Synthesis and anticonvulsant activity of some new pyrazolo[3,4-b]pyrazines and related heterocycles

Article abstract of DOI:10.1016/j.bmc.2014.02.019

A series of new pyrazolo[3,4-b]pyrazines containing, 1,2,4-oxadiazolyl, thiadiazolyl, imidazothiadiazolyl, thiazolidinonyl, substituents and other different substituents, was synthesized using 1,6-diphenyl-3-methyl-lH- pyrazolo[3,4-b]pyrazine-5-carbonitrile (2) as a starting material. Some of the newly prepared compounds were evaluated for their anticonvulsant activity. Compounds 9a, 13a-d and 14a at a dose of 10 mg/kg showed very significant anticonvulsant activity and increased the latency time of PTZ-induced tonic seizures. Compound 9b showed significant effect.

Full text of DOI:10.1016/j.bmc.2014.02.019

Bioorganic & Medicinal Chemistry 22 (2014) 2166–2175
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anticonvulsant activity of some new pyrazolo
[3,4-b]pyrazines and related heterocycles
Abdel-Rahman Farghaly a, , Sabah Esmail b, Ahmed Abdel-Zaher c, Ali Abdel-Hafez a, Hussein El-Kashef a,
⇑
a Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt
b Department of Chemistry, Faculty of Science, Ibb University, Yemen
c Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new pyrazolo[3,4-b]pyrazines containing, 1,2,4-oxadiazolyl, thiadiazolyl, imidazothiadiazolyl,
thiazolidinonyl, substituents and other different substituents, was synthesized using 1,6-diphenyl-3-
methyl-lH-pyrazolo[3,4-b]pyrazine-5-carbonitrile (2) as a starting material. Some of the newly prepared
compounds were evaluated for their anticonvulsant activity. Compounds 9a, 13a–d and 14a at a dose of
10 mg/kg showed very significant anticonvulsant activity and increased the latency time of PTZ-induced
tonic seizures. Compound 9b showed significant effect.
Received 21 November 2013
Revised 4 February 2014
Accepted 14 February 2014
Available online 25 February 2014
Keywords:
Pyrazolo[3,4-b]pyrazines
Oxadiazoles
Ó 2014 Elsevier Ltd. All rights reserved.
Thiadiazoles
Imidazothiadiazoles
Thiazolidinones
Anticonvulsant activity
1. Introduction
2. Results and discussion
Pyrazolo[3,4-b]pyrazine ring system is an interesting class of
heterocycles of great medical importance. It has been reported that
some of its derivatives are used as inhibitors of protein kinases,1
blood platelet aggregation,2 bone metabolism improvers,3 antiin-
flammatories,2 antifungal,4,5 antibacterial,4,6 antiparasitic,5 and
anticancer agents.7,8 On the other hand, pyrazolo[3,4-b]pyrazines
are also used as fluorescent9 and disperse dyes in dye chemistry.10
Certain derivatives were reported to possess antiviral, antineoplas-
tic and anti-fungal activities.11–13 Also pyrazolopyrazines were
used in the treatment of hematologic diseases,14 and reported as
adenosine antagonists.15,16 Recently, a microwave-assisted synthe-
sis of fused pyrazolo[3,4-b]pyrazines by the reaction of ortho-
aminonitrosopyrazoles and cyclic b-diketones was reported.17 In
continuation to our work directed toward the synthesis of new
fused pyrazines5,13,18–20 and pyrazole-based hetrocycles21 we
report herein the synthesis of new pyrazolo[3,4-b]pyrazines and
related heterocycles. Some derivatives of the compounds prepared
were tested for their anticonvulsant activity.
2.1. Chemistry
The starting material 1,6-diphenyl-3-methyl-lH-pyrazolo[3,4-b]
pyrazine-5-carbonitrile (2) was prepared via the reaction of the
readily available 5-amino-3-methyl-4-nitroso-l-phenylpyrazole
(1) with benzoyl acetonitrile in refluxing pyridine according to
our published procedure13 (Scheme 1).
When the cyano function of 2 was interacted with hydroxyl-
amine hydrochloride, the product obtained was the amidoxime 3.
Heating this latter compound with benzoylchloride or 4-chloro-
benzoyl chloride in refluxing pyridine afforded the corresponding
1,2,4-oxadiazolyl derivatives 4a,b. The IR spectrum of compound
ꢀ
3
showed
t
NH2 and OH bands at
m
¼ 3450; 3350 and
3200 cmꢀ1. These absorption bands disappeared in the IR spectra
of compounds 4a,b. Also, the 1H NMR spectrum of compound 3
showed a singlet at d 6.04 ppm (NH2) and another singlet at
d 9.55 ppm (OH). These two signals disappeared in the 1H NMR
spectra of compounds 4a,b (Scheme 2).
The alkaline hydrolysis of 2 gave the carboxylic acid 5 which
was esterified in refluxing absolute methanol in the presence of
concentrated sulfuric acid to give the corresponding methyl ester
6. The ester 6 was readily converted into the corresponding acid
hydrazide713 upon heating under reflux with ethanolic solution
of hydrazine hydrate. The reaction of 5 with thiosemicarbazide in
⇑
Corresponding author. Tel.: +20 1005075881; fax: +20 88 2342708.
E-mail addresses: elkashef15@hotmail.com, elkashef.hussein@yahoo.com
(H. El-Kashef).
Current address: Department of Chemistry, Faculty of Science, Jazan University,
Jazan 2097, Saudi Arabia.
http://dx.doi.org/10.1016/j.bmc.2014.02.019
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
2167
O
H3C
CN
NO
H3C
CN
N
Pyridine
Reflux
+
N
NH2
N
N
N
N
1
2
Scheme 1. Synthetic pathway of compound 2.
OH
N
H3C
N
O
N
N
H3C
N
H3C
N
N
N
CN
R
R
COCl
Pyridine
N
N
N
N
NH2OH.HCl
NH2
N
Ph
N
N
Ph
Ph
Ph
a, R = H
b, R = Cl
2
Ph
3
Ph
4a,b
Scheme2. Synthetic pathway of compounds 3 and 4a,b.
¼ 1720 cmꢀ1. Their 1H NMR showed
ꢀ
thiazolidinone ring around m
refluxing phosphorylchloride gave the corresponding amino-thi-
adiazolyl derivative 8 (Scheme 3).
Condensation of 8 with phenacylbromide and 4-bromo-phen-
acylbromide yielded the corresponding imidazothiadiazolyl deriv-
atives 9a,b (Scheme 4).
the characteristic two doublets of the CH2 protons of the
thiazolidinone ring around d 3.75 ppm (J = 15.6 Hz) and 3.84 ppm
( J = 15.6 Hz), a singlet corresponding to the proton at position 2
of the thiazolidinone ring within 5.48–6.09 ppm and the singlet
of the NH proton within d 9.17–9.01 ppm. 13C NMR spectrum of
compound 13a showed new signals at d 11.36 ppm (CH3), d
30.09 ppm (CH2 thiazolidinone), d 62.80 ppm (CH thiazolidinone)
and d 169.68 ppm (CO thiazolidinone).
On the other hand, when the above reaction was repeated using
the cycloalkylidenes 11a,b or isopropylidene derivative 12, the spi-
rothiazolidinones 14a,b and 15 were formed as shown in Scheme 7.
1H NMR spectra of compounds 14a,b and 15 showed disappearance
of a singlet corresponding to the proton at position 2 of the thiazo-
lidinone ring and appearance new signals of aliphatic protons.
Treatment of carbohydrazide7 with benzoyl chloride gave
N0-benzoyl-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-car-
bohydrazide (16) which underwent cyclodehydration when trea-
ted with phosphorylchloride giving the oxadiazolyl drerivative
17. The latter compound 17 could be synthesized unequivocally
Treatment of the carbohydrazide derivative 7 with some aro-
matic aldehydes, cyclic ketones and acetone gave the correspond-
ing arylidene 10a–d, cycloalkylidene 11a,b and isopropylidene 12
derivatives respectively (Scheme 5). The IR spectra of these com-
ꢀ1
ꢀ
m
pounds showed
tion to
t
NH bands within
¼ 3300-3210 cm in addi-
ꢀ1
ꢀ
m
t
C@O bands around
¼ 1690-1650 cm while their 1H
NMR spectra showed characteristic signals assigned to CH3, NH,
and CH@N protons.
The reaction of the arylidene compounds 10a–d with thiogly-
colic acid in boiling dry benzene, using a Dean–Stark apparatus
gave the corresponding N-(2-arylthiazolidin-4-on-3-yl)-1,6-diphe-
nyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxamide (13a–d)
(Scheme 6).The IR spectra of the latter compounds showed
t NH
ꢀ
absorption bands around
m
¼ 3300 and
t C@O bands around
ꢀ1
ꢀ
m
¼ 1690 cm in addition to the endocyclic
t
C@O band of the
H3C
N
N
CN
Ph
N
N
Ph
2
NaOH
H2O
N
N
O
O
H3C
N
H3C
H3C
NH2
N
N
CH3
N
S
O
OH
H2SO4 / MeOH
NH2NHCSNH2
POCl3
N
N
N
N
N
N
6
N
Ph
N
Ph
Ph
Ph
Ph
Ph
8
5
NH2NH2
O
H3C
N
N
NH2
N
H
N
N
Ph
Ph
7
Scheme 3. Synthetic pathway of compounds 5–8.

2168
A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
R
O
N
N
N
N
H3C
N
H3C
N
N
Br
NH2
R
N
N
N
S
S
K2CO3
/ DMF
N
N
Ph
N
Ph
a, R= H
Ph
Ph
b, R= Br
9 a,b
8
Scheme 4. Synthetic pathway of compounds 9a,b.
O
H3C
N
N
Ar
EtOH / dioxane
ArCHO
N
H
N
N
N
Ph
a, Ar = C6H5
b
Ph
, Ar = 4-ClC6H4
10 a-d
c
, Ar = 4-NO2C6H4
d, Ar = 3,4-OCH3C6H3
O
O
H3C
N
H3C
O
N
NH2
N
N
N
N
N
H
n
H
N
EtOH / dioxane
N
n
N
N
Ph
Ph
Ph
a
Ph
, n = 1
7
11 a,b
b, n = 2
O
H3C
N
N
N
N
CH3COCH3
H
N
N
Ph
Ph
12
Scheme 5. Synthetic pathway of compounds 10–12.
O
O
O
H3C
H3C
S
N
N
N
Ar
HSCH2COOH
N
N
H
N
H
N
H
N
Ar
N
N
N
Ph
dry/ benzene
N
Ph
Ph
Ph
10a-d
13a-d
a, Ar = C6H5
b, Ar = 4-ClC6H4
c, Ar = 4-NO2C6H4
d
, Ar = 3,4-OCH3C6H3
Scheme 6. Synthetic pathway of compounds 13a–d.
O
O
O
H3C
H3C
N
S
N
N
N
N
N
H
N
H
N
HSCH2COOH
dry/ benzene
N
N
Ph
n
N
N
Ph
Ph
n
Ph
11a,b
a, n = 1
b, n = 2
14a,b
O
O
O
H3C
N
N
N
H3C
N
S
N
N
HSCH2COOH
dry/ benzene
N
H
N
H
N
N
Ph
N
Ph
N
Ph
12
Ph
15
Scheme 7. Synthetic pathway of compounds 14,15.

A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
2169
O
N
N
H3C
N
H3C
N
H
N
Ph
N
H
N
N
N
H
POCl3
O
C6H5COOH
POCl3
O
N
N
N
Ph
Ph
Ph
19
Ph
17
HCOOH
N
POCl3
N
N
O
N
H3C
O
H3C
N
H3C
H
NH2
N
N
Ph
N
O
CH(OEt)3
N
PhCOCl
Pyridine
H
N
H
N
O
N
N
N
Ph
N
N
Ph
N
Ph
Ph
Ph
Ph
18
7
16
Scheme 8. Synthetic pathway of compounds 16–19.
NH
N
H3C
O
N
O
N
N
N
Ph
O
H3C
Ph
21
N
N
NH2
ClCOOEt
N
H
N
O
H3C
N
H
N
Ph
N
O
Ph
N
7
H
N
O
N
N
Ph
Ph
20
Scheme 9. Synthetic pathway of compounds 20,21.
H3C
N
O
H
N
H3C
N
O
H3C
N
O
N
NH2
N
N
H
NaNO2 / CH3COOH
H2O
N3
O
EtOH
N
N
N
Ph
N
N
Ph
N
N
Ph
Ph
23
Ph
7
Ph
22
NaOH 20%
H3C
N
H3C
N
H
N
N
Ph
N
NH2
Ph
PhCOCl
O
N
N
N
Ph
N
P
h
Ph
25
24
Scheme 10. Synthetic pathway of compounds 22–25.
when the carbohydrazide 16 was reacted with benzoic acid in
refluxing phosphorylchloride. Treatment of compound 7 with for-
mic acid yielded N0-formylcarbohydrazide derivative 19 which
failed to cyclize into the oxadiazole 18 upon boiling in phosphoryl-
chloride. However, 18 could be obtained when 7 was allowed to re-
act with triethylorthoformate (Scheme 8).
followed by alkaline hydrolysis of the produced carbamate 23 to
give the amino derivative 24. The reaction of the latter compound
with benzoylchloride gave the benzoylamino derivative 25
(Scheme 10).
2.2. Anticonvulsant activity
When compound 7 was reacted with ethyl chloroformate
whether neat or in refluxing ethanol (for 1 h or 24 h), the product
obtained was identified as the ethoxycarbonyl derivative 20 and
not the expected oxadiazolonyl derivative 21 (Scheme 9).
On the other hand, the substitution of the acid hydrazide func-
tion of 7 by an amino group could be performed by converting 7
into the corresponding acid azide 22 under diazotization condi-
tions, followed by heating the azide 22 in refluxing ethanol,
Certain derivatives of imidazothiadiazoles and thiazolidinones
were reported to possess anticonvulsant activity.22,23 Accordingly,
it deemed of interest to evaluate the anticonvulsant activity of
the imidazothiadiazole- and thiazolidinone derivatives of our pre-
pared compounds. The preliminary results of anticonvulsant test-
ing of the compounds listed in the Table
1 revealed that,
pretreatment of mice with compounds 9a,b, 13a–d and 14a at a

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A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
Table 1
Anticonvulsant activity+ (effect of compounds 9a–b, 13a–d, 14a, b and 15 on PTZ-induced seizures and mortality in mice)
Treatment
Total number of
animals
Clonic convulsions
Tonic convulsions
Survivors
(24 h)
% Survivors
(24 h)
Onset
(min)
% Animals showing
convulsions
Onset (min)
% Animals showing
convulsions
PTZ
8
8
8
8
8
8
8
8
8
8
8
4.62 0.30 87.5
10.00 0.60
0
0
50
0
0
4
8
8
7
7
6
6
6
5
2
0
50
Phenobarbital
Comp. 13d + PTZ
Comp. 13a + PTZ
Comp. 9a + PTZ
Comp. 13c + PTZ
Comp. 13b + PTZ
Comp. 14a + PTZ
Comp. 9b + PTZ
Comp. 15 + PTZ
Comp. 14b + PTZ
0
0
100
100**
87.5**
87.5**
75**
75**
75**
62.5*
25
4.50 0.38 87.5
4.28 0.35 75
4.85 0.29 87.5
5.25 0.42 87.5
4.83 0.32 75
5.28 0.41 100
5.12 0.41 87.5
4.25 0.38 100
4.12 0.36 100
15.90** 0.51 12.5**
14.61** 0.72 12.5**
16.21** 0.92 25**
16.12** 0.88 25**
16.32** 1.10 25**
18.00** 1.20 37.5*
9.12 0.72
8.56 0.62
75
100
0
The compounds under investigation were injected ip 20 min before ip injection of PTZ.
- PTZ (Pentylenetetrazole).
+
Compounds prepared were administered at a dose of 10 mg/kg.
Significantly compared with control (p <0.05 vs PTZ value).
Very significantly compared with control (P <0.01 vs PTZ value).
*
**
dose of 10 mg/kg, ip significantly increased the latency time of
PTZ-induced tonic seizures and decreased the number of animals
showing tonic seizures in response to PTZ injection. Also, these re-
sults indicated that pretreatment of mice with these compounds
significantly inhibited PTZ-induced mortality within 24 h. How-
ever, compounds 15 and 14b increased PTZ-induced tonic convul-
sions and mortality. All tested compounds showed insignificant
effect on PTZ-induced clonic convulsions. Phenobarbital com-
pletely prevented PTZ-induced convulsive seizures and mortality.
while compounds 14d and 15increased PTZ-induced tonic convul-
sions and mortality.
4. Experimental section
4.1. Chemistry
All melting points were measured on Stuart melting point appa-
ratus (Bibby Scientific) SMP3. The IR spectra were recorded on a
Shimadzu 470 IR-Spectrophotometer using KBr wafer technique.
The 1H NMR spectra were recorded on a Varian Mercury VX-300
NMR spectrometer (Varian, Palo Alto, CA, USA, 300 MHz for 1H
and 75 MHz for 13C) at the Faculty of Science, Cairo University,
and on a Jeol LA 400 MHz (400 MHz for 1H, 100 MHz for the 13C)
at Assiut university, 1H and 13C NMR chemical shifts (d) were re-
ported in parts per million (ppm) and were referenced to the sol-
vent peak; CDCl3 (7.26 ppm for 1H and 76.90 ppm for 13C) and
DMSO-d6 (2.50 ppm for 1H and 39.70 ppm for 13C). Multiplicities
are represented by s (singlet), d (doublet), t (triplet), q (quartet)
and m (multiplet). Coupling constants (J) are reported in Hertz
(Hz). Elemental analyses were carried out using a Perkin–Elmer
240C Microanalyzer at the Microanalytical Laboratory at Assiut
University, and the results obtained were in an acceptable range
( 0.4%). 1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-car-
bonitrile (2), 1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-
5-carboxylic acid (5), methyl 1,6-diphenyl-3-methyl-1H-pyrazolo
[3,4-b]pyrazine-5-carboxylate (6), 1,6-diphenyl-3-methyl-1H-pyr-
azolo[3,4-b]pyrazin-5-carbohydrazide (7), 1,6-diphenyl-3-methyl-
lH-pyrazolo[3,4-b]pyrazin-5-carboxylic acid azide (22) and ethyl
N-(1,6-diphenyl-3-methyl-lH-pyrazolo[3,4-b]pyrazin-5-yl) carba-
mate (23) were prepared in our earlier publication.13
2.3. Structure–activity relationship
Concerning the anticonvulsant activity against PTZ-induced
tonic seizures, compound 13a, in which the 2-phenyl group of
the thiazolidinone moiety is unsubstituted, showed very signifi-
cant protective activity. The introduction of two methoxyl groups
in that phenyl ring (compound 13d) rendered the compound hav-
ing the highest activity. The introduction of Cl or NO2 group in the
4-position of the phenyl group (13b,c) lowers the activity. Con-
trarily to compound 13d having the best activity, compound
(14b) which has two methyl substituents in the 2-position of the
thiazolidinone moiety caused 100% convulsion and 100% mortality.
The substitution of the two methyl groups of 14b by a spiro cyclo-
hexane ring (compound 15) also enhanced the convulsions and
mortality but with a lesser extent that 14b. However, when the
cyclohexane ring was substituted by a cyclopentane one (com-
pound 14a) an improvement of activity was observed, comparable
to that of 13b,c.
On the other hand, 1,6-diphenyl-3-methyl-5-(6-phenylimi-
dazo[2,1-b]-1,3,4-thiadiazol-2-yl)-1H-pyrazolo[3,4-b]pyrazine (9a)
showed very significant activity comparable to 13a. The introduc-
tion of Cl atom at the 4-position of the 6-phenyl group (9b) lowers
the acivity.
4.1.1. 1,6-Diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-
carboxamidoxime (3)
3. Conclusion
To a stirred mixture of 1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-
b]pyrazine-5-carbonitrile (2) (0.31 g, 1 mmol), and potassium
carbonate (0.7 g, 5 mmol) in dioxane (10 mL), was added hydroxyl-
amine hydrochloride (0.35 g, 5 mmol) in water (3.5 mL). The reac-
tion mixture was heated at reflux for 3 h, then suspended in water
(5 mL) and stirred for additional 1 h. The solid precipitate obtained
was filtered off, dried and recrystallized from ethanol/dioxanemix-
In conclusion, we have made available a series of new pyrazol-
o[3,4-b]pyrazinescontaining heterocyclic substituents, and their
anticonvulsant activity was evaluated. The anticonvulsant activity
against PTZ-induced convulsions in mice at a dose of 10 mg/kg for
each compound showed that seven compounds (9a,b, 13a–d and
14a) showed very significant anticonvulsant activity (p <0.01) with
respect to the control (see Table 1) with delayed the onset time of
PTZ-induced tonic seizer. Compound 13d showed best results
ture (1:1). Yield: 74%; mp: 226–228 °C; IR (m
, cmꢀ1): 3450, 3350,
3200 broad (NH, OH), 1650 (C@N);1H NMR (300 MHz, DMSO-d6)
d (ppm): 2.71 (s, 3H, CH3), 6.04 (s, 2H, NH2 exchangeable with

A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
2171
D2O), 7.35–7.37 (m, 1H, ArH), 7.47–7.61 (m, 5H, ArH), 7.86–7.89
4.1.4. General procedure for the synthesis of 1,6-diphenyl-3-
(m, 2H, ArH), 8.24–8.27 (m, 2H, ArH), 9.55 (s, 1H, OH exchangeable
with D2O); 13C NMR (75 MHz, DMSO-d6) d (ppm): 11.48 (CH3),
119.96 (C), 126.28 (CH), 128.39 (C), 128.44 (CH), 129.34 (2CH),
129.67 (2CH), 129.19 (2CH), 131.56 (2CH), 138.64 (C), 142.40 (C),
143.38 (C), 143.57 (C), 150.48 (C), 151.98 (C); Anal. Calcd for
methyl-5-(6-(substitutedphenyl)imidazo[2,1-b]-1,3,4-
thiadiazol-2-yl)-1H-pyrazolo[3,4-b]pyrazine 9a,b
A mixture of equimolar amounts of compound 8 (0.38 g,
1 mmol), phenacylbromide derivative (0.001 mol) and potassium
carbonate (0.5 g) was refluxed in DMF (10 mL) for 3 h. The excess
of solvent was removed under reduced pressure and the solid ob-
tained was collected by filtration, washed with water, dried and
recrystallized from ethanol.
C19H16N6O (344.37): C, 66.27; H, 4.68; N, 24.40. Found: C, 66.34;
H, 4.60; N, 24.33.
4.1.2. General procedure for the synthesis of 5-(aryl-1,2,4-
oxadiazol-3-yl)-1,6-diaryl-3-methyl-1H-pyrazolo[3,4-b]pyrazine
4a,b
Benzoyl chloride derivative (1 mmol) was added to a stirred
solution of 3 (0.34 g, 1 mmol) in pyridine (5 mL). The reaction mix-
ture was heated under reflux for 3 h, cooled and poured onto cold
water. The solid precipitate formed was filtered, washed with
water, dried and recrystallized from ethanol.
4.1.4.1. 1,6-Diphenyl-3-methyl-5-(6-phenylimidazo[2,1-b]-1,3,
4-thiadiazol-2-yl)-1H-pyrazolo[3,4-b]pyrazine (9a). Yield: 46%;
mp: 260–262 °C; IR (m
, cmꢀ1): 3130, 3050 (CH aromatic), 1600
(C@N); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.76 (s, 3H, CH3),
7.24–7.27 (m, 1H, ArH), 7.36–7.62 (m, 8H, ArH), 7.74 (d, J = 7.2 Hz,
2H, ArH), 7.85 (d, J = 7.6 Hz, 2H, ArH), 8.23 (d, J = 8.4 Hz, 2H, ArH),
8.52 (s, 1H, CH imidazole); Anal.Calcd for C28H19N7S (485.57): C,
69.26; H, 3.94; N, 20.19; S, 6.60. Found: C, 69.33; H, 3.90; N, 20.28;
S, 6.61.
4.1.2.1. 1,6-Diphenyl-3-methyl-5-(5-phenyl-1,2,4-oxadiazol-3-yl)
4.1.4.2. 1,6-Diphenyl-3-methyl-5-(6-(4-bromophenyl) imidazo
[2,1-b]-1,3,4-thiadiazol-2-yl)-1H-pyrazolo[3,4-b]pyrazine
-1H-pyrazolo[3,4-b]pyrazine (4a). Yield: 77%; mp: 204–206 °C; IR
(m
, cmꢀ1): 3050 (CH aromatic), 2900 (CH aliphatic); 1H NMR
(9b). Yield: 78%, mp: 292–294 °C; IR (m,
cmꢀ1): 3140 (CH
(300 MHz, DMSO-d6) d (ppm): 2.75 (s, 3H, CH3), 7.37–7.38 (m, 1H,
ArH), 7.45–7.73 (m,10H, ArH), 8.07–8.09 (d, 2H, ArH), 8.26 - 8.29
(d, 2H, ArH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 11.30 (CH3),
120.19 (C and 2CH), 122.99 (C), 126.24 (CH), 128.01 (2CH), 128.50
(C and CH), 128.70 (CH), 129.49 (2CH), 129.74 (2CH), 132.70 (2CH),
133.82(2CH),136.07(C),137.37(C),138.39(C), 144.18(C),153.20(C),
168.24(C), 175.61(C);Anal. Calcdfor C26H18N6O (430.46):C, 72.55; H,
4.21; N, 19.52. Found C, 72.50; H, 4.36; N, 19.49.
aromatic); 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.78 (s, 3H,
CH3), 7.38–7.41 (m, 1H, ArH), 7.54 (d, J = 7.5 Hz, 2H, ArH), 7.57–
7.64 (m, 5H, ArH), 7.76 (d, J = 6.8 Hz, 2H, ArH), 7.82 (d, J = 7.5 Hz,
2H, ArH), 8.24 (d, J = 8.4 Hz, 2H, ArH), 8.59 (s, 1H, CH imidazole);
Anal. Calcd for C28H18BrN7S (564.47):C, 59.58; H, 3.21; Br, 14.16; N,
17.37; S, 5.68. Found: C, 59.52; H, 3.22; Br, 14.11; N, 17.32; S, 5.71.
4.1.5. General procedure for the synthesis of N0-arylidene-1,6-
diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carbohyd-
razide 10a–d
4.1.2.2. 5-(5-(4-Chlorophenyl)-1,2,4-oxadiazol-3-yl)-1,6-diphe-
nyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine (4b). Yield: 79%;
A mixture of equimolar amounts (1 mmol) of 3-methyl-1,6-di-
phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carbohydrazide (7)13 and
the appropriate aromatic aldehyde in a mixture of ethanol/dioxane
(8:2 mL) was refluxed for 2 h. After cooling, the solid precipitate
formed was collected by filtration and recrystallized from etha-
nol/dioxane mixture (3:1).
mp: 182–184 °C; IR (m
, cmꢀ1): 3050 (CH aromatic), 1590 (C@N);
1H NMR (400 MHz, CDCl3) d (ppm): 2.78 (s, 3H, CH3), 7.24–7.28 (m,
1H, ArH), 7.36 (d, J = 8 Hz, 2H, ArH), 7.44–7.49 (m, 5H, ArH), 7.55–
7.57 (m, 2H, ArH), 8.01 (d, J = 8 Hz, 2H, ArH), 8.28 (d, J = 7.6 Hz, 2H,
ArH); 13C NMR (100 MHz, CDCl3) d (ppm): 11.69 (CH3), 120.24 (C
and 2CH), 122.22 (C), 126.23 (CH), 128.40 (C and CH), 129.28
(2CH), 129.57 (2CH), 129.65 (2CH), 133.01 (2CH), 136.35 (2CH),
137.67 (C), 138.87 (C), 139.58 (C), 142.87 (C), 144.46 (C), 153.20
(C), 168.72 (C), 175.18 (C); Anal. Calcd for C26H17ClN6O (464.91): C,
67.17; H, 3.69; Cl, 7.63; N, 18.08. Found: C, 67.27 ; H, 3.60; Cl, 7.59;
N, 18.18.
4.1.5.1.
[3,4-b]pyrazine-5-carbohydrazide (10a). Yield: 91%; mp: 248–
250 °C; IR (
, cmꢀ1): 3220 (NH), 3050 (CH aromatic), 2910, 2820
N0-Benzylidene-1,6-diphenyl-3-methyl-1H-pyrazolo
m
(CH aliphatic), 1680 (C@O); 1H NMR (400 MHz, DMSO-d6) d (ppm):
2.73 (s, 3H, CH3), 7.34–7.37 (m, 1H, ArH), 7.45–7.83 (m, 10H, ArH),
8.25–8.30 (m, 4H, ArH), 8.35 (s, 1H, CH@N), 12.11 (s, 1H, NH
exchangeable with D2O); 13C NMR (100 MHz, DMSO-d6) d (ppm):
11.28 (CH3), 119.95 (C), 120.01 (2CH), 126.71 (CH), 128.73 (C),
128.90 (CH), 128.98 (2CH), 129.52 (2CH), 129.73 (2CH), 129.80
(2CH), 130.44 (2CH), 131.29 (CH), 137.22 (C), 138.42 (C), 138.59
(C), 143.55 (C), 143.71 (CH), 148.74 (C), 150.19 (C), 168.61 (C@O);
Anal. Calcd forC26H20N6O (432.49):C, 72.21; H, 4.66; N, 19.43.
Found: C, 72.33; H, 4.59; N, 19.49.
4.1.3. 5-(5-Amino-1,3,4-thiadiazol-2-yl)-1,6-diphenyl-3-methyl-
1H-pyrazolo[3,4-b]pyrazine (8)
A mixture of the acid 1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]
pyrazine-5-carboxylic acid (5)13 (3 g, 9 mmol), and thiosemicarba-
zide (8.27 g, 90 mmol) in phosphorylchloride (15 mL) was refluxed
gently for 3 h. The reaction mixture was cooled and quenched (highly
exothermic) with cold water (27 mL), and the resulting solution was
refluxed for additional 4 h. The solution is allowed to cool and basi-
fied with aqueous potassium hydroxide solution. The solid that sep-
arated out was filtered, washed with water, dried and recrystallized
from dioxane/ DMF mixture (3:1). Yield: 77%; mp: 292–294 °C; IR
4.1.5.2.
pyrazolo[3,4-b]pyrazine-5-carbohydrazide (10b). Yield: 92%;
mp: 258–260 °C; IR (
, cmꢀ1): 3220 (NH), 3060 (CH aromatic),
N0-(4-Chlorobenzylidene)-1,6-diphenyl-3-methyl-1H-
m
2910, 2850 (CH aliphatic), 1685 (C@O); 1H NMR (400 MHz, DMSO-
d6) d (ppm): 2.73 (s, 3H, CH3), 7.24–7.79 (m, 10H, ArH), 8.25–8.29
(m, 4H, ArH), 8.34 (s, 1H, CH@N), 12.15 (s, 1H, NH exchangeable
with D2O); 13C NMR (100 MHz, DMSO-d6) d (ppm): 11.31 (CH3),
119.99 (C), 126.26 (2CH), 126.44 (CH), 128.65 (C), 128.39 (CH),
129.54 (2CH), 129.70 (2CH), 129.01 (2CH), 131.40 (2CH), 130.85
(2CH), 137.25 (C), 138.69 (C), 142.47 (C), 143.64 (CH), 147.47 (C),
150.30 (C), 151.31 (C), 162.97 (C), 168.76 (C@O); Anal. Calcd for
(m
, cmꢀ1): 3350, 3100 (NH2), 2950, 2900, 2830 (CH aliphatic); 1H
NMR (400 MHz, DMSO-d6) d (ppm): 2.68 (s, 3H, CH3), 7.31–7.34 (m,
1H, ArH), 7.44–7.47 (m, 5H, ArH and NH2), 7.54–7.61 (m, 4H, ArH),
8.19–8.21 (m, 2H, ArH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
11.28 (CH3), 108.09 (C), 119.85 (2CH), 126.23 (CH), 128.02 (CH),
129.03 (C), 129.49 (2CH), 129.60 (2CH), 131.87 (2CH), 138.62 (C),
139.81 (C), 141.61 (C), 143.46 (C), 151.59 (C), 155.80 (C), 170.01
(C); Anal. Calcd for C20H15N7S (385.45): C, 62.32; H, 3.92; N, 25.44;
S, 8.32. Found: C, 62.52; H, 3.73; N, 25.41; S, 8.11.
C26H19ClN6O (466.92): C, 66.88; H, 4.10; Cl, 7.59; N, 18.00. Found:
C, 66.90; H, 4.21; Cl, 7.50; N, 18.11.

2172
A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
4.1.5.3. N0-(4-Nitrobenzylidene)-1,6-diphenyl-3-methyl-1H-pyr-
(N@C); Anal. Calcd for C25H24N6O (424.5): C, 70.73; H, 5.70; N,
azolo[3,4-b]pyrazine-5-carbo-hydrazide (10c). Yield: 90%; mp:
19.80. Found; C, 70.92; H, 5.72; N, 19.71.
152–154 °C; IR (m
, cmꢀ1): 200 (NH), 3050 (CH aromatic), 2910-
2830 (CH aliphatic), 1680 (C@O); 1H NMR (400 MHz, DMSO-d6) d
(ppm): 2.74 (s, 3H, CH3), 7.35–7.39 (m, 1H, ArH), 7.45–7.82 (m, 5H,
ArH), 7.99 (d, J = 8.4 Hz, 2H, ArH), 8.12 (d, J = 8.8 Hz, 2H, ArH) 8.25–
8.31 (m, 4H, ArH),8.47 (s, 1H, CH@N), 12.39 (s, 1H, NH exchange-
able with D2O); 13C NMR (100 MHz, DMSO-d6) d (ppm): 11.33
(CH3), 120.06 (C), 123.99 (2CH), 124.12 (2CH), 126.18 (CH), 127.69
(2CH), 128.32 (C), 128.67 (CH), 129.52 (2CH), 129.78 (2CH), 139.53
(C),130.79 (2CH), 138.64 (C), 139.52 (C), 146.47 (C), 147.70 (CH),
148.10 (C), 150.32 (C), 151.34 (C), 162.92 (C), 168.91 (C@O); Anal.
Calcd for C26H19N7O3 (477.47): C, 65.40; H, 4.01; N, 20.53. Found:C,
65.51; H, 3.91; N, 20.49.
4.1.7. 1,6-Diphenyl-3-methyl-N0-(prop-2-ylidene)-1H-
pyrazolo[3,4-b]pyrazine-5-carbohydrazide (12)
A solution of carbohydrazide7 (0.34 g, 1 m mol) in acetone
(10 mL) was refluxed for 1 h. After cooling, the solid precipitate ob-
tained was collected by filtration and recrystallized from ethanol.
Yield: 82%; mp: 220–222 °C; IR(m
, cmꢀ1): 3300 (NH), 3050 (CH aro-
matic), 2910, 2820 (CH aliphatic), 1670 (C@O); 1H NMR (400 MHz,
CDCl3) d (ppm): 1.98 (s, 3H, N@CCH3), 2.16 (s, 3H, N@CCH3), 2.77 (s,
3H, CH3), 7.29–7.32 (m, 1H, ArH), 7.46–7.82 (m, 5H, ArH), 8.30 (d,
J = 7.8 Hz, 2H, ArH), 8.35 (d, J = 7.8 Hz, 2H, ArH), 9.88 (s, 1H, NH
exchangeable with D2O); 13C NMR (100 MHz, CDCl3) d (ppm):
11.63 (CH3), 16.79 (CH3), 25.61 (CH3), 120.08 (C and 2CH), 126.21
(CH), 128.30 (C), 128.11 (CH), 129.52 (2CH), 129.24 (2CH), 131.15
(2CH), 138.75 (C), 137.83 (C), 143.92 (C), 143.15 (C), 153.74 (C),
156.79 (C@O), 160.88 (N@C); Anal. Calcd for C22H20N6O (384.43):
C, 68.73; H, 5.24; N, 21.86. Found: C, 68.65; H, 5.29; N, 21.79.
4.1.5.4. N0-(3,4-Dimethoxybenzylidene)-1,6-diphenyl-3-methyl-
1H-pyrazolo[3,4-b]pyrazine-5-carbohydrazide
(10d). Yield:
72%; mp: 230–232 °C; IR (
m
, cmꢀ1): 3210 (NH), 3050 (CH aromatic),
2920-2810 (CH aliphatic), 1650 (C@O); 1H NMR (400 MHz, DMSO-
d6):d 2.73 (s, 3H, CH3), 3.67 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 6.81–
7.22 (m, 3H, ArH), 7.33–7.38 (m, 1H, ArH), 7.47–7.65 (m, 5H,ArH),
7.79–7.83 (m, 2H, ArH), 8.25–8.27 (m, 2H, ArH), 8.28 (s, 1H, CH@N),
12.19 (s, 1H, NH exchangeable with D2O); 13C NMR (100 MHz,
DMSO-d6) d: 11.28 (CH3), 55.46 (OCH3), 55.59 (OCH3), 108.10 (C),
108.25 (CH), 111.44 (CH), 119.77 (2CH), 122.26 (CH), 126.35 (CH),
128.63 (C), 128.96 (CH), 129.52 (2CH), 129.70 (2CH), 130.80 (2CH),
131.20 (C), 137.22 (C), 138.57 (C), 143.49 (C), 148.56 (C), 149.05
(C), 150.48 (C), 150.99 (C), 151.19 (C), 168.45 (C@O); Anal. Calcd
for C28H24N6O3 (492.53): C, 68.28; H, 4.91; N, 17.06. Found:C,
68.20; H, 4.98; N, 17.18.
4.1.8. General procedure for the synthesis of N-(2-aryl-4-oxo-
1,3-thiazolidin-3-yl)-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-
b]pyrazine-5-carboxamide 13a–d
A mixture of compounds 10a–d (1 mmol) and mercaptoacetic
acid (0.18 g, 2 mmol) in dry benzene (50 mL) was refluxed in
Dean–stark apparatus for (24 h for 13b,d, 48 h for 13a,c). The sol-
vent was evaporated and the residue obtained was triturated with
10% sodium bicarbonate solution. The precipitate formed was fil-
tered off, washed with water, air dried and recrystallized from
ethanol.
4.1.6. General procedure for the synthesis of N0-cychloalky
lidene-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-
carbohydrazide 11a,b
A mixture of equimolar amounts (1 mmol) of the carbohydraz-
ide7 and the appropriate cycloalkanone (cyclopentanone or cyclo-
hexanone) in a mixture of ethanol/dioxane (8:2 mL) was refluxed
for 2 h. After cooling, the solid precipitate obtained was collected
by filtration and recrystallized from ethanol.
4.1.8.1. N-(2-Phenyl-4-oxo-1,3-thiazolidin-3-yl)-1,6-diphenyl-3-
methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxamide (13a). Yield:
89%; mp: 206–208 °C; IR (m
, cmꢀ1): 3230 (NH), 3050 (CH aromatic),
2980, 2910 (CH aliphatic), 1720 (C@O thiazolidinone), 1680 (C@O);
1H NMR (400 MHz, CDCl3) d (ppm): 2.64 (s, 3H, CH3), 3.75 (d,
J = 15.6 Hz, 1H, CH2 thiazolidinone), 3.84 (d, J = 15.6 Hz, 1H, CH2
thiazolidinone), 6.01 (s, 1H, CH thiazolidinone), 7.25–7.29 (m, 1H,
ArH), 7.42–7.49 (m, 10H, ArH), 7.61–7.62 (m, 2H, ArH), 8.22–8.24
(d, J = 7.8 Hz, 2H, ArH), 9.03 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
d (ppm): 11.36 (CH3), 30.09 (CH2 thiazolidinone), 62.80 (CH
thiazolidinone), 120.09 (2CH and C), 126.31 (CH), 128.99-129.64
(12 CH), 131.12 (C), 137.04 (C), 137.71 (C), 138.55 (C), 142.87 (C),
144.27 (C), 153.96 (C), 163.18 (C@O), 169.68 (C@O thiazolidinone);
Anal. Calcd for C28H22N6O2S (506.58): C, 66.39; H, 4.38; N, 16.59; S,
6.33. Found: C, 66.42; H, 4.31; N, 16.63; S, 6.23.
4.1.6.1. N0-Cyclopentylidene-1,6-diphenyl-3-methyl-1H-pyraz-
olo[3,4-b]pyrazine-5-carbohydrazide (11a). Yield: 66%; mp:
216–218 °C; IR (m
, cmꢀ1): 3300 (NH), 3050 (CH aromatic), 2970,
2890 (CH aliphatic), 1690 (C@O); 1H NMR (400 MHz, CDCl3) d
(ppm): 1.56–2.02 (m, 4H, 2CH2), 2.34–2.61 (m, 4H, 2CH2), 2.77 (s,
3H, CH3), 7.26–7.31 (m, 1H, ArH), 7.46–7.54 (m, 5H, ArH), 7.76–
7.82 (m, 2H, ArH), 8.29–8.35 (m, 2H, ArH), 9.63 (s, 1H, NH); 13C
NMR (100 MHz, CDCl3) d (ppm): 11.63 (CH3), 24.81 (CH2), 27.54
(CH2), 33.61 (CH2), 38.41 (CH2), 120.11 (C and 2CH), 126.23 (CH),
128.27 (C), 128.16 (CH), 129.55 (2CH), 129.26 (2CH), 130.10 (2CH),
138.79 (C), 137.81 (C), 144.00 (C), 153.56 (C), 160.90 (C), 165.38
(C@O), 168.81 (N@C); Anal. Calcd for C24H22N6O (410.47):C, 70.23;
H, 5.40; N, 20.47. Found: C, 70.36; H, 5.49; N, 20.25.
4.1.8.2. N-(2-(4-Chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,6-
diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxamide
(13b). Yield: 98%; mp: 200–202 °C; IR (m
, cmꢀ1): 3360 (NH), 3070
(CH aromatic), 2950, 2850 (CH aliphatic), 1720 (C@O thiazolidi-
none), 1690 (C@O); 1H NMR (400 MHz, CDCl3) d (ppm): 2.68 (s, 3H,
CH3), 3.75 (d, J = 15.6 Hz, 1H, CH2 thiazolidinone), 3.84 (d,
J = 15.6 Hz, 1H, CH2 thiazolidinone), 5.98 (s, 1H, CH thiazolidinone),
7.29–7.31 (m, 1H, ArH), 7.39–7.41 (d, J = 8.8 Hz, 2H, ArH), 7.43–
7.45 (d, J = 8.8 Hz, 2H, ArH), 7.47–7.50 (m, 5H, CH ArH), 7.59 (m,
2H, CH ArH), 8.25 (d, J = 7.8 Hz, 2H, CH ArH), 9.03 (s, 1H, NH); 13C
NMR (100 MHz, CDCl3) d (ppm): 11.41 (CH3), 29.12 (CH2 thiazo-
lidinone), 60.76 (CH thiazolidinone), 120.01 (C), 126.38 (CH),
128.45 (CH), 128.65 (C), 129.06 (2CH), 129.50 (2CH), 129.60
(2CH), 129.74 (2CH), 130.77 (4CH), 136.81 (C), 137.47 (C), 138.32
(C), 141.98 (C), 142.40 (C), 143.70 (C), 151.17 (C), 164.98 (C@O),
4.1.6.2. N0-Cyclohexylidene-1,6-diphenyl-3-methyl-1H-pyrazol-
o[3,4-b]pyrazine-5-carbohydrazide (11b). Yield: 88%; mp:
218–219 °C; IR (m
, cmꢀ1): = 3300 (NH), 3050 (CH aromatic), 2910,
2820 (CH aliphatic), 1670 (C@O); 1H NMR (300 MHz, DMSO-d6) d
(ppm): 1.38–1.70 (m, 6H, 3CH2), 2.18–2.25 (m, 4H, 2CH2), 2.72 (s,
3H, CH3), 7.36–7.37 (m, 1H, ArH), 7.51–7.63 (m, 5H, ArH), 7.74–
7.83 (m, 2H, ArH), 8.25–8.28 (m, 2H, ArH), 10.85 (s, 1H, NH
exchangeable with D2O); 13C NMR (75 MHz, DMSO-d6) d (ppm):
11.29 (CH3), 24.97 (CH2), 26.66 (CH2), 26.88 (CH2), 27.43 (CH2),
34.63 (CH2), 119.85 (C), 119.91 (2CH), 126.23 (CH), 128.71 (C),
128.51 (CH), 129.63 (2CH), 129.51 (2CH), 130.89 (2CH), 138.56 (C),
146.25 (C), 150.77 (C), 158.68 (C), 162.70 (C), 164.61 (C@O), 168.68
168.91 (C@O thiazolidinone); Anal. Calcd for
C28H21ClN6O2S
(541.02): C, 62.16; H, 3.91; Cl, 6.55; N, 15.53; S, 5.93. Found: C,
62.22; H, 3.96; Cl, 6.50; N, 15.49; S, 5.90.

A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
2173
4.1.8.3.
diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxamide
(13c). Yield: 98%; mp: 234–236 °C; IR (
, cmꢀ1): = 3350 (NH),
N-(2-(4-Nitrophenyl)-4-oxo-1,3-thiazolidin-3-yl)-1,6-
1.53 (m, 2H, CH2), 1.65–1.73 (m, 2H, CH2), 2.76 (s, 3H, CH3), 3.61 (s,
2H, CH2 thiazolidinone), 7.37–7.40 (m, 1H, ArH), 7.53–7.63 (m, 5H,
ArH), 7.83–7.86 (m, 2H, ArH), 8.24–8.27 (d, 2H, ArH), 10.81 (s, 1H,
NH exchangeable with D2O); 13C NMR (75 MHz, DMSO-d6) d
(ppm): 11.43 (CH3), 22.78 (2CH2 cyclohexane), 23.75 (CH2 cyclo-
hexane), 27.82 (2CH2 cyclohexane), 36.72 (CH2 thiazolidinone),
72.41 (C thiazolidinone), 119.98 (C and CH), 126.35 (CH), 128.44
(CH), 129.42 (C), 129.50 (2CH), 129.69 (2CH), 131.12 (2CH), 137.14
(C), 138.39 (C), 143.26 (C), 143.75 (C), 151.34 (C), 165.82 (C@O),
167.65 (C@O thiazolidinone); Anal. Calcd for C27H26N6O2S (498.6):
C, 65.04; H, 5.26; N, 16.86; S, 6.43. Found: C, 65.15; H, 5.14; N,
16.94; S, 6.40.
m
3070 (CH aromatic), 2910, 2850 (CH aliphatic), 1720 (C@O
thiazolidinone), 1690 (C@O); 1H NMR (400 MHz, CDCl3):d 2.66 (s,
3H, CH3), 3.79 (d, J = 15.6 Hz, 1H, CH2 thiazolidinone), 3.86 (d,
J = 15.6 Hz, 1H, CH2 thiazolidinone), 6.09 (s, 1H, CH thiazolidinone),
7.27–7.31 (m, 1H, ArH), 7.46–7.52 (m, 5H, ArH), 7.56–7.58 (m, 2H,
ArH), 7.67–7.69 (m, 2H, ArH), 8.22–8.24 (m, 2H, ArH), 8.26–8.28
(m, 2H, ArH), 9.17 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm):
11.40 (CH3), 29.00 (CH2 thiazolidinone), 60.30 (CH thiazolidinone),
120.01 (2CH and C), 123.84 (2CH), 126.38 (CH), 128.78 (CH and C),
128.99 (2CH), 129.50 (2CH), 129.65 (2CH), 130.82 (2CH), 138.32
(C), 141.89 (C), 142.39 (C), 143.72 (C), 146.22 (C), 147.69 (C),
151.22 (C), 165.09 (C@O), 168.98 (C@O thiazolidinone); Anal. Calcd
for C28H21N7O4S (551.58): C, 60.97; H, 3.84; N, 17.78; S, 5.81.
Found: C, 60.89; H, 3.85; N, 17.84; S, 5.95.
4.1.10. N-(2,2-Dimethyl-4-oxo-1,3-thiazolidin-3-yl)-1,6-
diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxamide
(15)
A mixture of compound 12 (0.38 g, 1 mmol) and mercaptoacetic
acid (0.18 g, 2 mmol) in dry benzene (50 mL) was refluxed in
Dean–stark apparatus for 72 h. The solvent was then evaporated
under reduced pressure and the residue obtained was triturated
with 10% sodium bicarbonate solution. The precipitate formed
was filtered off, washed with water, air dried and recrystallized
4.1.8.4.
yl)-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-car-
boxamide (13d). Yield: 84%; mp: 212–214 °C; IR (
, cmꢀ1): =
N-(2-(3,4-Dimethoxyphenyl)-4-oxo-1,3-thiazolidin-3-
m
3300 (NH), 3070 (CH aromatic), 2950–2830 (CH aliphatic), 1715
(C@O thiazolidinone), 1680 (C@O); 1H NMR (400 MHz, CDCl3):d
2.67 (s, 3H, CH3), 3.74 (d, J = 15.6 Hz, 1H, CH2 thiazolidinone), 3.84
(d, J = 15.6 Hz, 1H, CH2 thiazolidinone), 3.89 (s, 6H, 2OCH3), 5.97 (s,
1H, CH thiazolidinone), 6.85 (m, 1H, ArH), 6.98–7.01 (m, 1H, ArH),
7.09 (m, 1H, ArH), 7.28–7.31 (m, 1H, ArH), 7.47–7.50 (m, 5H, ArH),
7.60–7.62 (m, 2H, ArH), 8.25 (d, J = 7.8 Hz, 2H, ArH), 9.01 (s, 1H,
NH); 13C NMR (100 MHz, CDCl3) d (ppm): 11.41 (CH3), 30.22 (CH2
thiazolidinone), 55.05 (OCH3), 55.96 (OCH3), 63.03 (CH thiazolid-
inone), 110.47 (CH), 110.65 (CH), 120.18 (C), 121.26 (CH), 126.38
(CH), 128.02 (CH), 128.83 (C), 129.24 (2CH), 129.42 (2CH), 129.52
(2CH), 131.15 (2CH), 137.63 (C), 138.57 (C), 142.39 (C), 144.28 (C),
149.51 (2C), 150.16 (C), 154.04 (C), 163.17 (C@O), 168.64 (C@O
thiazolidinone); Anal. Calcd for C30H26N6O4S (566.63):C, 63.59; H,
4.62; N, 14.83; S, 5.66. Found: C, 63.68; H, 4.59; N, 14.89; S, 5.76.
from ethanol. Yield: 96%; mp: 238–240 °C; IR (m
, cmꢀ1): 3200
(NH), 3050 (CH aromatic), 2980–2810 (CH aliphatic), 1725 (C@O
thiazolidinone), 1690 (C@O); 1H NMR (400 MHz, CDCl3) d (ppm):
1.64 (s, 6H, 2CH3), 2.70 (s, 3H, CH3), 3.61 (s, 2H, CH2 thiazolidi-
none), 7.22–7.25 (m, 1H, ArH), 7.40–7.45 (m, 5H, ArH), 7.64–7.67
(m, 2H, ArH), 8.22 (d, J = 8.8 Hz, 2H, ArH), 9.01 (s, 1H, NH exchange-
able with D2O); 13C NMR (100 MHz, CDCl3) d (ppm): 11.58 (CH3),
29.38 (2CH3), 29.60 (CH2 thiazolidinone), 66.95 (C thiazolidinone),
120.19 (C and 2CH), 126.36 (CH), 128.02 (C and CH), 129.26 (2CH),
129.55 (2CH), 131.15 (2CH), 137.80 (C), 138.65 (C), 143.00 (C),
144.31 (C), 154.38 (C), 164.06 (C@O), 168.11 (C@O thiazolidinone);
Anal. Calcd for C24H22N6O2S (458.54): C, 62.86; H, 4.84; N, 18.33; S,
6.99. Found: C, 62.80; H, 4.91; N, 18.43; S, 6.90.
4.1.11. N0-Benzoyl-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-
b]pyrazine-5-carbohydrazide (16)
4.1.9. General procedure for the synthesis of N-(cycloalkyl-
spiro-2-(4-oxo-1,3-thiazolidin-3-yl))-1,6-diphenyl-3-methyl-
1H-pyrazolo[3,4-b]pyrazine-5-carboxamide 14a,b
These compounds were prepared using the same procedure as
described before in synthesis of compounds 13a–d.
To a solution of carbohydrazide 7 (0.34 g, 1 mmol) in pyridine
(10 ml), benzoyl chloride (0.14 g, 1 mmol) was added, and the reac-
tion mixture was stirred at room temperature for 3 h. The solvent
was removed under reduced pressure and the residue obtained
was collected by filtration and recrystallized from dioxane. Yield:
96%; mp: 286–288 °C; IR (m
, cmꢀ1): 3270 (NH), 3050 (CH aromatic),
4.1.9.1. N-(Cyclopentane-spiro-2-(4-oxo-1,3-thiazolidin-3-yl))-
1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carbox-
1640 (C@O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.75 (s, 3H,
CH3), 7.38–7.64 (m, 9H, ArH), 7.94–7.96 (m, 2H, ArH), 8.06–8.09
(m, 2H, ArH), 8.27–8.29 (m, 2H, ArH), 10.67 (s, 1H, NH exchange-
able with D2O), 10.95 (s, 1H, NH exchangeable with D2O); 13C
NMR (75 MHz, DMSO-d6) d (ppm): 11.38 (CH3), 120.03 (2CH and
C), 126.33 (CH), 127.58 (2CH), 128.52 (CH and C), 128.80 (2CH),
129.54 (2CH), 129.82 (2CH), 130.80 (2CH), 132.02 (C), 132.38
(CH), 136.91 (C), 138.49 (C), 143.38 (C), 143.69 (C), 151.01 (C),
amide (14a). Yield: 85%; mp: 232–234 °C; IR (m
, cmꢀ1): 3250
(NH), 3050 (CH aromatic), 2950-2830 (CH aliphatic), 1720 (C@O
thiazolidinone), 1680 (C@O); 1H NMR (400 MHz, CDCl3) d (ppm):
1.79 (m, 4H, 2CH2), 2.01–2.05 (m, 2H, CH2), 2.17–2.24 (m, 2H, CH2),
2.75 (s, 3H, CH3), 3.66 (s, 2H, CH2 thiazolidinone) 7.27–7.31 (m, 1H,
ArH), 7.46–7.49 (m, 5H, ArH), 7.71–7.72 (m, 2H, ArH), 8.25–8.27
(m, 2H, ArH), 9.24 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm):
11.55 (CH3), 22.64 (2CH2 cyclopentane), 38.63 (2CH2 cyclopen-
tane), 29.40 (CH2 thiazolidinone), 66.70 (C thiazolidinone), 120.16
(C and 2CH), 126.35 (CH), 127.99 (C and CH), 129.24 (2CH), 129.55
(2CH), 131.07 (2CH), 137.85 (C), 138.60 (C), 142.93 (C), 144.20 (C),
154.20 (C), 164.09 (C@O), 168.60 (C@O thiazolidinone); Anal. Calcd
for C26H24N6O2S (484.57): C, 64.44; H, 4.99; N, 17.34; S, 6.62.
Found: C, 64.56; H, 4.91; N, 17.14; S, 6.72.
161.78 (C@O), 165.74 (C@O); Anal. Calcd for
C26H20N6O2
(448.48): C, 69.63; H, 4.49; N, 18.74. Found: C, 69.75; H, 4.50; N,
18.81.
4.1.12. 1,6-Diphenyl-3-methyl-5-(5-phenyl-1,3,4-oxadiazol-2-
yl)-1H-pyrazolo[3,4-b]pyrazine (17)
Procedure (1): A solution of compound 16 (0.37 g, 0.8 mmol) in
phosphorylchloride (5 mL) was heated under reflux at 100 °C for
5 h. After cooling, the solvent was removed in vacuo and the resi-
due obtained was poured into an ice-water mixture and neutral-
ized with ammonium hydroxide (20%). The solid product
obtained was filtered off and recrystallized from ethanol/dioxane
mixture (1:1). Yield: 80%; mp: 194–196 °C.
4.1.9.2. N-(Cyclohexane-spiro-2-(4-oxo-1,3-thiazolidin-3-yl))-
1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carbox-
amide (14b). Yield: 91.8%; mp: 240–242 °C; IR (m
, cmꢀ1): = 3230
(NH), 2920, 2850 (CH aliphatic), 1720 (C@O thiazolidinone), 1680
(C@O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.41 (m, 6H, 3CH2),

2174
A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
Procedure (2): To a cold mixture of equimolar amount of the
11.45 (OCH2CH3), 14.38 (CH3), 62.41 (OCH2CH3), 120.26 (2CH and
C), 126.33 (CH), 128.03 (2CH), 129.26 (2CH), 129.44 (CH), 129.48
(2CH), 130.48 (C), 137.99 (C), 138.79 (C), 143 (C), 144 (C), 154
(C), 157 (C@O), 164.26 (CO); Anal. Calcd for C22H20N6O3 (416.43):
Calcd: C, 63.45; H, 4.84; N, 20.18. Found: C, 63.22; H, 4.93; N,
20.09.
carbohydrazide7 (0.34 g, 1 mmol) and benzoic acid (0.12 g,
1 mmol) was added phosphorylchloride (5 mL) while stirring. The
reaction mixture was then heated under reflux for 48 h, cooled,
poured carefully to an equal volume of ice-water mixture followed
by neutralization with solid sodium bicarbonate. The precipitate
formed after standing for 1 h was filtered, washed with water
and recrystallized from ethanol/dioxane mixture (1:1). The prod-
uct obtained by using this method is identical in all respects (mp
mixed mp and spectral data) with that obtained using method
4.1.16. 5-Amino-1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-b]
pyrazine (24)
A
mixture of ethyl (1,6-diphenyl-3-methyl-lH-pyrazolo
(1). Yield: 91%; mp: 194–196 °C; IR (
m
, cmꢀ1): 3050 (CH aromatic);
[3,4-b]pyrazin-5-yl)carbamate (23)13 (0.37 g, 1 mmol) and sodium
hydroxide 20% (10 mL) was refluxed for 3 h. The solid product
formed was collected by filtration and recrystallized from
1H NMR (300 MHz, DMSO-d6) d (ppm): 2.73 (s, 3H, CH3), 7.35 (m,
1H, ArH), 7.49–7.64 (m, 10H, ArH), 7.79 (d, J = 8.1 Hz, 2H, ArH),
8.22 (d, J = 8.4 Hz, 2H, ArH); 13C NMR (75 MHz, DMSO-d6) d
(ppm): 11.36 (CH3), 120.01 (C), 122.77 (2CH), 126.54 (CH),
127.42 (2CH), 128.44 (2CH), 129.29 (4CH), 129.49 (2CH), 132.38
(C), 132.71 (2CH), 133.16 (C), 137.52 (C), 138.21 (C), 142.13 (C),
144.33 (C), 152.95 (C), 162.53 (C), 164.31 (C); Anal. Calcd for
ethanol/dioxane (1:1). Yield: 90%; mp: 180–182 °C; IR (m
, cmꢀ1):
3490, 3300, 3150 (NH2), 3050 (CH aromatic), 2900, 2820 (CH ali-
phatic); 1H NMR (400 MHz, CDCl3): d 2.65 (s, 3H, CH3), 4.85 (s,
2H, NH2), 7.20–7.26 (m, 1H, ArH), 7.44–7.55 (m, 5H, ArH), 7.84
(d, J = 7.8 Hz, 2H, ArH), 8.26 (d, J = 7.8 Hz, 2H, ArH); 13C NMR
(100 MHz, CDCl3) d: 11.33 (CH3), 100.53 (C), 119.62 (2CH),
125.23 (CH), 128.70 (C and CH), 129.08 (2CH), 129.70 (4CH),
137.14 (C), 139.58 (C), 141.94 (C), 149.17 (C), 154.70 (C), Anal.
Calcd for C18H15N5 (301.35): C, 71.74; H, 5.02; N, 23.24. Found:
C, 71.70; H, 5.12; N, 23.19.
C
26H18N6O (430.46): C, 72.55; H, 4.21; N, 19.52. Found: C, 72.46;
H, 4.20; N, 19.33.
4.1.13. 1,6-Diphenyl-3-methyl-5-(1,3,4-oxadiazol-2-yl)-1H-
pyrazolo[3,4-b]pyrazine (18)
A mixture of 7 (0.34 g, 1 mmol) and triethylorthoformate (3 mL)
was gently refluxed for 12 h. After cooling, the solid product
formed was collected and recrystallized from ethanol. Yield: 54%;
4.1.17. 5-Benzoylamino-1,6-diphenyl-3-methyl-1H-
pyrazolo[3,4-b]pyrazine (25)
mp: 194–196 °C; IR (m
, cmꢀ1): 3100 (CH aromatic); 1H NMR
A mixture of amine 24 (0.30 g, 1 mmol) in pyridine (10 ml) and
benzoyl chloride (0.14 g, 1 mmol) was stirred at room temperature
for 18 h. The reaction mixture was concentrated under reduced
pressure and the solid residue obtained was filtered, washed with
water, and recrystallized from ethanol. Yield: 83 %; mp:
(300 MHz, DMSO-d6) d (ppm): 2.74 (s, 3H, CH3), 7.38–7.40 (m,
1H, ArH), 7.48–7.63 (m, 7H, ArH), 8.23–8.26 (m, 2H, ArH), 9.39 (s,
1H, CH oxadiazole); 13C NMR (75 MHz, DMSO-d6) d (ppm): 11.33
(CH3), 120.06 (2CH and C), 128.50 (CH and C), 129.72 (2CH),
126.49 (CH), 132.61 (2CH), 133.19 (2CH), 137.19 (C), 138.21 (C),
142.32 (C), 144.28 (C), 152.85 (C), 162.27 (C), 155.06 (C); Anal.
Calcd for C20H14N6O (354.36): C, 67.79; H, 3.98; N, 23.72. Found:
C, 67.87; H, 3.89; N, 23.52.
206–208 °C; IR (m
, cmꢀ1): 3350 (NH), 3050 (CH aromatic), 2910,
2850 (CH aliphatic), 1690 (C@O).1H NMR (400 MHz, CDCl3) d
(ppm): 2.72 (s, 3H, CH3), 7.27–7.30 (m, 1H, ArH), 7.41–7.55 (m,
8H, ArH), 7.77 (d, J = 6.8 Hz, 2H, ArH), 7.85 (d, J = 5.9 Hz, 2H,
ArH), 8.29 (d, J = 7.8 Hz, 2H, ArH), 8.36 (s, 1H, NH); 13C NMR
(100 MHz, CDCl3) d (ppm): 11.43 (CH3), 119.98 (C and 2CH),
125.84 (CH), 127.37 (2CH), 128.75 (C and CH), 128.81 (2CH),
129.18 (2CH), 129.55 (2CH), 131.30 (2CH), 132.34 (CH), 133.57
(C), 137.49 (C), 139.13 (C), 142.08 (C), 142.85 (C), 148.20 (C),
165.95 (C@O); Anal. Calcd for C25H19N5O (405.45): C, 74.06; H,
4.72; N, 17.27. Found: C, 74.09; H, 4.68; N, 17.39.
4.1.14. 1,6-Diphenyl-N0-formyl-3-methyl-1H-pyrazolo[3,4-
b]pyrazine-5-carbohydrazide (19)
A mixture of carbohydrazide 7 (0.34 g, 1 mmol) and formic acid
(5 mL), was heated under reflux for 48 h. After cooling, the reaction
mixture was poured into ice-water and the precipitate thus ob-
tained was filtered off and recrystallized from ethanol. Yield:
78%;mp: 236–238 °C; IR (m
, m-1): 3250 (NH), 3050 (CH aromatic),
2920 (CH aliphatic), 1700 (C@O), 1660 (C@O); 1H NMR
(400 MHz, DMSO-d6) d (ppm): 2.71 (s, 3H, CH3), 7.34–7.38 (m,
1H, ArH), 7.51–7.61 (m, 5H, ArH), 7.94- (m, 2H, ArH), 8.13 (s, 1H,
CHO), 8.24–8.26 (d, 2H, ArH), 10.29 (s, 1H, NH), 10.93 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 11.36 (CH3), 120.02
(C), 120.07 (2CH), 126.33 (CH), 128.49 (C), 128.62 (CH), 129.51
(2CH), 129.73 (2CH), 130.75 (2CH), 136.85 (C), 138.43 (C), 143.69
(C), 147.35 (C), 150.92 (C), 159.62 (C@O), 165.03 (C@O); Anal.
Calcd for C20H16N6O2 (372.38): C, 64.51; H, 4.33; N, 22.57. Found:
C, 64.69; H, 4.23; N, 22.60.
5. Pharmacology
Male adult Swiss–Webster mice weighing 22–30 g from the
Animal House of Assiut University were used in these experiments.
Mice were housed in stainless steel cages under a 12 h light/dark
cycle at 25 °C and allowed water and food (laboratory show) ad
libidum. The research was conducted in accordance with the inter-
nationally accepted guidelines for laboratory animals Use and Care.
The experiments reported here were approved by our institutional
ethics committee.
4.1.15. Ethyl 2-(1,6-diphenyl-3-methyl-1H-pyrazolo[3,4-
b]pyrazine-5-oyl)hydrazine carboxylate (20)
6. PTZ-induced seizures24
A solution of carbohydrazide7 (0.34 g, 1 mmol) in ethanol
(10 mL) and ethylchloroformate (2 mL) was refluxed for 1 h. After
removal of the solvent, the solid product obtained was filtered
off and recrystallized from ethanol. Yield: 74%; mp: 224–
The animals were divided into groups, 8 mice each. The differ-
ent compounds were injected intraperitoneally (ip) at the dose of
10 mg/kg (0.2% in corn oil), each compound to one group of mice,
20 min before ip injection of pentylenetetrazole (PTZ) at the dose
of 75 mg/kg ((Minimal dose needed to induce convulsions) (1.5%
solution in saline). The animals were observed for 1 h after PTZ
injection for the appearance of convulsive seizures. Also, the mor-
tality was determined 24 h later, in each group of animals. Control
groups of animals were treated like wise with the pure vehicles.
226 °C;IR (m
, cmꢀ1): 3300 (NH), 3000 (CH aromatic), 2900 (CH ali-
phatic), 1710 (C@O ester), 1670 (C@O); 1H NMR (400 MHz, CDCl3)
d (ppm): 1.29 (t, J = 7.3 Hz, 3H, OCH2CH3), 2.77 (s, 3H, CH3), 4.23 (q,
J = 7.2 Hz, 2H, OCH2CH3), 6.74 (s, 1H, NH), 7.28–7.32 (m, 1H, ArH),
7.48–7.52 (m, 5H, ArH), 7.69–7.71 (m, 2H, ArH), 8.29 (d, J = 7.8 Hz,
2H, ArH), 9.09 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) d (ppm):

A.-R. Farghaly et al. / Bioorg. Med. Chem. 22 (2014) 2166–2175
2175
6. Foks, H.; Pancechowska-Ksepko, D.; Ke˛dzia, A.; Zwolska, Z.; Janowiec, M.;
Phenobarbital sodium was used as a reference drug and injected
into a group of animals at the dose of 30 mg/kg, 20 min before
PTZ administration.
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Supplementary data
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