Synthesis and pharmacological activities of novel 3-(isoxazol-3-yl)- quinazolin-4(3H)-one derivatives

Article abstract of DOI:10.1002/(SICI)1521-4184(19993)332:2<50::AID-ARDP50>3.0.CO;2-S

Several new 3-(Isoxazol-3-yl)-quinazolin-4(3H)-one derivatives were synthesized and tested for their analgesic and antiinflammatory activities, as well as for their acute toxicity and ulcerogenic effect. A few compounds were as active as phenylbutazone in the writhing and acetic acid peritonitis tests. They had a very low ulcerogenic effect.

Full text of DOI:10.1002/(SICI)1521-4184(19993)332:2<50::AID-ARDP50>3.0.CO;2-S

50
Daidone and co-workers
Synthesis and Pharmacological Activities of Novel
3-(Isoxazol-3-yl)-quinazolin-4(3H)-one Derivatives
a)
a)
a)
a)
b)
Giuseppe Daidone *, Demetrio Raffa , Benedetta Maggio , Fabiana Plescia , Vincenza Maria Catena Cutuli ,
b)
b)
Nunzio Guido Mangano , and Antonina Caruso
a) Dipartimento di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, via Archirafi, 32, 90123 Palermo. Italy
b) Istituto di Farmacologia, Facolta di Medicina e Chirurgia, Università degli Studi di Catania, viale Andrea Doria, 6, 95123 Catania, Italy
Key Words: 3-(Isoxazol-3-yl)-quinazolin-4(3H)-one; anti-inflammatory activity; analgesic activity
Summary
Pharmacology
All 4(3H)-quinazolinones 5a–n were evaluated for their
analgesic and antiinflammatory activities, as well as for their
ulcerogenic potential, behavioural effects, and acute toxicity.
Phenylbutazone (PBZ) was used as reference compound.
Pharmacological data are reported in Table 3.
Several new 3-(isoxazol-3-yl)-quinazolin-4(3H)-one derivatives
were synthesized and tested for their analgesic and antiinflamma-
tory activities, as well as for their acute toxicity and ulcerogenic
effect. A few compounds were as active as phenylbutazone in the
writhing and acetic acid peritonitis tests. They had a very low
ulcerogenic effect.
Results and Discussion
Behavioural Effects and Acute Toxicity in Mice
In mice the tested compounds did not induce remarkable
behaviour modification according to Irwin’s assay at a dose
of 700 mg/kg p.o. and 500 mg/kg i.p., whereas slight and
transient sedation was the only effect noted after administra-
tion of 1000 mg/kg p.o. and 800 mg/kg i.p.. Only compound
5d induced a dose-dependent decrease in spontaneous motor
activity and respiratory rate, ataxia, ptosis, piloerection, and
tremors. Death generally occurred at 1–3 h postdrug in 40–
60% of animals. The surviving mice appeared normal and
remained so throughout the seven-day observation period.
Introduction
In a previous paper we reported the synthesis and the study
of the analgesic, antipyretic, and antiinflammatory activities
of new 3-(isoxazol-5-yl)-quinazolin-4(3H)-ones. Among
these, 6-chloro-2-methyl-3-(3-methylisoxazol-5-yl)-quinaz-
olin-4(3H)-one showed percentage inhibition values in the
acetic acid peritonitis and carrageenin paw edema tests, as
well as LD that are comparable to those of ASA but with
50,
[1]
an ulceration index of approximately zero
Owing to this encouraging result and continuing our re-
.
search on new antiinflammatory agents with reduced side
Analgesic and Antiinflammatory Activities
[2,3]
effects
we carried out the synthesis of some substituted-
Analgesic and antiinflammatory activities were influenced
by substituents, following different trends for the studied
activities. In the phenylquinone writhing test, compounds
5e,f,l exhibited a remarkable analgesic action at 10 mg/kg po
and were twice as active as phenylbutazone (PBZ).
In the rat paw edema test, compounds 5a,d,l were equiac-
tive to the reference drug whereas in the acetic acid peritonitis
all the compounds were found to be more active than the PBZ,
with compounds 5a,l being very active.
quinazolin-4(3H)-ones 5a–n which bear in the position 3 of
the quinazolinone nucleus the 5-methylisoxazol-3-yl moiety
instead of the isomer 3-methylisoxazol-5-yl one, with the aim
of ascertaining whether this substitution could improve the
antiinflammatory and analgesic activities of the mentioned
derivatives.
Chemistry
Compounds 5a–n were prepared by a previously described
method , according to the route given in Scheme 1. The
Ulcerogenic Activity
[1]
appropriate N-(5-methylisoxazol-3-yl)-2-nitrobenzamide de-
rivatives 3a,e,i were thus synthesized starting from the 2-ni-
troaroyl chlorides 1a,e,i and 3-amino-5-methylisoxazole 2
in anhydrous chloroform. When compounds 3 were treated
with stannous chloride in aqueous hydrochloric acid the
corresponding N-(5-methylisoxazol-3-yl)-2-aminobenz-
amide derivatives 4a,e,i were obtained. By refluxing products
4 with the appropriate orthoester, the isoxazolylquinazoli-
nones 5a–n were obtained in very good yields.
Compounds 5a,l, which were overall the most active com-
pounds of the series, did not show any harmful effect on the
stomach when administered twice at a dose of 400 mg/kg in
fasted rats. In contrast, at lower doses phenylbutazone pro-
duced serious gastric ulcers in all animals.
[4]
Conclusion
Pharmacological screening showed that compounds 5a,l
The structures of the new compounds 3a,e,i, 4a,e,i, and are endowed with appreciable analgesic and antiinflamma-
5a-n were assigned on the basis of analytical as well as tory activities and, at the same time, with low systemic
spectroscopic data.
toxicity and very modest ulcerogenic effects.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0202/0050 $17.50 +.50/0

3-(Isoxazol-3-yl)-quinazolin-4(3H)-one Derivatives
51
Scheme 1
————————————————————————————————————————————————————————
a
b
c
d
e
f
g
h
i
l
m
n
————————————————————————————————————————————————————————
R
H
H
H
CH3
C2H5
C6H5
H
CH3
C2H5
C6H5
H
CH3
C2H5
C6H5
R1
R2
H
H
H
Cl
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
————————————————————————————————————————————————————————
in anhydrous chloroform (250 ml) was added. The mixture was treated with
triethylamine (3 ml) and then refluxed for 4 h. After the first hour of reflux,
three portion of triethylamine, 1, 4, and 4 ml each, were added at intervals of
1 hour. The solution was evaporated under reduced pressure, the residue was
washed until it became solid, filtered off, then left overnight under magnetic
stirring with an aqueous solution of KOH 20% (400 ml). The obtained
solution was filtered and the product was precipitated with a saturated
solution of ammonium chloride, washed with hot diethyl ether, and then
crystallized from ethanol; yields 45–70 %. Compounds 3a,e,i are listed in
Table 1.
Finally, comparison of the carrageenin paw oedema inhibi-
tion data for quinazolinones 5a–n with those reported for the
isomers bearing the 3-methylisoxazol-5-yl moiety [1] could
be performed. Generally, compounds of the isoxazol-3-yl
series were substantially more active than those of the isoxa-
zol-5-yl one.
With regard to the analgesic activity, an exact comparison
between the two series was not possible, as the pharmacologi-
cal data were obtained at different doses. However, the over-
all trend was higher activity for the isoxazol-3-yl series.
N-(5-methylisoxazol-3-yl)-2-aminobenzamides 4a,e,i
Compound 3a,e, or i (0.024 moles) was added to a cold (0–5 °C) magneti-
cally stirred suspension of stannous chloride (0.064 moles) in concentrated
HCl (25 ml) at such rate that the temperature of the slurry was maintained
below 10 °C. After complete addition, the mixture was stirred overnight with
a magnetic stirrer. The white slurry thus obtained was diluted with cold water
(500 ml) and sodium hydroxide (40%) was added till the solution became
alkaline.
Acknowledgement
This research was supported by a grant from M.U.R.S.T. (Rome).
Experimental Section
The solid which separated was filtered off and crystallized from ethanol;
yields 40–62%. Compounds 4a,e,i are listed in Table 1.
Chemistry
All melting points were determined on a Büchi 530 capillary melting point
apparatus and are uncorrected; IR spectra 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 (tetramethyl-
silane 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.
3-(5-methylisoxazol-3-yl)-4(3H)-quinazolinones 5a-n
Aminobenzamide 4a,e,i (10 mmoles) and the suitable triethyl orthoester
(20 ml) were heated under reflux for 5 h. After cooling, the crystalline solid
which separated out was recrystallized from ethanol; yields 71–88%. Com-
pounds 5a–n are listed in Table 2.
2-Nitroaroyl Chlorides 1a,e,i.
Pharmacology
Chloride 1a was commercially available; crude 2-nitroaroyl chlorides 1e,i
were obtained by refluxing the opportunely substituted 2-nitrobenzoic acids
(0.08 moles) with thionyl chloride (28.9 ml) for 5 h and by evaporation
Materials and Methods
under reduced pressure of the reaction mixture[4]
.
Swiss male mice (20–23 g) and Sprague-Dawley rats (130–160 g) were
used. The animals were starved for about 12 h before administration and
maintained at a temperature of 22±2 °C. The tested compounds and phenyl-
butazone (PBZ) were suspended in 0.5% aqueous methylcellulose solution
and administered orally or intraperitoneally. Control animals received the
same amounts of the vehicle.
N-(5-methylisoxazol-3-yl)-2-nitrobenzamides 4a,e,i.
Chloride 1a,e,i (0.08 moles) was dissolved in anhydrous chloroform
(200 ml) and then a solution of 5-methyl-3-aminoisoxazole 2 (0.08 moles)
Arch. Pharm. Pharm. Med. Chem. 332, 50–54 (1999)

52
Daidone and co-workers
Table 1. Physical and spectroscopic data of N-(5-methylisoxazol-3-yl)-benzamides 3 and 4
—————————————————————————————————————————————————————————————
Formulaa IR (cm–1) (nujol) 1H-NMR (δ)
Comp.
Mp °C
—————————————————————————————————————————————————————————————
3a
3e
3i
177–179
220–221
214–216
188–190
188–190
188–190
C11H9N3O4
3300–3020, 1695
3300–3000, 1695
3360–3020, 1680–1670
3480–3040, 1665
3480–3040, 1680
3500–3020, 1680
2.42 (s, 3H, CH3); 6.74 (s, 1H, isoxazole H-4);
7.77–8.18 (m, 4H, C6H4); 11.66 (s, 1H, NH, exchangeable)
C11H8N3O4Cl
C11H8N3O4Cl
C11H11N3O2
C11H10N3O2Cl
C11H10N3O2Cl
2.43 (s, 3H, CH3); 6.74 (s, 1H, isoxazole H-4);
7.82–8.22 (m, 3H, C6H3); 11.73 (s, 1H, NH, exchangeable)
2.42 (s, 3H, CH3); 6.74 (s, 1H, isoxazole H-4);
7.80–8.26 (m, 3H, C6H3); 11.73 (s, 1H, NH, exchangeable)
4a
4e
4i
2.40 (s, 3H, CH3); 6.55–7.77 (m, 7H, C6H4 isoxazole H-4
and exchangeable NH2); 10.92 (s, 1H, NH, exchangeable)
2.40 (s, 3H, CH3); 6.68–7.82 (m, 6H, C6H3 isoxazole H-4
and exchangeable NH2); 11.07 (s, 1H, NH, exchangeable)
2.40 (s, 3H, CH3); 6.53–7.75 (m, 6H, C6H3 isoxazole H-4
and exchangeable NH2); 10.99 (s, 1H, NH, exchangeable)
—————————————————————————————————————————————————————————————
a) All compounds are analyzed for C, H, N and results agreed to ±0.4% of theoretical values.
Table 2. Physical and spectroscopic data of 3-(5-methylisoxazol-3-yl)-4(3H)-quinazolinones 5a–n.
—————————————————————————————————————————————————————————————
Comp.
Mp °C
Formulaa
IR (cm–1) (nujol)
1H-NMR (δ)
—————————————————————————————————————————————————————————————
5a
5b
5c
5d
5e
5f
108–110
147–149
127–130
158–160
164–165
162–163
162–164
156–158
177–178
184–186
128–130
160–162
C12H9N3O2
1700–1690
1690
2.53 (s, 3H, CH3); 6.78 (s, 1H, isoxazole H-4);
7.52–8.53 (m, 5H, C6H4 and quinazolinone H-2)
C13H11N3O2
2.39 (s, 3H, CH3); 2.54 (s, 3H, CH3), 6.26 (s, 1H, isoxazole H-4);
7.43–8.24 (m, 4H, C6H4 )
C14H13N3O2
1680
1.25 (t, 3H, CH3); 2.51–2.62 (s+q, 5H, CH2 and CH3),
6.18 (s, 1H, isoxazole H-4); 7.40–8.22 (m, 4H, C6H4 )
C18H13N3O2
1690
2.39 (s, 3H, CH3); 6.16 (s, 1H, isoxazole H-4);
7.27–8.33 (m, 9H, C6H5 and C6H4)
C12H8N3O2Cl
C13H10N3O2Cl
C14H12N3O2Cl
C18H12N3O2Cl
C12H8N3O2Cl
C13H10N3O2Cl
C14H12N3O2Cl
C18H12N3O2Cl
1700
2.53 (s, 3H, CH3); 6.77 (s, 1H, isoxazole H-4);
7.73–8.57 (m, 4H, C6H3 and quinazolinone H-2)
1710
2.36 (s, 3H, CH3); 2.54 (s, 3H, CH3), 6.21 (s, 1H, isoxazole H-4);
7.56–8.14 (m, 3H, C6H3)
5g
5h
5i
1700
1.26 (t, 3H, CH3); 2.54–2.63 (s+q, 5H, CH2 and CH3),
6.20 (s, 1H, isoxazole H-4); 7.60–8.15 (m, 3H, C6H3 )
1700
2.42 (s, 3H, CH3); 6.16 (s, 1H, isoxazole H-4);
7.18–8.27 (m, 8H, C6H5 and C6H3)
1700
2.53 (s, 3H, CH3); 6.76 (s, 1H, isoxazole H-4);
7.47–8.59 (m, 4H, C6H3 and quinazolinone H-2)
5l
1700
2.36 (s, 3H, CH3); 2.54 (s, 3H, CH3), 6.19 (s, 1H, isoxazole H-4);
7.38–8.16 (m, 3H, C6H3)
5m
5n
1700
1.26 (t, 3H, CH3); 2.54–2.63 (s+q, 5H, CH2 and CH3),
6.21 (s, 1H, isoxazole H-4); 7.37–8.15 (m, 3H, C6H3 )
1690
2.37 (s, 3H, CH3); 6.15 (s, 1H, isoxazole H-4);
7.32–8.22 (m, 8H, C6H5 and C6H3)
—————————————————————————————————————————————————————————————
a) All compounds are analyzed for C, H, N and results agreed to ±0.4% of theoretical values
Arch. Pharm. Pharm. Med. Chem. 332, 50–54 (1999)

3-(Isoxazol-3-yl)-quinazolin-4(3H)-one Derivatives
53
Table 3. Pharmacological data of 3-(5-methylisoxazol-3-yl)-4(3H)-quinazolinones 5a–n..
——————————————————————————————————————————————————————
Acute toxicity
approximate LD50
mg/kg
Analgesic
activity
Phenylquinone
writhing-test
(%protection ± SE)(a)
Antiinflammatory Activity
——————————————–
Ulcerogenic
index
Carrageenin
paw oedema
Acetic acid
peritonitis
Comp.
(% inhibition ± SE)(a)
——————————————————————————————————————————————————————
po ip 10 mg/kg 100 mg/kg 10 mg/kg 400 mg/kg × 2
——————————————————————————————————————————————————————
5a
5b
5c
>1000
>1000
>1000
~700
>800
>800
>800
~300
>800
>800
>800
>800
>800
>800
>800
>800
~300
36 ± 4*
23 ± 5*
34 ± 4*
19 ± 5*
42 ± 3*
45 ± 3*
5 ± 8
68 ± 3*
47 ± 2*
25 ± 4*
58 ± 3*
45 ± 2*
43 ± 2*
33 ± 4*
40 ± 4*
48 ± 3*
60 ± 3*
36 ± 3*
44 ± 3*
59 ± 3*
43 ± 7*
13 ± 11
27 ± 9*
13 ± 11
15 ± 9
40
5d
5e
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
~700
5f
23 ± 10*
19 ± 10*
32 ± 4*
32 ± 9*
40 ± 8*
18 ± 9*
24 ± 11*
7 ± 7
5g
5h
5i
19 ± 5*
13 ± 5
5l
43 ± 4*
34 ± 4*
23 ± 5*
23 ± 5*
30
5m
5n
PBZ
250b
—————————————————————————————————————————————————————
Oral administration for all test.
(a) Values are percent of controls. * P < 0.05 Student-Newman-Keuls test versus controls.
(b) PBZ 2×100 mg.
Behavioural Effects and Acute Toxicity In Mice[5]
Acetic Acid Peritonitis [8]
Groups of 4 rats were tested. Peritonitis was produced by an i.p. injection
of acetic acid (10 ml/kg of 0.5% aqueous solution). 30 min later rats were
killed by ether and peritoneal exudate was collected and measured. The tested
compounds and PBZ were given orally at the dose of 10 mg/kg, 60 min
before the injection of the acetic acid. The antiexudative response was
expressed and the percentage of the exudate volume reduction comparedwith
controls.
Irwin’s multidimensional screening-evaluative procedure was used on
groups of 5 animals. The compounds were administered at three doses orally
(500–700–1000 mg/kg) or intraperitoneally (300–500–800 mg/kg). The ani-
mals were kept under blinder observation for 6 h and the symptomatology
was checked again 24 h later. The approximate LD50 was obtained from
mortality in the following 7-day period.
Ulcerogenic Activity[9]
Analgesic Activity: Phenylbenzoquinone Writhing Test[6]
Groups of 4 rats were used. Compounds 5a and 5l were given orally
(400 mg/kg) to animals fasted for 24 h and after 2 h the treatment was
repeated again. Six hours after the first dose, each rat was sacrificed by ether
inhalation, the stomach removed, opened along the greater curvature and
examined with a dissecting microscope for the presence of gastric ulcers. The
severity of mucosal damage (Ulcerogenic index) was graded by means of
scores from 0 (no lesion) to 4 (exceptionally severe lesions). In order to take
into account the percentage of rats having ulcers, an index of ulceration was
calculated on the basis of the following formula:
Analgesia was assessed by means of phenylbenzoquinone (PBQ)-induced
writhing on group of 4 male mice. Each mouse was given i.p. 0.25 ml of
0.02% PBQ in 6% ethanol and the number of writhes was counted for 5 min,
beginning 5 min after the injection. The tested compounds and PBZ were
orally administered (10 mg/kg) 60 min before PBQ. The analgesic effect was
expressed as percentage of protection in comparison with controls.
Antiinflammatory Activity
Carrageenin-Induced Oedema [7]
mean degree of ulceration × number of animals with ulcers
————————————————————————— × 100
number of animals
Groups of 4 rats were used. The tested compounds and PBZ were given
orally at 100 mg/kg. Sixty minutes later, 0.1 ml of 1% carrageenin solution
was injected into the sub-plantar tissue of the right hind paw. The volume
was measured by a mercury plethysmometer prior to the injection of car-
rageenin and 3 h later. The increase in volume of the paw 3 h after the
injection of carrageenin was adopted as a measure of oedema. Swelling in
treated animals was calculated as percentage of inhibition incomparison with
controls.
Statistical Analysis
The effects of drug treatments were expressed as percent inhibition vs.
controls. The approximate standard error of each percentage was calculated
by the following equation derived from Feiller’s theorem:
Arch. Pharm. Pharm. Med. Chem. 332, 50–54 (1999)

54
Daidone and co-workers
s.e. of % inhibition = 100 m √ (Cx) 2 + (Cy) 2
[3] G. Daidone, B. Maggio, S. Plescia, D. Raffa, D. Schillaci, A.Caruso,
V.M.C. Cutuli, M.Amico-Roxas, Farmaco 1998, 53, 350–356.
where Cx is the coefficient of variation of the control mean response, Cy the
coefficient of variation of the test mean response and m is the ratio between
the mean test response and the mean control response. The statistical signifi-
cance of test responses was evaluated by one way ANOVA followed by
Student-Newman-Keuls test.
[4] M.H. Palmer, G.J. McVie, J. Chem. Soc. (B) 1968, 745.
[5] S. Irwin, Psycopharmacologia (Berl) 1968, 13, 222.
[6] B.A. Berkowitz, A.D. Fink, S.H. Ngai, J. Pharmacol Exp. Ther. 1977,
203, 539-547.
[7] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. 1962,
111, 544-547.
References
[1] S. Plescia, G. Daidone, D. Raffa, M.L. Bajardi, A. Caruso, V.M.L.
[8] E.C.A. Arrigoni-Martelli, Boll. Chim. Farm. 1968, 107, 29–42.
[9] R. Domenioz, Ann. NY Acad. Sci. 1960, 86, 263.
Received: November 23, 1998 [FP350]
Cutuli, M.Amico-Roxas, Farmaco 1992, 47, 465.
[2] S. Plescia, M.L. Bajardi, D. Raffa, G. Daidone, M. Matera, A. Caruso,
M. Amico-Roxas, Eur. J. Med. Chem.-Chim. Ther. 1986, 21, 291–295.
Arch. Pharm. Pharm. Med. Chem. 332, 50–54 (1999)