Synthesis, Structural Elucidation, and In Vitro Antitumor Activities of Some Pyrazolopyrimidines and Schiff Bases Derived from 5-Amino-3-(arylamino)-1H-pyrazole-4-carboxamides

The reaction of 5-amino-3-(arylamino)-1H-pyrazole-4-carboxamides 1a,b with acetylacetone 2 and arylidenemalononitriles 5a–c yielded the pyrazolo[1,5-a]-pyrimidine derivatives 4a,b and 7a–f respectively. On the other hand, Schiff bases 9a,b and 12a–j were obtained upon treatment of carboxamides 1a,b with isatin 8 and some selected aldehydes 11a–e. The newly synthesized compounds were characterized by analytical and spectroscopic data. Representative examples of the synthesized products 4a,b, 7e, 7f, 9b, 12b–f, 12h, and 12j were screened for their in vitro antitumor activities against different human cancer cell lines and the structure-activity relationship (SAR) was discussed.

In view of these facts, we report herein the synthesis of a new series of substituted pyrazolo [1,5-a]pyrimidines and Schiff bases derived from 5-aminopyrazole derivatives for the examination of their antitumor activity.
The formation of compounds 4a,b was therefore assumed to proceed via initial attack of the exocyclic amino group of 1 on the keto group of the 1,3-dicarbonyl compound 2 followed by intramolecular cyclization via elimination of water.
The formation of compounds 7a-f was assumed to proceed via initial attack of the exocyclic amino function of the compounds 1a,b on the α,β-unsaturated system in compound 5, followed by intramolecular cyclization and spontaneous autooxidation through the loss of the H 2 molecule [17] (Scheme 1).
Condensation of 1a,b with isatin 8 in boiling ethanol gave 3-(arylamino)-5-[(2-oxoindolin-3ylidene)amino]-1H-pyrazole-4-carboxamides 9a,b in excellent yields (Scheme 2). Structures of compounds 9a,b were confirmed on the basis of elemental analysis and spectral data. As an example, the IR spectrum (KBr/cm −1 ) of 9a showed strong stretching bands at 3427, 3317, and 3181 for -NH 2 and -NH, two strong absorption bands at 1687 and 1623 due to two C=O groups, and a band at 1597 for the C=N group. Its 1 H NMR spectrum (DMSO-d 6 , δ ppm) showed a diffused multiplet at 6.89-7.53 due to aromatic and -NH 2 protons. Three singlets appeared at 9.14, 10.95, and 12.98 due to three -NH protons, which were D 2
The structures of compounds 12a-j were confirmed on the basis of their analytical and spectral data. As an example, the mass spectrum of compound 12e exhibited a molecular ion peak at m/z 413 (C 21

In vitro antitumor screening
Preliminary experiments were done to check the availability of the prepared compounds as antitumor agents. We selected different varieties of the newly synthesized compounds containing variable groups and then we evaluated their in vitro cytotoxic activities against the human breast cancer cell line (MCF7) where Doxorubicin was used as a standard drug [18]. The results were expressed as the IC 50 value, which corresponds to the concentration required for 50% inhibition of cell growth of the treated cells when compared to that of control cells.
From the results in Table 1, it was found that the IC 50 values of compounds 7f, 12j, and 12e were 0.085 µM, 9.294 µM, and 28.48 µM, respectively, which exhibited the highest cytotoxic activities, followed by compound 4a (IC 50 =122.9 µM) which also showed better activity than the reference drug Doxorubicin (IC 50 =96.41 µM), while compound 12d (IC 50 =280.0 µM) showed lower activity than the reference drug.

Tab. 1.
The cytotoxicity of the tested compounds on the MCF-7 tumor cell line.
Screening the cytotoxicity of the tested compounds on cervical carcinoma (KB), where Fluorouracil was used as a standard drug (IC 50 =4.46 nM), showed that the seven tested compounds 7e, 12b-f, and 12j were more potent than the standard. The most potent one was 12e (IC 50 =0.54 nM).
Studying the effects of cytotoxicity for the tested compounds on the CNS cancer (SF-268) cell line, using Cytarabine (IC 50 =7.68 nM) as a standard drug, revealed that compounds 4a, 7e, 7f, 12c, 12d, 12e, 12f, 12h, and 12j were more active than the standard drug, where 12e (IC 50 =0.30 nM) was the most promising one.
On the non-small cell lung cancer (NCl H460) cell line, the tested compounds, except for 12e and 12h (IC 50 =6.60 & 7.00 nM respectively), were more potent than the standard drug Gencitabine hydrochloride (IC 50 =2.13 nM).
On the colonadenocarcinoma (RKOP 27) cell line, the tested compounds, except for 12c, 12d, 12e, and 12h, were found to be less active than the standard drug Capecitabine (IC 50 =4.33 nM). Compounds 4b and 12j had a comparable activity to Capecitabine.
From the estimation of the cytotoxicity on the melanoma (SK-MEL-28) cell line, the tested compounds were less active than the standard drug Aldesleukin (IC 50 =3.45 nM), except 12b (IC 50 =3.20 nM) was slightly more active.
The cytotoxicity of the tested compounds on the HeLa (cervical) cell line showed that Tamoxifen (IC 50 =0.11 nM), the standard drug used, was more active than all of the tested compounds.
The cytotoxicity of the tested compounds on the HepG2 (liver) cell line showed that seven of the tested compounds were more bioactive than Tamoxifen (IC 50 =1.31 nM) in a decreasing order of 12e>12d>12j>12c>12f>7e>12h.  Based on these results, it is evident that there is a structure-activity relationship (SAR). Shown in Table 4, from the screening of the tested compounds against the HepG2 (liver) cell line, it was found that some derivatives, in which the amino group on the pyrazole ring is linked to a phenyl group, were more active than their respective analogues with a 4methoxyphenyl group on that nitrogen atom. Thus, compounds 12c (IC 50 =0.66 nM) and 12e (IC 50 =0.09 nM) were found to be more potent than 12h (IC 50 =0.99 nM) and 12j (IC 50 =0.62 nM), respectively. However, compounds 4a and 4b were found to be of a comparable potency.
On the other hand, the investigation confirmed the prominent biological activity of the ferrocenyl moiety over other substituents, where among the tested Schiff bases, compound 12e was found to be the most potent against the HepG2 (liver) cell line. Progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F 254 (Merck), viewed by short-wavelength UV lamp detection. All evaporations were carried out under reduced pressure at 40 °C. -3-(arylamino)-1H-pyrazole-4-carboxamides (1a,b).

5-Amino
A mixture of cyanoacetamide (0.01 mol) and 4-(methoxyphenyl)isothiocyanate or phenylisothiocyanate (0.01 mol) was heated for 5-10 min in ethanol (25 mL) containing potassium hydroxide (0.01 mol). After cooling, methyl iodide (0.01 mol) was added. The reaction mixture was stirred at room temperature for 1 h then poured onto ice-water. The precipitated product [3-(4-arylamino)-2-cyano-3-(methylthio)acrylamide] was filtered off and recrystallized from ethanol, then its mixture with hydrazine hydrate (0.01 mol) was refluxed for 4 h in ethanol (30 mL) containing triethylamine as a catalyst. After evaporating the solvent under reduced pressure, the resulting solid product was collected by filtration and recrystallized from ethanol.

Biological experiments
In vitro antitumor screening The tested compounds were subjected to in vitro disease-oriented primary antitumor screening. The different cell lines of tumor cell lines were utilized. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96-well microtiter plates in 100 mL at plating densities ranging from 5000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37°C, 5% CO 2 , 95% air, and 100% relative humidity for 24 h prior to the addition of the experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition. Experimental drugs were solubilized in DMSO at 400-fold of the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 mg/ml Gentamicin. Additional four 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus the control. Aliquots of 100 mL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 mL of medium, resulting in the required final drug concentrations. Following drug addition, the plates were incubated for an additional 48 h at 37°C, 5% CO 2 , 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 mL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4°C. The supernatant layer was discarded, and the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air-dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 mL of 80% TCA (final concentration, 16% TCA). The parameter used here was GI 50, which is the log10 concentration at which PG is 50, and was calculated for each cell line [19][20][21].

Conclusion
In conclusion, 5-amino-1H-pyrazole-4-carboxamide derivatives 1a,b were used as starting materials for the synthesis of some new pyrazolo[1,5-a]pyrimidine derivatives and Schiff bases. The new synthesized compounds were characterized by analytical and spectroscopic data. Some selected new compounds were screened for their potential antitumor activities. The results of the cytotoxicity for the tested compounds against different human cancer cell lines indicated that most of them exhibit a high cytotoxicity at very low concentrations in comparison with the reference drugs used, and pyrazolo[1,5-a]pyrimidine derivatives 4a, 7e, and 7f were found to have the most potent growth inhibitory activity against MCF7, ovarial carcinoma (SK OV-3), leukemia (K562), and HeLa (cervical) human tumor cell lines. On the other hand, Schiff bases 12b-e and 12j were found to be the most potent against cervical carcinoma (KB), CNS cancer (SF-268), non-small cell lung cancer (NCl H460), colonadenocarcinoma (RKOP 27), anti-leukemia (HL60, U937), melanoma (SK-MEL-28), neuroblastoma (GOTO, NB-1), HT1080 (fibrosarcoma), and HepG2 (liver) human tumor cell lines.