane, probably attributable to
the occurrence of both intercalative and groove geometry
of binding [6]. In addition, the new derivatives exert a sig-
nificant fluorescence signal, which is quenched upon addi-
tion of DNA. This property allowed for the determination
Figure 2

Nov-Dec 2003
Synthesis of Pyrimido[1,2-a]benzimidazol-4(10H)-one Derivatives
1093
Table 1
Physical Properties of Pyrimido[1,2-a]benzimidazole Derivatives 1-14
Compd.
R
R
Yield Mp [a]
Formula
Elemental Analysis
Calcd. %
1
(%)
(°C)
Found %
H
C
H
N
C
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
H
H
CONH(CH ) N(CH )
37
35
70
41
79
68
87
90
64
82
53
42
32
37
145-146
60-63
C
C
C
C
C
C
C
C
C
C
C
C
C
C
H
N O
60.19
62.37
61.33
63.32
67.85
69.21
62.18
63.14
64.03
64.85
61.60
63.29
64.76
66.05
5.72
6.47
6.11
6.79
5.95
6.29
6.14
6.48
6.79
7.07
7.07
7.59
8.03
8.42
23.40
21.39
22.35
20.51
17.98
16.81
17.06
16.36
15.72
15.12
22.68
21.09
19.70
18.48
59.94
62.04
61.09
63.13
67.68
69.00
61.98
62.98
63.90
64.82
61.32
63.01
64.52
65.82
5.93
6.24
6.35
6.93
6.13
6.45
6.16
6.38
6.51
7.12
7.16
7.49
7.97
8.60
23.37
21.37
22.13
20.60
17.92
17.07
16.99
16.11
15.63
14.91
22.42
21.08
19.55
18.44
2 2
3 2
15 17
5
2
2
2
2
2
2
3
3
3
3
2
2
2
2
CONH(CH ) N(C H )
H N O
2 2
2
5 2
17 21 5
CH
CH
CH C H
CH C H
(CH ) N(CH )
(CH ) N(CH )
(CH ) N(C H ) COOC H
(CH ) N(C H ) COOC H
(CH ) N(CH )
CONH(CH ) N(CH )
159-161
115-117
188-190
135-137
150-152
138-140
133-135
105-107
168-170
110-112
73-75
H N O
16 19 5
3
3
2 2
3 2
CONH(CH ) N(C H )
H N O
2 2
2
5 2
18 23 5
CONH(CH ) N(CH )
H N O
22 23 5
2
6
5
2 2
3 2
CONH(CH ) N(C H )
H N O
2
6
5
2 2
2
5 2
24 26 5
COOC H
H N O
17 20 4
2 2
3 2
2
5
5
5
5
COOC H
H N O
2 3
3 2
2
18 22 4
H N O
19 24 4
2 2
2
5 2
2
H N O
2 3
2
5 2
2
20 26 4
CONH(CH ) N(CH )
H N O
19 26 6
2 2
3 2
2 2
3 2
(CH ) N(CH )
(CH ) N(C H ) CONH(CH ) N(C H )
(CH ) N(C H ) CONH(CH ) N(C H )
CONH(CH ) N(CH )
H N O
2 3
3 2
2 3
3 2
21 30 6
H N O
23 34 6
2 2
2
5 2
2 2
2
5 2
106-108
H N O
2 3
2
5 2
2 3
2
5 2
25 38 6
[a] Recrystallization solvent: ethanol.
methylpyrimido[1,2-a]benzimidazol-4(10H)-one 16 [12], using
ethanol as the reaction solvent; 1-(2-dimethylaminoethyl)-2-
aminobenzimidazole 22 [6]; 1-(3-dimethylaminopropyl)-2-
aminobenzimidazole 23 [5]; 1-(2-diethylaminoethyl)-2-
aminobenzimidazole 24 [5]; 1-(3-diethylaminopropyl)-2-amino
benzimidazole 25 [5].
of intrinsic binding constants whose values appeared in the
5
5
-1
range from 0.34x10 to 0.60x10 (M ). Cellular growth
inhibition assays on two human tumor cell lines (HeLa e
HL-60) [7] revealed that all the considered compounds are
unable to exert a cytotoxic effect, despite their ability to
form a molecular complex with DNA. On the basis of
these results, it is possible to conclude that, despite an
intrinsic capacity of compounds 1-8, 10, 11 to interact with
DNA by forming a molecular complex, their low affinity
for the macromolecule can be considered responsible for
their inability to exert a detectable antiproliferative effect.
4-(Benzimidazol-2-ylimino)-3-ethoxycarbonyl-10H-pyrim-
ido[1,2-a]benzimidazole (21).
A solution of 2-aminobenzimidazole 18 (5.00 g, 37.5 mmol)
and diethyl ethoxymethylenemalonate (6.60 mL, 33.0 mmol) in
140 mL of ethanol was heated at reflux for 5 hours. After cooling,
the separated white solid was collected, washed with ethanol and
purified by recrystallization from DMF to give 6.38 g of the pure
imino derivative 21 (52%), mp 293-295° (dec); ir: 3250, 3100,
EXPERIMENTAL
-1
1
1700, 1600, 1550, 1240, 780 cm ; H nmr: δ 1.38 (t, 3H, CH , J
3
= 7.1 Hz); 4.30 (q, 2H, CH , J = 7.1 Hz); 6.97-7.02 (m, 2H, 5'-,
Melting points were determined using a Reichert Köfler hot-
stage apparatus and are uncorrected. Infrared spectra were
recorded with a PYE/UNICAM Infracord Model PU 9516 spec-
trophotometer in Nujol mulls. Routine nuclear magnetic reso-
2
and 6'-H); 7.19-7.23 (m, 2H, 4'-, and 7'-H); 7.25-7.33 (m, 1H, 8-
H); 7.38-7.47 (m, 1H, 7-H); 7.56 (dd, 1H, 9-H, J = 7.4, 0.5 Hz);
8.49 (dd, 1H, 6-H, J = 7.4, 0.7 Hz); 8.65 (s, 1H, 2-H); ms: m/z
257 (22), 212 (73), 133 (100).
nance spectra were recorded in DMSO-d solution on a Varian
6
Gemini 200 spectrometer operating at 200 MHz. Mass spectra
were obtained on a Hewlett-Packard 5988 A spectrometer using a
direct injection probe and an electron beam energy of 70 eV.
Evaporation was performed in vacuo (rotary evaporator).
Analytical tlc was carried out on Merck 0.2 mm precoated silica
gel aluminum sheets (60 F-254). Elemental analyses were per-
formed by our Analytical Laboratory and agreed with theoretical
values to within ± 0.4%.
Anal. Calcd. for C
Found: C, 64.31; H, 4.58; N, 22.33.
The same derivative 21 was also obtained following the syn-
thetic procedure described in references [9] and [10].
Recrystallization of 21 from AcOH gave a pure sample of com-
pound 15.
H N O : C, 64.51; H, 4.33; N, 22.57.
20 16 6 2
3-Ethoxycarbonylpyrimido[1,2-a]benzimidazol-4(10H)-one (15).
The following compounds were prepared in accordance with
reported procedures: 1-methyl-2-aminobenzimidazole 19 [11]; 1-
benzyl-2-aminobenzimidazole 20 [11]; 3-ethoxycarbonyl-10-
A suspension of 21 (3.91 g, 10.5 mmol) in 80 mL of dilute
hydrochloric acid was heated at 50° for 5 hours. After cooling, the
separated white solid was collected and purified by recrystallization

1094
A. Da Settimo, G. Primofiore, F. Da Settimo, A. M. Marini, S. Taliani,
S. Salerno and L. Dalla Via
Vol. 40

Nov-Dec 2003
Synthesis of Pyrimido[1,2-a]benzimidazol-4(10H)-one Derivatives
1095
Table 3
Calculated Values of the Average Orientation
Angle α for the Test Compounds 1-8, 10,
L
-3
11.[DNA]=1.6x10 M, [DNA]/[drug]=12.5
Compounds
α
L
1
2
3
4
5
6
7
8
10
11
68
68
80
82
64
65
76
73
77
83
from ethanol to give 2.56 g of pure 15 (95%), mp 292-293° (lit.
ref. [8]: mp 292-295°).
10-Benzyl-3-ethoxycarbonylpyrimido[1,2-a]benzimidazol-
4(10H)-one (17).
A solution of 2-amino-1-benzylbenzimidazole 20 (8.36 g, 37.5
mmol) and diethyl ethoxymethylenemalonate (6.60 mL, 33.0
mmol) in 140 mL of ethanol was heated at reflux for 7 hours. After
cooling, the separated white solid was collected, washed with
ethanol and purified by recrystallization from ethanol to give 8.85
g of pure 17 (68%), mp 192-194°; ir: 1730, 1650, 1520, 1280,
-1
1
1130, 700 cm ; H nmr: δ 1.30 (t, 3H, CH , J = 7.1 Hz); 4.26 (q,
3
2H, CH CH , J = 7.0 Hz); 5.63 (s, 2H, CH Ph); 7.30-7.57 (m, 7H,
2
3
2
ArH); 7.71 (d, 1H, 9-H, J = 7.2 Hz); 8.58 (d, 1H, 6-H, J = 7.0 Hz);
+
8.76 (s, 1H, 2-H); ms: m/z 347 (M , 4.7), 302 (3.7), 91 (100).
Anal. Calcd for C
H N O : C, 69.15; H, 4.93; N, 12.10.
20 17 3 3
Found: C, 68.99; H, 5.12; N, 12.04.
General Procedure for the Synthesis of 10-Substituted 3-[(N,N-
Dialkylaminoethyl)aminocarbonyl]pyrimido[1,2-a]benzimida-
zol-4(10H)-ones (1-6).
A suspension of 2.0 mmol of the appropriate 3-ethoxycarbonyl
pyrimido[1,2-a]benzimidazol-4-one 15-17 in an excess (44.0
mmol) of N,N-dimethylethylenediamine or N,N-diethylethylene-
diamine was heated at reflux for 6-12 hours (tlc analysis).
For derivatives 1-2, the solution obtained was evaporated in
vacuo to dryness. The oily residue was treated with acetone and
then filtered to give crude compounds 1-2, which were purified
by recrystallization from ethanol (Tables 1 and 2).
For derivatives 3-6, after cooling, the suspension obtained was
filtered to give the desired crude products. The filtrate was con-
centrated and the separated precipitate was collected to yield an
additional amount of crude products 3-6. The quantities of deriv-
atives 3-6 obtained from the initial insoluble precipitate or from
the concentrated solution were variable, depending upon the sol-
ubility of the various derivatives. The crude products 3-6 were
purified by recrystallization from ethanol (Tables 1 and 2).
General Procedure for the Synthesis of 3-Ethoxycarbonyl-10-
(N,N-dialkylaminoalkyl)pyrimido[1,2-a]benzimidazol-4(10H)-
ones (7-10).
Diethyl ethoxymethylenemalonate 0.45 mL (2.2 mmol) was
added to a solution of the appropriate 1-dialkylaminoalkyl-2-
aminobenzimidazole 22-25 (2.5 mmol) in ethanol and the reac-

1096
A. Da Settimo, G. Primofiore, F. Da Settimo, A. M. Marini, S. Taliani,
S. Salerno and L. Dalla Via
Vol. 40
[3] B. C. Baguley, Anti-Cancer Drug Des., 6, 1 (1991).
[4] D. Faulds, J. A. Balfour, P. Chrisp and H. D. Langtry, Drugs,
41, 440 (1991).
[5] A. Da Settimo, F. Da Settimo, A. M. Marini, G. Primofiore, S.
Salerno, G. Viola, L. Dalla Via and S. Marciani Magno, Eur. J. Med.
Chem., 33, 685 (1998).
[6] L. Dalla Via, O. Gia, S. Marciani Magno, A. Da Settimo, A.
M. Marini, G. Primofiore, F. Da Settimo and S. Salerno, Farmaco, 56,
159 (2001).
tion mixture was heated at reflux for 12 hours. After cooling,
the solution obtained was concentrated to precipitate the desired
crude products 7-10, which were collected and purified by
recrystallization from ethanol (Tables 1 and 2).
General Procedure for the Synthesis of 3-[(N,N-Dialkylamino-
alkyl) aminocarbonyl]-10-(N,N-dialkylaminoalkyl)pyrimido[1,2-
a]benzimidazol-4(10H)-ones (11-14).
A solution of 1.22 mmol of compounds 7-10 in an excess (13.4
mmol) of the appropriate diamine was heated at reflux for 5-12
hours (tlc analysis). After cooling, the suspension obtained was
filtered to give the crude compounds 11-14, which were purified
by recrystallization from ethanol (Tables 1 and 2).
[7] L. Dalla Via, O. Gia, S. Marciani Magno, A. Da Settimo, G.
Primofiore, F. Da Settimo, F. Simorini and A. M. Marini, Eur. J. Med.
Chem., 37, 475 (2002).
[8] H. Ogura, M. Kawano and T. Itoh, Chem. Pharm. Bull., 21,
2019 (1973).
[9] A. Richardson and F. J. McCarthy, J. Med. Chem., 15, 1203
(1972).
Acknowledgments.
[10] A. W. Chow, D. R. Jakas, B. P. Trotter, N. M. Hall and J. R. E.
Hoover, J. Heterocyclic Chem., 10, 71 (1973).
[11] P. Caroti, C. Ceccotti, A. Da Settimo, F. Palla and G.
Primofiore, J. Heterocyclic Chem., 23, 1833 (1986).
[12] J. J. Wade, R. F. Hegel and C. B. Toso, J. Org. Chem., 44,
1811 (1979).
This work was financially supported by MIUR (cofin 2002, ex
40%).
REFERENCES AND NOTES
[13] T. Denzel and H. Hoehn, US Patent 4,109,087 (1978); Chem.
Abstr., 90, 87510w (1979).
*
To whom correspondence should be addressed; E-mail: pri-
[14] B. Nordén, Appl. Spectrosc. Rev., 14, 157 (1978)
[15] B. Nordén, M. Kubista and T. Kurucsev, Q. Rev. Biophys., 25,
51 (1992)
[16] U. Sehlstedt, S. K. Kim, P. Carter, J. Goodisman, J. F. Vollano,
B. Nordén and J. C. Dabrowiak, Biochemistry, 33, 417 (1994).
mofiore@farm.unipi.it; Tel: +39-050-500209; Fax: +39-050-40517.
[1] W. A. Denny, Anti-Cancer Drug Des., 4, 241 (1989).
[2] L. P. G. Wakelin and M. J. Waring in Comprehensive
Medicinal Chemistry, Vol. 2, P. G. Sammes and J. B. Taylor Eds,
Pergamon Press, Oxford, 1990, pp. 703-723.