Synthesis and biological evaluation of some novel pyrido[1,2-a]pyrimidin-4- ones as antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.031

Novel pyrido[1,2-a]pyrimidin-4-ones have been synthesized and evaluated for their antimalarial activity by SYBR Green I assay against erythrocytic stages of chloroquine (CQ) sensitive Pf 3D7 strain. The antimalarial screening of 42 different compounds revealed that 3-Fluorobenzyl(4-oxo-4H-pyrido [1,2-a]pyrimidin-3-yl)carbamate (21, IC₅₀ value 33 μM) and 4-Oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (37, IC₅₀ value 37 μM) showed moderate antimalarial activity. Cytotoxicity study was performed against mammalian cell line (Huh-7) by using the MTT assay for the moderately active compounds. Structural activity relationship (SAR) studies displayed that B-ring unsubstituted pyrido[1,2-a]pyrimidine scaffold is responsible for the antimalarial activities of the evaluated derivatives. This SAR based antimalarial screening supported that pyrido[1,2-a]pyrimidin-4-one can be considered as a lead heterocyclic structure for further development of more potent derivatives for antimalarial activity.

Full text of DOI:10.1016/j.ejmech.2014.04.031

European Journal of Medicinal Chemistry 79 (2014) 422e435
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of some novel pyrido[1,2-a]
pyrimidin-4-ones as antimalarial agents
,
Uttam R. Mane a b, D. Mohanakrishnan c, Dinkar Sahal c, Prashant R. Murumkar a,
,
Rajani Giridhar a, Mange Ram Yadav a
*
a Pharmacy Department, Faculty of Tech. and Engg., The M. S. University of Baroda, Vadodara, 390 001 Gujarat, India
b Torrent Research Centre, Village Bhat, Dist. Gandhinagar, 382421 Gujarat, India
c Malaria Research Laboratory, ICGEB, Aruna Asaf Ali Marg, New Delhi 110067, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel pyrido[1,2-a]pyrimidin-4-ones have been synthesized and evaluated for their antimalarial activity
by SYBR Green I assay against erythrocytic stages of chloroquine (CQ) sensitive Pf 3D7 strain. The anti-
malarial screening of 42 different compounds revealed that 3-Fluorobenzyl(4-oxo-4H-pyrido [1,2-a]
Received 24 December 2013
Received in revised form
5 April 2014
Accepted 8 April 2014
Available online 13 April 2014
pyrimidin-3-yl)carbamate (21, IC50 value 33
m
M) and 4-Oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido
M) showed moderate antimalarial activity. Cyto-
[1,2-a]pyrimidine-3-carboxamide (37, IC50 value 37
m
toxicity study was performed against mammalian cell line (Huh-7) by using the MTT assay for the
moderately active compounds. Structural activity relationship (SAR) studies displayed that B-ring
unsubstituted pyrido[1,2-a]pyrimidine scaffold is responsible for the antimalarial activities of the eval-
uated derivatives. This SAR based antimalarial screening supported that pyrido[1,2-a]pyrimidin-4-one
can be considered as a lead heterocyclic structure for further development of more potent derivatives
for antimalarial activity.
Keywords:
Antimalarial
Plasmodium falcsiparum
Pyrido[1,2-a]pyrimidin-4-one
SYBR Green Assay
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
A large number of newer motifs like chalcones [5,6], (thio)sem-
icarbazones [7] and phenothiazines [8] have been reported in the
Malaria, a tropical disease caused by protozoan parasites of the
genus Plasmodium, has been of great concern to tropical countries
and is now a cause of concern to more than 40% of the world’s
population [1]. As per the recent WHO report malaria is having
devastating impact on the lives of more than 3 billion people of the
world, about half of the humanity [2]. There is an urgent and
compelling need for the discovery of new antimalarial drugs to save
lives and reduce morbidity and mortality [3]. The existing antima-
larial drugs are not adequate to combat malaria, primarily because of
resistance developed by the parasite. The drug resistance associated
parasitic mutations to antimalarial drugs in the malaria parasite is a
major contributor to the re-emergence of the disease and its spread
in new locations and populations. The problem has got further
compounded due to non-enrichment of antimalarial drug inventory
[4]. Unfortunately an effective antimalarial vaccine is also not avail-
able. WHO aims for the global roll back of malaria with intensified
efforts in providing access to affordable, safe and effective anti-
malarial treatments worldwide along with preventive measures [2].
literature to exhibit antimalarial activity. Unfortunately compounds
from none of these classes have crossed the clinical phase of eval-
uation as antimalarial agents. There are reports of some heterocyclic
[9] and polycyclic pyrimidines [10] exhibiting antimalarial activity.
Recently some of the potent antimalarial compounds containing 4-
Oxymethyl-1,2-dioxanes [14], 4-amino-quinoline-pyrimidine [12]
and quinolines trizoles [13] have been reported. Taking the lead
information from literature we have synthesized and reported [11]
some compounds of pyrido[1,2-a]pyrimidin-4-one class of hetero-
cycles (Fig. 1). In continuation of our previous work more novel
compounds belonging to the four series-(I to IV) of pyrido[1,2-a]
pyrimidin-4-ones were synthesized (Fig. 1) and evaluated against
Plasmodium falciparum for their antimalarial potential.
2. Results and discussion
2.1. Chemistry
The targeted compounds were prepared by coupling of suitable
isocyanate [14] (1) with amine derivatives of type R’NHA in toluene
under reflux conditions following the procedure as depicted in
* Corresponding author.
E-mail address: mryadav11@yahoo.co.in (M.R. Yadav).
http://dx.doi.org/10.1016/j.ejmech.2014.04.031
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
423
depicted in Scheme 1. The carbamates (21e25) were synthesized by
coupling of suitable isocyanate (1) with aryl alcohols in toluene
under reflux conditions [14] as depicted in Scheme 1. Some com-
pounds bearing ester (26, 27), amide (28, 29) and nitrile (30, 31)
groups were also synthesized following the reaction sequence as
outlined in Scheme 2.
The amides (33e49) were prepared by EDC coupling of a suit-
able acid derivative [14] (32) with suitable amine derivatives
following Scheme 3. The isocyanate derivatives (1) were synthe-
sized following the synthetic schemes reported in our earlier
publication [14].
In order to study the hydrophobic binding interactions it was
planned to prepare some compounds having aryl ring attached to
the B-ring (i.e. pyridine) of the pyridopyrimidine ring. Such com-
pounds (53e56) were synthesized by following the reaction
sequence outlined in Scheme 4. The ester (50) on Suzuki coupling
with p-methoxyphenylboronic acid yielded the ester (51) which on
acid hydrolysis gave the acid (52). The acid (52) on EDC coupling
with different amines offered compounds of type (IV) i.e. 53e56.
R1
N
O
H
O
H
R
N
A
N
N
A
A
R
N
N
N
O
O
N
N
II
I
O
O
O
H
R
R
O
N
N A
R1
N
N
O
N
N
IV
III
Where:
R = H, 8-CH3, 7-Cl, 4-C6H4OCH3
R1 = H, Alkyl
A = Alkyl/heteroaryl or alkyl
Fig. 1. Pyrido[1,2-a]pyrimidin-4-one (Series-I to IV) derivatives synthesized for po-
tential antimalarial activity.
2.2. Biological evaluation
The antimalarial activity of compounds (series IeIV) was
measured by SYBR Green I Assay against P. falciparum (3D7) and the
50% inhibitory concentrations (IC50) are given in Table 1. Different
substituents on the basic moiety of pyrido[1,2-a]pyrimidin-4-ones
have been explored to ascertain the structure activity relationship
(SAR) among the synthesized compounds. Analysis of the results
(Tables 1and 2) revealed some interesting points.
Scheme 1 to get the ureas (2e17). N-(4-Oxo-4H-pyrido[1,2-a]pyr-
imidin-3-yl)-4-(pyridin-2-yl)piperrazine-1-carboxamide
de-
rivatives (piperazine urea analogs) of the series-II (18e20) were
synthesized by coupling of the isocyanate with suitably substituted
piperazine derivatives under reflux conditions in toluene as
(2) R = R' = H,
A = 2-trifluoromethylbenzyl
A = 4-trifluoromethylbenzyl
A = 2,4-difluorobenzyl
R'
N
O
H
(3) R = R' = H,
(4) R = R' = H,
(5) R = R' = H,
(6) R = R' = H,
(7) R = R' = H,
(8) R = R' = H,
(9) R = H, R' = isopropyl,
N
A
N
R
A = 3,3-diphenylpropyl
O
A = 3-(n-pentyloxy)pyridin-2-yl
A = 3-(benzyloxy)pyridin-2-yl
A = 6-(morpholin-4-yl)pyridin-2-yl
A = benzyl
N
(2-17)
(10) R = 8-Me, R' = isopropyl, A = benzyl
(11) R = 7-Cl, R' = H,
(12) R = 7-Cl, R' = H,
(13) R = 7-Cl, R' = H,
(14) R = 7-Cl, R' = H,
(15) R = 7-Cl, R' = H,
A = (thiophen-2yl)methyl
A = 2-methoxyethyl
A = 4-trifluoromethylbenzyl
A = 3-trifluoromethybenzyl
A = 2-trifluoromethylbenzyl
R'HN-A
(16) R = 7-Cl, R' = isopropyl, A = benzyl
(17) R = 7-Cl, R' = benzyl,
A = phenethyl
A
N
O
H
O
N
HN
NA
N
N
O
N
N
R
N
R
O
N
(1)
(18-20)
(18) R = H, A = 3-(N-cyclopropylcarboxamido)pyridin-2-yl
(19) R = 7-Cl, A = tert-butyloxycarbonyl
HO-A
(20) R = 7-Cl, A = furan-2-carbonyl
O
N
H
N
O
(21) R = H,
(22) R = H,
(23) R = 7-Cl, A = pyridin-2-yl-methyl
(24) R = 7-Cl, A = 3-pyridinylmethyl
(25) R = 7-Cl, A = 3-fluorobenzyl
A = 3-fluorobenzyl
A = 3,4-methylenedioxybenzyl
A
N
R
O
(21-25)
Scheme 1. Synthesis of ureas (2e17), piperazine urea analogs (18e20) and carbamate derivatives (21e25).

424
U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
R'
R'
O
O
H
H
N
Me
O
N
O
N
H2N
N
n
O
N
O
O
N
Me
O
N
n
(26,27)
(26) n=1, R'= Ph
(27) n=0, R'=CH(CH3)2
(1)
Liq. ammonia
Cl
N
N
N
R'
N
O
N
R'
H
O
H
H
H
N
N
Cl
Cl
N
N
N
N
O
O
O
NH2
N
(30,31)
(28,29)
(
28, 30) R'= Ph
(29, 31) R'= CH(CH3)2
Scheme 2. Synthesis of ester (26e27), amide (28, 29) and nitrile (30, 31) derivatives.
Broadly two types of substitutions have been performed in the
inertness of urea derivatives was a bit surprise and disappointment
as this group had offered active compounds as falcipain-2/3 in-
hibitors as reported in our earlier publication [14]. Substitution of
8-(4-methoxyphenyl) on the pyrido[1,2-a]pyrimidine ring proved
to be another set back as earlier we had observed that 7-Cl and 8-
Me substituted derivatives yielded active falcipain-2/3 inhibitors
among the series of urea derivatives. Another interesting feature of
the study is that all the active compounds possessed fluoro/tri-
fluoromethyl groups attached to the benzyl moiety.
In nutshell, the bare pyrido[1,2-a]pyrimidin-4-one ring moiety
substituted with a carbamate function at C-3 position having 3-
fluorobenzyl side chain offered the most active compound (21)
The B-ring (i.e. pyridine) unsubstituted moiety is preferred over a
substituted one for antimalarial activity and carbamate or carbox-
amido groupings offered the most active compounds.
basic pyrido[1,2-a]pyrimidin-4-one ring system i.e. substitution in
the pyridine ring either at position 7 or 8 and substitution at po-
sition 3 in the pyrimidine ring of the pyrido[1,2-a]pyrimidinone
ring skeleton. Three types of functional groups have been attached
at C-3 position in the pyrimidine ring i.e. 1,3-disubstituted or 1,3,3-
trisubstituted urea, N-substituted carboxamide and carbamate.
Considering an IC50 of >100 mM in the category of inactives it can be
assumed that majority of the compounds are poorly active or
inactive. But some interesting observations could be made from the
study of Table 1. Substitution of 7-chloro or 8-methyl/8-(4-
methoxyphenyl) groups made the whole moiety inactive as some
of the compounds (9, 21, 34, 36, 37 and 38) having no substituents
at C-7 or C-8 showed antimalarial activity although of moderate
potency. Amongst the three functional groups attached at C-3 po-
sition, the carboxamide offered the highest number (34, 36, 37, 59,
70) of such compounds. One of the carbamates yielded the most
potent compound (21) amongst the whole lot inspite of the fact
that lesser number of carbamates were synthesized and evaluated
in comparison to the urea and carboxamide derivatives. Biological
Compounds (21, 36, 38 and 47) showing antimalarial activity in
the range of 33e70 mM were further evaluated for their cytotoxicity
potential by MTT assay against mammalian cell lines (Huh-7).
A selectivity index was calculated by dividing the IC50 value ob-
tained in mammalian cell by the IC50 value obtained in pf 3D7.
O
O
N
O
O
N
A
N
N
R
R'-NHA
OH
N
R
R'
(33-49)
(32)
(42)R = R' = H, A = (2,5-diethoxy)-4-morpholin-4-ylphenyl
(43)R = R' = H, A = 2,5-dimethyl-1,2-oxazol-4-yl
(44)R = R' = H, A = 1-(4-methoxyphenyl)ethyl
(45)R = R' = H, A = 1-benzylpiperidin-4-yl
(
33) R = R' = H, A = 4-methoxybenzyl
(34)R = R' = H, A = 3-fluorobenzyl
(35)R = R' = H, A = 2-trifluoromethylbenzyl
(36)R = R' = H, A = 3-trifluoromethylbenzyl
(37)R = R' = H, A = 4-trifluoromethylbenzyl
(38)R = R' = H, A = 2,4-difluorobenzyl
(39)R = R' = H, A = 2-(morpholin-4-yl)ethyl
(40)R = R' = H, A = 3-(n.pentyloxy)pyridin-2-yl
(41)R = R' = H, A = 3-(benzyloxypyridin-2-yl
(46)R = H, R' = cyclopropyl,
(47)R = 8-Me, R' = H,
(48)R = 7-Cl, R' = H,
A = 2-fluorobenzyl
A = 4-trifluoromethylbenzyl
A = 4-trifluoromethylbenzyl
(49)R = 7-Cl, R' = cyclopropyl, A = 2-fluorobenzyl
Scheme 3. Synthesis of amide derivatives (33e49).

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
425
H3CO
O
N
O
O
N
O
H3CO
B(OH)2
Cl
CH3
N
O
N
O
CH3
DME, P(Ph3)4, K2CO3,
Reflux
(51)
(50)
HCl,
Reflux
H3CO
O
N
O
H3CO
O
N
O
EDC, HOBT, DIEA,
A-NH2, 0OC/ RT
N
N A
H
OH
N
(53-56)
(52)
A =
CF3
(56) =
(53) =
(55) =
(54)
F
CF3
O
Scheme 4. Synthesis of 7-(4-methoxyphenyl) substituted derivatives (51e56).
Interestingly as shown in Table 2, all these compounds showed
selectivity index in the range of 1.4e5.4. Compound (37) showed
the highest selectivity index of >5.4 amongst the five tested com-
pounds. Compound (38, 21) showed selectivity index of >3 and
>3.3 respectively. Compounds (21 and 36) also showed selectivity
index of >3, whereas compound (47) showed >1.4 selectivity to-
wards Pf 3D7.
spectrometer) were recorded in DMSO-d6/CDCl3 (chemical shifts
in ppm). The assignments of exchangeable protons were
d
confirmed by the D2O exchange studies wherever required. Mass
spectral data was obtained on a Waters Micromass ESCi spec-
trometer. Elemental analyses were performed on Thermo fisher
scientific (Flash 2000) analyzer. Synthesis of isocyanate derivatives
(1), acids (32) and ethyl 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimi-
dine-3-carboxylate (50) was carried out as per the reported
method from our earlier publication [14].
3. Conclusion
In continuation of our efforts to design and synthesize novel
antimalarial compounds, we have examined the antiplasmodial
activity of forty two pyrido[1,2-a]pyrimidin-4-one derivatives by
SYBR Green Assay against Pf 3D7. Only two compounds viz. 3-
4.1.1. 1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-
trifluoromethylbenzyl)urea (2)
Compound (2) was prepared by refluxing the 3-isocyanato-4H-
pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g, 0.16 mmol) with 2-
trifluoromethylbenzylamine (0.281 g, 0.16 mmol) in toluene for
6 h. The reaction mixture was cooled to room temp. to get a solid
precipitate. The solid precipitate was filtered and washed with
hexane. The product was purified by column chromatography using
hexane (10%) in ethyl acetate as eluent to get the titled compound
(2) (0.22 g, 37.8%), m. p. 243-45 ꢀC. Rf 0.52 (AcOEt). IR (KBr): 3315,
3047, 1667, 1638, 1548, 1478, 1328 and 1224 cmꢁ1. 1H NMR: 4.51e
4.52 (d, 2H), 7.25e7.28 (t, 1H), 7.47e7.51 (m, 1H), 7.56e7.64 (m, 3H),
7.68e7.74 (m, 3H), 8.59 (s, 1H), 8.86e8.88 (d, 1H), 9.17 (s, 1H). MS:
(m/z) 361 (Mþꢁ1).
fluorobenzyl
(21, IC50 value of 33
4H-pyrido[1,2-a]pyrimidine-3-carboxamide (37, IC50 value of
37 M) showed moderate antimalarial activity in the whole series.
(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)carbamate
mM) and 4-oxo-N-[4-(trifluoromethyl)benzyl]-
m
Based on the activity profile of the reported compounds it could be
concluded that pyrido[1,2-a]pyrimidin-4-one ring skeleton can be
considered as a lead structure for further chemical optimization for
obtaining potential antimalarial compounds.
4. Experimental work
4.1. Chemistry
4.1.2. 1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-
trifluoromethylbenzyl)urea (3)
All the reagents and solvents required for synthesis were pu-
rified by general laboratory techniques before use. Compounds
were purified by passing them through silica gel H (100e200
mesh) purifying column using mixture of ethyl acetate and hexane
or methanol as eluent. Melting points were determined using a
Labindia make melting point apparatus (heating block type) and
are uncorrected. Purity of the compounds and completion of re-
actions were monitored by thin layer chromatography (TLC) on
silica gel plates (60 F254; Merck), visualizing with ultraviolet light
or iodine vapors. The yields reported here are un-optimized. The IR
spectra were recorded using KBr disc method on a Bruker FT-IR,
model alpha. The 1H and 13C NMR spectra (on a Bruker 400 MHz
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g,
0.16 mmol) was treated with 4-trifluoromethylbenzylamine
(0.281 g, 0.16 mmol) as described above for compound (2). The
crude product after chromatographic purification using hexane
(10%) in ethyl acetate as eluent offered the titled compound (3)
(0.21 g, 34.4%), m.p. 256e57 ꢀC. Rf 0.48 (AcOEt). IR (KBr): 3328,
3048, 1670, 1636, 1478, 1332, 1222 and 1107 cmꢁ1. 1H NMR: 4.42e
4.44 (d, 2H), 7.25e7.28 (m, 1H), 7.51e7.54 (d, 2H), 7.59e7.64 (m,
2H), 7.69e7.73 (m, 3H), 8.50 (s, 1H), 8.86e8.87 (d, 1H), 9.16 (s, 1H).
13C NMR (DMSO)
d: 155.28, 152.56, 144.29, 144.03, 138.38, 131.89,
127.85, 127.17, 125.97, 125.70, 124.87, 124.83, 120.81, 115.15, 42.50.
MS: (m/z) 363 (Mþþ1).

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U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
Table 1
Chloroquine sensitive P. falciparum (Pf 3D7) inhibition data of the title compounds.
Compound
R
R0
X
A
Pf 3D7 IC50 (mM)
2
H
H
100
3
5
H
H
H
H
>100
>100
7
9
H
H
H
>100
70
10
11
12
13
8-Me
7-Cl
7-Cl
7-Cl
>100
>100
>100
>100
H
H
H
14
15
7-Cl
7-Cl
H
H
>100
>100
16
18
7-Cl
H
>100
>100

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
427
Table 1 (continued )
Compound
R
R0
X
A
Pf 3D7 IC50
>100
(mM)
19
20
7-Cl
7-Cl
>100
21
22
23
24
25
H
33
>100
>100
>100
>100
H
7-Cl
7-Cl
7-Cl
26
28
H
H
H
H
>100
>100
29
30
H
H
H
H
>100
>100
31
33
34
35
H
H
H
H
H
H
H
H
>100
>100
43
>100
36
H
H
65
(continued on next page)

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U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
Table 1 (continued )
Compound
R
R0
X
A
Pf 3D7 IC50 (mM)
37
38
H
H
37
H
H
59
39
40
H
H
H
H
>100
>100
41
H
H
100
43
44
H
H
H
H
H
>100
>100
45
46
H
H
>100
>100
47
53
8-Me
H
H
70
7-(4-MeO-Ph)-
>100
55
56
7-(4-MeO-Ph)-
7-(4-MeO-Ph)-
H
H
>100
100
4.1.3. 1-(2,4-Difluorobenzyl)-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-
3-yl)urea (4)
(m, 1H), 8.50 (s, 1H), 8.85e8.87 (d, 1H), 9.14 (s, 1H). MS: (m/z) 331
(Mþþ1). Anal. Calcd. for C16H12F2N4O2: C, 58.18; H, 3.66; N,16.96.
Found: C, 58.34; H, 3.78; N, 16.85.
The coupling reaction of the 3-isocyanato-4H-pyrido[1,2-a]
pyrimidin-4-one [14] (1) (0.3 g, 0.16 mmol) was done with 2,4-
difluorobenzylamine (0.229 g, 0.16 mmol) as described above for
compound (2). Work-up of the reaction mixture followed by
chromatographic purification of the crude product using hexane
(5%) in ethyl acetate as eluent yielded compound (4) (0.1 g, 18.8%),
m.p. 170e71 ꢀC. Rf 0.48 (AcOEt). IR (KBr): 3328, 1676, 1558, 1483,
1236 and 1127 cmꢁ1. 1H NMR: 4.35e4.37 (d, 2H), 7.14e7.20 (m, 2H),
7.24e7.28 (m, 2H), 7.52e7.55 (t, 1H), 7.62e7.64 (d, 1H), 7.69e7.74
4.1.4. 1-(3,3-Diphenyl)propyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-
3-yl)urea (5)
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.25 g,
0.13 mmol) was reacted with 3,3-diphenylpropylamine (0.282 g,
0.13 mmol) as described above for compound (2). The chromato-
graphic purification of the crude product using hexane (10%) in
ethyl acetate as eluent afforded the urea derivative (5) (0.21 g,

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
429
Table 2
reacted as described above for compound (2). The crude product by
chromatographic purification using hexane (10%) in ethyl acetate
offered the titled compound (9) (0.15 g, 30.6%), m.p. 145e46 ꢀC. Rf
0.61 (AcOEt). IR (KBr): 3373, 1641, 1527, 1483, 1410, 1232, 1176, and
Selectivity index of moderately active compounds on human cell line (Huh7).
Compound
IC50
(
m
M)
Selectivity index
(IC50 Huh7/IC50 pf 3D7)
Pf 3D7
Huh7
940 cmꢁ1 1H NMR: 1.14e1.16 (d, 6H), 4.51e4.52 (m, 1H), 4.55 (s,
.
21
36
37
38
47
33
65
37
59
70
>100
>200
>200
>200
>100
>3
>3
>5.4
>3.3
>1.4
2H), 7.25e7.29 (m, 2H), 7.36e7.37 (d, 4H), 7.50 (s, 1H), 7.63e7.65 (d,
1H), 7.73e7.77 (m, 1H), 8.78e8.80 (d, 1H), 8.96 (s, 1H). MS: (m/z)
337 (Mþꢁ1).
4.1.9. 1-Benzyl-1-isopropyl-3-(8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)urea (10)
39.4%), m.p. 259e60 ꢀC. Rf 0.62 (AcOEt). IR (KBr): 3324, 3133, 3024,
1664, 1631, 1559, 1476 and 1241 cmꢁ1. 1H NMR: 2.18e2.23 (m, 2H),
2.97e3.05 (m, 2H), 4.01e4.05 (t, 1H), 7.11e7.34 (m, 12H), 7.60e7.63
(d, 1H), 7.67e7.74 (m, 1H), 8.32 (s, 1H), 8.85e8.92 (dd, 1H), 9.15(s,
3-Isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.12 mmol) in toluene was refluxed with N-isopropyl
benzylamine (0.185 g, 0.12 mmol) for 4 h to get a solid. The solid so
obtained were reacted as described above for compound (2). Chro-
matographic purification of the crude product using hexane (5%) in
ethyl acetate provided the titled compound (10) (0.16 g, 36.7%), m.p.
161e62 ꢀC. Rf 0.62 (AcOEt). IR (KBr): 3353, 3031, 2971, 1642, 1546,
1486, 1403 and 1242 cmꢁ1. 1H NMR: 1.13e1.15 (d, 6H), 2.41 (s, 3H),
4.49e4.51 (m, 1H), 4.54 (s, 2H), 7.13e7.15 (d, 1H), 7.25e7.26 (m, 1H),
7.35e7.36 (d, 4H), 7.45 (b, 2H), 8.70e8.72 (d,1H), 8.87 (s,1H).13C NMR
1H). 13C NMR (DMSO)
d: 155.20, 152.52, 144.20, 143.79, 138.01,
131.63, 128.14, 127.39, 125.96, 125.86, 125.64, 121.09, 115.07, 47.89,
37.64, 35.16. MS: (m/z) 399 (Mþþ1).
4.1.5. 1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-[3-
(n.pentyloxy)pyridin-2-yl]urea (6)
Compound (6) was prepared by reacting the 3-isocyanato-4H-
pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g, 0.16 mmol) with 3-
n.pentyloxy-2-aminopyridine (0.289 g, 0.16 mmol) as described
above for compound (2). The crude product after chromatographic
purification using methanol (1%) in ethyl acetate as eluent offered
the desired product (6) (0.2 g, 33.96%), m.p. 178-80 ꢀC. Rf 0.47
(CDCl3) d: 155.41,153.58,144.89,143.85,140.47,137.87,128.90,127.52,
126.45, 125.49, 124.49, 120.10, 118.15, 47.00, 45.76, 21.23, 20.82. MS:
(m/z) 349 (Mþ), 350.9 (Mþþ2). Anal. Calcd. for C20H22N4O2: C, 68.55;
H, 6.33; N, 15.99. Found: C, 68.76; H, 6.54; N, 15.89.
(AcOEt). IR (KBr): 3387, 3350, 3067,1665,1550,1478 and 1220 cmꢁ1
.
4.1.10. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-
(thiophen-2-yl)methylurea (11)
1H NMR: 0.90e0.94 (t, 3H), 1.32e1.47 (m, 4H), 1.78e1.85 (m, 2H),
4.07e4.11 (t, 2H), 7.05e7.08 (m, 1H), 7.30e7.34 (m, 1H), 7.44e7.46
(d, 1H), 7.67e7.69 (d, 1H), 7.76e7.81 (m, 1H), 7.89e7.91 (dd, 1H),
8.35 (s, 1H), 8.92e8.94 (d, 1H), 9.29 (s, 1H), 12.15 (s, 1H). MS: (m/z)
368 (Mþþ1).
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) in toluene was refluxed with (2-
thiophenyl)methylamine (0.127 g, 0.11 mmol) to get a solid. The
solid so obtained was filtered and washed with hexane. The crude
product was purified by column chromatography using 1% meth-
anol in ethyl acetate to obtain the compound (11) (0.15 g, 39.76%),
m.p. 285e87 ꢀC. Rf 0.6 (AcOEt). IR (KBr): 3272, 3024, 1652, 1562,
1472, 1337, 1240, and 1155 cmꢁ1. 1H NMR: 4.49e4.50 (d, 2H), 6.97e
6.98 (m, 1H), 7.00 (s, 1H), 7.40e7.41 (d, 1H), 7.56 (b, 1H), 7.64e7.66
4.1.6. 1-[3-(Benzyloxy)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)urea (7)
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g,
0.16 mmol) was treated with 2-amino-3-benzyloxypyridine
(0.319 g, 0.16 mmol) as described above for compound (2). The
crude product was purified by column chromatography using
methanol (1%) in ethyl acetate as eluent offering the titled com-
pound (7) (0.19 g, 30.6%), m.p. 249e-50 ꢀC. Rf 0.46 (AcOEt). IR (KBr):
3133, 1675, 1558, 1482, 1192 and 1118 cmꢁ1. 1H NMR: 5.29 (s, 2H),
7.07 (m, 1H), 7.33e7.42 (m, 3H), 7.51e7.57 (m, 3H), 7.70e7.76 (m,
1H), 7.77e7.79 (m,1H), 7.91e7.92 (d,1H), 8.46 (s,1H), 8.90e8.94 (m,
1H), 9.22e9.28 (d, 1H), 9.52 (s, 1H), 12.06 (s, 1H). MS: (m/z) 388
(Mþþ1).
(d, 1H), 7.72e7.74 (d, 1H), 8.51 (s, 1H), 8.89 (s, 1H), 9.18 (s, 1H). 13
NMR (CDCl3) : 159.19, 153.94, 152.81, 147.57, 144.08, 143.05, 139.60,
C
d
133.71, 127.73, 124.54, 123.79, 120.97, 117.26, 111.55, 43.93. MS: (m/
z) 335.34(Mþþ1), 337.38 (Mþþ2).
4.1.11. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-
methoxyethyl)urea (12)
A
mixture of 7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyr-
imidin-4-one [14] (1) (0.25 g, 0.11 mmol), 2-methoxyethylamine
(0.162 g, 0.11 mmol) and toluene refluxed for 4 h to get solid ppt.
The solid so obtained was filtered and washed with hexane.
Chromatographic purification of the product using hexane (15%) in
ethyl acetate accomplished the desired compound (12) (0.22 g,
65.74%), m.p. 254e56 ꢀC. Rf 0.65 (5% MeOH in AcOEt). IR (KBr):
3370, 3116, 2972, 1702, 1649, 1557, 1528, 1434, 1354, 1229, 1199,
1106, 817, 757 and 593 cmꢁ1. 1H NMR: 3.24e3.39 (m, 7H), 7.19e7.21
(m, 1H), 7.63e7.65 (d, 1H), 7.70e7.73 (dd, 1H), 8.51 (s, 1H), 8.83e
8.84 (b, 1H), 9.16 (s, 1H). MS: (m/z) 297.39 (Mþ), 299.38 (Mþþ2).
4.1.7. 1-[6-(Morpholin-4-yl)pyridin-2-yl]-3-(4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl)urea (8)
Compound (8) was prepared by treating the 3-isocyanato-4H-
pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g, 0.16 mmol) with 2-
amino-6-morpholinopyridine (0.287 g, 0.16 mmol) as described
above for compound (2). The chromatographic purification of the
crude product using methanol (1%) in ethyl acetate as eluent
offered compound (8) (0.25 g, 42.5%), m.p. 275e76 ꢀC. Rf 0.45
(AcOEt). IR (KBr): 3118, 1688, 1558, 1484, 1440, 1229 and 1113 cmꢁ1
.
1H NMR: 3.66e3.68 (b, 4H), 3.95e3.97 (b, 4H), 6.17e6.20 (d, 1H),
6.29e6.31 (d, 1H), 7.11e7.15 (m, 1H), 7.48e7.52 (m, 2H), 7.58e7.62
(m, 1H), 7.67e7.70 (d, 1H), 8.97e8.99 (d, 1H), 9.53 (s, 1H), 11.48 (s,
1H). MS: (m/z) 367 (Mþþ1).
4.1.12. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(4-
trifluoromethyl)benzylurea (13)
A mixture of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-
4-one [14] (1) (0.25 g, 0.11 mmol) and 4-trifluoromethylbenzylamine
(0.197 g, 0.11 mmol) in toluene was refluxed for 4 h. Solid precipitate
so obtained was filtered and washed with hexane. Chromatographic
purification of the crude product using 1% methanol in ethyl acetate
yielded the titled compound (13) (0.23 g, (51.4%), m.p. 287e89 ꢀC. Rf
0.61 (1% MeOH in AcOEt). IR (KBr): 3397, 3187,1692,1656,1632,1364
4.1.8. 1-Benzyl-1-isopropyl-3-(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl)urea (9)
3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.25 g,
0.13 mmol) and N-isopropyl benzylamine (0.199 g, 0.13 mmol) were

430
U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
and 1226 cmꢁ1. 1H NMR: 4.42e4.44 (d, 2H), 7.57e7.66 (m, 6H), 7.72e
7.75 (dd, 1H), 8.58 (s, 1H), 8.84e8.85 (d, 1H), 9.16 (s, 1H). MS: (m/z)
397.34(Mþ), 399.38 (Mþþ2).
chromatography using methanol (3%) in ethyl acetate as eluent to
get the desired compound (18) (0.22 g, 31.6%), m.p. 140e42 ꢀC. Rf
0.4 (AcOEt). IR (KBr): 3296, 3035, 1654, 1530, 1484, 1434, 1234 and
1134 cmꢁ1. 1H NMR: 0.55e0.58 (m, 2H), 0.69e0.72 (m, 2H), 2.82e
2.83 (m, 1H), 3.31e3.36 (t, 4H), 3.57e3.59 (t, 4H), 6.89e6.92 (m,
1H), 7.32e7.36 (m, 1H), 7.65e7.67 (m, 2H), 7.81e7.85 (m, 1H), 8.08
(s, 1H), 8.23e8.25 (dd, 1H), 8.51e8.53 (d, 1H), 8.76 (s, 1H), 8.91e8.93
4.1.13. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(3-
trifluoromethyl)benzylurea (14)
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol),3-trifluoromethylbenzylamine (0.197 g,
0.11 mmol) and toluene (50 ml) was refluxed for 4 h. The solid ppt
so obtained was filtered and washed with hexane. The crude
product was purified by column chromatography using hexane (5%)
in ethyl acetate to furnish the titled compound (14) (0.22 g, 49.1%),
m.p. 260e62 ꢀC. Rf 0.6 (1% MeOH in AcOEt). IR (KBr): 3312, 3282,
3085, 1677, 1646, 1564, 1488, 1433, 1331, 1243, 1160, 1107, 1016, 821,
765 and 644 cmꢁ1. 1H NMR: 4.42e4.43 (d, 2H), 7.51e7.53 (d, 2H),
7.61e7.63 (m, 2H), 7.70e7.75 (m, 3H), 8.60 (s, 1H), 8.84e8.85 (d,
1H), 9.17 (s, 1H). MS: (m/z) 397.34(Mþ), 399.32 (Mþþ2).
(d, 1H). 13C NMR (DMSO)
d: 168.35, 157.59, 154.70, 153.50, 148.34,
145.73, 142.89, 138.18, 133.52, 126.13, 126.01, 120.79, 119.17, 115.76,
115.46, 48.47, 43.43, 22.59, 5.61. MS: (m/z) 434 (Mþþ1).
4.1.18. N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-(4-
tert-butyloxycarbonyl)piperazine]-1-carboxamide (19)
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 11 mmol) was reacted with boc-piperazine (0.209 g,
0.11 mmol) as described above for compound (17). Chromato-
graphic purification of the crude product using hexane (10%) in
ethyl acetate provided the desired compound (19) (0.2 g, 43.4%),
m.p. 229e30 ꢀC. Rf 0.6 (1% MeOH in AcOEt). IR (KBr): 3393, 1691,
1650, 1629, 1490, 1422, 1233 and 1173 cmꢁ1. 1H NMR: 1.42 (s, 9H),
3.39 (b, 4H), 3.44e3.45 (b, 4H), 7.68e7.71 (d, 1H), 7.83e7.85 (d, 1H),
8.13 (s, 1H), 8.78 (s, 1H), 8.88e8.89 (d, 1H). MS: (m/z) 408.41, 410.39
(Mþꢁ1).
4.1.14. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-
trifluoromethyl)benzylurea (15)
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) in toluene was refluxed with 2-
trifluoromethylbenzylamine (0.197 g, 0.11 mmol) to get solid pre-
cipitate. The solid so obtained was filtered and washed with hex-
ane. Chromatographic purification of the crude product using
hexane (5%) in ethyl acetate furnished the titled compound (15)
(0.23 g, 51.4%), m.p. 276e78 ꢀC. Rf 0.62 (1% MeOH in AcOEt). IR
(KBr): 3362, 3126, 1707, 1622, 1558, 1461, 1311 and 1225 cmꢁ1. 1H
NMR: 4.51e4.52 (d, 2H), 7.47e7.51 (m, 1H), 7.59e7.75 (m, 6H), 8.68
4.1.19. N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
(furan-2-carbonyl)piperazine]-1-carboxamide (20)
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) was reacted with 1-(furan-2-carbonyl)
piperazine (0.203 g, 0.11 mmol) as described above for compound
(18). Chromatographic purification of the crude product using 1%
methanol in ethyl acetate offered the desired compound (20)
(0.19 g, 41.9%), m.p. 227e28 ꢀC. Rf 0.58 (1% MeOH in AcOEt). IR
(KBr): 3446, 3086, 1680, 1626, 1427, 1290, 1242 and 1199 cmꢁ1. 1H
NMR: 3.57e3.58 (b, 4H), 3.74 (b, 4H), 6.65 (s, 1H), 7.04e7.05 (d, 1H),
7.69e7.71 (d, 1H), 7.83e7.86 (m, 1H), 7.87 (s, 1H), 8.18 (s, 1H), 8.80 (s,
1H), 8.89 (b, 1H). MS: (m/z) 402 (Mþꢁ1).
(s, 1H), 8.85 (b, 1H), 9.17 (s, 1H). 13C NMR (DMSO)
d: 155.36, 152.10,
142.76, 138.32, 137.92, 133.49, 132.97, 129.25, 128.73, 127.85, 127.65,
126.54, 126.01, 125.96, 123.72, 123.24, 121.55. MS: (m/z) 397.34
(Mþ), 399.32, (Mþþ2).
4.1.15. 1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl)-1-isopropylurea (16)
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) in toluene (25 ml) was refluxed with N-
isopropylbenzylamine (0.168 g, 0.11 mmol) for 5 h to get solid
precipitate. Chromatographic purification of the crude product
using hexane (10%) in ethyl acetate offered the desired compound
(16) (0.2 g, 47.8%), m.p. 158e60 ꢀC. Rf 0.64 (AcOEt). IR (KBr): 3447,
1663, 1630, 1521, 1487, 1234, 1114 and 1070 cmꢁ1. MS: (m/z) 371.42
(Mþ), 373.41(Mþþ2). Anal. Calcd. for C19H19ClN4O2: C, 61.54; H,
5.16; N, 15.11. Found: C, 61.68; H, 5.28; N, 15.25.
Anal. Calcd. for C18H16ClN5O4: C, 53.81; H, 4.01; N, 17.43. Found:
C, 58.97; H, 4.17; N, 17.57.
4.1.20. 3-Fluorobenzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
carbamate (21)
A
mixture of 3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one
[14] (1) (0.25 g, 0.13 mmol) and 3-fluorobenzyl alcohol (0.169 g,
0.13 mmol) were refluxed together in toluene. The crude product
was purified by column chromatography using hexane (20%) in
ethyl acetate to get the titled compound (21) (0.11 g, 26.2%), m.p.
158e60 ꢀC. Rf 0.65 (AcOEt). IR (KBr): 3416, 3264, 1716, 1659, 1480,
1441, 1403, 1234, 1200, 1184, 1134, 796 and 773 cmꢁ1. 1H NMR: 5.19
(s, 2H), 7.14e7.19 (m, 1H), 7.27e7.37 (m, 3H), 7.41e7.47 (m, 1H),
7.69e7.71 (d, 1H), 7.85e7.89 (m, 1H), 8.72 (s, 1H), 8.93e8.95 (d, 1H),
9.16 (b, 1H). MS: (m/z) 314, (Mþꢁ1).
4.1.16. 1-Benzyl-3-(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl)-1-phenethylurea (17)
Reaction of 7-chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-
4-one [14] (1) (0.25 g, 0.11 mmol) and N-benzyl phenylethylamine
(0.238 g, 0.11 mmol) was done as described above for compound
(2). The crude product was purified by column chromatography
using 2% methanol in ethyl acetate to obtain the titled compound
(17) (0.18 g, 36.8%), m.p. 195e97 ꢀC. Rf 0.55 (1% MeOH in AcOEt). IR
(KBr): 3345, 3028, 1647, 1550, 1493, 1435, 1366 and 1233 cmꢁ1. 1H
NMR: 2.85e2.89 (t, 2H), 3.35e3.59 (t, 2H), 4.58 (s, 2H), 7.14e7.38
(m, 11H), 7.68e7.71 (d, 1H), 7.81e7.82 (b, 2H), 8.88 (b, 1H), 8.92 (s,
1H). MS: (m/z) 433.42 (Mþ), 435.41 (Mþþ2).
4.1.21. 3,4-Methylenedioxybenzyl(4-oxo-4H-pyrido[1,2-a]
pyrimdin-3-yl)carbamate (22)
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.5 g,
0.26 mmol) was reacted with 3,4-methylenedioxybenzyl alcohol
(0.244 g, 0.106 mmol) as described above for compound (21). The
chromatographic purification of the crude product using hexane
(10%) in ethyl acetate as eluent offered the titled compound (22)
(0.18 g, 33%), m. p. 175e76 ꢀC. Rf 0.5 (AcOEt). IR (KBr): 3242, 3031,
1717, 1666, 1485, 1443, 1235 and 1137 cmꢁ1. 1H NMR: 5.06 (s, 2H),
6.02 (s, 2H), 6.92 (s, 2H), 7.02 (s, 1H), 7.33e7.37 (m, 1H), 7.69e7.71
(d, 1H), 7.84e7.89 (m, 1H), 8.70 (b, 1H), 8.92e8.94 (d, 1H), 9.01 (b,
4.1.17. N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-[3-(N-
cyclopropylcarboxamido)pyridin-2-yl] piperazinecarboxamide (18)
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.3 g,
0.16 mmol) in toluene (25 ml) was refluxed with N-cyclopropyl-2-
(piperazine-1-yl)pyridine-3-carboxamide (0.319 g, 0.16 mmol) to
get a solid precipitate. The crude compound was purified by column
1H). 13C NMR (DMSO)
d: 153.60, 152.96, 147.29, 147.06, 146.12,

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
431
133.77, 129.67, 126.19, 126.00, 121.64, 117.94, 115.67, 108.50, 107.75,
IR (KBr): 3325, 3276, 1745, 1657, 1634, 1550, 1528 and 1191 cmꢁ1
.
100.78, 66.27, 52.00. MS: (m/z) 340 (Mþþ1).
MS: (m/z) 333 (Mþþ1).
4.1.27. (2S)-2-{[(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
carbamoyl]amino}-3-phenylpropanamide (28)
4.1.22. 2-Pyridinylmethyl(7-chloro-4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)carbamate (23)
A mixture of compound (26) (2 g, 0.52 mmol) and liq. ammonia
was stirred for 12 h. The solid precipitate so obtained was filtered,
washed with water and recrystallized from methanol to furnish
compound (28) (1.8 g, 97.4%), m. p. 239e40 ꢀC. Rf 0.35 (AcOEt). IR
(KBr): 3329, 1634, 1519, 1477, 1244 and 1191 cmꢁ1. MS: (m/z) 352
(Mþþ1).
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) was reacted with pyridine-2-methanol
(0.176 g, 0.135 mmol) as described above for compound (21). The
crude product was purified by column chromatography using 2%
methanol in ethyl acetate to accomplish the titled compound (23)
(0.05 g, 13.4%), m.p. 196e97 ꢀC. Rf 0.62 (2% MeOH in AcOEt). IR
(KBr): 3451, 3245, 1724, 1652, 1477, 1354, 1236 and 694 cmꢁ1 1H
.
4.1.28. (2S)-4-Methyl-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
carbamoyl]amino}pentanamide (29)
NMR: 5.23 (s, 2H), 7.33e7.36 (d, 1H), 7.51e7.53 (d, 1H), 7.71e7.73 (d,
1H), 7.83e7.87 (t, 1H), 7.88e7.91 (dd, 1H), 8.55e8.56 (d, 1H), 8.76 (s,
1H), 8.92e8.93 (d, 1H), 9.32 (s, 1H). MS: (m/z) 331.37, 333.35
(Mþꢁ1).
Compound (27) (2 g, 0.52 mmol) was stirred in liq. ammonia for
12 h to get the solid precipitate. The precipitated recrystallized
from methanol to obtain compound (29) (1.6 g 83.7%), m. p. 243e
44 ꢀC. Rf 0.4 (AcOEt). IR (KBr): 3361, 3320, 1672, 1628, 1528, 1469,
4.1.23. 3-Pyridinylmethyl(7-chloro-4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)carbamate (24)
1437 and 1234 cmꢁ1 13C NMR (DMSO)
. d: 154.31, 152.88, 145.02,
139.39, 135.64, 133.60, 129.65, 128.64, 127.38, 126.41, 119.92, 116.21,
7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14]
(1) (0.25 g, 0.11 mmol) and pyridine-3-methanol (0.123 g,
0.11 mmol) were reacted as described above for compound (21) to
get solid precipitate. The solid so obtained was filtered and washed
with hexane. Chromatographic purification of the crude product
using hexane (5%) in ethyl acetate provided the intended com-
pound (24) (0.15 g, 40.2%), m.p. 293e95 ꢀC. Rf 0.65 (AcOEt). IR (KBr):
3415, 3251, 1722, 1664, 1630, 1402 and 1232 cmꢁ1. 1H NMR: 5.22 (s,
2H), 7.41e7.45 (m, 1H), 7.70e7.73 (d, 1H), 7.86e7.91 (m, 2H), 8.54e
8.55 (d, 1H), 8.66 (b, 1H), 8.74 (b, 1H), 8.91e8.92 (d, 1H) 9.16 (s, 1H).
MS: (m/z) 331.37, 333.35, (Mþꢁ1).
42.80, 37.71. MS: (m/z) 318 (Mþþ1).
4.1.29. 1-[(1S)-1-(Cyano-2-phenyl)ethyl]-3-(4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl)urea (30)
The amide (28) (1.2 g, 0.34 mmol) was dissolved in dime-
thylformamide (2.5 ml) and cooled to 0 ꢀC. Cyanuric chloride (1.26 g,
0.68 mmol) was added in small portions and stirred for 2 h. The
reaction mixture was quenched in ice-cold water (20 ml) to get solid
precipitate. The solid precipitate was filtered and dried. The crude
product so obtained was purified bycolumn chromatography toearn
the compound (30) (0.34 g, 29.8%), m.p. 223e25 ꢀC. Rf 0.45 (AcOEt).
IR (KBr): 3303, 2210, 1686, 1631, 1530, 1433, 1227 and 1133 cmꢁ1. 1H
NMR: 3.09e3.19 (m, 2H), 4.92e4.98 (m, 1H), 7.26e7.34 (m, 2H),
7.36e7.37 (m, 4H), 7.63e7.67 (m, 2H), 7.72e7.77 (m,1H), 8.58 (s,1H),
4.1.24. 3-Fluorobenzyl(7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-
3-yl)carbamate (25)
Compound (25) was obtained by reaction of 7-Chloro-3-
isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (0.25 g,
0.11 mmol) with 3-fluorobenzyl alcohol (0.142 g, 0.11 mmol) to get
solid precipitate. The solid so obtained was filtered and washed
with hexane and purified by column chromatography using hexane
(10%) in ethyl acetate to give the titled compound (25) (0.06 g,
15.3%), m.p. 240e42 ꢀC. Rf 0.62 (1% MeOH in AcOEt). IR (KBr): 3431,
3276, 1726, 1660, 1546, 1229 and 1127 cmꢁ1. 1H NMR: 5.15 (s, 2H),
7.20e7.24 (m, 2H), 7.48e7.51 (m, 2H), 7.70e7.72 (d, 1H), 7.88e7.90
(d, 1H), 8.74 (s, 1H), 8.91 (s, 1H), 9.18 (s, 1H). MS: (m/z) 350 (Mþꢁ1),
348 (Mþꢁ3). Anal. Calcd. for C16H11ClFN3O3: C, 55.27; H, 3.19; N,
12.08. Found: C, 55.42; H, 3.31; N, 12.19.
8.85e8.87 (d, 1H), 9.10 (s, 1H). 13C NMR (CDCl3)
d: 175.09, 155.00,
153.44, 144.64, 140.88, 132.15, 126.65, 126.59, 120.96, 115.89, 52.37,
51.30, 42.05, 24.81, 22.80, 21.93. MS: (m/z) 334 (Mþþ1).
4.1.30. 1-[(1S) 1-(Cyano-3-methyl)butyl]-3-(4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl)urea (31)
Compound (30) was prepared by treating (29) (2.0 g,
0.63 mmol) with cyanuric chloride (2.33 g, 1.26 mmol) as described
above for compound (30). The chromatographic purification of the
crude product using hexane (25%) in ethyl acetate as eluent offered
compound (31) (0.35 g, 18%), m. p. 237e39 ꢀC. Rf 0.55 (AcOEt). IR
(KBr): 3342, 2210, 1642, 1557, 1510, 1465, 1434 and 1235 cmꢁ1. 1H
NMR: 0.91e0.96 (d, 6H), 1.64e1.80 (m, 3H), 4.65e4.71(m, 1H),
7.27e7.31 (m, 1H), 7.65e7.67 (d, 2H), 7.73e7.77 (m, 1H), 8.49 (s, 1H),
4.1.25. Ethyl (2S)-2-{[(4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
carbamoyl]amino}-3-phenylpropanoate (26)
8.86e8.88 (d, 1H), 9.12 (s, 1H). 13C NMR (DMSO)
d: 154.04, 152.54,
3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one [14] (1) (1.94 g,
144.48, 138.77, 132.57, 126.00, 125.86, 120.02, 119.64, 115.49, 40.73,
24.27, 21.74, 21.73. MS: (m/z) 300 (Mþþ1). Anal. Calcd. for
6.1 mmol) and -phenylalanine ethyl ester (2 g, 6.1 mmol) in
L
toluene (25 ml) were heated together at 80 ꢀC for 6 h. The reaction
mixture was cooled to RT to yield a solid precipitate. The solid
precipitate was filtered, washed with hexane and dried to get the
titled compound (26) (2 g, 62.5%), m. p. 158e59 ꢀC. Rf 0.75 (AcOEt).
IR (KBr): 3338, 3032, 1733, 1688, 1629, 1132 and 1185 cmꢁ1. MS:
381(Mþþ1).
C14H15N5O2: C, 58.94; H, 5.30; N, 24.55. Found: C, 58.86; H, 3.42; N,
24.58.
4.1.31. N-(4-Methoxybenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxamide (33)
In a two neck round bottom flask (100 ml) equipped with calcium
chloride guard tube, the acid derivative 4-oxo-4H-pyrido[1,2-a]-3-
carboxylic acid [14] (32) (0.5 g, 0.26 mmol) was dissolved in
dichloromethane (50 ml) and the solution was cooled to 0 ꢀC. 1-
Hydroxybenzotriazole hydrate (0.355 g, 0.26 mmol), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (0.603 g, 0.31 mmol)
and N,N-diisopropylethylamine (0.679 g, 0.52 mmol) were added to
the above solution. After 20 min 4-methoxybenzylamine (0.397 g,
0.26 mmol) was added and the reaction mixture was stirred for 1 h
4.1.26. Methyl (2S)-4-methyl-2-{[(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)carbamoyl]amino} pentanoate (27)
Compound (27) was obtained by reacting 3-isocyanato-4H-
pyrido[1,2-a]pyrimidin-4-one [14] (1) (2.85 g, 1.37 mmol) with -
L
leucine methyl ester (2 g, 1.37 mmol) as described above for com-
pound (26). The crude product was recrystallized from methanol to
yield compound (27) (2.1 g, 45.8%), m.p. 129e30 ꢀC. Rf 0.78 (AcOEt).

432
U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
at 0 ꢀC and for another 8 h at room temperature. The reaction mass
was quenched by adding cold water (20 ml) and the medium was
neutralized using dilute hydrochloric acid, extracted using ethyl
acetate (3 ꢂ 100 ml), dried over anhydrous sodium sulfate and
concentrated to get a dark brown product. The crude product was
purified by column chromatography using methanol (2%) in ethyl
acetate to offer the compound (33) (0.24 g, 28.2%), m. p. 192e94 ꢀC.
Rf 0.51 (AcOEt). IR (KBr): 3336, 1679, 1489, 1334, 1297, 1243 and
1030 cmꢁ1. 1H NMR: 3.80 (s, 3H), 4.62e4.64 (d, 2H), 6.86e6.90 (m,
2H), 7.31e7.33 (d, 2H), 7.34e7.38 (m, 1H), 7.84e7.86 (d, 1H), 7.92e
7.97 (m, 1H), 9.19e9.21 (d, 1H), 9.30 (b, 1H), 9.38 (s, 1H). 13C NMR
trifluoromethylbenzylamine (0.461 g, 0.26 mmol) as described
above for compound (33). The chromatographic purification of the
crude product using hexane (5%) in ethyl acetate as eluent yielded
compound (37) (0.25 g, 26.2%), m. p. 167-69 ꢀC. Rf 0.6 (AcOEt). IR
(KBr): 3336, 3115, 1663, 1600, 1570, 1500, 1325 and 1123 cmꢁ1. 1H
NMR: 4.75e4.76 (d, 2H), 7.38e7.41 (m, 1H), 7.49e7.51 (d, 2H), 7.58e
7.60 (d, 2H), 7.86e7.88 (d, 1H), 7.96e8.00 (m, 1H), 9.21e9.23 (d, 1H),
9.37e9.38 (d, 1H), 9.47 (b, 1H). 13C NMR (CDCl3)
d: 164.02, 158.60,
157.70, 152.48, 142.82, 138.50, 129.91, 128.95, 127.81, 125.61, 122.80,
120.10, 117.57, 106.81, 42.92. MS: (m/z) 348 (Mþþ1). Anal. Calcd. for
C17H12F3N3O2: C, 58.79, H,3.48, N, 12.10. Found: C, 58.91; H, 3.62; N,
(DMSO)
d: 162.98, 158.31, 157.24, 157.02, 152.07, 139.87, 131.16,
12.37.
128.79, 128.24, 126.38, 118.41, 113.81, 105.95, 55.03, 41.82. MS: (m/z)
310 (Mþþ1).
4.1.36. N-(2,4-Difluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-
3-carboxamide (38)
4.1.32. N-(3-Fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxamide (34)
Compound (38) was prepared by reacting 4-oxo-4H-pyrido[1,2-
a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 2,4-
difluoromethylbenzylamine (0.376 g, 0.26 mmol) as described
above for compound (33). The chromatographic purification of the
crude product using hexane (20%) in ethyl acetate as eluent affor-
ded the desired product (38) (0.21 g, 25.3%), m. p.172e75 ꢀC. Rf 0.62
Compound (34) was prepared by reacting compound 4-oxo-4H-
pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 3-
fluorobenzylamine (0.329 g, 0.26 mmol) as described above for
compound (33). The crude product was purified by column chro-
matography using hexane (5%) in ethyl acetate as eluent offering
the compound (34) (0.32 g, 40.9%), m.p. 168e70 ꢀC. Rf 0.64 (AcOEt).
IR (KBr): 3327, 3122, 1662,1623,1569,1533, 1500 and 1240 cmꢁ1. 1H
NMR: 4.59e4.61 (d, 2H), 7.06e7.11 (m,1H), 7.15e7.21 (m, 2H), 7.36e
7.41 (m, 1H), 7.59e7.63 (m, 1H), 7.90e7.92 (d, 1H), 8.19e8.23 (m,
1H), 9.05 (s, 1H), 9.19e9.21 (d, 1H), 9.43e9.46 (t, 1H). 13C NMR
(AcOEt). IR (KBr): 3328, 3125, 1664,1500,1430, 1184 and 1072 cmꢁ1
.
1H NMR: 4.60e4.62 (d, 2H), 7.14e7.21 (m, 2H), 7.24e7.29 (m, 1H),
7.59e7.63 (m, 1H), 7.89e7.92 (d, 1H), 8.19e8.23 (m, 1H), 9.03 (s, 1H),
9.20e9.23 (d, 1H), 9.42e9.45 (t, 1H). 13C NMR (CDCl3)
d: 164.03,
159.90, 159.87, 158.56, 157.95, 157.92, 155.54, 152.48, 138.52, 128.09,
127.58, 117.55, 116.44, 114.97, 106.78, 36.99. MS: (m/z) 316 (Mþþ1).
(DMSO)
d: 163.61, 161.19, 157.42, 152.33, 142.71, 140.14, 130.51,
128.48, 126.57, 123.51, 118.62, 114.28, 114.07, 113.86, 106.06, 42.04.
4.1.37. N-[2-(Morpholin-4-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (39)
MS: (m/z) 298 (Mþþ1).
Reaction of compound 4-oxo-4H-pyrido[1,2-a]-3-carboxylic
acid [14] (32) (0.5 g, 0.26 mmol) with 2-(4-morpholinyl)ethyl-
amine (0.342 g, 0.26 mmol) was effected as described above for
compound (33). The chromatographic purification of the crude
product using methanol (0.2%) in ethyl acetate as eluent offered the
titled compound (39) (0.18 g, 22.6%), m. p. 193e95 ꢀC. Rf 0.3 (1%
MeOH in AcOEt). IR (KBr): 3123, 1690, 1631, 1483, 1400, 1251 and
1115 cmꢁ1. 1H NMR: 2.43 (b, 4H), 2.50e2.51 (m, 2H), 3.45e3.50 (m,
2H), 3.59e3.61(t, 4H), 7.57e7.61 (m, 1H), 7.84e7.90 (d, 1H), 8.17e
8.21 (m, 1H), 9.02 (s, 1H), 9.15e9.16 (t, 1H), 9.20e9.22 (d, 1H). MS:
(m/z) 303 (Mþþ1).
4.1.33. 4-Oxo-N-[2-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (35)
Compound (35) was prepared by treating compound 4-oxa-4H-
pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 2-
trifluoromethylbenzylamine (0.461 g, 0.26 mmol) as described
above for compound (33). Chromatographic purification of the
crude product using hexane (5%) in ethyl acetate as eluent yielded
compound (35) (0.32 g, 35%), m.p. 158e60 ꢀC. Rf 0.6 (AcOEt). IR
(KBr): 3299, 3079, 1695, 1630, 1476, 1315, 1167 and 1094 cmꢁ1. 1H
NMR: 4.76e4.78 (d, 2H), 7.48e7.52 (m,1H), 7.59e7.69 (m, 3H), 7.75e
7.77 (d, 1H), 7.91e7.93 (d, 1H), 8.20e8.24 (m, 1H), 9.05 (s, 1H), 9.21e
9.23 (d, 1H), 9.48e9.51 (t, 1H). 13C NMR (DMSO)
d: 163.55, 157.49,
4.1.38. 4-Oxo-N-[3-(n.pentyloxy)pyridin-2-yl]-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (40)
152.36, 140.24, 137.61, 132.96, 129.49, 128.78, 127.74, 126.99, 126.69,
126.00,125.94,123.33,120.61,118.68,105.87. MS: (m/z) 348 (Mþþ1).
Compound (40) was prepared by reacting 4-oxo-4H-pyrido[1,2-
a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 2-amino-3-
n.pentyloxypyridine (0.474 g, 0.26 mmol) as described above for
compound (33). The chromatographic purification of the crude
product using methanol (2.5%) in ethyl acetate as eluent produced
the titled product (40) (0.3 g, 32.3%), m.p. 159e60 ꢀC. Rf 0.3 (AcOEt).
IR (KBr): 3292, 3237,1693,1595,1470,1333 and 1218 cmꢁ1. 1H NMR:
0.89e0.93 (t, 3H), 1.36e1.43 (m, 2H), 1.49e1.57 (m, 2H), 1.78e1.84
(m, 2H), 4.09e4.13 (t, 2H), 6.55 (s, 1H), 7.13e7.16 (m, 1H), 7.46e7.48
(d,1H), 7.66e7.70 (m,1H), 7.95e7.97 (d,1H), 8.24e8.28 (m,1H), 9.13
4.1.34. 4-Oxo-N-[3-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (36)
4-Oxo-4H-pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g,
0.26 mmol) was reacted with 3-trifluoromethylbenzylamine
(0.461 g, 0.26 mmol) as described above for compound (33). The
chromatographic purification of the crude product using hexane
(5%) in ethyl acetate as eluent furnished the compound (36) (0.33 g,
36.1%), m.p. 139e40 ꢀC. Rf 0.63 (AcOEt). IR (KBr): 3328, 3113, 1691,
1645, 1532, 1490, 1415 and 1335 cmꢁ1. 1H NMR: 4.66e4.67 (d, 2H),
7.56e7.63 (m, 3H), 7.66e7.68 (d, 1H), 7.72 (s, 1H), 7.90e7.92 (d, 1H),
8.18e8.23 (m, 1H), 9.05 (s, 1H), 9.20e9.21 (d, 1H), 9.50e9.53 (t, 1H).
(s, 1H), 9.21e9.24 (d, 1H), 11.51 (s, 1H). 13C NMR (CDCl3)
d: 160.31,
157.96, 152.14, 149.59, 144.25, 141.99, 139.28, 138.46, 127.75, 126.50,
119.20, 117.96, 115.44, 106.56, 68.26, 28.29, 27.49, 21.96, 13.74. MS:
(m/z) 353 (Mþþ1).
13C NMR (CDCl3)
d: 164.01, 158.60, 157.69, 152.48, 139.78, 138.50,
131.02, 130.70, 129.09, 128.06, 127.11, 125.44, 124.34, 122.73, 117.55,
106.83, 42.89. MS: (m/z) 348 (Mþþ1).
4.1.39. N-[3-(Benzyloxy)pyridin-2-yl]-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (41)
4.1.35. 4-Oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (37)
Compound (37) was prepared by reacting 4-oxo-4H-pyrido[1,2-
a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 4-
4-Oxo-4H-pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g,
0.26 mmol) and 2-amino-3-benzyloxypyridine (0.526 g, 0.26 mmol)
were reacted together as described above for compound (33). The
chromatographic purification of the crude product using methanol

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
433
(1%) in ethyl acetate as eluent offered compound (41) (0.23 g, 23.4%),
m.p. 193e95 ꢀC. Rf 0.55 (AcOEt). IR (KBr): 3480, 3106, 1699, 1598,
1482, 1285 and 1012 cmꢁ1. 1H NMR: 5.28 (s, 2H), 7.00e7.04 (dd, 1H),
7.23e7.26 (dd, 1H), 7.34e7.38 (m, 1H), 7.41e7.44 (m, 2H), 7.53e7.55
(m, 1H), 7.58e7.60 (d, 2H), 7.88e7.91 (d, 1H), 7.98e8.02 (m, 1H),
8.10e8.14 (m,1H), 8.16e8.18 (dd,1H), 9.27e9.31 (m,1H), 9.49 (s,1H),
(b, 4H), 7.60e7.64 (t, 1H), 7.90e7.92 (d, 1H), 8.19e8.23 (t, 1H), 9.04
(b, 2H), 9.20e9.22 (d, 1H). MS: (m/z) 363 (Mþþ1). Anal. Calcd. for
C
21H22N4O2: C, 69.59; H,6.12; N, 15.46. Found: C, 69.72; H, 6.24; N,
15.57.
4.1.44. N-Cyclopropyl-N-(2-fluorobenzyl)-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (46)
11.76 (s, 1H).13C NMR (DMSO)
d: 165.29, 160.66, 158.44, 157.84,
152.95, 145.05, 141.85, 140.58, 139.35, 136.53, 128.82, 127.97, 127.41,
The reaction of 4-oxo-4H-pyrido[1,2-a]-3-carboxylic acid [14]
(32) (0.5 g, 0.26 mmol) with N-cyclopropyl-2-fluorobenzylamine
(0.434 g, 0.26 mmol) was performed as described above for com-
pound (33). The chromatographic purification of the crude product
using hexane (5%) in ethyl acetate as eluent yielded the desired
compound (46) (0.24 g, 27%), m.p. 137e40 ꢀC. Rf 0.55 (AcOEt). IR
(KBr): 3110, 1669, 1492, 1404, 1090 and 1031 cmꢁ1 1H NMR: 0.58e
0.61 (d, 4H), 3.06 (b, 1H), 4.93 (s, 2H), 7.11e7.13 (m, 1H), 7.23e7.25
(m,1H), 7.32e7.34 (m, 2H), 7.67 (b,1H), 7.80e7.82 (d,1H), 7.90e7.94
126.69, 120.64, 119.00, 118.90, 106.37, 69.81. MS: (m/z) 373 (Mþþ1).
4.1.40. N-[(2,5-Diethoxy)-4-(morpholin-4-yl)phenyl]-4-oxo-4H-
pyrido[1,2-a]pyrimidine-3-carboxamide (42)
Compound (42) was prepared by treating compound 4-oxo-4H-
pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with
2,5-diethoxy-4-morpholin-4-yl-phenylamine (0.7 g, 0.26 mmol) as
described above for compound (33). The chromatographic purifi-
cation of the crude product using methanol (1%) in ethyl acetate as
eluent provided compound (42) (0.35 g, 30.3%), m. p. 166e67 ꢀC. Rf
(m, 1H), 8.60 (s, 1H), 9.21e9.23 (d, 1H). 13C NMR (CDCl3)
d: 167.90,
162.09, 159.65, 154.40, 153.22, 150.46, 138.44, 129.77, 128.86, 127.73,
125.62, 124.35, 116.04, 115.83, 44.40, 30.25, 14.20, 8.98. MS: (m/z)
338 (Mþþ1).
0.7 (AcOEt). IR (KBr): 3263, 1685, 1487, 1260, 1199, 1113, 1043 cmꢁ1
.
1H NMR: 1.44e1.47 (t, 3H), 1.54e1.58 (t, 3H), 3.09e3.10 (t, 4H),
3.89e3.91 (t, 4H), 4.12e4.17 (m, 4H), 6.60 (s, 1H), 7.37e7.41 (m, 1H),
7.85e7.87 (d, 1H), 7.95e7.99 (m, 1H), 8.40 (s, 1H), 9.33e9.34 (d, 1H),
9.42 (s, 1H), 11.49 (s, 1H). MS: (m/z) 439 (Mþþ1).
4.1.45. N-(4-Trifluorobenzyl)-8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (47)
Compound (47) was prepared by reacting 8-methyl-4-oxo-4H-
pyrido[1,2-a]pyrimidine-3-carboxylic acid [14] (32) (0.4 g,
0.19 mmol) with 4-trifluoromethylbenzylamine (0.343 g,
0.19 mmol) as described above for compound (33). Chromato-
graphic purification of the crude product using methanol (5%) in
methylene chloride as eluent yielded the titled product (47) (0.15 g,
21.2%), m.p. 175e77 ꢀC. Rf 0.4 (AcOEt). IR (KBr): 3414, 3294, 1695,
4.1.41. N-(3,5-Dimethyl-1,2-oxazol-4-yl)-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (43)
Compound (43) was prepared by reacting 4-oxo-4H-pyrido[1,2-
a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol) with 3,5-dimethyl-
1,2-oxazol-4yl-amine (0.295 g, 0.26 mmol) as described above for
compound (33). The chromatographic purification of the crude
product using hexane (10%) in ethyl acetate as eluent offered
compound (43) (0.34 g, 45.4%), m.p. 243e45 ꢀC. Rf 0.6 (AcOEt). IR
(KBr): 3253, 3209, 1694, 1486, 1332, 1235 and 1067 cmꢁ1. 1H NMR:
2.15 (s, 3H), 2.33 (s, 3H), 7.65e7.69 (m, 1H), 7.95e7.97 (d, 1H), 8.24e
8.29 (m, 1H), 9.09 (s, 1H), 9.26e9.27 (d, 1H), 10.20 (s, 1H). 13C NMR
1634, 1545, 1330, 1117, 1015 and 793 cmꢁ1 1H NMR: 2.55 (s, 3H),
.
4.65e4.67 (d, 2H), 7.46e7.49 (dd, 1H), 7.55e7.57 (d, 2H), 7.69e7.73
(m, 3H), 9.00 (s, 1H), 9.08e9.10 (d, 1H), 9.45e9.48 (t, 1H). 13C NMR
(CDCl3) d: 164.24, 158.88, 157.33, 152.33, 151.48, 142.91, 129.55,
129.23, 128.91, 127.81, 125.59, 122.81, 120.15, 105.95, 42.87, 21.66.
(DMSO) d: 162.45, 162.12, 157.60, 157.46, 152.27, 139.99, 128.20,
MS: (m/z) 362 (Mþþ1).
126.52, 118.50, 113.76, 105.44, 99.49, 11.04, 9.49. MS: (m/z) 285
(Mþþ1).
4.1.46. 7-Chloro-4-oxo-N-[4-(trifluoromethyl)benzyl]-4H-pyrido
[1,2-a]pyrimidine-3-carboxamide (48)
4.1.42. N-[1-(4-Methoxyphenyl)ethyl]-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (44)
Compound (48) was prepared by reacting 7-chloro-4-oxo-4H-
pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g, 0.22 mmol) with
4-trifluoromethylbenzylamine (0.39 g, 0.22 mmol) as described
above for compound (33). Chromatographic purification of the
crude product using hexane (10%) in ethyl acetate as eluent
offered compound (48) (0.2 g, 23.5%), m.p. 212e14 ꢀC. Rf 0.51
(AcOEt). IR (KBr): 3315, 3091, 1674, 1620, 1532, 1498, 1323 and
4-Oxo-4H-pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g,
0.26 mmol) was reacted with
a-methyl-4-methoxybenzylamine
(0.397 g, 0.26 mmol) as described above for compound (33) Chro-
matographic purification of the crude product using methanol
(0.2%) in ethyl acetate as eluent afforded compound (44) (0.24 g,
28.2%), m.p. 173e75 ꢀC. Rf 0.6 (AcOEt) IR (KBr): 3315, 3128, 1667,
1630, 1506, 1240 and 1025 cmꢁ1. 1H NMR: 1.61e1.66 (d, 3H), 3.79 (s,
3H), 5.28e5.35 (m, 1H), 6.87e6.91(d, 2H), 7.34e7.38 (m, 3H), 7.83e
7.86 (d, 1H), 7.92e7.97 (m, 1H), 9.20e9.22 (d, 1H), 9.33e9.36 (m,
1134 cmꢁ1 1H NMR: 4.67e4.68 (d, 2H), 7.55e7.57 (d, 2H), 7.70e
.
7.72 (d, 2H), 7.92e7.95 (d, 1H), 8.26e8.29 (dd, 1H), 9.04 (s, 1H),
9.17e9.18 (d, 1H), 9.43e9.46 (t, 1H). MS: (m/z) 382.2 (Mþþ1),
384.2 (Mþþ2).
2H). 13C NMR (CDCl3)
d: 162.70, 158.63, 158.39, 157.62, 152.31,
138.27, 135.96, 127.91, 127.30, 127.03, 117.43, 113.98, 107.17, 55.27,
48.36, 22.57. MS: (m/z) 324 (Mþþ1).
4.1.47. 7-Chloro-[N-Cyclopropyl-N-(2-fluorobenzyl)]-4-oxo-4H-
pyrido[1,2-a]pyrimidine-3-carboxamide (49)
4.1.43. N-(1-Benzylpiperidin-4-yl)-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (45)
Compound (49) was prepared by reacting the 7-chloro-4-oxo-
4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid [14] (32) (0.5 g,
0.22 mmol) with N-cyclopropyl-2-fluorobenzylamine (0.367 g,
0.22 mmol) as described above for compound (33). The chro-
matographic purification of the crude product using hexane (10%)
in ethyl acetate as eluent provided compound (49) (0.28 g, 33.8%),
m.p. 209e10 ꢀC. Rf 0.48 (AcOEt). IR (KBr): 3068, 3019, 1688, 1630,
1481,1344,1223 and 1030 cmꢁ1. 1H NMR: 0.51e0.55 (d, 4H), 2.87 (b,
1H), 4.75 (b, 2H), 7.21e7.24 (m, 2H), 7.33e7.36 (m, 1H), 7.63 (b, 1H),
7.84e7.87 (d, 1H), 8.14e8.17 (dd, 1H), 8.47 (s, 1H), 9.14 (s, 1H). MS:
(m/z) 372.4 (Mþþ1), 374.3 (Mþþ2).
Compound (45) was obtained by reaction of compound 4-oxo-
4H-pyrido[1,2-a]-3-carboxylic acid [14] (32) (0.5 g, 0.26 mmol)
with 4-amino-1-benzylpiperidine (0.5 g, 0.26 mmol) as described
above for compound (33). The chromatographic purification of
the crude product using hexane (10%) in ethyl acetate as eluent
earned the titled compound (45) (0.25 g, 5%), m. p. 180e83 ꢀC. Rf
0.56 (AcOEt). IR (KBr): 3288, 1695, 1544, 1479, 1335 and
.
1061 cmꢁ1 1H NMR: 1.52e1.58 (m, 2H), 1.89e1.92 (d, 2H), 2.18e
2.22 (t, 2H), 2.70 (b, 2H), 3.50 (s, 2H), 3.88 (b, 1H), 7.26 (b, 1H), 7.33

434
U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
4.1.48. Ethyl 7-(4-methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxylate (51)
m.p. 209e10 ꢀC. Rf 0.58 (AcOEt). IR (KBr): 3425, 3293, 1689, 1623,
1333, 1263, 792, 583 and 558 cmꢁ1. 1HNMR: 3.83 (s, 3H), 4.67e4.68
(d, 2H), 7.11e7.14 (d, 2H), 7.57e7.64 (m, 2H), 7.67e7.69 (m, 1H), 7.72
(b, 1H), 7.78e7.81 (d, 2H), 7.95e7.97 (d, 1H), 8.53e8.56 (dd, 1H),
9.04 (s, 1H), 9.26 (s, 1H), 9.51e9.54 (t, 1H). MS: (m/z) 454, (Mþꢁ1).
Ethyl 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(50) (10 g, 39.6 mmol) was dissolved in dimethoxyethane
(300 ml) and 4-methoxyphenylboronic acid (6 g, 39.6 mmol), po-
tassium carbonate (8.194 g, 59.4 mmol) and tetrakis(-
triphenylphosphene)palladium(0) (0.046 g, 0.039 mmol) were
added to the above reaction mixture under N2 atmosphere. The
reaction mixture was refluxed for 36 h at 90 ꢀC. The reaction mass
was filtered and the filtrate was washed with water and extracted
using methylene chloride (3 ꢂ 100 ml), dried over anhydrous so-
dium sulfate and concentrated to get a dark brown product. Chro-
matographic purification of the crude product using ethyl acetate
(15%) in hexane as eluent offered the titled compound [(51) (3.5 g,
27.3%)], m. p.166e67 ꢀC. Rf 0.7 (50% AcOEt in hexane). IR (KBr): 3087,
1744,1609,1569,1487,1296,1188,1113,1045, 826 and 795 cmꢁ1. MS:
(m/z) 325, (Mþꢁ1).
4.1.53. N-(Tetrahydrofuran-2-ylmethyl)-7-(4-methoxyphenyl)-4-
oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide (56)
Compound [(52) (0.25 g, 0.08 mmol)] was reacted with 2-
tetrahydrofuranylmethylamine (0.085 g, 0.08 mmol) as described
above for compound (31). Chromatographic purification of the
crude product using hexane (20%) in ethyl acetate as eluent pro-
vided compound [(56) (0.1 g, 31.2%)], m.p. 176e77 ꢀC. Rf 0.61(1%
MeOH in AcOEt). IR (KBr): 3453, 3306, 1689, 1611, 1545, 1337, 1264
and 1180 cmꢁ1 1H NMR: 1.54e1.61 (m, 1H), 1.80e1.89 (m, 2H),
.
1.90e1.99 (m, 1H), 3.38e3.39 (d, 1H), 3.52e3.57 (m, 1H), 3.66e3.69
(m,1H), 3.80e3.81 (d,1H), 3.84 (s, 3H), 3.96e3.99 (m,1H), 7.12e7.15
(d, 2H), 7.79e7.84 (d, 2H), 7.91e7.97 (d, 1H), 8.54e8.56 (dd, 1H),
4.1.49. 7-(4-Methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxylic acid (52)
9.03 (s, 1H), 9.17e9.19 (t, 1H), 9.29e9.30 (d, 1H). 13C NMR (CDCl3)
d:
163.95, 160.68, 157.81, 157.60, 151.01, 137.92, 131.16, 128.21, 126.95,
126.88, 124.07, 114.98, 106.95, 77.70, 68.34, 55.46, 43.25, 28.93,
25.98. MS: (m/z) 380, (Mþꢁ1). Anal. Calcd. for C21H21N3O4: C, 66.48;
H, 5.58; N, 11.07. Found: C, 66.63; H, 5.72; N, 11.19.
A solution of [(51) (5 g, 4.3 mmol)] in conc. HCl (15 ml) was
refluxed for 2 h. The reaction mixture was cooled to RT to get a solid
precipitate. The solid so obtained was filtered, washed with ether
(100 ml) and dried to obtain 7-(4-Methoxyphenyl)-4-oxo-4H-pyr-
ido[1,2-a]pyrimidine-3-carboxylic acid (52) (3.1 g, 99.8%), m.p.
211e12 ꢀC. Rf 0.17 (AcOEt). IR (KBr): 3317, 3087, 2474, 1791, 1772,
1606, 1289 and 1187 cmꢁ1. MS: (m/z) 297, (Mþꢁ1).
4.2. Measurement of in vitro antimalarial activity of pyrido[1,2-a]
pyrimidin-4-one derivatives
The in vitro antimalarial activities of pyrido[1,2-a]pyrimidin-4-
one derivatives were measured against erythrocytic stages of
chloroquine (CQ) sensitive P. falciparum strain (Pf 3D7). Parasite
strains were cultivated by the method of Trager and Jensen [15],
with minor modifications. Parasite cultures were maintained in
fresh Oþ human erythrocytes at 4% hematocrit in complete me-
dium (RPMI1640 with 0.2% sodium bicarbonate, 0.5% Albumax,
45 mg lꢁ1 hypoxanthine, and 50 mg lꢁ1 gentamicin) at 37 ꢀC under
reduced O2 (gas mixture contains 5% O2, 5% CO2, and 90% N2). Stock
solutions of CQ were prepared in water (Milli-Q grade) and pyrido
[1,2-a]pyrimidin-4-one derivatives (stock concentrations 50 mM)
were dissolved in dimethyl sulfoxide (DMSO). All stocks were then
diluted with complete culture medium to achieve the required
concentrations. In all cases, the final concentration of DMSO was
0.4% (v/v) which was found to be nontoxic to the parasite. Com-
pounds were then placed in triplicate wells of 96-well flat-bottom
tissue culture grade plates at concentrations ranging between 0 and
4.1.50. N-(3-Fluorobenzyl)-7-(4-methoxyphenyl)-4-oxo-4H-pyrido
[1,2-a]pyrimidine-3-carboxamide (53)
Compound 7-(4-Methoxyphenyl)-4-oxo-4H-pyrido[1,2-a]py-
rimidine-3-carboxylic acid (52) (0.25 g, 0.08 mmol) and 3-
fluorobenzylamine (0.105 g, 0.08 mmol) were reacted as
described above for compound (33). Chromatographic purification
of the crude product using hexane (15%) in ethyl acetate as eluent
afforded the titled compound (53) (0.16 g, 47.0%), m. p.171e73 ꢀC. Rf
0.55 (1% MeOH in AcOEt). IR (KBr): 3445, 3319, 3072, 1688, 1611,
1533, 1482, 1336, 1290, 1264, 1184, 1143, 1060, 941, 832, 793, 691,
649 and 556 cmꢁ1. 1H NMR: 3.83 (s, 3H), 4.60e4.62 (d, 2H), 7.07e
7.16 (m, 3H), 7.20e7.22 (d, 2H), 7.37e7.42 (d, 1H), 7.79e7.81(d, 2H),
7.95e7.98 (d, 1H), 8.54e8.57 (dd, 1H), 9.04 (s, 1H), 9.27e9.28 (d,
1H), 9.45e9.48 (t, 1H). MS: (m/z) 404, (Mþꢁ1). Anal. Calcd. for
C
23H18FN3O3: C, 68.48; H, 4.50; N,10.42. Found: C, 68.66; H, 4.63; N,
10.57.
100 mM in a final well volume of 100 mL for primary screening.
4.1.51. N-(3-Trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-
4H-pyrido[1,2-a]pyrimidine-3-carboxamide (54)
Chloroquine (100 nM) was used as a positive control in all experi-
ments. Parasite culture was synchronized at ring stage with 5%
sorbitol [16]. Synchronized culture was aliquoted to drug contain-
ing 96-well plates at 2% hematocrit and 1% parasitemia. After 48 h
of incubation under standard culture conditions, the plates were
harvested and read by the SYBR Green I fluorescence-based method
[17] using a 96-well fluorescence plate reader (Victor, Perkine
Elmer), with excitation and emission wavelengths of 497 and
520 nm, respectively. The fluorescence readings were plotted
against drug concentrations, and drawing a horizontal line from the
50% value on the growth axis till it intersected with the growth
inhibition curve. Then a vertical line was drawn from the point of
intersection and projected on the concentration axis to obtain IC50
values.
Compound (52) (0.3 g, 0.10 mmol) was reacted with 3-
trifluoromethylbenzylamine (0.177 g, 0.10 mmol) as described
above for compound (33). Chromatographic purification of the
crude product using hexane (5%) in ethyl acetate as eluent offered
the desired compound (54) (0.14 g, 30.49%), m.p. 197e98 ꢀC. Rf 0.54
(AcOEt). IR (KBr): 3414, 3290, 3110, 1692, 1563, 1478, 1333, 1262,
1163, 1112, 1066, 828 and 704 cmꢁ1. 1HNMR: 3.84 (s, 3H), 4.68e4.69
(d, 2H), 7.13e7.15 (d, 2H), 7.57e7.59 (d, 2H), 7.71e7.73 (d, 2H), 7.80e
7.82 (d, 2H), 7.96e7.99 (d, 1H), 8.55e8.58 (dd, 1H), 9.04 (s, 1H),
9.28e9.29 (d, 1H), 9.52e9.55 (t, 1H). MS: (m/z) 454, (Mþꢁ1).
4.1.52. N-(4-Trifluoromethylbenzyl)-7-(4-methoxyphenyl)-4-oxo-
4H-pyrido[1,2-a]pyrimidine-3-carboxamide (55)
Compound (55) was obtained by reacting compound (52) (0.3 g,
0.10 mmol) with 4-trifluoromethylbenzylamine (0.177 g,
0.10 mmol) as described above for compound (33). Chromato-
graphic purification of the crude product using hexane (5%) in ethyl
acetate as eluent yielded the titled compound (55) (0.16 g, 45.9%),
4.3. Measurement of in vitro cytotoxic activity of pyrido[1,2-a]
pyrimidin-4-one derivatives against mammalian cell line
Mammalian cell line (HUH-7) was used to determine pyrido[1,2-
a]pyrimidin-4-one derivatives toxicity by using MTT assay for cell

U.R. Mane et al. / European Journal of Medicinal Chemistry 79 (2014) 422e435
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in complete DMEM (cDMEM) containing 10% fetal bovine serum,
0.2% sodium bicarbonate, and 50
(104 cells/200
l well) were seeded into 96-well flat-bottom tissue-
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between 0 and 200 M) were added after overnight seeding and
m
g mlꢁ1 gentamicin. Briefly, cells
m
m
incubated for 24 h in a humidified atmosphere at 37 ꢀC and 5% CO2.
DMSO (final concentration 10%) was added as positive control. An
aliquot of a stock solution of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) (5 mg mLꢁ1 in 1ꢂ
phosphate-buffered saline) was added at 20 ml per well and incu-
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5 min, the supernatant was removed and 100 l of the stop agent
m
DMSO was added to the well to dissolve the formazan crystals.
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96-well plate reader (Versa Max), and IC50 values were determined
by analysis of doseeresponse curves as indicated earlier. The
therapeutic index was calculated as IC50 (mammalian cell)/IC50 (Pf
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Appendix A. Supplementary data
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