Charged π-conjugated molecules for the treatment of microbial infections

Abstract

In some embodiments, the present disclosure provides methods of using π-conjugated compounds having a charge as part of the π-conjugated structure to treat microbial infections. These methods can be used, in particular, to treat Gram-positive bacterial infections in patients. In another embodiment, the present disclosure provides methods of using π-conjugated compounds having a charge as part of the π-conjugated structure to treat cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 437,303, filed January 5, 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 1.Technical Field This disclosure relates to the fields of biology, biochemistry, chemistry, pharmacology, and medicine. In particular, new methods, compounds, and methods for treating cancer or microbial infections are disclosed.

[0003] 2. Related technologies In 2019, Staphylococcus aureus was the leading cause of bacterial death in 135 countries and the leading cause of death among people aged 15 years and older worldwide (Ikuta et al., 2019). Methicillin-resistant Staphylococcus aureus (MRSA) alone was responsible for more than 100,000 deaths in 2019 alone (Murray et al., 2022). In the United States, the number of deaths from MRSA now exceeds the number of deaths from HIV / AIDS and tuberculosis combined (Peterson et al., 2010). In addition to high mortality, MRSA infections result in an estimated $3.2 billion to $4.2 billion in additional healthcare costs each year (Bumahet et al., 2013). Vancomycin has been the cornerstone of MRSA treatment for over 20 years. However, in recent years, numerous cases of vancomycin-intermediately resistant Staphylococcus aureus infections (VISA) and vancomycin-resistant Staphylococcus aureus infections (VRSA) have been reported ( Weigel et al., 2003 ; Smith et al., 1999 ), calling into question the reliability of vancomycin in the treatment of MRSA.

[0004] Gram-positive vancomycin-resistant enterococci (VRE) cause a variety of nosocomial infections, including urinary tract infections, skin / wound infections, intra-abdominal infections, and bacteremia. Among enterococci, Enterococcus faecalis and Enterococcus faecium are the third and fourth most common nosocomial pathogens worldwide, respectively, and antibiotic resistance has developed and spread readily among these bacteria. Linezolid is generally the drug of choice for the treatment of VRE (Moellering et al., 2003), but it is expensive and can cause several serious side effects, including bone marrow suppression and peripheral neuropathy. It may also interact with psychiatric medications, particularly monoamine oxidase inhibitors. Worryingly, linezolid resistance in VRE has also been reported (Gonzales et al., 2001).

[0005] Streptococcus is a genus of Gram-positive, facultatively anaerobic, coccoid bacteria with over 50 species. They inhabit a variety of habitats, including human skin and mucous membranes. The epidemiology of streptococci is complex and can have serious impacts on human and animal health. In humans, they are one of the most frequent causes of bacterial infections, such as streptococcal pharyngitis, sinusitis, meningitis, pneumonia, and skin infections. Streptococcus pneumoniae species are the most lethal pathogens in children under 5 years of age (Ikuta et al., 2019). Meanwhile, Streptococcus pyogenes, also known as group A streptococcus, causes a wide range of diseases in people of all ages, including streptococcal pharyngitis, scarlet fever, rheumatic fever, and even necrotizing fasciitis (slaughterhouse fever) (Avire et al., 2021). In animals, streptococci are a major cause of mastitis in dairy cows, resulting in significant economic losses to the dairy industry. Streptococcus species are particularly susceptible to the development of antibiotic resistance through horizontal gene transfer and chromosomal point mutations, which can alter efflux pump activity and modify antimicrobial targets.

[0006] Therefore, there is an urgent need for alternative antibacterial therapies that have a different mode of action than existing antibiotics and are less prone to the development of resistance.

[0007] Cancer remains a leading cause of death worldwide, with approximately 20 million cases and 10 million deaths reported worldwide in 2020 (Xia et al., 2022). Therefore, therapeutic strategies that can kill cancer cells with minimal toxic non-target effects are urgently needed.

[0008] This invention was funded in part by Welch Grant No. C-2017 from the Robert A. Welch Foundation. Summary of the Invention [Means for solving the problem]

[0009] As provided herein, the present disclosure relates to methods for treating bacterial infections, including treatment with compounds comprising π-conjugated compounds having a charge as part of the π-conjugated structure, provided that at least a portion of the π-conjugated structure is non-aromatic. These methods may be used, in particular, to treat Gram-positive bacterial infections in patients. In some embodiments, the methods of the present disclosure may be used to treat cancer in patients. Charged π-conjugated compounds may include, but are not limited to, cyanine dyes.

[0010] In some aspects, the present disclosure provides methods for killing bacteria by treating them with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain. In some embodiments, the bacteria are Gram-positive bacteria. In other embodiments, the bacteria are Gram-negative bacteria. In another aspect, the present disclosure provides methods for killing cancer cells by treating them with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain. In another aspect, the present disclosure provides methods for killing fungal cells by treating them with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

[0011] In some embodiments, the method further comprises treating the bacteria with an adjunct agent, hi some embodiments, the adjunct agent is an antibiotic.

[0012] In some embodiments, the extended conjugated compound comprises a positive charge. In some embodiments, the conjugated compound has the formula: [ka] is further defined by During the ceremony, x is a positive or negative charge, n is an integer from 2 to 100, and each independent variable in n is independently selected; X1 and X2 are each independently a heteroatom selected from O, N, S, B, P, Ge, As, or Se, and R1, R2, R3, R4, R5, R6, and R7 are each independently hydrogen, alkyl, or (C≦18) , alkenyl (C≦18) , alkynyl (C≦18) , aryl (C≦18) , aralkyl (C≦18) , heteroaryl (C≦18) , heterocycloalkyl (C≦18) or a substitution of any of these groups, or R1 and R2, R1 and R5, R2 and R5, R3 and R4, R3 and R7, and R4 and R7 together form 1, 2, 3, 4, 5, or 6 aliphatic or aromatic rings containing at least 3 carbon atoms and not more than 36 carbon atoms, and optionally 1, 2, 3, 4, or 5 nitrogen, sulfur, or oxygen atoms.

[0013] In some embodiments, the compound is: [ka] is further defined as During the ceremony, x is a positive charge, n is an integer from 0 to 100, and each independent variable in n is independently selected; Each of R1, R2, R3, R4, R5, R6, and R7 is independently hydrogen, alkyl, (C≦18) , alkenyl (C≦18) , alkynyl (C≦18) , aryl (C≦18) , aralkyl (C≦18) , heteroaryl (C≦18) , heterocycloalkyl (C≦18) or a substitution of any of these groups, or each R1, R2, R3, R4, R5, R6, and R7 is independently a cell membrane targeting moiety, optionally including a linker; or Each of R1 and R2, R1 and R5, R2 and R5, R3 and R4, R3 and R7, R4 and R7, and R5 and R7, taken together, independently form 1, 2, 3, 4, 5, or 6 aliphatic or aromatic rings containing at least 3 carbon atoms and not more than 36 carbon atoms, and optionally 1, 2, 3, 4, or 5 nitrogen, sulfur, or oxygen atoms.

[0014] In some embodiments, X1 and X2 are the same. In some embodiments, X1 is N. In some embodiments, X2 is N. In some embodiments, X1 and X2 are N.

[0015] In some embodiments, R1 or R5 is symmetric with R3 or R4. In some embodiments, R1 together with R5 form 1, 2, 3, 4, or 5 rings. In some embodiments, R1 together with R5 form 2, 3, or 4 rings. In some embodiments, R1 together with R5 form 3 rings. In some embodiments, R1 together with R5 form 3 rings, one ring of which is aliphatic and two rings of which are aromatic.

[0016] In some embodiments, R2 is alkyl (C≦18) or substituted alkyl (C≦18)In some embodiments, R2 is alkyl (C≦18) In some embodiments, R2 is alkyl, such as methyl. (C≦8) is.

[0017] In some embodiments, R3 together with R7 form 1, 2, 3, 4, or 5 rings. In some embodiments, R3 together with R7 form 2, 3, or 4 rings. In some embodiments, R3 together with R7 form 3 rings. In some embodiments, R3 together with R7 form 3 rings, of which one ring is aliphatic and two rings are aromatic.

[0018] In some embodiments, R4 is alkyl (C≦18) or substituted alkyl (C≦18) In some embodiments, R4 is alkyl (C≦18) In some embodiments, R4 is alkyl, such as methyl. (C≦8) In some embodiments, R6 is hydrogen.

[0019] In some embodiments, R5 and R7 together form 1, 2, or 3 rings. In some embodiments, R5 and R7 together form a single ring. In some embodiments, the single ring is a 5-membered ring, a 6-membered ring, or a 7-membered ring. In some embodiments, n is an integer from 1 to 10. In some embodiments, n is an integer selected from 2, 3, 4, or 5. In some embodiments, n is 3.

[0020] In some embodiments, R4 is a cell targeting moiety having a linker. In some embodiments, the linker is an alkyl chain, an alkenyl chain, an aryl chain, a peptide chain, a polyethylene glycol chain, or a polypropylene chain. In some embodiments, the linker further comprises one or more linking functional groups selected from ether, amide, disulfide, ester, amine, or thioether. In some embodiments, the linker is two alkyl chains with an amide bond functional group. In some embodiments, the cell targeting moiety is a membrane-associated functional group, a carbohydrate or polysaccharide that binds to one or more membrane markers, a lipid that binds to one or more cell membrane markers, a small molecule that binds to one or more cell membrane markers, an aptamer that binds to one or more membrane markers, or a peptide or antibody that binds to one or more membrane markers. In some embodiments, the cell targeting moiety is a cell membrane-associated functional group. In some embodiments, the cell membrane-associated functional group is an amine. In some embodiments, the amine is protonated.

[0021] In some embodiments, the compound is further defined as a compound as depicted in Figure 1. In some embodiments, the compound is the following compound: [ka] TIFF2026501715000004.tif246170TIFF2026501715000005.tif224170TIFF2026501715 000006.tif218170TIFF2026501715000007.tif207170TIFF2026501715000008.tif68170 is selected from.

[0022] In some embodiments, the compound is the following compound: [ka] TIFF2026501715000010.tif227170TIFF2026501715000011.tif194170TIFF2026501715000012.tif207170TIFF2026501715000013.tif107170 is selected from.

[0023] In some embodiments, the compound is selected from the following group of compounds: [ka] is selected from.

[0024] In some embodiments, the compound is further defined as follows: [ka]

[0025] In yet another aspect, the present disclosure provides a method for treating a bacterial disease or disorder in a patient, comprising treating the patient with an extended conjugate compound having at least two conjugated double or triple bonds with a charge in the conjugated chain. In some embodiments, the method further comprises administering the compound with a different antibiotic. In some embodiments, the extended conjugate compound is sufficient to treat or prevent the disease or disorder. In some embodiments, the compound is further defined as a compound as described herein. In some embodiments, the patient is a mammal, such as a human.

[0026] In yet another aspect, the present disclosure provides a method of killing a virus by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

[0027] In yet another aspect, the disclosure provides a method of killing fungi by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

[0028] In another aspect, the disclosure provides a method of killing parasites by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

[0029] In some embodiments, the compound used in the method has a structure as described herein.In some embodiments, the method further comprises a second therapeutic agent.In some embodiments, the method is sufficient to treat or prevent a disease or disorder caused by cancer, virus, fungus, or parasite.

[0030] In another aspect, the present disclosure provides a compound of formula (IV): [ka] a method for forming a compound of During the ceremony, m is 0, 1, 2, or 3; X3 is Br, Cl, I, or F, and R8, R9, R 10 , and R 11 are each independently hydrogen, alkyl (C≦18) , alkenyl (C≦18) , alkynyl (C≦18) , aryl (C≦18) , aralkyl (C≦18) , heteroaryl (C≦18) , heterocycloalkyl (C≦18) or a substitution of any of these groups, The following steps: (a) Formula (III) [ka] obtaining a compound of During the ceremony, m is 0, 1, 2, or 3; X3 is Br, Cl, I, or F, and R8, R9, and R 10 are each independently hydrogen, alkyl (C≦18) , alkenyl (C≦18) , alkynyl (C≦18) , aryl (C≦18) , aralkyl (C≦18) , heteroaryl (C≦18) , heterocycloalkyl (C≦18) or a substitution of any of these groups; (b) reacting a compound of formula (III) with a catalyst and a base under conditions sufficient to form a compound of formula (IV).

[0031] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, for example, a compound synthesized in one method can be used to prepare a final compound by a different method.

[0032] The words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, can mean "one," but are also consistent with the meanings of "one or more," "at least one," and "one or more than one." The word "about" means ±5% of the stated numerical value.

[0033] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0035] [Figure 1] 1 shows the chemical structures of representative charged π-conjugated compounds that exhibit antibacterial activity herein. [Figure 2] We provide evidence that charged π-conjugated compounds are light-independent, Gram-positive bacteria-specific, narrow-spectrum antibacterial agents. A shows the chemical structures of the charged π-conjugated compounds, where "R" is a variable functional group. B shows the concentration-dependent growth inhibition (evaluated as OD600) of a representative Gram-positive bacterium (Staphylococcus aureus ATCC 25923) and a representative Gram-negative bacterium (Escherichia coli MG1655) under and without 48 J / cm-2 of 730 nm light. Data represent the mean ± standard error of at least three biological replicates. C shows the minimum inhibitory concentration (MIC) values ​​of various charged π-conjugated compounds in E. coli and S. aureus. Arrows next to the bars indicate MIC values ​​above the maximum tested concentration (80 μM). Dots represent individual data points. D. Growth curves (OD600) of representative Gram-positive bacterial strains Staphylococcus aureus ATCC 25923 and E. faecalis ATCC 29212 treated with increasing concentrations of representative charged π-conjugated compounds. Results are shown as the mean of three replicates (solid line) ± standard error of the mean (shaded area). [Figure 3] This further demonstrates that the charged π-conjugated compounds do not exhibit bacteriostatic potential against Gram-negative bacteria. In addition to E. coli (right side of Figure 2B, right side of Figure 2C), the charged π-conjugated compounds did not exhibit bacteriostatic activity against the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. The bar graphs represent results from at least three biological replicates. The arrows next to the bar graphs indicate that the MIC values ​​were higher than the maximum concentration tested (80 μM). [Figure 4] Figure 1 shows the susceptibility of various Gram-positive bacterial strains, including antibiotic-resistant strains, to a panel of 56 compounds of the present disclosure. Susceptibility was assessed using minimum inhibitory concentrations (MICs). Bar graphs show the average MIC values ​​calculated from at least three biological replicates. Dots represent individual data points. MICs exceeding the highest concentration of the compound tested are indicated by arrows. Lower MIC values ​​indicate greater antibacterial activity. [Figure 5] This table summarizes data on the antibacterial activity of charged π-conjugated compounds against Gram-positive bacteria. A shows the MIC50 values ​​(concentration at which 50% of strains are inhibited) of 56 different charged π-conjugated compounds against 126 bacterial strains (Figure 1, Table 2). Each bar graph shows the average MIC50 value for a given charged π-conjugated compound, while individual dots above the bar graph represent individual MIC50 data points for each bacterial strain tested. B shows the average MIC50 values ​​categorized by bacterial genus. The bar graph shows the average MIC50 value for all strains within a given genus, while the overlaid dots represent individual MIC data points for the strains tested within that genus. [Figure 6A] We provide evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. These are time-kill curves for a Gram-positive bacterial strain in the exponential phase after treatment with various charged π-conjugated compounds and standard antibiotics. The graph shows bacterial viability over time after treatment with various charged π-conjugated compounds at 4× the MIC compared to the standard antibiotics linezolid (LNZ) and vancomycin (VAN). Viability is expressed as log(N / N0), where N is the number of bacteria at a given time point and N0 is the initial number of bacteria. Data points indicate the average bacterial viability at each time point, and error bars indicate the standard error of the mean (SEM). The dashed line indicates the limit of detection, below which accurate quantification of bacterial numbers is not possible. [Figure 6B] This study provides evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. The effect of increasing concentrations of charged π-conjugated compounds on the growth (optical density at 600 nm) of MRSA USA300 is shown. [Figure 6C] We provide evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. We show the effect of increasing concentrations of charged π-conjugated compounds on the growth (optical density at 600 nm) of a representative vancomycin-resistant enterococcus (VRE) strain. [Figure 6D] This figure provides evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. Figure 1 shows time-kill curves for different Gram-positive bacterial strains treated with different charged π-conjugated compounds at 4× the MIC. Viability is expressed as log(N / N), where N is the number of bacteria at a given time point and N is the initial number of bacteria. The dashed line indicates the detection limit of this method. [Figure 6E] This figure provides evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. Figure 1 shows time-kill curves for different Gram-positive bacterial strains treated with different charged π-conjugated compounds at 8× the MIC. Viability is expressed as log(N / N), where N is the number of bacteria at a given time point and N is the initial number of bacteria. The dashed line indicates the detection limit of this method. [Figure 6F] This study provides evidence that charged π-conjugated compounds exhibit potent bactericidal activity against exponentially growing Gram-positive bacteria. Killing curves are shown for different Gram-positive bacterial strains treated with different charged π-conjugated compounds at 16×MIC over various time periods. Viability is expressed as log(N / N), where N is the number of bacteria at a given time point and N is the initial number of bacteria. The dashed line indicates the detection limit of this method. [Figure 7]We provide evidence that charged π-conjugated compounds effectively eradicate antibiotic-resistant persister cells. Time-kill curves for different Gram-positive bacterial strains of persister cells treated with different charged π-conjugated compounds and conventional antibiotics are shown. The graphs show bacterial survival over time after treatment with various charged π-conjugated compounds at four times their respective MICs, compared to the standard antibiotics linezolid (LNZ) and vancomycin (VAN). Survival is expressed as log(N / N0), where N is the bacterial count at a given time point and N0 is the initial bacterial count. Data points represent the average bacterial survival at each time point, and error bars represent the standard error of the mean (SEM). The dashed line indicates the limit of detection, below which accurate quantification of bacterial counts is not possible. [Figure 8] This study provides evidence that charged π-conjugated compounds exhibit various antibiofilm activities. The efficacy of various charged π-conjugated compounds at 8×MIC against established biofilms of various Gram-positive bacterial strains is shown. The bar graphs show the change in biofilm biomass, as measured by crystal violet assay, indicating the density and adherence of the biofilm matrix. The overlaid lines show the change in colony-forming units (CFU), indicating the change in the number of viable bacteria within the treated biofilm. Results are the mean of three independent replicates, and error bars indicate the standard error of the mean (SEM). Asterisks indicate significant differences between samples treated with charged π-conjugated compounds and those treated with DMSO. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 9] Figure 1 shows the antibiofilm activity of charged π-conjugated compounds or vehicle on various Gram-positive bacterial strains, assessed by quantifying the percentage of metabolically active cells as determined from biofilm ATP levels. Results are shown as mean ± SEM. Dots represent individual data points. Error bars indicate standard deviation. Asterisks indicate significant differences between samples treated with charged π-conjugated compounds and DMSO. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 10] Resistance development and mutation rates of bacterial strains exposed repeatedly to charged π-conjugated compounds are shown. (A) Stepwise development of resistance in Bacillus cereus CECT131123, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, and Streptococcus pneumoniae ATCC 49619. Bacterial strains were repeatedly exposed to charged π-conjugated compounds or the conventional antibiotics ciprofloxacin (CIP), linezolid (LNZ), and vancomycin (VAN) for over 30 cycles. The y-axis shows the fold change in MIC, reflecting the increase in resistance with repeated treatment. (B) Mutation rates in Staphylococcus aureus ATCC 25923 exposed repeatedly to charged π-conjugated compounds or the conventional antibiotic ciprofloxacin (CIP), expressed as the frequency of rifampicin-resistant cells. (C) Single-step resistance assessment of spontaneous mutants. Overnight cultures of S. aureus ATCC 25923 were inoculated onto media containing increasing concentrations of charged π-conjugated compounds (4x, 8x, or 16x the MIC). The table shows the frequency of spontaneous mutants that emerged at each charged π-conjugated compound concentration, expressed as the ratio of resistant colony-forming units (CFUs) to total CFUs. [Figure 11] The effect of various physicochemical parameters on the susceptibility of Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 to selected charged π-conjugated compounds is shown. A: Time-kill curves of S. aureus and E. faecalis treated with 4× the MIC of different charged π-conjugated compounds at 25°C and 37°C. B: MIC values ​​of different charged π-conjugated compounds against S. aureus and E. faecalis across a pH gradient. C: Variation in MIC values ​​with increasing serum concentration. D: Effect of varying NaCl concentration on the susceptibility of bacterial strains to charged π-conjugated compounds. E: Effect of Ca2+ concentration on MIC values ​​for both bacterial strains. F: MIC values ​​relative to the initial bacterial inoculum, expressed as optical density at 600 nm (OD600). [Figure 12]Figure 1 shows transcriptional changes in Staphylococcus aureus treated with sublethal concentrations of the representative charged π-conjugated compound BL 248, as assessed using RNA sequencing (RNA-seq). (A) Heatmap showing the expression patterns of transcripts showing the most significant changes upon treatment with BL 248 compared to DMSO vehicle. The color gradient represents log-transformed expression values, with red indicating upregulation and blue indicating downregulation. (B) Principal component analysis (PCA) plot showing the clustering pattern of samples treated with BL 248 and DMSO. Principal component 1 (PC1) explained 94% of the overall variance. Each point represents an individual sample, with cyan representing DMSO-treated samples and coral representing BL 248-treated samples. (C) Volcano plot showing the distribution of differentially expressed genes between BL 248 and DMSO treatments. The x-axis represents the log2 fold change in gene expression, and the y-axis represents the log10-transformed p-value. Genes whose expression changed significantly are highlighted and labeled. (D) Bar graph showing the Gene Ontology (GO) biological processes enriched in the transcripts showing the most significant changes with BL 248 treatment. The x-axis represents the fold enrichment for each biological process, and a biological process is considered significantly enriched if the false discovery rate (FDR) is P < 0.05. (E) Protein-protein interaction (PPI) network for proteins encoded by the transcripts showing the most significant changes with BL 248 treatment. Nodes represent proteins, and edges represent known interactions between them. Node colors distinguish proteins associated with the proton-translocating ATP synthase complex (purple), the plasma membrane (blue), and other membranes (green). [Figure 13] Figure 1 shows the effect of increasing concentrations of various cyanine dyes or vehicle (1% DMSO) on intracellular, extracellular, and total ATP levels normalized to protein levels in Staphylococcus aureus. Results are shown as the mean ± standard error of the mean (SEM) of three biological replicates. [Figure 14]Changes in membrane potential were determined using the voltage-sensitive dye DiBAC4(5) after treating Staphylococcus aureus with various concentrations of charged π-conjugated compounds or vehicle control (DMSO). Each plotted curve represents the average fluorescence value obtained from multiple experiments, and the shaded area around each curve indicates the standard error of the mean (SEM). An increase in DiBAC4(5) fluorescence is associated with bacterial membrane depolarization, while a decrease indicates hyperpolarization. [Figure 15A] This figure provides data on the interaction of charged π-conjugated compounds with conventional antibiotics. Representative checkerboard plates show the interaction of different charged π-conjugated compounds with conventional antibiotics with different mechanisms of action. The corresponding fractional inhibitory concentration index (FICI) is shown in red within the frame. Results are presented as a heat map, with white representing no growth (0%) and blue representing growth (100%). Growth was assessed as absorbance at 600 nm. Results are the average of three independent replicate experiments. FICI values ​​≤ 0.5 indicate a synergistic interaction, values ​​> 0.5 < 4.0 indicate an irrelevant (or additive) interaction, and values ​​> 4.0 indicate an antagonistic interaction. [Figure 15B] Figure 1 provides data on the interaction of charged π-conjugated compounds with conventional antibiotics. Figure 2 is a summary plot showing the FICI for the interaction of charged π-conjugated compounds with different antibiotics. Results are presented as mean ± standard deviation. Points represent individual values. [Figure 15C] This figure provides data on the interaction of charged π-conjugated compounds with conventional antibiotics. Time-kill curves for Staphylococcus aureus treated with 0.5×MIC of individual charged π-conjugated compounds, 0.5×MIC of antibiotics (gramicidin, GRAMI, or daptomycin, DAP), or a combination of 0.5×MIC of charged π-conjugated compounds and 0.5×MIC of antibiotics are shown. Viability is expressed as log(N / N), where N is the number of bacteria at a given time point and N is the initial number of bacteria. Data points represent the average bacterial survival rate at each time point, and error bars represent the standard error of the mean (SEM). The dashed line indicates the limit of detection, below which accurate quantification of bacterial numbers is not possible. [Figure 16] Data on the interactions between differently charged π-conjugated compounds are shown. (A) A representative checkerboard plate showing the interactions between differently charged π-conjugated compounds. The corresponding FICI is shown in red within the frame. The results are presented as a heat map, with white representing no growth (0%) and blue representing growth (100%). Results are the average of three independent replicate experiments. (B) Time-kill curves for S. aureus treated with individual charged π-conjugated compounds at 0.5×MIC or with a combination of differently charged π-conjugated compounds at 0.5×MIC. Viability is expressed as log(N / N0), where N is the number of bacteria at a given time point and N0 is the initial bacterial count. Data points represent the average bacterial viability at each time point, and error bars represent the standard error of the mean (SEM). The dashed line indicates the limit of detection, below which accurate quantification of bacterial counts is not possible. [Figure 17] We demonstrate the in vitro biocompatibility of charged π-conjugated compounds with mammalian cell lines. (A) Dose-response curves showing the effect of increasing concentrations of charged π-conjugated compounds on the viability of two mammalian cell lines (A549, HEK293) assessed using the Presto Blue assay. Data represent the mean ± standard error of the mean (SEM) of at least three independent biological replicates. The solid lines represent the approximation of the sigmoidal dose-response curve for each compound. (B) IC50s, i.e., the concentrations that reduce viability by 50% for various charged π-conjugated compounds calculated from the dose-response curves, and the therapeutic index (TI), calculated from the ratio of the average IC50 for the two cell lines tested to the MIC of each charged π-conjugated compound in MRSA USA300. [Figure 18A] Figure 1 shows the effect of increasing concentrations of charged π-conjugated compounds on the viability of various mammalian cell lines, as assessed using a clonogenic assay. Figure 2 shows the viability of A549 mammalian cells treated with increasing concentrations of different charged π-conjugated compounds, as assessed using a clonogenic assay. [Figure 18B]1 shows the effect of increasing concentrations of charged π-conjugated compounds on the viability of various mammalian cell lines, as assessed using a clonogenic assay. Figure 1 shows the viability of HEK-Blue™ hNOD1 mammalian cells treated with increasing concentrations of different charged π-conjugated compounds, as assessed using a clonogenic assay. [Figure 18C] 1 shows the effect of increasing concentrations of charged π-conjugated compounds on the viability of various mammalian cell lines, as assessed using a clonogenic assay. 2 shows the viability of HEK-Blue™ TLR mammalian cells treated with increasing concentrations of different charged π-conjugated compounds, as assessed using a clonogenic assay. [Figure 18D] Figure 1 shows the effect of increasing concentrations of charged π-conjugated compounds on the viability of various mammalian cell lines, as assessed using a clonogenic assay. Viability of HepG2 mammalian cells treated with increasing concentrations of different charged π-conjugated compounds, as assessed using a clonogenic assay. [Figure 19] Dose-response curves are provided for different mammalian cell lines (MH-S, HEK-Blue™, hNOD1, A549) treated with increasing concentrations of different charged π-conjugated compounds. Viability was assessed by quantifying ATP levels using a luminescence-based assay. Dots represent individual data points. Solid lines represent approximations of the sigmoidal dose-response curves for each compound. [Figure 20] This allows for the evaluation of the biocompatibility of charged π-conjugated compounds in vivo, which was assessed by monitoring the survival rate of Gallia mellonella (n = 9 individuals) after injection with increasing concentrations of the charged π-conjugated compounds or vehicle. [Figure 21] Figure 1 provides survival curves of Gallia mellonella (n=5 individuals) after MRSA infection and subsequent treatment with increasing concentrations of charged π-conjugated compounds. Uninfected worms and vehicle-treated worms were also used as controls. [Figure 22]Structures particularly relevant to the experiments described in Example 3 are shown. A is an exemplary, non-limiting design of a structure that can be used in cancer treatment. When Z is an atom other than carbon, "x" is a charge, such as a cation, anion, radical cation, radical anion, or radical. R1, R2, R3, and R4 can be alkyl-to-alkyl or aryl-to-aryl substituents. R1-R7 can be H, alkyl, aryl, aliphatic or aromatic cyclic rings, and the like. For cancer treatment, it is important that at least one of the pendant R groups possess a moiety that can promote cell association, such as an amine that is protonated at physiological pH and promotes association into lipid bilayers, thereby transferring the dye to the lipid bilayer. Alternatively, the addition of lipids, carbohydrates, polysaccharides, peptides, or antibodies can promote selective cell association and even cell entry. B is a more specific example of a charged π-conjugated compound of the present disclosure, which can be a cyanine dye, the general structure of which is provided in Figure 23B. R1, R2, R3, and R4 can be alkyl-to-alkyl or aryl-to-aryl substituents. R1-R7 can be H, alkyl, aryl, aliphatic, or aromatic cyclic rings, etc. To achieve the effects described in Example 3, it is important that at least one of the pendant R groups has a moiety that can promote cell association. For example, an amine that is protonated at physiological pH promotes association with lipid bilayers, thereby transferring the dye to the lipid bilayer. The structure of Cyanine 7.5-amine, also referred to herein as Cy7.5-amine, contains an amine that is protonated at physiological pH. This compound, as described in A and B, has a pendant ammonium ion for good cell association. [Figure 23] 1 shows the charged π-conjugated compounds studied in the experiments described in Example 3. [Figure 24]The effect of contact time on the cytotoxicity of charged π-conjugated compounds was demonstrated using a crystal violet assay. The contact time during incubation of charged π-conjugated compound molecules with A375 cells significantly impacts cytotoxicity. Cell viability was measured using a crystal violet assay. A: Cytotoxicity of Cy7.5-amine molecule at 40 minutes versus 1 day. B: Cytotoxicity of Cy5.5-amine molecule at 40 minutes versus 2 days. C: Cytotoxicity of molecule GL-261-2 at 50 minutes versus 2 days. D: Cytotoxicity of molecule GL-286 at 1 day versus 2 days. E: Cytotoxicity of molecule BL-142 at 1 day versus 2 days. F: Cytotoxicity of molecule GL-291-2 at 1 day versus 2 days. Note that the mild effect at short contact times (approximately 40-50 minutes) is due to a gradual mechanism of cytotoxicity (presumably apoptosis) because the cells were exposed for 40-50 minutes and then cultured for 2 days. For crystal violet analysis, each data point was repeated four times (n=4). Error bars represent standard deviation. [Figure 25] Figure 1 shows a crystal violet assay for calculating the toxicity IC50 without photoactivation and with prolonged contact (2-day incubation). The inhibitory concentration of charged π-conjugated compounds required to inhibit cell growth by 50% (toxicity IC50). [Figure 26] Figure 1 shows a crystal violet assay for calculating the toxicity IC50 without photoactivation and with prolonged contact (2-day incubation). The inhibitory concentration of charged π-conjugated compounds required to inhibit cell growth by 50% (toxicity IC50). [Figure 27] The data collected in Figures 25 and 26 to determine the cytotoxicity of charged π-conjugated compounds without photoactivation are summarized as the inhibitory concentration of charged π-conjugated compounds required to inhibit cell growth by 50% (toxicity IC50). [Figure 28]This paper provides a summary of evidence regarding the cytotoxicity of charged π-conjugated compounds without photoirradiation. Toxicity was measured using a clonogenic assay with 7 hours of incubation (contact time between molecules and A375 cells) and 7 days of colony formation. A is the lethal concentration (ToxLC50) of cyanine molecules that kills 50% of A375 cells without photoactivation. B is the chemical structure of an exemplary charged π-conjugated compound. On average, 300 cells, corresponding to 300 colonies, were seeded and analyzed for each data point. [Figure 29] Data are presented demonstrating the cytotoxicity of charged π-conjugated compounds without light irradiation. Both properties were measured using a clonogenic assay with a 7-hour incubation (contact time between the molecule and A375 cells) and a 7-day colony formation period. For each data point, 300 cells were seeded and analyzed. [Figure 30] Photographs of clonogenic assays for toxicity activity (without light irradiation) are provided. Toxicity was measured using a clonogenic assay with 7 hours of incubation (contact time between molecules and A375 cells) and 7 days of colony formation. A is molecule GL-261-2. B is molecule GL-286. C is molecule Cy7-amine. D is molecule GL-297-2. E is molecule BL-141-2. (Figure 30F) Molecule BL-204. G is molecule BL-141-1. On average, 300 cells were seeded and analyzed on each plate. [Figure 31] Photographs of clonogenic assays for toxicity activity (without light irradiation) are provided. Toxicity was measured using a clonogenic assay with 7 hours of incubation (contact time between molecules and A375 cells) and 7 days of colony formation. A is molecule BL-242. B is molecule BL-362-2. C is molecule BL-246-1. D is molecule BL-142. On average, 300 cells were seeded on each plate and analyzed. [Figure 32]Using 1-day incubation versus 2-day incubation, we provide evidence of the toxicity of charged π-conjugated compounds to HEMa (normal melanocytes) and A375 melanoma. BL-204, BL-141-2, and BL-304 are aminocyanines, while TS-1-32 and GL-261-2 are two names for carboxycyanine compounds, shown as GL-261-2 in Figure 23. [Figure 33] The treatment schedule for A375 tumors is shown. Cy7.5-Amine was injected intratumorally at a dose of 8 μg (equivalent to 50 μL of a 200 μM Cy7.5-Amine solution in PBS containing 2.5% DMSO). As a control, 50 μL of a PBS solution containing 2.5% DMSO was injected intratumorally. Blue dots indicate the days on which tumors were treated. [Figure 34] This figure provides evidence that Cy7.5-Amine induces the disappearance of A375 tumors in mice. (A) shows that intratumoral injection of 2.5% DMSO in PBS as a control did not induce tumor disappearance. Ten mice were injected. (B) shows that intratumoral injection of 200 μM Cy7.5-Amine in 2.5% DMSO in PBS was effective in tumor disappearance (n=10 mice). (C) shows that intratumoral injection of 200 μM Cy7.5-Amine improved the survival rate of A375 tumor-bearing mice (n=10 mice). Even after 7 months, two out of ten mice were tumor-free. (D) shows that Cy7.5-Amine is safe for mice. Cy7.5-Amine did not induce weight loss in mice compared to the control group injected with 2.5% DMSO. No weight loss occurred during treatment. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure relates to the use of several charged π-conjugated compounds with particularly potent antibacterial activity against Gram-positive bacteria, including MRSA, VRE, linezolid-resistant Staphylococcus epidermidis, and Streptococcus strains. These charged π-conjugated compounds may also be cyanine dyes. This is the first systematic investigation of the antibacterial activity of a library of different charged π-conjugated compounds. This study identified several molecules with potent antibacterial activity against a wide range of Gram-positive bacterial pathogens, including MRSA, VRE, and Streptococcus strains, via a novel mechanism of action. These molecules were found to be not only bacteriostatic but also bactericidal. The charged π-conjugated compounds described in this invention were also found to inhibit bacterial growth, prolong the lag phase of the growth curve, and reduce total bacterial biomass. Cells treated with the charged π-conjugated compounds of this invention showed a transient increase in ATP levels at sublethal concentrations, followed by a significant decrease in ATP levels at concentrations above the MIC, indicating that the charged π-conjugated compounds of this invention affect microbial metabolism. Treatment with the charged π-conjugated compounds of this invention also reduced the concentration-dependence of membrane potential. In several Gram-positive bacterial strains, treatment with the charged π-conjugated compounds of the present invention significantly reduced biofilm viability and biomass, and pretreatment of cells with the charged π-conjugated compounds of the present invention also enhanced the activity of the conventional antibiotic rifampin.

[0037] Biocompatibility assessment in various mammalian cell lines identified several candidate molecules with similar or even better therapeutic indices (Murray et al., 2022). In vivo toxicity assessment in invertebrate models revealed overall favorable biocompatibility. These and other details are described here.

[0038] In another aspect, the present disclosure relates to the use of several charged π-conjugated compounds with potent anticancer activity. These charged π-conjugated compounds may further be cyanine dyes. In some embodiments, direct injection of the charged π-conjugated compounds results in direct and efficient cell death, tumor shrinkage, and eventual elimination via biological mechanisms of cell death and clearance. In some embodiments, the charged π-conjugated compounds used herein may be advantageous as chemotherapeutic agents that do not require the addition of targeting additives, such as peptides for specific recognition of cancer cells. In some embodiments, the π-conjugated compounds are highly toxic to cancer cells but safe for surrounding healthy tissues or cells. Thus, the charged π-conjugated compounds may be useful as chemotherapeutic agents or for systemic treatment at concentrations lower than those used for similar compounds in treatments known in the art. Furthermore, the charged π-conjugated compounds of the present disclosure have unique pharmacological modes of action that are not predicted based on the known uses of such compounds. The use of the charged π-conjugated compounds of the present disclosure to treat cancer without photoactivation or generation of reactive oxygen species (ROS) represents a contribution to the known art. These aspects and others are discussed in more detail in the next section.

[0039] I. COMPOUNDS AND FORMULATIONS THEREOF A. Compound The compounds of the present disclosure are shown, for example, above, in the Summary of the Invention section, the Examples section, and in the claims below. They can be made using the synthetic methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by those skilled in the art. Such principles and techniques are taught, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2013), which is incorporated herein by reference. In addition, the synthetic methods can be further modified and optimized using the principles and techniques of process chemistry as applied by those skilled in the art for preparative, pilot, or mass production scale production, either batch or continuous. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development - A Guide for Organic Chemists (2012), which is incorporated herein by reference.

[0040] In some embodiments, all of the charged π-conjugated compounds of the present disclosure can be used for the prevention and treatment of one or more diseases or disorders discussed herein or elsewhere. In some embodiments, one or more of the compounds characterized or exemplified herein as intermediates, metabolites, and / or prodrugs may also be useful for the prevention and treatment of one or more diseases or disorders. Therefore, unless expressly stated to the contrary, all of the charged π-conjugated compounds of the present disclosure are considered "active compounds" and "therapeutic compounds" intended for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting public health by ensuring the safety, effectiveness, quality, and stability of human and veterinary drugs, vaccines, and other biological products, as well as medical devices.

[0041] The charged π-conjugated compounds of the present invention may be used in an amount of about 100 nM to about 10 mM, about 250 nM to about 5 mM, or about 500 nM to about 2 mM. The amount of compound used may be about 50 nM, 100 nM, 200 nM, 250 nM, 500 nM, 750 nM, 1 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 750 μM, 800 μM, 900 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM to about 10 mM, or any range derivable therein.

[0042] In some embodiments, the charged π-conjugated compounds of the present disclosure, whether for use in the indications described herein or not, have the advantage that they may be more effective, less toxic, longer acting, more potent, have fewer side effects, be more readily absorbed, be more metabolically stable, be more lipophilic, be more hydrophilic, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art.

[0043] The charged π-conjugated compounds of the present disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic forms. Therefore, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of the chemical formula are intended. The compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, single diastereomers are obtained. The chiral centers of the charged π-conjugated compounds of the present disclosure may have an S or R configuration. In some embodiments, the compounds of the present invention may contain two or more atoms with a defined stereochemical orientation.

[0044] The chemical formulas used to represent the charged π-conjugated compounds of the present disclosure typically represent only one of several different possible tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which tautomer is most common, all tautomers of a given chemical formula are intended. Charged π-conjugated compounds also have several resonance hybrid structures. Thus, the resonance structures of the molecules depicted herein will be permissible.

[0045] In addition, the atoms constituting the charged π-conjugated compounds of the present disclosure are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 Contains C.

[0046] In some embodiments, the charged π-conjugated compounds of the present disclosure function as prodrugs or can be derivatized to function as prodrugs. Because prodrugs are known to enhance many desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturability, etc.), the compounds used in some methods of the present invention can be delivered in prodrug form, if desired. Accordingly, the present disclosure contemplates prodrugs of the charged π-conjugated compounds of the present disclosure and methods of delivering prodrugs. Prodrugs of the compounds used in the present disclosure can be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved to the parent compound either by routine manipulation or in vivo. Thus, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that cleaves to form a hydroxy, amino, or carboxylic acid, respectively, when the prodrug is administered to a patient.

[0047] In some embodiments, the charged π-conjugated compounds of the present disclosure exist in salt or non-salt form. In some embodiments, with respect to the salt form(s), the specific anion or cation forming part of any salt form of the compounds provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their preparation and use methods are described in "Handbook of Pharmaceutical Salts: Properties, and Use (2002)," which is incorporated herein by reference.

[0048] It will be understood that many organic compounds can form complexes with solvents, react in solvents, or precipitate or crystallize from solvents. These complexes are known as "solvates." When the solvent is water, the complexes are known as "hydrates." It will also be understood that many organic compounds can exist in more than one solid form, including crystalline and amorphous forms. The methods of the present invention are intended to encompass all different polymorphs of the compounds used herein. All solid forms of the charged π-conjugated compounds provided herein, including any solvates of the compounds provided herein, are within the scope of the present invention.

[0049] B. Preparation In another aspect, for administration to a patient in need of such treatment, a pharmaceutical formulation (also referred to as a pharmaceutical preparation, pharmaceutical composition, pharmaceutical product, medicinal product, medicament, drug, or medicament) comprises a therapeutically effective amount of a charged π-conjugated compound disclosed herein formulated with one or more excipients and / or drug carriers appropriate for the indicated route of administration. In some embodiments, the charged π-conjugated compounds disclosed herein are formulated in a manner suitable for treating human and / or veterinary patients. In some embodiments, the formulation involves mixing or combining one or more of the charged π-conjugated compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acid, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, for example, for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the charged π-conjugated compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations such as sterilization and / or may contain drug carriers and / or excipients, for example, preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.

[0050] Pharmaceutical formulations can be administered in various ways, for example, topically, orally, by inhalation, or by injection (e.g., subcutaneously, intravenously, and intraperitoneally). Depending on the route of administration, the charged π-conjugated compounds disclosed herein can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. To administer the active compound by a method other than parenteral administration, it may be necessary to coat the compound with a material to prevent its inactivation, or to co-administer the compound with this material. In some embodiments, the active compound can be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions and conventional liposomes.

[0051] The charged π-conjugated compounds disclosed herein can also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally.Dispersions can be prepared in glycerol, liquid polyethylene glycol, and their mixtures, and in oil.Under normal storage and use conditions, these preparations can contain preservatives to prevent the growth of microorganisms.

[0052] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0053] The charged π-conjugated compounds disclosed herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's diet. For therapeutic oral administration, the charged π-conjugated compounds disclosed herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage (%) of therapeutic compound in compositions and formulations can, of course, vary. The amount of therapeutic compound in such pharmaceutical formulations is such that a suitable dosage is obtained.

[0054] Therapeutic compounds can also be administered topically to the skin, eye, ear, or mucous membranes. Topical administration of therapeutic compounds can include formulation of the compound as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When therapeutic compounds are formulated for topical administration, they can be combined with one or more agents that increase the permeability of the compound to the tissue to which they are administered. In other embodiments, topical administration is contemplated for ocular administration. Such administration can be applied to the surface of the cornea, conjunctiva, or sclera. While not wishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior segment of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration can also include administration to mucous membranes, such as the inside of the mouth. Such administration can be directly administered to a specific location within the mucous membrane, such as a tooth, a sore, or an ulcer. Alternatively, if local delivery to the lungs is desired, the therapeutic compound can be administered in a dry powder or aerosol formulation by inhalation.

[0055] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage for a patient to be treated, each unit containing a predetermined amount of a therapeutic compound calculated to produce a desired therapeutic effect in combination with the necessary pharmaceutical carrier. In some embodiments, the specifications for the dosage unit forms of the present invention are determined by and directly depend on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such therapeutic compounds to treat a selected condition in a patient. In some embodiments, the active compound is administered at a therapeutically effective dose sufficient to treat a condition associated with the patient's disease. For example, the efficacy of a compound can be evaluated in an animal model system that can predict its efficacy in treating a disease in humans or another animal.

[0056] In some embodiments, the effective dose range for a therapeutic compound can be extrapolated from the effective dose determined in animal studies for a variety of different animals. In some embodiments, the human equivalent dose (HED) in mg / kg can be calculated according to the following formula (see, e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, which is incorporated herein by reference): HED (mg / kg) = Animal dose (mg / kg) × (Animal K m / Human K m ) K in transformation m The use of the coefficients results in HED values ​​that are based on body surface area (BSA) rather than on body weight alone. m The value is well known. For example, the K for an average human weighing 60 kg (with a BSA of 1.6 m2) is m is 37, but the K of a 20 kg child (BSA is 0.8 m2) m is 25. K in some relevant animal models m are also well known, for example, mouse K m is 3 (body weight 0.02 kg, BSA 0.007), and the K m is 5 (body weight 0.08 kg, BSA 0.02), and the K m is 6 (body weight 0.15 kg, BSA 0.025), and the K m is 12 (for a body weight of 3 kg and a BSA of 0.24).

[0057] The exact amount of therapeutic composition depends on the judgment of the practitioner and is specific to each individual. However, the calculated HED dose provides a general guide. Other factors that affect the dose include the patient's physical and clinical condition, the route of administration, the intended goal of treatment, and the efficacy, stability, and toxicity of the particular therapeutic formulation.

[0058] The actual dosage of the charged π-conjugated compound of the present disclosure or the composition comprising the compound of the present disclosure administered to a patient can be determined by physical and physiological factors, such as the type of animal being treated, age, sex, weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the idiopathic nature of the patient, and the route of administration. These factors can be determined by those skilled in the art. The practitioner responsible for administration typically determines the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual patient. The dosage can be adjusted by the individual physician in the event of any complications.

[0059] In some embodiments, a therapeutically effective amount typically varies from about 0.001 mg / kg to about 1000 mg / kg, about 0.01 mg / kg to about 750 mg / kg, about 100 mg / kg to about 500 mg / kg, about 1 mg / kg to about 250 mg / kg, or about 10 mg / kg to about 150 mg / kg, administered in one or more doses daily for one or several days (depending, of course, on the mode of administration and the factors mentioned above). Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day. In some embodiments, the amount is less than 10,000 mg per day, in the range of 750 mg to 9,000 mg per day.

[0060] In some embodiments, the amount of active compound in the pharmaceutical formulation is from about 2 to about 75% by weight. In some of these embodiments, the amount is from about 25 to about 60% by weight.

[0061] The drug is intended to be administered in single or multiple doses.The desired time interval for the delivery of multiple doses can be determined by those skilled in the art using only routine experimentation.For example, the drug can be administered to patients twice a day, about 12 hours apart.In some embodiments, the drug is administered once a day.

[0062] The drug(s) can be administered on a regular schedule. As used herein, a regular schedule refers to a predetermined, specified period of time. A regular schedule can encompass periods of the same or different lengths, as long as the schedule is a predetermined schedule. For example, a regular schedule can involve administration twice a day, daily, every two days, every three days, every four days, every five days, every six days, weekly, monthly, or any set number of days or weeks therebetween. Alternatively, a predetermined regular schedule may include administration twice a day for the first week, followed by daily administration for several months. In other embodiments, the present invention provides that the drug(s) can be administered orally, and the timing may or may not depend on food intake. Thus, for example, the drug can be taken every morning and / or every evening, regardless of when the patient has eaten or will eat.

[0063] II. Indications A. Infectious diseases In some embodiments, the cell targeting moiety may target bacterial cells, protozoan cells, viruses, fungal cells, or another type of parasitic cell. In the present disclosure, the charged π-conjugated compounds described herein may be used to reduce the number of pathogenic cells and, therefore, potentially be used to treat various diseases or conditions associated with or caused by bacteria, protozoans, viruses, fungi, or other types of parasitic cells. In some embodiments, the charged π-conjugated compounds described herein are intended to permeabilize cell membranes, thereby allowing at least a second therapeutic agent to enter bacterial cells, protozoan cells, viruses, fungal cells, or other types of parasitic cells. In some aspects, it is anticipated that the charged π-conjugated compounds described herein may be used to treat virtually any malignancy associated with or caused by bacteria, protozoans, viruses, fungi, or other types of parasitic cells.

[0064] i. Bacterial pathogens Despite decades of efforts to develop antibiotics, hundreds of bacterial pathogens, both Gram-positive and Gram-negative, cause significant illness and death worldwide. Indeed, antibiotic resistance is an increasing challenge in the fight against bacterial diseases.

[0065] One of the bacterial diseases with the greatest disease burden is tuberculosis, caused by Mycobacterium tuberculosis and claiming approximately 2 million lives annually, primarily in sub-Saharan Africa. Non-limiting examples of Mycobacterium tuberculosis antigens include recombinant Ag85A, Ag85B, ESAT6, TB10.4, or fragments thereof, as taught in Ottenhoff and Kaufmann (2012), incorporated herein by reference. Pathogenic bacteria also contribute to other diseases of global importance, such as pneumonia caused by bacteria such as Streptococcus, Staphylococcus, Klebsiella, Pseudomonas, and Escherichia coli, and food poisoning caused by bacteria such as Shigella, Campylobacter, Salmonella, Listeria, Clostridium, and Vibrio. Pathogenic bacteria cause infectious diseases such as tetanus, typhoid, diphtheria, syphilis, and leprosy, as well as urinary tract infections, skin infections, and other types of illnesses.

[0066] Conditionally pathogenic bacteria become pathogenic only under certain conditions, such as when a wound facilitates their entry into the bloodstream or when the immune system is weakened. For example, Staphylococcus aureus and Streptococcus aureus are part of the normal human flora and typically reside on the skin or in the nose without causing disease. However, they can cause skin infections, pneumonia, meningitis, and even severe sepsis (a systemic inflammatory response leading to shock, massive vasodilation, and death). Some species of bacteria, such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium, are opportunistic pathogens that primarily cause disease in individuals with immunosuppression or cystic fibrosis.

[0067] Other bacteria, without exception, cause disease in humans, including obligate intracellular parasites (e.g., chlamydia, ehrlichia, and rickettsia) that can grow and reproduce only within the cells of other organisms and cause a variety of illnesses, from asymptomatic infections during incubation to more serious diseases such as meningitis, sepsis, and abscesses. For example, rickettsiae can cause typhus or Rocky Mountain spotted fever, while chlamydiae can cause pneumonia, genital infections, eye infections, and coronary artery disease. Other facultative (non-obligate) intracellular parasites include Mycobacterium, which can cause tuberculosis and leprosy; Brucella, which can cause brucellosis; Francisella, which can cause tularemia; Legionella, which can cause Legionnaires' disease; Listeria, which can cause listeriosis; Bordetella pertussis, which can cause whooping cough; Yersinia pestis, which can cause the plague; and Borrelia burgdorferi, which can cause Lyme disease. The cell membranes of these bacteria can be disrupted using the methods described herein.

[0068] ii. Viral pathogens Viral pathogens are a significant health problem. These pathogens include respiratory viruses such as adenoviruses, influenza A and B viruses, measles, parainfluenza viruses, respiratory syncytial virus (RSV), rhinoviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2; gastrointestinal viruses such as coxsackieviruses; enteroviruses such as poliovirus and rotavirus; hepatitis viruses such as hepatitis B virus (HBV), hepatitis C virus, and bovine viral diarrhea virus (BVDV); human papillomavirus (HPV); herpes simplex virus type 1 (HSV-1); herpes simplex virus type 2 (HSV-2); human cytomegalovirus (HCMV); and and herpesviruses such as varicella-zoster virus (VZV), retroviruses such as human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2), dengue virus, hantavirus, hemorrhagic fever virus, lymphocytic choriomeningitis virus, smallpox virus, norovirus, chikungunya virus, Ebola virus, rabies virus, West Nile virus envelope protein (E) and premembrane protein (prM), dengue virus E protein, HIV envelope proteins gp41 and gp120, rotavirus VP6 and enterovirus VP1, human papillomavirus type 1 (HPV-1), or fragments thereof. Non-limiting examples of viral antigens include hepatitis B virus (HBV) surface antigen and core antigen, influenza virus hemagglutinin antigen and neuraminidase antigen, West Nile virus envelope protein (E) and premembrane protein (prM), dengue virus E protein, Ebola virus glycoprotein, HIV envelope proteins gp41 and gp120, rotavirus VP6 and enterovirus VP1, human papillomavirus type 1 (HPV-1), or fragments thereof. Other HIV antigens can be found in de Taeye et al. (2016), which is incorporated herein by reference. The cell membranes of any of these viral pathogens may be disrupted using the methods described herein.

[0069] iii. Fungal pathogens Pathogenic fungi are fungi that cause disease in humans or other organisms. The following are just a few examples:

[0070] Candida species are important human pathogens, well known for causing opportunistic infections in immunocompromised hosts, such as transplant recipients, HIV-infected and AIDS patients, and cancer patients. These infections can be difficult to treat and can be very serious. Aspergillus species can cause disease by producing mycotoxins, inducing allergic reactions, and causing local or systemic infections. The type of infection varies depending on the host's immune status. The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus, but other species can also cause disease. Cryptococcus neoformans is the most common species that can cause severe meningitis and meningoencephalitis in HIV-infected and AIDS patients. Most Cryptococcus species reside in soil and do not cause disease in humans. However, Cryptococcus laurentii and Cryptococcus albidus are known to occasionally cause moderate to severe disease in immunocompromised patients. Cryptococcus gattii, endemic to tropical regions of Africa and Australia, can cause disease in both immunocompromised and immunocompetent individuals. Histoplasma capsulatum can cause histoplasmosis in humans, dogs, cats, birds, and reptiles, especially in areas with a high concentration of bird or bat droppings. Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in immunocompromised individuals, including premature infants, the elderly, transplant recipients, and AIDS patients, as well as in immunocompetent individuals. Stachybotrys charterum (also known as "black mold") can cause respiratory damage and severe headaches and is common in homes in chronically humid areas.Additionally, Coccidioides immitis can cause a severe infection known as valley fever, which is most prevalent in arid and semi-arid regions of the Americas; Paracoccidioides brasiliensis can cause paracoccidioidomycosis, the most common systemic mycosis in Latin America; Trichophyton rubrum can cause dermatophytosis, a type of fungal skin infection; and Alternaria alternata can cause allergic reactions such as asthma, hay fever, and rhinitis. The cell membranes of these fungi can be disrupted using the methods described herein.

[0071] iv. Parasites Parasites pose a major health problem worldwide, especially in developing countries. Important pathogenic parasites include Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Trypanosoma canobiensis, Trypanosoma rhodesiensis, Trypanosoma cruzi, Ascaris lumbricoides, Trichinella spiralis, Toxoplasma gondii, Leishmania donovani, Leishmania tropicalis, Leishmania braziliensis, Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Pneumocystis jiroveci, Ucheleria bancrofti, Ancylostoma duodenum, Necator americanus, Strongyloides starcoralis, Cryptosporidium parvum, Enterobius vermicularis, and Taenia solium. The cell membranes of these parasites can be disrupted using the methods described herein.

[0072] v. Cancer and other hyperproliferative diseases and disorders Hyperproliferative diseases can be associated with any condition in which cells begin to proliferate uncontrollably, with cancer being a common example. One of the key components of cancer is the disruption of the normal apoptotic cycle of cells, and therefore, agents that induce apoptosis in cells are important therapeutic agents in the treatment of these diseases. In the present disclosure, compounds of the present disclosure have been shown to kill cancer cells and, therefore, may be used to treat various types of cancer cell lines. In some embodiments, the method involves killing cancer cells through a programmed cell death mechanism, such as apoptosis. In some embodiments, the method is an improvement over known methods in that it does not require photoactivation. In some embodiments, the method is an improvement over known methods in that it does not require photothermal activation. In some embodiments, the method is an improvement over known methods in that it does not involve the generation of reactive oxygen species.

[0073] In some embodiments, the method involves selective targeting of cancer cells with a charged π-conjugated compound. Thus, the compounds of the present disclosure can be used to effectively treat cancers such as malignant melanoma. In various aspects, it is expected that the compounds of the present disclosure can be used to treat virtually any malignant tumor.

[0074] Cancer cells that may be treated with compounds of the present disclosure according to embodiments include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, oral cavity, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Furthermore, the cancer may specifically be of the following histological types, but is not limited to: malignant tumor, carcinoma, undifferentiated carcinoma, giant cell carcinoma and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, bile duct carcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and bile duct carcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma within an adenomatous polyp, adenocarcinoma in familial polyposis coli, solid carcinoma, malignant gastrinoma, Rutinoid tumor, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophilic carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-encapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrial carcinoma, skin adnexal carcinoma, apocrine gland carcinoma, sebaceous gland adenocarcinoma, ceruminous adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, breast Glandular Paget's disease, acinic cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant theca cell tumor, malignant granulosa cell tumor, malignant androblastoma, Sertoli cell carcinoma, malignant Leydig cell tumor, malignant adipocyte tumor, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomerular angiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant melanoma in giant pigmented nevus, epithelioid cell type malignant melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant Fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant ovarian stromatoid tumour, choriocarcinoma, malignant mesonephroma, angiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, paracortical osteosarcoma, chondrosarcoma, malignant chondroblastoma,Mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic odontoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroglioma, primitive neuroectodermal tumor, cerebellar sarcoma, ganglioneuroma, neuroblastoma, retinoblastoma, olfactory nerve tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemmoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's tumors include, but are not limited to, lymphoma, Hodgkin's lymphoma, paragranulomatous lymphoma, malignant lymphoma (small lymphocytic), malignant lymphoma (large cell, diffuse), malignant lymphoma (follicular), mycosis fungoides, other certain non-Hodgkin's lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia. In certain embodiments, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia.

[0075] III. Cell targeting site In some aspects, the present disclosure provides compounds linked directly or via a linker to a cell targeting moiety. In some embodiments, linking the compound to the cell targeting moiety allows the charged π-conjugated compound to more effectively bind and penetrate target cells, thereby increasing the specificity and efficacy of the compound in treating a disease or disorder, while avoiding non-target cells and reducing the toxicity of antibacterial therapy. The target cell can be a microbial pathogen itself or a mammalian cell infected with an intracellular pathogen.

[0076] In some embodiments, the cell targeting moiety may be, for example, an antibody, lipid, carbohydrate, polysaccharide, growth factor, peptide, aptamer, small molecule such as a hormone, imaging agent, cofactor, or cytokine. In some embodiments, the cell targeting moiety is a functional group that associates with the cell membrane, a carbohydrate or polysaccharide that binds to one or more markers on the cell membrane, a lipid that binds to one or more markers on the cell membrane, a small molecule that binds to one or more markers on the cell membrane, an aptamer that binds to one or more markers on the cell membrane, or a peptide or antibody that binds to one or more markers on the cell membrane. In some embodiments, the cell targeting moiety may target human cells infected with an intracellular pathogen, whereby the cell targeting moiety recognizes specific markers or molecules produced by the pathogen and present on the surface of infected cells and avoids markers or molecules present on healthy cells. These specific markers include adhesion molecules, cell surface receptors, surface enzymes, lipopolysaccharides, lipoproteins, peptidoglycans, and other pathogen-specific proteins and / or metabolites. Thus, in some embodiments, the compounds of the present disclosure may be used as conjugates with antibodies against specific antigens, for example, that are expressed on infected cells but not on healthy cells. The conjugates may be formed by covalent bonds, electrostatic interactions, or hydrophobic interactions between the cell targeting moiety and the antibody, allowing the antibody to specifically target and bind to infected cells.

[0077] In certain embodiments, the cell targeting group is a functional group, such as a positively charged group, such as amine.Positively charged group can be used to associate with the negatively charged group on the surface of cell membrane.It is contemplated that this group can be used to associate with other negatively charged groups, such as negatively charged phospholipids, proteins or nucleic acids.

[0078] Furthermore, specific cell surface molecules overproduced by infected mammalian cells can be used as cell targeting moieties for antimicrobial therapy using charged π-conjugated compounds. These include molecules such as lectins, cell adhesion molecules, and integrins, which are overexpressed on the surface of infected cells and can be used as cell-specific targeting moieties for antimicrobial therapy. Several cell surface receptors, such as Toll-like receptors, C-type lectins, and mannose receptors, are also overexpressed on infected mammalian cells, and these can be targeted to specifically deliver charged π-conjugated compounds to infected cells.

[0079] Additional cell targeting moieties that can be used include cofactors, sugars, drug molecules, imaging agents, or fluorescent dyes. Other molecules, such as cytokines, chemokines, and transcription factors, can also be used as cell targeting moieties for certain types of infections by binding to specific receptors on the surface of infected cells. For example, cytokines are proteins released from cells in response to infection. They can bind to receptors on infected cells and trigger various responses, such as increased inflammation, increased production of infection-fighting molecules, or increased migration of immune cells to the site of infection. Specific cytokines that can be used as cell targeting moieties in antibacterial therapy include tumor necrosis factor (TNF), interleukin-1 (IL-1), and interferon-γ (IFN-γ). Chemokines are molecules released from cells that attract and recruit other cells to the site of infection. Specific chemokines that can be used as cell targeting moieties include interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α). Transcription factors are molecules that control the expression of certain genes within cells and can be used to target specific genes essential for pathogen replication. By binding to these receptors, the cellular targeting moieties specifically target charged π-conjugated compounds to infected cells while sparing uninfected cells. Specific transcription factors that can be used as cellular targeting moieties include NF-κB and AP-1.

[0080] Furthermore, in some embodiments, the cell targeting moiety can be a peptide sequence or cyclic peptide designed to recognize and bind to a specific receptor or molecule on the surface of infected cells, thereby allowing the charged π-conjugated compound to be delivered directly to infected cells while avoiding uninfected cells. Examples of cell and tissue targeting peptides that can be used in accordance with the embodiments include peptides that recognize bacterial flagellin proteins, peptidoglycans, lipopolysaccharides, or lipoprotein molecules. Examples of such targeting peptides are described, for example, in U.S. Patent Nos. 9,072,793, 9,597,407, and U.S. Patent Application No. 2017 / 0362307, each of which is incorporated herein by reference.

[0081] Therefore, in some embodiments, cell targeting moiety is antibody or avimer.Antibody and avimer can be made against virtually any cell surface marker, thus providing a method for targeting charged π-conjugated compounds to virtually any cell population of interest.The method for making antibodies that can be used as cell targeting moiety is described in detail below.The method for making avimers that bind to given cell surface markers is described in detail in US Patent Publication No. 2006 / 0234299 and US Patent Publication No. 2006 / 0223114, each of which is incorporated herein by reference.

[0082] Furthermore, it is contemplated that the compounds described herein may be bound to nanoparticles or other nanomaterials.Some non-limiting examples of nanoparticles include metal nanoparticles such as gold or silver nanoparticles, or polymer nanoparticles such as poly-l-lactic acid or poly(ethylene)glycol polymers.Nanoparticles and nanomaterials that can be bound to the compounds of the present invention include those described in U.S. Patent Publication Nos. 2006 / 0034925, 2006 / 0115537, 2007 / 0148095, 2012 / 0141550, 2013 / 0138032, and 2014 / 0024610, and PCT Publication Nos. 2008 / 121949, 2011 / 053435, and 2014 / 087413, each of which is incorporated herein by reference.

[0083] IV. Therapy A. Treatment method In particular, disclosed herein are compositions that can be used to treat diseases or disorders in subjects (e.g., human subjects). The compositions described above are preferably administered to mammals (e.g., rodents, humans, non-human primates, dogs, cattle, sheep, horses, cats, etc.) in effective amounts, i.e., amounts capable of producing the desired results in the treated subject (e.g., slowing, halting, reducing, or eliminating one or more symptoms or underlying causes of the disease). The toxicity and therapeutic efficacy of the compositions used in the disclosed methods can be determined by standard pharmaceutical procedures. As is well known in the fields of medicine and veterinary medicine, the dosage administered to a single animal will vary depending on many factors, including the subject's size, body surface area, weight, age, the specific composition administered, the time and route of administration, general health, clinical symptoms, and other drugs administered concomitantly. In some embodiments, the amount of charged π-conjugated compound used is calculated to be between about 0.01 mg and about 10,000 mg per day. In some embodiments, the amount is between about 1 mg and about 1,000 mg per day. In some embodiments, these dosages may be reduced or increased based on specific patient biological factors, such as increased or decreased metabolic degradation of the drug or decreased absorption by the gastrointestinal tract when administered orally. Furthermore, charged π-conjugated compounds may be more effective, thus requiring lower doses to achieve similar effects. Such doses are typically administered once daily for several weeks or for a period long enough to achieve clinical benefit.

[0084] Therapeutic methods (including prophylactic treatments) of the present disclosure generally involve administering a therapeutically effective amount of a composition described herein to a subject in need thereof, including a mammal, particularly a human. Such treatments are suitably administered to subjects, particularly humans, who are suffering from, suffering from, susceptible to, or at risk of developing a disease, disorder, or a symptom thereof. Determining a subject as "at risk" can be made by objective or subjective determination, such as by diagnostic testing or the subject's or a healthcare provider's opinion (e.g., microbiological analysis, genetic testing, enzyme or protein markers, family history, etc.).

[0085] B. Combination Therapy It is envisioned that the charged π-conjugated compounds described herein may be used in combination with one or more additional therapies or compounds that alleviate one or more side effects experienced by patients.In the medical field, it is common to use multiple therapies in combination.The following is a general discussion of the therapies that can be used in combination with the therapies disclosed herein.

[0086] To treat a disease or disorder using the methods and compositions of the present disclosure, a cell or subject will typically be contacted with a charged π-conjugated compound and at least one other therapeutic agent. These therapeutic agents are provided in combination in amounts effective to achieve a reduction in one or more disease parameters. This process can include contacting a cell / subject with both agents / therapies simultaneously, for example, by using a single composition or pharmacological formulation containing both agents, or by contacting the cell / subject with two different compositions or formulations simultaneously, where one composition contains the compound and the other composition contains the other agent.

[0087] Alternatively, the compounds described herein may precede or follow the other treatment by intervals ranging from minutes to weeks. Generally, no significant time should elapse between each delivery, allowing the treatments to still exert their combined beneficial effect on the cells / subject. In such cases, it is contemplated that the two forms of contact with the cells will occur within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay of only about 1-2 hours. In some situations, it may be desirable to extend the treatment period significantly, with several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapsed between each administration.

[0088] It is also contemplated that more than one administration of either the compound or the other therapy may be desired. As exemplified below, where a compound of the present disclosure is "A" and the other therapy is "B," various combinations may be used. [ka] Other combinations are also contemplated. Other potential therapeutic modalities that may be used in combination with the compounds of the present disclosure are presented elsewhere herein.

[0089] V. Chemical background In some embodiments, the charged π-conjugated compounds of the present disclosure can be synthesized using the organic chemistry methods described herein. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by those skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated herein by reference.

[0090] A. Process Scale-Up In addition, the synthetic methods described herein can be further modified and optimized using the principles and techniques of process chemistry as applied by those skilled in the art for preliminary, pilot-scale, or large-scale production, either batch or continuous. Such principles and techniques are taught, for example, in Practical Process Research & Development (2000), which is incorporated herein by reference. The synthetic methods described herein can also be used to produce the compounds described herein in preliminary-scale quantities.

[0091] B. Definition of Chemical Substances When used in the context of chemical groups, "hydrogen" means -H, "hydroxy" means -OH, "oxo" means =O, "carbonyl" means -C(=O)-, "carboxy" means -C(=O)OH (also written as -COOH or -COH), "halo" means -F, -Cl, -Br, or -I, respectively, "amino" means -NH, "hydroxyamino" means -NHOH, "nitro" means -NO, "imino" means =NH, and "cyano" means -C. "Isocyanyl" means -N=C=O, "azido" means -N3, in the monovalent context "phosphate" means -OP(O)(OH)2 or its deprotonated form, and in the divalent context "phosphate" means -OP(O)(OH)O- or its deprotonated form, "mercapto" means -SH, and "thio" means =S, "thiocarbonyl" means -C(=S)-, "sulfonyl" means -S(O)2-, and "sulfinyl" means -S(O)-.

[0092] In the context of chemical formulas, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. [ka] represents an optional bond, which, if present, is either a single or double bond. [ka] represents a single or double bond. [ka] For example, [ka] It is understood that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when connecting one or two asymmetric atoms. Instead, it encompasses all stereoisomers and mixtures thereof. [ka] is drawn perpendicularly across the bond (e.g., for methyl, [ka] ) indicates the point of attachment of the group. Note that points of attachment are typically only identified in this manner for larger groups to aid the reader in unambiguously identifying the point of attachment. [ka] denotes a single bond where the group attached to the thick end of the wedge is "off the page". [ka] means a single bond, with the group attached to the thick end of the wedge "into the page". [ka] means a single bond where the geometry around the double bond (e.g., either E or Z) is undefined. Thus, both options and combinations thereof are contemplated. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented out of the plane of the paper.

[0093] When a variable is depicted as a "floating group" on a ring system, for example, the group "R" in the formula: [ka] A variable may replace any hydrogen atom bonded to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is formed. When a variable is depicted as a "floating group" on a fused ring system, for example, the group "R" in the formula: [ka] A variable may replace any hydrogen bonded to any of the ring atoms of any of the fused rings, unless otherwise specified. Replaceable hydrogens include depicted hydrogens (e.g., hydrogens bonded to nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and optional hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens bonded to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R can be on either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the parenthesized R represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than or equal to 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0094] In chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" or "C=n" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum being the smallest possible number for that group / class. For example, "alkyl (C≦8) ", "Alkanediyl (C≦8) ", "heteroaryl(C≦8) ", and "Acyl (C≦8) The minimum number of carbon atoms in the " group is 1, and " alkenyl (C≦8) ", "alkynyl (C≦8) ", and "heterocycloalkyl (C≦8) The minimum number of carbon atoms in the "cycloalkyl" group is two. (C≦8) The minimum number of carbon atoms in the "aryl" group is three. (C≦8) " and "Arrangeil (C≦8) It is understood that the minimum number of carbon atoms in a "Cn-n'" group is 6. "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Thus, "alkyl (C2~10) " denotes an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class that they modify, which may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C 1-4 -alkyl", "C 1-4 -alkyl," "alkyl (C1-4) " and "Alkyl (C≦4) " are all synonyms. Except as noted below, all carbon atoms are counted to determine whether a group or compound applies with a specified number of carbon atoms. For example, a dihexylamino group is a dialkylamino group. (C12) However, this is an example of a dialkylamino group. (C6) Similarly, phenylethyl is not an example of an aralkyl group. (C=8) is an example of a group. When any of the chemical groups or compound classes defined herein are modified by the term "substituted," any carbon atoms in the moiety that replace a hydrogen atom are not counted. Thus, methoxyhexyl, which has a total of 7 carbon atoms, is a substituted alkyl group. (C1-6) Unless otherwise specified, any chemical group or class of compounds recited in a claim group without a carbon atom limit has a carbon atom limit of 12 or less.

[0095] When used to modify a compound or chemical group, the term "saturated" means that the compound or chemical group has no carbon-carbon double bonds or carbon-carbon triple bonds, except as described below. When the term is used to modify an atom, it means that the atom is not part of a double or triple bond. In the case of substituted versions of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. When such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that the substance cannot be further dissolved in the solution.

[0096] The term "aliphatic" indicates that the compound or chemical group so modified is an acyclic or cyclic compound or group, but a non-aromatic compound or group. Within an aliphatic compound / group, the carbon atoms can be linked together in a straight chain, a branched chain, or a non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated, i.e., linked by a carbon-carbon single bond (alkane / alkyl), or unsaturated, linked by one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0097] The term "aromatic" indicates that the compound or chemical group so modified has a planar, unsaturated ring of atoms with 4n+2 electrons in a fully conjugated cyclic π system. An aromatic compound or chemical group may be represented as a single resonance structure; however, depiction of one resonance structure is considered to refer to other resonance structures as well. For example, [ka] What is that? [ka] It can also be interpreted as referring to

[0098] Aromatic compounds may also be depicted using circles to represent the delocalized nature of electrons within a fully conjugated cyclic π system, two non-limiting examples of which are shown below. [ka]

[0099] The term "alkyl" refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight or branched non-cyclic structure, and containing no atoms other than carbon and hydrogen, such as -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(-Pr, i Non-limiting examples of alkyl groups include -Pr or isopropyl), -CH2CH2CH2CH3(-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neopentyl). The term "alkanediyl" refers to a divalent saturated aliphatic group having one or two saturated carbon atom(s) as the point(s) of attachment, a straight-chain or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- groups are non-limiting examples of alkanediyl groups. The term "alkylidene" refers to the divalent group =CRR', where R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include =CH2, =CH(CH2CH3), and =C(CH3)2. "Alkane" refers to the class of compounds having the formula H-R, where R is alkyl as defined above.

[0100] The term "cycloalkyl" refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, which carbon atom forms part of one or more non-aromatic ring structures, and which has no carbon-carbon double or triple bonds and no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limits permitting) attached to a carbon atom of the non-aromatic ring structure. The term "cycloalkanediyl" refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Group [ka] is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula H-R, where R is cycloalkyl as defined above.

[0101] The term "alkenyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It should be noted that although alkenediyl groups are aliphatic, when attached at both ends, the group is not excluded from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula H-R, where R is alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene having only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule.

[0102] The term "alkynyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight-chain or branched non-cyclic structure, having at least one carbon-carbon triple bond, and having no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. -C≡CH, -C≡CCH3, and -CH2C≡CCH3 groups are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula H-R, where R is alkynyl.

[0103] The term "aryl" refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more aromatic ring structures, each of which has six ring atoms, all of which are carbon, and which consists of atoms other than carbon and hydrogen. When two or more rings are present, the rings may be fused or unfused. Unfused rings are connected by covalent bonds. As used herein, the term aryl does not exclude the presence of one or more alkyl groups (carbon number limits permitting) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and monovalent groups derived from biphenyl (e.g., 4-phenylphenyl). The term "arenadiyl" refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structures, each of the one or more six-membered aromatic ring structures having six ring atoms, all of which are carbon, and consisting of atoms other than carbon and hydrogen. As used herein, the term arenadiyl does not exclude the presence of one or more alkyl groups (carbon number limits permitting) attached to the first aromatic ring or any additional aromatic rings present. When two or more rings are present, the rings may be fused or unfused. Unfused rings are connected by a covalent bond. Non-limiting examples of arenadiyl groups include: [ka]

[0104] "Arene" refers to the class of compounds having the formula H-R, where R is aryl as defined above. Benzene and toluene are non-limiting examples of arenes.

[0105] The term "aralkyl" refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0106] The term "heteroaryl" refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, each of the one or more aromatic ring structures having from 3 to 8 ring atoms, wherein at least one of the ring atoms in the aromatic ring structure(s) is nitrogen, oxygen, or sulfur, and the heteroaryl group consists solely of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings are fused, but the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limits permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl, isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the point of attachment. "Heteroarene" refers to the class of compounds having the formula H-R, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0107] The term "heteroaralkyl" refers to the monovalent group -alkanediyl-heteroaryl, where the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are pyridinylmethyl and 2-quinolinyl-ethyl.

[0108] The term "heterocycloalkyl" refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom being part of one or more non-aromatic ring structures, each of which has 3 to 8 ring atoms, wherein at least one of the ring atoms in the non-aromatic ring structure(s) is nitrogen, oxygen, or sulfur, and the heterocycloalkyl group consists solely of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When more than one ring is present, the rings are fused rings. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limits permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds within the ring or ring system, provided that the resulting group is still non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the attachment point. N-pyrrolidinyl is an example of such a group.

[0109] The term "heterocyclic alkyl" refers to the monovalent group -alkanediyl-heterocyclic alkyl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples include morpholinylmethyl and piperidinylethyl.

[0110] The term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, or aryl, as defined above. The groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH(CH3), -C(O)CH(CH2), -C(O)CH5, and -C(O)CH4CH3 are non-limiting examples of acyl groups. "Thioacyl" is defined in an analogous manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkyl group, as defined above, attached to a -CHO group.

[0111] The term "alkoxy" refers to the group -OR, where R is alkyl as defined above. Non-limiting examples include -OCH (methoxy), -OCHCH (ethoxy), -OCHCHCH, -OCH(CH) (isopropoxy), or -OC(CH) (tert-butoxy). When used without the "substituted" modifier, the terms "cycloalkoxy," "alkenyloxy," "alkynyloxy," "aryloxy," "aralkoxy," "heteroaryloxy," "heterocycloalkoxy," and "acyloxy" refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The terms "alkylthio" and "acylthio" refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group.

[0112] The term "alkylamino" refers to the group -NHR, where R is alkyl as defined above. Non-limiting examples include -NHCH and -NHCHCH. The term "dialkylamino" refers to the group -NRR', where R and R' can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include -N(CH) and -N(CH)(CHCH). The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl as defined above. A non-limiting example of an amido group is -NHC(O)CH.

[0113] When a chemical group is used with the modifier "substituted," one or more hydrogen atoms have been replaced, independently in each occurrence, with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -COCHCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. For example, the following groups are non-limiting examples of substituted alkyl groups: -CHOH, -CHCl, -CF, -CHCN, -CHC(O)OH, -CHC(O)OCH, -CHC(O)NH, -CHC(O)CH, -CHOCH, -CHOC(O)CH, -CHNH, -CHN(CH), and -CHCHCl. ​​The term "haloalkyl" is a subset of substituted alkyl where the replacement of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or -I) such that no other atoms other than carbon, hydrogen, and halogen are present. The -CHCl group is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl where the replacement of hydrogen atoms is limited to fluoro such that no other atoms other than carbon, hydrogen, and fluorine are present. -CHF, -CF, and -CHCF groups are non-limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls include (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-eth-1-yl. -C(O)CHCF, -COH (carboxyl), -COCH (methylcarboxyl), -COCHCH, -C(O)NH (carbamoyl), and -CON(CH) groups are non-limiting examples of substituted acyl groups. -NHC(O)OCH and -NHC(O)NHCH groups are non-limiting examples of substituted amide groups.

[0114] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0115] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error due to the device or method used to determine the value, or the variation that exists between test subjects or patients. Unless otherwise specified, the term "about" is used to indicate a value of ±10% of the reported value, preferably ±5% of the reported value. It should be understood that whenever the term "about" is used, a specific reference to the exact numerical value indicated is also included.

[0116] An "active ingredient" (AI) or active pharmaceutical ingredient (API) (also called an active compound, active substance, active agent, pharmaceutical drug, drug, biologically active molecule, or therapeutic compound) is the component in a pharmaceutical drug that is biologically active.

[0117] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other unlisted steps.

[0118] The terms "effective" or "sufficient," as used herein and / or in the claims, mean sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means the amount of compound that, when administered to a patient or subject, is sufficient to effect such treatment or prevention of a disease, as defined below.

[0119] An "excipient" is a pharmaceutically acceptable substance formulated with the active ingredient(s) of a drug, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, extend the composition (and thus, when used for this purpose, are often referred to as "bulking agents," "fillers," or "diluents"), or to impart therapeutic enhancements to the active ingredient in the final dosage form (e.g., to promote drug absorption, reduce viscosity, or improve solubility). Excipients include anti-adherents, binders, coating agents, colorants, disintegrants, flavorings, glidants, lubricants, preservatives, adsorbents, sweeteners, and pharmaceutically acceptable forms of vehicles. The primary excipient, which functions as a vehicle for transporting the active ingredient, is usually referred to as the vehicle. Excipients may also be used to aid in in vitro stability, e.g., prevention of denaturation or aggregation, over the expected shelf life, as well as to aid in the manufacturing process, e.g., in handling of the active substance, e.g., by promoting powder flow or non-stick properties. The suitability of an excipient typically varies depending on the route of administration, dosage form, active ingredient, and other factors.

[0120] The term "hydrate," when used as a modifier of a compound, means that the compound has less than one (e.g., a hemihydrate), one (e.g., a monohydrate), or two or more (e.g., a dihydrate) water molecules associated with each compound molecule, e.g., in the solid form of the compound.

[0121] As used herein, "IC50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates the amount of a particular drug or other substance (inhibitor) required to inhibit a given biological, biochemical, or chemical process (or component of the process, i.e., enzyme, cell, cell receptor, or microorganism) by half. 50 The term "effective concentration" refers to the amount that produces a half-maximal response.

[0122] An "isomer" of a first compound is a distinct compound, each molecule of which contains the same constituent atoms as the first compound, but differs in the arrangement of those atoms in three dimensions.

[0123] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0124] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0125] "Pharmaceutically acceptable salts" refers to salts of the compounds disclosed herein that are pharmaceutically acceptable, as defined above, and that possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or salts formed with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]octen-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentane sulfonate, and the like. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as propionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, t-butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts, which can be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It will be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt as a whole is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their methods of preparation and uses are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0126] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable substance formulated with an active ingredient drug to carry, deliver, and / or transport the chemical agent. Drug carriers can be used to improve drug delivery and effectiveness, including, for example, controlled-release technologies to regulate drug bioavailability, reduce drug metabolism, and / or reduce drug toxicity. Some drug carriers can increase the effectiveness of drug delivery to specific target sites. Examples of carriers include liposomes, microspheres (e.g., those composed of poly(lactic-co-glycolic acid)), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes, and dendrimers.

[0127] A "pharmaceutical drug" (also referred to as a medicine, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicament, drug, medicine, or simply drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease; the composition contains an active pharmaceutical ingredient (API) (defined above) and, optionally, one or more inactive ingredients, also referred to as excipients (defined above).

[0128] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient, where the subject or patient may be at risk and / or predisposed to the disease, but has not yet experienced or exhibited any or all of the disease's pathology or symptomology, and / or (2) delaying the onset of a disease in a subject or patient, where the subject or patient may be at risk and / or predisposed to the disease, but has not yet experienced or exhibited any or all of the disease's pathology or symptomology.

[0129] "Prodrug" refers to a compound that can be metabolically converted into an active pharmaceutical ingredient of the present invention in vivo. The prodrug itself may or may not have activity in this prodrug form. For example, a compound containing a hydroxy group can be administered as an ester, and the ester is converted to the hydroxy compound in vivo by hydrolysis. Non-limiting examples of suitable esters that can be converted to the hydroxy compound in vivo include acetate, citrate, lactate, phosphate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-β-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyl tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinate, and esters of amino acids. Similarly, a compound containing an amine group can be administered as an amide, and the amide is converted to the amine compound in vivo by hydrolysis.

[0130] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the configuration of those atoms in three dimensions. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain chiral centers, also called stereogenic centers or asymmetric centers; chiral centers are any points within a molecule that bear groups, but not necessarily atoms, such that the interchange of any two groups results in a stereoisomer. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, but other atoms can be stereogenic centers in organic and inorganic compounds. Molecules can have multiple stereogenic centers, resulting in many stereoisomers. In compounds where stereoisomerism arises from a tetrahedral asymmetric center (e.g., a tetrahedral carbon), the total number of hypothetical possible stereoisomers does not exceed 2n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum number of possible stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched, such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or chirality axis where the stereochemistry is not defined, it is contemplated that the stereocenter or chirality axis can exist in the R-form, the S-form, or a mixture of the R- and S-forms, including racemic and non-racemic mixtures of the stereocenter or chirality axis. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer(s).

[0131] "Treatment" or "treating" includes (1) inhibiting the disease (e.g., halting further development of the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease, (2) ameliorating the disease (e.g., reversing the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease, and / or (3) causing any measurable decrease in the disease or its symptoms in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease.

[0132] The term "unit dose" refers to a formulation of a compound or composition prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that can be used include, but are not limited to, a single tablet, capsule, or other oral formulation, or a single vial or other injectable formulation containing a syringe-injectable liquid.

[0133] As used herein, the term "catalyst" refers to any compound or composition that promotes the progress of a reaction (i.e., increases the reaction rate) without being consumed by the reaction. The catalysts of the present disclosure may promote improved yields of a reaction under more favorable reaction conditions than corresponding methods known in the art. In some embodiments, the catalysts used in the methods disclosed herein are homogeneous catalysts. In some embodiments, the catalysts used in the methods disclosed herein are heterogeneous catalysts. Specific catalysts used in the methods disclosed herein are widely known in the art and commercially available. For example, one of skill in the art may find catalysts for use in the methods disclosed herein in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. Non-limiting examples of catalysts that can be used in the methods disclosed herein include tetrakis(triphenylphosphine)palladium (also referred to herein as Pd(PPh3)).

[0134] As used herein, the term "base" refers to any compound or composition that acts as an Allen-Hewis base, Bronsted base, or Lewis base in a reaction with another compound or composition, as understood by one of ordinary skill in the art. In some embodiments, a base according to the present disclosure reacts with another compound or composition to extract a proton (H) from the other compound or composition. + ) to accept or remove the base. In some embodiments, the base is an organic base. In some embodiments, the base is an inorganic base. The base used in accordance with the present disclosure may be selected based on the pKa of the base, or any other property the base possesses that is deemed preferable by the artisan. Specific bases for use in the methods of the present disclosure are widely known in the art and can be found by those of skill in the art in texts or literature such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. Non-limiting examples of bases that can be used in the methods of the present disclosure include K3PO4, K2HPO4, KF, K2CO3, NaOtBu, NaOAc, CsF, or Cs2CO3.

[0135] The above definitions supersede any conflicting definitions in any reference incorporated herein by reference. However, the fact that a particular term is defined should not be construed as indicating that any term not defined is indefinite. Rather, all terms used are intended to describe the invention in terms such that one skilled in the art can understand the scope and practice the invention. [Example]

[0136] The following examples are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for that practice. However, those skilled in the art will understand, in light of the present disclosure, that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0137] Example 1 - Synthesis of charged π-conjugated compounds i. Synthesis of charged π-conjugated compounds and characterization of charged π-conjugated compounds Charged π-conjugated compounds were synthesized and then characterized by NMR spectroscopy, high-resolution mass spectrometry, and Fourier transform infrared spectroscopy. Chemical shift information and observed mass data are given in the synthesis description.

[0138] General information: All glassware was oven dried overnight before use. All reactions were carried out under a N2 atmosphere unless otherwise stated. All other chemicals were purchased from commercial suppliers and used without further purification. [ka]

[0139] General procedure for the synthesis of heterocyclic salts: Method 1: Compound A (1 equiv.) and RX (X = halogen or other leaving group such as OTs, OMs, OTf, etc., below) (1.2-1.5 equiv.) were mixed with CH3CN in a screw-top vial. The mixture was stirred, heated to reflux, and left overnight. The solid was filtered, washed with ether, and dried in vacuo.

[0140] Method 2: Compound A (1 equiv.) and R-Br (1.0 equiv.) were mixed in an 8 mL screw-top vial. The mixture was stirred, heated to 125°C, and left overnight. The white solid was washed with ethyl acetate / ether and recrystallized from methanol / ether. [ka]

[0141] General procedure: The corresponding pyridinium salt B (1 equiv.) and 4-bromoaniline (1 equiv.) were dissolved in methanol (4 mL) in an 8 mL vial, and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt A (1 equiv.), C (1 equiv.), and sodium acetate (3 equiv.) were added. The reaction mixture was further stirred at room temperature overnight. The crude product D was purified by flash column chromatography (silica gel, dichloromethane / methanol). [ka]

[0142] The corresponding pyridinium salt 175 (206 mg, 0.48 mmol) and 4-bromoaniline (103 mg, 0.3 mmol) were dissolved in methanol (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt 144 (176 mg, 0.5 mmol), 302 (286 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, then 10:1) to give 58 mg of a green solid, GL308-2. Yield: 19%. 1H NMR(600MHz,メタノール-d4) δ 8.27(d,J=8.17Hz,1H),8.23(d,J=8.17Hz,1H),8.11(t,1H),8.06-7.98(m ,5H),7.69-7.63(m,4H),7.55(d,J=8.80Hz,1H),7.52(t,1H),7.48(t,1H) ,6.67-6.58(m,2H),6.42(d,J=13.82Hz,1H),6.27(d,J=13.82Hz,1H),4.2 9(t,2H),3.79(s,3H),2.77(t,2H),2.43(s,6H),2.02(d,J=4.66Hz,12H). 13 C NMR(150 MHz,メタノール-d4) δ 174.95,171.89,171.50,149.47,140.24,139.83,133.86,132.70,132.20 ,131.76,130.37,130.24,129.74,129.69,128.20,127.95,127.41,127.24 ,125.96,125.61,124.85,124.32,122.01,121.84,110.66,110.25,104.2 2,102.16,55.27,51.09,50.45,48.16,44.52,41.59,30.71,26.27,25.97. C 40 H 44 N3 + [M-Br] + のHRMS(ESI):566.3530. Measured value: 566.3534.

change

[0143] The corresponding pyridinium salt GL175 (209 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.65 mmol) were dissolved in pyridine (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, the heterocyclic salt GL220 (143 mg, 0.5 mmol), GL302 (286 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, 10:1). 80 mg of a green solid was obtained. Yield: 27.5%. 1 H NMR (600MHz, methanol-d4) δ 8.48(d,J=9.20Hz,1H),.8.23(s,J =9.15Hz,2H),8.13-8.00(m,4H),7.87(d,J=8.52Hz,2H),7.75(t,1H),7.62(t,1H),7.52(t,1H),7.33(m,3H),6.96(d,J=13.8 3Hz,1H),6.69(t,1H),6.42(t,1H),5.84(d,J=12.51Hz,1H),4.31(s,3H),4.08(t,2H),2.70(t,2H),2.45(s,6H),1.95(s,6H), 13 C NMR (150 MHz, methanol-d₄) δ 164.55, 155.60, 150.45, 141.82, 140.81, 140.41, 139.80, 133.82, 130.69, 129.88, 129.72, 129.58, 129.55, 128.69, 127.33, 126.82, 126.70, 126.28, 124.68, 122.86, 121.44, 119.68, 117.50, 114.24, 109.31, 98.05, 54.70, 48.70, 48.05, 47.88, 44.45, 40.41, 37.20, 26.46. C 35 H 38 N3 + [M-Br] + HRMS(ESI): 500.3060. Found: 500.3087. [ka]

[0144] The corresponding pyridinium salt GL175 (237 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, the heterocyclic salt GL176-1 (150 mg, 0.5 mmol), GL302 (286 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, 10:1) to produce 54 mg of a green solid in 18.1% yield. 1 H NMR (600MHz, methanol-d4) δ 8.24(d,J=8.43Hz,1H), 8.04-7.99(m,4H), 7.66-7.63(m,2H), 7.58(d,J=8.95Hz,1H), 7.51(m,2H), 7.43(m,1H), 7.32- 7.27(m,2H), 6.61(t,2H), 6.33(m,2H), 4.33(t,2H), 3.64(s,3H), 2.80(t,2H), 2.45(s,6H), 1.99(s,6H), 1.73(s,6H). 13 C NMR (150 MHz, methanol-d4) δ 142.95, 141.05, 139.67, 133.13, 131.97, 131.31, 130.31, 129.70, 128.94, 128.67, 128.36, 128.13, 127.31, 125.79, 124.79, 124.64, 124.53, 121.89, 121.26, 110.48, 110.40, 107.96, 103.83, 102.86, 70.74, 55.28, 50.70, 48.98, 44.48, 41.71, 30.20, 26.51, 26.25. 36 H 42 N3 + [M-Br] + HRMS(ESI): 516.3373. Found: 516.3380. [ka]

[0145] The corresponding pyridinium salt GL175 (103 mg, 0.24 mmol) and 4-bromoaniline (49 mg, 0.29 mmol) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature for 30 min. Next, the heterocyclic salt GL219 (105 mg, 0.3 mmol), GL344 (132 mg, 0.3 mmol), and sodium acetate (116 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, then 10:1) to produce GL365-2 as a green solid in 22% yield. 1 H NMR (600MHz, methanol-d4) δ 8.62(d,J=8.83Hz,1H),8.23(d,J=8.83Hz,1H),8.08-7.98(m,4H),7.88-7.82(m,2H),7.71-7.63(m,3H),7.58(m,2H),7.48(t,1H),6.69-6. 60(m,2H),6.51(d,J=13.55Hz,1H),6.31(d,J=13.55Hz,1H),4.34(t,2H),4.22(s,3H),2.84(t,2H),2.49(s,6H),2.01(s,6H),1.78(s,6H). 13 C NMR (150 MHz, methanol-d4) δ 174.57, 139.71, 139.02, 137.44, 135.06, 131.85, 130.30, 129.70, 129.40, 128.17, 127.29, 126.73, 126.57, 125.71, 124.44, 121.87, 121.64, 121.30, 118.98, 110.30, 55.13, 53.40, 50.58, 49.00, 44.39, 43.06, 41.49, 36.15, 28.93, 26.38, 26.24, 19.84. 35 H 38 N3 + [M-Br] + HRMS(ESI): 500.3060. Found: 500.3087. [ka]

[0146] The corresponding pyridinium salt 175 (206 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt 144 (176 mg, 0.5 mmol), 288 (252 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, 10:1, 8:1) to produce GL291-2 as a green solid in 19% yield. 1 H NMR (600MHz, methanol-d4) δ 8.13(m,1H),7.91(m,3H),7.54-7.48(m,2H),7.37(m,1H),6.52(m,1H),6.24(m,1H),3.38(m,2H) ),3.02(m,2H),2.79(m,12H),1.89(m,6H),1.81(m,2H),1.64(m,2H),1.42(m,2H),1.33(m,2H). 13 C NMR (150 MHz, methanol-d4) δ 140.36,139.80,136.85,133.37,133.30,131.899,131.86,130.91,1 30.30,130.27,129.71,129.68,128.28,128.13,128.02,127.31,127. 21,124.58,124.51,122.99,121.95,121.92,110.68,110.52,65.50,5 7.55,53.42,42.17,30.60,27.83,26.28,26.12,24.24,20.65,14.05. C 45 H 52 N3 + [M-Br] + HRMS(ESI): 622.4156. Found: 622.4162. [ka]

[0147] GL291-3 was produced as a green solid in 10% yield. 1 H NMR (600MHz, methanol-d4) δ 8.26(d,J=8.54Hz,2H),8.08(t,2H),8.03-8.00(m,3H),7.97-7.94(m,2H),7.78-7.77(m,2H) ,7.66(m,2H),7.60(d,J=8.64Hz,2H),7.51(t,2H),6.64(t,2H),6.37(d,J=13.71Hz,2H),4.25(t, 4H),3.03(m,4H),2.80(s,12H),2.03(s,12H),1.93(m,4H),1.73(m,4H),1.59(m,4H),1.50(m,4H). C 51 H 67 N3 + [M-Br] + HRMS(ESI): 735.5360. Found: 735.5368. [ka]

[0148] The corresponding pyridinium salt 183 (237 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt 144 (176 mg, 0.5 mmol), 288 (322 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 30:1, 20:1, 10:1) to produce GL297-2 as a green solid in 22% yield. The product was confirmed by LCMS with a M: 694.4 and [M2+] / 2: 347.8. 1H NMR (600MHz, methanol-d4) δ 8.28(m,2H),8.08-8.01(m,4H),7.86-7.76(m,2H),7,70-7,62(m,4H),7.55-7.5 1(m,2H),6.81-6.72(m,1H),6.61-6.57(m,1H),6.41-6.34(m,1H),4.34-4.28(m ,2H),4.19(s,3H),3.83(d,J=6.96Hz,3H),2.90(m,2H),2.68(m,6H),2.21(d,J= 12.22Hz, 3H), 1.97 (s, 12H), 1.95 (m, 2H), 1.70 (m, 2H), 1.61 (m, 2H), 1.50 (m, 2H). [ka]

[0149] 144 (105 mg, 0.3 mmol), 175 (103 mg, 0.24 mmol), 328 (154 mg, 0.3 mmol), 4-bromoaniline (49 mg), NaOAc (116 mg), and methanol (4 mL) were mixed. The mixture was stirred at room temperature overnight. Then, the methanol was removed by rotary evaporation. The crude product was then purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1 to 10:1) to produce GL328-2 as a green solid in 21% yield. 1 H NMR (600MHz, methanol-d4) δ 8.26-8.24(m,2H),8.11-7.99(m,6H),7.72-7.63(m,3H),7.61(t,2H), 7.49(m,2H),6.65(m,2H),6.36(d,J=13.67Hz,2H),4.25(t,2H),3.76( s,3H),3.41-3.38(m,2H),3.15(s,9H),2.01(d,J=1.89Hz,12H),1.96- 1.91(m,2H),1.87-1.82(m,2H),1.66-1.61(m,2H),1.54-1.48(m,2H). 13C NMR (150 MHz, methanol-d4) δ 140.35, 139.83, 133.45, 133.24, 132.05, 131.85, 130.29, 129.92, 129.72, 129.69, 128.15, 128.02, 127.99, 127.33, 127.32, 124.63, 124.51, 123.99 ,121.95,121.90,110.61,110.52,66.33,62.91,52.23,52.20,52.18,50. 84,50.67,43.46,30.55,29.32,27.21,26.27,26.09,26.05,25.79,22.48. C 47 H 56 N 32 + [M-Br] 2+ LCMS(ESI): 318.7. Found: 318.9. [ka]

[0150] The corresponding pyridinium salt 175 (206 mg, 0.5 mmol) and 4-bromoaniline (103 mg, 0.6 mmol) were dissolved in methanol (4 mL, 7 mL / mmol), and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt 144 (176 mg, 0.5 mmol), 162 (252 mg, 0.65 mmol), and sodium acetate (246 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. EtO (21 mL / mmol) was then added, and the mixture was placed in a freezer (-16 °C). The resulting precipitate was filtered, washed with water (2 × 10 mL / mmol), EtO (2 × 10 mL / mmol), and air-dried. The crude product was then purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1 to 10:1) to produce GL286 as a green solid in 18% yield. 1H NMR (600MHz, methanol-d4) δ 8.15-8.12(m,2H),7.99-7.87(m,6H),7.56-7.44(m,5H),7.40-7.36(m,2H),6.51(t,2H),6.24-6.20(m,2H) ,4.11(t,2H),3.64(s,3H),2.82(t,2H),2.26(s,2H),1.90(s,12H),1.81(m,2H),1.58(m,2H),1.43(m,2H). C 42 H 48 N3 + [M-Br-HBr] + HRMS(ESI): 594.3843. Found: 594.3846. [ka]

[0151] GL144 (210 mg, 0.6 mmol), GL175 (208 mg, 0.5 mmol), GL148 (265 mg, 0.6 mmol), 4-bromoaniline (100 mg), and NaOAc (232 mg) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature overnight (16 h). The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1 to 10:1) to produce compound GL261 (87 mg, 16% yield). 1 H NMR (600 MHz, methanol-d4) δ 8.24(d,J=8.86Hz,2H),8.09-7.99(m,6H),7.66(t,2H),7.59(t,2H),7.49(t,2H),6.62(t,2H),6.33(m ,2H),4.23(t,2H),3.74(s,3H),2.34(t,2H),2.01(s,12H),1.93(m,2H),1.74(m,2H),1.56(m,2H) were obtained. C 42 H 45 N2O2 + [M-Br] + HRMS(ESI): 609.3476. Found: 609.3481. [ka]

[0152] Compound GL261 (87 mg, 0.13 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 29 mg, 0.15 mmol), and N-hydroxysuccinimide (NHS, 20 mg, 0.16 mmol) were dissolved in 5 mL of DMF, and the resulting mixture was stirred at room temperature under a nitrogen atmosphere in the dark for 24 h. NH2(CH2)6NHBoc (41 mg, 0.19 mmol) and N,N-diisopropylethylamine (DIPEA, 49 mg, 0.38 mmol) were added to the reaction solution, and the mixture was stirred for an additional 6 h. The crude product was purified by silica gel column chromatography (dichloromethane / methane = 25:1 to 15:1) to yield a purple-red solid (80 mg, 69%). 1 H NMR (600 MHz, methanol-d4) δ 8.13(d,J=7.92Hz,2H),7.98-7.88(m,6H),7.54(t,2H),7.49-7.45(m,3H),7.38(t,2H),6.50(t,2H),6.24(m,2H),4.11(t,2H),3. 63(s,3H),2.98(t,2H),2.90(t,2H),2.10(t,2H),1.90(s,12H),1.78(m,2H),1.61(m,2H),1.38(m,2H),1.30(m,14H),1.17(m,6H). [ka]

[0153] In an 8 ml vial, 80 mg of the Boc compound was dissolved in 10 ml of DCM and 1 ml of TFA was added dropwise. After stirring the solution for 30 minutes, excess TFA was removed in vacuo to yield GL258-2 as a green solid. 1H NMR (600MHz, methanol-d4) δ 8.13(d,J=8.16Hz,2H),7.98-7.85(m,6H),7.57-7.45(m,5H),7.38(m,2H),6.50(t,2H),6.23(t,2H),4.10(t,2H),3.63(s,3H),3 .01(t,2H),2.78(t,2H),2.02(t,2H),1.89(s,12H),1.77(m,2H),1.60(m,2H),1.52(m,2H),1.42-1.32(m,4H),1.29-1.19(m,4H). [ka]

[0154] The corresponding pyridinium salt GL175 (103 mg, 0.24 mmol) and 4-bromoaniline (49 mg, 0.29 mmol) were dissolved in methanol (4 mL), and the mixture was stirred at room temperature for 30 min. Next, the heterocyclic salt GL144 (105 mg, 0.3 mmol), GL361 (129 mg, 0.3 mmol), and sodium acetate (116 mg) were added, and the reaction mixture was stirred at room temperature for an additional 16 h. The crude product was purified by flash column chromatography (silica gel, dichloromethane / methanol, 20:1, 10:1) to produce GL362-2 as a green solid in 23% yield. 1 H NMR (600MHz, methanol-d4) δ 8.24(d,J=8.51Hz,2H),8.09-7.99(m,6H),7.70-7.57(m,5H),7.49(t,2H),6.62(t,2H),6.34(d,J=14.40Hz,2 H),4.22(t,2H),3.74(s,3H),3.18(t,2H),2.01(s,12H),1.93(s,3H),1.88(m,2H),1.55(m,4H),1.46(m,2H). 13C NMR (150 MHz, methanol-d4) δ 171.84, 140.40, 139.78, 133.39, 133.28, 131.98, 131.92, 130.32, 130.26, 129.71, 128.11, 128.05, 127.31, 125.57, 124.56, 121.92, 110.61, 110.47, 53.40, 50.76, 50.73, 48.47, 48.22, 43.60, 38.88, 30.44, 28.82, 27.28, 26.26, 26.23, 26.11, 26.08, 21.16. 44 H 50 N3O + [M-Br] + HRMS(ESI): 636.3948. Found: 636.3954. [ka]

[0155] GL342 (830 mg, 2 mmol), Pd(PPh3)4 (250 mg, 0.2 mmol), and K2CO3 (1.1 mg, 6 mmol) were mixed in 15 mL of isopropanol and then stirred and heated at 90 °C overnight. The dark solution turned yellow. After cooling to room temperature, the solvent was removed by rotary evaporation. The crude product was purified on a flash silica gel column (hexane and ethyl acetate 10:1) to yield an orange solid, GL343. 24 H 28 N2[M+H] + LCMS(ESI): 345.23. Found: 345.3. [ka]

[0156] GL144 (176 mg, 0.5 mmol), GL343 (172 mg, 0.5 mmol), compound GL345 (286 mg, 0.65 mmol), sodium acetate (246 mg, 3 mmol), and acetic anhydride (4 ml) were stirred and heated at 80 °C overnight. The dark solution was cooled and poured into ether. The green solid was collected by filtration. The crude product was purified on a silica gel column (DCM and methanol 10:1) to produce GL349-2 as a green solid (yield: 10%). 1 H NMR (600MHz, methanol-d4) δ 8.27(d, J=8.82Hz, 1H), 8.23(d, J=8.82Hz, 1H), 8.07-7.98(m, 5H), 7.85(d, J=13.24Hz, 1H), 7.69- 7.59(m, 3H), 7.56-7.46(m, 4H), 6.32(d, J=14.42Hz, 1H), 6.23(d, J =14.22Hz, 1H), 4.36(t, 2H), 3.82(s, 3H), 2.92(m, 2H), 2.84(t, 2H), 2.50(s, 6H), 2.05(m, 12H), 2.01(s, 4H), 1.15(s, 9H). 13 C NMR (150 MHz, methanol-d4) δ 174.35,149.04,140.27,139.76,132.16,131.73,130.34,130.25,129.75,128.24,127.94,127.39,127.22,124.79,124.22,122.02,121 .84,110.62,110.07,100.05,98.09,54.94,51.06,50.32,44.40,43.67,41.11,32.06,30.72,26.55,26.29,26.14,26.06,25.92,19.64. C 47 H 56 N3 + [M-Br] + HRMS(ESI): 662.4469. Found: 662.4474. [ka]

[0157] GL144 (176 mg, 0.5 mmol), GL148 (202.2 mg, 0.5 mmol), (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (86.3 mg, 0.5 mmol), sodium acetate (86 mg, 1.05 mmol), and acetic anhydride (5 ml) were stirred and heated at 70 °C overnight. The dark solution was cooled and poured into ether. The green solid was collected by filtration. The crude product was purified on a silica gel column (DCM and methanol 10:1) to give GL149-2 as a green solid (yield: 26.5%). 1 H NMR (600MHz, methanol-d4) δ 8.58(t,1H),8.30(d,J=7.61Hz,1H),8.07-8.02(m,2H),7.69-7.63(m,2H),7.53(t,1H),6.35(d,J=12.17Hz,1H),4. 33(t,1H),3.83(s,3H),2.80(s,2H),2.34(t,1H),2.12(s,6H),2.04(m,2H),1.96(m,2H),1.75(m,2H),1.57(m,2H). 13 C NMR (150 MHz, methanol-d4) δ 176.40, 174.90, 174.24, 173.96, 149.19, 149.11, 143.33, 143.18, 143.05, 142.97, 140.32, 139.71, 133.83, 133.81, 132.23, 132.15, 130.49, 130.44, 129.76, 129.34, 128.05, 127.95, 127.44, 127.43, 126 .61,126.57,126.49,126.43,124.92,124.86,124.83,122.04,110.75,110.64,100.80,100.35,51.0 6,48.16,43.83,33.65,33.07,30.72,26.99,26.48,26.38,26.02,25.96,25.92,24.46,24.24,20.79. C 45 H 48 ClN2O2 + [M-Br] + HRMS(ESI): 683.3390. Found: 683.3312. [ka]

[0158] GL144 (176 mg, 0.5 mmol), GL162 (223.5 mg, 0.5 mmol), (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (86.3 mg, 0.5 mmol), sodium acetate (86 mg, 1.05 mmol), and acetic anhydride (3 mL) were stirred and heated at 70 °C overnight. The dark solution was cooled and poured into ether. The green solid was collected by filtration. The crude product was purified on a silica gel column (DCM and methanol 10:1) to give GL161-2 as a green solid (yield: 26.5%). 1 H NMR (600MHz, methanol-d4) 8.62-8.56(m,2H),8.30(m,2H),8.08-8.02(m,4H),7.70-7.63(m,4H),7.53(m,2H),6.35(m,2H),4.32(t,2H) ),3.83(s,3H),3.18(t,2H),2.80(m,4H),2.06(d,12H),2.03(m,2H),1.95(m,2H),1.55(m,4H),1.48(m,2H).

[0159] [Table 1] [a] The reaction was carried out under the following conditions: 1) 1 (0.25 mmol), Pd(PPh3)4 (10 mol%), and base (4 equivalents) were reacted in a solvent at 100 °C for 24 hours. [b] The yield was determined by HPLC using a C18 reverse-phase column. [c] The isolated yield.

[0160] General procedure: To a screw-capped vial were added 1 (0.25 mmol), Pd(PPh3)4 (0.025 mmol, 10 mol%), and base (1 mmol, 4 equiv). The vial was sealed with a PTFE septum and evacuated and backfilled with N2 three times, after which solvent was added via syringe and vigorously stirred. The sealed reaction was heated to 100 °C in an oil bath for 24 h. The reaction was then cooled to room temperature and concentrated under reduced pressure. 6 M HCl was then added at 0 °C and stirred at room temperature for 10 min. The precipitate was filtered, washed with HO, Et2O, and acetone, and dried under vacuum to yield compound 2 as a dark red solid.

[0161] N-((E)-(3-((E)-(phenylimino)methyl)cyclohex-2-en-1-ylidene)methyl)aniline (2): 1 H-NMR (600 MHz, CD3OD) δ 8.14(s,2H),7.79(s,1H),7.49-7.39(m,8H),7.28-7.23(m,2H),2.58(t,J=6.2Hz,4H),1.99-1.93(m,2H) 13 C NMR (151MHz, CD3OD) δ 163.58,153.18,140.74,130.98,127.16,119.97,119.18,22.96,21.58. [M+H,C 20 H 21 N2] + HRMS(ESI) calculated for: 289.1626. Found: 289.1707.

[0162] Scheme 1: Synthesis of charged π-conjugated compound 4 [ka]

[0163] General procedure: Compound 3 (1 equivalent) and alkyl halide (1.5 equivalents) were placed in a screw-cap vial and heated to reflux in CHCN until all of compound 3 was consumed. The mixture was then cooled to room temperature. Diethyl ether was then added to precipitate the product, which was collected by filtration and washed with diethyl ether to give compound 4.

[0164] Scheme 2: Synthesis of charged π-conjugated compound 5 [ka]

[0165] General synthetic procedures: All glassware was oven dried overnight before use. All reactions were carried out under a N2 atmosphere unless otherwise stated. All other chemicals were purchased from commercial suppliers and used without further purification.

[0166] General procedure: Compound 3 (1 equivalent) and alkyl halide (1.5 equivalents) were placed in a screw-cap vial and heated to reflux in CHCN until all of compound 3 was consumed. The mixture was then cooled to room temperature. Diethyl ether was then added to precipitate the product, which was collected by filtration and washed with diethyl ether to give compound 4.

[0167] Step b: Compound 4 (1 equivalent), 2 (1 equivalent), 4 (1 equivalent), and NaOAc (3 equivalents) were dissolved in absolute ethanol in a screw-cap vial. The mixture was heated to 80 °C overnight under a N2 atmosphere. The final product was purified by silica column chromatography to give compound 5. [ka]

[0168] 1,1,3-Trimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-141-1): Prepared according to the general procedure from BL-68 (176 mg, 0.5 mmol, 1 equiv.) and 2 (81 mg, 0.25 mmol, 1 equiv.). Yield: 88% yield (149 mg). 1 H-NMR (600MHz, CD3OD) δ 8.24-8.21(m,2H),8.00(d,J=8.8Hz,2H),7.97(d,J=8.6Hz,2H),7.87(d,J=14.2Hz,2H),7.64-7.60(m,2H),7.57(d,J=8. 8Hz,2H),7.53(s,1H),7.48-7.44(m,2H),6.19(d,J=14.1Hz,2H),3.72(s,6H),2.61(t,J=6.1Hz,4H),2.03-1.95(m,14H). [M,C 40 H 41 N2] + HRMS(ESI) calculated for: 549.3264. Found: 549.3275. [ka]

[0169] 3-(6-(Dimethylamino)hexyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-141-2): Prepared according to the general procedure from BL-68 (176 mg, 0.5 mmol, 1 equiv.), 2 (162 mg, 0.5 mmol, 1 equiv.), and BL-140 (248 mg, 0.5 mmol, 1 equiv.). Yield: 14% yield (45 mg). 1H-NMR(600MHz, CD3OD) δ 8.27-8.21(m,2H),8.05-7.96(m,4H),7.93-7.89(m,1H),7.85(d,J=14.0Hz,1H),7.67-7.61(m ,2H),7.60(d,J=8.8Hz,1H),7.57(d,J=8.8Hz,1H),7.52(s,1H),7.50-7.45(m,2H),6.21(dd,J =23.7,14.1Hz,2H),4.24(t,J=7.4Hz,2H),3.75(s,3H),3.04(t,J=9.0Hz,2H),2.80(s,6H),2. 63-2.60(m,4H),2.06-1.90(m,14H),1.75-1.70(m,2H),1.59-1.54(m,2H),1.51-1.47(m,2H). [M,C 47 H 56 N3] + HRMS(ESI) calculated for: 662.4469. Found: 662.4476. [ka]

[0170] 3-(6-(dimethylamino)hexyl)-2-((E)-2-((E)-3-((E)-2-(3-(6-(dimethylamino)hexyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-142): Prepared according to the general procedure from 2 (81 mg, 0.25 mmol, 1 equiv.) and BL-140 (248 mg, 0.5 mmol, 2 equiv.). Yield: 20% yield (40 mg). 1H-NMR (600MHz, CD3OD) δ 8.25-8.22(m,2H),8.03-7.96(m,4H),7.89(d,J=14.1Hz,2H),7.66-7.62(m,2H),7.6 0(d,J=8.8Hz,2H),7.56(s,1H),7.50-7.45(m,2H),6.24(d,J=14.1Hz,2H),4.27(t,J =7.4Hz,4H),4.27(t,J=7.4Hz,4H),2.82(s,12H),2.63(t,J=6.2Hz,4H),2.05-1.96( m, 12H), 1.95-1.90 (m, 6H), 1.76-1.72 (m, 4H), 1.60-1.55 (m, 4H), 1.52-1.47 (m, 4H). [M,C 54 H 71 N4] + HRMS(ESI) calculated for: 775.5673. Found: 775.5662. [ka]

[0171] 3-(2-(Dimethylamino)ethyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-204): Prepared according to the general procedure from BL-68 (70 mg, 0.2 mmol, 1 equiv.), 2 (65 mg, 0.2 mmol, 1 equiv.), and BL157 (107 mg, 0.2 mmol, 1 equiv.). Yield: 18% yield (26 mg). 1H-NMR (600MHz, CD3OD) δ 8.25(d,J=8.6Hz,1H),8.22(d,J=8.7Hz,1H),8.05-7.90(m,5H),7.82( d,J=13.9Hz,1H),7.67-7.59(m,3H),7.56-7.44(m,4H),6.28(d,J=14.2 Hz,1H),6.21(d,J=14.1Hz,1H),4.30(t,J=7.4Hz,2H),3.77(s,3H),2.7 7(t,J=7.4Hz,2H),2.66-2.59(m,4H),2.43(s,6H),2.06-1.95(m,14H). [M,C 43 H 48 N3] + HRMS(ESI) calculated for: 606.3843. Found: 606.3847. [ka]

[0172] 1,3,3-Trimethyl-2-((E)-2-((E)-3-((E)-2-(1,3,3-trimethyl-1,3-dihydro-2H-benzo[g]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-benzo[g]indol-1-ium (BL-205): Prepared according to the general procedure from BL-117 (70 mg, 0.2 mmol, 2 equiv.) and 2 (33 mg, 0.1 mmol, 1 equiv.). Yield: 74% yield (50 mg). 1 H-NMR (600MHz, CD3OD) δ 8.59(d,J=8.7Hz,2H),7.99(d,J=8.2Hz,2H),7.82(d,J=8.9Hz,4H),7.67-7.59(m,4H),7.58-7.52(m,2H), 7.50(s,1H),6.33(d,J=14.0Hz,2H),4.18(s,6H),2.63(t,J=6.2Hz,4H),2.02-1.96(m,2H),1.77(s,12H). [M,C 40 H 41 N2] + HRMS(ESI) calculated value: 549.3264, found value: 549.3247. [ka]

[0173] 3-(5-Carboxypentyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-242): Prepared according to the general procedure from BL-68 (117 mg, 0.33 mmol, 1 equiv.), 2 (107 mg, 0.33 mmol, 1 equiv.), and BL-85 (134 mg, 0.33 mmol, 1 equiv.). Yield: 18% yield (130 mg). 1 H-NMR (600MHz, CD3OD) δ8.12(d,J=8.5Hz,2H),7.91-7.85(m,4H),7.84-7 .69(m,2H),7.55-7.51(m,2H),7.50-7.32(m,5H),6.21-5.95(m,2H),4.20 -4.06(m,2H),3.63(s,3H),2.68-2.30(m,4H),2.23(t,J=7.3Hz,2H),1.9 9-1.84(m,14H),1.83-1.77(m,2H),1.65-1.60(m,2H),1.48-1.41(m,2H), 13 C-NMR(151 MHz,CD3OD) δ 177.49,174.88,173.98,163.31,163.08,162.84,149.06,148.67,141.83,141.22, 134.55,133.35,133.29,132.30,131.66,131.61,131.10,129.68,129.53,129.46, 128.66, 125.84, 124.39, 123.33, 113.51, 111.95, 111.83, 100.54, 100.28, 54.81, 52.05, 44.83, 34.81, 31.83, 28.22, 27.60, 27.50, 27.41, 25.78, 25.02, 22.78, 22.07. HRMS (ESI) calculated for [M,C45H49N2O2]+: 649.3789. Found: 649.3793. [ka]

[0174] 3-(5-carboxypentyl)-2-((E)-2-((E)-3-((E)-2-(3-(5-carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benz[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-243): Prepared according to the general procedure from 2 (33 mg, 0.1 mmol, 1 equiv.) and BL-85 (81 mg, 0.2 mmol, 2 equiv.). Yield: 45% yield (39 mg). 1 H-NMR (600MHz, CD3OD) δ 8.23(d,J=8.5Hz,2H),8.00(d,J=8.9Hz,2H),7.98(d,J=8.0Hz,2H),7.86(d,J=1 4.0Hz,2H),7.65-7.61(m,2H),7.57(d,J=8.8Hz,2H),7.50-7.44(m,3H),6.22(d, J=14.1Hz,2H),4.23(t,J=7.5Hz,4H),2.61(t,J=6.2Hz,4H),2.25(t,J=7.4Hz,4 H),2.03-1.96(m,12H),1.93-1.87(m,4H),1.75-1.70(m,4H),1.56-1.50(m,4H). [M,C 50 H 57 N2O4] + HRMS(ESI) calculated for: 749.4313. Found: 749.4312. [ka]

[0175] 3-(6-Methoxy-6-oxohexyl)-2-((E)-2-((E)-3-((E)-2-(3-(6-methoxy-6-oxohexyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-246-1): Prepared according to the general procedure from 2 (65 mg, 0.2 mmol, 1 equiv.) and BL-245 (167 mg, 0.4 mmol, 2 equiv.). Yield: 18% yield (30 mg). 1 H-NMR (600MHz, CD3OD) δ 8.23(d,J=8.5Hz,2H),8.01(d,J=8.9Hz,2H),7.99(d,J=8.1Hz,2H),7.87(d,J=14.1Hz, 2H),7.65-7.62(m,2H),7.57(d,J=8.8Hz,2H),7.51-7.46(m,3H),6.23(d,J=14.1Hz,2H) ,4.27-4.19(m,4H),3.69(s,3H),3.62(s,3H),2.62(t,J=6.2Hz,4H),2.36(dt,J=11.5,7 .3Hz,4H),2.06-1.96(m,12H),1.93-1.88(m,4H),1.76-1.70(m,4H),1.56-1.50(m,4H). [M,C 52 H 61 N2O4] + HRMS(ESI) calculated for: 777.4626. Found: 777.4626. [ka]

[0176] 2-((E)-2-((E)-3-((E)-2-(3-(5-carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohexyl-1-en-1-yl)vinyl)-3-(6-methoxy-6-oxohexyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-246-2): Prepared according to the general procedure from BL-85 (81 mg, 0.2 mmol, 1 equiv.), 2 (65 mg, 0.2 mmol, 1 equiv.), and BL-245 (83 mg, 0.2 mmol, 1 equiv.). Yield: 17% yield (28 mg). 1 H-NMR (600MHz, CD3OD) δ 8.23(d,J=8.5Hz,2H),8.21-7.96(m,4H),7.87(dd,J=14.1,9.3Hz,2H),7.65-7.61(m,2H) ,7.59-7.54(m,2H),7.51-7.45(m,3H),6.22(t,J=14.0Hz,2H),4.27-4.18(m,4H),3.70-3. 61(m,3H),2.61(t,J=6.4Hz,4H),2.36(dt,J=11.5,7.2Hz,2H),2.29(t,J=7.3Hz,2H),2.0 3-2.00(m,8H),1.97(s,6H),1.94-1.87(m,4H),1.76-1.68(m,4H),1.57-1.50(m,4H),[M,C 51 H 59 N2O4] + HRMS(ESI) calculated for: 763.4469. Found: 763.4455. [ka]

[0177] 2-((E)-2-((E)-3-(2-(3-(3-(dimethylamino)-3-oxopropyl)-1,1-dimethyl-2,3-dihydro-1H-benzo[e]indol-2-yl)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium (BL-248): Prepared according to the general procedure from BL-65 (70 mg, 0.2 mmol, 1 equiv.), 2 (65 mg, 0.2 mmol, 1 equiv.), and BL-65 (75 mg, 0.2 mmol, 1 equiv.). Yield: 20% yield (28 mg). 1 H-NMR (600MHz, CD3OD) δ 8.16-8.11(m,2H),7.94-7.88(m,2H),7.86-7.81(m,3H),7.69(d,J=13.8Hz,1H),7.56- 7.49(m,3H),7.43-7.38(m,2H),7.35-7.32(m,1H),7.30(d,J=8.8Hz,1H),6.17(d,J=14. 3Hz,1H),5.88(d,J=13.7Hz,1H),5.08(s,2H),3.67(s,3H),3.22(s,3H),2.94(s,3H),2. 50(t,J=6.2Hz,2H),2.46(t,J=6.2Hz,2H),1.96(s,6H),1.91(s,6H),1.88-1.82(m,2H). 13 C-NMR (151MHz, CD3OD) δ 174.77,172.68,165.98,154.83,149.06,146.27,140.60,140.25,133.71,13 2.79,132.18,132.13,131.72,130.32,129.93,129.74,129.66,128.19,127.9 6,127.38,127.08,124.76,124.06,122.02,121.85,110.61,110.18,100.14, 98.32,51.01,50.46,44.88,35.61,34.87,30.69,26.28,26.00,23.59,21.37. [M,C 43 H 46 N3O] + HRMS(ESI) calculated for: 620.3635. Found: 620.3634. [ka]

[0178] 2,2'-((1E,1'E)-1,3-phenylenebis(ethene-2,1-diyl))bis(1,1,3-trimethyl-1H-benzo[e]indol-3-ium) (BL-250): Prepared from m-phthalaldehyde (70 mg, 0.52 mmol, 1 equiv.) and BL-68 (365 mg, 1.04 mmol, 2 equiv.) according to the general procedure. Yield: 50% yield (208 mg). 1 H-NMR (600MHz, CD3OD) δ 8.64(d,J=16.5Hz,2H),8.48-8.44(m,2H),8.34-8.30(m,2H),8.27(d,J=8.9Hz,2H),8.19(d,J=8.2Hz,2H),8.04(d,J=8. 9Hz,2H),7.96(d,J=16.5Hz,2H),7.87-7.83(m,2H),7.81(t,J=7.7Hz,2H),7.76-7.73(m,2H),4.45(s,6H),2.18(s,12H). [M,C 40 H 38 N2] + HRMS(ESI) calculated for: 546.3024. Found: 546.3015. [ka]

[0179] 2,2'-((1E,1'E)-1,3-phenylenebis(ethene-2,1-diyl))bis(1,1,3-trimethyl-1H-benzo[e]indol-3-ium) (BL-262): Prepared according to the general procedure from 4-hydroxyisophthalaldehyde (50 mg, 0.33 mmol, 1 equiv.) and BL-68 (232 mg, 0.66 mmol, 2 equiv.). Yield: 80% yield (182 mg). 1H-NMR (600MHz, CD3OD) δ 8.82(s,1H),8.22(d,J=16.0Hz,1H),8.17(d,J=8.5Hz,1H),8.09(d,J=8.8Hz,1H),8.04(d,J=8.1Hz,1H),7.92(d ,J=8.6Hz,1H),7.85(d,J=16.0Hz,1H),7.81(dd,J=8.5,1.3Hz,1H),7.76(d,J=8.5Hz,1H),7.74-7.69(m,2H),7.6 6-7.57(m,2H),7.50(d,J=10.3Hz,1H),7.39-7.43(m,1H),7.26-7.22(m,1H),6.98(d,J=8.6Hz,1H),6.73(d,J=8 .4Hz,1H),5.89(d,J=10.3Hz,1H),4.56(s,3H),2.84(s,3H),2.06(s,3H),2.06(s,3H),1.62(s,3H),1.35(s,3H). [M,C 40 H 37 N2O] + HRMS(ESI) calculated for: 561.2900. Found: 561.2906. [ka]

[0180] 2-((E)-2-((E)-3-(2-(3-(3-(dimethylamino)propyl)-1,1-dimethyl-2,3-dihydro-1H-benzo[e]indol-2-yl)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium (BL-260): Prepared according to the general procedure from BL-68 (70 mg, 0.2 mmol, 1 equiv.), 2 (65 mg, 0.2 mmol, 1 equiv.), and BL-258 (91 mg, 0.2 mmol, 1 equiv.). Yield: 20% yield (28 mg). 1H-NMR (600MHz, CD3OD) δ 8.24(d,J=9.1Hz,1H),8.23(d,J=9.1Hz,1H),8.04-7.95(m,4H),7.91(d,J=14.2Hz,1H),7.85(d,J=13.9Hz,1H),7.67-7.56(m,4H),7.54-7 .43(m,3H),6.30-6.21(m,2H),4.27(t,J=7.2Hz,2H),3.76(s,3H),2.65-2.60(m,4H),2.58-2.51(m,2H),2.55(s,6H),2.09-1.96(m,14H). [M,C 44 H 50 N3] + HRMS(ESI) calculated for: 620.3999. Found: 620.4000. [ka]

[0181] 1,3,3-Trimethyl-2-((E)-2-((E)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (BL-264): Prepared according to the general procedure from BL-263 (60 mg, 0.2 mmol, 2 equiv.) and 2 (33 mg, 0.1 mmol, 1 equiv.). Yield: 87% yield (50 mg). 1 H-NMR (600MHz, CD3OD) δ 7.76(d,J=14.0Hz,2H),7.49-7.44(m,3H),7.39(td,J=7.7,1.2Hz,2H),7.27-7.21(m,4H),6 .15(d,J=14.0Hz,2H),3.60(s,6H),2.57(t,J=6.2Hz,4H),1.98-1.92(m,2H),1.71(s,12H). 13 C-NMR(151 MHz,CD3OD) δ 173.41,149.81,144.46,142.27,133.79,129.68,125.85,123.24,111.46,100.77,50.15,31.40,27.91,24.96,22.73. [M,C 32 H37 N2] + HRMS(ESI) calculated for: 449.2951. Found: 449.2946. [ka]

[0182] 3-(6-Aminohexyl)-1,1-dimethyl-2-((E)-2-((E)-3-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1H-benzo[e]indol-3-ium (BL-272): Prepared according to the general procedure from BL-68 (70 mg, 0.2 mmol, 1 equiv.), 2 (65 mg, 0.2 mmol, 1 equiv.), and BL-116 (94 mg, 0.2 mmol, 1 equiv.). Yield: 10% yield (14 mg). 1 H-NMR(600MHz, CD3OD) δ 8.24(d,J=4.8Hz,1H),8.22(d,J=4.9Hz,1H),8.03-7.95(m,4H),7.90(d,J=14.1Hz,1H), 7.85(d,J=14.0Hz,1H),7.65-7.61(m,2H),7.58(t,J=9.2Hz,2H),7.54(s,1H),7.49-7.44 (m,2H),6.25-6.17(m,2H),4.25(t,J=7.4Hz,2H),3.74(s,3H),2.95-2.90(m,2H),2.64-2 .58(m,4H),2.04-1.96(m,14H),1.94-1.88(m,2H),1.72-1.66(m,2H),1.59-1.48(m,4H). 13C-NMR(151 MHz,CD3OD) δ 175.21,173.65,156.10,149.40,148.36,141.78,141.29,134.67,134.44,133.83,133.5 1,133.40,133.22,131.64,131.11,131.09,129.99,129.54,129.42,128.69,128.68,125. 93,125.85,125.81,123.35,123.31,118.00,114.13,111.97,111.88,100.80,100.07,52 .13,51.97,44.80,40.64,31.90,28.52,28.40,27.62,27.56,27.47,27.29,25.04,22.80. [M,C 45 H 52 N3] + HRMS(ESI) calculated for: 634.4156. Found: 634.4153. [ka]

[0183] 3-(2-(dimethylamino)-2-oxoethyl)-2-((E)-2-((E)-3-((E)-2-(3-(2-(dimethylamino)-2-oxoethyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium (BL-273): Prepared according to the general procedure from 2 (33 mg, 0.1 mmol, 1 equiv.) and BL-247 (75 mg, 0.2 mmol, 2 equiv.). Yield: 26% yield (20 mg). 1H-NMR (600MHz, CD3OD) δ 8.24(d,J=8.5Hz,2H),7.98-7.94(m,4H),7.87(d,J=14.2Hz,2H),7.65-7.61(m,2H),7.51(s,1H),7.47(t,J=7.6Hz,2H),7.44(d,J= 8.8Hz,2H),6.06(d,J=14.0Hz,2H),5.24(s,4H),3.32(s,6H),3.04(s,6H),2.55(t,J=6.2Hz,4H),2.06(s,12H),1.96-1.90(m,2H). [M,C 46 H 51 N4O2] + HRMS(ESI) calculated for: 691.4007. Found: 691.4002. [ka]

[0184] 1-Methyl-2-((E)-2-((E)-3-(2-((E)-1-methylquinolin-2(1H)-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)quinolin-1-ium (BL-206): Prepared according to general procedure b from BL-69 (57 mg, 0.2 mmol, 2 equiv.) and 2 (33 mg, 0.1 mmol, 1 equiv.). Ochre solid. Yield: 70% (38 mg). 1 H-NMR(600MHz.DMSO-d6) δ 7.89(s,4H),7.79-7.73(m,4H),7.66-7.62(m,2H),7.54(d,J=13.6Hz,2H),7.38-7.34(m,2 H),7.10(s,1H),6.12(d,J=13.6Hz,2H),3.83(s,6H),2.52-7.50(m,4H),1.85-1.79(m,2H). 13 C-NMR(151 MHz,DMSO-d6) δ 150.63,149.52,143.90,140.09,135.16,132.19,130.46,128.73,124.64,124.54,120.05,116.15,104.06,36.21,24.24,21.34. [M,C 30 H 29 N2]+ HRMS(ESI) calculated for: 417.2325. Found: 417.2329. [ka]

[0185] 1,4,4-Trimethyl-5-((E)-2-((E)-3-((E)-2-(1,3,3-trimethylpyrrolidin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,4-dihydro-2H-pyrrol-1-ium (BL-328): Prepared from BL-327 (50 mg, 0.3 mmol, 2 equiv.) and BL-111 (50 mg, 0.15 mmol, 1 equiv.) according to the general procedure. Yield: 50% (30 mg). 1 H-NMR (600MHz, CD3OD) δ 7.49(d,J=14.2Hz,2H),7.08(s,1H),5.52(d,J=14.1Hz,2H),3.70(t,J=7.2Hz,4H),3. 15(s,6H),2.40(t,J=6.2Hz,4H),2.00-1.94(m,4H),1.88-1.82(m,2H),1.47(s,12H). [ka]

[0186] 1,4,4-Trimethyl-5-((1E,3E,5E,7E)-7-(1,3,3-trimethylpyrrolidin-2-ylidene)hepta-1,3,5-trien-1-yl)-3,4-dihydro-2H-pyrrol-1-ium (BL-329): Prepared according to the general procedure from BL-327 (50 mg, 0.3 mmol, 2 equiv.) and glutacondianilic acid hydrochloride (44 mg, 0.15 mmol, 1 equiv.). Yield: 50% (30 mg). 1H-NMR (600MHz, CD3OD) δ 7.49(d,J=14.2Hz,2H),7.08(s,1H),5.52(d,J=14.1Hz,2H),3.70(t,J=7.2Hz,4H),3. 15(s,6H),2.40(t,J=6.2Hz,4H),2.00-1.94(m,4H),1.88-1.82(m,2H),1.47(s,12H). [ka]

[0187] 1-(2-(Dimethylamino)ethyl)-3,3-dimethyl-2-((E)-2-((E)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (BL-305): Prepared according to the general procedure from 2 (97 mg, 0.3 mmol, 1 equiv.), BL-263 (90 mg, 0.3 mmol, 1 equiv.), and BL-304 (117 mg, 0.3 mmol, 1 equiv.). Yield: 26% yield (49 mg). 1 H-NMR (600MHz, CD3OD) δ 7.84(d,J=14.2Hz,1H),7.73(d,J=13.9Hz,1H),7.54-7.49(m,2H),7.48-7.43(m,2H), 7.43-7.38(m,1H),7.34-7.32(m,1H),7.31-7.27(m,1H),7.25-7.20(m,2H),6.25(d,J= 14.2Hz,1H),6.18(d,J=13.8Hz,1H),4.20(t,J=7.4Hz,2H),3.67(s,3H),2.72(t,J=7. 4Hz, 2H), 2.63-2.57 (m, 4H), 2.42 (s, 6H), 1.99-1.94 (m, 2H), 1.74 (s, 6H), 1.73 (s, 6H). [ka]

[0188] 1-(6-(Dimethylamino)hexyl)-3,3-dimethyl-2-((E)-2-((E)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (BL-294): Prepared according to the general procedure from BL-263 (90 mg, 0.3 mmol, 1 equiv.), 2 (97 mg, 0.3 mmol, 1 equiv.), and BL-293 (134 mg, 0.3 mmol, 1 equiv.). Yield: 25% yield (52 mg). 1 H-NMR (600 MHz, CD3OD) δ 7.82-7.72(m,2H),7.54(s,1H),7.50-7.45(m,2H),7.42-7.37(m,2H),7.2 9-7.20(m,4H),6.22-6.12(m,2H),4.13(t,J=7.4Hz,2H),3.62(s,3H),3.0 9-3.04(m,2H),2.82(s,6H),2.58(t,J=6.2Hz,4H),1.98-1.92(m,2H),1.8 9-1.83(m,2H),1.78-1.67(m,14H),1.58-1.52(m,2H),1.51-1.45(m,2H). [ka]

[0189] 1-(5-Carboxypentyl)-3,3-dimethyl-2-((E)-2-((E)-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (BL-296): Prepared according to the general procedure from 2 (65 mg, 0.2 mmol, 1 equiv.), BL-263 (60 mg, 0.2 mmol, 1 equiv.), and BL-295 (71 mg, 0.2 mmol, 1 equiv.). Yield: 30% yield (41 mg). 1H-NMR (600MHz, CD3OD) δ 7.83-7.69(m,2H),7.55-7.43(m,3H),7.42-7.36(m,2H),7.29-7.18(m,4H),6.24-6.08(m,2H),4.15-4.06(m,2H),3.69-3.54( m,3H),2.68-2.45(m,4H),2.30(t,J=7.3Hz,2H),1.98-1.91(m,2H),1.86-1.81(m,2H),1.78-1.60(m,14H),1.53-1.48(m,2H). [ka]

[0190] General procedure: The corresponding pyridinium salt B (1 equiv.) and 4-bromoaniline (1 equiv.) were dissolved in methanol (4 mL) in an 8 mL vial, and the mixture was stirred at room temperature for 30 min. Next, heterocyclic salt A (1 equiv.), C (1 equiv.), and sodium acetate (3 equiv.) were added. The reaction mixture was further stirred at room temperature overnight. The crude product D was purified by flash column chromatography (silica gel, dichloromethane / methanol). [ka]

[0191] 2-((1E,3Z,5E)-4-cyano-7-((E)-1,3,3-trimethylindolin-2-ylidene)-hepta-1,3,5-trien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium iodide: TS-17 was produced in 90% yield as a green solid. 1 H NMR (600MHz, methanol-d4) δ 7.96(t,J=16.2Hz,2H),7.41(td,J=9.6,1.8Hz,2H),7.39(d,J=9.0Hz,2H),7.28( t,J=9.0Hz,2H),7.17(d,J=9.0Hz,2H),6.88(bro,2H),3.78(s,6H),1.71(s,12H). [ka]

[0192] 1-(5-Carboxypentyl)-2-((1E,3Z,5E)-4-cyano-7-((E)-1,3,3-trimethylindolin-2-ylidene)-hepta-1,3,5-trien-1-yl)-3,3-dimethyl-3H-indol-1-ium iodide: TS-31 was obtained in 41% yield as a green solid. 1 H NMR (600MHz, methanol-d4) δ 8.09(td,J=15.6,7.8Hz,2H),7.59-7.56(m,2H),7.50-7.46(m,2H),7.41(t,J=10.2Hz,2H),7.38-7.33 (m,2H),6.83(dd,J=15.0,4.8Hz,2H),6.57(dd,J=16.8,9.6Hz,2H),4.20(t,J=9.0Hz,2H),3.73(s,3H) ,2.33(t,J=9.0Hz,2H),1.89-1.86(m,2H),1.75(d,t,J=4.2Hz,12H),1.73-1.68(m,2H),1.56-1.51(m,2H). [ka]

[0193] 2-((1E,3Z,5E,7E)-4-cyano-7-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)-hepta-1,3,5-trien-1-yl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium iodide: TS-21 was produced in 78% yield as a green solid. 1H NMR (600MHz, methanol-d4) δ 8.29(d,J=10.2Hz,2H),8.16(t,J=16.2Hz,2H),8.07(d,J=10.8Hz,2H),8.03(d,J=10.2Hz,2H),7.67(t,J=10.8Hz,4H),7.53(t, J=9.0Hz,2H),6.83(d,J=9.0Hz,2H),6.58(d,J=16.2Hz,2H),6.57(dd,J=16.8,9.6Hz,2H),3.82(s,6H),2.02(d,J=25.2Hz,12H). [ka]

[0194] 3-(5-Carboxypentyl)-2-((1E,3Z,5E,7E)-4-cyano-7-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)-hepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-benzo[e]indol-3-ium iodide: TS-34 was produced in 34% yield as a green solid. 1 H NMR (600MHz, methanol-d4) δ 8.29(dd,J=9.6,4.2Hz,2H),8.17(d,J=8.4Hz,2H),8.06(dd,J=10.8,6.0Hz,2H),8.03(dd,J=10.2,3.6Hz,2H),7.69-7.65(m 4H),7.55-7.51(m,2H),6.85(dd,J=15.6,8.4Hz,2H),6.60(dd,J=16.8,6.0Hz,2H),4.30(t,J=9.0Hz,2H), 2.31(t,J=9.0Hz,2H),2.02(d,t,J=1.2Hz,12H),1.94-1.89(m,2H),1.73-1.69(m,2H),1.59-1.54(m,2H). [ka]

[0195] 6-(2-((1E,3Z,5E,7E)-7-(3-(5-carboxypentyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)-4-cyanohepta-1,3,5-trien-1-yl)-1,1-dimethyl-1H-3,4-benzo[e]indol-3-yl)-hexanoic acid bromide: TS-32 was produced in 42% yield as a green solid. 1 H NMR (600MHz, methanol-d4) δ 7.93(bro,2H),7.44(d,J=7.2Hz,2H),7.36-7.33(m,2H),7.30(d,J=7.8Hz,2H),7.21(t,J=7.2Hz,2H),6.73(d,J=13.2Hz,2H),6.47( d,J=13.8Hz,2H),4.07(t,J=7.8Hz,4H),2.09(t,J=7.8Hz,4H),1.76-1.73(m,4H),1.61(s,12H),1.60-156(,4H),1.41-1.38(m,4H).

[0196] Example 2 - Charged π-conjugated compounds as narrow-spectrum antimicrobial agents against antibiotic-resistant Gram-positive pathogens The data presented below provide evidence that charged π-conjugated compounds possess potent bacteriostatic and bactericidal activity against various Gram-positive pathogens, including antibiotic-resistant strains of Staphylococcus aureus and Enterococcus faecalis, even in the absence of light. In addition to logarithmic growth phase cells, charged π-conjugated compounds are also effective in eradicating antibiotic-resistant phenotypes, such as bacterial persisters and established biofilms of some Gram-positive strains, demonstrating superior efficacy compared to conventional antibiotics. In contrast, the disclosed charged π-conjugated compounds did not exhibit significant inhibitory effects against Gram-negative bacteria. The data presented below indicate that repeated exposure to charged π-conjugated compounds results in lower levels of resistance compared to standard antibiotics. Without being bound by any particular theory, mechanistic studies revealed that charged π-conjugated compounds inhibit bacterial membrane-associated metabolic processes, particularly energy metabolism. This was revealed by observing changes in gene expression related to ATP synthesis and membrane transport induced by the charged π-conjugated compounds. Without being bound by any particular theory, the charged π-conjugated compounds were also found to exhibit synergistic activity with conventional antibiotics, demonstrating their unique mechanism of antibacterial action. Biocompatibility assessment revealed dose-dependent cytotoxicity in mammalian cell lines. Preliminary in vivo evaluation in Gallia mellonella demonstrated that low doses of the charged π-conjugated compounds exhibited a protective effect on helminth survival, suggesting promising therapeutic efficacy against MRSA infections. For at least these reasons, the disclosed charged π-conjugated compounds represent a promising new class of narrow-spectrum antibacterial agents for combating antibiotic-resistant Gram-positive pathogens.

[0197] a. Evaluation of the antibacterial properties of charged π-conjugated compounds We constructed a library of 56 compounds with various amine groups in different regions of the molecule (Fig. 1 and Fig. 2A). Because amine groups are typically positively charged under physiological conditions in the unbound state, various amine groups were incorporated into the molecules to enhance their affinity for negatively charged bacterial membranes.

[0198] In the initial studies, two bacterial strains were used: Staphylococcus aureus as a representative Gram-positive bacterium and Escherichia coli as a representative Gram-negative bacterium. Bacterial cell suspensions were prepared and subsequently treated with increasing concentrations (from 16 to 80 μM) of representative charged π-conjugated compounds. The charged π-conjugated compound-treated cell suspensions were then subjected to a 500 mW cm field. -2 was exposed to near-infrared (NIR) radiation of 48 J cm for 10 minutes. -2 The dose was equivalent to 1000 s of irradiation (UHP-F-730; Prizmatix, Israel). A DMSO-treated control was included to evaluate the potential effect of the solvent. After irradiation, the cell suspension was harvested and inoculated into cation-adjusted Mueller-Hinton medium (CA-MHB) and incubated overnight at 37°C. The samples were then evaluated for growth. The minimum inhibitory concentration (MIC) was determined as the compound concentration at which no clear bacterial growth was observed.

[0199] Figure 2B shows the concentration-dependent growth inhibition patterns of E. coli and S. aureus treated with representative charged π-conjugated compounds. In S. aureus, increasing the concentration of charged π-conjugated compounds resulted in a gradual decrease in bacterial growth, which is consistent for both irradiated and non-irradiated samples. Interestingly, in the case of E. coli, treatment with charged π-conjugated compounds reduced bacterial growth by up to approximately 40%, even at the highest concentration tested. This trend is consistent for both irradiated and non-irradiated samples.

[0200] Based on these initial observations, we further investigated the potential antibacterial activity of the entire library of 56 charged π-conjugated compounds against both Escherichia coli and Staphylococcus aureus in the absence of light. The MICs of various charged π-conjugated compounds in E. coli and S. aureus are shown in Figure 2C. Several molecules exhibited potent bacteriostatic activity against the Gram-positive bacterium S. aureus, with average MIC values ​​ranging from 1.1 μM to over 80 μM (Figure 2C). Against the Gram-negative bacterium E. coli, all molecules exhibited MIC values ​​higher than the highest concentration tested. Similar results were obtained with two other Gram-negative bacteria, Pseudomonas aeruginosa and Acinetobacter baumannii (Figure 3).

[0201] The bacteriostatic effects of charged π-conjugated compounds were further investigated by monitoring the growth curves of Staphylococcus aureus and Enterococcus faecalis treated with increasing concentrations of representative charged π-conjugated compounds (Figure 2D). As the concentration of the charged π-conjugated compounds increased, the maximum OD 600 A significant reduction in bacterial growth was observed, characterized by a decrease in pH and a prolonged lag phase. At the highest concentration tested (10 μM), bacterial growth was completely inhibited, indicating a clear bacteriostatic and / or bactericidal effect.

[0202] Without being bound by theory, these results suggest that charged π-conjugated compounds are narrow-spectrum antibacterial agents specific to Gram-positive bacteria. Gram-negative bacteria have an inner and outer membrane surrounding a thin peptidoglycan layer. In contrast, Gram-positive bacteria have a dense peptidoglycan cell wall but lack an outer membrane (Silhavy et al., 2010). The increased permeability of the Gram-positive cell wall, again without being bound by theory, allows for more efficient uptake of positively charged π-conjugated compounds, which explains their effectiveness against Gram-positive bacteria such as Staphylococcus aureus but their ineffectiveness against Gram-negative species.

[0203] Importantly, the charged π-conjugated compounds exhibit antimicrobial activity regardless of the presence of light, which distinguishes them from previously described photosensitized cyanine derivatives used in photodynamic therapy (PDT). In PDT, light-absorbing molecules called photosensitizers (such as some cyanine derivatives) are excited by specific wavelengths of light, generating reactive oxygen species (ROS), such as singlet oxygen, which can cause cell damage and death (Lange et al., 2021). Accordingly, recent studies have reported a new series of cyclohexene-based cyanine dyes containing two or three indolenine, benzothiazole, or benzoselenazole terminal groups, which are highly efficient near-infrared photosensitizers for antimicrobial PDT (aPDT) (Prakash et al., 2023). However, aPDT also has several limitations (Niculescu & Grumezescu, 2011). One of the main limitations is the depth of light penetration. Effective aPDT requires the interaction of the photosensitizer, oxygen, and light in the target area. When bacteria are located deep within tissues, it can be difficult for the necessary wavelengths of light to reach the bacteria, especially when using visible light. Near-infrared (NIR) light penetrates deeper but is not absorbed by all photosensitizers. Another significant limitation is that aPDT relies on the presence of molecular oxygen to generate reactive oxygen species (ROS) that mediate bacterial killing. Therefore, the effectiveness of aPDT may be reduced in hypoxic or anaerobic environments. The disclosed charged π-conjugated compounds do not require light to exert their antibacterial effects, thereby avoiding the limitations associated with poor light penetration and oxygen dependency that limit the application of aPDT.

[0204] The bacteriostatic potential of the disclosed compounds was extended to a wide range of Gram-positive bacteria from various genera, particularly Aerococcus, Bacillus, Corynebacterium, Enterococcus, Listeria, Micrococcus, Propionibacterium, Rothia, Staphylococcus, and Streptococcus. Additionally, the antibacterial effects of the charged π-conjugated compounds on Mycobacterium smegmatis, a species of Mycobacterium tuberculosis, were also evaluated. Mycobacteria are characterized by a unique cell wall structure with a thick peptidoglycan layer and high mycolic acid concentration, which makes them unstainable using traditional Gram staining. They are classified using alternative methods, such as Ziehl-Neelsen staining and acid-fast staining. Despite this resistance, Mycobacterium tuberculosis is sometimes associated with Gram-positive bacteria because it shares a thick peptidoglycan layer with them (Maitra et al., 2019).

[0205] Antibacterial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and by monitoring the time-dependent bacterial killing of exponentially growing cells. Bacterial cell suspensions (OD in cation-adjusted Mueller-Hinton broth) were cultured at 100°C for 1 hour. 600 A total of approximately 0.02% of the 1000-kJ / ml spores were treated with increasing concentrations of various molecules. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of a molecule that completely stopped bacterial growth. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of a molecule that killed 99.9% of the inoculum. A molecule was determined to be bactericidal if the MBC:MIC ratio was less than 4:6. The MIC values ​​of various charged π-conjugated compounds against various representative bacterial strains are shown in Figure 4. Several molecules exhibited potent antibacterial activity against all Gram-positive strains tested, with average MIC values ​​ranging from 0.88 μM to ≥80 μM. The antibacterial activity of charged π-conjugated compounds against Mycobacterium smegmatis was generally low. However, several molecules exhibited significant antibacterial activity, with MIC values ​​ranging from 5 μM to ≥80 μM.

[0206] Without being bound by theory, the differential activity of charged π-conjugated compounds against Gram-positive bacteria and Mycobacterium smegmatis can be attributed to differences in cell wall structure and composition. The cell wall of Gram-positive bacteria is primarily composed of a relatively porous and thick peptidoglycan layer, potentially allowing large molecules such as charged π-conjugated compounds to easily penetrate and exert their effects. The cell wall of Mycobacterium tuberculosis, including Mycobacterium smegmatis, is unique. While possessing a thick peptidoglycan layer similar to that of Gram-positive bacteria, it also contains a high concentration of mycolic acids. These mycolic acids form a hydrophobic barrier that can be a significant obstacle for many drugs, potentially reducing their penetration and efficacy (Batt et al., 2020). Furthermore, without being bound by theory, it is expected that positively charged π-conjugated compounds have a higher affinity for negatively charged bacterial membranes. While Gram-positive bacterial cell walls contain negatively charged teichoic acids, the Mycobacterium tuberculosis cell wall, with its complex layers of mycolic acids, glycolipids, and other lipids, may not provide the same degree of charge attraction to the disclosed compounds, without being bound by theory (Maitra et al., 2019).

[0207] Importantly, the charged π-conjugated compounds exhibit potent bacteriostatic effects against antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), linezolid-resistant Staphylococcus epidermidis (S. epidermidis), and vancomycin-resistant enterococci (VRE) (Figure 4).

[0208] Using the MIC values ​​of all tested Gram-positive strains (Table 2), MIC 50 The MIC, i.e., the minimum inhibitory concentration required to inhibit the growth of 50% of the tested isolates, was calculated (Figure 5A). 50 According to the results, the molecules that showed the strongest antibacterial activity were Cy5.5amine, BL 248, BL 273, Cy7.5amine, and GL 356-2. Mean MIC 50 When the mean MIC values ​​were evaluated, the most susceptible bacterial genera were Streptococcus, followed by Staphylococcus. In contrast, Propionibacterium had the highest mean MIC values. 50 was shown (Figure 5B).

[0209] MIC 50 To evaluate the bactericidal activity of the most potent charged π-conjugated compounds identified from the calculated values, time-dependent bactericidal experiments were performed. Cell suspensions of various representative Gram-positive bacterial strains in the exponential growth phase were exposed to different charged π-conjugated compounds for increasing times. The conventional antibiotics linezolid and vancomycin were used as controls. Treatment with the charged π-conjugated compounds effectively eradicated bacterial populations, including antibiotic-resistant strains, within 2–12 h (i.e., approximately 5 log 10 The efficacy of these antibiotics exceeded that of conventional antibiotics in all strains tested (Figure 6).

[0210] We also investigated the potential of charged π-conjugated compounds to eradicate bacterial persister cells. Persister cells are a small subset of dormant, highly antibiotic-resistant phenotypic variants present in bacterial populations that are genetically indistinguishable from wild-type populations (Huemer et al., 2020). The charged π-conjugated compounds disclosed herein effectively eradicated persister cells, including those from antibiotic-resistant strains, within 2 to 24 hours. This bactericidal activity exceeded that of conventional antibiotics, which only achieved a maximum bacterial population reduction of approximately 2 log after 24 hours of treatment. 10 (Figure 7).

[0211] The effectiveness of the charged π-conjugated compounds in removing established biofilms was also examined by quantifying biofilm biomass using a crystal violet assay and counting colony-forming units (CFUs) within the treated biofilms. Biofilms are dense communities of microorganisms bound to each other and to various surfaces and encased in a self-sustaining extracellular polymer matrix. This complex structure and unique physiological properties make biofilms highly resistant to antibiotics and immune responses (Stewart & Costerton, 2001). The ability of charged π-conjugated compounds to remove biofilms varied among bacterial strains. The average reduction in biofilm biomass by charged π-conjugated compounds ranged from 5.8% for Listeria monocytogenes CECT93 to 52.1% for Bacillus subtilis. Similarly, the reduction in viable cell numbers within biofilms by charged π-conjugated compounds ranged from an average of 6.9% for Staphylococcus saprophyticus CECT235 to 81.5% for Rothia dentocariosa CECT4829, indicating different susceptibilities of biofilms to charged π-conjugated compounds (Figure 8). Similar results were obtained when evaluating the effect of charged π-conjugated compounds on the percentage of metabolically active cells within established biofilms (Figure 9).

[0212] b. Resistance development and mutation frequency Both stepwise and single-step testing methods were used to assess the potential for resistance development following exposure to charged π-conjugated compounds. In the stepwise evaluation of resistance development, bacterial cells were exposed to increasing concentrations of either the charged π-conjugated compounds or conventional antibiotics. Surviving cells were repeatedly exposed to either the charged π-conjugated compounds or the antibiotic, and the MICs were monitored over time. For all strains tested, cells treated with the charged π-conjugated compounds showed an increase in MIC over 30 cycles, from cycle 4 to cycle 16. This was comparable to the results obtained with the antibiotics vancomycin and linezolid, but was much lower than that obtained with ciprofloxacin (Figure 10A). Furthermore, the mutation frequency (assessed as the frequency of rifampicin-resistant colonies) was measured for Staphylococcus aureus ATCC 25923 cells repeatedly treated with either the charged π-conjugated compounds or the conventional antibiotic ciprofloxacin. The mutation rate of ciprofloxacin showed a significant increase throughout the treatment cycles, whereas the mutation rate of cells repeatedly treated with the charged π-conjugated compound remained relatively stable throughout the exposure cycles, showing a low mutation rate ( Figure 10B ).

[0213] To isolate resistant mutants in a single step, overnight cultures (approximately 10 CFU mL) of different bacterial strains were cultured. -1 ) were plated onto LB agar containing 4×, 8×, and 16× the MIC of the charged π-conjugated compounds Cy5.5amine, BL248, BL273, Cy7.5amine, and GL 356-2. The range of spontaneous mutation rates was ≤10 on plates containing Cy7.5amine. -6 from the remaining molecules <10 -9The results were not significant (Figure 10C). Spontaneous mutants resistant to Cy7.5 amine could be isolated from overnight cultures. However, these strains failed to grow on fresh plates supplemented with charged π-conjugated compounds, and even when they did, their MICs were indistinguishable from those of the wild-type (WT) strain. There are several possible explanations for this. Without being bound by theory, it is possible that the mutations acquired in the Cy7.5 amine-resistant mutants were unstable, and when transferred to a new environment, they reverted to the wild-type genotype, resulting in the observed MICs similar to those of the WT strain (Dan et al., 2021). Another possibility, without being bound by theory, is that the spontaneous mutants evolved compensatory mutations or mechanisms that counteracted the effects of the original mutations. Without being bound by theory, these compensatory mechanisms could restore bacterial fitness and sensitivity to charged π-conjugated compounds to the same level as the wild-type (Martinez & Baquero, 2000). Furthermore, without being bound by theory, changes in antimicrobial susceptibility may not necessarily be due to genetic mutations but may instead be due to epigenetic modifications. Such changes may be transient and not persist in subsequent generations, resulting in the absence of observed phenotypic differences in susceptibility to charged π-conjugated compounds in WT strains (Adam et al., 2008).

[0214] c. Effect of various physicochemical factors on the antibacterial activity of charged π-conjugated compounds The effects of various physicochemical factors on the antibacterial activity of charged π-conjugated compounds were tested by measuring the minimum inhibitory concentration (MIC) under various conditions, including temperature, pH, serum, NaCl, and calcium concentration. Additionally, the effect of the initial inoculum size was also investigated.

[0215] The effect of temperature on the activity of charged π-conjugated compounds was evaluated by comparing the time-kill curves of two representative bacterial strains at two different temperatures (25°C and 37°C) (Figure 11A). For most charged π-conjugated compounds, the reduction in bacterial population size (log N / N0) was more pronounced at 37°C than at 25°C. This observation, without being bound by theory, suggests that the antibacterial efficacy of charged π-conjugated compounds is enhanced at 37°C, a physiologically relevant temperature. The enhanced antibacterial activity of charged π-conjugated compounds at elevated temperatures may be associated with accelerated bacterial metabolism, which could result in increased absorption of or increased susceptibility to charged π-conjugated compounds. Furthermore, without being bound by theory, temperature fluctuations may affect the fluidity and permeability of bacterial membranes, which may affect the absorption and efficacy of charged π-conjugated compounds (Hajdu et al., 2010).

[0216] The effect of pH on the antibacterial activity of charged π-conjugated compounds was evaluated by measuring the MICs of various charged π-conjugated compounds in CA-MHB adjusted to different pH values. For both Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, the MIC values ​​of all tested molecules tended to decrease as the pH increased from 5 to 9 (Figure 11B). This suggests, without being bound by theory, that as the environment becomes more alkaline, charged π-conjugated compounds become more effective at inhibiting bacterial growth. This may be due to the increased stability, improved absorption, or other pH-sensitive bacterial responses of charged π-conjugated compounds (Thomas et al., 2012; Kincses et al., 2021). Without being bound by theory, the presence of terminal amine groups in charged π-conjugated compounds may be one of the reasons for the observed pH-dependent activity. Amine groups carry protons (H + ) and in an acidic environment, it becomes positively charged and accepts the ammonium ion (-NH3 +As the pH increases (toward alkalinity), amines become less likely to be protonated and tend to exist in an uncharged state. This change in charge state has several implications. The charge of a molecule can affect its ability to cross bacterial cell membranes. In general, uncharged molecules tend to cross lipid bilayers more easily than charged molecules. Therefore, alkaline conditions, where more charged π-conjugated compounds exist in an uncharged state, may enhance their penetration into bacterial cells. The binding of a molecule to a target site within bacteria may also be affected by its charge. Without being bound by theory, the protonation state of a charged π-conjugated compound may determine its binding affinity for a particular bacterial target. Without being bound by theory, protonation or deprotonation of the amine group may result in structural changes in the charged π-conjugated compound molecule, affecting its interaction with the bacterial target. The chemical stability of charged π-conjugated compounds may depend on pH. Under certain pH conditions, these molecules may become more stable, less susceptible to degradation, and exhibit improved efficacy.

[0217] The effect of serum on the antibacterial activity of charged π-conjugated compounds was investigated by examining the effect of increasing serum concentration on the MIC of the charged π-conjugated compounds (Figure 11C). The MIC increased significantly as serum concentration increased from 0% to 50%. This suggests, without being bound by theory, that higher serum concentrations result in reduced efficacy. One possible reason for this, without being bound by theory, is reduced bioavailability, where serum proteins bind to the charged π-conjugated compounds, reducing their free concentration and, therefore, their ability to act on pathogens. Another possible explanation, without being bound by theory, is the inactivation of charged π-conjugated compounds. Some components present in serum may chemically inactivate or promote the degradation of charged π-conjugated compounds. Finally, without being bound by theory, serum may provide a protective environment for bacteria, reducing their susceptibility to the antibacterial activity of charged π-conjugated compounds (Kaplan et al., 2013).

[0218] Next, we tested the effect of NaCl concentration on the activity of the charged π-conjugated compounds by measuring the MIC of CA-MHB supplemented with increasing concentrations of NaCl (Figure 11D). For Staphylococcus aureus, the MIC either increased or remained constant with increasing NaCl concentration, whereas for Enterococcus faecalis, the MIC either remained constant or decreased. While not bound by theory, the variation in MIC with increasing NaCl concentration may be attributed to the effect of NaCl on the ionic environment. This effect may affect bacterial membrane permeability or the stability of the charged π-conjugated compounds, resulting in altered antibacterial efficacy. While not bound by theory, the different responses of the two bacterial strains to NaCl suggest inherent differences in their response to osmotic stress or variations in cell wall structure. The presence of free amine groups on the charged π-conjugated compounds may also play an important role in influencing the MIC in the presence of NaCl. Without being bound by any particular theory, the ionic strength of the environment, altered by the addition of NaCl, may affect the electrostatic interactions between the positively charged amine groups on the molecule and the negatively charged bacterial cell surface. As a result, the binding affinity of the charged π-conjugated compound to bacterial cells may be affected, enhancing or reducing the molecule's ability to exert antibacterial activity. The electrostatic shield formed by the salt, again without being bound by theory, may hinder the interaction of the charged π-conjugated compound molecule with its target on the bacterial cell, thereby requiring a higher molecule concentration to achieve the same inhibitory effect, leading to an increased MIC. Conversely, without being bound by theory, in certain scenarios, a more favorable interaction may be observed due to the change in charge distribution (Li et al., 2021; Yoon et al., 2013).

[0219] Next, we examined the effect of increasing calcium ion concentrations (Figure S1E). The different responses of the two strains to increasing calcium concentrations indicate different effects of calcium on the MIC, which may be mediated by effects on bacterial cell wall stability, uptake mechanisms, or efflux pump activity (Xie & Yang, 2016).

[0220] Finally, we evaluated the effect of inoculum size on the antibacterial activity of charged π-conjugated compounds (Figure 11F). We found that increasing the inoculum size, i.e., the initial number of bacterial cells exposed to the charged π-conjugated compounds, increased the concentration of compound required to inhibit growth. Without being bound by theory, this phenomenon can be explained by considering the antibacterial action of charged π-conjugated compounds at the population level. When the initial inoculum of bacteria is high, even if the charged π-conjugated compounds can kill some cells, the remaining viable cells can grow and restore the bacterial population. Therefore, a higher concentration of charged π-conjugated compounds is required to inhibit growth. Without being bound by theory, at high cell densities, individual cells may be exposed to a microenvironment with a lower compound concentration per cell (Morrissey & George, 1999).

[0221] d. Mechanism of action test The mechanism of action of charged π-conjugated compounds was investigated using RNA sequencing (RNA-seq) by treating S. aureus with sublethal concentrations of the charged π-conjugated compound BL-248 (Figure 12). RNA-seq analysis of S. aureus treated with sublethal concentrations of charged π-conjugated compounds revealed distinct transcriptional responses. Several genes showed significant up- or down-regulation in response to treatment with charged π-conjugated compounds (Figure 12A). Principal component analysis (PCA) further distinguished gene expression profiles between treatments. Notably, 94% of the variance was captured by PC1, indicating significant differences in gene expression profiles between treated and untreated samples (Figure 12B). Protein-protein interaction networks indicated proteins associated with the proton-transporting ATP synthase complex and membranes as key regulatory and metabolic nodes affected by treatment with charged π-conjugated compounds (Figure 12C). Gene Ontology (GO) biological process analysis revealed that the processes most affected by treatment with charged π-conjugated compounds were proton motive force-driven ATP synthesis, threonine biosynthesis, nitrate metabolism, histidine biosynthesis, and proton membrane transport (Figures S12D and S12E). Without being bound by theory, these results suggest that treatment with charged π-conjugated compounds affects energy production pathways, amino acid synthesis, and ion transport mechanisms.

[0222] To investigate the effect of treatment with charged π-conjugated compounds on bacterial energy metabolism, we examined the effect of increasing concentrations of charged π-conjugated compounds on total ATP levels (intracellular and extracellular) in S. aureus (Figure 13). Treatment with charged π-conjugated compounds consistently decreased total ATP levels, and both intracellular and extracellular ATP levels decreased with increasing concentrations of charged π-conjugated compounds. Without being bound by theory, this suggests that charged π-conjugated compounds may inhibit bacterial metabolic pathways related to ATP production, such as oxidative phosphorylation and glycolysis, thereby affecting bacterial growth and viability.

[0223] Next, we examined the effects of different charged π-conjugated compounds on membrane potential using the fluorescent probe DiBAC4(5). DiBAC4(5) is a voltage-sensitive fluorescent dye commonly used to measure changes in membrane potential (Clementi et al., 2014). Most of the tested charged π-conjugated compounds had a detectable effect on the membrane potential of S. aureus. Higher concentrations of the charged π-conjugated compounds consistently resulted in greater membrane depolarization, suggesting a dose-dependent effect (Figure 14). This suggests that the charged π-conjugated compounds may affect bacterial membrane integrity or disrupt ion channels or transporters, preventing bacteria from maintaining an electrochemical proton gradient. These observations are consistent with the RNA-seq data.

[0224] e. Interactions with conventional antibiotics The interactions of charged π-conjugated compounds with various conventional antibiotics with different mechanisms of action were evaluated using a checkerboard assay. Generally, the combination of charged π-conjugated compounds with conventional antibiotics yielded fractional inhibitory concentration index (FICI) values ​​below 0.5, demonstrating synergistic interactions between conventional antibiotics and various charged π-conjugated compounds (Figure 15A). Such consistent synergistic interactions indicate that the mechanisms of action of charged π-conjugated compounds differ from those of conventional antibiotics and that they can cooperate to inhibit bacterial growth. Notably, the interactions between charged π-conjugated compounds and the antibiotics gramicidin and daptomycin yielded the lowest average FICI, suggesting a particularly strong synergistic relationship (Figure 15B). The interactions between charged π-conjugated compounds and antibiotics were further examined using a time-kill assay. When Staphylococcus aureus was treated with sublethal concentrations (0.5 × MIC) of individual charged π-conjugated compounds, gramicidin, or daptomycin, bacterial populations were reduced by a maximum of approximately 3 log , even 12 hours after treatment. 10 In contrast, sublethal concentrations of charged π-conjugated compounds combined with antibiotics resulted in complete eradication of bacterial populations (i.e., approximately 5 log 10 (reduction of 1000 kJ / kg) was achieved (Figure 15C).

[0225] Gramicidin and daptomycin primarily target bacterial membranes. Daptomycin is known to integrate into bacterial membranes in a calcium-dependent manner, causing potassium ion efflux from bacterial cells. This phenomenon disrupts the membrane potential and can lead to cell death (Silverman et al., 2003). Conversely, gramicidin forms ion channels in bacterial membranes, facilitating ion movement and disrupting proton and ion gradients essential for bacterial function (Wenzel et al., 2018). The strong synergistic effects observed between gramicidin and daptomycin and charged π-conjugated compounds suggest, without being bound by theory, that these antibiotics may complement the mechanisms of action of charged π-conjugated compounds. Furthermore, one antibacterial agent may promote the intracellular uptake or retention of another, resulting in a stronger combined effect than the individual agents. Therefore, without being bound by theory, it is possible that the synergistic effect of the charged π-conjugated compounds with daptomycin and gramicidin results from a multifaceted attack on the bacterial membrane, causing severe dysfunction and resulting in bacterial death.

[0226] The checkerboard assay was also used to examine the interactions between different charged π-conjugated compounds. Interestingly, some molecules exhibit synergistic interactions (FICI ≤ 0.5) (Figure 16A). Accordingly, the time-kill curves of S. aureus treated with sublethal combinations of charged π-conjugated compounds showed complete elimination of bacteria (approximately 5 log 10 Even after 12 hours of treatment with equal concentrations of each compound, the maximum reduction was approximately 3 log 10(Figure 16B). Without being bound by theory, these results suggest that differently charged π-conjugated compound derivatives may have complementary mechanisms of action that enhance their combined antibacterial activity. Without being bound by theory, it is contemplated that these molecules may bind to different membrane components or induce unique membrane structural changes, collectively exerting a more pronounced disruptive effect. Without being bound by theory, these variants may also have different binding affinities or kinetics. As a non-limiting example, one variant may rapidly bind and destabilize membranes, whereas another variant may induce slower but more sustained integration. Without being bound by theory, these combined effects may enhance antibacterial efficacy in some embodiments. Furthermore, in some embodiments, combined variants may affect membrane function by altering membrane protein composition or inhibiting membrane-based efflux pumps, amplifying perturbations through their individual interactions.

[0227] f. Biocompatibility The biocompatibility of charged π-conjugated compounds with mammalian cell lines was evaluated by testing the effect of different concentrations of the charged π-conjugated compounds on the viability of two mammalian cell lines, A549 (epithelial-like lung cells) and HEK293 (epithelial-like kidney cells). In both cell lines, an inverse correlation was observed between the concentration of the charged π-conjugated compounds and viability, with viability decreasing as the concentration of the charged π-conjugated compounds increased. This indicates that these molecules are cytotoxic, especially at high concentrations (Figure 17A). These results are consistent with those obtained with complementary techniques and other mammalian cell lines (Figures 18 and 19). IC values ​​were calculated using dose-response curves. 50 The IC value, i.e., the concentration of charged π-conjugated compound required to inhibit cell viability by 50%, was determined. 50 IC values ​​ranged from 2.5 μM (GL 356-2) to 14.7 μM (BL 248), whereas in the HEK293 cell line, IC 50The values ​​ranged from 6.3 μM (Cy7.5 amine) to 13.7 μM (BL 273). The safety of the molecules was assessed by measuring the therapeutic index, i.e., the concentration at which the drug becomes toxic (IC 50 The therapeutic index was further quantified by calculating the ratio of the MIC to the effective drug concentration (MIC). The therapeutic index was calculated using the MIC of MRSA USA300. MRSA USA300 is a major cause of community-acquired MRSA infections, including skin and soft tissue infections, bacteremia, endocarditis, bone and joint infections, and hospital-acquired infections. MRSA USA300 has attracted attention due to increasing multidrug resistance, including resistance to antibiotics such as erythromycin, levofloxacin, mupirocin, and tetracycline. Furthermore, its high transmissibility has been associated with outbreaks in various environments, including prisons and sports teams. The therapeutic index is 1.3 for Cy7.5 amine and 9.3 for BL248 (Figure 17B).

[0228] The in vivo biocompatibility of charged π-conjugated compounds was further investigated using the invertebrate model Gallia mellonella (Serrano et al., 2023). Worms were injected with increasing concentrations of the charged π-conjugated compounds or vehicle, and survival rates were monitored. An interesting finding emerged: moderate concentrations of the charged π-conjugated compounds improved survival rates compared to the vehicle control (1% DMSO) (Figure 20). Without being bound by theory, these results suggest the possibility of inducing a hormetic response, in which moderate doses of the charged π-conjugated compounds can confer a protective effect by activating the worm's innate defense mechanisms.

[0229] g. In vivo anti-infective activity The in vivo anti-infective potential of charged π-conjugated compounds was evaluated in the invertebrate model Gallia mellonella infected with MRSA USA300. Survival curves, shown in Figure 21, demonstrate that two charged π-conjugated compounds, BL 248 and BL 273, are effective against MRSA USA300 infection in Gallia mellonella. As concentrations of both molecules increased, survival rates were significantly improved compared to vehicle-treated controls. Specifically, for BL 248, doses of 5–40 μM extended the survival period of infected worms from at least 1 day for vehicle-treated worms to more than 4 days for worms treated with the charged π-conjugated compounds. For BL 273, treatment with the charged π-conjugated compounds extended survival from 1 day for vehicle-treated worms to more than 3 days for worms treated with 10–80 μM charged π-conjugated compounds. These results highlighted the promising anti-infective activity of the ambivalently charged π-conjugated compounds against the MRSA USA300 strain.

[0230] h. Materials and Methods i Synthetic chemistry The synthesis and characterization of the charged π-conjugated compounds used in this study have been reported elsewhere (Ayala-Orozco et al., 2023), including above. Figure 1 shows the chemical structures of these molecules.

[0231] ii Strains and reagents A complete list of strains used in this study and their origins is shown in Table 2. Unless otherwise stated, all reagents were purchased from MedChem Express (Princeton, NJ, USA), Cayman Chemical Company (Ann Arbor, MI, USA), or MilliporeSigma (St. Louis, MO, USA) and dissolved in 100% DMSO or appropriate solvent according to the manufacturer's instructions. [Table 2] TIFF2026501715000086.tif254170TIFF2026501715000087.tif244170 CA-MH: Cation-adjusted Mueller-Hinton. BHI: Brain Heart Infusion

[0232] iii. Preparation of cell suspension Bacterial cells recovered from glycerol stocks stored at -80°C were streaked onto blood agar plates to isolate individual colonies. Single colonies were cultured overnight in the liquid media shown in Table 2 at the appropriate temperature with agitation at 220 rpm. The overnight culture was then diluted 1:50 with fresh medium and analyzed for optical density at 600 nm (OD ). 600 The cells were centrifuged at 5,000 × g for 10 minutes, washed, and resuspended in phosphate-buffered saline (1 × PBS). 6 CFU mL -1 The density was set to .

[0233] iv Irradiation settings The cell suspension prepared as described above was transferred to a 6-well plate and placed at the center of a 730 nm light-emitting diode (UHP-F-730 nm, Prizmatix, Israel). The distance from the light source was verified using a ThorLabs Sensor S415C (Thorlabs, Newton, MA, USA), and the output power was 80 mW cm. -2 The sample was adjusted to obtain a strength of 48 J / cm 2 The irradiation was carried out for 10 minutes at a fluence of 1.0.

[0234] v Minimum inhibitory concentration MICs of charged π-conjugated compounds were determined using the broth microdilution method according to CLSI guidelines. Charged π-conjugated compound stock solutions (16 mM) were prepared in dimethyl sulfoxide (DMSO). Serial two-fold dilutions of the charged π-conjugated compounds were freshly prepared in the appropriate liquid medium (Table 2), and 100 μL of each dilution was added to the appropriate wells of a 96-well microtiter plate. The bacterial inoculum was further diluted in liquid medium, and 100 μL was added to each well to achieve a final cell density of approximately 5 × 10. 5 CFU mL -1A negative control (without cells) and a positive control (without charged π-conjugated compound) were also prepared. After 16–20 h of incubation at the appropriate temperature (Table 2), OD600 was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of a compound that completely stopped bacterial growth. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of a molecule that killed 99.9% of the inoculum. A molecule was considered bactericidal if the ratio of MBC to MIC was less than 4:6. All experiments were repeated at least three times.

[0235] vi Growth curve As previously described, the cells were cultured overnight in CA-MH broth and then incubated for approximately 10 min. 6 CFU mL -1 The cells were cultured at a density of 1000 x 1000 μg / ml. Cells were treated with 1% DMSO or increasing concentrations of charged π-conjugated compounds. These samples were transferred to a 96-well microtiter plate (Corning, NY, USA) and covered with mineral oil to reduce evaporation during incubation. Growth trajectories were recorded by monitoring OD600 every 10 min at 37°C using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Each experiment was repeated at least three times.

[0236] vii Time-dependent sterilization experiments Reference bacterial strains representing each species listed in Table 2 (including antibiotic-resistant strains of known clinical significance) were used in time-kill assays using both exponentially growing and persister cells.

[0237] Exponentially growing cells from a fresh overnight culture on a blood agar plate were suspended in the appropriate medium (Table 2) and grown at an OD 600 To isolate persistently growing cells, bacterial cultures were grown at an appropriate temperature in the appropriate medium (Table 2) with stirring (220 rpm) until the OD 600The cells were cultured until the β-glucan concentration reached approximately 0.1. The cells were then diluted 1:1000 in fresh medium and cultured under the same conditions for an additional 16 hours. To eliminate antibiotic-sensitive cells, linezolid was added at a concentration equivalent to 10× the MIC. After 24 hours of treatment, the cells were harvested, washed to remove residual antibiotic, and diluted 1:10 in fresh medium (Table 2). 6 CFU mL -1 (Santos et al., 2022).

[0238] Charged π-conjugated compounds were added at concentrations of 4x, 8x, and 16x the MIC. Samples treated with conventional antibiotics (linezolid and vancomycin) were used as controls. After antibiotic exposure, samples were collected at different time points, diluted with PBS, and plated on appropriate agar media (Table 2). After 18–24 h of incubation at the appropriate temperature, colony counts were measured (Table 2). Time-kill curves were generated, and bactericidal activity was measured as CFU mL compared to the initial inoculum. -1 is 3 logs 10 A reduction of ≥100 colonies was defined as a reduction of ≥100 colonies. Only dilutions yielding 10–100 colonies were used. Results were expressed as log(N / N0), where N is the number of CFU mL at each observation time point. -1 , N0 is the initial CFU mL of each sample -1 Represents.

[0239] viii Anti-biofilm activity Bacterial strains were grown overnight in the appropriate medium (Table 2) at the appropriate temperature and shaken at 200 rpm. The cultures were monitored for optical density (OD 600 The pH was adjusted to approximately 0.1. 100 μL of these cultures were then transferred to 96-well polystyrene microtiter plates. After 24 hours of static incubation at 30°C or 37°C (Table 2), biofilm formation was allowed, and nonadherent cells were washed with phosphate-buffered saline (PBS). Established biofilms were exposed to 8x the MIC of the charged π-conjugated compounds or reference antibiotics in the appropriate medium (Table 2). After 24 hours, the supernatant was removed, and the biofilms were washed with PBS and dried (Santos et al., 2022).

[0240] For quantification, biofilms were stained with 0.1% (w / v) crystal violet solution (100 μL) at room temperature for 30 minutes. The stain was then removed, and the wells were washed with water. The bound dye was dissolved in 30% acetic acid, and the absorbance at 550 nm was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). In a separate group of similarly treated samples, biofilms were mechanically disrupted with PBS, diluted, and plated on appropriate media for viable cell counts. These results were expressed as viable CFU mL. -1 The results were expressed as mean values ​​(mg / mL) and compared with those of untreated controls. In a separate group of similarly treated samples, metabolically active cells were assessed by quantifying ATP levels in the biofilm using the BacTiter-Glo Microbial Cell Viability Assay (Promega, WI, USA). All experiments were repeated at least three times.

[0241] ix. Emergence of antibiotic resistance and antibiotic mutation frequency The development of resistance was tested using serial passage experiments. The cell suspension was cultured at approximately 10 6 CFU mL -1 The cells were prepared at 100°C and treated with increasing concentrations of charged π-conjugated compounds or antibiotics (linezolid, vancomycin, or ciprofloxacin) (Figures 10A-10C). After 18 hours of incubation at 30°C or 37°C (Table 2), cells grown at 0.5×MIC were harvested and re-exposed to various concentrations of charged π-conjugated compounds. This procedure was repeated for 30 days. Samples harvested from each cycle were stored at -80°C in 25% glycerol for later mutation frequency assessment.

[0242] Mutation frequency was measured using rifampicin resistance as a marker. The preserved cells were cultured overnight in LB medium and then treated with rifampicin (100 mg mL -1 The mutants were plated on LB agar with and without rifampicin (Schaaff et al., 2002). The mutation frequency was calculated as the percentage of rifampicin-resistant colonies relative to the total number of colonies.

[0243] To estimate the frequency of spontaneous mutants resistant to charged π-bonding compounds, overnight cultured S. aureus cells were resuspended in PBS and incubated for 10 min. 10 pieces / mL -1 The resulting mixture was then plated onto selective LB agar plates (containing charged π-binding compounds) and non-selective LB agar plates. After 24-48 hours of incubation at 37°C, the number of colonies was counted and the frequency of charged π-binding compound-resistant mutants was calculated. All experiments were repeated at least three times.

[0244] Influence of physicochemical factors on the antibacterial activity of x-charged π-conjugated compounds The antibacterial activity of charged π-conjugated compounds was evaluated under various environmental conditions, including temperature, pH, serum concentration, NaCl concentration, calcium concentration, and inoculum size. The effect of temperature was examined by conducting time-dependent killing experiments at both 25°C and 37°C as previously described. The effect of pH was measured using CA-MHB buffered to various pH values ​​(5–9). The role of serum was examined by adding various concentrations of serum (0–50% v / v) to CA-MHB. The effects of NaCl and calcium were examined by adding different concentrations of NaCl or CaCl2 to MHB. The effect of bacterial population size was examined using suspensions with varying cell densities.

[0245] xi RNA sequencing Three different S. aureus colonies were cultured in CA-MHB and grown to mid-logarithmic phase with agitation at 37°C as previously described. Cells were treated with 0.5×MIC of charged π-binding compounds or 1% DMSO for 8 hours. Cells were then filtered and fixed with RNAprotect (QIAGEN, Hilden, Germany). RNA extraction was performed using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) (Santos et al., 2022). RNA-seq and data processing were performed by Azenta Life Sciences (Leipzig, Germany). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Kit with Ribo-Zero Plus rRNA Depletion and sequenced on an Illumina NovaSeq 6000 platform. An average of 70 million reads were generated per sample. For data analysis, reads were trimmed and matched to the S. aureus reference genome to identify differentially expressed genes. Gene annotation was based on the National Center for Biotechnology Information (NCBI) database. Functional enrichment analysis of differentially expressed genes was performed using Panther (https: / / pantherdb.org / ).

[0246] xii Membrane potential Dilute an overnight culture of S. aureus 1:100 in fresh Mueller-Hinton medium until it reaches mid-logarithmic growth phase (OD 600 The cells were then harvested by centrifugation, washed with phosphate-buffered saline (PBS), and incubated at 0.8°C for 1 hour until the OD reached 0.4–0.6. 600 The cells were resuspended in PBS (containing glucose) until the δ was 0.1, and increasing concentrations of a charged π-conjugated compound were added. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control at a concentration of 10 μM because it is a proton ionophore that reduces membrane potential.

[0247] After 6 hours of treatment, the fluorescent probe DiBAC4(3) was added at 1 μg mL-1 (final concentration) (Clementi et al., 2012). The mixture was placed in the dark and incubated for an additional 15 minutes. Samples were transferred to a black 96-well microplate, and fluorescence was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA) set at excitation / emission wavelengths of 495 / 516 nm. Relative fluorescence units (RFU) were recorded after subtracting blank values. All experiments were performed in triplicate.

[0248] xiii Intracellular and extracellular ATP Staphylococcus aureus cell suspension (approximately 10 6 CFU mL -1 ) were prepared as described above and treated with increasing concentrations of charged π-conjugated compounds for 8 hours. Cells were harvested by centrifugation at 12,000 × g for 10 minutes. Extracellular ATP was quantified in the supernatant. The pellet was sonicated (using a Misonix S-4000) and the resulting supernatant was used to measure intracellular ATP after centrifugation. ATP was quantified using the luminescent BacTiter-Glo Microbial Cell Viability Assay (Promega, WI, USA) according to the manufacturer's instructions. Luminescence measurements were performed using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). ATP content was determined by linear regression based on an ATP standard curve. Protein concentrations were determined using both the supernatant and pellet using the Pierce assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, MA, USA). The ATP content of each sample was then normalized to protein concentration. All experiments were performed in triplicate.

[0249] xiv Checkerboard assay The interactions of charged π-conjugated compounds with conventional antibiotics, as well as between differently charged π-conjugated compounds, were tested using a modified microdilution checkerboard assay in an 8 × 8 well format (Santos et al., 2022). Bacterial cell suspensions (approximately 106 CFU mL -1 ) were prepared as described above and treated with increasing concentrations of a charged π-conjugated compound along the x-axis of the microplate. Next, increasing concentrations of an antibiotic (or another charged π-conjugated compound) in Mueller-Hinton medium were added along the y-axis of the microplate. The plates were sealed and incubated at 37°C for 18 hours, after which the OD 600 Bacterial growth was determined by measuring the fractional inhibitory concentration index (FICI), which was calculated by dividing the sum of the MICs of the charged π-conjugated compounds and antibiotics used in combination by the MICs of the antibiotics used alone. FICI values ​​of ≤0.5 indicate synergistic interactions, 0.5–4 indicate additive interactions, and values ​​above 4 indicate antagonistic interactions (Odds 2003). All experiments were performed at least in triplicate. The interactions between charged π-conjugated compounds and conventional antibiotics, or between differently charged π-conjugated compounds, were further characterized in time-dependent killing experiments, as previously described.

[0250] xv Toxicity profiling and therapeutic index calculation The biocompatibility of charged π-conjugated compounds with mammalian cell lines was evaluated by determining their effects on mitochondrial activity in human alveolar basal adenocarcinoma cells (A549) and human embryonic kidney cells (HEK293T) using the Presto Blue assay according to the manufacturer's protocol. Mammalian cell lines obtained from ATCC were cultured in DMEM. Cells were treated with various concentrations of charged π-conjugated compounds. After 8 hours, Presto Blue cell viability reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells, and after 2 hours of incubation, fluorescence was measured using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The IC values ​​were calculated from the resulting dose-response curves. 50 The therapeutic index was calculated as IC 50 The concentration was calculated from the ratio of MIC to MIC.

[0251] The biocompatibility of the charged π-conjugated compounds was further investigated in additional mammalian cell lines, specifically epithelial-like hepatocellular carcinoma cells (HepG2), mouse alveolar macrophage cell line (MHS), and HEK293 variants HEK-Blue™ TLR and HEK-Blue™ NOD, as well as A549 cells and wild-type HEK293T, by quantifying ATP levels. Mammalian cells were treated with increasing concentrations of various molecules for 8 hours. Cell viability was then determined using the CellTiter-Glo™ Luminescent Cell Viability Assay (Promega, WI, USA) to quantify the level of intracellular ATP. The concentration of molecules that reduces cell viability by 50% (i.e., IC 50 ) was determined.

[0252] Finally, biocompatibility was also assessed by measuring the effect of the charged π-conjugated compounds on the replicative capacity of mammalian cells using a clonogenic assay. Briefly, cells were first cultured under standard conditions until they reached approximately 80% confluence. Then, the cells were harvested and treated with increasing concentrations of the charged π-conjugated compounds for 8 hours. After treatment, the cells were seeded in fresh medium and cultured for 5 days. The medium was changed every 48 hours during culture. After incubation, the medium was carefully aspirated, and colonies were fixed with a mixture of methanol and acetic acid (3:1, v / v). After removing the fixative, the cells were stained with 1% crystal violet (w / v).

[0253] xvi Animal Experiments Animal experiments were conducted using the invertebrate model Gallia mellonella (Santos et al., 2022). Larvae at a stage in their life cycle that does not require feeding were commercially obtained. Larvae were sorted onto Petri dishes lined with Whatman filter paper (Thermo Fisher Scientific, Pittsburgh, PA, USA) and stored at 4°C until use.

[0254] Working solutions of charged π-conjugated compounds were prepared at increasing concentrations in sterile phosphate-buffered saline (PBS). For in vivo biocompatibility assessment, cohorts of nine larvae were injected with 10 μL of each charged π-conjugated compound formulation into the hemocoel of the most posterior left proleg using a Hamilton syringe and needle. 1% DMSO (in PBS) was injected as a negative control. Injected larvae were incubated at 37°C and subsequently monitored for survival.

[0255] To evaluate the in vivo anti-infective activity of the charged π-conjugated compounds, MRSA USA300 was cultured overnight in tryptic soy broth and incubated at OD 600 Approximately 2 x 10 cells in sterile phosphate-buffered saline based on 7 CFU mL -1 A cohort of five Gallia mellonella larvae (approximately 300 mg each) was inoculated with the diluted bacterial suspension (2 × 10 5 CFU mL -1 ) was injected into the most posterior left proleg. One hour after infection, larvae were injected with increasing concentrations of the charged π-conjugated compound or 1% DMSO (in PBS) as a negative control. Uninfected larvae were also used as controls. Larvae were cultured at 37°C and then monitored for survival. Death was defined as the time when the larval body became completely melanized and no longer moved when touched.

[0256] xvii Statistical analysis Unless otherwise noted, arithmetic means and standard errors across multiple biological and technical replicates were used as indicators of median and spread. The number of replicates for each experimental type is listed in the respective figure legends, if applicable. All statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). Where appropriate, data were normalized by minimum-maximum. Depending on the sample size, the Anderson-Darling normality test, D'Agostino-Pearson omnibus normality test, Shapiro-Wilk normality test, or Kolmogorov-Smirnov normality test was used to assess data normality, and the Dalal-Wilkinson-Lilyfors test was used for P values. Comparisons between two groups were performed using t-tests for parametric data and Mann-Whitney U-tests for nonparametric data. Comparisons between multiple groups were performed using analysis of variance (ANOVA) or Kruskal-Wallis and Dunn's multiple comparison tests. The Mantel-Cox test was used to determine statistical significance in the Gallia mellonella survival assay. Statistical significance was set at p<0.05. Asterisks are used to indicate significant differences where appropriate: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Unless otherwise noted, all figures were generated using GraphPad Prism 8.0 (San Diego, CA, USA).

[0257] Example 3: Charged π-conjugated compounds for cancer treatment i. Cytotoxicity is dependent on exposure time and concentration: quantification by crystal violet assay Structures relevant to the experiments described below and elsewhere in this example are shown in Figures 22A-22C and 23. Most of the charged π-conjugated compounds are aminocyanines functionalized with alkylamine groups. The charged π-conjugated compounds were added to cell cultures of A375 cancer cells (human melanoma), and their cytotoxicity was quantified by crystal violet testing and / or clonogenic assays for cell viability. As shown in Figures 24A-24C, the charged π-conjugated compounds (Cy7.5-Amine, Cy5.5-Amine, and GL-261-2) were safe and exhibited low cytotoxicity when exposed to cancer cells for short periods of time (e.g., approximately 30-50 minutes). Figures 24A-C also show that the charged π-conjugated compounds exhibited cytotoxicity to A375 cells when exposed to the compounds for extended periods of time (e.g., 1 or 2 days). The longer the exposure time, the stronger the cytotoxicity of the charged π-conjugated compounds to A375 cells. In Figure 24D–F, other charged π-conjugated compounds (GL-286, BL-142, and GL-291-2) also exhibited cytotoxicity against A375 cells. Similar behavior was observed, with longer exposure times resulting in stronger cytotoxicity. All of the molecules presented here are aminocyanines, with the exception of GL-261-2, a carboxyl cyanine. Surprisingly, GL-261-2 has a protective effect on A375 cells when used for short exposure times (approximately 50 min), even at relatively high concentrations (1–4 μM). Furthermore, we observed that some aminocyanines, such as GL-286, can increase cell viability at short incubation times (1 day instead of 2 days) and relatively low concentrations below 1 μM. This suggests for the first time that a tunable therapeutic window may exist to selectively kill and / or protect cancer cells. To find this therapeutic window, concentration and exposure time are key variables and require fine tuning.

[0258] Based on the above findings, a wider range of charged π-conjugated compounds were evaluated for cytotoxicity against A375 cancer cells, as shown in Figures 25 and 26. The results of these studies are summarized in Figure 27 and Table 3. [Table 3]

[0259] The results shown in Table 3 demonstrate that the charged π-conjugated compounds are highly potent cytotoxic to A375 cells, with IC 50 The IC is approximately 0.125 to 0.5 μM. 50 is the concentration required to inhibit cell viability by 50%. These molecules, shown in Figures 25 and 26, were evaluated by crystal violet assay under 2-day exposure conditions. In summary, Cy7.5-Amine and GL-308-2 showed IC values ​​of 0.01 and 0.02 in A375 cells after 2-day exposure. 50 The molecules BL-141-1 and GL-176 showed potent cytotoxicity at IC = 0.5 μM. 50 = 0.125 μM (BL-141-1) and IC 50 = 0.25 μM (GL-176), making it the most cytotoxic charged π-conjugated compound.

[0260] j. Quantification of cytotoxicity by clonogenic assay Further cytotoxicity testing was performed on several additional molecules using a clonogenic assay for cell viability (Figure 28). A375 cells were exposed to the charged π-conjugated compounds for 7 hours, unlike the previous assay, which used a crystal violet assay and exposed for 2 days. Data points showing quantification of the cytotoxicity of each individual molecule are shown in Figure 29, and the resulting IC 50 The values ​​are summarized in Table 4. [Table 4]

[0261] According to the results of the clonogenic assay, as shown in Table 4, BL-141-1 (IC 50 = 0.14 μM) was the most toxic charged π-conjugated compound in this group, followed by GL-362-2 (IC 50=0.19 μM). Figures 30 and 31 show images of the clonogenic assay. It is observed that at lethal concentrations, the killed cancer cells are unable to form colonies, indicating the high efficacy of these charged π-conjugated compounds in eradicating cancer cells.

[0262] k. Dose range for selectively killing cancer cells Charged π-conjugated compounds such as BL-204, BL-141-2, and BL-304 were shown to exhibit selectivity for specifically killing cancer cells (A375) at a concentration of 1 μM, while remaining safe for normal skin melanocyte cells (HEMa) (Figure 32). Thus, charged π-conjugated compounds can have a protective effect on normal skin melanocytes at very low concentrations (below 0.5 μM), exhibit selective toxic effects against cancer cells at relatively weak concentrations (approximately 0.5–1 μM), and exhibit toxic effects against both healthy and cancer cells at relatively high concentrations (above 2 μM). This characterization is accurate for long exposure times (approximately 24 hours) and at 200,000 cells / mL. The limit of toxic concentrations (referred to herein as the therapeutic window) may vary depending on the exposure time and the total number of cells present during testing. For example, a 1 μM concentration of charged π-conjugated compounds is safe when used in a cell culture of 200,000 cells / mL for 30 minutes of exposure. However, at 200,000 cells / mL, concentrations above approximately 2 μM of cyanine produce toxicity (approximately 8 μM) even after short exposure times (approximately 30 minutes). In contrast, for solid tumors, this lethal concentration is expected to be higher because small solid tumors contain hundreds of millions of cells. The dose can be calibrated and normalized by the total number of cells. The exact therapeutic range for killing cancer cells must be expressed in μg of charged π-conjugated compound per million cells or μg of charged π-conjugated compound per gram of cells. In summary, the same molecule has been demonstrated to have both agonist and antagonist effects depending on different concentrations and cell types. Interestingly, the charged π-conjugated compounds BL-204 and BL-141-2 enhanced cell viability in normal skin melanocytes. Therefore, the charged π-conjugated compounds disclosed in this invention may be useful in clinical applications to patients, where they can protect normal tissues and selectively exert toxicity on cancer cells.

[0263] l. Charged π-conjugate compound therapy in mouse tumors Next, Cy7.5-Amine (Figure 22C) was applied to A375 tumors by intratumoral injection, as shown in Figure 33. The results are summarized in Figure 34. Cy7.5-Amine caused a 20% reduction in A375 tumors, and 2 out of 10 mice were observed to be cancer-free even 7 months after the start of the study (Figure 34C). Furthermore, Cy7.5-Amine showed no signs of toxicity to the mice, as evidenced by the lack of effect on body weight compared to the control in Figure 34D. This is a promising treatment for safe and selective tumor remission in cancer patients. * * * * * * * * * * * * *

[0264] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made in the compositions and / or methods described herein, and in the steps or order of steps of the methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure, as defined by the appended claims.

[0265] VI. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Adam et al.,BMC Evol.Biol.8,52(2008). Ayala Orozco et al.,ACS Appl.Mater.Interfaces.12,410-417(2020). Ayala-Orozco et al., bioRxiv (2023). Avire et al.,Pathogens 2021,10(2),248。 Batt et al.,Biochem.J.477,1983-2006(2020)。 Bumah et al.,Photomed.Laser Surg.2013,31(11),547-553。 Clementi et al.,J.Vis.Exp.,e51008(2014)。 Dan et al.,Proc.Natl.Acad.Sci.USA118(2021)。 Gonzales et al.,Lancet 2001,357(9263),1179。 Hajdu et al.,Antimicrob.Agents Chemother.54,4078-4084(2010)。 Huemer et al.,EMBO Rep.21,e51034(2020).Ikuta et al.,Lancet 2022。 Kincses et al.,Antibiot.(Basel,Switzerland).10(2021)。 Kaplan et al.,J.Chemother.25,18-25(2013)。 Lange et al.,Pharmaceutics.13(2021)。 Li, et al., Front. Microbiol. 12, 705326 (2021). Maitra et al.,FEMS Microbiol.Rev.43,548-575(2019)。 Martinez&Baquero,Antimicrob.Agents Chemother.44,1771-1777(2000)。 Moellering et al.,Ann.Intern.Med.2003,138(2),135-142。 Morrissey&George,J.Antimicrob.Chemother.43(1999),pp.423-425。 Murray et al.,Lancet 2022,399(10325),629-655。 Niculescu&Grumezescu,Appl.Sci.11(2021)。 Odds,J.Antimicrob.Chemother.52,1(2003)。 Peterson et al.,J.Clin.Microbiol.2010,48(3),683-689。 Prakash et al.,Dye.Pigment.211,111053(2023)。 Weigel et al.,Science 2003,302(5650),1569-1571。 Smith et al.,N.Engl.J.Med.1999,340(7),493-501。 Santos,et al.,Adv.Sci.,2203242(2022)。 Santos et al.,Adv.Sci.10,2205781(2023)。 Schaaff et al.,Antimicrob.Agents Chemother.46,3540-3548(2002)。 Serrano et al.,Antibiotics.12,505(2023)。 Silhavy,et al.,Cold Spring Harb.Perspect.Biol.2,a000414(2010)。 Silverman et al.,Antimicrob.Agents Chemother.47,2538-2544(2003)。 Stewart&Costerton,Lancet.358,135-138(2001)。 Thomas et al.,Int.Wound J.9,428-435(2012)。 Wenzel et al.,MBio.9(2018)。 Xie&Yang,Sci.Rep.6,20628(2016)。 Yoon et al.,Biomed Res.Int.2013,274096(2013)。

Claims

1. A method of killing bacteria by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

2. The method of claim 1 , wherein the bacterium is a gram-positive bacterium.

3. The method of claim 1 , wherein the bacterium is a gram-negative bacterium.

4. A method of killing cancer cells by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

5. A method of killing fungal cells by treatment with an extended conjugated compound having at least two conjugated double or triple bonds with a charge in the conjugated chain.

6. The method of any one of claims 1 to 5, wherein the extended conjugated compound comprises a positive charge.

7. The conjugated compound has the formula: 【Chemistry 1】 is further defined as During the ceremony, x is a positive or negative charge; n is an integer from 2 to 100, and each independent variable in n is independently selected; X １ and X ２ are each independently a heteroatom selected from O, N, S, B, P, Ge, As, or Se, and R １ , R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ are each independently hydrogen, alkyl （Ｃ≦１８） , alkenyl （Ｃ≦１８） , alkynyl （Ｃ≦１８） , aryl （Ｃ≦１８） , aralkyl （Ｃ≦１８） , heteroaryl （Ｃ≦１８） , heterocycloalkyl （Ｃ≦１８） or a substitution of any of these groups, or R １ and R ２ , R １ and R ５ , R ２ and R ５ , R ３ and R ４ , R ３ and R ７ , and R ４ and R ７ together form 1, 2, 3, 4, 5, or 6 aliphatic or aromatic rings containing at least 3 carbon atoms and no more than 36 carbon atoms, and optionally containing 1, 2, 3, 4, or 5 nitrogen, sulfur, or oxygen atoms; The compound according to any one of claims 1 to 6.

8. The compound is 【Chemistry 2】 is further defined as During the ceremony, x is a positive charge, n is an integer from 0 to 100, and each independent variable in n is independently selected; Each R １ , R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ are each independently hydrogen, alkyl （Ｃ≦１８） , alkenyl （Ｃ≦１８） , alkynyl （Ｃ≦１８） , aryl （Ｃ≦１８） , aralkyl （Ｃ≦１８） , heteroaryl （Ｃ≦１８） , heterocycloalkyl （Ｃ≦１８） or a substitution of any of these groups, or Each R １ , R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ are each independently a cell membrane targeting moiety, said cell targeting moiety optionally comprising a linker, or Each R １ and R ２ , R １ and R ５ , R ２ and R ５ , R ３ and R ４ , R ３ and R ７ , R ４ and R ７ , and R ５ and R ７ are taken together to form, independently, 1, 2, 3, 4, 5, or 6 aliphatic or aromatic rings containing at least 3 carbon atoms and no more than 36 carbon atoms, and optionally containing 1, 2, 3, 4, or 5 nitrogen, sulfur, or oxygen atoms.

9. X １ is N or X ２ The method of claim 7 , wherein is N.

10. R １ is R ５ The method of any one of claims 7 to 9, wherein, together with

11. R ３ is R ７ The method of any one of claims 7 to 10, wherein, together with

12. R ２ or R ４ is alkyl （Ｃ≦１８） or substituted alkyl （Ｃ≦１８） The method according to any one of claims 7 to 11, wherein

13. R ２ The compound according to any one of claims 7 to 12, wherein is methyl.

14. R ４ The compound according to any one of claims 7 to 13, wherein is methyl.

15. R ５ and R ７ The method of any one of claims 7 to 9 and 12 to 14, wherein together form a single ring, and said single ring is a 5-membered ring, a 6-membered ring, or a 7-membered ring.

16. 16. The method of any one of claims 7 to 15, wherein n is an integer selected from 2, 3, 4, or 5.

17. R ４ The method of any one of claims 7 to 13, 15, and 16, wherein is a cell targeting moiety with a linker.

18. The compound is 【Transformation 3】 【change】 【change】 【change】 【change】 The method of any one of claims 1 to 17, further defined as:

19. 19. The method of any one of claims 1 to 18, which is sufficient to treat or prevent a disease or disorder in a patient caused by either a bacteria, a cancer cell, or a fungal cell.

20. Formula (IV) 【Chemistry 4】 1. A method for forming a compound of the formula: During the ceremony, m is 0, 1, 2, or 3; X ３ is Br, Cl, I, or F, and R ８ , R ９ , R １０ , and R １１ are each independently hydrogen, alkyl （Ｃ≦１８） , alkenyl （Ｃ≦１８） , alkynyl （Ｃ≦１８） , aryl （Ｃ≦１８） , aralkyl （Ｃ≦１８） , heteroaryl （Ｃ≦１８） , heterocycloalkyl （Ｃ≦１８） or a substitution of any of these groups, (a) Formula (III) 【Transformation 5】 obtaining a compound of During the ceremony, m is 0, 1, 2, or 3; X ３ is Br, Cl, I, or F, and R ８ , R ９ , and R １０ are each independently hydrogen, alkyl （Ｃ≦１８） , alkenyl （Ｃ≦１８） , alkynyl （Ｃ≦１８） , aryl （Ｃ≦１８） , aralkyl （Ｃ≦１８） , heteroaryl （Ｃ≦１８） , heterocycloalkyl （Ｃ≦１８） or a substitution of any of these groups; (b) reacting said compound of formula (III) with a catalyst and a base under conditions sufficient to form said compound of formula (IV); A method comprising:

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