Design, synthesis, antimicrobial, anti-inflammatory and analgesic activity of novel isoxazolyl pyrimido[4,5-b]quinolines and isoxazolyl chromeno[2,3-d]pyrimidin-4-ones

Article abstract of DOI:10.1016/j.ejmech.2012.07.029

Novel series of 2-methyl-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4- isoxazolyl}-3,4-dihydropyrimido[4,5-b]quinolin-4-ones 5 and 3-{3-methyl-5-[(E)- 2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d] pyrimidin-4-ones 7 have been synthesized from isoxazolyl cyanoacetamide synthon 2. Compound 2 was obtained by reaction of 4-amino-3-methyl-5-styrylisoxazole 1 with ethyl cyanoacetate. Isoxazolyl pyrimido[4,5-b]quinolin-4-ones 5 were obtained from compounds 2 by condensation with o-nitro benzaldehyde followed by treatment with SnCl₂ and subsequent tandem N-acetylation and cyclodehydration with acetic anhydride. Compounds 2 were converted to isoxazolyl chromeno[2,3-d]pyrimidin-4-ones 7 by reaction with salicylaldehydes and subsequent cyclization with formaldehyde. Compounds 2-7 were characterized by IR, ¹H NMR, ¹³C NMR, and Mass spectral data. The title compounds 5a-f and 7a-g were evaluated for their antimicrobial, anti-inflammatory and analgesic activity. Compounds 5d and 7e exhibited significant antimicrobial activity, potent anti-inflammatory and analgesic activities as that of standard drugs.

Full text of DOI:10.1016/j.ejmech.2012.07.029

European Journal of Medicinal Chemistry 55 (2012) 273e283
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, antimicrobial, anti-inflammatory and analgesic activity of
novel isoxazolyl pyrimido[4,5-b]quinolines and isoxazolyl chromeno[2,3-d]
pyrimidin-4-ones
,
a
a
a
b
c
E. Rajanarendar a , M. Nagi Reddy , S. Rama Krishna , K. Rama Murthy , Y.N. Reddy , M.V. Rajam
*
a Department of Chemistry, Kakatiya University, Vidyaranyapuri, Warangal 506 009, A.P., India
b Department of Pharmacology and Toxicology, Kakatiya University, Warangal 506009, India
c Department of Genetics, University of Delhi, South Campus, New Delhi 110021, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel series of 2-methyl-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-dihydropyrimido
[4,5-b]quinolin-4-ones and 3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-dihydro-2H-
Received 1 March 2012
Received in revised form
10 July 2012
Accepted 17 July 2012
Available online 24 July 2012
5
chromeno[2,3-d]pyrimidin-4-ones 7 have been synthesized from isoxazolyl cyanoacetamide synthon 2.
Compound 2 was obtained by reaction of 4-amino-3-methyl-5-styrylisoxazole 1 with ethyl cyanoacetate.
Isoxazolyl pyrimido[4,5-b]quinolin-4-ones 5 were obtained from compounds 2 by condensation with o-
nitro benzaldehyde followed by treatment with SnCl2 and subsequent tandem N-acetylation and
cyclodehydration with acetic anhydride. Compounds 2 were converted to isoxazolyl chromeno[2,3-d]
pyrimidin-4-ones 7 by reaction with salicylaldehydes and subsequent cyclization with formaldehyde.
Compounds 2e7 were characterized by IR, 1H NMR, 13C NMR, and Mass spectral data. The title
compounds 5aef and 7aeg were evaluated for their antimicrobial, anti-inflammatory and analgesic
activity. Compounds 5d and 7e exhibited significant antimicrobial activity, potent anti-inflammatory and
analgesic activities as that of standard drugs.
Keywords:
Isoxazolyl pyrimido[4,5-b]quinolines
Isoxazolyl chromeno[2,3-d] pyrimidin-4-
ones
Antimicrobial activity
Anti-inflammatory activity
Analgesic activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
synthesis of title compounds to improve specificity and efficacy of
these scaffolds against micro organisms. In this context, we have
The recent literature is enriched with progressive findings about
the synthesis and pharmacological action of fused heterocycles.
Since the discovery of 5-deaza flavin derivatives (A) as one of the
naturally occurring essential redox coenzymes [1], extensive
research in this field has been done to explore its functions in
biological systems [2] and to develop a biomimetic model pos-
sessing the same basic skeleton [3,4]. 5-Deaza-10-oxaflavin (B)
structure which is isosteric with 5-deaza flavin showed interesting
biological activity [5] (Fig. 1). Similarly, the biological activity of
substituted isoxazoles has made them a focus of medicinal chem-
istry over the years. Isoxazoles are potent analgesic, anti-
inflammatory [6], antimicrobial [7], COX-2 inhibitory [8], antitu-
bercular [9], and anticancer agents [10].
utilized isoxazolyl cyanoacetamides as useful synthons, to build
different heterocyclic systems, because the carbonyl and cyano
functions of this synthon are suitably situated to enable reactions
with common bidentate reagents. Moreover, the active hydrogen on
C-2 of this compound can take part in a variety of condensation and
substitution reactions. The main objective of the present study is to
provide various organic heterocycles which can evolve as better
chemotherapeutic agents. As a sequel to our studies on the synthesis
and biological activity of heterocycles linked to isoxazole moiety
[11e16], we undertook the synthesis of isoxazolyl pyrimido[4,5-b]
quinolines and isoxazolyl chromeno[2,3-d]pyrimidin-4-ones in order
to explore the pharmacological activity of these compounds. Herein,
we report the discovery of pyrimidoquinolines and chromenoqui-
nolines linked to isoxazole from isoxazolyl cyanoacetamide synthon
and their antimicrobial, anti-inflammatory and analgesic activities.
In view of this, we are of the opinion that, several pyr-
imidoquinolines (5-deazaflavinsystem)andchromenoquinolines (5-
deaza-10-oxaflavin system) linked to isoxazole may possess
remarkable pharmacological activity. Hence, we embarked on the
2. Chemistry
* Corresponding author. Tel.: þ91 870 2456363; fax: þ91 870 2438800.
E-mail addresses: rajanarendareligeti@gmail.com, eligeti_rajan@yahoo.co.in
(E. Rajanarendar).
Thekeyintermediate, N1-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-
4-isoxazolyl}-2-cyanoacetamide 2 required for synthesis of target
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.029

274
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
compounds was obtained by reacting4-amino-3-methyl-5-
styrylisoxazole [17] 1 with ethyl cyanoacetate in refluxing ethanol.
Compound 2 yielded the desired N1-{3-methyl-5-[(E)-2-phenyl-1-
ethenyl]-4-isoxazolyl}-(E)-2-cyano-3-(2-nitrophenyl)-2-propeana-
mides 3 on Knoevenagel condensationwith o-nitro benzaldehyde in
refluxingethanol under the catalytic influence of piperidine. Further,
compound 3 underwent reductive cyclization to afford the corre-
sponding isoxazolyl quinoline derivatives viz., N3-{3-methyl-5-[(E)-
bacteria used in the present investigation. The compounds 7 having
coumarin ring showed maximum activity by inhibiting the growth
of all the bacteria under investigation to a greater extent in
comparison with compounds 5, and the standard drug Cipro-
floxacin, and compounds 5d and 7e can be exploited for the
formulation of bactericides after detailed study.
The title compounds 5aef and 7aeg were also evaluated for
their antifungal activity against Fusarium oxysporum, Verticillium
dahale, Alternaria solani, Rhizoctonia solani, Colletotrichum capsici
and Pythium aphanidermatum in acetone by agar cup bioassay
method [19], using Clotrimazole as the standard drug. The anti-
fungal activity results (Table 2) indicate that compounds 5aef and
7aeg are significantly toxic towards all the fungi under investi-
gation. In series 5, compounds 5d, 5e and 5f, exhibited high
antifungal activity by inhibiting the growth of fungi to a remark-
able extent, which may be due to the presence of chloro substit-
uents on the benzene ring. In series 7, compounds 7d, 7e, 7c and
7b possessing bromo, chloro and methyl substituents on benzene
ring are highly toxic towards all the fungi. However, the degree of
spore germination inhibition varied with the test compound as
well as with the fungi under investigation. Compounds 5aef with
pyrimidoquinoline ring is found to have high toxicity compared
with that of compounds 7 bearing coumarin ring. The antifungal
activity of the compounds 5aef and 7aeg has shown better
activity, when compared with the standard drug clotrimazole. It is
note worthy that compounds 5d, 5e, 7e and 7b may be exploited
for control of wilt diseases of different crops as fungicides after
detailed study.
2-phenyl-1-ethenyl]-4-isoxazolyl}-2-amino-3-quinoline
carbox-
amides 4aef on treatment with SnCl2eEtOH. Finally, compounds 4
underwent tandem N-acetylation and cyclodehydration involving
intramolecular cyclization to afford the title compounds viz., 2-
methyl-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-
dihydro pyrimido[4,5-b]quinolin-4-ones 5 on heating with acetic
anhydride for 5 h (Scheme 1). The structures of the newly synthe-
sized compounds 2e5 have been established based on their spectral
(IR, 1H NMR, 13C NMR and MS) and analytical data.
Cyclocondensation of isoxazolyl cyanoacetamides 2 with salicy-
laldehydes in boiling absolute ethanol containing a catalytic amount
of piperidine afforded the coumarin derivatives viz., N3-[3-methyl-
5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-2-imino-2H-3-chromene
carboxamides 6 in good yields (73e86%). The scope and generality of
this reaction is illustrated by utilizing different substituted salicy-
laldehydes in the reaction to afford corresponding derivatives 6aeg
without any difficulty. Compounds 6 on heating with formalin in
ethanol afforded the corresponding 3-{3-methyl-5-[(E)-2-phenyl-
1-ethenyl]-4-isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]pyrimi-
din-4-ones (7) in good yields (Scheme 2). The structure of
compounds 6 and 7 were confirmed by their spectroscopic (IR, 1H
NMR, 13C NMR and MS) and analytical data.
3.2. Anti-inflammatory activity
The anti-inflammatory activity of the newly synthesized
compounds, 5aef and 7aeg, were evaluated by applying
carrageenan-induced paw edema test in rats [20], using Indo-
methacin as a reference drug for comparison. Results were
expressed as mean ꢀ S.E. Difference between vehicle control and
treatment groups were tested using one way Analysis of variance
(ANOVA) followed by the least significant difference (L.S.D.)
Methods of statistical analysis were done according to Armitage et
al. [21]. The anti-inflammatory activity data (Table 3) indicated
that all the tested compounds in 60 min prior to carrageenan
injection at dose of 10 mg/kg weight caused significant inhibition
of paw edema response. A few among them have significant acute
as well as residual anti-inflammatory activity. Compounds 7b, 7d,
7e and 5e caused significant decrease in paw edema after, 2, 3, 4 h
drug administration, while 5b, 5c and 5d gave their response after
2 h of administration and continued to the third hour. Compounds
7f and 7c showed the effect only after 2 h, but compounds 7g and
5f significantly decreased the paw edema after 4 h of post
administration. On the other hand, compounds 5a and 7a were
inactive towards carrageenan-induced edema in comparison to
the reference drug Indomethacin, which markedly and signifi-
cantly inhibited the paw edema after 2, 3, 4 h, of carrageenan
injection. Thus compounds 7b, 7d, 7e, 5e, 5b, 5c, 5d, 7f, 7c and 5f
have good anti-inflammatory activity. Among all the test
compounds, it is interesting to note that compounds 5d and 7e
showed best anti-inflammatory activity. The results are illustrated
in Table 3.
3. Biological screening
3.1. Antimicrobial activity
The newly synthesized compounds 5aef and 7aeg were eval-
uated for their in vitro antibacterial activity against Gram-positive
bacteria viz. Bacillus subtilis (MTCC 441), Bacillus sphaericus (MTCC
511) and Staphylococcus aureus (MTCC 96) and Gram-negative
bacteria viz. Pseudomonas aeruginosa (MTCC 741), Klebsiella aero-
genes (MTCC 39) and Chromobacterium violaceum (MTCC 2656) at
100 mg/mL concentration. The in vitro antibacterial activity of the
tested compounds was assessed by minimum inhibitory concen-
tration (MIC) using broth dilution method [18]. Ciprofloxacin was
used as standard drug for comparison. The results of antibacterial
screening (Table 1) reveal that the compounds 5aef and 7aeg
displayed a better activity and more active than the standard
Ciprofloxacin. In series 5, compounds 5d, 5e, and 5f possessing
chloro group as substituent on the benzene ring showed a better
activity. Compound 5a exhibited least activity because it has no
substituent on benzene ring. In series 7, compounds 7d, 7e and 7b,
7c carrying bromo and chloro groups as substituents on the
benzene ring imparted a remarkable activity. However, the degree
of inhibition varied both with the test compound and with the
A
B
O
N
O
N
R
R
N
N
3.3. Analgesic activity
N
R
O
O
O
The analgesic activity of the newly synthesized compounds
5aef and 7aeg were also evaluated by applying the Hot plate test
[22] (central analgesic activity), using Indomethacin as a reference
drug (10 mg/kg-Po). The analgesic activity was measured at 0, 1 and
Fig. 1. Structures of biologically active 5-deaza flavine and 5-deaza-10-oxaflavin as
design templates.

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
275
O
H
H3C
NH2
H3C
CN
H3C
N
NO2
N
H
CN
(i)
(ii)
N
O
N
N
O
O
O
Ar
Ar
Ar
R
1
3
2
(iii)
H3C
O
NH2
N
H3C
N
H3C
N
N
H
N
(iv)
N
O
N
O
O
R
Ar
Ar
R
5
4
2: a, Ar = C6H5; b, Ar = 4-CH3C6H4; c, Ar = 2-CH3C6H4; d, Ar = 4-OCH3C6H4;
e, Ar = 4-ClC6H4; f, Ar = 2-ClC6H4
3, 4 and 5: a, Ar = C6H5, R = H; b, Ar = 4-CH3C6H4, R = H; c, Ar = 4-CH3OC6H4, R = H;
d, Ar = C6H5, R = Cl; e, Ar = 4-CH3C6H4, R = Cl; f, Ar= 4-CH3OC6H4, R = Cl;
Scheme 1. Synthesis of isoxazolyl pyrimido[4,5-b]quinolines (5aef). Reagents and conditions. (i) Ethyl cyanoacetate, ethanol,
(iii) SnCl2-ethanol; (iv) acetic anhydride,
D; (ii) o-nitro benzaldehyde, ethanol, piperidine, D;
D.
2 h time intervals after pain induction. The results were recorded as
the average value of six administrations and the percentage
increase of the reaction time in comparison with the basal values.
The results were presented in Table 4, and expressed as mean ꢀ S.E.
Difference between vehicle control and treatment groups were
tested using one way Analysis of variance (ANOVA) followed by the
least significant difference (L.S.D.) Methods of statistical analysis
were done according to Armitage et al. [21]. A comparative study of
the analgesic activity of the test compounds relative to the refer-
ence drug at different time intervals indicated that test compounds
exhibited moderate analgesic activity at 60 min of reaction time
(Table 4).
Compounds 7e, 7c and 5f showed significant analgesic activity
higher than that obtained by reference drug Indomethacin, after 1 h
and 2 h post administration. While the compounds 5b, 5c and 5d
exhibited equipotent analgesic effects or slightly less than that of
Indomethacin, after 1 and 2 h of their administration. Compounds
7b, 7d and 5e exhibited significant analgesic activity higher than or
slightly equipotent to Indomethacin only after 2 h of administra-
tion. Compounds 7g and 7f exhibited the analgesic effect after 1 h
of administration only. Compounds 5a and 7a have no analgesic
activity in comparison to the base line of same group 1 h and 2 h
post administration. Therefore, compounds 7b, 7c, 5b, 5c, 7e, 7d
and 5f have significant analgesic activity. Among all the test
H
O
H3C
O
N
H3C
CN
R'
H3C
N
R'
H
N
N
(ii)
O
(i)
N
O
N
HN
Ar
O
O
N
O
O
R
R
Ar
Ar
2
6
7
6 and 7; a, Ar = C6H5, R =R'=H; b, Ar = 4-CH3C6H4, R=H, R'= Cl; c, Ar = C6H5, R=R'= Cl;
d, Ar = 4-OCH3C6H4, R=H, R'= Br; e, Ar = C6H5, R =R'= Br; f, Ar = C6H5, R=H,
R'= CH3; g, Ar = C6H5, R=H, R'= 4-OCH3
Scheme 2. Synthesis of isoxazolyl chromeno[2,3-d]pyrimidin-4-ones (7aeg). Reagents and conditions; (i) Salicylaldehyde, absolute ethanol, piperidine, D; (ii) Formalin, ethanol, D.

276
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
Table 1
Antibacterial activity of isoxazolyl pyrimido[4,5-b]quinolines (5aef) and isoxazolyl chromeno[2,3-d]pyrimidin-4-ones (7aeg).
Compound
Minimum inhibitory concentration in mg/mL (MIC)
Gram þve bacteria
Gram ꢂve bacteria
B. subtilis
B. sphaericus
S. aureus
P. aeruginosa
K. aerogenes
C. violaceum
5a
5b
5c
5d
5e
5f
7a
7b
20
19
18
8
12
12
18
14
15
12
9
22
23
22
16
11
10
12
16
14
10
7
20
25
25
9
10
13
16
15
16
8
24
23
22
10
10
14
14
11
15
15
6
20
18
20
7
14
11
18
18
17
13
5
20
15
16
5
12
14
18
18
16
10
8
7c
7d
7e
8
7f
7g
15
18
20
18
12
24
16
22
28
12
24
25
15
17
22
18
18
20
Ciprofloxacin
Negative control (acetone) e No activity.
Values are indicated in g/mL.
m
compounds, it is interesting to note that, compound 7e showed
good analgesic activity. The results are illustrated in Table 4.
performed on Merck precoated 60 F254 silica gel plates. Visuali-
zation was done by exposure to iodine vapor. IR spectra (KBr
pellet) were recorded on a PerkineElmer BX series FT-IR spec-
trometer. 1H NMR spectra were recorded on a Varian Gemini
300 MHz spectrometer. 13C NMR spectra were recorded on
a Bruker 75 MHz spectrometer. Chemical shift values are given in
4. Conclusion
In conclusion, we report the synthesis of novel isoxazolyl pyr-
imido[4,5-b]quinolines and isoxazolylchromeno[2,3-d]pyrimidin-
4-ones, using inexpensive and commercially available materials
with potential medicinal properties. This synthesis benefits from
a simple method of purification, which does not require chroma-
tography. This ease of purification compliments this synthetic
technology practical, easy to perform and facile. The newly
synthesized novel isoxazolyl pyrimido[4,5-b]quinolines and iso-
xazolyl chromeno[2,3-d]pyrimidin-4-ones 5aef and 7aeg were
evaluated for their antimicrobial, anti-inflammatory and analgesic
activity. It has been found that the derivatives 7b, 7d, 7e, 5d and 5f
exhibited good antimicrobial, anti-inflammatory and analgesic
activity compared to the reference drugs. Among all the test
compounds, it is interesting to note that compounds 5d and 7e
showed best antimicrobial, anti-inflammatory and analgesic
activity.
ppm (d) with tetramethyl silane as an internal standard. Mass
spectral measurements were carried out by EI method on a Jeol
JMC-300 spectrometer at 70 eV. Elemental analyses were per-
formed on a Carlo Erba 106 and PerkineElmer model 240
analyzers.
5.1. General procedure for the synthesis of N1-{3-methyl-5-[(E)-2-
phenyl-1-ethenyl]-4-isoxazolyl}-2-cyanoacetamides (2aef)
A mixture of compound 1 (1 mmol) and ethyl cyanoacetate
(1 mmol) in ethanol (15 mL) were refluxed for 5 h. The reaction
mixture was cooled and the solid obtained was filtered off and
recrystallized from ethanol to give isoxazolyl cyanoacetamide 2.
5.1.1. N1-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-2-
cyanoacetamide (2a)
5. Experimental section
Yield 75%, mp 142e146 ꢁC. IR (KBr) cmꢂ1: 3250 (NH), 2195
(C^N), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.23 (s, 3H,
All the melting points were determined on a Fisher-Johns
melting point apparatus and are uncorrected. Analytical TLC was
CH3), 3.72 (s, 2H, CH2CN), 6.74 (d, 1H, CH]CH, J ¼ 12 Hz), 6.91 (d,
1H, CH]CH, J ¼ 12 Hz), 7.03e7.78 (m, 5H, ArH), 8.51 (s, 1H, CONH,
Table 2
Antifungal activity of isoxazolyl pyrimido[4,5-b]quinolines (5aef) and isoxazolyl chromeno[2,3-d]pyrimidin-4-ones (7aeg) mg/mL.
Compound
Minimum inhibitory concentration in mg/mL (MIC)
F. oxysporum
V. dahale
A. solani
R. solani
C. capsici
P. aphanidermatum
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
16
12
10
8
10
11
16
9
16
18
9
20
12
28
14
12
14
9
11
13
18
10
18
14
8
10
10
16
10
13
12
20
8
14
12
7
20
18
22
12
14
14
11
12
14
12
11
15
14
6
15
18
16
11
14
16
20
12
16
12
10
18
17
20
18
15
18
15
10
17
18
9
22
20
8
22
23
30
22
20
26
24
12
25
7g
Clotrimazole
Negative control (acetone) e No activity.

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
277
Table 3
C4), 120.05 (]CH), 120.45 (]CH), 125.20 (AreC), 127.01 (AreC),
128.15 (AreC), 128.55 (AreC), 128.90 (AreC), 129.98 (AreC),
132.15 (CN), 156.40 (Isoxazole-C3), 159.50 (Isoxazole-C5), 182.10
(C]O). EI-MS (70 eV) m/z: 282 [M þ H]þ. Anal. Calcd for
C16H15N3O2: C, 68.32; H, 5.33; N, 14.94. Found: C, 68.37; H, 5.28; N,
14.90%.
Anti-inflammatory activity of the isoxazolyl pyrimido[4,5-b]quinolines (5aef) and
isoxazolyl chromeno[2,3-d]pyrimidin-4-ones (7aeg).
Groupa
Volume of edema (mL)b
1 h
2 h
3 h
4 h
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
74.2 ꢀ 5.4
53.6 ꢀ 7.1
53.1 ꢀ 5.4
50.7 ꢀ 6.1
60.2 ꢀ 6.8
59.4 ꢀ 7.2
65.1 ꢀ 7.2
62.4 ꢀ 8.1
64.2 ꢀ 6.4
59.6 ꢀ 8.4
51.5 ꢀ 7.8
86.2 ꢀ 3.8
51.8 ꢀ 2.9
59.2 ꢀ 6.4
48.8 ꢀ 5.0
86.0 ꢀ 8.6
70.8 ꢀ 5.7c
58.2 ꢀ 3.9c
60.5 ꢀ 6.7c
65.4 ꢀ 5.9c
72.6 ꢀ 3.8
82.1 ꢀ 6.9
72.4 ꢀ 6.3c
75.1 ꢀ 3.5c
64.2 ꢀ 7.8c
64.2 ꢀ 8.4c
76.4 ꢀ 8.1c
88.4 ꢀ 5.9
89.4 ꢀ 7.3
43.6 ꢀ 4.9c
88.1 ꢀ 7.5
74.4 ꢀ 4.6c
64.6 ꢀ 2.6c
66.3 ꢀ 6.4c
69.6 ꢀ 8.1c
78.4 ꢀ 2.4
83.9 ꢀ 2.3
80.3 ꢀ 7.2c
80.3 ꢀ 3.1
67.4 ꢀ 6.5c
59.2 ꢀ 7.2c
86.3 ꢀ 7.1
82.3 ꢀ 4.2
96.2 ꢀ 2.5
46.4 ꢀ 4.3c
90.4 ꢀ 6.3
86.2 ꢀ 5.1
79.5 ꢀ 2.4
69.2 ꢀ 4.2c
71.8 ꢀ 8.3
74.3 ꢀ 6.2c
79.4 ꢀ 7.3
76.5 ꢀ 6.7c
85.2 ꢀ 2.6
70.6 ꢀ 6.1c
54.6 ꢀ 6.8c
88.5 ꢀ 2.4
77.4 ꢀ 2.3c
91.2 ꢀ 2.3
47.8 ꢀ 5.4c
5.1.3. N1-{3-Methyl-5-[(E)-2-(2-methylphenyl)-1-ethenyl]-4-isoxazol-
yl}-2-cyanoacetamide (2c)
Yield 84%, mp 155e157 ꢁC. IR (KBr) cmꢂ1: 3300 (NH), 2180
(C^N), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.34 (s, 3H,
CH3), 2.51 (s, 3H, CH3), 3.75 (s, 2H, CH2CN), 6.62 (d, 1H, CH]CH,
J ¼ 12 Hz), 6.81 (d, 1H, CH]CH, J ¼ 12 Hz), 6.95e7.42 (m, 4H, ArH),
8.67 (s, 1H, CONH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d
ppm: 11.75 (CH3), 26.45 (AreCH3), 28.75 (CH2), 100.75 (Isoxazole-
C4), 119.95 (]CH), 120.15 (]CH), 124.95 (AreC), 126.10 (AreC),
128.10 (AreC), 128.65 (AreC), 128.90 (AreC), 130.11 (AreC),
132.40 (CN), 156.40 (Isoxazole-C3), 159.75 (Isoxazole-C5), 182.55
(C]O). EI-MS (70 eV) m/z: 282 [M þ H]þ. Anal. Calcd for
C16H15N3O2: C, 68.32; H, 5.33; N, 14.94. Found: C, 68.30; H, 5.35; N,
14.97%.
7g
Control
Indomethacin
a
Dose levels, po: test compounds (10 mg/kg b.wt.), Indomethacin (10 mg/kg
b.wt.).
b
c
Values are expressed as means ꢀ S.E. (number of animals N ¼ 6 rats).
Statistically significant compared to respective control values, P < 0.05 (Dun-
nett’s test).
5.1.4. N1-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-isoxaz-
olyl}-2-cyanoacetamide (2d)
Yield 74%, mp 133e136 ꢁC. IR (KBr) cmꢂ1: 3275 (NH), 2190
D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.25 (CH3),
(C^N), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.23 (s, 3H,
CH3), 3.84 (s, 3H, OCH3), 3.96 (s, 2H, CH2CN), 6.72 (d, 1H, CH]CH,
J ¼ 12 Hz), 6.93 (d, 1H, CH]CH, J ¼ 12 Hz), 7.04e7.52 (m, 4H,
ArH), 8.81 (s, 1H, CONH, D2O exchangeable). 13C NMR (75 MHz,
28.62 (CH2), 100.55 (Isoxazole-C4), 119.40 (]CH), 120.35 (]CH),
125.00 (AreC), 126.22 (AreC), 128.00 (AreC), 128.72 (AreC), 128.85
(AreC), 130.05 (AreC), 132.23 (CN), 156.55 (Isoxazole-C3), 159.80
(Isoxazole-C5), 182.20 (C]O). EI-MS (70 eV) m/z: 268 [M þ H]þ.
Anal. Calcd for C15H13N3O2: C, 67.41; H, 4.86; N, 15.73. Found: C,
67.45; H, 4.84; N, 15.76%.
CDCl3)
d ppm: 11.15 (CH3), 28.10 (CH2), 63.75 (OCH3), 100.15
(Isoxazole-C4), 119.35 (]CH), 120.40 (]CH), 125.11 (AreC), 126.10
(AreC), 128.09 (AreC), 128.58 (AreC), 128.76 (AreC), 130.15
(AreC), 132.16 (CN), 156.45 (Isoxazole-C3), 159.75 (Isoxazole-C5),
181.98 (C]O). EI-MS (70 eV) m/z: 298 [M þ H]þ. Anal. Calcd for
C16H15N3O3: C, 64.64; H, 5.05; N, 14.14. Found: C, 64.69; H, 5.01;
N, 14.19%.
5.1.2. N1-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-2-cyanoacetamide (2b)
Yield 80%, mp 167e169 ꢁC. IR (KBr) cmꢂ1: 3270 (NH), 2200
(C^N), 1685 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.26 (s, 3H,
CH3), 2.52 (s, 3H, CH3), 3.94 (s, 2H, CH2CN), 6.63 (d, 1H, CH]CH,
J ¼ 12 Hz), 6.82 (d, 1H, CH]CH, J ¼ 12 Hz), 6.94e7.53 (m, 4H, ArH),
8.31 (s, 1H, CONH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
5.1.5. N1-{5-[(E)-2-(4-Chlorophenyl)-1-ethenyl]-3-methyl-4-isoxaz-
olyl}-2-cyanoacetamide (2e)
Yield 78%, mp 185e187 ꢁC. IR (KBr) cmꢂ1: 3280 (NH), 2150
d
ppm: 11.01 (CH3), 26.52 (AreCH3), 28.69 (CH2), 100.20 (Isoxazole-
(C^N), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.34
(s, 3H, CH3), 3.73 (s, 2H, CH2CN), 6.65 (d, 1H, CH]CH, J ¼ 12 Hz),
6.84 (d, 1H, CH]CH, J ¼ 12 Hz), 6.92e7.54 (m, 4H, ArH), 8.56
(s, 1H, CONH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
Table 4
Analgesic activity of isoxazolyl pyrimido[4,5-b]quinolines (5aef) and isoxazolyl
chromeno[2,3-d]pyrimidin-4-ones (7aeg).
d
ppm: 11.55 (CH3), 28.75 (CH2), 100.35 (Isoxazole-C4), 119.15
(]CH), 120.33 (]CH), 125.45 (AreC), 127.23 (AreC),
128.11 (AreC), 128.65 (AreC), 128.90 (AreC), 130.17 (AreC),
132.23 (CN), 156.45 (Isoxazole-C3), 159.75 (Isoxazole-C5),
182.11 (C]O). EI-MS (70 eV) m/z: 302 [M þ H]þ. Anal. Calcd for
C15H12N3O2Cl: C, 59.80; H, 3.98; N, 13.95. Found: C, 59.83; H,
4.01; N, 13.92%.
Groupa
Reaction time (S)b
0 h
1 h
2 h
5a
5b
5c
5d
5e
5f
7a
7b
7c
7d
7e
7f
13.4 ꢀ 0.85
13.8 ꢀ 1.13
13.7 ꢀ 0.70
13.4 ꢀ 1.02
17.5 ꢀ 1.35
16.8 ꢀ 1.15
15.9 ꢀ 0.84
16.8 ꢀ 0.97
17.2 ꢀ 0.87
14.5 ꢀ 1.05c
15.1 ꢀ 0.93
13.3 ꢀ 1.02
13.8 ꢀ 1.13
16.4 ꢀ 0.65
17.3 ꢀ 0.80
14.8 ꢀ 0.58
21.3 ꢀ 1.54c
21.7 ꢀ 1.35c
21.2 ꢀ 1.13c
20.8 ꢀ 1.20
25.1 ꢀ 2.49c
17.7 ꢀ 0.78
19.1 ꢀ 1.04
28.3 ꢀ 1.67c
18.8 ꢀ 0.95c
23.3 ꢀ 1.48c
21.2 ꢀ 1.13c
21.3 ꢀ 1.57c
16.2 ꢀ 0.85
21.8 ꢀ 0.34c
16.5 ꢀ 1.19
21.1 ꢀ 1.27c
21.4 ꢀ 1.13c
21.5 ꢀ 1.15c
22.3 ꢀ 1.38c
22.6 ꢀ 0.71c
15.7 ꢀ 0.87
24.7 ꢀ 1.21c
21.9 ꢀ 2.64c
22.6 ꢀ 1.49c
23.4 ꢀ 1.02c
21.5 ꢀ 1.15
13.4 ꢀ 0.91
16.4 ꢀ 1.20
22.4 ꢀ 0.30c
5.1.6. N1-{5-[(E)-2-(2-Chlorophenyl)-1-ethenyl]-3-methyl-4-isoxazo-
lyl}-2-cyanoacetamide (2f)
Yield 80%, mp 190e192 ꢁC. IR (KBr) cmꢂ1: 3250 (NH), 2150
(C^N), 1675 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.24 (s, 3H,
CH3), 3.71 (s, 2H, CH2CN), 6.63 (d, 1H, CH]CH, J ¼ 12 Hz), 6.82 (d,
1H, CH]CH, J ¼ 12 Hz), 6.93e7.42 (m, 4H, ArH), 8.65 (s, 1H, CONH,
D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.20 (CH3),
7g
Control
Indomethacin
28.61 (CH2), 100.65 (Isoxazole-C4), 119.38 (]CH), 120.33 (]CH),
125.11 (AreC), 127.45 (AreC), 128.15 (AreC), 128.45 (AreC), 128.93
(AreC), 130.17 (AreC), 132.01 (CN), 156.25 (Isoxazole-C3), 159.78
(Isoxazole-C5), 182.03 (C]O). EI-MS (70 eV) m/z: 302 [M þ H]þ.
Anal. Calcd for C15H12N3O2Cl: C, 59.80; H, 3.98; N, 13.95. Found: C,
59.76; H, 4.02; N, 13.91%.
a
Dose levels, po: test compounds (10 mg/kg b.wt.), Indomethacin (10 mg/kg
b.wt.).
b
c
Values are expressed as means ꢀ S.E. (number of animals N ¼ 6 rats).
Statistically significant compared to respective control values, P < 0.05 (Dun-
nett’s test).

278
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
5.2. General procedure for the synthesis of N1-{3-methyl-5-[(E)-2-
phenyl-1-ethenyl]-4-isoxazolyl}-(E)-2-cyano-3-(2-nitrophenyl)-2-
propenamides (3aef)
CH]CH, J ¼ 12 Hz), 6.82 (d, 1H, CH]CH, J ¼ 12 Hz), 6.93e7.56
(m, 8H, ArH), 8.82 (s, 1H, CONH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3)
d ppm: 11.23 (CH3), 100.39 (isoxazole-C4), 108.79
(]CH), 119.11 (]CH), 120.28 (]CeCN), 121.15 (]CH), 125.11
(AreC), 125.48 (AreC), 126.21 (AreC), 126.28 (AreC), 127.53
(AreC), 128.03 (AreC), 128.51 (AreC), 128.72 (AreC),
129.98 (AreC), 130.07 (AreC), 132.09 (AreC), 132.26 (AreC),
145.73 (]CHeAr), 156.43 (isoxazole-C3), 159.65 (isoxazole-C5),
182.20 (C]O). EI-MS (70 eV) m/z: 435 [M þ H]þ. Anal. Calcd for:
C22H15N4O4Cl: C, 60.82; H, 3.45; N, 12.90. Found: C, 60.89; H,
3.40; N, 12.97%.
A mixture of compound 2 (1 mmol) and o-nitro benzaldehyde
(1 mmol) in ethanol (15 mL) containing a few drops of piperidine (3
drops) was refluxed for 4 h. The reaction mixture was cooled and
the solid obtained was filtered off and recrystallized from ethanol
to give 3.
5.2.1. N1-{3-Methyl-5-[(E)-2-(2-phenyl)-1-ethenyl]-4-isoxazolyl}-
(E)-2-cyano-3-(2-nitro phenyl)-2-propenamide (3a)
Yield 80%, mp 161e163 ꢁC. IR (KBr) cmꢂ1: 3200 (NH), 2175 (C^N),
5.2.5. N1-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-(E)-3-(5-chloro-2-nitrophenyl)-2-cyano-2-
propenamide (3e)
1680 (C]O),1560,1350 (NO2).1H NMR (300 MHz, CDCl3)
d ppm: 2.20
(s, 3H, CH3), 6.01 (s, 1H, CH]CeCN), 6.62 (d, 1H, CH]CH, J ¼ 12 Hz),
6.85 (d, 1H, CH]CH, J ¼ 12 Hz), 6.93e7.62 (m, 9H, ArH), 8.51 (s, 1H,
Yield 71%, mp 200e202 ꢁC. IR (KBr) cmꢂ1: 3215 (NH), 2181
CONH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm:
(C^N), 1680 (C]O), 1560, 1365 (NO2). 1H NMR (300 MHz, CDCl3)
11.26 (CH3), 100.43 (isoxazole-C4), 108.92 (]CH), 119.32 (]CH),
120.35 (]CeCN), 121.25 (]CH), 125.00 (AreC), 125.52 (AreC),
126.22 (AreC), 126.30 (AreC), 126.50 (AreC), 128.00 (AreC), 128.62
(AreC), 128.85 (AreC), 130.05 (AreC), 130.08 (AreC), 132.06 (AreC),
132.23 (AreC), 145.80 (]CHeAr), 156.55 (isoxazole-C3), 159.72
(isoxazole-C5),182.11 (C]O). EI-MS (70 eV) m/z: 401 [M þ H]þ. Anal.
Calcd for C22H16N4O4: C, 66.00; H, 4.00; N, 14.00. Found: C, 66.05; H,
4.04; N, 13.95%.
d
ppm: 2.32 (s, 3H, CH3), 2.54 (s, 3H, CH3), 6.20 (s, 1H, CH]CeCN),
6.61 (d, 1H, CH]CH, J ¼ 12 Hz), 6.83 (d, 1H, CH]CH, J ¼ 12 Hz),
6.95e7.62 (m, 7H, ArH), 8.84 (s, 1H, CONH, D2O exchangeable). 13
NMR (75 MHz, CDCl3) ppm: 11.21 (CH3), 29.01 (AreCH3), 100.21
C
d
(isoxazole-C4), 108.65 (]CH), 119.23 (]CH), 121.01 (]CeCN),
121.25 (]CH), 125.09 (AreC), 125.52 (AreC), 126.28 (AreC),
126.30 (AreC), 127.42 (AreC), 128.06 (AreC), 128.43 (AreC),
128.52 (AreC), 129.99 (AreC), 130.08 (AreC), 132.03 (AreC),
132.17 (AreC), 145.76 (]CHeAr), 156.41 (isoxazole-C3), 159.45
(isoxazole-C5), 182.26 (C]O). EI-MS (70 eV) m/z: 449 [M þ H]þ.
Anal. Calcd for C23H17N4O4Cl: C, 61.60; H, 3.79; N, 12.50. Found: C,
61.68; H, 3.71; N, 12.57%.
5.2.2. N1-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-(E)-2-cyano-3-(2-nitrophenyl)-2-propenamide (3b)
Yield 82%, mp 182e185 ꢁC. IR (KBr) cmꢂ1: 3215 (NH), 2167
(C^N), 1670 (C]O), 1560, 1370 (NO2). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.21 (s, 3H, CH3), 2.43 (s, 3H, CH3), 6.08 (s, 1H, CH]CeCN),
6.62 (d, 1H, CH]CH, J ¼ 12 Hz), 6.84 (d, 1H, CH]CH, J ¼ 12 Hz),
6.93e7.52 (m, 8H, ArH), 8.81 (s, 1H, CONH, D2O exchangeable). 13
NMR (75 MHz, CDCl3) ppm: 11.28 (CH3), 28.75 (AreCH3), 100.11
5.2.6. N1-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-
isoxazolyl}-(E)-3-(5-chloro-2-nitrophenyl)-2-cyano-2-
propenamide (3f)
C
d
Yield 84%, mp 188e190 ꢁC. IR (KBr) cmꢂ1: 3200 (NH), 2188
(isoxazole-C4),108.75 (]CH),118.99 (]CH),120.11 (]CeCN),121.10
(]CH), 125.26 (AreC), 125.72 (AreC), 126.17 (AreC), 126.47 (AreC),
126.43 (AreC), 128.10 (AreC), 128.53 (AreC), 128.72 (AreC),
130.03 (AreC), 130.10 (AreC), 132.09 (AreC), 132.17 (AreC),
145.75 (]CHeAr), 156.43 (isoxazole-C3), 159.70 (isoxazole-C5),
182.19 (C]O). EI-MS (70 eV) m/z: 415 [M þ H]þ. Anal. Calcd for
C23H18N4O4: C, 66.66; H, 4.34; N, 13.52. Found: C, 66.62; H, 4.30; N,
13.55%.
(C^N), 1675 (C]O), 1570, 1365 (NO2). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.21 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.10 (s, 1H, CH]CeCN),
6.64 (d, 1H, CH]CH, J ¼ 12 Hz), 6.82 (d, 1H, CH]CH, J ¼ 12 Hz),
6.94e7.63 (m, 7H, ArH), 8.62 (s, 1H, CONH, D2O exchangeable). 13
NMR (75 MHz, CDCl3) ppm: 11.29 (CH3), 64.23 (OCH3), 100.21
C
d
(isoxazole-C4), 108.63 (]CH), 119.18 (]CH), 120.15 (]CeCN),
121.32 (]CH), 125.01 (AreC), 125.48 (AreC), 126.18 (AreC),
126.36 (AreC), 127.41 (AreC), 128.03 (AreC), 128.53 (AreC),
128.76 (AreC), 130.11 (AreC), 130.20 (AreC), 132.09 (AreC),
132.31 (AreC), 145.71 (]CHeAr), 156.45 (isoxazole-C3), 159.61
(isoxazole-C5), 182.16 (C]O). EI-MS: m/z 465 [M þ H]þ. Anal. Calcd
for C23H17N4O5Cl: C, 59.48; H, 3.66; N, 12.06. Found: C, 59.52; H,
3.71; N, 12.01%.
5.2.3. N1-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-
isoxazolyl}-(E)-2-cyano-3-(2-nitro phenyl)-2-propenamide (3c)
Yield 76%, mp 170e172 ꢁC. IR (KBr) cmꢂ1: 3210 (NH), 2180
(C^N), 1670 (C]O), 1570, 1360 (NO2). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.21 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.10 (s, 1H, CH]CeCN),
6.64 (d, 1H, CH]CH, J ¼ 12 Hz), 6.81 (d, 1H, CH]CH, J ¼ 12 Hz),
6.92e7.54 (m, 8H, ArH), 8.56 (s, 1H, CONH, D2O exchangeable). 13
NMR (75 MHz, CDCl3) ppm: 11.20 (CH3), 63.82 (OCH3), 100.40
5.3. General procedure for the synthesis of N3-{3-methyl-5-[(E)-2-
phenyl-1-ethenyl]-4-isoxazolyl}-2-amino-3-quinolinecarboxamides
(4aef)
C
d
(isoxazole-C4), 109.01 (]CH), 119.11 (]CH), 120.31 (]CeCN),
121.29 (]CH), 125.07 (AreC), 125.50 (AreC), 126.28 (AreC),
126.42 (AreC), 126.63 (AreC), 128.11 (AreC), 128.63 (AreC),
128.70 (AreC), 130.01 (AreC), 130.09 (AreC), 132.08 (AreC),
132.15 (AreC), 145.75 (]CHeAr), 156.40 (isoxazole-C3), 159.68
(isoxazole-C5), 182.01 (C]O). EI-MS (70 eV) m/z: 431 [M þ H]þ.
Anal. Calcd for C23H18N4O5: C, 64.18; H, 4.18; N, 13.02. Found: C,
64.24; H, 4.12; N, 13.08%.
A mixture of compound 3 (1 mmol) and SnCl2$2H2O (2 mmol) in
ethanol (15 mL) was refluxed for 6 h. After cooling down to room
temperature, the reaction mixture was poured in to ice-cold water.
The precipitate that formed was filtered off, washed with ethanol
and recrystallized from ethanol to get product 4.
5.3.1. N3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-2-
amino-3-quinolinecarboxamide (4a)
5.2.4. N1-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-(E)-
3-(5-chloro-2-nitrophenyl)-2-cyano-2-propenamide (3d)
Yield 70%, mp 185e187 ꢁC. IR (KBr) cmꢂ1: 3300 (CONH), 3290,
3210 (NH2), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.23 (s,
Yield 70%, mp 156e158 ꢁC. IR (KBr) cmꢂ1: 3210 (NH), 2176
3H, CH3), 6.21 (s, 2H, NH2, D2O exchangeable), 6.74e7.63 (m, 10H,
(C^N), 1685 (C]O), 1550, 1350 (NO2). 1H NMR (300 MHz, CDCl3)
ArH & 2H, CH]CH), 9.20 (s, 1H, CONH, D2O exchangeable). 13C NMR
d
ppm: 2.24 (s, 3H, CH3), 6.00 (s, 1H, CH]CeCN), 6.61 (d, 1H,
(75 MHz, CDCl3) d ppm: 11.21 (CH3), 100.36 (isoxazole-C4), 108.72

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
279
(]CH), 120.42 (]CH), 121.21 (AreC), 125.06 (AreC), 125.56 (AreC),
126.18 (AreC), 126.28 (AreC), 126.52 (AreC), 126.73 (AreC), 128.00
(AreC),128.56 (AreC),128.96 (AreC),130.05 (AreC),130.25 (AreC),
132.08 (quinoline ring-C3), 132.33 (quinoline ring-C4), 145.72
(quinoline ring-C2), 153.25 (isoxazole-C3), 159.80 (isoxazole-C5),
181.90 (C]O). EI-MS (70 eV) m/z: 371 [M þ H]þ. Anal. Calcd for
C22H18N4O2: C, 71.35; H, 4.86; N, 15.13. Found: C, 71.41; H, 4.80; N,
15.18%.
121.19 (AreC), 125.16 (AreC), 125.63 (AreC), 126.21 (AreC), 126.28
(AreC), 126.81 (AreC), 127.47 (AreC), 128.06 (AreC), 128.41 (AreC),
128.58 (AreC), 130.19 (AreC), 130.41 (AreC), 132.16 (quinoline ring-
C3), 132.46 (quinoline ring-C4), 145.58 (quinoline ring-C2), 153.09
(isoxazole-C3), 159.61 (isoxazole-C5), 181.51 (C]O). EI-MS (70 eV)
m/z: 419 [M þ H]þ. Anal. Calcd for C23H19N4O2Cl: C, 66.02; H, 4.54;
N, 13.39. Found: C, 66.09; H, 4.48; N, 13.31%.
5.3.6. N3-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-
isoxazolyl}-2-amino-6-chloro-3-quinolinecarboxamide (4f)
Yield 81%, mp 218e220 ꢁC; IR (KBr) cmꢂ1: 3350 (CONH), 3320,
5.3.2. N3-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-2-amino-3-quinolinecarboxamide (4b)
Yield 78%, mp 210e212 ꢁC. IR (KBr) cmꢂ1: 3325 (CONH), 3300,
3200 (NH2), 1675 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.31 (s,
3200 (NH2), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.24 (s,
3H, CH3), 3.84 (s, 3H, OCH3), 6.12 (s, 2H, NH2, D2O exchangeable),
3H, CH3), 2.41 (s, 3H, CH3), 6.53 (s, 2H, NH2, D2O exchangeable),
7.04e7.85 (m, 8H, ArH & 2H, CH]CH), 9.21 (s, 1H, CONH, D2O
6.82e7.86 (m, 9H, ArH & 2H, CH]CH), 9.02 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.21 (CH3), 65.09
exchangeable). 13C NMR (75 MHz, CDCl3)
d
ppm: 11.30 (CH3). 28.63
(OCH3), 100.28 (isoxazole-C4), 108.51 (]CH), 120.56 (]CH), 121.19
(AreC), 125.11 (AreC), 125.35 (AreC), 126.15 (AreC), 126.42 (AreC),
126.53 (AreC), 127.59 (AreC), 128.01 (AreC), 128.33 (AreC), 128.72
(AreC), 130.11 (AreC), 130.39 (AreC), 132.22 (quinoline ring-C3),
132.53 (quinoline ring-C4), 145.78 (quinoline ring-C2), 153.11 (iso-
xazole-C3), 159.61 (isoxazole-C5), 181.43 (C]O). EI-MS (70 eV) m/z:
435 [M þ H]þ. Anal. Calcd for C23H19N4O3Cl: C, 63.59; H, 4.37; N,
12.90. Found: C, 63.50; H, 4.30; N, 12.96%.
(AreCH3), 100.23 (isoxazole-C4), 108.53 (]CH), 120.38 (]CH),
121.20 (AreC), 125.11 (AreC), 125.73 (AreC), 126.26 (AreC), 126.32
(AreC), 126.67 (AreC), 126.79 (AreC), 128.04 (AreC), 128.43
(AreC), 128.69 (AreC), 130.17 (AreC), 130.33 (AreC), 132.11 (quin-
oline ring-C3), 132.42 (quinoline ring-C4), 145.65 (quinoline ring-
C2), 153.11 (isoxazole-C3), 159.73 (isoxazole-C5), 181.68 (C]O). EI-
MS (70 eV) m/z: 385 [M þ H]þ. Anal. Calcd for C23H20N4O2: C,
71.87; H, 5.20; N, 14.58. Found: C, 71.80; H, 5.14; N, 14.64%.
5.4. General procedure for the synthesis of 2-methyl-3-{3-methyl-
5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-dihydropyrimido[4,5-
b]quinolin-4-ones (5aef)
5.3.3. N3-{-5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-
isoxazolyl}-2-amino-3-quinolinecarboxamide (4c)
Yield 72%, mp 194e196 ꢁC. IR (KBr) cmꢂ1: 3350 (CONH), 3325,
3200 (NH2), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.32 (s,
A solution of 4 (1 mmol) in acetic anhydride (15 mL) was
refluxed for 5 h. The reaction mixture was allowed to cool down to
room temperature The precipitate that formed was filtered off,
washed with ethanol and recrystallized from ethanol to give
compound 5.
3H, CH3), 3.81 (s, 3H, OCH3), 5.94 (s, 2H, NH2, D2O exchangeable),
6.38e7.85 (m, 9H, ArH & 2H, CH]CH), 9.02 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.28 (CH3), 65.66
(OCH3), 100.31 (isoxazole-C4), 108.58 (]CH), 120.38 (]CH), 121.17
(AreC), 125.03 (AreC), 125.48 (AreC), 126.11 (AreC), 126.30 (AreC),
126.49 (AreC), 126.70 (AreC), 128.11 (AreC), 128.43 (AreC), 128.82
(AreC), 130.09 (AreC), 130.33 (AreC), 132.17 (quinoline ring-C3),
132.43 (quinoline ring-C4), 145.76 (quinoline ring-C2), 153.13 (iso-
xazole-C3), 159.73 (isoxazole-C5), 181.56 (C]O). EI-MS (70 eV) m/z:
401 [M þ H]þ. Anal. Calcd for C23H20N4O3: C, 69.00; H, 5.00; N,
14.00. Found: C, 68.08; H, 5.06; N, 14.05%.
5.4.1. 2-Methyl-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-
isoxazolyl}-3,4-dihydropyrimido[4,5-b]quinolin-4-one (5a)
Yield 85%, mp 220e222 ꢁC. IR (KBr) cmꢂ1: 1670 (C]O), 1640
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.23 (s, 3H, isoxazole
CH3), 2.41 (s, 3H, pyrimidine-CH3), 6.63e7.54 (m, 10H, ArH & 2H,
CH]CH). 13C NMR (75 MHz, CDCl3):
11.44 (isoxazole-CH3),
d
22.45 (pyrimidine-CH3), 108.03 (isoxazole-C4), 110.15 (]CH), 111.12
(]CH),123.05 (AreC),125.00 (AreC),126.42 (AreC),126.95 (AreC),
127.93 (AreC), 128.83 (AreC), 128.91 (AreC), 129.34 (AreC), 131.15
(AreC), 133.70 (AreC), 134.65 (AreC), 135.11 (AreC), 135.88 (AreC),
140.44 (AreC), 147.08 (AreC), 153.55 (isoxazole-C3), 156.57 (iso-
xazole-C5),162.80 (pyrimidine ring-C),172.50 (C]O). EI-MS (70 eV)
m/z: 395 [M þ H]þ. Anal. Calcd for C24H18N4O2: C, 73.09; H, 4.56; N,
14.21. Found: C, 73.03; H, 4.61; N, 14.26%.
5.3.4. N3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-2-
amino-6-chloro-3-quinolinecarboxamide (4d)
Yield 65%, mp 200e202 ꢁC. IR (KBr) cmꢂ1: 3315 (CONH), 3300,
3210 (NH2), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.24 (s,
3H, CH3), 5.93 (s, 2H, NH2, D2O exchangeable), 6.82e7.54 (m, 9H,
ArH & 2H, CH]CH), 8.80 (s, 1H, CONH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3)
d ppm: 11.23 (CH3), 100.28 (isoxazole-C4), 108.63
(]CH), 120.35 (]CH), 121.15 (AreC), 125.07 (AreC), 125.43 (AreC),
126.26 (AreC), 126.30 (AreC), 126.49 (AreC), 127.45 (AreC), 128.15
(AreC), 128.47 (AreC), 128.73 (AreC), 130.11 (AreC), 130.23 (AreC),
132.11 (quinoline ring-C3), 132.42 (quinoline ring-C4), 145.62
(quinoline ring-C2), 153.15 (isoxazole-C3), 159.76 (isoxazole-C5),
181.70 (C]O). EI-MS (70 eV) m/z: 405 [M þ H]þ. Anal. Calcd for:
C22H17N4O2Cl: C, 65.34; H, 4.20; N, 13.86. Found: C, 65.30; H, 4.24;
N, 13.82%.
5.4.2. 2-Methyl-3-{3-methyl-5-[(E)-2-(4-methylphenyl)-1-
ethenyl]-4-isoxazolyl}-3,4-dihydropyrimido[4,5-b]quinolin-4-one
(5b)
Yield 82%, mp 208e210 ꢁC. IR (KBr) cmꢂ1: 1680 (C]O), 1645
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.24 (s, 3H, isoxazole
CH3), 2.42 (s, 3H, CH3), 2.51 (s, 3H, pyrimidine-CH3), 6.84e7.52 (m,
9H, ArH & 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
11.32 (iso-
d
xazole-CH3), 22.28 (pyrimidine-CH3), 23.35 (AreCH3), 107.04 (iso-
xazole-C4), 109.10 (]CH), 111.02 (]CH), 122.08 (AreC), 125.10
(AreC), 125.40 (AreC), 126.49 (AreC), 126.90 (AreC), 128.43
(AreC), 128.31 (AreC), 129.14 (AreC), 130.25 (AreC), 133.20 (AreC),
133.25 (AreC), 134.21 (AreC), 135.48 (AreC), 141.24 (AreC), 146.08
(AreC), 152.55 (isoxazole-C3), 156.07 (isoxazole-C5), 161.70
(pyrimidine ring-C), 171.40 (C]O). EI-MS (70 eV) m/z: 409
[M þ H]þ. Anal. Calcd for C25H20N4O2: C, 73.52; H, 4.90; N, 13.72.
Found: C, 73.50; H, 4.82; N, 13.80%.
5.3.5. N3-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-2-amino-6-chloro-3-quinolinecarboxamide (4e)
Yield 85%, mp 178e180 ꢁC. IR (KBr) cmꢂ1: 3315 (CONH), 3300,
3220 (NH2), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.20 (s,
3H, CH3), 2.42 (s, 3H, CH3), 6.04 (s, 2H, NH2, D2O exchangeable),
7.05e7.83 (m, 8H, ArH & 2H, CH]CH), 8.92 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d
ppm: 11.28 (CH3), 28.53
(AreCH3), 100.17 (isoxazole-C4), 108.41 (]CH), 120.33 (]CH),

280
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
5.4.3. 3-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]3-methyl–4-
isoxazolyl}-2-methyl-3,4-dihydropyrimido[4,5-b]quinolin-4-one (5c)
Yield 74%, mp 215e217 ꢁC. IR (KBr) cmꢂ1: 1670 (C]O), 1640
5.5. General procedure for the synthesis of N3-{3-methyl-5-[(E)-2-
phenyl-1-ethenyl]-4-isoxazolyl}-2-imino-2H-3-
chromenecarboxamides (6aeg)
(C]N). 1H NMR (300 MHz, CDCl3)
d ppm: 2.21 (s, 3H, isoxazole
CH3), 2.45 (s, 3H, CH3), 2.52 (s, 3H, pyrimidine-CH3), 3.86 (s, 3H,
To a solution of compound 2 (1 mmol) in absolute ethanol
(20 mL) containing piperidine (0.5 mL), salicylaldehyde (1 mmol)
was added. The reaction mixture was heated under reflux for 3 h
and then allowed to cool. The precipitate obtained on pouring the
reaction mixture in to crushed ice was filtered off, washed with
ethanol, dried and recrystallized from ethanol to afford 6.
OCH3), 6.82e7.83 (m, 9H, ArH & 2H, CH]CH). 13C NMR (75 MHz,
CDCl3):
d 11.34 (isoxazole-CH3), 22.23 (pyrimidine-CH3), 64.33
(OCH3), 108.11 (isoxazole-C4), 110.23 (]CH), 111.23 (]CH), 123.10
(AreC), 125.04 (AreC), 126.56 (AreC), 126.82 (AreC), 127.81
(AreC), 128.72 (AreC), 128.83 (AreC), 129.17 (AreC), 131.03
(AreC), 133.54 (AreC), 134.51 (AreC), 135.06 (AreC), 135.67
(AreC), 140.36 (AreC), 147.17 (AreC), 153.61 (isoxazole-C3), 156.43
(isoxazole-C5), 162.69 (pyrimidine ring-C), 172.99 (C]O). EI-MS
(70 eV) m/z: 425 [M þ H]þ. Anal. Calcd for C25H20N4O3: C,
70.75; H, 4.71; N, 13.20. Found: C, 70.70; H, 4.76; N, 13.25%.
5.5.1. N3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-2-
imino-2H-3-chromenecarboxamide (6a)
Yield 73%, mp 235e237 ꢁC. IR (KBr) cmꢂ1: 3300 (CONH), 3215
(C]NH), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.42 (s, 3H,
CH3), 6.24 (s, 1H, ]CH), 6.91e7.63 (m, 9H, ArH & 2H, CH]CH), 9.24
5.4.4. 7-Chloro-2-methyl-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-
4-isoxazolyl}-3,4-dihydropyrimido[4,5-b]quinolin-4-one (5d)
Yield 79%, mp 206e208 ꢁC. IR (KBr) cmꢂ1: 1675 (C]O), 1640
(s, 1H, ]NH), 10.20 (s, 1H, CONH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3)
d ppm: 11.25 (CH3), 108.35 (isoxazole-C4), 110.96
(]CH), 115.12 (]CH), 121.26 (AreC), 123.43 (AreC), 126.01 (AreC),
126.15 (AreC), 126.41 (AreC), 127.56 (AreC), 128.80 (AreC), 129.06
(AreC), 130.08 (AreC), 130.72 (AreC), 131.19 (AreC), 132.73 (AreC),
134.00 (AreC), 136.56 (AreC), 155.15 (isoxazole-C3), 159.01 (iso-
xazole-C5), 160.01 (C]NH), 181.01 (C]O). EI-MS (70 eV) m/z: 372
[M þ H]þ. Anal. Calcd for C22H17N3O3: C, 71.15; H, 4.58; N, 11.32.
Found: C, 71.20; H, 4.53; N, 11.37%.
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.24 (s, 3H, isoxazole
CH3), 2.51 (s, 3H, pyrimidine-CH3), 6.92e7.84 (m, 9H, ArH & 2H,
CH]CH). 13C NMR (75 MHz, CDCl3):
11.23 (isoxazole-CH3),
d
24.66 (pyrimidine-CH3), 108.18 (isoxazole-C4), 110.26 (]CH),
111.08 (]CH), 123.17 (AreC), 125.08 (AreC), 126.51 (AreC),
126.85 (AreC), 127.87 (AreC), 128.91 (AreC), 129.01 (AreC),
129.26 (AreC), 131.09 (AreC), 133.65 (AreC), 134.57 (AreC),
135.19 (AreC), 135.78 (AreC), 140.31 (AreC), 147.10 (AreC),
153.35 (isoxazole-C3), 156.50 (isoxazole-C5), 162.76 (pyrimidine
ring-C), 172.86 (C]O). EI-MS (70 eV) m/z: 429 [M þ H]þ. Anal.
Calcd for C24H17N4O2Cl: C, 67.28; H, 3.97; N, 13.08. Found: C,
67.37; H, 3.90; N, 13.02%.
5.5.2. N3-{3-Methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-4-
isoxazolyl}-6-chloro-2-imino-2H-3-chromenecarboxamide (6b)
Yield 86%, mp 228e230 ꢁC. IR (KBr) cmꢂ1: 3300 (CONH), 3200
(C]NH), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.34 (s, 3H,
CH3), 2.51 (s, 3H, CH3), 6.20 (s, 1H, ]CH), 6.92e7.80 (m, 7H, ArH &
2H, CH]CH), 9.02 (s, 1H, ]NH), 10.01 (s, 1H, CONH, D2O
5.4.5. 7-Chloro-2-methyl-3-{3-methyl-5-[(E)-2-(4-methylphenyl)-
1-ethenyl]-4-isoxazolyl}-3,4-dihydropyrimido[4,5-b]quinolin-4-one
(5e)
exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.28 (CH3), 26.43
(AreCH3),109.01 (isoxazole-C4), 110.93 (]CH),115.11 (]CH),121.15
(AreC), 123.46 (AreC), 126.00 (AreC), 126.09 (AreC), 126.36
(AreC), 127.43 (AreC), 128.72 (AreC), 129.00 (AreC), 130.01 (AreC),
130.56 (AreC), 131.20 (AreC), 132.56 (AreC), 134.11 (AreC), 136.43
(AreC),155.11 (isoxazole-C3),159.00 (isoxazole-C5),160.11 (C]NH),
181.23 (C]O). EI-MS (70 eV) m/z: 420 [M þ H]þ. Anal. Calcd for
C23H18N3O3Cl: C, 65.87; H, 4.29; N, 10.02. Found: C, 65.80; H, 4.22;
N, 10.08%.
Yield 74%, mp 188e190 ꢁC. IR (KBr) cmꢂ1: 1680 (C]O), 1640
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.14 (s, 3H, isoxazole
CH3), 2.42 (s, 3H, CH3), 2.53 (s, 3H, pyrimidine CH3), 7.04e7.85 (m,
8H, ArH & 2H, CH]CH); 13C NMR (75 MHz, CDCl3):
11.36 (iso-
d
xazole-CH3), 25.22 (pyrimidine-CH3), 28.55 (AreCH3), 107.11 (iso-
xazole-C4), 109.19 (]CH), 111.11 (]CH), 122.17 (AreC), 125.20
(AreC),125.35 (AreC),126.51 (AreC),126.85 (AreC),128.55 (AreC),
128.51 (AreC), 129.18 (AreC), 130.17 (AreC), 133.17 (AreC), 133.14
(AreC), 134.27 (AreC), 135.33 (AreC), 141.21 (AreC), 146.16 (AreC),
152.43 (isoxazole-C3), 156.15 (isoxazole-C5), 161.64 (pyrimidine
ring-C), 171.83 (C]O). EI-MS (70 eV) m/z: 409 [M þ H]þ. EI-MS
(70 eV) m/z: 443 [M þ H]þ. Anal. Calcd for C25H19N4O2Cl: C,
67.87; H, 4.29; N, 12.66. Found: C, 67.80; H, 4.22; N, 12.71%.
5.5.3. N3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-6,8-
dichloro-2-imino-2H-3-chromenecarboxamide (6c)
Yield 85%, mp 194e196 ꢁC. IR (KBr) cmꢂ1: 3251 (CONH), 3220
(C]NH), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.41 (s, 3H,
CH3), 6.30 (s, 1H, ]CH), 6.90e7.84 (m, 7H, ArH & 2H, CH]CH), 9.31
(s, 1H, ]NH), 10.20 (s, 1H, CONH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3)
d ppm: 11.21 (CH3), 108.45 (isoxazole-C4), 111.01
5.4.6. 7-Chloro-3-{5-[(E)-2-(4-methoxyphenyl)-1-ethenyl]-3-
methyl-4-isoxazolyl}-2-methyl-3,4-dihydropyrimido[4,5-b]
quinolin-4-one (5f)
(]CH), 115.20 (]CH), 121.25 (AreC), 123.41 (AreC), 126.06 (AreC),
127.08 (AreC), 127.48 (AreC), 127.56 (AreC), 128.73 (AreC), 129.10
(AreC), 130.01 (AreC), 130.55 (AreC), 131.23 (AreC), 132.65 (AreC),
134.08 (AreC), 136.63 (AreC), 155.09 (isoxazole-C3), 159.06 (iso-
xazole-C5), 160.03 (C]NH), 181.20 (C]O). EI-MS (70 eV) m/z: 440
[M þ H]þ. Anal. Calcd for C22H15N3O3Cl2: C, 60.13; H, 3.41; N, 9.56.
Found: C, 60.17; H, 3.45; N, 9.52%.
Yield 86%, mp 200e202 ꢁC. IR (KBr) cmꢂ1: 1670 (C]O), 1640
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.24 (s, 3H, isoxazole
CH3), 2.52 (s, 3H, pyrimidine CH3), 3.81 (s, 3H, OCH3), 6.84e7.52 (m,
8H, ArH & 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
11.29 (iso-
d
xazole-CH3), 22.32 (pyrimidine-CH3), 64.28 (OCH3), 108.17 (iso-
xazole-C4), 110.29 (]CH), 111.21 (]CH), 123.17 (AreC), 125.13
(AreC), 126.43 (AreC), 126.71 (AreC), 127.88 (AreC), 128.65 (AreC),
128.78 (AreC), 129.13 (AreC), 131.11 (AreC), 133.49 (AreC), 134.59
(AreC), 135.03 (AreC), 135.58 (AreC), 140.29 (AreC), 147.21 (AreC),
153.56 (isoxazole-C3), 156.50 (isoxazole-C5), 162.73 (pyrimidine
ring-C), 173.01 (C]O). EI-MS (70 eV) m/z: 459 [M þ H]þ. Anal. Calcd
for C25H19N4O3Cl: C, 65.50; H, 4.14; N, 9.17. Found: C, 65.58; H, 4.07;
N, 9.10%.
5.5.4. N3-{5-[(E)-2-(4-Methoxyphenyl)-1-ethenyl]-3-methyl-4-
isoxazolyl}-6-bromo-2-imino-2H-3-chromenecarboxamide (6d)
Yield 76%, mp 209e211 ꢁC. IR (KBr) cmꢂ1: 3250 (CONH), 3155
(C]NH), 1675 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.20 (s, 3H,
CH3), 3.62 (s, 3H, OCH3), 6.13 (s, 1H, ]CH), 6.80e7.64 (m, 7H, ArH &
2H, CH]CH), 9.42 (s, 1H, ]NH), 10.24 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d
ppm: 11.30 (CH3), 64.73
(OCH3), 108.31 (isoxazole-C4), 110.88 (]CH), 115.11 (]CH), 121.15

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
281
(AreC), 123.41 (AreC),126.03 (AreC), 126.18 (AreC), 127.43 (AreC),
5.6.1. 3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-3,4-
127.51 (AreC), 128.61 (AreC), 129.11 (AreC), 130.01 (AreC), 131.21
(AreC), 132.71 (AreC), 133.62 (AreC), 134.01 (AreC), 136.43 (AreC),
155.16 (isoxazole-C3), 159.08 (isoxazole-C5), 160.03 (C]NH), 181.11
(C]O). EI-MS (70 eV) m/z: 480 [M þ H]þ. Anal. Calcd for
C23H18N3O4Br: C, 57.62; H, 3.75; N, 8.76. Found: C, 57.68; H, 3.70; N,
8.70%.
dihydro-2H-chromeno [2,3-d]pyrimidin-4-one (7a)
Yield 70%, mp 226e228 ꢁC. IR (KBr) cmꢂ1: 1660 (C]O), 1640
(C]N). 1H NMR (300 MHz, CDCl3)
CH3), 4.52 (s, 2H, CH2), 6.51 (s, 1H, ]CH), 6.84e7.63 (m, 9H, ArH
& 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
11.02 (CH3), 65.30
d ppm: 2.20 (s, 3H, isoxazole
d
(NeCH2eN), 109.87 (isoxazole-C4), 111.05 (]CH), 115.20 (]CH),
121.35 (AreC), 123.55 (AreC), 126.07 (AreC), 126.10 (AreC),
126.33 (AreC), 127.55 (AreC), 128.88 (AreC), 129.07 (AreC),
130.05 (AreC), 130.88 (AreC), 131.19 (AreC), 132.80 (AreC),
134.02 (AreC), 136.52 (AreC), 155.28 (isoxazole-C3),
159.00 (isoxazole-C5), 160.05 (pyrimidine ring-OeC]N), 175.05
(C]O). EI-MS (70 eV) m/z: 384 [M þ H]þ. Anal. Calcd for
C23H17N3O3: C, 72.06; H, 4.43; N, 10.96. Found: C, 72.10; H, 4.47;
N, 10.92%.
5.5.5. N3-{3-Methyl-5-[(E)-2-phenyl-1-ethenyl]-4-isoxazolyl}-6,8-
dibromo-2-imino-2H-3-chromenecarboxamide (6e)
Yield 80%, mp 216e218 ꢁC. IR (KBr) cmꢂ1: 3250 (CONH), 3200
(C]NH), 1680 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.42 (s, 3H,
CH3), 6.20 (s, 1H, ]CH), 6.81e7.74 (m, 7H, ArH & 2H, CH]CH), 9.02
(s, 1H, ]NH), 10.34 (s, 1H, CONH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3)
d ppm: 11.28 (CH3), 108.16 (isoxazole-C4), 110.86
(]CH), 115.16 (]CH), 121.32 (AreC), 123.41 (AreC), 126.06 (AreC),
126.43 (AreC), 127.51 (AreC), 127.56 (AreC), 128.73 (AreC), 129.01
(AreC), 130.11 (AreC), 130.61 (AreC), 131.10 (AreC), 132.53 (AreC),
134.03 (AreC), 136.51 (AreC), 155.10 (isoxazole-C3), 159.00 (iso-
xazole-C5), 160.07 (C]NH), 181.21 (C]O). EI-MS (70 eV) m/z: 528
[M þ H]þ. Anal. Calcd for C22H15N3O3Br2: C, 50.09; H, 2.84; N, 7.96.
Found: C, 50.01; H, 2.90; N, 7.90%.
5.6.2. 7-Chloro-3-{3-methyl-5-[(E)-2-(4-methylphenyl)-1-ethenyl]-
4-isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]pyrimidin-4-one
(7b)
Yield 78%, mp 203e204 ꢁC. IR (KBr) cmꢂ1: 1680 (C]O), 1660
(C]N). 1H NMR (300 MHz, CDCl3)
d ppm: 2.20 (s, 3H, isoxazole
CH3), 2.53 (s, 3H, CH3), 4.52 (s, 2H, CH2), 6.40 (s, 1H, ]CH),
6.80e7.64 (m, 7H, ArH & 2H, CH]CH); 13C NMR (75 MHz,
5.5.6. 2-Imino-6-methyl-N-(3-methyl-5-styrylisoxazol-4-yl)-2H-
chromene-3-carboxamide (6f)
CDCl3): d 10.72 (CH3), 22.04 (AreCH3), 64.20 (NeCH2eN), 109.37
(isoxazole-C4), 110.09 (]CH), 116.40 (]CH), 121.05 (AreC),
122.25 (AreC), 126.47 (AreC), 126.50 (AreC), 126.13 (AreC),
127.05 (AreC), 128.48 (AreC), 129.67 (AreC), 130.00 (AreC),
130.28 (AreC), 131.59 (AreC), 132.20 (AreC), 134.42 (AreC),
137.32 (AreC), 154.28 (isoxazole-C3), 158.00 (isoxazole-C5),
161.05 (pyrimidine ring-OeC]N), 176.25 (C]O). EI-MS (70 eV)
m/z: 432 [M þ H]þ. Anal. Calcd for C24H18N3O3Cl: C, 66.82; H,
4.17; N, 9.74. Found: C, 66.78; H, 4.21; N, 9.70%.
Yield 75%, mp 204e208 ꢁC. IR (KBr) cmꢂ1: 3280 (CONH), 3215
(C]NH), 1670 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.40 (s,
3H, CH3), 2.50 (s, 3H, CH3), 6.24 (s, 1H, ]CH), 6.90e7.64 (m, 8H,
ArH & 2H, CH]CH), 9.24 (s, 1H, ]NH), 10.20 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.22 (CH3), 26.53
(AreCH3), 109.03 (isoxazole-C4), 110.86 (]CH), 115.23 (]CH),
121.11 (AreC), 123.23 (AreC), 126.11 (AreC), 126.18 (AreC), 126.31
(AreC), 127.40 (AreC), 128.73 (AreC), 129.03 (AreC), 130.11
(AreC), 130.55 (AreC), 131.21 (AreC), 132.51 (AreC), 134.08
(AreC), 136.44 (AreC), 155.10 (isoxazole-C3), 159.06 (isoxazole-C5),
160.13 (C]NH), 181.30 (C]O). EI-MS (70 eV) m/z: 386 [M þ H]þ.
Anal. Calcd for C23H19N3O3: C, 71.68; H, 4.93; N, 10.90. Found: C,
71.72; H, 4.89; N, 10.85%.
5.6.3. 7,9-Dichloro-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-
isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]pyrimidin-4-one (7c)
Yield 80%, mp 220e223 ꢁC. IR (KBr) cmꢂ1: 1675 (C]O), 1660
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.24 (s, 3H, isoxazole
CH3), 4.20 (s, 2H, CH2), 6.51 (s, 1H, ]CH), 6.92e7.64 (m, 7H, ArH
& 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
11.25 (CH3), 65.44
d
5.5.7. 2-Imino-6-methoxy-N-(3-methyl-5-styrylisoxazol-4-yl)-2H-
chromene-3-carboxamide (6g)
(NeCH2eN), 109.79 (isoxazole-C4), 111.16 (]CH), 115.26 (]CH),
121.41 (AreC), 123.61 (AreC), 126.23 (AreC), 126.18 (AreC),
126.43 (AreC), 127.65 (AreC), 128.81 (AreC), 129.02 (AreC),
130.13 (AreC), 130.75 (AreC), 131.23 (AreC), 132.61 (AreC),
134.00 (AreC), 136.48 (AreC), 155.20 (isoxazole-C3),
159.03 (isoxazole-C5), 160.09 (pyrimidine ring-OeC]N), 175.45
(C]O). EI-MS (70 eV) m/z: 452 [M þ H]þ. Anal. Calcd for
C23H15N3O3Cl2: C, 61.19; H, 3.32; N, 9.31. Found: C, 61.23; H,
3.36; N, 9.27%.
Yield 74%, mp 217e219 ꢁC. IR (KBr) cmꢂ1: 3275 (CONH), 3200
(C]NH), 1675 (C]O). 1H NMR (300 MHz, CDCl3)
d ppm: 2.40 (s, 3H,
CH3), 3.62 (s, 3H, OCH3), 6.20 (s, 1H, ]CH), 6.94e7.62 (m, 8H, ArH &
2H, CH]CH), 9.21 (s, 1H, ]NH), 10.24 (s, 1H, CONH, D2O
exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 11.26 (CH3), 66.03
(OCH3), 108.28 (isoxazole-C4), 110.73 (]CH), 115.23 (]CH), 121.10
(AreC), 123.39 (AreC), 126.02 (AreC), 126.17 (AreC), 126.40 (AreC),
127.41 (AreC), 128.51 (AreC), 129.01 (AreC), 130.10 (AreC), 131.26
(AreC), 132.69 (AreC), 134.08 (AreC), 136.40 (AreC), 155.15 (iso-
xazole-C3),159.06 (isoxazole-C5),160.08 (C]NH),181.20 (C]O). EI-
MS (70 eV) m/z: 402 [M þ H]þ. Anal. Calcd for C23H19N3O4: C, 68.82;
H, 4.73; N, 10.47. Found: C, 68.75; H, 4.78; N, 10.40%.
5.6.4. 7-Bromo-3-{3-methyl-5-[(E)-2(4-methoxyphenyl)-1-
ethenyl]-4-isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]
pyrimidin-4-one (7d)
Yield 82%, mp 200e204 ꢁC. IR (KBr) cmꢂ1: 1687 (C]O), 1655
(C]N). 1H NMR (300 MHz, CDCl3)
d ppm: 2.40 (s, 3H, isoxazole
CH3), 3.62 (s, 3H, OCH3), 4.64 (s, 2H, CH2), 6.40 (s, 1H, ]CH),
6.80e7.62 (m, 7H, ArH & 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
5.6. General procedure for the synthesis of 3-{3-methyl-5-[(E)-2-
phenyl-1-ethenyl]-4-isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]
pyrimidin-4-ones (7aeg)
d 11.24 (CH3), 64.23 (OCH3), 65.45 (NeCH2eN), 109.72 (isoxazole-
C4), 111.10 (]CH), 115.23 (]CH), 121.37 (AreC), 123.41 (AreC),
126.01 (AreC), 126.13 (AreC), 126.39 (AreC), 127.52 (AreC),
128.83 (AreC), 129.09 (AreC), 130.10 (AreC), 130.81 (AreC),
131.10 (AreC), 132.79 (AreC), 134.00 (AreC), 136.39 (AreC), 155.17
(isoxazole-C3), 159.06 (isoxazole-C5), 160.15 (pyrimidine ring-
OeC]N), 175.55 (C]O). EI-MS (70 eV) m/z: 492 [M þ H]þ. Anal.
Calcd for C24H18N3O4Br: C, 58.65; H, 3.66; N, 8.55. Found: C, 58.69;
H, 3.62; N, 8.51%.
A mixture of compound 6 (1 mmol) and formalin (37%, 1 mmol)
was refluxed in ethanol (15 mL) for 4 h. The reaction mixture after
cooling to room temperature was poured in to ice-cold water. The
separated solid was filtered and recrystallized from ethanol to get
pure compound 7.

282
E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
5.6.5. 7,9-Dibromo-3-{3-methyl-5-[(E)-2-phenyl-1-ethenyl]-4-
isoxazolyl}-3,4-dihydro-2H-chromeno[2,3-d]pyrimidin-4-one (7e)
Yield 86%, mp 199e201 ꢁC. IR (KBr) cmꢂ1: 1670 (C]O), 1655
The medium was poured in to sterile petri-dishes under aseptic
conditions in a Laminar flow chamber. When the medium in the
plates solidified, 0.5 mL of the culture (one-week-old) of fungal
spore suspension was inoculated and uniformly spread over the
agar surface with a sterile L-shaped rod. Solutions were prepared
by dissolving test compound in acetone. Agar inoculation, cups
were scooped out with 6 mm sterile cork borer and the lids of the
dishes were replaced. To each cup, different concentrations of test
solution were added. Controls were maintained with acetone and
clotrimazole. The treated and the controls were kept at room
temperature for 72e96 h. The Minimum inhibitory Concentration
(C]N). 1H NMR (300 MHz, CDCl3)
d
ppm: 2.30 (s, 3H, isoxazole
CH3), 4.42 (s, 2H, CH2), 6.61 (s, 1H, ]CH), 6.92e7.80 (m, 7H, ArH &
2H, CH]CH). 13C NMR (75 MHz, CDCl3):
11.22 (CH3), 65.40
d
(NeCH2eN), 109.71 (isoxazole-C4), 111.10 (]CH), 115.21 (]CH),
121.38 (AreC), 123.60 (AreC), 126.27 (AreC), 126.10 (AreC), 127.41
(AreC), 127.69 (AreC), 128.78 (AreC), 129.08 (AreC), 130.21 (AreC),
130.64 (AreC), 131.42 (AreC), 132.58 (AreC), 134.12 (AreC), 136.35
(AreC), 155.18 (isoxazole-C3), 159.15 (isoxazole-C5), 160.21
(pyrimidine ring-OeC]N), 175.65 (C]O). EI-MS (70 eV) m/z: 540
[M þ H]þ. Anal. Calcd for C23H15N3O3Br2: C, 51.20; H, 2.78; N, 7.79.
Found: C, 51.24; H, 2.74; N, 7.75%.
(MIC) was recorded in mg/mL. Three to four replicates were main-
tained for each treatment.
5.7.3. Anti-inflammatory analysis
5.6.6. 2,3-Dihydro-7-methyl-3-(3-methyl-5-styrylisoxazol-4-yl)
chromeno[2,3-d]pyrimidin-4-one (7f)
The albino rats weighing 150e180 g were used throughout the
assay. They were kept in the animal house under standard condi-
tion of light and temperature with free access to food and water.
The rats were randomly divided into groups, each of six rats. One
group of six rats was kept as a control and another group received
the standard drug Indomethacin (at a dose of 10 mg/kg body
weight po). All the test compounds were dissolved in 0.05 mL
solution of DMSO and given 60 min before the commencement of
the study. After that 0.1 mL of 1% W/V carrageenan solution in
normal saline was injected into the subplanter region of the left
hind paw under light ether anesthesia 1 h after oral administration
(po) of the test compound. The paw volume of each rat was
measured immediately by mercury plethysmo meter and re-
measured again 1, 2, 3, and 4 h after administration of DMSO. The
edema was expressed as an increase in the volume of paw, and the
% of edema inhibition for each rat and each group was calculated as
follows:
Yield 78%, mp 221e223 ꢁC. IR (KBr) cmꢂ1: 1660 (C]O), 1640
(C]N). 1H NMR (300 MHz, CDCl3)
d ppm: 2.24 (s, 3H, isoxazole
CH3), 2.52 (s, 3H, CH3), 4.52 (s, 2H, CH2), 6.51 (s, 1H, ]CH),
6.84e7.63 (m, 8H, ArH & 2H, CH]CH). 13C NMR (75 MHz, CDCl3):
d
11.34 (CH3), 23.04 (AreCH3), 64.26 (NeCH2eN),109.30 (isoxazole-
C4), 110.13 (]CH), 116.46 (]CH), 121.02 (AreC), 122.23 (AreC),
126.47 (AreC), 126.59 (AreC), 126.10 (AreC), 127.15 (AreC),
128.40 (AreC), 129.63 (AreC), 130.02 (AreC), 130.21 (AreC),
131.66 (AreC), 132.27 (AreC), 134.45 (AreC), 137.39 (AreC),
154.32 (isoxazole-C3), 158.10 (isoxazole-C5), 161.15 (pyrimidine
ring-OeC]N), 176.68 (C]O). EI-MS (70 eV) m/z: 398 [M þ H]þ.
Anal. Calcd for C24H19N3O3: C, 72.54; H, 4.78; N, 10.57. Found: C,
72.59; H, 4.73; N, 10.50%.
5.6.7. 2,3-Dihydro-7-methoxy-3-(3-methyl-5-styrylisoxazol-4-yl)
chromeno[2,3-d]pyrimidin-4-one (7g)
Yield 74%, mp 211e213 ꢁC. IR(KBr)cmꢂ1:1660(C]O),1640(C]N).
ðVt ꢂ VoÞcontrol ꢂ ðVt ꢂ VoÞtested compound
%Inhibition ¼
1H NMR (300 MHz, CDCl3)
d
ppm: 2.20 (s, 3H, isoxazole CH3), 3.60 (s,
3H, OCH3), 4.50(s,2H, CH2), 6.54 (s,1H, ]CH), 6.80e7.62 (m, 8H, ArH &
2H, CH]CH); 13C NMR (75 MHz, CDCl3):
11.31 (CH3), 64.13 (OCH3),
ðVt ꢂ VoÞcontrol
ꢃ 100
d
Where
65.34 (NeCH2eN),109.61 (isoxazole-C4), 111.17 (]CH),115.31 (]CH),
121.42 (AreC), 123.55 (AreC), 126.09 (AreC), 126.21 (AreC), 126.43
(AreC), 127.61 (AreC), 128.75 (AreC), 129.21 (AreC), 130.20 (AreC),
130.79 (AreC), 131.21 (AreC), 132.65 (AreC), 134.21 (AreC), 136.44
(AreC), 155.23 (isoxazole-C3), 159.18 (isoxazole-C5), 160.21 (pyrimi-
dine ring-OeC]N), 175.66 (C]O). EI-MS (70 eV) m/z: 414 [M þ H]þ.
Anal. Calcd for C24H19N3O4: C, 69.73; H, 4.60; N,10.16. Found: C, 69.77;
H, 4.56; N, 10.12%.
Vt ¼ Volume of edema at specific time interval and Vo ¼ Volume
of edema at zero time interval.
5.7.4. Analgesic analysis
Albino rats weighing 150e180 g were used throughout this
assay. They were kept in the animal house under standard condi-
tion of light and temperature with free access to food and water.
The animals were randomly divided into groups each of six rats.
One group of six rats was kept as a control and another group
received the standard drug Indomethacin (at a dose of 10 mg/kg
body weight po). The tested compounds were administrated orally
at a dose of 10 mg/kg and Indomethacin was used as a reference
drug (10 mg/kg). The recorded values were the average of six
5.7. Pharmacological screening
5.7.1. Antibacterial assay
The ready-made nutrient broth medium (Himedia, 24 g) was
suspended in distilled water (100 mL) and heated until it dissolved
completely. The medium and test tubes were autoclaved at a pres-
sure of 15 lb/inc2 for 20 min. A set of sterilized test tubes with
nutrient broth medium was capped with cotton plugs. The test
administrations
comparison with the basal values.
ꢀ
S.E. and the percentage increase of the
compound was dissolved in acetone and a concentration of 100
mg/
Acknowledgments
mL of the test compound was added in the first test tube, which
was serially diluted. A fixed volume of 0.5 mL of overnight culture
was added in all the test tubes which were incubated at 37 ꢁC for
24 h. After 24 h these tubes were measured for turbidity.
The authors are thankful to the Head, Department of Chemistry,
Kakatiya University, Warangal for providing the facilities, the
Director, Indian Institute of Chemical Technology, Hyderabad for
recording 1H NMR, 13C NMR, and Mass Spectral data. Financial
assistance from the UGC SAP (phase-1)-DRS programme, New
Delhi, India, is greatly acknowledged. We are grateful to Prof. J.R.
Falck, Departments of Biochemistry and Pharmacology, University
of Texas, Southwestern Medical Center, Texas, USA for helpful
discussions.
5.7.2. Antifungal assay
For the antifungal assay, the ready-made potato dextrose agar
medium (Himedia, 39 g) was suspended in distilled water (100 mL)
and heated until it dissolved completely. The medium and glass
petri-dishes were autoclaved at a pressure of 15 lb/inc2 for 20 min.

E. Rajanarendar et al. / European Journal of Medicinal Chemistry 55 (2012) 273e283
283
[11] E. Rajanarendar, E. Kalyan Rao, Firoz Pasha Shaik, M. Nagi Reddy,
M. SrinivasReddy, J. Sulphur Chem. 31 (2010) 263.
References
[12] E. Rajanarendar, M. Nagi Reddy, K. Rama Murthy, K. Govardhan Reddy, S. Raju,
M. Srinivas, B. Praveen, M. Srinivasa Rao, Bioorg. Med. Chem. Lett. 20 (2010)
6052.
[13] E. Rajanarendar, M. Nagi Reddy, S. Rama Krishna, K. Rama Murthy,
P. Surendar, R.N. Reddy, Y.N. Reddy, Bioorg. Med. Chem. Lett. 22 (2012) 149.
[14] E. Rajanarendar, S. Raju, M. Nagi Reddy, S. Rama Krishna, L. Hari Kiran,
A. RamNarasimha Reddy, Y. Narasimha Reddy, Eu. J. Med. Chem. 50 (2012) 274.
[15] E. Rajanarendar, M. Nagi Reddy, S. Rama Krishna, K. Govardhan Reddy,
Y.N. Reddy, M.V. Rajam, Eu. J. Med. Chem. 50 (2012) 344.
[1] L.D. Eirich, G.D. Vogels, R.S. Wolfe, Biochemistry 17 (1978) 4583.
[2] C. Walsh, Enzymatic Reaction Mechanism, W. H. Freeman and Co., San Fran-
sisco, 1979, p. 373.
[3] F. Yoneda, K. Tanaka, Med. Res. Rev. 7 (1987) 477.
[4] S. Shinkai, A. Kawase, T. Yamaguchi, O. Manabe, J. Chem. Soc. Chem. Commun.
(1988) 457.
[5] N.M. Sarbry, H.M. Mohamed, E.S.A.E.H. Kattab, S.S. Motlar, A.M. EI-Agrody, Eu.
J. Med. Chem. 46 (2011) 765.
[6] G. Daidone, D. Raffa, B. Maggio, F. Plescia, V.M.C. Cutuli, N.G. Mangano,
A. Caruso, Arch. Pharm. Med. Chem. 332 (1999) 50.
[7] K. Tomita, Y. Takahi, R. Ishizuka, S. Kamamura, M. Nakagawa, M. Ando,
T. Nakanishi, T. Nakamura, H. Udaira, Ann. Sankyo Res. Lab. 1 (1973) 25;
Chem. Abstr. 80 (1974) 120808.
[16] E. Rajanarendar, M. Nagi Reddy, K. Govardhan Reddy, S. Rama Krishna,
Tetrahedron Lett. 53 (2012) 2909.
[17] A.K. Murthy, K.S.R.K.M. Rao, N.V.S. Rao, J. Indian Chem. Soc. 53 (1976) 1047.
[18] National committee for clinical laboratory standards (NCCLS), Standard
Methods Fordilution Antimicrobial Susceptibility Tests for Bacteria, Which
Grows Aerobically, Nat. Comm. Clini. Lab. Stands. Ltd., Villanova, 1982, p. 242.
[19] E. Margery Linday, Practical Introduction to Microbiology, E. & F.N. Spon Ltd.,
1962, p. 177.
[20] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. III (1962) 544.
[21] P. Armitage, Statistical Methods in Medical Research, first ed., Blackwell
Scientific Publ. Oxford, London, 1971, p. 147.
[8] (a) J.J. Talley, Prog. Med. Chem. 13 (1999) 201;
(b) J.J. Talley, D.L. Brown, J.S. Carter, M.J. Graneto, C.M. Koboldt, J.L. Masferrer,
W.E. Perkins, R.S. Rogers, A.F. Shaffer, Y.Y. Zhang, B.S. Zweifel, K. Seibert,
J. Med. Chem. 43 (2000) 775.
[9] K. Haripara, S. Patel, A. Joshi, H. Parekh, Indian J. Heterocycl. Chem. 13(2004)221.
[10] W.-T. Li, D.-R. Hwang, C.-P. Chen, C.-W. Shen, C.-L. Huang, T.-W. Chen, C.-H. Lin, Y.-
L. Chang, Y.-Y. Chang, Y.-K. Lo, H.-Y. Tseng, C.-C. Lin, J.-S. Song, H.-C. Chen, S.-J. Chen,
S.-H. Wu, C.-T. Chen, J. Med. Chem. 46 (2003) 1706.
[22] R.A. Turner, Analgesics, in: R.A. Turner (Ed.), Screening Methods in Pharma-
cology, Academic Press, London, 1965, p. 100.