Compositions and methods for the treatment and prevention of bacterial infections caused by gram-positive bacteria

Abstract

The invention relates to photoactive compounds and compositions including same, and to their use in the treatment and prevention of infections caused by gram-positive bacteria. In embodiments, the invention relates to methods of administering compounds and compositions, and in particular, topical compositions, for the treatment and prevention of bacterial infections caused by gram-positive bacteria, including diseases and conditions caused by gram-positive bacteria and infections of skin lesions.

Description

[0001] Compositions and Methods for the Treatment and Prevention of Bacterial Infections caused by Gram-Positive Bacteria

[0002] The present invention relates to compounds and compositions, and to their use in the treatment and prevention of infections caused by gram-positive bacteria. In embodiments, the invention relates to methods of administering compounds and compositions, and in particular, topical compositions, for the treatment and prevention of bacterial infections caused by gram-positive bacteria, including diseases and conditions caused by gram-positive bacteria and infections of skin lesions.

[0003] The discovery of, and introduction into clinical care of antibiotics is arguably one of the greatest breakthroughs in 20th century medicine. The discovery of penicillin in the 1920s marked the beginning of the golden age of antimicrobial discovery, which continued with the rapid discovery of multiple classes of natural antibiotics during a relatively condensed period up to the 1960s. This era also saw widespread antibiotic use, and overuse, providing selective pressure for acquisition and dissemination of antimicrobial resistance (AMR) across various species, resulting in a global health problem often heralded as the "silent pandemic". In 2019 alone, AMR was responsible for around 1.3 million deaths globally. Additionally, there is clear evidence that bacteria are becoming increasingly resistant to existing antibiotics. However, with the rate at which resistance is developing out-pacing the discovery of new antibiotics, additional therapeutic approaches for microbial infections are urgently required.

[0004] Light-activated, cytotoxic compounds, which can be used to trigger cell-killing capabilities when exposed to light, have advantages in minimising systemic, off-target toxicity in humans.

[0005] Photoactive cytotoxic activity, often via the generation of reactive oxygen species (ROS), is a promising area of research since bacteria are understood to be unable to develop resistance towards ROS. However, a major roadblock to the use of photoactive compounds in therapy is the photosensitisers themselves. Amongst other problems, most known photosensitizing compounds are high molecular weight porphyrin-based structures that are generally insoluble or poorly soluble, leading to difficulties in formulating them into useful therapeutic forms. Known compounds also exhibit, amongst other properties, poor or inadequate pharmacokinetic characteristics, limited or no ability to penetrate bacterial cells, and limited selectivity between cell types. In combination, these difficulties limit the use of such compounds in antimicrobial therapies.

[0006] Consequently, it would be advantageous to provide compounds which mitigate one or more of these disadvantages, and which could be used as antibacterial agents in the treatment of bacterial infections caused by gram-positive bacteria. Compounds which have, alongside efficacious antibacterial action against gram-positive bacteria, good physical properties, such as good solubility in commonly-used formulation solvents, would be useful. Compositions comprising such compounds, would also be beneficial.

[0007] Summary of the Invention

[0008] Accordingly, the present invention relates generally to compounds of formula I, in free or salt form, for use in the treatment and prevention of an infection caused by gram-positive bacteria. Formula I in which R is C1-C4 alkyl.

[0009] Throughout this specification, the term "alkyl" refers to a fully saturated, branched, unbranched or cyclic hydrocarbon moiety, i.e. primary, secondary, or tertiary alkyl or, where appropriate, cycloalkyl or alkyl substituted by cycloalkyl. Where not otherwise indicated, an alkyl group comprises 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, or more preferably 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- heptyl, n-octyl, n-nonyl and n-decyl.

[0010] In formula I, R is C1-C4 alkyl. R may be methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so-butyl, or tert-butyl. In an embodiment, R is methyl or butyl. In an embodiment, R is methyl or tert-butyl.

[0011] In an embodiment, R is methyl or tert-butyl.

[0012] In an embodiment, R is tert-butyl.

[0013] Compounds for use in the invention include salts thereof, and reference to a compound of Formula I is intended to include reference to salts thereof, unless otherwise stated. Suitable salts include for instance, acidic salts formed with inorganic and / or organic acids, basic salts formed with inorganic and / or organic bases, as well as zwitterions ("inner salts") which may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts may be useful in certain circumstances. Exemplary acid addition salts which may be useful include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, formates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates,) triflates, and the like. Exemplary basic salts which may be useful include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternerized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others.

[0014] In an embodiment, the salt may be selected from hydrochloride, hydrobromide, hydroiodide, acetate, trifluoroacetate, para-toluenesulfonate, trifluoromethanesulfonate, triflate, tetrafluoroborate, formate and sulfate.

[0015] In an embodiment, the salt is formed with an anion comprising at least two oxygen atoms. The salt may be, for example, an acetate, trifluoroacetate, triflate, tosylate bistriflimide, ethyl sulfate, hydrogen sulfate, bis(fluorosulfonyl)imide, or dimethyl phosphate.

[0016] In an embodiment, the salt may be selected from acetate, trifluoroacetate, triflate, hydrobromide and hydroiodide.

[0017] The term "gram-positive bacteria" refers to bacteria that give a positive result in the Gram stain test. These bacteria take up the crystal violet stain used in the Gram stain test, and retain the stain after it is washed away from the rest of the sample, due to the presence of the thick peptidoglycan layer in the bacterial cell wall.

[0018] Gram-positive bacteria include Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Community-Acquired Methicillin-resistant Staphylococcus aureus (community acquired MRSA), Staphylococcus epidermidis, Staphylococcus haemolyticus, Micrococcus spp including Micrococcus luteus, Corynebacterium minutissimum, Group A Streptococcus (GAS) including Streptococcus pyogenes, Group C Streptococcus of the skin, Group G Streptococcus of the skin, Enterococcus faecalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leptromatosis, Mycobacterium marinum, and Mycobacterium ulcerans.

[0019] The term "infection caused by gram-positive bacteria" encompasses both diseases and conditions caused by gram-positive bacteria, and bacterial (gram-positive) infections of skin lesions. Diseases and conditions caused by gram-positive bacteria include, but are not limited to cellulitis, erysipelas, impetigo, folliculitis, carbuncles, furuncles, erthrasma, leprosy and cutaneous tuberculosis; while infections of skin lesions include infections of ulcers, skin wounds, burns and insect bites. As a skilled person would appreciate, many bacterial infections are associated with skin lesions, in which the lesions themselves have arisen from a variety of causes. In this case, the compounds or compositions of the invention can be used to treat or prevent a bacterial infection of the lesion caused by gram-positive bacteria. Skin lesions which are susceptible to infection by gram-positive bacteria include ulcers, skin wounds, burns and insect bites. The term "ulcers" includes arterial ulcers, diabetic foot ulcers, pressure ulcers and Buruli ulcers. Skin wounds which may be susceptible to bacterial infection include wounds arising from injury, ischemic wounds, and surgical wounds. Burns include heat burns and chemical burns.

[0020] In an embodiment, the compounds or compositions of the invention are for use in the treatment of an infection caused by gram-positive bacteria, wherein the infection caused by gram-positive bacteria is selected from cellulitis, erysipelas, impetigo, folliculitis, erthyrasma, leprosy and cutaneous tuberculosis, or the gram-positive infection is an infection of a skin lesion selected from arterial ulcers, pressure ulcers, Buruli ulcers, infectious wounds, ischemic wounds, surgical wounds, chemical burns and insect bites.

[0021] The invention also relates to pharmaceutical compositions comprising a compound of formula I.

[0022] The term "pharmaceutical composition" refers to a composition suitable for administration to a patient. Thus, the term "pharmaceutical composition" refers to compositions which comprise a compound of formula I of the invention, or salts thereof, solvates, isomers or tautomers thereof, optionally in conjunction with one or more pharmaceutically acceptable excipients, carriers, or diluents. The term "pharmaceutical composition" is also intended to encompass both the bulk composition (i.e., in a form that has not yet been formed into individual dosage units) and individual dosage units. Such individual dosage units include impregnated dressings, transdermal patches, ampoules, pre-filled syringes, packets, and the like. The composition includes a therapeutically effective amount of a compound of formula I. The term "therapeutically effective" amount or "effective amount" refers to the quantity of the compound or composition of the present invention which is effective in producing the desired therapeutic effect.

[0023] The pharmaceutical composition comprises a compound of formula I optionally in conjunction with one or more pharmaceutically acceptable excipients, carriers, or diluents for use in the treatment of an infection caused by gram-positive bacteria. The composition may optionally comprise one or more additional therapeutic agents.

[0024] In an embodiment, the pharmaceutical composition is in the form of an aqueous solution, suspension, milk, cream, foam, paste, ointment, gel, or hydrogel. The inventors have advantageously determined that the compound of formula I can be formulated in commonly- used pharmaceutical excipients, exhibiting good stability, whilst retaining its efficacy against gram-positive bacteria.

[0025] In an embodiment, the pharmaceutical composition is in the form of a gel or hydrogel.

[0026] Suitable dosages for administering compounds of the invention to patients may be determined by those skilled in the art, e.g., by an attending physician, pharmacist, or other skilled worker and may vary according to factors such as patient weight, health, age, frequency of administration, mode of administration, the presence of any other active ingredients, and the condition for which the compounds are being administered.

[0027] Suitable aqueous solutions or formulations comprise the compound of formula I (in free base or salt forms) in concentrations ranging from 0.001 to 20% (w / w), from 0.05 to 10% (w / w), or from 0.1 to 5% (w / w). Suitable excipients for aqueous solutions include, amongst others, propylene glycol, dipropylene glycol, hexylene glycol, butylene glycol, benzyl alcohol, glycerol, sodium chloride and sodium hydroxide. Suitable surfactants include betaines and foaming agents including cocamidopropylbetaine.

[0028] Alternatively, the compound or pharmaceutical composition may be formulated as a cream or ointment. The cream or ointment may comprise the compound of formula I (in free base or salt forms) in concentrations ranging from 0.001 to 20% (w / w), from 0.05 to 10% (w / w), or from 0.1 to 5% (w / w). Suitable excipients include, but are not limited to, petrolatum / mineral oil base, paraffin, alginate, ceresin, glycerin, emulsifiers, and emollients such as glyceryl stearate panthenol.

[0029] Alternatively, the compound or pharmaceutical composition may be formulated as a gel or hydrogel. The gel or hydrogel may comprise the compound of formula I (in free base or salt forms) in concentrations ranging from 0.001 to 20% (w / w), from 0.05 to 10% (w / w), or from 0.1 to 5% (w / w). Suitable excipients include, but are not limited to, celluloses, polyacrylates, polyvinyl alcohol, polyethylene glycol, alginate, chitosan, hyaluronic acid, and polyacrylamide preparations.

[0030] In an embodiment, the pharmaceutical composition may be formulated as a gel or hydrogel. The gel may be a cellulose gel. The gel composition may comprise hydroxyethylcellulose or carboxymethylcellulose.

[0031] The compound or pharmaceutical composition may be formulated into, or impregnated in a dressing or patch. Suitable dressings include hydrocolloid dressing preparations derived from solid hydrocolloid matrices such as gelatin, pectin, carboxymethyl cellulose, and combinations thereof. Alternative dressings include hydrogel dressing preparations including, but not limited to alginate, chitosan, hyaluronic acid, polyvinyl alcohol, polyacrylamide and polyethylene glycol; alginate dressings; electrospun nanofibre dressings derived from materials including, but not limited to gelatin, collagen, chitosan, hyaluronic acid, alginate, cellulose, and polymers such as polyvinylpyrrolidone, polycaprolactone, polyvinyl alcohol, polyethylene glycol and poly(lactic-co-glycolic acid); and film dressings, such as those derived from polyurethane, polylactic acid, cellulose, starch, alginate and hyaluronic acid. Suitable patches include fabric patches comprising non-woven, woven or knitted materials.

[0032] The mode of formulation of the compound or pharmaceutical composition may depend on the condition for which the compound is being administered, and a variety of formulation types is envisaged, allowing an attending physician, pharmacist, or other skilled worker to select the most suitable administration type, taking into account any relevant factors, such as the condition being treated, the area of treatment, any co-administration requirements, etc. In an embodiment, the pharmaceutical composition is a topical composition.

[0033] Topical compositions are applied directly to the skin. Topical compositions can be advantageous for therapeutic use as they can allow an increased dose of the compound to be applied directly where it is needed. Topical compositions may also exhibit reduced sideeffects and toxicity to organs compared with systemic therapeutics.

[0034] In an embodiment, the compound of formula I orthe composition comprising a compound of formula I is administered topically.

[0035] In an embodiment, the compound of formula I orthe composition comprising a compound of formula I is exposed to light.

[0036] In an embodiment, the compound of formula I orthe composition comprising a compound of formula I is administered topically and exposed to light.

[0037] The compounds of formula I have been demonstrated to exhibit cytotoxic activity against gram-positive bacteria when activated by light.

[0038] In an embodiment, the compound or composition is exposed to light of a wavelength between 365 - 460 nm.

[0039] The compound or composition may be exposed to light for from 1 to 30 minutes. In an embodiment the compound or composition is exposed to light for from 1 to 10 minutes, from 2 to 8 minutes, or for around 5 minutes.

[0040] As an example of the administration of the compound or composition of the invention, a gel formulation could be applied directly to an area of affected tissue, and left for a period of time of from 1 minute to 120 minutes, before the formulation is activated by light at a wavelength between 365 - 460 nm emitted from a conventional LED light source, to activate the antibacterial effect against the gram-positive infection. The formulation can then be removed from the area.

[0041] Alternatively, the compound, or a composition comprising the compound, could be applied to the affected area, and then covered by a transparent dressing or bandage (or a transparent window in the dressing or bandage), through which the light can be applied to activate the compound. The dressing can then be left in place to aid wound healing.

[0042] The compound or composition of the invention could also be applied to a skin lesion to inhibit or delay the growth of gram-positive bacteria, i.e., to prevent infection of a skin lesion by reducing bacterial load. This embodiment is particularly suitable for skin lesions susceptible to infection arising from gram-positive bacteria that colonise mammalian skin, such as ulcers, skin wounds, burns and insect bites.

[0043] In an embodiment, there is provided a dressing or patch comprising a compound of formula I or a pharmaceutical composition comprising same.

[0044] In this embodiment, the compound or composition can be impregnated into a topical delivery device such as a dressing or patch, which can be applied to the affected area, bringing the compound into contact with the infection. Advantageously, at least a portion of the dressing or patch can be transparent, allowing the compound to be exposed to light through the dressing or patch, or through a window in the dressing or patch.

[0045] In an aspect of the invention there is provided a method of treatment of a patient with a bacterial infection caused by gram-positive bacteria, the method comprising administering to the patient a therapeutically effective amount of a compound of formula I or a salt thereof. The administering may be topical. The compound of formula I or salt thereof may be formulated in a pharmaceutical composition, optionally a gel composition. The method may further comprise exposing the therapeutically effective amount of a compound of formula I or a salt thereof to light.

[0046] In an aspect of the invention, there is provided a compound of formula II:

[0047] in free or salt form, in which R is C1-C3 alkyl.

[0048] In an embodiment, in Formula II, R is methyl.

[0049] In an embodiment, the compound of formula II is in salt form, and the salt is an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

[0050] Examples:

[0051] The invention will now be described by way of example only with reference to the accompanying figures, in which:

[0052] Figure 1 illustrates the synthesis of compound la, a representative compound of formula I;

[0053] Figure 2 illustrates the synthesis of compound lb, a representative compound of formula I and formula II;

[0054] Figure 3 shows (A) normalised absorption spectra of compound la in dimethyl sulfoxide and chloroform (10 pM) and (B) emission spectra of compound la in toluene (100 nM), chloroform (100 nM) and dimethyl sulfoxide (2.65 pM) ;

[0055] Figure 4 shows (A) normalised absorption spectra of compound lb in toluene (5 pM), chloroform (5 pM) and dimethylsulfoxide (5 pM) and (B) emission spectra of compound lb in toluene (100 nM), chloroform (100 nM) and dimethyl sulfoxide (5 pM) ;

[0056] Figure 5 illustrates the synthesis of salts of compounds la (Figure 5A) and lb (Figure 5B);

[0057] Figure 6 shows an excitation spectrum of an exemplar formulation of compound la at 1% w / w in a mixture of benzyl alcohol (50%), dimethyl sulfoxide (30%) and propylene glycol (20%);

[0058] Figure 7 shows an emission spectrum of an exemplar formulation of compound la at 1% w / w in a mixture of benzyl alcohol (50%), dimethyl sulfoxide (30%) and propylene glycol (20%);

[0059] Figure 8 demonstrates action of compound la against the growth of gram-positive bacteria (B. subtilis and 5. epidermidis) and gram-negative bacteria (£. coli and P. fluorescens);

[0060] Figure 9 shows the effect of compound la on the growth of gram-positive bacteria (B. subtilis and 5. epidermidis); Figure 10 shows the effect of compound la on the viability of gram-positive bacteria (B. subtilis and 5. epidermidis) and gram-negative bacteria (£. coli and P. fluorescens);

[0061] Figure 11 shows the effect of compound la on the viability of gram-positive ESKAPE bacteria E. faecalis, methicillin-sensitive 5. aureus (SH1000) and methicillin-resistant 5. aureus (USA300);

[0062] Figure 12 shows the effect of compound la on the membrane integrity of gram-positive bacteria (B. subtilis and 5. epidermidis) and gram-negative bacteria (£. coli and P. fluorescens);

[0063] Figure 13 shows the real time change in propidium iodide (PI) after photoactivation to monitor membrane integrity of Gram-positive bacteria B. subtilis and 5. epidermidis during treatment with compound la;

[0064] Figure 14 illustrates the localisation of compound la in gram-positive bacteria (B. subtilis and 5. epidermidis).

[0065] Experimental Examples

[0066] The following examples are intended to be illustrative only.

[0067] Materials and Methods

[0068] Reagents were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesar and Fluorochem, and purified, if required, by recrystallisation or distillation / sublimation under vacuum. Solvents were used as supplied from Fisher Scientific or Sigma Aldrich, and dried before use if required with appropriate drying agents. Thin-layer chromatography (TLC) was conducted using Merck Millipore silica gel 60G F254 25 glass plates and / or TLC-PET foils of aluminium oxide with fluorescent indicator 254 nm (40 x 80 mm) with visualisation by UV lamp or appropriate staining agents. Flash column chromatography was performed using SiCh from Sigma-Aldrich (230-400 mesh, 40-63 pM, 60 A), and monitored using TLC. NMR spectra were recorded using Bruker Avance Neo-700, Bruker Avance-600 or Bruker Avance-400 spectrometers operating at ambient probe temperature. NMR peaks are reported as singlet (s), doublet (d), triplet (t), quartet (q), broad (br), septet (sept), combinations thereof, or as a multiplet (m), with reference to the following deuterated solvent signals: CDCI3 (1H = 7.26 ppm,13C = 77.0 ppm), (CDshSO (1H = 2.50 ppm,13C = 39.5 ppm). Electrospray Mass Spectrometry (ESMS) was performed using a TQD (Waters Ltd., UK) mass spectrometer with an Acquity UPLC (Waters Ltd., UK), and accurate mass measurements were obtained using a QtoF Premier mass spectrometer with an Acquity UPLC (Waters Ltd., UK). IR spectra were recorded using a Perkin Elmer FTIR spectrometer.

[0069] Example 1: Synthesis of compounds of formula I and exemplar formulation

[0070] 1.1 Synthesis of compound la

[0071] The synthesis of compound la is shown in Figure 1, and described below. 1.1.1 Synthesis of 5-lodothiophene-2-carbaldehyde, 2

[0072] To a solution of 2-thiophenecarboxaldehyde (37.4 mL, 400.0 mmol) in ethanol (EtOH) (300 mL) at 50° C was added / V-iodosuccinimide (NIS) (99.0 g, 440.0 mmol) and p-toluenesulfonic acid monohydrate (pTSA) (7.60 g, 40.0 mmol), whereupon the resultant solution was stirred at 50 ° C for 50 minutes. The solution was cooled to 35 °C, whereupon IM hydrochloric acid (500 mL) was added, and the mixture was then extracted with ethyl acetate, washed with sat. Na2S2O3, sat. NaHCCh, H2O and brine, dried (MgSC ) and evaporated to give compound 2 as a brown oil that slowly crystallised (85.2 g, 90%) :1H NMR (400 MHz, CDCI3) 6 7.39 (s, 2H), 9.77 (s, 1H);13C NMR (101 MHz, CDCI3) 6 87.8, 137.0, 138.2, 149.6, 181.1.

[0073] 1.1.2 Synthesis of 5-f2-(Trimethylsilyl)ethynyl]thiophene-2-carbaldehyde, 3

[0074] Compound 2 (78.3 g, 329 mmol) was dissolved in triethylamine (Et3N)(l,000 mL) and the resulting solution was degassed by sonication under vacuum, replacing the atmosphere with Ar (lOx). Trimethylsilylacetylene ( -TMS) (50.1 mL, 362 mmol), Pd(PPh3)2Cl2 (2.31 g, 3.29 mmol) and Cui (0.64 g, 3.29 mmol) were then added under argon and the resultant suspension was stirred at 40 °C for 18 h. The mixture was diluted with diethyl ether and passed through Celite / SiCh to give a crude brown oil (72 g). This was purified by silica chromatography (cyclohexane / ethyl acetate, 9:1) to give compound 3 as a light brown oil that slowly crystallised (64.22 g, 94%):3H NMR (400 MHz, CDCI3) 6 0.26 (s, 9H), 7.25 (d, J = 3.9 Hz, 1H), 7.61 (d, J = 3.9 Hz, 1H), 9.84 (s, 1H);13C NMR (101 MHz, CDCI3) 6 -0.5, 26.9, 96.3, 104.6, 132.5, 133.1, 135.7, 143.8, 182.4; IR (ATR) Vmax / crn’12960w, 2899w, 2833w, 2148m, 1666s, 1438s, 1249s, 1223s, 1207s, 838s; MS (ES) m / z = 209.0 [M+H]+; HRMS (ES) calcd. for CioHi3SOSi [M+H]+: 209.0451, found 209.0454.

[0075] 1.1.3 Synthesis of Tert-butyl (2E)-3-{5-[2-(trimethylsilyl)ethvnyl]thiophen-2-yl}prop-2-enoate, 4

[0076] Tert-butyl diethylphosphonoacetate (40.6 mL, 173 mmol) and LiCI (7.33 g, 173 mmol) were added to anhydrous tetrahydrofuran (THF) (700 mL) at 0 °C and the resultant solution was stirred for 15 min, whereupon compound 3 (30.0 g, 144 mmol) was added. To this solution was slowly added l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (25.9 mL, 173 mmol), and the resultant slurry was stirred at room temperature for 16 h. This was poured into crushed ice and extracted with ethyl acetate. The organics were washed with H2O and brine, dried (MgSC ) and evaporated to give a crude brown oil (49.6 g). This was purified by silica chromatography (cyclohexane / ethyl acetate, 95:5) to give compound 4 as an orange oil, containing approximately 25% of the TMS-deprotection product, compound 5 (37.47 g, 85%):XH NMR (400 MHz, CDCI3) 6 0.25 (s, 8H), 1.51 (s, 9H), 6.13 (dd, J = 15.7, 0.4 Hz, 1H), 7.05 (d, J = 3.9 Hz, 1H), 7.12 (d, J = 3.9 Hz, 1H), 7.57 (dd, J = 15.7, 0.4 Hz, 1H). Note: the percentage yield of this step was calculated on the basis of compound 4; TMS deprotection was observed during purification on SiCh.

[0077] 1.1.4 Synthesis of Tert-butyl (2E)-3-(5-ethvnylthiophen-2-yl)prop-2-enoate, 5

[0078] Compound 4 (37.47 g, 122 mmol) was dissolved in a mixture of dichloromethane (DCM) (300 mL) and methanol (MeOH) (30 mL), whereupon K2CO3 (33.7 g, 244 mmol) was added. The resultant suspension was stirred at room temperature (RT) for 18 h before being diluted with dichloromethane and H2O. The organics were washed with sat. NH4CI and H2O, dried (MgSC ) and evaporated to give a crude orange oil (28 g). This was purified by SiC chromatography (95:5, cyclohexane / ethyl acetate) to give compound 5 as an orange oil that slowly darkens (26.33 g, 83%):XH NMR (300 MHz, CDCI3) 6 1.51 (s, 9H), 3.44 (s, 1H), 6.15 (d, J = 15.7 Hz, 1H), 7.07 (d, J = 3.8 Hz, 1H), 7.17 (d, J = 3.8 Hz, 1H), 7.58 (dd, J = 15.7, 0.6 Hz, 1H);13C NMR (101 MHz, CDCI3) 6 26.9, 28.1, 80.8, 83.4, 120.2, 124.0, 130.0, 133.8, 135.1, 141.2, 165.7; IR (ATR) vmax / cm12978w, 2933w, 1702m, 1623m, 1447m, 1392w, 1368m, 1148s, 753m; MS (ES) m / z = 235.1 [M+H]+; HRMS (ES) calcd. for C13H15SO2 [M+H]+: 235.0787, found 235.0786.

[0079] 1.1.5 Synthesis of Tert-butyl (2E)-3-(5-{2-[4-(piperazin-l-yl)phenyl]ethvnyl}thiophen-2- yl)prop-2-enoate, compound la (representative compound of formula I)

[0080] Compound 5 (26.33 g, 112.4 mmol) was dissolved in triethylamine (EtsN) (750 mL) and the solution was degassed by sparging with Ar for 1 h. l-(4-lodophenyl)piperazine (29.45 g, 102.2 mmol), Pd(PPh3)2CI2(3.59 g, 5.11 mmol) and Cui (0.97 g, 5.11 mmol) were then added under Ar and the resultant suspension was stirred at 60 °C for 72 h (hours). The resultant suspension was diluted with dichloromethane and washed with sat. NaHCOs and water, dried (MgSC ) and evaporated to give a crude orange solid (57 g). This was purified by SiC chromatography (95:5, dichloromethane / methanol, 1% triethylamine) followed by recrystallisation from acetonitrile to give compound la as a yellow / orange solid (27.01 g, 67%):1H NMR (400 MHz, CDCI3) 6 1.51 (s, 9H), 2.98 - 3.05 (m, 4H), 3.17 - 3.25 (m, 4H), 6.12 (d, J = 15.7 Hz, 1H), 6.80 - 6.88 (m, 2H), 7.08 (d, J = 3.9 Hz, 1H), 7.10 (d, J = 3.8 Hz, 1H), 7.36 - 7.43 (m, 2H), 7.59 (dd, J = 15.7, 0.6 Hz, 1H);13C NMR (101 MHz, CDCI3) 6 28.1, 45.9, 49.1, 80.6, 81.1, 96.4, 112.0, 114.8, 119.2, 126.4, 130.6, 131.8, 132.6, 135.5, 140.1, 151.6, 165.9; IR (ATR) Vmax / cm’12977w, 2929w, 2820w, 2194w, 1698s, 1617m, 1602m, 1526w, 1323m, 1141s, 812w; MS(ES): m / z = 395.3 [M+H]+; HRMS (ES) calcd. for C23H27N2O2S [M+H]+: 395.1793, found 395.1792.

[0081] 1.2 Synthesis of Compound lb

[0082] The synthesis of compound lb is shown in Figure 2, and described below.

[0083] 1.2.1 Methyl (E)-3-(5-iodothiophen-2-yl)acrylate, 9

[0084] Trimethyl phosphonoacetate (19.8 mL, 122.2 mol) was dissolved in cyclopentyl methyl ether (CPME) (250 mL) along with LiCI (5.2 g, 122.2 mmol) and the mixture was left stirring at 0 °C for 20 minutes. 5-lodothiophene-2-carbaldehyde (26.5 g, 111.1 mmol) was added to the stirring solution followed by dropwise addition of l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (18.3 mL, 122.2 mmol). The mixture was stirred for 18 hours at room temperature. The mixture was diluted with water (400 mL) and extracted with ethyl acetate (400 mL x 2). Organics were combined and dried over Na2SO4 before concentrating under reduced pressure to give compound 9 as a beige solid (31.6 g, 97%) Rf = 0.59 (Hexane / EtOAc 9:1);TH NMR (CDCI3, 300 MHz) 6 3.77 (s, 3H), 6.13 (d, J = 15.7 Hz, 1H), 6.87 (d, J = 3.8 Hz, 1H), 7.19 (d, J = 3.8 Hz, 1H), 7.67 (d, J = 15.7 Hz, 1H).

[0085] 1.2.2 l-(4-((Trimethylsilyl)ethynyl)phenyl)piperazine, 10 l-(4-lodophenyl)piperazine (10.0 g, 34.7 mmol) was added to a 3-neck round bottom flask. Triethylamine (EtsN) (200 mL) was added to the flask and the solution was degassed by vacuum sonication, replacing the atmosphere with Ar ten times. Under positive Ar flow, Pd(PPh3)2CI2(0.60 g, 0.86 mmol), Cui (0.16 g, 0.86 mmol) and trimethylsilylacetylene (TMSA) (5.3 mL, 38.2 mmol) were added to the solution. The mixture was stirred at 40 °C for 18 hours. The mixture was cooled to room temperature and solvent was evaporated under reduced pressure. Residue was extracted with dichloromethane (200 mL) and washed with sat. NaHCCh (200 mL). Organics were then combined and dried over Na2SO4 before concentrating to give a crude orange-brown crystalline solid which was purified further via column chromatography (dichloromethane / methanol, 9:1) to give compound 10 (8.9 g, 100 %) as a brown crystalline solid:XH NMR (CDCI3, 300 MHz) 6 0.22 (s, 3H), 3.01 (t, J = 5.0 Hz, 4H), 3.17 (t, J= 5.0 Hz, 4H), 6.79 (d, J = 8.9 Hz, 2H), 7.35 (d, J = 8.9 Hz, 2H).

[0086] 1.2.3 l-(4-Ethynylphenyl)piperazine, 11

[0087] Compound 10 (8.9 g, 34.4 mmol) was dissolved in MeOH (150 mL), whereupon K2CO3 (9.51 g, 68.87 mmol) was added. The mixture was stirred at room temperature for 18 hours. The solvent was evaporated under reduced pressure and the residue was extracted with dichloromethane (500 mL) and washed with water (500 mL) and NH4CI (300 mL). Organics were collected and dried over Na2SO4 before concentrating to give compound 11 (4.9 g, 76%) as a beige solid:XH NMR (CDCI3, 300 MHz) 6 2.99 (s, 1H), 2.99-3.06 (m, 4H), 3.15-3.22 (m, 4H), 6.79-6.86 (m, 2H), 7.35-7.41 (m, 2H).

[0088] 1.2.4 Methyl (E)-3-(5-((4-(piperazin-l-yl)phenyl)ethynyl)thiophen-2-yl)acrylate, compound

[0089] Compound 9 (6.8 g, 23.3 mmol) and compound 11 (4.9 g, 26.3 mmol) were added to a 3 neck round bottom flask along with triethylamine (Et3N) (150 mL). The mixture was degassed by vacuum sonication, replacing the atmosphere with Ar ten times. Under positive Ar flow, Pd(PPh3)2CI2(0.41 g, 0.59 mmol) followed by Cui (0.11 g, 0.59 mmol) were added and the mixture was stirred at 65 ° C for 72 hours. The solvent was evaporated under reduced pressure and residue was extracted with dichloromethane (600 mL) and washed with sat. NaHCCh (500 mL x 2). Organics were collected and dried over Na2SO4 before concentrating to give an orange solid (8.5 g) which was recrystallised in acetonitrile (250 mL) to give a brick red solid which was further purified via column chromatography (dichloromethane / methanol, 9:1) to give compound lb (4.9 g, 60%) as an orange solid: m.p. 174-176 °C;1H NMR (CDCI3 300 MHz) 6 3.03 ( t, J= 5.0 Hz, 4H), 3.22 (t, J = 5.0 Hz, 4H), 3.79 (s, 3H), 6.19 (d, J = 15.7 Hz, 1H), 6.85 (d, J = 8.9 Hz, 2H), 7.12 (s, 2H), 7.40 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 15.7 Hz, 1H);13C NMR (CDCI3300 MHz) 6 45.8, 49.0, 51.7, 81.1, 96.7, 112.1, 11.9, 116.6, 127.0, 131.1, 131.8, 132.7, 136.7, 139.7, 151.6, 167.1.

[0090] Example 2: Preparation of salt forms of compounds of formula I and of formula II

[0091] Figure 5A shows the preparation of salts of compounds of formula I; Figure 5B shows the preparation of salts of compounds of formula l / formula II;

[0092] 2.1 (E)-4-(4-((5-(3-(Tert-butoxy)-3-oxoprop-l-en-l-yl)thiophen-2-yl)ethvnyl)phenyl) piperazin -1-ium trifluoroacetate, 6

[0093] Compound la (2.0 g, 5.06 mmol) was dissolved in diethyl ether (50 mL). Trifluoroacetic acid (0.39 mL, 5.06 mmol) was added dropwise to the stirring solution. Reaction was left stirring at room temperature for 1 hour. Upon completion, the resultant suspension was filtered off under vacuum and washed further with diethyl ether and dried to afford compound 6 (2.48 g, 100%) as a light yellow solid.3H NMR (300 MHz CDCI3) 6 1.52 (s, 9H), 3.36-3.32 (m, 4H), 3.52-3.48 (m, 4H), 6.14 (d, J = 15.6 Hz, 1H), 6.88 (d, J = 9.2 Hz, 2H), 7.12 (q, 7 = 5.2 Hz, 2H), 7.45 (d, 7=9.2 Hz, 2H), 7.60 (d, 7=15.6 Hz, 2H).13C NMR (CDCI3 300MHz) 6 15.6, 28.2, 42.9, 44.9, 65.3, 80.6, 81.5, 96.7, 111.7, 115.6, 117.6 (q, 7 = 299 Hz), 119.4, 125.6, 132.5, 132.9, 133.3, 135.9, 140.2, 150.5, 159.0 (q, J= 31 Hz, 1C), 165.5;19F NMR (DMSO 300MHz) 6 73.48; Abs. Xmax DCM = 372 nm, DMSO = 385 nm, Toluene = 383 nm; Emm. Xmax DCM = 512 nm, DMSO = 591 nm, Toluene = 481 nm.

[0094] 2.2 (E)-4-(4-((5-(3-(Tert-butoxy)-3-oxoprop-l-en-l-yl)thiophen-2-yl)ethvnyl)phenyl) piperazin-l-ium acetate, 7

[0095] Compound la (2.0 g, 5.06 mmol) was dissolved in diethyl ether (50 mL). Acetic acid (0.289 mL, 5.06 mmol) was added dropwise to the stirring solution. Reaction was left stirring at room temperature for 1 hour. Upon completion, the resultant suspension was filtered off under vacuum and washed further with diethyl ether and dried to afford compound 7 (2.25 g, 98%) as a light yellow solid:TH NMR (DMSO-c / 6300 MHz) 6 1.48 (s, 9H), 1.90 (s, 3H), 2.84-2.81 (m, 4H), 3.18- 3.15 (m, 4H), 6.18 (d, J = 15.7 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 7.31 (d, J = 3.8 Hz, 1H), 7.38 (d, 7=8.9 Hz, 2H), 7.49 (d, 7=3.9 Hz, 1H), 7.67 (d, 7=15.7 Hz, 1H);13C NMR (DMSO-c / 6300 MHz) 6 22.0, 28.2, 45.3, 48.0, 80.6, 81.3, 97.2, 110.2, 114.8, 119.3, 125.9, 132.6, 132.9,

[0096] 133.1, 136.0, 140.0, 151.9, 165.5, 172.9; Abs. XmaxDCM = 387 nm, DMSO = 388 nm, Toluene = 390 nm; Emm. XmaxDCM = 555 nm, DMSO = 589 nm, Toluene = 487 nm.

[0097] 2.3 (E)-4-(4-((5-(3-(tert-butoxy)-3-oxoprop-l-en-l-yl)thiophen-2-yl)ethvnyl)phenyl) piperazin-l-ium triflate, 8

[0098] Compound la (2.0 g, 5.06 mmol) was dissolved in diethyl ether (50 mL). Trifluoromethanesulfonic acid (0.447 mL, 5.06 mmol) was added dropwise to the stirring solution using a glass syringe. Reaction was left stirring at room temperature for 1 hour. Upon completion, the resultant suspension was filtered off under vacuum and washed further with diethyl ether and dried to afford compound 8 (2.68 g, 97%) as a light yellow solid1H-NMR (DMSO-c / 6300MHz) 6 1.48 (s, 9H), 3.24-3.20 (m, 4H), 3.47-3.44 (m, 4H), 6.20 (d, 7=15.7 Hz, 1H), 7.03 (d, 7=9.0 Hz, 2H), 7.33 (d, 7=3.8 Hz, 1H), 7.46 (d, 7=9.0 Hz, 2H), 7.51 (d, 7=3.9 Hz, 1H), 7.67 (d, 7=15.7 Hz, 1H), 8.74 (br, 1H);13C NMR (DMSO-c / 6300MHz) 6 28.2, 31.7, 43.0, 44.8, 80.6, 81.6, 96.7, 111.8, 115.7, 119.4, 121.2 (q, 7=322 Hz), 125.6, 132.5, 133.0, 133.3, 135.9,

[0099] 140.2, 150.5, 165.5 ;19F NMR (DMSO-c / 6300MHz) 6 77.73. Abs. XmaxDCM = 369 nm, DMSO = 384 nm, Toluene = 372 nm; Emm. AmaXDCM = 492 nm, DMSO = 586 nm, Toluene = 469 nm.

[0100] 2.4 (E)-4-(4-((5-(3-methoxy-3-oxoprop-l-en-l-yl)thiophen-2-yl)ethvnyl)phenyl) piperazin-l-ium trifluoracetate, 13

[0101] Compound lb (0.50 g, 1.42 mmol) was dissolved in a mixture of diethyl ether (10 mL) and dichloromethane (80 mL), whereupon trifluoroacetic acid (0.109 mL, 1.42 mmol) was added and the solution was stirred for 45 mins. The solution was evaporated and the residue was resuspended in diethyl ether (100 mL) and filtered to give a crude orange solid. This was recrystallised from ethyl acetate to give salt 13 as an orange solid (0.29 g, 44%):1H NMR (300 MHz, DMSO-C / 6) 6 8.95 (br, 2H), 7.79 (d, 7 = 15.8 Hz, 1H), 7.54 (d, 7 = 3.9 Hz, 1H), 7.45 (d, 7 = 8.9 Hz, 2H), 7.34 (d, J = 3.8 Hz, 1H), 7.03 (d, 7 = 8.9 Hz, 2H), 6.31 (d, 7 = 15.8 Hz, 1H), 3.72 (s, 3H), 3.47 (t, 7 = 5.1 Hz, 5H), 3.23 (t, 7 = 5.1 Hz, 4H);13C NMR (176 MHz, DMSO-c / 6) 6 166.2, 158.0 (q, J = 31 Hz), 150.1, 139.6, 136.5, 132.9, 132.6, 132.5, 125.5, 117.2 (q, J = 300 Hz), 116.8,

[0102] 114.7, 111.2, 96.5, 81.1, 51.6, 44.4, 42.5.

[0103] Example 3: Synthesis of exemplar formulation - compound la (1% w / w) in benzyl alcohol (50%), dimethyl sulfoxide (30%), propylene glycol (20%)

[0104] An exemplar formulation was prepared by mixing benzyl alcohol (5 mL), dimethyl sulfoxide (3 mL) and propylene glycol (2 mL), and the resultant mixture was stirred using a magnetic stirrer for 5 minutes to give a benzyl alcohol (50% v / v), dimethyl sulfoxide (30% v / v) and propylene glycol (20% v / v) solution. A formulation of compound la was prepared by weighing 30 mg of compound la and dissolving this in 2.97 g of the solvent mixture, giving a 1% w / w formulation of compound I in benzyl alcohol (50% v / v), dimethyl sulfoxide (30% v / v) and propylene glycol (20% v / v). In this way, the compound of formula la was demonstrated to be formulatable in common pharmaceutical excipients. Characterisation of the formulation was carried out in Example 5.2.

[0105] Example 4: Manufacture of gel formulations

[0106] To a 10 mL glass vial, 1 to 25 mg of compound la, compound lb, or salts 6, 7, 8, or 13 was added followed by the addition of 1,2-propanediol (1.0 g). The vial was placed in an ultrasonic bath to completely dissolve the material. Deionised (DI) water (8.85 g) was then added, and the resulting mixture was stirred vigorously until complete homogenisation. Hydroxyethyl cellulose (0.15 g) was then added and the mixture was stirred until complete incorporation. One drop of triethanolamine was added into the resulting mixture and after 2-3 minutes of vigorous manual stirring a change in consistency of the mixture was observed from liquid to gel.

[0107] Table 1: Gel formulations

[0108] In this way, the preparation of gel formulations comprising compounds of the invention was demonstrated. Example 5: Characterisation of compounds la and lb and of exemplar formulation

[0109] 5.1 Absorption and emission spectra - compounds la and lb

[0110] Absorption spectra were obtained using a Perkin Elmer Cary 60 spectrometer and emission spectra using an Agilent Cary Eclipse spectrometer. For absorption spectra, 5 pM solutions of the compound of formula I in CHCI3, and in DMSO, were added to 10 mm path length quartz optical cuvettes (Hellma) and absorbances recorded at 1 nm intervals. Extinction coefficient measurements were determined in triplicate using measurements obtained from absorption readings at the respective Xmax exhibited by the compound of formula I in each solvent at concentrations from 5-30 pM. Extinction coefficient values are expressed as an average of the three replicates, with the standard deviation. Emission spectra were obtained at 1 nm intervals from 100 nM solutions in quartz cuvettes, as specified above, using excitation at X = 380 nm, and normalised according to the respective maximal intensity values. Absorption and emission spectra of compound la are shown in Figure 3A and 3B, respectively, and absorption and emission spectra of compound lb are shown in in Figure 4A and 4B, respectively.

[0111] 5.2 Excitation and emission spectra of compound la prepared in an exemplar formulation

[0112] The excitation spectrum of the exemplar formulation of Example 3 in 10 mm path length quartz optical cuvettes (Hellma) was recorded using an Agilent Cary Eclipse spectrometer, with an emission wavelength at 600 nm. The excitation spectrum is shown in Figure 6. The emission spectrum of compound la at 1% w / w in a mixture of benzyl alcohol (50%), dimethyl sulfoxide (30%) and propylene glycol (20%) is shown in Figure 7 and was recorded in 10 mm path length quartz optical cuvettes (Hellma) was recorded using an Agilent Cary Eclipse spectrometer with excitation wavelength at 380 nm.

[0113] 5.3 Quantum yield measurement

[0114] The quantum yield from one-photon excitation was determined using (2E)-3-(4-{2-[4,4- dimethyl-l-(propan-2-yl)-l,2,3,4- tetrahydroquinolin-6-yl]-ethynyl}phenyl)prop-2-enoic acid (Quantum yield 0.67) in toluene as standard. Compound la was measured at varying concentrations in each of the solvents aiming for absorbances of 0.1 and below, these corresponded to concentrations in the 0.50 - 2 pM range. Absorbances of each compound in solution were recorded between 300-1000 nm and the corresponding fluorescence intensity was measured between 250-700 nm.

[0115] Quantum yield was calculated using the relative method. The absorbance and fluorescence of compounds in solution were measured at multiple concentrations and compared to a reference via the following equation: where Grad is the gradient obtained by plotting the integrated fluorescence, / , against the absorbance, 1 - 10-.4, and n is the refractive index of the solvents. The absorbance was measured at 390nm using a Cary 60 UV-Vis spectrometer, aiming for absorbances 0.1 and below. The fluorescence was measured using the same excitation wavelength (390 nm) by a Cary Eclipse spectrometer and integrated between 400-700 nm. (2£)-3-(4-{2-[4,4-dimethyl- l-(propan-2-yl)-l,2,3,4- tetrahydroquinolin-6-yl]-ethynyl}phenyl)prop-2-enoic acid in toluene was used as the reference for all calculations (Chisholm et al., Chem. Sci. 17 (2019)). Between 5-7 concentrations were measured with 3 repeats. The results are shown in Table 2 below.

[0116] Table 2: Photophysical characterisation of compound la Example 6: Characterisation of compound la in gram-positive bacteria

[0117] 6.1 Experimental

[0118] 6.1.1 Bacteria

[0119] Bacillus subtilis 168 (ATCC 23857), Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus SH1000 (ATCC 6538P), Staphylococcus aureus USA300 (ATCC BAA-1556), Enterococcus faecalis (ATCC 29212), Pseudomonas fluorescens (ATCC 13525) and Escherichia coli FDA strain Seattle 1946 20 (ATCC 25922) were obtained from the American Type Culture Collection.

[0120] 6.1.2 Photoactivated bacterial rowth inhibition

[0121] All bacteria were cultivated in LB (Miller) broth in an orbital shaker (VWR) at 30-37 °C. Overnight cultures were prepared by inoculating a single, isolated colony into 10 ml of LB broth followed by incubation with shaking for 16-20 h. Bacteria exposed to photoactivatable compound were placed in a LightOx PhotoReact 365™ Lightbox (Merck) and exposed to light at a wavelength of 365 nm for 5 min, with an energy intensity of 13 mW / cm2(total energy delivered: 3.9 J / cm2). For assays requiring half of a 96-well or agar plate to be irradiated, a section of black card was used to mask the relevant samples.

[0122] For bacterial overlays, 50 ml of 1.5% LB agar was poured into a 100 x 100 x 20 mm square petri dish (Sarstedt) and once solidified, 15 ml of 0.75% LB soft agar inoculated with 200 pl of bacteria from an overnight culture was poured onto the surface. Serially diluted concentrations of compound were applied to the overlay in 6 pl volumes. Plates to be photoactivated were then exposed to light at 365 nm and incubated at 30 °C for 24 h before imaging in a Bio-Rad Gel Doc XR+ System.

[0123] For growth curves, 5 ml of LB broth in a 15 ml Falcon tube (Sarstedt) was inoculated with 50 pl of bacteria from an overnight culture. Compound I was added to give a final concentration of 2 pM and incubated in the dark with shaking at 30 °C for 30 min. Samples (100 pl) were pipetted into the wells of a 96-well plate, with 8 repeats per sample. Half of the plate was covered, and the rest irradiated at 365 nm. Growth was monitored at OD600nm every 5 min for 24 h in a plate reader (Biotek Synergy HT) and data normalised against the negative control containing media alone.

[0124] 6.1.3 Viability assays

[0125] A semi-microcuvette (Sarstedt) containing 2 ml of LB broth inoculated with 50 pl of bacteria from an overnight culture was incubated with shaking at 30 °C until early log phase, ODeoonm 0.2. Samples of 1 ml were transferred to a 24-well plate and incubated with Compound la at 2 pM for 30 min at 30 °C. Serial dilutions (10-fold) were performed, 30 pl sample in 270 pl LB, in a 96-well plate. Samples in the 24-well plate were irradiated, incubated for 15 min at room temperature and 30 pl samples removed and serial 10-fold dilutions made as before. Samples (10 pl) were applied to an LB agar plate in triplicate and incubated for 16-20 h. Colonies at appropriate dilutions were enumerated and viability determined in colony forming units (CFU) per ml.

[0126] 6.1.4 Monitoring loss of membrane integrity using propidium iodide

[0127] Bacteria were cultivated as in viability assays and 500 pl of the culture transferred to a 1.5 ml microcentrifuge tube containing compound la to give a final concentration of 2 pM. A control sample with 500 pl of the culture and 0.2% DMSO was set up in parallel. Samples were incubated at 30 °C for 30 min and cells pelleted by centrifugation at 17,000 g for 4 min. Cell pellets were resuspended in 200 pl lx PBS containing 7.5 pM propidium iodide (PI; ThermoFisher). 3 x 50 pl volumes of each sample were applied to a 96-well plate, transferred to a Biotek Synergy HT and fluorescence measurements made every 2 min for 20 min with excitation at 485 nm and emission at 645 nm. PBS (50 pl) was then added to sample wells and 50 pl 100% ethanol added to additional samples as a positive control for loss of membrane integrity and cell killing. The plate was placed in the PhotoReact 365 lightbox, irradiated for 5 min, returned immediately to the plate reader and fluorescence monitored every 2 min for 1 h. 6.1.5 Confocal microscopy

[0128] For confocal microscopy, cell pellets were prepared as above in section 6.1.4 with 500 pl of the culture transferred to a 1.5 ml microcentrifuge tube containing compound la to give a final concentration of 2 pM. Samples were incubated at 30 °C for 30 min and cells pelleted by centrifugation at 17,000 g for 4 min, then resuspended in 200 pl of Baclight solution containing 10 pM SYTO9 and 60 pM PI. A 10 pl sample was applied to a 1 cm x 1 cm 1.5% agarose (Bioline) pad on a microscope slide with a cover slip placed on top. The slide was imaged using a 63x lens on a confocal microscope (Zeiss 800 Airyscan) with compound I being imaged with the airyscan function using a 405 nm laser and an emission filter of 450-550 nm, SYTO 9 using a 488 nm laser and 550-580 nm filter and PI using a 488 nm laser and an emission filter of 600-650 nm. Samples were irradiated using the 405 nm laser at 30% power for 1 minute (total energy: 90mJ / cm2) on the microscope.

[0129] 6.1.6 Screening of compound la for activity against gram-positive and gram-negative bacteria

[0130] Compound la was screened for activity against two species of gram-positive bacteria (B. subtilis and 5. epidermidis) and two species of gram-negative bacteria (£. coli and P. fluorescens). Two-fold dilutions of compound la were applied to the surface of an overlay containing each bacterial species and were subsequently activated by exposure to light at 365 nm, followed by incubation for 24 hours. 5 structurally-related compounds (shown in Table 3 below), were also tested. Figure 8A shows irradiated bacterial overlays treated with compound la (shown in lane 2) and the structurally related compounds (shown in lanes 1 and 3-6). Figure 8B shows unirradiated bacterial overlays treated with compound la (shown in lane 2) and the 5 structurally-related compounds (shown in lanes 1 and 3-6). Figure 8A demonstrates that compound la restricts the bacterial growth of both gram-positive bacterial species (B. subtilis and 5. epidermidis) after irradiation. With clearer growth inhibition observed at 0.1 pM than was observed at 1 pM of the next best compound (shown in lane 1), Compound la was observed to be at least lOx more potent than the next best compound. No significant growth restriction is observed in gram-negative species (£. coli and P. fluorescens). Unirradiated samples did not show any restriction of bacterial growth (Figure 8B).

[0131] 21

[0132] Table 3: Compound la and reference compounds.

[0133] 6.1.7 Effect of compound la on bacterial growth Compound la was examined for its ability to inhibit the growth of gram-positive bacteria (B. subtilis and 5. epidermidis) and gram-negative bacteria (£. coli ). Bacterial growth in response to treatment with 2pM compound la was followed by optical density measurements over a 24-hour period with half of the samples being irradiated with 365 nm light at the outset and the remaining half being unirradiated controls. Figure 9 shows irradiated and unirradiated bacterial cells treated with 2 pM compound I and DMSO (control). Figure 9 demonstrates that both species of gram-positive bacteria (B. subtilis and 5. epidermidis) exhibit growth restriction after treatment with compound la and irradiation. Gram-negative E. coli experienced an initial lag in growth relative to the unirradiated control followed by a resumption in growth. No difference in growth was observed in control cells treated with DMSO.

[0134] 6.1.8 Effect of compound la on bacterial viability

[0135] Cell viability assays were conducted to determine whether compound la exhibits any bacteriostatic or bactericidal properties in gram-positive bacteria (B. subtilis and 5. epidermidis) and gram-negative bacteria (£. coli and P. fluorescens). The four species of bacteria were cultured to early log-phase priorto addition of 2 pM compound la and exposure to 365 nm light. Appropriate controls without irradiation and equivalent concentrations of DMSO were conducted in parallel. Serial dilutions of the bacteria were applied to the surface of agar plates and CFU / ml calculated. Figure 10 illustrates that both gram-positive species show a 6-log reduction in survival after treatment with compound la and exposure to light compared to the non-irradiated controls. No such reduction was observed in the gramnegative species.

[0136] Bacterial viability assays were also conducted in two gram-positive ESKAPE pathogens - Enterococcus faecium and Staphylococcus aureus (strains SH1000 and USA300). ESKAPE pathogens are a group of highly virulent and multidrug resistant bacterial pathogens and are major causes of nosocomial infections. Figure 11 shows a 4-log reduction in viable cells following photoactivation of compound la. 6.1.9 Effect of compound la on bacterial membrane integrity

[0137] Propidium iodide (PI) was used to assess loss of membrane integrity in cells exposed to compound la. PI is a membrane-impermeable dye that fluoresces in the presence of chromosomal DNA only when it can penetrate the cell envelope and thus serves as a reporter, with some caveats, for cell death. The two gram-positive bacterial species (B. subtilis and 5. epidermidis) and two gram-negative bacteria (£. co / / and P. fluorescens) were incubated in the presence of 2 pM compound la and then exposed to 365 nm light after 20 minutes. PI fluorescence was monitored throughout the experiment from 0-80 minutes. Figure 12 demonstrates that a significant increase in PI fluorescence was evident with both grampositive bacterial species following photoactivation, consistent with rapid loss of cell viability as demonstrated in Figure 10. Only minor increases in fluorescence were evident in the gramnegative species after light activation. A small increase in PI fluorescence was noted in both gram-positive species prior to irradiation, indicating that there may be a slight loss of membrane integrity, especially with B. subtilis.

[0138] 6.1.10 Real-time monitoring of bacterial membrane integrity

[0139] To provide further evidence that membrane disruption by compound la is dependent on photoactivation, a real time BacLight assay was employed. The assay utilises SYTO 9, a membrane permeable dye that fluoresces when bound to chromosomal DNA, while PI, as mentioned above, only enters cells when membrane integrity is compromised and displaces SYTO 9 due to its higher affinity for DNA. Hence, comparing the proportion of PI positive and SYTO 9 positive cells provides an indication of bacterial membrane integrity and of bacterial viability. Bacteria were grown to early-log phase, treated with 2 pM compound la in the presence of the two dyes and visualised by microscopy. Compound la was activated by light at 405 nm and images were captured over 10 minutes to monitor changes in fluorescence. Figure 13 shows that most of the 5. epidermidis (panel A) and B. subtilis (panel B) bacteria were SYTO 9 positive prior to light activation indicating these cells possessed intact cell envelopes and were alive. Photoactivation resulted in a rapid fluorescence change in both gram-positive species (B. subtilis and 5. epidermidis), with all cells PI positive after 10 minutes. These changes indicate significant membrane damage, and likely cell death, due to the activated compound la, allowing the influx of and subsequent fluorescence from PI. In contrast, neither of the gram-negative species (£. coli and P. fluorescens) showed a change in fluorescence after photoactivation and therefore showed no signs of membrane disruption.

[0140] 6.1.11 Visualisation of compound la in bacterial cells

[0141] Utilising the fluorescence properties of compound la, intracellular localisation of the compounds in two gram-positive bacterial species (B. subtilis and 5. epidermidis) and two gram-negative bacterial species (£. coli and P. fluorescens) was visualised. Bacteria were grown to mid-log phase and visualised by microscopy. Figure 14 demonstrates some association of the compound with the cell surface in both gram-positive species. Although there are also dense patches evident, either resulting from (potential) aggregates at the surface or accumulation within the cytosol. Clustering of compound la at the poles of B. subtilis cells (panel A) was particularly apparent, notably between cells undergoing division potentially because these areas are more accessible. These concentrated patches were entirely absent in the two gram-negative species where compound la appeared to associate solely with the cell surface.

Claims

Claims:

1. A compound of formula I:Formula I in which R is C1-C4 alkyl, in free or salt form, for use in the treatment or prevention of an infection caused by grampositive bacteria.

2. A compound of formula I as claimed in claim 1, wherein R is methyl or tert-butyl.

3. A compound of formula I as claimed in claim 1 or claim 2, wherein the infection caused by gram- positive bacteria is a disease or condition caused by gram-positive bacteria, or is an infection of a skin lesion.

4. A compound of formula I as claimed in any preceding claim, in salt form, wherein the salt is an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

5. A pharmaceutical composition comprising the compound of formula I as claimed in any preceding claim.

6. A pharmaceutical composition as claimed in claim 5, wherein the composition is in the form of an aqueous solution, suspension, milk, cream, foam, paste, ointment, gel, or hydrogel.

7. A pharmaceutical composition as claimed in claim 5 or claim 6, wherein the pharmaceutical composition is a topical composition.

8. A compound of formula I as claimed in any of claims 1 to 4 or a pharmaceutical composition as claimed in any of claims 5 to 7 for use in the treatment or prevention of an infection caused by gram-positive bacteria, wherein the compound or composition is administered topically.

9. A compound or pharmaceutical composition as claimed in claim 8, wherein the compound or composition is administered topically and exposed to light.

10. A compound or pharmaceutical composition as claimed in claim 9, wherein the compound or composition is exposed to light of a wavelength between 365 - 460 nm.

11. A dressing or patch comprising a compound of formula I as claimed in any of claims 1 to 4 or 8 to 10, or comprising a pharmaceutical composition as claimed in any of claims 5 to 10.

12. A dressing or patch as claimed in claim 11, wherein at least a portion of the dressing or patch is transparent.

13. A compound of formula I as claimed in any of claims 1 to 4 or 8 to 10, or a pharmaceutical composition as claimed in any of claims 5 to 10 for use in the treatment or prevention of a disease or condition caused by gram-positive bacteria, or an infection of a skin lesion, wherein the disease or condition caused by gram-positive bacteria is selected from cellulitis, erysipelas, impetigo, folliculitis, carbuncles, furuncles, erthrasma, leprosy and cutaneous tuberculosis; and wherein an infection of a skin lesion is selected from a grampositive infection of an ulcer, skin wound, burn, or insect bite.

14. A compound of formula I as claimed in any of claims 1 to 4 or 8 to 10 or a pharmaceutical composition as claimed in any of claims 5 to 10 for use in the treatment of an infection caused by gram-positive bacteria, wherein the infection is caused b Staphylococcusaureus, Methicillin resistant Staphylococcus aureus (MRSA), Community-Acquired Methicillin resistant Staphylococcus aureus (CA-MRSA), Staphylococcus epidermidis, Staphylococcus haemolyticus, Micrococcus luteus, Corynebacterium minutissimum, Enterococcus faecalis, Group A Streptococcus, Group C Streptococcus, Group G Streptococcus, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leptromatosis, Mycobacterium marinum and Mycobacterium ulcerans.

15. A method of treatment of a patient with a bacterial infection caused by gram-positive bacteria, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I or a salt thereof as claimed in any of claims 1 to 4.

16. A method of treatment as claimed in claim 15, wherein the administering is topical administration.

17. A method of treatment as claimed in claim 15 or claim 16, wherein the method further comprises exposing the compound of formula I to light.

18. A compound of formula II:in free or salt form, in which R is C1-C3 alkyl.

19. A compound of formula II as claimed in claim 18, wherein the salt is an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

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