Synthesis and antimicrobial ealuation of some annelated phthalazine deriaties and acyclo C-nucleosides from 1-chloro-4-(2,4,6-trimethylphenyl) phthalazine precursor

Article abstract of DOI:10.3906/kim-1111-52

A highly efficient and ersatile synthetic approach to the synthesis of annelated phthalazine deriaties iz. 1,2,4-triazolo[3,4-a]phthalazine 11a,b, 14, 18, 19a,b, 29-31, 33, 1,2,4-triazino[3,4-a]phthalazine 25a,b-28, 1,3,5-triazino[4,3-a]phthalazine 22, tetrazolo[5,1-a] phthalazine 23, imidazophthalazine 9a,b,15, and pyrimidinophthlazine 6, 10, 16, 17, 20 is presented. Moreoer, acyclo C -nucleoside and double headed acyclo C-nucleoside of 1,2,4-triazolo[3,4-a]phthalazine 12, 13 were obtained ia heterocyclization reaction of 1-chloro-4-(2,4,6-trimethylphenyl)phthalazine (4) with gluconic acid hydrazide and galactaric acid bis hydrazide, respectiely. The new compounds were synthesized with the objectie of studying their antimicrobial actiity. TUeBITAK.

Full text of DOI:10.3906/kim-1111-52

Turk J Chem
36 (2012) , 347 – 366.
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ꢀ TUBITAK
doi:10.3906/kim-1111-52
Synthesis and antimicrobial evaluation of some annelated
phthalazine derivatives and acyclo C-nucleosides from
1-chloro-4-(2,4,6-trimethylphenyl) phthalazine precursor
Maher Abdel Aziz EL-HASHASH1, Ahmed Youssef SOLIMAN2, Ibrahim Essam EL-SHAMY3,∗
1Chemistry Department, Faculty of Science, Ain Shams University, Cairo-EGYPT
2Chemistry Department, College of Science, King Khalid University, Abha-SAUDI ARABIA
3Chemistry Department, Faculty of Science, Fayoum University, Fayoum-EGYPT
e-mail: iei00@fayoum.edu.eg
Received: 30.11.2011
A highly efficient and versatile synthetic approach to the synthesis of annelated phthalazine derivatives
viz. 1,2,4-triazolo[3,4-a]phthalazine 11a,b, 14, 18, 19a,b, 29-31, 33, 1,2,4-triazino[3,4-a]phthalazine
25a,b-28, 1,3,5-triazino[4,3-a]phthalazine 22, tetrazolo[5,1-a] phthalazine 23, imidazophthalazine 9a,b,15,
and pyrimidinophthlazine 6, 10, 16, 17, 20 is presented. Moreover, acyclo C -nucleoside and double
headed acyclo C -nucleoside of 1,2,4-triazolo[3,4-a]phthalazine 12, 13 were obtained via heterocyclization
reaction of 1-chloro-4-(2,4,6-trimethylphenyl)phthalazine (4) with gluconic acid hydrazide and galactaric
acid bis hydrazide, respectively. The new compounds were synthesized with the objective of studying their
antimicrobial activity.
Key Words: Triazinophthalazine, pyrazolylphthalazine, triazolophthalazine, antimicrobial activity
Introduction
The synthesis of new compounds and testing their biological and pharmacological activities are the major
goals of drug development projects. Nitrogen-containing heterocyclic compounds have received much at-
tention as shown by the numerous studies published on their applicability in different areas, especially as
drugs.1,2 Phthalazines are examples of nitrogen heterocycles that possess exciting biological properties.3−5
They form the structural profile for several biologically active compounds and hence they are considered
∗Corresponding author
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
important key elements. Several reports in the literature have focused on the pharmacology of phthalazine
derivatives. These reports have resulted in a great number of contributions in diverse areas of interest.6−11
Phthalazines have been reported to possess, anticonvulsant,12 cardiotonic,13 antimicrobial,14 antitumor,15−18
antihypertensive,19,20 antithrombotic,21 antidiabetic,22,23 antitrypanosomal,24 anti-inflammatory,25−31 and
vasorelaxant activities.20,32 Additionally, phthalazines have recently been reported to potentially inhibit sero-
tonin reuptake and are considered anti-depression agents.33 Therefore, a number of methods have been reported
for the synthesis of phthalazine derivatives.12,34−40 Nevertheless, the development of new synthetic methods
for the efficient preparation of heterocycles containing phthalazine ring fragment is an interesting challenge. In
view of the aforementioned facts, it seemed most interesting to study the chemical behavior of 1-chloro-4-(2,4,6-
trimethylphenyl) phthalazine (2) towards some nitrogen and carbon nucleophiles to produce new phthalazine
derivatives with the aim to evaluate their antimicrobial activities.
Experimental
Melting points were determined on a MEL-TEMP II melting point apparatus in open glass capillaries and are
uncorrected. The infrared spectra IR were recorded for potassium bromide disks on a Pye-Unicam SP1025
spectrophotometer. NMR spectra were obtained at ambient temperature (∼25 ◦ C) with a Bruker AC-250
spectrometer or with a Varian Gemini 200 spectrometer at 250 MHz using tetramethylsilane (TMS) as an
internal standard. Mass spectra (MS) were obtained on a Hewlett-Packard 5995 gas chromatography-mass
spectrometer system or on a Shimadzu GCMS-QP 1000 EX mass spectrometer. Homogeneity of the products
and follow up of the reactions were checked by ascending thin-layer chromatography TLC on plates precoated
with silica gel G (E. Merck; layer thickness 0.25 mm), used without pretreatment. Elemental microanalyses
were performed at the Microanalytical Unit, Cairo University, Cairo, Egypt.
4-(2,4,6-Trimethylphenyl)lphthalazin-1(2H)-one (3)
Hydrazine hydrate (0.3 mL, 98%) was added to a solution of compound 2 (0.01 mol) in absolute ethanol and the
reaction mixture was refluxed for 2 h. After cooling, the mixture to room temperature, a solid was obtained.
This crude product was filtered off and recrystallized from ethanol to give 3. Yield: 65%; mp 235-237 ◦ C;
1 H-NMR (DMSO-d6) δ: 2.39 (s, 3H, CH3), 2.50 (s, 6H, 2CH3), 7.27-8.50 (m, 6H, Ar–H), 11.17 (s, 1H, NH,
exchangeable with D2 O); IR (KBr) v: 3212 (NH), 1654 cm−1 (CO); MS (70 eV) m/z (%): 264 (M+ , 18).
Anal. calcd. for C17 H16 N2 O: C 77.25, H 6.10, N 10.60; found C 77.16, H 6.18, N 10.70.
1-Chloro-4-(2,4,6-trimethylphenyl)phthalazine (4)
A suspension of compound 3 (0.01 mol) and PCl5 (0.01 mol) in 8 mL of POCl3 was heated under reflux on a
water bath for 2 h. Then the mixture was cooled to room temperature and poured into crushed ice slowly. The
solid formed was filtered off, washed with cold water, dried and recrystallized from benzene to give 4. Yield:
70%; mp 160-161 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.40 (s, 3H, CH3), 2.51 (s, 6H, 2CH3), 7.01-8.04 (m, 6H,
Ar–H); IR (KBr) v: 1605 (C=N), 847 cm−1 (C-Cl); MS (70 eV) m/z (%): 284 (M+ Cl37 , 1), 282 (M+ Cl35
,
4). Anal. calcd. for C17 H15 ClN2 : C, 72.21; H, 5.35; N, 9.91; found C, 72.30; H, 5.41; N, 9.89.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
5-(2,4,6-Trimethylphenyl)-8H-phthalazino[1,2-b]quinazolin-8-one (6)
In a fusion tube provided with an air condenser, a mixture of 4 (0.001 mol) and anthranilic acid (0.005 mol) was
heated in an oil bath at 190-191 ◦ C for 2 h. Then the mixture was cooled (to room temperature) and poured
into 40 mL of cold water. The obtained solid product was collected and recrystallized from benzene to give 6.
Yield: 64%; mp. 220-223 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.38 (s, 3H, CH3), 2.45 (s, 6H, 2CH3), 6.98-8.13 (m,
10H, Ar–H); 13 C-NMR (DMSO-d6) δ: 20.9, 23.5, 122.2, 125.8, 126.5, 127.1, 127.6, 127.9, 128.5, 128.9, 132.0,
132.6, 133.3, 134.9, 136.2, 140.6, 145.7, 146.3, 147.8, 155.5, 170.1; IR (KBr) v: 1670 (CO), 1614 cm−1 (C=N);
%MS (70 eV) m/z (%): 365 (M+ , 8). Anal. calcd. for C24 H19 N3 O: C, 78.88; H, 5.24; N, 11.50; found C,
78.94; H, 5.19; N, 11.44.
General procedure for the synthesis of phthalazinylamino acids 7a,b and 8
The corresponding amino acid (0.01 mol) and sodium carbonate (0.005 mol) were dissolved in water (15 mL),
and then adjusted to pH 9-9.5. Then compound 4 (0.005 mol) was added to it and refluxed at the same pH for
8 h. The reaction mixture was left overnight at room temperature, and then treated with cold hydrochloric acid
(pH 0.5). The solid product obtained was filtered off, washed with water, and recrystallized from an appropriate
solvent to give the target compound.
2-(4-(2,4,6-Trimethylphenyl)phthalazin-1-ylamino)acetic acid (7a)
Yield, 70% (dioxane); mp 243-244 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.33 (s, 3H, CH3), 2.42 (s, 6H, 2CH3), 4.61
(s, 2H, CH2), 7.03-8.17 (m, 7H, ArH and NH), 10.81 (brs, 1H, OH, exchangeable with D2 O); IR (KBr) v:
3280-2510 (OH, NH), 1702 cm−1 (CO); MS (70 eV) m/z (%): 321 (M+ , 7). Anal. calcd. for C19 H19 N3 O2 :
C, 71.01; H, 5.96; N, 13.08; found C, 71.10; H, 6.02; N, 13.00.
2-(4-(2,4,6-Trimethylphenyl)-1-ylamino)propanoic acid (7b)
Yield, 68% (dioxane); mp 233-234 ◦ C; 1 H-NMR (DMSO-d6) δ: 1.53 (d, J = 10 Hz, 3H, CH3), 2.39 (s, 3H,
CH3), 2.51 (s, 6H, 2CH3), 4.50 (q, J = 10 Hz, 1H, CH), 7.11-8.12 (m, 7H, ArH and NH), 10.60 (brs, 1H, OH,
exchangeable with D2 O); IR (KBr) v: 3240-2500 (OH, NH), 1694 (CO), 1605 cm−1 (C=N); MS (70 eV) m/z
(%): 335 (M+ , 6). Anal. calcd. for C20 H21 N3 O2 : C, 71.62; H, 6.31; N, 12.53; found C, 71.57; H, 6.38; N,
12.60.
3-(4-(2,4,6-Trimethylphenyl)phthalazin-1-ylamino)propanoic acid (8)
Yield, 70% (ethanol); mp 244-245 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.44 (s, 6H, 2CH3), 2.81
(t, J = 10 Hz, 2H, NCH2), 3.77 (t, J = 10 Hz, 2H, CH2 CO), 7.00-8.18 (m, 7H, ArH and NH), 11.00 (brs, 1H,
OH, exchangeable with D2 O); IR (KBr) v: 3288-2511 (OH, NH), 1700 (CO), 1605 cm−1 (C=N); MS (70 eV)
m/z (%): 335 (M+ , 4). Anal. calcd. for C20 H21 N3 O2 : C, 71.62; H, 6.31; N, 12.53; found C, 71.71; H, 6.23;
N, 12.50.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
General procedure for the synthesis of imidazophthalazines 9a,b and 10
A mixture of 7a, 7b, or 8 (0.01 mol), acetic anhydride (30 mL), and anhydrous sodium acetate (0.82 g, 0.01
mol) was heated under reflux for 3 h. The solvent was removed under reduced pressure and the residue washed
with water, filtered, dried, and crystallized from appropriate solvent to give 9a,b and 10.
6-(2,4,6-Trimethylphenyl)imidazo[2,1-a]phthalazin-3(2H)-one (9a)
Yield, 60% (DMF); mp 179-180 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.40 (s, 6H, 2CH3), 5.03 (s,
2H, CH2), 6.98-8.05 (m, 6H, ArH); IR (KBr) v: 1685 (CO), 1610 cm−1 (C=N); MS (70 eV) m/z (%): 303
(M+ , 19). Anal. calcd. for C19 H17 N3 O: C, 75.23; H, 5.65; N, 13.85; found C, 75.19; H, 5.71; N, 13.92.
6-(2,4,6-Trimethylphenyl)-2-methylimidazo[2,1-a]phthalazin-3(2H)-one (9b)
Yield, 63% (benzene); mp 170-171 ◦ C; 1 H-NMR (DMSO-d6) δ: 1.64 (d, J = 10 Hz, 3H, CH3), 2.31 (s,
3H, CH3), 2.44 (s, 6H, 2CH3), 4.90 (q, J = 10 Hz, 1H, methine proton), 7.12-8.13 (m, 6H, ArH); 13 C-NMR
(DMSO-d6 , 300 MHz) δ: 19.3, 20.4, 23.0, 66.2, 127.0, 127.8, 129.2, 129.9, 130.4, 132.0, 132.5, 132.9, 137.1,
139.2, 144.0, 158.3, 180.5; IR (KBr) v: 1670 (CO), 1604 cm−1 (C=N). Anal. calcd. for C20 H19 N3 O: C, 75.69;
H, 6.03; N, 13.24; found C, 75.77; H, 6.10; N, 13.14.
7-(2,4,6-Trimethylphenyl)-2H-pyrimido[2,1-a]phthalazin-4(3H)-one (10)
Yield, 65% (ethanol); mp 199-200 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.34 (s, 3H, CH3), 2.46 (s, 6H, 2CH3), 3.04
(t, J = 10 Hz, 2H, CH2), 4.02 (t, J = 10 Hz, 2H, CH2), 6.99-7.90 (m, 6H, ArH); IR (KBr) v: 1675 (CO),
1610 cm−1 (C=N); MS (70 eV) m/z (%): 317 (M+ , 20). Anal. calcd. for C20 H19 N3 O: C, 75.69; H, 6.03; N,
13.24; found C, 75.60; H, 6.11; N, 13.19.
Reaction of 1-chloro-4-(2,4,6-trimethylphenyl)phthalazine (4) with some acid hydrazides
General procedure
A mixture of chlorophthalazine 4 (2.82 g, 0.01 mol) and appropriate acid hydrazides (0.01 mol) namely acetic
acid hydrazide, benzoic acetic acid hydrazide and/or gluconic acid hydrazide in ethanol (40 mL) was heated
under reflux for 4 h. The solid separated after concentrating and cooling, and recrystallized from the appropriate
solvent to give 11a,b and 12
6-(2,4,6-Trimethylphenyl)-3-methyl-[1,2,4]triazolo[3,4-a]phthalazine (11a)
Yield, 70% (ethanol); mp 189-190 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.44 (s, 6H, 2CH3), 2.48
(s, 3H, CH3), 7.03-7.97 (m, 6H, ArH); 13 C-NMR (DMSO-d6) δ: 10.1, 20.3, 22.2, 122.0, 126.1, 126.8, 127.2,
128.2, 129.0, 134.1, 134.9, 137.0, 139.2, 139.8, 149.0, 155.1; IR (KBr) v: 1611 cm−1 (C=N); MS (70 eV) m/z
(%): 302 (M+ , 16). Anal. calcd. for C19 H18 N4 :C 75.47, H 6.00, N 18.53; found C 75.50, H 5.91, N 18.45.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
6-(2,4,6-Trimethylphenyl)-3-phenyl-[1,2,4]triazolo[3,4-a]phthalazine (11b)
Yield, 73% (ethanol); mp 201-202 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.29 (s, 3H, CH3), 2.42 (s, 6H, 2CH3), 6.98-
8.12 (m, 11H, ArH); IR (KBr) v: 1609 cm−1 (C=N); MS (70 eV) m/z (%): 364 (M+ , 20). Anal. calcd. for
C24 H20 N4 : C 79.10, H 5.53, N 15.37; found C 79.08, H 5.55, N 15.39.
1-[6-(2,4,6-Trimethylphenyl)]-[1,2,4]triazolo[3,4-a]phthalazin-3-yl)pentane-1,2,3,4,5-pentaol (12)
Yield, 60% (ethanol); mp 280-281 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.44 (s, 6H, 2CH3), 3.19-
3.64 (m, 4H, 4CH), 3.70-3.84 (m, 2H, CH2 OH), 4.41-5.11 (m, 5H, 5OH, D2 O exchangeable), 6.98-8.00 (m, 6H,
ArH); IR (KBr) v: 3425-3222 (OH), 1608 cm−1 (C=N). Anal. calcd. for C23 H26 N4 O5 : C, 63.00; H, 5.98; N,
12.78; found C, 64.01; H, 6.03; N, 12.69.
1,4-Bis{6-(2,4,6-trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3-yl}butane-1,2,3,4-tetraol (13)
A mixture of 4 (2.82 g, 0.01 mol) and galactaric acid bishydrazide (0.005 mol) in 20 mL of absolute ethanol
containing a few drops of acetic acid was refluxed for 5 h. The solid separated after concentrating and cooling,
and recrystallized from H2 O-EtOH to give compound 13 in 71% yield; mp 291-292 ◦ C; 1 H-NMR (DMSO-d6)
δ: 2.33 (s, 6H, 2CH3), 2.48 (s, 12H, 4CH3), 3.79-4.84 (2d, 4H, 4CH), 5.10-5.45 (2d exchangeable, 4H, 4OH),
6.99- 8.08 (m, 12H, ArH); 13 C-NMR (DMSO-d6) δ: 20.0, 23.2, 70.93, 71.23, 122.3, 127.1, 127.7, 128.0, 128.6,
129.5, 135.0, 136.2, 136.9, 137.4, 150.4, 153.1, 156.2; IR (KBr) v: 3488-3210 (OH), 1610 cm−1 (C=N); MS (70
eV) m/z (%): 694 (M+ , 0.55). Anal. calcd. for C40 H38 N8 O4 : C, 69.15; H, 5.51; N, 16.13; found C, 69.24;
H, 5.41; N, 16.17.
Bis{6-(2,4,6-Trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3-yl}methane (14)
A solution of malonic acid dihydrazide (0.005 mol) in benzene (10 mL) was added to the solution of 4 (0.01
mol) in 40 mL of benzene and the mixture was heated under reflux for 8 h. The product that separated upon
cooling was filtered, washed with benzene, and crystallized from chloroform to give 14 in 67% yield; mp 300-301
◦ C; 1 H-NMR (DMSO-d6) δ: 2.32 (s, 6H, 2CH3), 2.46 (s, 12H, 4CH3), 5.03 (s, 2H, CH2), 7.02-8.04 (m, 12H,
ArH); IR (KBr) v: 1602 cm−1 (C=N); MS (70 eV) m/z (%): 588 (M+ , 7). Anal. calcd. for C37 H32 N8 : C,
75.49; H, 5.48; N, 19.03; found C, 75.57; H, 5.40; N, 19.10.
5-(2,4,6-Trimethylphenyl)benzo[4,5]imidazo[2,1-a]phthalazine (15)
A mixture of compound 4 (2.8 g, 0.01 mol) and o-phenylenediamine (1.0 g, 0.01 mol) was heated in a fusion
tube provided with an air condenser in an oil bath at 170-180 ◦ C for 2 h. After cooling (to room temperature),
the reaction mixture was poured into cold water (80 mL). The solid obtained was filtered off and recrystallized
from n-butanol to give 15 in 64% yield; mp 204-205 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.35 (s, 3H, CH3), 2.48 (s,
6H, 2CH3), 7.01- 8.10 (m, 10H, ArH); IR (KBr) v: 1610 cm−1 (C=N); MS (70 eV) m/z (%): 337 (M+ , 37).
Anal. calcd. for C23 H19 N3 : C, 81.87; H, 5.68; N, 12.45; found C, 81.94; H, 5.60; N, 12.51.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
7-(2,4,6-Trimethylphenyl)-2,3-dimethyl-4H-thieno-[2’,3’:4,5]-pyrimido[2,3-a]pyridazin-4-one (16)
To a solution of 4 (0.01 mol) in absolute ethanol (50 ml), ethyl 2-amino-4,5-dimethylthiophene-3-carboxylate
(0.01 mol) was added and the reaction mixture was refluxed for 5 h. The solid obtained upon cooling was
collected by filtration, dried, and crystallized from ethanol to give 16 in 69% yield; mp 311-312 ◦ C; 1 H-NMR
(DMSO-d6) δ: 1.83 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.48 (s, 6H, 2CH3), 6.94-8.02 (m,
6H, ArH); IR (KBr) v: 1695(CO), 1614 cm−1 (C=N); MS (70 eV) m/z (%): 399 (M+ , 43). Anal. calcd. for
C24 H21 N3 OS: C, 72.15; H, 5.30; N, 10.52; S, 8.03; found C, 72.22; H, 5.21; N, 10.57; S, 8.10.
2-(2,4,6-Trimethylphenyl)-10,11,12,13-tetrahydro-14H-[1]-benzothieno-[2’,3’:4,5]-pyrimido [2,3-a]
pyridazin-14-one (17)
A mixture of 4 (0.001 mol) and ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (0.005 mol)
was heated in a fusion tube provided with an air condenser in an oil bath at 180-182 ◦ C for 2 h. Then the
reaction mixture was cooled to room temperature and added to 50 mL of cold water. The solid product obtained
was collected and recrystallized from benzene to give 17 in 63% yield; mp 325-326 ◦ C; 1 H-NMR (DMSO-d6)
δ: 2.21-2.26 (m, 4H, 2CH2), 2.30-2.34 (m, 4H, 2CH2), 2.41 (s, 3H, CH3), 2.50 (s, 6H, 2CH3), 7.00-8.05 (m,
6H, Ar–H); 13 C-NMR (DMSO-d6) δ: 20.2, 22.5, 24.1, 24.9, 26.3, 27.4, 119.0, 126.3, 126.9, 128.1, 128.5, 129.0,
130.4, 133.2, 134.2, 136.7, 136.9, 139.8, 142.2, 142.9, 153.1, 158.3, 165.6; IR (KBr) v: 1711 (CO), 1622 cm−1
(C=N); MS (70 eV) m/z (%): 425 (M+ , 3). Anal. calcd. for C26 H23 N3 OS: C, 73.38; H, 5.45; N, 9.87; S,
7.54; found C, 73.27; H, 5.55; N, 9.90; S, 7.60.
6-(2,4,6-Trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazine-3-amine (18)
A mixture of compound 4 (2.82 g, 0.01 mol) and thiosemicarbazide (0.01 mol) in absolute ethanol was refluxed
for 8 h. The solid product obtained upon cooling was filtered off, dried, and crystallized from ethanol to give
18 in 73% yield; mp 239-240 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.34 (s, 3H, CH3), 2.45 (s, 6H, 2CH3), 6.24 (s, 2H,
NH2 , exchangeable with D2 O), 7.3-8.01 (m, 6H, ArH); IR (KBr) v: 3422, 3380 (NH2), 1605 cm−1 (C=N).
Anal. calcd. for C18 H17 N5 : C, 71.27; H, 5.65; N, 23.09; found C, 71.31; H, 5.74; N, 23.00.
Reaction of 6-(2,4,6-trimethylphenyl)-3-amino-[1,2,4]triazolo[3,4-a]phthalazine (18) with some
aromatic aldehydes
General procedure
A mixture of 18 (0.001 mol) and an appropriate aromatic aldehyde, namely benzaldehyde or p-chlorobenzaldehyde
(0.001 mol), was refluxed in 20 mL of absolute ethanol for 7 h. The solid obtained upon cooling was collected by
filtration, dried, and crystallized from the appropriate solvent to give the title compounds 19a,b, respectively.
N -Benzylidene-6-(2,4,6-trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3-amine (19a)
Yield, 70% (ethanol); mp 210-211 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.36 (s, 3H, CH3), 2.47 (s, 6H, 2CH3),
7.00-8.10 (m, 11H, ArH), 8.51 (s, 1H, CH); IR (KBr) v: 1618 cm−1 (C=N); MS (70 eV) m/z (%): 391 (M+
,
10). Anal. calcd. for C25 H21 N5 : C, 76.70; H, 5.41; N, 17.89; found C, 76.63; H, 5.37; N, 17.96.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
N -(4-Chlorobenzylidene)-6-(2,4,6-trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3-amine (19b)
Yield, 73% (ethanol); mp 200-201 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.39 (s, 3H, CH3), 2.49 (s, 6H, 2CH3),
6.98-8.01 (m, 10H, ArH), 9.05 (s, 1H, CH); IR (KBr) v: 1621 cm−1 (C=N). Anal. calcd. for C25 H20 ClN5 : C,
70.50; H, 4.73; N, 16.44; found C, 70.66; H, 4.70; N, 16.51.
7-(2,4,6-Trimethylphenyl)-13H-phthalazino[2,1-a]quinazoline (20)
A mixture of compound 4 (2.82 g, 0.01 mol) and o-chlorobenzylamine (0.01 mol) in pyridine (20 mL) was
refluxed for 7 h. After cooling, the reaction mixture was poured on ice water (80 mL). The solid obtained was
filtered off and recrystallized from ethanol to give 20 in 63% yield; mp 185-186 ◦ C; 1 H-NMR (DMSO-d6) δ:
2.37 (s, 3H, CH3), 2.49 (s, 6H, 2CH3), 4.90 (s, 2H, CH2 quinazoline), 6.97-8.14 (m, 10H, ArH); IR (KBr) v:
1610 cm−1 (C=N). Anal. calcd. for C24 H21 N3 : C, 82.02; H, 6.02; N, 11.96; found C, 82.10; H, 6.11; N, 11.90.
7-(2,4,6-Trimethylphenyl)-3-phenyl-2-thioxo-2,3-dihydro-[1,3,5]triazino[2,1-a] phthalazin-4-one (22)
The solution of ammonium thiocyanate (0.005 mol) in dry acetone was added to a stirred solution of chlorophtha-
lazine 4 (0.005 mol) in dry acetone. The reaction mixture was stirred for 1 h at room temperature. Ammonium
chloride was precipitated during the progress of the reaction. After filtration of the ammonium chloride, phenyl
isocyanate (0.6 g, 0.005 mol) was added to the filtrate. The reaction mixture was heated under reflux for 30
min. The solid product that separated after cooling was crystallized from ethanol to give 22 in 55% yield;
mp 239-240 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.33 (s, 3H, CH3), 2.46 (s, 6H, 2CH3), 6.99-8.12 (m, 11H, ArH);
13 C-NMR (DMSO-d6) δ: 20.1, 23.2, 125.6, 126.9, 127.8, 128.5, 128.8, 129.9, 130.7, 132.0, 132.7, 134.1, 134.8,
136.0, 136.8, 139.0, 144.2, 155.8, 157.0, 180.5; IR (KBr) v: 1675 (CO), 1618 (C=N), 1265 cm−1 (C=S). Anal.
calcd. for C25 H20 N4 OS: C, 70.73; H, 4.75; N, 13.20; S, 7.55; found C, 70.77; H, 4.69; N, 13.27; S, 7.61.
6-(2,4,6-Trimethylphenyl)-tetrazolo[5,1-a]phthalazine (23)
A mixture of compound 4 (2.82 g, 0.01 mol) and sodium azide (0.01 mol) in acetic acid (30 mL) was refluxed
for 5 h. After cooling, the reaction mixture was poured on ice water (80 mL). The solid obtained was filtered
off and recrystallized from ethanol to give 23 in 70% yield; mp 175-176 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.33 (s,
3H, CH3), 2.40 (s, 6H, 2CH3), 7.01- 8.00 (m, 6H, ArH); IR (KBr) v: 1607 cm−1 (C=N); MS (70 eV) m/z
(%): 289 (M+ , 39). Anal. calcd. for C17 H15 N5 : C, 70.57; H, 5.23; N, 24.21; found C, 70.66; H, 5.20; N, 24.30.
1-[(2,4,6-Trimethylphenyl)-1,2-dihydrophthalazin-1yl]hydrazine (24)
To a solution of 4 (0.01 mol) in 15 mL of absolute ethanol was added 0.3 mL of hydrazine hydrate (98%) and
the reaction mixture was refluxed for 5 h. After cooling, the obtained solid was filtered off and crystallized
from ethanol to give 24 in 71% yield; mp 266-267 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.33 (s, 3H, CH3), 2.45 (s,
6H, 2CH3), 5.31 (s, 2H, NH 2 exchangeable with D2 O), 7.27-8.50 (m, 6H, Ar–H), 9.55 (s, 1H, NH exchangeable
with D2 O); IR (KBr) v: 3390-3112 cm−1 (NHNH2); MS (70 eV) m/z (%): 278 (M+ , 21). Anal. calcd. for
C17 H18 N4 : C, 73.35; H, 6.52; N, 20.13; found C, 73.44; H, 6.43; N, 20.10.
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Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
7-(2,4,6-Trimethylphenyl)-3-methyl/phenyl-2H-[1,2,4]triazino[3,4-a]phthalazine (25a,b)
A mixture of 24 (2.78 g, 0.01 mol) and α-haloketones (0.01 mol) (viz. chloroacetone and phenacyl bromide) in
dry xylene (40 mL) was heated under reflux for 8 h. The solid that separated upon cooling was filtered off and
recrystallized from the appropriate solvent to give 25a,b.
25a; Yield, 61%; mp 200-201 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.11 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.50
(s, 6H, 2CH3), 5.88 (s, 1H, CH of triazine), 7.10-8.28 (m, 7H, ArH and NH of triazine); IR (KBr) v: 3190
(NH), 1615 cm−1 (C=N); Anal. calcd. for C20 H20 N4 : C, 75.92; H, 6.37; N, 17.71; found C, 76.01; H, 6.40; N,
17.66.
25b; Yield, 66%; mp 212-213 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.47 (s, 6H, 2CH3),
6.50 (s, 1H, CH of triazine), 7.03-8.31 (m, 12H, ArH and NH of triazine); IR (KBr) v: 3222 (NH), 1610 cm−1
(C=N); MS (70 eV) m/z (%): 378 (M+ , 11). Anal. calcd. for C25 H22 N4 : C, 79.34; H, 5.86; N, 14.80; found
C, 79.29; H, 5.90; N, 14.88.
7-(2,4,6-Trimethylphenyl)-2H-[1,2,4]triazino[3,4-a]phthalazin-3(4H)-one (26)
A mixture of 24 (2.78 g, 0.01 mol) and ethyl bromoacetate (0.03 mol) in absolute ethanol (30 mL) was heated
under reflux for 15 h. The solid that separated after cooling and recrystallization from ethanol gave 26 in 53%
yield; mp 246-247 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.29 (s, 3H, CH3), 2.44 (s, 6H, 2CH3), 4.57 (s, 2H, CH2 of
triazine), 6.97-8.25 (m, 7H, ArH and NH of triazine); IR (KBr) v: 3198 (NH), 1677 (CO), 1613 cm−1 (C=N);
MS (70 eV) m/z (%): 318 (M+ , 5). Anal. calcd. for C19 H18 N4 O: C, 71.68; H, 5.70; N, 17.60; found C, 71.57;
H, 5.77; N, 17.55.
7-(2,4,6-Trimethylphenyl)-2H-[1,2,4]triazino[3,4-a]phthalazine-3,4-dione (27)
A mixture of 24 (2.78 g, 0.01 mol) and diethyl oxalate (0.01 mol) in absolute ethanol (40 mL) was heated under
reflux for 18 h. After cooling, the separated solid produced was collected and recrystallized from ethanol to give
27 in 65% yield; mp 259-260 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.33 (s, 3H, CH3), 2.47 (s, 6H, 2CH3), 7.02-8.32
(m, 6H, ArH and NH of triazine); IR (KBr) v: 3228 (NH), 1691-1680 (CO), 1607 cm−1 (C=N); MS (70 eV)
m/z (%): 332 (M+ , 1.5). Anal. calcd. for C19 H16 N4 O2 : C, 68.66; H, 4.85; N, 16.86; found C, 68.75; H, 4.90;
N, 16.79.
7-(2,4,6-Trimethylphenyl)-3-methyl-4H-[1,2,4]triazino[3,4-a]phthalazin-4-one(28)
A mixture of 24 (2.78 g, 0.01 mol) and pyruvic acid (0.01 mol) was heated in oil bath at 180 ◦ C for 1 h. After
cooling, the formed precipitate was heated with ethanol and the precipitate was collected and crystallized from
acetic acid to give 28 in 47% yield; mp 269-270 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.01 (s, 3H, CH3), 2.28 (s, 3H,
CH3), 2.43 (s, 6H, 2CH3), 7.01-8.00 (m, 6H, ArH); 13 C-NMR (DMSO-d6) δ: 16.8, 20.7, 22.4, 126.0, 126.7,
129.0, 129.6, 130.8, 130.9, 133.1, 133.9, 135.0, 140.2, 143.3, 158.1, 158.9, 166.0; IR (KBr) v: 1679 (CO), 1603
cm−1 (C=N); MS (70 eV) m/z (%): 330 (M+ , 15). Anal. calcd. for C20 H18 N4 O: C, 72.71; H, 5.49; N, 16.96;
found C, 72.75; H, 5.57; N, 16.88.
354

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
6-(2,4,6-Trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazine (29)
A solution of 24 (2.78 g, 0.01 mol) in formic acid (20 mL) was heated under reflux for 15 h. After cooling and
dilution with water, the formed precipitate was collected and crystallized from dioxane to give 29 in 66% yield;
mp 220-221 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3), 2.48 (s, 6H, 2CH3), 6.98-7.89 (m, 6H, ArH),
9.57 (s, 1H, =CH triazol); IR (KBr) v: 1601 cm−1 (C=N). Anal. calcd. for C18 H16 N4 : C, 74.98; H, 5.59; N,
19.43; found C, 75.10; H, 5.57; N, 19.38.
6-(2,4,6-Trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazine-3(2H)-thione (30)
To an ice cooled solution of 24 (2.78 g, 0.01 mol) in absolute ethanol (10 mL) containing potassium hydroxide
(0.01 mol), carbon disulfide (2 mL) was added dropwise with stirring. The mixture was diluted with absolute
ethanol (20 mL) and refluxed for 8 h. The reaction mixture was filtered, concentrated, diluted with water, and
neutralized with acetic acid. The precipitated product was crystallized from dioxane to give 30 in 60% yield;
mp 310-311 ◦ C; 1 H-NMR (DMSO-d6) δ: 2.35 (s, 3H, CH3), 2.50 (s, 6H, 2CH3), 7.01-7.90 (m, 6H, ArH), 9.32
(s, 1H, NH exchangeable with D2 O); IR (KBr) v: 3218(NH), 1601 cm−1 (C=N); MS (70 eV) m/z (%): 320
(M+ , 10). Anal. calcd. for C18 H16 N4 S: C, 67.47; H, 5.03; N, 17.49; S, 10.01; found C, 67.56; H, 5.10; N,
17.38; S, 10.00.
6-(2,4,6-Trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3(2H)-one (31)
A mixture of 24 (2.78 g, 0.01 mol) and ethylchloroformate (0.02 mol) in 20 mL of pyridine was heated on a
water bath for 12 h. After cooling, the reaction mixture was poured onto ice water. The precipitated solid was
collected by filtration, dried and crystallized from ethanol to give 31 in 61% yield; mp 320-321 ◦ C; 1 H-NMR
(DMSO-d6) δ: 2.36 (s, 3H, CH3), 2.48 (s, 6H, 2CH3), 6.98-7.94 (m, 6H, ArH), 8.78 (s, 1H, NH exchangeable
with D2 O); IR (KBr) v: 3211 (NH), 1664 (CO), 1608 cm−1 (C=N); MS (70 eV) m/z (%): 304 (M+ , 16).
Anal. calcd. for C18 H16 N4 O: C, 71.04; H, 5.30; N, 18.41; found C, 71.11; H, 5.36; N, 18.32.
2-(4-(2,4,6-Trimethylphenyl)phthalazin-1-yl)-N-phenylhydrazinecarbothioamide (32)
A mixture of 24 (2.78 g, 0.01 mol) and phenyl isothiocyanate (0.01 mol) in absolute ethanol (30 mL) was
heated under reflux for 8 h. After cooling, the separated solid produced was collected and recrystallized from
ethanol to give 32 in 73% yield; mp 177-176 ◦ C; 1 H-NMR (DMSO-d6) δ: 1.96 (s, 1H, CSNH, exchangeable
with D2 O), 2.33 (s, 3H, CH3), 2.44 (s, 6H, 2CH3), 6.99-8.00 (m, 11H, ArH), 12.52 (s, 1H, NHPh, exchangeable
with D2 O); IR (KBr) v: 3211 cm−1 (NH); MS (70 eV) m/z (%): 413 (M+ , 6). Anal. calcd. for C24 H23 N5 S:
C, 69.71; H, 5.61; N, 16.94; S, 7.75; found C, 69.60; H, 5.70; N, 16.91; S, 7.81.
6-(2,4,6-Trimethylphenyl)-N-phenyl-[1,2,4]triazolo[3,4-a]phthalazin-3-amine (33)
A mixture of 32 (0.01 mol) and 3 molar equivalents of mercuric chloride in dry chloroform (50 mL) was heated
under reflux, while stirring, for 24 h. Chloroform was evaporated, and the residue boiled with ethanol (20
mL) containing 10% aqueous hydrochloric acid solution (30 mL) and saturated with hydrogen sulfide gas. The
mercuric sulfide formed was filtered, and the filtrate evaporated until all alcohol was removed. After cooling, it
355

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
was extracted with benzene; the benzene was then removed to give uncyclized products. The acidic layer was
made distinctly alkaline with sodium hydrogen carbonate to separate compound 33 in 50% yield; mp 163-164
◦ C; 1 H-NMR (DMSO-d6) δ: 2.36 (s, 3H, CH3), 2.48 (s, 6H, 2CH3), 7.01-8.10 (m, 11H, ArH), 9.25 (s, 1H,
NHPh, exchangeable with D2 O); IR (KBr) v: 3211 cm−1 (NH); MS (70 eV) m/z (%): 379 (M+ , 6). Anal.
calcd. for C24 H21 N5 : C, 75.97; H, 5.58; N, 18.46; found C, 76.10; H, 6.00; N, 18.38.
Results and discussion
Aroylation of an aromatic system by reaction with phthalic anhydride under Friedel Craft’s conditions yields
o-aroylbenzoic acid.41,42 Thus, reaction of mesitylene with phthalic anhydride in the presence of anhydrous
aluminum chloride was carried out to produce 2-(2,4,6-trimethyl benzoyl)benzoic acid (2). Merchant et al.43
prepared phthalazin-l-ones via the condensation of the aroyl benzoic acid with hydrazine hydrate in boiling
ethanol. Accordingly, adopting the Merchant et al. procedure, condensation of benzoic acid derivative 2
with hydrazine hydrate in boiling ethanol afforded the 4-(2,4,6-trimethyl phenyl)-2H -phthalazin-l-one (3) in
65% yield. The IR spectrum showed a characteristic absorption band at 1654 cm−1 corresponding to the
CO group. The 1 H-NMR spectrum of compound 3 showed a NH signal at 11.17 ppm. Compound 3 was
treated with a mixture of phosphorous oxychloride and phosphorous pentachloride to afford the corresponding
chlorophthalazine 4, which is a promising intermediate for the synthesis of diverse annelated phthalazine
derivatives (Scheme 1). The structure of compound 4 was confirmed on the basis of its elemental analysis
and spectral data. The IR spectrum revealed no absorption for NH and CO groups.
Scheme 1. Synthesis of 1-chloro-4-(2,4,6-trimethylphenyl)phthalazine (4).
The reactivity of chlorophthalazine 4 towards some nitrogen nucleophiles was investigated for the con-
struction of a novel heteroaromatic system. Thus, interaction of chlorophthalazine 4 with anthranilic acid under
fusion conditions afforded the corresponding quinazolinone derivative 6 as the only isolable product. The for-
mation of 6 was explained by the formation of intermediate 5, which undergoes intramolecular ring closure to
form the final product 6. The IR spectrum of compound 6 showed an absorption band at 1670 cm−1 assigned
to the CO group. The 13 C-NMR spectrum of 6 exhibited the expected number of signals for the aromatic
carbons as well as 3 methyl signals and a carbonyl signal at 20.9, 23.5 and 170.1 ppm. Treatment of compound
4 with the sodium salt of various amino acids, namely glycine, alanine, and β-alanine under reflux conditions
356

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
Scheme 2. Synthesis of acyclo C -nucleosides 12, 13 and some annelated phthalazine derivatives.
357

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
produced the corresponding phthalazinaylamino acids (7a,b and 8). The IR spectrum of compounds 7a,b and
8 revealed the presence of a CO group at 1694-1702 cm−1 . The 1 H-NMR spectrum of compound 7a displayed
CH2 protons as a singlet signal at δ 4.61, and exchangeable NH and OH protons as singlet signals at δ 8.01
and 10.81, respectively. The 1 H-NMR spectrum of compound 7b displayed a CH proton as a quartet signal
at δ 4.50 and CH3 protons as a doublet signal at δ 1.53. The 1 H-NMR spectrum of compound 8 showed a
triplet signal at δ 2.81 assigned for NCH2 and a triplet signal at 3.77 assigned for CH2 CO beside NH and
OH protons. The amino acid derivatives 7a,b and 8 were easily cyclized44 via 1,3-tautomerism in boiling
acetic anhydride in the presence of anhydrous sodium acetate to yield imidazophthalazine derivatives 9a,b and
pyrimidinophthalazine 10. The 1 H-NMR spectrum of compounds 9a,b and 10 showed the absence of NH and
OH signals. The reaction of chlorophthalazine 4 with different acid hydrazide, namely acetic acid hydrazide and
benzoic acid hydrazide afforded 1,2,4-triazolo[3,4-a]phthalazine derivatives 11a,b, respectively. The 13 C-NMR
spectrum of 11a exhibited the expected number of signals for the aromatic carbons as well as 4 methyl signals
at 10.1, 20.3 and 22.2 ppm. Treatment of compound 4 with gluconic acid hydrazide afforded 1,2,4-triazolo[3,4-
a]phthalazine acyclo C -nucleoside derivative 12. The IR spectrum of compound 12 revealed a broad absorption
band at 3425-3222 cm−1 attributable to OH groups. The 1 H-NMR spectrum showed the presence of the sugar
protons. On the other hand, treatment of 2 equivalents of 4 with galactaric acid bishydrazide and malonic
acid dihydrazide yielded double headed 1,2,4-triazolo[3,4-a]phthalazine acyclo C -nucleoside derivative 13 and
bis{6-(2,4,6-trimethylphenyl)-[1,2,4]triazolo[3,4-a]phthalazin-3-yl}methane (14), respectively (Scheme 2). The
1 H-NMR spectrum of compound 13 displayed tetritolyl 4 exchangeable OH protons as 2 doublet signals at δ
5.45 and 5.10 and tetritolyl 4 CH protons as 2 doublet signals at 4.84 and 3.79 ppm. The 13 C-NMR spectrum
of compound 13 gave characteristic signals in accordance with the assigned structure. The 1 H-NMR spectrum
of compound 14 showed δ 5.03 ppm for assigned CH2 and aromatic protons.
Fusion of chlorophthalazine 4 with o-phenylenediamine afforded benzo[4,5]imidazo[2,1-a]phthalazine
derivative 15. Interaction of chlorophthalazine 4 with 2-amino-3-carboxy-4,5-dimethylthiophene afforded the
corresponding dimethylthienopyrimidinone derivative 16. Similarly, 4 reacted with 2-amino-3-carbethoxy
tetrahydrobenzothiophene to afford tetrahydrobenzothienopyrimidinone derivative 17. The IR spectrum of
compound 16 and 17 revealed the presence of a CO group at 1695 and 1711 cm−1 , respectively (Scheme 3).
The 13 C-NMR spectrum of compound 17 gave characteristic signals in accordance with the assigned structure.
Treatment of chlorophthalazine 4 with thiosemicarbazide in boiling ethanol afforded triazolo derivative
18. The 1 H-NMR spectrum of compound 18 displayed 2 exchangeable NH2 protons as a singlet signal at 6.24
ppm. Condensation of 18 with different aromatic aldehydes, namely benzaldehyde and p-chlorobenzaldehyde
in absolute ethanol afforded the corresponding Schiff bases 19a,b, respectively. The IR spectrum of compounds
19a,b showed a characteristic absorption band at 1618 and 1621 cm−1 corresponding to the C=N group,
respectively. The 1 H-NMR spectra of compounds 19a,b showed the presence of azomethin (CH=N) at δ
8.51 and 9.05 ppm, respectively. Interaction of compound 4 with o-chlorobenzylamine in pyridine afforded
phthalazino[2,1-a]quinazoline derivative 20. The 1 H-NMR spectrum of compound 20 showed δ 2.37 (s, 3H,
CH3), 2.49 (s, 6H, 2CH3), 4.90 (s, 2H, CH2 quinazoline), and aromatic protons. Furthermore, the reaction of
chlorophthalazine 4 with ammonium thiocyanate in dry acetone afforded the nonisolable intermediate 21 that
reacted in situ with phenyl isocyanate via 2+4 cycloaddition reaction to yield triazinophthalazine derivative 22.
The IR spectrum of compound 22 revealed the presence of a CO group at 1675 cm−1 . The 13 C-NMR spectrum
358

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
Scheme 3. Synthesis of annelated phthalazine derivatives 14-17.
of 22 exhibited the expected number of signals for the aromatic carbons as well as 3 methyl signals, carbonyl
and thiocarbonyl signals at 20.1, 23.2, 155.8, and 180.5 ppm. Treatment of chlorophthalazine 4 with sodium
azide in boiling acetic acid afforded tetrazolo derivative 23 (Scheme 4). The mass spectrum of 23 showed a
peak corresponding to its molecular ion at m/z 289.
Treatment of chlorophthalazine 4 with hydrazine hydrate in boiling ethanol afforded hydrazine derivative
24. The 1 H-NMR spectrum of 24 showed signals at 5.31 for assigned NH2 (exchangeable with D2 O) and 9.55
for assigned NH (exchangeable with D2 O). Cyclo-condensation of compound 24 with different α-haloketons,
namely chloroacetone and phenacylbromide in dry xylene afforded the corresponding hydrazone, which under-
went 1,3-tautomerism followed by ring closure to give 1,2,4-triazino[3,4-a]phthalazine derivatives 25a,b. The IR
spectra of compounds 25a,b showed a characteristic band at 1615 and 1610 cm−1 corresponding to the C=N
group. The 1 H-NMR spectrum of compound 25a showed the presence of a NH signal at 7.92 ppm. Moreover,
the reaction of compound 4 with ethylbromoacetate yielded 7-(2,4,6-trimethylphenyl)-2H-[1,2,4]triazino[3,4-
a]phthalazin-3(4H)-one (26). Compound 26 was obtained by nucleophilic attack of NH2 of hydrazino moiety
359

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
Scheme 4. Synthesis of annelated phthalazine derivatives 18-23.
to acyl carbon of the ester group through a tetrahedral mechanism followed by 1,3-tautomerism and ring closure
via a S2N mechanism. The IR spectrum of compound 26 showed the presence of an absorption band for CO
group at 1677 cm−1 . The 1 H-NMR spectrum of compound 26 showed a singlet signal at 4.57 for assigned CH2
(triazine moiety). Moreover, 1,2,4-triazino[4,3-c]phthalazine 27 was obtained from the reaction of compound 4
with diethyl oxalate in absolute ethanol by a ring closure happening via consecutive 2 tetrahedral mechanisms.
The IR spectrum of compounds 27 showed the presence of absorption bands for CO groups at 1680-1691 cm−1
.
The 1 H-NMR spectrum of compound 27 showed a signal at 8.32 for assigned NH (exchangeable with D2 O).
Condensation of compound 24 with pyruvic acid by heating at 180 ◦ C gave the corresponding triazino deriva-
360

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
Scheme 5. Synthesis of 1,2,4-triazino[3,4-a]phthalazine 25a,b-28.
tive 28 in 47% yield (Scheme 5). The IR spectrum of compound 28 revealed the presence of a CO group at
1679 cm−1 . The 13 C-NMR spectrum of 28 exhibited the expected number of signals for the aromatic carbons
as well as 4 methyl signals and a carbonyl signal at 16.8, 20.7, 22.4, and 166.0 ppm.
Cyclocondensation of compound 24 with formic acid gave s-triazolo derivative 29. Its IR revealed no
absorption for NHNH2 . The 1 H-NMR spectrum of compound 29 showed a CH signal of the triazole ring at 9.57
ppm. Cyclization of 24 using carbon disulfide in alcoholic potassium hydroxide gave the corresponding triazolo
derivative 30. The IR spectrum of compound 30 revealed the presence of a NH group at 3218 cm−1 . On the
other hand, cyclization of 24 using ethylchloroformate in pyridine gave the corresponding triazolo derivative 31.
The IR spectrum of compound 31 revealed the presence of a CO group at 1664 cm−1 . The 1 H-NMR spectrum
of compound 31 showed a NH signal at 8.78 ppm. Refluxing of compound 24 with phenyl isothiocyanate in
absolute ethanol afforded the thiocarbamate derivative 32. Cyclodesulfurization reaction of compound 32 with
mercuric chloride afforded the phenylamino derivative 33 (Scheme 6). The 1 H-NMR spectrum of compound
361

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
Scheme 6. Synthesis of 1,2,4-triazolo[3,4-a]phthalazines 29- 31 and 33.
32 showed δ 1.96 (s, 1H, CSNH, exchangeable with D2 O) and 12.52 (s, 1H, NHPh, exchangeable with D2 O)
ppm. The 1 H-NMR spectrum of compound 33 showed δ 9.26 assigned for NHPh (exchangeable with D2 O).
Antimicrobial assay
The antimicrobial activity of the newly synthesized compounds 6, 9-23 and 25-31, 33 were evaluated against
Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi bacterial strains and Aspergillums
niger and Candida albicans fungal strains by disk diffusion method. Amoxicillin and Ketoconazole were used
as standard drugs for the bacteria and fungi, respectively. Preliminary screening of phthalazine-derivatives and
standard drugs was performed at fixed concentrations of 500 μg/mL. Inhibition was recorded by measuring the
diameter of the inhibition zone at the end of 24 h for bacteria and 72 h for fungi. Each experiment was repeated
362

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
twice. Based on the results of zone of inhibition, the minimum inhibitory concentration (MIC) of compounds 6,
9-23 and 25-31, 33 against all bacterial and fungal strains was determined by liquid dilution method. Stock
solutions of tested compounds with 500, 250, 200, 100, 50, 25, 12.5, and 6.25 μg mL−1 concentrations were
prepared with DMSO solvent. The solutions of standard drugs, Amoxicillin and Ketoconazole, were prepared
Table. Antimicrobial activity of compounds 6, 9-23 and 25-31, 33.
Minimum inhibitory concentration (MIC) in μg/mL
Compounds
Bacterial strains
Fungal strains
S. aureus B. subtilis S. typhi E. coli A. niger C. albican
6
500
200
250
500
50
200
-
-
500
500
50
-
250
250
500
125
62.5
62.5
62.5
-
500
500
500
250
125
100
125
62.5
250
500
250
250
125
125
62.5
250
62.5
62.5
-
9a
250
200
250
50
9b
500
250
100
50
10
100
50
11a
11b
25
50
25
12
25
50
25
50
13
50
25
50
25
14
200
-
200
500
250
500
50
250
250
500
250
100
25
200
500
250
250
50
15
-
16
200
200
50
250
500
125
125
125
500
125
62.5
-
17
18
19a
25
25
50
19b
25
50
25
25
20
500
25
200
50
250
25
200
25
22
23
25
25
25
25
25a
250
100
500
200
200
50
-
500
50
500
200
-
25b
50
250
500
250
500
250
62.5
125
250
-
500
-
26
250
50
-
27
100
100
50
100
200
25
500
250
125
125
250
125
-
28
250
50
29
30
31
25
50
25
100
50
100
100
6.25
-
200
50
50
33
50
50
Amoxicillin
Ketoconazole
6.25
-
6.25
-
6.25
-
31.25
31.25
363

Synthesis and antimicrobial evaluation of some annelated phthalazine..., M. A. A. EL-HASHASH, et al.
in the same concentrations. Inoculums of the bacterial and fungal culture were also prepared. To a series of
tubes containing 1 mL each of phthalazine compound solution with different concentrations and 0.2 mL of the
inoculums was added. Further 3.8 mL of sterile water was added to each of the test tubes. These test tubes were
incubated for 24 h at 37 ◦ C and observed for the presence of turbidity. This method was repeated by changing
phthalazine compounds with standard drugs Amoxicillin and Ketoconazole for comparison. The minimum
inhibitory concentration at which no growth was observed was taken as the MIC value (Table). The comparison
of the MICs (in μg/mL) of potent compounds and standard drugs against tested strains are presented in the
Table.
Investigation of the antibacterial screening data (Table) showed that some of the compounds were
active against 4 pathogenic bacteria. 1,2,4-Triazolo[3,4-a]phthalazine derivatives 11b, 12, 19a,b, 30, 1,3,5-
triazino[4,3-a]phthalazine 22, and tetrazolo[5,1-a] phthalazine 23 exhibited good activity against S. aureus.
Similarly 1,2,4-triazolo[3,4-a]phthalazine derivatives 13, 19a, and tetrazolo[5,1-a] phthalazine 23 exhibited
good activity against B. subtilis. 1,2,4-Triazolo[3,4-a]phthalazine derivatives 12, 19a,b, 30, 1,3,5-triazino[4,3-
a]phthalazine 22, and tetrazolo[5,1-a] phthalazine 23 exhibited good activity against S. typhi. 1,2,4-Triazolo[3,4-
a]phthalazine derivatives 11b, 13, 19b, 29, 1,3,5-triazino[4,3-a]phthalazine 22, and tetrazolo[5,1-a] phthalazine
23 exhibited good activity against E. coli.
The antifungal results (Table) revealed that the synthesized compounds showed variable degrees of inhibi-
tion against the tested fungi. Compounds 11b, 12, 13, 19b, 22, 23, and 30 possessed good antifungal activity
against A. niger and C. albican. From the results it was concluded that the 1,2,4-triazolo[3,4-a]phthalazine
derivatives, 1,3,5-triazino[4,3-a]phthalazine 22, and tetrazolo[5,1-a] phthalazine 23 showed better activity.
Conclusions
In this article we report the synthesis of annelated phthalazine derivatives and acyclo C-nucleoside starting from
1-chloro-4-(2,4,6-trimethylphenyl)phthalazine (4). Investigation of their antimicrobial activity revealed that
1,2,4-triazolo[3,4-a]phthalazine derivatives, 1,3,5-triazino[4,3-a]phthalazine 22, and tetrazolo[5,1-a] phthalazine
23 were the most active compounds although the activity was significantly less than that of the positive control.
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