Delocalized lipophilic cationic compounds and methods of use thereof

Abstract

The present invention provides a delocalized lipophilic cation (DLC) compound and methods of using such compounds. Also provided are pharmaceutical compositions comprising the DLC compounds. The methods provided include methods of killing cells by contacting the cells with a DLC compound of the present disclosure and methods of fluorescently labeling mitochondria. Also provided are methods of imaging mitochondria of a cell, methods of determining whether a patient has a mitochondria-related disease, and methods of treating a mitochondria-related disease in a patient. Also provided are kits comprising a compound of the present disclosure.

Description

[0001] Government rights

[0002] This invention was made with government funding granted by the U.S. Department of Energy under grant number DE-SC0008397. The government holds certain rights to this invention.

[0003] Cross-references to related applications

[0004] Pursuant to 35 U.S.SC §119(e), this application claims priority to U.S. Provisional Patent Application No. 62 / 792,703, filed January 15, 2019, which is incorporated herein by reference in its entirety. Background Technology

[0005] Cancer places a tremendous burden on society. In fact, there were approximately 18.1 million new cancer cases worldwide in 2018. Traditionally, cancer treatment involves chemical or biological compounds (chemotherapy), radiation (radiotherapy), or surgery. However, while these treatments are effective in some cases, they are ineffective in others. Furthermore, surgical removal of cancerous tissue sometimes results in inadequate margin removal, leading to cancer recurrence.

[0006] Delocalized lipophilic cations (DLCs) have been used as fluorescent dyes and have been investigated as potential chemotherapeutic agents for cancer treatment. Some DLCs have been shown to be cytotoxic to cancer cells and accumulate within the mitochondria of cancer cells. However, many DLCs exhibit low cytotoxicity to cancer cells, and their structure-activity relationships remain poorly understood. Summary of the Invention

[0007] This invention provides a delocalized lipophilic cationic (DLC) compound and a method of using this compound. Pharmaceutical compositions comprising the DLC compound are also provided. The provided methods include: a method for killing cells by contacting cells with the disclosed DLC compound and a method for fluorescently labeling mitochondria. Methods for imaging mitochondria in cells, determining whether a patient has mitochondrial-related diseases, and treating patients with mitochondrial-related diseases are also provided. Kits comprising the compounds of this disclosure are also provided. Brief description of the attached diagram

[0009] The invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. Each patent or application document contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and at the necessary cost. It should be emphasized that, by convention, the various features in the drawings are not drawn to scale. Instead, for clarity, the dimensions of the various features have been arbitrarily enlarged or reduced. The following figures are included in the drawings.

[0010] Figure 1 A schematic diagram of the synthesis of compounds (1) to (11) is shown.

[0011] Figure 2A The fluorescence absorption spectra of compounds (1) to (11) are shown.

[0012] Figure 2B The fluorescence emission spectra of compounds (1) to (11) are shown.

[0013] Figure 2C The fluorescence quantum yields of compounds (1) through (11) are shown.

[0014] Figure 2D The colocalization of compound (3) with mitochondrial dyes was shown.

[0015] Figure 3A Compound (3) was shown to be effective at low or high K. + Fluorescence in H838 cells pre-incubated at a certain concentration.

[0016] Figure 3B Compound (3) was shown to have been reacted with K + Fluorescence in H838 cells after pre-incubation following the addition of carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP).

[0017] Figure 3C Showing data from already low or high K + The average fluorescence density of compound (3) in cells pre-incubated at a given concentration.

[0018] Figure 3D Showing along Figure 3B The cross-sectional fluorescence intensity distribution diagram is shown by the white dashed line in the figure.

[0019] Figure 4 The cytotoxicity of compounds (1) to (11) on T24, H838 and 3T3 cells was demonstrated.

[0020] Figure 5A The cytotoxic IC50 of compound (3) in nine lung cancer cell lines was shown. 50 .

[0021] Figure 5B Representative in vivo fluorescence images of mice carrying human lung cancer HCC827 are shown 2 hours after injection (PI) of compound (3).

[0022] Figure 5C The effect of compound (3) on the growth of HCC827 tumors in nude mice (n=10 per group) was shown.

[0023] Figure 5D The effect of compound (3) (15 mg / kg) on ​​the nude weight of tumor-bearing nude mice was shown compared with the control group (PBS).

[0024] Figure 6 The effects of compounds (1) to (11) on water and penicillin-streptomycin were shown.

[0025] Figure 7 The fluorescent labeling of Escherichia coli with compound (3) is shown.

[0026] Figure 8 Fluorescent labeling of Arabidopsis thaliana roots was shown 1 hour after treatment with compound (6).

[0027] Figure 9 Fluorescent labeling of Arabidopsis roots was shown 20 hours after treatment with compound (6).

[0028] Figure 10 The experimental setup used to capture images of whole Arabidopsis thaliana plants is shown.

[0029] definition

[0030] The terms “treatment (noun),” “treatment (gerund),” “treatment (verb),” etc., are used herein to generally refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative, i.e., complete or partial prevention of aspects of the disease or its symptoms, and / or therapeutic, i.e., partial or complete stabilization or cure of aspects of the disease and / or adverse effects attributable to the disease. The term “treatment” includes any treatment of diseases in mammals (particularly humans) and includes: (a) preventing the occurrence of disease and / or symptoms in subjects who may be susceptible to the disease or symptoms but have not yet been diagnosed with it; (b) suppressing the disease and / or symptoms, i.e., preventing the development of the disease and / or related symptoms; or (c) alleviating the disease and related symptoms, even if the disease and / or symptoms subside. Subjects requiring treatment may include those who have already been harmed (e.g., those with cancer, such as those with a tumor) and those requiring prevention (e.g., those susceptible to cancer; those who have cancer; those suspected of having cancer; etc.).

[0031] The terms “recipient,” “individual,” “object,” “host,” and “patient” are used interchangeably herein to refer to any mammalian object, particularly a human, that requires diagnosis, treatment, or therapy. For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, domesticated and farm animals, as well as zoo, sporting, or pet animals such as dogs, horses, cats, cattle, sheep, goats, pigs, camels, etc. In some implementations, the mammal is a human.

[0032] The terms "co-administration" and "combination" encompass the simultaneous, concurrent, or sequential administration of two or more therapeutic agents without a specific time limit. In one embodiment, the agent is present in cells or simultaneously within the subject's body, or exerts its biological or therapeutic effects simultaneously. In one embodiment, the therapeutic agents are contained in the same composition or unit dosage form. In other embodiments, the therapeutic agents are contained in separate compositions or unit dosage forms. In some implementations, the first agent may be administered before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second therapeutic agent.

[0033] As used herein, the term "sample" refers to a material or mixture of materials containing one or more components of interest, typically, but not necessarily, in fluid form, i.e., an aqueous solution. Samples can be derived from a variety of sources, such as food, environmental materials, biological samples, or solids, such as tissues or body fluids isolated from an individual, including but not limited to: e.g., plasma, serum, cerebrospinal fluid, semen, lymph; external sections of skin, respiratory tract, intestine, and genitourinary tract; tears, saliva, breast milk, blood cells, tumors, organs, and samples of in vitro cell culture components (including but not limited to conditioned media produced by growing cells in cell culture media, presumed virus-infected cells, recombinant cells, and cell components). In some embodiments of the method, the sample comprises cells. In some cases of the method, the cells are in vitro. In some cases of the method, the cells are in vivo.

[0034] "Therapeutic effective amount," "therapeutic effective dose," or "therapeutic dose" is an amount sufficient to achieve the desired clinical outcome (i.e., to achieve a therapeutic effect, to achieve a desired therapeutic response, etc.). A therapeutic effective dose can be administered in the form of a single or multiple doses. For the purposes of this disclosure, a therapeutic effective dose of a composition is an amount sufficient, when administered to an individual, to reduce, improve, stabilize, reverse, prevent, slow, or delay the progression of a disease state (e.g., cancer, etc.) present in the subject.

[0035] Numerous general references, which provide useful information on known chemical synthetic schemes and conditions for synthesizing the compounds of this invention (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structures, 5th Edition, Wiley-Interscience, 2001; or Volgo, Practical Organic Chemistry Textbook, Including Qualitative Organic Analysis, 4th Edition, New York: Longman, 1978), are available.

[0036] When the compounds described herein contain one or more chiral centers and / or double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers, all possible enantiomers and stereoisomers of the compound (including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), and mixtures of enantiomers and stereoisomers) are included in the description of the compounds of the present invention. Mixtures of enantiomers and stereoisomers can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. The compounds may also exist in several tautomeric forms, including enol forms, ketone forms, and mixtures thereof. Therefore, the chemical structures described herein include all possible tautomeric forms of the compounds. The described compounds also include isotopically labeled compounds, wherein the atomic mass of one or more atoms differs from that commonly found in nature. Examples of isotopes that may be incorporated into the compounds disclosed herein include, but are not limited to, those listed below. 2 H, 3 H, 11 C 13 C 14 C 15 N、 18 O、 17 O, etc. The compound can exist in both non-solventized and solvated forms, including hydrated forms. Generally, the compound can be hydrated or solvated. Some compounds can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the purposes considered herein and fall within the scope of this invention.

[0037] As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group (i.e., a monoalkyl group) that typically, but not necessarily, contains 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, octyl, decyl, etc., and cycloalkyl, such as cyclopentyl, cyclohexyl, etc. Typically, and although not necessarily, the alkyl groups of the present invention may contain 1 to 18 carbon atoms, and such groups may contain 1 to 12 carbon atoms. The term "lower alkyl" means an alkyl group having 1 to 6 carbon atoms. "Substituted alkyl" means an alkyl group substituted with one or more substituents, including cases where two hydrogen atoms from the same carbon atom in an alkyl substituent are substituted, for example in a carbonyl group (i.e., substituted alkyl groups may include -C(=O)- groups). As described in further detail below, the terms "heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl substituents in which at least one carbon atom is substituted with a heteroatom. Unless otherwise specified, the terms "alkyl" and "lower alkyl" respectively include straight-chain, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkyl or lower alkyl groups.

[0038] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain have been optionally replaced by heteroatoms such as -O-, -N-, -S-, -S(O). n - (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) substituted, and having 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, aminoacyl, aminoacyloxy, oxamido, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NR a R b R' and R" can be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0039] As used herein, the term "alkenyl" refers to a straight-chain, branched, or cyclic hydrocarbon group comprising 2 to 24 carbon atoms containing at least one double bond, such as vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosene, 24-carbonenyl, etc. Typically, and although not strictly necessary, the alkenyl groups of the present invention may comprise 2 to 18 carbon atoms, for example, 2 to 12 carbon atoms. The term "lower alkenyl" means an alkenyl group having 2 to 6 carbon atoms. The term "substituted alkenyl" refers to an alkenyl group substituted by one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to alkenyl groups in which at least one carbon atom is substituted by a heteroatom. Unless otherwise specified, the terms "alkenyl" and "lower alkenyl" respectively include straight-chain, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl groups.

[0040] "Substituted alkylene" refers to a group having 1 to 3 hydrogen atoms substituted with substituents as described in the definition of "substituted" for carbon below.

[0041] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2 to 24 carbon atoms, such as ethynyl, n-propynyl, etc. Typically, and although not necessarily, the alkynyl group of the present invention may contain 2 to about 18 carbon atoms, and such a group may further contain 2 to 12 carbon atoms. The term "lower alkynyl" means an alkynyl group having 2 to 6 carbon atoms. The term "substituted alkynyl" means an alkynyl group substituted by one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroatom-containing alkynyl" mean an alkynyl group in which at least one carbon atom is substituted by a heteroatom. Unless otherwise specified, the terms "alkynyl" and "lower alkynyl" respectively include straight-chain, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl groups.

[0042] The terms “alkylaryl” or “aryl” refer to the groups -alkylene-aryl and -substituted alkylene-aryl, wherein the alkylene, substituted alkylene and aryl are as defined herein.

[0043] "Alkoxy" refers to the -O-alkyl group, where alkyl is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, etc. The term "alkoxy" also refers to the groups: alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and ynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and ynyl are as defined herein.

[0044] The term “substituted alkoxy” refers to a group such as substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein the substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0045] The term "halogenated alkyl" refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been replaced by a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0046] The term “alkylalkoxy” refers to the group: -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, wherein the alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0047] "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms and having at least 1, preferably 1 to 2, unsaturated double bond sites. This term includes, for example, divinyl, allyl, and but-3-en-1-yl. The term also includes cis and trans isomers or mixtures of these isomers.

[0048] The term "substituted alkenyl" refers to an alkenyl group as defined herein, having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxamido, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0049] "Acyl" refers to the following groups: HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, etc. The groups are alkyl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclic-C(O)-, and substituted heterocyclic-C(O)-, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, the acyl group includes the "acetyl" group CH3C(O)-.

[0050] "Acylamino" refers to -NR 20 C(O)alkyl, -NR 20 C(O) substituted alkyl groups, NR 20 C(O)cycloalkyl, -NR 20 C(O)-substituted cycloalkyl groups, NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O) alkenyl, NR 20 C(O)-substituted alkenyl, -NR 20 C(O) ynyl group, -NR 20 C(O)-substituted alkynyl group, -NR 20 C(O)aryl, -NR 20 C(O)-substituted aryl, -NR 20 C(O) heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O) heterocycle and -NR 20 C(O)-substituted heterocycles, where R 20 It is hydrogen or alkyl, and wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle are as defined herein.

[0051] "Amino carbonyl" or the term "amino acyl" refers to the group -C(O)NR. 21 R 22 , where R 21 and R 22Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, wherein R 21 and R 22 Optionally linked with nitrogen to form a heterocyclic or substituted heterocyclic group, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle are as defined herein.

[0052] "Amino carbonyl amino" refers to the group -NR 21 C(O)NR 22 R 23 , where R 21 R 22 and R 23 It is independently selected from hydrogen, alkyl, aryl or cycloalkyl, or two of the R groups are linked to form a heterocyclic group.

[0053] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, wherein the alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic groups are as defined herein.

[0054] The term “acyloxy” refers to a group such as alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O-, wherein the alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic groups are as defined herein.

[0055] As used herein, the term "aryl," unless otherwise stated, refers to an aromatic substituent that typically, and though not necessarily, comprises 5 to 30 carbon atoms and contains a single aromatic ring or multiple aromatic rings fused together, directly or indirectly linked (such that different aromatic rings are bonded to a common group, such as methylene or ethylene). An aryl group may, for example, comprise 5 to 20 carbon atoms, and as a further example, may comprise 5 to 12 carbon atoms. For example, an aryl group may comprise one aromatic ring, or two or more fused or linked aromatic rings (i.e., biaryl, aryl-substituted aryl, etc.). Examples include phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, etc. "Substituted aryl" refers to an aryl group substituted with one or more substituents, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom is substituted with a heteroatom, as will be described in more detail below. Aryl groups are intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated C3-C groups. 14 Groups, such as but not limited to phenyl, biphenyl, naphthyl, pyridyl, furanyl, thiophene, imidazolyl, pyrimidinyl, and oxazolyl; which may be further substituted by one to five members selected from the group consisting of: hydroxyl, C1-C8 alkoxy, C1-C8 branched or straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro, halogen, trifluoromethyl, cyano, and carboxyl (see, for example, Katritzky, Handbook of Heterocyclic Chemistry). Unless otherwise stated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0056] The term "aralkyl" refers to an alkyl group having an aryl substituent, while the term "alkylaryl" refers to an aryl group having an alkyl substituent, wherein "alkyl" and "aryl" are as defined above. Typically, aralkyl and alkylaryl groups as used herein contain 6 to 30 carbon atoms. Aralkyl and alkylaryl groups may, for example, contain 6 to 20 carbon atoms, and as a further example, these groups may contain 6 to 12 carbon atoms.

[0057] "Aryloxy" refers to the group: -O-aryl, wherein the aryl group as defined herein includes, for example, phenoxy, naphthoxy, etc., and also includes optionally substituted aryl groups as defined herein.

[0058] "Amino" refers to the group -NH2.

[0059] The term “substituted amino” refers to a group -NRR, wherein each R is independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic, provided that at least one R is not hydrogen.

[0060] The term "azido" or "azide" refers to the -N3 group.

[0061] "Carboxyl" or "carboxylate" refers to -CO2H or its salt.

[0062] The terms "carboxyl ester" or "carboxy ester," or "carboxyalkyl" or "carboxylalkyl," refer to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-ynyl, -C(O)O-substituted ynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, and -C(O)O-alkyl. -substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic and -C(O)O-substituted heterocyclic, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0063] "(Carboxylate)oxy" or "carbonate" refers to the following groups: -OC(O)O alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-ynyl, -OC(O)O-substituted ynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl The terms "alkyl", "substituted alkyl", "alkenyl", "substituted alkenyl", "alkynyl", "substituted alkynyl", "cycloalkyl", "substituted cycloalkyl", "cycloalkenyl", "substituted cycloalkenyl", "aryl", "substituted aryl", "heteroaryl", "substituted heterocyclic", "heterocyclic" and "substituted heterocyclic" are as defined herein.

[0064] "Cyano" or "nitrile" refers to the -CN group.

[0065] As used herein, “carbocycle” or “carbocyclic ring” is intended to refer to any stable monocyclic, bicyclic, or tricyclic ring having a defined number of carbon atoms, any of which may be saturated, unsaturated, or aromatic. For example, a C3-14 carbocycle is intended to refer to a monocyclic, bicyclic, or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, and tetrahydronaphthyl. Bridged rings are also included in the definition of carbocyclic rings, including, for example, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane, and [2.2.2]bicyclooctane. A bridged ring occurs when two non-adjacent carbon atoms in a ring are connected by a covalent bond or one or more carbon atoms. In one embodiment, the bridged ring is one or two carbon atoms. It should be noted that bridging always transforms a monocyclic ring into a bicyclic ring. When rings are bridged, substituents listed for the ring can also be present on the bridge. Fused rings (e.g., naphthyl and tetrahydronaphthyl) and spirocyclic rings are also included.

[0066] "Cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms and one or more rings, including fused rings, bridged rings, and spirocyclic systems. Suitable examples of cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl.

[0067] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxamidoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0068] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group with 3 to 10 carbon atoms, having a monocyclic or polycyclic structure and having at least one double bond, preferably 1 to 2 double bonds.

[0069] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxamidoacyl, azide, cyano, halogen, hydroxy, ketone, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0070] "Cycloynyl" refers to a non-aromatic cycloalkyl group with 5 to 10 carbon atoms, which has a monocyclic or polycyclic structure and at least one triple bond.

[0071] “Cycloalkoxy” refers to -O-cycloalkyl.

[0072] "Cycloalkenyl group" refers to -O-cycloalkenyl group.

[0073] "Halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0074] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0075] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, for example 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from oxygen, nitrogen, and sulfur within a ring. Such a heteroaryl group may have a monocyclic ring (e.g., pyridyl, imidazolyl, or furanyl) or multiple fused rings in a ring system (e.g., in groups such as indoindole, quinolinyl, benzofuran, benzimidazolyl, or benzothiophene), wherein at least one ring in the ring system is aromatic, provided that the connecting point is an atom through an aromatic ring. In some embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to form an N-oxide (N→O), sulfinyl, or sulfonyl group. The term includes, for example, pyridyl, pyrroleyl, indoyl, thiophene, and furanyl. Unless limited by the definition of a heteroaryl substituent, such heteroaryl group may optionally be substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxyl, mercapto, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amide, alkylaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, aminoacyloxy, oxyamide, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl and trihalomethyl.

[0076] The term "heteroaryl" refers to the group: -alkylene-heteroaryl, wherein the alkylene and heteroaryl groups are defined herein. This term includes, for example, pyridinemethyl, pyridineethyl, indolemethyl, etc.

[0077] "Heteroaryl group" refers to -O-heteroaryl group.

[0078] "Heterocyclic," "heterocyclic," "heterocyclic alkyl," and "heterocyclic group" refer to a saturated or unsaturated group having a monocyclic or multiple fused rings, including fused, bridged, and spirocyclic ring systems, and having 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, wherein in fused ring systems, one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the connection point is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to yield an N-oxide, -S(O)-, or -SO2- group.

[0079] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azahexacyclic butanes, pyrroles, imidazoles, pyrazoles, pyridines, pyrazines, pyrimidines, pyridazines, indoleazines, isoindole, indole, dihydroindole, indazoles, purines, quinazines, isoquinoline, quinoline, phthalazines, naphthylpyridine, quinoxaline, quinazoline, cinnamic acid, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroxazine, isothiazine, phenoxazine, imidazoline, imidazoline, and piperidine. Piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazoline, thiophene, benzo[b]thiophene, morpholino, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0080] Unless limited by the definition of a heterocyclic substituent, such a heterocyclic group may optionally be substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxamido, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl and fused heterocycles.

[0081] "Nitro" refers to the group -NO2.

[0082] "O" refers to an atom (=O).

[0083] "Sulfonyl" refers to the following groups: SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl groups include, for example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0084] "Thiol group" refers to the -SH group.

[0085] The term "thio" or "thionyl group" refers to the atom (=S).

[0086] "alkylthio" or the term "thioalkoxy" refers to the group: -S-alkyl, wherein the alkyl group is as defined herein. In some embodiments, sulfur may be oxidized to -S(O)-. Sulfoxides may be present in one or more stereoisomers.

[0087] The term "substituted thioalkoxy" refers to a group: -S-substituted alkyl.

[0088] The term “thioaryloxy” refers to the group: aryl-S-, wherein the aryl group is as defined herein and includes optionally substituted aryl groups, which are also defined herein.

[0089] In addition to what is disclosed herein, the term “substituted” when used to modify a particular group or radical can also refer to one or more hydrogen atoms of a particular group or radical being independently substituted by the same or different substituents as defined below.

[0090] Except for groups disclosed in particular terms herein, unless otherwise specified, the group used to replace one or more hydrogen atoms on a saturated carbon atom in a designated group or radical (any two hydrogen atoms on a carbon atom can be represented by =O, =NR) 70 =N-OR 70 The substituent group for (e.g., =N2 or =S substitution) is: -R 60 Halogen, =O, -OR 70 -SR 70 -NR 80 R 80 Trihalomethyl, -CN, -OCN, -SON, -NO, -NO2, =N2, -N3, -SO2R 70 -SO2O - M + -SO2OR 70 -OSO2R 70 -OSO2O - M + -OSO2OR 70 -P(O)(O) - )2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R70 -C(O)O - M + -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OC(O)O - M + -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 70 -NR 70 CO2 - M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 Selected from the group consisting of: optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, and heteroarylalkyl; each R 70 Independently hydrogen or R 60 ; Each R 80 Independently for R 70 Alternatively, two R 80 Together with the nitrogen atom bonded thereto, they form a 5-, 6-, or 7-membered heterocyclic alkyl group, which may optionally include 1 to 4 identical or different additional heteroatoms selected from O, N, and S, wherein N may be substituted with -H or C1-C3 alkyl groups; and each M + It is a counterion with a net single positive charge. Each M + It can be independently, for example, a base ion, such as K+. + Na + Li + Ammonium ions, for example+ N(R 60 )4; or alkaline earth ions, such as [Ca 2 + ] 0.5 、[Mg 2+ ] 0.5 or[Ba 2+ ] 0.5 ("The subscript 0.5 indicates that one of the counter ions of this divalent alkaline earth ion can be the ionized form of the compound of the present invention, and the other is a typical counter ion such as a chloride ion, or two ionized compounds disclosed herein can serve as counter ions of this divalent alkaline earth ion, or the double-ionized compound of the present invention can serve as a counter ion of this divalent alkaline earth metal ion.") In a specific example, -NR 80 R 80 The intended group includes -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl and N-morpholinyl.

[0091] In addition to the disclosure of this invention, unless otherwise stated, the substituents for hydrogen on the unsaturated carbon atom in the "substituted" olefin, alkyne, aryl, and heteroaryl groups are: -R 60 halogen, -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80 Trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 -SO3 - M + -SO3R 70 -OSO2R 70 -OSO3 - M + -OSO3R 70 -PO3 -2 (M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -CO2 - M + -CO2 - R 70-C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OCO2 - M + -OCO2R 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 70 -NR 70 CO2 - M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As defined above, the prerequisite is that, in the case of substituted alkenes or alkynes, the substituent is not -O. - M + -OR 70 -SR 70 、or -S - M + .

[0092] Except for the groups disclosed with respect to individual terms herein, unless otherwise stated, the substituents for hydrogen on the nitrogen atom in “substituted” heteroalkyl and cycloalkyl groups are: -R 60 -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80Trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 -S(O)2O - M + -S(O)2OR 70 -OS(O)2R 70 -OS(O)2O - M + -OS(O)2OR 70 -P(O)(O) - )2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 (OR) 70 -C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 70 -NR 70 C(O)OR 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As defined above.

[0093] In addition to the disclosure of this invention, in one embodiment, the substituted group has 1, 2, 3 or 4 substituents, 1, 2 or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0094] It should be understood that, among all the substituents defined above, polymeric forms obtained by defining substituents that have further substituents on themselves (e.g., a substituted aryl group having a substituted aryl group as a substituent that is itself substituted by a substituted aryl group, which can be further substituted by a substituted aryl group, etc.) are not intended to be included herein. In this case, the maximum number of such substitutions is three. For example, the successive substitutions of substituted aryl groups specifically considered herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0095] Unless otherwise stated, the nomenclature of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group and then moving towards the adjacent functional group at the junction point. For example, the substituent “aranekoxycarbonyl” refers to the group: (aryl)-(alkyl)-OC(O)-.

[0096] Regarding any group disclosed herein containing one or more substituents, it is understood that such group does not contain any sterically unrealizable and / or synthetically infeasible substitution or substitution pattern. Furthermore, the compounds of this invention include all stereochemical isomers resulting from the substitution of these compounds.

[0097] The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient (e.g., a mammal) (a salt containing an anti-ion that has acceptable mammalian safety for a given dosage regimen). Such a salt can be derived from pharmaceutically acceptable inorganic or organic bases, as well as from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound derived from a variety of organic and inorganic anti-ions well known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and, when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochlorides, hydrobromic acids, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc.

[0098] "Pharmaceutical effective amount" and "therapeutic effective amount" refer to the amount of a compound sufficient to produce the desired therapeutic effect (e.g., treating one or more of a particular condition or disease or its symptoms, and / or preventing the occurrence of a disease or condition). In the context of polyglutamine diseases, a pharmaceutically effective or therapeutically effective amount includes, among other substances, an amount sufficient to prevent or result in a reduction of protein deposits in the subject's brain.

[0099] The term "its salt" refers to a compound formed when the proton of an acid is replaced by a cation (e.g., a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds not intended for administration to patients. For example, salts of the compounds of the present invention comprise compounds in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of said inorganic or organic acid serves as the anionic component of the salt.

[0100] A solvate is a complex formed by the combination of solvent molecules and solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0101] "Stereoisomers" and "stereoisomers (complex)" refer to compounds that have the same atomic connections but different spatial arrangements of atoms. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0102] "Tautomers" refer to alternative forms of molecules that differ only in the electronic bonding and / or proton positions of atoms, such as enol-ketone and imine-enamine tautomers, or tautomers of heteroaryl groups containing a -N=C(H)-NH- ring arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetraazoles. Those skilled in the art will recognize that other tautomer arrangements of ring atoms are possible.

[0103] The term "functional group" refers to a chemical group, such as halogen, hydroxyl, mercapto, C1-C24 alkoxy, C2-C24 alkenoxy, C2-C24 alkynoxy, C5-C20 aryloxy, acyl (including C2-C24 alkyl carbonyl (-CO-alkyl) and C6-C20 aryl carbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxy carbonyl (-(CO)-O-alkyl), C6-C20 aryloxy carbonyl (-(CO)-O-aryl), halocarbonyl (-(CO)-X, where X is a halogen), C2-C24 alkyl carbonate (-O-( C6-C20 aryl carbonate (-O-(CO)-O-aryl), carboxyl (-COOH), carboxylic acid ester (-COO-), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkyl carbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkyl carbamoyl (-(CO)-N(C1-C24 alkyl)2), monosubstituted aryl carbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), urea (-NH-(CO)-NH2) ), cyano (-C≡N), isocyano (-N+≡C-), cyanoxy (-OC≡N), isocyanoxy (-O-N+≡C-), isothiocyano (-SC≡N), azide (-N=N+=N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino groups, mono- and di-(C5-C20 aryl)-substituted amino groups, C2-C24 alkylamides (-NH-(CO)-alkyl), C5-C20 arylamides (-NH-(CO)-aryl), imine alkyl (-CR=NH, where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkylaryl, C6-C20 arylalkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkylaryl, etc.), arylimino (-CR=N(aryl)), where R=hydrogen, alkyl, aryl, alkylaryl, etc.), nitro (-NO2), nitroso (-NO), sulfonamide (-SO2-OH), sulfonate (-SO2-O-), C1-C24 alkylthio (-S-alkyl; also called "alkylthio"), arylthio (-S-aryl;Also known as "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphonocarboxyl (-P(O)(OH)2), phosphonocarboxyl ester (-P(O)(O-)2), hypophosphonate (-P(O)(O-)), dioxophosphoryl (-PO2) and phosphonyl (-PH2), mono- and di-(C1-C24 alkyl)-substituted phosphonyl, mono- and di-(C5-C20 aryl)-substituted phosphonyl. Furthermore, if specific groups permit, the above functional groups can be further substituted by one or more additional functional groups or one or more hydrocarbon groups (such as those specifically listed above).

[0104] In the context of terms like "linking group" and "linking moiety," "linker" refers to the linking moiety that connects two groups via covalent bonds. The linker can be linear, branched, cyclic, or monatomic. Examples of such linking groups include alkyl, alkenyl, ynyl, aryl, alkylenearyl, and arylalkylene groups, and linking moiety containing functional groups, including but not limited to: amide (-NH-CO-), urein (-NH-CO-NH-), imide (-CO-NH-CO-), epoxy (-O-), cyclic sulfur (-S-), cyclic dioxy (-OO-), cyclic disulfide (-SS-), carbonyl dioxy (-O-CO-O-), and alkyl dioxy (-O-(CH2)). n-O-), epoxy imino (-O-NH-), cycloimino (-NH-), carbonyl (-CO-), etc. In some cases, one, two, three, four, or five or more carbon atoms in the backbone of the linking group may optionally be substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and typically no more than one, two, or three unsaturated bonds exist in the backbone of the linking group. The linking group may include one or more substituents, such as alkyl, aryl, or alkenyl. The linking group may include, but is not limited to: poly(ethylene glycol) units (e.g., -(CH2-CH2-O)-); ethers; thioethers; amines; alkyl groups (e.g., (C1-C12)alkyl), which may be straight-chain or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linking group backbone may include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein two or more atoms (e.g., 2, 3, or 4 atoms) of the cyclic group are included in the backbone. The linking group may be cleavable or incleavable. The linking group can be attached using any convenient orientation and / or connection method.

[0105] As used herein, the terms “terminal group” or “terminal group” are used interchangeably and refer to a group located at the end of an endosomal disruptor (e.g., as described herein). Terminal groups of interest include, but are not limited to, terminal capping groups such as H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the terminal group may be defined as a chemoselective group (e.g., as described herein).

[0106] As used herein, the term "hydrophilic group" itself refers to a monovalent or polyvalent group comprising a hydrophilic group and an optional linking group. In some cases, the hydrophilic group is attached to a hydrophilic moiety and a hydrophilic tail group of the target endosome disruptor. The hydrophilic moiety is a group that is well solubilized in an aqueous environment, for example, under reversed-phase (RP) chromatographic conditions, such that the water solubility of the group to which it is attached or bound (e.g., the linking group) increases. In some cases, the hydrophilic group is referred to as a hydrophilic functional group. In some cases, the hydrophilic group is heterocyclic. In some cases, the hydrophilic group is heteroaryl. In some cases, the hydrophilic group is charged (e.g., ionic). In some cases, the hydrophilic group is polar and neutral (e.g., nonionic). It should be understood that certain functional groups may exist in ionic or nonionic forms depending on surrounding conditions, such as solvent, pH, etc., and all such forms of the hydrophilic groups described herein are intended to be included within the scope of this disclosure. For example, the hydrophilic group may be a basic group that is neutral before protonation (e.g., under aqueous conditions with a suitable pH), or the hydrophilic group may be an acidic group that is neutral before deprotonation (e.g., under aqueous conditions with a suitable pH).

[0107] Compared to a control group lacking hydrophilic groups, hydrophilic groups can increase the solubility of the groups they are attached to in predominantly aqueous solutions. Hydrophilic groups differ from hydrophobic groups, which do not solubilize well in aqueous environments. In some cases, hydrophilic groups include at least one neutral polar functional group per 5 carbons, or at least one charged functional group per 7 carbons. In some cases, the solubility of hydrophilic groups (e.g., hydrophilic groups in discrete molecule form) in water is at least 1% by weight.

[0108] The hydrophilic groups and moieties of interest include, but are not limited to: nitrogen-containing heterocycles, amides, carbamates, carboxylic acids, carboxylic esters, methyl ethers, cyano groups, amines, sulfonamides, sulfonates, urea, thiourea, sulfonic acids, carboxylates, phosphonates, phosphates, sulfates, sulfinates, sulfonates, sulfonium, polyethylene glycol (PEG) and modified PEG, hydroxyl groups, ammonium groups, guanidines, pyridines, polyamines and sulfonates, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, phosphonate groups, hypophosphonate groups, ascorbate groups, glycols, including polyethers, -COOM', -SO3M', -PO3M', -NR 3+ Y', (CH2CH2O) p R and mixtures thereof, wherein Y' can be any halogen, sulfate, sulfonate or oxyanion, p can be 1 to 500, each R can be independently H or alkyl (e.g. methyl), M' can be a cationic counterion or hydrogen, -(CH2CH2O). yyCH2CH2XR yy --(CH2CH2O) yy CH2CH2X-, -X(CH2CH2O) yy CH2CH2-, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR. zz R zz and R yy Independently selected from H and C 1-3 Alkyl group. In some cases, the hydrophilic group is (CH2). x (OCH2CH2) y OCH3, where each x is an independent integer from 0 to 20, and each y is an independent integer from 0 to 50.

[0109] Interesting nitrogen-containing heterocycles that have been identified as usable as hydrophilic groups include, but are not limited to: azacyclic butane, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indoleazine, isoindole, indole, dihydroindole, indazole, purine, quinazine, isoquinoline, quinoline and their substituted forms.

[0110] As used herein, the term "PEG" refers to polyethylene glycol or modified polyethylene glycol. Modified polyethylene glycol polymers include methoxy polyethylene glycol and unsubstituted polymers or polymers substituted at one end with an alkyl, substituted alkyl, or functional group (e.g., as described herein). Any convenient linking group may be used at the end of the PEG to connect it to a group of interest, including but not limited to: alkyl, aryl, hydroxyl, amino, acyl, acyloxy, carboxyl ester, and amide terminal groups and / or substituents.

[0111] It should be understood that the term "or its salt or its solvate or its stereoisomer" is intended to include all forms of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the compounds of the present invention.

[0112] In some embodiments, substituents may contribute to the optical and / or stereoisomerism of the compound. Salt, solvate, hydrate, and prodrug forms of the compound are also of interest. This disclosure includes all such forms. Therefore, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomers, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers. In some embodiments, the compound may be metabolized into a pharmaceutically active derivative.

[0113] Unless otherwise stated, references to an atom are intended to include isotopes of that atom. For example, references to H are intended to include... 1 H, 2 H (i.e., D) and 3 H (i.e. T), mentioning C is intended to include12 All isotopes of C and carbon (e.g.) 13 C).

[0114] Definitions of other terms and concepts appear in the following specific implementation. Detailed Implementation

[0115] A delocalized lipophilic cationic (DLC) compound and a method of using this compound are provided. Pharmaceutical compositions comprising the DLC compound are also provided. The provided methods include methods for killing cells by contacting cells with the disclosed DLC compound and methods for fluorescently labeling mitochondria therefrom. Methods for imaging cellular mitochondria, determining whether a patient has mitochondrial-related diseases, and treating patients with mitochondrial-related diseases are also provided. Kits comprising the compounds of this disclosure are also provided.

[0116] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as these embodiments can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0117] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit, unless the context clearly indicates otherwise, as well as any other stated or intermediate value within the stated range, is included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also within the scope of this invention, subject to any express exclusions within the stated range. When a stated range includes one or both limits, the range excluding any one or both of those included limits is also included in the scope of this invention.

[0118] Certain ranges in this document are presented with the term "approximately" preceding the numerical value. The term "approximately" is used here to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximate to that number. In determining whether a number is close to or approximates a specifically listed number, the close to or approximate listed number can be a number that, in the context in which it appears, provides a substantially equivalent nature to the specifically listed number.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, representative exemplary methods and materials are described hereafter.

[0120] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually designated as incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to these referenced publications. References to any publication refer to its publication prior to the filing date and should not be construed as an admission that the present invention does not claim any rights based on a prior invention as presented in that publication. Furthermore, the date of the publication provided may differ from the actual publication date, which may require separate verification.

[0121] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural indicators. It should also be noted that claims may be drafted to exclude any optional elements. Similarly, this statement is intended as a priori expression in order to use such exclusive terms related to the formulation of claim elements, such as “solely,” “merely,” etc., or to use the limiting term “negative.”

[0122] Upon reading this disclosure, those skilled in the art will recognize that each individual embodiment described and illustrated herein has its own components and features, which may be separated from or combined with features of any other embodiment without departing from the scope and spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events or in any other logically practicable order.

[0123] Although the apparatus and method have been or will be described for grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly formulated in accordance with 35 U.SC §112, the claims should not be construed as necessarily being subject to any limitation on the construction of “means” or “steps”, but should be given the meaning and full scope of the definition provided by the claims under the judicial principle of equivalence, and where the claims are expressly formulated in accordance with 35 U.SC §112, they should include the full legal equivalent form in accordance with 35 U.SC §112.

[0124] As summarized above, this disclosure provides compounds comprising delocalized lipophilic cationic compounds. Such delocalized lipophilic cationic compounds can be used in a variety of different fields, including, but not limited to, killing cells by contacting the compound, fluorescently labeling cells or subparts thereof by contacting the compound, and treating a condition of a subject by administering the compound or a pharmaceutical composition comprising the compound.

[0125] In some cases, the compounds of this disclosure selectively accumulate in the mitochondria of cancer cells. "Selective accumulation in the mitochondria of cancer cells" generally means that the compounds accumulate to a greater extent in the mitochondria of cancer cells compared to the mitochondria of normal (i.e., non-cancerous) cells. Without being bound by theory, DLC can partially or completely dissipate the proton gradient across the inner mitochondrial membrane and, in some cases, cause cancer cell necrosis, induce cancer cell apoptosis, reduce cancer cell growth rate, reduce cancer cell division, or a combination thereof.

[0126] The present invention also provides pharmaceutical compositions comprising the compounds of the present invention, wherein the compounds disclosed herein can be formulated with pharmaceutically acceptable excipients. Formulations can be obtained in unit doses, wherein said dose provides an amount of compound that effectively achieves the desired result. The desired result may vary and may include, but is not limited to, partially or completely dissipating the proton gradient across the internal mitochondrial membrane, inducing cancer cell necrosis, inducing cancer cell apoptosis, reducing cancer cell growth rate, reducing cancer cell division rate, or combinations thereof.

[0127] As described above, these compounds and methods can be used in a variety of applications. Non-limiting examples of such applications include the partial or complete dissipation of proton gradients across the internal mitochondrial membrane, cancer treatment, or both. These compounds can also be used in other applications, such as, but not limited to, photodynamic therapy, photothermal therapy, optical imaging, fluorescence image-guided surgery, etc.

[0128] Composition

[0129] This disclosure provides delocalized lipophilic cationic compounds. The lipophilic cationic compounds of this disclosure comprise an aryl group, a linking group, and a positively charged third group, wherein the delocalized lipophilic cationic compound is a π-conjugated system. The aryl group is linked to the third group via the linking group. As used herein, the term "π-conjugated system" refers to a delocalized lipophilic cationic compound having π-conjugation, comprising an aryl group, a linking group, and a positively charged third group. In some cases, the aryl group may be a substituted aryl, a heteroaryl, or a substituted heteroaryl.

[0130] In some cases, the delocalized lipophilic cationic compound is a compound of formula (I):

[0131] ALZ +

[0132] (I)

[0133] in:

[0134] A is selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl;

[0135] L is a linking group;

[0136] Z + It is a positively charged group.

[0137] The compound described therein is a π-conjugated system.

[0138] Group A

[0139] In some embodiments of formula (I), group A has a structure (A1):

[0140]

[0141] in:

[0142] X is selected from CR 8 and N;

[0143] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0144] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0145] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms attached to it together form a fused ring selected from aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0146] In some embodiments of formula (I), group A is indole or a substituted indole. In some cases, group A has the structure (A2):

[0147]

[0148] in:

[0149] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0150] R 2 R 3 and R 4Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0151] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms attached to it together form a fused ring selected from aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0152] In some cases, group A has a structure (A3):

[0153]

[0154] in:

[0155] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0156] R 2 R 4 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano.

[0157] In some cases where group A has the structure A3, R 1 It is H. In some cases, R 1 It is an alkyl group, such as a methyl group. In some cases, R 2 It is H. In some cases, R 2 It is an alkyl group, such as a methyl group. In some cases, R 6 It is H. In some cases, R 4 It is an alkyl group, such as a methyl group. In some cases, R 4 It is a halogen, such as bromine. In some cases, R 4 It's a nitro group. In some cases, R... 4 It is a nitrile group. In some cases, R 6 It is H. In some cases, R 6 It is an alkyl group, such as methyl.

[0158] In some cases where group A has the structure A3, R 3 It is an electron-withdrawing group. In some cases, R 4 It is an electron-donating group.

[0159] In some embodiments of formula (I), group A has the structure (A4):

[0160]

[0161] in:

[0162] X is selected from CR 8 and N;

[0163] R 1 and R 8 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0164] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano.

[0165] In some cases where group A has the structure A4, X is CH. In some cases, R... 1 R 2 R 3 and R 4 Both are H.

[0166] Linking group L

[0167] In some cases, group L is selected from alkenyl, alkyneyl, aryl, alkyleneyl, and arylenealkyl. In some cases, group L has the structure (L1):

[0168]

[0169] in:

[0170] j is an integer between 0 and 10, and

[0171] Wavy lines (i.e.) ) indicates that a carbon-carbon double bond can be any combination of cis or trans.

[0172] In some cases where group L has structure L1, j is 0, 1, 2, 3, 4, or 5. In some cases, j is 0. In some cases, j is 0 and the carbon-carbon double bond is trans (i.e., group L has structure (L2)):

[0173]

[0174] Group Z +

[0175] In some implementations, Z + Selected from positively charged heteroaryl groups or substituted heteroaryl groups. In some cases, Z + It is pyridinium or a substituted pyridinium. In some cases, Z + It has a structure (Z1):

[0176]

[0177] in:

[0178] R 11 R 12 R 13 R 15 and R 16 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0179] In Z + In some cases with structure Z1, R 11 It is an alkyl group, such as a methyl group. In some cases, R 11 R 12 R 13 R 15 and R 16 Both are H. In some cases, R 11 It is a methyl group, and R 12 R 13 R 14 and R 15 All are H (i.e. Z) + Having a structure (Z4):

[0180]

[0181] In some cases, Z + It is pyridinium or a substituted pyridinium. In some cases, Z + It has a structure (Z3):

[0182]

[0183] in:

[0184] R 11 R 12 R 13 R 15 and R 16 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0185] In some cases, Z +It is pyridinium or a substituted pyridinium. In some cases, Z + It has a structure (Z4):

[0186]

[0187] in:

[0188] R 11 R 12 R 13 R 15 and R 16 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0189] Delocalized lipophilic cationic compounds

[0190] In some embodiments of formula (I), the delocalized lipophilic cationic compound has formula (II):

[0191]

[0192] in:

[0193] X - It is a counter ion;

[0194] L is a linking group;

[0195] Z + It is a group containing a positive charge;

[0196] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0197] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0198] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms attached to it together form a fused ring selected from aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0199] In some embodiments of formula (II), L has a structure selected from L1 and L2. In some embodiments of formula (II), Z + It has a structure selected from Z1, Z2, Z3, and Z4. In some embodiments of formula (II), L has a structure selected from L1 and L2, and Z... + It has a structure selected from Z1, Z2, Z3 and Z4.

[0200] In some embodiments of formula (I) or (II), the delocalized lipophilic cationic compound has formula (III):

[0201]

[0202] in:

[0203] X - It is a counter ion;

[0204] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0205] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0206] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms bonded to it together form a fused ring selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

[0207] R 11 It is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0208] In some embodiments of formula (III), R 1 R 2 R 3 R 4 R 5 and R 6 At least one of them is a substituent other than hydrogen. In some embodiments of formula (II), X - It is an iodide ion.

[0209] In some embodiments of formulas (I) to (III), the delocalized lipophilic cationic compound has formula (IV):

[0210]

[0211]

[0212] in:

[0213] X - It is a counter ion;

[0214] R 1 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0215] R 2 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0216] R 6 Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms bonded to it together form a fused ring selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

[0217] R 11 It is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0218] In some embodiments of formula (IV), R 1 R 2 R 4 and R 6 At least one of them is a substituent other than hydrogen. In some embodiments of formula (III), X - It is an iodide.

[0219] In some embodiments of formulas (I) to (IV), the delocalized lipophilic cationic compound is a derivative of F16, wherein the F16 has the structure (F16):

[0220]

[0221] In some embodiments of formulas (I) to (III), the delocalized lipophilic cationic compound has a structure selected from the following:

[0222]

[0223]

[0224] As used herein, compound (3) may also be referred to interchangeably as 5BMF.

[0225] In some embodiments of formulas (I) to (IV), the delocalized lipophilic cationic compound has the following structure:

[0226]

[0227] In some cases, the delocalized lipophilic cationic compounds exhibit cytotoxicity. In some cases, the compounds exhibit selective cytotoxicity against cancer cells, meaning they are more cytotoxic to cancer cells than compounds that are cytotoxic to normal cells. In some cases, the selective cytotoxicity factor of the compounds is 2 or higher, for example, 3 or higher, 4 or higher, 5 or higher, 10 or higher, 20 or higher, 50 or higher, or 100 or higher.

[0228] In some cases, the compound exhibits a half-maximal inhibitory concentration (IC50) for cytotoxicity against cancer cells. 50 The concentration is 100 μM or less, for example, 50 μM or less, 25 μM or less, 10 μM or less, 5 μM or less, 1 μM or less, 500 nM or less, 250 nM or less, or 100 nM or less. In some cases, the cytotoxicity IC50 of said compound against cancer cells is [not specified]. 50 Greater than the cytotoxic IC50 of normal cells 50 In some cases, the cytotoxic IC50 of cancer cells... 50 cytotoxic IC50 with normal cells 50 The ratio is 2 or higher, such as 3 or higher, 4 or higher, 5 or higher, 10 or higher, 20 or higher, 50 or higher, or 100 or higher.

[0229] In some cases, the compound has a fluorescence absorption peak between 350 nm and 500 nm, for example, between 375 nm and 475 nm, between 400 nm and 450 nm, or between 400 nm and 425 nm. In some cases, the compound has a fluorescence emission peak between 475 nm and 575 nm, for example, between 490 nm and 550 nm, or between 500 nm and 525 nm.

[0230] In some cases, the compound has a fluorescence quantum yield of 5% or higher relative to a reference standard. Suitable reference standards may vary and may include a variety of fluorescent molecules, including but not limited to, for example, fluorescein, rhodamine, N,N,N',N'-tetramethylacridin-3,6-diamine (acridin orange), allophycocyanin, chlorophyll, malondiiodide, 4',6-diamidinyl-2-phenylindole (DAPI), Hoechst 33342, R-phycoerythrin, etc. In some cases, the compound has a fluorescence quantum yield of 5% or higher relative to a rhodamine 6G reference, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, or 40% or more. In some cases, the compound has a fluorescence quantum yield of 10% or higher relative to a rhodamine 6G reference. In some cases, the compound has a fluorescence quantum yield of 10% or higher relative to a rhodamine 6G reference in ethanol solvent.

[0231] In some cases, the compound accumulates in the mitochondria of cells. In some cases, the compound selectively accumulates in the mitochondria of cancer cells. Selective accumulation of the compound can refer to a greater accumulation of the compound in the mitochondria of cancer cells than in the mitochondria of normal cells. In some cases, the factor for selective accumulation of the compound in the mitochondria of cancer cells is 2 or higher, such as 3 or higher, 4 or higher, 5 or higher, 10 or higher, 20 or higher, 50 or higher, or 100 or higher.

[0232] In some cases, the compounds can induce apoptosis in cancer cells. In some cases, the compounds can induce necrosis in cancer cells. In some cases, the apoptosis and / or necrosis induced by the compounds of this disclosure can be selective for cancer cells, i.e., the apoptosis and / or necrosis induced by the compounds of this disclosure is more prevalent in cancer cells than in normal (i.e., non-cancerous) cells. In some cases, the compounds can reduce the growth rate of tumors. In some cases, the compounds can reduce the size of tumors.

[0233] Pharmaceutical Composition

[0234] Pharmaceutical compositions comprising the compounds of the present invention (i.e., delocalized lipophilic cationic compounds or combinations thereof) may be administered to patients alone or in combination with other complementary active agents. The pharmaceutical compositions may be manufactured using any of a variety of processes, including but not limited to: conventional mixing, dissolving, granulation, pill-coating, suspension, emulsification, encapsulation, embedding, and lyophilization. The pharmaceutical compositions may take any of a variety of forms, including but not limited to: sterile solutions, suspensions, emulsions, lyophilized products, tablets, pills, granules, capsules, powders, syrups, elixirs, or any other suitable dosage form for administration.

[0235] The compounds of the present invention can be administered to the host in any convenient manner that can desiredly alleviate the symptoms or signs of the disease. Therefore, the compounds of the present invention can be incorporated into a variety of formulations for therapeutic administration. More specifically, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with suitable pharmaceutically acceptable carriers or diluents, and can be formulated into formulations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, creams, solutions, suppositories, injections, inhalers, and aerosols.

[0236] Formulations of pharmaceutical compositions are well known in the art. For example, E.W. Martin's *Remington's Pharmaceutical Sciences* (Mack Publishing Co., Easton, PA, 19th edition, 1995) describes exemplary formulations (and their components) suitable for drug delivery of the disclosed compounds. Pharmaceutical compositions comprising at least one compound of the present invention can be formulated for use as human or veterinary medicine. Specific formulations of the disclosed pharmaceutical compositions may depend on, for example, the route of administration and / or the site of the infection to be treated. In some embodiments, the formulation includes, in addition to at least one active ingredient (such as a compound of the present invention), a pharmaceutically acceptable carrier. In other embodiments, other drugs or agents having similar, related, or complementary effects on the disease being treated may also be included as active ingredients in the pharmaceutical composition.

[0237] Pharmaceutically acceptable carriers that can be used in the disclosed methods and compositions are conventional in the art. The nature of the drug carrier depends on the specific route of administration employed. For example, parenteral preparations typically include injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc., as carriers. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional nontoxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may optionally contain small amounts of nontoxic excipients (e.g., excipients), such as wetting agents or emulsifiers, preservatives, and pH buffers; for example, sodium acetate or sorbitol monolaurate. Other non-limiting excipients include nonionic solubilizers, such as levofloxacin, or proteins (e.g., human serum albumin or plasma preparations).

[0238] Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter, suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; (10) diols (11) Polyols, such as propylene glycol; (12) Esters, such as ethyl oleate, ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide, aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; and (22) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0239] The disclosed pharmaceutical compositions can be formulated into pharmaceutically acceptable salts of the disclosed compounds. Pharmaceutically acceptable salts are non-toxic salts of the compounds in their free base form, possessing the desired pharmacological activity of the free base. These salts can be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids include hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids include acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc. Other suitable pharmaceutically acceptable salts are listed in E.W. Martin's *Remington's Pharmaceutical Sciences* (Mack Publishing Co., Easton, PA, 19th edition, 1995). Pharmaceutically acceptable salts can also be used to adjust the osmotic pressure of compositions.

[0240] The compounds of this invention can be used alone or in combination with suitable additives to formulate tablets, powders, granules, or capsules, for example with conventional additives such as lactose, mannitol, corn starch, or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; with disintegrants such as corn starch, potato starch, or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, may be added diluents, buffers, wetting agents, preservatives, and flavoring agents. Such formulations can be administered orally.

[0241] The compounds of this invention can be formulated into injectable preparations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol; conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives, may be added if desired. The formulations can also be emulsified or the active ingredient can be encapsulated in a liposome carrier. Suitable injectable formulations can be administered via intravitreal, intraocular, intramuscular, subcutaneous, sublingual, or other routes of administration, such as injection into the gingival tissue or other oral tissues. Such formulations are also suitable for topical administration.

[0242] In some embodiments, the compounds of the present invention can be delivered by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system,” which includes a continuous (e.g., controlled) delivery device (e.g., a pump) combined with a catheter, injection device, etc., of which many such delivery systems are known in the art.

[0243] The compounds of this invention can be used in aerosol formulations administered by inhalation. The compounds of this invention can be formulated with pressurizable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0244] Furthermore, the compounds of the present invention can be formulated into suppositories by mixing with various matrices (such as emulsified matrices or water-soluble matrices). The compounds of the present invention can be administered rectally via suppositories. The suppositories may include carriers such as cocoa butter, carbon wax, and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0245] As used herein, the term "unit dose form" refers to a physically separated unit suitable as a unit dose for use in humans and animal subjects, each unit containing a predetermined amount of the compound of the invention, calculated by means of an amount sufficient to produce the desired effect when used with a pharmaceutically acceptable diluent, carrier, or medium. The specifications of the compounds of the invention depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0246] The dosage form of the disclosed pharmaceutical composition will depend on the chosen route of administration. For example, in addition to injectable fluids, topical or oral dosage forms may be used. Topical formulations may include eye drops, creams, sprays, etc. In some cases, topical formulations of the pharmaceuticals available in the methods described herein may include, for example, creams that include one or more excipients in addition to one or more additional active agents, such as mineral oil, paraffin, propylene carbonate, white petrolatum, beeswax, etc.

[0247] Oral formulations can be liquids (e.g., syrups, solutions, or suspensions) or solids (e.g., powders, pills, tablets, or capsules). Methods for preparing these dosage forms are known or will be obvious to those skilled in the art.

[0248] Some embodiments of pharmaceutical compositions comprising the compounds of the present invention can be formulated into unit dosage forms suitable for precise dosing. The amount of active ingredient administered depends on the subject being treated, the severity of the ailment, and the route of administration, and is known to those skilled in the art. Within these ranges, the formulation to be administered comprises an amount of the disclosed extract or compound that is effective in achieving the desired effect in the subject being treated.

[0249] Each therapeutic compound can be administered independently in any dosage form, such as those described herein, and can also be administered in various ways as described herein. For example, compounds can be formulated together in a single dose unit (i.e., combined in one form, such as capsules, tablets, powders, or liquids) as a combination product. Alternatively, when not formulated together in a single dose unit, individual compounds of the present invention can be administered simultaneously or sequentially with another therapeutic compound in any order.

[0250] In some cases, the pharmaceutical composition may include a delocalized lipophilic cationic compound and a pharmaceutically acceptable excipient.

[0251] method

[0252] As described above, the compositions of the present invention can be used to treat cancer in certain situations. The compositions of the present invention can be used to treat various cancers, including but not limited to: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendiceal cancer, astrocytoma, atypical teratoma / rhabdomyosarcoma, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma, etc.), brainstem glioma, brain tumors (e.g., astrocytoma, embryonal tumors of the central nervous system, germ cell tumors of the central nervous system, craniopharyngioma, ependymoma, etc.), breast cancer (e.g., female breast cancer, male breast cancer). Breast cancer, childhood breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors (such as those in children and the gastrointestinal tract), cancers of unknown primary origin, cardiac tumors, central nervous system tumors (such as atypical teratomas / rhabdomyomas, embryonal tumors, germ cell tumors, lymphomas, etc.), cervical cancer, childhood cancers, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma (e.g., bile duct, extrahepatic, etc.), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, neuroblastoma. Tumors, Ewing sarcoma, extracranial germ cell tumors, external germ cell tumors, extrahepatic bile duct carcinoma, ocular cancer (e.g., intraocular melanoma, retinoblastoma, etc.), osteofibrous histiocytoma (e.g., malignant osteosarcoma, etc.), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, gonadal, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular carcinoma, histiocytosis (e.g., Langerhans cells, etc.), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (e.g., pancreatic neuroendocrine tumors). Kaposi's sarcoma, renal cell carcinoma (e.g., renal cell, nephroblastoma, pediatric renal tumor, etc.), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), hairy cell, etc.), lip and oral cancer, primary liver cancer, lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma (e.g., AIDS-related, Burkitt's, cutaneous T-cell, Hodgkin's, non-Hodgkin's, primary central nervous system (CNS), etc.), macroglobulinemia (e.g., Waldenström's). (etc.), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous cell carcinoma, midline tube carcinoma involving the NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia (e.g., chronic (CML) etc.), myeloid leukemia (e.g., acute (A... Myeloproliferative neoplasms (e.g., chronic), nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer (e.g., lip cancer), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., epithelial cell tumor, germ cell tumor, low-potency tumor, etc.), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papilloma, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, Penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterus, etc.), Sezary syndrome, skin cancer (e.g., childhood melanoma, Merkel cell carcinoma). Cancers include: small cell lung cancer, non-melanoma, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer (e.g., occult primary, metastatic, etc.), gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis cancer, urethral cancer, uterine cancer (e.g., endometrial cancer), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, nephroblastoma, etc.

[0253] As summarized above, in some cases, the compositions of the present invention can be used to treat cancer. The compositions of the present invention can be used to treat various cancers, including but not limited to: acinar carcinoma, acinar cell carcinoma, acinoid epithelial carcinoma, acinar cystic carcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical carcinoma, alveolar carcinoma, ameloblastic carcinoma, apocrine carcinoma, basal cell carcinoma, bronchioloalveolar carcinoma, bronchial carcinoma, cholangiocarcinoma, choriocarcinoma, clear cell carcinoma, colloidal carcinoma, cribriform carcinoma, ductal carcinoma in situ, embryonal carcinoma, capsule carcinoma, endometrioid carcinoma, epidermoid carcinoma, malignant mixed tumor, pleomorphic carcinoma adenoma, thyroid follicular carcinoma, hepatocellular carcinoma, carcinoma in situ, and ductal carcinoma. Intraductal carcinoma, Hurthle cell carcinoma, inflammatory breast cancer, large cell carcinoma, invasive lobular carcinoma, lobular carcinoma, lobular carcinoma in situ (LCIS), medullary carcinoma, meningeal carcinoma, Merkel cell carcinoma, mucinous carcinoma, mucoepidermoid carcinoma, nasopharyngeal carcinoma, non-small cell carcinoma, non-small cell lung cancer (NSCLC), oat cell carcinoma, papillary carcinoma, renal cell carcinoma, scleroderma, sebaceous gland carcinoma, simple carcinoma, signet ring cell carcinoma, small cell carcinoma, small cell lung cancer, spindle cell carcinoma, squamous cell carcinoma, terminal ductal carcinoma, transitional cell carcinoma, tubular carcinoma, verrucous cell carcinoma, etc.

[0254] In some cases, the methods of this disclosure include treating the cancer of the subject by administering an effective amount of one or more delocalized lipophilic compounds to the subject.

[0255] Any useful delocalized lipophilic cationic compound may be used in the method of the present invention. Non-limiting examples of useful delocalized lipophilic cationic compounds include compounds (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), and (11).

[0256] The subjects to whom the compounds of this disclosure may be applied may vary and may include those who have or are suspected of having a disease. The diseases that subjects to whom the compounds of this disclosure may have or are suspected of having may vary and may include, but are not limited to, neoplasia. In some cases, the individual treated according to this method will be an individual with neoplasia. As used herein, “neoplasia” includes any form of abnormal new tissue formation, etc. In some cases, the individual has recently received treatment for neoplasia (e.g., cancer, tumor, etc.) and is therefore at risk of recurrence. In some cases, the individual has not recently or previously received treatment for neoplasia (e.g., cancer, tumor, etc.) but is newly diagnosed with neoplasia. Any and all neoplasms are suitable for treatment by the methods of this invention (e.g., using delocalized lipophilic cationic compounds).

[0257] The compositions disclosed herein (e.g., compositions comprising one or more delocalized lipophilic cationic compounds) may be provided as pharmaceutical compositions. Any suitable pharmaceutical composition may be used, which is described in more detail below. Thus, in some cases, the methods of this disclosure may include administering a delocalized lipophilic cationic compound in a composition comprising an excipient (e.g., an isotonic excipient), said composition being prepared under sufficiently sterile conditions, to a mammal, such as a human.

[0258] As described herein, delocalized lipophilic cationic compounds can be administered to subjects via a variety of routes of administration. The route of administration can be selected based on a variety of factors, including, but not limited to, the condition to be treated, the formulation and / or device used, and the patient to be treated. Possible routes of administration include, but are not limited to, oral and parenteral routes, such as oral, intravenous (iv), intraperitoneal (ip), rectal, topical, ocular, nasal, and transdermal. Formulations of these dosage forms are described herein.

[0259] The effective amount of the compounds of this invention depends at least on the specific method of use, the subject being treated, the severity of the disease, and the route of administration of the therapeutic composition. The "therapeutic effective amount" of a composition is the amount of a specific compound sufficient to achieve the desired effect in the subject (host) receiving treatment.

[0260] The therapeutically effective amount of the compounds or pharmaceutical compositions of the present invention can be determined by those skilled in the art to achieve a local (e.g., tissue) concentration at least equal to the IC50 of the applicable compounds disclosed herein. 50 They are the same height.

[0261] The specific dosage level and frequency of administration for any particular subject can vary and depend on a variety of factors, including the activity of the compound of the invention, the metabolic stability and duration of action of the compound, the subject's age, weight, general health condition, sex and diet, route and timing of administration, excretion rate, drug combination and the severity of the host's condition.

[0262] In some cases, the conversion of animal doses to human equivalent doses (HEDs) can be performed using conversion tables and / or algorithms provided by the U.S. Department of Health and Human Services, the Food and Drug Administration, and the Center for Drug Evaluation and Research (CDER), such as industry guidance: Estimating the maximum safe starting dose for treatment in an initial clinical trial in healthy adult volunteers (2005) FDA (5600 Fishers Lane, Rockville, MD 20857); (available at www.fda.gov / cder / guidance / index.htm, the public content of which is incorporated herein by reference) (Table 1).

[0263]

[0264] Table 1. Conversion between animal doses and human equivalent doses based on body surface area.

[0265] The method of treating the subject according to the present invention includes administering a delocalized lipophilic cationic compound or a composition comprising such a delocalized lipophilic compound to the subject in need. In some cases, the pharmaceutical composition is administered by intravenous injection, intramuscular injection, or intraperitoneal injection.

[0266] In some cases, the patient requiring treatment with a delocalized lipophilic cationic compound suffers from a condition. In some cases, the condition is a bacterial infection. In some cases, the condition is tumor formation. In some cases, the tumor formation is a tumor. In some cases, the tumor formation is cancer. In some cases, the cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, brain tumor, thyroid cancer, bladder cancer, head and neck cancer, lung cancer, or leukemia.

[0267] In some cases, the method of treating the subject includes: administering a therapeutically effective amount of a delocalized lipophilic cationic compound and treating the subject's condition. In some cases, the administration of a therapeutically effective amount of the delocalized lipophilic cationic compound can inhibit growth in the subject, such as the growth of bacterial infections or cancer in the subject.

[0268] In some embodiments, the delocalized lipophilic cationic compound can be used to treat a subject via photodynamic therapy or photothermal therapy. This disclosure provides a method of treating a subject comprising: administering a therapeutically effective amount of the delocalized lipophilic cationic compound to the subject, and exposing the delocalized lipophilic cationic compound in vivo to light. In some cases, exposing the delocalized lipophilic cationic compound to light results in the generation of reactive oxygen species. In some cases, the method kills at least one cancer cell. In some cases, the method reduces the size of a tumor. In some cases, the method reduces tumor growth.

[0269] The delocalized lipophilic cationic compound of the present invention is fluorescent. Therefore, the delocalized lipophilic cationic compound of the present invention can be used for fluorescent endoscopy or fluorescent-guided surgery.

[0270] This invention provides a method for treating a subject, comprising: administering a delocalized lipophilic cationic compound to the subject and detecting a fluorescence signal generated by the administered delocalized lipophilic cationic compound. In some cases, the compound is administered to the subject in an amount sufficient to produce fluorescence in vivo. In some cases, in vivo fluorescence imaging includes fluorescence endoscopy, such as upper gastrointestinal endoscopy. In some cases, the fluorescence endoscopy includes colonoscopy. In some cases, the method includes obtaining a biopsy sample from the subject and detecting a fluorescence signal of the administered compound from the cells of the biopsy. In some cases, the biopsy is a cancer biopsy. In some cases, the cancer has been removed and the method further includes: assessing the surgical margins of the removed cancer by visualizing the fluorescence signal of the administered compound.

[0271] This invention provides a method for killing cells, comprising: contacting the cells with an effective amount of a delocalized lipophilic cationic compound. In some cases, the cells are prokaryotic cells. In some cases, the cells are eukaryotic cells, such as plant cells or animal cells. In some cases, the cells are cancer cells. In some cases, the eukaryotic cells are mammalian cells. In some cases, the mammalian cells are cancer cells.

[0272] In some embodiments, the method of the present invention provides fluorescently labeled mitochondria of cells. This method may include contacting the cells with an effective amount of a delocalized lipophilic cationic compound. Various cells can be labeled according to this method. In some cases, the cells are cancer cells. In some cases, the cells are prokaryotic cells. In some cases, the cells are eukaryotic cells, such as plant cells, animal cells, etc. In some cases, the eukaryotic cells are mammalian cells. In some cases, the mammalian cells are cancer cells.

[0273] This invention provides a method for imaging mitochondria in cells, comprising: contacting the cells with an effective amount of a delocalized lipophilic cationic compound and detecting a fluorescence signal from said compound. In some cases, the detection is in vitro. In some cases, the detection includes fluorescence imaging, including, for example, fluorescence microscopy. This method can be used with a variety of different cells. In some cases, the cells are cancer cells. In some cases, the cells are prokaryotic cells. In some cases, the cells are eukaryotic cells, such as plant cells, animal cells, etc. In some cases, the eukaryotic cells are mammalian cells. In some cases, the mammalian cells are cancer cells.

[0274] This invention provides a method for determining whether a patient has a mitochondrial-related disease, comprising: contacting the patient's cells with an effective amount of a delocalized lipophilic cationic compound and detecting a fluorescent signal of the compound. In some cases, the contact includes administering the delocalized lipophilic cationic compound to the patient. In some cases, the contact includes obtaining cells from a biopsy and contacting the cells with the delocalized lipophilic cation in vitro. In some cases, the detection includes in vivo fluorescence imaging. In some cases, the in vivo fluorescence imaging includes fluorescence endoscopy. In some cases, the fluorescence endoscopy includes upper gastrointestinal endoscopy. In some cases, the fluorescence endoscopy includes colonoscopy.

[0275] Among the methods for determining whether a patient has a mitochondrial-related disease are mitochondrial myopathy; diabetes and deafness (DAD); Leber hereditary optic neuropathy (LHON); Leigh syndrome; myoneurogenic gastrointestinal encephalopathy (MNGIE); myoclonic epilepsy with unequal red fibers (MERRF); neurological disorders, ataxia, retinitis pigmentosa, and ptosis (NARP); mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like symptoms (MELAS), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), or Friedrieich ataxia.

[0276] This invention provides a method for treating a patient with mitochondrial-related disease, comprising: determining whether a patient has mitochondrial-related disease, wherein the determination includes contacting the patient's cells with a delocalized lipophilic cationic compound and detecting a fluorescence signal of the compound; and if the patient is determined to have mitochondrial-related disease, treating the patient for the disease. In some cases, the contact includes administering the delocalized lipophilic cationic compound to the patient. In some cases, the contact includes obtaining cells from a biopsy and contacting the cells with the delocalized lipophilic cation in vitro. In some cases, the detection includes in vivo fluorescence imaging. In some cases, the in vivo fluorescence imaging includes fluorescence endoscopy. In some cases, the fluorescence endoscopy includes upper gastrointestinal endoscopy. In some cases, the fluorescence endoscopy includes colonoscopy.

[0277] Among the methods for treating patients with mitochondrial-related diseases are mitochondrial myopathy; diabetes and deafness (DAD); Leber hereditary optic neuropathy (LHON); Leigh syndrome; myoneurogenic gastrointestinal encephalopathy (MNGIE); myoclonic epilepsy with unequal red fibers (MERRF); neurological disorders, ataxia, retinitis pigmentosa, and ptosis (NARP); mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like symptoms (MELAS), mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), or Friedrieich ataxia.

[0278] Reagent test kit

[0279] The present invention also provides a kit for carrying out one or more of the above-described methods and / or producing one or more of the above-described compositions. The kit of the present invention can vary considerably. The reagents and devices included in the kit of the present invention may include the reagents and devices mentioned above for the methods described, including, for example, methods for treating a subject, methods for labeling cells, methods for killing cells, etc. In the kit of the present invention, one or more components may be present in the same or different containers, depending on convenience or need.

[0280] The kits disclosed herein may include one or more reagents and / or devices for preparing samples for processing and / or assaying, including, for example, processing and / or assaying as described herein. Useful reagents and / or devices that may be included in the kits of this invention include, but are not limited to, immobilization reagents, immobilization solutions comprising at least one immobilization reagent, homogenization devices, devices for generating cell suspensions, devices for culturing cell suspensions, DNA labeling reagents, and devices for obtaining samples (e.g., blood collection devices, biopsy devices, aspiration needles, etc.).

[0281] The kits disclosed herein may include one or more reagents and / or devices for imaging the compounds of the present invention. Such devices may vary and may include, but are not limited to, devices and / or reagents for fluorescence endoscopy, devices and / or reagents for fluorescence-guided surgery, etc.

[0282] In addition to the components described above, the kit of the present invention may also include (in some embodiments) instructions for carrying out the method of the present invention. These instructions may exist in various forms in the kit of the present invention, one or more of which may be present in the kit. One form of these instructions may be as printed information on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, in the packaging of the kit, in a packaging insert, etc. Another form of these instructions is as a computer-readable medium, such as a disk, optical disc (CD), flash drive, etc., on which the information has been recorded. Another possible form of these instructions is as a website address, which allows access to information on a remote site via the Internet.

[0283] practicality

[0284] The delocalized lipophilic cationic compounds of this invention have a variety of applications, including but not limited to: labeling cells, killing cells (including eukaryotic and prokaryotic cells), and treating tumors such as cancers, masses, etc. Not bound by theory, the anticancer activity of the delocalized lipophilic cationic compounds can involve the selective accumulation of the delocalized lipophilic cationic compounds relative to normal cells in the mitochondria of cancer cells. In some cases, administration of delocalized lipophilic cationic compounds to subjects with cancer can kill cancer cells, reduce proliferation, and / or additionally lead to a reduction in cancer size. In some cases, administration of delocalized lipophilic cationic compounds to subjects with cancer can lead to a reduction in cancer growth rate.

[0285] Furthermore, many delocalized lipophilic cationic compounds are fluorescent. Therefore, such delocalized lipophilic cationic compounds can be used in a variety of applications utilizing fluorescence signal detection, such as, but not limited to, optical imaging, fluorescence image-guided surgery, fluorescence endoscopy, etc. Methods and systems for fluorescence endoscopy are known in the art, for example, as described in U.S. Patent Nos. 4,821,117, 5,092,331, and 5,749,830, which are incorporated herein by reference. Methods and systems for fluorescence image-guided surgery are known in the art, for example, as described in U.S. Patent Application Publications 2014 / 0276008, 2008 / 010339, and 2014 / 0276008, which are incorporated herein by reference.

[0286] The following embodiments are provided in an illustrative rather than limiting manner.

[0287] Example

[0288] The following embodiments are provided to offer a complete disclosure and description of how to make and use the invention to those skilled in the art, and are not intended to limit the scope of the inventors' invention, nor to represent that the following experiments were all or only those conducted. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at atmospheres or near atmospheres.

[0289] General methods of molecular and cellular biochemistry can be found in standard textbooks such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); *Short Protocols in Molecular Biology*, 4th edition (Ausubel et al., eds., John Wiley & Sons 1999); *Protein Methods* (Bollag et al., John Wiley & Sons 1996); *Nonviral Vectors for Gene Therapy* (Wagner et al., eds., Academic Press 1999); *Viral Vectors* (Kaplift & Loewy eds., Academic Press 1995); and *Immunology Methods Manual* (I. Lefkovits ed., Academic Press). The contents of these publications are incorporated herein by reference in *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons, 1998). Reagents, cloning vectors, cells, and kits used in the methods mentioned or related to this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and repositories such as Addgene, Inc., and the American Type Culture Collection (ATCC).

[0290] General Information

[0291] All air- and moisture-sensitive reactions were performed in flame-dried glassware under a nitrogen atmosphere. Reactive liquid compounds were measured and transferred using a gas-tight syringe and added to the reaction flask via a rubber septum. Tetrahydrofuran (THF) was freshly distilled from sodium benzophenone carbonyl. Dichloromethane, toluene, and DMF were distilled from CaH2. All standard synthetic reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and were ready for use without further purification. Cell lines were obtained from the American Center for Type Culture Collection (Manassas, VA). Female athymic nude mice (nu / nu) were purchased from Charles River Laboratories (Boston, MA). Thin-layer chromatography was performed on glass-backed silica gel plates with F254 indicator. Compounds were observed under UV light, or by color development in iodine, vanillin, and phosphomolybdic acid solutions, or in potassium permanganate solution followed by heating on a hot plate to approximately 350°C. Rapid chromatography is performed on 230-400 mesh silica gel using industrial-grade solvents that have been distilled before use. 1 ¹H NMR spectra were recorded at 400 MHz on a Bruker AV400, with a CDCl₃ solution containing tetramethylsilane (δ = 0 ppm) as an internal standard. 13 C60 spectra were obtained on the same instrument at 100 MHz, with CDCl3 (δ = 77 ppm) as an internal control. Chemical shifts were reported in parts per million (ppm). Multiplicity was reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet), etc. High-resolution mass spectrometry was performed on a Bruker APEX III 7.0 Tesla Ion Spec 4.7 Tesla FTMS and a Thermo Scientific LTQ ORBITRAP XL. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was provided by the Stanford University Protein and Nucleic Acid Biotechnology Facility. Analytical or preparative high-performance liquid chromatography (HPLC) was performed on a DIONX ultimate 3000 instrument with PDA detection.

[0292] Example 1: Synthesis of delocalized lipophilic cationic compounds

[0293] Compounds (1) to (11) according to Figure 1The schematic diagram shows the synthesis. 1,4-Dimethylpyridine iodide (1.1 mmol) and various indole-3-carboxaldehyde derivatives (1 mmol) were added to 10 mL of methanol. After stirring for ten minutes, a catalytic amount of piperidine (0.2 mmol) was added to the mixture. Under nitrogen protection, the reaction solution was heated to reflux and maintained at reflux for 4 to 24 hours, depending on the derivative. The reaction solution changed from light yellow to dark brown, and a brown precipitate formed. Thin-layer chromatography was used to detect the reaction endpoint. The reaction was stopped when the indole-3-carboxaldehyde derivatives disappeared.

[0294] The product was purified by recrystallization from diethyl ether and semi-preparative HPLC. For recrystallization, the precipitate was collected, washed with methanol, and then recrystallized in acetonitrile to obtain an orange or brown powder as the product. For semi-preparative HPLC purification, a Dionex Summit high-performance liquid chromatography (HPLC) system (Dionex Corporation, Sunnyvale, CA) with a 340U four-channel UV-Vis absorbance detector and a reversed-phase semi-preparative HPLC column, a Zorbax SB (C18, 9.4 mm × 250 mm), was used. The mobile phase was water and acetonitrile (both containing 0.1% TFA). The flow rate was 3 mL / min, with gradient elution starting from 5% acetonitrile and ending at 95% acetonitrile at 27 min. 254 nm and 650 nm were used as detection wavelengths.

[0295] The purity of compounds (1) through (11) was determined by analytical HPLC using a Dionex Acclaim 120 (C18, 4.6 mm × 250 mm) column, a flow rate of 1 mL / min, and the same gradient. All products had a purity above 98%. All NMR spectra ( 1 H, 13 C) All tests were performed on a Varian XL-400 (Varain, Palo Alto, CA).

[0296] Electron spray ionization (ESI) mass spectrometry was performed.

[0297] Example 2: Absorbance and fluorescence of delocalized lipophilic cationic compounds

[0298] The absorbance and fluorescence of delocalized lipophilic cationic compounds (1) to (11) were measured. The UV absorbance of the compounds at a concentration of 31 μM in water was recorded using an Agilent 8453 UV spectrophotometer. The fluorescence at a concentration of 7.8 μM in water was recorded using a Fluoromax-3 fluorescence spectrometer (Jobin Yvon).

[0299] The absorbance of the compound between wavelengths of 350 nm and 500 nm is as follows Figure 2AAs shown. The absorption peaks of all compounds are approximately 425 nm, except for compounds (5) and (9), whose maximum absorption values ​​are approximately 450 nm. The fluorescence emission spectra of the compounds are shown below. Figure 2B As shown, the compound's maximum fluorescence intensity is approximately 525 nm.

[0300] The quantum yields of compounds (1) through (11) were measured in ethanol using rhodamine 6G as a reference. The observed quantum yields are as follows: Figure 2C As shown, compound (4) exhibits the highest quantum yield of 49.1%.

[0301] Example 3: Photostability of Delocalized Lipophilic Cationic Compounds

[0302] Compounds (1) to (5), (7) to (9), and (11), and the mitochondrial green fluorescent probe ( ) were evaluated. The photostability of compounds (2), (3), (4), (5), (7) and (11) is similar to that of Rhodamine 6G and mitochondrial green fluorescent probes, while (1), (8) and (9) are relatively easy to bleach.

[0303] A 75W xenon arc lamp (Flamamatsu, San Jose, California, USA) equipped with a 420-470 nm pass-through filter (MF445-45, Thorlabs, Newton, New Jersey, USA) was used as the light source for F16 derivatives, Rhodamine 6G, and the mitochondrial green fluorescent probe. Similarly, a 540-580 nm pass-through filter (MF559-34, Thorlabs, Newton, New Jersey, USA) was used to excite the mitochondrial red fluorescent probe (Mitotracker Red). All dyes were dissolved in water at a concentration of 10 μM in a 700 μL miniature quartz cuvette (10 mm, Sigma-Aldrich, St. Louis, MO, USA). They were continuously excited for 50 minutes. During this period, fluorescence intensity was measured every 5 minutes at 525 nm (F16s, Rhodamine 6G, mitochondrial green fluorescent probe) or 600 nm (mitochondrial red fluorescent probe). The change in relative fluorescence intensity over time was monitored.

[0304] Example 4: Quantum yield of delocalized lipophilic cationic compounds

[0305] The quantum yields of compounds (1) through (11) and rhodamine 6G were evaluated. Compound (4) exhibited the highest quantum yield (49.1%). The quantum yields of compounds (3), (7), and (9) were approximately 20%, (1) and (2) were approximately 12%, and (5), (6), (8), (10), and (11) were less than 10%.

[0306] The fluorescence quantum yield of the compounds was determined using the formula Φx = Φs(Fx / Fs)(As / Ax), where Φ is the quantum yield, F is the integrated area under the corrected emission spectrum, and A is the absorbance at the excitation wavelength; the subscripts x and s refer to the F16 compound and the standard, respectively. An ethanol solution of rhodamine 6G (ΦF = 95%) was used as the standard. All compounds and rhodamine 6G were dissolved in ethanol at five different concentrations, all with absorbance less than 0.1 at 425 nm. The corresponding five different fluorescence values ​​of the compounds and rhodamine 6G were excited at 425 nm, and emission spectra from 450 nm to 650 nm were obtained for the following integrated area measurements. All data were analyzed using Origin Pro 9.0 software (OriginLab, Northampton, Massachusetts, USA) to obtain the final quantum yield of the compounds.

[0307] Example 5: Mitochondrial targeting properties of delocalized lipophilic cationic compounds

[0308] The mitochondrial targeting properties of compounds (1) through (11) were tested. 3 μM of the compound was added to cells grown in MatTek glass-bottomed dishes (Ashland, Massachusetts) and maintained for 1 hour, followed by washing three times with PBS (phosphate-buffered saline). After changing the culture medium, the cells were imaged using a fluorescence microscope (Zeiss) with a 63× oil or 20× objective lens (excited in the GFP channel).

[0309] Follow the manufacturer's instructions. Red (molecular probe) and Hoechst (ThermoScientific) were added to the culture medium to stain mitochondria and nuclei. Cells were photographed using a 63× oil lens or a 20× objective lens.

[0310] All compounds were found to stain mitochondria in the test cell lines. Figure 2D Compound (3) was shown to colocalize with MitoTrack in NIH-353, H838 and T24 cell lines.

[0311] Example 6: Detection of whether the accumulation of delocalized lipophilic cationic compounds in mitochondria is influenced by mitochondrial membrane potential (ΔΨm) causes

[0312] To detect whether mitochondrial membrane potential (ΔΨm) leads to the accumulation of delocalized lipophilic cationic compounds in mitochondria, H838 cells were treated with a high concentration of K+. + Pre-incubation in a medium containing 137 mM ions (i.e., 137 mM) to depolarize the mitochondrial membrane, or incubation with a medium containing a low concentration of K+. + Pre-incubation was performed with a medium containing 3 mM of K+. Then, 3 μM of compound (3) was added and the cells were incubated for 30 minutes. Fluorescence analysis showed that low concentrations of K+... + Ions lead to the accumulation of more compound (3) in mitochondria, while in high K + Less accumulation was observed at the concentration (see) Figure 3A , 3C ).

[0313] Furthermore, when carbonyl cyano(trifluoromethoxy)phenylhydrazone (FCCP) (a proton carrier that dissipates mitochondrial membrane potential) is added to the cell, mitochondrial staining immediately decreases and diffuses into the cytoplasm. Figure 3B and 3D Therefore, the selective accumulation of compound (3) in mitochondria is caused by the negative transmembrane potential of mitochondria.

[0314] Example 7: Cytotoxicity and Structure-Activity Relationship of Delocalized Lipophilic Cationic Compounds

[0315] The in vitro cytotoxicity of compounds (1) to (11) was tested using bladder cancer T24 cells, non-small cell lung cancer (NSCLC) H838 cells, and normal HIN-3T3 cells. Figure 4 All test cell lines were incubated overnight in 96-well plates at a density of 3000 cells / well. The medium was replaced with 200 μL of medium containing the test compounds at various concentrations (1.95–500 μM). After 3 days of incubation, the number of viable cells was examined and counted directly under a microscope (10×). A minimum area of ​​1 mm × 1 mm was counted from at least three completely independent regions of each cell culture. Cell proliferation was calculated using the following formula: Cell proliferation (%) = (Average number of cells in sample wells / Average number of cells in control wells) × 100%. Intact culture medium was used as a control for evaluation.

[0316] Six of the eleven compounds showed strong antitumor activity, IC50. 50 The values ​​ranged from 0.36 to 6.21 μM (2, 3, 4, 5, 7, 10). Two of them showed selectivity indices greater than 10 (3, 7). Compound (3) exhibited the best antitumor activity and relatively high selectivity, with IC50 values ​​in T24 and H838 cells. 50 The values ​​were 0.82 μM and 0.36 μM, respectively.

[0317] Based on these cytotoxicity studies, structure-activity relationships (SORs) for delocalized lipophilic cationic compounds are proposed. The following SORs are described based on delocalized lipophilic cationic compounds of formula (IV), where R... 11 It is a methyl group. R 1 Methyl substitution reduces antitumor activity (ATAC) and normal cytotoxicity (NCT), for example, comparing the toxicity of (1) and (7). 2 Instead of adding NCT, for example, compare (5) and (8). R 4 Substitution with electron-withdrawing groups increases ATAC, and this is the largest factor in ATAC, for example, comparing (4), (6), (7), and (11). R 6 Substitution with electron-donating groups increases ATAC, for example, compare (3), (4), and (10). Conjugation of the indole ring with the extended π system increases NCT and ATCA.

[0318] Therefore, altering the distribution of electron density in the indole ring of delocalized lipophilic cationic compounds appears to play a crucial role in determining the antitumor activity of such compounds.

[0319] Example 8: In vivo cytotoxicity and effects on body weight of compound (3)

[0320] In vivo studies were conducted on the effects of compound (3) on tumor growth and body weight in a mouse model. Female athymic nude mice (nu / nu) aged 4–6 weeks were obtained from Charles River Laboratories (Boston, MA, USA) and maintained under sterile conditions. Compound (3) was suspended in 150 μL of PBS at a concentration of 5 × 10⁻⁶ ppm. 6 HCC827 cells of individual lung adenocarcinoma were subcutaneously inoculated into the right shoulder of nude mice. When the tumor diameter reached 3–5 mm, tumor-bearing mice were intravenously administered compound (3) (dissolved in 150 pL PBS containing 2 pL DMSO) at a concentration of 15 mg / kg on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. Correspondingly, untreated tumor-bearing mice served as controls and were intravenously administered 150 pL of 1×PBS pH 7.4 (containing 2 pL DMSO) on the same day as the treated mice. Ten mice were used in each group. After each intravenous injection, the length and width of the elliptical tumor were measured using calipers. The modified ellipsoid formula 1 / 2 (length × width) was used. 2 Calculate tumor size. Tumor growth is calculated as follows: (Final volume (FV) minus initial volume (IV)) to initial volume (IV).

[0321] In vivo fluorescence imaging was performed 40 minutes and 2 hours after injection of compound (3) at 15 mg / kg. An IVIS spectrometer was used. The excitation wavelength was set to 450 nm. The acquisition wavelength was set to 550 nm, and the acquisition time was 3 s. Four mice were used in each group. After in vivo imaging in each group, the mice were sacrificed and the organs were harvested. Fluorescence images of the organs were taken under the same settings.

[0322] Figure 5A The results of cytotoxicity studies on the non-small cell lung cancer (NSCLC) cell line HCC827, as well as the NSCLC cell lines H838, HCC4006, H1693, H2030, H2228, A549, H1437, and H1944, are presented. Compound (3) showed very high ATCA values ​​in all these cell lines. In particular, the H2228 cell line showed an IC50 of 48.9 nM. 50 Furthermore, regarding the differences between 3T3 and normal cells (3T3, ...), ... Figure 4 a) The ratio is approximately 225 ( Figure 5A ).

[0323] In addition, the HCC827 cell line was selected for further in vivo studies. Figure 5B Typical fluorescence images are shown 2 hours after intravenous injection of 15 mg / kg of compound (3) into nude mice with subcutaneous HCC827 tumors (PI). The tumors were clearly visible in the surrounding background tissue, with a tumor-to-background ratio of approximately 2, sufficient for fluorescence image-guided surgery. More of the compound (3) accumulated in the tumor, kidney, bone, stomach, intestine, pancreas, and skin compared to the liver, spleen, heart, lung, and brain. The tumor-to-normal organ ratios (approximately 2 to approximately 7) were high, particularly the observed high tumor / lung ratio, which implies potentially low lung tissue toxicity and applicability for tumor detection and fluorescence image-guided lung cancer surgery.

[0324] also, Figure 5C The therapeutic response of HCC827 xenograft to intravenously (IV)-injected compound (3) was demonstrated. As shown by the mean tumor size in the control and treatment groups, compound (3) exhibited significant ATCA on day 21 of PI. The tumor volume in the compound (3) treatment group (drug group) was significantly smaller than that in the PBS treatment group (control group), which was statistically significant (p = 0.0426). Meanwhile, there was no significant difference in body weight between the control and treatment groups (p = 0.477). Figure 5D This indicates that compound (3) has low in vivo toxicity. Flow cytometry results showed that compound (3) inhibited cancer cell growth in a time-dependent manner. H&E staining of tumor pathological sections showed that compound (3) induced inflammatory cell infiltration, fibrous tissue proliferation, and apoptosis in tumors.

[0325] Example 9: Cytotoxicity and Cell Staining of Delocalized Lipophilic Cationic Compounds

[0326] The cytotoxicity of compounds (1) to (11) to *E. coli* was evaluated by contacting *E. coli* bacteria already cultured on culture plates with the compounds. *E. coli* was contacted with water and penicillin-streptomycin samples as controls. Results of the cytotoxicity assays were shown... Figure 6 middle.

[0327] The ability of compound (3) to stain E. coli was further evaluated, such as Figure 7 As shown. Plates A and B show staining as indicated by GFP and bright-field images. Plates C and D show staining at pH 7.4, 1×PBS (blank) using GFP and bright-field images.

[0328] The ability of compound (6), namely “CN-F16”, to act on stained Arabidopsis thaliana roots was further evaluated by contacting the roots with compound (6). Figure 8 and 9 As shown. Figure 8 The images were taken one hour after the contact, and Figure 9 Images were taken 20 hours after contact. In both cases, a 10× microscope and an exposure time of 20 ms were used. In both images, plate AC shows the root in contact with compound (6), while plate DF is the control. Figure 10 The experimental setup used to capture images of whole Arabidopsis thaliana plants is shown.

[0329] Notwithstanding the appended claims, this disclosure is also defined by the following provisions:

[0330] Clause 1. A compound of formula (I):

[0331] ALZ +

[0332] (I)

[0333] in:

[0334] A is selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl;

[0335] L is a linking group;

[0336] Z + It is a positively charged group.

[0337] The compound described therein is a π-conjugated system.

[0338] Clause 2. The compound of claim 1, wherein A is selected from indole and substituted indole.

[0339] Clause 3. The compound as claimed in claim 1 or 2, wherein Z + Selected from positively charged heteroaryl groups or substituted heteroaryl groups.

[0340] Clause 4. The compound of claim 3, wherein Z + Selected from pyridinium or substituted pyridinium.

[0341] Clause 5. The compound of any one of claims 1 to 4, wherein L is selected from alkenyl, alkyneyl, aryl, alkyleneyl, and arylalkylene.

[0342] Clause 6. The compound according to any one of claims 1 to 5, having formula (II):

[0343]

[0344] in,

[0345] X - It is a counter ion;

[0346] Z + It is a group containing a positive charge;

[0347] L is a linking group;

[0348] X is selected from CR 8 and N;

[0349] R 1 and R 8 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0350] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0351] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms attached to it together form a fused ring selected from aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0352] Clause 7. The compound according to any one of claims 1 to 6, having formula (III):

[0353]

[0354] in,

[0355] X - It is a counter ion;

[0356] X is selected from CR 8 and N;

[0357] R 1 and R 8 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0358] R 2 R 3 and R 4 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro and cyano;

[0359] R 5 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; or R 5 and R 6 The carbon atoms bonded to it together form a fused ring selected from aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

[0360] R 11 It is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0361] Clause 8. The compound as claimed in claim 6 or 7, wherein R 1 R 2 R 4 R 5 and R 6 One or more of them are substituents other than hydrogen.

[0362] Clause 9. The compound according to any one of claims 1 to 7, having formula (IV):

[0363]

[0364] in,

[0365] X - It is a counter ion;

[0366] R 1Selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

[0367] R 2 R 4 and R 6 Each is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, nitro, and cyano; and

[0368] R 11 It is selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl.

[0369] Clause 10. The compound of any one of claims 1 to 9, wherein R 1 For H.

[0370] Clause 11. The compound according to any one of claims 1 to 10, wherein R 4 It is bromine.

[0371] Clause 12. The compound according to any one of claims 1 to 11, wherein R 6 It is a methyl group.

[0372] Clause 13. The compound of any one of claims 6 to 12, wherein the counter ion is an iodide ion.

[0373] Clause 14. The compound according to any one of claims 1 to 13, selected from the structure:

[0374]

[0375] Clause 15. The compound of claim 14, having the following structure:

[0376]

[0377] Clause 16. The compound of any one of claims 1 to 15, wherein the compound has selective cytotoxicity against cancer cells.

[0378] Clause 17. The compound of any one of claims 1 to 16, wherein the IC50 of said compound is effective against cancer cells. 50 Less than 10 μM.

[0379] Clause 18. The compound of claim 17, wherein the IC50 of the cancer cells 50 It is 500 nM or smaller.

[0380] Clause 19. The compound of any one of claims 1 to 18, wherein the IC50 of the compound is effective against non-cancer cells. 50 At least IC targeting cancer cells 50 Twice as much.

[0381] Clause 20. The compound of any one of claims 1 to 19, wherein the compound has a fluorescence absorption peak between 375 nm and 475 nm and a fluorescence emission peak between 475 nm and 575 nm.

[0382] Clause 21. The compound of any one of claims 1 to 20, wherein the compound has a fluorescence quantum yield of 10% or higher relative to the rhodamine 6G reference.

[0383] Clause 22. The compound of any one of claims 1 to 21, wherein the compound is a derivative of F16.

[0384] Clause 23. A pharmaceutical composition comprising:

[0385] The compound according to any one of claims 1-22; and

[0386] Pharmaceutically acceptable excipients.

[0387] Clause 24. A method comprising administering the pharmaceutical composition of claim 23 to a subject in need of it.

[0388] Clause 25. The method of claim 24, wherein the pharmaceutical composition is administered by intravenous injection, intramuscular injection, or intraperitoneal injection.

[0389] Clause 26. The method of claim 24 or 25, wherein the object has a disease.

[0390] Clause 27. The method of claim 26, wherein the condition is a bacterial infection.

[0391] Clause 28. The method of claim 26, wherein the condition is cancer.

[0392] Clause 29. The method of claim 28, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, brain tumor, thyroid cancer, bladder cancer, head and neck cancer, lung cancer, or leukemia.

[0393] Clause 30. The method of any one of claims 26 to 29, wherein the method comprises administering a therapeutically effective amount of the composition and treating the condition of the subject.

[0394] Clause 31. The method of claim 30, wherein the therapeutically effective amount inhibits the growth of bacterial infection or cancer in the subject.

[0395] Clause 32. The method of any one of claims 24 to 31, wherein the method comprises: detecting the fluorescence signal of the applied compound.

[0396] Clause 33. The method of claim 32, wherein the composition is applied to the subject in an amount sufficient for in vivo fluorescence imaging.

[0397] Clause 34. The method of claim 33, wherein the in vivo fluorescence imaging includes fluorescence endoscopy.

[0398] Clause 35. The method of claim 34, wherein the fluorescence endoscopy includes upper gastrointestinal endoscopy.

[0399] Clause 36. The method of claim 34, wherein the fluorescence endoscopy includes colonoscopy.

[0400] Clause 37. The method of any one of claims 24 to 36, wherein the method further comprises obtaining a biopsy from the subject and detecting a fluorescent signal of a compound applied to the biopsy cells.

[0401] Clause 38. The method of claim 37, wherein the biopsy is a cancer biopsy.

[0402] Clause 39. The method of any one of claims 28 to 38, wherein the cancer is removed and the method further comprises: evaluating the surgical margin of the removed cancer by visualizing the fluorescent signal of the applied compound.

[0403] Clause 40. A method of killing cells, the method comprising: contacting the cells with an effective amount of the compound according to any one of claims 1-22.

[0404] Clause 41. The method of claim 40, wherein the cell is a prokaryotic cell.

[0405] Clause 42. The method of claim 40, wherein the cell is a eukaryotic cell.

[0406] Clause 43. The method of claim 42, wherein the eukaryotic cell is a plant cell.

[0407] Clause 44. The method of claim 42, wherein the eukaryotic cell is a mammalian cell.

[0408] Clause 45. The method of claim 44, wherein the mammalian cell is a cancer cell.

[0409] Clause 46. A method for fluorescently labeling mitochondria of cells, the method comprising: contacting the cells with an effective amount of the compound according to any one of claims 1-22.

[0410] Clause 47. The method of claim 46, wherein the cell is a prokaryotic cell.

[0411] Clause 48. The method of claim 46, wherein the cell is a eukaryotic cell.

[0412] Clause 49. The method of claim 48, wherein the eukaryotic cell is a plant cell.

[0413] Clause 50. The method of claim 48, wherein the eukaryotic cell is a mammalian cell.

[0414] Clause 51. The method of claim 50, wherein the mammalian cell is a cancer cell.

[0415] Clause 52. The method of any one of claims 46 to 51, wherein the method further comprises: exposing the contacted cells to light of an excitation wavelength to detect a fluorescence signal of the emission wavelength of the compound, thereby visualizing the fluorescently labeled mitochondria of the cells.

[0416] Clause 53. A kit comprising any one of claims 1-22.

[0417] Clause 54. The kit of claim 53, wherein the kit further comprises a pharmaceutically acceptable excipient.

[0418] Clause 55. The kit as claimed in claim 53 or 54 further includes a delivery device.

[0419] Although the invention has been described in some detail by way of illustration and examples for the purpose of clarity, it will be apparent to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims, based on the teachings of the invention.

[0420] Therefore, the foregoing only illustrates the spirit of the invention. It should be understood that, although not explicitly described or shown herein, those skilled in the art will be able to make various arrangements that embody the spirit of the invention and are included within its spirit and scope. Furthermore, all embodiments and conditional language referenced herein, primarily intended to aid the reader in understanding the spirit and concepts of the invention contributed by the inventors to the field, should be interpreted as not being limited to these specifically referenced embodiments and conditions. Moreover, all statements herein referencing the spirit, aspects, and implementations of the invention and their specific embodiments are intended to cover their structural and functional equivalents. Additionally, this equivalent is intended to include both currently known equivalents and future developments, i.e., any member of a development performing the same function, regardless of its structure. Furthermore, whatever is disclosed herein, whether or not it is expressly referenced in the claims, is not intended as a donation to the public.

[0421] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35U.SC §112(f) or 35U.SC §112(6) is explicitly defined as follows: the provision is invoked for the limitation in the claims only when the exact phrase “means” or the exact phrase “step” is used at the beginning of such limitation in the claims; if such an exact phrase is not used in the limitation of the claims, then 35U.SC §112(f) or 35U.SC §112(6) is not invoked.

Claims

1. A compound having the structure of formula (IV): in, X - It is an iodide ion; R 1 For H; R 2 For H; R 4 It is bromine; R 6 Alkyl groups containing 1 to 6 carbon atoms; R 11 It is an alkyl group containing 1 to 6 carbon atoms.

2. The compound according to claim 1, characterized in that, R 6 Selected from methyl, ethyl, n-propyl, and isopropyl; and R 11 Selected from methyl, ethyl, n-propyl and isopropyl.

3. The compound according to claim 1, characterized in that, Selected from structure:

4. A pharmaceutical composition, characterized in that, include: a) The compound according to any one of claims 1 to 3; and c) Pharmaceutically acceptable excipients.

5. Use of the compound according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4, characterized in that, This is used to prepare a pharmaceutical composition for treating cancer, wherein the cancer is selected from bladder cancer, lung adenocarcinoma, and non-small cell lung cancer.

6. Use of the compound according to any one of claims 1 to 3, characterized in that, Compositions for preparing fluorescently labeled mitochondria of cells.

7. A kit comprising the compound of any one of claims 1 to 3.

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