Targeted prodrugs for the resolution of bacterial infections

Abstract

The present invention relates to compounds comprising a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, or a mitomycin C, one or more antibacterial agents, and a biodegradable linker linking the glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, or the mitomycin C and the one or more antibacterial agents.

Description

[0001] Targeted prodrugs for the resolution of bacterial infections

[0002] Technical field

[0003] The present invention relates to compounds comprising a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, or a mitomycin C, one or more antibacterial agents, and a biodegradable linker linking the glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, or the mitomycin C and the one or more antibacterial agents.

[0004] Background

[0005] Bacterial colonization of implanted biomaterials leads to infections that are a serious complication with a high socio-economic and healthcare burden. Despite advances in surgery, infection remains a risk, with incidence rates of 1 -2% for prosthetic knees and hips (Springer et al., 2017), 1 -5% for prosthetic vascular grafts (Darouiche, 2004) and up to 8.5% for spinal implants (Kurtz et al., 2012). Post-operative prophylaxis often has little to no effect on the implant-associated infections (Thornley et al., 2015), and surgical intervention is often required to cure the patient (Ciofu et al., 2022). Patients ineligible for surgery are faced with either amputation of limbs or life-long suppressive antibiotic therapy, which is also associated with significant morbidity.

[0006] The resilience of implant-associated infections is linked to the formation of bacterial biofilms on and around the implant (Ciofu et al., 2022; Arciola et al., 2018). Bacteria in biofilms are embedded in a shared extracellular matrix, which offers protection from the immune system. Within the biofilm, slow-growing or dormant sub-populations emerge, and these populations are called “persister cells” because they survive extremely high concentrations of all the antibiotics in current clinical use (Anderson et al., 2008). Treatment of implant-associated infections therefore remains a major healthcare challenge and requires a novel treatment paradigm (Haddad et al., 2017).

[0007] Novel therapies developed to specifically tackle biofilm infections often rely on the discovery of new antibiotics (Miethke et al., 2021 ) or the delivery of a high local dose of current antibiotics (Jones, 2005). Alternatively, prodrug therapies can activate a circulating inactive drug at site of infection (Cao-Milan et al., 2020; Walther et al., 2018; Yu et al., 2023). Promising results have very recently been obtained when prodrug activation was mediated by the host enzymes (typically proteases) (Ngambenjawong et al., 2022). Summary

[0008] The present inventors have developed compounds for targeted delivery of antibacterial agents, such as mitomycin C. The present inventors have discovered that a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, can be used as a targeting agent. Vancomycin is an antibacterial agent on its own which targets the synthesis of the bacterial peptidoglycan cell wall. Almost all bacteria contain a peptidoglycan cell wall, and vancomycin is therefore a broad -spectrum antimicrobial agent and a broad-spectrum targeting agent. Alternatively, the compound can comprise mitomycin C and one or more antibacterial agents.

[0009] The main object of the present disclosure is to provide a compound which takes advantage of drug repurposing and prodrug therapy to deliver an antibacterial agents and / or anti-neoplastic drug that is highly effective against biofilms and persister cells. Furthermore, the compounds of the present disclosure is formulated into a prodrug which might minimize side effects.

[0010] In an aspect, the present disclosure relates to a compound comprising: a. a glycopeptide antibiotic; b. one or more antibacterial agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0011] Thus, in an aspect, the present disclosure relates to a compound comprising: a. a vancomycin; b. one or more antibacterial agents; and c. a biodegradable linker linking the vancomycin and the one or more antibacterial agents.

[0012] In another aspect, the present disclosure relates to a compound comprising: a. a mitomycin C; b. one or more antibacterial agents; and c. a biodegradable linker linking the mitomycin C and the one or more antibacterial agents. In another aspect, the present disclosure relates to a pharmaceutical composition comprising the compound according to the present disclosure or a pharmaceutically acceptable salt thereof.

[0013] In another aspect, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use as a medicament.

[0014] In another aspect, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use in the treatment of a bacterial infection.

[0015] In another aspect, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use in the treatment of a bacterial biofilm.

[0016] In another aspect, the present disclosure relates to use of the compound of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treatment of a bacterial infection.

[0017] In another aspect, the present disclosure relates to a method for treatment of bacterial infection comprising administration of a therapeutically effective amount of the compound of the present disclosure or the pharmaceutical composition of the present disclosure to an individual in need thereof.

[0018] In another aspect, the present disclosure relates to a kit of parts comprising: a. the compound of the present disclosure; and b. instructions for use.

[0019] In another aspect, the present disclosure relates to a method of manufacturing the compound of the present disclosure, the method comprising the steps of: a. providing a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and one or more antibacterial agents; and b. conjugating the glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and the one or more antibacterial agents with a biodegradable linker, optionally wherein the biodegradable linker is a disulfide linker.

[0020] In another aspect, the present disclosure relates to a method of manufacturing the compound of the present disclosure, the method comprising the steps of: a. providing a mitomycin C, and one or more antibacterial agents; and b. conjugating the mitomycin C and the one or more antibacterial agents with a biodegradable linker, optionally wherein the biodegradable linker is a disulfide linker.

[0021] Description of Drawings

[0022] Figure 1 : (A) Schematic overview of one embodiment of the technology wherein the vancomycin targets the antibacterial agent, exemplified by mitomycin, to the bacteria, which results in the release of the antibacterial agent by degradation of the biodegradable linker. (B) Chemical structures of vancomycin (V) and mitomycin (M), and preferred the sites of bioconjugation (marked with arrows).

[0023] Figure 2. (A) Schematic illustration of synthesis of the V-M conjugate using the vancomycin carboxylic group (referred to as V-C-M herein). HPLC chromatogram of release study compared with HPLC chromatograms of the untreated V-C-M conjugate, (B) compound 2 (here named V-C-TP) and (C) commercial MMC. (D) Unspecific drug release from V-C-M. (E)1H NMR spectrum and (F)13C NMR spectrum of 2-(pyridin-2- yldisulfaneyl)ethan-1 -ol. (G)1H NMR spectrum and (H)13C NMR spectrum of compound 1 . (I)1H NMR spectrum of compound 2. (J) RP-HPLC chromatogram of Compound 2. (K) RP-HPLC chromatogram of V-C-M.

[0024] Figure 3. Schematic illustration of synthesis of the V-M conjugates via the primary and secondary amine groups of vancomycin (referred to as V-Np-M and V-Ns-M, respectively). (B) HPLC chromatogram of V-Ns-M release study compared with HPLC chromatograms of the untreated conjugate and commercial MMC. (C) HPLC chromatogram of V-Np-M (Van-Np-MMC) release study compared with HPLC chromatograms of the untreated conjugate, and commercial MMC. (D)1H NMR spectrum and (E)13C NMR spectrum of 3-(pyridin-2-yldisulfaneyl)propanoic acid. (F)1H NMR spectrum and (G)13C NMR spectrum of 3-(pyridin-2-yldisulfaneyl)propanoic acid. (H) RP-HPLC chromatogram of V-Ns-M. (I) RP-HPLC chromatogram of V-Np-M. (J) MS fragmentation. (K) Ninhydrin stain.

[0025] Figure 4. Schematic illustration of synthesis of control, non-targeted compounds, (A) a mitomycin dimer and (B) a conjugate between GSH and mitomycin. Throughout the text, these are referred to as M-M and G-M conjugates, respectively. (C) HPLC chromatogram of M-M release study compared with HPLC chromatograms of the untreated conjugate, and commercial MMC. (D) HPLC chromatogram of G-M release study compared with HPLC chromatograms of the untreated conjugate, and commercial MMC. (E) RP-HPLC chromatogram of M-M. (F) RP-HPLC chromatogram of G-M.

[0026] Figure 5. (A) HPLC data that illustrate that the conjugates were of high purity and did not contain residual (free) mitomycin, and that upon the addition of a reducing agent (DTT), retention time of the conjugate degradation product was identical to that of mitomycin. Compound detection was performed at 360 nm, at which wavelength light is absorbed only by mitomycin and derivatives thereof (not vancomycin); (B) quantitative data for V-C-M conjugate that illustrate that incubation with 5 mM NAC afforded a near- quantitative drug release within the first 15 minutes of incubation; conjugate incubation in buffers (PBS or mM9) showed no spontaneous drug release within the first hour of incubation; spontaneous drug release was observed over 24 of incubation.

[0027] Figure 6. Vancomycin binds to both (A) planktonic and (B) biofilm S. aureus: CLSM images of planktonic and 48 h old biofilm S. aureus incubated with vancomycin-FITC or fluorescein . Bacterial cells were visualized with DNA-binding SYTO60 staining. Scale bars are 20 pm.

[0028] Figure 7. Antimicrobial effect through bacterial targeting V-M (vancomycin-C- mitomycin) and non-targeting M-M and G-M (mitomycin dimer and the conjugate with glutathione), as well as the free mitomycin (M). Planktonic S. aureus were treated with 2 mg / L (equivalent content of MMC) for 15 min followed by wash and suspension in mM9 buffer with and without NAC or DTT added. Antimicrobial effect was determined through quantification of the colony forming units after 24 hours of incubation. This protocol will be henceforth be referred to as 15:24. Statistical evaluation was performed using the two-way ANOVA. ***, p < 0.001 ; **, p < 0.01. Figure 8. Quantification of antimicrobial effect of V-C-M and M-M conjugates on planktonic S. aureus following a 24 h incubation, determined through quantification of the colony forming units. Statistical evaluation was performed using a two-way ANOVA. ***, p < 0.001; **, p < 0.01.

[0029] Figure 9. Antibacterial activity of the V-M conjugates against S. aureus as a function of the conjugation site on vancomycin using 15:24 protocol. Planktonic S. aureus were treated with three different V-M conjugates (V-C-M, V-Ns-M and V-Np-M). The concentration of MMC in the conjugates was 0.5 mg / L (determined by UV measurement) and treatment lasted 15 min followed by wash and suspension in mM9 buffer. Antimicrobial effect was determined through quantification of the colony forming units count. Statistical evaluation was performed using one-way ANOVA. ***, p < 0.001 ; *, p < 0.05.

[0030] Figure 10. (A)1H NMR spectrum and (B)13C NMR spectrum of disulfanediylbis(ethane-2,1 -diyl) bis(4-nitrophenyl ) bis(carbonate).(C)1H NMR spectrum of Compound 4. (D) V-S-M was analyzed by analytical RP-HPLC. (E) HPLC chromatogram of V-S-M release study compared with HPLC chromatograms of the untreated conjugate, and commercial MMC. (F) Schematic illustration of synthesis of VM conjugates re-designed for scale up purposes; (G) Quantification of antimicrobial effects by the V-s-M conjugate in S. aureus cell culture. Bacteria were exposed to the drug for 15 minutes, thereafter washed via centrifugation, and thereafter incubated for additional 24 hours in mM9 buffer. Antimicrobial effect was determined through quantification of the colony forming units count. Statistical evaluation was performed using one-way ANOVA. ***, p < 0.001 ; *, p < 0.05.

[0031] Figure 11. Antibacterial efficacy of mitomycin and the V-S-M conjugate against a panel of Gram-positive and Gram-negative bacteria using the 15:24 protocol. Bacteria were exposed to the drug or the conjugate for 15 minutes, thereafter washed via centrifugation, and thereafter cultured for additional 24 hours in mM9 buffer, optionally in the presence of 5 mM NAC. Antimicrobial effect was determined through quantification of the colony forming units count; statistical evaluation was performed using one-way ANOVA. ***, p < 0.001 ; *, p < 0.05. Figure 12. Chemical structure of A) dalbavancin , B) teicoplanin with the different lipophillic R-groups shown, C) mitomycin C and D) floxuridine.

[0032] Figure 13. Overview of the synthesis of dalbavancin conjugated to mitomycin through the amine (D-N-M) and teicoplanin conjugated to mitomycin C through the amine (T-N- M). A) synthesis of the amine reactive mitomycin C linker compound 2. B) Synthesis of D-N-M using 2. C) One-pot synthesis of TC9-N-M, Tmix-N-M and TC10-N-M using 2. Figure 14. HPLC chromatogram showing the purity of TC9-N-M, Tmix-N-M and TC10- N-M and D-N-M conjugates. Absorbance is measured at 210 nm.

[0033] Figure 15. HPLC chromatogram showing TC9-N-M, Tmix-N-M and TC10-N-M and D- N-M conjugates before and after cleavage with DTT. Absorbance is measured at 360 nm and the conjugates are compared with commercial MMC. Full release of MMC is observed for all conjugates.

[0034] Figure 16. 15:24 protocol of TC9-N-M conjugate against S. aureus 29213 using 1 mg / L mitomycin C equiv. concentration. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 (GC), mM9 + 5.6 mg / L teicoplanin + 1 mg / L mitomycin C (T+M), mM9 + 1 mg / L mitomycin C (M) or 1 mg / L TC9-N-M conjugate (mitomycin C equiv. concentration) (TC9-N-M) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in either 100 pL mM9 (no NAC) or 100 pL mM9 + 5 mM NAC (with NAC). Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified, n = 4 biological replicates, LOD: limit of detection.

[0035] Figure 17. 15:24 protocol of TC10-N-M conjugate against S. aureus 29213 using 1 mg / L mitomycin C equiv. concentration. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 (GC), mM9 + 5.6 mg / L teicoplanin + 1 mg / L mitomycin C (T+M), mM9 + 1 mg / L mitomycin C (M) or 1 mg / L TC10-N-M conjugate (mitomycin C equiv. concentration) (TC10-N-M) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in either 100 pL mM9 (no NAC) or 100 pL mM9 + 5 mM NAC (with NAC). Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified, n = 4 biological replicates, LOD: limit of detection.

[0036] Figure 18. 15:24 protocol of Tmix-N-M conjugate against S. aureus 29213 using 1 mg / L mitomycin C equiv. concentration. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 (GC), mM9 + 5.6 mg / L teicoplanin + 1 mg / L mitomycin C (T+M), mM9 + 1 mg / L mitomycin C (M) or 1 mg / L Tmix-N-M conjugate (mitomycin C equiv. concentration) (Tmix-N-M) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in either 100 pL mM9 (no NAC) or 100 pL mM9 + 5 mM NAC (with NAC). Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified, n = 4 biological replicates, LOD: limit of detection.

[0037] Figure 19. 15:24 protocol of the D-N-M conjugate against S. aureus 29213 using 1 mg / L mitomycin C equiv. concentration. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 (GC), mM9 + 5.4 mg / L dalbavancin + 1 mg / L mitomycin C (D+M), mM9 + 1 mg / L mitomycin C (M) or 1 mg / L D-N-M conjugate (mitomycin C equiv. concentration) (D-N-M) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in either 100 pL mM9 (no NAC) or 100 pL mM9 + 5 mM NAC (with NAC). Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified. mM9 was supplemented with 0.002 % tween 80 throughout this experiment due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces, n = 4 biological replicates, LOD: limit of detection.

[0038] Figure 20. Synthesis scheme for the synthesis of dalbavancin coupled to mitomycin C through the carboxylic acid (D-C-M).

[0039] Figure 21. HPLC chromatogram showing the purity of the D-C-M conjugate. Absorbance is measured at 210 nm.

[0040] Figure 22. HPLC chromatogram showing the D-C-M conjugate before and after cleavage with DTT. Absorbance is measured at 360 nm and the conjugate is compared with commercial MMC. Full release of mitomycin C (MMC) from the conjugate is observed.

[0041] Figure 23. 15:24 protocol of D-C-M conjugate against S. aureus 29213 with and without NAC. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 + 0.002% tween 80 (GC), mM9 + 0.002% tween 80 + 0.5, 1 or 2 mg / L mitomycin C (M) or 0.5, 1 or 2 mg / L D-C-M conjugate (mitomycin C equiv. concentrations) (D-C-M) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 + 0.002% tween 80 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in either 100 pL mM9 + 0.002% tween 80 (A) or 100 pL mM9 + 0.002% tween 80 + 5 mM NAC (B). Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified. mM9 was supplemented with 0.002 % tween 80 throughout this experiment due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces, n = 4 biological replicates, LOD: limit of detection. All bacterial counts below the limit of detection are coloured grey.

[0042] Figure 24. Synthesis scheme for the synthesis of dalbavancin conjugated to floxuridine through the carboxylic acid (D-C-F). Figure 25. HPLC chromatogram showing the purity of the D-C-F conjugate.

[0043] Absorbance is measured at 210 nm.

[0044] Figure 26. 15:24 protocol of D-C-F conjugate against S. aureus 29213 with NAC. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 + 0.002% tween 80 (GC), mM9 + 0.002% tween 80 + 0.125, 0.25, 0.5, 1 , 2 or 4 mg / L floxuridine (F), 0.925, 1 .85, 3.7, 7.4, 14.8, 29.5 mg / L dalbavancin with 0.125, 0.25, 0.5, 1 , 2, 4 mg / L floxuridine, respectively (D+F) or 0.125, 0.25, 0.5, 1 , 2 or 4 mg / L D-C-F conjugate (floxuridine equiv. concentrations) (D-C-F) yielding a bacterial turbidity of OD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 + 0.002% tween 80 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in 100 pL mM9 + 0.002% tween 80 + 5 mM NAC. Samples were incubated for 24 h before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified. mM9 was supplemented with 0.002 % tween 80 throughout this experiment due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces, n = 3 biological replicates. All bacterial counts below the limit of detection are coloured grey.

[0045] Figure 27. 15:24 protocol of D-C-F conjugate against S. aureus 29213 without NAC. S. aureus was grown overnight in TSB and subsequently washed and diluted in modified M9 (mM9) buffer to OD600 = 0.1 . The diluted cultures were inoculated into either mM9 + 0.002% tween 80 (GC), mM9 + 0.002% tween 80 + 4, 8 or 16 mg / L floxuridine (F) or 4, 8 or 16 mg / L DF conjugate (floxuridine equiv. concentrations) (D-C-F) yielding a bacterial turbidity of GD600 = 0.05. Samples were incubated for 15 min at 37 °C, 100 rpm. Subsequently, samples were washed by adding 900 pL mM9 + 0.002% tween 80 to the 100 pL samples, centrifuging for 5 min at 14000 rpm, removing 1000 pL supernatant and resuspending in 100 pL mM9 + 0.002% tween 80. Samples were incubated for 22 h 30 min before being serially diluted and spotted onto agar plates. Agar plates were incubated at 30 °C for 20 h and CFU was quantified. mM9 was supplemented with 0.002 % tween 80 throughout this experiment due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces, n = 3 biological replicates, LOD: limit of detection. Detailed description

[0046] Definitions

[0047] The term “bacterial persister cells” refers to a bacterial cell population that may escape the effects of antibiotics without undergoing genetic change; these cells are also known as dormant cells. Unlike resistant cells that grow in the presence of antibiotics, persister cells do not grow in the presence of antibiotics, but instead become antimicrobial tolerant by changing to a state of dormancy or quiescence. When treatment has stopped the state of dormancy can be reversed and the cells can reactivate and multiply.

[0048] The term “DNA alkylating agents” as used herein generally refers to an agent giving an alkyl group in the alkylation reaction in which a hydrogen atom of an organic compound is substituted with an alkyl group. DNA alkylating agents are compounds that generally work by adding an alkyl group to one or more guanine bases of the DNA molecule.

[0049] As used herein "antibacterial agent" refers to an agent that either kills or inhibits the growth of a microorganism, such as bacteria.

[0050] A wavy line drawn over a chemical bond in a formula, as used herein, indicates a variable bond or connection to another unspecified chemical entity. For example, indicates a variable bond from the chemical entity X to another unspecified chemical entity. Such notation is commonly used in chemistry.

[0051] The term “vancomycin” refers to a compound having the structure according to Formula (1 )

[0052] and / or derivates thereof and / or salts thereof.

[0053] The terms “mitomycin C” and “MMC” refer to a compound having the structure according to Formula (4) and / or derivates thereof and / or salts thereof.

[0054] The term “dalbavancin” refers to a compound having the structure according to Formula (15)

[0055] and / or derivates thereof and / or salts thereof.

[0056] The term “teicoplanin” refers to a compound having the structure according to Formula wherein R is: and / or derivates thereof and / or salts thereof.

[0057] The term “floxuridine” refers to a compound having the structure according to Formula (17) and / or derivates thereof and / or salts thereof.

[0058] The present disclosure relates to compounds comprising a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, one or more antibacterial agents, and a biodegradable linker linking the glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and the one or more antibacterial agents. Compounds of the present disclosure are further described in the section “Glycopeptide antibiotic compounds”.

[0059] The present disclosure further relates to compounds comprising a vancomycin or a mitomycin C, one or more antibacterial agents, and a biodegradable linker linking the vancomycin or the mitomycin C and the one or more antibacterial agents. Compounds of the present disclosure are further described in the sections “Vancomycin compounds” and “Mitomycin C compounds”.

[0060] In some embodiments, the compound binds to or is capable of binding to a bacterial cell. In some embodiments, the bacterial cell is a Gram-positive bacterial cell, such as Staphylococcus aureus. In some embodiments, the bacterial cell is a planktonic bacterial cell. In some embodiments, the bacterial cell is a bacterial cell capable of forming biofilm. In some embodiments, the bacterial cell is a bacterial persister cell. In some embodiments, the bacterial cell is selected from the group consisting of: Staphylococcus spp., Bacillus spp., Listeria spp., Streptococcus spp., Corynebacterium spp., and Enterococcus spp.. In some embodiments, the one or more antibacterial agents has a low off target effect compared to one or more antibacterial agents not linked to the vancomycin. In some embodiments, the off target effect is killing non- bacterial cells. In some embodiments, the non-bacterial cells are derived from cat, dog, rabbit, donkey, cow, fish, sheep, horse, goat, or pig. In some embodiments, the non- bacterial cells are derived from a human or a non-human primate.

[0061] In some embodiments, the present disclosure relates to a compound comprising: a. a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin; b. one or more antibacterial agents, such as one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0062] In some embodiments, the present disclosure relates to a compound comprising: a. a glycopeptide antibiotic selected from vancomycin, dalbavancin or teicoplanin; b. one or more antibacterial agents selected from one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0063] In some embodiments, the present disclosure relates to a compound comprising: a. a glycopeptide antibiotic selected from vancomycin, dalbavancin or teicoplanin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents. In some embodiments, the glycopeptide antibiotic is vancomycin. In some embodiments, the glycopeptide antibiotic is dalbavancin. In some embodiments, the glycopeptide antibiotic is teicoplanin. In some embodiments, the glycopeptide antibiotic is oritavancin.

[0064] In some embodiments, the present disclosure relates to a compound comprising: a. a vancomycin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0065] In some embodiments, the present disclosure relates to a compound comprising: a. a dalbavancin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0066] In some embodiments, the present disclosure relates to a compound comprising: a. a teicoplanin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0067] In some embodiments, the present disclosure relates to a compound comprising: a. a dalbavancin; b. a mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0068] In some embodiments, the present disclosure relates to a compound comprising: a. a teicoplanin; b. a mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker. In some embodiments, the present disclosure relates to a compound comprising: a. a dalbavancin; b. a floxuridine; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0069] In some embodiments, the “dalbavancin” has the structure according to Formula (15)

[0070] In some embodiments, the teicoplanin has the structure according to Formula (16)

[0071]

[0072] In some embodiments, the compound comprises or consists of a structure of formula

[0073] or a pharmaceutically acceptable salt thereof.

[0074] In some embodiments, the compound comprises or consists of a structure of formula

[0075] R= wherein the group “TEI” represents where teicoplanin is connected to the R group, or a pharmaceutically acceptable salt thereof.

[0076] In some embodiments, the compound comprises or consists of a structure of formula

[0077] Vancomycin compounds

[0078] In one embodiment, the present disclosure relates to a compound comprising: a. a vancomycin; b. one or more antibacterial agents; and c. a biodegradable linker linking the vancomycin and the one or more antibacterial agents.

[0079] In some embodiments, the vancomycin has the structure of formula (1 )

[0080]

[0081] In some embodiments, the compound comprises or consists of a structure of formula (2) wherein one or more of Ri, R2 and R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0082] In some embodiments, R1 comprises or consists of the biodegradable linker and the one or more antibacterial agents. In some embodiments, R2 comprises or consists of the biodegradable linker and the one or more antibacterial agents. In some embodiments, R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0083] In some embodiments, the compound comprises or consists of a structure of formula (3) wherein R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents. In some embodiments, R1 comprises or consists of a structure of formula (8)

[0084] (8).

[0085] In some embodiments, R2 comprises or consists of a structure of formula (9)

[0086]

[0087] In some embodiments, R3 comprises or consists of a structure of formula (9)

[0088] In some embodiments, the compound comprises or consists of a structure of formula (10) or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments, the compound comprises or consists of a structure of formula (1 1 )

[0090] or a pharmaceutically acceptable salt thereof.

[0091] In some embodiments, the compound comprises or consists of a structure of formula (12) or a pharmaceutically acceptable salt thereof.

[0092] In some embodiments, the compound comprises or consists of a structure of formula (13) or a pharmaceutically acceptable salt thereof.

[0093] In one embodiment, the present disclosure relates to a compound comprising: a. a vancomycin; b. one or more mitomycin C; and c. a biodegradable linker linking the vancomycin and the one or more mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0094] In one embodiment, the present disclosure relates to a compound comprising: a. a vancomycin; b. a mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0095] In some embodiments, the present disclosure relates to a compound comprising: a. a vancomycin; b. one or more fluorophores; and c. a biodegradable linker linking the vancomycin and the one or more fluorophores, optionally wherein the biodegradable linker is a disulfide linker.

[0096] In some embodiments, the present disclosure relates to a method of manufacturing the compound of the present disclosure, the method comprising the steps of: a. providing a vancomycin and one or more antibacterial agents; and b. conjugating the vancomycin and the one or more antibacterial agents with a biodegradable linker, optionally wherein the biodegradable linker is a disulfide linker.

[0097] Mitomycin C compounds

[0098] The present disclosure further concerns compounds comprising mitomycin C linked to one or more antibacterial agents. In some embodiments, such compounds may be homodimers of mitomycin linked by a biodegradable linker.

[0099] In another embodiments, the present disclosure relates to a compound comprising: a. a mitomycin C; b. one or more antibacterial agents; and c. a biodegradable linker linking the mitomycin C and the one or more antibacterial agents.

[0100] In some embodiments, the mitomycin C has the structure of formula (4)

[0101] In some embodiments, the mitomycin C has the structure of formula (5) wherein R comprises or consists of the biodegradable linker and the one or more antibacterial agents. In some embodiments, the compound comprises or consists of a structure of formula (14) or a pharmaceutically acceptable salt thereof.

[0102] In one embodiment, the present disclosure relates to a compound comprising: a. a first mitomycin C; b. one or more further mitomycin C; and c. a biodegradable linker linking the a first mitomycin C and the one or more further mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0103] In one embodiment, the present disclosure relates to a compound comprising: a. a first mitomycin C; b. a further mitomycin C; and c. a biodegradable linker linking the first mitomycin C and the further mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0104] In some embodiments, the present disclosure relates to a method of manufacturing the compound of the present disclosure, the method comprising the steps of: a. providing a mitomycin C and one or more antibacterial agents; and b. conjugating the mitomycin C and the one or more antibacterial agents with a biodegradable linker.

[0105] Antibacterial agents

[0106] The antibacterial agents described in this section can be used for all compounds of the present disclosure, i.e., compounds described in the sections “Glycopeptide antibiotic compounds”, “Vancomycin compounds” and “Mitomycin C compounds”. In some embodiments, the one or more antibacterial agents is one or more antineoplastic drugs, such as one or more antineoplastic antibiotics. In some embodiments, the one or more antineoplastic drugs is floxuridine. In some embodiments, the one or more antineoplastic drugs is mitomycin C. In some embodiments, the one or more antibacterial agents is one or more DNA alkylating agents. In some embodiments, the one or more DNA alkylating agents is an Aminoquinone, such as streptonigrins or mitomycin C. In some embodiments, the one or more DNA alkylating agents is mitomycin C.

[0107] In some embodiments, the mitomycin C has the structure of formula (4)

[0108] In some embodiments, the mitomycin C has the structure of formula (5) wherein R comprises or consists of the biodegradable linker and the vancomycin.

[0109] In some embodiments, the floxuridine has the structure according to Formula (17) In some embodiments, the one or more DNA alkylating agents is selected from the group consisting of: Duocarmycins and Pyrrolobenzodiazepines. In some embodiments, the one or more Pyrrolobenzodiazepines is selected from the group consisting of: SG3199, SJG-136, SG2057, Tomaymycin DM, Hemiasterlin, Aniline- MPB-amino-C3-PBD and Py-MPB-amino-C3-PBD.

[0110] Biodegradable linker

[0111] The biodegradable linkers and release-agents described in this section can be used for all compounds of the present disclosure, i.e., compounds described in the sections “Glycopeptide antibiotic compounds”, “Vancomycin compounds” and “Mitomycin C compounds”.

[0112] In some embodiments, the biodegradable linker can release the one or more antibacterial agents from the vancomycin or the mitomycin C upon contact with a thiol group. In some embodiments, the biodegradable linker can release the one or more antibacterial agents from the vancomycin or the mitomycin C upon contact with a bacterial cell. In some embodiments, the biodegradable linker can release the one or more antibacterial agents from the vancomycin or the mitomycin C without contact with a release-agent.

[0113] In some embodiments, the biodegradable linker is a disulfide linker.

[0114] In some embodiments, the disulfide linker comprises or consists of the structure of formula (6)

[0115] (6).

[0116] In some embodiments, the disulfide linker comprises or consists of the structure of formula (7)

[0117] (7). In some embodiments, the biodegradable linker can release the one or more antibacterial agents from the vancomycin or the mitomycin C upon contact with a release-agent. In some embodiments, the biodegradable linker can release the one or more antibacterial agents from the vancomycin or the mitomycin C without contact with a release-agent. In some embodiments, the release-agent is N-acetyl cysteine (NAC). In some embodiments, the release-agent is dithiothreitol (DTT).

[0118] Pharmaceutical compositions and uses

[0119] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising the compound according to the present disclosure or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0120] Such pharmaceutically acceptable excipients include any commonly used excipients in pharmaceutics and should be selected on the basis of compatibility and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like.

[0121] "Pharmaceutically compatible carrier materials" may comprise, e.g., acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.

[0122] In some embodiments, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use as a medicament.

[0123] In some embodiments, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use in the treatment of a bacterial infection. In some embodiments, the bacterial infection is an implant associated bacterial infection. In some embodiments, the present disclosure relates to the compound of the present disclosure or the pharmaceutical composition of the present disclosure for use in the treatment of a bacterial biofilm.

[0124] In some embodiments, the present disclosure relates to use of the compound of the present disclosure or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treatment of a bacterial infection. In some embodiments, the bacterial infection is an implant associated bacterial infection.

[0125] In some embodiments, the present disclosure relates to a method for treatment of bacterial infection comprising administration of a therapeutically effective amount of the compound of the present disclosure or the pharmaceutical composition of the present disclosure to an individual in need thereof. In some embodiments, the bacterial infection is an implant associated bacterial infection.

[0126] In some embodiments, the present disclosure relates to a kit of parts comprising: a. the compound of the present disclosure; and b. instructions for use.

[0127] Examples

[0128] Examples - General: Instruments and materials

[0129] Abbreviation list

[0130] HOBt: Hydroxybenzotriazole

[0131] HBTLI: Hexafluorophosphate Benzotriazole Tetramethyl Uranium

[0132] EDC: 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0133] DIPEA: N,N-Diisopropylethylamine"

[0134] MQ: Milli Q water purified by a Milli Q direct 8 system (Millipore).

[0135] DMF: Dimethylformamide

[0136] DMSO: Dimethylsulfoxide

[0137] Et20: Diethyl ether

[0138] MeOH: Methanol

[0139] EtOAc: Ethyl acetate

[0140] TEA: Triethylamine

[0141] DTT: Dithiotreitol

[0142] DTBA: Dithiobutylamine DCM: Dichloromethane

[0143] DCC: dicyclohexyl carbodiimide

[0144] NHS: N-Hydroxysuccinimide

[0145] MMC / M: Mitomycin C

[0146] Van / V: Vancomycin

[0147] GSH / G: Glutathione

[0148] V-C-M: Mitomycin C conjugated to the carboxylic acid of vancomycin using a self- immolative disulfide linker.

[0149] V-Np-M: Mitomycin C conjugated to the primary amine of vancomycin using a self- immolative disulfide linker.

[0150] V-Np-M: Mitomycin C conjugated to the secondary amine of vancomycin using a self- immolative disulfide linker.

[0151] M-M: A mitomycin C dimer connected using a self-immolative disulfide linker.

[0152] G-M: Mitomycin C connected to glutathione using a self-immolative disulfide linker.

[0153] Instruments and materials

[0154] All reaction were carried out under inert atmosphere (argon or nitrogen) in flame-dried glass equipment unless otherwise stated or water is used as a solvent. Reaction completion was monitored by analytical thin layer chromatography (TLC) or by analytical HPLC. TLC was performed using pre-coated aluminum-backed plates (Merck Kieselgel 60 F254) and visualized by staining with potassium permanganate, ninhydrin, Ellman’s reagent or exposure to ultraviolet light.

[0155] NMR spectra were acquired on a Broker AVANCE III HD spectrometer running at 400 MHz for 1 H-NMR and 100 MHz. for 13C-NMR. Chemical shifts (5) are reported in ppm relative to residual solvent signals (CDCI3, 7.26 ppm for 1 H NMR and 77.16 for 13C NMR), (deuterated DMSO, 2.50 ppm for 1 H NMR), (CD3OD, 3.31 ppm for 1 H NMR and 49.00 for 13C NMR). The following abbreviations are used to indicate the multiplicity in NMR spectra: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet. 13C NMR spectra were acquired on a broad band decoupled mode. The Mass spectrum was recorded on a Broker Maxis Impact-TOF-MS with electrospray ionization (ESI+).

[0156] Unless otherwise noted, analytical grade solvents and commercially available reagents were used without further purification. For flash column chromatography silica gel (SiO2 60, 230-400 mesh, Sigma-Aldrich) was used. Automated flash purification was performed on an Advion Interchim puriFlash XS 530Plus system with a UV detector. For especially water sensitive reactions the solvents used were purified by a Solvent Purification System (SPS). Milli Q water (MQ) was purified by a Milli Q direct 8 system (Millipore). An ice bath was used for cooling systems to 0°C.

[0157] Analytical HPLC was performed on an Agilent 1260 Inifinity II analytical HPLC system with the solvents and additives stated for the specific compounds and a flow of 0.4 mL / min. Preparative HPLC was performed on an Agilent 1260 Infinity II preparative HPLC system with the solvents and additives stated for the specific compounds and a flow of 10 mL / min. Analytical HPLC chromatograms, used to evaluate compound purity, were recorded at 210 nm to show all potential impurities, but have been zoomed in to not show the injection peaks.

[0158] Lyophilization was carried out on a Christ freeze-drier beta 2-8 LSCpIus. UV measurements were carried out on a NanoDropTM 2000c Spectrophotometer. For LC- MS the LC system consisted of an RS Pump, RS autosamples, RS column compartment and Diode Array detector all from the UltiMate 3000 series from Thermo Scientific, and the MS system was a Broker Maxis Impact TOF mass spectrometer.

[0159] Bacterial culture

[0160] Bacterial strain. The Staphylococcus aureus (S. aureus) strain used throughout this project is S. aureus ATCC29213. The stock was stored in 15 % glycerol at -80 °C. Single colonies were grown on brain-heart-infusion (BHI) agar plates every month and stored at 4-8 °C.

[0161] Media and buffers. The growth media used in the experiments was Brain Heart Infusion Broth (BHI) or modified M9 minimal media (mM9 media) consisting of modified M9 buffer (mM9 buffer) supplemented with 1 % glucose and 1 % casamino acids. Modified M9 buffer (mM9) containing M9 minimal salts 1 X, 2 mM MgSC , 0.1 mM CaCl2, 1 mM Thiamine-HCI, 0.05 mM nicotinamide and 1 mL / L trace metal solution TMS3. The pH of mM9 was adjusted to 7.4. After autoclavation components were sterile filtered and added to the buffer. Assays involving dithiothreitol (DTT) or N-acetyl cysteine (NAC) contained 5 mM.

[0162] Overnight cultures. All S. aureus overnight cultures were prepared by adding single colonies to BHI (20 mL) in Erlenmeyer flasks. They were incubated at 37 °C, 180 rpm for 16-20 h. MIC and MBC assays for mitomycin C and vancomycin (Table 3) in nutrient-rich media: Serial dilutions of mitomycin C and vancomycin in BHI were prepared in a 96- well plate with a volume of 180 pL per well. Each well was inoculated with 20 pL of overnight grown culture of S. aureus in BHI (OD600 = 0.05) and incubated at 37°C, 50 rpm for 18 hours. Samples showing no apparent growth were spotted (10 pL) onto BHI agar plates and incubated at 37°C for 24 hours. The MIC was defined as the lowest concentration that inhibited visible bacterial growth after 18 hours of incubation in the microplate, while the MBC was determined as the lowest concentration that resulted in no bacterial growth on the agar plates.

[0163] MIC and MBC assays for mitomycin C and vancomycin (Table 3) in nutrient-free buffer: Serial dilutions of mitomycin C and vancomycin in modified M9 (mM9) buffer were prepared in a 96-well plate with a volume of 180 pL per well. S. aureus was grown overnight in BHI, then washed and diluted in mM9 buffer to an OD600 of 0.05. Twenty microliters of the diluted culture were inoculated into each well containing the antibiotic serial dilutions and incubated at 37°C, 50 rpm for 18 hours. Five microliters of each sample were then spotted onto BHI agar plates and incubated at 37°C for 24 hours. The MBC was determined as the lowest concentration that resulted in no bacterial growth on the agar plates.

[0164] MBC assays for dalbavancin and floxuridine (table 4) in nutrient-free buffer.

[0165] Serial dilutions of floxuridine, dalbavancin and a combination of equimolar floxuridine and dalbavancin in modified M9 (mM9) buffer were prepared in a 96-well plate with a total volume of 180 pL per well. S. aureus was grown overnight in BHI, then washed and diluted in mM9 buffer to an OD600 of 0.05. Twenty microliters of the diluted culture were inoculated into each well containing the antibiotic serial dilutions and incubated at 37°C, 50 rpm for 18 hours. The bacteria were diluted, washed and resuspended in mM9. Ten microliters of each sample were then spotted onto BHI agar plates and incubated at 37°C for 24 hours. The MBC was determined as the lowest concentration that resulted in no bacterial growth on the agar plates. mM9 was supplemented with 0.002 % tween 80 during treatment with floxuridine and dalbavancin due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces. MIC and MBC assay for floxuridine in nutrient-rich minimal media (table 4). Serial dilutions of floxuridine in modified M9 (mM9) media (containing MgSC , CaCh, nicotinamide, thiamine and glucose) were prepared in a 96-well plate with a total volume of 180 pL per well. S. aureus was grown overnight in BHI, then washed and diluted in mM9 media to an OD600 of 0.05. Twenty microliters of the diluted culture were inoculated into each well containing the antibiotic serial dilutions and incubated at 37°C, 50 rpm for 18 hours. OD600 measurements were used to determine MIC. The bacteria were then diluted, washed and resuspended in mM9. Ten microliters of each sample were then spotted onto BHI agar plates and incubated at 37°C for 24 hours. The MBC was determined as the lowest concentration that resulted in no bacterial growth on the agar plates.

[0166] MIC and MBC assays for gemcitabine, floxuridine and dalbavancin (Table 4) in nutrient-rich BHI media: Serial dilutions of gemcitabine, floxuridine and dalbavancin in BHI were prepared in a 96-well plate with a volume of 180 pL per well. Each well was inoculated with 20 pL of overnight grown culture of S. aureus in BHI (OD600 = 0.05) and incubated at 37°C, 50 rpm for 18 hours. Samples showing no apparent growth were spotted (10 pL) onto BHI agar plates and incubated at 37°C for 24 hours. The MIC was defined as the lowest concentration that inhibited visible bacterial growth after 18 hours of incubation in the microplate, while the MBC was determined as the lowest concentration that resulted in no bacterial growth on the agar plates. BHI was supplemented with 0.002 % tween 80 during treatment with dalbavancin due to the recommendation from EUCAST for using dalbavancin to prevent adsorption of the molecule to plastic surfaces.

[0167] Example 1 conjugate via the carboxylate.

[0168] Synthesis of 2-(Dyridin-2-yldisulfaneyl)ethan-1 -ol (2-(pyridin-2-yldisulfaneyl)ethan-1 -ol) 2,2-ditiodipyridine (590 mg, 2.68 mmol, 2 equiv.) was dissolved in a mixture of AcOH (0.5 mL) and MeOH (2 mL). In a separate flask 3-mercaptoethanol (89.3 pL, 1 .28 mmol, 1 equiv.) was dissolved in MeOH (0.3 mL) and added to the stirring solution containing 2,2-ditiodipyridine. The reaction mixture was left stirring at r.t. for 1 hour and 40 minutes. The crude mixture was concentrated and purified with flash column chromatography (1 .5:8.5 EtOAc:pentane to 3:7 EtOAC:pentane) yielding the product as a bone white solid (164.03 mg, 0.876 mmol, 68 %).

[0169] 1H NMR (400 MHz, Chloroform-d) 5 8.52 (d, J = 5.0 Hz, 1 H), 7.59 (td, J = 7.7, 1.8 Hz, 1 H), 7.40 (d, J = 8.1 Hz, 1 H), 7.20 - 7.09 (m, 1 H), 3.81 (t, J = 5.0 Hz, 2H), 2.96 (t, J = 5.3 Hz, 2H) (Figure 2E).13C NMR (100 MHz, Chloroform-d) 5 159.27, 150.00, 137.00, 122.12, 121.68, 58.40, 42.92 (Figure 2F). HRMS (ESI+) m / z calculated (calcd.) for C7H9NOS2 + H+: 188.0911 , found: 188.0209.

[0170] Synthesis of 2-(pyridin-2-yldisulfaneyl)ethyl 2-(4-nitrophenyl)acetate 2-(pyridin-2-yldisulfaneyl)ethan-1 -ol (25 mg, 0.133 mmol, 1 equiv.) was dissolved in dry CH2CI2 (0.4 ml) followed by addition of TEA (40 zL, 0,287 mmol, 2.1 equiv.). In a separate flask p-nitrophenyl chloroformate (38.96 mg, 0.193 mmol, 1 .5 equiv.) was dissolved in dry CH2CI2 (0.2 mL) followed by dropwise addition of the solution to the reaction mixture. The reaction mixture was stirred for 28 hours, diluted with CH2CI2 (4 mL), quenched with NH4CI (2 mL), washed with brine three times, dried over sodium sulphate and concentrated. TLC confirmed product formation and the crude was used directly without further purification.

[0171] Synthesis of compound 1 (compound 1 )

[0172] A crude mixture of 2-(pyridin-2-yldisulfaneyl)ethyl 2-(4-nitrophenyl)acetate (47 mg, 0.133 mmol, 1 equiv.) was dissolved in dry DMF (1 ml) under N2. In a separate flask MMC (50.24 mg, 0.2 mmol, 1 .1 equiv.) and TEA (40 zL, 0.287 mmol, 1 equiv.) were dissolved in dry DMF (1 mL) followed by dropwise addition to the stirring reaction mixture. HOBt (48.74 mg, 0.361 mmol, 2.7 equiv.) was subsequently added and the reaction was left stirring in the dark for 24 hours. The crude was concentrated and purified with flash column chromatography (1 :1 EtOAc:pentane to 4:1 EtOAc:pentane) yielding the product as a purple solid (39.5 mg, 0,072 mmol, 50 %).

[0173] 1H NMR (400 MHz, Chloroform-d) 5 8.44 (d, J = 4.9 Hz, 1 H), 7.66 - 7.55 (m, 2H), 7.11 - 7.03 (m, 1 H), 4.93 (s, 2H), 4.84 (dd, J = 10.8, 4.6 Hz, 1 H), 4.48 - 4.18 (m, 4H), 3.65 (dd, J = 11 .1 , 4.6 Hz, 1 H), 3.53 - 3.40 (m, 2H), 3.29 (dd, J = 4.6, 1 .8 Hz, 1 H), 3.17 (s, 3H), 3.10 - 2.95 (m, 2H), 1.74 (s, 3H), 1.28 - 1.19 (m, 1 H) (Figure 2G).13C NMR (101 MHz, Chloroform-d) 5 178.43, 176.07, 160.66, 159.55, 156.55, 154.44, 149.86, 147.26, 137.21 , 121.11 , 120.22, 110.55, 105.58, 105.24, 64.71 , 62.23, 49.86, 48.77, 43.65, 42.05, 40.11 , 36.96, 8.01 (Figure 2H). HRMS (ESI+) m / z calcd. for C23H25N5O7S2

[0174] + H+: 548.1268, found: 548.1297 m / z calcd. for C23H25N5O7S2 + Na+: 570.1087, found: 570.1103.

[0175] Synthesis of compound 2 (Compound 2)

[0176] Dithiodipyridine (856 mg, 3.88 mmol, 1.5 equiv.) was dissolved in a dry MeOH (10 ml). Cysteamine (200 mg, 2.59 mmol, 1 equiv.) was dissolved in MeOH (5 ml) and added dropwise to the stirring solution followed by addition of TEA (0.54 mL, 3.87 mmol, 1 .5 equiv.). The mixture was left to stir overnight and purified with flash column chromatography (column packed in 19:1 DCM:MeOH with 1 % Ammonium hydroxide (25% solution in water) and run in 9:1 DCM:MeOH with 1 % ammonium hydroxide to yield 2-(pyridin-2-yldisulfaneyl)ethan-1 -amine as a crude mixture.

[0177] Vancomycin (121 .2 mg, 81 .58 pmol, 1 .01 equiv.) was dissolved in DMSO (1 .5 mL) followed by addition of DIPEA (0.20 mL, 1 .2 mmol, 15 equiv.) and TEA (30 pL, 0.22 mmol, 2.7 equiv.). The crude 2-(pyridin-2-yldisulfaneyl)ethan-1 -amine mixture (121.5 mg, 652.2 pmol, 8.07 equiv.) was dissolved in dry DMF (0.5 mL) and DMSO (0.5 mL) and added to the vancomycin mixture. The reaction mixture was cooled to 0°C, HBTU (15.4 mg, 243 mmol, 3.0 equiv.) was added and the reaction was left stirring overnight slowly heating to r.t. The reaction turned yellow. The product was precipitated by addition of cool Et20 and cooling in the freezer overnight. The solid was then washed with Et20 (3x) and dissolved in DMSO (4 mL). The crude reaction mixture was purified by preparative RP-HPLC equipped with a ZORBAX Eclipse CDB-C18 column and using a solvent system consisting of A: MQ with 0.1 % formic acid and B: acetonitrile with 0.1 % formic acid.

[0178] Method: 5 % B for 4 min, gradient 5% B to 22.5 % B over 10 min, gradient 22.5 % B to 100 % B over 3 min, followed by washing and equilibrating. The product eluted at 12 min and 14.56 mg (8.80 pmol, 10.9 % yield) was isolated.

[0179] 1H NMR (400 MHz, MeOD) 5 8.40-8.52, (broad s, 2H), 8.38z (d, 1 H), 7.77-7.85 (m, 2H), 7.67 (s, 1 H), 7.57-7.64 (m, 2H), 7.18-7.26 (mf, 2H), 7.05 (d, J=1 .5 Hz, 1 H), 6.97-6.88 (m, 1 H), 6.84-6.76 (m, 1 H), 6.43 (d, J=2.3 Hz, 1 H), 6.38 (d, J=2.3 Hz, 1 H), 5.72-5.89 (m, 1 H), 5.45-5.51 (m, 1 H), 5.40-5.45 (m, 1 H), 5.34-5.39 (m, 2H), 5.28-5.33 (m, 1 H), 4.68 (s, 1 H), 4.62-4.67 (m, 1 H), 4.19 (s, 1 H), 3.80-3.91 (m, 1 H), 3.71 -3.80 (m, 2H), 3.60-4.68 (m, 2H), 3.51 -3.59 (m, 2H), 3.00 (t, J=6.5 Hz, 2H), 2.78-2.87 (m, 1 H), 2.63 (s, 3H), 2.04 (s, 1 H), 1.89-1.98 (m, 1 H), 1.68-1.81 (m, 2H), 1.55-1.64 (m, 1 H), 1.48 (s, 3H), 1.21 (d, J=5.6, 3H), 0.95 (dd, J=1 1 .6, 5.8 Hz, 6H) (Figure 2I). HRMS(ESI): calcd. for C73H83CI2N11O23S2+2H: m / z 808.7314 found 808.7320. Treatment with 26 equiv. of DTT in MQ followed by MS analysis yielded the expected cleavage product: calcd. for C68H80CI2N10O23S+2H: m / z 754.2321 found 754.2316.

[0180] Evaluation of purity: Compound 2

[0181] (Van-C-Thiopyridine) was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate. Method: 5 % B for 6 minutes, gradient 5% B to 20 % B over 10 min, gradient 20 % B to 95 % B over 10 minutes followed by washing and equilibrating for a total run time of 48 min. The product was detected at 210 nm and eluted after 24.2 min (Figure 2J).

[0182] Synthesis of V-C-M conjugate

[0183] Solutions of compound 2 in MeOH (4 mM), DTBA in MQ (40 mM) and compound 1 in DMF (20 mM) were prepared. From these solutions 250 uL of the compound 2 conjugate solution (1 .00 pmol, 1 .62 mg, 1 equiv.) was mixed with 250 uL of PBS buffer (50 mM, pH = 7.8) and 25 uL of the DTBA solution (1 .00 pmol, 0.174 mg, 1 equiv.) and allowed to react while stirring for 30 min. Afterwards 100 uL of the compound 1 solution (2.00 pmol, 1 .1 mg, 2 equiv.) was added and the mixture was stirred for an additional 4 hours in the dark. The final product mixture was purified by preparative RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0184] Method: 7% B for 2 min, gradient 7% B to 22 % B over 3 min, gradient 22% B to 30% B over 10 min, gradient 30% B to 50 % B over 2.5 min followed by washing and equilibrating. The product eluted at 10.5 min and 0.68 mg (0.35 pmol, 35% yield) was isolated.

[0185] HRMS(ESI): calcd. for C86H100CI2N14O30S2+2H: m / z 972.2847 found 972.2850 calcd. for CseHiooCLNuOst^+H+Na: 983.2757 found 983.2757

[0186] Evaluation of purity: V-C-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0187] Method: 5 % B for 6 minutes, gradient 5% B to 20% B over 10 min, gradient 20% B to 95% B over 10 minutes followed by washing and equilibrating for a total run time of 48 min. The product was detected at 210 nm and eluted after 24.5 min (Figure 2K).

[0188] Release studies of V-C-M:

[0189] V-C-M in DMSO was treated with 10 equiv. of DTT for 30 minutes and analyzed by an analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0190] Method: 5 % B for 6 minutes, gradient 5% B to 20 % B over 10 min, gradient 20 % B to 95 % B over 10 minutes followed by washing and equilibrating for a total run time of 48 min.

[0191] The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated V-C-M conjugate, commercial MMC and compound 2 (here named V-C-TP), the last of which had also been treated with 10 equiv. of DTT.

[0192] With detection at 210 nm and comparison with V-C-TP the release of the vancomycin adduct was observed (Figure 2B).

[0193] With detection at 360 nm and comparison with MMC release of MMC was observed (Figure 2C). Unspecific drug release from V-C-M

[0194] V-C-M in MQ at a concentration of 32 mg / L (17 pM) was diluted 1 :3 with either PBS (50 mM, pH = 7.4), mM9 buffer, RPM1640 cell media or NAC (5 mM) in MQ to a final concentration of Van-C-MMC of 8.0 mg / L. The resulting mixtures were shaken at 800 rpm and 37 degrees for 24 h. 50 pL were taken out of each sample at timepoints 15 min, 1 h and 24 h, frozen and then analyzed by analytical HPLC. For each sample the HPLC peak corresponding to released MMC was integrated at 360 nm and the integral was compared to V-C-M at 8.0 mg / mL without incubation.

[0195] The results are based on three independent experiments (Figure 2D).

[0196] Results

[0197] For bioconjugation via the vancomycin carboxylate functionality, the thiol-containing derivatives of vancomycin and mitomycin were synthesized and then finally coupled the two via th iol-disulf ide exchange (Figure 2A). In brief, mercaptoethanol was first reacted with dithiodipyridine, thereafter with nitrophenyl chloroformate, and finally with mitomycin to obtain compound 1. In turn, a reaction between cysteamine and dithiodipyridine afforded a functionalized linker, which was immediately reacted with vancomycin, specifically via the carboxylic acid group, to afford compound 2. The latter was treated with a stoichiometric amount of dithiobutylamine to produce the derivative of vancomycin with an exposed thiol which was reacted in situ (without purification) with compound 1 , to afford the desired conjugate, herein termed V-C-M (whereby C indicates the conjugation via the vancomycin carboxylic group).

[0198] Example 2: conjugate via the amine groups.

[0199] Synthesis of 3-(Dyridin-2-yldisulfaneyl)DroDanoic acid -(pyridin-2-yldisulfaneyl)propanoic acid) Dithiodipyridine (2.10 g, 9.54 mmol, 1 .5 equiv.) was dissolved in ethanol (20 mL) followed by addition of glacial acetic acid (350 uL, 1 v / v % of final volume) while stirring. 3-mercaptopropanoic acid (554 uL, 6.36 mmol, 1 equiv.) was dissolved in EtOH (15 ml) and added dropwise to the stirring solution over 5 minutes. The mixture was left to stir overnight and purified with flash column chromatography (EtOAc / Hexane, 2:3 with 0.1 % acetic acid added) to yield an off-white solid after freeze drying (1 .06 g, 4.91 mmol, 77% yield). HRMS(ESI): calcd. for C8H9NO2S2+H: m / z 216.0148 found 216.0145.

[0200] 1H NMR (400 MHz, CDCI3) 5 8.48 (ddd, J = 5.0, 1.8, 0.9 Hz, 1 H, thiopyridyl), 7.67 (td, J = 8.1 , 7.2, 1 .8 Hz, 1 H, thiopyridyl), 7.61 (dt, J = 8.1 , 1.1 Hz, 1 H, thiopyridyl), 7.17 (ddd, J = 7.2, 5.0, 1 .3 Hz, 1 H, thiopyridyl), 3.08 (t, J = 6.7 Hz, 2H, S-CH2-CH2), 2.80 (t, J = 6.7 Hz, 2H, CH2-CH2-COOH) (Figure 3D).13C NMR (101 MHz, CDCI3) 5 175.23 (COOH), 159.12 (thiopyridyl), 149.33 (thiopyridyl), 137.59 (thiopyridyl), 121.37 (thiopyridyl), 120.84 (thiopyridyl), 34.26 (CH2), 34.18 (CH2) (Figure 3E).

[0201] Synthesis of compound 3 (Compound 3)

[0202] 3-(pyridin-2-yldisulfaneyl)propanoic acid (1 .05 g, 4.86 mmol, 1 equiv.) was dissolved in dry DCM (20 mL). DCC (1 .51 g, 7.32 mmol,1 .5 equiv.) and NHS (839 mg, 7.29 mmol, 1 .5 equiv.) were added, and the reaction mixture was stirred at room temperature for 2 h. The product mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude product as a clear oil. The product was then purified by silica gel column chromatography (EtOAc / Pentane, 1 :1 v / v) to give the product as an off- white oil (1 .09 g, 3.48 mmol, 72 % yield).

[0203] HRMS(ESI): calcd. for C12H12N2O4S2+H : m / z 313.031 1 found 313.0333. calcd. for Ci2Hi3N2O4S2-i-Na: 335.0131 found 335.0136.

[0204] 1H NMR (400 MHz, CDCI3) 5 8.50 (dt, J= 4.8, 1 .4 Hz, 1 H, thiopyridyl), 7.73 - 7.61 (m, 2H, thiopyridyl), 7.12 (td, J= 5.2, 3.1 Hz, 1 H, thiopyridyl), 3.17 - 3.03 (m, 4H, S-CH2- CH2-COOH), 2.84 (s, 4H, NHS) (Figure 3F).

[0205] 13C NMR (101 MHz, CDCh) 5 167.85 (NHS C=O), 165.97 (COO), 158.12 (thiopyridyl), 148.75 (thiopyridyl), 136.57 (thiopyridyl), 136.34 (thiopyridyl), 120.05 (thiopyridyl), 1 19.02 (thiopyridyl), 31 .83 (CH2), 29.96 (CH2), 24.56 (NHS CH2) (Figure 3G). Synthesis of Van-Ns-Thiopyridine (Compound 3)

[0206] Compound 3 (300 mg, 864 pmol, 1 equiv.) was dissolved in DMSO (3 mL) and added to HBTLI (327 mg, 864 pmol, 1 equiv) dissolved in DMSO (8 mL). The contents of this flask was added dropwise to another flask containing vancomycin (1 .54 g, 1 .04 mmol, 1 .2 equiv.) dissolved in DMSO (10 mL) and pyridine (342 pL, 4.32 mmol, 5 equiv.). The mixture was allowed to stir overnight followed by purification by preparative RP-HPLC equipped with a Zorbax Eclipse XDB-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1% formic acid. Method: 5% B for 4 min, gradient 5% B to 22.5% B over 10 min, gradient 22.5% B to 40 % B over 4 min, followed by washing and equilibrating. Both products with attachment at the secondary and primary amine of vancomycin were isolated in this way. The secondary amine product, Van-Ns-Thiopyridine, was detected at 210 nm and eluted after 17.0 min. Impurities were still present, but the product was used for the next reaction without further purification. 104 mg of crude product were isolated. HRMS(ESI): calcd. for C74H82N10O25S2CI2+2H: m / z 823.2208 found 823.2213 No fragmentation peak at m / z 1305 observed confirming attachment at secondary amine Synthesis of V-Ns-M (Compound V-Ns-M)

[0207] Crude Van-Ns-thiopyridine mixture (2.1 mg, 1 .3 nmol, 1 equiv.) was dissolved in DMF (328 pL) and diluted with PBS buffer (50 mM, pH = 7.8, 328 pL). Dithiobutylamine (0.3 mg, 1 .7 pmol, 1 .3 equiv.) was dissolved in PBS (50 mM, pH = 7.8, 39 pL) and added to first solution. The reaction mixture was allowed to react while shaking for 40 min followed by addition of compound 1 (1 .4 mg, 2.6 pmol, 2 equiv.), synthesized as described in Example 1 , in DMF (131 pL). The solution was shaken for an additional 3.5 hours in the dark and then purified by preparative RP-HPLC equipped with ZORBAX Eclipse XDB-C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0208] Method: 7% B for 4 min, gradient 7% B to 40% B over 20 min, gradient 40% B to 100% B over 2 min followed by washing and equilibrating. The V-Ns-M eluted at 19.4 min and the total mass was determined by UV measurement to 0.2 mg (0.1 nmol, <1% yield over 2 steps).

[0209] HRMS(ESI): calcd. for C87H99N13O32S2CI2+2H: m / z 987.2766 found 987.2756 calcd. for C87H99Ni3O32S2Cl2+H+Na: m / z 998.2668 found 998.2662.

[0210] No fragmentation ion with m / z 1305 observed confirming secondary amine attachment (see S2). Evaluation of purity: V-Ns-M in DMSO was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0211] Method: 7% B for 5 minutes, gradient 7% B to 40% B over 10 min, gradient 40% B to 95% B over 10 minutes, gradient 95% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 39 min. The product was detected at 210 nm and eluted after 20.3 min (Figure 3H).

[0212] Release studies of V-Ns-M:

[0213] V-Ns-M was diluted 1 :2 with DTT (7.5 mM) in PBS (50 mM, pH 7.8) and stirred at room temperature for 5 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0214] Method: 7% B for 5 minutes, gradient 7% B to 40% B over 10 min, gradient 40% B to 95% B over 10 minutes, gradient 95% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 39 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated V-Ns-M conjugate and commercial MMC (Figure 3B).

[0215] Synthesis of Van-Np-ThioDyridine (Compound Van-Np-Thiopyridine)

[0216] Compound 3 (100 mg, 288 pmol, 1 equiv.) was dissolved in DMSO (1 mL) and added to HBTLI (109 mg, pmol, 1 equiv) dissolved in DMSO (6 mL). The contents of this flask was added dropwise to another flask containing vancomycin (513 mg, 346 pmol, 1 .2 equiv.) dissolved in DMSO (4 mL) and pyridine (117 pL, 1440 pmol, 5 equiv.). The mixture was allowed to stir overnight followed by purification by preparative RP-HPLC equipped with a Zorbax Eclipse XDB-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1 % formic acid. Method: 5% B for 4 min, gradient 5% B to 22.5% B over 10 min, gradient 22.5% B to 40% B over 4 min, followed by washing and equilibrating. Both products with attachment at the secondary and primary amine of vancomycin were isolated in this way. The primary amine product, Van-Np-Thiopyridine, was detected at 210 nm and eluted after 14.5 min.

[0217] Impurities were still present after purification, but the crude product was used for the next reaction. 7 mg were isolated.

[0218] HRMS(ESI): calcd. for C74H82N10O25S2CI2+2H: m / z 823.2208 found 823.2209 Fragmentation ion to confirm primary amine attachment, calcd. for C59H61N8O22CI2+H: m / z 1305.3429 found 1305.3474. (see S2)

[0219] Confirmed attachment at primary amine by ninhydrin stain (see S3).

[0220] S2: Differentiating between attachment at the secondary and primary amine of vancomycin

[0221] During purification of V-S-M by preparative HPLC two peaks were isolated. The peak with the lower retention time was expected to correspond to attachment at the primary amine, while the peak with the higher retention time was expected to correspond to attachment at the secondary amine, as this was observed for previous vancomycin conjugates and in literature. The product mass is the same for the two products, however MS can be used to differentiate between them. When attachment is at the primary amine an expected major fragmentation reaction is cleavage at the vancosamine sugar.

[0222] If the equivalent fragmentation reaction happens for the product with attachment at the secondary amine the resulting fragment ion would still have MMC attached, and therefore no ion with m / z of 1305 should be observed. Both Van-S-MMC products were analyzed by LC-MS with peak fragmentation. Analysis of the product with attachment expected to be at the primary amine, showed a compound with m / z 1990.5426 (V-S-M [M+H]+product peak) and this compound fragmented into a compound with m / z 1305.3432, corresponding to the vancosamine sugar cleavage described above. Analysis of the product with attachment expected to be at the secondary amine, showed a compound with m / z 1990.5409 (V-S-M [M+H]+product peak) and 995.2717 (V-S-M [M+2H]+product peak) and these compounds showed no sign of fragmentation ions with m / z of 1305.

[0223] S3: Ninhydrin stain to differentiate between primary and secondary amine attachment Solutions of Van-Np-Thiopyridine and Van-Ns-Thiopyridine in a H2O:MeCN 1 :1 mixture with 0.1 % formic acid (taken directly after purification by preparative HPLC ) were spotted on a TLC plate 6-8 times followed by Ninhydrin staining (3% ninhydrin in ethanol by mass). Reaction of ninhydrin with primary amines yields a purple color, while reaction with secondary amines yields a pale yellow color.

[0224] Synthesis of V-Np-M

[0225] Van-Np-Thiopyridine (13.6 mg, 8.4 pmol, 1 equiv.) was dissolved in DMF (1 mL) and DTBA (1 .9 mg, 10.9 pmol, 1 .3 equiv.) dissolved in PBS (1 mL, 50 mM, pH 7.8) was added. The mixture was left to stir for 35 min. Compound 1 (Synthesized as described in Example 1 ) (7.83 mg, 14.3 pmol, 1.7 equiv.) dissolved in DMF (0.5 mL) was added and the mixture was allowed to react in the dark for 2 hours. The resulting product mixture was purified by preparative HPLC equipped with ZORBAX Eclipse XDB-C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate. Method: 7% B for 4 min, gradient 7% B to 14% B over 1 min, gradient 14% B to 24% B over 19 min, gradient 24% B to 100% B over 2 min followed by washing and equilibrating. Van-Np-MMC eluted at 16.8 min and its mass was determined by UV- measurement to 1 .5 mg (0.77 pmol, <1 % yield over 2 steps).

[0226] HRMS(ESI): calcd. for C87H99Ni3O32S2Cl2+H+Na: m / z 997.7651 found 997.7659 calcd. for C87H99N13O32S2CI2+2H: m / z 987.2758 found 987.2766

[0227] Fragmentation peak to confirm primary amine attachment, calcd. for C59H61N8O22CI2+H: m / z 1305.3429 found 1305.3504 (see S2).

[0228] Evaluation of purity: V-Np-M in DMSO was analyzed by analytical RP-HPLC equipped with an InfinityLab Poroshell 120 EC-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1 % formic acid. Method: 5% B for 2 minutes, gradient 5% B to 25% B over 3 min, gradient 25% B to 72% B over 10 minutes, gradient 72% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 27 min. The product was detected at 210 nm and eluted after 11 .4 min (Figure 3I).

[0229] Release studies of V-Np-M:

[0230] V-Np-M was diluted 1 :3 with NAC (6.7 mM) in PBS (50 mM, pH 7.8) for a final NAC concentration of 5 mM and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with an InfinityLab Poroshell 120 EC-C18 column and using a solvent system consisting of A: MQ with 0.1 % formic acid and B: acetonitrile with 0.1 % formic acid.

[0231] Method: 5% B for 2 minutes, gradient 5% B to 25% B over 3 min, gradient 25% B to 72% B over 10 minutes, gradient 72% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 27 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated Van-Np-MMC conjugate and commercial mitomycin C (Figure 3C).

[0232] Results

[0233] For conjugation via the vancomycin amine groups, mercaptopropionic acid was reacted with dithiodipyridine and thereafter with N-hydroxysuccinimide to afford a heterobifunctional reagent 3. This compound can react with vancomycin via both, the primary and the secondary amine functionality, and the two compounds can be purified and separated from each other by preparative HPLC. The products were differentiated by the HPLC retention times, the MS fragmentation (S2, Figure 3 J) or with ninhydrin stain (S3, Figure 3K). The vancomycin derivatives were then reacted with stoichiometric dithiobutylamine (DTBA) and thereafter with compound 1 via thioldisulfide exchange to form the vancomycin conjugates V-Ns-M and V-Np-M (whereby Ns and Np indicate conjugate via the vancomycin secondary and primary amines, respectively) (Figure 3A).

[0234] Example 3 Control conjugate

[0235] Synthesis of M-M

[0236] M-M was isolated as a bi-product from the synthesis of V-C-M. Solutions of Van-C- Thiopyridine conjugate in MeOH (4 mM), DTBA in MQ (40 mM) and compound 1 synthesized as described in Example 1 in DMF (20 mM) were prepared. From these solutions 250 uL of the Van-C-Thiopyridine conjugate solution (1 .00 pmol, 1 .62 mg, 1 equiv.) was mixed with 250 uL of PBS buffer (50 mM, pH = 7.8) and 25 uL of the DTBA solution (1 .00 pmol, 0.174 mg, 1 equiv.) and allowed to react while stirring for 30 min. Afterwards 100 uL of the compound 1 synthesized as described in Example 1 solution (2.00 pmol, 1 .1 mg, 2 equiv.) was added and the mixture was stirred for an additional 4 hours in the dark. The final product mixture was purified by preparative RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0237] Method: 7% B for 2 min, gradient 7% B to 22% B over 3 min, gradient 22% B to 30% B over 10 min, gradient 30% B to 50 % B over 2.5 min followed by washing and equilibrating. M-M eluted at 18.9 min.

[0238] HRMS(ESI): calcd. for C36H42N8Oi4S2+Na: m / z 897.2154 found 897.2161 .

[0239] Evaluation of purity: M-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ and B: acetonitrile, both without additives.

[0240] Method: 5% B for 6 minutes, gradient 5% B to 20 % B over 10 min, gradient 20% B to 95% B over 10 min, gradient 95% B to 100% B over 4 min followed by washing and equilibrating for a total run time of 42 min. The product was detected at 210 nm and eluted after 26.5 min (Figure 4E). Release studies of M-M

[0241] M-M was mixed 1 :1 by volume with DTT (10 mM) in PBS (50 mM, pH = 7.8) and allowed to react while stirring for 30 minutes. The resulting mixture was analyzed by analytical RP-HPLC using a solvent system consisting of A: MQ and B: acetonitrile, both without additives.

[0242] Method: 5% B for 6 minutes, gradient 5% B to 20 % B over 10 min, gradient 20% B to 95% B over 10 min, gradient 95% B to 100% B over 4 min followed by washing and equilibrating for a total run time of 42 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated M-M conjugate and commercial mitomycin C (Figure 4C).

[0243] Synthesis of G-M

[0244] Reduced glutathione (3.92 mg, 12.8 pmol, 3.2 equiv.) was dissolved in PBS buffer (200 uL, 50 mM, pH = 7.8). Compound 1 (4.19 mg, 7.7 pmol , 1 equiv.) synthesized as described in Example 1 was dissolved in DMF (150 uL) and added to the glutathione solution. The reaction was left to stir in the dark for 4 hours. The resulting mixture was purified by preparative RP-HPLC equipped with a ZORBAX Eclipse CDB-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1% formic acid.

[0245] Method: 5% B for 4 min, gradient 5% B to 40% B over 20 min, gradient 40% B to 100 % B over 2 min, followed by washing and equilibrating. The product eluted at 14.4 min and 3.21 mg (4.32 pmol, 56% yield) of G-M was isolated.

[0246] HRMS(ESI): calcd. for C28H37N7O13S2+H: m / z 744.1963 found 744.1995

[0247] HPLC purity: G-M in DMSO was analyzed by analytical RP-HPLC equipped with an InfinityLab Poroshell 120 EC-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1% formic acid.

[0248] Method: 5% B for 6 minutes, gradient 5% B to 20% B over 10 min, gradient 20% B to 95 % B over 10 min, gradient 95% B to 100 % B over 4 min followed by washing and equilibrating for a total run time of 42 min. The product was detected at 210 nm and eluted after 22.8 min (Figure 4F).

[0249] Release studies of G-M:

[0250] G-M was treated with DTT (15 equiv.) in PBS (50 mM, pH = 7.8) and stirred at room temperature for 5 min. The resulting product was analyzed by analytical RP-HPLC equipped with an InfinityLab Poroshell 120 EC-C18 column and using a solvent system consisting of A: MQ with 0.1% formic acid and B: acetonitrile with 0.1 % formic acid. Method: 5% B for 6 minutes, gradient 5% B to 20% B over 10 min, gradient 20% B to 95% B over 10 min, gradient 95% B to 100% B over 4 min followed by washing and equilibrating for a total run time of 42 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated G-M conjugate and commercial MMC (Figure 4D).

[0251] Results

[0252] For control purposes, we also synthesized the conjugates of mitomycin (G-M) with glutathione and with another molecule of mitomycin (M-M). In these conjugates, the scissile bond is identical to the one in the V-M conjugates whereas the targeting agent (V) was replaced with the non-targeting glutathione (G) or the second copy of mitomycin (Figure 4A).

[0253] Example 4 Drug Release

[0254] Results

[0255] Drug release from the conjugates was monitored via HPLC and is illustrated in Figure 5A on an example of the V-C-M conjugate and the M-M dimer. Here, compound detection was performed at 360 nm. At this wavelength, vancomycin and all other molecules except mitomycin exhibit no absorbance. Thus, only mitomycin and derivatives thereof were detected. Figure 5A illustrates that mitomycin was well resolved from its V-C-M conjugate and that the preparation of V-C-M had no residual free mitomycin. Upon addition of a common biochemical reducing agent dithiothreitol (DTT), the retention time of the compound in the HPLC elution profile became identical to that of the pristine mitomycin, and thus validated drug release from the prodrug via th iol-disulf ide exchange. The same conclusions was made for the M-M dimer. HPLC was also used to quantify the drug release using a biocompatible trigger, N- acetylcysteine, as well as the non-specific drug release (Figure 5B, Figure 2D). NAC is an FDA approved drug and is a biologically safe trigger of thiol -disulfide exchange. As shown in Figure 2D, 5 mM NAC afforded a near quantitative release of mitomycin from the V-C-M conjugate within minutes of incubation. In contrast, the non-specific drug release over the initial hour of incubation in physiological buffers was insignificant. Nonspecific drug release proved to be non-negligible over a 24 h incubation, reaching appr. 20% for PBS and 40% for mM9 media.

[0256] Example 5 Fluorescence conjugation

[0257] Bacterial cell culture

[0258] Bacterial strain. The Staphylococcus aureus (S. aureus) strain used throughout this were S. aureus ATCC29213. The stock was stored in 15 % glycerol at -80 °C. Single colonies were grown on brain-heart-infusion (BHI) agar plates every month and stored at 4-8 °C.

[0259] Media and buffers. The growth media used in the experiments was Brain Heart Infusion Broth (BHI) or modified M9 minimal media (mM9 media) consisting of modified M9 buffer (mM9 buffer) supplemented with 1 % glucose and 1 % casamino acids. Modified M9 buffer (mM9) containing M9 minimal salts 1 X, 2 mM MgSC , 0.1 mM CaCl2, 1 mM Thiamine-HCI, 0.05 mM nicotinamide and 1 mL / L trace metal solution TMS3. The pH of mM9 was adjusted to 7.4. After autoclavation components were sterile filtered and added to the buffer. Assays involving dithiothreitol (DTT) or N-acetyl cysteine (NAC) contained 5 mM.

[0260] Overnight cultures. All S. aureus overnight cultures were prepared by adding single colonies to BHI (20 mL) in Erlenmeyer flasks. They were incubated at 37°C, 180 rpm for 16-20 h.

[0261] Visualization of vancomycin binding to planktonic and biofilm S. aureus by confocal laser scanning microscopy:

[0262] Overnight cultures were resuspended in mM9 and OD6oo adjusted to 1 . Samples and controls were prepared for imaging by adding vanco-FITC or fluorescein (25 mg / L) and SYTO60 (20 pM) to the OD-adjusted bacteria in Eppendorf tubes. Samples were then vortexed and incubated for 30 min in the dark, 50 rpm. Sample and controls were then washed (145000 rpm 10 min) to remove unbound stain and resuspended in PBS corresponding to Vs of original volume to concentrate cells. 20 pL of sample and control was added to a superfrost ultra plus slide for 10 min. Unbound cells were washed off and a drop of antifade added to the slide. Bacteria were then visualized with confocal laser scanning microscopy (CLSM, Zeiss LSM700) through a 100 x oil objective using 488 and 639 nm wavelengths for excitation.

[0263] Biofilm samples were grown in BHI in IBIDI 96 well black p-plate (IBIDI 89621 ) for 48 h, 37 °C, 50 rpm. Media was exchanged after 24 h. BHI was removed and replaced with 1% bovine serum albumin in BHI and incubated for 1 h. Wells were then washed three times in PBS and vanco-FITC or fluorescein (25 pM) and SYTO60 (20 pM) added to samples and controls respectively. Sample and control were then washed 3 times in PBS to remove unbound conjugate and stain and imaged as described for planktonic cells.

[0264] Results

[0265] Independently, it was validated that vancomycin conjugates bind to the nominated target pathogen, S. aureus. To this end, the commercially available conjugate between vancomycin and fluorescein was used, in this conjugate the fluorophore attachment is at the primary amine of vancomycin. Planktonic S. aureus were incubated with Vancomycin-Fluorescein and thereafter imaged using confocal laser scanning microscopy. Green fluorescence on the cell surface indicated the binding of the vancomycin derivative to planktonic S. aureus cells and to a 48 h old S. aureus biofilm (Figure 6), confirming the ability of vancomycin to act as a targeting agent.

[0266] Example 6 V-M conjugates are targeted to S. aureus

[0267] Quantifying antimicrobial effect of vanco-MMC conjugate after washing off unbound conjugate: S. aureus overnight cultures were harvested by centrifugation (14 000 rpm, 5 min), resuspended in mM9 buffer and diluted to OD6oo = 0.05. OD-adjusted cultures were added to Eppendorf tubes to obtain an OD6oo of 0.005. Samples were incubated with 2 mg / L vanco-MMC, GSH-MMC, MMC-MMC or free MMC (commercial MMC) for 15 min. Wash was performed by centrifuging 14500 and resuspending in either mM9 buffer, mM9 buffer + NAC or mM9 buffer + DTT. Samples were then incubated at 37 °C, 100 rpm in darkness for 24 h. Ten-fold dilutions of the samples and controls were made in mM9 buffer, after which 20 pL from each well was plated on agar plates. The plates were subsequently incubated for 19 h at 37 °C followed by CFU enumeration. The antimicrobial effect was evaluated by comparing the CFU of treated samples with the CFU of the growth control.

[0268] Results

[0269] Next, the aim was to validate if the V-M conjugates was targeted to S. aureus. The V- M conjugate wherein conjugation to vancomycin was performed through carboxylic acid (V-C-M) was used in this example. To validate drug targeting, a following protocol consisting of the following steps was employed: 1 ) a binding step where the bacteria were exposed to the conjugate in buffer for 15 minutes, 2) a washing step where unbound conjugate was removed by centrifugation and resuspension of the cells, and 3) an incubation step where the washed bacteria were then incubated in buffer for 24 h to allow time for the cell-bound conjugate to perform its antimicrobial activity. In these experiments, the non-targeting control conjugates G-M and M-M, as well as the free mitomycin was used. The incubation step optionally included reducing agents to trigger drug-release (DTT or N-acetylcysteine (NAC)). NAC was used as a biocompatible, FDA-approved disulfide reducing agent, for potential use in vivo. In these experiments, the G-M and the M-M conjugates exhibited no antibacterial activity, regardless of the incubation conditions. In the absence of reducing agents, the V-M conjugate was also devoid of activity. However, upon the addition of NAC and especially upon exposure to DTT, the VM conjugate exhibited strong bacterial cell killing. These results clearly illustrated that V-M, but not G-M and M-M, exhibited association with the bacterial cell surface even within the short incubation time of 15 minutes (Figure 7). These results validated targeting of the conjugate to bacteria.

[0270] Example 7 planktonic S. aureus to the V-M and M-M conjugates over 24 h of incubation

[0271] Quantifying antimicrobial effect of ranging vanco-MMC or MMC-MMC conjugate concentrations:

[0272] S. aureus overnight cultures were harvested by centrifugation (14 000 rpm, 5 min), resuspended in mM9 buffer and diluted to QD600 = 0.05. A two-fold serial dilution of vancomycin-MMC and MMC-MMC 0,25 - 2 mg / L (commercial MMC) was prepared in 96-well plates in mM9-buffer. OD-adjusted cultures were added to wells to obtain an ODeoo of 0.005. All wells were thoroughly mixed before adding 5 mM NAC / DTT to relevant wells. 96-well plates were incubated at 37 °C, 50 rpm in darkness for 24 h. Ten-fold dilutions of the samples and controls were made in mM9 buffer, after which 20 pL from each well was plated on agar plates. The plates were subsequently incubated for 19 h at 37 °C followed by CFU enumeration. The antimicrobial effect was evaluated by comparing the CFU of treated samples with the CFU of the growth control.

[0273] Results

[0274] To increase the releasable content of the drug, planktonic S. aureus was exposed to the V-C-M and M-M conjugates over 24 h of incubation. Under these conditions, the MM conjugate revealed minimal bacterial cell killing (Figure 8). At the same time, the V- C-M conjugate revealed statistically significant antibacterial activity. At matched equivalent concentrations of mitomycin, V-C-M conjugate was significantly more active than the M-M conjugate, highlighting that the antibacterial effect was due to the presence of vancomycin as a targeting agent. Most importantly, in this experiment no external agent was used, and drug release was therefore limited to the spontaneous mechanism and / or the bacteria-mediated drug release.

[0275] Example 8 carboxylate vs amine, primary or secondary Results

[0276] Encouraged by the above data, it was analyzed if antibacterial activity of the V-M conjugate could be optimized by the architecture of the conjugate and specifically, the site of vancomycin (carboxylate vs amine, primary or secondary) used for drug conjugation. Here, the bacteria were exposed to the conjugates for 15 minutes, thereafter washed, and then incubated for additional 24 h before quantification of the bacteria colony forming units (Figure 9). Note that in this experiment the equivalent concentration of mitomycin was 0.5 mg / L as compared to 2 mg / L used in Figure 7, which explains the diminished activity of mitomycin drug used for control purposes. Results presented in Figure 9 illustrate that the site of conjugation may affect the antibacterial effect. Thus, conjugation via the carboxylic acid group makes up the V-M conjugate that registered lowest activity (at this low concentration and with the given protocol). At the same conditions, the two conjugates via the vancomycin amine groups exhibited higher bacterial cell killing activity, and the conjugate via the primary amine afforded the killing of over 99% of bacteria in cell culture. Example 9 Symmetrical disulfide linker

[0277] Synthesis of disulfanediylbis(ethane-2,1 -diyl) bis(4-nitrophenyl) bis(carbonate) (disulfanediylbis(ethane-2,1 -diyl) bis(4-nitrophenyl) bis(carbonate))

[0278] 2-hydroxy-ethyldisulfide (0.94 mL, 7.8 mmol, 1 .0 equiv.) was dissolved in dry DCM (8 ml) followed by addition of TEA (3.25 mL, 23.3 mmol, 3.0 equiv.). In another flask paranitrophenyl chloroformate (3.656 g, 18.14 mmol, 2.3 equiv.) was dissolved in DCM (8 ml), cooled to 0 degrees and the mixture containing TEA and 2-hydroxytheyldisulfide was added dropwise to the solution over 10 min. After addition the ice was removed, and the reaction was left stirring at room temperature for 24 hours. The reaction was diluted with DCM (52 mL) and quenched with saturated ammonium chloride (25 mL). The product was washed with brine (3x) and dried over sodium sulphate. The crude product was purified by flash column chromatography packed in pentane:DCM 3:7 and eluted in pure DCM. The product was isolated as a thick slightly yellow oil (3.1 g, 6.4 mmol, 82 % yield).

[0279] HRMS(ESI): calcd. for Ci8Hi6N2Oi0S2+Na: m / z 507.0139 found 507.0130 calcd. for C18H16N2O10S2+NH4: m / z 502.0585 found 502.0577

[0280] 1H NMR (400 MHz, CDCI3) 5 8.27 (dt, J = 9.2, 3.2 Hz, 4H, aromatic), 7.38 (dt, J = 9.3,

[0281] 3.2 Hz, 4H, aromatic), 4.57 (t, J = 6.5 Hz, 4H, S-CH2-CH2-O), 3.08 (t, J = 6.5 Hz, 4H, S- CH2-CH2-O) (Figure 10A).

[0282] 13C NMR (100 MHz, CDCI3) 5 155.34 (Ar-O), 152.33 (carbonyl), 145.51 (Ar-NO2), 125.36 (Ar-H), 121.78 (Ar-H), 66.76 (S-CH2-CH2-O), 36.77 (S-CH2-CH2-O) (Figure 10B).

[0283] Synthesis of compound 4 (Compound 4)

[0284] Disulfanediylbis(ethane-2,1 -diyl) bis(4-nitrophenyl) bis(carbonate) (0.82 g, 1.7 mmol, 2.7 equiv.) was dissolved in dry DMF (15 mL) and stirred under N2. In another flask MMC (207.7 mg, 0.6212 mmol. 1 .0 equiv.) and TEA (0.14 mL, 1 .0 mmol, 1 .6 equiv.) were dissolved in dry DMF (5 mL) followed by dropwise addition to the stirring reaction mixture. HOBt (521 .2 mg, 3.857 mmol, 6.2 equiv.) was subsequently added and the reaction was left stirring in the dark overnight. The resulting mixture was diluted with DCM (50 mL), washed with ammonium chloride (x2), and then brine (x2). The resulting organic phase was dried over sodium sulphate and then concentrated under vacuum. The concentrate was then purified by flash column chromatography (1 :1 pentane:EtOAc to 3:7 pentane:EtOAc) to yield the product as a purple powder (170 mg, 0.25 mmol, 40% yield).

[0285] HRMS(ESI): calcd. for C27H29N5O12S2+H : m / z 680.1327 found 680.1323 calcd. for C27H29N5Oi2S2+Na: m / z 702.1146 found 702.1168

[0286] 1H NMR (400 MHz, CDCI3) 5 8.30 (dt, J = 9.1 , 3.1 Hz, 2H, p-nitrophenyl), 7.41 (dt, J = 9.2, 3.1 Hz, 2H, p-nitrophenyl), 5.20 (broad s, 2H), 4.83 (dd, J = 10.9, 4.7 Hz, 1 H), 4.70 (broad zs, 2H), 4.53 (t, J = 6.7 Hz, 2H, O-CH2-CH2), 4.46 (d, J = 13.4 Hz, 1 H), 4.35 (td, J = 6.6, 1 .1 Hz, 2H, O-CH2-CH2), 4.30 (t, J = 11 .2 Hz, 1 H), 3.92 (q, J = 3.8 Hz, 2H), 3.68 (dd, J = 1 1 .1 , 4.6 Hz, 1 H), 3.49 (dd, J = 13.3, 2.0 Hz, 1 H), 3.44 (d, J = 4.8 Hz, 1 H), 3.33 (dd, J = 4.6, 1 .9 Hz, 1 H), 3.20 (s, 3H), 3.03 (t, J = 6.6 Hz, 2H, CH2-CH2-S), 2.97 (t, J = 6.6 Hz, 2H, CH2-CH2-S), 2.89 (t, J = 5.8 Hz, 2H), 1 .76 (s, 3H) (Figure 10C).

[0287] Synthesis of V-S-M

[0288] Vancomycin (854 mg, 575 pmol, 5 equiv.) was dissolved in DMSO (5 mL) followed by addition of DIPEA (300 uL, 1725 pmol, 15 equiv.) and stirring for 5 minutes.

[0289] Compound 4 (78.5 mg, 115 pmol, 1 equiv.) was dissolved in DMSO (3.27 mL, concentration of 24 mg / mL) and added to the stirring solution. The mixture was stirred overnight followed by purification by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0290] Method: 7% B for 4 min, gradient 7% B to 20% B over 1 min, constant 20% B for 9 min, gradient 20% to 100% B over 1 min followed by washing and equilibrating. The product eluted at 13 min and the total product mass was determined using UV-absorbance measurements (36.7 mg, 18.5 pmol, 16.0% yield).

[0291] HRMS(ESI): calcd. for C87H99Ni3O33S2Cl2+H+Na: m / z 1006.2643 found 1006.2638 calcd. for C87H99Ni3O33S2Cl2+H: m / z 1989.5393 found 1989.5458.

[0292] For determination of amine attachment location see S2

[0293] Evaluation of purity: V-S-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10% MQ and 8 mM ammonium acetate.

[0294] Method: 7% B for 4 minutes, gradient 7% B to 25% B over 2 min, gradient 25% B to 80% B over 14 min, gradient 80% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 30 min. The product was detected at 210 nm and eluted after 14.3 min (Figure 10D).

[0295] S4: Optimization of coupling agents and bases used in vancomycin conjugation Solutions of compound 4 in DMSO (12 mg / mL, 17.7 mM), vancomycin in DMSO (87.67 mg / mL, 59 mM), HOBt in DMSO (5.74 mg / mL, 42.4 mM), HBTU in DMSO (16.12 mg / mL, 42.4 mM), pyridine in DMSO (531 mM) and DIPEA in DMSO (531 mM) were prepared. To each test vial 100 uL of the compound 4 solution was added (1 equiv. 1.77 pmol) followed by addition 50 pL of HOBt or HBTU solutions (2.12 pmol, 1.2 equiv.) (if applicable) and stirring for 5 minutes. Afterwards 30 pL of vancomycin solution was added (1.77 pmol, 1 equiv.) immediately followed by addition of 10 pL of DIPEA or pyridine solutions (5.31 pmol, 3 equiv.) (if applicable). After 18h each sample was diluted with H2O:MeCN 1 :1 by volume to a final volume of 250 uL before analysis by preparative HPLC. The integrals of the peaks corresponding to attachment at the secondary and primary amine of vancomycin were compared and the ratio of secondary : primary amine attachment was recorded in the table. Table 1 Ratio of vancomycin product conjugated at secondary amine to product conjugated at primary amine (secondary : primary)

[0296] S5: Optimization of base and vancomycin equivalents for vancomycin conjugation

[0297] Solutions of compound 4 in DMSO (24 mg / mL, 35.4 mM), vancomycin in DMSO (100 mg / mL, 67.3 mM) and DIPEA in DMSO (531 mM) were prepared . To each test vial 39.5 pL (2.66 pmol, 1 .5 equiv.), 131 .5 pL (8.85 pmol, 5 equiv.) or 263 pL (17.7 pmol, 10 equiv.) of the vancomycin solution was added followed by addition of either 16.6 pL (8.85 pmol, 5 equiv.), 33 pL (17.7 pmol, 10 equiv.) or 50 uL (26.6 17.7 pmol, 15 equiv.) of the DIPEA solution and stirring for 5 minutes. Afterwards 50 pL of the compound 4 solution was added (1 equiv., 1 .77 pmol) to each vial and left stirring overnight. The contents of the vials were diluted with FfeChMeCN 1 : to 500 pL and then further 1 :4 with the same solution before analysis by analytical HPLC. The integrals of the peaks corresponding to attachment at the primary amine of vancomycin were measured. The amount of product formed relative to reaction the reaction with 1 .5 equiv. vancomycin and 5 equiv. base was recorded in the table below.

[0298] Table 2: Relative amount of product formed with attachment at the primary amine of vancomycin Release studies of V-S-M:

[0299] V-S-M was mixed 1 :1 with DTT (10 mM) in PBS (50 mM, pH 7.8) and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0300] Method: 7% B for 4 minutes, gradient 7% B to 25 % B over 2 min, gradient 25% B to 80% B over 14 min, gradient 80% B to 100% B over 1 minute followed by washing and equilibrating for a total run time of 30 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated V-S-M conjugate and commercial MMC (Figure 10E).

[0301] Results

[0302] The translational path of therapeutic molecules often requires that the original, small- scale synthesis of the drug molecule is revisited and optimized as regards the protocols safety, reagent costs, and the compound yields. In the case of the V-M conjugates, the synthetic schemes shown in Figure 2 and 3 were robust, but required that the compounds are repeatedly purified via preparative HPLC, which decreased the overall yields of syntheses. For reagent scale up, the synthesis of the V-M conjugate via the vancomycin primary amine was revisited. The synthesis was carried out starting with an oxidized, dimeric form of mercaptoethanol (Figure 10F), via the reaction with nitrophenylchloroformate, thereafter with mitomycin and finally with vancomycin. The reaction with vancomycin was optimized, first by varying the base and coupling agents used (S4), and next by varying the equivalents of vancomycin and base (S5), to maximize attachment at the primary amine of vancomycin. The final compound (herein termed VsM, where “s” denotes that the disulfide linker is symmetrical) was purified via preparative HPLC. Antibacterial effects of VsM were quantified against S. aureus via a 15 minutes exposure to the drug, removal of the excess drug via washing, and a subsequent 24 h incubation in mM9 buffer. The conjugate afforded potent, efficacious antibacterial effect, which was statistically significant at a concentration as low as 0.5 mg / L (equivalent mitomycin concentration). At all concentrations tested, antimicrobial activity of the VsM conjugate was significantly greater than that for mitomycin, reflecting the benefits of drug targeting (Figure 10G). Example 10 V-S-M against other gram positive bacteria

[0303] V-S-M showed great activity against S. aureus (strain 29213), but since vancomycin is active against all gram positive bacteria, this activity should be expandable to a panel of bacterial strains. The V-S-M conjugate was therefore tested against the gram positive bacteria: S. aureus (strain SAU060112), E. faecalis, Methicillin resistant S. aureus and S. epidermidis (Figure 1 1 ). The equivalent mitomycin C dose was adjusted according to the MBC value for each given strain (Table 3). The V-S-M conjugate alone was equally or more potent than equimolar mitomycin C against all strains, however by addition of N-acetyl cysteine (NAC), to ensure full release of mitomycin C, the potency of the conjugate was increased in all cases. The V-S-M conjugate was then tested against vancomycin intermediate S. aureus and vancomycin resistant E. faecalis. Against both of these strains, that are resistant to the targeting agent vancomycin, we surprisingly observed excellent activity for the V-S-M conjugate. This is likely because the MBC of mitomycin C is so much lower in buffer than the MBC of vancomycin (Table 3), so minimal binding of vancomycin to the bacteria is required for it to be used as a targeting agent. This result opens up for the use of these drug conjugates as general broad spectrum antibiotics against resistant bacterial strains, as conjugation of mitomycin C has effectively reversed the bacterial resistance against vancomycin. Lastly V-S-M was tested against the gram negative bacteria E. Coli and P. aeruginosa. Here the activity of V-S-M was comparable to or slightly higher than equimolar mitomycin C.

[0304] Table 3. Minimum Inhibitory Concentrations and Minimum Biocidal Concentrations of mitomycin C and vancomycin in a nutrient-rich and nutrient-free environment.

[0305] Example 11: Synthesis of new conjugates

[0306] See the section “Instruments and material” for information concerning instruments and material.

[0307] Synthesis of compound 1 in Figure 13A

[0308] 2-Hydroxy-ethyldisulfide (0.94 mL, 7.8 mmol, 1 .0 equiv.) was dissolved in dry DCM (8 ml) followed by addition of TEA (3.25 mL, 23.3 mmol, 3.0 equiv.). In another flask paranitrophenyl chloroformate (3.656 g, 18.14 mmol, 2.3 equiv.) was dissolved in DCM (8 ml), cooled to 0 degrees and the mixture containing TEA and 2-hydroxytheyldisulfide was added dropwise to the solution over 10 min. After addition the ice was removed, and the reaction was left stirring at room temperature for 24 hours. The reaction was diluted with DCM (52 mL) and quenched with saturated ammonium chloride (25 mL). The product was washed with brine (3x) and dried over sodium sulphate. The crude product was purified by flash column chromatography packed in pentane:DCM 3:7 and eluted in pure DCM. The product was isolated as a thick slightly yellow oil (3.1 g, 6.4 mmol, 82 % yield).

[0309] HRMS(ESI): calcd. for Ci8Hi6N2Oi0S2+Na: m / z 507.0139 found 507.0130 calcd. for C18H16N2O10S2+NH4: m / z 502.0585 found 502.0577.1H NMR (400 MHz, CDCI3) 5 8.27 (dt, J = 9.2, 3.2 Hz, 4H, aromatic), 7.38 (dt, J = 9.3, 3.2 Hz, 4H, aromatic), 4.57 (t, J = 6.5 Hz, 4H, S-CH2-CH2-O), 3.08 (t, J = 6.5 Hz, 4H, S- CH2-CH2-O).

[0310] 13C NMR (100 MHz, CDCI3) 5 155.34 (Ar-O), 152.33 (carbonyl), 145.51 (Ar-NO2), 125.36 (Ar-H), 121.78 (Ar-H), 66.76 (S-CH2-CH2-O), 36.77 (S-CH2-CH2-O).

[0311] Synthesis of compound 2 in Figure 13A

[0312] Compound 1 (0.82 g, 1.7 mmol, 2.7 equiv.) was dissolved in dry DMF (15 mL) and stirred under N2. In another flask MMC (207.7 mg, 0.6212 mmol. 1.0 equiv.) and TEA (0.14 mL, 1 .0 mmol, 1 .6 equiv.) were dissolved in dry DMF (5 mL) followed by dropwise addition to the stirring reaction mixture. HOBt (521.2 mg, 3.857 mmol, 6.2 equiv.) was subsequently added and the reaction was left stirring in the dark overnight. The resulting mixture was diluted with DCM (50 mL), washed with ammonium chloride (x2), and then brine (x2). The resulting organic phase was dried over sodium sulphate and then concentrated under vacuum. The concentrate was then purified by flash column chromatography (1 :1 pentane:EtOAc to 3:7 pentane:EtOAc) to yield the product as a purple powder (170 mg, 0.25 mmol, 40 % yield).

[0313] HRMS(ESI): calcd. for C27H29N5O12S2+H: m / z 680.1327 found 680.1323 calcd. for C27H29N5Oi2S2+Na: m / z 702.1146 found 702.1168.

[0314] 1H NMR (400 MHz, CDCI3) 5 8.30 (dt, J = 9.1 , 3.1 Hz, 2H, p-nitrophenyl), 7.41 (dt, J = 9.2, 3.1 Hz, 2H, p-nitrophenyl), 5.20 (broad s, 2H), 4.83 (dd, J = 10.9, 4.7 Hz, 1 H), 4.70 (broad zs, 2H), 4.53 (t, J = 6.7 Hz, 2H, O-CH2-CH2), 4.46 (d, J = 13.4 Hz, 1 H), 4.35 (td, J = 6.6, 1 .1 Hz, 2H, O-CH2-CH2), 4.30 (t, J = 1 1 .2 Hz, 1 H), 3.92 (q, J = 3.8 Hz, 2H), 3.68 (dd, J = 11 .1 , 4.6 Hz, 1 H), 3.49 (dd, J = 13.3, 2.0 Hz, 1 H), 3.44 (d, J = 4.8 Hz, 1 H), 3.33 (dd, J = 4.6, 1 .9 Hz, 1 H), 3.20 (s, 3H), 3.03 (t, J = 6.6 Hz, 2H, CH2-CH2-S), 2.97 (t, J = 6.6 Hz, 2H, CH2-CH2-S), 2.89 (t, J = 5.8 Hz, 2H), 1 .76 (s, 3H).

[0315] 13C NMR (100 MHz, CDCI3) 5 178.51 , 176.08, 160.73, 156.43, 155.51 , 154.43, 152.50, 147.10, 145.65, 125.51 , 121.99, 110.56, 105.57, 105.39, 66.98, 64.74, 62.40, 49.93, 48.79, 43.70, 41.97, 41.36, 40.12, 36.79 (doublet, J =12 Hz), 8.04. Synthesis of D-N-M

[0316] Dalbavancin hydrochloride (brand name Xydalba) was dissolved in dry DMSO to a final concentration of 100 mg / mL. 0.25 mL of dalbavancin (25 mg, 1.0 equiv., 0.0134 mmol) were taken out of this solution and added to a flame dried flask followed by addition of DIPEA (7.05 pL, 0.0402 mmol, 3.0 equiv.). In another vial compound 2 (9.16 mg, 0.0134 mmol, 1 .0 equiv.) was solubilized in DMSO (0.5 mL). This solution was added to the Dalbavancin containing solution and the resulting mixture was stirred overnight at room temperature. The product was purified by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0317] Method: 7 % B for 4 min, gradient 7% B to 40 % B over 1 min, constant 40 % B for 19 min, gradient 40 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 16 min (broad peak) and the total product mass was determined using UV-absorbance measurements (4.43 mg, 1 .88 pmol, 14.0 % yield).

[0318] HRMS(ESI): calcd. for C109H124N14O37S2CI2+2H: m / z 1178.8625 found 1178.8644 calcd. for C87H99N13O33S2CI2+H+NH4: m / z 1186.8741 found 1186.8765.

[0319] Evaluation of purity: D-N-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate. Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 19.2 min. The product was found to be of sufficient purity.

[0320] Release studies: D-N-M was treated with DTT (3 mM) in PBS (10 mM, pH 7.8) and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0321] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated D-N-M conjugate and commercial MMC. DTT treatment of D-N-M resulted in a peak at the same time as commercial MMC (Figure 15).

[0322] Synthesis of TC9-N-M

[0323] Teicoplanin (26.3 mg, 2.7 equiv. 14.0 pmol) was solubilized in dry DMSO (0.25 mL) in a flame dried vial and DIPEA (56 pmol, 11 equiv., 9.7 |_1L). In another vial compound 2 (1 .0 equiv., 3.5 mg, 5.1 pmol) was solubilized in DMSO (0.5 mL). The compound 2 solution was added dropwise to the teicoplanin containing solution and the resulting mixture was stirred overnight at room temperature. TC9-N-M alongside Tmix-N-M and TC10-N-M was purified by preparative HPLC equipped with a ZORBAX Eclipse XDB- C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0324] Method: 7 % B for 4 min, gradient 7% B to 30 % B over 1 min, gradient 30 % B to 45 % B over 19 min, gradient 45 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 16.7 min and the total product mass was determined using UV-absorbance measurements (2.1 mg, 0.87 pmol, 17 % yield).

[0325] HRMS(ESI): calcd. for C109H123N13O42S2CI2+2H: m / z 1210.3365 found 1210.3386 calcd. for Cio9Hi23Ni3042S2Cl2+H+Na: m / z 1121.3275 found 1221.3312.

[0326] Evaluation of purity: TC9-N-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0327] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 18.6 min. The product was found to be of sufficient purity.

[0328] Release studies: TC9-N-M was treated with DTT (3 mM) in PBS (10 mM, pH 7.8) and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0329] Method: Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 11 min followed by washing and equilibrating for a total run time of 31 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated TC9-N-M conjugate and commercial MMC. DTT treatment of TC9-N-M resulted in a peak at the same time as commercial MMC (Figure 15).

[0330] Synthesis of Tmix-N-M mix

[0331] Teicoplanin (26.3 mg, 2.7 equiv. 14.0 nmol) was solubilized in dry DMSO (0.25 mL) in a flame dried vial and DIPEA (56 nmol, 11 equiv., 9.7 nL). In another vial compound 2 (1 .0 equiv., 3.5 mg, 5.1 nmol) was solubilized in DMSO (0.5 mL). The compound 2 solution was added dropwise to the teicoplanin containing solution and the resulting mixture was stirred overnight at room temperature. This product alongside TC9-N-M and TC10-N-M was purified by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0332] Method: 7 % B for 4 min, gradient 7% B to 30 % B over 1 min, gradient 30 % B to 45 % B over 19 min, gradient 45 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 18.9 min and the total product mass was determined using UV-absorbance measurements (0.34 mg, 0.14 pmol, 2.7 % yield).

[0333] HRMS(ESI): calcd. for C109H123N13O42S2CI2+2H: m / z 1210.3365 found 1210.3401 calcd. for CiogH^NisC^Ch+H+Na: m / z 1121 .3275 found 1 121 .3323 calcd. for C110H125N13O42S2CI2+2H: m / z 1217.3443 found 1217.3471 calcd. for CnoHi25Ni3042S2Cl2+H+Na: m / z 1128.3353 found 1228.3404.

[0334] Evaluation of purity: Tmix-N-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0335] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 18.3 min. The product was found to be of sufficient purity.

[0336] Release studies: Tmix-N-M was treated with DTT (3 mM) in PBS (10 mM, pH 7.8) and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0337] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated Tmix-N-M conjugate and commercial MMC. DTT treatment of Tmix-N-M resulted in a peak at the same time as commercial MMC (Figure 15).

[0338] Synthesis of TC10-N-M

[0339] Teicoplanin (26.3 mg, 2.7 equiv. 14.0 pmol) was solubilized in dry DMSO (0.25 mL) in a flame dried vial and DIPEA (56 pmol, 11 equiv., 9.7 |_1L). In another vial compound 2 (1 .0 equiv., 3.5 mg, 5.1 pmol) was solubilized in DMSO (0.5 mL). The compound 2 solution was added dropwise to the teicoplanin containing solution and the resulting mixture was stirred overnight at room temperature. This product alongside TC9-N-M and Tmix-N-M was purified by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0340] Method: 7 % B for 4 min, gradient 7% B to 30 % B over 1 min, gradient 30 % B to 45 % B over 19 min, gradient 45 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 20.2 min and the total product mass was determined using UV-absorbance measurements (0.24 mg, 0.10 pmol, 2.0 % yield). HRMS(ESI): calcd. for C110H125N13O42S2CI2+2H: m / z 1217.3443 found 1217.3473 calcd. for CnoHi25Ni3042S2Cl2+H+Na: m / z 1128.3353 found 1228.3393.

[0341] Evaluation of purity: TC10-N-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0342] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 18.6 min. The product was found to be of sufficient purity.

[0343] Release studies: TC10-N-M was treated with DTT (3 mM) in PBS (10 mM, pH 7.8) and stirred at room temperature for 15 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0344] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated TC10-N-M conjugate and commercial MMC. DTT treatment of TC10-N-M resulted in a peak at the same time as commercial MMC (Figure 15).

[0345] Synthesis of compound 3 in Figure 20

[0346] 2.2-Dithiodipyridine (590 mg, 2.68 mmol, 2.1 equiv.) was dissolved in a mixture of AcOH (0.5 mL) and MeOH (2 mL). In a separate flask 3-mercaptoethanol (89.3 zL, 1 .28 mmol, 1 .0 equiv.) was dissolved in MeOH (0.3 mL) and added to the stirring solution containing

[0347] 2.2-ditiodipyridine. The reaction mixture was left stirring at r.t. for 1 hour and 40 minutes. The crude mixture was concentrated and purified with flash column chromatography (gradient 3:17 EtOAc:pentane to 3:7 EtOAC:pentane) yielding the product as a bone white solid (164.03 mg, 0.876 mmol, 68 %).1H NMR (400 MHz, Chloroform-d) 5 8.52 (d, J = 5.0 Hz, 1 H), 7.59 (td, J = 7.7, 1.8 Hz, 1 H), 7.40 (d, J = 8.1 Hz, 1 H), 7.20 - 7.09 (m, 1 H), 3.81 (t, J = 5.0 Hz, 2H), 2.96 (t, J = 5.3 Hz, 2H).

[0348] 13C NMR (100 MHz, Chloroform-d) 5 159.27, 150.00, 137.00, 122.12, 121.68, 58.40, 42.92.

[0349] HRMS (ESI+) m / z calculated (calcd.) for C7H9NOS2 + H+: 188.091 1 , found: 188.0209. 0

[0350] 2-(Pyridin-2-yldisulfaneyl)ethan-1 -ol (25 mg, 0.133 mmol, 1 equiv.) was dissolved in dry CH2CI2 (0.4 ml) followed by addition of TEA (40 zL, 0.287 mmol, 2.2 equiv.). In a separate flask p-nitrophenyl chloroformate (38.96 mg, 0.193 mmol, 1.5 equiv.) was dissolved in dry CH2CI2 (0.2 mL) followed by dropwise addition of the solution to the reaction mixture. The reaction mixture was stirred for 28 hours, diluted with CH2CI2 (4 mL), quenched with NH4CI (2 mL), washed with brine three times, dried over sodium sulphate and concentrated. TLC confirmed product formation, and the crude was used directly without further purification.

[0351] A crude mixture of 2-(pyridin-2-yldisulfaneyl)ethyl 2-(4-nitrophenyl)acetate (47 mg, 0.133 mmol, 1 equiv.) was dissolved in dry DMF (1 ml) under N2. In a separate flask MMC (50.24 mg, 0.2 mmol, 1 .5 equiv.) and TEA (40 zL, 0.287 mmol, 2.2 equiv.) were dissolved in dry DMF (1 mL) followed by dropwise addition to the 2-(pyridin -2-yldisu lfaneyl)ethyl 2- (4-nitrophenyl)acetate mixture. HOBt (48.74 mg, 0.361 mmol, 2.7 equiv.) was subsequently added and the reaction was left stirring in the dark for 24 hours. The crude was concentrated and purified with flash column chromatography (1 :1 EtOAc:pentane to 4:1 EtOAc:pentane) yielding the product as a purple solid (39.5 mg, 0,072 mmol, 54 %).

[0352] 1H NMR (400 MHz, Chloroform-d) 5 8.44 (d, J = 4.9 Hz, 1 H), 7.66 - 7.55 (m, 2H), 7.11 - 7.03 (m, 1 H), 4.93 (s, 2H), 4.84 (dd, J = 10.8, 4.6 Hz, 1 H), 4.48 - 4.18 (m, 4H), 3.65 (dd, J = 11 .1 , 4.6 Hz, 1 H), 3.53 - 3.40 (m, 2H), 3.29 (dd, J = 4.6, 1 .8 Hz, 1 H), 3.17 (s, 3H), 3.10 - 2.95 (m, 2H), 1.74 (s, 3H), 1.28 - 1.19 (m, 1 H).

[0353] 13C NMR (101 MHz, Chloroform-d) 5 178.43, 176.07, 160.66, 159.55, 156.55, 154.44, 149.86,

[0354] 147.26, 137.21 , 121.11 , 120.22, 110.55, 105.58, 105.24, 64.71 , 62.23, 49.86, 48.77, 43.65, 42.05, 40.1 1 , 36.96, 8.01.

[0355] HRMS (ESI+) m / z calcd. for C23H25N5O7S2 + H+: 548.1268, found: 548.1297 m / z calcd. for C23H25N5O7S2 + Na+: 570.1087, found: 570.1103

[0356] Synthesis of compound 6 in Figure 20

[0357] Compound 5 (43.3 mg, 0.0791 mmol, 1 equiv.) was solubilized in MeOH (1 mL). Cysteamine hydrochloride (10.1 mg, 0.0899 mmol, 1 .12 equiv.) and TEA (0.025 mL, 0.18 mmol, 2.3 equiv.) were solubilized in MeOH (1 mL) and the resulting mixture was added to the compound 5 solution over 14 min. The reaction was stirred for 1 hour and then diluted with with DCM (approx. 20 mL) followed by washing with MQ water (2x). The product was brought into the aqueous phase with sat. ammonium chloride and extracted with sat. ammonium chloride (2x). The pH was adjusted to >8 with sat. NaHCOs and the product was extracted with DCM (5x) with TEA (0.1 %) added. The organic phase was combined and solvent was evaporated under reduced pressure to yield compound 6 as a purple solid (12.4 mg, 0.0256 mmol, 32 % yield, adjusted for leftover DCM and EtOAc).

[0358] 1H NMR (400 MHz, CDCI3) 5 5.11 (s, 1 H), 4.82 (dd, J = 10.8, 4.6 Hz, 1 H), 4.45 (dd, J = 13.4, 2.7 Hz, 1 H), 4.40 - 4.19 (m, 3H), 3.75 - 3.61 (m, 2H), 3.51 (dd, J = 13.4, 2.0 Hz, 1 H), 3.45 (t, J = 4.3 Hz, 1 H), 3.33 (dd, J = 4.6, 1 .9 Hz, 1 H), 3.21 (d, 3H), 3.04 (t, J = 6.2 Hz, 1 H), 2.91 (q, 2H), 2.83 (t, J = 6.2 Hz, 1 H), 2.30 (s, 4H), 1 .77 (s, 3H).

[0359] HRMS (ESI+) m / z calcd. for C20H27N5O7S2 + H+: 514.1425, found: 514.1434. Synthesis of D-C-M

[0360] Dalbavancin hydrochloride (brand name Xydalba) was dissolved in dry DMSO to a final concentration of 100 mg / mL. 0.21 mL of this dalbavancin solution (21 mg, 0.01 13 mmol, 1 .0 equiv.) was added to hydroxybenzotriazole (2.7 mg, 0.020 mmol, 1 .8 equiv.) and 1 - Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.0 mg, 0.021 mmol, 1.8 equiv.). In another flask compound 6 (6.5 mg, 0.0127 mmol, 1 .1 equiv.) was dissolved in DMSO (1 mL) and added to the dalbavancin solution. The resulting mixture was stirred overnight, diluted 1 :1 with H2O:MeCN 1 :1 v / v and purified by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0361] Method: 7 % B for 4 min, gradient 7% B to 35 % B over 1 min, gradient 35 % B to 40 % B over 19 min, gradient 40 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 21 min (broad peak) and the total product mass was determined using UV-absorbance measurements (4.68 mg, 0.0020 mmol, 18 % yield). HRMS(ESI): calcd. for C108H127N15O34S2CI2+2H: m / z 1155.8739 found 1155.8736.

[0362] Evaluation of purity: D-C-M was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0363] Method: 7% B for 6 minutes, gradient 7% B to 50 % B over 3 min, gradient 50 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 16.6 min. The product was found to be of sufficient purity.

[0364] Release studies: 15 pL of D-C-M in DMSO was treated with 35 pL of DTT (10 mM) in PBS (10 mM, pH 8.0) and stirred (600 rpm) at room temperature for 5 min. The resulting product was analyzed by analytical RP-HPLC equipped with a ZORBAX Eclipse Plus C18 column and using a solvent system consisting of A: MQ with 8 mM ammonium acetate pH adjusted to 5.4 and B: acetonitrile with 10 % MQ and 8 mM ammonium acetate.

[0365] Method: 7% B for 6 minutes, gradient 7% B to 50 % B over 3 min, gradient 50 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The resulting HPLC chromatogram was compared with HPLC chromatograms of the untreated D-C-M conjugate and commercial MMC. DTT treatment of D-C-M resulted in a peak at the same time as commercial MMC (Figure 22).

[0366] Synthesis of compound 7 in Figure 24

[0367] Floxuridine (21 1.1 mg, 0.857 mmol,1 equiv.) was dissolved in dioxane (5 mL) and pyridine (5.8 equiv.) was added followed by cooling to 0 °C. A solution of p-nitrophenyl chloroformate (197.3 mg, 0.979 mmol, 1.14 equiv.) in dioxane (3 mL) was prepared and added dropwise to the floxuridine solution over 15 min forming a slurry. The resulting mixture was allowed to heat to room temperature and stirred for 23 hours. The crude mixture was loaded (20 % MeOH in DCM) unto an automated flash purification system using Interchim SI-HP 30 pm puriflash silica and a solvent system consisting of A: DCM and B: MeOH. Method: 0% B for 3 column volumes, gradient 0 % B to 5 % B over 7 column volumes, constant 5 % B for 3 column volumes, gradient 5 % B to 10 % B over 4 column volumes. The product (28.1 mg, 0.0683 mmol, 8.0 % yield) eluted at 12.5 column volumes.

[0368] HRMS(ESI): calcd. for C16H14N3O9F+H: m / z 412.0787 found 412.0787 calcd. for C HuNsOgF+Na: m / z 434.0606 found 434.0606.

[0369] 1H NMR (400 MHz, CD3CN) 5 9.34 (bs, 1 H), 8.33 - 8.22 (m, 2H), 7.69 (d, J = 7.0 Hz, 1 H), 7.49 - 7.40 (m, 2H), 6.19 (td, J = 6.7, 1 .8 Hz, 1 H), 4.49 (dd, = 1 1 .9, 3.3 Hz, 1 H), 4.45 - 4.34 (m, 2H), 4.10 (q, = 4.0 Hz, 1 H), 2.28 (ddd, J = 13.9, 6.4, 4.2 Hz, 1 H), 2.22 - 2.12 (m, 2H, coincides with H2O peak).

[0370] Synthesis of compound 8 in Figure 24

[0371] Cysteamine dihydrochloride (64.4 mg, 0.286 mmol, 4.2 equiv.) was solubilized in DMF (2 mL) followed by addition of DIPEA (0.12 mL, 0.689 mmol, 10 equiv.). Compound 7 (28.1 mg, 0.0683 mmol, 1 .0 equiv.) was solubilized in DMF (1 mL + 1 mL for transferring) and added dropwise to the cysteamine solution over 19 min. The resulting mixture was stirred for 19h hours and solvent was evaporated under reduced pressure. The crude was suspended in DCM and washed with DCM and dioxane followed by solubilization in MeOH. Solvent was evaporated under reduced pressure and the crude mixture was loaded (20 % MeOH in DCM) unto an automated flash purification system using Interchim SI-HP 30 pm puriflash silica and a solvent system consisting of A: DCM with 0.1 % TEA and B: MeOH with 0.1 % TEA.

[0372] Method: 5% B for 3 column volumes, gradient 5 % B to 30 % B over 10 column volumes. The product (8.1 mg, 0.683 mmol, 28 % yield corrected for TEA) eluted at 11 .3 column vol.

[0373] Rf = 0.23 in 3:5 MeOH:DCM with 5 % TEA.

[0374] HRMS(ESI): calcd. for C14H21N4O6S2F+H: m / z 425.0959 found 425.0958.1H NMR (400 MHz, MeOD): 5 7.82 (d, J = 6.7 Hz, 1 H), 6.24 (t, J = 5.9 Hz, 1 H), 4.41 - 4.32 (m, 2H), 4.21 (dd, J= 12.0, 4.3 Hz, 1 H), 4.08 (q, J= 3.1 Hz, 1 H), 3.45 (t, J= 6.8 Hz, 2H), 3.29 (t, 6.8 Hz, 2H, partly obscured by H2O peak) 2.99 (t, J= 6.8 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.32 (ddd, J= 13.7, 6.1 , 3.0 Hz, 1 H), 2.28 - 2.18 (m, 1 H).

[0375] Synthesis of D-C-F

[0376] Compound 8 (7.3 mg, 0.0173 mmol, 1.0 equiv.) was solubilized in DMSO (1 mL) and morpholine (8 uL, 0.0728 mmol, 4.2 equiv.) was added. Dalbavancin hydrochloride (brand name Xydalba) was dissolved in DMSO to a final concentration of 100 mg / mL. 0.32 mL of this dalbavancin solution (32 mg, 0.0176 mmol, 1.0 equiv.) was mixed with EDC (6.6 mg, 0.0344 mmol, 2.0 equiv.) and HOBt (5.2 mg, 0.0385 mmol, 2.2 equiv.) and added dropwise to the compound 8 solution using DMSO (0.3 mL) for transferring. The resulting mixture was stirred for 17 h and then diluted with H2O:MeCN 1 :1 before purification by preparative HPLC equipped with a ZORBAX Eclipse XDB-C8 column and using a solvent system consisting of A: MQ with 0.1 % formic acid and B: acetonitrile with 0.1 % formic acid.

[0377] Method: 5 % B for 4 min, gradient 5% B to 23 % B over 1 min, gradient 23 % B to 28 % B over 19 min, gradient 28 % to 100 % B over 2 min followed by washing and equilibrating. The product eluted at 17 min (broad peak) and the total product mass was determined using gravimetric measurements to (11 .93 mg, 0.005366 mmol, 31 % yield). HRMS(ESI): calcd. for C102H119N14O33S2CI2+2H: m / z 11 11 .3506 found 11 11 .3523. Evaluation of purity: D-C-F was analyzed by analytical RP-HPLC equipped with a InfinityLab Poroshell 120 EC-C18 column and using a solvent system consisting of A: MQ with 0.1 % formic acid and B: acetonitrile with 0.1 % formic acid.

[0378] Method: 7% B for 6 minutes, gradient 7% B to 30 % B over 3 min, gradient 30 % B to 100 % B over 1 1 min followed by washing and equilibrating for a total run time of 31 min. The product was detected at 210 nm and eluted after 16.9 min.

[0379] Example 12 Conjugates comprising teicoplanin and dalbavancin against S. aureus With the broad spectrum of activity confirmed for V-S-M, the present inventors set out to develop further conjugates by changing the glycopeptide targeting agent.

[0380] Dalbavancin and teicoplanin (figure 12A and 12B) are examples of glycopeptides with the same mechanism of action as vancomycin, but different binding affinities, pharmacokinetics and antibacterial activities (Butler, M.S et al 2014). We therefore envisioned that both teicoplanin and dalbavancin could be used as targeting agents analogously to vancomycin in the V-S-M conjugate. Teicoplanin and dalbavancin both have a carboxylic acid and a secondary amine that could be used for conjugation of mitomycin C. Due to ease of synthesis the inventors started by conjugation through the secondary amines forming conjugates between teicoplanin and mitomycin C (T-N-M) and dalbavancin and mitomycin C (D-N-M) (Figure 13). The syntheses started with the formation of a previously used amine reactive disulfide linker in 2 steps that has mitomycin C in one end and p-nitrophenyl carbonate in the other end. This linker was added to teicoplanin and dalbavancin, and stirred under basic conditions followed by purification using preparative RP-HPLC. The teicoplanin conjugate purification was further complicated by teicoplanin being supplied as a mixture with different lengths of the alkyl tail (figure 12B). Conjugates with different lipophilic tails can be differentiated by their mass using MS spectroscopy. Three different versions of the teicoplanin - mitomycin C conjugate were therefore isolated: teicoplanin with a 9 carbon long tail (TC9-N-M), teicoplanin with a 10 carbon long tail (TC10-N-M) and teicoplanin with a mixture of 9 and 10 carbon long tails (Tmix-N-M). All conjugates (TC9-N-M, TC10-N-M, Tmix-N-M and D-N-M) were isolated in excellent purity (Figure 14) and could be cleaved with DTT to release mitomycin C (Figure 15).

[0381] The new conjugates were tested against S. aureus using the previously established protocol consisting of: 1 ) treatment of the bacteria in buffer with the conjugates, mitomycin C or control for 15 min, 2) washing to remove unbound compounds and 3) a 24h incubation followed by quantification of colony forming units. The activity of TC9-N- M (Figure 16), TC10-N-M (Figure 17) and Tmix-N-M (Figure 18) against the bacteria showed a similar pattern. Without the addition of NAC the reduction in bacterial load was less than 1 -log fold, which was not significantly different from treatment with equimolar mitomycin C, however upon treatment with NAC the conjugates were significantly more potent than treatment with mitomycin C. All the three T-N-M conjugates show around a 2-log reduction in CFU and as expected no major difference between conjugates with different tail lengths was observed. Treatment with the D-N-M conjugate was also ineffective without addition of NAC, but with NAC the conjugate caused an impressive 4-log reduction in CFU (Figure 19), which was significantly more than treatment with equimolar mitomycin C and dalbavancin.

[0382] Next we set out to see if changing the conjugation site from the secondary amine to the carboxylic acid of dalbavancin would significantly alter the activity of the drug. The synthesis (Figure 20) starts from 2-mercaptoethanol and goes through a heterobifunctional disulfide linker (compound 4) to form mitomycin C with an amine handle (compound 6), which is coupled to the carboxylic acid of dalbavancin using HOBt and EDC in the final step resulting in the conjugate named D-C-M. The final conjugate showed excellent purity by HPLC analysis (Figure 21 ) and could be cleaved using DTT to release pristine mitomycin C (Figure 22).

[0383] D-C-M was tested against S. aureus in the previously used protocol consisting of 15 min treatment, washing and then 24h incubation (Figure 23). This new conjugate exhibited an antibacterial activity that was significant even without the addition of NAC and it was superior to equimolar mitomycin C treatment when treating with 0.5 mg / L. With addition of NAC the activity of D-C-M was further enhanced and the bacterial load was reduced below the detection limit in 3 out of 4 replicates at the lowest tested concentration of 0.5 mg / L. Both the carboxylic acid site on dalbavancin and the optimal primary amine site on vancomycin used for V-S-M are located on the vancosamine sugar on their respective glycopeptide. Thus it is a general trend that this conjugation site is preferred when using these glycopeptides as targeting agents. Earlier data from V-S-M under similar experimental conditions showed that at the concentration of 0.5 mg / L (mitomycin C equivalent concentration) only around a 2-log reduction in CFU is observed meaning that the D-C-M is more potent and therefore the new lead drug candidate. Example 13 Conjugates comprising different antibacterial agent against S. aureus Having successfully shown that multiple different glycopeptide targeting agents yielded functional and potent drug conjugates we turned our attention to the other side of the conjugates: The potent antibiotic agent. Mitomycin C was chosen at first because it had been shown to kill persister cells at very low concentrations (Kwan, B.W et al 2015). Other studies however show that other antineoplastic drugs such as floxuridine (see structure in Figure 12D) and gemcitabine also exhibit potent antibacterial activity (Sandrini, M.P.B et al 2007 and Pertusati, F. et al 2020) against gram positive bacteria. S. aureus were therefore treated with both floxuridine and gemcitabine at varying concentrations to determine MIC and MBC values in nutrient rich media (live, actively growing bacteria) (Table 4). Here we saw the highest potency for floxuridine, particularly in the nutrient-rich minimal media where bacteria need to synthesize their own nucleotides and cannot acquire them from the media. We then set out to synthesize a new conjugate using the most promising targeting agent, dalbavancin with conjugation at the carboxylic acid, to deliver this new antineoplastic and antibiotic agent, floxuridine, to bacteria.

[0384] Table 4. MIC / MBC of floxuridine, gemcitabine, dalbavancin and the combination of equimolar dalbavancin and floxuridine against S. aureus (29213) after 24 h incubation in nutrient-rich BHI, nutrient-rich minimal media and nutrient-free buffer. Fields containing have not been measured or are not relevant.

[0385] The synthesis started by reacting floxuridine with p-nitrophenyl chloroformate forming a reactive carbonate on one of the alcohols (Figure 24, compound 7). This carbonate was then reacted with 2,2'-dithiobisethanamine forming a urea bond and resulting in a disulfide reactive self-immolative linker with an amine handle (compound 8). This amine handle was used for coupling to the carboxylic acid of dalbavancin with HOBt and EDC forming the final conjugate between dalbavancin and floxuridine, D-C-F. The isolated compound showed excellent purity on HPLC (Figure 25).

[0386] First D-C-F was used to treat S. aureus at different concentrations in nutrient-free buffer with addition of NAC using the established 15:24 protocol with 15 min treatment followed by washing and then a 24h incubation period (Figure 26). The conjugate proved highly potent causing a 3-log or higher reduction in CFU for all samples at a dose of only 0.25 mg / L (equivalent floxuridine). In the same experimental setup no antibacterial activity was observed for equimolar floxuridine nor the combination of floxuridine and dalbavancin, highlighting the benefit of targeted drug delivery through conjugation. This result is especially noteworthy when compared with the MBC values for floxuridine, dalbavancin and an equimolar combination of the two in nutrient-free buffer (table 4). The MBC of floxuridine is >32 mg / L, for dalbavancin it is 128 mg / L, and the combination of the two had an MBC of 8 mg / L floxuridine and 59 mg / L dalbavancin. The conjugate on the other hand had a significant bactericidal activity at a concentration of only 0.125 mg / L, which is 64 times lower than the treatment with free unconjugated dalbavancin and floxuridine, despite only having a 15 min time frame to bind to the bacteria in the experimental setup used. This vast increase in potency is unlikely to be explained simply by up concentration of floxuridine at the bacterial surface due to targeting. More likely it is the interaction between dalbavancin and the bacterial membrane that aids in uptake of floxuridine in the inactive persister cells.

[0387] It was also observed that floxuridine is exceptionally potent, with an MBC value of only 0.05 mg / L, when treating bacteria in minimal media (table 4), which contains the necessary components to allow for bacterial growth but does not contain nucleotides that could potentially compete with floxuridine. This is promising for the use of the D-C- F conjugate as a general antibiotic agent. Next we set out to see if NAC was necessary to see an antibacterial activity (Figure 27). Even at a concentration of 16 mg / L (floxuridine equivalent) the conjugate exhibited no antibacterial activity without NAC showing that drug release is essential for activity. References

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[0406] Walther R, Nielsen SM, Christiansen R, Meyer RL, Zelikin AN. Combatting implant- associated biofilms through localized drug synthesis. J Control Release. 2018 Oct 10;287:94-102. doi: 10.1016 / j. jeon rel.2018.08.025. Epub 2018 Aug 20. PMID: 30138714; PMCID: PMC6176123.

[0407] Yu L, Shang Z, Jin Q, Chan SY, Hong W, Li N, Li P. Antibody-Antimicrobial Conjugates for Combating Antibiotic Resistance. Advanced Healthcare Materials. 2023 Jan;12(1 ):2202207.

[0408] Items

[0409] 1. A compound comprising: a. a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin; b. one or more antibacterial agents, such as one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0410] 2. A compound comprising: a. a glycopeptide antibiotic selected from vancomycin, dalbavancin or teicoplanin; b. one or more antibacterial agents selected from one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0411] 3. A compound comprising: a. a glycopeptide antibiotic selected from vancomycin, dalbavancin or teicoplanin; b. one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

[0412] 4. A compound comprising: a. a vancomycin; b. one or more antibacterial agents, such as such as one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the vancomycin and the one or more antibacterial agents.

[0413] 5. A compound comprising: a. a mitomycin C; b. one or more antibacterial agents, such as one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the mitomycin C and the one or more antibacterial agents.

[0414] 6. The compound according to any one of the preceding items, wherein the glycopeptide antibiotic is vancomycin.

[0415] 7. The compound according to any one of the preceding items, wherein the glycopeptide antibiotic is dalbavancin.

[0416] 8. The compound according to any one of the preceding items, wherein the glycopeptide antibiotic is teicoplanin. 9. The compound according to any one of the preceding items, wherein the vancomycin has the structure of formula (1 )

[0417] 10. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (2) wherein one or more of Ri, R2 and R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents. 1 1 . The compound according to any one of the preceding items, wherein R1 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0418] 12. The compound according to any one of the preceding items, wherein R2 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0419] 13. The compound according to any one of the preceding items, wherein R3comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0420] 14. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (3) wherein R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0421] 15. The compound according to any one of the preceding items, wherein the dalbavancin has the structure of formula (15)

[0422]

[0423] 16. The compound according to any one of the preceding items, wherein the teicoplanin has the structure of formula (16) wherein R is:

[0424] 17. The compound according to any one of the preceding items, wherein the mitomycin C has the structure of formula (4)

[0425] 18. The compound according to any one of the preceding items, wherein the mitomycin C has the structure of formula (5) and wherein R comprises or consists of the biodegradable linker and the one or more antibacterial agents.

[0426] 19. The compound according to any one of the preceding items, wherein the one or more antibacterial agents is one or more antineoplastic drugs, such as one or more antineoplastic antibiotics.

[0427] 20. The compound according to any one of the preceding items, wherein the one or more antineoplastic drugs is floxuridine. 21 . The compound according to any one of the preceding items, wherein the floxuridine has the structure of formula (17)

[0428] 22. The compound according to any one of the preceding items, wherein the one or more antibacterial agents is one or more DNA alkylating agents.

[0429] 23. The compound according to any one of the preceding items, wherein the one or more DNA alkylating agents is an Aminoquinone, such as streptonigrins or mitomycin C.

[0430] 24. The compound according to any one of the preceding items, wherein the one or more DNA alkylating agents is mitomycin C.

[0431] 25. The compound according to any one of the preceding items, wherein the mitomycin C has the structure of formula (4)

[0432] 26. The compound according to any one of the preceding items, wherein the mitomycin C has the structure of formula (5) and wherein R comprises or consists of the biodegradable linker and the glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin.

[0433] 27. The compound according to any one of the preceding items, wherein the one or more DNA alkylating agents is selected from the group consisting of: Duocarmycins and Pyrrolobenzodiazepines.

[0434] 28. The compound according to any one of the preceding items, wherein the one or more Pyrrolobenzodiazepines is selected from the group consisting of: SG3199, SJG-136, SG2057, Tomaymycin DM, Hemiasterlin, Aniline-MPB-amino-C3- PBD and Py-MPB-amino-C3-PBD.

[0435] 29. The compound according to any one of the preceding items, wherein the biodegradable linker can release the one or more antibacterial agents from the vancomycin upon contact with a thiol group.

[0436] 30. The compound according to any one of the preceding items, wherein the biodegradable linker can release the one or more antibacterial agents from the vancomycin upon contact with a bacterial cell.

[0437] 31 . The compound according to any one of the preceding items, wherein said biodegradable linker can release the one or more antibacterial agents from the vancomycin without contact with a release-agent.

[0438] 32. The compound according to any one of the preceding items, wherein the biodegradable linker is a disulfide linker. 33. The compound according to any one of the preceding items, wherein the disulfide linker comprises or consists of the structure of formula (6)

[0439] 34. The compound according to any one of the preceding items, wherein the disulfide linker comprises or consists of the structure of formula (7)

[0440] (7).

[0441] 35. The compound according to any one of the preceding items, wherein said biodegradable linker can release the one or more antibacterial agents from the vancomycin upon contact with a release-agent.

[0442] 36. The compound according to any one of the preceding items, wherein said biodegradable linker can release the one or more antibacterial agents from the vancomycin without contact with a release-agent.

[0443] 37. The compound according to any one of the preceding items, wherein the release-agent is N-acetyl cysteine (NAC).

[0444] 38. The compound according to any one of the preceding items, wherein the release-agent is dithiothreitol (DTT).

[0445] 39. The compound according to any one of the preceding items, wherein Ri comprises or consists of a structure of formula (8)

[0446] The compound according to any one of the preceding items, wherein R2 comprises or consists of a structure of formula (9) The compound according to any one of the preceding items, wherein R3comprises or consists of a structure of formula (9) The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (10)

[0447] or a pharmaceutically acceptable salt thereof. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (11 ) or a pharmaceutically acceptable salt thereof. The compound according to any one of the preceding items , wherein the compound comprises or consists of a structure of formula (12)

[0448] or a pharmaceutically acceptable salt thereof. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (13) or a pharmaceutically acceptable salt thereof. The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a vancomycin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker. 47. The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a dalbavancin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0449] 48. The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a teicoplanin; b. one or more antineoplastic drugs, such as floxuridine, and / or one or more DNA alkylating agents, such as mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0450] 49. The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a dalbavancin; b. a mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0451] 50. The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a teicoplanin; b. a mitomycin C; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0452] 51 . The compound according to any one of the preceding items, wherein the compound comprises or consists of a. a dalbavancin; b. a floxuridine; and c. a biodegradable linker linking the vancomycin and the mitomycin C, optionally wherein the biodegradable linker is a disulfide linker.

[0453] 52. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (18) or a pharmaceutically acceptable salt thereof. 53. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (19)

[0454] R=

[0455] (19), wherein the group “TEI” represents where the compound via teicoplanin is connected to the R group, or a pharmaceutically acceptable salt thereof. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (20)

[0456] or a pharmaceutically acceptable salt thereof.

[0457] 55. The compound according to any one of the preceding items, wherein the compound comprises or consists of a structure of formula (14) or a pharmaceutically acceptable salt thereof.

[0458] 56. The compound according to any one of the preceding items, wherein the compound binds to or is capable of binding to a bacterial cell.

[0459] 57. The compound according to any one of the preceding items, wherein the bacterial cell is a Gram-positive bacterial cell, such as Staphylococcus aureus.

[0460] 58. The compound according to any one of the preceding items, wherein the bacterial cell is a planktonic bacterial cell.

[0461] 59. The compound according to any one of the preceding items, wherein the bacterial cell is a bacterial cell capable of forming biofilm. 60. The compound according to any one of the preceding items, wherein the bacterial cell is a bacterial persister cell.

[0462] 61 . The compound according to any one of the preceding items, wherein the bacterial cell is selected from the group consisting of: Staphylococcus spp., Bacillus spp., Listeria spp., Streptococcus spp., Corynebacterium spp., and Enterococcus spp.

[0463] 62. The compound according to any one of the preceding items, wherein the one or more antibacterial agents has a low off target effect compared to one or more antibacterial agents not linked to said vancomycin.

[0464] 63. The compound according to any one of the preceding items, wherein the off target effect is killing non-bacterial cells.

[0465] 64. The compound according to any one of the preceding items, wherein the non- bacterial cells are derived from cat, dog, rabbit, donkey, cow, fish, sheep, horse, goat, or pig.

[0466] 65. The compound according to any one of the preceding items, wherein the non- bacterial cells are derived from a human or a non-human primate.

[0467] 66. A pharmaceutical composition comprising the compound according to any one of the preceding items or a pharmaceutically acceptable salt thereof.

[0468] 67. A pharmaceutical composition comprising the compound according to any one of the preceding items and one or more pharmaceutically acceptable excipients.

[0469] 68. The compound or the pharmaceutical composition according to any one of the preceding items for use as a medicament.

[0470] 69. The compound or the pharmaceutical composition according to any one of the preceding items for use in the treatment of a bacterial infection. 70. The compound or the pharmaceutical composition for use according to any one of the preceding items, wherein the bacterial infection is an implant associated bacterial infection.

[0471] 71 . The compound or the pharmaceutical composition according to any one of the preceding items for use in the treatment of a bacterial biofilm.

[0472] 72. Use of the compound or the pharmaceutical composition according to any one of the preceding items in the manufacture of a medicament for treatment of a bacterial infection.

[0473] 73. A method for treatment of bacterial infection comprising administration of a therapeutically effective amount of the compound or the pharmaceutical composition according to any one of the preceding items to an individual in need thereof.

[0474] 74. The method or use according to any one of the preceding items, wherein the bacterial infection is an implant associated infection.

[0475] 75. A kit of parts comprising: a. the compound according to any one of the preceding items; and b. instructions for use.

[0476] 76. A method of manufacturing the compound according to any one of the preceding items, the method comprising the steps of: a. providing a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and one or more antibacterial agents; and b. conjugating said glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and said one or more antibacterial agents with a biodegradable linker.

Claims

Claims1. A compound comprising: a. a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin; b. one or more antibacterial agents selected from one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; and c. a biodegradable linker linking the glycopeptide antibiotic and the one or more antibacterial agents.

2. The compound according to claim 1 , wherein the glycopeptide antibiotic is vancomycin.

3. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (2)wherein one or more of Ri, R2 and R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

4. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (3)wherein R3 comprises or consists of the biodegradable linker and the one or more antibacterial agents.

5. The compound according to claim 1 , wherein the glycopeptide antibiotic is dalbavancin.

6. The compound according to claim 5, wherein the dalbavancin has the structure of formula (15)7. The compound according to claim 1 , wherein the glycopeptide antibiotic is teicoplanin.

8. The compound according to claim 7, wherein the teicoplanin has the structure of formula (16)wherein R is:

9. The compound according to any one of the preceding claims, wherein the one or more DNA alkylating agents is an Aminoquinone, such as streptonigrins or mitomycin C.

10. The compound according to any one of the preceding claim, wherein the one or more DNA alkylating agents is mitomycin C.11 . The compound according to any one of the preceding claims, wherein the mitomycin C has the structure of formula (5)and wherein R comprises or consists of the biodegradable linker and the glycopeptide antibiotic selected from vancomycin, dalbavancin or teicoplanin.

12. The compound according to any one of the preceding claims, wherein the one or more antineoplastic drugs is floxuridine.

13. The compound according to claim 12, wherein the floxuridine has the structure of formula (17)14. The compound according to any one of the preceding claims, wherein the biodegradable linker can release the one or more antibacterial agents from the vancomycin upon contact with a bacterial cell and / or wherein the biodegradable linker can release the one or more antibacterial agents from the vancomycin without contact with a release-agent.

15. The compound according to any one of the preceding claims, wherein the biodegradable linker is a disulfide linker, optionally wherein the disulfide linker comprises or consists of the structure of formula (6)16. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (12)or a pharmaceutically acceptable salt thereof.

17. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (18)or a pharmaceutically acceptable salt thereof.

18. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (19)R=(19), wherein the group “TEI” represents where the compound via teicoplanin is connected to the R group, or a pharmaceutically acceptable salt thereof.

19. The compound according to any one of the preceding claims, wherein the compound comprises or consists of a structure of formula (20)(20), or a pharmaceutically acceptable salt thereof.

20. A pharmaceutical composition comprising the compound according to any one of the preceding claims or a pharmaceutically acceptable salt thereof.21 . The compound or the pharmaceutical composition to any one of the preceding claims for use as a medicament.

22. The compound or the pharmaceutical composition according to any one of the preceding claims for use in the treatment of a bacterial infection, such as an implant associated bacterial infection.

23. The compound or the pharmaceutical composition according to any one of the preceding claims for use in the treatment of a bacterial biofilm.

24. A method of manufacturing the compound according to any one of the preceding claims, the method comprising the steps of: a. providing a glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and one or more antibacterial agents selected from one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents; andb. conjugating said glycopeptide antibiotic, such as vancomycin, dalbavancin or teicoplanin, and said one or more antibacterial agents selected from one or more antineoplastic drugs, such as one or more antineoplastic antibiotics, and / or one or more DNA alkylating agents with a biodegradable linker.

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