Antibacterial sulfonyl oxadiazoles

Abstract

A method of treating or preventing a bacterial infection in a subject in need thereof is described. The method includes administering a therapeutically effective amount of a compound according to formula I to the subject; wherein R1 is is a benzyl or lower alkyl group and R2 -R6 are independently selected from H, methyl, and halogen, or a pharmaceutically acceptable salt thereof.

Description

ANTIBACTERIAL SULFONYL OXADIAZOLESGOVERNMENT FUNDING

[0001] The present invention was made with government support under Grant No. 1 R01AG076699 awarded by the National Institutes of Health. The US government has certain rights in this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 574,290, filed April 4, 2024, and U.S. Provisional Patent Application Serial No. 63 / 641,451, filed May 2, 2024, the disclosures of which are both incorporated herein by reference.BACKGROUND

[0003] Methicillin-resistant Staphylococcus aureus (MRSA) infections affect hundreds-of- thousands of Americans every year, resulting in additional $1.7 billion in attributable healthcare costs and tens-of-thousands of deaths. Although several new drugs have been approved in the past twenty years, there are few antibiotics currently in the clinical trial pipeline and mortality rates associated with MRSA infections have not significantly decreased. For this reason, researchers continue searching for new antibiotics based on structure types that differ from currently utilized drugs such as P-lactams, glycolipopeptides, etc.

[0004] All the Staphylococcus aureus strains have penicillin-binding proteins (PBPs) from PBP1 to PBP4, but MRSA express a special PBP (PBP2 or PBP2a) from the mecA gene PBP2a takes over the biosynthetic function of normal PBPs in the presence of inhibitory concentration of P-lactams because PBP2 has a decreased binding affinity to P-lactams.

[0005] Recently, several groups have reported successful studies on the use of various heterocycle-based compounds, namely quinazolinones and oxadiazoles. Qian Y, et al., J Med Chem 63(10):5287-5296 (2020); Verma SK, et al., Eur J Med Chem 219:1 13442 (2021). The former were found to exhibit remarkably minimum inhibitory concentration (MIC) values as well as water solubility. Furthermore, they were demonstrated to inhibit penicillin binding protein 2a (PBP2a) allosterically. PBPs are involved in cell-wall synthesis and are generallythe target of β-lactam antibiotics. MRSA, however, expresses PBP2a, which contains a closed active site until it has been allosterically activated by cell wall peptide fragments. This allosteric mechanism is targeted by the only two (3-lactams with significant anti-MRSA activity: ceftaroline and ceftobiprole. Mahasenan KV, et al., J Am Chem Soc 139(5):2102-2110 (2017).

[0006] The same group that investigated the use of quinazolinones (Bouley R, et al., J Med Chem 59(10):5011-5021 (2016)) also found that that oxadiazoles could also serve as antibacterial agents against MRSA. Ceballos S, et al., Antimicrob Agents Chemother 62(8) (2018). They found, through another screening and synthesis study, that the 1 ,2,4-oxadiazoles are also active against MRSA. O'Daniel PI, et al., J Am Chem Soc 136(9): 3664-3672 (2014). The identified lead compound was 4-(3-(4-(4-(Trifluoromethy l)phenoxy)phenyl)- 1 ,2,4- oxadiazol-5-yl)aniline, which exhibited an MIC of 2 pg / mL (5 pM). Although they were unable to confirm, crystallographically, that these compounds also bind to the same allosteric site as the quinazolinones or the 5th generation anti-MRSA cephalosporins (Zhanel GG, et al., Am J Clin Dermatol 9(4): 245-254 (2008)), the authors could demonstrate potent synergism between P-lactams and this new class of antibiotics. This synergism is in all likelihood due to allosteric inhibition by the oxadiazole, followed by competitive inhibition by the (3 lactam. Chang M, Mahasenan KV, Hermoso JA, & Mobashery S, Acc Chem Res 54(4):917-929 (2021). This work was followed by further research into this structure type (Biernacki K, et al., Pharmaceuticals (Basel) 13(6) (2020)), and several compounds with lower MIC values down to 0.5 pg / mL (1 pM) were reported. Boudreau MA, et al., ACS Med Chem Lett 11(3):322- 326 (2020). Although effective, some of these compounds exhibited noticeable cytotoxicity and their efficacy did not translate well to animal studies. Leemans E, et al., Bioorg Med Chem Lett 26(3):1011-1015 (2016).

[0007] Importantly, the 1 ,2,4-oxadiazoles only represent one possible set of structural isomers, with 1,2,5- and 1,3,4-oxadiazoles also being investigated by medicinal chemists. Although both the 1,2,4- and 1,3,4-oxadiazoles are highly studied, scientists at AstraZeneca published a report indicating that the 1,3,4-isomers are expected to exhibit greater metabolic stability as compared to matched 1,2,4-oxadiazole regio-isomers. Bostrom et al., J Med Chem 55(5): 1817-1830 (2012). They suggest that this is in part due to decreased lipophilicity of 1,3,4 analogues as compared to their 1,2,4 counterparts, higher dipole moments, and decreased cytochrome P450 recognition. Since then, there have been only a few reports on the antibacterial properties of new 1,3,4-oxadiazoles. Yang P, et al., ChemistrySelect 6(46):13209-13214 (2021). Many ofthese compounds are ineffective against MRSA, including those which were bound to fluoroquinolones. Of those which demonstrated potent antibacterial activity, these typically had an inert, aromatic group directly attached at C2 of the 1 ,3,4-oxadiazole ring and aryl-amide derivatives at C5. Naclerio et al., ACS Infect Dis 8(4):865-877 (2022); Naclerio et al., J Med Chem 63(20): 11934-11944 (2020).

[0008] Based on these findings, this class remains open to further investigation as many other alternative substituent patterns have not been thoroughly considered. For example, the Shoichet group published a report detailing the identification of 2 sulfonyl- 1 ,3 ,4-oxadiazoles as potential covalent p-lactamase inhibitors. Babaoglu K, et al., J Med Chem 51 (8):2502-2511 (2008). Both compounds were found to be potent AmpC inhibitors with IC50 <1 μM. and the mechanism was suggested to be nucleophilic aromatic substitution with the sulfonyl substituent acting as the leaving group.SUMMARY OF THE INVENTION

[0009] The present invention provides a method of treating or preventing a bacterial infection in a subject in need thereof. The method includes administering a therapeutically effective amount of a compound according to formula I:to the subject; wherein R1is is a benzyl or lower alkyl group and R2-R6are independently selected from H, methyl, and halogen, or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTION OF THE FIGURES

[0010] The present invention may be more readily understood by reference to the following drawings wherein:

[0011] Figure 1 provides a graph and chemical schematics showing the general chemical scaffold and single-dose activity screening against MRSA. Structure-Activity Relationship among the 1,3,4-Oxadiazoles series with single-dose cell-based assay against MRSA pathogen.

[0012] Figure 2 provides graphs showing the half-maximal cytotoxic concentrations (CC50) of both L- and D- isomers against human HEK239 cells.

[0013] Figure 3 provides images showing the inhibition zone assay comparing PEG vehicle control and 0.6% D-O30.

[0014] Figures 4a-4e provide graphs and images showing skin wound infection mouse model study design and results: a) study design; b) imaging results; c) ROI quantification for mice treated with the vehicle control; d) ROI quantification for mice treated with 0.6% D-O30; e) CFU counts from biopsied tissue at the conclusion of the experiment. *** p < 0.001

[0015] Figure 5 shows a chemical schematic and image of LC-MS / MS analysis and determined chemical reaction mechanism.

[0016] Figure 6 provides an image showing a covalent inhibition model of D-isomer #30 for the allosteric site of PBP2a.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0017] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0018] As used herein, the term "organic group" is used to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for the compounds of this invention are those that do not interfere with the antibacterial activity of the compounds. In the context of the present invention, the term "aliphatic group"means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0019] As used herein, the terms "alkyl", "alkenyl", and the prefix "alk-" are inclusive of straight chain groups and branched chain groups. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In some embodiments, these groups have a total of at most 10 carbon atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4 carbon atoms. Alkyl groups including 4 or fewer carbon atoms can also be referred to as lower alkyl groups. Alkyl groups can also be referred to by the number of carbon atoms that they include (i.e., Ci - C4 alkyl groups are alky groups including 1-4 carbon atoms).

[0020] Cycloalkyl, as used herein, refers to an alkyl group (i.e., an alkyl, alkenyl, or alkynyl group) that forms a ring structure. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. A cycloalkyl group can be attached to the main structure via an alkyl group including 4 or less carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbornyl, and norbornenyl.

[0021] Unless otherwise specified, "alkylene" and "alkenylene" are the divalent forms of the "alkyl" and "alkenyl" groups defined above. The terms, "alkylenyl" and "alkenylenyl" are used when "alkylene" and "alkenylene", respectively, are substituted. For example, an arylalkylenyl group comprises an alkylene moiety to which an aryl group is attached.

[0022] The term "haloalkyl" is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of other groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like. Halo moieties include chlorine, bromine, fluorine, and iodine.

[0023] The term "aryl" as used herein includes single aromatic rings or multiring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may be substituted or unsubstituted.

[0024] Unless otherwise indicated, the term "heteroatom" refers to the atoms O, S, or N.

[0025] When a group is present more than once in any formula or scheme described herein, each group (or substituent) is independently selected, whether explicitly stated or not. For example, for the formula -C(O)-NR2 each R group is independently selected.

[0026] As a means of simplifying the discussion and the recitation of certain terminology used throughout this application, the terms "group" and "moiety" are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not so allow for substitution or may not be so substituted. Thus, when the term "group" is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and that group with nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well as carbonyl groups or other conventional substituents. Where the term "moiety" is used to describe a chemical compound or substituent, only an unsubstituted chemical material is intended to be included. For example, the phrase "alkyl group" is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tertbutyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, "alkyl group" includes ether groups, haloalkyls, nitroalkyls, carboxy alkyls, hydroxy alky Is, cyanoalkyls, etc. On the other hand, the phrase "alkyl moiety" is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, and the like.

[0027] A subject, as defined herein, is an animal such as a vertebrate or invertebrate organism. In other embodiments, the subject is a mammal such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). More preferably, the subject is a human.

[0028] “Treat", "treating", and "treatment", etc., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as bacterial infection, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, or delay in the onset of the disease, etc.

[0029] “Preventing,” as used herein, refers to any action that decreases the risk that a subject will develop an infection, or that will decrease the risk of symptoms should an infection nonetheless occur. Preventing infection can be done in subjects who have an increased risk of developing an infection. Subjects can have an increased risk of developing an infection as aresult of, for example, being immunosuppressed or having recently been exposed to other individuals who are infected.

[0030] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0031] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses.Treating a Bacterial Infection

[0032] A method of treating or preventing a bacterial infection in a subject in need thereof, comprising administering a therapeutically effective amount of a compound according to formula I:

[0033] to the subject; wherein R1is a benzyl or lower alkyl group and R2-R6are independently selected from H, methyl, and halogen, or a pharmaceutically acceptable salt thereof.

[0034] The in ventors have carried out structure- activity studies to evaluate the effect of varying the core structure of the compound of formula I on activity. For instance, R1is a benzyl or lower alkyl group and R2- R6are independently selected from H, methyl, and halogen In some embodiments, Rlis a benzyl group. In further embodiments, Rlis a lower alkyl group, such as a methyl, ethyl, propyl, or butyl group. In some embodiments, R2- R6are the same. In further embodiments, R2- R6are selected from hydrogens and halogens. For example, in some embodiments, R3and R4are chloro substituents.

[0035] In some embodiments, the compound is the L-isomer:

[0036] In further embodiments, the compound is the D-isomer:

[0037] The present invention provides a method of treating or preventing bacterial infection in a subject. Bacterial infection refers to infection of the subject by pathogenic bacteria. The bacteria can be either gram-negative bacteria or gram-positive bacteria. A wide variety of pathogenic bacteria are known to those skilled in the art. Examples of pathogenic bacterial species include Mycobacterium tuberculosis, Bordella pertussis, Chlamydia trachomatis, Salmonella Typhi, Escherichia coli, Francisella tularensis, Helicobacter pylori, Vibrio cholerae, Clostridium botulinum, Streptococcus pneumoniae, Yersinia enterocolitica, and Staphylococcus aureus. In some embodiments, only treatment is provided, while in other embodiments, administration of the compound is prophylactic. Preventive treatment can be administered to a subject who has an increased risk of developing a bacterial infection.

[0038] In some embodiments, the bacterial infection is an antibiotic resistant pathogen. Antibiotic resistant pathogens (e.g., pathogenic bacteria) are pathogens having the ability to resistant treatment with an antibiotic, such that the antibiotic is much less effective against that pathogen than a corresponding non-resistant pathogen. Resistance mechanisms include the ability to restrict access of the antibiotic, remove antibiotic that enters the cell, change or destroy the antibiotic, change the target of the antibiotic, or bypass the effects of the antibiotic. For example, methicillin-resistant Staphylococcus aureaus (MRSA) is known to have amutated pencilillin binding protein which cannot be targeted by β-lactam antibiotics. In some embodiments, the bacterial infection is a multidrug-resistant bacterial infection. Multidrugresistant bacteria are those that are resistant to one or more classes of antimicrobial agents. The Center for Disease Control defines a multidrug-resistant bacteria as one that is resistant to at lest one antibiotic in three or more drug classes. In some embodiments, the multidrug-resistant bacterial infection is by methicillin-resistant Staphylococcus aureaus. Other examples of multidrug-resistant bacteria include drug-resistant tuberculosis, drug-resistant Neisseria gonorrhoeae, and Carbapenem-resistant Aceinetobacter.

[0039] The compounds of Formula I can be administered together with an antibiotic compound (i.e., a second compound) to provide more effective treatment or prevention of bacterial infection. Suitable antibiotics include bactericidal or bacteriostatic compounds already known in the art. Examples of known antibiotics include agents that target the bacterial cell wall, such as penicillins, cephalosporins, agents that target the cell membrane such as polymyxins, agents that interfere with essential bacterial enzymes, such as quinolones and sulfonamides, and agents that that target protein synthesis such as the aminoglycosides, macrolides and tetracyclines. Additional known antibiotics include cyclic lipopeptides, glycylcyclines, and oxazolidinones. In some embodiments, the additional antibiotic is a fluoroquinolone compound such as ciprofloxacin.Antibacterial Compounds

[0040] In a further aspect, the present invention provides an antibacterial compound having the structure:

[0041] In some embodiments, the compound is the L-isomer:

[0042] In further embodiments, the compound is the D-isomer:

[0043] The invention is inclusive of the compounds described herein in any of their pharmaceutically acceptable forms, including, tautomers, salts, solvates, polymorphs, prodrugs, and the like. In particular, if a compound isoptically active, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated).

[0044] The compounds disclosed herein may contain one or more asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. The compounds can be, for example, racemates or optically active forms. The optically active forms can be obtained by resolution of the racemates or by asymmetric synthesis. In some instances, the compounds disclosed herein are R enantiomers. In other instances, the compounds disclosed herein are S enantiomers. In some instances, the compounds disclosed herein are varying mixtures of enantiomers.Formulation and Administration

[0045] The present invention provides a method for administering one or more anti-bacterial compounds together with a pharmaceutically acceptable carrier. Examples of pharmaceuticalcarriers or compositions include those for oral, intravenous, intramuscular, subcutaneous, or intraperitoneal administration, or any other route known to those skilled in the art, and generally involves providing a compound formulated together with a pharmaceutically acceptable carrier.

[0046] When preparing the compounds described herein for oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate. The active ingredient may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.

[0047] For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as sealed ampoules or vials.

[0048] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in nonaqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.

[0049] In some embodiments, the compound is administered topically. Suitable topical formulations for use in the present embodiments may also include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, gels, and the like. In some embodiments, the topical formulation is a polyethylene glycol (PEG) based ointment.

[0050] The dosage form and amount can be readily established by reference to known treatment or prophylactic regiments. The amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this invention depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the unwanted proliferating cells, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg / kg body weight, preferably between about 0.1 and about 50 mg / kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

[0051] The anti-bacterial compounds can also be provided as pharmaceutically acceptable salts. The phrase “pharmaceutically acceptable salts” connotes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2- hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, y-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds described herein include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Alternatively, organicsalts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl glucamine) and procaine may be used form base addition salts of the compounds described herein. All of these salts may be prepared by conventional means from the corresponding compounds described herein by reacting, for example, the appropriate acid or base with the compound.Preparation of Antibacterial Compounds

[0052] Compounds of the invention may be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, particularly in light of the description contained herein. Preparation of the compounds is also described in the Example herein. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wisconsin, USA) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.) and similar texts known to those skilled in the art.

[0053] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLESExample 1: Development of Non-h-Lactam Allosteric Inhibitors Targeting PBP2a in Methicillin-Resistant Staphylococcus aureus

[0054] Based on previous work with P-lactamase inhibitors, the inventors set the 2-sulfonyl- 1 ,3,4-oxadiazole ring and substituent as our template core from which we began our screening. Herein, we report the computational screening, stereoselective synthesis and characterization of a new set of 2-sulfonyl-l,3,4-oxadiazoles, demonstrating their potent anti-MRSA activity in vitro and in vivo.Results and Discussion

[0055] Following several existing reports on oxadiazoles as non-|3-lactam antibiotics with activity against various bacteria and drug-resistant enzymes, we utilized a commerciallyavailable compound library with the general structure depicted in Figure 1 for initial screening. For the initial investigation, the list of 69 compounds selected was broadly classified into a group of compounds including 2-thio-l,3,4-oxadizoles and 2-sulfonyl-l,3,4-oxadiazoles cores.

[0056] The bacteria cell inhibition efficacy of these initial 1,3,4-oxadiazole compounds (50 pM) was tested against MRSA (ATCC BAA-44, 5 x 105CFU / mL; Fig 1) using a single-dose assay. Amoxicillin and meropenem were included as control groups, showing significant resistance with survival rates of 85% and 81%, respectively, after 18 hours of treatment. Twenty-one of these compounds reduced the survival rate of MRSA cells by more than 80%, and the antibacterial activity of those selected in the primary screening was further characterized. In our initial screening, it is important that all 21 compounds selected for further research are comprised of 2-sulfonyl- 1,3,4-oxadiazole, indicating that other heteroatoms severely inhibit activity. To further understand the activity of the remaining 21 compounds, minimum inhibitory concentrations (MIC) were determined using the same ATCC BAA-44 MRSA strain (Table 1).

[0057] Table 1. MIC determination for the MRSA strain of the 21 initially selected 2-sulfonyl- 1,3,4-oxadiazole compounds.

[0059] Several observations were made from the MIC measurements. First, the lowest MIC (12 pg / mL) was observed for compound 30 after 18 hours of treatment. Second, the R2 substituent appeared to have a relatively weak correlation with activity because almost every R2, benzyl, group had a combination that demonstrated some activity. Third, the Ri, 3,4- dichloro benzyl, group appeared to have a stronger effect on antibacterial efficacy; the majority of active compounds have an electron-withdrawing dichlorobenzyl Ri group (Table 1).

[0060] From such structure- activity relationship analysis, it became clear that stereochemistry could act as an additional variable affecting the inhibition activity of the compounds due to the stereocenter at the carbon attached to both the Ri and tert-butoxycarbonyl (NHBOC) groups. Therefore, a new stereochemically selective synthesis was developed to produce both L(R) and D(S) enantiopure forms of compound 30, a 2-sulfonyl-l,3,4-oxadiazole derivative (Scheme 1).

[0061] Scheme 1. Synthesis of enantiopure D(S)-O30 (5).

[0062] A combination of NMR, mass spectrometry, and Optical rotation was used to confirm the purity of both the D(S) and L(R) enantiomers. Subsequently, the overall MIC values for both enantiomer compounds were measured and compared against all in-house laboratory strains of MRS A, including ATCC BAA-44 bioluminescent S. aureus, to analyze their inhibition activity (Table 2).

[0063] Table 2. MIC determination and comparison using L- and D-O30 against MRSA strains.

[0064] Through repeated experiments, we demonstrated that treatment with compound D- isomer (S) 30 exhibited significantly increased MIC activity compared to its L-isomer (R) enantiomer. Furthermore, similar results were observed in the MIC values between the bioluminescent SA strain and other MRSA laboratory strains following treatment with D-O30, showing a 4~8 fold increase in overall cell inhibition activity.Enzyme kinetics study

[0065] Noncompetitive inhibition, a type of allosteric regulation, is a specific type of enzyme inhibition characterized by an inhibitor binding to an allosteric site resulting in decreased efficacy of the enzyme. An allosteric site is simply a site that differs from the active site- where the substrate binds.

[0066] Structures of apo-PBP2a and nitrocefin-acylated PBP2a revealed a closed active site, such that an intact (3-lactam could not gain access to the active site. This suggested the need for a conformational change at the active site in order to accommodate the antibiotic. The closed active site can explain resistance, but not the physiological reaction of PBP2a. The enzyme is able to efficiently perform the peptidoglycan-crosslinking reaction, which requires the ability of the active site to accommodate the two strands of the peptidoglycan, which entail a volume in excess of 1000 A3. This indicates that the protein should undergo conformational changes in the course of catalysis, which has been documented to be the case, as we will elaborate.

[0067] The allosteric domain is found 60 A distant from the active site. In the X-ray structure of PBP2a with ceftaroline bound at both the allosteric and active sites, a large conformational change was seen through the protein when compared to the structure of the apo enzyme (PDB 1VQQ). This allosteric triggering propagates to the active site through a network of salt bridges, akin to dominoes falling from one site to the other (a span of 60 A).Cytotoxicity study

[0068] To determine whether this compound could be utilized in further in vivo studies, an MTT assay was carried out with human kidney HEK239 cells to determine potential cell toxicity. Both the D- and L-isomers exhibited similar 50% cytotoxicity concentrations (CC50) of 61.9±0.4 pM and 73.2±14.8 pM, respectively, indicating a cytotoxicity range that is sufficiently acceptable compared to several conventional antibiotics in use. (Figure 2).Application of PEG-based ointment to the skin wound infection model.

[0069] Based on these promising results, an in vivo study was planned to validate the antibacterial efficacy of this new compound in a more complex biological model. However, this compound is insoluble in water and its current solubility is limited to polar organic solventssuch as DMSO. For this reason, two steps were taken. First, a topical PEG-based ointment was prepared with 0.6% D-O30 and its in vitro efficacy was validated to ensure there was no measurable reduction in activity (Figure 3).

[0070] By comparing both plates, it could be clearly observed that the 0.6% D-O30 PEG-based ointment retained its antibacterial activity, exhibiting a clear inhibition zone surrounding the location where a small pellet (50 pL) had been placed. In comparison, the vehicle control did not demonstrate any inhibition.

[0071] The in vivo study was then carried out using a skin wound infection mouse model. Dorsal biopsies (5 mm) were created on JAX Swiss albino mice and subsequently infected with S. aureus (Xen 36; 40 pL, 5 x 105CFU / mL; Figure 4a). The extent of infections (and treatment) was monitored using the IVIS Lumina system in the bioluminescence imaging mode (Figure 4b-d). Treatments were applied once daily for three days and images were taken prior to each treatment. From these images and from further region of interest (ROI) quantification, it could be clearly observed, in real time, that the D-30 topical ointment quickly and significantly decreased bacterial burden. After one treatment, the image (day 2) demonstrates a more than lOx decrease in bacterial bioluminescence as compared to the control. However, to quantify the decrease in bacterial concentration, the infected tissue with an additional 1 mm margin (6 mm biopsy) was biopsied, homogenized, and plated to count the number of colonies forming units (CFU). After 18 hours, a clear and significant (p<0.001) difference could be observed between the D-O30 and vehicle control treated mice (Figure 4e).Confirmation of the allosteric site covalent binding mechanism for PBP2a.

[0072] Sequential LC-MS / MS studies and computer analyses were conducted to analyze the potential binding mechanism of the D-isomer 30 to the PBP2a protein, which is responsible for the high resistance of MRS A, and to understand its inhibitory activity against various S. aureus strains. The recently published crystal structure of the complex of PBP2a with ceftaroline, a cephalosporin antibiotic that shows efficacy against MRSA, has revealed the allosteric site at 60- A distance from the transpeptidase domain.

[0073] According to numerous research reports, the activity of the PBP2a protein is known to be regulated by allosteric sites distinct from the active site where cell wall cross-linking occurs.

[0074] Our LC-MS / MS analysis, conducted after treating PBP2a with compound 30, revealed the formation of a covalent bond at tyrosine 223 / 272 located in the allosteric site of PBP2a. Specifically, it was confirmed that compound 30 forms a covalent bond at tyrosine 223 / 272 located in the allosteric site of PBP2a, increasing the mass of each tyrosine residue at the peptide terminus by precisely 287 g / mol. This finding provides clear support for the proposed nucleophilic aromatic substitution (SNAr) reaction-based mechanism of allosteric site covalent binding and its association with the closure of the key active site of PBP2a, as anticipated (Fig 5).

[0075] Based on the analyzed LC-MS / MS data, a predictive model for the covalent binding between compound 30 and tyrosine 223 / 272 located at the PBP2a allosteric site was constructed (Fig 6). The protein model of PBP2a bound with compound 30 was subjected to long-term molecular dynamic simulations using the Schrodinger package, Desmond, to perform spatiotemporal and dynamic analysis of structural changes in the major active site regulated by binding at the allosteric site.Clinical Isolate MRSA Pathogen test

[0076] We received support for 10 clinical isolates of MRSA from the group of Dr. Robert Bonomo at the VA Northeast Ohio Healthcare System, and further tests using compound D- isomer 30 were conducted. Most of the clinical isolates of MRSA were found to exhibit strong resistance to both Methicillin and Oxacillin. Treatment with our compound 30 against these resistant S. aureus strains demonstrated highly potent inhibitory activity, with MIC values ranging from 1.8 to 3.6 pg / mL. Furthermore, the measured IC50 values also remained within a low micromolar range, indicating the potential application of our compound as a novel and potent non-beta-lactam antibiotic candidate against Gram-positive MRSA infections.

[0077] Table 4. MIC determination against MRSA Clinical isolate strain using D-isomer 30.Materials and MethodsSynthesis:

[0078] All reactions were carried out in oven-dried glassware (unless water was present in the reaction mixture) with magnetic stirring under a positive pressure of argon unless otherwise indicated. ACS reagent grade solvents were used. Reactions were monitored by thin layer chromatography (TLC) carried out on 250 pm Merck silica gel plates (60 F254) containing a fluorescent indicator (254 nm). TLC plates were visualized under an iodine chamber or UV lamp before treatment with the cerium ammonium molybdate stain and development with heat. Flash column chromatography was performed using Silicycle SiliaFlash P60 silica gel (60 A pore size, 40 - 63 pm particle size, 230 - 400 mesh) and ACS reagent grade solvents. Melting points were determined with a Mel-Temp Digital Melting Point Apparatus. Optical rotation was measured using LAXCO POL-200 Series Automatic Polarimeter. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer and reported as wavenumber ( cnr i. High- resolution mass spectra (HRMS) were recorded using Thermo Scientific Exactive Plus Orbitrap Mass Spectrometer. 1H NMR and13C NMR spectra were recorded in (CDshSO or CDCh on a Bruker Av 400 MHz NMR-spectrometer operating at 400 MHz (1H), 101 MHz(13C) and Agilent 500 MHz NMR-spectrometer operating at 500 MHz (1H) and 125 MHz (13C). Chemical shifts (8) are reported in ppm and are referenced to the chemical shift of the residual solvent proton(s) present in dimethyl sulfoxide 8[ppm] = ((CHahSO) = 2.50 ppm for the ’H NMR spectra and 8[ppm] = ((CDalSO) = 39.52 ppm for the13C NMR spectra, or chloroform8[ppm] = (CDC13) = 7.26 ppm for the 1H NMR spectra and 8[ppm] = (CDC13) = 77.16 ppm for the13C NMR spectra. The corresponding peak multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. Coupling constant values were extracted assuming first-order coupling and shown in hertz (Hz).

[0079] D-Boc-phenylalanine methyl ester (la)

[0080] To the solution of carboxylic acid 1 (18.85 mmol, 5.00 g) in acetone (0.60 M, 30 mL) in a 100 mL RB flask, K2CO3(37.70 mmol, 5.21 g) and CH3I (56.54 mmol, 3.50 mL) were added, and the mixture was stirred overnight. The solvent was removed under reduced pressure, diluted with NH4CI (15 mL), extracted with EtOAc (2 X 20 mL), washed with brine (15 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give a pure colorless oily methyl ester la (5.37 g, 100 %).

[0081] N-Boc-D- phenylalanine hydrazide (2)

[0082] In a 100 mL RB flask, the solution of methyl ester la (15.60 mmol, 4.37 g), ethanol (0.60 M, 26 mL), and N2H4.H2O (47.00 mmol, 2.40 mL) was prepared and was stirred for 8 hours until TLC analysis indicated complete consumption of starting material. The solvent was removed under reduced pressure to give the white residue which on recrystallization in EtOAc / hexanes afforded white powder hydrazide 2 (2.63 g, 60%).

[0083] tert-butyl N-[(lR)-l-(4,5-dihydro-5-thioxo-l,3,4-oxadiazol-2-yl)-2-phenylethyl] carbamate (3)

[0084] To the solution of hydrazide 2 (13.96 mmol, 3.90 g) in dry ethanol (0.18 M, 75 mL) in a 250 mL RB flask, KOH (16.76 mmol, 940 mg) was added under Argon at room temperature and was stirred for an hour. The solution of CS2 (16.76 mmol, 1.01 mL) was prepared in dry ethanol (5 mL) and slowly added to the above solution and heated to reflux overnight. The solvent was evaporated and CH3COOH (2 mL) was added to get the white precipitate which was recrystallized in EtOAc / hexanes to give 3 (2.47 g, 55 %). However, on flash chromatography at 20 % EtOAc in hexanes, 3 can be obtained up to 81 %. Note: Anhydrous ethanol was prepared in oven-dried RB flask with 4 A molecular sieves under Argon.

[0085] tert-butyl N-[(lR)-l-[5-[[(3,4-dichlorophenyl) methyl] thio]-l,3,4-oxadiazol-2-yl]-2- phenylethyl] carbamate (4)

[0086] The mixture of thio-oxadiazole 3 (10.39 mmol, 2.30 g) and deionized water (0.92 M, 20 mL) was prepared in a 100 mL RB flask. Then, NaOH (12.47 mmol, 500 mg) and 3,4- dichlorobenzyl chloride (15.59 mmol, 2.16 mL) were added and heated to reflux for 36 hours. The reaction mixture was cooled and then diluted with NH4CI (10 mL) and 20 mL EtOAc. The layers were separated, and the aqueous layer was extracted with two additional portions of EtOAc (20 mL each). The combined organics were washed with brine (10 mL) before drying over anhydrous Na2SO4. The solvent was removed under reduced pressure to afford a benzylated product 4 (2.98 g, 87 %).

[0087] tert-butyl N-[(lR)-l-[5-[[(3,4-dichlorophenyl) methyl] sulfonyl]-!, 3, 4-oxadiazol-2- yl]-2-phenylethyl] carbamate (5)

[0088] To the solution of sulfide 5 (377 mg, 0.78 mmol) in CH2Q2 (0.05 M, 15 mL), 70 % m- CPBA (3.12 mmol, 700 mg) was added at 0 °C and stirred for 2 days at room temperature. The reaction mixture was diluted with 2 M NaOH (10 mL), extracted with CH2CI2 (2 X 20 mL), washed with brine (10 mL), dried over anhydrous Na2SC>4, and concentrated under reduced pressure to get crude (411 mg) which on recrystallization in methanol gave pure sulfone 5 (213 mg, 60 %).

[0089] 5-[[(3,4-Dichlorophenyl) methyl] thio]-a-(phenylmethyl)-l,3,4-oxadiazole-2- methanamine trifluoroacetic acid salt (6)

[0090] The solution of sulfide 4 (1.16 mmol, 577 mg) in CH2CI2 (0.08 M, 15 mL) was prepared in a 50 mL RB flask. To it, trifluoroacetic acid (TFA) (11.6 mmol, 0.90 mL) was added and heated to reflux for 24 hours. The solvent was removed under reduced pressure to give the white residue which was recrystallized in ether to get the pure salt 6 (235 mg, 65 %). Alternatively, the reaction mixture was neutralized with 1 M NaOH (10 mL), extracted with CH2Q2 (2 X 15 mL), washed with brine (10 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford the oily product. The product was dissolved in ether (10 mL) and a few drops of 1 M HC1 were added slowly until a white precipitate was formed (upto 37 % yield).

[0091] 5-[[(3,4-Dichlorophenyl) methyl] sulfoxide]-a-(phenylmethyl)-l,3,4-oxadiazole-2- methanamine (7)

[0092] To the suspension of salt 6 (100 mg, 0.26 mmol) in CH2C12(0.05 M, 5 mL), 70 % mCPBA (0.78 mmol, 175 mg) was added at 0 °C and stirred for 3 days at room temperature. The reaction mixture was diluted with 2 M NaOH (10 mL), extracted with CH2C12 (2 X 15 mL), washed with brine (10 mL), dried over anhydrous Na2S()|. and concentrated under reduced pressure to give crude (115 mg) which was recrystallized in methanol to afford white powder sulfoxide 7 (17 mg, 16 %).Expression and Purification of Staphylococcus aureus PBP2a

[0093] A gene fragment containing PBP2a (residues 23-668) from the Staphylococcus aureus strain was amplified using PCR and subsequently cloned into a PET28a plasmid via Ndel / Xhol restriction digestion, resulting in a PET28a-PBP2a construct. This construct features an octahistidine (6x His) tag at the C-terminus of PBP2a. The resulting plasmid was then transformed into E. coll BL21(DE3) cells (Novagen) for protein expression. The bacterial cells were cultured in Luria broth (LB), supplemented with an antibiotic (100 pg / ml ampicillin), and incubated at 37 °C until the optical density of the culture reached 0.6 at a wavelength of 600 nm (OD600). The expression of PBP2a was induced with 0.1 mM isopropyl-[3-D- thiogalactopyranoside (IPTG) and incubated at 25 °C for 16 hours.

[0094] The cultures were harvested by centrifugation, and the cell pellets were resuspended in a purification buffer (20 mM Tris-HCl pH 7.5 and 150 mM NaCl), supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF, Sigma- Aldrich). The cells were lysed by passing them three times through a cell disrupter (ATS Engineering Ltd), and cell debris was removed by centrifugation at 18,000 x g for 45 minutes at 4 °C. The solubilized protein suspension was ultracentrifuged at 100,000 x g for 1 hour before being loaded onto a 5 ml HisTrap HP column (GE Healthcare). The column was washed with a purification buffer supplemented with 20 mM imidazole, and the bound protein was eluted with a purification buffer supplemented with 250 mM imidazole. The protein eluted from the HisTrap HP column was further purified by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 column (GE Healthcare) equilibrated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl. The purity of the protein fractions was analyzed by SDS-PAGE. Fractions with the highest purity were collected and concentrated to 5 mg / ml for further study.Docking studies:

[0095] Modeling and docking studies were carried out with the Maestro Schrodinger 2022-2 software package. Computational studies were based on NMR and X-ray structures (PDB: 6TTA and 4C1D). Proteins were prepared through the standard workflow, which includes adding missing sidechains and hydrogens as well as energy minimization using the OPLS-2005 force field. Glide docking was utilized to generate docking scores and compare models.Biochemical assays:

[0096] Nitrocefin was used as the substrate as this is a chromogenic cephalosporin. The activity of purified VIM-2 was measured spectrophotometrically (Cytation 3, BioTek) in potassium phosphate buffer (PBS, pH = 7.4). Nitrocefin concentrations ranging from 8 pM to 120 pM were utilized. The formation of the hydrolyzed product is measured at 486 nm at intervals of 10 s for at least 30 min until a plateau in product formation is observed. The Km and kcat values were determined by plotting initial velocity measurements, followed by fitting with a non-linear regression Michaelis-Menten kinetic model in Origin 2022b.

[0097] The inhibitor constants (Ki) were determined following a similar approach, where the inhibitor was fixed at 500 pM with nitrocefin ranging from 5-100 pM. A blank consisting of DMSO, PBS, and the inhibitor was utilized to subtract any potential absorbance from the colored inhibitors at 486 nm. Each inhibitor was pre-incubated with the VIM-2 enzyme for 10 min at room temperature and then the absorbance at 486 nm was measured and evaluated using a competitive inhibition model in Origin 2022b using the previously determined Km value.

[0098] Single Dose enzyme inhibition was determined by comparing the absorbance of the hydrolyzed nitrocefin (7=486 nm) after several time points (e.g. 5 min, 30 min, 1 hr.) between various samples of purified TEM-1 (10 nM) with and without inhibitors. Quercetin analogue inhibitors were tested at concentrations of 500 pM, whereas known covalent inhibitors (clavulanate, sulbactam, tazobactam, vaborbactam) had concentrations of 50 pM. Enzymes were pre-incubated with inhibitors for 10 min at room temperature prior to the their addition to nitrocefin (final nitrocefin concentration of 120 pM)Bacteria transformation:

[0099] VIM-2 plasmid was isolated using the protocol provided by New England BioLabs, Inc. with their Monarch Plasmid Miniprep Kit. Transformation was carried out following apreviously published procedure utilizing electroporation. Woodall CA (2003) Electroporation of E. coli. in E. coli Plasmid Vectors: Methods and Applications, eds Casali N & Preston A (Humana Press, Inc.) After incubation in recovery media, bacteria (100 pL) were plated on Luria-Bertani Agar (LB A) containing 50 pg / mL of kanamycin and incubated overnight at 37 °C. The following day, samples were taken and added to LB broth and incubated overnight at 37 °C with agitation (120 rpm). To test whether VIM-2 was expressed or not disc diffusion assays with ethylenediaminetetraacetic acid (EDTA) and single dose inhibition assays were carried out.Disc diffusion assay:

[0100] Overnight cultured bacteria were diluted to an optical density of 0.08-0.1 at 600 nm. Kirby-Bauer discs containing either 10 pg meropenem or 10 pg meropenem with 930 pg EDTA were added to the inoculated Muller-Hinton Agar (MHA) plates and incubated overnight at 37 °C. The following day photos and luminescence images were recorded to determine the inhibition zones around each disc.Single Dose inhibition assay:

[0101] Bacteria were diluted to 5 x 105CFU / mL in MH broth (MHB). A growth control containing only bacteria and a negative control containing only MHB were added. Test wells were treated with either 25 pM meropenem, ceftazidime, amoxicillin, or doripenem as well as 1: 1 combinations of the previous antibiotics with vaborbactam. Each set was repeated in triplicate and the final viability was determined in reference to the growth control.Luminescence Imaging:

[0102] Luminescent images were obtained using the IVIS® Lumina XRMS Series III (PerkinElmer) in bioluminescence imaging mode. Exposure times were automatically determined by the Livingimage Software with medium binning. Mice were kept under anesthesia using the attached XGI-8 Gas Anesthesia System from Caliper LifeSciences.Minimum inhibitory concentration (MIC) assay:

[0103] MIC assays were carried out following established guidelines using MHB-2 and bacterial concentrations of 5 x 105CFU / mL. The MIC was reported as the concentration at which no growth was observed.In vivo treatment of murine skin wound model:

[0104] PAX5 VIM-2 was grown overnight in LB broth. The overnight culture was then centrifuged, the media removed, and the pellet was resuspended in PBS. This suspension was later used to inoculate the mice. Anesthesia was induced with 3-5% isoflurane and maintained with 2-3% isoflurane. Mice were then shaved, and the dorsal skin was scrubbed with povidone and washed away with 70% ethanol. A clear plastic template with a square 1 cm2cutout was used to demarcate the corners of the planned needle-scratch grid. A 25G, 1 V2 needle was then used to carefully create the abrasive wound, with caution being paid not to deeply lacerate. Having created the two sets of wounds, PAX5VIM-2 (20 pL) was added via pipette to each wound. IVIS images were taken after 24 hours, and this was determined to be the start point of the experiment (t=0). Treatments were given while animals were anesthetized by adding either the QATC (4: 1:2, 50 pM amoxicillin, 12.5 pM clavulanate, 25 pM taxifolin in DMSO) or DMSO directly to the wound area at t=0, 2, 4, 8, 24 hours. IVIS images were taken once per day prior to any planned treatment. Following the final treatment at t=24, IVIS images were taken for an additional 2 days. After which point, biopsies of the infected tissue were taken using a 6 mm biopsy punch. To each biopsy, 1 mL of PBS was added, and the tissue was homogenized. The slurry was diluted 10,000x and 50 pL plated onto LBA and incubated overnight at 37 °C. Colonies were counted the following day.

[0105] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. In particular, while various theories are presented describing possible mechanisms through with the compounds are effective, the compounds are effective regardless of the particular mechanism employed and the inventors are therefore not bound by theories described herein. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or preventing a bacterial infection in a subject in need thereof, comprising administering a therapeutically effective amount of a compound according to formula I:to the subject; wherein R1is a benzyl or lower alkyl group and R2-R6are independently selected from H, methyl, and halogen, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the method is treating a bacterial infection.

3. The method of claim 1 , wherein the method is preventing a bacterial infection.

4. The method of claim 1, wherein R1is a benzyl group.

5. The method of claim 1 , wherein R3and R4are chloro substituents.

6. The method of claim 1, wherein the compound is:

7. The method of claim 1, wherein the compound is:

8. The method of claim 1, wherein the compound is administered together with a pharmaceutically acceptable carrier.

9. The method of claim 1, wherein the compound is administered topically.

10. The method of claim 1, wherein the subject is human.1 1. The method of claim 1, wherein the bacterial infection is a multi drug-resistant bacterial infection.

12. The method of claim 11 , wherein the multidrug-resistant bacterial infection is by methicillin-resistant staphylococcus aureaus.

13. An antibacterial compound having the structure:

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