Synthesis of some triazolophthalazine derivatives for their anti-inflammatory and antimicrobial activities

Article abstract of DOI:10.1002/ardp.201100053

Several novel series of triazolophthalazine derivatives namely; pyrazolylethenyltriazolophthalazinones (4a-d), styryltriazolophthalazinones (5a,b), aryloxopropenyltriazolophthalazinones (7a,b), pyrazolinyl-(8a,b), (9a,b) and (10a-f), pyrazolyl-(11a-d), (1,2-oxazol-5-yl)-1,2,4-triazolo[3,4-a] phthalazin-6(5H)-ones (14a,b), triazolo[3,4-a]phthalazin-3-yl-pyridine-3- carbonitriles (12a,b), triazolo[3,4-a]phthalazin-3-yl)ethylthioacetic acids (13a,b) and 2-aryl-5-arylamino-1H,5H-pyrazolo[2a€,3a€-1a5] imidazo[3,4-1,5]-1,2,4-triazolo[3,4-a]phthalazin-12(13H)-ones (15a-c) have been synthesized. The anti-inflammatory activity of representative compounds has been studied. Compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a showed anti-inflammatory activities comparable to that of the reference standard, indomethacin. They exhibit also minimal ulcerogenic effect relevant to the reference standard and were found to be non-toxic up to 120 mg/kg orally or up to 75/kg through parenteral route. Concerning the antimicrobial activity; compounds 12b and 13b were found to be equipotent to ampicillin against Staphylococcus aureus, while compounds 10a and 10f were found to be as potent as ampicillin against E. coli, whereas compound 14b exhibited equipotency to clotrimazole against Candida albicans. Compounds 8b, 10f, 11b, 12a, and 13b exhibited, besides their antimicrobial activity, moderate to potent anti-inflammatory profiles. This represents a fruitful matrix for the development of a new class of dual non-acidic anti-inflammatory/antimicrobial agents. Several novel series of triazolophthalazine derivatives has been synthesized. The results reveal that compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a showed anti-inflammatory activities comparable to indomethacin. Compounds 8b, 10f, 11b, 12a, and 13b exhibited, besides their antimicrobial activity, moderate to potent anti-inflammatory profiles.

Full text of DOI:10.1002/ardp.201100053

530
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Full Paper
Synthesis of Some Triazolophthalazine Derivatives for Their
Anti-Inflammatory and Antimicrobial Activities
Nargues S. Habib1, Ahmed M. Farghaly1, Fawzia A. Ashour1, Adnan A. Bekhit1,
Heba A. Abd El Razik1, and Tarek Abd El Azeim2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
2 Department of Pharmacology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
Several novel series of triazolophthalazine derivatives namely; pyrazolylethenyltriazolophthalazinones
(4a–d), styryltriazolophthalazinones (5a,b), aryloxopropenyltriazolophthalazinones (7a,b), pyrazolinyl-
(8a,b), (9a,b) and (10a–f), pyrazolyl- (11a–d), (1,2-oxazol-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones
(14a,b), triazolo[3,4-a]phthalazin-3-yl-pyridine-3-carbonitriles (12a,b), triazolo[3,4-a]phthalazin-3-yl)-
ethylthioacetic acids (13a,b) and 2-aryl-5-arylamino-1H,5H-pyrazolo[200,300-10,50]imidazo[30,40-1,5]-1,2,4-
triazolo[3,4-a]phthalazin-12(13H)-ones (15a–c) have been synthesized. The anti-inflammatory activity
of representative compounds has been studied. Compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a showed
anti-inflammatory activities comparable to that of the reference standard, indomethacin. They exhibit
also minimal ulcerogenic effect relevant to the reference standard and were found to be non-toxic up to
120 mg/kg orally or up to 75 mg/kg through parenteral route. Concerning the antimicrobial activity;
compounds 12b and 13b were found to be equipotent to ampicillin against Staphylococcus aureus, while
compounds 10a and 10f were found to be as potent as ampicillin against E. coli, whereas compound 14b
exhibited equipotency to clotrimazole against Candida albicans. Compounds 8b, 10f, 11b, 12a, and 13b
exhibited, besides their antimicrobial activity, moderate to potent anti-inflammatory profiles. This
represents a fruitful matrix for the development of a new class of dual non-acidic anti-inflammatory/
antimicrobial agents.
Keywords: Acute toxicity / Anti-inflammatory/antimicrobial activities / Triazolophthalazines / Ulcerogenic effect
Received: February 13, 2011; Revised: March 12, 2011; Accepted: April 6, 2011
DOI 10.1002/ardp.201100053
Introduction
known as selective COX-2 enzyme inhibitor [8]. The discovery
that the naturally occurring pyrazole C-nucleoside, pyrazo-
furin (4-hydroxy-3-b-D-ribofuranosyl-1H-pyrazole-5-carboxa-
mide), possesses broad spectra of antimicrobial and antiviral
activities in addition of being active against several tumor
cell lines [9, 10] has enlightened the way for many pyrazole
derivatives to be screened for comparable activities [11–15].
Motivated by the afore-mentioned findings, and as a continu-
ation of our ongoing program in the field of anti-inflamma-
tory/antimicrobial agents [16–21], it was designed to
synthesize novel series of pyrazole derivatives that would
have dual function with minimal GI disorders and high
safety margin. For this reason, in addition of subjecting
the target compounds to anti-inflammatory and antimicro-
bial screening, the ulcerogenic and acute toxicity profiles
were also determined. Some substituted isoxazole derivatives
Several triazolophthalazines were found to exhibit antifun-
gal and antibacterial activities [1–3]. In addition, phthala-
zines and triazolophthalazines were found to exhibit anti-
inflammatory profiles [4–7]. Among the already marketed
COX-2 inhibitors that comprise pyrazole nucleus, celecoxib
(4-[5-(4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfon-
amide), occupies a unique position as a potent and GI-safe
anti-inflammatory and analgesic agent. It is considered as a
typical model of the diaryl heterocycle template that is
Correspondence: Heba A. Abd El Razik, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.
E-mail: heba_attia75@yahoo.com
Fax: 002-03-4873273
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
531
were found to possess anti-inflammatory [22, 23] and anti-
microbial [24, 25] activities. This prompted us to synthesize
compounds having both triazolophthalazine and isoxazole
ring systems hoping to obtain compounds with dual anti-
inflammatory and antimicrobial activities. Moreover, design
to synthesize several pyridyltriazolophthalazinones was
rationalized on the fact that some pyridine derivatives were
reported to possess anti-inflammatory [26, 27] and antimicro-
bial activities [1, 28]. Furthermore, arylacetic acid derivatives
as indomethacin and ibuprofen are receiving intensive inter-
est as anti-inflammatory agents [29]. Therefore, it became of
interest to synthesize triazolophthalazylthioacetic acid
derivatives hoping to go a step forward in the field of anti-
inflammatory agents.
the key starting material, 3-methyl-1,2,4-triazolo[3,4-a]phthal-
azin-6(5H)-one 2 [30], which was reacted with 1,3-diarylpyr-
azole-4-carbaldehydes 3a-d yielding 3-[2-(1,3-diaryl-1H-pyrazol-
4-yl)ethenyl]-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones (4a–d).
Refluxing 2 with 4-substituted benzaldehydes afforded 3-
(4-substituted styryl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones
(5a,b), whereas refluxing 2 with 4-substituted arylglyoxals
afforded 3-(3-aryl-3-oxopropenyl)-1,2,4-triazolo[3,4-a]phthala-
zin-6(5H)-ones (7a,b). The latter compounds were subjected
to cyclization using hydrazine hydrate to yield 3-(3-aryl-2-
pyrazolin-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones (8a,b).
These were subjected to benzoylation giving rise to 3-(3-aryl-
1-benzoyl-2-pyrazolin-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-
ones (9a,b). Refluxing 7a,b with hydrazine hydrate in excess
carboxylic acids afforded 3-(1-formyl or acyl-3-aryl-2-pyrazo-
lin-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones (10a–f).
In addition, refluxing 7a,b with substituted hydrazines yielded
3-(1-substituted 3-aryl-1H-pyrazol-5-yl)-1,2,4-triazolo[3,4-a]phthal-
azin-6(5H)-ones (11a–d), with malononitrile/ammonium
acetate afforded 6-aryl-2-cyanomethylidene-4-(6-oxo-5,6-dihy-
dro-1,2,4-triazolo[3,4-a]phthalazin-3-yl)-1,2-dihydropyridine-
Results and discussion
Chemistry
To achieve the synthesis of the target compounds, the steps
outlined in Scheme 1 were adopted. Thus, reaction of 1-
hydrazinophthalazinone 1 with acetic anhydride afforded
Scheme 1. Synthetic pathway.
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532
N. S. Habib et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
–
–
3-carbonitriles (12a,b), with hydroxylamine 3-(3-aryl-1,2-oxazol-
5-yl)-1,2,4-triazolo[3,4-a]phthalazin-6(5H)-ones (14a,b) were
obtained whereas, with substituted thiosemicarbazides yielded
2-aryl-5-arylamino-1H,5H-pyrazolo[200,300-10,50]imidazo[30,40-1,5]-
1,2,4-triazolo[3,4-a]phthalazin-12(13H)-ones (15a–c). Moreover,
the prepared chalcones 7a,b were allowed to react with thio-
glycolic acid to give 2-(4-substituted benzoyl)-1-(6-oxo-5,6-dihy-
dro-1,2,4-triazolo[3,4-a]phthalazin-3-yl)ethylthioacetic acids
(13a,b). The structures of the newly synthesized compounds
were confirmed by IR, 1H-NMR, microanalyses as well as mass
spectra for some representative examples.
1715 cmꢀ1. Ketone C O and phthalazinone C O overlapped
–
–
together and appeared as one band at 1686-1683 cmꢀ1
.
1H-NMR of 13b showed a singlet at 3.51 ppm assigned for
SCH2 proton, and three double doublets; two assigned for
CH2CO and the third assigned for CH proton. MS of 13a did
not show the molecular ion peak (Mþ) at m/z 408 and showed
the base peak at m/z 287. FAB mass spectrum of 13a showed a
peak at m/z 409 corresponding to (Mþ þ H).
IR spectra of 14a,b showed several stretching C–O–C
vibration bands due to isoxazole in addition to the band
1
–
–
–
–
due to NH, C O and C N. H-NMR of 14b showed a singlet
1H-NMR of compound 4d was characterized by the presence
at 8.02 ppm assigned for isoxazole C4-H.
of two doublets at 7.31 and 7.84 (J ¼ 16.8 Hz) assigned for
1H-NMR of 15b showed two singlets at 2.23 and 4.19 ppm
assigned for CH3 and CH2 protons and two deuterium
exchangeable singlets at 12.57 and 12.95 assigned to two
NH protons, while that of 15c revealed three singlets at
2.25, 2.32 and 4.19 ppm assigned for two CH3 groups and
CH2 protons, respectively. MS of 15a, 15b, and 15c showed the
molecular ion peaks at m/z 433, 447 and 461 corresponding
to C25H19N7O, C26H21N7O and C27H23N7O, respectively, which
are also the base peaks.
–
CH CH, indicating E configuration.
–
1H-NMR of 5a showed two doublets at 7.38 and 7.46 ppm
due to CH ¼ CH. 1H-NMR of 7a showed two triplets and a
doublet assigned for phenyl protons, two doublets at 8.25 and
–
8.52 ppm characteristic for CH CH.
–
1H-NMR of 8b showed a singlet at 2.33 assigned for CH3
protons and two double doublets at 3.48 and 3.80 ppm
assigned for pyrazoline-C4 protons and another double
doublet at 5.34 ppm characteristic for pyrazoline-C5-H.
–
IR spectra of 9a,b showed additional C O absorption band
Biology
Anti-inflammatory activity
–
at 1694-1692 cmꢀ1 not found in their precursors due to the
1
benzoyl C O. H-NMR of 9a showed two double doublets at
–
–
The anti-inflammatory activity of twenty two representatives
of the synthesized compounds 2, 7b, 8a,b, 10a–f, 11a–d, 12a,b,
13b, 14a,b, and 15a–c was evaluated in vivo using the
sponge implantation model of inflammation in rats with
indomethacin as a standard. Polyester sponge implantation,
the animal model chosen in this study, has the merit of
triggering a non-immune acute type of inflammatory response.
This model was used to assess the possibility of some of
the synthesized compounds altering the course of inflam-
mation. This was assessed by determining their effects on
the inflammatory exudate parameters: Exudate volume, total
leukocyte cell count (TLC), differential leukocyte cell count
(DLC), reduced cytochrome C and interleukin-1 beta (IL-1b)
levels (Tables 1 and 2).
3.57 and 3.95 characteristic for pyrazoline-C4 protons and
another double doublet at 6.24 assigned for pyrazoline-C5-H,
a triplet at 7.52, a multiplet between 7.71–7.74 and a doublet
at 7.85 ppm assigned for benzoyl protons.
–
IR spectra of 10a–f showed additional C O absorption
–
–
band or the two C O absorption bands overlapped together
–
and appeared as broad band. 1H-NMR of 10a showed a singlet
at 8.10 ppm for CHO, 1H-NMR of 10b and 10e showed
additional singlet-not found in their precursors-assigned
1
for COCH3, whereas H-NMR of 10f showed a triplet at 1.00
and a quartet at 2.69 ppm characteristic for COCH2CH3.
1H-NMR of 11a showed two triplets and a doublet assigned
for N-phenyl protons, a multiplet assigned for the second
phenyl protons and a singlet at 7.63 ppm characteristic for
pyrazole-C4-H. 1H-NMR of the aminosulphonyl derivative 11b
showed two multiplets assigned for phenyl protons, two
doublets at 7.58 and 7.88 ppm assigned for C6H4SO2- and a
deuterium exchangeable singlet assigned for NH2.
The neutrophil phagocytic function was determined
as one of the most important indices used to assess the
different neutrophilic functions. This is because it is the
ultimate and major step in neutrophilic response to acute
inflammation. The main step in this function is the respir-
atory burst that reflects an important face of immune system
integrity. This was evaluated by the cytochrome C reduction
test, where the reduction of cytochrome C by superoxide
anion will change its light absorption properties, as detected
spectrophotometrically.
IR spectra of 12a,b showed two C N at 2259-2255 and
2213-2208 cmꢀ1 absorption bands in addition to NH,
C O and C N absorption bands. 1H-NMR spectra of 12a
–
–
–
–
and 12b showed two singlets at 6.92–7.02 and 7.32–
–
7.40 ppm assigned for CH and pyridine C protons, respect-
–
5
ively. MS of 12a and 12b showed the molecular ion peaks at
m/z 403 and 417, respectively, which are also the base peaks.
IR spectra of 13a,b revealed OH broad absorption band
Effect on exudate volume
Results presented in Table 1 revealed that pre-treatment with
indomethacin significantly reduced the exudate volume in
–
between 3100 and 2300 and acid C O band at 1718–
–
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Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
533
Table 1. Effect of inflammation induced by sponge implantation, in drug-pretreated rats, on non-immunological parameters
Compd.
Exudate volumea
TLCb
DLC (neutrophil %)c
X (ꢁ SE)
X (ꢁ SE)
X (ꢁ SE)
Control
Indomethacin
2
7b
8a
8b
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
12a
0.349 (ꢁ 0.016)
0.221 (ꢁ 0.012)
0.319 (ꢁ 0.015)
0.316 (ꢁ 0.015)
0.308 (ꢁ 0.014)
0.256 (ꢁ 0.012)ꢃ
0.310 (ꢁ 0.016)
0.299 (ꢁ 0.014)
0.248 (ꢁ 0.013)ꢃ
0.332 (ꢁ 0.017)
0.298 (ꢁ 0.015)
0.236 (ꢁ 0.013)ꢃ
0.329 (ꢁ 0.014)
0.234 (ꢁ 0.011)ꢃ
0.315 (ꢁ 0.017)
0.356 (ꢁ 0.016)
0.264 (ꢁ 0.013)ꢃ
0.297 (ꢁ 0.014)
0.248 (ꢁ 0.013)ꢃ
0.328 (ꢁ 0.015)
0.305 (ꢁ 0.016)
0.229 (ꢁ 0.011)ꢃ
0.307 (ꢁ 0.017)
0.332 (ꢁ 0.018)
261.82 (ꢁ 17.24)
122.38 (ꢁ 13.21)
231.17 (ꢁ 16.28)
251.24 (ꢁ 16.28)
247.29 (ꢁ 17.21)
139.42 (ꢁ 12.28)ꢃ
241.56 (ꢁ 15.27)
237.26 (ꢁ 16.51)
143.18 (ꢁ 13.16)ꢃ
199.26 (ꢁ 15.37)
233.63 (ꢁ 16.27)
143.25 (ꢁ 14.21)ꢃ
228.36 (ꢁ 16.28)
137.52 (ꢁ 14.23)ꢃ
205.33 (ꢁ 16.37)
238.11 (ꢁ 17.28)
139.22 (ꢁ 13.16)ꢃ
209.28 (ꢁ 17.16)
147.19 (ꢁ 12.71)ꢃ
216.24 (ꢁ 16.28)
213.51 (ꢁ 17.67)
129.36 (ꢁ 14.11)ꢃ
213.29 (ꢁ 17.51)
213.26 (ꢁ 17.86)
89.32 (ꢁ 3.37)
47.16 (ꢁ 2.16)
76.39 (ꢁ 3.37
81.56 (ꢁ 3.21)
77.26 (ꢁ 3.16)
48.27 (ꢁ 2.24)ꢃ
84.16 (ꢁ 3.56)
72.16 (ꢁ 3.39)
51.23 (ꢁ 2.21)ꢃ
76.37 (ꢁ 4.10)
79.36 (ꢁ 3.57)
52.39 (ꢁ 2.63)ꢃ
75.36 (ꢁ 3.27)
49.28 (ꢁ 2.37)ꢃ
80.33 (ꢁ 3.58)
72.27 (ꢁ 3.36)
53.18 (ꢁ 3.39)ꢃ
71.25 (ꢁ 4.16)
46.17 (ꢁ 3.51)ꢃ
70.16 (ꢁ 3.96)
78.21 (ꢁ 4.28)
52.31 (ꢁ 2.28)ꢃ
74.36 (ꢁ 3.36)
72.18 (ꢁ 3.91)
12b
13b
14a
14b
15a
15b
15c
P > 0.05 unless otherwise stated; ꢃP < 0.001. Exudate Volume (expressed in mL). Total leukocytic count (TLC) (expressed as
a
b
cell/cm3). Differential leukocytic count (DLC) (expressed as exudate neutrophil %).
c
comparison to the inflammatory control group (P < 0.001).
The mean values were 0.221 ꢁ 0.012 and 0.349 ꢁ 0.016 mL
respectively.
Effect on differential leukocyte cell count (DLC)
Table 1 clearly shows that pre-treatment with indomethacin
significantly reduced the exudate neutrophil
%
Pre-treatment with compounds 8b, 10c, 10f, 11b, 12a, 13b,
and 15a significantly reduced the exudate volume as com-
pared to the inflammatory control group (P < 0.001). The
mean values were 0.256 ꢁ 0.012, 0.248 ꢁ 0.013, 0.236 ꢁ
0.013, 0.234 ꢁ 0.011, 0.264 ꢁ 0.013, 0.248 ꢁ 0.013, and
0.229 ꢁ 0.011 mL, respectively, compared to a control mean
value of 0.349 ꢁ 0.016 mL.
(47.16 ꢁ 2.16%) as compared to the inflammatory control
group (89.32 ꢁ 3.37%) (P < 0.001). Pre-treatment with com-
pounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a significantly
reduced the exudate neutrophil %. The mean values were
48.27 ꢁ 2.24, 51.23 ꢁ 2.21, 52.39 ꢁ 2.63, 49.28 ꢁ 2.37,
53.18 ꢁ 3.39, 46.17 ꢁ 3.51, and 52.31 ꢁ 2.28%, compared
to a control mean value of 89.32 ꢁ 3.37%.
Effect on total leukocyte cell count (TLC)
Effect on neutrophil phagocyte function
From Table 1 it is clearly shown that pre-treatment with
indomethacin significantly reduced the exudate TLC
(122.38 ꢁ 13.21) as compared to the inflammatory control
group (261.82 ꢁ 17.24) cell/cm3 (P < 0.001). Pre-treatment
with compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a signifi-
cantly reduced the exudate TLC as compared to the inflam-
matory control group (P < 0.001). The mean values were
139.42 ꢁ 12.28, 143.18 ꢁ 13.16, 143.25 ꢁ 14.21, 137.52 ꢁ
14.23, 139.22 ꢁ 13.16, 147.19 ꢁ 12.71, and 129.36 ꢁ
14.11 cell/cm3, respectively, compared to a control mean
value of 261.82 ꢁ 17.24 cell/cm3.
Neutrophil phagocyte function was measured using the cyto-
chrome C reduction test. In this test, both the basal level
of reduced cytochrome C (unstimulated), as well as its level
after stimulation of granulocytes by means of Zymosan
particles (stimulated) was estimated. Results are expressed
as nmol O2ꢀ/(2 ꢂ 106 PMN h). Table 2 revealed that pre-
treatment with indomethacin significantly reduced the
unstimulated reduced cytochrome C level (0.711 ꢁ 0.012)
as compared to the inflammatory control group (1.416 ꢁ
0.019) (P < 0.001). Results showed that pre-treatment
with the investigated compounds 8b, 10c, 10f, 11b,
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N. S. Habib et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Table 2. Effect of inflammation induced by sponge implantation, in drug-pretreated rats, on immunological parametersa,b
Compd.
Unstimulated reduced
cytochrome C levels
X (ꢁ SE)
Stimulated reduced
cytochrome C levels
X (ꢁ SE)
Unstimulated
IL-1b levels
X (ꢁ SE)
LPS stimulated
IL-1b levels
X (ꢁ SE)
Control
Indomethacin
2
7b
8a
8b
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
12a
1.416 (ꢁ 0.019)
0.711 (ꢁ 0.012)
1.173 (ꢁ 0.019)
1.316 (ꢁ 0.018)
1.256 (ꢁ 0.019)
0.781 (ꢁ 0.011)ꢃ
1.217 (ꢁ 0.018)
1.196 (ꢁ 0.017)
0.796 (ꢁ 0.013)ꢃ
1.186 (ꢁ 0.016)
1.213 (ꢁ 0.019)
0.786 (ꢁ 0.013)ꢃ
1.136 (ꢁ 0.017)
0.786 (ꢁ 0.014)ꢃ
1.263 (ꢁ 0.017)
1.181 (ꢁ 0.014)
0.756 (ꢁ 0.011)ꢃ
1.271 (ꢁ 0.018)
0.781 (ꢁ 0.013)ꢃ
1.296 (ꢁ 0.019)
1.196 (ꢁ 0.018)
0.806 (ꢁ 0.013)ꢃ
1.168 (ꢁ 0.017)
1.127 (ꢁ 0.018)
1.516 (ꢁ 0.026)
0.821 (ꢁ 0.015)
1.282 (ꢁ 0.022)
1.392 (ꢁ 0.021)
1.361 (ꢁ 0.024)
0.856 (ꢁ 0.015)ꢃ
1.311 (ꢁ 0.025)
1.286 (ꢁ 0.026)
0.854 (ꢁ 0.015)ꢃ
1.256 (ꢁ 0.024)
1.326 (ꢁ 0.025)
0.868 (ꢁ 0.017)ꢃ
1.246 (ꢁ 0.023)
0.912 (ꢁ 0.016)ꢃ
1.326 (ꢁ 0.023)
1.287 (ꢁ 0.022)
0.846 (ꢁ 0.016)ꢃ
1.381 (ꢁ 0.023)
0.876 (ꢁ 0.014)ꢃ
1.376 (ꢁ 0.023)
1.273 (ꢁ 0.022)
0.891 (ꢁ 0.016)ꢃ
1.251 (ꢁ 0.021)
1.234 (ꢁ 0.023)
194.36 (ꢁ 14.21)
106.21 (ꢁ 6.29)
175.24 (ꢁ 13.31)
173.27 (ꢁ 13.51)
181.21 (ꢁ 13.57)
120.36 (ꢁ 5.38)ꢃ
176.17 (ꢁ 14.59)
169.28 (ꢁ 13.36)
119.37 (ꢁ 7.35)ꢃ
166.36 (ꢁ 12.56)
174.27 (ꢁ 13.81)
124.36 (ꢁ 7.23)ꢃ
168.45 (ꢁ 13.28)
122.76 (ꢁ 7.26)ꢃ
175.86 (ꢁ 13.52)
176.2 (ꢁ 14.21)
119.36 (ꢁ 7.36)ꢃ
170.18 (ꢁ 12.56)
118.31 (ꢁ 6.16)ꢃ
187.36 (ꢁ 13.91)
194.36 (ꢁ 14.21)
175.24 (ꢁ 13.31)ꢃ
173.27 (ꢁ 13.51)
176.17 (ꢁ 14.59)
281.21 (ꢁ 17.31)
141.29 (ꢁ 7.28)
263.41 (ꢁ 16.31)
255.17 (ꢁ 16.53)
259.24 (ꢁ 16.27)
164.18 (ꢁ 7.36)ꢃ
236.39 (ꢁ 17.27)
206.26 (ꢁ 16.27)
163.62 (ꢁ 6.28)ꢃ
232.41 (ꢁ 16.21)
263.72 (ꢁ 16.69)
171.39 (ꢁ 8.19)ꢃ
227.26 (ꢁ 15.21)
176.34 (ꢁ 8.16)ꢃ
251.36 (ꢁ 16.36)
244.27 (ꢁ 16.21)
162.31 (ꢁ 8.16)ꢃ
261.23 (ꢁ 15.22)
171.23 (ꢁ 18.24)ꢃ
235.17 (ꢁ 15.28)
246.23 (ꢁ 14.37)
186.22 (ꢁ 8.18)ꢃ
256.24 (ꢁ 17.11)
266.16 (ꢁ 16.18)
12b
13b
14a
14b
15a
15b
15c
P > 0.05 unless otherwise stated; ꢃP < 0.001. Neutrophil Phagocyte Function (expressed in nmol O.2/2X106 PMN/h) on both
a
b
unstimulated and stimulated reduced cytochrome C level. The assay of Interleukin-1b (IL-1b) (expressed in pg/mL) on both
unstimulated IL-1b level and LPS stimulated IL-1b level.
12a, 13b, and 15a significantly reduced the cytochrome C
level as compared to the inflammatory control group
(P < 0.001). The mean values were 0.781 ꢁ 0.011, 0.796 ꢁ
0.013, 0.786 ꢁ 0.013, 0.786 ꢁ 0.014, 0.756 ꢁ 0.011, 0.781 ꢁ
0.013, and 0.806 ꢁ 0.013, respectively, compared to a control
value of 1.416 ꢁ 0.019.
taneously released as well as the lipo-polysaccharide (LPS)
stimulated release of IL-1b). The results were expressed
in pg/mL.
Results, presented in Table 2, revealed that pre-treatment
with indomethacin significantly decreased the unstimulated
IL-1b level (106.21 ꢁ 6.29) as compared to the inflammatory
control group (194.36 ꢁ 14.21) (P < 0.001). Pre-treatment
with compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a signifi-
cantly decreased the unstimulated IL-1b levels in comparison
to the inflammatory control group (P < 0.001). The mean
values were 120.36 ꢁ 5.38, 119.37 ꢁ 7.35, 124.36 ꢁ 7.23,
122.76 ꢁ 7.26, 119.36 ꢁ 7.36, 118.31 ꢁ 6.16, and 175.24 ꢁ
13.31, respectively, compared to a control mean value of
194.36 ꢁ 14.21. Results presented in Table 2 also showed that
pre-treatment with indomethacin significantly decreased the
LPS-stimulated IL-1b levels (141.29 ꢁ 7.28) as compared to the
inflammatory control group (281.21 ꢁ 17.31) (P < 0.001).
Pre-treatment with compounds 8b, 10c, 10f, 11b, 12a, 13b,
and 15a significantly decreased the LPS-stimulated IL-1b
levels in comparison to the inflammatory control group
(P < 0.001). The mean values were 164.18 ꢁ 7.36,
Furthermore, results clearly show that pre-treatment with
indomethacin significantly reduced the stimulated cyto-
chrome C level (0.821 ꢁ 0.015) as compared to the inflam-
matory control group (1.516 ꢁ 0.026) (P < 0.001). Pre-
treatment with compounds 8b, 10c, 10f, 11b, 12a, 13b, and
15a significantly decreased the stimulated reduced cyto-
chrome C level in comparison to the inflammatory control
group (P < 0.001). Their mean value were 0.856 ꢁ 0.015,
0.854 ꢁ 0.015, 0.868 ꢁ 0.017, 0.912 ꢁ 0.016, 0.846 ꢁ 0.016,
0.876 ꢁ 0.014, and 0.891 ꢁ 0.016, respectively, compared to
a control mean value of 1.516 ꢁ 0.026.
Effect on interleukin-1b (IL-1b) level
The assay of interleukin-1b using an IL-1b ELISA kit was done
by estimating both the unstimulated level of IL-1b (spon-
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Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
535
163.62 ꢁ 6.28, 171.39 ꢁ 8.19, 176.34 ꢁ 8.16, 162.31 ꢁ 8.16,
171.23 ꢁ 8.24, and 186.22 ꢁ 8.18, respectively, compared to
a control mean value of 281.21 ꢁ 17.31.
its tolyl analogue 12b was not. Among the pyrazole deriva-
tives 11a–d only 11b which contains a sulphone moiety was
active anti-inflammatory agent. Among the acylpyrazoline
series 10a–f, the propanoyl derivatives 10c and 10f
were active whereas their formyl and acetyl derivatives
were not.
It can be concluded that the sponge implantation model of
inflammation seemed to be a reliable method for collection
of an adequate amount of exudate necessary for the evalu-
ation of different inflammatory markers. Results revealed
that pre-treatment with the investigated compounds 8b,
10c, 10f, 11b, 12a, 13b, and 15a significantly altered the
inflammatory markers, not only, the non-immunological
parameters (exudate volume, TLC and DLC), but also the
immunological ones (reduced cytochrome C and IL-1b levels).
Therefore, the indicated compounds are considered capable
of modulating the inflammatory response and are assumed
to have in vivo anti-inflammatory activity similar to that of
indomethacin.
Antimicrobial activity
Twenty representative compounds were selected for antimi-
crobial screening by measuring their minimal inhibitory
concentration (MIC) against Staphylococcus aureus as Gram-
positive bacteria, Escherichia coli as Gram-negative bacteria
and Candida albicans as a fungal strain (Table 3) [34].
It deserves mentioning that the tolylpyridyl derivative 12b
was found to be as potent as ampicillin against S. aureus (MIC
12.5 mg/mL), whereas its phenyl analogue 12a was less active.
Moreover, both 12a and 12b exhibited half the potency of
the reference against E. coli. Among the acylpyrazoline series
10a–f, the formylpyrazoline 10a and 10d exhibited half the
potency of the reference standard against S. aureus, However,
the formyl derivative 10a and propanoyl derivative 10f were
found to be as potent as ampicillin against E. coli and the
acetyl analogue 10e exhibited half the potency of the refer-
ence against E. coli. Among the pyrazole series 11a–d, 11c
exhibited half the potency of the reference standard S. aureus,
Ulcerogenic effects
Compounds which exhibited moderate to potent anti-
inflammatory profiles in the pre-mentioned animal models
(8b, 10c, 10f, 11b, 12a, 13b, and 15a) were evaluated for their
ulcerogenic potential in rats [31]. Phenylbutazone and indo-
methacin were tested as reference drugs. It was found
that the incidence of ulcers with these compounds was
30%, 30%, 20%, 40%, 10%, 20%, and 30%, respectively, com-
pared with phenylbutazone (60% ulceration) or indometha-
cin (100% ulceration).
Acute toxicity
Table 3. Minimum inhibitory concentrations (MIC) of
representative compounds.
Compounds 8b, 10c, 10f, 11b, 12a, 13b, and 15a which
showed promising anti-inflammatory activities were
further investigated for their oral as well as parenteral
route acute toxicity in male mice [32, 33]. All of the tested
compounds proved to be non-toxic up to 120 mg/kg
when given orally. In addition, the same compounds proved
to be non-toxic up to 75 mg/kg when administered
parenterally.
Compd.
MIC (mg/mL)
E. coli
S. aureus
C. albicans
7b
8b
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
12a
12b
13b
14a
100
50
25
>200
>200
>200
50
25
>200
50
>200
>200
50
50
50
>200
>200
100
>200
50
>200
>200
25
100
100
25
100
100
>200
>200
25
>200
>200
100
50
100
>200
100
>200
50
100
>200
>200
>200
>200
>200
25
12.5
50
>200
>200
–
It is clear that compounds 8b, 10c, 10f, 11b, 12a, 13b, and
15a under test were found to exhibit minimum ulcerogenic
activity as compared to phenylbutazone and indomethacin.
They are well tolerated by experimental animals and showed
high safety margin as revealed from their LD50 values
(>500 mg kgꢀ1). These speculations remain to be further
expanded in other models of experimental inflammation
and confirmed by clinical trials before a final drug design
is to be made.
50
50
12.5
12.5
>200
>200
>200
50
It is to be noted down that the tolylpyrazoline derivative
8b showed anti-inflammatory activity whereas its phenyl
analogue 8a was devoid of activity. Similarly, for the
tolylthioacetic acid derivative 13b against its phenyl
analogue 13a. On the other hand, its phenylpyridyl derivative
12a was found to elicit anti-inflammatory activity whereas
14b
15a
15b
15c
50
Ampicillin
Clotrimazole
25
–
12.5
–
12.5
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536
N. S. Habib et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
whereas 11b exhibited half the potency of the reference Experimental
standard against E. coli.
The tolylthioacetic acid derivative 13b was found to be as
potent as ampicillin against S. aureus but it showed half its
potency against E. coli. The most active compound against
C. albicans was the isoxazolyl derivative 14b which was found
to be equipotent to clotrimazole, whereas the 3-phenyl analogue
14a showed half its potency. The pyrazoline 8b exhibited half
the potency of ampicillin against E. coli. Moreover, only the
tolylpyrazoloimidazotriazolophthalazinone 15c showed half
the potency of the reference against E. coli.
Chemistry
Melting points were performed on a Gallen Kamp apparatus and
are uncorrected. IR spectra (KBr) were measured on a Perkin-
Elmer 1430 Spectrophotometer. 1H-NMR spectra were measured
on a Varian Gemini 200 spectrometer, 200 MHz with TMS as
internal standard. The chemical shifts are given in d (ppm).
Microanalyses were performed at the Microanalytical Unit,
Faculty of Science, Assiut University, Egypt. All the values of
C, H, N, and S were within ꢁ0.4% of the calculated data.
3-[2-(1,3-Diaryl-1H-pyrazol-4-yl)ethenyl]1,2,4-triazolo[3,4-
a]phthalazin-6(5H)-ones 4a–d
A mixture of 2 (0.4 g, 2 mmol) and the appropriate 3 (2 mmol) in
Ac2O (5 mL) was heated under reflux for 5 h then allowed to cool.
The obtained precipitate was filtered, washed with EtOH, dried,
and crystallized from gl. HOAc (Table 4). IR (KBr, cmꢀ1): 3413–
It can be concluded that compounds 8b, 10f, 11b, 12a,
and 13b possess dual anti-inflammatory/antimicrobial
activities. These compounds would represent a fruitful
matrix for the development of a new class of dual non-acidic
anti-inflammatory/antimicrobial agents.
Table 4. Physicochemical data of compounds 4–10
Compd.
R
R1
Yield
(%)
M.p. (8C)
Cryst. solv.
Mol. formula
(Mol. wt.)
4a
H
CH3
Cl
–
51
53
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
C26H18N6O
(430.47)
1
4b
–
C27H20N O ꢄ = H O
2
6
2
(453.51)
1
4c
–
63
C26H17ClN O ꢄ = H O
2
6
2
(473.93)
1
4d
5a
Br
–
70
C26H17 BrN O ꢄ = H O
2
6
2
(518.38)
C17H11BrN4O
(367.21)
Br
–
63
5b
NO2
H
–
87
C17H11N5O3
(333.31)
C18H12N4O2
(316.32)
C19H14N4O2
(330.35)
C18H14N6O
(330.35)
C19H16N6O
(344.38)
C25H18N6O2
(434.46)
C26H20N6O2
(448.49)
C19H14N6O2
(358.36)
7a
–
70
7b
CH3
H
–
70
8a
–
91
8b
CH3
H
–
90
9a
–
–
48
9b
CH3
H
56
10a
10b
10c
10d
10e
10f
H
75
286–288
DMF
297–299
DMF
188–190
DMF/H2O
>300
DMF/H2O
201–203
HOAc/H2O
188–190
EtOH/H2O
H
CH3
CH2CH3
H
90, 91ꢃ
90
C20H16N6O2
(372.39)
H
C21H18N6O2 ꢄ 2 H2O
(422.45)
1
CH3
CH3
CH3
67
C20H16N6O2 ꢄ = H O
2
2
(381.40)
C21H18N6O2 ꢄ H2O
CH3
CH2CH3
95, 97ꢃ
98
(404.43)
C22H20N6O2 ꢄ 2 H2O
(436.47)
ꢃ
Yield according to Method B.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
537
–
–
–
3127 (NH); 1663–1661 (C O); 1638–1626 (C N); 1599–1595,
7.84–8.24 (m, 2H, triazolophthalazine-C8,9-H); 8.43 (d, J ¼ 7.8 Hz,
1H, triazolophthalazine-C10-H); 8.52 (d, J ¼ 8.1 Hz, 1H, triazolo-
phthalazine-C7-H); 13.2 (s, 1H, NH, D2O exchangeable).
–
–
–
1503–1501 (C C); 1537–1519 (d NH); 1415–1411, 1349–1333
(C–N lactam). 1H-NMR (DMSO-d6), (d ppm), (Jeol 500 MHz) of 4d:
7.31 (d, J ¼ 16.8 Hz, 1H, CH ¼ CH); 7.35 (t, J ¼ 7.6 Hz, 1H, phe-
nyl-C4-H); 7.53 (t, J ¼ 7.6 Hz, 2H, phenyl-C3,5-H); 7.67, 7.72 (two d,
J ¼ 8.4 Hz, each 2H, bromophenyl-H); 7.84 (d, J ¼ 16.8 Hz, 1H,
3-(3-Aryl-1-benzoyl-2-pyrazolin-5-yl)-1,2,4-triazolo[3,4-
a]phthalazin-6(5H)-ones 9a,b
–
–
CH CH ); 7.85 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C -H); 7.94
8
To a suspension of the appropriate 8 (1 mmol) in gl. HOAc (3 mL),
benzoyl chloride (0.15 g, 0.13 mL, 1.1 mmol) was added. The
reaction mixture was heated under reflux while stirring for
1 h then allowed to cool. The obtained precipitate was filtered,
washed with EtOH, dried, and crystallized from DMF/EtOH (Table
(d, J ¼ 7.6 Hz, 2H, phenyl-C2,6-H); 7.99 (t, J ¼ 7.65 Hz, 1H, triazo-
lophthalazine-C9-H); 8.17 (d, J ¼ 8.4 Hz, 1H, triazolophthalazine-
C10-H); 8.43 (d, J ¼ 7.65 Hz, 1H, triazolophthalazine-C7-H); 9.15
(s, 1H, pyrazole-C5-H); 13.23 (s, 1H, NH, D2O exchangeable).
4). IR (KBr, cmꢀ1): 3307 (NH); 1694–1692 (COC H ); 1663 (C O);
–
–
6
5
–
–
–
3-(4-Substituted styryl)-1,2,4-triazolo[3,4-a]phthalazin-
6(5H)-ones 5a,b
1644, 1631–1627 (C N); 1599–1593, 1501 (C C); 1533 (d NH);
–
1427–1411, 1341–1313 (C–N lactam). 1H-NMR (DMSO-d6), (d ppm),
(Jeol 500 MHz) of 9a: 3.57 (dd, J ¼ 17.57, 5.35 Hz, 1H, pyrazoline-
C4-H); 3.95 (dd, J ¼ 17.57, 12.2 Hz, 1H, pyrazoline-C4-H); 6.24
(dd, J ¼ 12.2, 5.35 Hz, 1H, pyrazoline-C5-H); 7.43–7.5 (m, 5H,
C6H5); 7.52 (t, J ¼ 6.9 Hz, 1H, benzoyl-C4-H); 7.71–7.74 (m, 2H,
benzoyl-C3,5-H); 7.85 (d, J ¼ 7.65 Hz, 2H, benzoyl-C2,6-H); 7.87
(t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C8-H); 7.99 (t, J ¼ 7.65 Hz,
1H, triazolophthalazine-C9-H); 8.19 (d, J ¼ 7.65 Hz, 1H, triazolo-
phthalazine-C10-H); 8.41 (d, J ¼ 7.65 Hz, 1H, triazolophthalazine-
C7-H); 13.28 (s, 1H, NH, D2O exchangeable).
A mixture of 2 (0.4 g, 2 mmol) and p-bromo- or p-nitrobenzalde-
hyde (2 mmol) in Ac2O (5 mL) was heated under reflux for 5 h
then allowed to cool. The obtained precipitate was filtered,
washed with EtOH, dried, and crystallized from the proper solvent
(Table 4). IR (KBr, cmꢀ1): 3391–3327 (NH); 1660 (C O); 1642, 1624–
–
–
–
–
–
–
1618 (C N); 1595–1594, 1515–1513, 1488 (C C); 1554 (d NH);
1461, 1334 (C–N lactam). For 5a, 700 (C-Br), for 5b, 1554, 1343
(NO2). 1H-NMR (DMSO-d6), (d ppm), (Varian Gemini 200 MHz)
–
of 5a: 7.38, 7.46 (two d, J ¼ 10 Hz, each 1H, CH CH); 7.6–7.8
–
(m, 4H, bromophenyl-H); 7.88 (t, J ¼ 7.8 Hz, 1H, triazolophthala-
zine-C8-H); 8.02 (t, J ¼ 7.8 Hz, 1H, triazolophthalazine-C9-H);
8.21(d, J ¼ 8 Hz, 1H, triazolophthalazine-C10-H); 8.47 (d, J ¼ 7.9 Hz,
1H, triazolophthalazine-C7-H); 13.01 (s, 1H, NH, D2O exchangeable).
5.1.6. 3-(1-Formyl or acyl-3-aryl-2-pyrazolin-5-yl)-1,2,4-
triazolo[3,4-a]-phthalazin-6(5H)-ones 10a–f
Method A:
A mixture of the appropriate 7 (1 mmol), NH2NH2 ꢄ H2O (0.2 g,
0.19 mL, 4 mmol) and the appropriate carboxylic acid (10 mL)
was heated under reflux for 8–12 h then allowed to cool. The
obtained precipitate was filtered, washed with EtOH and crystal-
lized from the proper solvent (Table 4).
3-(3-Aryl-3-oxopropenyl)-1,2,4-triazolo[3,4-a]phthalazin-
6(5H)-ones 7a,b
A mixture of 2 (2 g, 10 mmol) and phenyl or p-tolylglyoxal 6a or b
(11 mmol) in Ac2O (10 mL) was heated under reflux for 1 h then
allowed to cool. The obtained precipitate was filtered, washed
with EtOH, and crystallized from dioxane. IR (KBr, cmꢀ1): 3375–
Method B:
A suspension of the appropriate 8 (1 mmol) in Ac2O (3 mL) was
heated under reflux for 1 h during which dissolution and re-
precipitation occurred. The reaction mixture was allowed to cool.
The obtained precipitate was filtered, washed with EtOH, and
crystallized from the proper solvent. IR (KBr, cmꢀ1): 3414–3175
–
–
3332 (NH); 1668–1665 (C O); 1647, 1616 (C N); 1599–1594, 1516–
–
–
–
1512 (C C); 1570–1553 (d NH); 1411, 1341–1337 (C–N lactam).
–
1H-NMR (DMSO-d6), (d ppm), (Varian Gemini 200 MHz) of 7a:
7.62 (t, J ¼ 7.63 Hz, 2H, phenyl-C3,5-H); 7.72 (d, J ¼ 7.63 Hz,
2H, phenyl-C2,6-H); 7.85 (t, J ¼ 7.63 Hz, 1H, phenyl-C4-H); 7.93
(t, J ¼ 7.6 Hz, 1H, triazolophthalazine-C8-H); 8.07 (t, J ¼ 7.6 Hz,
1H, triazolophthalazine-C9-H); 8.16 (t, J ¼ 8 Hz, 1H, triazolo-
–
–
(NH); 1694–1691, 1660 (C O); 1643–1621 (C N); 1609–1595, 1520–
–
–
–
–
1500 (C C); 1555–1533 (d NH); 1439–1412, 1336–1311 (C–N lac-
tam). 1H-NMR (DMSO-d6), (d ppm), (Jeol 500 MHz) of 10a: 2.85 (dd,
J ¼ 17.98, 6.5 Hz, 1H, pyrazoline-C4-H); 3.20 (dd, J ¼ 17.98,
11.85 Hz, 1H, pyrazoline-C4-H); 5.35 (dd, J ¼ 11.85, 6.5 Hz, 1H,
pyrazoline-C5-H); 6.61–6.7 (m, 3H, phenyl-C3,4,5-H); 7.01–7.10
(m, 3H, phenyl-C2,6-H and triazolophthalazine-C8-H); 7.16
(t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C9-H); 7.49 (d, J ¼
7.65 Hz, 1H, triazolophthalazine-C10-H); 7.65 (d, J ¼ 7.65 Hz, 1H,
triazolophthalazine-C7-H); 8.10 (s, 1H, CHO). 1H-NMR (DMSO-d6),
(d ppm), (Varian Gemini 200 MHz) of 10b: 2.33 (s, 3H, CH3); 3.55
(dd, J ¼ 20, 4.7 Hz, 1H, pyrazoline-C4-H); 3.95 (dd, J ¼ 20, 11 Hz,
1H, pyrazoline-C4-H); 6.04 (dd, J ¼ 11, 4.7 Hz, 1H, pyrazoline-C5-H);
7.45–7.56 (m, 3H, phenyl-C3,4,5-H); 7.78–7.86 (m, 2H, phenyl-C2,6-H);
7.88 (t, J ¼ 7.7 Hz, 1H, triazolophthalazine-C8-H); 8.02
(t, J ¼ 7.7 Hz, 1H, triazolophthalazine-C9-H); 8.21 (d, J ¼
7.94 Hz, 1H, triazolophthalazine-C10-H); 8.44 (d, J ¼ 7.88 Hz,
1H, triazolophthalazine-C7-H); 13.22 (s, 1H, NH, D2O exchange-
able). 1H-NMR (DMSO-d6), (d ppm), (Jeol 500 MHz) of 10e: 2.26
(s, 3H, CH3); 2.32 (s, 3H, CH3); 3.48 (dd, J ¼ 18, 5.35 Hz, 1H, pyrazo-
line-C4-H); 3.87 (dd, J ¼ 18, 12.2 Hz, 1H, pyrazoline-C4-H); 5.97 (dd,
J ¼ 12.2, 5.35 Hz, 1H, pyrazoline-C5-H); 7.27, 7.66 (two d, J ¼ 8 Hz,
each 2H, tolyl-H); 7.85 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C8-H);
–
–
–
phthalazine-C10-H); 8.25 (d, J ¼ 7.68 Hz, 1H, CH CH-C O); 8.48
–
(t, J ¼ 8.3 Hz, 1H, triazolophthalazine-C7-H); 8.52 (d, J ¼ 7.68 Hz,
–
–
–
1H, CH CH-C O); 13.3 (s, 1H, NH, D O exchangeable). MS, m/z (%)
–
2
of (7a): 316 (16) (Mþ), 287 (100).
3-(3-Aryl-2-pyrazolin-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-
6(5H)-ones 8a,b
To a suspension of the appropriate 7 (1 mmol) in abs. EtOH
(5 mL), NH2NH2 ꢄ H2O (0.2 g, 0.19 mL, 4 mmol) was added. The
reaction mixture was heated under reflux for 30 min then
allowed to cool. The obtained precipitate was filtered, washed
with EtOH, dried, and crystallized from DMF/EtOH, 1:2 (Table 4).
IR (KBr, cmꢀ1): 3269–3260, 3149 (NH); 1677–1663 (C O); 1647,
–
–
–
–
–
–
1627–1626 (C N); 1596–1595, 1519–1481 (C C); 1532–1530
(d NH); 1419–1412, 1333–1308 (C–N lactam). 1H-NMR (DMSO-d6),
(d ppm), (Varian 300 MHz) of 8b: 2.33 (s, 3H, CH3); 3.48 (dd,
J ¼ 16.5, 10.2 Hz, 1H, pyrazoline-C4-H, under DMSO); 3.80 (dd,
J ¼ 16.5, 6 Hz, 1H, pyrazoline-C4-H); 5.34 (dd, J ¼ 10.2, 6 Hz, 1H,
pyrazoline-C5-H,); 7.23, 7.59 (two d, J ¼ 8.1 Hz, each 2H, tolyl-H);
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538
N. S. Habib et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
7.98 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C9-H); 8.17 (d, J ¼
7.65 Hz, 1H, triazolophthalazine-C10-H); 8.39 (d, J ¼ 7.65 Hz, 1H,
triazolophthalazine-C7-H); 13.75 (s, 1H, NH, D2O exchangeable). 1H-
NMR (DMSO-d6), (d ppm), (Jeol 500 MHz) of 10f: 1.00 (t, J ¼ 7.65 Hz,
3H, CH2CH3); 2.32 (s, 3H, CH3); 2.69 (q, J ¼ 7.65 Hz, 2H, CH2CH3);
3.48 (dd, J ¼ 17.55, 5.35 Hz, 1H, pyrazoline-C4-H); 3.86 (dd,
J ¼ 17.55, 12.2 Hz, 1H, pyrazoline-C4-H); 5.99 (dd, J ¼ 12.2,
5.35 Hz, 1H, pyrazoline-C5-H); 7.26, 7.66 (two d, J ¼ 8 Hz, each
2H, tolyl-H); 7.84 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C8-H);
7.98 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C9-H); 8.17 (d, J ¼
7.65 Hz, 1H, triazolophthalazine-C10-H); 8.39 (d, J ¼ 7.65 Hz, 1H,
triazolophthalazine-C7-H); 13.26 (s, 1H, NH, D2O exchangeable). MS,
m/z (%) of 10f: 400 (20) (Mþ), 158 (100).
7.63 (s, 1H, pyrazole-C4-H); 7.86 (t, J ¼ 7.65 Hz, 1H, triazolo-
phthalazine-C8-H); 7.95 (d, J ¼ 7.6 Hz, 2H, N-phenyl-C2,6-H); 8.01
(t, J ¼ 7.65 Hz, 1H, triazolophthalazine-C9-H); 8.15 (d, J ¼ 7.65 Hz,
1H, triazolophthalazine-C10-H); 8.44 (d, J ¼ 7.65 Hz, 1H, triazolo-
phthalazine-C7-H); 13.16 (s, 1H, NH, D2O exchangeable). 1H-NMR
(DMSO-d6), (d ppm), (Jeol 500 MHz) of 11b: 7.32–7.4 (m, 3H, phenyl-
C3,4,5-H); 7.43 (s, 2H, NH2, D2O exchangeable); 7.58, 7.88 (two d,
J ¼ 8.4 Hz, each 2H, C6H4SO2-); 7.76 (s, 1H, pyrazole-C4-H); 7.82–
7.84 (m, 2H, phenyl-C2,6-H); 7.85 (t, J ¼ 7.65 Hz, 1H, triazolo-
phthalazine-C8-H); 7.98 (t, J ¼ 7.65 Hz, 1H, triazolophthalazine-
C9-H); 8.23 (d, J ¼ 8.4 Hz, 1H, triazolophthalazine-C10-H); 8.41
(d, J ¼ 8.4 Hz, 1H, triazolophthalazine-C7-H).
6-Aryl-2-cyanomethylidene-4-(6-oxo-5,6-dihydro-1,2,4-
triazolo[3,4-a]-phthalazin-3-yl)-1,2-dihydropyridine-3-
carbonitriles 12a,b
A mixture of the appropriate 7 (1 mmol), malononitrile (0.07 g,
1.1 mmol), and NH4OAc (0.31 g, 4 mmol) in abs. EtOH (10 mL)
was heated under reflux for 6 h then allowed to cool. The obtained
precipitate was filtered, washed with EtOH then with H2O, dried,
and crystallized from DMF/EtOH (Table 5). IR (KBr, cmꢀ1): 3380,
3235–3234, 3113–3111 (NH); 2259–2255, 2213–2208 (C N); 1662–
3-(1-Substituted 3-aryl-1H-pyrazol-5-yl)-1,2,4-triazolo[3,4-
a]phthalazin-6(5H)-ones 11a–d
A mixture of the appropriate 7 (1 mmol), phenyl hydrazine ꢄ HCl
or 4-hydrazinobenzenesulphonamide ꢄ HCl (1.1 mmol) and
NaOAc (0.09 g, 1.1 mmol) in abs. EtOH (10 mL) or abs. EtOH
containing few drops gl. HOAc was heated under reflux for
10 h then allowed to cool. The obtained precipitate was filtered,
washed with EtOH then with H2O, dried, and crystallized from
dioxane/H2O (Table 5). IR (KBr, cmꢀ1): 3398–3263 (NH); 1663–1661
–
–
–
1660 (C O); 1642, 1626 (C N); 1587, 1520, 1484 (C C); 1551 (d NH);
–
–
–
1416, 1321 (C–N lactam). 1H-NMR (DMSO-d6), (d ppm), (Varian
–
–
–
(C O); 1644–1643, 1627–1615 (C N); 1599–1594, 1513–1500 (C C);
–
–
–
1570–1532 (d NH); 1411–1405, 1342–1332 (C–N lactam); for 11b
and 11d 1386–1383, 1162–1160 (SO2). 1H-NMR (DMSO-d6), (d ppm),
(Jeol 500 MHz) of 11a: 7.31 (t, J ¼ 6.9 Hz, 1H, N-phenyl-C4-H); 7.34–
7.42 (m, 5H, phenyl-H); 7.47 (t, J ¼ 7.6 Hz, 2H, N-phenyl-C3,5-H);
–
300 MHz) of 12a: 7.02 (s, 1H, CH); 7.40 (s, 1H, pyridine-C -H);
5
–
7.52–7.6 (m, 3H, phenyl-C3,4,5-H); 7.66–7.72 (m, 2H, phenyl-C2,6-H);
7.93 (t, J ¼ 7.6 Hz, 1H, triazolophthalazine-C8-H); 8.08
(t, J ¼ 7.6 Hz, 1H, triazolophthalazine-C9-H); 8.22 (d, J ¼ 7.6 Hz,
Table 5. Physicochemical data of compounds 11–15.
Compd.
R
R1
Yield
(%)
M.p. (8C)
Cryst. solv.
Mol. formula
(Mol. wt.)
11a
11b
11c
11d
12a
12b
13a
13b
14a
14b
15a
15b
15c
H
H
C6H5
75
80
89
87
45
47
59
69
69
87
93
95
94
292–294
263–265
294–295
>300
C24H16N6O ꢄ H2O
(422.45)
1
4-C6H4SO2NH2
C24H17N O S ꢄ = H O
2
7
3
2
(492.52)
C25H18N6O
CH3
CH3
H
C6H5
(418.46)
C25H19N O S ꢄ 11= H O
4-C6H4SO2NH2
2
7
3
2
(524.56)
C23H13N7O ꢄ H2O
–
>300
(421.42)
1
CH3
H
–
>300
C24H15N O ꢄ = H O
2
7
2
(426.44)
C20H16N4O4S
(408.44)
–
284–286
276–278
284–286
>300
CH3
H
–
C21H18N4O4S
(422.47)
–
C18H11N5O2 ꢄ H2O
(347.34)
C19H13N5O2
(343.35)
CH3
H
–
C6H5
266–268
DMF/H2O
224–226
EtOH/H2O
218–220
EtOH/H2O
C25H19N7O ꢄ H2O
(451.49)
C26H21N7O ꢄ H2O
(465.52)
H
C6H4CH3(2)
C6H4CH3(2)
CH3
C27H23N7O ꢄ H2O
(479.55)
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Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
539
1H, triazolophthalazine-C10-H); 8.55 (d, J ¼ 7.6 Hz, 1H, triazolo-
phthalazine-C7-H); 13.2 (s, 1H, NH, D2O exchangeable). 1H-NMR
(DMSO-d6), (d ppm), (Varian 300 MHz) of 12b: 2.39 (s, 3H, CH3);
solution. The obtained precipitate was filtered, washed with
H2O, dried, and crystallized from the proper solvent (Table 5).
IR (KBr, cmꢀ1): 3378–3326, 3212–3149 (NH); 1661–1660 (C O);
–
–
–
–
1644–1642, 1625–1617 (C N); 1595–1593, 1517–1503 (C C);
–
–
6.92 (s, 1H, CH); 7.14, 13.18 (two s, each 1H, 2-NH); 7.32 (s, 1H,
–
–
1
pyridine-C5-H); 7.37, 8.06 (two d, J ¼ 7.2 Hz, each 2H, tolyl-H); 7.58
(t, J ¼ 7.8 Hz, 1H, triazolophthalazine-C8-H); 7.92 (t, J ¼ 7.8 Hz,
1H, triazolophthalazine-C9-H); 8.20 (d, J ¼ 8.1 Hz, 1H, triazolo-
phthalazine-C10-H); 8.52 (d, J ¼ 7.5 Hz, 1H, triazolophthalazine-
C7-H). MS, m/z (%) of (12a): 404 (28) (Mþ þ 1), 403 (100) (Mþ). MS,
m/z (%) of (12b): 418 (29) (Mþ þ 1), 417 (100) (Mþ).
1551–1536 (d NH); 1414–1403, 1338–1333 (C–N lactam). H-NMR
(DMSO-d6), (d ppm), (Jeol 500 MHz) of 15b: 2.23 (s, 3H, CH3);
4.19 (s, 2H, CH2); 6.44 (t, J ¼ 7.65 Hz, 1H, tolyl-C4-H); 6.81
(t, J ¼ 7.65 Hz, 1H, tolyl-C5-H); 6.92 (d, J ¼ 6.1 Hz, 1H, tolyl-C6-
H); 7.36 (t, J ¼ 6.9 Hz, 1H, phenyl-C4-H); 7.4–7.51 (m, 4H, phenyl-
C3,5-H, tolyl-C3-H and pyrazoloimidazotriazolophthalazine-C5-H);
7.69 (d, J ¼ 6.9 Hz, 2H, phenyl-C2,6-H); 7.81 (t, J ¼ 7.65 Hz,
1H, pyrazoloimidazotriazolophthalazine-C10-H); 7.95 (t,
J ¼ 7.65 Hz, 1H, pyrazoloimidazotriazolophthalazine-C9-H); 8.13
(d, J ¼ 7.65 Hz, 1H, pyrazoloimidazotriazolophthalazine-C8-H);
8.37 (d, J ¼ 7.65 Hz, 1H, pyrazoloimidazotriazolophthalazine-
C11-H); 12.57, 12.95 (two s, each 1H, 2-NH, D2O exchangeable).
1H-NMR (DMSO-d6), (d ppm), (Varian 300 MHz) of 15c: 2.25, 2.32
(two s, each 3H, 2 CH3); 4.21 (s, 2H, CH2); 6.49 (t, J ¼ 6.9 Hz, 1H, o-
tolyl-C4-H); 6.85 (t, J ¼ 6.9 Hz, 1H, o-tolyl-C5-H); 6.95 (d, J ¼ 7.2 Hz,
1H, o-tolyl-C6-H); 7.28 (d, J ¼ 8 Hz, 3H, p-tolyl-C3,5-H and o-tolyl-C3-H);
7.40 (m, 1H, pyrazoloimidazotriazolophthalazine-C5-H); 7.57 (d,
J ¼ 8 Hz, 2H, p-tolyl-C2,6-H); 7.85 (t, J ¼ 7.5 Hz, 1H, pyrazoloimida-
zotriazolophthalazine-C10-H); 7.99 (t, J ¼ 7.5 Hz, 1H, pyrazoloimid-
azotriazolophthalazine-C9-H); 8.17 (d, J ¼ 7.5 Hz, 1H, pyrazolo-
imidazotriazolophthalazine-C8-H); 8.41 (d, J ¼ 7.5 Hz, 1H, pyrazo-
loimidazotriazolophthalazine-C11-H). MS, m/z (%) of 15a: 434 (55)
(Mþ þ 1), 433 (100) (Mþ). MS, m/z (%) of 15b: 448 (56) (Mþ þ 1), 447
(100) (Mþ). MS, m/z (%) of 15c: 462 (41) (Mþ þ 1), 461 (100) (Mþ).
2-(4-Substituted benzoyl)-1-(6-oxo-5,6-dihydro-1,2,4-
triazolo[3,4-a]phthalazin-3-yl)ethylthioacetic acids 13a,b
To a suspension of the appropriate 7 (1 mmol) in dry dioxane or
gl. HOAc (10 mL), thioglycolic acid (0.18 g, 0.14 mL, 2 mmol) was
added. The reaction mixture was under reflux for 12 h then
filtered while hot. The obtained precipitate was washed with
dioxane, dried, and crystallized from DMF/EtOH (Table 5). IR
(KBr, cmꢀ1): br 3100–2300 (OH); 3100 (NH); 1718–1715 (C O acid);
–
–
–
–
–
–
1686–1683 (C O ketone, phthalazinone); 1625 (C N); 1595, 1515–
–
1514 (C C); 1551–1532 (d NH); 1412–1409, 1333–1331 (C–N lactam);
–
1279–1267, 1061–1057 (C–S–C); 1120 (C–O). 1H-NMR (DMSO-d6),
(d ppm), (Jeol 500 MHz) of 13b: 2.34 (s, 3H, CH3); 3.51 (s, 2H, SCH2);
3.86 (dd, J ¼ 19.15, 5.35 Hz, 1H, CH2CO); 4.29 (dd, J ¼ 19.15, 9.15 Hz,
1H, CH2CO); 5.02 (dd, J ¼ 9.15, 5.35 Hz, 1H, CH); 7.31, 7.86 (two d,
J ¼ 8.4 Hz, each 2H, tolyl-H); 7.84 (t, J ¼ 7.7 Hz, 1H, triazolophthal-
azine-C8-H); 7.95 (t, J ¼ 7.7 Hz, 1H, triazolophthalazine-C9-H); 8.16
(d, J ¼ 7.7 Hz, 1H, triazolophthalazine-C10-H); 8.37 (d, J ¼ 7.7 Hz,
1H, triazolophthalazine-C7-H); 12.99 (s, 1H, NH, D2O exchange-
able). MS, m/z (%) of 13a: 408 (absent), 344 (4), 317 (10), 316 (20),
288 (25), 287 (100), 272 (12), 271 (20), 213 (10), 195 (13), 140 (10),
130 (26), 129 (10), 128 (10), 105 (13), 104 (6), 103 (16), 102 (21), 77
(22), 76 (11), 51 (7). FAB mass spectrum of 13a: m/z ¼ 409
(Mþ þ H).
Biology
Anti-inflammatory Screening
The anti-inflammatory activity of twenty two representatives of
the synthesized compounds 2, 7b, 8a,b, 10a–f, 11a–d, 12a,b, 13b,
14a,b, and 15a–c was evaluated in vivo using the sponge implan-
tation model of inflammation in rats with indomethacin as a
standard.
3-(3-Aryl-1,2-oxazol-5-yl)-1,2,4-triazolo[3,4-a]phthalazin-
6(5H)-ones 14a,b
A mixture of the appropriate 7 (1 mmol), NH2OH ꢄ HCl (0.1 g,
1.5 mmol), and NaOH (0.08 g, 2 mmol) in n-butanol (5 mL) was
heated under reflux for 8 h then allowed to cool. The obtained
precipitate was filtered, washed with EtOH then with H2O, dried,
and crystallized from dioxane/H2O (Table 5). IR (KBr, cmꢀ1): 3266–
Materials and methods
One hundred and forty-four male albino rats weighing 150–
200 g were used throughout the study. They were kept in the
animal house under standard conditions of light and
temperature with free access to food and water. The animals
were randomly divided into twenty four groups each of six
rats as follows:
–
–
3213, 3188–3134 (NH); 1687–1660 (C O); 1644, 1627 (C N); 1596–
–
–
1591, 1507–1482 (C C); 1552–1525 (d NH); 1407, 1340–1333 (C–N
–
–
lactam); 1265–1227, 1240, 1189–1163, 1140–1104, 1086–1083
1
(C–O–C). H-NMR (DMSO-d6), (d ppm), (Jeol 500 MHz) of 14b: 2.34
(s, 3H, CH3); 7.33, 7.83 (two d, J ¼ 7.65 Hz, each 2H, tolyl-H); 7.91
(t, J ¼ 7.6 Hz, 1H, triazolophthalazine-C8-H); 8.02 (s, 1H, isoxazole-
C4-H); 8.04 (t, J ¼ 7.6 Hz, 1H, triazolophthalazine-C9-H); 8.22
(d, J ¼ 7.6 Hz, 1H, triazolophthalazine-C10-H); 8.49 (d, J ¼ 7.6 Hz,
1H, triazolophthalazine-C7-H); 13.43 (s, 1H, NH, D2O exchangeable).
MS, m/z (%) of 14a: 329 (4) (Mþ), 213 (100).
Group I (control group): Received 1% gum acacia orally
(suspending vehicle) and served as inflammatory control
group, to estimate reference values of the parameters
studied.
Group II (indomethacin pre-treated inflammatory group):
Received indomethacin (Indomethacin-Parco Pharmaceuti-
cals, Egypt) suspended in 1% gum acacia, in a dose of
10 mg/kg body weight per day orally, divided into two
equal doses, for three successive days [34]. On the third
day, the first dose was administrated 30 min before sponge
implantation and the second dose one hour before its
removal.
2-Aryl-5-arylamino-1H,5H-pyrazolo[200,300-
10,50]imidazo[30,40-1,5]-1,2,4-triazolo[3,4-a]phthalazin-
12(13H)-ones 15a–c
To a mixture of 7 (1 mmol) and the appropriate thiosemicarba-
zide (1.1 mmol) in abs. EtOH (5 mL), a few drops conc. H2SO4 were
added. The reaction mixture was heated under reflux for 6 h
then filtered while hot. The filtrate was neutralized with NaHCO3
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

540
N. S. Habib et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Group III to XXIV (pre-treated inflammatory groups): each
being treated with the appropriate synthesized compound.
Each compound was suspended in 1% gum acacia, and was
given in a dose of 10 mg/kg body weight per day, orally,
divided into two equal doses, for three successive days, as
indomethacin.
The granulocyte cell layer was washed 3 times in RPMI-
1640 complete medium (Grand Island Biological Company,
NY) containing 2 mmol glutamine, 100 units/mL penicillin,
100 mg/mL streptomycin, and 20 mmol N1-hydroxyethylpi-
perazine-N2-ethane sulphonic acid (HEPES buffer) (Nalge,
Sybron Corporation-Rochester, NY). The supernatant was
aspirated and the cell pellet was vigorously tapped and
resuspended in a known volume of complete RPMI medium
supplemented with 10% inactivated fetal bovine serum (FBS),
heat inactivated at 568C for 30 min. (Biofluid-Inc., Tockville,
USA). The granulocyte count was then adjusted to 2 ꢂ 106
cells/mL by complete RPMI-1640 supplemented with 10% heat
inactivated FBS (RPMI-HIFBS). This constituted the granulo-
cyte cell suspension that was stored at -208C until tested for
IL-1b levels.
Induction of inflammation
Inflammation was induced by subcutaneous implantation of
dry polyester sponge (type E41-blue-kay Modeler, UK) [35]. The
polyester sponge was cut to a standard size of 15 mm
diameter, 4 mm thickness, and a weight of 17–17.5 mg.
The sponges were sterilized in 70% (v/v) ethanol, washed
thoroughly twice with distilled water and antiseptic solution
(Cereal), and were then dried in an oven (Galena, Poland) at
1008C for 1.5 h.
For implantation, rats were anaesthetized with diethyl
ether and the skin of the ventral aspect of the abdominal
surface was shaved and swabbed with a 1% solution of salon
in 70% (v/v) ethanol/water [36–38]. Two small medial incisions
each of 1 cm length were made on either side of the midline
of the ventral abdominal surface, and two cavities were
performed by blunt dissection, separating the dermis from
the muscular layers. Lastly one dry sponge was implanted in
each side, the incisions were then closed with silk sutures
(Merrill 3/0-sterile silk suture, Ethic on, UK) [36] and the rats
were kept in their cages for 6 h.
Stimulation of granulocytes by lipopolysaccharides (LPS) as
a mitogen (lyophilized E coli, Sigma, USA) [45] 100 mL of the
prepared cell suspension were added to LPS (10 mg/mL)
(reconstituted in 5 mL sterile complete RPMI-1640 medium
and stored at ꢀ208C until used) in a flat-bottomed well of a
microtitre plate. Another 100 mL of the cell suspension (with-
out stimulant) were pipetted into a flat-bottomed well to
serve as a control. Each sample was placed in triplicate well
to obtain enough supernatants for measuring IL-1b levels.
Complete RPMI medium was added to all wells to obtain a
final volume of 200 mL per well. The cell cultures were incu-
bated for 24 h at 378C, 10% CO2, and 100% humidity in a CO2
jar (Angelantoni Scientifica, Italy). After 24 h, the cultures
were centrifuged at 700 ꢂ g for 10 min and the supernatant
containing putative IL-1b were collected and stored at ꢀ208C
until tested for the cytokine by ELISA kit.
Collection of exudates
After 6 h, animals were reanesthetized with ether. The
ventral incisions were quickly opened and the sponges
were carefully dissected out to avoid local bleeding.
Both sponges were gently squeezed with a 5-mL syringe
plunger and the total exudates were collected in polyethy-
lene centrifuge tubes containing 0.1 mL of 1.5% EDTA [36].
This was used to determine non-immunological markers,
namely: Exudates volume, total and differential leukocyte
cell counts, in addition to immunological markers,
namely: Cytochrome C reduction test and interleukin-1 beta
levels.
Statistical methods
Data are expressed as means with their corresponding stand-
ard errors. Data were evaluated by the one way analysis
of variance and were subjected to the least significant differ-
ence ‘LSD’ test [46].
Ulcerogenic effects
Compounds which exhibited moderate to potent anti-inflam-
matory profiles in the pre-mentioned animal models (8b, 10c,
10f, 11b, 12a, 13b, and 15a) were evaluated for their ulcero-
genic potential in rats [31]. Phenylbutazone and indometha-
cin were tested as reference drugs. One hundred male albino
rats (150–200 g) were fasted for 12 h prior to drug adminis-
tration. Water was given ad libitum. The animals were divided
into ten groups each of ten rats as follows:
Cell isolation and preparations
Granulocyte cell suspension was prepared using a modifi-
cation of the method of Lehrer and Cline [39].
Neutrophil phagocyte function estimation [39–43]
The isolated cells were resuspended in 1 mL Hank’s bal-
anced salt solution (HBSS, free from phenol red-Gibco,
England) and were adjusted to
a
concentration of
Group I (control group): Received 2% gum acacia orally.
Group II: Received phenylbutazone suspended in 2% gum
acacia at a dose of 10 mg/kg per day orally.
2 ꢂ 105 cells/mL. This was stored at ꢀ208C until tested for
neutrophilic function by evaluation of the cytochrome C
reduction test as detected spectrophotometrically.
Interleukin-1 beta level estimation (IL-1b) [44]
Group III: Received indomethacin suspended in 2% gum
acacia at a dose of 10 mg/kg per day orally.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 344, 530–542
Synthesis of some triazolophthalazine derivatives
541
Groups IV–X: Received test compounds suspended in 2%
gum acacia at a dose of 10 mg/kg per day orally.
Minimum inhibitory concentration (MIC) determination
1- For each compound four sets of two-fold dilutions of
the compound in a sterile broth were prepared. In each
set, the concentration gradient starts from 800 mg/mL
to 1.5 mg/mL (i.e.10 tubes/set).
2- Each tube of the first set of dilution was inoculated
with one drop (0.02 mL) of a diluted overnight broth
culture of Escherichia coli (ATCC 25922) (i.e., a drop
containing 106 colony forming unit, CFU). The second
and the third sets were similarly inoculated with refer-
ence strains Staphylococcus aureus (ATCC 19433) and
Candida albicans, respectively. The fourth set received
no test organisms and served as broth sterility control
(control a).
The compounds were administrated orally in two equal
doses at 0 and 12 h for three successive days. Animals were
sacrificed after 6 h of the last dose. The stomach from each
animal was removed. An opening at the greater curvature
was made and the stomach was washed with cooled saline
and inspected with 3 X magnifying lens for any evidence for
hyperemia and hemorrhage, definite hemorrhagic erosion or
ulcer. The percentage ulceration for each group was calcu-
lated as follows:
% Ulceration
Number of animals bearing ulcer in a group
¼
ꢂ 100
3- For each run three controls were also included testing
the following:
Total number of animals in the same group
It was found that the incidence of ulcers with these com-
pounds was 30%, 30%, 20%, 40%, 10%, 20%, and 30%, respect-
ively, compared with phenylbutazone (60% ulceration) or
indomethacin (100% ulceration).
- The activity of ampicillin trihydrate against the refer-
ence strain of Escherichia coli (control b).
- The activity of ampicillin trihydrate against the refer-
ence strain of Staphylococcus aureus (control c).
- The activity of clotrimazole against the reference
strain of Candida albicans (control d).
Acute toxicity
The promising compounds 8b, 10c, 10f, 11b, 12a, 13b, and
15a were further investigated for their oral acute toxicity in
male mice [32, 33]. Eight groups of mice each consisting of
six animals, were used. The compounds were given orally in
doses of 1, 10, 40, 80, 100, and 120 mg/kg, respectively.
Twentyfour hours later, the % mortality in each group and
for each compound was recorded.
4- The tubes were incubated for 18–24 h, except for
C. albicans where incubation was left for at least 48 h,
at 378C and examined for turbidity due to bacterial
growth. Control sets were examined first. Control a was
used as indicator of broth sterility (clear tubes) and
control sets (b–d) were used as indicators of reprodu-
cibility of the results.
5- Tubes containing the lowest concentration of the test
compound that prevents visible growth of the respect-
ive organism were judged to contain the MIC of that
organism (Table 3).
Moreover, these compounds were tested for their toxicity
through parenteral route. Groups of mice each consisting of
six animals were used. The compounds or their vehicle pro-
pylene glycol (control) were given intraperitoneal injection
in doses 10, 25, 50, and 75 mg/kg, respectively. Survival was
followed up to 7 days.
The authors have declared no conflict of interest.
All of the tested compounds proved to be non-toxic up to
120 mg/kg when given orally. In addition, the same com-
pounds proved to be non-toxic up to 75 mg/kg when admin-
istered parenterally.
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