Synthesis and biological evaluation of new fused isoxazolo[4,5-d]pyridazine derivatives

Article abstract of DOI:10.1002/jccs.201190076

Three new heterocyclic ring systems, isoxazolo[4,5-d]-1,2,4- triazolopyridazines 12-15, tetrazolo-[4,3-b]pyridazine 18 and isoxazolo[4,5-d]pyridazino[2,3-c]2H-triazines 16,17 along with isoxazolo[4,5-d]pyridazines 2, 5-10 have been synthesized. Preliminar

Full text of DOI:10.1002/jccs.201190076

Journal of the Chinese Chemical Society, 2011, 58, 191-198
191
Synthesis and Biological Evaluation of New Fused Isoxazolo[4,5-d]
Pyridazine Derivatives
Hassan M. Faidallah,* Khalid A. Khan and Mohammed S. I. Makki
Department of Chemistry, Faculty of Science, King Abdulaziz University,
Box 80203 Jeddah-21589, Saudi Arabia
Received May 30, 2010; Accepted December 27, 2010; Published Online January 18, 2011
Three new heterocyclic ring systems, isoxazolo[4,5-d]-1,2,4-triazolopyridazines 12-15, tetrazolo-
[4,3-b]pyridazine 18 and isoxazolo[4,5-d]pyridazino[2,3-c]2H-triazines 16,17 along with isoxazolo[4,5-
d]pyridazines 2, 5-10 have been synthesized. Preliminary screening of these tricyclic heterocycles re-
vealed that some of them possess significant antibacterial and antifungal activity.
Keywords: Isoxazoles; Triazoles; Pyridazines.
INTRODUCTION
Isoxazoles,1-6 triazoles7-10 and pyridazines11-16 are bi-
4. The 13C NMR data of compounds 2 and 4 are summa-
rized in Table 2 and the chemical shift values conform to
the suggested structures.
ologically important heterocyclic compounds. Some of
these compounds have antibacterial activity better than
carbenicillin against Gram-negative strains17 others are
used as antimicrobial agents.18,19 A variety of condensed
triazolo20-23 and triazino7,22,24 derivatives show a wide
range of pharmacological and biological properties. Prompted
by these observations and in continuation to our work on
the synthesis of biologically active heterocyclic com-
pounds,25-29 it was felt interesting to synthesize fused
heterocyclic systems incorporating isoxazole, triazole and
pyridazine moieties and evaluate their antimicrobial activ-
ity.
The MS of the pyridazinone 2a showed a molecular
ion as the base peak at m/z 165 while for 2b the base peak
was the [M-NHSO2C6H4NCO] species and the molecular
ion was only 25%. On the other hand, the mass spectrum of
oxazine derivative 4 exhibited the molecular ion peak at
m/z 166 while the base peak is at m/z 41.
Condensation of the pyridazine derivative 2a with
isocyanates and isothiocyanates afforded the correspond-
ing carbamoyl 5a-c and thiocarbamoyl 6a-c derivatives re-
spectively. The IR spectra of the carbamoyl derivatives 5
exhibited two distinguishable carbonyl absorptions at
1668-1672 cm-1 and 1654-1659 cm-1 whereas compounds
6a-c revealed distinct thiocarbonyl band at 1060-1150 cm-1
as well as the pyridazinone carbonyl band at 1655-1658
cm-1. The 13C NMR data of compounds 5 and 6 as summa-
rized in Table 2 is in agreement with the suggested struc-
tures.
RESULTS AND DISCUSSION
The readily available ethyl 5-acetyl-3-methylisoxa-
zole-4-carboxylate 1 served as starting material for the syn-
thesis (Scheme I). Reaction of 1 with hydrazine derivatives
afforded the 1-substituted 3,6-dimethyl-7-oxoisoxazolo-
[4,5-d]pyridazines 2a,b, while reaction with hydroxyl-
amine hydrochloride 1 gave oxime 3 or 3,6-dimethyl-7-
oxoisoxazolo[4,5-d]-1,2-oxazine 4 depending on the reac-
tion conditions (see Experimental). Consistent with the as-
signed structure the 1H NMR spectrum of oxime 3 exhib-
ited a triplet and quartet at d 1.40 and 4.33 for the CH3 and
CH2 of the ester group, respectively. These signals are
missing in the 1H NMR spectrum of the oxazine derivative
Refluxing of 2a with phosphorus oxychloride gave
the 4-chloro derivative 7, which on reaction with ethanolic
hydrazine hydrate at room temperature yielded the 3,6-
dimethyl-7-hydrazinoisoxazolo[4,5-d]pyridazine 8 in 86%
yield. Reaction of 7 with thiourea gave the thiol derivative
9, which on treatment with an aqueous solution of sodium
hydrogen sulfate and sodium nitrite yielded the disulfide
10. In accordance with the suggested structure, the 13C
* Corresponding author. E-mail: hfaidallahm@hotmail.com

192 J. Chin. Chem. Soc., Vol. 58, No. 2, 2011
Faidallah et al.
Scheme I
NMR spectrum of 9 exhibited the expected number of sig-
at d 10.48 and 15.82 (Table 2).
Reaction of the hydrazino derivative 8 with acid chlo-
nals for the aromatic carbons as well as two methyl signals

Fused Isoxazolo[4,5-d]pyridazines
J. Chin. Chem. Soc., Vol. 58, No. 2, 2011 193
Table 1. Physical and analytical data
Yield
Analysis
M.P
(°C)
Compound
R or R¢
Formula
Calcd. %
Found %
(%)
C
H
N
S
C
H
N
S
2a
2b
3
H
82
88
70
65
82
85
88
80
78
76
84
86
78
82
76
82
70
74
75
72
66
78
64
62
257
176
58
C7H7N3O2
50.91 4.27 25.44
50.95 4.30 25.60
p-NH2SO2C6H4
C13H12N4O4S
C9H12N2O4
C7H6N2O3
48.74 3.77 14.49 10.01 48.82 3.88 14.52 10.21
50.94 5.70 13.20
50.61 3.64 16.86
57.92 6.25 19.30
59.15 4.25 19.71
52.76 3.48 17.58
51.00 5.90 13.31
50.75 3.80 16.78
58.02 6.36 19.42
59.26 4.37 19.90
52.86 3.50 17.62
4
212
220
228
252
186
204
162
102
160
230
258
145
156
245
170
236
305
>300
246
230
207
5a
5b
5c
6a
6b
6c
7
Cyclohexyl
C6H5
C14H18N4O3
C14H12N4O3
C14H11ClN4O3
C14H12N4O2S
p-ClC6H4
C6H5
55.99 4.03 18.65 10.67 56.00 4.12 18.70 10.81
9.57 50.30 3.42 16.84 9.68
52.82 3.48 17.60 10.07 52.92 3.53 17.72 10.14
p-ClC6H4
p-FC6H4
C14H11ClN4O2S 50.33 3.31 16.73
C14H11FN4O2S
C7H6ClN3O
C7H9N5O
45.78 3.29 22.88
46.92 5.06 39.08
45.87 3.30 22.00
46.91 5.13 39.20
8
9
C7H7N3OS
C14H12N6O2S2
C9H12ClN5O2
C15H16ClN5O3
C9H9N5O
46.39 3.89 23.19 17.69 46.40 3.92 23.25 17.71
46.65 3.35 23.32 17.79 46.62 3.42 23.30 17.82
10
11a
11b
12
13
14
15
16
17a
17b
18
CH3
41.94 4.69 27.17
51.50 4.61 20.02
53.19 4.46 34.46
61.00 4.43 23.71
38.31 2.86 24.82
61.10 5.13 32.39
46.35 3.02 30.03
64.50 4.69 25.07
55.29 5.10 32.23
44.21 3.18 44.19
41.02 4.72 27.25
51.65 4.72 20.15
53.21 4.56 34.56
60.99 4.52 23.86
38.19 2.90 24.76
61.21 5.23 32.40
46.38 3.10 30.15
64.62 4.72 25.18
55.31 5.23 32.30
44.30 3.22 44.20
p-CH3OC6H4
CH3
p-CH3OC6H4
CH2Br
C15H13N5O2
C9H8BrN5O
C11H11N5O3
C9H7N5O3
COOEt
C6H5
CH3
C15H13N5O
C10H11N5O
C7H6N6O
Table 2. 13C NMR spectral data of isoxazolo[4,5-d]pyridazine derivatives
Compd
Aromatic C
CH3 and other C
CO & CS
2a
2b
133.12, 156.75, 158.63
15.64, 14.61
12.74, 14.90
163.77
107.81, 111.52, 125.41, 130.67, 134.28
145.95, 158.73, 160.52
168.51
4
115.32, 134.20, 154.82, 158.93
112.37, 132.39, 155.95, 159.57
11.02, 15.83
170.20
5a
9.09, 14.61
168.89, 178.20
23.29, 24.15, 32.45, 46.34
(cyclohexyl C)
10.89, 15.06
5b
6a
6b
7
111.83, 121.23, 124.72, 127.78, 128.20, 134.68, 149.26, 158.73
109.71, 121.38, 124.81, 127.09, 129.05, 136.28, 152.48, 158.42
110.35, 122.36, 124.10, 125.99, 133.42, 147.09, 156.28, 159.36
114.82, 135.06, 155.20, 157.43, 162.03
169.33, 180.12
10.55, 14.59
167.85, 205 (CS)
168.85, 200.20 (CS)
12.02, 15.13
10.56, 15.48
9
113.80, 136.55, 146.70, 155.04, 158.36
10.48, 15.82
12
15
16
17b
18
114.46, 133.25, 144.78, 152.68, 156.70, 158.45
113.66, 134.02, 146.60, 154.28, 155.89, 159.32
113.86, 132.98, 148,33, 152.98, 158.72
11.21, 13.03, 15.68
11.34, 13.74, 18.10, 60.54 (CH3) 170.33
10.78, 15.90
168.78, 170.50
112.98, 132.56, 136.42, 138.05, 146.35, 157.56, 162.78
113.63, 133.42, 145.32, 156.78, 158.14
10.66, 13.35, 15.84
10.47, 15.82
a Solution in DMSO–d6/CDCl3, d in ppm.
rides afforded the corresponding acyl hyrazides hydro-
3-substituted 6,9-dimethylisoxazolo[4,5-d]l,2,4-triazolo-
chlorides 11 which readily cyclized in hot pyridine to the
[4,3-b]pyridazines 12 and 13 (Scheme II). The IR spectra

194 J. Chin. Chem. Soc., Vol. 58, No. 2, 2011
Faidallah et al.
Scheme II
of the acyl hydrazides 11 exhibited secondary carbonyl ab-
sorption at 1662-1672 cm-1, which is missing in the IR
spectra of the cyclic derivatives, 12 and 13.
Moreover, refluxing the hydrazino compound 8 with
b-bromoketones in acetic acid afforded the corresponding
3-substituted 7,10-dimethylisoxazolo[4,5-d]pyridazino-
[2,3-c]-2H-1,2,4-triazines 17a,b. The 1H NMR spectra of
the triazines 17a and 17b showed, besides the CH3 and aro-
matic protons, an exchangeable NH signal at d 11.95-
12.46. The structure of 17a was further confirmed by its
MS analyses. A molecular ion peak at m/z 279 was de-
tected, while the base peak is at m/z 162.
Cyclocondensation of 8 with ethyl bromoacetate
gave the 3-(bromomethyl)-1,2,4-triazolo derivative 14. Its
IH NMR exhibited a singlet of two protons intensity at d
4.78 for the CH2 as well as two methyl singlets at d 2.47 and
2.85.
In 1969 Polts et al30 reported that the reaction of an
excess of oxalic acid with 1-hydrazinophthalazine at 160
°C to give a 50% yield of s-triazolo[3,4-a]phthalazine, pre-
sumably arising from the decarboxylation of the originally
formed s-triazolo[3,4-a]phthalazine-3-carboxylic acid.
Zimmer et al31 isolated the 2H-as-triazolo[3,4-a]phthal-
azine-3,4-dione in 90% yield instead of the expected ethyl
s-triazolo[3,4-a]phthalazine-3-carboxylate from the reac-
tion of hydralazine with excess ethyl oxalate. However, in
our case, when we refluxed 8 with ethyl oxalate, the prod-
uct isolated was ethyl 6,9-dimethylisoxazolo[4,5-d]-1,2,4-
triazolo[4,3-b]pyridazine-3-carboxylate 15 or 7,10-di-
methylisoxazolo[4,5-d]pyridazino[2,3-c]-2H-l,2,4-tri-
azine-3,4-dione 16 depending on the reaction conditions
(see Experimental) as evidenced by analytical and spectral
data (Tables 1 & 2).
Finally, when a solution of 8 in acetic acid was al-
lowed to react with sodium nitrite the product was found to
be the tetrazolo derivative 18, a reaction similar to what’s
reported by Gatta et al.23
ANTIMICROBIAL ACTIVITY
Antimicrobial testing of compounds 2-8, 12-15 and
17 was carried out (Table 3) and it was found that com-
pounds 5a, 6c and 17a showed maximum activity (+++)
(MIC = 25 mg/mL) against S. aureus, compounds 5c, 6b
and 14 showed moderate activity (++) (MIC = 50 mg/mL),
while compounds 5b, 7, 8 and 17b exhibited feeble activity
(+) (MIC = 75 mg/mL). Compounds 6c and 17a showed the
maximum activity against Gram-negative strain E. coli,
while 5c, 6b and 14 exhibited moderate activity and com-

Fused Isoxazolo[4,5-d]pyridazines
J. Chin. Chem. Soc., Vol. 58, No. 2, 2011 195
Table 3. Antimicrobial activities of synthesized compounds (+++ for maximum activity, MICa = 25; ++ for moderate activity, MIC =
50; + for slight activity MIC = 75; and – for inactive compounds)
Organism
2a
2b
3
4
5a
5b
5c
6a
6b
6c
7
8
12
13
14
15 17a 17b
S aureus
-
-
-
-
+++
+
++
++ +++
++ +++
+
+
-
-
++
-
-
-
+++
+++
+
-
(Gram-positive)
E. coli
-
-
-
-
-
+
-
-
-
++
-
+
+
-
-
-
-
-
++
++
(Gram-negative)
C. albicans
(Fungi)
+
+
+++
-
-
+
++ ++
a Minimum inhibitory concentration (MIC in mg/mL)
pounds 5a and 7 showed weak activity. In the antifungal ac-
tivity profile against C. albicans compound 3 revealed
maximum inhibition (+++), compounds 14, 17a and 17b
showed moderate inhibition (++) and compounds 2a, 2b, 7
and 8 were slightly active (+). However, other compounds
were found inactive towards various strains of bacteria.
Quantitative assays were done on active compounds only.
intensity) 320 (M+, 25), 292 (M-CO, 28), 240 (M-SO2NH2,
14), 184 (N2C6H4SO2NH2, 21), 170 (+NC6H4SO2NH2, 16),
164 (M-C6H4SO2NH2, 10), 156 (C6H4SO2NH2, 40), 142
(40), 123 (100), 108 (9), 107 (14), 92(44), 81(5), 85 (11),
80 (12), 75 (37), 66 (15), 50 (29).
Ethyl 5-acetyl-3-methylisoxazole-4-carboxylate oxime
(3)
Asolution of 1 (0.39 g, 0.002 mol) in ethanol (20 mL)
was refluxed with hydroxylamine hydrochloride (0.15 g,
0.0022 mol) and sodium acetate (0.18 g, 0.0022 mol) for 3
h. The oxime which separated after dilution with water was
recrystallized from diluted ethanol as needles.
IR (KBr, nmax, cm-1) 1735 (CO), 3310 (OH); lH NMR
(CDCl3) d 1.38 (t, J = 6 Hz, 3H, CH3), 2.45 (s, 3H, CH3),
2.56 (s, 3H, CH3), 4.32 (q, J = 6 Hz, 2H, CH2).
EXPERIMENTAL
Melting points were determined on Gallenkamp melt-
ing point apparatus and are uncorrected. IR spectra were
obtained on Perkin-Elmer 297 infrared spectrophotometer
using potassium bromide pellets. 1H NMR and 13C NMR
spectra were recorded on Bruker DPX-400-FT spectrome-
ter using TMS as internal standard. MS were obtained on a
Kratos MS 30. Elemental analyses were performed by
Microanalyses unit, King Abdulaziz University, Jeddah,
Saudi Arabia. Ethyl 5-acetyl-3-methylisoxazole-4-car-
boxylate was obtained from Maybridge Chemical Com-
pany Limited, UK.
3,6-Dimethyl-7-oxoisoxazolo[4,5-d]-1,2-oxazine (4)
A solution of 1 (0.39 g, 0.002 mol) in pyridine (10
mL) was refluxed with hydroxylamine hydrochloride (0.15
g, 0.0022 mol) for 6 h. The oxazinone 4 separated on dilu-
tion with water and was recrystallized from ethanol as nee-
dles. IR (KBr, nmax, cm-1) 1750 (CO); lH NMR (CDCl3) d
2.48 (s, 3H, CH3), 2.68 (s, 3H, CH3); MS m/z (relative in-
tensity) 166 (M+, 65), 125 (M-CH3CN, 41), 109 (10), 97
(5), 98 (25), 81 (10), 66 (18),44 (22), 41 (100).
1-Substituted-3,6-dimethyl-7-oxoisoxazolo[4,5-d]pyrid-
azines (2a,b)
A solution of ethyl 5-acetyl-3-methylisoxazole-4-
carboxylate 1 (0.39 g, 0.002 mol) in ethanol (25 mL) was
refluxed with the appropriate hydrazine for 2 h. The pyr-
idazines which separated after concentration of the reac-
tion mixture were filtered, washed with cold ethanol, and
recrystallized from ethanol as needles.
2a: IR (KBr, nmax, cm-1) 1662 (CO), 3285 (NH); lH
NMR (DMSO-d6) d 2.49 (s, 3H, CH3), 2.58 (s, 3H, CH3),
12.90 (s, 1H, NH); MS m/z (relative intensity) 165 (M+,
100), 137 (M-CO, 30), 123 (M-NCO, 25), 109 (22), 94 (4),
81 (8), 66 (18) 50(12), 43 (8).
1-Substituted-carbamoyl-3,6-dimethyl-7-oxoisoxazolo-
[4,5-d]pyridazines (5a-c)
A mixture of the pyridazine derivative 2a (0.91 g,
0.005 mol), anhydrous K2CO3 (0.01 mol) in dry acetone
(50 mL) was stirred. A solution of the appropriate isocya-
nate (0.005 mol) in dry acetone (5 mL) was added dropwise
with a dropping funnel while stirring the reaction mixture.
After addition, the reaction mixture was stirred and re-
fluxed for 16 h. The reaction mixture was cooled to room
temperature and acetone was removed under reduced pres-
sure. The solid residue obtained after the evaporation of the
solvent was dissolved in H2O. The crude product was sepa-
2b: IR (KBr, nmax, cm-1) 1665 (CO), 3337 and 3200
(NH2); lH NMR (DMSO-d6) d 2.35 (s, 3H, CH3), 2.44 (s,
3H, CH3), 7.39-8.20 (m, 6H, Ar + NH2). MS m/z (relative

196 J. Chin. Chem. Soc., Vol. 58, No. 2, 2011
Faidallah et al.
rated upon acidification with dilute HCl, which was filtered
and recrystallized from ethanol.
which was separated during stirring was filtered and crys-
tallized from methanol as needles; IR (KBr, nmax, cm-1)
1
3192 and 3342 (NHNH2), H NMR (DMSO-d6) d 8.65-
5a: IR (KBr, nmax, cm-1) 1654 (CO), 1668 (CO), 3289
l
(NH); H NMR (DMSO-d6) d 0.99-1.92 (m, 11H, cyclo-
9.25 (broad s, 3H, NH and NH2), 2.39 (s, 3H, CH3), 2.68 (s,
3H, CH3).
hexyl H), d 2.46 (s, 3H, CH3), 2.56 (s, 3H, CH3), 10.23 (s,
1H, NH).
3,6-Dimethylisoxazolo[4,5-d]pyridazine-7-thiol (9)
Asolution of 7 (0.84 g, 0.005 mol) in acetone (25 mL)
was refluxed with thiourea (0.76 g, 0.010 mol) for 1 h. The
reaction was then cooled to 0 °C. A solution of 0.5 M
Na2CO3 (10 mL) was added slowly while keeping the tem-
perature below 5 °C. The mixture was stirred overnight.
The resulting reaction mixture was neutralized with 5%
HCl (v/v). The precipitated product was collected, washed
with water, and recrystallized from DMSO as pale yellow
needles; IR (KBr, nmax, cm-1) 2610 (SH); 1H NMR (DMSO-
d6) d 2.54 (s, 3H, CH3), 2.60 (s, 3H, CH3).
5b: IR (KBr, nmax, cm-1) 1656 (CO), 1670 (CO), 3275
(NH); lH NMR (DMSO-d6) d 2.42 (s, 3H, CH3), 2.58 (s,
3H, CH3), 7.11-7.35 (m, 5H, C6H5), 9.88 (s, 1H, NH).
5c: IR (KBr, nmax, cm-1) 1655 (CO), 1672 (CO), 3282
(NH); lH NMR (DMSO-d6) d 2.39 (s, 3H, CH3), 2.50 (s,
3H, CH3), 6.98-7.30 (m, 4H, ArH), 9.85 (s, 1H, NH).
1-Substituted-thiocarbamoyl-3,6-dimethyl-7-oxoisoxa-
zolo[4,5-d]pyridazines (6a-c)
A mixture of the pyridazine derivative 2a (0.91 g,
0.005 mol), anhydrous K2CO3 (0.01 mol) in dry acetone
(50 mL) was stirred. A solution of the appropriate isocya-
nate (0.005 mol) in dry acetone (5 mL) was added dropwise
with a dropping funnel. After stirring and refluxing the
mixture for 10 h, acetone was removed under reduced pres-
sure. The resulting solid residue was dissolved in water and
acidified with dilute HCl. The precipitated crude product
was filtered and recrystallized from ethanol.
6a: IR (KBr, nmax, cm-1) 1655 (CO), 1060 (CS), 3290
(NH); lH NMR (DMSO-d6) d 2.42 (s, 3H, CH3), 2.54 (s,
3H, CH3), 7.12-7.35 (m, 5H, C6H5), 10.22 (s, 1H, NH).
6b: IR (KBr, nmax, cm-1) 1657 (CO), 1150 (CS), 3268
(NH); lH NMR (DMSO-d6) d 2.41 (s, 3H, CH3), 2.49 (s,
3H, CH3), 7.00-7.45 (m, 4H, ArH), 9.88 (s, 1H, NH).
6c: IR (KBr, nmax, cm-1) 1658 (CO), 1078 (CS), 3288
(NH).
Bis(3,6-dimthylisoxazolo[4,5-d]pyridazin-7-yl)disulfide
(10)
To a suspension of 9 (0.36 g, 0.002 mol) and NaHSO4
(2 g) in water (15 mL), NaNO2 (2 g) was gradually added
(30 min) with stirring at 20 °C. The disulfide which sepa-
rated was recrystallized from DMSO as pale yellow nee-
dles; 1H NMR (DMSO-d6) d 2.54 (s, 3H, CH3), 2.70 (s, 3H,
CH3).
3-Substituted-6,9-dimethylisoxazolo[4,5-d]-1,2,4-tri-
azolo[4,3-b]-pyridazines (12) and (13)
The hydrazino derivative 8 (0.45 g, 0.0025 mol) was
heated on a water bath with the appropriate acid chloride
(0.003 mol) for 1 h. The acylhydrazides 11 that separated
out in the form of hydrochlorides were recrystallized from
ethanol as needles (Table 1). Cyclization to the triazolo de-
rivatives 12 and 13 was performed by refluxing these acyl-
hydrazides (0.001 mol) in dry pyridine (8 mL) for one hour.
The reaction mixture was then poured onto 10% Na2CO3
solution and the separated solid was recrystallized from
methanol as needles.
11a; IR (KBr, nmax, cm-1) 1668 (CO), 3270 (NH).
11b; IR (KBr, nmax, cm-1) 1562 (CO), 3265 (NH).
12; 1H NMR (DMSO-d6): d 2.48 (s, 3H, CH3), 2.62
(s, 3H, CH3), 2.68 (s, 3H, CH3).
7-Chloro-3,6-dimethylisoxazolo[4,5-d]pyridazine (7)
Compound 2a (0.83 g, 0.005 mol) was suspended in
phosphorus oxychloride (10 mL) and the mixture was
refluxed for 1 h. The excess of phosphorus oxychloride
was distilled under reduced pressure and the residual syrup
was poured onto finely crushed ice (~50 g), extracted with
ether and the solvent was removed to give pale yellow solid
of the chloro derivative 7 which was recrystallized from
methanol as needles; 1H NMR (CDCl3) d 2.44 (s, 3H, CH3),
2.60 (s, 3H, CH3).
13; 1H NMR (DMSO-d6): d 2.44 (s, 3H, CH3), 2.80
3,6-Dimethyl-7-hydrazinoisoxazolo[4,5-d]pyridazine
(8)
(s, 3H, CH3), 7.42-7.72 (m, 5H, C6H5).
3-Bromomethyl-6,9-dimethylisoxazolo[4,5-d]-1,2,4-tri-
azolo[4,3-b]-pyridazines (14)
A solution of the 4-chloro derivative 7 (0.84 g, 0.005
mol) and 94% hydrazine hydrate (1 g) in absolute ethanol
(50 mL) was stirred at room temperature for 2 h. The solid
A solution of 8 (0.45 g, 0.0025 mol) in ethanol (20
mL) was refluxed with ethyl bromoacetate (0.43 g, 0.0026

Fused Isoxazolo[4,5-d]pyridazines
J. Chin. Chem. Soc., Vol. 58, No. 2, 2011 197
mol) for 2 h. The reaction mixture was then concentrated to
yield white solid which was recrystallized from ethanol as
needles; 1H NMR (DMSO-d6) d 2.42 (s, 3H, CH3), 2.88 (s,
3H, CH3), 4.78 (s, 2H, CH2).
CH3), 2.70 (s, 3H, CH3).
BIOLOGICAL TESTING
Compounds 2-8, 12-16 and 17 were screened for their
antibacterial and antifungal activity following agar-diffu-
sion method32 using Gram-positive bacteria Staphylococ-
cus aureus and Gram-negative bacteria Escherichia coli.
The antifungal testing was carried out against Candida
albicans.
Ethyl 6,9-dimethylisoxazolo[4,5-d]-1,2,4-triazolo[4,3-
b]pyridazine-3-carboxylate (15)
A mixture of 8 (0.45 g, 0.0025 mol) and excess of
ethyl oxalate (10 mL) was refluxed for 3 h. The reaction
mixture was cooled to room temperature. A solid was sepa-
rated which was collected and recrystallized from DMF as
pale yellow needles; IR (KBr, nmax, cm-1) 1725 (CO); 1H
NMR (DMSO-d6) d 1.45 (t, J = 6 Hz, 3H, CH3), 2.50 (s, 3H,
CH3), 2.70 (s, 3H, CH3), 4.42 (q, J = 6 Hz, 2H, CH2).
7,10-Dimethylisoxazolo[4,5-d]pyridazino[2,3-c]-2H-
l,2,4-triazine-3,4-dione (16)
ACKNOWLEDGMENT
The authors wish to thank Department of Biology
Faculty of Science, University of Alexandria for their help-
ful contribution and for facilities.
A mixture of 8 (0.45 g, 0.0025 mol), sodium acetate
(1.0 g) and ethyl oxalate (0.38 g, 0.0027 mol) in ethanol (30
mL) was refluxed for 8 h. The reaction mixture was then
poured into ice-cold water. The precipitated solid was
recrystallized from DMF as needles; IR (KBr, nmax, cm-1)
1692 (CO), 1740 (CO), 3182 (NH); 1H NMR (DMSO-d6) d
2.52 (s, 3H, CH3), 2.64 (s, 3H, CH3), 8.48 (broad s, 1H,
NH).
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