Antibacterial evaluation and molecular properties of pyrazolo[3,4-b] pyridines and thieno[2,3-b]pyridines

A series of pyrazolo[3,4-b]pyridines (6a-h) and thieno[2,3-b]pyridines (8a-h) was synthesized for the evaluation of their in vitro antibacterial activities against four bacteria species (namely Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) and compared the result with the standard drug (Tetracycline). The result of the antibacterial evaluation showed that some pyrazolo[3,4-b]pyridines and thieno[2,3-b]pyridines display moderate antibacterial activity against the four bacteria species in this study. Furthermore, the physicochemical, pharmacokinetic, and drug-likeness properties were carried out using SwissADME website. The results of molecular properties show that all the pyrazolopyridines 6a-h and thienopyridines 8a-h showed agreement with the Lipinski and Veber rules. The two pyrazolo[3,4-b]pyridines 6b and 6c are almost in the range of the bioavailability radar pink area. Also, pyrazolo[3,4-b]pyridine derivatives 6a-h show high gastrointestinal absorption, all the derivatives except 6c are nonsubstrates for P-glycoprotein, and most of the derivatives show CYP isoform inhibition. 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 multidrug resistance caused by human pathogenic microbes. Therefore, the design of new compounds acting as antibacterial agents is an essential approach to overcome the problem of drug resistance (Shaaban et al., 2019).

Antibacterial activities
In vitro antibacterial activities of pyrazolo[3,4-b] pyridines 6a-h and thieno[2,3-b]pyridines 8a-h were measured against B. subtilis and S. aureus as Gram-positive bacteria and also against Escherichia coli and Pseudomonas aeruginosa as   Gram-negative bacteria species using a modified Kirby-Bauer disk diffusion method (Bauer et al., 1966;Osman et al., 2012). The bacteria were maintained on Meuller-Hinton agar. Dimethyl sulfoxide (DMSO) showed no inhibition zone (IZ). The agar media were incubated at 35°C-37°C for 24-48 hours for bacteria such as B. subtilis, S. aureus, E. coli, and P. aeruginosa. The diameter of the IZ (mm) was measured. Tetracycline is used as a reference for antibacterial activities.
The structure of 6a-h was established on the basis of spectral data. The IR spectrum of compound 6d shows bands at ν 3,577, 3,565, 3,414, and 3,296 cm −1 due to NH 2 and NH groups. Also, 1 H-NMR of 6d shows a broad signal at δ = 5.50 ppm assigned to an NH 2 group and another broad signal at δ = 12.0 ppm assigned to an NH group.
The second way was the reaction of pyridine-2-thione 4a-h with phenacyl bromide in dry ethanol to give the intermediate 7a-h which was cyclization to form the corresponding thieno[2,3-b] pyridines 8a-h.
The structure of thieno[2,3-b]pyridines 8a-h was established on the basis of spectral data. The IR spectrum of 8a revealed the bands at ν 3,577 and 3,285 for the NH 2 group and the band at ν 1,696 cm −1 for the carbonyl group. The 1HNMR spectrum contained a broad signal at δ = 7.26 ppm assignable to an amino function and a multiplet at δ = 7.36-7.88 ppm assigned to the aromatic protons.

In vitro antibacterial activities
Pyrazolo[3,4-b]pyridines 6a-h and thieno[2,3-b] pyridines 8a-h were screened in vitro for their antibacterial activities against four bacteria species (namely B. subtilis, S. aureus, E. coli, and P. aeruginosa) and compared with tetracycline as the standard drug. The results of antibacterial activities are shown in Table 2 and Figure 3, and we can found the following.

Physicochemical properties
The results of the computed physicochemical properties of the pyrazolo[3,4-b]pyridines 6a-h and thieno[2,3-b]pyridines 8a-h are shown in Table 3.  Drug-likeness was used for finding the oral drug candidates and was established based on the physicochemical properties. Lipinski's filter and Veber's filter are rule-based filters (Daina et al., 2017;Hassan et al., 2020;Lipinski et al., 2001;Veber et al., 2002).
From Table 3, all the pyrazolopyridines 6a-h and thienopyridines 8a-h showed agreement to Lipinski's rule and Veber's rule. Therefore, the two series 6a-h and 8a-h may be used as oral drug candidates.
The bioavailability radar was including lipophilicity, size, polarity, solubility, saturation, and flexibility of the physicochemical properties.
The optimal range of these properties was presented by the pink area (Lovering et al., 2009;Ritchie et al., 2011) and the properties of pyrazolo[3,4-b]pyridines 6a-h and thieno[2,3-b] pyridines 8a-h were presented by the red line.
From this study, we can conclude that the red line of two pyrazolo[3,4-b]pyridines 6b and 6c is almost in the range of the pink area. Therefore, the two compounds are nearly predicted orally bioavailable ( Fig. 4a and b) and we will modify them to obtain more active antibacterial agents.
In the future, these results provide the lead for the design of new derivatives of pyrazolo[3,4-b]pyridine and thieno [2,3-b] pyridine with advanced studies to obtain more potent antibacterial agents.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

FUNDING
There is no funding to report.

CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.

ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.

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