Pyrido[1,2-a]pyrimidin-4-ones as antiplasmodial falcipain-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.09.008

Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is a promising target for antimalarial chemotherapy and inhibition of this protease affects the growth of parasite adversely. A series of pyrido[1,2-a]pyrimidin-4-ones were synthesized and evaluat

Full text of DOI:10.1016/j.bmc.2012.09.008

Bioorganic & Medicinal Chemistry 20 (2012) 6296–6304
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrido[1,2-a]pyrimidin-4-ones as antiplasmodial falcipain-2 inhibitors
U. R. Mane a,b, H. Li c, , J. Huang c, , R. C. Gupta b, S. S. Nadkarni b, R. Giridhar a, P. P. Naik a, M. R. 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 East China University of Science and Technology, 130 Meilong Road, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2012
Revised 4 September 2012
Accepted 6 September 2012
Available online 13 September 2012
Plasmodium falciparum cysteine protease falcipain-2 (FP-2) is a promising target for antimalarial
chemotherapy and inhibition of this protease affects the growth of parasite adversely. A series of
pyrido[1,2-a]pyrimidin-4-ones were synthesized and evaluated for their in vitro FP-2 inhibitory poten-
tial. Compounds (14,17) showed excellent FP-2 inhibition and can serve as lead compounds for further
development of potent FP-2 inhibitors as potential antimalarial drugs.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pyrido[1,2-a]pyrimidin-4-one
Cysteine protease
Falcipain-2 inhibitor
Malaria
1. Introduction
Hemoglobin degradation is essential to the survival of the par-
asite, as interruption of this process with a variety of protease
Parasite born diseases were having extirpating impact on
human civilizations. However, none of the parasitic diseases has
had as an enormous impact on humans as that of malaria, which
is one of the major causes of morbidity and mortality of human civ-
ilizations in the past, and even today, it remains one of the deadli-
est diseases on the planet.1 Malaria, particularly the one caused by
Plasmodium falciparum, remains a serious health problem in Africa,
South America and many parts of Asia.2 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.3
An estimated 600 million people are at risk of contracting ma-
laria infection and the disease kills more than 1 million children
each year as well as a large number of pregnant women. About
90% of these casualties occur in tropical Africa and the great major-
ities are children under the age of five. Exacerbated by poverty,
malaria exerts a high toll on populations that can least afford a
cure.4 Despite the toll of tropical parasitic diseases on human life
in the developing world, present therapies still rely on drugs that
were developed decades ago. The P. falciparum genome sequencing
has revealed a number of new targets for drug and vaccine devel-
opment. Development of newer antimalarial drugs only remains an
economically and environmentally viable alternative to fight out
the menace of the disease.5
inhibitors leads to parasite’s death.6 The hemoglobin degradation
pathway involving several parasite specific enzymes like plasmep-
sins and falcipains plays an important role in providing food to the
parasite.7 One possibility to strike against the parasite is to inhibit
an enzyme within this pathway, interrupting the nutrition source
and therefore eliminating the parasite by starvation.8 Erythrocytic
malaria parasite degrades hemoglobin as the principal source of
amino acids for parasite protein synthesis. One of the potential
targets for antimalarial chemotherapy includes an enzyme in-
volved in the processing of hemoglobin that is, cysteine protease
enzyme FP-2.9
Very few peptides and peptidomimetic compounds have been
reported to exhibit FP-2 inhibitory activity in the literature other
than some chalcones and semicarbazones. 2-(3,4-Dihydro-4-oxo-
thieno[2,3-d]pyrimidin-2-ylthio)acetamides10 and dihydroarte-
misinin based thiosemicarbazones11 were reported as FP-2
inhibitors. Some 4H-pyrido[1,2-a]pyrimidin-4-ones12 have been
reported earlier in the patent literature for antimalarial and anti-
inflammatory activity and 2-chloro-4H-pyrido[1,2-a]pyrimidin-4-
one has been used as a synthone for the synthesis of some new
polycyclic heteroaromatic compounds for antimalarial activity.13
Herein we report some pyrido[1,2-a]pyrimidin-4-ones as
potential FP-2 inhibiting antimalarials. It has been tried to incorpo-
rate the pharmacophoric features of
a,b-unsaturated system of
chalcones14 and of semicarbazones/thiosemicarbazones15 as urea/
amide/carbamates in the designed compounds. The compounds
so designed have been synthesized and evaluated for inhibition
of FP-2 enzyme (see Fig. 1).
⇑
Corresponding author. Tel.: +91 265 2434187; fax: +91 265 2418927.
E-mail address: mryadav11@yahoo.co.in (M.R. Yadav).
Tel./fax: +86 21 64253681.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.008

U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
6297
O
N
S
R
X
A
R1
R2
N
R
R
N
N
H
N
H
O
R1
X = Urea, carbamate, amide
A = Alkyl/aryl/heteroaryl
Pyrido[1,2-a]pyrimidin-4-ones
Thiosemicarbazones
Chalcones
Figure 1. FP-2 inhibiting scaffolds
cysteine proteases (papain family), namely E-64 was used as posi-
tive control. Assays were performed to determine the percentage
inhibition of the enzyme at a concentration of 10 lM. FP-2 activity
2. Results and discussion
2.1. Chemistry
is assessed by cleavage of the fluorogenic substrate Cbz-Phe-Arg-
AMC releasing the fluorescent AMC group. Hence decrease in
fluorescence intensity in a sample represents inhibition of enzyme
activity. The IC50 values were determined for those compounds
The targeted compounds were prepared by adopting synthetic
strategies outlined in Schemes 1–3. As depicted in Scheme 1
substituted 2-aminopyridines (1a–c) by Gould and Jacob coupling
reaction with diethyl ethoxymethylenemalonate yielded (2a–c),
which on cyclization by refluxing in diphenyl ether resulted
into pyrido[1,2-a]pyrimidin-4-one monoesters (3a–c). The esters
(3a–c) on acid catalyzed hydrolysis offered free acids (4a–c). The
acids (4a–c) when reacted with ethylchloroformate in presence
of triethylamine yielded anhydrides which on further in situ reac-
tion with sodium azide yielded the azides (5a–c). The azides (5a–c)
offered isocyanate derivatives (6a–c) (Scheme 1) by refluxing them
in toluene.
only which showed more than 40% inhibition at 10
concentration.
lM
The enzyme inhibition data given in Table 1 shows that the pyr-
ido[1,2-a]pyrimidin-4-ones show FP-2 inhibition in micromolar
range. Based on these results a broad structure–activity relation-
ship could be deduced for this series of compounds. Urea moiety
has the potential to provide potent FP-2 inhibitors as the carba-
mate and carboxamide derivatives are much less active in
comparison to the disubstituted ureas.
Urea derivatives of ethylmorpholino (17, IC50
methoxyethyl (14, IC50 M) proved to be the most potent com-
pounds among the whole lot. 2-Thiophenethyl (12, IC50 14
16, IC50 21 M; 18, IC50 23 M), acetamidoethyl (10, IC50 16
and 4-methoxyphenylpiperazine (22, IC50 19
7 lM) and
The ureas (7–26) (Scheme 2) were prepared by coupling of suit-
able isocyanate (6a–c) with amine derivatives in toluene under
reflux conditions following the procedure as depicted in Scheme 2.
The carbamates (27–29) were synthesized by coupling of suitable
isocyanate (6a–c) with aryl alcohols in toluene under reflux
conditions. The amides (30–32) (Scheme 3) were prepared by
EDC coupling of a suitable acid (4a–c) with amine derivatives.
6
l
l
l
M;
M)
l
l
l
M) were the other
groups which gave active compounds when attached to one end
of urea moiety. 2-Pyridylpiperazine is another moiety which
offered quite active compounds (24, IC50 12 lM; 19, IC50 15 lM).
In general 8-methyl and 7-chloro substituted derivatives of-
fered more potent compounds indicating that increasing the steric
size of the group attached to the B-ring (i.e., pyrido) is likely to pro-
vide more potent FP-2 inhibitors. An electron rich environment at
the end of the carbon chain attached to the urea nitrogen also
seems to be conducive for better activity as indicated by the com-
pounds having morpholino, 2-thiophenenyl, 2-pyridyl, 4-methoxy-
phenyl, acetamidoethyl and 2-methoxyethyl groupings. But bulky
(23, benzhydrylpiperazine) and (8, 4-morpholinopropyl) groupings
may not offer potent derivatives.
2.2. Biology
Compounds (7–32) were evaluated for their inhibitory activity
against recombinant FP-2 using Cbz-Phe-Arg-AMC as fluorogenic
substrate.9 Preliminary screening was performed at 10
lM concen-
tration. An equivalent concentration of DMSO was used as negative
control and the irreversible standard inhibitor of clan CA family C1
H3C
O
O
O
N
R
R
N
i
O
3. Conclusion
NH2
1 (a-c)
N
H
CH3
There is a need for development of new drug molecules for the
control of malaria parasite as the parasite is developing resistance
to the older drugs. Inhibition of FP-2 could be an efficient strategy
to kill the parasite by starvation due to the blockade of catabolic
chain, essential for obtaining growth nutrients. In this regards we
planned to develop parasitic-enzyme-specific inhibitors. For
identification of FP-2 inhibitors, we have synthesized thirty two
pyrido[1,2-a]pyrimidin-4-one derivatives, which showed good
2 (a-c)
ii
a. R = H, b. R = 4-CH3, c. R = 5-Cl
O
O
O
O
R
R
iii
N
OH
N
O
O
N
N
CH3
3 (a-c)
4 (a-c)
iv
FP-2 inhibitory potential. The compounds like (10, IC50 16
(12, IC50 14 M), (14, IC50 M), (16, IC50 21 M), (17, IC50
M), (18, IC50 23 M), (19, IC50 15 M), (22, IC50 19 M), (24,
IC50 12 M) and (29, IC50 18 M) are showing excellent FP-2 inhi-
lM),
l
6
l
l
O
N
O
N
R
7
l
l
l
l
N
N
O
R
v
N+
N
N
l
l
N
bition. Based on the activity data of the synthesized compounds,
pyrido[1,2-a]pyrimidin-4-one can serve as a lead series for future
investigations. By appropriate structural optimizations the potency
of this series of compounds can be improved substantially against
FP-2 enzyme which in turn can serve to be potential antimalarial
agents.
5 (a-c)
6 (a-c)
a. R = H, b. R = 8-CH3, c. R = 7-Cl
Scheme 1. Reagents and conditions: (i) EMME, reflux (ii) DPE, reflux (iii) HCl, reflux
(iv) (a) ECF, TEA, DMF, 0 °C (b). NaN3, H2O, 0 °C (v) toluene, reflux.

6298
U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
O
H
N
H
N
X = H2N-A
R
A
N
7. R = H, A = CH2-(4-PhCl)
O
8. R = H, A = (CH2)3-(4-morpholine)
9. R = H, A = (CH2)2-OCH3
10. R = H, A = (CH2)2-NHCOCH3
11. R = H, A = CH2-(4-PhOCH3)
N
(7-18)
12. R = H, A = (CH2)2-(2-thiophene)
13. R = 8-CH3, A = (CH2)2-(4-morpholine)
14. R = 8-CH3, A = (CH2)2-OCH3
15. R = 8-CH3, A = (CH2)2-NHCOCH3
16. R = 8-CH3, A = (CH2)2-(2-thiophene)
17. R = 7-Cl, A = (CH2)2-(4-morpholine)
18. R = 7-Cl, A = (CH2)2-(2-thiophene)
O
N
O
N
X= HN
N A
H
N
N
O
R
R
A
N
N
N
i
N
19. R = H, A = 2-Pyridyl
20. R = H, A = 4-PhOCH3
O
21. R = 8-CH3, A = 2-Pyridyl
22. R = 8-CH3, A = 4-PhOCH3
23. R = 8-CH3, A = CH(Ph)2
(19-25)
24. R = 7-Cl, A = 2-Pyridyl
25, R = 7-Cl, A = 4-PhOCH3
O
N
H
A
R
O
N
X = HO-A
26. R = H, A = CH2Ph
O
27. R = H, A = CH2-(2-Pyridine)
28. R = 8-CH3, A = CH2Ph
29. R = 8-CH3, A = CH2-(2-Pyridine)
N
(26-29)
Scheme 2. Reagents and conditions: (i) X, toluene, reflux.
O
N
O
O
O
R
i
R
N
H
A
N
N
OH
30. R = H, A = 3,4-MethylenedioxyBn
31. R = 8-CH3, A = CH2-(4-PhF)
32. R = 7-Cl, A = 3,4-MethylenedioxyBn
N
4
30-32
Scheme 3. Reagents and conditions:(i) X, EDC, HOBT, DIEA, MDC, 0 °C, RT.
CH2), 7.14–7.17 (m, 1H, Ar-H), 7.39–7.41 (d, 1H, Ar-H), 7.79–7.83
4. Experimental work
4.1. Chemistry
(m, 1H, Ar-H), 8.36–8.37 (d, 1H, Ar-H), 9.05–9.08 (d, 1H, –NH–
CH@C), 10.79–10.82 (d, 1H, N–H). MS: (m/z) 265 (M++1).
4.1.2. Ethyl 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(3a)
All the reagents and solvents required for synthesis were puri-
fied by general laboratory techniques before use. Compounds were
purified by passing them through silica gel H (100–200 mesh) puri-
fying column using mixture of ethyl acetate and hexane or metha-
nol as eluent. Melting points were determined using a Labindia
make melting point apparatus (heating blok type) and are uncor-
rected. Purity of the compounds and completion of reactions 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 NMR spectra (on a Bruker 400 MHz spectrometer) were
recorded in DMSO-d6 (chemical shifts in d ppm). The assignments
of exchangeable protons were confirmed by the D2O exchange
studies wherever required. Mass spectral data was obtained on a
Waters Micromass ESCi spectrometer. Elemental analyses were
performed on Perkin-Elmer/Carlo-Erba elemental analyzer.
The pyridine derivative (2a) (13 g, 5.9 mmol) was refluxed in di-
phenyl ether (100 ml) using air condenser for 8 h in a two-neck
round-bottom flask (250 ml). The reaction mixture was cooled to
RT and added hexane (500 ml) in to it to get a solid precipitate.
The precipitated solid was filtered, washed with hexane and dried
to obtain compound (3a) (10 g, 93.1%), mp 111–13 °C (Lit.16 110–
11 °C). Rf 0.48 (EtOAc). IR (KBr): 1730, 1482, 1288, 1227 and
1104 cmꢀ1. MS: (m/z) 219 (M++1).
4.1.3. 4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4a)
A solution of the ester (3a) (10 g, 5.2 mmol) in conc. HCl (15 ml)
was refluxed for 6 h. The reaction mixture was cooled to RT to get a
solid precipitate. The solid precipitate was filtered, washed with
ether and dried to get compound (4a) as a dark brown solid (8 g,
85.5%), mp. 268 °C (decomp.) [Lit.16 265 °C (decomp.)]. Rf 0.1
(EtOAc). IR (KBr): 3091, 1757, 1677, 1636, 1519, 1483, 1275,
1247 cmꢀ1 1H NMR: 7.67–7.71 (m, 1H, Ar-H), 7.97–8.00 (d, 1H,
.
4.1.1. Diethyl 2-pyridylaminomethylenemalonate (2a)
Ar-H), 8.31–8.35 (m, 1H, Ar-H), 8.96 (s, 1H, Ar-H), 9.22–9.23 (d,
A mixture of 2-aminopyridine (1a) (10 g, 10.6 mmol) and diethyl
ethoxymethylenemalonate (24.47 g, 10.6 mmol) was refluxed for
2 h. The reaction mixture was cooled to RT to get (2a) as a light
brown solid (14 g, 50%), mp 68–69 °C (Lit.16,17 67.5–68 °C). Rf 0.85
1H, Ar-H). MS: (m/z) 191 (M++1).
4.1.4. 4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonylazide (5a)
In a two-neck round-bottom flask (250 ml), compound (4a) (8 g,
4.2 mmol) and triethylamine (8.5 g, 8.4 mmol) were dissolved in
(EtOAc). IR (KBr): 3274, 1686, 1649, 1601, 1248 cmꢀ1 1H NMR:
.
1.22–1.29 (m, 6H, CH3), 4.12–4.18 (q, 2H, CH2), 4.20–4.25 (q, 2H,

U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
6299
Table 1
Inhibition of FP-2 in percentage and IC50 compounds of 7–32
O
N
R
X
A
N
Sr. No.
7
R
X
A
Falcipain-2 inhibition (%)
39
IC50
nd
(lM)
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
Cl
O
N
8
9
H
25
31
46
18
44
27
63
15
45
68
42
46
43
37
46
nd
nd
16
nd
14
nd
6
N
H
N
H
O
CH3
H
N
H
N
H
H
N
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
H
N
H
N
H
O
S
H
CH3
N
H
N
H
O
O
H
N
H
N
H
N
O
8-CH3
8-CH3
8-CH3
8-CH3
7-Cl
7-Cl
H
N
H
N
H
CH3
N
H
N
H
H
N
CH3
nd
21
7
N
H
N
H
O
S
N
H
N
H
O
N
N
H
N
H
23
15
nd
nd
19
S
N
H
N
H
N
N
N
N
N
N
N
N
N
N
N
N
H
O
CH3
H
N
H
8-CH3
8-CH3
N
N
H
O
CH3
N
H
N
H
23
8-CH3
23
nd
O
O
24
25
7-Cl
7-Cl
41
36
12
nd
N
N
N
N
N
N
H
O
CH3
N
H
(continued on next page)

6300
U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
Table 1 (continued)
Sr. No.
R
X
A
Falcipain-2 inhibition (%)
12
IC50
nd
(lM)
O
O
O
O
26
27
28
29
H
N
H
O
H
18
31
42
nd
nd
18
N
N
N
H
O
O
O
8-CH3
8-CH3
N
H
N
H
O
O
O
30
31
32
H
7
nd
nd
nd
N
O
O
8-CH3
7-Cl
6
N
N
F
O
24
O
33
34
E-64
DMSO
—
—
—
—
92(2
0
l
M)
0.02
—
nd = not determined
DMF (25 ml). The reaction mixture was cooled to 0 °C. Ethyl chlo-
roformate (5.03 g, 4.6 mmol) was added to the reaction mixture
and stirred for 15 min followed by addition of aqueous solution
of sodium azide (10.95 g, 16.8 mmol) resulting into a yellow solid
precipitate. The solid precipitate was filtered, washed with cold
water; hexane and then air dried affording the compound (5a) as
brown solid (6.8 g, 75.1%) mp 130–31 °C. Rf 0.28 (EtOAc) IR
4.1.8. 8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (4b)
A solution of the ester (3b) (10 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 get 8-methyl-4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-carboxylic acid (4b) (8.5 g, 96.7%), mp 225 °C
[Lit.18 223 °C]. Rf 0.11(EtOAc). IR (KBr): 2645, 1764, 1696, 1620,
(KBr): 2310, 1725, 1472, 1185, 1012 and 787 cmꢀ1 1H NMR:
.
7.61–7.64 (m, 1H, Ar-H), 7.87–7.91 (d, 1H, Ar-H), 8.24–8.35 (m,
1H, Ar-H), 8.94 (s, 1H, Ar-H), 9.10–9.28 (d, 1H, Ar-H). MS: (m/z)
214 (M+ꢀ1).
1522, 1393, 1268, 1204 and 1152 cmꢀ1 1H NMR: 2.62 (s, 3H,
.
CH3), 7.62–7.64 (d, 1H, J = 8.0 Hz, Ar-H), 7.89 (s, 1H, Ar-H), 8.90
(s, 1H, Ar-H), 9.14–9.16 (d, 1H, J = 8.0 Hz, Ar-H), 10.9 (b, 1H, COOH).
MS: (m/z) 205 (M++1).
4.1.5. 3-Isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one (6a)
The acid azide (5a) (4 g, 1.8 mmol) in toluene was refluxed for
2 h. Work up of the reaction mixture was done by removing
toluene under vacuum to get a light brown solid compound (6a)
(3.2 g, 40.6%), mp 130–31 °C. Rf 0.5 (EtOAc). IR (KBr): 2300,
1687, 1650, 1575, 1550 1490 and 1190 cmꢀ1. MS: (m/z) 188
(M++1).
4.1.9. 8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carbonylazide (5b)
Compound (5b) was prepared by reacting the acid (4b) (8 g,
3.9 mmol) with ethyl chloroformate (4.68 g, 4.3 mmol) and sodium
azide (10.19 g, 15.7 mmol) as described above for compound (5a)
yielding the compound (5b) (7 g, 77.9%), mp 148–50 °C. Rf 0.35
(EtOAc). IR (KBr): 2142, 1712, 1640, 1569, 1485, 1285 and
4.1.6. Diethyl(4-methyl-2-pyridylamino)methylene-malonate
(2b)
1172 cmꢀ1 1H NMR: 2.58 (s, 3H, CH3), 7.49–7.51 (d, 1H,
.
J = 8.0 Hz, Ar-H), 7.75 (s, 1H, Ar-H), 8.83 (s, 1H, Ar-H), 9.09–9.11
4-Methyl-2-aminopyridine (10 g, 9.25 mmol) was reacted with
diethyl ethoxymethylenemalonate (21.29 g, 9.25 mmol) as de-
scribed above for compound (2a), affording (2b) (15 g, 58.2%),
mp 73–74 °C (Lit.16 72–73 °C). Rf 0.9 (EtOAc). IR (KBr): 1731,
1682, 1648, 1605, 1548, 1374 and 1218 cmꢀ1. MS: (m/z) 279
(M++1).
(d, 1H, J = 8.0 Hz, Ar-H). MS: (m/z) 230 (M++1).
4.1.10. 3-Isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-
one (6b)
The azide (5b) (4 g) was refluxed in toluene for 2 h. The reaction
mixture was cooled to RT and the solvent was removed under va-
cuo to get 3-isocyanato-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-
one (6b) (3.34 g, 95.1%), mp 234–35 °C. Rf 0.61 (EtOAc). IR (KBr):
2223, 1670, 1639, 1482, 811 and 764 cmꢀ1. MS: (m/z) 202 (M++1).
4.1.7. Ethyl 8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxylate (3b)
In analogy to compound (3a), (2b) (10 g, 4.31 mmol) was
refluxed in diphenylether to obtain (3b) (10 g, 88.4%), mp 174–
76 °C (Lit.16 171–72 °C). Rf 0.55 (EtOAc). IR (KBr): 1689, 1639,
4.1.11. Diethyl (5-chloro-2-pyridylamino)methylene malonate
(2c)
1556, 1501, 1284 and 1143 cmꢀ1
.
1H NMR: 1.28–1.32 (t, 3H,
Compound (2c) was prepared by reacting the compound (1c)
(10 g, 7.8 mmol) with diethyl ethoxymethylenemalonate (17.96 g,
7.8 mmol) as described above for compound (2a), affording the
compound (2c) (15 g, 42.9%), mp 132–34 °C (Lit.19 131–32 °C). Rf
0.91 (EtOAc). IR (KBr): 3270, 1730, 1676, 1644, 1588, 1552, 1465,
J = 7.2 Hz, CH3), 2.51 (s, 3H, CH3), 4.23–4.29 (q, 2H, J = 7.2 Hz,
CH2), 7.42–7.44 (d, 1H, J = 7.2 Hz, Ar-H), 7.67 (s, 1H, Ar-H), 8.82
(s, 1H, Ar-H), 9.04–9.05 (d, 1H, J = 7.2 Hz, Ar-H). MS: (m/z) 233
(M++1).

U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
6301
1395, 1219 and 1007 cmꢀ1. 1H NMR: 1.22–1.29 (m, 6H, CH3), 4.13–
4.26 (m, 4H, CH2), 7.47–7.49 (d, 1H, J = 8.0 Hz, Ar-H), 7.91–7.93 (d,
1H, J = 8.0 Hz, Ar-H), 8.41 (s, 1H, Ar-H), 8.94–8.97 (d, 1H,
J = 12.0 Hz, –NHCH@C), 10.79–10.82 (d, 1H, J = 12.0 Hz, N–H). MS:
(m/z) 299 (M++1).
6H, NCH2), 3.10–3.15 (m, 2H, CH2), 3.56–3.58 (t, 4H, J = 4.6 Hz,
OCH2), 7.01–7.04 (t, 1H, J = 7.4 Hz, N–H), 7.23–7.27 (m, 1H, Ar-H),
7.60–7.63 (d, 1H, J = 8.8 Hz, Ar-H), 7.67–7.71 (m, 1H, Ar-H), 8.29
(s, 1H, N–H), 8.84–8.86 (d, 1H, J = 6.8 Hz, Ar-H), 9.17 (s, 1H,
Ar-H). MS: (m/z) 332 (M++1).
4.1.12. Ethyl 7-chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxylate (3c)
4.1.18. 1-(2-Methoxyethyl)-3-(4-oxo-4H-pyrido[1,2-
a]pyrimidin-3-yl)urea (9)
With a procedure similar to that adopted for compound (3a)
compound (3c) was prepared from compound (2c) (15 g,
5.03 mmol) affording the titled compound (3c) (10 g, 78.8%), mp
146–47 °C (Lit.19 147–48 °C). Rf 0.54 (EtOAc). IR (KBr): 1681,
1645, 1591, 1552, 1466, 1366, 1222 and 1091 cmꢀ1. MS: (m/z)
253 (M++1).
Compound (9) was prepared by reacting the isocyanate (6a)
(0.3 g,
0.16 mmol)
with
2-methoxyethylamine
(0.119 g,
0.16 mmol) as previously described for compound (7). Chromato-
graphic purification of the crude product using methanol (0.9%)
in ethyl acetate as eluant yielded the compound (9) (0.5 g,
61.1%), mp 195–97 °C. Rf 0.65 (1% MeOH in EtOAc). IR (KBr):
3362, 1696, 1639, 1548, 1463, 1226, 1192 and 1099 cmꢀ1
.
1H
4.1.13. 7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxylic acid (4c)
NMR: 3.25–3.28 (m, 5H, NCH2 & OCH3), 3.38–3.39 (t, 2H,
J = 5.2 Hz, OCH2), 7.15–7.18 (t, 1H, J = 5.4 Hz, N–H), 7.23–7.27 (m,
1H, Ar-H), 7.60–7.63 (d, 1H, J = 9.2 Hz, Ar-H), 7.67–7.72 (m, 1H,
Ar-H), 8.41 (s, 1H, N–H), 8.84–8.86 (d, 1H, J = 6.8 Hz, Ar-H),
9.16(s, 1H, Ar-H). MS: (m/z) 263 (M++1).
In analogy to compound (4a), (4c) was obtained by employing
the same procedure using (3c) (10 g, 3.9 mmol) to get 7-chloro-
4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid (4c) (8 g,
89.9%), mp 256–57 °C. Rf 0.12 (EtOAc). IR (KBr): 3063, 1759,
1661, 1612, 1510, 1418, 1263 and 1163 cmꢀ1. MS: (m/z) 225
(M++1).
4.1.19. N-{2-[3-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
ureido]ethyl}acetamide (10)
In analogy to compound (7), (10) was obtained by refluxing the
isocyanate (6a) (0.3 g, 0.16 mmol) with 2-acetamidoethylamine
(0.62 g, 0.16 mmol). Chromatographic purification of the crude
product using methanol (0.9%) in ethyl acetate as eluant offered
compound (10) (0.5 g, 61.1%), mp 247–49 °C. Rf 0.45 (5% MeOH
in EtOAc). IR (KBr): 3317, 3251, 1726, 1633, 1536, 1467, 1219
4.1.14. 7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carbonylazide (5c)
Compound (5c) was prepared by reacting compound (4c) (8 g,
6.25 mmol) with ethyl chloroformate (6.78 g, 6.87 mmol) and so-
dium azide (16.25 g, 25 mmol) as described above for compound
(5a) affording the compound (5c) (6.5 g, 74.2%), mp 158–60 °C. Rf
0.25 (EtOAc). IR (KBr): 2158, 1732, 1563, 1472, 1296, 1201 and
1023 cmꢀ1. MS: (m/z) 250 (M++1).
and 1120 cmꢀ1 1H NMR: 1.81 (s, 3H, CH3), 3.11–3.16 (m, 4H,
.
NCH2), 7.09–7.11 (t, 1H, J = 5.0 Hz, N–H), 7.23–7.27 (m, 1H, Ar-H),
7.61–7.63 (d, 1H, J = 8.8 Hz, Ar-H), 7.68–7.72 (m, 1H, Ar-H), 7.94
(b, 1H, N–H), 8.35 (s, 1H, N–H), 8.84–8.86 (d, 1H, J = 7.2 Hz,
Ar-H), 9.17 (s, 1H, Ar-H). MS: (m/z) 290 (M+ꢀ1).
4.1.15. 7-Chloro-3-isocyanato-4H-pyrido[1,2-a]pyrimidin-4-one
(6c)
(5c) (4 g, 1.8 mmol) was refluxed in toluene for 2 h. The reaction
mixture was concentrated to get 7-chloro-3-isocyanato-4H-pyr-
ido[1,2-a]pyrimidin-4-one (6c) (3.34 g, 98.5%), mp 312–13 °C. Rf
0.65 (EtOAc). IR (KBr): 2243, 1668, 1625, 1547, 1525, 1475 and
1182 cmꢀ1. MS: (m/z) 222 (M++1).
4.1.20. 1-(4-Methoxybenzyl)-3-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)urea (11)
Compound (11) was prepared by treating the isocyante (6a)
(0.3 g, 0.16 mmol) with 4-methoxybenzylamine (0.172 g,
0.16 mmol) as described above for compound (7). The crude prod-
uct was purified by column chromatography using methanol (1%)
in ethyl acetate as eluant offering the compound (11) (0.15 g,
51.9%), mp 228–30 °C. Rf 0.68 (1% MeOH in EtOAc). IR (KBr):
4.1.16. 1-(4-Chlorobenzyl)-3-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)urea (7)
The isocyanate (6a) (0.5 g, 0.27 mmole) and 4-chlorobenzyl-
amine (0.227 g, 0.16 mmol) in toluene (25 ml) were refluxed
together for 6 h. The reaction mixture was cooled to RT to get a so-
lid precipitate. The solid precipitate was filtered and washed with
hexane. The product was purified by column chromatography
using ethyl acetate (80%) in hexane as eluant to get the titled com-
pound (7) (0.2 g, 37.9%), mp 250–51 °C. Rf 0.6 (EtOAc). IR (KBr):
3361, 3277, 1639, 1531, 1242, 1220, 1180 and 1122 cmꢀ1 1H
.
NMR: 3.73 (s, 3H, OCH3), 4.24–4.25 (d, 2H, J = 6 Hz, CH2), 6.89–
6.91 (d, 2H, J = 8.8 Hz, Ar-H), 7.22–7.27 (m, 3H, N–H & Ar-H),
7.40–7.43 (m, 1H, Ar-H), 7.61–7.63 (d, 1H, J = 9.2 Hz, Ar-H), 7.68–
7.72 (m, 1H, Ar-H), 8.39 (s, 1H, N–H), 8.84–8.86 (d, 1H, J = 7.2 Hz,
Ar-H), 9.18 (s, 1H, Ar-H). MS: (m/z) 325 (M++1).
3318, 1693, 1637, 1536, 1479, 1220 and 1136 cmꢀ1
.
1H NMR:
4.1.21. 1-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-
thiophen-2-ylethyl)urea (12)
4.39–4.41 (d, 2H, J = 6.0 Hz, CH2), 7.26–7.35 (m, 3H, N–H & Ar-H),
7.41–7.56 (m, 3H, Ar-H), 7.61–7.64 (d, 1H, J = 9.2 Hz, Ar-H), 7.68–
7.71 (m, 1H, Ar-H), 8.55 (s, 1H, N–H), 8.85–8.87 (d, 1H, J = 7.2 Hz,
Ar-H), 9.16 (s, 1H, Ar-H). MS: (m/z) 328.9 (M+).
A mixture of isocyanate (6a) (0.3 g, 0.16 mmol), 2-(2-thio-
phenyl)ethylamine (0.204 g, 0.16 mmol) and toluene was refluxed
for 6 h. The reaction mixture was cooled to RT to get solid precip-
itate. The solid precipitate so obtained was filtered and the crude
product was purified by column chromatography using methanol
(2%) in ethyl acetate as eluant to get the compound (12) (0.2 g,
39.6%), mp 216–19 °C. Rf 0.5 (1% MeOH in EtOAc). IR (KBr): 3213,
4.1.17. 1-[3-(Morpholin-4-yl)propyl]-3-(4-oxo-4H-pyrido [1,2-
a]pyrimidin-3-yl)urea (8)
A mixture of (6a) (0.5 g, 0.27 mmol), 3-(4-morpholino)propyl-
amine (0.227 g, 0.16 mmol) and toluene (25 ml) was refluxed for
6 h to get a solid precipitate. The solid precipitate was filtered
and washed with hexane. The crude product was purified by
column chromatography using ethyl acetate (90%) in hexane as
eluant to obtain the titled compound (8) (0.18 g, 40.6%), mp 188–
90 °C. Rf 0.67 (EtOAc). IR (KBr): 3297, 1663, 1549, 1479, 1228
3033, 1656, 1525, 1469, 1218, 1182, 1127, 758 and 695 cmꢀ1 1H
.
NMR: 2.97–3.01 (t, 2H, J = 6.6 Hz, CH2), 3.37–3.40 (m, 2H, CH2),
6.92–6.93 (d, 1H, J = 2.4 Hz, Ar-H), 6.97–6.99 (m, 1H, Ar-H), 7.15–
7.18 (t, 1H, J = 5.4 Hz, N–H), 7.23–7.27 (m, 1H, Ar-H), 7.35–7.37
(d, 1H, J = 5.2 Hz, Ar-H), 7.61–7.63 (d, 1H, J = 8.8 Hz, Ar-H), 7.68–
7.72 (m, 1H, Ar-H), 8.41 (s, 1H, N–H), 8.84–8.85 (d, 1H, J = 7.2 Hz,
Ar-H), 9.19 (s, 1H, Ar-H). MS: (m/z) 315 (M++1).
and 1118 cmꢀ1 1H NMR: 1.55–1.62 (m, 2H, CH2), 2.28–2.34 (m,
.

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U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
4.1.22. 1-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-
[2-(morpholin-4-yl)ethyl]urea (13)
1H NMR: 2.56 (b, 6H, NCH2), 3.24–3.27 (m, 2H, CH2), 3.60 (b, 4H,
OCH2), 7.08 (b, 1H, N–H), 7.63–7.65 (d, 1H, J = 9.6 Hz, Ar-H),
7.70–7.73 (d, 1H, J = 9.6 Hz, Ar-H), 8.57 (s, 1H, N–H), 8.84 (s, 1H,
Ar-H), 9.17 (s, 1H, Ar-H). MS: (m/z) 352 (M++1).
The isocyanate derivative (6b) (0.3 g, 0.149 mmol) was reacted
with 2-(4-morpholino)ethylamine (0.163 g, 0.149 mmol) as de-
scribed above for compound (7). Chromatographic purification of
the crude product using methanol (0.5%) in ethyl acetate as eluant
offered the compound (13) (0.21 g, 42.5%), mp 225–26 °C. Rf 0.64
(1% MeOH in EtOAc). IR (KBr): 3334, 1632, 1569, 1460, 1385,
4.1.27. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-
(2-thiophen-2-ylethyl)urea (18)
Compound (18) was obtained by treating the compound (6c)
(0.3 g, 0.135 mmol) with 2-(2-thiophenyl)ethylamine (0.17 g,
0.135 mmole) as described above for compound (7). The chromato-
graphic purification of the crude product using ethyl acetate (90%)
in hexane as eluant yielded the titled compound (18) (0.24 g,
50.8%), mp 215–17 °C. Rf 0.75 (1% MeOH in EtOAc). IR (KBr):
1235, 1104 and 1002 cmꢀ1 1H NMR: 2.37–2.41 (m, 9H, CH3
. &
NCH2), 3.21–3.24 (m, 2H, CH2), 3.58–3.59 (m, 4H, CH2), 6.99 (b,
1H, N–H), 7.11–7.12 (d, 1H, J = 7.2 Hz, Ar-H), 7.42 (s, 1H, Ar-H),
8.37 (s, 1H, N–H), 8.76–8.78 (d, 1H, J = 7.2 Hz, Ar-H), 9.09 (s, 1H,
Ar-H) MS: (m/z) 332 (M++1).
3387, 1651, 1544, 1510, 1470, 1224 and 1189 cmꢀ1 1H NMR:
.
4.1.23. 1-(2-Methoxyethyl)-3-(8-methyl-4-oxo-4H-pyrido [1,2-
a]pyrimidin-3-yl)urea (14)
2.97–3.00 (m, 2H, CH2), 3.37–3.40 (m, 2H, CH2), 6.92–6.93 (d, 1H,
J = 2.8 Hz, Ar-H), 6.97–6.99 (m, 1H, Ar-H), 7.18–7.20 (t, 1H,
J = 5.6 Hz, N–H), 7.35–7.37 (d, 1H, J = 5.2 Hz, Ar-H), 7.63–7.66 (d,
1H, J = 9.6 Hz, Ar-H), 7.71–7.74 (dd, 1H, J = 2.6 Hz and J = 9.6 Hz,
Ar-H), 8.51 (s, 1H, N–H), 8.83–8.84 (d, 1H, J = 2.6 Hz, Ar-H), 9.19
(s, 1H, Ar-H). MS: (m/z) 349 (M+).
Compound (14) was prepared by reacting compound (6b) (0.3 g,
0.149 mmol) with 2-methoxyethylamine (0.111 g, 0.149 mmol) as
described above for compound (7). Work up of the reaction mixture
followed by chromatographic purification using ethyl acetate (95%)
in hexane as eluant afforded compound (14) (0.16 g, 38.84%). mp
232–34 °C. Rf 0.75 (1% MeOH in EtOAc). IR (KBr): 3388, 3305,
4.1.28. N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(pyridin-2-
yl)piperazine-1-carboxamide (19)
1633, 1534, 1452, 1228 cmꢀ1 1H NMR: 2.39 (s, 3H, CH3), 3.22–
.
3.25 (m, 2H, CH2), 3.26 (s, 3H, OCH3), 3.35 (m, 2H, CH2), 7.09–7.11
(m, 1H, N–H), 7.41 (s, 1H, Ar-H), 8.30 (s, 1H, N–H), 8.73–8.75 (d,
1H, J = 7.6 Hz, Ar-H), 9.06 (s, 1H, Ar-H). MS: (m/z) 276 (M+ꢀ1).
Compound (19) was prepared by reacting the compound (6a)
(0.3 g, 0.16 mmole) with 4-(2-pyridyl)piperazine (0.262 g,
0.16 mmol) as described above for compound (7). The crude prod-
uct was purified by column chromatography using ethyl acetate
(90%) in hexane as eluant to afford compound (19) (0.19 g,
33.8%), mp 192–95 °C. Rf 0.51 (1% MeOH in EtOAc). IR (KBr):
4.1.24. N-{2-[3-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin- 3-
yl)ureido]- ethyl}acetamide (15)
Compound (15) was prepared by reacting the compound (6b)
(0.3 g, 0.149 mmol) with 2-acetamidoethylamine (0.151 g,
0.149 mmol) as described above for compound (7). Chromato-
graphic purification of the crude product was done using ethyl ace-
tate (95%) in hexane as eluant to get the desired product (15)
(0.2 g, 44.2%), mp 269–70 °C. Rf 0.7 (1% MeOH in EtOAc). IR
3393, 1659, 1596, 1537, 1482, 1238 and 762 cmꢀ1 1H NMR: 3.58
.
(b, 8H, NCH2), 6.65–6.69 (m, 1H, Ar-H), 6.86–6.88 (d, 1H,
J = 8.8 Hz, Ar-H), 7.31–7.35 (m, 1H, Ar-H), 7.54–7.58 (m, 1H, Ar-
H), 7.67–7.69 (m, 1H, Ar-H), 7.80–7.84 (m, 1H, Ar-H), 8.08 (s, 1H,
N–H), 8.13–8.14 (d, 1H, J = 3.6 Hz, Ar-H), 8.77 (s, 1H, Ar-H) 8.91–
8.93 (d, 1H, J = 7.2 Hz, Ar-H). MS: (m/z) 351 (M++1).
(KBr): 3356, 3289, 1633, 1524, 1451, 1239 and 1004 cmꢀ1 1H
.
NMR: 1.81 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.11–3.15 (m, 4H, CH2),
7.04–7.06 (t, 1H, J = 5.0 Hz, N–H) 7.11–7.13 (d, 1H, J = 7.2 Hz, Ar-
H), 7.43 (s, 1H, Ar-H), 7.93 (b, 1H, N–H), 8.26 (s, 1H, N–H), 8.76–
8.78 (d, 1H, J = 7.2 Hz, Ar-H), 9.09 (s, 1H, Ar-H). MS: (m/z) 304
(M++1).
4.1.29. N-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-(4-
methoxyphenyl)piperazine-1-carboxamide (20)
The urea derivative (20) was obtained by refluxing the isocya-
nate (6a) (0.3 g, 0.16 mmol) with 4-(4-methoxyphenyl)piperazine
(0.308 g, 0.16 mmol) in toluene for 6 h. The reaction mixture was
cooled to RT and the solid precipitate so obtained was filtered.
Chromatographic purification of the crude product using ethyl ace-
tate (90%) in hexane as eluant yielded the titled compound (20)
(0.19 g 31.2%), mp 143–45 °C. Rf 0.52 (1% MeOH in EtOAc). IR
4.1.25. 1-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-
(2-thiophen-2-ylethyl)urea (16)
Compound (6b) (0.3 g, 0.149 mmol) was treated with 2-(2-thio-
phenyl)ethylamine (0.188 g, 0.149 mmol) as described above for
compound (7). Chromatographic purification of the crude product
was done using methanol (0.2%) in ethyl acetate as eluant to obtain
1-(8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-(2-thio-
phen-2-yl-ethyl)urea (16) (0.2 g, 40.8%), mp 248–51 °C. Rf 0.78 (1%
MeOH in EtOAc). IR (KBr): 3278, 1634, 1518, 1449, 1237 and
(KBr): 3396, 1649, 1522, 1479, 1437, 1229 and 1021 cmꢀ1 1H
.
NMR: 2.99 (b, 4H, CH2), 3.61(b, 4H, CH2), 3.81 (s, 3H, OCH3),
6.87–7.01 (m, 4H, Ar-H), 7.32–7.35 (m, 1H, Ar-H), 7.67–7.69 (d,
1H, J = 9.2 Hz, Ar-H), 7.80–7.84 (m, 1H, Ar-H), 8.06 (s, 1H, N–H),
8.77 (s, 1H, Ar-H), 8.91–8.93 (d, 1H, J = 7.2 Hz, Ar-H). MS: (m/z)
380 (M++1).
684 cmꢀ1 1H NMR: 2.41 (s, 3H, CH3), 2.95–2.98 (t, 2H, J = 6.4 Hz,
.
CH2), 3.55–3.77 (m, 2H, NCH2), 6.92 (b, 1H, Ar-H), 6.97–6.98 (m,
1H, Ar-H), 7.11–7.12 (m, 1H, NH & Ar-H), 7.35–7.37 (d, 1H,
J = 4.8 Hz, Ar-H), 7.43 (s, 1H, Ar-H), 8.32 (s, 1H, N–H), 8.75–8.77
(d, 1H, J = 7.2 Hz, Ar-H), 9.11 (s, 1H, Ar-H). MS: (m/z) 329 (M++1).
4.1.30. N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
(pyridin-2-yl)piperazine-1-carboxamide (21)
With a procedure similar to that adopted for compound (7), (6b)
(0.3 g, 0.149 mmol) was reacted with 4-(2-pyridyl)piperazine
(0.243 g, 0.149 mmol). Chromatographic purification of the crude
product was done using methanol (2%) in ethyl acetate as eluant
to get the product (21) (0.18 g, 33.1%), mp 160–61 °C. Rf 0.75 (1%
MeOH in EtOAc). IR (KBr): 3395, 1654, 1596, 1531, 1484, 1245
4.1.26. 1-(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3-
[2-(morpholin-4-yl)ethyl]urea (17)
With a procedure similar to that adopted for compound (7), (17)
was prepared by reacting the compound (6c) (0.3 g, 0.135 mmol)
with 2-(4-morpholino)ethylamine (0.176 g, 0.135 mmol). Chro-
matographic purification of the crude product using methanol
(2%) in ethyl acetate as eluant yielded the titled compound (17)
(0.22 g, 46.2%), mp 226–27 °C. Rf 0.5 (2% MeOH in EtOAc). IR
and 1192 cmꢀ1 1H NMR: 2.45 (s, 3H, CH3), 3.57 (b, 8H, CH2),
.
6.65–6.68 (m, 1H, Ar-H), 6.86–6.88 (d, 1H, J = 8.8 Hz, Ar-H),
7.20–7.22 (d, 1H, J = 7.2 Hz, Ar-H), 7.50 (s, 1H, Ar-H), 7.54–7.58
(m, 1H, Ar-H), 8.05 (s, 1H, N–H), 8.13–8.14 (d, 1H, J = 4.4 Hz,
Ar-H), 8.66 (s, 1H, Ar-H), 8.82–8.84 (d, 1H, J = 7.2 Hz, Ar-H). MS:
(m/z) 365 (M++1).
(KBr): 3285, 1729, 1646, 1552, 1464, 1284, 1247 and 1117 cmꢀ1
.

U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
6303
4.1.31. N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
[(4-methoxyphenyl)]piperazine-1-carboxamide (22)
the titled compound (26) (0.5 g, 61.1%), mp 158–60 °C (Lit.20
153–55 °C). Rf 0.6 (1% MeOH in EtOAc). IR (KBr): 3239, 1718,
Compound (22) was prepared by reacting the compound (6b)
(0.3 g, 0.149 mmol) with 4-(4-methoxyphenyl)piperazine (0.287
g, 0.149 mmol) as described above for compound (7). Chromato-
graphic purification of the crude product using methanol (2%) in
ethyl acetate as eluant offered the titled compound (22) (0.195 g,
33.2%), mp 134–35 °C. Rf 0.78 (1% MeOH in EtOAc). IR (KBr):
1650, 1461, 1274, 1228, 1180, 1116 cmꢀ1 1H NMR: 5.17 (s, 2H,
.
CH2), 7.32–7.50 (m, 6H, Ar-H), 7.69–7.71 (d, 1H, J = 8.8 Hz, Ar-H),
7.84–7.89 (m, 1H, Ar-H), 8.70 (br s, 1H, Ar-H), 8.92–8.94 (d, 1H,
J = 6.8 Hz, Ar-H), 9.01 (br s, 1H, N–H). MS: (m/z) 296 (M++1).
4.1.36. 2-Pyridinylmethyl (4-oxo-4H-[1,2-a]pyrimidin-3-yl)
carbamate (27)
3393, 1648, 1530, 1483, 1241, 1188 and 1153 cmꢀ1 1H NMR:
.
2.44 (s, 3H, CH3), 2.97–2.99 (t, 4H, J = 4.6 Hz, CH2), 3.58–3.61 (t,
4H, J = 4.6 Hz, CH2), 3.80 (s, 3H, O-CH3), 6.87–7.00 (m, 4H, Ar-H),
7.19–7.22 (d, 1H, J = 7.4 Hz, Ar-H), 7.50 (s, 1H, Ar-H), 8.00 (s, 1H,
NH), 8.66 (s, 1H, Ar-H), 8.82–8.84 (d, 1H, J = 7.4 Hz, Ar-H). MS:
(m/z) 394 (M++1).
Compound (27) was prepared by reacting (6a) (0.5 g,
0.26 mmol) with pyridin-2-methanol (0.175 g, 0.16 mmol) as de-
scribed above for compound (26). The crude product was subjected
to chromatographic purification using ethyl acetate (90%) in hex-
ane as eluant affording the titled compound (27) (0.24 g, 50.5%),
mp.178–80 °C. Rf 0.6 (1% MeOH in EtOAc). IR (KBr): 3245, 3070,
4.1.32. N-(8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
benzhydrylpiperazine-1-carboxamide (23)
1713, 1666, 1549, 1477, 1234 and 1125 cmꢀ1 1H NMR: 5.22 (s,
.
2H, CH2), 7.33–7.38 (m, 2H, Ar-H), 7.51–7.53 (d, 1H, J = 8.0 Hz,
Ar-H), 7.69–7.72 (d, 1H, J = 8.8 Hz, Ar-H), 7.83–7.89 (m, 2H, Ar-
H), 8.55–8.57 (d, 1H, J = 4.4 Hz, Ar-H), 8.72 (s, 1H, Ar-H), 8.94–
8.95 (d, 1H, J = 7.2 Hz, Ar-H), 9.21 (b, 1H, N–H). MS: (m/z) 297
(M++1).
The isocyanate derivative (6b) (0.3 g, 0.149 mmole) and ben-
zhydrylpiperazine (0.376 g, 0.147 mmol) were reacted together
as described above for compound (7). The crude product was puri-
fied by chromatographic purification using methanol (0.5%) in
ethyl acetate as eluant to obtain the compound (23) (0.2 g,
29.5%), mp 233–35 °C. Rf 0.54 (EtOAc). IR (KBr): 3000, 1644,
4.1.37. Benzyl (8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-
yl)carbamate (28)
1529, 1481, 1237 and 1186 cmꢀ1 1H NMR: 2.32–2.34 (t, 4H,
.
J = 4.4 Hz, CH2), 2.43 (s, 3H, CH3), 3.46–3.48 (t, 4H, J = 4.4 Hz,
CH2), 4.34 (s, 1H, CH), 7.18–7.22 (m, 3H, Ar-H), 7.29–7.33 (m, 4H,
Ar-H), 7.44–7.48 (m, 5H, Ar-H), 7.87 (s, 1H, N–H), 8.63 (s, 1H, Ar-
H), 8.79–8.81 (d, 1H, J = 7.6 Hz, Ar-H). MS: (m/z) 454 (M++1).
Compound (28) was prepared by treating the isocyanate deriv-
ative (6b) (0.5 g, 0.26 mmol) with benzyl alcohol (0.161 g,
0.149 mmol) as described above for compound (26). Chromato-
graphic purification of the crude product using ethyl acetate
(90%) in hexane as eluant yielded the titled compound (28)
(0.18 g, 39%), mp 168–70 °C (Lit.20 177–79 °C). Rf 0.7 (1% MeOH
in EtOAc). IR (KBr): 3242, 1717, 1635, 1546, 1465, 1224 and
4.1.33. N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
(pyridin-2-yl)piperazine]-1-carboxamide (24)
In analogy to compound (7), (24) was prepared by reacting 6c
(0.3 g, 0.135 mmol) with 4-(2-pyridyl)piperazine (0.221 g,
0.135 mmol). Chromatographic purification of the crude product
using methanol (2%) in ethyl acetate as eluant offered the com-
pound (24) (0.21 g, 40.3%), mp 189–90 °C. Rf 0.45 (10% MeOH in
EtOAc). IR (KBr): 3397, 3088, 1729, 1643, 1531, 1482, 1243
1056 cmꢀ1 1H NMR: 2.45 (s, 3H, CH3), 5.15 (s, 2H, CH2), 7.21–
.
7.23 (d, 1H, J = 7.4 Hz, Ar-H), 7.31–7.44 (m, 5H, Ar-H), 7.52 (s, 1H,
Ar-H), 8.60 (s, 1H, Ar-H), 8.83–8.85 (d, 1H, J = 7.4 Hz, Ar-H), 8.97
(b, 1H, N–H). MS: (m/z) 310 (M++1).
4.1.38. 2-Pyridinylmethyl (8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)carbamate (29)
and 1124 cmꢀ1 1H NMR: 3.57–3.58 (b, 8H, CH2), 6.65–6.68 (m,
.
1H, Ar-H), 6.86–6.88 (d, 1H, J = 8.4 Hz, Ar-H), 7.54–7.58 (m,
1H, Ar-H), 7.69–7.71 (d, 1H, J = 9.4 Hz, Ar-H), 7.83–7.86 (dd, 1H,
J = 2.2 Hz and J = 9.4 Hz, Ar-H), 8.13–8.14 (d, 1H, J = 3.2 Hz, Ar-H),
8.16 (s, 1H, N–H), 8.82 (s, 1H, Ar-H), 8.89–8.90 (d, 1H, J = 2.2 Hz,
Ar-H). MS: (m/z) 385 (M++1).
In analogy to compound (26), (6b) (0.5 g, 0.26 mmol) was re-
acted with pyridine-2-methanol (0.163 g, 0.149 mmol). Chromato-
graphic purification of the crude product using ethyl acetate (90%)
in hexane as eluant offered the titled compound (29) (0.19 g, 41%),
mp 180–82 °C. Rf 0.75 (1% MeOH in EtOAc). IR (KBr): 3429, 3247,
1721, 1667, 1644, 1553, 1480, 1244 and 1117 cmꢀ1 1H NMR
.
4.1.34. N-[(7-Chloro-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-4-
(4-methoxyphenyl) piperazine]-1-carboxamide (25)
(CDCl3): 2.45 (s, 3H, CH3), 5.20 (s, 2H, CH2), 7.22–7.24 (d, 1H,
J = 7.4 Hz, Ar-H), 7.33–7.36 (m, 1H, Ar-H), 7.50–7.53 (m, 2H, Ar-
H), 7.83–7.87 (m, 1H, Ar-H), 8.55–8.56 (d, 1H, J = 4.0 Hz, Ar-H),
8.62 (s, 1H, Ar-H), 8.85–8.87 (d, 1H, J = 7.4 Hz, Ar-H), 9.14 (b, 1H,
N–H). MS: (m/z) 311 (M++1).
With a similar procedure, compound (6c) (0.3 g, 0.135 mmol)
was reacted with 4-(4-methoxyphenyl)piperazine (0.26 g, 0.135
mmol) as described previously for compound (7). Chromatographic
purification of the crude product was done using methanol (2%) in
ethyl acetate as eluant yielding the titled compound (25) (0.25 g,
44.72%), mp 163–64 °C. Rf 0.75 (10% MeOH in EtOAc). IR (KBr):
4.1.39. N-(3,4-Methylenedioxybenzyl)-4-oxo-4H-pyrido[1,2-
a]pyrimidine-3-carboxamide (30)
3393, 1656, 1626, 1525, 1483, 1347, 1236 and 1065 cmꢀ1
.
1H
Compound (4a) (0.5 g, 0.26 mmol) was suspended in dichloro-
methane (50 ml) and added 3,4-methylenedioxybenzylamine
(0.397 g, 0.26 mmol), the reaction mixture 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 above solution. The reaction mixture was stirred for 1h 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 HCl, extracted using ethyl acetate
(3 ꢁ 100 ml), dried over anhydrous sodium sulfate and concen-
trated to get a dark brown product. Chromatographic purification
of the crude product using ethyl acetate (80%) in hexane as eluant
offered the compound (30) (0.3 g, 35.2%), mp 193–95 °C. Rf 0.6
NMR: 2.97–2.99 (t, 4H, J = 4.2 Hz, CH2), 3.60–3.61(t, 4H, J = 4.2 Hz,
CH2), 3.80 (s, 3H, O–CH3), 6.87–6.93 (m, 2H, Ar-H), 6.95–7.00 (m,
2H, Ar-H), 7.69–7.71 (d, 1H, J = 9.6 Hz, Ar-H), 7.83–7.86 (t, 1H,
J = 9.6 Hz, Ar-H), 8.13 (s, 1H, N–H), 8.82 (s, 1H, Ar-H), 8.90 (s, 1H,
Ar-H). MS: (m/z) 414 (M++1).
4.1.35. Benzyl (4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)
carbamate (26)
Compound (6a) (0.3 g, 16 mmol) and benzyl alcohol (0.173 g,
16 mmol) in toluene (25 ml) were refluxed for 6 h to get solid pre-
cipitate. The solid precipitate was filtered and washed with hexane
and the crude product so obtained was purified by column chro-
matography using ethyl acetate (90%) in hexane as eluant to get

6304
U. R. Mane et al. / Bioorg. Med. Chem. 20 (2012) 6296–6304
(EtOAc). IR (KBr): 3260, 3071, 1665, 1627, 1490, 1434, 1246 and
1224 cmꢀ1 1H NMR: 4.46–4.48 (d, 2H, J = 6.0 Hz, CH2), 5.98 (s,
2H, CH2), 6.82–6.93 (m, 3H, Ar-H), 7.58–7.62 (m, 1H, Ar-H), 7.89–
7.91 (d, 1H, J = 8.8 Hz, Ar-H), 8.17–8.22 (m, 1H, Ar-H), 9.05 (s, 1H,
Ar-H), 9.17–9.19 (d, 1H, J = 7.6 Hz, Ar-H), 9.31–9.33 (t, 1H,
J = 6.0 Hz, N–H). MS: (m/z) 324 (M++1).
355 nm; emission 460 nm) for 10 min at room temperature with
a Synergy™ 4 Multi-Mode Microplate Reader (BioTek). The inhibi-
tion rate (%) is calculated using the given equation:
.
%Inhibition ¼ ½1 ꢀ ðF test=F controlÞꢂo100
where (F test is the fluorescence intensity of the test compound, F
control is the fluorescence intensity of the standard (E64). All values
are the means of three independent determinations and the devia-
tions are <10% of the mean value. The IC50 values were determined
for those compounds only which showed 40% or more of enzyme
4.1.40. N-(4-Fluorobenzyl)-8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carboxamide (31)
Compound (31) was prepared by treating (4b) (0.5 g,
0.26 mmol) with 4-fluorobenzylamine (0.306 g, 0.24 mmol) as de-
scribed above for compound (30). Chromatographic purification of
the crude product using ethyl acetate (90%) in hexane as eluant
afforded the desired product (31) (0.15 g, 51.9%), mp 185–87 °C.
Rf 0.68 (1% MeOH in EtOAc). IR (KBr): 3287, 3014, 1677, 1633,
inhibition at 10 lM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.008.
1481, 1279, 1222 and 1146 cmꢀ1 1H NMR (CDCl3): 2.58 (s, 3H,
.
CH3), 4.65–4.66 (d, 2H, J = 4.0 Hz, CH2), 7.00–7.04 (m, 2H, Ar-H),
7.18–7.21 (d, 1H, Ar-H), 7.34–7.37 (m, 2H, Ar-H), 7.63 (s, 1H,
Ar-H), 9.08–9.09 (d, 1H, J = 4.0 Hz, Ar-H), 9.33 (s, 1H, Ar-H), 9.35
(br s, 1H, N–H). MS: (m/z) 312 (M++1).
References and notes
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4.1.41. N-(3,4-Methylenedioxybenzyl)-7-chloro-4-oxo-4H-
pyrido[1,2-a]pyrimidine-3-carboxamide (32)
With a similar procedure, (4c) (0.5 g, 0.22 mmol) was reacted
with 3,4-methylenedioxybenzylamine (0.336 g, 0.22 mmol) as de-
scribed previously for compound (30). Chromatographic purifica-
tion of the crude product using ethyl acetate (90%) in hexane as
eluant yielded the product (32) (0.4 g, 50.2%), mp 199–201 °C. Rf
0.4 (EtOAc). IR (KBr): 3300, 1672, 1490, 1434, 1248 and
1036 cmꢀ1 1H NMR: 4.46–4.48 (d, 2H, J = 6 Hz, CH2), 5.98 (s, 2H,
.
CH2), 6.81–6.92 (m, 3H, Ar-H), 7.90–7.93 (d, 1H, J = 8.8 Hz, Ar-H),
8.24–8.26 (m, 1H, Ar-H), 9.04 (s, 1H, Ar-H), 9.16 (s, 1H, Ar-H),
9.25 (b, 1H, N–H). MS: (m/z) 358.3 (M++1).
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4.2. Falcipain-2. enzyme assay
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The diluted soluble Plasmodium falciparum FP-2 (30 nM) was
incubated for 10 min at room temperature in 100 mM sodium ace-
tate, pH 5.5, 10 mM DTT, with a fixed different concentration of the
compounds to be tested or the standard (E64). Compound solu-
tions were prepared from stock in DMSO. After 10 min incubation,
the substrate Z-Phe-Arg-AMC was added to a final concentration of
25 lM. The fluorescence intensity was monitored (excitation