Design, synthesis, and evaluation the anti-β-lactamase activity of new sulphathiazole-derived monobactam compounds

Objectives: β-Lactams are the most successful antibiotics for the management of infectious diseases. Unfortunately, the bacterial production of β-lactamase that hydrolyzes the β-lactam ring can inactivate these drugs. The use of β-lactamase inhibitors like (clavulanic acid) in combination with the β-lactams may reduce this inactivation. The prevalent β-lactamase phenotype is the TEM-1 of class A released by Gram-positive and Gram-negative bacteria. Methods: The docking study with TEM-1 β-lactamase lead to synthesize of new 5 monobactam compounds as the acid chloride derivatives reacted with the Schiff bases compound forming the monobactam ring. The final 5 synthesized compounds were characterized using physical and spectroscopic methods and tested biologically by evaluating their MIC values against 4 strains of β-lactamase Gram-positive and Gram-negative bacteria. The results were compared with those acquired from using clavulanic acid as a co-inhibiter with amoxicillin against the tested bacteria. Results: The results revealed that 2 synthesized compounds showed an anti βlactamase effect resemble to that of clavulanic acid. Conclusion: As conclusion; the β-lactamase active pocket prefers hydrophobic substituents, as the synthesized products with these groups appeared to have the highest affinity.


Introduction:
ntibiotics, particularly the βlactam classes are a cornerstone of modern medicine for fighting the infectious diseases 1 . Their mechanism of action involves inhibition of the synthesis of peptidoglycan (the chief constituent of the bacterial cell wall), thus producing permanent damage and death of the bacteria (bactericidal) 1,2 . The β-lactam antibiotics group includes four families of antibiotics: the penicillins, cephalosporins, carbapenems and monobactams. All of them are containing a fourmembered β-lactam ring (azetidin-2one), which is essential for their antibacterial activity 1,3 .
Of the several manifestations of β-lactams bacterial resistance currently known, the production of beta-lactamases is the most important 2,3 The Ambler system makes use of amino acid sequence to categorize βlactamases into four broad classes: A, B, C, and D 3 . The A, C, and D classes are serine β-lactamases (SBLs) whereas the class B enzymes are metallo β-lactamases (MBLs) 3,4 .
The β-Lactamase inhibitors are affect the capability of the bacteria to inactivate β-lactam antibiotic, and their administration in combination with β-lactam antibiotics by co-administration are now the most effective procedures to fight a particular resistance mechanism 4,5 . However, their restricted spectrum of effectiveness, which is restricted to class A enzymes necessitates the search for further broadly effective βlactamase inhibitors 5 . Therefore, there is an urgent need to discover and develop new antibiotics or anti βlactamases to handle this situation.
Monocyclic β-lactams are more stable to hydrolysis by βlactamases compared to other βlactams 4 .The monobactams like Aztreonam belong to a subclass of monocyclic beta-lactam moieties are the only U.S. Food and Drug Administration-approved monobactam in clinical use 5,6 . This study aims to design, dock (computer calculation), synthesize, and study the A biological activities of new βlactamase inhibitors.

Material and methods:
All employed chemicals were purchased from commercial sources and their suppliers are Fluka (Switzerland), Alpha (India), Scharlau (Spain), and Merck (Germany). Melting points of the synthesized compound were determined by the Electrochemical CIA 9300 melting point apparatus (UK) by using an open capillary method and they were uncorrected. FTIR spectra were recorded on a PerkinElmer infrared spectrophotometer. 1H NMR and 13C NMR spectra in DMSO-d6 on a Bruker Avance DPX 400 MHz spectrometer using TMS as an internal reference. All the products were synthesized by the method given in the literature and identified by 1 H NMR 13 C NMR and IR spectra and microanalyses of these compounds were in satisfactory agreement with the structures. The purity of the compounds and the completion of the reactions were monitored by TLC using pre-coated silica gel plate. Docking Study: The Computational Docking Study was carried out using the online platform Mcule by AutoDockVina logarithm (https://mcule.com/ apps/1click-docking/). The structure of the bacterial Penicillin Binding Proteins PBP (1qmf) and β-lactamases TEM-1 (1pzo) were retrieved from PDB 7 .

Docking Study on Penicillin Binding Protein (PBP):
The docking study of the investigated products on PBP (1qmf) was conducted to test the antimicrobial activities of investigated products 8 , and to be compared with reference antibiotics of various activities to validate this approach. Docking Study on β-Lactamases TEM-1: The docking study of the investigated products on β-lactamases TEM-1 (1pzo) was conducted to investigates the affinity toward the TEM-1 β-lactamases to be compared with reference anti β-lactamases of various activities. The docking scores of the binding energies due to the interaction between the active ligands (inhibitors) and the enzymes pockets were chosen with the highest score based on geometric shape complementarity 9 .

Chemical study:
General procedure for the preparation of Schiff base derivatives (A1-A4) 10,11 : A (0.004) mole of benzaldehyde derivatives was dissolved by heating in 20 ml absolute ethanol before mixing with (0.004) mole of sulphathiazole which was also dissolved by heating in 20 ml absolute ethanol, then the mixture was refluxed with stirring for 10-15h (the proceeding of the reaction was monitored by TLC using 1:1:1 methanol: diethylether : DCM as eluent), after cooling the precipitate was filtered, dried and then washed once with cold ethanol and 3 times with distilled water and dried.

General
procedure for the preparation of acid chloride derivatives (A 5 and A6) 12 : A (3-5 mole) of individual carboxylic acids was dissolved in 10-15 ml of thionyl chloride, the mixture was reflxed for 30 min in the hood, then the excess of thionyl chloride was distilled under reduced pressure, after cooling the yielded products was taken and used freshly in the next step.
General procedure for the preparation of monobactam derivatives (A7-A11) 13 : The corresponding acid chloride (0.0016) mole in 10 ml dichloroethane was added drop by drop to a mixture of a corresponding Schiff base (0.0016) mole with (0.0016) mole pyridine in 30 ml dichloroethane at 0˚C. The resulting mixture was reflexed for 20-25 h (as monitored by TLC using 1:2:2 ethanol: diethyl ether: DCE as eluent). After cooling the solution was washed twice with water (30 mL), saturated aqueous NaHCO3 (30 mL) and finally with saturated solution of NaCl (30 mL). The organic layer was removed and dried using anhydrous sodium sulphate. After solvent evaporation, the dry product was washed with cold ethanol.

Antimicrobial Study Detection of β-lactamases in bacterial isolate:
The detection of β-lactamases in bacterial isolates was done for Gram-positive (Staphylococcus aurous) and 3 Gram-negative (Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeroginosa) pathogenic bacteria isolates using acidimetric method 14 . Were 2 mL of 0.5% (w/v) aqueous phenol red solution was diluted with 16.6 mL distilled water and 1.2 g of benzylpenicillin is added. The pH is adjusted to 8.5 with 1 M NaOH. Then 100 μl of resulting violet solution was distributed into tubes and inoculated with several colonies of bacteria from culture to get a dense suspension. A yellow color within 5 min indicates βlactamase activity. Positive controls were run in parallel 15 . 16

Determination of anti β-lactamase activities:
The anti β-lactamase activity of the synthesized compounds against human pathogenic bacterial isolates was evaluated by measuring the zones of inhibition in the disk diffusion method 16 . Each tested compound was used as co-inhibiter with 1000 or 2000 µg of amoxicillin prepared as disks (5µl/disk) at a concentration equal or below their MIC, and then placed on Petri dish with Mueller-Hinton agar medium (previously inoculated with the tested bacterial strains by sterile cotton swabs). After incubation at 37°C for 24 h, zones of microbial growth produced around the tested substances were measured and recorded as the diameters of inhibition 18 . Disks containing 1000 or 2000 µg of amoxicillin (5µl/disk) was prepared and used as control.
In all the above microbial study DMSO was used as a solvent for the synthesized compounds with a final concentration of less than 2 % in order to ensure that it has no effect on bacterial growth.

Results and discussion: Molecular Docking Study for standard inhibitors:
The docking for both clavulanic acid and sulbactam were carried out on both bacterial PBP (1qmf) and βlactamases TEM-1 (1pzo). The results are (-6.1) and (-5.1) for clavulanic acid and for sulbactam are (-6.6) and (-6.2) respectively. Docking for monobactam derivatives was carried out on both bacterial PBP (1qmf) and β-lactamases TEM-1 (1pzo). The chemical structures of the designated substitutions are listed in Table ( 1), while the results of docking are listed in Table (2).  The docking results showed that as the hydrophobicity increases in the tested compounds the score will decrease. The compounds bound variably to the region located between H10, H11 and H12 helices of TEM-1, indicating that the pocket is mostly hydrophobic in nature 19 , and the residue participated in the binding with our compounds were almost the same as those combined with the standard inhibitors (clavulanic acid and sulbactam). In general these compounds were disposed the absence of the N-H (NH2 group) bond stretching at 3272 cm -1 of the sulphathiazol and appearance of absorption bond at 1580-1590 cm -1 related to the C=N bond stretching which indicating the formation of Schiff base. There was an addition of characteristics peaks for each substitution of the aldehydes used.

Preparation of acid chloride derivatives (A5 and A6):
This reaction was occurred via nucleophilic addition-elimination mechanism which involves, firstly displacement of chloride from the sulfur by the hydroxyl group to form the acyl chloro-sulfite intermediate. In the second step the chloride ion (Cl -) attacks the carbonyl carbon via addition step to form a tetrahedral intermediate, which losses SO2 and HCl molecules in the elimination step to form the acid chloride (Table 4).

Preparation of monobactam compounds (A7-A11):
This reaction was occurred as the nitrogen of the imine (Schiff base) acts as a nucleophile attacking the acid chloride carbonyl and eliminating the chloride ion as a result. This anion attacks the electron seeking carbonyl carbon via intramolecular nucleophilic addition reaction to form the four membered ring azetidine moiety (monobactam) 20 .
The physical properties and the most characteristic peaks of the FT-IR spectrum for these compounds are shown in the Table (5).   (Table 6).
While in the 13 C-NMR and for compounds A7-11 the appearance of the carbonyl C2 at 162.98-166.61 ppm, and the appearance of C1 and C4 at 59.06-74.16 ppm and 57.79-60.77 ppm respectively also confirm the formation of the monobactam ring (Table 6).

Antimicrobial study: Determination of minimum inhibitory concentration (MIC):
After The detection of βlactamases in bacterial isolates, a broth microdilution method was used to evaluate the (MIC), as a serial of 10 doubling dilution of the synthesized compounds and standard antibacterial (Amoxiclav) (Amoxicillin 2000mg + clavulanic acid 400mg), Amoxicillin, Cefotaxime, Ceftriaxone, and Ciprofloxacin) was tested with final concentration starting from 2000 mg/ml (and from 5000 mg/ml for Amoxicillin). The results were summarized in table (7). Compounds A8 had an antibacterial activity and has Cl group which may be responsible for its antibacterial activity 21 .

Determination of anti β-lactamase activities:
Firstly amoxicillin and amoxiclav (2000/400mg) were incubated with the bacterial isolates by using three concentrations 3000, 2000, and 1000 µg/ml, the results would be conceders as a control for the results of the incubation of the synthesized compounds table (8). Depending on the above results, amoxicillin was used in two concentrations in the next step, 1000µg/ml for Gram (+)ve bacteria and 2000µg/ml for Gram (-)ve bacteria Table (9).  In general, the results indicated that all the synthesized compounds had no activity as anti β-lactamase against Staph. aureus, K. pneumonia, and P. aeroginosa.
Concerning the anti βlactamase activities against E. coli bacteria, 2 compounds which are A7, and A11 showed strong anti βlactamase activities resembling that of clavulanic acid, although both of them have no antibacterial activities. These compounds having one or more hydrophobic residue in its structure, this is coming true with the docking results which indicate that the selectivity for the β-lactamase enzyme increases as the compounds became more hydrophobic, as the active binding site pocket of the β-lactamase are mostly hydrophobic in nature 22 .

Conclusion:
It was concluded that the βlactamase TEM-1 active pocket prefers hydrophobic substituents, as the 2 synthesized active anti βlactamase compounds having hydrophobic residue. So that the selectivity for the β-lactamase enzyme increases as the compounds became more hydrophobic, and the Cl group will increase the antibacterial activity of the synthesized compounds.