Synthesis, In Silico Study and Antibacterial Evaluation of New Cyanopyridine Based Scaffold

Abstract Synthesis, antibacterial evaluation, and in silico study of new series of cyanopyridines, and fused cyanopyridines are described. One-pot reaction of N-(2-ethylphenyl)-2-cyanoacetamide (1) with arylmethylketones and various aldehydes afforded 4, 6-diaryl-2-oxopyridine-3-carbonitriles, 4–9. Cyclocondensation of 1 using various aldehydes and diethyl malonate/malononitrile afforded 3-esters, 10–12 and pyridine-3, 5-dicarbonitrile derivatives, 13–15. Then, compounds 13, 14 were employed for the synthesis of various fused compounds, namely pyrazolo[3,4-d]pyridines, 16–18 and pyrido[2,3-d]pyrimidines, 21–26. These target compounds were tested in vitro for their antibacterial activity (minimum inhibitory concentration [MIC]) against Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA) using amoxicillin and trimethoprim/sulfamethoxazole (TMP/SMX) as reference standards. Compounds 4, 8, and 10–12 exhibited remarkable antibacterial profile compared to the standard drugs. Besides, in silico absorption, distribution, metabolism, and excretion (ADME) prediction studies indicated that most of the potent compounds are orally bioavailable with no permeation to the blood–brain barrier (BBB).


Introduction
In past decades, the challenge of bacterial resistance is rising around the world, leading to great mortality and increased healthcare expenses. The World Health Organization has designated the antibiotic resistance as one of the three principal public health menaces of the 21st century. Indeed, it remains necessary to develop new antibacterial agents with novel structures and modes of action to tackle this issue. [1][2][3][4][5][6][7] Staphylococcus aureus is a Gram-positive, prevalent bacterial pathogen that can entirely modify and can live in diverse states. It has been declared that S. aureus can hold up in an inanimate setting, prevailing as a colonizer and may construct biofilms. 8,9 In addition; S. aureus is an important cause of a broad array of infections including bacteremia and infective endocarditis, osteoarticular, skin, soft tissue, and pleuropulmonary infections. 10 The dissemination of methicillin-resistant Staphylococcus aureus (MRSA), particularly in nosocomial health surroundings, is a notable health threat and economic load worldwide. 11 The overall universal trend manifests an alarming climb and expansion of MRSA. 12 Preventive strategies explicitly aimed MRSA could reduce healthcare costs and improve patient outcomes. 13,14 The currency of MRSA is fast accelerating worldwide but is a prime public health concern in developing countries. 15,16 On the other hand, Gram-negative bacteria, namely Escherichia coli diarrheal disease in humans can be originated by various pathotypes, each showing unique sets of virulence factors that adapt each pathotype for a specific function and assign to a typical disease syndrome. 17 Lack of surveys and gathering of epidemiological data and absence of efficient health control protocols are imparting to the spread of bacterial infections.
Keeping in mind our previous efforts 27 on synthesis and evaluation of cyanopyridines as anticancer agents, to further assess, the biological profile of this class of compounds. Our ongoing studies aimed at the discovery of new cyanopyridine derivatives acting as antibacterial agents. Thus, our target compounds were designed to encounter various functionalities at p-4 and p-6 of pyridine core in monocyclic 1,4,6-tri-arylpyridines. Additionally, the importance of p-5 will be explored via incorporation of CN or COOEt functionality in 1,4-diarylpyridines. As a part of our ongoing endeavor to synthesize novel heterocyclic rings through simple and straightforward convenient routes, therefore; some pyridine derivatives can be utilized as efficient scaffolds for constructing fused-ring system, pyrazolo [3,4-d]pyridines and pyrido [2,3-d]pyrimidines as an interesting structural variation, hoping to improve the anticipated antibacterial activity. Furthermore, the physicochemical properties and absorption, distribution, metabolism, and excretion (ADME) prediction for the new hits will be studied.

In vitro antimicrobial screening
In vitro antimicrobial screening Antimicrobial susceptibility testing of the newly synthesized compounds was detected against Gram-positive bacterial isolates namely S. aureus ATCC 25923, Gramnegative bacterial isolates namely (E. coli ATCC 25922) and MRSA, amoxicillin and trimethoprim/ sulfamethoxazole (TMP/SMX) were used as reference standards and dimethyl sulfoxide (DMSO) was used as a negative control. Standard bacterial strains were obtained from The Regional Center for Mycology and Biotechnology, Al-Azhar University Cairo, Egypt. Antimicrobial susceptibility testing was performed by broth microdilution assay to determine minimum inhibitory concentration (MIC) according to guidelines of CLSI2018 standard methodology. 29 For the determination of MIC of the tested chemical compounds, microdilution test was performed in 96-well microtiter plates. Two-fold dilution of each compound were prepared in the test wells with Muller Hinton Broth (Oxoid), 100 ml\well, the final drug concentration being (125-0.004) mg/ml. A constant volume (100 ml/well) of bacterial suspension adjusted to match 0.5 McFarland's turbidity standard and diluted (1/100) in case of S. aureus and (1/1000) in case of E. coli. Three control wells were prepared containing sterile Muller Hinton Broth (background control), bacterial suspension with DMSO (growth control) and bacterial suspension alone without drug (growth control). The plates were sealed and incubated for 24 h at 37 C; then the wells were examined for turbidity. MIC was detected as the lowest sample concentration showing no visible turbidity that prevented microbial growth. Each MIC was determined three times. The test compounds were also compared with reference broad spectrum antibiotics Amoxicillin and TMP/SMX.

Evaluation of minimum inhibitory concentration (MIC)
Many antimicrobial agents have been introduced into remedy; however, the field still needs vast efforts for the evolution of recent antimicrobial agents to master the extremely resistant strains of microorganisms. The wide range of activity profile of cyanopyridines and fused pyridines probes us to test and inspect the antibacterial activity of our novel cyanopyridine analogs via MIC determination. 21 The test compounds 4-26 were examined in vitro for their antibacterial activity against E.coli, S. aureus, and MRSA using Amoxicillin and TMP/SMX as reference standards. After 24 h incubation at 37 C, the MICs were measured in mg/mL (Table 2). Results revealed that, 17 out of the 24 screened compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative microorganisms. With regard to the activity against S. aureus, 4, 6-diarylpridone, 4 (MIC 7.8 mg/mL) is the most potent compound; it is two folds more active than TMP/SMX (MIC 15.6 mg/mL) and it also exhibited half the potency of Amoxicillin. Compounds, 11, 15, 16, and 23 displayed equipotent antibacterial activity to TMP/SMX (MIC 15.6 mg/mL). Moderate activity was noticed for the rest of compounds (MIC 31.25 mg/ml), however compounds 17, 18, 21, 24 and 25 were inactive. Compound 8 was the most potent antibacterial one. The structure-activity relationship (SARs) Scheme 2. Synthesis of cyanopyridine derivatives 10-15. Scheme 3. Synthesis of cyanopyridine derivatives 16-18. shows the contribution of lipophilic and electronic factors on the antibacterial activity of cyanopyridine compounds.
Concerning the antibacterial activity against MRSA, the ester 11 exhibited 20 times (MIC 3.9 mg/mL) the activity of amoxicillin (MIC >80mg/mL) and 16 folds the activity of TMP/SMX (MIC 62.5 mg/mL). In addition, compounds 8, 10 (MIC 7.8 mg/mL) showed 8 folds the activity of TMP/SMX and compound 4 displayed 4 folds of the same reference standard. Regarding compounds 12, 20, and 23, they elicited two folds, while the other compounds were equipotent to the antibacterial activity of TMP/SMX. What is more, most of compounds showed better antibacterial activity than amoxicillin (Table 2 and Figure 2).

Structure-activity relationships
It is worth noting from SARs study that, among 4,6-diarylpyridines, the pair 5 (R2 4-ClPh), 7 (R 2 ¼4-OCH 3 Ph) bearing the same R 1 (4-NO 2 Ph) have similar activity profile which means that electronic effect is not important, however for 6: R 2 ¼ 4-OHPh, the activity is abolished. This may be due OH group imparted high polar character; it is interesting to mention that the high   Concerning ring fusion as in pyrazolo [3,4-b]pyridines, [16][17][18]; this fusion has a negative impact on the antibacterial activity either through decreasing it as in analog 16 or abolishing it as in 17, 18. Ultimately, the antibacterial profile of N-fromamidates 19, 20 is close to their parents 13, 14, but in a reversed pattern. In regard of N-aryl-pyrido [2,3-d]pyrimidines, 23-26, it is noticed that compounds 24, 25 are inactive; however, compound 23 bearing N-4-OCH 3 Ph elicited enhanced activity (MIC 15.6 mg/mL) against S. aureus and diminished activity against E. coli compared to the parent 13. Concerning N-tolyl derivative, 26 the MIC range remains constant (MIC 31.25 mg/ mL) toward E. coli but the enhanced result was demonstrated against S. aureus compared to its parent, 14. Our hits elicited pronounced activity against S. aureus than E. coli bacterial strains. A close examination of the structures of the active compounds revealed that their antibacterial activity is strongly bound to the nature of the substituent at p-4 and p-6 of pyridine core.

In silico studies
In silico prediction of physicochemical properties SWISS ADME 33 was used to evaluate the pharmacokinetic properties of the most active cyanopyridine derivatives 4, 8, 10-12 ( Table 3). According to the data, the molar mass of the indole chalcones is less than 500 ranging from 392.45 to 478.49, indicating that they can be easily metabolized in comparison to larger molecules. While the Log P suggests the lipophilicity and values for 4, 8, 10-12 are in the range of 1.73 À 4.44. Log p value of them are in the   34 founding that the calculated % ABS of all these hits ranged between 63.01% and 90.02%, demonstrating that these synthesized derivatives may have the required cell membrane permeability and bioavailability. All compounds have rotatable bonds between 4 and 9 which indicating molecular flexibility to their bio target. 4, 8, 10-12 are found to have drug-like molecular properties and the probability of being lead candidates.
In silico prediction of absorption, distribution, metabolism, and excretion (ADME) properties The newly synthesized compound should have high biological activity in lower effective concentration with low toxicity and should be active until the desired action takes place, to be an effective drug. The drug discovery sector now utilizes the computational prediction of pharmacokinetic ADME properties as a useful tool to identify drug candidates.
In this study, various computational methods, SwissADME website, 33 and pkCSM, 35 were applied to assess whether the most active compounds 4, 8, 10-12 possess the optimum drug-likeness parameters or not.
The ADME properties of the active derivatives are given in Table 4. Drug absorption was evaluated using solubility measurement and intestinal permeability. The aqueous solubility of the compounds is given as the logarithm of molar concentration and the solubility of designed compounds ranges from À5.712 to À7.361. The compounds are moderately water-soluble due to the presence of lipophilic functionalities aimed at improved cell permeability. As the absorption of an orally administrated drug occurs mostly through the small intestine, the percentage absorption of the compounds was evaluated. In general, Caco-2 permeability can predict the intake of oral drugs as Caco-2 from human colon carcinoma resembles intestinal epithelial cells. It is important to mention that the synthesized derivatives should have Papp > 8 Â 10 À6 cm/s for high permeability. Interestingly, 8, 10, 11, and 12 show high cell permeability (0.76 À 1.111), while 4 Another important consideration during preclinical analysis trial was to analyses the P-glycoprotein (P-gp) non-substrate candidature. P-gp works as efflux transporter which pumps drugs and other compounds and its substrate out of the cell, which was found to be one of explanation for its resistance to various chemotherapeutics for cancer. It has been found that P-gp is a critical transporter of drug which consequently results in resistance to anticancer drugs like Lonafarnib, and Taxanes. 36 It was found that, all the screened derivatives are not substrates of P-gp protein except 4 and 8 (c.f. Table 4), indicating that, these hits have a very low chance to efflux out of the cell, thus resulting in a maximum effect. Additionally, the distribution profile of the tested hits was predicted using a volume of distribution (Vd), fraction unbound, and blood-brain barrier (BBB) permeability. Higher VDss indicates better distribution of the drug in the tissues than in plasma, and if Log VDss > 0.45, it shows the greater distribution in the tissues. All the compounds are moderately distributed in the tissues. Drug efficacy calculated by fraction bound indicates that it is less bound to blood proteins and is free to diffuse. The BBB permeability was calculated by both SwissADME and pkSCM. The BBB permeation is very important as it affects the central nervous system as in the case of tuberculosis meningitis. All the screened derivatives are unable to cross BBB. Also, all the compounds interact with cytochromes either as substrates or as inhibitors. The total clearance of drugs (both hepatic and renal) was also studied and all the tested cyanopyridones show a total clearance of 0.124 À 0.311 logmL/min/kg. It can be concluded that 4, 8, 10-12 show good ADME properties and can be considered as probable lead candidates.

Conclusion
In summary, we utilized straightforward and fruitful methods to synthesize 4,6-diarylpyridines, pyridine-3,5-dicarbonitriles, and fused pyridine-3-carbonitriles. The fusion of pyridine-3,5-dicarbonitriles to pyrazolo [3,4-b]pyridines was not beneficial for antibacterial activity. It worth to mention that compounds 4, 8, 10, and 11 exhibited significant (maximum) antibacterial activities, which may develop into the potential class of antibacterial agents. The activity results indicated that some of the test compounds displayed the most promising broad-spectrum antibacterial activities. Finally, in silico studies revealed that these active derivatives considered as an active antibacterial framework and further studies are in progress in our laboratories and will be reported upon in the future.

Disclosure statement
No potential conflict of interest was reported by the authors.