SYNTHESIS OF SOME NEW AZOLE DERIVATIVES AS ANTIBACTERIAL AGENTS

Antibiotic resistance is a growing global health threat and requires extensive research to combat this urgent problem. Phenylthiazoles, known for their diverse biological activities including anthelmintic, insecticidal, and antimicrobial properties, have recently gained particular attention as potential anti-MRSA lead compounds. This class of compounds is an established pharmacophore for the development of new antibacterial agents, particularly against multidrug-resistant bacteria such as MRSA, a notorious pathogen resistant to most first-line antibiotics. In-depth structure-activity relationship (SAR) studies of phenylthiazoles revealed two key features critical to their antibacterial activity: a nitrogen-containing head and a lipophilic tail. In this study, we aimed to reduce the lipophilicity of phenylthiazoles and improve their overall physicochemical and pharmacokinetic profiles by synthesizing a new series bearing primary amines at the phenyl-4 position. Notably, compounds 5m exhibited bactericidal activity against MRSA, exhibiting a minimum inhibitory concentration (MIC) of only 8 µg/mL against the prevalent MRSA strain USA300.


Introduction
The number of fatal infections caused by antibiotic-resistant bacteria has steadily increased in recent years and represents a major global health challenge(Ventola 2015, Aslam, Wang et al. 2018, Biondo 2023).Staphylococcus aureus, a notorious bacterial pathogen, is responsible for a wide range of infections, from minor skin lesions to lifethreatening invasive diseases such as soft tissue infections, pneumonia, endocarditis, and osteomyelitis.The emergence of methicillin-resistant S. aureus (MRSA) in the 1960s, triggered by resistance to lactam antibiotics, particularly methicillin, triggered numerous hospital epidemics (Shanson, Kensit et al. 1976, Levy and Marshall 2004, Rivera and Boucher 2011, Thati, Shivannavar et al. 2011, Elsebaei, Mohammad et al. 2018).Physicians today face enormous difficulties in treating MRSA infections due to the emergence of strains resistant to various classes of antibiotics, including tetracyclines, aminoglycosides, macrolides, lincosamides and fluoroquinolones (Jones, Karlowsky et al. 2003, Jones, Draghi et al. 2004, Pantosti, Sanchini et al. 2007).The situation has been further exacerbated by the recent emergence of drug-resistant S. aureus strains with intermediate susceptibility or resistance to vancomycin (VISA or VRSA) (Howden, Davies et al. 2010).Until recently, vancomycin served as the primary treatment option for MRSA infections (Tang, Hu et al. 2015).
As antimicrobial resistance reaches alarming levels, personal responsibility for combating it is becoming increasingly important.Our laboratory set out to find effective antibacterial agents, starting with the promising phenylthiazole scaffold.Phenylthiazoles were originally identified as a thiazole core with a guanidine head and an alkyl chain tail (Elsebaei, Mohammad et al. 2018, Elsebaei, Abutaleb et al. 2019, Elsebaei, Mohammad et al. 2019, Hosny, Abutaleb et al. 2020) have undergone extensive research and optimization to improve their effectiveness and metabolic profile.While they showed anti-biofilm activity, the poor permeability of the early phenylthiazoles likely contributed to their narrow spectrum (Eid, Elsebaei et al. 2017, Helal, Sayed et al. 2019, Mancy, Abutaleb et al. 2019, Elsebaie, El-Din et al. 2022, Sayed, Abutaleb et al. 2023).Consequently, changes to the tail led to the third generation, which showed intracellular effectiveness but required further optimization of the metabolic profile(El-Din, Elsebaie et al. 2023, Omara, Hagras et al. 2023, Shahin, Mohamed et al. 2023, Elbakry, Harras et al. 2024).This study delves deeper into scaffold modifications to elucidate their influence on activity against various microbial organisms and expands our understanding of the structure-activity relationship within this promising class of antibacterial agents.Therefore, this study aims to achieve two goals: first, to modify the scaffold by inserting a polar nitrogen atom into the lipophilic part to understand its activity against various microbial organisms, and second, to improve our knowledge of the structure-activity relationship to expand within this new class of antibacterial agents.The underlying concept for the current scaffold modification is to replace the benzylic carbon responsible for the short half-life with a nitrogen atom, which, according to previous studies, possesses broad-spectrum antibacterial activity and improves its physicochemical properties.An initial investigation of the SAR of the phenylthiazole compounds prompted us to cyclize the terminal n-butyl moiety of the lead drug 1a to enhance its antibacterial activity (Mohammad, Mayhoub et al. 2014).Unfortunately, while this modification indeed enhanced potency, it compromised the physicochemical properties and pharmacokinetic profile of the resulting analogues (Figure 1).However, this increase in potency was accompanied by a significant increase in lipophilicity, as evidenced by the increased clogP value.This led to a drastic decrease in water solubility, which dropping from 65 µg/mL for 1a to mere 3 µg/mL for 1b.Consequently, formulation of 1b for oral or parenteral administration became a major challenge due to its poor solubility.The introduction of nitrogen atom to 1b afford compound 1c, which enhances its solubility and physicochemical properties by 160-fold higher than 1b.Scheme (1).

Chemistry
Treatment of para -iodobenzothioamide (2) with α-chloroacetylacetone yielded the key starting compound 3, as previously reported (Mohammad, Mayhoub et al. 2014).The corresponding 1ry-amine derivatives 4a-q were obtained using the Buchwald carbon-nitrogen cross-coupling conditions, using the Pd-catalyzed protocol, in which Pd(II) and X-Phos ligand in a polar aprotic solvent provided the best yields of compounds 4a-q (Scheme 1).Condensation of 4a-q with semicarbazide hydrochloride gave the final products 5-21 (Scheme 1).

Antimicrobial Evaluation
The newly synthesized phenylthiazole derivatives 5a-q were initially tested against methicillin-resistant Staphylococcus aureus (MRSA) USA300, which is a significant source of MRSA skin and soft tissue infections (SSTIs) worldwide.The phenylthiazole derivatives were also tested against an Acinetobacter baumannii AB5075 strain to examine the activity of the compounds against gram-negative bacteria.In this study, vancomycin was used as a positive control in the antibacterial evaluation.The initial screening against MRSA USA300 revealed an abundance of structure-activity relationships (SAR) as MIC values ranged from 8 to over 32 µg/mL (Table 1).The 2-ethylhexyl amine analog (compound 5m) represented the best side chain as it provided the most potent analog from this series (MIC value of 8 µg/mL) Table 1, meaning that the target protein is at this point cannot adjust specific position for side chains with more than 8 carbon atoms.The SAR appears clear because it relates to the size of the side chain on the nitrogen atom of the primary amine.Briefly, the shrinkage of the nitrogen-containing side chain gradually impaired the anti-MRSA activity of the compounds, as the MIC value increased to four times 32 µg/mL for the 2ethylhexylamine-containing derivative 5m and for the sec-butylamine-containing one derivative was completely abolished analogue 5g.Similarly, extension of the nitrogencontaining side chain yielded the nonylamine-containing derivatives 10 without inhibition for MRSA Table 1.This observation confirms our previous hypothesis that the active site of the target receptor cannot accommodate side chains larger than octylamine.The MIC for the control antibiotic vancomycin against MRSA USA300 was 1 µg/ml.

Computational ADMET study
The ability to predict pharmacokinetics is an important step in drug research development.It helps drug manufacturers get the optimal edge themselves, or even encourages others to continue the fight.In the last two decades (Amin, El-Saadi et al. 2021), there have been online applications and tools for predicting absorption, distribution, metabolism and excretion (ADME).In this particular study, those properties for the best three candidates in our study were calculated and the results are summed up in the tables 1-3.To study the ADME, probability and physiochemical properties of these three candidates, three different programs were used: PreAdME, SwissAdme and Molsoft.To evaluate the contribution of molecular rigidity to oral bioavailability, the number of rotatable bonds was chosen as the most appropriate parameter.In general, the number of rotatable bonds corresponds to the molecular weight, i.e. if this number increases as the molecular weight increases, and vice versa.The molecular weight of our candidates is less than 500, the logP (calculated octanol/water partition coefficient) is less than 5, indicating their initial good oral bioavailability.A further division was made by dividing the number of rotatable bonds: this number measures the molecule flexibility and indicates the oral bioavailability.It can be calculated from the number of individual non-ring bonds that are not bonded to either a terminal or a hydrogen atom.It is divided into three groups: number of rotatable bonds 7 or less, 8-10 and more than 10.Our research has shown that the number of rotatable bonds for the three compounds was over 10, which means that it has predominantly low bioavailability.The number of Hbond donors and acceptors is less than 5 for all candidates.Therefore, all three compounds plausibly have good absorption (Veber, Johnson et al. 2002).
In this study, we also measured the topological polar surface area (tPSA) which allows us to predict the permeability of polar atoms through membranes.The tPSA values for the three compounds are all similar, at 120.64.This suggests that the compounds are all similarly polar (Veber, Johnson et al. 2002).Since a molecules that have a PSA value above 140 Å usually also have poor permeability (Lipinski, Lombardo et al. 2012), all three investigated compounds showed a PSA value low 140 Å indicating their moderated permeability.Swiss ADME was the software of choice to predict the physicochemical parameters.The data retrieved are listed in Table 1.NORTB is a measure of the flexibility of a molecule.The NORTB values for the three compounds are 11, 11, and 12, respectively.This suggests that the compounds differ slightly in their flexibility.Compound 5m has the most rotatable bonds, which suggests that it may be the most flexible of the three compounds.Log P is a measure of the lipophilicity of a molecule.Lipophilic molecules are soluble in nonpolar solvents and insoluble in polar solvents.The log P values for the three compounds are 1.95, 1.4, and 0.8, respectively.This suggests that the compounds differ in their lipophilicity, with compound 5m being the least lipophilic.
Lipiniski rule (Lipinski, Lombardo et al. 2012): states that the oral drug must have only one violation of the following criteria: not more than 5 HBD and not more than 10 HBA, molecular weight less than 500 Daltons and logP is less than the value of 5.At using SwissADME and Molsoft, our compounds met Lipinski's rules for drug similarity; For this reason, we believe they have good absorption.Drug solubility is a critical factor in determining the rate of dissolution that enables oral bioavailability; Therefore, the solubility of the drug is tested, which improves when the solubility values are less than 10 mg/L.Since our compounds have values of 2-4 mg/L, they are poorly soluble in water (Ustaoğlu, Taş et al. 2021).3.62 0.9 0 0.55 Caco-2 Permeability: This measures how easily a compound can pass through the intestinal wall and enter the bloodstream.Compound 5d has the highest bCaco-2 permeability, followed by compound 5m and then compound 5l.HIA (%): This measures the percentage of a compound that is absorbed from the intestine.Compound 8 has the highest CHIA, followed by compound 5m and then compound 5l.MDCK permeability: This measures how easily a compound can pass through the kidney membrane and enter the bloodstream.Compounds 5d and 5m have similar MDCK permeability, while compound 16 has the lowest MDCK permeability.PPB (%): This measures the percentage of a compound bound to plasma proteins.Compound 5m has the highest PPB, followed by compound 5d and then compound 5l.CYP2D6 Metabolism: This measures whether a compound is metabolized by the enzyme CYP2D6.Compound 5d is not metabolized by CYP2D6, while compound 5l is a substrate for CYP2D6 and compound 5m is not metabolized by CYP2D6.In order to determine those properties, PreADMET[133] is used as a tool to investigate as deeply as we can from those pharmacokinetics.Compound 8 has the highest permeability and absorption, followed by compound 5m and then compound 5l.Compound 5m has the highest plasma protein binding, followed by compound 5d and then compound 5l.Compound 5l is metabolized by CYP2D6 while compound 5d is not the case and compound 5m is not metabolized by CYP2D6.These differences in physicochemical properties may impact the biological activity and pharmacokinetics of the compounds.For example, compound 5d may have a longer half-life in the body than compound 16 due to its lower plasma protein binding.The pre-ADMET program was used to generate the data in Table 3.All biologically tested compounds are with purity of 98% or more. 1 H NMR spectra were run at 400 MHz and 13 CNMR spectra were determined at 100 MHz in deuterated dimethyl sulfoxide (DMSO-d6) on a Varian Mercury VX-400 NMR scale.Chemical shifts were calibrated relative to those of the solvents.Column chromatography was performed on 230-400 mesh silica.The progress of reactions was monitored with Merck silica gel IB2-F plates (0.25 mm thickness).Melting points were determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected.All yields reported refer to isolated yields.Compound (3) was prepared as reported (Mohammad, Mayhoub et al. 2014).

4.3.Plate preparation
Add 75 uL double strength MH broth to each well.Add 75 uL of each tested compound dilution to the broth in the wells, using the same tip starting from the low to the high concentration.Add 15 uL of the adjusted cultures to each well using the same tip starting from the low to the high concentration (5 × 10 5 CFU/mL).Positive control: Media+ DMSO "the highest used conc"+ organism.Negative control "sterility check": Media only.All compounds were prepared from a stock concentration of 512 ug/mL, starting from a concentration of 64 ug/mL and up to 0.125 ug/mL.Compounds that showed ppt in the 512 ug/mL concentration were directly prepared from the original stock 5000 ug/mL, starting from a concentration of 64 ug/mL and up to 0.5 ug/mL.DMSO was used in the same concentrations used in the compounds.All MIC was done twice in duplicates.TTC was used to distinguish compounds with low solubility or color from those with positive bacterial growth.Antimicrobial activity was detected by adding 10 µL of 0.5% TTC (2,3,5-triphenyl tetrazolium chloride) aqueous solution.The viable bacterial cells reduced the yellow TTC to pink/red 1,3,5-triphenylformazan (TPF) (Radaelli, et al., 2016).MIC was defined as the lowest concentration of the tested compounds that inhibited visible growth, as indicated visually or by the TCC staining (yellow= no growth, red= microbial growth).

Colony counts validation of inoculum suspensions
Laboratories are encouraged to perform colony counts on inoculum suspensions at least quarterly to ensure that the final inoculum concentration routinely obtained closely approximates 5 × 10 5 CFU/mL.Do this by removing a 0.01-mL aliquot from the growth control well or tube immediately after inoculation and diluting it in 10 mL of saline (1:1000 dilution).After mixing, remove a 0.1-mL aliquot and spread it over the surface of a suitable agar medium.After incubation, the presence of approximately 50 colonies indicates an inoculum density of 5 × 10 5 CFU/mL.The MIC was done in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI documents M100-S24, and M07-A9) (Wayne, 2014).

Conclusion
From the above-mentioned results in table (3) it was found that (for the on MRSA-US300 activity) the highest activity obtained with compounds 5m with MIC value about 8 µg/mL followed by compound 5b, 5c, 5d, 5k, 5l, 5n, 5o and 5p with MIC value about 16 µg/mL.The other compounds show very low activity.On the other hand, most synthesized compounds gave very weak activity against Acinetobacter baumannii AB5075 except compounds 5b, 5k, 5n and 5o which has moderate activity with MIC 32 µg/mL followed by compound 5h, 5j and 5q with MIC value about 64 µg/mL.This means that the presence of terminal hydrogen bond acceptor group is essential for activity against resistant gram-positive bacteria.

Figure 1 .
Figure 1.Progress of phenylthiazole antibiotics development and the general idea of the present work.

Table 1 :
Physiochemical properties prediction of selected analogs.

Table 2 :
Predicting drug-likeness of selected analogs.