WO2014173904A1

WO2014173904A1 - Compounds having antibacterial activity

Info

Publication number: WO2014173904A1
Application number: PCT/EP2014/058149
Authority: WO
Inventors: Han REMAUT; Alvin Lo; Gabriel Waksman; David Selwood; Paul Gane
Original assignee: Vib Vzw; Vrije Universiteit Brussel; Ucl Business Plc
Priority date: 2013-04-22
Filing date: 2014-04-22
Publication date: 2014-10-30
Also published as: GB201307233D0

Abstract

The present invention relates to compounds of formula (I) and their uses. In particular, though not exclusively, it concerns heterocyclic compounds that inhibit the biogenesis of adhesive pili in Gram- negative bacteria, and their use in the prevention or treatment of conditions arising from colonisation by bacterial pathogens.

Description

COMPOUNDS HAVING ANTIBACTERIAL ACTIVITY

The present invention relates to compounds and their uses. In particular, though not exclusively, it concerns heterocyclic compounds that inhibit the biogenesis of adhesive pili in Gram- negative bacteria, and their use in the prevention or treatment of conditions arising from colonisation by bacterial pathogens.

Gram-negative bacteria possess a wide variety of proteinaceous structures on their surfaces. These include flagella, responsible for cell motility; injectosomes, thin needles that inject effector molecules into host cells; curli, involved in adhesion to surfaces and bio film formation; and pili (or fimbriae), a broad category which encompasses a range of different structures and functions (Fronzes et al, EMBO J 2008, 27:2271-2280; Erhardt et al, Cold Spring Harb Perspect Biol 2010, 2:a000299; Epstein et al, Cell Microbiol 2008, 10: 1413-1420; Ayers et al, Future Microbiol 2010, 5: 1203-1218; and Craig et al, Curr Opin Struct Biol 2008, 18:267- 277).

In pathogenic bacterial species, pili are often crucial virulence factors, determining establishment and persistence of infection. Pili mediate bacterial attachment and invasion of host cells and tissues, and are involved in evasion of host immune systems and/or in biofilm formation. In these pathogenic bacteria, the genetic disruption of pilus formation seriously compromises or abolishes their ability to cause infection. As a result, there is great interest in understanding their structure and mechanism of assembly, since they represent an attractive target for the development of new therapeutics (Durand et al, Infect Disord Drug Targets 2009, 9:518-547; and Cusumano et al, IDrugs 2009, 12:699-705).

Pili can be categorised into five major classes depending on the biosynthetic pathway involved: (i) chaperone-usher pili (CU pili), (ii) curli, (iii) type IV pili, (iv) the type III secretion needle, and (v) type IV secretion pili. Of these five classes, CU pili are the most common and most well characterised. CU pili are assembled at the outer membrane by two proteins, a periplasmic chaperone and an outer-membrane, pore-forming protein called the usher (Sauer et al, Biochim Biophys Acta 2004, 1694:259-267). The chaperone facilitates folding of pilus subunits (Barnhart et al, Proc Natl Acad Sci 2000, 97:7709-7714), prevents them from polymerising in the periplasm, and targets them to the usher (Dodson et al, Proc Natl Acad Sci 1993, 90:3670- 3674; and Thanassi et al., Proc Natl Acad Sci 1998, 95:3146-3151). In turn, the usher acts as an assembly platform, recruiting chaperone-subunit complexes from the periplasm and coordinating their assembly into a non-covalent polymer that is translocated to the extracellular surface of the bacterium through the usher pore.

In particular, type 1 pili are implicated in bacterial adherence to the human bladder epithelium and the gastrointestinal tract by binding terminal D-mannose residues on high mannose glycoprotein receptors. In addition, type 1 pili are known to mediate bio film formation on non- biological materials such as plastics and steel. As such, they have been proven to be a prime virulence factor in the establishment and persistence of E. co //-caused urinary tract infections (UTIs). For example, via the targeted inactivation of type 1 pilus-dependent adherence, Escherichia coli (E. coli) are prevented from binding the host tissue or from forming bio films on medical devices such as urinary catheters, a major risk factor for the development of hospital-associated UTIs (up to 40% of nosocomial infections).

At present, two compound classes exist that target type 1 pili: (i) D-mannosides which function as inhibitory receptor analogs (e.g., Han et al., J Med Chem 2012, 55:3945-3959), and (ii) pilicides which work by inhibiting subunit recruitment from the periplasm to the pilus assembly platform in the outer membrane (Aberg et al., Org Biomol Chem 2007, 5: 1827-1834; Pinkner et al, Proc Natl Acad Sci USA 2006, 103: 17897-902). Of these, D-mannosides have been most extensively studied, including the evaluation of orally delivered D-mannosides by means of in vivo mouse trials (Klein et al., J Med Chem 2010, 53:8627-8641; Cusumano et al., Sci Transl Med 2011, 3: 109ral l5) and in the prevention of catheter-associated UTIs (Guiton et al., Antimicrob Agents Chemother 2012, 56:4738-45). However, due to their mode of action, there are significant limitations to the use of D-mannosides and pilicides.

D-mannosides function by preventing the adhesion of uropathogenic E. coli (UPEC) to urothelial cell surfaces by means of the lectin FimH, which is located at the tip of bacterial type 1 pili. Therefore, since D-mannosides are potential ligands for all mannose receptors of the human host system, the lack of target selectivity of these antagonists poses a significant concern. Given that mammalian mannose receptors are present on many tissues throughout the body and are involved in a number of important biological processes, non-selective interactions of such antagonists could potentially have a major impact on these processes and could cause harmful side-effects. Furthermore, one provision of current applications is the inclusion of the disclosed pilus biogenesis inhibitors in catheter plastics for the prevention of catheter- associated UTIs. However, due to their mode of action, D-mannosides, when incorporated in catheter plastic, lead to the risk of opposing effects such as enrichment of type-1 piliated bacteria.

Known pilicides target the interaction between chaperone-subunit complexes and the N- terminal domain of the usher at the periplasmic side of the outer membrane. Pilicides, by blocking chaperone and usher functions, therefore have the potential to inhibit pili formation in a broad spectrum of pathogenic bacteria, and thus prevent critical host-pathogen interactions necessary for many diseases. However, pilicides have not been described to dissociate preformed pili. Instead, they only prevent pilus formation (Pinkner et al., Proc Natl Acad Sci USA 2006, 103: 17897-902). Furthermore, pilicides have shown apparently pleiotropic effects in non-related biosynthesis pathways (Cegelski et al, Nat Chem Biol 2009, 5:913-9), suggesting a poor target specificity and potential problems with toxicity.

As a result, it is an object of the present invention to provide therapeutic agents that inhibit the adherence and/or colonisation of bacterial pathogens by targeting aspects of the CU pili system. Such therapeutics have the potential to prevent or treat medical conditions associated with CU pili-mediated bacterial infections, either by direct therapeutic treatment (e.g. drug administration), or by their use to prevent adherence and/or colonisation of bacterial pathogens on medical devices employed in a number of medical procedures.

In this regard, the present inventors have found a set of structurally distinct compounds that exhibit a novel mode of action in targeting CU pilus assembly. The result of this new targeting mechanism is that the biogenesis of new pili is prevented, or pre-existing pili are removed from the bacterial surface. In particular, these compounds are proposed to function by inhibiting the pilus subunit polymerization step during type 1 pilus assembly, thereby preventing the formation of type 1 pili on the bacterial surface. In addition, they have been found to potentially efface pre-assembled pili from the bacterial cell surface.

Given that these compounds potentially eliminate the presence of pili from the bacterial cell surface, the possibility of attachment, and thus colonisation, is reduced. Therefore, the prospective utility of such compounds is particularly wide-ranging and includes, but is by no means limited to, (i) the non-antibiotic treatment and prophylaxis of human urinary tract infections caused by bacterial pathogens such as E. coli (e.g. in a hospital environment: catheterised patients who are susceptible to recurrent infections), (ii) the prevention of bacterial biofilm formation on medical devices, primarily urinary catheters, as a means to reduce the risk of catheter-associated infections, and (iii) the inhibition of type 1 pilus formation in adherent invasive bacterial strains, for example those associated with Peyer's patch attachment in colitis.

According to the present invention, there is provided a compound of the formula (I):

wherein

ring A is an optionally substituted 5- or 6-membered heterocyclyl, aryl, heteroaryl, or cycloalkyl group;

ring B is an optionally substituted 5-membered heterocyclyl or heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is a bond, -0-, -N(R₃)-, -CH₂-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is a bond, -0-, -N(R₄ , or -S-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group; Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

Preferably, there is provided a compound of the formula (I):

wherein

ring A is an optionally substituted 6- or 5- membered aryl, heterocyclyl, heteroaryl, or cycloalkyl group;

ring B is an optionally substituted 5-membered heteroaryl or heterocyclyl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is a -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted

Ci_6 alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Y is -S-, a bond, -0-, or -N(R-i)-. - wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Z is a Ci_6 alkyl carbamoyl group, a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_6 alkoxy carbonyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6- membered cycloalkenyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

The molecular target of the disclosed compounds is believed to be a defined hydrophobic pocket on the chaperone:subunit complexes, referred to as the P5 pocket (Remaut et al., Mol Cell 2006, 2:831-42). Accessibility of this pocket may be a determining factor in the initiation and progression of subunit polymerisation during pilus assembly (Remaut et al., Mol Cell 2006, 2:831-42; Verger et al, EMBO Rep 2006, 7: 1228-32; Verger et al, Structure 2008, 16: 1724- 31). This principle may be conserved throughout chaperone-usher pilus systems such that compounds targeting accessibility of the pocket can be expected to have an inhibitory effect in pilus biogenesis of chaperone-usher pili other than the type 1 pilus system. Although the molecular principle is conserved, the detailed 3-dimensional structural landscape of the pocket can differ between different subunits and chaperone-usher pilus systems. Specificity and tuning towards different chaperone-usher pilus systems can be attained by chemical modification of the specific chemical entity.

As such, the compounds of formula (I) have been found to represent novel, virulence-targeted inhibitors that target CU pilus subunits, thereby inhibiting pilus subunit polymerisation during a process called 'donor-strand exchange' (DSE). The structure and mechanism of chaperone- assisted pilus assembly via DSE is well-known in the art, and reference is made, for example, to Sauer et al, Cell 2002, 111 :543-51 and Remaut et al, Mol Cell 2006, 22:831-42. Given the widespread conservation of the DSE reaction mechanism and the ubiquitous presence of the pocket in the chaperone-usher assembly of a diverse array of cell surface-associated virulence determinants in many important bacterial pathogens, the present inhibitors have applications beyond the inhibition of pilus-mediated adhesion.

As compared to the traditional antibiotics, anti-virulence therapies work differently. Antivirulence drugs disarm pathogens rather than killing them. In this way, these drugs prevent a disease by neutralising virulence factors, the specific proteins or toxins that a pathogen uses to establish an infection. Anti-virulence therapy is a particularly advantageous strategy since it minimises selective pressure that perpetuates drug resistance through horizontal gene transfer, resulting in slowing down the rate of resistance evolution. Moreover, because of their selective nature, anti-virulence therapeutics leave the commensal microbiota untouched, unlike the broad-acting bacteriostatic or bacteriolytic antibiotics commonly in use. The collateral damage to the host microbiota inflicted by broad-acting antibiotics brings a dysbiosis that can lead to negative effects on the patient, including the risk of bacterial blooms of opportunistic pathogens, for example Clostridium difficile. There is increasing evidence that antibiotic- related dysbiosis can have long lasting metabolic effects (Dethlefsen et al., PLoS Biol 2008, 6:e280; Cho et al, Nature 2012, 488:621-626).

The term 'C_x__y alkyl' as used herein refers to a linear or branched saturated hydrocarbon group containing from x to y carbon atoms. For example, Ci_₆ alkyl refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. Examples of Ci_₆ alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, hexyl, and isohexyl. The term 'C_x__y alkylene' as used herein refers to a divalent hydrocarbon group obtained by removing one hydrogen atom from 'C_x__y alkyl' above. Examples of Ci_₆ alkylene groups include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

The term 'C_x__y alkenyl' as used herein refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds and having from x to y carbon atoms. Examples of C₂_₆ alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, and 5-hexenyl. The term 'C_x__y alkynyl' as used herein refers to a divalent hydrocarbon group containing one or more carbon-carbon triple bonds and having from x to y carbon atoms. Examples of C₂_₆ alkynyl groups include ethynyl, propynyl, butynyl and pentynyl.

The term 'C_x__y alkoxy' as used herein refers to an -0-C_x__y alkyl group wherein C_x__y alkyl is as defined herein. Examples of Ci_₆ alkoxy groups include methoxy, ethoxy, propoxy, iso- propoxy, butoxy, tert-butoxy, pentoxy and hexoxy.

The term 'x- to y-membered cycloalkyl' as used herein refers to a saturated monocyclic hydrocarbon ring of x to y carbon atoms. For example, 3- to 6-membered cycloalkyl refers to a saturated monocyclic hydrocarbon ring of 3 to 6 carbon atoms. Examples of 3- to 6-membered cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term 'x- to y-membered cycloalkenyl' as used herein refers to a monocyclic hydrocarbon ring containing one or more carbon-carbon double bonds of x to y carbon atoms. For example, C₃_6 cycloalkenyl refers to an unsaturated monocyclic hydrocarbon ring of 3- to 6-carbon atoms. Examples of C₃_₆ cycloalkenyl groups include 2-cyclopenten-l-yl, 3-cyclopenten-l-yl, 2- cyclohexen- 1 -yl, and 3-cyclohexen- 1 -yl.

The term 'aryl' as used herein refers to a 5- to 6-membered monocyclic hydrocarbon ring containing x to y carbon atoms, wherein the ring is aromatic. An example of such an aryl group is phenyl. Alternatively, the term 'C_x__y aryl' as used herein refers to a monocyclic or bicyclic ring containing from x to y carbon atoms, wherein at least one ring is aromatic. Examples of C₆-i4 aryl groups include phenyl, naphthyl, tetrahydronaphthalenyl, anthryl, phenanthryl, acenaphthylenyl, biphenylyl, anthracenyl, phenanthrenyl, and phenalenyl.

The term 'heteroaryl' as used herein refers to a 5- to 6-membered monocyclic aromatic ring in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur. Examples of such monocyclic aromatic rings include thienyl, furyl, furazanyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, triazinyl, and tetrazinyl.

The term 'heterocyclyl' refers to a 5- to 6-membered monocyclic ring which may be saturated or partially unsaturated, in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur. Examples of such monocyclic rings include aziridinyl, oxiranyl, pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dioxolanyl, dioxanyl, oxathiolanyl, oxathianyl, dithianyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, diazepanyl and azepanyl. The term 'amino' as used herein refers to an organonitrogen compound with the connectivity - N(R')(R"), where R' and R" are each independently a hydrogen or an optional substituent as defined below in relation to formula (I). The term 'C_x__y alkyl carbonyl' as used herein refers to an alkyl group wherein C_x__y alkyl is as defined herein and at least one methylene group (i.e. -CH₂-) is replaced with a carbonyl group (i.e. >C=0). Examples of Ci_₆ alkyl carbonyl groups include ethanoyl, propanoyl, butanoyl, pentanoyl, and hexanoyl. The term 'carbonyl' as used herein refers to a single carbonyl group of the formula: >C=0.

The term 'C_x__y alkoxy carbonyl' as used herein refers to an alkyl group wherein C_x__y alkyl is as defined herein and at least one methylene group (i.e. -CH₂-) is replaced with an ester group (e.g. -OC(O)-). Examples of Ci_₆ alkyl carbonyl groups include ethyl oxycarbonyl, propyl oxycarbonyl, butyl oxycarbonyl, pentyl oxycarbonyl, and hexyl oxycarbonyl. The term 'oxycarbonyl' as used herein refers to a single oxycarbonyl group of the formula: -OC(O)-.

The term 'C_x__y alkyl carbonyloxy' as used herein refers to an alkyl group wherein C_x__y alkyl is as defined herein and at least one methylene group (i.e. -CH₂-) is replaced with an ester group (e.g. -C0₂-). Examples of Ci_₆ alkyl carbonyloxy groups include ethanoate, propanoate, butanoate, pentanoate, and hexanoate. The term 'carbonyloxy' as used herein refers to a single carbonyloxy group of the formula: -C0₂-.

The term 'C_x__y alkyl carbamoyl' as used herein refers to an alkyl group wherein C_x__y alkyl is as defined herein and at least one methylene group (i.e. -CH₂-) is replaced with an amide group (e.g. -C(0)NR-, where R is a hydrogen atom, a 5- or 6-membered heterocyclyl group, a 5- or 6- membered heteroaryl group, a 3- to 6-membered cycloalkyl group, a Ci_₆ alkyl group, a Ci_₆ alkoxy group, or a C_6-14 aryl group, preferably a hydrogen atom). Examples of Ci_₆ alkyl carbamoyl groups include ethyl carbamoyl, propyl carbamoyl, butyl carbamoyl, pentyl carbamoyl, and hexyl carbamoyl. The term 'carbamoyl' as used herein refers to a single carbamoyl group of the formula: -C(0)NR-, where R is as defined above.

The term 'C_x__y alkyl carbamate' as used herein refers to an alkyl group wherein C_x__y alkyl is as defined herein and at least one methylene group (i.e. -CH₂-) is replaced with a carbamate group (e.g. -OC(0)NR-, where R is a hydrogen atom, a 5- or 6-membered heterocyclyl group, a 5- or 6-membered heteroaryl group, a 3- to 6-membered cycloalkyl group, a Ci_₆ alkyl group, a Ci_₆ alkoxy group, or a C_6-14 aryl group, preferably a hydrogen atom). Examples of Ci_₆ alkyl carbamate groups include ethyl carbamate, propyl carbamate, butyl carbamate, pentyl carbamate, and hexyl carbamate. The term 'carbamate' as used herein refers to a single carbamate group of the formula: -OC(0)NR-, where R is as defined above.

Hereto, the present invention is in particular captured by any one or any combination of one or more of the below aspects and embodiments and numbered statements 1 to 79.

1. A compound of the formula (I):

wherein

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxy carbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is a bond, -0-, -N(R₄ , or -S-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group; Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

A compound according to statement 1 , wherein

ring A is an optionally substituted 5- or 6-membered heterocyclyl, aryl, or heteroaryl group; ring B is an optionally substituted 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is -0-, -N(R₃)-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group;

Z is a bond, a Ci_₆ alkylene carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_₆ alkyl carbamoyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

A compound according to statement 1 or statement 2, wherein

ring A is an optionally substituted 6-membered heterocyclyl or aryl group.

A compound according to any one of statements 1 to 3, wherein

ring B is an optionally substituted 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S.

A compound according to any one of statements 1 to 4, of the formula (la):

wherein

Xi, X₂, and X₃ are each independently selected from CR₅R₅, CR₅, O, N, NR7, and S, and at least one is selected from O, N, NR7, and S, the possibility of CR₅R₅ or CR₅, and NR7 or N depending on the degree of saturation of ring B,

wherein R₅, 5, and R7 are each independently a hydrogen atom, an optionally substituted Ci_6 alkyl group, or an optionally substituted Ci_₆ alkyl carbonyloxy group.

6. A compound according to any one of statements 1 to 5, of the formula (lb):

wherein

Xi is selected from O, N, and S, and X₂ and X₃ are both N.

7. A compound according to any one of statements 1 to 6, wherein

V is -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group.

8. A compound according to any one of statements 1 to 7, wherein

W is a bond, a carbonyl group, an oxycarbonyl group, a carbonyloxy group, or a carbamoyl group.

9. A compound according to any one of statements 1 to 8, wherein

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group.

10. A compound according to any one of statements 1 to 9, wherein Y is a bond, -0-, or -S-.

A compound according to statement 1, wherein

ring A is an optionally substituted 6-membered heterocyclyl, aryl, or heteroaryl group; ring B is a 5-membered heteroaryl group containing 3 heteroatoms selected from O, N, and S;

Y is a -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C₆-i4 aryl group;

W is a bond;

Y is -S-;

Z is a Ci_6 alkyl carbamoyl group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group;

or a pharmaceutically acceptable salt thereof.

A compound according to statement 1, wherein

ring A is an optionally substituted 6-membered heterocyclyl, aryl, or heteroaryl group; ring B is a 5-membered heteroaryl group containing from 3 heteroatoms selected from O, N, and S;

W is a carbamoyl group;

Y is a bond;

Z is a bond;

or a pharmaceutically acceptable salt thereof. A compound of the formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group;

or a pharmaceutically acceptable salt thereof,

for use in therapy.

A compound of the formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group; Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group;

or a pharmaceutically acceptable salt thereof,

for use in the prophylaxis or treatment of bacterial infections.

15. A compound of formula (I) for use according to statement 14, in sequential or simultaneous combination with at least one anti-infective agent or anti- virulence agent.

16. A pharmaceutical composition comprising a compound of the formula (I):

wherein

W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxy carbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group; Y is a bond, -0-, -N(R4 , or -S-,

wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof, and

a pharmaceutically acceptable diluent, excipient or carrier.

A pharmaceutical composition according to statement 16, further comprising one or more antimicrobial agents.

A medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material, incorporating a compound of the formula (I):

wherein

V is a bond, -0-, -N(R₃)-, -CH₂-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

W is a bond, a C₁.3 alkylene group, a C₁.3 alkyl carbonyl group, a carbonyl group, a C₁.3 alkoxy carbonyl group, an oxy carbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is a bond, -0-, -N(R₄ , or -S-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

A medical device according to statement 18, wherein the device is for in vivo implantation. A medical device according to statement 18 or statement 19, wherein the device is a catheter, cannula, stent, shunt, or hypodermic needle.

A medical device, laboratory apparatus, food preparation surface-bearing device, or packaging material, according any of statements 18 to 20, wherein the compound according to formula (I) is disposed as a coating on a surface thereof which preferably, in use, comes into contact with a biological material or a subject to be treated with the device.

A method of preventing or treating bacterial infections, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group; Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_ 6 alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

A method of prevention or treatment according to statement 22, wherein the compound of formula (I) is administered prior to, concurrent with, or following administration of at least one antibiotic.

A method of preventing or inhibiting biofilm formation on a surface, the method comprising the treatment of the surface, or a device bearing the surface, with a compound according to formula (I):

wherein

Y is a bond, -0-, -N(R4 , or -S-, wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof.

A compound of the formula (I):

wherein

ring A is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered aryl group, an optionally substituted 5- or 6-membered heteroaryl group, or an optionally substituted 5- or 6-membered cycloalkyl group;

ring B is an optionally substituted 5-membered heterocyclyl group containing from 1 to 3 heteroatoms selected from O, N, and S; or is an optionally substituted 5- or 6-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S;

Y is a bond, -0-, -N(R4 , or -S-,

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-₆ alkenyl group, an optionally substituted C₂-₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

A compound according to any one of statements 1 to 15, 25, or as recited in any one of statements 16 to 24, wherein

V is a -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6- membered cycloalkenyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group. mpound of the formula (I):

wherein

ring A is an optionally substituted 6- or 5- membered aryl group, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, or an optionally substituted 5- or 6-membered cycloalkyl group;

ring B is an optionally substituted 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S; or is an optionally substituted 5-membered heterocyclyl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is a -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Y is -S-, a bond, -0-, or -N(R-i)-. - wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Z is a Ci_6 alkyl carbamoyl group, a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_6 alkoxy carbonyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group; Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6- membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

A compound of the formula (I):

wherein

V is a -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

Y is -S-, a bond, -0-, or -N(R-i)-. - wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_6 alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6- membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

provided that the following compounds are excluded

32

The compound according to any one of statements to 1 to 15, 25 to 28, or as recited in any one of statements 16 to 24; wherein

ring A is an optionally substituted 6 or 5- or membered aryl, heterocyclyl, or heteroaryl group;

ring B is an optionally substituted 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is -SO2-, -N(R₃)-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6- 14 aryl group;

Z is a Ci_6 alkyl carbamoyl group, a bond, a Ci_₆ alkylene carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof. The compound according to any one of statements to 1 to 15, 25 to 29, or as recited in any one of statements 16 to 24; wherein

ring A is an optionally substituted 6-membered aryl or heterocyclyl group.

The compound according to any one of statements to 1 to 15, 25 to 30, or as recited in any one of statements 16 to 24; wherein

The compound according to any one of statements to 1 to 15, 25 to 31 , or as recited in any one of statements 16 to 24; of the formula (la):

wherein

Xi, X₂, and X₃ are each independently selected from CR₅R₅, CR₅, O, N, NR₇, and S, and at least one is selected from O, N, NR₇, and S, the possibility of CR₅R₅ or CR₅, and NR₇ or N depending on the degree of saturation of ring B,

wherein R₅, 5, and R₇ are each independently a hydrogen atom, an optionally substituted Ci_6 alkyl group, or an optionally substituted Ci_₆ alkyl carbonyloxy group.

The compound according to any one of statements to 1 to 15, 25 to 32, or as recited in any one of stat ments 16 to 24; of the formula (lb):

wherein

Xi is selected from O, N, and S, and X₂ and X₃ are both N.

The compound according to any one of statements to 1 to 15, 25 to 33, or as recited in any one of statements 16 to 24; wherein

V is -SO2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

The compound according to any one of statements to 1 to 15, 25 to 34, or as recited in any one of statements 16 to 24; wherein

The compound according to any one of statements to 1 to 15, 25 to 35, or as recited in any one of statements 16 to 24; wherein

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group.

The compound according to any one of statements to 1 to 15, 25 to 36, or as recited in any one of statements 16 to 24, wherein

Y is -S-, a bond, -O- .

The compound according to any one of statements to 1 to 15, 25 to 37, or as recited in any one of statements 16 to 24, wherein

ring A is an optionally substituted 6-membered aryl, heterocyclyl, or heteroaryl group;

ring B is a 5-membered heteroaryl group containing 3 heteroatoms selected from O, N, and S;

Y is a -S0₂-, -N(R₃)S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group;

W is a bond;

Y is -S-;

Z is a Ci_6 alkyl carbamoyl group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof.

The compound according to any one of statements to 1 to 15, 25 to 38, or as recited in any one of statements 16 to 24, wherein

ring B is a 5-membered heteroaryl group containing from 3 heteroatoms selected from O, N, and S;

V is a -S0₂-,-N(R3)S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group;

W is a carbamoyl group;

Y is a bond;

Z is a bond;

or a pharmaceutically acceptable salt thereof.

A compound of the formula (I):

wherein ring A is an optionally substituted 6- or 5-membered aryl, heterocyclyl, heteroaryl, or cycloalkyl group;

ring B is an optionally substituted 5-membered heteroaryl heterocyclyl or group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted

C₆-i4 aryl group;

Y is -S-, a bond, -0-, or -N(R₄)-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted

C₆-i4 aryl group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6- membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

or use in therapy

rovided that the following compounds are excluded

A compound of the formula (I):

wherein

ring A is an optionally substituted 6- or 5-membered aryl, heterocyclyl, heteroaryl, or cycloalkyl group;

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

Y is -S-, a bond, -0-, or -N(R4 ,

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted C₆-i4 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6- membered cycloalkenyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof,

se in the prophylaxis or treatment of bacterial infections

provided that the following compounds are excluded

A compound of formula (I) for use according to statement 41, in sequential or simultaneous combination with at least one anti-infective agent or anti- virulence agent.

A pharmaceutical composition comprising a compound of the formula (I):

wherein

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

Y is -S-, a bond, -0-, or -N(R4 , wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6- membered cycloalkenyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof, and

a pharmaceutically acceptable diluent, excipient or carrier

rovided that the following compounds are excluded

A pharmaceutical composition according to statement 43, further comprising one or more antimicrobial agents.

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof.

46. A medical device according to statement 45, wherein the device is for in vivo implantation.

47. A medical device according to statement 45 or statement 46, wherein the device is a catheter, cannula, stent, shunt, or hypodermic needle.

48. A medical device, laboratory apparatus, food preparation surface-bearing device, or packaging material, according any one of statements 45 to 47, wherein the compound according to formula (I) is disposed as a coating on a surface thereof which preferably, in use, comes into contact with a biological material or a subject to be treated with the device.

49. A method of preventing or treating bacterial infections, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to formula (I):

wherein

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

W is a bond, a C₁.3 alkylene group, a C₁.3 alkyl carbonyl group, a carbonyl group, a Ci_3 alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is -S-, a bond, -0-, or-N(R4 ,

wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof rovided that the following compounds are excluded

A method of prevention or treatment according to statement 50, wherein the compound of formula (I) is administered prior to, concurrent with, or following administration of at least one antibiotic.

A method of preventing or inhibiting bio film formation on a surface, the method comprising the treatment of the surface, or a device bearing the surface, with a compound according to formula (I):

wherein

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group; W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is a bond, -0-, -N(R4 , or -S-,

wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-₆ alkenyl group, an optionally substituted C₂-₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

The compound according to any one of statements to 1 to 15, 25 to 42, or as recited in any one of statements 16 to 24, 43 to 51; wherein ring A is an optionally substituted 5- or 6- membered heterocyclyl, an optionally substituted 5- or 6-membered aryl, an optionally substituted 5- or 6-membered heteroaryl, or an optionally substituted 5- or 6-membered cycloalkyl group. In a preferable aspect, ring A is an optionally substituted 6-membered heterocyclyl or an optionally substituted 6-membered aryl group. Even more preferably, ring A is an optionally substituted 6-membered aryl group.

The compound according to any one of statements 1 to 15, 25 to 42, 52, or as recited in any one of statements 16 to 24, 43 to 51, wherein ring B is an optionally substituted 5- membered heterocyclyl or heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S. In a preferable aspect, ring B is a 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 53, or as recited in any one of statements 16 to 24, 43 to 51 , wherein V is a bond, -0-, -N(R₃)-, -CH₂-, - N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group. Preferably V is -N(R₃)-, -CH₂-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group. Preferably, V is -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is as defined above. Preferably, R₃ is a hydrogen atom, an optionally substituted 3- to 6-membered cycloalkyl group, or an optionally substituted Ci_₆ alkyl group.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 54, or as recited in any one of statements 16 to 24, 43 to 51 , wherein W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group. Preferably, W is a bond, a carbonyl group, an oxycarbonyl group, a carbonyloxy group, or a carbamoyl group.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 55, or as recited in any one of statements 16 to 24, 43 to 51 , wherein Y is a bond, -0-, -Ν(Ρ )-, or -S-, wherein R₄ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group. Preferably, R₄ is a hydrogen atom, an optionally substituted 3- to 6-membered cycloalkyl group, or an optionally substituted Ci_₆ alkyl group. Even more preferably, Y is a bond, -O- , or -S-. Most preferably, Y is a bond or -S-. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 56, or as recited in any one of statements 16 to 24, 43 to 51 , wherein Z is a bond, a Ci_₆ alkylene group, a Ci_ 6 alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_₆ alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group. Preferably, Z is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a Ci_₃ alkoxy carbonyl group, a Ci_₃ alkyl carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a Ci_₃ alkyl carbamate group. Even more preferably, Z is a bond, a Ci_₃ alkyl carbonyl group, a Ci_₃ alkyl carbonyloxy group, or a Ci_₃ alkyl carbamoyl group.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 57, or as recited in any one of statements 16 to 24, 43 to 51 , wherein Ri is an optionally substituted 5- or 6- membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group. In a preferred aspect, Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 58, or as recited in any one of statements 16 to 24, 43 to 51 , wherein said compound is a compound of formula (la):

wherein

wherein R5, R^, and R₇ are each independently a hydrogen atom, an optionally substituted Ci_₆ alkyl group, or an optionally substituted Ci_₆ alkyl carbonyloxy group, and each of the other groups is as defined above in relation to formula (I). The compound according to statement 51, wherein if Xi, X₂, and X₃ form saturated bonds with neighbouring atoms, then they are each independently selected from CR₅R₀, O, NR7, and S.

The compound according to statement 51, wherein if X_{l s} X₂, and X₃ form unsaturated bonds with neighbouring atoms, then they are each independently selected from CR₅ and N. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 61, or as recited in any one of statements 16 to 24, 43 to 51, wherein said compound is a compound of formula (l

wherein

Xi is selected from O, N, and S, and X₂ and X₃ are both N,

and each of the other groups is as defined above in relation to formula (I). For example, preferred heteroaryl rings include triazolyl, oxadiazolyl, and thiadiazolyl (preferably oxadiazolyl).

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 62, or as recited in any one of statements 16 to 24, 43 to 51, wherein each of the optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted cycloalkenyl, optionally substituted hydroxy, and optionally substituted amino groups, may be substituted by:

(1) one or two groups selected from -J-aryl, -J-heteroaryl, -J- heterocyclyl and -J-C₃_₆ cycloalkyl, wherein J represents a bond or Ci_₆ alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and thiazolidinyl, and said C₃_₆ cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and/or

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl), (b) Ci_6 alkenyl (preferably propenyl),

(c) Ci_6 alkynyl (preferably ethynyl or propynyl),

(d) halogen (preferably CI or Br),

(e) haloCi_6 alkyl (preferably trifluoromethyl),

(f) cyano,

(g) amino, optionally mono-or di-substituted with Ci_₆ alkyl, tert-butoxycarbonyl or benzyl,

(h) Ci_6 alkoxy (preferably methoxy),

(i) Ci_6 alkyl carbonyl, including ketones and derivatives thereof such as ketals and hemiketals, and aldehydes (e.g. formyl) and derivatives thereof such as acetals and hemiacetals (preferably acetyl),

(j) Ci_6 alkoxy carbonyl,

(k) Ci_6 alkyl carbonyloxy, including carboxyl,

(1) Ci_6 alkyl carbamoyl, including carbamoyl,

(m) Ci_₆ thioether, or

(n) nitro.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 63, or as recited in any one of statements 16 to 24, 43 to 51, wherein

V is -N(R₃)-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆_ 14 aryl group;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group; R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

and each of the other groups is as defined in any one of statements 1 to 15, 25 to 42, 52 to

63, or as recited in any one of statements 16 to 24, 43 to 51 , or a pharmaceutically acceptable salt thereof.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 64, or as recited in any one of statements 16 to 24, 43 to 51 , wherein

V is a -N(R3)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group;

W is a bond;

Y is -S-;

Z is a Ci_6 alkyl carbamoyl group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group; or a pharmaceutically acceptable salt thereof.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 65, or as recited in any one of statements 16 to 24, 43 to 51 , wherein

ring A is an optionally substituted 6-membered heterocyclyl, aryl, or heteroaryl group; ring B is a 5-membered heteroaryl group containing from 3 heteroatoms selected from O, N, and S; V is a -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group;

W is a carbamoyl group;

Y is a bond;

Z is a bond;

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 66, or as recited in any one of statements 16 to 24, 43 to 51, wherein

each of the optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted cycloalkenyl, optionally substituted hydroxy, and optionally substituted amino groups, may be substituted by:

(1) a group selected from -J-aryl, -J- heteroaryl, -J-heterocyclyl and -J-C₃_₆ cycloalkyl, wherein J represents a bond or Ci_₃ alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, and thiazolidinyl, and said C₃_6 cycloalkyl is selected from cyclopropyl, cyclopentyl and cyclohexyl; or

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci_₃ alkenyl (preferably propenyl),

(c) halogen (preferably CI or Br),

(e) haloCi_6 alkyl (preferably trifluoromethyl), (d) cyano,

(e) amino, optionally mono-or di-substituted with Ci_₃ alkyl, tert-butoxycarbonyl or benzyl,

(f) Ci_₃ alkoxy (preferably methoxy),

(g) Ci_6 alkyl carbonyl (preferably acetyl),

(h) Ci_₃ alkoxy carbonyl,

(i) Ci_₃ alkyl carbonyloxy, including carboxyl,

(j) Ci_₃ alkyl carbamoyl, including carbamoyl.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 67, or as recited in any one of statements 16 to 24, 43 to 51 , wherein

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group; and

wherein each of the optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted cycloalkenyl, optionally substituted hydroxy, and optionally substituted amino groups, may be substituted by:

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci_6 alkenyl (preferably propenyl),

(c) Ci_6 alkynyl (preferably ethynyl or propynyl),

(d) halogen (preferably CI or Br), (e) haloCi_6 alkyl (preferably trifluoromethyl),

(f) cyano,

(h) Ci_6 alkoxy (preferably methoxy),

(j) Ci_6 alkoxy carbonyl,

(k) Ci_6 alkyl carbonyloxy, including carboxyl,

(1) Ci_6 alkyl carbamoyl, including carbamoyl,

(m) Ci_₆ thioether, or

(n) nitro.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 68, or as recited in any one of statements 16 to 24, 43 to 51, wherein

R₂ is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group; preferably R² is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6- membered cycloalkenyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group; preferably R² is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group; and wherein each of the optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted cycloalkenyl, optionally substituted hydroxy, and optionally substituted amino groups, may be substituted by:

(1) one or two groups selected from -J-aryl, -J-heteroaryl, -J- heterocyclyl and -J-C3-6 cycloalkyl, wherein J represents a bond or Ci_₆ alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and thiazolidinyl, and said C3-6 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and/or

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci_6 alkenyl (preferably propenyl),

(c) Ci_6 alkynyl (preferably ethynyl or propynyl),

(d) halogen (preferably CI or Br),

(e) haloCi_6 alkyl (preferably trifluoromethyl),

(f) cyano,

(h) Ci_6 alkoxy (preferably methoxy),

(j) Ci_6 alkoxy carbonyl,

(k) Ci_6 alkyl carbonyloxy, including carboxyl,

(1) Ci_6 alkyl carbamoyl, including carbamoyl,

(m) Ci_₆ thioether, or

(n) nitro.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 69, or as recited in any one of statements 16 to 24, 43 to 51, wherein heteroaryl refers to a 5- to 6-membered monocyclic aromatic ring in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur.

71. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 70, or as recited in any one of statements 16 to 24, 43 to 51, wherein heteroaryl is selected from the group comprising thienyl, furyl, furazanyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, triazinyl, and tetrazinyl.

72. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 71, or as recited in any one of statements 16 to 24, 43 to 51, wherein heterocyclyl refers to a 5- to 6- membered monocyclic ring which may be saturated or partially unsaturated, in which the monocyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur.

73. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 72, or as recited in any one of statements 16 to 24, 43 to 51, wherein said heterocyclyl is selected from the group comprising aziridinyl, oxiranyl, pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dioxolanyl, dioxanyl, oxathiolanyl, oxathianyl, dithianyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, diazepanyl and azepanyl.

74. A compound selected from the group consisting of N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin-

1- ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, ethyl 2-((5-(4-((4- methylpiperidin- 1 -yl)sulfonyl)phenyl)- 1 ,3 ,4-oxadiazol-2-yl)thio)acetate, 1 -(4-(4- methoxyphenyl)piperazin- 1 -yl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- 1 ,3,4-oxadiazol-

2- yl)thio)ethan-l-one, N-(4-chlorophenyl)-2-((5-(4-(N-cyclopentylsulfamoyl)phenyl)-l,3,4- oxadiazol-2-yl)thio)acetamide, N-(4-chlorophenyl)-2-((5-(4-(N- cyclohexylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N-(4-chlorophenyl)-2- [[5-[4-(4-phenylcyclohexyl)sulfonylphenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5- [4-(4-benzylcyclohexyl)sulfonylphenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]-N-(4- chlorophenyl)acetamide, N-(3-chlorophenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide, N-(3,4-dichlorophenyl)-2-[[5-(4-pyrrolidin-l- ylsulfonylphenyl)-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5-(4-pyrrolidin-l- ylsulfonylphenyl)-l,3,4-oxadiazol-2-yl]sulfanyl]-N-[4-(trichloromethyl)phenyl]acetamide, N-(4-methoxyphenyl)-2-[[5-(4-pyrrolidin-l-ylsulfonylphenyl)-l,3,4-oxadiazol-2- yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-(4-pyrrolidin-l-ylsulfonylphenyl)-l,3,4- thiadiazol-2-yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-(4-pyrrolidin- 1 - ylsulfonylphenyl)-4H- 1 ,2,4-triazol-3-yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-[4- (cyclopentylsulfonylamino)phenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5-[4- (benzenesulfonamido)phenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]-N-(4-chlorophenyl)acetamide, N-(4-chlorophenyl)-2-[[5-[4-(cyclohexylsulfonylamino)phenyl]-l,3,4-oxadiazol-2- yl]sulfanyl]acetamide, N-[5-(4-tert-butylphenyl)-l,3,4-thiadiazol-2-yl]-l-(p- tolylsulfonyl)piperidine-4-carboxamide, N-[5-(l,3-benzodioxol-5-yl)-l,3,4-thiadiazol-2-yl]-

1- (p-tolylsulfonyl)piperidine-4-carboxamide, N-[5-(l,3-benzodioxol-5-yl)-l,3,4-thiadiazol-

2- yl]- 1 -(4-chlorophenyl)sulfonyl-piperidine-4-carboxamide, 2-((5-(4-(pyrrolidin- 1 - ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(m-tolyl)acetamide, 2-((5-(4-(pyrrolidin-l- ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(p-tolyl)acetamide, N-(3-methoxyphenyl)- 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N- cyclohexyl-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide

1 -(4-phenylpiperidin- 1 -yl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- 1 ,3,4-oxadiazol-2- yl)thio)ethan- 1 -one, N-(4-ethylphenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- 1,3,4- oxadiazol-2-yl)thio)acetamide, N-(4-methoxyphenyl)-2-((5-(4-(pyrrolidin- 1 - ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, 2-((5-(4-(pyrrolidin-l- ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(4-(trifluoromethyl)phenyl)acetamide, N- (4-benzylphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide, N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)-4H- 1 ,2,4- triazol-3-yl)thio)acetamide, 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)-N-(thiazol-2-ylmethyl)acetamide, N-propyl-2-((5-(4-(N-(thiophen-2- ylmethyl)sulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N-(4-chlorophenyl)-2- ((5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N-(4- chlorophenyl)-2-((5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetami 2-((5-(4-((4-(tert-Butyl)piperidin-l-yl)sulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(4- chlorophenyl)acetamide, N-(4-chlorophenyl)-2-((5-(4-(N,N-dibutylsulfamoyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide. Preferably, the compound is selected from the group of N-(4-chlorophenyl)-2-((5-(4-(N-cyclopentylsulfamoyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide, N-(4-chlorophenyl)-2-((5-(4-(N-cyclohexylsulfamoyl)phenyl)-l,3,4- oxadiazol-2-yl)thio)acetamide, N-(4-chlorophenyl)-2-[[5-[4-(4- phenylcyclohexyl)sulfonylphenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5-[4-(4- benzylcyclohexyl)sulfonylphenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]-N-(4- chlorophenyl)acetamide, N-(3-chlorophenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide, N-(3,4-dichlorophenyl)-2-[[5-(4-pyrrolidin-l- ylsulfonylphenyl)-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5-(4-pyrrolidin-l- ylsulfonylphenyl)-l,3,4-oxadiazol-2-yl]sulfanyl]-N-[4-(trichloromethyl)phenyl]acetamide, N-(4-methoxyphenyl)-2-[[5-(4-pyrrolidin-l-ylsulfonylphenyl)-l,3,4-oxadiazol-2- yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-(4-pyrrolidin-l-ylsulfonylphenyl)-l,3,4- thiadiazol-2-yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-(4-pyrrolidin- 1 - ylsulfonylphenyl)-4H- 1 ,2,4-triazol-3-yl]sulfanyl]acetamide, N-(4-chlorophenyl)-2-[[5-[4- (cyclopentylsulfonylamino)phenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]acetamide, 2-[[5-[4- (benzenesulfonamido)phenyl]-l,3,4-oxadiazol-2-yl]sulfanyl]-N-(4-chlorophenyl)acetamide, N-(4-chlorophenyl)-2-[[5-[4-(cyclohexylsulfonylamino)phenyl]-l,3,4-oxadiazol-2- yl]sulfanyl]acetamide, 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)- N-(m-tolyl)acetamide, 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)- N-(p-tolyl)acetamide, N-(3-methoxyphenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide, N-cyclohexyl-2-((5-(4-(pyrrolidin-l- ylsulfonyl)phenyl)- 1 ,3,4-oxadiazol-2-yl)thio)acetamide, 1 -(4-phenylpiperidin- 1 -yl)-2-((5- (4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- 1 ,3 ,4-oxadiazol-2-yl)thio)ethan- 1 -one, N-(4- ethylphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetami N-(4-methoxyphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide, 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N- (4-(trifluoromethyl)phenyl)acetamide, N-(4-benzylphenyl)-2-((5-(4-(pyrrolidin- 1 - ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N-(4-chlorophenyl)-2-((5-(4- (pyrrolidin- 1 -ylsulfonyl)phenyl)-4H- 1 ,2,4-triazol-3-yl)thio)acetamide, N-(4-chlorophenyl)- 2-((5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide, N-(4- chlorophenyl)-2-((5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetami 2-((5-(4-((4-(tert-Butyl)piperidin-l-yl)sulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(^ chlorophenyl)acetamide, N-(4-chlorophenyl)-2-((5-(4-(N,N-dibutylsulfamoyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide.

75. The compound according to statement 74, for use as a medicament.

76. The compound according to any one of statements 1 to 15, 25 to 42, 52 to 75, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49, 50, for use in the prophylaxis or treatment of a bacterial infection caused by pathogenic bacteria.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 76, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49,

50, for use in the prophylaxis or treatment of a bacterial infection caused by pathogenic bacteria of the family of Enterobacteriaceae.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 77, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49,

50, for use in the prophylaxis or treatment of a bacterial infection caused by one or more pathogenic bacteria of the family of Enterobacteriaceae selected from the genera Escherichia, Enterobacter Klebsiella, Salmonella, Shigella, and Yersinia

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 78, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49, 50, for use in the prophylaxis or treatment of a bacterial infection caused by Escherichia coli.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 79, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49, 50, wherein said bacterial infection is selected from a urinary tract infection, including cystitis (infection of the bladder), pyelonephritis (infection of the kidney), and bacteriuria (infection of the urine), as well as infectious complications thereof, such as those resulting in acute renal failure.

The compound according to any one of statements 1 to 15, 25 to 42, 52 to 80, or as recited in any one of statements 16 to 24, 43 to 51, or a pharmaceutical composition according to any one of statements 16, 17, 43, 44, or a method according to any of statements 22, 23, 49, 50, wherein said bacterial infection is selected from an infection of the gastrointestinal tract, in particular the gastrointestinal mucosa including inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, as well as infectious complications thereof, such as colorectal cancer and irritable bowel syndrome. Each symbol in formula (I) is described in detail in the following.

Ring A is an optionally substituted 5- or 6-membered heterocyclyl, aryl, heteroaryl, or cycloalkyl group. In a preferable aspect, ring A is an optionally substituted 6-membered heterocyclyl or aryl group. Even more preferably, ring A is an optionally substituted 6- membered aryl group.

Ring B is an optionally substituted 5 -membered heterocyclyl or heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S. In a preferable aspect, ring B is a 5- membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S. It is believed that when ring B is a 5 -membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S, binding of the compound in the P5 pocket of the CU pili subunit is further enhanced. V is a bond, -0-, -N(R₃)-, -CH₂-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group. Preferably, V is -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is as defined above. Preferably, R₃ is a hydrogen atom, an optionally substituted 3- to 6-membered cycloalkyl group, or an optionally substituted Ci_₆ alkyl group.

W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group. Preferably, W is a bond, a carbonyl group, an oxycarbonyl group, a carbonyloxy group, or a carbamoyl group.

Y is a bond, -0-, -Ν(Ρ )-, or -S-, wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group. Preferably, R4 is a hydrogen atom, an optionally substituted 3- to 6-membered cycloalkyl group, or an optionally substituted Ci_₆ alkyl group. Even more preferably, Y is a bond, -0-, or -S-. Most preferably, Y is a bond or -S-. Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group. Preferably, Z is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a Ci_₃ alkoxy carbonyl group, a Ci_₃ alkyl carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a Ci_₃ alkyl carbamate group. Even more preferably, Z is a bond, a Ci_₃ alkyl carbonyl group, a Ci_₃ alkyl carbonyloxy group, or a Ci_₃ alkyl carbamoyl group.

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-₆ alkenyl group, an optionally substituted C₂-₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group. In a preferred aspect, Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group.

In a preferred aspect of the invention, there is provided a compound according to the formula (la):

wherein

Xi, X₂, and X₃ are each independently selected from CR₅R₅, CR₅, O, N, NR₇, and S, and at least one is selected from O, N, NR₇, and S, the possibility of CR₅R₅ or CR₅, and NR₇ or N depending on the degree of saturation of ring B, wherein R₅, 5, and R₇ are each independently a hydrogen atom, an optionally substituted Ci_₆ alkyl group, or an optionally substituted Ci_₆ alkyl carbonyloxy group,

and each of the other groups is as defined above in relation to formula (I). In this embodiment, if Xi, X₂, and X₃ form saturated bonds with neighbouring atoms, they may be selected from CR₅R₀, O, NR₇, and S. Alternatively, if Xi, X₂, and X₃ form unsaturated bonds with neighbouring atoms, they may be selected from CR₅ and N.

In a particularly preferred embodiment, there is provided a compound according to the formula (lb):

wherein

Xi is selected from O, N, and S, and X₂ and X₃ are both N,

(1) one or two groups selected from -J-aryl, -J-heteroaryl, -J-heterocyclyl and -J-C₃_₆ cycloalkyl, wherein J represents a bond or Ci_₆ alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and thiazolidinyl, and said C₃_6 cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and/or

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci_6 alkenyl (preferably propenyl), (c) Ci_6 alkynyl (preferably ethynyl or propynyl),

(d) halogen (preferably CI or Br),

(e) haloCi_6 alkyl (preferably trifluoromethyl),

(f) cyano,

(h) Ci_6 alkoxy (preferably methoxy),

(j) Ci_6 alkoxy carbonyl,

(k) Ci_6 alkyl carbonyloxy, including carboxyl,

(1) Ci_6 alkyl carbamoyl, including carbamoyl,

(m) Ci_₆ thioether, or

(n) nitro.

The term 'halogen' as used herein refers to a fluorine, chlorine, bromine or iodine atom, and any radioactive isotope thereof, including fluorine-18, iodine-123, iodine-124, iodine-125, iodine- 131, and astatine-211, unless otherwise specified.

In another aspect of the invention, there is provided a compound of the formula (I), wherein ring A is an optionally substituted 5- or 6-membered heterocyclyl, aryl, or heteroaryl group; ring B is an optionally substituted 5-membered heteroaryl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is -0-, -N(R₃)-, -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-,

Z is a bond, a Ci_₆ alkylene carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_₆ alkyl carbamoyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group; Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_6 alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

and each of the other groups is as defined above in relation to formula (I),

or a pharmaceutically acceptable salt thereof.

In another particularly preferred aspect of the invention, there is provided a compound according to formula (I), wherein

ring A is an optionally substituted 6-membered heterocyclyl, aryl, or heteroaryl group;

V is a -N(R3)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group;

W is a bond;

Y is -S-;

Z is a Ci_6 alkyl carbamoyl group;

Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof. In another particularly preferred aspect of the invention, there is provided a compound according to formula (I), wherein

V is a -N(R₃)S0₂-, -S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C_6-14 aryl group; W is a carbamoyl group;

Y is a bond;

Z is a bond;

or a pharmaceutically acceptable salt thereof.

Preferably, each of the optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted cycloalkenyl, optionally substituted hydroxy, and optionally substituted amino groups, may be substituted by:

(1) a group selected from -J-aryl, -J- heteroaryl, -J-heterocyclyl and -J-C3-6 cycloalkyl, wherein J represents a bond or Ci_₃ alkylene, and said aryl is selected from phenyl, said heteroaryl is selected from triazolyl, thiazolyl, thienyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, and pyridyl, said heterocyclyl is selected from pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, and thiazolidinyl, and said C3-6 cycloalkyl is selected from cyclopropyl, cyclopentyl and cyclohexyl; or

(2) one to three substituents selected from

(a) Ci_6 alkyl (preferably methyl, ethyl or isopropyl),

(b) Ci_3 alkenyl (preferably propenyl),

(c) halogen (preferably CI or Br),

(e) haloCi_6 alkyl (preferably trifluoromethyl),

(d) cyano,

(f) Ci_3 alkoxy (preferably methoxy),

(g) Ci_6 alkyl carbonyl (preferably acetyl),

(h) Ci_3 alkoxy carbonyl,

(i) Ci_3 alkyl carbonyloxy, including carboxyl,

(j) Ci_3 alkyl carbamoyl, including carbamoyl. Given the evident biological activity of the claimed compounds, in another aspect of the invention, there is provided a compound of the formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

or a pharmaceutically acceptable salt thereof,

for use in therapy. Any of the preferred structural variants mentioned above in relation to formula (I) also represent preferred aspects of this embodiment as therapeutic agents.

In particular, the compounds of the present invention are believed to function by inhibiting the pilus subunit polymerization step during type 1 pilus assembly, thereby preventing the formation of type 1 pili on the bacterial surface. In addition, they have been found to efface pre-assembled pili from the bacterial cell surface. As such, they are potentially effective for the prophylaxis or treatment of medical conditions associated with bacterial infections.

Accordingly, there i

wherein

Y is a bond, -0-, -N(R4 , or -S-, wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂-6 alkenyl group, an optionally substituted C₂-6 alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a

Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group;

or a pharmaceutically acceptable salt thereof,

for use in the prophylaxis or treatment of bacterial infections.

In particular, the compounds of the present invention are for use in the prophylaxis or treatment of a bacterial infection caused by pathogenic bacteria, preferably pathogenic bacteria of the family of Enterobacteriaceae, including the genera Escherichia, Enterobacter Klebsiella, Salmonella, Shigella, and Yersinia. More preferably, the compounds of the present invention are for use in the prophylaxis or treatment of uropathogenic bacteria, such as Escherichia coli. The bacterial infection may be hospital acquired or community acquired.

More specifically, a bacterial infection may be a urinary tract infection, including cystitis (infection of the bladder), pyelonephritis (infection of the kidney), and bacteriuria (infection of the urine), as well as infectious complications thereof, such as those resulting in acute renal failure. In addition, a bacterial infection may be an infection of the gastrointestinal tract, in particular the gastrointestinal mucosa. Infections of the gastrointestinal tract include, but are by no means not limited to, inflammatory bowel disease (IBD), such as Crohn's disease or ulcerative colitis, as well as infectious complications thereof, such as colorectal cancer and irritable bowel syndrome. Any of the structural variants mentioned above in relation to formula (I) also represent preferred aspects of this embodiment as agents for use in the prophylaxis or treatment of the same conditions.

The compound of formula (I) may be used as the sole therapeutic agent, or may be employed in conjunction with at least one further pharmacologically active agent, such as anti- infectives (e.g. disinfectants, antiseptics, antibacterials, antifungals, and antivirals) or anti-virulence agents. For example, since the present compounds are effective in the treatment of conditions associated with bacterial infections, they exhibit a particular synergy when used in combination with one or more antimicrobial agents, including antibiotics and antibacterials. Such antimicrobial agents include, but are by no means limited to, amikacin, gentamicin, tobramycin, amoxicillin, amoxicillin/clavulanate, amphotericin B, ampicillin, ampicillin/sulbactam, atovaquone, azithromycin, cefazolin, cefepime, cefotaxime, cefotetan, cefpodoxime, ceftazidime, ceftizoxime, ceftriaxone, cefuroxime, cephalexin, chloramphenicol, clotrimazole, ciprofloxacin, clarithromycin, clindamycin, cicloxacillin, coxycycline, echincandins, erythromycin (including estolate, ethylsuccinate, gluceptate, lactobionate, and stearate), fluconazole, foscarnet, imipenem/cilastatin (Primaxin), isoniazid, itraconazole, ketoconazole, metronidazole, nafcillin, nitrofurantoin, nystatin, penicillin (including G benzathine, G potassium, G procaine, V potassium), pentamidine, piperacillin/tazobactam, rifampin, ticarcillin/clavulanate, trimethoprim, trimethoprim sulfate, valacyclovir, vancomycin, aztreonam, levofloxacin, meropenem, tobramycin, cephaiothin, mezlocillin, nalidixic acid, netilmicin, minocycline, ofloxacin, norfloxacin, sulfamethoxazole, tetracycline, neomycin, streptomycin, ticarcillin, carbenicillin, cloxacillin, cefoxitin, ceforanide, teicoplanin, ristocetin, viomycin, capreomycin, bacitracin, gramicidin, gramicidin S, tyrocidine, tachyplesin, kanamycin, methicillin, oxacillin, azocillin, bacampicillin, carbenicillin indanyl, cephapirin, cefaxolin, cephradine, cefradoxil, cefamandole, cefaclor, cefuromime axetil, cefonicid, cefoperazone, demeclocytetracycline, methacycline, oxytetracycline, spectinomycin, ethambutol, aminosalicylic acid, pyrazinamide, ethionamide, cycloserine, dapsone, sulfoxone sodium, clofazimine, sulfanilamide, sulfacetamide, sulfadiazine, sulfixoxazole, cinoxacin, methenamine, phenazopyridine, and various human or animal antibacterial peptides such as defensins, magainins, cathelidicins, or histatins. Other suitable pharmacologically active agents which can be used in combination with the compound of formula (I) include one or more other anti- virulence drugs, such as pilicides and D-mannosides (see Background section), one or more anti-viral drugs, such as famciclovir and ganciclovir, one or more anti-inflammatory drugs, such as steroid anti-inflammatory drugs (e.g. glucocorticoids) and non-steroid anti-inflammatory drugs (for example, aspirin, ibuprofen, naproxen, and mesalazine), one or more immunosuppressants (for example, prednisone, TNF inhibition, azathioprine, methotrexate, 6-mercaptopurine), or one or more chemotherapeutic drugs (for example, fluorouracil, capecitabine, UFT (tegafur-uracil), leucovorin, irinotecan, oxaliplatin, and paclitaxel), amongst others.

The present invention also relates to a pharmaceutical composition comprising a compound according to formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, excipient or carrier. The pharmaceutical composition may further comprise one or more other pharmacologically active agents (as above), such as one or more antimicrobial agents. In particular, the composition may contain one or more antibiotics (as above).

Pharmaceutically acceptable diluents, excipients and carriers that may be used in the pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.

One skilled in the art would appreciate that a pharmaceutical composition can be administered to a subject by various routes including, for example, oral administration; intramuscular administration; intravenous administration; anal administration; vaginal administration; parenteral administration; nasal administration; intraperitoneal administration; subcutaneous administration; (intra)urethral administration and topical administration. One skilled in the art would select an effective dose and administration regimen taking into consideration factors such as the patient's weight and general health, and the particular condition being treated, etc.

The pharmaceutical compositions of this invention may be administered orally, parenterally, topically, rectally, buccally, urethrally or via an implanted reservoir. Preferably, the pharmaceutical compositions are administered orally, rectally, urethrally or by injection. The pharmaceutical compositions may contain any conventional non-toxic pharmaceutically- acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intravaginal, intraurethral, intra- articular, intrasynovial, intrasternal, intrathecal, intraocular, intralesional and intracranial injection or infusion techniques. Preferably, the route of administration of the composition is oral or rectal administration. The pharmaceutical composition could be in the form of a liquid, gel, lotion, tablet, capsule, or ointment, etc. In a highly preferred aspect, the following compounds:

or pharmaceutically acceptable salts thereof,

are for use in the prophylaxis or treatment of bacterial infections, either alone or in combination with one or more other pharmacologically active agents, as mentioned above.

In another aspect of the invention, a compound of formula (I) may be incorporated in a medical device, or an article on which bacterial colonisation may occur, in order to reduce or prevent the colonisation of bacterial pathogens on the device or article before or whilst it is being used. In particular, the present compounds inhibit the formation of biofilms by either preventing the formation of adhesive pili or removing adhesive pili, the means by which Gram-negative bacteria adhere to biological or non-biological surfaces and cause infection.

The term "biofilm" is known in the art. Typically, a biofilm is an aggregate of microorganisms in which cells adhere to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix generally composed of extracellular DNA, proteins, and polysaccharides in various configurations. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae. However, monospecies biofilms occur as well. Microorganisms living in a biofilm usually have significantly different properties from free-floating (planktonic) microorganisms of the same species, as a result of the dense and protected environment of the film. For example, increased resistance to detergents and antibiotics is often observed, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. Accordingly, the present invention also concerns a medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material, incorporating a compound of the formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

or a pharmaceutically acceptable salt thereof. Any of the structural variants mentioned above in relation to formula (I) also represent preferred aspects of this embodiment, which may be incorporated in such articles to prevent bacterial spread.

Where the article incorporating a compound of formula (I) is a medical device, the device is preferably for in vivo implantation. In this manner, the claimed compounds mitigate the likelihood of bacterial infections being introduced by, or resulting from, medical procedures involving medical devices. Such medical devices include, for example, catheters, cannulas, stents, shunts, or hypodermic needles. In particular, the present compounds show distinct utility when applied to or incorporated in a catheter, since they can efficiently reduce the occurrence of UTIs (especially hospital-associated UTIs).

The compound of formula (I) may be incorporated in the medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material as a component of the material making up the body of the article, or may be disposed as a coating or outer layer thereon. Incorporation may include absorption of the compound into the matrix of the medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material, or covalent chemical attachment of the compound thereto. In a preferred aspect, the compound according to formula (I) is disposed as a coating on a surface of the medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material, which preferably, in use, comes into contact with a biological material or a subject to be treated with the device.

In a further aspect of the invention, there is provided a method of preventing or treating bacterial infections, cystitis, inflammatory bowel diseases, or irritable bowel syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to formula (I):

wherein ring A is an optionally substituted 5- or 6-membered heterocyclyl, aryl, heteroaryl, or cycloalkyl group;

C₆-i4 aryl group;

Y is a bond, -0-, -N(R₄ , or -S-,

C₆-i4 aryl group;

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group; Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof. Any of the structural variants mentioned above in relation to formula (I) also represent preferred aspects of this embodiment, which may be employed in this method. In particular, a subject may be a human or an animal patient. An animal may be a horse, cow, dog, cat, sheep, rat, mouse, pig, or bird, etc. Preferably, a subject is a human patient.

In addition, this method of prevention or treatment is for use in bacterial infections, as mentioned above. Thus, the compound of formula (I) may be used as the sole therapeutic agent, or may be employed in conjunction with at least one further pharmacologically active agent (as above). For example, the present compounds exhibit a particular synergy when used in combination with one or more antibiotics, and may be administered prior to, concurrently with, or following administration of at least one antibiotic. Such antibiotics include, but are by no means limited to, those listed above.

In a further aspect of the invention, there is provided a method of preventing or inhibiting biofilm formation on a surface, the method comprising the treatment of the surface, or a device bearing the surface, with a compound according to formula (I):

wherein

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkenyl group, an optionally substituted Ci_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group; Y is a bond, -0-, -N(R4 , or -S-,

wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkenyl group, an optionally substituted Ci_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

Z is a bond, a Ci_₆ alkylene group, a Ci_₆ alkyl carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_6 alkyl carbonyloxy group, a Ci_₆ alkyl carbamoyl group, or a Ci_₆ alkyl carbamate group; Ri and R₂ are each independently an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkenyl group, an optionally substituted Ci_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

Any of the structural variants mentioned above in relation to formula (I) also represent preferred aspects of this embodiment, which may be employed in this method. The surface may be treated, for example, by immersion in a solution comprising the compound of formula (I), or by spraying a solution comprising the compound of formula (I) thereon. In addition or alternatively, the compound of formula (I) may be covalently attached to the surface, either directly or via a suitable linker moiety.

In a related aspect, the invention provides a method of functionalising a surface, or a device bearing the surface, the method comprising covalently attaching the compound of formula (I) to the surface, directly or via a suitable linker moiety. Certain embodiments of the invention will now be described in more detail by way of example only, and with reference to the following figures. Brief Description of the Figures

Figure 1

Identification of pilus polymerisation inhibitors: (a) structure and schematic representation (b) of the FimH pilin domain (residues 158-279; PDB: 1ZE3 and shown as molecular surface) in complex with the FimC Fl-Gl strands (left side) or FimG Nte peptide (right side; taken from 3JWN). The pocket is labeled and the binding residue (Vain) is shown as sticks. PI to P5 pockets are shown as white circles and the interacting residues are labeled (c) SDS-PAGE showing FimC:FimH DSE with FimGNte after a 24 hour incubation (d) structure of HP5AL1 (compound 1), N-(4-Chloro-phenyl)-2-{5-[4-(pyrrolidine-l-sulfonyl)-phenyl]-[l,3,4]oxadiazol- 2-yl sulfanyl}-acetamide. (e) Dose response curve for in vitro FimC:FimH - FimGNte DSE in presence of compound 1 (0.001 - 2 mM). DSE progression is presented as adjusted volume of pixels of the DSE product (FimH: FimGNte) normalised over the adjusted volume of pixels of the total protein (FimC + FimH + FimH:FimGNte) and shown as a function of compound 1 concentration.

Figure 2

In vivo pilus biogenesis inhibition by compound 1: (a) Inhibition of UTI89 type 1 pili dependent-bio film formation in the presence of compound 1 and compound 1 * (commercial and synthesised N-(4-Chloro-phenyl)-2- {5-[4-(pyrrolidine- 1 -sulfonyl)-phenyl]- [l,3,4]oxadiazol-2-yl sulfanyl}-acetamide, respectively) at varying concentrations, plotted as percent biofilm formation relative to non-treated UTI89. Data are presented as sample mean ± s.e.m. (standard error of mean), n = 5 independent experiments with 3 replicates each. UTI89A zm was deficient in biofilm formation under the same static growth condition (Figure 6). (b) The abundance of surface-exposed FimH on UTI89 grown in the presence of compound 1 at 50 μΜ or 200 μΜ, as monitored by anti-FimH whole-cell ELISA (WCE) and plotted as percentage versus non-treated UTI89. Data are mean ± s.e.m, n = 2 independent experiments of

3 replicates each. Statistical analyses were performed using two-tailed unpaired Student's -^test to compare the percentage of surface-exposed FimH of non-treated UTI89 to that of compound 1 -treated UTI89 at 50 μΜ and 200 μΜ (*: P = 0.0004 and P = 0.0002, respectively), (c) Representative electron micrographs of UTI89 grown in the presence of 50 μΜ or 200 μΜ compound 1 + 1% DMSO, 1% DMSO, and non-supplemented LB respectively (d) Adherence of compound 1 -treated (200 μΜ, 1% DMSO) UTI89 and UTI89 Afim to human urinary bladder epithelial cell line 5637. Data expressed as mean percentage relative to non-treated UTI89 ± s.e.m., n = 2 independent experiments with 3 replicates each (*: P = 0.0003; two-tailed unpaired Student's latest against non-treated UTI89).

Figure 3

Timing of compound 1 activity: (a) Degree of piliation as represented by FimH surface- exposure on UTI89 grown on LB, measured by anti-FimH whole cell ELISA on samples taken before (t₀) and 1, 30 and 60 mins following the addition of 200 μΜ compound 1 + 1% DMSO or 1% DMSO alone, shown as mean percentage relative to UTI89 to ± s.e.m., n = 2 independent experiments with 2 samples each.(*: P = 0.0004, P = 0.0005 and P = 0.0004 for samples taken at 1, 30 and 60 mins respectively; two-tailed unpaired Student's -^test against UTI89 t₀).

(b) Electron microscopy and quantitative analyses of piliated and non-piliated UTI89 cells 1 minute following the addition of 200 μΜ compound 1 + 1% DMSO, 1% DMSO or UTI89 controls. Figure 4

(a) Schematic presentation of type 1 pilus CU pathway and its subunits undergoing donor- strand-exchange (DSE). CU pilus subunit translocate to the periplasmic space through the SEC translocon, where they form a binary complex with a periplasmic chaperone (FimC). Chaperone: subunit complexes are recruited to the usher (FimD), the pilus assembly platform in the outer membrane. At the usher, subunits undergo dono-strand-exchange and are built into the growing fiber. During DSE, the N-terminal extension on an incoming subunit displaces the chaperone bound to the subunit at the base of the fibre, (b) X-ray structures the FimH pilin domain (residues 158-279; shown as molecular surface) in complex with the FimC chaperone (left; taken from PDB: 1ZE3; shown in ribbon representation) or FimG Nte (right; taken from PDB:3JWN; shown in ribbon representation).

Figure 5

Molecular docking of small compounds. (A) The docking of a small compound against the P5 pocket of FimC-FimH complex. (B) Docking of the full binding groove with a hydrophobic anchor, but in the absence of FimC. Figure 6

(a) Percent biofilm formation of E. coli strain UTI89 and UTI89A zm under the same static growth condition as compound 1 -treated cells in 96-well microtitre plate for 48 h at room temperature, (b) Growth curves of E. coli strain UTI89 and UTI89A zm grown on LB at 37 °C, monitored as OD600. UTI89 grown in LB medium containing 250 μΜ compound 1, 10 % DMSO or 10% DMSO exhibited comparable growth kinetics as then grown on LB alone.

Figure 7

1H-NMR spectrum of compound 1. Table 1

List of strains, plasmids and primers used.

Strain, plasmid or Relevant description or sequence (5'-3' ) Reference(s) or source primer

Strains

Chen et al,

UTI89 E. coli strain UTI89, a human cystitis isolate Proc. Natl Acad. Sci. USA

2006, 103:5977-82.

Derous et al, BMC Res Notes

UTI89Afim mmAfimBEAICDFGH

2011, 4:213

F ompT gal dcm Ion hsdSB(rB^~ mB ) (laclq /ac£/ 5- 77 Studier & Moffat J Mol Biol, BL21(DE3)

indl sam7 nin5 clts857) 1986, 189: 113-30

Plasmids

fimC with six C-terminal His-tag codons inSaulino et al EMBO J 1998, pETSlOOO

pMON6235Acat, Spec^r 17: 2177-85 pDONRP4-PlR-/7»iC with six C-terminal His-tag codons,_™ . ,

pKVWcl J- l lS SXUCIV

Kan ^J pENT53 pDONR221 -fimH, Kan^r This study

pENT105 pDONRP2R-P3-g >+, Kan^r A gift from Henri De Greve pDEST22a This study

pDEST22a-/¾wC with six C-terminal His-tag codons fimH^- . ,

pKVWel r ms sxuciy

gjupv, Amp

Primers

GGGGACAACTTTGTATAGAAAAGTTGTCGAAGGA

FimCf GATAGAACCATGAGTAATAAAAACGTCAATGTAA This study

GG

GGGGACTGCTTTTTTGTACAAACTTGCTTAGTGAT

FimCr GGTGATGGTGATGTTCCATTACGCCCGTCATTTTG This study

G

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGA

FimH3 AGGAGATATATCATGAAACGAGTTATTACCCTGTT This study

TGCTGT Strain, plasmid or Relevant description or sequence (5'-3' ) Reference(s) or source primer

GGGGACCACTTTGTACAAGAAAGCTGGGTTTTATT GATAAACAAAAGTCACGCCAATA 1 Hⁱs study

GGGGACAGCTTTCTTGTACAAAGTGGTCGAAGGA

gfp7 GATAGAACCATGAGTAAAGGAGAAGAACTTTTCA This study

C

GGGGACAACTTTGTATAATAAAGTTGCCTTATTTG„ .

g^P TAGAGCTCATCCATGCCATG 1 Hⁱs study

Examples

Compound Screen

CU pilus subunits are characterised by an incomplete Ig-fold, lacking the C-terminal β-strand, and by the presence of a disordered 10-20 residues extension at the N-terminus (Choudhury, et al. Science 1999, 285: 1061-1066; Sauer, et al. Science 1999, 285: 1058-1061). In the periplasm, pilus subunits are stabilised by the chaperone, which donates an extended β-strand (strand Gl) to complement the missing structural information in the subunit Ig-fold (Choudhury, et al. Science 1999, 285: 1061-1066; Sauer, et al. Science 1999, 285: 1058-1061). At the usher, subunits undergo non-covalent polymerisation through a similar fold complementation mechanism, now involving the N-terminal extension peptide (Nte) of the newly incoming pilus subunit (Figure 4). These Nte sequences contain a conserved motif of alternating hydrophobic residues termed 'P2-P5 residues' that make "knobs-in-hole" interactions with the equivalent hydrophobic pockets in the acceptor groove of the pilus subunit (Fig. la, b). In the chaperone-subunit interaction, the Gl strand occupies pockets PI to P4 and leaves P5 accessible to the solvent (Fig. la, b). During subunit polymerisation, the chaperone Gl donor strand bound to the subunit at the base of the pilus is exchanged for the Nte of the newly recruited chaperone: subunit complex, a process called 'donor-strand exchange' (DSE) (Sauer, et al. Cell 2002, 111 : 543-551). DSE occurs in a concerted 'zip-in zip-out' mechanism that involves the formation of a ternary complex between the chaperone: subunit complex and the incoming Nte (Remaut, et al. Mol Cell 2006, 22: 831-842). DSE ternary complex formation is initiated by the docking of the Nte P5 residue to the P5 pocket on the acceptor chaperone- subunit complex (Remaut, et al. Mol Cell 2006, 22: 831-842).

It was reasoned that chemical compounds which are able to competitively bind the P5 pocket could serve as pilus polymerisation inhibitors. In the search for type 1 pilus inhibitors, an assay was developed to screen for compounds that bind the P5 pocket in the FimC:FimH chaperone:adhesin complex. FimH constitutes the first subunit to be incorporated, is present in a single copy and is crucial to activate the FimD usher for pilus assembly (Nishiyama, et al. Science 2008, 320: 376-379). In addition, genetic inactivation of FimG and/or FimF, the subunits succeeding FimH and forming the link between the adhesin and the FimA pilus shaft leads to a polymerization arrest and the accumulation of FimD:FimC:FimH complexes unable to promote mannose-sensitive hemagglutination (Saulino, et al. Proc Natl Acad Sci USA 2000, 97: 9240-9245). Hence it was speculated that the chemical inhibition of DSE reaction between FimH and FimG would prevent FimG incorporation in the pilus and that of downstream subunits.

To screen for type 1 pilus DSE inhibitors an SDS-PAGE based assay was developed that monitors the formation of a SDS-stable complex between FimH and the FimG Nte (Fig. lc). In particular, compound 1 was identified (Fig. Id), and showed a dose-dependent inhibition of FimG-FimH DSE with IC₅₀ values of 253 μΜ (R²=0.94) (Fig. le).

In vivo Validation

In order to verify that the in vitro inhibition of FimG:FimH DSE could be translated into an in vivo inhibition of pilus formation, uropathogenic E. coli UTI89, a cystitis isolate, were grown in presence of compound 1 and type 1 pilus formation was monitored using a mannose-sensitive biofilm assay (Anderson, et al. Science 2003, 301 : 105-107; Justice, et al. Proc Natl Acad Sci U SA 2004, 101 : 1333-1338; Pratt, et al. Mol Microbiol 1998, 30: 285-293) (Fig. 2a). Compound 1 had no adverse effects on bacterial growth in rich media (Fig. 6) and inhibited type 1 pili dependent-bio film formation of UTI89 in a titratable fashion. Under the conditions tested, compound 1 inhibition of UTI89 biofilms showed an IC₅₀ of 37 μΜ (R²=0.98). Thus, compound 1 efficiently reaches its biological target in the E. coli periplasm. Moreover, the mid-inhibitory concentrations for biofilm formation showed approximately 7-fold lower IC₅₀ compared to that of the in vitro DSE assay. In vivo, FimG:FimH DSE is catalysed by the FimD usher and occurs on a sub-minute timescale under roughly equimolar concentrations of FimG and FimH (Jacob-Dubuisson, et al. J Biol Chem 1994, 269: 12447-12455). In contrast, in vitro, the non-catalysed DSE reaction requires an 8-fold excess of FimG Nte in order to reach completion over a time scale of 12 hours. This excess of FimG Nte likely explains the higher IC₅₀ values observed in the DSE assay. Activity on type 1 Pili

To further confirm that the bio film-deficient phenotype stems from the loss of type 1 pili, the presence of surface-exposed FimH and type 1 pili on UTI89 grown in presence of compound 1 were analysed by negative stain EM and anti-FiniH whole-cell ELISA (WCE) (Fig. 2b). WCE revealed that compared to DMSO-treated controls, co-culturing of UTI89 with 200 μΜ or 50 μΜ compound 1 reduced the abundance of surface-exposed FimH by approximately 97% and

85%, respectively (yP-value < 0.001) (Fig. 2b). Representative electron micrographs showed peritrichous piliation of bacteria in DMSO-control, whereas treatment with 200 μΜ and 50 μΜ of compound 1 resulted in non- and pauci-piliated bacteria, respectively (Fig. 2c). Moreover, the few pili that were present on the bacteria at 50 μΜ of compound 1 were aberrantly long (Fig.2c). A similar phenotype is observed in fimH mutant strains, where the accumulated periplasmic pool of FimA pilus subunits is directed to those few pilus assembly platforms in the outer membrane that non-specifically become activated for pilus assembly in absence of FimH (Ronald, et al. Proc Natl Acad Sci U S A 2008, 105: 10937-10942). This leads to non- physiologically long pili that have an increased tendency to break under shear stress. The lack of type 1 pili renders UPEC avirulent by abrogating its ability to colonise and invade during early events of infection (Mulvey, et al. Science 1998, 282: 1494-1497). UTI89 cultured in the presence of 250 μΜ compound 1 exhibited a 74% reduction in the ability to bind to human bladder cells compared to non-treated and DMSO-treated bacteria (yP-value < 0.001) (Fig. 2d). Under the same conditions bladder cell binding of UTI89 fim reduced 84% relative to wild-type

(WT) (rvalue < 0.001).

Duration of Action

In each of the above experiments, compound 1 was present throughout bacterial growth. In order to determine the time frame under which compound 1 becomes active, UTI89 was cultured in rich medium and treated with 200 μΜ of compound 1 during the exponential growth phase. Cells were harvested at 1, 30 and 60 minutes post treatment and assayed for FimH surface exposure using ELISA and type 1 pilus counts through EM visualisation (Fig. 3a, b). Based on a model in which compound 1 exclusively acts on nascent pili, it was predicted that levels of surface-exposed FimH would drop by 50% per generation time (20 min). Unexpectedly, it was found that surface-exposed FimH was reduced to background levels within 1 minute after the addition of compound 1 (Fig. 3a). Furthermore, EM inspection revealed the compound 1 treated cells lost their type 1 pili (Fig. 3b). Type 1 pilus expression is phase variable and under the tested culture conditions, roughly 15% of WT bacteria showed type 1 pilus expression. In compound 1 -treated cultures the number of piliated bacteria fell back to 2%, whilst DMSO-treated controls maintained near WT levels of pili (Fig. 3b). These observations indicate that pre-existing type 1 pili are shed from the bacterial surface upon addition of compound 1 and suggest that the compound retains an inhibitory effect downstream of the FimH-FimG DSE reaction. Chaperone-usher pili are tethered to the bacterial surface by anchoring to the outer membrane usher via the last incorporated chaperone:subunit complex. Possibly, compound 1 binding to the extended P5 pocket of the basal subunit destabilises the chaperone:subunit interaction leading to the dissociation of the pili.

Compounds

General Procedures

The following synthetic procedures represent general protocols for the synthesis of a range of structurally diverse compounds according to the invention. In these schema, R corresponds to Ri-V- in formula (I) and R' corresponds to -R₂ in formula (I).

5-Phenyl-l,3,4-oxadiazole-2-thioether analogues

l,3,4-Oxadiazole-2-thiols can be accessed from the corresponding hydrazides by refluxing in ethanol with a base such as potassium hydroxide, triethylamine or 4-methyl morpholine in the presence of carbon disulfide. Treatment with a suitable acid, such as hydrochloric acid, gives the l,3,4-oxadiazole-2-thiol.

The hydrazides required for the cyclisation step can be formed from the coupling of t-butyl carbazate with a carboxylic acid using a suitable coupling reagent such as l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (EDC), benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBop) or O-(Benzotriazol-l-yl)- A .A .N'.N'-tetramethyluronium hexafluorophosphate (HBTU). The Boc protecting group can then be removed by treatment under acidic conditions such as with trifluoroacetic acid to give the corresponding h drazide.

An alternative strategy to synthesise hydrazides is by refluxing an ester with hydrazine hydrate in a suitable solvent, such as ethanol or methanol.

The l,3,4-oxadiazole-2-thiol core can be functionalised by, for example, alkylation. This can be achieved by using an alkylating agent, such as bromoacetic acid, in the presence of a suitable base, such as sodium hydroxide, potassium hydroxide or 4-methyl morpholine.

The synthesis may be completed by a final amide coupling reaction with a suitable amine coupling partner and a coupling reagent such as EDC, PyBOP or HBTU.

Sulfonamide side-chains can be introduced at an early stage in the synthesis by the reaction of a suitable amine with a sulfonyl chloride, in the presence of a base, such as sodium hydroxide or pyridine.

Alternatively, sulphonamides having a reversed configuration can be synthesised with the appropriate reaction partners and using a suitable base, such as sodium hydroxide or pyridine.

1 ,3,4-oxadiazole-2-amino analogues

l,3,4-oxadiazole-2-amino analogues can be accessed by reacting the corresponding hydrazide with cyanogen bromide in methanol in the presence of a suitable base, such as potassium carbonate, potassium h drogen carbonate or sodium hydro en carbonate.

The l,3,4-oxadiazole-2-amino core can then be reacted with a carboxylic acid coupling partner in the presence of a suitable amide coupling reagent, such as EDC, PyBop or HBTU, to give access to l,3,4-oxadiazole-2-amino analogues.

1 ,3,4-thiadiazole-2-thiol analogues

l,3,4-thiadiazole-2-thiol analogues can be synthesised from the corresponding hydrazide by refluxing with carbon disulfide in ethanolic base, such as potassium carbonate or potassium hydroxide followed by treatment with cone, sulphuric acid.

1 ,3,4-thiadiazole-2-thiol analogues The l,3,4-thiadiazole-2-thiol can then be alkylated using an alkylating agent, such as bromoacetic acid, in the presence of a base, such as sodium hydroxide, to give access to 1,3,4- thiadiazole-2-thiol analogues. This is then followed by amide bond formation with a suitable amine in the presence of a amide coupling reagent, such as EDC, PyBop or HBTU, to give 1 ,3,4-thiadiazole-2-thiol analogues. l,2,4-triazole-3-thiol analo ues

l,2,4-triazole-3-thiol analogues can be synthesised from the corresponding thiosemicarbazide, which can be prepared from the reaction of an acid chloride with thiosemicarbazide in the presence of a base such as pyridine. This benzothiosemicarbazide can then be cyclised to the l,2,4-triazole-3-thiol by refluxing in ethanol in the presence of a base, such as sodium hydroxide or potassium hydroxide.

1,2,4-triazole-3-thiol analogues

Alkylation of this thiol can be achieved by reaction with a suitable alkylating agent such as bromoacetic acid, in the presence of a base. This is then followed by amide bond formation with a suitable amine in the presence of an amide coupling reagent, such as EDC, PyBop or HBTU to give l,2,4-triazole-3-thiol analogues.

Alternatively, the l,2,4-triazole-3-thiol core can be synthesised by refluxing the hydrazide compound with trimethylsilyl isothiocyanate in ethanol, followed by cyclisation in the presence of base to give the l,2,4-triazole-3-thiol core.

Example Syntheses

All commercially available solvents and reagents were used without further treatment as received unless otherwise noted. NMR spectra were measured with a Bruker DRX 500 or 600 MHz spectrometer; chemical shifts are expressed in ppm relative to TMS as an internal standard and coupling constants (J) in Hz. Mass spectra were obtained using a Waters ZQ2000 single quadrupole mass spectrometer with electrospray ionisation (ESI). High resolution mass spectra were acquired on a Waters LCT time of flight mass spectrometer with electrospray ionisation (ESI) or chemical ionization (CI). Analysis of intermediates by reverse-phase LCMS was carried out on an analytical C18 column (Phenomenex Gemini, 50 x 3.0 mm, 5 μιη) and an AB gradient of 5-95 % for B at a flow rate of 1 mL/minute, where eluent A was 0.1 % formic acid/water and eluent B was 0.1 % formic acid/acetonitrile. Routine analytical thin layer chromatography was performed on pre-coated plates (60F254, Machery-Nagel).

Synthesis of N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyPphenyl)- 1 ,3,4-oxadiazol-2- yl)thio)acetamide (compound 1)

4-(Pyrrolidin-l-ylsulfonyl)benzoic acid

To a stirred solution of pyrrolidine (0.75 mL, 9.00 mmol) in NaOH (10 mL, aq. 10% soln.) was added 4-(chlorosulfonyl)benzoic acid (l .OOg, 4.50 mmol). The reaction was stirred at rt for 16 hours, then acidified with cone. HCl. The resulting precipitate was filtered, washed with more HCl (1M), and dried on frit to give 4-(Pyrrolidin-l-ylsulfonyl)benzoic acid as a white solid (0.64 g, 56%).

1H NMR (500 MHz, (CD₃)₂SO)) δ; 13.50 (1H, s, COOH), 8.15 (2H, d, J = 8.6 Hz, Ar H), 7.92 (2H, d, J = 8.6 Hz, Ar H), 3.16 (4H, t, J = 6.74 Hz, NC¾), 1.65 (4H, m, NCH₂C¾); ¹³C NMR (150 MHz, (CD₃)₂SO)) δ; 166.3 (COOH), 139.9, 134.6, 130.3, 127.6, 47.9 (CH₂), 24.78 (CH₂); m/z (HR ES-) found [M-H]^~ 254.0486. CnHi₂N0₄S requires 254.0487. Tert-butyl 2-(4-(pyrrolidin-l-ylsulfonyl)benzoyl)hydrazinecarboxylate

To a stirred solution of 4-(Pyrrolidin-l-ylsulfonyl)benzoic acid (3.84 g, 15.6 mmol) in CH₂CI₂ (15 mL) was added t-butyl carbazate (2.14 g, 16.2 mmol) and l-[3-(dimethylamino)propyl]-3- ethyl carbodiimide hydrochloride (3.11 g, 16.2 mmol). The reaction was stirred for 16 hours at rt. The reaction mixture was then washed with NaHC0₃ (3 x 15 mL, satd. soln.), NaCl (15 mL, satd. soln.) extracted with CH₂CI₂ (50 mL) and dried over MgSC^. The product was purified by flash silica chromatography (EtOAc:hexane, 2:8) to give tert-butyl 2-(4-(pyrrolidin-l- ylsulfonyl)benzoyl)hydrazinecarboxylate as a white crystalline solid (3.60 g, 71% yield).

1H NMR (500 MHz, (CDC1₃)) δ; 9.18 (1H, s, COOH), 7.92 (2Η, d, J= 8.3 Hz, Ar H), 7.78 (2Η, d, J = 8.3 Hz, Ar H), 3.20 (4Η, t, J = 6.7 Hz, NC¾), 1.73 (4H, m, NCH₂CH₂); 1.45 (9Η, s, CH₃); ¹³C NMR (150 MHz, (CDC1₃)) δ; 171.5 (C=0), 156.2 (C=0), 140.2, 135.5, 128.3, 127.7, 82.5 (C-(CH₃)₃), 48.1 (CH₂), 28.3 (( H₃)₃), 25.3 (CH₂); m/z (HR CI+) found [MH]⁺ 370.1417. Ci₆Hi₃N₃0₅S requires 370.1431.

4-(pyrrolidin-l-ylsulfonyl)benzohydrazide

Tert-butyl 2-(4-(pyrrolidin-l-ylsulfonyl)benzoyl)hydrazinecarboxylate (0.93g, 2.50 mmol) was cooled to 0°C and trifluoroacetic acid (5 mL) was added dropwise. The mixture was stirred and allowed to warm to room temperature over 1 hour. After this time NaHC0₃ (satd. soln.) was added until effervescence ceased. Product was then extracted with CH₂CI₂ (50 mL) and washed with further NaHC0₃ (4 X 25 mL, satd. soln.). Solvent was then removed under reduced pressure to give 4-(pyrrolidin-l-ylsulfonyl)benzohydrazide as a white solid (0.48 g, 72% yield). Product was carried through to cyclisation step without further purification. 5-(4-(pyrrolidin- l-ylsulfonyl)phenyl)- 1 ,3 ,4-oxadiazole-2-thiol

To a stirred solution of 4-(pyrrolidin-l-ylsulfonyl)benzohydrazide (0.51 g, 1.90 mmol) in ethanol (20 mL) was added potassium hydroxide (0.1 1 g, 2.00 mmol) and the mixture was stirred at rt for 5 minutes. Carbon disulfide (0.18 mL, 3.00 mmol) was then slowly added to the mixture while the reaction was heated to reflux. The reaction was then stirred under reflux for 18 hours. After this time the reaction was allowed to cool to rt and solvent was removed under reduced pressure. The residue was then dissolved in water (25 mL) and HC1 (1M) added until a white precipitate formed. The precipitate was filtered and dried on frit to give 5-(4-(pyrrolidin-

1- ylsulfonyl)phenyl)-l ,3,4-oxadiazole-2-thiol as a white solid (0.45g, 76%).

1H NMR (600 MHz, (CD₃CN)) δ; 12.15 (1H, s, SH), 8.08 (2Η, d, J = 8.1 Hz, Ar H), 7.95 (2Η, d, J = 8.1 Hz, Ar H), 3.22 (4Η, t, J = 6.3 Hz, NC¾), 1.71 (4H, t, J = 6.3 Hz, N-CH₂-CH₂-); ¹³C NMR (150 MHz, (CDC1₃) δ; 179.4 (OCSH), 160.7 (OC=N), 140.9, 129.2, 127.9, 127.6, 49.0 (CH₂), 25.9 (CH₂); m/z (HR CI+) found [M]⁺ 31 1.0383. (Ci₂Hi₃N₃0₃S₂) requires 31 1.0392.

2- ((5-(4-(pyrrolidin-l-ylsulfon l)phenyl)-l A-oxadiazol-2-yl)thio)acetw acid

To a stirred solution of 5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazole-2-thiol (0.05 g, 0.15 mmol) in ethanol (5 mL) was added N-methylmorpholine (0.02 mL, 0.20 mmol) and bromoacetic acid (0.03 g, 0.17 mmol). Reaction was stirred at room temperature for 72 hours. After this time solvent was removed under reduced pressure, and the residue redissolved in CH₂C1₂ (25 mL) and washed with HC1 (1 M, 20 mL). The organic layer was separated, dried over magnesium sulphate and filtered. Solvent was removed under reduced pressure to give 2- ((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetic acid as a white sold (0.03 g, 54%). Product was used without further purification. N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 1

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetic acid (0.03 g, 0.08 mmol) in CH₂C1₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.020 g, 0.10 mmol) and para- chloroaniline (0.01 g, 0.10 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HC1 (1M) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography and solvent removed under reduced pressure to give N-(4- chlorophenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (0.03 g, 78%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.61 (1H, s, NH), 8.18 (2Η, d, J = 8.3 Hz, Ar H), 7.99 (2Η, d, J = 8.3 Hz, Ar H), 7.62 (2Η, d, J = 8.8 Hz, Ar H), 7.39 (2Η, d, J = 8.8 Hz, Ar H), 4.38 (2Η, s, SCH₂), 3.18 (4Η, t, J = 6.6 Hz, NCH₂), 1.65 (4Η, t, J = 6.6 Hz, NCH₂CH₂); ¹³C NMR (150 MHz, (CD₃)₂SO)) δ: 165.1 (CONH), 164.4 (CS), 164.2 (CON), 138.9 (CS0₂), 137.6 (C- NH), 128.9, 128.3, 127.3 (signals overlapping), 126.7 (CCl), 120.7, 47.94 (NCH₂), 36.83 (SCH₂), 24.80 (CH₂CH₂N); m/z (HR CI+) found [MH]⁺ 479.0610. (C₂oH₂oClN₄0₄S₂) requires 479.0609 (See Figure 7). Synthesis of N-(4-chlorophenyl)-2-((5-(4-(N-cyclopentylsulfamoyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 4)

4- (N-cyclopentylsulfamoy I) benzoic acid

Cyclopentylamine (894 μί, 9.07 mmol) was added to a stirred solution of 4- (chlorosulfonyl)benzoic acid (l .OOg, 4.53 mmol) in NaOH (10 mL, 10% aq.). The reaction was stirred at RT for 16 h before being acidified with cone. HC1. The resulting white precipitate was filtered, washed with HC1 (1M aq.) and dried to give 4-(N-cyclopentylsulfamoyl)benzoic acid as a white solid (580 mg, 48%).

δ_Η (500 Hz, DMSO-dg) 8.14-8.06 (2H, m, Ar CH), 7.94-7.87 (2Η, m, Ar CH), 3.48-3.36 (1Η, m, CHNH), 1.60-1.46 (4H, m, CH₂), 1.42-1.20 (4Η, m, CH₂); 5_C (125 Hz, DMSO-d₆) 166.37 (COOH), 145.4 (Ar Q, 134.0 (Ar Q, 130.1 (Ar CH), 126.7 (Ar CH), 54.5 (CHNH), 32.4 (CH₂), 22.8 (CH₂); HRMS found 270.079508; Ci₂Hi₆N0₄S (MH⁺) requires 270.07946. tert-butyl 2-(4-(N-cyclopentylsul amoyl)benzoyl)hydrazine-l-carboxylate

To a stirred solution of 4-(N-cyclopentylsulfamoyl)benzoic acid (500 mg, 1.86 mmol) in DCM (0.20 mL) was added tert-butyl carbazate (255 mg, 1.93 mmol) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (370 mg, 1.93 mmol). The reaction was stirred for 16 h at RT before being washed with NaHC0₃ (15 mL, sat. aq.) and NaCl (15 mL, sat. aq.) and dried (MgS0₄). Purification by flash chromatography (Si0₂, 20→60% EtOAc in cyclohexane) gave tert-butyl 2-(4-(N- cyclopentylsulfamoyl)benzoyl)hydrazine-l-carboxylate as a white crystalline solid (710 mg,

100%).

δ_Η (500 Hz, CDC1₃) 7.86 (2H, d, 8.5 Hz, Ar CH), 7.82 (2Η, d, J 8.5 Hz, Ar CH), 5.43 (1Η, d, J 7.0 Hz, NH), 3.55 (1Η, q, J 7.0 Hz, CHNH), 1.77-1.70 (2H, m, CHH') 1.65-1.56 (2H, m, CHH'), 1.53-1.43 (11Η, m, CHH' and C(CH₃)₃), 1.39-1.31 (2Η, m, CHH'); 5_C (125 Hz, CDC1₃) 166.75 (CONH), 156.1 (COC(CH₃)₃), 144.3 (Ar Q, 135.4 (Ar Q, 128.31 (Ar CH), 127.2 (Ar CH), 82.5 (C(CH₃)₃) 55.3 (CHNH), 33.4 (CH₂), 28.2 (C(CH₃)₃), 23.2 (CH₂); HRMS found 382.1441; Ci₇H₂₄N₃0₅S ([M-H]^") requires 382.1437. N-cyclopentyl-4- (hydrazinecarbon l)benzenesulfonamide

Tert-butyl 2-(4-(N-cyclopentylsulfamoyl)benzoyl)hydrazine-l-carboxylate (640 mg, 1.67 mmol) was cooled to 0°C and trifluoroacetic acid (3.34 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h before being quenched with NaHC0₃ (sat. aq.) until effervescence ceased. The solution was extracted with DCM (40 mL), the aqueous taken to pH13 with NaOH (10% aq.) and extracted again with DCM (40 mL). The combined organics were dried (MgS0₄) and evaporated in vacuo to give N-cyclopentyl-4- (hydrazinecarbonyl)benzenesulfonamide as a white solid (280 mg, 59%) which was used without further purification.

δ_Η (500 Hz, DMSO-dg) 7.97-7.94 (2H, m, Ar CH), 7.85 (2Η, d, J 8.5 Hz, Ar CH), 3.46-3.37 (1Η, m, CHNH), 1.63-1.46 (4H, m, CH₂), 1.42-1.21 (4Η, m, CH₂).

N-cyclopentyl-4- ( 5-mercapto-l, 3 4-oxadiazol-2-yl)benzenesulfonamide

Potassium hydroxide (34 mg, 0.60 mmol) was added to a solution of N-cyclopentyl-4- (hydrazinecarbonyl)benzenesulfonamide (160 mg, 0.57 mmol) in ethanol (6.0 mL) and the mixture stirred at RT for 5 min before carbon disulfide (55 μί, 0.90 mmol) was added. The reaction was heated at reflux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (25 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give N-cyclopentyl-4-(5-mercapto- l,3,4-oxadiazol-2-yl)benzenesulfonamide as a white solid (120 mg, 65%>).

δ_Η (500 Hz, DMSO-dg) 8.07 (2H, dt, J 8.5, 2.0 Hz, Ar CH), 7.97 (2Η, dt, J 8.5, 2.0 Hz, Ar CH), 3.48-3.40 (1Η, m, CHNH), 1.64-1.48 (2H, m, CH₂), 1.41-1.23 (2Η, m, CH₂); 5_C (125 Hz, DMSO-de) 177.6 (CSH), 159.4 (CO), 144.5 (Ar Q, 127.5 (Ar CH), 126.9 (Ar CH), 125.7 (Ar Q, 54.5 (CHNH), 32.5 (CH₂), 22.8 (CH₂); HRMS found 326.061936; Ci₃Hi₆N30₃S₂ (MH⁺) requires 326.06276.

2- ((5- (4- (N-cyclopentylsulfamoyl)phenyl)- 1 , 3, 4-oxadiazol-2-yl) thio) acetic acid

To a stirred solution of N-cyclopentyl-4-(5-mercapto-l,3,4-oxadiazol-2-yl)benzenesulfonamide (80 mg, 0.25 mmol) in ethanol (8.0 mL) was added N-methylmorpholine (35 μί, 0.32 mmol) and bromoacetic acid (38 mg, 0.27 mmol). The reaction was stirred at RT for 72 h before being evaporated in vacuo and the residue dissolved in DCM (25 mL) and washed with HC1 (1 M aq., 20 mL). The organic layer was dried (MgS0₄) and evaporated in vacuo to give 2-((5-(4-(N- cyclopentylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid as a white solid (90 mg, 94%), which was used without further purification.

δ_Η (500 MHz, DMSO-dg) 8.16 (2H, d, J 8.5 Hz, ArCH), 7.98 (2Η, d, J 8.5 Hz, Ar CH), 4.25 (2Η, s, SCH₂), 3.47-3.38 (1Η, m, NHCH), 1.53-0.94 (8Η, m, 4 ^χ CH₂); HRMS found 384.068578; Ci₅Hi₈N₃0₅S₂ (MH⁺) requires 384.06824.

N- ( 4-chlorophenyl)-2- ((5- (4- (N-cyclopentylsulfamoyl)phenyl)- 1 , 3, 4-oxadiazol-2- yl)thio)acetamide (compound 4

To a solution of 2-((5-(4-(N-cyclopentylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid (85 mg, 0.22 mmol) in DCM (4.5 mL) was added l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (56 mg, 0.29 mmol) and 4-chloroaniline (28 mg, 0.22 mmol). The reaction was stirred at RT for 1 h before being washed with HC1 (1M aq., 30 mL) and NaHC0₃ (sat. aq., 30 mL). Purification by flash chromatography (Si0₂, 20→70% ethyl acetate in cyclohexane) gave N-(4-chlorophenyl)-2-((5-(4-(N-cyclopentylsulfamoyl)phenyl)-l,3,4- oxadiazol-2-yl)thio)acetamide as a white solid (43 mg, 40%).

δ_Η (500 Hz, DMSO-dg) 10.59 (IH, br s, CONH), 8.14 (2Η, d, J 8.5 Hz, Ar CH), 7.96 (2Η, d, J 8.5 Hz, Ar CH), 7.85 (1Η, d, J 7.0 Hz, S0₂NH), 7.61 (2Η, d, J 9.0 Hz, Ar CH), 7.37 (2Η, d, J 9.0 Hz, Ar CH), 4.36 (2Η, s, SCH₂), 3.44 (1Η, sext, J 7.0 Hz, CHNH), 1.63-1.48 (4H, m, 2 x CH₂), 1.42-1.21 (4 H, m, 2 x CH₂); 5_C (125 Hz, DMSO-dg) 165.0 (CONH), 164.2 (2 peaks overlapping, CS and OCN), 144.4 (Ar CS0₂), 137.6 (Ar CNH), 128.8 (Ar CH), 127.5 (Ar CH), 127.3 (Ar Q, 127.2 (Ar CH), 126.1 (Ar Q, 120.7 (Ar CH), 54.5 (CHNH), 36.8 (SCH₂), 32.4 (CH₂), 22.8 (CH₂); HRMS found 493.075805; C₂iH₂₂ClN₄0₄S₂ (MH⁺) requires 493.07655.

Synthesis of N-(4-chlorophenyl)-2-((5-(4-(N-cyclohexylsulfamoyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 5) 4-(N-cyclohexylsulfamoyl)benzoic acid

Cyclohexylamine (1.04 mL, 9.07 mmol) was added to a stirred solution of 4- (chlorosulfonyl)benzoic acid (l .OOg, 4.53 mmol) in NaOH (10 mL, 10% aq.). The reaction was stirred at RT for 16 h before being acidified with cone. HCl. The resulting precipitate was filtered, washed with HCl (1M aq.) and dried to give 4-(N-cyclohexylsulfamoyl)benzoic acid as a white solid (870 mg, 68%).

δ_Η (500 MHz, DMSO-dg) 13.36 (1H, br s, COOH), 8.09 (2Η, d, J 8.5 Hz, Ar CH), 7.90 (2Η, d, J 8.5 Hz, Ar CH), 3.00-2.88 (1Η, m, NHCH), 1.90-0.95 (10Η, m, 5 CH₂); 5_C (125 MHz, DMSO-de) 166.27 (CO), 146.03 (Ar Q, 133.91 (Ar Q, 130.07 (Ar CH), 126.49 (Ar CH), 52.18 (NHCH), 33.20 (CH₂), 24.79 (CH₂), 24.31 (CH₂); HRMS found 284.094555; Ci₃Hi₆N0₄S (MH⁺) requires 284.09511. tert-butyl 2- ( 4- (N-cyclohexylsul amoyl)benzoyl)hydrazine-l-carboxylate

To a stirred solution of 4-(N-cyclohexylsulfamoyl)benzoic acid (800 mg, 2.83 mmol) in DCM (0.20 mL) was added tert-butyl carbazate (389 mg, 2.94 mmol) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (564 mg, 2.94 mmol). The reaction was stirred for 16 h at RT before being washed with NaHC0₃ (15 mL, sat. aq.) and NaCl (15 mL, sat. aq.) and dried (MgS0₄). Purification by flash chromatography (Si0₂, 20→60% EtOAc in cyclohexane) gave tert-butyl 2-(4-(N- cyclohexylsulfamoyl)benzoyl)hydrazine-l-carboxylate as a white solid (780 mg, 69%).

δ_Η (500 MHz, CDC1₃) 8.92 (1H, br s, NH), 7.89-7.82 (4H, m, Ar CH), 6.96 (1H, br s, NH), 5.26 (1H, br s, NH), 3.16-3.05 (1H, m, NHCH), 1.78-1.68 (2Η, m, 2 x CHH'), 1.65-1.60 (2H, m, 2 x CHH'), 1.52 (9Η, s, C(CH₃)₃), 1.30-1.03 (6Η, m, 3 x CH₂); 5_C (125 MHz, CDC1₃) 165.6 (CO), 156.0 (NCO), 144.9 (Ar Q, 135.4 (Ar Q, 128.3 (Ar CH), 127.0 (Ar CH), 82.5 (C(CH₃)₃), 53.0 (NHCH), 34.0 (CH₂), 28.3 (C(CH₃)₃), 25.1 (CH₂), 24.7 (CH₂); HRMS found 398.174477; Ci₈H₂₈N₃0₅S (MH⁺) requires 398.17442. N-cyclohexyl-4-(hydrazinecarbon l)benzenesulfonamide

7¾rt-butyl 2-(4-(N-cyclohexylsulfamoyl)benzoyl)hydrazine-l-carboxylate (710 mg, 1.79 mmol) was cooled to 0°C and trifluoroacetic acid (3.58 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h before being quenched with NaHC0₃ (sat. aq.) until effervescence ceased. The solution was extracted with DCM (40 mL), dried (MgS0₄) and evaporated in vacuo to give N-cyclohexyl-4-(hydrazinecarbonyl)benzenesulfonamide as a white solid (270 mg, 51%) which was used without further purification.

δ_Η (500 MHz, DMSO-dg) 7.99-7.94 (2H, m, ArCH), 7.90-7.84 (2Η, m, Ar CH), 2.98-2.89 (1Η, m, NHCH), 2.00-1.00 (10Η, m, 5 CH₂).

N-cyclohexyl-4- ( 5-mercapto-l, 3, 4-oxadiazol-2- l)benzenesulfonamide

Potassium hydroxide (53 mg, 0.95 mmol) was added to a solution of N-cyclohexyl-4- (hydrazinecarbonyl)benzenesulfonamide (270 mg, 0.91 mmol) in ethanol (9.5 mL) and the mixture stirred at RT for 5 min before carbon disulfide (87 μί, 1.44 mmol) was added. The reaction was heated at refiux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (25 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give N-cyclohexyl-4-(5-mercapto- l,3,4-oxadiazol-2-yl)benzenesulfonamide as a white solid which was used without further purification.

δ_Η (500 MHz, DMSO-dg) 8.11-8.03 (2H, m, ArCH), 8.01-7.93 (2Η, m, Ar CH), 3.03-2.87 (1Η, m, NHCH), 1.65-0.85 (10Η, m, 5 CH₂); HRMS found 340.077873; Ci₄Hi₈N₃0₃S₂ (MH⁺) requires 340.07841. 2- ((5- (4- (N-cyclohexylsulfamoyl)phenyl)- 1 , 3, 4-oxadiazol-2-yl) thio) acetic acid

To a stirred solution of N-cyclopentyl-4-(5-mercapto-l,3,4-oxadiazol-2-yl)benzenesulfonamide (100 mg, 0.29 mmol) in ethanol (7.3 mL) was added N-methylmorpholine (42 ^L, 0.38 mmol) and bromoacetic acid (45 mg, 0.32 mmol). The reaction was stirred at RT for 72 h before being evaporated in vacuo and the residue dissolved in DCM (25 mL) and washed with HC1 (1 M aq., 20 mL). The organic layer was dried (MgS0₄) and evaporated in vacuo to give 2-((5-(4-(N- cyclohexylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid as a white solid (105 mg, 91%), which was used without further purification.

δ_Η (500 MHz, DMSO-dg) 8.15 (2H, d, J 8.5 Hz, ArCH), 7.99 (2Η, d, J 8.5 Hz, Ar CH), 4.23 (2Η, s, SCH₂), 3.02-2.90 (1Η, m, NHCH), 1.84-0.97 (10Η, m, 5 CH₂); HRMS found 398.083635; Ci₆H₂oN₃0₅S₂ (MH⁺) requires 398.08389.

N- ( 4-chlorophenyl)-2- ((5- (4- (N-cyclohexylsulfamoyl)phenyl)- 1 , 3, 4-oxadiazol-2- yl)thio)acetamide (compound 5

To a solution of 2-((5-(4-(N-cyclohexylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid (100 mg, 0.25 mmol) in DCM (5.0 mL) was added l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (64 mg, 0.33 mmol) and 4-chloroaniline (32 mg, 0.25 mmol). The reaction was stirred at RT for 1 h before being washed with HC1 (1M aq., 30 mL) and NaHC0₃ (sat. aq., 30 mL). Purification by flash chromatography (Si0₂, 20→70% ethyl acetate in cyclohexane) gave N-(4-chlorophenyl)-2-((5-(4-(N-cyclohexylsulfamoyl)phenyl)-l,3,4- oxadiazol-2-yl)thio)acetamide as a white solid (20 mg, 16%).

δ_Η (500 Hz, DMSO-dg) 10.57 (IH, br s, CONH), 8.13 (2Η, d, J 8.5 Hz, Ar CH), 7.97 (2Η, d, J 8.5 Hz, Ar CH), 7.85 (1Η, d, J 7.5 Hz, S0₂NH), 7.60 (2Η, d, J 9.0 Hz, Ar CH), 7.37 (2Η, d, J 9.0 Hz, Ar CH), 4.36 (2Η, s, SCH₂), 3.01-2.91 (1Η, m, CHNH), 1.60-1.50 (4H, m, 4 x CHH'), 1.46-1.39 (IH, m, CHH'), 1.16-0.96 (5H, m, 5 ^χ CHH'); 5_C (125 Hz, DMSO-dg) 165.0 (CONH), 164.2 (2 peaks overlapping, CS and OCN), 145.1 (Ar CS0₂), 137.6 (Ar CNH), 128.8 (Ar CH), 127.3 (2 peaks overlapping, Ar C and Ar CH), 127.2 (Ar CH), 126.0 (Ar Q, 120.7 (Ar CH), 52.2 (CHNH), 36.8 (SCH₂), 33.2 (CH₂), 24.8 (CH₂), 24.3 (CH₂); HRMS found 507.090723; C₂₂H₂₄C1N₄0₄S₂ (MH⁺) requires 507.09220.

N-(3-chlorophenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetamide (compound 8

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂C1₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and m- chloroaniline (0.04 g, 0.30 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HCl (1 M) and NaHC0₃ (satd.soln.) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography to give N-(3-chlorophenyl)-2-((5-(4- (pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (0.08 g, 52%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.66 (1H, s, NH), 8.18 (2Η, d, J= 8.4 Hz, Ar H), 7.99 (2Η, d, J = 8.4 Hz, Ar H), 7.79 (1Η, s, Ar H), 7.44 (1Η, d, J = 8.1 Hz, Ar H), 7.37 (1Η, t, J = 8.1 Hz, Ar H), 7.15 (1Η, d, J = 8.1 Hz, Ar H), 4.38 (2Η, s, SCH₂), 3.18 (4Η, t, J = 6.6 Hz, NC¾), 1.70 - 1.60 (4H, t, J = 6.6 Hz, NCH₂CH₂); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 165.3 (CONH), 164.4 (CS), 164.2 (CON), 140.1 , 138.9, 133.2, 130.7, 128.3, 127.4, 126.7, 123.5, 1 18.6, 1 17.6, 47.9 (NCH₂), 36.8 (SCH₂), 24.8 (CH₂CH₂N); m/z (HR CI+) found [MH]⁺ 479.0591. (C₂oH₂₀ClN₄0₄S₂) requires 479.0609. 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)-N-(m-tolyl)acetamide (compound 20)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and m- toluidine (0.03 mL, 0.30 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HCl (1 M) and NaHC0₃ (satd.soln.) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography and solvent removed under reduced pressure to give 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)-N-(m- tolyl)acetamide as a white solid (0.07 g, 51%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.38 (1H, s, NH), 8.18 (2Η, d, J= 8.4 Hz, Ar H), 7.43 (1Η, s, Ar H), 7.36 (1Η, d, J = 8.2 Hz, Ar H), 7.20 (1Η, t, J = 8.2 Hz, Ar H), 6.90 (1Η, d, J = 8.2 Hz, Ar H), 4.38 (2Η, s, SCH₂), 3.18 (4Η, t, J = 6.6 Hz, NC¾), 2.26 (3H, s, Ph-C¾), 1.81 - 1.50 (4H, t, J = 6.6 Hz, NCH₂CH₂), ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.8 (CONH), 164.5 (CS), 164.2 (CON), 138.9, 138.6, 138.1 , 128.8, 128.3, 127.4, 126.7, 124.4, 1 19.7, 1 16.4, 47.9 (NCH₂), 36.9 (SCH₂), 24.8 (CH₂CH₂N), 21.2 (CH₃); m/z (HR CI+) found [MH]⁺ 459.1 145. (C₂iH2₃N40₄S2) requires 459.1 155. 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)-N-(p-tolyl)acetamide (compound 21)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and p- toluidine (0.03 mL, 0.30 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HCl (1 M) and NaHC0₃ (satd.soln.) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography to give 2-((5-(4-(pyrrolidin-l- ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)-N-(m-tolyl)acetamide as a white solid (0.06 g, 40%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.38 (s, 1H, NH), 8.18 (2Η, d, J= 8.3 Hz, Ar H), 7.99 (2Η, d, J = 8.3 Hz, Ar H), 7.47 (2Η, d, J = 8.2 Hz, Ar ), 7.13 (2H, d, J = 8.2 Hz, Ar H), 4.36 (2Η, s, SCH2 ), 3.18 (4Η, t, J = 6.5 Hz, NC¾ ), 2.25 (3H, s, C¾), 1.66 (4H, t, J = 6.5 Hz, NCH₂C¾); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 171.2 (CONH), 164.5 (CS), 164.2 (CON), 138.9, 136.2, 132.7, 129.3, 128.3, 127.3, 126.7, 1 19.8, 47.9 (NCH₂), 36.9 (SCH₂), 24.8 (CH₂CH₂N), 20.5 (CH₃); m/z (HR CI+) found [MH]⁺ 459.1 159. (C₂iH₂₃N₄0₄S₂) requires 459.1 155.

N-(3-methoxyphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetamide (compound 22)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂C1₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and m- anisidine (0.03 mL, 0.30 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HCl (1M) and NaHC0₃ (satd. soln.) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography and solvent removed under reduced pressure to give 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)-N-(m- tolyl)acetamide as a white solid (0.06 g, 44%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.48 (s, 1H), 8.18 (d, J = 8.3 Hz, 2H), 7.98 (d, J = 8.3 Hz, 2H), 7.28 (s, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.1 Hz, 1H), 4.37 (s, 2H), 3.72 (s, 3H), 3.18 (t, J = 6.5 Hz, 4H), 1.66 (t, J = 6.5 Hz, 4H); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.9 (CONH), 164.4 (CS), 164.2 (CON), 159.6 (COMe), 139.8, 138.9, 129.8, 128.3, 127.4, 126.7, 1 1 1.4, 109.1 , 105.0, 55.0, 47.9 (CH₂CH₂N), 36.9 (SCH₂), 24.8 (CH₂CH₂N); HRMS (m/z): [CI]⁺ calcd. for C₂iH₂₃N₄0₅S₂, 475.1 104; found 475.1 100. N-cyclohexyl-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetamide (compound 23)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and cyclohexylamine 0.03 mL, 0.30 mmol). Reaction was stirred at room temperature for 18 hours before being washed with HCl (1 M) and NaHC0₃ (satd. so In.) and extracted with further CH₂CI₂ (50 mL). Product was purified by flash silica chromatography to give N-cyclohexyl-2- ((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (0.02 g, 16%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 8.23 (1H, d, J = 7.7 Hz, NH), 8.20 (2Η, d, J = 8.3 Hz, Ar H), 8.01 (2Η, d, J = 8.3 Hz, Ar H), 4.1 1 (2Η, s, SCH₂), 3.60-3.47 (1Η, m, NHCH), 3.19 (4Η, t, J = 6.6 Hz, NCH₂ ), 1.71 (2Η, dd, J = 13.0 Hz, 10.2 Hz,); 1.69-1.61 (m, 4H, NCH₂CH₂), 1.58- 1.48 (m 1Η), 1.30-1.20 (m, 2Η), 1.20-1.06 (m, 3H); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.8 (CONH), 164.6 (CS), 164.1 (CON), 139.0, 128.3, 127.4, 126.8, 48.2, 47.9 (CH₂CH₂N), 36.1 (SCH₂), 32.2, 25.2, 24.8 (CH₂CH₂N), 24.4; m/z (HR CI+) found [MH]⁺ 451.1474. C20H27N4O4S2 requires: 451.1468. 1 -(4-phenylpiperidin- 1 -yl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyPphenyl)- 1 ,3,4-oxadiazol-2- yl)thio)ethan-l-one (compound 24

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and 4- phenylpiperidine (0.05 g, 0.30 mmol). Reaction was stirred at room temperature for 18 hours before being washed with HCl (1 M) and NaHC0₃ (satd. so In.) and extracted with further CH₂CI₂ (50 mL). Product was purified by flash silica chromatography to give l-(4- phenylpiperidin- 1 -yl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)- 1 ,3,4-oxadiazol-2- yl)thio)ethan-l-one as a white solid (0.06 g, 38%).

1H NMR (600 MHz, DMSO) δ 8.22 (2H, d, J = 8.3 Hz, Ar H), 8.01 (2Η, d, J = 8.3 Hz, Ar H), 7.31 (2Η, t, J = 7.6 Hz, Ar H), 7.26 (2Η, d, J = 7.6 Hz, Ar H), 7.21 (1Η, t, J = 7.6 Hz, Ar H), 4.65 (2Η, q, J = 15.4 Hz, SC¾), 4.50 (1H, d, J = 13.1 Hz), 4.03 (1H, d, J = 1 1.6 Hz), 3.27 - 3.20 (1H, m), 3.19 (4H, t, J = 6.7 Hz, S0₂NCH₂), 2.81 (1Η, m), 2.74 (1Η, m), 1.88 - 1.78 (2Η, m), 1.74 (1Η, m), 1.69 - 1.62 (4Η, m, S0₂NCH₂CH₂ ), 1.50 (1Η, m); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.8 (CONH), 164.3 (CS), 164.0 (CON), 145.6, 138.9, 128.5, 128.4, 127.4, 126.8, 126.7, 126.3, 47.9 (CH₂CH₂N), 46.1 , 42.4, 41.5, 37.3 (SCH₂), 33.1 , 32.6, 24.8 (CH₂CH₂N); m/z (HR CI+) found [MH]⁺ 513.1609. C₂₅H₂9N40₄S₂ requires: 513.1625.

N-(4-ethylphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetamide (compound 25)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂C1₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and 4- ethylaniline (0.04 mL, 0.30 mmol). Reaction was stirred at room temperature for 18 hours before being purified by flash silica chromatography to give N-(4-ethylphenyl)-2-((5-(4- (pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (0.07 g, 48%).

'H NMR (600 MHz, (CD₃)₂SO)) δ: 10.39 (s. 1H), 8.18 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.36 (s, 2H), 3.18 (t, J = 6.6 Hz, 4H), 2.58 - 2.52 (m, 2H), 1.70 - 1.60 (m, 4H), 1.15 (t, J = 7.6 Hz, 3H); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.6 (CONH), 164.5 (CS), 164.2 (CON), 139.2, 138.9, 136.4, 128.3, 128.1 , 127.3, 126.7, 1 19.3, 47.9 (NCH₂), 36.9 (SCH₂), 27.6 (CH₂), 24.8 (CH₂CH₂N), 15.7 (CH₃); HRMS (m/z): [CI]⁺ calcd. for C₂₂H₂₄N₄0₄S₂, 472.1233; found 472.1228 N-(4-methoxyphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l ,3,4-oxadiazol-2- yl)thio)acetamide (com ound 26)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and p- anisidine (0.03 mL, 0.30 mmol). Reaction was stirred at room temperature for 1 hour before being washed with HCl (IM) and NaHC0₃ (satd. soln.) and extracted with further CH₂C1₂ (50 mL). Product was purified by flash silica chromatography and solvent removed under reduced pressure to give N-(4-methoxyphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4- oxadiazol-2-yl)thio)acetamide as a light brown solid (0.05 g, 35%).

1H NMR (600 MHz, (CD₃)₂SO)) δ: 10.33 (s, 1H), 8.18 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.34 (s, 2H), 3.72 (s, 3H), 3.18 (t, J = 6.7 Hz, 4H), 1.66 (m, 4H); ¹³C NMR (150 MHz, (CD₃)₂SO))) δ: 164.5 (CONH), 164.3 (CS), 164.2 (CON), 155.5, 138.9, 131.8, 128.3, 127.3, 126.7, 120.7, 114.0, 55.2, 47.9 (NCH₂), 36.8 (SCH₂), 24.8 (CH₂CH₂N), 20.5 (CH₃); HRMS (m/z): [CI]⁺ calcd. for C₂iH₂₃N₄0₅S₂, 475.1104; found 475.1104.

2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)-N-(4- (trifluoromethyDphenyl acetamide (compound 27)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added (Benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (0.16 g, 0.30 mmol), Et₃N (0.04 mL, 0.30 mmol) and p-(trifluoromethyl)aniline (0.04 mL, 0.30 mmol). Reaction was stirred at room temperature for 18 hours before being washed with HCl (IM) and NaHC0₃ (satd. soln.) and extracted with further CH₂CI₂ (50 mL). Product was purified by flash silica chromatography and solvent removed under reduced pressure to give 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)-N-(4-(trifluoromethyl)phenyl)acetamide as a light brown solid (0.03 g, 20%).

1H NMR (600 MHz, DMSO) δ 10.8 (s, 1H), 8.20 - 8.15 (m, 2H), 8.01 - 7.96 (m, 2H), 7.80 (d, J= 8.6 Hz, 2H), 7.71 (d, J= 8.6 Hz, 2H), 4.42 (s, 2H), 3.18 (t, J= 6.7 Hz, 4H), 1.69 - 1.60 (m, 4H); ¹³C NMR (150 MHz, DMSO) δ 165.6, 164.4, 164.2, 142.2, 138.9, 128.3, 127.3, 126.7, 126.3 (q, ³JcF = 3.0 Hz), 124.3 (q, ¹J_CF = 270.0 Hz), 123.7 (q, ²J_CF = 37.5 Hz), 119.1, 47.9, 36.9, 24.8; HRMS (m/z): [CI]⁺ calcd. for C₂₁H₂₀F₃N₄S₂, 513.0878; found 513.0869.

N-(4-benzylphenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 28)

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetic acid (0.10 g, 0.30 mmol) in CH₂CI₂ (5 mL) was added l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (0.07 g, 0.40 mmol) and 4- benzylaniline (0.06 g, 0.30 mmol). Reaction was stirred at room temperature for 18 hours before being purified by flash silica chromatography to give N-(4-benzylphenyl)-2-((5-(4- (pyrrolidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (0.05 g,

30%).

1H NMR (600 MHz, DMSO) δ 10.41 (s, 1H), 8.20 - 8.16 (m, 2H), 8.00 - 7.95 (m, 2H), 7.49 (d, J = 8.5 Hz, 2H), 7.27 (t, J = 7.5 Hz, 2H), 7.19 (m, 5H), 4.35 (s, 2H), 3.89 (s, 2H), 3.18 (t, J = 6.7 Hz, 4H), 1.67 - 1.63 (m, 4H); ¹³C NMR (151 MHz, DMSO) δ 164.7, 164.5, 164.2, 141.4, 138.9, 136.7 (signals overlapping), 129.1, 128.6, 128.5, 128.3, 127.3, 126.7, 126.0, 119.4, 47.9, 40.5, 36.8, 24.8; [CI]⁺ calcd. for C27H27N4O4S2, 535.14737; found 535.1478. Synthesis of N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin- 1 -ylsulfonyl)phenyl)-4H- 1 ,2,4-triazol-3- yl)thio)acetamide (compound 29) 5-(4-(pyrrolidin- l-ylsulfonyl)phen -4H- 1 ,2 ,4-triazole-3-thiol

To a stirred solution of 4-(pyrrolidin-l-ylsulfonyl)benzohydrazide (0.10 g, 0.30 mmol) in ethanol (15 mL) was added (trimethylsilyl)isothiocyanate (0.04 mL, 0.30 mmol). The reaction was stirred at reflux for 8 hours, after which time further (trimethylsilyl)isothiocyanate (0.04 mL, 0.30 mmol) was added and the reaction stirred for a further 18 hours under reflux. After this time NaOH (2 mL, 4 M) was added and the reaction stirred at reflux for a further 4 hours. The reaction was then allowed to cool to room temperature, concentrated under vacuum and redissolved in water (5 mL). Glacial acetic acid (0.50 mL) was then added and a white precipitate formed which was then filtered to give 5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-4H- l,2,4-triazole-3-thiol as an off-white solid (35 mg, 38%).

2-((5-(4-(pyrrolidin-l-yl lfonyl)phenyl)-4H-l,2A-triazo acid

H

To a stirred solution of 5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-4H-l,2,4-triazole-3-thiol (35 mg, 0.11 mmol) in ethanol (10 mL) was added NMM (0.02 mL, 0.2 mmol) and bromoacetic acid (0.025 g, 0.17 mmol). The reaction was stirred for 18 hours at room temperature before being concentrated, redissolved in EtOAc and washed with 1 M HC1. The product was extracted with further EtOAc, and then concentrated to give 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-4H- l,2,4-triazol-3-yl)thio)acetic acid as a colourless oil (35 mg, 95%). N-(4-chlorophenyl)-2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)

yl)thio)acetamide (compound 29

To a stirred solution of 2-((5-(4-(pyrrolidin-l-ylsulfonyl)phenyl)-4H-l,2,4-triazol-3- yl)thio)acetic acid (0.08 g, 0.20 mmol) in CH₂C1₂ (5 mL) was added EDC.HC1 (0.040 g, 0.20 mmol) and /?-chloroaniline (0.02 g, 0.20 mmol). The reaction was stirred at room temperature for 1 hour before being washed with HCl (1 M) and extracted with further CH₂C1₂ (50 mL). The product was purified by flash silica chromatography (EtOAcxyclohexane, 2:8) to give 6 as a white solid (0.01 g, 10%).

1H NMR (600 MHz, (CD₃)₂SO)) δ; 10.48 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.92 (m, 3H), 7.62 (d, J = 8.8 Hz, 2H), 7.41-7.32 (m, 2H), 4.17 (s, 2H), 3.16 (t, J = 6.3 Hz, 4H), 1.64 (m, 4H); ¹³C NMR (150 MHz, (CD₃)₂SO)) δ: 165.1 (CONH), 164.4 (CS), 164.2 (CON), 138.9 (CS0₂), 137.6 (C-NH), 128.9, 128.3, 127.3 (signals overlapping), 126.7 (CC1), 120.7, 47.94 (NCH₂), 36.83 (SCH₂), 24.80 (CH₂CH₂N); HRMS (m/z): [MH]⁺ calcd. for C₂₀H₂iClN₅O₃S₂, 478.0769; found 478.0766.

Synthesis of N-(4-Chlorophenyl)-2-((5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 32) 4- (Morpholinosulfonyl) benzoic acid

Morpholine (793 μί, 9.07 mmol) was added to a stirred solution of 4-(chlorosulfonyl)benzoic acid (1.00 g, 4.53 mmol) in NaOH (10 mL, 10% aq.). The reaction was stirred at RT for 16 h before being acidified with cone. HCl. The resulting white precipitate was filtered, washed with HCl (1M aq.) and dried to give 4-(morpholinosulfonyl)benzoic acid as a white solid (650 mg, 53%>) which was used without further purification. δ_Η (600 MHz, dg-DMSO) 13.52 (IH, br s, COOH), 8.17 (2Η, d, J 8.5 Hz, Ar CH), 7.85 (2Η, d, J 8.5 Hz, Ar CH), 3.62 (4Η, dd, J 5.0, 4.0 Hz, C¾), 2.89 (4H, dd, J 5.0, 4.0 Hz, CH₂); 5_C (150 MHz, de-DMSO) 166.2 (COOH), 138.1 (Ar Q, 135.0 (Ar Q, 130.3 (Ar CH), 128.0 (Ar CH), 65.3 (CH₂), 45.9 (CH₂); HRMS (CI +ve) found 272.058602; CnHi₄N0₅S (MH⁺) requires 272.05872. tert-Butyl 2-(4-(morpholinosulfon l)benzoyl)hydrazine-l-carboxylate

HBoc

To a stirred solution of 4-(morpholinosulfonyl)benzoic acid (600 mg, 2.21 mmol) in DCM (0.60 mL) was added tert-bvXy\ carbazate (304 mg, 2.30 mmol) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (441 mg, 2.30 mmol). The reaction was stirred for 16 h at RT before being diluted in DCM (30 mL), washed with NaHC0₃ (10 mL, sat. aq.) and NaCl (10 mL, sat. aq.) and dried (MgS0₄) to give tert-butyl 2- (4-(morpholinosulfonyl)benzoyl)hydrazine-l-carboxylate as a white solid which was used without further purification.

δ_Η (600 MHz, CDC1₃) 8.46 (IH, br s, NH), 7.99 (2Η, d, 8.0 Hz, Ar CH), 7.80 (2Η, d, 8.0 Hz, Ar CH), 6.77 (1Η, br s, NH), 3.74 (4Η, app t, 4.5 Hz, CH₂), 3.05-2.96 (4Η, m, CH₂), 1.51 (9Η, s, C(CH₃)₃). 4-(Morpholinosulfonyl)benzohydrazide

tert-Butyl 2-(4-(morpho lino sulfonyl)benzoyl)hydrazine-l-carboxy late (4.53 mmol) was cooled to 0°C and trifluoroacetic acid (9.06 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h before being quenched with NaOH (10% aq.) until effervescence ceased. The solution was extracted with DCM (3 x 100 mL) and the combined organics were dried (MgS0₄) and evaporated in vacuo to give 4-(Morpholinosulfonyl)benzohydrazide as a white solid (410 mg, 32% over three steps) which was used without further purification. δ_Η (600 MHz, dg-DMSO) 10.06 (1H, s, NH), 8.05 (2Η, d, J 8.5 Hz, Ar CH), 7.81 (2Η, d, J 8.5 Hz, Ar CH), 3.63 (4Η, app t, J 5.0 Hz, C¾), 2.88 (4H, app t, J 5.0 Hz, CH₂); 5_C (150 MHz, dg- DMSO) 164.4 (CONHNH₂), 137.6 (Ar Q, 136.5(Ar Q, 128.1 (Ar CH), 127.8 (Ar CH), 65.3 (CH₂), 45.9 (CH₂); HRMS (EI +ve) found 285.077778; CnHi₅N₃0₄S (M⁺) requires 285.07778.

(5- (4- (Morpholinosulfonyl)phenyl - 1 , 3, 4-oxadiazole-2-thiol

Potassium hydroxide (85 mg, 1.51 mmol) was added to a solution of 4-(morpholinosulfonyl)benzohydrazide (410 mg, 1.44 mmol) in ethanol (15 mL) and the mixture stirred at RT for 5 min before carbon disulfide (138 μί, 2.28 mmol) was added. The reaction was heated at reflux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (10 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give (5-(4- (morpholinosulfonyl)phenyl)-l,3,4-oxadiazole-2-thiol as a white solid (190 mg, 40 %) which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 8.14 (2H, d, J 8.0 Hz, Ar CH), 7.93 (2Η, d, J 8.0 Hz, Ar CH), 3.65- 3.62 (4Η, m, 2 x CH₂), 2.93-2.89 (4Η, m, 2 x CH₂); 5_C (150 MHz, dg-DMSO) 177.7 (Ar Q, 159.3 (Ar Q, 137.3 (Ar Q, 128.7 (Ar CH), 127.2 (Ar CH), 126.7 (Ar Q, 65.3 (2 x CH₂), 45.9 (2 x CH₂); HRMS (EI +ve) found 327.034121; Ci₂Hi₃N₃0₄S₂ requires 327.03420.

2- ((5- (4- (Morpholinosulfonyl)phenyl)- 1 , 3, 4-oxadiazol-2-yl) thio) acetic acid

To a stirred solution of (5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazole-2-thiol (140 mg, 0.43 mmol) in ethanol (10.6 mL) was added N-methylmorpholine (61 μί, 0.56 mmol) and bromoacetic acid (65 mg, 0.47 mmol). The reaction was stirred at RT for 72 h before being evaporated in vacuo and the residue dissolved in DCM (25 mL) and washed with HC1 (1 M aq., 20 mL). The organic layer was dried (MgS0₄) and evaporated in vacuo to give 2-((5-(4- (morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid as a white solid which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 8.23 (2H, d, J 8.5 Hz, Ar CH), 7.94 (2Η, d, J 8.5 Hz, Ar CH), 4.26 (4Η, s, SCH₂), 3.64 (4Η, app t, J 4.5 Hz, 2 x C¾), 2.92 (4H, app t, J 4.5 Hz, 2 x CH₂); 5_C (150 MHz, de-DMSO) 168.9 (C=0), 164.4 (Ar Q, 164.1 (Ar Q, 137.3 (Ar Q, 128.8 (Ar CH), 127.4 (Ar CH), 127.1 (Ar Q, 65.3 (2 x CH₂), 45.9 (2 x CH₂), 34.3 (SCH₂); HRMS (EI +ve) found 386.0422; Ci₄Hi₆N30₆S₂ (MH⁺) requires 386.0481.

N-(4-Chlorophenyl)-2-((5-(4-(morpholinosulfony

(compound 32)

To a solution of 2-((5-(4-(morpholinosulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid (0.43 mmol theoretical) in DCM (10 mL) was added l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (109 mg, 0.57 mmol) and 4-chloroaniline (55 mg, 0.43 mmol). The reaction was stirred at RT for 1 h before being diluted in DCM, washed with HC1 (1M aq., 30 mL) and NaHC0₃ (sat. aq., 30 mL). Purification by flash chromatography (Si0₂, 20→70% ethyl acetate in cyclohexane) gave N-(4-chlorophenyl)-2-((5-(4-(morpholinosulfonyl)phenyl)- l,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (135 mg, 63%).

δ_Η (600 MHz, dg-DMSO) 10.25 (1H, br s, NH), 8.73 (2Η, J 8.5 Hz, Ar CH), 8.43 (2Η, d, J 8.5 Hz, Ar CH), 8.16 (2Η, d, J 8.5 Hz, Ar CH), 7.82 (2Η, d, J 8.5 Hz, Ar CH), 4.85 (2Η, s, SCH₂), 4.10 (4Η, app t, J 5.0 Hz, 2 x CH₂), 3.47 (4Η, app t, J 4.5 Hz, 2 x CH₂); 5_C (150 MHz, dg- DMSO) 164.9 (Q, 164.8 (Q, 164.6 (Q, 138.2 (Ar Q, 137.7 (Ar Q, 128.79 (Ar CH), 128.78 (Ar CH), 128.2 (Ar Q, 127.7 (Ar Q, 127.2 (Ar CH), 120.9 (Ar CH), 65.7 (2 CH₂), 46.2 (2 x CH₂), 36.9 (SCH₂); HRMS (EI +ve) found 494.048057; C₂oHi₉Cl ₄0₅S₂ requires 494.04799.

Synthesis of N-(4-Chlorophenyl)-2-((5-(4-(piperidin- 1 -ylsulfonyPphenyl)- 1 ,3 ,4-oxadiazol-2- yl)thio)acetamide (compound 33) 4-(Piperidin-l-ylsulfonyl)benzoic acid

Piperidine (449 μί, 4.53 mmol) was added to a stirred solution of 4-(chlorosulfonyl)benzoic acid (1.00 g, 4.53 mmol) and triethylamine (63 μί, 0.45 mmoL) in DCM (45 mL). The reaction was stirred at RT for 16 h before being washed with HCl (1M aq.) and dried (MgS0₄) to give 4- (piperidin-l-ylsulfonyl)benzoic acid as a white solid which was used without further purification.

HRMS (ES -ve) found 268.0632; Ci₂Hi₄N0₄S ([Μ-ΗΓ) requires 268.0644. tert-Butyl 2- (4- (piperidin- 1 -ylsulfony I) benzoyl) hydrazine- 1 -carboxylate

To a stirred solution of 4-(piperidin-l-ylsulfonyl)benzoic acid (4.53 mmol theoretical) in DCM (0.30 mL) was added tert-bvXy\ carbazate (622 mg, 4.71 mmol) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (903 mg, 4.71 mmol). The reaction was stirred for 16 h at RT before being diluted in DCM (30 mL), washed with NaHC0₃ (10 mL, sat. aq.) and NaCl (10 mL, sat. aq.) and dried (MgS0₄) to give tert-butyl 2- (4-(piperidin-l-ylsulfonyl)benzoyl)hydrazine-l -carboxylate as a white solid which was used without further purification.

δ_Η (600 MHz, CDC1₃) 8.41-8.28 (1H, br s, NH), 7.94 (2Η, d, J 8.0 Hz, Ar CH), 7.80 (2H, d, J 8.0 Hz, Ar CH), 6.85-6.71 (1H, br s, NH), 2.98 (2Η, t, J 5.5 Hz, 2 x CH₂), 1.63 (2H, quin, J 5.5 Hz, 2 x CH₂), 1.50 (9H, s, C(C¾)₃), 1.45-1.39 (2H, m, CH₂); 5_C (150 MHz, CDC1₃) 165.6 (CO), 155.6 (C0₂t-Bu), 140.0 (Ar Q, 135.7 (Ar Q, 128.1 (Ar CH), 128.0 (Ar CH), 82.6 (C(CH₃)), 47.0 (NCH₂), 28.2 (CH₂), 25.2 (CH₂), 23.5 (CH₂); HRMS (ES -ve) found 382.1446; CivH₂₄N₃0₅S ([M-H]^") requires 382.1437. 4- (Piperidin- 1 -ylsulfonyl) benzohydrazide

tert-Butyl 2-(4-(piperidin-l-ylsulfonyl)benzoyl)hydrazine-l-carboxylate (290 mg, 0.76 mmol) was cooled to 0°C and trifluoroacetic acid (1.51 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h before being quenched with NaOH (10% aq.) until effervescence ceased. The solution was extracted with DCM (3 x 50 mL) and the combined organics were dried (MgS0₄) and evaporated in vacuo to give 4-(piperidin-l- ylsulfonyl)benzohydrazide as a white solid (65 mg, 30% over three steps) which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 10.04 (1H, s, NH), 8.02 (2Η, d, J 8.5 Hz, Ar CH), 7.80 (2Η, d, J 8.5 Hz, Ar CH), 2.90 (2Η, t, J 8.3 Hz, 2 x CH₂CH₂CH₂N), 1.53 (4Η, app. quin., J 5.0 Hz, 2 x CH₂CH₂CH₂N), 1.39-1.33 (2H, m, CH₂CH₂CH₂N); HRMS (CI +ve) found 284.107577; C₂iHi₈N30₃S (MH⁺) requires 284.10634. 5-(4-(Piperidin- l-ylsulfonyl)phenyl)- 1 ,3 ,4-oxadiazole-2-thiol

Potassium hydroxide (12.4 mg, 0.22 mmol) was added to a solution of 4-(piperidin-l- ylsulfonyl)benzohydrazide (60 mg, 0.21 mmol) in ethanol (2.2 mL) and the mixture stirred at RT for 5 min before carbon disulfide (20 μί, 0.33 mmol) was added. The reaction was heated at reflux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (10 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give 5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazole- 2-thiol as a white solid (45 mg, 66 %) which was used without further purification.

δ_Η (600 MHz, de-acetone) 8.19 (2H, d, J 8.0 Hz, Ar CH), 7.99 (2Η, d, J 8.0 Hz, Ar CH), 3.09- 2.97 (4Η, m, 2 x CH₂CH₂CH₂N), 1.67-1.57 (4Η, m, 2 x CH₂CH₂CH₂N), 1.48-1.40 (2H, m, CH₂CH₂CH₂N) ; 5_C (150 MHz, d₆-acetone) 179.3 (C), 160.5 (C), 140.5 (Ar C), 129.5 (Ar CH), 127.8 (Ar CH), 127.6 (Ar C), 47.8 (CH₂CH₂CH₂N), 26.0 (CH2CH₂CH₂N), 24.1 (CH₂CH₂CH₂N); HRMS (ES +ve) found 326.0628; Ci₃Hi₆N₃0₃S₂ (MH⁺) requires 326.0633. 2- ((5- (4- (Piperidin-l-ylsulfon l)phenyD-l, 3, 4-oxadiazol-2-yl) thio) acetic acid

To a stirred solution of 5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazole-2-thiol (41 mg, 0.13 mmol) in ethanol (3.2 mL) was added N-methylmorpholine (19 μί, 0.17 mmol) and bromoacetic acid (19.3 mg, 0.14 mmol). The reaction was stirred at RT for 72 h before being evaporated in vacuo and the residue dissolved in DCM (25 mL) and washed with HCl (1 M aq., 20 mL). The organic layer was dried (MgS0₄) and evaporated in vacuo to give 2-((5-(4- (piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid as a white solid which was used without further purification.

δ_Η (600 MHz, dg-acetone) 8.27 (2H, d, J 8.5 Hz, Ar CH), 7.98 (2Η, d, J 8.5 Hz, Ar CH), 4.32 (2Η, s, SCH₂), 3.04 (4Η, app. t, J 5.5 Hz, 2 x CH₂CH₂CH₂N), 1.63 (4Η, app. quin., J 5.5 Hz, 2 x CH₂CH₂CH₂N), 1.45 (2H, t, J 5.5 Hz, CH₂CH₂CH₂N); 5_C (150 MHz, dg-acetone) 168.7 (COOH), 165.5 (Q, 165.3 (Q, 140.2 (Ar Q, 129.4 (Ar CH), 128.2 (Ar Q, 128.0 (Ar CH), 47.7 (CH₂CH₂CH₂N), 34.9 (SCH₂), 25.9 (CH₂CH₂CH₂N), 24.0 (CH₂CH₂CH₂N); HRMS (EI -ve) found 382.0542; Ci₅Hi₆N₃0₅S₂ ([M-H]^") requires 382.0531.

N-(4-Chlorophenyl)-2-((5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 33

To a solution of give 2-((5-(4-(piperidin-l-ylsulfonyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid (0.13 mmol theoretical) in DCM (2.6 mL) was added (benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (88 mg, 0.17 mmol) and 4-chloroaniline (17 mg, 0.13 mmol). The reaction was stirred at RT for 1 h before being diluted in DCM, washed with HCl (1M aq., 30 mL) and NaHC0₃ (sat. aq., 30 mL). Purification by flash chromatography (Si0₂, 10→50% ethyl acetate in cyclohexane) gave N-(4-chlorophenyl)-2-((5- (4-(piperidin-l-ylsulfonyl)-l,3,4-oxadiazol-2-yl)thio)acetamide as a white solid (10 mg, 16%). δ_Η (600 MHz, dg-DMSO) 10.61 (IH, s, NH), 8.18 (2Η, d, J 8.5 Hz, Ar CH), 7.91 (2Η, d, J 8.5 Hz, Ar CH), 7.62 (2Η, d, J 9.0 Hz, Ar CH), 7.39 (2Η, d, J 9.0 Hz, Ar CH), 4.38 (2Η, s, SCH₂), 2.93 (4Η, app. t, J 5.5 Hz, CH₂CH₂CH₂N), 1.54 (4Η, dt, J 11.5, 6.0 Hz, CH₂CH₂CH₂N), 1.38- 1.33 (2H, m, CH₂CH₂CH₂N); 5_C (150 MHz, dg-DMSO) 165.1 (Q, 164.5 (Q, 164.2 (Q, 138.4 (Ar Q, 137.6 (Ar Q, 128.9 (Ar CH), 128.5 (Ar CH), 127.32 (Ar CH), 127.30 (Ar Q, 126.8 (Ar Q, 120.7 (Ar CH), 46.6 (CH₂CH₂CH₂N), 36.8 (SCH₂), 24.7 (CH₂CH₂CH₂N), 22.8 (CH₂CH₂CH₂N); HRMS (EI -ve) found 491.0598; C₂iH₂iN₄0₄S₂ ([M-H] ) requires 491.0615.

Synthesis of 2-((5-(4-((4-(tert-Butyl)piperidin-l-yl)sulfonyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)- N-(4-chlorophenyl)acetamide (compound 34)

4-((4-(tert-Butyl)piperidin-l-yl)sul onyl)benzoic acid

tert-Butylpiperidine hydrochloride (1.00 g, 7.08 mmol) was added to a stirred solution of 4-(chlorosulfonyl)benzoic acid (1.56 g, 7.08 mmol) and triethylamine (1.09 mL, 7.79 mmol) in DCM (45 mL). The reaction was stirred at RT for 16 h before being washed with HC1 (1M aq.) and dried (MgS0₄) to give 4-(piperidin-l-ylsulfonyl)benzoic acid as a white solid which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 8.15 (2H, d, J 8.5 Hz, Ar CH), 7.84 (2Η, d, J 8.5 Hz, Ar CH), 3.73 (2Η, d, J 11.5 Hz, 2 x NCHH'), 2.16 (2H, app. t, J 11.5 Hz, NCHH'), 1.66 (2Η, d, J 12.0 Hz, NCH₂CHH'), 1.17 (2H, app. t, J 7.0 Hz, NCH₂CHH'), 0.91 (1Η, tt, J 12.0, 3.0 Hz, CH(t-Bu)), 0.76 (9Η, s, C(CH₃)₃); 5_C (150 MHz, dg-DMSO) 167.3 (COOH), 140.6 (Ar Q, 136.2 (Ar Q, 130.1 (Ar CH), 127.7 (Ar CH), 46.7 (CH₂N), 44.7 (CH(t-Bu)), 31.8 (C(CH₃)), 27.0 (C(CH₃)₃), 25.6 (CH₂CH₂N); HRMS (ES +ve) found 326.1439; Ci₆H₂₄N0₄S (MH⁺) requires 326.1426. tert-Butyl 2-(4-((4-(tert-butyl)pi eridin-l-yl)sulfonyl)benzoyl)hydraz

To a stirred solution of 4-((4-(tert-butyl)piperidin-l-yl)sulfonyl)benzoic acid (2.11 g, 6.50 mmol) in DCM (0.60 mL) was added tert-butyl carbazate (892 mg, 6.75 mmol) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (1.29 g, 6.75 mmol). The reaction was stirred for 16 h at RT before being diluted in DCM (30 mL), washed with NaHC0₃ (15 mL, sat. aq.) and NaCl (15 mL, sat. aq.) and dried (MgS0₄) to give tert-butyl 2-(4-((4-(tert- butyl)piperidin-l-yl)sulfonyl)benzoyl)hydrazine-l-carboxylate as a white solid which was used without further purification.

δ_Η (600 MHz, CDC1₃) 7.97 (2H, d, J 8.0 Hz, Ar CH), 7.80 (2Η, d, J 8.0 Hz, Ar CH), 3.86 (2Η, d, J 11.5 Hz, NCHH'), 2.18-2.12 (2H, m, NCHH'), 1.72 (2Η, d, J 13.2 Hz, NCH₂CHH'), 1.50 (9H, s, C0₂C(CH₃)₃), 1.36 (2Η, qd, J 12.5, 3.8 Hz, NCH₂CHH'), 0.90-0.85 (1Η, m, CH(t-Bu)), 0.81 (9Η, s, CHC(CH₃)₃; 5_C (150 MHz, CDC1₃) 268.5 (C=0), 155.6 (COt-Bu), 140.7 (Ar Q, 139.7 (Ar Q, 128.1 (Ar CH), 127.5 (Ar CH), 82.5 (C(CH₃)₃), 47.2 (CH₂CH₂N), 45.9 (CH(t- Bu)), 32.2 CHC(CH₃)₃, 28.3 (C(CH₃)₃), 27.3 (C(CH₃)₃), 26.3 (CH₂CH₂N); HRMS (ES +ve) found 439.2697; C₂iH₃₄N₃0₅S (MH⁺) requires 439.2219.

4-((4-(tert-Butyl)piperidin-l-yl)sul onyl)benzohydrazide

tert-Eutyl 2-(4-((4-(tert-butyl)piperidin- 1 -yl)sulfonyl)benzoyl)hydrazine- 1 -carboxylate (6.50 mmol) was cooled to 0°C and trifluoroacetic acid (13.0 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h before being quenched with NaOH (10% aq.) until effervescence ceased. The solution was extracted with DCM (3 x 100 mL) and the combined organics were dried (MgS0₄) and evaporated in vacuo to give 4-((4-(tert-butyl)piperidin-l- yl)sulfonyl)benzohydrazide as a white solid (1.60 g, 72% over four steps) which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 10.69 (1H, s, NH), 8.02 (2Η, d, J 8.5 Hz, Ar CH), 7.80 (2Η, d, J 8.5 Hz, Ar CH), 4.70-4.50 (2Η, br m, NH₂), 3.74 (2Η, d, J 11.5 Hz, NCHH'), 2.14 (2H, dd, J 12.0, 10.0 Hz, NCHH'), 1.66 (2Η, d, J 12.0 Hz, NCH₂CHH'), 1.17 (2H, qd, J 12.5, 4.0 Hz, NCH₂HH'), 0.96-0.86 (1Η, m, CH(t-Bu)), 0.77 (9Η, C(CH₃)₃); 5_C (150 MHz, dg-DMSO) 164.4 (C=0), 137.6 (Ar Q, 137.2 (Ar Q, 127.9 (Ar CH), 127.6 (Ar CH), 46.7 (NCH₂), 44.8 (CH(t- Bu)), 31.8 (C(CH₃)₃), 27.0 (C(CH₃)₃), 25.6 (NCH₂CH₂); HRMS (EI -ve) found 338.1541; Ci₆H₂₄N₃0₃S requires 338.1538. 5-(4-((4-( tert-Butyl)piperidin-l-yl)sulfonyl)phenyl)-l, 3, 4-oxadiazole-2-thiol

Potassium hydroxide (278 mg, 4.95 mmol) was added to a solution of 4-((4-(tert- butyl)piperidin-l-yl)sulfonyl)benzohydrazide (1.60 g, 4.71 mmol) in ethanol (47 mL) and the mixture stirred at RT for 5 min before carbon disulfide (455 μί, 7.54 mmol) was added. The reaction was heated at reflux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (20 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give 5-(4-((4-(tert-butyl)piperidin- l-yl)sulfonyl)phenyl)-l,3,4-oxadiazole-2-thiol as a white solid which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 8.11 (2H, d, J 7.5 Hz, Ar CH), 7.92 (2Η, d, J 7.5 Hz, Ar CH), 3.70 (2Η, obscured by water peak, 2 x NCHH'), 2.24-2.10 (2H, 2 x NCHH'), 1.81- 1.56 (2Η, 2 x NCH₂CHH'), 1.35-1.09 (2 x NCH₂CHH'), 0.98-0.88 (1Η, m, CH(t-Bu)), 0.77 (9Η, s, C(CH₃)₃); 5c (150 MHz, de-DMSO) 177.7 (Q, 159.3 (Q, 138.4 (Ar Q, 128.5 (Ar CH), 127.0 (Ar CH), 126.4 (Ar Q, 46.7 (NCH₂), 44.7 (CH(t-Bu)), 31.8 (C(CH₃)₃), 27.0 (C(CH₃)₃), 25.7 (NCH₂CH₂); HRMS (CI +ve) found 382.125631; Ci₇H₂₄N₃0₃S₂ (MH⁺) requires 382.12591.

2-((5-(4-((4-( tert-Butyl)piperidin-l- l)sulfonyl)phenyl)-l, 3, 4-oxadiazol-2-yl) thio) acetic acid

δ_Η (600 MHz, dg-DMSO) 13.40-12.80 (1H, br s, COOH), 8.20 (2Η, d, J 8.5 Hz, Ar CH), 7.93 (2Η, d, J 8.5 Hz, Ar CH), 4.25 (2Η, s, SCH₂), 3.78-3.73 (2H, m, 2 x NCHH'), 2.19 (2H, t, J 11.5 Hz, 2 x NCHH'), 1.71-1.64 (2Η, m, 2 x NCH₂CHH'), 1.17 (2H, td, J 12.5, 3.5 Hz, NCH₂CHH'), 0.97-0.89 (1Η, m, CH(t-Bu)), 0.77 (9Η, s, C(CH₃)₃); 5_C (150 MHz, dg-DMSO) 168.9 (Q, 164.4 (Q, 164.1 (Q, 138.3 (Ar Q, 128.5 (Ar CH), 127.3 (Ar CH), 126.8 (Ar Q, 46.7 (NCH₂CH₂), 44.7 (CH(t-Bu)), 34.4 (SCH₂), 31.8 (C(CH₃)₃), 27.0 (C(CH₃)₃), 25.7 (NCH₂CH₂); HRMS (ES +ve) found 440.1347; Ci₉H₂gN₃0₅S₂ (MH⁺) requires 440.1314.056 2-((5-(4-((4-( tert-Butyl)piperidin-l-yl)sulfonyl)phenyl)-l, 3, 4-oxadiazol-2-yl) thio)-N- ( 4- chlorophenyDacetamide compound 34)

To a solution of give 2-((5-(4-((4-(tert-butyl)piperidin-l-yl)sulfonyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetic acid (250 mg, 0.57 mmol) in DCM (11.4 mL) was added (benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (385 mg, 0.74 mmol) and 4- chloroaniline (73 mg, 0.57 mmol). The reaction was stirred at RT for 1 h before being diluted in DCM, washed with HC1 (1M aq., 30 mL) and NaHC0₃ (sat. aq., 30 mL). Purification by flash chromatography (Si0₂, 50→100% ethyl acetate in cyclohexane) gave 2-((5-(4-((4-(tert- butyl)piperidin- 1 -yl)sulfonyl)phenyl)- 1 ,3 ,4-oxadiazol-2-yl)thio)-N-(4-chlorophenyl)acetamide as a white solid (15 mg, 5%).

δ_Η (600 MHz, dg-DMSO) 10.62 (1H, s, NH), 8.18 (2Η, d, J 8.0 Hz, Ar CH), 7.91 (2Η, d, J 8.0 Hz, Ar CH), 7.62 (2Η, J 8.5 Hz, Ar CH), 7.40 (2Η, d, J 8.5 Hz, Ar CH), 4.38 (2Η, s, SCH₂), 3.76 (2Η, d, J 12.0 Hz, NCHH'), 2.18 (2H, dd, J 12.5, 11.5 Hz, NCHH'), 1.67 (2Η, d, J 12.5 Hz, NCH₂CHH'), 1.22-1.13 (2H, m, NCH₂CHH'), 0.96-0.89 (1Η, m, CH(t-Bu)), 0.77 (9H, s, C(C¾)₃); 5c (150 MHz, d₆-acetone) 170.0 (C=0), 165.54 and 165.52 (2 x Q, 143.9 (Ar Q, 129.7 (Ar Q, 129.5 (Ar Q, 129.2 (Ar Q, 128.0 (2 peaks, 2 x Ar CH), 125.5 (Ar CH), 121.8 (Ar Q, 119.8 (Ar CH), 49.8 (NCH₂), 47.9 (CH(t-Bu)), 37.6 (SCH₂), 32.6 (C(CH₃)₃), 27.4 (C(CH₃)₃), 26.97 (NCH₂CH₂); HRMS (ES -ve) found 547.1253; C₂₅H₂₈N₄0₄S₂C1 ([M-H]^") requires 547.1246.

Synthesis of N-(4-Chlorophenyl)-2-((5-(4-(N,N-dibutylsulfamoyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 35)

4-(N,N-Dibutylsulfamoyl)benzoic

Dibutylamine (11.5 mL, 68.0 mmol) and triethylamine (63 μί, 0.45 mmol) was added to a stirred solution of 4-(chlorosulfonyl)benzoic acid (1.00 g, 4.53 mmol) in DCM (45 mL). The reaction was stirred at RT for 16 h before being diluted in DCM (20 mL) and washed with aq. HC1 (1.0 M, 2 x 50 mL). The combined organics were dried (MgS0₄) and evaporated in vacuo to give 4-(N,N-dibutylsulfamoyl)benzoic acid as a white solid (1.23 g, 87%).

δ_Η (600 MHz, dg-DMSO) 13.71-13.31 (1H, br s, COOH), 8.06 (2Η, d, J 8.5 Hz, Ar CH), 7.90 (2Η, d, J 8.5 Hz, Ar CH), 3.08 (4Η, dd, J 8.0, 7.0 Hz, 2 x NC¾), 1.42 (4H, app. quin., J 7.5 Hz, 2 x C¾), 1.23 (4H, app. sext., J 7.5 Hz, 2 x C¾), 0.84 (6H, J 7.5 Hz, 2 x C¾); 5_C (150 MHz, DMSO) 166.3 (COOH), 143.1 (Ar Q, 134.3 (Ar Q, 130.3 (Ar CH), 127.1 (Ar CH), 47.6 (NCH₂), 30.4 (CH₂), 19.3 (CH₂), 13.6 (CH₃); HRMS (ES -ve) found 312.1279; Ci₅H₂₂N0₄S ([M-H]^") requires 312.1270. tert-Butyl 2-(4-iN,N-dibutylsulfamoyl)benzoyl)hydrazine-l-carboxylate

To a stirred solution of 4-(N-(sec-butyl)sulfamoyl)benzoic acid (1.23 mg, 3.93 mmol) in DCM (0.10 mL) was added tert-butyl carbazate (3 portions of 200 mg, 1.51 mmol each) and l-[3- (dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (3 portions of 290 mg, 1.51 mmol each). The reaction was stirred for 16 h at RT before being diluted in DCM (30 mL), washed with NaHC0₃ (25 mL, sat. aq.) and NaCl (25 mL, sat. aq.) and dried (MgS0₄) to give tert-butyl 2-(4-(N,N-dibutylsulfamoyl)benzoyl)hydrazine-l-carboxylate as a white crystalline solid (1.68 g, quant.) which was used without further purification.

δ_Η (600 MHz, CDC1₃) 7.93 (2H, d, J 8.0 Hz, Ar CH), 7.81 (2Η, d, J 8.0 Hz, Ar CH), 3.09 (4Η, dd, J 8.0, 7.0 Hz, 2 x NCH₂), 1.52-1.41 (13Η, m, 2 x CH₂ and C(CH₃)₃), 1.27 (4Η, dq, J 15.0, 7.5 Hz, 2 x CH₂), 0.88 (6Η, t, J 7.5 Hz, 2 x CH₃); 5_C (150 MHz, CDC1₃) 165.7 (NCON), 155.7 (NC0₂), 143.6 (Ar Q, 135.3 (Ar Q, 128.3 (Ar CH), 127.4 (Ar CH), 82.4 (C(CH₃)₃), 48.1 (NCH₂), 30.8 (CH₂), 28.2 (CH₂), 20.0 (C(CH₃)₃), 13.8 (CH₃); HRMS (CI +ve) found 428.221014; C₂₀H₃₄N₃O₅S (MH⁺) requires 428.22192. N,N-Dibutyl-4-(hydrazinecarbonyl)benzenesulfonamide

tert-Butyl 2-(4-(N,N-dibutylsulfamoyl)benzoyl)hydrazine- 1 -carboxylate (3.93 mmo 1 theoretical) was cooled to 0°C and trifluoroacetic acid (5.51 mL) was added dropwise. The mixture was allowed to warm to RT over 1 h 30 before being quenched and taken to pH 13 with NaOH (10% aq.) and extracted with DCM (3 x 40 mL). The combined organics were dried (MgS0₄) and evaporated in vacuo to give N,N-dibutyl-4- (hydrazinecarbonyl)benzenesulfonamide as a white solid which was used without further purification.

δ_Η (600 MHz, dg-DMSO) 10.02 (1H, s, NH), 7.99 (2Η, d, J 8.5 Hz, Ar CH), 7.86 (2Η, d, J 8.5 Hz, Ar CH), 4.55-4.63 (2Η, br s, 2 x NH), 3.07 (4Η, t, J 7.5 Hz, 2 x NC¾), 1.42 (4H, app. quin., J 7.5 Hz, 2 x CH₂), 1.23 (4H, app. sext., J 7.5 Hz, 2 x CH₂), 0.84 (6H, t, J 7.5 Hz, 2 x CH₃); 5c (150 MHz, d₆-DMSO) 164.5 (CO), 141.5 (Ar C), 136.9 (Ar C), 128.0 (Ar CH), 126.9 (Ar CH), 47.6 (NCH₂), 30.4 (CH₂), 19.3 (CH₂), 13.6 (CH₃); HRMS (CI +ve) found 328.168913; Ci₅H₂₆N₃0₃S (MH⁺) requires 328.16949.

N,N-Dibutyl-4- ( 5-mercapto-l, 3, 4-oxadiazol-2- l)benzenesulfonamide

Potassium hydroxide (312 mg, 5.56 mmol) was added to a solution of N,N-dibutyl-4- (hydrazinecarbonyl)benzenesulfonamide (1.65 g, 5.05 mmol) in ethanol (51 mL) and the mixture stirred at RT for 5 min before carbon disulfide (488 μί, 8.07 mmol) was added. The reaction was heated at reflux for 18 h before being cooled to RT and the solvent removed in vacuo. The residue was dissolved in water (50 mL) and cone. HC1 was added until a white precipitate formed. The precipitate was filtered and dried to give N,N-dibutyl-4-(5-mercapto- l,3,4-oxadiazol-2-yl)benzenesulfonamide as a white solid (1.40 g, 75%).

δ_Η (600 MHz, dg-DMSO) 15.40-14.50 (1H, br s, SH), 8.07 (2Η, d, J 8.5 Hz, Ar CH), 7.98 (2Η, d, J 8.5 Hz, Ar CH), 3.09 (4Η, app. t, J 7.5 Hz, 2 x NCH₂), 1.43 (4Η, tt, J 8.0, 7.0 Hz, 2 x CH₂),

1.24 (4Η, app. sext., J 7.5 Hz, 2 x CH₂), 0.85 (6Η, t, J 7.5 Hz, 2 x C¾); 5_C (150 MHz, DMSO) 177.7 (Q, 159.4 (Q, 142.2 (Ar Q, 127.8 (Ar CH), 127.1 (Ar CH), 126.0 (Ar Q, 47.6 (NCH₂), 30.4 (CH₂), 19.3 (CH₂), 13.6 (CH₃); HRMS (ES +ve) found 370.1260; Ci₆H₂₄N₃0₃S₂ (MH⁺) requires 370.1259. 2- ((5- (4- (N,N-Dibutylsulfamo l)phenyl)-l , J, 4-oxadiazol-2-yl) thio) acetic acid

To a stirred solution of N,N-dibutyl-4-(5-mercapto-l,3,4-oxadiazol-2-yl)benzenesulfonamide (1.35 g, 3.66 mmol) in ethanol (100 mL) was added N-methylmorpholine (523 μί, 4.76 mmol) and bromoacetic acid (559 mg, 4.02 mmol). The reaction was stirred at RT for 20 h before being evaporated in vacuo and the residue dissolved in DCM (100 mL) and washed with HC1 (1 M aq., 75 mL). The organic layer was dried (MgS0₄) and evaporated in vacuo to give 2-((5-(4- (N,N-dibutylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid as a white solid (1.57 g, quant.), which was used without further purification. δ_Η (600 MHz, dg-DMSO) 8.16 (2H, d, J 8.5 Hz, Ar CH), 7.99 (2H, d, J 8.5 Hz, Ar CH), 4.25 (2H, s, SCH₂), 3.10 (4H, t, J 7.5 Hz, 2 x NCH₂), 1.43 (4H, app. quin., J 7.5 Hz, 2 x CH₂), 1.24 (4H, app. sext., J 7.5 Hz, 2 x CH₂), 0.85 (6H, t, J 7.0 Hz, 2 x CH₃); 5_C (150 MHz, DMSO) 168.9 (Q, 164.3 (Q, 164.2 (Q, 142.1 (Ar Q, 127.9 (Ar CH), 127.4 (Ar CH), 126.4 (Ar Q, 47.6 (NCH₂), 34.3 (SCH₂), 30.4 (CH₂), 19.3 (CH₂), 13.6 (CH₃); HRMS (CI +ve) found 428.131946; Ci₈H₂₆N₃0₅S₂ (MH⁺) requires 428.13139.

N-(4-Chlorophenyl)-2-((5-(4-(NM-dibutylsulfamoyl)phenyl)-l,3,4-oxadiazol-2- yl)thio)acetamide (compound 35)

To a solution of 2-((5-(4-(N,N-dibutylsulfamoyl)phenyl)-l,3,4-oxadiazol-2-yl)thio)acetic acid (250 mg, 0.59 mmol) in DCM (9.0 mL) was added l-[3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride (146 mg, 0.76 mmol) and 4-chloroaniline (75 mg, 0.59 mmol). The reaction was stirred at RT for 16 h before being diluted in DCM (20 mL), with NaHC0₃ (sat. aq., 50 mL) and brine (sat. aq., 50 mL). Trituration of the residue with ethyl acetate gave N-(4- chlorophenyl)-2-((5-(4-(N,N-dibutylsulfamoyl)phenyl)-l ,3,4-oxadiazol-2-yl)thio)acetamide as a white solid, insoluble in the ethyl acetate solution (73 mg, 22%).

δ_Η (600 MHz, dg-DMSO) 10.63 (1H, s, NH), 8.15 (2Η, d, J 8.5 Hz, Ar CH), 7.97 (2Η, d, J 8.5 Hz, Ar CH), 7.62 (2Η, d, J 9.0 Hz, Ar CH), 7.39 (2Η, d, J 9.0 Hz, Ar CH), 4.38 (2Η, s, CH₂), 3.09 (4Η, dd, J 8.0, 7.0 Hz, 2 x NC¾), 1.46-1.40 (4H, m, 2 x C¾), 1.23 (4H, app. sext., J 7.5 Hz, 2 x C¾), 0.85 (6H, t, J 7.5 Hz, 2 x C¾); 5_C (150 MHz, DMSO) 165.1 (Q, 164.4 (Q, 164.2 (Q, 142.1 (Ar Q, 137.6 (Ar Q, 128.8 (Ar CH), 127.9 (Ar CH), 127.34 (Ar CH), 127.29 (Ar Q, 126.4 (Ar Q, 120.7 (Ar CH), 47.6 (NCH₂), 36.8 (SCH₂), 30.3 (CH₂), 19.3 (CH₂), 13.6 (CH₃); HRMS (CI +ve) found 537.138513; C₂4H₃oCl ₄0₄S₂ (MH⁺) requires 537.13970.

Inhibitory Activity (Biofilm assay)

Conclusions The targeting compounds identified herein represent novel, virulence-targeted inhibitors. The concept of such anti-virulence therapies has gained significant momentum in recent years due to the increasing realisation of the adverse effect of broad-acting bacteriostatic or bacteriolytic antibiotics (Cegelski, et al. Nat Chem Biol 2009, 5: 913-919; Dethlefsen, et al. PLoS Biol 2008, 6: e280). Moreover, targeted antibacterials minimise selective pressure that perpetuates drug resistance through horizontal gene transfer. Given the widespread conservation of DSE reaction mechanism, it is anticipated that these inhibitors will have applications beyond the inhibition of FimH-mediated adhesion.

Experimental Details

Bacterial strains

Uropathogenic E. coli UTI89, UTI89 ΔβτηΑ-Η and E. coli BL21 (DE3) are described in the Table 1.

Construction, expression and purification of FimCc-ms-FimH

To facilitate the cloning of fimH and fimC for co-expression, the MultiSite Gateway^® Three- Fragment Vector Construction Kit (Invitrogen) was used. The full-coding sequence of fimC with C-terminal His-tag was amplified from pETSlOOO (Saulino et al, EMBO J 1998, 17:2177- 2185) using primers FimCf and FimCr (Table 1). The amplicon was cloned into pDONRP4- P1R donor vector via the BP recombination (Invitrogen) resulting in pKVWcl . FimH3 and FimH7 primers were used to amplify carrying full-coding sequence of fimH. The amplicon was cloned into pDONR221 via the BP recombination (Invitrogen) yielding plasmid pENT53. In order to monitor the protein complex yield, green fluorescent protein gene gfp^ was amplified from pWH1012gfp+ (Scholz et al., Eur J Biochem 2000, 267: 1565-70) in the presence of gfp7 and gfp8 primers and the resulting amplicon was cloned into pDONRP2R-P3 to give rise to plasmid pENT105. Subsequently, pKVWcl, pENT53 and pENT105 underwent LR reaction (Invitrogen) in the presence of pDEST22a destination vector to generate pKVWel . The pDEST22a was derived from pET22b (Novagen) digested with Xbal and EcoRI and followed by fill-in reaction to generate blunt ends. The attR4-ccdB-CmI-attR3 cassette was amplified from pDEST R4-R3 using primers FP24 and RP24 and then ligated to the linearised PET22b resulting in pDEST22a.

E. coli BL21 (DE3) cells harbouring pKVWel were grown at 37 °C in minimal medium supplemented with 0.4 % glucose, 20 mM MgCl₂, 100 μg mL^"1 ampicilin and 1 % (v/v) vitamin solution. Cells were induced at OD₆oonm of 1 with 1 mM IPTG at 37 °C. After overnight incubation, cells were harvested and periplasmic extraction of these cells was carried out. FimC_hi_s:FimH complex was purified using Ni-NTA column (Qiagen). The complex was eluted with 200 mM imidazole in 20 mM Tris-HCl pH8.8, 250 mM NaCl buffer. Fractions containing the FimC_hi_s:FimH complex were as judged by SDS-PAGE were pooled and dialysed against 20 mM Mes pH6, 10 mM NaCl overnight at 4 °C. The final polishing step was done using a HiTrap SP FF column (GE Healthcare) at room temperature and subjected to 10 mM - 1 M NaCl gradient elution. Fractions were analysed by SDS-PAGE, FimCc-Hi_s:FimH was eluted at 400 mM NaCl and dialysed against 20 mM Tris pH8.8, 10 mM NaCl overnight at 4 °C.

In vitro donor strand exchange (DSE) inhibition screening assay

FimG:FimH DSE reactions were initiated by addition of 15 μΜ FimG N-terminal extension peptide (FimG_Nte), ADVTITVNGKVVAKP-amide (Bio-Synthesis) to 2 μΜ of FimC:FimH complex in 100 mM Tris HC1 pH8.8, 20 mM NaCl buffer and allowed to proceed for 12 hours at 37 °C. To screen for DSE inhibitors, DSE assays were performed in the presence of the preselected compounds to a final concentration of 500 μΜ each. A total of 20 μΐ_^ each of the DSE reaction was diluted in NuPage LDS loading buffer (Invitrogen) and loaded onto SDS-PAGE. The intensities of the coomassie-stained bands corresponding to FimC, FimH (the DSE substrate) and the SDS-resistant FimH:FimGNte complex (DSE product) (Fig. lc) were visualised using Biorad Quantity One^® imaging densitometer, and quantified and background corrected with Image Lab software (Biorad). In order to determine the IC₅₀ value of the identified hit, further DSE assays were carried out in the presence of the hit compound at varying concentrations ranging from 0.001 mM to 2 mM in 20 % (v/v) DMSO. The ratio of the FimH:FimGNte product band intensity to the sum of the total non-reacted FimC:FimH complex and FimH:FimGNte) protein band intensities was plotted as a function of compound concentration. The IC₅₀ values were calculated by nonlinear regression analysis. DMSO of equivalent concentration (20 % v/v) was the negative control.

Biofilm formation assay

Static biofilm formation was assayed as previously described by O' Toole and Kolter (Mol Microbiol 1998, 28:449-461), with slight modifications. An overnight culture grown at 37 °C was diluted 1000-fold with fresh LB medium. Aliquots of 150 μΐ, of the diluted cells suspension were added to 96-well microtiter plate and incubated at room temperature (RT) for 48 h. After incubation, wells were washed twice with 200 μΐ, phosphate-buffered saline (PBS) and stained with 175 μΐ, of 0.1 % (w/v) crystal violet (CV). The plate was then washed twice with PBS and destained with 175 of ethanol. 150 of the ethanol-solubilised CV was transferred to a new microtiter plate, and the absorbance at 595 nm was measured. Biofilm formation was qualitatively determined to be proportional to the absorbance of the CV. Data were normalized to percent biofilm inhibition relative to non-treated cultures using the equation (A595 X - A595 background / A₅₉₅ control), where "A595" is the absorbance at 595 nm for data point X, for non- inoculated LB medium (background) or for non-treated culture (control). Mid inhibitory concentrations (IC₅₀'s) of compounds with respect to biofilm formation were determined by non-linear regression fitting of the normalized data, using a fixed Hill slope of - 1.4.

Enzyme-linked immunosorbent assay (ELISA) with anti-FimH antibody

A whole-bacterial cell ELISA was performed to detect the expression of type 1 pili by UTI89 cultures incubated with the compound, as measured by the abundance of surface-exposed FimH. Bacterial strains were grown in the presence of 50 μΜ and 200 μΜ of the compound at RT for 48 h. Nunc MaxiSorp™ flat-bottom 96 well plates (eBioscience) were coated with 75 μΐ_^ of washed bacteria in 1 X PBS pH 7.4, OD₆oon_m of each well was measured and plates were incubated at RT for 2 h. The wells were washed twice in lx PBS and blocked with 1 % bovine serum albumin for 30 min. After washing, the presence of type 1 pili was detected using a rabbit polyclonal anti-FimH antibody followed by alkaline phosphate-conjugated goat anti- rabbit IgG (Sigma). The reaction was developed in the presence of NBT (5-bromo-4-chioro-3- indo !y l-phosphatc )/BC I P (nitro blue tetrazoiium) substrate (Roche) and absorbance was read at 405 nm.

Pilus dislocation assays

Overnight bacterial cell culture was diluted 1000-fold with fresh LB medium and grown to exponential growth phase (OD₆oonm of 0.5) at 37 °C. Bacteria were then treated with 200 μΜ of compound in a total of 8 mL of culture. Cultures were sampled (800 μί) at 1, 30 and 60 minutes after addition of compound or 2% DMSO for control samples. Samples were washed and suspended in equivalent volume of PBS. Whole cell ELISA was performed using anti- FimH antibody to detect the presence of type 1 pili as described above. The compound treated and non-treated samples were fixed and stained with 0.5 % (w/v) uranyl acetate for visualisation by electron microscopy in order to quantify the amount of piliated versus non- piliated bacterial cells in each sample. Approximately 100 bacterial cells were counted for each sample. Human urothelial cell adherence assay

The adhesion of compound-treated UTI89 to human urinary bladder epithelial cell line 5637 (American Type Culture Collection HTB-9) was assessed using protocol as described in Wellens et ah, 2008 (Wellens et al., PLoS One 2008, 3:e2040) with a few minor modifications. Briefly, bacteria were grown in the presence or absence of 200 μΜ of compound. Bacteria were washed and suspended in sterile PBS to an OD₅9_5nm of 0.5. A total of 20 μΙ_, of this suspension, corresponding to 10⁶ to 10⁷ cells, was added to each well of a 24-well plate (Falcon BD) containing a confluent culture of bladder cells. The plates were incubated at RT under gentle shaking for 15 minutes to allow binding of the bacteria to the bladder cells. Unattached bacterial cells were removed by five washes in sterile PBS. Bladder cell lysis and detachment from culture plates was induced by incubation with 0.5 mL of 0.25 % w/v trypsin - 2 mM of EDTA for 15 minutes and then terminated by addition of 10 % fetal calf serum. Bacterial colony forming units (cfu) in the bladder cells lysates were determined by 10-fold serial dilutions in sterile PBS and plating onto LB agar plates. UTI89 AfimA-H and UTI89 grown in the presence of DMSO 2 % (v/v) were used as controls.

Claims

1. A compound of the formula (I):

wherein

ring B is an optionally substituted 5 -membered heteroaryl or heterocyclyl group containing from 1 to 3 heteroatoms selected from O, N, and S;

V is a -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

R₂ is an optionally substituted C_6-14 aryl group, an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted 5- or 6-membered cycloalkenyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted hydroxy group, or an optionally substituted amino group; or a pharmaceutically acceptable salt thereof,

rovided that the following compounds are excluded

A compound according to claim 1 , wherein

V is -SO2-, -N(R₃)-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

Z is a Ci_6 alkyl carbamoyl group, a bond, a Ci_₆ alkylene carbonyl group, a Ci_₆ alkoxy carbonyl group, a Ci_₆ alkyl carbonyloxy group, or a Ci_₆ alkyl carbamate group; Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted

5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group, an optionally substituted hydroxy group, or an optionally substituted amino group;

or a pharmaceutically acceptable salt thereof.

A compound according to claim 1 or claim 2, wherein

ring A is an optionally substituted 6-membered aryl or heterocyclyl group.

A compound according to any preceding claim, wherein

A compound according to any preceding claim, of the formula (la):

wherein

wherein R5, R^, and R₇ are each independently a hydrogen atom, an optionally substituted Ci_₆ alkyl group, or an optionally substituted Ci_₆ alkyl carbonyloxy group. A compound according to any preceding claim, of the formula (lb):

wherein Xi is selected from O, N, and S, and X₂ and X₃ are both N.

7. A compound according to any preceding claim, wherein

V is -SO2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

8. A compound according to any preceding claim, wherein

9. A compound according to any preceding claim, wherein

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C₆-i4 aryl group.

10. A compound according to any preceding claim, wherein

Y is -S-, a bond, or -O- .

11. A compound according to claim 1 , wherein

ring A is an optionally substituted 6-membered aryl, heterocyclyl, or heteroaryl group; ring B is a 5-membered heteroaryl group containing 3 heteroatoms selected from O, N, and S;

V is a -S0₂-, -N(R₃)S0₂-, or -S0₂N(R₃)-, wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6- membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, or an optionally substituted C₆-i4 aryl group;

W is a bond;

Y is -S-; Z is a Ci_6 alkyl carbamoyl group;

Ri an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6- membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

Ri and R₂ are each independently an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof.

12. A compound according to claim 1 , wherein

ring A is an optionally substituted 6-membered aryl, heterocyclyl, or heteroaryl group; ring B is a 5-membered heteroaryl group containing from 3 heteroatoms selected from O, N, and S;

W is a carbamoyl group;

Y is a bond;

Z is a bond;

Ri is an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6- membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C_6-14 aryl group;

R₂ is an optionally substituted 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkoxy group, an optionally substituted C₆-i4 aryl group;

or a pharmaceutically acceptable salt thereof.

13. A compound of the formula (I):

wherein

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

Y is -S-, a bond, -0-, or -N(R-i)-.

wherein R4 is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group;

or a pharmaceutically acceptable salt thereof,

for use in therapy

provided that the followin compounds are excluded

14. A compound of the formula (I):

wherein

Y is -S-, a bond, -0-, or -N(R₄ ,

pharmaceutically acceptable salt thereof, for use in the prophylaxis or treatment of bacterial infections

provided that the followin compounds are excluded

15. A compound of formula (I) for use according to claim 14, in sequential or simultaneous combination with at least one anti-infective agent or anti- virulence agent.

16. A pharmaceutical composition comprising a compound of the formula (I):

wherein

V is -SO2-, -N(R₃)-, -CH2-, -N(R₃)S0₂-, or -S0₂N(R₃)-,

wherein R₃ is a hydrogen atom, an optionally substituted 5- or 6-membered heterocyclyl group, an optionally substituted 5- or 6-membered heteroaryl group, an optionally substituted 3- to 6-membered cycloalkyl group, an optionally substituted Ci_₆ alkyl group, an optionally substituted C₂_₆ alkenyl group, an optionally substituted C₂_₆ alkynyl group, an optionally substituted Ci_₆ alkoxy group, or an optionally substituted C_6-14 aryl group; W is a bond, a Ci_₃ alkylene group, a Ci_₃ alkyl carbonyl group, a carbonyl group, a Ci_₃ alkoxy carbonyl group, an oxycarbonyl group, a Ci_₃ alkyl carbonyloxy group, a carbonyloxy group, a Ci_₃ alkyl carbamoyl group, or a carbamoyl group;

Y is -S-, a bond, -0-, or -N(R4 ,

a pharmaceutically acceptable salt thereof, and

a pharmaceutically acceptable diluent, excipient or carrier

provided that the followin compounds are excluded

, and

17. A pharmaceutical composition according to claim 16, further comprising one or more antimicrobial agents.

18. A medical device, laboratory apparatus, food preparation surface-bearing device, nanoparticle material, or packaging material, incorporating a compound of the formula (I):

wherein

Y is a bond, -0-, -N(R₄ , or -S-,

or a pharmaceutically acceptable salt thereof.

19. A medical device according to claim 18, wherein the device is for in vivo implantation.

20. A medical device according to claim 18 or claim 19, wherein the device is a catheter, cannula, stent, shunt, or hypodermic needle.

21. A medical device, laboratory apparatus, food preparation surface-bearing device, or packaging material, according any of claims 18 to 20, wherein the compound according to formula (I) is disposed as a coating on a surface thereof which preferably, in use, comes into contact with a biological material or a subject to be treated with the device.

22. A method of preventing or treating bacterial infections, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to formula (I):

wherein

Y is -S-, a bond, -0-, or-N(R4 ,

or a pharmaceutically acceptable salt thereof provided that the followin compounds are excluded

23. A method of prevention or treatment according to claim 22, wherein the compound of formula (I) is administered prior to, concurrent with, or following administration of at least one antibiotic.

24. A method of preventing or inhibiting bio film formation on a surface, the method comprising the treatment of the surface, or a device bearing the surface, with a compound according to formula (I):

wherein

Y is a bond, -0-, -N(R4 , or -S-,

a pharmaceutically acceptable salt thereof.