Synthesis and anticancer evaluation of novel isoxazole/pyrazole derivatives

Article abstract of DOI:10.1007/s11164-015-2091-5

The key intermediate 3-amino-5-methylisoxazole (1) was allowed to react with phthalic anhydride and/or maleic anhydride under different conditions to produce different isoxazole products. Schiff bases 9a-c obtained via the reaction of 1 with different aldehydes were condensed with thioglycolic acid to afford the corresponding thiazolidin-4-one derivatives 10a, b. Furthermore, condensation of the Schiff bases 9a, c with various secondary amines produced the corresponding 5-substituted pyrazole derivatives 11a-d, respectively. The anticancer activity of some of the newly synthesized compounds was evaluated against Panc-1 and Caco-2 cell lines using doxorubicin as a standard drug. Most of the tested derivatives exhibited high cytotoxic potency against Panc-1 carcinoma cell lines, but moderate to weak activity was obtained against Caco-2 cell lines.

Full text of DOI:10.1007/s11164-015-2091-5

Res Chem Intermed
DOI 10.1007/s11164-015-2091-5
Synthesis and anticancer evaluation of novel isoxazole/
pyrazole derivatives
1
2
•
Sherifa M. Abu Bakr
3
•
Somaia S. Abd El-Karim
4
Mahmoud M. Youns
Medhat M. Said
•
Received: 27 December 2014 / Accepted: 30 April 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract The key intermediate 3-amino-5-methylisoxazole (1) was allowed to
react with phthalic anhydride and/or maleic anhydride under different conditions to
produce different isoxazole products. Schiff bases 9a–c obtained via the reaction of
1
with different aldehydes were condensed with thioglycolic acid to afford the
corresponding thiazolidin-4-one derivatives 10a, b. Furthermore, condensation of
the Schiff bases 9a, c with various secondary amines produced the corresponding
5
-substituted pyrazole derivatives 11a–d, respectively. The anticancer activity of
some of the newly synthesized compounds was evaluated against Panc-1 and Caco-
cell lines using doxorubicin as a standard drug. Most of the tested derivatives
2
exhibited high cytotoxic potency against Panc-1 carcinoma cell lines, but moderate
to weak activity was obtained against Caco-2 cell lines.
Keywords Isoxazole Á Pyrazole Á Anticancer Á Caco-2 Á Panc-1
Introduction
Cancer is presently responsible for about 25 % of deaths in developed countries and
for 15 % of all deaths worldwide. It can, therefore, be considered one of the
foremost health problems with about 1.45 million new cancer cases expected yearly
&
Somaia S. Abd El-Karim
somaia_elkarim@hotmail.com
1
2
3
4
Chemistry of Natural and Microbial Products, National Research Centre, Dokki, Cairo, Egypt
Therapeutical Chemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
Organometallic and Organometaloid Chemistry, National Research Centre, Dokki, Cairo, Egypt
Biochemistry and Molecular Biology Department, Faculty of Pharmacy, Helwan University,
Cairo, Egypt
123

S. M. Abu Bakr et al.
[
1]. The treatment of cancer is associated with various side effects which include
bone marrow depression, alopecia, drug-induced cancer, hepatotoxicity, and many
more [2].
Because of the need for and value of anticancer drugs, many laboratories are
intensively investigating the chemistry and biology of novel anticancer agents. Also,
the development of resistance against existing anticancer drugs and cytotoxicity and
genotoxicity of anticancer drugs to the normal cells are other major problems in
cancer therapy, keeping the research window open in search for newer anticancer
molecules [3]. But the window passage has become narrower because it is rather
hard to search a molecule that can selectively inhibit the proliferation of abnormal
cells with the least or no effect on the normal cells.
A literature survey revealed that the isoxazole rings have been widely used as key
building blocks for drugs. Its derivatives are endowed with a broad spectrum of
pharmacological properties, for example, hypoglycemic, analgesic, anti-inflamma-
tory, antibacterial, anti-HIV, and anticancer activity [4–9]. In addition, pyrazole
derivatives have received considerable attention owing to their diverse
chemotherapeutic potentials including versatile antineoplastic activities such as
antileukemic [10–12], antitumor [13–16], and anti-proliferative [17, 18] in addition
to their capability to exert remarkable anticancer effects through inhibiting different
types of enzymes that play important roles in cell division [19–21].
Therefore, it was an attractive challenge in this work to synthesize novel
molecules carrying the isoxazole moiety in combination with different side chains
and with pyrazole ring aiming to get potent anticancer drugs.
Experimental
Chemistry
All melting points are uncorrected and were taken in open capillary tubes using an
Electrothermal IA 9100 digital melting point apparatus. Elemental microanalyses
were carried out at Microanalytical Laboratory, Central Services Laboratory,
National Research Centre, Dokki, Cairo, Egypt, on a Vario Elementar analyzer and
were found within ± 0.5 % of the theoretical values. Infrared spectra were recorded
on a FT/IR-6100, Fourier transform infrared spectrometer using a KBr disc
1
13
technique. H NMR (500 MHz) and C NMR (125 MHz) were determined by
using a JEOL AS-500 NMR spectrometer at Central Services Laboratory, National
Research Centre, Dokki, Cairo, Egypt, chemical shifts are expressed in d (ppm)
downfield from TMS as an internal standard. The mass spectra were measured with
a Finnigan MAT SSQ-7000 mass spectrometer. Follow up of the reactions and
checking the purity of the compounds were made by TLC on silica gel precoated
aluminium sheets (Type 60, F 254, Merck, Darmstadt, Germany) and the spots were
detected using UV lamp at k254 nm. The chemical names given for the prepared
compounds are according to the IUPAC system. 3-Amino-5-methylisoxazole (1)
was purchase from Sigma Aldrich. The biological evaluation of some of the new
1
23

Synthesis and anticancer evaluation of novel…
compounds was screened at Functional Genome Analysis, German Cancer Research
Center (DKFZ), INF 580, 69120 Heidelberg, Germany.
General procedure for the synthesis of 2-(5-methylisoxazol-3-yl)isoindoline-
1
,3-dione (2) and 1-(5-methylisoxazol-3-yl)-1H-pyrrole-2,5-dione (3)
A mixture of 3-amino-5-methylisoxazole (1) (0.49 g, 5 mmol) and phthalic
anhydride (0.82 g, 5.5 mmol) or maleic anhydride (0.54 g, 5.5 mmol) in dry
toluene (20 mL) was refluxed using a Dean Stark apparatus at 140 °C for 12 h. The
solvent was evaporated and the resulting residue was recrystallized from ethanol to
give the title compounds 2 and 3, respectively.
2
-(5-Methylisoxazol-3-yl)isoindoline-1,3-dione (2) Yield 70 %, white, mp.
1
-
1
1
67–179 °C; IR (KBr, cm ): 1792, 1738(2 CO); H NMR (DMSO-d ): d 2.44
6
1
3
(
3H, s, CH ), 7.01–7.97 (5H, m, Ar-H and isoxazole protons); C NMR (CDCl ): d
3
3
1
1
62.9 (2 C=O), 137.5 (C=N), 164.9, 134.2, 132.5, 131.8, 127.7, 127.5, 95.2 (C-Ar),
?
3.3 (CH ); MS, m/z (%): 228 [M ] (46), 104 [C H O] (100); Anal. For C H N O
3
7
4
12
8
2
3
(
228.20): Calcd. C, 63.16; H, 3.53; N, 12.28. Found: C, 63.24; H, 3.72; N, 12.51.
1
-(5-Methylisoxazol-3-yl)-1H-pyrrole-2,5-dione (3) Yield 60 %, pale yellow, mp.
-
1
1
1
CH ), 6.32–7.23 (3H, m, isoxazole and pyrrole protons); MS, m/z (%): 178 [M ]
42–145 °C; IR (KBr, cm ): 1716 (2 CO); H NMR (DMSO-d ): d 2.42 (3H, s,
6
?
3
(61), 82 [C H NO] (100); Anal. For C H N O (178.14): Calcd. C, 53.94; H, 3.39;
4 4 8 6 2 3
N, 15.73. Found: C, 53.73; H, 3.52; N, 15.93.
General procedure for the synthesis of 2-(5-methylisoxazol-3-
ylcarbamoyl)benzoic acid (4) and 3-(5-methylisoxazol-3-
ylcarbamoyl)acrylic acid (5)
A mixture of 3-amino-5-methylisoxazole (1) (0.49 g, 5 mmol) and phthalic
anhydride (0.82 g, 5.5 mmol) or maleic anhydride (0.54 g, 5.5 mmol) in glacial
acetic acid (20 mL) was refluxed for 10 h. The glacial acetic acid was removed
under vacuum and the residue was poured onto water. The formed precipitate was
filtered, washed several times with water, dried, and recrystallized from ethanol to
give the title compounds 4 and 5, respectively.
2
-(5-Methylisoxazol-3-ylcarbamoyl)benzoic acid (4) Yield 55 %, pale yellow,
-
1
mp. 121–123 °C; IR (KBr, cm ): Broad band centered at 3456 (CO H), 3214
2
1
(
NH), 1725, 1604 (2 CO); H NMR (DMSO-d ): d 2.43 (3H, s, CH ), 6.51, 11.02
6
3
(
2H, s, NH, COOH, D O exchangeable), 7.91–7.97 (5H, m, Ar-H and isoxazole
3
2
1
protons); C NMR (CDCl ): d 170.5, 157.8 (2 C=O), 165.0 (C=N), 158.8, 134.1,
1
OH] (5), 98 [C H N O] (100); Anal. Calcd. for C H N O (246.22): C, 58.54; H,
3
?
32.1, 131.6, 130.4, 129.8, 128.4, 96.3 (C-Ar), 12.0 (CH ); MS, m/z (%): 229 [M –
3
4
6
2
12 10 2 4
4
.09; N, 11.38. Found: C, 58.72; H, 3.87; N, 11.11.
3
8
-(5-Methylisoxazol-3-ylcarbamoyl)acrylic acid (5) Yield 50 %, dark brown, mp.
-
1
6–88 °C; IR (KBr, cm ): Broad band centered at 3434 (CO H), 3220 (NH), 1729,
2
123

S. M. Abu Bakr et al.
1
1
CH=CH–), 7.57 (1H, s, isoxazole proton), 8.21, 11.32 (2H, s, NH, COOH, D O
622(2 CO); H NMR (DMSO-d ): d 2.46 (3H, s, CH ), 6.84, 7.11 (2H, 2d, –
6
3
2
?
1
exchangeable); MS, m/z (%): 197 [M ] (10), 98 [C H N O] (100); Anal. Calcd. for
4
6 2
C H N O (196.16): C, 48.98; H, 4.11; N, 14.28. Found: C, 48.63; H, 4.32; N,
8
8 2 4
1
4.46.
General procedure for the synthesis of 2-(hydroxy(5-methylisoxazol-3-
ylimino)methyl)benzoic acid (6) and 4-hydroxy-4-(5-methylisoxazole-3-
ylimino)but-2-enoic acid (7)
A mixture of 3-amino-5-methylisoxazole (1) (0.49 g, 5 mmol) and phthalic
anhydride (0.82 g, 5.5 mmol) or maleic anhydride (0.54 g, 5.5 mmol) in dry
dioxane (20 mL) was refluxed for 9 h. After cooling, the formed precipitate was
filtered, washed with ethylacetate, dried, and recrystallized from ethanol to give the
title compounds 6 and 7, respectively.
2
-(Hydroxy(5-methylisoxazol-3-ylimino)methyl)benzoic acid (6) Yield 73 %,
-
1
pale brown, mp. 241–243 °C; IR (KBr, cm ): Broad band centered at 3433
1
(
CO H, OH), 1736(CO), 1604 (C=N); H NMR (DMSO-d ): d 2.43 (3H, s, CH ),
2
6
3
7
exchangeable); MS, m/z (%): 229 [M – OH] (10), 104 [C H O] (100); Anal. Calcd.
.32–7.79 (5H, s, Ar-H and isoxazole protons), 9.30, 10.23 (2H, s, OH, COOH, D O
2
?
7
4
for C H N O (246.22): C, 58.54; H, 4.09; N, 11.38. Found: C, 58.22; H, 4.26; N,
2 10 2 4
1
1
1.53.
4
-Hydroxy-4-(5-methylisoxazole-3-ylimino)but-2-enoic acid (7) Yield 65 %, pale
-
1
brown, mp. 207–209 °C; IR (KBr, cm ): Broad band centered at 3432 (CO H),
3
2
1
261(OH), 1711(CO), 1624 (C=N); H NMR (DMSO-d ): d 2.34 (3H, s, CH ), 6.32,
6
3
6
.37 (2H, 2d, –CH=CH–), 6.62 (1H, s, isoxazole proton), 11.24, 12.90 (2H, s, OH,
1
3
COOH, D O exchangeable); C NMR (CDCl ): d 169.8 (C–OH), 168.6 (C=O),
1
2
3
?
1
66.4 (C=N), 165.9, 128.4, 126.5, 93.7 (C-Ar), 13.5 (CH ); MS, m/z (%): 196 [M ]
3
(8), 98 [C H N O] (100); Anal. Calcd. for C H N O (196.16): C, 48.98; H, 4.11;
4 6 2 8 8 2 4
N, 14.28. Found: C, 49.13; H, 4.35; N, 14.52.
General procedure for the synthesis of 5-chloro-3-methyl-1H-pyrazole-4-
carbaldehyde (8a), phenyl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde
(
(
8b) and/or 4-nitrophenyl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde
8c)
5
-Chloro-3-methyl-1H-pyrazole-4-carbaldehyde (8a) and 1-aryl-5-chloro-3-methyl-
H-pyrazole-4-carbaldehyde 8b, c were prepared according to a literature procedure
1
[22] by Vilsmeier–Haack with a reaction of 3-methyl-1H-pyrazol-5(4H)-one and/
or1-aryl-3-methyl-1Hpyrazol-5(4H)-one.
Phosphorus oxychloride (0.4 mol) was added dropwise with stirring to ice-cold
dimethylformamide (0.4 mol) over a period of 30 min; stirring was continued for a
further 45 min keeping the reaction mixture at 0–5 °C. Appropriate 3-methyl-1H-
pyrazol-5(4H)-one and/or 1-aryl-3-methyl-1H-pyrazol-5(4H)-one (0.08 mol) was
1
23

Synthesis and anticancer evaluation of novel…
then added, and the reaction mixture was allowed to attain room temperature. The
mixture was then heated at 90 °C for 4 h, allowed to cool, and poured onto mixture
of crushed ice and water. The precipitates obtained were filtered, dried, and
crystallized from ethanol to obtain the required 5-chloro-3-methyl-1H-pyrazole-4-
carbaldehyde (8a) and 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde 8b, c.
General procedure for the synthesis of N-((5-chloro-3-methyl-1-substituted-
1
H-pyrazol-4-yl)methylene)-5-methylisoxazol-3-amine 9a–c
A mixture of 3-amino-5-methylisoxazole (1) (0.98 g, 0.01 mol) and different
aldehydes, namely 5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde, 5-chloro-3-
methyl-1-phenyl-1H-pyrazole-4-carbaldehyde, and/ or 5-chloro-3-methyl-1-(4-ni-
trophenyl)-1H-pyrazole-4-carbaldehyde 8a–c (0.01 mol), in glacial acetic acid
(20 mL) was refluxed for 18 h. After cooling, the formed precipitate was filtered,
washed with diethylether, dried, and recrystallized from ethanol to give the title
compounds 9a–c respectively.
N-((5-chloro-3-methyl-1H-pyrazol-4-yl)methylene)-5-methylisoxazol-3-amine (9a)
-
1
Yield 40 %, brown, mp. 158–159 °C; IR (KBr, cm ): 3217 (NH), 3092 (CH-
1
arom.), 2982, 2869 (CH-aliph.), 1624 (N=CH); H NMR (DMSO-d ): d 2.18 (3H, s,
6
CH ), 2.38 (3H, s, CH ), 6.58 (1H, s, isoxazole proton), 7.41 (1H, s, –N=CH
3
3
-
1
proton), 10.82 (1H, br.s, NH, D O exchangeable); MS, m/z (%): 223, 225 [M ] (68,
2
8
4
1), 168, 170 [C H ClN ] (100, 70); Anal. Calcd. for C H ClN O (224.65): C,
6 5 4 9 9 4
8.12; H, 4.04; N, 24.94. Found: C, 48.75; H, 3.83; N, 24.68.
N-((5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)methylene)-5-methylisoxazol-3-
-
1
amine (9b) Yield 40 %, brown, mp. 155–156 °C; IR (KBr, cm ): 3090 (CH-
1
arom.), 2928, 2840 (CH-aliph.), 1620 (N=CH); H NMR (DMSO-d ): d 2.21 (3H, s,
6
CH ), 2.40 (3H, s, CH ), 6.61 (1H, s, isoxazole proton), 7.07–7.68 (6H, m, –N=CH
3
3
-
and Ar-H protons); MS, m/z (%): 298, 300 [M ] (42, 58), 155 [C H N ] (100);
2
1
0 7 2
Anal. Calcd. for C H ClN O (300.74): C, 59.91; H, 4.36; N, 18.63. Found: C,
1
5
13
4
5
9.70; H, 4.55; N, 18.91.
N-((5-chloro-3-methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl)methylene)-5-methylisoxa-
-1
zol-3-amine (9c) Yield 45 %, brown, mp 180–182 °C; IR (KBr, cm ): 3097 (CH-
1
arom.), 2923, 2854 (CH-aliph.), 1618 (N=CH); H NMR (DMSO-d ): d 2.19 (3H, s,
6
CH ), 2.39 (3H, s, CH ), 6.59 (1H, s, isoxazole proton), 7.07–8.31 (5H, m, –N=CH and
3
3
13
Ar-H protons); C NMR (CDCl ): d 166.5, 153.7, 135.4 (3 C=N), 165.7, 146.5, 141.8,
3
1
30.6, 127.8, 125.4, 119.3, 92.9 (C-Ar), 14.2 (CH C-O), 13.6 (CH C-N); MS, m/z (%):
3 3
?
344, 346 [M ] (33, 70), 155 [C H N ] (100); Anal. Calcd. for C H ClN O
5 3
10
7
2
15 12
(345.74): C, 52.11; H, 3.50; N, 20.26. Found: C, 52.38; H, 3.85; N, 20.39.
General procedure for the synthesis of 2-(5-chloro-3-methyl-1-substituted-
H-pyrazol-4-yl)-3-(5-methylisoxazol-3-yl)thiazolidin-4-one 10a, b
1
A mixture of compounds 9b, c (0.002 mol) and thioglycolic acid (0.2 mL,
.002 mol) in dry benzene (20 mL) was heated under reflux for 6–10 h. The solvent
0
123

S. M. Abu Bakr et al.
was evaporated under reduced pressure and the formed residue was treated with a
dilute solution of sodium carbonate (10 %). The formed precipitate was filtered,
dried, and recrystallized from ethanol to give the title compounds 10a, b,
respectively.
2
-(5-Chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-(5-methylisoxazol-3-yl)thiazolid-
-
1
in-4-one (10a) Yield 57 %, dark brown, mp[300 °C; IR (KBr, cm ): 3078 (CH-
arom.), 2923, 2851 (CH-aliph.), 1714 (CO); H NMR (CDCl ): d 1.63 (3H, s, CH ),
1
3
3
2
proton), 6.65 (1H, s, isoxazole proton), 7.27–7.93 (5H, m, Ar-H protons); MS, m/z
.39 (3H, s, CH ), 3.34 (2H, m CH -thiazole protons), 5.21 (1H, m, CH-thiazole
3 2
?
1
(
H, 4.03; N, 14.95; S, 8.55. Found: C, 54.63; H, 4.35; N, 14.74; S, 8.31.
%): 375, 377 [M ] (67, 100); Anal. Calcd. for C H ClN O S (374.84): C, 54.47;
17 15 4 2
2
-(5-Chloro-3-methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl)-3-(5-methylisoxazol-3-
yl)thiazolidin-4-one (10b) Yield 52 %, dark brown, mp 190–191 °C; IR (KBr,
-
1
1
cm ): 3089 (CH-arom.), 2923, 2856 (CH-aliph.), 1712 (CO); H NMR (CDCl ): d
3
1
m, CH-thiazole proton), 6.67 (1H, s, isoxazole proton), 7.34–8.29 (4H, m, Ar-H
.61 (3H, s, CH ), 2.40 (3H, s, CH ), 3.33 (2H, m CH -thiazole protons), 5.28 (1H,
3 3 2
?
protons); MS, m/z (%): 419, 421 [M ] (53, 25), 80 [C H NO] (100); Anal. Calcd.
4
2
for C H ClN O S (419.84): C, 48.63; H, 3.36; N, 16.68; S, 7.64. Found: C, 48.87;
5 4
1
7
14
H, 3.92; N, 16.46; S, 7.83.
General procedure for the synthesis of 5-methyl-N-((3-methyl-5-
(
substituted)-1-(substituted)-1H-pyrazol-4-yl)methylene)isoxazol-3-amine
1a–d
1
A mixture of compounds 9a, c (0.005 mol) and different amines, namely
morpholine, 2-aminothizole, and/ or piperazine (0.005 mol), in dry acetone
(20 mL) was refluxed for 24 h. After cooling, the reaction mixture was poured
onto ice/cold water. The formed precipitate was filtered, dried, and recrystallized
from ethanol to give the title compounds 11a–d, respectively.
5
-Methyl-N-((3-methyl-5-morpholino-1H-pyrazol-4-yl)methylene)isoxazol-3-amine
-
1
(
(
(
11a) Yield 51 %, brown, mp 136–137 °C; IR (KBr, cm ): 3218 (NH), 3095
1
CH-arom.), 2923, 2858 (CH-aliph.), 1624 (N=CH); H NMR (DMSO-d ): d 1.69
6
3H, s, CH ), 1.94-2.41 (11H, m, CH , 4 (CH ) morpholine protons), 6.62 (1H, s,
3
3
2
isoxazole proton), 7.51 (1H, s, –N=CH proton), 10.52 (1H, br.s, NH, D O
2
?
exchangeable); MS, m/z (%): 267 [M ] (80), 275 [M ] (66), 74 [C H O] (100);
1
?
4
10
Anal. Calcd. for C H N O (275.31): C, 56.71; H, 6.22; N, 25.44; Found: C,
3 17 5 2
1
5
6.37; H, 6.40; N, 25.19.
4
-((5-Methylisoxazol-3-ylimino)methyl)-3-methyl-N-(thiazol-2-yl)-1H-pyrazol-5-
-
1
amine (11b) Yield 52 %, brown, mp 143-144 °C; IR (KBr, cm ): 3217 (NH),
3
1
093 (CH-arom.), 2981, 2861 (CH-aliph.), 1623 (N=CH); H NMR (DMSO-d ): d
6
2
m, –N=CH and thiazole protons), 8.34, 10.20 (2H, 2br.s, 2NH, D O exchangeable);
.02 (3H, s, CH ), 2.41 (3H, s, CH ), 6.53 (1H, s, isoxazole proton), 6.91–7.65 (3H,
3
3
2
1
3
C NMR (CDCl ): d 167.8, 151.2, 144.8, 129.9 (4C=N), 165.8, 143.8, 137.2, 115.2,
3
1
23

Synthesis and anticancer evaluation of novel…
?
5.7, 92.9 (C-Ar), 13.6 (CH C-O), 13.3 (CH C-N); MS, m/z (%): 288 [M ] (82),
9
2
4
3 3
?
89 [M ] (60), 191 [C H N O] (100); Anal. Calcd. for C H N OS (288.33): C,
1
9
11
4
12 12 6
9.99; H, 4.19; N, 29.15; S, 11.12. Found: C, 49.81; H, 4.35; N, 29.41; S, 11.39.
5
-Methyl-N-((3-methyl-5-morpholino-1-(4-nitrophenyl)-1H-pyrazol-4-yl)methyle-
ne)isoxazol-3-amine (11c) Yield 56 %, dark brown, mp 109–110 °C; IR (KBr,
cm ): 3090 (CH-arom.), 2926, 2859 (CH-aliph.), 1611 (N=CH); H NMR (DMSO-
-
1
1
d ): d 1.63 (3H, s, CH ), 1.96–2.35 (11H, m, CH , 4 (CH ) morpholine protons),
6
3
3
2
6
.59 (1H, s, isoxazole proton), 7.21–8.34 (5H, m, –N=CH and Ar-H protons); MS,
m/z (%): 351 [M- NO ] (2), 98 [C H N O] (100); Anal. Calcd. for C H N O
19 20 6 4
2
4
6
2
(
396.40): C, 57.57; H, 5.09; N, 21.20. Found: C, 57.21; H, 5.34; N, 21.41.
5
-Methyl-N-((3-methyl-1-(4-nitrophenyl)-5-(piperazin-1-yl)-1H-pyrazol-4-yl)methy-
lene)isoxazol-3-amine (11d) Yield 53 %, dark brown, mp 168–169 °C; IR (KBr,
cm ): 3085 (CH-arom.), 2935, 2862 (CH-aliph.), 1618 (N=CH); H NMR (DMSO-
-
1
1
d ): d 1.64 (3H, s, CH ), 1.84–2.36 (11H, m, CH , 4 (CH ) piperazine protons), 6.65
6
3
3
2
(
1H, s, isoxazole proton), 7.24–8.45 (5H, m, –N=CH and Ar-H protons), 11.01 (1H,
1
3
br.s, NH, D O exchangeable); C NMR (CDCl ): d 165.8, 152.3, 138.1 (3 C=N),
2
3
1
piperazine), 15.3 (CH C-O), 13.5 (CH C-N); MS, m/z (%): 393 [M ] (49), 75
35.8, 150., 144.8, 142.4, 128.8, 125.3, 99.1, 92.5 (C-Ar), 50.2, 45.7 (4 CH
2
-
2
3
3
[C H ] (100); Anal. Calcd. for C H N O (395.42): C, 57.71; H, 5.35; N, 24.80.
6 3 19 21 7 3
Found: C, 57.93; H, 5.05; N, 24.47.
Anticancer evaluation
Cell culture and treatment
All reagents were handled as previously described in our work [23–29] in a sterile
fume hood. DMEM medium and fetal bovine serum (FBS) were purchased from
Gibco; phosphate-buffered saline pH 7.4 (PBS) and trypsin-EDTA were obtained
from Sigma–Aldrich. Alamar Blue or Resazurin (Promega, Mannheim, Germany)
reduction assay was used to assess the cytotoxicity of the studied samples. The
growth medium (DMEM medium with 10 % FBS, 100 U/mL penicillin, and
1
00 mg/L streptomycin), and Alamar Blue were stored at 48 °C, while trypsin–
EDTA and FBS were stored frozen at -208 °C and thawed before use; PBS was
stored at room temperature. Caco-2 and Panc-1 were obtained from the German
2
Cancer Research Center (DKFZ). Cells were cultured in 50 cm culture flasks
(
Corning) using DMEM medium supplemented with 10 % FBS, penicillin (100 IU/
mL), and streptomycin (100 mg/mL). The culture was maintained at 37 °C in an
atmosphere of 5 % CO and 95 % relative humidity. The cells were transferred to a
2
new flask every 2 days and treated with trypsin-EDTA to detach them from the
flask. Cells were counted under a microscope using a hemacytometer (Hausser
Scientific). Cell solutions were diluted with growth medium to a concentration of
5
1
9 10 cells/mL and transferred to a 96-well plate, and treated with gradient
concentrations of test compounds.
123

S. M. Abu Bakr et al.
Resazurin cell growth inhibition assay
Alamar Blue or Resazurin (Promega, Mannheim, Germany) reduction assay was
used to assess the cytotoxicity of the studied samples. The assay tests the cellular
viability and mitochondrial function. Briefly, adherent cells were grown in tissue
culture flasks, and then harvested by treating the flasks with 0.025 % trypsin and
0
.25 mM EDTA for 5 min. Once detached, cells were washed, counted, and an
3
aliquot (5 9 10 cells) was placed in each well of a 96-well cell culture plate in a
total volume of 100 lL. Cells were allowed to attach overnight and then treated
with samples. The final concentration of samples ranged from 0–100 lM. After
4
8 h, 20 lL Resazurin 0.01 % w/v solution was added to each well and the plates
were incubated at 37 °C for 1–2 h. Fluorescence was measured on an automated
TM
9
6-well Infinite M2000 Pro plate reader (Tecan, Crailsheim, Germany) using an
excitation wavelength of 544 nm and an emission wavelength of 590 nm. After
8 h incubation, plates were treated with resazurin solution as above mentioned.
4
Doxorubicin was used as a positive control. Each assay was done at least three
times, with two replicates each. The viability was compared based on a comparison
with untreated cells. IC50 (on cancer cells) were the concentration of the sample
required to inhibit 50 % of the cell proliferation and were calculated from a
calibration curve by a linear regression using Microsoft Excel.
Caspase-glo 3/7 assay
The influence of our test samples on caspase 3/7 activity in pancreatic cancer
resistant cells (Panc-1) was detected using Caspase-Glo 3/7 Assay kit (Promega).
Cells cultured in DMEM were seeded in 96-well plates and treated with the sample
(
2 9 IC ; IC ; ‘ 9 IC ) or DMSO (solvent control). After 24 h treatment,
5
0
50
50
1
00 ll of caspase 3/7 reagent were added to each well, mixed, and incubated for 1 h
TM
at room temperature. Luminescence was measured using well Infinite M2000 Pro
instrument (Tecan). Caspase 3/7 activity was expressed as percentage of the
untreated control as previously described [23–29].
Results and discussions
Chemistry
The target compounds were synthesized according to the steps outlined in
(
Schemes 1, 2). The key intermediate 3-amino-5-methylisoxazole (1) was allowed
to react with phthalic anhydride and/or maleic anhydride under different conditions
to produce different isoxazole products. Treatment of key intermediate 1 with
phthalic anhydride and/or maleic anhydride in toluene afforded the cyclic imide
isoindoline-1,3-dione derivative 2 and the pyrrole-2,5-dione derivative 3, respec-
tively. While the reaction of key intermediate 1 with phthalic anhydride and/or
maleic anhydride in acetic acid yielded the corresponding benzoic acid derivative 4
and acrylic acid derivative 5, respectively. Also, condensation of the intermediate 1
1
23

Synthesis and anticancer evaluation of novel…
O
H
N
H
N
Maleic anhydride
Dioxane
Phthalic anhydride
Toluene
N
OH
N
O
H
3
C
O
O
OH
H
3
C
O
2
7
O
N
N
NH
2
Phthalic anhydride
Dioxane
Maleic anhydride
Toluene
N
OH
N
O
N
H
3
C
O
O
OH
H
3
C
O
3
H C
O
6
3
1
H
O
H
N
H
H
N
Maleic anhydride
AcOH
Phthalic anhydride
AcOH
N
O
N
O
H
3
C
O
OH
H
C
O
O
OH
3
5
4
Scheme 1 Synthesis of isoxazole derivatives
with phthalic anhydride and/or maleic anhydride in dioxane gave the corresponding
hydroxyiminobenzoic acid derivative 6 and hydroxyimino butenoic acid derivative
7
, respectively (Scheme 1).
On the other hand, Schiff bases 9a–c were obtained by the condensation of
-amino-5-methylisoxazole (1) with different aldehydes, namely 5-chloro-3-
3
methyl-1H-pyrazole-4-carbaldehyde, 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde, and/or 5-chloro-3-methyl-1-(4-nitrophenyl)-1H-pyrazole-4-carbalde-
hyde 8a–c, in glacial acetic acid, respectively. The target thiazolidin-4-one
derivatives 10a, b were synthesized by the reaction of Schiff bases 9b, c with
thioglycolic acid in dry benzene. Furthermore, condensation of the chloro-pyrazole
derivatives 9a, c with secondary amines, namely morpholine, 2-aminothizole and/or
piperazine, produced the corresponding 5-substituted pyrazole derivatives 11a–d,
respectively (Scheme 2).
Anticancer screening
Some of the synthesized compounds were screened for their in vitro cytotoxic and
growth inhibitory activities against two different types of carcinoma cell lines,
namely human pancreatic carcinoma, epithelial-like cell line (Panc-1) and
heterogeneous human epithelial colorectal adenocarcinoma (Caco-2), in comparison
with the activity of the known anticancer reference drug doxorubicin. The cytotoxic
activities of our tested compounds were expressed as IC50 values lM (the dose that
reduces survival to 50 %) (Table 1).
Regarding to Caco-2 cells, it is evident that all of the tested compounds showed
antitumor activities with IC50 values ranging from 9.441 ± 3.425 to
55.589 ± 4.9 lM. Interestingly, the conjugation of thiazolidinone ring with both
isoxazole and pyrazole rings as compound 10a produced much higher cytotoxic
123

S. M. Abu Bakr et al.
O
S
N
CH
3
N
N
H
3
C
O
Cl
N
R
1
a, R
1
=
=
1
0a,b
b, R
1
NO
2
OH
HS
O
H
C
NH
2
OHC
Cl
CH3
N
CH3
N
+
N
N
N
H
3
C
O
N
3
H C
O
Cl
N
R
1
R
1
a, R
1
= H
=
1
8
a-c
9a-c
b, R
1
a, R
1
= H
=
c, R
1
=
NO
2
2 2
R NH
b, R
1
c, R
1
=
NO
2
H
C
N
CH
3
N
N
O
H
3
C
O
R
2
N
R
1
1
1a-d
a, R
1
= H
= H
R
2
=
N
N
b, R
1
R
2
=
HN
S
c, R
1
=
NO
2
R
2
=
N
O
d, R
1
=
NO
2
R
2
=
N
NH
Scheme 2 Syntesis of isoxazole-pyrazole derivatives
potency than that of the reference drug doxorubicin (IC : 9.44, 27.40 lM,
5
0
respectively). A slight decrease in the activity was observed by the derivatives
bearing phthalic-isoxazole and pyrazole-isoxazole conjugates 2, 11a (IC : 13.114,
5
0
15.304 lM, respectively). The substitution of the latter pyrazole ring with p-
nitrophenyl ring at 3-position as compounds 11c, 11d and the conjugation of the
parent isoxazole ring with maleic anhydride and/or benzoic acid nuclei, compounds
3
and 6 produced further reduction in the potency, but still higher than that of
doxorubicin (IC50: 23.57, 21.40, 23.96, 24.6 lM, respectively). Compounds 4, 5, 7,
1
5
0b, and 11b exhibited remarkable decrease in their cytotoxic activity (IC50: 31.3-
5.5 lM) to be much less than that obtained by the standard drug doxorubicin.
Regarding to Panc-1 cells, it is evident that the tested compounds showed antitumor
activities with IC50 values ranging from 19.653 ± 5.123 to 75.292 ± 4.553 lM. It is
noteworthy that the phthalic-isoxazole derivative 2 in addition to its high efficiency
1
23

Synthesis and anticancer evaluation of novel…
Table 1 In vitro anticancer
Compound no.
Cell lines
activity of the tested compounds
against Caco-2 and Panc-1 cell
lines
Caco-2 IC50/lM
Panc-1 IC50/lM
Doxorubicin
27.40 ± 0.42
13.114 ± 2.816
23.968 ± 6.122
39.523 ± 8.276
31.309 ± 3.785
24.609 ± 2.334
42.198 ± 13.511
9.441 ± 3.425
55.589 ± 4.9
24.10 ± 0.35
19.653 ± 5.123
24.854 ± 2.334
64.255 ± 4.485
[100
2
3
4
5
6
7
49.318 ± 2.573
25.308 ± 0.845
53.012 ± 5.451
52.401 ± 1.385
36.905 ± 4.471
68.292 ± 12.053
68.994 ± 3.152
75.292 ± 4.553
10a
10b
11a
11b
11c
11d
15.304 ± 5.131
32.363 ± 1.787
23.575 ± 3.262
21.404 ± 14.095
against Caco-2 cells, also showed higher activity than doxorubicin against Panc-1
carcinoma cells (IC50: 19.653, 24.10 lM, respectively). At the same time, compounds
3
and exhibited an approximate equipotent activity to that produced by the standard
drug (IC50: 23.968, 24.854 lM, respectively). The rest of the examined derivatives
appeared to produce weak cytotoxic activity.
The resultant data showed that the synthesized compounds are mostly effective as
cytotoxic agents against Caco-2 carcinoma cell lines, but they produced lower
potency or lack their cytotoxic effect against Panc-1 carcinoma cell lines.
Fig. 1 Caspase 3/7activity of the tested compounds (2, 3, 10a, and 11a)
123

S. M. Abu Bakr et al.
Caspase 3/7 assay
It is well established that the induction of the apoptotic cascade is one of the main
mechanisms of chemotherapy-induced cell death. To determine whether the
chemosensitizing effect of our compounds demonstrated above is secondary to
their ability to activate the apoptotic cascade, the pancreatic resistant cell lines
Panc-1 were treated with test compounds 2, 3, 10a, and 11a (2 9 IC ; IC ;
5
0
50
‘
9 IC ) or DMSO (solvent control). Six hours after treatment, the activity of
50
caspase 3/7 was measured using the Caspase-Glo 3/7 assay. Figure 1 shows that our
samples (2, 3, 10a, and 11a) caused significant increase in activation of caspase 3/7
in a dose-dependent manner. These results suggest that samples-induced apoptosis
was in part due to activation of caspases 3/7.
Conclusion
This study represented the synthesis and characterization of new 5-methylisoxazole
derivatives. Some of the novel compounds were selected as representative examples
to examine their cytotoxic activity against two different types of carcinoma cell
lines, human pancreatic carcinoma, epithelial-like cell line (Panc-1) and heteroge-
neous human epithelial colorectal adenocarcinoma (Caco-2), using doxorubicin as a
reference drug. The obtained results exhibited that the tested compounds are more
active against Caco-2 than Panc-1 cell lines.
References
1. E.S. Al-Abdullah, Molecules 16, 3410–3419 (2011)
2. M.J. Ahsan, H. Khalilullah, S. Yasmin, S.S. Jadav, J. Govindasamy, BioMed. Res. Int. (2013).
doi:10.1155/2013/239354
3. N. Aydemir, R. Bilaloglu, Mutat. Res. 537, 43–51 (2003)
4. K.D. Shin, M.Y. Lee, D.S. Shin, S. Lee, K.H. Son, S. Koh, Y.K. Paik, B.M. Kwon, D.C. Han, J. Biol.
Chem. 280, 41439–41448 (2005)
5
6
. J. Demers, W. Hageman, S. Johnson, D. Klaubert, R. Look, J. Moore, Bioorg. Med. Chem. Lett. 4,
2451–2459 (1994)
. D. Simoni, M. Roberti, I.F. Paolo, R. Rondanin, R. Baruchello, C. Malagutti, A. Mazzali, M. Rossi,
S. Grimaudo, F. Capone, L. Dusonchet, M. Meli, M.V. Raimondi, M. Landino, N. D’Alessandro, M.
Tolomeo, D. Arindam, S. Lu, D.M. Benbrook, J. Med. Chem. 44, 2308–2318 (2001)
7
8
9
. X.H. Liu, P. Cui, B.A. Song, P.S. Bhadury, H.L. Zhu, S.F. Wang, Bioorg. Med. Chem. 16,
4
075–4082 (2008)
. S. Velaparthi, M. Brunsteiner, R. Uddin, B. Wan, S.G. Franzblau, P.A. Petukhov, J. Med. Chem. 51,
999–2002 (2008)
1
. I.V. Magedov, M. Manpadi, S. van Slambrouck, W.F.A. Steelant, E. Rozhkova, N.M. Przheval’skii,
S. Rogelj, A. Kornienko, J. Med. Chem. 50, 5183–5192 (2007)
1
0. G. Daidone, B. Maggio, D. Raffa, S. Plescia, D. Schillaci, M.V. Raimondi, Il Farmaco 59, 413–417
2004)
1. L.-C. Chou, L.-J. Huang, J.-S. Yang, F.-Y. Lee, C.-M. Teng, S.-C. Kuo, Bioorg. Med. Chem. 15,
732–1740 (2007)
(
1
1
1
23

Synthesis and anticancer evaluation of novel…
12. F. Manetti, C. Brullo, M. Magnani, F. Mosci, B. Chelli, E. Crespan, S. Schenone, A. Naldini, O.
Bruno, M.L. Trincavelli, G. Maga, F. Carraro, C. Martini, F. Bondavalli, M. Botta, J. Med. Chem. 51,
1252–1259 (2008)
1
3. J. Li, Y.F. Zhao, X.L. Zhao, X.Y. Yuan, P. Gong, Arch. Pharm. Chem. Life Sci. 339, 593–597 (2006)
4. Y. Xia, Z.-W. Dong, B.-X. Zhao, X. Ge, N. Meng, D.-S. Shin, J.-Y. Miao, Bioorg. Med. Chem. 15,
1
6
893–6899 (2007)
5. Y. Xia, C.-D. Fan, B.-X. Zhao, J. Zhao, D.-S. Shin, J.-Y. Miao, Eur. J. Med. Chem. 43, 2347–2353
2008)
1
(
1
6. A.M. Farag, A.S. Mayhoub, S.E. Barakat, A.H. Bayomi, Bioorg. Med. Chem. 16, 881–889 (2008)
7. S. Schenone, O. Bruno, A. Ranise, F. Bondavalli, C. Brullo, P. Fossa, L. Mosti, G. Menozzi, F.
Carraro, A. Naldini, C. Bernini, F. Manettic, M. Botta, Bioorg. Med. Chem. Lett. 14, 2511–2517
1
(
2004)
8. G. Daidone, D. Raffa, B. Maggio, M.V. Raimondi, F. Plescia, D. Schillaci, Eur. J. Med. Chem. 39,
19–224 (2004)
1
1
2
2
2
9. N.C. Warshakoon, S. Wu, A. Boyer, R. Kawamoto, S. Renock, K. Xu, M. Pokross, A.G. Evdokimov,
S. Zhou, C. Winter, R. Walter, M. Mekel, Bioorg. Med. Chem. Lett. 16, 5687–5690 (2006)
0. S. Huang, R. Lin, Y. Yu, Y. Lu, P.J. Connolly, G. Chiu, S. Li, S.L. Emanuel, S.A. Middleton, Bioorg.
Med. Chem. Lett. 17, 1243–1245 (2007)
1. G.-D. Zhu, J. Gong, V.B. Gandhi, K. Woods, Y. Luo, X. Liu, R. Guan, V. Klinghofer, E.F. Johnson,
V.S. Stoll, M. Mamo, Q. Li, S.H. Rosenberg, V.L. Giranda, Bioorg. Med. Chem. 15, 2441–2452
(
2007)
2. H.Q. Xiao, G.P. Ouyang, X.D. Sun, X.D. Yao, G.Q. Bao, C.Z. Qi, Chin. J. Syn. Chem. 13, 600–602
2005)
2
(
2
3. L. Pinto-Garcia, T. Efferth, A. Torres, J.D. Hoheisel, M. Youns, Planta Med. 76, 1155–1161 (2010)
4. M. Youns, T. Efferth, J. Reichling, K. Fellenberg, A. Bauer, J.D. Hoheisel, Biochem. Pharmacol. 78,
2
273–283 (2009)
2
2
2
2
2
5. M. Youns, G.M. Fathy, J. Cell. Biochem. 114, 2654–2665 (2013)
6. T. Nasr, S. Bondock, M. Youns, Eur. J. Med. Chem. 76, 539–548 (2014)
7. M.S. Mohamed, M.M. Youns, N.M. Ahmed, Eur. J. Med. Chem. 69, 591–600 (2013)
8. M.F. Ahmed, M. Youns, Arch. Pharm. (Weinheim). 346, 610–617 (2013)
9. M.S. Alhamdani, M. Youns, M. Buchholz, T.M. Gress, M.C. Beckers, D. Mar e´ chal, A. Bauer, C.
Schr o¨ der, J.D. Hoheisel, J. Proteomics 75, 3747–3759 (2012)
123