WO2018134412A1

WO2018134412A1 - Antimicrobial compounds and uses thereof

Info

Publication number: WO2018134412A1
Application number: PCT/EP2018/051476
Authority: WO
Inventors: Urs Spitz; Lukas Wick; Julian Ihssen; Thomas Mayer
Original assignee: Biosynth Ag
Priority date: 2017-01-20
Filing date: 2018-01-22
Publication date: 2018-07-26

Abstract

The present invention generally relates to a class of antimicrobial compounds, to compositions containing said compounds and to the use of said compounds for medical and non-medical purposes. More specifically, the present invention relates to antimicrobial compounds capable of changing microbiota composition and function by selectively inhibiting distinct microbial species.

Description

ANTIMICROBIAL COMPOUNDS AND USES THEREOF

FIELD OF THE INVENTION

[0001 ] The present invention generally relates to a new class of antimicrobial compounds, to compositions containing said compounds and to the use of said compounds for medical and non-medical purposes. More specifically, the present invention relates to antimicrobial compounds capable of changing microbiota composition and function by selectively inhibiting distinct microbial species.

BACKGROUND OF THE INVENTION

[0002] Over hundreds of millions of years of co-evolution, microbial communities (microbiota) have developed that live in close association with their hosts (e.g., animals or plants). It is now generally recognized that microbiota colonizing humans and animals play a key role in the host's health and disease state. The human gut, for example, harbors one of the most complex microbiota, and disturbance of the normal gut microbiology have been implicated in many health and disease issues. These findings have spurred intensive research to find ways of influencing microbiota to result in human health benefits.

[0003] Early attempts to alter microbiota composition primarily involved the use of probiotic bacteria (e.g., Lactobacilli or Bifidobacteria) or prebiotic food components (e.g., nondigestable oligosaccharides) as food or feed additives. However, these approaches are limited in that they target undefined clinical indications by unknown mechanisms of action and often do not evoke the desired positive health effect. In case of probiotics, there is also a potential risk for transfer of drug-resistant genes or harmful infection and the problem of inconsistent product quality, leading to differing results.

[0004] Another approach is based on a medical procedure known as fecal microbiota transplantation (FMT). This procedure involves the infusion of a fecal suspension from a healthy donor into the gastrointestinal (Gl) tract of a patient to restore the intestinal microbiota. It was found that FMT can have some therapeutic effects on gastrointestinal disorders, e.g. infections caused by Clostridium difficile. However, the efficacy of FMT remains in question. In addition, FMT is cumbersome, unpleasant and carries the risk of spreading infectious diseases.

[0005] A more recent strategy to modulate the microbiota composition and function is based on the use of antimicrobial peptides (AMPs). AMPs are promising alternatives to conventional antibiotics because of their natural, broad-spectrum antimicrobial properties and low propensity for development of induced resistance. AMPs, for example, have been demonstrated to positively modulate the intestinal microbiota and to alter the oral microbiota for caries prevention. However, AMPs have disadvantages that limit their use, including hemolytic activity toward human cells, rapid turnover in the human body, reduced activity due to their fragile chemical nature and/or sensitivity to the environment, limited scope of application and/or high cost of production.

[0006] Due to the above limitations, it was also continued to make intensive efforts to develop small-molecule antimicrobials as alternatives to existing antimicrobial compounds. However, the discovery of novel small-molecule antimicrobial compounds by conventional high throughput screening (HTS) approaches using small-molecule libraries has been disappointingly slow. Therefore, scientists were compelled to look for new options for identifying novel antimicrobials. Among other things, they explored strategies for exploiting beneficial and commensal bacteria in microbiota, including the development of generic systems based on the principle of microbial auto-inhibition (i.e. the self-inhibition of a given microbial species by an inhibitory substance generated by the species itself).

[0007] A feasible auto-inhibitory strategy is, for example, based on the use of a microbial inhibitor (also referred as "microbial biocide" or "antimicrobial compound") that is masked with an enzyme-labile chemical group to abolish the compound's inhibitory activity. Once such a masked inhibitor is contacted with a mixed population or community of microorganisms (e.g., with a microbiota), the masked inhibitor will be selectively unmasked by the enzymatic action of some microbial species of the microbial population or community, thereby resulting in self-inflicted inhibition of the growth of the respective microbial species (i.e. leading to "auto-inhibition"). However, this auto-inhibitory approach suffers from at least two major drawbacks.

[0008] A first drawback is the difficulty of masking a microbial inhibitor such as to fully eliminate its inhibitory activity. For example, US 2014/0178923 proposes masking of the phenolic group of triclosan with enzyme-labile chemical groups such as sugars of the pyranose type. However, as is evident from the relatively low MIC (minimum inhibitory concentration) values reported for glycoside derivatives of triclosan in bacterial species lacking the enzymatic activity required for cleaving the enzyme-labile group, e.g. Salmonella spp., the inhibitory activity of triclosan is not eliminated but only reduced.

[0009] The unwanted residual inhibitory activity may be explained by the fact that the phenol groups of triclosan are not the sole parts of the structure that contribute to inhibitory activity. Hence, in order to be a promising auto-inhibitor, the microbial inhibitor must contain a structural element which (i) is crucial for its antimicrobial activity and (ii) can be masked by a chemical group so as to fully eliminate the inhibitor's activity.

[0010] Another drawback is that the free microbial inhibitor that is released by the enzymatic action of targeted microorganisms may diffuse into the surrounding habitat to indiscriminately exert its antimicrobial effects. This may lead to the undesired inhibition of non-targeted microorganisms, thereby compromising the desired selective inhibition of one or some microbial species in the respective microbiota.

[001 1 ] Despite the efforts in the art as described above, there remains a continuous need for new antimicrobial compounds which are suitable for medical use and/or non-medical use. For example, it would be highly desirable to have small- molecules which can be manufactured at low cost and exhibit a high antimicrobial efficacy while avoiding all or some of the drawbacks associated with known antimicrobials such as insufficient efficacy, lack of specificity, toxicity, microbial resistance, unfavorable pharmacokinetics and side-effects.

OBJECT OF THE INVENTION

[0012] It is therefore an object of the present invention to provide new antimicrobial agents for medical and/or non-medical use.

SUMMARY OF THE INVENTION

[0013] According to the present invention, the above object is achieved by a compound comprising an enzyme-labile group as a first moiety and an antimicrobial compound (also referred to as "inhibitor (structure)", "microbial inhibitor (structure)", or "biocide"), wherein the first moiety is covalently linked to the second moiety via an enzyme-labile bond. Optionally, the compound may comprise a spacer (linker), wherein the first moiety is covalently linked to the spacer by an enzyme-labile bond, and wherein the spacer is further covalently linked to the second moiety.

[0014] The first moiety, optionally together with the optional spacer, preferably substantially eliminates the antimicrobial activity of the second moiety. The first moiety is, for example, an amino acid, peptide or carbohydrate (e.g., a sugar), and the enzyme-labile bond is susceptible to cleavage by an enzyme (e.g., a hydrolase). Upon uptake of the compound of the present invention into a target microbial cell, the compound is "unmasked" by cleavage of the enzyme-labile bond through an enzyme of the target microbial cell, thereby releasing the second moiety (the "enzyme-labile group") or, if the compound of the present invention includes a spacer, a spacer- substituted second moiety. The spacer-substituted second moiety is not a stable intermediate and typically undergoes self-immolation, resulting in the release of the second moiety. The released (or "free") second moiety is now capable of exerting its antimicrobial action to specifically inhibit the target microbial cell. [0015] In a first aspect, the present invention relates to a compound as described above, wherein the second moiety has the formula (I)

wherein:

R¹ is O, S or NH;

X is C or N, wherein if X is C, then R¹ is O;

R² forms together with X and the carbon atom bonded to R¹ a 5-, 6- or 7- membered partially saturated or unsaturated heterocyclic ring containing one, two or three heteroatoms independently selected from N, O and S, which 5-, 6- or 7-membered heterocyclic ring may optionally be fused with a 5- or 6 membered saturated, partially unsaturated or unsaturated carbocyclic or heterocyclic ring, wherein said 5-, 6- or 7-membered heterocyclic ring and/or said fused 5- or 6-membered carbocyclic or heterocyclic ring may be unsubstituted or substituted by one or more R³;

R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C₃-₆cycloalkylC_0-6alkyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl, C_0-6alkylNR¹⁴R¹⁵, OCi- 6alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵, SR⁵, (SO₂)NR⁵R⁶, (SO)NR⁵R⁶, SO₃R⁵, SO₂R⁵, SOR⁵ or a 5- or 6-membered heterocyclic saturated, partially unsaturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic ring containing one or more heteroatoms independently selected from N, O or S; and

R⁴, R⁵ and R⁶ are independently selected from the group consisting of H, d- 4alkyl, heteroarylC_0-6alkyl and arylC_0-6alkyl;

or tautomers or salts thereof. [0016] Preferred second moieties include moieties having formula (II) or formula (III)

wherein:

X is C, O, N or S, wherein if X is C, then R⁸ is defined as R³, wherein if X is N, then R⁸ is absent or defined as R³, wherein if X is O or S, then R⁸ is absent; R⁷ is O, S or NH;

R⁹, R¹⁰, and R¹¹ are each independently defined as R³;

or tautomers or salts thereof.

[0017] In a second aspect, the present invention provides a composition comprising a compound of the present invention. The composition may be, for example, a pharmaceutical composition.

[0018] In a third aspect, the present invention provides a kit comprising a compound of the present invention and, optionally, instructions for use.

[0019] In a fourth aspect, the present invention provides a method for changing the microbial composition of microbiota comprising contacting said microbiota with a compound of the present invention.

[0020] In a fifth aspect, the present invention relates to an in vitro use of a compound of the present invention as an antimicrobial compound.

[0021 ] In a sixth aspect, the present invention relates to a use of a compound of the present invention

for the selective culturing, plating, enrichment or growth of microorganisms, for example in food, water or environmental safety testing; to discover, determine, isolate, or investigate pathogenic microbial species, for example in research or clinical diagnostics;

in culturing of animal, human and plant cells or tissues and plans to avoid contamination with unwanted organisms;

as a microbiota-modifying agent in in the processing (fermentation) of feed, food or other organic matter where the development of chemical components, smells, flavors, colors, shelf live and/or consistency depends on the diversity and composition of microbiota;

as a microbiota-altering agent in the treatment of waste water or solid waste, composting, or the production of fuel from organic matter where optimized microbiota are relevant to yield, efficacy and process quality; as a microbiota-optimizing agent in farming of animals and plants; and as a supplement to pre- and/or probiotics to improve microbiota of animals and humans.

[0022] In a seventh aspect, the present invention relates to a use of a compound of the present invention for cosmetic applications, in particular cosmetic applications caused by dysbiosis of the skin, scalp or oral cavity, such as seborrheic dermatitis, acne vulgaris, acne rosacea, caries, periodontal diseases, and halitosis.

[0023] In an eighth aspect, the present invention provides a compound for use in therapy, in particular for use in the treatment or prevention of

gastrointestinal diseases, such as inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), enterocolitis, and colorectal cancer;

bacterial gastrointestinal infections, such as salmonellosis, listeriois, shigellosis, infection by enterohemorrhagic or enteroinvasive E. coli, or Clostridium difficile infections (CDI);

cardiovascular diseases, such as cerebrovascular disease, myocardial infarction, and stroke;

autoimmune diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, multiple sclerosis, and Celiac disease; neurological disorders, such as Alzheimer's disease, autistic spectrum disorders, and Parkinson's disease;

metabolic diseases, such as metabolic syndrome, autoimmune type 1 diabetes (T1 D), and insulin resistant type 2 diabetes (T2D);

neoplastic diseases, such as ulcers, adenocarcinomas, gastric B-cell lymphomas, and colorectal carcinoma;

conditions caused by dysbiosis of vaginal microbiota (VMB), such as vaginal discharge, poor pregnancy outcomes, pelvic inflammatory disease, post-operative infections, endometritis following elective abortions, and sexually transmitted diseases (STDs); and

other disorders like obesity, chronic fatigue syndrome, and atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above mentioned and other features and objects of this invention and the manner of achieving them will become more apparent and this invention itself will be better understood by reference to the following detailed description of this invention taken in conjunction with the accompanying drawings, in which:

[0025] Figure 1 shows the chemical formulas of triclosan (1 ), deferasirox (2), deferiprone (3), oxypyrion (4), pyrithione (5), and 2-aminopyridine-N-oxide (6);

[0026] Figure 2 shows the chemical formulas of different beta-D-galacto- pyranosides, i.e. triclosan-beta-D-galactopyranoside (1a), deferasirox-beta-D- galactopyranoside (2a), deferiprone-beta-galactopyranoside (3a), oxypyrion-beta-D- galactopyranoside (4a), pyrithione-0-beta-D-galactopyranoside (5a), and pyrithione- S-beta-D-galactopyranoside (5b);

[0027] Figure 3 shows the chemical formulas of 4-(pyrithione-S-methyl)phenyl- beta-D-galactopyranoside (5Sa) and L-alanine(4-(pyrithione-S-methyl)phenyl)amide (5Sb), both of which contain a self-immolative spacer (-C₆H₄-CH₂-) separating the enzyme-labile group (beta-D-galactopyranose) from the pyrithione inhibitor; [0028] Figure 4 shows the general formulas of possible tautomeric forms of microbial inhibitor structures used in the present invention (la-d, lla-b, llla-b);

[0029] Figure 5 shows the chemical formulas of preferred microbial inhibitors and tautomers thereof for use in the present invention (3/3', 4/4', 5/5', 6/6');

[0030] Figure 6 shows the spacer structures between the inhibitor structure (pyrithione) and the enzyme-labile group (beta-D-galactopyranoside and L-alanine, respectively) of the exemplary compounds 5Sa and 5Sb;

[0031 ] Figure 7 is a scheme showing the synthesis of triclosan-beta-D- galactopyranoside (1a);

[0032] Figure 8 is a scheme showing the synthesis of deferasirox-beta-D- galactopyranoside (2a);

[0033] Figure 9 is a scheme showing the synthesis of deferiprone-beta-D- galactopyranoside (3a), oxypyrion-beta-D-galactopyranoside (4a), pyrithione-0-beta- D-galactopyranoside (5a), and pyrithione-S-beta-D-galactopyranoside (5b);

[0034] Figure 10 is a scheme showing the synthesis of 4-(pyrithione-S- methyl)phenyl-beta-D-galactopyranoside (5Sa) ;

[0035] Figure 11 is a scheme showing the synthesis of L-alanine(4-(pyrithione-S- methyl)phenyl)amide (5Sb);

[0036] Figure 12 is a diagram showing the effect of selected enzymes on mixtures containing enzyme-responsive (i.e. masked) inhibitor compounds and iron (III) (the released inhibitor forms coloured iron complexes which increase absorption of light); (a): compound 3a with and without beta-galactosidase; (b): compound 4a with and without beta-galactosidase; (c): compound 5a with and without beta-galactosidase; (d): compound 5Sb with and without aminopeptidase M;

[0037] Figure 13 is a representation showing the effect of the enzyme-responsive compound triclosan-beta-D-galactopyranoside (1a) on beta-galactosidase negative Salmonella enteritidis (S.e.) and beta-galactosidase positive Escherichia coli {E.c); shown is the growth of a mixture of S.e. and E.c. and of S.e. alone on nutrient agar without 1a (control; left part) and with 1a (right part) for 24 h; and

[0038] Figure 14 is a representation showing the effect of the enzyme-responsive compound deferiprone-beta-D-galactopyranoside (3a) on beta-galactosidase negative Salmonella enteritidis (S.e.) and beta-galactosidase positive Escherichia coli {E.c.) after growth on nutrient agar for 23 hours (right part) in comparison to growth of S.e. and E.c. without 3a (control; left part); for comparison strains were also inoculated on a plate containing unmasked inhibitor compound 3 (middle part).

[0039] Figure 15 shows the effect of the enzyme-responsive compound oxypyrion- beta-D-galactopyranoside (4a) on beta-galactosidase negative Salmonella enteritidis (S.e.) and beta-galactosidase positive Escherichia coli {E.c); (a): growth in nutrient broth with 4a and without 4a (control, Ctrl.) after 24 h; for comparison cultures supplemented with unmasked inhibitor compound 4 are also shown; (b): growth of S.e. and E.c. on nutrient agar with 4a (right part) and without 4a (control; left part) after 24 h (Miles-Misra test with individual strains); (c): growth of a mixture of the same S.e. and E.c. on nutrient agar with 4a (right part) and without 4a (control; left part) after 24 h; for comparison the same number of S.e. as in the mixture of S.e. & E.c. was inoculated on a second control plate (middle part) (the agar medium contained iron (III) and Tween 80);

[0040] Figure 16 shows the effect of the enzyme-responsive compound pyrithione- O-beta-D-galactopyranoside (5a) on beta-galactosidase negative Salmonella enteritidis (S.e.) and beta-galactosidase positive Escherichia coli {E.c); (a): growth in nutrient broth with 5a and without 5a (control; left part) after 17 h; for comparison cultures supplemented with unmasked inhibitor compound 5 are also shown; (b): growth of a mixture of the same S.e. and E.c. strains and individual S.e. and E.c. strains on Rambach agar with 5a (right part) and without 5a (control; left part) after 24 h;

[0041 ] Figure 17 shows the effect of the spacer-containing enzyme-responsive compound 4-(pyrithione-S-methyl)phenyl-beta-D-galactopyranoside (5Sa) on beta- galactosidase negative Salmonella enteritidis (S.e.) and beta-galactosidase positive Escherichia coli {E.c); (a): growth in nutrient broth with 5Sa and without 5Sa (control) after 20 h; (b): growth of a mixture of the same S.e. and E.c. strains and individual S.e. and E.c. strains on Rambach agar with 5Sa (right part) and without 5Sa (control; left part) after 24 h (the medium contained 5-bromo-4-chloro-3-indoxyl-beta-D- glucuronic acid for visualization of E.c. (blue colonies));

[0042] Figure 18 shows the effect of the spacer-containing enzyme-responsive compound L-alanine(4-(pyrithione-S-methyl)phenyl)amide (5Sb) on L-alanine aminopeptidase negative Staphylococcus aureus (S.a.), L-alanine aminopeptidase positive Citrobacter freundii (C.f.), and L-alanine aminopeptidase positive Escherichia coli {E.c); (a): growth in nutrient broth with 5Sb and without 5Sb (control) after 24 h, for comparison cultures supplemented with unmasked inhibitor compound 5 are also shown; (b): growth of S.a. and E.c. on nutrient agar with 5Sb and without 5Sb (control) after 24 h (Miles-Misra test);

[0043] Figure 19 shows the effect of the spacer-containing enzyme-responsive compound 4-(pyrithione-S-methyl)phenyl-beta-D-glucopyranosiduronic acid (5Sc) on beta-glucuronidase negative Salmonella enteritidis (S.e.), beta-glucuronidase negative Escherichia coli 0157 LMG 21756 (E.c. 1 ), beta-glucuronidase positive Escherichia coli NM1 (E.c. 2), and beta-glucuronidase positive Escherichia coli ATCC 25922 (E.c. 3); (a) growth in liquid mineral medium with 5Sc and without 5Sc (control) after 24 h, mean values of 3 replicate cultures, error bars represent standard deviations; (b) growth of bacteria on nutrient agar with 5Sc and without 5Sc (control) after 24 h; [0044] Figure 20 shows the effect of the spacer-containing enzyme-responsive compound L-pyroglutamyl(4-pyrithione-S-methyl)phenylamide (5Sd) on pyroglutamyl aminopeptidase negative Salmonella enteritidis (S.e.) and pyroglutamyl aminopeptidase positive Citrobacter freundii (C.f.) bacteria; (a) growth in nutrient broth with 5Sd and without 5Sd (control) after 24 h; (b) growth on nutrient agar with 5Sd and without 5Sd (control) after 24 h; and

[0045] Figure 21 shows the effect of different concentrations of the spacer- containing enzyme-responsive compound 4-(pyrithione-S-methyl)phenyl-phosphate (5Se) on phosphatase positive Staphylococcus aureus.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is based on the surprising finding that compounds of the present invention, comprising a first (masking) moiety, optionally a spacer, and a second (inhibitor) moiety, are capable of controlling the composition and/or diversity of microbiota in vitro and in vivo by selectively inhibiting microbial species. The compounds of the present invention represent a new class of microbial compounds, which are selectively activated by susceptible microbial species or strains (target microbial species or strains) expressing the necessary enzyme function to cleave an enzyme-labile bond. The cleavage of the enzyme-labile bond results in the release of the first moiety, leaving behind the second moiety or a spacer-substituted second moiety which undergoes self-immolation to release the second moiety. The released ("free") second moiety is then capable of exerting its antimicrobial effect to selectively inhibit the growth of said susceptible microbial species or strains.

[0047] The compounds of the present invention are advantageous in that they can be produced in a cost-efficient manner, have a high efficacy and are specific for selected microbial species. Thus, the present invention offers the benefit of providing a broad range of selective antimicrobial compounds for targeting different selected microbial species by inhibiting their growth while not affecting other microbial species. The compounds of the present invention further have the potential to enable the development of personal therapies based on real-time analysis of the composition of a patient's microbiota.

[0048] Another advantage of the compounds of the present invention is that they can be administered or applied in a wide variety of ways. For example, for medical uses, the compounds can be conveniently administered orally, e.g., as a food or feed supplement, or topically. Also, due to their inherent stability, the compounds can be used in a wide range of non-medical applications such as for the selective enrichment of microbial samples, the avoidance of contamination in microbial culturing, and the isolation of microorganisms on plating media.

[0049] In a first aspect, the present invention relates to a compound comprising a first moiety, a second moiety, and optionally a spacer that covalently links the first moiety to the second moiety, wherein the first moiety is covalently linked to the second moiety or to the optional spacer via an enzyme-labile bond, and wherein the second moiety is an antimicrobial compound, and further wherein the second moiety has the formula (I)

wherein:

R¹ is O, S or NH, preferably O or S;

X is C or N, wherein if X is C, then R¹ is O;

or tautomers or salts thereof.

[0050] Additionally, the first moiety, optionally together with the spacer, substantially eliminates the antimicrobial activity of the second moiety. The term "substantially eliminates", as used herein, means that the antimicrobial activity of the second moiety, when part of the compound of the present invention is preferably less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1 %, 0.5%, less than 0.1 % or 0% of the antimicrobial activity of the second moiety that is released after cleavage of the enzyme-labile bond, and optionally self-immolation of the spacer, i.e. of the "free" second moiety. The antimicrobial activity of the (masked) compound of the present invention can be determined as known to those skilled in the art. For example, the Fe(lll) chelation assay described in Example 1 1 may be used. This assay quantifies the ability of the compounds to form colored Fe(lll) complexes and is an indirect measure for their microbial activity. Alternatively, a biological assay as that used in Examples 12 to 19 may be used. Generally, in these assays a bacterial species may be used as test microorganism that is sensitive to the free antimicrobial second moiety but lacks an enzyme that is capable of cleaving the enzyme-labile bond of the compound. The read-out (e.g., growth of microorganisms) in the presence of the free (unmasked) second moiety and of the same but covalently-linked (masked) second moiety (i.e. the compound of the present invention including the same second moiety) is then compared.

[0051 ] The terms "microorganism", "microbial species", "microbial strains" and the like, as used herein, refers to bacteria, blue-green algae, fungi, yeast, protozoa and algae, preferably bacteria. Further, the term "inhibition", as used herein, generally refers to inhibition of the growth of microbial cells (e.g., bacterial cells) by decreasing, slowing or stopping growth of cells. Thus, a compound of this invention usually inhibits cells by preventing cells from dividing and replicating and increasing in number.

[0052] Preferably, R² forms together with X and the carbon atom bonded to R¹ a 5- or 6-membered partially saturated or unsaturated heterocyclic ring containing one or two heteroatoms selected from N, O and S, at least one of the two heteroatoms being N, which 5- or 6-membered heterocyclic ring may optionally be fused with a 5- or 6 membered saturated, partially unsaturated or unsaturated carbocyclic or heterocyclic ring, wherein said 5- or 6-membered heterocyclic ring and/or said fused 5- or 6- membered carbocyclic or heterocyclic ring may be unsubstituted or substituted by one or more R³.

[0053] More preferably, R² forms together with X and the carbon atom bonded to R¹ a 6-membered partially saturated or unsaturated heterocyclic ring containing one or two heteroatoms selected from N, O and S, wherein if X is C, then said 6- membered heterocyclic ring contains N as the only heteroatom, and if X is N, then said 6-membered heterocyclic ring contains said N as the only heteroatom or said N and a second heteroatom selected from N, O and S, which 6-membered heterocyclic ring may optionally be fused with a 5- or 6 membered saturated, partially unsaturated or unsaturated carbocyclic or heterocyclic ring, wherein said 6-membered heterocyclic ring and/or said fused 5- or 6-membered carbocyclic or heterocyclic ring may be unsubstituted or substituted by one or more R³. [0054] Particularly preferred, the second moiety of formula (I) is selected from a compound represented by formula (II) or formula

wherein:

X is C, O, N or S, wherein if X is C, then R⁸ is defined as R³, wherein if X is N, then R⁸ is absent or defined as R³, wherein if X is O or S, then R⁸ is absent;

R⁷ is O, S or NH, preferably O or S;

R⁹, R¹⁰, and R¹¹ are each independently defined as R³, or

R⁹ and R¹⁰, or if X is not O or S, R⁸ and R⁹ or R⁸ and R¹¹ , form together a 5- or

6-membered, preferably 6-membered, saturated, partially saturated or unsaturated carbocyclic ring or heterocyclic ring containing one or two heteroatoms independently selected from N, O or S, preferably from N and O or only O, which 5- or 6-membered carbocyclic ring or 5- or 6-membered heterocyclic ring is unsubstituted or substituted by one or more R³;

or tautomers or salts thereof.

[0055] Preferably, X of the second moiety of formula (II) or formula (III) is C, O or N, more preferably C or N, and most preferably N in case of formula (II) and C in case of formula (III). Furthermore, in a preferred embodiment, R⁹, R¹⁰, and R¹¹ are each independently defined as R³, and R⁹ and R¹⁰, and, if X is not O or S, R⁸ and R⁹ and R⁸ and R¹¹ do not form together a carbocyclic or heterocyclic ring.

[0056] R³ of the second moiety (e.g., of formulas (I) to (III)) is preferably halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C₃-₆cycloalkyl C_0-6alkyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl, C_0-6alkylNR¹⁴R¹⁵, OC_{1 -6}alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵, SR⁵, (SO₂)NR⁵R⁶, (SO)NR⁵R⁶, SO₃R⁵, SO₂R⁵, or SOR⁵; wherein R⁴, R⁵ and R⁶ are independently selected from the group consisting of H, Ci- ₄alkyl, heteroarylC_0-6alkyl and arylC_0-6alkyl.

[0057] More preferably, R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C₂- ₆alkenyl, C_2-6alkynyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl, C_0-6alkylNR¹⁴R¹⁵, OC₁- ₆alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵, SR⁵, (SO₂)NR⁵R⁶, (SO)NR⁵R⁶, SO₃R⁵, SO₂R⁵, SOR⁵; wherein R⁴, R⁵ and R⁶ are independently selected from the group consisting of H and C_{1 -4}alkyl. It is further preferable that R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C_{1 -4}haloalkyl, OC₁- ₄haloalkyl, C_0-6alkylNR¹⁴R¹⁵, OC_{1 -6}alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵; and R⁴, R⁵ and R⁶ are independently selected from the group consisting of H and C_{1 -4}alkyl. It is still further preferable that R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl, CO₂R⁵, O(CO)R⁵; and R⁴ and R⁵ are independently selected from the group consisting of H and C_{1 -4}alkyl.

[0058] Particularly preferably, R³ is halo, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2- ₆alkynyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl or CO₂R⁵; wherein R⁴ and R⁵ are independently selected from the group consisting of H and C_{1 -4}alkyl. Most preferably, R³ is H, halo, OH, C_{1 -6}alkyl, C_{1 -4}alkyl, C_2-6alkenyl, C_2-4alkenyl or OC_{1 -4}alkyl, particularly H, OH or C_{1 -4}alkyl.

[0059] Preferred examples of the second moiety include compounds 3, 4, 5 and 6, in particular 3, 4 and 5) or tautomers (e.g. 3', 4', 5' and 6', or 3', 4' and 5') or salts thereof:

[0060] In accordance with the present invention, the first moiety may be selected from the group consisting of amids, amino acids, peptides, nitro compounds and other groups which by the action of certain microbial enzymes are converted to amines, sulfides and disulfides and other groups which by the action of certain enzymes are converted to mercaptanes, organic esters (i.e. esters derived from an organic acid, in particular a carboxylic acid having, e.g., 2 to 10 carbon atoms, and an alcohol) such as fatty acid esters (e.g., esters of short, medium and long chain fatty acids), inorganic esters (i.e. esters derived from an inorganic acid, in particular, and an alcohol) such as sulfate esters and phosphate esters (e.g., inositol phosphates and choline phosphates), ethers, and carbohydrates such as sugars (e.g., pyranoses) and other groups which are converted by the action of certain microbial enzymes to hydroxyl compounds. The first moiety may also be a phosphate, more specifically phosphoryl. Preferably, the first moiety is a carbohydrate.

[0061 ] Suitable amino acids include, for example, a-amino acids like any one of the 22 proteinogenic (protein-building) amino acids, preferably one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Pyroglutamic acid is another amino acid that may be used within the present invention. In one embodiment, the suitable amino acid may be as defined herein, with the proviso that it is not L-cysteine and/or R-cysteine. In another embodiment, the compound of the present invention is not pyrithion-L-cystein. Preferred a-amino acids include those with a hydrophobic side chains, such as alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryphtophan. Exemplary peptides comprise 2 to 50 amino acids, in particular 3 to 10 or 4 to 6 amino acids.

[0062] The carbohydrate may be a mono-, di- or polysaccharide moiety and is preferably a monosaccharide. A preferred carbohydrate is a sugar. A particularly preferred first sugar moiety is a pyranose such as arabinose, galactose, glucose and maltose. The pyranoses may be bonded to the second moiety or the spacer of the compound of the present invention via an enzyme-labile O- or N-linkage. The second moiety and the optional spacer are generally bonded to the anomeric carbon atom of the pyranoses. Exemplary compounds of the present invention include, but are not limited to, cc-D-arabinopyranosides, β-D-arabinopyranosides, cc-D- galactopyranosides, β-D-galactopyranosides, cc-D-glucopyranosides, β-D- glucopyranosides, and a-D-mannopyranosides. Other exemplary compounds include β-D-glucopyranosiduronic acids, -D-(acetylamino)-2-deoxy-glucopyranosides, and N-acetylneuraminic acids.

[0063] Within the context of the present invention, the first moiety is usually linked via the enzyme-labile bond or via the optional spacer to:

(i) the oxygen atom bonded to X of the second moiety having formula (I),

(ii) the exocyclic heteroatom at position R¹ of the second moiety having formula (I),

(iii) the oxygen atom of the OH group of the second moiety shown in formula (II) or (III),

(iv) the exocyclic oxygen atom of the second moiety shown in formula (II) that is attached to the ring carbon atom next to X by a double bond, or

(v) the heteroatom of R⁷ of the second moiety having formula (III).

[0064] The enzyme-labile bond is cleavable by an enzyme and, preferably, is susceptible to cleavage by hydrolases, for example by glycosidases, proteases, peptidases, ureidases, esterases, lipases, phosphatases, sulfatases, phospholipases, dealkylases, decarboxylases, or nitroreductases.

[0065] In accordance with the present invention, the compound described herein may optionally comprise a spacer. The spacer is preferably a self-immolative spacer (i.e. a self-eliminating spacer). The use of a spacer may be advantageous since the spacer generally allows increasing the stability of the compounds of the present invention, in particular the stability against non-enzymatic hydrolysis. As a result, unspecific inhibition by spontaneous hydrolysis is reduced, leading to an extended shelf life of the compounds. In addition, the chemical synthesis of a first and a second moiety linked by an enzyme-labile bond is very difficult and laborious in many cases. The use of a spacer, however, often enables the skilled person to readily link the first (enyme-labile) moiety with a second (inhibitor) moiety and, thus, allows for the expansion of the basic principle underlying the present invention to a wide variety of masking groups (herein "first moieties").

[0066] The basic concept of self-immolative (or self-eliminating) spacers is well known in the art (see, e.g., Senter et al., JOC (1990), 55, 2975; Li et al., JACS (2003), 125, 1051 6; Sella et al., JACS (2009), 131, 9934; de Groot et al., JOC (2001 ), 66, 8815; Amir et al., Angew. Chem Int. Ed. (2003), 42, 4494; DeWit et al., JACS (2009), 131, 18327; Meyer at al., Org. Lett. (2008), 10, 1517; Seo et al., JACS (2010), 132, 9234; Carl et al., J. Med. Chem. (1981 ), 24, 479; Schmidt et al., JOC (2012), 77, 4363; and Robbins et al., JOC (2013), 78, 3159). In the present case, 4- hydroxybenzylalcohols and 4-aminobenzylalcoholes may, for example, be used as spacer units. As described in the examples, for preparation of a preferred compound of the present invention, the phenolic hydroxyl group may be converted to the beta-D- galactopyranoside. Then, the benzylic hydroxyl group may be activated by tosylation and the resulting intermediate may be coupled with an inhibitor structure like pyrithione. For the resulting structure, the removal of the galactose by enzymatic cleavage triggers a 1 ,6-elimination of the benzyl structure to indirectly release the pyrithione. Using this "spacer approach", it does not matter whether the inhibitor structure provides a sulfur (more difficult to handle under chemical synthesis aspects) or oxygen atom, and hydrolytic stability of the compound is generally improved. Furthermore, the spacer approach readily allows the skilled person to use numerous other masking groups than sugar moieties, such as amino acids or peptides.

[0067] Suitable self-immolative spacers for use herein are well-known in the art and are not particularly limited for use herein. However, preferred self-immolative spacers are derived from unsubstituted or substituted hydroxybenzylalcohol (e.g., optionally substituted 2- or 3- or 4-hydroxybenzylalcohol), unsubstituted or substituted aminobenzylalcohol (e.g., optionally substituted 2- or 3- or 4- aminobenzylalcohol), unsubstituted or substituted aminobenzyloxycarbonyl (e.g., optionally substituted 2- or 3- or 4-aminobenzyloxycarbonyl), and unsubstituted or substituted hydroxybenzyloxycarbonyl (e.g., optionally substituted 2- or 3- or 4- hydroxybenzyloxycarbonyl).

[0068] The substituents attached to benzene ring of the self-immolative spacer have been described to influence the rate of the self-immolation process. For example, Schmidt et al. {supra) reported that methoxy substituents accelerate the benzylic self-immolation process several orders of magnitude. Further, in case of a benzyloxycarbonyl spacer instead of a benzyl spacer, self-immolation has been reported to occur in conjunction with the release of one equivalent carbon dioxide which provides a strong driving force, thereby accelerating the process (Carl et al., supra). Hence, the selection of an appropriate substitution pattern allows one to specifically tailor the self-immolative characteristics as needed.

[0069] Preferably, the spacer has a structure as shown in general formula (IV) or (V)

wherein:

X-l represents the second moiety;

ELG (enzyme labile group) represent the first moiety, wherein the bond between ELG and Y is the enzyme-labile bond;

Y is selected from NH or O;

X is selected from O, NH, S or 0-C(=0), preferably from O, NH and 0-C(=0),or from O and NH, or form O and 0-C(=0); R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from the group consisting of hydrogen, C_{1 -4}alkyl, C_{1 -4}alkoxy, fused heteroaryl, fused aryl, heteroarylC₀- 4alkyl, arylC₀-₄alkyl, halo, cyano, nitro, formyl, and optionally substituted amino, carboxy, carbonyl, hydroxy and sulfonyl.

[0070] R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are preferably independently selected from the group consisting of hydrogen, C_{1 -4}alkyl, C_{1 -4}alkoxy, halo, cyano, nitro, formyl, and optionally substituted amino, carboxy, carbonyl, hydroxy and sulfonyl, wherein R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are more preferably independently selected from the group consisting of hydrogen, C_{1 -4}alkyl, C_{1 -4}alkoxy, halo, formyl, NH₂ and hydroxyl, and wherein R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are most preferably independently selected from the group consisting of hydrogen, C_{1 -4}alkyl, and C_{1 -4}alkoxy, and are even more preferable all hydrogen.

[0071 ] Preferred examples of the compound of the present invention include compounds selected from 3a, 4a, 5a, 5b, 5Sa and 5Sb:

[0072] Other preferred compounds of the present invention are compounds 5Sc, 5Sd and 5Se (see examples).

[0073] As used herein, "C_{1 -6}" means a carbon group having 1 , 2, 3, 4, 5 or 6 carbon atoms, "C_{1 -4}" means a carbon group having 1 , 2, 3 or 4 carbon atoms, "C_0-6" means a carbon group having 0 (i.e. no carbon group), 1 , 2, 3, 4, 5 or 6 carbon atoms, "Co-₄" means a carbon group having 0 (i.e. no carbon group), 1 , 2, 3 or 4 carbon atoms, " C_2-6" means a carbon group having 2, 3, 4, 5 or 6 carbon atoms, and "C₃-6" means a carbon group having 3, 4, 5 or 6 carbon atoms.

[0074] The term "alkyl", as used herein, includes both straight chain and branched chain alkyl groups and may be, but is not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, neo-pentyl, n-hexyl, i-hexyl or t-hexyl. The term "C_{1 -4}alykl" or " C_{1 -6}alykl" includes Ci_-3alkyl having 1 to 3 carbon atoms that may be methyl, ethyl, n-propyl or i-propyl.

[0075] The term "alkenyl", as used herein, includes both straight chain and branched chain alkenyl groups. The C_2-6alkenyl having 2 to 6 carbon atoms may have one or two double bonds and may be, but is not limited to, vinyl, allyl, propenyl, butenyl, crotyl, pentenyl, or hexenyl.

[0076] The term "alkynyl", as used herein, includes both straight chain and branched chain alkynyl groups. The C_2-6alkynyl having 2 to 6 carbon atoms may have one or two trippel bonds and may be, but is not limited to, etynyl, propargyl, pentynyl or hexynyl.

[0077] The terms "aryl" and "heteroaryl", as used herein, preferably refer to an optionally substituted monocyclic unsaturated aromatic ring system. An example of "aryl" is phenyl, and examples of "heteroaryl" include furan, thiophene, pyrrole, triazole, pyrazole, pyridazine, pyrimidine, and pyrazine. The terms "arylalkyl" and "heteroarylalkyl", as used herein, refer to a substituent that is attached via the alkyl to an aryl or heteroaryl group.

[0078] The terms "halo", as used herein means halogen and may be fluoro, iodo, chloro or bromo. The term "haloalkyl", as used herein, means an alkyl group as defined above, which is substituted with halo. The term "C_{1 -6}haloalkyl" may be, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, difluoroethyl or bromopropyl. The term "OC_{1 -4}haloalkyl", as used herein, may be, but is not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, fluoroethoxy or difluoroethoxy.

[0079] For the avoidance of doubt, it is to be understood that the present invention relates to any and all tautomeric forms of the compounds described herein. Further, some compounds described herein may have chiral centres and/or geometric isomeric centres, and it is to be understood that the present invention encompasses all such optical, diastereoisomeric and geometric isomers, unless otherwise stated. Also, the present invention relates to, and encompasses, any and all salts of the compounds, tautomers and optical, diastereoisomeric and geometric isomers.

[0080] In a second aspect, the present invention relates to a composition comprising a compound of the present invention. The composition may be, for example, a pharmaceutical composition.

[0081 ] The composition may be in any form including, but not limited to, solid, liquid, semi-solid and gel form. In case the composition is a pharmaceutical composition, it comprises as active ingredient a therapeutically effective amount of a compound according to the present invention or tautomers or salts thereof, in association with one or more pharmaceutically acceptable diluents, excipients and/or inert carriers. The pharmaceutical composition may be in a form suitable for oral administration, for example as a tablet, pill, syrup, powder, granule or capsule, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) as a sterile solution, suspension or emulsion, for topical administration, e.g. as an ointment, patch or cream, or for rectal administration, e.g. as a suppository.

[0082] In a third aspect, the present invention relates to a kit comprising a compound of the present invention and, optionally, instructions for use. The compound may be contained in a container such as a vial. Buffer and other solutions may also be included in the kit.

[0083] In a fourth aspect, the present invention relates to a method for changing the microbial composition of microbiota comprising contacting said microbiota with a compound of the present invention. This method results, e.g., in the inhibition of distinct sub-populations of microbiota.

[0084] The method may further comprise the step of adding non-toxic transition metals to said microbiota. In a preferred embodiment, Fe(lll) ions are added to the microbiota, for example in an amount so as to achieve a final concentration of 0.01 mM to 10 mM Fe(lll) ions, in particular 0.1 mM to 5 mM Fe(lll) ions.

[0085] Surprisingly, the addition of Fe(lll) ions was found to reduce or avoid unspecific inhibition. It is believed that supplementation of cultures of microbial species (e.g., microbiota) with Fe(lll) results in the formation of chelates of the Fe(lll) and the unmasked inhibitor to form insoluble precipitates. Hence, the addition of nontoxic transition metals (i.e. Fe(lll)) to microbiota, e.g., microbiota in a culture medium, is an optional step in all (medical or non-medical; in vitro and in vivo) uses described herein.

[0086] In a fifth aspect, the present invention relates to the in vitro use of a compound of the present invention as an antimicrobial compound. In one embodiment, the compound of the present invention, or a composition comprising the compound of the present invention, may be included in enrichment medium or growth medium for microorganisms, in particular in a bacterial growth medium. [0087] In a sixth aspect, the present invention relates to the use of a compound of the present invention for the selective culturing, plating, enrichment or growth of microorganisms. For example, a compound of the present invention may be used to supplement media for the selective enrichment or growth of certain bacterial species or strains, e.g., Pseudomonas spp. like Pseudomonas aeruginosa. The selective enrichment and culturing of microorganisms is of great importance in food, water and environmental safety testing, in food and feed quality assurance, environmental quality control, hygiene and clinical diagnostics.

[0088] In addition, the compound according to the present invention may be used to discover, determine, isolate, or investigate pathogenic microbial species. This is, for example, of great benefit in clinical diagnostics and highly advantageous for research purposes. In addition, the compound of the present invention may be used in culturing of plants and cells or tissues of animals, humans and/or plants to avoid contamination with unwanted organisms.

[0089] Furthermore, the compound according to the present invention may be used

as a microbiota-modifying agent in in the processing (fermentation) of feed, food or other organic matter where the development of chemical components, smells, flavors, colors, shelf live and/or consistency depends on, or is substantially affected by, the diversity and composition of microbiota;

as a microbiota-altering agent in the treatment of waste water or solid waste, composting, or the production of fuel from organic matter where optimized microbiota are relevant to yield, efficacy and process quality; as a microbiota-optimizing agent in farming of animals and plants, in particular in high intensity farming of animals and plants where microbial infections affects quality and value of products and possibly inflicts financial burden of farmers, environmental damage and/or public health hazards due to application of antibiotics and biocides; and as a supplement to pre- and/or probiotics to improve microbiota of animals and humans, in particular to inhibit unwanted microorganisms, thereby enhancing the effects of pre- and/or probiotics.

[0090] In a seventh aspect, the present invention relates to the use of a compound of the present invention for cosmetic applications, in particular cosmetic conditions caused by dysbiosis, i.e. cosmetic conditions linked to composition and diversity of microbiota, especially cosmetic conditions caused by impaired microbiota. In particular, the compound of the present invention may be used in the treatment of seborrheic dermatitis, acne vulgaris, acne rosacea, caries, periodontal disease including gingivitis and periodontitis, and halitosis.

[0091 ] In an eighth aspect, the present invention relates to a compound of the present invention for use in therapy, in particular for the treatment or prevention of gastrointestinal diseases like inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), enterocolitis, and colorectal cancer;

cardiovascular diseases like cerebrovascular disease, myocardial infarction and stroke, (Chlamydia, Helicobacter pylori or periodontopathic bacteria have been considered to increase the risk of development of cardiovascular disease);

autoimmune diseases like rheumatoid arthritis, juvenile idiopathic arthritis, multiple sclerosis and Celiac disease,

neurological disorders like Alzheimer's disease, autistic spectrum disorders, and Parkinson's disease;

metabolic diseases, such as metabolic syndrome, autoimmune type 1 diabetes (T1 D) and insulin resistant type 2 diabetes (T2D); neoplastic diseases like ulcers, adenocarcinomas, gastric B-cell lymphomas, colorectal carcinoma (in particular, ulcers, adenocarcinomas and gastric B-cell lymphomas caused by H. pylori) (since commensal bacteria strongly affect inflammation locally and systemically and inflammation is believed to be an important factor in tumor formation, dysbiosis in microbiota must be considered a potential factor in carcinogenesis. Hence, successful treatment of such may be of importance to future oncology);

conditions caused by dysbiosis of vaginal microbiota (VMB) like vaginal discharge, poor pregnancy outcomes, pelvic inflammatory disease, postoperative infections, endometritis following elective abortions and sexually transmitted diseases (STDs); and

other disorders like obesity, chronic fatigue syndrome, and atherosclerosis.

[0092] The compound of the present invention may be particularly suitable in the therapy of conditions linked to composition and/or diversity of microbial colonization of the gastrointestinal tract (Gl), such as cardiovascular disease caused by plasma trimethylamine-N-oxide (TMAO), irritable bowel syndrome (IBS) linked to butyrate producing Clostridia cluster, Clostridium difficile infections (CDI ; a notorious side effect of antibiotic therapy), inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis linked to low diversity of microbiome and increased abundance of Enterobacteriaceae.

[0093] Other preferred indications for treatment by the compounds of the present invention include multiple sclerosis linked to increased levels of Methanobrevibactor, Akkermansia and Butyricimonas, obesity linked to a number of microbial effects such as a lack of acetate, propionate, butyrate production, increased lipoprotein lipase activity, suppression of adenosine monophosphate protein kinase (AMPK) and microbiota induced release of digestive enzymes, and Parkinson's disease linked to microglia activation through imbalances in the gut levels of short chain fatty acids (SCFA's). Moreover, cardiovascular diseases like cerebrovascular disease, myocardial infarction and stroke are considered to be linked to bacteria. [0094] Furthermore, since commensal bacteria strongly affect inflammation locally and systemically and inflammation is believed to be an important factor in tumor formation, dysbiosis in microbiota also plays a role in carcinogenesis. Hence, the compounds of the present invention are also expected to be useful in the treatment of neoplastic diseases, in particular neoplastic diseases for which a link to microorganisms, such as certain bacterial species (e.g., Helicobacter pylori) as underlying cause has been established.

[0095] In the following, the present invention will be described and explained with additional specificity and detail by reference to specific non-limiting examples.

EXAMPLES

[0096] As stated above, in order for the general concept of auto-inhibition to be successful, the compound according to the present invention should (1 ) be taken up by the targeted microbial cells and not be released into the environment by, e.g. lysis of microbial cells, (2) be inactive in its masked state, and (3) be such that the unmasked (i.e. "free") biocide cannot pass microbial cell membranes.

[0097] Moreover, it would be of great advantage if various building blocks (e.g., different inhibitors, enzyme-labile groups, and optionally spacer groups) would be available to allow for the generation of a high number of different compounds specifically tailored for the intended purpose. Although this is an exceptionally demanding and difficult technical problem, the inventors succeeded to solve this problem, as demonstrated below.

[0098] In order to ease understanding of the experiments conducted, an overview of the compounds tested together with references to the corresponding examples and figures is provided in Table I. st of compounds and corresponding figures and examples

[0099] As can be seen, deferasirox (2), deferiprone (3), oxypyrion (4), pyrithione (5), and 2-amino-pyridine-N-oxide (6) (see Figure 1 ) were identified and selected as potentially suitable inhibitor (biocide) candidates. These candidate inhibitor compounds are transition metal chelators and known to have low toxicity.

Discussion of preparation Examples 1 to 1 1

[00100] In a next step, compounds 1 (triclosan; reference), 2, 3, 4, and 5 were masked with D-galactose to give beta-D-galactoside derivatives 1 a, 2a, 3a, 4a, 5a and 5b (see Figure 2) In addition, compound 5 was coupled to a self-immolative spacer that is connected via an enzyme-labile bond to either beta-D- galactopyranoside, L-alanine, beta-D-glucuronyl, L-pyroglutamyl and phosphoryl to give compounds 5Sa, 5Sb, 5Sc, 5Sd, and 5Se, respectively (with respect to 5Sa and 5Sb, see Figure 3). The chemical synthesis of these compounds is described in Examples 1 to 11 and the synthesis schemes are shown in Figures 7 to 11 (not for 5Sc, 5Sd and 5Sd). The chemical synthesis is based on standard procedures well established in the art, unless otherwise stated.

Discussion of MIC and strain specificity (Examples 12 and 13)

[00101 ] The minimal inhibitory concentration (MIC) of candidate compounds 2 to 6 was determined for the assessment of antimicrobial properties (Example 12). For this purpose compounds 2 to 6 were inoculated with approximately 10⁶ CFU/mL of freshly grown cells of Escherichia coli ATCC 25922. Growth was recorded by measuring optical density at 600 nm after incubation for 24 h at 37°C and 150 rpm. Minimal inhibitory concentration was defined as the concentration leading to at least 3-fold reduction in the optical density compared to control cultures without added compounds. The results are shown in Table II.

Table II. Minimal inhibitory concentrations (MIC), E. coli

[00102] Biocidal /inhibitory effect decreases in the order of 5 » 4 > 3. Therefore, it is established that 5 is a very potent growth inhibitor (MIC EC 0.006 mM), followed by 4 (MIC EC 1 mM) and 3 (MIC EC 3 mM). In contrast, MIC of deferasirox (2) and 2- amino-pyridine-N-oxide (6), the nitrogen analogue of oxypyrion (4) and pyrithione (5) was too high to be determined under the chosen experimental conditions. Thus, 2 and 6 exhibited no significant biocidal activity.

[00103] In order to assess the breadth of scope of inhibitory/biocidal property of 5, nutrient agar plates containing 0.2 mM of 5 were prepared (Example 13) along with control plates without 5. Agar plates were inoculated (streak-outs) from freshly grown colonies of test strains and incubated for 24 h at 37°C. Growth was inspected visually and rated from "vigorous growth" [++++] to "no growth detectable" [-]. The results are shown in Table III. Table III. Effect of pyrithione (5) on growth of different bacteria

[00104] The data confirm that 5 is active against a very broad range of bacterial species with the notable exception of Pseudomonas aeruginosa. This surprising discovery is expected to be useful for the development of Pseudomonas aeruginosa media which, if supplemented with 5, will eliminate competing bacterial species without harming growth of Pseudomonas aeruginosa. In other words, pyrithione (5) can be used as a selective inhibitor to improve commercial P. aeruginosa enrichment, growth and plating media, which, due to the medical importance of this pathogen, is a discovery of significant commercial potential. Discussion of Fe(lll)/enzymatic assay of Example 14

[00105] In a next step, the effect of masking the inhibitors/biocides was studied. It is relevant in current context that metal ion-chelating inhibitors/biocides 3, 4 and 5 form colored complexes or precipitates in the presence of iron(lll) in aqueous solution at neutral pH (Example 14). Absorbance maxima were found at 420 nm (compound 3), 455 nm (compound 4) and 555 nm (compound 5), respectively. The corresponding potential enzyme responsive inhibitors 3a, 4a, 5a and 5Sb did not exhibit color formation under the same conditions. Therefore, it is established that iron chelating ability is completely eliminated in masked inhibitors/biocides such as 3a, 4a, 5a and 5Sb.

[00106] Change in absorbance at 420, 455 or 555 nm was used to monitor the enzymatic unmasking and activation of metal ion-chelating inhibitors (Example 14, Figure 12). Compounds 3a, 4a, 5a, 5Sb were incubated with the corresponding enzyme (beta-galactosidase, see Figure 12a, 12b, 12c) or L-alanine aminopeptidase (see Figure 12d), and the change in absorbance over time was recorded with a spectrophotometer (Figure 12; data points are shown as solid triangles) and compared to controls without enzyme (open triangles).

[00107] As can be seen from Figure 12, absorbance increased in reactions with added enzyme while absorbance in reactions without enzyme remained constant. Firstly, it is hereby established that enzymatic unmasking of inhibitor/biocides proceeds efficiently. Secondly, this represents proof-of-concept for the use of a self- immolative spacer connecting a biocide/inhibitor structure (e.g., compound 5) with an enzyme-labile group (ELG) such as L-alanine (Example 14, Figure 12d). Furthermore, it was conformed that compound 5b could not be unmasked by the action of beta-galactosidase. Discussion of microbial assays of Examples 15 to 22

[00108] As a comparison to the prior art as disclosed in US 2014/0178923, Rambach agar plates containing 2 mg/L triclosan-beta-D-galactopyranoside (1a) and 1 mM IPTG were inoculated with (i) a mixed cell suspension (50 μΙ_) containing approximately 10³ CFU/ml S. enteritidis {S.e.) and 10⁴ CFU/ml E. coli {E.c.) and (ii) a cell suspension (50 μΙ_) containing 10³ CFU/ml S. enteritidis. After incubation at 37°C for 24 h, growth was recorded by digital imaging (Example 15, Figure 13). The data show that, while S. enteritidis was not inhibited by 2 mg/L triclosan-beta-D- galactopyranoside (1a) when plated as pure culture, S.e. was inhibited under the same conditions when plated in a mixture with excess E.c. Evidently, triclosan (1 ) produced from 1a by enzymatic activity of E.c. is diffusing into the medium inhibiting colonies indiscriminately. Clearly, this demonstrates that triclosan-beta-D- galactopyranoside (1a) is not suited for use as a selective microbial inhibitor, emphasizing the importance of the present disclosure.

[00109] In a next attempt, nutrient broth with 1 mM IPTG and 0.1 to 5 mM of 2a was prepared (Example 16). Test tubes were inoculated with freshly grown, overnight cultures of Escherichia coli ATCC 25922 (beta-galactosidase positive) and incubated at 37°C and 150 rpm. Growth was inspected by measurement of optical density (600 nm) after 24 h. No difference in growth was observed between tubes containing 2a and control tubes. Hence, 2a showed no activity against the strains tested.

[001 10] For the testing of compound 3a, plates containing 5 mM of 3 and 20 mM of 3a were prepared and inoculated with freshly grown cultures of the S. enteritidis (S.e.) and E. coli {E.c), and growth was recorded by digital imaging after 23 h of incubation at 37°C (Example 17, Figure 14). The data show that 3a significantly inhibits the growth of E. coli {E.c.) but does not at all affect the growth of S. enteritidis {S.e.). Therefore, these experiments show that the auto-inhibition concept underlying the present invention works. [001 1 1 ] After successful testing of compound 3a, additional experiments were conducted with compound 4a. Based on the results of the previous experiments, it was expected that upon contact of 4a with beta-galactosidase positive bacteria, inhibitor compound 4 would be released, selectively inhibiting growth of said beta- galactosidase positive bacteria. Indeed, 4a in combination with further supplements almost completely suppressed growth of beta-galactosidase positive bacteria (E.c.) on plates, while growth of S.e. was not visibly affected by 4a (Example 18, Figures 15b and 15c). The effect was similar but less pronounced in liquid culture (Figure 15a). These results further confirm that, in the presence of a suitable inhibitor/biocide (such as compound 4) masked with a suitable enzyme-labile group (ELG), bacterial enzymes such as beta-galactosidase can trigger auto-inhibition of bacteria producing said enzyme.

[001 12] In further experiments, compound 5a was tested in an analogous fashion (Example 19). In view of the high inhibitory potency of 5 (see Table II above), especially good auto-inhibitory results were expected. Rambach Salmonella agar plates were prepared, containing 0.2 mM of 5a and 1 mM IPTG, and tube cultures were prepared containing compounds 5 and 5a along with 1 mM IPTG at concentrations of 0.1 mM and 0.2 mM, respectively. Agar plates and tube cultures were inoculated with S. enteritidis (S.e.) and E. coli (E.c). A mixed cell suspension (50 μΙ_ was plated) which contained approximately 10³ CFU/ml S.e. and 3x10⁴ CFU/ml E.c. was prepared and inoculated in parallel. Plates and tubes were incubated at 37°C, and tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 17 h, and growth on agar plates was recorded by digital imaging after 24 h.

[001 13] The recorded data reveal that 5a is indeed a very efficient and highly selective inhibitor of beta-galactosidase positive bacteria (see Figure 16). In fact, the combination 5a with further supplements (deoxycholate, propylene glycol), allowed the complete inhibition of beta-galactosidase positive organisms on agar plates, while growth of beta-galactosidase negative bacteria was not affected. [001 14] Thus, the above results show that the 0-beta-D-galactopyranosides of deferiprone (3a), oxypyrion (4a), pyrithione (5a) were active against all strains testing positive for beta-D-galactosidase activity with MIC values corresponding to those obtained for the unmasked structures. No activity against beta-D-galactosidase negative strains was observed. The S-beta-D-galactopyranoside of pyrithione (5b), which cannot be readily cleaved by most beta-D-galactosidases, was also tested with negative results for all strains.

[001 15] Surprisingly, the results obtained for pyrithione-0-beta-D-galactoside (5a) and oxypyrion-beta-D-galactoside (4a) in mixed cultures of E. coli {beta-D- galactosidase positive) and S. enteritidis (beta-D-galactosidase negative), in liquid and on plating media, also show that there was little to no unspecific inhibition. While E. coli was inhibited to various degrees depending on the inhibitor and its concentrations, the growth of S. enteritidis was not substantially affected. This proves that the unmasked inhibitors are well contained within the E. coli cells. This also demonstrates that these inhibitors do not kill E.coli but slow or stop cell division and hence colony growth, which may explain the observed containment effect.

[001 1 6] The above findings clearly validate the original concept since (1 ) effective microbial growth inhibitors were found that (2) can be masked with groups labile to metabolic enzymes specific to certain species and strains of bacteria and that (3) masking metal binding sites of these inhibitors completely eliminates their biocidal properties and (4) unmasking the inhibitors in situ by metabolic enzymes proceeds efficiently. Finally, (5) the unmasked inhibitors are well contained within the targeted bacterial cells thereby avoiding unspecific inhibition.

[001 17] For the purpose of comparison of 5a and 5Sa, which differ by the presence of a self-immolative spacer inserted between the inhibitor and ELG in case of 5Sa, further experiments were carried out (Example 20). Rambach Salmonella agar was prepared containing 0.5 mM of 5Sa, 1 mM IPTG, 0.5 mM 1 -O-methyl-glucuronic acid and 0.2 mM 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid. Agar plates and tube cultures were inoculated with S. enteritidis (S.e.) and E. coli (E.c). A mixed cell suspension containing 10³ CFU/ml S.e. and 3x10⁴ CFU/ml E.c. was prepared and inoculated in parallel. Plates and tubes were incubated at 37°C, and tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 20 h (Figure 17a), and growth on agar plates was recorded by digital imaging after 24 h (Figure 17b). Evidently, growth of E. coli (colonies on agar plates stained blue due to enzymatic transformation of 5-bromo-4-chloro-3-indoxyl- beta-D-glucuronic acid) was strongly suppressed by 5Sa.

[001 18] These results are in line with the results obtained using compounds 3a, 4a and 5a in broth and on plates, and extends the basic auto-inhibitory principle to the extended modular concept of linking different inhibitors and enzyme-labile groups with self-immolative spacers. In addition, it was observed that 5Sa remains remarkably stable in aqueous environments for weeks. This is a very desirable property, in particular if commercial products require a certain minimum shelf life. Thus, the self-immolative spacer technology provides significant added benefits.

[001 19] A further experiment was designed to test whether the auto-inhibitory concept could be used to improve the notoriously challenging and time limiting enrichment process of samples mandatory in food testing methods of foodborne pathogens (Example 21 ). Nutrient broth containing 25 mM sodium phosphate buffer (pH 7.3), 1 mM IPTG, 1 mM Fe(lll) chloride heptahydrate and either 0.5 or 1 .0 mM 5Sa was prepared. Addition of Fe(lll) in combination with phosphate lead to the formation of iron phosphate particles. A control medium was prepared similarly without 5Sa. S. enteritidis (S.e.) and E. coli {E.c.) were used for testing. Tube cultures with 3-4 ml_ medium were inoculated with 50 μΙ_ of a mixed cell suspension containing 1 x10³ CFU/mL S. enteritidis {S.e.) and 1 x10⁴ CFU/mL E. coli {E.c). Broth cultures were incubated for 20 h at 37°C and 150 rpm, followed by plating of appropriate dilutions on trypticase soy agar containing 0.5 mM 1 -O-methyl-glucuronic acid and 0.2 mM 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid. Colony forming units (CFU) were analyzed after an incubation of 22 h at 37°C. Blue colonies were counted as (beta-glucuronidase positive) E. coli and white colonies were counted as (beta-glucuronidase negative) S. enteritidis. The percentage of S. enteritidis relative to the total number of colonies is given in Table IV.

Table IV. Enrichment of S. enteritidis in broth culture by 5Sa

[00120] The data demonstrate that best results were obtained with 0.5 mM 5Sa, phosphate and FeCI₃, changing the ratio of S.e. to E.c. from 1 :10 to 1 :1 . The optimal concentration of 5Sa was, unexpectedly, found to be relatively low. Thus, the results confirm the general validity of the modular concept of auto-inhibition, i.e. use of a compound made of (1 ) an inhibitor in combination with (2) an enzyme-labile "masking" group and, optionally, (3) a self-immolative spacer.

[00121 ] Moreover, the results prove evidence that the use of Fe(lll) in dissolved form and/or as particulate salts is effective in the prevention of nonspecific inhibition. Without being bound by theory, it is believed that this surprising effect is due to the fact that the added Fe(lll) scavenges excess inhibitor/biocide that is present and/or released into the medium.

[00122] In addition, experiments were conducted to examine whether the concept of enzyme responsive inhibitors and the use of self-immolative spacers can be extended to enzyme-labile groups other than glycosides (Example 22). Nutrient agar and nutrient broth containing 0.2 and 1 .0 mM of compound 5Sb, containing L-alanine as enzyme-labile group, a -C₆H₄-CH₂- group as self-immolative spacer and pyrithione as inhibitor structure, were prepared and inoculated with Staphylococcus aureus ATCC 29213 (S.a., aminopeptidase negative), Citrobacter freundii RKI NM8 {C.f., aminopeptidase positive) and E. coli {E.c, aminopeptidase positive). For comparison, some tubes contained 0.2 mM of free inhibitor compound 5 instead of masked compound 5b. Plates and tubes were incubated at 37°C, and tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 24 h (Figure 18a), and growth on agar plates was recorded by digital imaging after 24 h (Figure 18b).

[00123] Similar to the results describe above for inhibitors masked by beta-D- galactopyranosyl as enzyme-labile groups, L-alanine aminopeptidase positive bacteria were inhibited by 5Sb which is masked by an L-alanyl enzyme-labile group with a spacer. In contrast, the growth of L-alanine aminopeptidase negative bacteria (S.a.) was not affected by 5Sb, in spite of being sensitive to unmasked inhibitor 5. Thus, the results show that masking of inhibitors/biocides is possible with different enzyme labile groups, verifying the broad applicability of the described basic principle underlying the present invention.

[00124] Furthermore, the results show the great advantage of using self-immolative spacer groups. A version of 5Sb without spacer is chemically not possible (no N atom available for the required amide bond). Therefore, the embodiment of present invention using self-immolative spacer technology greatly enhances the number of compounds according to the present invention and their potential applications by multiplying the number of suitable enzyme-labile groups. For example, the principle design of the inventive compounds as exemplified by 5Sb can easily be adopted and extended to amino acids other than L-alanine or to include peptides and/or proteins as enzyme labile groups.

[00125] What is more, the experimental data also show that the use of the auto- inhibitory compounds of the present invention, e.g. of 5Sb, generally allows for the selective inhibition of Gram negative bacteria (e.g., E. coli) without affecting the growth of Gram positive bacteria (e.g., Staphylococcus aureus) in mixed culture, which is a breakthrough achievement and an important contribution to the art. Hence, the present invention provides promising compounds for specifically altering microbiota. [00126] Additional experiments show that the concept of enzyme responsive inhibitors including a self-immolative spacer also works if beta-D-glucuronyl, L- pyroglutamyl or phosporyl is selected as the enzyme-labile group (Examples 23, 24, 25). For example, these enzyme responsive inhibitors allow for the selective enrichment of Salmonella spp. by suppressing the growth of other bacteria such as Escherichia coli, Citrobater freundii, Staphylococcus aureus etc.

[00127] In the following, Examples 1 to 25 are described in detail. An overview of the examples conducted together with references to the respective Figures and Tables is shown in Table V.

Table V. List of examples

Example 1

Synthesis of triclosan-beta-D-galactopyranoside (1a, Figure 7)

[00128] At 5°C, boran trifluoride diethyl etherate (1 ml, 7.9 mmol) was added to a solution of triclosan (1 ) (22.4 g, 77.4 mmol) and 2,3,4,6-tetra-O-acetyl-a/b-D- galactopyranosyl tnchloroacetimidate (47.8 g, 97.3 mmol) in dichloromethane (1 15 ml) and the mixture was stirred for 2 hours. Concentration of the reaction mixture in vacuum and crystallization from ethanol gave 1 b (43 g; 89 %, 69.3 mmol) as colorless crystals.

[00129] ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1 .86 (s, 3 H) 1 .97 (s, 3 H) 2.09 (s, 3 H) 2.17 (s, 3 H) 4.02 - 4.08 (m, 1 H) 4.12 - 4.23 (m, 2 H) 5.00 - 5.08 (m, 2 H) 5.36 - 5.45 (m, 2 H) 6.72 (d, J=8.7 Hz, 1 H) 6.79 (d, J=8.7 Hz, 1 H) 7.03 (dd, J=8.7, 2.5 Hz, 1 H) 7.14 (dd, J=8.7, 2.5 Hz, 1 H) 7.24 (d, J=2.4 Hz, 1 H) 7.45 (d, J=2.5 Hz, 1 H)

[00130] To a suspension of 1 b (42.2 g; 68 mmol) in anhydrous methanol (210 ml) was added sodium methoxide (5.4 M in methanol; 1 .3 ml) and the reaction mixture was stirred for 5 hours. The resulting solution was neutralized by stirring with ion exchange resin (Dowex™ Monosphere 650C H+; 49 g), filtered and concentrated in vacuum to provide 1a (30.1 g, 66.6 mmol, 98 %) as a white foam.

[00131 ] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.36 - 3.56 (m, 4 H) 3.61 (t, J=x Hz, 1 H) 3.67 (t, J=3.8 Hz, 1 H) 4.52 (d, J=4.5 Hz, 1 H) 4.67 (t, J=5.4 Hz, 1 H) 4.82 (d, J=5.8 Hz, 1 H) 4.90 (d, J=6.1 Hz, 1 H) 4.97 (d, J=7.6 Hz, 1 H) 6.88 (d, J=8.9 Hz, 1 H) 6.95 (d, J=8.6 Hz, 1 H) 7.04 (dd, J=8.3, 2.3 Hz, 1 H) 7.32 (dd, J=8.9, 2.6 Hz, 1 H) 7.36 (d, J=2.4 Hz, 1 H) 7.68 (d, J=2.7 Hz,1 H)

[00132] ¹³C NMR (100 MHz, DMSO-d6) δ ppm 60.3, 68.1 , 70.1 , 73.5, 75.7, 101 .1 , 1 17.3, 120.1 , 121 .5, 122.2, 124.2, 127.5, 128.6, 128.9, 129.8, 143.5, 149.2, 151 .6

Example 2

Synthesis of deferasirox^-D-galactopyranoside (2a, Figure 8)

[00133] To a suspension of deferasirox (1 6 g, 42 mmol), tetra-butylammonium bromide (5 g, 20 mmol) and 2,3,4,6-tetra-0-acetyl-beta-D-galactopyranosyl bromide (43 g, 100 mmol) in dichloromethane (250 ml) was added water (250 ml) and 10 N sodium hydroxide solution (15 ml) and the reaction mixture was vigorously stirred for 18 hours at room temperature. The pH of the reaction mixture was adjusted to 4.5 with acetic acid, the organic layer was separated and concentrated in vacuum. The residue was purified by flash chromatography [petroleum ether/ ethyl acetate (1 :1 )] affording 2b (29 g, 67 %, 28 mmol) as a white foam.

[00134] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .56 (s, 3 H) 1 .86 (s, 3 H) 1 .94 (s, 3H) 1 .96 (s, 3H) 1 .98 (s, 3H), 1 .99 (s, 3H) 2.12 (s, 3 H) 2.14 (s, 3 H) 3.90- 4.09 (m, 4 H) 4.29 (t, J=6.5 Hz, 1 H) 4.50 (t, J=6.4 Hz, 1 H) 4.81 (dd, J=10.4, 8.0 Hz, 1 H) 5.10 (dd, J=10.5, 3.6 Hz, 1 H) 5.20 - 5.26 (m, 2 H) 5.30 -5.35 (m, 2 H) 5.43 (dd, J=10.5, 3.5 Hz, 1 H) 6.12 (d, J=7.9 Hz, 1 H) 6.98 - 7.04 (m, 2 H) 7.1 6 (d, J=8.0 Hz, 1 H) 7.29 (t, J=7.3 Hz, 1 H) 7.34 - 7.40 (m, 1 H) 7.56 (d, J= 8.8 Hz, 2H) 7.61 - 7.65 (m, 1 H) 7.69 (d, J=7.5 Hz, 1 H) 7.97 (d, J=8.0 Hz, 2 H) 8.05 (d, J=8.0 Hz, 1 H) 10.60 (s, 1 H)

[00135] At 30°C 2b (28 g; 27.2 mmol) was dissolved in anhydrous methanol (300 ml) and sodium methoxide (5.4 M in methanol; 4.9 g) was added. The reaction mixture was stirred for 18 hours. The suspension was cooled to room temperature and the precipitate was collected, washed with cold methanol and dried in vacuum to yield pure 2c (1 1 .6 g, 76 %, 21 .1 mmol) as colourless crystals.

[00136] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.24 - 3.38 (m, 3 H) 3.44 - 3.49 (m, 2 H) 3.61 (br. s., 1 H) 3.84 (s, 3 H) 4.31 (d, J=4.4 Hz, 1 H) 4.50 - 4.61 (m, 1 H) 4.70 - 4.86 (m, 3 H) 6.96 - 7.05 (m, 2 H) 7.12 (t, J=7.1 Hz, 1 H) 7.25 (d, J=7.8 Hz, 1 H) 7.36 (t, J= 8.3 Hz, 1 H) 7.47 - 7.55 (m, 2 H) 7.60 (d, J=8.3 Hz, 2 H) 7.96 (d, J=8.6 Hz, 2 H) 8.04 (d, J=7.3 Hz, 1 H) 10.76 (s, 1 H)

[00137] To a suspension of 2c (12 g, 21 .8 mmol) in methanol (180 ml) and water (180 ml) was added 10 N sodium hydroxide solution (4.6 ml) and the mixture was stirred for 4 hours at 45°C. The resulting solution was concentrated in vacuum, redissolved in methanol (150 ml) and passed through an ion exchange column (Dowex™ MS 650C H+-form, 200 ml, washed with methanol). The column was rinsed with methanol and product containing fractions were collected and concentrated in vacuum. The residue was crystallized from ethanol giving 2a (10.5 g, 90 %, 19.6 mmol) as colorless crystals.

[00138] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.28 - 3.53 (m, 5 H) 3.64 (br. s., 1 H) 4.34 (br. s., 1 H) 4.59 (br. s., 1 H) 4.69 - 4.92 (m, 3 H) 6.96 - 7.05 (m, 2 H) 7.12 (t, J=7.0 Hz, 1 H) 7.28 (d, J=8.4 Hz, 1 H) 7.36 (t, J=7.3 Hz, 1 H) 7.46 (d, J= 7.2 Hz, 1 H) 7.52 (t, J= 7.6 Hz, 1 H) 7.60 (d, J=8.4 Hz, 2H) 7.95 (d, J=8.4 Hz, 2 H) 8.05 (d, J=7.3 Hz, 1 H) 10.78 (br. s., 1 H) 13.14 (br. s., 1 H)

[00139] ¹³C NMR (100 MHz, DMS0-d6) δ ppm 60.2, 67.9, 70.0, 73.3, 75.6, 101 .5, 1 13.8, 1 16.3, 1 17.1 , 1 17.5, 1 19.7, 122.2, 124.5, 126.8, 130.3, 130.8, 131 .2, 131 .4, 132.6, 140.6, 151 .4, 155.4, 156.4, 159.8, 1 66.5

Example 3

Synthesis of deferiprone-beta-D-galactopyranoside (3a, Figure 9)

[00140] Deferiprone (12.4 g, 89.1 mmol) was dissolved in a solution of sodium methoxide (4.9 g, 90.7 mmol) in methanol (75 ml). The resulting red solution was cooled to 5°C and 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (36.6 g, 89.1 mmol) in dichloromethane (60 ml) was added dropwise. The reaction mixture was stirred for 24 hours at room temperature, neutralized with acetic acid (5 ml), concentrated in vacuum and purified by flash chromatography [ethyl acetate/ methanol (2:1 ) to (1 :1 )] giving the title compound as a foam, which was crystallized from ethanol/ water (5:1 ) to provide pure 3a (8.2 g, 31 %, 27.2 mmol) as colorless crystals.

[00141 ] ¹H NMR (400 MHz, DMSO-d6) δ ppm 2.4 (s, 3 H) 3.26 - 3.37 (m, 2 H) 3.42 - 3.61 (m, 4 H) 3.66 (s, 3H) 4.29 (d, J=7.8 Hz, 1 H) 4.43 (br. s., 1 H) 4.53 (br. s., 1 H) 4.4.8 (br. d, 1 H) 6.27 (d, J=7.2 Hz, 1 H) 7.1 1 (s, 1 H) 7.75 (d, J=7.1 Hz, 1 H)

[00142] ¹³C NMR (100 MHz, DMS0-d6) δ ppm 13.2, 41 .5, 60.4, 67.9, 71 .1 , 73.9, 75.9, 107.5, 1 15.3, 141 .3, 144.1 , 144.9, 171 .9

Example 4

Synthesis of oxypyrion^-D-galactopyranoside (4a, Figure 9)

[00143] To a solution of oxypyrion (47 g, 423 mmol), tetra-butylammonium bromide (38 g, 1 18 mmol) and 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (90 g, 219 mmol) in 1 .25 I dichloromethane were added water (1 .25 I) and 10 N sodium hydroxide solution (100 ml) and the reaction mixture was vigorously stirred for 6 hours at reflux temperature. pH was kept constant at 12 to 13 by adding 10 N sodium hydroxide solution. The reaction mixture was cooled to room temperature and diluted with dichloromethane (150ml). The organic layer was separated, washed with water (200 ml) and concentrated in vacuum. Purification via flash chromatography [toluene/ ethyl acetate (1 :1 )] afforded 4b (28 g, 29 %, 63.4 mmol) as a white foam.

[00144] ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1 .99 (s, 3 H) 2.01 (s, 3 H) 2.18 (s, 3 H) 2.22 (s, 3 H) 3.89 (t, J=6.6 Hz, 1 H) 4.07 (dd, J=1 1 .3, 6.6 Hz, 1 H) 4.22 (dd, J=1 1 .3, 6.7 Hz, 1 H) 5.1 1 (dd, J=10.4, 3.4 Hz, 1 H) 5.19 (d, J=8.2 Hz, 1 H) 5.37 (dd, J=8.4, 10.4 Hz, 1 H) 5.43 (d, J=3.2 Hz, 1 H) 6.05 (t, J=6.8 Hz, 1 H) 6.63 (d, J=9.3 Hz, 1 H) 7.27 - 7.33 (m, 1 H) 7.56 (d, J=7.0 Hz, 1 H)

[00145] At 5°C, 4b (27 g; 61 .2 mmol) was added to a solution of sodium methoxide (5.4 M in methanol; 0.6 ml) in anhydrous methanol (108 ml) and the reaction mixture was stirred for 2 hours. The formed precipitate was collected by suction, washed with cold methanol and dried in vacuum. The crude material thus obtained was recrystallized from ethanol/ water (1 :1 ) to yield pure 4a (15.1 g, 90 %, 55.3 mmol) as colorless crystals.

[00146] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.39 - 3.58 (m, 5 H) 3.62 (t, J=3.4 Hz, 1 H) 4.55 - 4.62 (m, 2 H) 4.88 (d, J=7.5 Hz, 1 H) 4.97 (d, J=5.3 Hz, 1 H) 5.41 (d, J=2.9 Hz, 1 H) 6.26 (t, J=6.8 Hz, 1 H) 6.59 (d, J=9.2 Hz, 1 H) 7.48 (ddd, J=9.1 , 6.8, 2.0 Hz, 1 H) 7.85 (d, J=7.0 Hz, 1 H)

[00147] ¹³C NMR (100 MHz, DMSO-d6) δ ppm 60.4, 67.8, 69.1 , 72.7, 76.3, 105.1 , 107.7, 121 .1 , 139.1 , 140.2, 158.4

Example 5

Synthesis of pyrithione^-D-O-galactopyranoside (5a, Figure 9)

[00148] Synthesis was conducted according to Hartung et al. Eur. J. Org. Chem., 1999, 97-106 for the D-glucose analogue: pyrithione (7.5 g, 59 mmol) was dissolved in anhydrous acetonitrile (105 ml). Anhydrous potassium carbonate (25 g, 180 mmol), tetra-butylammonium hydrogensulfate (2.0 g, 5.9 mmol) and 2,3,4,6-tetra-O-acetyl-a- D-galactopyranosyl bromide (20 g, 49 mmol) were added and the slurry was vigorous stirred for 3 hours at room temperature. The yellow reaction mixture was diluted with methyl tert-butyl ether (150 ml) and ice cold water (400 ml) and the pH was adjusted to 13 with 1 N sodium hydroxide solution. A part of the title compound precipitated as a yellow solid, which was removed by suction. The organic phase was separated, diluted with ethanol (150 ml), the precipitated solid added and concentrated in vacuum to a thick yellow suspension, which was cooled to 0°C. The solid was isolated by filtration, washed with ice cold ethanol and dried in vacuum to yield 5e (13 g, 58 %, 28.4 mmol) as a yellow solid.

[00149] ¹H NMR (400 MHz, chloroform-d) δ ppm 1 .96 (s, 3 H) 2.01 (s, 3 H) 2.19 (s, 3H) 2.21 (s, 3H) 3.90 (t, J=6.4 Hz, 1 H) 4.03 (dd, J=1 1 .3, 6.1 Hz, 1 H) 4.23 (dd, J=1 1 .3, 6.9 Hz, 1 H) 5.15 (dd, J=10.4, 3.3 Hz, 1 H) 5.37 - 5.48 (m, 2 H) 5.69 (d, J=8.2 Hz, 1 H) 6.49 (t, J=6.7 Hz, 1 H) 7.13 (t, J=7.5 Hz, 1 H) 7.62 (d, J=8.0 Hz, 1 H) 7.78 (d, J=6.9 Hz, 1 H)

[00150] At 5°C, 5e (1 6 g, 35 mmol) was added to a solution of sodium methoxide (5.4 M in methanol; 1 ml) in anhydrous methanol (160 ml) and the reaction mixture was stirred for 3 hours. The formed precipitate was collected by suction, washed with cold methanol and dried in vacuum to afford 5a (9.6 g, 94 %, 33 mmol) as slightly yellow crystals. An analytical sample was recrystallized from ethanol/ water (1 :1 ).

[00151 ] ¹H NMR (400 MHz, D₂0) δ ppm 3.68 - 3.83 (m, 4 H) 3.89 (dd, J=8.1 Hz, J=9.8 Hz, 1 H) 3.97 (d, J=3.3 Hz, 1 H) 5.44 (d, J=8.0 Hz, 1 H) 7.00 (t, J=6.9 Hz, 1 H) 7.54 (t, J=7.8 Hz, 1 H) 7.69 (d, J=8.7 Hz, 1 H) 8.30 (d, J=6.8 Hz, 1 H)

[00152] ¹³C NMR (100 MHz, DMSO-d6/D₂0 (1 :1 )) δ ppm 61 .5, 69.0, 70.8, 73.8, 77.1 , 107.9, 1 1 6.0, 136.8, 137.4, 142.7, 174.0

Example 6

Synthesis of pyrithione-S^-galactopyranoside (5b, Figure 9)

[00153] To a solution of pyrithione (3.24 g, 25.5 mmol) and 2,3,4,6-tetra-O-acetyl- beta-D-galactopyranosyl trichloroacetimidate (9.0 g, 18.2 mmol) in dichloromethane (50ml) boron trifluoride diethyl etherate (0.56 ml, 4.6 mmol) was added and the mixture was stirred overnight at room temperature. Concentration of the reaction mixture in vacuum and purification via flash chromatography [toluene/ ethyl acetate (2:1 ) to (1 :1 )] gave 5f (7.2 g; 86 %, 15.7 mmol) as a white foam.

[00154] ¹H NMR (400 MHz, chloroform-d) δ ppm 1 .97 (s, 3 H) 2.02 (s, 3 H) 2.14 (s, 3 H) 2.17 (s, 3 H) 4.05 - 4.17 (m, 3 H) 5.00 (d, J=10.2 Hz, 1 H) 5.14 (dd, J=9.9, 3.3 Hz, 1 H) 5.44 - 5.51 (m, 2 H) 7.09 - 7.18 (m, 1 H) 7.18 - 7.27 (m, 1 H) 7.45 (d, J=8.0 Hz, 1 H) 8.22 (d, J=4.9 Hz, 1 H)

[00155] At 5°C, 5f (7.2 g; 15.7 mmol) was added to a solution of sodium methoxide (5.4 M in methanol; 0.4 ml) in anhydrous methanol (60 ml) and the reaction mixture was stirred for 5 hours. The formed precipitate was collected by suction, washed with cold methanol and dried in vacuum. The crude material thus obtained was recrystallized from ethanol/ water (1 :1 ) to yield pure 5b (3.7 g, 12.8 mmol, 84 %) as colorless crystals.

[00156] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.39 - 3.64 (m, 5 H) 3.74 (br. s., 1 H) 4.59 (d, J=4.3 Hz, 1 H) 4.66 (t, J=5.4 Hz, 1 H) 4.73 (d, J=9.7 Hz 1 H) 4.99 (d, J=5.5 Hz, 1 H) 5.43 (d, J=6.0 Hz, 1 H) 7.20 (t, J=6.7 Hz, 1 H) 7.31 (t, J=7.8 Hz, 1 H) 7.55 (d, J=8.1 Hz, 1 H) 8.27 (d, J=6.2 Hz, 1 H)

[00157] ¹³C NMR (100 MHz, DMSO-d6) δ ppm 60.9, 68.6, 69.4, 74.6, 79.8, 83.1 , 122.2, 123.9, 127.5, 138.5, 150.9

Example 7

4-(pyrithione-S-methyl)phenyl-beta-D-galactopyranoside (5Sa, Figure 10)

[00158] To a suspension of 4-hydroxybenzyl alcohol (15 g, 120.8 mmol), tetra- butylammonium bromide (4 g, 12.4 mmol) and 2,3,4,6-tetra-O-acetyl-alpha-D- galactopyranosyl bromide (40 g, 97 mmol) in dichloromethane (150 ml) were added water (100 ml) and 10 N sodium hydroxide solution (20 ml) and the reaction mixture was vigorously stirred for 18 hours at room temperature. The organic phase was separated and the aqueous phase extracted with dichloromethane (100 ml). The combined organic phases were dried (sodium sulfate) and concentrated in vacuum. Purification [flash chromatography, petroleum ether/ ethyl acetate (1 .5:1 ) to (1 :1 )] afforded 7 (1 6 g, 36 %, 35 mmol) as a white foam.

[00159] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .94 (s, 3 H) 2.00 (s, 3H) 2.03 (s, 3H) 2.14 (s, 3 H) 4.08 - 4.10 (m, 2 H) 4.37-4.45 (m, 3H) 5.10 (t, J=5.7 Hz, 1 H) 5.20 (dd, J=7.8 Hz, J=10.3 Hz, 1 H) 5.28 (dd, J=3.4 Hz, J=10.3 Hz, 1 H) 5.33 (d, J=3.4 Hz, 1 H) 5.41 (d, J=7.8 Hz, 1 H) 6.93 (m, J=8.7 Hz, 2 H) 7.26 (m, J=8.7 Hz, 2 H)

[001 60] To a solution of 7 (1 6 g, 35 mmol), 4-dimethylaminopyridine (1 .4 g, 1 1 mmol) and trimethylamine (7.2 ml, 52 mmol) in dry dichloromethane (150 ml) was added p-toluenesulfonyl chloride (9.5 g, 50 mmol) and the reaction mixture was stirred for 4 hours at room temperature. The mixture was concentrated in vacuum and purified by flash chromatography (petroleum ether/ ethyl acetate (2:1 )) giving 7a (8.9 g, 54 %, 19 mmol) as a sirup.

[001 61 ] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .94 (s, 3 H) 2.00 (s, 3H) 2.03 (s, 3H) 2.14 (s, 3 H) 4.08 - 4.10 (m, 2 H) 4.43 (t, J=6.3 Hz, 1 H) 4.72 (s, 2H) 5.21 (dd, J=7.7 Hz, J=10.3 Hz, 1 H) 5.28 (dd, J=3.4 Hz, J=10.3 Hz, 1 H) 5.34 (d, J=3.4 Hz, 1 H) 5.48 (d, J=7.7 Hz, 1 H) 6.98 (m, J=8.7 Hz, 2 H) 7.40 (m, J=8.7 Hz, 2 H)

[001 62] 7a (8.3 g, 18 mmol) was dissolved in anhydrous acetonitrile (50 ml). Anhydrous potassium carbonate (8.9 g, 64 mmol), tetra-butylammonium hydrogensulfate (0.72 g, 2.1 mmol) and pyrithione (2.7 g, 21 mmol) were added and the slurry was vigorously stirred for 18 hours at room temperature. The reaction mixture was filtered, the solid was washed with dichloromethane (2 x 100 ml) and the filtrate was concentrated in vacuum. The residue was crystallized from ethanol giving 7b (6.4 g, 63 %, 1 1 .4 mmol) as colorless crystals.

[001 63] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .94 (s, 3 H) 1 .98 (s, 3H) 2.02 (s, 3H) 2.13 (s, 3 H) 4.04 - 4.13 (m, 2 H) 4.20 (s, 2H) 4.41 (t, J=6.3 Hz, 1 H) 5.20 (dd, J=7.8 Hz, J=10.2 Hz, 1 H) 5.27(dd, J=3.3 Hz, J=10.3 Hz, 1 H) 5.33 (d, J=3.0 Hz, 1 H) 5.45 (d, J=7.7 Hz, 1 H) 6.96 (d, J=8.6 Hz, 2 H) 7.14 - 7.23 (m, 1 H) 7.32 (t, J=7.9 Hz, 1 H) 7.38 - 7.48 (m, 3 H) 8.28 (d, J=6.4 Hz, 1 H)

[001 64] At room temperature, a solution of 7b (6.4 g, 1 1 .4 mmol) in anhydrous dichloromethane (25 ml) was added to a solution of sodium methoxide (5.4 M in methanol; 0.2 ml) in anhydrous methanol (25 ml) and the reaction mixture was stirred for 2 hours. The formed precipitate was collected by suction, washed with cold ethanol and dried in vacuum. The crude material thus obtained was recrystallized from ethanol/water (1 :1 ) to yield pure 5Sa (4.2 g, 93 %, 10.6 mmol) as colorless crystals.

[001 65] ¹H NMR (400 MHz, D20/DMSO-d6) δ ppm 3.44 - 3.60 (m, 5 H) 3.73 (d, J=3.2 Hz, 1 H) 4.13 (s, 2 H) 4.78 (d, J=7.7 Hz, 1 H) 6.97 d, J=8.6 Hz, 2H) 7.13 - 7.22 (m, 1 H) 7.32 (d, J=8.6 Hz, 2 H) 7.41 (d, J=4.8 Hz, 2 H) 8.17 (d, J=6.4 Hz, 1 H)

[001 66] ¹³C NMR (100 MHz, D20/DMSO-d6) δ ppm 34.6, 61 .6, 69.2, 71 .3, 73.8, 76,2, 101 .7, 1 17.7, 122.5, 123.7, 129.9, 131 .3, 139.6, 152.4, 157.7

Example 8

L-Alanine(4-(pyrithione-S-methyl)phenyl)amide (5Sb, Figure 11)

[001 67] To a solution of 4-aminobenzyl alcohol (5 g, 40.6 mmol) and N-(tert- butoxycarbonyl)-L-alanine (9.8 g, 52 mmol) in anhydrous dioxane (60 ml) was added N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (13 g, 54 mmol) and the reaction mixture was stirred for 3 days at room temperature. The reaction mixture was diluted with water (600 ml), washed with petroleum ether/ methyl tert-butyl ether (100 ml, 1 :1 ) and ethyl acetate (3 x 150 ml). The combined ethyl acetate extracts were dried (sodium sulfate), concentrated in vacuum and crystallized from toluene to yield N- Boc-L-alanyl(4-(hydroxymethyl)phenyl)amide 8 (9.8 g, 82 %, 33.3 mmol) as a waxy solid. [001 68] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .24 (d, J= 7.1 Hz, 3 H) 1 .37 (s, 9 H) 4.09 (m, 1 H) 4.42 (d, J=5.5 Hz, 2 H) 5.08 (t, J=5.6 Hz, 1 H) 7.03 (d, J=7.2 Hz, 1 H) 7.22 (m, J=8.5 Hz, 2 H) 7.53 (m, J=8.5 Hz, 2 H) 9.84 (s, 1 H)

[001 69] To a solution of 8 (9.8 g, 33.3 mmol) and 4-dimethylaminopyridine (6.1 g, 50 mmol) in dry dichloromethane (50 ml) was added p-toluenesulfonyl chloride (9.5 g, 50 mmol) and the reaction mixture was stirred for 48 hours at reflux temperature. The reaction mixture was cooled to room temperature, diluted with dichloromethane (100 ml), washed with water (200 ml), dried (sodium sulfate) and concentrated in vacuum affording crude activated compound (17.1 g), which was dissolved in anhydrous acetonitrile (70 ml).

[00170] Anhydrous potassium carbonate (1 6 g, 1 1 6 mmol), tetra-butylammonium hydrogensulfate (2 g, 5 mmol) and pyrithione (5 g, 39 mmol) were added and the slurry was vigorous stirred for 7 hours at room temperature. The reaction mixture was filtered, the solid washed with warm dichloromethane (2 x 100 ml) and the filtrate was concentrated in vacuum. The residue was purified by flash chromatography (toluene/ acetone (2:1 ) to (1 :1 .5)) and crystallized from 2-propanol giving 8a (3.6 g, 27 %, 8.9 mmol) as colorless crystals.

[00171 ] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .23 (d, J= 7.1 Hz, 3 H) 1 .36 (s, 9 H) 4.09 (m, 1 H) 4.19 (s, 2 H) 7.05 (d, J=7.1 Hz, 1 H) 7.18 (t, J=6.8 Hz, 1 H) 7.31 (t, J=7.8 Hz, 1 H) 7.38 (d, J=8.4 Hz, 2H) 7.43 (d, J=8.0 Hz, 1 H) 7.57 (d, J=8.4 Hz, 2 H) 8.28 (d, J=6.4 Hz, 1 H) 9.94 (s, 1 H) [00172] At 40°C, a solution of 8a (3.5 g, 8.6 mmol) in ethanol (35 ml) and 6.7 M ethanolic HCI (17.5 ml) was stirred for 2 hours. The solvent was removed in vacuum and the residue was crystallized from 2-propanol. The crude product was recrystallized by dissolving in methanol, diluting with 2-propanol and concentrating in vacuum to afford 5Sb (2.85 g, 96 %, 8.3 mmol) as off-white crystals.

[00173] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .48 (d, J=6.2 Hz, 3 H) 4.1 6 (br. s., 1 H) 4.26 (br. s., 2 H) 7.26 (br. s., 1 H) 7.40 (d, J=8.0 Hz, 2H), 7.48 (br. t., J=7.4 Hz, 1 H) 7.55 (br. d., J=7.7 Hz, 1 H) 7.66 (d, J=7.9 Hz, 2H) 7.66 (d, J=7.8 Hz, 6 H) 8.41 (d, J=5.6 Hz, 1 H) 8.53 (br. s., 3 H) 1 1 .15 (s., 1 H)

[00174] ¹³C NMR (100 MHz, DMSO-d6) δ ppm 17.4, 33.7, 49.0, 1 19.6, 121 .6, 123.0, 128.7, 129.6, 130.7, 137.9, 139.0, 151 .6, 1 68.4

Example 9

4-(pyrithione-S-methyl)phenyl-beta-D-glucopyranosiduronic acid (5Sc)

[00175] At 5°C, boran trifluoride diethyl etherate (4 ml, 31 .6 mmol) was added to a solution of p-cresol (45 g, 416 mmol) and 2,3,4-tri-O-acetyl-alpha-D-glucuronic acid methylester trichloroacetimidate (200 g, 418 mmol) in dichloromethane (650 ml) and the mixture was stirred for 1 hour. Concentration of the reaction mixture in vacuum and crystallization from 2-propanol gave 9 (140 g; 80 %, 332 mmol) as colorless crystals.

[00176] ¹H NMR (400 MHz, DMS0-d6) δ ppm 1 .96 - 2.06 (m, 9 H) 2.25 (s, 3 H) 3.64 (s, 3 H) 4.69 (d, J=9.90 Hz, 1 H) 5.02 - 5.15 (m, 2 H) 5.47 (t, J=9.60 Hz, 1 H) 5.59 (d, J=7.95 Hz, 1 H) 6.89 (d, J=7.82 Hz, 2 H) 7.13 (d, J=8.19 Hz, 2 H).

[00177] To a suspension of 9 (100 g, 240 mmol) and N-bromosuccinimide (43 g, 240 mmol) in dry carbon tetrachloride (500 ml) was added 2,2'-azobis(2- methylpropionitrile) (0.7 g, 4 mmol) and the reaction mixture was stirred for 1 hour at reflux temperature. The hot reaction mixture was filterd, cooled to room temperature and concentrated in vacuum affording crude activated compound, which was dissolved in anhydrous acetonitrile (1000 ml).

[00178] Anhydrous potassium carbonate (120 g, 850 mmol), tetra-butylammonium hydrogensulfate (9.5 g, 28 mmol) and 2-mercaptopyridine N-oxide (36 g, 280 mmol) were added and the slurry was vigorous stirred for 18 hours at room temperature. The reaction mixture was filtered, the solid was washed with dichloromethane (2 x 1000 ml) and the filtrate was concentrated in vacuum. The residue was crystallized from ethanol to give 9a (75 g, 58 %, 137 mmol) as colorless crystals.

[00179] ¹H NMR (400 MHz, DMS0-d6) δ ppm 1 .98 - 2.05 (m, 9 H) 3.64 (s, 3 H) 4.22 (s, 2 H) 4.72 (d, J=9.90 Hz, 1 H) 5.04 - 5.1 6 (m, 2 H) 5.48 (t, J=9.60 Hz, 1 H) 5.68 (d, J=7.95 Hz, 1 H) 7.00 (d, J=7.93 Hz, 2 H) 7.20 (t, J=6.79 Hz, 1 H) 7.33 (t, J=7.83 Hz, 1 H) 7.40 - 7.50 (m, 3 H) 8.29 (d, J=6.29 Hz, 1 H).

[00180] At room temperature, a sodium methoxide solution (5.4 M in methanol; 5.4 ml) was added to a suspension of 9a (75 g, 140 mmol) in anhydrous methanol (330 ml) and the reaction mixture was stirred for 1 hour. The formed precipitate was collected by suction, washed with cold methanol and gradually added to a ice-cold 0.25 M sodium hydroxide solution (140 mmol, 560 ml). After 30 minutes the resulting solution was acidified to pH 1 .8 (1 M hydrochloric acid), filtered and the crude material thus obtained was recrystallized from ethanol/water (1 :1 ) to yield 5Sc (37 g, 66 %, 90 mmol) as colorless crystals.

[00181 ] ¹H NMR (400 MHz, DMSO-d6) δ ppm 3.24 - 3.49 (m, 3 H) 3.92 (d, J=9.54 Hz, 1 H) 4.20 (s, 2 H) 5.05 (d, J=7.34 Hz, 1 H) 5.24 (br. s., 1 H) 5.45 (br. s., 2 H) 7.00 (d, J=8.68 Hz, 2 H) 7.14 - 7.25 (m, 1 H) 7.30 - 7.49 (m, 4 H) 8.30 (d, J=6.35 Hz, 1 H) 12.80 (br. S., 1 H).

[00182] ¹³C NMR (100.6 MHz, DMS0-d6) δ ppm 33.7, 71 .8, 73.4, 75.9, 76.3, 100.4, 1 1 6.8, 121 .7. 122.7, 125.9, 129.6, 130.6, 138.6, 151 .0, 156.8, 170.6.

Example 10

L-pyroglutamyl(4-(pyrithione-S-methyl)phenyl)amide (5Sd)

[00183] To a solution of 4-aminobenzyl alcohol (62 g, 504 mmol) and N-(tert- butoxycarbonyl)-L-pyroglutamic acid (1 16 g, 504 mmol) in anhydrous dioxane (500 ml) was added N-ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (130 g, 526 mmol) and the reaction mixture was stirred for 12 hours at room temperature. To complete precipitation the reaction mixture was cooled to 10°C. Compound 10 (1 14 g, 68 %, 340 mmol) was collected by suction, washed with cold dioxane and dried in vacuum.

[00184] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .34 (s, 9 H) 1 .83 - 1 .94 (m, 1 H) 2.23 - 2.26 (m, 1 H) 2.37 - 2.48 (m, 2 H) 4.42 (s, 2 H) 4.63 (dd, J=8.99, 3.61 Hz, 1 H) 5.10 (bt, 1 H) 7.25 (d, J=8.56 Hz, 2 H) 7.53 (d, J=8.44 Hz, 2 H) 10.23 (s, J=4.76 Hz, 1 H). [00185] To a cooled suspension of 10 (1 13 g, 338 mmol) and p-toluenesulfonyl chloride (71 g, 372 mmol) in dry dichloromethane (500 ml) was added 4- dimethylaminopyridine (3 g, 25 mmol). Under cooling triethylamine (100 ml, 718 mmol) was added dropwise during 15 minutes. To complete conversion the reaction mixture was stirred for 2 hours at room temperature. The solution was concentrated in vaccum and the residue was purified by flash chromatography (toluene/acetone (4:1 )) to afford crude activated compound (61 g, 50 %, 173 mmol), which was dissolved in anhydrous acetonitrile (350 ml).

[00186] Anhydrous potassium carbonate (80 g, 579 mmol), tetra-butylammonium hydrogensulfate (8 g, 20 mmol) and 2-mercaptopyridine N-oxide (26.4 g, 208 mmol) were added and the slurry was vigorous stirred over night at room temperature. The reaction mixture was diluted with dichloromethane (500 ml) and water (2000 ml). The organic layer was separated and the aqueous layer was extracted with dichloromethane (500 ml). The combined organic layers were concentrated in vacuum. Purification via flash chromatography [acetone] afforded 10a (38 g, 25 %, 86 mmol) as off-white solid.

[00187] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .36 (s, 9 H) 1 .85 - 1 .95 (m, 1 H) 2.22 - 2.37 (m, 1 H) 2.39 - 2.49 (m, 2 H) 4.22 (s, 2 H) 4.65 (dd, J=8.86, 3.24 Hz, 1 H) 7.14 - 7.27 (m, 1 H) 7.34 (t, J=7.77 Hz, 1 H) 7.40 - 7.48 (m, 3 H) 7.58 (d, J=8.19 Hz, 2 H) 8.30 (d, J=6.08 Hz, 1 H) 10.32 (s, 1 H) [00188] A mixture of 10a (36.5 g, 82.3 mmol) in ethanol (380 ml) and 6.7 M ethanolic HCI (180 ml) was stirred for 1 hour at reflux temperature. The solvent was removed in vacuum, the residue was coevaporated twice with 2-propanol (400 ml) and fractional crystallized from ethanol. The crude product was recrystallized from methanol/water (1 :1 ) to afford 5Sd (1 6 g, 57 %, 46.6 mmol) as off-white crystals.

[00189] ¹H NMR (400 MHz, DMSO-d6) δ ppm 1 .94 - 2.04 (m, 1 H) 2.10 - 2.26 (m, 2 H) 2.29 - 2.39 (m, 1 H) 4.1 6 - 4.25 (m, 3 H) 7.20 (t, J=6.67 Hz, 1 H) 7.31 - 7.48 (m, 4 H) 7.61 (d, J=8.44 Hz, 2 H) 7.89 (bs, 1 H) 8.30 (d, J=6.29 Hz, 1 H) 10.10 (s, J=5.03 Hz, 1 H)

[00190] ¹³C NMR (100.6 MHz, DMSO-d6) δ ppm 25.81 , 29.73, 33.92, 56.88, 1 19.99, 121 .68, 122.70, 125.81 , 129.90, 131 .18, 138.58, 138.63, 150.99, 171 .81 , 177.92.

Example 11

4-(pyrithione-S-methyl)phenyl-phosphate (5Se)

[00191 ] To a solution of p-cresol phosphate (450 mg, 2.39 mmol) in dry acetonitrile (5.5 ml_, 100 mmol) was added NBS (426 mg, 2.39 mmol). The reaction mixture was stirred for 1 6 h at room temperature and concentrated in vacuum to afford crude activated compound 11.

[00192] To a solution of crude 11 in dry acetonitrile (4.8 mL, 92 mmol) were added potassium carbonate (1 .25 g, 9.04 mmol), tetrabutylammonium hydrogensulfate (80 mg, 0.24 mmol) and 2-mercaptopyridine N-oxide (280 mg, 2.20 mmol) and the slurry was vigorously stirred at room temperature. Then, 15 mL water were added and the pH-value was adjusted to 1 with 8 mL of a 2N HCI solution. The aqueous layer was separated, washed with dichloromethane (15 mL), concentrated in vacuum and ethanol (6 mL) was added. The precipitated solid was filtered and the solution was concentrated in vacuum. The residue was purified via preparative HPLC (YMC Triart 150 x 20 mm, S-5 μπι, 12 nm; H₂0/MeCN 98:2^MeCN ) to yield 5Se (1 10 mg, 20 %, 0.35 mmol) as yellow solid.

[00193] ¹H NMR (400 MHz, DMSO-d6) δ ppm 4.20 (s. 2 H), 4.20 (s, 2 H) 5.05 (d, J=7.34 Hz, 1 H) 5.24 (br. s., 1 H) 5.45 (br. s., 2 H) 7.00 (d, J=8.68 Hz, 2 H) 7.17 - 7.21 (m, 1 H) 7.30 - 7.49 (m, 4 H) 8.30 (d, J=6.35 Hz, 1 H)

Example 12 Minimal inhibitory concentration of compounds used for synthesis of enzyme responsive inhibitors (Table II)

[00194] Nutrient broth (5 g/l peptone, 5 g/l NaCI, 2 g/L yeast extract, 1 g/l beef extract, pH 7.4) was autoclaved in glass tubes (20 ml_ total volume, 3 ml_ liquid volume) and allowed to cool to room temperature. A series of concentrations of metal-ion chelating compounds 2 to 6 with potential antimicrobial effect were added as concentrated stock solution and tubes were inoculated with approximately 10⁶ CFU/mL of freshly grown cells of Escherichia coli ATCC 25922. Growth was recorded by measuring optical density at 600 nm after incubation for 24 h at 37°C and 150 rpm. Minimal inhibitory concentration was defined as the concentration leading to at least 3-fold reduction in the optical density compared to control cultures without added compounds.

Example 13

Inhibition of diverse bacteria by pyrithione (5) and resistance of Pseudomonas aeruginosa (Table III)

[00195] Nutrient agar (5 g/l peptone, 5 g/l NaCI, 2 g/L yeast extract, 1 g/l beef extract, 15 g/L agar, pH 7.4) was autoclaved and allowed to cool to 50°C. The inhibitor / biocide 5 was added to yield a final concentration of 0.2 mM (from a 100 mM stock solution in dimethyl formamide) then plates were poured. Control plates were prepared similarly, but without inhibitor 5. Agar plates were inoculated (streak- outs) from freshly grown colonies of test strains and incubated for 24 h at 37°C. Growth was inspected visually and rated from "vigorous growth" [++++] to "no growth detectable" [-].

Example 14

Enzymatic release of iron-chelating inhibitors (Figure 12)

[00196] Metal ion-chelating inhibitors 3, 4, and 5 formed colored complexes or precipitates in the presence of iron (III) in aqueous solution at neutral pH. Absorbance maxima were 455 nm (3), 420 nm (4), and 555 nm (5), respectively. The corresponding enzyme responsive inhibitors / masked inhibitors 3a, 4a, 5a and 5Sb did not exhibit color formation under the same conditions.

[00197] Change in absorbance at 420, 455 or 555 nm was used to monitor the enzymatic release of metal ion-chelating inhibitors. Enzymatic reactions were performed in 100 mM Na₂HP0₄ buffer, pH 7.3 containing 1 mM MgCI₂ and 1 mM FeCI₃-6H₂0 in a microtiter plate heated to 37°C (0.3 ml reaction volume). Enzyme responsive inhibitors / masked inhibitors were added to final concentrations of 1 mM (4a, 5a, 5Sb) and 10 mM (3a). After the addition of enzyme and a pre-incubation period of 2 - 6 min the change in absorbance against air over time was recorded with a spectrophotometer. Control reactions without enzyme contained similar concentrations. Absorbance increased in reactions with added enzyme (black symbols) while absorbance in reactions without enzyme (open symbols) remained constant. Furthermore, and as expected within the art, it was observed that compound 5b could not be unmasked by the action of beta-galactosidase. Hence, S- glycosides cannot be considered suitable for the purpose.

Example 15

Inhibition of beta-galactosidase negative bacteria by surrounding bacteria degrading triclosan-beta-D-galactopyranoside 1a (Figure 13)

[00198] Rambach agar (5 g/l peptone, 2 g/l yeast extract, 1 g/L sodium deoxycholate, 10 g/l propylene glycol, 4.5 mg/l neutral red, 15 g/l agar, pH 7.4) was autoclaved and allowed to cool to 50°C. Then 1 mM IPTG, 0.5 mM 1 -O-methyl- glucuronic acid and 0.2 mM 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid were added; followed by the addition of triclosan-beta-D-galactopyranoside (1a) to yield a final concentration of 2 mg/L (from a 10 mg/mL stock solution in dimethyl formamide - concentration taken from GB 2509159A). Agar plates were inoculated with S. enteritidis RKI 05/07992 (S.e., beta-galactosidase negative) and E. coli ATCC 25922 (E.c, beta-galactosidase positive). Either a mixed cell suspension (50 μΙ_) containing approximately 10³ CFU/ml S. enteritidis and 1 0⁴ CFU/ml E. coli or a cell suspension (50 μΙ_) containing only 10³ CFU/ml S. enteritidis were plated. Agar plates were incubated at 37°C and growth was recorded by digital imaging after 24 h (Fig. 13). Sefa-glucuronidase positive E. coli appeared as blue colonies while beta- glucuronidase negative S. enteritidis appeared as white colonies.

Example 16

No inhibition of beta-galactosidase positive bacteria by deferasirox-beta-D- galactopyranoside (2a)

[00199] Nutrient broth with 1 mM IPTG was prepared as described above. Compound 2a was added to yield final concentrations of 0.1 to 5 mM (from 100 mM stock solution in dimethyl formamide). Test tubes were inoculated with freshly grown overnight cultures of Escherichia coli ATCC 25922 (beta-galactosidase positive). Tubes were incubated at 37°C and 150 rpm and growth was inspected by measurement of optical density (600 nm) after 24 h. No difference in growth was observed between tubes containing 2a and control tubes.

Example 17

Selective inhibition of beta-galactosidase positive bacteria by enzyme responsive inhibitor 3a (Figure 14)

[00200] Nutrient Agar with 1 mM IPTG was prepared as described above. Iron- chelating inhibitor 3 and enzyme responsive inhibitor 3a were added to yield final concentrations of 5 mM and 20 mM, respectively (from 50 mM and 200 mM filter- sterilized stock solutions in H₂0), then plates were poured. Agar plates were inoculated with freshly grown cultures of the same bacterial strains as in Example 15 (S. enteritidis, E. coli). Growth was recorded by digital imaging after 23 h of incubation at 37°C (Figure 14).

Example 18

Selective inhibition of beta-galactosidase positive bacteria by enzyme responsive inhibitor 4a (Figure 15) [00201 ] Nutrient broth was prepared as described above. IPTG was added to yield a final concentration of 1 mM (from 1 M filter-sterilized stock solution in H₂0). Compound 4 and corresponding enzyme responsive inhibitor 4a were added to yield final concentrations of 1 mM and 5 mM, respectively (from 100 mM filter-sterilized stock solution in H₂0). Nutrient Agar was prepared as described above I PTG and 4a were added to yield final concentrations of 1 mM and 5 mM, respectively. Control plates were prepared similarly, but without 4a. Another set of nutrient agar plates was prepared with 10 mM 4a and supplemented in addition with 0.8 mM 5-Bromo-6- chloro-3-indoxyl caprylate, 0.4% v/v Tween 80 and 0.3 mM FeCI₃-6H₂0 (added from concentrated stock solutions, prepared either in dimethyl formamide or in ultrapure water and filter-sterilized). Plates and tubes were inoculated from freshly grown overnight cultures of Salmonella enteritidis RKI 05/07992 (S.e., beta-galactosidase negative) and Escherichia coli ATCC 25922 (E.c, beta-galactosidase positive) which had been diluted in sterile saline (NaCI 0.9%). A mixed cell suspension (50 μΙ_ was plated) contained approximately 10³ CFU/ml S.e. and 3 0⁴ CFU/ml E.c. A reference cell suspension with only S.e. also contained approximately 10³ CFU/ml. Agar plates and tube cultures were incubated at 37°C, and tubes were shaken continuously with an agitation of 150 rpm. Growth in nutrient broth was inspected after 24 h by measurement of optical density (600 nm) (Figure 15a). Growth on nutrient agar was recorded by digital imaging after 23-24 h (Figures 15b and 15c).

Example 19

Selective inhibition of beta-galactosidase positive bacteria by enzyme responsive inhibitor 5a (Figure 16)

[00202] Nutrient broth with 1 mM IPTG was prepared as described above. Metal ion-chelating inhibitor pyrithione (5) and enzyme responsive inhibitor pyrithione-beta- D-1 -O-galactopyranoside (5a) were added to yield final concentrations of 0.1 mM and 0.2 mM, respectively (from 100 mM stock solutions in dimethyl formamide). Rambach Salmonella agar (5 g/l peptone, 2 g/l yeast extract, 1 g/L sodium deoxycholate, 10 g/l propylene glycol, 4.5 mg/l neutral red, 15 g/l agar, pH 7.4) was autoclaved and allowed to cool to 50°C. Then IPTG and 5a were added to yield final concentrations of 1 mM and 0.2 mM, respectively, then plates were poured. Agar plates and tube cultures were inoculated with the same bacterial strains of S. enteritidis and E. coli as specified above. A mixed cell suspension (50 μΙ_ was plated) contained approximately 10³ CFU/ml S.e and 3 0⁴ CFU/ml E.c. Plates and tubes were incubated at 37°C, tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 17 h (Figure 16a). Growth on agar plates was recorded by digital imaging after 24 h (Figure 16b).

Example 20

Selective inhibition of beta-galactosidase positive bacteria by enzyme responsive inhibitor 5Sa (Figure 17)

[00203] Nutrient broth with 1 mM IPTG was prepared as described above. Enzyme responsive inhibitor 5Sa was added to yield a final concentration of 1 mM (from 200 mM stock suspension in dimethyl formamide). Rambach Salmonella agar was prepared as described above, except that neutral red was omitted. After autoclaving and cooling to 50°C, 1 mM IPTG, 0.5 mM 1 -O-methyl-glucuronic acid, 0.2 mM 5- bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid and 0.5 mM enzyme responsive inhibitor 5Sa were added from concentrated stock solutions (either prepared in ultrapure water and filter-sterilized or in dimethyl formamide) then plates were poured. Agar plates and tube cultures were inoculated with the same bacterial strains (S. enteritidis, E. coli) as specified above. A mixed cell suspension (50 μΙ_ was plated) contained approximately 10³ CFU/ml S.e. and 3 0⁴ CFU/ml E.c. Plates and tubes were incubated at 37°C, tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 20 h (Figure 17a). Growth on agar plates was recorded by digital imaging after 24 h (Figure 17b).

Example 21

Enrichment of S. enteritidis in broth by the action of enzyme responsive inhibitor 5Sa

(Table IV) [00204] Nutrient broth with 1 mM IPTG was prepared as described above. Sodium phosphate buffer (pH 7.3) and Fe (III) chloride heptahydrate were added to yield final concentrations of 25 mM and 1 mM, respectively (from 100 mM, sterilized stock solutions in ultrapure water), which resulted in the formation of iron phosphate particles. Enzyme responsive 5Sa was added to yield a final concentration of either 0.5 or 1 .0 mM. A control medium was prepared similarly, but without 5Sa. Test strains S. enteritidis and E. coli as specified above were used. Tube cultures with 3 - 4 ml_ medium were inoculated with 50 μΙ_ of a mixed cell suspension containing approximately 1 x 10³ CFU/mL S. enteritidis and 1 x 10⁴ CFU/mL E. coli. Broth cultures were incubated for 20 h at 37°C and 150 rpm, followed by plating of appropriate dilutions on trypticase soy agar containing 0.5 mM 1 -O-methyl-glucuronic acid and 0.2 mM 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic. Colony forming units (CFU) were analysed after an incubation of 22 h at 37°C. Blue colonies were counted as (beta-glucuronidase positive) E. coli and white colonies were counted as (beta-glucuronidase negative) S. enteritidis. The percentage of S. enteritidis relative to the total number of colonies is given in Table IV, mean values of two replicate plates.

Example 22

Selective inhibition of aminopeptidase positive bacteria by enzyme responsive inhibitor 5Sb (Figure 18)

[00205] Nutrient broth and nutrient agar were prepared as described above. Compounds 5Sb and 5 were added to yield a final concentration of 0.2 mM (from 100 mM stock suspension/solution in dimethyl formamide) in nutrient broth and concentration of 1 .0 mM in nutrient agar (from 100 mM stock solution in ultrapure water). Test tubes with nutrient broth were inoculated with freshly grown overnight cultures of Staphylococcus aureus ATCC 29213 (S.a., aminopeptidase negative), Citrobacter freundii RKI NM8 (C.f., aminopeptidase positive) and Escherichia coli ATCC 25922 (E.c, aminopeptidase positive) that had been diluted in sterile saline (NaCI 0.9%). Nutrient agar plates were inoculated with a dilution series (prepared with sterile saline) of freshly grown S. aureus and E. coli cultures (Miles-Misra test, 10 μΙ_ per drop). Tubes and plates were incubated at 37°C, tubes were shaken at 150 rpm. Growth in tube cultures was inspected by measurement of optical density (600 nm) after 24 h (Figure 18a). Growth on agar plates was recorded by digital imaging after 24 h (Figure 18b).

Example 23

Selective inhibition of beta-glucuronidase positive bacteria by enzyme responsive inhibitor 5Sc (Figure 19)

[00206] Mineral medium (2.7 g/L potassium dihydrogen phosphate, 0.5 g/L ammonium sulfate, 1 g/L casamino acids, 4 g/L glycerol, pH set to 7.4 with NaOH) was autoclaved, allowed to cool to room temperature and supplemented with 0.5 mM magnesium sulfate, 0.05 mM calcium chloride, trace elements and 1 mM 1 -O-Methyl- beta-D-glucuronic acid. Enzyme responsive inhibitor 5Sc (4-(pyrithione-S- methyl)phenyl-beta-D-glucopyranosiduronic acid; 4-((1 -oxypyridin-1 -ium-2yl) sulfanylmethy)phenyl-beta-D-glucuronic acid) was added to a final concentration of 1 mM (from 200 mM stock solution in dimethyl formamide). Rambach Agar (5 g/L peptone, 5 g/L yeast extract, 1 g/L sodium deoxycholate, 10 g/L propylene-glycol, 15 g/L agar, pH 7.4) was autoclaved and allowed to cool to 50°C, then 1 mM 5Sc and 1 mM 1 -O-Methyl-beta-D-glucuronic were added from concentrated stock solutions. Controls did not contain enzyme responsive inhibitor. Overnight pre-cultures of Salmonella enteritidis RKI 05/07992 (S.e., beta-glucuronidase negative), Escherichia coli O157 LMG 21756 (E.c. 1 , 0157, beta-glucuronidase negative), Escherichia coli NM1 (E.c. 2, non-0157, beta-glucuronidase positive) and Escherichia coli ATCC 25922 (E.c. 3, non-0157, beta-glucuronidase positive) were diluted in sterile saline. Tubes cultures (in triplicate) were inoculated with approximately 10⁴ CFU/mL and incubated for 24 h at 37°C and 150 rpm. Growth was analysed by measuring optical density at 600 nm after 24 h (Figure 19a). Cell suspensions with approximately 10⁸ CFU/mL were streaked-out on agar plates and growth was documented after 24 h at 37°C (Figure 19b). As can be seen from the figures, growth of beta-glucuronidase positive bacteria is inhibited by enzyme responsive inhibitor 5Sc, while growth ofbeta-glucuronidase bacteria is not affected.

Example 24

Selective inhibition of pyroglutamyl aminopeptidase positive bacteria by enzyme responsive inhibitor 5Sd (Figure 20)

[00207] Nutrient broth (5 g/L peptone, 5 g/L sodium chloride, 2 g/L yeast extract, 1 g/L meat extract, pH 7.4) was autoclaved and allowed to cool to room temperature. Nutrient agar (similar to nutrient broth, with 15 g/L agar) was autoclaved and allowed to cool to 50°C. Enzyme responsive inhibitor 5Sd (L-pyroglutamyl(4-pyrithione-S- methyl)phenylamide; (2S)-N-[4-[(1 -oxidopyridin-1 -ium-2-yl)sulfanylmethyl]phenyl]-5- oxo-pyrrolidine-2-carboxamide) was added to a final concentration of 1 mM (from 100 mM stock suspension in dimethyl formamide). Controls did not contain enzyme responsive inhibitor. Overnight pre-cultures of Salmonella enteritidis ATCC 13076 (S.e., pyroglutamyl aminopeptidase negative) and Citrobacter freundii ATCC 8090 (C.f., pyroglutamyl aminopeptidase positive) were diluted in sterile saline. Tubes cultures with nutrient broth were inoculated with approximately 10⁶ CFU/mL and incubated for 24 h at 37°C and 150 rpm. Growth was analysed by measuring optical density at 600 nm after 24 h (Figure 20a). Cell suspensions with approximately 10⁸ CFU/mL were streaked-out on agar plates and growth was documented after 24 h at 37°C (Figure 20b). As can be seen from the figures, growth of pyroglutamase positive bacteria is inhibited by enzyme responsive inhibitor 5Sd, while growth of pyroglutamase negative bacteria is not affected, i.e. 5Sd facilitates suppression of Citrobacter freundii in Salmonella enrichment cultures.

Example 25

Selective inhibition of phosphatase positive bacteria by enzyme responsive

inhibitor 5Se (Figure 21)

[00208] Nutrient broth (5 g/L peptone, 5 g/L sodium chloride, 2 g/L yeast extract, 1 g/L meat extract, 15 g/L agar, pH 7.4) was autoclaved and allowed to cool to 50°C. Enzyme responsive inhibitor 5Se (4-(pyrithione-S-methyl)phenyl-phosphate; 4-((1 - oxypyridin-1 -ium-2yl)sulfanylmethy)phenyl-phosphate) was added to final concentrations of 0.5, 1 .0 and 1 .5 mM, respectively (from sterile-filtered 25 mM stock solution in dimethyl sulfoxide). A control plate did not contain enzyme responsive inhibitor 5Se. An overnight culture of Staphylococcus aureus ATCC 29213 was serially diluted in sterile saline and spotted on agar plates (Miles-Misra test). Growth was documented after 24 h at 37°C (Figure 21 ). As can be seen from the figure, growth of phosphatase positive bacteria is inhibited by enzyme responsive inhibitor 5Se in a concentration-dependent manner.

Claims

CLAIMS 1. A compound comprising a first moiety, a second moiety, and optionally a spacer that covalently links the first moiety to the second moiety, wherein the first moiety is covalently linked to the second moiety or to the optional spacer via an enzyme-labile bond, and wherein the second moiety is an antimicrobial compound, and further wherein the second moiety has the formula (I)

wherein:

R¹ is O, S or NH;

X is C or N, wherein if X is C, then R¹ is O;

R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C₃-₆cycloalkylC_0-6alkyl, C_{1 -4}haloalkyl, OC_{1 -4}haloalkyl, C_0-6alkylNR¹⁴R¹⁵, OCi- 6alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵, SR⁵, (SO₂)NR⁵R⁶, (SO)NR⁵R⁶, SO₃R⁵, SO₂R⁵, SOR⁵ or a 5- or 6-membered heterocyclic saturated, partially unsaturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic ring containing one or more heteroatoms independently selected from N, O or S; and R⁴, R⁵ and R⁶ are independently selected from the group consisting of H, Ci- 4alkyl, heteroarylC_0-6alkyl and arylC_0-6alkyl;

or tautomers or salts thereof.

2. The compound of claim 1 , wherein the second moiety has the formula (II) or (III)

wherein:

R⁹, R¹⁰, and R¹¹ are each independently defined as R³, or

6-membered saturated, partially saturated or unsaturated carbocyclic ring or heterocyclic ring containing one or two heteroatoms independently selected from N, O or S, which 5- or 6-membered carbocyclic ring or 5- or 6-membered heterocyclic ring is unsubstituted or substituted by one or more R³,

or tautomers or salts thereof.

3. The compound of claim 1 or 2, wherein R³ is halo, nitro, CHO, CN, OR⁴, COR⁵, R⁵, C_{1 -6}alkyl, C_2-6alkenyl, C_2-6alkynyl, C₃-₆cycloalkylC_0-6alkyl, C_{1 -4}haloalkyl, OC1- 4haloalkyl, C_0-6alkylNR¹⁴R¹⁵, O C_{1 -6}alkylNR⁵R⁶, CO₂R⁵, CONR⁵R⁶, NR⁵(CO)R⁶, O(CO)R⁵, SR⁵, (SO₂)NR⁵R⁶, (SO)NR⁵R⁶, SO₃R⁵, SO₂R⁵, SOR⁵; and R⁴, R⁵ and R⁶ are independently selected from the group consisting of H, C_{1 -4}alkyl, heteroaryl C_0-6alkyl and arylC_0-6alkyl; or wherein R³ is H, halo, OH, C_{1 -6}alkyl, C1- 4alkyl, C_2-6alkenyl, C_2-4alkenyl or OC_{1 -4}alkyl, particularly OH, C_{1 -6}alkyl or C_2- ₄alkenyl.

4. The compound of any one of claims 1 to 3, wherein the second moiety is selected from compounds 3, 4, 5 and 6:

or tautomers or salts thereof.

5. The compound of any one of claims 1 to 4, wherein the first moiety, optionally together with the optional spacer, substantially eliminates the antimicrobial activity of the second moiety, and/or

wherein the first moiety is selected from the group consisting of amids, amino acids, peptides, nitro compounds and other groups which by the action of certain microbial enzymes are converted to amines, sulfides and disulfides and other groups which by the action of certain enzymes are converted to mercaptanes, organic esters such as fatty acid esters, inorganic esters such as sulfate esters and phosphate esters including inositol phosphates and choline phosphates, ethers, and carbohydrates such as sugars including pyranoses and other groups which are converted by the action of certain microbial enzymes to hydroxyl compounds, and/or

wherein the first moiety is linked via the enzyme-labile bond or via the optional spacer to:

(i) the oxygen atom bonded to X of the second moiety having formula (I),

(iii) the oxygen atom of the OH group of the second moiety shown in formula (II) or (III), (iv) the exocyclic oxygen atom of the second moiety shown in formula (II) that is attached to the ring carbon atom next to X by a double bond, or

(v) the heteroatom of R⁷ of the second moiety having formula (III).

6. The compound of any one of claims 1 to 5, wherein the enzyme-labile bond is susceptible to cleavage by hydrolases, in particular by an enzyme selected from the group consisting of glycosidases, proteases, peptidases, ureidases, esterases, lipases, phosphatases, sulfatases, phospholipases, dealkylases, decarboxylases, and nitroreductases.

7. The compound of any one of claims 1 to 6, wherein the spacer is a self- immolative spacer, particularly a self-immolative spacer derived from hydroxybenzylalcohol, aminobenzylalcohol, aminobenzyloxycarbonyl, and hydroxybenzyloxycarbonyl, more particularly a spacer having a structure as shown in general formula (IV) or (V)

wherein:

X-l represents the second moiety;

Y is selected from NH or O;

X is selected from O, NH, S or 0-C(=0);

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from the group consisting of hydrogen, C_{1 -4}alkyl, C_{1 -4}alkoxy, fused heteroaryl, fused aryl, heteroarylC₀- 4alkyl, arylC₀-₄alkyl, halo, cyano, nitro, formyl, and optionally substituted amino, carboxy, carbonyl, hydroxy and sulfonyl.

8. The compound of any one of claims 1 to 7, where the compound is selected from compounds 3a, 4a, 5a, 5b, 5Sa or 5Sb:

9. A composition comprising a compound according to any one of claims 1 to 8, wherein the composition may be a pharmaceutical composition.

10. A kit comprising a compound according to any one of claims 1 to 8 and, optionally, instructions for use.

1 1 . A method for changing the microbial composition of microbiota comprising contacting said microbiota with a compound according to any one of claims 1 to 8, and optionally further comprising the step of adding non-toxic transition metals to said microbiota, particularly adding Fe(l ll) ions to said microbiota in an amount so as to result in a concentration of 0.1 mM to 10 mM Fe(lll) ions.

12. In vitro use of a compound according to any one of claims 1 to 8 as an antimicrobial compound.

13. Use of a compound according to any one of claims 1 to 8

for the selective culturing, plating, enrichment or growth of microorganisms; to discover, determine, isolate, or investigate pathogenic microbial species; in culturing of animal, human and plant cells or tissues and plans to avoid contamination with unwanted organisms;

as a microbiota-altering agent in the treatment of waste water or solid waste, composting, or the production of fuel from organic matter where optimized microbiota are relevant to yield, efficacy and process quality; as a microbiota-optimizing agent in farming of animals and plants; and as a supplement to pre- and/or probiotics to improve microbiota.

14. Use of a compound according to any one of claims 1 to 8 for cosmetic applications, including treatment of cosmetic conditions caused by dysbiosis of the skin, scalp or oral cavity, such as seborrheic dermatitis, acne vulgaris, acne rosacea, caries, periodontal disease, and halitosis.

15. A compound according to any one of claims 1 to 8 for use in therapy, particularly for use in the treatment or prevention of

gastrointestinal diseases, such as inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), enterocolitis, and colorectal cancer; bacterial gastrointestinal infections, such as salmonellosis, listeriois, shigellosis, infection by enterohemorrhagic or enteroinvasive E. coli, or Clostridium difficile infections (CDI);

autoimmune diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, multiple sclerosis, and Celiac disease;

neurological disorders, such as Alzheimer's disease, autistic spectrum disorders, and Parkinson's disease;

other disorders like obesity, chronic fatigue syndrome, and atherosclerosis.