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

Abstract

The present invention relates to photoactive compounds and compositions containing the same, and to the use of them for the treatment and prevention of infections caused by Gram-positive bacteria. In several embodiments, the present invention relates to methods for administering compounds and compositions, particularly 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

[Technical Field]

[0001] The present invention relates to compounds and compositions, and their use in the treatment and prevention of infections caused by Gram-positive bacteria. In several embodiments, the present invention relates to compounds and compositions, particularly methods for administering 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.

[0002] The discovery and introduction of antibiotics into clinical care is almost certainly one of the greatest breakthroughs in 20th-century medicine. The discovery of penicillin in the 1920s marked the beginning of a golden age of antimicrobial discovery, which continued with the rapid discovery of several classes of natural antibiotics in a relatively concentrated period until the 1960s. This era also saw widespread use and overuse of antibiotics, which created selective pressure for the acquisition and spread of antimicrobial resistance (AMR) in various species, leading to a global health problem often referred to as a "silent pandemic." In 2019 alone, AMR caused approximately 1.3 million deaths worldwide. Furthermore, there is clear evidence that bacteria are increasingly acquiring resistance to existing antibiotics. However, because the rate of resistance development is outpacing the rate of new antibiotic discovery, there is an urgent need for additional therapeutic approaches to microbial infections.

[0003] Photoactivatable cytotoxic compounds can be used to induce cell-killing activity upon exposure to light, thus offering the advantage of minimizing systemic and extratarget toxicity in humans.

[0004] Photoactive cytotoxic activity is a promising area of ​​research because it is often expressed through the generation of reactive oxygen species (ROS), and bacteria are thought not to acquire resistance to ROS. However, the main obstacle to the therapeutic use of photoactivated compounds lies with the photosensitizers themselves. Among other problems, many known photosensitizers are high molecular weight porphyrin-based structures and are generally poorly or poorly soluble, making formulation into useful therapeutic forms difficult. Known compounds also exhibit other characteristics such as inadequate or unsuitable pharmacokinetic properties, limited or absent ability to penetrate bacterial cells, and limited selectivity between cell types. These difficulties, combined, limit the use of such compounds in antimicrobial therapy.

[0005] Therefore, it is advantageous to mitigate one or more of these disadvantages and provide compounds that can be used as antimicrobial agents in the treatment of bacterial infections caused by Gram-positive bacteria. Compounds that have effective antimicrobial activity against Gram-positive bacteria and also possess good physical properties, such as good solubility in commonly used formulation solvents, are useful. Compositions containing such compounds are also beneficial. [Overview of the project]

[0006] Therefore, the present invention generally relates to compounds of formula I, either in free form or salt form, for use in the treatment and prevention of infections caused by Gram-positive bacteria: [ka] In the formula, R is a C1-C4 alkyl group.

[0007] Throughout the present specification, the term "alkyl" refers to a fully saturated, branched, unbranched or cyclic hydrocarbon moiety, that is, primary, secondary or tertiary alkyl, or optionally cycloalkyl or alkyl substituted with cycloalkyl. Unless otherwise stated, an alkyl group contains 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, 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.

[0008] In Formula I, R is C1-C4 alkyl. R may be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In one embodiment, R is methyl or butyl. In one embodiment, R is methyl or tert-butyl.

[0009] In one embodiment, R is methyl or tert-butyl.

[0010] In one embodiment, R is tert-butyl.

[0011] The compounds used in the present invention also include salts thereof, and reference to the compound of formula I is intended to include reference to the salt thereof, unless otherwise specified. Suitable salts include, for example, acid salts formed with inorganic acids and / or organic acids, and basic salts formed with inorganic bases and / or organic bases; when a zwitterion ("inner salt") can be formed, this is also included in the term "salt" as used herein. Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, but other salts may also be useful under certain circumstances. Examples of acid addition salts that may be useful include acetate, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate, naphthalenesulfonate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also called tosylate), triflate, and the like. Examples of basic salts that 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 (e.g., organic amines) such as dicyclohexylamine and t-butylamine, as well as salts with amino acids such as arginine and lysine. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chloride, bromide, and iodide), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfate), long chain halides (e.g., decyl, lauryl, and stearyl chloride, bromide, and iodide), arylalkyl halides (e.g., benzyl and phenethyl bromide), and the like.

[0012] In one embodiment, the salt may be selected from hydrochloride, hydrobromide, hydroiodide, acetate, trifluoroacetate, p-toluenesulfonate, trifluoromethanesulfonate, triflate, tetrafluoroborate, formate, and sulfate.

[0013] In one embodiment, the salt is formed from an anion containing at least two oxygen atoms. The salt may be, for example, an acetate, trifluoroacetate, triflate, bistriflimide tosylate, ethyl sulfate, bis(fluorosulfonyl)imide, or dimethyl phosphate.

[0014] In one embodiment, the salt may be selected from acetate, trifluoroacetate, triflate, hydrobromide, and hydroiodide.

[0015] "Gram-positive bacteria" refer to bacteria that test positive in the Gram staining test. These bacteria absorb the crystal violet stain used in the Gram staining test and retain it even after the staining solution has been washed away from the rest of the sample, due to the presence of a thick peptidoglycan layer in the bacterial cell wall.

[0016] Gram-positive bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), community-acquired methicillin-resistant Staphylococcus aureus (community-acquired MRSA), Staphylococcus epidermidis, hemolytic Staphylococcus, Micrococcus species (including Luteus), Corynebacterium minutisimum, Group A Streptococcus (GAS) (including Streptococcus pyogenes), cutaneous Group C Streptococcus, cutaneous Group G Streptococcus, Enterococcus faecalis, Mycobacterium tuberculosis, Mycobacterium bovine, Mycobacterium lepromatosis, Mycobacterium marinum, and Mycobacterium ulcerans.

[0017] "Infections caused by Gram-positive bacteria" encompass both diseases and conditions caused by Gram-positive bacteria, as well as 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, boils, erthrasma, leprosy, and cutaneous tuberculosis. On the other hand, infections of skin lesions include ulcers, skin wounds, burns, and insect bites. As technicians will understand, many bacterial infections are associated with skin lesions, and the lesions themselves arise from a variety of causes. In this case, the compounds or compositions of the present invention can be used to treat or prevent bacterial infections of lesions caused by Gram-positive bacteria. Skin lesions susceptible to infection by Gram-positive bacteria include ulcers, skin wounds, burns, and insect bites. "Ulcers" include arterial ulcers, diabetic foot ulcers, pressure ulcers, and Buruli ulcers. Skin wounds susceptible to bacterial infection include traumatic wounds, ischemic wounds, and surgical wounds. Burns include burns and chemical burns.

[0018] In one embodiment, the compounds or compositions of the present invention are used to treat infections caused by Gram-positive bacteria, the infections caused by Gram-positive bacteria being selected from cellulitis, erysipelas, impetigo, folliculitis, erthyrasma, leprosy, and cutaneous tuberculosis, or the Gram-positive bacterial infection is an infection of a skin lesion selected from arterial ulcers, pressure ulcers, Buruli ulcers, infected wounds, ischemic wounds, surgical wounds, chemical burns, and insect bites.

[0019] The present invention also relates to a pharmaceutical composition comprising a compound of formula I.

[0020] "Pharmaceutical composition" refers to a composition suitable for administration to a patient. Therefore, "pharmaceutical composition" refers to a composition comprising the compound of Formula I of the present invention, or a salt thereof, its solvates, isomers, or tautomers, optionally combined with one or more pharmaceutically acceptable excipients, carriers, or diluents. "Pharmaceutical composition" is also intended to encompass both bulk compositions (i.e., forms not molded into individual dosage units) and individual dosage units. Such individual dosage units include impregnated dressings, transdermal patches, ampoules, filled syringes, packets, etc. A composition contains a therapeutically effective amount of the compound of Formula I. "Therapeutably effective" or "effective amount" refers to the amount of the compound or composition of the present invention that is effective in producing the desired therapeutic effect.

[0021] The pharmaceutical composition comprises a compound of formula I, optionally combined with one or more pharmaceutically acceptable excipients, carriers, or diluents, for use in the treatment of infections caused by Gram-positive bacteria. The composition may optionally contain one or more additional therapeutic agents.

[0022] In one 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 found that the compound of formula I can be formulated into commonly used pharmaceutical excipients and exhibits good stability while maintaining efficacy against Gram-positive bacteria.

[0023] In one embodiment, the pharmaceutical composition is in the form of a gel or hydrogel.

[0024] The appropriate dosage for administering the compound of the present invention to a patient can be determined by a person skilled in the art, such as a physician, pharmacist, or other expert, and may vary depending on factors such as the patient's weight, health condition, age, frequency of administration, route of administration, presence or absence of other active ingredients, and the conditions under which the compound is administered.

[0025] A suitable aqueous solution or formulation contains the compound of formula I (in the form of a free base or salt) at a concentration of 0.001–20% (w / w), 0.05–10% (w / w), or 0.1–5% (w / w). Suitable excipients for aqueous solutions include, in particular, propylene glycol, dipropylene glycol, hexylene glycol, butylene glycol, benzyl alcohol, glycerol, sodium chloride, and sodium hydroxide. Suitable surfactants include foaming agents, including betaine and cocamidopropyl betaine.

[0026] Alternatively, the compound or pharmaceutical composition may be formulated as a cream or ointment. The cream or ointment may contain the compound of formula I (in the form of a free base or salt) at concentrations of 0.001–20% (w / w), 0.05–10% (w / w), or 0.1–5% (w / w). Suitable excipients include, but are not limited to, petrolatum / mineral oil bases, paraffin, alginates, ceresin, glycerin, emulsifiers, and emollients such as glyceryl stearate and panthenol.

[0027] Alternatively, the compound or pharmaceutical composition may be formulated as a gel or hydrogel. The gel or hydrogel may contain the compound of formula I (in the form of a free base or salt) at concentrations of 0.001 to 20% (w / w), 0.05 to 10% (w / w), or 0.1 to 5% (w / w). Suitable excipients include, but are not limited to, celluloses, polyacrylates, polyvinyl alcohol, polyethylene glycol, alginates, chitosan, hyaluronic acid, and polyacrylamide formulations.

[0028] In one embodiment, the pharmaceutical composition may be formulated as a gel or hydrogel. The gel may be a cellulose gel. The gel composition may contain hydroxyethylcellulose or carboxymethylcellulose.

[0029] Compounds or pharmaceutical compositions may be formulated or impregnated into dressings or patches. Suitable dressings include hydrocolloid dressing formulations derived from solid hydrocolloid matrices such as gelatin, pectin, carboxymethylcellulose, and combinations thereof. Alternative dressings include, but are not limited to, hydrogel dressing formulations containing alginates, chitosan, hyaluronic acid, polyvinyl alcohol, polyacrylamide, and polyethylene glycol; alginate dressings; electrospun nanofiber dressings derived from polymers such as gelatin, collagen, chitosan, hyaluronic acid, alginates, cellulose, and polyvinylpyrrolidone, polycaprolactone, polyvinyl alcohol, polyethylene glycol, and poly(lactic acid-coglycolic acid); and film dressings derived from polyurethane, polylactic acid, cellulose, starch, alginates, and hyaluronic acid. Suitable patches include fabric patches containing nonwoven, woven, or knitted materials.

[0030] The formulation of a compound or pharmaceutical composition may depend on the condition in which the compound is administered, and various formulations are possible. This allows the attending physician, pharmacist, or other expert to select the most appropriate formulation, taking into account relevant factors such as the condition being treated, the treatment site, and any requirements for concomitant administration.

[0031] In one embodiment, the pharmaceutical composition is a topical composition.

[0032] Topical compositions are applied directly to the skin. Topical compositions can be advantageous in therapeutic applications because they allow for the direct application of compounds in high doses to the required area. Topical compositions may also have reduced side effects and organ toxicity compared to systemic therapeutics.

[0033] In one embodiment, a compound of formula I or a composition containing a compound of formula I is administered topically.

[0034] In one embodiment, a compound of formula I or a composition containing a compound of formula I is exposed to light.

[0035] In one embodiment, a compound of formula I or a composition containing a compound of formula I is administered topically and exposed to light.

[0036] The compound of formula I has been shown to exhibit cytotoxic activity against Gram-positive bacteria when activated by light.

[0037] In one embodiment, the compound or composition is exposed to light with a wavelength of 365 to 460 nm.

[0038] The compound or composition may be exposed to light for 1 to 30 minutes. In one embodiment, the compound or composition is exposed to light for 1 to 10 minutes, 2 to 8 minutes, or about 5 minutes.

[0039] As an example of administering the compound or composition of the present invention, the gel formulation can be directly applied to the affected tissue area, left for 1 to 120 minutes, and then activated with light of a wavelength of 365 to 460 nm emitted from a conventional LED light source to exhibit an antibacterial effect against Gram-positive bacterial infections. The formulation can then be removed from the area.

[0040] Alternatively, the compound or a composition containing the compound can be applied to the affected area, covered with a transparent dressing or bandage (or a transparent window in the dressing or bandage), and then exposed to light to activate the compound. The dressing may be left in place to promote wound healing.

[0041] The compounds or compositions of the present invention can also be applied to skin lesions to suppress or delay the growth of Gram-positive bacteria, that is, to prevent infection of skin lesions by reducing the bacterial load. This embodiment is particularly suitable for skin lesions susceptible to infection caused by Gram-positive bacteria that are normally present on mammalian skin, such as ulcers, skin wounds, burns, and insect bites.

[0042] In one embodiment, a dressing or patch is provided that comprises a compound of formula I or a pharmaceutical composition containing the same.

[0043] In this embodiment, the compound or composition can be impregnated into a topical delivery device such as a dressing or patch and applied to the affected area to bring the compound into contact with the infection site. Advantageously, if at least a portion of the dressing or patch is transparent, the compound can be exposed to light through the dressing or patch, or through a window in the dressing or patch.

[0044] One aspect of the present invention provides a method for treating a patient having a bacterial infection caused by Gram-positive bacteria, the method comprising administering to the patient a therapeutically effective amount of the compound of formula I or a salt thereof. The administration may be topical. The compound of formula I or a salt thereof may be formulated into a pharmaceutical composition, or optionally into a gel composition. The method may further comprise exposing the therapeutically effective amount of the compound of formula I or a salt thereof to light.

[0045] In one aspect of the present invention, a compound of formula II is provided in the form of a free or salt: [ka] In the formula, R is a C1-C3 alkyl group.

[0046] In one embodiment, in formula II, R is methyl.

[0047] In one embodiment, the compound of formula II is in the form of a salt, and the salt is an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

[0048] Examples The present invention will be described below with reference to the attached drawings, but only as an example. [Brief explanation of the drawing]

[0049] [Figure 1]This figure shows the synthesis of compound Ia, a representative compound of formula I. [Figure 2] This figure shows the synthesis of compound Ib, a representative compound of formulas I and II. [Figure 3] Figure 3A shows the normalized absorption spectra of compound Ia in dimethyl sulfoxide and chloroform (10 μM). Figure 3B shows the emission spectra of compound Ia in toluene (100 nM), chloroform (100 nM), and dimethyl sulfoxide (2.65 μM). [Figure 4] Figure 4A shows the normalized absorption spectra of compound Ib in L-toluene (5 μM), chloroform (5 μM), and dimethyl sulfoxide (5 μM). Figure 4B shows the emission spectra of compound Ib in toluene (100 nM), chloroform (100 nM), and dimethyl sulfoxide (5 μM). [Figure 5A] This figure shows the synthesis of the salt of compound Ia. [Figure 5B] This figure shows the synthesis of the salt of compound Ib. [Figure 6] This figure shows the excitation spectrum of a typical formulation of compound Ia at 1% (w / w) in a mixture of benzyl alcohol (50%), dimethyl sulfoxide (30%), and propylene glycol (20%). [Figure 7] This figure shows the emission spectrum of a typical formulation of compound Ia at 1% (w / w) in a mixture of benzyl alcohol (50%), dimethyl sulfoxide (30%), and propylene glycol (20%). [Figure 8A] This figure shows the effect of compound Ia on the growth of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). [Figure 8B] This figure shows the effect of compound Ia on the growth of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). [Figure 9] This figure shows the effect of compound Ia on the growth of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis). [Figure 10] This figure shows the effect of compound Ia on the survival rates of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). [Figure 11] This figure shows the effect of compound Ia on the survival rates of Gram-positive ESKAPE bacteria (Enterococcus faecalis), methicillin-sensitive Staphylococcus aureus (SH1000), and methicillin-resistant Staphylococcus aureus (USA300). [Figure 12] This figure shows the effect of compound Ia on the membrane integrity of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). [Figure 13] This figure shows the real-time changes in propidium iodide (PI) after photoactivation to monitor the membrane integrity of Gram-positive bacteria Bacillus subtilis and Staphylococcus epidermidis during treatment with compound Ia. [Figure 14] This figure shows the localization of compound Ia in Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis). [Modes for carrying out the invention]

[0050] Experimental example The following examples are for illustrative purposes only.

[0051] material and method Reagents were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesar, and Fluorochem and purified as needed by recrystallization or distillation / sublimation under vacuum. Solvents supplied by Fisher Scientific or Sigma-Aldrich were used and dried with appropriate desiccants before use as needed. Thin-layer chromatography (TLC) was performed using Merck Millipore silica gel 60G F254 25 glass plates and / or aluminum oxide (with 254 nm fluorescent indicator) TLC-PET foil (40 × 80 mm) and visualized with a UV lamp or appropriate stain. Flash column chromatography was performed using Sigma-Aldrich SiO2 (230–400 mesh, 40–63 μM, 60 Å) and monitored using TLC. NMR spectra were recorded using a Bruker Avance Neo-700, Bruker Avance-600, or Bruker Avance-400 spectrometer 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 multiplet (m), relative to the following deuterated solvent signal: CDCl3 (1H = 7.26 ppm, 13 C=77.0ppm), (CD3)2SO(1H=2.50ppm, 13 (C=39.5 ppm). Electrospray mass spectrometry (ESMS) was performed using a TQD (Waters Ltd., UK) mass spectrometer and an Acquity UPLC (Waters Ltd., UK), and accurate mass measurements were obtained using a QtoF Premier mass spectrometer and an Acquity UPLC (Waters Ltd., UK). IR spectra were recorded using a Perkin Elmer FTIR spectrometer.

[0052] Example 1: Synthesis of compound I and representative formulations 1.1 Synthesis of Compound Ia The synthesis of compound Ia is shown in Figure 1 and is explained below.

[0053] 1.1.1 Synthesis of 5-iodothiophene-2-carbaldehyde, 2 To a solution of 2-thiophenecarboxaldehyde (37.4 mL, 400.0 mmol) at 50°C in ethanol (EtOH) (300 mL), N-iodosuccinimide (NIS) (99.0 g, 440.0 mmol) and p-toluenesulfonic acid monohydrate (pTSA) (7.60 g, 40.0 mmol) were added, and the resulting solution was stirred at 50°C for 50 minutes. The solution was cooled to 35°C, 1 M hydrochloric acid (500 mL) was added, and the mixture was extracted with ethyl acetate. The mixture was washed with saturated Na2S2O3, saturated NaHCO3, water, and saline solution, dried (MgSO4), and evaporated to obtain compound 2 as a gradually crystallizing brown oil (85.2 g, 90%). 1 H NMR (400MHz, CDCl3)δ 7.39(s, 2H), 9.77(s, 1H); 13 C NMR (101MHz, CDCl3) δ 87.8, 137.0, 138.2, 149.6, 181.1.

[0054] 1.1.2 Synthesis of 5-[2-(trimethylsilyl)ethynyl]thiophene-2-carbaldehyde, 3 Compound 2 (78.3 g, 329 mmol) was dissolved in triethylamine (Et3N) (1,000 mL), and the resulting solution was degassed under vacuum by sonication and substituted with an Ar atmosphere (10 times). 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 added under argon, and the resulting suspension was stirred at 40°C for 18 hours. The mixture was diluted with diethyl ether and passed through Celite / SiO2 to obtain a crude brown oil (72 g). This was purified by silica chromatography (cyclohexane / ethyl acetate, 9:1) to obtain Compound 3 as a gradually crystallizing light brown oil (64.22 g, 94%). 1 H NMR (400MHz, CDCl3)δ 0.26(s, 9H), 7.25(d, J=3.9Hz, 1H), 7.61(d, J=3.9Hz, 1H), 9.84(s, 1H);13 ¹³C NMR (101 MHz, CDCl₃) δ -0.5, 26.9, 96.3, 104.6, 132.5, 133.1, 135.7, 143.8, 182.4; IR (ATR) v max / cm -1 2960w, 2899w, 2833w, 2148m, 1666s, 1438s, 1249s, 1223s, 1207s, 838s; MS (ES) m / z = 209.0 [M+H] + ; HRMS (ES) Calcd. for C 10 H 13 SOSi [M+H] + : 209.0451, found 209.0454.

[0055] 1.1.3 Synthesis of tert-butyl (2E)-3-{5-[2-(trimethylsilyl)ethynyl]thiophen-2-yl}prop-2-enoate, 4 tert-Butyl diethylphosphonoacetate (40.6 mL, 173 mmol) and LiCl (7.33 g, 173 mmol) were added to anhydrous tetrahydrofuran (THF) (700 mL) at 0°C, the resulting solution was stirred for 15 minutes, then compound 3 (30.0 g, 144 mmol) was added. To this solution, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (25.9 mL, 173 mmol) was gradually added, and the resulting slurry was stirred at room temperature for 16 hours. This was poured into crushed ice and extracted with ethyl acetate. The organic phase was washed with water and brine, dried (MgSO₄) 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 about 25% of the TMS deprotected product (compound 5) (37.47 g, 85%): 1 ¹H NMR (400 MHz, CDCl₃) δ 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 yield of this step was calculated based on compound 4; TMS deprotection was observed during purification on SiO₂.

[0056] 1.1.4 Synthesis of tert-butyl(2E)-3-(5-ethynylthiophen-2-yl)prop-2-enoate, 5 Compound 4 (37.47 g, 122 mmol) was dissolved in a mixture of dichloromethane (DCM) (300 mL) and methanol (MeOH) (30 mL), and K2CO3 (33.7 g, 244 mmol) was added. The resulting suspension was stirred at room temperature (RT) for 18 hours, and then diluted with dichloromethane and water. The organic matter was washed with saturated NH4Cl and water, dried (MgSO4), and evaporated to obtain crude orange oil (28 g). This was purified by SiO2 chromatography (95:5, cyclohexane / ethyl acetate) to obtain compound 5 as an orange oil that gradually darkened (26.33 g, 83%). 1 H NMR (300MHz, CDCl3)δ 1.51(s, 9H), 3.44(s, 1H), 6.15(d, J=15.7Hz, 1H), 7.07(d, J=3.8Hz, 1H), 7.17(d, J=3.8Hz, 1H), 7.58(dd, J=15.7, 0.6Hz, 1H); 13 C NMR(101MHz, CDCl3)δ 26.9, 28.1, 80.8, 83.4, 120.2, 124.0, 130.0, 133.8, 135.1, 141.2, 165.7;IR(ATR)v max / cm -1 2978w, 2933w, 1702m, 1623m, 1447m, 1392w, 1368m, 1148s, 753m;MS(ES)m / z=235.1[M+H] + HRMS(ES) calculated value C 13 H 15 SO2[M+H] + : 235.0787, measured value 235.0786.

[0057] 1.1.5 Synthesis of tert-butyl(2E)-3-(5-{2-[4-(piperazin-1-yl)phenyl]ethynyl}thiophen-2-yl)prop-2-enoate, compound Ia (a representative compound of formula I) Compound 5 (26.33 g, 112.4 mmol) was dissolved in triethylamine (Et3N) (750 mL), and the solution was degassed by spraying with Ar for 1 hour. Then, 1-(4-iodophenyl)piperazine (29.45 g, 102.2 mmol), Pd(PPh3)2Cl2 (3.59 g, 5.11 mmol), and CuI (0.97 g, 5.11 mmol) were added under Ar, and the resulting suspension was stirred at 60°C for 72 hours. The resulting suspension was diluted with dichloromethane, washed with saturated NaHCO3 and water, dried (MgSO4), and evaporated to obtain a crude orange solid (57 g). This was purified by SiO2 chromatography (95:5, dichloromethane / methanol, 1% triethylamine), and then recrystallized from acetonitrile to obtain compound Ia as a yellow / orange solid (27.01 g, 67%). 1 H NMR (400MHz, CDCl3)δ 1.51(s, 9H), 2.98-3.05(m, 4H), 3.17-3.25(m, 4H), 6.12(d, J=15.7Hz, 1H), 6.80-6.88(m, 2H), 7.08(d, J=3.9Hz, 1H), 7.10(d, J=3.8Hz, 1H), 7.36-7.43(m, 2H), 7.59(dd, J=15.7, 0.6Hz, 1H); 13 IR(ATR)v max / cm -1 2977w, 2929w, 2820w, 2194w, 1698s, 1617m, 1602m, 1526w, 1323m, 1141s, 812w;MS(ES):m / z=395.3[M+H] + HRMS(ES) calculated value C 23 H 27 N2O2S[M+H] + : 395.1793, measured value 395.1792.

[0058] 1.2 Synthesis of Compound Ib The synthesis of compound Ib is shown in Figure 2 and is explained below.

[0059] 1.2.1 Methyl(E)-3-(5-iodothiophen-2-yl)acrylate, 9 Trimethyl phosphonoacetate (19.8 mL, 122.2 mmol) was dissolved in cyclopentyl methyl ether (CPME) (250 mL) with LiCl (5.2 g, 122.2 mmol), and the mixture was stirred at 0°C for 20 minutes. 5-iodothiophene-2-carbaldehyde (26.5 g, 111.1 mmol) was added to the stirring solution, followed by dropwise addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (18.3 mL, 122.2 mmol). The mixture was stirred at room temperature for 18 hours. The mixture was diluted with water (400 mL) and extracted with ethyl acetate (400 mL x 2). The organic matter was dried together over Na2SO4 and concentrated under reduced pressure to obtain compound 9 as a beige solid (31.6 g, 97%). Rf = 0.59 (hexane / siRNA 9:1); 1 H NMR (CDCl3, 300MHz) δ 3.77(s, 3H), 6.13(d, J=15.7Hz, 1H), 6.87(d, J=3.8Hz, 1H), 7.19(d, J=3.8Hz, 1H), 7.67(d, J=15.7Hz, 1H).

[0060] 1.2.2 1-(4-((trimethylsilyl)ethynyl)phenyl)piperazine, 10 1-(4-iodophenyl)piperazine (10.0 g, 34.7 mmol) was added to a three-necked round-bottom flask. Triethylamine (Et3N) (200 mL) was added to the flask, and the solution was degassed by vacuum sonication and replaced with an Ar atmosphere 10 times. Under a positive Ar flow, Pd(PPh3)2Cl2 (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 the solvent was evaporated under reduced pressure. The residue was extracted with dichloromethane (200 mL) and washed with saturated NaHCO3 (200 mL). The organic matter was dried together over Na2SO4, concentrated, and a crude orange-brown crystalline solid was obtained. This was further purified by column chromatography (dichloromethane / methanol, 9:1) to obtain compound 10 (8.9g, 100%) as a brown crystalline solid: 1 H NMR (CDCl3, 300MHz) δ 0.22 (s, 3H), 3.01 (t, J=5.0Hz, 4H), 3.17 (t, J=5.0Hz, 4H), 6.79 (d, J=8.9Hz, 2H), 7.35 (d, J=8.9Hz, 2H).

[0061] 1.2.3 1-(4-ethynylphenyl)piperazine, 11 Compound 10 (8.9 g, 34.4 mmol) was dissolved in MeOH (150 mL), and 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 NH4Cl (300 mL). The organic matter was dried together over Na2SO4, concentrated, and compound 11 (4.9 g, 76%) was obtained as a beige solid: 1 H NMR (CDCl3, 300MHz) δ 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).

[0062] 1.2.4 Methyl(E)-3-(5-((4-(piperazine-1-yl)phenyl)ethynyl)thiophen-2-yl)acrylate, compound Ib (representative compound of formulas I and II) Compound 9 (6.8 g, 23.3 mmol) and Compound 11 (4.9 g, 26.3 mmol) were added to a three-necked round-bottom flask with triethylamine (Et3N) (150 mL). The mixture was degassed by vacuum sonication and purged with an Ar atmosphere 10 times. Under a positive Ar flow, Pd(PPh3)2Cl2 (0.41 g, 0.59 mmol), followed by CuI (0.11 g, 0.59 mmol), was added, and the mixture was stirred at 65°C for 72 hours. The solvent was evaporated under reduced pressure, and the residue was extracted with dichloromethane (600 mL) and washed with saturated NaHCO3 (500 mL x 2). The organic matter was dried together over Na2SO4 and concentrated to obtain an orange solid (8.5 g). This was recrystallized with acetonitrile (250 mL) to obtain a brick-red solid. This was further purified by column chromatography (dichloromethane / methanol, 9:1) to obtain compound Ib (4.9g, 60%) as an orange solid: melting point 174-176°C; 1 H NMR(CDCl3300MHz)δ 3.03(t, J=5.0Hz, 4H), 3.22(t, J=5.0Hz, 4H), 3.79(s, 3H), 6.19(d, J=15.7Hz, 1H), 6.85(d, J=8.9Hz, 2H), 7.12(s, 2H), 7.40(d, J=8.9Hz, 2H), 7.70(d, J=15.7Hz, 1H); 13 C NMR(CDCl3300MHz)δ 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.

[0063] Example 2: Preparation of salt forms of compounds of formulas I and II Figure 5A shows the preparation of the salt of the compound of formula I, and Figure 5B shows the preparation of the salt of the compound of formula I / formula II.

[0064] 2.1 (E)-4-(4-((5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)thiophen-2-yl)ethynyl)phenyl)piperazine-1-ium trifluoroacetate, 6 Compound Ia (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. The reaction was continued with stirring at room temperature for 1 hour. After completion, the resulting suspension was filtered under vacuum, further washed with diethyl ether, and dried to obtain compound 6 (2.48 g, 100%) as a pale yellow solid. 1 H NMR(300MHz CDCl3)δ 1.52(s, 9H), 3.36-3.32(m, 4H), 3.52-3.48(m, 4H), 6.14(d, J=15.6Hz, 1H), 6.88(d, J=9.2Hz, 2H), 7.12(q, J=5.2Hz, 2H), 7.45(d, J=9.2Hz, 2H), 7.60(d, J=15.6Hz, 2H). 13 C NMR(CDCl3300MHz)δ 15.6, 28.2, 42.9, 44.9, 65.3, 80.6, 81.5, 96.7, 111.7, 115.6, 117.6 (q, J=299Hz), 11 9.4, 125.6, 132.5, 132.9, 133.3, 135.9, 140.2, 150.5, 159.0 (q, J=31Hz, 1C), 165.5; 19 F NMR(DMSO 300MHz)δ 73.48; Absorption λ max DCM = 372 nm, DMSO = 385 nm, toluene = 383 nm; emission λ max DCM = 512nm, DMSO = 591nm, toluene = 481nm.

[0065] 2.2 (E)-4-(4-((5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)thiophen-2-yl)ethynyl)phenyl)piperazine-1-ium acetate, 7 Compound Ia (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. The reaction was continued with stirring at room temperature for 1 hour. After completion, the resulting suspension was filtered under vacuum, further washed with diethyl ether, and dried to obtain compound 7 (2.25 g, 98%) as a pale yellow solid. 1 H NMR(DMSO-d6300MHz)δ 1.48(s, 9H), 1.90(s, 3H), 2.84-2.81(m, 4H), 3.18-3.15(m, 4H), 6.18(d, J=15.7Hz, 1H), 6.94(d, J=8. 9Hz, 2H), 7.31(d, J=3.8Hz, 1H), 7.38(d, J=8.9Hz, 2H), 7.49(d, J=3.9Hz, 1H), 7.67(d, J=15.7Hz, 1H); 13 ¹³C NMR (DMSO-d6 300MHz) δ 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, 133.1, 136.0, 140.0, 151.9, 165.5, 172.9; absorption λ max DCM = 387 nm, DMSO = 388 nm, toluene = 390 nm; emission λ max DCM = 555nm, DMSO = 589nm, toluene = 487nm.

[0066] 2.3 (E)-4-(4-((5-(3-(tert-butoxy)-3-oxoprop-1-en-1-yl)thiophen-2-yl)ethynyl)phenyl)piperazine-1-ium triflate, 8 Compound Ia (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 syringe. The reaction was continued with stirring at room temperature for 1 hour. After completion, the resulting suspension was filtered under vacuum, further washed with diethyl ether, and dried to obtain compound 8 (2.68 g, 97%) as a pale yellow solid. 1H NMR(DMSO-d6300MHz)δ 1.48(s, 9H), 3.24-3.20(m, 4H), 3.47-3.44(m, 4H), 6.20(d, J=15.7Hz, 1H), 7.03(d, J=9.0Hz, 2H), 7.3 3(d, J=3.8Hz, 1H), 7.46(d, J=9.0Hz, 2H), 7.51(d, J=3.9Hz, 1H), 7.67(d, J=15.7Hz, 1H), 8.74(br, 1H); 13 C NMR(DMSO-d6300MHz)δ 28.2, 31.7, 43.0, 44.8, 80.6, 81.6, 96.7, 111.8, 115.7, 119.4, 121.2 (q, J=322Hz), 125.6, 132.5, 133.0, 133.3, 135.9, 140.2, 150.5, 165.5; 19 F NMR (DMSO-d6 300MHz) δ 77.73; absorption λ max DCM = 369 nm, DMSO = 384 nm, toluene = 372 nm; emission λ max DCM = 492nm, DMSO = 586nm, toluene = 469nm.

[0067] 2.4 (E)-4-(4-((5-(3-methoxy-3-oxoprop-1-en-1-yl)thiophen-2-yl)ethynyl)phenyl)piperazine-1-ium trifluoroacetate, 13 Compound Ib (0.50 g, 1.42 mmol) was dissolved in a mixture of diethyl ether (10 mL) and dichloromethane (80 mL), trifluoroacetic acid (0.109 mL, 1.42 mmol) was added, and the solution was stirred for 45 minutes. The solution was evaporated, the residue was resuspended in diethyl ether (100 mL), and filtered to obtain a crude orange solid. This was recrystallized from ethyl acetate to obtain salt 13 as an orange solid (0.29 g, 44%). 1H NMR (300MHz, DMSO-d6)δ 8.95(br, 2H), 7.79(d, J=15.8Hz, 1H), 7.54(d, J=3.9Hz, 1H), 7.45(d, J=8.9Hz, 2H), 7.34(d, J=3.8Hz, 1H) , 7.03(d, J=8.9Hz, 2H), 6.31(d, J=15.8Hz, 1H), 3.72(s, 3H), 3.47(t, J=5.1Hz, 5H), 3.23(t, J=5.1Hz, 4H); 13 C NMR (176MHz, DMSO-d6)δ 166.2, 158.0 (q, J=31Hz), 150.1, 139.6, 136.5, 132.9, 132.6, 132.5, 125. 5, 117.2 (q, J=300Hz), 116.8, 114.7, 111.2, 96.5, 81.1, 51.6, 44.4, 42.5.

[0068] Example 3: Preparation of a typical formulation - Compound Ia (1% w / w) in benzyl alcohol (50%), dimethyl sulfoxide (30%), and propylene glycol (20%) A representative formulation was prepared by mixing benzyl alcohol (5 mL), dimethyl sulfoxide (3 mL), and propylene glycol (2 mL). The resulting mixture was stirred for 5 minutes using a magnetic stirrer to obtain a solution of benzyl alcohol (50% v / v), dimethyl sulfoxide (30% v / v), and propylene glycol (20% v / v). The formulation of compound Ia was prepared by weighing 30 mg of compound Ia and dissolving it in 2.97 g of a solvent mixture to obtain 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, it was demonstrated that the compound of formula Ia can be formulated in common pharmaceutical excipients. The characterization of the formulations was performed in Example 5.2.

[0069] Example 4: Manufacturing of a gel formulation 1 to 25 mg of compound Ia, compound Ib, or salts 6, 7, 8, or 13 were added to a 10 mL glass vial, followed by 1.0 g of 1,2-propanediol. The vial was placed in an ultrasonic bath to completely dissolve the substances. Then, 8.85 g of deionized (DI) water was added, and the resulting mixture was vigorously stirred until completely homogenized. 0.15 g of hydroxyethylcellulose was added, and the mixture was stirred until completely incorporated. One drop of triethanolamine was added to the resulting mixture, and it was vigorously stirred by hand for 2 to 3 minutes, causing the viscosity of the mixture to change, transforming it from a liquid to a gel. [Table 1-1] [Table 1-2]

[0070] Thus, the preparation of a gel formulation containing the compound of the present invention has been demonstrated.

[0071] Example 5: Characterization of Compounds Ia and Ib and Representative Formulations 5.1 Absorption and emission spectra - Compounds Ia and Ib Absorption spectra were obtained using a Perkin Elmer Cary 60 spectrometer, and emission spectra were obtained using an Agilent Cary Eclipse spectrometer. For absorption spectra, 5 μM solutions of the compound of formula I in CHCl3 and DMSO were placed in a 10 mm path length quartz optical cuvette (Hellma), and absorbance was recorded at 1 nm intervals. The extinction coefficient was measured for each solvent at concentrations of 5 to 30 μM, corresponding to the λ value of the compound of formula I. maxThree different values ​​were determined using the measurements obtained from the absorption measurements at [location]. The extinction coefficient value was expressed as the average of three replicates, along with the standard deviation. Emission spectra were acquired at 1 nm intervals from a 100 nM solution in a quartz cuvette using excitation at λ = 380 nm, as described above, and normalized according to the respective maximum intensity values. The absorption and emission spectra of compound Ia are shown in Figures 3A and 3B, respectively, and the absorption and emission spectra of compound Ib are shown in Figures 4A and 4B, respectively.

[0072] 5.2 Excitation and emission spectra of prepared compound Ia in representative formulations The excitation spectra of representative formulations from Example 3 in a 10 mm pass length quartz optical cuvette (Hellma) were recorded at an emission wavelength of 600 nm using an Agilent Cary Eclipse spectrometer. The excitation spectra are shown in Figure 6. The emission spectra of 1% w / w compound Ia in a mixture of 50% benzyl alcohol, 30% dimethyl sulfoxide, and 20% propylene glycol are shown in Figure 7 and were recorded at an excitation wavelength of 380 nm using an Agilent Cary Eclipse spectrometer in a 10 mm pass length quartz optical cuvette (Hellma).

[0073] 5.3 Measurement of Quantum Yield The quantum yield by one-photon excitation was determined using (2E)-3-(4-{2-[4,4-dimethyl-1-(propan-2-yl)-1,2,3,4-tetrahydroquinoline-6-yl]-ethynyl}phenyl)prop-2-enoic acid in toluene (quantum yield 0.67) as a standard. Compound Ia was measured at varying concentrations in each solvent, aiming for absorbances of 0.1 or less, corresponding to concentrations in the range of 0.50 to 2 μM. The absorbance of each compound in solution was recorded at 300 to 1000 nm, and the corresponding fluorescence intensity was measured at 250 to 700 nm.

[0074] The quantum yield was calculated using the relative method. The absorbance and fluorescence of the compound in solution were measured at multiple concentrations and compared with a reference substance using the following formula:

number

[0075] Example 6: Characterization of compound Ia in Gram-positive bacteria 6.1 Experiment 6.1.1 Bacteria Bacillus subtilis 168 (ATCC 23857), Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus SH1000 (ATCC 6538P), Staphylococcus aureus USA300 (ATCC BAA-1556), Enterococcus faecalis (ATCC 29212), Fluorescent bacterium (ATCC 13525), and Escherichia coli FDA strain Seattle 1946 20 (ATCC 25922) were obtained from the American Type Culture Collection.

[0076] 6.1.2 Inhibition of bacterial growth by photoactivation All bacteria were cultured in LB (Miller) broth in an orbital shaker (VWR) at 30–37°C. Overnight cultures were prepared by seeding a single isolated colony into 10 mL of LB broth and incubating with shaking for 16–20 hours. Bacteria to be exposed to the photoactivating compound were placed in a LightOx PhotoReact 365® Lightbox (Merck) at 13 mW / cm². 2 Energy intensity (total energy supply: 3.9 J / cm²) 2 The samples were then exposed to light at a wavelength of 365 nm for 5 minutes. In assays where only half of a 96-well or agar plate was irradiated, a section of black card was used to mask the relevant samples.

[0077] For bacterial overlay, 50 mL of 1.5% LB agar was poured into a 100 × 100 × 20 mm square petri dish (Sarstedt). After solidification, 15 mL of 0.75% LB soft agar inoculated with 200 μL of bacteria obtained from overnight culture was poured onto the surface. 6 μL of sequentially diluted compounds were spread onto the overlay. The photoactivatable plates were exposed to 365 nm light and incubated at 30°C for 24 hours, after which they were imaged using a Bio-Rad Gel Doc XR+ System.

[0078] For growth curve analysis, 50 μL of bacteria obtained from an overnight culture were inoculated into 5 mL of LB broth in a 15 mL Falcon tube (Sarstedt). Compound I was added to a final concentration of 2 μM, and the mixture was incubated with shaking at 30°C in the dark for 30 minutes. The sample (100 μL) was dispensed into 8 repeats of wells in a 96-well plate. Half of the plate was covered, and the other half was irradiated at 365 nm. Growth was monitored every 5 minutes for 24 hours at OD600 nm using a plate reader (Biotek Synergy HT), and the data were normalized against a negative control containing only culture medium.

[0079] 6.1.3 Survival Rate Assay A semi-micro cuvette (Sarstedt) containing 2 mL of LB broth inoculated with 50 μL of bacteria obtained from an overnight culture was subjected to logarithmic growth at 30°C, followed by OD (Oxygen-Drug) growth.600nm The mixture was shaken to 0.2 and incubated. A 1 mL sample was transferred to a 24-well plate, compound Ia (2 μM) was added, and the mixture was incubated at 30°C for 30 minutes. In a 96-well plate, a 10-fold dilution series was performed on 30 μL of sample in 270 μL LB. The sample in the 24-well plate was irradiated and incubated at room temperature for 15 minutes, then 30 μL of the sample was removed, and the 10-fold dilution series was performed similarly. Three different samples (10 μL each) were spread onto LB agar plates and incubated for 16-20 hours. The number of colonies for each appropriate dilution was measured, and the viability was calculated in terms of colony-forming units (CFU) / mL.

[0080] 6.1.4 Monitoring of membrane integrity loss using propidium iodide Bacteria were cultured in the same manner as in the viability assay. 500 μL of culture was transferred to a 1.5 mL microcentrifuge tube, and compound Ia was added to a final concentration of 2 μM. A control sample containing 500 μL of culture and 0.2% DMSO was also set up simultaneously. The samples were incubated at 30°C for 30 minutes and then centrifuged at 17,000 g for 4 minutes to pellet the cells. The cell pellet was resuspended in 200 μL of 1 × PBS containing 7.5 μM propidium iodide (PI; ThermoFisher). 3 × 50 μL from each sample was dispensed into a 96-well plate and transferred to a Biotek Synergy HT. Fluorescence measurements were performed every 2 minutes for 20 minutes at excitation 485 nm and emission 645 nm. Subsequently, PBS (50 μL) was added to the sample wells, and 50 μL of 100% ethanol was added to the additional samples to serve as a positive control for membrane integrity loss and cell death. The plates were placed in a PhotoReact 365 lightbox, irradiated for 5 minutes, and immediately returned to the plate reader. Fluorescence was monitored every 2 minutes for 1 hour.

[0081] 6.1.5 Confocal Microscopy Observation For confocal microscopy observation, a cell pellet was prepared in the same manner as described in section 6.1.4 above. 500 μL of culture medium was transferred to a 1.5 mL microcentrifuge tube, and compound Ia was added to a final concentration of 2 μM. The sample was incubated at 30°C for 30 minutes, and the cells were pelleted by centrifugation at 17,000 g for 4 minutes. The cells were then resuspended in 200 μL of Baclight solution containing 10 μM SYTO9 and 60 μM PI. 10 μL of the sample was dropped onto a 1 cm × 1 cm 1.5% agarose (Bioline) pad on a microscope slide, and a coverslip was placed on top. Slides were imaged using a confocal microscope (Zeiss 800 Airyscan) with a 63x lens. Compound I was imaged using the airyscan function with 405nm laser excitation and a 450-550nm emission filter, SYTO 9 with a 488nm laser and a 550-580nm filter, and PI with a 488nm laser and a 600-650nm emission filter. Samples were excited on the microscope with a 405nm laser at 30% power for 1 minute (total energy: 90mJ / cm²). 2 ) was irradiated.

[0082] 6.1.6 Activity screening of compound Ia against Gram-positive and Gram-negative bacteria Compound Ia was screened for its activity against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and two Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). Compound Ia, diluted 2-fold, was applied to the surface of an overlay containing each bacterial species, activated by exposure to 365 nm light, and incubated for 24 hours. Five structurally similar compounds (shown in Table 3 below) were also tested. Figure 8A shows irradiated bacterial overlays treated with compound Ia (shown in lane 2) and structurally similar compounds (shown in lanes 1 and 3-6). Figure 8B shows unirradiated bacterial overlays treated with compound Ia (shown in lane 2) and the five structurally similar compounds (shown in lanes 1 and 3-6). Figure 8A shows that compound Ia inhibits bacterial growth of both Gram-positive species (Bacillus subtilis and Staphylococcus epidermidis) after irradiation. Growth inhibition at 0.1 μM was clearer than that observed with the next-best compound (shown in lane 1) at 1 μM, indicating that compound Ia is at least 10 times potent. No significant growth inhibition was observed in Gram-negative bacterial species (E. coli and fluorescent bacteria). No inhibition of bacterial growth was observed in non-irradiated samples (Figure 8B). [Table 3-1] [Table 3-2]

[0083] 6.1.7 Effects of Compound Ia on Bacterial Growth The inhibitory effect of compound Ia on the growth of Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli) was investigated. Bacterial growth in response to treatment with 2 μM compound Ia was tracked over 24 hours by optical density measurement, with half of the sample irradiated with 365 nm light at the start and the other half used as a non-irradiated control. Figure 9 shows bacterial cells from the irradiated and non-irradiated groups treated with 2 μM compound I and DMSO (control). From Figure 9, it is clear that both Gram-positive bacterial species (Bacillus subtilis and Staphylococcus epidermidis) showed inhibited growth after treatment with compound Ia and irradiation. Gram-negative E. coli showed an initial growth delay compared to the non-irradiated control, but then resumed growth. No difference in growth was observed in control cells treated with DMSO.

[0084] 6.1.8 Effect of Compound Ia on bacterial survival Cell viability assays were performed to investigate whether compound Ia exhibits bacteriostatic or bactericidal effects in Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). Four bacterial species were cultured to the initial logarithmic growth phase before the addition of 2 μM compound Ia and exposure to 365 nm light. Appropriate controls without irradiation and DMSO controls at equivalent concentrations were also performed in parallel. Serial dilutions of bacteria were spread on the surface of agar plates, and CFU / ml were calculated. Figure 10 shows that both Gram-positive bacterial species exhibited a 6-log decrease in viability compared to the unirradiated control after compound Ia treatment and light exposure. No such decrease was observed in Gram-negative bacterial species.

[0085] Bacterial survival assays were also performed on two Gram-positive ESKAPE pathogens, namely *Acetigo faecium* and *Staphylococcus aureus* (strains SH1000 and USA300). ESKAPE pathogens are highly pathogenic and multidrug-resistant bacterial pathogens, and are a major cause of hospital-acquired infections. Figure 11 shows that the number of viable cells decreases by 4 logs after photoactivation of compound Ia.

[0086] 6.1.9 Effect of Compound Ia on Bacterial Membrane Integrity To evaluate the loss of membrane integrity in cells exposed to compound Ia, propidium iodide (PI) was used. PI is a membrane-impermeable dye that fluoresces only when it can pass through the cell epithelium in the presence of chromosomal DNA, thus functioning as a reporter for cell death (with some caveats). Two Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and two Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus) were incubated in the presence of 2 μM compound Ia and exposed to 365 nm light after 20 minutes. PI fluorescence was monitored for 0–80 minutes throughout the experiment. Figure 12 shows a significant increase in PI fluorescence after photoactivation in both Gram-positive bacterial species, consistent with the rapid decline in cell viability shown in Figure 10. In Gram-negative bacterial species, the increase in fluorescence after photoactivation was slight. A slight increase in PI fluorescence was also observed in both Gram-positive bacterial species before irradiation, suggesting the possibility of mild loss of membrane integrity, particularly in Bacillus subtilis.

[0087] 6.1.10 Real-time monitoring of bacterial membrane integrity To further confirm that membrane disruption by compound Ia is photoactivation-dependent, a real-time BacLight assay was used. This assay utilizes SYTO 9, a membrane-permeable dye that fluoresces upon binding to chromosomal DNA, and PI, which, as mentioned above, enters the cell only when membrane integrity is compromised and replaces SYTO 9 due to its high affinity for DNA. Therefore, comparing the ratio of PI-positive cells to SYTO 9-positive cells provides an indicator of bacterial membrane integrity and viability. Bacteria were grown to the initial logarithmic growth phase, then treated with 2 μM compound Ia in the presence of both dyes and visualized by microscopy. Compound Ia was activated with 405 nm light, and imaging was performed for 10 minutes to monitor fluorescence changes. Figure 13 shows that the majority of Staphylococcus epidermidis (Panel A) and Bacillus subtilis (Panel B) were SYTO 9-positive before photoactivation, indicating that these cells possessed intact cell epithelium and were viable. Upon photoactivation, both Gram-positive bacterial species (Bacillus subtilis and Staphylococcus epidermidis) exhibited rapid fluorescence changes, with all cells becoming PI-positive after 10 minutes. These changes suggest significant membrane damage and cell death induced by activated compound Ia, enabling PI influx and subsequent fluorescence expression. In contrast, Gram-negative bacterial species (Escherichia coli and fluorescent bacteria) showed no fluorescence changes after photoactivation, indicating no signs of membrane disruption.

[0088] 6.1.11 Visualization of compound Ia in bacterial cells The fluorescence properties of compound Ia were used to visualize the intracellular localization of the compound in two Gram-positive bacteria (Bacillus subtilis and Staphylococcus epidermidis) and two Gram-negative bacteria (Escherichia coli and Bacillus fluorescentus). The bacteria were grown to the logarithmic metaphase and visualized by microscopic observation. Figure 14 shows that the compound has some association with the cell surface in both Gram-positive bacterial species. However, clearly high-density patches are also observed, although it is unclear whether this is due to (potential) aggregates on the surface or accumulation within the cell matrix. Clustering of compound Ia is particularly pronounced in the polar regions of Bacillus subtilis cells (Panel A), and this tendency is especially high among dividing cells, which is thought to be because these regions are more accessible. These high-density patches were not observed at all in the two Gram-negative bacteria, suggesting that compound Ia is associated only with the cell surface.

Claims

1. Compounds of formula I, in free form or salt form, for use in the treatment or prevention of infections caused by Gram-positive bacteria: 【Chemistry 1】 In the formula, R is C １ -C ４ It is alkyl.

2. The compound of formula I according to claim 1, wherein R is methyl or tert-butyl.

3. The compound of formula I according to claim 1 or 2, wherein the infection caused by the Gram-positive bacteria is a disease or condition caused by Gram-positive bacteria, or an infection of a skin lesion.

4. The compound of formula I according to any one of claims 1 to 3, wherein the salt is in the form of an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

5. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, which is in the form of an aqueous solution, suspension, milk, cream, foam, paste, ointment, gel, or hydrogel.

7. A pharmaceutical composition according to claim 5 or 6, which is a topical composition.

8. A compound of formula I according to any one of claims 1 to 4, or a pharmaceutical composition according to any one of claims 5 to 7, for use in the treatment or prevention of infections caused by Gram-positive bacteria, which is administered topically.

9. The compound or pharmaceutical composition according to claim 8, which is administered topically and exposed to light.

10. The compound or pharmaceutical composition according to claim 9, which is exposed to light with a wavelength of 365 to 460 nm.

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

12. The dressing or patch according to claim 11, wherein at least a portion of it is transparent.

13. A compound of formula I according to any one of claims 1 to 4 or 8 to 10, or a pharmaceutical composition according to any one of claims 5 to 10, for use in the treatment or prevention of diseases or conditions caused by Gram-positive bacteria, or infections of skin lesions, wherein the disease or condition caused by Gram-positive bacteria is selected from cellulitis, erysipelas, impetigo, folliculitis, carbuncle, boil, erthrasma, leprosy, and cutaneous tuberculosis, and the infection of skin lesions is selected from Gram-positive bacterial infections caused by ulcers, skin wounds, burns, or insect bites.

14. A compound of formula I according to any one of claims 1 to 4 or 8 to 10, or a pharmaceutical composition according to any one of claims 5 to 10, for use in the treatment of an infection caused by Gram-positive bacteria, wherein the infection is caused by Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), Staphylococcus epidermidis, hemolytic Staphylococcus, Luteus, Corynebacterium minutisimum, Enterococcus faecalis, Group A Streptococcus, Group C Streptococcus, Group G Streptococcus, Mycobacterium tuberculosis, Mycobacterium bovine, Mycobacterium leptromatosis, Mycobacterium marinum, and Mycobacterium ulcerans.

15. A method for treating a patient having a bacterial infection caused by Gram-positive bacteria, comprising administering to the patient a therapeutically effective amount of a compound of formula I or a salt thereof as described in any one of claims 1 to 4.

16. The therapeutic method according to claim 15, wherein the administration is local administration.

17. The therapeutic method according to claim 15 or 16, further comprising exposing the compound of formula I to light.

18. Compounds of formula II in free form or salt form: 【Chemistry 2】 In the formula, R is C １ -C ３ It is alkyl.

19. The compound of formula II according to claim 18, wherein the salt is an acetate, trifluoroacetate, triflate, hydrobromide, or hydroiodide.

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