Discovery of New Schiff Bases Tethered Pyrazole Moiety: Design, Synthesis, Biological Evaluation, and Molecular Docking Study as Dual Targeting DHFR/DNA Gyrase Inhibitors with Immunomodulatory Activity

A series of Bis-pyrazole Schiff bases (6a–d and 7a–d) and mono-pyrazole Schiff bases (8a–d and 9a–d) were designed and synthesized through the reaction of 5-aminopyrazoles 1a–d with aldehydes 2–5 using mild reaction condition with a good yield percentage. The chemical structure of newly formed Schiff bases tethered pyrazole core was confirmed based on spectral and experimental data. All the newly formed pyrazole Schiff bases were evaluated against eight pathogens (Gram-positive, Gram-negative, and fungi). The result exhibited that, most of them have good and broad activities. Among those, only six Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) displayed MIC values (0.97–62.5 µg/mL) compared to Tetracycline (15.62–62.5 µg/mL) and Amphotericin B (15.62–31.25 µg/mL), MBC values (1.94–87.5 µg/mL) and selectivity to tumor cell than normal cells. Immunomodulatory activities showed that the promising Schiff bases increase the immunomodulator effect of defense cell and the Schiff base 8a is the highest one by (Intra. killing activity = 136.5 ± 0.3%) having a pyrazole moiety as well as amide function (O=C-NH2) and piperidinyl core. Furthermore, the most potent one exhibited broad activity depending on both MIC and MBC values. Moreover, to study the mechanism of these pyrazole Schiff bases, two active Schiff bases 8a and 9b from six derivatives were introduced to study the enzyme assay as dihydrofolate reductase (DHFR) on E. coli organism and DNA gyrase with two different organisms, S. aureus and B. subtilis, to determine the inhibitory activities with lower values in the case of DNA gyrase (8a and 9b) or nearly as DHFR compound 9b, while pyrazole 8a showed excellent inhibitory against all enzyme assay. The molecular docking study against dihydrofolate reductase and DNA gyrase were performed to study the binding between active site in the pocket with the two Schiff bases (8a and 9b) that exhibited good binding affinity with different bond types as H-bonding, aren-aren, and arene-cation interaction as well as study the physicochemical and pharmacokinetic properties of the two active Schiff bases 8a and 9b.


Introduction
One of the most common causes of death all over the world is the microbial infections and multidrug-resistant bacteria. According to the World Health Organization (WHO), the resistance of microbes to antibiotics drugs is one of the dangerous health problems that threaten humans. Therefore, the production of novel bioactive compounds acting as antimicrobial agents for overcoming the resistance problem is an urgent topic that configures interest between the organic and medicinal chemistry researchers and is considered one of the greatest achievements over time [1][2][3]. Antibiotic-resistant infection problem arises from the broad use and abuse of conventional antibiotics, already, every year an estimated 700,000 people around the world die of drug-resistant diseases, 10 million people could die each year from diseases that have grown resistant [4][5][6].
Dihydrofolate reductase (DHFR) enzyme is a target for several anticancer and antibacterial drugs and of high importance in medicinal chemistry [26,27] because of its function as a cofactor in the biosynthesis of nucleic acids and amino acids. Mechanism of action for DHFR is blocking the synthesis of DNA, RNA, and proteins, causing cell growth arrest [28,29]. Also, DNA gyrase is an enzyme that belongs to type II topoisomerases and catalyzes changes in DNA topology [30]. In addition, it is composed of two chains GyrA and GyrB subunits that are responsible for the transient break of two strands of DNA, as well as introducing negative supercoils in DNA during replication. Drugs that target DNA gyrase exhibited their antibacterial activity by two mechanisms as gyrase poisoning as in Ciprofloxacin or by blocking the ATP binding site as in Novobiocin [31] and depending on the previous important function, it has become an attractive target for the development of antibacterial drug [32]. Therefore, DHFR and DNA topoisomerases have a proven track record in supporting their role in microbial diseases and cancer chemotherapy [33,34]. The immune system is a remarkably advanced vertebrate defense system and its main function is defense against invaders by producing cell and molecular varieties that can recognize and thus eliminate unlimited varieties of foreign and harmful Molecules 2020, 25,2593 3 of 23 agents [35,36]. In general, immunomodulators can be classified as two different types depending on their effects: immunosuppressants and Immunostimulants, and both could mount an immune response or defend against pathogens or tumors [37]. Immunomodulation is the process produced by the administration of a drug or compound to alter an immune response positive or negative. A number of proteins, amino acids, and natural compounds showed an important ability to regulate immune responses, including interferon-γ (IFN-γ) steroids [38]. Dihydrofolate reductase (DHFR) enzyme is a target for several anticancer and antibacterial drugs and of high importance in medicinal chemistry [26,27] because of its function as a cofactor in the biosynthesis of nucleic acids and amino acids. Mechanism of action for DHFR is blocking the synthesis of DNA, RNA, and proteins, causing cell growth arrest [28,29]. Also, DNA gyrase is an enzyme that belongs to type II topoisomerases and catalyzes changes in DNA topology [30]. In addition, it is composed of two chains GyrA and GyrB subunits that are responsible for the transient break of two strands of DNA, as well as introducing negative supercoils in DNA during replication. Drugs that target DNA gyrase exhibited their antibacterial activity by two mechanisms as gyrase poisoning as in Ciprofloxacin or by blocking the ATP binding site as in Novobiocin [31] and depending on the previous important function, it has become an attractive target for the development of antibacterial drug [32]. Therefore, DHFR and DNA topoisomerases have a proven track record in supporting their role in microbial diseases and cancer chemotherapy [33,34]. The immune system is a remarkably advanced vertebrate defense system and its main function is defense against invaders by producing cell and molecular varieties that can recognize and thus eliminate unlimited varieties of foreign and harmful agents [35,36]. In general, immunomodulators can be classified as two different types depending on their effects: immunosuppressants and Immunostimulants, and both could mount an immune response or defend against pathogens or tumors [37]. Immunomodulation is the process produced by the administration of a drug or compound to alter an immune response positive or negative. A number of proteins, amino acids, and natural compounds showed an important ability to regulate immune responses, including interferon-γ (IFN-γ) steroids [38].
Also, its 13 C NMR (101 MHz) spectrum showed the characteristic signals for CH 3 , -N-CH 3 and OCH 3 at δ 11.50, 33.93, and 55.21 ppm, respectively. The carbons for the carbonyl groups of amide function and antipyrine moiety appeared at δ 162. 54  protons. Furthermore, two singlet singlets at δ 7.19 and 7.80 ppm for NH2-amide function.
Also, its 13 C NMR (101 MHz) spectrum showed the characteristic signals for CH3, -N-CH3 and OCH3 at δ 11.50, 33.93, and 55.21 ppm, respectively. The carbons for the carbonyl groups of amide function and antipyrine moiety appeared at δ 162.54 and 166.76 ppm, respectively. The mass spectrum showed its [M + ] peak (m/z 445) corresponding to C23H23N7O3 (molecular formula) with the relative intensity = 13.44%. Also, the synthesis of Schiff bases tethered pyrazole moiety 8a-d or 9a-d were performed by the condensation of 5-aminopyrazole derivatives 1a-d with 4-(piperidin-1-yl) benzaldehyde (4) or 3, 4, 5-trimethoxybenzaldehyde (5), respectively, in good yield (Scheme 3). The 1 H NMR spectrum of compound 8b revealed four singlet signals at δ 8.71, 8.77, 10.14, and 12.56 ppm for the protons of -CH=N-and 3NH groups, respectively. The protons of the methoxy group appeared at δ 3.72 ppm as a singlet signal. Moreover, the piperidine moiety protons appeared at δ 1.60 ppm for six protons as a broad signal and 3.43 ppm for four protons. The aromatic protons appear in region δ 6.89-7.85 ppm for thirteen protons and represented as three doublet, one triplet, and one multiplet signals at δ 6.89(d), 7.68(d), 7.85(d), 7.08 (t), and 7.35-7.48 (m) ppm respectively, with coupling constant (J) ranging between 8.1-8. 3 Hz. Its 13 C NMR spectrum showed three characteristic signals at δ 23.95, 24.93, and 47.65 ppm referred to as the carbons of piperidinyl moiety. In addition, two signals at δ 55.20 and 163.06 ppm for the carbons of -OCH3 and C=O, respectively, as well as aromatic carbons and C=N appeared from δ 92.57 to 160.09 ppm. The mass spectrum exhibited the molecular ion peak at m/z = 494 [M + , 32.82%] corresponding to C29H30N6O2 (molecular formula).

NH
In the same way, treatment of 5-aminopyrazole derivative 1c with 3,4,5-trimethoxybenzaldehyde (5) afforded 3-(p-methoxyphenylamino)-N-p-tolyl-5-(3,4,5-trimethoxybenzylideneamino)-1H-pyrazole-4carboxamide (9c) having four methoxy groups, as well as N-p-tolyl 4-carboxamide based on elemental analysis and spectral data. IR spectrum of Schiff base 9c showed characteristic bands related to NH and carbonyl groups at 3437, 3285, and 1655 cm −1 , respectively. The 1 H NMR spectrum of Schiff base 9c exhibited four singlet signals at δ 2.27, 3.73, 3.79, and 3.92 ppm for one methyl and four methoxy groups. Furthermore, four singlet signals at upfield can be classified as the signal at δ 8.70 ppm related to -CH=N and three singlet signals at δ 8.96, 9.95, and 12.64 ppm for three NH protons as well as ten aromatic Also, the synthesis of Schiff bases tethered pyrazole moiety 8a-d or 9a-d were performed by the condensation of 5-aminopyrazole derivatives 1a-d with 4-(piperidin-1-yl) benzaldehyde (4) or 3, 4, 5-trimethoxybenzaldehyde (5), respectively, in good yield (Scheme 3). The 1 H NMR spectrum of compound 8b revealed four singlet signals at δ 8.71, 8.77, 10.14, and 12.56 ppm for the protons of -CH=N-and 3NH groups, respectively. The protons of the methoxy group appeared at δ 3.72 ppm as a singlet signal. Moreover, the piperidine moiety protons appeared at δ 1.60 ppm for six protons as a broad signal and 3.43 ppm for four protons. The aromatic protons appear in region δ 6.89-7.85 ppm for thirteen protons and represented as three doublet, one triplet, and one multiplet signals at δ 6.89(d), 7.68(d), 7.85(d), 7.08 (t), and 7.35-7.48 (m) ppm respectively, with coupling constant (J) ranging between 8.1-8. 3 Hz. Its 13 C NMR spectrum showed three characteristic signals at δ 23.95, 24.93, and 47.65 ppm referred to as the carbons of piperidinyl moiety. In addition, two signals at δ 55.20 and 163.06 ppm for the carbons of -OCH 3 and C=O, respectively, as well as aromatic carbons and C=N appeared from δ 92.57 to 160.09 ppm. The mass spectrum exhibited the molecular ion peak at m/z = 494 [M + , 32.82%] corresponding to C 29 H 30 N 6 O 2 (molecular formula).
In the same way, treatment of 5-aminopyrazole derivative 1c with 3,4,5-trimethoxybenzaldehyde (5) afforded 3-(p-methoxyphenylamino)-N-p-tolyl-5-(3,4,5-trimethoxybenzylideneamino)-1H-pyrazole-4-carboxamide (9c) having four methoxy groups, as well as N-p-tolyl 4-carboxamide based on elemental analysis and spectral data. IR spectrum of Schiff base 9c showed characteristic bands related to NH and carbonyl groups at 3437, 3285, and 1655 cm −1 , respectively. The 1 H NMR spectrum of Schiff base 9c exhibited four singlet signals at δ 2.27, 3.73, 3.79, and 3.92 ppm for one methyl and four methoxy groups. Furthermore, four singlet signals at upfield can be classified as the signal at δ 8.70 ppm related to -CH=N and three singlet signals at δ 8.96, 9.95, and 12.64 ppm for three NH protons as well as ten aromatic protons between δ 6.91 to 7.59 ppm. The 13 C NMR spectrum of 9c displayed a singlet signal at 21.31 ppm for methyl group and three singlet signals at 55.04, 56.79, and 60.65 related to four methoxy groups as well as one signal at 162.88 for carbonyl group beside aromatic carbons that appeared in region 91.93-161.74 ppm.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 24 protons between δ 6.91 to 7.59 ppm. The 13 C NMR spectrum of 9c displayed a singlet signal at 21.31 ppm for methyl group and three singlet signals at 55.04, 56.79, and 60.65 related to four methoxy groups as well as one signal at 162.88 for carbonyl group beside aromatic carbons that appeared in region 91.93-161.74 ppm.

In Vitro Antimicrobial Activities
Antimicrobial activities of the newly formed Schiff bases (6a-d, 7a-d, 8a-d, and 9a-d) and reference drugs were evaluated against three Gram-positive, three Gram-negative, and two fungi strain at Al-Azhar University, Cairo, Egypt. The inhibition zones (IZ, in mm ± standard deviation) and the minimal inhibitory concentration (MIC) (µg/mL) were determined by the conventional paper disk diffusion method [54,55]. The broad-spectrum antibiotics, Tetracycline and Amphotericin B were used as positive controls. The results are presented in Tables 1-3. Table 1: Inhibition zone (IZ) in mm ± standard deviation of Schiff bases (6a-d, 7a-d, 8a-d, and 9a-d) and reference drugs against pathogenic microbes.

In Vitro Antimicrobial Activities
Antimicrobial activities of the newly formed Schiff bases (6a-d, 7a-d, 8a-d, and 9a-d) and reference drugs were evaluated against three Gram-positive, three Gram-negative, and two fungi strain at Al-Azhar University, Cairo, Egypt. The inhibition zones (IZ, in mm ± standard deviation) and the minimal inhibitory concentration (MIC) (µg/mL) were determined by the conventional paper disk diffusion method [54,55]. The broad-spectrum antibiotics, Tetracycline and Amphotericin B were used as positive controls. The results are presented in Tables 1-3.   Table 1. Inhibition zone (IZ) in mm ± standard deviation of Schiff bases (6a-d, 7a-d, 8a-d, and 9a-d) and reference drugs against pathogenic microbes.   From the inhibition zone measurements (IZ, Table 1), we can conclude that there are six Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) that displayed inhibition zone more than or near to the reference drugs (Tetracycline and Amphotericin B) against pathogenic microbes. For Gram positive and Gram negative, it was found that most of the Schiff bases exhibited moderate to potent anti-bacterial activities with inhibition zone (IZ) ranging between 12 and 33 mm for all pathogen tested in this study compared to Tetracycline that have IZ ranging between 20 and 25 mm. Moreover, most of the newly synthesized Schiff bases showed IZ between 9 and 27 mm, in comparison to Amphotericin B as a therapeutic abroad anti-fungal agent (18-22 mm). This result encouraged us to complete the study and measure the minimal inhibitory concentrations (MIC, µg/mL) of the potent Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b). The results are represented in Tables 2 and 3.
Schiff base 8a bearing amide group in position four of pyrazole moiety exhibited minimal inhibitory concentrations (0.97 and 5.57 µg/mL) with 16 and 11.2-folds more than Tetracycline (15.62 and 62.5 µg/mL) against E. coli (Ec) and P. aeruginosa (Pa), respectively. From Table 3, among the tested pyrazole Schiff bases that exhibited anti-fungal activities, the two Schiff bases 8a and 9b revealed the best antifungal results with MIC values (7.81 and 15.62 µg/mL) against C. albicans (Ca) and F. oxysporum (Fo), respectively, that reveled 2-fold activity in comparison with Amphotericin B (15.62, 31.25 µg/mL).

Minimal Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)
The MBC is complementary to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that greatly inhibits growth, but the MBC determines the lowest level of antimicrobial agent that leads to the death of microbial organisms. In other words, if a MIC shows inhibition only, i.e., the antimicrobial activity does not cause death, in contrast to MBC that causes death [56]. MBC is usually presented as MBC 50 , which means the drug concentration kills 50% of the initial bacterial population [57]. The promising pyrazole Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b  On the other hand, for Gram negative bacteria, Schiff base 7b showed the best MBC value among the six Schiff bases (6.63 µg/mL with nearly 6.6 folds potent than the standard drug) against S. typhi (St). But, Schiff base 7c exhibited the worst results to Gram negative bacterial strains. In the same way, four from six of the promising Schiff bases known as 7b, 8a, 8d, and 9b showed a good fungicidal activity with MFC values ranging from (12.94-56.25 µg/mL) in comparison with Amphotericin B (34.62-65.62 µg/mL). However, both 6b and 7c showed slightly increased activity than reference drug.
Previous studies reported that there is a relation between the MIC and MBC to decide whether the newly designed compounds have cidal or statical activities for bacteria and fungi [58]; antibacterial agent that has MBC/MIC ratios higher than or equal to 8 is considered as a bacteriostatic agent, and by contrast, the MBC/MIC ratio between 1 and 2 is regarded as bactericidal agent according to Clinical and Laboratory Standards Institute (CLSI) standards [59,60]. As shown in Tables 2 and 3 and by applying MBC/MIC and MFC/MIC ratios, the values obtained ranged between one and nearly two and that expressed all the promising compounds show bactericidal and fungicidal activities with lower concentration depending on MBC, MFC, and MIC values.

Immunomodulatory Activity
In this section, we gain insight into the in vitro immunomodulatory potential of the active compounds. Innate immunity is the first line of defense against pathogens present in our environment such as fungi, bacteria, and viruses and eliminates damaged cells and tumor cells characterized by rapid response, infection halting. The innate immunity system may include macrophage, mast cells, dendritic cells (DCs), eosinophils, basophils, neutrophils as well as invariant natural killer cells (NK cells) [64]. The neutrophil can cause microorganisms intracellular killing [65]. The immunomodulatory activity expressed by percentage (%) values for the intracellular killing was performed using nitro blue tetrazolium (NBT) reductase assay, where the increasing percentage is related to improvement in the killing ability of the neutrophils, and was performed according to the reported method [66]. The results are represented in Table 5.
The potent Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b were evaluated as immunomodulatory agents by percentage values that ranged between (49.6 ± 0.14%) and (136.5 ± 0.3%) and depending on the obtained results the pyrazole derivatives generally induced the immune system to defend against pathogens or tumors by variable percentage. The highest immunostimulatory action was shown by compound 8a (136.5 ± 0.3%) that has a pyrazole moiety as well as amide function (O=C-NH2) and piperidinyl core followed by compound 9b with a ratio (115.2 ± 0.5%) having pyrazole and benzamide (Ph-NH-C=O) in position four in addition to three methoxy groups.

Drug Resistance
The synthesized pyrazole Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b were tested for their in vitro antibacterial activity against a panel of multidrug-resistant bacteria (MDRB) classified as one clinical strain S. aureus (ATCC 43300) (MRSA) with three standard strain S. aureus (ATCC 33591), E. coli (ATCC BAA-196), and P. aeruginosa (ATCC BAA-2111). The broad-spectrum antibiotic Norfloxacin was used as a positive control. The obtained results from Table 6 revealed that the synthesized To determine the safety and selectivity of the most potent Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b, the cytotoxicity of promising compounds was determined on healthy non-cancer cells (Vero cells, ATCC CCL-81) from an African green monkey kidney continuous cell by colorimetric MTT assay. The new Schiff bases showed very lower toxicity on healthy non-cancer cells Vero cells with (IC 50 > 120), and that proved that these new Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b displayed safety and revealed selectivity toward cancerous cells.
Finally, from all the previously studied and because of the efficiency of the promising six Schiff bases as antimicrobial and antiproliferative potential agents, these derivatives were chosen for further investigation.

Immunomodulatory Activity
In this section, we gain insight into the in vitro immunomodulatory potential of the active compounds. Innate immunity is the first line of defense against pathogens present in our environment such as fungi, bacteria, and viruses and eliminates damaged cells and tumor cells characterized by rapid response, infection halting. The innate immunity system may include macrophage, mast cells, dendritic cells (DCs), eosinophils, basophils, neutrophils as well as invariant natural killer cells (NK cells) [64]. The neutrophil can cause microorganisms intracellular killing [65]. The immunomodulatory activity expressed by percentage (%) values for the intracellular killing was performed using nitro blue tetrazolium (NBT) reductase assay, where the increasing percentage is related to improvement in the killing ability of the neutrophils, and was performed according to the reported method [66]. The results are represented in Table 5. The potent Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b were evaluated as immunomodulatory agents by percentage values that ranged between (49.6 ± 0.14%) and (136.5 ± 0.3%) and depending on the obtained results the pyrazole derivatives generally induced the immune system to defend against pathogens or tumors by variable percentage. The highest immunostimulatory action was shown by compound 8a (136.5 ± 0.3%) that has a pyrazole moiety as well as amide function (O=C-NH 2 ) and piperidinyl core followed by compound 9b with a ratio (115.2 ± 0.5%) having pyrazole and benzamide (Ph-NH-C=O) in position four in addition to three methoxy groups.

Drug Resistance
The synthesized pyrazole Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b were tested for their in vitro antibacterial activity against a panel of multidrug-resistant bacteria (MDRB) classified as one clinical strain S. aureus (ATCC 43300) (MRSA) with three standard strain S. aureus (ATCC 33591), E. coli (ATCC BAA-196), and P. aeruginosa (ATCC BAA-2111). The broad-spectrum antibiotic Norfloxacin was used as a positive control. The obtained results from Table 6 revealed that the synthesized pyrazole Schiff bases showed varying degrees of activities with strong activities (inhibition zone higher than 15 mm) against the tested microorganisms and therefore, MIC and MBC were performed and are shown in Table 7.
From Table 7, it is observed that the MIC values range, for the most promising six Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b, between 1.95 and 15.62 µg/mL as well as MBC values between 3.31 and 31.25 µg/mL against MDRB strains compared with Norfloxacin MIC (0.78-3.13 µg/mL) and MBC (1.56-4.69 µg/mL). Most of the tested pyrazole Schiff bases 6b, 7b, 7c, 8a, 8d, and 9b showed remarkable broad-spectrum activities against both Gram-positive and Gram-negative bacteria for inhibitory or bactericidal activity and among them Schiff bases 8a and 9b showed the highest activities in comparison with other derivatives; compound 8a with mono-pyrazole, piperidinyl, and p-tolyl-amide derivative demonstrated MIC values ranging between (1.95 and 7.8 µg/mL). Besides, Schiff base 9b having a mono pyrazole nucleus and benzamide in position four also showed MIC values (1.95-6.25 µg/mL) and MBC values (3.31-11.87 µg/mL). According to the Clinical and Laboratory Standards Institute (CLSI) standards and by applying the MBC/MIC ratio, we found that all the tested pyrazole Schiff bases exhibited values less than two, meaning bactericidal property.  In continuation of our previous efforts, we targeted the most two active Schiff bases (8a and 9b) depending on previous studies as antimicrobial and anticancer agents from the previous results to evaluate their activities and mechanism against DHFR and DNA gyrase enzymes. The two active Schiff bases (8a and 9b) were introduced to enzyme assay against dihydrofolate reductase (DHFR) on E. coli organism and DNA gyrase with two different organisms as S. aureus and B. subtilis to determine the inhibitory activities expressed as IC 50 (µM), using Trimethoprim and Ciprofloxacin, respectively, as the reference drugs. The result is shown in Table 8 and Figure 3. Schiff bases (8a and 9b) were introduced to enzyme assay against dihydrofolate reductase (DHFR) on E. coli organism and DNA gyrase with two different organisms as S. aureus and B. subtilis to determine the inhibitory activities expressed as IC50 (µM), using Trimethoprim and Ciprofloxacin, respectively, as the reference drugs. The result is shown in Table 8 and Figure 3.
Finally, it is noticed that designing new Schiff bases with pyrazole moiety (mono or Bis) with different amide and aniline derivatives in position four and three respectively, based on pyrazole nucleus prepared from cyan-acetanilide derivatives, and the reaction of amino pyrazole obtained with different aldehyde may improve the antimicrobial activity (MIC, MBC, MDRB); therefore, the enzyme inhibitory activity (DNA gyrase) and dihydrofolate reductase (DHFR) and anticancer activity against the two different cell lines, HepG-2 and MCF-7, determine the safety and selectivity on Vero cell line.

Docking and Molecular Modeling Study
In recent years, molecular modeling study is considered as an important study because it provides a more accurate picture of biologically active molecules at the atomic level and plays a  1.32 µM). Moreover, Schiff base 8a (3.98 ± 0.61 µM) with pyrazole core and piperidinyl pharmacophore showed DHFR enzyme with 1.3-fold higher potential than the positive control (Trimethoprim, 5.17 ± 0.12 µM) while the Schiff base 9b with one pyrazole moiety showed IC 50 value (6.48 ± 0.33 µM) with (0.80 fold), respectively.
Finally, it is noticed that designing new Schiff bases with pyrazole moiety (mono or Bis) with different amide and aniline derivatives in position four and three respectively, based on pyrazole nucleus prepared from cyan-acetanilide derivatives, and the reaction of amino pyrazole obtained with different aldehyde may improve the antimicrobial activity (MIC, MBC, MDRB); therefore, the enzyme inhibitory activity (DNA gyrase) and dihydrofolate reductase (DHFR) and anticancer activity against the two different cell lines, HepG-2 and MCF-7, determine the safety and selectivity on Vero cell line.

Docking and Molecular Modeling Study
In recent years, molecular modeling study is considered as an important study because it provides a more accurate picture of biologically active molecules at the atomic level and plays a major role in the drug designing process [67]. Docking process was performed using Molecular Operating Environment software 10.2008 (MOE), Chemical Computing Group Inc., Montreal, Quebec, Canada. Two proteins were obtained from the protein data bank (https://www.rcsb.org/) for this purpose. First one (PDB: 1DLS) for dihydrofolate reductase enzyme (DHFR) that co-crystallized with Methotrexate (MTX) [68], and another one is S. aureus DNA gyrase enzyme (PDB: 2XCT) and Ciprofloxacin was found co-crystallized inside the pocket [57,60]. The docking results involving all energy score (S) (Kcal/mol), binding of amino acids inside pocket with interacting groups of the ligand in docking compounds, and lengths of the bond are summarized in Table 9 and represented in Figure 4a-d.

(-) indicate arene-cation interaction; (-)indicate arene-arene interaction
Docking of the promising compounds 8a, 9b inside the active site of (PDB: 1DLS) for the dihydrofolate reductase enzyme (DHFR) revealed that compounds 8a, 9b provide a docking pose with an energy score (S= −18.96 and −26.13 Kcal/mol) compared to Methotrexate (MTX) (S = −27.31 Kcal/mol) that forms five hydrogen bonds, two hydrogen bond donor with Ile 7 and Glu 30 two amino of pyrimidine, and three hydrogen bond acceptor two with Arg 70 and the third one with Asn 64 with bond length (3.12, 2.68 and 2.64 Å). It was observed that the two Schiff bases 8a and 9b (Figure 4a,b) mainly interacts with the target enzyme by showing hydrogen bonding interaction for example, Schiff base 8a with IC 50 values (3.98 ± 0.61 µM) showed two hydrogen bonds with one hydrogen bond donor between Asp 21 and NH of pyrazole nucleus and another hydrogen bond acceptor between Lys 55 and the oxygen atom of the methoxy group of the p-anisidine amine with bond length 3.10 and 3.33 Å respectively. On the other hand, compound 9b showed energy score near to Methotrexate (MTX), with three hydrogen bond acceptor between Arg 28 and two oxygen of methoxy groups of tri-methoxy benzaldehyde derivatives with (2.84 and 3.07 Å), and the third methoxy group forms a bond with Ars 64 with bond length (3.00 Å) as well as two arene-arene interaction with two amino acids Phe 34 and Phe 31, beside hydrophobic interaction.
Molecules 2020, 25, x FOR PEER REVIEW 13 of 24 major role in the drug designing process [67]. Docking process was performed using Molecular Operating Environment software 10.2008 (MOE), Chemical Computing Group Inc., Montreal, Quebec, Canada. Two proteins were obtained from the protein data bank (https://www.rcsb.org/) for this purpose. First one (PDB: 1DLS) for dihydrofolate reductase enzyme (DHFR) that co-crystallized with Methotrexate (MTX) [68], and another one is S. aureus DNA gyrase enzyme (PDB: 2XCT) and Ciprofloxacin was found co-crystallized inside the pocket [57,60]. The docking results involving all energy score (S) (Kcal/mol), binding of amino acids inside pocket with interacting groups of the ligand in docking compounds, and lengths of the bond are summarized in Table 9 and represented in Figure 4a-d.    Table 9) [57,60]. As for amino acid interactions, Schiff base 8a showed two hydrogen bond donors with Asp 508 through the NH of pyrazole, in addition to hydrogen bond between Pro 1080 and amino of amide group ( Figure  4c). Schiff base 9b formed two hydrogen bonds (Figure 4d) Lys 1043 and Ser 1085 through the oxygen of methoxy group and NH of pyrazole as well as two arene-cation interactions between phenyl derivatives of tri-substituted aldehyde and amide derivatives with Lys 460.
Finally, all the docked compounds showed desirable interaction, especially hydrogen bond and arene-cation or arene-arene interaction with two protein targets, and that proceeds mainly through NH of pyrazole or oxygen of methoxy groups, present in our new promising compounds. It can be concluded that the newly designed pyrazole Schiff bases, mainly mono pyrazole Schiff bases 8a and 9b could exert their antimicrobial, anticancer activity with selectivity to the cancer cell and induced increased immunity against pathogen and tumor via two different mechanisms as DHFR and DNA gyrase inhibitors.  Table 9) [57,60]. As for amino acid interactions, Schiff base 8a showed two hydrogen bond donors with Asp 508 through the NH of pyrazole, in addition to hydrogen bond between Pro 1080 and amino of amide group (Figure 4c). Schiff base 9b formed two hydrogen bonds (Figure 4d) Lys 1043 and Ser 1085 through the oxygen of methoxy group and NH of pyrazole as well as two arene-cation interactions between phenyl derivatives of tri-substituted aldehyde and amide derivatives with Lys 460.
Finally, all the docked compounds showed desirable interaction, especially hydrogen bond and arene-cation or arene-arene interaction with two protein targets, and that proceeds mainly through NH of pyrazole or oxygen of methoxy groups, present in our new promising compounds. It can be concluded that the newly designed pyrazole Schiff bases, mainly mono pyrazole Schiff bases 8a and 9b could exert their antimicrobial, anticancer activity with selectivity to the cancer cell and induced increased immunity against pathogen and tumor via two different mechanisms as DHFR and DNA gyrase inhibitors.

Predication of the Physicochemical and Pharmacokinetics Properties
The physicochemical properties to evaluate the drug-likeness of the two pyrazole Schiff bases (8a and 9b) were generated in silico using Swiss ADME (http://swissadme.ch/index.php#undefined). Also, the reference drugs (Norfloxacin, Ciprofloxacin, and Trimethoprim) were generated, and the results are shown in Table 10.
From Table 10, the number of hydrogen bond acceptors (nHBA), donors (nHBD), and lipophilicity property for the two pyrazole Schiff bases were in agreement with the Lipinski's rule of five (Ro5). The molecular weight of the Schiff base (9b) is more than 500 g/mol. The pyrazole Schiff bases must not be violated more than one parameter to become an oral drug concerning bioavailability [70]. Therefore, the two pyrazole Schiff bases (8a and 9b) may become an oral drug. Veber (GSK) filter includes two parameters: (a) nRB ≤ 10 as rotatable bonds, (b) TPSA ≤ 140 as topological polar surface area [71]. According to this rule, the pyrazole Schiff base 8a is in agreement with Veber filter.
The pharmacokinetics properties of the two pyrazole Schiff bases (8a and 9b), and the reference drugs (Norfloxacin, Ciprofloxacin, and Trimethoprim) were generated in silico using Swiss ADME (http://swissadme.ch/index.php#undefined), shown in Table 11 and we can deduce that: -Schiff base 8a and the three reference drugs show a high gastrointestinal absorption (GI). - The two pyrazole Schiff bases (8a and 9b) and the reference drugs did not show any effect on the central nervous system as none of the Schiff bases show blood-brain barrier (BBB) permeation. -None of the Schiff bases are predicted as non-substrate for the permeability of glycoprotein (P-gp).
The in vitro enzyme assay of the most promising compounds 8a and 9b was carried out against DNA gyrase and dihydrofolate reductase (DHFR) enzymes using Ciprofloxacin and Trimethoprim as reference drugs according to previous work [68].

Molecular Docking Study
Docking simulations were performed using Molecular Operating Environment (MOE) software version 2008.10, Chemical Computing Group Inc., Montreal, Quebec, Canada. The docking process methodology of new compounds was performed according to previous work [57,58,68].

Conclusions
In conclusion, a new series of mono and bis-pyrazole Schiff bases 6a-d-9a-d were synthesized successfully and evaluated preliminary for their in vitro antimicrobial activities. From inhibition zones values, it was found that six Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) displayed more than or near to the reference drugs (Tetracycline and Amphotericin B). Therefore, Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) were evaluated for their minimal inhibitory, minimal bactericidal concentrations, antiproliferative activities against two cell lines (HepG-2 and MCF-7), immunomodulatory and drug resistance properties as well as antiproliferative activity against healthy non-cancer Vero cells that exhibited IC 50 values (≥120 µM). The results of evaluation exhibited that, the two Schiff bases containing mono-pyrazole 8a and 9b were more potent among the Schiff bases (6b, 7b, 7c, 8a, 8d and 9b) in this study. The results of enzyme assay for the two Schiff bases 8a and 9b against dihydrofolate reductase (DHFR) and DNA gyrase exhibited that IC 50 values of the both Schiff bases 8a and 9b were stronger than Trimethoprim and Ciprofloxacin drugs, respectively. The molecular docking revealed that the two Schiff bases 8a and 9b showed desirable interaction, especially hydrogen bond and arene-cation or arene-arene interaction with the two protein targets 1DLS and 2XCT. Finally, the physicochemical and pharmacokinetic properties predication displayed that the two pyrazole Schiff bases may show drug-likeness and considered as candidates for the discovery or development of new drugs.