Biological Evaluation and Molecular Docking with In Silico Physicochemical, Pharmacokinetic and Toxicity Prediction of Pyrazolo[1,5-a]pyrimidines

Pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i were synthesized for evaluation of their in vitro antimicrobial properties against some microorganisms and their immunomodulatory activity. The biological activities of pyrazolo[1,5-a]pyrimidines showed that the pyrazolo[1,5-a]pyrimidines (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h) displayed promising antimicrobial and immunomodulatory activities. Studying the in silico predicted physicochemical, pharmacokinetic, ADMET and drug-likeness properties for the pyrazolo[1,5-a]pyrimidines 5a–c, 9a–c and 13a–i confirmed that most of the compounds (i) were within the range set by Lipinski’s rule of five, (ii) show higher gastrointestinal absorption and inhibition of some CYP isoforms, and (iii) have a carcinogenicity test that was predicted as negative and hERG test that presented medium risk. Moreover, the molecular docking study demonstrated that the compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h are potent inhibitors of 14-alpha demethylase, transpeptidase and alkaline phosphatase enzymes. This study could be valuable in the discovery of a new series of drugs.


Introduction
Treatment of infectious diseases remains a worldwide problem because of the increasing multi-drug resistance caused by human pathogenic microbes. Therefore, the design of new compounds acting as antimicrobial agents is an essential approach to overcome the problem of drug resistance [1].

In Vitro Antimicrobial Evaluation
The antimicrobial activities inhibition zone (IZ, mm ± standard deviation) of the pyrazolo[1,5-a]pyrimidines (5a-c, 9a-c and 13a-i) were evaluated using the agar plate diffusion method [27,28]. The results of the inhibition zone are listed in Table 1.
In order to further study and conclude which of the above promising pyrazolopyrimidines compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h may be bactericidal or show bacteriostatic action, the action of the compounds will be deduced from the relationship between MIC and MBC or MFC and from a comparison between the values of the compounds ( Table 2). The range ratio between MIC-MBC was 1-2 ratios; antibacterial agents are generally regarded as bactericidal if the MBC is no more than four times the MIC [32,33], and the values of the compounds are in this range. Therefore, the results indicated that the pyrazolopyrimidine compounds exhibited bactericidal and fungicidal properties in comparison to Amoxicillin/Clavulanic acid as an antibacterial standard and Nystatin as an antifungal standard.

Immunomodulatory Activity for Active Compounds
In this study, the immunomodulatory activity of the active pyrazolopyrimidine compounds was investigated by in vitro test. The most potent compounds depending on the previous antimicrobial results were chosen to evaluate their immunomodulatory activity for it was predicted that these compounds may have a dual function. The neutrophils play a primary role as an effecting or killer cell for many types of infections [34].
The active pyrazolopyrimidine compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h were evaluated by nitroblue tetrazolium (NBT) reduction test, and the results were presented as intracellular killing percentage % values and listed in Table 3. The highest immunostimulatory action were 13d, 13e and 9a with 136.5 ± 0.3, 129.8 ± 0.47 and 125.6 ± 0.44, respectively. In various in vitro and in vivo bioassays, Zymosan represents an efficient chemo-attractant parameter, where the nucleophile can cause microorganism intracellular killing.
From Table 3, an increase in the intracellular killing activity of neutrophils can be observed. Therefore, the effectiveness of the body's immune system may be activated by these compounds, as the neutrophils play a primary role as an effecting or killer cell for many types of infections.

Structure-Activity Relationship (SAR)
From the results ( Table 2) of in vitro antimicrobial activities of pyrazolopyrimidine compounds 5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h against the screening organisms, it was found that some pyrazolopyrmidine derivatives bearing X = Cl (Chloro atom, electron withdrawing group) were more active than those bearing X = CH 3 (Methyl group, electron donating group), whereas 13h was more active than 13e. Furthermore, 9c was more active than 9a, where the derivatives bearing X = Cl (Chloro atom) were more active than those bearing X = H (Hydrogen atom, without substitutions) against some of the screening organisms ( Figure 3).  Figure 3. A brief structure-activity relationship (SAR) study of the active pyrazolopyrimidines. Lipinski's rule of five were calculated using SwissADME web (http://swissadme.ch/index.php#undefined) and are shown in Table 4.

Pharmacokinetic Properties of
All the compounds 5a-c, 9a-c and 13a-i show high gastrointestinal absorption (GI absorption) except 13i. All the compounds 5a-c, 9a-c and 13a-i are not predicted to penetrate the blood-brain barrier (BBB) and are non-substrates for P-glycoprotein (P-gp). Therefore, they have no effect on the central nervous system. Inhibition of the five major CYP isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4) is certainly one major cause of pharmacokinetic-related drug-drug interactions. The compounds 13b-i are non-inhibitors of the CYP1A2 enzyme, the two compounds 9a and 9b are non-inhibitors of the CYP2C19 enzyme and all compounds 5a-c, 9a-c and 13a-i are inhibitors and are active against the CYP2C9 enzyme. The series 9a-c are non-inhibitors of the CYP2D6 enzyme and compound 9a is a non-inhibitor of the CYP3A4 enzyme [36].
The results of the in vitro Caco-2 cell permeability indicated that all pyrazolo[1,5-a]pyrimidines 5a-c, 9a-c and 13a-i exhibited moderate permeation.
On the in vitro MDCK cell permeability test, all the pyrazolo[1,5-a]pyrimidines 5a-c, 9a-c and 13a-i showed permeation less than 25 nm/s, indicating low permeability.
For the in vitro skin permeability test for the delivery of drugs via transdermal administration, all the pyrazolo[1,5-a]pyrimidines 5a-c, 9a-c and 13a-i exhibited negative values.
For the in vitro plasma protein binding (PPB) test, most of the pyrazolo[1,5-a]pyrimidines were predicted more than 90%, which indicates decreased excretion and increased half-life.
On the Ames test that assesses mutagenicity, all the pyrazolo[1,5-a]pyrimidines were predicted to be mutagens. Moreover, on analyzing carcinogenicity in animals (mouse), all the compounds were predicted as positive, except compound 5b which presented negative, while for the carcinogenicity test in animals (rat), the compounds 5c, 9a-c, 13c and 13f-i were predicted as negative.
In the case of the hERG encodes potassium channels test, compound 9a presented high risk; 13a presented ambiguous risk and the rest of the compounds presented medium risk.

Bacteria
There are about 30 enzymes involved in the biochemistry of the cell wall of bacteria. Antibiotics such as the penicillin series work on the inhibition of the final cross link step by inhibiting the transpeptidase enzyme. The E-score (energy score), considered as one of the most important factors, reflects the interaction between the ligand and enzyme. The molecular docking validation explains the interaction (E-score) between the reference ligand, 2-[N-cyclohexylamino]ethane sulfonic acid, (E-score = −5.23) and the compounds (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h) with a transpeptidase enzyme as −6.56, −6.47, −6.82, −6.44, −6.50, −7.17, −6.97 and −6.87, respectively. Furthermore, Figure 4 shows the 2D and 3D interaction diagrams of compound 13d with transpeptidase.

Fungi
Ergosterol biosynthesis is considered as a very important step in the building of the fungal cell membrane. 14-Alfa demethylase is the responsible enzyme that converts lanosterol to ergosterol. The molecular docking study (E-score) between the reference ligand, lanosterol, (E-score = −8.06) and the synthesized compounds (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h) Figure 5 shows 2D and 3D interaction diagrams of compound 5c with 14-α demethylase.

Immunity Docking
Nitro blue tetrazolium (NBT) is an organic salt compound purchased as the chloride salt based on two tetrazole moieties. These moieties have sensitivity to alkaline phosphatase (ALP) enzyme and therefore are used as a test in immunology to detect the reactivity of organic compounds on the immunity system. ALP enzyme is an ahomodimeric metalloenzyme promoting the unspecified hydrolysis of the phosphate monoesters process. This enzymatic promotion proceeds by a phosphoseryl intermediate to give inorganic phosphate and an alcohol. ALP enzyme structure as a phosphate has been determined by X-ray technique [38]. The inhibition of ALP presents a unique challenge since the active site pocket is characteristically shallow and, in continuation of our work [39][40][41] to discover new drug enzyme interactions, we present this calculated part.
The active site analysis of the ALP protein was performed from a database of similar amino acid residues (Glu411, Arg166, His331, Asp269, Lys328, Asn263, Asp153, Asp327, His412, Ser102, Gly150, Asp51, His370 and Tyr169). The 2D pocket in the alkaline phosphatase (ALP) complex with 13e as a ligand explained that the interaction between them is potent through hydrogen bonds. Furthermore, Figure 6 presents the 2D the interaction between 13e as a ligand with alkaline phosphatase (ALP) residues.

Fungi
Ergosterol biosynthesis is considered as a very important step in the building of the fungal cell membrane. 14-Alfa demethylase is the responsible enzyme that converts lanosterol to ergosterol. The molecular docking study (E-score) between the reference ligand, lanosterol, (E-score = −8.06) and the synthesized compounds (5c, 9a, 9c, 13a, 13c, 13d, 13e and 13h)

Immunity Docking
Nitro blue tetrazolium (NBT) is an organic salt compound purchased as the chloride salt based on two tetrazole moieties. These moieties have sensitivity to alkaline phosphatase (ALP) enzyme and therefore are used as a test in immunology to detect the reactivity of organic compounds on the immunity system. ALP enzyme is an ahomodimeric metalloenzyme promoting the unspecified hydrolysis of the phosphate monoesters process. This enzymatic promotion proceeds by a phosphoseryl intermediate to give inorganic phosphate and an alcohol. ALP enzyme structure as a phosphate has been determined by X-ray technique [38]. The inhibition of ALP presents a unique challenge since the active site pocket is characteristically shallow and, in continuation of our work [39][40][41] to discover new drug enzyme interactions, we present this calculated part.
The active site analysis of the ALP protein was performed from a database of similar amino acid residues (Glu411, Arg166, His331, Asp269, Lys328, Asn263, Asp153, Asp327, His412, Ser102, Gly150, Asp51, His370 and Tyr169). The 2D pocket in the alkaline phosphatase (ALP) complex with 13e as a ligand explained that the interaction between them is potent through hydrogen bonds. Furthermore,  The selection of pyrazolopyrimidine scaffold analogs (5a-c, 9a-c, 13a-i) to build a pharmacophore for potential alkaline phosphatase (ALP) inhibitors was based on the high potency superposition of 13e that generated a pharmacophore with H-bond acceptors (Acc, Acc2), with

Pharmacophore and Electrostatic Map of 13e
The selection of pyrazolopyrimidine scaffold analogs (5a-c, 9a-c, 13a-i) to build a pharmacophore for potential alkaline phosphatase (ALP) inhibitors was based on the high potency superposition of 13e that generated a pharmacophore with H-bond acceptors (Acc, Acc2), with H-bond donor projection (Don), an aromatic center (Aro) and which was hydrophobic (hyd) (Figure 7). The electrostatic map of compound 13e shows the hydrophilic section as a violet color and the lipophilic part as a blue color (Figure 8).
The antimicrobial activities inhibition zone (IZ, mm ± standard deviation) was measured according to the agar plate diffusion method [27,28] (see Supplementary Material).

Molecular Docking
The standard docking protocol was carried out using MOE 2015.10 software. The proteins in the mdb file downloaded from the PDB (protein data bank) were transpeptidase (PDB code: 4ZTK), 14-alpha demethylase (PDB code: 4LXJ) and alkaline phosphatase ALP (PDB code: 1EW8) (http://www.rcsb.org/pdb/home/home.do).

Conclusions
In this work, a series of pyrazolo[1,5-a]pyrimidine derivatives 5a-c, 9a-c and 13a-i were synthesized for evaluation of their in vitro antimicrobial and immunomodulatory activities. The result of pyrazolo [1,5-a]pyrimidines exhibited that most of the compounds displayed significant antimicrobial (bactericidal and fungicidal properties) and immunomodulatory activities. Furthermore, the in silico predicted physicochemical, pharmacokinetic, ADMET properties and drug-likeness studies of the pyrazolo[1,5-a]pyrimidines 5a-c, 9a-c and 13a-i revealed that the compounds fulfill Lipinski's rule requirements and have good drug score values, particularly in 13c (DLS = 1.31) and 13i (DLS = 1.44). Furthermore, the molecular docking study was compatible with antimicrobial and immunomodulatory activities. These preliminary results of pyrazolo[1,5-a]pyrimidines as antimicrobial activities and the structure-activity relationship with molecular docking could provide an exceptional model that may lead to the discovery of new drugs.
Supplementary Materials: The following are available online, Tables S1-S3, Spectral data of compounds (1a-c, 5a-c, 9a-c and 13a-i) and biological methods are available online.

Conflicts of Interest:
The authors declare no conflicts of interest.