Synthesis, antimicrobial, and mosquito larvicidal activity of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6, 10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones

Article abstract of DOI:10.1016/j.bmcl.2010.08.060

A series of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6, 10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones (6a-h) have been synthesized by cyclization of ethyl-3-aryl-4-(2-chlorophenyl)-6-methyl-1-(5-methylisoxazol-3- yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 4a-h with 3-amino-5-methylisoxazole 5. Compounds 4a-h were obtained by Biginelli reaction, by condensation of aromatic aldehyde 1, ethyl acetoacetate 2, and isoxazolyl thioureas 3 in a one-pot reaction catalyzed by ceric ammonium nitrite (CAN). Compounds 6a-h were tested for their antibacterial and antifungal activities against various bacterial and fungal strains. The results showed that these compounds exhibited good antibacterial and antifungal activity compared with that of standard antibiotics. Mosquito larvicidal activity of the newly synthesized compounds 6a-h is also studied against fourth instar larvae Culex quinquefasciatus. Some of the compounds are proved to be lethal for mosquito larvae.

Full text of DOI:10.1016/j.bmcl.2010.08.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6052–6055
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antimicrobial, and mosquito larvicidal activity of
1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-
1H-pyrimido[5,4-c]quinolin-5-ones
E. Rajanarendar a, , M. Nagi Reddy a, K. Rama Murthy a, K. Govardhan Reddy a, S. Raju a, M. Srinivas a,
⇑
B. Praveen b, M. Srinivasa Rao b
a Department of Chemistry, Kakatiya University, Warangal 506 009, AP, India
b Division of Biology, Indian Institute of Chemical Technology, Hyderabad 500 007, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimi-
Received 3 April 2010
Revised 24 July 2010
Accepted 12 August 2010
Available online 16 August 2010
do[5,4-c]quinolin-5-ones (6a–h) have been synthesized by cyclization of ethyl-3-aryl-4-(2-chlorophenyl)-
6-methyl-1-(5-methylisoxazol-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 4a–h with
3-amino-5-methylisoxazole5. Compounds4a–hwereobtainedbyBiginellireaction, bycondensationofaro-
matic aldehyde 1, ethyl acetoacetate 2, and isoxazolyl thioureas 3 in a one-pot reaction catalyzed by ceric
ammonium nitrite (CAN). Compounds 6a–h were tested for their antibacterial and antifungal activities
against various bacterial and fungal strains. The results showed that these compounds exhibited good anti-
bacterial and antifungal activity compared with that of standard antibiotics. Mosquito larvicidal activity of
the newly synthesized compounds 6a–h is also studied against fourth instar larvae Culex quinquefasciatus.
Some of the compounds are proved to be lethal for mosquito larvae.
Keywords:
Isoxazolyl pyrimidoquinolines
Biginelli reaction
Antibacterial activity
Antifungal activity
Mosquito larvicidal test
Ó 2010 Elsevier Ltd. All rights reserved.
The increasing incidence of bacterial and fungal resistance to a
large number of antimicrobial agents have prompted studies on
the development of new potential antimicrobial compounds. The
molecular manipulation of promising lead compounds is still a ma-
jor line of approach to develop new drugs. So, the discovery of no-
vel and potent antimicrobial agents is the best way to overcome
microbial resistance and develop effective therapies.1
Isoxazole derivatives represent an interesting class of com-
pounds possessing a wide spectrum of biological activities. A large
number of isoxazole derivatives exhibited antibacterial,2 anti-
fungal,3 anticonvulsant,4 analgesic,5 and anticancer6 activity. Vari-
ous substituted dihydropyrimidines have attracted considerable
interest because of their promising activity as calcium channel
PLT antagonists,21 and antimalarial activity.22 New hybrid moieties
secured by linking isoxazoles with pyrimidines and quinolines
promise to offer fascinating scaffolds. Design of synthetic methods
for the efficient preparation of these tri heterocyclic compounds is
necessary. Hence, we embarked on the synthesis and bioassay of
the compounds having isoxazole, pyrimidine, and quinoline moie-
ties embedded in a fused molecular frame work to improve specific-
ity and efficacy of these scaffolds against microorganisms. As a
sequel to our work on the synthesis of fused isoxazoles,23–26 we re-
port herein, the synthesis and biological evaluation of a novel series
of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,
10b-tetrahydro-1H-pyrimido[5,4-c]-quinolin-5-ones.
Biginelli’s one-pot condensation of 2-chlorobenzaldehyde 1,
ethyl acetoacetate 2, and 1-(5-methylisoxazol-3-yl)-3-phenyl-
thioureas 327 in presence of 10 mol % of ceric ammonium nitrite
(CAN) in methanol (10 mL) at 80 °C with stirring for 3 h afforded
ethyl-3-aryl-4-(2-chlorophenyl)-6-methyl-1-(5-methylisoxazol-3-
yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 4 in 80–
90% yields.28
blockers, antihypertensive agents, a-1a-antagonists, and neuropep-
tide Y (NPY), antagonists.7 Pyrimidine derivatives and heterocyclic
annulated pyrimidines displays a wide variety of interesting phar-
macological properties such as antiproliferative,8 antiviral,9 antitu-
mor,10 anti-inflammatory,11 antibacterial,12 antifungal,13 and
antitubercular14 activity. Similarly, the structural core of quinoline
is frequently associated with medicinal applications such as anti-
cancer,15 antimicrobial,16 HIV-1 integrase inhibition,17 HIV protease
Encouraged by these results, several isoxazolyl thioureas were
examined under the optimized conditions: refluxing in ethanol
with 10 mol % CAN for 3 h. In all the cases, the three-component
reaction proceeded smoothly to give the corresponding isoxazolyl
dihydropyrimidine-thione carboxylates 4a–h in high yields. Many
of the pharmacologically relevant substitution patterns on the
isoxazolyl thiourea were introduced with high efficiency.
inhibitors,18 antileishmanial activity,19 NK-3 receptor antagonists20
,
⇑
Corresponding author. Tel.: +91 870 2456363; fax: +91 870 2438800.
E-mail address: eligeti_rajan@yahoo.co.in (E. Rajanarendar).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.060

E. Rajanarendar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6052–6055
6053
Isoxazolyl dihydropyrimidine-thione carboxylates 4 on heating
with 3-amino-5-methylisoxazole 5 for 10 h in diphenyl ether at
200 °C under nitrogen atmosphere underwent cyclization to give
the title compounds viz; 1-aryl-4-methyl-3,6-bis-(5-meth-
ylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-
c]quinolin-5-ones 6 in good yields (70–80%) (Scheme 1).29
The structures of the newly synthesized compounds 4a–h and
6a–h were confirmed by analytical and spectral data (IR, 1H
NMR, 13C NMR, ESI-MS). The IR spectra of 4a–h showed absorption
bands around 1740 cmÀ1 for ester and 1220 cmÀ1 for C@S func-
tional groups, respectively. The 1H NMR spectra of 4a–h displayed
the methyl proton of pyrimidine ring around d 2.42 confirming
cyclization. The ESI-MS of compound 4a displayed molecular ion
peak at m/z 467.97, which confirmed the molecular weight. Ab-
sence of the ester frequency in IR spectra of 6a–h provided definite
proof for the formation of title compounds. Moreover, the amide
mobacterium violaceum (MTCC 2656). The antibacterial activity re-
sults showed that compounds 6a–h exhibited moderate to good
antibacterial activity and are more active than standard drug Cip-
rofloxacin (Table 1). The activity was expressed in terms of mini-
mum inhibitory concentration (MIC). The compounds 6d and 6h
are highly active, because the activity is considerably affected by
the presence of groups like bromo and chloro as substitutes on
benzene ring, when compared to unsubstituted compound 6a. It
has been observed that exceptional activity of compound 6b is
due to the presence of methyl group on para position of phenyl
ring. Rest of the compounds showed moderate activity. However,
the degree of inhibition varied both with the test compound as
well as with the bacteria used in the present investigation. The
antibacterial activity of 6b, 6d, and 6h is promising compared to
standard Ciprofloxacin, and they can be exploited for formulation
of bacteriocide after further study.
carbonyl appeared as
a
strong absorption band around
Fungal strains tested in the present investigation are Aspergillus
niger (MTCC 282), Chrysosporium tropicum (MTCC 2821), Rhizopus
oryzae (MTCC 262), Fusarium moniliformae (MTCC 1848), and Curvu-
laria lunata (MTCC 2030). The antifungal activity of the compounds
6a–h showed that they are significantly toxic towards all the five
1640 cmÀ1 in IR spectra of 6a–h. In the 1H NMR spectra of 6a–h,
the disappearance of ethoxy proton signals (present in 4a–h) and
appearance of additional methyl protons signal of isoxazole at d
2.33 clearly evidences the formation of new products. Electron im-
pact mass spectra of 6a showed an accurate molecular ion peak at
m/z 483.55 confirming cyclization.
pathogenic fungi and they are lethal even at 100
tion (Table 2). The activity data is indicated as zone of inhibition at
30 g and 100 g/mL concentration. Compounds 6b and 6c exhib-
lg/mL concentra-
The newly synthesized compounds 6a–h were evaluated for
in vitro antibacterial and antifungal activity against various
Gram-positive, Gram-negative bacteria, and fungal strains using
broth dilution method30 and agar cup bioassay method,31 respec-
tively. The results are shown in Tables 1 and 2. Ciprofloxacin and
Clotrimazole were used as standard drugs for comparison. Com-
pounds 6a–h were also tested for toxicity against fourth instar
mosquito larvae, Culex quinquefasciatus. Mosquito larval bioassay
was performed according to standard methodology.32,33
Microbial strains were obtained from Institute of Microbial
Technology, Chandigarh, India. The bacterial strains used in the
present investigation are Bacillus subtilis (MTCC 441), Bacillus sph-
aericus (MTCC 511), Staphylococcus aureus (MTCC 96), Pseudomonas
aeruginosa (MTCC 741), Klebsiella aerogenes (MTCC 39), and Chro-
l
l
ited high activity and they inhibited the growth of fungi to a
remarkable extent, which may be due to the presence of methyl
and methoxy substituents on para position of benzene ring. These
compounds are highly toxic compared to that of standard Clotrim-
azole. Compound with benzyl substituent 6e showed remarkable
toxicity against the fungi used in the present investigation. How-
ever, the degree of spore germination inhibition varied with the test
compound as well as with the fungi under study. Compounds 6b,
6c, and 6e are highly toxic towards the fungi under investigation
and they are lethal even at 100 lg/mL concentration in comparison
with standard Clotrimazole at the same concentration.
Fourth instar mosquito larvae, C. quinquefasciatus is tested in
the current investigation. The toxicity of test compounds 6a–h to
S
O
S
2
NH
C
NH
Ar
Ar
3
O
O
1
3
"
4
"
H
N
N
.
.
CAN ( 10 mol )
/
6"
5
4
"
"
2
N
CH3
+
OEt
ethanol
,3h.
+
CH3
6
7
N
Cl
O
5
H3C
O
"
1
Cl
OCH2CH3
1
O
2
3 a-h
8
9
4 a-h
NH2
diphenyl ether
a,
b,
Ar = C6H5
CH C H
4
4,6
4,6
N
H3C
200º C,10h.
O
Ar = 4-
3
6
N2 atm.
4,6c,
5
Ar = 4-CH3OC6H4
4,6
4,6
d,
e,
Ar = 4-
BrC6H4
C H CH
S
Ar =
6
5
2
2
1
Ar
3
"
4"
f,
g,
N
N
4
Ar = 4-ClC6H4
4,6
4,6
4,6
3
2
5
6
"
"
N
Ar = 3-
CH3
CH3OC6H4
ClC6H4
O
5
CH3
7
1
"
6
"
h, Ar = 2-
O
N
3
"
4"
5"
"
2
"
6"
N
CH3
O
1
6 a-h
Scheme 1. Synthesis of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones (6a–h).

6054
E. Rajanarendar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6052–6055
Table 1
Antibacterial activity data (MIC (in
do[5,4-c]quinolin-5-ones (6a–h)
l
g/mL) values) of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimi-
Compds
Gram positive
Gram negative
B. subtilis
B. sphaericus
S. aureus
P. aeruginosa
K. aerogenes
C. violaceum
6a
6b
6c
6d
19
16
20
6
15
11
15
8
15
10
10
9
20
10
15
9
13
8
14
7
18
6
18
5
6e
6f
6g
6h
22
16
18
8
18
15
18
10
20
25
15
12
9
20
20
8
5
30
15
15
13
8
18
13
11
6
Ciprofloxacin
20
25
25
25
Negative control (acetone)—no activity. Values are indicated in
lg/mL.
Table 2
Antifungal activity results of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5,4-c]quinolin-5-ones
(6a–h)
Compds
Concn (
lg/mL)
Zone of inhibition (mm)
A. niger
C. tropicum
R. oryzae
F. moniliformae
C. lunata
6a
30
100
30
100
30
100
30
100
30
100
30
100
30
100
30
100
100
25
45
40
60
35
60
40
50
31
61
25
40
20
40
30
55
26
30
45
40
65
40
60
25
45
30
60
30
45
28
40
25
40
30
40
50
45
65
45
65
39
45
35
59
40
50
40
65
35
50
33
30
45
45
60
35
60
25
40
32
55
30
45
35
60
30
45
25
35
40
45
65
40
60
35
45
30
55
35
50
35
55
40
55
35
6b
6c
6d
6e
6f
6g
6h
Clotrimazole
Negative control (acetone)—no activity.
fourth instar larvae of C. quinquefasciatus is reported in Table 3.
Toxicity and activity of the compounds were reported as LC50
and LC90 representing the concentration in ppm that killed 50%
and 90% of larvae, respectively. From the data it appears that com-
pounds 6b, 6d, and 6h are most toxic to larvae at LC50 value, fol-
lowed by compounds 6a and 6f. Rest of the compounds (6c, 6e,
and 6g) are moderately toxic to larvae.
these were condensed with isoxazole amine to obtain the title
compounds (6a–h). The antibacterial activity data indicated that
compounds 6d and 6h exhibited maximum activity against all
the six organisms (Gram +ve and Àve) when compared with the
standard drug Ciprofloxacin and they can be exploited for the for-
mulation of bacteriocide after detailed study. Compounds 6a–h are
highly toxic toward all the fungi used in the present experiments
In conclusion, we report the synthesis, antimicrobial, and mos-
quito larvicidal test of a novel series of 1-aryl-4-methyl-3,6-bis-(5-
methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimi-
do[5,4-c]quinolin-5-ones 6a–h. The compounds 4a–h were pre-
pared by Biginelli one-pot condensation using CAN as catalyst,
and they are lethal even at 100 lg/mL concentration. It is notewor-
thy that 6b and 6c may be exploited for control of wilt diseases of
different crops as fungicides after detailed study. Compounds 6b
and 6d are proved to be lethal for mosquito larvae, hence can be
useful as more toxic substances to kill mosquito larvae.
Acknowledgments
Table 3
Toxicity of 1-aryl-4-methyl-3,6-bis-(5-methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tet-
rahydro-1H-pyrimido[5,4-c]quinolin-5-ones (6a–h) against fourth instar larvae Culex
quinquefasciatus
The authors are thankful to Head, Department of Chemistry, Ka-
katiya University, Warangal, India for providing necessary facilities
and to the Director, Indian Institute of Chemical Technology (IICT),
Hyderabad, India for providing Spectral data. K. Govardhan Reddy
thanks Council of Scientific and Industrial Research (CSIR), New
Delhi, India for financial support.
Compds
LC50
LC90
CHI square
Reg. coeff.
6a
6b
6c
6d
6e
6f
1.02
0.85
2.09
0.88
2.27
1.01
1.25
0.98
3.10
2.41
3.65
1.80
1.59
4.00
2.41
1.00
4.85
5.19
5.87
0.55
3.21
5.25
3.78
0.40
5.01
5.24
5.79
4.10
4.01
6.04
4.09
4.50
Supplementary data
6g
6h
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.060.
Negative control (acetone)—no activity.

E. Rajanarendar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6052–6055
6055
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(1 mmol), isoxazolyl thiourea
3 (1 mmol), and ceric ammonium nitrate
(10 mol %) in ethanol (10 mL) were heated at 100 °C, while stirring for 3 h.
After completion of the reaction (monitored by TLC), the reaction mixture was
cooled and poured in to ice water. The separated solid was filtered and
recrystallized from ethanol to give pure product (4a–h).
29. General procedure for the preparation of 1-aryl-4-methyl-3,6-bis-(5-
methylisoxazol-3-yl)-2-thioxo-2,3,6,10b-tetrahydro-1H-pyrimido[5.4-
c]quinolin-5-ones (6a–h): A mixture of isoxazolyl o-chloro phenyl pyrimidine
carboxylate 4a (1 mmol) and 3-amino-5-methylisoxazole 5 (1 mmol) were
heated in diphenyl ether (15 mL) at 200 °C for 10 h under N2 atmosphere. The
progress of the reaction was monitored by TLC. After the completion of the
reaction, the solvent was removed by vacuum distillation and the contents are
poured in ice cold water. The separated solid was filtered and recrystallized
from ethanol to give the pure product (6a–h).
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