Introduction

The important tasks of the researchers in the medical, pharmaceutical, and chemical fields are the design and preparation of bioactive compounds to overcome some health problems such as cancer and bacterial infectious diseases. Therefore, there is a growing interest in the preparation of pyrazolo[1,5-a]pyrimidines derivatives due to their biological applications as promising antimicrobial, anticancer, antitubercular, and enzyme inhibitors [1,2,3,4,5,6]. For example, pyrazolo[1,5-a]pyrimidine-3-carbonitrile derivative 1 was synthesized by Attia et al., as a promising anti-proliferative agent against the MCF-7 cell line [7]. The two water-soluble pyrazolo[1,5-a]pyrimidine derivatives (2 and 3) were synthesized and characterized with antibacterial activity against Escherichia coli [8]. Also, from our previous studies, the two pyrazolo[1,5-a]pyrimidine derivatives {5,7-dimethylpyrazolo[1,5-a]pyrimidine derivative 4 and 7-phenylpyrazolo[1,5-a]pyrimidine derivative 5} were prepared and displayed a promising antimicrobial agent [9] and antitumor agent against HepG-2 cells [10], respectively (Fig. 1). Moreover, the pyrazolopyrimidine structure is present in drugs such as Dinaciclib, Anagliptin, and Ocinaplon [11].

Fig. 1
figure 1

Biological applications of pyrazolopyrimidines 1–5 and pyrazole Schiff bases 6–8

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Besides, pyrazole Schiff bases (pyrazole-azomethine) are known to have a vast spectrum of biological activities such as antimicrobial, antitumor, immunomodulatory, analgesic, DNA binding, and DHFR/DNA gyrase inhibitors [12,13,14,15,16,17,18]. In 2013, Malladi and co-workers have described a synthesis of compound triazole-pyrazole Schiff base 6 which exhibited promising antibacterial activity against Staphylococcus aureus [19]. Recently, in 2020, Hassan et al., prepared two furan-pyrazole Schiff bases 7 and 8 as Nitrofurantoin® analogues which exhibited antibacterial activities against Escherichia coli and Salmonella typhimurium bacteria [20] (Fig. 1).

Because of the biological importance of pyrazolo[1,5-a]pyrimidines, pyrazole Schiff bases, and in continuation of our program for synthesis bioactive compounds [21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40]. In this work, we have synthesized 5-amino-N-aryl-1H-pyrazole-4-carboxamides 9a, b [41] as starting materials for the preparation of new 5-aryl-7-amino-pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j for evaluation of their antimicrobial properties as well as the structure–activity relationship (SAR), the molecular docking, the electrostatic potential maps (MEP), and in silico properties prediction were studied (Fig. 2).

Fig. 2
figure 2

The target compounds (14a–j and 16a–j) and their studies

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Result and discussion

Chemistry

The target compounds, pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j, are shown in Schemes 1 and 2. The starting materials, 5-amino-pyrazole derivatives 9a, b and 2-(arylidene)malononitriles 10a–e, were prepared according to previously reported methods [42, 43] and are described in Scheme 3.

Scheme 1
scheme 1

Synthesis of pyrazolo[1,5-a]pyrimidines 14a–j

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Scheme 2
scheme 2

Synthesis of Schiff bases 16a–j

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Scheme 3
scheme 3

Synthesis of starting materials (9a, b and 10a–e)

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A series of 7-amino-pyrazolo[1,5-a]pyrimidines 14a–j were prepared via the reaction of 5-amino-N-aryl-1H-pyrazole-4-carboxamides 9a, b with 2-(arylidene)malononitriles 10a–e in refluxing ethanol (Scheme 1). This reaction involves nucleophilic addition of amino group of 9a, b to double bond of 2-(arylidene)malononitriles 10a–e to produce the intermediate 5-((2,2-dicyano-1-phenylethyl)amino)-1H-pyrazole derivative 11a–j. Furthermore, the cyclic imino group in intermediate 1H-pyrazole derivative 11a–j acts as nucleophile to cyano group to obtain 7-imino-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivatives 12a–j that underwent proton shift and oxidation to afford the corresponding 7-amino-pyrazolo[1,5-a]pyrimidine derivatives 14a–j.

The structure of 7-amino-pyrazolo[1,5-a]pyrimidines structures 14a–j was confirmed by spectral data and elemental analysis. The IR spectrum of 7-amino-pyrazolo[1,5-a]pyrimidine derivative 14a was characterized by appearing new band for C≡N group at ν 2213 cm−1 and two bands at ν 3428 and 3304 cm−1 for NH2 and 2NH groups. Also, the C=O amide group appeared at ν 1641 cm−1. The mass spectrum of pyrazolo[1,5-a]pyrimidine derivative 14a was exhibited a molecular ion pack at m/z 489 with intensity 17.65% corresponding to the formula C28H23N7O2 (489.53) and the base peak at m/z 396 (100%). The 1H NMR (δ/ppm) spectrum of 14a exhibited two signals at δ 2.43 and 3.74 ppm assigned to the protons of CH3 and OCH3 groups, respectively. Besides, the NH2 group appeared as a broad signal at δ 9.00 ppm, and two exchangeable singlet signals at δ 9.23 and 10.14 ppm corresponding to two NH groups.

The three aromatic rings (Figure S1, Supplementary Material) were interpreted as the following:

  1. (i)

    Phenyl ring (five protons) appeared as two triplets at δ 7.09 ppm (1H, J = 7.5 Hz) and δ 7.37 ppm (2H, J = 7.8 Hz) and one doublet at δ 7.60 ppm (2H, J = 7.7 Hz).

  2. (ii)

    4-Methoxyphenyl ring (4 protons, AB-system) appeared as two doublets at δ 6.90 (2H) and 7.84 (2H) ppm (each with J (coupling constant) = 9.0 Hz).

  3. (iii)

    4-Methylphenyl ring (4 protons, AB-system) appeared as two doublets at δ 7.44 (2H) and 7.90 (2H) ppm (each with J (coupling constant) = 8.2 Hz).

Additionally, the 13C NMR spectrum of 14a exhibited characterized signals of CH3 and –OCH3 carbon at δ 21.19 and 55.26 ppm, respectively. The rest of carbon atoms (26 carbon) of 7-amino-pyrazolo[1,5-a]pyrimidine derivative 14a have appeared ranged between δ 74.88–145.70 ppm as well as signals at δ 149.49, 153.98, 156.32, 161.47, and 162.29 ppm corresponding to two C=N, carbonyl group, carbon attached to methoxy (C–OMe), and carbon attached to an amino group (C-NH2), respectively.

Moreover, the 5-(2,5-dimethoxyphenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14j was another example of the 1H NMR spectra, exhibited three singlet signals at δ 3.70, 3.75, and 3.78 ppm for the three methoxy groups. The aromatics protons appeared at δ 6.86 (d, 2H, J = 9.5 Hz, p-methoxyphenyl ring), 7.10–7.17 (m, 3H, 2, 5-dimethoxyphenyl ring), 7.34 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), 7.48 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), and 7.78 (d, 2H, J = 9.3 Hz, p-methoxyphenyl ring). Further, three exchangeable singlet signals appeared as the broad peak at δ 8.97 ppm, and two signals at δ 9.11 and 10.04 ppm refer to the protons of the amino group (NH2) as well as two NH groups, respectively. The 13C NMR spectrum of 14j exhibited characterized signals of three methoxy carbons at δ 55.12, 55.68, and 55.93 ppm. The rest of carbon atoms (26 carbon) of 14j have appeared at δ 78.02, 88.84, 104.95, 113.21, 114.13, 115.17, 117.23, 118.96, 120.38, 126.91, 128.90, 133.25, 137.20, 145.72, 148.51, 150.65, 153.07, 153.91, 156.07, 159.97, 159.99, and 162.09 ppm. Also, the structure of 7-amino-pyrazolo[1,5-a]pyrimidine derivatives 14a–j and reaction mechanism pathway was proved from our previous study through X-ray single-crystal analysis of analogues [44].

Schiff bases 16a–j were prepared via the reaction of 5-amino-pyrazoles 9a, b with various aldehydes 15a–e in refluxing absolute ethanol (Scheme 2). Schiff bases structure 16a–j was confirmed by spectral analysis. The IR spectrum of azomethine derivative 16d displayed bands at ν 3431, 3235, 1635, and 1615 cm−1 characteristic for 3NH, carbonyl (C=O), and imino (C=N) groups, respectively. The 1H NMR (δ/ppm) spectrum of 5-(4-chloro-3-nitrobenzylideneamino)-1H-pyrazole-4-carboxamide derivative 16d exhibited one signal at δ 3.70 ppm for the methoxy group. The aromatic protons have appeared at δ 6.89 (d, 2H, J = 8.3 Hz, p-methoxyphenyl ring), 7.06 (t, 1H, J = 7.3 Hz, phenyl ring), 7.31–7.34 (m, 4H, phenyl ring), 7.67 (d, 2H, J = 7.9 Hz, p-methoxyphenyl ring), 8.00 (d, 1H, J = 8.3, 4-chloro-3-nitrophenyl ring), 8.30 (d, 1H, J = 8.3, 4-chloro-3-nitrophenyl ring), and 8.67 (s, 1H, 4-chloro-3-nitrophenyl ring). The methine proton (–CH=N–) has appeared at δ 8.76 ppm. Besides, the three exchangeable signals appeared at δ 9.13, 9.79, and 12.80 ppm related to three NH protons.

Additionally, the IR spectrum of pyrazole Schiff base derivative 16f was characterized by bands for 3NH, C=O, C=N, and C=C groups at ν 3434, 3229, 1656, 1612, and 1587 cm−1, respectively. The 1H NMR (δ/ppm) spectrum of pyrazole derivative 16f exhibited two signals at δ 3.73 and 3.87 ppm for the protons of two methoxy groups. Besides, the proton of –CH=N– function appeared at δ 8.66 ppm and the protons of the three NH appeared as single signals at δ 9.03, 10.02, and 12.78 ppm. The spectrum has exhibited the protons of the three aromatic rings (Figure S2, Supplementary Material) as.

  1. (i)

    Two doublets appeared at δ 6.90 (2H, J = 8.9 Hz) and 7.69 (2H, J = 8.9 Hz) for the p-methoxyphenyl ring.

  2. (ii)

    One doublet at δ 7.40 (4H) for the p-chlorophenyl ring with J = 8.9 Hz.

  3. (iii)

    m-Methoxyphenyl ring; one proton as a doublet–doublet (dd) at δ 7.23 (J = 8.2 & 1.8 Hz), one proton as a triplet at δ 7.53 (J = 7.9 Hz), single signal at δ 7.59 for one proton, and one doublet for one proton at δ 7.62 (J = 2.4 Hz).

The 13C NMR spectrum of 16f exhibited characterized signals of two methoxy carbons at δ 55.19 and 55.23 ppm. The rest of 23 carbon atoms appeared at δ 92.61, 112.24, 114.39, 119.22, 119.53, 120.41, 122.48, 126.69, 128.34, 128.73, 130.28, 133.79, 136.17, 137.46, 151.21, 154.04, 159.74, 162.03, and 162.65 ppm.

Antimicrobial evaluation

Pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j were evaluated in vitro for their antimicrobial activity against four microbes using the agar diffusion method [45, 46], and the results are listed in Table 1 and represented in Fig. 3.

Table 1 Antimicrobial activities represented by the mean of Inhibition Zone (IZ) in (mm) of pyrazolo[1,5-a]pyrimidines 14a–j, pyrazole Schiff bases 16a–j, and standard antibiotics using the agar diffusion method
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Fig. 3
figure 3

Showed the antimicrobial activities (Inhibition Zone (IZ) in mm) of pyrazolo[1,5-a]pyrimidines 14a–j, pyrazole Schiff bases 16a–j, and standard antibiotics a bacteria, b fungi

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From the results obtained in Table 1 and Fig. 3, it was found that most of the pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j have a reasonable antimicrobial undertaking impact against most of the tested pathogens in this study. Besides, most of the pyrazolopyrimidines 14a–j and pyrazole Schiff bases 16a–j confirmed a diverse inhibitory effect against all pathogenic microorganisms used.

In the case of Bacillus subtilis (G+) bacteria: the three pyrazolopyrimidines [14b, 14e (inhibition zone (IZ) = 20 mm), and 14j (IZ = 21 mm)] and the pyrazole Schiff base 16i (IZ = 19 mm) confirmed a strong inhibition effect more than the antibacterial standard used (Tetracycline, IZ = 18 mm). Furthermore, pyrazolopyrimidine 14d and the pyrazole Schiff bases 16a (IZ = 18 mm) showed comparable antibacterial activity of the Tetracycline drug (IZ = 18 mm). Both the pyrazolopyrimidine derivatives (14c and 14g–i) and pyrazole Schiff bases (16c–h and 16j) showed a good inhibition with range = 15–17 mm.

In the case of Escherichia coli (G−) bacteria: all the pyrazolo[1,5-a] pyrimidines 14a–j and pyrazole Schiff bases 16a–j (IZ = 13 to 16 mm) presented from good to an excellent inhibition compared to the Novobiocin (IZ = 16 mm).

Aspergillus niger (Filamentous fungi): Pyrazolopyrimidine 14f and four pyrazole Schiff bases (16c, 16d, 16h, and 16i) revealed IZ from (18 mm) to (20 mm) and confirmed a robust inhibition impact, while, four pyrazolopyrimidines (14d, 14e, 14g, and 14i) and three Schiff bases (16a, 16e, and 16j) with IZ = 15 mm showed a reasonable inhibitory activity compared to Clotrimazole (IZ = 23 mm) as a positive control. It is worth mentioning that Aspergillus niger is one of the fungal pathogens that can affect the respiratory tract. A. niger is a causative agent of pulmonary diseases inclusive of aspergillosis, bronchial asthma, and acute allergic alveolitis. The fungus colonizes ancient tuberculosis or bronchi static cavities, in which its varieties a large colony (aspergilloma); or it can also without a doubt invade the lung tissue to produce hemorrhagic and necrotizing pneumonia [47].

In the case of Candida albicans (Yeast): the pyrazolopyrimidines (14b, 14d, and 14f) and Schiff base 16i (IZ rang = 17–20 mm) showed a good inhibition effect. Moreover, the pyrazolopyrimidines (14c, 14e, 14g, and 14i) and Schiff bases (16b, 16c, 16d, 16e, 16g, and 16h) showed a moderate inhibition with inhibition zone (IZ) = 15 mm. Further, the pyrazolopyrimidine 14a (IZ = 13 mm) and two Schiff bases 16a (IZ = 13 mm) and 16j (IZ = 12 mm) showed a weak effect in contrast to the antibacterial standard antibiotic Cyclohexamide (IZ = 32 mm).

From the above, the three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and four pyrazole Schiff bases (16c, 16d, 16h, and 16i) proved to be potent against the four microbes tested, possessed a broad spectrum of activity, and in several cases, their activities exceeded the reference drugs used.

Structure activity relationship study

Depending on the structure represented on Schemes 1, 3, and Fig. 4, our synthesis strategy depends on the preparation of two 2-cyano-N-(aryl)acetamide, and the aryl represented as phenyl or 4-chlorophenyl. The cyanoacetanilide derivatives reacted with 4-methoxyphenyl isothiocyante and finally refluxed with hydrazine hydrate to produce 5-amino-pyrazole derivatives 9a, b with two variable aryl groups. The 5-amino-pyrazole derivatives 9a, b obtained used as key starting materials to form pyrazolo[1,5-a]pyrimidine derivatives 14a–j and azomethine pyrazole derivatives 16a–j. Generally, most of the target compounds 14a–j and 16a–j showed moderate to favorable activity against the tested pathogens.

Fig. 4
figure 4

Illustrate the structure of different substituent in both pyrazolo[1,5-a]pyrimidine derivatives 14a–j and pyrazole-azomethine derivatives 16a–j

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Firstly, the 7-amino-6-cyano-2-((4-methoxyphenyl)amino)-N-phenyl-5-(aryl)pyrazolo[1,5-a]pyrimidine-3-carboxamide 14a–e displayed good antibacterial results, moderate antifungal activity, and the activity depends on the aryl group at position five and aromatic amide at position three of pyrazolo[1,5-a]pyrimidine derivatives 14. The presence of a dimethoxy group at positions two and five in the aryl group and meta methyl group in pyrazolo[1,5-a]pyrimidine 14e & 14b exhibited antibacterial activity with IZ ranged between 16–20 mm compared with Tetracycline 18 mm and Novobiocin 16 mm. Additionally, these two derivatives displayed moderate antifungal activity with IZs ranged between 15–17 mm in comparison with Clotrimazole (IZ = 23 mm) and Cycloheximide (IZ = 32 mm). Further, replacement N-phenyl by N-(4-chlorophenyl) exhibited equipotent in case of 5-(2,5-di-CH3O-C6H3)pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14j with IZs ranged between 16–21 mm, while 5-(3-CH3-C6H4)pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14g revealed slight decrease with IZs 16 mm, and 15 mm against B. subtilis and E. Coli, respectively. Additionally, it was found that the phenyl ring in pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14d had higher activity than the corresponding member incorporating chloro group as N-(4-chlorophenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14j against B. subtilis strain. Moreover, replacement N-phenyl by N-(4-chlorophenyl) of 3-carboxamide group in pyrazolo[1,5-a]pyrimidine derivatives 14h and 14j demonstrated no activity against fungal strains. Besides, addition of the electron-withdrawn group as chlorine atom in pyrazolo[1,5-a]pyrimidine-3-carboxamide derivatives 14f and 14g displayed increased the activity with IZ values from zero to 18 and 15 mm, respectively.

Similarly, the azomethine of 5-amino-pyrazole derivatives 16a–j showed variation in activity related to the different substituents in both two-aryl groups at positions three and five in pyrazole derivatives 16a–j. Regarding the series of 5-((substituted benzylidene)amino)-1H-pyrazole-4-carboxamide derivatives 16a–j, changing N-(phenyl) or N-(4-chlorophenyl) has little effect on antimicrobial activity. Furthermore, introducing more electron-withdrawing group as N-(4-chlorophenyl) to pyrazole derivatives 16a–j increase activity against B. subtilis from 12–18 mm for pyrazoles 16a–e to 15–19 mm for pyrazoles 16f–j. Additionally, changing of 5-((substituted benzylidene)amino) by heterocyclic aldehyde as 5-((thiophen-2-ylmethylene)amino) in 1H-pyrazole-4-carboxamide derivatives 16a–d and 16f–i not cause significant enhancement in antimicrobial activity. The most active azomethine pyrazole derivatives 16a–j among the synthesized derivatives that having 5-((4-chloro-3-nitrobenzylidene)amino) and N-(4-chlorophenyl)-4-carboxamide derivative is pyrazole derivative 16i demonstrated antimicrobial activity IZs ranging between 15–19 mm and antifungal activity IZs 20 mm. Unfortunately, the pyrazole derivatives 16b, 16f, and 16g are inactive against A. Niger except for moderate activity for other pyrazole derivatives. While the pyrazole derivative 16f showed no activity against C. albicans.

Finally, in the general presence of the chloro atom in amide as N-(4-chlorophenyl) in position three in pyrazolo[1,5-a]pyrimidine derivatives 14a–j and position four in pyrazole derivatives 16a–j causes increases the activity with significant results against B. subtilis, while in the case of E. Coli nearly results were obtained. Besides, N-(phenyl)-1H-pyrazolo[1,5-a]pyrimidin-3-carboxamide derivatives 14a–j and N-(phenyl)-1H-pyrazole-4-carboxamide derivatives 16a–j revealed abroad antifungal activity. Moreover, it can be concluded that the pyrazolo[1,5-a]pyrimidine derivatives 14a–j more sensitive to antibacterial activity while 5-((substituted benzylidene)amino)-1H-pyrazole-4-carboxamide derivatives 16a–j displayed better antifungal activity.

In silico studies

Physicochemical properties and drug-likeness model score

The physicochemical properties and drug-likeness (Lipinski and Veber rules) of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) were predicted by using the SwissADME web (http://swissadme.ch/index.php#undefined) [48, 49]. Also, the drug-likeness model score was predicted by using the Molsoft web (https://www.molsoft.com/mprop/) [50]. The predicted results are listed in Table 2.

Table 2 Physicochemical properties and drug-likeness model score of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and pyrazole Schiff bases (16c, 16d, 16h, and 16i)
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From Table 2, the pyrazolo[1,5-a]pyrimidine 14b and Schiff bases (16c, 16d, 16h, and 16i) agreement with Lipinski's rule of five and with Veber’s rule. While the two pyrazolo[1,5-a]pyrimidines (14e and 14j) do not match with each of Lipinski's rule of five and Veber’s rule by the violation in molecular weight and TPSA.

The rule for the drug-likeness model score estimation is based on positive and negative values, when the drug-likeness score was positive value; the compound was like a drug. But, if the drug-likeness score was negative value; the compound was non-drug [51].

Accordingly, all the tested compounds except 16c showed positive values in the range from 0.05 to 0.44; therefore, these compounds may be considered to be drug-like. Figure 5 represents the drug-likeness model score of the two pyrazolo[1,5-a]pyrimidines 14b and 14j.

Fig. 5
figure 5

Displayed the drug-likeness model score plotting of two pyrazolo[1,5-a]pyrimidines 14b and 14j. The green curve represents non-drug, and the blue curve represents drug

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The bioactivity scores prediction

The bioactivity scores of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) toward G protein-coupled receptor (GPCR) ligand, ion channel modulator, a kinase inhibitor, nuclear receptor ligand, protease inhibitors, and enzyme inhibitor were predicted by using the Molinspiration bioactivity score v2018.03 web (https://www.molinspiration.com/cgi-bin/properties) [52]. The predicted results are written in Table 3.

Table 3 The bioactivity scores prediction of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and pyrazole Schiff bases (16c, 16d, 16h, and 16i)
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The rule for the bioactivity scores estimation is the following: when the bioactivity score was more than 0.00; the compound was considered active. While if the bioactivity score in the range between − 0.50 and 0.00; the compound was moderately active. But if the bioactivity score was less than − 0.50, the compound was inactive.

The following observations are concluded from Table 3:

  • G protein-coupled receptor (GPCR) ligand: all the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) were found to be moderately active with the bioactivity scores in the range from − 0.10 to − 0.44.

  • Ion channel modulator: the two pyrazolo[1,5-a]pyrimidines 14b and 14e were found to be moderately active with the bioactivity scores − 0.32 and − 0.44, respectively, while the other compounds were found to be inactive.

  • Kinase inhibitor: the three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and the two Schiff bases (16c and 16h) were found to be active with bioactivity scores in the range from 0.06 to 0.045. While the two Schiff bases (16d and 16i) were found to be moderately active with the bioactivity scores − 0.02 and − 0.03, respectively.

  • Nuclear receptor inhibitor: all the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) were found to be inactive with the bioactivity scores less than − 0.50 (the range from − 0.59 to − 0.77).

  • Protease inhibitor: the three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and the two Schiff bases (16c and 16h) were found to be moderately active (range from − 0.37 to − 0.42). But, the two Schiff bases (16d and 16i) were found to be inactive with the bioactivity scores -0.56 and -0.55, respectively.

  • Enzyme inhibitor: all the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) were found to be moderately active (range from − 0.23 to − 0.38).

From the above, the pyrazolo[1,5-a]pyrimidine 14b almost shows the highest bioactivity score compared to the other compounds.

ADME and toxicity properties prediction

ADME properties and toxicity properties prediction of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) were predicted by using the Pre-ADMET web (https://preadmet.bmdrc.kr/) [53]. The predicted results are written in Table 4.

Table 4 ADME and toxicity properties prediction of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and pyrazole Schiff bases (16c, 16d, 16h, and 16i)
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The following observations are concluded from Table 4:

  • Cytochrome P450 isoforms determine the pharmacokinetic properties, bioactivation, and safety persistence of drugs. In the case of all the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schiff bases (16c, 16d, 16h, and 16i) are an inhibitor of the two enzymes (CYP2C9 and CYP3A4). But all the compounds are non-inhibitor of the two enzymes (CYP2C19 and CYP2D6).

  • P-glycoprotein (P-gp) has two functions the first is a barrier for delivering drugs properly and it is responsible for extruding toxins. All the compounds (14b, 14e, 14j, 16c, 16d, 16h, and 16i) were found to be effective.

  • The rule for the plasma protein binding (PPB) estimation is the following: when the value was less than 90%; the compound was low activity. While if the value was more than or equal to 90%; the compound was high activity. The two pyrazolo[1,5-a]pyrimidines (14b and 14e) and two Schiff bases (16d and 16i) had plasma protein binding (PPB) more than 90% (range from 91.17 to 98.23%, highly active), which affects the excretion of the drugs and its efficiency.

  • Ames test (mutagenicity), the three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and three Schiff bases (16c, 16d, and 16h) were predicted to be mutagens.

  • Carcinogenicity (mouse) test and the two Schiff bases (16d and 16i) were predicted as positive, while the three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and the two Schiff bases (16c and 16h) presented negatively.

  • Carcinogenicity (rat) test, the pyrazolo[1,5-a]pyrimidine 14j, and the three Schiff bases (16d, 16h, and 16i) were predicted as negative.

  • In the case of the hERG inhibition, the two compounds 14e and 16c showed high risk and the three compounds 14j, 16h, and 16i presented a medium risk. While compounds 14b and 16d presented an ambiguous risk.

From the above, all compounds are an inhibitor of the two cytochrome P450 isoforms (CYP2C9 and CYP3A4 enzymes) and P-glycoprotein (P-gp). Besides, some compounds showed highly active toward plasma protein binding (PPB), were predicted as negative against Carcinogenicity (mouse and rat) test, and presented medium risk in the case of the hERG inhibition.

Computational study of the binding mode

One of the most important tools in structure-based drug discovery is molecular docking simulation. It can describe newly designed derivatives' behavior in the binding site of target proteins [54]. Docking study plays an important role and considers one of the most applied simulated screening methods due to its ability to predict and get insights into a protein–ligand complex structure, which is useful information for the successful development of drug targeting and lead optimization [55]. The preferring structure selected from one to another in the docking study related to the highest binding affinity between the ligand and the protein [56]. Our study involved simulation of the most active derivatives inside both DNA gyrase for bacterial and secreted aspartic protease for fungal promising derivatives using Molecular Operating Environmental (MOE) software version 2008.10. The docking result is represented in Table 5 and Fig. 6a–d.

Table 5 Molecular modeling study of the most promising derivatives inside the active sites of S. aureus DNA gyrase (PDB: 2XCT) and secreted aspartic protease from c. albicans (PDB: IZAP)
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Fig. 6
figure 6figure 6

a 2D and 3D binding mode of pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14j inside the active site of S. auras DNA gyrase (PDB: 2XCT). b 2D and 3D binding mode of pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14b inside the active site of S. auras DNA gyrase (PDB: 2XCT). c 2D and 3D binding mode of 1H-pyrazole-4-carboxamide derivative 16i inside the active site of secreted aspartic protease (Saps) (PDB: 1ZAP). d 2D and 3D binding mode of 1H-pyrazole-4-carboxamide derivative 16c inside the active site of secreted aspartic protease (Saps) (PDB: 1ZAP)

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Study the most active derivatives against bacterial strains inside DNA gyrase

The most active derivatives 14b, 14e, 14j, and 16i against Bacillus subtilis and Escherichia coli were docked inside the active site of S. auras DNA gyrase (PDB: 2XCT) [57, 58]. The co-crystallized ligand (Ciprofloxacin) redocked inside S. aureus Gyrase's active site revealed binding energy S = − 9.87 kcal mol−1. The ligand formed one hydrogen bond donor between Tyr580 and nitrogen of piperazine ring as well as hydrogen bond acceptor between His1081 and oxygen of carboxylate group with bond length 2.35, 2.30 Å, respectively. The most promising pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14j with inhibition zone (IZ) ranged between 16-21 mm showed binding energy S = − 25.35 kcal mol−1, through two hydrogen bond and one arene–cation interaction between His1061 and pyrazole ring. The residue Asp512 formed a side-chain hydrogen bond donor with the amino group of pyrimidine as well as a side-chain hydrogen bond acceptor between Ser1028 and oxygen of methoxy group with bond length and strength 2.83  Å (11%), 2.99  Å (38%), respectively Fig. 6a.

Furthermore, N-phenyl-5-(3-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14b exhibited binding affinity S = − 23.05 kcal mol−1, through two hydrogen bonds from a side-chain. These two bonds created between the Ser1028 with the cyano in pyrimidine ring (H-acceptor), Ser1028 with the amino of pyrimidine (H-donor) with bond length 2.89  Å, and 3.05 Å. Besides, arene–cation interaction between His1061 and pyrazole ring Fig. 6b. Similarly, N-phenyl-5-(2,5-dimethoxyphenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14e displayed binding energy S = − 22.76 kcal mol−1, by one hydrogen bond side-chain acceptor between Ser1028 and cyano group attached to pyrimidine ring as well as one arene–cation interaction between His1061 and pyrazole ring. Moreover, N-(4-chlorophenyl)-1H-pyrazole-4-carboxamide 16i displayed one hydrogen bond donor between Asp510 and NH of pyrazole ring with bond length 2.76  Å and strength 45%.

Finally, the active derivatives 14b, 14e, 14j, and 16i showed a good binding affinity inside the pocket sites of DNA gyrase ranged between − 25.35 to − 20.18 kcal mol−1 with different binding modes and therefore suggested that these derivatives act as gyrase inhibitors.

Study the most active derivatives against fungal strains inside secreted aspartic protease

As new possible antifungal drug goals, secreted aspartic protease (Saps) obviously hold promise. Saps are important virulence factors because it includes a wide variety of physiological processes of fungal as well as the various aspects of the interactions between host and fungal [59]. Similarly, the most active four pyrazole derivatives 16c, 16d, 16h, and 16i against A. niger and C. albicans were docked inside the active site of secreted aspartic protease (Saps) (PDB: 1ZAP) [58]. The co-crystallized ligand (L-Norleucinamide derivative) showed binding energy S = − 30.98 kcal mol−1 through two hydrogen bond side-chain donor between the residue Thr221 and NH of amide derivative as well as Asp218 with hydroxyl group with bond length 3.05 Å (22%) and 2.31 Å (23%), respectively. The most promising derivative against fungal strains 1H-pyrazole-4-carboxamide derivative 16i exhibited binding energy S = − 28.03 kcal mol−1 through one side-chain hydrogen bond acceptor. The pyrazole derivative 16i indicated that a hydrogen bond was formed between the oxygen of the methoxy group and the residue Asn301 (distance 3.18 Å and strength 13%). Meanwhile, an arene–arene interaction was observed between the 4-chlorobenzamide derivative and Tyr84 (Fig. 6c). (All docking study figures in supplementary material, Figures S3-S22).

Additionally, the NH of pyrazole and NH of anisidine derivatives in pyrazole 16d could form two hydrogen bonds donor between Asp218 and Gly34, respectively. Besides, the aryl group of aldehyde derivative created an arene–cation interaction with Tyr225. Further, the docking pose of 5-((2,5-dimethoxybenzylidene)amino)-3-((4-methoxyphenyl)amino)-N-(4-chlorophenyl)-1H-pyrazole-4-carboxamide (16h) demonstrated three favorable hydrogen bonds with binding energy S = − 27.66 kcal mol−1. Two of them between the nitrogen of pyrazole derivative with the oxygen of carboxylate and oxygen of carbonyl of Asp32 (distance 2.81, 2.84 Å). Besides, one hydrogen bond side-chain donor between the NH of anisidine moiety and Asp218 with bond length 2.41 Å. Furthermore, N-phenyl-1H-pyrazole-4-carboxamide 16c displayed binding energy S = − 27.52 kcal mol−1 with three hydrogen bonds between Asp218, Asp32, and Ser88 with NH of anisidine, the nitrogen of pyrazole, and oxygen of methoxy group, respectively (Fig. 6d). (All docking study figures in supplementary material, Figures S3-S22).

Finally, it can be concluded that the docking results revealed that the 1H-pyrazole-4-carboxamide derivative 16c, 16d, 16h, and 16i binding to the active site of secreted aspartic protease (Saps) (PDB: 1ZAP) and may act as inhibitors. Besides, the other derivatives 14b, 14e, 14j, and 16i were suggested to be DNA gyrase inhibitors with good bind energy scores and different binding modes. Overall, these derivatives are suggested to be inhibitors for bacterial and fungal by a different mode of actions and thus may participate in antimicrobial activity.

Molecular electrostatic potential (MEP) maps

Our work was extended to investigate the reasons behind a good binding of the most promising derivatives inside the two active pockets. Molecular electrostatic potential (MEP) maps were generated to determine the charge distribution around a molecule. MEP maps provide information about the regions with electron-rich regions (red) and electron deficiency regions, i.e., hydrophobic regions that responsible for forming interactions (hydrogen bonds, arene–arene, and arene–cation interactions) with the residue inside the active site. The MEP was performed using MOE 2008.10, where the active derivatives' geometry optimized using the MMFF94x force field according to the previously reported method [60]. As represented in Fig. 7, the positive charge (blue) was located on the nitrogen of pyrimidine and pyrazoles as well as amino, cyano, and NH of both benzamide and anisidine derivatives. Simultaneously, the negative charge (red) was displayed on the oxygen of carbonyl and methoxy groups. Finally, the presence of nucleophiles (red) and electrophile (blue) regions showed that these regions could form hydrogen bonding donors and acceptors and illustrate the binding mode in the docking study (All Molecular Electrostatic Potential (MEP) maps figures in supplementary material, Figures S23-S29).

Fig. 7
figure 7

Showed molecular electrostatic potential maps of most promising derivatives to determine the charge distribution around molecules

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Conclusion

In summary, new bioactive pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j have been designed and synthesized by the reaction of 5-amino-pyrazoles 9a, b with 2-(arylidene)malononitriles 10a–e or various aldehydes 15a–e, respectively. The structures of new bioactive pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j were confirmed using different spectroscopic techniques. The pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schiff bases 16a–j were screened for their in vitro antimicrobial activities against four microbes. The three pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and four pyrazole Schiff bases (16c, 16d, 16h, and 16i) proved to be potent against the four microbes tested, have a wide spectrum effect, and in several cases, their activities exceeded that of the reference drugs used. The structure–activity relationship (SAR) was performed. As well as, physicochemical properties, drug-likeness model score, bioactivity scores, ADME, and toxicity properties were predicted in silico. The prediction result showed the five compounds (14b, 16c, 16d, 16h, and 16i) agreement with Lipinski's rule of five and with Veber’s rule. Also, in the case of a drug-likeness model score, all the tested compounds (14b, 14e, 14j, 16d, 16h, and 16i) except 16c showed positive values (range 0.05 to 0.44). Therefore, these compounds may be considered to be drug-like. The pyrazolo[1,5-a]pyrimidine 14b almost shows the highest bioactivity score compared to the other compounds. All compounds are an inhibitor of CYP2C9, CYP3A4 enzymes, and P-glycoprotein (P-gp). Also, some compounds showed highly active (≥ 90%) toward plasma protein binding (PPB), were predicted as negative against carcinogenicity (mouse and rat) test, and presented medium risk in the hERG inhibition test. Furthermore, molecular docking studies were performed for the most active derivatives against S. auras DNA gyrase (PDB: 2XCT) and secreted aspartic protease (Saps) (PDB: 1ZAP) as antibacterial and antifungal targets, respectively, to determine the binding patterns. The most active antibacterial derivatives 14b, 14e, 14j, and 16i against DNA Gyrase (PDB:2XCT) showed binding energy S = − 25.35 to − 20.18 kcal mol−1. Besides, for promising fungal derivatives 16c, 16d, 16h, and 16i displayed binding energy S = − 28.03 to − 24.91 kcal mol−1, and both of them showed different types of binding mode. The results suggested that these derivatives could act as good antimicrobial agents. MEP maps were performed to provide information about the regions with electron-rich regions (red) and electron deficiency regions that responsible for forming interactions with the residue inside the active site and therefore confirmed the binding mode in the docking study. In the future, these compounds are possibly suitable temples for the development of a novel antibacterial and antifungal therapeutic drug.

Materials and methods

Chemistry

All melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. The IR spectra were recorded (KBr disk) on a PerkinElmer 1650 FTIR instrument. 1H NMR (500 or 400 MHz) and 13C NMR (125 or 100 MHz) spectra were recorded on a JEOL spectrometer using DMSO-d6 as solvent and tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ-values) are reported in ppm. Mass spectra were recorded on a Varian MAT 112 spectrometer at 70 eV. Elemental analyses were performed at the Micro Analytical Center, Cairo University, Egypt.

The progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F254 (Merck), viewing under a short-wavelength UV lamp effected detection. All evaporations were performed under reduced pressure at 40 °C.

3-Methoxybenzaldehyde (15a), 4-(dimethylamino)benzaldehyde (15b), 2,5-dimethoxybenzaldehyde (15c), 4-chloro-3-nitrobenzaldehyde (15d), and thiophene-2-carbaldehyde (15e) were of Merck AR grade, Germany.

Synthesis of 5-amino-N-aryl-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (9a, b) [41] and 2-(arylidene)malononitriles (10a–e) [42, 43] were prepared according to the literature procedure.

Synthesis of 7-amino-6-cyano-5-aryl-2-(4-methoxyphenylamino)-N-aryl-pyrazolo[1,5-a]pyrimidine-3-carboxamides (14a–j)

A mixture of 5-amino-N-aryl-1H-pyrazole-4-carboxamide derivatives 9a, b (0.01 mol) with 2-(arylidene)malononitriles 10a–e (0.01 mol) as [2-(4-methylbenzylidene)malononitrile (10a), 2-(3-methylbenzylidene)malononitrile (10b), 2-(3-methyoxybenzylidene)malononitrile (10c), 2-(3-chlorobenzylidene)malononitrile (10d), and 2-(2,5-dimethoxybenzylidene)malononitrile (10e)] and a catalytic amount of triethylamine (four drops) in absolute ethanol (30 mL) was refluxed for 6 h. After cooling, the solvent was concentrated under reduced pressure and the solid obtained was collected and recrystallized from ethanol to give the corresponding pyrazolo[1,5-a]pyrimidines 14a–j.

7-Amino-6-cyano-2-(4-methoxyphenylamino)-N-phenyl-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14a)

Yellow crystals; M.p.: > 300 °C; Yield (80%); IR (KBr) νmax/cm−1 3428, 3304 (NH2, 2NH), 2213 (C≡N), 1641 (C=O), 1597 (C=N), 1551 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 2.43 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.90 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.09 (t, 1H, J = 7.5 Hz, phenyl ring), 7.37 (t, 2H, J = 7.8 Hz, phenyl ring), 7.44 (d, 2H, J = 8.2 Hz, p-methylphenyl ring), 7.60 (d, 2H, J = 7.7 Hz, phenyl ring), 7.84 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.90 (d, 2H, J = 8.2 Hz, p-methylphenyl ring), 9.00 (s, br, 2H, NH2 exchangeable by D2O), 9.23, 10.14 (2s, 2H, 2NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ ppm): 21.15 (C, CH3), 55.26 (C, OCH3), 74.88, 89.09, 114.26, 118.87, 119.06, 123.54, 123.78, 128.69, 129.24, 129.34, 133.40, 133.98, 138.46, 141.08, 145.70, 149.49, 153.98, 156.32, 161.47, 162.29 (26C); MS (m/z, %): 489 (M+, 17.65%), 396 (base peak, 100%); Anal. Calcd. (%) for C28H23N7O2 (489.53): C, 68.70; H, 4.74; N, 20.03. Found: C, 68.98; H, 4.57; N, 20.29%.

7-Amino-6-cyano-2-(4-methoxyphenylamino)-N-phenyl-5-(3-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14b)

Yellow crystals; M.p.: > 300 °C; Yield (77%); IR (KBr) νmax/cm−1 3428, 3319 (NH2, 2NH), 2216 (C≡N), 1656 (C=O), 1599 (C=N), 1554 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 2.48 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.94 (d, 2H, J = 9.5 Hz, p-methoxyphenyl ring), 7.13 (d, 1H, J = 7.7 Hz, m-methylphenyl ring), 7.39 (t, 2H, J = 8.2 Hz, phenyl ring), 7.50 (d, 1H, J = 7.9 Hz, m-methylphenyl ring), 7.56 (t, 1H, J = 7.9 Hz, phenyl ring), 7.62 (d, 4H, J = 8.6 Hz, m-methylphenyl ring + phenyl ring), 7.86 (d, 2H, J = 9.2 Hz, p-methoxyphenyl ring), 9.12 (s, br, 2H, NH2 exchangeable by D2O), 9.23, 10.21 (2s, 2H, 2NH exchangeable by D2O); Anal. Calcd. (%) for C28H23N7O2 (489.53): C, 68.70; H, 4.74; N, 20.03. Found: C, 68.53; H, 4.48; N, 20.27%.

7-Amino-6-cyano-5-(3-methoxyphenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (14c)

Yellow crystals; M.p.: 258–260 °C; Yield (72%); IR (KBr) νmax/cm−1 3433, 3332 (NH2, 2NH), 2212 (C≡N), 1652 (C=O), 1595 (C=N), 1547 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.74, 3.86 (2s, 6H, 2OCH3), 6.90 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.09 (t, 1H, J = 7.4 Hz, phenyl ring), 7.18–7.21 (m, 1H, m-methoxyphenyl ring), 7.36 (t, 2H, J = 7.9 Hz, phenyl ring), 7.54–7.56 (m, 3H, m-methoxyphenyl ring), 7.59 (d, 2H, J = 7.7 Hz, phenyl ring), 7.85 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 9.09 (s, br, 2H, NH2 exchangeable by D2O), 9.23, 10.14 (2s, 2H, 2NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.43, 56.11 (2C, 2OCH3), 75.09, 89.19, 113.86, 114.24, 115.98, 116.90, 118.89, 119.06, 120.98, 123.51, 129.14, 129.87, 133.30, 137.95, 138.38, 145.57, 149.40, 154.00, 156.36, 159.26, 160.78, 162.17 (26C); MS (m/z, %): 505 (M+, 41.43%), 93 (base peak, 100%); Anal. Calcd. (%) for C28H23N7O3 (505.53): C, 66.52; H, 4.59; N, 19.39. Found: C, 66.40; H, 4.31; N, 19.65%.

7-Amino-5-(3-chlorophenyl)-6-cyano-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (14d) [61]

Orange crystals; M.p. > 300 °C; Yield (74%); IR (KBr) νmax/cm−1 3442, 3299 (NH2, 2NH), 2212 (C≡N), 1665 (C=O), 1632 (C=N), 1598 (C=C); 1H NMR (400 MHz, DMSO-d6, δ/ppm) 3.78 (s, 3H, OCH3), 6.89 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.07 (t, 1H, J = 7.4 Hz, phenyl ring), 7.33 (t, 2H, J = 8.3 & 7.5 Hz, phenyl ring), 7.60 (d, 2H, J = 8.0 Hz, phenyl ring), 7.62 (d, 1H, J = 2.9 Hz, m-chlorophenyl ring), 7.76 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.93–7.96 (m, 1H, m-chlorophenyl ring), 8.02 (s, 1H, m-chlorophenyl ring), 8.06 (d, 1H, J = 2.8 Hz, m-chlorophenyl ring), 8.93 (s, br, 2H, NH2 exchangeable by D2O), 9.28, 10.06 (2s, 2H, 2NH exchangeable by D2O); 13C NMR (100 MHz, DMSO-d6, CDCl3, δ/ppm): 55.45 (1C, OCH3), 75.14, 89.98, 114.40, 115.97, 119.20, 119.29, 123.66, 127.23, 128.77, 129.21, 130.48, 130.86, 133.59, 134.16, 138.73, 138.78, 145.75, 149.60, 154.38, 157.00, 159.45, 162.53 (26C); MS (m/z, %): 511 (M+2, 2.69), 509 (M+, 8.45), 368 (base peak, 100%); Anal. Calcd. (%) for C27H20ClN7O2 (509.95): C, 63.59; H, 3.95; N, 19.23. Found: C, 63.50; H, 4.00; N, 19.27%.

7-Amino-6-cyano-5-(2,5-dimethoxyphenyl)-2-(4-methoxyphenylamino)-N-phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (14e)

Orange crystals; M.p. 210–212 °C; Yield (75%); IR (KBr) νmax/cm−1 3429, 3321 (NH2, 2NH), 2217 (C≡N), 1654 (C=O), 1595 (C=N), 1552 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.72, 3.82, 3.88 (3s, 9H, 3OCH3), 6.90 (d, 2H, J = 7.5 Hz, p-methoxyphenyl ring), 7.07–7.22 (m, 4H, 2,5-dimethoxyphenyl ring + phenyl ring), 7.34 (t, 2H, J = 7.3 Hz, phenyl ring), 7.51 (d, 1H, J = 7.8 Hz, 2,5-dimethoxyphenyl ring), 7.65 (d, 2H, J = 7.8 Hz, p-methoxyphenyl ring), 7.85 (d, 1H, J = 8.3 Hz, 2,5-dimethoxyphenyl ring), 8.68 (s, br, 2H, NH2 exchangeable by D2O), 9.34, 10.06 (2s, 2H, 2NH exchangeable by D2O); Anal. Calcd. (%) for C29H25N7O4 (535.55): C, 65.04; H, 4.71; N, 18.31. Found: C, 65.24; H, 4.45; N, 18.65%.

7-Amino-N-(4-chlorophenyl)-6-cyano-2-(4-methoxyphenylamino)-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14f)

Yellow crystals; M.p.: > 300 °C; Yield (76%); IR (KBr) νmax/cm−1 3439, 3296, 3234 (NH2, 2NH), 2214 (C≡N), 1639 (C=O), 1595 (C=N), 1564 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 2.38 (s, 3H, CH3), 3.70 (s, 3H, OCH3), 6.85 (d, 2H, J = 7.3 Hz, p-methoxyphenyl ring), 7.36 (d, 4H, J = 7.5 Hz, ArH), 7.55 (d, 2H, J = 6.2 Hz, ArH), 7.74 (d, 2H, J = 7.1 Hz, ArH), 7.83 (d, 2H, J = 7.7 Hz, p-methoxyphenyl ring), 8.76 (s, br, 2H, NH2), 9.06, 10.15 (2s, 2H, NH); MS (m/z, %): 525 (M+2, 25.41), 523 (M+, 76.85), 93 (base peak, 100%); Anal. Calcd. (%) for C28H22ClN7O2 (523.97): C, 64.18; H, 4.23; N, 18.71. Found: C, 64.35; H, 4.05; N, 18.48.

7-Amino-N-(4-chlorophenyl)-6-cyano-2-(4-methoxyphenylamino)-5-(3-methylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14g)

Yellow crystals; M.p.: > 300 °C; Yield (71%); IR (KBr) νmax/cm−1 3419, 3335 (NH2, 2NH), 2216 (C≡N), 1651 (C=O), 1592 (C=N), 1554 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 2.44 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.89 (d, 2H, J = 8.9 Hz, p-methoxyphenyl ring), 7.40 (d, 2H, J = 8.9 Hz, p-chlorophenyl ring), 7.44 (d, 1H, J = 7.7 Hz, m-methylphenyl ring), 7.49 (t, 1H, J = 7.6 Hz, m-methylphenyl ring), 7.58 (d, 2H, J = 8.8 Hz, p-chlorophenyl ring), 7.77 (d, 1H, J = 7.7 Hz, m-methylphenyl ring), 7.80 (s, 1H, m-methylphenyl ring), 7.82 (d, 2H, J = 8.9 Hz, p-methoxyphenyl ring), 9.00 (s, br, 2H, NH2 exchangeable by D2O), 9.12, 10.21 (2s, 2H, 2NH exchangeable by D2O); Anal. Calcd. (%) for C28H22ClN7O2 (523.97): C, 64.18; H, 4.23; N, 18.71. Found: C, 64.02; H, 4.54; N, 18.52%.

7-Amino-N-(4-chlorophenyl)-6-cyano-5-(3-methoxyphenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14h)

Yellow crystals; M.p.: > 300 °C; Yield (73%); IR (KBr) νmax/cm−1 3436, 3307 (NH2, 2NH), 2213 (C≡N), 1643 (C=O), 1603 (C=N), 1544 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.74, 3.86 (2s, 6H, 2OCH3), 6.90 (d, 2H, J = 9.1 Hz, p-methoxyphenyl ring), 7.18–7.20 (m, 1H, m-methoxyphenyl ring), 7.42 (d, 2H, J = 8.8 Hz, p-chlorophenyl ring), 7.51–7.55 (m, 3H, J = 7.7 Hz, m-methoxyphenyl ring), 7.62 (d, 2H, J = 8.9 Hz, p-chlorophenyl ring), 7.84 (d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 8.99 (s, br, 2H, NH2 exchangeable by D2O), 9.17, 10.18 (2s, 2H, 2NH exchangeable by D2O); Anal. Calcd. (%) for C28H22ClN7O3 (539.97): C, 62.28; H, 4.11; N, 18.16. Found: C, 62.35; H, 4.05; N, 18.34%.

7-Amino-5-(3-chlorophenyl)-N-(4-chlorophenyl)-6-cyano-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14i) [61]

Yellow crystals; M.p. 252–254 ºC; Yield (71%); IR (KBr) νmax/cm−1 3397, 3308 (NH2, 2NH), 2211 (C≡N), 1652 (C=O), 1613 (C=N), 1547 (C=C); 1H NMR (400 MHz, DMSO-d6, CDCl3, δ/ppm): 3.73 (s, 3H, OCH3), 6.82 (d, 2H, J = 8.8 Hz, ArH), 7.16–7.22 (m, 2H, ArH), 7.52 (d, 2H, J = 7.8 Hz, ArH), 7.60 (d, 2H, J = 8.8 Hz, ArH), 7.66 (d, 1H, J = 8.8 Hz, ArH), 7.81–7.91 (m, 3H, ArH), 8.63 (s, br, 2H, NH2 exchangeable by D2O), 9.14, 10.02 (2s, 2H, 2NH exchangeable by D2O); 13C NMR (100 MHz, DMSO-d6, CDCl3, δ/ppm): 55.52 (1C, OCH3), 75.48, 89.92, 114.36, 115.87, 119.28, 120.51, 127.13, 127.89, 128.34, 129.01, 130.38, 131.00, 133.49, 134.24, 134.93, 137.49, 145.89, 149.54, 154.42, 156.98, 161.21, 163.20 (26C); Anal. Calcd. (%) for C27H19Cl2N7O2 (544.39): C, 59.57; H, 3.52; N, 18.01. Found: C, 59.49; H, 3.60; N, 17.95%.

7-Amino-N-(4-chlorophenyl)-6-cyano-5-(2,5-dimethoxyphenyl)-2-(4-methoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (14j)

Yellow crystals; M.p.: > 300 °C; Yield (74%); IR (KBr) νmax/cm−1 3446, 3310 (NH2, 2NH), 2215 (C≡N), 1637 (C=O), 1596 (C=N), 1568 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.70, 3.75, 3.78 (3s, 9H, 3OCH3), 6.86 (d, 2H, J = 9.5 Hz, p-methoxyphenyl ring), 7.10–7.17 (m, 3H, 2,5-dimethoxyphenyl ring), 7.34 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), 7.48 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), 7.78 (d, 2H, J = 9.3 Hz, p-methoxyphenyl ring), 8.97 (s, br, 2H, NH2 exchangeable by D2O), 9.11, 10.04 (2s, 2H, 2NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.12, 55.68, 55.93 (3C, 3OCH3), 78.02, 88.84, 104.95, 113.21, 114.13, 115.17, 117.23, 118.96, 120.38, 126.91, 128.90, 133.25, 137.20, 145.72, 148.51, 150.65, 153.07, 153.91, 156.07, 159.97, 159.99, 162.09 (26C); Anal. Calcd. (%) for C29H24ClN7O4 (570.00): C, 61.11; H, 4.24; N, 17.20. Found: C, 60.90; H, 4.39; N, 17.48%.

Synthesis of Schiff bases 16a-j

A mixture of 5-amino-N-aryl-1H-pyrazole-4-carboxamide derivatives 9a, b (0.01 mol) and various aldehydes 15a–e (0.01 mol) {namely: 3-methoxybenzaldehyde (15a), 4-(dimethylamino)benzaldehyde (15b), 2,5-dimethoxybenzaldehyde (15c), 4-chloro-3-nitrobenzaldehyde (15d), or thiophene-2-carbaldehyde (15e)} with a catalytic amount of glacial acetic acid (1 mL) in absolute ethanol (25 mL) was refluxed for 2 h and then left to cool. The solid product was filtered off, dried and finally recrystallized from ethanol to afford the corresponding Schiff bases 16a–j.

5-(3-Methoxybenzylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (16a)

Yellow crystals; M.p.: 201–203 °C; Yield (75%); IR (KBr) νmax/cm−1 3434, 3229 (3NH), 1656 (C=O), 1612 (C=N), 1587 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.73, 3.88 (2s, 6H, 2OCH3), 6.91 (d, 2H, J = 8.3 Hz, p-methoxyphenyl ring), 7.08 (t, 1H, J = 7.4 Hz, phenyl ring), 7.23 (d, 1H, J = 7.5 Hz, m-methoxyphenyl ring), 7.36 (t, 2H, J = 7.9 Hz, phenyl ring), 7.55 (t, 1H, J = 7.8 Hz, m-methoxyphenyl ring), 7.62–7.64 (m, 4H, phenyl ring + m-methoxyphenyl ring), 7.68 (d, 2H, J = 8.0 Hz, p-methoxyphenyl ring), 8.71 (s, 1H, –CH=N–), 9.05, 9.99, 12.80 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.27, 55.34 (2C, 2OCH3), 83.17, 112.33, 114.51, 118.98, 119.82, 123.30, 128.54, 129.08, 129.37, 130.53, 135.02, 138.54, 146.21, 152.25, 155.38, 156.89, 159.83, 161.70, 162.69 (23C); MS (m/z, %): 441 (M+, 25.67%), 83 (base peak, 100%); Anal. Calcd. (%) for C25H23N5O3 (441.48): C, 68.01; H, 5.25; N, 15.86. Found: C, 68.17; H, 5.06; N, 15.69%.

5-(4-(Dimethylamino)benzylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (16b) [62]

Yellow crystals; M.p.: 216–218 °C; Yield (87%); IR (KBr) νmax/cm−1 3445, 3292 (3NH), 1652 (C=O), 1614 (C=N), 1592 (C=C); 1H NMR (400 MHz, DMSO-d6, δ/ppm): 3.01 (s, 6H, N(CH3)2), 3.66 (s, 3H, OCH3), 6.83 (d, 4H, J = 8.9 Hz, ArH), 7.02 (t, 1H, ArH), 7.33 (t, 2H, ArH), 7.48 (2H, ArH), 7.60 (d, 2H, J = 8.4 Hz, ArH), 7.80 (d, 2H, J = 8.7 Hz, ArH), 8.67 (s, 1H, –N=CH–), 9.03, 10.07, 12.59 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (100 MHz, DMSO-d6, δ/ppm) δ 40.10 (2C, N (CH3)2 under solvent DMSO), 55.48 (OCH3), 92.74, 112.34, 115.12, 120.08, 121.82, 126.58, 128.07, 128.43, 129.19, 132.02, 132.84, 135.21, 152.78, 153.40, 153.84, 154.45, 160.57, 162.06 (23C); Anal. Calcd. (%) for C26H26N6O2 (454.52): C, 68.70; H, 5.77; N, 18.49. Found: C, 68.75; H, 5.70; N, 18.55%.

5-(2,5-Dimethoxybenzylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (16c)

Orange crystals; M.p.: 234–236 °C; Yield (69%); IR (KBr) νmax/cm−1 3389, 3320 (3NH), 1654 (C=O), 1594 (C=N), 1552 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.72, 3.83, 3.89 (3s, 9H, 3OCH3), 6.91 (d, 2H, J = 8.0 Hz, p-methoxyphenyl ring), 7.08–7.35 (m, 7H, Ar–H), 7.50 (s, 1H, 2,5-dimethoxyphenyl ring), 7.65 (d, 2H, J = 8.3 Hz, p-methoxyphenyl ring), 8.69 (s, 1H, –CH=N–), 9.47, 10.18, 13.01 (3s, 3H, 3NH exchangeable by D2O); Anal. Calcd. (%) for C26H25N5O4 (471.51): C, 66.23; H, 5.34; N, 14.85. Found: C, 66.62; H, 5.17; N, 14.68%.

5-(4-Chloro-3-nitrobenzylideneamino)-3-(4-methoxyphenylamino)-N-phenyl-1H-pyrazole-4-carboxamide (16d)

Buff crystals; M.p.: 252–254 °C; Yield (72%); IR (KBr) νmax/cm−1 3431, 3235 (3NH), 1653 (C=O), 1615 (C=N), 1513 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.70 (s, 3H, OCH3), 6.89 (d, 2H, J = 8.3 Hz, p-methoxyphenyl ring), 7.06 (t, 1H, J = 7.3 Hz, phenyl ring), 7.31–7.34 (m, 4H, phenyl ring), 7.67 (d, 2H, J = 7.9 Hz, p-methoxyphenyl ring), 8.00 (d, 1H, J = 8.3, 4-chloro-3-nitrophenyl ring), 8.30 (d, 1H, J = 8.3, 4-chloro-3-nitrophenyl ring), 8.67 (s, 1H, 4-chloro-3-nitrophenyl ring), 8.76 (s, 1H, –CH=N–), 9.13, 9.79, 12.80 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.23 (1C, OCH3), 96.78, 114.61, 118.92, 119.73, 121.51, 123.06, 125.26, 126.27, 128.87, 132.72, 133.81, 134.09, 138.39, 147.84, 152.02, 153.37, 157.62, 160.86, 162.42 (23C); Anal. Calcd. (%) for C24H19ClN6O4 (490.90): C, 58.72; H, 3.90; N, 17.12; Found: C, 58.99; H, 3.79; N, 17.28%.

3-(4-Methoxyphenylamino)-N-phenyl-5-(thiophen-2-ylmethyleneamino)-1H-pyrazole-4-carboxamide (16e)

Yellow crystals; M.p.: 212–214 °C; Yield (67%); IR (KBr) νmax/cm−1 3433, 3272 (3NH), 1652 (C=O), 1594 (C=N), 1547 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.73 (s, 3H, OCH3), 6.90 (d, 2H, J = 7.1 Hz, p-methoxyphenyl ring), 7.09 (d, 1H, J = 5.3 Hz, thiophene ring), 7.33–7.40 (m, 5H, phenyl ring), 7.72 (d, 2H, J = 7.0 Hz, p-methoxyphenyl ring), 7.91 (d, 1H, J = 3.6 Hz, thiophene ring), 8.05 (s, 1H, thiophene ring), 8.68 (s, 1H, –CH=N–), 9.19, 9.98, 12.50 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.79 (1C, OCH3), 89.70, 115.01, 119.23, 123.68, 129.62, 129.74, 133.89, 134.14, 134.29, 136.99, 139.23, 141.83, 155.79, 156.45, 157.46, 159.54, 163.28 (21C); MS (m/z, %): 417 (M+, 18.85%), 186 (base peak, 100%); Anal. Calcd. (%) for C22H19N5O2S (417.48): C, 63.29; H, 4.59; N, 16.78; Found: C, 63.45; H, 4.39; N, 16.85%.

N-(4-Chlorophenyl)-5-(3-methoxybenzylideneamino)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (16f)

Yellow crystals; M.p.: 205–207 °C; Yield (73%); IR (KBr) νmax/cm−1 3434, 3229 (3NH), 1656 (C=O), 1612 (C=N), 1587 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.73, 3.87 (2s, 6H, 2OCH3), 6.90 (d, 2H, J = 8.9 Hz, p-methoxyphenyl ring), 7.21, 7.23 (dd, 1H, J = 8.2, 1.8 Hz, m-methoxyphenyl ring), 7.40 (d, 4H, J = 8.9 Hz, p-chlorophenyl ring), 7.53 (t, 1H, J = 7.9 Hz, m-methoxyphenyl ring), 7.59 (s, 1H, m-methoxyphenyl ring), 7.62 (d, 1H, J = 2.4 Hz, m-methoxyphenyl ring), 7.69 (d, 2H, J = 8.9 Hz, p-methoxyphenyl ring), 8.66 (s, 1H, –CH=N–), 9.03, 10.02, 12.78 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.19, 55.23 (2C, 2OCH3), 92.61, 112.24, 114.39, 119.22, 119.53, 120.41, 122.48, 126.69, 128.34, 128.73, 130.28, 133.79, 136.17, 137.46, 151.21, 154.04, 159.74, 162.03, 162.65 (23C); Anal. Calcd. (%) for C25H22ClN5O3 (475.93): C, 63.09; H, 4.66; N, 14.72; Found: C, 63.34; H, 4.53; N, 14.43%.

N-(4-Chlorophenyl)-5-(4-(dimethylamino)benzylideneamino)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (16g) [62]

Yellow crystals; M.p.: 209–211 °C, Yield (86%); IR (KBr) νmax/cm−1 3431, 3275 (3NH), 1662 (C=O), 1609 (C=N), 1581 (C=C); 1H NMR (400 MHz, DMSO-d6, δ ppm): 3.38 (s, 6H, N(CH3)2), 3.71 (s, 3H, OCH3), 6.85 (d, 2H, J = 9 Hz, p-dimethylaminophenyl ring), 6.88 (d, 2H, J = 8.9 Hz, p-methoxyphenyl ring), 7.40 (d, 2H, J = 8.6 Hz, p-chlorophenyl ring), 7.45 (d, 2H, J = 8.5 Hz, p-chlorophenyl ring), 7.68 (d, 2H, J = 8.6 Hz, p-methoxyphenyl ring), 7.82 (d, 2H, J = 8.5 Hz, p-dimethylaminophenyl ring), 8.66 (s, 1H, –CH=N–), 8.74, 10.24, 12.24 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (100 MHz, DMSO-d6, δ ppm): 40.06 (2C, N (CH3)2 under solvent DMSO), 55.67 (1C, OCH3), 92.48, 112.27, 114.78, 120.87, 121.08, 122.12, 127.29, 129.43, 132.02, 133.35, 135.00, 152.88, 153.70, 153.89, 154.25, 161.19, 163.66 (23C); Anal. Calcd. (%) for C26H25ClN6O2 (488.97): C, 63.86; H, 5.15; N, 17.19; Found: C, 63.95; H, 5.10; N, 17.25%.

N-(4-Chlorophenyl)-5-(2,5-dimethoxybenzylideneamino)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (16h)

Orange crystals; M.p.: 250–252 °C; Yield (74%); IR (KBr) νmax/cm−1 3432, 3251 (3NH), 1653 (C=O), 1597 (C=N), 1544 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.73, 3.82, 3.88 (3s, 9H, 3OCH3), 6.89, 6.91 (dd, 2H, J = 2.8 & 9.5 Hz, p-methoxyphenyl ring), 7.18 (d, 1H, J = 2.8 Hz, 2,5-dimethoxyphenyl ring), 7.21 (d, 1H, J = 2.7 Hz, 2,5-dimethoxyphenyl ring), 7.38 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), 7.42 (d, 2H, J = 9.0 Hz, p-chlorophenyl ring), 7.61 (s, 1H, 2,5-dimethoxyphenyl ring), 7.66, 7.68 (dd, 2H, J = 9.2, 2.7 Hz, p-methoxyphenyl ring), 8.64 (s, 1H, –CH=N–), 9.34, 10.13, 11.74 (3s, 3H, 3NH exchangeable by D2O); 13C NMR (125 MHz, DMSO-d6, δ/ppm): 55.26, 55.59, 56.47 (3C, 3OCH3), 92.55, 109.44, 114.21, 114.42, 119.00, 120.58, 121.94, 123.30, 126.66, 128.83, 134.18, 137.62, 153.41, 153.81, 154.87, 156.70, 156.90, 160.72, 162.88 (23C); Anal. Calcd. (%) for C26H24ClN5O4 (505.95): C, 61.72; H, 4.78; N, 13.84; Found: C, 61.90; H, 4.51; N, 13.98%.

5-(4-Chloro-3-nitrobenzylideneamino)-N-(4-chlorophenyl)-3-(4-methoxyphenylamino)-1H-pyrazole-4-carboxamide (16i)

Yellow crystals; M.p.: 280–282 °C; yield (71%); IR (KBr) νmax/cm−1 3440, 3280, 3230 (3NH), 1653 (C=O), 1615 (C=N), 1561 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.72 (s, 3H, OCH3), 6.89 (d, 2H, J = 8.7 Hz, p-methoxyphenyl ring), 7.35 (d, 4H, J = 8.8 Hz, p-chlorophenyl ring), 7.70 (d, 2H, J = 8.8 Hz, p-methoxyphenyl ring), 7.96 (d, 1H, J = 8.4 Hz, 4-chloro-3-nitrophenyl ring), 8.27 (d, 1H, J = 8.3 Hz, 4-chloro-3-nitrophenyl ring), 8.26 (s, 1H, 4-chloro-3-nitrophenyl ring), 8.72 (s, 1H, –CH=N–), 9.14, 9.87, 12.94 (3s, 3H, 3NH exchangeable by D2O); Anal. Calcd. (%) for C24H18Cl2N6O4 (525.34): C, 54.87; H, 3.45; N, 16.00; Found: C, 55.05; H, 3.28; N, 16.19%.

N-(4-Chlorophenyl)-3-(4-methoxyphenylamino)-5-(thiophen-2-ylmethyleneamino)-1H-pyrazole-4-carboxamide (16j)

Yellow crystals; M.p.: 221–223 °C; yield (68%); IR (KBr) νmax/cm−1 3432, 3278 (3NH), 1653 (C=O), 1598 (C=N), 1562 (C=C); 1H NMR (500 MHz, DMSO-d6, δ/ppm): 3.73 (s, 3H, OCH3), 6.91 (d, 2H, J = 8.1 Hz, p-methoxyphenyl ring), 7.39–7.45 (m, 5H, p-chlorophenyl ring + thiophene ring), 7.74 (d, 2H, J = 8.0 Hz, p-methoxyphenyl ring), 7.91 (d, 1H, J = 2.3 Hz, thiophene ring), 8.05 (d, 1H, J = 4.8 Hz, thiophene ring), 8.64 (s, 1H, –CH=N–), 9.19, 10.04, 12.97 (3s, 3H, 3NH exchangeable by D2O); Anal. Calcd. (%) for C22H18ClN5O2S (451.93): C, 58.47; H, 4.01; N, 15.50; Found: C, 58.58; H, 4.19; N, 15.21%.

In vitro antimicrobial evaluation

Microorganisms

The microbes used in this study were Gram-positive bacterial strain (Bacillus subtilis ATCC-6633), Gram-negative bacterial strain (Escherichia coli ATCC-25922), yeast (Candida albicans ATCC-10231), and Aspergillus niger NRRL-3 as fungi. These microorganisms were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and Northern Utilization Research and Development Division, United State Department of Agriculture, Peoria, Illinois, USA (NRRL). The bacterial strains were revived for bonsai by sub-culturing fresh nutrient agar (NA) medium (Merck, Darmstadt, Germany) for 24 h before the test, while, fungi were cultured on potato dextrose agar (PDA) (Lab M., Bury, Lancashire, UK) for 7 days at 28 °C before the experiment was carried out.

Inoculum preparation

Stock cultures were maintained at 4 °C on slopes of nutrient agar and potato dextrose agar. Active cultures for experiments were prepared by transferring a lapful of cells from the stock cultures to test tubes of Mueller–Hinton broth (MHB) (Lab M Limited, Bury, Lancashire, UK) for bacteria and Sabouraud dextrose broth (SDB) (Lab M., Bury, Lancashire, UK) for fungi that were incubated without agitation for 24 h at 37 °C and 25 °C, respectively. To 5 mL of MHB and SDB, 0.2 mL of culture was inoculated and incubated (or diluted) till it reached the turbidity equal to that of the standard 0.5 McFarland solution at 625 nm (A = 0.08 to 0.1) which is equivalent to 1.5 × 108 cfu.mL−1.

Bioassay method

Agar diffusion technique as described by Perez et al. [45] was used to determine the in vitro antibacterial activity of the powder samples. A 0.1 mL aliquot of 18 h broth culture of the above-mentioned bacteria that had been adjusted to the turbidity equivalent of 0.5 McFarland standards [63] was dispensed into sterile Petri dishes previously labeled with the test bacteria. Molten sterile Muller-Hinton was aseptically poured into the plates and gently rotated for the bacteria to be homogeneously distributed in the medium. The agar plates were allowed to solidify. The antibacterial screening bioassay was made by the agar well diffusion method described by Jorgensen and Turnidge [64] using Mueller–Hinton agar (Lab M Limited, Bury, Lancashire, UK), then the plates allowed to diffuse for two hours at 4 °C. The experiment was conducted in triplicates. All plates were incubated at 37 °C for 24 h for bacterial strains and 28–30 °C for 48 h for fungal strain. Clearance zones around the wells were noted and measured in millimeters [65]. Standard bacterial antibiotics as Tetracycline (30 µg) and Novobiocine (30 µg) and fungal antibiotic as Clotrimazole (50 µg), and Cyclohexamide (50 µg) were used as a positive control for bacteria and fungi, respectively.

Molecular docking

All the molecular docking studies were performed using Molecular Operating Environmental (MOE) software 2008.10. The most active derivatives against the tested bacterial strains (14b, 14e, 14j, and 16i) and fungal pathogens (16c, 16d, 16h, and 16i) were builders using ChemDraw 2014. The structure of the most active derivatives was protonated and minimized energy using MMFF94X force field with a root mean standard deviation (RMSD) gradient of 0.05 kcal mol−1 Å as well as the partial charged calculated. For promising antibacterial derivatives S. aureus Gyrase complex with Ciprofloxacin (PDB:2XCT), and for promising fungal derivatives secreted aspartic protease from C. albicans (PDB: 1ZAP) were selected for this study [57, 58]. The enzyme with its co-crystallized ligand was retrieved from the protein data bank. For DNA gyrase (PDB:2XCT), the docking process performed using only one chain G chain only according to the reported method. For fungal, the protein structure (PDB: 1ZAP) contains only one chain (A). The enzyme was prepared according to default protocol by removing the water molecules, hydrogen atom added. The alpha site finder was used to generate the active site. For docking studies, the trigonal matcher placement and the London dG scoring function were selected after redocking the co-crystalized ligand in both enzymes.