Synthesis of new 2-substituted pyrido[2,3-d]pyrimidin-4(1H)-ones and their antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2008.05.025

2-Substituted-5,7-dimethyl pyrido[2,3-d]pyrimidin-4(1H)-ones (8) were synthesized by oxidation of 2-substituted-5,7-dimethyl dihydropyrido[2,3-d]pyrimidin-4(1H)-ones (7) which were in turn prepared from 2-amino-4,6-dimethyl nicotinamide (5) and substituted aryl aldehydes (6). 2-Amino-4,6-dimethyl nicotinamide (5) was prepared from ethyl cyanoacetate (1) via malonamamidine hydrochloride (3). The compounds were characterized by IR, NMR, MS and elemental analyses. Compounds 7 and 8 were screened for antibacterial activity against Gram positive and Gram negative bacteria. Dehydrogenated compounds (8) showed less antibacterial activity than the compounds 7. Among all the test compounds screened for antibacterial activity 7c (1.25 μg/ml) showed greater activity. All the synthesized compounds were found inactive when screened for antifungal activity at the concentration of 200 μg/ml.

Full text of DOI:10.1016/j.ejmech.2008.05.025

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1369e1376
http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis of new 2-substituted pyrido[2,3-d]pyrimidin-4(1H )-ones
and their antibacterial activity*
a
b
B. Lakshmi Narayana a, , A. Raghu Ram Rao , P. Shanthan Rao
*
a Department of Pharmaceutical Chemistry, University Institute of Pharmaceutical Sciences, Panjab University, Sector-14, Chandigarh 160014, India
b BASR Fine Chemicals Pvt. Ltd, Jeedimetla, Hyderabad, India
Received 2 January 2008; received in revised form 16 April 2008; accepted 23 May 2008
Available online 7 July 2008
Abstract
2-Substituted-5,7-dimethyl pyrido[2,3-d]pyrimidin-4(1H )-ones (8) were synthesized by oxidation of 2-substituted-5,7-dimethyl
dihydropyrido[2,3-d]pyrimidin-4(1H )-ones (7) which were in turn prepared from 2-amino-4,6-dimethyl nicotinamide (5) and substituted
aryl aldehydes (6). 2-Amino-4,6-dimethyl nicotinamide (5) was prepared from ethyl cyanoacetate (1) via malonamamidine hydrochloride
(3). The compounds were characterized by IR, NMR, MS and elemental analyses. Compounds 7 and 8 were screened for antibacterial
activity against Gram positive and Gram negative bacteria. Dehydrogenated compounds (8) showed less antibacterial activity than the
compounds 7. Among all the test compounds screened for antibacterial activity 7c (1.25 mg/ml) showed greater activity. All the
synthesized compounds were found inactive when screened for antifungal activity at the concentration of 200 mg/ml.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrimidines; Pyrido[2,3-d]pyrimidines; Antibacterial activity; Nitrobenzene; Oxidation
1. Introduction
to possess antifungal, antibacterial and antiinflammatory
activities.
Pyrimidine analogues are used as chemotherapeutic agents
for the treatment of diseases resulting from different microor-
ganisms and now there has been a major expansion in the
number of pyrimidine derivatives as drugs. The design of
pyrimidine and purine analogues as antimetabolites involves
changes in the substituents on ring, isosteric replacement of
rings, changes in ring size, attached sugars and phosphate
residues, etc. Many useful drugs containing these skeletal
structures with diverse biological activities have now emerged
and are widely used against tumors, viral diseases and found
Pyrimidine derivatives like pyrimethamine and trimetho-
prim are well known to inhibit the enzyme, dihydrofolate
reductase (DHFR), thereby blocking the reduction to tetrahy-
drofolic acid from its dihydro precursor, an essential coen-
zyme in nucleic acid synthesis. Both of them exhibit a far
higher affinity for protozoal than for mammalian DHFR, so
that they are used for malarial prophylaxis without adversely
affecting the human (host) enzyme which otherwise results
in toxic effects. They are active against both Plasmodium
falciparum and Plasmodium vivax, in combination with an
appropriate sulfonamide. They are effective because the
sulfonamide acts synergistically by arresting the production
of dihydrofolic acid from p-aminobenzoic acid, thus achieving
a double (sequential) blockade of the folate pathway in the mi-
croorganism, which (unlike man) cannot use preformed folic
acid. Diaminopyrimidine derivatives trimetrexate, piritrexim
and several fused pyrimidines and pyrido[2,3-d]pyrimidines
*
Part of the thesis (M. Pharm.) submitted to Kakatiya University, Warangal,
India, 2006 and presented in the Scientific Sessions, Medicinal Chemistry dur-
ing 58th Indian Pharmaceutical Congress held in Mumbai, India, December
2006 (BP23).
* Corresponding author. Fax: þ91 172 254 1142.
E-mail address: lakshman1021@gmail.com (B. Lakshmi Narayana).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.025

1370
B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
were synthesized and evaluated for antibacterial [1e3] and
DHFR inhibition [4e8].
aldehydes (9a and 9b) gave respective dihydropyrido[2,3-
d]pyrimidin-4(1H )-ones (10a and 10b) (Scheme 3). The prod-
ucts were purified by passing through silica gel column using
n-hexane, ethyl acetate (95:5) as mobile phase. They are char-
OCH3
OCH3
OCH3
OCH3
NH2 CH3
NH2
CH3
1
acterized using physical, spectral (IR, H NMR and MS) and
N
N
NH
elemental analyses. The IR spectra of compounds (7 and 10)
showed signals around 3200 cmꢀ1 (eNH) and 1620 cmꢀ1
(C]O) and 1H NMR spectra of compounds gave broad signal
at d 8.2e8.8 ppm indicating the presence of two eNH-protons.
The proton of 2-position gave a prominent singlet at d 5.8 ppm.
The physical, spectral and analytical data of compounds 7 and
10 were presented in Table 1.
OCH3
H2N
N
H2N
N
Trimetrexate
Piritrexim
Pyrido[2,3-d]pyrimidine ring system is present in a number
of biologically active compounds which includes antitumor
[9], antipyretic [10], analgesic [11], antihistaminic [12],
PDE4 inhibitor [13], adenosine kinase inhibitor [14], tyrosine
kinase inhibitor [15] and diuretic [16,17] activities. More
specifically pyrido[2,3-d]pyrimidines were considered as in-
hibitors of Pneumocystis carinii, Toxoplasma gondii of tumor
cells in culture. The activity is mainly due to inhibition of
DHFR enzyme.
The synthesis of pyrido[2,3-d]pyrimidines is mainly by two
ways, i.e. annulation of pyrimidine ring over pyridine or vice
versa. The wide range of activity profile of pyrido[2,3-d]
pyrimidines prompted us to probe the synthesis of novel
analogues and to study their antibacterial, antifungal and
cytotoxic activities.
In the subsequent step different oxidation methods were
attempted using potassium permanganate [22] and manganese di-
oxide [23,24], but the yields were poor. Taking the clue from the
facile nature of oxidation process using nitrobenzene from litera-
ture [25], an attempt to oxidize 7 and 10 to 2-substituted-5,7-di-
methyl pyrido[2,3-d]pyrimidin-4(1H )-ones (8 and 11) resulted
in good yields (65e92%). The method was found to be superior
(Scheme 4). A comprehensive account of the percentageyields of
three different methods tried have been tabulated (Table 3). A
mixture of 2-substituted-5,7-dimethyl dihydropyrido[2,3-d]pyri-
midin-4(1H )-ones (7 and 10) and nitrobenzenewas treated under
reflux for 5 h. The ease of removal of nitrobenzene by steam dis-
tillation made it very facile. The structures of compounds 8and11
were deduced through physical, spectral (IR, 1H NMR and MS)
and elemental analyses. The formation of 8 was confirmed by
the absence C-2 proton (at d 5.8 ppm) was conspicuous in the
1H NMR spectra of 8. Further confirmation of the structure was
through 13C NMR of 8a (2-C signal at d 69.1 ppm disappeared).
The physical, spectral and analytical data of compounds 8 and 11
were tabulated (Table 2).
2. Chemistry
2-Amino-4,6-dimethyl nicotinamide (5) was prepared from
ethyl cyanoacetate (1) via malonamamidine hydrochloride (3)
(Scheme 1) [18,19]. On a reaction of 5 with aryl aldehydes
(6aek) in the presence of catalytic amounts of glacial acetic
acid gave 2-substituted-5,7-dimethyl dihydropyrido[2,3-d]pyr-
imidin-4(1H )-ones (7aek) in quantitative yields. Substituted
aryl aldehydes (6aek) were used as intermediates for the nu-
cleophilic addition reaction with primary amines resulted in
Schiff base formation, which spontaneously cyclised in acidic
conditions to give various 2-substituted-5,7-dimethyl dihydro-
pyrido[2,3-d]pyrimidin-4(1H )-ones (7aek) (Scheme 2)
[20,21]. Compound 5 in analogous reaction with heterocyclic
3. Antibacterial assays (in vitro)
Four bacterial test organisms such as Bacillus subtilis
(MTCC 441), Staphylococcus aureus (MTCC 96), Escherichia
coli (MTCC 722) and Klebsiella pneumoniae (MTCC 109)
CONH2
COOC2H5
H2C
HN
H2C
H2
C
NH.HCl
OC2H5.HCl
HN
COOC2H5
NC
1
2
3
CH3
H2C
H3C
O
O
4
CH3
N
O
NH2
NH2
H3C
5
Scheme 1.

CHO
R3
R1
R2
CH3
N
O
R4
CH3
N
O
NH
R1
R4
NH2
NH2
(6a- k)
R2
R3
H3C
N
H
H3C
5
(7a- k)
Ph-NO2
CH3
N
O
NH
N
R1
R4
R2
R3
H3C
(8a- k)
R1
R2
R3
R4
a
b
c
d
e
f
g
h
i
H
H
H
OCH3
Cl
CH3
H
H
OCH3
H
OCH3
H
Cl
CH3
H
OCH3
CH3
H
OCH3
OCH3
OCH3
OCH3
H
H
OAc
OH
H
H
OCH3
H
H
H
H
H
CH3
H
H
H
OH
OH
OH
OCH3
H
j
k
H
Scheme 2.
CH3
N
O
CH3
N
O
NH2
NH2
NH
+
OHC
X
H3C
H3C
N
H
X
5
9a (X = O)
9b (X = S)
10a (X = O)
10b (X = S)
Ph-NO2
CH3
O
NH
H3C
N
N
X
11a (X = O)
11b (X = S)
Scheme 3.

1372
B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
Table 1
Physical, spectral and analytical data of 2-substituted-5,7-dimethyl dihydropyrido[2,3-d]pyrimidin-4(1H )-ones (7 and 10)
S.
No.
Compound
code
Mol. for.
(Mol. wt.)
Melting point
(ꢁC) (yield %)
IR (cmꢀ1
KBr discs
)
1H NMR (CDCl3/DMSO-d6) d
Mass
m/z (Mþ)
CHN analyses
found (calcd)
1
7aa
C17H19N3O3 (313)
190 (77.8)
3202 (NH)
1650 (C]O)
2.28 (s, 3H, CH3), 2.6 (s, 3H, CH3), 3.9
(s, 6H, OCH3), 5.8 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.8e7.2 (m, 3H, AreH), 8.4
(s, br, 1H, NH, D2O exchangeable), 8.7
(s, br, 1H, NH, D2O exchangeable).
13C NMR (DMSO-d6, 400 MHz):
d (ppm) ¼ 21.4 (CH3); 24.5 (CH3);
56.5 (2C), 69.1, 112.4, 112.9, 113.1,
115.4, 123.5, 140.1, 148.2, 150.2,
152.2, 153.4, 161.2, 168.3
314
C, 65.49, H,
6.43, N, 13.12
(C, 65.15, H,
6.12, N, 13.42)
2
7b
C16H17N3O2 (283)
209 (79)
3202 (NH)
1640 (C]O)
2.28 (s, 3H, CH3), 2.6 (s, 3H, CH3), 3.9
(s, 3H, OCH3), 5.8 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.9e7.5 (d, 4H, AreH), 8.3
(s, br, 1H, NH, D2O exchangeable),
8.52 (s, br, 1H, NH, D2O
284
C, 67.49, H, 6.43,
N, 14.82 (C, 67.81,
H, 6.05, N, 14.84)
exchangeable)
3
7c
7d
7e
7f
C18H21N3O4 (343)
C17H19N3O3 (313)
C15H11N3OCl2 (320)
C17H19N3O (281)
C17H17N3O3 (311)
C16H17N3O3 (299)
C17H19N3O2 (287)
C16H17N3O3 (299)
212 (86)
189 (77.8)
218 (77.1)
192 (75)
192 (75)
204 (72)
198 (85)
184 (83)
3219 (NH)
1662 (C]O)
2.2 (s, 3H, CH3), 2.6 (s, 3H, CH3), 3.8
(s, 9H, OCH3), 5.8 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.8 (s, 2H, AreH), 8.3 (s, br,
1H, NH, D2O exchangeable), 8.6 (s, br,
1H, NH, D2O exchangeable)
344
314
322
282
312
300
288
300
C, 62.69, H, 6.51,
N, 12.53 (C, 62.95,
H, 6.17, N, 12.24)
4
3202 (NH)
1640 (C]O)
2.28 (s, 3H, CH3), 2.6 (s, 3H, CH3), 3.9
(s, 6H, OCH3), 5.9 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.8e7.2 (m, 3H, AreH), 8.5
(s, br, 1H, NH, D2O exchangeable), 8.7
(s, br, 1H, NH, D2O exchangeable)
2.28 (s, 3H, CH3), 2.6 (s, 3H, CH3), 5.8
(s, 1H, H-2), 6.6 (s, 1H, H-6), 6.9e7.5
(d, 3H, AreH), 8.2 (s, br, 1H, NH, D2O
exchangeable), 8.5 (s, br, 1H, NH, D2O
exchangeable)
C, 65.29, H, 6.25,
N, 13.22 (C, 65.15,
H, 6.12, N, 13.42)
5
3200 (NH)
1620 (C]O)
C, 55.98, H, 3.25,
N, 13.32 (C, 56.25,
H, 3.46, N, 13.14)
6
3208 (NH)
1620 (C]O)
2.2 (s, 6H, CH3), 2.5 (s, 3H, CH3), 2.8
(s, 3H, CH3), 5.8 (s, 1H, H-2), 6.4 (s,
1H, H-6), 7.4 (s, 3H, AreH), 8.3 (s, br,
1H, NH, D2O exchangeable), 8.6 (s, br,
1H, NH, D2O exchangeable)
C, 72.89, H, 6.45,
N, 14.82 (C, 72.57,
H, 6.80, N, 14.94)
7
7g
7h
7i
3202 (NH)
1608 (C]O)
2.2 (s, 3H, CH3), 2.8 (s, 3H, CH3), 4.2
(s, 3H, OCH3), 5.7 (s, 1H, H-2), 6.6 (s,
1H, H-6), 6.9e7.5 (d, 4H, AreH), 8.4
(s, br, 1H, NH, D2O exchangeable), 8.6
(s, br, 1H, NH, D2O exchangeable)
2.28 (s, 3H, CH3), 2.6 (s, 3H, CH3), 4.0
(s, 3H, OCH3), 5.8 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.9e7.4 (d, 3H, AreH), 8.5
(s, br, 1H, NH, D2O exchangeable), 8.7
(s, br, 1H, NH, D2O exchangeable)
2.2 (s, 6H, CH3), 2.5 (s, 3H, CH3), 2.8
(s, 3H, CH3), 5.8 (s, 1H, H-2), 6.5 (s,
1H, H-6), 6.9 (s, 2H, AreH), 8.3 (s, br,
1H, NH, D2O exchangeable), 8.6 (s, br,
1H, NH, D2O exchangeable)
C, 65.29, H, 5.25,
N, 13.82 (C, 65.57,
H, 5.51, N, 13.50)
8
3200 (NH)
1600 (C]O)
C, 64.26, H, 5.56,
N, 14.32 (C, 64.19,
H, 5.73, N, 14.04)
9
3208 (NH)
1602 (C]O)
C, 68.39, H, 6.25,
N, 13.92 (C, 68.65,
H, 6.44, N, 14.14)
10
7j
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3), 3.8
(s, 3H, OCH3), 5.2 (s, 1H, OH), 5.7 (s,
1H, H-2), 6.9 (s, 1H, H-6), 7.2e7.4 (s,
3H, AreH), 8.6 (s, br, 1H, NH, D2O
exchangeable), 8.8 (s, br, 1H, NH, D2O
exchangeable)
C, 64.42, H, 5.84,
N, 14.13 (C, 64.20,
H, 5.72, N, 14.04)
11
7k
C15H15N3O2 (269)
193 (75)
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3), 5.2
(s, 1H, OH), 5.7 (s, 1H, H-2), 6.9 (s,
1H, H-6), 7.2e7.4 (s, 4H, AreH), 8.7
(s, br, 1H, NH, D2O exchangeable), 8.8
(s, br, 1H, NH, D2O exchangeable)
270
C, 66.88, H, 5.55,
N, 15.73 (C, 66.90,
H, 5.61, N, 15.60)

B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
1373
Table 1 (continued)
S.
No.
Compound
code
Mol. for.
(Mol. wt.)
Melting point
(ꢁC) (yield %)
IR (cmꢀ1
KBr discs
)
1H NMR (CDCl3/DMSO-d6) d
Mass
m/z (Mþ)
CHN analyses
found (calcd)
12
10a
C13H13N3O2 (243)
203 (83)
3208 (NH)
1602 (C]O)
2.3 (s, 3H, CH3), 2.6 (s, 3H, CH3), 6.3
(s, 1H, H-6), 6.6 (s, 1H, H-2), 6.06 (d,
1H, H2-furyl), 6.36 (m, 1H, H3-furyl),
7.3 (d, 1H, H4-furyl), 8.2 (s, br, 1H,
NH, D2O exchangeable), 8.5 (s, br, 1H,
NH, D2O exchangeable)
244
C, 64.39, H, 5.25,
N, 17.62 (C, 64.17,
H, 5.39, N, 17.28)
13
10b
C13H13N3OS (259)
210 (85)
3208 (NH)
1602 (C]O)
2.4 (s, 3H, CH3), 2.8 (s, 3H, CH3), 6.3
(s, 1H, H-6), 6.6 (s, 1H, H-2), 6.06 (d,
1H, H2-thiophenyl), 6.72 (m, 1H, H3-
thiphenyl), 6.71 (d, 1H, H4-
260
C, 60.39, H, 5.25,
N, 16.42 (C, 60.21,
H, 5.06, N, 16.21)
thiophenyl), 8.3 (s, br, 1H, NH, D2O
exchangeable), 8.5 (s, br, 1H, NH, D2O
exchangeable)
a
13C NMR data.
were obtained from Institute of Microbial Technology (IM-
TECH), Chandigarh. Cultures of test organisms were main-
tained on nutrient agar slants and were subcultured in petri
dishes prior to testing. The media used was nutrient agar
(procured from Himedia Laboratories, Mumbai). The mini-
mum inhibitory concentration (MIC) was determined by the
test tube dilution technique using ciprofloxacin as standard.
exhibited lesser antibacterial activity than dihydro analogues
(7 and 10). Among all pyridopyrimidines, C-2 substitutions
having electron releasing groups exhibited better antibacterial
activity compared to substitutions having electron withdraw-
ing groups. Compounds having more number of methoxy
groups showed better activity, replacing the methoxy groups
with methyl, hydroxyl and hydrogen found to be less active.
Replacing substituted six-member ring with unsubstituted
five-member ring at C-2 exhibited less activity. The MIC
values of the compounds were compared with ciprofloxacin
sample. The details of the test compounds and their activity
against different microorganisms are given in Tables 4 and 5.
All the test compounds were screened for antifungal activ-
ity by using Aspergillus niger and Candida albicans and found
inactive (maximum concentration of 200 mg/ml), Subse-
quently all the test compounds 7, 8, 10 and 11 were screened
for their cytotoxicity in vitro on MCF-7 cell lines as per the
method described by Moon et al. [26] and found to be non-
toxic up to 150 mg/ml concentration.
4. Results and discussion
All the synthesized compounds (7, 8, 10 and 11) were
screened for their in vitro antibacterial activity against Gram
þve (B. subtilis and S. aureus) and Gram eve (E. coli and
K. pneumoniae) bacteria. All the dihydro compounds (7)
showed significantly better activity over their oxidized ana-
logues (8) against Gram þve and Gram eve bacteria. Com-
pound 7c showed potent activity against all species of Gram
þve and Gram eve bacteria compared to other compounds.
Enhanced activity was observed in the case of 7c due to the
presence of methoxy groups. Replacing methoxy group with
methyl, hydroxyl and chloro groups resulted in lower activity.
Compounds 7a and 7i showed better activity against Gram
þve bacteria compared to Gram ꢀve bacteria. Compound
7h was more active against S. aureus. Compounds 7bed, 7f,
7g, 7j and 7k showed moderate activity against both Gram
þve and Gram eve bacteria. Compound 7e was the least
active among the test compounds and inactive against E. coli
(100 mg/ml concentration). Compounds 10a and 10b showed
potent antibacterial activity against Gram þve than Gram
ꢀve bacteria. All the oxidized compounds (8 and 11)
5. Experimental
Melting points were recorded on Casiaa siamea (VMP-AM)
melting point apparatus and are uncorrected. IR spectra were
recorded on a PerkineElmer FT-IR 240-C spectrophotometer
1
using KBr discs. H NMR spectra were recorded on Gemini
Varian 200 MHz and 400 MHz spectrometer in CDCl3 and
DMSO-d6 using TMS as internal standard. 13C NMR was
recorded on Bruker Avance II 400 MHz spectrometer in
DMSO-d6 using TMS as internal standard. Mass spectra
CH3
O
CH3
O
NH
R1
R4
NH
R1
R4
R2
R3
R2
R3
H3C
N
N
H
H3C
N
N
KMnO4 / MnO2 / Ph-NO2
(7a- k)
(8a- k)
Scheme 4.

1374
B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
Table 2
Physical, spectral and analytical data of 2-substituted-5,7-dimethyl pyrido[2,3-d]pyrimidin-4(1H )-ones (8 and 11)
S.
No.
Compound
code
Mol. for.
(Mol. wt.)
Melting point
(ꢁC) (yield %)
IR (cmꢀ1
KBr discs
)
1H NMR (CDCl3/DMSO-d6) d
Mass
m/z (Mþ)
CHN analyses
found (calcd)
1
8aa
C17H17N3O3 (311)
254 (84)
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
3.9 (s, 6H, OCH3), 6.5 (s, 1H, H-6),
6.9e7.6 (d, 3H, AreH), 8.9 (s, br,
1H, NH, D2O exchangeable). 13C
NMR (DMSO-d6, 400 MHz):
d (ppm) ¼ 21.5 (CH3); 24.3 (CH3);
55.5 (2C), 110.1, 110.4, 110.5, 112.1,
121.4, 123.5, 124.1, 148.2, 150.8,
151.6, 154.3, 163.4, 170.8
312
C, 65.49, H, 5.53,
N, 13.82 (C, 65.57,
H, 5.51, N, 13.50)
2
8b
8c
8d
8e
8f
C16H15N3O2 (281)
C18H19N3O4 (341)
C17H17N3O3 (311)
C15H9N3OCl2 (318)
C17H17N3O (279)
C17H15N3O3 (309)
C16H15N3O3 (297)
C17H17N3O2 (295)
C16H15N3O3 (297)
230 (82)
202 (92)
230 (86)
230 (60)
192 (70)
260 (82)
250 (86)
252 (80)
258 (68)
3200 (NH)
1602 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
3.9 (s, 3H, OCH3), 6.5 (s, 1H, H-6),
6.9e7.4 (d, 4H, AreH), 8.8 (s, br,
1H, NH, D2O exchangeable)
282
342
312
320
280
310
298
296
298
C, 68.42, H, 5.52,
N, 14.74 (C, 68.30,
H, 5.38, N, 14.94)
3
3206 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
3.9 (s, 9H, OCH3), 6.9 (s, 1H, H-6),
7.4 (s, 2H, AreH), 8.8 (s, br, 1H,
NH, D2O exchangeable)
C, 63.46, H, 5.43,
N, 12.02 (C, 63.32,
H, 5.61, N, 12.31)
4
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
3.9 (s, 6H, OCH3), 6.9 (s, 1H, H-6),
7.6e7.9 (d, 3H, AreH), 8.9 (s, br,
1H, NH, D2O exchangeable)
C, 65.49, H, 5.53,
N, 13.82 (C, 65.57,
H, 5.51, N, 13.50)
5
3210 (NH)
1600 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
6.9 (s, 1H, H-6), 7.2e7.4 (d, 3H,
AreH), 8.8 (s, br, 1H, NH, D2O
exchangeable)
C, 56.54, H, 2.56,
N, 13.73 (C, 56.61,
H, 2.85, N, 13.51)
6
3208 (NH)
1620 (C]O)
2.2 (s, 6H, CH3), 2.5 (s, 3H, CH3),
2.8 (s, 3H, CH3), 6.5 (s, 1H, H-6), 7.4
(s, 3H, AreH), 8.9 (s, br, 1H, NH,
D2O exchangeable)
C, 73.23, H, 6.43,
N, 14.82 (C, 73.09,
H, 6.13, N, 15.04)
7
8g
8h
8i
3210 (NH)
1606 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
4.2 (s, 3H, OCH3), 6.9 (s, 1H, H-6),
6.9e7.5 (d, 4H, AreH), 8.8 (s, br,
1H, NH, D2O exchangeable)
C, 65.89, H, 5.03,
N, 13.82 (C, 66.00,
H, 4.89, N, 13.59)
8
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
4.0 (s, 3H, OCH3), 6.9 (s, 1H, H-6),
7.2e7.4 (d, 3H, AreH), 8.9 (s, br,
1H, NH, D2O exchangeable)
C, 64.48, H, 5.33,
N, 14.52 (C, 64.62,
H, 5.09, N, 14.44)
9
3200 (NH)
1610 (C]O)
2.2 (s, 6H, CH3), 2.6 (s, 3H, CH3),
2.8 (s, 3H, CH3), 6.9 (s, 1H, H-6), 7.2
(s, 2H, AreH), 8.7 (s, br, 1H, NH,
D2O exchangeable)
C, 69.44, H, 5.62,
N, 14.04 (C, 69.12,
H, 5.81, N, 14.23)
10
8j
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
3.8 (s, 3H, OCH3), 5.2 (s, 1H, OH),
6.9 (s, 1H, H-6), 7.2e7.4 (s, 3H, Are
H), 8.6 (s, br, 1H, NH, D2O
C, 64.42, H, 5.01,
N, 14.22 (C, 64.63,
H, 5.08, N, 14.13)
exchangeable)
11
12
8k
C15H13N3O2 (267)
C13H11N3O2 (241)
250 (65)
203 (68)
3200 (NH)
1610 (C]O)
2.6 (s, 3H, CH3), 2.8 (s, 3H, CH3),
5.2 (s, 1H, OH), 6.9 (s, 1H, H-6),
7.2e7.4 (s, 4H, AreH), 8.7 (s, br,
1H, NH, D2O exchangeable)
268
242
C, 67.28, H, 5.01,
N, 15.83 (C, 67.40,
H, 4.90, N, 15.72)
11a
3208 (NH)
1602 (C]O)
2.3 (s, 3H, CH3), 2.6 (s, 3H, CH3),
6.3 (s, 1H, H-6), 6.06 (d, 1H, H2-
furyl), 6.36 (m, 1H, H3-furyl), 7.3 (d,
1H, H4-furyl), 8.9 (s, br, 1H, NH,
D2O exchangeable)
C, 64.54, H, 4.55,
N, 17.53 (C, 64.71,
H, 4.60, N, 17.42)
13
11b
C13H11N3OS (257)
210 (65)
3208 (NH)
1602 (C]O)
2.4 (s, 3H, CH3), 2.8 (s, 3H, CH3),
6.3 (s, 1H, H-6), 6.6 (d, 1H, H2-
thiophenyl), 6.72 (m, 1H, H3-
thiphenyl), 6.71 (d, 1H, H4-
258
C, 60.39, H, 4.25,
N, 16.42 (C, 60.69,
H, 4.31, N, 16.34)
thiophenyl), 8.9 (s, br, 1H, NH, D2O
exchangeable)
a
13C NMR data.

B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
1375
Table 3
Percentage yields of compounds (8) by different oxidation methods
was slowly added to methanol under nitrogen atmosphere at
20e25 ꢁC. Malonamamidine hydrochloride (3) was added to
it at 20e25 ꢁC to remove salt form and acetyl acetone was
added to it at 25e30 ꢁC under nitrogen environment and the
reaction mixture was stirred at room temperature for 24 h.
The resulted solid (KCl) was filtered off and the filtrate was
concentrated by removing methanol. The viscous residue
was cooled to 10 ꢁC to get a precipitate which was filtered
and dried. Compound (5) was purified by recrystallized from
Compounds
Oxidation by
KMnO4
Oxidation by
MnO2
Oxidation by
nitrobenzene
8a
8b
8c
8d
8e
8f
48
41
50
46
39
33
30
42
40
40
43
41
42
46
41
33
37
33
41
39
33
30
84
82
92
86
60
70
82
86
80
68
65
ethyl acetate. Yield 82%; m.p. 182 ꢁC; IR (KBr) cmꢀ1
:
8g
8h
8i
1
3450, 3200, 1640 (eCONH2). H NMR (CDCl3) d: 2.4 (3H,
s, CH3), 2.6 (3H, s, CH3), 4.2 (2H, s, br, eNH2), 6.2 (2H, s,
br, eCONH2), 6.8 (1H, s, H-5). MS m/z: 166 (Mþ). Anal.
Calcd for C8H11N3O: C, 58.17, H, 6.71, N, 25.44. Found: C,
58.29, H, 6.53, N, 25.31.
8j
8k
(EI and CI) were recorded on a VG 7070 H instrument at
70 eV. All the reactions were monitored by thin layer chroma-
tography (TLC) on precoated silica gel 60 F254 (mesh) (E
Merck, Mumbai), spots were visualized under UV light
(254 nm). Merck silica gel (100e200 mesh) was used for
chromatography and CHN analyses were recorded on a Vario
EL analyzer.
5.2. 2-Substituted-5,7-dimethyl dihydropyrido[2,3-d]
pyrimidin-4(1H )-ones (7)
General procedure. 2-Amino-4,6-dimethyl nicotinamide
(5) (0.00606 mol) was dissolved in glacial acetic acid
(15 ml) and to it was added suitably substituted aryl
aldehyde (6aek) (0.00606 mol). The mixture was heated un-
der reflux in an oil bath for 7 h. After complete conversion
of starting materials (monitored by TLC) the reaction mixture
was cooled, poured onto crushed ice and neutralized with
aqueous ammonia to give a precipitate. It was filtered and
dried (in vacuo). The resulted solid product was purified by
column chromatography using n-hexane and ethyl acetate as
mobile phase (95:5).
5.1. 2-Amino-4,6-dimethyl nicotinamide (5)
Ethylcyanoacetate (1), absolute alcohol were cooled to
0e5 ꢁC, to this was added dry ether and maintained 0e5 ꢁC
while passing dry HCl until its weight increased. Then the re-
action mass was kept aside for 24 h at 0 ꢁC. A colorless solid
precipitated out, filtered and dried under nitrogen atmosphere.
Compound (2) was found to very unstable; all the procedures
were carried out under nitrogen environment. Absolute alcohol
was saturated by passing gaseous ammonia at 0e5 ꢁC for 1 h.
To this was added ethyl-b-emino-b-ethoxy acrylate hydrochlo-
ride (2) at once, pH was maintained at w10 and further
ammonia gas was passed at 0e5 ꢁC for 4 h. The reaction mix-
ture was kept aside for 5 days at room temperature, which was
filtered and dried (in vacuo). Compound (3) was purified by
recrystallization from ethyl acetate. Potassium hydroxide
5.3. 2-Substituted-5,7-dimethyl pyrido[2,3-d]pyrimidin-
4(1H )-ones (8)
General procedure. A mixture of 7 (0.5 g) and nitrobenzene
(5 ml) was refluxed for 5 h. The reaction mixture was sub-
jected to steam distillation to remove nitrobenzene and the
solid obtained was filtered, washed with petroleum ether and
dried.
Table 4
MIC (in mg/ml) values of 2-substituted-5,7-dimethyl dihydropyrido[2,3-d]pyrimidin-4(1H)-ones (7 and 10)
Compounds
Molecular formula
Microorganisms
Gram positive
Bacillus subtilis
Gram negative
Staphylococcus aureus
Escherichia coli
Klebsiella pneumoniae
7a
7b
C17H19N3O3
C16H17N3O2
C18H21N3O4
C17H19N3O3
C15H11N3OCl2
C17H19N3O
C17H17N3O3
C16H17N3O3
C17H19N3O2
C16H17N3O3
C15H15N3O2
C13H13N3O2
C13H13N3OS
3.25
6.25
3.25
12.50
1.25
6.25
3.25
12.50
6.25
1.25
7c
7d
3.25
3.25
3.25
25.00
e
12.50
50.00
12.50
12.50
1.25
12.50
100.00
25.00
50.00
50.00
12.50
25.00
50.00
12.50
25.00
0.78
7e
25.00
6.25
6.25
7f
7g
12.50
25.00
50.00
6.25
7h
7i
12.50
3.25
3.25
7j
7k
25.00
25.00
3.25
25.00
50.00
12.50
12.50
0.39
50.00
50.00
25.00
12.50
0.39
10a
10b
6.25
0.78
Ciprofloxacin

1376
B. Lakshmi Narayana et al. / European Journal of Medicinal Chemistry 44 (2009) 1369e1376
Table 5
MIC (in mg/ml) values of 2-substituted-5,7-dimethyl pyrido[2,3-d]pyrimidin-4(1H)-ones (8 and 11)
Compounds
Molecular formula
Microorganisms
Gram positive
Bacillus subtilis
Gram negative
Staphylococcus aureus
Escherichia coli
Klebsiella pneumoniae
8a
8b
C17H17N3O3
C16H15N3O2
C18H19N3O4
C17H17N3O3
C15H9N3OCl2
C17H17N3O
C17H15N3O3
C16H115N3O3
C17H17N3O2
C16H15N3O3
C15H13N3O2
C13H11N3O2
C13H11N3OS
6.25
25.00
12.50
12.50
50.00
12.50
25.50
25.00
12.50
50.00
50.00
12.50
12.50
0.78
6.25
12.50
6.25
12.50
12.50
25.00
25.00
e
12.50
25.00
6.25
8c
8d
25.00
100.00
12.50
25.00
12.50
6.25
50.00
e
8e
8f
8g
12.50
25.00
50.00
6.25
50.00
50.00
50.00
12.50
50.00
100.00
50.00
50.00
0.78
8h
8i
8j
8k
50.00
50.00
25.00
25.00
0.39
50.00
50.00
25.00
12.50
0.39
11a
11b
Ciprofloxacin
[3] L.F. Kuyper, J.M. Garvey, D.P. Baccanari, J.N. Champness,
4 (1996)
5.4. Antibacterial assays (in vitro)
D.K. Stanmmers, C.R. Beddell, Bioorg. Med. Chem.
593e602.
All the test compounds was assayed in vitro for antibacte-
rial activity against different strains of Gram ꢀve [E. coli
(MTCC 722), and K. pneumoniae (MTCC 109)] and Gram
þve [B. subtilis (MTCC 441) and S. aureus (MTCC 96)] bac-
teria using standard protocol [27]. The minimum inhibitory
concentration (MIC) was determined by the test tube dilution
technique using ciprofloxacin as standard. The stock solution
(1 mg/ml) of test compounds was prepared in DMSO. The
stock solution was sterilized by passing through a 0.2 mm pol-
ycarbonate sterile membrane (Nuclepore) filter. Further the se-
rial dilution of test compounds was carried out and the
following concentrations were used: 100, 50, 25, 12.5, 6.25,
3.25, 1.25 mg/ml. Test compounds at various concentrations
were added to culture medium in a sterilized borosilicate
tube and different bacterial strains were inoculated at 106 ba-
cilli/ml concentration. The tubes were incubated at 37 ꢁC for
24 h and then examined for the presence or absence of growth
of the test organisms. All experiments were performed in trip-
licate. The MIC values were obtained from the lowest concen-
tration of the test compound where the tubes remained clear,
indicated that the bacterial growth was completely inhibited
at this concentration.
[4] A. Gangjee, O.O. Adair, S.F. Queener, J. Med. Chem. 46 (2003) 5074e
5082.
[5] A. Rosowsky, H. Chen, H. Fu, S.F. Queener, Bioorg. Med. Chem. 11
(2003) 59e67.
[6] A. Rosowsky, H. Chen, H. Fu, S.F. Queener, Bioorg. Med. Chem. 9
(2001) 2929e2935.
[7] A. Rosowsky, H. Chen, H. Fu, S.F. Queener, J. Med. Chem. 42 (1999)
2447e2455.
[8] A. Gangjee, A. Vasudevan, S.F. Queener, R.L. Kisliuk, J. Med. Chem. 39
(1996) 1438e1446.
[9] L. Cordeu, E. Cubedo, E. Bandres, A. Rebollo, X. Saenz, M. Font,
Bioorg. Med. Chem. 15 (2007) 1659e1669.
[10] J.R. Piper, G.S. McCaleb, J.A. Montgomery, R.L. Kisliuk, Y. Gaumont,
F.M. Sirotnaks, J. Med. Chem. 29 (1986) 1080e1087.
[11] B.R. Robins, G. Hitciiings, J. Am. Chem. Soc. 80 (1958) 3449e3458.
[12] J.M. Quintela, C. Peinador, L. Botana, M. Estevez, R. Riguera, Bioorg.
Med. Chem. 5 (1997) 1543e1553.
[13] G. Nam, C. Min Yoon, E. Kim, C.K. Rhee, J. Hyup Kim, J. Hyu Shin,
S. Hoon Kim, Bioorg. Med. Chem. Lett. 11 (2001) 611e614.
[14] G. Zhu Zheng, Y. Mao, C. Hung Lee, J.K. Patt, J.R. Koenig, R.J. Perner,
Bioorg. Med. Chem. Lett. 13 (2003) 3041e3044.
[15] C.J. Connolly, J.M. Hamby, M.C. Schroeder, M. Barvian, G.H. Lu,
R.L. Panek, Bioorg. Med. Chem. Lett. 7 (1997) 2415e2420.
[16] A. Monge, V. Martinez-Merino, C. Sanmartin, F.J. Fernandez,
M.C. Ochoa, C. Bellver, Eur. J. Med. Chem. 24 (1989) 209e216.
[17] H.A. Parish Jr., R.D. Gilliom, J. Med. Chem. 25 (1982) 98e102.
[18] R. Troschutz, J. Troschutz, M. Sollhuber-Kretzer, Arch. Pharm. 318
(1985) 777e781.
Acknowledgements
[19] C.H. Senanayake, L.E. Fredenburgh, R.A. Reamer, J. Liu, R.D. Larsen,
P.J. Reider, Tetrahedron Lett. 35 (1994) 5775e5778.
The authors wish to thank the Principal, University College
of Pharmaceutical Sciences, Kakatiya University, Warangal,
Andhra Pradesh, India for providing the facilities. One of
the authors (BLN) is grateful to AICTE, New Delhi for provid-
ing fellowship (JRF).
[20] S. Ravikanth, G. Venkat Reddy, D. Maitraie, V.V.V.V.N.S. Ramarao,
P. Shanthan Rao, B. Narsaiah, Synth. Commun. 34 (2004) 4463e4469.
[21] A. Monge, V. Martinez-Merino, M.A. Simon, C. Sanmartin, J. Hetero-
cycl. Chem. 29 (1992) 1545e1549.
[22] K.R. Devi, M.S. Reddy, Indian J. Chem. 33B (1994) 1013e1016.
[23] U.T. Bhalerao, A. Krishaiah, Indian J. Chem. 34B (1995) 587e590.
[24] B. Bonnaud, D.C.H. Bigg, Synthesis 34 (1994) 465e467.
[25] L. Zachariah, M.S. Reddy, Indian J. Chem. 32B (1993) 826e829.
[26] S.O.K. Moon, J. Lee, T. Lee, Exp. Mol. Med. 30 (1998) 29e40.
[27] NCCLS, Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria, Which Grows Aerobically Approved Standard M7-A5, fifth
ed. NCCLS, Villanova, PA, 2000.
References
[1] S. Ravikanth, G.V. Reddy, K.H. Kishore, P. Shanthanrao, B. Narsaiah,
S.N. Murthy, Eur. J. Med. Chem. 41 (2006) 1011e1016.
[2] N. Kumar, G. Singh, A.K. Yadav, Heteroat. Chem. 12 (2001) 52e56.