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

Hussein El-Kashef, Talaat El-Emary, Pierre Verhaeghe, P. Vanelle, Maha Samy

► To cite this version:

Hussein El-Kashef, Talaat El-Emary, Pierre Verhaeghe, P. Vanelle, Maha Samy. Anticancer and anti-inflammatory activities of some new pyrazolo[3,4-b]pyrazines. Molecules, 2018, 23 (10), pp.2657. 10.3390/molecules23102657. [https://hal.science/hal-01954194](https://hal.science/hal-01954194)

HAL Id: hal-01954194

https://hal.science/hal-01954194

Submitted on 25 Oct 2019

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Article

# Anticancer and Anti-Inflammatory Activities of Some New Pyrazolo[3,4-*]pyrazines

Hussein El-Kashef <fnref n="1" />*, Talaat El-Emary <fnref n="1" />, Pierre Verhaeghe <fnref n="2" />, Patrice Vanelle <fnref n="3" /> and Maha Samy <fnref n="1" />

Received: 8 August 2018; Accepted: 3 September 2018; Published: 16 October 2018

<figure><img src="image_2.png" /></figure>

check for
updates

**Abstract:** 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.

**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 heterocycles. 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 [4]. Some reports indicated their use as blood platelet aggregation inhibitors [5], anti-inflammatories [6], in controlling herbicides [7], and anticancer agents with low toxicity [8,9]. 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 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].

<footnote n="1">¹ Chemistry Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt; emarytalaat@yahoo.com (T.E.-E.); maha11292@yahoo.com (M.S.)</footnote>

<footnote n="2">² LCC-CNRS Université de Toulouse, CNRS, UPS, 205 Route de Narbonne, 3107 Toulouse, France; pierre.verhaeghe@lcc-toulouse.fr</footnote>

<footnote n="3">³ Aix-Marseille Université, CNRS, Institut de Chimie Radicalaire ICR, UMR 7273, Laboratoire de Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin CS 30064, 13385 Marseille CEDEX 05, France; patrice.vanelle@univ-amu.fr</footnote>

<footnote>* Correspondence: elkashef@aun.edu.eg; Tel.: +20-1005075881</footnote>
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 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 (5). The synthetic route for this compound involved the use of *o*-aminonitrosopyrazole **1** as a starting material, which was reacted with the active methylene benzoylacetonitrile to give **2**. Hydrolysis of the cyano group of the latter compound gave the carboxylic acid **3**, which was converted into the acid chloride **4**, followed by intramolecular Friedel-Crafts cyclization giving **5** (Scheme 1).

<figure><img src="image_3.png" /><figcaption>**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.</figcaption></figure>

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 v OH and v 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 **7** [13], followed by decomposition of the resulting diazonium salt. The alkaline hydrolysis of **6a** gave the hydroxycarboxylic acid **8**, which was esterified in refluxing absolute ethanol in the presence of concentrated H₂SO₄ to give the corresponding hydroxyester **9**. Contrary to the work of Rangnekar et al. [22], all attempts to prepare the latter compound **9** directly through the reaction of **1** with diethylmalonate under various conditions 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 **1** was reacted with thiobarbituric acid in refluxing pyridine, the reaction product 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).
<figure><img src="image_4.png" /><figcaption>**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.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**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.</figcaption></figure>

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).

<figure><img src="image_6.png" /><figcaption>**Scheme 4.** Synthesis of compound 15. Reagents and conditions: (i) CH₂(COCH₃)₂, dry pyridine, reflux 8 h.</figcaption></figure>

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
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).

<figure><img src="image_7.png" /><figcaption>**Scheme 5.** Synthesis of compound **16–21**. Reagents and conditions: (i) NH₂NHCONH₂·HCl, EtOH, reflux 8 h; (ii) NH₂NHCSNH₂, EtOH, reflux 8 h; (iii) NH₂OH·HCl, sodium acetate, EtOH, reflux 3–4 h; (iv) NH₂NH₂, reflux 6 h; (v) isatin, (C₂H₅)₂NH, EtOH, reflux overnight; and (vi) EtOH, HCl, reflux 15 min.</figcaption></figure>

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).

<figure><img src="image_8.png" /><figcaption>**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.</figcaption></figure>

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.
<figure><img src="image_9.png" /><figcaption>**Scheme 7.** Synthesis of chalcones 25a–k. Reagents and conditions: (i) ArCHO, EtOH, 25% aq. NaOH, rt overnight. *a*, Ar = C₆H₅; *b*, Ar = 2-OHC₆H₄; *c*, Ar = 2-NO₂C₆H₄; *d*, Ar = 4-NO₂C₆H₄; *e*, Ar = 3-NO₂C₆H₄; *f*, Ar = 4-CNC₆H₄; *g*, Ar = 4(CH₃O)C₆H₄; *h*, Ar = 4-N(CH₃)₂C₆H₄; *i*, Ar = 4-ClC₆H₄; *j*, Ar = 3,4-(CH₃O)₂C₆H₃; and *k*, Ar = CH=CH-C₆H₄.</figcaption></figure>

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).

<figure><img src="image_10.png" /><figcaption>**Scheme 8.** Synthesis of compounds 26–30. Reagents and conditions: (i) NHNH₂·H₂O, EtOH, reflux 10 h; (ii) PhNHNH₂, EtOH, reflux 15 min; (iii) NH₂OH·HCl, CH₃COONa, EtOH, reflux 6 h; (iv) NH₂NHCSNH₂, ethanolic NaOH (25%), reflux 3 h; and (v) NH₂C=NHNH₂·H₂SO₄, ethanolic KOH (25%), reflux 3–4 h.</figcaption></figure>

## 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
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><caption>**Table 1.** Anti-inflammatory Activity of Tested Compounds (15, 25a, 26–30) against Acute Carrageenan-Induced Paw Oedema in Rats (Statistical analysis).</caption><thead><tr><th rowspan="3">Compound <sup>a</sup></th><th colspan="7">Paw Oedema Inhibition (Swell ± S.E.M.) <sup>a,b,c</sup> (%)</th></tr><tr><th>30 min</th><th>1 (h)</th><th>2 (h)</th><th>3 (h)</th><th>4 (h)</th><th>5 (h)</th><th>Potency <sup>d</sup></th></tr><tr><th colspan="7">Edema Induced by Carrageenan (% Edema Inhibition Relative to Control)</th></tr></thead><tbody><tr><td>15</td><td>0.40 ± 0.00</td><td>0.55 ± 0.03</td><td>0.48 ± 0.02</td><td>0.42 ± 0.02</td><td>0.38 ± 0.02</td><td>0.40 ± 0.00</td><td>44.44</td></tr><tr><td>25a</td><td>0.63 ± 0.03</td><td>0.66 ± 0.03</td><td>0.65 ± 0.03</td><td>0.66 ± 0.02</td><td>0.63 ± 0.02</td><td>0.63 ± 0.03</td><td>12.50</td></tr><tr><td>26</td><td>0.55 ± 0.03</td><td>0.68 ± 0.02</td><td>0.66 ± 0.02</td><td>0.62 ± 0.02</td><td>0.62 ± 0.02</td><td>0.55 ± 0.03</td><td>23.61</td></tr><tr><td>27</td><td>0.61 ± 0.02</td><td>0.68 ± 0.02</td><td>0.60 ± 0.03</td><td>0.60 ± 0.03</td><td>0.63 ± 0.02</td><td>0.62 ± 0.02</td><td>15.07</td></tr><tr><td>28</td><td>0.46 ± 0.02</td><td>0.63 ± 0.02</td><td>0.63 ± 0.02</td><td>0.60 ± 0.03</td><td>0.48 ± 0.02</td><td>0.47 ± 0.02</td><td>34.72</td></tr><tr><td>29</td><td>0.43 ± 0.00</td><td>0.45 ± 0.03</td><td>0.43 ± 0.02</td><td>0.42 ± 0.02</td><td>0.38 ± 0.03</td><td>0.43 ± 0.02</td><td>40.27</td></tr><tr><td>30</td><td>0.51 ± 0.02</td><td>0.61 ± 0.02</td><td>0.55 ± 0.05</td><td>0.45 ± 0.03</td><td>0.45 ± 0.03</td><td>0.52 ± 0.02</td><td>27.77</td></tr><tr><td>Drug *</td><td>0.40 ± 0.00</td><td>0.45 ± 0.03</td><td>0.42 ± 0.02</td><td>0.38 ± 0.0</td><td>0.37 ± 0.02</td><td>0.40 ± 0.00</td><td>44.44</td></tr><tr><td>Negative Control</td><td>0.72 ± 0.02</td><td>0.73 ± 0.02</td><td>0.73 ± 0.02</td><td>0.72 ± 0.02</td><td>0.73 ± 0.02</td><td>0.72 ± 0.02</td><td>-</td></tr></tbody></table>

The percent oedema inhibition was calculated from the mean effect shown by the control and treated animals according to the following equation:

$$ \text{Percent oedema inhibition} = \frac{v_c - v_t}{v_c} \times 100 $$

where $v_c$ represents the mean increase in paw volume in the control group of rats and $v_t$ 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 ± standard error of the mean (S.E.M.). Statistical evaluations were performed using graph pad prism program software version 5.00 through One-way ANOVA.

### 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 α,β-unsaturated ketonic function, 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

<footnote>(a) Dose 28 µM/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).</footnote>

<footnote>* Drug = indomethacin (28 µM/kg).</footnote>
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 µM. The reference drug Paclitaxel showed an IC₅₀ of 1.02 µM (Figure 2).

<figure><img src="image_11.png" /><figcaption>**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.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**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 ± standard error of the mean (SEM). Two-tailed paired student test *p*-values as determined by Graphpad Prism software is indicated as *** *p* < 0.001.</figcaption></figure>
### 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 (ν, cm⁻¹): 3197 (OH), 3073 (CH aromatic), 2234 (C≡N). ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 2.51 (s, 3H, CH₃), 7.38 (t, 1H, phenyl), 7.57 (t, 2H, phenyl), 8.08 (d, 2H, phenyl), 8.43 (s, 1H, OH). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 10.99 (CH₃), 114.58 (C), 115.73 (C≡N), 120.28 (2CH), 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 COOH), 1667 (C=O). ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 2.57 (s, 3H, CH₃), 7.33 (t, 1H, phenyl), 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,
2H, phenyl), 12.08 (s, 1H, OH),<sup>13</sup>C-NMR (100 MHz, CDCl<sub>3</sub>): 11.64 (CH<sub>3</sub>), 14.20 (CH<sub>3</sub>), 63.48 (CH<sub>2</sub>), 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<sup>+</sup>). Anal. calcd. for C<sub>15</sub>H<sub>14</sub>N<sub>4</sub>O<sub>3</sub> (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 (ν, cm<sup>-1</sup>): 3438 (NH), 2922, 2784 (CH aliph.), 1655 (C=O). <sup>1</sup>H-NMR (400 MHz, DMSO-d<sub>6</sub>) δ (ppm): 2.40 (s, 3H, CH<sub>3</sub>), 2.60 (s, 3H, CH<sub>3</sub>), 7.33 (t, 1H, phenyl), 7.52 (t, 2H, phenyl), 7.6 (s, 1H, NH), 8.11 (d, 2H, phenyl). <sup>13</sup>C-NMR (100 MHz, DMSO-d<sub>6</sub>) δ (ppm): 11.26 (CH<sub>3</sub>), 21.17 (CH<sub>3</sub>), 119.87 (2CH), 125.79 (CH, C), 129.19 (2CH, C), 138.82 (2C), 155.33 (C=O). Anal. calcd. for C<sub>13</sub>H<sub>12</sub>N<sub>4</sub>O (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 (ν, cm<sup>-1</sup>): 3075 (NH), 2921, 2781 (CH aliph.), 1641 (C=O). <sup>1</sup>H-NMR (400 MHz, DMSO-d<sub>6</sub>) δ (ppm): 2.76 (s, 3H, CH<sub>3</sub>), 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). <sup>13</sup>C-NMR (100 MHz, DMSO-d<sub>6</sub>) δ (ppm): 14.08 (CH<sub>3</sub>), 120.327 (C), 120.99 (2CH), 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<sup>+</sup>). Anal. calcd. for C<sub>18</sub>H<sub>14</sub>N<sub>4</sub>O (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 (ν, cm<sup>-1</sup>): 3416 (NH), 3068 (CH arom.), 2891 (CH aliph.), 1693 (C=O). <sup>1</sup>H-NMR (400 MHz, DMSO-d<sub>6</sub>), δ (ppm): 2.70 (s, 1H, CH<sub>3</sub>), 7.27 (t, 1H, phenyl), 7.45 (t, 2H, phenyl), 8.13 (d, 2H, phenyl), 11.76 (s, 1H, NH), 11.91 (s, 1H, NH), <sup>13</sup>C-NMR (100 MHz, DMSO-d<sub>6</sub>) δ (ppm): 11.29 (s, 3H, CH<sub>3</sub>), 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<sup>+</sup>). Anal. calcd. for C<sub>14</sub>H<sub>10</sub>N<sub>5</sub>OS (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 K<sub>2</sub>CO<sub>3</sub> (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 (ν, cm<sup>-1</sup>): 2922, 2784 (CH aliph.), 1655 (C=O). <sup>1</sup>H-NMR (400 MHz, CDCl<sub>3</sub>) δ (ppm): 1.47 (t, *J* = 7.1 Hz, 3H, CH<sub>3</sub>), 1.53 (t, *J* = 7.4 Hz, 3H, CH<sub>3</sub>), 2.86 (s, 3H, CH<sub>3</sub>), 3.51 (q, *J* = 7.4 Hz, 2H, CH<sub>2</sub>), 4.34 (q, *J* = 7.0 Hz, 2H, CH<sub>2</sub>), 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). <sup>13</sup>C-NMR (100 MHz, CDCl<sub>3</sub>) δ (ppm): 11.75 (CH<sub>3</sub>), 12.93 (CH<sub>3</sub>), 13.68 (CH<sub>3</sub>), 27.05 (CH<sub>2</sub>), 40.44 (CH<sub>2</sub>), 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<sup>+</sup>). Anal. calcd. for C<sub>18</sub>H<sub>18</sub>N<sub>6</sub>OS (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 K<sub>2</sub>CO<sub>3</sub> (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
crystals, yield 0.10 g (71.4%), m.p. 243–245 °C. IR (ν, cm⁻¹): 2980, 2923 (CH aliph.), 1737 (C=O), 1686 (C=O). ¹H-NMR (400 MHz, CDCl₃) 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) δ (ppm): 11.79 (CH₃), 14.57 (CH₃), 33.31 (CH₂), 61.73 (CH₂), 120.21 (2CH), 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) δ (ppm): 2.70 (s, 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) δ (ppm): 11.32 (CH₃), 25.15 (CH₃), 27.79 (CH₃), 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 (93%), m.p. 210–212 °C. IR (ν, 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): 3434, 3237, 3152 (NH₂ + NH), 1690 (C=O). ¹H-NMR (300 MHz, CDCl₃) δ (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) δ (ppm): 11.49 (CH₃), 25.00 (CH₃), 27.96 (CH₃), 120.29 (C, 2CH), 126.7 (CH), 129.7 (2CH), 131.11 (C), 138.89 (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%), m.p. 223–225 °C, IR (ν, cm⁻¹): 3281 broad (OH), 2900 (CH aliph.). ¹H-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) δ (ppm): 11.01 (s, 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
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 (ν, cm⁻¹): 3453, 3342 (NH₂), 3050 (CH arom.). ¹H-NMR (400 MHz, CDCl₃) δ (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) δ (ppm): 11.36 (CH₃), 12.56 (CH₃), 25.65 (CH₃), 120.03 (CH), 120.14 (CH), 125.60 (CH), 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 filtered and recrystallized from ethanol-water mixture (1:1) as buff crystals, yield 0.18 g (90%), m.p. 167–169 °C. IR (ν, cm⁻¹), 3322 (NH), 1745 (C=O), 1697 (C=O). ¹H-NMR (400 MHz, CDCl₃) 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), 3444 (NH), 3163, 3074 (CH arom.), 1716 (C=O), 1670 (CO-NH). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 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, and recrystallized from acetone-water mixture (1:1) as buff crystals, yield 0.05 g (17%), m.p. 222–224 °C, IR (ν, cm⁻¹): 3178 (NH), 3067 (CH arom.), 2921, 2852 (CH aliph.). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 2.36 (s, 3H, CH₃), 2.59 (s, 3H, CH₃), 2.74 (s, 3H, CH₃), 3.06 (s, 3H, CH₃), 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) δ (ppm): 11.46 (CH₃), 14.02 (CH₃), 16.71 (CH₃), 26.43 (CH₃), 103.13 (CH), 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,
$cm^{-1}$): 3435 (NH), 3065 (CH arom.), 2924 (CH aliph.). $^1$H-NMR (400 MHz, CDCl$_3$) $\delta$ (ppm): 2.53 (s, 3H, CH$_3$), 2.73 (s, 3H, CH$_3$), 3.09 (s, 3H, CH$_3$), 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 $M^+$). $^{13}$C-NMR (100 MHz, CDCl$_3$) $\delta$ (ppm): 11.49 (CH$_3$), 24.85 (CH$_3$), 29.07 (CH$_3$), 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 C$_{24}$H$_{21}$N$_7$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 crystals, yield 0.12 g (50%), m.p. 308–310 °C. IR (v, $cm^{-1}$): 3446 (NH), 2850 (CH aliph.), 1705 (C=O). MS: *m/z* (379 $M^+$). Anal. calcd. for C$_{18}$H$_{17}$N$_7$OS (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 (v, $cm^{-1}$): 3059 (CH arom.), 2900 (CH aliph.), 1670 (C=O). $^1$H-NMR (400 MHz, CDCl$_3$) $\delta$ (ppm): 2.80 (s, 3H, CH$_3$), 3.06 (s, 3H, CH$_3$), 7.20–8.30 (m, 10H, phenyl), $^{13}$C-NMR (100 MHz, CDCl$_3$) $\delta$ (ppm): 11.49 (CH$_3$), 24.91 (CH$_3$), 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 C$_{22}$H$_{18}$N$_4$O (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%), m.p. 194–196 °C. IR (v, $cm^{-1}$): 3213 (OH), 2923.40 (CH aliph.), 1657 (C=O). $^1$H-NMR (400 MHz, DMSO-d$_6$) $\delta$ (ppm): 2.69 (s, 3H, CH$_3$), 2.89 (s, 3H, CH$_3$), 6.90 (t, 1H, Ar-H), 6.96 (d, *J* = 8.1 Hz, 1H, CH), 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). $^{13}$C-NMR (100 MHz, DMSO-d$_6$): 11.59 (CH$_3$), 24.68 (CH$_3$), 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 C$_{22}$H$_{18}$N$_4$O$_2$ (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 (v, $cm^{-1}$): 3066 (CH arom.), 1672 (C=O). $^1$H-NMR (400 MHz, CDCl$_3$) $\delta$ (ppm): 2.84 (s, 3H, CH$_3$), 3.10 (s, 3H, CH$_3$), 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). $^{13}$C-NMR (100 MHz, CDCl$_3$) $\delta$ (ppm): 11.52 (CH$_3$), 25.11 (CH$_3$), 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 C$_{22}$H$_{17}$N$_5$O$_3$ (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 (v, $cm^{-1}$): 3051 (CH arom.), 1672 (C=O). $^1$H-NMR (400 MHz, CDCl$_3$) $\delta$ (ppm): 2.84 (s, 3H, CH$_3$), 3.10 (s, 3H, CH$_3$), 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). $^{13}$C-NMR (100 MHz, CDCl$_3$) $\delta$ (ppm): 11.52 (CH$_3$), 25.11 (CH$_3$), 120.37 (2CH), 124.21 (2CH), 126.40 (CH), 126.86 (CH), 129.18 (2CH), 129.26 (2CH),
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 (ν, cm⁻¹): 3086 (CH arom.), 2925 (CH aliph.), 1674 (C=O); ¹H-NMR (400 MHz, CDCl₃) 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, 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) δ (ppm): 11.58 (CH₃), 25.09 (CH₃), 120.36 (2CH), 122.80 (CH), 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 (ν, cm⁻¹): 3079 (CH arom.), 2224 (CN), 1672 (C=O). ¹H-NMR (400 MHz, CDCl₃) 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) δ (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), 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 C₂₃H₁₇N₅O₃ (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%), m.p. 210–212 °C. IR (ν, cm⁻¹): 3069 (CH arom.), 2934, 2836 (CH aliph.), 1663 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ (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) δ (ppm): 11.49 (CH₃), 24.85 (CH₃), 55.41 (CH₃), 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-dimethylaminobenzylideneacetyl)-1-phenyl-1H-pyrazolo[3,4-b]pyrazine (25h):** Yield 0.14 g (70%), m.p. 227–229 °C. IR (ν, cm⁻¹): 2921 (CH aliph.), 1659 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ (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 C₂₄H₂₃N₅O₃ (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%), m.p. 228–230 °C; IR (ν, cm⁻¹): 3081 (CH arom.), 1669 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 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 C₂₂H₁₇ClN₄O₃ (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 (ν, cm⁻¹): 3078 (CH arom.), 2988, 2837 (CH aliph.), 1663 (C=O); ¹H-NMR (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:
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 (85%), m.p. 211–213 °C, IR (ν, cm⁻¹): 2922 (CH aliph.), 1662 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ (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) 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. 146–148 °C. IR (ν, cm⁻¹): 3351 (NH), 2921 (CH aliph.). ¹H-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) δ (ppm): 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 g (74%), m.p. 200–202 °C. IR (ν, cm⁻¹): 2926 (CH aliph.). ¹H-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) δ (ppm): 11.38 (CH₃), 23.15 (CH₃), 46.08 (CH₂), 102.88 (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 recrystallized 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.).
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) δ (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 (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, cm⁻¹): 3302, 3182 (NH₂), 3061.37 (CH arom.), 2919, 2849 (CH aliph.). ¹H-NMR (400 MHz, CDCl₃) δ (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) δ (ppm): 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 ± 2C with a 12 h light/dark cycle. All the compounds under test and the indomethacin reference drug [30], in 3% Tween 80 solution in normal saline as a vehicle, were administered at a dose of 28 µM/kg bodyweight, while control group received only a vehicle. The difference between the 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 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 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 µL DMSO were added to each well. 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).
**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 Verhaeghe) 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.

## References

1. Akahane, A.; Kuroda, S.; Itani, H.; Tabuchi, S.; Sato, Y.; Matsuoka, N.; Tada, M.; Matsuoka, H.; Oku Tanaka, T.A. Pyrazolopyrazines and Their Use as Adenosine Antagonists. *Chem. Abstr.* **2001**, *135*, 33489a.

2. Matsuoka, N.; Moriguchi, A.; Tada, M.; Mihara, T. Novel Use of Adenosine A₁A₂a Receptor Dual Antagonists. *Chem. Abstr.* **2000**, *134*, 533m.

3. Akahane, A.; Tanaka, A. Pyrazolopyrazine Compound and Pharmaceutical Use Thereof. *Chem. Abstr.* **2003**, *138*, 24732a.

4. Imaizumi, K.; Sado, T. Bone Metabolism Improvers Containing Pyrazolopyrazine. *Chem. Abstr.* **1994**, *121*, 91797.

5. Sado, T.; Inoue, A. Preparation of 1H-pyrazolo[3,4-b]pyrazines as blood platelet aggregation inhibitors and anti-inflammatories. *Chem. Abstr.* **1990**, *113*, 78422k.

6. Abdelmohsen, S.A.; El-Emary, T.I.; El-Kashef, H. Synthesis, anti-inflammatory and antibacterial activities of novel pyrazolo[4,3-g]pteridines. *Chem. Pharm. Bull.* **2016**, *64*, 476–482. [CrossRef] [PubMed]

7. Takabe, F.; Shibayama, A.; Yanaguchi, M.; Yamaji, M.; Hanai, R.; Sadohara, H. Preparation of pyrazolopyridinones and analogs as herbicides. *Chem. Abstr.* **1997**, *126*, 277477a.

8. Suzuki, S.; Inoue, A. Anticancer agents containing 1H-pyrazolo[3,4-b]pyrazines. *Chem. Abstr.* **1990**, *113*, 218276t.

9. Suzuki, S. Preparation of 1,5-disubstituted-1H-pyrazolo[3,4-b]pyrazines as anticancer agents. *Chem. Abstr.* **1991**, *114*, 178382m.

10. Hofmann, J.; Sickerand, D.; Mann, G. Preparation of 1,3,5-trisubstituted-6-hydroxypyrazolo[3,4-b]pyrazines. *Chem. Abstr.* **1990**, *113*, 231409h.

11. Rangnekar, D.W.; Dhamnaskar, S.V. Synthesis of pyrazolo[4',3':5,6]pyrazino[2,3-c] pyrazoles and pyrazolo[4',3':5,6]pyrazino[2,3-d]pyrimidines and their application to polyester fibres. *J. Chem. Technol. Biotechnol.* **1990**, *49*, 311–320. [CrossRef]

12. El-Emary, T.I.; Kamal El-Dean, A.M.; El-Kashef, H.S. Facile synthesis of some new pyrazolo[3,4-b]pyrazines and their antifungal activity. *Il Farmaco* **1998**, *53*, 383–388. [CrossRef]

13. El-Kashef, H.S.; El-Emary, T.I.; Gasquet, M.; Timon-David, P.; Maldonado, J.; Vanelle, P. New pyrazolo[3,4-b]pyrazines: Synthesis and biological activity. *Pharmazie* **2000**, *55*, 572–576. [PubMed]

14. Suzuki, S.; Suzuki, K.; Honda, H. 1-Phenyl-1H-pyrazolo[3,4-b]pyrazine derivatives. *Chem. Abstr.* **1984**, *100*, 68319z.

15. Svetlik, J. New condensed tri- and tetracyclic pyrazole ring system. *Heterocycles* **1984**, *22*, 2513–2516. [CrossRef]

16. Farghaly, A.R.; Esmail, S.; Abdel-Zaher, A.; Hafez, A.A.; El-Kashef, H. Synthesis and anticonvulsant activity of some new pyrazolo[3,4-b]pyrazines and related heterocycles. *Bioorg. Med. Chem.* **2014**, *22*, 2166–2175. [CrossRef] [PubMed]

17. El-Emary, T.I. Synthesis and biological activity of some new pyrazolo[3,4-b] pyrazines. *J. Chin. Chem. Soc.* **2006**, *53*, 391–401. [CrossRef]

18. El-Emary, T.; El-Kashef, H. First synthesis and biological evaluation of indeno[2,1-e]pyrazolo[3,4-b]pyrazin-5-one and related derivatives. *Eur. J. Med. Chem.* **2013**, *62*, 478–485. [CrossRef] [PubMed]

19. Siegel, S.; Wilmen, A.; Roehrig, S.; Svenstrup, N.; Gnoth, N.; Heitmeier, M.J.; Rester, S. Preparation of Imidazopyrazines, Pyrazolopyrazines, and Imidazotriazines for Treatment of Hematologic Diseases. DE Patent 102007032349A1, 15 January 2009.
20. Tasker, A.; Pettus, L.H.; Wurz, R. Pyrazolo[3,4-b]pyrazine Compounds as p38 Modulators and Methods of Use as Anti-Inflamatory Agents. U.S. Patent 8367671B2, 5 February 2013.

21. Quiroga, J.; Sanchez, N.E.; Acosta, P.; Insuasty, B.; Abonia, R. Microwave-assisted synthesis of fused pyrazolo[3,4-b]pyrazines by the reaction of ortho-aminonitrosopyrazoles and cyclic β-diketones. *Tetrahedron Lett.* **2012**, *53*, 3181–3187. [CrossRef]

22. Rangnekar, D.W.; Dhamnaskar, S.V. Synthesis of 5-hetarylpyrazolo[3,4-b]pyrazines and their use as disperse dyes for polyester fibres. *Dyes Pigment.* **1990**, *13*, 241–250. [CrossRef]

23. Gomes, M.N.; Muratov, E.N.; Pereira, M.; Peixoto, J.C.; Rosseto, L.P.; Cravo, P.V.L.; Andrade, C.H.; Neves, B.J. Chalcone derivatives: Promising starting points for drug design. *Molecules* **2017**, *22*, 1210. [CrossRef] [PubMed]

24. Mirossay, L.; Varinská, L.; Mojžiš, J. Antiangiogenic effect of flavonoids and chalcones: An update. *Int. J. Mol. Sci.* **2018**, *19*, 27. [CrossRef] [PubMed]

25. Kotha, R.R.; Kulkarni, R.G.; Garige, A.K.; Nerella, S.G.; Garlapati1, A. Synthesis and cytotoxic activity of new chalcones and their flavonol derivatives. *Med. Chem. 2017*, *7*, 353–360. [CrossRef]

26. Champelovier, P.; Chauchet, X.; Hazane-Puch, F.; Vergnaud, S.; Garrel, C.; Laporte, F.; Boutonnat, J.; Boumendjel, A. Cellular and molecular mechanisms activating the cell death processes by chalcones: Critical structural effects. *Toxicol. In Vitro* **2013**, *27*, 2305–2315. [CrossRef] [PubMed]

27. Lipinski, C.F.; Oliveira, A.A.; Honorio, K.M.; Oliveira, P.R.; Ferreira da Silva, A.B. A molecular modeling study of combretastatin-like chalcones as anticancer agents using PLS, ANN and consensus models. *Struct. Chem.* **2018**, *29*, 957–965. [CrossRef]

28. Bhale, P.S.; Chavan, H.V.; Dongare, S.B.; Shringare, S.N.; Mule, Y.B.; Nagane, S.S.; Bandgar, B.P. Synthesis of extended conjugated indolyl chalcones as potent anti-breast cancer, anti-inflammatory and antioxidant agents. *Bioorg. Med. Chem.* **2017**, *27*, 1502–1507. [CrossRef] [PubMed]

29. Karthikeyan, C.; Moorthy, N.S.H.N.; Ramasamy, S.; Vanam, U.; Manivann, E.; Karunagaran, D.; Trivedi, P. Recent Patents on Anti-Cancer. *Drug Discov.* **2015**, *10*, 97–115.

30. Winter, C.A.; Risly, E.A.; Nuss, G.W. Carrageenin-induced edema in hind paw of the rat as an assay for antiinflammatory drugs. *Proc. Soc. Exp. Biol. Med.* **1962**, *111*, 544–547. [CrossRef] [PubMed]

31. Ismail, I.A.; Kang, H.S.; Lee, H.-J.; Chang, H.; Yun, J.; Lee, C.W.; Kim, N.H.; Kim, H.S.; Yook, J.I.; Hong, S.-H.; Kwon, B.-M. 2-Hydroxycinnamaldehyde inhibits the epithelial-mesenchymal transition in breast cancer cells. *Breast Cancer Res. Treat.* **2013**, *137*, 697–708. [CrossRef] [PubMed]

**Sample Availability:** Samples of the compounds **25a–k** are available from the authors.

<figure><img src="image_13.png" /></figure>

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).