Efficient regioselective synthesis and potential antitumor evaluation of isoxazolo[5,4-b]pyridines and related annulated compounds

Article abstract of DOI:10.1002/ardp.201100258

The reaction of 5-amino-3-methylisoxazole with appropriate α,β-unsaturated ketones gave the corresponding isoxazolo[5,4-b] pyridines. Treatment of 1 with 2,6-dibenzylidenecyclohexanone or 2-benzylidenedimedone afforded the corresponding isoxazolo[5,4-b]quinoline derivatives. 4,6,8,9-Tetrahydroisoxazolo[5,4-b]quinolin-5-one derivative was also obtained by multicomponent condensation reaction of 1 with dimedone and benzaldehyde. Heterocyclic annulation of the isoxazolo[5,4-b]pyridine system was achieved via reaction of 1 with benzylidene derivatives of indandione, quinuclidone, pyrazolone, and oxazolone. A representative of some newly synthesized compounds was evaluated as antitumor agents.

Full text of DOI:10.1002/ardp.201100258

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Efficient Regioselective Synthesis and Potential Antitumor
Evaluation of Isoxazolo[5,4-b]pyridines and Related
Annulated Compounds
Wafaa S. Hamama, Mona E. Ibrahim, and Hanafi H. Zoorob
Faculty of Science, Department of Chemistry, Mansoura University, Mansoura, Egypt
The reaction of 5-amino-3-methylisoxazole with appropriate a,b-unsaturated ketones gave the
corresponding isoxazolo[5,4-b]pyridines. Treatment of 1 with 2,6-dibenzylidenecyclohexanone
or 2-benzylidenedimedone afforded the corresponding isoxazolo[5,4-b]quinoline derivatives.
4,6,8,9-Tetrahydroisoxazolo[5,4-b]quinolin-5-one derivative was also obtained by multicomponent
condensation reaction of 1 with dimedone and benzaldehyde. Heterocyclic annulation of the
isoxazolo[5,4-b]pyridine system was achieved via reaction of 1 with benzylidene derivatives
of indandione, quinuclidone, pyrazolone, and oxazolone. A representative of some newly
synthesized compounds was evaluated as antitumor agents.
Keywords: Annulation / Antitumor activity (in vitro) / Biselectrophiles / Isoxazole / Regioselective
Received: July 18, 2011; Revised: December 12, 2011; Accepted: December 20, 2011
DOI 10.1002/ardp.201100258
Introduction
direct multicomponent procedure may lead to the formation
of different products [4], connected to the complex reaction
mechanism of these transformations [5, 6]. It was felt inter-
esting to synthesize fused heterocyclic systems incorporating
isoxazole moiety and evaluate their biological activity. Our
interest directed to synthesize pyridine carboxylic acid fused
to isoxazole rings is mainly caused by the known biological
activity of these systems [7].
The isoxazole rings are frequently present in biologically
active compounds and are used as building blocks in
the synthesis of new potential drugs. Therefore, 5-amino-3-
methylisoxazole (1) acts as a building block for construction
of isoxazole-annulated heterocyclic systems. Compound 1
has multiple competing sites for ring-annulation reaction
toward the biselectrophiles. The 4-position of the isoxazole
is the most nucleophilic and attacks the most electrophilic
carbon atom of the reactants.
Indandiones are very important synthons for synthesis of
fused compounds containing the indandione moiety, which
are evaluated as inhibitors of human papillomavirus type II
[8, 9] and as antimicrobial agents [10].
Cyclocondensation of aminoazoles and aminoazines with
a,b-unsaturated ketones or their synthetic precursor alde-
hydes and ketones containing at least two active hydrogen Results and discussion
atoms are the most widespread and investigated pathways to
fused dihydroazaheterocycles [1, 2]. In most cases both types
of reaction pathways, that is, a sequential protocol involving
the initial synthesis of the a,b-unsaturated ketonic com-
pounds and the other pathway is the three-component reac-
tion, yield the same products [3]. However, in rare cases the
Chemistry
5-Amino-3-methylisoxazole (1) was reacted with (E)-3-(4-
methoxyphenyl)-1-phenylprop-2-en-1-one (2) in DMF via cyclo-
condensation reaction to furnish the isoxazolo[5,4-b]pyridine
3. We have found that condensation between 1 and (E)-3-(4-
methoxyphenyl)-1-phenylprop-2-en-1-one (2) was regioselec-
tive and afforded 3 (TLC monitoring) which is concordant
with similar reactions of aminoisoxazole and biselectro-
philes [11] (Scheme 1).
Correspondence: Wafaa S. Hamama, Faculty of Science, Department of
Chemistry, Mansoura University, El-Gomhoria Street, Mansoura 35516,
Egypt.
E-mail: wshamama@yahoo.com
Fax: (þ2050)2246254
The plausible reaction mechanism for the formation of 3 is
displayed in Scheme 2.
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W. S. Hamama et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
OCH3
OCH3
H3CO
Ph
H3C
N
H3C
N
O
Auto-oxidation
2
O
N
Ph
O
N
H
Ph
OCH3
3
H3CO
CH3
H3C
H3C
N
O
4
N
NH2
O
1
O
N
H
CH3
5
Ph
Ph
Ph
Ph
H3C
H3C
N
O
6
Auto-oxidation
N
Scheme 1. Cyclocondensation reactions of 1
with a,b-unsaturated ketones.
O
O
Ph
N
7
Ph
N
H
In a similar manner, 4-(4-methoxyphenyl)-3,6-dimethyl-4,7-
dihydro-isoxazolo[5,4-b]pyridine (5) was synthesized by the
reaction of 1 with (E)-4-(4-methoxyphenyl)but-3-en-2-one (4)
in DMF. An efficient method for selective synthesis of 6-
styrylisoxazolo[5,4-b]pyridine derivatives 7 was achieved by
the Micheal addition of 1 to 1,5-diphenyl-1,4-pentadiene-3-
one (6) in DMF via cyclocondensation reaction (Scheme 1).
The dihydropyridine adduct from Hantzsh synthesis afforded
the fully oxidized (aromatized) pyridyl product presumably
due to the highly conjugated system formed. The consti-
tution of 7 was affirmed through its spectral data.
Moreover, we have provided an efficient method for the
synthesis of fused heterocyclic compound containing the
dihydroisoxazolo[5,4-b]pyridin-6-one skeleton 11. Therefore,
refluxing equimolar amounts of
1
and anisylidene
Meldrum’s acid (10) [12, 13] for 30 min afforded 11 in modest
yield. In principle, the amine 1 attack on b-C of the a,b-
unsaturated cyclic ester 10 was followed by cyclization reac-
tion of the intermediate adduct to form 11. The formation of
11 was in line with that reported in the literature to synthes-
ize pyrazolo[5,4-b]pyridin-6-one [14], while an alternative
route to synthesize 11 was reported by Tu et al. [15]. We
assumed that formation of 11 was directed via initial
addition of the b-carbon of the enamino group in compound
1 to the b-C of the cyclic ester and then loss of one molecule of
Treatment of (E)-4-(4-methoxyphenyl)-2-oxobut-3-enoic acid
(8) with 1 in acetic acid led to 4-(4-methoxyphenyl)-3-methyl-
isoxazolo[5,4-b]pyridine-6-carboxylic acid (9, Scheme 3).
OCH3
OCH3
OCH3
O
H3C
H3C
H3C
Ph
OH
N
N
N
NH2
O
NH2
O
O
N
H
O
1
2
-H2O
OCH3
OCH3
H3C
H3C
Auto-oxidation
Scheme 2. The plausible mechanism for the
formation of isoxazolo[5,4-b]pyridine deriva-
tive 3.
N
N
O
N
H
Ph
N
Ph
O
3
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Isoxazolo[5,4-b]pyridines: Synthesis and Antitumor Activity
3
OCH3
OCH3
H3CO
O
OH
H3C
H3C
N
O
8
Auto-oxidation
N
O
O
N
O
O
N
H
OH
OH
9
O
OCH3
OCH3
O
a
b
O
O
O
O
10
H3C
N
H3C
N
_
CO2
O
H3CO
1
C
_
·
·
O
(CH3)2CO
O
O
O
NH2
NH2
O
O
H3CO
O
O
H3C
X
O
12
H
O
O
H3C
H
a
N
N
O
H
HC(OCH3)3
N
b
H
13
O
O
N
O
H
_
_
(CH3)2CO
CO2
11
O
H3C
N
O
H3C
PhNO2
N
O
Scheme 3. Pyridine ring formation through
auto-oxidation, nucleophile attack, and cycli-
zation reactions.
N
H
O
N
H
14
acetone and CO2 which leads to the formation of ketene
intermediate followed by its cyclization. This behavior is
well known for the thermolysis of Meldrum’s acid derivatives
[16, 17], and it is shown in Scheme 3. Spectroscopic data for 11
showed the expected distinctive features. Furthermore, follow-
ing the procedure reported by Tu et al. [15] for 5-amino-3-aryl
pyrazoles, refluxing a solution of Meldrum’s acid 12 [18] and
methyl orthoformate (1:5) followed by immediate addition
of 1 in an equimolecular amount relative to Meldrum’s
acid furnished the corresponding 2,2-dimethyl-5-[(3-methyl-
isoxazol-5-ylamino)methylene]-1,3-dioxane-4,6-dione (13). Its
reflux in nitrobenzene afforded the dihydroisoxazolopyridine
compound 14.
by multicomponent condensation reaction of 1 with
dimedone and benzaladehyde in ethanol/acetic acid mixture
gave 18 [19a]. The objective of this work was directed to
annulate 1 via one-step cyclocondensation reaction with 19
which leads to regioselective linear tetracyclic derivative 20.
Thus, Michael addition of 1 to 2-benzylidene indandione (19)
in ethanol/acetic acid mixture afforded 3-methyl-4-phenyl-
4,10-dihydroisoxazolo[40,50:5,6]pyrido[2,3-a]inden-5-one (20)
in high yield and not the angular tetracyclic derivative
20a (Scheme 4). Similarly, multicomponent condensation
of 1 with indandione and benzaldehyde led to dihydropyr-
ido[2,3-a]inden-5-one derivative 20.
It is worthy to mention that the dihydropyridine derivative
20 is easily accessed via Hantzsch reaction and does not
undergo auto-oxidation. Alternatively, Tu et al. [12, 15]
obtained the annulated isoxazolo[5,4-b]pyridines through a
feasible economic strategy by one-pot tandem reaction under
microwave irradiation in water.
In addition, the reaction of 1 with 2,6-dibenzylidenecyclo-
hexanone (15) in DMF afforded 8-benzylidene-4,5,6,7,8,9-
hexahydro-3-methyl-4-phenyl isoxazolo [5,4-b]quinoline
(16). Dimedone derivative 17 is an attractive synthetic inter-
mediate to develop an efficient and versatile synthesis of
linear 3,7,7-trimethyl-4-phenyl-4,6,8,9-tetrahydroisoxazolo-
[5,4-b]quinolin-5-one (18) and not the angular tricyclic
(18a). Accordingly, the regioselective reaction of 1 with 2-
benzylidenedimedone (17) in ethanol/acetic acid mixture, or
Linear heterocyclic annulation of isoxazolo[5,4-b]pyridine
system through fusion to the pyridine moiety of the system
was achieved through cycloaddition of 1 with a,b-unsatu-
rated heterocycles. Compounds containing quinuclidine ring
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W. S. Hamama et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
O
Ph
Ph
Ph
Ph
O
H3C
H3C
N
15
N
O
N
O
O
O
N
H
20a
H
Ph
16
Ph
O
19 or
1
H3C
N
O
CHO
O
O
+
Ph
O
N
H
Ph
H3C
N
O
O
O
or
CHO
18a
17
O
N
H
20
Ph
+
Ph
O
H3C
N
O
Scheme 4. The pyridine ring formation
through reaction of 1 with 2,6-dibenzylidene-
cyclohexanone and multicomponent reaction.
O
N
H
18
were reported to exhibit pharmacological activity and this
enthused us to synthesize fused isoxazole moiety bearing a
quinuclidine nucleus [19b]. Thus, Michael addition of 1 with
p-chlorobenzylidene-3-quinuclidinone (21) in ethanol/acetic
acid mixture led to the formation of 22 (Scheme 5). No
attention has been paid to the similar reaction with benzyl-
idenepyrazolone derivatives 23 and 24 or benzylideneoxazo-
lone derivative 25 which can be used as a key intermediate for
building of a pyridoisoxazole moiety fused to pyrazole or
oxazole nucleus (26–28, respectively). Hence, the reaction of 1
with 23, 24, or 25 in ethanol/glacial acetic acid mixture gave
the linear tricyclic derivatives 26–28, respectively. Moreover,
when compound 1 was reacted with 2-(benzo[d]thiazol-2-yl)-3-
(4-chlorophenyl) acrylonitrile (29), a Michael adduct 30
was obtained instead of the fused isoxazolo[5,4-b]pyridine
derivative 30a.
Assignment of the newly synthesized compounds was
1
based on elemental analyses, IR, H NMR, and mass spectral
data (cf. Experimental Section).
Biological activity
Effect of drugs on the viability of Ehrlich ascites carcinoma
cells (EAC) in vitro
Thirteen oxazolopyridines and its annulated analogues were
tested for potential antitumor activity against EAC in vitro
[20]. Results for the IC100, IC50, and IC25 values of the active
compounds are summarized in Table 1. The data showed
clearly that all the tested compounds showed more toxicity
Cl
Ph
N
N
Ph
H3C
N
Ph
O
O
25
H3C
N
Cl
O
N
O
21
N
Ph
Cl
O
N
H
O
N
H
22
28
Ar
1
N
S
H3C
H3C
N
NC
Ar
N
N
S
Ar
O
H3C
N
O
N
30a
CH3
NH2
N
Ph
29
N
23, Ar = 4-OCH3C6H4
24, Ar = 3,4(OCH2O)C6H4
O
N
Cl
N
H
Ph
26, Ar = 4-OCH3C6H4
27, Ar = 3,4(OCH2O)C6H4
N
H3C
N
S
CN
O
Scheme 5. Reaction of 1 with arylidenes of
different heterocyclic compounds.
NH2
30
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Isoxazolo[5,4-b]pyridines: Synthesis and Antitumor Activity
5
Table 1. In vitro potential antitumor activity of isoxazole analogues
using EAC assay
Based on these preliminary screening results, compound 13
showed significant activities in certain cancer cells and have
been targeted for further studies. Additional research, includ-
ing mode of action studies, is planned to accurately establish
relative activity for SARs and rational design. Recently, the
cancer chemopreventive effects of oxazole derivatives have
been intensively investigated. Oxazole derivatives exhibited
pronounced antitumor activities by triggering apoptosis in
human tumor cells [24]. Studies are underway to investigate
the apoptosis-inducing activity of compounds found to be
cytotoxic in this study.
Compound no.
% Dead
IC100 (mM)
IC50 (mM)
IC25 (mM)
5-FU
5
7
0.74
0.32
0.23
0.26
0.30
0.07
0.49
0.21
0.32
0.23
0.19
0.20
0.24
0.198
0.52
0.18
0.12
0.14
0.16
0.04
0.25
0.11
0.28
0.12
0.099
0.10
0.13
0.11
0.33
0.11
0.05
0.07
0.099
0.02
0.13
0.06
0.22
0.07
0.04
0.06
0.08
0.066
9
11
13
14
16
20
22
26
27
28
30
Experimental
All melting points are in degree centigrade (uncorrected)
and were determined on Gallenkamp electric melting point
apparatus. Elemental analyses were carried out at the Micro
Analytical Center, Faculty of Science, Cairo University. IR spectra
were recorded (KBr), (v cmꢀ1) on a Mattson 5000 FTIR spectro-
photometer at the Micro Analytical Center Faculty of Science,
IC100, IC50, and IC25 are the inhibitive concentration at 25, 50,
and 100 mM, respectively, of the compounds used. The dead %
refers to the % of the dead tumor cells and 5-FU is 5-fluorouracil
as a well-known cytotoxic agent.
1
Mansoura University. The H-NMRꢁ spectra were measured on
a Varian spectrophotometer at 300 MHz, using TMS as an
internal reference and DMSO-d6 or CDCl3 as solvent at the
Chemistry Department, Faculty of Science, Cairo University.
1
The H-NMRꢁꢁ and 13C-NMR spectra were acquired on a
than the 5-FU in vitro studies. Interestingly, compounds 13, 26,
and 30 were around 5–10 times more toxic than the 5-FU.
Experimental potential antitumor activity of the compounds
reported in this study to their structures, the following
structure activity relationships (SAR’s) were postulated: (a)
Compound 13 contains Meldrum’s acid moiety and this is in
agreement with that reported by Lukevics et al. [21]. (b) Also,
in case of compound 26, the high cytotoxic activity may be
attributed to the presence of the pyrazole moiety which is in
agreement with that reported by Perchellet et al. [22]. (c)
Moreover, the high cytotoxic activity of compound 30 may
be attributed to the presence of a benzothiazole moiety,
similar results have been reported by Kok et al. [23] (Fig. 1).
JEOL ECX-400 spectrometer at the Chemistry Department,
School of Engineering and Science, Jacobs University of
Bremen, Germany, operating at 400 MHz for 1H-NMR and
100 MHz for 13C-NMR at room temperature in CDCl3, and
DMSO-d6 using a 5 mm probe. The chemical shifts (d) are
reported in parts per million and where referenced to the residual
solvent peak. High resolution mass spectra (HRMS) were recorded
using both a Bruker HCT ultra and a high resolution (Bruker
Daltonics micrOTOF) instrument from methanol or dichloro-
methane solutions using the positive electrospray ionization
1
mode (ESI). The H-NMRꢁꢁꢁ spectra were measured on a
Varian spectrophotometer at 500 MHz, using TMS as an internal
reference and CDCl3 as solvent at the National Research Center,
Cairo. Mass spectra were recorded on (Kratos, 70 eV) MS
equipment and/or a Varian MAT 311 A spectrometer, at the
Microanalytical Center, Faculty of Science, Cairo University.
Reaction mixtures were monitored by thin layer chromatography
(TLC) using EM science silica gel coated plates with visualization
by irradiation with ultraviolet lamp. Biological testing was carried
out at the Drug Department, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt.
Conclusions
Modification of oxazole derivatives produced compounds
with potential for further development as anticancer agents.
Cl
OCH3
N
H3C
N
O
O
H3C
N
CH3
N
H3C
O
O
S
N
CN
O
N
H
O
O
N
NH2
N
H
Ph
Figure 1. Structures of the most potent com-
pounds 13, 26, and 30.
26
13
30
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W. S. Hamama et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Yield 80%; gray crystals; m.p. 2188C (ethanol); Rf ¼ 0.3 [pet.
Reaction of 1 with a,b-unsaturated ketones
General procedure
ether (40–60): ethyl acetate (1:3); IR (KBr): n/cmꢀ1 ¼ 3434 (OH),
1
2840, 2939 (CH, str.), 1677 (CO), 1598 (C C); H-NMR (DMSO-d )
ꢁ
–
–
(ppm): 2.32 (s, 3H, CH3), 3.8 (s, 3H, OCH3), 6.77
6
A mixture of 5-amino-3-methylisoxazole (1) (0.40 g, 4 mmol) and
a,b-unsaturated ketones namely; (E)-3-(4-methoxyphenyl)-1-phe-
nylprop-2-en-1-one (2) (0.95 g, 4 mmol), (E)-4-(4-methoxyphenyl)-
but-3-en-2-one (4) (0.7 g, 4 mmol) or 1,3-dibenzalacetone (6)
(0.94 g, 4 mmol) in dimethylformamide (25 mL) was refluxed
for appropriate time and then left to cool. Ethanol (25 mL)
was added to the reaction mixture, then left in a refrigerator
overnight. The formed precipitate was filtered and recrystallized
from the appropriate solvents to give the corresponding pyridine
derivatives 3, 5, and 7.
d
(d, J ¼ 8.2 Hz, 2H, aromatic-CH), 6.9 (d, J ¼ 8.3 Hz, 2H,
–
aromatic-CH), 8.0 (s, 1H, pyridine-CH C), 13.0 (s, 1H,
–
COOH); HRMS (micrOTOF): m/z for C15H12N2O4, Calcd.:
284.2700. Found: 285.1000 (MþþH), 307.0000 [MþþNa];
MS (EI, 70 eV) m/z (%) ¼ 285 (MþþH, 12.4), 284 (Mþ,
100.0), 283 (MþꢀH, 7.8), 239 (7.8), 132 (12.4), 117 (10.9),
107 (8.5).
4-(4-Methoxyphenyl)-3-methyl-4,5-dihydroisoxazolo[5,4-b]-
pyridin-6(7H)-one (11)
A mixture of 1 (0.49 g, 5 mmol) and (1.30 g, 5 mmol) of p-anisyl-
idene Meldrum’s acid [26], (10) in dry nitrobenzene (5 mL) was
heated under reflux for 30 min, left to cool, the formed precipi-
tate was filtered off, washed with ethanol, and recrystallized
from ethanol to afford 11.
4-(4-Methoxyphenyl)-3-methyl-6-phenylisoxazolo[5,4-b]-
pyridine (3)
Reaction time 15 h; yield 60%; yellow crystals; m.p. 199–2008C
(ethanol); Rf ¼ 0.4 [pet. ether (40–60): ethyl acetate (1:3)]; IR (KBr):
1
n/cmꢀ1 ¼ 2967, 2933 (CH, str.), 1606 (C C); H-NMR (DMSO-d )
ꢁ
–
–
d (ppm): 2.31 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 7.13–8.27
6
Yield 57%; yellow crystals; m.p.1908C (ethanol); Rf ¼ 0.5 [pet.
–
ether (40–60): ethyl acetate (2:5)]; IR (KBr): n/cmꢀ1 ¼ 3430 (NH),
(m, 10H, aromatic-CH and pyridine-CH C); HRMS(micrOTOF):
–
1
1720 (CO), 1644, 1622 (C–N), (C C); H-NMRꢁꢁꢁ(CDCl3) d (ppm):
m/z for C20H16N2O2, Calcd.: 316.3500. Found: 317.0000
(Mþþ1), 339.0000 (MþþNa), 655.0000 (2MþþNa); MS
(EI, 70 eV) m/z (%) ¼ 317 (MþþH, 24.7), 316 (Mþ, 100.0),
315 (Mþꢀ1, 10.9), 301 (3.1), 288 (13.4), 285 (4.0), 273 (38.3),
239 (1.4), 209 (0.3), 177 (7.6), 107 (4).
–
–
2.31 (s, 3H, CH3), 3.04 (dd, Ja,x ¼ 9.2 Hz, 1H, 5-Ha), 3.18 (dd,
Ja,b ¼ 5.4 Hz, 1H, 5-Hb), 3.6 (t, 1H, 4-Hx), 3.77 (s, 3H, OCH3),
6.83 (d, J ¼ 8.4 Hz, 2H, aromatic-CH), 6.99 (d, J ¼ 8.5 Hz, 2H,
aromatic-CH), 8.07 (s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 260
(Mþþ2, 2.3), 259 (MþþH, 11.8), 258 (Mþ, 43.4), 243 (15.6),
215 (70.3), 217 (11.7), 151 (4.2), 148 (13.5), 147 (100.0), 135
(8.4), 107 (4.4). Anal. Calcd. for C14H14N2O3 (258.28): C, 65.11;
H, 5.46%. Found: C, 65.33; H, 5.54%.
4-(4-Methoxyphenyl)-3,6-dimethyl-4,7-dihydroisoxazolo-
[5,4-b]pyridine (5)
Reaction time 10 h; yield 40%; yellow crystals; m.p. 299–3008C
(DMF); Rf ¼ 0.5 [pet. ether (40–60)/ethyl acetate (1:4)]; IR (KBr):
1
n/cmꢀ1 ¼ 3409, 3369 (NH), 1664, 1610 (C C), (C N); H-NMRꢁ
2,2-Dimethyl-5-[(3-methylisoxazol-5-ylamino)methylene]-
1,3-dioxane-4,6-dione (13)
A mixture of Meldrum’s acid (12) (0.72 g, 5 mmol) and methyl
orthoformate (25 mmol) was refluxed for 2.5 h, and then (0.49 g,
5 mmol) of 1 was added. The reaction mixture was heated for
15 min. The formed precipitate was filtered off and recrystallized
from EtOH/DMF (1:1) to furnish 13.
–
–
–
–
(DMSO-d6) d (ppm): 2.29 (s, 3H, CH3), 2.60 (s, 3H, CH3),
3.76 (s, 3H, OCH3), 7.50 (d, J ¼ 8.6 Hz, 2H, aromatic-CH)
and 7.70 (d, J ¼ 8.4 Hz, 2H, aromatic); HRMS(micrOTOF):
m/z for (C15H14N2O2þH), Calcd.: 255.1134. Found: 255.2000
(MþþH).
Yield 60%; yellow crystals; m.p. 2038C [EtOH-DMF (1:1)];
Rf ¼ 0.4 [pet. ether (40–60): ethyl acetate (1:3)]; IR (KBr):
3-Methyl-4-phenyl-6-styrylisoxazolo[5,4-b]pyridine (7)
Reaction time 10 h, yield 50%; yellow crystals; m.p. 270–2718C
(ethanol); Rf ¼ 0.3 [pet. ether (40–60)/ethyl acetate (1:4)]; IR (KBr):
n/cmꢀ1 ¼ 3294 (NH), 2925 (CH, str.), 1743, 1693 (2C O), 1643
–
–
1
(C N), 1609 (C C); H-NMRꢁ (CDCl3) d (ppm): 1.75 (s, 6H,
–
–
–
–
n/cmꢀ1 ¼ 3409 (NH), 2969, 2929 (CH, str.), 1644 (C N), 1600
–
–
2CH3), 2.29 (s, 3H, CH3), 5.7(s, 1H, isoxazole-CH), 7.20 (s, 1H,
þ
1
(C C); H-NMRꢁ (DMSO-d6) d (ppm): 2.5 (s, 3H, CH3), 6.55
–
–
–
NH), 8.53 (s, 1H, C CH); MS (EI, 70 eV) m/z (%) ¼ 252 (M þH,
–
–
(d, J ¼ 5.4 Hz, 1H, Ph-CH CH), 6.60 (d, J ¼ 5.6 Hz, 1H,
–
5.3), 251 (Mþ, 20.0), 194 (28.3), 193 (74.7), 108 (91.9), 81
(56.6), 80 (100.0), 54 (37.9), 52 (58.7). Anal. Calcd. for
C11H12N2O5 (252.07): C, 52.38; H, 4.80%. Found: C, 52.25;
H, 4.675%.
–
Ph-CH CH), 7.16–8.07 (m, 11H, aromatic-CH and pyridine-
–
þ
–
–
CH C); MS (EI, 70 eV) m/z (%) ¼ 312 (M , 2.1), 324 (4.6),
236 (7.1), 132 (13.4), 131 (100.0), 130 (12.6), 104 (26.8), 103
(61.5), 102 (17.6), 91 (10), 77 (47.3), 51 (16.3). Anal. Calcd.
for C21H16N2O (312.37): C, 80.75; H, 5.16%. Found: C, 80.83;
H, 5.22%.
3-Methyl-4,7-dihydroisoxazolo[5,4-b]pyridin-4-one (14)
Compound 13 (1.26 g, 5 mmol) in nitrobenzene was refluxed for
30 min. The formed precipitate after cooling was filtered,
washed with ethanol, and crystallized from ethanol/DMF (1:1)
to afford 14.
4-(4-Methoxyphenyl)-3-methylisoxazolo[5,4-b]pyridine-6-
carboxylic acid (9)
A mixture of 1 (0.49 g, 5 mmol) and (E)-4-(4-methoxyphenyl)-2-
oxobut-3-enoic acid (8) [25], (1.03 g, 5 mmol) in glacial acetic acid
(5 mL) was refluxed for 30 min, then allowed to cool, ethanol
(10 mL) was added, and the reaction mixture was allowed to
stand overnight. The precipitate formed was filtered and recrys-
tallized from ethanol to give 9.
Yield 77%; yellow crystals; m.p. 2038C [EtOH-DMF (1:1)];
Rf ¼ 0.5 [pet. ether (40–60): ethyl acetate (1:3)]; IR (KBr):
n/cmꢀ1 ¼ 3294 (NH), 2983, 2933 (CH, str.), 1769 (C O), 1633
–
–
1
(C N), 1610 (C C); H-NMRꢁ (CDCl3) d (ppm): 2.63 (s, 3H,
–
–
–
–
CH3), 6.70 (s, 1H, NH), 7.58 (d, 1H, CH5 ¼ CH6), 8.26
(d, 1H, CH5 ¼ CH6); MS (EI, 70 eV) m/z (%) ¼ 151 (MþþH,
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Isoxazolo[5,4-b]pyridines: Synthesis and Antitumor Activity
7
1
5.3), 150 (Mþ, 20.0), 122 (5.6), 97 (14.7), 81 (60.6), 80 (100.0).
Anal. Calcd. for C7H6N2O2 (150.14): C, 56.00; H, 4.03%.
Found: C, 56.18; H, 4.11%.
(NH), 2926 (CH, str.), 1719 (C O), 1594 (C C); H-NMRꢁꢁ
–
–
–
–
(DMSO-d6) d (ppm): 1.8 (s, 3H, CH3), 4.9 (s, 1H, H-4), 7.22–
7.51 (m, 9H, aromatic-CH), 12.02 (s, 1H, NH); 13C-NMR
(CDCl3) d (ppm): 10.48 (CH3), 39.63 (C-4), 97.97 (C-4a),
108.82 (C-3a), 119.79, 121.06 (C-9), 121.41 (C-7), 127.16,
128.85, 130.92 (C-6), 132.50, 133.94 (C-8), 133.95, 144.84
(E)-8-Benzylidene-3-methyl-4-phenyl-4,5,6,7,8,9-
hexahydroisoxazolo[5,4-b] quinoline (16)
–
(C-9a), 155.76 (C-3),159.65 (C-6a), 160 (C-1a), 190.94 (C O);
–
A mixture of 1 (0.40 g, 4 mmol) and 2,6-dibenzalcyclohexanone
(15) (4 mmol) in dimethylformamide (25 mL) was refluxed for
10 h, then left to cool. Ethanol (25 mL) was added to the reaction
mixture then left in a refrigerator overnight. The formed pre-
cipitate was filtered and recrystallization from ethanol gave 16.
Yield 40%; yellow crystals; m.p. 270–2728C (ethanol); Rf ¼ 0.3
HRMS (micrOTOF): m/z for C20H13 N2O2, Calcd.: 314.1100.
Found: 313.0975 (MþꢀH); MS (EI, 70 eV) m/z (%) ¼ 315
(MþþH, 0.3), 314 (Mþ, 2.7), 313 (MþꢀH, 29.3), 312 (Mþꢀ2,
100.0), 311 (38.7), 297 (9.3), 217 (3.0), 124 (8.3), 123 (7.3),
97 (5.7), 98 (17.0).
[pet. ether (40–60)/ethyl acetate (1:4)]; IR (KBr): n/cmꢀ1 ¼ 3370
1
(NH), 2923, 2852 (CH, str.), 1654, 1611 (C N), (C C), H-NMRꢁ
Reaction of 1 with a,b-unsaturated carbonyl compounds
A mixture of 1 (0. 49 g, 5 mmol), a,b-unsaturated carbonyl
derivatives namely; p-chlorobenzylidene-3-quinuclidinone (21)
(1.24 g, 5 mmol), 23 (1.46 g, 5 mmol), 24 (1.53 g, 5 mmol)
or 25 (1.25 g, 5 mmol) in ethanol (50 mL) and glacial acetic
acid (20 mL) was refluxed for the necessary time. The reaction
mixture was allowed to stand overnight at room temperature
to give the linear fused tricyclic systems 22 and 26–28,
respectively.
–
–
–
–
(CDCl3) d (ppm): 2.53 (s, 3H, CH3), 2.72–2.93 (m, 6H, 3 CH2),
–
7.4 (s, 1H, CH C), 7.50–7.91(m, 10H, aromatic CH); MS (EI,
–
70 eV) m/z (%) ¼ 354 (Mþþ2, 2.3), 352 (Mþ, 2.7), 264 (2.67),
194 (16.6), 193 (73.0), 177 (10.7), 176 (17.9), 105 (12.2), 104
(11.7), 98 (3.2), 80 (14.9), 44 (100.0). Anal. Calcd. for
C24H20N2O (352.43): C, 81.79; H, 5,72%%; Found: C, 81.52;
H, 5.48%.
General procedure for preparation of 18 and 20
3-Methyl-4-(4-chlorophenyl)-4,11-dihydrooxazolo-
[40,50:5,6]pyrido[2,3-b]quinuclidine (22)
Reaction time 5 h, yield 60%; brown crystals; m.p. 256–2578C
Method (A)
A mixture of 1 (0. 49 g, 5 mmol) and 2-benzylidenedimedone (17)
[25] or 2-benzylideneindandione (19) (5 mmol) in ethanol (50 mL)
and glacial acetic acid (1 mL) was refluxed for 3 h, the reaction
mixture was allowed to stand overnight. The precipitate formed
was filtered and recrystallized from the proper solvent to give 18
and 20, respectively.
(DMF); Rf ¼ 0.6 [pet. ether (40–60): ethyl acetate (1:3)]; IR (KBr):
n/cmꢀ1 ¼ 3439 (NH), 2954 (CH, str.), 1605 (C C); H-NMRꢁ
1
–
–
(DMSO-d6) d (ppm): 1.57–1.98 (m, 4H, (CH2)2–C), 2.68
(s, 3H, CH3), 3.15 (m, 4H, (CH2)2–N), 3.22 (q, 1H, bridgehead),
3.74 (s, 1H, NH), 7.53 (d, J ¼ 8.1 Hz, 2H, aromatic-CH),
8.12 (d, J ¼ 8.1 Hz, 2H, aromatic-CH); MS (EI, 70 eV)
m/z (%) ¼ 327/329 (Mþ, 28.3:9.2), 214 (6.3), 137 (36.6), 125
(19.5), 111 (22.4), 76 (43.9), 51 (100.0). Anal. Calcd. for
C18H18ClN3O (327.82): C, 65.95; H, 5.53%. Found: C, 65.68;
H, 5.70%.
Method (B)
A mixture of 1 (0.49 g, 5 mmol), dimedone (0.7 g, 5 mmol) or
indandione (0.73 g, 5 mmol) and benzaldehyde (0.5 mL,
5 mmol) in ethanol (15 mL) and glacial acetic acid (1 mL) was
heated under reflux for 3 h. The solvent was evaporated under
vacuum to give yellow precipitate, which was washed with
water then recrystallized from proper solvent to give 18 and
20, respectively.
4-(4-Methoxyphenyl)-3,5-dimethyl-7-phenyl-4a,7-dihydro-
4H-pyrazolo [40,30:5,6]pyrido[3,2-d]isoxazole (26)
Reaction time 4 h, yield 40%; white crystals; m.p. 2208C
(ethanol); Rf ¼ 0.6 [pet. ether (40–60)/ethyl acetate (1:3)]; IR
(KBr): n/cmꢀ1 ¼ 3300 (NH), 3000, 2990 (CH, str.); H-NMRꢁ
1
3,7,7-Trimethyl-4-phenyl-4,6,8,9-tetrahydroisoxazolo-
[5,4-b]quinolin-5-one (18)
(DMSO-d6) d (ppm): 2.19 (s, 3H, CH3), 2.39 (s, 3H, CH3),
3.61 (s, 3H, OCH3), 5.04 (s, 1H, CH), 6.74 (d, J ¼ 8.6 Hz,
2H, aromatic-CH), 6.78 (d, J ¼ 9. 2 Hz, 2H, aromatic-
CH)7.04–7.28 (m,5H, aromatic-CH), 11.38 (s, 1H, NH); MS
(EI, 70 eV) m/z (%) ¼ 373 (MþþH, 0.3), 372 (Mþ, 0.6), 292
(10.0), 158 (1.0), 116 (6.8), 92 (8.1), 82 (18.7), 77 (100.0). Anal.
Calcd. for C22H20N4O2 (372.43): C, 70.95; H, 5.41%. Found: C,
70.69; H, 5.56%.
Yield 60% [A]; 88% [B]; yellow crystals; m.p. 222–2248C (benzene);
Rf ¼ 0.5 [pet. ether (40–60): ethyl acetate (3:8)]; IR (KBr):
n/cmꢀ1 ¼ 3207 (NH), 2958, 2931, 2877 (CH, str.), 1670 (CO),
1
1610 (C C); H-NMRꢁꢁꢁ (CDCl3) d (ppm): 1.9 (s, 6H, 2CH3),
–
–
2.1 (s, 2H, 8-CH2), 2.16–2.2 (dd, 2H, 6-CH2), 2.4 (s, 3H,
CH3), 5.05 (s, 1H, 4-H), 7.2 (s, 1H, 9-NH), 7.22–7.25
(m, 5H, aromatic-CH); HRMS (micrOTOF): m/z for
C19H20N2O2, Calcd.: 308.3700. Found: 307.0000 (Mþ ꢀ H),
331.1000 (MþþNa), 639.3000 (2MþþNa); MS (EI, 70 eV)
m/z (%) ¼ 308 (Mþ, 20.6), 307 (MþꢀH, 19.5), 234 (2.1),
293 (10.1), 251 (3.3), 239 (4.4), 231 (100.0).
4-(Benzo[d][1,3]dioxol-5-yl)-3,5-dimethyl-7-phenyl-7,8-
dihydro-4H-pyrazolo [40,30:5,6]pyrido[3,2-d]isoxazole (27)
Reaction time 3 h, yield 50%; white crystals; m.p. 2298C
(ethanol); Rf ¼ 0.6 [pet. ether (40–60): ethyl acetate (1:3)];
IR (KBr): n/cmꢀ1 ¼ 3320 (NH), 2997, 2995 CH, str.); H-NMRꢁ
1
3-Methyl-4-phenyl-4,10-dihydroisoxazolo[40,50:5,6]pyrido-
[2,3-a]inden-5-one (20)
(DMSO-d6) d (ppm): 2.19 (s, 3H, CH3), 2.41 (s, 3H, CH3),
4.74 (s, 1H, CH), 5.92 (s, 2H, O–CH2–O), 6.67–7.66 (m, 8H,
aromatic-CH), 11.34 (s, 1H, NH); MS (EI, 70 eV) m/z (%) ¼ 387
(MþþH, 3.65), 386 (Mþ, 71.1), 359 (52.0), 356 (47.7),
Yield 90% [A]; 95% [B]; red crystals; m.p. 261–2638C (DMF); Rf ¼ 0.6
[pet. ether (40–60)/ethyl acetate (1:3)]; IR (KBr): n/cmꢀ1 ¼ 3340
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
W. S. Hamama et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
266 (45.0) 307 (3.9), 124 (55.6), 105 (100.0), 99 (65.9). Anal.
Calcd. for C22H18N4O3 (386.41): C, 68.38; H, 4.70%. Found: C,
68.51; H, 4.62%.
[3] V. V. Lipson, S. M. Desenko, M. G. Shirobokova, V. V. Borodina,
Chem. Heterocycl. Compd. (Engl. Transl.) 2003, 39, 1213–1217.
[4] S. M. Desenko, V. D. Orlov, N. V. Getmanskii, O. V. Shishkin,
S. V. Lindeman, Y. T. Struchkov, Chem. Heterocycl. Compd. (Engl.
Transl.) 1993, 29, 406–410.
1-Methyl-6-phenyl-8-phenyl-7,8-dihydro-oxazolo-
[40,50:5,6]pyrido[3,2-d]isoxazole (28)
[5] K. Wermann, M. Hartman, Synthesis 1991, 189–191.
Reaction time 5 h, yield 70%; white crystals; m.p. 1458C
[6] S. M. Desenko, V. D. Orlov, N. V. Getmanskii, O. V. Shishkin,
S. V. Lindeman, Y. T. Struchkov, Dokl. Akad. Nauk 1992, 324,
801.
(ethanol); Rf ¼ 0.6 [pet. ether (40–60): ethyl acetate (1:3)];
1
IR (KBr): n/cmꢀ1 ¼ 3264 (NH), 1643 (C N); H-NMRꢁꢁ (CDCl3) d
–
–
(ppm): 2.50 (s, 3H, CH3), 4.20 (s, 1H, CH), 7.35–8.02 (m, 10H,
aromatic), 10.12 (s, 1H, NH). Anal. Calcd. for C20H15N3O2
(329.36): C, 72.94; H, 4.59%. Found: C, 72.76; H, 4.63%.
[7] T. Yamamori, Y. Hiramatu, K. Sakai, I. Adachi, Tetrahedron
1985, 41, 913–917.
[8] C. Yoakim, W. W. Ogilvie, N. Goudreau, J. Naud, B. Hache,
J. A. O’Meara, M. G. Cordingley, J. Archambault, P. W. White,
Bioorg. Med. Chem. Lett. 2003, 13, 2539–2541.
3-(5-Amino-3-methylisoxazol-4-yl)-2-(benzo[d]thiazol-2-yl)-
3-(4-chlorophenyl) propiononitrile (30)
[9] N. Goudreau, D. R. Cameron, R. Deziel, B. Hache, A. Jakalian,
E. Malenfant, J. Naud, W. W. Oglivie, J. O’Meara, P. W. White,
C. Yoakim, Bioorg. Med. Chem. 2007, 15, 2690–2700.
A mixture of 1 (0.49 g, 5 mmol), 2-(benzo[d]thiazol-2-yl)-3-(4-
chlorophenyl) acrylonitrile (29) (1.50 g, 5 mmol) was refluxed
for 3 h in ethanol (50 mL) and triethylamine (few drops) for
3 h, then allowed to stand overnight at room temperature.
The formed precipitate was collected by filtration to give 30.
Yield 55%; white crystals; m.p. 2408C (ethanol); Rf ¼ 0.6 [pet. ether
(40–60): ethyl acetate (1:3)]; IR (KBr): n/cmꢀ1 ¼ 3392 (NH2), 3061,
[10] Y. A. El-Ossaily, Phosphorus, Sulfur Silicon 2007, 182, 1109–
1117.
[11] M. Suzuki, H. Iwasaki, Y. Fujikawa, M. Sakashita, M. Kitahara,
R. Sakoda, Bioorg. Med. Chem. Lett. 2001, 11, 1285–1288.
1
ꢁ
[12] J. Quiroga, A. Hormaza, B. Insuasty, M. Nogueras, A. Sanchez,
N. Hanold, H. Meier, J. Heterocycl. Chem. 1997, 34, 521–524.
–
–
–
2926 (CH, str.), 2261 (CN), 1632 (C N), 1594 (C C); H-NMR (CDCl )
–
3
d (ppm): 1.86 (s, 3H, CH3), 5.40 (s, 2H, NH2), 7.08 (d, J ¼ 6.1 Hz,
2H, aromatic-CH), 7.26 (d, J ¼ 6.5 Hz, 2H, aromatic-CH), 7.59
(d, J ¼ 6.1 Hz, 2H, aromatic-CH), 8.12 (d, J ¼ 7.8 Hz, 2H,
aromatic-CH), 8.22 (d, J ¼ 8.1 Hz, 2H, aromatic-CH); MS (EI,
70 eV) m/z (%) ¼ 394/396 (Mþ, 35.1/11.0), 134 (6.5), 111 (8.3),
69 (100.0), 66 (14.9). Anal. Calcd. for C20H15ClN4OS (394.88):
C, 60.83; H, 3.83%. Found: C, 60.96; H, 3.68%.
[13] J. Quiroga, A. Hormaza, B. Insuasty, C. Saitz, A. Can˜ete,
C. Jullian, J. Heterocycl. Chem. 1998, 35, 409–412.
[14] F. Shi, J. Zhang, S. Tu, R. Jia, Y. Zhang, B. Jiang, H. Jiang,
J. Heterocycl. Chem. 2007, 44, 1013–1017.
[15] S.-J. Tu, X.-H. Zhang, Z.-G. Han, X.-D. Cao, S.-S. Wu, S. Yan,
W.-J. Hao, G. Zhang, N. Ma, J. Comb. Chem. 2009, 11, 428–432.
[16] H. McNab, Chem. Soc. Rev. 1978, 7, 345–358.
Antitumor activity
Cytotoxic evaluation of the tested compounds
[17] R. F. C. Brown, F. W. Eastwood, K. J. Harrington, Aust. J. Chem.
1974, 27, 2373–2384.
EAC were obtained from National Cancer Institute, Cairo, Egypt.
To examine whether the synthesized compounds have a direct
cytotoxic effect on EAC [20], viability and the percentage of viable
cells was estimated by the trypan blue [27] exclusion test. The
desired concentration of tumor cells (2 ꢂ 106 cells per 0.2 mL)
was obtained by dilution with saline solution (0.9% sodium
chloride). The viability of the tumor cells obtained and used
in this experiment was always higher than 90%. Below this
percentage, the cells were discarded and the entire procedure
was repeated for three times.
[18] S. Claudio, J. Carolina, C. Alvaro, J. Heterocycl. Chem. 1998, 35,
61–64.
[19] (a) J. Quiroga, A. Hormaza, B. Insuasty, A. J. Ortiz, A. Sanchez,
M. Nogueras, J. Heterocycl. Chem. 1998, 35, 231–233; (b) P.
Sauerberg, P. H. Olesen, US patent 1996, 5,527,813; Chem.
Abstr. 1996, 125, 167989r.
[20] N. Dashora, V. Sodde, J. Bhagat, K. S. Prabhu, R. Lobo, Pharm.
Crops 2011, 2, 1–7.
[21] E. Lukevics, L. Ignatovich, I. Shestakova, Appl. Organomet.
Chem. 2003, 17, 898–905.
I deeply appreciate the assistance of Prof. Dr. N. Kuhnert, Professor of
Organic Chemistry, Chemistry Department, School of Engineering and
Science, Jacobs University, Bremen, Germany funded by DFG, for the
award of a fellowship for spectral measurement (HRMS, 1H-NMR,
13C-NMR, and MS) of some of our samples.
[22] E. M. Perchellet, M. M. Ward, A.-L. Skaltsounis, I. K. Kostakis,
N. Pouli, P. Marakos, J.-P. H. Perchellet, Anticancer Res. 2006,
26, 2791–2804.
[23] S. H. L. Kok, R. Gambari, C. H. Chui, M. C. W. Yuen, E. Lin, R. S.
M. Wong, F. Y. Lau, G. Y. M. Cheng, W. S. Lam, S. H. Chan,
K. H. Lam, C. H. Cheng, P. B. S. Lai, M. W. Y. Yu, F. Cheung, J. C.
O. Tang, A. S. C. Chan, Bioorg. Med. Chem. 2008, 16, 3626–3631.
The authors have declared no conflict of interest.
[24] H. Weinmann, E. Ottow, Comprehensive Med. Chem. II 2007, 7,
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ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com