Bis-aminoquinoline and nanoformulations for treatment of cancer

Abstract

The present invention provides diaminoquinoline compounds of formula (I). The invention also provides nanocarriers comprising the compounds of the invention, as well as methods of treating disease and imaging using the nanocarriers.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 18, 2020, application number 202080080553.6, and invention name “Bisaminoquinoline and nanoformulations for cancer treatment”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 902,156, filed on September 18, 2019, which is incorporated herein in its entirety for all purposes.

[0004] STATEMENT AS TO RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] This invention was made with government support under Grant Nos. R01CA199668 and 5R01CA232845 awarded by the National Institutes of Health and the National Cancer Institute, Grant No. R01DE029237 awarded by the National Institute of Dental and Craniofacial Research, and Grant No. R01HD086195 awarded by the National Institutes of Health and the National Institute of Child Health and Human Development. The U.S. Government has certain rights in this invention. Background Art

[0006] Over the past few decades, the continuous exploration of nanomedicine has made great contributions to cancer treatment. Carrier-assisted nanomedicines and carrier-free nanomedicines are being developed to improve drug intrinsic kinetics and safety. However, there are some limitations to these nanotherapeutic approaches. First, the complexity and toxicity caused by their multi-component nature have seriously hindered the clinical transformation of many nanoformulations. Second, most of the drugs used for conventional delivery studies were approved decades ago, and some are no longer first-line therapeutic drugs. Third, nanoformulations designed for recently developed therapeutic agents (especially new chemical entities) may encounter patent issues. Finally, not all drugs can be structurally modified. These limitations can be addressed in the context of medicinal chemistry. Compared with nanomedicine, which focuses on the development of drug delivery properties, medicinal chemistry is committed to discovering drug entities at an early stage. Although drug discovery technology has produced many drug leads and candidates, issues surrounding pharmacokinetics, metabolism and toxicology remain challenging. These challenges can also be relatively easily addressed by nanotechnology in the field of nanomedicine. In order to take advantage of this interdisciplinary connection, the principles of nanotechnology are integrated into initial drug design, and a single-component non-prodrug nanomedicine (ONN) strategy ( Figure 1 ). In this strategy, drug design follows both conventional drug design strategies and the principle of molecular self-assembly, so that the designed drugs have advantages from the perspectives of drug discovery and drug delivery.

[0007] Lysosomes are selected as targets for cancer therapy. Cancer cell lysosomes are enlarged, easily ruptured, and more fragile than normal lysosomes. Lysosomal membrane permeabilization (LMP) can directly trigger cell death by enabling proteolytic enzymes (i.e., cathepsins) to be released into the cytoplasm; therefore, lysosomal detergents that can induce LMP have been developed for tumor therapy. In addition, lysosomal inhibition has considerable potential as an anticancer strategy because it interferes with autophagy, which is an important pathway for the stress response and drug resistance of established tumors. The lysosomal alkalinizing agents chloroquine (CQ) and hydroxychloroquine (HCQ) are commonly used autophagy inhibitors that have been tested in multiple clinical trials for various cancer types. However, their efficacy is considered insufficient, especially when they are used as a single agent.

[0008] A series of lipophilic cationic BAQ derivatives ( Figure 1 and Figure 7). BAQ12 and BAQ13 were chosen to construct ONNs because of their potential as therapeutic agents and self-assembling building blocks. These BAQ ONNs showed excellent anticancer activity in vitro, with enhanced effects on lysosomal disruption, lysosomal dysfunction, and autophagy inhibition. In addition, as nanomedicines, BAQ ONNs exhibited expected self-delivery properties. From both drug discovery and drug delivery perspectives, these advantages ultimately contribute to the significant anticancer activity of these compounds as single agents in in vivo gastrointestinal cancer models. In addition, BAQ ONNs show promise for use in combination therapy with nabuccal, as they play a dual role as a therapeutic agent and a delivery vehicle. With its multidisciplinary integration and ingenious functional superposition, BAQ ONNs will become a good alternative for improving cancer treatment.

[0009] The integration of unique advantages in the field of drug discovery and drug delivery is very valuable for the progress of drug development. This paper describes a self-delivered single-component new chemical entity nanomedicine (ONN) strategy to improve cancer treatment by incorporating self-assembly principles into drug design. Lysosomal detergents (MSDH) and autophagy inhibitors (Lys05) are hybridized to develop bisaminoquinoline derivatives that can inherently form nanoassemblies. The selected BAQ12 and BAQ13 ONNs are very effective in inducing lysosomal destruction, lysosomal dysfunction and autophagy blocking, and show 30 times higher antiproliferative activity than hydroxychloroquine used in clinical trials. These single drug nanoparticles show excellent pharmacokinetic and toxicological properties and significant anti-tumor efficacy in vivo. In addition, they can encapsulate additional drugs and deliver them to the tumor site, and are therefore promising agents for combined therapies based on autophagy inhibition. Considering their interdisciplinary advantages, these BAQ ONNs have great potential for improving cancer treatment. What is needed is a new BAQ ONN. Surprisingly, the present invention meets this demand and other needs. Summary of the invention

[0010] In one embodiment, the present invention provides a compound of formula (I):

[0011]

[0012] or a pharmaceutically acceptable salt thereof, wherein: R 1 For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R1c Substitution; W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl substitution; each L is independently absent, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkyne; each Y is independently absent, –O–, –NH–, –NHC(O)–, –NHC(O)NH–, –NHSO2–, –OC(O)–, –OC(O)NH–, –C(O)–, or –SO2–; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, a sterol, C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl having 1 to 4 heteroatoms each independently being N, O or S, C 6-12 Aryl, 5- to 12-membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, -OH or -NH2; R 1a C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-40 Alkoxy, hydroxyl or –NR 1b R 1c Replacement; R 1b For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl; R 1c For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl or -LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, halogen, -CN or -NO2; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40alkoxy, halogen, -CN or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; and each X is independently absent or -O - , where when X does not exist, R 2a and R 2b is hydrogen, and R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-40 Alkyl, C 2-40 Alkenyl, C 4-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , and when X does not exist, R 1 is CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

[0013] In another embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds of the present invention or pharmaceutically acceptable salts thereof, wherein each compound self-assembles in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed in the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0014] In another embodiment, the invention provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the invention.

[0015] In another embodiment, the present invention provides a method of imaging, comprising administering to a subject to be imaged an effective amount of a nanocarrier of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Schematic diagrams of the proposed drug design strategy and the current work are shown. Path (a) shows that an interdisciplinary drug design strategy is proposed to integrate the conventional fields of medicinal chemistry and nanomedicine. The drugs are named single-component non-prodrug nanomedicines (ONNs), which are designed according to the strategies of conventional drug design and molecular self-assembly, so that they can gain advantages from the perspectives of drug discovery and drug delivery at the same time. Path (b) shows a proof-of-concept experiment in this work: self-delivered lysosomal bisaminoquinoline (BAQ) derivatives are found for cancer treatment. BAQ derivatives produced by hybridization of lysosomal detergents and BAQ-based autophagy inhibitors can self-assemble into BAQ ONNs, which show enhanced in vitro function, excellent delivery characteristics and significant in vivo therapeutic effects as single agents. In addition, they also have high drug loading efficiency to deliver additional drugs to tumor sites, thereby generating promising applications for combined therapies.

[0017] Figures 2A to 2G Characterization of BAQ ONNs is shown. Figure 2A Shown are the size changes of BAQ NPs (10 μM) in acetate buffers with different pH values; data are mean ± SD; n = 3 independent nanoparticle samples. Figure 2B Shown is the pH-dependent hemolysis induced by BAQ NPs (50 μM, 4 h) in PBS buffer; data are mean ± SD; n = 3 independent nanoparticle samples. Figure 2C Shown is the pH change of BAQ NPs (1 mM) in hydrochloric acid (HCl, 0.1 M) titration. FIG. 2D to FIG. 2E Representative TEM micrographs at pH 7.4 are shown ( Figure 2D ) and representative TEM micrographs at pH 5.0 ( Figure 2E ); Insets show the size distribution (left) and the Tyndall effect (right); The experiments were repeated three times independently. Figure 2F In vitro drug release patterns at pH 7.4 and pH 5.0 are shown; data are mean ± SD; n = 3 independent nanoparticle samples. Figure 2G The count rates of BAQ NPs at various concentrations in water are shown; the intersection of the two lines refers to the CAC of BAQ12NP (0.45 μg mL -1 , 0.76 μM) and CAC of BAQ13 NPs (0.15 μg mL -1 , 0.25 μM); data are mean ± SD; n = 3 independent nanoparticle samples.

[0018] FIG. 3A to FIG. 3K Shown is BAQ ONN-induced lysosomal disruption and inhibited autophagy in MIA PaCa-2 cells. Figure 3ARepresentative images of cellular uptake of nanoparticles are shown; cells loaded with dextran-AF488 were incubated with DiD-labeled BAQ ONNs for 2 h; the experiment was repeated three times independently. Figure 3B Shown are cells treated as indicated (10 μM, 2 h) and stained with LysoTracker Green; experiments were repeated three times independently. Figure 3C Shown are AO staining of cells in the indicated treatments (5 μM, 12 h); experiments were repeated three times independently. Figure 3D Shown are representative images of cells loaded with dextran-AF488 treated as indicated (5 μM, 12 h); experiments were repeated three times independently. Figure 3E Shown is the release of cathepsin B from isolated lysosomes after the indicated treatments (25 μM, 12 h); data are presented as mean±SD; n=3. Figure 3F Western blots are shown. Figure 3G Shows Figure 3F Normalized quantitative analysis of gel blots; data are presented as mean ± SD; n = 3. Figure 3H Representative LC3B-GFP images of the indicated 4 h treatments are shown; experiments were repeated three times independently. Fig. 3I Quantification of LC3B-GFP puncta per cell is shown in h; data are expressed as mean ± SD; n = 3. Figure 3J Representative TEM images of cells treated as indicated (2 μM, 48 h) are shown; orange rectangle: region of interest; purple arrows: autophagic vesicles; red arrows: lysosomes. Figure 3K The average diameter of lysosomes is shown; data are expressed as mean ± SD; n = 7. All statistical p values ​​were calculated by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. ns., not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0019] FIG. 4A to FIG. 4J BAQ ONN was shown to alter the expression of lysosomal genes and cause cell death by apoptosis. Figure 4A Shown is a GSEA demonstrating the enrichment of lysosomal gene sets in MIA PaCa-2 cells treated with BAQ13 NPs (5 μM, 24 h). GSEA was performed with n=1,000 permutations, with p-adjustment < 0.05 and FDR < 0.05 considered significant. Figure 4B Representative upregulated lysosomal genes from a are shown. Figure 4C Comparison of gene upregulation between Lys05 and BAQ13 NPs is shown. FIG. 4D to FIG. 4E The V-ATPase genes involved in the indicated treatments (5 μM, 24 h) are shown. Figure 4D) and Cl- channel genes ( Figure 4E ) were analyzed by qPCR; data are mean ± SD; n = 3. Figure 4F Viability curves and corresponding IC50 values ​​of cells exposed to 48 h treatment are shown; data are mean ± SD; n = 3. Figure 4G Shown are the growth curves of MIA PaCa-2 (1.5 μM) and HT29 (1.0 μM) cells in sequential treatments; data are mean ± SD; n = 3 independent experiments. Figure 4H Shown are clonogenic assays of MIAPaCa-2 cells and HT29 cells; n=3. Fig. 4I Shown are caspase 3 / 7 activities in MIA PaCa-2 cells and HT29 cells treated for 6 h and 12 h, respectively; data are mean ± SD; n = 4. Figure 4J The percentage of apoptotic population of MIA PaCa-2 cells (left) and HT29 cells (right) treated for 24 h is shown. All statistical p values ​​were calculated by one-way ANOVA and Tukey's multiple comparison test; ns., not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0020] FIG. 5A to FIG. 5H The pharmacokinetics, biodistribution, and in vivo antitumor effects of BAQ ONN are shown. Figure 5A Shown are plasma concentration-time curves of DiD-loaded BAQ ONN and free DiD after intravenous injection; data are mean ± SD; n = 3. Figure 5B Shown are the in vivo and ex vivo biodistribution of BAQ13NPs in HT29 tumor-bearing mice 24 h after injection. Figure 5C Quantitative fluorescence intensity of tissues obtained 12 h and 24 h after injection is shown; data are mean ± SD; n = 3. Figure 5D Shown are MIA PaCa-2 tumor growth curves in mice treated every three days as indicated; data are mean ± SD; n = 6 tumors per group. Figure 5E Body weights of mice during treatment are shown; data are mean ± SD; n = 6 mice per group. Fig. 5F Shown are the weights of tumors harvested at the end of treatment; data are mean ± SD; n = 6 tumors per group. Figure 5G to Figure 5H Representative H&E results of tumors harvested at the end of treatment are shown ( Figure 5G )、IHC results( Figure 5H ) Figure 5G to Figure 5H All experiments were repeated three times independently. All statistical p values ​​were calculated by one-way ANOVA and Tukey's multiple comparison test; *p < 0.05; ****p < 0.0001.

[0021] FIG. 6A to FIG. 6K BAQ ONN was shown to have a dual effect in combination therapy. Fig. 6A Shown is the establishment of a patient-derived pancreatic cancer stem cell (PCSC) model. Figure 6B Shown is histological analysis showing high levels of stroma in PCSC tumors; experiments were repeated three times independently. Figure 6C Viability curves and IC50 values ​​of PCSCs treated for 48 h are shown; n=3 independent experiments. Fig.6D AO staining is shown to reveal LMPs of PCSCs treated for 12 h; the experiment was repeated three times independently. Fig. 6E Shown are immunoblot analyses of autophagic proteins in PCSCs treated as indicated (2.5 μM, 48 h); experiments were repeated three times independently. Fig. 6F The synergistic effect of BAQ13 NP and Nabucacin (48h) was shown. Figure 6G Shown are tumor growth curves in a subcutaneous MIA PaCa-2 xenograft model administered intravenously every three days; data are mean ± SD; n = 10 tumors per group. Figure 6H Images of tumors harvested at the end of treatment are shown. Fig.6I Shown are changes in mouse body weight during treatment; data are mean ± SD; n = 5 mice per group. Figure 6J Representative images of PCSC tumor sections are shown; the experiment was repeated three times independently. Intravenous injection (10 mg kg -1 ) 48 h after in vivo and ex vivo fluorescence imaging of BAQ13 NPs co-loaded with nabucacin and DiD in the PCSC model. Figure 6K Shows Figure 6J Quantitative fluorescence intensity of tissues in the middle, data are mean ± SD; n = 3 mice per group. All statistical p values ​​were calculated by two-tailed Student's t-test. ns., not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0022] Figure 7 The chemical structures of the compounds involved in this work and the synthetic routes of BAQ12-BAQ18 are shown.

[0023] FIG. 8A to FIG. 8C In vitro evaluation of BAQ NPs is shown. Fig. 8A The size distribution is shown. Figure 8B Observation of pH-dependent hemolysis is shown; erythrocytes were treated as indicated (50 μM, 4 h). Figure 8C Viability curves of various cell lines exposed to different compounds for 24 h are shown. Data are presented as mean ± SD; n = 3 independent experiments.

[0024] FIG. 9A to FIG. 9G Stability measurements and TEM characterization of BAQ NPs are shown. FIG. 9A to FIG. 9B Shown are dynamic light scattering (DLS) measurements of BAQ12 NPs and BAQ13 NPs under neutral conditions; data are presented as mean ± SD; n = 3. Fig. 9C The overall appearance of BAQ12 NPs and BAQ13 NPs at day 10 and day 30 are shown. FIG. 9D to FIG. 9E Shown are DLS measurements of BAQ12 NPs and BAQ13 NPs in the presence of 10% FBS; data are presented as mean ± SD; n = 3. Fig.9F Shown are the size distributions of BAQ12 NPs (left) and BAQ13 NPs (right) 24 h after incubation with 0.5 mM BSA. Figure 9G Representative TEM images of BAQ13 NPs loaded with different agents are shown; the experiments were repeated three times independently.

[0025] FIG. 10A to FIG. 10F Shown are the effects of BAQ NPs on autophagy at the lysosomal and cellular levels. Fig. 10A Shown for Figure 3A Pearson correlation coefficient of colocalization analysis. Fig. 10B Shown are quantifications of LysoTracker Green fluorescence in MIA PaCa-2 cells treated as indicated; data are presented as mean ± SD; n = 3 independent experiments; statistical significance was calculated by two-tailed Student's t-test; **p < 0.01. Fig. 10C Shown are HT29 cells treated as indicated (10 μM, 2 h) and then stained with LysoTracker Red; the experiments were repeated three times independently. Fig. 10D Shown are AO staining of HT29 cells treated as indicated (5 μM, 12 h); experiments were repeated three times independently. Fig.10E Shown are the ratios of fluorescence intensities at 525 nm and 650 nm in MIA PaCa-2 cells treated for 12 h as indicated; data are expressed as mean ± SD; n = 3 independent experiments; statistical significance was calculated by one-way ANOVA and Tukey's multiple comparison test; ns., not significant; ****p < 0.0001. Fig.10F Representative LC3B-GFP images in cells under corresponding treatment (5 μM, 4 h) are shown; the experiments were repeated three times independently.

[0026] FIG. 11A to FIG. 11F Shown are the effects of BAQ NPs on gene expression and lipid metabolism. Fig.11A Shown are volcano plots of RNA-seq showing differentially expressed genes in MIA PaCa-2 cells induced by Lys05 (lower panel) and BAQ13 NPs (upper panel). Fig. 11B Shown are changes in autophagy-related genes according to RNA-seq results. Fig. 11C qPCR analysis of representative autophagy-related genes and apoptosis-related genes of MIA PaCa-2 cells treated as indicated (5 μM, 24 h) is shown; data are expressed as mean ± SD; n = 3; statistical significance was calculated by one-way ANOVA and Tukey's multiple comparison test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fig.11D Shown are changes in apoptosis-related genes in MIA PaCa-2 cells treated as indicated (5 μM, 12 h); data are expressed as mean ± SD; n = 3; statistical significance was calculated by two-tailed Student's t-test; *p < 0.05; **p < 0.01. FIG. 11E to FIG. 11F The expression of acid sphingomyelinase (ASM) substrate ( Fig.11E ) and phospholipase A (PLA) substrates ( Fig.11F ) concentration changes; data are expressed as mean ± SD; n = 4.

[0027] Fig.12 Viability curves of various cell lines treated for 48 h as indicated are shown. Data are presented as mean ± SD; n = 3 independent experiments.

[0028] FIG. 13A to FIG. 13F Biodistribution and toxicity studies of BAQ NPs are shown. Fig.13A Shown are in vivo and ex vivo imaging of HT29 tumor-bearing mice 12 h after injection (iv) with free DiD (upper panels) and DiD-loaded BAQ13 NPs (lower panels), n=3 mice per group. Fig. 13B Quantitative fluorescence intensity of the organs in a is shown; data are expressed as mean±SD; n=3 mice per group; data are expressed as mean±SD; n=3; statistical significance was calculated by two-tailed Student's t-test; ***p<0.001; ****p<0.0001. Fig. 13C Concentration-dependent hemolysis induced by corresponding treatments at physiological pH is shown; data are expressed as mean ± SD; n = 3 independent nanoparticle samples; statistical significance was calculated by one-way ANOVA and Tukey's multiple comparison test; ns., not significant; ***p < 0.001; ****p < 0.0001. Fig.13D Shown are the survival of FVB / n mice injected intravenously with the corresponding agents every two days; n = 6 mice per group. Fig.13E Shown are body weights of mice treated every two days as indicated; data are expressed as mean ± SD; n = 4 mice per group. Fig.13FDynamic light scattering (DLS) measurements and representative TEM images of Liposome@Lys05 are shown; the experiments were repeated three times independently.

[0029] FIG. 14A to FIG. 14E Analysis of tissue sections and hematology are shown. Fig.14A The results from the treatment with vehicle (saline), LysO5 (20 mg kg -1 , ip), BAQ12 NP (20 mg kg -1 , iv) and BAQ13 NP (20 mg kg -1 iv) H&E analysis of major organs of mice treated for 24 days. Scale bar: 100 μm. The experiment was repeated three times independently. Fig. 14B The corresponding serum chemistry analysis of the mice in a is shown. 1. Alanine aminotransferase UL -1 , 2. Aspartate aminotransferase UL -1 , 3. Blood urea nitrogen mg dL -1 , 4. Creatinine mg dL -1 , 5. Total bilirubin mg dL -1 , 6. Hemolysis. FIG. 14B to FIG. 14E The corresponding complete blood count (CBC) analysis of the mice in a is shown; data are expressed as mean ± SD; n = 3 mice per group. 1. Absolute neutrophils (k μL -1 ), 2. Absolute monocytes (kμL -1 ), 3. Absolute eosinophils (kμL -1 ), 4. Absolute basophils (kμL -1 ), 5. Eosinophil%, 6. Basophil%, 7. MPV (fL), 8. Absolute lymphocytes (kμL -1 ), 9.WBC (kμL -1 ), 10.RBC (MμL -1 ), 11. Hemoglobin (g dL -1 ), 12. MCH (pg), 13. Monocytes (%), 14. RDW (%), 15. Neutrophils%, 16. Lymphocytes%, 17. Hematocrit%, 18. MCV (fL), 19. MCHC (g dL -1 ), 20. Platelets (kμL -1 ), 21. The presence of blood clots.

[0030] FIG. 15A to FIG. 15C Shown is the in vivo evaluation of BAQ NPs in the HT29 mouse model. Fig.15ATumor growth curves in a subcutaneous HT29 xenograft model administered intravenously every two days are shown; data are expressed as mean ± SD; n = 12 tumors per group. Statistical significance was calculated by two-tailed Student's t-test; ns., not significant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Fig. 15B Body weights of mice during treatment are shown; data are expressed as mean ± SD; n = 6 mice per group. Fig. 15C Survival curves of mice are shown; n=6 mice per group. Treatment was terminated on day 24.

[0031] Fig.16 Shown is the gating strategy for isolating pancreatic cancer stem cells (PCSCs).

[0032] FIG. 17A to FIG. 17F Shown is the evaluation of BAQ NPs in a model of pancreatic cancer stem cells (PCSCs). Fig.17A Lysosomal deacidification assay is shown. PCSCs were treated for 2 h (10 μM) and then stained with LysoTracker Red; the experiment was repeated three times independently. Fig. 17B Shown are representative images of PCSCs expressing LC3B-GFP treated as indicated (5 μM, 4 h); experiments were repeated three times independently. Fig. 17C Shown are the percentages of apoptotic populations of PCSCs in the indicated treatments (5 μM, 24 h). Fig.17D Shown are immunoblot assays of PCSCs treated for 8 h as indicated; experiments were repeated three times independently. Fig.17E Shown are H&E analyses of major organs harvested at the end of treatment; experiments were repeated three times independently. Fig.17F Shown is the biodistribution of free DiD and BAQ13 NP@nabukacin+DiD in mice 24 h after tail vein injection; red circle: tumor.

[0033] FIG. 18A to FIG. 18G Characterization of PBC NPs at physiological and lysosomal pH is shown. Fig.18A The chemical structure of the PBC monomer is shown. Fig.18B Schematic representation of convertibility at various pHs is shown. Fig.18C Ultrafiltration analysis and TEM observation of PBC NPs at pH 7.4 and pH 5.0 are shown. Fig.18D pH-dependent DLS measurements of PBC nanoparticles are shown. Fig.18E Time course DLS measurements of PBC nanoparticles at pH 5.0. Fig.18F pH-dependent TEM measurements of PBC nanoparticles are shown. Figure 18G Shown are the time-dependent TEM measurements of PBC nanoparticles.

[0034] FIG. 19A to FIG. 19H The convertibility of PBC nanoparticles was shown to induce lysosomal dysfunction and cause apoptosis in OSC-3 cells. Fig.19A The chemical structures of convertible PBC nanoparticles and non-convertible BAQ nanoparticles are shown. Fig.19B shows cell viability, Fig.19C shows the autophagy analysis, Fig.19D Apoptosis analysis is shown, and Fig.19E Shown are TEM images showing PBC nanofibers formed in lysosomes. Fig.19F Acridine orange (AO) staining is shown to visualize lysosomal membranes (LMPs) induced by PBC NPs. Figure 19G Dextran staining is shown to demonstrate PBC NP-induced LMP. (h) PBC NPs induced significant vacuolation in OSC-3 cells.

[0035] FIG. 20A to FIG. 20L PBC NPs were shown to display high photodynamic therapy efficacy and overcome autophagy-related drug resistance. FIG. 20A to FIG. 20B Figure 2 shows the induction of autophagy by the traditional photosensitizer pheophorbide (Pa) in OSC-3 cells, as imaged by LC3-GFP-RFP ( Fig. 20A ) and immunoblotting ( Fig. 20B ) confirmed. FIG. 20C to FIG. 20D showed that Pa-mediated photodynamic therapy was sensitized by the autophagy inhibitor Lys05, which was significantly increased in terms of cell viability ( Fig. 20C ) and immunoblotting ( Fig.20D ) was confirmed. Fig.20E The production of singlet oxygen is shown. Fig.20F Shown is the production of ROS in OSC-3 cells. Figure 20G Shown is DiO / PI live / dead staining of OSC-3 oral cells after different treatments. Fig. 20H Cell viability is shown. Fig.20I Apoptosis assay is shown. Fig. 20J Immunoblot assays of apoptotic pathways are shown. Figure 20K Shown are TEM images of OSC-3 cells after different treatments. Figure 20L Immunoblot assay of the autophagy process is shown.

[0036] Fig.21 Other synthetic BAQ derivatives and their cell viability results in OSC-3 cells (48 h) are shown.

[0037] FIG. 22A to FIG. 22B A preliminary screen of BAQO derivatives is shown. Fig.22A The chemical structures are shown. Fig. 22BViability results of pancreatic cancer stem cells (PCSCs) treated for 72 hours are shown.

[0038] FIG. 23A to FIG. 23F Characterization of BAQ12O NPs is shown. Fig.23A Shown is the DLS measurement of BAQ12O NPs. Fig. 23B Representative TEM images of BAQ12O NPs are shown. Fig.23C Shown is the measurement of the critical aggregation concentration (CAC) of BAQ12O NPs. FIG. 23D to FIG. 23E The absorbance of BAQ12O NPs and free BAQ12O solutions is shown ( Fig.23D ) and fluorescence ( Fig.23E )spectrum. Fig.23F Stability of BAQ12O NPs.

[0039] FIG. 24A to FIG. 24F Shown is the evaluation of the antitumor activity of BAQ12O NPs in PCSC tumor-bearing mice. Fig.24A Shown are tumor growth curves in mice treated every three days (iv). Fig. 24B Tumor weights were collected at the end of treatment. Fig.24C Body weight of mice during treatment. Fig.24D Immunoblotting of autophagy in mice. FIG. 24E to FIG. 24F Representative HE images of tumors in different groups are shown ( Fig.24E ) and Ki67-IHC images ( Fig.24F ).

[0040] Fig.25 The chemical structures of the designed BAQO derivatives are shown. DETAILED DESCRIPTION

[0041] I. Overview

[0042] The present invention provides bisaminoquinoline derivative compounds, and nanocarriers formed from these compounds, which can be used to treat diseases. These compounds and nanocarriers can target lysosomes, leading to lysosomal destruction, lysosomal dysfunction and / or autophagy inhibition. In addition, nanocarriers can be used for combined therapy by encapsulating additional drugs, or co-administered with additional drugs, which can be used to overcome drug resistance. Nanocarriers can also be used to image cells or organisms of interest.

[0043] I I.Definition

[0044] Unless otherwise expressly stated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. In addition, any method or material similar or equivalent to the method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

[0045] As used herein, "a," "an," or "the" include not only aspects having one member, but also aspects having more than one member. For example, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth.

[0046] "Alkyl" refers to a straight or branched chain saturated aliphatic group having the indicated number of carbon atoms. Alkyl groups may include any number of carbon atoms, such as C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 1-20 , C 1-30 , C 1-40 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C 3-5 , C 3-6 , C 4-5 , C 4-6 and C 5-6 For example, C 1-6 Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl may also refer to an alkyl group having up to 40 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl may be substituted or unsubstituted.

[0047] "Alkylene" refers to a straight or branched saturated aliphatic group, i.e., a divalent hydrocarbon group, having the indicated number of carbon atoms and connecting at least two other groups. The two parts connected to the alkylene group can be connected to the same atom or different atoms of the alkylene group. For example, a straight chain alkylene group can be -(CH2) n-a divalent radical wherein n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. -alkylene groups may be substituted or unsubstituted.

[0048] "Alkenyl" refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl groups may include any number of carbon atoms, such as C2, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C 2-20 , C 2-30 , C 2-40 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6 and C6. The alkenyl group may have any suitable number of double bonds, including but not limited to 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include but are not limited to ethenyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl or 1,3,5-hexatrienyl. The alkenyl group may be substituted or unsubstituted.

[0049] "Alkenylene" refers to an alkenyl group as defined above that is connected to at least two other groups, i.e., a divalent hydrocarbon group. The two parts connected to the alkenylene group can be connected to the same atom or different atoms of the alkenylene group. Alkenylene groups include, but are not limited to, vinylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene, and hexenylene. Alkenylene groups can be substituted or unsubstituted.

[0050] "Alkynyl" refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl groups may include any number of carbon atoms, such as C2, C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 2-7 , C 2-8 , C 2-9 , C 2-10 , C 2-20 , C 2-30, C 2-40 , C3, C 3-4 , C 3-5 , C 3-6 , C4, C 4-5 , C 4-6 , C5, C 5-6 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentenyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. The alkynyl group may be substituted or unsubstituted.

[0051] "Alkynylidene" refers to an alkynyl group as defined above, i.e., a divalent hydrocarbon group, connected to at least two other groups. The two moieties connected to the alkynylene group may be connected to the same atom or different atoms of the alkynylene group. Alkynylidene groups include, but are not limited to, ethynylene, propynylene, isopropynylene, butynylene, sec-butynylene, pentynylene, and hexynylene. -Alkynylidene groups may be substituted or unsubstituted.

[0052] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic ring assembly containing 3 to 12 ring atoms or the number of atoms indicated. Cycloalkyl may include any number of carbon atoms, such as C 3-6 , C 4-6 , C 5-6 , C 3-8 , C 4-8 , C 5-8 , C 6-8 , C 3-9 , C 3-10 , C 3-11 and C 3-12 . Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane, decalin, and adamantane. Cycloalkyl groups may also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3-isomers and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-isomers, 1,4-isomers, and 1,5-isomers), norbornene, and norbornadiene. When the cycloalkyl group is a saturated monocyclic C 3-8 When cycloalkyl is a saturated monocyclic C 3-6In the case of cycloalkyl, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl group may be substituted or unsubstituted.

[0053] "Heterocycloalkyl" refers to a saturated ring system having 3 to 12 ring members and 1 to 4 N, O and S heteroatoms. The heteroatoms may also be oxidized, such as, but not limited to -S(O)- and -S(O)2-. Heterocycloalkyl may include any number of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11 or 3 to 12 ring members. Any suitable number of heteroatoms may be included in the heterocycloalkyl, such as 1, 2, 3 or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4 or 3 to 4. Heterocycloalkyl can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepan, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-isomers, 1,3-isomers and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiazolidine (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane or dithiane. Heterocycloalkyl can also be fused with aromatic or non-aromatic ring systems to form members including but not limited to dihydroindole. Heterocycloalkyl can be unsubstituted or substituted. For example, heterocycloalkyl can be C 1-6 alkyl or oxo (=O) or the like.

[0054] "Aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl can include any suitable number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, and 6 to 10, 6 to 12 or 6 to 14 ring members. Aryl can be monocyclic, fused to form a bicyclic or tricyclic group, or connected by a bond to form a biaryl. Representative aryl includes phenyl, naphthyl and biphenyl. Other aryl includes benzyl with a methylene linking group. Some aryl has 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl has 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl has 6 ring members, such as phenyl. Aryl can be substituted or unsubstituted.

[0055] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, of which 1 to 5 ring atoms are heteroatoms such as N, O or S. The heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. The heteroaryl group may include any number of ring atoms, such as 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11 or 3 to 12 ring members. Any suitable number of heteroatoms may be included in the heteroaryl group, such as 1, 2, 3, 4 or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4 or 3 to 5. Heteroaryl can have 5 to 8 ring members and 1 to 4 heteroatoms, or 5 to 8 ring members and 1 to 3 heteroatoms, or 5 to 6 ring members and 1 to 4 heteroatoms, or 5 to 6 ring members and 1 to 3 heteroatoms.Heteroaryl can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-isomer, 1,2,4-isomer and 1,3,5-isomer), thiophene, furan, thiazole, isothiazole, oxazole and isoxazole.Heteroaryl can also be fused with aromatic ring system such as benzene ring to form including but not limited to benzopyrrole such as indole and isoindole, benzopyridine such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazine such as phthalazine and cinnoline, benzothiophene and benzofuran members. Other heteroaryl groups include heteroaryl rings connected by bonds, such as bipyridine. The heteroaryl group can be substituted or unsubstituted.

[0056] "Alkoxy" refers to an alkyl group with an oxygen atom connecting the alkyl group to the point of attachment: alkyl-O-. As with the alkyl group, the alkoxy group can have any suitable number of carbon atoms, such as C 1-6 Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. Alkoxy groups may be further substituted with various substituents described herein. Alkoxy groups may be substituted or unsubstituted.

[0057] "Hydroxyl" refers to an -OH functional group.

[0058] "Halogen" refers to fluorine, chlorine, bromine and iodine.

[0059] "Fluorophore" refers to a compound that emits light, typically in the range of 300nm-700nm, upon excitation of the compound. Upon absorbing transferred light energy (e.g., photons), the fluorophore enters an excited state. When the molecule exits the excited state, it emits light energy in the form of lower energy photons (e.g., emits fluorescence) and returns the dye molecule to its ground state. Fluorophores can be natural compounds or synthetic compounds. Fluorophores include, but are not limited to, DAPI, ethidium bromide, acridine orange, GFP, mCherry, hydroxycoumarins, fluorescein, LysoTracker (red & green), dextran-Alexa Fluor 488, Premo TM Autophagy sensors LC3B-GFP and Ac-DEVD-AMC.

[0060] "Photosensitizer" refers to a compound that can be activated by light to generate reactive free radicals, which are usually reactive oxygen species (ROS) used in photodynamic therapy, but can also generate reactive free radicals for polymerization, crosslinking or degradation. Photosensitizers can be used to treat diseases by generating singlet oxygen to destroy tumors. Photosensitizers include, but are not limited to, porphyrins, dyes, and chlorophylls.

[0061] "Porphyrin" refers to any compound having a porphine core as follows:

[0062]

[0063] The porphin core may be substituted or unsubstituted.

[0064] "Sterols" are compounds with the following core structure:

[0065]

[0066] The core can be further replaced.

[0067] "Drug" refers to an agent that can treat and / or improve a condition or disease. The drug can be a hydrophobic drug, i.e., any drug that repels water. Hydrophobic drugs that can be used in the present invention include, but are not limited to, hydrochloroquine (HCQ), Lys05, bortezomib, β-lapachone, JQ1, nabucasin, rapamycin, paclitaxel, SN38, etoposide, lenalidomide, and apoazole. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids, such as vinblastine and vincristine. The drugs of the present invention also include prodrug forms. Those skilled in the art will appreciate that other drugs can be used in the present invention.

[0068] "Imaging agent" or "contrast agent" refers to a compound that increases the contrast of structures within a cell or body part and is used in imaging methods including, but not limited to, MRI, PET, SPECT, and CT. Imaging agents can emit radiation, fluorescence, magnetic fields, or radio waves. Imaging agents include, but are not limited to, radiometal chelators, radiometal atoms or ions, and fluorophores.

[0069] "Chemotherapeutic agent" refers to a chemical drug that can be used to treat diseases such as, but not limited to, cancer, tumors, and neoplasms. In some embodiments, the chemotherapeutic agent can be in the form of a prodrug that can be activated into a cytotoxic form. Chemotherapeutic agents commonly known to those of ordinary skill in the art can be used in the present invention. Chemotherapeutic agents include, but are not limited to, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoazole, carboplatin, cisplatin, oxaliplatin, vinblastine, and vincristine.

[0070] "Molecular targeted agents" refer to drugs that can target specific molecules involved in the evolution, growth, and spread of tumors and cancers. Targeting specific molecules involved in the evolution of tumors and cancers can kill or inhibit the growth and spread of tumors and cancers. Molecular targeted agents include, but are not limited to, trastuzumab, erlotinib, imatinib, nilotinib, and vemurafenib.

[0071] "Immunotherapeutic agents" refer to a class of drugs that can alter immune responses by stimulating or suppressing the immune system. Immunomodulators include, but are not limited to, HCQ, Lys05, JQ1, rapamycin, nabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0072] "Radiotherapeutic agent" refers to a drug that can be used to treat a disease by radiation therapy. Radiation therapy is a disease treatment method that uses radiation to kill or inhibit tumors and cancer cells. Radiotherapeutic agents include, but are not limited to, beta-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, and tirapazamine.

[0073] "Nanocarrier" or "nanoparticle" refers to a micelle produced by the aggregation of a compound of the invention. The nanocarrier of the invention may have a hydrophobic core and a hydrophilic exterior.

[0074] "Inhibit" and "inhibitor" refer to a compound that inhibits a particular effect or function or a method of inhibiting a particular effect or function.

[0075] "Treatment" refers to any indication of success in treating or ameliorating an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter, such as reducing, alleviating, alleviating, or making symptoms, injury, pathology, or condition more tolerable to the patient; reducing the frequency or duration of symptoms or condition; or, in some cases, preventing the onset of symptoms. Treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, for example, the results of a physical examination.

[0076] "Disease" refers to abnormal cellular function in an organism that is not the direct result of physical or external injury. Disease can refer to any condition that causes suffering, dysfunction, disability, disorder, infection, pain, or even death. Diseases include, but are not limited to, genetic diseases such as hereditary and non-hereditary diseases, infectious diseases, non-infectious diseases such as cancer, defective diseases, and physiological diseases.

[0077] "Administer" means administering to a subject orally, in the form of a suppository, topically, parenterally, intravenously, intraperitoneally, intramuscularly, intralesionally, intranasally or subcutaneously, intrathecally, or by implanting a sustained-release device, such as a mini-osmotic pump. --

[0078] "Subject" refers to an animal such as a mammal, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the subject is a human.

[0079] "Therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refers to a dose that produces the therapeutic effect for which it is administered. The exact dose will depend on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th edition, 2003, Gennaro, ed., Lippincott Williams Wilkins Publishers). In sensitized cells, the therapeutically effective dose may generally be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0080] "Target" or "targeting" refers to the use of a compound, protein or antibody that specifically or preferentially binds to a cell, viral particle, viral protein, antigen or biomolecule, or localizes to a particular cell type, tissue type, microorganism type or virus type.

[0081] "Imaging" refers to the use of a device outside the body of a subject to determine the location of an imaging agent (such as a compound of the invention). Examples of imaging tools include, but are not limited to, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), x-ray computed tomography (CT). Positron emission tomography detects radiation from positrons emitted by the imaging agent.

[0082] II I. Compounds

[0083] In some embodiments, the present invention provides compounds of formula (I):

[0084]

[0085] or a pharmaceutically acceptable salt thereof, wherein: R 1 For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c Substitution; W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl substitution; each L is independently absent, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkyne; each Y is independently absent, –O–, –NH–, –NHC(O)–, –NHC(O)NH–, –NHSO2–, –OC(O)–, –OC(O)NH–, –C(O)–, or –SO2–; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, a sterol, C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl having 1 to 4 heteroatoms each independently being N, O or S, C 6-12Aryl, 5- to 12-membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, -OH or -NH2; R 1a C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-40 Alkoxy, hydroxyl or –NR 1b R 1c Replacement; R 1b For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl; R 1c For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl or -LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 alkoxy, halogen, -CN or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; and each X is independently absent or -O - , where when X does not exist, R 2a and R 2b are each independently hydrogen, and R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-40 Alkyl, C 2-40 Alkenyl, C 4-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , and when X does not exist, R 1 is –CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

[0086] In some embodiments, the present invention provides compounds of formula (I), wherein: R1 For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c Substitution; W is C 3-12 Cycloalkyl, C 6-12 Aryl or C 4-12 heteroaryl, wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl substitution; each L is independently absent, C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne; each Y is independently absent, –O–, –NH–, –NHC(O)–, –NHC(O)NH–, –NHSO2–, –OC(O)–, –OC(O)NH–, –C(O)–, or –SO2–; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, or a sterol; R 1a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, wherein each alkyl, alkenyl and alkynyl is optionally substituted by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c Replacement; R 1b For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, and R 1c For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl or -LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, halogen, -CN or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; each X is independently absent or -O - , where when X does not exist, R 2a and R 2b is hydrogen, and R3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-20 Alkyl, C 2-20 Alkenyl, C 4-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a ; and where when X is absent, R 1 is –CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

[0087] In some embodiments, R 1 For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c Substitution; W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl, wherein the 5 to 12 membered heteroaryl has 1 to 4 heteroatoms which are N, O and S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl substitution; each L is independently absent, C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkyne; each Y is independently absent, –O–, –NH–, –NHC(O)–, –NHC(O)NH–, –NHSO2–, –OC(O)–, –OC(O)NH–, –C(O)–, or –SO2–; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, or a sterol; R 1a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-20 Alkoxy, hydroxyl or –NR1b R 1c Replacement; R 1b For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R 1c For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl or -LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 alkoxy, halogen, -CN or -NO2; m and n are independently integers from 1 to 10; p is independently an integer from 1 to 20; and each X is independently absent or -O - , where when X does not exist, R 2a and R 2b is hydrogen, and R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-20 Alkyl, C 2-20 Alkenyl, C 4-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a ; and where when X is absent, R 1 is –CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

[0088] In some embodiments, R 1 For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a In some embodiments, R 1 C 1-40 Alkyl, C 2-40 Alkenyl, -(LY)p -Z or –C(O)R 1a .

[0089] In some embodiments, R 1 C 1-40 In some embodiments, R 1 C 1-25 In some embodiments, R 1 C 1-20 In some embodiments, R 1 C 10-25 In some embodiments, R 1 C 10-20 In some embodiments, R 1 C 12-22 In some embodiments, R 1 C 12-18 alkyl.

[0090] In some embodiments, R 1 C 2-40 In some embodiments, R 1 C 20-40 In some embodiments, R 1 C 30-40 In some embodiments, R 1 C 2-40 In some embodiments, R 1 C 20-40 In some embodiments, R 1 C 30-40 Alkynyl.

[0091] In some embodiments, R 1 is W. In some embodiments, W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 In some embodiments, W is C 5-12 Cycloalkyl, C 6-12 aryl or a 5- to 12-membered heteroaryl group having 1 to 4 heteroatoms each independently being N, O or S. In some embodiments, W is C 5-12 In some embodiments, W is C 5-8 In some embodiments, W is cyclopentyl or cyclohexyl.

[0092] In some embodiments, R 1 -(LY) p -Z. In some embodiments, p is an integer from 1 to 20. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is 1, 2, 3, 4, or 5. In some embodiments, p is 1.

[0093] In some embodiments, L is C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 In some embodiments, L is C 1-20 In some embodiments, L is C 1-10 In some embodiments, L is C 1-5 Alkylene.

[0094] In some embodiments, Y is absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-. In some embodiments, Y is absent, -NH-, -NHC(O)-, -NHC(O)NH-, -OC(O)-, -OC(O)NH-, or -C(O)-. In some embodiments, Y is absent, -NH-, -NHC(O)-, or -NHC(O)NH-. In some embodiments, Y is absent, -NH-, -NHC(O)-, or -NHC(O)NH-. In some embodiments, Y is absent, -NH-, or -NHC(O)-.

[0095] In some embodiments, Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, a sterol, a C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl having 1 to 4 heteroatoms each independently being N, O or S, C 6-12 Aryl, 5- to 12-membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, -OH or -NH2.

[0096] Photosensitizers that can be used in the present invention include, but are not limited to, porphyrins, benzoporphyrins, corrins, chlorins, bacteriochlorophyll, corphins, or their derivatives. Representative photosensitizers are shown below:

[0097]

[0098]

[0099]

[0100] In some embodiments, the photosensitizer is a porphyrin, a benzoporphyrin, a corrin, a chlorin, a bacteriochlorophyll, a corrin, or a derivative thereof. In some embodiments, the photosensitizer compound is a porphyrin, a pyropheophorbide a, a pheophorbide, a chlorin e6, a purpurin, a purpurin diimide, a verteporfin, a photoporphyrin, a rotaporfin, a talporfin, or a temoporfin. In some embodiments, the photosensitizer is a pyropheophorbide a. In some embodiments, the photosensitizer is a pheophorbide a. In some embodiments, the photosensitizer is a porphyrin.

[0101] Any suitable porphyrin can be used in the compounds of the present invention. Representative porphyrins suitable for use in the present invention include, but are not limited to, pyropheophorbide a, pheophorbide, chlorin e6, purpurin, or purpurin diimide. In some embodiments, the porphyrin can be pheophorbide a. In some embodiments, the porphyrin can be pyropheophorbide a.

[0102] In some embodiments, Z is porphyrin, sterol, 6- to 12-membered heterocycloalkyl, 8- to 12-membered heteroaryl, -OH, or -NH2, wherein the 6- to 12-membered heterocycloalkyl and 8- to 12-membered heteroaryl have 1 to 4 heteroatoms that are N, O, and S. In some embodiments, Z is porphyrin, cholic acid, indoline, isoindoline, 1-isoindolinone, phthalimide, phthalic anhydride, -OH, or -NH2.

[0103] In some embodiments, p is 1; L is C 4-5 Alkylene; Y is absent, -NH- or -NHC(O)-; Z is porphyrin, cholic acid, isoindoline, phthalimide, -OH or -NH2.

[0104] In some embodiments, R 1 for –C(O)R 1a In some embodiments, R 1a C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-40 Alkoxy, hydroxyl or –NR 1b R 1c In some embodiments, R 1a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c In some embodiments, R 1a C 1-10Alkyl, C 2-10 Alkenyl or C 2-10 In some embodiments, R 1a C 1-10 In some embodiments, R 1a C 1-5 In some embodiments, R 1a It is methyl, ethyl, propyl or butyl.

[0105] In some embodiments, R 1b C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 In some embodiments, R 1b C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 In some embodiments, R 1b C 1-10 In some embodiments, R 1b C 1-5 In some embodiments, R 1b It is methyl, ethyl, propyl or butyl.

[0106] In some embodiments, R 1c C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 In some embodiments, R 1c C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 In some embodiments, R 1c C 1-10 In some embodiments, R 1c C 1-5 In some embodiments, R 1c It is methyl, ethyl, propyl or butyl.

[0107] In some embodiments, R 2a and R 2b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 In some embodiments, R 2a and R 2b are independently hydrogen, C 1-20 In some embodiments, R 2a and R2b Each is independently hydrogen or halogen. In some embodiments, R 2a and R 2b Each is independently hydrogen, fluorine, chlorine, bromine or iodine. 2a and R 2b are each independently hydrogen.

[0108] In some embodiments, R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 In some embodiments, R 3a and R 3b are independently hydrogen, C 1-20 In some embodiments, R 3a and R 3b Each is independently hydrogen or halogen. In some embodiments, R 3a and R 3b Each is independently hydrogen, fluorine, chlorine, bromine or iodine. 3a and R 3b are each independently chlorine.

[0109] In some embodiments, m and n are independently integers from 1 to 10. In some embodiments, m and n are independently integers from 1 to 5. In some embodiments, m and n are independently 1, 2, 3, 4, or 5. In some embodiments, m and n are each independently 1.

[0110] In some embodiments, each X is independently absent or -0. - In some embodiments, each X is absent. In some embodiments, each X is -0 - .

[0111] In some embodiments, each X is absent. In some embodiments, the compound is of formula (Ia):

[0112]

[0113] In some embodiments, R 1 C 1-20 Alkyl, and the compound is of formula (Ia):

[0114]

[0115] In some embodiments, the compound has the structure:

[0116]

[0117] wherein n is an integer from 1 to 7.

[0118] In some embodiments, the compound is selected from the group consisting of:

[0119]

[0120]

[0121] In some embodiments, the compound is:

[0122]

[0123] In some embodiments, the compound is selected from the group consisting of:

[0124]

[0125]

[0126] In some embodiments, each X is -0 - In some embodiments, the compound is a compound of formula (Ib):

[0127]

[0128] In some embodiments, the compound is selected from the group consisting of:

[0129]

[0130]

[0131] In some embodiments, the compound is selected from the group consisting of:

[0132]

[0133]

[0134]

[0135] The present invention includes all tautomers and stereoisomers of the compound of the present invention, which are in the form of mixtures or in pure or substantially pure forms. The compound of the present invention can have an asymmetric center on a carbon atom, so the compound of the present invention can exist in diastereomeric form or enantiomeric form or its mixture. All conformational isomers (for example, cis and trans isomers) and all optical isomers (for example, enantiomers and diastereomers), racemic mixtures of such isomers, diastereomeric mixtures and other mixtures, and solvates, hydrates, isomorphs, polymorphs and tautomers are all within the scope of the present invention. Compounds according to the present invention can be prepared using diastereomers, enantiomers or racemic mixtures as starting raw materials. In addition, diastereoisomer products and enantiomeric products can be separated by chromatography, fractional crystallization or other methods known to those skilled in the art.

[0136] The present invention also includes isotopically labeled compounds of the present invention, wherein one or more atoms are replaced by one or more atoms having a specific atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present invention include, but are not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, sulfur, and chlorine (such as 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 18 F. 35 S and 36 Cl). The isotopically labeled compounds of the present invention can be used to determine the tissue distribution of the compounds and their prodrugs and metabolites; preferred isotopes for such determinations include 3 H and 14 C. In addition, in some cases, heavier isotopes such as deuterium ( 2 H) substitution can provide increased metabolic stability, which provides therapeutic advantages such as extended in vivo half-life or reduced dosage requirements. Isotopically labeled compounds of the present invention can generally be prepared by replacing non-isotopically labeled reagents with isotopically labeled reagents according to methods known to those skilled in the art. The compounds of the present invention can be isotopically labeled at positions adjacent to basic amines in the aromatic ring and at the methyl group of a methoxy substituent.

[0137] The compounds of the present invention can also be in the form of pharmaceutically acceptable salts, such as acid salts or alkaline salts of the compounds of the present invention. Illustrative examples of pharmaceutically acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, etc.) salts, organic acid (fumaric acid, acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It should be understood that pharmaceutically acceptable salts are non-toxic. Other information about suitable pharmaceutically acceptable salts can be found in "Remington's Pharmaceutical Sciences", 17th edition, Mack Publishing Company of Easton, Pennsylvania, 1985, which is incorporated herein by reference.

[0138] IV. Nanocarriers

[0139] In some embodiments, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds of the present invention or pharmaceutically acceptable salts thereof, wherein each compound self-assembles in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed in the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

[0140] The diameter of the nanocarrier of the present invention can be any suitable size. In some embodiments, the nanocarrier may have a diameter of 5nm to 200nm. In some embodiments, the nanocarrier may have a diameter of 10nm to 150nm. In some embodiments, the nanocarrier may have a diameter of 50nm to 150nm. In some embodiments, the nanocarrier may have a diameter of 100nm to 150nm. In some embodiments, the nanocarrier may have a diameter of about 80nm, 90nm, 100nm, 110nm, 120nm, or 130nm. In some embodiments, the nanocarrier may have a diameter of about 100nm.

[0141] The exterior of the nanocarrier can be used for cell or lysosomal targeting. The nanocarrier can target cells or lysosomes to inhibit autophagy. In some embodiments, the nanocarrier can target lysosomal destruction, lysosomal dysfunction, autophagy inhibition, or a combination thereof. In some embodiments, the nanocarrier targets lysosomes.

[0142] In some embodiments, the hydrophobic pocket is formed by R 1 In some embodiments, the nanocarrier further comprises one or more hydrophobic drugs or imaging agents sequestered in the hydrophobic pocket of the nanocarrier.

[0143] The hydrophobic drug that can be used in the present invention can be any hydrophobic drug known to those skilled in the art. The hydrophobic drug that can be used in the present invention includes but is not limited to deoxycholic acid, deoxycholate, resiquimod, gardiquimod, imiquimod, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, baccatin III, 10-deacetylbaccatin, yew A, yew B or yew C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, carmustine, amphotericin, ixabepilone, patupirone (epothilones), rapamycin and platinum drugs. Other drugs include nonsteroidal anti-inflammatory drugs and vinca alkaloids, such as vinblastine and vincristine.

[0144] Other hydrophobic drugs that can be used in the present invention include, but are not limited to, chemotherapeutic agents, molecular targeted agents, immunomodulatory agents, immunotherapeutic agents, radiotherapeutic agents, or combinations thereof.

[0145] In some embodiments, the hydrophobic drug is a chemotherapeutic agent, a molecular targeted agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof. In some embodiments, the hydrophobic drug is an immunotherapeutic agent. Immunotherapeutic agents that can be used in the present invention include, but are not limited to, HCQ, Lys05, JQ1, rapamycin, nabucasin, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab.

[0146] In some embodiments, the hydrophobic drug is a radiotherapeutic agent. Radiotherapeutic agents useful in the present invention include, but are not limited to, β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, and tirapazamine.

[0147] In some embodiments, the hydrophobic drug is a chemotherapeutic agent or a molecular targeted agent. Chemotherapeutic agents or molecular targeted agents include, but are not limited to, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoazole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, and vemurafenib.

[0148] In some embodiments, the hydrophobic drug is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a MAP kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopalanib, GSK690693, RTA744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinib, nilotinib, decatanib, panitumumab, amrubicin, ogavuzumab, Lep-etu, nolatrexed, AZD2171, batabulin, ofatumumab, zalimumab, etakaline, tetrandrine, rubitecan, tesmilifen, oblimersen, tesimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR; INO 1001, IPdR1KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposome doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidine [(5-(methylsulfonylpiperidinylmethyl)-indolyl)-oxazolidinone]-disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperidinylmethyl)-indolyl-oxazolidinone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t)6,Azgly 10] acetate (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84 N 18 O 14-(C2H4O2)X, wherein X=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, actinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, cyclohexyl lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, Oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozotocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uramustine, estramustine, hexamethylmelamine, floxuridine, 5-deoxyuridine, cytarabine, 6-thiopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974 , interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimetidine, trastuzumab, denileukin, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without hydrogenated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipenoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrast im, darbepoetin alfa, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrochloride cortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol acetate, immunoglobulin, nitrogen mustard, methylprednisolone, ibritumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netol pipitant, NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabitril, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilimumab, vemurafenib, or a combination thereof. In some embodiments, the hydrophobic drug is HCQ, Lys05, JQ1, rapamycin, nabucaccine, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, β-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, tirapazamine, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoazole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, vemurafenib, or a combination thereof.

[0149] In some embodiments, the nanocarrier comprises a plurality of compounds of the present invention, having the compound structures described above.

[0150] V. Formulation and Administration

[0151] The compounds, nanocarriers and compositions of the present invention can be prepared into a variety of oral, parenteral and topical dosage forms. Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by patients. The compositions of the present invention can also be administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal or intraperitoneal injection. In addition, the compositions described herein can be administered by inhalation, such as intranasal administration. In addition, the compositions of the present invention can be administered transdermally. The compositions of the present invention can also be administered via intraocular, intravaginal and intrarectal routes, including suppositories, insufflations, powders and aerosol formulations (e.g., steroid inhalers, see Rohatagi, J. Clin. Pharmacol., Vol. 35: 1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol., Vol. 75: 107-111, 1995). Therefore, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of the present invention.

[0152] In order to prepare pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances, which may also be used as diluents, flavoring agents, binders, preservatives, tablet disintegrants, or encapsulating materials. Details on formulation and administration techniques are described in detail in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania ("Remington's").

[0153] In powders, the carrier is a finely divided solid which is mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain from 5% or 10% to 70% of the compound of the invention.

[0154] Suitable solid excipients include, but are not limited to, magnesium carbonate, magnesium stearate; talc; pectin; dextrin; starch; tragacanth; low melting wax; cocoa butter; carbohydrates; sugars, including but not limited to lactose, sucrose, mannitol or sorbitol, starch from corn, wheat, rice, potato or other plants; cellulose, such as methylcellulose, hydroxypropyl methylcellulose or sodium carboxymethylcellulose; and gums, including gum arabic and tragacanth; and proteins, including but not limited to gelatin and collagen. If desired, disintegrators or solubilizers may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[0155] The dragee cores are provided with a suitable coating, such as a concentrated sugar solution, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or dragee coating for product identification or to characterize the amount (i.e., dosage) of the active compound. The pharmaceutical preparations of the invention may also be used orally, for example using push-fit capsules made of gelatin, and sealed soft capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules may contain the compound of the invention mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsule, the compound of the invention may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or a liquid polyethylene glycol with or without a stabilizer.

[0156] To prepare suppositories, a low melting point wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the compound of the present invention is uniformly dispersed therein by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby solidified.

[0157] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in solution in aqueous polyethylene glycol solution.

[0158] By dissolving the compound of the present invention in water and adding suitable colorants, flavoring agents, stabilizers and thickeners as needed, an aqueous solution suitable for oral use can be prepared. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active component in water containing a viscous substance and a dispersant or wetting agent, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth and gum arabic, and the dispersant or wetting agent is such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain fatty alcohols (e.g., 17-ethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The formulation may be adjusted for osmolarity.

[0159] Also included are preparations in solid form that are converted into preparations in liquid form for oral administration shortly before use. Such liquid forms include solutions, suspensions, and emulsions. In addition to the active ingredient, these preparations may contain coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, etc.

[0160] Oily suspensions can be prepared by suspending the compound of the present invention in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil), or in a mineral oil (such as liquid paraffin), or in a mixture thereof. Oily suspensions may contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweeteners may be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These preparations can be preserved by adding antioxidants (such as ascorbic acid). As an example of an injectable oil solvent, see Minto, Journal of Pharmacology and Experimental Therapeutics (J.Pharmacol.Exp.Ther.), Vol. 281: pp. 93-102, 1997. The pharmaceutical preparations of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil or a mineral oil as described above, or a mixture of these. Suitable emulsifiers include naturally occurring gums such as gum arabic and gum tragacanth; naturally occurring phospholipids such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate; and condensation products of these partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. Emulsions may also contain sweeteners and flavorings, such as in the preparation of syrups and elixirs. Such preparations may also contain a demulcent, a preservative, or a coloring agent.

[0161] In another embodiment, the composition of the present invention can be formulated for parenteral administration, such as intravenous (IV) administration or administration into a body cavity or organ lumen. The preparation for administration will generally include a solution of the composition of the present invention dissolved in a pharmaceutically acceptable carrier. Acceptable solvents and solvents that can be used are water and Ringer's solution (an isotonic sodium chloride). In addition, sterile fixed oils can generally be used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injections. These solutions are sterile and generally do not contain undesirable substances. These preparations can be sterilized by conventional, well-known sterilization techniques. The preparation can contain pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as pH regulators and buffers, toxicity regulators, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the composition of the present invention in these preparations can vary widely, and will be mainly based on fluid volume, viscosity, body weight, etc. and selected according to the needs of the selected specific mode of administration and the patient. For IV administration, the preparation can be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. The suspension can be formulated according to known techniques using those suitable dispersants or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.

[0162] The compositions of the present invention can be delivered by any suitable means, including oral, parenteral and topical methods. Transdermal administration methods by topical routes can be formulated into applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, paints, powders and aerosols.

[0163] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the compound of the invention. The unit dosage form can be a packaged formulation containing discrete quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. Additionally, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these packaged forms.

[0164] The compounds and nanocarriers of the present invention may be present in any suitable amount and may depend on a variety of factors, including but not limited to the weight and age of the subject, the state of the disease, etc. Suitable dosage ranges for the compounds of the present invention include about 0.1 mg to about 10,000 mg, or about 1 mg to about 1000 mg, or about 10 mg to about 750 mg, or about 25 mg to about 500 mg, or about 50 mg to about 250 mg. Suitable dosages for the compounds of the present invention include about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg.

[0165] The compounds of the present invention and nanocarriers can be applied at any suitable frequency, interval and duration. For example, the compounds of the present invention can be applied once per hour, or twice, three times or more per hour, once a day, or twice, three times or more per day, or once every 2, 3, 4, 5, 6 or 7 days, to provide a preferred dosage level. When the compounds of the present invention are applied more than once a day, representative intervals include 5, 10, 15, 20, 30, 45 and 60 minutes, and 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours. The compounds of the present invention can be applied once, twice or three times or more, for 1 hour, 1 to 6 hours, 1 to 12 hours, 1 to 24 hours, 6 to 12 hours, 12 to 24 hours, 1 day, 1 to 7 days, 1 week, 1 to 4 weeks, 1 month, 1 to 12 months, 1 year or longer, or even indefinitely.

[0166] The compositions may also contain other compatible therapeutic agents.The compounds described herein may be used in combination with each other, with other active agents known to be useful in modulating the glucocorticoid receptor, or with adjunct agents that may not be effective alone but may aid in the efficacy of the active agent.

[0167] The compounds of the present invention may be co-administered with another activating agent. Co-administration includes administering the compounds of the present invention and activating agents within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20 or 24 hours of each other. Co-administration also includes administering the compounds of the present invention and activating agents simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20 or 30 minutes of each other) or sequentially in any order. In addition, the compounds of the present invention and activating agents may be administered once a day, or twice, three times or more a day, so as to provide a preferred daily dosage level.

[0168] In some embodiments, co-administration can be accomplished by co-formulation, ie, preparing a single pharmaceutical composition containing both the compound of the invention and the active agent. In other embodiments, the compound of the invention and the active agent can be formulated separately.

[0169] The compounds of the present invention and the activating agent can be present in the compositions of the present invention in any suitable weight ratio, such as about 1:100 to about 100:1 (w / w), or about 1:50 to about 50:1, or about 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1:5 to about 5:1 (w / w). The compounds of the present invention and the other activating agent can be present in any suitable weight ratio, such as about 1:100 (w / w), 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1 or 100:1 (w / w). Other doses and dose ratios of the compounds of the present invention and the activating agent are also applicable to the compositions and methods of the present invention.

[0170] VI. Treatment Methods

[0171] In some embodiments, the present invention provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a nanocarrier of the present invention.

[0172] In some embodiments, the method further comprises a combination therapy using an additional agent for treating the disease. The additional agent is a therapeutic agent. The combination therapy of the present invention includes but is not limited to the use of the nanocarrier of the present invention and one or more additional agents.

[0173] Combination therapy may include, but is not limited to, immunotherapy, radiation therapy, chemotherapy, molecular targeted therapy, or a combination thereof.

[0174] In some embodiments, the method further comprises one or more additional agents, wherein the additional agent is a chemotherapeutic agent, a molecular targeting agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof. In some embodiments, the additional agent is an immunotherapeutic agent. The immunotherapeutic agents that can be used in the present invention are listed above. In some embodiments, the additional agent is a radiotherapeutic agent. The radiotherapeutic agents that can be used in the present invention are listed above. In some embodiments, the additional agent is a chemotherapeutic agent or a molecular targeting agent. The chemotherapeutic agents and molecular targeting agents that can be used in the present invention are listed above.

[0175] In some embodiments, the one or more additional agents include two additional agents. In some embodiments, the additional agent is an immunotherapeutic agent and a radiotherapeutic agent. In some embodiments, the additional agent is an immunotherapeutic agent and a chemotherapeutic agent. In some embodiments, the additional agent is an immunotherapeutic agent and a molecular targeting agent. In some embodiments, the additional agent is a radiotherapeutic agent and a chemotherapeutic agent. In some embodiments, the additional agent is a radiotherapeutic agent and a molecular targeting agent.

[0176] In some embodiments, the additional agent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a MAP kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopalanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinib, nilotinib, decatanib, panitumumab, amrubicin, ogavuzumab, Lep-etu, nolatrexed, AZD2171, batabulin, ofatumumab, zalimumab, etakaline, tetrandrine, rubitecan, tesmilifen, oblimersen, tesimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR; INO 1001, IPdR1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposome doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidine [(5-(methylsulfonylpiperidinylmethyl)-indolyl)-oxazolidinone]-disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperidinylmethyl)-indolyl-oxazolidinone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t)6,Azgly 10] acetate (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84 N 18 O 14-(C2H4O2)X, wherein X=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, actinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, cyclohexyl lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, Oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozotocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uramustine, estramustine, hexamethylmelamine, floxuridine, 5-deoxyuridine, cytarabine, 6-thiopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974 , interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimetidine, trastuzumab, denileukin, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without hydrogenated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipenoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787 / ZK222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-fi lgrastim, darbepoietin alfa, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol acetate, immune globulin, nitrogen mustard, methylprednisolone, ibritumomab tiuxetan, androgens, decitabine avena cava, altretamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netupitant, NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabidiol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilimumab, vemurafenib, or a combination thereof. In some embodiments, the additional agent is HCQ, Lys05, JQ1, rapamycin, nabucaccine, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, beta-lapachone, cisplatin, nimorazole, cetuximab, misonidazole, tirapazamine, daunorubicin, doxorubicin, paclitaxel, docetaxel, abraxane, bortezomib, etoposide, lenalidomide, apoazole, carboplatin, cisplatin, oxaliplatin, vinblastine, vincristine, trastuzumab, erlotinib, imatinib, nilotinib, vemurafenib, or a combination thereof.

[0177] Diseases treatable by the methods of the invention include coronavirus, malaria, antiphospholipid antibody syndrome, lupus, rheumatoid arthritis, chronic urticaria or Sjögren's syndrome and cancers such as, but not limited to: carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer and Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, Cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penis cancer, urethra cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin cancer, retinoblastoma, multiple myeloma, Hodgkin lymphoma and non-Hodgkin lymphoma (for additional cancers see, CANCER: PRINCIPLES AND PRACTICE (DeVita, VT et al., eds., 2008)).

[0178] Other diseases that can be treated by the nanocarriers of the present invention include: (1) inflammatory or allergic diseases, such as systemic anaphylaxis or hypersensitivity reactions, drug allergies, and insect bite allergies; inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis, and enteritis; vaginitis; psoriasis and inflammatory skin diseases, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, and urticaria; vasculitis; spondyloarthropathies; scleroderma; respiratory allergic diseases, such as asthma, allergic rhinitis, and allergic lung disease, etc., (2) autoimmune diseases, such as arthritis (rheumatoid arthritis), and psoriasis), osteoarthritis, multiple sclerosis, systemic lupus erythematosus, diabetes, glomerulonephritis, etc., (3) transplant rejection (including allogeneic transplant rejection and graft-versus-host disease), and (4) other diseases requiring suppression of undesirable inflammatory responses (e.g., atherosclerosis, myositis, neurological disorders such as stroke and closed head injury, neurodegenerative diseases, Alzheimer's disease, encephalitis, meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis, sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonary disease, sinusitis and Behcet's syndrome).

[0179] In some embodiments, the disease is cancer. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and uterine cancer. In some embodiments, the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, and uterine cancer.

[0180] In some embodiments, the disease is coronavirus, malaria, antiphospholipid antibody syndrome, lupus, rheumatoid arthritis, chronic urticaria, or Sjögren's syndrome.

[0181] In some embodiments, the method for treating a disease comprises targeting autophagy and / or lysosomes. Targeting autophagy can result in autophagy inhibition or autophagy activation. Targeting lysosomes can result in lysosomal destruction, lysosomal dysfunction, or both.

[0182] In some embodiments, the treatment methods target lysosomal destruction, lysosomal dysfunction, and / or autophagy inhibition. In some embodiments, the treatment methods target lysosomes.

[0183] In some embodiments, the nanocarrier targets lysosomal destruction, lysosomal dysfunction, and / or autophagy inhibition. In some embodiments, the nanocarrier targets lysosomes.

[0184] VII. Imaging Methods

[0185] In some embodiments, the present invention provides methods of imaging, comprising administering to a subject to be imaged an effective amount of a nanocarrier of the present invention.

[0186] The imaging agent that can be used in the present invention can be any imaging agent known to those skilled in the art. Imaging agents include, but are not limited to, paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are imaging agents that have magnetism under an applied magnetic field. Examples of paramagnetic agents include, but are not limited to, iron particles including nanoparticles. Optical probes are fluorescent compounds that can be detected by excitation at one radiation wavelength and detection at a second, different radiation wavelength. Optical probes that can be used in the present invention include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo radioactive decay. Radionuclides that can be used in the present invention include, but are not limited to 3 H. 11 C. 13 N. 18 F. 19 F. 60 Co. 64 Cu, 67 Cu, 68 Ga, 82 Rb, 90 Sr. 90 Y. 99 Tc, 99m Tc, 111In, 123 I. 124 I. 125 I. 129 I. 131 I. 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu and 241 Am.

[0187] Imaging methods useful in the present invention include, but are not limited to, fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT), x-ray computed tomography (CT), echocardiography, and functional near-infrared spectroscopy.

[0188] VIII. Examples

[0189] Example 1: Compound

[0190] Materials and instruments. Chemicals such as diethylenetriamine, dodecanal, fatty alcohols, pyridinium dichromate, sodium cyanoborohydride, ammonium hydroxide solution, deuterated solvents, anhydrous solvents, and Z-Arg-Arg-AMC were purchased from Millipore-Sigma, MO, USA. 4,7-Dichloroquinoline, anhydrous sulfate, and most solvents were purchased from Thermo Fisher Scientific, MA, USA. All solvents were used without further purification. Water used in all experiments was purified using a Mill-Q filtration system. Other reagents or drugs were purchased as indicated: tridecanal (Alfa Aesar), HCQ (Spectrum), Lys05 (MCE (MedchemExpress)), bortezomib (eNovation chemical), DiD perchlorate and β-lapachone (Tocris Bioscience), JQ1 and nabuccal (ApExBIO), rapamycin, paclitaxel and vinblastine (LC Laboratory), CN38 (AcrosOrganics), etoposide (Adipogen), lenalidomide (Matrix Scientific), nabuccal (ApExBIO) and apoxetine (Selleck). Lysosomal enrichment kit, LysoTracker (red & green), acridine orange, dextran-AlexaFluor 488, Premo TMThe autophagy sensor LC3B-GFP was purchased from Thermo Fisher Scientific, Massachusetts, USA. Homogeneous AMC caspase 3 / 7 assay kit and FITC-Annexin V / PI apoptosis detection kit were purchased from AnaSpec, California, USA and Biolegend, California, USA, respectively. The compounds were characterized by measuring NMR spectra with a 600MHz NMR spectrometer (Bruker, Germany) and measuring ESI-HRMS spectra with a LTQ-Orbitrap XL hybrid ion trap mass spectrometer (Thermo Fisher, Massachusetts, USA). Cell imaging studies were performed by fluorescence microscopy (Olympus, Tokyo, Japan) or LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany). Absorbance and fluorescence intensity were measured with a SpectraMax M2 microplate reader (Molecular Devices, California, USA). Western blots were developed by Power Pac200 electrophoresis instrument (Bio-Rad, California, USA). Studies were conducted on ChemiDocTM MP imaging system (Bio-Rad, California, USA), including WB imaging, in vivo and ex vivo fluorescence imaging. DLS experiments were performed with ZetasizerNano ZS (Malvern Instruments, Worcestershire, UK). TEM was performed with 80 kV accelerating voltage on Talos L120C TEM (FEI, Oregon, USA (OR, USA)). Apoptosis assays were performed by using BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Cancer stem cells were separated by BD FACSAria II cell sorter (BD Biosciences, New Jersey, USA (BD Biosciences)). Matrigel (catalog number 354230) for 3D culture and Matrigel (catalog number 354234) for xenograft model establishment were purchased from Corning, NY, USA.LC3B antibody (1:1000, catalog number 2775), SQSTM1 / p62 antibody (1:1000, catalog number 39749) and β-actin antibody (1:1000, catalog number 4970) were purchased from Cell Signaling, and Pacific Blue anti-CD44 antibody (5 μL per million cells, 100 μL volume, catalog number 338823), APC anti-CD326 (EpCAM) antibody (5 μL per million cells, 100 μL volume, catalog number 324207), PE / Cy7 anti-CD24 antibody (5 μL per million cells, 100 μL volume, catalog number 311119) were all purchased from Biolegend.

[0191]

[0192] Synthesis of O-methyl-serine-dodecylamide hydrochloride (MSDH). MSDH was synthesized according to the published literature (Bioorg. Med. Chem. Lett., 1995, Vol. 5, pp. 893-898). 1 H NMR (600MHz, CDCl3): δ7.40(s,1H),3.63(m,1H),3.58(m,2H),3.70(s,3H),3.26( m,2H),1.74(s,2H),1.51(m,2H),1.29(m,18H),0.89(t,3H,J=7.2Hz).ESI-HRMS: C 16 H 35 N2O2 + m / z[M+H] + The calculated value is 287.2693 and the measured value is 287.2690.

[0193]

[0194] Synthesis of BAQ. 4,7-Dichloroquinoline (1.2 g, 6.00 mmol) in a 10 mL flask was kept at 80 ° C for 2 h without stirring, and then diethylenetriamine (0.22 mL, 2.00 mmol) was added. The reaction solution was stirred at 130 ° C for 6 h. The residue was dissolved in 30 mL of methanol to obtain a white solid, which was a BAQ compound. Yield: 470 mg, 55%. 1H NMR (600MHz, DMSO-d6): δ8.38(d,2H,J=6.0Hz), 8.23(d,2H,J=10.8Hz), 7.78(d,2H,J=1.8Hz), 7.42(dd,2H,J1=1 0.8Hz, J2=2.4Hz), 7.25 (s, 1H), 6.51 (d, 2H, J=6.6Hz), 3.40 (t, 4H, J=7.2Hz), 2.94 (t, 4H, J=7.8Hz). ESI-HRMS: C 22 H 22 Cl2N5 + m / z[M+H] + The calculated value is 426.1247 and the measured value is 426.1243.

[0195] General Synthesis of BAQ12-18. To a solution of BAQ (426 mg, 1.0 mmol) in 30 mL of anhydrous methanol and 10 mL of anhydrous dichloromethane, the corresponding aldehyde (2 mmol) and acetic acid (20 μL) were added, followed by stirring at room temperature for 20 minutes, followed by the addition of sodium cyanoborohydride (126 mg, 2 mmol). The mixture was stirred for 12 h and diluted with chloroform (100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate overnight. The crude product was purified by silica gel chromatography using an eluent containing 0.1% triethylamine (dichloromethane: methanol = 30: 1-10: 1) to give the corresponding compound.

[0196]

[0197] BAQ12. Yield: 320 mg, 53.8%. 1 H NMR (600MHz, CD3OD): δ8.27(d,2H,J=5.4Hz), 7.67(d,2H,J=2.4Hz), 7.55(d,2H,J=9.0Hz), 6.95(dd,2H,J1=9.0Hz, J2=1.8Hz), 6.46(d ,2H,J=5.4Hz),3.41(t,4H,J=6.0Hz),2.90(t,4H,J=6.0Hz),2.66(t,2H,J=6.6Hz),1.55(m,2H),1.33(m,20H),0.91(t,3H,J=7.2Hz). 13C NMR (150MHz, CD3OD): δ150.9,150.8,147.9,134.8,126.3,124.4,121.9,117.0,98.4,5 4.1,51.9,40.3,31.6,29.5,29.5,29.4,29.4,29.1,27.4,27.3,22.4,13.1.ESI-HRMS: C 34 H 46 Cl2N5 + m / z[M+H] + The calculated value is 594.3125 and the measured value is 594.3134.

[0198] BAQ13. Yield: 350 mg, 57.5%. 1 H NMR (600MHz, CD3OD): δ8.27 (d, 2H, J = 5.4Hz), 7.67 (d, 2H, J = 1.8Hz), 7.56 (d, 2H, J = 9.0Hz), 6.96 (dd, 2H, J1 = 9.0Hz, J2 = 2.4Hz), 6.46 (d ,2H,J=5.4Hz),3.42(t,4H,J=6.0Hz),2.90(t,4H,J=6.0Hz),2.66(t,2H,J=7.2Hz),1.56(m,2H),1.32(m,23H),0.92(t,3H,J=7.2Hz). 13 CNMR (150MHz, CD3OD): δ151.0,150.7,147.8,134.8,126.1,124.5,121.9,117.0,98.4,5 4.6,51.9,40.3,31.7,29.5,29.5,29.5,29.4,29.1,27.4,27.3,22.3,13.1.ESI-HRMS: C 35 H 48 Cl2N5 + m / z[M+H] + The calculated value is 608.3281 and the measured value is 608.3274.

[0199] BAQ14. Yield: 295 mg, 47.4%. 1H NMR (600MHz, CD3OD): δ8.56(d,2H,J=9.0Hz), 8.48(d,2H,J=4.8Hz), 7.86(d,2H,J=1.2Hz), 7.60(dd,2H,J1=9.0Hz, J2=1.2Hz) ,7.06(d,2H,J=5.4Hz),4.16(s,4H),3.82(s,4H),3.48(s,2H),1.92(s,2H),1.44(s,2H),1.35(m,23H),0.93(t,3H,J=6.6Hz). 13 C NMR (150MHz, CD3OD): δ155.9,143.0,139.8,138.2,127.4,125.3,118.7,115.5,98.9,54.6,51.1 ,38.3,31.5,29.2,29.2,29.2,29.2,29.1,29.0,28.9,28.7,26.1,23.0,22.2,12.9.ESI-HRMS: C 36 H 50 Cl2N5 + m / z[M+H] + The calculated value is 622.3438 and the measured value is 622.3505.

[0200] BAQ15. Yield: 290 mg, 45.5%. 1 H NMR (600MHz, CD3OD): δ8.56 (d, 2H, J = 9.0Hz), 8.48 (d, 2H, J = 6.0Hz), 7.86 (d, 2H, J = 1.2Hz), 7.59 (dd, 2H, J1 = 9.0Hz, J2 = 1.2Hz) ,7.07(d,2H,J=6.0Hz),4.16(s,4H),3.82(s,4H),3.48(s,2H),1.92(s,2H),1.44(s,2H),1.35(m,25H),0.93(t,3H,J=6.6Hz). 13 C NMR (150MHz, CD3OD): δ155.8,143.0,139.7,138.1,127.3,125.3,118.7,115.4,98.9,54.5,5 1.0,38.2,31.5,29.2,29.2,29.2,29.1,29.0,28.9,28.7,26.1,23.0,22.2,12.9.ESI-HRMS: C 37 H 52 Cl2N5 + m / z[M+H] +The calculated value is 636.3594 and the measured value is 636.3661.

[0201] BAQ16. Yield: 280 mg, 43.0%. 1 H NMR (600MHz, CD3OD): δ8.56 (d, 2H, J = 9.0Hz), 8.48 (d, 2H, J = 6.6Hz), 7.86 (d, 2H, J = 1.8Hz), 7.59 (dd, 2H, J1 = 9.0Hz, J2 = 1.8Hz), 7.0 7(d,2H,J=7.2Hz),4.17(m,4H),3.84(m,4H),3.50(t,2H,J=7.8Hz),1.92(m,2H),1.44(m,2H),1.30(m,27H),0.93(t,3H,J=7.2Hz). 13 C NMR (150MHz, CD3OD): δ155.9,143.0,139.7,138.1,127.3,125.2,118.7,115.4,98.8,54.5,5 1.0,38.2,31.5,29.2,29.2,29.2,29.1,28.9,28.9,28.7,26.1,23.0,22.1,12.9.ESI-HRMS: C 38 H 54 Cl2N5 + m / z[M+H] + The calculated value is 650.3751 and the measured value is 650.3774.

[0202] BAQ18. Yield: 290 mg, 42.7%. 1 H NMR (600MHz, CD3OD): δ8.56 (d, 2H, J = 9.0Hz), 8.48 (d, 2H, J = 5.4Hz), 7.86 (s, 2H, J = 1.8Hz), 7.56 (d, 2H, J1 = 8.4Hz), 7.06(d,2H,J=6.6Hz),4.16(s,4H),3.82(m,4H),3.48(s,2H),1.91(s,2H),1.43(m,2H),1.30(m,29H),0.93(s,3H). 13 C NMR (150MHz, CD3OD): δ155.9,143.0,139.8,138.2,127.4,125.2,118.7,115.5,98.8,5 4.5,51.1,38.2,31.5,29.2,29.1,28.9,28.9,28.7,26.1,23.0,22.2,12.9.ESI-HRMS: C 38 H54 Cl2N5 + m / z[M+H] + Calculated value: 650.3751, found: 650.3774. ESI-HRMS: C 40 H 56 Cl2N5 + m / z[M+H] + The calculated value is 678.4064 and the measured value is 678.4069.

[0203]

[0204] Synthesis of compound 1. To a solution of 4,4-diethoxybutylamine (1.84 mL, 10.7 mmol, 1.00 equiv) in THF (30 mL) was added N-ethoxycarbonylphthalimide ethyl ester (2.34 g, 10.7 mmol, 1.00 equiv) and triethylamine (1.49 mL, 10.7 mmol, 1.00 equiv). The resulting reaction mixture was stirred at room temperature for 12 h. After the solvent was removed under reduced pressure, the resulting crude material was purified on a silica gel column, eluted with 1:20 EtOAc / hexane to give a clear oil. (2.9 g, 94.0 mmol, 93% yield). 1 H NMR (600MHz, DMSO-d6) δ7.87-7.83(m,4H),4.47(t,J=5.4Hz,1H),3.58-3.51(m,4H),3.43-3.38(m,2H),1.61-1.60(m,2H),1.54-1.51(m,2H).ESI-HRMS m / z 314.1361[M+Na] + .

[0205]

[0206] Synthesis of Compound 2. A solution of Compound 1 (2.2 g, 7.6 mmol, 1.00 equiv) in acetone (20 mL) and 1 M aqueous HCl (15.2 mL, 15.2 mmol, 2.00 equiv) was vigorously stirred at reflux (80 ° C) for 1 h. Acetone was evaporated under reduced pressure, and the resulting aqueous layer was extracted 3 times with Et2O. The combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure and purified by column chromatography eluting with 1:2 EtOAc / hexane to give a waxy white solid. (1.2 g, 74% yield). 1H NMR (600MHz, DMSO-d6) δ9.64(t,J=1.2Hz,1H,NH2),7.88-7.83(m,4H),3.61(t,J=7.2Hz,2H),2.54-2.51(m,2H),1.85-1.83(m,2H).

[0207]

[0208] Synthesis of BAQ4q. Compound 2 (434 mg, 2 mmol, 2.0 equiv) and acetic acid (10 μL) were added to a solution of BAQ (426.5 mg, 1.0 mmol, 1.00 equiv) in methanol (40 mL) and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (126 mg, 2 mmol, 2 equiv) was slowly added and stirred for 24 h. The reaction solution was diluted with dichloromethane (150 mL), then washed with saturated sodium carbonate, water and brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the mixture was purified by silica gel chromatography, eluted with 20:1 dichloromethane / methanol to give a white solid. (520 mg, 83% yield). 1 H NMR(600MHz,DMSO-d6)δ8.27(d,J=5.4Hz,2H),7.97(d,J=9.0Hz,2H),7.78(s,4 H),7.67(d,J=2.4Hz,2H),7.19(dd,J1=9.0Hz,J2=2.4Hz,2H),7.09(m,2H),6.3 9(d,J=6.0Hz,2H),3.51(t,J=7.2Hz,2H),3.31(t,J=6.0Hz,4H),2.77(t,J=6.6 Hz,4H),2.58(t,J=6.6Hz,2H),1.60-1.55(m,2H),1.45-1.40(m,2H).ESI-HRMS 627.2035[M+H] + .

[0209]

[0210] Synthesis of BAQ4a. BAQ4q (314 mg, 0.5 mmol, 1.0 equiv) was dissolved in ethanol, and after adding hydrazine (2.5 mmol, 5.0 equiv), the reaction solution was refluxed and stirred for 12 h. The reaction mixture was then allowed to stand at room temperature. After filtering to remove the precipitate, the filtrate was concentrated and then added to ether (100 mL) to produce a white precipitate, which was collected as compound 5. (200 mg, 80% yield). 1H NMR 8.30(d,J=5.4Hz,2H),8.03(d,J=9.6Hz,2H),7.78(s,4H),7.71(d,J=2.4Hz,2H),7.2 7(dd,J1=9.0Hz,J2=2.4Hz,2H),7.05(t,J=5.4Hz,2H),6.40(d,J=5.4Hz,2H),3.31(t ,J=6.6Hz,2H),3.31(t,J=6.0Hz,4H),2.78(t,J=7.2Hz,4H),2.54(t,J=7.2Hz,2H),2 .42(t,J=7.2Hz,2H),1.41-1.38(m,2H),1.29-1.26(m,2H)..ESI-HRMS497.1981[M+H] + .

[0211]

[0212] Synthesis of PBC. Pheophorbide a (296 mg, 0.5 mmol, 1.0 equiv.), 6-chloro-1-hydroxybenzotriazole (102 mg, 0.6 mmol, 1.2 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (93 mg, 0.6 mmol, 1.2 equiv.) and N,N-diisopropylethylamine (174 μL, 1.0 mmol, 2 equiv.) were suspended in anhydrous dichloromethane (75 mL) and stirred at room temperature for 30 minutes. Compound 5 (314 mg, 0.5 mmol, 1.0 equiv.) was added to the reaction mixture and then stirred for 48 h. The mixture was purified by silica gel chromatography, eluted with 20:1 dichloromethane / methanol to give a black solid. (200 mg, 37% yield). 1H NMR (600MHz, CD3OD) δ8.94 (s, 1H), 8.78 (s, 1H), 8.58 (s, 1H), 7.86 (dd, J1=6.0Hz, J2=3.6Hz ,2H),7.86(dd,J1=6.0Hz,J2=3.6Hz,2H),7.81(dd,J1=18.0Hz,J2=11.4Hz,2H),7.63(m,1H) ,7.54(d,J=6.0Hz,2H),7.18(dd,J1=9.0Hz,J2=2.4Hz,1H),6.88(m,2H),6.55(d,J=8.4Hz, 2H),6.22(dd,J1=8.4Hz,J2=1.8Hz,2H),6.17(d,J=18.0Hz,1H),6.10(d,J=11.4Hz,1H),5.5 5(d, J=18.0Hz,1H),4.52-4.51(m,1H),4.15-4.14(m,1H),3.88(s,3H),3.72-3.67(m,2H),3.30(s,3H),3.2(q, J2=7.2Hz,4H,triethylamine),2.89(m,1H),2.80(s,3H),2.75(m,1H),2.63-2.61( m, 2H), 2.56-2.53 (m, 1H), 2.50-2.43 (m, 4H), 2.3-2.16 (m, 8H), 1.94 (s, 1H), 1.82 (d, J = 7.2 Hz, 3H), 1.47 (t, J = 7.8 Hz, 3H), 1.35 (m, 10H + 6H triethylamine), 1.11 (m, 4H). ESI-HRMS 1071.4576 [M + H] + .

[0213]

[0214] Synthesis of CAB. Cholic acid (204 mg, 0.5 mmol, 1.0 equiv), 6-chloro-1-hydroxybenzotriazole (102 mg, 0.6 mmol, 1.2 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (93 mg, 0.6 mmol, 1.2 equiv) and N,N-diisopropylethylamine (174 μL, 1.0 mmol, 2 equiv) were suspended in anhydrous dichloromethane (75 mL) and stirred at room temperature for 30 minutes. BAQ4a (314 mg, 0.5 mmol, 1.0 equiv) was added to the reaction mixture, which was then stirred for 48 h. The mixture was purified by silica gel chromatography eluting with 20:1 dichloromethane / methanol to give a black solid. (220 mg, 49.5% yield). ESI-HRMS 887.4774 [M+H] + .

[0215]

[0216] Synthesis of BAQ5h. To a solution of BAQ (426.5 mg, 1.0 mmol, 1.00 equiv) in methanol (40 mL) was added glutaraldehyde (211 μL, 2.0 mmol, 2.00 equiv) and acetic acid (10 μL), which was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (126 mg, 2 mmol, 2 equiv) was slowly added and stirred for 24 h. The reaction solution was diluted with dichloromethane (150 mL), then washed with saturated sodium carbonate, water and brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the mixture was purified by silica gel chromatography, eluted with 20:1 dichloromethane / methanol to give a white solid. (520 mg, 83% yield). 1 H NMR (600MHz, CD3OD) δ8.23(d,J=5.4Hz,2H),7.60-7.56(m,4H),7.02(dd,J1=9.0Hz,J2=1.8Hz,2H),6.44(t,J=5.4Hz,2H),3.58( t,J=6.6Hz,2H),3.42(t,J=5.4Hz,4H),2.91(t,J=6.0Hz,4H),2.75-2.72(m,2H),1.66-1.57(m,4H),1.51-1.47(m,2H).ESI-HRMS 512.1961[M+H] + .

[0217]

[0218] Synthesis of DCQO. 4,7-Dichloroquinoline (DCQ) (2 g, 10 mmol) was dissolved in 50 mL and stirred vigorously (500 rpm) on an ice-water bath for 15 minutes. mCPBA (2.7 g, 12 mmol) was carefully added (4 times) to the reaction solution in an ice-water bath. The resulting reaction mixture was stirred (500 rpm) at room temperature for 12 hours. TLC showed that the starting material was completely converted to one main spot. Dichloromethane (100 mL) and potassium carbonate (4.1 g, 30 mmol) were added to the reaction solution, which was stirred (300 rpm) at room temperature for 1 hour. The mixture was poured into a 500 mL beaker containing 200 mL of water and stirred (300 rpm) for another hour. The organic phase was collected, washed with saturated sodium carbonate (75 mL×3), water (75 mL×3), and brine (75 mL×3), respectively, and dried over anhydrous sodium sulfate overnight. After filtration, the solvent was evaporated under reduced pressure, and the resulting crude material was recrystallized with 80 mL of acetonitrile. The resulting solid product was collected by filtration and then dried under vacuum to yield 1.8 g of DCQO as a white solid.1 HNMR(600MHz, CDCl3) δ8.79(d,J=1.8Hz,2H),8.44(d,J=6.6Hz,2H),8.44(d,J=8.4Hz,2H ),7.71(d,J1=9.0Hz,J2=2.4Hz,2H),7.38(d,J=6.0Hz,2H).HRMS(ESI): C9H6Cl2NO[M+H] + The calculated m / z value is 213.9821 and the found value is 213.9834.

[0219]

[0220] Synthesis of BAQO. Sodium bicarbonate (840 mg, 10 mmol) and diethylenetriamine (432 μL, 4 mmol) were added to a solution of DCQO (2.14 g, 10 mmol) in 30 mL of anhydrous ethanol. The mixture was refluxed at 95°C for 48 hours. TLC was used to indicate the production of the target substance (TM, yellow spot). The ethanol was evaporated under reduced pressure, and the residue was resuspended with 30 mL of methanol and slowly dripped into a 300 mL beaker containing a mixed solution of 100 mL of hydrochloric acid (1 M) and 50 mL of dichloromethane. The aqueous phase was collected, washed with dichloromethane (50 mL×2), and alkalized to pH 10 with 10 M NaOH (12 mL) to produce a yellow precipitate. The precipitate was collected, washed with water (30 mL×3) and dried in vacuo to obtain 850 mg of BAQO as a yellow solid. 1 HNMR (600MHz, CD3OD) δ8.51(d,J=1.8Hz,2H),8.35(d,J=7.2Hz,2H),8.14(d,J=9.0Hz,2H),7.55(d,J1= 9.0Hz, J2=2.4Hz, 2H), 6.63 (d, J=7.2Hz, 2H), 3.58 (t, J=6.0Hz, 4H), 2.94 (t, J=6.0Hz, 4H). HRMS (ESI): C 22 H 22 Cl2N5O2[M+H] + The calculated m / z value is 458.1145 and the found value is 458.1126.

[0221]

[0222] Synthesis of BAQ12O. A mixture of BAQO (916 mg, 2 mmol), dodecanal (1.8 mL, 8 mmol) and acetic acid (20 μL) was vigorously stirred (500 rpm) at room temperature for 30 minutes. Sodium cyanoborohydride (377 mg, 6 mmol) was then slowly added. The reaction mixture was stirred for another 12 hours at room temperature. TLC showed that the starting material was completely converted to one main spot. The solvent was concentrated to 25 mL, and the resulting residue was diluted with 75 mL of dichloromethane. The organic phase was washed three times with 100 mL of saturated sodium bicarbonate. The emulsion layer was collected and then filtered to obtain a yellow solid, which was washed with water (30 mL×3) and ether (30 mL×3). The collected yellow solid was dried in vacuo to obtain 1.2 g of BAQ12O. 1 HNMR(CD3OD,600MHz)δ8.43(d,J=1.8Hz,2H),8.31(d,J=7.2Hz,2H),7.82(d,J=9.0Hz,2H),7.31(d,J1=9.0Hz, J2=2.4Hz,2H),6.56(d,J=7.2Hz,2H),3.50(t,J=6.0Hz,4H),2.94(t,J=6.0Hz,4H),2.71(t,J=7.2Hz,2H),1.5 5(m,2H),1.32(m,18H),0.92(t,J1=6.6Hz,3H).CNMR(CD3OD,150MHz)δ148.0,140.1,139.9,138.4,127.6,123 .9,118.7,117.9,98.09,54.9,52.3,41.5,32.4,30.2,30.1,30.1,29.8,28.1,27.7,23.1,13.8.HRMS (ESI): C 34 H 46 C l2 The calculated m / z value of N5O2[M+H]+ is 626.3023, and the found value is 626.3060.

[0223]

[0224] Synthesis of BAQ1O. This compound was prepared using the method of BAQ12O. Formaldehyde was used as the starting material. ESI-HRMS 472.1325 [M+H] + .

[0225]

[0226] Synthesis of BAQAO. A solution of BAQO (229 mg, 0.5 mmol) in 5 mL of acetic anhydride was refluxed for 6 h. Excess acetic anhydride was removed under reduced pressure. The residue was dissolved in cold ether to give a yellow solid as the product (180 mg). ESI-HRMS 500.1246 [M+H] + .

[0227]

[0228] Synthesis of BAQ5hO. This compound was prepared using the method of BAQ5h. BAQO was used as the starting material. ESI-HRMS 544.1870 [M+H] + .

[0229]

[0230] Synthesis of BAQ4qO. This compound was prepared using the method of BAQ4q. BAQO was used as the starting material. ESI-HRMS 659.1946 [M+H] + .

[0231]

[0232] Synthesis of BAQ4aO. This compound was prepared using the method of BAQ4a. BAQ4qO was used as the starting material. ESI-HRMS 529.1884 [M+H] + .

[0233]

[0234] Synthesis of BAQ130. This compound was prepared using the method of BAQ120. Tridecaldehyde was used as the starting material. 1 HNMR(800MHz,DMSO-d6)δ9.79(s,2H),8.91(d,J=7.2Hz,2H),8.78(d,J=9.6Hz,2H),8.16(d,J=1.6Hz,2H),7.80(dd,J1=9.6Hz, J1=1.8Hz,2H),6.97(d,J=8.0Hz,2H),4.04(s,4H),3.68-3.62(m,6H),1.70(s,2H),1.27-1.17(m,21H),0.87(t,J=7.2Hz,3H).

[0235] Synthesis of BAQ14O. This compound was prepared using the method of BAQ12O. Tetradecaldehyde was used as the starting material. 1HNMR(800MHz,DMSO-d6)δ9.62(s,2H),8.88(d,J=8.0Hz,2H),8.70(d,J=9.6Hz,2H),8.15(d,J=2.4Hz,2H),7.77(dd,J1=8.8Hz, J1=1.6Hz,2H),6.95(d,J=7.2Hz,2H),4.02(s,4H),3.68-3.62(m,6H),1.68(s,2H),1.27-1.15(m,23H),0.86(t,J=5.4Hz,3H).

[0236] Synthesis of BAQ150. This compound was prepared using the method of BAQ120. Pentadecaldehyde was used as the starting material. 1 HNMR(800MHz,DMSO-d6)δ9.63(s,2H),8.88(d,J=8.0Hz,2H),8.70(d,J=8.8Hz,2H),8.15(d,J=2.4Hz,2H),7.77(dd,J1=8.8Hz, J1=1.6Hz,2H),6.95(d,J=8.8Hz,2H),4.01(s,4H),3.68-3.62(m,6H),1.68(s,2H),1.28-1.15(m,25H),0.86(t,J=5.4Hz,3H).

[0237] Synthesis of BAQ16O and BAQ18O. This compound was prepared using the method of BAQ12O. Hexadecanal was used as the starting material for BAQ16O and octadecanal was used as the starting material for BAQ18O.

[0238] Using the method of BAQ12O, additional BAQO derivatives can be prepared using appropriate aldehyde and ketone starting materials.

[0239] Example 2: Nanocarriers

[0240] Preparation and characterization of BAQ ONN. NPs were prepared by reprecipitation. A methanol solution of BAQ derivative was added dropwise to MilliQ water while stirring for 5 min (volume ratio, 1:10), and then uniform NPs were obtained after rotary evaporation (40 °C, 20 min), which were subsequently characterized by Zetasizer Nano ZS (Malvern Instruments). TEM samples were prepared by dropping 0.5 mM NPs on a carbon square mesh and dried naturally, and then observed under a Talos L120C TEM (FEI) at an accelerating voltage of 80 kV. In order to determine the drug content in the nanoformulation, the prepared drug-loaded NPs were cut off by a centrifugal filter (ultracel-10 kDa, Mil lipore), and the absorbance of the filtrate (diluted with DMSO, 1:10, volume ratio) was measured to calculate the drug concentration.

[0241] Discovery of BAQ derivatives as potential ONNs. BAQ12-BAQ18 were designed by hybridizing the key structural elements of the lysosomal autophagy inhibitor Lys05 and the lysosomal detergent MSDH to achieve pharmacological fusion ( Figure 1 Based on the principle of self-assembly, it is envisioned that the inclusion of a long hydrophobic tail with a cationic BAQ head would drive them to form nanoparticles (NPs). Since the calculated pKa of the BAQ head is 8.4, this self-assembly should be dependent on the pH of the environment, with NPs forming under neutral conditions and dissociating into free building blocks after protonation in an acidic environment.

[0242] Compounds (BAQ12-BAQ18) were synthesized and 1 H NMR spectrum, 13 C NMR and HRMS spectra confirmed the structure ( Figure 7 ). In contrast to Lys05 and MSDH, BAQ12-BAQ18 are not completely soluble in water either as free base or hydrochloride salt. However, the lipophilic cations allow spontaneous self-assembly in water via nanoprecipitation, resulting in a homogeneous milky white NP solution. The assembled NPs of BAQ12-BAQ18 have similar nanoscale features, including their size (100 nm-140 nm), polydispersity index (PDI) values ​​less than 0.1, and positive surface charge (approximately +40 mV) (Table 1 and Fig. 8A The pH-responsive dissociation behavior was then evaluated by monitoring the size changes of the particles ( Figure 2A). All BAQ NPs are intact under near-neutral conditions and dissociate only under relatively acidic conditions. The critical dissociation pH of BAQ12-BAQ14 is 5.5-6.0, and the critical dissociation pH of BAQ15-BAQ18 is 5.0-5.5. When protonated in an acidic environment, the BAQ12-BAQ18 lipophilic cations become amphiphilic molecules and acquire surface activity. The hemolysis test was then used to evaluate the pH-responsive biofilm disruption ability of the compounds. Under near-neutral conditions (pH ≥ 6.5), none of the NPs had a hemolytic effect, but when the pH was below 6.0, the NPs began to induce hemolysis ( Figure 2B and Figure 8B ). Among these compounds, BAQ12 NP and BAQ13 NP exhibited the strongest hemolytic activity, inducing up to 90% hemolysis under simulated lysosomal conditions (pH 4.0-5.5); in contrast, BAQ14 NP induced moderate hemolysis (70%), and BAQ15-BAQ18 NP produced only about 50% hemolysis. In the control group, the conventional lysosomal detergent MSDH showed only a weak hemolytic response to pH, and Lys05, which has no detergent ability, did not cause observable hemolysis at the same concentration and over the entire pH range. Because LMP is a potential stimulator of apoptosis, BAQ12 and BAQ13, whose detergents can be activated in lysosomes, may directly and effectively induce cancer cell death. In addition, after titration with hydrochloric acid (HCl), BAQ12 NP and BAQ13 NP showed a clear pH plateau in a narrow pH range (at about pH 6.0), indicating their strong pH buffering capacity ( Figure 2C ). In contrast, the pH values ​​of the other NPs (BAQ14-BAQ18) decreased proportionally, and only a brief pH plateau was observed for Lys05 (pH 7.2) and MSDH (pH 6.2). Since lysosomal degradation requires sufficient acidification, BAQ12 and BAQ13 were preferred due to their strong H + buffering capacity, showing a greater potential than other compounds to induce lysosomal dysfunction and thus potentially impair tumor cell growth.

[0243] Table 1: Nanoparticle characterization of BAQ derivatives and IC50 values ​​against cancer cells.

[0244]

[0245] To validate the therapeutic efficacy of BAQ12-BAQ18, a preliminary screening was performed on various cancer cell lines using the MTS assay. Within 24 h of treatment, these derivatives exhibited varying degrees of antiproliferative effects. BAQ12 and BAQ13 were highly potent and showed approximately 3-fold, 20-fold, and 10-fold greater potency than Lys05, HCQ, and MSDH, respectively, but the activity steadily decreased as the hydrophobic tail was extended from 14 to 18 carbons (Table 1 and Figure 8C ). This decrease is due to the detergency of the compound and the H + Based on the above results, BAQ12 and BAQ13 were then selected as representatives for constructing BAQ ONNs in subsequent studies.

[0246] pH-responsive assembly and high drug loading efficiency. The pH-responsive assembly-dissociation phase transition of BAQ ONNs was then determined by transmission electron microscopy (TEM). At pH 7.4, the NPs exhibited a strong Tyndall effect and displayed a liposome-like nanostructure with a diameter of approximately 100 nm and a bilayer thickness of approximately 5 nm ( Figure 2D ). These results are consistent with dynamic light scattering (DLS) measurements. In contrast, at pH 5.0, the solution loses its Tyndall effect and no vesicles are present under TEM, demonstrating that the NPs are dissociated under this condition ( Figure 2E Then, the release behavior of BAQ ONN at physiological pH (7.4) and lysosomal pH (5.0) was studied. Figure 2F As shown, BAQ12 NP and BAQ13 NP released almost completely (about 90%) within 8 h at pH 5.0, but only about 10% of the agent was released within 24 h under neutral conditions. Considering that lysosomes maintain the pH in the range of 4.0-5.5, it is believed that BAQ ONNs dissociate into free small molecules upon reaching these compartments, thereby exerting their therapeutic effects. The critical aggregation concentration (CAC) of BAQ12NP and BAQ13 NP was measured to be 0.76 μM and 0.25 μM, respectively ( Figure 2G The 3-fold difference observed between them suggests that BAQ13 can form NPs more easily than BAQ12, despite the difference of only one methylene unit between their molecular structures. Both NPs exhibited good particle size stability at room temperature for a relatively long duration, even in the presence of 10% serum or 0.5 mM bovine serum albumin ( 9A to 9F ). Moreover, BAQ13 NPs showed higher stability than BAQ12 NPs in this long-term storage, which may be due to their different CAC.

[0247] The next study was whether the liposome-like BAQ ONNs could encapsulate additional pharmaceutical agents. After nanoprecipitation of BAQ13 and various pharmaceutical agents, uniform NPs with a unimodal size distribution were spontaneously formed (Table 2 and Figure 9G BAQ13 NPs exhibited high drug loading (up to 50%, by mass) and drug encapsulation efficiency of approximately 90% (Table 2), suggesting that BAQ13 NPs can exceed the drug loading limits of conventional liposome-based and polymer-based drug delivery systems. It is very encouraging that these simple NPs composed of a single small molecule therapeutic entity exhibit such a strong drug loading capacity.

[0248] Table 2: Drug loading parameters using BAQ13 NPs.

[0249]

[0250] Accumulation in lysosomes and lysosomal destruction. To confirm the lysosomal accumulation of BAQ ONNs, the near-infrared fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldi-carbocyanine (DiD) was loaded for labeling and tracking. As expected, lysosomal spots (green) in MIA PaCa-2 cells stained with dextran-Alexa Fluor 488 (AF488) consistently overlapped with DiD-labeled NPs (red), indicating that BAQ ONNs were rapidly taken up by cells and accumulated in lysosomes ( Figure 3A and Fig. 10A After this accumulation, BAQ ONNs reduced LysoTracker-positive spots, showing their ability to deacidify lysosomes similar to Lys05 and MSDH ( Figure 3B and FIG. 10B to FIG. 10C ).

[0251] The induction of LMP by BAQ12 and BAQ13 was investigated by live cell staining with the dye acridine orange (AO). Compared with cells treated with Lys05 and MSDH, cells treated with BAQ12 NP or BAQ13 NP showed a reduced number of red spots and an increased ratio of green fluorescence to red fluorescence, indicating that BAQ ONN has an increased ability to induce lysosomal disruption in cancer cells. Figure 3C and FIG. 10D to FIG. 10E The LMP effect was further confirmed by detecting the release of dextran-AF488 from lysosomes. Figure 3D As shown, treatment with BAQ ONNs resulted in a diffuse staining pattern throughout the cytoplasm, indicative of lysosomal leakage, whereas fluorescence in control cells appeared confined to punctate structures, representing intact lysosomes. Using their LMP function, BAQ ONNs were shown to induce the release of cathepsin B from isolated lysosomes, an important trigger of apoptosis ( Figure 3ESince no LMP was observed in MSDH-treated cells, the results suggest that BAQ12 and BAQ13 represent the next generation of lysosomal detergents.

[0252] Autophagy inhibition. To explore the effects of BAQ ONN on autophagy, the levels of microtubule-associated protein 1 light chain 3 (LC3) and Sequestosome 1 (SQSTM1) / p62 proteins, which are commonly used to monitor changes during autophagy, were measured. During autophagy, the cytosolic form of LC3 (LC3-I) is converted into a lipid-modified form (LC3-II), which is then recruited to the autophagosome membrane. At the same time, the autophagic substrate SQSTM1 / p62 protein is degraded by selective incorporation into autophagosomes. Therefore, when autophagy is inhibited, increased levels of LC3-II and SQSTM1 / p62 should be observed, while if autophagy is activated, increased levels of LC3-II and decreased levels of SQSTM1 / p62 should be observed. Figure 3F to Figure 3G As shown, MIA PaCa-2 cells treated with BAQ ONN showed significant concentration-dependent increases in both LC3B-II and SQSTM1 / p62 protein levels compared to untreated cells and cells treated with Lys05. This increase was also observed after treatment with bafilomycin A1 (BfA1), a known autophagy inhibitor. These findings suggest that BAQ ONN can inhibit cellular autophagy more effectively than Lys05.

[0253] The autophagy inhibition was then confirmed by imaging with LC3B-GFP, as the formation of fluorescent LC3-II puncta in cells can be used to visualize the accumulation of autophagosomes. Cells treated with BAQ ONNs generated distinct LC3B-GFP puncta in a concentration-dependent manner ( Figure 3H and Fig.10F Quantification of LC3B-GFP puncta per cell revealed that BAQ ONNs were more potent in inhibiting autophagy than Lys05 ( Fig. 3I ). For further verification, TEM was used to monitor the micromorphological changes of the cells. As expected, BAQ ONN induced the formation of larger autophagic vesicles (AV) or autophagosomes in cells compared with Lys05 and MSDH, which further confirmed the improvement of BAQ ONN autophagy inhibition ( Figure 3J to Figure 3K ). In summary, the findings indicate that BAQ12 NPs and BAQ13 NPs outperform the parent Lys05 in inhibiting autophagy; thus, BAQ ONNs represent a new generation of nanoformulated autophagy inhibitors.

[0254] Proton sponge effect and lysosomal dysfunction. As cationic molecules, BAQ12 and BAQ13 have strong H + Buffering capacity, which is a fundamental characteristic of materials with proton sponge effect ( Figure 2C ). The above TEM results show that BAQ ONN can significantly enlarge lysosomes ( Figure 3J to Figure 3K ), demonstrating the proton sponge effect of BAQ ONN. To further investigate these effects, RNA sequencing (RNA-seq) was used to characterize the transcriptomic changes of MIA PaCa-2 cells after treatment. A total of 13,234 genes were tested, and their expression levels in the vehicle, Lys05, and BAQ13 groups were compared. Using volcano plot analysis, 165 differentially expressed genes (DEGs) were found in the Lys05 group compared with the vehicle group (fold change ≥ 2 and p-value ≤ 0.05), including 62 upregulated genes and 103 downregulated genes. In contrast, 390 DEGs were found in BAQ13-treated cells, including 209 upregulated genes and 181 downregulated genes ( Fig.11A Enrichment analysis of this gene set using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that BAQ13 NP induced a strong upregulation of lysosome-related genes, such as V-ATPase genes, Cl-channel genes, protease genes, and lysosomal-associated membrane protein (LAMP) genes ( FIG. 4A to FIG. 4C and Fig. 11B ). qPCR analysis also confirmed the upregulation of V-ATPase genes and Cl- channel genes, which significantly indicated that BAQ ONNs have strong proton sponge properties ( FIG. 4D to FIG. 4E ).

[0255] The upregulation of important lysosomal enzyme genes such as cathepsin and NEU1 also emphasized the lysosomal dysfunction caused by BAQ ONN ( FIG. 4B to FIG. 4C LAMP genes, which are thought to be partially responsible for maintaining lysosomal integrity, were also upregulated, suggesting a function for BAQ ONN in lysosomal disruption ( Fig. 11C BAQ ONN-treated groups showed high transcriptional levels of pro-apoptotic genes (BAX, BAK1, BAD, BIM, and PUMA), revealing an enhanced pro-apoptotic effect ( FIG. 11C to FIG. 11D BAQ13 NPs induced the accumulation of acid sphingomyelinase (ASM) precursor sphingomyelin (SM) and led to a decrease in the level of its product ceramide (Cer) ( Fig.11E In addition, the levels of phospholipase A (PLA) precursors including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PI) were decreased, and the levels of their corresponding products lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), and lysophosphatidylserine (LPI) were increased ( Fig.11F ).

[0256] The nanocarrier of the present invention may also include 1 It is a conjugate of pheophorbide a to form pheophorbide a bisaminoquinoline conjugate (PBC). PBC nanoparticles are lysosome-targeted, morphologically transformable nanoassemblies. PBC nanoparticles have a liposome-like morphology under physiological conditions and can be converted into nanofibers after accumulation in lysosomes. The nanofibers formed in lysosomes can cause lysosomal dysfunction and trigger apoptosis of cancer cells. Due to the inclusion of photosensitive groups in the structure, this nanoparticle also supports efficient lysosome-based photodynamic therapy, which can essentially overcome autophagy-related drug resistance.

[0257] Example 3: In vitro studies

[0258] Cell lines and cell culture. Human pancreatic cancer cell lines (MIA PaCa-2, BXPC3, and PANC-1) were originally purchased from ATCC and were kindly provided by the laboratory of Dr. Shiro Urayama. HT29, HCT116, H460, MCF7, NIH / 3T3, and IMR-90 cell lines were purchased from ATCC. Bone marrow cells were collected from the leg bone marrow of FVB / N mice. Cells were cultured according to ATCC protocols at 37°C in a humidified atmosphere of 5% CO2 / 95% air using 10% fetal bovine serum, 100 μg mL -1 Penicillin and 100 units mL -1 All cells were cultured in the appropriate medium containing streptomycin. All cell lines were routinely tested for mycoplasma contamination.

[0259] Establishment of patient-derived pancreatic cancer stem cells (PCSCs). Pancreatic patient tissue was donated by the laboratory of Dr. Shiro Urayama at the University of California, Davis (UC Davis) Medical Center. Patient consent for the use of “residual clinical biospecimens” was obtained in accordance with the Institutional Review Board (UC Davis IRB protocol #244896). Patient tumor tissue was collected using collagenase IV and dispase (Stem Cell Technologies, Vancouver, Canada) and filtered through a 70 μm filter. Cells were isolated by labeling with the following antibodies (Biolegend, CA, USA): anti-CD44 (IM7, catalog number 338823), anti-CD326 (9C4, catalog number 324207), and anti-CD24 (ML5, catalog number 311119), and using a BD FACSAria II cell sorter ( Fig.16). Purified PCSCs were collected and maintained in Essential 8Flex medium (Thermo Fisher Scientific) and trypsinized using a gentle cell dissociation reagent (Stem Cell Technologies, Vancouver, Canada). In the tumor sphere formation assay, cold single-cell suspensions of PCSCs in Essential 8Flex medium were mixed with cold Matrigel (1:1, volume ratio), and then they (100 μL) were slowly and evenly dripped into the center of the wells on a 24-well plate (5,000 cells per well). Matrigel was allowed to solidify in a humidified incubator at 37°C for 45 min-60 min, and warm culture medium (500 μL) was added to each well. Tumor spheres were allowed to form within two weeks. For in vivo tumorigenicity, PCSCs were counted and resuspended in a mixture of PBS and Matrigel (1:1) and subsequently injected subcutaneously into the flank of NRG mice.

[0260] Cell viability, cell growth and colony formation. Cell viability was assessed by MTS assay. Briefly, cells in 96-well plates (4,000 cells per well) were treated as indicated and then incubated with MTS reagent for 4 h. OD values ​​(490 nm) were measured by microplate reader. Results are shown as given by the formula [(OD 治疗 -OD 空白 ) / (OD 对照 -OD 空白 )×100%] calculated average cell viability. Drug combination data were analyzed by Combenefit 2.02. In the cell growth assay, cells in 6-well plates (50,000 cells per well) were treated as indicated and manually counted every 24 h. Colony formation assays were also performed on 6-well plates with a starting density of 1,000-2,000 cells per well. After incubation for 10-20 days as indicated, the cells were washed with PBS and stained with a solution of crystal violet and methanol for 20 minutes.

[0261] Apoptosis and caspase 3 / 7 activity. Cell apoptosis was measured using the FITC-Annexin V / PI apoptosis detection kit (AnaSpec). Briefly, treated cells were stained according to the manufacturer's instructions and detected on a BD FACSCanto II flow cytometer. Data were analyzed by FlowJo 7.6.1. In the caspase 3 / 7 activity assay, cells in 96-well plates (10,000 cells per well) were treated as indicated, followed by the addition of the AMC caspase 3 / 7 assay kit (50 μL per well, AnaSpec). Fluorescence intensity (λ) was recorded by a microplate reader. ex =356nm, λem =442nm).

[0262] Cellular uptake and deacidification. For cellular uptake, Alexa Fluor 488-dextran (10 kDa, 100 μg mL -1 Lysosomes were labeled with BAQ NPs (10 μM, 1:10, mass ratio) for 36 h and then incubated with DiD-loaded BAQ NPs (10 μM, 1:10, mass ratio) for 2 h. In the lysosomal deacidification assay, cells were treated for 2 h and incubated with LysoTracker Red (100 nM, Thermo Fisher) for 1 h. Cell images were obtained using a Zeiss confocal microscope and analyzed by Zen 2.3 and ImageJ 1.51s.

[0263] Lysosomal integrity. Lysosomal integrity in living cells was measured by staining with AO (Thermo Fisher Scientific) or AlexaFluor 488-dextran (10 kDa). For AO staining, treated cells were incubated with AO (2 μg mL -1 ) and incubated for 1 h. For dextran staining, cells loaded with dextran were exposed to treatment for 12 h. Images were captured under a Zeiss confocal microscope and analyzed by Zen2.3 and ImageJ 1.51s.

[0264] LC3B-GFP imaging. Cells in 96-well plates (5,000 cells per well) were transfected with the autophagy sensor LC3B-GFP (Thermo Fisher) for 12 h. After 4 h of treatment as indicated, cells were visualized by fluorescence microscopy (Olympus). Spots in each well were quantified using ImageJ 1.51s.

[0265] Lysosome isolation and cathepsin release. Lysosomes were isolated using a lysosome enrichment kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Aliquots of isolated lysosomes were incubated at 37°C for 12 h as indicated and then centrifuged at 15,000×g for 30 min at 4°C to pellet intact lysosomes. After incubation with 200 μM fluorescent cathepsin B substrate III (Z-Arg-Arg-AMC) for 2 h, the amount of cathepsin B released into the supernatant was determined (Ex=380 mm, Em=460 mm).

[0266] Hemolysis. Red blood cells (2%) dissolved in PBS (10 mM, pH 7.4) were incubated with NPs at 37°C for 4 h. After centrifugation at 500 × g for 5 min, the extent of hemolysis was determined spectrophotometrically based on the amount of hemoglobin (540 nm) in the supernatant. The hemolysis test was used to evaluate the pH-dependent decontamination ability and toxicity of the NPs.

[0267] Western blotting. Cells or tumor samples were lysed with RIPA buffer (Thermo Fisher Scientific). After centrifugation at 4°C (15 min, 12,000×g), the concentration of protein in the supernatant was determined by Bradford protein assay dye (Bio-Rad). Immunoblotting was performed routinely and ChemiDoc TM MP Imaging System was used for imaging.

[0268] TEM of cells and tumor tissues. MIA PaCa-2 cells (30,000 cells per well, Lab-Tek) in 8-well slides were treated for 48 h as indicated. Freshly harvested tumors were cut into 1 mm 3 The samples were fixed with 0.1 M cacodylate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde, transferred to carbon square grids, and then observed under a Talos L120C TEM.

[0269] RNA-seq. Total RNA was extracted from treated MIAPaCa-2 cells (5 μM, 24 h) using the RNeasy mini kit (Qiagen, Germany). Samples were submitted to the Genomics Shared Resource (GSR) of the UC Davis Comprehensive Cancer Center for RNA-Seq analysis. Stranded RNA-seq libraries were prepared from 100 ng of total RNA using the NEBNext Ultra Directional RNA Library Preparation Kit (New England BioLabs). Subsequently, the libraries were combined for multiplex sequencing (2×150 bp, double-end, >20×10 6 Reads / sample). Data of normalized gene read counts were analyzed using fold change and t-test. Differentially expressed genes (DEGs) were collected for signaling pathway enrichment by Funrich software 3.1.3. Gene sets were from the MSigDB database (Broad Institute). GSEA was performed online using GSEA version 3.0 in the KEGG gene set category with the following parameters: n = 1,000 permutations, with p-adjustment < 0.05 and FDR < 0.05 considered significant.

[0270] qPCR. Total RNA was isolated using TRIZOL reagent (Invitrogen) and phenol-chloroform extraction. cDNA was synthesized using 2 μg of total RNA in a 20 μL reaction using SuperScript II reverse transcriptase (Invitrogen). The resulting cDNA was diluted 1:20 in nuclease-free water, and 4 μL was used for each qPCR reaction in triplicate. qPCR was performed on a CFX96 real-time PCR detection system (Bio-Rad) including a non-template negative control using Power SYBR Green PCR premix (Thermo Fisher). Amplification of GAPDH was used to normalize the level of mRNA expression. Primer sequences are listed in Table 3.

[0271] Table 3: Primers and sequences used for RT-PCR analysis.

[0272]

[0273]

[0274] Lipidomics.MIA PaCa-2 cells were treated with compounds (2.5 μM) for 48 h, and 1.5 million cells per group were collected to prepare routine samples for RPLC-QTOF analysis. Samples were run on a Vanquish UHPLC system, followed by data acquisition using a Q-Exactive HF hybrid quadrupole-orbitrap mass spectrometer. LC-MS data were processed using MS-DIAL 3.70. Statistical analysis was performed by first normalizing the data using the sum of known values ​​or mTIC normalization to scale each sample. The normalized peak heights were then submitted to R 3.5.1 for statistical analysis. ANOVA analysis was performed with FDR correction and post hoc tests.

[0275] In vitro antitumor activity of BAQ derivatives. To systematically study the in vitro antitumor effect, three pancreatic cancer cell lines (MIA PaCa-2, BxPC-3, and PANC-1) and two colon cancer cell lines (HT29 and HCT116) were selected for 48 h MTS assay. BAQ ONN showed an IC of 1 μM to 3 μM. 50 values, and thus the potency is approximately 5, 30, and 20 times that of Lys05, HCQ, and MSDH, respectively ( Figure 4F , Fig.12 These results also showed that treatment with BAQ12 or BAQ13 alone was more effective than treatment with Lys05 and MSDH combined, indicating that significant pharmacodynamic synergy occurred when the pharmacophores were fused. The results of cell growth and colony formation assays further demonstrated the inhibitory effect of BAQ ONN on tumor cells ( Figures 4G to 4H ). The improved anticancer activity of BAQ ONNs can be attributed to their multiple functions of inducing LMP, lysosomal dysfunction, and autophagy inhibition in cancer cells; these effects are considered to be important triggers of apoptosis. To examine the pro-apoptotic effects of BAQ ONNs, apoptotic signals were subsequently detected in MIA PaCa-2 and HT29 cells. BAQ ONN treatment resulted in a significant increase in caspase 3 / 7 activity and apoptosis levels ( FIG. 4I to FIG. 4J ). The control Lys05 increased apoptotic signal in a concentration-dependent manner, but its 50 The effects at high concentrations of BAQ ONN were still milder than those at low concentrations of BAQ ONN. These results suggest that cancer cells are more likely to undergo apoptosis when treated with the multifunctional BAQ entity than when treated with Lys05, whose primary function is autophagy inhibition. In addition, non-cancer cell lines, including IMR-90 cells, NIH / 3T3 cells, and bone marrow cells, showed relative insensitivity to BAQ ONN compared to a panel of cancer cell lines, indicating the relatively high safety profile of these compounds ( Fig.12 and Table 4).

[0276] Table 4: Calculated IC50 values. Data are presented as mean ± SD.

[0277]

[0278] In vitro antitumor activity of BAQO derivatives. The in vitro antitumor effect of BAQO derivatives was investigated in pancreatic cancer stem cells (PCSCs). BAQ12O ONN showed an IC of less than 5 μM 50 value( Fig. 22B ), while other BAQO derivatives showed no toxicity at concentrations up to 100 μM. These results suggest that BAQO derivatives can be versatile and used as therapeutic agents or drug delivery agents without causing cytotoxicity.

[0279] Example 4: In vivo studies

[0280] Animal model. To establish a subcutaneous xenograft model, 5 × 10 6 MIA PaCa-2 cells, 2×10 6 HT29 cells or 2 × 10 4 PCSCs were injected subcutaneously into the right flank of nude or NRG mice.

[0281] Animal husbandry. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of California, Davis (#20265). Female mice (4-6 weeks) including BALB / c nude mice (Envigo), NRG mice (Jackson Laboratory), and FVB / N mice (Charles River) were purchased and group-housed under standard conditions (22 ± 1 °C, 50%-60% humidity, 12 h light / 12 h dark cycle, free access to food and water).

[0282] In vivo treatment plan. MIA PaCa-2 xenograft tumors (approximately 100 mm 3 ) were randomly divided into 5 groups (n=6) and then injected iv every three days as indicated. For the HT29 xenograft model, 100 mm 3 Six groups of nude mice (n=6) with tumors were administered vehicle (saline, iv), Lys05 (ip), BAQ12 NP (iv), BAQ13 NP (iv), and irinotecan (ip). Treatment of the HT29 model was stopped on day 24, and then the survival of each group of mice was recorded. 3 Mice with tumors were considered dead. For co-delivery studies, NRG mice bearing PCSC tumors (n=5) were treated every three days with vehicle (saline, iv), nabucasin (ip), BAQ13 NPs (iv), BAQ13 NPs + nabucasin (iv and ip, respectively), and BAQ13 NPs @ nabucasin (iv). Tumor volume and body weight were recorded before each drug administration. At the end of treatment, mice were sacrificed and tumors were collected for further analysis.

[0283] In vivo toxicity study. The toxicity of BAQ NPs was studied in female FVB / N mice by iv injection. The mice were administered various concentrations (10 mg kg -1 , 20mg kg -1 or 40 mg kg -1 ) of Lys05, liposome@Lys05, BAQ12 NP and BAQ13NP. The status of the mice was monitored daily and their body weight was recorded every two days. Blood samples were collected and sent to the UCD Comparative Pathology Laboratory for complete blood count (CBC) and serum chemistry tests.

[0284] In vivo pharmacokinetic study. A catheter for drug injection and blood collection (Harland, Indianapolis, IN, USA) was implanted in the jugular vein of female Sprague-Dawley rats (200 g-250 g). Rats (n = 3) were injected with free DiD, BAQ12 NP@DiD (10:1, mass ratio) and BAQ13 NP@DiD (10:1, mass ratio) containing an equivalent dose of DiD (0.5 mg kg -1 ). Blood samples were collected at designated time points and then centrifuged to obtain plasma. Plasma was diluted with DMSO (1:100) and the fluorescence intensity (λ Ex =595nm,λ Em =665nm).

[0285] In vivo / in vitro biodistribution. Nude mice bearing HT29 tumors were treated with 1.0 mg kg -1 BAQ13NP@DiD (10:1, mass ratio) was administered iv. In vivo imaging studies were performed at corresponding time points. Organs (brain, heart, lung, liver, spleen, kidney, intestine, and muscle) and tumors were collected from mice for ex vivo imaging. BAQ13 NP@NAPA+DiD (10 / 2.5 / 1.0 mg kg -1 , iv) Biodistribution. In vivo and ex vivo imaging studies were performed as described above.

[0286] Statistics. Statistical analysis was performed using GraphPad Prism 7.0. Data are presented as mean ± SD, n = biological replicates or independent nanoparticle sample replicates. p values ​​were calculated using one-way ANOVA with Tukey's multiple comparison test or two-tailed Student's t-test as indicated in each figure legend. ns., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0287] Data availability. RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession code GSE154323 [https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE154323].

[0288] Pharmacokinetics, Biodistribution, and Toxicity. The pharmacokinetics of BAQ ONN were studied in Sprague-Dawley rats by intravenous (iv) injection. Figure 5AAs shown in Table 5, the serum concentration of BAQ ONN was higher than that of free DiD at the same time points until 48 h, indicating that the plasma clearance of BAQ ONN was slower than that of DiD due to the nanoscale characteristics of BAQ ONN. DiD-labeled BAQ13 NPs were also used to study the biodistribution of NPs in nude mice bearing HT29 tumors. As expected, both in vivo and ex vivo imaging showed that the fluorescence signal of BAQ13 NPs was clearly discernible in the tumor area but not in the surrounding normal tissues at 12 h and 24 h after injection, indicating the tumor-targeted biodistribution of BAQ ONN ( FIG. 5B to FIG. 5C and FIG. 13A to FIG. 13B This targeting ability may be due to the relatively high permeability of tumor blood vessels, which enables the passive accumulation of nanotherapeutics. The free DiD (control) group showed high signals in the lungs but not in the tumor sites ( FIG. 13A to FIG. 13B ). In particular, the fluorescence ratio of tumor to lung in the BAQ13 NP group was about 4 times that in the free DiD group. These results indicate that BAQ ONN has tumor-targeted biodistribution.

[0289] Table 5. Pharmacokinetic parameters calculated using Kinetica 5.0.

[0290]

[0291] Then, a hemolysis test was performed to evaluate the safety of BAQ ONN. -1 -1mg mL -1 Lys05, BAQ12 NP, or BAQ13 NP were used to treat erythrocytes, close to the working concentration used in animal treatment studies ( Fig. 13C ). Use with concentrations higher than 0.5 mg mL -1 Compared with the treatment with BAQ12 NP or BAQ13NP, the treatment with the control drug Lys05 resulted in a significantly higher hemolysis rate, indicating that BAQ ONN is safer than Lys05. In the following animal toxicity study on FVB / N mice, it was found that after iv administration, even at 10 mg kg -1 Lys05 treatment also resulted in acute death of mice at low concentrations; in contrast, BAQ ONN treatment resulted in low mortality and no weight loss, revealing that BAQ ONN is safe when administered by iv injection ( FIG. 13D to FIG. 13E ). Since BAQ13 NP is at 40 mg kg -1BAQ13 NPs were better tolerated by mice than BAQ12 NPs, as they did not cause any mortality. This result may be due to the high stability and low CAC of BAQ13 NPs. Liposomes were used to encapsulate Lys05 (Liposome@Lys05), and the formulation was found to be safe for iv injection ( FIG. 13E to FIG. 13F Because autophagy plays an important role in intestinal homeostasis, intraperitoneal (ip) administration of BAQ12 NP or BAQ13 NP, which results in enhanced autophagy inhibition, may cause intestinal disturbances and weight loss in mice. - 1 H&E staining of tissue sections and hematological indices did not show obvious abnormal changes in BAQ NP-treated mice, further indicating that iv administration of BAQ ONN was well tolerated ( FIG. 14A to FIG. 14E ). Therefore, BAQ ONNs should be administered by iv injection rather than ip injection to study their advantages in vivo.

[0292] Antitumor effect in mice as a single agent. After demonstrating the safety of BAQ ONN, the anticancer efficacy of NP was evaluated in a pancreatic xenograft model of MIA PaCa-2 cells. 3 ) were randomly divided into five groups (n=6): saline (iv) group, Lys05 (ip) group, liposome@Lys05 (iv) group, BAQ12NP (iv) group and BAQ13NP (iv) group. Then, 20 mg kg -1 The mice were treated with a dose of FIG. 5D to FIG. 5F The results in showed that treatment with BAQ12 NPs or BAQ13NPs significantly slowed tumor growth without interfering with body weight. The control drug Lys05 showed no therapeutic effect under this condition, but its nanoformulation liposome@Lys05 caused increased tumor inhibition, which highlights the advantages of nanomedicines in drug delivery. It should also be emphasized that the single-component formulations of self-assembled BAQ12 NPs or BAQ13 NPs were significantly more effective than free Lys05 or nanoformulated Lys05. These findings clearly illustrate the potential advantages of BAQ ONN in drug discovery and drug delivery.

[0293] To further understand the in vivo effects of BAQ ONN, tumor tissues were harvested for histological evaluation. Significant cell destruction, increased levels of cleaved caspase-3, and decreased Ki67 expression were observed in both BAQ ONN groups, indicating that tumors treated with BAQ ONN tended to die or become apoptotic or quiescent ( Figure 5G to Figure 5H LC3 expression was increased in both BAQ ONN groups ( Figure 5H); This finding is an important clue to explain the in vivo autophagy inhibition effect of the two BAQ ONNs. Subsequent immunoblot analysis further demonstrated that autophagy in tumors was blocked by BAQ ONN. In addition, tissue ultrastructure was observed by TEM and it was found that tumors treated with BAQ ONN contained more large AVs than the tumor group. In the above assays, the Lys05 nanoformulation liposomes@Lys05 also exhibited some effects that were not observed with solvent or free Lys05. However, the effect of liposomes@Lys05 was much weaker than that of BAQ12 NP or BAQ13 NP. These tissue-level results reveal the excellent autophagy inhibition effect of BAQ ONN in vivo.

[0294] The therapeutic effect of BAQ ONN in vivo was further demonstrated in another animal model consisting of mice bearing colon HT29 tumors. BAQ ONN administration significantly inhibited tumor growth compared to vehicle or Lys05 administration ( FIG. 15A to FIG. 15B ). And BAQ13 NPs showed better efficacy than BAQ12 NPs. Interestingly, this result contradicts the results obtained in the in vitro proliferation assay, which demonstrated that BAQ12 NPs were more effective than BAQ13 NPs. This discrepancy could be explained by the differences in self-assembly behavior and pharmacokinetic properties between BAQONNs ( Figure 2G , Figure 5A ). In addition, BAQ13 NP was also more effective than FDA-approved irinotecan at reported therapeutic doses, while BAQ12 NP showed similar effects to irinotecan. Survival analysis revealed that BAQ13 NP treatment resulted in significantly longer survival (median survival of 48 days) than the vehicle group and irinotecan group (median survival of 21 days or 36 days, respectively) ( Fig. 15C and Table 6). Due to the integration of their multiple advantages in pharmacodynamic effects and pharmacokinetic properties, hybrid BAQ ONNs show great potential in in vivo cancer treatment as a single agent.

[0295] Table 6: Median survival of mice bearing HT29 tumors. n = 6 mice per group.

[0296]

[0297] Dual Role of BAQ ONN in Combination Therapy. Combination therapy based on autophagy inhibition can sensitize tumors to conventional treatments, but the current limitation is the insufficient efficacy of autophagy inhibitors. In addition, the different pharmacokinetics and different dosing regimens of the drugs used in combination therapy are inconvenient. Considering the 30-fold higher anticancer potency of BAQ ONN than HCQ and their considerable potential to encapsulate additional drugs, BAQ ONN may be able to simultaneously address both pharmacodynamic and pharmacokinetic issues. To test this hypothesis, a xenograft model with high heterogeneity and high tumor stroma ratio was established by using a pancreatic cancer stem cell (PCSC) line from patient-derived pancreatic adenocarcinoma tissue ( FIG. 6A to FIG. 6B and Fig.16 In vitro results showed that BAQ ONNs have similar functions in inhibiting lysosomes and autophagy in PCSCs, thus exhibiting potent pro-apoptotic and anti-proliferative activities ( FIG. 6C to FIG. 6E and 17A to 17C ). Nabucacin, a STAT3 inhibitor that can be encapsulated in BAQ13 NPs, was selected for combination therapy because it can induce autophagy and synergize with BAQ13 NPs ( Fig. 6F and Fig.17D The mice were randomly divided into 5 groups, including vehicle (saline) group, nabucasin group, BAQ13 NP group, mixture (BAQ13 NP+nabucasin) group and BAQ13 NP@nabucasin group ( FIG. 6G to FIG. 6I ). BAQ13 NPs modestly inhibited tumor growth, while nabucasin itself did not show antitumor effects under these conditions. Although in vitro synergistic effects of nabucasin and BAQ13 NPs were observed, the mixed group did not show enhanced effects in vivo. This lack of in vivo effect may be due to the poor solubility and low delivery efficiency of nabucasin. When loaded in BAQ13 NPs (BAQ13 NP@nabucasin), nanoformulated nabucasin achieved satisfactory antitumor effects by synergizing with BAQ13 NPs. Significant changes in tumor histology were also observed in the BAQ13 NP@nabucasin group, with cells showing low proliferation activity ( Figure 6J In addition, no mice in the treatment group showed significant systemic toxicity ( Fig.6I and Fig.17E To further validate the ability of BAQ13 NPs to deliver nabucasin, another imaging study was performed on the PCSC model by using DiD-labeled BAQ13 NP@nabucasin. The results showed that NPs accumulated significantly at tumor sites rather than in normal organs ( Figures 6J to 6K and Fig.17F ). These interesting results suggest that BAQ13 NPs may be used not only as therapeutic agents but also as delivery vehicles in combination therapies; thus, they show promise for improving cancer treatment.

[0298] Antitumor effects of BAQO derivatives in mice. BAQO derivatives can be formed into nanoparticles and used in in vivo mouse studies. For example, BAQ12O NPs were used to treat mice bearing PCSC tumors. Fig.24A As shown, mice treated with BAQ100 NPs had lower tumor volumes, with significant differences in tumor volume by day 27. Fig. 24B The tumor weight at the end of treatment with BAQ12O NPs was shown to be 50% lighter than that of the control. These interesting results suggest that BAQO derivatives can be used as promising agents for drug discovery and drug delivery.

[0299] Based on the ONN strategy and the principles of pharmacophore hybridization and molecular self-assembly, self-delivering new chemical entities BAQONN were developed. In addition to the properties of improved drug delivery and tumor-targeted biodistribution, these entities also have enhanced abilities to induce lysosomal disruption, lysosomal dysfunction, and autophagy blockade; thus, they exhibit significant anticancer efficacy both in vitro and in vivo. Strikingly, simple BAQ13 NPs were found to display high drug loading efficiency and can effectively synergize and deliver additional drugs, showing promise for use in combination therapies.

[0300] Compared to conventional NPs, which typically have less than 20% active pharmaceutical ingredient (API) content and are complex to synthesize, BAQONNs have 100% API content and are easy to synthesize and scale up. Since they are non-prodrug chemical entities, they are also superior to emerging single-component prodrug NPs. All of these advantages will greatly facilitate their translation into clinical trials. This is an important attempt to expand nanotechnology into the design of new chemical entities. The seamless connection between drug discovery and nanotechnology-assisted drug delivery will enable researchers to develop increasingly advanced nanomedicines that have a wide range of therapeutic and commercial benefits for cancer targeting.

[0301] Although the foregoing invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, it will be appreciated by those skilled in the art that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were incorporated by reference individually. In the event of a conflict between the present application and the references provided herein, the present application shall prevail.

Claims

1. A compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: R 1 For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c replace; W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl substitution; Each L is independently absent, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene; each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, photosensitizer, porphyrin, chemotherapeutic drug, sterol, C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl having 1 to 4 heteroatoms each independently being N, O or S, C 6-12 Aryl, 5- to 12-membered heteroaryl having 1 to 4 heteroatoms each independently being N, O or S, -OH or -NH2; R 1a C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-40 Alkoxy, hydroxyl or –NR 1b R 1c replace; R 1b For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl or C 2-40 Alkynyl; R 1c For hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl or –LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-40 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, halogen, -CN or -NO2; m and n are independently an integer from 1 to 10; p is independently an integer from 1 to 20; and Each X is independently absent or -0 - ， When X does not exist, R 2a and R 2b is hydrogen, and R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-40 Alkyl, C 2-40 Alkenyl, C 4-40 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a ,and When X does not exist, R 1 is –CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-40 Alkenyl, C 2-40 Alkynyl, C 1-40 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a , wherein each alkyl, alkenyl and alkynyl group is optionally replaced by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c replace; W is C 3-12 Cycloalkyl, C 6-12 aryl or 5 to 12 membered heteroaryl, wherein the 5 to 12 membered heteroaryl has 1 to 4 heteroatoms which are N, O or S, and wherein each cycloalkyl, aryl and heteroaryl is optionally replaced by C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl substitution; Each L is independently absent, C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 Alkynylidene; each Y is independently absent, -O-, -NH-, -NHC(O)-, -NHC(O)NH-, -NHSO2-, -OC(O)-, -OC(O)NH-, -C(O)-, or -SO2-; Z is a fluorophore, a photosensitizer, a porphyrin, a chemotherapeutic drug, or a sterol; R 1a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, wherein each alkyl, alkenyl and alkynyl group is optionally substituted by C 1-20 Alkoxy, hydroxyl or –NR 1b R 1c replace; R 1b For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl; R 1c For hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl or –LW; R 2a , R 2b , R 3a and R 3b are independently hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 Alkoxy, halogen, -CN or -NO2; m and n are independently an integer from 1 to 10; p is independently an integer from 1 to 20; and Each X is independently absent or -0 - ， When X does not exist, R 2a and R 2b is hydrogen, and R 3a and R 3b are each independently hydrogen, -OMe, fluorine, chlorine, bromine or -NO2, then R 1 C 2-20 Alkyl, C 2-20 Alkenyl, C 4-20 Alkynyl, -W, -(LY) p -Z or –C(O)R 1a ,and When X does not exist, R 1 is –CH2CH2NH(7-chloro-4-quinolyl), and R 2a and R 2b is hydrogen, then R 3a and R 3b are independently selected from hydrogen, C 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 1-20 Alkoxy, fluorine, bromine, iodine, -CN or -NO2.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 C 1-40 Alkyl, C 2-40 Alkenyl, -(LY) p -Z or –C(O)R 1a .

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 1 C 1-25 alkyl.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein R 1 C 10-25 alkyl.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R 1 C 12-18 alkyl.

7. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 1 C 20-40 Alkenyl.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein R 1 C 30-40 Alkenyl.

9. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 1 -(LY) p -Z. 10 . The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein p is 1.

11. The compound according to claim 9 or 10 or a pharmaceutically acceptable salt thereof, wherein L is C 1-10 Alkylene. 12 . The compound or pharmaceutically acceptable salt thereof according to claim 9 , wherein Y is absent, —NH—, —NHC(O)—, or —NHC(O)NH—.

13. A compound according to any one of claims 9 to 12, or a pharmaceutically acceptable salt thereof, wherein Z is a porphyrin, a sterol, a 6- to 12-membered heterocycloalkyl, an 8- to 12-membered heteroaryl, -OH or -NH2, wherein the 6- to 12-membered heterocycloalkyl and the 8- to 12-membered heteroaryl have 1 to 4 heteroatoms which are N, O and S.

14. The compound according to any one of claims 9 to 13 or a pharmaceutically acceptable salt thereof, wherein: p is 1; L is C 4-5 Alkylene; Y is absent, -NH- or -NHC(O)-; and Z is porphyrin, cholic acid, isoindoline, phthalimide, -OH or -NH2.

15. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 1 for –C(O)R 1a .

16. The compound according to claim 15 or a pharmaceutically acceptable salt thereof, wherein R 1a C 1-10 alkyl.

17. A compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof, wherein R 2a and R 2b Each is independently hydrogen, fluorine, chlorine, bromine or iodine.

18. A compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, wherein R 2a and R 2b are each independently hydrogen.

19. A compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, wherein R 3a and R 3b Each is independently hydrogen, fluorine, chlorine, bromine or iodine.

20. A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein R 3a and R 3b are each independently chlorine.

21. The compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein m and n are each independently an integer from 1 to 5.

22. The compound according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof, wherein m and n are each independently 1.

23. The compound according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (Ia):

24. A compound according to any one of claims 1 to 6 or 17 to 23, or a pharmaceutically acceptable salt thereof, wherein the compound has the following structure: wherein n is an integer from 1 to 7.

25. A compound according to any one of claims 1 to 6 or 17 to 24, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

26. A compound according to any one of claims 1 to 3, 9 to 14 or 17 to 23, or a pharmaceutically acceptable salt thereof, wherein the compound is 27. A compound according to any one of claims 1 to 3, 9 to 14 or 17 to 23, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

28. A compound according to any one of claims 1 to 3 or 4 to 22, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (Ib):

29. The compound according to claim 28 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

30. The compound according to claim 28 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

31. A nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds according to any one of claims 1 to 30, or pharmaceutically acceptable salts thereof, wherein each compound self-assembles in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed in the interior of the nanocarrier and hydrophilic groups self-assemble on the exterior of the nanocarrier.

32. The nanocarrier of claim 31 , wherein the nanocarrier further comprises one or more hydrophobic drugs or imaging agents sequestered in the hydrophobic pocket of the nanocarrier.

33. The nanocarrier of claim 32, wherein the hydrophobic drug is a chemotherapeutic agent, a molecular targeting agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof.

34. The nanocarrier of claim 33, wherein the hydrophobic drug is the immunotherapeutic agent.

35. The nanocarrier of claim 33, wherein the hydrophobic drug is the radiotherapeutic agent.

36. The nanocarrier of claim 33, wherein the hydrophobic drug is the chemotherapeutic agent or a molecular targeting agent.

37. The nanocarrier of any one of claims 32 to 36, wherein the hydrophobic drug is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a MAP kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, albumin paclitaxel (abraxane), TLK286, AV-299, DN-101, pazopalanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinib, nilotinib, decatanib, panitumumab, amrubicin, ogavuzumab, Lep-etu, nolatrexed, AZD2171, batabulin, ofatumumab, zalimumab, etakaline, tetrandrine, rubitecan, tesmilifen, oblimersen, tesimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR; INO 1001, IPdR1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposome doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidine [(5-(methylsulfonylpiperidinylmethyl)-indolyl)-oxazolidinone]-disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperidinylmethyl)-indolyl-oxazolidinone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t)6,Azgly 10] acetate (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84 N 18 O 14 -(C2H4O2)X, wherein X=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, cyclohexyl lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide , oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozotocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, retinoic acid, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uramustine, estramustine, hexamethylmelamine, floxuridine, 5-deoxyuridine, cytarabine, 6-thiopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EM D121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimetidine, trastuzumab, denileukin, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without hydrogenated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipenoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxyfene, TSE-424, HMR-3339,ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim (filgras tim), darbepoietin alfa, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone pine, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol acetate, immunoglobulin, nitrogen mustard, methylprednisolone, ibritumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netupitant, NK- 1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabitril, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilimumab, vemurafenib, or a combination thereof.

38. The nanocarrier of any one of claims 31 to 37, wherein the nanocarrier comprises a plurality of compounds of any one of claims 24 to 30.

39. A method of treating a disease, comprising administering a therapeutically effective amount of the nanocarrier of any one of claims 31 to 38 to a subject in need thereof.

40. The method of claim 39, further comprising one or more additional agents, wherein the additional agent is a chemotherapeutic agent, a molecular targeted agent, an immunotherapeutic agent, a radiotherapeutic agent, or a combination thereof.

41. The method of claim 40, wherein the additional agent is the immunotherapeutic agent.

42. The method of claim 40, wherein the additional agent is the radiotherapeutic agent.

43. The method of claim 40, wherein the additional agent is the chemotherapeutic agent or a molecular targeted agent.

44. The method of any one of claims 40 to 43, wherein the additional agent is a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an Aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK / STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a MAP kinase (mek) inhibitor, a VEGF capture antibody, everolimus, trabectedin, albumin paclitaxel (abraxane), TLK286, AV-299, DN-101, pazopalanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinib, nilotinib, decatanib, panitumumab, amrubicin, ogavuzumab, Lep-etu, nolatrexed, AZD2171, batabulin, ofatumumab, zalimumab, etakaline, tetrandrine, rubitecan, tesmilifen, oblimersen, tesimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR; INO 1001, IPdR1 KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposome doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidine [(5-(methylsulfonylpiperidinylmethyl)-indolyl)-oxazolidinone]-disodium salt heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258; 3-[5-(methylsulfonylpiperidinylmethyl)-indolyl-oxazolidinone, vatalanib, AG-013736, AVE-0005, [D-Ser(Bu t)6,Azgly 10] acetate (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH2 acetate [C. 59 H 84 N 18 O 14 -(C2H4O2)X, wherein X=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, canertinib, ABX-EGF antibody, Erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, Gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, cyclohexyl lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide , oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozotocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, retinoic acid, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uramustine, estramustine, hexamethylmelamine, floxuridine, 5-deoxyuridine, cytarabine, 6-thiopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EM D121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimetidine, trastuzumab, denileukin, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without hydrogenated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipenoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxyfene, TSE-424, HMR-3339,ZK186619, PTK787 / ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim (filgras tim), darbepoietin alfa, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone pine, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol acetate, immunoglobulin, nitrogen mustard, methylprednisolone, ibritumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, edwina-asparaginase, strontium 89, casopitant, netupitant, NK- 1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabitril, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilimumab, vemurafenib, or a combination thereof.

45. The method of claim 39, wherein the disease is cancer.

46. ​​The method of claim 45, wherein the cancer is bladder cancer, brain cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, intestinal cancer, head and neck cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, and uterine cancer.

47. The method of claim 39, wherein the disease is coronavirus, malaria, antiphospholipid antibody syndrome, lupus, rheumatoid arthritis, chronic urticaria, or Sjögren's syndrome.

48. The method of any one of claims 39 to 47, wherein the treatment method targets lysosomal destruction, lysosomal dysfunction and / or autophagy inhibition.

49. The method of any one of claims 39 or 48, wherein the treatment method targets the lysosome.

50. A method of imaging, comprising administering to a subject to be imaged an effective amount of a nanocarrier according to any one of claims 31 to 38.

Images