Synthesis, in vitro antiplatelet activity and molecular modelling studies of 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones

Article abstract of DOI:10.1016/j.ejmech.2013.01.026

The multistep preparation of the new 10-substituted 2-(1-piperazinyl) pyrimido[1,2-a]benzimidazol-4(10H)-ones 6a-o, and of the two isomers 10-ethyl-2-(diethylamino)pyrimido[1,2-a]benzimidazol-4(10H)-one 6p and 10-ethyl-4-(diethylamino)pyrimido[1,2-a]benzimidazol-2(10H)-one 13, as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca ²⁺ ionophore A23187 were here described. Nine out of fifteen 2-(1-piperazinyl)derivatives (6g-o) showed good inhibitory properties towards all the platelet aggregation agonists used. Moreover, a molecular modelling study has been performed on two of the best compounds of this series (6i and 6o) to confirm in silico their interactions with the catalytic site of human platelet PDE3, using the X-ray data of the PDE3B isoform in complex with an inhibitor.

Full text of DOI:10.1016/j.ejmech.2013.01.026

European Journal of Medicinal Chemistry 62 (2013) 564e578
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro antiplatelet activity and molecular modelling studies of 10-
substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones
,
a
,
b
b
a
Mario Di Braccio a , Giancarlo Grossi
, Maria Grazia Signorello , Giuliana Leoncini , Elena Cichero ,
*
**
Paola Fossa a, Silvana Alfei c, Gianluca Damonte d
a Dipartimento di Farmacia, Sezione Chimica Farmaceutica, Università di Genova, Viale Benedetto XV, 16132 Genoa, Italy
b Dipartimento di Medicina Sperimentale, Sezione Biochimica, Università di Genova, Viale Benedetto XV, 16132 Genoa, Italy
c Dipartimento di Farmacia, Sezione Chimica e Tecnologie Farmaceutiche e Alimentari, Università di Genova, Via Brigata Salerno 13, 16147 Genova, Italy
d Dipartimento di Medicina Sperimentale e Centro di Eccellenza per la Ricerca Biomedica, Università di Genova, Viale Benedetto XV, 16132 Genoa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The multistep preparation of the new 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-
4(10H)-ones 6aeo, and of the two isomers 10-ethyl-2-(diethylamino)pyrimido[1,2-a]benzimidazol-
4(10H)-one 6p and 10-ethyl-4-(diethylamino)pyrimido[1,2-a]benzimidazol-2(10H)-one 13, as well as the
in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich
plasma by ADP, collagen or the Ca2þ ionophore A23187 were here described. Nine out of fifteen 2-(1-
piperazinyl)derivatives (6geo) showed good inhibitory properties towards all the platelet aggregation
agonists used. Moreover, a molecular modelling study has been performed on two of the best com-
pounds of this series (6i and 6o) to confirm in silico their interactions with the catalytic site of human
platelet PDE3, using the X-ray data of the PDE3B isoform in complex with an inhibitor.
Received 5 September 2012
Received in revised form
12 December 2012
Accepted 21 January 2013
Available online 29 January 2013
Keywords:
10-Substituted 2-(1-piperazinyl)pyrimido
[1,2-a]benzimidazol-4(10H)-ones
Synthesis
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antiplatelet activity
Molecular modelling study
1. Introduction
aminochromones 1, have been reported as highly effective
in vitro antiplatelet agents [9,10].
Since long time we have been interested in the synthesis of
novel heterocyclic compounds endowed with human platelet ag-
gregation in vitro inhibitory properties. In the course of our studies
we synthesized and tested for their in vitro antiplatelet activity
The antiplatelet activity data of the above-mentioned com-
pounds 1e5 towards all the platelet aggregation agonists used [i.e.
adenosine diphosphate (ADP), collagen and the Ca2þ ionophore
A23187 (calcimycin)] allow us to draw the following conclusions:
a
number of substituted 2-aminochromones 1 [1e3], 4-
aminocoumarins 2 [2e5] and their angular and linear benzo-
fused derivatives [2,3]. Also several N-substituted 2-amino-4H-
pyrido[1,2-a]pyrimidin-4-ones 3 [3,6e8], and 4-amino-2H-pyrido
[1,2-a]pyrimidin-2-ones 4 [7], isosteric analogues of compounds 1
and 2, respectively, and their benzo-fused angular derivatives [7]
were synthesized and evaluated by us for their in vitro antiplate-
let properties. Within these studies, also some other nitrogen
bridgehead compounds 5, differing from 3 for proper modifications
of the pyridine ring, were prepared and tested [3,8] (Fig. 1).
Furthermore, it must be pointed out that also several 7-
- The bicyclic compounds 1e4 were more active than their linear
and angular benzo-fused derivatives.
- In each structural class, the 1-piperazinyl group was the most
effective amino substituent among all those tested, even
R
though such a substitution (
N
N
NH) was not possible
=
R1
in the case of 4-amino-2H-pyrido[1,2-a]pyrimidin-2-ones 4
and their benzo-fused derivatives [7,8].
- The pyrido[1,2-a]pyrimidine derivatives 3 and, specially, the
coumarins 2 were more effective in vitro antiplatelet agents in
comparison with their isomers 4 and 1, respectively.
substituted
or
7,8-disubstituted
2-(dialkylamino)-4H-1,3-
R
- The coumarin scaffold of compounds 2 (
N
N
=
NH
)
benzoxazin-4-ones, isosteric analogues of the substituted 2-
R1
appeared particularly suitable for inserting proper substituents
in positions 7 and 8, in order to increase the antiplatelet ac-
tivity potency. In fact, compounds 2a, 2b and 2c (Fig. 2),
obtained following this rationale, were the most potent in vitro
antiplatelet agents synthesized by us [4,5].
* Corresponding author. Tel.: þ39 010 3538374; fax: þ39 010 3538358.
** Corresponding author. Tel.: þ39 010 3538397; fax: þ39 010 3538358.
E-mail addresses: dbraccio@unige.it (M. Di Braccio), grossig@unige.it (G. Grossi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.026

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
565
R3
Starting from these results, we have pursued our studies to
obtain new potent antiplatelet agents also in the field of 1,2-fused
pyrimidine derivatives. Our interest was attracted by the struc-
ture of the 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzi-
midazol-4(10H)-ones 6. In fact, even though generally, in each
structural class studied, the bicyclic compounds 1e4 were more
active than their benzofused analogues, some tricyclic nitrogen
bridgehead compound such as the 7-chloro-2-(1-piperazinyl)-4H-
pyrimido[2,1-b]benzoxazol-4-one (5a), which structure is analo-
gous to compounds 6, had previously shown very interesting
in vitro antiplatelet properties [3]. Moreover, compounds 6, bearing
a NH group in position 10 were suitable for preparing a series of
new promising derivatives by attaching in this position several
proper substituents, preferring those that had given the best results
when they were inserted on the coumarin scaffold of compounds 2.
(Fig. 3).
R3
R
R1
O
N
O
O
R2
R2
R
R1
1
N
N
O
N
2
R
R1
O
N
R3
N
N
R2
R
R1
N
O
4
3
Therefore, in this paper we report the synthesis of a series of 10-
N
O
N
NH
substituted
2-(1-piperazinyl)-pyrimido[1,2-a]benzimidazol-
4(10H)-ones 6aeo and their antiplatelet activity. It must be noted
that until now, no dialkylamino derivative of pyrimido[1,2-a]ben-
zimidazol-4(10H)-one has been already described in the literature.
N
5
Fig. 1. Structures of compounds 1e5.
2. Chemistry
The synthetic route to compounds 6aeo is shown in Scheme 1.
Thus, the reaction of 2-aminobenzimidazole (7) with proper alkyl
halides in refluxing acetone in the presence of anhydrous K2CO3/
KOH gave good yields of 1-substituted 2-aminobenzimidazoles 8ae
f,leo (Method A). On the contrary, in the case of compounds 8gek
this method was not satisfactory. Therefore, in these cases, 2-
aminobenzimidazole (7) was treated with an equimolar amount
of EtONa in anhydrous EtOH to give the sodium salt derivative; after
removal of solvent, this salt was taken up in acetone and treated
with an equimolar amount of the proper alkyl halide, refluxing the
mixture for 30 min to obtain the desired compounds 8gek
(Method B). The 1-substituted 2-aminobenzimidazoles 8aeo
were then condensed with diethyl malonate (excess 10:1) in the
presence of EtONa (2.5 eq) (150 ꢀC, 3 h) to afford good yields of
crystalline sodium salts of compounds 9aeo. The nearly pure 10-
substituted 2-hydroxypyrimido[1,2-a]benzimidazol-4(10H)-ones
9aeo (most likely as zwitterionic structure in solid state [14e16])
were then obtained, as white solids, after a suitable acidification of
the aqueous solution of these salts (compounds 9leo, bearing
a tertiary amino group on the side chain, were precipitated as
water-insoluble perchlorates). The subsequent treatment of 9aeo
with excess refluxing POCl3 (2 h) afforded the corresponding 2-
chloroderivatives 10aeo, in satisfactory yields (35e93%, average
59%). The last step of the synthesis was the reaction of 2-
chloroderivatives 10aeo with excess piperazine in refluxing etha-
nol (3 h) to give usually good yields (52e92%, average 71%) of the
desired 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimi-
dazol-4(10H)-ones 6aeo.
- In regard to the mechanism involved in the antiplatelet activity
of compounds 1e5, it is interesting to point out that 2-(dieth-
ylamino)-7-hydroxychromone 1a [11], the 2-(1-piperazinyl)-
4H-pyrido[1,2-a]pyrimidin-4-one 3a [12] as well as the cou-
marin derivative 2a [13] (Fig. 2) proved to exert their platelet
antiaggregating effect by specifically inhibiting the activity of
the platelet PDE3 enzyme, producing an increase in the intra-
cellular cAMP concentration. This finding confirmed the
expected bioisosterism of the benzopyran derivatives 1, 2 and
1,2-fused pyrimidine derivatives 3e5. Moreover, it was shown
that the coumarin derivative 2a was able to increase nitric
oxide formation [13].
- The substituted 4-(1-piperazinyl)coumarins 2a and 2c (Fig. 2)
displayed a very potent inhibitory effect on the activity of PDE3
isolated from human platelets (IC50 values ¼ 37 nM and 78 nM,
respectively), better than that of milrinone (IC50 ¼ 280 nM) and
cilostazol (IC50 ¼ 210 nM) [5].
N
CH3
C2H5
O
O
N
O
HO
O
N
C2H5
1a
2a
NH
O
CH3
CH3
S
O
O
N
O
O
O
N
O
It must be pointed out that the critical step of this synthetic
route was the preparation of 2-hydroxyderivatives 9aeo. Our first
CH3
N
O
NH
NH
2c
2b
H (R)
9
10
O
N
N
8
N 1
N
N
O
N
NH
7
5N
Cl
N
NH
N
NH
6
2
N
3
4
O
5a
O
6
3a
Fig. 3. Structural analogy between compound 5a [3] and the 10-substituted 2-(1-
piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones 6.
Fig. 2. Structures of compounds 1a, 2aec and 3a.

566
M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
Scheme 1. Synthetic route to compounds 6aeo.
attempts using the malonamate approach of Hermecz et al. [17], or
the di(2,4,6-trichlorophenyl) malonate method [18,3], gave sat-
isfactory results only in few cases (compounds 9a,b,d,f,o). However,
checking the literature about this topic, our attention was attracted
by a Japanese patent [19], that claimed the synthesis of a series of
3,10-disubstituted 4-hydroxypyrimido[1,2-a]benzimidazol-2(10H)-
ones, through the condensation of properly 1-substituted 2-
aminobenzimidazoles with 2-substituted diethyl malonates in the
presence of EtONa. In contrast to the Japanese patent, we thought
that the isomeric structure of 3,10-disubstituted 2-
two carbonyl groups, the enolization always involves the carbonyl
group far from the bridgehead nitrogen [14e16,20]. However, the
reaction reported in the Japanese patent [19] was an excellent
method to prepare the 10-substituted 2-hydroxypyrimido[1,2-a]
benzimidazol-4(10H)-ones 9aeo in very good yields (58e94%,
average 78%).
In order to give a definitive chemical demonstration of the
structure of compounds 6, we prepared the 10-ethyl-4-(dieth-
ylamino)pyrimido[1,2-a]benzimidazol-2(10H)-one 13 through the
univocal route reported in Scheme 2.
hydroxypyrimido[1,2-a]benzimidazol-4(10H)-ones
was
more
Thus, the one-pot reaction of N,N-diethylmalonamic acid 11 [7]
with PCl5 in CH2Cl2 (room temperature for 3 h) followed by con-
densation of the resulting acyl chloride with 1-ethyl-2-
aminobenzimidazole (8a) (in refluxing CH2Cl2 for 1 h) afforded
likely for these compounds, in agreement with previous literature
reports in the field of nitrogen bridgehead pyrimidine derivatives,
which demonstrated that, when on the pyrimidine ring there are

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
567
Scheme 2. Synthesis of isomers 6p and 13.
the intermediate malonamide 12, in moderate yield (31%). The
subsequent cyclization of the malonamide 12 by treatment with
POCl3 (refluxing 1,2-dichloroethane, 1 h) afforded a satisfactory
yield (56%) of the desired 10-ethyl-4-(diethylamino)pyrimido[1,2-
a]benzimidazol-2(10H)-one 13.
On the other hand, we prepared in very good yield (89%) the
isomer 6p through the standard method (excess diethylamine,
refluxing ethanol, 16 h) starting from 2-chloroderivative 10a. Since
the structure of compound 13 is secure, due to its synthetic path-
way, to isomer 6p must be attributed the structure of 10-ethyl-2-
(diethylamino)pyrimido[1,2-a]benzimidazol-4(10H)-one. In such
way, also the structures of compounds 6aeo (obtained through the
same route) and of their intermediates 10aeo and 9aeo are
validated.
Furthermore, the availability of 6p and 13 allowed us to compare
the antiplatelet activity of a pair of the two possible isomers of this
new class of compounds, even though for this purpose it would
have been better to compare two isomers bearing the 1-piperazinyl
group. Unfortunately, the synthesis of the pharmacologically more
interesting 4-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-2(10H)-
one isomers type-13 was not possible. A similar problem was
previously encountered also in the case of other nitrogen bridge-
head compounds reported by us [7,8].
coplanar 4-CO. The chemical shift average values (
d
) are: 8.44
(DMSO-d6) for the 2-hydroxyderivatives 9, 8.66 (CDCl3) for the 2-
chloroderivatives 10 and 8.56 (CDCl3) for the 2-(1-piperazinyl)de-
rivatives 6, respectively. These data confirm that compounds 9, 10
and 6 possess the same pyrimido[1,2-a]benzimidazol-4(10H)-one
scaffold. Moreover, in the 1H NMR spectrum of the 4-(dieth-
ylamino)-2-one derivative 13, the H-6 signal is clearly less
deshielded (
of the 2-(diethylamino)-4-one isomer 6p (
a literature report for compounds belonging to the same hetero-
cyclic system [21]. Another characteristic difference between the 1H
NMR spectra of the two isomers is the chemical shift of the py-
rimidine ring proton (H-3), more deshielded in the 4-(dieth-
d
¼ 7.92) in comparison with the corresponding signal
d
¼ 8.54), according to
ylamino)-2-one derivative 13 (
(diethylamino)-4-one isomer 6p (
features perfectly agree with those previously reported by us for
analogous nitrogen bridgehead compounds [7].
d
¼ 5.87) in comparison with its 2-
d
¼ 5.19). These spectroscopic
Moreover, for a further confirmation, a good number of repre-
sentative examples of compounds 9,10 and 6 were subjected also to
13C NMR spectrometry analysis and electrospray ionization mass
spectrometry (ESIeMS) full scan and MS2 analysis (see
Experimental protocols). The results of these analyses are perfectly
consistent with the proposed structures.
The structures attributed to the compounds described in this
paper are supported by the results of elemental analyses, as well as
by the IR and 1H NMR spectral data (see Experimental protocols)
which agree with the ones previously reported by us for analogous
compounds [3,6e8].
3. Biological results and discussion
The seventeen novel compounds 6aep and 13 were tested
in vitro for their inhibitory activity on the human platelet aggre-
gation induced in platelet-rich plasma (PRP) by adenosine
diphosphate (ADP), collagen, or the Ca2þ ionophore A23187 (cal-
cimycin) (see Experimental protocols). Acetylsalicylic acid (ASA),
In the 1H NMR spectra, particularly meaningful are the charac-
teristic H-6 signals (multiplets with Jortho ¼ 7.8 Hz) of compounds 9,
10 and 6, shifted downfield due to deshielding effect of the nearly

568
M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
Table 1
In vitro inhibitory activity of the new pyrimido[1,2-a]benzimidazole derivatives 6aep and 13 on platelet aggregation induced in human PRP by ADP, collagen and A23187. The
IC50 values previously described by us [3] for the pyrimido[2,1-b]benzoxazole derivative 5a are herein reported for comparison.
R
R
O
N
N
N
N
N
N
N
N
R1
R1
Cl
O
N
N
NH
6a-p
5a
13
R1
O
O
N
R1
R1
R1
Compound
R
IC50
(
m
M) ꢁ SDa
N
ADP (5.0
m
M)
Collagen (5.0
m
g/mL)
A23187 (20.0 mM)
5a [3]
e
e
3.6 ꢁ 1.2
8.8 ꢁ 5.6
13.0 ꢁ 5
6a
CH2CH3
127 ꢁ 49
62 ꢁ 13
55 ꢁ 28
50 ꢁ 14
77 ꢁ 10
206 ꢁ 89
50 ꢁ 8
N
NH
NH
NH
6b
6c
CH2CH2CH3
N
N
CH3
116 ꢁ 17
142 ꢁ 38
CH CH3
6d
6e
90 ꢁ 20
27 ꢁ 15
172 ꢁ 7
143 ꢁ 27
175 ꢁ 12
N
N
NH
NH
CH2
CH3
CH
163 ꢁ 11
6f
53 ꢁ 3
15 ꢁ 2
75 ꢁ 21
N
NH
CH2CH2
6g
6h
26 ꢁ 3
19 ꢁ 1
31 ꢁ 7
60 ꢁ 15
38 ꢁ 7
CH2
CH2
S
CH3
N
N
NH
NH
N
30 ꢁ 10
N
6i
6j
15 ꢁ 6
15 ꢁ 6
9 ꢁ 1
23 ꢁ 13
32 ꢁ 9
N
N
N
NH
NH
NH
CH2
CH2
CH2
21 ꢁ 9
N
N
CH3
OCH3
6k
33 ꢁ 10
49 ꢁ 15
56 ꢁ 18
H3C
6l
CH2CH2N(CH3)2
CH2CH2N(C2H5)2
CH2CH2N
36 ꢁ 5
16 ꢁ 7
16 ꢁ 2
12 ꢁ 5
8 ꢁ 1
13 ꢁ 2
28 ꢁ 7
15 ꢁ 7
41 ꢁ 4
13 ꢁ 5
42 ꢁ 11
11 ꢁ 6
N
N
N
N
NH
NH
NH
NH
6m
6n
6o
O
CH2CH2N
6p
13
ASA
Cilostazol
Milrinone
N(C2H5)2
N(C2H5)2
CH2CH3
CH2CH3
>1000
>1000
>1000
73 ꢁ 19
11 ꢁ 4
75 ꢁ 15
75 ꢁ 17
183 ꢁ 4
40 ꢁ 9
>1000
415 ꢁ 150
>1000
109 ꢁ 35
14 ꢁ 5
12 ꢁ 4
a
IC50 ¼ compound concentration which inhibits platelet aggregation by 50%.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
569
N
N
cilostazol and milrinone were also tested under the same condi-
CH3
tions as reference compounds. The IC50 values obtained are
reported in Table 1 along with the IC50 values previously described
by us for the 7-chloro-2-(1-piperazinyl)-4H-pyrimido[2,1-b]ben-
zoxazol-4-one (5a) [3], taken as a lead compound.
O
O
N
O
N
N
N
N
NH
These data suggest the following remarks:
NH
O
2a
6i
- Also in this class of 1,2-fused pyrimidine derivatives, the 1-
piperazinyl group confirmed to be the best dialkylamino sub-
stituent, as shown by the comparison of IC50 values of 6a and
those of its diethylamino analogue 6p.
- The 10-alkylsubstituted derivatives 6aec displayed a poor
antiplatelet activity: among these three compounds, the
highest activity was shown by the 10-propyl derivative 6b,
which performed better than the corresponding isopropyl
analogue 6c.
O
O
N
N
CH3
O
O
N
O
N
N
N
N
NH
2c
NH
O
I
6o
O
- Concerning, the 10-(arylalkyl)derivatives 6def, it can be
remarked that the substitution of the alkyl group with the
benzyl one (6d) afforded only a modest antiplatelet activity,
differently from what happened when this group was inserted
on the 7-OH substituent of coumarins 2. In fact, in that case we
had obtained very interesting antiplatelet agents [3,4]. Slightly
better results were afforded by the 10-(2-phenylethyl)deriva-
tive 6f, whereas the branched chain derivative 6e was the
worst among these three compounds.
N
H
CH3
Merck 1
N
N
H
O
- The isosteric replacement of benzyl group with the three iso-
meric (pyridylmethyl)substituents afforded a small set of novel
interesting antiplatelet agents (6hej), with the (3-
pyridylmethyl)derivative 6i being the most active within
these three compounds. It must be pointed out that the (3-
pyridylmethyl)group was present also in the coumarin 2a
(Fig. 2), i.e. one of the most powerful antiplatelet agents pre-
viously synthesized by us [4,5]. Therefore, this result confirms
the hypothesized bioisosterism between the 10-substituted 2-
Fig. 4. Structures of compounds subjected to molecular modelling study.
induced by collagen (IC50 ¼ 75
m
M for both compounds). The
keeping of a very poor activity against the platelet aggregation
M) by the 4-(diethylamino)-2-one
induced by A23187 (IC50 ¼ 415
m
isomer 13 was the only difference observed between the two
isomers.
On the basis of the structural analogies between the
(1-piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones
6
10-substituted
2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-
and the 7-substituted 4-(1-piperazinyl)coumarins 2. When the
pyridyl ring of the 10-substituent was hindered with further
methyl and/or methoxy groups (compound 6k), the anti-
platelet potency decreased remaining anyway significant
(Table 1).
4(10H)-ones 6aeo and the 4-(1-piperazinyl)coumarins 2 (Fig. 2)
[5], it is very likely that also compounds 6aeo could exert an
inhibitory effect on the activity of human platelet PDE3.
- Also the introduction in 10-position of a proper 2-(dia-
lkylamino)ethyl substituent revealed to be efficacious, lead-
ing to some very interesting compounds (6leo) endowed
with an antiplatelet potency comparable with that of (pyr-
idylmethyl)derivatives 6hej. In particular the 2-(4-
morpholinyl)ethylderivative 6o was nearly equiactive with
the (3-pyridylmethyl)derivative 6i, showing a slightly lower
effect against the aggregation induced by collagen, but
a somewhat better efficacy against the aggregation induced
by ADP and A23187. It’s noteworthy that the 2-(4-
morpholiny)ethyl substituent of compound 6o was present
also in the structure of the very active coumarin 2c (Fig. 2)
[5]. Moreover, also the 2-(diethylamino)ethyl derivative 6m
exhibited an antiplatelet activity very similar to those of
compounds 6i and 6o.
- On the contrary, the introduction of a CH2SCH3 group in the
position 10 of compounds
6 afforded compound 6g,
endowed with only moderate antiplatelet activity
a
(Table 1), while the analogous substitution was very effica-
cious when applied on the coumarin scaffold (compound 2b,
Fig. 2 [5]).
Fig. 5. The dihydropyridazinone inhibitor X-ray pose (pdb code 1SO2) into the PDE3B
catalytic site is reported. The ligand C atoms are coloured in magenta. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Finally, also the platelet antiaggregating properties of isomers
6p and 13 were evaluated: both compounds were almost inactive,
showing a mild activity only against the platelet aggregation

570
M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
Fig. 6. Selected docking pose of compound 2a into the PDE3B catalytic site (pdb code
1SO2). The ligand C atoms are coloured in white. Metal ions are represented as green
spheres. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
4. Molecular modelling studies
In order to confirm the hypothesized PDE3 inhibition by the
compounds 6, a molecular modelling study was performed on
compounds 6i and 6o, chosen as examples, due to their high an-
tiplatelet activity and their analogy to coumarin derivatives 2a and
2c, which were previously evaluated by us in silico for their ability
to interact with the PDE3 catalytic site [5].
Fig. 8. Selected docking pose of compound 6i into the PDE3B catalytic site (pdb code
1SO2). The ligand C atoms are coloured in white. Metal ions are represented as green
spheres. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
the two nitrogen atoms of the dihydropyridazinone ring and the
First of all, since compounds 2a and 2c proved to be very potent
PDE3 inhibitors [5], they were submitted to docking studies using
the X-ray data of the PDE3B isoform in complex with a dihydropyr-
idazinone inhibitor [Merck 1 (Fig. 4), Protein Data Bank (pdb) code
1SO2] [22]. On the basis of these results, compounds 6 were eval-
uated by us as potential PDE3 inhibitors: in particular we focused
our attention on 6i and 6o, being the two compounds of this series
more structurally related with the above mentioned 2a and 2c.
Thus, the 6i and 6o docking poses were compared with those of
the two PDE3 inhibitors 2a and 2c in the 1SO2 complex (Fig. 4).
PDE3 residue Q315, (ii) the dihydropyridazinone ring oxygen atom
and H275. The inhibitor is also engaged in pep stacking with Y78,
F286, and F318 through the aminophenyl group, and with the F303
residue, by means of the iodobenzyl ring. Furthermore, Van der
Waals contacts with L222, I265, L314 and I322 are detected.
According to our molecular docking results, the PDE3 inhibitors
2a (Fig. 6) and 2c (Fig. 7) display one H-bond with the key residue
Q315, through the pyridine and the morpholine ring, respectively,
and are also engaged in pep stacking with the Y78, F286 and F318
residues. In addition, the two compounds orient the piperazine ring
towards the catalytic site metal ions, being also engaged in one H-
bond with D149 side chain.
4.1. Discussion
In the 1SO2 X-ray pdb, the dihydropyridazinone inhibitor bio-
active conformation (Fig. 5), displays H-bonds between: (i) one of
Fig. 7. Selected docking pose of compound 2c into the PDE3B catalytic site (pdb code
1SO2). The ligand C atoms are coloured in white. Metal ions are represented as green
spheres. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
Fig. 9. Selected docking pose of compound 6o into the PDE3B catalytic site (pdb code
1SO2). The ligand C atoms are coloured in white. Metal ions are represented as green
spheres. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
571
Interestingly, compounds 6i (Fig. 8) and 6o (Fig. 9) prove to
spectrometer; chemical shifts (
d
) are reported in ppm using tetra-
show the same binding mode which has been previously observed
for compounds 2a and 2c, respectively.
methylsilane as an internal reference (
d
¼ 0). Spin multiplicities are
given as follows: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). 13C NMR spectra were acquired on a Bruker DPX
spectrometer at 75.5 MHz with tetramethylsilane as internal ref-
erence. Electrospray ionization mass spectrometry (ESIeMS) full
scan and MS2 analysis were performed in positive or negative ion
mode on a LCQ-Fleet ion trap mass spectrometer (ThermoFisher
Scientific, UK) in direct infusion (DIA) using the infusion pump
Thus, the 6i pyridine ring and the 6o morpholine one are involved
in a H-bond with the key residue Q315, while the piperazine moiety
is projected towards the enzyme metal ions and residue D149. In
particular, the H-bond displayed by the 6i pyridine ring seems to be
ꢀ
slightly weaker (H-bond distance: 3.00 A) than that of the 6o mor-
ꢀ
pholine ring (H-bond distance: 2.70 A), probably because of the
shorter linker which is located between the 6i tricyclic ring and the
pyridine group, in comparisonwith the linker placed between the 6o
scaffold and the morpholine moiety. Accordingly, compound 6o
displays an inhibitory activity on human platelet aggregation
induced by ADP higher than that displayed by 6i.
Notably, compounds 6 bearing no H-bond acceptor groups at
the R substituent (compounds 6aef, Table 1) showed lower
inhibitory activity values, being probably unable to act as PDE3
inhibitors, due to the absence of an anchoring H-bond with Q315
residue.
included in the instrument. The flow rate was set at 5 mL/min. Each
sample was diluted in methanol at a concentration of 1 mg/mL and
then diluted 100 fold in methanol:water 50:50 containing 0.1%
formic acid to get a final concentration of 0.01 mg/mL. Analyses of
all new compounds, indicated by the symbols of the elements, were
within ꢁ0.4% of the theoretical values and were performed by the
Laboratorio di Microanalisi, Dipartimento di Scienze Farm-
aceutiche, University of Genoa.
Thin-layer chromatograms were run on Merck silica gel 60 F254
precoated plastic sheets (layer thickness 0.2 mm). Column chro-
matography was performed using Carlo Erba silica gel (0.05e
0.20 mm).
5. Conclusions
Taking into account the biological results of the new compounds
6aep and 13 described in the present paper, the following con-
clusions can be drawn:
6.1.1. General procedure for the synthesis of 1-substituted 1H-
benzimidazol-2-amines 8aeo
6.1.1.1. Method
A
(compounds 8aef,leo). A mixture of 2-
aminobenzimidazole 7 (1.33 g, 10.0 mmol), 10.0 mmol of the
proper alkyl halide, 1.0 g of finely powdered KOH mixed with 2.0 g
of anhydrous K2CO3 and acetone (50 mL) was heated at reflux for
3 h, with stirring. The solvent was removed in vacuo and the
residue was partitioned between water (100 mL) and CH2Cl2
(100 mL), and the aqueous phase was further extracted twice with
CH2Cl2. The combined extracts, dried over anhydrous Na2SO4, after
removal of solvent afforded an oily or solid residue which was
treated with a small amount of ethyl ether to give compounds 8ae
e,leo as whitish solids which were crystallized from the proper
- The 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimi-
dazol-4(10H)-ones 6aeo proved to be a new interesting class of
in vitro antiplatelet agents.
- Nine out of fifteen 2-(1-piperazinyl)derivatives 6aeo (i.e.
compounds 6geo) showed good inhibitory properties towards
all the platelet aggregation inducers used; among these com-
pounds, 6i,m,o were active in the low micromolar range. As
regards the in vitro antiplatelet efficacy, compounds 6i,m,o
were comparable with the 7-chloro-2-(1-piperazinyl)-4H-
pyrimido[2,1-b]benzoxazol-4-one (5a) [3] and other (1-
piperazinyl)substituted nitrogen bridgehead compounds, pre-
viously synthesized by us [3,7], even though inferior to the
substituted 4-(1-piperazinyl)coumarins 2 [3e5].
solvent. Only in the case of compound 8f,
a preliminary
purification by column chromatography [SiO2/ethyl acetate-
acetone (1:1)] was necessary to obtain a crystalline compound.
- The (1-piperazinyl)substituent inserted in a
b
-enaminonic
6.1.1.2. Method B (compounds 8gek). 2-Aminobenzimidazole 7
(1.33 g, 10.0 mmol) was dissolved in an ethanolic solution of
sodium ethoxide [10.0 mmol (0.23 g) of sodium in 40 mL of
anhydrous EtOH (preparation of 8g) or 20.0 mmol (0.46 g) of
sodium in 40 mL of anhydrous EtOH (preparation of 8hek),
because in these cases the alkylating reagents were as
hydrochlorides] and the mixture was evaporated to dryness in
vacuo. The solid residue was suspended in acetone (50 mL) and
treated with 10.0 mmol of the proper alkyl halide, refluxing the
mixture for 30 min. The solvent was removed in vacuo and the
residue was partitioned between water (100 mL) and CH2Cl2
(100 mL), and the aqueous phase was further extracted twice
with CH2Cl2. The combined extracts, dried over anhydrous
moiety confirmed to be a structural feature essential for dis-
playing a good antiplatelet activity in this kind of compounds:
in fact the two (diethylamino)substituted isomers 6p and 13
were nearly inactive.
- A molecular modelling study showed that compounds 6i and
6o (two of the best compounds of the series) possess the same
binding mode to the PDE3 catalytic site which has been already
observed for the substituted 4-(1-piperazinyl)coumarins 2a
and 2c, respectively. Since in a previous study compounds 2a
and 2c proved to be very potent inhibitors of human platelet
PDE3 [5], we can hypothesize that also compounds 6aeo could
exert their in vitro antiplatelet activity through the same
mechanism of action.
Na2SO4, after removal of solvent afforded
a solid residue
(compounds 8hek) which was treated with a small amount of
ethyl ether to give compounds 8hek as whitish solids which
were crystallized from the proper solvent. Only in the case of
compound 8g, was obtained an oil which was purified by column
chromatography [SiO2/ethyl acetate-acetone (1:1)] to give 8g as
a crystalline compound.
6. Experimental protocols
6.1. Chemistry
Melting points were determined using a Fisher-Johns apparatus
and are uncorrected. IR spectra were recorded on a PerkineElmer
“Spectrum One” spectrophotometer (abbreviations relative to IR
bands: br ¼ broad, s ¼ strong, w ¼ weak, sh ¼ shoulder). 1H NMR
spectra were recorded partly on a Varian Gemini 200 (200 MHz)
According to these two procedures the following compounds 8
were prepared (IR and 1H NMR data are reported only for the new
compounds and for 8h,i,n, due to their incomplete literature data).
6.1.1.3. 1-Ethyl-1H-benzimidazol-2-amine (8a). Obtained (1.05 g,
65%) from reaction of 7 with iodoethane (1.56 g); white crystals,
spectrometer and partly on
a Bruker WM-300 (300 MHz)

572
M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
m.p. 158e159 ꢀC (diisopropyl ether) (lit. [23] m.p. 158e159 ꢀC).
Anal. C9H11N3 (C, H, N).
H-4,5,6,7 þ pyridyl H-30,50), 8.50 (m, 2H, pyridyl H-20,60). Anal.
C13H12N4 (C, H, N).
6.1.1.4. 1-Propyl-1H-benzimidazol-2-amine (8b). Obtained (1.07 g,
61%) from reaction of 7 with 1-iodopropane (1.70 g); white crystals,
m.p. 131e132 ꢀC (diisopropyl ether) (lit. [23] m.p. 130 ꢀC). Anal.
6.1.1.13. 1-[(4-Methoxy-3,5-dimethyl-2-pyridyl)methyl]-1H-benzimi-
dazol-2-amine (8k). Obtained (1.79 g, 63%) from reaction of 7 with
2-(chloromethyl)-4-methoxy-3,5-dimethylpyridine hydrochloride
(2.22 g); white crystals, m.p. 206e207 ꢀC (EtOAc/petroleum ether).
IR (KBr, cmꢂ1): 3390, 3341 and 3164 br (NH2), 1669 s, 1619, 1594,
C
10H13N3 (C, H, N).
6.1.1.5. 1-Isopropyl-1H-benzimidazol-2-amine
(8c). Obtained
1570 w, 1547 s. 1H NMR (200 MHz, CDCl3, ppm):
d 2.27 and 2.44 (2 s,
(0.84 g, 48%) from reaction of 7 with 2-iodopropane (1.70 g); white
crystals, m.p. 195e196 ꢀC (EtOAc/petroleum ether) (lit. [24] m.p.
195e196 ꢀC). Anal. C10H13N3 (C, H, N).
3H þ 3H, 2 CH3), 3.76 (s, 3H, OCH3), 5.20 (s, 2H, NCH2), 5.93 (broad s,
2H, NH2; disappeared with D2O), 7.02e7.50 (m, 4H, H-4,5,6,7), 8.23
(s, 1H, pyridyl H-60). Anal. C16H18N4O (C, H, N).
6.1.1.6. 1-Benzyl-1H-benzimidazol-2-amine (8d). Obtained (1.72 g,
77%) from reaction of 7 with benzyl chloride (1.26 g); white crys-
tals, m.p. 194e195 ꢀC (EtOAc/petroleum ether) (lit. [23] m.p. 195e
196 ꢀC). Anal. C14H13N3 (C, H, N).
6.1.1.14. 2-Amino-N,N-dimethyl-1H-benzimidazole-1-ethanamine
(8l). Obtained (1.23 g, 60%) from reaction of 7 with (2-chloroethyl)
dimethylamine hydrochloride (1.44 g); white crystals, m.p. 147e
149 ꢀC (EtOAc/petroleum ether) (lit. [28] m.p. 140e142 ꢀC). Anal.
C11H16N4 (C, H, N).
6.1.1.7. 1-(1-Phenylethyl)-1H-benzimidazol-2-amine
(8e).
Obtained (1.74 g, 73%) from reaction of 7 with (1-bromoethyl)
benzene (1.85 g); white crystals, m.p. 120e122 ꢀC (EtOAc/petro-
leum ether) (lit. [25] m.p. 120e122 ꢀC). Anal. C15H15N3 (C, H, N).
6.1.1.15. 2-Amino-N,N-diethyl-1H-benzimidazole-1-ethanamine
(8m). Obtained (1.49 g, 64%) from reaction of with (2-
chloroethyl)diethylamine hydrochloride (1.72 g); white crystals,
m.p. 134e135 ꢀC (EtOAc/petroleum ether) (lit. [29] m.p. 134e
135 ꢀC). Anal. C13H20N4 (C, H, N).
7
6.1.1.8. 1-(2-Phenylethyl)-1H-benzimidazol-2-amine
(8f).
Obtained (0.71 g, 30%) from reaction of 7 with (2-bromoethyl)
benzene (1.85 g); white crystals, m.p. 125e126 ꢀC (Et2O) (lit. [26]
m.p. 120e122 ꢀC). Anal. C15H15N3 (C, H, N).
6.1.1.16. 1-[2-(1-Pyrrolidinyl)ethyl]-1H-benzimidazol-2-amine (8n).
Obtained (1.64 g, 71%) from reaction of 7 with 1-(2-chloroethyl)
pyrrolidine hydrochloride (1.70 g); white crystals, m.p. 172e173 ꢀC
(diisopropyl ether) (lit. [30], no m.p. reported). IR (KBr, cmꢂ1): 3321
br and 3134 br (NH2), 1659 s, 1618 w, 1548 s. 1H NMR (200 MHz,
6.1.1.9. 1-[(Methylthio)methyl]-1H-benzimidazol-2-amine
(8g).
Obtained (0.60 g, 31%) from reaction of 7 with (chloromethyl) methyl
sulphide (0.97 g); whitish crystals, m.p. 142e144 ꢀC (EtOAc/petro-
leum ether). IR (KBr, cmꢂ1): 3322 br and 3110 br (NH2), 1670, 1635,
DMSO-d6, ppm):
d 1.60e1.75 (m, 4H, pyrrolidinyl b-CH2’s), 2.44e
2.57 (m, 4H, pyrrolidinyl NeCH2’s), 2.68 (t, 2H, NCH2CH2Ne
pyrrolidinyl), 4.75 (t, 2H, NCH2CH2Nepyrrolidinyl), 6.39 (s, 2H,
NH2; disappeared with D2O), 6.78e6.99 (m, 2H, H-5,6), 7.10e7.15
(m, 2H, H-4,7). Anal. C13H18N4 (C, H, N).
1557 s, 1538. 1H NMR (200 MHz, CDCl3, ppm):
d 2.13 (s, 3H,
NCH2SCH3), 3.95(broads,2H, NH2;disappeared with D2O), 5.14 (s, 2H,
NCH2SCH3), 7.07e7.53 (m, 4H, H-4,5,6,7). Anal. C9H11N3S (C, H, N, S).
6.1.1.10. 1-(2-Pyridylmethyl)-1H-benzimidazol-2-amine
(8h).
6.1.1.17. 1-[2-(4-Morpholinyl)ethyl]-1H-benzimidazol-2-amine (8o).
Obtained (1.61 g, 65%) from reaction of 7 with 1-(2-chloroethyl)
morpholine hydrochloride (1.86 g); white crystals, m.p. 190e191 ꢀC
(EtOAc) (lit. [29] m.p. 190e191 ꢀC). Anal. C13H18N4O (C, H, N).
Obtained (1.51 g, 67%) from reaction of 7 with 2-(chloromethyl)
pyridine hydrochloride (1.64 g); white crystals, m.p. 153e154 ꢀC
(EtOAc/petroleum ether) (lit. [27], no m.p. reported). IR (KBr,
cmꢂ1): 3330 br and 3055 br (NH2), 1660, 1615 w, 1596, 1571 sh, 1554
s. 1H NMR (300 MHz, CDCl3, ppm):
d
5.17 (s, 2H, NCH2), 5.78 (broad
6.1.2. General procedure for the synthesis of 10-substituted 2-
hydroxypyrimido[1,2-a]benzimidazol-4(10H)-ones 9aeo
s, 2H, NH2; disappeared with D2O), 7.03e7.27 and 7.42 (2 m,
5H þ 1H, H-4,5,6,7 þ pyridyl H-30,50), 7.65 (m, 1H, pyridyl H-40),
8.56 (m, 1H, pyridyl H-60). 13C NMR (75.5 MHz, CDCl3, ppm):
Sodium (0.29 g,12.5 mmol) was dissolved in 15 mL of anhydrous
ethanol and this solution was concentrated to a small volume (2e
3 mL), then 5.0 mmol of the proper 1-substituted 1H-benzimida-
zol-2-amine 8 and 50.0 mmol (8.00 g) of diethyl malonate were
added and the resulting mixture was heated at 150 ꢀC (under
reflux) with stirring for 3 h. After cooling, the sodium salt of
compound 9, which precipitated as a whitish solid during the re-
action, was collected, crushed in a mortar, suspended in Et2O, fil-
tered and dried. This solid was then dissolved in water and the
resulting solution was acidified with an excess of 6 N aq HCl (or
carefully acidified with 6 N aq HCl down to pH 4, in the case of the
pyridyl derivatives compounds 9hek): this way compounds 9aek
separated out as white or whitish solids that were collected by
filtration, washed with water, dried and crystallized from the
proper solvent. Only in the case of compounds 9leo (bearing
a tertiary amino group on the side chain), the aqueous solution of
sodium salt was treated with an excess of 70% aq. HClO4 to get the
precipitation of compounds 9leo as water-insoluble perchlorates
which were collected by filtration, washed with water, dried and
crystallized from ethanol. According to this procedure the following
compounds 9 were obtained (IR and 1H NMR data are reported only
for the new compounds).
d
48.24, 107.09, 116.56, 119.73, 121.57, 122.31, 123.28, 134.10, 137.57,
142.09, 149.68, 154.84, 155.72. Anal. C13H12N4 (C, H, N).
6.1.1.11. 1-(3-Pyridylmethyl)-1H-benzimidazol-2-amine
(8i).
Obtained (1.28 g, 57%) from reaction of 7 with 3-(chloromethyl)
pyridine hydrochloride (1.64 g); white crystals, m.p. 247e248 ꢀC
(EtOAc) (lit. [27], no m.p. reported). IR (KBr, cmꢂ1): 3309 br and
3107 br (NH2), 1672, 1618 w, 1595, 1578 sh, 1556 s. 1H NMR
(300 MHz, DMSO-d6, ppm):
d 5.32 (s, 2H, NCH2), 6.63 (s, 2H, NH2;
disappeared with D2O), 6.82e6.98 (m, 2H, H-5,6), 7.10e7.18 (m, 2H,
H-4,7), 7.34 (m,1H, pyridyl H-50), 7.55 (m,1H, pyridyl H-40), 8.46 (m,
Jo ¼ 4.8 Hz, Jm ¼ 1.6 Hz, 1H, pyridyl H-60), 8.51 (m, Jm ¼ 1.6 Hz, 1H,
pyridyl H-20). Anal. C13H12N4 (C, H, N).
6.1.1.12. 1-(4-Pyridylmethyl)-1H-benzimidazol-2-amine
(8j).
Obtained (0.88 g, 39%) from reaction of 7 with 4-(chloromethyl)
pyridine hydrochloride (1.64 g); white crystals, m.p. 247e249 ꢀC
(acetone). IR (KBr, cmꢂ1): 3265 br and 3031 br (NH2), 1672, 1618
w, 1605, 1554 s. 1H NMR (200 MHz, DMSO-d6, ppm):
d
5.33 (s, 2H,
NCH2), 6.58 (s, 2H, NH2; disappeared with D2O), 6.78e7.22 (m, 6H,

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
573
6.1.2.1. 10-Ethyl-2-hydroxypyrimido[1,2-a]benzimidazol-4(10H)-one
(9a). Obtained (0.94 g, 82%) from 8a (0.81 g); white crystals, m.p.
308e310 ꢀC dec. (EtOH) (lit. [31] m.p. 281e283 ꢀC). Anal.
1H, H-3), 5.59 (s, 2H, NCH2), 7.26e7.54 (m, 5H, H-7,8,9 þ pyridyl H-
30,50), 7.79 (m, 1H, pyridyl H-40), 8.42e8.49 (m, 2H, H-6 þ pyridyl H-
60), 11.47 (s, 1H, OH; disappeared with D2O). 13C NMR (75.5 MHz,
C
12H11N3O2 (C, H, N).
DMSO-d6, ppm): d 47.25, 81.89, 111.04. 116.05, 122.55, 123.34,
123.88, 126.29, 126.35, 131.96, 138.13, 149.75, 150.31, 155.58, 161.52,
170.65. Anal. C16H12N4O2 (C, H, N).
6.1.2.2. 2-Hydroxy-10-propylpyrimido[1,2-a]benzimidazol-4(10H)-
one (9b). Obtained (0.99 g, 81%) from 8b (0.88 g); white crystals,
m.p. 298 ꢀC dec. (MeOH) (lit. [31] m.p. 253e255 ꢀC). Anal.
6.1.2.9. 2-Hydroxy-10-(3-pyridylmethyl)pyrimido[1,2-a]benzimida-
zol-4(10H)-one (9i). Obtained (1.20 g, 82%) from 8i (1.12 g); white
crystals, m.p. 234e235 ꢀC (acetone). IR (KBr, cmꢂ1): 3250e2300 br
(zwitterionic form NHþ), 1674 br, s (CO), 1613, 1585, 1547 s, 1499. 1H
C
13H13N3O2 (C, H, N).
6.1.2.3. 2-Hydroxy-10-isopropylpyrimido[1,2-a]benzimidazol-
4(10H)-one (9c). Obtained (1.13 g, 93%) from 8c (0.88 g); white
crystals, m.p. 285e287 ꢀC dec. (acetone). IR (KBr, cmꢂ1): 3180e
2200 br (zwitterionic form NHþ), 1670 sh and 1631 (CO), 1603,
NMR (300 MHz, DMSO-d6, ppm):
d 5.22 (s, 1H, H-3), 5.52 (s, 2H,
NCH2), 7.31e7.49 (m, 3H, H-7,8 þ pyridyl H-50), 7.65 (m, Jo ¼ 7.8 Hz,
1H, H-9), 7.79 (m, 1H, pyridyl H-40), 8.43 (m, Jo ¼ 7.8 Hz, 1H, H-6),
8.51 (m, Jo ¼ 4.8 Hz, Jm ¼ 1.6 Hz, 1H, pyridyl H-60), 8.74 (m,
Jm ¼ 1.6 Hz, 1H, pyridyl H-20), 11.50 (broad s, 1H, OH; disappeared
with D2O). Anal. C16H12N4O2 (C, H, N).
1558 s,1529 w,1496. 1H NMR (300 MHz, DMSO-d6, ppm):
d 1.62 and
1.68 [2d, J ¼ 7 Hz, 3H þ 3H, NCH(CH3)2], 5.11 [m, 1H, NCH(CH3)2],
5.17 (s, 1H, H-3), 7.34e7.49 (m, 2H, H-7,8), 7.79 (m, Jo ¼ 8 Hz, 1H, H-
9), 8.48 (m, Jo ¼ 7.8 Hz, 1H, H-6), 11.35 (s, 1H, OH; disappeared with
D2O). Anal. C13H13N3O2 (C, H, N).
6.1.2.10. 2-Hydroxy-10-(4-pyridylmethyl)pyrimido[1,2-a]benzimida-
zol-4(10H)-one (9j). Obtained (1.38 g, 94%) from 8j (1.12 g); white
crystals, m.p. 271e273 ꢀC (EtOH). IR (KBr, cmꢂ1): 3089e2133 br
(zwitterionic form NHþ), 1677 s (CO),1610, 1591 w, 1568 w, 1546. 1H
6.1.2.4. 10-Benzyl-2-hydroxypyrimido[1,2-a]benzimidazol-4(10H)-
one (9d). Obtained (1.33 g, 91%) from 8d (1.12 g); white crystals,
m.p. 256e257 ꢀC dec. (acetone) (lit. [19,32], no m.p. reported). Anal.
NMR (200 MHz, DMSO-d6, ppm):
d 5.22 (s, 1H, H-3), 5.53 (s, 2H,
C
17H13N3O2 (C, H, N).
NCH2), 7.23e7.61 (m, 5H, H-7,8,9 þ pyridyl H-30,50), 8.39e8.62 (m,
3H, H-6 þ pyridyl H-20,60), 11.50 (broad s, 1H, OH; disappeared with
D2O). MS (ESI) m/z: monoisotopic expected MW ¼ 292.10 exper-
imental full scan m/z [M þ H]þ ¼ 293.17. Fragment ions MS2
[M þ H]þ 293.17: m/z 251.08 (24%) ([M þ H]þ e CH2]C]O), 223.17
6.1.2.5. 2-Hydroxy-10-(1-phenylethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (9e). Obtained (1.03 g, 67%) from 8e (1.19 g); white
crystals, m.p. 232e234 ꢀC dec. (EtOH/petroleum ether). IR (KBr,
cmꢂ1): 3167e2378 br (zwitterionic form NHþ), 1671 s (CO), 1644 w,
(59%) ([M
þ
H]þ
e
CH2]C]O,
). Anal. C16H12N4O2 (C, H, N).
N H
e C]O), 93.08 (100%)
+
1607, 1532 s. 1H NMR (200 MHz, DMSO-d6, ppm):
d
2.00 [d, J ¼ 7 Hz,
(
CH2
3H, NCH(CH3)C6H5], 5.23 (s, 1H, H-3), 6.22 [q, J ¼ 7 Hz, 1H,
NCH(CH3)C6H5], 7.07e7.75 (m, 8H, H-7,8,9 þ C6H5), 8.47 (m,
Jo ¼ 7.8 Hz, 1H, H-6), 11.45 (broad s, 1H, OH; disappeared with D2O).
Anal. C18H15N3O2 (C, H, N).
6.1.2.11. 2-Hydroxy-10-[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]
pyrimido[1,2-a]benzimidazol-4(10H)-one (9k). Obtained (1.07 g,
61%) from 8k (1.41 g); white crystals, m.p. 232e234 ꢀC dec. (ace-
tone/petroleum ether). IR (KBr, cmꢂ1): 3200e2330 br (zwitterionic
form NHþ), 1679 s (CO), 1610, 1586. 1H NMR (200 MHz, DMSO-d6,
6.1.2.6. 2-Hydroxy-10-(2-phenylethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (9f). Obtained (1.09 g, 71%) from 8f (1.19 g); white
crystals, m.p. 260e261 ꢀC dec. (MeOH/acetone). IR (KBr, cmꢂ1):
3170e2380 br (zwitterionic form NHþ), 1680 sh and 1632 (CO),
ppm):
d
2.14 and 2.36 (2 s, 3H þ 3H, 2 CH3), 3.76 (s, 3H, OCH3), 5.18
(s, 1H, H-3), 5.54 (s, 2H, NCH2), 7.23e7.57 (m, 3H, H-7,8,9), 7.96 (s,
1H, pyridyl H-60), 8.44 (m, Jo ¼ 7.8 Hz, 1H, H-6), 10.40e11.80 (broad
signal, 1H, OH; disappeared with D2O). Anal. C19H18N4O3 (C, H, N).
1610, 1556 s, 1500. 1H NMR (300 MHz, DMSO-d6, ppm):
d 3.12 (t,
J ¼ 7.3 Hz, 2H, NCH2CH2C6H5), 4.46 (t, J ¼ 7.3 Hz, 2H, NCH2CH2C6H5),
5.19 (s, 1H, H-3), 7.10e7.60 (m, 8H, H-7,8,9 þ C6H5), 8.39 (m,
6.1.2.12. 10-[2-(Dimethylamino)ethyl]-2-hydroxypyrimido[1,2-a]
benzimidazol-4(10H)-one (9l). Obtained as perchlorate hemihy-
drate (9l$HClO4$0.5H2O) (1.11 g, 58%) from 8l (1.02 g); white crys-
tals, m.p. 260 ꢀC dec. (EtOH). IR (KBr, cmꢂ1): 3470e2490 br
(NHþ þ OH þ crystallization H2O),1674 s (CO),1612,1566 s,1490. 1H
Jo ¼ 7.8 Hz, 1H, H-6), 11.43 (s, 1H, OH; disappeared with D2O). 13
C
NMR (75.5 MHz, DMSO-d6, ppm):
d 33.26, 43.01, 80.63, 109.81,
114.88, 122.03, 125.05 (2C), 126.48, 128.31, 128.75, 130.55, 137.80,
148.17, 160.46, 169.56. Anal. C18H15N3O2 (C, H, N).
NMR (300 MHz, DMSO-d6, ppm): d
2.96 [s, 6H, NHþ(CH3)2], 3.60 [m,
6.1.2.7. 2-Hydroxy-10-[(methylthio)methyl]pyrimido[1,2-a]benzimi-
dazol-4(10H)-one (9g). Obtained (1.16 g, 89%) from 8g (0.97 g);
white crystals, m.p. 252e254 ꢀC dec. (EtOH). IR (KBr, cmꢂ1): 3180e
2195 br (zwitterionic form NHþ), 1675 sh and 1634 (CO), 1609,
2H, NCH2CH2NHþ(CH3)2], 4.61 [m, 2H, NCH2CH2NHþ(CH3)2], 5.22
(s, 1H, H-3), 7.32e7.55 (m, 2H, H-7,8), 7.75 (m, Jo ¼ 7.8 Hz, 1H, H-9),
8.42 (m, Jo ¼ 7.8 Hz, 1H, H-6), 9.13 (broad s, 1H, NHþ; disappeared
with D2O), 11.41 (broad s, 1H, OH; disappeared with D2O). MS (ESI)
m/z: monoisotopic expected MW ¼ 272.13, experimental MS full
scan m/z [M þ H]þ ¼ 273.17. Fragment ions MS2 [M þ H]þ 273.17:
m/z 228.08 (100%) ([M þ H]þ e HNMe2). Anal. C14H16N4O2$H-
ClO4$0.5H2O (C, H, N).
1558 s, 1530 w, 1496. 1H NMR (300 MHz, DMSO-d6, ppm):
d 2.17 (s,
3H, NCH2SCH3), 5.20 (s, 1H, H-3), 5.44 (s, 2H, NCH2SCH3), 7.34e7.53
(m, 2H, H-7,8), 7.76 (m, Jo ¼ 8 Hz, 1H, H-9), 8.42 (m, Jo ¼ 7.8 Hz, 1H,
H-6), 11.51 (broad s, 1H, OH; disappeared with D2O). MS (ESI) m/z:
monoisotopic expected MW ¼ 261.06, experimental full scan m/z
[M ꢂ H]ꢂ ¼ 260.08. Fragment ions MS2 [M ꢂ H]ꢂ 260.08: m/z
216.00 (100%), 200.08 (28%) ([M ꢂ H]ꢂe CH2]S]CH2). Anal.
6.1.2.13. 10-[2-(Diethylamino)ethyl]-2-hydroxypyrimido[1,2-a]benzi-
midazol-4(10H)-one (9m). Obtained as perchlorate hemihydrate
(9m/HClO4/0.5H2O) (1.41 g, 69%) from 8m (1.16 g); white crys-
tals, m.p. 266e267 ꢀC dec. (EtOH). IR (KBr, cmꢂ1): 3470e2510 br
(NHþ þ OH þ crystallization H2O), 1667 br, s (CO), 1615, 1562 br, s,
C
12H11N3O2S (C, H, N, S).
6.1.2.8. 2-Hydroxy-10-(2-pyridylmethyl)pyrimido[1,2-a]benzimida-
zol-4(10H)-one (9h). Obtained (1.04 g, 71%) from 8h (1.12 g); white
crystals, m.p. 247e248 ꢀC dec. (acetone). IR (KBr, cmꢂ1): 3200e
2280 br (zwitterionic form NHþ), 1680 sh and 1634 (CO), 1610,
1493. 1H NMR (300 MHz, DMSO-d6, ppm):
d
1.18 [t, J ¼ 7 Hz, 6H,
NHþ(CH2CH3)2], 3.33 [m, 4H, NHþ(CH2CH3)2], 3.61 [m, 2H,
NCH2CH2NHþ(CH2CH3)2], 4.60 [m, 2H, NCH2CH2NHþ(CH2CH3)2],
5.22 (s, 1H, H-3), 7.32e7.57 (m, 2H, H-7,8), 7.78 (m, Jo ¼ 7.8 Hz, 1H,
1558 s, 1530 sh, 1505. 1H NMR (300 MHz, DMSO-d6, ppm):
d 5.22 (s,

574
M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
H-9), 8.43 (m, Jo ¼ 7.8 Hz, 1H, H-6), 8.88 (broad s, 1H, NHþ; dis-
appeared with D2O), 11.42 (broad s, 1H, OH; disappeared with D2O).
Anal. C16H20N4O2$HClO4$0.5H2O (C, H, N).
J ¼ 7 Hz, 2H, NCH2CH2CH3), 6.21 (s, 1H, H-3), 7.36e7.55 (m, 3H, H-
7,8,9), 8.65 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal. C13H12ClN3O (C, H, N).
6.1.3.3. 2-Chloro-10-isopropylpyrimido[1,2-a]benzimidazol-4(10H)-
one (10c). Obtained (0.97 g, 93%) from 9c (0.97 g); white crystals
m.p. 202e203 ꢀC (EtOAc). IR (KBr, cmꢂ1): 1687 (CO),1615 w,1605 w,
6.1.2.14. 2-Hydroxy-10-[2-(1-pyrrolidinyl)ethyl]pyrimido[1,2-a]ben-
zimidazol-4(10H)-one (9n). Obtained as perchlorate (9n/HClO4)
(1.64 g, 82%) from 8n (1.15 g); white crystals, m.p. 266e267 ꢀC dec.
(EtOH). IR (KBr, cmꢂ1): 3545e2510 br (NHþ þ OH), 1669 s (CO),
1578 s, 1527. 1H NMR (200 MHz, CDCl3, ppm):
d
1.72 [d, J ¼ 7 Hz, 6H,
NCH(CH3)2], 5.30 [m, 1H, NCH(CH3)2], 6.22 (s, 1H, H-3), 7.32e7.47
(m, 3H, H-7,8,9), 8.72 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal. C13H12ClN3O
(C, H, N).
1614, 1562 s, 1492 w. 1H NMR (300 MHz, DMSO-d6, ppm):
d
1.70e
2.20 (m, 4H, pyrrolidinyl
b-CH2’s), 3.10e3.40 and 3.60e3.90 (2 m,
2H þ 2H, pyrrolidinyl NHþeCH2’s), 3.76 [m, 2H, NCH2CH2NHþe
pyrrolidinyl], 4.60 [m, 2H, NCH2CH2NHþepyrrolidinyl], 5.22 (s,
1H, H-3), 7.35e7.56 (m, 2H, H-7,8), 7.76 (m, Jo ¼ 7.8 Hz, 1H, H-9),
8.44 (m, Jo ¼ 7.8 Hz, 1H, H-6), 9.17 (broad s, 1H, NHþ; disappeared
with D2O), 11.39 (broad s, 1H, OH; disappeared with D2O). Anal.
6.1.3.4. 10-Benzyl-2-chloropyrimido[1,2-a]benzimidazol-4(10H)-one
(10d). Obtained (0.76 g, 61%) from 9d (1.17 g); white crystals, m.p.
256e257 ꢀC (EtOAc). IR (KBr, cmꢂ1): 1674 s (CO), 1614 w, 1576 s,
1523. 1H NMR (300 MHz, CDCl3, ppm):
d 5.48 (s, 2H, NCH2), 6.25 (s,
C
16H18N4O2/HClO4 (C, H, N).
1H, H-3), 7.27e7.47 (m, 8H, H-7,8,9 þ C6H5), 8.64 (m, Jo ¼ 7.8 Hz, 1H,
H-6). Anal. C17H12ClN3O (C, H, N).
6.1.2.15. 2-Hydroxy-10-[2-(4-morpholinyl)ethyl]pyrimido[1,2-a]ben-
zimidazol-4(10H)-one (9o). Obtained as perchlorate (9o/HClO4)
(1.64 g, 79%) from 8o (1.23 g); white crystals, m.p. 276 ꢀC dec.
(EtOH). IR (KBr, cmꢂ1): 3450e2500 br (NHþ þ OH), 1678 sh and
1634 (CO), 1612, 1566 s, 1528 w, 1504. 1H NMR (300 MHz, DMSO-d6,
6.1.3.5. 2-Chloro-10-(1-phenylethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (10e). Obtained (1.00 g, 77%) from 9e (1.22 g); white
crystals, m.p. 164e164.5 ꢀC (EtOAc/petroleum ether). IR (KBr,
cmꢂ1): 1681 s (CO), 1608 w, 1575 s, 1524. 1H NMR (200 MHz, CDCl3,
ppm):
d
3.10e3.32 and 3.43e3.60 (2 m, 2H þ 2H, NHþemorpholinyl
ppm):
d
2.04 [d, J ¼ 7 Hz, 3H, NCH(CH3)C6H5], 6.29 (s, 1H, H-3), 6.45
CH2’s), 3.64 [m, 2H, NCH2CH2NHþemorpholinyl], 3.82e4.12 (m, 4H,
morpholinyl OeCH2’s), 4.64 [m, 2H, NCH2CH2NHþemorpholinyl],
5.21 (s, 1H, H-3), 7.35e7.57 (m, 2H, H-7,8), 7.78 (m, Jo ¼ 7.8 Hz,
1H, H-9), 8.44 (m, Jo ¼ 7.8 Hz, 1H, H-6), 9.17 (broad s, 1H, NHþ;
disappeared with D2O), 11.39 (broad s, 1H, OH; disappeared with
[q, J ¼ 7 Hz, 1H, NCH(CH3)C6H5], 6.98e7.08 and 7.26e7.52 (2 m,
1H þ 7H, H-7,8,9 þ C6H5), 8.71 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal.
C18H14ClN3O (C, H, N).
6.1.3.6. 2-Chloro-10-(2-phenylethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (10f). Obtained (0.59 g, 45%) from 9f (1.22 g); white
crystals, m.p. 176e177 ꢀC (EtOAc/petroleum ether). IR (KBr, cmꢂ1):
1682 s (CO), 1614 w, 1580 s, 1519. 1H NMR (300 MHz, CDCl3, ppm):
D2O). 13C NMR (75.5 MHz, DMSO-d6, ppm):
d 35.93, 51.49, 54.18,
63.26, 80.84, 110.03, 114.96, 122.60, 125.30, 125.63, 130.10, 149.06,
160.53, 169.26. Anal. C16H18N4O3/HClO4 (C, H, N).
d
3.16 (t, J ¼ 7.2 Hz, 2H, NCH2CH2C6H5), 4.50 (t, J ¼ 7.2 Hz, 2H,
6.1.3. General procedure for the synthesis of 10-substituted 2-
chloropyrimido[1,2-a]benzimidazol-4(10H)-ones 10aeo
NCH2CH2C6H5), 6.18 (s, 1H, H-3), 7.03e7.43 (m, 8H, H-7,8,9 þ C6H5),
8.59(m, Jo ¼ 7.8 Hz,1H, H-6).13C NMR(75.5MHz, CDCl3, ppm):
d34.40,
A mixture of the proper 2-hydroxyderivative 9 (4.0 mmol) and
an excess of POCl3 (15.0 mL) was stirred at 130 ꢀC (under reflux) for
2 h. The excess POCl3 was removed in vacuo, and the thick and dark
residue was treated with 5% NaHCO3 (100 mL) and CH2Cl2 (100 mL)
until it was completely dissolved. The mixture was stirred at room
temperature for 30 min, treated with charcoal and filtered. The
organic phase was then collected and the aqueous one was
exhaustively extracted with CH2Cl2. The combined extracts, dried
over anhydrous Na2SO4 were concentrated in vacuo and chroma-
tographed on a silica gel column [eluants: CH2Cl2 for compounds
10aeg, CH2Cl2eEtOAc (1:1) for compounds 10hek, acetoneeEtOAc
(1:1) for compounds 10leo]. The eluate collected, after removal of
solvents, afforded the desired 2-chloroderivative 10 as a solid
which was taken up in a small amount of petroleum ether, recov-
ered by filtration, then crystallized from the proper solvent. Ac-
cording to this procedure the following compounds 10 were
obtained.
44.14,101.31,109.04,116.86,123.24,125.12,126.51,127.10,128.77 (2C),
130.53, 137.11, 147.33, 158.00, 159.13. Anal. C18H14ClN3O (C, H, N).
6.1.3.7. 2-Chloro-10-[(methylthio)methyl]pyrimido[1,2-a]benzimida-
zol-4(10H)-one (10g). Obtained (0.51 g, 45%) from 8g (1.05 g);
white crystals, m.p. 256e257 ꢀC (EtOAc). IR (KBr, cmꢂ1): 1680 s
(CO), 1605 w, 1581 s, 1524. 1H NMR (200 MHz, CDCl3, ppm):
d 2.24
(s, 3H, NCH2SCH3), 5.41 (s, 2H, NCH2SCH3), 6.28 (s, 1H, H-3), 7.39e
7.64 (m, 3H, H-7,8,9), 8.67 (m, Jo ¼ 7.8 Hz, 1H, H-6). MS (ESI) m/z:
monoisotopic expected MW ¼ 279.02; experimental full scan m/z
[M ꢂ H]ꢂ ¼ 278.00. Fragment ions MS2 [M ꢂ H]ꢂ 278.00: m/z
242.00 (100%) ([M ꢂ H]ꢂ e HCl), 231.00 (81%). Anal. C12H10ClN3OS
(C, H, N, S).
6.1.3.8. 2-Chloro-10-(2-pyridylmethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (10h). Obtained (0.78 g, 63%) from 9h (1.17 g); white
crystals, m.p. 194e195 ꢀC (EtOAc). IR (KBr, cmꢂ1): 1686 s (CO), 1613
w, 1583 s, 1525. 1H NMR (300 MHz, CDCl3, ppm):
d 5.60 (s, 2H,
6.1.3.1. 2-Chloro-10-ethylpyrimido[1,2-a]benzimidazol-4(10H)-one
(10a). Obtained (0.88 g, 89%) from 9a (0.92 g); white crystals, m.p.
199e201 ꢀC (EtOAc/petroleum ether). IR (KBr, cmꢂ1): 1686 s (CO),
NCH2), 6.24 (s,1H, H-3), 7.21e7.47 (m, 5H, H-7,8,9 þ pyridyl H-30,50),
7.67 (m, 1H, pyridyl H-40), 8.49e8.63 (m, 2H, H-6 þ pyridyl H-60).
13C NMR (75.5 MHz, CDCl3, ppm):
d 47.85, 101.70, 110.25, 116.79,
1605 sh, 1583 s, 1526. 1H NMR (200 MHz, CDCl3, ppm):
d
1.48 (t,
122.18, 123.32, 123.56, 125.32, 126.73, 130.64, 137.23, 147.73, 149.75,
154.03, 158.04, 159.12. Anal. C16H11ClN4O (C, H, N).
J ¼ 7 Hz, 3H, NCH2CH3), 4.36 (q, J ¼ 7 Hz, 2H, NCH2CH3), 6.21 (s, 1H,
H-3), 7.30e7.60 (m, 3H, H-7,8,9), 8.65 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal.
C
12H10ClN3O (C, H, N).
6.1.3.9. 2-Chloro-10-(3-pyridylmethyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (10i). Obtained (0.65 g, 52%) from 9i (1.17 g); white
crystals, m.p. 243e244 ꢀC (EtOAc). IR (KBr, cmꢂ1): 1672 s (CO), 1613
w, 1605 w, 1579 s, 1571 s, 1523. 1H NMR (200 MHz, CDCl3, ppm):
6.1.3.2. 2-Chloro-10-propylpyrimido[1,2-a]benzimidazol-4(10H)-one
(10b). Obtained (0.85 g, 81%) from 9b (0.97 g); white crystals, m.p.
162e163 ꢀC (EtOAc/petroleum ether). IR (KBr, cmꢂ1): 1673 s (CO),
d
5.53 (s, 2H, NCH2), 6.30 (s, 1H, H-3), 7.25e7.80 (m, 5H, H-
1611, 1575 s, 1523. 1H NMR (300 MHz, CDCl3, ppm):
d
1.02 (t,
7,8,9 þ pyridyl H-40,50), 8.55e8.91 (m, 3H, H-6 þ pyridyl H-20,60).
J ¼ 7 Hz, 3H, NCH2CH2CH3), 1.95 (m, 2H, NCH2CH2CH3), 4.26 (t,
Anal. C16H11ClN4O (C, H, N).

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
575
6.1.3.10. 2-Chloro-10-(4-pyridylmethyl)pyrimido[1,2-a]benzimida-
6.1.4. General procedure for the synthesis of 10-substituted 2-(1-
zol-4(10H)-one (10j). Obtained (0.53 g, 44%) from 9j (1.17 g); white
piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones 6aeo
crystals, m.p. 265e267 ꢀC dec. (EtOAc). IR (KBr, cmꢂ1): 1671 s (CO),
A mixture of 2.0 mmol of the proper chloroderivative 10,
piperazine (1.72 g, 20.0 mmol) and ethanol (60 mL) was heated at
reflux for 3 h. The solution obtained was poured into water
(250 mL): only in the case of reaction with the 10-benzylderivative
10d, pure compound 6d separated out as a white solid which was
collected by filtration, dried and crystallized from proper solvent. In
all other cases an emulsion was obtained which was exhaustively
extracted with CH2Cl2. The combined extracts, after drying and
removal of solvents, afforded an oily or nearly solid residue which,
in most cases, was treated with a small amount of EtOAc and diethyl
ether to give pure compound 6 as a whitish solid which was then
crystallized from the suitable solvent (compounds 6a,f,gel,n,o).
Compounds 6b,c required a preliminary purification by chroma-
tography on a silica gel column, eluting with THF to remove some
impurities, then with MeOH to recover pure compound 6. In the
case of compounds 6e,m the final oily residue was treated with
a solution of maleic acid (0.29 g, 2.5 mmol) in anhydrous ethanol to
give the corresponding pure maleates (6e/C4H4O4 or
6m/2C4H4O4) as a white solid which was then crystallized from
anhydrous ethanol.
1582 s, 1526. 1H NMR (200 MHz, CDCl3, ppm):
d 5.52 (s, 2H, NCH2),
6.31 (s, 1H, H-3), 7.15e7.30 and 7.38e7.57 (2 m, 3H þ 2H, H-
7,8,9 þ pyridyl H-30,50), 8.55e8.75 (m, 3H, H-6 þ pyridyl H-20,60).
MS (ESI) m/z: monoisotopic expected MW ¼ 310.06, experimental
full scan m/z [M þ H]þ ¼ 311.17. Fragment ions MS2 [M þ H]þ
311.17: m/z 275.17 (100%) ([M
þ
H]þ
e
HCl), 247.17 (15%)
+
([M þ H]þ e HCl, e C]O), 93.08 (26%) (
). Anal.
CH2
N
H
C
16H11ClN4O (C, H, N).
6.1.3.11. 2-Chloro-10-[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]
pyrimido[1,2-a]benzimidazol-4(10H)-one (10k). Obtained (0.87 g,
59%) from 9k (1.40 g); white crystals, m.p. 214e215 ꢀC (EtOAc/pe-
troleum ether). IR (KBr, cmꢂ1): 1683 s (CO), 1612 w, 1580 s, 1529. 1H
NMR (200 MHz, CDCl3, ppm):
d
2.30 and 2.36 (2 s, 3H þ 3H, 2 CH3),
3.83 (s, 3H, OCH3), 5.69 (s, 2H, NCH2), 6.27 (s, 1H, H-3), 7.38e7.58
(m, 3H, H-7,8,9), 8.20 (s, 1H, pyridyl H-60), 8.68 (m, Jo ¼ 7.8 Hz,
1H, H-6). Anal. C19H17ClN4O2 (C, H, N).
6.1.3.12. 2-Chloro-10-[2-(dimethylamino)ethyl]pyrimido[1,2-a]ben-
zimidazol-4(10H)-one (10l). Obtained (0.44 g, 38%) from
9l$HClO4$0.5H2O (1.53 g); white crystals, m.p. 159e160 ꢀC (diiso-
propyl ether). IR (KBr, cmꢂ1): 1679 s (CO), 1615 w, 1578 s, 1521. 1H
According to procedures above described, the following com-
pounds were prepared.
NMR (200 MHz, CDCl3, ppm):
d
2.35 [s, 6H, N(CH3)2], 2.81 [t,
Hz, 2H,
6.1.4.1. 10-Ethyl-2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (6a). Obtained (0.44 g, 74%) from 10a (0.50 g); white
crystals, m.p. 212e214 ꢀC (EtOAc). IR (KBr, cmꢂ1): 3316 w and 3287
w (NH), 1661 s (CO), 1609, 1585 s, 1528. 1H NMR (200 MHz, CDCl3,
J
¼
7
Hz, 2H, NCH2CH2N(CH3)2], 4.42 [t,
J
¼
7
NCH2CH2N(CH3)2], 6.24 (s,1H, H-3), 7.35e7.63 (m, 3H, H-7,8,9), 8.68
(m, Jo ¼ 7.8 Hz, 1H, H-6). MS (ESI) m/z: monoisotopic expected
MW ¼ 290.09, experimental full scan m/z [M þ H]þ ¼ 291.17.
Fragment ions MS2 [M þ H]þ 291.17: m/z 228.08 (10%), 210.08
ppm):
d
1.42 (t, J ¼ 7 Hz, 3H, NCH2CH3), 2.39 (s, 1H, NH; disappeared
with D2O), 2.97 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.64 [t,
J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne], 4.21 (q, J ¼ 7 Hz, 2H, NCH2CH3),
5.30 (s, 1H, H-3), 7.19e7.41 (m, 3H, H-7,8,9), 8.55 (m, Jo ¼ 7.8 Hz, 1H,
H-6). Anal. C16H19N5O (C, H, N).
(100%) ([M þ H]þ e HNMe2, e HCl), 182.17 (72%) ([M þ H]þ
e
HNMe2, e HCl, e C]O), 155.08 (21%). Anal. C14H15ClN4O (C, H, N).
6.1.3.13. 2-Chloro-10-[2-(diethylamino)ethyl]pyrimido[1,2-a]benzi-
midazol-4(10H)-one (10m). Obtained (0.45 g, 35%) from
9m$HClO4$0.5H2O (1.64 g); white crystals, m.p. 125e127 ꢀC (dii-
sopropyl ether). IR (KBr, cmꢂ1): 1680 s (CO), 1614 w, 1605 w, 1587 s,
6.1.4.2. 2-(1-Piperazinyl)-10-propylpyrimido[1,2-a]benzimidazol-
4(10H)-one (6b). Obtained (0.35 g, 56%) from 10b (0.52 g); white
crystals, m.p. 188e189 ꢀC (EtOAc/diethyl ether). IR (KBr, cmꢂ1):
3319 w and 3288 w (NH), 1668 s (CO), 1610, 1584, 1529. 1H NMR
1524. 1H NMR (300 MHz, CDCl3, ppm):
d
0.90 [t, J ¼ 7 Hz, 6H,
N(CH2CH3)2], 2.55 [q, J ¼ 7 Hz, 4H, N(CH2CH3)2], 2.87 [t, J ¼ 7 Hz, 2H,
NCH2CH2N(CH2CH3)2], 4.34 [t, J ¼ 7 Hz, 2H, NCH2CH2N(CH2CH3)2],
6.19 (s, 1H, H-3), 7.33e7.55 (m, 3H, H-7,8,9), 8.63 (m, Jo ¼ 7.8 Hz, 1H,
H-6). Anal. C16H19ClN4O (C, H, N).
(200 MHz, CDCl3, ppm):
d
1.01 (t, J ¼ 7 Hz, 3H, NCH2CH2CH3), 1.75e
2.15 (m, 3H, NCH2CH2CH3 þ NH; 2H after treatment with D2O), 2.99
[t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.64 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 4.16 (t, J ¼ 7 Hz, 2H, NCH2CH2CH3), 5.32 (s,
1H, H-3), 7.15e7.48 (m, 3H, H-7,8,9), 8.58 (m, Jo ¼ 7.8 Hz, 1H, H-6).
Anal. C17H21N5O (C, H, N).
6.1.3.14. 2-Chloro-10-[2-(1-pyrrolidinyl)ethyl]pyrimido[1,2-a]benzi-
midazol-4(10H)-one (10n). Obtained (0.70 g, 55%) from 9n/HClO4
(1.60 g); white crystals, m.p. 142e143 ꢀC (EtOAc/petroleum ether).
IR (KBr, cmꢂ1): 1682 s (CO), 1589 s, 1526. 1H NMR (200 MHz, CDCl3,
6.1.4.3. 10-Isopropyl-2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (6c). Obtained as monohydrate (6c$H2O) (0.46 g, 70%)
from 10c (0.52 g); white crystals m.p. 152e153 ꢀC (EtOAc). IR (KBr,
cmꢂ1): 3465 (crystallization H2O), 3232 (NH), 1659 s (CO), 1608,
ppm): d 1.82 (m, 4H, pyrrolidinyl b-CH2’s), 2.66 (m, 4H, pyrrolidinyl
NeCH2’s), 2.98 [t, J ¼ 7 Hz, 2H, NCH2CH2Nepyrrolidinyl], 4.44 [t,
J ¼ 7 Hz, 2H, NCH2CH2Nepyrrolidinyl], 6.23 (s, 1H, H-3), 7.34e7.62
(m, 3H, H-7,8,9), 8.67 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal. C16H17ClN4O (C,
H, N).
1571, 1531. 1H NMR (200 MHz, CDCl3, ppm):
d
1.64 [d, J ¼ 7 Hz, 6H,
NCH(CH3)2], 2.19 (s, 3H, NH þ crystallization H2O; disappeared with
D2O), 2.99 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.64 [t, J ¼ 5 Hz,
4H, piperazinyl (CH2)2Ne], 5.04 [m, 1H, NCH(CH3)2], 5.31 (s, 1H, H-
3), 7.16e7.43 (m, 3H, H-7,8,9), 8.59 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal.
6.1.3.15. 2-Chloro-10-[2-(4-morpholinyl)ethyl]pyrimido[1,2-a]benzi-
midazol-4(10H)-one (10o). Obtained (0.69 g, 52%) from 9o/HClO4
(1.66 g); white crystals, m.p. 189e191 ꢀC (EtOAc). IR (KBr, cmꢂ1):
C17H21N5O$H2O (C, H, N).
1682 s (CO), 1586 s, 1527. 1H NMR (300 MHz, CDCl3, ppm):
d
2.54 (t,
6.1.4.4. 10-Benzyl-2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-
4(10H)-one (6d). Obtained (0.53 g, 74%) from 10d (0.62 g); white
crystals, m.p. 165e166 ꢀC (EtOAc/petroleum ether). IR (KBr, cmꢂ1):
3215 (NH), 1667 s (CO), 1610, 1583, 1532. 1H NMR (300 MHz, CDCl3,
J ¼ 4.6 Hz, 4H, morpholinyl NeCH2’s), 2.80 [t, J ¼ 6.3 Hz, 2H,
NCH2CH2Nemorpholinyl], 3.60 (t, J ¼ 4.6 Hz, 4H, morpholinyl Oe
CH2’s), 4.40 [t, J ¼ 6.3 Hz, 2H, NCH2CH2Nemorpholinyl], 6.19 (s,
1H, H-3), 7.31e7.58 (m, 3H, H-7,8,9), 8.62 (m, Jo ¼ 7.8 Hz, 1H, H-6).
ppm):
d
1.89 (s, 1H, NH; disappeared with D2O), 2.96 [t, J ¼ 5 Hz, 4H,
13C NMR (75.5 MHz, CDCl3, ppm):
d
39.94, 53.70, 55.96, 66.83,
piperazinyl (CH2)2NH], 3.64 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne],
5.33 (s, 1H, H-3), 5.36 (s, 2H, NCH2), 7.11e7.36 (m, 8H, H-
7,8,9 þ C6H5), 8.56 (m, Jo ¼ 7.8 Hz,1H, H-6). Anal. C21H21N5O (C, H, N).
101.19, 109.19, 116.97, 123.32, 125.36, 126.54, 130.63, 147.79, 157.94,
159.17. Anal. C16H17ClN4O2 (C, H, N).

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
6.1.4.5. 10-(1-Phenylethyl)-2-(1-piperazinyl)pyrimido[1,2-a]benzimi-
dazol-4(10H)-one (6e). Obtained as maleate (6e/C4H4O4) (0.68 g,
70%) from 10e (0.65 g); white crystals, m.p. 146e148 ꢀC (anhydrous
EtOH/Et2O). The 1H NMR and IR spectra were recorded on the free
amine obtained (as amorphous solid, m.p. 110e112 ꢀC) from the
analytical sample of maleate, by treatment with aqueous NaHCO3,
exhaustive extraction with CH2Cl2 and removal of solvent. IR (KBr,
cmꢂ1): 3220 (NH), 1663 s (CO), 1610, 1579, 1533. 1H NMR (200 MHz,
6.1.4.10. 2-(1-Piperazinyl)-10-(4-pyridylmethyl)pyrimido[1,2-a]ben-
zimidazol-4(10H)-one (6j). Obtained as hemihydrate (6j$0.5H2O)
(0.60 g, 81%) from 10j (0.62 g); white crystals, m.p. 202e204 ꢀC
(EtOAc). IR (KBr, cmꢂ1): 3422 (crystallization H2O), 3320 (NH),
1669 s (CO), 1615, 1593, 1539. 1H NMR (200 MHz, CDCl3, ppm):
d
1.84 (s, 2H, NH þ crystallization H2O; disappeared with D2O), 2.96
[t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.62 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 5.36 (s, 1H, H-3), 5.39 (s, 2H, NCH2), 7.00e
7.40 (m, 5H, H-7,8,9 þ pyridyl H-30,50), 8.47e8.67 (m, 3H, H-
CDCl3, ppm):
d
2.00 [d, J ¼ 7 Hz, 3H, NCH(CH3)C6H5], 2.67 (s,1H, NH;
disappeared with D2O), 3.19 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH],
3.88 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne], 5.42 (s,1H, H-3), 6.27 [q,
J ¼ 7 Hz, 1H, NCH(CH3)C6H5], 6.91e7.03 and 7.15e7.44 (m, 1H þ 7H,
6
þ
pyridyl H-20,60). MS (ESI) m/z: monoisotopic expected
MW ¼ 360.17, experimental full scan m/z [M þ H]þ ¼ 361.25.
Fragment ions MS2 [M þ H]þ 361.25: m/z 318.17 (100%) ([M þ H]þ
e
H-7,8,9
þ
C6H5), 8.61 (m, Jo
¼
7.8 Hz, 1H, H-6). Anal.
CH2]CHeNH2), 226.08 (32%) ([M þ H]þ e CH2]CHeNH2, e
C
22H23N5O/C4H4O4 (C, H, N).
). Anal. C20H20N6O$0.5H2O (C, H, N).
N
CH2
6.1.4.6. 10-(2-Phenylethyl)-2-(1-piperazinyl)pyrimido[1,2-a]benzimi-
dazol-4(10H)-one (6f). Obtained (0.63 g, 84%) from 10f (0.65 g);
white crystals, m.p. 137e138 ꢀC (EtOAc/diisopropyl ether). IR (KBr,
cmꢂ1): 3237 (NH), 1675 s (CO), 1612, 1584 s, 1530. 1H NMR
6.1.4.11. 2-(1-Piperazinyl)-10-[(4-methoxy-3,5-dimethyl-2-pyridyl)
methyl]pyrimido[1,2-a]benzimidazol-4(10H)-one (6k). Obtained as
hemihydrate (6k$0.5H2O) (0.79 g, 92%) from 10k (0.74 g); white
crystals, m.p. 206e208 ꢀC (EtOAc). IR (KBr, cmꢂ1): 3440 (crystal-
lization H2O), 3283 (NH), 1670 s (CO), 1612, 1590, 1540. 1H NMR
(300 MHz, CDCl3, ppm):
d 2.37 (s, 1H, NH; disappeared with D2O),
2.97 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.11 (t, J ¼ 7.3 Hz, 2H,
NCH2CH2C6H5), 3.59 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne], 4.36 (t,
J ¼ 7.3 Hz, 2H, NCH2CH2C6H5), 5.27 (s, 1H, H-3), 7.03e7.32 (m, 8H,
H-7,8,9 þ C6H5), 8.52 (m, Jo ¼ 7.8 Hz, 1H, H-6). 13C NMR (75.5 MHz,
(200 MHz, CDCl3, ppm): d 2.27 and 2.29 (2 s, 8H, 2
CH3 þ NH þ crystallization H2O; 6H after treatment with D2O), 3.13
[t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.75 (s, 3H, OCH3), 3.82 [t,
J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne], 5.39 (s, 1H, H-3), 5.49 (s, 2H,
NCH2), 7.20e7.42 (m, 3H, H-7,8,9), 8.22 (s, 1H, pyridyl H-60), 8.56
(m, Jo ¼ 7.8 Hz, 1H, H-6). Anal. C23H26N6O2$0.5H2O (C, H, N).
CDCl3, ppm):
d 34.27, 43.57, 45.74, 45.81, 78.67, 108.10, 116.11,
122.24, 124.72, 126.01, 126.87, 128.70, 128.72, 130.66, 137.77, 148.23,
161.25, 162.47. Anal. C22H23N5O (C, H, N).
6.1.4.12. 10-[2-(Dimethylamino)ethyl]-2-(1-piperazinyl)pyrimido
[1,2-a]benzimidazol-4(10H)-one (6l). Obtained (0.45 g, 66%) from
10l (0.58 g); white crystals, m.p. 124e125 ꢀC (EtOAc/petroleum
ether). IR (KBr, cmꢂ1): 3311 (NH), 1672 s (CO), 1612, 1587, 1537. 1H
6.1.4.7. 10-[(Methylthio)methyl]-2-(1-piperazinyl)pyrimido[1,2-a]
benzimidazol-4(10H)-one (6g). Obtained (0.34 g, 52%) from 10g
(0.56 g); white crystals, m.p. 175e177 ꢀC (EtOAc/Et2O). IR (KBr,
cmꢂ1): 3330 (NH), 1668 s (CO), 1611, 1585, 1537. 1H NMR (300 MHz,
NMR (200 MHz, CDCl3, ppm):
d 2.01 (s, 1H, NH; disappeared with
CDCl3, ppm):
d
2.18 (s, 4H, NCH2SCH3 þ NH; 3H after treatment with
D2O), 2.36 [s, 6H, N(CH3)2], 2.76 [t, J ¼ 7 Hz, 2H, NCH2CH2N(CH3)2],
2.98 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.64 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 4.30 [t, J ¼ 7 Hz, 2H, NCH2CH2N(CH3)2], 5.33
(s, 1H, H-3), 7.22e7.47 (m, 3H, H-7,8,9), 8.57 (m, Jo ¼ 7.8 Hz, 1H, H-
6). MS (ESI) m/z: monoisotopic expected MW ¼ 340.20, exper-
imental full scan m/z [M þ H]þ ¼ 341.25. Fragment ions MS2
[M þ H]þ 341.25: m/z 298.17 (43%) ([M þ H]þ e CH2]CHeNH2),
296.00 (43%) ([M þ H]þ e HNMe2), 270.33 (100%) ([M þ H]þe
CH2]CHeNH2, e C]O), 155.08 (21%). Anal. C18H24N6O (C, H, N).
D2O), 2.98 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.64 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 5.27 (s, 2H, NCH2SCH3), 5.31 (s, 1H, H-3),
7.28e7.43 (m, 3H, H-7,8,9), 8.55 (m, Jo ¼ 7.8 Hz,1H, H-6). MS (ESI) m/
z: monoisotopic expected MW ¼ 329.13, experimental full scan m/z
[M þ H]þ ¼ 330.17. Fragment ions MS2 [M þ H]þ: m/z 282.17 (100%)
([M þ H]þ e CH3SH), 270.17 (52%), 239.17 (8%) ([M þ H]þ e CH3SH, e
CH2]CHeNH2). Anal. C16H19N5OS (C, H, N, S).
6.1.4.8. 2-(1-Piperazinyl)-10-(2-pyridylmethyl)pyrimido[1,2-a]benzi-
midazol-4(10H)-one (6h). Obtained (0.49 g, 68%) from 10h (0.62 g);
white crystals, m.p. 175e176 ꢀC (EtOAc). IR (KBr, cmꢂ1): 3240 (NH),
1665 s (CO), 1612, 1584, 1532. 1H NMR (300 MHz, CDCl3, ppm):
6.1.4.13. 10-[2-(Diethylamino)ethyl]-2-(1-piperazinyl)pyrimido[1,2-
a]benzimidazol-4(10H)-one (6m). As dimaleate (6m/2H4C4O4)
(0.68 g, 57%) from 10m (0.64 g); whitish crystals, m.p. 156e158 ꢀC
(anhydrous EtOH). The 1H NMR and IR spectra were recorded on
the free amine obtained (as a whitish resin) from the analytical
sample of maleate as above described for compound 6e. IR (CHCl3,
cmꢂ1): 1667 s (CO), 1613, 1590 s, 1520 (NH stretching was not
d
2.17 (s, 1H, NH; disappeared with D2O), 2.94 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2NH], 3.62 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne],
5.34 (s, 1H, H-3), 5.49 (s, 2H, NCH2), 7.13e7.32 (m, 5H, H-
7,8,9 þ pyridyl H-30,50), 7.63 (m,1H, pyridyl H-40), 8.53e8.61 (m, 2H,
H-6 þ pyridyl H-60). 13C NMR (75.5 MHz, CDCl3, ppm):
d
45.75,
observable). 1H NMR (300 MHz, CDCl3, ppm):
d
0.97 [t, J ¼ 7 Hz, 6H,
45.87, 47.41, 78.87, 109.11, 116.08, 121.60, 122.66, 123.01, 124.96,
126.18, 130.73, 137.16, 148.58, 149.61, 155.19, 161.27, 162.51. Anal.
N(CH2CH3)2], 2.12 (s, 1H, NH; disappeared with D2O), 2.59 [q,
J ¼ 7 Hz, 4H, N(CH2CH3)2], 2.84 [t, J ¼ 7 Hz, 2H, NCH2CH2N(C2H5)2],
3.02 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.67 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 4.24 [t, J ¼ 7 Hz, 2H, NCH2CH2N(C2H5)2],
5.30 (s, 1H, H-3), 7.23e7.44 (m, 3H, H-7,8,9), 8.58 (m, Jo ¼ 7.8 Hz, 1H,
H-6). Anal. C20H28N6O/2 H4C4O4 (C, H, N).
C
20H20N6O (C, H, N).
6.1.4.9. 2-(1-Piperazinyl)-10-(3-pyridylmethyl)pyrimido[1,2-a]benzi-
midazol-4(10H)-one (6i). Obtained as hemihydrate (6i$0.5H2O)
(0.45 g, 61%) from 10i (0.62 g); white crystals, m.p. 185e186 ꢀC
(EtOAc). IR (KBr, cmꢂ1): 3391 (crystallization H2O), 3316 (NH),
1675 s (CO), 1613, 1592, 1539. 1H NMR (200 MHz, CDCl3, ppm):
6.1.4.14. 2-(1-Piperazinyl)-10-[2-(1-pyrrolidinyl)ethyl]pyrimido[1,2-
a]benzimidazol-4(10H)-one (6n). Obtained (0.63 g, 86%) from 10n
(0.63 g); white crystals, m.p. 152e153 ꢀC (EtOAc). IR (KBr, cmꢂ1):
3280 (NH), 1671 s (CO), 1610, 1585, 1537. 1H NMR (300 MHz, CDCl3,
d
1.82 (s, 2H, NH þ crystallization H2O; disappeared with D2O), 2.94
[t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.61 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2Ne], 5.31 (s, 1H, H-3), 5.36 (s, 2H, NCH2), 7.11e
7.37 (m, 4H, H-7,8,9 þ pyridyl H-50), 7.63 (m, 1H, pyridyl H-40),
8.50e8.62 (m, 2H, H-6 þ pyridyl H-60), 8.71 (s, 1H, pyridyl H-20).
Anal. C20H20N6O$0.5H2O (C, H, N).
ppm):
d 1.80 (m, 4H, pyrrolidinyl b-CH2’s), 2.16 (s, 1H, NH; dis-
appeared with D2O), 2.63 (m, 4H, pyrrolidinyl NeCH2’s), 2.90 [t,
J ¼ 7 Hz, 2H, NCH2CH2Nepyrrolidinyl], 2.96 [t, J ¼ 5 Hz, 4H,
piperazinyl (CH2)2NH], 3.62 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2Ne],

M. Di Braccio et al. / European Journal of Medicinal Chemistry 62 (2013) 564e578
577
4.31 [t, J ¼ 7 Hz, 2H, NCH2CH2Nepyrrolidinyl], 5.30 (s, 1H, H-3),
7.23e7.39 (m, 3H, H-7,8,9), 8.54 (m, Jo ¼ 7.8 Hz, 1H, H-6). Anal.
114.73, 122.24, 125.12, 125.67, 131.00, 150.28, 152.02, 171.88. MS
(ESI) m/z: monoisotopic expected MW ¼ 284.16, experimental full
scan m/z [M þ H]þ ¼ 285.25. Fragment ions MS2 [M þ H]þ 285.25:
m/z 257.17 (84%) ([M þ H]þ e CH2]CH2), 241.17 (48%), 162.17
(100%), 124.00 (30%). Anal. C16H20N4O (C, H, N).
C
20H26N6O (C, H, N).
6.1.4.15. 10-[2-(4-Morpholinyl)ethyl]-2-(1-piperazinyl)pyrimido[1,2-
a]benzimidazol-4(10H)-one (6o). Obtained (0.59 g, 77%) from 10o
(0.66 g); white crystals, m.p. 172e173.5 ꢀC (EtOAc). IR (KBr, cmꢂ1):
3265 (NH),1686 s (CO),1611,1593 s,1535. 1H NMR (300 MHz, CDCl3,
6.1.7. 10-Ethyl-2-(diethylamino)pyrimido[1,2-a]benzimidazol-
4(10H)-one (6p)
ppm):
d
2.04 (s, 1H, NH; disappeared with D2O), 2.55 (t, J ¼ 4.6 Hz,
A mixture of 2-chloro-10-ethylpyrimido[1,2-a]benzimidazol-
4(10H)-one (10a) (0.37 g, 1.5 mmol) and a large excess of diethyl-
amine (5 mL) in ethanol (25 mL) was refluxed for 16 h. The resulting
solution was evaporated to dryness in vacuo and the residue was
partitioned between CH2Cl2 and aq 5% NaHCO3. The combined
extracts, dried over Na2SO4, afforded an oily residue which was
taken up in a little diisopropyl ether to give pure compound 6p as
a crystalline solid (0.38 g, 89%); white crystals, m.p. 152e153 ꢀC
(EtOAc/petroleum ether). IR (KBr, cmꢂ1): 1671 s (CO), 1615, 1594,
4H, morpholinyl NeCH2’s), 2.78 [t, J ¼ 6.6 Hz, 2H, NCH2CH2Ne
morpholinyl], 2.95 [t, J ¼ 5 Hz, 4H, piperazinyl (CH2)2NH], 3.53e
3.70 [m, 8H, morpholinyl OeCH2’s þ piperazinyl (CH2)2Ne], 4.29
[t, J ¼ 6.6 Hz, 2H, NCH2CH2Nemorpholinyl], 5.30 (s, 1H, H-3), 7.23e
7.40 (m, 3H, H-7,8,9), 8.55 (m, Jo ¼ 7.8 Hz, 1H, H-6). 13C NMR
(75.5 MHz, CDCl3, ppm):
d 39.31, 45.82, 45.87, 53.75, 55.86, 66.82,
78.71, 108.21, 116.24, 122.36, 124.75, 126.21, 130.79, 148.59, 161.26,
162.52. Anal. C20H26N6O2 (C, H, N).
1538. 1H NMR (300 MHz, CDCl3, ppm):
d
1.22 [t, J ¼ 7 Hz, 6H,
6.1.5. N,N-Diethyl-N0-2-(1-ethylbenzimidazolyl)propanediamide
(12)
N(CH2CH3)2], 1.42 ( t, J ¼ 7 Hz, 3H, NCH2CH3), 3.52 [q, J ¼ 7 Hz, 4H,
N(CH2CH3)2], 4.20 (q, J ¼ 7 Hz, 2H, NCH2CH3), 5.19 (s,1H, H-3), 7.18e
7.36 (m, 3H, H-7,8,9), 8.54 (m, Jo ¼ 7.8 Hz, 1H, H-6). 13C NMR
PCl5 (1.46 g, 7.0 mmol) was slowly added to an ice-cooled so-
lution of N,N-diethylmalonamic acid 11 [7] (1.05 g, 6.6 mmol) and
1-ethyl-1H-benzimidazol-2-amine (8a) (0.97 g, 6.0 mmol). The
mixture was stirred at room temperature for 3 h, then heated at
reflux for 1 h and finally poured into cold water. After alkalinization
with Na2CO3, the mixture was exhaustively extracted with CH2Cl2.
The combined extracts, dried over Na2SO4, afforded an oily residue
which was chromatographed on a silica gel column eluting with
acetone/EtOAc (1:1). The eluate collected, after removal of solvent,
gave a thick oil from which pure compound 12 separated out as
a whitish solid, after treatment with a small amount of Et2O/dii-
sopropyl ether (1:1) (0.56 g, 31%); white crystals, m.p. 94e95 ꢀC
(Et2O/diisopropyl ether). IR (KBr, cmꢂ1): 3268 (NH), 1628 s (CO),
(75.5 MHz, CDCl3, ppm):
d 13.06, 13.24, 36.79, 43.05, 77.26, 107.81,
116.08, 121.97, 124.42, 126.34, 130.40, 148.18, 161.18, 161.28. MS (ESI)
m/z: monoisotopic expected MW ¼ 284.16, experimental full scan
m/z [M þ H]þ ¼ 285.25. Fragment ions MS2 [M þ H]þ 285.25: m/z
257.17 (86%) ([M þ H]þ e CH2]CH2), 214.08 (18%), 188.08 (100%),
162.08 (56%). Anal. C16H20N4O (C, H, N).
6.2. Biology
6.2.1. Platelet aggregation
Human blood from healthy volunteers was added to a 130 mM
trisodium citrate aqueous solution (volume ratio 9:1), then cen-
trifuged at 100 g for 30 min to give platelet-rich plasma (PRP).
1599 s (CO), 1560. 1H NMR (200 MHz, CDCl3, ppm):
d 1.19 and 1.26
[2t, J ¼ 7 Hz, 3H þ 3H, CON(CH2CH3)2], 1.39 (t, J ¼ 7 Hz, 3H,
NCH2CH3), 3.44 and 3.46 [2q, J ¼ 7 Hz, 2H þ 2H, CON(CH2CH3)2],
3.64 (s, 2H, COCH2CO), 4.20 (q, J ¼ 7 Hz, 2H, NCH2CH3), 7.18e7.42
(m, 4H, H-4,5,6,7), 9.20e10.70 (broad signal, 1H, NH; disappeared
with D2O). MS (ESI) m/z: monoisotopic expected MW ¼ 302.17,
experimental full scan m/z [M þ H]þ ¼ 303.17. Fragment ions MS2
[M þ H]þ 303.17: m/z 230.17 (51%) ([M þ H]þ e HNEt2), 188.17 (26%)
([M þ H]þ e CH3CONEt2), 162.17 (100%) ([M þ H]þ e O]C]CHe
CONEt2). Anal. C16H22N4O2 (C, H, N).
PRP (500
(dimethylsulfoxide, 5
platelet aggregation agent. PRP aggregation was induced by ADP
(Sigma) (final concentration 5.0 M), collagen from bovine tendon
(Mascia Brunelli) at the final concentration of 5.0 g/mL, or A23187
(Sigma) (final concentration 20.0 M). Before each experiment the
m
L) was preincubated at 37 ꢀC for 2 min with solvent
mL) or drug solution before the addition of the
m
m
m
stock solutions of ADP, collagen, and A23187 were diluted in saline.
Platelet aggregation, performed in an Aggrecoder PA-3210
aggregometer (A. Menarini, Florence, Italy), was measured follow-
ing the Born’s turbidimetric method [33] and quantified by the light
transmission reached after 3 min.
6.1.6. Cyclization of compound 12 to 10-ethyl-4-(diethylamino)
pyrimido[1,2-a]benzimidazol-2(10H)-one 13
POCl3 (0.18 mL, 2.0 mmol) was added to an ice-cooled solution
of compound 12 (0.45 g, 1.50 mmol) in 10 mL of 1,2-dichloroethane.
The mixture was stirred at room temperature for 45 min, then
refluxed for 6 h. The mixture was then treated with an aqueous
solution (5 g/20 mL) of CH3COONa$3H2O, refluxing for further
45 min. The mixture was then exhaustively extracted with CH2Cl2.
The combined extracts, dried over Na2SO4, afforded an oily residue
which was chromatographed on a silica gel column eluting with
CH2Cl2/EtOAc (1:1) to remove some impurities, then with the
mixture acetoneeEtOAceMeOH (9:9:2). The eluate collected, after
removal of solvent, gave a thick oil from which, after treatment
with a small amount of EtOAc, pure compound 13 separated out as
a crystalline solid (0.24 g, 56%); white crystals, m.p. 186e187 ꢀC
(EtOAc). IR (KBr, cmꢂ1): 1651 and 1620 s (CO), 1604, 1543. 1H
6.2.2. Calculation of inhibition
In order to calculate the percentage of inhibition, the extent of
aggregation measured in the presence of the compounds tested
was always compared with that measured for a control sample
containing the solvent, in an experiment carried out under the
same conditions. From each series of experiments, in which the
inhibitors were tested in at least five concentrations, a percentage
inhibitioneconcentration curve was derived. From this curve the
IC50 value was calculated as the concentration of inhibitor causing
a 50% inhibition of the aggregation. The IC50 values reported in
Table 1 are averages (ꢁstandard deviation) of those obtained from
at least five different batches of platelets (usually 5e8 batches).
6.3. Molecular modelling
NMR (300 MHz, CDCl3, ppm):
d
1.16 [t, J ¼ 7 Hz, 6H, N(CH2CH3)2],
1.44 (t, J ¼ 7 Hz, 3H, NCH2CH3), 3.18 and 3.25 [2q, J ¼ 7 Hz, 2H þ 2H,
N(CH2CH3)2], 4.31 (q, J ¼ 7 Hz, 2H, NCH2CH3), 5.87 (s, 1H, H-3),
7.19e7.44 (m, 3H, H-7,8,9), 7.92 (m, Jo ¼ 7.8 Hz, 1H, H-6). 13C NMR
Compounds 2a, 2c, 6i and 6o were built, parameterized
(GasteigereHuckel method) and energy minimized within MOE
using MMFF94 forcefield (MOE: Chemical Computing Group Inc.
Montreal. H3A 2R7 Canada. http://www.chemcomp.com).
(75.5 MHz, CDCl3, ppm):
d 10.70, 13.17, 37.03, 44.90, 100.49, 109.13,

578
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