4-(4-chlorophenyl)-5-methylene-pyrrol-2-one and 5-methylene-4-(p-tolyl)pyrrol-2-one for use in the treatment of gram negative bacterial infections

4-(4-Chlorophenyl)-5-methylene-pyrrol-2-one and 5-methylene-4-(p-tolyl)pyrrol-2-one for use in the treatment of gram-negative bacterial infections, in particular infections in which Pseudomonas is implicated.

This application is a national phase filing under 35 USC 371 of International Application No. PCT/EP2017/067783, filed on Jul. 13, 2017, which claims priority from EP16180590.8 filed Jul. 21, 2016, the contents of which are incorporated herein in their entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to certain lactam compounds for use in a method of treatment of gram negative bacterial infections.

BACKGROUND

The emergence of drug-resistant bacteria and fungi presents a significant medical and public health problem. Consequently, there is an urgent need for the development of antimicrobial agents that can overcome drug resistance problems. Bacteria and fungi generally develop drug resistance in four ways: producing metabolizing enzymes for the degradation of the drugs, modifying their targets to render the drugs ineffective, expressing a high level of efflux proteins that “pump” the drug out in order to lower its concentration, and inducing biofilm formation to prevent permeation of drugs into the bacteria.

WO2007/085042 (Biosignal Limited) describes certain lactam structures and their use in the treatment of bacterial infections.

WO2014/118240 (Unilever) describes antimicrobial compositions comprising a lactam and a hydrotrope.

WO2014/183164 (Kumar, Perry and Kit) describes certain N-functionalised dihydropyrrolone compounds and methods for preparing surfaces to which the dihydropyrrolone compounds are attached.

SUMMARY OF THE INVENTION

The invention is based on the inventors' understanding and insight into the unusual biological properties of certain lactam compounds, and the utility of these compounds in methods of treatment owing to their biological profile.

The lactams described herein are antibacterial. Their particular biological profile makes them surprisingly suitable for the treatment of gram-negative bacterial infections, in particular in long term treatment.

In a first aspect, the invention therefore relates to a lactam for use in a method of treatment of an infection caused by gram-negative bacteria.

Suitably, the infection is chronic (not cleared within 12 weeks) or an infection that is considered at risk of becoming chronic.

Suitably, the infection is a bacterial infection in whichPseudomonas, (usually but not necessarilyP. aeruginosa) is implicated.

Accordingly, in a first aspect, the invention may provide a lactam selected from

for use in a method of treatment of an infection caused by a gram-negative bacteria.

The lactams of Formula I and II act to prevent the formation of, retard or prevent the growth and development of, and/or reduce the extent of a bacterial biofilm.

Pseudomonas, typicallyP. aeruginosa, may be implicated in the bacterial biofilm.

In the present invention, the subject being treated is preferably a mammalian subject and most preferably a human.

The lactams of the invention may be used as the first line of treatment of an infection, or may be used in subjects already treated with other antibiotics for the same infection, for example those who have not shown a satisfactory response to those other antibiotics.

If appropriate, the lactams of the invention may be administered together with other treatments, including other antibiotic treatments.

In a second aspect, the invention provides a method of treatment of an infection caused by a gram-negative bacteria, the method comprising administering to a subject a therapeutically effective amount of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) or 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491).

In a third aspect, the invention provides the use of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) or 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491) in the manufacture of a medicament for the treatment of bacterial infections of skin lesion.

It will be appreciated that all preferences described with respect to the first aspect apply similarly to the second and third aspects, as appropriate.

The proteobacteria are a major group of gram-negative bacteria, includingEscherichia coli(E. coli),Salmonella, Shigella, and other Enterobacteriaceae,Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria,Legionellaetc. Other notable groups of gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria.

Medically relevant gram-negative cocci include the four organisms that cause a sexually transmitted disease (Neisseria gonorrhoeae), a meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis, Haemophilus influenzae).

Pseudomonas aeruginosa

There are a number ofP. aeruginosastrains, including PA01, PA7, USBPP-PA14 and strain 2192. Except where indicated otherwise, a reference toP. aeruginosais intended to refer to any and all strains.

The methods described herein may be directed to treatment of infections in whichP. aeruginosais implicated. TheP. aeruginosamay be a strain that produces AQs (alkylquinoline compounds). TheP. aeruginosamay be a strain that produces one or both of PQS (Pseudomonasquinolone signal; 2-heptyl-3-hydroxy-4-(1H)-quinolone) and HHQ (4-hydroxy-2-heptylquinoline). TheP. aeruginosamay be a strain belonging to one of the two majorP. aeruginosagenomic groups (PAO1 and PA14).

Quorum Sensing

Quorum sensing (QS) is a mechanism whereby microorganisms, and in particular bacteria, communicate with each other and exhibit community-wide behaviour coordination through the secretion and detection of chemical signals called autoinducers (AIs). Quorum sensing has been demonstrated in a large number of bacteria species/strains and important in regulating bacterial virulence, drug resistance, expression of efflux transporters, and biofilm formation, and therefore is attracting attention in the antimicrobial field. (Frontiers in microbiology6 (1036) September 2015).

Quorum sensing is a cell-density based intercellular communication system to regulate collective behaviour, which plays a key role in regulation of bacterial virulence and biofilm formation. The process relies on the production, release and group-wide detection of signal molecules called autoinducing peptides (AIPs), which in gram-negative bacteria are typically homoserine lactones, (HSLs), especially N-acyl-homoserine lactones (AHLs). Other quorum sensing molecules are known, including epinephrine/norepinephrine.

Biofilm formation enables the bacteria to resist antibiotics because once the bacteria sense that the outer layer of the biofilm is being destroyed, the inner layers will grow stronger to re-establish the community. The present invention is based on the inventors' investigation into the properties of certain lactams as described herein and their insight into the way in which said lactams influence QS in gram negative bacteria such asP. aeruginosa.

The QS network ofP. aeruginosais organised in a multi-layered hierarchy consisting of at least four interconnected signalling mechanisms and demonstrates plasticity, in that it can respond to bacterial population changes and possibly also environmental stress cues.P. aeruginosaorchestrates biofilm formation—and production of virulence factors—by reliance on two QS systems, both part of the Luxl/R signalling cascade, the Las and Rh1 systems.

InPseudomonas aeruginosa, the acyl-homoserine lactone (AHL) and alkyl quinolone (AQ) QS systems are important for virulence and biofilm formation. One acyl-HSL QS regulator is LasR. A non-AHL signalling molecule produced byP. aeruginosais known as PQS (Pseudomonasquinolone signal), which is 2-heptyl-3-hydroxy-4-(1H)-quinolone.

The term “biofilm” as used herein refers to biological films that develop and persist at interfaces in aqueous environments. These biological films are composed of microorganisms embedded in organic gelatinous matrices composed of one or more matrix polymers that are secreted by the resident microorganisms. Biofilms can develop into macroscopic structures and are also capable of trapping nutrients and particulates that can contribute to their enhanced development and stability. Biofilms can also prevent penetration of antimicrobial agents, which may lead to persistent infections. Formation of biofilms provides bacteria with a protected environment can withstand various stresses, including many antibiotic treatments.

Methods of Treatment

It will be appreciated that the term “methods of treatment” as used herein includes prophylaxis, treatment that hamper bacterial colony population growth, treatments that keep a bacterial colony population stable, and treatments that reduce or eradicate a bacterial population.

Owing to their unusual biological profile, the lactams of the invention may be useful in the long term treatment of infections. This is because, owing to their selective interactions with the various bio-pathways of the bacteria, and in particularP. aeruginosa, the lactams may permit population control and reduction without triggering the mechanisms that are thought to lead to evolutionary resistance.

In some cases, the bacterial infection is an infection in whichPseudomonas, (usually but not necessarilyP. aeruginosa) is implicated.

The methods described herein may be suitable for long term use. Accordingly, the methods may include regular administration of the lactam to a subject for a period of at least several weeks, several months, at least one year, at least two years, at least three years, at least 5 years, at least 8 years, or at least 10 years.

EXAMPLES

To elucidate the mechanism of action of compounds described herein, the impact of each lactam on both N-acylhomoserine lactone (AHL) and 2-alkyl-4-quinolone-(AQ)-dependent quorum sensing (QS) inP. aeruginosawas explored.

The expression of rhl and las AHL- and the pqsA AQ-synthase genes was evaluated using lux-based lasl (FIG. 1), rhll (FIG. 2) and pqsA (FIG. 3) chromosomally integrated promoter fusions. Luminescence was quantified as a function of bacterial growth. The applicant further determined that none of the lactams is growth inhibitory at 100 μM using a large number of strains (figure not shown).

TheP. aeruginosaPAO1-N tac::lux strain was used as a positive control biosensor as it constitutively expresses luxCDABE and hence light (FIG. 4). Any reduction in light output in this strain in the presence of a lactam will show whether or not the compound has an adverse effect on luminescence per se. The tac::lux expression profile shows that there is no interference between the compounds and the enzymes involved in light generation. Any effects observed on incubation of the QS reporter strains with the lactams will therefore be due to alterations in promoter expression.

The qualitative effect of compound 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) on pyocyanin production by PAO1-L was then investigated.

Pyocyanin is a blue redox-active secondary metabolite and a putative signalling molecule inP. aeruginosa(Jayaseelan et al, 2014). The pyocyanin biosynthetic (phz) genes are regulated by QS and in part via the transcriptional regulator PqsR acting through PqsE (Rampioni et al 2011). Pyocyanin production was visually assessed after 8 h of incubation at 37° C. inP. aeruginosastrain PAO1-L in the presence and absence of compound 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488), whichFIG. 3shows was the most potent inhibitor of the PqsR-dependent pqsA::lux promoter fusion.FIG. 5shows the culture supernatants of PAO1-L grown in the absence or presence of lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488).

The inhibition of pyocyanin production by lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) was observed as the absence of green pigmentation in culture supernatant in vial 2 (FIG. 5). Although qualitative, this observation offers further confirmation of the inhibitory effects of lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) on AQ-dependent QS.

The inhibitory properties of compounds of the invention on AQ-dependent QS inP. aeruginosawere then investigated.

InP. aeruginosa, the pqsABCDE genes code for the biosynthetic pathway required for the synthesis and action of 2-alkyl-4(1H)-quinolones (AQs) (Heeb et al 2010). The transcriptional activator PqsR regulates the expression of the pqs biosynthetic pathway that in turn controls secondary metabolites such as elastase, pyocyanin and phospholipase as well as biofilm maturation and swarming motility. In this QS system, the primary AQ signal molecules are 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS) both of which act as co-inducers of PqsR (Heeb et al2010; Ilangovan et al 2013).

The results from the first screening experiments (FIG. 1-3) using AHL and AQ biosensor strains clearly show that inhibition of the pqs-system is the main effect of lactams 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) and 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491) on QS inP. aeruginosa. To determine the relative inhibitory activities (IC50s) of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) and 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491), dose response curves were constructed using the same biosensor strain (P. aeruginosaPAO1 pqsA::lux; Fletcher et al, 2007; Ilangovan et al 2013). This strain produces light in response to the endogenous production of AQs such as PQS and HHQ. The data obtained are presented inFIG. 6andFIG. 7from which IC50swere calculated as 22 μM for 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) and 44 μM for 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491) respectively. While 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) is the more potent of the two compounds, 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491) showed the desired effect. It can also be seen that 5-methylene-4-(p-tolyl)pyrrol-2-one (Ref. 491) did not inhibit pqsA expression as strongly as 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488); at high concentrations while 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) completely inhibited light output and hence pqsA expression.

FIGS. 6 and 7demonstrate the binding and inhibition of the PQS quorum sensing inpseudomonas, with compound 488 showing the ability to fully knock out the PQS. The 491 compound inFIG. 7whist proving inhibition did not give complete knock out at higher concentration.

The mode of action on AQ-dependent QS was investigated.

The major QS molecules in the pqs system are the AQs, PQS and its precursor HHQ (Williams & Camara 2009). Their biosynthesis requires the regulator PqsR and the biosynthetic proteins, PqsABCD and the mono-oxygenase, PqsH (Heeb et al 2010).

The inhibitory action of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) may involve inhibition of AQ biosynthesis or 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) may behave like an antagonist and block the PQS receptor, PqsR. These experiments do not discriminate between inhibition of PqsR activation and inhibition of AQ biosynthesis because the biosensor strain used maintains an intact AQ-dependent QS system. Experiments were therefore designed to determine the nature and degree of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) inhibitory activity. Firstly, the possibility that 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) is an inhibitor of the response regulator protein, PqsR that is essential for expression of the pqsABCDE genes and hence AQ production was explored.

By using the AQ-non producingP. aeruginosastrain PAO1-L ΔpqsA, pqsA::lux, the EC50value for the native PqsR agonist, PQS was calculated as ˜2 μM (Ilangovan et al. 2013). In the presence of a fixed concentration of lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) and a range of PQS concentrations, the EC50value increased ˜15 fold to 31 μM (FIG. 8). This suggests that 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) acts as a competitive antagonist of AQ signalling potentially inhibiting the interaction between PqsR and its co-inducer, PQS.

The experiment shown inFIG. 8was then repeated using 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) concentrations of 0, 50, 100 and 200 μM. The results obtained are shown inFIG. 9which shows that as 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) concentrations are increased, maximal light output is substantially reduced. The shape of the dose response curves indicates that 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) is a competitive antagonist as increasing the PQS concentration, restores pqsA::lux expression.

These data demonstrate the selective binding and competitive antagonist activity of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488).

A PAO1-L ΔpqsA pqsA′::lux biosensor-based assay was performed to determine whether lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) exhibits any partial agonist activity at higher concentrations. A partial agonist is a molecule that can bind to and activate a receptor resulting in a non-complete response compared with a full agonist. Graphically, an antagonist that is also a partial agonist exhibits agonist activity at high concentrations.FIG. 10shows that 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) has no partial agonist activity as it was unable to activate pqsA expression at concentrations up to 800 μM. This contrasts with its activity as an antagonist in the presence of 10 or 40 μM PQS. This demonstrates that at higher concentrations there is no activation of receptor binding, offering low or even no toxicity at higher doses.

PQS is capable of weakly activating pqsA in the absence of PqsR via a mechanism that is not fully understood but appears to depend on the iron chelating properties of PQS. The aim of this experiment was to clarify whether lactam 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) is also inhibitory for pqsA expression via the PqsR independent pathway. As previously described, the pqsA′::lux biosensor based on the triple mutant PAO1-N ΔpqsAHR cannot produce AQs and lacks pqsR but still responds to exogenous PQS. This response is however much weaker that the PqsR-dependent response.

FIG. 11shows the impact of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) on the PqsR-independent expression of pqsA in the presence or absence of 40 μM PQS. 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) has a small inhibitory effect on the PqsR-independent pathway. This suggests that while the compounds of the invention may have some effect onP. aeruginosathat do not express AQs, they show most promise for in the treatment of disorders in which AQ-producing strains are implicated.

Indeed for 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) the PQS pathway is the dominant inhibitory route. Taking into account the role of quorum sensing in toxicity and virulence ofpseudomonas, then this technology is most effective against those strains capable of quorum sensing.

To investigate the inhibitory effect of 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) onP. aeruginosaPA14, a pqsA′::lux fusion was introduced onto the chromosome. Comparable inhibitory effects with those previously described for strain PAO1 were observed. The IC50extrapolated from the inhibition curve inFIG. 12was calculated to be 17.9 μM.

To determine whether 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) is capable of inhibiting the enzymes (PqsABCD) involved in AQ biosynthesis, theP. aeruginosaPAO1-N ΔpqsAHR was exploited. This mutant cannot produce AQs because the genes coding for the key biosynthetic enzymes (PqsA and PqsH) and regulation (PqsR) have been deleted. To restore AQ production in a PqsR-independent manner in PAO1-N ΔpqsA, the pqsABCD genes were introduced on a plasmid (pBBR pqsABCD; Niewerth et al. 2011).P. aeruginosaPAO1-N ΔpqsAHR andP. aeruginosaPAO1-N ΔpqsAHR pBBR pqsABCD were both incubated overnight in LB with and without 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) (200 μM), resuspended to an OD6001.0. The cultures were extracted with acidified ethyl acetate, the organic phase removed, dried and resuspended in methanol subjected to thin layer chromatography (TLC).

After chromatography using a dichloromethane-methanol mobile phase, the TLC plates were overlaid with a thin agar layer containing the AQ biosensor strain PAO1-L ΔpqsA CTX::pqsA′-luxCDABE (Fletcher et al. 2007) incubated and examined for reporter output (FIG. 13).FIG. 13panel A shows the TLC plate under UV illumination after chromatography. The biosensor strain produces light (FIG. 13panel C) and pyocyanin (FIG. 13panel B) in response to AQs that bind to PqsR and activate the pqsA promoter.

The biosensor revealed the presence of HHQ both in the samples of PAO1-N ΔpqsAHR pBBR-pqsABCD without (FIG. 13panels B and C; lane 5) and with (FIG. 13panels B and C lane 6) compound 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one (Ref. 488) indicating that the lactam did not inhibit HHQ biosynthesis.

Sources of Bacterial Strains

The bacterial strains were obtained or made as follows:

The examples demonstrate that compounds of the invention have been shown to:inhibit alkylquinolone (AQ) dependent quorum sensing (QS) inP. aeruginosa.inhibit PqsR in representative strains belonging to the majorP. aeruginosagenomic groups (PAO1 and PA14 respectively).interact antagonistically with the LysR-type regulator PqsR (in a competitive manner without partial-agonist activity).do not appear to directly inhibit AQ biosynthesis but block AQ synthesis by acting as a PqsR antagonist.potentially interact with the co-inducer binding domain of PqsR (PqsRCBD) acting an allosteric inhibitors.

This provides an unusual biological profile that may enable compounds of the invention to be useful in the treatment of chronic bacterial infections. This is because the compounds of the invention have a biological activity profile that controls multiplication and biofilm formation, but does not trigger the bio-pathways that are associated with developing antibacterial resistance.

REFERENCES