Synthesis and characterization of some novel 2-(trifluoromethyl)pyrimido- [1,2-a]benzimidazoles and pyrimido[1,2-a]benzimidazol-2H-ones of biological interest

Article abstract of DOI:10.1055/s-2006-942444

The synthesis of some potentially active 2-(trifluoromethyl)pyrimido [1,2-a]benzimidazoles and pyrimido[1,2-a]benzimidazol-2(1H)-ones by the cyclization of 4-alkoxyvinyl trifluoro(chloro)methyl ketones with 2-aminobenzimidazole is described. The structure

Full text of DOI:10.1055/s-2006-942444

PAPER
2305
Synthesis and Characterization of Some Novel 2-(Trifluoromethyl)pyrimido-
[1,2-a]benzimidazoles and Pyrimido[1,2-a]benzimidazol-2H)-ones of
Biological Interest
S
ynthesis of Some
i
N
ove
l
l
Pyrimi
o
do[1, 2-
a
]benzimid
Z
azole
s
anatta,*a Simone S. Amaral,a Andressa Esteves-Souza,b Aurea Echevarria,*b Patrícia B. Brondani,a
Darlene C. Flores,a Helio G. Bonacorso,a Alex F. C. Flores,a Marcos A. P. Martinsa
a
Núcleo de Química de Heterociclos (NUQUIMHE), Departamento de Química, Universidade Federal de Santa Maria,
97.105-900, Santa Maria, RS, Brazil
Fax +55(55)2208031; E-mail: zanatta@base.ufsm.br
Departamento de Química, Universidade Federal Rural do Rio de Janeiro, 23851-970, Seropédica, RJ, Brazil
b
E-mail: echevarr@ufrrj.br
Received 24 February 2006
traditional methods to prepare pyrimidobenzimidazoles:
Abstract: The synthesis of some potentially active 2-(trifluoro-
methyl)pyrimido[1,2-a]benzimidazoles and pyrimido[1,2-a]benz-
imidazol-2(1H)-ones by the cyclization of 4-alkoxyvinyl
trifluoro(chloro)methyl ketones with 2-aminobenzimidazole is de-
(i) they are easily prepared by acylation of enol ethers13
and acetals,14 (ii) the reactions are more regioselective
than 1,3-dicarbonyl compounds, and (iii) they allow one
scribed. The structure of the products was assigned based on 1H and to introduce a wider range of substituents in the final prod-
13C NMR as well as 2D-NMR experiments. Some of the obtained
uct, compared with propiolic esters and diethyl ethoxy-
products exhibited significant DNA-topoisomerase I inhibitory ac-
tivity.
methylenemalonates. In addition, considering that two
possible structures can be formed in the cyclization step,
a concise characterization of the regioisomers, based on
modern NMR spectral data such as 2D HMBC and NOE-
SY, is limited in the literature.15
Key words: 2-aminobenzimidazole,
pyrimidobenzimidazole,
enones, halogenated heterocycles, DNA-topoisomerase I inhibitory
activity
The DNA-topoisomerases are ubiquitous nuclear en-
zymes that play crucial roles in DNA metabolism events
such as replication, transcription, recombination, repair,
chromatin assembly and chromosome segregation.16,17
DNA-topoisomerase type I enzymes are monomeric and
catalyze an ATP-independent relaxation of DNA super-
coils by transiently breaking and relegating single-strand-
ed DNA. Topoisomerase inhibitors have gained wide
clinical significance due to their efficacy as the principal
intracellular targets of important antimicrobial and antitu-
mor agents.18
Imidazopyrimidines and their analogues have been found
to be of pharmacological interest for a long time. More
specifically, imidazo[1,2-a]pyrimidine derivatives have
already been described as benzodiazepine receptor ago-
nists,1 as well as antiviral,2 antitumor,3 and antimicrobial
agents,4 and calcium-channel blockers.5
Trifluoromethyl-substituted pyrimidines and their con-
densed cyclic derivatives are well known to possess great
importance in the medicinal and agricultural fields.6 Py-
rimidobenzimidazoles bearing a trifluoromethyl group
have been synthesized from the cyclization reaction of 2-
aminoazole with trifluoromethylated b-dicarbonyl com-
pounds,7 3-trifluoroacetyl lactams,8 4-trifluoroacetyl-2,3-
dihydropyrroles9 or b-trifluoroacetyl vinyl sulfones.6
Based on these facts, and as an extension of the research
developed in our laboratory,19 we wish to report the syn-
thesis and structure assignment of some novel 2-(trifluo-
romethyl)pyrimido[1,2-a]benzimidazoles and pyrimido-
[1,2-a]benzimidazol-2(1H)-ones from the cycloconden-
sation reactions of 2-aminobenzimidazole hydrobromide
(2) and 2-aminobenzimidazole (3) with 4-alkoxyvinyl tri-
halomethyl ketones 1a–f and 5a–e. The obtained products
were tested for DNA-topoisomerase inhibitory activity.
Pyrimido[1,2-a]benzimidazol-2-ones have been synthe-
sized by the reaction of propiolic esters10 and a,b-unsatur-
ated esters10a,11 with 2-aminobenzimidazole. Other
synthetic methods to obtain pyrimidobenzimidazoles with
other type of substituents, not directly related to this work,
have also been reported.12
Scheme 1 outlines the synthesis of novel 2-(trifluorome-
thyl)pyrimido[1,2-a]benzimidazoles 4–f from the cycliza-
tion reaction of 4-alkoxyvinyl trifluoromethyl ketones
1a–f with 2-aminobenzimidazoles 2 and 3. The best con-
ditions to obtain the trifluoromethylpyrimidines 4a–f
were achieved using 2-aminobenzimidazole hydrobro-
mide (2) with triethylamine in toluene (acetonitrile for
4e). This procedure promoted a significant reduction in
reaction times (see Table 1), if compared with the cycliza-
tion using the free base, 2-aminobenzimidazole (3). We
believe that the triethylamine salt formed in situ when the
Despite the variety of methods available for the synthesis
of pyrimidobenzimidazoles,10–12 the synthetic potential of
4-alkoxy-1,1,1-trihaloalk-3-ene-2-ones 1a–f and 5a–e as
precursors of this class of heterocycles has not been tested
yet. Enones 1 and 5 have the following advantage over the
SYNTHESIS 2006, No. 14, pp 2305–2312
x
x.
x
x
.
2
0
0
6
Advanced online publication: 28.06.2006
DOI: 10.1055/s-2006-942444; Art ID: M00906SS
© Georg Thieme Verlag Stuttgart · New York

2306
N. Zanatta et al.
PAPER
benzimidazole hydrobromide 2 was used increases the Table 1 Preparation of 2-(Trifluoromethyl)pyrimido [1,2-a]benz-
imidazoles 4a–f
medium polarity, allowing a more efficient dehydration
that occurs on the last step of the formation of products 4
Compound 2-Aminobenzimidazole Time (h)a Yield (%) Product
and, as a consequence, the reaction proceeded faster than
when the free base benzimidazole (3) was used. In both
cyclizations, the reaction was regiospecific, showing the
formation of only the 2-trifluoromethylpyrimido[1,2-
a]benzimidazole derivatives, except for the compound 4c
that furnished the 4-trifluoromethylpyrimido isomer in
smaller amount (70:30%, respectively). The reaction
mechanism suggests a Michael addition of an imidazo ni-
trogen on the b-carbon of the 4-alkoxyvinyl trifluoro-
methyl ketones 1a–f, followed by the attack of the
exocyclic amino group of the 2-aminobenzimidazole on
the carbonyl of the ketones 1a–f, as the general five- and
six-membered cyclization mechanism proposed by the re-
action of 4-alkoxyvinyl trifluoromethyl ketones with di-
nucleophiles of the N–N and N–C–N type, respectively.20
1a
1a
1b
1b
1c
1c
1d
1e
1f
2
3
2
3
2
3
2
2
2
1
95
95
80
4a
4a
4b
4b
4cc
4cc
4d
4e
2
6
b
72
10
72
24
72
60
–
70
b
–
65
25
65
4f
a Reaction condition: toluene (MeCN for 4e), Et3N, reflux.
b Incomplete reaction.
10
N
9
9a
5a
c Regioisomer mixture: 4c/4c¢ (70:30%, respectively).
R2
OR3
R1
8
7
i
10a
N 1
2
25–95%
N
O
5
CF3
6
before. Thus, this work additionally aims to explore the
synthetic potential of the enones 5a–e for the synthesis of
pyrimido[1,2-a]benzimidazol-2(1H)-ones. The cycliza-
tion reaction of 5a–e with 2-aminobenzimidazoles 2 and
3 furnished different compounds and regioisomers de-
pending on the substituents of the enones 5a–e (e.g. R1
and R2) and the reaction conditions. Table 2 shows the
composition products obtained as a function of different
enone structures and reaction conditions.
3
4
CF3
R1
R2
1a–f
4a–f
4e: R2 = (CH2)2OH
4f: R2 = (CH2)3OH
Compound R1 R2 R3
a
b
c
d
e
f
H
H
Et
H
Me Et
Me
Ph
H
H
H
Me
Me
-(CH2)2-
-(CH2)3-
H
The results show that, in general, the cyclization of 5 with
the 2-aminobenzimidazole free base 3 gave better yields
than its hydrobromide form 2 but the reaction times when
using either 2 or 3 were basically the same. One can also
observe that substituents in both R1 and R2 positions, such
as in 5b–d, increased the reaction times and raised the
possibility of regioisomer formation. An interesting result
was given by the reaction of the enone 5a with 2-ami-
nobenzimidazole (3). When this reaction was carried out
in toluene it gave only the pyrimido-2-one derivative 6a
but, when carried out in acetonitrile, only the dihydropy-
rimido-2-one product 8a was obtained; both products 6a
and 8a, in high yields. For the reaction of the enones 5b–
d with 2 or 3, a mixture of isomers 6 and 7 was usually ob-
tained, with the reaction favoring the formation of com-
pounds 6 (Table 2). In our hands, the mixture of products
6c/7c and 6d/7d could not be isolated. For these com-
pounds only the NMR data (taken from the mixture of iso-
mers) of the major compounds 6c and 6d were reported.
The composition product of the reaction of the enone 5e
Scheme 1 Reagents and conditions: i) 2-aminobenzimidazole hy-
drobromide (2) or 2-aminobenzimidazole (3), Et3N, toluene or
MeCN, reflux, 1–72 h.
Scheme 2 shows the reaction of 4-alkoxyvinyl trichlo-
romethyl ketones 5a–f with 2-aminobenzimidazoles 2 and
3. The reactions were carried out using the same condi-
tions for the trifluorinated analogues 1a–f. The cyclization
was achieved through the elimination of the trichloro-
methyl group, giving pyrimido[1,2-a]benzimidazol-2(1H)-
ones 6 and 7 or the dihydropyrido analogues 8 as the reac-
tion products. The elimination of the trichloromethyl
group in the cyclization reaction of 4-alkoxyvinyl trichlo-
romethyl ketones with other amidine derivatives has been
reported previously.21 Although the synthesis of pyrimi-
do[1,2-a]benzimidazol-2(1H)-ones has been extensively
explored,10–12 4-alkoxyvinyl trichloromethyl ketones 5a–
e had never been used as precursors of these compounds
R2
OR3
R1
N
N
N
N
N
NH
R2
NH
NH
R2
i
N
+
R1
O
O
O
R3O
CCl3
R1
O
H
R1
R2
5a–e
6
7
8
Scheme 2 Reagents and conditions: i) 2-aminobenzimidazole hydrobromide (2) or 2-aminobenzimidazole (3), Et3N, reflux, 0.25–72 h.
Synthesis 2006, No. 14, 2305–2312 © Thieme Stuttgart · New York

PAPER
Synthesis of Some Novel Pyrimido[1,2-a]benzimidazoles
2307
Table 2 Preparation of Pyrimido[1,2-a]benzimidazol-2(1H)-ones 6–8
Compound R1
R2
R3
Benzimidazole Reaction conditionsa
Productsb
Yield (%)c
6
7
8
5a
5a
5a
5a
5b
5b
5b
5b
5c
5c
5d
5d
5e
5e
5e
5e
H
H
Et
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
toluene, reflux, 0.5 h
toluene, reflux, 2 h
MeCN, r.t., 0.25 h
MeCN, r.t., 0.25 h
toluene, reflux, 24 h
toluene, reflux, 16 h
MeCN, reflux, 24 h
MeCN, reflux, 24 h
toluene, reflux, 48 h
toluene, reflux, 48h
toluene, reflux, 24h
toluene, reflux, 24 h
toluene, reflux, 72 h
toluene, reflux, 72 h
MeCN, reflux, 8 h
MeCN, reflux, 1 h
100
100
–
–
–
90
90
80
90
25
65
6
H
H
Et
–
–
H
H
Et
–
100
100
–
H
H
Et
–
–
H
Me
Et
70
70
100
100
70
70
70
70
–
30
30
–
H
Me
Et
–
H
Me
Et
–
H
Me
Et
–
–
20
25
60
14
50
25
Me
Me
Ph
Ph
H
H
Et
30
30
30
30
–
H
Me
Me
Me
–
H
H
–
-(CH2)3-
-(CH2)3-
-(CH2)3
-(CH2)3-
100
–
d
H
100
–
–
–
H
–
100
–
12
60
H
100
–
a Molar proportion between reagents 5a–e/Et3N/2-aminobenzimidazole) = 1:1:1.
b Isolated isomer proportion (%).
c Isolated yield.
d Incomplete reaction.
with 2 and 3 seems to be more sensitive to the 2-ami- 4a,b and 6a,b. Deshielded hydrogens attached to shielded
nobenzimidazoles 2 and 3 used. When the free base 2- carbons in both the 4 and 6 positions of compounds 4 and
aminobenzimidazole (3) was used, only product 6e was 6 suggest a steric hindrance effect between H-4 and H-6.24
isolated and, when the 2-aminobenzimidazole hydrobro-
Table 3 1H and 13C NMR Data for Compounds 4a,b, 6a,b and Stan-
dard I and II
mide (2) was used, only product 8e was obtained.
In order to determine the pyrimido[1,2-a]benzimidazole
1
structure, H and 13C NMR data of 4a,b and 6a,b were
Compound
d
compared with the NMR data of representative com-
pounds, such as 2-amino-4-trifluoromethyl pyrimidine
(standard I)22 and 5-H-thiazolo[3,2-a] pyrimidin-5-one
(standard II),20a to be used as the model compounds for the
H-4 and C-4 nuclei of 4a,b and 6a,b (Table 3). The 2-ami-
no-N-methylbenzimidazole (standard III)23 was taken as
the model of comparison of the same molecular residue of
the pyrimido[1,2-a]benzimidazoles (Table 4). Table 3
shows that the H-6 of standard I and the H-7 of standard
II are, on average, 1.3 ppm more shielded than the corre-
sponding H-4 of compounds 4a,b and 6a,b. Table 3 also
shows that the C-6 of standard I and the C-7 of standard II
are, on average, 24.4 ppm more deshielded than the corre-
sponding C-4 of the compounds 4a,b and 6a,b. Standard
III (Table 4) shows that the H-7 and C-7 of 2-aminobenz-
imidazole have a trend similar to that shown by the pyrim-
ido moiety. Here, H-7 is also more shielded and C-7 is
more deshielded than the corresponding H-6 and C-6 of
H-n
C-n
8.5 (H-6)
161.4 (C-6)
CF3
H
4
3N
5
2
6
1
H
N
NH2
Standard I22
4a
9.91 (H-4)
9.74 (H-4)
7.93 (H-7)
139.4 (C-4)
137.8 (C-4)
160.6 (C-7)
4b
O
Me
H
N
7
S
N
Standard II20a
6a
9.79 (H-4)
8.73 (H-4)
138.8 (C-4)
130.5 (C-4)
6b
Synthesis 2006, No. 14, 2305–2312 © Thieme Stuttgart · New York

2308
N. Zanatta et al.
PAPER
Table 4 1H and 13C NMR Data for Compounds 4a,b, 6a,b and Stan-
dard III
thesized from the N-alkylation of 6a with 4,4-diethoxy-
1,1,1-trifluorobut-3-en-2-one (9) using potassium carbon-
ate in acetone. Compound 6a was brominated using NBS
in chloroform to give compound 11 (Scheme 3).
Compound
d
H-n
C-n
The alkylation reaction of 6e furnished a mixture of N-
alkylated and O-alkylated products. In order to obtain
only the N-alkylated product of 6e, the OH group was first
protected using p-toluenesulfonyl chloride and pyridine in
acetonitrile to give compound 12. Without further purifi-
cation, compound 13 was synthesized through the N-alky-
lation reaction of 12 with 4,4-diethoxy-1,1,1-trifluorobut-
3-en-2-one (9) using potassium carbonate in acetone
(Scheme 4).
7.70 (H-7)
115.4 (C-7)
N
4
5
6
3
NH2
2
1
7
N
Me
Standard III23
4a
8.48 (H-6)
8.35 (H-6)
8.42 (H-6)
7.85 (H-6)
113.4 (C-6)
113.1 (C-6)
113.7 (C-6)
109.9 (C-6)
4b
6a
6b
F3C
EtO
N
N
N
O
N
N
NH
ii
i
Furthermore, these two hydrogens should also experience
deshielding by a ring current effect; H-4 from the benzim-
idazole portion and H-6 from the pyrimido residue.24 This
trend was considered to be the key information in assign-
ing the correct structure of the title compounds as 2-(tri-
fluoromethyl)pyrimido[1,2-a]benzimidazoles and pyrimi-
do[1,2-a]benzimidazol-2(1H)-ones.
H
6e
O
90%
65%
O
H
(CH2)3
H
(CH2)3
TsO
12
TsO
13
Scheme 4 Reagents and conditions: i) TsCl, MeCN, Py, reflux, 16
h; ii) 4,4-diethoxy-1,1,1-trifluorobut-3-en-2-one (9), K2CO3, acetone,
reflux, 4 h.
In order to confirm our hypothesis, a NOESY experiment
of compound 4b was carried out. In the NOESY spec-
trum, a distinct cross-peak between H-4 and H-6 was ob-
served, which confirmed the spatial proximity of these
two nuclei. In addition, compound 4a had already been
synthesized by Nenajdenko et al.6 and their NOE results
are in agreement with our proposed structure.
Unfortunately, compounds 10 and 13 decomposed when
submitted to purification and could not be tested in the bi-
ological assays.
DNA-Topoisomerase Inhibitory Activity
The hexahydropyranopyrimido ring closure of 8e seems
to be accomplished with cis-configuration. This indica-
tion is obtained from the coupling constants between H-3
and H-4 of 3.8 Hz, which suggests an axial-equatorial re-
lationship between these nuclei. The trans configuration
for related hexahydropyranopyrimidines reports coupling
constants of 9.1 Hz.21a
The conversion of supercoiled plasmid DNA to relaxed
DNA by topoisomerase I was examined in the presence of
4a, 4b, 4d, 4f, 6a, 6e, 8a, 8e, 11 and 12 derivatives. Cam-
pothecin, a well-known DNA-topoisomerase I enzyme in-
hibitor, was used as a positive control.25,26
The results were observed by the alteration of the electro-
phoretic mobility of pBR322 plasmid DNA by the com-
bined action of the enzyme and the drugs. The results
were analyzed after development with ethidium bromide
in UV light, and the record was photographed with a dig-
ital camera. The preliminary assays were performed at
200 mM as the higher drug concentration, and the deriva-
tives with inhibitory activity were screened at 20 mM and
2 mM. The results indicate the dose-dependent inhibition
of DNA-topoisomerase I catalytic activity and the impor-
tance of the steric effect of the b-carbonyl moiety. For the
trifluoromethylated compounds 4a, 4b, 4d and 4f, the re-
sults indicated significant inhibitory activity for all deriv-
atives at 200 mM. However, only compound 4a
(unsubstituted derivative) did not show catalytic effect
against DNA-topoisomerase I at 2 mM indicating the im-
portance of the hydrophobic effect.
Compounds 6a and 6e, which were isolated in higher
yields and purities, were derivatized in an attempt to im-
prove their solubility in organic solvents and to increase
their scope for biological assays. Compound 10 was syn-
F3C
EtO
O
i
N
N
N
90%
H
O
H
H
6a
10
N
N
ii
NH
65%
O
H
Br
In the pyrimidinones series 6a, 6e, 8a, 8e, 11 and 12, only
compound 8a did not show significant catalytic effect
against the enzyme at 200 mM. When screened for 20 mM
and 2 mM, all compounds showed a catalytic effect against
11
Scheme 3 Reagents and conditions: i) 4,4-diethoxy-1,1,1-trifluoro-
but-3-en-2-one (9), K2CO3, acetone, reflux, 4 h; ii) NBS, CHCl3, r.t.,
24 h.
Synthesis 2006, No. 14, 2305–2312 © Thieme Stuttgart · New York

PAPER
Synthesis of Some Novel Pyrimido[1,2-a]benzimidazoles
2309
1
5a), 122.6 (C-7), 121.8 (C-9), 120.5 (q, JC,F = 274.1 Hz, CF3),
113.4 (C-6), 102.0 (C-3).
DNA-topoisomerase I at 2 mM, except compound 12. The
results indicate the dose-dependent inhibition of DNA-to-
poisomerase I catalytic activity. Thus, the pyrimido[1,2- 19F NMR (DMSO-d6/fluorobenzene): d = –66.93 (CF3).
a]benzimidazol-2(1H)-one derivatives showed a DNA-
topoisomerase I inhibitory effect, indicating a significant
performance for this class. The potential in vitro and in
vivo cytotoxic effects are under investigation.
Anal. Calcd for C11H6F3N3 (237.18): C, 55.70; H, 2.55; N, 17.72.
Found: C, 55.78; H, 2.77; N, 17.53.
3-Methyl-2-(trifluoromethyl)pyrimido[1,2-a]benzimidazole
(4b)
In conclusion, a series of 2-(trifluoromethyl)pyrimido
[1,2-a]benzimidazoles 4a–f and pyrimido[1,2a]benzimi-
dazol-2(1H)-ones 6a–e, 8a, and 8e were synthesized from
the 4-alkoxyvinyl trifluoro(chloro)methyl ketones 1a–f
and 5a–e via cyclocondensation reactions with 2-ami-
nobenzimidazoles 2 and 3. Several conditions were tested
in order to improve the reaction times, compound purities,
yields, and selectivity. The synthesis of different com-
pounds was possible by maintaining the same reagents
and making simple variation on the reaction conditions.
The conversion of supercoiled plasmid DNA to relaxed
DNA by topoisomerase I was examined in the presence of
4a, 4b, 4d, 4f, 6a, 6e, 8a, 8e, 11 and 12 derivatives in 200
mM, 20 mM and 2 mM concentrations. The assays results
showed a significant inhibitory effect for both series of
compounds, indicating a significant performance for this
class. All compounds were active in 2 mM concentrations,
except for compound 4a in the trifluoromethylated series
and for compounds 8a (>200 mM) and 12 (20 mM) in the
pyrimidinone series.
Mp 299–301 °C (CHCl3–MeOH).
1H NMR (DMSO-d6/TMS): d = 9.74 (s, 1 H, H-4), 8.35 (d, 1 H,
J = 8.4 Hz, H-6), 7.95 (d, 1 H, J = 8.4 Hz, H-9), 7.64 (t, 1 H, J = 8.4
Hz, H-8), 7.53 (t, 1 H, J = 8.4 Hz, H-7), 2.49 (s, 3 H, CH3).
13C NMR (DMSO-d6/TMS): d = 149.8 (q, JC,F = 33.8 Hz, C-2),
2
147.3 (C-10a), 144.5 (C-9a), 137.8 (C-4), 126.9 (C-8), 126.5 (C-
1
5a), 122.5 (C-7), 120.91 (q, JC,F = 275.4 Hz, CF3), 119.7 (C-9),
113.1 (C-6), 112.1 (C-3), 14.2 (CH3).
19F NMR (DMSO-d6/fluorobenzene): d = –65.58 (CF3).
Anal. Calcd for C12H8F3N3 (251.21): C, 57.37; H, 3.21; N, 16.73.
Found: C, 57.48; H, 3.36; N, 16.70.
4-Methyl-2-(trifluoromethyl)pyrimido[1,2-a]benzimidazole
(4c) (Major Isomer)
Isomer proportion: 70:30.
1H NMR (DMSO-d6/TMS): d = 8.37 (d, 1 H, J = 8.3 Hz, H-6), 7.99
(d, 1 H, J = 8.3 Hz, H-9), 7.68 (t, 1H, J = 8.3 Hz, H-8), 7.53–7.49
(m, 2 H, H-3, H-7), 3.21 (s, 3 H, CH3).
13C NMR (DMSO-d6/TMS):
d = 154.1 (C-10a), 150.2 (q,
2JC,F = 35.0 Hz, C-2), 149.2 (C-9a), 144.9 (C-4), 127.6 (C-5a),
126.8 (C-8), 122.5 (C-7), 120.5 (q, 1JC,F = 275.0 Hz, CF3), 119.6 (C-
9), 116.7 (C-6), 102.6 (C-3), 20.7 (CH3).
The syntheses of compounds 1a–f, 5a–e13,14 and 227 were reported
in the literature. All melting points were determined on a MQAPF-
301 apparatus and are uncorrected. The CHN microanalyses were
performed on a PerkinElmer 2400 elemental analyzer from the De-
partment of Chemistry of the São Paulo University (USP), São Pau-
lo, SP, Brazil. Mass spectra were recorded on a HP 5973 MSD
connected to a HP 6890 GC. The GC was equipped with a split-
splitless injector, auto-sampler, cross-linked HP-5 capillary column
(30 m, 0.32 mm of internal diameter), and He was used as the carrier
gas. 1H and 13C NMR spectra were recorded on a Bruker DPX200
or DPX400 spectrometer in DMSO-d6 with TMS as the internal ref-
erence. 19F and 2D-NMR spectra such as COSY, HMQC, HMBC,
and NOESY were recorded on a Bruker DPX400 spectrometer. Flu-
orobenzene was used as the internal reference for the 19F NMR
spectra.
19F NMR (DMSO-d6/fluorobenzene): d = –66.26 (CF3).
4-Phenyl-2-(trifluoromethyl)pyrimido[1,2-a]benzimidazole
(4d)
Mp 167–169 °C (CHCl3–MeOH) (Lit.28 mp 188 °C).
1H NMR (DMSO-d6/TMS): d = 8.44–8.39 (m, 2 H, H-6, H-9), 8.22
(s, 1 H, H-3), 8.04–7.95 (m, 2 H, H-7, H-8), 7.67–7.48 (m, 5 H,
C6H5).
13C NMR (DMSO-d6/TMS): d = 159.8 (C-10a), 150.5 (C-9a), 144.8
2
(C-4), 135.3 (C6H5), 133.8 (q, JC,F = 37.1 Hz, C-2), 131.9, 129.0,
127.8 (C6H5), 126.4 (C-8), 125.5 (C-5a), 123.1 (C-7), 120.0 (C-9),
119.8 (q, 1JC,F = 272.4 Hz, CF3), 113.9 (C-6), 104.2 (C-3).
GC/MS (EI, 70 eV): m/z (%) = 313 (M+, 100), 244 (21), 90 (33).
3-(1-Hydroxyethyl)-2-(trifluoromethyl)pyrimido[1,2-a]benz-
imidazole (4e)
The reaction was carried out in MeCN instead of toluene; mp 147–
2-(Trifluoromethyl)pyrimido[1,2-a]benzimidazole (4a);
Typical Procedure
Et3N (0.2 mL, 1.4 mmol) was added to a solution of 2 (0.3 g, 1.4
mmol) in toluene (10 mL). The mixture was stirred for 15 min at r.t.
After that, the ketone 1a (0.24 g, 1.4 mmol) was added and the mix-
ture was stirred under reflux until the reaction was complete (see
Table 1). The solvent was evaporated under reduced pressure. A
mixture of CHCl3–H2O (10:30 mL) was added to the residual solid
to dissolve the Et3N salt and the product was filtered and dried in a
desiccator under vacuum; mp 307–308 °C (CHCl3–MeOH) (Lit.6
mp 290–292 °C).
1H NMR (DMSO-d6/TMS): d = 9.91 (d, 1 H, J = 7.0 Hz, H-4), 8.48
(d, 1 H, J = 8.0 Hz, H-6), 7.99 (d, 1 H, J = 8.0 Hz, H-9), 7.69 (t, 1
H, J = 8.0 Hz, H-8), 7.64 (d, 1 H, J = 7.0 Hz, H-3), 7.57 (t, 1 H,
J = 8.0 Hz, H-7).
151 °C (CHCl3–MeOH).
1H NMR (DMSO-d6/TMS): d = 9.72 (s, 1 H, H-4), 8.45 (d, 1 H,
J = 8.3 Hz, H-6), 7.97 (d, 1 H, J = 8.3 Hz, H-9), 7.66 (t, 1 H, J = 8.3
Hz, H-8), 7.50 (t, 1 H, J = 8.3 Hz, H-7), 3.74 (t, 2 H, J = 5.4 Hz, H-
1¢), 2.98 (t, 2 H, J = 5.4 Hz, H-2¢).
2
13C NMR (DMSO-d6/TMS): d = 149.6 (q, JC,F = 33.8 Hz, C-2),
147.1 (C-10a), 144.6 (C-9a), 138.7 (C-4), 127.0 (C-8), 126.4 (C-
1
5a), 122.4 (C-7), 120.9 (q, JC,F = 275.8 Hz, CF3), 119.6 (C-9),
113.7 (C-3), 113.2 (C-6), 60.4 (C-1¢), 31.2 (C-2¢).
GC/MS (EI, 70 eV): m/z (%) = 281 (M+, 40), 250 (100), 102 (14),
76 (4.5).
2
13C NMR (DMSO-d6/TMS): d = 150.8 (q, JC,F = 35.7 Hz, C-2),
3-(1-Hydroxypropyl)-2-(trifluoromethyl)pyrimido[1,2-a]benz-
imidazole (4f)
148.0 (C-10a), 144.5 (C-9a), 139.4 (C-4), 127.2 (C-8), 126.9 (C-
Mp 83–85 °C (CHCl3–MeOH).
Synthesis 2006, No. 14, 2305–2312 © Thieme Stuttgart · New York

2310
N. Zanatta et al.
PAPER
1H NMR (DMSO-d6/TMS): d = 9.68 (s, 1 H, H-4), 8.42 (d, 1 H,
J = 8.1 Hz, H-6), 7.92 (d, 1 H, J = 8.1 Hz, H-9), 7.61 (t, 1 H, J = 8.1
Hz, H-8), 7.50 (t, 1 H, J = 8.1 Hz, H-7), 4.65 (s, 1 H, OH), 3.55 (t,
2 H, J = 5.4 Hz, H-1¢), 2.87 (t, 2 H, J = 5.4 Hz, H-3¢), 1.87 (m, 2 H,
H-2¢).
1H NMR (DMSO-d6/TMS): d = 12.57 (br s, 1 H, NH), 8.73 (s, 1 H,
H-4), 7.85 (d, 1 H, J = 7.1 Hz, H-6), 7.50 (d, 1 H, J = 7.1 Hz, H-9),
7.33–7.19 (m, 2 H, H-7, H-8), 2.00 (s, 3 H, CH3).
13C NMR (DMSO-d6/TMS): d = 162.9 (C-2), 147.1 (C-10a), 140.4
(C-9a), 130.5 (C-4), 128.3 (C-5a), 123.8 (C-8), 120.8 (C-7), 116.8
(C-9), 113.9 (C-3), 109.9 (C-6), 13.2 (CH3).
2
13C NMR (DMSO-d6/TMS): d = 149.5 (q, JC,F = 33.4 Hz, C-2),
147.1 (C-10a), 144.6 (C-9a), 137.8 (C-4), 127.0 (C-8), 126.6 (C-
1
5a), 122.4 (C-7), 120.98 (q, JC,F = 275.6 Hz, CF3), 119.6 (C-9),
4-Methylpyrimido[1,2-a]benzimidazol-2(1H)-one (6c); Typical
Procedure B
116.8 (C-3), 113.4 (C-6), 59.9 (C-1¢), 33.2 (C-3¢), 24.5 (C-2¢).
19F NMR (DMSO-d6/fluorobenzene): d = –63.96 (CF3).
GC/MS (EI, 70 eV): m/z (%) = 295 (M+, 88), 250 (100), 182 (16).
1H NMR (DMSO-d6/TMS): d = 12.70 (br s, 1 H, NH), 8.38 (d, 1 H,
J = 7.9 Hz, H-6), 7.53 (d, 1 H, J = 7.9 Hz, H-9), 7.43 (t, 1 H, J = 7.9
Hz, H-7), 7.29 (t, 1 H, J = 7.9 Hz, H-8), 5.84 (s, 1 H, H-3), 2.31 (s,
3 H, CH3).
Pyrimido[1,2-a]benzimidazol-2(1H)-one (6a); Typical
Procedure A
13C NMR (DMSO-d6/TMS): d = 159.2 (C-2), 148.2 (C-9a, C-10a),
126.7 (C-4, C-5a), 125.4 (C-8), 121.2 (C-7), 115.0 (C-9), 113.5 (C-
6), 98.4 (C-3), 21.6 (CH3).
Et3N (0.2 mL, 1.4 mmol) was added to a solution of 2 (0.3 g, 1.4
mmol) in toluene (10 mL). The mixture was stirred for 15 min at r.t.
After that, the ketone 5a (0.31 g, 1.4 mmol) was added and the mix-
ture was stirred under reflux until the reaction time was complete
(see Table 2). The solvent was evaporated under reduced pressure.
A mixture of CHCl3–H2O (10:30 mL) was added to the residual sol-
id to dissolve the Et3N salt and the product was filtered and dried in
a desiccator under vacuum.
3-Phenylpyrimido[1,2-a]benzimidazol-2(1H)-one (6d);11b Typi-
cal Procedure B
1H NMR (DMSO-d6/TMS): d = 12.97 (br s, 1 H, NH), 8.49 (d, 1 H,
J = 8.0 Hz, H-6), 8.15–8.10 (m, 2 H, H-8, H-9), 7.49–7.52 (m, 6 H,
H-7, C6H5), 6.63 (s, 1 H, H-3).
13C NMR (DMSO-d6/TMS): d = 159.8 (C-2), 149.5 (C-10a), 137.0
(C-9a), 130.2 (C-4), 129.0, 128.6, 128.4, 127.0 (C6H5), 126.2 (C-8),
125.7 (C-5a), 121.8 (C-7), 115.7 (C-9), 111.0 (C-6), 97.1 (C-3).
Pyrimido[1,2-a]benzimidazol-2(1H)-one (6a); Typical
Procedure B
Et3N (0.2 mL, 1.4 mmol) was added to a solution of 3 (0.19 g, 1.4
mmol) in toluene (10 mL). The mixture was stirred for 15 min at r.t.
After that, the ketone 5a (0.31 g, 1.4 mmol) was added and the mix-
ture was stirred under reflux until the reaction time was complete
(see Table 2). The solvent was evaporated under reduced pressure.
A mixture of CHCl3–H2O (10:30 mL) was added to the residue to
dissolve the Et3N and the product was filtered and dried in a desic-
cator under vacuum; mp 254–257 °C (CHCl3–MeOH, dec.) {Lit.10b
mp 336–339 °C (probably, this is the pyrimido[1,2-a]benzimidazol-
4(1H)-one isomer!)}.
1H NMR (DMSO-d6/TMS): d = 12.26 (br s, 1 H, NH), 9.79 (d, 1 H,
J = 7.3 Hz, H-4), 8.42 (d, 1 H, J = 8.1 Hz, H-6), 7.94 (d, 1 H, J = 8.1
Hz, H-9), 7.77 (d, 1 H, J = 7.3 Hz, H-3), 7.63 (t, 1 H, J = 8.1 Hz, H-
8), 7.53 (t, 1 H, J = 8.1 Hz, H-7).
3-(3-Hydroxypropyl)pyrimido[1,2-a]benzimidazol-2(1H)-one
(6e); Typical Procedure C
Mp 269–270 °C (CHCl3–MeOH).
1H NMR (DMSO-d6/TMS): d = 11.70 (br s, 1 H, NH), 7.80 (s, 1 H,
H-4), 7.43–7.39 (m, 2 H, H-6, H-9), 7.09–7.04 (m, 2 H, H-7, H-8),
4.04 (t, 2 H, J = 5.0 Hz, H-1¢), 2.31 (t, 2 H, J = 5.0 Hz, H-3¢), 1.84
(quintet, 2 H, J = 5.0 Hz, H-2¢).
13C NMR (DMSO-d6/TMS): d = 167.2 (C-2), 153.3 (C-4), 147.8
(C-9a, C-10a), 120.9 (C-5a, C-7, C-8, C-9), 113.7 (C-6), 108.6 (C-
3), 66.1 (C-1¢), 20.7 (C-3¢), 18.9 (C-2¢).
GC/MS (EI, 70 eV): m/z (%) = 243 (M+, 18), 213 (28), 111 (100),
83 (28).
13C NMR (DMSO-d6/TMS): d = 161.1 (C-2), 147.2 (C-10a), 143.4
(C-9a), 138.8 (C-4), 127.1 (C-8), 126.8 (C-5a), 123.2 (C-7), 119.1
(C-9), 113.7 (C-6), 102.9 (C-3).
Anal. Calcd for C13H13N3O2 (243.26): C, 64.19; H, 5.39; N, 17.27.
Found: C, 64.19; H, 5.10; N, 17.58.
2a,6a,3,4,5,6-Hexahydropyran[2¢,3¢:6,5]pyrimido[1,2-a]benz-
imidazol-2-one (8e); Procedure A
Mp 260–262 °C (CHCl3–MeOH).
4-Ethoxy-3,4-dihydropyrimido[1,2-a]benzimidazol-2(1H)-one
(8a); Typical Procedure C
1H NMR (DMSO-d6/TMS): d = 11.70 (br s, 1 H, NH), 7.49 (dd, 1H,
J = 3.4 Hz, 2.2 Hz, H-6), 7.43 (dd, 1 H, J = 3.4 Hz, 2.3 Hz, H-9),
7.15–7.13 (m, 2 H, H-8, H-7), 5.92 (d, 1 H, J = 3.6 Hz, H-4), 3.89–
3.87 (m, 1 H, H-1¢), 3.75–3.70 (m, 1 H, H-1¢), 3.23–3.20 (m, 1 H,
H-3), 2.49–2.45 (m, 1 H, H-3¢), 1.83–1.77 (m, 1 H, H-3¢), 1.47–1.43
(m, 2 H, H-2¢).
13C NMR (DMSO-d6/TMS): d = 168.5 (C-2), 147.4 (C-10a), 141.8
(C-9a), 131.8 (C-5a), 122.1 (C-8), 121.2 (C-7), 117.4 (C-9), 109.1
(C-6), 77.6 (C-4), 66.5 (C-1¢), 39.2 (C-3), 21.6 (C-3¢), 21.4 (C-2¢).
Et3N (0.2 mL, 1.4 mmol) was added to a solution of 3 (0.19 g, 1.4
mmol) in MeCN (10 mL). The mixture was stirred for 15 min at r.t.
After that, 5a (0.31 g, 1.4 mmol) was added and the mixture was
stirred at r.t. (under reflux for 5b and 5e) until the reaction time was
complete (see Table 2). The solvent was evaporated under reduced
pressure. A mixture of CHCl3–H2O (10:30 mL) was added to the
residue to dissolve the Et3N and the product was filtered and dried
in a desiccator under vacuum; mp 339–342 °C (CHCl3–MeOH).
1H NMR (DMSO-d6/TMS): d = 11.63 (br s, 1 H, NH), 7.58 (d, 1 H,
J = 8.2 Hz, H-6), 7.43 (d, 1 H, J = 8.2 Hz, H-9), 7.16–7.13 (m, 2 H,
H-7), 6.00–5.8 (m, 1 H, H-4), 3.71–3.67 (m, 1 H, OCH2), 3.41–3.38
(m, 1 H, OCH2), 3.32–3.27 (m, 1 H, H-3), 2.82–2.50 (m, 1 H, H-3),
1.02 (t, 3 H, J = 7.0 Hz, CH3).
CG/MS (EI, 70 eV): m/z (%) = 243 (M+, 34), 213 (18), 111 (100),
83 (18).
Anal Calcd for C13H13N3O2 (243.26): C, 64.19; H, 5.39; N, 17.27.
Found: C, 64.62; H, 5.44; N, 16.91.
13C NMR (DMSO-d6/TMS): d = 167.2 (C-2), 147.3 (C-10a), 141.6
(C-9a), 132.9 (C-5a), 122.1 (C-8), 121.4 (C-7), 117.6 (C-9), 109.4
(C-6), 77.6 (C-4), 64.1 (OCH2), 37.7(C-3), 14.9 (CH3).
1-(1-Ethoxy-4,4,4,-trifluoro-3-oxobuten-1-yl)pyrimido[1,2-
a]benzimidazol-2(1H)-one (10)
K2CO3 (1.38 g, 1.0 mmol) was added to a solution of 6a (0.185 g,
1.0 mmol) in acetone (15 mL). The mixture was stirred for 15 min
at r.t. Ketone 9 (0.212 g, 1.0 mmol) was added and the mixture was
3-Methylpyrimido[1,2-a]benzimidazol-2(1H)-one (6b); Typical
Procedure C
Mp 284–285 °C (CHCl3–MeOH, dec.).
Synthesis 2006, No. 14, 2305–2312 © Thieme Stuttgart · New York

PAPER
Synthesis of Some Novel Pyrimido[1,2-a]benzimidazoles
2311
stirred under reflux for 4 h. The solvent was partially evaporated un-
der reduced pressure. The product was collected by filtration and
dried in a desiccator under vacuum. From the recrystallization of 10
from CHCl3–MeOH, only 6a was recovered.
1H NMR (DMSO-d6/TMS): d = 9.84 (d, 1 H, J = 7.1 Hz, H-4), 8.44
(d, 1 H, J = 7.9 Hz, H-6), 7.95 (d, 1 H, J = 7.9 Hz, H-9), 7.75 (d, 1
H, J = 7.1 Hz, H-3), 7.64 (t, 1 H, J = 7.9 Hz, H-8), 7.52 (t, 1 H,
J = 7.9 Hz, H-7), 4.57 (s, 1 H, H-2 enone), 3.91 (q, 2 H, J = 7.1 Hz,
OCH2), 1.13 (t, 3 H, J = 7.1 Hz, CH3).
13C NMR (DMSO-d6/TMS): d = 167.9 (C-2), 166.5 (q, 2JC,F = 27.2
Hz, C-3 enone), 160.5 (C-1 enone), 147.4 (C-10a), 144.5 (C-9a),
138.4 (C-4), 126.9 (C-8), 126.7 (C-5a), 122.5 (C-7), 119.7 (qua,
1JC,F = 291.8 Hz, CF3), 119.5 (C-9), 113.1 (C-6), 102.2 (C-3), 76.2
(C-2 enone), 56.1 (OCH2), 14.7 (CH3).
(Ar), 119.5 (q, 1JC,F = 280.0 Hz, CF3) 108.5 (C-3), 76.4 (C-2 enone),
66.0 (C-1¢), 56.2 (OCH2), 20.6 (C-2¢, CH3), 18.7 (C-3¢), 14.6
(OCCH3).
Biological Assays; Materials for DNA-relaxation
The DNA-topoisomerase I drug screening kit from TopoGEN con-
tained supercoiled (form I) plasmid substrate DNA (25 mg in 10 mL
of TE buffer). TE buffer (10 mM tris-HCl at pH 7.5 and 1 mM ED-
TA), and the assay buffers 10 mM tris-HCl pH 7.9, 1 mM EDTA,
0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol were
used. The DNA utilized was supercoiled pBR322 plasmid pur-
chased from Sigma. The loading buffer contained 25% bromophe-
nol blue, 50% glycerol and 10% SDS. The agarose and the
substances utilized in these assays were purchased from the Sigma.
Topo I Assay
3-Bromopyrimido[1,2-a]benzimidazol-2(1H)-one (11)
NBS (0.178 g, 1.0 mmol) was added to a solution of 6a (0.185 g, 1.0
mmol) in CHCl3 (20 mL). The mixture was stirred at r.t. for 24 h.
The mixture was extracted with H2O (3 × 10 mL) in order to elimi-
nate the residual NBS. The product was collected by filtration as a
reddish solid and dried in a desiccator under vacuum; mp 270–274
°C (CHCl3–MeOH).
1H NMR (DMSO-d6/TMS): d = 11.60 (br s, 1 H, NH), 10.21 (s, 1
H, H-4), 8.46 (d, 1 H, J = 8.0 Hz, H-6), 7.96 (d, 1 H, J = 8.0 Hz, H-
9), 7.67 (t, 1 H, J = 8.0 Hz, H-8), 7.56 (t, 1 H, J = 8.0 Hz, H-7).
The topo I inhibition was determined by relaxation assay and was
carried out as described in the TopoGEN screening kit. For topo I,
one unit of the enzyme was utilized to relax 0.25 mg of the super-
coiled pBR322 plasmid DNA. The reaction mixture (10 mL) con-
tained the drug, DNA, assay buffer, 1 U of topo I and H2O. The
mixture was incubated at 37 °C for 30 min, and the reaction was fi-
nalized by the addition of 1 mL of dye solution containing 25% bro-
mophenol blue, 50% glycerol and 10% SDS.
Reaction products were loaded onto a 1% agarose gel containing
ethidium bromide. Electrophoresis was carried out in tris-acetate-
EDTA pH 8.5 at 15 V for 3.5 h and then photographed with a digital
camera by illumination.
13C NMR (DMSO-d6/TMS): d = 161.5 (C-2), 147.9 (C-10a), 144.5
(C-9a), 134.1 (C-4), 128.5 (C-8), 126.4 (C-5a), 124.0 (C-7), 122.1
(C-9), 113.6 (C-6), 111.1 (C-3).
Acknowledgment
3-(1-Tosylpropyl)pyrimido[1,2-a]benzimidazol-2(1H)-one (12)
p-Toluenesulfonyl chloride (0.190 g, 1.0 mmol) and pyridine (0.1
mL, 1.0 mmol) were added to a solution of 6e (0.243 g, 1.0 mmol)
in MeCN (15 mL). The mixture was stirred under reflux for 16 h.
The solvent was evaporated under reduced pressure. A mixture of
CHCl3–H2O (10:30 mL) was added to the residual solid to dissolve
the Et3N salt and the product was filtered and dried in a desiccator
under vacuum; mp 174–176 °C (CHCl3–MeOH).
The authors thank the financial support from the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (Universal grant No.
477682/01-4) and fellowship from CAPES (S.S.A.) and CNPq
(P.B.B., D.C.F., A.E.S, and A.E.).
References
1H NMR (DMSO-d6/TMS): d = 12.73 (br s, 1 H, NH), 7.96 (s, 1 H,
H-4), 7.69 (m, 2 H, H-6, H-8), 7.55 (d, 2 H, J = 8.0 Hz, Ar), 7.40
(m, 2 H, H-9, H-7), 7.14 (d, 2 H, J = 7.8 Hz, Ar), 4.14 (t, 2 H,
J = 5.6 Hz, H-1¢), 2.34 (t, 2 H, J = 5.6 Hz, H-3¢), 2.29 (s, 3 H, CH3),
1.89 (quin, 2 H, J = 5.6 Hz, H-2¢).
13C NMR (DMSO-d6/TMS): d = 165.7 (C-2), 156.1 (C-4), 144.7
(C-10a), 143.8 (C-9a), 138.1 (Ar), 128.5 (C-5a, Ar), 128.1 (C-8, C-
7), 125.4 (C-9, C-6), 124.5, 113.2 (Ar), 107.6 (C-3), 66.7 (C-1¢),
20.6 (CH3), 20.3 (C-2¢), 18.5 (C-3¢).
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1-(1-Ethoxy-4,4,4-trifluoro-3-oxobuten-1-yl)-3-(1-tosylpro-
pyl)pyrimido[1,2-a]benzimidazol-2(1H)-one (13)
K2CO3 (0.138 g, 1.0 mmol) was added to a solution of 12 (0.398 g,
1.0 mmol) in acetone (15 mL). The mixture was stirred for 15 min
at r.t. The enone 9 (0.212 g, 1.0 mmol) was added and the mixture
was stirred under reflux for 4 h. The solvent was partially evaporat-
ed under reduced pressure. The product was collected by filtration
and dried in a desiccator under vacuum. From the recrystallization
of 13 from CHCl3–MeOH, only 12 was recovered.
1H NMR (DMSO-d6/TMS): d = 7.81 (s, 1 H, H-4), 7.50–7.40 (m, 2
H, Ar), 7.12–7.04 (m, 2 H, Ar), 4.53 (s, 1 H, H-2 enone), 4.04 (t, 2
H, J = 5.0 Hz, H-1¢), 3.88 (q, 2 H, J = 7.0 Hz, OCH2), 2.27–2.30
(m, 5 H, H-3¢, CH3), 1.84 (quin, 2 H, J = 5.0 Hz, H-2¢), 1.11 (t, 3 H,
J = 7.0 Hz, OCCH3).
(6) Krasovsky, A. L.; Hartulyari, A. S.; Nenajdenko, V. G.;
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