FIELD OF THE INVENTION The present invention relates to hamamelitannin analogues, pharmaceutical compositions comprising the same, and combinations thereof with antimicrobial agents such as antibiotics or disinfectants. It in particular relates to the use of the compounds, compositions and combinations according to this invention in human or veterinary medicine, more in particular for use in the prevention and/or treatment of bacterial infections, such as Staphylococcus aureus infections, in humans or animals.

BACKGROUND TO THE INVENTION

For a long time, microorganisms such as bacteria, yeasts or fungi were considered non-complex organisms that could easily be eradicated with antimicrobials. However, today we are confronted with an alarming growth of disease-causing microbes that can no longer efficiently be controlled by available antibiotics or antifungals. This phenomenon is generally referred to as "antimicrobial resistance" or AMR. Clinicians in many different disciplines are experiencing difficulties to treat patients due to ineffective antimicrobials.

To combat the spread of AMR, there is an increasing pressure towards a more rational use of antibiotics. As a result, almost every Western country has launched campaigns to make people aware that misuse of antimicrobials makes them ineffective. In fact, the problem of AMR has grown to such a dramatic level that the World Health Organization moved it to the top-3 of global health risks. In a recently published report, commissioned by the British government, economist Jim O'Neill and co-authors estimate that by 2050 10 million lives a year will be at risk due to the rise of drug-resistant infections (1 ). Antibiotic resistance is not only a future threat, but posing a serious global problem to patients and care providers right now. Methicillin-resistant Staphylococcus aureus (MRSA), often referred to as a superbug, is responsible for nosocomial as well as a communal infections and represents a rapidly growing health threat. Compounding the problem even further is the fact that Staphylococcus aureus (S. aureus) is notorious for its ability to form biofilms, surface-attached encasements of cells in a polymer-based matrix. Biofilm formation provides a multi-level protection to microbes against antibiotics. It alters growth rate and metabolism, raises the fraction of dormant persister cells and hampers the penetration of antimicrobial agents. The global problem of AMR is further aggravated by a lack of novel antibiotic development, which is fraught with scientific risk and business challenges. The fact that resistance development is unavoidable with agents that target bacterial viability, prompted the scientific community to explore alternative approaches, such as neutralizing virulence mechanisms. Therapeutic strategies to reduce the expression of bacterial virulence factors would render pathogens more susceptible to natural host defenses and would maintain normal microbiota. Microorganisms such as bacteria use quorum sensing to coordinate certain behaviors such as biofilm formation, virulence, and antibiotic resistance, based on the local density of the bacterial population. Quorum sensing (QS) can occur within a single bacterial species as well as between diverse species, and can regulate different processes, in essence, serving as a simple indicator of population density or the diffusion rate of the cell's immediate environment.

There is a continuous need for new antimicrobial compositions, or agents that increase sensitivity of micro-organisms towards existing antimicrobials in order to combat the spread of AMR. A particular way of sensitizing microorganisms for certain antimicrobials is by interfering with their quorum sensing system. Several non-peptide small molecules, peptides and proteins have been shown to affect quorum sensing in bacteria and are suitable in the prevention and/or treatment of bacterial infections. For example hamamelitannin (HAM) or 2',5-di-O-galloyl-D-hamamelose, a natural compound found in the bark and leaves of Hamamelis virginana (witch hazel), was found to act as a quorum sensing inhibitor (QSI) (WO2007147098). HAM interferes with a quorum sensing system in bacteria. It was found that when combining HAM with an antibiotic, a potentiating or synergistic effect is observed, in particular HAM increases the susceptibility of bacterial biofilms to antibiotics in vitro as well as in vivo (2, 3). The natural HAM product is metabolically unstable and only moderately active, and therefore less preferable in the treatment of microbial infections. Nevertheless, interesting results obtained with this molecule provide a conspicuous lead for further optimization, and present an opportunity for the development of a novel class of pharmaceutical compounds, capable of combating microbial infections. HAM analoguous have been explored in JP06234784 and WO16005340. It was an object of the present invention to provide further HAM analogues with an improved metabolic and chemical stability and increased activity. Interesting compounds with improved synthetic accessibility were identified as potentiators for antibiotics in the treatment of microbial infections.

SUMMARY OF THE INVENTION

The invention includes a method of preventing or reducing the growth or proliferation of microorganisms or biofilm-embedded microorganisms, e.g. on a surface of a medical device or in or on subject's body.

In a first aspect, the present invention provides a compound of Formula I, or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof

Wherein

R-i is selected from -Ci_ ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri6; wherein said -d- ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₅ , -Het ₅ , -CN, and -CF ₃ ;

or R ₃ taken together with R ₅ forms -C ₃ . ₆ cycloalkyl or Het- ;

R ₄ is selected from -H, -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₆ , -Het ₆ , -CN, and -CF ₃ ;

R ₅ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₇ , -Het ₇ , -CN, and -CF ₃ ;

R ₆ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₈ , -Het ₈ , -CN, and -CF ₃ ;

or R ₆ taken together with R ₉ forms -C ₃ . ₆ cycloalkyl or Het ₁₂ ;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₆ ;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

R ₉ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₉ , -Het ₉ , -CN, and -CF ₃ ;

R-io is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₁₀ , -Het ₁₀ , -CN, and -CF ₃ ;

R-| ₄ , Ri5, Ri6, R ₂ 5, and R ₂₆ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i ,

Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci_

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R ₁₂ and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ .

₆ cycloalkyl, -Het ₂ and -Ar ₂ ;

Ri ₇ , Ris, Rig, R ₂₀ , R ₂ i , R ₂₂ , R ₂₃ and R ₂₄ are each independently selected from -H, and -Ci_ ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , - halo, -Ci- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR21 R22, and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH, -halo, -d- ₆ alkyl, -O-C1- 6alkyl, -CN, -CF ₃ , or -NR ₂ i R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het ₁₀ , Het-n and Het ₁₂ are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N , O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het-io, Het-n and Het ₁₂ is optionally and independently substituted with from 1 to 5 substituents selected from =0, -OH, -halo, -C _1-6 alkyl, -0-Ci _-6 alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH, -halo, -C _1-6 alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ . In a particular embodiment, the present invention provides a compound according to formula I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R-i is selected from -Ci_ ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri ₆ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is -H;

R ₄ is -H;

R ₅ is -H;

R ₆ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₈ , -Het ₈ , -CN and -CF ₃ ;

or R ₆ taken together with R ₉ forms -C ₃ . ₆ cycloalkyl or Het ₁₂ ;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₆ ;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

R ₉ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₉ , -Het ₉ , -CN, and -CF ₃ ;

R-io is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ari ₀ , -Heti ₀ , -CN, and -CF ₃ ;

Ri ₄ , R-I5, R16, R ₂ 5, and R ₂₆ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i ,

Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1-

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ; R-ι-ι , R-12 and R ₁₃ are each independently selected from -d- ₆ alkyl , -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -N H- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH , -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN , -NR ₁₉ R ₂₀ , -C ₃ . 6cycloalkyl, -Het ₂ and -Ar ₂ ;

R-I7, Ri8, Ri9, R ₂ o, R21 , R22, R ₂₃ and R ₂₄ are each independently selected from -H , and -Ci_ ₆ alkyl ; Ar-ι , Ar ₂ , Ar ₃ , Ar ₄ , Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar ₉ , and Ar ₁₀ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , Ar ₄ , Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar ₉ , and Ar ₁₀ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , - halo, -Ci- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN , -CF ₃ , -NR ₂₁ R ₂₂ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH , -halo, -Ci_ ₆ alkyl, -O-C1- 6alkyl, -CN , -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het ₁₀ , Het-n and Het ₁₂ are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N , O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het-io, Het-n and Het ₁₂ is optionally and independently substituted with from 1 to 5 substituents selected from =0, -OH , -halo, -C _1-6 alkyl, -0-Ci _-6 alkyl , -CN , -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH , - halo, -C _{1 -6} alkyl, -0-Ci_ ₆ alkyl, -CN , -CF ₃ , or -NR ₂₃ R ₂₄ . In yet a further embodiment the present invention provides a compound according to formula I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R ₂ is -H ;

R ₃ is -H ;

R ₄ is -H ;

R ₅ is -H ;

R ₆ is -H ;

R ₈ is -H ;

R ₉ is -H ;

Ar-ι , and Ar ₂ , are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN , -CF ₃ , -NR ₂₁ R ₂₂ , and -phenyl;

R ₂₁ , and R ₂₂ are each independently selected from -H , and -Ci_ ₆ alkyl. In another specific embodiment, the present invention provides a compound according to formula

I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof,

Wherein

R ₂ is -H;

R ₃ is -H;

R ₄ is -H;

R ₅ is -H;

R ₆ is -H;

R ₈ is -H;

R ₉ is -H;

Ar-ι , and Ar ₂ , are each phenyl; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 -halo.

In yet a further embodiment, the present invention provides a compound according to formula I wherein the compound has the stereoisomeric conformation as represented in formula la:

In another aspect, the present invention provides a compound of Formula II, or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof

Wherein

R-i is selected from -d- ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri ₆ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ; R ₂ is selected from -H and -d- ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is selected from -H, -OH , and -F;

R ₄ is selected from -H, -OH , and -F;

R ₅ is selected from -H, -OH, and -F;

R ₆ is selected from -H, -OH , -F, -C _1-6 alkyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, and -CN;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -SO2-R13; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂ 6;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

Ri4, Ri5, R16, R25, and R ₂ 6 are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i , Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1- 6alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R11 , R12 and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ . 6cycloalkyl, -Het ₂ and -Ar ₂ ;

R16, Ri7, R18, Rig, R20, R21 , R22 and R ₂₃ are each independently selected from -H, and -Ci_ ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and - phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , and He are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N, O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , and Het ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH , - halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ .

In another embodiment, the present invention provides a compound according to formula II or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R-i is selected from -Ci_ ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri ₆ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is -OH; R ₄ is -OH;

R ₅ is -H;

R ₆ is -H;

R ₇ is selected from -d- ₆ alkyl, -Ar ₄ , and -SO2-R13; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂ 6;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

Ri4, Ri5, R16, R25, and R ₂ 6 are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i , Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1-

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R-12 and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ . 6cycloalkyl, -Het ₂ and -Ar ₂ ;

R16, Ri7, R18, Rig, R ₂₀ , R21 , R ₂₂ and R ₂₃ are each independently selected from -H, and -Ci_ ₆ alkyl;

Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and - phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , and He are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N, O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , and Het ₄ is optionally and independently substituted with from 1 to 5 substituents selected from -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH, -halo, - Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ .

In yet a further embodiment, the present invention provides a compound according to formula II or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R ₂ is -H;

R ₃ is -OH;

R ₄ is -OH;

R ₅ is -H;

R ₆ is -H;

R ₇ is -(C=0)-Rn ;

R ₈ is -H;

Ar-i , and Ar ₂ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -C _{1 -6} alkyl, -0-Ci _-6 alkyl, -CN, -CF ₃ , -NR ₂ iR ₂₂ , and -phenyl.

In another particular embodiment, the present invention provides a compound according to formula II or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof,

Wherein

R ₂ is -H;

R ₃ is -OH;

R ₄ is -OH;

Rs is -H;

R ₆ is -H;

Ar-ι , and Ar ₂ , are each phenyl; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 -halo.

In yet a further embodiment, the present invention provides a compound according to formula II wherein the compound has the stereoisomeric conformation as represented in formula Ila:

In a specific embodiment, the present invention encompasses a compound as defined herein, such as any and all compounds represented by formulas I, la, II, Ila or as represented in Table 1 , or stereoisomer, tautomer, racemic, salt, metabolite, pre- or prodrug, hydrate, or solvate thereof, and more specific a stereoisomer, tautomer, racemic, salt, hydrate, or solvate thereof. Such compound is for example a compound according to formula I and la, and represented as

Alternatively such compound is for example a compound according to formula II and lla, and represented as follows:

The present invention also provides a pharmaceutical composition comprising a compound according to the present invention and a pharmaceutically acceptable excipient, diluent and/or carrier. Said composition may be administered oral, systemic or topical.

In a further aspect, the present invention provides a combination of a compound according to the present invention with an antimicrobial agent, in particular an antibiotic or disinfectant.

In another specific, the present invention provides a medical device comprising one or more compounds, a combination or a pharmaceutical composition according to the present invention. Such medical device is for example selected from the group consisting of grafts, membranes, tubes, connectors, surgical instruments, intra-aortic balloons, stents (including drug-eluting vascular stents), blood bags, catheters, sutures, prostheses, heart valves, tissue adhesives, cardiac pacemaker leads, artificial organs, lenses for the eye, blood handling equipment, apheresis equipment, (bio)sensors, dental devices, skin patches, wound dressings (including drug-eluting wound dressings), implantable devices, tampons, bandages, drug delivery systems, bodily implants, and protein-eluting scaffolds for tissue regeneration. In a specific embodiment, the one or more compounds, the combination or pharmaceutical composition according to the present invention is formulated in or applied on the medical device.

In a specific aspect, the present invention provides a compound, composition, medical device or combination according to the present invention, for use in human or veterinary medicine, more in particular, for use in the preventing, reducing the risk of and/or treating bacterial infections in humans or animals. In a particular embodiment, said bacterial infection is a Staphylococcus aureus infection. In a specific aspect, the present invention provides a compound, composition, medical device or combination according to the present invention, for use in human or veterinary medicine, more in particular, for use in the preventing, reducing the risk of and/or treating bacterial infections in humans or animals. In a particular embodiment, said bacterial infection is a Staphylococcus aureus infection.

Alternatively, the present invention provides a compound, composition, medical device or combination according to the present invention, for usein preventing, reducing the risk of and/or treating wound infections, skin ulcers, diabetic foot ulcers, burn wound infections, catheter- or stent-associated infections, infections resulting from animal bites, mastitis, pneumonia or sepsis.

In another specific aspect, the present invention provides a compound, composition, medical device or combination according to the present invention, for use in preventing or reducing biofilm formation. In a further aspect, the present invention provides a method for the prevention and/or treatment of bacterial infections; said method comprising administering to a subject in need thereof a therapeutic effective amount of a compound, a composition or a combination as according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. U nless a context dictates otherwise, asterisks are used herein to indicate the point at which a mono- or bivalent radical depicted is connected to the structure to which it relates and of which the radical forms part. Recently, HAM and analogous compounds were shown to be able to potentiate the effect of several classes of antibiotics in vitro and to increase the effect of cefalexin in a murine mastitis model of S. aureus infection (3,4). Previous studies have focused on the structural optimization of the substituents at both the 5- and the 2'-position of the molecule (4,5,6). Several hamamelitannin analogues have been identified, some of which proved to be active potentiators of vancomycin (VAN).

After having investigated the structure-activity relationship (SAR) of both 'wings' of HAM , the present inventors have explored alternatives for the central hamamelose-like scaffold in search for derivatives with improved synthetic accessibility and/or potentiator activity.

In a first aspect the present invention provides compounds of Formula I , or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof

Wherein

Ri is selected from -C^alkyl, -Ar ₃ , -(C=0)-R ₁₄ , -(C=S)-R ₁₅ , and -S0 ₂ -Ri ₆ ; wherein said -C^alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -d- ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is selected from -H , -Ci_ ₆ alkyl , -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl , -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₅ , -Het ₅ , -CN , and -CF ₃ ;

or R ₃ taken together with R ₅ forms -C ₃ . ₆ cycloalkyl or Het- ;

R ₄ is selected from -H , -Ci_ ₆ alkyl , -C ₂ . ₆ alkenyl , -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl , -0-C ₃ . ₆ alkenyl , -S-C ₃ .

₆ alkenyl, -Ar ₆ , -Het ₆ , -CN , and -CF ₃ ;

R ₅ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₇ , -Het ₇ , -CN , and -CF ₃ ;

R ₆ is selected from -H , -Ci_ ₆ alkyl , -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl , -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₈ , -Het ₈ , -CN ; and -CF ₃

or R ₆ taken together with R ₉ forms -C ₃ . ₆ cycloalkyl or Het ₁₂ ;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂ 6;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -d- ₆ alkyl;

R ₉ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₉ , -Het ₉ , -CN, and -CF ₃ ;

R-io is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₁₀ , -Het ₁₀ , -CN, and -CF ₃ ;

Ri4, R-I5, Ri6, R25, and R ₂ 6 are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i ,

Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1-

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R-12 and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH-

Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ . 6cycloalkyl, -Het ₂ and -Ar ₂ ;

R-I7, R18, Rig, R ₂ o, R21 , R22, R ₂₃ and R ₂₄ are each independently selected from -H, and -Ci_ ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , - halo, -Ci- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -O-C1-

₆ alkyl, -CN, -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het ₁₀ , Het-n and Het ₁₂ are each independently a

5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N , O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ ,

Het-io, Het-n and Het ₁₂ is optionally and independently substituted with from 1 to 5 substituents selected from =0, -OH, -halo, -Ci. ₆ alkyl, -0-Ci. ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH,

-halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ .

In a particular embodiment, the invention relates to a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate of the compounds provided herein, and especially any and each of the compounds represented by Formula I, la, II or Ma or compounds as provided in Table 1 . More specific, the invention also encompasses a stereoisomer, tautomer, racemic, salt, hydrate, or solvate of said compounds.

When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise. The term "alkyl" by itself or as part of another substituent refers to a fully saturated hydrocarbon of Formula C _x H _2x+ i wherein x is a number greater than or equal to 1 . Generally, alkyl groups of this invention comprise from 1 to 20 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, d- ₄ alkyl means an alkyl of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, butyl, and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers. C C6 alkyl includes all linear, branched, or cyclic alkyl groups with between 1 and 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers, cyclopentyl, 2-, 3-, or 4-methylcyclopentyl, cyclopentylmethylene, and cyclohexyl.

The term "optionally substituted alkyl" refers to an alkyl group optionally substituted with one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3, or 4 substituents or 1 to 2 substituents) at any available point of attachment. Non-limiting examples of such substituents include halo, hydroxyl, carbonyl, nitro, amino, oxime, imino, azido, hydrazino, cyano, aryl, heteroaryl, cycloalkyl, acyl, alkylamino, alkoxy, thiol, alkylthio, carboxylic acid, acylamino, alkyl esters, carbamate, thioamido, urea, sullfonamido and the like.

The term "cycloalkyl" by itself or as part of another substituent is a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 , 2, or 3 cyclic structures. Cycloalkyl includes all saturated or partially saturated (containing 1 or 2 double bonds) hydrocarbon groups containing 1 to 3 rings, including monocyclic, bicyclic, or polycyclic alkyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 15 atoms. The further rings of multi-ring cycloalkyls may be either fused, bridged and/or joined through one or more spiro atoms. Cycloalkyl groups may also be considered to be a subset of homocyclic rings discussed hereinafter. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantanyl and cyclodecyl with cyclopropyl being particularly preferred. An "optionally substituted cycloalkyl" refers to a cycloalkyl having optionally one or more substituents (for example 1 to 3 substituents, for example 1 , 2, 3 or 4 substituents), selected from those defined above for substituted alkyl. When the suffix "ene" is used in conjunction with a cyclic group, hereinafter also referred to as "Cycloalkylene", this is intended to mean the cyclic group as defined herein having two single bonds as points of attachment to other groups. Cycloalkylene groups of this invention preferably comprise the same number of carbon atoms as their cycloalkyl radical counterparts. Where alkyl groups as defined are divalent, i.e., with two single bonds for attachment to two other groups, they are termed "alkylene" groups. Non-limiting examples of alkylene groups includes methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethylethylene, 1 ,2-dimethylethylene, pentamethylene and hexamethylene. Similarly, where alkenyl groups as defined above and alkynyl groups as defined above, respectively, are divalent radicals having single bonds for attachment to two other groups, they are termed "alkenylene" and "alkynylene" respectively.

Generally, alkylene groups of this invention preferably comprise the same number of carbon atoms as their alkyl counterparts. Where an alkylene or cycloalkylene biradical is present, connectivity to the molecular structure of which it forms part may be through a common carbon atom or different carbon atom, preferably a common carbon atom. To illustrate this applying the asterisk nomenclature of this invention, a C ₃ alkylene group may be for example *-CH ₂ CH ₂ CH ₂ -*, * *, or *-CH ₂ CH(-CH ₃ )-*. Likewise a C ₃ cycloalkylene group may be

The terms "heterocycle", "heterocyclyl" or "heterocyclo" as used herein by itself or as part of another group refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 13 member monocyclic, 7 to 17 member bicyclic, or 10 to 20 member tricyclic ring systems, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1 , 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multi- ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms. An optionally substituted heterocyclic refers to a heterocyclic having optionally one or more substituents (for example 1 to 4 substituents, or for example 1 , 2, 3 or 4), selected from those defined above for substituted aryl.

Exemplary heterocyclic groups include piperidinyl, azetidinyl, imidazolinyl, imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidyl, succinimidyl, 3H- indolyl, isoindolinyl, chromenyl, isochromanyl, xanthenyl, 2H-pyrrolyl, 1 -pyrrolinyl, 2-pyrrolinyl, 3- pyrrolinyl, pyrrolidinyl, 4H-quinolizinyl, 4aH-carbazolyl, 2-oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, pyranyl, dihydro-2H-pyranyl, 4H-pyranyl, 3,4- dihydro-2H-pyranyl, phthalazinyl, oxetanyl, thietanyl, 3-dioxolanyl, 1 ,3-dioxanyl, 2,5- dioximidazolidinyl, 2,2,4-piperidonyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, indolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrehydrothienyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1 ,3- dioxolanyl, 1 ,4-oxathianyl, 1 ,4-dithianyl, 1 ,3,5-trioxanyl, 6H-1 ,2,5-thiadiazinyl, 2H-1 ,5,2-dithiazinyl, 2H-oxocinyl, 1 H-pyrrolizinyl, tetrahydro-1 ,1 -dioxothienyl, N- formylpiperazinyl, and morpholinyl. The term "aryl" as used herein refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthalene or anthracene) or linked covalently, typically containing 6 to 10 atoms; wherein at least one ring is aromatic. The aromatic ring may optionally include one to three additional rings (either cycloalkyl, heterocyclyl, or heteroaryl) fused thereto. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein. Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, 1 -, 2-, 3-, 4-, 5-, 6-, 7-, or 8-azulenyl, 1 - or 2-naphthyl, 1 -, 2-, or 3-indenyl, 1 -, 2-, or 9-anthryl, 1 - 2-, 3-, 4-, or 5-acenaphtylenyl, 3-, 4-, or 5-acenaphtenyl, 1 -, 2-, 3-, 4-, or 10-phenanthryl, 1 - or 2-pentalenyl, 1 , 2-, 3-, or 4-fluorenyl, 4- or 5-indanyl, 5-, 6-, 7-, or 8-tetrahydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl, dibenzo[a,d]cylcoheptenyl, and 1 -, 2-, 3-, 4-, or 5-pyrenyl.

The aryl ring can optionally be substituted by one or more substituents. An "optionally substituted aryl" refers to an aryl having optionally one or more substituents (for example 1 to 5 substituents, for example 1 , 2, 3 or 4) at any available point of attachment. Non-limiting examples of such substituents are selected from halogen, hydroxyl, oxo, nitro, amino, hydrazine, aminocarbonyl, azido, cyano, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkylalkyl, alkylamino, alkoxy, -S0 ₂ -NH ₂ , aryl, heteroaryl, aralkyl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkylaminocarbonyl, heteroarylalkyl, alkylsulfonamide, heterocyclyl, alkylcarbonylaminoalkyl, aryloxy, alkylcarbonyl, acyl, arylcarbonyl, aminocarbonyl, alkylsulfoxide, -S0 ₂ R ^a , alkylthio, carboxyl, and the like, wherein R ^a is alkyl or cycloalkyl.

Where a carbon atom in an aryl group is replaced with a heteroatom, the resultant ring is referred to herein as a heteroaryl ring. The term "heteroaryl" as used herein by itself or as part of another group refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 3 rings which are fused together or linked covalently, typically containing 5 to 8 atoms; at least one of which is aromatic in which one or more carbon atoms in one or more of these rings can be replaced by oxygen, nitrogen or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of such heteroaryl, include: pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,1 - b][1 ,3]thiazolyl, thieno[3,2-b]furanyl, thieno[3,2-b]thiophenyl, thieno[2,3-d][1 ,3]thiazolyl, thieno[2,3- d]imidazolyl, tetrazolo[1 ,5-a]pyridinyl, indolyl, indolizinyl, isoindolyl, benzofuranyl, benzopyranyl, 1 (4H)-benzopyranyl, 1 (2H)-benzopyranyl, 3,4-dihydro-1 (2H)-benzopyranyl, 3,4-dihydro-1 (2H)- benzopyranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, 1 ,3-benzoxazolyl, 1 ,2-benzisoxazolyl, 2,1 -benzisoxazolyl, 1 ,3-benzothiazolyl, 1 ,2- benzoisothiazolyl, 2,1 -benzoisothiazolyl, benzotriazolyl, 1 ,2,3-benzoxadiazolyl, 2,1 ,3- benzoxadiazolyl, 1 ,2,3-benzothiadiazolyl, 2,1 ,3-benzothiadiazolyl, thienopyridinyl, purinyl, imidazo[1 ,2-a]pyridinyl, 6-oxo-pyridazin-1 (6H)-yl, 2-oxopyridin-1 (2H)-yl, 6-oxo-pyridazin-1 (6H)-yl, 2-oxopyridin-1 (2H)-yl, 1 ,3-benzodioxolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl. Within the context of the instant application the heteroaryls are in particular selected from furanyl, pyrazolyl, imidazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, and isoindolyl.

An "optionally substituted heteroaryl" refers to a heteroaryl having optionally one or more substituents (for example 1 to 4 substituents, for example 1 , 2, 3 or 4), selected from those defined above for substituted aryl, such as for example 1 ,3-dioxoindolyl.

The term "oxo" as used herein refers to the group =0.

The term "alkoxy" or "alkyloxy" as used herein refers to a radical having the Formula -OR ^b wherein R ^b is alkyl. Preferably, alkoxy is C Ci ₀ alkoxy, C C ₆ alkoxy, or C C ₄ alkoxy. Non-limiting examples of suitable alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy. Where the oxygen atom in an alkoxy group is substituted with sulfur, the resultant radical is referred to as thioalkoxy. "Haloalkoxy" is an alkoxy group wherein one or more hydrogen atoms in the alkyl group are substituted with halogen. Non- limiting examples of suitable haloalkoxy include fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 1 ,1 ,2,2-tetrafluoroethoxy, 2-fluoroethoxy, 2-chloroethoxy, 2,2-difluoroethoxy, 2,2,2-trichloroethoxy; trichloromethoxy, 2-bromoethoxy, pentafluoroethyl, 3,3,3-trichloropropoxy, 4,4,4-trichlorobutoxy.

The term "aryloxy" as used herein denotes a group -O-aryl, wherein aryl is as defined above. The term "arylcarbonyl" or "aroyl" as used herein denotes a group -C(0)-aryl, wherein aryl is as defined above.

The term "carboxy" or "carboxyl" or "hydroxycarbonyl" by itself or as part of another substituent refers to the group -C0 ₂ H. Thus, a carboxyalkyl is an alkyl group as defined above having at least one substituent that is -C0 ₂ H.

The term "alkoxycarbonyl" by itself or as part of another substituent refers to a carboxy group linked to an alkyl radical i.e. to form -C(=0)OR ^e , wherein R ^e is as defined above for alkyl.

The term "alkylcarbonyloxy" by itself or as part of another substituent refers to a -0-C(=0)R ^e wherein R ^e is as defined above for alkyl.

The term "alkylcarbonylamino" by itself or as part of another substituent refers to an group of Formula -NH(C=0)R or -NR'(C=0)R, wherein R and R' are each independently alkyl or substituted alkyl. The term "thiocarbonyl" by itself or as part of another substituent refers to the group -C(=S)-. The term "alkoxy" by itself or as part of another substituent refers to a group consisting of an oxygen atom attached to one optionally substituted straight or branched alkyl group, cycloalkyl group, aralkyl, or cycloalkylalkyl group. Non-limiting examples of suitable alkoxy group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, hexanoxy, and the like.

The term "halo" or "halogen" as a group or part of a group is generic for fluoro, chloro, bromo, or iodo.

The term "haloalkyl" alone or in combination, refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1 -bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1 ,1 ,1 -trifluoroethyl, and the like.

The term "haloaryl" alone or in combination, refers to an aryl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. The term "haloalkoxy" alone or in combination refers to a halo-O-alkyl group wherein the alkyl group is substituted by 1 , 2, or 3 halogen atoms. For example, "haloalkoxy" includes -OCF ₃ , - OCHF ₂ , -OCH2F, -O-CF2-CF3, -O-CH2-CF3, -O-CH2-CHF2, and -O-CH2-CH2F.

Whenever the term "substituted" is used in the present invention, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated group, provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.

Where groups may be optionally substituted, such groups may be substituted once or more, and preferably once, twice or thrice. Substituents may be selected from, for example, the group comprising halogen, hydroxyl, oxo, nitro, amido, carboxy, amino, cyano haloalkoxy, and haloalkyl.

As used herein the terms such as "alkyl, aryl, or cycloalkyl, each being optionally substituted with" or "alkyl, aryl, or cycloalkyl, optionally substituted with" refers to optionally substituted alkyl, optionally substituted aryl and optionally substituted cycloalkyl.

As described herein, some of the compounds of the invention may contain one or more asymmetric carbon atoms that serve as a chiral center, which may lead to different optical forms (e.g. enantiomers or diastereoisomers). The invention comprises all such optical forms in all possible configurations, as well as mixtures thereof.

More generally, from the above, it will be clear to the skilled person that the compounds of the invention may exist in the form of different isomers and/or tautomers, including but not limited to geometrical isomers, conformational isomers, E/Z-isomers, stereochemical isomers (i.e. enantiomers and diastereoisomers) and isomers that correspond to the presence of the same substituents on different positions of the rings present in the compounds of the invention. All such possible isomers, tautomers and mixtures thereof are included within the scope of the invention.

Whenever used in the present invention the term "compounds of the invention" or a similar term is meant to include the compounds of general Formula I, la, II or II, and any subgroup thereof. This term also refers to the compounds as depicted in Table 1 , their derivatives, /v-oxides, salts, solvates, hydrates, stereoisomeric forms, racemic mixtures, tautomeric forms, optical isomers, analogues, pro-drugs, esters, and metabolites, as well as their quaternized nitrogen analogues. The v-oxide forms of said compounds are meant to comprise compounds wherein one or several nitrogen atoms are oxidized to the so-called /v-oxide.

As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound.

The terms described above and others used in the specification are well understood to those in the art.

In a particular embodiment, the present invention provides a compound according to formula I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R-i is selected from -d- ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri6; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is -H;

R ₄ is -H;

R ₅ is -H;

R ₆ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₈ , -Het ₈ , -CN, and -CF ₃ ;

or R ₆ taken together with R ₉ forms -C ₃ . ₆ cycloalkyl or Het ₁₂ ;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₆ ;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

R ₉ is selected from -H , -Ci_ ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

6alkenyl, -Ar ₉ , -Het ₉ , -CN, and -CF ₃ ; R-ιο is selected from -H , -d- ₆ alkyl, -C ₂ . ₆ alkenyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, -0-C ₃ . ₆ alkenyl, -S-C ₃ .

₆ alkenyl, -Ar ₁₀ , -Het ₁₀ , -CN, and -CF ₃ ;

Ri4, Ri5, Ri6, R25, and R ₂ 6 are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i ,

Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1-

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R-12 and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH-

Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ . 6cycloalkyl, -Het ₂ and -Ar ₂ ;

Ri7, R18, Ri9, R ₂ o, R21 , R22, R ₂₃ and R ₂₄ are each independently selected from -H, and -Ci_ ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , Ar _4, Ar ₅ , Ar ₆ , Ar ₇ , Ar ₈ , Ar _9, and Ar ₁₀ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , - halo, -Ci- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH, -halo, -Ci_ ₆ alkyl, -O-C1-

₆ alkyl, -CN, -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ , Het ₁₀ , Het-n and Het ₁₂ are each independently a

5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N , O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , Het ₄ , Het ₅ , Het ₆ , Het ₇ , Het ₈ , Het ₉ ,

Het-io, Het-n and Het ₁₂ is optionally and independently substituted with from 1 to 5 substituents selected from =0, -OH, -halo, -Ci. ₆ alkyl, -0-Ci. ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH, - halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ .

In yet a further embodiment the present invention provides a compound according to formula I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R ₂ is -H;

R ₃ is -H;

R ₄ is -H;

R ₅ is -H;

R ₆ is -H;

R ₇ is -(C=0)-Rn ;

R ₈ is -H;

R ₉ is -H;

R ₁₄ is -Ar-i R-ι -ι is -Ar ₂

Ar-ι , and Ar ₂ , are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -d- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN , -CF ₃ , -NR21 R22, and -phenyl;

R ₂ i , and R ₂ 2 are each independently selected from -H, and -Ci_ ₆ alkyl.

In another specific embodiment, the present invention provides a compound according to formula

I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof,

Wherein

R ₂ is -H;

R ₃ is -H;

R ₄ is -H;

Rs is -H;

R ₆ is -H;

R ₈ is -H;

R ₉ is -H;

R ₁₀ is -H;

Ar-ι , and Ar ₂ , are each phenyl; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 -halo.

In yet a further embodiment, the present invention provides a compound according to formula I wherein the compound has the stereoisomeric conformation as represented in formula la:

la

Such compound is for example a compound according to formula I and la, and represented follows:

ln another aspect, the present invention provides a compound of Formula II, or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof

Wherein

R-i is selected from -d- ₆ alkyl, -Ar -S0 ₂ -Ri6; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is selected from -H, -OH , and -F;

R ₄ is selected from -H, -OH , and -F;

R ₅ is selected from -H, -OH, and -F;

R ₆ is selected from -H, -OH , -F, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -S-Ci_ ₆ alkyl, and -CN;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₆ ;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

Ri ₄ , Ri5, R-I6, R ₂ 5, and R ₂₆ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i , Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci_ 6alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R ₁₂ and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ . ₆ cycloalkyl, -Het ₂ and -Ar ₂ ;

Ri6, Ri7, -I8, Ri9, R20, R21 , R22 and R ₂ 3 are each independently selected from -H, and -d- ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and - phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from, -OH, -halo, -C _1-6 alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₁ R ₂₂ ;

Het-ι , Het ₂ , Het ₃ , and He are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N, O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , and Het ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH, -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH , - halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ . In another embodiment, the present invention provides a compound according to formula II or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof, Wherein

R-i is selected from -Ci_ ₆ alkyl, -Ar ₃ , and -S0 ₂ -Ri ₆ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₅ ;

R ₂ is selected from -H and -Ci_ ₆ alkyl;

or R-i taken together with R ₂ forms Het ₃ ;

R ₃ is -OH;

R ₄ is -OH;

R ₅ is -H;

R ₆ is -H;

R ₇ is selected from -Ci_ ₆ alkyl, -Ar ₄ , and -S0 ₂ -Ri ₃ ; wherein said -Ci_ ₆ alkyl may be further substituted with -R ₂₆ ;

or R ₇ taken together with R ₈ forms Het ₄ ;

R ₈ is selected from -H and -Ci_ ₆ alkyl;

Ri ₄ , R-I5, R16, R ₂ 5, and R ₂₆ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar-i ,

Het-ι , -NH-Ci_ ₆ alkyl, -NH-Het-ι and -NH-Ar-ι ; wherein each of said -Ci_ ₆ alkyl is optionally and independently substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -O-C1-

₆ alkyl, -S-Ci_ ₆ alkyl, -CN, -NR ₁₇ R ₁₈ , -C ₃ . ₆ cycloalkyl, -Het-ι and -Ar-i ;

R-i -i , R ₁₂ and R ₁₃ are each independently selected from -Ci_ ₆ alkyl, -C ₃ . ₆ cycloalkyl, -Ar ₂ , Het ₂ , -NH- Het ₂ and -NH-Ar ₂ ; wherein each of said -Ci_ ₆ alkyl is optionally substituted with from 1 to 3 substituents selected from =0, -OH, -halo, -0-Ci. ₆ alkyl, -S-Ci. ₆ alkyl, -CN, -NR ₁₉ R ₂₀ , -C ₃ .

₆ cycloalkyl, -Het ₂ and -Ar ₂ ;

R16, R-I7, R18, Ri9, R20, R21 , R22 and R ₂₃ are each independently selected from -H, and -Ci_ ₆ alkyl; Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , Ar ₂ , Ar ₃ , and Ar ₄ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , -halo, -d- ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR21 R22, and - phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH , -halo, -Ci_ ₆ alkyl , -0-Ci_ ₆ alkyl , -CN, -CF ₃ , or -NR ₂ iR ₂₂ ;

Het-ι , Het ₂ , Het ₃ , and He are each independently a 5-10 membered mono-or bicyclic heterocycle comprising from 1 to 3 heteroatoms selected from N, O and S; wherein each of said Het-ι , Het ₂ , Het ₃ , and Het ₄ is optionally and independently substituted with from 1 to 5 substituents selected from -OH , -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₃ R ₂₄ , and -phenyl; wherein said phenyl is optionally substituted with from 1 to 3 substituents selected from -OH , -halo, - Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , or -NR ₂₃ R ₂₄ .

In yet a further embodiment, the present invention provides a compound according to formula I I or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof,

Wherein

R ₂ is -H ;

R ₃ is -OH ;

R ₄ is -OH ;

R ₅ is -H ;

R ₆ is -H ;

R ₇ is -(C=0)-Rn ;

R ₈ is -H ;

Ar-ι , and Ar ₂ are each independently a 5-10 membered aromatic mono-or bicycle; wherein each of said Ar-ι , and Ar ₂ is optionally and independently substituted with from 1 to 5 substituents selected from, -OH , -halo, -Ci_ ₆ alkyl, -0-Ci_ ₆ alkyl, -CN, -CF ₃ , -NR ₂₁ R ₂₂ , and -phenyl.

In another particular embodiment, the present invention provides a compound according to formula II or a stereoisomer, tautomer, racemic, metabolite, pro- or predrug, salt, hydrate, or solvate thereof,

Wherein

R ₂ is -H ;

R ₃ is -OH ;

R ₄ is -OH ;

R ₅ is -H ;

R ₆ is -H ;

R ₇ is -(C=0)-Rn ; R ₈ is -H;

R is -Ar

R is -Ar ₂

Ar and Ar ₂ , are each phenyl; wherein each of said Ar and Ar ₂ is optionally and independently substituted with from 1 to 5 -halo.

In yet a further embodiment, the present invention provides a compound according to formula II wherein the compound has the stereoisomeric conformation as represented in formula Ila:

In a specific embodiment, the present invention encompasses a compound as defined herein, such as any and all compounds represented by formulas I, la, II, Ila or as represented in Table 1 , or stereoisomer, tautomer, racemic, salt, metabolite, pre- or prodrug, hydrate, or solvate thereof, and more specific a stereoisomer, tautomer, racemic, salt, hydrate, or solvate thereof.

Such compound is for example a compound according to formula II and Ila, and represented as follows:

The compounds of the present invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention and that the compounds of this invention can be prepared by any of several standard synthetic processes commonly used by those skilled in the art of organic chemistry. MEDICAL USE

It is an aim of the present invention to provide compounds that interfere with quorum sensing (QS) in bacteria and/or inhibit or prevent biofilm formation. The present invention thus further provides a pharmaceutical composition comprising a compound according to the present invention and a pharmaceutically acceptable excipient, diluent and/or carrier.

In a further aspect, the present invention provides a combination, as part of a composition or in use, of a compound according to the present invention with an antimicrobial agent, in particular an antibiotic or disinfectant. Hence, one or more compounds as described herein and one or more antimicrobial agents can be part of a single composition or can be used in a separate or sequential application. In a specific aspect, the present invention provides a compound, composition or combination according to the present invention, for use as a human or veterinary medicament, more in particular, for use in the prevention, reduction and/or treatment of bacterial infections in humans or animals. In a particular embodiment, said bacterial infection is a Staphylococcus aureus infection.

The compounds of the present invention can be used as an alternative to replace antibiotics for combating (bacterial) infections or can be used as an adjunct therapy in combination with e.g. antibiotics so that lower doses of the conventional antibiotics are required. In a particular embodiment, the present invention provides a method of preventing, reducing the risk of and/or treating a bacterial infection, more in particular a disorder associated with biofilm formation in a subject; said method comprising administering to a subject in need thereof a therapeutic effective amount of a compound, a composition or a combination according to the present invention. The present invention thus also provides a compound, combination or pharmaceutical composition as defined herein for use in preventing or reducing biofilm formation.

The term "biofilm" as used herein refers to biological films that develop and persist at interfaces in aqueous environments, on medical implants, or as foci of chronic infections. Biofilms may also form on biological surfaces such as teeth, or any other natural or artificial surfaces that may be exposed to or are in contact with non-sterile aqueous environments that may include nutrients suitable for the colonization and proliferation of the microorganisms. These biological films are composed of microorganisms embedded in organic gelatinous matrices composed of one or more matrix polymers that are secreted by the resident microorganisms. Biofilms can develop into macroscopic structures several millimeters or centimeters in thickness and can cover large surface areas. Biofilms are also capable of trapping nutrients and particulates that can contribute to their enhanced development and stability. Biofilms can also prevent penetration of antimicrobial agents, which may lead to persistent infections. Disorders associated with biofilm formation include but are not limited to, dental caries, periodontitis, otitis media, muscular skeletal infections, necrotizing fasciitis, biliary tract infection, osteomyelitis, bacterial prostatitis, endocarditis, native valve endocarditis, cystic fibrosis pneumonia, meloidosis, or skin lesions associated with bullous impetigo, atopic dermatitis and pemphigus foliaceus or implanted device-related infections. In some embodiments, the disorder is a nosocomial infection, including but not limited to, pneumonia, sepsis (including SIRS (Systemic Inflammatory Response Syndrome), severe sepsis and MODS (multiorgan dysfunction syndrome)), or an infection associated with sutures, exit sites, arteriovenous sites, scleral buckles, contact lenses, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis, lUDs, endotracheal tubes, Hickman catheters, central venous catheters, mechanical heart valves, vascular grafts, biliary stent blockage, and orthopedic devices. The compounds of the present invention are of particular interest to prevent, limit the risk of and/or treat post-operative wound infections, skin ulcers, diabetic foot ulcers, pressure ulcers such as decubitus ulcers or bedsores, burn wound infections, catheter associated infections, and infections resulting from animal bites. In a further embodiment, the compounds as described herein are effective in preventing and/or treating mastitis, i.e. inflammation of the breast tissue. S. aureus is the most common etiological organism responsible, but S. epidermidis and streptococci are occasionally isolated as well. Mastitis in dairy cattle is the persistent, inflammatory reaction of the udder tissue.

The present invention further provides a method for the prevention, reducing the risk of and/or treatment of microbial infections, in particular bacterial infections, more in particular infections with bacteria of the genus Staphylococcus such as Staphylococcus aureus infections. The method further comprises contacting a surface with the composition in an amount sufficient to prevent or reduce the growth or proliferation of microorganisms or biofilm-embedded microorganisms on said surface. In the invention, particular preference is given to compounds of Formula I, la, II or II, or any subgroup thereof, that in the inhibition assay as described in the examples, inhibit with an IC ₅₀ value of less than 100μΜ, preferably less than 50 μΜ, and more preferably less than 10 μΜ.

Said inhibition may be effected in vitro and/or in vivo, and when effected in vivo, is preferably effected in a selective manner, as defined above. In some embodiments, the bacterium is contacted with the compound as provided herein ex vivo. In such an embodiment, for example, the contacting comprises administering the compound to a surface in an amount effective to inhibit biofilm formation on said surface, including but not limited to a medical device. For pharmaceutical use, the compounds of the invention may be used as a free acid or base, and/or in the form of a pharmaceutically acceptable acid-addition and/or base-addition salt (e.g. obtained with non-toxic organic or inorganic acid or base), in the form of a hydrate, solvate and/or complex, and/or in the form or a pro-drug or pre-drug, such as an ester. As used herein and unless otherwise stated, the term "solvate" includes any combination which may be formed by a compound of this invention with a suitable inorganic solvent (e.g. hydrates) or organic solvent, such as but not limited to alcohols, ketones, esters and the like. Such salts, hydrates, solvates, etc. and the preparation thereof will be clear to the skilled person; reference is for instance made to the salts, hydrates, solvates, etc. described in US-A-6,372,778, US-A-6,369,086, US-A- 6,369,087 and US-A-6, 372,733.

The pharmaceutically acceptable salts of the compounds according to the invention, i.e. in the form of water-, oil-soluble, or dispersible products, include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalene-sulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. In addition, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl-bromides and others. Other pharmaceutically acceptable salts include the sulfate salt ethanolate and sulfate salts.

Generally, for pharmaceutical use, the compounds of the invention may be formulated as a pharmaceutical preparation or pharmaceutical composition comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including ocular), for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is again made to for instance US- A-6, 372,778, US-A-6,369,086, US-A-6,369,087 and US-A-6, 372,733, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, eye drops, sterile injectable solutions, and sterile packaged powders (which are usually reconstituted prior to use) which may be administered as a bolus and/or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof.

Furthermore, the compounds, combinations or pharmaceutical compositions may also be formulated in or may be applied on a medical device, such as skin patches, implantable devices, catheters, tampons, wound dressings, gauges and bandages. In a particular embodiment, one or more compounds as described hereinbefore is used to inhibit biofilm formation on a medical device by contacting the device with said compound (or a combination or pharmaceutical composition comprising the same) in an amount effective to inhibit biofilm formation. Percutaneous devices (such as catheters) and implanted medical devices (including, but not limited to, pacemakers, vascular grafts, stents, and heart valves) commonly serve as foci for bacterial infection. The tendency of some microorganisms to adhere to and colonize the surface of the device promotes such infections, which increase the morbidity and mortality associated with use of the devices.

For example, one or more compounds of the present invention can be used or applied on substrates used to manufacture medical devices associated with non-invasive and invasive medical procedures. Such substrates include, without limitation, tubular, sheet, rod and articles of proper shape for use in a number of medical devices such as vascular grafts, aortic grafts, arterial, venous, or vascular tubing, vascular stents, dialysis membranes, tubing or connectors, blood oxygenator tubing or membranes, surgical instruments, ultrafiltration membranes, intra- aortic balloons, stents, blood bags, catheters, sutures, soft or hard tissue prostheses, synthetic prostheses, prosthetic heart valves, tissue adhesives, cardiac pacemaker leads, artificial organs, endotracheal tubes, lenses for the eye such as contact or intraocular lenses, blood handling equipment, apheresis equipment, diagnostic and monitoring catheters and sensors, biosensors, dental devices, drug delivery systems, or bodily implants of any kind. In particular, one or more compounds of the present invention can be used or applied on drug-eluting medical implants. These are active implants that induce healing effects in addition to their regular task of support. This effect is achieved by controlled release of the active agent, including the compounds of the present invention, into the surrounding tissue. Examples are drug-eluting vascular stents, drug- eluting wound dressings and protein-eluting scaffolds for tissue regeneration. As such, the present invention encompasses a medical device as mentioned herein comprising one or more compounds of the present invention. The compounds can be incorporated in or applied on the surface of the medical device.

In a further embodiment, the present invention provides dressings or (cotton) gauges loaded with the compounds or compositions as described herein. Said dressing or guzes are typically used to prevent wound infection as well as treat infected wounds.

In a particular embodiment, the composition or formulation of the present invention can optionally contain other pharmaceutically active substances, especially antimicrobial agents such as antibiotics or disinfectants (which may or may not lead to a synergistic effect with the compounds of the invention) and/or other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow regulators, release agents, etc..

In a specific embodiment, the method and use as described herein further comprises the step of administering a standard of care antimicrobial agent to the subject. "Standard of care" as used herein refers to a treatment that is generally accepted by clinicians for a certain type of patient diagnosed with a type of illness. In the context of the present invention, exemplary standard of care antimicrobial agents include, but are not limited to, (i) β-lactam antibiotics like penicillins either alone (including but not limited to penicillin G, penicillin V, flucloxacillin, oxacillin, ampicillin, methicillin, amoxicillin, temocillin, and piperacillin), or combined with a β-lactamase inhibitors (including but not limited to amoxicillin + clavulanic acid, and piperacillin + tazobactam), carbapenems (including but not limited to biapenem, meropenem and imipenem), monobactams (including but not limited to aztreonam) and cephalosporins (including but not limited to cefadroxil, cefalexine, cefazoline, cefuroxime, cefotaxim, ceftazidim, ceftriaxone, cefepim and ceftarolin), (ii) macrolides (including but not limited to erythromycin, azithromycin, clarithromycin, roxithromycin, and spiramycin), (iii) tetracyclines and glycyclcyclines (including but not limited to doxycycline, lymecycline, minocycline, and tigecycline), (iv) lincosamides (including but not limited to clindamycin and lincomycin), (v) (fluoro-)quinolones (including but not limited to ciprofloxacin, levofloxacin, nemonoxacin, garenoxacin, moxifloxacin, norfloxacin, ofloxacin, parfloxacin, gemifloxacin, zabofloxacin and cinafloxacin), (vi) trimethoprim + sulfamethoxazole, (vii) aminoglycosides (including but not limited to streptomycin, amikacin, gentamicin, paromomycin, kanamycin, spectinomycin and tobramycin), (viii) glycopeptides (including but not limited to oritavancin, LY-333328, dalbavancin, teicoplanin and vancomycin), (ix) polymyxins (including but not limited to polymyxin B and colistin), (x) anti-tuberculosis drugs (including but not limited to isoniazide, rifampicin, pyrazinamide, ethambutol and bedaquilin and p-aminosalicylic acid), (xi) oxazolidinones (including but not limited to tedizolid, AZD-2563 and linezolid), (xii) lipopeptides (including but not limited to daptomycin and ramoplanin), (xiii) streptogramins (including but not limited to quinupristin/dalfopristin, pristinamycin, virginiamycin and NXL-103), (xiv) ketolides (including but not limited to telithromycin and solithromycin), (xv) various other antibiotics, including but not limited to cycloserine, fosfomycin, nitrofurantoin, nifurtoinol, thiamphenicol, chloramphenicol, metronidazole, bacitracin and mupirocin, and (xvi) locally applied disinfectants including but not limited to chlorhexidine, PVP-l _{2 (} Povidone-iodine), colloidal silver, Manuka honey, ozonated olive oil, silver nitrate, silver sulfadiazine, ethanol, isopropanol, hydrogen peroxide, chloroxylenol, and cetrimide, benzalkoniumchloride and other quaternary ammonium compounds.

Combination therapy comprising a compound of the present invention and an antimicrobial agent, e.g. an antibiotic or disinfectant, described herein for the treatment of a bacterial infection is specifically contemplated. For example, in one embodiment, the invention provides a method of treating a bacterial infection or a disorder associated with bacterial QS and/or biofilm formation in a subject comprising administering to the subject a therapeutically-effective amount of a combination therapy comprising (a) a compound of formula I, la, II or II or any subgroup thereof such as for example a compound as depicted in table 1 , and (b) an antimicrobial agent.

Such combination therapy would be provided in a combined amount effective to prevent or inhibit QS and/or biofilm formation of the bacteria and/or prevent or treat the bacterial infection and/or treat the disorder associated with biofilm formation. This process involves administering to a subject in need thereof the compound and a (standard of care) therapeutic agent at the same time, which may be achieved by administering a single composition or pharmacological formulation that includes both the compound of the invention and a therapeutic agent, or by administering two distinct compositions or formulations, at the same time, wherein one composition includes the compound of the invention and the other includes a (standard of care) therapeutic agent. In another embodiment, the combination therapy involves administering to a subject in need thereof the compound of the invention and a (standard of care) therapeutic agent at different times, which may be achieved by administering two distinct compositions or formulations, at different time intervals, wherein one composition includes the compound of the invention and the other includes a (standard of care) therapeutic agent.

The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein, for example using liposomes or hydrophilic polymeric matrices based on natural gels or synthetic polymers. In order to enhance the solubility and/or the stability of the compounds of a pharmaceutical composition according to the invention, it can be advantageous to employ α-, β- or γ-cyclodextrins or their derivatives. An interesting way of formulating the compounds in combination with a cyclodextrin or a derivative thereof has been described in EP-A-721 ,331 . In particular, the present invention encompasses a pharmaceutical composition comprising an effective amount of a compound according to the invention with a pharmaceutically acceptable cyclodextrin. A specific example is a nontoxic hydroxypropyl-β- cyclodextrin-functionalized cellulose gauze loaded with one or more of the compounds as described herein, optionally in combination with an antimicrobial agent.

In addition, co-solvents such as alcohols may improve the solubility and/or the stability of the compounds. In the preparation of aqueous compositions, addition of salts of the compounds of the invention can be more suitable due to their increased water solubility.

Particular reference is made to the compositions, formulations (and carriers, excipients, diluents, etc. for use therein), routes of administration etc., which are known per se for analogous pyridinocarboxamides, such as those described in US-A-4,997,834 and EP-A-0 370 498.

More in particular, the compositions may be formulated in a pharmaceutical formulation comprising a therapeutically effective amount of particles consisting of a solid dispersion of the compounds of the invention and one or more pharmaceutically acceptable water-soluble polymers.

The term "a solid dispersion" defines a system in a solid state (as opposed to a liquid or gaseous state) comprising at least two components, wherein one component is dispersed more or less evenly throughout the other component or components. When said dispersion of the components is such that the system is chemically and physically uniform or homogenous throughout or consists of one phase as defined in thermodynamics, such a solid dispersion is referred to as "a solid solution". Solid solutions are preferred physical systems because the components therein are usually readily bioavailable to the organisms to which they are administered.

It may further be convenient to formulate the compounds in the form of nanoparticles which have a surface modifier adsorbed on the surface thereof in an amount sufficient to maintain an effective average particle size of less than 1000 nm. Suitable surface modifiers can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifiers include nonionic and anionic surfactants.

Yet another interesting way of formulating the compounds according to the invention involves a pharmaceutical composition whereby the compounds are incorporated in hydrophilic polymers and applying this mixture as a coat film over many small beads, thus yielding a composition with good bio-availability which can conveniently be manufactured and which is suitable for preparing pharmaceutical dosage forms for oral administration. Materials suitable for use as cores in the beads are manifold, provided that said materials are pharmaceutically acceptable and have appropriate dimensions and firmness. Examples of such materials are polymers, inorganic substances, organic substances, and saccharides and derivatives thereof.

The preparations may be prepared in a manner known per se, which usually involves mixing at least one compound according to the invention with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is again made to US-A-6, 372,778, US-A- 6,369,086, US-A-6,369,087 and US-A-6,372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

The pharmaceutical preparations of the invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the invention, e.g. about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage. The compounds can be administered by a variety of routes including the oral, rectal, ocular, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used and the condition to be treated or prevented, and with oral and intravenous administration usually being preferred. At least one compound of the invention will generally be administered in an "effective amount", by which is meant any amount of a compound of the Formula I, la, II or II or any subgroup thereof, such as a compound as depicted in table 1 , that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered. More specific, it refers to an amount of the compound at least sufficient to achieve a desired modulation of the activity or physiological property of a microbial population as exemplified in the present examples. The effective amount is determined, at least in part, upon the compound used, the microbial species present, the structure, system, or host, and the desired level of regulation. Modulating the activity or physiological property of the microbial population includes, but is not limited to, slowing, attenuating, inhibiting, or enhancing the colonization of a surface or proliferation of bacteria, inhibiting the formation of a biofilm, and the like. Modulation includes slowing the formation of bacteria or new bacteria if some bacteria are already present, inhibiting the formation of a biofilm. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram body weight day of the patient per day, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight day of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses, or essentially continuously, e.g. using a drip infusion. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is again made to US-A- 6,372,778, US-A-6, 369, 086, US-A-6,369,087 and US-A-6, 372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. In accordance with the method of the present invention, said pharmaceutical composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.

For an oral administration form, the compositions of the present invention can be mixed with suitable additives, such as excipients, stabilizers, or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the invention or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. For subcutaneous administration, the compound according to the invention, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds of the invention can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally- acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

In preferred embodiments, the compounds and compositions of the invention are used locally, for instance topical or in both absorbed and non-adsorbed applications.

In a particular embodiment, compositions comprising a compound of the present invention, either alone or in combination as described herein, can be used as a topical agent. The topical agent is a solution, that is, in one aspect, a liquid formulation comprising the compound and a carrier. Other suitable forms include semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water, provided that the carrier does not deleteriously react with the compound in the composition. Suitable formulations include, but are not limited to, lip balms, suspensions, emulsions, creams, ointments, powders, liniments, salves and the like. If desired, these compositions may be sterilized or mixed with auxiliary agents, including but not limited to, preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure and the like well known in the art. Preferred vehicles for semi-solid or solid forms topical preparations include ointment bases, conventional ophthalmic vehicles; creams; and gels. These topical preparations optionally contain emollients, perfumes, and/or pigments to enhance their acceptability for various usages, provided that the additives do not deleteriously react with the compound in the composition.

Also suitable for topical application are sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a Freon (chlorofluorocarbon) or environmentally acceptable volatile propellant. Such compositions are used in one aspect, for application to the skin or to mucous membranes. The aerosol or spray preparations optionally contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compound.

The compositions of the present invention are further of value in the veterinary field, which for the purposes herein not only includes the prevention and/or treatment of diseases in animals, but also - for economically important animals such as cattle, pigs, sheep, chicken, fish, etc. - enhancing the growth and/or weight of the animal and/or the amount and/or the quality of the meat or other products obtained from the animal. Thus, in a further aspect, the invention relates to a composition for veterinary use that contains at least one compound of the invention and at least one suitable carrier (i.e. a carrier suitable for veterinary use). The invention also relates to the use of a compound of the invention in the preparation of such a composition.

As used herein, the term "subject" includes humans, mammals (e.g., cats, dogs, horses, chicken, pigs, hogs, cows, fish, crabs, shrimps, cattle, and other), and other living species that are in need of treatment. In particular, the term "host" includes humans.

In related variations of the preceding embodiments, a composition comprising a compound of the present invention packaged alone, e.g., in a kit or package or unit dose, or is optionally arranged to permit co-administration with one or more other (therapeutic) agents as described herein, but the compound and the agent are not in admixture. In an alternative variation, the compound and the agent are in admixture. In some embodiments, the two components to the kit/unit dose are packaged with instructions for administering the two agents to a human subject for treatment of one of the above-indicated disorders and diseases. The kit may comprise a composition described herein in combination with a vehicle in a cream or gel base, as a pump-spray, as an aerosol, on an impregnated bandage, or in a dropper.

This invention will be better understood by reference to the experimental details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Particular embodiments and examples are not in any way intended to limit the scope of the invention as claimed. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

EXAMPLES

Compound synthesis - Reaction schemes The compounds of the invention may be prepared by methods well known to those skilled in the art, and as described in the synthetic and experimental procedures shown below.

An overview of the selected scaffolds is provided below.

HAM-171

A first scaffold was obtained by transposing the 2-benzamidomethyl moiety to position 1 of the tetrahydrofuran core. A 2,5-substituted 1 ,4-dioxane core was put forward as possible bio-isostere of the tetrahydrofuran ring present in HAM-171 . Antimicrobial potentiator HAM-171 consists of a 5-ortho-chlorobenzamidomethyl and a 2'- benzamidomethyl moiety, linked to a central (3S,4R)-tetrahydrofuran-3,4-diol scaffold. While maintaining both aromatic moieties intact, the impact of transposing the 2-benzamidomethyl fragment to position 1 was investigated. The synthesis of 7, a rather conservative scaffold hop (reflected in a Tc of 0.909), starts from the known azidolactole 1 (1 1 ) (Scheme 1 ). Wittig olefination of 1 with methyl (triphenylphosphoranylidene)acetate and treatment of the resulting α,β-unsaturated carbonyl intermediate with a methanolic sodium methoxide solution afforded methyl esters 2 as an inseparable mixture of cis and trans epimers (12). Saponification yielded a mixture of epimeric carboxylic acids 3, which were converted into the separable Boc-protected amines by Curtius rearrangement. TFA-mediated Boc-deprotection and subsequent EDC- mediated acylation with benzoic acid gave benzamide 6. The latter was subjected to Staudinger reduction and the resulting amine was converted into an ortho-chlorobenzamide. Acidic hydrolysis of the acetonide gave 7, an isomer of HAM-171 (Scheme 1 ).

NaOH, I— ». 3 R = H 4 (46%) 5 (13%) H ₂ 0/dioxane

[i] TFA, CI(CH ₂ ) ₂ CI, OX

(q )

(a) [i] PMe ₃ , THF, 3h [ii] PhCOOH, EDC.HCI,

DMF, (80%)

SCHEME 1 Synthesis of epimer 9

A 1 ,4-dioxane motif was explored as central scaffold as well. Derivative 15 contains a similar atom connectivity as HAM-1 71 . The synthesis of 15 starts with the reaction of benzyl alcohol with (R)- epichlorohydrin (Scheme 2). The resulting (R)-1 -(benzyloxy)-3-chloropropan-2-ol was treated with (S)-glycidyl tosylate to give intermediate 9. Treatment of the latter with aqueous NaOH solution lead to intramolecular ring closure in a 6-exo-tet process to give monoprotected cis- dioxanedimethanol intermediate 10. Subsequent transformation of 10 into heterobisbenzamide 15 occurred via known procedures (Scheme 2).

[i] BF ₃ .OEt ₂ ,

H ₂ ) ₂ CI,

SCHEME 2 Synthesis of 1 ,4-dioxane derivative 29

Chemistry - general

All reactions described were performed under an argon atmosphere and at ambient temperature unless stated otherwise. All reagents and solvents were purchased from Sigma-Aldrich (Diegem, Belgium), Acros Organics (Geel Belgium), TCI Europe (Zwijndrecht, Belgium) or Carbosynth Ltd (Compton Berkshire, United Kingdom) and used as received. NMR solvents were purchased from Eurisotop (Saint-Aubin, France). Reactions were monitored by TLC analysis using TLC aluminium sheets (Macherey-Nagel, Alugram Sil G/UV254) with detection by UV or by spraying with a solution of (ΝΗ ₄ )6Μθ7θ24-4Η ₂ 0 (25 g/L) and (NH ₄ ) ₄ Ce(S0 ₄ )4-2H ₂ 0 (10 g/L) in H ₂ S0 ₄ (10 %) followed by charring or an aqueous solution of KMn0 ₇ (20 g/L) and K ₂ C0 ₃ (10 g/L) or an ethanolic solution of ninhydrin (2 g/L) and acetic acid (1 % v/v) followed by charring. Silica gel column chromatography was performed manually using Grace Davisil 60A silica gel (40-63 μιη) or automated using a Grace Reveleris X2 system and the corresponding flash cartridges. High resolution spectra were recorded with a Waters LCT Premier XE Mass spectrometer. H- and ³ C-NMR spectra were recorded with a Varian Mercury-300BB (300/75 MHz) spectrometer. Chemical shifts are given in ppm (δ) relative to tetramethylsilane as an internal standard ( H NMR) or the NMR solvent ( ³ C NMR). In ⁹ F NMR, signals have been referred to CDCI ₃ or DMSO-d ₆ lock resonance frequency according to lUPAC referencing with CFCI ₃ set to 0 ppm. Coupling constants are given in Hz. Preparative HPLC purifications were carried out using a Laprep preparative HPLC system equipped with a Xbridge Prep C18 column (19x250 mm, 5 micron) using a water/acetonitrile/formic acid gradient system. General procedure 1 : EDC-mediated amide formation.

To a solution of the crude amine obtained from 4 in DMF (25 mL/mmol) were added the appropriate organic acid (1 .5 equiv per amine), EDC.HCI (2 equiv per amine), diisopropylethylamine (4 equiv per amine) and a catalytic amount of 1 -hydroxybenzotriazole. The reaction mixture was stirred overnight at rt. The mixture was concentrated and partioned between water and EtOAc. The organic layer was dried over sodium sulphate, filtered and concentrated in vacuo. The products were purified by column chromatography with appropriate eluents.

General procedure 2: Staudinger reduction and subsequent EDC-mediated acylation of the resulting amine with the appropriate benzoic acid.

A solution of compound 6, 11 , or 14 (0.4 to 1 .4 mmol) in THF (10 mL/mmol) was treated with Me ₃ P (1 M solution, 5 equiv) and the reaction mixture was stirred for 3h. Water (13 equiv) was added and the solution was stirred for another hour, after which it was concentrated. The residue was co-evaporated with toluene. The obtained crude amine was used without further purification. To a solution of this crude amine in DMF (25 mL/mmol) were added the appropriate organic acid (1 .5 equiv), EDC.HCI (2 equiv), diisopropylethylamine (4 equiv) and a catalytic amount of 1 - hydroxybenzotriazole and the reaction mixture was stirred overnight at rt. The reaction mixture was concentrated and partioned between water and EtOAc. The organic layer was dried over sodium sulphate, filtered and evaporated. The products were then purified by column chromatography with appropriate eluents.

General procedure 3: TFA in water - mediated deprotection.

A known amount of the isopropylidene protected crude obtained from 8 was treated with a 35% aq. CF ₃ COOH solution (30 mL/mmol) overnight at room temperature. When TLC indicated that the deprotection was complete, the reaction mixture was concentrated and, if required, purified by column chromatography.

(3aR, 6R, 6aR)-6-(azidomethyl)-2, 2-dimethyltetrahydrofuro[3, 4-d][1 ,3]dioxol-4-ol (1 )

See reference 4 and 1 1 . Spectroscopy data for 1 are consistent with those published previously (4; 1 1 ).

Methyl 2-((3aS, 6R, 6aR)-6-(azidomethyl)-2, 2-dimethyltetrahydrofuro[3, 4-d][1 ,3]dioxol-4-yl)acetate (2)

A solution of lactol 1 (4.00 g, 18.6 mmol) in acetonitrile (60 mL) was flushed with N ₂ gas. Methyl (triphenylphosphoranylidene)acetate (6.84 g, 20.5 mmol) was added and the reaction mixture was heated to reflux for 90 minutes. Then, the mixture was concentrated in vacuo and the residue was taken up in MeOH (40 mL), after which a methanolic sodium methoxide solution (5.4 M, 370 μί) was added. This mixture was stirred for another 90 minutes and then neutralized with Amberlite (IR 120 H-form). The suspension was filtered and the filtrate was concentrated. FCC of the residue (toluene/EtOAc 1 :1 ) gave methyl esters 2 as a pale oil in 46% yield. Epimeric ratio = 94:6. Major epimer: H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .34 (s, 3 H) 1 .54 (s, 3 H) 2.65 (dd, J = 15.8, 7.0 Hz, 1 H) 2.74 (dd, J = 15.8, 5.4 Hz, 1 H) 3.34 (dd, J = 13.2, 4.7 Hz, 1 H) 3.54 (dd, J = 13.2, 4.0 Hz, 1 H) 3.71 (s, 3 H) 4.05 - 4.12 (m, 1 H) 4.27 - 4.33 (m, 1 H) 4.52 - 4.63 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.6, 27.5, 38.1 , 52.0, 52.4, 81 .0, 82.2, 83.2, 84.3, 1 15.1 , 170.9. Minor epimer: H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .33 (s, 3 H) 1 .49 (s, 3 H) 2.76 - 2.79 (m, 2 H) 3.24 - 3.47 (m, 2 H) 3.71 (s, 3 H) 4.18 - 4.24 (m, 1 H) 4.41 (td, J = 6.8, 4.0 Hz, 1 H) 4.64 (dd, J = 6.2, 1 .2 Hz, 1 H) 4.80 (dd, J = 6.2, 4.1 Hz, 1 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.1 , 26.4, 34.4, 51 .7, 51 .9, 77.5, 81 .4, 82.8, 83.4, 1 15.1 , 170.9. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for

272.12410; Found 272.1240.

2-((3aS,6R,6aR)-6-(azidomet yl)-2,2-dimet yltetra ydrofuro[3 -d][1,3fi acid (3)

To a solution of methyl esters 2 (0.950 g, 3.50 mmol) in dioxane (10 mL), was added a 1 M NaOH solution (4.20 mL). After 150 minutes of stirring, TLC analysis (toluene/EtOAc 4:1 ) showed complete consumption of starting material and presence of a lower-running spot. The pH of the reaction mixture was adjusted to approximately 2 by addition of HCI (1 M solution). The reaction mixture was extracted with EtOAc. The organic layers were pooled, dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. The product was obtained as a transparent oil without further purification (q.). Epimeric ratio = 76:24. Major epimer: H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .35 (s, 3 H) 1 .55 (s, 3 H) 2.63 - 2.74 (dd, J = 16.1 , 7.3 Hz, 1 H) 2.74 - 2.82 (m, 1 H) 3.35 (dd, J = 13.1 , 4.5 Hz, 1 H) 3.56 (dd, J = 13.1 , 3.8 Hz, 1 H) 4.08 - 4.13 (m, 1 H) 4.26 - 4.34 (m, 1 H) 4.52 - 4.58 (dd, J = 6.7, 4.4 Hz, 1 H) 4.58 - 4.63 (dd, J = 6.7, 4.1 Hz, 1 H) 10.82 (br. s., 1 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.5, 27.4, 38.1 , 52.3, 80.6, 82.0, 83.1 , 84.1 , 1 15.2, 176.1 . Minor epimer: H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .34 (s, 3 H) 1 .50 (s, 3 H) 2.72 - 2.89 (m, 2 H) 3.32 (dd, J = 12.9, 4.7 Hz, 1 H) 3.44 (d, J = 12.9, 6.2 Hz, 1 H) 4.18 - 4.24 (m, 1 H) 4.37 - 4.44 (m, 1 H) 4.66 (dd, J = 6.2, 1 .2 Hz, 1 H) 4.81 (dd, J = 6.2, 4.1 Hz, 1 H) 10.82 (br. s., 1 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.0, 26.2, 34.4, 51 .8, 77.3, 81 .3, 82.7, 83.3, 1 13.2, 176.8. HRMS (ESI-TOF) m/z: [M-H] ^~ Calcd for doH^f- 256.09389; Found 256.0939. Tert-butyl (((3aS,4S, 6R, 6aR)-6-(azidomethyl)-2, 2-dimethyltetrahydrofuro[3,4-d][1 ,3]dioxol-4- yl)methyl)carbamate (4)

A flask containing carboxylic acids 3 (1 .20 g, 4.66 mmol) and molecular sieves in iert-butanol (90 mL) was purged with N ₂ gas. Tiethylamine (0.715 mL, 5.13 mmol) and diphenylphosphoryl azide (1 .10 mL, 5.13 mmol) were added and the reaction mixture was heated to reflux for 20 hours. TLC analysis (hexane/EtOAc 85:15) showed complete consumption of starting material. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was taken up in EtOAc and washed with brine. The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. This residue was adsorbed onto celite and purified via FCC (hexane/EtOAc 10:0→ 8:2). Both compound 4 (46% yield) and its trans epimer 5 (13% yield) were obtained as transparent oils. H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .33 (s, 3 H) 1 .45 (s, 9 H) 1 .53 (s, 3 H) 3.29 - 3.49 (m, 3 H) 3.60 (dd, J = 13.0, 3.7 Hz, 1 H) 3.98 - 4.09 (m, 2 H) 4.46 - 4.57 (m, 2 H) 4.91 (br. s., 1 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.5, 27.5, 28.4, 42.5, 52.4, 79.6, 81 .8, 82.4, 83.1 , 83.5, 1 14.9, 156.1 . HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for

329.18195; Found 329.1829.

Tert-butyl (((3aS, 4R, 6R, 6aR)-6-(azidomethyl)-2, 2-dimethyltetrahydrofuro[3, 4-d][1,3]dioxol-4- yl)methyl)carbamate (5)

Compound 5 was obtained according to the procedure described for compound 4. HRMS (ESI- TOF) m/z: [M+H] ⁺ Calcd for 329.18195; Found 329.1831 .

N-(((3aS, 4S, 6R, 6aR)-6-(azidomethyl)-2, 2-dimethyltetrahydrofuro[3, 4-d][1 ,3]dioxol-4- yl)methyl)benzamide (6)

Boc-protected amine 4 (100 mg, 0.305 mmol) was taken up in dry 1 ,2-dichloroethane (2 mL) and molecular sieves (3 A rods) were added. The flask was purged with N ₂ gas and cooled on ice to 0 °C. Trifluoroacetic acid (0.179 mL, 2.34 mmol) was added and the reaction mixture was stirred for 3 hours. After filtration, the filtrate was taken to dryness and the residue was adsorbed onto celite. FCC (CH ₂ CI ₂ /MeOH/NH ₄ OH 100:0:0.1 → 90:10:0.1 ) gave the corresponding primary amine, which was subjected to general procedure 1 . Transparent oil, 80%. H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .32 (s, 3 H) 1 .53 (s, 3 H) 3.44 (dd, J = 13.0, 4.0 Hz, 1 H) 3.64 - 3.76 (m, 2 H) 3.85 (ddd, J = 14.1 , 6.6, 5.1 Hz, 1 H) 4.06 - 4.14 (m, 1 H) 4.16 - 4.23 (m, 1 H) 4.51 - 4.60 (m, 2 H) 6.69 (br. s., 1 H) 7.37 - 7.55 (m, 3 H) 7.75 - 7.87 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 25.5, 27.5, 41 .6, 52.4, 81 .6, 82.5, 83.1 , 83.3, 1 15.0, 127.1 , 128.7, 131 .7, 134.3, 167.8. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₁₆ H ₂ i N ₄ 04 ⁺ 333.15573; Found 333.1563.

N-(((2R, 3S,4R, 5S)-5-(benzamidomethyl)-3, 4-dihydroxytetrahydrofuran-2-yl)methyl)-2- chlorobenzamide (7)

Compound 6 was subjected to general procedure 2. H NMR (300 MHz, CDCI ₃ ) δ ppm 1 .32 (s, 3 H) 1 .53 (s, 3 H) 3.52 - 3.86 (m, 4 H) 4.10 - 4.21 (m, 2 H) 4.49 - 4.60 (m, 2 H) 6.88 (br. s., 1 H) 7.00 (br. s., 1 H) 7.21 - 7.41 (m, 5 H) 7.42 - 7.51 (m, 1 H) 7.55 - 7.65 (m, 1 H) 7.75 - 7.81 (m, 2 H). 3C NMR (75 MHz, CDCI ₃ ) δ ppm 25.5, 27.4, 42.16, 42.22, 82.79, 82.84, 83.6, 83.8, 1 14.7, 127.19, 127.23, 128.6, 130.1 , 130.4, 130.7, 131 .5, 131 .6, 134.1 , 135.1 , 167.3, 167.9. HRMS (ESI- TOF) m/z: [M+H] ⁺ Calcd for C ₂₃ H ₂₅ CIN ₂ 05 ⁺ 445.15248; Found 445.1523. The isopropylidene protected derivative was subjected to general procedure 3. White foam, 80% from 6. H NMR (300 MHz, CDCI ₃ ) δ ppm 3.21 - 3.55 (m, 4 H) 3.73 - 3.90 (m, 4 H) 4.86 (br. s., 2 H) 7.29 - 7.58 (m, 7 H) 7.77 - 7.88 (m, 2 H) 8.37 - 8.52 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 41 .3, 41 .8, 72.1 , 72.3, 81 .4, 81 .6, 127.0, 127.2, 128.2, 128.9, 129.5, 129.9, 130.7, 131 .1 , 134.4, 137.0, 166.5, 166.6. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₂₀ H ₂₂ CIN ₂ O ₅ ⁺ 405.121 18; Found 405.1213. (R)-1-(benzyloxy)-3-chloropropan-2-ol (8)

To a flame-dried, three neck round bottomed flask was added dry 1 ,2-dichloroethane (45 mL). R- (-)-epichlorohydrin (3.92 mL, 50.0 mmol) and benzyl alcohol (10.4 mL, 100 mmol) were added and the flask was purged with N ₂ gas. The solution was cooled on ice to 0 °C under N ₂ . The stirring solution was then treated with boron trifluoro etherate (0.272 mL, 2.20 mmol) and again purged with N ₂ . The reaction mixture was allowed to attain room temperature overnight (16h). Subsequently, the mixture was heated to reflux for 2h and then allowed to cool and washed with NaHC0 ₃ (sat. aq. soln.). The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. Excess benzyl alcohol was removed under reduced pressure overnight in an oil bath at 50 °C. FCC (hexane/EtOAc 10:0→ 7:3) of the residue afforded the title compound as a colorless liquid in 48% yield. H NMR (300 MHz, CDCI ₃ ) δ ppm 2.54 (d, J = 5.9 Hz, 1 H) 3.55 - 3.69 (m, 4 H) 3.94 - 4.06 (m, 1 H) 4.56 (s, 2 H) 7.26 - 7.40 (m, 5 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 46.2, 70.4, 70.9, 73.7, 127.9, 128.1 , 128.6, 137.7. (S)-3-(((R)-1-(benzyloxy)-3-chloropropan-2-yl)oxy)-2-hydroxy propyl 4-methylbenzenesulfonate (9) A solution of compound 8 (1 .87 g, 9.32 mmol) in dry 1 ,2-dichloroethane (40 mL) was purged with N ₂ . S-glycidyl tosylate (0.710 g, 3.1 1 mmol) was added and the resulting solution was back- flushed and cooled on ice to 0 °C. Boron trifluoro etherate (catalytic quantity, 100 μί) was added and the reaction mixture was allowed to attain room temperature overnight. The mixture was washed with NaHC0 ₃ (sat. aq. soln.) and brine. The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated under reduced pressure. FCC (hexane/EtOAc 10:0→ 4:6) afforded compound 9 as a colorless liquid in 50% yield. H NMR (300 MHz, CDCI ₃ ) δ ppm 2.44 (s, 3 H) 2.94 (d, J = 5.0 Hz, 1 H) 3.47 - 3.65 (m, 5 H) 3.67 - 3.76 (m, 2 H) 3.92 - 4.12 (m, 3 H) 4.53 (s, 2 H) 7.27 - 7.40 (m, 7 H) 7.74 - 7.83 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 21 .8, 44.0, 68.7, 69.8, 70.3, 71 .3, 73.7, 79.9, 127.9, 128.09, 128.15, 128.7, 130.1 , 132.8, 137.6, 145.2. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₂₀ H ₂₆ CIO ₆ S ⁺ 429.1 1331 ; Found 429.1 139.

((2R, 5R)-5-((benzyloxy)methyl)-1,4-dioxan-2-yl)methanol (10)

To a flask containing compound 9 (0.740 g, 1 .73 mmol) was added an aqueous NaOH solution (1 M, 6 mL, 6 mmol) and the resulting biphasic mixture was stirred vigorously at room temperature. After 150 minutes, the reaction mixture was heated to 90 °C for 4h and then cooled to room temperature again for reaction overnight (16h). Then, the mixture was heated to 90 °C and stirred for 2h. After that, the mixture was neutralized with HCI (1 M) and extracted with CH ₂ CI ₂ (3 x 15 mL). The combined organic layers were rewashed with a saturated aqueous solution of NaHC0 ₃ and brine, then dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. Silica-gel chromatography (CH ₂ CI ₂ /MeOH 100:0→ 96:4) afforded the title compound as a colorless oil in 66% yield. H NMR (300 MHz, CDCI ₃ ) δ ppm 2.36 (br. s., 1 H) 3.50 - 3.86 (m, 10 H) 4.51 - 4.59 (m, 2 H) 7.23 - 7.39 (m, 5 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 61 .0, 63.9, 64.3, 68.2, 72.2, 73.5, 73.8, 127.77, 127.81 , 128.5, 137.9. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₁₃ H ₁₉ 0 ₄ ⁺ 239.12779; Found 239.1279.

(2R,5R)-2-(azidomet yl)-5-((benzyloxy)met yl)-1,4-dioxane (11 )

To a solution of compound 10 (530 mg, 2.22 mmol) and triethylamine (0.618 mL, 4.44 mmol) in CH ₂ CI ₂ (12 mL) stirred at 0 °C, methanesulfonyl chloride (0.205 mL, 2.66 mmol) was added dropwise. The reaction mixture was allowed to attain ambient temperature. After 3 hours, TLC analysis (hexane/EtOAc 1 :1 ) showed complete consumption of the starting material. The reaction mixture was washed with saturated sodium bicarbonate solution and water. The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo to afford the mesylate as a yellow to orange colored oil. To this crude mesylate, dissolved in DMF (12 mL), was added sodium azide (0.722 g, 1 1 .1 mmol). After overnight reaction at 60 °C, TLC analysis (hexane/EtOAc 1 :1 ) showed the presence of one major product. The solvent was evaporated and the residue was taken up in EtOAc. The resulting solution was washed with saturated NaHC0 ₃ solution and water. The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo. This crude material was purified by flash column chromatography (hexane/EtOAc 10:0 → 5:5) to afford azide 11 as a colorless liquid (91 % over two steps). H NMR (300 MHz, CDCI ₃ ) δ ppm 3.26 (dd, J = 12.9, 5.0 Hz, 1 H) 3.47 - 3.85 (m, 9 H) 4.55 (s, 2 H) 7.21 - 7.39 (m, 5 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 49.9, 64.0, 64.7, 68.2, 71 .9, 72.5, 73.4, 127.71 , 127.74, 128.4, 137.8. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₁₃ H ₁₈ N ₃ 04 ⁺ 264.13427; Found 264.1349.

N-(((2R,5R)-5-((benzyloxy)methyl)-1,4-dioxan-2-yl)methyl) benzamide (12)

Compound 11 was subjected to general procedure 2. Colorless liquid, 90%. H NMR (300 MHz, CDCI3) δ ppm 3.52 - 3.88 (m, 10 H) 4.56 (s, 2 H) 6.53 (br. s., 1 H) 7.27 - 7.55 (m, 8 H) 7.72 - 7.82 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 39.5, 64.2, 65.2, 68.4, 72.0, 72.4, 73.6, 127.1 , 127.87, 127.93, 128.6, 128.7, 131 .7, 134.5, 138.0, 167.8. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C20H24NO 342.16998; Found 342.1703.

N-(((2R,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)benz amide (13)

A solution of benzamide 12 (440 mg, 1 .29 mmol) in MeOH (50 mL) was placed under an N ₂ atmosphere. Palladium black (catalytic amount) was added and the reaction vessel was purged again with N ₂ . Hydrogen gas was bubbled through the solution for 3h (MS analysis for conversion). The vessel was purged with nitrogen gas and the reaction mixture was filtered over a Whatman fiberglass filter. The filtrate was concentrated in vacuo and the residue (i.e. the product) was obtained as a colorless oil, quantitatively. H NMR (300 MHz, CDCI ₃ ) δ ppm 3.45 - 3.84 (m, 1 1 H) 7.17 (br. s., 1 H) 7.32 - 7.53 (m, 3 H) 7.70 - 7.86 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 39.2, 60.7, 63.3, 64.9, 71 .6, 73.9, 127.0, 128.5, 131 .6, 134.1 , 168.1 . HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C ₁₃ H ₁₈ N0 ₄ ⁺ 252.12303; Found 252.1236. N-(((2R,5R)-5-(azidomethyl)-1,4-dioxan-2-yl)methyl)benzamide (14)

Following a similar procedure described for compound 11 , compound 13 (325 mg, 1 .29 mmol) gave azide 14 as a pale oil in 69% yield. H NMR (300 MHz, CDCI ₃ ) δ ppm 3.27 - 3.35 (m, 1 H) 3.48 - 3.59 (m, 1 H) 3.60 - 3.89 (m, 8 H) 6.76 (t, J = 4.8 Hz, 1 H) 7.37 - 7.54 (m, 3 H) 7.75 - 7.84 (m, 2 H). ³ C NMR (75 MHz, CDCI ₃ ) δ ppm 39.4, 50.0, 64.2, 64.7, 72.0, 72.1 , 127.0, 128.6, 131 .6, 134.2, 167.8. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for Ο^ _π Ν ₄ 0 ₃ ⁺ 277.12952; Found 277.1299.

N-(((2R,5R)-5-(benzamidomethyl)-1,4-dioxan-2-yl)methyl)-2 -chlorobenzamide (15)

Compound 14 was subjected to general procedure 2. White foam, 74%. H NMR (300 MHz, MeOD) δ ppm 3.47 - 3.58 (m, 2 H) 3.64 - 3.89 (m, 8 H) 7.33 - 7.56 (m, 7 H) 7.79 - 7.85 (m, 2 H). 3C NMR (75 MHz, MeOD) δ ppm 40.5, 40.6, 65.8, 66.0, 73.3, 73.4, 128.1 , 128.3, 129.6, 129.9, 131 .0, 131 .9, 132.2, 132.7, 135.6, 137.6, 170.3, 170.5. HRMS (ESI-TOF) m/z: [M+H] ⁺ Calcd for C20H22CIN2O 389.12626; Found 389.1264. Microbiology

Reagents used

Hamamelitannin (HAM) and vancomycin (VAN) were purchased from Sigma Aldrich (Bornem, Belgium). HAM was stored in DMSO at -20°C. VAN was dissolved in ultrapure water and stored at 4°C.

Strains and culture conditions

Methicillin-resistant Staphylococcus aureus Mu50 (MRSA Mu50) was cultured in Mueller-Hinton broth (MH , Oxoid, Basingstoke, England) at 37°C under aerobic conditions.

Determination of the MIC

MICs of HAM analogues used against S. aureus Mu50 were determined in triplicate using flat- bottom 96-well microtiter plates (TPP, Trasadingen, Switzerland) as previously described (2). Effect of pretreatment and co-treatment on biofilm susceptibilty

S. aureus Mu50 biofilms were formed and HAM analogues were evaluated as previously described (3,4,6). In brief, overnight cultures in MH were centrifuged, the pellet was resuspended in double-concentrated MH (2 x MH) and diluted to an OD590 nm of 0.2. Fifty microliter of the diluted bacterial suspension was transferred to the wells of a round-bottom 96-well microtiter plate (TPP). Control wells received 50 μΙ MilliQ. Wells used to evaluate pre-treatment received 50 μΙ of HAM-analogue solution. Bacteria were allowed to adhere and grow without agitation for 4 h at 37 °C. After 4 h, medium was removed, and the adhered cells were washed with sterile physiological saline (0.9% NaCI; PS). After this washing step, control wells were filled with 50 μΙ 2 x MH and 50 μΙ MilliQ. Other wells were filled with 50 μΙ 2 ^χ MH and 50 μΙ of HAM analogue solution, and the plate was incubated for 20 h at 37 °C. To evaluate the effect of co-treatment on mature biofilms, control biofilms were formed in the absence of HAM analogues, as described above. After 24 h of biofilm formation, the medium was removed and the wells were rinsed with PS. Control wells were either filled with 100 μΙ PS (untreated controls) or with 50 μΙ PS and 50 μΙ antibiotic solution. Wells used to evaluate the effect of pre-treatment were also filled with 50 μΙ PS and 50 μΙ antibiotic solution while wells used to evaluate combination treatment were filled with 50μΙ of a HAM analogue solution and 50 μΙ antibiotic solution. The plates were then incubated for an additional 24 h at 37 °C. After biofilm formation and treatment of the biofilms, the number colony forming units (CFU) per biofilm were determined by conventional plating. To collect the cells for plating, plates were rinsed with PS, sessile cells were removed from the microtiter plate by two cycles of vortexing (5 min) and sonication (5 min) and the number of CFU/biofilm was determined by plating the resulting suspensions. The number of CFU/biofilm (for plating) of the control biofilms was set to 100% and the results of the treated biofilms were compared to this. Each condition was tested in at least three wells in each assay, and each assay was carried out at least in triplicate (n > 9).

Statistical evaluation

The normal distribution of the data was checked by using the Shapiro-Wilk test. Normally distributed data were analyzed using a one-way ANOVA. Non-normally distributed data were analyzed using the Kruskal-Wallis test. Statistics were determined using SPSS software, version 22.0.

Results and discussion Biological evaluation

For all final compounds, minimum inhibitory concentrations (MIC) against S. aureus Mu50 were determined to rule out a direct effect on growth. In all cases, MIC values were higher than 500 μΜ, being the highest concentration tested (data not shown). Subsequently, the HAM derivatives were tested for their in vitro effect on S. aureus biofilm susceptibility to vancomycin (VAN), both under pretreatment and combination treatment regimens. VAN is considered a drug of last resort against MRSA. To evaluate the effect of pretreatment, S. aureus Mu50 was allowed to form a biofilm in the presence of the HAM analogues, after which the biofilm was treated with VAN (20 μg/ml). In the combination treatment setup, the bacteria were allowed to form a mature biofilm after which a HAM analogue and VAN were administered simultaneously. When used alone, VAN resulted only in a minor reduction of the number of S. aureus sessile cells (30 ± 14% compared to an untreated control, Table 1 ). In contrast, combined treatment of VAN with 2 resulted in significantly more killing of bacterial biofilm cells, both under pretreatment and under combined treatment regimens (Table 1 ). Initially, all of the compounds with alternative scaffolds were tested in a concentration of 100 μΜ . For the most active derivatives, the effect on biofilm susceptibility towards VAN was tested in lower concentrations, which allowed us to determine an EC ₅₀ value. The latter is defined as the concentration of the analogue needed to double the activity of VAN , as measured by the number of surviving cells.

TABLE 1 Microbiological evaluation of HAM analogues with different scaffold. ^a : Percentage reduction in Colony Forming Units (CFU's) per biofilm when biofilms are treated with VAN alone (20 μg ml) or in combination with HAM or a HAM-analogue (100 μΜ) compared to the untreated (negative) control. * significantly different from treatment with HAM + VAN (p < 0.05).

Reduction in CFU's compared to

Rj = oCl Ph

control (%) ^*

Compound

R ₂ = Ph Combination Combination

Pretreatment Pretreatment

treatment treatment

VAN alone - 30 ± 14* 30 ± 14* - -

HAM, 1 57 ± 13 57 ± 22 145.5 165.1

HAM-171 88 ± 2 92 ± 4 0.3890 7.976

O O

7 93 ± 6 95 ± 2 2.598 3.708

HO OH

15 95 ± 3 91 ± 1 5.568 13.12 o

When comparing the potentiating activity of lead compound HAM-171 with its isomer 7, it can be concluded that this scaffold rearrangement is well-tolerated (Table 1 ). Bisbenzamide 7 shows very good potentiating activity in the combination treatment setup (EC ₅₀ = 3.708 μΜ, compared to 7.976 μΜ for HAM-171 ). This is encouraging, as clinicians in daily practice are often confronted with already infected and biofilm-related wound infections. Moreover, the improved synthetic accessibility (compared to a longer route towards HAM-171 ) makes compound 7 even more interesting . In the combination treatment regimen, dioxane derivative 15 caused little change in activity compared to HAM-171 and 7. This compound contains to a large extend the same connectivity of atoms relative to HAM-171 and 7. With 15 being almost equipotent in de combination treatment setup, this scaffold hop proves that it is possible to replace the central hamamelose-like central scaffold. Two practical pathways towards the synthesis of novel HAM analogues with a different central scaffold were successfully pioneered. The resulting compounds were tested for their ability to potentiate the activity of VAN in S. aureus biofilms in vitro. The readily accessible 2,5-anhydro-D- allitol derivative 7 and dioxane derivative 15 showed comparable activity to that of lead compound HAM-171 in the combination treatment setup.

Jim O'Neill: Tackling drug-resistant infections globally: final report and recommendations (2016)

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