Anticancer and anti-inflammatory activities of some new pyrazolo[3,4-b]pyrazines

Article abstract of DOI:10.3390/molecules23102657

New derivatives of pyrazolo[3,4-b]pyrazines and related heterocycles were synthesized using 5-Amino-3-methyl-4-nitroso-1-phenyl-pyrazole (1) as a starting material. The 5-Acetyl derivative 15 was shown to be a useful key intermediate for the synthesis of several derivatives of pyrazolopyrazines. Some of the prepared compounds were evaluated for their anti-inflammatory and anti-breast cancer MCF-7 cell line activities. SAR study showed that compounds 15 and 29 exhibited remarkable anti-inflammatory activity, where 15 showed the same activity as that of the reference drug indomethacin. On the other hand, compounds 25i, 25j showed very significant inhibitory activity (p < 0.001) against MCF-7 breast cancer cell line.

Full text of DOI:10.3390/molecules23102657

molecules
Article
Anticancer and Anti-Inflammatory Activities of Some
New Pyrazolo[3,4-b]pyrazines
Hussein El-Kashef 1,*, Talaat El-Emary 1, Pierre Verhaeghe 2, Patrice Vanelle 3 and Maha Samy 1
1
Chemistry Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt;
emarytalaat@yahoo.com (T.E.-E.); maha11292@yahoo.com (M.S.)
LCC-CNRS Université de Toulouse, CNRS, UPS, 205 Route de Narbonne, 3107 Toulouse, France;
2
pierre.verhaeghe@lcc-toulouse.fr
Aix-Marseille Université, CNRS, Institut de Chimie Radicalaire ICR, UMR 7273,
3
Laboratoire de Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin CS 30064,
13385 Marseille CEDEX 05, France; patrice.vanelle@univ-amu.fr
*
Correspondence: elkashef@aun.edu.eg; Tel.: +20-1005075881
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 August 2018; Accepted: 3 September 2018; Published: 16 October 2018
Abstract: New derivatives of pyrazolo[3,4-b]pyrazines and related heterocycles were synthesized
using 5-amino-3-methyl-4-nitroso-1-phenyl-pyrazole ( ) as a starting material. The 5-acetyl derivative
1
15 was shown to be a useful key intermediate for the synthesis of several derivatives of
pyrazolopyrazines. Some of the prepared compounds were evaluated for their anti-inflammatory and
anti-breast cancer MCF-7 cell line activities. SAR study showed that compounds 15 and 29 exhibited
remarkable anti-inflammatory activity, where 15 showed the same activity as that of the reference
drug indomethacin. On the other hand, compounds 25i, 25j showed very significant inhibitory
activity (p < 0.001) against MCF-7 breast cancer cell line.
Keywords: pyrazolo[3,4-b]pyrazines; o-aminonitrosopyrazole; anticancer; anti-inflammatory;
anti-breast cancer
1. Introduction
Although pyrazolo[3,4-b]pyrazines are not highly sited in the literature, they proved to be an
interesting class of pyrazolopyrazine heterocyles. Therapeutic importance has been reported for these
heterocycles, such as their use for the treatment and/or prevention of a wide variety of diseases
related to adenosine receptors, depression, anxiety, Parkinson’s disease, pain, dementia, heart failure,
and cerebrovascular disease [1–3].
They are also used as therapeutic agents for periodontosis, hypercalcemia, osteoporosis,
rheumatoid arthritis, Paget’s disease, and as bone metabolism improvers [
their use as blood platelet aggregation inhibitors [ ], anti-inflammatories [ ], in controlling herbicides [
and anticancer agents with low toxicity [ ]. In the domain of dye chemistry, they are used as
fluorescent [10] and disperse dyes [11]. Certain derivatives were reported to possess antiviral,
antineoplastic, antiparasitic, and anti-fungal properties [12 15]. Others showed anticonvulsant [16
and antibacterial activities [17 18]. Certain derivatives are useful for the treatment of hematologic
4]. Some reports indicated
5
6
7],
8,9
–
]
,
diseases [19], also for the prophylaxis and treatment of protein kinase-mediated diseases, including
inflammation and other related diseases. They are also used for the treatment of p38 map
kinase-mediated diseases including rheumatoid arthritis, psoriasis, chronic obstructive pulmonary
disease, pain, and other inflammatory disorders [20]. A microwave-assisted synthesis of fused
pyrazolo[3,4-b]pyrazines via the reaction of o-aminonitrosopyrazoles with cyclic β-diketones was also
reported [21].
Molecules 2018, 23, 2657; doi:10.3390/molecules23102657
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2657
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In continuation with our interest in the synthesis of pyrazolo[3,4-b]pyrazins [5,12,13,16–18],
we report herein the synthesis of other derivatives and related heterocycles. Certain newly synthesized
derivatives were screened for their anti-inflammatory and and anti-breast cancer MCF-7 cell
line activities.
2. Results
2.1. Chemistry
In a preceding paper of our group [18], we have described the first synthesis of 3-methyl-1-phenyl-
1H-indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one (
use of o-aminonitrosopyrazole as a starting material, which was reacted with the active methylene
benzoylacetonitrile to give . Hydrolysis of the cyano group of the latter compound gave the carboxylic
acid , which was converted into the acid chloride , followed by intramolecular Friedel-Crafts
cyclization giving 5 (Scheme 1).
5). The synthetic route for this compound involved the
1
2
3
4
Scheme 1. Synthesis of compound
5. Reagents and conditions: (i) Benzoylacetonitrile, pyridine, reflux
3 h; (ii) NaOH 20%, reflux 6 h; (iii) SOCl2, reflux 2 h; and (iv) AlCl3, CS2, reflux 6 h.
In the present work, when o-aminonitrosopyrazole
1 was reacted with ethyl cyanoacetate in
refluxing pyridine, the expected reaction product could be either 6a or 6b (Scheme 2). The structure
6b was immediately ruled out by examining the ir spectrum of the product, which showed no
bands corresponding to -NH2 and -COOEt groups. However, the spectrum showed two bands at
3197 and 2254 cm−1 corresponding to
ν OH and ν CN, respectively confirming the structure 6a.
Further confirmation of this structure was obtained from the 1H-NMR spectral analysis which showed,
in addition to the phenyl protons, two characteristic signals assigned to CH3 and OH protons at
2.51 and 8.43 ppm, respectively. Alternatively, compound 6a was obtained through unequivocal
synthesis via diazotization of the amino group of the derivative
of the resulting diazonium salt. The alkaline hydrolysis of 6a gave the hydroxycarboxylic acid
which was esterified in refluxing absolute ethanol in the presence of concentrated H2SO4 to give the
corresponding hydroxyester . Contrary to the work of Rangnekar et al. [22], all attempts to prepare
the latter compound directly through the reaction of with diethylmalonate under various conditions
7 [13], followed by decomposition
8,
9
9
1
were unsuccessful (Scheme 2).
On the other hand, the interaction of 1 with α-haloketones such as chloroacetone and phenacyl
bromide gave the corresponding imidazo[4,5-c]pyrazole derivative 10 and 11 respectively. When the
aminonitrosopyrazole was reacted with thiobarbituric acid in refluxing pyridine, the reaction product
1
was identified as 3-methyl-1-phenyl-7-thioxo-7,8-dihydro-1H-pyrazolo[4,3-g]pteridin- 5(6H)-one (12).
Alkylation of 12 with excess ethyl iodide in DMF in the presence of anhydrous K2CO3 yielded
6-ethyl-7-(ethylthio)-3-methyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (13), however when
12 was interacted with one mole of ethyl chloroacetate, the ethyl mercaptoacetate derivative 14 was
obtained (Scheme 3).

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Scheme 2. Synthesis of compounds 6–9. Reagents and conditions: (i) CH2(CN)(COOC2H5), pyridine,
reflux overnight; (ii) NaNO2, H2SO4, room temperature (rt) at 0 ◦C; (iii) NaOH 20%, reflux 5 h;
(iv) EtOH, HCl, reflux 6 h; and (v) CH2(COOC2H5)2, pyridine, reflux overnight.
Scheme 3. Synthesis of compounds 10–14. Reagents and conditions: (i) CH3COCH2Cl, pyridine,
reflux overnight; (ii) PhCOCH2Br, pyridine, reflux overnight; (iii) thiobarbituric acid, pyridine, reflux
overnight; (iv) C2H5I, DMF, rt 5 h; and (v) ClCH2COOC2H5, DMF, rt 5 h.
The interaction of
1 with acetylacetone under the same reaction conditions, the expected product
5-acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (15) was obtained (Scheme 4).
Scheme 4. Synthesis of compound 15. Reagents and conditions: (i) CH2(COCH3)2, dry pyridine, reflux
8 h.
The acetyl derivative 15 was shown to be a useful key intermediate for the synthesis of
several derivatives of the title compounds. Thus, the condensation of 15 with semicarbazide
and thiosemicarbazide in boiling ethanol afforded the corresponding semicarbazone and

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thiosemicarbazone 16 and 17, respectively. On the other hand, when the acetyl function of 15 was
interacted with hydroxyl amine the reaction product was identified as the oxime 18. The hydrazone
19 was obtained via the interaction of 15 with hydrazine hydrate. Crossed aldol condensation
between 5-acetylpyrazolo[3,4-b]pyrazine 15 and isatin was carried out in the presence of diethyl
amine as a basic catalyst to give the 3-hydroxy-3-(2-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]
pyrazine-5-yl)-2-oxoethyl)indolin-2-one (20). Dehydration of 20 by heating its ethanolic solution
under reflux in the presence of concentrated HCl afforded the corresponding chalcone 21 (Scheme 5).
Scheme 5. Synthesis of compound 16
reflux 8 h; (ii) NH2NHCSNH2, EtOH, reflux 8 h; (iii) NH2OH
(iv) NH2NH2, reflux 6 h; (v) isatin, (C2H5)2NH, EtOH, reflux overnight; and (v) EtOH, HCl, reflux
15 min.
–
21. Reagents and conditions: (i) NH2NHCONH2
·
HCl, EtOH,
·
HCl, sodium acetate, EtOH, reflux 3–4 h;
When the thiosemicarbazone 17 was allowed to react with α-haloketones, such as chloroacetone
and phenacyl bromide, the corresponding thiazolines 22, 23 were obtained, while its reaction with
α-chloroacetic acid gave the thiazolidinone 24 (Scheme 6).
Scheme 6. Synthesis of compound 22–24. Reagents and conditions: (i) ClCH2COCH3, anhyd. CH3COONa,
EtOH reflux 5 h; (ii) PhCOCH2Br, anhyd. CH3COONa, EtOH, reflux 5 h; and (iii) ClCH2COOH, anhyd.
CH3COONa, EtOH, reflux 5 h.
Chalcones are known by their biological activities and in particular by their anticancer
activities [23–29]. Accordingly, a series of chalcones 25a–k was synthesized via the Claisen-Schmidt
reaction of 15 with a number of aromatic aldehydes (Scheme 7) for the sake of their evaluation against
MCF-7 breast cancer cells.

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Scheme 7. Synthesis of chalcones 25a
NaOH, rt overnight. , Ar = C6H5; , Ar = 2-OHC6H4;
Ar = 3-NO2C6H4; , Ar = 4-CNC6H4; , Ar = 4(CH3O)C6H4;
j, Ar = 3,4-(CH3O)2C6H3; and k, Ar = CH=CH-C6H4.
–
k
. Reagents and conditions: (i) ArCHO, EtOH, 25% aq.
, Ar = 2-NO2C6H4; , Ar = 4-NO2C6H4;
, Ar = 4-N(CH3)2C6H4; Ar = 4-ClC6H4;
a
b
g
c
d
e,
f
h
i,
On the other hand, the α,β-unsaturated ketonic function of chalcones renders them ready to
undergo reaction with bidentate nucleophiles to give five- and six-membered heterocyclic rings. Thus,
the reaction of 25a with hydrazine hydrate and phenyl hydrazine in ethanol gave the corresponding
pyrazolinyl derivatives 26 and 27, respectively. Also, the reaction of 25a with hydroxylamine
hydrochloride in the presence of anhydrous sodium acetate led to the formation of the dihydroisoxazole
28. Moreover, the interaction of 25a with thiosemicarbazide in an ethanolic sodium hydroxide solution
(25%) yielded the pyrazolylthioamide 29. Finally, the treatment of 25a with guanidine sulfate in
ethanolic potassium hydroxide solution (10%) yielded the 2-aminopyrimidine 30 (Scheme 8).
Scheme 8. Synthesis of compounds 26
reflux 10 h; (ii) PhNHNH2, EtOH, reflux 15 min; (iii) NH2OH
h; (iv) NH2NHCSNH2, ethanolic NaOH (25%), reflux 3 h; and (v) NH2C=NHNH2
KOH (25%), reflux 3–4 h.
–
30
.
Reagents and conditions: (i) NHNH2
·
H2O, EtOH,
·
HCl, CH3COONa, EtOH, reflux 6
·
H2SO4, ethanolic
2.2. Biology
2.2.1. Anti-Inflammatory Activity
Anti-inflammatory activity of compounds (15, 25a, 26–30) was evaluated against the
carrageenan-induced rat oedema using indomethacin as the reference drug [30]. Mean changes

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in paw oedema thickness of the animals pretreated with the tested compounds after 0.5, 1, 2, 3, 4,
and 5 h from induction of inflammation was measured, and the inhibition percent of oedema by the
tested compounds was calculated. The relative potencies % of the tested compounds compared with
indomethacin at the fifth hour was also calculated (Table 1). Amongst all the tested pyrazolopyrazines,
the starting compound 5-acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b] pyrazine (15) showed the
highest anti-inflammatory activity, compared to that of indomethacin (44.44%). This activity was
decreased when the acetyl derivative 15 was converted into its corresponding chalcone 25a, displaying
12.5% inhibition. An increase of this latter activity was shown by compounds 26 and 27, having
pyrazolinyl substituent at 5-position of the pyrazolopyrazine nucleus (23.6%, 15.07% respectively).
Substituting the 5-pyrazolinyl moiety by aminopyrimidinyl ring (compound 30) results in more
increase in activity (27%). Higher activity (34%) was observed when the latter aminopyrimidinyl ring
was replaced by isoxazolinyl moiety (compound 28). Noticeable increase of potency (close to that of
indomethacin) was shown by the pyrazolylthioamide derivative 29 (40%).
Table 1. Anti-inflammatory Activity of Tested Compounds (15, 25a, 26–30) against Acute
Carrageenan-Induced Paw Oedema in Rats (Statistical analysis).
Paw Oedema Inhibition (Swell ± S.E.M.) a,b,c (%)
Compound a
Potency d
30 min
1 (h)
2 (h)
3 (h)
4 (h)
5 (h)
Edema Induced by Carrageenan (% Edema Inhibition Relative to Control)
15
25a
26
27
28
29
30
0.40
±
±
±
±
±
±
±
±
±
0.00 0.55
±
±
±
±
±
±
±
±
±
0.03 0.48
0.03 0.65
0.02 0.66
0.02 0.60
0.02 0.63
0.03 0.43
0.02 0.55
0.03 0.42
0.02 0.73
±
±
±
±
±
±
±
±
±
0.02 0.42
0.03 0.66
0.02 0.62
0.03 0.60
0.02 0.60
0.02 0.42
0.05 0.45
±
±
±
±
±
±
±
0.02 0.38
0.02 0.63
0.02 0.62
0.03 0.63
0.03 0.48
0.02 0.38
0.03 0.45
±
±
±
±
±
±
±
±
±
0.02 0.40
0.02 0.63
0.02 0.55
0.02 0.62
0.02 0.47
0.03 0.43
0.03 0.52
0.02 0.40
0.02 0.72
±
±
±
±
±
±
±
±
±
0.00
0.03
0.03
0.02
0.02
0.02
0.02
0.00
0.02
44.44
12.50
23.61
15.07
34.72
40.27
27.77
44.44
-
0.63
0.55
0.61
0.46
0.43
0.51
0.40
0.03 0.66
0.03 0.68
0.02 0.68
0.02 0.63
0.00 0.45
0.02 0.61
0.00 0.45
0.02 0.73
Drug *
Negative Control 0.72
(a) Dose 28
0.02
0.38 ± 0.0
0.37
0.02 0.73
0.02 0.72
±
µ
M/kg. (b) n = 6. (c) Statistically significant from the indomethacin at p < 0.05. (d) Potency was expressed
as % oedema inhibition of the tested compounds relative to % oedema inhibition of indomethacin (reference drug).
* Drug = indomethacin (28 µM/kg).
The percent oedema inhibition was calculated from the mean effect shown by the control and
treated animals according to the following equation:
vc − vt
Percent oedema inhibition =
× 100
vc
where vc represents the mean increase in paw volume in the control group of rats and vt represents the
mean increase in paw volume in rats treated with tested compounds. The potency was calculated as
the percentage of the change of the standard and tested compounds, as depicted in Table 1. All the
results are expressed as the mean
performed using graph pad prism program software version 5.00 through One-way ANOVA.
±
standard error of the mean (S.E.M.). Statistical evaluations were
2.2.2. Cytotoxic Activity
The chalcones 25a–k along with their starting compound 15 were evaluated for their cytotoxic
activity against MCF-7 breast cancer cells using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium
bromide (MTT) cell viability assay according to literature procedure [31]. The results obtained revealed
that the parent acetyl compound 15 exhibited an inhibitory activity with IC50 value of 9.42
µM
(Figure 1). The chalcone 25a, with unsubstituted phenyl group in the -unsaturated ketonic function,
α,β
showed higher activity than that of the parent compound 15. An increase in activity was observed
upon replacement of this phenyl ring by a phenyl ethen-2-yl group (25k). The effect of substitution
in the phenyl group of 25a on the cytotoxic activity was studied. Thus, the introduction of an OH

Molecules 2018, 23, 2657
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group in the 2-position of this phenyl group (25b) led to an improvement of the activity. On the other
hand, the introduction of an NO2 group showed variable activities according to its position, where the
activity was in the order 4- > 2- > 3-nitro isomer (25d, 25c, 25e). Replacement of the 4-NO2 group by
a CN or OCH3 function (25f and 25g) lowered the cytotoxic activity. However, its replacement by
4-N,N-dimethylamino group (25h) resulted in higher cytotoxic activity than all the former derivatives
with IC50 of 3.66 µM. A further higher activity was shown by the 4-Cl derivative (25i), while the highest
activity was shown by the 3,4-dimethoxy derivative (25j) with IC50 value of 2.22
drug Paclitaxel showed an IC50 of 1.02 µM (Figure 2).
µM. The reference
12
10
8
6
4
2
0
Samples (15, 25a–k) and the Paclitaxel drug
Figure 1. Effect of compounds (15, 25a–k) on the proliferation of MCF-7 breast cancer cells using
3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay. The IC50 of
the tested compounds was calculated after 24 h.
120
25i
100
80
60
40
20
0
Concentration µM
Figure 2. Cytotoxic effect of compounds 25i, 25j against the proliferation of MCF-7 breast cancer cells.
Cells were treated with the indicated doses and cell viability was obtained using MTT cell viability
assay. *** p < 0.001 calculated by comparing each concentration with the control. The data were
normally distributed and were expressed as the mean
paired student test p-values as determined by Graphpad Prism software is indicated as *** p < 0.001.
±
standard error of the mean (SEM). Two-tailed

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3. Experimental
3.1. Chemistry
All melting points were determined on a Stuart melting point apparatus SMP3 (Sigma-Aldrich,
Saint Louise, MI, USA). IR spectra were recorded on a Nicolet iS10 FT-IR spectrometer (Thermo Fisher,
Waltham, MA, USA) using KBr wafer technique. The 1H-NMR spectra were recorded on Bruker AV500
(400 MHz) (Bruker, Billerica, MA, USA) and Bruker Avance III (400 MHz) spectrometers (Bruker).
1H- and 13C-NMR chemical shifts
δ were reported in parts per million (ppm) and were referenced
to the solvent 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). Mass spectra were taken on
a Jeol JMS-600 mass spectrometer (Jeol Inc., Peabody, MA, USA). Elemental analyses were carried
out using a Perkin Elmer 240 C Micro analyzer (Perkin Elmer, Waltham, MA, USA) and they were
found to be within
±
0.4% of the theoretical values. Their results were found in good agreement with
the calculated values. Paclitaxel and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide
(MTT) were purchased from Sigma Aldrich, more detailed IR and NMR data can be found in the
Supplementary Materials.
6-Hydroxy-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carbonitrile (6a). Procedure (1): A mixture
of compound 1 (3.0 g, 1.5 mmol) and ethyl cyanoacetate (1.8 mL, 1.5 mmol) in dry pyridine (20 mL)
was heated under reflux for overnight. After cooling, the solid product obtained was filtered, washed
with water, and dried. Recrystallization from ethanol-water mixture (2:1) gave fine yellow needles,
yield 0.68 g (68%), m.p. 240–242 ◦C. Procedure (2): Compound
7 [13] (0.5 g, 2 mmol) was dissolved in
cold H2SO4 (5 mL), then a cold sodium nitrite solution (0.4 g in 2 mL H2O) was added dropwise under
stirring to the above solution during 0.5 h in an ice bath at 0–5 ◦C. The reaction mixture was allowed
to stir at room temperature (rt) for 1 h further, then it was poured onto ice-water mixture. The solid
product obtained was filtered, washed with water, and recrystallized from ethanol-water mixture (2:1)
as yellow crystals, yield 0.3 g (60%), m.p. 240–242 ◦C. IR (
2234 (C (ppm): 2.51 (s, 3H, CH3), 7.38 (t, 1H, phenyl), 7.57 (t, 2H,
N). 1H-NMR (400 MHz, DMSO-d6)
phenyl), 8.08 (d, 2H, phenyl), 8.43 (s, 1H, OH). 13C-NMR (100 MHz, DMSO-d6)
(ppm): 10.99 (CH3),
ν
, cm−1): 3197 (OH), 3073 (CH aromatic),
≡
δ
δ
114.58 (C), 115.73 (C N), 120.28 (2 CH), 126.55 (CH), 129.01 (C), 129.31 (2CH), 137.89 (C), 141.84 (C),
≡
144.48 (C), 160.40 (C) MS: m/z (251 M+). Anal. calcd. for C13H9N5O (251.24): C, 62.15; H, 3.61; N, 27.87.
Found: C, 62.30; H, 3.50; N, 27.72%.
6-Hydroxy-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid (8): The hydroxy cyano
compound 6a (0.5 g) was dissolved in EtOH (10 mL), then aqueous solution of NaOH (20%, 10 mL)
was added. The reaction mixture was heated under reflux for 5 h, then it was evaporated to half of its
volume. After cooling, it was neutralized with dil. HCl, giving a solid precipitate which was filtered
off, washed with water, dried, and recrystallized from dioxane as yellow crystals, yield 0.4 g (80%),
m.p. 190–192 ◦C. IR (
ν
, cm−1): 3493 (OH), 2969, 2920, 2862 (CH aliph.), 3350-2493 (characteristic of
(ppm): 2.57 (s, 3H, CH3), 7.33 (t, 1H, phenyl),
COOH), 1667 (C=O). 1H-NMR (400 MHz, DMSO-d6)
δ
7.55 (t, 2H, phenyl), 8.12 (d, 2H, phenyl), 8.38 (s, 1H, OH). 13C-NMR (100 MHz, DMSO-d6): 11.76 (CH3),
119.67 (2CH, C), 125.59 (CH, C), 129.53 (2CH, C), 139.55 (2C), 144.77 (C), 165.16 (C). Anal. calcd. for
C13H10N4O3 (270.24): C, 57.78; H, 3.73; N, 20.73. Found. C, 57.66; H, 3.88; N, 20.56%.
Ethyl 6-hydroxy-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylate (9): To the hydroxy acid 8
(0.5 g, 1 mmol) in anhydrous ethanol (20 mL), conc. HCl (15 mL) was added, and the mixture was
heated under reflux for 6 h. The reaction mixture was then cooled, poured on ice-cold solution of
NaHCO3, and the solid precipitate was filtered, washed with water, and dried. Crystallization from
◦
dioxane-water mixture (2:1) gave pale brown crystals, yield 0.35 g (70%), m.p. 143–145 C. IR (ν
, cm−1):
3117 (OH), 2974 (CH aliph.), 1677 (C=O). 1H-NMR (400 MHz, CDCl3) δ (ppm): 1.57 (t, J = 7.1 Hz, 3H,
CH3), 2.76 (s, 3H, CH3), 4.64 (q, J = 7.1 Hz, 2H, CH2), 7.33 (t, 1H, phenyl), 7.53 (t, 2H, phenyl), 8.25 (d,

Molecules 2018, 23, 2657
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2H, phenyl), 12.08 (s, 1H, OH),13C-NMR (100 MHz, CDCl3): 11.64 (CH3), 14.20 (CH3), 63.48 (CH2),
120.32 (2 CH), 123.05 (C) 126.37 (CH), 129.23 (2 CH), 130.58 (C), 138.39 (C), 143.09 (C), 145.8 (C),
161.27 (C), 169.07 (C=O). MS: m/z (298 M+). Anal. calcd. for C15H14N4O3 (298.30): C, 60.40; H, 4.73; N,
18.78. Found: C, 60.30; H, 4.50; N, 18.92%.
5-Acetyl-3-methyl-1-phenyl-1H,6H-imidazo[4,5-c]pyrazole (10): A mixture of compound
1 (1.0 g, 5 mmol)
and chloro acetone (0.46 mL, 5 mmol) in dry pyridine (15 mL) was heated under reflux for
overnight. After cooling the solid product was filtered, washed with water, and recrystallized from
◦
ethanol-dioxane mixture (2:1) as fine yellow needles, yield 0.52 g (98%), m.p. 288–290 C. IR (
3438 (NH), 2922, 2784 (CH aliph.), 1655 (C=O). 1H-NMR (400 MHz, DMSO-d6)
(ppm): 2.40 (s, 3H,
CH3), 2.60 (s, 3H, CH3), 7.33 (t, 1H, phenyl), 7.52 (t, 2H, phenyl), 7.6 (s, 1H, NH), 8.11 (d, 2H, phenyl).
13C-NMR (100 MHz, DMSO-d6)
(ppm): 11.26 (CH3), 21.17 (CH3), 119.87 (2CH), 125.79 (CH, C),
ν
, cm−1):
δ
δ
129.19 (2CH, C), 138.82 (2C), 155.33 (C=O). Anal. calcd. for C13H12N4O (240. 26): C, 64.99; H, 5.03; N,
23.32. Found: C, 64.60; H, 5.21; N, 23.40%.
5-Benzoyl-3-methyl-1-phenyl-1H,6H-imidazo[4,5-c]pyrazole (11): A mixture of compound
1 (1.0 g, 5 mmol)
and phenacyl bromide (0.46 g, 5 mmol) in dry pyridine (15 mL) was heated under reflux for overnight.
After cooling the solid product was filtered, washed with water, dried, and recrystallized from
◦
ethanol-dioxane mixture (1:1) as yellow needles, yield 0.8 g (88%), m.p. 304–306 C. IR (
3075 (NH), 2921, 2781 (CH aliph.), 1641 (C=O). 1H-NMR (400 MHz, DMSO-d6)
(ppm): 2.76 (s, 3H,
CH3), 7.33 (t, 1H, phenyl), 7.41 (t, 1H, phenyl), 7.65–7.50 (m, 5H, phenyl), 8.12–8.31 (m, 3H, phenyl),
12.85 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
(ppm): 14.08 (CH3), 120.327 (C), 120.99 (2CH),
ν
, cm−1):
δ
δ
123.08 (CH), 126.78 (CH), 127.02 (CH), 127.96 (CH), 128.45 (CH), 129.57 (CH), 129.77 (CH), 129.88 (CH),
131.49 (C), 135.14 (C), 139.11 (C), 154.56 (C), 155.46 (C), 177.73 (C). MS: m/z (302 M+). Anal. calcd. for
C18H14N4O (302.33): C, 71.51; H, 4.67; N, 18.53. Found: C, 71.62; H, 4.25; N, 18.72%.
3-Methyl-1-phenyl-7-thioxo-7,8-dihydro-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (12): A mixture of amino
nitroso
1 (1.01 g, 5 mmol) and thiobarbituric acid (0.72 g, 5 mmol) in dry pyridine (20 mL) was
heated under reflux for overnight. After cooling, the solid product was filtered, washed with water,
and recrystallized from ethanol-dioxane mixture (1:3) as fine yellow needles, yield 0.15 g (65%),
m.p. 332–334 ◦C. IR (
(400 MHz, DMSO-d6),
phenyl), 11.76 (s, 1H, NH), 11.91 (s, 1H, NH), 13C-NMR (100 MHz, DMSO-d6)
ν
, cm−1): 3416 (NH), 3068 (CH arom.), 2891 (CH aliph.), 1693 (C=O). 1H-NMR
δ
(ppm): 2.70 (s, 1H, CH3), 7.27 (t, 1H, phenyl), 7.45 (t, 2H, phenyl), 8.13 (d, 2H,
(ppm): 11.29 (s, 3H,
δ
CH3), 119.60 (2CH), 126.36 (CH), 126.99 (C), 129.37 (2CH), 132.78 (C), 138.13 (C), 142.42 (C), 145.60 (C),
147.27 (C), 158.31 (C=O), 175.95 (C=S). MS: m/z (310 M+). Anal. calcd. for C14H10N5OS (310.3): C,
54.18; H, 3.25; N, 27.08 Found: C, 54.02; H, 3.21; N, 26.82%.
6-Ethyl-7-(ethylthio)-3-methyl-1-phenyl-1H-pyrazolo[4,3-g]pteridin-5(6H)-one (13): A mixture of the thione
12 (0.25 g, 0.8 mmol), ethyl iodide (0.24 g, 1.6 mmol), and anhydrous K2CO3 (0.41 g, 3 mmol) in
DMF(15 mL) was stirred at 80 ◦C for 5 h. After cooling the solid product was filtered, washed with
water, dried, and recrystallized from ethanol-acetone mixture (2:1) to give yellow crystals, yield 0.25 g
(86%), m.p. 238–240 ◦C. IR (
(ppm): 1.47 (t, J = 7.1 Hz, 3H, CH3), 1.53 (t, J = 7.4 Hz, 3H, CH3), 2.86 (s, 3H, CH3), 3.51 (q, J = 7.4 Hz,
2H, CH2), 4.34 (q, J = 7.0 Hz, 2H, CH2), 7.35 (t, J = 7.3 Hz, 1H, phenyl), 7.57 (t, J = 7.8 Hz, 2H, phenyl),
8.33 (d, J = 8.4 Hz, 2H, phenyl). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.75 (CH3), 12.93 (CH3),
ν
, cm−1): 2922, 2784 (CH aliph.), 1655 (C=O). 1H-NMR (400 MHz, CDCl3)
δ
δ
13.68 (CH3), 27.05 (CH2), 40.44 (CH2), 120.25 (2CH), 125.89 (CH), 128.22 (C), 129.21 (2CH), 135.53 (C),
138.67 (C), 144.52 (C), 145.96 (C), 151.08 (C), 160.43 (C), 162.82 (C=O). MS: m/z (366 M+). Anal. calcd.
for C18H18N6OS (366.44): C, 59.00; H, 4.95; N, 22.93. Found: C, 59.25; H, 4.72; N, 22.65%.
Ethyl 2-(3-methyl-5-oxo-1-phenyl-5,6-dihydro-1H-pyrazolo[4,3-g]pteridin-7-ylthio)acetate. (14): A mixture
of the thione 12 (0.15 g, 0.4 mmol), ethyl chloroacetate (0.1 g, 0.8 mmol), and anhydrous K2CO3
◦
(0.44 g, 3 mmol) in DMF (15 mL) was stirred at 80 C for 5 h. After cooling the solid product was
filtered, washed with water, dried, and recrystallized from dioxane-water mixture (2:1) to give yellow

Molecules 2018, 23, 2657
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◦
crystals, yield 0.10 g (71.4%), m.p. 243–245 C. IR (
1686 (C=O). 1H-NMR (400 MHz, CDCl3)
ν
, cm−1): 2980, 2923 (CH aliph.), 1737 (C=O),
δ
(ppm): 1.28 (t, J = 17.3, 10.2 Hz, 3H, CH3), 2.78 (s, 3H, CH3),
4.34–4.11 (m, 4H, 2CH2), 7.27 (t, 1H, phenyl), 7.47 (t, 2H, phenyl), 8.23 (d, 2H, phenyl), 9.64 (s, 1H, NH).
13C-NMR (100 MHz, CDCl3)
(ppm): 11.79 (CH3), 14.57 (CH3), 33.31 (CH2), 61.73 (CH2), 120.21 (2CH),
δ
126.65 (CH), 129.78 (2CH), 130.09 (C), 134.73 (C), 138.76 (C), 144.31 (C), 145.79 (C), 160.60 (C), 161.41 (C),
162.100 (C=O), 168.64 (C=O). Anal. calcd. for C18H16N6O3S (396.42): C, 54.54; H, 4.07; N, 21.20. Found:
C, 54.66; H, 4.12; N, 21.13%.
5-Acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (15): A mixture of amino nitroso compound
1
(6.06 g, 30 mmol) and acetylacetone (3.0 g, 30 mmol) in dry pyridine (20 mL) was heated under reflux for
8 h. The reaction mixture was then evaporated to one-half volume and after cooling it was neutralized
with dil. HCl (10%). The solid precipitate was filtered off, washed with water, and recrystallized
◦
from ethanol-water mixture (1:3) as pale brown needles, yield 6.4 g (80%), m.p. 130–132 C. IR (
ν
,
cm−1): 3050 (CH arom.), 2900 (CH aliph.), 1689 (C=O). 1H-NMR (400 MHz, CDCl3)
3H, CH3), 2.80 (s, 3H, CH3), 3.00 (s, 3H, CH3), 7.27–7.23 (t, 1H, phenyl), 7.51–7.56 (t, 2H, phenyl),
8.23–8.31 (d, 2H, phenyl). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.32 (CH3), 25.15 (CH3), 27.79 (CH3),
δ (ppm): 2.70 (s,
δ
120.15 (2CH), 126.16 (CH), 129.18 (2CH), 131.83 (C), 138.85 (C), 142.66 (C), 143.18 (C), 144.82 (C),
154.14 (C), 200.54 (C=O). MS: m/z (266 M+). Anal. calcd. for C15H14N4O (266. 12): C, 67.65; H, 5.30; N,
21.04. Found: C, 67.63; H, 5.32; N, 21.08%.
(3,6-Dimethyl-1-phenyl-5-acetyl-1H-pyrazolo[3,4-b]pyrazine)semicarbazone (16): A mixture of the acetyl
derivative
5 (0.72 g, 3 mmol), semicarbazide hydrochloride (0.3 g, 3 mmol), and sodium acetate (0.6 g,
1 mol) in ethanol (15 mL) was heated under reflux for 8 h. After cooling, the product was filtered,
washed with water, and recrystallized from dioxane-water mixture (3:1) as yellow crystals, yield 0.53 g
ν
(93%), m.p. 210–212 ◦C. IR ( , cm−1): 3487, 3204 (NH2 + NH), 1739 (C=O). MS: m/z 323 (M+). Anal.
calcd. for C16H17N7O (323.35): C, 59.43; H, 5.30; N, 30.32. Found: C, 59.33; H, 5.41; N, 30.26%.
No numerical data could be obtained for this compound due its insolubility in deuterated solvents.
(3,6-Dimethyl-1-phenyl-5-acetyl-1H-pyrazolo[3,4-b]pyrazine)thiosemicarbazone (17): A mixture of 15 (1.0 g,
4 mmol) and thiosemicarbazide (0.34 g, 4 mmol) in ethanol (10 mL) was heated under reflux for 8 h.
After cooling, the solid product was filtered, washed with water, and dried. Crystallization from
dioxane-water mixture (1:1) gave yellow crystals, yield 0.28 g (87%); m.p. 230–232 ◦C; IR (
ν
, cm−1):
(ppm): 2.40 (CH3), 2.82 (CH3),
2.93 (CH3), 6.40 (s, 2H, NH2), 7.32–7.35 (t, 1H, phenyl), 7.51–7.56 (t, 2H, phenyl), 8.27–8.31 (d, 2H,
phenyl), 8.88 (s, 1H, NH). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.49 (CH3), 25.00 (CH3), 27.96 (CH3),
3434, 3237, 3152 (NH2 + NH), 1690 (C=O).1H-NMR (300 MHz, CDCl3)
δ
δ
120.29 (C, 2CH), 126.7 (CH), 129.7 (2CH), 131 (C), 138 (C), 142.6 (C), 143.8 (C), 144.77 (C), 153.8 (C),
200.19 (C=S). Anal. calcd. for C16H17N7S (339.42): C, 56.62; H, 5.05; N, 28.89. Found: C, 56.33; H, 5.20;
N, 28.66%.
(5-Acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine)oxime (18): A mixture of 15 (0.25 g, 0.9 mmol),
hydroxyl amine (0.07 g, 1 mmol), and sodium acetate (0.25 g, 3 mmol) in ethanol (10 mL ethanol)
was heated under reflux for 3 h. The reaction mixture was then evaporated to half volume and
after cooling it was neutralized with dil. HCl. The solid precipitate was filtered off, washed with
water, and recrystallized from ethanol-water mixture (3-1) as fine buff needles, yield 0.16 g (89%),
1
m.p. 223–225 ◦C, IR (
ν
, cm−1): 3281 broad (OH), 2900 (CH aliph.). H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 2.29 (s, 3H, CH3), 2.63 (s, 3H, CH3) 2.81 (s, 3H, CH3), 7.31–7.36 (t, 1H, phenyl), 7.54–7.59 (t, 2H,
phenyl), 8.20–8.23 (d, 2H, phenyl), 11.59 (s, 1H, OH). 13C-NMR (100 MHz, DMSO)
(ppm): 11.01 (s,
δ
3H, CH3), 13.08 (s, 3H, CH3), 25.11 (s, 3H, CH3), 119.52 (2CH), 125.81 (CH), 129.29 (2CH), 131.11 (C),
138.69 (C), 141.37 (C), 143.08 (C), 146.15 (C), 151.87 (C), 154.14 (C). MS: m/z (281 M+). Anal. calcd. for
C15H15N5O (281.31): C, 64.04; H, 5.37; N, 24.90. Found: C, 64.11; H, 5.42; N, 24.97%.
(5-Acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine)hydrazone (19): A mixture of 15 (0.25 g,
0.9 mmol) and hydrazine hydrate (0.5 mL, 80%) in ethanol (10 mL) was heated under reflux for

Molecules 2018, 23, 2657
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6 h. After cooling the solid product was filtered, washed with water, and recrystallized from ethanol
as buff needles, yield 0.2 g (87%), m.p. 136–138 ◦C. IR ( , cm−1): 3453, 3342 (NH2), 3050 (CH arom.).
1H-NMR (400 MHz, CDCl3)
(ppm): 2.3 (s, 3H, CH3), 2.70 (s, 3H, CH3) 2.90 (s, 3H, CH3), 5.58 (s,
2H, NH2), 7.27–7.31 (t, 1H, phenyl), 7.49–7.54 (t, 2H, phenyl), 8.20–8.31 (d, 2H, phenyl). 13C-NMR
(100 MHz, CDCl3) (ppm): 11.36 (CH3), 12.56 (CH3), 25.65 (CH3), 120.03 (CH), 120.14 (CH), 125.60 (CH),
ν
δ
δ
129.12 (CH), 129.18 (CH), 131.52 (C), 139.37 (C), 141.94 (C), 143.48 (C), 147.35 (C), 147.78 (C), 152.34 (C).
MS: m/z (280 M+). Anal. calcd. for C15H16N6 (280.33): C, 64.27; H, 5.75; N, 29.98. Found: C, 64.11; H,
5.42; N, 29.93%.
5-(3-Hydroxy-2,3-dihydroindol-2-on-3-ylacetyl)-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine
(20):
A mixture of 15 (0.25 g, 0.9 mmol) and isatin (0.14 g, 0.9 mmol) and diethyl amine (0.5 mL) in
absolute ethanol (15 mL) was stirred at rt for overnight. The solid product obtained was filtere◦d and
recrystallized from ethanol-water mixture (1:1) as buff crystals, yield 0.18 g (90%), m.p. 167–169 C. IR
, cm1), 3322 (NH), 1745 (C=O), 1697 (C=O). 1H-NMR (400 MHz, CDCl3)
(ν δ (ppm): 2.77 (d, J = 9.7 Hz,
1H, CH2), 2.84 (s, 6H, 2CH3), 3.01 (d, J = 11.4 Hz, 1H, CH2), 6.90 (s, 1H, OH), 7.07 (s, 1H, NH), 7.38–7.29
(m, 2H, Ar-H), 7.44 (d, 2H, Ar-H), 7.55 (t, 2H, Ar-H), 8.34–8.24 (m, 3H, Ar-H). 13C-NMR (100 MHz,
CDCl3) δ (ppm): 11.25 (CH3), 25.02 (CH3), 27.46 (CH2), 45.61 (C), 110.11 (C), 115.52 (C), 120.31 (CH),
120.40 (CH), 123.03 (CH), 124.52 (CH), 126.19 (CH), 126.41 (CH), 129.20 (2CH), 129.98 (CH), 131.45 (C),
138.65 (C), 142.78 (C), 144.86 (C), 148.28 (C), 151.96 (C), 169.03 (C), 191.12 (C=O). Anal. calcd. for
C23H21N5O3 (413.4): C, 66.82; H, 4.63; N, 16.94. Found: C, 66.51; H, 5.43; N, 16.93%.
2,3-Dihydroindol-2-on-3-ylideneacetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (21): Compound
20 (0.18 g, 4 mmol) was heated in a mixture of ethanol (7.5 mL) and conc. HCl (2.5 mL) for 15 min.
After cooling the solid product obtained was filtered, washed with water, and recrystallized from
ethanol-dioxane mixture (1:3) as orange needles, yield 0.13 g (81%), m.p. 248–250 ◦C. IR (
ν
, cm−1),
δ (ppm):
3444 (NH), 3163, 3074 (CH arom.), 1716 (C=O), 1670 (CO-NH). 1H-NMR (300 MHz, CDCl3)
2.90 (s, 3H, CH3), 3.20 (s, 3H, CH3), 6.85–6.89 (d, 1H, phenyl), 7.10 (t, 1H, phenyl), 7.34–7.37 (t, 2H,
phenyl), 7.52–7.55 (t, 2H, phenyl), 7.50 (s, 1H, NH), 8.30–8.33 (d, 2H, phenyl), 8.56 (s, 1H, CH), 8.68–8.71
(d, 1H, phenyl). 13C-NMR (100 MHz, DMSO-d6)
δ (ppm): 11.72 (CH3), 25.32 (CH3), 110.80 (CH),
120.43 (CH), 120.58 (2CH), 122.29 (CH), 126.73 (CH), 126.87 (CH), 128.37 (CH), 129.87 (2CH), 131.56 (C),
133.90 (C), 137.79 (C), 138.71 (C), 142.55 (C), 144.31 (C), 145.09 (C), 145.88 (C), 155.16 (C), 168.97 (C=O),
190.75 (C=O) MS: m/z (395 M+). Anal. calcd. for C23H19N5O2 (395.4): C, 69.86; H, 4.33; N, 17.71.
Found: C, 69.43; H, 4.64; N, 17.83%.
N-(4-Methyl-2,3-dihydrothiazol-2-ylidene)-(5-acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine)
hydrazine (22): A mixture of the thiosemicarbazone 17 (0.4 g, 1 mmol), chloro acetone (0.1 mL,
1 mmol), and anhydrous sodium acetate (0.5 g) in absolute ethanol was heated under reflux for
5 h. After cooling, the solid product obtained was filtered the reaction mixture was cooled, washed
with water, an◦d recrystallized from acetone-water mixture (1:1) as buff crystals, yield 0.05 g (17%),
m.p. 222–224 C, IR (
(400 MHz, CDCl3)
5.66 (s, 1H, NH), 6.29 (s, 1H, CH), 7.35–7.27 (m, 1H, phenyl), 7.54 (t, 2H, phenyl), 8.33 (d, 2H, phenyl).
13C-NMR (100 MHz, CDCl3)
(ppm): 11.46 (CH3), 14.02 (CH3), 16.71 (CH3), 26.43 (CH3), 103.13 (CH),
ν
, cm−1): 3178 (NH), 3067 (CH arom.), 2921, 2852 (CH aliph.). 1H-NMR
δ
(ppm): 2.36 (s, 3H, CH3), 2.59 (s, 3H, CH3), 2.74 (s, 3H, CH3), 3.06 (s, 3H, CH3),
δ
120.08 (2CH), 125.76 (CH), 128.74 (2CH), 131.52 (C), 141.26 (C), 139.02 (C), 143.79 (C), 145.19 (C),
146.05 (C), 149.26 (C), 152.70 (C), 169.00 (C). Anal. calcd. for C19H19N7S (377.47): C, 60.46; H, 5.07; N,
25.98. Found: C, 60.33; H, 5.11; N, 25.21%.
N-(4-Phenyl-2,3-dihydrothiazol-2-ylidene)-(5-acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine)
hydrazine (23): A mixture of thiosemicarbazide 17 (0.25 g, 0.7 mmol), phenacyl bromide (0.3 g,
1 mmol), and anhydrous sodium acetate (0.25 g) in absolute ethanol (15 mL) was heated under
reflux for 5 h. After cooling, the solid product was filtered, washed with water, and recrystallized
◦
from ethanol-dioxane mixture (3:1) as brown crystals, yield 0.11 g (40%), m.p. 256–258 C. IR (
ν
,

Molecules 2018, 23, 2657
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1
cm−1): 3435 (NH), 3065 (CH arom.), 2924 (CH aliph.). H-NMR (400 MHz, CDCl3)
δ
(ppm): 2.53 (s,
3H, CH3), 2.73 (s, 3H, CH3), 3.09 (s, 3H, CH3), 6.90 (s, 1H, CH), 7.30–7.35 (t, 2H, phenyl), 7.51–7.56
(t, 2H, phenyl), 7.39–7.44 (t, 2H, phenyl), 7.82–7.84 (d, 2H, phenyl), 8.31–8.33 (d, 2H, phenyl) 8.90
(bs, 1H, NH). MS: m/z (439 M+). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 11.49 (CH3), 24.85 (CH3),
29.07 (CH3), 114.30 (CH), 120.23 (2CH), 121.02 (CH), 126.13 (CH), 127.81 (CH), 129.19 (2CH), 130.52
(2CH), 131.67 (C), 138.92 (C), 142.67 (C), 144.47 (2C), 144.59 (C), 144.71 (C), 154.59 (2C), 161.76 (C).
Anal. calcd. for C24 H21 N7 S (439.54): C, 65.58; H, 4.82; N, 22.31%. Found: C, 65.51; H, 4.75; N, 22.21%.
N-(Thiazolidin-4-on-2-ylidene)-(5-Acetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine) hydrazine (24):
A mixture of 17 (0.5 g, 1 mmol), chloro acetic acid (0.1 g, 0.8 mol), and anhydrous sodium acetate (0.5 g)
in absolute ethanol was heated under reflux for 5 h. After cooling, the solid product obtained was
filtered, washed with water, and dried. Recrystallization from ethanol-dioxane mixture (1:3) gave buff
ν
crystals, yield 0.12 g (50%), m.p. 308–310 ◦C. IR ( , cm−1): 3446 (NH), 2850 (CH aliph.), 1705 (C=O).
MS: m/z (379 M+). Anal. calcd. for C18H17N7OS (379.44): C, 56.98; H, 4.52; N, 25.84. Found: C, 56.88; H,
4.65; N, 25.91%.
General procedure for the synthesis of 5-arylideneacetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b] pyrazine
(25a–k): To a mixture of equimolar amounts (0.27, 1 mmol) of 5-acetyl-3,6-dimethyl-1-phenyl-1H-
pyrazolo[3,4-b]pyrazine (15) and an aromatic aldehyde in ethanol (15 mL), aqueous NaOH (0.5 mL,
25%) was added. The reaction mixture was stirred at room temperature for overnight, then it was
poured onto ice-cold water. The solid precipitate formed was collected by filtration, washed with
water, and recrystallized from dioxane-water mixture.
5-Benzylideneacetyl-3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25a): Yield 1.68 g (84%), m.p.
167–169 ◦C. IR ( , cm−1): 3059 (CH arom.), 2900 (CH aliph.), 1670 (C=O). 1H-NMR (400 MHz, CDCl3)
ν
δ
(ppm): 2.80 (s, 3H, CH3), 3.06 (s, 3H, CH3), 7.20–8.30 (m, 10H, phenyl),13C-NMR (100 MHz, CDCl3)
δ
(ppm): 11.49 (CH3), 24.91 (CH3), 120.34 (2CH), 123.29 (CH), 126.23 (CH), 128.74 (2CH), 128.95 (2CH),
129.23 (2CH), 130.60 (CH, C), 135.09 (2C), 142.69 (C), 144.55 (CH, C), 145.82 (2C), 190.23 (C=O). MS:
m/z (354 M+). Anal. calcd. for C22H18N4O (354): C, 74.56; H, 5.12; N, 15.81. Found: C, 74.63; H, 5.14;
N, 15.60%.
3,6-Dimethyl-5-(2-hydroxylbenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25b): Yield 0.14 g (70%),
◦
1
m.p. 194–196 C. IR (
DMSO-d6) (ppm): 2.69 (s, 3H, CH3), 2.89 (s, 3H, CH3), 6.90 (t, 1H, Ar-H), 6.96 (d, J = 8.1 Hz, 1H, CH),
ν
, cm−1): 3213 (OH), 2923.40 (CH aliph.), 1657 (C=O). H-NMR (400 MHz,
δ
7.30 (t, 1H, Ar-H), 7.37 (t, 1H, Ar-H), 7.59 (t, 2H, Ar-H), 7.70 (d, J = 7.6 Hz, 1H, CH), 8.03 (s, 2H, Ar-H),
8.23 (d, 2H, phenyl), 10.32 (s, 1H, OH).13C-NMR (100 MHz, DMSO-d6): 11.59 (CH3), 24.68 (CH3),
116.80 (C), 120.00 (CH), 120.31(2CH), 121.73 (C), 123.33 (CH), 126.67 (CH), 129.61 (CH), 129.82 (2CH),
131.49 (CH), 132.70 (C), 138.87 (C), 140.71 (C), 142.60 (C), 144.73 (CH), 145.18 (C), 154.09 (C), 157.95 (CH),
190.71 (C=O). Anal. calcd. for C22H18N4O2 (370.40): C, 71.34; H, 4.90; N, 15.13. Found: C, 71.23; H,
4.85; N, 15.25%.
3,6-Dimethyl-5-(2-nitrobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25c): Yield 0.17 g (85%),
m.p. 263–265 ◦C. IR (
ν
, cm−1): 3066 (CH arom.), 1672 (C=O). 1H-NMR (400 MHz, CDCl3)
δ
(ppm):
2.84 (s, 3H, CH3), 3.10 (s, 3H, CH3), 7.37 (d, J = 6.5 Hz, 2H, CH, Ar-H), 7.57 (t, 2H, Ar-H), 7.90 (t, 3H,
Ar-H), 8.41–8.30 (m, 4H, CH, Ar-H). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.52 (CH3), 25.11 (CH3),
δ
120.38 (2CH), 124.20 (2CH), 126.40 (CH), 126.85 (CH), 129.18 (2CH), 129.26 (2CH, C), 138.76 (C),
140.86 (2C), 141.31 (C), 144.93 (CH, C), 155.35 (2C), 189.20 (C=O). Anal. calcd. For C22H17N5O3 (399.40):
C, 66.16; H, 4.29; N, 17.53. Found: C, 66.22; H, 4.18; N, 17.41%.
3,6-Dimethyl-5-(4-nitrobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25d): Yield 0.12 g (80%),
m.p. 263–265 ◦C. IR (
ν
, cm−1): 3051 (CH arom.), 1672 (C=O). 1H-NMR (400 MHz, CDCl3)
δ
(ppm):
2.84 (s, 3H, CH3), 3.10 (s, 3H, CH3), 7.35–7.37 (t, J= 7.4 Hz, 1H, CH), 7.55–7.59 (t, 2H, Ar-H), 7.87–7.93 (t,
J = 11.9 Hz, 2H, CH, Ar-H), 8.32–8.40 (m, 5H, Ar-H). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.52 (CH3),
25.11 (CH3), 120.37 (2CH), 124.21 (2CH), 126.40 (CH), 126.86 (CH), 129.18 (2CH), 129.26 (2CH),
δ

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131.89 (C), 138.75 (C), 140.86 (CH), 141.31 (C), 142.67 (C), 143.25 (C), 144.94 (C), 148.57 (C), 155.35 (C),
189.20 (C=O). Anal. calcd. for C22H17N5O3 (399.40): C, 66.16; H, 4.29; N, 17.53. Found: C, 66.11; H,
4.17; N, 17.42%.
3,6-Dimethyl-5-(3-nitrobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25e): Yield 0.15 g (75%);
m.p. 223–225 ◦C; IR (
CDCl3)
Ar-H), 7.91 (d, J = 16.0 Hz, 1H, CH), 8.02 (d, J = 7.5 Hz, 1H, CH), 8.42–8.27 (m, 4H, Ar-H), 8.59 (s,
1H, Ar-H).13C-NMR (100 MHz, CDCl3)
(ppm): 11.58 (CH3), 25.09 (CH3), 120.36 (2CH), 122.80 (CH),
ν
, cm−1): 3086 (CH arom.), 2925 (CH aliph.), 1674 (C=O); 1H-NMR (400 MHz,
δ
(ppm): 2.85 (s, 3H, CH3), 3.10 (s, 3H, CH3), 7.36 (t, 1H, Ar-H), 7.57 (t, 2H, Ar-H), 7.66 (t, 1H,
δ
124.64 (CH), 125.79 (CH), 126.35 (CH), 129.25 (2CH), 129.97 (CH), 131.89 (CH), 134.38 (C), 136.93 (C),
138.73 (C), 141.06 (C), 142.67 (C), 143.36 (C), 144.98 (C), 148.97 (C), 155.25 (C), 189.29 (C=O). Anal. calcd.
For C22H17N5O3 (399.40): C, 66.16; H, 4.29; N, 17.53. Found: C, 66.24; H, 4.17; N, 17.66%.
3,6-Dimethyl-5-(4-cyanobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25f): Yield 0.16 g (80%),
m.p. 254–256 ◦C. IR ( , cm−1): 3079 (CH arom.), 2224 (CN), 1672 (C=O). 1H-NMR (400 MHz, CDCl3)
ν
δ
(ppm): 2.84 (s, 3H, CH3), 3.09 (s, 3H, CH3), 7.37 (t, 1H, Ar-H), 7.57 (d, J = 7.4 Hz, 2H, CH), 7.81 (m,
6H, Ar-H), 8.39–8.30 (d, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.50 (CH3), 25.10 (CH3),
113.45 (C),118.46 (C N), 120.32 (2CH), 126.16 (CH), 126.94 (CH), 128.94 (CH), 129.24 (2CH), 130.02 (CH),
δ
≡
131.85 (CH), 132.66 (CH), 138.77 (C), 139.45 (C), 140.34 (C), 141.42 (C), 142.64 (C), 143.32 (C), 144.89 (CH),
155.28 (C), 189.27 (C=O). Anal. calcd. for C23H17N5O (379.41): C, 72.81; H, 4.52; N, 18.46. Found: C,
72.76; H, 4.40; N, 18.55%.
3,6-Dimethyl-5-(4-methoxybenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25g): Yield 0.18 g (90%),
◦
1
m.p. 210–212 C. IR (
(400 MHz, CDCl3) (ppm): 2.82 (s, 3H, CH3), 3.05 (s, 3H, CH3), 3.89 (s, 3H, CH3), 6.98 (d, 2H, Ar-H),
7.34 (t, 1H, Ar-H), 7.56 (t, 2H, Ar-H), 7.69 (d, 2H, Ar-H), 7.84 (d, 1H, CH), 8.00 (d, J = 15.9 Hz, 1H,
CH), 8.34 (d, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.49 (CH3), 24.85 (CH3), 55.41 (CH3),
ν
, cm−1): 3069 (CH arom.), 2934, 2836 (CH aliph.), 1663 (C=O); H-NMR
δ
δ
114.40 (2CH), 120.23 (2CH), 121.02 (C), 126.13 (CH), 127.81 (CH), 129.19 (2CH), 130.52 (2CH), 131 (C),
138.92 (C), 142.67 (C), 144.47 (CH), 144.59 (C), 144.71 (C), 154.59 (C), 161.76 (C), 190.26 (C=O). Anal.
calcd. for C23H20N4O2 (384.43): C, 71.86; H, 5.24; N, 14.57. Found: C, 71.75; H, 5.33; N, 14.63%.
3,6-Dimethyl-5-(4-N,N-dim◦ethylaminobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25h): Yield
0.14 g (70%), m.p. 227–229 C. IR (ν δ
, cm−1): 2921 (CH aliph.), 1659 (C=O); 1H-NMR (400 MHz, CDCl3)
(ppm): 2.81 (s, 3H, CH3), 3.03 (s, 3H, CH3), 3.08 (s, 6H, 2CH3), 6.79 (d, 2H, Ar-H), 7.38–7.25 (t, 1H, Ar-H),
7.69–7.50 (m, 4H, CH, Ar-H), 7.83 (s, 2H, Ar-H), 8.34 (d, 2H, phenyl). 13C-NMR (100 MHz, CDCl3)
δ
(ppm): 11.48 (CH3), 24.64 (CH3), 40.08 (2CH3), 111.78 (2C), 118.36 (C), 120.17 (2CH), 122.77 (CH),
126.02 (CH), 129.16 (2CH), 130.75 (2CH), 131.57 (C), 139.06 (C), 142.81 (C), 144.61 (C), 146.06 (2C),
152.12 (CH), 154.100 (C), 190.60 (C=O). Anal. calcd. For C24H23N5O (397.47): C, 72.52; H, 5.83; N, 17.62.
Found: C, 72.63; H, 5.94; N, 17.45%.
3,6-Dimethyl-5-(4-chlorobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25i): Yield 0.23 g (92%),
ν δ (ppm):
m.p. 228–230 ◦C; IR ( , cm−1): 3081 (CH arom.), 1669 (C=O); 1H-NMR (400 MHz, CDCl3)
2.82 (s, 3H, CH3), 3.07 (s, 3H, CH3), 7.33–7.37 (t, 1H, Ar-H), 7.43–7.45 (d, 2H, Ar-H), 7.54–7.58 (t, 2H,
Ar-H), 7.65–7.67 (d, 2H, Ar-H), 7.81–7.85 (d, J = 16.0 Hz, 1H, CH), 8.14–8.18 (d, J = 16.0 Hz, 1H, CH),
8.32–8.34 (d, 2H, Ar-H).13C-NMR (100 MHz, CDCl3)
δ (ppm): 11.50 (CH3), 24.98 (CH3), 120.31 (2CH),
123.61 (CH), 126.26 (CH), 129.23 (2CH), 129.63 (2CH), 129.86 (2CH), 131.77 (C), 133.60 (C), 136.46 (C),
138.85 (C), 142.67 (C), 142.85 (C), 43.92 (C), 144.82 (CH), 154.96 (C), 189.83 (C). Anal. calcd. for
C22H17ClN4O (388.85): C, 67.95; H, 4.41; Cl, 9.12; N, 14.41. Found: C, 67.8; H, 4.55; Cl, 9.07; N, 14.54%.
3,6-Dimethyl-5-(3,4-Dimethoxybenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25j): Yield 0.17 g
(85%), m.p. 199–201 ◦C. IR ( , cm−1): 3078 (CH arom.), 2988, 2837 (CH aliph.), 1663 (C=O); 1H-NMR
ν
(400 MHz, CDCl3)
δ (ppm): 2.82 (s, 3H, CH3), 3.05 (s, 3H, CH3), 3.98 (d, J = 9.8 Hz, 6H, 2CH3), 6.95 (d,
J = 8.2 Hz, 1H, CH), 7.40–7.24 (m, 3H, CH, Ar-H), 7.56 (t, 2H, Ar-H), 7.82 (d, 1H, Ar-H), 7.96 (d, 1H,
Ar-H), 8.34 (d, 2H, Ar-H). Anal. calcd. for C24H22N4O3 (414.46): C, 69.55; H, 5.35; N, 13.52. Found:

Molecules 2018, 23, 2657
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C, 69.43; H, 5.47; N, 13.67%. No 13C-NMR data could be obtained due to the precipitation of the
compound while running the measurement.
3,6-Dimethyl-5-(Ethene-2-ylbenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25k): Yield 0.17 g
◦
1
(85%), m.p. 211–213 C, IR (
(ppm): 2.81 (s, 3H, CH3), 3.05 (s, 3H, CH), 7.13 (m, 2H, CH, Ar-H), 7.43–7.33 (m, 4H, CH, Ar-H),
7.55 (d, 4H, CH, Ar-H), 7.70 (t, 2H, Ar-H), 8.33 (d, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3)
ν
, cm−1): 2922 (CH aliph.), 1662 (C=O); H-NMR (400 MHz, CDCl3)
δ
δ
(ppm): 11.45 (CH3), 24.88 (CH3), 120.32 (2CH), 126.20 (CH), 126.84 (CH), 127.35 (2CH), 128.86 (2CH),
129.22 (2CH), 136.20 (CH), 138.90 (C), 142.02 (CH, C), 144.29 (2C), 144.58 (CH, C),144.78 (CH, C),
154.72 (C), 189.32 (C=O). Anal. calcd. for C24H20N4O for (380.44): C, 75.77; H, 5.30; N, 14.73. Found: C,
75.83; H, 5.18; N, 14.66%.
3,6-Dimethyl-5-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (26): A mixture
of chalcone 25a (0.25 g, 7 mmol), hydrazine hydrate (80%, 0.3 mL), in ethanol (15 mL) was heated
under reflux for 10 h. After cooling the solid product obtained was filtered, washed with water,
and recrystallized from dioxane-water mixture (3:1) as yellow crystals, yield 0.23 g (50%), m.p.
1
146–148 ◦C. IR (
ν
, cm−1): 3351 (NH), 2921 (CH aliph.). H-NMR (400 MHz, CDCl3)
δ
(ppm): 2.63 (s,
3H, CH3), 3.10 (s, 3H, CH3), 3.73 (s, 3H, CH, CH2), 5.08 (s, 1H, NH), 7.33 (m, 3H, phenyl), 7.45–7.37
(m, 3H, phenyl), 7.55 (t, 2H, phenyl), 8.32 (d, 2H, phenyl). 13C-NMR (100 MHz, CDCl3)
(ppm):
δ
11.27 (CH3), 25.00 (CH3), 43.00 (CH2), 67.10 (CH), 120.04 (2CH), 125.69 (2CH), 126.3 (2CH), 128.86
(2CH), 129.1 (2CH), 129.8 (C), 131 (C), 139.7 (C), 142 (C), 143.3 (C), 143.5 (C), 144.5 (C), 154 (C). Anal.
calcd. for C22H20N6 (368.43): C, 71.72; H, 5.47; N, 22.81. Found: C, 71.63; H, 5.53; N, 22.60%.
3,6-Dimethyl-5-(5-phenyl-4,5-dihydro-1-phenyl-1H-pyrazol-3-yl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (27):
A mixture of chalcone 25a (0.25 g, 7 mmol), phenyl hydrazine (0.1 g, 7 mmol) in ethanol (15 mL) was
heated under reflux for 15 min. After cooling, the reaction mixture was filtered, washed with water,
and recrystallized from ethanol as yellow crystals, yield 0.15 g (62%), m.p. 162–164 ◦C. IR (
ν
, cm−1):
2900 (CH aliph.); 1H-NMR (400 MHz, CDCl3)
δ (ppm): 2.81 (s, 3H, CH3), 3.06 (s, 3H, CH3), 3.70 (t, 1H,
CH), 7.34–7.44 (d, 2H, phenyl), 7.45–7.46 (t, 2H, phenyl), 7.52–7.58 (t, 2H, phenyl), 7.71–7.74 (m, 6H,
phenyl), 7.85 (d, 1H, CH2), 8.15 (d, 1H, CH2), 8.30–8.33 (d, 3H, phenyl). 13C-NMR (100 MHz, CDCl3)
δ
(ppm): 11.50 (CH3), 24.93 (CH3), 38.13 (CH2), 59.51 (CH), 102.18 (C), 120.33 (2CH), 123.25 (2CH),
126.23 (2CH), 128.73 (2CH), 128.94 (2CH), 129.23 (2CH), 130.60 (2CH), 131.75 (CH), 135.06 (C), 138.86 (C),
142.68 (C), 144.20 (C), 144.55 (2C), 144.82 (C), 154.82 (C). MS: m/z (443 M+-1). Anal. calcd. for C28H24N6
(444.5): C, 75.65; H, 5.44; N, 18.91. Found: C, 75.41; H, 5.21; N, 19.20%.
3,6-Dimethyl-5-(5-phenyl-4,5-dihydro[1,2]oxazol-3-yl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (28): A mixture
of chalcone 25a (0.25 g, 7 mmol), hydroxylamine hydrochloride (0.1 g, 7 mmol), and sodium acetate
(0.3 g, 2 mmol) in ethanol (10 mL) was heated under reflux for 6 h. The reaction mixture was then
evaporated to half of its volume. After cooling, it was neutralized with dil. HCl and the solid precipitate
was filtered off, washed with water, and recrystallized from ethanol-water mixture (1:1), yield 0.17
1
g (74%), m.p. 200–202 ◦C. IR (
ν
, cm−1): 2926 (CH aliph.). H-NMR (400 MHz, CDCl3)
δ
(ppm): 2.69
(s, 3H, CH3), 2.73 (s, 3H, CH3), 2.79 (d, 1H, CH2), 3.14 (d, 1H, CH2), 3.72–3.77 (t, 1H, CH), 7.28–8.33
(m, 10H, Ar-H). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.38 (CH3), 23.15 (CH3), 46.08 (CH2), 102.88
δ
(CH cyclic), 106.66 (C), 120.33 (2CH), 123 (CH), 125.95 (CH), 126.17 (CH), 126.95 (CH), 128.71 (CH),
128.92 (CH), 129.00 (CH), 129.19 (CH), 130 (C), 139.01 (C), 144.53 (C), 151.42 (C), 156.52 (C), 162.43 (C),
169.64 (C). Anal. calcd. for C22H19N5O (369.42): C, 71.53; H, 5.18; N, 18.96. Found: C, 71.41; H, 5.21; N,
18.91%.
5-Phenyl-4,5-dihydro-3-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-yl) pyrazole-1-carbothioamide
(29): A mixture of compound 25a (0.25 g, 7 mmol) and thiosemicarbazide (0.1 g, 7 mmol) in ethanolic
NaOH (25%, 10 mL) was heated under reflux for 3 h. After cooling, the reaction mixture was filtered,
washed with water, and rec◦rystallized from ethanol-dioxane mixture (1:1) as yellow crystals, yield
0.08 g (50%), m.p. 234–236 C. IR (ν
, cm−1): 3407, 3280 (NH2), 3165 (CH arom.), 2851 (CH aliph.).

Molecules 2018, 23, 2657
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1H-NMR (400 MHz, CDCl3)
δ
(ppm): 2.67 (s, 3H, CH3), 2.70 (s, 3H, CH3), 3.09–3.22 (dd, J = 27.5,
21.8 Hz, 2H), 7.03 (s, 1H, CH), 7.31–7.39 (m, 6H, phenyl), 7.52–7.60 (m, 2H, phenyl), 8.30–8.40 (d, 2H,
phenyl), 9.06 (2H, NH2). 13C-NMR (100 MHz, CDCl3)
(ppm): 11.48 (CH3), 26.86 (CH3), 44.7(cyclic
CH2), 66.85 (CH cyclic), 120.25 (2CH), 125.7 (CH, C), 126.5 (2CH), 127.4 (CH, C), 129.02 (2CH), 129.8
δ
(2CH), 131 (C), 138 (C), 141.60 (C), 155.80 (C), 156.27 (C), 165 (C), 178 (C=S). Anal. calcd. for C23H19N7S
(425.5): C, 64.92; H, 4.50; N, 23.04. Found: C, 64.94; H, 4.44; N, 23.30%.
2-Amino-4-(4-phenyl)-6-(3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-b]pyrazine-5-yl)pyrimidine (30): A mixture
of chalcone 25a (7 mmol), guanidine sulfate (0.12 g, 7 mmol) in ethanolic KOH (30%, 5 mL) was heated
under reflux for 3–4 h. The reaction mixture was then evaporated to half volume and after cooling it was
neutralized with dil. HCl. The solid precipitate was filtered off, washed with water, and recrystallized
◦
from dioxane-acetone mixture (1:3) as yellow crystals, yield 0.1 g (40%), m.p. 150–152 C. IR (
ν,
δ
cm−1): 3302, 3182 (NH2), 3061.37 (CH arom.), 2919, 2849 (CH aliph.).1H-NMR (400 MHz, CDCl3)
(ppm): 2.77 (s, 3H, CH3), 2.99 (s, 3H, CH3), 5.27 (d, 2H, NH2), 7.34 (t, 3H, phenyl), 7.53 (t, 3H, phenyl),
7.67 (s, 1H, CH), 8.23 (d, 2H, phenyl), 8.34–8.36 (d, 2H, phenyl). 13C-NMR (100 MHz, CDCl3)
(ppm):
δ
11.11 (CH3), 24.58 (CH3), 107.81 (C), 119.92 (CH), 120.21 (2CH), 125.86 (CH), 127.22 (2CH), 128.77 (2CH),
129.14 (2CH), 130.66 (CH), 132.31 (C), 137.40 (C), 139.23 (C), 142.91 (C), 144.02 (C), 146.52 (C), 152.54 (C),
162.87 (C), 166.64 (C). Anal. calcd. for C23H21N7 (395): C, 69.85; H, 5.35; N, 24.79. Found: C, 69.63; H,
5.44; N, 24.60%.
3.2. Biological Evaluations
3.2.1. Anti-Inflammatory Assay
Adult albino rats, weighing 150–200 g, were divided into ten groups, each of three animals.
The animals were allowed food and water ad libitum, except during the experiment. They were housed
in a room at 23
reference drug [30], in 3% Tween 80 solution in normal saline as a vehicle, were administered at a dose
of 28 M/kg bodyweight, while control group received only a vehicle. The difference between the
±
2C with a 12 h light/dark cycle. All the compounds under test and the indomethacin
µ
thicknesses of the paw at zero time and different time intervals after injection was taken as a measure of
edema. The measurement was carried out at 0.5, 1, 2, 3, and 4 h after injection of the tested compounds,
reference drug, and control. The right paw volume was measured using a digital plethysmometer
(Model 7150, Ugo Basile, Varese, Italy), directly before and after carrageenan injection to detect the
carrageenan induced inflammation.
3.2.2. Cytotoxicassay
◦
A number of 1
×
104 cells/well were cultured in 96-well plates and then incubated at 37 C,
under conditions of 5% CO2 and 95% air for 24 h. On the following day, the compounds under test,
diluted to the desired concentrations, were added to the wells with the respective negative control and
then the cells were incubated for 24 h. Next, 50
tetrazolium bromide) solution (2 mg/mL of MTT in PBS) was added to each well and left for 3–4 h.
Formazan crystals formed after incubation were dissolved in 150 L DMSO were added to each well.
µL of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl
µ
The 96-well plates were shaken for 10 min and then were read at A570 with reference filter at A650
using Elisa plate reader. Paclitaxel was used as a positive control.
4. Conclusions
In conclusion, several new derivatives of pyrazolo[3,4-b]pyrazines and related heterocycles were
prepared and evaluated for their anti-inflammatory and anti-breast cancer MCF-7 cell line activities.
Compounds 15 and 29 showed the higher anti-inflammatory activity, where the potency of the former
compound was comparable to that of the indomethacin reference drug (cf. Table 1). On the other hand,
compounds 25i
, 25j exhibited very significant anticancer activity against MCF-7 breast cancer cell line
with respect to the positive control Paclitaxel (cf. Figures 1 and 2).

Molecules 2018, 23, 2657
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Supplementary Materials: The spectral data are available online Figures S1–S111.
Author Contributions: Conceptualization, H.E.-K., T.E.-E., P.V. (Pierre Verhaeghe) and P.V. (Patrice Vanelle);
Data curation, H.E.-K., P.V. (Pierre Verhaeghe) and P.V. (Patrice Vanelle); Formal analysis, T.E.-E.; Investigation,
M.S.; Methodology, H.E.-K. and P.V. (Pierre Verhaeghe); Supervision, H.E.-K. and T.E.-E.; Validation, H.E.-K.,
P.V. (Pierre Verhraeghe) and P.V. (Patrice Vanelle); Writing–original draft, M.S.; Writing–review & editing, H.E.-K.
All authors have read and approved the final manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 25a–k are available from the authors.
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