Microwave assisted synthesis of dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4- ones; Synthesis, in vitro antimicrobial and anticancer activities of novel coumarin substituted dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones

Article abstract of DOI:10.1016/j.ejmech.2013.07.015

The present article describes the synthesis of dihydrobenzo[4,5]imidazo[1, 2-a]pyrimidin-4-one (2a-h) under microwave irradiation. The product was obtained in excellent yield (74-94%) in a shorter reaction time (2 min). These molecules (2a, b) further reacted with various substituted 4-bromomethylcoumarins (3a-f) to yield a new series of coumarin substituted dihydrobenzo[4,5]imidazo[1,2-a] pyrimidin-4-ones (4a-h). The structure of all the synthesized compounds were confirmed by spectral studies and screened for their in vitro antibacterial activity against three Gram-positive bacteria viz., Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and three Gram-negative bacteria viz., Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and antifungal activity against Candida albicans, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Fusarium oxysporum, Penicilliumchrysogenum and anticancer activity against Dalton's Ascitic Lymphoma (DAL) cell line. In general, all the compounds possessed better antifungal properties than antibacterial properties. The coumarin substituted dihydrobenzo[4,5]imidazo[1,2- a]pyrimidin-4-one (4g) (R = i-Pr, R₁ = 6-Cl) was found to be the most potent cytotoxic compound (88%) against Dalton's Ascitic Lymphoma cell line at the concentration of 100 μg/mL.

Full text of DOI:10.1016/j.ejmech.2013.07.015

European Journal of Medicinal Chemistry 69 (2013) 316e322
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave assisted synthesis of dihydrobenzo[4,5]imidazo[1,2-a]
pyrimidin-4-ones; synthesis, in vitro antimicrobial and anticancer
activities of novel coumarin substituted dihydrobenzo[4,5]imidazo
[1,2-a]pyrimidin-4-ones
,
b
b
Kallimeledoddi B. Puttaraju a, Kalegowda Shivashankar a , Chandra , M. Mahendra ,
Vijaykumar P. Rasal c, Ponnuru N. Venkata Vivek c, Khushboo Rai c,
Maibam Beebina Chanu c
*
a P.G. Department of Chemistry, Central College Campus, Bangalore University, Bangalore 560 001, Karnataka, India
b Department of Studies in Physics, Manasagangothri, University of Mysore, Mysore 570 006, Karnataka, India
c Department of Pharmacology, KLE University College of Pharmacy, Belgaum 590 010, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The present article describes the synthesis of dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-one (2aeh)
under microwave irradiation. The product was obtained in excellent yield (74e94%) in a shorter reaction
time (2 min). These molecules (2a, b) further reacted with various substituted 4-bromomethylcoumarins
(3aef) to yield a new series of coumarin substituted dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones
(4aeh). The structure of all the synthesized compounds were confirmed by spectral studies and screened
for their in vitro antibacterial activity against three Gram-positive bacteria viz., Staphylococcus aureus,
Enterococcus faecalis, Streptococcus mutans and three Gram-negative bacteria viz., Escherichia coli, Kleb-
siella pneumonia, Pseudomonas aeruginosa and antifungal activity against Candida albicans, Aspergillus
niger, Aspergillus fumigatus, Aspergillus flavus, Fusarium oxysporum, Penicillium chrysogenum and anti-
cancer activity against Dalton’s Ascitic Lymphoma (DAL) cell line.
Received 19 February 2013
Received in revised form
17 July 2013
Accepted 22 July 2013
Available online 15 August 2013
Keywords:
4-Bromomethylcoumarins
Coumarins
Benzimidazole
Pyrimidine
In general, all the compounds possessed better antifungal properties than antibacterial properties. The
coumarin substituted dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-one (4g) (R ¼ i-Pr, R1 ¼ 6-Cl) was
found to be the most potent cytotoxic compound (88%) against Dalton’s Ascitic Lymphoma cell line at the
concentration of 100 mg/mL.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Melanoma cell lines. QSAR analyses of 2-aminobenzimidazole de-
rivatives [5] were studied on the relation between acute toxicity
and the octanol/water partition coefficient.
Dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-one is a class of
fused tricyclic system having three nitrogen atoms. The design
concept of dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones has
arised from the broad spectrum and the wide range of biological
activities of the benzimidazole and pyrimidine.
Benzimidazole derivatives [1] exhibited high cytotoxicity
against HepG-2 cells and good EGFR inhibitory activity. 2-
Substituted-5-amino-benzimidazoles [2] possessed significant
cytotoxicity against breast cancer cell line MCF-7. 1,2,5-
Trisubstituted benzimidazoles [3] and benzimidazolo pyrimidine
conjugates [4] were found to be antitumor agents against
6-Butylfuro[2,3-d]pyrimidine derivatives [6] showed the high-
est cytostatic activity against Malignant leukemia and T-lympho-
cyte cells. 5-Benzylidine barbiturate derivatives [7] inhibited the
growth of mushroom tyrosinase and Gram-positive bacteria
Staphylococcus aureus. Pyrimidine bases [8] exerted pronounced
antiproliferative activity against the HeLa and MiaPaCa-2 cell lines.
1-Adamantyl thiopyrimidines [9] displayed the significant cyto-
toxic activity particularly against H69AR cell line. Pyrimidine ana-
logs of indane-1,3-diones [10] showed significant reduction
in ulcerogenic activity when compared to standard drug
Indomethacin.
The fusion of benzimidazole and pyrimidine pharmacophores in
a single molecular frame work and the study of subsequent influ-
ence on the biological activities are of current interest.
* Corresponding author. Tel.: þ91 80 22961249.
E-mail address: shivashankark@gmail.com (K. Shivashankar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.015

K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
317
Table 1
The known synthetic method of dihydrobenzo[4,5]imidazo[1,2-
Synthesis of reported compounds (2deh) under microwave irradiation.
Compounds Thermal (reported) Microwave
Temperature Yield Time Temperature Yield
a]pyrimidin-4-one derivatives [11] had many demerits such as long
reaction time, drastic condition, tedious experimental procedure
and low yield. Hence, there is a need for a simple and straight
forward method for the synthesis of dihydrobenzo[4,5]imidazo
[1,2-a]pyrimidin-4-one derivatives. On the otherhand, to date,
neither the synthesis nor the biological evaluation of dihydrobenzo
[4,5]imidazo[1,2-a]pyrimidin-4-ones has been reported in the
literature. For all these reasons, we have done laboratory work on
the synthesis of fused dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-
4-ones.
Time
(%)
(%)
2d
2e
2f
2g
2h
60 min 140 ꢀC
60 min 140 ꢀC
60 min 140 ꢀC
60 min 140 ꢀC
60 min 140 ꢀC
55
75
87
80
87
3 min 130 ꢀC
3 min 130 ꢀC
3 min 130 ꢀC
3 min 130 ꢀC
3 min 130 ꢀC
74
87
91
90
94
Synthesis of reported compounds (2deh) under microwave irradiation.
Coumarins are known to be biologically versatile compounds
possessing several biological properties. Coumarin Mannich bases
[12] inhibited carraggeenin-induced hind paw edema and found to
possess protective properties against adjuvant-induced arthritis in
rats. 4-Amino-3-(2-methylbenzyl)coumarin derivatives [13]
exhibited potent estrogenic activity on the estrogen receptor pos-
itive (ERþ) human MCF-7 breast cancer cell line. 4-Hydroxy
coumarin derivatives [14] showed pronounced prolongation of
prothrombin time with anticoagulant values similar to that of
warfarin. Benzothiazolyl coumarin acetamide derivatives [15]
exhibited strong in vitro anti-HIV effect against the wild-type
HIV-1 cell line. The in vitro antioxidant activities of 4-schiff bases-
7-benzyloxy coumarin derivatives [16] revealed that DPPH and
ABTSþ radical scavenging activities were better than that of the
commercial antioxidant BHT.
coumarins (3aef) (R1 ¼ a; 6-OMe, b; 6-F, c; 6-CH3, d; 6,8-dimethyl,
e; 6-Cl, f; 6-Br) with dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-
ones (2a, b) (R ¼ a; i-Pr, b; 4-CF3C6H4) in the presence of anhydrous
K2CO3 in dry acetone at room temperature for 24 h. Removal of
solvents under reduced pressure afforded the title compounds
(4aeh) as solids which were purified by routine methods. The
numbering of the skeleton is shown in Fig. 1.
2.1. Result and discussion
In the IR spectrum of the compound 2-isopropyl-10H-benzo
[4,5]imidazo[1,2-a]pyrimidin-4-one (2a) (R ¼ i-Pr), the carbonyl
stretching frequency was observed at 1664 cmꢁ1, where as the NeH
stretching frequency showed
3230 cmꢁ1. The 1H NMR spectrum of compound (2a) exhibited a
singlet in the region 5.87 due to presence of C3eH proton. A
multiplet was observed at 2.79 due to methine proton of isopropyl
group. The methyl protons of isopropyl group and C7eH proton
were found to be a doublet at
1.22 (J ¼ 9 Hz) and 8.38 (J ¼ 9 Hz)
respectively. A triplet was appeared at
7.27 (J ¼ 6 Hz) due to the
presence of C8eH proton. The multiplet was observed in the region
between 7.42e7.50 due to the presence of C9eH and C10eH pro-
tons. The NeH proton was resonated as a singlet at 12.88 which
a strong absorption band at
Based on the survey of recent literature studies on benzimid-
azoles, pyrimidines and coumarins and in our effort to discover
novel antimicrobial and anticancer agents, the aim of our work is
synthesis of coumarin substituted dihydrobenzo[4,5]imidazo[1,2-
a]pyrimidin-4-ones and to evaluate them for their therapeutic
importance.
d
d
d
d
2. Chemistry
d
d
The synthesis of compounds (2aeh) (R: a; i-Pr, b; 4-CF3C6H4, c;
3-FC6H4, d; CF3, e; C6H5, f; 4-FC6H4, g; 3-ClC6H4, h; 4-CH3OC6H4)
was accomplished by synthetic sequence shown in Scheme 1. The
preparation of dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-ones
was further confirmed by its D2O exchange. The mass spectrum
(ESI-MS) of the compound (2a) showed a [M þ 1] peak at 228. The
13C NMR spectral data of all the compounds is given
in Experimental section.
was carried out by the condensation of
b
-ketoesters (1aeh) with
The IR spectrum of the compound 10-(6-methoxy-2-oxo-2H-
chromen-4-ylmethyl)-2-trifluoromethyl-10H-benzo[4,5]imidazo
[1,2-a]pyrimidin-4-one (4a) (R ¼ CF3 & R1 ¼ 6-OCH3) displayed the
benzimidazopyrimidine carbonyl stretching frequency at
1685 cmꢁ1, where as the lactone carbonyl stretching frequency
appeared at 1712 cmꢁ1. The 1H NMR spectrum of the compound
2-aminobenzimidazole under microwave irradiation. The best
conditions to obtain these compounds were achieved at 130 ꢀC
using DMF as a reaction media. This method gave the higher yield
(74e94%) and required a shorter reaction time (3 min). The com-
pounds (2deh) have already been reported by the thermal method
(Table 1) in the literature [11].
(4a) showed a singlet at
d 3.50, 5.82, 6.02 and 6.26 due to 6-OCH3,
4-Bromomethyl coumarins [17] (3aef) were synthesized by
Pechmann cyclization of phenols with 4-bromoethylacetoacetate
[18] using conc. H2SO4 as a cyclizing agent. The synthesis of
coumarin substituted dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-
4-ones (Scheme 2) were carried out by reaction of 4-bromomethyl
NeCH2, C3eH of coumarin and C3eH of benzimidazopyrimidine
protons respectively. The aromatic protons resonated as a multiplet
in the range of
d 7.45e8.32. The mass spectrum (ESI-MS) of the
compound (4a) displayed a [M þ 1] peak at 442. The 13C NMR
spectral data of all the compounds is given in Experimental section.
O
O
O
N
DMF
N
NH2
R
R
O
N
MW, 3 min
N
H
N
H
1a-h
2a-h
R; a; i-Pr, b; 4-CF3C6H4, c; 3-FC6H4, d; CF3, e; C6H5, f; 4-FC6H4, g; 3-ClC6H4, h; 4-OCH3C6H4
Scheme 1.

318
K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
O
N
Br
O
N
R
N
Anhydrous K2CO3
N
R1
N
R1
Dry Acetone
RT, 24 hr, Stirr
R
O
O
N
H
O
O
a-h
4
2a-b
3a-f
Compounds
4a
4b
4c
CF3
4d
4e
4f
4g
4h
R
CF3
CF3
CF3
i-Pr
i-Pr
i-Pr i-Pr
R1
6-OMe 6-F 6-CH3 6,8-dimethyl 6-F 6-OMe 6-Cl 6-Br
Scheme 2.
The molecular structure of the compound (4b) is also established
by single crystal analysis [19] as shown in Fig. 2.
to be highly active against A. fumigatus and A. flavus with MIC of
0.2
m
g/mL. The compounds (2b) (R ¼ 4-CF3C6H4), (2d) (R ¼ CF3),
(2e) (R ¼ C6H5), (2g) (R ¼ 3-ClC6H4), (2h) (R ¼ 4-CH3OC6H4) and
(4aeh) were found to be highly active against F. oxysporum with
2.2. Antimicrobial activity
MIC of 0.2
m
g/mL. The compounds (2a) (R ¼ i-Pr), (2b) (R ¼ 4-
CF3C6H4), (2c) (R ¼ 3-FC6H4), (2e) (R ¼ C6H5), (4b) (R ¼ CF3,
R1 ¼ 6-F), (4c) (R ¼ CF3, R1 ¼ 6-CH3), (4d) (R ¼ CF3, R1 ¼ 6,8-
dimethyl) and (4h) (R ¼ i-Pr, R1 ¼ 6-Br) were found to be highly
All the newly synthesized compounds (2aeh) and (4aeh) were
screened for their antibacterial and antifungal activity at different
concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.6, 0.8, 0.4 and
active against P. chrysogenum with MIC of 0.2 mg/mL (Fig. 4). It is
0.2
mg/mL by the broth micro dilution method. The minimum
interesting to note that all the tested compounds were found to be
most potent against A. fumigatus, F. oxysporum and P. chrysogenum
when compared to standard drug Fluconazole.
inhibitory concentrations (MIC) were determined by serial dilution
method [20].
Antibacterial activity was carried out against three Gram-
positive bacteria viz., Staphylococcus. aureus, Enterococcus faecalis,
Streptococcus mutans and three Gram-negative bacteria viz.,
Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa.
Ciprofloxacin was used as a standard. Antifungal activity was carried
out against six fungi viz., Candida albicans, Aspergillus niger, Asper-
gillus fumigatus, Aspergillus flavus, Fusarium oxysporum and Peni-
cillium chrysogenum. Fluconazole was used as a standard.
The investigation of antibacterial data (Table 2) showed that
most of the tested compounds exhibited good bacterial inhibition.
The compounds (2aeh) were found to be highly active against
2.3. In vitro cell cytotoxicity
The newly synthesized compounds (2aeh) and (4aeh) were
determined in vitro cell cytotoxicity by using trypan blue dye
exclusion assay method [21]. In this test, only the dead cells took up
the dye due to lack of intact membranes. Dalton’s Ascitic Lym-
phoma (DAL) cells (0.2 mL, 106 cells/mL), ice cold phosphate buffer
saline (1 mL, pH ¼ 7.4) and one of the compounds (2aeh) and (4ae
h) (0.2 mL) were taken in an Eppendorf tube. They were incubated
in CO2 incubator at 37 ꢀC with continuous flow of 5% CO2 for 3 h.
E. faecalis with MIC of 0.2
R1 ¼ 6,8-dimethyl) was found to be highly active against S. mutans
with MIC of 0.2
g/mL. The compound (4e) (R ¼ i-Pr, R1 ¼ 6-F) was
found to be most active against S. mutans with MIC of 0.8 g/mL
mg/mL. The compound (4d) (R ¼ CF3,
m
m
(Fig. 3). It is interesting to note that all the tested compounds were
found to be most potent against E. faecalis when compared to
standard drug Ciprofloxacin. The rest of the compounds were found
to be inactive.
The investigation of antifungal data (Table 3) showed that most
of the tested compounds exhibited good fungal inhibition. The
compounds (2f) (R ¼ 4-FC6H4) and (2g) (R ¼ 4-ClC6H4) were found
O
3
4
15
16
17
7
5
N
14
6
8
9
CF3
2
N
12
N
H
19 18
13
1
11
10
Fig. 2. ORTEP diagram with the displacement at 50% probability level (4b).
Fig. 1. Numbering of the compound (2b).

K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
319
Table 2
Results of antibacterial activities of compounds 2aeh and 4aeh MICs (
mg/mL).
Compounds
Gram-positive
Gram-negative
S. aureus
E. faecalis
S. mutans
E. coli
K. penumoniae
P. aeruginosa
12.5
>100
25
100
100
100
100
100
>100
100
>100
100
100
>100
100
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
4e
4f
4g
4h
100
>100
100
100
100
100
100
100
100
100
100
100
100
100
100
>100
2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.4
0.8
0.4
0.4
0.4
0.4
0.4
0.8
2
25
>100
12.5
50
100
12.5
25
100
12.5
12.5
12.5
0.2
12.5
>100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1
100
>100
100
100
100
100
100
100
100
100
100
100
100
100
100
>100
1
0.8
3.16
12.5
>100
2
>100
2
Ciprofloxacin
Then, previous mixture (0.2 mL), ice cold phosphate buffer saline
(0.3 mL, pH ¼ 7.4) and trypan blue solution (0.5 mL, 0.4% in normal
saline) were taken in an Eppendorf tube and kept for 5e15 min at
room temperature. The percentage of dead cells was calculated
with the following formula using Neubauer chamber.
spectra (KBr disc) were recorded on a Shimadzu-8400S FT-IR
Spectrophotometer. 1H NMR spectra were recorded on Bruker
300 MHz spectrometer. 13C NMR, HeH Cosy, HSQC and 19F NMR
spectra were recorded on Bruker 400 MHz spectrometer by using
DMSO-d6 as a solvent and TMS as an internal standard. The
chemical shifts are expressed in
d ppm. The mass spectra were
Number of dead cells
Sum of dead cells and living cells
recorded using Agilent-Single Quartz ESI-MS and Agilent-Single
Quartz LC-MS. The purity of the compounds was checked by TLC.
Milestone laboratory’s microwave reactor was used to carry out the
microwave reactions. The elemental analyses were carried out us-
ing Elemental Vario Micro Cube CHN Rapid Analyzer. All the com-
pounds gave satisfactory elemental analysis.
% Dead cell ¼
ꢂ 100
The investigation of in vitro cell cytotoxicity (Table 4) revealed
that most of the tested compounds exhibited good activity. The
compounds (2b) (R ¼ 4-CF3C6H4), (2d) (R ¼ CF3), (2fb) (R ¼ 4-
FC6H4), (2g) (R ¼ 4-ClC6H4), (4b) (R ¼ CF3, R1 ¼ 6-F), (4d)
(R ¼ CF3, R1 ¼ 6,8-dimethyl), (4e) (R ¼ i-Pr, R1 ¼ 6-F) and (4g) (R ¼ i-
Pr, R1 ¼ 6-Cl) were found to be highly active (>70%) against DAL cell
3.1. General procedure for the preparation of compounds
at the concentration of 100
found to be moderately active (>40%) against DAL cell at the con-
centration of 100 g/mL (Fig. 5).
mg/mL. The rest of the compounds were
3.1.1. Synthesis of 10H-benzo[4,5]imidazo[1,2-a]pyrimidin-4-ones
(2aeh)
m
An equimolar mixture of 2-aminobenzimadzole (0.5 g,
3. Experimental section
3.75 mmol) and b-ketoesters (1aeh) (3.75 mmol) in DMF (10 mL)
was added to a microwave tube equipped with a magnetic stir bar.
The microwave tube was fitted with a reflux condenser and irra-
diated in a microwave reactor at a temperature of 130 ꢀC for 3 min
The melting points were determined by open capillary method
using electric melting point apparatus and are uncorrected. The IR
0.125
0.25
0.5
1
S.aureus
E.faecalis
S.mutans
E.coli
2
4
K.penumoniae
P.aeruginosa
8
16
32
64
128
Tested compounds
Fig. 3. Antibacterial activity of compounds (2aeh) and (4aeh).

320
K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
Table 3
Results of antifungal activities of compounds (2aeh) and (4aeh) MICs (
mg/mL).
Compounds
C. albicans
A. niger
A. fumigatus
A. flavus
F. oxysporum
P. chrysogenum
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
4e
4f
4g
4h
3.12
6.25
1.6
1.6
1.6
1.6
1.6
6.25
50
50
50
50
25
25
100
12.5
6.25
6.25
1.6
25
25
25
25
12.5
3.12
1.6
50
12.5
25
1.6
1.6
0.8
0.8
0.8
0.2
0.2
1.6
6.25
1.6
1.6
1.6
0.8
0.8
0.8
0.2
0.2
0.8
3.12
1.6
12.5
3.12
1.6
0.8
1.6
0.8
8
0.4
0.2
0.4
0.2
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
8
0.2
0.2
0.2
0.4
0.2
0.4
0.4
0.4
0.4
0.2
0.2
0.2
0.4
0.8
0.4
0.2
8
25
1.6
0.8
0.8
0.8
0.8
8
25
50
50
16
Fluconazole
8
at a maximum power of 320 W. Then, the reaction mixture was
poured on to crushed ice. The solid was filtered and washed with
100 mL of cold water. The crude product was dried and recrystal-
lized from 1:3 ethyl acetate and chloroform.
DMSO-d6): 98.18, 111.02, 115.69, 117.46, 120.56, 121.93, 125.45,
2
126.30, 126.92, 127.71, 129.12, 129.89 (q, JCF ¼ 32 Hz), 140.99,
148.14, 149.54, 158.93, 166.44 ppm; ESI-MS: m/z [M þ 1] 330; Anal.
C17H10 F3N3O. Calcd for: C, 62.01; H, 3.06; N, 12.76. Found: C, 61.92;
H, 2.91; N, 12.60.
3.1.1.1. 2-Isopropyl-10H-benzo[4,5]imidazo[1,2-a]pyrimidin-4-one
(2a). Colorless solid, Yield: 93%. Mp: 189e191 ꢀC, IR (KBr, cmꢁ1):
3230 cmꢁ1 (NeH), 1664 cmꢁ1 (C]O); 1H NMR (300 MHz, DMSO-
3.1.1.3. 2-(3-Fluoro-phenyl)-10H-benzo[4,5]imidazo[1,2-a]pyr-
imidin-4-one (2c). Colorless solid, Yield: 94%. Mp: 213e215 ꢀC, IR
(KBr, cmꢁ1); 3237 cmꢁ1 (NeH), 1681 cmꢁ1 (C]O); 1H NMR
d6):
d
1.22 (d, 6H, 2-CH3 of i-Pr, J ¼ 6.9 Hz), 2.79 (m, 1H, CH of i-Pr),
5.87 (s, 1H, C3eH), 7.27 (t, 1H, C8eH, J ¼ 7.8 Hz), 7.42e7.50 (m, 2H,
(300 MHz, DMSO-d6) d 6.65 (s, 1H, C3eH), 7.31e7.38 (m, 3H, AreH),
C9eH & C10eH), 8.38 (d, 1H, C7eH, J ¼ 8.1 Hz), 12.88 (s, 1H, NeH,
7.49 (d, 2H, J ¼ 3.9 Hz), 8.17e8.21 (m, 2H, AreH), 8.46 (d, 1H,
D2O exchangeable) ppm; 13C NMR (100 MHz, DMSO-d6):
d
21.65,
J ¼ 8.1 Hz, AreH), 13.11 (s, 1H, NeH) ppm; 13C NMR (100 MHz,
2
34.87, 98.42, 113.59, 115.08, 121.28, 125.92, 126.81, 135.16, 148.25,
158.95, 159.22 ppm; ESI-MS: m/z [M þ 1] 228; Anal. C13H13N3O.
Calcd for: C, 68.70; H, 5.77; N, 18.49. Found: C, 68.60; H, 5.71; N,
18.37.
DMSO-d6):
d
97.62, 110.97, 113.42, (d, JCF ¼ 23 Hz), 115.67, 116.81,
121.89, 123.0, 125.69, 126.25, 130.47, 130.58, 139.56, 139.64, 149.46,
159.06, 159.71, 161.20, 163.61 ppm; ESI-MS: m/z [M þ 1] 280; Anal.
C16H10FN3O. Calcd for: C, 68.81; H, 3.61; N, 15.05. Found: C, 68.70;
H, 3.55; N, 14.97.
3.1.1.2. 2-(4-Trifluoromethyl-phenyl)-10H-benzo[4,5]imidazo[1,2-a]
pyrimidin-4-one (2b). Colorless solid, Yield: 87%. Mp: 233e235 ꢀC,
IR (KBr, cmꢁ1): 3236 cmꢁ1 (NeH), 1687 cmꢁ1 (C]O); 1H NMR
3.1.2. Synthesis of 10-(2-oxo-2H-chromen-4-ylmethyl-10H-benzo
[4,5]imidazo[1,2-a])pyrimidin-4-ones (4aeh)
(300 MHz, DMSO-d6):
d
6.82 (s, 1H, C3eH), 7.34e7.39 (m, 1H, C10e
A mixture of 10H-benzo[4,5]imidazo[1,2-a]pyrimidin-4-ones
(2a, b) (2.20 mmol) and anhydrous K2CO3 (0.6 g, 4.4 mmol) was
stirred in 30 mL of dry acetone for 20 min. 4-
Bromomethylcoumarins (3aef) (2.20 mmol) was added and
H), 7.51e7.53 (d, 2H, C8eH & C9eH, J ¼ 3.9 Hz), 8.05 (d, 2H, C15eH &
C
19eH, J ¼ 6.0 Hz), 8.48 (d, 1H, C7eH, J ¼ 8.1 Hz), 8.73 (d, 2H, C16eH
& C18eH, J ¼ 3.6 Hz), 13.14 (s, 1H, NeH) ppm; 13C NMR (100 MHz,
0.125
0.25
0.5
1
2
C.Albican
A.niger
4
8
A.fumigatus
A.flaves
16
32
64
128
F.oxysporum
P.chrysogenum
Tested compounds
Fig. 4. Antifungal activity of compounds (2aeh) and (4aeh).

K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
321
1673 cmꢁ1 (C]O), 1738 cmꢁ1 (lactone C]O); 1H NMR (300 MHz,
DMSO-d6): 5.86 (s, 2H, NeCH2), 6.28 (s, 1H, C3eH of coumarin),
6.64 (s, 1H, C3eH of benzimidazopyrimidine), 7.47e8.58 (m, 7H,
AreH); 13C NMR (100 MHz, DMSO-d6):
42.44, 100.88, 110.54,
Table 4
In vitro cytotoxicity of compounds (2aeh) and (4aeh).
d
Type of cancer
Concentration of
compounds
(mg/ml)
Number of
compounds
Number of
cells
% of dead
cells
cell (1 ꢂ 105)
d
111.44, 113.12, 116.01, 118.16, 118.46, 118.55, 119.49, 119.74, 123.38,
Live
Dead
2
125.39, 126.83, 130.71, 148.15, 149.44, 150.03 (q, JCF ¼ 34 Hz),
Dalton’s Ascitic
Lymphoma
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
4e
4f
43
21
46
20
42
17
29
36
42
21
31
22
19
38
12
53
12
57
79
54
80
58
83
71
64
58
79
69
78
81
62
88
47
88
57
79
54
80
58
83
71
64
58
79
69
78
81
62
88
47
88
156.91, 158.96, 159.23, 159.30 ppm; ESI-MS: m/z [M þ 1] 430; Anal.
C21H11F4N3O3. Calcd for: C, 58.75; H, 2.58; N, 9.79. Found: C, 58.63;
H, 2.47; N, 9.56.
3.1.2.3. 10-(6-Methyl-2-oxo-2H-chromen-4-ylmethyl)-2-trifluoro-
methyl-10H-benzo[4,5]Imidazo
[1,2-a]pyrimidin-4-one
(4c).
Colorless solid, Yield: 92%. Mp: 211e213 ꢀC, IR (KBr, cmꢁ1):
1665 cmꢁ1 (C]O), 1734 cmꢁ1 (lactone C]O); 1H NMR (300 MHz,
DMSO-d6):
of coumarin), 6.64 (s, 1H, C3eH of benzimidazopyrimidine), 7.37e
8.58 (m, 7H, AreH) ppm; 13C NMR (100 MHz, DMSO-d6):
20.31,
d 2.45 (s, 3H, CH3), 5.86 (s, 2H, NeCH2), 6.15 (s, 1H, C3eH
d
4g
4h
e
42.63, 100.81, 110.23, 111.70, 115.73, 116.63, 119.74, 121.15, 122.32,
123.34, 125.17, 125.32, 125.56, 127.16, 130.81, 133.18, 134.38, 148.81,
150.04 (q, 2JCF ¼ 34 Hz),158.93 ppm; ESI-MS: m/z [M þ 1] 426; Anal.
5-Flourouracil
C23H16F3N3O3. Calcd for: C, 62.12; H, 3.32; N, 9.88. Found: C, 62.01;
H, 3.21; N, 9.74.
stirring was continued for 24 h. The reaction mixture was
concentrated to one fourth volume and poured on to crushed ice.
The solid separated was filtered and washed with 5% HCl (10 mL).
Then, it was washed with 50 mL of cold water. The crude product
was dried and recrystallized from ethanol.
3.1.2.4. 10-(6,8-Dimethyl-2-oxo-2H-chromen-4-ylmethyl)-2-
trifluoromethyl-10H-benzo[4,5] imidazo[1,2-a]pyrimidin-4-one (4d).
Colorless solid, Yield: 91%. Mp: 216e218 ꢀC, IR (KBr, cmꢁ1):
1670 cmꢁ1 (C]O), 1730 cmꢁ1 (lactone C]O); 1H NMR (300 MHz,
DMSO-d6): d 2.36 (s, 3H, 6-CH3), 2.41 (s, 3H, 8-CH3), 5.84 (s, 2H, Ne
CH2), 6.13 (s, 1H, C3eH of coumarin), 6.64 (s, 1H, C3eH of benzi-
3.1.2.1. 10-(6-Methoxy-2-oxo-2H-chromen-4-ylmethyl)-2-trifluoro-
midazopyrimidine), 7.43e8.58 (m, 6H, AreH) ppm; 13C NMR
methyl-10H-benzo[4,5]
imidazo[1,2-a]pyrimidin-4-one
(4a).
Colorless solid, Yield: 93%. Mp: 228e230 ꢀC, IR (KBr, cmꢁ1):
(100 MHz, DMSO-d6):
d 15.16, 20.30, 42.62, 100.08, 110.52, 111.69,
1685 cmꢁ1 (C]O), 1712 cmꢁ1 (lactone C]O); 1H NMR (300 MHz,
115.98, 116.62, 122.46, 122.31, 123.33, 125.16, 125.31, 126.86, 130.80,
2
133.17, 134.37, 148.79, 149.35, 149.46, 150.03 (q, JCF ¼ 35 Hz),
DMSO-d6):
H of coumarin), 6.26 (s,1H, C3eH of benzimidazopyrimidine), 7.45e
8.32 (m, 7H, AreH) ppm; 13C NMR (100 MHz, DMSO-d6)
42.64,
d 3.50 (s, 3H, OCH3), 5.82 (s, 2H, NeCH2), 6.02 (s,1H, C3e
158.92, 159.53 ppm; ESI-MS: m/z [M þ 1] 440; Anal. C23H16F3N3O3.
Calcd For: C, 62.87; H, 3.67; N, 9.56. Found: C, 62.67; H, 3.58; N,
9.40.
d
55.86, 100.84, 107.75, 100.52, 112.32, 116.02, 117.56, 117.70, 119.87,
123.36, 125.36, 126.84, 130.74, 147.37, 148.48, 149.41 (q,
2JCF ¼ 64 Hz), 150.05, 155.64, 158.96, 159.57 ppm; ESI-MS: m/z
[M þ 1] 442; Anal. C22H14F3N3O4. Calcd for: C, 59.87; H, 3.20; N,
9.52. Found: C, 59.68; H, 3.06; N, 9.30.
3.1.2.5. 10-(6-Fluoro-2-oxo-2H-chromen-4-ylmethyl)-2-isopropyl-
10H-benzo[4,5]imidazo[1,2-a] pyrimidin-4-one (4e). Colorless solid,
Yield: 88%. Mp: 230e232 ꢀC, IR (KBr, cmꢁ1): 1664 cmꢁ1 (C]O),
1736 cmꢁ1 (lactone C]O); 1H NMR (300 MHz, DMSO-d6):
d 1.20 (d,
6H, 2CH3 of i-Pr, J ¼ 6.0 Hz), 2.80 (m, 1H, CH of i-Pr), 5.82 (s, 2H, Ne
CH2), 6.03 (s, 1H, C3eH of coumarin), 6.24 (s, 1H, C3eH of benzi-
midazopyrimidine), 7.45e8.50 (m, 7H, AreH) ppm; 13C NMR
3.1.2.2. 10-(6-Fluoro-2-oxo-2H-chromen-4-ylmethyl)-2-trifluoro-
methyl-10H-benzo[4,5]imidazo
[1,2-a]pyrimidin-4-one
(4b).
Colorless solid, Yield: 90%. Mp: 240e245 ꢀC, IR (KBr, cmꢁ1):
100
s
90
l
l
e
c
80
d
a
e
70
d
f
o
60
e
g
a
50
t
n
e
40
c
r
e
P
30
20
10
0
Tested Compounds
Fig. 5. In vitro cell cytotoxicity of (2aeh) and (4aeh).

322
K.B. Puttaraju et al. / European Journal of Medicinal Chemistry 69 (2013) 316e322
(100 MHz, DMSO-d6):
112.88, 116.92, 117.76, 121.47, 122.76, 123.85, 125.91, 128.52, 131.27,
d
21.77, 33.95, 42.98, 96.88, 101.97, 110.54,
properties than antibacterial properties. The coumarin substituted
dihydrobenzo[4,5]imidazo[1,2-a]pyrimidin-4-one (4g) (R ¼ i-Pr,
R1 ¼ 6-Cl) was found to be the most potent cytotoxic compound
(88%) against Dalton’s Ascitic Lymphoma cell line at the concentra-
2
132.69, 142.60, 144.96, 145.10, 152.93 (d, JCF ¼ 66 Hz), 153.66,
161.23 ppm; LC-MS: m/z [M þ 1] 404; Anal. C23H18FN3O3. Calcd for:
C, 68.48; H, 4.50; N, 10.42. Found: C, 68.34; H, 4.40; N, 10.29.
tion of 100 mg/mL.
3.1.2.6. 10-(6-Methoxy-2-oxo-2H-chromen-4-ylmethyl)-2-isopropyl-
10H-benzo[4,5]imidazo[1,2-a] pyrimidin-4-one (4f). Colorless solid,
Yield: 94%. Mp: 210e213 ꢀC, IR (KBr, cmꢁ1): 1670 cmꢁ1 (C]O),
Acknowledgments
We are grateful to the University Grant Commission, New Delhi,
India for the financial support [F.NO.37-76/2009 (SR)]. We are
thankful to Prof. Y.S. Bhat, Bangalore Institute of Technology, Ban-
galore, for providing Microwave Reactor facility and for his
encouragement. We are also thankful to Indian Institute of Science,
Bangalore for the spectral analysis.
1740 cmꢁ1 (lactone C]O); 1H NMR (300 MHz, DMSO-d6):
d 1.20 (d,
6H, 2-CH3 of i-Pr, J ¼ 6.0 Hz), 2.80 (m, 1H, CH of i-Pr), 3.52 (s, 3H,
OCH3), 5.82 (s, 2H, NeCH2), 6.01 (s, 1H, C3eH of coumarin), 6.10 (s,
1H, C3eH of benzimidazopyrimidine), 7.25e8.52 (m, 7H, AreH)
ppm; 13C NMR (100 MHz, DMSO-d6):
d 23.90, 32.27, 42.37, 55.85,
101.49, 110.01, 112.38, 114.46, 115.06, 115.67, 116.20, 117.42, 117.73,
119.86, 122.49, 122.60, 126.01, 130.59, 147.39, 149.04, 155.65, 159.52,
163.87 ppm; ESI-MS: m/z [M þ 1] 416; Anal. C24H21N3O4. Calcd for:
C, 69.39; H, 5.10; N, 10.11. Found: C, 69.17; H, 4.86; N, 10.01.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.015.
3.1.2.7. 10-(6-Chloro-2-oxo-2H-chromen-4-ylmethyl)-2-isopropyl-
10H-benzo[4,5]imidazo[1,2-a] pyrimidin-4-one (4g). Colorless solid,
Yield: 89%. Mp: 237e239 ꢀC, IR (KBr, cmꢁ1): 1669 cmꢁ1 (C]O),
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1732 cmꢁ1 (lactone C]O): 1H NMR (300 MHz, DMSO-d6):
d
1.20 (d,
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