Synthesis of 1,2,4-triazolopyridazines, isoxazolofuropyridazines, and tetrazolopyridazines as antimicrobial agents

Article abstract of DOI:10.1002/jhet.4065

Through current and previous researches, it was found that the derivatives of pyridazine, isoxazole, tetrazole, quinazoline, hydrazinyl, and 1,2,4-triazole have many pharmacological activities. Thus, a series of novel furopyridazinones (7), isoxazolopyridazine (8), sub-benzylidene-furopyridazinones (9a-c), isoxazolofuropyridazines (10a-c), 3-chloro-(pyridin-4-ylmethylene)-dihydropyridazines (11), tetrazolopyridazines (12), pyridazinoquinazolinones (13), piperazinyl/morpholino-pyridazines (14a,b), hydrazinyl-pyridazines (15), and 1,2,4-triazolo-pyridazines (16a,b) in good yields (72%-90%) were synthesized from substituted ethyl 4-oxo-4-phenylbutanoate (2), 6-phenyl-4,5-dihydropyridazinone (3), and 6-phenyl-4-(pyridin-4-ylmethylene)-4,5-dihydropyridazinone (4) as beginning materials. All the chemical structures of the new compounds have been demonstrated by different spectroscopy analyses such as infrared, NMR, mass spectrum, and elemental analysis. Also, the activities of the newly prepared compounds were tested against many types of bacteria and fungi in vitro. Hence, 1,2,4-triazolopyridazines (16a,b), isoxazolofuropyridazines (10a-c), tetrazolopyridazines (12), Piperazinyl/morpholinyl-pyridazines (14a,b) displayed the most efficient antimicrobial activities compared with the cefotaxime sodium and nystatin as standard drugs.

Full text of DOI:10.1002/jhet.4065

Received: 3 April 2020
DOI: 10.1002/jhet.4065
Revised: 12 May 2020
Accepted: 10 June 2020
A R T I C L E
Synthesis of 1,2,4-triazolopyridazines,
isoxazolofuropyridazines, and tetrazolopyridazines
as antimicrobial agents
Ameen A. Abu-Hashem1,2
| Usama Fathy3
| Moustafa A. Gouda4,5
1Photochemistry Department
(Heterocyclic Unit), National Research
Centre, Giza, Egypt
2Chemistry Department, Faculty of
Science, Jazan University, Jazan,
Saudi Arabia
3Applied Organic Chemistry Department,
National Research Centre, Giza, Egypt
4Department of Chemistry, Faculty of
Science and Arts, Taibah University, Ulla,
Saudi Arabia
Abstract
Through current and previous researches, it was found that the derivatives
of pyridazine, isoxazole, tetrazole, quinazoline, hydrazinyl, and 1,2,4-
triazole have many pharmacological activities. Thus, a series of novel furo-
pyridazinones (7), isoxazolopyridazine (8), sub-benzylidene-furopyridazinones
(9a-c), isoxazolofuropyridazines (10a-c), 3-chloro-(pyridin-4-ylmethylene)-
dihydropyridazines (11), tetrazolopyridazines (12), pyridazinoquinazolinones
(13), piperazinyl/morpholino-pyridazines (14a,b), hydrazinyl-pyridazines
(15), and 1,2,4-triazolo-pyridazines (16a,b) in good yields (72%-90%) were
synthesized from substituted ethyl 4-oxo-4-phenylbutanoate (2), 6-phenyl-4,5-
dihydropyridazinone (3), and 6-phenyl-4-(pyridin-4-ylmethylene)-4,5-dihydro-
pyridazinone (4) as beginning materials. All the chemical structures of
the new compounds have been demonstrated by different spectroscopy ana-
lyses such as infrared, NMR, mass spectrum, and elemental analysis. Also, the
activities of the newly prepared compounds were tested against many types
of bacteria and fungi in vitro. Hence, 1,2,4-triazolopyridazines (16a,b),
isoxazolofuropyridazines (10a-c), tetrazolopyridazines (12), Piperazinyl/
morpholinyl-pyridazines (14a,b) displayed the most efficient antimicrobial
activities compared with the cefotaxime sodium and nystatin as standard
drugs.
5Department of Chemistry, Faculty of
Science, Mansoura University, Mansoura,
Egypt
Correspondence
Ameen A. Abu-Hashem, Photochemistry
Department (Heterocyclic Unit), National
Research Centre, Dokki, Giza 12622,
Egypt.
Email: aminaliabuhashem@yahoo.com
1 | INTRODUCTION
Azolopyridazine derivatives showed pharmaceutical and
biological activity such as anxiolytic activity without
Lately, the biological, pharmacological, and chemical
aspects of pyridazinones were discussed in many
reviews.[1,2] The pyridazinones are especially a significant
class of compounds due mainly to their many biological
activities.[3] Also, the pyridazinone and pyridazine
derivatives have possessed different types of biological
and pharmacological activities such as antimicrobial,[4]
antihypertensive,[5] anti-tubercular,[6] anti-secretory,
anti-ulcer, antihistamine,[7] analgesic activity,[8] anti-
feedant, herbicidal, and molluscicidal activities,[9] treat of
cardiovascular,[10] anticonvulsant,[11,12] and antiepileptic.[13]
the side effects. Thus, 1,2,4-triazolo-pyridazine deriva-
tives are used as a sedative and anticonvulsant[14–16]
and lowered blood pressure with no effects on the heart
rate.[17] Moreover, pyridazino-pyrazino-pyrimidine deriv-
atives are used as anti-inflammatory and analgesic activ-
ity.[18] Furthermore, a series imidazo [1,5-b] pyridazine
derivatives have used as inhibited activity against the
reverse transcriptase of HIV-1 (RT) with their efficiency
to inhibit the growth of infected (MT-4) cells.[19]
Also, aryl-pyridazinone derivatives have possessed
many pharmacological properties such as anticancer,[20]
J Heterocyclic Chem. 2020;1–14.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC.
1

2
ABU-HASHEM ET AL.
antiplatelet activity,[21] and antimicrobial.[4,22] In special,
indolylpyridazinone derivatives showed as antimicrobial
agents,[23] inflammation inhibitors,[24] antipyretics,[25]
antiphlogistics,[26] antihypertensive, and blood platelet
gathering inhibitors with treat of cardiovascular.[27]
Pyridazines are scarce in nature, probably reflecting
the rarity of natural occur hydrazines. Therefore, the
researchers have synthesized many heterocyclic com-
pounds that contain the pyridazine moiety, some of
the pyridazine structures are used as a drug to treat
many diseases such as, cefozopran is a fourth-generation
cephalosporin, which is used as an antibiotic, is a type
of antimicrobial.[4] Also, cadralazine and apresoline or
hydralazine are antihypertensive drugs, used to treat
high blood pressure and heart fiasco,[5] minaprine is an
antidepressant,[28] pipofezine is an anti-depressants, anti-
histamine, and anticholinergic[29] (shown in Figure 1).
6-phenyl-4,5-dihydropyridazin-3 (2H)-one (3)[8,39] was
obtained by treating compound (1) or (2) with hydrazine
hydrate in boiling ethanol. Also, the reaction of compound
(3) with isonicotinaldehyde in methanol containing sodium
hydroxide or dioxane containing piperidine afforded
the corresponding 6-phenyl-4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazin-3(2H)-one (4). The IR spectrum of 2 dis-
played absorption bands at ν 1790, 1750 cm−1 for the two
carbonyl groups. The 1H-NMR spectrum of 2 indicated one
triplet signal at δ 1.30 ppm for the three protons of CH3
and two triplet signal at δ 2.15, 3.70 ppm for the four pro-
tons of (2CH2) and one quartet signal at δ 4.12 ppm for
the two protons of CH2 and multiplet signals at δ 7.55
to 7.92 ppm for the five protons of phenyl. The mass spec-
trum of 2 demonstrated the molecular ion peak at m/z
206 (100%). The infrared spectrum of 4 revealed absorption
bands at ν 3315 cm−1 for the NH group and 1682 cm−1 for
1
Our research group has published
a
lot of
the carbonyl group. The H-NMR spectrum of 4 exposed
scientific research that include many biological and
pharmaceutical activities.[18,30–38] Furthermore, with
the continuation of the research work in this manuscript,
we have prepared many of heterocyclic compounds
such as furopyridazinones, isoxazolopyridazines, tetrazolo-
pyridazines, pyridazinoquinazolinones, piperazinyl and
one singlet signal at δ 10.93 ppm for the one proton of NH
(D2O exchangeable) and the mass spectrum of 4 showed
the molecular ion peak at m/z 263 (100%) (Scheme 1).
Moreover, the reaction of 6-phenyl-4,5-dihydro-
pyridazin-3(2H)-one (3) with ethyl bromo-acetate[39] or
chloroacetic acid under reflux in dry acetone and anhy-
drous potassium carbonate with the control by (TLC)
afforded the compounds ethyl 2-([6-phenyl-4,5-dihydro-
pyridazin-3-yl] oxy) acetate (5) and 2-([6-phenyl-
4,5-dihydropyridazin-3-yl] oxy) acetic acid (6) in good
yields, respectively. The IR spectrum of compound 6 indi-
cated the presence of broadband absorption at 3355 cm−1
of one OH group, 1755 cm−1 of the acid carbonyl group.
morpholinopyridazines,
hydrazinylpyridazines,
and
1,2,4-triazolopyridazines with studying their activities as
antimicrobials in vitro.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
1
The H-NMR spectrums of 6 displayed one singlet broad
signal at 12.55 ppm corresponding to one proton of one
OH group (with D2O exchangeable). Also, condensation
of (5) or (6) in dimethylformamide and anhydrous potas-
sium carbonate with refluxing for 12 to 16 hours (long
time) under control (TLC) produced 3-phenyl-4,4a-
Esterification of 3-benzoylpropionic acid (1)[8] by using
refluxed absolute ethanol (40 mL) containing a few
drops of the concentrated sulphuric acid solution with
3-benzoylpropionic acid (1) yielded ethyl 4-oxo-4-
phenylbutanoate (2) in 75% yield. On the other hand,
1
dihydrofuro[2,3-c] pyridazin-5(6H)-one (7). The H-NMR
spectrums of 7 revealed three doublet signals at 2.43, 2.70
and 4.60 ppm corresponding to the four protons of the
FIGURE 1 The chemical structure of many drugs that contain
SCHEME 1 Synthesis of 6-phenyl-4-(pyridin-4-ylmethylene)-
pyridazine derivatives
pyridazinone derivatives

ABU-HASHEM ET AL.
3
two CH2 and one triplet signals at 5.85 ppm conforming
to the one proton of the CH-furanone ring. The mass
spectra of 5, 6, and 7 showed molecular ion peaks at m/z
260 (M+, 100%), 232 (M+, 100%), and 214 (M+, 100%),
respectively (Scheme 2).
the isoxazole and a broad singlet signal at 11.20 ppm for
one proton of NH group (with D2O exchangeable). The
mass spectra of 9a, 9b, 9c, 10a, 10b, and 10c revealed
molecular ion peaks at m/z 302 (M+, 85%), 336 (M+,
92%), 332 (M+, 91%), 317 (M+, 88%), 351 (M+, 90%),
and 347 (M+, 92%), respectively. All spectroscopy data of
new compounds are described in the experimental part
(Scheme 3).
Also, by using cycloaddition reaction, reacted of
6-phenyl-4-(pyridin-4-ylmethylene)-4,5-dihydro pyridazin-3
(2H)-one (4) with hydroxylamine hydrochloride in glacial
acetic acid and anhydrous sodium acetate under stirring
and refluxing to give 5-phenyl-3-(pyridin-4-yl)-2,3,3a,
4-tetrahydroisoxazolo [5, 4-c] pyridazine (8). The infrared
spectrum of compound 8 displayed the presence of broad-
band absorption at 3325 cm−1 of one NH group and the
1H-NMR spectrum of 8 showed one singlet broad signals
at 11.10 ppm identically to the one proton of the one
NH group (with D2O exchangeable). Likewise, stirring
and refluxing of 3-phenyl-4,4a-dihydrofuro[2,3-c] pyridazin-
5(6H)-one (7) with an appropriate aromatic aldehyde,
namely, benzaldehyde, 4-chlorobenzaldehyde or 4-meth-
oxybenzaldehyde, respectively, and anhydrous sodium
acetate in glacial acetic acid with acetic anhydride for
7 to 10 hours gave the products 6-(4-substituted-
benzylidene)-3-phenyl-4,4a-dihydrofuro [2,3-c] pyridazin-5
(6H)-one (9a-c) in good yields. Additionally, the same com-
pounds (9a-c) reacted with hydroxylamine hydrochloride
in glacial acetic acid and anhydrous sodium acetate by
using cycloaddition reaction to yield 3-(4-substituted-
phenyl)-7-phenyl-2,3,8,8a-tetrahydroisoxazolo[50,40:4,5]furo
[2,3-c] pyridazine (10a-c), hence the formation of com-
pounds 10a-c of compounds 9a-c by position 1, 4-the addi-
tion of hydroxylamine on the methylenic double bond
followed the exit of the water.[18] The 1H-NMR spectrum of
9a exhibited one doublet signal at 2.66 ppm, J = 5.01 Hz
corresponding to the two protons of the one CH2 group
and one singlet at 8.06 ppm conforming to the one methine
Recently, it is well known in pyridazinones chemistry
that position number of three in the pyridazinone ring
and fused pyridazinone displayed distinct activities toward
nucleophiles. Therefore, 3-chloro-6-phenyl-4-(pyridin-4-
ylmethylene)-4,5-dihydropyridazine (11) was synthesized
from the reaction of (4) with phosphorus oxychloride
(POCl3)[36] or phosphorus pentachloride (PCl5) and then
heat and reflux on a steam bath for 8 to 12 hours. The IR
spectrum of 11 exposed the absence of NH and carbonyl
groups, and the 1H-NMR spectrum of 11 showed the
absence of NH group, while the two singlet signals at 2.90
and 8.10 for corresponding to the two protons of (CH2)
and one proton of (CH) methine. The compound 11 is
activity to nucleophiles such as primary aromatic amines,
secondary amines, and hydrazine hydrates were inspected.
Moreover, heating and refluxing of compound 11 with
sodium azide in dimethylformamide and water afforded
6-phenyl-8-(pyridin-4-ylmethylene)-7, 8-dihydrotetrazolo
[1,5-b] pyridazine (12). Also, compound 11 reacted with
anthranilic acid in dimethylformamide or in dry benzene
and refluxed for 8 to 10 hours under control (TLC)
to give 2-phenyl-4-(pyridin-4-ylmethylene)-3,4-dihydro-
10H-pyridazino[6,1-b]quinazolin-10-one (13) in high
yield. Likewise, heating the latter compound 11 under
reflux with secondary amines such as piperazine or mor-
pholine in glacial acetic acid or absolute methanol gave
6-phenyl-3-(piperazin-1-yl)-4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (14a) and 4-(6-phenyl-4-(pyridin-4-
ylmethylene)-4,5-dihydropyridazin-3-yl) morpholine (14b).
The IR spectrum of 14a indicated the presence of a
broadband absorption at 3375 cm−1 of (NH) group and
1H-NMR spectrum of 14a proved one singlet broad
signal at δ 10.20 ppm corresponding to the one proton
of the (NH) group (with D2O exchangeable). Similarly,
the 3-hydrazinyl-6-phenyl-4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (15) was synthesized from 11 and
hydrazine hydrate by heating in dry dioxane. The IR of
15 showed a broadband absorption at 3435 to 3355 cm−1
of (NH–NH2) groups and 1H-NMR of 15 proved two
singlet signals at δ 6.70, 10.90 ppm agreement to the
two and one protons of the (NH2) and (NH) groups
(with D2O exchangeable), respectively. Also, compound
15 reacted with formic and acetic acid to yield the 3-
substituted-6-phenyl-8-(pyridin-4-ylmethylene)-7,8-dihydro-
[1,2,4]triazolo[4,3-b]pyridazine (16a,b). Besides, the 13C-NMR
1
proton (CH). The H-NMR spectrum of 10a indicated a
doublet signal at δ 5.05 ppm conforming to one proton of
SCHEME 2 Synthesis of 3-phenyl-furo [2,3-c] pyridazin-5
(6H)-one derivatives

4
ABU-HASHEM ET AL.
SCHEME 3 Synthesis of
substituted-isoxazolo[50,40:4,5]
furo[2,3-c]pyridazine derivatives
SCHEME 4 Synthesis of
quinazoline, piperazinyl,
morpholinyl, hydrazinyl, and
1,2,4-triazolopyridazine
derivatives
spectrum of 16b revealed absorption signals at δ 18.5 ppm,
conforming to one carbon atom of the methyl group,
33.4 ppm, corresponding to one carbon atom of (CH2)
group, 137.8 ppm, corresponding to one carbon atom
of (CH, methine), and 124.2, 128.1, 128.9, 131.2, 135.2,
141.4, 143.5, 144.7, 149.9, 150.6, and 163.8 ppm due
to 15 sp2 carbons atom of the aromatic carbons. The MS of
11, 12, 13, 14a, 14b, 15, 16a, and 16b revealed molecular ion
peaks at m/z 281 (M+, 100%), 288 (M+, 85%), 364 (M+,
80%), 331 (M+, 92%), 332 (M+, 82%), 277 (M+, 100%),
287 (M+, 90%), and 301 (M+, 95%), respectively. The spec-
troscopy (IR, NMR, and Mass) and elemental analysis of
new compounds were illustrated in the Experimental
Section (Scheme 4).

ABU-HASHEM ET AL.
5
3 | BIOLOGICAL ACTIVITIES
3.1 | Antimicrobial activity
pneumoniae. The MIC values in μmol mL−1 to the com-
pounds were as the follows, 16a,b (1-5), 10 a-c (2-8), 12
(5-8) and 14a,b (6-10). Also, other compounds as 13, 15,
8, and 9a-c showed moderate antimicrobial activities.
Also, the compounds 10a-c, 12, 16a,b, and 14a,b gave a
potent antifungal activity with MIC in μmol /cm3 of
10a-c (2-7), 12 (5-8), 16a,b (6-10), and 14a,b (8-12)
when compared with the positive control, nystatin
(MIC 3-6 μmol mL−1). Other compounds showed mod-
erate antifungal activity when compared with nystatin
(MIC 3-6 μmol mL−1): 13 (10-13), 15 (11-14), 8 (12-15),
and 9a-c (13-18). The tested fungi were Aspergillus
niger, Alternaria alternata, Curvularia lunata, and
Candida albicans.
The new compounds were investigated in vitro for
microbial activity (bacteria and fungi) and all the
results were presented in Tables 1 and 2. Many of the
compounds were shown the best antimicrobial activity
when compared with cefotaxime sodium (MIC =
2-5 μmol mL−1). The new compounds 16a,b, 10a-c, 12,
and 14a,b evidenced the better efficient as an antimi-
crobial activity to against gram-positive bacteria;
Staphylococcus aureus, Streptococcus pyogenes, and
gram-negative bacteria; Escherichia coli and Klebsiella
TABLE 1 Minimum inhibitory concentrations against bacteria of the new compounds
MIC (μmol mL−1
)
Microorganisms
Gram-positive bacteria
Gram-negative bacteria
Klebsiella pneumoniae
Compounds
Staphylococcus aureus
Streptococcus pyogenes
E. coli
22
21
19
18
16
17
15
10
13
11
12
6
1
24
23
21
20
18
19
17
12
15
13
14
8
23
22
20
19
17
18
16
11
14
12
13
7
21
20
18
17
15
16
14
9
2
3
4
5
6
7
8
9a
9b
9c
10a
10b
10c
11
12
13
14a
14b
15
16a
16b
12
10
11
4
6
5
4
2
7
6
5
3
16
8
15
7
14
7
13
5
11
9
10
8
9
8
7
6
10
11
5
9
8
7
10
4
10
3
9
1
4
3
2
1
Cefotaxime sodium
Negative control
5
4
3
2
NI
NI
NI
NI
Note: DMSO was used as the negative control and as the solvent for test compounds and the reference drug.
Abbreviation: NI, no inhibition.

6
ABU-HASHEM ET AL.
TABLE 2 Minimum inhibitory concentrations against fungi of the new compounds
MIC (μmol mL−1
)
Microorganisms
Compounds
Aspergillus niger
Alternaria alternata
Curvularia lunata
Candida albicans
1
27
26
25
23
21
22
20
15
18
16
17
7
26
25
24
22
20
21
19
14
17
15
16
6
25
24
23
21
19
20
18
13
16
14
15
5
24
23
22
20
18
19
17
12
15
13
14
4
2
3
4
5
6
7
8
9a
9b
9c
10a
10b
5
4
3
2
10c
6
5
4
3
11
19
8
18
7
17
6
16
5
12
13
13
11
12
14
10
9
12
10
11
13
9
11
9
10
8
14a
14b
10
12
8
9
15
11
7
16a
16b
8
7
6
Nystatin
6
5
4
3
Negative control
NI
NI
NI
NI
Note: DMSO was used as the negative control and as the solvent for test compounds and the reference drug.
Abbreviation: NI, no inhibition.
3.2 | Structure-activity relationships
of the transformation of the (NH–NH2) group into
1,2,4-triazole ring linkage with numerous functional
groups and other of rings such as isoxazole, tetrazole,
and furo-pyridazine. Hence, pyridazine link with many
functional groups such as pyridine, phenyl, methoxy,
methyl, ethyl, hydroxy, chloro, triazole, isoxazole, tetrazole,
and quinazolinone moieties. Also, some of the compounds
13, 15, 8, and 9a-c exhibited mild antimicrobial activity and
the rest of the compounds showed a weak antimicrobial
activity compared with the standard drugs (cefotaxime
sodium and nystatin). Also, the pyridazines, triazole,
1,2,4-triazolopyrimidines, furopyrimidine, quinazolinone,
tetrazole, piperazinyl, morpholinyl, thiadiazole, and spiro-
pyrimidine derivatives have efficient as antimicrobial
agents in previous articles.[4,30,31,35,37] In this
In the study of the relationship among chemical struc-
tures and microbial activity (Structure-activity relation-
ships [SARs]) of the synthesized compounds, the next
conclusion was obtained, first maybe: very good antimi-
crobial activity were found to compounds; 1,2,4-triazolo-
pyridazine (16a,b), isoxazolo-furo-pyridazine (10a-c),
tetrazolo-pyridazine (12), piperazinyl or morpholinyl-
pyridazine (14a,b) derivatives against the test microor-
ganisms, because they contain 1,2,4-triazole ring as an
essential nucleus with an increasing number of nitrogen
atoms according to past and new scientific articles.[30,31]
Second, maybe the same compounds 16a, b, 10a-c, 12,
and 14a-b possess highest antimicrobial activities because

ABU-HASHEM ET AL.
7
manuscript and previous articles generally, the bacte-
ria and fungi types are influenced through this cate-
gory of heterocyclic compounds.[30,31,35,37] Moreover,
past results and our findings in this article corroborate
the promising antimicrobial activity of 1,2,4-triazolo-
pyridazine, isoxazolofuropyridazine, tetrazolopyridazine,
and piperazinyl or morpholinyl-pyridazine derivatives
which can be developed to the highest antimicrobial
activities.
(CH alkyl), 1790 (C O), 1750 (C O), 1570 (C C).
1H-NMR (DMSO-d6, ppm) δ 1.30 (t, 3H, J = 4.20 Hz,
CH3), 2.15 (t, 2H, J = 4.25 Hz, CH2), 3.70 (t, 2H,
J = 4.27 Hz, CH2), 4.12 (q, 2H, J = 4.29 Hz, CH2), 7.55 to
7.92 (m, 5H, phenyl); 13C NMR (DMSO-d6) δ 20.1 (1C,
CH3), 30.5, 34.6, 63.4 (3C, 3CH2), 128.1, 128.5, 130.9,
134.8 (6C, Ar–C), 168.7, 183.5 (2C, 2C O), MS (70 eV, %)
m/z 206 (M+, 100%); Anal. Calc. (Found) for C12H14O3
(206.24): C, 69.89 (69.80), H, 6.84 (6.78).
4 | EXPERIMENTAL
4.1 | Materials
4.4 | Synthesis of
6-phenyl-4,5-dihydropyridazin-3(2H)-
one (3)
All materials used were obtained from Sigma Aldrich
(Saint Lewis, USA).
To a mix of 4-oxo-4-phenylbutanoic acid (1) (1.78 g,
0.01 mol) or ethyl 4-oxo-4-phenyl butanoate (2) (2.06 g,
0.01 mol) in 30 mL ethanol, (2 mL) of hydrazine hydrate
(80%) was added.[39] The reaction mixture was heated under
reflux for 8 to 15 hours, after completion of the reaction
(TLC monitoring). The precipitate obtained after cooling
was filtered off and crystallized from methanol in 90% yield.
4.2 | Equipments
All melting points are in degree centigrade (uncorrected)
and were determined on Gallenkamp electric melting
point apparatus. TLC analysis was carried out on silica gel
60 F254 precoated aluminum sheets. The IR spectra were
recorded (KBr) on a Perkin-Elmer 1430 spectrometer
4.5 | Synthesis of 6-phenyl-4-(pyridin-
4-ylmethylene)-4,5-dihydropyridazin-3
(2H)-one (4)
1
(λ, cm−1) in National Research Centre, Egypt. H and 13C-
NMR Spectra were measured on JEOL-ECA 500 and JEOL
JNM-LA-400 FT NMR Spectrometers at 500, 125 MHz,
respectively, using tetramethylsilane (TMS) as an internal
reference and DMSO-d6 as the solvent at the Microana-
lytical Center in National Research Centre, Egypt. The
mass spectra (EI) were recorded on GCMS-QP 1000 EX
(Shimadzu) at National Research Centre, Egypt. Elemen-
tal analyses (C, H, and N) were carried out at the Micro-
analytical Center in National Research Centre, Egypt.
The elemental analyses were found to agree favorably
with the calculated values. Biological evaluations were
done by the antimicrobial unit, Department of Chemistry
of Natural and Microbial Products, National Research
Centre, Egypt.
Method A: A mixture of pyridazinone (3) (1.74 g,
0.01 mol) and isonicotinaldehyde (1.07 g, 0.01 mol) in
methanol containing sodium hydroxide (0.4 g, 0.01 mol)
was refluxed for 12 to 15 hours. The reaction mixture
was cooled by poured into ice-cold water, then filtered
and recrystallized to get the yellowish crystals. Method
B: A solution of pyridazinone (3) (1.74 g, 0.01 mol) and
isonicotinaldehyde (1.07 g, 0.01 mol) in dioxane (40 mL)
containing piperidine (0.5 mL) as a catalyst, was stirred
and heated under reflux for 6 to 8 hours. The reaction
mixture was cooled, the formed precipitate filtered off,
dried and recrystallized from benzene in 85% yield
as yellowish crystals, m. p. 270ꢀC to 272ꢀC; IR (KBr):
ν
= 3315 (NH), 3058 (CH aryl), 2952 (CH alkyl),
max
1682 (C O), 1632 (C N), 1578 (C C) cm−1; H-NMR
(DMSO-d6): δ = 2.88 (s, 2H, CH2), 7.35 to 7.41 (m, 3H,
phenyl), 7.44 to 7.50 (dd, 2H, J = 5.05 Hz, pyridine),
7.71 to 7.77 (dd, 2 H, J = 5.06 Hz, phenyl), 7.80 to 7.86
(dd, 2H, J = 5.08 Hz, pyridine), 8.05 (s, 1H, CH,
methine), 10.93 (s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6) δ = 32.5 (1C, CH2), 138.3 (1C, CH,
methine), 124.1, 128.4129.1, 131.4, 134.2, 136.1, 141.5,
144.7, 149.9 (13C, Ar–C), 166.5 (1C, 1C O); MS (70 eV): m/
z = 263 (M+, 100%); Anal. Calc. (Found) for C16H13N3O
(263.30): C, 72.99 (72.91); H, 4.98 (4.92); N, 15.96 (15.88).
1
4.3 | Synthesis of ethyl 4-oxo-
4-phenylbutanoate (2)
A mixture of 3-benzoylpropionic acid 1 (1.78 g, 0.01 mol)
in absolute ethanol (40 mL) containing a few drops of
concentrated sulphuric acid was refluxed for 3 to 5 hours
under control (TLC). The oil formed was separated
by using a rotary system and the purification by chloro-
form and sodium sulphate to give compound 2 as a liquid
oil in 75% yield, IR (ν, cm−1): 3055 (CH aryl), 2960

8
ABU-HASHEM ET AL.
4.6 | Synthesis of ethyl 2-([6-phenyl-4,5-
dihydropyridazin-3-yl] oxy) acetate (5) and
2-([6-phenyl-4,5-dihydropyridazin-3-yl] oxy)
acetic acid (6)
cm−1; 1H-NMR (DMSO-d6): δ = 2.41 to 2.44 (t, 2H,
J = 5.01 Hz, CH2), 2.91 to 2.94 (t, 2H, J = 5.03 Hz, CH2),
4.43 (s, 2H, CH2), 7.38 to 7.44 (m, 3H, phenyl), 7.77 (d,
1H, J = 5.03 Hz, phenyl), 7.78 (d, 1H, J = 5.04 Hz, phe-
nyl), 12.55 (br, 1H, OH, D2O exchangeable). 13C NMR
(DMSO-d6) δ = 21.3, 25.4, 60.1 (3C, 3CH2), 128.1, 128.7,
132.2, 137.3, 160.7, 162.9 (8C, Ar–C), 171.5 (1C, 1C O);
MS (70 eV): m/z = 232 (M+, 100%); Anal. Calc. (Found)
for C12H12N2O3 (232.24): C, 62.06 (62.12); H, 5.21
(5.28); N, 12.06 (12.15).
General procedure: Method A: A mix of compound
3 (1.74 g, 0.01 mol), (ethyl bromoacetate, 0.01 mol or
chloroacetic acid 0.94 g, 0.01 mol) and anhydrous potas-
sium carbonate (0.015 mol) in dry acetone (30 mL) was
refluxed for 4 to 8 hours. The reaction mixture was
allowed to cool to room temperature. Cold water (30 mL)
was added, and then the solid that separated was filtered
off, dried, and crystallized from appropriate solvent
to give 5 and 6, respectively. Method B: A solution of
compound 3 (1.74 g, 0.01 mol), (ethyl bromoacetate,
0.01 mol or chloroacetic acid 0.94 g, 0.01 mol), and
(0.02 mol) anhydrous sodium acetate was stirred under
reflux in (40 mL) of glacial acetic acid and (30 mL) of
acetic anhydride for 6 to 9 hours under (TLC). The reac-
tion solution was cooled and poured into cold water
(100 mL). The product deposited precipitate was filtered
off and crystallized from suitable solvent to yield 5 and 6,
respectively.
4.9 | Synthesis of 3-phenyl-4,4a-
dihydrofuro [2,3-c] pyridazin-5(6H)-one (7)
Method A: A mix of 5 (2.60 g, 0.01 mol) or 6 (2.32 g,
0.01 mol) and anhydrous potassium carbonate (0.015 mol)
was stirred under reflux in dimethylformamide (45 mL) for
12 to 16 hours under control (TLC). The solution was
allowed to cool to room temperature and poured into cold
water (100 mL) and neutralized. The solid formed was fil-
tered off, dried, and crystallized from the proper solvent to
produce 7. Method B: A solution of compound 5 (2.60 g,
0.01 mol) or 6 (2.32 g, 0.01 mol) in dioxane (35 mL) con-
taining a catalyst amount of piperidine (0.5 mL) was stirred
and heated under reflux for 9 to 13 hours (TLC control).
The reaction mixture was cooled, the formed precipitate fil-
tered off, dried, and recrystallized from the methanol to
afford (7) in 80% yield as white crystals, m. p. 220ꢀC to
222ꢀC; IR (KBr): νmax = 3056 (CH aryl), 2950 (CH alkyl),
4.7 | Synthesis of ethyl 2-([6-phenyl-4,5-
dihydropyridazin-3-yl] oxy) acetate (5)
The compound was obtained from the reaction of 3 with
ethyl bromoacetate as white crystals, crystallized from
dioxane in 80% yield, m. p. 170ꢀC to 172ꢀC. IR (KBr):
1
1715 (C O), 1635 (C N), 1584 (C C) cm−1; H NMR
ν
= 3057 (CH aryl), 2952 (CH alkyl), 1780 (C O,
(DMSO-d6): δ = 2.43 (d, 1H, J = 5.08 Hz, CH2), 2.70
(d, 1 H, J = 5.09 Hz, CH2), 4.60 (d, 2H, J = 5.06 Hz, CH2),
5.85 (t, 1H, J = 5.06 Hz, CH-furanone), 7.42 to 7.48 (m, 3H,
phenyl), 7.80 (d, 1H, J = 5.02 Hz, phenyl), 7.82 (d, 1H,
J = 5.03 Hz, phenyl). 13C NMR (DMSO-d6) δ = 22.1 (1C,
CH2), 52.5 (1C, 1CH), 72.8 (1C, CH2), 128.3, 128.9, 132.6,
137.7, 161.5, 163.5 (8C, Ar–C), 190.2 (1C, 1C O); MS
(70 eV): m/z = 214 (M+, 100%); Anal. Calc. (Found) for
C12H10N2O2 (214.22): C, 67.28 (67.22); H, 4.71 (4.65); N,
13.08 (13.14).
max
ester), 1634 (C N), 1580 (C C) cm−1; H-NMR (DMSO-
d6): δ = 1.26 (t, 3H, J = 4.25 Hz, CH3), 2.40 to 2.43 (t, 2H,
J = 5.02 Hz, CH2), 2.90 to 2.93 (t, 2H, J = 5.04 Hz, CH2),
4.25 (q, 2H, J = 4.30 Hz, CH2), 4.40 (s, 2H, CH2), 7.39 to
7.45 (m, 3H, phenyl), 7.75 (d, 1H, J = 5.03 Hz, phenyl),
7.76 (d, 1H, J = 5.04 Hz, phenyl), 13C NMR (DMSO-d6)
δ = 18.4 (1C, CH3), 21.5, 25.7, 56.8, 60.5 (4C, 4CH2),
128.3, 128.9, 132.4, 137.1, 160.5, 162.8 (8C, Ar–C), 170.2
(1C, 1C O); MS (70 eV): m/z = 260 (M+, 100%);
Anal. Calc. (Found) for C14H16N2O3 (260.29): C, 64.60
(64.52); H, 6.20 (6.25); N, 10.76 (10.68).
1
4.10 | Synthesis of 5-phenyl-3-(pyridin-
4-yl)-2,3,3a, 4-tetrahydroisoxazolo [5,4-c]
pyridazine (8)
4.8 | Synthesis of 2-([6-phenyl-4,5-
dihydropyridazin-3-yl] oxy) acetic acid (6)
A mix of 4 (2.63 g, 0.01 mol), hydroxylamine hydrochlo-
ride (0.695 g, 0.01 mol), and anhydrous sodium acetate
(0.82 g, 0.01 mol) was stirred under reflux in glacial acetic
acid (35 mL) for 8 to 10 hours. The solution was allowed
to cool to room temperature and poured into cold water
(100 mL). The final solid product was filtered off, dried,
The compound was obtained from the reaction of 3
with chloroacetic acid as yellowish crystals, crystallized
from ethanol in 83% yield, m. p. 188ꢀC to 190ꢀC. IR
(KBr): ν
= 3355 (br, OH), 3058 (CH aryl), 2953
max
(CH alkyl), 1755 (C O, acid), 1631 (C N), 1581 (C C)

ABU-HASHEM ET AL.
9
and crystallized from dimethylformamide to give 8 in
78% yield as yellowish crystals, m. p. = 290ꢀC to 292ꢀC;
IR (KBr): νmax = 3325 (NH), 3062 (CH aryl), 2954
4.13 | Synthesis of 6-(4-
chlorobenzylidene)-3-phenyl-4,4a-
dihydrofuro [2,3-c] pyridazin-5(6H)-
one (9b)
(CH alkyl), 1630 (C N), 1580 (C C) cm−1 1H-NMR
;
(DMSO-d6): δ = 2.30 (d, 2H, CH2), 4.10 (q, 1H, isoxazolo
proton), 5.15 (t, 1H, isoxazolo proton), 7.32 to 7.38
(m, 3H, phenyl), 7.41 to 7.47 (dd, 2H, J = 5.07 Hz, pyri-
dine), 7.70 to 7.76 (dd, 2H, J = 5.08 Hz, phenyl), 7.79 to
7.85 (dd, 2H, J = 5.05 Hz, pyridine), 11.10 (brs., NH,
isoxazolo proton, D2O exchangeable); 13C NMR (DMSO-
d6) δ = 28.8 (1C, CH2), 50.2, 70.4 (2C, 2CH, isoxazole),
124.5, 128.1128.9, 131.2, 136.8, 144.5, 149.6, 161.8, 164.9
(13C, Ar–C); MS (70 eV): m/z = 278 (M+, 90%); Anal.
Calc. (Found) for C16H14N4O (278.32): C, 69.05
(69.12); H, 5.07 (5.15); N, 20.13 (20.08).
The compound was obtained from the reaction of 7 with
4-chlorobenzaldehyde as yellowish crystals, crystallized
from dioxane in 80% yield, m. p. 330ꢀC to 332ꢀC. IR
(KBr): νmax = 3057 (CH aryl), 2954 (CH alkyl), 1719
(C O), 1632 (C N), 1580 (C C) cm−1
;
1H-NMR
(DMSO-d6): δ = 2.68 (d, 2H, J = 5.05 Hz, CH2), 5.58 (t,
1H, J = 5.06 Hz, CH-furanone), 7.36 to 7.42 (m, 3H, phe-
nyl), 7.60 to 7.66 (dd, 2H, J = 5.01 Hz, 4-chlorophenyl),
7.69 to 7.75 (dd, 2H, J = 5.04 Hz, 4-chlorophenyl), 7.88 to
7.93 (dd, 2H, J = 5.08 Hz, phenyl), 8.08 (s, 1H,CH,
methine).13C NMR (DMSO-d6) δ = 22.8 (1C, CH2), 52.9
(1C, CH), 101.4 (1C, CH), 128.4, 128.6, 128.8, 129.1,
130.7, 131.5, 134.1, 137.3, 162.2, 164.1, 170.5 (15C, Ar–C),
192.7 (1C, 1C O); MS (70 eV): m/z = 336 (M+, 92%);
Anal. Calc. (Found) for C19H13ClN2O2 (336.77): C, 67.76
(67.68); H, 3.89 (3.80); N, 8.32 (8.25).
4.11 | Synthesis of 6-(4-substituted-
benzylidene)-3-phenyl-4,4a-dihydrofuro
[2,3-c] pyridazin-5(6H)-one (9a-c)
General procedure: A mix of compound 7 (2.14 g, 0.01 mol),
aromatic aldehydes namely; benzaldehyde (1.06 g, 0.01 mol),
4-chlorobenzaldehyde (1.40 g, 0.01 mol), or 4-methoxy benz-
aldehyde (1.36 g, 0.01 mol), and (0.01 mol) of anhydrous
sodium acetate was stirred under heated and reflux in a
mixture of glacial acetic acid (40 mL) and acetic anhydride
(20 mL) for 7 to 10 hours. The reaction solution was
allowed to cool to room temperature, poured into cold water
(100 mL). The product precipitate was filtered off, dried,
and crystallized from suitable solvent to give 9a-c,
respectively.
4.14 | Synthesis of 6-(4-
methoxybenzylidene)-3-phenyl-4,4a-
dihydrofuro [2,3-c] pyridazin-5(6H)-
one (9c)
The compound was obtained from the reaction of 7 with
4-methoxybenzaldehyde as white crystals, crystallized from
hexane in 78% yield, m. p. 345ꢀC to 347ꢀC. IR (KBr):
νmax = 3059 (CH aryl), 2957 (CH alkyl), 1717 (C O), 1631
1
(C N), 1581 (C C) cm−1; H-NMR (DMSO-d6): δ = 2.70
(d, 2H, J = 5.04 Hz, CH2), 3.90 (s, 3H, CH3), 5.54 (t, 1H,
4.12 | Synthesis of 6-benzylidene-
3-phenyl-4,4a-dihydrofuro [2,3-c]
pyridazin-5(6H)-one (9a)
J = 5.02 Hz, CH-furanone), 7.10 to 7.16 (dd, 2H,
J = 5.07 Hz, 4-methoxyphenyl), 7.37 to 7.43 (m, 3H, phe-
nyl), 7.65 to 7.71 (dd, 2H, J = 5.03 Hz, 4-methoxyphenyl),
7.90 to 7.95 (dd, 2H, J = 5.06 Hz, phenyl), 8.09 (s, 1H,CH,
methine).13C NMR (DMSO-d6) δ = 22.3 (1C, CH2), 52.6
(1C, CH), 57.1 (1C, OCH3), 101.6 (1C, CH), 122.8, 125.4,
128.3, 128.7, 131.6, 133.1137.2157.5162.7164.5171.2 (15C,
Ar–C), 193.1 (1C, 1C O); MS (70 eV): m/z = 332 (M+,
91%); Anal. Calc. (Found) for C20H16N2O3 (332.36): C,
72.28 (72.35); H, 4.85 (4.75); N, 8.43 (8.50).
The compound was obtained from the reaction of 7
with benzaldehyde as brownish crystals, crystallized from
acetone in 77% yield, m. p. 300ꢀC to 302ꢀC. IR (KBr):
ν
= 3058 (CH aryl), 2951 (CH alkyl), 1718 (C O),
max
1633 (C N), 1582 (C C) cm−1 1H-NMR (DMSO-d6):
;
δ = 2.66 (d, 2 H, J = 5.01 Hz, CH2), 5.55 (t, 1H,
J = 5.04 Hz, CH-furanone), 7.35 to 7.55 (m, 6 H, phenyl),
7.70 to 7.75 (dd, 2H, J = 5.05 Hz, phenyl), 7.85 to 7.90
(dd, 2H,
J
= 5.07 Hz, phenyl), 8.06 (s, 1H,CH,
4.15 | Synthesis of 3-(4-substituted-
phenyl)-7-phenyl-2,3,8,8a-
Methine).13C NMR (DMSO-d6) δ = 22.5 (1C, CH2), 52.8
(1C, CH), 101.2 (1C, CH), 128.1, 128.3, 128.5, 128.9,
130.1, 131.6, 132.8, 135.9, 161.5, 163.8, 170.2 (15C, Ar–C),
192.4 (1C, 1C O); MS (70 eV): m/z = 302 (M+, 85%);
Anal. Calc. (Found) for C19H14N2O2 (302.33): C, 75.48
(75.40); H, 4.67 (4.58); N, 9.27 (9.32).
tetrahydroisoxazolo [50,40:4,5] furo[2,3-c]
pyridazine (10a-c)
A mixture of 9a-c (0.01 mol), respectively, hydroxylamine
hydrochloride (0.695 g, 0.01 mol) and anhydrous sodium

10
ABU-HASHEM ET AL.
acetate (0.82 g, 0.01 mol) was stirred under heat and
reflux in glacial acetic acid (40 mL) for 4 to 8 hours. The
reaction mixture was allowed to cool to room tempera-
ture and poured into ice and cold water (100 mL). The
resulting precipitate was filtered off, dried, and crystal-
lized from proper solvent to give 10a-c.
4.18 | Synthesis of 3-(4-methoxyphenyl)-
7-phenyl-2,3,8,8a-tetrahydroisoxazolo
[50,40:4,5] furo [2,3-c] pyridazine (10c)
The compound was obtained from the reaction of 9c (3.32 g,
0.01 mol) with hydroxylamine hydrochloride as yellowish
crystals, crystallized from dimethyl-formamide in 75% yield,
m. p. >350ꢀC. IR (KBr): νmax = 3332 (NH), 3064 (CH aryl),
1
4.16 | Synthesis of 3,7-diphenyl-2,3,8,8a-
tetrahydroisoxazolo[50,40:4,5]furo[2,3-c]
pyridazine (10a)
2951 (CH alkyl), 1631 (C N), 1582 (C C) cm−1; H NMR
(DMSO-d6): δ = 2.79 (d, 2H, J = 5.10 Hz, CH2), 3.94 (s, 3H,
OCH3), 5.09 (d, 1H, J = 5.07 Hz, isoxazolo proton), 5.61
(t, 1H, J = 5.09 Hz, CH-furan), 7.08 to 7.14 (dd, 2H,
J = 5.14 Hz, 4-methoxyphenyl), 7.16 to 7.22 (dd, 2H,
J = 5.13 Hz, 4-methoxyphenyl), 7.42 to 7.47 (m, 3H, phenyl),
7.87 to 7.93 (dd, 2H, J = 5.15 Hz, phenyl), 11.30 (brs., NH,
isoxazolo proton, D2O exchangeable); 13C NMR (DMSO-d6)
δ = 22.9 (1C, CH2), 33.6 (1C, CH), 53.1 (1C, OCH3), 72.7 (1C,
CH, isoxazole), 122.8, 125.9, 128.4, 128.7, 131.2, 134.4, 136.1,
144.2, 146.4, 155.8, 162.5, 164.7 (16C, Ar–C); MS (70 eV):
m/z = 347 (M+, 92%); Anal. Calc. (Found) for C20H17N3O3
(347.37): C, 69.15 (69.24); H, 4.93 (4.85); N, 12.10 (12.17).
The compound was obtained from the reaction of 9a
(3.02 g, 0.01 mol) with hydroxylamine hydrochloride as
yellowish crystals, crystallized from methanol in 75%
yield, m. p. >350ꢀC. IR (KBr): νmax = 3328 (NH), 3062
(CH aryl), 2950 (CH alkyl), 1630 (C N), 1588 (C C)
1
cm−1; H-NMR (DMSO-d6): δ = 2.74 (d, 2H, J = 5.10 Hz,
CH2), 5.05 (d, 1H, J = 5.03 Hz, isoxazolo proton), 5.60
(t, 1H, J = 5.12 Hz, CH-furan), 7.23 to 7.28 (dd, 2H,
J = 5.15 Hz, phenyl), 7.30 to 7.53 (m, 6H, phenyl), 7.88 to
7.93 (dd, 2H, J = 5.14 Hz, phenyl), 11.20 (brs., NH,
isoxazolo proton, D2O exchangeable); 13C NMR (DMSO-
d6) δ = 22.7 (1C, CH2), 33.6 (1C, CH), 72.1 (1C, CH, iso-
xazole), 126.8, 127.2, 128.3, 128.6, 128.7, 131.7, 136.8,
142.6, 144.2, 146.8, 162.3, 164.1 (16C, Ar–C); MS (70 eV):
m/z = 317 (M+, 88%); Anal. Calc. (Found) for
C19H15N3O2 (317.35): C, 71.91 (71.98); H, 4.76 (4.70); N,
13.24 (13.19).
4.19 | Synthesis of 3-chloro-6-phenyl-
4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (11)
A mix of compound 4 (2.63 g, 0.01 mol) and phosphorous
oxychloride (20 mL, POCl3), or phosphorus pentachloride
(20 mL, PCl5) was heated and refluxed on a steam bath for
8 to 12 hours. After heating, the mixture was cautiously
poured on crushed ice and create alkaline by addition of
sodium bicarbonate. The resulting precipitate was filtered
off, dried, and crystallized from benzene in 81% yield as yel-
lowish crystals, m. p. 233ꢀC to 235ꢀC. IR (KBr): νmax = 3058
(CH aryl), 2950 (CH alkyl), 1632 (C N), 1588 (C C),
4.17 | Synthesis of 3-(4-chlorophenyl)-
7-phenyl-2,3,8,8a-tetrahydroisoxazolo
[50,40:4,5] furo [2,3-c] pyridazine (10b)
The compound was obtained from the reaction of 9b
(3.36 g, 0.01 mol) with hydroxylamine hydrochloride
as yellow crystals, crystallized from dioxane in 79% yield,
m. p. >350ꢀC. IR (KBr): νmax = 3330 (NH), 3065
(CH aryl), 2954 (CH alkyl), 1633 (C N), 1586 (C C)
1
715 (C–Cl) cm−1; H NMR (DMSO-d6): δ = 2.90 (s, 2H,
CH2), 7.37 to 7.43 (m, 3H, phenyl), 7.48 to 7.54 (dd, 2H,
J = 5.08 Hz, pyridine), 7.76 to 7.82 (dd, 2H, J = 5.05 Hz,
phenyl), 7.85 to 7.91 (dd, 2H, J = 5.07 Hz, pyridine), 8.10
(s, 1H, CH, methine); 13C NMR (DMSO-d6) δ = 32.8 (1C,
CH2), 138.6 (1C, CH, methine), 124.5, 125.9, 128.6129.4, 131.8,
134.7, 143.5, 149.8, 150.4, 162.1 (14C, Ar–C); MS (70 eV):
m/z = 281 (M+, 100%); Anal. Calc. (Found) for C16H12ClN3
(281.74): C, 68.21 (68.30); H, 4.29 (4.21); N, 14.91 (14.85).
1
cm−1; H NMR (DMSO-d6): δ = 2.77 (d, 2H, J = 5.13 Hz,
CH2), 5.07 (d, 1H, J = 5.05 Hz, isoxazolo proton), 5.65
(t, 1 H, J = 5.14 Hz, CH-furan), 7.24 to 7.30 (dd, 2H,
J = 5.11 Hz, 4-chlorophenyl), 7.32 to 7.38 (dd, 2H,
J = 5.12 Hz, 4-chlorophenyl), 7.40 to 7.45 (m, 3H, phe-
nyl), 7.85 to 7.91 (dd, 2H, J = 5.13 Hz, phenyl), 11.25
(brs., NH, isoxazolo proton, D2O exchangeable); 13C
NMR (DMSO-d6) δ = 22.8 (1C, CH2), 33.7 (1C, CH), 72.5
(1C, CH, isoxazole), 126.4, 128.4, 128.7, 128.9, 131.4,
132.5, 136.7, 142.2, 144.5, 146.6, 162.8, 164.5 (16C, Ar–C);
MS (70 eV): m/z = 351 (M+, 90%); Anal. Calc. (Found)
for C19H14ClN3O2 (351.79): C, 64.87 (64.80); H, 4.01
(4.10); N, 11.94 (11.85).
4.20 | Synthesis of 6-phenyl-8-(pyridin-
4-ylmethylene)-7,8-dihydrotetrazolo [1,5-b]
pyridazine (12)
Method A: A mixture of compound 11 (2.81 g, 0.01 mol),
sodium azide (0.65 g, 0.01 mol), water (5 mL), and

ABU-HASHEM ET AL.
11
dimethylformamide (25 mL) was heated and refluxed for
4 to 6 hours. The solid product was obtained upon dilu-
tion with (100 mL) of water was filtered off and rec-
rystallized with suitable solvent. Method B: A solution of
compound 11 (2.81 g, 0.01 mol), sodium azide (NaN3,
0.65 g, 0.01 mol), in (30 mL) of dimethyl formamide
(DMF) was heated and refluxed for 10 to 12 hours. The
reaction solution was evaporated to dryness in vacuo
and the residue was recrystallized from dioxane in 77%
yield as white crystals, m. p. 315ꢀC to 317ꢀC. IR (KBr):
νmax = 3052 (CH aryl), 2957 (CH alkyl), 1634 (C N),
4.22 | Synthesis of 6-phenyl-3-(piperazin-
1-yl)-4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (14a) and 4-(6-phenyl-
4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazin-3-yl) morpholine (14b)
General procedure: In a heated mixture of compound 11
(2.81 g, 0.01 mol) in glacial acetic acid (35 mL) or absolute
methanol (35 mL), freshly distilled secondary amines such
as piperazine (0. 86 g, 0.01 mol), or morpholine (0.87 mL,
0.01 mol) were added. The reaction mixture was stirred
under heat and reflux for 7 to 9 hours with TLC; then, it
was allowed to cool to zeroꢀC for 3 hours. The precipitate
obtained was filtered, washed with water (100 mL), dried,
and recrystallized from a suitable solvent to produce 14a,b.
1
1582 (C C)cm−1; H-NMR (DMSO-d6): δ = 2.94 (s, 2H,
CH2), 7.39 to 7.45 (m, 3H, phenyl), 7.50 to 7.56 (dd, 2H,
J = 5.05 Hz, pyridine), 7.78 to 7.84 (dd, 2H, J = 5.09 Hz,
phenyl), 7.90 to 7.96 (dd, 2H, J = 5.08 Hz, pyridine), 8.09
(s, 1H, CH, methine); 13C NMR (DMSO-d6) δ = 34.5 (1C,
CH2), 137.2 (1C, CH, methine), 124.3, 128.1, 128.7, 131.5,
134.4, 141.1, 143.9, 146.6, 149.5, 162.7 (14C, Ar–C); MS
(70 eV): m/z = 288 (M+, 85%); Anal. Calc. (Found) for
C16H12N6 (288.31): C, 66.66 (66.73); H, 4.20 (4.27); N,
29.15 (29.10).
4.23 | Synthesis of 6-phenyl-3-(piperazin-
1-yl)-4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (14a)
The compound was obtained from the reaction of com-
pound 11 with piperazine as white crystals, crystallized
from dimethyl-formamide in 78% yield, m. p. >350ꢀC. IR
(KBr): νmax = 3375 (br, NH), 3056 (CH aryl), 2948
4.21 | Synthesis of 2-phenyl-4-(pyridin-
4-ylmethylene)-3,4-dihydro-10H-pyridazino
[6,1-b] quinazolin-10-one (13)
(CH alkyl), 1633 (C N), 1585 (C C) cm−1 1H-NMR
;
(DMSO-d6): δ = 2.88 (s, 2H, CH2), 3.10 to 3.23 (m, 8H,
4CH2-piperazine), 7.39 to 7.45 (m, 3H, phenyl), 7.50 to
7.55 (dd, 2H, J = 5.14 Hz, pyridine), 7.79 to 7.85 (dd, 2H,
J = 5.16 Hz, phenyl), 7.90 to 7.96 (dd, 2H, J = 5.15 Hz,
pyridine), 8.15 (s, 1H, CH, methine), 10.20 (br, 1NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ = 32.4 (1C,CH2),
50.1, 54.7 (4C, piperazine ring), 136.9 (1C, CH, methine),
124.2, 125.7, 128.1, 128.6, 131.5, 134.4, 143.8, 149.5, 154.6,
162.5 (14 C, Ar–C); MS (70 eV): m/z = 331 (M+, 92%);
Anal. Calc. (Found) for C20H21N5 (331.42): C, 72.48
(72.55); H, 6.39 (6.32); N, 21.13 (21.21).
Method A: A mixture of compound 11 (2.81 g, 0.01 mol)
and anthranilic acid (1.37 g, 0.01 mol), in (35 mL)
of dimethyl formamide (DMF) was heated and refluxed
on oil bath at 150 to 180ꢀC for 10 to 13 hours. The reac-
tion mixture was cooled and triturated with ethanol.
The solid product was filtered off, dried, and crystal-
lized. Method B: Anthranilic acid (1.37 g, 0.01 mol) was
added to a mixture of 11 (2.81 g, 0.01 mol) in dry ben-
zene (30 mL) and the reaction mixture was heated
and refluxed for 8 to 10 hours, under control (TLC).
The solid product that separated on cooling was rec-
rystallized from benzene in 72% yield as brownish crys-
tals, m. p. >350ꢀC. IR (KBr): νmax = 3055 (CH aryl),
2956 (CH alkyl), 1673 (C O), 1636 (C N), 1590 (C C)
4.24 | Synthesis of 4-(6-phenyl-
4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazin-3-yl) morpholine (14b)
1
cm−1; H-NMR (DMSO-d6): δ = 2.92 (s, 2H, CH2), 7.36
to 7.42 (m, 3H, phenyl), 7.46 to 7.52 (dd, 2H,
J = 5.02 Hz, pyridine), 7.56 to 7.65 (m, 4H, phenyl),
7.75 to 7.82 (dd, 2H, J = 5.03 Hz, phenyl), 7.88 to 7.95
(dd, 2H, J = 5.05 Hz, pyridine), 8.11 (s, 1H, CH,
methine); 13C NMR (DMSO-d6) δ = 34.8 (1C, CH2),
137.6 (1C, CH, methine), 122.1124.5, 125.7, 126.1, 126.5,
127.6, 128.4, 128.9, 131.2, 133.1, 134.2, 141.5144.2,
146.1, 149.3159.5 (20C, Ar–C), 163.1 (1C, C O); MS
(70 eV): m/z = 364 (M+, 80%); Anal. Calc. (Found) for
C23H16N4O (364.41): C, 75.81 (75.90); H, 4.43 (4.35); N,
15.38 (15.31).
The compound was obtained from the reaction of com-
pound 11 with morpholine as yellowish crystals, crystal-
lized from dioxane in 73% yield, m. p. >350ꢀC. IR (KBr):
νmax = 3057 (CH aryl), 2951 (CH alkyl), 1630 (C N),
1
1582 (C C) cm−1; H-NMR (DMSO-d6): δ = 2.85 (s, 2H,
CH2), 3.24 to 3.35 (m, 8H, 4CH2-morpholine), 7.41 to
7.47 (m, 3H, phenyl), 7.55 to 7.60 (dd, 2H, J = 5.10 Hz,
pyridine), 7.75 to 7.82 (dd, 2H, J = 5.13 Hz, phenyl), 7.87
to 7.93 (dd, 2H, J = 5.14 Hz, pyridine), 8.11 (s, 1H, CH,
methine); 13C NMR (DMSO-d6) δ = 32.1 (1C,CH2), 55.8,

12
ABU-HASHEM ET AL.
68.5 (4C, morpholine ring), 136.2 (1C, CH, methine),
124.3, 125.5, 128.4, 128.7, 131.8, 134.2, 144.1, 149.3, 155.1,
162.3 (14 C, Ar–C); MS (70 eV): m/z = 332 (M+, 82%);
Anal. Calc. (Found) for C20H20N4O (332.41): C, 72.27
(72.35); H, 6.06 (6.14); N, 16.86 (16.77).
from methanol in 83% yield, m. p. >350ꢀC. IR (KBr):
νmax = 3050 (CH aryl), 2945 (CH alkyl), 1627 (C N),
1
1578 (C C) cm−1; H-NMR (DMSO-d6): δ = 2.90 (s, 2H,
CH2), 7.46 to 7.52 (m, 3H, phenyl), 7.56 to 7.63 (dd, 2H,
J = 5.10 Hz, pyridine), 7.84 to 7.91 (dd, 2H, J = 5.11 Hz,
phenyl), 7.92 to 7.98 (dd, 2H, J = 5.12 Hz, pyridine), 8.20
(s, 1H, CH, methine), 8.45 (s, 1H, triazole ring); 13C NMR
(DMSO-d6) δ = 33.1 (1C, CH2), 137.6 (1C, CH, methine),
124.5, 128.4129.1, 131.5, 135.3, 141.2, 143.7, 144.8, 145.2,
149.8, 163.4 (15C, Ar–C); MS (70 eV): m/z = 287 (M+,
90%); Anal. Calc. (Found) for C17H13N5 (287.33): C, 71.06
(71.15); H, 4.56 (4.62); N, 24.37 (24.30).
4.25 | Synthesis of 3-hydrazinyl-6-phenyl-
4-(pyridin-4-ylmethylene)-4,5-
dihydropyridazine (15)
A solution of compound 11 (2.81 g, 0.01 mol) and hydra-
zine hydrate (98%-100%, 25 mL) was stirred under heated
and refluxed in dioxane (45 mL) and ethanol (15 mL) for
15 to 18 hours. The reaction solution was allowed to cool
to zero ꢀC for 4 hours, and the solid precipitate was col-
lected via filtration and crystallized from methanol in 86%
yield as yellow crystals, m. p. 280ꢀC to 282ꢀC. IR (KBr):
νmax = 3435 to 3355 (brs, NH–NH2), 3050 (CH aryl), 2945
(CH alkyl), 1629 (C N), 1584 (C C), cm−1; 1H-NMR
(DMSO-d6): δ = 2.94 (s, 2H, CH2), 6.70 (br, 2H, NH2, D2O
exchangeable), 7.41 to 7.47 (m, 3H, phenyl), 7.50 to 7.57
(dd, 2H, J = 5.09 Hz, pyridine), 7.81 to 7.88 (dd, 2H,
J = 5.07 Hz, phenyl), 7.90 to 7.96 (dd, 2H, J = 5.06 Hz, pyr-
idine), 8.17 (s, 1H, CH, methine), 10.90 (br, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ = 32.4 (1C, CH2),
137.3 (1C, CH, methine), 124.1, 125.7128.5129.2, 131.6,
135.2, 145.4, 149.5, 156.1, 162.8 (14C, Ar C); MS (70 eV):
m/z = 277 (M+, 100%); Anal. Calc. (Found) for C16H15N5
(277.33): C, 69.29 (69.20); H, 5.45 (5.53); N, 25.25 (25.19).
4.28 | Synthesis of 3-methyl-6-phenyl-
8-(pyridin-4-ylmethylene)-7,8-dihydro-
[1,2,4] triazolo [4,3-b] pyridazine (16b)
The compound was obtained from the reaction of com-
pound 15 with glacial acetic acid as yellowish crystals,
crystallized from dioxane in 85% yield, m. p. >350ꢀC. IR
(KBr): νmax = 3055 (CH aryl), 2948 (CH alkyl), 1629
1
(C N), 1580 (C C) cm−1; H-NMR (DMSO-d6): δ = 2.27
(s,3H,CH3), 2.95 (s, 2H,CH2), 7.50 to 7.54 (m, 3H, phenyl),
7.60 to 7.67 (dd, 2H, J = 5.15 Hz, pyridine), 7.81 to 7.88
(dd, 2 H, J = 5.13 Hz, phenyl), 7.90 to 7.96 (dd, 2H,
J = 5.16 Hz, pyridine), 8.18 (s, 1H, CH, methine); 13C
NMR (DMSO-d6) δ = 18.5 (1C, CH3), 33.4 (1C, CH2), 137.8
(1C, CH, methine), 124.2, 128.1128.9, 131.2, 135.2, 141.4,
143.5, 144.7149.9, 150.6, 163.8 (15C, Ar–C); MS (70 eV):
m/z = 301 (M+, 95%); Anal. Calc. (Found) for C18H15N5
(301.35): C, 71.74 (71.80); H, 5.02 (5.10); N, 23.24 (23.17).
4.26 | Synthesis of 3-substituted-
6-phenyl-8-(pyridin-4-ylmethylene)-7,
8-dihydro-[1,2,4]triazolo [4,3-b] pyridazine
(16a,b)
5 | MATERIALS AND METHODS
5.1 | Biological screening
General procedure: A mix of compound 15 (2.77 g,
0.01 mol) and formic acid (40 mL) or glacial acetic acid
(40 mL) was heated under reflux for 20 to 25 hours under
control (TLC). The reaction solution was allowed to cool
to room temperature and poured into water (100 mL).
The solid precipitate formed was collected via filtration,
washed with ethanol (30 mL), dried, and recrystallized
from an appropriate solvent to produce 16a,b.
The antimicrobial activity of the newly prepared com-
pounds was tested in vitro against gram-positive bacteria
Staphylococcus aureus, Streptococcus pyogenes, gram-
negative bacteria Escherichia coli, Klebsiella pneumoniae,
and the fungi Aspergillus niger, Alternaria alternate,
Curvularia lunata, and Candida albicans. The newly pre-
pared compounds were dissolved in dimethyl sulfoxide
(DMSO) and tested for their antimicrobial activity by the
agar disk diffusion technique. Cefotaxime sodium and
nystatin[30–31,35,37] were used as the standard drugs for
antibacterial and antifungal assays, respectively. A solu-
tion of 100 μg mL−1 of the tested compound was practi-
cal and microplate wells, 1 cm in diameter, were used.
Zones of inhibition were measured with calipers or auto-
mated scanners and were paralleled with those of the
4.27 | Synthesis of 6-phenyl-8-(pyridin-
4-ylmethylene)-7,8-dihydro-[1,2,4]triazolo
[4,3-b] pyridazine (16a)
The compound was obtained from the reaction of com-
pound 15 with formic acid as white crystals, crystallized

ABU-HASHEM ET AL.
13
standards. Cefotaxime sodium (0.15 μmol mL−1) and
nystatin (0.037 μmol mL−1) were used as the standard
drugs for antibacterial and antifungal activity, respec-
tively. Compound-impregnated disks were placed on an
agar plate containing a standard suspension of microor-
ganisms. The plate was incubated for 24 hours at 37ꢀC.
For the assessment of the minimum inhibitory concen-
tration (MIC) by the serial plate dilution way,[30–31,35,37]
5 mg of each tested compound was dissolved in 1 mL of
DMSO separately to prepare stock solutions. Serial dilu-
tions were prepared from each stock solution. The plates
were incubated at 37ꢀC for 24 hours. MIC is defined as
the lowest concentration (μmol mL−1) of the tested com-
pound that results in no visible growth on the plates.
DMSO was used as the solvent control to ensure that the
solvent had no effect on bacterial growth. The results are
shown in Tables 1 and 2.
Research Centre for helping us to screen the newly
synthesized compounds for antimicrobial activity. The
present work was supported by the Departments of
Photochemistry (Heterocyclic Unit) and Applied Organic
Chemistry, Chemical Industries Research Division,
National Research Centre in Cairo, Egypt.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial, or
otherwise.
ETHICS STATEMENT
All the procedures adopted were approved and agreed
upon by the Animal Ethical Committee of the National
Research Centre, Department of Chemistry of Natural
and Microbial Products, Giza12622, Egypt.
HUMAN AND ANIMAL RIGHTS
No humans or animals were used in the study. The
research was conducted in accordance with ethical
standards.
6 | CONCLUSION
The new compounds synthesized in this research have
proven to be of great importance as an antimicrobial activ-
ity by studying the minimum inhibitor concentration
against bacteria and fungi as shown in Tables 1 and 2. Thus,
we obtained new heterocyclic compounds in good yields
by using new procedures which offers rapid and efficient,
these compounds include ethyl 4-oxo-4-phenyl-butanoate
(2), 6-phenyl-pyridazinone (3), phenyl-4-(pyridin-4-ylmet-
hylene)-pyridazinone (4), ethyl ([pyridazinyl] oxy) acetate
(5), ([pyridazinyl] oxy) acetic acid (6), 3-phenyl-furo[2,3-c]
pyridazinone (7), isoxazolo[5,4-c]pyridazine (8), substituted-
furo [2,3-c] pyridazinone (9a-c), substituted-isoxazolo
[50,40:4,5]furo[2,3-c]pyridazine (10a-c), 3-chloro-6-phenyl-
4-(pyridin-4-ylmethylene)-pyridazine (11), tetrazolo
[1,5-b]pyridazine (12), pyridazino[6,1-b]quinazolinone
(13), piperazinyl or morpholinyl-pyridazine (14a,b),
3-hydrazinyl-pyridazine (15) and substituted-1,2,4-triazolo
[4,3-b] pyridazine (16a,b). The new compounds 1,2,4-
triazolopyridazines (16a,b), isoxazolofuro[2,3-c]pyridazines
(10a-c), tetrazolopyridazines (12) and piperazinyl/
morpholinyl-pyridazines (14a,b) showed the highest effi-
ciency as antimicrobial activity, probably because these
compounds containing 1,2,4-triazole ring as the fundamen-
tal nucleus and different functional groups as pyridine, phe-
nyl, methoxy, methyl, ethyl, hydroxy, chloro, triazole,
isoxazole, tetrazole, and quinazolinone moieties when com-
pared with the standard drugs (cefotaxime sodium and
nystatin).
ORCID
Ameen A. Abu-Hashem https://orcid.org/0000-0001-
8557-2806
Usama Fathy https://orcid.org/0000-0002-0821-5271
Moustafa A. Gouda https://orcid.org/0000-0002-9508-
8089
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How to cite this article: Abu-Hashem AA,
Fathy U, Gouda MA. Synthesis of 1,2,4-
triazolopyridazines, isoxazolofuropyridazines, and
tetrazolopyridazines as antimicrobial agents.
J Heterocyclic Chem. 2020;1–14. https://doi.org/10.
1002/jhet.4065