Synthesis and antileukemic activity of new 3-(5-methylisoxazol-3-yl) and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones

Article abstract of DOI:10.1016/j.farmac.2003.10.006

3-(3-Methylisoxazol-5-yl) and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)- ones 8a-l and 9a,c-e,h-l were synthesized by refluxing in acetic acid the corresponding 2-methylquinazolinones 6 and 8 with the opportune benzoic aldehyde for 12 h. The synthesized styrylquinazolinones 8a-l and 9a,c-e,h-l were tested in vitro for their antileukemic activity against L-1210 (murine leukemia), K-562 (human chronic myelogenous leukemia) and HL-60 (human leukemia) cell lines showing in some cases good activity.

Full text of DOI:10.1016/j.farmac.2003.10.006

IL FARMACO 59 (2004) 451–455
www.elsevier.com/locate/farmac
Synthesis and antileukemic activity of new 3-(5-methylisoxazol-3-yl)
and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones
Demetrio Raffa *, Giuseppe Daidone, Benedetta Maggio, Stella Cascioferro,
Fabiana Plescia, Domenico Schillaci
Dipartimento di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, Palermo 90123, Italy
Received 13 August 2003; accepted 31 October 2003
Available online 08 May 2004
Abstract
3-(3-Methylisoxazol-5-yl) and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones 8a–l and 9a,c–e,h–l were synthesized by refluxing in
acetic acid the corresponding 2-methylquinazolinones 6 and 8 with the opportune benzoic aldehyde for 12 h. The synthesized styrylquinazoli-
nones 8a–l and 9a,c–e,h–l were tested in vitro for their antileukemic activity against L-1210 (murine leukemia), K-562 (human chronic
myelogenous leukemia) and HL-60 (human leukemia) cell lines showing in some cases good activity.
© 2004 Published by Elsevier SAS.
Keywords: 3-(3-Methylisoxazol-5-yl)-2-styrylquinazolin-4(3H)-ones; 3-(Pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones; Antileukemic activity
1. Introduction
2. Chemistry
Quinazolinones are very interesting drugs with antiprolif-
erative activity known to bind to tubulin and interfering with
its polymerization [1,2]; the 2-styrylquinazolinones are the
best example of this class of antimitotic agents [3].
Owing to the antitumoral activity described for the
styrylquinazolinones, our group has been recently engaged
in the synthesis of some new 6-R-3-(1-phenyl-3-methyl-
pyrazol-5-yl)-2-styrylquinazolin-4(3H)-ones 2 and 3 [4]
(Fig. 1).
The synthesized styrylquinazolinones 2 and 3 were tested
in vitro for their antileukemic activity against L-1210 (mu-
rine leukemia), K-562 (human chronic myelogenous leuke-
mia) and HL-60 (human leukemia) cell lines showing in
some cases good activity (IC50 z 2 µM).
Pursuing our research in this field, in order to further
explore the structure–activity relationships of this class of
antileukemic agents, we refer now the synthesis and the
biological activity of new analogs of styrylquinazolinones 2
and 3.
In particular, in the new synthesized 2-styrylquinazolino-
nes 8 and 9, we substituted N-3 pyrazole with the 5-methyl-
isoxazol-2-yl and with the pyrimidin-2-yl moieties.
6-Cl-3-(5-methylisoxazol-5-yl)-2-styrylquinazolinones
8a–l and 6-Cl-3-(pyrimidin-2-yl)-2-styrylquinazolinones
9a,c–e,h–l were obtained starting from the corresponding
2-methylquinazolinones 6 and 7 by condensation with the
opportune benzaldehyde (Scheme 1); the reaction was per-
formed by refluxing equimolar amounts of the opportune
2-methylquinazolinone 6,7 and benzaldehyde in glacial ace-
tic acid [5].
The structures of new compounds were elucidated by
analytical as well as spectroscopic measurements. In particu-
1
lar, as reported for derivatives 2 and 3 [4], the H NMR
spectra of compounds 8 and 9 are consistent with a E-olefinic
structure: b-olefinic protons appeared as doublets at d 6.48–
6.88 (J = 15.3–15.9 Hz) for compounds 8 and at d 5.92–6.38
(J = 14.9–15.7 Hz) for compounds 9 as requested for a E
structure [6] while the a-olefinic hydrogens were found
along with aromatic multiplet because of the deshielding of
two quinazolinone nitrogens; only for compound 8i the
a-olefinic hydrogens were found at d 8.41.
6-Cl-3-(5-methylisoxazol-5-yl)-2-methylquinazolinone 6
[7] and 6-Cl-3-(pyrimidin-2-yl)-2-methylquinazolinones 7
[8] were known and were obtained, as reported, by fusion of
the 6-chloro-2-methyl-benzoxazin-4(3H)-one 5 [9] with the
opportune amine according with the Scheme 1.
* Corresponding author.
E-mail address: demraffa@unipa.it (D. Raffa).
© 2004 Published by Elsevier SAS.
doi:10.1016/j.farmac.2003.10.006

452
D. Raffa et al. / IL FARMACO 59 (2004) 451–455
CH3
O
R
N
N
N
C6H5
N
CH
HC
R4
R3
R1
R2
2 R = H
3 R = Cl
a
b
c
d
e
f
g
h
i
l
m
n
o
p
q
H
H
H
H
OCH3
H
H
OCH3
H
H
H
OCH3 OCH3
H
H
OCH3
CH3
H
H
CH3
H
H
H
NO2
H
H
H
H
OH
OCH3
H
R1
R2
R3
R4
H
OCH3 OCH3 OCH3 OCH3
H
OCH3 OCH3
H
H
OCH3 OCH3 OCH3
OCH3
H
CH3
H
H
Cl
H
H
H
H
H
H
H
H
H
H
Fig. 1. Structural formulas of compounds 2 and 3
3. Biological results and discussion
Table 1. In Table 2 the IC50 values for compounds that
exhibited at least 50% of growth inhibition at screening
concentration are reported.
Among the tested new 2-styrylquinazolinones 8 and 9,
several compounds were active at the microgram level even if
their antileukemic activity (Table 1) was lower than the
reference compound (colchicine 10).
The synthesized styrylquinazolinones 8a–l and 9a,c–e,
h–l were tested in vitro for their antileukemic activity against
L-1210 (murine leukemia), K-562 (human chronic myelog-
enous leukemia) and HL-60 (human leukemia) cell lines.
Colchicine 10, whose antileukemic activity is well known,
was used as reference compound. The results as percent of
growth inhibition at 1 µg/ml concentration are reported in
A comparison between the antileukemic activity of both
2,3 [4] and 8,9 (Table 1) series, showed that there are no
substantial difference among these groups of compounds;
compounds 2,3 and 8,9 were characterized by the same
degree of activity against L-1210, K-562 and HL-60 cell
lines.
Table 1
Percent growth inhibiti on recorded on K-562, HL-60 and L-1210 cell lines
at 1 µg/ml concentration of 8a–l and 9a,c–e,h–l compounds
Compounds
K-562
n.s.
HL-60
n.s.
L-1210
30.0
20.4
n.s.
8a
8b
n.s.
n.s.
In particular, the antileukemic activity was not positively
affected by substituting the N-3 pyrazole (compounds 2,3)
with the 5-methyl-isoxazol-2-yl (compounds 8) and the
pyrimidin-2-yl (compounds 9) moieties.
The most active compounds 8d, 8e and 9l (Table 2) were
characterized by the presence of two methoxyl groups (8d,e)
and a chlorine atom (9l) in the styryl moiety.
8c
8d
8e
8f
n.t.
34.0
22.0
n.s.
50.0
58.0
37.0
n.s.
88.0
20.0
27.0
18.2
21.0
n.s.
n.s.
8g
n.s.
8h
44
n.s.
8i
n.s.
n.s.
8l
9a
9c
9d
9e
9h
9i
9l
36.5
26.1
n.s.
n.s.
29.0
25.0
n.s.
n.s.
36.8
20.6
19.4
19.4
36.8
40.0
84.8
Table 2
n.s.
n.s.
IC50 recorded on K-562 and L-1210 cell lines of compounds 8d,e and 9l
n.t.
n.s.
Compounds
IC50 (µM)
0.20 0.03
<0.025
n.s.
16.4
28.4
39.4
79.0
L-1210
K-562
8d
39.0
63.0
84.7
Colchicine
8d
8e
9l
2.50 0.22
1.61 0.13
1.21 0.18
<0.025
Colchicine
Values are the means of at least three independent determinations; coeffi-
cient of variation was less than 15%.
n.s.: not significant, % inhibition <10%; n.t.: not tested.
Colchicine

D. Raffa et al. / IL FARMACO 59 (2004) 451–455
453
O
Cl
COOH
NH2
Cl
(CH3CO)2O
O
N
5
CH3
4
R-NH2
CHO
R1
O
N
O
Cl
R
R4
R2
R3
N
Cl
R
N
CH
N
CH3
HC
R4
R3
O
CH3
N
6 R =
R1
N
R2
7 R =
O
CH3
N
N
8a-l R =
N
9a,c-e,h-l R =
N
a
b
c
d
e
f
g
h
i
l
OCH3
H
H
OCH3
H
H
H
OCH3 OCH3
H
H
OCH3
NO2
H
H
H
R1
R2
R3
R4
H
OCH3 OCH3 OCH3 OCH3
H
OCH3 OCH3
H
H
OCH3 OCH3 OCH3
OCH3
H
Cl
H
H
H
H
H
H
H
H
Scheme 1. General synthetic route to compounds 8a-l and 9a, c-e, h-l
4. Conclusion
were recorded with a Jasco IR-810 spectrophotometer as
Nujol Mull supported on NaCl disks; 1H NMR spectra were
obtained using a Bruker AC-E 250 MHz spectrometer (tet-
ramethylsilane as internal standard). Microanalyses (C, H,
N) performed in the laboratories of the Dipartimento di
Scienze Farmaceutiche—Università di Catania, were
within 0.4% of the theoretical values.
6-Cl-3-(5-methylisoxazol-5-yl)-2-styrylquinazolinones
8a–l and 6-Cl-3-(pyrimidin-2-yl)-2-styrylquinazolinones
9a,c–e,h–l.
A series of 2-styrylquinazolinone derivatives have been
synthesized and evaluated for their antileukemic activity.
Although most of the new synthesized compounds exhib-
ited moderate antileukemic activity they were less active than
the reference compound (colchicine 10) and, on the basis of
the obtained results, it can be concluded that there are no
advantages in replacing the N-3 pyrazole with the 5-methyl-
isoxazol-2-yl and the pyrimidin-2-yl moieties.
Equimolar amounts (10 mmol) of 6-Cl-3-(5-methyl-
isoxazol-5-yl)-2-methylquinazolinone
6
and 6-Cl-3-
5. Experimental section
(pyrimidin-2-yl)-2-methylquinazolinone 7 and the oppor-
tune benzoic aldehyde were reacted in acetic acid (10 ml)
under reflux for 12 h.
5.1. Chemistry
All melting points were determined on a Büchi 530 capil-
lary melting point apparatus and are uncorrected; IR spectra
The solid product, which separated, was filtered and crys-
tallized to give pure 8a–l and 9a,c–e,h–l. Yield 22–96%.

454
D. Raffa et al. / IL FARMACO 59 (2004) 451–455
Table 3
Physical data for compounds 8a–l and 9a,c–e,h–l
Compounds
Melting point (°C)
210–212
213–216
225–228
214–215
248–249
233–235
245–247
202–203
>250
Crystallization solvent
Ethyl acetate
Dioxane
Analyses
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
Formula
Yield (%)
73
8a
8b
8c
8d
8e
8f
8g
8h
8i
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
21H16N3O3Cl
21H16N3O3Cl
21H16N3O3Cl
22H18N3O4Cl
22H18N3O4Cl
22H18N3O4Cl
23H20N3O5Cl
23H20N3O5Cl
20H13N4O4Cl
20H13N3O2Cl2
21H15N4O2Cl
21H15N4O2Cl
22H17N4O3Cl
22H17N4O3Cl
23H19N4O4Cl
20H12N5O3Cl
20H12N4OCl2
80
Ethyl acetate
Dioxane
41
38
Dioxane
41
Dioxane
59
Dioxane
52
Dioxane
37
Dioxane
29
8l
247–249
212–214
220–222
208–209
205–208
179–180
218–220
>250
Dioxane
24
9a
9c
9d
9e
9h
9i
Dioxane
44
Dioxane
15
Ethanol
12
Dioxane
12
Ethanol
44
Dioxane
15
9l
Dioxane
41
Compounds 8a–l and 9a,c–e,h–l are listed in Table 3.
aromatic protons and CH). 9a: IR (cm–1): 1691, 1677 (mul-
tiple bands, CO). 1H NMR (CDCl3) (d): 3.74 (3H, s, OCH3);
6.28 (1H, d, CH, J = 15.1 Hz); 6.82–9.02 (11H, a set of
signals, aromatic protons and CH). 9c: IR (cm–1): 1672 (CO).
1H NMR (DMSO-d6) (d): 3.77 (3H, s, OCH3); 5.96 (1H, d,
CH, J = 15.2 Hz); 6.88–9.18 (11H, a set of signals, aromatic
protons and CH). 9d: IR (cm–1): 1684 (CO). 1H NMR
(CDCl3) (d): 3.71 (3H, s, OCH3); 3.80 (3H, s, OCH3); 6.17
(1H, d, CH, J = 15.4 Hz); 6.37–9.04 (10H, a set of signals,
1
8a: IR (cm–1): 1694 (CO). H NMR (CDCl3) (d): 2.59
(3H, s, CH3); 3.85 (3H, s, OCH3); 6.24 (1H, s, isoxazole
H-4); 6.76 (1H, d, CH, J = 15.7 Hz); 6.88–8.28 (8H, a set of
signals, aromatic protons and CH). 8b: IR (cm–1): 1702
(CO). 1H NMR (CDCl3) (d): 2.60 (3H, s, CH3); 3.86 (3H, s,
OCH3); 6.25 (1H, s, isoxazole H-4); 6.56 (1H, d, CH,
J = 15.4 Hz); 6.88–8.22 (8H, a set of signals, aromatic
protons and CH). 8c: IR (cm–1): 1696 (CO). 1H NMR
(CDCl3) (d): 2.60 (3H, s, CH3); 3.83 (3H, s, OCH3); 6.24
(1H, s, isoxazole H-4); 6.43 (1H, d, CH, J = 15.3 Hz);
6.86–8.21 (8H, a set of signals, aromatic protons and CH).
8d: IR (cm–1): 1693 (CO). 1H NMR (CDCl3) (d): 2.58 (3H, s,
CH3); 3.80 (3H, s, OCH3); 3.82 (3H, s, OCH3); 6.24 (1H, s,
isoxazole H-4); 6.40–8.18 (8H, a set of signals, aromatic
1
aromatic protons and CH). 9e: IR (cm–1): 1685 (CO). H
NMR (CDCl3) (d): 3.73 (3H, s, OCH3); 3.85 (3H, s, OCH3);
6.38 (1H, d, CH, J = 15.7 Hz); 6.88–9.02 (10H, a set of
signals, aromatic protons and CH). 9h: IR (cm–1): 1681
(CO). 1H NMR (CDCl3) (d): 3.73 (3H, s, OCH3); 3.81 (3H, s,
OCH3); 3.84 (3H, s, OCH3); 6.24 (1H, d, CH, J = 15.5 Hz);
6.57–9.01 (9H, a set of signals, aromatic protons and CH). 9i:
IR (cm–1): 1685 (CO). 1H NMR (DMSO-d6) (d): 6.22 (1H, d,
CH, J = 15.2 Hz); 7.57–9.15 (11H, a set of signals, aromatic
protons and CH). 9l: IR (cm–1): 1672 (CO). 1H NMR
(DMSO-d6) (d): 6.21 (1H, d, CH, J = 15.6 Hz); 7.22–9.18
(11H, a set of signals, aromatic protons and CH).
1
protons and 2XCH). 8e: IR (cm–1): 1688 (CO). H NMR
(CDCl3) (d): 2.58 (3H, s, CH3); 3.82 (3H, s, OCH3); 3.87
(3H, s, OCH3); 6.25 (1H, s, isoxazole H-4); 6.80 (1H, d, CH,
J = 15.9 Hz); 6.89–8.22 (7H, a set of signals, aromatic
protons and CH). 8f: IR (cm–1): 1687 (CO). 1H NMR
(CDCl3) (d): 2.59 (3H, s, CH3); 3.88 (3H, s, OCH3); 3.90
(3H, s, OCH3); 6.26 (1H, s, isoxazole H-4); 6.42 (1H, d, CH,
J = 15.9 Hz); 6.82–8.20 (7H, a set of signals, aromatic
protons and CH). 8g: IR (cm–1): 1695 (CO). 1H NMR
(CDCl3) (d): 2.59 (3H, s, CH3); 3.87 (9H, s, 3XOCH3); 6.26
(1H, s, isoxazole H-4); 6.48 (1H, d, CH, J = 15.6 Hz);
6.67–8.23 (6H, a set of signals, aromatic protons and CH).
8h: IR (cm–1): 1697 (CO). 1H NMR (CDCl3) (d): 2.58 (3H, s,
CH3); 3.85 (3H, s, OCH3); 3.86 (3H, s, OCH3); 3.88 (3H, s,
OCH3); 6.26 (1H, s, isoxazole H-4); 6.64–8.20 (7H, a set of
signals, aromatic protons and 2XCH). 8i: IR (cm–1): 1698
(CO). 1H NMR (CDCl3) (d): 2.57 (3H, s, CH3); 6.28 (1H, s,
isoxazole H-4); 6.56 (1H, d, CH, J = 15.3 Hz); 7.48–8.44
(8H, a set of signals, aromatic protons and CH); 8.41 (1H, d,
5.2. Biology
5.2.1. Antiproliferative activity in vitro
Compounds 8a–l and 9a,c–e,h–l were tested in vitro for
antileukemic activity against L-1210 (murine leukemia),
K-562 (human chronic myelogenous leukemia) and HL-60
(human leukemia) cell lines. These cell lines were grown at
37 °C in a humidified atmosphere containing 5% CO2, in
RPMI-1640 medium (Biochrom KG) supplemented with
10% fetal calf serum and antibiotics.
Cells were suspended at a density of 1 × 105 (L-1210 and
K-562) or 2 105 (HL-60) cells per ml in growth medium,
transferred to 24-well plate (1 ml per well), cultured with or
without (control wells) screening concentration of com-
pounds and incubated at 37 °C for 48 h (HL-60, K-562) or
1
CH, J = 15.3 Hz). 8l: IR (cm–1): 1703 (CO). H NMR
(CDCl3) (d): 2.60 (3H, s, CH3); 6.27 (1H, s, isoxazole H-4);
6.54 (1H, d, CH, J = 15.5 Hz); 7.34–8.22 (8H, a set of signals,

D. Raffa et al. / IL FARMACO 59 (2004) 451–455
455
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