Synthesis, In Vitro Antimicrobial and Cytotoxic Activities of Some New Pyrazolo[1,5-a]pyrimidine Derivatives

A new series of pyrazole 4–7 and pyrazolo[1,5-a]pyrimidine 8–13 were synthesized by using a simple, efficient procedure, and screened for their in-vitro antimicrobial and antitumor activities. Symmetrical and asymmetrical 3,6-diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine were synthesized by the conventional method and also subjected to microwave irradiation and under ultrasound conditions. The biological results revealed that most of the tested compounds proved to be active as antibacterial and antifungal agents. The antitumor activity of the synthesized compounds was evaluated against human cancer cell lines, MCF-7, HCT-116, and HepG-2, as compared with Doxorubicin as a control.


Introduction
Pyrazole, a heterocyclic five-membered ring system, is one of the most significant heterogeneous compounds. Pyrazole derivatives have been found to possess a broad spectrum of biological properties including antitumor activity [1,2]. Pyrazoles have attracted much attention from researchers due to their high antibacterial and antifungal values [3]. In addition, pyrazoles exhibit anti-inflammatory [4], anti-HIV [5], antiviral [6], anti-diabetic [7], and anti-tubercular activities [8]. It gained great attention since the privileged structure was commonly found as an active constituent in commercial drugs (Figure 1), such as lonazolac nonsteroidal anti-inflammatory drug (NSAID), pyrazofurin (anticancer), difenamizole (analgesic), and deracoxib (NSAID). A great deal of interest has been paid to pyrazole derivatives in the last few years in agrochemical and chemical industries [9], as some pyrazole derivatives have exhibited insecticidal [10] and herbicidal activities [11]. The great importance of pyrazole is due to its ability to construct fused pyrazole compounds that have been used as intermediates for the synthesis of dyestuffs mainly for heterocyclic azo pyrazole disperse dyes [12]. Also, it has gained considerable attention from researchers in the past few decades since it is a major component in some marketing drugs such as Ocinaplon and Lorediplon [12]. Based on the above facts, the purpose of this paper is to design and synthesize new compounds containing pyrazole moieties and study their antimicrobial activity.

Chemistry
2-Arylazomalononitriles 2a-c were synthesized by diazotization of aniline derivatives 1a-c followed by coupling with malononitrile according to the reported method [13] (Scheme 1). The structure of 2- [4-(trifluoromethyl) phenylazo]malononitrile (2b) was established by analytical and spectral data. The IR spectrum of (2b) revealed the presence of absorption bands at 3141 and 2227 cm −1 characteristic for NH and CN groups, respectively. The 1 H-NMR spectrum revealed two doublets at δ 7.64 and 7.76 ppm assignable to the aromatic protons and a broad signal at δ 10.36 ppm indicating the presence of the NH2 group, please find more detailed data in the supplementary materials.
Refluxing of arylazomalononitrile 2a with piperidine or morpholine in ethanol afforded the amino analogs 3a,b (Scheme 1). The structures for 3a,b were confirmed by spectral data. IR spectra of compounds 3a,b showed the appearance of NH at 3301-3303 cm −1 and CN stretch at 2179-2184 cm −1 . The 1 H and 13 C-NMR spectrum of compound 3b revealed the presence of two characteristic signals aliphatic groups N-(CH2)2 and O-(CH2)2 of morpholine moiety.

Chemistry
2-Arylazomalononitriles 2a-c were synthesized by diazotization of aniline derivatives 1a-c followed by coupling with malononitrile according to the reported method [13] (Scheme 1). The structure of 2- [4-(trifluoromethyl) phenylazo]malononitrile (2b) was established by analytical and spectral data. The IR spectrum of (2b) revealed the presence of absorption bands at 3141 and 2227 cm −1 characteristic for NH and CN groups, respectively. The 1 H-NMR spectrum revealed two doublets at δ 7.64 and 7.76 ppm assignable to the aromatic protons and a broad signal at δ 10.36 ppm indicating the presence of the NH 2 group, please find more detailed data in the Supplementary Materials.
Refluxing of arylazomalononitrile 2a with piperidine or morpholine in ethanol afforded the amino analogs 3a,b (Scheme 1). The structures for 3a,b were confirmed by spectral data. IR spectra of compounds 3a,b showed the appearance of NH at 3301-3303 cm −1 and CN stretch at 2179-2184 cm −1 . The 1 H and 13 C-NMR spectrum of compound 3b revealed the presence of two characteristic signals aliphatic groups N-(CH 2 ) 2 and O-(CH 2 ) 2 of morpholine moiety.

Chemistry
2-Arylazomalononitriles 2a-c were synthesized by diazotization of aniline derivatives 1a-c followed by coupling with malononitrile according to the reported method [13] (Scheme 1). The structure of 2-[4-(trifluoromethyl) phenylazo]malononitrile (2b) was established by analytical and spectral data. The IR spectrum of (2b) revealed the presence of absorption bands at 3141 and 2227 cm −1 characteristic for NH and CN groups, respectively. The 1 H-NMR spectrum revealed two doublets at δ 7.64 and 7.76 ppm assignable to the aromatic protons and a broad signal at δ 10.36 ppm indicating the presence of the NH2 group, please find more detailed data in the supplementary materials.
Acetylation of 3,5-diamino-1H-pyrazole derivatives 4a,b in refluxing acetic acid afforded the corresponding 4-arylazo-3,5-diacetamidopyrazole derivatives 7a,b; (Scheme 2). The structures of 1H-pyrazole derivatives 7a,b were supported by elemental and spectral data. The 1 H-NMR spectrum for compound 7a exhibited two singlet signals at δ 2.12 and 2.17 ppm corresponding to the presence of methyl protons and two broad signals at δ 6.56 and 10.27 ppm related to two (NH) protons, while its mass spectrum revealed a molecular ion peak at m/z 304 (M + , 100%).
3,5-Diaminopyrazoles 4 was employed as a key intermediate for the synthesis of several poly substituted fused pyrazolopyrimidines. Pyrazolo [1,5-a]pyrimidines are of considerable pharmacological important as purine analogs [15][16][17]. Thus, it was important to study the reactivity of diaminopyrazole derivatives 4a,b toward a variety of nucleophilic reagents. Symmetrical 3,6-diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine (8a,b) were synthesized by the reaction of hydrazone 2a,b [13,18]   Moreover, the synthesis of pyrazolopyrimidine derivatives 8a,b and 9a,b were achieved under microwave irradiation and ultrasound reactions conditions. TLC monitored the progress of the reactions, and the yields of the products were noted. Comparisons between conventional, microwave, and ultrasound techniques are given in (Table 1). The analytical and spectral data of the newly synthesized compounds 8a,b and 9a,b were compatible with their structures. For instance, IR spectrum for 6-[(4-trifluoromethyl)phenylazo]-3-(4fluorophenylazo)-2,5,7-triaminopyrazolo[1,5-a] pyrimidine (9a) showed absorption bands at 3474, 3276, and 3123 cm −1 , representing the presence of the amino groups, while 1 H-NMR spectrum showed signals at δ 6.98 and 8.22 ppm referring to two amino groups, and its mass spectrum showed a molecular ion peak at m/z 458 (M + , 100%), which corresponds with its molecular formula C19H14F4N10. Moreover, the synthesis of pyrazolopyrimidine derivatives 8a,b and 9a,b were achieved under microwave irradiation and ultrasound reactions conditions. TLC monitored the progress of the reactions, and the yields of the products were noted. Comparisons between conventional, microwave, and ultrasound techniques are given in (Table 1). The analytical and spectral data of the newly synthesized compounds 8a,b and 9a,b were compatible with their structures. For instance, IR spectrum for 6-[(4-trifluoromethyl)phenylazo]-3-(4fluorophenylazo)-2,5,7-triaminopyrazolo[1,5-a] pyrimidine (9a) showed absorption bands at 3474, 3276, and 3123 cm −1 , representing the presence of the amino groups, while 1 H-NMR spectrum showed signals at δ 6.98 and 8.22 ppm referring to two amino groups, and its mass spectrum showed a molecular ion peak at m/z 458 (M + , 100%), which corresponds with its molecular formula C 19 H 14 F 4 N 10 .
Sulfonamide group can be considered as the main component in some drugs used in clinical treatment.
It is reported that many of the sulfonamide derivatives act as antibacterial, antifungal, antihypertensive, anticancer, antimalarial, and antiprotozoal agents [13].
Thus, the reaction of 3,5-diaminopyrazole 4a,b with several hydrazones of sulfa drugs (N- [4- in ethanol under reflux in the presence of a catalytic amount of pyridine afforded pyrazolo[1,5-a]pyrimidine derivatives 10a,b-12a,b, respectively (Scheme 4). Aiming to modify the yields and reduce the reactions time, we applied microwave irradiation conditions to resynthesize the targeted compounds 10a,b-12a,b, and the comparison of the reactions times and yields between the conventional and microwave irradiation conditions are summarized in Table 2.
The structures of 10a,b-12a,b were confirmed by their elemental analysis and spectral data (MS, IR, 1 H-NMR, and 13 C-NMR). The IR spectrum revealed an absorption band at 3474-3207 cm −1 that corresponds to NH and NH 2 groups and at 2960-2835 and 1369-1347 cm −1 characteristic to aliphatic CH 2 and SO 2 groups, respectively. 1 H-NMR spectrum of (12b), as an example, revealed signals at δ 1.36, 1.55, and 2.91 which refer to the presence of (3CH 2 ) and (2NCH 2 ) groups, respectively, while its 13 C-NMR spectrum exhibited signals representing the presence of piperidine protons at δ 22.85, 24.66, and 46.58. Mass spectrum of (12b), as an example, gives the molecular ion peak at m/z 587 (M + ; 44%), corresponding to the molecular formula C 24 (13)

In Vitro Antimicrobial Evaluation
The newly synthesized compounds were evaluated for their antimicrobial activity toward six bacterial strains, three Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus mutants), and three Gram-negative (Enterococcus faecalis, Proteus vulgaris, and Escherichia coli), using the standard antibiotic Gentamycin (5 mg/mL) as a reference. Also, their antifungal activity was evaluated against three fungal strains (Aspergillus fumigates, Aspergillus flavus, and Candida albicans) to determine the zone of inhibition using the standard antibiotic Ketoconazole (5 mg/mL). Their antibacterial activities were tested for their activities at a concentration of (5 mg/mL) using inhibition zone diameter in mm as a criterion for the antimicrobial activity [19,20], and the results are shown in (Table 3).
Compounds 2a, 2c, and 11b demonstrated more potent significant activity against tested Enterococcus faecalis and Proteus vulgaris with a diameter of inhibition zone between 25 and 40 mm; further, compound 2a showed high antimicrobial activity on Bacillus subtilis with a zone of inhibition of 40 mm, and these compounds had moderate antimicrobial activity on other bacterial strains with an inhibition zone ranging from 13 to 25 mm. Moreover, compound 2a has significant antifungal activity towards three fungal strains, and compound 11b showed a moderate effect on fungal strains, while compound 2c was not active on fungal strains. Also, compound 4b had a moderate effect on bacterial strains with an inhibition zone of 20-24 mm diameter, as well as on fungal strains with an inhibitory region ranging from 13 to 20 mm.
Furthermore, compounds 5a, 7a, 7b, and 11a showed mild antibacterial activity on the tested Gram-positive and Gram-negative bacterial strains with an inhibition zone of 9 to 18 mm, except compound 5a was NA on Streptococcus mutants strain. Meanwhile, compounds 5a, 7b, and 11a had no antifungal activity towards the three fungal strains.

In Vitro Cytotoxic Screening
The antitumor activity of the target compounds was screened against three human cancer cell lines-breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and human colon carcinoma (HCT-116). The IC 50 values of the tested compounds are listed in Table 4 and Figure 2.
From the obtained results, compounds 2a,b, 4b, 5b, 8b, and 12b showed the most potent cytotoxic profile on cancer cells (MCF-7, HepG2, and HCT-116) with IC 50 values ranging from 0.3 to 3.4 µg while the compound 8b is most effective among all of them on all types of cancer cells.
Compounds 2c, 11b, and 13 have demonstrated a significant effect on MCF-7 and HePG2 cells with IC 50 values ranging from 2.7 to 9.8 µg, and 2c and 11b compounds significantly increased on HCT 116 with IC 50 of 1.2 µg and 2.4, respectively. Moreover, the compounds 4a, 8a, and 10a showed moderate effects on cancer cells with IC 50 values ranging from 6.7 to 12.9 µg, while the compound 8a was the most significant on HePG2 cells with IC 50 of 4.9 µg; on the other hand, the effect of the 4a compound was low on MCF-7 cells. In addition, the compounds 3a,b, 5a, 7a,b, 9b, 11a, and 12a had slightly significant effects with IC 50 values ranging from 14.1 to 38.5 µg, the compound 3a indicated a slightly noticeable effect on HePG2 cells with IC 50 of 6.1 µg, and the compound 12a had weak toxicity on HCT 116 cells with IC 50 of 46.1 µg. Furthermore, the 10b compound had a low cytotoxic effect on MCF-7 and HePG2 cells with IC 50 of 43.9 and 85.9 µg, respectively, and a moderate effect with IC 50 of 29.4 µg. Compound 9a showed very low cellular toxicity with IC 50 higher than 100 µg compared to all other compounds on cancer cells.

General Information
All melting points were determined with a Stuart Digital Melting Point apparatus SMP10 and are uncorrected. Elemental analyses were performed on a Perkin-Elmer 240 microanalyser, PE 2400 Series II CHNS/O Analyzer, carried out at the regional center for mycology and biotechnology, Al-Azhar University, cairo, Egypt. IR spectra were determined as KBr pellets on a Thermo Nicolet apparatus (Thermo Scientific, Madison, WI, USA) at Postgraduate campus for Girls at Lassan, King Khalid University, Abha, Saudi Arabia. The NMR spectra were recorded on a Bruker NMR spectrometer (Bruker, Billerica, MA, USA) in DMSO-d6, as solvent at 300, 500 MHz for 1 H-NMR and 75, 125 MHz for 13 C-NMR at Faculty of Science, Cairo University, Egypt and King Khalid University, Abha, respectively. The chemical shifts (δ) are reported in parts per million (ppm). Mass spectra were measured on GC/MS-QP5 spectrometer at regional center for mycology and biotechnology, Al-Azhar University, Egypt. Antimicrobial activity was measured at the regional center for mycology and biotechnology, Al-Azhar University, Egypt. Follow-up of the reactions and checking the purity of the compounds were carried out using TLC on silica gel-precoated aluminum sheets (Fluorescent indicator 254 nm, Fluka, Germany) and the spots were detected by exposure to UV lamp at λ 254/366 nm for a few seconds or under iodine vapor. Compounds 2-(4-fluorphenylazo)malononitrile 2a and 3,5-diamino-4-(4-fluorophenylazo)-1H-pyrazole 4b were prepared according to the reported procedure [21][22][23] To a solution of aniline derivatives 1a-c (0.01 mol) in hydrochloric acid (6 mL), a solution of sodium nitrite (0.72 g, 0.0105 mol) in water (3 mL) was added portion wise with stirring at 0-5 °C for 1 h. The clear diazonium salt was added to a stirred solution of malononitrile (0.66 g, 0.01 mol) and sodium acetate (4.6 g) in aqueous ethanol 50% (50 mL) with continued stirring at 0-5 °C for 2 h. The reaction mixture was allowed to stand overnight at room temperature, then the precipitate that formed was filtered off, washed several times with water, dried, and recrystallized from ethanol to afford 2-arylazomalononitrile 2a-c.

General Information
All melting points were determined with a Stuart Digital Melting Point apparatus SMP10 and are uncorrected. Elemental analyses were performed on a Perkin-Elmer 240 microanalyser, PE 2400 Series II CHNS/O Analyzer, carried out at the regional center for mycology and biotechnology, Al-Azhar University, cairo, Egypt. IR spectra were determined as KBr pellets on a Thermo Nicolet apparatus (Thermo Scientific, Madison, WI, USA) at Postgraduate campus for Girls at Lassan, King Khalid University, Abha, Saudi Arabia. The NMR spectra were recorded on a Bruker NMR spectrometer (Bruker, Billerica, MA, USA) in DMSO-d 6 , as solvent at 300, 500 MHz for 1 H-NMR and 75, 125 MHz for 13 C-NMR at Faculty of Science, Cairo University, Egypt and King Khalid University, Abha, respectively. The chemical shifts (δ) are reported in parts per million (ppm). Mass spectra were measured on GC/MS-QP5 spectrometer at regional center for mycology and biotechnology, Al-Azhar University, Egypt. Antimicrobial activity was measured at the regional center for mycology and biotechnology, Al-Azhar University, Egypt. Follow-up of the reactions and checking the purity of the compounds were carried out using TLC on silica gel-precoated aluminum sheets (Fluorescent indicator 254 nm, Fluka, Germany) and the spots were detected by exposure to UV lamp at λ 254/366 nm for a few seconds or under iodine vapor. Compounds 2-(4-fluorphenylazo)malononitrile 2a and 3,5-diamino-4-(4-fluorophenylazo)-1H-pyrazole 4b were prepared according to the reported procedure [21][22][23].

General Procedure for the Synthesis of 2-Arylazomalononitrile 2a-c:
To a solution of aniline derivatives 1a-c (0.01 mol) in hydrochloric acid (6 mL), a solution of sodium nitrite (0.72 g, 0.0105 mol) in water (3 mL) was added portion wise with stirring at 0-5 • C for 1 h. The clear diazonium salt was added to a stirred solution of malononitrile (0.66 g, 0.01 mol) and sodium acetate (4.6 g) in aqueous ethanol 50% (50 mL) with continued stirring at 0-5 • C for 2 h. The reaction mixture was allowed to stand overnight at room temperature, then the precipitate that formed was filtered off, washed several times with water, dried, and recrystallized from ethanol to afford 2-arylazomalononitrile 2a-c. To a solution of 2-arylazomalononitrile 2a,b (0.01 mol) in ethanol (30 mL), hydrazine hydrate (0.01 mol) and pyridine (0.5 mL) were added. The reaction mixture was heated under reflux for 2 h (monitored by TLC). After completion of the reaction, the precipitate that formed was filtered off, dried, and recrystallized from ethanol to afford 3,5-diamino-4-(arylazo)pyrazole 4a,b. A solution of 3,5-diamine-4-arylazo-1H-pyrazole 4a,b (0.01 mol) in acetic acid (30 mL) was heated under reflux for 10 h (monitored by TLC). The reaction mixture was left to cool to room temperature, and then poured onto cooled water (50 mL) portion wise with continuous stirring for 1 h. The separated yellow compound was filtered off, washed with water, and, finally, dried and recrystallized from ethanol to afford 3,5-diacetamio-4-arylazo-1H-pyrazole 7a,b. Method B: A mixture of 4-arylazo-3,5-diaminopyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile 2a,b (0.01 mol) in ethanol (30 mL) and in the presence of pyridine (0.5 mL) was heated under reflux for 4-5 h, (monitored by TLC). After completion of the reaction, the precipitates those formed were filtered off, dried, and recrystallized from 1,4-dioxane to afford 8a and 8b, respectively.

B. Under Microwave Irradiation:
A mixture of 5-diamino-4-arylazopyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile 2a,b (0.01 mol) was grinded carefully in a porcelain mortar using a pestle and transferred to a pyrex test tube; then, ethanol (4 mL) was added, followed by pyridine (0.5 mL). The reaction mixture was heated under microwave irradiation at 50% power for 2 min at 140 • C, monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and the precipitated solid was filtered off, washed with MeOH, and recrystallized from dioxane to afford 8a and 8b, respectively.

B. Under Microwave Irradiation:
A mixture of 5-diamino-4-(arylazo)pyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile 2a,b (0.01 mol) was grinded carefully in a porcelain mortar using a pestle and transferred to a pyrex test tube, and ethanol (4 mL) was added, followed by pyridine (0.5 mL). The reaction mixture was heated under microwave irradiation at 50% power for 2 min at 140 • C, monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and the precipitated solid was filtered off, washed with MeOH, and recrystallized from ethanol to afford 9a,b.
Cytotoxicity assessment: The cytotoxicity of different compounds was tested against human tumor cells using Sulphorhodamine B assay (SRB). Healthy growing cells were cultured in a 96-well tissue culture plate (3000 cells/well) for 24 h before treatment with the synthesized compounds to allow attachment of the cells to the plate. Cells were exposed to the five different concentrations of tested compounds (0.01, 0.1, 1, 10, and 100 µM/mL); untreated cells (control) were added. Triplicate wells were incubated with the different concentrations for 72 h and subsequently fixed with TCA (10% w/v) for 1 h at 4 • C. After several washing, cells were stained by 0.4% (w/v) SRB solution for 10 min in a dark place. Excess stain was washed with 1% (v/v) glacial acetic acid. After drying overnight, the SRB-stained cells were dissolved with tris-HCl and the color intensity was measured in a microplate reader at 540 nm. The linear relation between viability percentage of each tumor cell line and extracts concentrations were analyzed to get the IC 50 (dose of the drug which reduces survival to 50%) using Sigma Plot 12.0 software [25].

Conclusions
In conclusion, 4-arylazo-3,5-diaminopyrazole 4-6 was synthesized and used as a building block for the synthesis of pyrazolopyrimidine derivatives such as 3,6-diarylazo-2,5,7-triaminopyrazolo[1,5a]pyrimidine, 8 and 9, 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine 10-12 and 13. The structures of newly synthesized compounds were confirmed by spectral data (IR, 1 H-NMR, 13 C-NMR, and mass spectra) and elemental analysis. Most of the newly synthesized compounds were assessed as antimicrobial agents against a number of Gram-positive, Gram-negative, and fungal strains. Some of the tested compounds showed excellent antimicrobial activity compared to the standard antibiotics. The obtained data reflected that compound 2b exhibited the best antimicrobial activity against most tested microorganisms and the rest of the compounds were moderately active or inactive for all the microorganisms. Most of the synthesized compounds exhibited good cytotoxic activity towards the examined three cell lines. From the obtained results, compounds 2a,b, 4b, 5b, 8b, and 12b showed the most potent cytotoxic profile on cancer cells (MCF-7, HepG2, and HCT-116) with IC 50 values ranging from 0.3 to 3.4 µg, while the compound 8b is most effective among all of them on all types of cancer cells.