Organic fluorescent compounds and methods thereof

Abstract

This disclosure concerns organic fluorescent compounds, synthesis methods and applications thereof. The compounds may be used for treating a microbial disease or disorder.

Description

[0001] Organic Fluorescent Compounds and Methods Thereof

[0002] Technical Field

[0003] The present invention relates, in general terms, to organic fluorescent compounds and a synthesis method and application thereof.

[0004] Background

[0005] Sepsis is a disorder of the immune response to infection that often leads to organ dysfunction and death. Sepsis has an alarmingly high mortality rate and has been recognized as a major global healthcare challenge, placing a heavy burden on patients, caregivers and healthcare systems alike. Clinically, antibiotic therapy is the standard treatment in sepsis guidelines, but antibiotic resistance is widespread due to the overuse and long-term abuse of antibiotics, as shown by the high clinical mortality caused by persistent infections during sepsis, so there is an urgent need for new therapeutic agents that may destroy pathogens and improve the prognosis of sepsis without developing resistance.

[0006] Clinical data show that more than 60% of patients with sepsis initially survive the inflammatory storm, but then progress to a long-term immunosuppressed state. The paralysis and death of immune cells may result in impaired ability to clear invading pathogens. Therefore, effort has been made to identify potential therapeutic targets for suppressing infection-induced immune dysregulation, such as the removal of reactive oxygen species (ROS) such as H2O2, O2-, OH-, etc., or some immunoreactive glycoproteins such as the allergic toxin C5a. Recently, strategies aimed at restoring immune function have been developed and tested in patients with sepsis. One immunotherapy strategy that has attracted a lot of attention is adoptive cell transfer (ACT), which may potentially enhance pathogen clearance, reduce excessive inflammation, and restore immune balance. Various immune cells, such as T cells, macrophages, NK cells, and neutrophils, have been studied in ACT sepsis therapy, with promising results in preclinical and early-stage clinical studies. Among them, macrophages appear to play a crucial role as effective pathogen scavenger during infection. Therefore, restoring or enhancing the immune function of macrophages during ACT may promote infection eradication. Current nanomaterials for adoptive cell transfer therapy are mainly loaded in the lysosomes of cells. However, many bacteria, such as Staphylococcus aureus and Escherichia coii, have evolved immune escape mechanisms to prevent lysosomal killing, leading to intracellular survival and recurrent infections. Therefore, the selection of other organelles loaded with nanomaterials may provide beter results for adoptive cell transfer treatment of bacterial infections in vivo.

[0007] It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0008] Summary

[0009] The present disclosure concerns a compound comprising two rigid hydrophobic structures and a cation. The present disclosure also concerns a synthesis method of the compound.

[0010] The present disclosure concerns a compound of Formula (la), or a solvate thereof: wherein

[0011] R1and R2are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;

[0012] R3is selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy;

[0013] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore; and R5, R6, R7and R8independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl.

[0014] In some embodiments, R1and R2are independently selected from optionally substituted alkyl.

[0015] In some embodiments, R4is selected from H and optionally substituted alkyl.

[0016] In some embodiments, R4is selected from optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy. In some embodiments, Ft, is selected from: where denotes a bond to the phenyl ring; wherein

[0017] R9, R10, and R12are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy;

[0018] R13 and R14 are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore.

[0019] In some embodiments, R9, R10, R11, and R12are independently selected from H and optionally substituted alkyl.

[0020] In some embodiments, R13and R14are independently selected from H and optionally substituted alkyl.

[0021] In some embodiments, R5, R6, R8, and Rs are independently selected from H and optionally substituted alkyl.

[0022] In some embodiments, the compound of Formula (la) is selected from:

[0023]

[0024] In some embodiments, the compound is characterised by an antimicrobial activity.

[0025] In some embodiments, the compound is characterised by an antibacterial activity against Gram-negative and / or Gram-positive bacterial cells.

[0026] In some embodiments, the compound is characterised by an antibacterial activity against antibiotics resistant bacterial cells.

[0027] In some embodiments, the compound is characterised by a minimum inhibitory concentration (MIC) against bacterial cells of about 1 μM to about 10 μM. In some embodiments, the compound is characterised by an antifungal activity against drug resistant fungal cells. In some embodiments, the compound of Formula (la) is characterised by a MIC against fungus of about 1 μM to about 10 μM.

[0028] In some embodiments, the compound of Formula (la) is characterized by a minimum fungal concentration (MFC) of about 1 μM to about 10 μM.

[0029] The present disclosure also concerns a method of synthesising a compound of Formula

[0030] (la), or a salt or solvate thereof, comprising: a) reacting compound of Formula (II) with a haloalkane:

[0031] In some embodiments, a mole ratio of compound of Formula (II) to haloalkane is about 1 : 1.5 to about 1:3.

[0032] In some embodiments, the haloalkane is iodomethane.

[0033] In some embodiments, the method further comprises a step before step a) of reacting a compound of Formula (III) with a heterocyclic acid to form compound of Formula (II): wherein X is halo; wherein the reaction is conducted in the presence of a catalyst and a base.

[0034] In some embodiments, a mole ratio of compound of Formula (II) to heterocyclic acid is about 2:3 to about 2:7.

[0035] In some embodiments, the heterocyclic acid is pyridine-4-boronic acid. In some embodiments, the method further comprises a step before step a) of reacting a compound of Formula (IV) with benzyl bromide to form compound of Formula (III):

[0036] In some embodiments, a mole ratio of compound of Formula (IV) to benzyl bromide is about 1 :0.5 to about 1:2.

[0037] In some embodiments, the benzyl bromide is selected from:

[0038] The present disclosure also concerns a method of labelling and / or imaging bacterial cells and / or fungal cells, comprising contacting the bacterial cells and / or fungal cells with a compound of Formula (Ia), or a salt or solvate thereof.

[0039] The present disclosure also concerns a compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof for use in therapy.

[0040] The present disclosure also concerns a compound of Formula (la) or a salt or solvate thereof for use in the imaging and / or treatment of a microbial disease or disorder.

[0041] The present disclosure also concerns a use of compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the imaging and / or treatment of a microbial disease or disorder.

[0042] The present disclosure also concerns a method of imaging and / or treating a microbial disease or disorder in a subject thereof, comprising administering to the subject a therapeutically effective amount of compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof. In some embodiments, the microbial disease or disorder has a resistance against drugs.

[0043] In some embodiments, the microbial disease or disorder is selected from skin infection, a respiratory disease, food poisoning or any other life-threatening systemic disease, sepsis, urinary tract infection, meningitis, wound infection, tuberculosis, diarrhoea, Legionnaires' disease, meningococcal disease, Q fever, strep throat, whooping cough (pertussis), aspergillosis, blastomycosis, Candida auris, candidiasis, chromoblastomycosis, cryptococcosis, fungal eye infection, histoplasmosis, mucormycosis, mycetoma, paracoccidioidomycosis, pneumocystis pneumonia, ringworm and nail infection, sporotrichosis, talaromycosis, and valley fever.

[0044] Brief description of the drawings

[0045] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which :

[0046] Figure 1 shows (A) a normalised UV-VIS absorption and emission spectrum of PBS solution of TPA2PyPh; (B) a normalised UV-VIS absorption and emission spectrum of PBS solution of TPA2PyBiPh; (C) a normalised UV-VIS absorption and emission spectrum of PBS solution of TPA2PyProp.

[0047] Figure 2 shows (A) antibacterial activity of TPA2PyPh against Staphylococcus aureus; (B) antibacterial activity of TPA2PyBiPh against Staphylococcus aureus; (C) antibacterial activity of TPA2PyProp against Staphylococcus aureus; (D) antibacterial activity of TPA2PyPh against Escherichia coll. ; (E) antibacterial activity of TPA2PyBiPh against Escherichia coli; (F) antibacterial activity of TPA2PyProp against Escherichia coll; (G) cytotoxicity of TPA2PyPh on Raw 264.7 cells; (H) cytotoxicity of TPA2PyBiPh on Raw 264.7 cells; and (I) cytotoxicity of TPA2PyProp on Raw 264.7 cells.

[0048] Figure 3 shows (A) a confocal fluorescence image of Raw 264.7 cells incubated with TPA2PyPh and infected with GFP-S. aureus for 2 hours; (B) number of colonies of Raw 264.7 cells incubated with media with different concentrations of TPA2PyPh; (C) survival rate of intracellular bacteria in Raw 264.7 incubated with different concentrations of TPA2PyPh, and the ratio of cells to bacteria is 1 : 20.

[0049] Figure 4 shows (A) survival rate of mice in the PBS injection group, the untreated Raw 264.7 cell injection group and the Raw 264.7 cell injection group incubated with TPA2PyPh; (B) change of mouse weight in the Raw 264.7 cell injection group incubated with TPA2PyPh after injection of liquid within 10 days.

[0050] Figure 5 shows the chemical structure of TPW, TPY (TPA2PyProp), and TPZ (TPA2PyBiPh) and schematic representation of the experimental approach for fungi killing in vitro and in vivo. Created in BioRender. Figure 6 shows the synthetic route and photophysical properties of TPW, TPY, and TPZ.

[0051] (A) Synthetic route of TPW, TPY, and TPZ. (B) Molecular docking model of TPW, TPY, and TPZ with a DNA fragment (PDB code: 4U8C). (C) Normalized UV-vis absorption spectra of TPW, TPY, and TPZ in PBS (l x, pH = 7.4) at a concentration of 10 μM. (D-F) Photoluminescence (PL) spectra of TPW (D), TPY (E), and TPZ (F) upon addition of ctDNA at a concentration of 10 μM. (G) Fluorescence quenching of the Hoechst-ctDNA complex by addition of TPW, TPY, and TPZ. (H,I) PL spectra of TPW in glycerol / H2O mixtures and plots of the relative emission intensity versus glycerol fraction.

[0052] Figure 7 shows antifungal and fungal uptake properties of TPW, TPY, and TPZ. (A-C) Changes in UV-vis absorption spectra of supernatants after incubation of TPW (A), TPY

[0053] (B), and TPZ (C) with C. albicans (107 CFU / mL) for different time intervals (PBS solution (l x , pH = 7.4), contains 0.1% DMSO, dye concentration = 10 pM). (D) Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of TPW, TPY, and TPZ against C. albicans. (E) Confocal images of C. albicans upon incubation with TPW, TPY, and TPZ at a concentration of 10 pM . Scale bar: 10 pm. (F-H) Fungicidal efficiency of flucytosine (F), TPY (G), and TPZ (H) against mature biofilms. (I) 3D confocal images of C. albicans biofilms incubated with TPW (5 x MIC) at different time points (interval of x-axis and y-axis: 50 pm; z-axis: 10 pm).

[0054] Figure 8 shows antifungal mechanism. (A) Time-lapse fluorescence confocal imaging of the Hoechst prelabeled C. albicans killing process upon addition of TPZ (5 x MIC). Scale bar: 10 pm. (B) Fluorescence intensity profiles of TPZ and Hoechst in area A at 10 min. (C,D) Fluorescence change of TPZ and Hoechst from 5 to 60 min in area A (C) and area B (D). (E) Fungal cell wall disturbance testing of TPW, TPY, TPZ, and Triton X- 100 treated C. albicans. (F,G) Volcano plots and heat map of DEGs between TPZ-treated groups and control groups.

[0055] Figure 9 shows biosafety. (A) Confocal images of NIH / 3T3 cells upon incubation with TPW (10 pM) for 12 or 24 h. Scale bar: 10 pm. (B) Fluorescence intensity profiles of TPZ and Hoechst in NIH / 3T3 cells at 48 h. (C) Confocal images of NIH / 3T3 cells costained with TPZ (10 pM) and BODIPY 493 / 503 (5 pg mL1). Cells were preincubated with TPZ at 37 °C for 12 h before staining with BODIPY 493 / 503. (D-G) Hemolytic properties and selectivity index of TPY and TPZ. (H-K) Cytotoxicity selectivity index of TPY and TPZ. Selectivity index of TPY or TPZ calculated by the ratio of HC50 and IC50 to MFC, respectively.

[0056] Figure 10 shows in vivo experiments. (A) Schematic illustration of the experimental approach for vaginitis treatment. Created in BioRender. (B,C) Fungal burden in blank control group, PBS-treated group, TPY-treated group, and TPZ-treated group after 3 days treatment. **p < 0.01. (D) Histological analysis of H&E and PAS staining after various treatments. Scale bar: 100 pm. (E) Histological analysis of H&E staining in major organs after various treatments. Scale bar: 50

[0057] Detailed description

[0058] "Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, / so-propyl, n-butyl, iso- butyl, n-hexyl, and the like.

[0059] "Alkenyl" refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), / so-propenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH=CHCH3), and the like.

[0060] "Alkoxy" refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, / so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0061] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

[0062] "Oxo / hydroxy" refers to groups =0, HO-.

[0063] "Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.

[0064] "Heteroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Hiickel criteria for aromaticity (ie. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N- oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).

[0065] Examples of heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine and the like.

[0066] "Cycloalkyl" refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 11 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, indanyl, 1,2,3,4-tetrahydronapthalenyl and the like.

[0067] "Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, R2or R1is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.

[0068] Examples of heterocyclyl and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2, 3, 4-tetra hydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, and the like.

[0069] In this specification "optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.

[0070] The present disclosure concerns a compound of Formula (I), or a solvate thereof: wherein

[0071] R is independently selected from H, alkyl, unsaturated alkyl, heteroatomic alkyl, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, alkoxy, or one or more chromophores capable of conjugation with one or more fluorescent substances.

[0072] The present disclosure concerns a compound of Formula (I), or a solvate thereof: wherein

[0073] R is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a chromophore. In some embodiments, the compound of Formula (I) is represented by Formula (la) or a solvate thereof: wherein

[0074] R1and R2are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;

[0075] R3is selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy;

[0076] R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore; and R9, R10, R11ar ande R12independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl.

[0077] In some embodiments, R1and R2are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, and optionally substituted heterocyclyl. In some embodiments, R1and R2are independently selected from optionally substituted alkyl, and optionally substituted alkenyl. In some embodiments, R1and R2are independently selected from optionally substituted alkyl. In some embodiments, R1and R2are independently selected from optionally substituted C1-C6alkyl. In some embodiments, R1and R2are independently selected from optionally substituted methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl and tert-butyl.

[0078] In some embodiments, Rs is selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy. In some embodiments, R3is selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, and optionally substituted alkoxy. In some embodiments, R3is selected from H, optionally substituted alkyl, and optionally 1ubstituted alkenyl. In some embodiments, R3is selected from H and optionally substituted alkyl. In some embodiments, R3is selected from H, and optionally substituted C1-C6alkyl. In some embodiments, R3is selected from H and optionally substituted methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl and tert-butyl. In some embodiments, R3is selected from H.

[0079] In some embodiments, R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy. In some embodiments, R4is selected from optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy.

[0080] In some embodiments, R4is selected from: where denotes a bond to the phenyl ring; wherein R9, R10, R11and R12are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy;

[0081] R13and R14are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore.

[0082] In some embodiments, R9, R10, R11and R12are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy. In some embodiments, R9, R10, R11and R12are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, and optionally substituted alkoxy. In some embodiments, R9, R10, R1,1and

[0083] R12are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl. In some embodiments, R9, R10, R111 a2re independently selected from H and optionally substituted alkyl. In some embodiments, and R12are independently selected from H, and optionally substituted Ci-Cs alkyl. In some embodiments, Rs, R10, Rn, and R12are independently selected from H and optionally substituted methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl and tert-butyl. In some embodiments, R9, R10, R11, and R12are independently selected from H.

[0084] In some embodiments, R13and R14are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy. In some embodiments, R13and R14are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, and optionally substituted heterocyclyl. In some embodiments, R13and R1 4re independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl. In some embodiments, R13and R14are independently optionally substituted C1-C10alkyl and optionally substituted C2-C10alkenyl. In some embodiments, R13and R14are independently selected from H.

[0085] In some embodiments, R5, R6, R7, and R8 are independently selected from H and optionally substituted alkyl. In some embodiments, R9, R10, R11are and R in12dependently selected from H and optionally substituted C1-C6alkyl. In some embodiments, R5, R6, R7, and R8are independently selected from H and optionally substituted methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl and tert-butyl. In some embodiments, Rs, R6, R7, and Rs are independently selected from H.

[0086] The optional substituent in R1-R14may be independently selected from halo, hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, nitro, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, and aminoacyloxy.

[0087] A fluorophore or chromophore is a molecule or a functional group within a larger molecule that is capable of fluorescing or emitting light when excited by light of a specific wavelengths. When a fluorophore is attached to another molecule, compound or structure, it may allow that target to be detected and visualised using fluorescence- based techniques. The fluorophore may act as a label or tag that enables the identification and tracking of the molecule (e.g. bacterial cells) it is attached to. For example, fluorescein and / or quinine may be conjugated via the -OH moiety (such that R4is optionally substituted alkoxy) or alkenyl moiety.

[0088] Compound of Formula (I) and (la) are charged at the pyridinyl moieties. To balance this charges, anions may be associated with the compound. The anion may be halide, hydroxide, sulfate, sulfide, sulfite, nitride, nitrite, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, hydrogen sulfate, perchlorate, iodate, chlorate, bromate, chlorite, hypochlorite, hypobromite, carbonate, chromate, bicarbonate, dichromate, acetate, formate, or oxalate. In some embodiments, the compound of Formula (I) and (la) is selected from:

[0089] In some embodiments, the compound of Formula (I) is characterised by an absorption wavelength of about 250 nm to about 600 nm. In some embodiments, the absorption wavelength is about 350 nm to about 500 nm. In some embodiments, the absorption wavelength is about 320 nm to about 500 nm. In some embodiments, the compound of Formula (I) is characterised by an emission wavelength of about 400 nm to about 850 nm. In some embodiments, the emission wavelength of about 500 nm to about 750 nm. In some embodiments, the compound of Formula (I) is characterised by an emission peak wavelength of about 600 nm to about 650 nm. In some embodiments, the emission peak wavelength is about 620 nm.

[0090] In some embodiments, the compound of Formula (I) is characterised by an antimicrobial activity. The antimicrobial activity may be against bacteria, fungi, viruses or parasites. The antimicrobial activity may be against bacteria or fungi. Antimicrobials may act through various mechanisms, such as disrupting cell membranes, inhibiting protein synthesis.

[0091] In some embodiments, the compound of Formula (I) is characterised by an antibacterial activity. The antibacterial activity may be against Gram-negative and / or Gram-positive bacterial cells. The bacteria may be Staphylococcus aureus (S. aureus}, Escherichia coli (E. coli}, or P. aeruginosa. In some embodiments, the compound is characterised by an antibacterial activity against drug resistant bacterial cells. In some embodiments, the compound is characterised by an antibacterial activity against antibiotics resistant bacterial cells.

[0092] In some embodiments, the compound is characterised by a minimum inhibitory concentration (MIC) against bacterial cells of about 1 μM to about 10 μM. In other embodiments, the MIC is about 1 μM to about 8 μM, about 1 μM to about 5 μM, about 1 μM to about 3 μM, about 3 μM to about 10 μM, about 3 μM to about 8 μM, about 3 μM to about 5 μM, about 5 μM to about 1 μM, about 5 μM to about 8 μM, or about 8 μM to about 10 μM.

[0093] The compounds of the present disclosure are also advantageous for use as antifungal agents. The fungus may be C. albicans, Cryptococcus neoformans or Saccharomyces cerevisiae. In some embodiments, the compound is characterised by an antimicrobial activity against drug resistant fungal cells.

[0094] In some embodiments, the compound of Formula (I) is characterized by a MIC against fungus of about 1 μM to about 10 μM. In other embodiments, the MIC is about 1 μM to about 8 μM, about 1 μM to about 5 μM, about 1 μM to about 3 μM, about 3 μM to about 10 μM, about 3 μM to about 8 μM, about 3 μM to about 5 μM, about 5 μM to about 1 μM, about 5 μM to about 8 μM, or about 8 μM to about 10 μM. In some embodiments, the compound of Formula (I) is characterized by a minimum fungal concentration (MFC) of about 1 μM to about 10 μM. In other embodiments, the MFC is about 1 μM to about 8 μM, about 1 μM to about 5 μM, about 1 μM to about 3 μM, about 3 μM to about 10 μM, about 3 μM to about 8 μM, about 3 μM to about 5 μM, about 5 μM to about 1 μM, about 5 μM to about 8 μM, or about 8 μM to about 10 μM. In some embodiments, the MFC is about 2 μM to about 4 μM.

[0095] The synthesis method of the above compounds includes the following step:

[0096] Step 1: Mix the reactant, the first base and pyridine-4-boronic acid, add tetra- (triphenylphosphine) palladium (Pd(PPh3)4) as catalyst, add 1,4-dioxane aqueous solution, stir under the under nitrogen atmosphere at 90~105°c for 10~15h, cool to room temperature, add into ethyl acetate, extract with water, dry the resulting organic layer and evaporate the solution under pressure. The yellow solid is TPA2Py skeleton, in which the ratio of reactant to pyridine-4-boronic acid is 2: (4~6) according to the amount of substance, and the ratio of pyridine-4-boronic acid to the first base is 1: (1~1.5) according to the amount of substance.

[0097] The TPA2Py skeleton is as follows: wherein R is independently selected from H, alkyl, unsaturated alkyl, heteroatomic alkyl, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, alkoxy, or one or more chromophores capable of conjugation with one or more fluorescent substances.

[0098] The structural formula of the reactants is as follows: wherein R is independently selected from H, alkyl, unsaturated alkyl, heteroatomic alkyl, cycloalkyl, heterocyclic alkyl, aryl, heteraryl, alkoxy and one or more groups capable of conjugation with one or more fluorescent substances; and wherein R' is

[0099] In Step 1, the first base is K2CO3, Na2CO3, potassium acetate, caesium carbonate, or sodium acetate.

[0100] In Step 1, the ratio of the reactant to tetra-(triphenylphosphine) palladium (Pd(PPh3)4) is 2: (0.1 ~0.2) in terms of the amount of substance.

[0101] In Step 1, the ratio of the quantity of the substance of the reactant to the volume of the 1,4-dioxane aqueous solution is less than or equal to 2:20, the unit of the quantity of the substance is mmol, and the unit of the volume is mL.

[0102] In Step 1, 1,4-dioxane aqueous solution is a mixture of 1, 4-dioxane and water, and the ratio of 1,4-dioxane to water in 1,4-dioxane aqueous solution is (4~5) : 1 by volume.

[0103] In Step 1, the ratio of the quantity of the substance of the reactant to the volume of the ethyl acetate is less than or equal to 2:40, the quantity of the substance is measured in mmol, and the volume of the volume is measured in mL.

[0104] In Step 1, purification was performed by silica gel chromatography using a mixture of ethyl acetate and n-hexane as eluent, the ratio of ethyl acetate to n-hexane by volume is (3 to 5) : 1.

[0105] In the above technical scheme, the organic layer in Step 1 is dried by NaSO4.

[0106] In the above technical scheme, when the structural formula of the reactant is

[0107] the method of preparing the reactant is: Compound b2 (1 part of substance), potassium carbonate (2 to 5 part of substance) and benzyl bromide (1 to 1.2 part of substance) were added to 15 to 30 part volume of acetone, stirred at 55 to 65°C for 10 to 15 h. After the reaction, the solids were removed by vacuum extraction and a filter cake was obtained. An organic phase (crude product) was collected and dried by washing the cake with acetone. A white solid crude product was obtained and purified by silica gel chromatography to obtain a white solid as the reactant. The unit of the quantity of the substance is mmol and the unit of the volume is mL.

[0108] The structural formula of benzyl bromide is as follows:

[0109] The structural formula of compound b2 is as follows:

[0110] Step 2: Mix the TPA2Py skeleton with a first solvent, add iodomethane under the nitrogen or inert atmosphere, stir at 60~80°C for 10~15 h, remove the solvent under pressure to obtain a first crude orange solid, distribute the first crude product evenly in MeOH, and then add ether to obtain organic fluorescent compounds. The ratio of TPA2Py skeleton to iodomethane is 1 : (2~2.5) according to the amount of substance. In Step 2, the first solvent is selected from acetonitrile, tetra hydrofuran and N,N- d I methylformamide.

[0111] In Step 2, the mass of the MeOH is the minimum amount capable of dissolving the first crude product.

[0112] In Step 2, the ratio of the quantity of the substance of the TPA2Py skeleton to the volume of the first solvent is 1: (50~150), the unit of the quantity of the substance is mmol, and the unit of the volume is mb

[0113] In Step 2, the volume ratio of the first solvent to the ether is 1 : (1~5).

[0114] Accordingly, the present disclosure also concerns a method of synthesising a compound of Formula (I) or Formula (la), or a salt or solvate thereof, comprising: a) reacting compound of Formula (II) with a haloalkane:

[0115] In some embodiments, a mole ratio of compound of Formula (II) to haloalkane is about 1 : 1.5 to about 1:3. In other embodiments, the mass ratio is about 1:1.5 to about 1:2.5, about 1: 1.5 to about 1:2, about 1:2 to about 1:3, about 1:2 to about 1:2.5, or about 1 :2.5 to about 1:3. In some embodiments, the mass ratio is about 1:2 to about 1:2.5.

[0116] In some embodiments, the haloalkane is iodomethane.

[0117] In some embodiments, the reaction is conducted at a temperature of about 40 °C to about 100 °C. In other embodiments, the temperature is about 40 °C to about 90 °C, about 40 °C to about 80 °C, about 40 °C to about 70 °C, about 50 °C to about 100 °C, about 50 °C to about 90 °C, about 50 °C to about 80 °C, about 50 °C to about 70 °C, about 60 °C to about 100 °C, about 60 °C to about 90 °C, about 60 °C to about 80 °C, about 60 °C to about 70 °C, about 70 °C to about 100 °C, about 70 °C to about 90 °C, about 70 °C to about 80 °C, about 80 °C to about 100 °C, or about 80 °C to about 90 °C. In some embodiments, the temperature is about 60 °C to about 80 °C. In some embodiments, the reaction is conducted for a duration of about 5 hours to about 20 hours. In other embodiments, the duration is about 5 hours to about 18 hours, about 5 hours to about 15 hours, about 5 hours to about 12 hours, about 5 hours to about 10 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 15 hours, or about 15 hours to about 20 hours. In some embodiments, the duration is about 10 hours to about 15 hours.

[0118] In some embodiments, the compound of Formula (II) is mixed with a first solvent. The solvent may be acetonitrile, tetra hydrofuran or / V, / V-dimethylformamide. In some embodiments, a ratio of compound of Formula (II) to solvent is about 1 mmol:40 mL to about 1 mmol:200 mL. In other embodiments, the ratio is about 1 mmol:40 mLto about 1 mmol:175 mL, about 1 mmol:40 mL to about 1 mmol: 150 mL, about 1 mmol:80 mL to about 1 mmol:200 mL, about 1 mmol:80 mL to about 1 mmol:175 mL, about 1 mmol:80 mL to about 1 mmol: 150 mL, about 1 mmol: 100 mL to about 1 mmol:200 mL, about 1 mmol: 100 mL to about 1 mmol: 175 mL, about 1 mmol: 100 mL to about 1 mmol: 150 mL, about 1 mmol:150 mL to about 1 mmol:200 mL, or about 1 mmol: 150 mL to about 1 mmol:175 mL. In some embodiments, the ratio is about 1 mmol:50 mL to about 1 mmol:150 mL.

[0119] In some embodiments, the reacted product is mixed with a second solvent to precipitate compound of Formula (I) or Formula (la) as a solid. In some embodiments, the second solvent is ether.

[0120] In some embodiments, a volume ratio of the first solvent to the second solvent is about 1 :0.5 to about 1 :8. In other embodiments, the ratio is about 1:0.5 to about 1:5, about 1 : 1 to about 1:8, about 1:1 to about 1:5, about 1 :2 to about 1:8, about 1:2 to about 1 :5, about 1:4 to about 1:8, or about 1:4 to about 1: 5. In some embodiments, the ratio is about 1: 1 to about 1:5.

[0121] In some embodiments, the method further comprises a step before step a) of reacting a compound of Formula (III) with a heterocyclic acid to form compound of Formula (II): wherein X is halo.

[0122] In some embodiments, the reaction is conducted in the presence of a catalyst. The catalyst may be Pd(PPhs)4. In some embodiments, the reaction is conducted in the presence of a base. The base may be K2CO3, Na2COs, potassium acetate, caesium carbonate, or sodium acetate.

[0123] In some embodiments, a mole ratio of compound of Formula (III) to heterocyclic acid is about 2:3 to about 2:7. In other embodiments, the mole ratio is about 2:3 to about 2:6, about 2:3 to about 2:5, about 2:3 to about 2:4, about 2:4 to about 2:7, about 2:4 to about 2:6, about 2:4 to about 2: 5, about 2:5 to about 2:7, about 2:5 to about 2:6, or about 2:6 to about 2:7. In some embodiments, the mole ratio is about 2:4 to about 2:6.

[0124] In some embodiments, a mole ratio of heterocyclic acid to base is about 1 :0.5 to about 1 :2. In other embodiments, the mole ratio is about 1:0.5 to about 1: 1.5, about 1:0.5 to about 1 :1, about 1: 1 to about 1:2, about 1 : 1 to about 1: 1.5, or about 1: 1.5 to about 1 :2. In some embodiments, the mole ratio is about 1:1 to about 1 : 1.5.

[0125] In some embodiments, a mole ratio of compound of Formula (III) to catalyst is about 2:0.05 to about 2:1. In other embodiments, the mole ratio is about 2:0.05 to about 2:0.5, about 2:0.05 to about 2:0.2, about 2:0.05 to about 2:0.1, about 2:0.1 to about 2: 1, about 2:0.1 to about 2:0.5, about 2:0.1 to about 2:0.2, about 2:0.2 to about 2: 1, or about 2:0.2 to about 2:0.5. In some embodiments, the mole ratio is about 2:0.1 to about 2:0.2.

[0126] In some embodiments, the heterocyclic acid is pyridine-4-boronic acid.

[0127] In some embodiments, the reaction is conducted at a temperature of about 60 °C to about 150 °C. In other embodiments, the temperature is about 60 °C to about 120 °C, about 60 °C to about 100 °C, about 60 °C to about 80 °C, about 80 °C to about 150 °C, about 80 °C to about 120 °C, about 80 °C to about 100 °C, about 100 °C to about 150 °C, or about 100 °C to about 120 °C. In some embodiments, the temperature is about 90 °C to about 105 °C.

[0128] In some embodiments, the reaction is conducted for a duration of about 5 hours to about 20 hours. In other embodiments, the duration is about 5 hours to about 18 hours, about 5 hours to about 15 hours, about 5 hours to about 12 hours, about 5 hours to about 10 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 15 hours, or about 15 hours to about 20 hours. In some embodiments, the duration is about 10 hours to about 15 hours.

[0129] In some embodiments, the compound of Formula (III) is mixed with an aqueous solution. The aqueous solution may be 1,4-dioxane. In some embodiments, a ratio of compound of Formula (III) to aqueous solution is less than or equal to about 2 mmol: 15 mL. In other embodiments, the ratio is less than or equal to about 2 mmol: 18 mL, about 2 mmol:20 mL, about 2 mmol:25 mL. In some embodiments, the ratio is less than or equal to about 2 mmol:20 mL.

[0130] In some embodiments, the aqueous solution is a mixture of 1,4-dioxane and water. In some embodiments, a volume ratio of 1,4-dioxane to water in the aqueous solution is about 2: 1 to about 8:1. In other embodiments, the volume ratio is about 2: 1 to about 6: 1, about 2: 1 to about 5: 1, about 2: 1 to about 4: 1, about 2: 1 to about 3: 1, about 3: 1 to about 8: 1, about 3:1 to about 6: 1, about 3:1 to about 5: 1, about 3:1 to about 4: 1, about 4: 1 to about 8: 1, about 4:1 to about 6: 1, about 4: 1 to about 5: 1, about 5: 1 to about 8: 1, or about 5: 1 to about 6: 1. In some embodiments, the volume ratio is about 4: 1 to about 5:1.

[0131] In some embodiments, the reacted product is mixed with ethyl acetate to obtain an organic layer. In some embodiments, a ratio of compound of Formula (III) to volume of ethyl acetate is less than or equal to about 2 mmol: 30 mL. In other embodiments, the ratio is less than or equal to about 2 mmol:35 mL, about 2 mmol:40 mL, about 2 mmol:45 mL. In some embodiments, the ratio is less than or equal to about 2 mmol:40 mL.

[0132] In some embodiments, the reacted product is purified using silica gel chromatography to obtain the compound of Formula (II).

[0133] In some embodiments, the method further comprises a step before step a) of reacting a compound of Formula (IV) with benzyl bromide to form compound of Formula (III):

[0134] In some embodiments, the compound of Formula (III) is selected from

[0135]

[0136] In some embodiments, a mole ratio of compound of Formula (IV) to benzyl bromide is about 1:0.5 to about 1:2. In other embodiments, the ratio is about 1:0.5 to about 1: 1.5, about 1:0.5 to about 1: 1, about 1: 1 to about 1:2, about 1: 1 to about 1: 1.5, or about 1 : 1.5 to about 1:2. In some embodiments, the ratio is about 1: 1 to about 1:1.5.

[0137] In some embodiments, the benzyl bromide is selected from

[0138] In some embodiments, the reaction is conducted at a temperature of about 30 °C to about 80 °C. In other embodiments, the temperature is about 30 °C to about 70 °C, about 30 °C to about 65 °C, about 30 °C to about 60 °C, about 40 °C to about 80 °C, about 40 °C to about 70 °C, about 40 °C to about 65 °C, about 40 °C to about 60 °C, about 50 °C to about 80 °C, about 50 °C to about 70 °C, about 50 °C to about 65 °C, about 50 °C to about 60 °C, about 60 °C to about 80 °C, about 60 °C to about 70 °C, or about 60 °C to about 65 °C. In some embodiments, the temperature is about 55 °C to about 65 °C.

[0139] In some embodiments, the reaction is conducted for a duration of about 5 hours to about 20 hours. In other embodiments, the duration is about 5 hours to about 18 hours, about 5 hours to about 15 hours, about 5 hours to about 12 hours, about 5 hours to about 10 hours, about 10 hours to about 20 hours, about 10 hours to about 18 hours, about 10 hours to about 15 hours, or about 15 hours to about 20 hours. In some embodiments, the duration is about 10 hours to about 15 hours.

[0140] In some embodiments, the reacted product is purified using silica gel chromatography to obtain the compound of Formula (III).

[0141] The compound of Formula (I) and / or Formula (la) may be used as antimicrobial drugs. For example, the compound may be used in selective imaging of bacteria cells and / or fungal cells. The compound may be used as drugs in the treatment of bacteria-induced sepsis. The compound may be used as drugs in the treatment of fungal infections.

[0142] Compared with the prior art, the disclosed compounds have the following beneficial effects:

[0143] The TPA2Py skeleton may significantly enhance the penetration of organic fluorescent compounds on the bacterial membrane through the modification of rigid hydrophobic groups. After penetrating the bacterial membrane, the TPA2Py skeleton may bind to the DNA in the bacterioid nucleus and induce the DNA to aggregate, thus realizing the antibacterial effect. The MIC of organic fluorescent compounds was 1 μM for Grampositive bacteria S. aureus and 5 μM for Gram-negative bacteria E. coli. At the same time, organic fluorescent compounds may be effectively loaded in the lipid droplets of mammalian cells, but may not produce toxicity to mammalian cells, so it may be possible to achieve adoptive transfer of macrophages through mammalian cell loading to treat bacterial-induced sepsis.

[0144] The compound may be selectively introduced into fungal nuclei while preserving the integrity of mammalian cell nuclei (Figure 5). The compound may penetrate the fungal membrane, leading to a rapid death of fungal cells and may reduce drug resistance. By targeting the fungal nuclei, the compound may induce DNA disorder or directly damage DNA, thus minimising possibility of resistance arising from genetic mutations. The incorporation of isopropylbenzene and biphenyl groups into the DNA-binding domain may enhance the penetration of the compound into fungal cells and nuclear membranes while also imparting a mechanism of action on fungal membranes. The compound may eliminate fungal cells through this dual mechanism and may depolarise fungal membranes and induce DNA damage with minimal toxicity to mammalian cells. The compound may be able to combat fungal infections and mitigate antifungal resistance. The present disclosure also concerns a method of labelling and / or imaging bacterial cells, comprising contacting the bacterial cells with a compound of Formula (I) or (la), or a salt or solvate thereof.

[0145] The bacteria may be Gram-negative and / or Gram-positive bacterial cells. The bacteria may be selected from E. coli, S. aureus, P. aeruginosa and a combination thereof.

[0146] The bacterial cells may be detected via the fluorescence emitted when the compound binds with the bacterial cells.

[0147] The method may further comprise a step of irradiating a sample or the bacterial cells to electromagnetic radiation of about 300 nm to about 800 nm, or about 350 nm to about 500 nm.

[0148] The present disclosure also concerns a method of detecting the presence of bacterial cells in a sample, comprising contacting the sample with a compound of Formula (I) or (la), or a salt or solvate thereof. The sample may be obtained from a patient having a disease or disorder associated with bacteria.

[0149] The present disclosure also concerns a method of labelling and / or imaging fungal cells, comprising contacting the fungal cells with a compound of Formula (I) or (la), or a salt or solvate thereof.

[0150] The fungus may be selected from C. albicans, Cryptococcus neoformans, Saccharomyces cerevisiae, and a combination thereof.

[0151] The fungal cells may be detected via the fluorescence emitted when the compound binds with the fungal cells.

[0152] The method may further comprise a step of irradiating a sample or the fungal cells to electromagnetic radiation of about 300 nm to about 800 nm, or about 350 nm to about 500 nm.

[0153] The present disclosure also concerns a method of detecting the presence of fungal cells in a sample, comprising contacting the sample with a compound of Formula (I) or (la), or a salt or solvate thereof. The sample may be obtained from a patient having a disease or disorder associated with fungus. The present disclosure also concerns a compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof for use in therapy.

[0154] The present disclosure also concerns a compound of Formula (I) or (la) or a salt or solvate thereof for use in the imaging and / or treatment of a microbial disease or disorder.

[0155] The present disclosure also concerns a use of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the imaging and / or treatment of a microbial disease or disorder.

[0156] The present disclosure also concerns a method of imaging and / or treating a microbial disease or disorder in a subject thereof, comprising administering to the subject a therapeutically effective amount of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof.

[0157] The present disclosure also concerns a compound of Formula (I) or (la) or a salt or solvate thereof for use in the imaging and / or treatment of a disease or disorder associated with bacteria.

[0158] The present disclosure also concerns a use of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the imaging and / or treatment of a disease or disorder associated with bacteria.

[0159] The present disclosure also concerns a method of imaging and / or treating a disease or disorder associated with bacteria in a subject thereof, comprising administering to the subject a therapeutically effective amount of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof.

[0160] The disease or disorder associated with bacteria may be a bacterial infection. The disease or disorder associated with bacteria may have a resistance against antibiotics. The disease or disorder may be skin infection (e.g. boils, abscesses, cellulitis), soft tissue infection, respiratory disease (e.g. pneumonia, sinusitis), bloodstream infections (e.g. sepsis), heart valve infections, ear infections, urinary tract infections, wound infections, food poisoning, sexually transmitted infections, meningitis, gastroenteritis (diarrhoea), tuberculosis, meningococcal disease, Legionnaires' disease, Q fever, strep throat, whooping cough (pertussis) or any other life-threatening systemic disease. The present disclosure also concerns a compound of Formula (I) or (la) or a salt or solvate thereof for use in the imaging and / or treatment of a disease or disorder associated with fungus.

[0161] The present disclosure also concerns a use of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the imaging and / or treatment of a disease or disorder associated with fungus.

[0162] The present disclosure also concerns a method of imaging and / or treating a disease or disorder associated with fungus in a subject thereof, comprising administering to the subject a therapeutically effective amount of compound of Formula (I) or (la) or pharmaceutically acceptable salt or solvate thereof.

[0163] The compound may selectively target fungal cells and disrupt the fungal membrane and induce DNA damage, resulting in the death of the fungal cells. By inducing DNA damage, the possibility of resistance arising from genetic mutations may be minimised.

[0164] The disease or disorder associated with fungus may be a fungal infection. The disease or disorder may be aspergillosis, blastomycosis, Candida auris, candidiasis, chromoblastomycosis, cryptococcosis, fungal eye infection, histoplasmosis, mucormycosis, mycetoma, paracoccidioidomycosis, pneumocystis pneumonia, ringworm and nail infection, sporotrichosis, talaromycosis, and valley fever. The disease or disorder may be or may lead to skin infection, lung infection multi-drug resistant illness, limb swelling, brain infection, eye infection, and / or nail infection.

[0165] The present disclosure pertains to compounds and their various forms, including ionic forms, tautomers, isomers, polymorphs, pseudopolymorphs, amorphous forms, solvates, co-crystals, chelates, esters, prodrugs, and protected forms. The disclosure also encompasses methods of utilizing these compounds for various purposes. It should be noted that terms like "crystalline form," "polymorph," can be used interchangeably to include all crystalline and amorphous forms, such as polymorphs, pseudopolymorphs, solvates (including hydrates), co-crystals, unsolvated polymorphs (including anhydrates), conformational polymorphs, amorphous forms, and mixtures thereof, unless a specific crystalline or amorphous form is specified. In certain embodiments, the compounds and their subgroups include polymorphs, solvates, co-crystals, isomers, tautomers, and / or oxides. In other embodiments, they may include polymorphs, solvates, and / or co-crystals. The compound of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0166] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0167] Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

[0168] It will be appreciated that any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention. Thus, the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art. Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al., Chapter 5, (Harwood Academic Publishers).

[0169] The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

[0170] The compound of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration. The term "therapeutic effect" refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology. "Therapeutically effective amount" as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0171] As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 1000 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 800 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage, such as up to 250 mg per body weight per dosage.

[0172] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

[0173] The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0174] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0175] The compound of the invention can be administered to the vitreous of the eye using any intravitreal or transscleral administration technique. For example, the compound can be administered to the vitreous of the eye by intravitreal injection. Intravitreal injection typically involves administering a compound of the invention or a pharmaceutically acceptable salt, solvate or prodrug in a total amount between 0.1 ng to 10 mg per dose.

[0176] Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. By way of example, the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1 / 2 or 3 / 8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).

[0177] A person skilled in the art will appreciate that other means for injecting and / or administering the compound, composition or combinations to the vitreous of the eye can also be used. These other means can include, for example, intravitreal medical delivery devices. These devices and methods can include, for example, intravitreal medicine delivery devices, and biodegradable polymer delivery members that are inserted in the eye for long term delivery of medicaments. These devices and methods can further include transscleral delivery devices. Other modes of administration including topical or intravenous administration may also be possible. For example, solutions or suspensions of the compound, composition or combinations of the invention may be formulated as eye drops, or as a membranous ocular patch, which is applied directly to the surface of the eye. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.

[0178] The compound or composition of the invention may also be suitable for intravenous administration. For example, a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 100 mg / m2.

[0179] The compound or composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.

[0180] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0181] The compound of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0182] The compound of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention.

[0183] The compound of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0184] Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

[0185] It should be understood that in addition to the active ingredients particularly mentioned above, the composition or combination of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0186] Examples

[0187] The organic fluorescent compound disclosed may efficiently load into macrophages and treat bacterial-induced sepsis by cell adoptive transfer. The synthesis method obtains the organic fluorescent compounds by modifying the TPA2Py skeleton with rigid hydrophobic groups (benzene, biphenyl, isopropyl benzene, etc.), and the organic fluorescent compounds have high membrane penetration and strong bacterial affinity.

[0188] The organic fluorescent compound, which is TPA2PyProp, TPA2PyBiPh or TPA2PyPh. The general structure of TPA2PyBiPh is as follows:

[0189] The general structure of TPA2PyProp is as follows:

[0190] The general structure of TPA2PyPh is as follows:

[0191] wherein R is independent, R is H, alkyl, unsaturated alkyl, heteroatomic alkyl, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, alkoxy and one or more groups that can be conjugated with one or more fluorescent substances.

[0192] The technical scheme of the invention is further explained in combination with specific embodiments. The synthesis route of TPA2PyPh in the following embodiments is as follows:

[0193] The synthesis route of TPA2PyBiPh / TPA2PyProp in the following embodiments is as follows:

[0194]

[0195] Purity and source of purchase of the drug in the following embodiments:

[0196] Diphenylamine, 97%, Sigma Aldrich; 4-methoxytrianiline, 97%, BLDpharm; N- bromosuccinimide, 99%, Sigma Aldrich; Boron tribromide, 1.0 M in DCM, Sigma Aldrich; K2CO3, 99%, Sigma Aldrich; Pyridine-4-boronic acid, 90%, Sigma Aldrich; Tetri (triphenylphosphine) palladium, 99%, Sigma Aldrich; 4-bromomethyl biphenyl, 98%, BLDpharm; P-isopropyl bromobenzyl, 98%, BLDpharm; lodomethane, 99%, Sigma Aldrich; Raw 264.7 cells purchased from ATCC, TIB-71; S. aureus purchased from ATCC (Strain designation: NCTC 8532); E. coll purchased from ATCC (Strain designation: HB101); Staphylococcus aureus labeled with green fluorescent protein. Gentamicin: Purchased from sigma aidrich; MRSA bacteria: Purchased from ATCC (Strain designation: F-182).

[0197] The temperature of saturated salt water is 21-25 ° C. The pH of PBS is 7.4.

[0198] Example 1

[0199] The synthesis of TPA2PyPh consists of the following steps:

[0200] SI, compound a2 (954 mg, 2 mmol) (i.e., the reactant), K2CO3(i.e., the first base) (828 mg, 6 mmol), and pyridine-4-boronic acid (541.2 mg, 4.4 mmol) were added to a 100 ml round-bottled flask, followed by tetri (triphenylphosphine) palladium (115.6 mg, 115.6 mg). 0.1 mmol) as the catalyst, add 25 ml of 1, 4-dioxane aqueous solution, wherein the 1, 4-dioxane aqueous solution is a mixture of 1, 4-dioxane and water. The volume ratio of 1, 4-dioxane and water is 4: 1. The round-bottom flask is sealed and stirred under nitrogen atmosphere for 12 h at 90°C. After cooling to room temperature, the solution poured into lOOmL ethyl acetate for extraction with water. The extracted organic layer is dried with NaSCU and evaporated under reduced pressure to obtain the crude product, which is purified by silica gel chromatography. The eluent used for purification is a mixture of ethyl acetate and n-hexane (by volume, the ratio of ethyl acetate to n-hexane is 4: 1). The yellow solid obtained after purification is TPA2Py skeleton (compound a3, N, n-BIS (4-(pyridin-4-yl)phenyl)-[l,l'-biphenyl]-4-amine), the yield is 683 mg, the yield is 71.9%, the structural formula of compound a2 is:

[0201] S2, dissolve the TPA2Py skeleton (compound a3) (475 mg, 1 mmol) in 50 mL of the first solvent (acetonitrile) in a 100 ml round-bottom flask, seal the round-bottom flask under nitrogen atmosphere and add iodomethane (0.137 mL, 2.2 mmol). After stirring at 70°C for 12 h and removing solvent under reduced pressure, the first crude of orange solid is obtained. The first crude is evenly distributed in MeOH (the mass of MeOH is the minimum amount that may dissolve the first crude product), and then lOOmL ether is added to obtain TPA2PyPh (orange solid). The yield of TPA2PyPh is 676 mg. The yield is 95%.

[0202] The nuclear magnetic data of TPA2PyPh are as follows: 1H NMR (400 MHz, Methanol- d4) 6 8.77 (d, J = 6.3Hz, 4H), 8.32 (d, J = 6.4Hz, 4H), 8.00-7.96 (m, 4H), 7.38 (d, J = 7.9 Hz, 3 h), 7.30 7.26 (m, 7 h), 7.19 7.17 (m, 2 h), 7.12 7.09 (m, 2 h), 4.34 (d, J = 1.8 Hz, 6 h).

[0203] Preparation method of compound a2: Compound al (1.6g, 5 mmol) and 30 mL DCM were added to a 100 ml round -bottomed flask, cooled to 0°c with an ice bath, N- bromosuccinimide (1.72g, 10 mmol) was dissolved in 30 ml DCM, and then added to the round-bottomed flask through a drip funnel, and stirred in a 0°C ice bath for 1 hour away from light. The extracted organic layer was washed with water and dried using NaSCU. After evaporation of solvent under reduced pressure, the crude product was obtained. The crude product was purified by silica gel chromatography. The eluent used for purification was a mixture of DCM and hexane (Hex) by volume. The ratio of DCM to hexane (Hex) is 1 / 10 to 1 / 4 (gradient elation). The white solid obtained after purification is compound a2 (N, n-BIS (4-bromophenyl)-[l,l'-biphenyl]-4-amine), the yield of compound a2 is 2.23 g, the yield is 94%. Preparation method of compound al: Diphenylamine (1.69 g, 10 mmol), 4-iodobiphenyl (2.8g, 10 mmol), bistriphenylphosphine dichloride (112 mg, 0.5 mmol), potassium tert- buok (t-BuOK, 20 mmol) and tritert-butylphosphine tetrafluoroborate (290 mg, 1 mmol) were mixed in a 100 mL round-bottom flask, and 50 mL toluene was added to dissolve it. The round-bottom flask was sealed under nitrogen atmosphere, heated in an oil bath at 90°C for 12 h, cooled to room temperature after the reaction, then added into 100 mL ethyl acetate. The organic phase was extracted with saturated salt water. The organic phase was dried with anhydrous sodium sulfate and spun dry to obtain the crude product, and the crude product is purified by silica gel column chromatography. The eluent used for purification is a mixture of DCM and n-hexane, and the volume ratio of DCM and n-hexane is 1 : 10. The white solid obtained is compound al (N, n-diphenyl - [l,l'-biphenyl]-4-amine), the yield of compound al is 1.8g, the yield is 56.8%.

[0204] Example 2

[0205] The synthesis of TPA2PyBiPh / TPA2PyProp involves the following steps:

[0206] SI, add 0.5 mmol compound b3 (i.e., the reactant), 1.5 mmol K2CO3(i.e., the first base) and 1.1 mmol pyridine-4-boronic acid to a 100 ml round-bottled flask, and add tetri (triphenylphosphine) palladium (0.025 mmol) as catalyst. Add 25 ml of 1,4-dioxane aqueous solution, wherein the 1,4-dioxane aqueous solution is a mixture of 1,4-dioxane and water. The volume ratio of 1,4-dioxane and water in the 1,4-dioxane aqueous solution is 4: 1. The round-bottom flask was sealed and stirred under nitrogen atmosphere for 12 h at 90°C, and then added into lOOmL ethyl acetate after cooling to room temperature for extraction with water. The extracted organic layer was dried by and evaporated under reduced pressure. The organic layer was purified by silica gel chromatography. The eluent used for purification is a mixture of ethyl acetate and n-hexane (by volume, the ratio of ethyl acetate to n-hexane is 4: 1). The yellow solid obtained after purification is the TPA2Py skeleton (compound b4).

[0207] The structural formula of compound b3 is: when R' is the yield of compound b4 is 73%, and when R1the yield of compound b4 is 68%. S2, the 0.2mmol TPA2Py skeleton (compound b4) was dissolved in 50ml of the first solvent (acetonitrile), placed in a 100ml round-bottom flask, sealed the round-bottom flask under nitrogen atmosphere, added 0.44mmol iodomethane into the round-bottom flask under nitrogen atmosphere, stirred at 70°C for 12 h, and the solvent was removed under reduced pressure. The first crude orange solid is obtained, and the first crude product is evenly distributed in MeOH (the mass of MeOH is the minimum amount that can dissolve the first crude product), and then lOOmL diethyl ether is added to obtain organic fluorescent compounds (orange solid).

[0208] When R1s the organic fluorescent compounds obtained are TPA2PyBiPh. The yield of TPA2PyBiPh is 95%. When R1is the organic fluorescent compounds obtained are TPA2PyProp, and the yield of TPA2PyProp is 91%.

[0209] Preparation method of compound b3: 1 mmol of compound b2, 3 mmol of potassium carbonate and 1.1 mmol of benzyl bromide were added to a round-bottomed flask containing 20 mL of acetone and stirred at 60°C for 12 h. After the reaction, the solids were removed by vacuum extraction to obtain filter cake, and the filter cake was washed with acetone (10 mL, 3 times) to collect organic phase and spin dry. The white solid crude product is obtained, and the white solid crude product is purified by silica gel chromatography. The eluent used for purification is a mixture of DCM and Hex (the ratio of DCM to Hex is 1: 10 by volume), and the white solid obtained after purification is compound b3.

[0210] The structural formula of benzyl bromide is when

[0211] R1is the yield of compound b3 is 91%, and when R1is the yield of compound b3 is 94%.

[0212] Preparation method of compound b2: Compound b1 (866 mg, 2 mmol) was added to a 50 mL round-bottomed flask, and 20 mL ultra-dry DCM was added to dissolve compound b1 and cooled to 0°C with ice bath. 3 mmol boron tribromide was added drop by drop and the mixture was stirred in the ice bath for 30 min and then increased to room temperature for 12 h. The methanol was slowly added to the solution to quench the reaction, and the organic phase was collected and concentrated to obtain the gray crude product. The gray crude product was purified by silica gel chromatography. The eluent used for purification was the mixture of DCM and hexane (Hex) (the ratio of DCM and Hex) by volume was 1 : 1), the gray solid obtained after purification was the compound b2 (4-(bis(4-Bromophenyl)amino)phenol) with a yield of 544 mg and a yield of 64.8%.

[0213] Preparation method of compound b1 : 4-methoxytrianiline (1.38 g, 5 mmol) and 30 mL DCM were added to 100 ml round-bottomed flask, cooled to 0°c with ice bath. N- bromosuccinimide (1.72 g, 10 mmol) was dissolved in 30 ml DCM, and then added to the round-bottomed flask through a dropping funnel. The mixture was stirred in ice bath at 0°c for 1 hour without light, heated up to room temperature and stirred away from light for 12 h, quenched with water, extracted with DCM, cleaned the organic layer with water and dried on Na SO, evaporated the solvent under pressure to obtain a crude product, purified the crude product by silica gel chromatography to obtain a white solid. The eluent used for purification is a mixture of DCM and hexane (Hex), measured by volume. The ratio of DCM to hexane (Hex) was l / 10~l / 4 (gradient elute), and the white solid was compound b1 (4-bromo-N-(4-bromophenyl)-N-(4-methoxyphenyl)aniline), the yield of compound b1 was 1.96g, and the yield was 91%.

[0214] Organic fluorescent compounds and DMSO were mixed and configured into DMSO mixtures with a concentration of organic fluorescent compounds of ImM, and the organic fluorescent compounds were one of TPA2PyPh, TPA2PyBiPh and TPA2PyProp. The photophysical properties of organic fluorescent compounds were measured by adding 10 pL DMSO mixture to 0.99 mL PBS to prepare PBS solution with 10 μM concentration of organic fluorescent compounds. As shown in Figure 1A to 1C, Figure 1A and 1B show the normalized UV-VIS spectral absorption, PL is the emission spectrum. TPA2PyPh, TPA2PyBiPh and TPA2PyProp dissolve well in PBS solutions and exhibit a wide absorption spectrum from 350nm to 500nm. In PBS solution, TPA2PyPh, TPA2PyBiPh and TPA2PyProp have a wide emission spectrum, with emission coverage ranging from 500 nm to 750 nm, and the peak value is near 620 nm.

[0215] To evaluate the antibacterial effects of organic fluorescent compounds (TPA2PyPh, TPA2PyBiPh, and TPA2PyProp), the minimum inhibitory concentrations (MIC) of organic fluorescent compounds against Gram-negative and Gram-positive bacteria were measured. MIC is defined as the lowest concentration of organic fluorescent-like compounds with no significant bacterial growth after 14 hours of culture at 37°C. 105 CFU Staphylococcus aureus or Escherichia coli were inoculated in 100 pL LB medium containing different concentrations of organic fluorescent compounds, such as TPA2PyPh, TPA2PyBiPh or TPA2PyProp. Then the absorbance (OD600) of LB medium at 600 nm was measured after 14 hours of incubation at 37°C at 220rpm. As shown in Figure 2A to 2C (S. aureus is Staphylococcus aureus in the figure), TPA2PyPh, TPA2PyBiPh and TPA2PyProp showed a general inhibitory effect on the growth of Staphylococcus aureus. The MIC of TPA2PyPh, TPA2PyBiPh and TPA2PyProp are all IμM. On the other hand, TPA2PyPh, TPA2PyBiPh and TPA2PyProp can significantly inhibit the growth of gram-negative Escherichia coii, as shown in Figure 2D to 2F. The MIC of TPA2PyPh, TPA2PyBiPh and TPA2PyProp is 5 μM. This suggests that the antibacterial activity of TPA2PyPh, TPA2PyBiPh and TPA2PyProp is not specific to Gram- negative / positive bacteria.

[0216] Raw 264.7 cells were incubated in DMEM medium containing different concentrations of organic fluorescent compounds as dyes in a 5 % carbon dioxide incubator at 37°C for 48 h. The organic fluorescent compounds were one of TPA2PyPh, TPA2PyBiPh and TPA2PyProp. The incubated Raw 264.7 cells were placed in different concentrations of dyes: O. luM, 0.2uM, 0.5uM, luM, 2uM, 5uM, lOuM, 20μM and 50uM, respectively. The growth of Raw 264.7 cells incubated in the dye concentration below 20 μM was not significantly inhibited (Figure 2G to 21), indicating the low cytotoxicity of TPA2PyPh, TPA2PyBiPh and TPA2PyProp.

[0217] In order to verify the bacterial-targeting ability of organic fluorescent compounds (TPA2PyPh, TPA2PyBiPh, or TPA2PyProp) after being loaded into macrophages, fluorescence co-localization experiments were performed by confocal microscopy using TPA2PyPh as an example. Raw 264.7 cells (500,000-600,000 / well) were inoculated with DMEM medium in confocal petri dishes and incubated in a 37°C, 5% carbon dioxide cell incubator for 10 h. DMEM medium was discarded and DMEM medium containing 5μM TPA2PyPh was added, and incubated for 3 h under the same conditions. TPA2PyPh is fully loaded into Raw 264.7 cells. After 3 hours of incubation, the DMEM medium was discarded, washed three times with PBS and DMEM medium containing 5μM TPA2PyPh was added. Staphylococcus aureus labelled with green fluorescent protein (GFP-S. aureus) was added according to the ratio of infection number to 20. After 2 hours of bacterial infection, the DMEM medium containing 5μM TPA2PyPh was discarded and washed three times with PBS. Finally, the nucleus was labelled with DMEM medium containing Hoechst (IμM). As shown in Figure 3A (Hoechst, GFP-SA, TPA2PyPh, merged, Bright field), the green fluorescence of green fluorescent protein (GFP) overlaps well with the red fluorescence of TPA2PyPh. These results indicated that TPA2PyPh loaded in Raw 264.7 cells could still bind to bacteria well.

[0218] Further, in order to prove that TPA2PyPh loaded in macrophages still maintains its killing ability against bacteria, Raw 264.7 cells (500,000-600,000 / well) were first inoculated in 6-well plates and incubated with DMEM medium containing different concentrations of TPA2PyPh (0, 0.5, 1, 2, 5, 10 μM) for 3 h. Raw 264.7 cells were infected with Staphylococcus aureus in the ratio of infection number to 20. Two hours after bacterial infection, the bacteria were washed three times with PBS, and then DMEM medium containing 50 |jg / mL gentamicin was added and incubated for 1 h to remove the residual bacteria of Raw 264.7 extracellular. After three times of cleaning with PBS, PBS cells containing 10% Triton X-100 were used for 10 min and the number of bacteria in Raw 264.7 cells was counted by plate counting method. As shown in Figure 3B and 3C, with the increase of TPA2PyPh concentration, the number of colonies of TPA2PyPh incubated macrophages (TPP-RAW) significantly decreased, and the survival rate of bacteria in Raw 264.7 cells significantly decreased, indicating that macrophages may kill phagocytic bacteria more effectively after loading TPA2PyPh. These results indicate that TPA2PyPh may still have the binding ability and antibacterial effect on bacteria after being loaded in macrophages.

[0219] In order to prove that macrophages incubated with TPA2PyPh may treat sepsis mice, a mouse sepsis model was constructed. The incubation mode of TPA2PyPh is as follows: Raw 264.7 cells (500,000-600,000 I well) were inoculated in plastic culture dishes with DMEM medium and incubated in a cell incubator at 37°C, 5% carbon dioxide until the cells were fully covered at the bottom 80-90%. DMEM medium was discarded and DMEM medium containing 10 μM TPA2PyPh was added. The Raw 264.7 cells incubated with TPA2PyPh were obtained by continuing incubation for 3 h under the same conditions, and TPA2PyPh was fully loaded in Raw 264.7 cells.

[0220] Mice were divided into three groups: PBS injection group (PBS), untreated Raw 264.7 cell injection group (PBS-RAW), and TPA2PyPh incubated Raw 264.7 cell injection group (TPP-Raw). Three groups of mice were injected with cyclophosphamide (dose: 100 mg / kg, frequency:) on Day 1 to Day 3 for three consecutive days. The mice were inoculated with MRSA bacteria (0.1 mL, 5 X 107 CFU) by intrabitoneal injection on Day 4 to cause mouse sepsis, followed by the same dose of 0.1 mL fluid through the abdominal and tail veins in the three groups of mice, respectively. The fluids of PBS injection group (PBS), untreated Raw 264.7 cell injection group (PBS-RAW), and TPA2PyPh incubated Raw 264.7 cell injection group (TPP-Raw) were: PBS, PBS containing 106 Raw 264.7 cells, and PBS containing 106 Raw 264.7 cells incubated with TPA2PyPh. As shown in Figure 4A, mice treated with TPP-Raw had a significantly higher survival rate after 10 days than mice treated with PBS injection and PBS-RAW. In addition, as shown in Figure 4B, the body weight of mice treated with TPP-Raw increased significantly and gradually returned to normal level after the third day, indicating that macrophages loaded with TPA2PyPh may effectively alleviate the state of sepsis in mice and significantly improve the survival rate of sepsis mice. Based on these findings, TPA2PyPh may be loaded into lipid droplets of macrophages and enhance the immune function of macrophages during adoptive transfer to treat bacterial-induced sepsis, thus overcoming immune escape and drug resistance. TPA2PyPh is designed as a cationic compound with a highly hydrophobic biphenyl unit, which may help TPA2PyPh efficiently penetrate cell membranes and load into lipid droplets of macrophages. After macrophages engulf the bacteria, TPA2PyPh may be absorbed and further inserted into the bacteria's nucleic acid, causing DNA aggregation and inducing bacterial death.

[0221] Combating Fungal Infections and Resistance with a Dual-Mechanism Luminoqen to Disrupt Membrane Integrity and Induce DNA Damage

[0222] Fungal infections are a global health concern, affecting over one billion individuals annually. The efficacy of existing antifungal treatments is limited by the increasing prevalence of antifungal drug resistance, driven by overuse and misuse of antifungal agents. Moreover, poor selectivity of antifungal drugs arisen from the eukaryotic nature of both fungal and mammalian cells further exacerbates this issue. Recent efforts have begun to reinvigorate antifungal research, but most of the work has resulted in compounds that function via mechanisms similar to those of traditional antifungal agents. For example, voriconazole, a triazole antifungal drug approved in 2002, is more effective than other triazoles (fluconazole and itraconazole) due to its broad antifungal spectrum. However, voriconazole is still susceptible to the cross-resistance in fluconazole-resistant Candida spp. arose. Therefore, there is an urgent need to develop new therapeutic agents with distinct mechanisms of action (MoA) that can selectively and effectively combat fungal pathogens without inducing drug resistance.

[0223] Over the past years, significant efforts have been made to identify new antifungal targets, including fungus-specific components of the cell wall or cell membrane, as well as processes such as metabolism, DNA synthesis, mitochondrial function, and stress response. However, antifungal agents targeting a single site often lose efficacy due to genetic variations at the pathogen’s target site. Dual-targeting antifungal agents hold promise for substantially reducing the likelihood of fungal resistance by simultaneously acting on multiple pathways. This strategy, validated in antibacterial drug design, integrates two antimicrobial mechanisms into a single molecule. However, the similarity between fungal and mammalian cells makes it extremely difficult to develop dualmechanism drugs that can selectively kill fungi. As a result, the dual-targeting MoA approach in antifungal therapy remains limited to the combined use of two separate drugs, such as the clinical use of fluconazole and amphotericin B (AMB) in combination to treat drug-resistant Candida infections. Nevertheless, the differing in vivo distribution and pharmacokinetics of the two drugs make it difficult to achieve the desired synergistic effect.

[0224] Membrane targeting represents an ideal antifungal target to prevent the development of resistance. The benefit of such a strategy arises from the crucial role of membrane in maintaining morphology and cell viability in both active and metabolically inactive pathogens. As a result, disruption of the fungal membrane may lead to a rapid death of fungal cells, thereby reducing the drug resistance. Furthermore, fungal membranes consist of various components such as chitin, ^-glucans, and glycosylated proteins, whose synthesis involves various metabolic and physiological processes. Consequently, disrupting fungal membranes engages multiple mechanisms, making it challenging for pathogens to develop resistance through single-gene mutations. Additionally, DNA has long been recognized as an excellent target for treating various diseases, including cancer and microbial infections, due to its role in storing genetic information and ensuring organismal growth and metabolism. Thus, antifungal agents targeting fungal nuclei acid to induce DNA disorder or directly damage DNA may minimize the possibility of resistance arising from genetic mutations. Based on these findings, integrating the dual-targeting MoA of simultaneous disruption of fungal membranes and DNA into a single antifungal agent holds significant promise for minimizing resistance and combating fungal infections. However, small molecules that eliminate fungi through such a dual mechanism have not been reported, and the potential of such small molecules for treating fungal infections remains underestimated.

[0225] In this study, the inventors designed and synthesized two dual-targeting luminescent compounds, TPY (TPA2PyProp) and TPZ (TPA2PyBiPh), based on the same DNA-binding domain. This design selectively introduces TPY and TPZ into fungal nuclei while preserving the integrity of mammalian cell nuclei (Figure 5). The incorporation of isopropylbenzene and biphenyl groups into the DNA-binding domain may effectively enhanced the penetration of both luminogens into fungal cells and nuclear membranes while also imparting a mechanism of action on fungal membranes. Consequently, TPY and TPZ may exhibit dual mechanisms of action. Both compounds demonstrated robust fungicidal activity by depolarizing fungal membranes and inducing DNA damage, with minimal toxicity to mammalian cells. Furthermore, TPY and TPZ showed potent therapeutic efficacy against C. albicans infections in the vaginitis mouse model. This dual-targeting strategy offers a new approach for developing potent antifungal agents to combat fungal infections and mitigate antifungal resistance.

[0226] Results and Discussion Design and Characterisation. The structures and synthetic routes of TPW, TPY, and TPZ are depicted in Figure 6A. TPW features a Y-branched structure with two positively charged pyridinium units, which may have good solubility in biological environments and may facilitate its insertion into DNA grooves through electrostatic interactions (Figure 6B). Based on this foundation, TPY and TPZ were developed to enhance the membrane penetration abilities of the DNA binding backbone. This may be achieved by attaching highly rigid hydrophobic termini: isopropylbenzene for TPY and biphenyl for TPZ. These modifications enabled efficient insertion and transmembrane crossing of TPY and TPZ through fungal cell walls and nuclear membranes, disrupting membrane integrity. Molecular docking simulations revealed that, regardless of its initial orientation, TPZ consistently interacts with the phospholipid bilayer by inserting its biphenyl group after 30 ns of simulation time, highlighting the crucial role of hydrophobic groups in facilitating compound penetration through fungal membranes. Additionally, the electron-withdrawing effect of the pyridinium salt moiety induced an effective donor-acceptor (D-A) interaction within the molecule, which led to nearinfrared emission to facilitate its molecular imaging function.

[0227] The photophysical properties of TPW, TPY, and TPZ were subsequently investigated. Due to their similar fluorescent backbone, all three molecules showed similar absorption spectra, ranging from 320 to 500 nm (Figure 6C). In PBS (l x, pH = 7.4) solution, all three compounds showed negligible luminescence due to their good water solubility and flexible intramolecular motions. As shown in Figure 6D-6F, the addition of dsDNA to the TPW, TPY, and TPZ solution resulted in a marked enhancement in fluorescence intensity. To further explore the mechanism behind the fluorescence enhancement, the luminescence behaviour of TPZ in high viscosity solvents was investigated. As shown in Figure 6H and 61, increasing the viscosity of the solvent environment restricted the intramolecular movement of TPZ, leading to a gradual enhancement of fluorescence intensity. This enhancement was more than 35-fold in 99% glycerol (v / v) compared to that in a PBS (lx, pH = 7.4) solution. Thus, the photoluminescence (PL) enhancement may be attributed to the strong binding affinity of these luminogens for dsDNA, which may limit their intramolecular motions and, thereby, activate fluorescence processes. Additionally, a competitive displacement experiment against the commercial DNA minor-groove-binding dye Hoechst showed more than 75% fluorescence quenching of Hoechst upon the addition of TPW, TPY, and TPZ. In contrast, dyes lacking DNA-binding capability, such as BODIPY and Ce6, do not cause the fluorescence quenching of Hoechst when added to a Hoechst-DNA complex solution (Figures 6G). Circular dichroism (CD) studies were further performed to investigate the DNA-binding properties of the compounds. After adding TPW, TPY, and TPZ to ctDNA or Hoechst-DNA PBS solution, the intensity of the negative band of ctDNA at 247 nm was enhanced, reflecting that the helicity of ctDNA was increased, which suggested a more winding helix caused by the groove binding of TPW, TPY, and TPZ to ctDNA. These results indicate that these compounds may bind efficiently to ctDNA. To further investigate the binding capacity between these compounds and DNA, isothermal titration calorimetry (ITC) traces were measured by adding dsDNA into the TPW and TPZ aqueous solutions to further investigate the binding affinity between these compounds and dsDNA. The binding constants ( / Ca) of the dsDNA to TPW and TPZ were determined to be 4.02 x 107 and 1.16 x 107 M-1, respectively. Notably, TPZ exhibits significantly lower affinity for ssDNA compared to that for dsDNA, which may be attributed to the structural features of TPZ that may enhance its affinity for the minor grooves of dsDNA. These results indicate the efficient DNA-targeting property of these luminogens.

[0228] Fungal Imaging and Antifungal Activities. Subsequently, the inventors evaluated the antifungal activities of TPW, TPY, and TPZ by determining their minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). As depicted in Figure 7D, TPY and TPZ showed potent antifungal activity against C. albicans with an MIC of 2 juM and MFC ranging from 2 / μM (for TPZ) to 4 μM (for TPY). The inventors further assessed the fungicidal activity of these compounds against other fungi such as Cryptococcus neoformans and Saccharomyces cerevisiae. TPY and TPZ exhibited potent antifungal activity against Cryptococcus neoformans and Saccharomyces cerevisiae, with MIC values ranging from 1 to 2 pM. In contrast, although TPW has a strong affinity toward DNA, it exhibited weak antimicrobial activity, with an MIC of 20 / μM and an MFC over 50 / JM against C. albicans. To further explore the reasons behind the difference in antifungal efficiency, a cell uptake experiment to investigate the internalization of TPW, TPY, and TPZ in C. albicans was carried out. Confocal laser scanning microscopy (CLSM) was utilized first to observe the cell uptake of TPW, TPY, and TPZ by C. albicans. Figure 7E reveals substantial fluorescence within fungal cells upon incubation with TPY and TPZ, indicating their efficient cell uptake. TPW showed weak fluorescent signals upon incubation with C. albicans, indicating a low penetration of TPW into fungal cells. To further investigate the efficiency of this uptake process, C. albicans at a concentration of -107CFU was incubated with 10 / JM luminogens for different time intervals. Subsequently, the supernatant was analysed using a UV-vis spectrophotometer. As shown in Figure 7A-7C, incubating fungal cells with TPW for 30 min resulted in a slight decrease in the absorbance of the supernatant. Conversely, TPY and TPZ exhibited a decrease in the absorbance of the supernatant within 15 min and reached saturation around 30 min, indicating rapid uptake of TPY and TPZ by C. albicans. These results demonstrated that the modification of the hydrophobic terminus may benefit the membrane penetration ability of TPY and TPZ. The efficient antifungal performance of TPY and TPZ prompted further investigation into their activities against fungal biofilms, which are a persistent challenge in healthcare due to heightened antimicrobial resistance. As depicted in Figure 7F-7H, mature C. albicans biofilms were treated with flucytosine, TPY, and TPZ, followed by incubation with MTT. The gradually faded purple colour indicated the potent biofilm clearance activities of TPY and TPZ, with TPZ being more effective, as evidenced by the lightest purple colour in solution. In contrast, flucytosine, although highly effective against free C. albicans, was not effective in killing fungal biofilms. Inspired by TPZ's efficient antibiofilm activity, the inventors monitored its penetration into C. albicans biofilms using CLSM. As shown in Figure 71, the red fluorescence from TPZ rapidly increased in intensity within the biofilm, reaching saturation within 90 min. Notably, TPZ also showed superior performance in mitigating antifungal resistance. C. albicans did not acquire resistance even after continuous treatment of 30 passages compared to fluconazole (32-fold MIC change). These results indicate the potential of TPZ in combating fungal infections and drug resistance.

[0229] Antifungal Mechanism. Having demonstrated the effective antifungal properties of TPY and TPZ, the inventors further investigated their antifungal mechanisms. To understand how TPZ affects the fungal nucleoid in live cells, the inventors utilized fungal cytological profiling to provide insights into the mechanism of action of TPZ. Hoechst-stained C. albicans cells were incubated with TPZ, and time-lapse CLSM images were captured. Within 5 min, TPZ accumulated on the fungal surface and gradually permeated the entire cytoplasm over approximately 60 min. As shown in Figure 8A and 8B, TPZ incrementally entered the fungal nucleus after enriching the fungal cytoplasm. After TPZ had been in the fungal nucleus for 15 min, the fluorescent signal from Hoechst began to diffuse from the nucleus into the cytoplasm, indicating disruption of the nuclear membrane and efflux of nuclear DNA. After that, the fluorescence from the Hoechst-DNA complex gradually faded, suggesting effective binding between TPZ and DNA (Figure 8A, 8C, 8D). This observation is consistent with the results shown in Figure 6G. Based on these findings, the inventors further investigated the effect of TPZ on the outer membrane of C. albicans cells. The inventors utilized 1-W-phenylnaphthylamine (NPN) and DiSC3(5) to provide information on the fungal cell wall and membrane integrity, respectively. As depicted in Figure 8E, TPZ caused negligible perturbation of the fungal cell wall but significant depolarization of the cytoplasmic membrane, indicating that TPZ targets the fungal cell membrane. The inventors also observed a similar process of membrane integrity disruption caused by TPZ through confocal laser scanning microscopy. After coincubation with TPZ and PI for approximately 30 min, fluorescence from PI began to be detected within the fungal cells, indicating that the permeability of the fungal membrane was compromised. As TPZ shows a good membrane penetration ability and high affinity for DNA, it may have the potential to affect multiple fungal functions and biological processes. To understand the details, the inventors conducted a comparative transcriptome analysis on C. albicans to elucidate the gene expression differences between TPZ-treated and untreated groups, thereby gaining insight into its antibacterial mechanism. As shown in Figure 8F and 8G, the analysis revealed 43 differentially expressed genes (DEGs) in the TPZ-treated groups compared with the untreated groups. Further gene ontology (GO) enrichment analysis revealed that the DEGs mainly associated with biological process, plasma membrane, and molecular functions. These changes at the gene level suggest adaptation mechanisms of C. albicans cells in response to TPZ treatment. Collectively, these findings indicate that TPZ may achieve fungal elimination by targeting both the fungal cell and nucleus membranes as well as fungal DNA.

[0230] Biocompatibility. To investigate the potential toxicity of TPY and TPZ on mammalian cells, the inventors incubated TPZ with NIH / 3T3 cells and used CLSM to monitor its effects on the cell morphology and internalization. Fluorescence images revealed that NIH / 3T3 cells exhibited a slower uptake of TPZ compared to C. albicans. As shown in Figure 9A, after 6 h incubation with 10 jμM TPZ, only a weak fluorescent signal was detected in NIH / 3T3 cells, indicating minimal cellular uptake of TPZ. After 48 h, the TPZ signal was gradually intensified and localized precisely within the cytoplasm. Notably, the fluorescence pattern of TPZ did not overlap with nuclear staining by Hoechst, and the nuclear region labeled by Hoechst remained intact (Figures 9A and 9B). In contrast, treating C. albicans under the same conditions for 10 min, TPZ was significantly enriched in the nucleus of fungal cells and led to DNA leakage within 25 min (Figure 8A). To further investigate the reasons for this difference, the inventors examined the distribution of TPZ in NIH / 3T3 cells by using confocal laser scanning microscopy. As shown in Figure 9C, TPZ primarily accumulates in the lipid droplets of NIH / 3T3 cells, as evidenced by its colocalization with the widely used lipid droplet tracker BODIPY 493 / 503. Thus, when TPZ is taken up by mammalian cells, lipid droplets in the cytoplasm may slow the diffusion of TPZ throughout the cell, further impeding its ability to disrupt the mammalian cell nucleus. These results indicate that TPZ may not affect the nuclear membrane morphology in the mammalian cells. To further investigate the biosafety of TPY and TPZ for in vivo red blood cells, the inventors evaluated their hemolytic effects on mouse red blood cells. As shown in Figures 9D and 9E, TPY and TPZ exhibited low hemolysis ratios within concentrations ranging from 10 to 60 / μM, with an HC50 value (the minimum concentration causing 50% hemolysis) in the range of 80-100 / μM. Additionally, cytotoxicity assays demonstrated low growth inhibition toward NIH / 3T3 cells within the concentration range of 2-20 / μM (Figures 9H and 91). Minimal growth inhibition was observed at concentrations exceeding 40 μM, which is much higher than their MICs and MFCs against C. albicans (20-fold difference for TPY’s MIC; 10-fold difference for TPY ’ s MFC; 20-fold difference for TPZ ’ s MIC and MFC, respectively). These results collectively indicate that TPY and TPZ exhibit potent antifungal activity against C. albicans while maintaining excellent biocompatibility, making them promising candidates for in vivo antifungal applications due to their superior antifungal selectivity.

[0231] In Vivo Experiment. Given the potent antifungal activities and superior selectivity of TPY and TPZ, the inventors evaluated their therapeutic efficacy in a murine model of C. a / b / cans-induced vaginitis. To mimic clinical conditions, estradiol (0.3 mg in 100 pL of sesame oil) was subcutaneously injected for three consecutive days, creating a susceptibility to fungal infection, as evidenced by vulvar swelling and increased discharge. Mice were then intravagina I ly infected with 2.5 x 10sCFUs of C. albicans. Three days postinfection, TPY and TPZ were administered intravaginally to treat the local infection. As shown in Figures 10B and 10C, TPY and TPZ treatment for 3 days resulted in a significant reduction in fungal load (>99.9%) compared to the PBS-treated group. Notably, TPY and TPZ exhibited superior clearance rates against C. albicans in the vaginitis model compared with the commercially available antifungal drug flucytosine. The histological images of mouse vaginal tissue sections stained with Hematoxylin and Eosin (HE) and Periodic Acid-Schiff (PAS) demonstrate the therapeutic effects of the antifungal agents TPY and TPZ on vaginitis (Figure 10D). The blank control group shows healthy tissue with intact epithelium and normal glycogen distribution. In contrast, the PBS group, representing untreated infected mice, exhibits inflammatory infiltration and a disrupted epithelial structure. Treatment with TPY reduces inflammation and partially reduces the inflammatory infiltration and restores glycogen content. TPZ treatment results in a reduction in inflammation and near-normal restoration glycogen structures. These results indicate that both TPY and TPZ are effective in treating vaginitis, with TPZ showing superior therapeutic efficacy. Finally, the inventors systematically investigated the in vivo toxicity of TPY and TPZ after intravenous injection. The levels of the important liver and kidney function biomarkers, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (UREA), and creatinine (CREA), are like those in the control group, indicating no obvious hepatotoxicity and nephrotoxicity. For hematological analysis, white blood cells (WBCs) and lymphocytes (Lyms) in TPY and TPZ-treated groups were normal in comparison with the control group. Subsequently, the inventors also carried out histopathological analysis for major organs, including heart, lung, spleen, liver, and kidney. The H&E staining images showed no obvious organ damage (Figure 10E). These findings collectively indicate that TPY and TPZ may efficiently eliminate fungi and alleviate the infection conditions in vivo with a high biocompatibility.

[0232] Conclusions

[0233] In this study, the inventors introduced a dual-targeting approach to combat C. albicans infection by disrupting the fungal membrane and inducing DNA damage. TPY and TPZ were designed and synthesized by enhancing the hydrophobicity and rigidity of a DNA- binding domain. These compounds selectively target fungal cells and exhibit robust fungicidal activity with minimal toxicity to mammalian cells. In a murine model of Candida albicans-induced vaginitis, both TPY and TPZ have significantly reduced the fungal loads and alleviated infection symptoms. Additionally, TPY and TPZ demonstrated excellent biocompatibility with no observed hepatotoxicity, nephrotoxicity, or organ damage in treated animals. These findings highlight the potential of dual-targeting strategies in developing antifungal agents that address the limitations of current therapies such as adverse side effects and the emergence of drug-resistant strains. Systematic studies revealed that both TPY and TPZ are promising candidates for the treatment of fungal infections, offering a valuable approach to overcoming antifungal resistance and improving treatment outcomes.

[0234] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0235] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0236] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0237] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A compound of Formula (la), or a solvate thereof:whereinR1and R2are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl;Rs is selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy;R4is selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore; and R5, R6, R7, and Re are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl.

2. The compound according to claim 1, wherein R1and R2are independently selected from optionally substituted alkyl .

3. The compound according to claim 1 or 2, wherein Rs is selected from H and optionally substituted alkyl.

4. The compound according to any one of claims 1 to 3, wherein R4is selected from optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted alkoxy.

5. The compound according to any one of claims 1 to 4, wherein R4is selected from:wheredenotes a bond to the phenyl ring; wherein R9, R10, R11, and R12are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy;R13and R14are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkoxy, and a fluorophore.

6. The compound according to claim 5, wherein R9, R10, R11, and R12are independently selected from H and optionally substituted alkyl.

7. The compound according to claim 5 or 6, wherein R13and R14are independently selected from H and optionally substituted alkyl.

8. The compound according to any one of claims 1 to 7, wherein R5, R6, R7, and Rs are independently selected from H and optionally substituted alkyl.

9. The compound according to any one of claims 1 to 8, wherein the compound of Formula (la) is selected from:

10. The compound according to any one of claims 1 to 9, wherein the compound is characterised by an antimicrobial activity.

11. The compound according to any one of claims 1 to 10, wherein the compound is characterised by an antibacterial activity against Gram-negative and / or Gram-positive bacterial cells.

12. The compound according to any one of claims 1 to 11, wherein the compound is characterised by an antibacterial activity against antibiotics resistant bacterial cells.

13. The compound according to any one of claims 1 to 12, wherein the compound is characterised by a minimum inhibitory concentration (MIC) against bacterial cells of about 1 μM to about 10 μM.

14. The compound according to any one of claims 1 to 13, wherein the compound is characterised by an antifungal activity against drug resistant fungal cells.

15. The compound according to any one of claims 1 to 14, wherein the compound of Formula (la) is characterised by a MIC against fungus of about 1 μM to about 10 μM.

16. The compound according to any one of claims 1 to 15, wherein the compound of Formula (la) is characterized by a minimum fungal concentration (MFC) of about 1 μM to about 10 μM.

17. A method of synthesising a compound of Formula (la), or a salt or solvate thereof, comprising: a) reacting compound of Formula (II) with a haloalkane:

18. The method according to claim 17, wherein a mole ratio of compound of Formula (II) to haloalkane is about 1: 1.5 to about 1:3.

19. The method according to claim 17 or 18, wherein the haloalkane is iodomethane.

20. The method according to any one of claims 17 to 19, wherein the method further comprises a step before step a) of reacting a compound of Formula (III) with a heterocyclic acid to form compound of Formula (II): wherein X is halo;wherein the reaction is conducted in the presence of a catalyst and a base.

21. The method according to claim 20, wherein a mole ratio of compound of Formula (II) to heterocyclic acid is about 2:3 to about 2:7.

22. The method according to claim 20 or 21, wherein the heterocyclic acid is pyridine-4-boronic acid.

23. The method according to any one of claims 17 to 22, wherein the method further comprises a step before step a) of reacting a compound of Formula (IV) with benzyl bromide to form compound of Formula (III):

24. The method according to claim 23, wherein a mole ratio of compound of Formula (IV) to benzyl bromide is about 1:0.5 to about 1:2.

25. The method according to claim 23 or 24, wherein the benzyl bromide is selected from:

26. A method of labelling and / or imaging bacterial cells and / or fungal cells, comprising contacting the bacterial cells and / or fungal cells with a compound of Formula (la), or a salt or solvate thereof.

27. A compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof for use in therapy.

28. A compound of Formula (la) or a salt or solvate thereof for use in the imaging and / or treatment of a microbial disease or disorder.

29. Use of compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the imaging and / or treatment of a microbial disease or disorder.

30. A method of imaging and / or treating a microbial disease or disorder in a subject thereof, comprising administering to the subject a therapeutically effective amount of compound of Formula (la) or pharmaceutically acceptable salt or solvate thereof.

31. The compound for use, use or method according to any one of claims 23 to 26, wherein the microbial disease or disorder has a resistance against drugs.

32. The compound for use, use or method according to any one of claims 23 to 27, wherein the microbial disease or disorder is selected from skin infection, a respiratory disease, food poisoning or any other life-threatening systemic disease, sepsis, urinary tract infection, meningitis, wound infection, tuberculosis, diarrhoea, Legionnaires' disease, meningococcal disease, Q fever, strep throat, whooping cough (pertussis), aspergillosis, blastomycosis, Candida auris, candidiasis, chromoblastomycosis, cryptococcosis, fungal eye infection, histoplasmosis, mucormycosis, mycetoma, paracoccidioidomycosis, pneumocystis pneumonia, ringworm and nail infection, sporotrichosis, talaromycosis, and valley fever.

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