Synthesis of some novel pyrazolo[3,4-b]pyridine and pyrazolo[3,4-d]pyrimidine derivatives bearing 5,6-diphenyl-1,2,4-triazine moiety as potential antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2009.05.031

The reaction of 5,6-diphenyl-3-hydrazino-1,2,4-triazine (1) with bis(methylthio)methylene]malononitrile (2) afforded 5-amino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-(methylthio)-1H-pyrazole-4-carbonitrile (3). Compound 3 reacted with thiourea to give 3,4-di

Full text of DOI:10.1016/j.ejmech.2009.05.031

European Journal of Medicinal Chemistry 44 (2009) 4385–4392
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some novel pyrazolo[3,4-b]pyridine and pyrazolo[3,4-d]pyrimidine
derivatives bearing 5,6-diphenyl-1,2,4-triazine moiety as potential
antimicrobial agents
Tarik El-Sayed Ali
Department of Chemistry, Faculty of Education, Ain Shams University, Roxy, 11711 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 August 2008
Received in revised form
The reaction of 5,6-diphenyl-3-hydrazino-1,2,4-triazine (1) with bis(methylthio)methylene]malononi-
trile (2) afforded 5-amino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-(methylthio)-1H-pyrazole-4-carbonitrile
(
3). Compound 3 reacted with thiourea to give 3,4-diaminopyrazolo[3,4-d]pyrimidine 5, which was
6
December 2008
treated with benzoyl chloride to give pyrazolo[5,4,3-kl]pyrimido[4,3-d]pyrimidine 6. Treatment of 3 with
acetic anhydride produced 3-methylthio-pyrazolo[3,4-d]pyrimidine derivative 7, which was allowed to
react with hydrazine hydrate to give the corresponding hydrazino derivative 8. Heterocyclization of 8
with benzoyl chloride and sodium pyruvate afforded the polyfused heterocycles 9 and 10, respectively.
Reaction of 3 with benzoylacetone yielded pyrazolo[3,4-b]pyridine 12, which was allowed to react with
malononitrile and acetanilide to get heterocyclic systems 13 and 14, respectively. Interaction of 3 with
cyanoacetone gave pyrazolo[3,4-b]pyridine 15, which was refluxed in formic acid to yield pyr-
Accepted 29 May 2009
Available online 12 June 2009
Keywords:
1,2,4-Triazine
Pyrazolo[3,4-b]pyridines
Pyrazolo[3,4-d]pyrimidines
Antimicrobial activity
0
0
azolo[4 ,3 :5,6]pyrido[4,3-d]pyrimidine 16. Reaction of 3 with 2 afforded the triazinylpyrazole derivative
0
0
1
7, which was reacted with hydrazine hydrate to give dipyrazolo[1,5-a:3 ,4 -d]pyrimidine 19. Further-
more, treatment of the latter compound with methyl anthranilate furnished tetraheterocyclic compound
1. Structures of the products have been determined by elemental analysis and spectral studies. All
2
compounds have been screened for their antibacterial and antifungal activities. Compounds 9, 10, 13, 19
and 21 showed maximum activity comparable to the standard drugs with lower toxicity in the case of 9
and 10.
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Moreover, in recent years, 1,2,4-triazine compounds have been
reported to possess biological activities as anti-AIDS [11],
Pyrazole, pyridine and pyrimidine derivatives attracted organic
chemists very much due to their biological and chemotherapeutic
importance. Pyrazolopyridine, pyrazolopyrimidine and related
fused heterocycles are of interest as potential bioactive molecules.
They are known to exhibit pharmacological activities such as CNS
depressant [1,2], neuroleptic [3] and turberculostatic [4]. Pyr-
azolo[3,4-b]pyridines were reported as antimicrobial agents [5],
inhibitors of glycogen synthase kinase-3 (GSK-3) [6] and potent
antitumor agents [7]. Also, pyrazolo[3,4-d]pyrimidines were iden-
tified as a general class of adenosine receptors [8–10]. In the liter-
ature, we have found that replacement of 1H of pyrazole of
pyrazolo[3,4-b]pyridine and pyrazolo[3,4-d]pyrimidine systems by
some other bioactive moieties drastically alter their pharmacolog-
ical properties.
anticancer [12] and antimicrobial activities [13–16]. Prompted by
the varied biological activities of pyrazolo[3,4-b]pyridine, pyr-
azolo[3,4-d]pyrimidine derivatives and 1,2,4-triazine compounds,
we envisioned our approach toward the synthesis of novel pyr-
azolo[3,4-b]pyridine and pyrazolo[3,4-d]pyrimidine that are fused
with nitrogen heterocycles and bearing 5,6-diphenyl-1,2,4-triazine
moiety to study the biological activity.
2. Results and discussions
The starting material 5-amino-1-(5,6-diphenyl-1,2,4-triazin-3-
yl)-3-(methylthio)-1H-pyrazole-4-carbonitrile (3) was prepared in
good yield by the reaction of 5,6-diphenyl-3-hydrazino-1,2,4-
triazine (1) and [bis(methylthio)methylene]malononitrile (2) in
1
boiling methanol (Scheme 1). The H NMR spectrum of the product
3
showed SCH
3
protons as a singlet signal around
d 3.17 ppm, in
E-mail address: tarik_elsayed1975@yahoo.com
addition to NH
2
protons as a broad signal near 3.58 ppm. Also, its
d
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.031

4386
T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
Ph
Ph
N
Ph
Ph
N
N
N
NC
NC
SCH3
SCH3
N
+
MeOH
NH2
N
N
N
N
SCH3
H
1
2
H N
2
CN
3
NH CSNH2
2
EtONa-EtOH
Ph
N
N
N
Ph
N
N
N
H
N
Ph
N
N
H
Ph
N
N
N
S
S
H N
N
2
H N
NH2
2
H N
2
N
5
4
PhCOCl
pyridine
Ph
N
N
N
N
H
Ph
N
N
S
N
N
N
H
Ph
6
Scheme 1.
1
IR spectrum revealed absorptions bands due to NH
groups near 3172 and 2216 cm , respectively.
2
and C^N
data. Their IR and H NMR spectra proved disappearance of hydrazino
group (see Experimental section).
ꢀ1
When compound 3 was treated with thiourea in ethanolic sodium
ethoxide, it furnished a single product identified as 3,4-diamino-
-(5,6-diphenyl-1,2,4-triazin-3-yl)-2,7-dihydro-6H-pyrazolo[3,4-d]
pyrimidine-6-thione (5) (Scheme 1), which was established on the
On the other hand, treatment of compound 3 with benzoyla-
cetone in basic medium gave the corresponding pyrazolo[3,4-
b]pyridine derivative 12 (Scheme 3). The IR spectrum of the isolated
2
ꢀ
1
product showed absorption bands at 3180 and 1673 cm corre-
1
basis of its elemental analysis and spectral data. For example, its IR
sponding to NH
trum showed a singlet signal at
a broad signal at 7.08 ppm for NH
2
2
and C]O groups, respectively. Its H NMR spec-
2.17 ppm for CH CO protons and
protons. The formation of 12
ꢀ
1
spectrum showed absorptions bands at 3483–3219 cm for NH
2
d
3
groups, beside disappearance of C^N group band of compound 3.
Treatment of the diamino derivative 5 with benzoyl chloride in
pyridine resulted in the formation of the fused triheterocyclic
d
indicated that the benzoyl group condensed with amino group to
give the corresponding nonisolable arylidene 11, which underwent
cyclization via addition of active CH CO on the nitrile group.
2
1
derivative 6 (Scheme 1). The H NMR spectrum of 6 exhibited broad
signals at
d
8.91 and 10.68 ppm due to NH protons [17–19].
Incorporation of various functionally-substituted nitrogen
heterocyclic ring into pyrazolo[3,4-b]pyridine structure, was ach-
ieved by treating 12 with different reagents. Thus, treatment of 12
with malononitrile and acetanilide under basic conditions afforded
the pyrazolo[3,4-h][1,6]naphthyridine derivative 13 and pyr-
azolo[5,4,3-kl]pyrido[4,3-d]pyrimidine derivative 14, respectively
Refluxing of compound 3 in acetic anhydride in the presence of
acetic acid afforded a pale brown product that was identified as 1-
(
5,6-diphenyl-1,2,4-triazin-3-yl)-6-methyl-3-(methylthio)-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7) (Scheme 2). Its
IR spectrum showed disappearance of NH and C^N groups and
2
ꢀ
1
appearance of new band at 1668 cm for C]O group. Moreover,
(Scheme 3). In IR spectrum of compound 13, the absorptions bands at
1
ꢀ1
its H NMR spectrum revealed two characteristic singlet signals at
3182 and 2209 cm correspond to NH
2
and C^N groups, respec-
d
2.50 and 13.59 ppm due to methyl protons at C-2 of pyrimidine
tively, were observed. Besides, the normal signals that correspond to
the different protons of aromatic moieties in H NMR spectrum,
1
moiety and NH proton, respectively [20]. Hydrazinolysis of
compound 7 in boiling ethanol afforded the corresponding
hydrazino derivative 8 (Scheme 2). The H NMR of compound 8
there are three signals at
SCH and NH protons, respectively. Also, H NMR spectrum of 14
confirmed participation of SCH and NH groups in cyclization,
beside it showed two singlet signals at 2.38 and 2.68 ppm assigned
to CH CO and CH protons, respectively.
3
d 2.71, 3.17 and 6.80 ppm correspond to CH ,
1
1
3
2
showed hydrazino protons at
d
4.78 (NH
2
) and 10.45 (NH) ppm.
3
2
The 3-hydrazinopyrazolo[3,4-d]pyrimidine 8 could be cyclized
under different conditions to form a variety of triheterocyclic
compounds. Thus, cyclocondensation of 8 with benzoyl chloride in
pyridine and/or sodium pyruvate in ethanol containing few drops of
d
3
3
On the other hand, interaction of compound 3 with cyanoace-
tone in dry DMF gave the corresponding pyrazolo[3,4-b]pyridine
0
0
concentrated
pyrazolo[3,4-d]pyrimidine
sulfuric
acid
9
yielded
1,2,4-triazolo[4 ,3 :1,5]
derivative 15 (Scheme 3). Bands of NH
compound 15 appeared in IR spectrum, while its H NMR spectrum
confirmed the presence of CH , SCH and NH functionalities at
2.36, 3.41 and 5.05 ppm, respectively [18]. Cyclization of 15 upon
2
, C^N and C]N groups of
0
0
1
and pyrimido[4 ,5 :3,4]pyrazolo[5,1-
c][1,2,4]triazine 10, respectively (Scheme 2). The structures of 9 and 10
were established on the basis of their elemental analysis and spectral
3
3
2
d

T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
4387
Ph
Ph
N
N
methyl anthranilate at the methylthio moiety to give the intermediate
0, which underwent an intramolecular cyclizationwith elimination of
methanol molecule. Both elemental analysis and spectral data of 21
Ph
Ph
N
N
N
N
2
N
N
N
SCH3
N
SCH3
Ac O
were consistent with the assigned structure. Thus, its IR spectrum
2
ꢀ
1
1
revealed appearance of C]O group at 1686 cm . Furthermore, its H
NMR spectrum confirmed disappearance of SCH protons and showed
two broad signals 8.87 and 9.82 ppm assigned to two NH groups of
pyrazolo[1,5-a]pyrimidine moiety.
H N
AcOH
N
2
O
CN
3
N
H
d
2
3
H C
3
7
NH NH2
2
EtOH
3. Biological activity
Ph
3.1. Antimicrobial activity
Ph
Ph
N
N
Ph
N
N
N
N
N
NH2
N
All the newly synthesized compounds were evaluated in vitro for
their antimicrobial activity. The antimicrobial activities are carried
out against three bacterial strains, Staphylococcus aureus (MTCCB
737), Staphylococcus epidermidis (MTCCB 1824) and Escherichia coli
(MTCCB 1652) and three fungal strains, namely Aspergillus fumiga-
tus, Aspergillus niger and Alternaria alternata. The preliminary
screening results indicated that the most synthesized compounds
showed antimicrobial activity from moderate to good. From the
inhibition zone diameter data analysis (Table 1):
N
N
N
PhCOCl
pyridine
Ph
N
N
H
O
N
N
O
N
N
H
H
H C
3
H C
3
8
9
EtOH
CH COCOONa
3
drops H SO4
2
CH3
N
1) It can be noted that the most synthesized compounds showed
a greater inhibitory effect against both the bacterial and fungal
strains compared to the starting material 3 which confirmed
improving biological properties by buildingof the fused nitrogen
heterocycles on triazinylpyrazole moiety.
O
Ph
Ph
N
N
N
N
N
N
2
) Compounds 6, 7 and 8 showed a moderate inhibition against
S. aureus (MTCCB 737) and lower inhibition against S. epidermidis
(MTCCB 1824), E. coli (MTCCB 1652) and the three species of
fungal strains.
) Also, compounds 12 and 14–17 showed in general good inhibi-
tions against S. aureus (MTCCB 737) and S. epidermidis (MTCCB
N
O
N
H
H C
3
10
3
Scheme 2.
1
824). However, they showed lower inhibitions against the three
0
0
refluxing in formic acid afforded the pyrazolo[4 ,3 :5,6]pyrido[4,3-
d]pyrimidine derivative 16 (Scheme 3). IR spectrum of 16 revealed
appearance of new band characteristic for C]O group at
species of fungal strains which suggest that they have more
comprehensive bacteria inhibitory prosperities than fungicidal
activity due to the presence of pyrazolo[3,4-b]pyridine moiety in
compounds 12, 14, 15, 16 and methylthio and nitrile groups in
compound 17.
ꢀ
1
1
1
672 cm , while its H NMR spectrum exhibited a broad signal at
d
11.59 ppm assigned to NH proton.
Equimolar ratio of compounds 3 and 2 were refluxed in ethanol
4) Compounds 9, 10, 13, 19 and 21 showed in general good inhi-
bitions against all species of bacterial and fungal strains with
respect to the standard drugs. Compounds 9 and 10 are good
antibacterial agents due to presence of pyrazolo[3,4-b]pyrimi-
dine fused with bioactive heterocycles as 1,2,4-triazole and
1,2,4-triazine moieties while 19 and 21 are good antifungal
agents due to presence of dipyrazolopyrimidine system.
5) We can conclude from preliminary antimicrobial screening that
the pyrazolo[3,4-b]pyridine and pyrazolo[3,4-d]pyrimidine
systems bearing 5,6-diphenyl-1,2,4-triazin-3-yl moiety enhance
the biological properties especially when these systems are
fused with some certain bioactive moieties as pyrazole, 1,2,4-
triazole and 1,2,4-triazine.
to afford the corresponding pyrazole derivative 17 in 76% yield
Scheme 4). The formation of 17 was supported by IR spectrum
which showed absorption vibrations at 3123 (NH) and 2269–2211
(
ꢀ
1
1
(
3 C^N) cm . Also, its H NMR spectrum exhibited appearance of
two singlet signals due to two SCH groups at 3.06 and 3.07 ppm.
3
d
Interestingly, compound 17 seemed to be a useful candidate for
further chemical transformations. Thus, hydrazinolysis of 17 in
DMF gave the nonisolable intermediate 18, which underwent an
intramolecular cyclization via addition of NH functional group of
0
0
pyrazole moiety at C^N group to give the pyrazolo[1,5-a:3 ,4 -d]
pyrimidine derivative 19 as the final product (Scheme 4). The IR
spectrum of 19 exhibited many characteristic absorption bands at
ꢀ
1
3
300–3127, 2210 and 1598 cm that corresponding to NH
2
, NH,
C^N and C]N groups, respectively. Besides, H NMR spectrum of
9 revealed a singlet signal at 3.08 ppm correspond to SCH
protons and two doublet signals at 8.84 and 8.95 ppm assigned to
two NH groups of pyrazolo[1,5-a]pyrimidine moiety [21].
Finally, 2,13-diamino-5-(5,6-diphenyl-1,2,4-trazin-3-yl)-7-oxo-
,14-dihydro pyrazolo[1,5-a][quinazolino[3 ,2 -5,1](3-pyrzolino)[3,4-
1
1
d
3
d
3.2. The minimum inhibitory concentration (MIC)
2
The minimum inhibitory concentration (MIC,
mg/mL) of the
0
0
6
most active compounds 9, 10, 13, 19 and 21 against two species of
bacteria (S. epidermidis (MTCCB 1824) and E. coli (MTCCB 1652)
and also two species of fungi (A. niger and A. alternata)) were
determined (Table 2). Compounds 9 and 10 demonstrated good
inhibitions against the selected bacterial and fungal strains.
d]pyrimidine-3-carbonitrile (21) was achieved from refluxing
compound 19 with methyl anthranilate in boiling DMF containing
a catalytic amount of piperidine (Scheme 4). The reaction mechanism
2
was postulated to proceed through nucleophilic attack of NH group of

4388
T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
Ph
Ph
N
N
Ph
Ph
N
N
N
N
CH COCH CN
N
N
3
2
N
SCH3
N
SCH3
N
DMF
H N
N
2
CN
3
Ph
Ph
N
N
NH2
N
Ph
2
PhCOCH COCH3
N
H C
SCH3
NH2
3
CN
N
-H O EtOH-EtONa
2
13
N
CH (CN)2
EtOH
piperidine
2
H C
3
CN
Ph
Ph
N
N
Ph
Ph
N
N
1
5
N
N
N
N
SCH3
N
N
SCH3
HCOOH
N
CN
N
NH2
Ph
Ph
N
Ph
N
O
Ph
N
H C
3
O
N
N
SCH3
N
H C
3
11
12
N
Ph
Ph
N
N
N
CH CONHPh
pyridine
H C
N
3
3
N
Ph
N
H
O
N
16
CH3
N
N
Ph
O
H C
3
14
Scheme 3.
ꢀ1
1
3.3. Cytotoxicity activity
spectrophotometer (cm ), using KBr disks. H NMR spectra were
measured on Gemini-200 spectrometer (200 MHz), using DMSO-d
as a solvent and TMS ( ) as the internal standard. Elemental
6
The LC50 values of the tested compounds 9, 10, 13, 19 and 21
were found to be 3.54, 6.49, 0.58, 2.31 and 1.39 g/mL, respectively
Table 3). The standard drug Bleomycin has LC50 value at 0.41 g/mL.
The lowest LC50 value was found in the case of compound 13, indi-
cating its higher cytotoxicity than the other compounds. Compounds
d
m
microanalyses were performed at microanalysis center in Bulgarian
Academy of Science, Sofia, Bulgaria. The purity of the synthesized
compounds was checked by thin layer chromatography (TLC). 5,6-
Diphenyl-3-hydrazino-1,2,4-triazine (1) [22] and [bis(methylth-
io)methylene]malononitrile (2) [23] were prepared by published
methods in literature.
(
m
9
and 10 showed potent biocidal activity against brine shrimp due to
their lower cytotoxicity that agreement with preliminary antimi-
crobial screening and the minimum inhibitory concentration (MIC).
So, we can conclude that the pyrazolo[3,4-d]pyrimidine system
bearing 5,6-diphenyl-1,2,4-triazin-3-yl moiety at 1H of pyrazole
moiety that are fused with 6-methyl-1,2,4-triazin-5-one moiety is
most active potent antibacterial and antifungal agent with lower
toxicity in host cells.
5.1. Synthesis of 5-amino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-
(methylthio)-1H-pyrazole-4-carbonitrile (3)
A
mixture of 5,6-diphenyl-3-hydrazino-1,2,4-triazine (1)
(
(
0.005 mol, 1.31 g) and [bis(methylthio)methylene]malononitrile
2) (0.005 mol, 0.85 g) in methanol (30 mL) was refluxed for 4 h.
4
. Conclusion
After cooling, the resulting precipitate which separated out was
collected and crystallized from methanol to afford pale yellow
ꢁ
ꢀ1
2
n (cm ): 3172 (NH ),
We have successfully synthesized a series of novel pyrazolo
crystals. Yield 72%; mp 212–214 C. IR (KBr),
3025 (C–Harom), 2945 (C–Haliph), 2216 (C^N), 1607 (C]N), 1444
[3,4-b]pyridines and pyrazolo[3,4-d]pyrimidines fused with
1
nitrogen heterocycles and bearing 5,6-diphenyl-1,2,4-triazine
moiety. The antimicrobial activity data of the prepared compounds
showed that some fused heteropolycyclic rings showed good
antimicrobial activity with lower toxicity. In our study, we have
replaced the 1H-atom of the pyrazole of pyrazolo[3,4-b]pyridine
and pyrazolo[3,4-d]pyrimidine by 5,6-diphenyl-1,2,4-triazine
moiety as bioactive moiety. These structural changes made
increasing activity against the tested organisms.
(NH
2
def). H NMR (DMSO-d
), 7.35–7.49 (m, 10H, aromatic protons). Anal. Calcd for
S (385.44): C, 62.32; H, 3.92; N, 25.44; Found: C, 61.93; H,
3.70; N, 24.98.
6 3
), d: 3.17 (s, 3H, SCH ), 3.58 (br, 2H,
NH
2
20 15 7
C H N
5
2
.2. Synthesis of 3,4-diamino-2-(5,6-diphenyl-1,2,4-triazin-3-yl)-
,7-dihydro-6H-pyrazolo[3,4-d]pyrimidine-6-thione (5)
A mixture of compound 3 (0.005 mol, 1.92 g) and thiourea
5
. Experimental
(0.005 mol, 0.38 g) in ethanolic sodium ethoxide solution
(prepared by dissolving sodium metal (0.005 mol, 0.12 g) in
[
Melting points were determined on a digital Stuart SMP-3
apparatus. Infrared spectra were measured on Perkin–Elmer 293
absolute ethanol (30 mL))], was refluxed for 4 h. After cooling, the
resulting precipitate which separated out was collected and

T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
4389
O
Ph
Ph
N
N
N
N
Ph
Ph
N
N
N
N
N
N
N
N
SCH3
NH2
N
H N
2
NC
N
CN
3
NH2
2
1
NC
NC
SCH3
SCH3
EtOH
-
CH OH
3
2
O
Ph
N
N
Ph
Ph
N
N
MeO
N
N
H
N
N
N
Ph
N
SCH3
N
HN
N
CN
SCH3
NH2
N
NC
NC
N
CN
NH2
1
7
2
0
NH NH
2
2
COOMe
DMF
-MeSH
DMF-piperidine
NH2
Ph
Ph
N
N
N
Ph
Ph
N
N
N
N
N
SCH3
N
N
SCH
3
HN
CN
N
NH
2
NH
NC
N
N
NC
N
H N
2
NH2
1
8
1
9
Scheme 4.
ꢁ
ꢀ1
n (cm ): 3422, 3183 (NH, NH),
crystallized from ethanol to afford pale green crystals. Yield 74%;
Yield 95%; mp 186–188 C. IR (KBr),
3057 (C–Harom), 1627 (C]N), 1254 (C]S). H NMR (DMSO-d
7.21–7.93 (m, 15H, aromatic protons), 8.91 (br, 1H, NH), 10.68 (br,
1H, NH). Anal. Calcd for C27 S (499.54): C, 64.92; H, 3.43; N,
25.23; Found: C, 64.69; H, 3.22; N, 24.95.
ꢁ
ꢀ1
1
mp 168–170 C. IR (KBr),
NH ), 3195 (NH), 3052 (C–Harom), 1633 (C]N), 1427 (NH
223 (C]S). H NMR (DMSO-d
0H, aromatic protons), 9.50 (s, 2H, NH
Calcd for C20 S (413.45): C, 58.10; H, 3.66; N, 30.49; Found: C,
n
(cm ): 3483, 3321 (NH
2
), 3245, 3219
def),
), 7.10–7.55 (m,
2
), 10.71 (s, 1H, NH). Anal.
6
), d:
(
1
2
2
1
6
),
d
: 4.16 (s, 2H, NH
2
17 9
H N
1
15 9
H N
57.78; H, 3.41; N, 29.98.
5.4. Synthesis of 1-(5,6-diphenyl-1,2,4-triazin-3-yl)-6-methyl-3-
(methylthio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (7)
5.3. Synthesis of 1-(5,6-diphenyl-1,2,4-triazin-3-yl)-7-phenyl-
pyrazolo[5,4,3-kl]pyrimido[4,3-d]pyrimidine-4(2H)-thione (6)
A solution of compound 3 (0.005 mol, 1.92 g) in glacial acetic
acid (10 mL) and acetic anhydride (20 mL) was heated for 6 h. The
solvent was evaporated under reduced pressure to give solid, which
was crystallized from diluted methanol to afford pale brown crys-
A mixture of compound 5 (0.005 mol, 1.84 g) and benzoyl
chloride (0.005 mol, 1 g) in pyridine (30 mL) was refluxed for 8 h.
The reaction mixture was cooled at room temperature, poured into
ice water, and neutralized with dilute HCl. The isolated solid was
filtered off and crystallized from ethanol to afford buff crystals.
ꢁ
ꢀ1
tals. Yield 47%; mp 145–147 C. IR (KBr),
(C–Harom), 2980 (C–Haliph), 1668 (C]O), 1590 (C]N). H NMR
n
(cm ): 3192 (NH), 3054
1
6 3 3
(DMSO-d ), d: 2.50 (s, 3H, CH ), 3.37 (s, 3H, SCH ), 7.28–7.43 (m,

4390
T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
Table 1
The antimicrobial activity of the synthesized compounds at 100
m
g/mL concentration.
Compd. No.
Diameter of the inhibition zonea (mm)
Bacteria
Fungi
Staphylococcus aureus
Staphylococcus epidermidis
Escherichia coli
Aspergillus fumigatus
Aspergillus niger
Alternaria alternata
MTCCB 737
MTCCB 1824
MTCCB 1652
3
5
6
7
8
9
12
9
7
11
13
11
10
27
26
18
22
13
16
17
21
22
23
25
–
3
13
9
5
8
26
29
13
19
10
12
10
11
17
20
28
–
8
11
9
12
10
17
19
10
16
3
6
11
9
22
21
–
6
7
11
9
11
12
15
4
11
7
7
10
6
18
19
–
0
2
5
10
13
8
16
5
9
9
5
12
8
16
14
18
27
28
16
25
18
19
17
17
24
26
30
–
10
12
13
14
15
16
17
19
21
16
19
–
b
Tetracycline
Ketoconazoleb
18
20
21
a
1
2 mm or less: resistant or no inhibition1, 13–17 mm: moderate inhibition, 18 mm or more: maximum inhibition.
b
The concentration of used standard drugs was 30 g/mL.
m
1
10H, aromatic protons), 13.59 (s, 1H, NH). Anal. Calcd for
3055 (C–Harom), 2986 (C–Haliph), 1667 (C]O), 1584 (C]N). H NMR
C
22
H
17
N
7
OS (427.48): C, 61.81; H, 4.01; N, 22.94; Found: C, 61.42; H,
(DMSO-d
protons), 13.45 (br, 1H, NH). Anal. Calcd for C28
7.60; H, 3.85; N, 25.34; Found: C, 67.31; H, 3.58; N, 25.02.
6
),
d
: 2.50 (s, 3H, CH
3
), 7.28–7.53 (m, 15H, aromatic
3
.79; N, 22.63.
19 9
H N
O (497.51): C,
6
5.5. Synthesis of 1-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-hydrazino-6-
methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (8)
5.7. Synthesis of 6-(5,6-diphenyl-1,2,4-triazin-3-yl)-3,8-
dimethylpyrimido[4 ,5 :3,4]pyrazolo[5,1-c][1,2,4]triazine-4,10-
(6H,9H)-dione (10)
0
0
A mixture of compound 7 (0.005 mol, 2.13 g) and hydrazine
hydrate (0.007 mol, 0.35 mL) in ethanol (30 mL) was refluxed for
4
h. After cooling, the resulting precipitate was filtered off and
A mixture of compound 8 (0.005 mol, 2.05 g) and sodium
pyruvate (0.005 mol, 0.55 g) in ethanol (30 mL) containing
concentrated H SO (0.5 mL) was refluxed for 6 h. After cooling, the
crystallized from ethanol to afford pale yellow crystals. Yield 83%;
ꢁ
ꢀ1
2
n (cm ): 3341, 3266 (NH ), 3216 (NH),
mp 170–172 C. IR (KBr),
056 (C–Harom), 2930 (C–Haliph), 1676 (C]O), 1604 (C]N). H NMR
DMSO-d ), : 2.78 (s, 3H, CH ), 7.18–
), 4.78 (d, 2H, J ¼ 5.6 Hz, NH
2
4
1
3
(
obtained solid was filtered off and crystallized from ethanol to afford
ꢁ
ꢀ1
n (cm ):
6
d
3
2
pale brown crystals. Yield 66%; mp 256–258 C. IR (KBr),
3166 (NH), 3037 (C–Harom), 2935 (C–Haliph), 1686 (C]Opyrimidinone),
7.84 (m, 10H, aromatic protons), 10.45 (s, 1H, NH), 10.74 (s, 1H, NH).
1
Anal. Calcd for C21
H
17
N
9
O (411.41): C, 61.31; H, 4.16; N, 30.64;
1650 (C]Otriazinone), 1605 (C]N). H NMR (DMSO-d ),
d
: 2.49 (s, 3H,
6
Found: C, 60.99; H, 3.79; N, 30.38.
CH ), 2.89 (s, 3H, CH ), 7.11–8.12 (m, 10H, aromatic protons), 11.23
3
3
17 9 2
(br, 1H, NH). Anal. Calcd for C24H N O (463.45): C, 62.20; H, 3.70;
5
.6. Synthesis of 5-(5,6-diphenyl-1,2,4-triazin-3-yl)-7-methyl-3-
N, 27.20; Found: C, 61.84; H, 3.41; N, 26.83.
0
0
phenyl-5H-[1,2,4]triazolo[4 ,3 : 1,5]pyrazolo[3,4-d]pyrimidin-9-
(
8H)-one (9)
5
6
.8. Synthesis of 5-acetyl-4-amino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-
-phenyl-3-(methylthio)-1H-pyrazolo[3,4-b]pyridine (12)
A mixture of compound 8 (0.005 mol, 2.05 g) and benzoyl
chloride (0.005 mol, 0.70 g) in pyridine (30 mL) was refluxed for
h. The reaction mixture was cooled at room temperature, poured
A solution of compound 3 (0.005 mol,1.92 g) and benzoylacetone
8
(
0.005 mol, 0.81 g) in ethanolic sodium ethoxide solution [(prepared
by dissolving sodium metal (0.005 mol, 0.12 g) in absolute ethanol
30 mL))], was refluxed for 8 h. After cooling, add some water
into ice water, and neutralized with dilute HCl. The isolated solid
was filtered off and crystallized from ethanol to afford yellow
(
ꢁ
ꢀ1
n (cm ): 3194 (NH),
crystals. Yield 54%; mp 208–210 C. IR (KBr),
Table 3
Table 2
Cytotoxicity activity of the synthesized compounds 9, 10, 13, 19 and 21.
The minimum inhibitory concentration (MIC,
, 10, 13, 19 and 21.
mg/mL) of the synthesized compounds
Samples
95% confidence limit ppm
Regression equation
X2 (df)
9
LC50
Lower
Upper
The selected organisms
The minimum inhibitory concentration (MIC)
9
3.54
6.49
0.58
2.31
1.39
0.41
4.53
2.08
4.15
0.19
1.30
0.69
0.27
3.33
6.02
10.15
1.77
4.10
2.82
0.62
6.15
y ¼ 3.98 þ 1.85x
y ¼ 3.17 þ 2.27x
y ¼ 4.08 þ 1.22x
y ¼ 4.36 þ 1.78x
y ¼ 3.54 þ 1.29x
y ¼ 3.16 þ 2.98x
y ¼ 3.93 þ 1.62x
3.38(2)
0.35(2)
0.20(2)
0.32(2)
0.41(2)
0.62(2)
1.25(2)
9
10
13
19
21
Standarda
10
Staphylococcus aureus (MTCCB 737) 50 25
50
50
50
6.25
13
19
21
Escherichia coli (MTCCB 1652)
Aspergillus niger
Alternaria alternata
25 12.5 25
50 25
50 50
>100 >100 12.5
>100 25
>100 25
>100
50
6.25
6.25
a
Bleomycin
Gallic acid
a
a
Tetracycline and ketoconazole were used as standard drugs against bacterial
and fungal strains, respectively.
a
Bleomycin and gallic acid were used as standard drugs in cytotoxicity activity.

T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
4391
(
10 mL), the resulting precipitate was filtered off and crystallized
d
6
),
aromatic protons), 8.51 (br, 1H, C
Anal. Calcd for C25 OS (478.52): C, 62.75; H, 3.79; N, 23.42;
d
: 2.33 (s, 3H, CH
3
), 3.39 (br, 3H, SCH
3
), 7.38–7.95 (m, 10H,
from diluted ethanol to afford yellow crystals. Yield 67%; mp 188–
1
H
COCH
aromatic protons). Anal. Calcd for C30
4
2
–Hpyrimidine), 11.59 (brs, 1H, NH).
ꢁ
ꢀ1
91 C. IR (KBr),
n
(cm ): 3180 (br, NH
2
), 3050 (C–Harom), 2910 (C–
), : 2.17 (s, 3H,
), 7.30–7.48 (m, 15H,
OS (529.61): C, 68.03; H,
.38; N, 18.51; Found: C, 67.79; H, 4.09; N, 18.19.
18 8
H N
1
aliph), 1673 (C]O), 1603 (C]N). H NMR (DMSO-d
6
d
Found: C, 62.43; H, 3.52; N, 23.08.
3 3 2
), 3.30 (s, 3H, SCH ), 7.08 (br, 2H, NH
H N
23 7
5.13. Synthesis of [{[4-cyano-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-3-
(methylthio)-1H-pyrazol-5-yl]amino}(methylthio)
methylene]malononitrile (17)
5.9. Synthesis of 2-amino-7-(5,6-diphenyl-1,2,4-triazin-3-yl)-4-
methyl-9-(methylthio)-5-phenyl-7H-pyrazolo[3,4-
h][1,6]naphthyridine-3-carbonitrile (13)
A mixture of compound 3 (0.005 mol, 1.92 g) and 2 (0.005 mol,
0.85 g) in ethanol (30 mL) was refluxed for 4 h. The reaction
mixture was cooled at room temperature to isolate the solid, which
was filtered off and crystallized from ethanol to afford yellow
A mixture of compound 12 (0.005 mol, 2.65 g) and malononitrile
ꢁ
ꢀ1
n (cm ): 3123 (NH),
(
0.005 mol, 0.33 g) in ethanol (30 mL) containing few drops of
crystals. Yield 76%; mp 175–178 C. IR (KBr),
3014 (C–Harom), 2971, 2898 (C–Haliph), 2269–2211 (3 C^N), 1623
piperidine was refluxed for 10 h. The obtained solid was filtered off
and crystallized from DMF/ethanol to afford brown crystals. Yield
1
(C]N), 1597 (C]C). H NMR (DMSO-d
(s, 3H, SCH ), 4.15 (s, 1H, NH), 7.44–7.71 (m, 10H, aromatic protons).
Anal. Calcd for C25 (507.59): C, 59.16; H, 3.38; N, 24.83;
6 3
), d: 3.06 (s, 3H, SCH ), 3.07
ꢁ
ꢀ1
8
H
1%; mp 210–211 C. IR (KBr),
n
(cm ): 3182 (br, NH
arom), 2928 (C–Haliph), 2209 (C^N), 1625 (C]N), 1443 (NH
), : 2.71 (s, 3H, CH ), 3.17 (s, 3H, SCH ), 6.80 (br, 2H,
), 7.35–7.52 (m,15H, aromatic protons). Anal. Calcd for C33
577.66): C, 68.61; H, 4.01; N, 21.82; Found: C, 68.31; H, 3.83; N, 21.51.
2
), 3057 (C–
3
1
2
def). H
17 9 2
H N S
NMR (DMSO-d
NH
(
6
d
3
3
Found: C, 58.89; H, 3.19; N, 24.59.
2
23 9
H N S
5.14. Synthesis of 4,7-diamino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-
0 0
-(methylthio)-1H-dipyrazolo[1,5-a:3 ,4 -d]pyrimidine-8-
3
5.10. Synthesis of 6-acetyl-3,7diphenyl-1-(5,6-diphenyl-1,2,4-
carbonitrile (19)
triazin-3-yl)-4-methyl-pyrazolo[5,4,3-kl]pyrido[4,3-d]
pyrimidine (14)
A mixture of compound 17 (0.005 mol, 2.53 g) and hydrazine
hydrate (0.005 mol, 0.25 g) in DMF (30 mL) was refluxed for 8 h.
The reaction mixture was cooled at room temperature, poured into
ice water to isolate the solid, which was filtered off and crystallized
from diluted ethanol to afford yellow crystals. Yield 71%; mp 212–
A mixture of compound 12 (0.005 mol, 2.65 g) and acetanilide
(
0.005 mol, 0.67 g) in pyridine (30 mL) was refluxed for 12 h. The
reaction mixture was cooled at room temperature, poured into ice
water, and neutralized with dilute HCl. The isolated solid was
filtered off and crystallized from diluted DMF to afford yellow
ꢁ
ꢀ1
2
n (cm ): 3300, 3127 (NH , NH), 3062 (C–Harom),
214 C. IR (KBr),
2940 (C–Haliph), 2210 (C^N), 1598 (C]N), 1446 (NH
(DMSO-d ), : 3.08 (s, 3H, CH ), 7.22–7.77 (m, 10H, aromatic
protons), 8.84 (d, 2H, NH2pyrazole), 8.95 (d, 2H, NH2pyrimidine). Anal.
Calcd for C24 11S (491.53): C, 58.64; H, 3.49; N, 31.35; Found: C,
58.41; H, 3.22; N, 31.09.
1
2
def). H NMR
ꢁ
ꢀ1
crystals. Yield 69%; mp 234–237 C. IR (KBr),
n
(cm ): 3056 (C–
6
d
3
1
Harom), 2919 (C–Haliph), 1683 (C]O), 1620 (C]N). H NMR (DMSO-
d
6
),
d
: 2.38 (s, 3H, COCH
3
), 2.68 (s, 3H, CH
O (598.65): C, 74.23; H,
.38; N, 18.72; Found: C, 73.91; H, 4.08; N, 18.39.
3
), 7.27–7.54 (m, 20H,
17
H N
aromatic protons). Anal. Calcd for C37
4
26 8
H N
5
.15. Synthesis of 2,13-diamino-5-(5,6-diphenyl-1,2,4-trazin-3-yl)-
0
0
5.11. Synthesis of 4-amino-1-(5,6-diphenyl-1,2,4-triazin-3-yl)-6-
7-oxo-6,14-dihydropyrazolo[1,5-a]quinazolino[3 ,2 -5,1](3-
methyl-3-(methylthio)-1H-pyrazolo[3,4-b]pyridine-5-
pyrzolino)[3,4-d]pyrimidine-3-carbonitrile (21)
carbonitrile (15)
A mixture of compound 19 (0.005 mol, 2.45 g) and methyl
anthranilate (0.005 mol, 0.76 g) in DMF (30 mL) containing few
drops of piperidine, was refluxed for 12 h. The reaction mixture was
cooled at room temperature, poured into ice water to isolate the
A mixture of chloroacetone (0.005 mol, 0.42 g) and potassium
cyanide (0.005 mol, 0.33 g) in dry DMF (10 mL), was heated for
10 min. A solution of compound 3 (0.005 mol, 1.92 g) in DMF
(
20 mL) was added to the above mixture and refluxed for 10 h. The
solid, which was filtered off and crystallized from diluted DMF to
ꢁ
reaction mixture was cooled at room temperature, poured into ice
water to isolate the solid, which was filtered off and crystallized
from diluted ethanol to afford yellow crystals. Yield 59%; mp 176–
afford pale brown crystals. Yield 53%; mp 286–288 C. IR (KBr),
n
ꢀ
1
(cm ): 3376, 3314, 3181 (NH2, NH), 3050 (C–Harom), 2273 (C^N),
1
1686 (C]O), 1629, 1602 (C]N). H NMR (DMSO-d
(m, 14H, aromatic protons), 8.87 (br, 2H, NH2pyrazole), 9.82 (br, 2H,
NH2pyrimidine). Anal. Calcd for C30 12O (562.54): C, 64.05; H,
6
), d: 7.34–8.14
ꢁ
ꢀ1
1
78 C. IR (KBr),
n
(cm ): 3381, 3124 (NH
2
), 3010 (C–Harom), 2815
), : 2.36 (s,
), 7.43–7.90 (m, 10H,
S (450.51): C, 63.98; H,
.03; N, 24.87; Found: C, 63.69; H, 3.86; N, 24.51.
1
(
C–Haliph), 2119 (C^N), 1621 (C]N). H NMR (DMSO-d
6
d
18
H N
3
H, CH
3
), 3.41 (br, 3H, SCH
3
), 5.05 (s, 2H, NH
2
3.23; N, 29.88; Found: C, 63.81; H, 3.11; N, 29.61.
aromatic protons). Anal. Calcd for C24
4
18 8
H N
5.16. Antimicrobial screening
5
.12. Synthesis of 7-(5,6-diphenyl-1,2,4-triazin-3-yl)-5-methyl-9-
All the newly synthesized compounds were evaluated in vitro
for their antimicrobial activity. The antimicrobial activities are
carried out against three bacterial strains, S. aureus (MTCCB 737),
S. epidermidis (MTCCB 1824) and E. coli (MTCCB 1652) and three
fungal strains, A. fumigatus, A. niger and A. alternata employing the
0
0
(
methylthio)-3,7-dihydro-4H-pyrazolo[4 ,3 : 5,6]pyrido[4,3-
d]pyrimidin-4-one (16)
A solution of compound 15 (0.005 mol, 2.25 g) in formic acid
(
20 mL), was heated for 4 h. The reaction mixture was cooled at
nutrient agar disc diffusion method [24,25] at 100 mg/mL concen-
room temperature, poured into ice water and neutralized with
NaOH solution (5%) to isolate the solid, which was filtered off and
crystallized from DMF/ethanol to afford pale brown crystals. Yield
tration. DMSO was used as blank exhibited no activity against any
of the used organisms. The antimicrobial activity was determined
by measuring of the inhibition zone (Table 1), after 16–20 h of
incubation at 37 C for bacterial strains and 3–4 days at 37 C for
fungal strains. Tetracycline and ketoconazole were used as standard
ꢁ
ꢀ1
ꢁ
ꢁ
5
8%; mp 199–201 C. IR (KBr),
n
(cm ): 3182 (br, NH), 3057 (C–
1
Harom), 2925 (C–Haliph), 1672 (C]O), 1599 (C]N). H NMR (DMSO-

4392
T. El-Sayed Ali / European Journal of Medicinal Chemistry 44 (2009) 4385–4392
drugs against bacterial and fungal strains, respectively at 30
concentration.
mg/mL
Acknowledgment
The author is thankful to Department of Microbiology, Bulgarian
Academy of Science, Sofia, Bulgaria, for helping in evaluating anti-
microbial activity.
5.17. The minimum inhibitory concentration (MIC)
A current definition of the minimum inhibitory concentration
MIC is ‘‘the lowest concentration which resulted in maintenance or
reduction of inoculum viability’’. The determination of the MIC
References
involves
a semi-quantitative test procedure which gives an
[
[
1] M. Julino, M.F.G. Stevens, J. Chem. Soc., Perkin Trans. 1 (1998) 1677–1684.
2] M. Ibrahim Abdou, A.M. Saleh, H.F. Zohdi, Molecules 9 (2004) 109–116.
approximation to the least concentration of antimicrobial agent
needed to prevent microbial growth. The method displays tubes of
growth broth containing a test level of preservatives, into which an
inoculum of microbes was added. The end result of the test was the
minimum concentration of antimicrobial. The serial dilution tech-
nique [26] was applied for the determination of MIC of the tested
compounds 9, 10, 13, 19 and 21 against two species of bacterial
strains (S. aureus MTCCB 737 and E. coli MTCCB 1652) and two
species of fungal strains (A. niger and A. alternata). Dilution series
[3] R. Filler, Chem. Technol. 4 (1974) 752.
[
[
[
4] M.M. Ghorab, Z.H. Ismail, S.M. Abdel-Gawad, A. Abdel-Aziem, Heteroat. Chem.
5 (2004) 57–62.
1
5] F.E. Goda, A.A.M. Abdel-Aziz, O.A. Attef, Bioorg. Med. Chem. Lett. 12 (2004)
1845–1852.
6] J. Witherington, V. Bordas, S.L. Garland, D.M.B. Hickey, R.J. Lfe, J. Liddle,
M. Saunders, D.G. Smith, R.W. Ward, Bioorg. Med. Chem. Lett. 13 (2003)
1577–1580.
[7] R. Lin, P.J. Connolly, Y. Lu, G. Chin, S. Li, Y. Yu, S. Huang, X. Li, S.L. Emanuel,
S.A. Middleton, R.H. Gruninger, M. Adams, A.R. Fuentes-Pesquera,
L.M. Greenberger, Bioorg. Med. Chem. Lett. 17 (2007) 4292–4302.
were set up with 6.25, 12.5, 25, 50 and 100 mg/mL of nutrient broth
medium to each tube, 100
microbes (10 cells/mL) were added and incubated at 37 C for 24 h
[
8] S. Schenore, G. Bruno, P. Fossa, A. Ranise, G. Menozzi, L. Mosti, F. Bondavalli,
C. Martini, L. Trincavelli, Bioorg. Med. Chem. Lett. 11 (2001) 2529–2531.
9] L.P. Davies, S.C. Chow, J.H. Skerritt, D.J. Brown, G.A.R. Johuston, Life Sci. 34
m
L of standardized suspension of the test
7
ꢁ
[
(1984) 2117–2128.
(Table 2).
[
10] M.A. Timoteo, L. Oliveira, E.C. Campesatto-Mella, A. Barroso, C. Silva,
M.T. Magalhaes-Cardoso, W. Alves-do-Prado, P. Correia-de-sa, Neurochem. Int.
52 (2008) 834–845.
[
[
11] R.M. Abdel-Rahman, Pharmazie 56 (2001) 275.
12] Z. El-Gendy, J.M. Morsy, H.A. Allimony, W.R. Abdel-Monem, R.M. Abdel-
Rahman, Phosphorus, Sulfur Silicon Relat. Elem. 178 (2003) 2055–2071.
5.18. Cytotoxicity bioassay
Brine shrimp lethality bioassay [27,28] is a recent development
[13] T.E. Ali, Phosphorus, Sulfur Silicon Relat. Elem. 182 (2007) 1717–1726.
[
[
14] T.E. Ali, W.R. Abdel-Monem, Int. J. Chem. 17 (2007) 303–314.
15] T.E. Ali, S.A. Abdel-Aziz, H.M. El-Shaaer, F.I. Hanafy, A.Z. El-Fauomy,
Phosphorus, Sulfur Silicon Relat. Elem. 183 (2008) 2139–2160.
in the assay which indicates cytotoxicity as well as a wide range of
pharmacological activities (e.g. antimicrobial, anticancer, antiviral,
insecticidal, pesticidal, AIDS, etc). In this method, the eggs of the
brine shrimp, Artemia salina leach, were hatched for 48 h to mature
shrimp. 38 g of sea salt was weighed, dissolved in one liter of
distilled water, filtered off and was kept in a small tank. The eggs
were then added to the divided tank. Constant oxygen supply was
[16] F. El-Mariah, M. Hosny, A. Deeb, Phosphorus, Sulfur Silicon Relat. Elem. 181
2006) 2505–2517.
(
[17] B.S. Holla, M. Mahalinga, M.S. Karthikeyan, P.M. Akberali, N.S. Shetty, Bioorg.
Med. Chem. 14 (2006) 2040–2047.
[18] H.M. Gaber, S.M. Sherif, F.A. Abu-Shanab, J. Sulfur Chem. 26 (2005) 393–403.
[19] D.C. Kim, Y.R. Lee, B.S. Yang, K.J. Shin, D.J. Kim, B.Y. Chung, K.H. Yoo, Eur. J. Med.
Chem. 38 (2003) 525–532.
ꢁ
provided and temperature 37 C was maintained for 48 h to hatch
[20] A.M. El-Agrody, H.A. Emam, M.H. El-Hakim, M.S. Abdel-Latif, A.H. Fakery,
J. Chem. Res., Miniprint (1997) 2039–2048.
and mature the shrimp called as nauplii (Larvae). The solutions of
compounds 9, 10, 13, 19 and 21 were prepared by dissolving 10 mg
of each compound in 2 mL of DMSO. From this stock, a series of
[21] A.M. Farag, K.M. Dawood, H.A. Elmenoufy, Heteroat. Chem. 15 (2004) 508–514.
[
[
[
22] P.V. Laakso, R. Robinson, H.P. Vandrewala, Tetrahedron 1 (1957) 103–118.
23] H. Junjappa, H. Ila, C.V. Asokan, Tetrahedron 46 (1990) 5423–5506.
24] B.A. Arthington, M. Motly, D.W. Warnock, C.J. Morrison, J. Clin. Microbiol. 38
(2000) 2254–2260.
solutions 5, 10, 20, 40 and 80
three for each dilutions were used for each test sample and LC50 is
the mean of three values) and one vial was kept as control having
mL of DMSO. Then about 10 brine shrimp nauplii were applied to
mg/mL were transferred to fifteen vials
(
[
[
25] E.J. Baron, S.M. Finegolg, Baily and Scott’s Diagnostic Microbiology, C.V. Mosby,
St Louis, 1990, 184–188.
26] C. Nishina, N. Enoki, S. Tawata, A. Mori, K. Kobayashi, M. Fukushima, Agric.
Biol. Chem. 51 (1987) 139.
2
each of all experimental vials and control vial. The number of the
nauplii that died after 24 h was counted. The resulting data were
transformed to the probit analysis [29] for the determination of
LC50 values for the five tested compounds (Table 3).
[
[
27] B. Jaki, J. Orjala, H.R. Burji, O. Sticher, J. Pharm. Biol. 37 (1999) 138–143.
28] B.N. Mayer, N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols,
J.L. Mclaudhlin, Planta Med. 45 (1982) 31–34.
[29] D.J. Finney, Probit Analysis, third ed. Cambridge University Press, UK, 1972.