Biguanide-vancomycin conjugates as effective broad-spectrum antibiotics

Abstract

Compositions and methods are provided for the synthesis and use of biguanide-conjugated antibiotics, which provide a generalizable strategy for rapidly synthesizing potent antibiotics derived from clinically relevant agents.

Description

BIGUANIDE-VANCOMYCIN CONJUGATES AS EFFECTIVE BROAD-SPECTRUM ANTIBIOTICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 645,425, filed May 10, 2024, the disclosure of which application is herein incorporated by reference.GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract GM1 17278 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] In 2019, 4.95 million deaths were attributed to antibiotic resistant infections. Without interventions, this global figure is predicted to rise to 10 million by 2050. Many of the highest- threat bacterial pathogens are covered under the ESKAPE acronym, which stands for E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species, but this acronym overlooks mycobacteria like Mycobacteria tuberculosis which alone was responsible for 1.3 million deaths globally in 2022. The CDC places the Enterobacteriaceae family and A. baumannii in the highest threat level category of “urgent” threats to human health, with the rest of the ESKAPE pathogens and M. tuberculosis being defined as “serious” threats. However, despite the critical need for more drugs against these high-priority pathogens, only 38% of antibiotics currently in development are expected to be active against ESKAPE pathogens. Modifying existing antibiotics to improve and expand efficacy is one attractive strategy to expedite introduction of drugs to treat high-priority pathogens and minimize the scientific, regulatory, and safety barriers associated with the development of de novo therapeutics. The cell wall inhibitor vancomycin is a promising candidate for this strategy, and vancomycin-conjugates have gained attention for rapid approval for clinical treatment of challenging ESKAPE pathogens.

[0004] Vancomycin is a potent glycopeptide antibiotic administered intravenously as a first- line therapy to treat Methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections. It inhibits cell wall synthesis in bacteria by binding to the D-Ala-D-Ala termini of Lipid II at the cell surface, effectively sequestering key cell wall precursors and inhibiting cell wall assembly. However, vancomycin is ineffective against pre-formed biofilms, and the emergence of resistance in vancomycin intermediate S. aureus (VISA), vancomycin resistance S. aureus (VRSA) and vancomycin resistance enterococci (VRE) emphasizes the need for new treatment options. Furthermore, despite the conserved target, the D-Ala-D-Alatermini of Lipid II, across Gram-positive bacteria, Gram-negative bacteria, and mycobacteria vancomycin is only highly effective against Gram-positive bacteria. The Gram-negative outer membrane prevents sufficient antibiotic accumulation in the periplasm where cell wall synthesis occurs. Similarly, vancomycin is not considered to be highly effective against mycobacteria because of the high concentration required for efficacy. This reduced potency is generally attributed to ineffective penetration of the complex mycobacterial cellular envelope which inhibits the binding of vancomycin to cell-wall precursors.

[0005] Promising vancomycin derivatives that address some of these shortcomings have been introduced, with oritavancin being a prominent example of an FDA-approved semisynthetic derivative with demonstrated in vitro antibacterial activity against biofilm- associated S. aureus. Analogues of vancomycin commonly incorporate cationic and lipophilic functionalities to enhance affinity for D-ala-D-ala, membrane localization, membrane depolarization, and / or membrane permeabilization, resulting in antibiotics with multiple modes of action. The cationic functional group is frequently a quaternary ammonium like those found in the Maxamycins of the Boger group, or peptide-derived ammonium residues like those reported by the Cooper group. These studies also incorporated lipophilic moieties like chlorobiphenyl-, n-alkyl-, and 4-phenylbenzoyl- groups to enhance membrane interactions. Similarly, our groups have reported that vancomycin modified with one or more arginines or guanidinium groups exhibit profoundly improved efficacy and an expanded spectrum of activity. Subsequently, others have explored persistently charged guanidinium modifications with notable advances in several cases.

[0006] Improvement in antibiotic efficacy is of great interest. Antibiotic conjugates with enhanced efficacy are provided herein.SUMMARY OF THE INVENTION

[0007] Compositions and methods are provided for the synthesis and use of biguanide- conjugated antibiotics. The synthetic methods disclosed herein provide a generalizable strategy for rapidly synthesizing potent antibiotics derived from clinically relevant agents. The resulting biguanide drug conjugates are useful in treating difficult-to-treat bacterial infections including biofilms, stationary and persister cells, and multi-drug resistant bacteria, as well as intracellular bacteria.

[0008] In an embodiment, a biguanide conjugate of the disclosure has a structure:wherein:D is an antibiotic drug; n is from 1 to 10; e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, and in some embodiments n is 6; and R is selected from H, alkyl, for example an alkyl of from C2 to about C24 in length, e.g.2, 4, 6, 8, 10, 12, 14, 16, 18, 20, etc., a substituted alkyl, phenyl, diphenyl, substituted phenyl, substituted diphenyl, furan, furfuryl, etc.

[0009] In some embodiments the antibiotic, D, is a glycosylated cyclic or polycyclic nonribosomal peptide, including without limitation vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, oritavancin, dalbavancin, etc. In some embodiments the antibiotic is vancomycin or a derivative thereof. In some embodiments the biguanide is conjugated to vancomycin or a derivative thereof at the C-terminus, for example at the readily derivatizable carboxylic acid functional group that is not involved in vancomycin’s mode of action.

[0010] In some embodiments R is selected from:where X is selected from OH, H, F, Br, Cl, I, CF3, NO2, and PhCI;(l) furfuryl; and(m) 1 ,1 -diphenylmethyl.

[0011] In some embodiments, a biguanide conjugate of the disclosure has a structure:where R is as defined above.

[0012] In some embodiments the biguanide-conjugated drug is at least 2-fold more active than the unconjugated drug against a pathogen of interest, including without limitation E. faecium, E. coli, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter sp., Mycobacteria sp.; etc., and including biofilms and persister cells of such pathogens. In some embodiments the bacteria are Gram-positive bacteria. In some embodiments the bacteria are methicillin-resistant Staphylococcus aureus (MRSA).

[0013] In some embodiments the biguanide-conjugated drug is at least 4-fold more active, at least 10-fold more active, at least 25-fold more active, at least 50-fold more active, and may be greater than 100-fold more active than the unconjugated drug.

[0014] The biguanide-conjugated drug can provide benefits in delivering an effective dose of antibiotic for treatment of, for example, stationary and persistent bacterial cells, biofilms, intracellular infections, vancomycin-resistant cells, stationary-phase and persistent MRSA cells, MRSA biofilms, etc. at concentrations that are non-toxic to human skin and red blood cells. The biguanide-conjugated drug can be provided in an effective topical dose.

[0015] In some embodiments, a method of treating a bacterial infection is provided, where the infection may be present in a mammalian host, the method comprising: determining that an infection is caused at least in part by the presence of a pathogen of interest, including without limitation biofilm, stationary and / or persistent bacteria, and contacting the bacteria causing the infection with an effective dose of a biguanide-conjugated antibiotic. In some embodimentsthe efficacy in eradicating the infection at the effective dose is at least 2-fold higher than the efficacy of the unconjugated antibiotic at the same dose. In some such embodiments, the antibiotic is vancomycin or a derivative thereof.

[0016] In some embodiments, a method of treating a bacterial infection is provided, where the infection may be present in a mammalian host, the method comprising: determining that an infection is caused at least in part by a pathogen of interest as discussed above, and contacting the bacteria causing the infection with an effective dose of a biguanide-conjugated antibiotic for a period of time sufficient to treat the infection.

[0017] In some embodiments a pharmaceutical composition comprising a conjugated antibiotic of the invention as an active agent and a pharmaceutically acceptable excipient is provided. The formulation can be provided, for example, as a unit dose formulation, in a dose that is effective for treating persister or stationary bacteria, biofilms, MRSA, MRSE, etc. The formulation may be administered to a patient suffering from a microbial infection.

[0018] In some embodiments a method is provided for synthesis of a biguanide-conjugated antibiotic. The synthesis may involve the biguanidinylation of an amine with an activated pyrazole-biguanide intermediate. The resulting boc-aminobiguanide is converted with acid to an aminobiguanide that is reacted with an antibiotic, e.g. vancomycin, to yield an antibiotic- biguanide conjugate.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0020] Figure 1 . A) Biguanides can bind anionic functional groups through Coulombic and chelative hydrogen bonding interactions B) Vancomycin-biguanide conjugates are proposed to retain their D-ala-D-ala binding while also engaging surface anions through Coulombic and hydrogen-bonding interactions, synergistic interactions that increase efficacy. C) Previously explored ammonium and guanidinium cations and the biguanidinium cations in this study differ in their pKa values, number and spatial array of hydrogen bond donors, and charge distributions.

[0021] Figure 2. Synthesis of Boc-aminobiguanides 3a-e. A) synthesis of Boc-aminobiguanide 3a from commercially available cyanoguanidine and Boc-protected 1 ,6-diaminohexane 1. B)synthesis of lipophilic Boc-aminobiguanides 3b-e from 1 , NaN(CN)2 and, ammonium salts, through a fusion procedure. C) alternative synthesis of Boc-aminobiguanides 3a-e using pyrazole-biguanides 9a-e as biguanidinylation reagents. Note: compounds 3a-e do not have any counterions because they are purified in the presence of ammonium hydroxide (See SI).

[0022] Figure 3. Synthesis of vancomycin-biguanide conjugates, 5a-e, and control acetamide- biguanides 6a-e from Boc-aminobiguanides 3a-e.

[0023] Figure 4. Time-kill kinetics analysis of V-C6-Bg-PhCI 5e and controls in A) S. aureus 29213, B) E. coli 25922, and C) M. smegmatis 700084.

[0024] Figure 5. Accumulation of peptidoglycan precursors in A) S. aureus, C) E. coli, and E) M. smegmatis. B), D), and F) HPLC chromatogram showing peptidoglycan precursor accumulation for treated cells. Mass spectrum of peptidoglycan precursor UDP-MuRNAc pentapeptide detected at the 4.7 min, 4.7 min, and 3.9 min retention time.

[0025] Figure 6. Evaluation of membrane permeability with fluorescent probes. (A, Left) PI fluorescence of S. aureus 29213. (B, Right) NPN fluorescence of E. coli 25922.

[0026] Figure 7: A) Microscopy of S. aureus 29213 treated with 5 uM of Fl-V and Fl-5e, with GFP exposure time of 40 ms. B) FACS analysis of S. aureus 29213 treated with 5 uM of Fl-V and Fl-5e. C) Microscopy of E. coli 25922 treated with 5 uM of Fl-V and Fl-5e, with GFP exposure time of 40 ms. D) FACS analysis of E. coli 25922 treated with 10 uM of Fl-V and Fl- 5e. E) Microscopy of M. smegmatis 700084 treated with 20 uM of Fl-V and Fl-5e, with GFP exposure time of 80 ms. F) FACS analysis of M. smegmatis 700084 treated with 30 uM of Fl- V and Fl-5e. Error bars represent the range of normalized fluorescence values obtained in three experiments.

[0027] FIG. 8 provides an overview of the synthetic scheme.

[0028] Table 1 . Minimum inhibitory concentrations (MICs) of Vancomycin V, Oritivancin, Metformin (a control biguanide), vancomycin-biguanide conjugates 5a-e, control biguanide 6e, and an equimolar mixture of V and 6e, against Gram(+) (green) and Gram(-) (red) ESKAPE pathogens and mycobacteria (blue). MICs are bolded when they are single digit M and more potent than the parent compound, vancomycin.

[0029] Table 2. Minimum biofilm eradication concentrations (MBECs) of vancomycin- biguanide conjugates 5a-e against pre-formed biofilms of S. aureus 29213, E. coli 25922, M. smegmatis 700084.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to an animal, including, but not limited to, human and non-human primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines;canines; and the like. "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, e.g., non-human primates, and humans. Non-human animal models, e.g., mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0031] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0032] Antibiotics. Any of a number of antibiotics are suitable for conjugation, or can be modified to be rendered suitable for use in the subject compounds. Classes of antibiotics include, for example, penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with p-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; glycopeptides; etc.

[0033] Glycopeptide antibiotics are a class of drugs of microbial origin that are composed of glycosylated cyclic or polycyclic nonribosomal peptides. Significant glycopeptide antibiotics include the anti-infective antibiotics vancomycin, teicoplanin, telavancin, ramoplanin and decaplanin. Derivatives of vancomycin include, for example, oritavancin and dalbavancin (both lipoglycopeptides). Telavancin is a semi-synthetic lipoglycopeptide derivative of vancomycin (approved by FDA in 2009). Other vancomycin analogs are disclosed, for example, in WO 2015022335 A1 and Chen et al. (2003) PNAS 100(10): 5658-5663, each herein specifically incorporated by reference.

[0034] Gram-positive bacteria. Gram-positive organisms (including bacteria of the genera Staphylococcus, Streptococcus and Enterococcus) are among the most common bacterial causes of clinical infection. This is primarily due to their association with a diverse spectrum of pathology, ranging from mild skin and soft tissue infections (SSTIs) to life-threatening systemic sepsis and meningitis. Although a number of antimicrobial agents already exist for the treatment of such diseases, emerging issues such as antimicrobial resistance (AMR) and and an increase in hospital-acquired infections have created a need for antimicrobials with novel spectra of activity and pharmacokinetic (PK) profiles.

[0035] Included as organisms of particular concern are Staphylococci sp., Streptococci sp., Enterococci sp., C. diptheriae, B. anthracis, C. difficile', and specifically may include methicillin- resistant Staphylococcus aureus (MRSA), which is resistant to almost all [3-lactam antibiotics; glycopeptide-resistant Enterococci (GRE), multidrug resistant (MDR) Streptococcuspneumoniae MDR Streptococcus agalactiae Streptococcus pyogenes Enterococcus faecium, Staphylococcus aureus, multidrug-resistant Staphylococcus epidermidis (MRSE), etc.

[0036] The term "MRSA" as used herein refers generally to a strain of Staphylococcus aureus that is resistant to a large group of antibiotics called the beta-lactams, which include the penicillins and the cephalosporins. Specific examples of beta-lactam antibiotics include methicillin, dicloxacillin, nafcillin, and oxacillin. MRSA is sometimes referred to as multidrugresistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA).

[0037] Staphylococcus aureus (S. aureus) is a cause of a variety of conditions in humans, including skin infections (e.g. folliculitis, styes, cellulitis, impetigo, and furunculosis), pneumonia, mastitis, phlebitis, meningitis, scalded skin syndrome, osteomyelitis, urinary tract infections, and food poisoning. Methicillin resistance is caused by the acquisition of an exogenous gene mecA that encodes penicillin-binding protein (PBP2a or PBP2'), which exhibits a low affinity for (3-lactam antibiotics. The mecA gene also is found in coagulasenegative Staphylococcus strains that are less pathogenic than S. aureus. These strains include S. epidermidis, S. haemolyticus, S. saprophyticus, S. capitis, S. warned, S. sciuri and S. caprae. An additional mec gene, named mecC, was discovered which also confers betalactam resistance.

[0038] Vancomycin-resistant Enterococcus is another significant threat to public health. Six different types of vancomycin resistance are shown by enterococcus: Van-A, Van-B, Van-C, Van-D, Van-E and Van-G. The mechanism of resistance to vancomycin found in enterococcus involves the alteration of the peptidoglycan synthesis pathway. The D-alanyl-D-lactate variation results in the loss of one hydrogen-bonding interaction (four, as opposed to five for D-alanyl-D-alanine) being possible between vancomycin and the peptide. The D-alanyl-D- serine variation causes a six-fold loss of affinity between vancomycin and the peptide, likely due to steric hindrance.

[0039] Gram-negative bacteria. Gram-negative bacteria are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane. They are an important medical challenge, as their outer membrane protects them from many antibiotics.

[0040] The Gram-negative bacteria include Escherichia coli, and many pathogenic bacteria, such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Chlamydia trachomatis, Yersinia pestis, and Vibrio cholerae. Pathogens of interest in this group include, without limitation, Acinetobacter baumannii] Pseudomonas aeruginosa] Klebsiella pneumoniae] and Enterobacter spp.

[0041] Gram-negative bacteria are intrinsically resistant to vancomycin because their outer membranes are impermeable to large glycopeptide molecules, with the exception of some non-gonococcal Neisseria species. Surprisingly, vancomycin conjugates described herein are effective against certain Gram-negative bacteria, e.g. E. coli, Acinetobacter baumannii, etc.

[0042] Biofilm. A biofilm is an accumulation of microorganisms (bacteria, fungi, and / or protozoa, with associated bacteriophages and other viruses) embedded in a polysaccharide matrix and adherent to solid biological or non-biotic surfaces. Biofilms are medically important, accounting for over 80 percent of hospital-acquired microbial infections in the body. Examples include infections of the oral soft tissues, teeth and dental implants; middle ear; gastrointestinal tract; urogenital tract; airway / lung tissue; eye; urinary tract prostheses; peritoneal membrane and peritoneal dialysis catheters, indwelling catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman catheters); cardiac implants such as pacemakers, prosthetic heart valves, ventricular assist devices, and synthetic vascular grafts and stents; prostheses, internal fixation devices, percutaneous sutures; and tracheal and ventilator tubing. The microorganisms tend to be far more resistant to antimicrobial agents and to be particularly difficult for the host immune system to render an appropriate response.

[0043] Biofilms are remarkably difficult to treat with antimicrobials. Antimicrobials may be readily inactivated or fail to penetrate into the biofilm. In addition, bacteria within biofilms have increased (up to 1000-fold higher) resistance to antimicrobial compounds, even though these same bacteria are sensitive to these agents if grown under planktonic conditions.

[0044] Biofilms play a significant role in the transmission and persistence of human disease and have emerged as virulence hallmarks of serious and persistent infectious diseases, including cystic fibrosis pneumonia, infective endocarditis, urinary tract infection (UTI), periodontitis, chronic infections of the middle ear, and infections of medical devices such as intravenous catheters and artificial joints. Currently available antibiotics often fail to eradicate biofilm-associated bacteria, necessitating multiple and intense antibiotic treatment regimens that drive the evolution of resistant pathogens and the exhaustion of last-resort antibiotics. As a consequence, biofilm-associated infections are the cause of significant morbidity and mortality in the clinic.

[0045] A biofilm is an assemblage of microbial cells that is closely associated with a surface and enclosed in a matrix of material, including polysaccharides, DNA, and proteins. Noncellular materials such as mineral crystals, corrosion particles, clay or silt particles, or blood components, depending on the environment in which the biofilm has developed, may also be found in the biofilm matrix. Biofilm-associated organisms also differ from their planktonic (freely suspended) counterparts with respect to the genes that are transcribed.Biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical devices, industrial or potable water system piping, or natural aquatic systems.

[0046] The solid-liquid interface between a surface and an aqueous medium provides an ideal environment for the attachment and growth of microorganisms. The solid surface may have several characteristics that are important in the attachment process. The extent of microbial colonization appears to increase as the surface roughness increases. This is because shear forces are diminished, and surface area is higher on rougher surfaces. The physicochemical properties of the surface may also exert a strong influence on the rate and extent of attachment. Microorganisms attach more rapidly to hydrophobic, nonpolar surfaces such as Teflon and other plastics than to hydrophilic materials such as glass or metals.

[0047] Other characteristics of the aqueous medium, such as pH, nutrient levels, ionic strength, and temperature, may play a role in the rate of microbial attachment to a substratum. Several studies have shown a seasonal effect on bacterial attachment and biofilm formation in different aqueous systems. This effect may be due to water temperature or to other unmeasured, seasonally affected parameters.

[0048] Cell surface hydrophobicity, presence of fimbriae and flagella, and production of EPS all influence the rate and extent of attachment of microbial cells. The hydrophobicity of the cell surface is important in adhesion because hydrophobic interactions tend to increase with an increasing nonpolar nature of one or both surfaces involved (i.e., the microbial cell surface and the substratum surface). Most bacteria are negatively charged but still contain hydrophobic surface components. Fimbriae, i.e., nonflagellar appendages other than those involved in transfer of viral or bacterial nucleic acids, contribute to cell surface hydrophobicity. Most fimbriae that have been examined contain a high proportion of hydrophobic amino acid residues. Fimbriae play a role in cell surface hydrophobicity and attachment, probably by overcoming the initial electrostatic repulsion barrier that exists between the cell and substratum. A number of aquatic bacteria possess fimbriae, which have also been shown to be involved in bacterial attachment to animal cells.

[0049] Other cell surface properties may also facilitate attachment. Several studies have shown that treatment of adsorbed cells with proteolytic enzymes caused a marked release of attached bacteria, providing evidence for the role of proteins in attachment. The O antigen component of lipopolysaccharide (LPS) has also been shown to confer hydrophilic properties to Gram-negative bacteria.

[0050] Persisters are dormant variants of regular cells that form stochastically in microbial populations and are highly tolerant to antibiotics. Persisters may be the main culprit responsible for the recalcitrance of chronic infectious disease to antimicrobial therapy.Persister cells usually comprise about 1 % of the populations in the stationary-phase growth state and in biofilms.

[0051] These persister cells comprise a subpopulation of bacteria that become highly tolerant to antibiotics and reach this state without undergoing genetic change. Also, the number of persister cells depends on the growth stage. Persister cells in biofilms appear to be responsible for the recalcitrance of chronic infections, since antibiotics kill the majority of cells; however, persisters remain viable and repopulate biofilms when the level of antibiotics drops.

[0052] A model for the formation of persister cells is that toxin-antitoxin (TA) pairs are primarily responsible, as they induce a state of dormancy that enables cells to escape the effects of antibiotics. TA systems typically consist of a stable toxin (always a protein) that disrupts an essential cellular process (e.g., translation via mRNA degradation) and a labile antitoxin (either RNA or a protein) that prevents toxicity. For example high persistence (hip) mutants have been identified. The hipBA locus constitutes a toxin-antitoxin locus, and the HipA toxin inactivates the translation factor EF-Tu by phosphorylating it.

[0053] Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. For example, see Andrews (2001 ) J Antimicrob Chemother. 48 Suppl 1 :5-16’

[0054] Minimal biofilm eradication concentration (MBEC) is defined as the lowest concentration of an antimicrobial agent required to eradicate a biofilm.Compositions

[0055] In some embodiments a biguanide conjugate of the disclosure has a structure:wherein:D is an antibiotic drug; n is from 1 to 16; e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, and in some embodiments n is 6; andR is selected from H, alkyl, for example an alkyl of from C2 to about C24 in length, e.g. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, etc., a substituted alkyl, phenyl, diphenyl, substituted phenyl, substituted diphenyl, furan, furfuryl, etc.

[0056] In some embodiments the antibiotic, D, is a glycosylated cyclic or polycyclic nonribosomal peptide, including without limitation vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, oritavancin, dalbavancin, etc. In some embodiments the antibiotic is vancomycin or a derivative thereof. In some embodiments the biguanide is conjugated to vancomycin or a derivative thereof at the C-terminus, for example at the readily derivatizable carboxylic acid functional group that is not involved in vancomycin’s mode of action.

[0057] In some embodiments R is selected from:where X is selected from OH, H, F, Br, Cl, I, CF3, NO2, and PhCI;(l) furfuryl; and(m) 1 ,1 -diphenylmethyl.Synthesis

[0058] In some embodiments, methods are provided for synthesis of a biguanide-conjugated drug. Isolatable biguanide transfer reagents are provided, which reagents comprise a linker with a free amino group that reacts with a carboxy group present on the drug to form a peptide bond. In some embodiments where the drug is a peptide, the carboxy group is present at the C-terminus.

[0059] In one aspect, the method utilizes a direct Pinner-like addition of an amine to an appropriate cyanoguanidine at elevated temperatures in the presence or absence of solvent.

[0060] In another aspect the method utilizes biguanidinylation of an amine with an activated pyrazole-biguanide intermediate. The resulting boc-aminobiguanides are converted with acid to the aminobiguanides and subsequently conjugated to the drug, as described in the Examples, and depicted in FIGs. 2 and 3. The amine is biguanidinylated with an activated pyrazole-biguanide intermediate, for example a structure:where R is selected from H, alkyl, for example an alkyl of from C2 to about C24 in length, e.g. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, etc., a substituted alkyl, phenyl, diphenyl, substituted phenyl, substituted diphenyl, furan, furfuryl, etc. and in some embodiments R is selected fromwhere X is selected from OH, H, F, Br, Cl, I, CF3, NO2, and PhCI;(l) furfuryl; and(m) 1 ,1 -diphenylmethyl.

[0061] A biguanide transfer reaction utilizes the pyrazole-biguanide intermediate above to biguanidinylate a linker comprising an amino group under mild-conditions which yields a transfer reagent (see 3a-e) that can be used to biguanylate the drug.Methods of Use

[0062] Methods are provided for the use of biguanide-conjugated antibiotics as antimicrobial agents, including without limitation conjugates of vancomycin and vancomycin derivatives and analogs. In some embodiments the conjugate is as shown in structure I or structure II. Such conjugates can be administered alone or in combination with other active agents to a patient suffering from or predisposed to infections that are resistant or tolerant to conventional antibiotics, including infections resistant to vancomycin, methicillin, etc. The infection is treated by contacting the infectious bacterial cell population with a dose and for a period of timesufficient to reduce the population of microbial pathogens, in vivo or in vitro, including for example medical surfaces.

[0063] An effective dose may be the dose that achieves substantial depletion or eradication of the bacterial cell population, which result in the killing of substantially all of the bacterial cells, e.g. at least about 99%, at least about 99.9%, at least about 99.99%, or more. The effective dose may be based on the MIC, or MBEC, although is typically a higher dose to ensure eradication. The effective dose of a conjugated antibiotic is generally at least about 5- fold less than the effective dose for the corresponding non-conjugated antibiotic, and may be 10-fold less, 50-fold less, 100-fold less, or less. The effective time for eradication is also decreased, for example decreased at least 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, or more.

[0064] An effective dose of a conjugated antibiotic may be a dose that achieves a concentration at the target site of at least about 0.01 y.M, at least about 0.1 LM, at least about 1 piM, at least about 5 |j.M, at least about 10 y.M, at least about 50 p.M, at least about 100 p.M, at least about 500 p.M, at least about 1 mM, at least about 5 mM, at least about 10 mM.

[0065] In some embodiments, the effective daily flat dose for an individual adult can range from about 0.5 mg to about 500 g, for example at least about 0.5 mg, at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 50 mg, at least about 100 mg, at least about 500 mg, at least about 1 g, at least about 5 g, at least about 10 g, at least about 50 g, at least about 100 g, and not more than about 500 g.

[0066] In some embodiments an infection for treatment comprises a bacterial cell population in which at least about 5% of the bacteria are resistant or tolerant to antibiotics, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 85%, at least about 95% resistant or tolerant bacteria, where resistant or tolerant bacteria may include, for example: persister cells; MRSA; MRSE; GRE; ORSA; Gramnegative bacteria when the antibiotic is vancomycin or a derivative thereof; biofilms; vancomycin resistant bacteria; etc.

[0067] In some embodiments the infection is present on the skin, i.e. a wound. In such embodiments, a topical formulation is optionally utilized for treatment. An advantage of the conjugated antibiotic provided herein is the enhanced bioavailability for topical formulations that is provided.

[0068] In some embodiments the antibiotic resistant or tolerant bacteria are present as a biofilm. In some embodiments the biofilm is substantially comprised of Gram-positive bacteria. In some embodiments a biofilm is present on implantable medical devices, which are particularly susceptible to biofilm formation.

[0069] In some embodiments, the effective daily dose is provided in a unit dosage formulation in any increment. As non-limiting illustrative examples: administration of one 1.6 mg capsule, two 800 |ig capsules, etc. can be performed twice in one day to deliver a daily dose of 3.2 mg; or thrice in one day to deliver a daily dose of 4.8 mg. As another non-limiting example, the use of 1 mg capsules facilitates any dose (e.g., a daily dose) with a multiple of (1 mg) (e.g., 2 mg, 3 mg, 4 mg, etc.)

[0070] A treatment regime can entail administration daily (e.g., once, twice, thrice, etc. daily), every other day (e.g., once, twice, thrice, etc. every other day), semi-weekly, weekly, once every two weeks, once a month, etc. In another example, treatment can be given as a continuous infusion. Unit doses are usually administered on multiple occasions. Intervals can also be irregular as indicated by monitoring clinical symptoms. Alternatively, the unit dose can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the patient. It will be understood by one of skill in the art that such guidelines will be adjusted for localized administration, e.g. intranasal, inhalation, rectal, etc., or for systemic administration, e.g. oral, rectal (e.g., via enema), i.m. (intramuscular), i.p. (intraperitoneal), i.v. (intravenous), s.c. (subcutaneous) transurethrally, and the like.

[0071] The conjugated antibiotic can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment. In some embodiments, pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention or pharmaceutically acceptable salts, esters or solvates thereof. In some other embodiments, the use of biguanide-antibiotic conjugate includes use in combination with another therapeutic agent, e.g., a bacteriocidal or bacteriostatic agent. Therapeutic formulations can be prepared for storage by mixing the biguanide-antibiotic conjugate with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. The biguanide-antibiotic conjugate composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. The "effective amount" to be administered will be governed by considerations such as those cited above (e.g., severity of disease etc.), and is the minimum amount necessary to prevent and / or reduce the targeted biofilm.

[0072] Formulations of biguanide-antibiotic conjugates are administered to a host suffering from or predisposed to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism, preferably it will be localized. Generally the dose of biofilm inhibitor will be sufficient to decrease the microbial population in the biofilmby at least about 50%, usually by at least 1 log, and may be by 2 or more logs of release. The compounds of the present invention are administered at a dosage that reduces the microbial population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician for in vivo use.

[0073] Biguanide-antibiotic conjugates are also useful for in vitro formulations to dissolve microbial biofilms. For example, biofilm inhibitors may be added to hospital equipment, e.g. ventilation, water processing, etc.

[0074] The susceptibility of a particular microbe to biguanide-antibiotic conjugates may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with agents at varying concentrations for a period of time sufficient to allow the biguanide-antibiotic conjugates to act, usually between about one hour and one day. The attached microbes are then counted, and the level of viability determined.

[0075] Various methods for administration may be employed. The formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific biofilm inhibitor to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously.Formulations

[0076] The biguanide-antibiotic conjugate can be incorporated into a variety of formulations for therapeutic administration. In some embodiments the formulation comprises a biguanide- antibiotic conjugate of Formula I or II.

[0077] More particularly, the compounds can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, transurethral, etc., administration. Thebiofilm inhibitors may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.

[0078] The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., antibiotics, etc.) In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting.

[0079] For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, 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 carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0080] The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0081] The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0082] The compounds can be used as lotions, for example to prevent infection of burns, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0083] Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[0084] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present inventionin a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[0085] Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and / or glycolic acid form an erodible polymer that is well- tolerated by the host. The implant containing biofilm inhibitors is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body.

[0086] The term “unit dosage form”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host.

[0087] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[0088] Typical dosages for systemic administration range from 0.1 jig to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.

[0089] Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.

[0090] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for.Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.EXPERIMENTALVancomycin-Biguanide Conjugates are Effective Dual-mode Broad-Spectrum Antibiotics against ESKAPE Pathogens and Mycobacteria

[0091] Strategies to increase the efficacy and / or expand the spectrum of activity of existing antibiotics provide a potentially fast path to clinically address the growing crisis of antibioticresistant infections. Here, we report the synthesis of an unprecedented class of antibiotic- biguanide conjugates and their resulting activity against ESKAPE pathogens, mycobacteria, and pre-formed biofilms. Our lead conjugate, V-C6-Bg-PhCI (5e), induces highly effective cell killing with up to a 2 orders-of-magnitude improvement over its parent compound, vancomycin (V), against vancomycin-resistant enterococcus. V-C6-Bg-PhCI (5e) also shows activity against a spectrum of bacterial strains and efficacy against pre-formed S. aureus, E. coli, and M. smegmatis biofilms. Mode-of-action studies suggest that the bactericidal effects of 5e are due to association with the bacterial cell surface and vancomycin-like cell wall synthesis disruption. Due to its potency, expanded activity and lack of acute mammalian cell toxicity, V- C6-Bg-PhCI (5e) is a promising candidate for treating antibiotic resistant infections and difficult-to-treat bacterial biofilms associated with chronic infections.

[0092] The genesis of our interest in guanidinylated antibiotics arose in seminal studies in 2000, in which we reported that oligomers of arginine (e.g. D- and L-octa- and nona-arginines), and more generally, polyguanidinylated oligomers (e.g. N-Arg-peptoids) readily cross cell membranes and other biological barriers including human skin. These and related guanidinylated transporter systems, referred to by some as cell-penetrating peptide, have subsequently been used to deliver a wide range of cargoes including therapeutic agents, peptides, protein optical probes, metals, RNA, and DNA readily through cell membranes.

[0093] We proposed that the positively-charged guanidinium groups associate with negatively-charged cell surface phosphates, carboxylates and sulfates through electrostatic and chelative hydrogen bonding interactions and that this association converts an otherwise polar polycation into a less polar complex that is driven inward by the cell membrane potential or through endocytotic pathways. In previous work, we reported that a cell-penetrating guanidinium-rich molecular transporter (octaarginine) conjugate of vancomycin, V-r8, kills vancomycin resistant organisms and eradicates S. aureus biofilms and persister cells. We subsequently showed that a single amino acid addition to vancomycin affording the conjugate V-R, effectively kills Gram-negative ESKAPE pathogens both in vitro and in vivo. Our lab thendemonstrated the addition of this same cationic moiety also increases the efficacy of vancomycin against multiple species of mycobacteria through an improved peptidoglycan binding mechanism via favorable interaction of the guanidinium with the meso-diaminopimelic acid (mDAP) amino acid residue.

[0094] Building on these foundational insights into guanidinium-based transporters, here we report a new cationic modifier of antibiotic activity, the biguanidinium functional group. The biguanidinium and guanidinium functional groups have some properties in common such as similar pKa values under physiological conditions and the ability to engage in electrostatic cation-anion and hydrogen bonding interactions with anionic and neutral hydrogen bond accepting substrates, including cell surface anions (phosphates, sulfates, and carboxylates, Figure 1A). The resulting association with biguanidiniums can be stronger than those of guanidiniums. This was quantified in a synthetic ADP / ATP carrier protein mimic where exchange of a guanidinium for a biguanidinium moiety resulted in a 100-fold increase in the affinity of the carrier protein mimic’s for AMP, ADP, and ATP. Additionally, biguanide- functionalized small molecules have been shown to enter cells through cationic transporter assistance.

[0095] Mono- and poly-biguanidinium agents have been utilized alone as antibiotics and membrane permeabilizing reagents and bis-biguanides when co-administered with vancomycin have been shown to sensitize pathogens to antibiotic accumulation through membrane disruption. Notwithstanding these contributions, covalent chimeras of antibiotic- biguanidinium dual-function conjugates have not been explored.

[0096] Here, we report the synthesis and activity of a novel series of vancomycin-biguanide conjugates with broad-spectrum activity against both planktonic and biofilm-associated ESKAPE pathogens and mycobacteria. Significantly, the lead conjugate, 5e, shows highly effective cell killing against S. aureus, E. faecium, E. coli, A. baumannii, K. pneumoniae, P. aeruginosa, M. smegmatis, and M. abscessus with up to 2 orders-of-magnitude improvement over the parent compound, vancomycin. Conjugate 5e additionally eradicates pre-formed S. aureus, E. coli, and M. smegmatis biofilms. Our studies suggest a multifunctional mode-of- action involving D-ala-D-ala binding, increased accumulation, and membrane disruption.RESULTS AND DISCUSSION

[0097] Design and Synthesis of V-Biguanide Conjugates. We devised two synthetic routes to boc-aminobiguanides 3a-e (Figure 2). The first method involves the direct Pinner-like addition of an amine to an appropriate cyanoguanidine (2 or 8a) at elevated temperatures in the presence (Figure 2A) or absence (Figure 2B) of solvent. The second and preferred route to boc-aminobiguanides 3a-e involves the biguanidinylation of an amine with an activatedpyrazole-biguanide intermediate, 9a-e. The resulting boc-aminobiguanides 3a-e were then converted with acid to the aminobiguanides 4a-e and subsequently conjugated to vancomycin to yield antibiotic-biguanide conjugates V-C6-Bg-R 5a-e or N-acetylated to yield control compounds Ace-CeBiguan-R 6a-e, respectively. (Figure 3).

[0098] A hexamethylene linker was selected to conjugate vancomycin’s C-terminus to the biguanide moiety by a peptide bond (FIG. 1 ). This strategy required a terminal amine which prompted our selection of the monoprotected diamine 1. Boc-aminobiguanides 3a-e were synthesized by two strategies differing principally by whether an activated pyrazole-biguanide 9a-e is used as an intermediate. The strategy 1 (FIG. 2A, FIG. 2B), while atom- and step- economical, suffered from poor selectivity and only serviceable yields. Purification of 3a-e from the reaction mixture required silica-gel chromatography to remove unidentified side-products. Strategy 2 (Figure 2C), based on the work of Bardovskyi, et. al, involves a biguanide transfer reaction utilizing pyrazole-biguanides 9a-e to biguanidinylate 1 under mild-conditions which yields 3a-e upon precipitation from diethyl ether. This method (Figure 2C) requires an additional synthetic operation to generate the activated intermediates 9a-e but removes a chromatographic purification and increases yields of 3a-e by 1 .3- to 2.0- fold over strategy 1 .

[0099] Cyanoguanidine 8a is commercially available while lipidated cyanoguanidines 8b-d were synthesized from the appropriate amine using NaN(CN)2and trifluoroacetic acid (TFA) in DMF. The completed reaction was then diluted with 1 M NaOH, the organic soluble compounds were extracted with ethyl acetate, and concentrated in vacuo. From the crude concentrate, N-alkylated cyanoguanidines were crystallized from diethyl ether (8b) or THF and hexanes (8c, 8d) and then collected by filtration in 71 -91% yield. Cyanoguanidine 8e was synthesized in water from NaN(CN)2, chloroaniline, and aqueous HCI following a reported procedure. The product precipitated from the reaction mixture and 8e was collected by filtration in 92% yield.

[0100] Pyrazole-biguanide 9a was synthesized in water following a published procedure, but further development was required to acquire functionalized pyrazole-biguanides 9b-e. N- alkylated pyrazole-biguanides 9b-d were synthesized from 8b-d with stoichiometric pyrazolium chloride in hot DMF. The hot reaction mixtures were added to stirring diethyl ether from which the products precipitated and were collected by filtration in 86-95% yields. The products were further purified by recrystallization from boiling acetonitrile. Because the addition reaction proceeded slowly in DMF, water, pyridine, and methanol, acetonitrile proved to be a suitable solvent to synthesize the chlorophenyl analogue 9e in good yields. Upon heating the reactant solution to reflux at 0.05 M, 9e rapidly crystallized from solution and was collected by filtration in 63% yield (98% BRSM). The starting materials can be recovered from the filtrate and resubjected to yield additional product.

[0101] Mono-protected diamine 1 was dissolved in pyridine (1 M) with the appropriate biguanidinylation reagent 9a-e and stirred at 60°C for 18 hours. We observed clean conversion to corresponding Boc-aminobiguanide 3a-e. Dropwise addition of the crude reaction into diethyl ether resulted in precipitation of the product which was purified by column chromatography to yield 3a-e in 69-83% isolated yield.

[0102] The Boc-aminobiguanides 3a-e were deprotected with TFA in DCM (1 :1 v / v) to yield aminobiguanides 4a-e (Figure 3). Each aminobiguanide 4a-e and vancomycin were conjugated in the presence of HBTU and DIPEA in a DMSO / DMF cosolvent solution (1 :1 v / v). The resulting antibiotic-biguanide conjugates, V-C6Biguan-R 5a-e, were purified by reversephase HPLC to yield the corresponding trifluoroacetate salts and conjugation to vancomycin’s C-terminus was confirmed with HSQC and HMBC NMR experiments. Additionally, to investigate antimicrobial activities of the biguanide-moiety itself (without vancomycin), we acetylated the aminobiguanides 4a-e with acetic anhydride to yield the acetamide-biguanides Ace-CeBiguan-R 6a-e. In summary, antibiotic-biguanide conjugates 5a-e were synthesized in 5 steps in 21-40% overall yield and acetamide-biguanides 6a-e were synthesized in 5 steps in 38-63% yield. Full synthetic details, high-resolution mass analysis,1H-NMR,13C-NMR, and 2D-NMR spectra are provided in supporting information.

[0103] Evaluation of V-Biguanide Conjugates in Antimicrobial Susceptibility Assays with ESKAPE Pathogens. The vancomycin conjugates 5a-e and acetamide-biguanides 6a- e were first evaluated for antimicrobial activity against a panel containing Gram-positive, Gram-negative, and mycobacterial pathogens (Table 1 ). Significantly, the conjugation of a single unfunctionalized biguanide to vancomycin, 5a, resulted in a 2- to 32- fold increase in potency. More generally, all conjugates except 5d were significantly better than vancomycin (V), exhibiting excellent killing activity against multiple organisms that are resistant or insensitive to vancomycin. S. aureus 29213 is sensitive to vancomycin with an MIC of 1 pM, and the V-biguanide conjugates 5a-c and 5e maintain this activity with MICs similar to or slightly lower than vancomycin. E. faecium 51559 is a vanA-type resistant strain that produces Lipid II terminating in D-Ala-D-Lac. This modification results in greatly diminished vancomycin sensitivity (V MIC of 512 pM), but conjugates 5a and 5b have 16- to 32- fold lower MICs against this strain (16-32 pM) and more hydrophobic conjugates 5c, 5d, and chlorophenyl conjugate 5e demonstrate potent single-digit MICs from 2-4 pM. Gram-negative organisms, including ESKAPE pathogen strains E. coli, A. baumannii, K. pneumoniae, and P. aeruginosa are inherently insensitive to vancomycin. This resistance primarily stems from the presence of the outer membrane which is absent in Gram-positive organisms and prevents vancomycin from accumulating in the periplasmic space where cell wall synthesis occurs. As a result, the typical MICs for vancomycin against Gram-negative organisms are 128 pM or higher. The V-biguanide conjugates 5a-c and 5e all showed significantly lower MICs against Gram-negative organisms, including an improvement of 32-fold for 5e against E. coli. Finally, in two mycobacteria strains, M. smegmatis 700084 and M. abscessus 19977, three V-biguanide conjugates 5a, 5b, and 5e exhibited more potent antimicrobial activity than vancomycin, including the non-lipidated conjugate 5a which demonstrated 16-fold improvement over the parent compound in M. smegmatis. In M. abscessus 5e was most effective, improving upon vancomycin by 4-8 fold. Interestingly, biguanide modification of vancomycin, regardless of the lipid, resulted in decreased efficacy against M. avium. This is not surprising because its cationic functionalization of vancomycin has previously been reported to decrease antibiotic conjugate efficacy against this strain of bacteria.

[0104] T aken together, compound 5e represents the most effective conjugate in this collection with MICs of 4-8 piM, 8 pM, 32 pM and 64-128 pM against E. coli, A. baumannii and M. abscessus, K. pneumoniae, and P. aeruginosa, respectively. Compound 5e surpasses the MIC of vancomycin in S. aureus with a value of 0.25-0.5 pM and matches our other lead compounds against VRE with an MIC of 4 pM. Beyond ESKAPE pathogens, vancomycin- biguanide conjugate 5e is capable of enhanced activity against mycobacteria with up to 4-8 fold greater potency than vancomycin in the MIC assay. In all 5e is a potent antibiotic with truly broad spectrum activity and the potential to combat many classes of bacterial pathogens.

[0105] We observed covalent conjugation of vancomycin to the aminobiguanide was essential for the effective killing. Acetamide- biguanides 6a, 6b, and 6e controls displayed no antimicrobial activity alone, with MICs > 256 pM in all strains (Table 1 , Table 2). For these three compounds, the unconjugated combination of acetamide-biguanides and vancomycin (1 :1 ratio) displayed activity indistinguishable from vancomycin alone (Table 2), indicating the importance of covalent conjugation for the improvements in MICs discussed above. For the aminobiguanides with longer lipophilic chains, we observed noticeable antimicrobial activity in the acetamide-biguanide transporters by themselves. The combination of V + 6c matched MICs for either V alone or 6c alone, whichever was lower for a given strain, this antimicrobial activity from the medium to long chain amphiphiles is potentially due to the acetamide- biguanide’s surfactant-like structure.

[0106] In Vitro Evaluation of Mammalian Cell Toxicity. With pre-clinical assays in mind, we evaluated Vancomycin-biguanide conjugates for their toxicity in mammalian cells using an MTS assay with HeLa cells. Interestingly, we observed significant cytotoxicity from 5c at concentrations equal to or greater than 40 pM. Mild cytotoxicity was observed with 5b and 5d starting at 80 pM and 160 pM, respectively. No significant cytotoxicity was observed from 5a or 5e up to 160 pM. The cytotoxicity demonstrated by 5c potentially arises from the membrane disrupting activities associated with medium-chain lipids, as previously reported.

[0107] Overall, these studies indicate that the therapeutic window for these biguanide conjugates could be expanded by variations in the lipid attachment. The MIC and mammalian cell cytotoxicity results show V-C6-Bg-PhCI (5e) as having activities matching or superior to the other tested agents with no acute toxicity, thus supporting further mode of action characterization of this lead derivative.

[0108] Mode of Action Characterization of Lead Compound 5e. To dissect the enhanced activity observed for 5e, we employed a time resolved viability assay based on enumeration of viable cells in colony forming units (CFU) per milliliter (mL) following antibiotic treatment. V- C6-Bg-PhCI (5e) displayed similar killing kinetics to vancomycin in S. aureus, in line with MIC data. An equimolar mixture of V + 6e against S. aureus 29213 (Figure 5A) was indistinguishable from vancomycin alone. In E. coli, where vancomycin is ineffective at clinically relevant concentrations, 5e was approximately 16-fold more effective than V, displaying killing kinetics at 16 pM that is comparable to a high dose of V (256 pM) (Figure 5B). In contrast, V (16 pM) and equimolar V + 6e (16 pM each), showed growth indistinguishable from the untreated control. These results are consistent with the hypothesis that in E. coli 5e demonstrates a vancomycin-like inhibition of cell wall synthesis at a lower effective dose than Vancomycin. In the mycobacteria M. smegmatis, 5e (pM) displays killing kinetics slightly better to V (16 pM) and the equimolar V + 6e (16 pM each). This data aligns with the comparable MIC values of these conditions in this strain. This is consistent with a vancomycin-like mode of action whereby cell wall synthesis inhibition results in a relatively slow killing profile.

[0109] To explore whether 5e exhibits vancomycin-like inhibition of cell wall synthesis, we examined whether treatment with the conjugate would result in intracellular accumulation of peptidoglycan precursors in all three model strains. The build-up of peptidoglycan precursor UDP-MurNAc pentapeptide is expected after treatment with vancomycin in S. aureus, E. coli, M. smegmatis as a result of the cell-wall precursor targeting mode of action of vancomycin. Indicative of a Vancomycin-like mode of action in all strains we observed that 5e, too, resulted in accumulation of UDP-MurNAc pentapeptide. In S. aureus we observed comparable accumulation of UDP-MurNAc pentapeptide following treatment with vancomycin and treatment with 5e (Figure 6A). In E. coli treated with conjugate 5e (16 pM), we observed significantly higher accumulation of the UDP-MurNAc pentapeptide than in E. coli treated with vancomycin (256 pM) at a 16-fold greater concentration (Figure 6C). Lastly, similar to the timekill kinetics data, we observe in M. smegmatis slightly greater, but generally comparable accumulation of this key cell-wall precursor following treatment with 5e when compared to cells treated with vancomycin (Figure 6E). All treatment conditions showed significantly more peptide accumulation in treated cells when compared to untreated cells. (Figure 6).

[0110] The expanded and potent Gram-negative activity observed for our previous guanidine- modified Vancomycin conjugates and the biguanide conjugates herein could be associated with increased membrane disruption and cell permeability. We, therefore, evaluated lead conjugate 5e for membrane permeabilization using a fluorescent probe, propidium iodide (PI). PI is unable to penetrate healthy viable cells, but if membrane integrity is perturbed, PI can enter the cytoplasm and bind to DNA, resulting in increased fluorescence. Membrane integrity was evaluated in exponential-phase cells suspended in HEPES-Glucose buffer to maintain metabolic activity.5758Lysostaphin was included as a positive control which disrupts cell membrane integrity through peptidoglycan digestion. Treatment of S. aureus with V-C6-Bg- PhCI was accompanied by increased PI fluorescence (Figure 7A), consistent with membrane disruption. Vancomycin, the acetamide-chlorophenylbiguanide 6e and unconjugated equimolar combination of V + 6e all showed no increase in fluorescence. These results suggest that covalent conjugation is key to the membrane disrupting properties of lead conjugate V-C6-Bg-PhCI (5e). These additional membrane disruption effects against S. aureus may help contribute to the excellent S. aureus biofilm activity seen with this conjugate.

[0111] Next, we evaluated the membrane disrupting properties of 5e against E. coli. We hypothesize that in E. coli, the vancomycin-conjugated biguanide groups would engage the membrane surface by binding LPS and disrupting local membrane architecture thereby enhancing uptake of vancomycin conjugates. Thus, we examined whether treatment with 5e would increase intercalation of the probe molecule 1 -N-phenylnapthylamine (NPN) into the outer membrane. NPN is a sensitive reporter that exhibits fluorescence upon access to and incorporation into the hydrophobic environment of the phospholipid inner leaflet of the outer membrane and is typically indicative of LPS destabilization and increased membrane permeability. Polymyxin B at 80 pM was included as a control compound known to increase cell permeability. Treatment with 5e at 80 pM (10x MIC) in E. coli 25922 resulted in increased NPN fluorescence, consistent with outer membrane destabilization (Figure 7B). Vancomycin, the acetamide-chlorophenylbiguanide 6e and unconjugated equimolar combination of V + 6e all showed no increase in fluorescence, again highlighting the necessity of covalent conjugation.

[0112] These collective results reveal membrane disruption and permeabilization activity associated with lead conjugate 5e. The distinction between 5e and unconjugated combination of 6e and V + 6e suggests that covalent conjugation to the chlorophenylbiguanide is necessary to achieve membrane permeabilization. Importantly, the bacterial membrane disruption effects of 5e occur without an accompanying increase in mammalian cell toxicity.

[0113] Evaluation of Cellar Localization of V-C6-Bg-PhCI, 5e, using Fluorescent Conjugates. The readily-scaled synthesis of 5e was also used to make a fluorescein-taggedderivative (Fl-5e) to optically evaluate uptake and localization of the vancomycin conjugates in all three representative bacteria strains.

[0114] Evaluation of V-Biguanide Conjugates Against Biofilm-Associated Pathogens. Having established the efficacy of the V-biguanide conjugates against planktonic Grampositive and Gram-negative bacteria, and mycobacteria in MIC assays, we evaluated their efficacy against preformed biofilms of representative pathogens. Multiple vancomycin- biguanide conjugates show anti-bacterial activity against pre-formed biofilms of S. aureus 29213, against which vancomycin has no activity (Table 2, MBEC >512 pM). 5b, 5c, and 5e had the most potent activity here, with minimum biofilm eradication concentrations (MBECs) of 4-8, 16-32 and 8-16 pM. These results are comparable to the control anti-biofilm antibiotics rifampicin (16-32 pM MBEC) and V-r8 (8-16 pM MBEC). 5a had an MBEC of 64-128 in this strain, indicating that the addition of hydrophobic components enhanced anti-S. aureus biofilm activity. However, we observed the C16-functionalized biguanide conjugate 5d possess no activity against biofilm-associated S. aureus (MBEC > 128 pM) indicating too large of a hydrophobic moiety can impede efficacy against biofilm-associated pathogens.

[0115] We next evaluated the biguanide conjugates for activity against pre-formed biofilms of E. coli 25922 (Table 2). Gram-negative strains like E. coli are inherently insensitive to vancomycin due to their outer membrane, and vancomycin has a high MBEC value of 256 pM in this assay. All conjugates except 5d demonstrated significant biofilm eradication in this assay with MBECs ranging from 32-64 pM, comparable to the control compound polymyxin B (16-32 pM MBEC).

[0116] Lastly, we evaluated vancomycin-biguanide conjugates against pre-formed biofilms of M. smegmatis 700084 (Table 2). In line with the MIC assay results, we observed an inverse correlation between biguanide hydrophobicity and antibiotic potency. For example, conjugates with larger lipid substituents showed lower biofilm eradication concentrations. As previously observed and expected, 5d showed no activity, but 5a demonstrated 32-fold greater potency than vancomycin and 4-fold greater potency than mycobacterial pathogens V-R. In this same biofilm associated pathogen, Oritavancin showed no demonstrable activity (>256 pM MBEC).

[0117] It is not surprising that variation in the lipophilicity of the conjugates can affect the antibiotic-conjugate’s activity against biofilm-associated pathogens. The varying extracellular matrices of each biofilm would be expected to interact with each hydrophobic moiety differently. Of the conjugates evaluated in this study, 5e consistently outperforms the parent compound, Vancomycin, and displays remarkable activity across all three biofilm-associated pathogens. While Oritavancin demonstrates strong activity against S. aureus biofilm- associated pathogens, it is ineffective against E. coli and M. smegmatis biofilm-associated pathogens where multiple V-biguanide conjugates are effective.

[0118] We face a future where drug-resistant microbes and difficult-to-treat biofilms render our currently available antimicrobials ineffective, and routine surgeries and other modes of exposure carry the risk of a life-threatening infection. The design or discovery of more effective antibiotics is urgently needed to address this issue. Generalizable strategies to increase the potency of existing antibiotics and expand their spectrum of activity provide a fast path to clinical evaluation as needed to address the growing threat of antibiotic-insensitive infections and biofilms. Here, we report the design and synthesis of unprecedented antibiotic-biguanide conjugates and their resulting activity against ESKAPE pathogens, mycobacteria and preformed biofilms. Our lead conjugate, V-C6-Bg-PhCI (5e), demonstrates excellent cell killing with up to 2 orders-of-magnitude improvement over the parent compound - vancomycin, against vancomycin-resistant enterococcus. V-C6-Bg-PhCI also shows potency against a broad spectrum of bacterial ESKAPE pathogens and mycobacteria, and eradicates S. aureus,E. coli, and M. smegmatis biofilms. Our studies show a multifunctional mode of action including cell wall and membrane disruption, with no acute mammalian cell toxicity. Thus, V-C6-Bg- PhCI represents a promising agent against difficult-to-treat biofilm-associated infections and urgent threat bacterial pathogens.REFERENCES

[0119] Murray et al. Global Burden of Bacterial Antimicrobial Resistance in 2019: ASystematic Analysis. Lancet 2022, 399 (10325), 629-655.

[0120] O’Neill, J. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. Rev. Antimicrob. Resist. 2016, 84.

[0121] O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations. Rev. Antimicrob. Resist. 2014.

[0122] World Health Organization. Global Tuberculosis Report 2023', World Health Organization, 2023; Vol. Global tub.

[0123] Thomas, D.; Wessel, C. The State of Innovation in Antibacterial Therapeutics. BIO Ind. Anal. 2022, No. February, 3-5.

[0124] Boyd, N. K.; Teng, C.; Frei, C. R. Brief Overview of Approaches and Challenges in New Antibiotic Development: A Focus On Drug Repurposing. Front. Cell. Infect. Microbiol. 2021 , 11 (May), 1-12.

[0125] Acharya, Y.; Bhattacharyya, S.; Dhanda, G.; Haidar, J. Emerging Roles of Glycopeptide Antibiotics: Moving beyond Gram-Positive Bacteria. ACS Infect. Dis. 2021.

[0126] Liu, C.; Bayer, A.; Cosgrove, S. E.; Daum, R. S.; Fridkin, S. K.; Gorwitz, R. J.; Kaplan, S. L.; Karchmer, A. W.; Levine, D. P.; Murray, B. E.; Rybak, M. J.; Talan, D. A.; Chambers, H.F. Clinical Practice Guidelines by the Infectious Diseases Society of America for the Treatmentof Methicillin-Resistant Staphylococcus Aureus Infections in Adults and Children. Clin. Infect. Dis. 2011 , 52 (3), e18-e55.

[0127] Levine, D. P. Vancomycin : A History. Clin Infect. Dis. 2006, 42 (Suppl 1 ), 5-12.

[0128] Nour El-Din, H. T.; Elsebaie, M. M.; Abutaleb, N. S.; Kotb, A. M.; Attia, A. S.; Seleem,M. N.; Mayhoub, A. S. Expanding the Structure-Activity Relationships of Alkynyl Diphenylurea Scaffold as Promising Antibacterial Agents. RSC Med. Chem. 2023, No. Cdc, 367-377.

[0129] Reynolds, P. E. Structure, Biochemistry and Mechanism of Action of Glycopeptide Antibiotics. Eur. J. Clin. Microbiol. Infect. Dis. 1989, 8 (11 ), 943-950.

[0130] Barna, J. C. J.; Williams, D. H. The Structure and Mode of Action of Glycopeptide Antibiotics of the Vancomycin Group. Annu Rev Microbiol. 1984, 38 (1), 339-357.

[0131] Walsh, C. T.; Fisher, S. L.; Park, I. S.; Prahalad, M.; Wu, Z. Bacterial Resistance to Vancomycin: Five Genes and One Missing Hydrogen Bond Tell the Story. Chem Biol. 1996, 3 (1 ), 21-28.

[0132] Cantoni, L.; Glauser, M. P.; Bille, J. Comparative Efficacy of Daptomycin, Vancomycin, and Cioxacillin for the Treatment of Staphylococcus Aureus Endocarditis in Rats and Role of Test Conditions in This Determination. Antimicrob. Agents Chemother. 1990, 34 (12), 2348- 2353.

[0133] Bhattacharya, M.; Wozniak, D. J.; Stoodley, P.; Hall-Stoodley, L. Prevention and Treatment of Staphylococcus Aureus Biofilms. Expert Rev. Anti. Infect. Ther. 2015, 13 (12), 1499-1516.

[0134] Antonoplis, A.; Zang, X.; Huttner, M. A.; Chong, K. K. L.; Lee, Y. B.; Co, J. Y.; Amieva, M. R.; Kline, K. A.; Wender, P. A.; Cegelski, L. A Dual-Function Antibiotic-Transporter Conjugate Exhibits Superior Activity in Sterilizing MRSA Biofilms and Killing Persister Cells. J Am Chem Soc. 2018, 140 (47), 16140-16151.

[0135] Dhanda, G.; Sarkar, P.; Samaddar, S.; Haidar, J. Battle against Vancomycin-Resistant Bacteria: Recent Developments in Chemical Strategies. J. Med. Chem. 2019, 62 (7), 3184- 3205.

[0136] Antonoplis, A.; Zang, X.; Wegner, T.; Wender, P. A.; Cegelski, L. Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. Coli and Targets Cell-Wall Synthesis. ACS Chem. Biol. 2019, 14 (9), 2065-2070.

[0137] Srivastava, S.; Chapagain, M.; van Zyl, J.; Deshpande, D.; Gumbo, T. Potency of Vancomycin against Mycobacterium Tuberculosis in the Hollow Fiber System Model. J. Glob. Antimicrob. Resist. 2021 , 24, 403-410.

[0138] Brcic, J.; Tong, A.; Wender, P. A.; Cegelski, L. Conjugation of Vancomycin with a Single Arginine Improves Efficacy against Mycobacteria by More Effective Peptidoglycan Targeting. J. Med. Chem. 2023, 66 (15), 10226-10237.

[0139] Soetaert, K.; Rens, C.; Wang, X. M.; De Bruyn, J.; Laneelle, M. A.; Laval, F.; Lemassu, A. ; Daffe, M.; Bifani, P.; Fontaine, V.; Lefevre, P. Increased Vancomycin Susceptibility in Mycobacteria: A New Approach to Identify Synergistic Activity against Multidrug-Resistant Mycobacteria. Antimicrob. Agents Chemother. 2015, 59 (8), 5057-5060.

[0140] Belley, A.; Neesham-Grenon, E.; McKay, G.; Arhin, F. F.; Harris, R.; Beveridge, T.; Parr, T. R.; Moeck, G. Oritavancin Kills Stationary-Phase and Biofilm Staphylococcus Aureus Cells in Vitro. Antimicrob. Agents Chemother. 2009, 53 (3), 918-925.

[0141] Wu, Z.-C.; Cameron, M. D.; Boger, D. L. Vancomycin C-Terminus Guanidine Modifications and Further Insights into an Added Mechanism of Action Imparted by a Peripheral Structural Modification. ACS Infect. Dis. 2020.

[0142] Wu, Z.; Boger, D. L. Maxamycins: Durable Antibiotics Derived by Rational Redesign of Vancomycin. Acc. Chem. Res. 2020, 53 (1 1 ), 2587-2599.

[0143] Wu, Z. C.; Isley, N. A.; Boger, D. L. N-Terminus Alkylation of Vancomycin: Ligand Binding Affinity, Antimicrobial Activity, and Site-Specific Nature of Quaternary Trimethylammonium Salt Modification. ACS Infect. Dis. 2018, 4 (10), 1468-1474.

[0144] Muhlberg, E.; Umstatter, F.; Dornhan, C.; Hertlein, T.; Ohlsen, K.; Krause, A.; Kleist, C.; Beijer, B.; Zimmermann, S.; Haberkorn, U.; Mier, W.; Uhl, P. Vancomycin-Lipopeptide Conjugates with High Antimicrobial Activity on Vancomycin-Resistant Enterococci. Pharmaceuticals 2020, 13 (6).

[0145] Blaskovich, M. A. T.; Hansford, K. A.; Gong, Y. ; Butler, M. S.; Muldoon, C.; Huang, J. X.; Ramu, S.; Silva, A. B.; Cheng, M.; Kavanagh, A. M.; Ziora, Z.; Premraj, R.; Lindahl, F.; Bradford, T. A.; Lee, J. C. ; Karoli, T.; Pelingon, R.; Edwards, D. J.; Amado, M.; Elliott, A. G.; Phetsang, W. ; Daud, N. H. ; Deecke, J. E.; Sidjabat, H. E.; Ramaologa, S.; Zuegg, J.; Betley, J. R. ; Beevers, A. P. G.; Smith, R. A. G.; Roberts, J. A.; Paterson, D. L.; Cooper, M. A. Protein- Inspired Antibiotics Active against Vancomycin- and Daptomycin-Resistant Bacteria. Nat. Common. 2018, 9 (1 ).

[0146] Chosy, M. B. ; Sun, J.; Rahn, H. P.; Liu, X.; Brcic, J.; Wender, P. A.; Cegelski, L. Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S. Aureus. ACS Infect. Dis. 2024, 10 (2), 384-397.

[0147] Okano, A.; Isley, N. A.; Boger, D. L. Peripheral Modifications of [l4J[CH2NH]Tpg4]Vancomycin with Added Synergistic Mechanisms of Action Provide Durable and Potent Antibiotics. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (26), E5052-E5061 .

[0148] Umstatter, F.; Dornhan, C.; Hertlein, T.; Ohlsen, K. ; Muhlberg, E.; Kleist, C.; Zimmermann, S.; Beijer, B.; Klika, K. D.; Haberkorn, U.; Mier, W.; Uhl, P. VancomycinResistance Is Overcome by Conjugation of Polycationic Peptides. Angew. Chemie Int. Ed. 2020, 59, 8823-8827.

[0149] Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman, L.; Rothbard, J. B. The Design, Synthesis, and Evaluation of Molecules That Enable or Enhance Cellular Uptake: Peptoid Molecular Transporters. Proc Natl Acad Sci USA. 2000, 97 (24), 13003- 13008.

[0150] Stanzl, E. G.; Trantow, B. M.; Vargas, J. R.; Wender, P. A. Fifteen Years of Cell- Penetrating, Guanidinium-Rich Molecular Transporters: Basic Science, Research Tools, and Clinical Applications. Acc. Chem. Res. 2013, 46 (12), 2944-2954.

[0151] Vargas, J. R.; Stanzl, E. G.; Teng, N. N. H.; Wender, P. A. Cell-Penetrating, Guanidinium-Rich Molecular Transporters for Overcoming Efflux-Mediated Multidrug Resistance. Mol. Pharm. 2014, 11 (8), 2553-2565.

[0152] Rothbard, J. B.; Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider, E.; McGrane, P. L.; Wender, P. A.; Khavari, P. A. Conjugation of Arginine Oligomers to Cyclosporin A Facilitates Topical Delivery and Inhibition of Inflammation. Nat. Med. 2000, 6 (11 ), 1253-1257.

[0153] Gongalves, E.; Kitas, E.; Seelig, J. Binding of Oligoarginine to Membrane Lipids and Heparan Sulfate: Structural and Thermodynamic Characterization of a Cell-Penetrating Peptide. Biochemistry 2005, 44 (7), 2692-2702.

[0154] Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. Role of Membrane Potential and Hydrogen Bonding in the Mechanism of Translocation of Guanidinium-Rich Peptides into Cells. J. Am. Chem. Soc. 2004, 726 (31 ), 9506-9507.

[0155] Rothbard, J. B.; Jessop, T. C.; Wender, P. A. Adaptive Translocation: The Role of Hydrogen Bonding and Membrane Potential in the Uptake of Guanidinium-Rich Transporters into Cells. Adv. Drug Deliv. Rev. 2005, 57(4 SPEC.ISS.), 495-504.

[0156] Dubikovskaya, E. A.; Thorne, S. H.; Pillow, T. H.; Contag, C. H.; Wender, P. A. Overcoming Multidrug Resistance of Small-Molecule Therapeutics through Conjugation with Releasable Octaarginine Transporters. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (34), 12128— 12133.

[0157] Neville, L. F.; Shalit, I.; Warn, P. A.; Scheetz, M. H.; Sun, J.; Chosy, M. B.; Wender, P. A.; Cegelski, L.; Rendell, J. T. In Vivo Targeting of Escherichia Coli with Vancomycin-Arginine . Antimicrob. Agents Chemother. 2021 , 65 (4), 1-7.

[0158] Neville, L. F.; Shalit, I.; Warn, P. A.; Rendell, J. T. Vancomycin-Arginine (STM-001) Abrogates ESBL Carrier and Carbapenem-Resistant Escherichia Coli Burden in a Murine Complicated Urinary Tract Infection Model. J Antimicrob Chemother. 2022, 77(6), 1706-1709.

[0159] Kathuria, D.; Raul, A. D.; Wanjari, P.; Bharatam, P. V. Biguanides: Species with Versatile Therapeutic Applications. Eur. J. Med. Chem. 2021 , 219, 113378.

[0160] Bharatam, P. V.; Patel, D. S.; Iqbal, P. Pharmacophoric Features of Biguanide Derivatives: An Electronic and Structural Analysis. J. Med. Chem. 2005, 48 (24), 7615-7622.

[0161] Chen, H. Y.; Zhao, M.; Tan, J. H.; Huang, Z. S.; Liu, G. F.; Ji, L. N.; Mao, Z. W. |3- Biguanidinium-Cyclodextrin: A Supramolecular Mimic of Mitochondrial ADP / ATP Carrier Protein. Tetrahedron 2014, 70 (14), 2378-2382.

[0162] Sowlati- Hashjin, S.; Karttunen, M.; Carbone, P. Insights into the Polyhexamethylene Biguanide (PHMB) Mechanism of Action on Bacterial Membrane and DNA: A Molecular Dynamics Study. J. Phys. Chem. B 2020, 124 (22), 4487-4497.

[0163] Rzycki, M.; Drabik, D.; Szostak-Paluch, K.; Hanus-Lorenz, B.; Kraszewski, S. Unraveling the Mechanism of Octenidine and Chlorhexidine on Membranes: Does Electrostatics Matter? Biophys. J. 2021 , 720 (16), 3392-3408.

[0164] Kathuria, D.; Bankar, A. A.; Bharatam, P. V. “What’s in a Structure?” The Story of Biguanides. J. Mol. Struct. 2018, 1152, 61-78.

[0165] Fortun, S.; Schmitzer, A. R. The Chemistry of Biguanides: From Synthetic Routes to Applications in Organic Chemistry. Can. J. Chem. 2020, 98 (6), 251-260.

[0166] Grytsai, O.; Ronco, C.; Benhida, R. Synthetic Accesses to Biguanide Compounds. Beilstein J. Org. Chem. 2021 , 77(i), 1001-1040.

[0167] Obianom, O. N.; Coutinho, A. L.; Yang, W.; Yang, H.; Xue, F.; Shu, Y. Incorporation of a Biguanide Scaffold Enhances Drug Uptake by Organic Cation Transporters 1 and 2. Mol. Pharm. 2017, 14 (8), 2726-2739.

[0168] Chawner, J. A. ; Gilbert, P. Interaction of the Bisbiguanides Chlorhexidine and Alexidine with Phospholipid Vesicles : Evidence for Separate Modes of Action. J. Appl. Bacteriol. 1989, 66, 253-258.

[0169] Lei, E.; Tao, H.; Jiao, S.; Yang, A.; Zhou, Y.; Wang, M.; Wen, K.; Wang, Y.; Chen, Z.; Chen, X.; Song, J.; Zhou, C.; Huang, W.; Xu, L.; Guan, D.; Tan, C.; Liu, H.; Cai, Q.; Zhou, K.; Modica, J.; Huang, S. Y.; Huang, W.; Feng, X. Potentiation of Vancomycin: Creating Cooperative Membrane Lysis through a “Derivatization-for-Sensitization” Approach. J. Am. Chem. Soc. 2022, 744 (23), 10622-10639.

[0170] Bardovskyi, R.; Fabre, M.; Ronco, C.; Benhida, R. Mild Biamidine-Transfer Conditions for the Synthesis of Aliphatic Biguanides. SynOpen 2021 , 5 (4), 314-320.

[0171] Loesche, A.; Wiese, J.; Sommerwerk, S.; Simon, V.; Brandt, W.; Csuk, R. Repurposing N,N’-Bis-(Arylamidino)-1 ,4-Piperazinedicarboxamidines: An Unexpected Class of Potent Inhibitors of Cholinesterases. Eur. J. Med. Chem. 2017, 725, 430-434.

[0172] Chen, A.; Karanastasis, A.; Casey, K. R.; Necelis, M.; Carone, B. R.; Caputo, G. A.; Palermo, E. F. Cationic Molecular Umbrellas as Antibacterial Agents with Remarkable Cell- Type Selectivity. ACS Appl. Mater. Interfaces 2020, 72 (19), 21270-21282.

[0173] Wu, C.; Lim, J. Y.; Fuller, G. G.; Cegelski, L. Disruption of Escherichia Coli Amyloid- Integrated Biofilm Formation at the Air-Liquid Interface by a Polysorbate Surfactant. Langmuir 2013, 29 (3), 920-926.

[0174] Morstein, J.; Capecchi, A.; Hinnah, K.; Park, B.; Petit-Jacques, J.; Van Lehn, R. C.; Reymond, J.-L.; Trauner, D. Medium-Chain Lipid Conjugation Facilitates Cell-Permeability and Bioactivity. J. Am. Chem. Soc. 2022.

[0175] Peng, B.; Su, Y. Bin; Li, H.; Han, Y.; Guo, C.; Tian, Y. M.; Peng, X. X. Exogenous Alanine and / or Glucose plus Kanamycin Kills Antibiotic-Resistant Bacteria. Cell Metab. 2015, 21 (2), 249-262.

[0176] Allison, K. R.; Brynildsen, M. P.; Collins, J. J. Metabolite-Enabled Eradication of Bacterial Persisters by Aminoglycosides. Nature 2011 , 473 (7346), 216-220.

[0177] Muheim, C.; Gbtzke, H.; Eriksson, A. U.; Lindberg, S.; Lauritsen, I.; Norholm, M. H. H.; Daley, D. O. Increasing the Permeability of Escherichia Coli Using MAC13243. Sc / . Hep. 2017, 7(1 ), 1-11.

[0178] Trauble, H.; Overath, P. The Structure of Escherichia Coli Membranes Studied by Fluorescence Measurements of Lipid Phase Transitions. Biochim Biophys Acta. 1973, 307 (3), 491-512.

[0179] Helander, I. M.; Mattila-Sandholm, T. Fluorometric Assessment of Gram-Negative Bacterial Permeabilization. J. Appl. Microbiol. 2000, 88 (2), 213-219.

[0180] Chindera, K.; Mahato, M.; Sharma, A. K.; Horsley, H.; Kloc-muniak, K.; Kamaruzzaman, N. F.; Kumar, S.; Mcfarlane, A. The Antimicrobial Polymer PHMB Enters Cells and Selectively Condenses Bacterial Chromosomes. Nat. Publ. Gr. 2016, No. February, 1-13.

[0181] Hao, X.; Wang, H.; Zhao, W.; Wang, L.; Peng, F.; Yan, Q. Dynamic Macro- and Microgels Driven by Adenosine Triphosphate-Fueled Competitive Host-Guest Interaction. CCS Chem. 2022, 4 (3), 838-846.

[0182] Verderosa, A. D.; Totsika, M.; Fairfull-Smith, K. E. Bacterial Biofilm Eradication Agents: A Current Review. Front. Chem. 2019, / (November), 1-17..

[0183] Michaelis, C.; Grohmann, E. Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms. Antibiotics 2023, 12 (2).

[0184] Camara, M.; Green, W.; MacPhee, C. E.; Rakowska, P. D.; Raval, R.; Richardson, M. C.; Slater-Jefferies, J.; Steventon, K.; Webb, J. S. Economic Significance of Biofilms: A Multidisciplinary and Cross-Sectoral Challenge, npj Biofilms Microbiomes 2022, 8 (1), 1-8.

[0185] Carson, L.; Merkatz, R.; Martinelli, E.; Boyd, P.; Variano, B.; Salient, T.; Malcolm, R. K. The Vaginal Microbiota, Bacterial Biofilms and Polymeric Drug-Releasing Vaginal Rings. Pharmaceutics 2021 , 13 (5), 1-28.

[0186] Campoccia, D.; Montanaro, L; Arciola, C. R. The Significance of Infection Related to Orthopedic Devices and Issues of Antibiotic Resistance. Biomaterials 2006, 27 (11 ), 2331- 2339.

[0187] Sahoo, J.; Sarkhel, S.; Mukherjee, N.; Jaiswal, A. Nanomaterial-Based Antimicrobial Coating for Biomedical Implants: New Age Solution for Biofilm-Associated Infections. ACS Omega 2022, 7(50), 45962-45980..

[0188] Shahid, A.; Aslam, B.; Muzammil, S.; Aslam, N.; Shahid, M.; Almatroudi, A.; Allemailem, K. S.; Saqalein, M.; Nisar, M. A.; Rasool, M. H.; Khurshid, M. The Prospects of Antimicrobial Coated Medical Implants. J. Appl. Biomater. Funct. Mater. 2021 , 19.

[0189] Sarkar, P.; Basak, D.; Mukherjee, R.; Bandow, J. E.; Haidar, J. Alkyl-Aryl- Vancomycins: Multimodal Glycopeptides with Weak Dependence on the Bacterial Metabolic State. J. Med. Chem. 2021 , 64 (14), 10185-10202.

[0190] All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0191] The present invention has been described in terms of particular embodiments found or proposed by the inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. Moreover, due to biological functional equivalency considerations, changes can be made in methods, structures, and compounds without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:1 . A biguanide antibiotic drug conjugate having the structure:wherein:D is an antibiotic drug; n is from 1 to 10; andR is selected from H, alkyl, substituted alkyl, phenyl, diphenyl, substituted phenyl, substituted diphenyl, furan, and furfuryl.

2. The biguanide drug conjugate of claim 1 , wherein the antibiotic drug is a glycosylated cyclic or polycyclic nonribosomal peptide.

3. The biguanide drug conjugate of claim 1 or claim 2, wherein the drug is selected from vancomycin, teicoplanin, telavancin, ramoplanin, decaplanin, oritavancin, and dalbavancin.

4. The biguanide drug conjugate of any of the preceding claims, wherein n is 6.

5. The biguanide drug conjugate of any of the preceding claims, wherein R is selected from:furfuryl,1 ,1 -diphenylmethyl; and where X is selected from6. The biguanide drug conjugate of any of the preceding claims, wherein the drug is vancomycin or a derivative thereof.

7. The biguanide drug conjugate of any of the preceding claims, wherein the conjugate has the structure:

8. A pharmaceutical formulation comprising an effective dose of a biguanide conjugate according to any of the preceding claims; and a pharmaceutically acceptable excipient.

9. A method of reducing bacterial load, the method comprising contacting a bacterial cell population with an effective dose of a formulation of claim 8.

10. The method of claim 9, wherein the bacterial cell population comprises one or more of: E. faecium, E. coli, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter sp., and Mycobacteria sp.1 1 . The method of claim 9 or claim 10, wherein the bacterial cell population comprises one or more of: persister cells, vancomycin-resistant bacteria, MRSA, MRSE, GRE and biofilms.

12. The method of any of claims 9-1 1 , wherein the bacterial cell population is tested for the presence of one or more of E. faecium, E. coli, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter sp., Mycobacteria sp.; persister cells, vancomycin-resistant bacteria, MRSA, MRSE, GRE and biofilms prior to said contacting step.

13. The method of claim 9, wherein an individual with a bacterial infection is treated by administering an effective dose of the formulation.

14. The method of claim 13, wherein the effective dose achieves a concentration at the target site of from 0.01 (iM to 10 mM of the conjugate.

15. The method of claim 13, wherein the effective dose achieves a concentration at the target site of from 0.1 u.M to 1 mM of the conjugate.

16. The method of claim 13, wherein the effective dose is from 0.5 mg to 50 mg / day.

17. The method of any of claims 13-16, wherein the bacterial population in the infection comprises at least about 5% bacteria that are resistant or tolerant to antibiotics.

18. A method for synthesis of a biguanide drug conjugate of claim 1 , comprising: biguanidinylation of an amine with an activated pyrazole-biguanide intermediate to generate a boc-aminobiguanide; and reacting the boc-aminobiguanide with an antibiotic drug to generate a biguanide drug conjugate.

Images