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

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.


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][2][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][13][14][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].
In continuation with our interest in the synthesis of pyrazolo [3,4-b]pyrazins [5,12,13,[16][17][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.

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).
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 1 H-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). Scheme 1. Synthesis of compound 5. Reagents and conditions: (i) Benzoylacetonitrile, pyridine, reflux 3 h; (ii) NaOH 20%, reflux 6 h; (iii) SOCl 2 , reflux 2 h; and (iv) AlCl 3 , CS 2 , 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 -NH 2 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 1 H-NMR spectral analysis which showed, in addition to the phenyl protons, two characteristic signals assigned to CH 3 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 H 2 SO 4 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 K 2 CO 3 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). 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). 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 αchloroacetic acid gave the thiazolidinone 24 (Scheme 6). 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). Chalcones are known by their biological activities and in particular by their anticancer activities [23][24][25][26][27][28][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. Chalcones are known by their biological activities and in particular by their anticancer activities [23][24][25][26][27][28][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. Chalcones are known by their biological activities and in particular by their anticancer activities [23][24][25][26][27][28][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. 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 2aminopyrimidine 30 (Scheme 8). 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).

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 [

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

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 IC 50 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 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 NO 2 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-NO 2 group by a CN or OCH 3 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 IC 50 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 IC 50 value of 2.22 µM. The reference drug Paclitaxel showed an IC 50 of 1.02 µM (Figure 2).  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.

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 1 H-NMR spectra were recorded on Bruker AV500 (400 MHz) (Bruker, Billerica, MA, USA) and Bruker Avance III (400 MHz)  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.

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 1 H-NMR spectra were recorded on Bruker AV500 (400 MHz) (Bruker, Billerica, MA, USA) and Bruker Avance III (400 MHz) 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.