CC BY
22
CHEMICAL PROBLEMS 2024 no. 2 (22) ISSN 2221-8688
139
UDC 547-327
DOCKING, SYNTHESIS AND p-LACTAMASE INHIBITORY ACTIVITY EVALUATION
FOR NEW AMIDE COMPOUNDS
Ahmed A. J. Mahmood and Safaa P. Bahnam
Pharmaceutical Chemistry Department, Pharmacy College, Mosul University, Iraq e-mail: [email protected]
Received 23.11.2023 Accepted 16.01.2024
Abstract: The beta-lactams are recognized as effective antibiotics for treating infections, yet bacterial production of [¡-lactamase, which hydrolyzes the beta-lactam ring, can render these drugs inactive. Combining these antibiotics with a [-lactamase inhibitor, such as clavulanic acid, mitigates this resistance. In a docking study involving Temoniera-1 (TEM-1), 1pzp, we induced the synthesis of eight amide compounds by reacting acid chloride derivatives with sulphathiazol or oxadiazol amine, forming an amide bond. The newly synthesized compounds were differentiated using physical and spectroscopic methods and verified biologically by estimating their minimum inhibitory concentration (MIC) against four strains of [ -lactamase G(+)ve and G(-)ve bacteria. Their anti [-lactamase behaviors were then compared with that of clavulanic acid as a co-inhibitor with amoxicillin against the same four strains of bacteria. The results indicate that four of the new amides exhibit excellent anti-[-lactamase activity and contain one or more hydrophobic residues in their structures. Halogen atoms enhance the selectivity of tower [-lactamases. Derivatives of both oxadiazole and sulfathiazole scaffolds show promising anti-[-lactamase activity as non-[-lactam inhibitors.
Keywords: Sulfathiazole, Oxadiazole, TEM-1 [-lactamase, amides, docking, anti- [-lactamase. DOI: 10.32737/2221-8688-2024-2-139-149
Introduction
P-lactam antibiotics, the original native antibacterial compounds still distinguish an exceptional class of antibiotics, gratitude to both their exceptional antibacterial action and selectivity. Nevertheless, their resistance can quickly extend on a universal level [1]. With chief bacterial pathogens, this bacterial resistance has nowadays touched terrifying stages [2]. Illnesses initiated by drug-resistant bacteria have developed international hazards to community healthiness [3].
P-lactamase indicates the chief associated procedure of resistance [4]. Consequently, two approaches to overwhelm P- lactamase-mediated resistance: (a) improving the persisting P- lactamase-resistant antibiotics and (b) initiation of P-lactamase specifying inhibitors (BLIS) in order to co-administer together with a P-lactam antibiotics [4,5]. Consequently, here is an critical demand to
purpose and improve additional antimicrobial or anti-P-lactamases to control this condition [4,6].
Serine P-lactamases were classified under the classes A, C, and D. They are accountable for the braking of the P-lactam moiety. This backing route includes the creation of a covalent acyl-intermediate among the catalytic serine and the P-lactam moiety in the enzyme's bocket [7]. Alternatively, the metallo-P-lactamases of class B utilize single or double of the zinc ions for accomplishing the P-lactam braking [7,8].
The P-lactamases Temoniera-1 (TEM-1) 1pzp belongs to the serine P-lactamases of the category A which braking the P-lactam group by developing an acyl-intermediate structure with the catalytic serine in the enzyme pocket [7,8].
BLIS targeting the P-lactamase enzymes when co-administer beside the P-lactam antibiotics. Nevertheless, because the presently consumed BLIS comprise P-lactam ring, they
www.chemprob.org
CHEMICAL PROBLEMS 2024 no. 2 (22)
were prone to time-limited appliance as the P-lactam antibiotics themselves [9]. Moreover, the P-lactamases pocket is subjected to transmutations that equivocates the attachment of BLIS. Inhibitor-resistant P-lactamases (IRTs) have been established and continuously increasing by time [10].
Talented approaches include the initiation of non-P-lactam inhibitors; they will be effective versus a broad range of P-lactamases. Various inhibitors have now been defined [11]. Boronic acid derivatives are effective for P-lactamases categories A, C and D [12]. A new acylated phenoxyaniline derivatives presenting P-lactamase TEM-171 inhibition and theoretically attached to the allosteric site of enzyme was newly defined [13,14].
Indeed, searching the various heterocyclic molecules has been widely performed, however sulfathiazole and oxadiazoles are continuing as reserved platforms in different medicinal discovery for invention of a new candidates [15]. Sulfathiazoles are original structure with
numerous activities [16]. Several heterocyclic sulfonamide compounds was therapeutically applied, of which around 30 medications are currently available [15,16]. Sulfathiazole derivatives have many pharmacological implications as antiglaucoma, antimicrobial, diuretics, antiparasitic, anti-Alzheimer, antioxidant, antiviral, antiobesity,
anticonvulsant, antiinflammatory,
antitubercular, antidiabetic, anticancer, etc. [1620]. While heterocyclic oxadiazole platform was described to show a wide variety of biological events as anticonvulsant, antiinflammatory, antimalarial, and anticancer [2024].
Considering the extensive
pharmacological profiles of sulfathiazole and oxadiazoles, this research aims to purposefully (through docking) synthesize original non-P-lactam inhibitors derived from sulfathiazole and oxadiazole scaffolds. The study also involves assessing their ability to inhibit P-lactamases.
Materials and Methods
Docking analysis results:
Docking analysis was accomplished utilizing the online Mcule platform (https://mcule.com/ apps/1-click-docking/) [3]. The docking was generated with P-lactamase TEM-1 (1PZP ). The chemical constructions of all compounds in this work were drown by ChemDraw program (PerkinElmer company), version 16.0.0.82(68). The choice of the best compounds will realize on the docking scores of the binding energies besides the geometric shape complementarity visualized by discovery
studio visualizer (BIOVIA) v20.1.0.19295. Synthesis:
All substances utilized in this work were bought from commercial sours and their sources are Scharlau (Spain), Merck (Germany), Alpha (India) and Fluka (Switzerland). Melting points were calculated using open capillaries. FTIR spectra were verified on a PerkinElmer infrared spectrophotometer, NMR and 13C NMR spectra in DMSO-d6 on a Bruker, Avance DPX 400 MHz spectrometer (TMS was used as an internal reference).
Preparation of acid chloride derivatives [25]:
The carboxylic acids (2-3) mmol were dissolved in thionyl chloride (10-15 ml), the resultant combination was reflexed (30 min) within the hood (which contain the activated
carbon fibers that trapped the resultant HCl and SO2 byproducts). The excess amount of thionyl chloride was removed under reduced pressure. The remaining yields was occupied after cooling and consumed freshly in the subsequent
step of the amides synthesis.
Preparation of amides products (Ad1-Ad8)
[26]:
The consequent acid chloride (2.5 x 10-3) mole dissolved in dichloromethane (5 ml) and adds up drop by drop to a mixture of sulfathiazole or oxadiazole (2.5 x 10-3) mole plus pyridine (2.5 x 10-3) mole dissolved in dichloromethane (30 ml) in ice bath. The yielded mixture mixed at 25 °C to the next day. Afterward the solvent was evaporated and the yield was washed 2-3 times with cold ethanol for removing the west products. Biological study
Bacterial selection (^-lactamases detection):
Acidimetric method was used to identify P-lactamases in one G(+)ve (Staphylococcus aureus) and 3 G(-)ve (E.coli and K. pneumonia, P. aeroginosa) isolates of pathogenic bacteria
[27]. Converting the mixture color from pink^ yellow in 5 min reveals P-lactamase existence. (+)ve control was participated in equivalence
[28]. Additionally these isolates were selected to become resistant to amoxicillin and sensitive to Augmentin for one certain strength, this is confirming that the clavulanic acid (in the Augmentin) will mask the P-lactamases enzymes in the tested bacteria and without its action there is no effects for amoxicillin as antibiotic.
Minimum inhibitory concentration (MIC) determination [29]:
To evaluate the MIC a broth microdilution method was utilized. A consecutive of ten replication diluted concentrations belong to the new amides and the typical antibacterial (Augmentin and Amoxicillin) was formulated, the dilution initiated from concentration of 2000 |ig/ml. Bacterial colonies were inserted to the previous test tubes at of 5x105 CFU/ml concentration next to the adding 1 ml of Mueller-Hinton media. Afterward at 37°C they incubated for 18 h [20,21]. The agar plus the bacterial isolates was used as a (+)ve control, while the (-)ve control including the agar only. By utilizing this technique, investigators were capable to establish the MIC, which characterizes the lowermost strength of stuff necessary to inhibit the growth of specific
bacteria. The MIC determining stage was performed to check the antibacterial activity for both the standard and the synthesized compounds. The MIC for the standard compound will represent the strength at which the Augmentin will be sensitive and surely the amoxicillin will be resistance as indicated in the prior step. The MIC estimation for the new compounds (synthesized) judged their antibacterial activities (if there is any), and to identify the intensity of these compounds that will be utilized in the following step [30,31]. This concentration must be lower than their MIC and overhead the clavulanic acid MIC of the Augmentin. This is to guarantee that the antibacterial activities of the amoxicillin that performed in the subsequent phase will be initiated by the inhibition of the P-lactamase by either the clavulanic acid or the newly synthesized compound when inoculating individually with the amoxicillin [31]. Anti P-lactamase activities evaluation:
In the paper, the disk diffusion method was occupied to evaluate the anti P-lactamase activity of the new amides [32,33]. To develop the experimentation, every investigated compound was employed as a co-inhibitor along with 1000 |ig of amoxicillin (equal to that of Augmentin) formulated as disks (5^l per disk). These disks including the amoxicillin and the investigated compound (in their sub MIC concentration) were subsequently positioned on Mueller-Hinton agar medium in a Petri dish (inoculated with the bacterial strains in earlier times) using sterile cotton swabs. The whole setup was then incubated for of 24 hours at 37°C. The zones of microbial progress inhibition that performed around the disk were restrained and documented as the diameters of inhibition [34,35]. For control, disks comprising 1000 |ig of amoxicillin alone (5^l/disk) were also organized and involved in the trial. Dimethyl sulfoxide was employed to dissolve the new compounds, with a guarantee of its final concentration less than 2% to certify that it did not undesirably influence bacterial growth. This precaution was occupied to preserve the reliability and precision of the experiment [32,36].
Results an
Docking Study results:
The designed amides along with clavulanic acid, sulbactam, tazobactam, avibactam were docked with P-lactamase TEM-1 (1pzp). The substituents used for the
discussion
designing the amides are listed in Table (1), whereas their docking results were representing in Table (2).
Table 1. The substituents used for amides c esignation
Item R Item R Item R
R1 R11 R21 Cl^ Cl
R2 R12 ch3 & R22 CI—1—CI Cl
R3 R13 m'o R23
R4 XT R14 qJOCQ R24 Br BA
R5 f cy R15 V no2 R25
R6 FTX R16 XT R26 CH3
R7 jy R17 cv R27 Br
R8 jy R18 "'V R28 Cl
R9 JX R19 R29 qV-
R10 R20 ci—j—CH3 R30 06
Table 2. The docking results for the oxadiazole and the sulfathiazole amides compounds with TEM-1 P- lactamase (1PZP) enzymes (kcal/mol)
No. Docking Score No. Docking Score No. Docking Score
oxadiazole Sulfathiazole oxadiazole sulfathiazole oxadiazole sulfathiazole
R1 -7.2 -8.0 R11 -7.0 -6.0 R21 -6.8 -6.5
R2 -7.0 -9.1 R12 -7.4 -9.0 R22 -7.6 -6.1
R3 -8.0 -8.1 R13 -7.8 -8.4 R23 -6.4 -6.6
R4 -7.6 -9.4 R14 -8.1 -8.3 R24 -6.8 -6.5
R5 -7.5 -7.0 R15 -7.4 -8.0 R25 -9.2 -9.0
R6 -8.5 -7.1 R16 -7.4 -8.4 R26 -7.0 -7.7
R7 -8.5 -7.0 R17 -7.8 -7.0 R27 -6.4 -6.6
R8 -7.0 -7.7 R18 -7.4 -8.0 R28 -7.8 -8.3
R9 -7.4 -9.0 R19 -9.2 -9.1 R29 -7.2 -8.0
R10 -8.4 -8.4 R20 -7.3 -7.9 R30 -7.0 -7.7
The docking of the standard inhibitor sulbactam, avibactam, tazobactam and clavulanic acid were -5.5, -5.4, -4.3 and -6.1 kcal/mol respectively. The standard inhibitors docking was not performed for achieving its score as reference control only but moreover
their binding poses consumed for the definition of the active pocket site amino acids for P-lactamases TEM-1 (1pzp). The amino acids residues (LEU 196, ALA 199, IEU 200, GLY 211, ALA 253 and GLY 256) were found to bind mainly all of the standard inhibitors in the
(1pzp) P-lactamase [34]. The overhead 6 residues will describe the active binding site for the connection with this enzyme.
Regarding the collaboration of the tested amides with enzymes (Table 2), the results demonstrated that 8 compounds with the substituents (R19 and R25) for the oxadiazole and the substituents (R2, R4, R9, R12, R19 and
Chemistry:
Table (3) demonstrates the most important physical characteristic and the documented
R25) for the sulfathiazole represent the best docking scores, additionally they bound 4-5 out of the 6 amino acids that defined the binding site of P-lactamases TEM-1 (1pzp), besides to 3-5 additional amino acids residues for every compound, those may play a role in the potentiation the contact with the enzyme (Figure 1).
representative FT-IR values as v cm-1 belongs to the newly synthesized amides.
Table 3. The most important physical characteristic and the documented representative FT-IR values as v cm-1 belongs to the amides (Ad1-Ad8). O
R HN-R,
Amides R1 R m.p(°C) Yield % Color N-H v cm-1 C=O v cm-1 Other v cm-1
Ad1 N-™ I1,ciçr 225-227 72 Off White w 3397 m, sh 1675
Ad2 N.....f™ I >=s jy^ 247-249 85 White w 3359 m , sh 1679 C-Cl m 400 C-O-C s, sh 1228
Ad3 O H 218-220 65 Pinkish off white w 3370 m, sh 1668 C-Cl m 406
Ad4 H rjy 271-273 70 Pinkish off white w 3391 m, sh 1676 C-F m 507
Ad5 h . JX 254-256 87 Pink w 3396 m, sh 1661 C-F m 513
Ad6 CH3 & 160-162 63 Off white w 3313 m, sh 1694
A
Fig. 1. 2 dimensional poses for selected R9 (A) R19 (B) with the 1pzp enzyme.
Ad7 O H JQT.X) HîCrr CH3 266-268 82 White w 3370 m, sh 1688
Ad8 ^ Vv\ Xr^ Jf 269-271 74 White w 3372 m, sh 1693 C-O-C m 1238 C-Cl m 400
The FTIR denote the amide bonds creation by the vanishing of the 1° NH2 at 3356 cm-1 and 3259 cm-1 of the original oxadiazole and sulfathiazole compounds respectively, and switched by the 2° amine NH at 3313-3397 cm" 1, in addition to the emergence of carbonyl at 1661-1694 cm-1 which document amide establishments. Besides to additional peaks for every compound that identify to the substitutions of the acid chlorides consumed (Table 3).
1H-NMR and 13C-NMR of amides (Ad1-Ad8) designate the development of the amide bonds, as the HNMR performance the lone proton emergence belongs to the NH oxadiazoles amide (compounds Ad1 and Ad2) at 7.81-7.82 ppm and the NH of the sulfathiazole amides (compounds A3-A8) at 8.34-10.49 ppm.
Whereas in the 13C-NMR the attendance of the amide carbonyl C=O of the oxadiazoles (compounds Ad1 and Ad2) at 172.09 ppm, and the sulfathiazole amide carbonyl C=O (compounds Ad3-Ad8) at 161.34-168.85 ppm. This also will authorize the development of amide bond.
The spectral representation of the new amides compounds Ad1-Ad8
Ad1 [3,5 -dimethyl-N-(4-(5 -thioxo-4,5 -dihydro-1,3,4-oxadiazol-2-yl)phenyl)benzamide] The 1H-NMR of Ad1 (Ô, ppm) (DMSO-J6): 5 14.69 (s, 1H,
5 12), 5 10.49 (s, 1H, N 13), 5 7.87 (s, 4H, C 2, 3, 5
6 6), 5 7.65 (d, 2H, C 17 & 21), 5 7.12 (m, 1H, C 19), 5 2.30 (s, 6H, C 22 & 23).The 13C-NMR of Ad1 (Ô, ppm) (DMSO-J6): 5 179.14 ppm (C9), 5 166.60 ppm (C14), 5 161.07 ppm (C7), 5 140.34 ppm (C1), 5 137.23 ppm (C18,20), 5 132.24 ppm (C16), 5 131.91 ppm (C19), 5 127.82 ppm (C3,5), 5 127.71 ppm (C17,21), 5119.77 ppm (C4), 5 119.63 ppm (C2,6), 5 20.92 ppm (C22,23).
Ad2 [2-(4-chlorophenoxy)-N-(4-(5 -thioxo-4,5 -dihydro-1,3,4-oxadiazol -2-
yl)phenyl)acetamide] .The 1H-NMR of Ad2 (Ô, ppm) (DMSO-J6): 5 14.69 (s, 1H, S 12), 5 10.49 (s, 1H, N 13), 5 7.87 (s, 4H, C 2, 3, 5 & 6), 5 7.37 (d, 2H, C 20 & 22), 5 7.04 (d, 2H, C 19 & 23), 5 4.77 (s, 2H, C 16). The 13C-NMR of Ad2 (ô, ppm) (DMSO-
d6): 5 179.14 ppm (C9), 5 168.04 ppm (C14), 5 161.07 ppm (C7), 5 157.21 ppm (C18), 5 138.40 ppm (C1), 5 129.54 ppm (C20,22), 5 127.75 ppm (C3,5), 5 127.29 ppm (C21), 5 119.77 ppm (C4), 5 119.09 ppm (C2,6), 5 116.05 ppm (C19,23), 5 67.49 ppm (C16).
Ad3 [3-chloro-N-(4-(N-(thiazol-2-
yl)sulfamoyl)phenyl)benzamide].The 1H-NMR of Ad3 (ô, ppm) (DMSO-d6): 5 12.72 (s, 1H, N 11), 5
10.45 (s, 1H, N 1), 5 8.10 (s, 1H, C 21), 5 8.04 (s, 1H, C 25), 5 7.92 (d, 2H, C 3 & 7), 5 7.78 (d, 2H, C
4 & 6), 5 7.48 (s, 1H, C 24), 5 7.45 (s, 1H, C 23), 5 7.20 (d, 1H, C 15), 5 6.80 (d, 1H, C 16). The 13C-NMR of Ad3 (ô, ppm) (DMSO-d6): 5 166.24 ppm (C18), 5 162.17 ppm (C10), 5 142.23 ppm (C2), 5 138.93 ppm (C15), 5 133.34 ppm (C20), 5 132.77 ppm (C5), 5 132.63 ppm (C22), 5 130.99 ppm (C23), 5 129.96 ppm (C24), 5 128.85 ppm (C4,6), 5 127.64 ppm (C21), 5 126.57 ppm (C25), 5 120.26 ppm (C3,7), 5 113.94 ppm (C16).
Ad4 [4-fluoro-N-(4-(N-(thiazol-2-
yl)sulfamoyl)phenyl)benzamide]. The 1H-NMR of Ad4 (ô, ppm) (DMSO-d6): 5 12.70 (s, 1H, N 9), 5 9.02 (s, 1H, N 1), 5 8.00 (d, 2H, C 21 & 25), 5 7.837.89 (m, 4H, C 3, 4, 6 & 7), 5 7.24 (d, 1H, C 15), 5 7.13 (d, 2H, C 22 & 24), 5 6.69 (d, 1H, C 16). The 13C-NMR of Ad4 (ô, ppm) (DMSO-d6): 5 166.34 ppm (C18), 5 164.82 ppm (C23), 5 162.17 ppm (C10), 5 142.43 ppm (C2), 5 138.93 ppm (C15), 5 132.77 ppm (C5), 5 130.64 ppm (C20), 5 129.84 ppm (C21,25), 5 128.85 ppm (C4,6), 5 120.26 ppm (C3,7), 5 115.97 ppm (C22,24), 5 113.94 ppm (C16). Ad5 [2-methyl-N-(4-(N-(thiazol-2-
yl)sulfamoyl)phenyl)benzamide]. The 1H-NMR of Ad5 (ô, ppm) (DMSO-d6): 5 12.70 (s, 1H, N 11), 5
10.46 (s, 1H, N 1), 5 7.92 (d, 2H, C 3 & 7), 5 7.82 (s, 1H, C 25), 5 7.78 (d, 2H, C 4 & 6), 5 7.51 (dd, 1H, C 23), 5 7.32 (s, 1H, C 24), 5 7.27 (s, 1H, C 22), 5 7.24 (d, 1H, C 15), 5 6.81 (d, 1H, C 16). The 13C-NMR of Ad5 (ô, ppm) (DMSO-d6): 5 164.26 ppm (C18),
5 162.17 ppm (C10), 5 160.22 ppm (C21), 5 142.14 ppm (C2), 5 138.93 ppm (C15), 5 132.93 ppm (C23), 5 132.77 ppm (C5), 5 129.88 ppm (C25), 5 128.85 ppm (C4,6), 5 124.54 ppm (C24), 5 120.92 ppm (C20), 5 120.14 ppm (C3,7), 5 115.93 ppm (C22), 5 113.94 ppm (C16).
Ad6 [4-nitro-N-(4-(N-(thiazol-2-
yl)sulfamoyl)phenyl)benzamide]. The 1H-NMR of Ad6 (ô, ppm) (DMSO-d6): 5 12.70 (s, 1H, N 9), 5 9.00 (s, 1H, N 1), 5 8.31 (d, 2H, C 22 & 24), 5 8.15
(d, 2H, C 21 & 25), 5 7.83- 7.89 (m, 4H, C 3, 4, 6 & 7), 5 7.13 (d, 1H, C 15), 5 6.69 (d, 1H, C 16). The 13C-NMR of Ad6 (ô, ppm) (DMSO-d6): 5 166.37 ppm (C18), 5 162.17 ppm (C10), 5 149.31 ppm (C23), 5 142.23 ppm (C2), 5 138.93 ppm (C15), 5 138.62 ppm (C20), 5 132.77 ppm (C5), 5 129.66 ppm (C21,25), 5 128.85 ppm (C4,6), 5 123.66 ppm (C22,24), 5 120.26 ppm (C3,7), 5 113.94 ppm (C16). Ad7 [3,5 -dimethyl -N-(4 -(N-(thiazol -2 -yl)
sulfamoyl) phenyl) benzamide]0. The 1H-NMR of Ad7 (ô, ppm) (DMSO-d6): 5 12.70 (s, 1H, N 11), 5 10.47 (s, 1H, N 1), 5 7.92 (d, 2H, C 3 & 7), 5 7.78 (d, 2H, C 4 & 6), 5 7.56 (s, 2H, C 21 & 25), 5 7.26 (s, 1H, C 23), 5 7.24 (d, 1H, C 15), 5 6.82 (d, 1H, C 16), 5 2.36 (s, 6H, C 26 & 27). The 13C-NMR of Ad7 (ô, ppm) (DMSO-d6): 5 166.65 ppm (C18), 5 162.17 ppm (C10), 5 142.43 ppm (C2), 5138.93 ppm (C15), 5 137.23 ppm (C22,24), 5 132.77 ppm (C5), 5 132.24 ppm (C20), 5 131.91 ppm (C23), 5 128.85 ppm (C4,6), 5 127.71 ppm (C21,25), 5 120.26 ppm (C3,7), 5 113.94 ppm (C16), 5 20.92 ppm (C26,27). Ad8 [2-(4-chlorophenoxy)-N-(4-(N-(thiazol-2-yl) sulfamoyl) phenyl) acetamide]. The 1H-NMR of Ad8 (ô, ppm) (DMSO-d6): 5 12.70 (s, 1H, N 11), 5 10.44 (s, 1H, N 1), 5 7.45 (d, 1H, C 15), 5 7.36 (d, 2H, C 4 & 6), 5 7.24 (d, 2H, C 3 & 7), 5 7.02 (d, 2H, C 24 & 26), 5 6.18 (d, 2H, C 23 & 27), 5 6.56 (d,
145
1H, C 16), 5 3.37 (s, 2H, C 20). The 13C-NMR of Ad8 (8, ppm) (DMSO-J6): 5 168.05 ppm (C18), 5 162.17 ppm (C10), 5 157.21 ppm (C22), 5 140.26 ppm (C2), 5 138.93 ppm (C15), 5 132.77 ppm (C5), 5 129.54 ppm (C24,26), 5 128,84 ppm (C4,6), 5 127.29 ppm (C25), 5 120.11 ppm (C3,7), 5 116.05 ppm (C23,27), 5 113.94 ppm (C16), 5 67.49 ppm (C20).
Biological study results:
The MIC results indicate that Augmentin exhibits significant activity against all bacterial strains, whereas Amoxicillin shows negligible antibacterial activity against all bacterial strains except for K. pneumonia. This implies that clavulanic acid (in Augmentin) acts as an anti-ß-lactamase agent, protecting Amoxicillin from enzyme action. However, the new amides exhibit minimal antibacterial activity. MIC analysis was conducted for the synthesized amides to ensure that when co-administered with Amoxicillin as an anti-ß-lactamase, they do not develop any antibacterial activity (their own sub MIC strengths of 400 |g and 800 |g will be used in the subsequent incubation step) (Table
4).
Tab
e 4. MIC outcomes for the standard antibacterial and the newly amides compounds
Minimum inhibitory concentration Minimum inhibitory concentration
Compds G (-)ve G (+)ve Compds G (-)ve G (+)ve
E. coli Klep. Pnem. Psed. Aerog. Staph. Aur. E. coli Klep. Pnem. Psed. Aerog. Staph. Aur.
Augmentin 750 1000 500 500 Ad4 1000 1000 1000 1000
Amoxicillin >2000 1000 >2000 >2000 Ad5 >2000 >2000 >2000 >2000
Ad1 >2000 1000 >2000 >2000 Ad6 1000 >2000 1000 >2000
Ad2 >2000 >2000 >2000 >2000 Ad7 >2000 1000 >2000 >2000
Ad3 1000 1000 1000 1000 Ad8 1000 >2000 >2000 >2000
In this phase, the P-lactamase inhibitory action of the new amides against the tested pathogenic bacterial isolates was assessed [29]. Each tested amides was utilized as co-inhibitor with 1000 |ig of amoxicillin formulated as disks
(5^l/disk) at 400 |ig and 800 |ig strengths.
Amoxicillin 1000 |ig alone and Amoxiclav (1000/200mg) were incubated too, the outcomes would be respects as a control for the newly synthesized amides (Table 5).
Table 5. The zones of inhibition for the amides when with the Amoxicillin against bacterial isolates
Diameter of Inhib. zones (mm) Diameter of Inhib. zones (mm)
G (+)ve G (-)ive G (+)ve G (-)ive
Compds Staph. Aureus E. coli K. pneumonia P. aeruginosa Compds Staph. Aureus E. coli K. pneumonia P. aeroginosa
1:1 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 1:2 1:1 1:2
Augmentin 18 18 21 22 20 22 21 22 Ad4 15 18 17 19 9 10 5 8
Amoxicilli n 9 10 0 0 0 0 0 0 Ad5 8 10 5 8 18 19 0 5
Ad1 18 19 19 19 18 21 20 20 Ad6 10 12 10 14 16 19 8 10
Ad2 18 20 18 20 9 12 20 22 Ad7 19 21 18 20 19 19 18 20
Ad3 20 21 21 22 12 15 21 21 Ad8 9 12 21 22 9 10 8 8
1:1 = 1000 |g/ml Amoxicillin: 400 |g/ml amides
1:2 = the same 1000 |g/ml Amoxicillin: 800 |g/ml amides
Generally, the above outcomes specified the fact that totally the new amides developed good action as P-lactamase inhibitors versus all bacterial strains signified that these compounds stop the P-lactamases in these bacterial strains.
Notably, two compounds, Ad1 and Ad7 (oxadiazole and sulfathiazole with R19), demonstrated promising activities resembling those of clavulanic acid, despite having no antibacterial activities when used alone at this strength. Conversely, compounds Ad2 and Ad3 (oxadiazole with R25 and sulfathiazole with R2) exhibited negative anti-P-lactamase activities against K. pneumonia only, with beneficial activities against the remaining strains. The
remaining amides demonstrated P-lactamase inhibition in only one or two strains, classifying them as weak inhibitors. All these amides incorporate one or more hydrophobic residues in their structures, along with halogen atoms or a nitro group in some compounds. This aligns with docking results and other research, indicating that increased hydrophobicity enhances selectivity for the P-lactamase enzyme. These findings suggest promising anti-P-lactamase activities for both oxadiazole and sulfathiazole scaffolds as non-P-lactam inhibitors, emphasizing their potential in combating bacterial resistance.
Conclusion
In conclusion, our findings suggest that the active pocket of P-lactamases favors hydrophobic residues, supported by the observation that four of the new amides with effective P-lactamase inhibitory activities contain one or more hydrophobic components. Additionally, the presence of halogens enhances the affinity towards P-lactamases. Derivatives
from both oxadiazole and sulfathiazole scaffolds exhibit promising anti-ß-lactamase activities, positioning them as potential non-ß-lactam inhibitors. These results underscore the significance of hydrophobicity and halogen entities in designing effective compounds to combat ß-lactamase-mediated bacterial resistance.
References
1. Olsen O. New promising b-lactamase inhibitors for clinical use // Eur. J. Clin. Microbiol. Infect Dis. 2015, V. 34, p. 13031308.
2. King D.T., Sobhanifar S., Strynadka N.C.J. One ring to rule them all: current trends in combating bacterial resistance to the b-lactams // Protein Sci. 2016, V. 25, p. 787803.
3. Mahmood A.A.J., Al-Iraqi M.A., Abachi F.T. Design, synthesis and anti-B-lactamase activity for new monobactam Compounds. Materials Today // Proceedings, 2021, V. 42, pp. 1860-1866.
4. Sakurai Y. Yoshida Y., Saitoh K., Nemoto M., Yamaguchi A., Sawai T. Characteristics of aztreonam as a substrate, inhibitor and inducer for beta-lactamases // J Antibiot (Tokyo), 1990, V. 43, p. 403-410.
5. Araoka H., Baba M., Tateda K., Ishii Y., Oguri T., Okuzumi K., Oishi T., Mori S., Mitsuda T., Moriya K., et al. Monobactam
and aminoglycoside combination therapy against metallo-b-lactamase-producing multidrug resistant Pseudomonas aeruginosa screened using a 'break-point checkerboard plate // Jpn. J. Infect. Dis. 2012, V. 65, p. 84-87.
6. Rongfeng Li, Ryan A.O., Craig A.T. Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster // Cell Chemical Biology, 2017, V. 24, no. 1, p. 24-34. http://dx.doi.org/10.1016/j.chembiol.2016.1 1.010.
7. Grigorenko V.G., Andreeva I.P., Rubtsova M.Yu., Deygen I.M., Antipin R.L., Majouga A.G., Egorov A.M., Beshnova D.A., Kallio J., Hackenberg C., Lamzin V.S. Novel non-ß-lactam inhibitor of ß-lactamase TEM-171 based on acylated phenoxyaniline // Biochimie, 2017, V. 132, p. 45-53, https://doi.org/10.1016/j.biochi.2016.10.011.
8. Mahmood A.A.J., Zainal A.A., Khalaf MM.
Cholinesterase activity in non-alcoholic-fatty liver disease in diabetic patients taking oral antidiabetic drugs // Current Topics in Pharmacology, 2021, V. 25, pp. 43-51
9. Baquero F., Tedim A.P. , Coque T.M. Antibiotic resistance shaping multilevel population biology of bacteria // Front. Microbiol., 2013, V. 15, no. 4, pp. 1-15
10. Tang S., Apisarnthanarak A., Hsu L.Y. Mechanisms of P-lactam antimicrobial resistance and epidemiology of major community-and healthcare-associated multidrug-resistant bacteria // Adv. Drug Deliv. Rev., 2014, V. 78, pp. 3-13. doi: 101016/j addr201408003
11. Mahmood A.A.J., Khalaf M.M., Zainal A.A. Cholinesterases activities in diabetic and hyperlipidemic patient // Indian Journal of Public Health Research and Development, 2019, V. 10, no. 10, pp. 2812-2816
12. Pimenta A.C., Fernandes R., Moreira I.S. Evolution of drug resistance: insight on TEM P-lactamases structure and activity and P-lactam antibiotics // Mini Rev. Med. Chem., 2014, V. 14, pp. 111-122
13. Bujor A., Hanganu A., Tecuceanu V., Madalan A.M., Tudose M., Marutescu L., Popa M., Chifiriuc C.M., Zarafu I., Ionita P. Biological Evaluation and Structural Analysis of Some Aminodiphenylamine Derivatives // Antioxidants, 2023, V. 12, 713.
https://doi .org/10.3390/antiox 12030713
14. Ehmann D.E., Jahic H., Ross P.L., Gu R.F., Hu J., Kern G., Walkup G.K., Fisher S.L. Avibactam is a covalent, reversible, non-P-lactam P-lactamase inhibitor // PNAS, 2012, V. 109, pp. 11663-11668
15. Bryant B., Knights K., editors. Pharmacology for health professionals, 3rd ed. Pharmacology, Australia: Elsevier; 2011, vol 3., 645 p.
16. Zhao C., Rakesh K.P., Ravidar L., Fang W.Y., Qin H.L. Pharmaceutical and medicinal significance of sulfur (SVI)-containing motifs for drug discovery: a critical review // Eur. J. Med. Chem. 2019, V. 162, p. 679-734.
17. Kha A., Mushtaq S., Naz S., Farooq U., Zaidi A., Bukhari S.M. Sulfonamides as potential bioactive scaffolds // Curr. Org. Chem. 2018, V.22, p. 818-30.
147
18. Yurtta§ L., Akalin-£ift9i G. New quinoline based sulfonamide derivatives // Anti Canc Agents Med Chem. 2018, V.18, p. 1122-30.
19. Naim M.J., Alam O., Farah Nawaz M., Alam J., Alam P. Current status of pyrazole and its biological activities // J Pharm Bioallied Sci. 2016, no.8, p. 2-17. https://doi.org/10.4103/0975-7406.171694
20. Ahn M., Gunasekaran P., Rajasekaran G., Kim E.Y., Lee S.J., Bang G., Cho K., Hyun J.K., Lee H.J., Jeon Y.H., Kim N.H. Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity // Eur J Med Chem. 2017, V.125, p. 551-564. https://doi. org/10.1016/j.ejmech.2016.09.071
21. Mihailovi'c N., Markovi'c V., Mati'I Z., Stanisavljevi'c N.S., Jovanovi'c Z.S., Trifunovi S.Z., Joksovi'c L. Synthesis and antioxidant activity of 1,3,4-oxadiazoles and their diacylhydrazine precursors derived from phenolic acids // RSC Adv. 2017, no. 7, p. 8550-8560
22. Gaba M., Mohan C. Development of drugs based on imidazole and benzimidazole bioactive heterocycles: recent advances and future directions // Med Chem Res. 2016, V.25, p. 173-210. https://doi.org/10.1007/s00044-015-1495-5
23. Feng M., Tang B., Liang S.H., Jiang X. Sulfur containing scaffolds in drugs: synthesis and application in medicinal chemistry // Curr Top Med Chem 2016, V. 16, p. 1200-1216
24. Nitulescu G.M., Draghici C., Olaru O.T. Thiede J.M., Kordus S.L., Turman B.J., Buonomo J.A., Aldrich C.C., Minato Y., Baughn A.D. Targeting intracellular paminobenzoic acid production potentiates the anti-tubercular action of antifolates // Sci Rep. 2016, V. 6, 38083. https://doi.org/10.1038/ srep38083
25. Alwan S.M. Computational Calculations of Molecular Properties and Molecular Docking of New and Reference Cephalosporins on Penicillin Binding Proteins and Various P-Lactamases // Journal of Pharmacy and Pharmacology, 2016, no. 4, p. 212-225.
26. Rafiq Z., Saranya S., Vaidyanathan R. Computational Docking and In Silico Analysis Of Potential Efflux Pump Inhibitor
Punigratane // International Journal of Pharmacy And Pharmaceutical Sciences. 2018, V. 10, no. 3, p. 27-34.
27. Bidya S., Suman R.S. Comparative Study of Three ß Lactamase Test Methods in Staphylococcus aureus Isolated from Two Nepalese Hospitals // Open Journal of Clinical Diagnostics, 2014, no. 4, p. 47-52. http://dx.doi.org/10.4236/ojcd.2014.41009
28. Mahmood A.A.J., Al-Iraqi M.A., Abachi F.T. New 4, 4'-Methylenedianiline Monobactame Compounds: Synthesis, Antioxidant and Antimicrobial Activities Evaluation // AIP Conference Proceedings, 2022, V. 2660, 020024
29. Kowalska-Krochmal B., Dudek-Wicher R. The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance // Pathogens. 2021, V. 10, no. 2, 165. doi: 10.3390/pathogens10020165. PMID: 33557078; PMCID: PMC7913839.
30. Sperling D., Karembe H., Zouharova M., Nedbalcova K. Examination of the minimum inhibitory concentration of amoxicillin and marbofloxacin against Streptococcus suis using standardised methods // Vet Med -Czech, 2020, V. 65, no. 9, p. 377-386 DOI: 10.17221/111/2020-VETMED
31. Mahmood A.A.J. Synthesis, antioxidant and antimicrobial activities for new 4,4'-
methylenedianiline amide compounds // Egyptian Journal of Chemistry, 2021, V. 64, no. 12, pp. 6999-7005
32. Balouiri M., Sadiki M., Ibnsouda S.K. Methods for in vitro evaluating antimicrobial activity: A review // J Pharm Anal. 2016, V. 6, no. 2, p. 71-79. doi: 10.1016/j jpha.2015.11.005.
33. Saleh A.A., Mahmood A.A. New p-aminodiphenylamine amide compounds: design, synthesis and anti ß-lactamases activity evaluation. Chemical Problems, 2024, V. 22, no. 1, p. 20-32
34. Arun K., Sharma L., Kaura M. Synthesis and Comparative Antimicrobial Study of Beta-Lactam Derivatives // International Journal of Pharmaceutical and Chemical Sciences 2013, Vol. 2, no.2, p. 560-565
35. Bahnam S.P., Jasim Mahmood H.M., Mahmood A.A.J. In silico study of new schiff base-and amide-boronic acid derivatives as potential inhibitors of ß-lactamases. Indian Drugs, 2023, V. 60, no. 9, p. 37. D0I:10.53879/id.60.09.13937
36. Avci FG., Altinisik F.E., Vardar Ulu D., Ozkirimli O.E., Akbulut B.S. An evolutionarily conserved allosteric site modulates beta-lactamase activity // J. Enzyme Inhib. Med. Chem. 2016, V. 31, p. 33-40.
DOI:10.1080/14756366.2016.1201813.
YENi AMID BiRL9§M9L9RiNiN p-LAKTAMAZ iNHiBiTOR AKTiVLiYiNiN QiYMaTLaNDiRiLMaSi, SiNTEZi V9 DOKiNQi
9hmad A. J. Mahmud, Safaa P. Bahnam
Mosul Universiteti, dczagiliq Kolleci, dczagiliq Kimyasi Departamenti, iraq e-pogt: [email protected]. iq
Xulasa: Beta-laktamlar infeksiyalarin mualicasi u9un effektiv antibiotiklar kimi taninir, lakin beta-laktam halqasini hidroliz edan P-laktamazanin bakterial istehsali bu darmanlari tasirsiz hala sala bilar. Bu antibiotiklari klavulan tur§usu kimi bir P-laktamaz inhibitoru ila kopmbinasiyasi bu muqavimati azaldir. Temoniera-1 (TEM-1), 1pzp-ni ahata edan tadqiqatlarda biz tur§u xlorid toramalarini sulfatiazol va ya oksadiazol amin ila reaksiyaya daxil edarak, amid rabitasi yaradaraq sakkiz amid birla§masinin sintezini tamin etmi§ik. Yeni sintez edilmi§ birla§malar fiziki va spektroskopik usullarla differensialla§dirilmi§, P-laktamaz G(+) va G(-) bakteriyalarinin dord §tamina qar§i onlarin minimum inhibitor qatiligi (MiK) qiymatlandirilmi§ va bioloji cahatdan yoxlanilmi§dir. Daha sonra onlarin anti P-laktamaz davrani§lari eyni dord bakteriya §tamina qar§i
amoksisillinla birga inhibitor kimi klavulan tur§usu ila muqayisa edilmi§dir. Naticalar gostarir ki, yeni amidlardan dordu ala anti-P-laktamaz aktivliyi numayi§ etdirir va strukturlarinda bir va ya daha 9ox hidrofobik qaliq ehtiva edir. Halogen atomlari artiqligi P-laktamazlarin se?iciliyini artirir. Ham oksadiazol, ham da sulfatiazol karkaslarinin toramalari qeyri-P-laktam inhibitorlari kimi perspektivli anti-P-laktamaz xassasi gostarir.
A?ar sozlar: Sulfatiazol, Oksadiazol, TEM-1 P-laktamaza, amidlar, dokinq, anti-P-laktamaza.
ДОКИНГ, СИНТЕЗ И ОЦЕНКА р-ЛАКТАМАЗНОЙ ИНГИБИРУЮЩЕЙ АКТИВНОСТИ НОВЫХ АМИДНЫХ СОЕДИНЕНИЙ
Ахмед А.Дж. Махмуд, Сафаа П. Бахнам
Кафедра фармацевтической химии, Фармацевтический колледж, Мосульский университет, Ирак e-mail: ahmedsot@,uomosul. edu. iq
Резюме: Бета-лактамы признаны эффективными антибиотиками для лечения инфекций, однако бактериальная продукция Р-лактамазы, которая гидролизует бета-лактамное кольцо, может сделать эти препараты неактивными. Сочетание этих антибиотиков с ингибитором Р-лактамаз, таким как клавулановая кислота, снижает эту резистентность. В исследовании стыковки с участием Temoniera-1 (TEM-1), 1pzp, мы индуцировали синтез восьми амидных соединений путем взаимодействия производных хлорангидридов с сульфатиазолом или оксадиазоламином, образуя амидную связь. Вновь синтезированные соединения были дифференцированы физическими и спектроскопическими методами и биологически проверены путем оценки их минимальной ингибирующей концентрации (МПК) в отношении четырех штаммов бактерий Р-лактамаз G(+)ve и G(-)ve. Затем их анти-Р-лактамазное поведение сравнивали с действием клавулановой кислоты в качестве со-ингибитора с амоксициллином против тех же четырех штаммов бактерий. Результаты показывают, что четыре из новых амидов проявляют превосходную анти-Р-лактамазную активность и содержат в своей структуре один или несколько гидрофобных остатков. Увеличение атомов галогенов повышают селективность Р-лактамаз. Производные как оксадиазольных, так и сульфатиазольных каркасов демонстрируют многообещающую анти-Р-лактамазную активность в качестве не-Р-лактамных ингибиторов.
Ключевые слова: сульфатиазол, оксадиазол, Р-лактамазы ТЕМ-1, амиды, докинг, анти-Р-лактамазы.
