Synthesis, characterization and biological activity of new cyclization products of 3-(4-substituted benzylidene)-2H-pyrido[1,2-a]pyrimidine 2,4-(3H)-diones

Article abstract of DOI:10.1007/s12039-013-0367-0

A method is presented for the synthesis of 4-(substituted phenyl)-3-(3-substituted phenyl)4H-spiro[isoxazole-5,3′-pyrido[1,2-a] pyrimidine]-2′,4′-dione (3), 3-(4-substituted phenyl)-3H- isoxazole[3, 4-d]pyrido[1,2-a]pyrimidin-4-(3aH)-one (4) and 3-(4-substituted phenyl) 3,3a-dihydropyrazolo[3,4-d]pyrido[1,2-a]pyrimidin-4-(2H)-one (5) which consists of the conversion of 2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione (1) to chalcones (2) and their 1,3-dipolar cycloaddition with appropriate aldoximes to give spiro compounds and heterocyclization using amines to yield isoxazolines and pyrazolines. All the compounds were screened for their antimicrobial and antitubercular activity.

Full text of DOI:10.1007/s12039-013-0367-0

c
J. Chem. Sci. Vol. 125, No. 2, March 2013, pp. 305–312. ꢀ Indian Academy of Sciences.
Synthesis, characterization and biological activity of new cyclization
products of 3-(4-substituted benzylidene)-2H-pyrido[1,2-a]pyrimidine
2,4-(3H)-diones
ABHA BISHNOIa,∗, SURUCHI SINGHa, ANIL K TIWARIa, KRISHNA SRIVASTAVAb,
RAM RAGHUVIRc and CHANDRAKANT M TRIPATHIc
aDepartment of Chemistry, University of Lucknow, University Road, Lucknow 226 007, India
bDepartment of Chemistry, Sri Ramswaroop College of Engineering and Management, Faizabad Road,
Lucknow 226 001, India
cDivision of Pharmacology, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226 001, India
e-mail: dr.abhabishnoi@gmail.com; abhabishnoi5@gmail.com; anilk.tiwari04@gmail.com
MS received 10 March 2012; revised 10 June 2012; accepted 25 July 2012
Abstract. A method is presented for the synthesis of 4-(substituted phenyl)-3-(3-substituted phenyl)4H-
spiro[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione (3), 3-(4-substituted phenyl)-3H-isoxazole[3,
4-d]pyrido[1,2-a]pyrimidin-4-(3aH)-one (4) and 3-(4-substituted phenyl) 3,3a-dihydropyrazolo[3,4-
d]pyrido[1,2-a]pyrimidin-4-(2H)-one (5) which consists of the conversion of 2H-pyrido[1,2-a]pyrimidine-
2,4(3H)-dione (1) to chalcones (2) and their 1,3-dipolar cycloaddition with appropriate aldoximes to give
spiro compounds and heterocyclization using amines to yield isoxazolines and pyrazolines. All the compounds
were screened for their antimicrobial and antitubercular activity.
Keywords. Antimicrobial; antitubercular; pyrimidine; spiroisoxazolines; pyrazoline.
1. Introduction
antiviral,20 analgesic,21 antidiabetic22 and anticancer23
activities. In addition to this, isoxazoline derivatives
have played a crucial role in the theoretical deve-
lopment of heterocyclic chemistry and are also used
extensively in organic synthesis.
Spiroisoxazolines are heterocyclic nuclei which have
stimulated much interest in the field of medicinal
and biological chemistry.24 They display interesting
plant growth regulatory,25 herbicidal26 and antitu-
mour27 activities. Antitubercular and antimicrobial acti-
vity28,29 of some spiroisoxazoline derivatives have also
been reported recently. The results clearly show that
spiroisoxazoline-based compounds have the ability to
become drugs of immense use in the above mentioned
areas.
Pyrazolopyrimidines and fused heterocycles related
to it have also been identified as bioactive molecules.
They are known to function as CNS depressants,30 neu-
roleptic,31 antihypertensive,32 analgesic,33 antimicro-
bial34 and tuberculostatic35 agents and have also been
identified as adenosine receptors.36
Encouraged by the diverse biological activities of
isoxazoline, pyrazoline, spiroisoxazoline and pyrimi-
dine compounds, in our investigation we found an inter-
esting approach to synthesize these ring systems fused
with pyrimidine nucleus.
Compounds incorporating heterocyclic ring systems
continue to attract considerable interest due to the wide
range of biological activities they possess. Amongst
them, five-membered heterocyclic compounds occupy
a unique place in the realm of natural and synthetic
organic chemistry. Five-membered heterocycles like
isoxazoline and pyrazoline have found wide applica-
tions in the fields of pharmaceutical and agrochemical
chemistry.
The value of pyrimidine derivatives is significant
among various heterocycles, as they are found to
possess antineoplastic,1–3 antiviral,4–6 antibiotic,7 anti-
inflammatory,8 antifungal,9 antitumour10,11 and other
diverse biological activities.12–15 Many pyrimidine
derivatives are used for the production of thyroid drugs
and in the treatment of leukemia also.
In recent years, attention has been increasingly drawn
to the synthesis of isoxazolines and pyrazolines as
a new source of antibacterial agents. Isoxazoline
derivatives have been reported to possess antifun-
gal,16 antibacterial,17 antitumour,18 antiinflammatory,19
∗For correspondence
305

306
Abha Bishnoi et al.
benzylidene), 8.82 (d, 1H, C6H); 13C NMR (CDCl3) δ:
2. Experimental
117.4, 125.7, 128.2, 128.5, 131.6, 132.9, 133.4, 133.7,
135.1, 138.4, 147.1, 148.9, 157.2, 178.7, 180.7; Anal.
Calcd. C: 63.27, H: 3.16, N: 9.84, Found C: 63.45, H:
3.08, N: 9.89%.
All melting points are uncorrected. IR spectra were
recorded on a Perkin-Elmer 1430 spectrophotometer
using KBr pellets. Mass spectra were recorded on a Va
1
70-70H spectrophotometer at 70 eV. H and 13C spec-
tra were recorded in CDCl3 or DMSO using TMS as an
internal standard on a Bruker F 400 MHz NMR spec-
trophotometer, respectively. Elemental analyses were
carried out on Carlo Erba EA-1108 element analyzer.
2.2c 3-(Benzo[d][1,3]dioxol-5-ylmethylene)-2H-
pyrido[1,2-a]pyrimidine-2,4(3H)-dione
2c: Light
brown crystals, yield 78%, mp 202◦C; Anal. Calcd. C:
65.30, H: 3.40, N: 9.52, Found C: 65.0, H: 3.37, N:
9.49%.
2.1 General procedure for the preparation
of 2H-pyrido[1,2-a]pyrimidine-2,4(3H)-dione 1
2.3 General procedure for the preparation
of 4-(substituted phenyl)-3-(substituted phenyl)-4H-
spiro[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-
dione 3a–f
2-Amino pyridine (2 g, 0.0212 mol) and malonic acid
(2.208 g, 0.0211 mol) were refluxed in absolute ethanol
(20 mL) under anhydrous conditions for 12 h and excess
solvent was then distilled off. Crystalline solid which
separated on cooling was collected and recrystallized
with methanol. mp 180◦C ; IR (KBr)cm−1: 3438 (O–H),
3100 (C–H), 2930 (C–H aliphatic), 1700 (C=O), 1656
(C=N); 1H NMR (CDCl3) δ: 3.29 (CH2 protons), 7.28
(t, 1H, C7H), 7.5 (d, 1H, C9H), 7.7 (t, 1H, C8H), 8.82 (d,
1H, C6H); 13C NMR (CDCl3) δ: 40.20 (CH2 carbons),
121.7, 124.20, 125.2, 130.3, 149.5, 170.08, 171.1. Anal.
Calcd. C: 59.26, H: 3.70, N: 17.28, Found C: 60.00, H:
3.58, N: 17.14%.
A mixture of arylidene 2a–c (0.068 mol) and para
substituted benzadoximes (0.07 mol) was stirred in
chloroform (20 ml) at cold temperature (0–5◦C). To
this mixture was added 15 ml of sodium hypochlorite
(degree = 12 g NaClO4/100 g solution) drop-wise over
20 min keeping the temperature below 5◦C. The reac-
tion mixture was then stirred for 4–8 h at room tem-
perature, until TLC indicated complete disappearance
of reactants. The chloroform layer was then separated,
washed with water and dried over anhydrous sodium
sulphate. Excess of chloroform was then distilled off.
The compounds 3(a–f) obtained, were recrystallized
from ethanol.
2.2 General procedure for the preparation
of 3-(4-substituted benzylidene)-2H-pyrido[1,2-
a]pyrimidine-2,4(3H)-dione 2a–c
2.3a 3-(3-Nitrophenyl)-4-phenyl-4H-spiro[isoxazole-
The desired compounds 2a–c were prepared via Claisen
condensation of 1 with substituted benzaldehydes in the
presence of NaOH by known procedure.37–41
5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione
3a: Pale
yellow crystals, yield 66%, mp 167◦C; IR (KBr)cm−1:
1
1710 (C=O), 1533 (C=N), 1310 (C–O); H NMR
(CDCl3) δ: 5.36 (s, 1H, 4-CHAr), 6.05–6.96 (m, 9H,
Ar-H), 7.21–8.05 (m, 3H, protons of pyridine ring),
8.72 (d, 1H, proton adjacent to N-atom); 13C NMR
(CDCl3) δ: 33.4 (CH2 of oxazole), 94.2 (C–O of oxa-
zole), 121, 124.9, 125.1, 125.5, 125.8, 126.3, 127.2,
127.4, 128.5, 128.8, 130.9, 131.3, 134.2, 134.6, 135.7,
140.6, 150.4, 166.4, 175.3, 195.8; Mass: M+ 414, 164,
160, 122, 93, 90, 78, 28; Anal. Calcd. C: 66.33, H:
3.52, N: 14.07, Found C: 66.26, H: 3.49, N: 13.96%.
2.2a 3-Benzylidene-2H-pyrido[1,2-a]pyrimidine-
2,4(3H)-dione 2a: Yellowish white crystals, yield
75%, mp 180◦C; IR (KBr)cm−1: 3016 (Ar-H), 1680
1
(C=O), 1634 (C=N); H NMR (CDCl3) δ: 6.05–7.09
(m, 5H, Ar-H), 7.18–7.80 (m, 3H, C7–9), 8.25 (s, 1H,
benzylidene), 8.80 (d, 1H, C6H); 13C NMR (CDCl3) δ:
118.5, 125.6, 127.7, 127.9, 128.4, 128.7, 128.8, 133.2,
134.6, 138.4, 145.2, 148.3, 156.4, 179, 181.3; Anal.
Calcd. C: 72.00, H: 4.03, N: 11.20, Found C: 71.89, H:
3.94, N: 11.17%.
2.3b 3-(4-Chlorophenyl)-4-phenyl-4H-spiro[isoxazole-
5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione 3b: Light
yellow crystals, yield 68%, mp 171◦C; IR (KBr)cm−1:
2.2b 3-(4-Chlorobenzylidene)-2H-pyrido[1,2-a]
pyrimidine-2,4(3H)-dione 2b: Brown crystals, yield 1722 (C=O), 1530 (C–N), 1318 (C–O), 737 (C–Cl);
79 %, mp 168◦C; IR (KBr)cm−1: 3009 (Ar-H), 1676 1H NMR (CDCl3) δ: 5.33 (s, 1H, 4-CHAr), 6.12–6.89
1
(C=O), 1630 (C=N); H NMR (CDCl3) δ: 6.15–7.09 (m, 9H, Ar-H), 6.99–7.94 (m, 3H, protons of pyri-
(m, 4H, Ar-H), 7.45–7.97 (m, 3H, C7–9), 8.30 (s, 1H, dine ring), 8.75 (d, 1H, proton adjacent to N-atom);

New pyridopyrimidin-diones/ones
307
13C NMR (CDCl3) δ: 32.6, 93.3, 119.5, 125.6, 125.7, dione 3f: White crystals, yield 67%, mp 206◦C; IR
126.2, 127.4, 127.6, 128.2, 128.3, 128.5, 128.7, 128.8, (KBr)cm−1: 1698 (C=O), 1538 (C=N), 1250 (C–O),
128.9, 131.8, 135.6, 136.2, 140.9, 151.2, 165.6, 174.8, 756 (C–Cl); 1H NMR (CDCl3) δ: 5.11 (s, 1H, 4-CHAr),
192.1; Mass: M+ 403, 405(M+2), 160, 155, 153, 113, 6.05 (s, 2H, -CH2 of piperonal ring), 7.05–7.86 (m, 7H,
111, 93, 90, 78, 28; Anal. Calcd. C: 65.43, H: 3.47, N: Ar-H), 7.94–8.34 (m, 3H, protons of pyridine ring),
10.41, Found C: 65.50, H: 3.51, N: 10.45%.
8.70 (d, 1H, proton adjacent to N-atom); 13C NMR
(CDCl3) δ: 33.1, 93.9, 100.80, 108.4, 108.9, 116.9,
120.6, 125.1, 125.4, 126.3, 129.3, 130.5, 130.7, 133.9,
134.6, 135.7, 135.8, 145.8, 148.1, 151.5, 164.9, 176.3,
190.5; Mass: M+ 447, 449 (M+2), 160, 155, 153, 134,
113, 111, 93, 78, 28; Anal. Calcd. C: 61.68, H: 3.13, N:
9.39, Found C: 62.00, H: 3.23, N: 9.42%.
2.3c 4-(4-Chlorophenyl)-3-(3-nitrophenyl)-4H-spiro
[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione 3c:
Yellow crystals, yield 66.5%, mp 189◦C; IR (KBr)
cm−1: 1726 (C=O), 1528 (C–N), 1314 (C–O), 741
1
(C–Cl); H NMR (CDCl3) δ: 5.69 (s, 1H, 4-CHAr),
6.76–7.18 (m, 8H, Ar-H), 7.26–8.09 (m, 3H, protons of
pyridine ring), 8.81 (d, 1H, proton adjacent to N-atom);
13C NMR (CDCl3) δ: 34.2, 93.9, 120.6, 124.8, 125.1,
125.3, 126.2, 128.5, 128.7, 129.2, 129.6, 130.8, 131.4,
131.7, 134.5, 134.6, 135.0, 139.1, 148.5, 164.7, 174.9,
2.4 General procedure for the preparation
of 3-(4-substituted phenyl)-3H-isoxazolo[3,4-d]
pyrido[1,2-a]pyrimidin-4(3aH)-one 4a–c
192.6; Mass: M+ 448, 500 (M+2), 160, 155, 153, 124, A mixture of compound 2 (0.01 mol) and hydroxy-
122, 113, 111, 93, 78, 28; Anal. Calcd. C: 61.04, H: lamine hydrochloride (0.01 mol) in 2% ethanolic
3.00, N: 12.95, Found C: 60.87, H: 2.98, N: 13.02%.
sodium hydroxide solution (5 ml) was heated to reflux
for 6–7 h. The progress of the reaction was monitored
by TLC. After completion of the reaction, the solvent
was removed under reduced pressure and the residue
was added to ice water (15 ml). The resulting solution
was neutralized by dilute HCl and extracted with CHCl3
(20 ml). Chloroform layer was dried over anhydrous
sodium sulphate, filtered and concentrated to obtain
the product. The crude product was recrystallized with
appropriate solvent.
2.3d 3-(4-Chlorophenyl)-4-(4-chlorophenyl)-4H-spiro
[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione 3d:
Yellowish white crystals, yield 69.2%, mp 185–186◦C;
IR (KBr)cm−1: 1706 (C=O), 1539 (C=N), 1313
1
(C–O), 760 (C–Cl); H NMR (CDCl3) δ: 5.61 (s, 1H,
4-CHAr), 6.70–7.09 (m, 8H, Ar-H), 7.55–8.18 (m, 3H,
protons of pyridine ring), 8.79 (d, 1H, proton adjacent
to N-atom); 13C NMR (CDCl3) δ: 32.1, 93.1, 116.0,
125.3, 125.7, 126.4, 128.5, 128.8, 128.9, 129.4, 129.7,
130.8, 131.4, 131.6, 133.8, 135.1, 135.4, 139.0, 150.2,
165.1, 174.8, 190.2; Mass: M+ 437, 160, 155, 153,
124, 113, 111, 93, 78, 28; Anal. Calcd. C: 60.27, H:
2.97, N: 9.59, Found C: 60.16, H: 3.0, N: 9.60%.
2.4a 3-Phenyl-3H-isoxazolo[3,4-d]pyrido[1,2-a]
pyrimidin-4(3aH)-one 4a: Light brown crystals,
yield 77%, mp >290◦C; Recrystallization solvent:
Ethanol; IR (KBr) cm−1: 1726 (C=O), 1545 (C=N),
1
1312 (C–O); H NMR (CDCl3) δ: 2.65 (d, 1H, C3),
4.01 (d, 1H, C3a), 7.22–7.38 (m, 5H, Ar-H), 7.45–8.0
(m, 3H, protons of pyridine ring), 8.24 (d, 1H, proton
adjacent to N-atom,); 13C NMR (CDCl3) δ: 40.5, 82.9,
120.6, 125.6, 125.8, 126.8, 127.2, 127.6, 128.3, 128.4,
135.4, 140.7, 150.4, 164.3, 173.2; Mass: M+ 265, 188,
160, 93, 78, 77; Anal. Calcd. C: 67.92, H: 4.15, N:
15.85, Found C: 68.15, H: 4.06, N: 15.92%.
2.3e 4-(Benzo[d][1,3]dioxol-5-yl)-3-(3-nitrophenyl)-
4H-spiro[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,
4ꢁ-dione 3e: Shiny white crystals, yield 66%, mp
212◦C; IR (KBr)cm−1: 1692 (C=O), 1540 (C=N),
1
1246 (C–O); H NMR (CDCl3) δ: 5.03 (s, 1H, 4-
CHAr), 6.07 (s, 2H, -CH2 of piperonal ring), 6.96–7.44
(m, 7H, Ar-H), 7.69–8.27 (m, 3H, protons of pyridine
ring), 8.7 (d, 1H, proton adjacent to N-atom); 13C NMR
(CDCl3) δ: 33.7, 94.5, 108.3, 109.2, 117.4, 121.6,
125.3, 125.6, 125.8, 126.2, 130.7, 131.2, 133.8, 134.0,
134.6, 135.3, 146.5, 147.9 (Ar-C), 151.8, 165.1, 178.2,
195.7; Mass: M+ 458, 164, 160, 134, 122, 93, 78, 28;
Anal. Calcd. C: 60.26, H: 3.06, N: 12.23, Found C:
60.18, H: 3.12, N: 12.18%.
2.4b 3-(4-Chlorophenyl)-3H-isoxazolo[3,4-d]pyrido
[1,2-a]pyrimidin-4(3aH)-one 4b: Brownish white
crystals, yield 76.5%, mp 188◦C; Recrystallization
solvent: Ethanol; IR (KBr) cm−1: 1729 (C=O), 1542
(C=N), 1319 (C–O), 754 (C–Cl); 1H NMR (CDCl3) δ:
2.3 (d, 1H, C3), 3.75 (d, 1H, C3a), 7.36–7.41 (m, 4H,
Ar-H), 7.43–7.50 (m, 3H, protons of pyridine ring),
8.28 (d, 1H, proton adjacent to N-atom); 13C NMR
(CDCl3) δ: 40.3, 83.2, 119.7, 125.4, 125.7, 126.3,
2.3f 4-(Benzo[d][1,3]dioxol-5-yl)-3-(3-chlorophenyl)-
4H-spiro[isoxazole-5,3ꢁ-pyrido[1,2-a]pyrimidine]-2ꢁ,4ꢁ-

308
Abha Bishnoi et al.
126.6, 129.1, 129.4, 133.4, 135.8, 138.6 (Ar-C), 150.3, 126, 124, 113, 111, 93, 78, 42; Anal. Calcd. C: 60.30,
164.0, 172.5; Mass: M+ 299, 301 (M+2), 188, 160, H: 3.69, N: 18.76, Found C: 60.17, H: 3.72, N: 18.69%.
126, 124, 113, 111, 93, 78; Anal. Calcd. C: 60.10, H:
3.33, N: 14.02, Found C: 59.84, H: 3.30, N: 14.16%.
2.5c 3-(Benzo[d][1,3]dioxol-4-yl)-3,3a-dihydropyra-
zolo[3,4-d]pyrido[1,2-a]pyrimidin-4(2H)-one
5c:
2.4c 3-(Benzo[d][1,3]dioxol-4-yl)-3H-isoxazolo[3,4-
White crystals, yield 76%, mp 179–180◦C; IR (KBr)
d]pyrido[1,2-a]pyrimidin-4(3aH)-one
4c: Dirty
cm−1: 3310 (N–H), 1720 (C=O), 1529 (C=N); H
1
white crystals, yield 71%, mp 168–169◦C; Recrys-
NMR (CDCl3) δ: 3.60 (d, 1H, C3), 5.00 (d, 1H, C3a),
6.09 (s, 2H, O-CH2-O-), 7.51 (d, 2H, Ar-H), 7.64 (d,
1H, Ar-H), 7.93–8.27 (m, 3H, protons of pyridine ring),
8.68 (d, 1H, proton adjacent to N-atom); 13C NMR
(CDCl3) δ: 45.4, 53.4, 101.6, 110.2, 112.4, 116.6,
118.0, 125.3, 125.7, 135.4, 135.9, 145.7, 148.6, 151.2,
155.9, 172.7; Mass: M+ 308, 187, 145, 121, 93, 78, 70,
42; Anal. Calcd. C: 62.34, H: 3.90, N: 18.18, Found C:
63.14, H: 3.86, N: 18.07%.
tallization solvent: Methanol; IR (KBr)cm−1: 1731
1
(C=O), 1542 (C=N), 1310 (C–O); H NMR (CDCl3)
δ: 2.25 (d, 1H, C3), 3.80 (d, 1H, C3a), 6.12 (s, 2H,
O-CH2-O-), 7.37 (d, 2H, Ar-H), 7.43 (d, 1H, Ar-H),
7.5–8.19 (m, 3H, protons of pyridine ring), 8.56 (d, 1H,
proton adjacent to N-atom); 13C NMR (CDCl3) δ: 40.5,
83.5, 101.3, 109.0, 110.1, 116.5, 119.4, 125.4, 125.7,
133.9, 135.2, 146.9, 149.3, 150.8, 164.5, 172.2; Mass:
M+ 309, 188, 160, 121, 93, 78; Anal. Calcd. C: 62.13,
H: 3.56, N: 13.59, Found C: 62.18, H: 3.51, N: 13.60%.
2.6 Chemistry
2.5 General procedure for the preparation
of 3-(4-substituted phenyl)-3,3a-dihydropyrazolo[3,4-
d]pyrido[1,2-a]pyrimidin-4(2H)-one 5a–c
The starting material 2H-pyrido[1,2-a]pyrimidine-
2,4(3H)dione 1 was prepared via cyclization reaction
between 2-amino pyridine and malonic acid in the
presence of ethanolic sodium hydroxide. This under-
went Claisen condensation with aromatic aldehydes
to afford chalcones 2a–c. The chalcones were reacted
successfully with aromatic oximes, hydroxylamine and
hydrazine hydrate under different reaction conditions
to give cyclized products 4-(substituted phenyl)-3-
(3-substituted phenyl)4H-spiro[isoxazole-5,3ꢁ-pyrido
[1,2-a]pyrimidine]-2ꢁ,4ꢁ-dione 3a–f, 3-(4-substituted
phenyl)-3H-isoxazole[3,4-d]pyrido[1,2-a]pyrimidin-
4-(3aH)-one 4a–c and 3-(4-substituted phenyl)
3,3a-dihydropyrazolo[3,4-d]pyrido[1,2-a]pyrimidin-4-
(2H)-one 5a–c. In compounds 3a–f, the oxygen atom
of oxime is attached at the most substituted carbon of
dipolarophile 2a–c as generally observed for reaction
of nitrones and ethylenic dipolarophiles42,43 whereas
compounds 4a–c and 5a–c are formed presumably by
way of the hydrazones, followed by cyclization and
proton transfer.
A mixture of compound 2 (0.01 mol) and hydrazine
hydrate (0.013 mol) in absolute ethanol (15 ml) was
refluxed for 10–12 h. The reaction mixture was concen-
trated and the crude products thus obtained were then
purified by column chromatography using a mixture of
n-hexane-ethyl acetate (2:1).
2.5a 3-Phenyl-3,3a-dihydropyrazolo[3,4-d]pyrido[1,
2-a]pyrimidin-4(2H)-one 5a: Wheatish brown crys-
tals, yield 75%, mp 175◦C; IR (KBr) cm−1: 3323 (N–
1
H), 1716 (C=O), 1531 (C=N); H NMR (CDCl3) δ:
3.50 (d, 1H, C3), 4.45 (d, 1H, C3a), 7.06 (s, 1H, -N-H,
D2O exchangeable), 7.3–7.54 (m, 5H, Ar-H), 7.57–
8.32 (m, 3H, protons of pyridine ring), 8.35 (d, 1H,
proton adjacent to N-atom); 13C NMR (CDCl3) δ: 45.8,
53.5, 118.4, 125.3, 125.5, 125.6, 125.9, 126.4, 128.7,
128.9, 135.2, 143.5, 150.7, 156.2, 172.8 (C=O); Mass:
M+ 264, 145, 93, 78, 77, 42; Anal. Calcd. C: 68.18, H:
4.55, N: 21.21, Found C: 67.97, H: 4.52, N: 20.99%.
The structures of new compounds were assigned on
1
the basis of their analytical and spectral data (IR, H,
13C NMR and Mass). The characteristic C=O bands
appeared in the 1700–1680 cm−1 region in the FT-IR
spectra of 1 and 2a–c. The FT-IR spectra of compounds
3a–f, 4a–c and 5a–c showed the presence of C=N band
in the region 1585–1520 cm−1. Compound 1 displayed
2.5b 3-(4-Chlorophenyl)-3,3a-dihydropyrazolo[3,4-d]
pyrido[1,2-a]pyrimidin-4(2H)-one 5b: Muddy brown
crystals, yield 72%, mp 193◦C; IR (KBr) cm−1: 3286
1
(N–H), 1712 (C=O), 1527 (C=N), 744 (C–Cl); H
NMR (CDCl3) δ: 3.54 (d, 1H, C3), 5.01 (d, 1H, C3a),
7.33–7.40 (m, 4H, Ar-H), 7.48–8.15 (m, 3H, protons of
pyridine ring), 8.42 (d, 1H, proton adjacent to N-atom);
13C NMR (CDCl3) δ: 45.1, 53.1, 116.9, 125.7, 125.9,
127.3, 127.6, 127.8, 128.4, 132.3, 135.1, 141.6, 150.5,
155.7, 172.3 (C=O); Mass: M+ 298, 300 (M+2), 139,
1
in its H NMR spectra, in addition to ethylenic pro-
tons, a doublet at δ 3.29 ppm due to resonance of CH2
protons. In the benzylidene derivatives 2a–c, this sig-
nal was absent, confirming the condensation had taken
place. Regarding compounds 3a–f, the 1H NMR spectra

New pyridopyrimidin-diones/ones
309
O
O
HO
OH
N
N
N
NH2
Abs. EtOH
Reflux, 12 h
O
O
1
ArCHO, EtOH, NaOH
Ref. 37-41
NH2.NH2.2H2O,
Glacial AcOH
N
N
NH2OH,
2% NaOH
N
N
N
N
O
N
O
O
O
N
N
Ar
O
H
H
Ar
Ar
5 (a-c)
2 (a-c)
4 (a-c)
R
, NaOCl, CHCl3
NOH
N
O
N
O
O
N
Ar
R
3 (a-f)
Scheme 1. Synthetic route for cyclization products of 3-(4-substituted benzylidene)-
2H-pyrido[1,2-a]pyrimidine 2,4-(3H)-diones.
showed the presence of one singlet, H4 of the isoxa- as a solvent by Disc Diffusion method.44 The activ-
zolineic ring at 5.30–5.90 ppm. Compounds 4a–c and ity was compared with ciprofloxacin and gentamycin.
5a–c revealed in their 1H NMR spectra, two doublets in The results are presented in table 1. Antifungal activ-
the region 2.25–5.01 ppm due to one proton on each 3a– ities of these compounds were evaluated against Can-
CH and 3–CH, respectively, showing that cyclization dida albicans (ATCC24433) using ethanol as solvent
had occurred. 13C NMR of 3a–f showed distinct reso- by the same method.44 The activity was compared with
nances in agreement with the proposed structure. Spiro fluconazole and the results are presented in table 1. The
carbon resonated at δ 71.93 ppm and C=N carbon at zone of inhibition was measured after 24 h of incuba-
∼162-8 ppm. All the compounds have shown an excel- tion at 28◦C. The zone of inhibition developed, if any,
lent agreement between calculated and experimentally was then measured and recorded. Zone of inhibition for
obtained data for C, H, N analysis (scheme 1).
ethanol was done separately and found that there was
no activity.
2.7 Bioassay
2.7b Evaluation of antitubercular activity: All the
2.7a Evaluation of antimicrobial activity: Compounds compounds (3a–f, 4a–c and 5a–c) have been tested for
3a–f, 4a–c and 5a–c were evaluated in vitro for their their antitubercular activity through Microplate Alamar
antibacterial activity against Staphylococcus aureus Blue Assay (MABA).45 The compounds were dissolved
(ATCC9144), Bacillus subtilis (ATCC6633), Pseu- in dimethyl sulphoxide (DMSO) to make 5 mg/ml
domonas aeruginosa (ATCC25619), Klebsiella pneu- stock solutions. Serial dilutions from stocks were also
moniae and Escherichia coli (MTCC739) using ethanol made in DMSO. Standard anti-TB drugs (isoniazid and

310
Abha Bishnoi et al.
Table 1. Antimicrobial activity of the synthesized compounds.
Zone of inhibition (mm)
of fungal strain
Zone of inhibition (mm) of bacterial (Gram+ and Gram−) strains
Compounds
S. aureus
B. subtilis
P. aeruginosa
E. coli
K. pneumoniae
C. albicans
3a
3b
3c
3d
3e
3f
4a
4b
4c
5a
5b
5c
10
11
9
12
18
24
10
11
9
8
8
9
6
6
7
8
12
13
6
6
7
7
8
8
6
6
7
7
7
9
9
9
8
11
7
6
6
6
7
10
9
9
10
9
12
15
11
19
27
30
15
17
27
16
16
28
6
7
21
20
8
7
8
9
9
11
6
8
7
10
10
6
9
8
6
12
14
6
Control
Ciprofloxacin/
Gentamycin
Fluconazole
20/19
20/17
29
Table 2. Antitubercular activity of all the synthesized compounds.
Compounds %Inhibition@12.5 μg/mL %Inhibition@6.25 μg/mL %Inhibition@3.12 μg/mL
3a
3b
4
2
4
3
5
4
3c
3d
3e
3f
9
12
14
16
11
0
9
27
28
23
0
15
10
16
0
4a
4b
4c
0
2
0
2
0
2
5a
5b
0
0
0
0
0
0
5c
Isoniazid
0
100
2
100
3
100
rifampicin) were used as positive control and the vehi- S. aureus (18 and 24 mm), B. subtilis (21 and 20 mm)
cle (DMSO) was used as negative control. The per-
cent inhibition at different concentrations is presented
in table 2.
and the fungus C. albicans (27 and 30 mm, respec-
tively). It is noteworthy, that these two compounds
also inhibited the multiresistant P. aeruginosa to some
extent which is very encouraging. Rest of the synthe-
sized spiro compounds were moderately active. The
results were almost similar for isoxazole 4a–c and pyra-
zole derivatives 5a–c also. The benzo[d] [1,3]dioxole
substituted compounds 4c and 5c were more active than
the compounds with other substituents. These com-
pounds showed a marvelous zone of inhibition of 27
and 28 mm against the fungal strain C. albicans. The
increased activity of benzo[d] [1,3]dioxole substitution
3. Results and discussion
3.1 Antimicrobial activity
Among the spiro compounds 3a–f, the one having
benzo[d] [1,3]dioxole as substituents i.e., 3e and 3f
showed excellent activity against both bacterial and
fungal strains. The zone of inhibition of these sub-
stituents was quite considerable against the Gram+ve might be due to its structure leading to a better fit at

New pyridopyrimidin-diones/ones
311
Figure 1. Graphical representation of antimicrobial activity.
The isoxazole 4a–c and pyrazole 5a–c derivatives were
found to be more or less inactive. The percentage inhi-
bition at different concentration is expressed in μg/mL
and the results are shown in table 2 and, the graphical
representations are shown in figure 2.
4. Conclusion
In summary, we tested the new series of heterocycles
at several concentrations to evaluate their antimicro-
bial and antitubercular activity. Our results have shown
that among the spiro compounds 3a–f, the one having
benzo[d] [1,3]dioxole as a substituent showed excellent
activity against both bacterial and fungal strains, how-
ever no active antitubercular compound was identified.
The active compounds of the series would be promising
structural templates for the development of novel and
more efficient antimicrobial agents.
Figure 2. Graphical representation of antitubercular
activity.
the receptor site. The Diameter of zone of inhibition is
expressed in mm and the results are shown in table 1.
The graphical representations are shown in figure 1.
3.2 Antitubercular activity
Acknowledgements
The compounds of the spiro series were somewhat
active as they inhibited the mycobacteria to a consider-
able percentage (23–28%). Compounds 3d, 3e and 3f
showed good inhibition at a concentration of 12 μg/mL
which was maximum among all the synthesized com-
pounds. It was quite interesting to observe that many
compounds viz. 3a, 3b and 5c showed a decreased per-
centage of inhibition of the mycobacterium when their
concentration was increased. The activity of compound
3b was quite abnormal on increasing its concentration
from 3.12 μg/mL to 6.25 μg/mL, the percentage inhi-
bition increased from 9 to 12 but at 12.5 μg/mL the per-
centage inhibition was again 9 which is a considerable
decrease. This may be due to the toxicity of these com-
pounds at a higher concentration or may be due to some
other reason. However, it is a matter of further research.
The authors express their sincere thanks to the
Head, Department of Chemistry, Lucknow University,
Lucknow, for providing laboratory facilities. They are
also thankful to the Director, Central Drug Research
Institute, Lucknow for elemental analysis, spectral data
and biological activity studies.
References
1. Giller S A, Zhuk R A and Lidak M I U 1967 Dokl. Akad.
Nauk. SSR. 176 332
2. Curreri A R, Ansfield R J, McIver F H, Waisman H A
and Hedelberger C 1958 Cancer Res. 18 478
3. Chaudhary N K, Montag B J and Heidelberger C 1958
Cancer Res. 18 318

312
Abha Bishnoi et al.
4. Malik V, Singh P and Kumar S 2006 Tetrahedron 62 25. Howe R K and Shelton B R 1990 J. Org. Chem. 55
5944 4603
5. Holý A, Votruba I, Masojídková M, Andrei G, Snoeck 26. Smietana M, Gouverneur V and Mioskowski C 1999
R, Naesens L, De Clercq E and Balzarini J 2002 J. Med.
Chem. 45 1918
Tetrahedron Lett. 40 1291
27. Najim N, Bathich Y, Zain M M, Hamzah A H and
Shaameri Z 2010 Molecules 15(12) 9340
28. Kumar R R, Perumal S, Manju S C, Bhatt P, Yogeeswari
P and Sriram D 2009 Bioorg. Med. Chem. Lett. 19 3461
29. (a) Wright A D, King G M and Sticher O 1990 J. Nat.
Prod. 53 1615. (b) Abdou I M, Ayman M S and Hussein
F Z 2004 Molecules 9(3) 109
6. Andres P and Marhold A 1996 J. Fluorine Chem. 77 93
7. Reddick J J, Saha S, Lee J, Melnick J S, Perkins J and
Begley T P 2001 Bioorg. Med. Chem. Lett. 11 2245
8. Simoni D, Grisolia G, Giannini G, Roberti M, Rondanin
R, Piccagli L, Baruchello R, Rossivz M, Romagnoli R,
Invidiata F P, Grimaudo S, Jung M K, Hamel E, Gebbia
N, Crosta L, Abbadessa V, Di Cristina A, Dusonchet L,
Meli M and Tolomeo M 2005 J. Med. Chem. 48 723
9. Gauthier B 1963 Ann. Pharm. Fr. 21 655
10. O’ Reilly B C and Atwal K S 1987 Heterocycles 26 1185
11. Overman L E, Rabinowitz M H and Renhowe P A 1995
J. Am. Chem. Soc. 117 2657
12. Ghoneim K M and Youssef R 1986 J. Indian Chem. Soc.
53 914
13. Atwal K S and Moreland S 1991 Bioorg. Med. Chem.
Lett. 1 291
14. Godfraind T, Miller R and Wbo M 1986 Pharmacol.
Rev. 38 321
15. Ertan M, Balkan A, Sarac S, Uma S, Renaud J F and
Rolland Y 1991 Arch. Pharm. 324 135
30. Filler R 1974 Chem. Technol. 4 752
31. EI-Feky A and Abd-Ei-Samii Z K 1996 Pharmazie 51
540
32. Vijaya Raj K K, Narayan B V, Ashalatha N and Kumari
S 2006 J. Pharmacol. Toxicol. 1(6) 559
33. Bazgir A, Khanaposthani M M and Soorki A A 2008
Bioorg. Med. Chem. Lett. 18 5800
34. Moustafa M G, Zeinab H I and Soad A A 2004 Hetero-
cyclic Chem. 1 57
35. Stewart M, Steinig A G, Ma C, Song J P, Kibben B
M, Castelhano A L and Lennan S 2004 J. Biochem.
Pharmacol. 68 305
36. Cacciarini M, Cordero F M, Faggi C and Goti A 2000
Molecules 5 637
37. Vorlander D and Kunze K 1926 Ber. 59 2078
38. Garland C E and Reid E E 1925 J. Am. Chem. Soc. 47
2333
39. Vorlander D 1925 Ber. 58 118
40. Huitric A C and Kumler W D 1956 J. Am. Chem. Soc.
78 614
16. Korgaokar S S, Patil P H, Shab M T and Parekh H H
1996 Indian J. Pharm. Sci. 58 222
17. Cali P, Naerum L, Mukhija S and Hjelmencrantz A 2004
Bioorg. Med. Chem. Lett. 14 5997
18. Getal J 1975 Antibiot. 28(1) 91
19. Rapposelli S, Lapucci A, Minutolo F, Orlandini E,
Ortore G, Pinza M and Balsamo A 2004 Farmaco 59 25
20. Sechi M, Sannia L, Carta F, Palomba M, Dallocchio R,
Dessi A, Derudas M, Zawahir Z and Neamati N 2005
Antiviral Chem. Chemother. 16 41
41. Pfeiffer P, Angerf O, Backes P, Fitz W, Prahi E,
Rheinboldt H and Stoll W 1925 Ann. 441 228
42. Rastelli A, Gandolfi R and Amade M S 2000 Adv.
Quantum Chem. 36 151
21. Carr J B, Durham H G and Hass D K 1977 J. Med. Chem.
20 934
22. Cottineau B, Toto P, Marot C, Pipaud A and Chenault J
2002 Bioorg. Med. Chem. Lett. 12 2105
43. Sahm D F and Washington 1991 Antibacterial suscepti-
bility tests: dilution methods, Manual of Clinical Micro-
biology, 5th ed., (eds) A Balows, W J Hausler Jr., K L
Herrmann, H D Isenberg and H J Shadomy, Washington
DC, American Society for Microbiology, 1105–1116
23. Simoni D, Manfredini S, Tabrizi M A, Bazzanini R,
Guarinini M, Ferroni R, Traniello F, Nastruzzi C G and 44. Collins L A and Franzblau S G 1997 Antimicrob. Agents
Gambari R 1991 Top. Mol. Organ. Eng. 8 119
24. Faulkner D 2001 J. Nat. Prod. Rep. 18 1
Chemother. 41 1004
45. Vorländer D and Hobohm K 1886 Ber. 29 1836