Small molecule inhibitors of Id protein

Abstract

The present technology relates generally to compounds, compositions, and methods useful for treating, preventing, and / or ameliorating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic vascular proliferative disease in a subject.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 909,036, filed on October 1, 2019, the content of which is incorporated herein by reference in its entirety for all purposes. Support of the U.S. government This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in this invention. This technology generally relates to compounds, compositions, and methods that inhibit Id (inhibitor of differentiation) proteins useful for treating, preventing, and / or ameliorating pathogenic cell proliferation, angiogenesis, cancer, metastatic diseases, and / or pathogenic angiogenic diseases in a subject.

Summary of the Invention

[0002] In one aspect, the present disclosure provides a compound according to Formula I

Chemical formula

[0003] In related embodiments, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula I and a pharmaceutically acceptable carrier for treating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease in a subject.

[0004] In one embodiment, a method is provided for treating a condition of a subject, comprising administering to the subject an effective amount of a compound of formula I, wherein the condition includes one or more of pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease. Further aspects and embodiments of this technology are described herein. [Brief explanation of the drawing]

[0005] [Figure 1A-1C]We demonstrate that gene deletion of Id1 or Id3 suppresses angiogenesis in Id1- / - (Figure 1A) or Id3- / - (Figure 1B) mice, and littermates of control Id1+ / + or Id3+ / + (n=12 in each group), of the exudative age-related macular degeneration (AMD) mouse model characterized by laser-induced rupture of Bruch's membrane in three locations in one eye. After 14 days, the mean area of ​​choroidal neovascularization (NV) was significantly smaller in Id1- / - or Id3- / - mice than in the corresponding controls (* indicates p<0.03 by unpaired t-test, and error bars represent SEM). Figure 1C shows ischemia-induced retinal neovascularization in wild-type, Id1- / -, or Id3- / - mice. Id1- / - or Id3- / - mice and littermate control Id1+ / + or Id3+ / + mice were placed in a 75% O2 chamber for 5 days on postnatal day 7 to induce retinopathy of prematurity (ROP). Animals were euthanized on postnatal day 17, and the area of ​​retinal neovascularization was evaluated by Griffonia simplicifolialectin staining (selective for vascular cells) of choroidal flat mounts (representative images of staining are shown) (white scale bar = 500 μm). Quantification of the mean total area of ​​NV per group is plotted with n = number of offspring, * indicates p < 0.0001 by unpaired t-test, and error bars represent SEM. [Figure 2A-2G]The identification of AGX51 is shown. Figure 2A shows the crystal structures of Id1 and E47, with hydrogen bonds indicated by black dashed lines, salt bridges by gray dashed lines, and the residues involved labeled. Figure 2B shows the hydrophobic gap analysis of Id1. Black arrows and gray areas indicate identified gaps. Figure 2C shows the in vitro electrophoretic mobility shift assay (EMSA) of compounds A, B, and C. The wedge shape indicates an increase in small molecule concentration from 1 to 100 μM: lanes 3-5: compound A at 20, 50, and 100 μM; lanes 6-8: compound B at 20, 50, and 100 μM; lanes 10-16: compound C at 1, 5, 10, 20, 30, 50, and 100 μM. Figure 2D shows the structures of compounds A, B, and C from Figure 2C, where C is AGX51, and the red asterisks indicate the chiral center. Figure 2E shows the prediction of the AGX51 docking site (shown in gray) in the Id1 HLH domain (shown in green). Figure 2F shows the prediction of Id1 residues interacting with AGX51. Figure 2G shows the circular dichroism (CD) of Id1 using DMSO (left plot), or AGX51 (0, 10, 20, and 50 μM) (center plot), and E47 (0, 10, 20, and 50 μM) with AGX51 (right plot). See also Figures 8A-8C and 8A-8C and 8A-8C. [Figure 3A-3D] The effects of AGX51 on ID protein levels, E protein interactions, and ID1 ubiquitination are shown. Figure 3A shows Western blots of ID1 and ID3 in whole cell lysates from HUVEC treated with 0–40 μM AGX51 for 24 hours. Figure 3B shows a Western blot of Flag in whole cell lysates from HCT116 cells (expressing Flag-tagged ID1) treated with 60 μM AGX51 for 0–24 hours. Figure 3C shows ubiquitination assays in U87MG and HCT116 cells treated with MG132 and AGX51. Figure 3D shows immunoprecipitation (IP) of endogenous ID1 and E47 in HCT116 cells treated with 60 μM AGX1 for 1 hour, with the corresponding immunoblot of whole cell lysates to the right of the IP blot. See also Figures 11A–11C. [Figures 4A-4F] The effect of AGX51 on HUVEC proliferation is shown. Figure 4A shows cell proliferation of HUVEC treated with DMSO or 20 μM AGX51 for 5 days. Figure 4A shows cell cycle analysis of HUVEC treated with DMSO or 20 μM AGX51 for 24 hours. Figure 4C shows Western blotting of cyclin D1 on total cell lysates of HUVEC treated with 0–40 μM AGX51 for 24 hours. Tubulin is used as a protein loading control. See also Figure 12. Figure 4D shows that HUVEC branching was observed after 18–20 hours of culture on Matrigel in the absence or presence of 0–40 μM AGX51, and images were taken at 10x magnification. Figure 4E shows quantification of nodes, junctions, mesh, and total branch length (n=4, replicated for each concentration tested). * indicates p<0.05 by Wilcoxon test. Figure 4F shows the effect of AGX51 on HUVEC migration. After scratching the HUVEC monolayer, the culture medium was replaced with a medium containing 0-40 μM of AGX51. Migration was observed after 24 hours, and images were taken at 20x magnification. [Figures 5A-5F]This report demonstrates that AGX51 treatment suppresses ocular angiogenesis in mouse models of AMD and ROP. Figure 5A shows the effect of AGX51 on laser-induced choroidal angiogenesis. In wild-type mice, Bruch's membrane ruptured at three locations in each eye, and 10 μg of AGX51 or vehicle was intravitreal-injected into one eye immediately and 7 days later (n=10 per group). Fourteen days after laser-induced rupture, the mean area of ​​CNV was significantly smaller in the AGX51-injected eye than in the control eye (* indicates p<0.05 by ANOVA with Bonferroni correction for multiple comparisons, error bars indicate SEM). Figure 5B shows representative choroidal flat mounts stained with Griffonia simplicifolialectin (to mark vascular cells) from the control-injected eye and the AGX51-injected eye (bar = 100 μm). Figure 5C shows that CNV was significantly suppressed by twice-daily intravitreal injections of 500 μg AGX51 (n=10 mice per group, * indicates p<0.05 by unpaired t-test, error bars indicate SEM). Figure 5D shows representative Griffonia simplicifolialectin-stained choroidal flat mounts of the eyes from mice treated with AGX51 (10 μg) or vehicle by twice-daily intravitreal injections (bar=100 μm). Figure 5E shows images showing immunofluorescence of Id1 in the CNV region from mice treated with AGX51 or DMSO by intravitreal injection (bar=50 μm). Figure 5F shows the effect of AGX51 on ischemia-induced retinal neovascularization. To induce ROP, pups (n=15) were placed in a 75% O2 chamber for 5 days on postnatal day 7. On day 12 after birth, mice were returned to room air and received intravitreal injections of 10 μg of AGX51 or FE with DMSO into one eye. On day 17 after birth, mice were euthanized and the area of ​​retinal neovascularization (RNV) was evaluated (* indicates p<0.01 by unpaired t-test, and error bars indicate SEM). See also Figures 13 and 17. [Figures 6A-6D]The effects of AGX51 enantiomer alone and in combination with aflibercept are shown. Figure 6A shows the effect of AGX51 enantiomer on laser-induced CNV. Laser-induced CNV was induced in mice and treated with DMSO, AGX51 racemate, or enantiomer (AGX51E1 or AGX51E2) (n=10 mice per group). Mice were treated by intraperitoneal administration. Treatment was by ip injection twice daily with 50 μL of vehicle or 10 mg / mL of the compound. On day 14, the animals were euthanized and the area of ​​CNV was measured as described (ANOVA and Bonferroni correction for multiple comparisons, * indicates p<0.05, ** indicates p=0.0014, and error bars indicate SEM). Figure 6B shows a representative choroidal flat mount stained with griffonia simplicifolialectin (a vascular cell marker) from the eye of a mouse treated with (a) (bar = 100 μm). Figure 6C (upper panel) shows a choroidal flat mount stained with griffonia simplicifolialectin from the eye of a mouse subjected to laser-induced CNV and treated with AGX51E2. Eight mice per group were treated with intravitreal injection of 1, 3, 10, or 30 μg of AGX51E2 on days 1 and 7 after laser-induced CNV. Mice were euthanized on day 14, and the area of ​​the CNV was measured. A representative choroidal flat mount of an eye stained with griffonia simplicifolialectin is shown. Figure 6C (lower panel) shows that the quantification of data is plotted, with * indicating p<0.05 by ANOVA and error bars indicating SEM (bar = 100 μm). Figure 6D shows the effects of AGX51 alone and in combination with aflibercept on laser-induced CNV. Laser-induced CNV was induced in mice, and on days 1 and 7, n=10 mice per group were treated with DMSO, AGX51 (10 μg), aflibercept (A) (40 μg), or AGX51 + aflibercept. The animals were euthanized on day 14, and the area of ​​the CNV was measured as described.In Figures 6C-6D, FE refers to the "sister eye" and is defined as the untreated eye of an animal whose two eyes have undergone laser treatment (ANOVA indicates p<0.05 for *, p=0.0014 for **, p<0.0001 for ***, and error bars indicate SEM). See also Figure 14. [Figures 7A-7E] This document describes the properties of AGX-A, the Id inhibitor compound of this technology, which exhibits surprisingly and unexpectedly superior efficacy compared to known Id inhibitors such as AGX51. Figure 7A shows the chemical structure of AGX-A. Figure 7B shows the circular dichroism (CD) spectrum showing the interaction between AGX-A (0–110 μM in DMSO) and Id1. Figure 7C shows a Western blot of Id1 in whole cell lysates from HCT116 cells treated with 0–10 μM AGX-A for 24 hours. IC50 is shown. The IC50 of AGX51 was 22.28 μM. Figure 7D shows a Western blot of Id1 in whole cell lysates from 4T1 cells treated with 0–20 μM AGX-A for 24 hours. IC50 is shown. The IC50 of AGX51 was 26.66 μM. Figure 7E shows the effect of AGX-A or AGX51 on laser-induced choroidal angiogenesis. Laser-induced choroidal angiogenesis (NV) was induced in mice, and the mice were treated with intravitreal injection of DMSO, 1 or 5 μg AGX-A, or 1 or 5 μg AGX51. The animals were euthanized on day 14, and the area of ​​CNV was measured as described. (** indicates p<0.01 by ANOVA, *** indicates p<0.0001, and error bars indicate SEM). [Figures 8A-8C]This report describes an investigation into the physical interaction between AGX51-XL2 and Id1. Figure 8A shows the conservation of the Id HLH domain across Id family members (Id1-Id4) in humans (hs) and mice (mm). The Drosophila melanogaster (dm) Id orthologue (emc) is also shown. Figure 8B shows the structure of AGX51-XL2. Figure 8C shows a schematic diagram of the Id1 helix and loop regions. Black dots indicate amino acids predicted to be adjacent to the Id1 binding pocket, and orange dots indicate amino acids identified as covalently bound to Id1 by mass spectrometry. One sample was analyzed per group (+ / - UV; + / - AGX51-XL2). The experiment was performed twice, 3 months apart. See also Figures 17-18. [Figure 9] The circular dichroism of AGX51 and Id3 is shown. The circular dichroism (CD) of Id3 using 0 or 100 μM AGX51 is shown. [Figure 10A-10C] This document describes the NanoBRET® assay using NanoLuc-ID1 and AGX tracers. Figure 10A shows the structure of the AGX51 tracer. Figure 10B shows the results of the NanoBRET® assay. 293T cells transfected with NanoLuc-ID1 were treated with 0–4 μM AGX fluorescent tracer. The bioluminescence resonance energy transfer (BRET) ratio was plotted as a function of AGX51 tracer concentration. Figure 10C shows the results of the NanoBRET® assay performed in the presence of AGX51 tracer and the indicated drug. 293T cells transfected with NanoLuc-ID1 were treated with 2 μM AGX fluorescent tracer and 0–60 μM AGX51 or AGX-A in the presence of 50 μg / mL digitonin. Complete substrate was added to the cells, and the emission wavelengths and BRET ratios of the donor and acceptor were determined using a GloMax Discover System illuminometer. [Figure 11A-11C]The effect of AGX51 on ID proteins in HCT116 cells is shown. Figure 11A shows Western blots of ID1, ID2, ID3, and ID4 in whole cell lysates from HCT116 cells treated with 40 μM AGX51 for 0–48 hours. Tubulin was used as a protein loading control. Figure 11B shows qRT-PCR analysis of ID1 and ID3 in HCT116 cells treated with 40 μM AGX51 for 24 hours. The mean magnification difference is accompanied by error bars indicating technical replication SEM. Figure 11C shows EMSA of whole cell lysates from HCT116 cells treated with 40 μM AGX51 for 1 hour or 24 hours. Spaces indicate that samples were performed on different gels or different portions of gels. [Figure 12A-12C] The effects of AGX51 on HCT116 cells in culture are shown. Figure 12A shows the MTT cell viability assay in HCT116 cells treated with 40 μM AGX51 for 24 hours. Mean values ​​of technical triplicates are plotted, and error bars represent standard deviation. Figure 12B shows the cell cycle analysis in HCT116 cells treated with 40 μM AGX51 for 4–24 hours. Figure 12C shows the Western blot of cyclin D1 in lysates from HCT116 cells treated with 40 μM AGX51 or vehicle for 0–48 hours. Tubulin was used as a protein loading control. Spaces indicate that samples were performed on different gels or different portions of gels. [Figure 13] The dose titration of AGX51 is shown. Laser-induced CNV was induced in mice, and the mice were treated on days 1 and 7 by intravitreal injection with DMSO (control) (n=7), AGX51 (1 μg) (n=8), or AGX51 (5 μg) (n=7). On day 14, the animals were euthanized, and the area of ​​the CNV was measured as described (* indicates 0.05 by ANOVA, and error bars indicate SEM). [Figure 14A-14C] The circular dichroism of the AGX51 enantiomer is shown. The circular dichroism (CD) of Id1 using 0 or 100 μM AGX51 (Figure 14A), AGX51-E2 (Figure 14B), and AGX51-E1 (Figure 14C) is shown. [Figure 15-1] The reagents used in the studies described in the examples and their sources are shown. [Figure 15-2] The reagents used in the studies described in the examples and their sources are shown (continued). [Figure 16] A summary of crystallographic diffraction data collection and refinement statistics based on the examples is shown. [Figure 17] The Id1 amino acid identified as covalently bound to AGX51-XL2 by mass spectrometry, as per the example, is shown. [Figure 18] The Id1 amino acids identified as covalently bound to AGX51-XL2 by mass spectrometry, with and without AGX51 competition, as per the examples, are shown. [Figure 19] The results of a toxicity study following a two-week treatment with AGX51 administered twice daily by intraperitoneal injection at a dose of 60 mg / kg, as described in the example, are shown. [Figure 20] The X-ray structure of R-(+)-3-(benzo[d][1,3]dioxol-5-yl)-N-benzyl-3-(2-methoxyphenyl)propan-1-amine, as described in the examples, is shown. [Figure 21] The comparative effects of AGX51 and AGX-A on Id1 knockdown in TFK1 cell cultures, as described in the examples, are shown. [Figure 22] The comparative effects of AGX51 and AGX-A on Id3 knockdown in TFK1 cell cultures, as described in the examples, are shown. [Figure 23] The dose-dependent effects of AGX51 and AGX-A on TFK1 cell viability over 24 hours, as described in the examples, are shown. [Figure 24] This document provides results from a mouse model of cholangiocarcinoma demonstrating the in vivo efficacy of AGX-A in DMSO and combination therapy utilizing gemcitabine and AGX-A in DMSO, according to the examples. [Figures 25A-25B]We provide the results of a 24-hour cell viability study of SNU1079 cells (Figure 25A) and SNU1196 cells (Figure 25B) treated with AGX-A in an aqueous solution containing 2-hydroxypropyl-β-cyclodextrin ("HPBCD"), according to the examples. [Figure 26] This provides results from a mouse model of cholangiocarcinoma demonstrating the in vivo efficacy of AGX-A in an aqueous solution (containing 12.5% ​​by mass of HPBCD) and a combination therapy using an aqueous solution of gemcitabine and AGX-A, according to the examples. [Figure 27] To support future comparisons with compounds of this technology as described in the examples, we provide the results of an Allamer Blue viability assay of 4T1 cells treated with AGX51. It is expected that the compounds of this technology will exhibit similar or significantly improved effects. [Figure 28] The effects of AGX51 on mouse pancreatic organoid cell lines T7 and T8 are shown to support future comparisons with compounds of the present technology, as described in the examples. It is expected that compounds of the present technology will show similar or significantly improved effects. [Figure 29] The effects of AGX51 on mouse pancreatic cancer strains 806 (KrasG12D;Ink4a- / -;Smad4- / -), NB44 (KrasG12D;Ink4a- / -), and 4279 (KrasG12D;Ink4a- / -) are shown to support future comparisons with compounds of the present technology, as described in the examples. Similar or significantly improved effects are expected with compounds of the present technology. [Figure 30] The effects of AGX51 on human pancreatic cancer cell lines Panc1 and A21 are shown to support future comparisons with compounds of this technology, as described in the examples. It is expected that compounds of this technology will show similar or significantly improved effects. [Figure 31] The effects of AGX51 (with and without paclitaxel) on primary tumors are shown to support future comparisons with compounds of the present technology, as described in the examples. It is expected that compounds of the present technology will show similar or significantly improved effects. [Figure 32]The effects of AGX51 on lung implantation are shown to support future comparisons with compounds of the present technology, as described in the examples. It is expected that compounds of the present technology will show similar or significantly improved effects. [Figure 33] The effect of AGX51 on establishing lung metastases is shown to support future comparisons with compounds of the present technology, as described in the examples. It is expected that compounds of the present technology will show similar or significantly improved effects. [Figures 34A-34B] The results of a study evaluating the inhibition of tumor progression of extravasation and early dissemination or extravasation cancer cell proliferation in secondary sites by treatment with AGX51, in accordance with the examples, are presented to support future comparisons with compounds of the present technology. The effect of AGX51 on early dissemination of cancer cells in secondary sites is shown in Figure 34A for all cells and in Figure 34B for all tissue area. Similar or significantly improved effects are expected with compounds of the present technology. [Figures 35A-35D] The effects of AGX51 on sporadic tumors are shown to support future comparisons with compounds of the present technology, as per the examples. Figure 35A provides results regarding the total number of tumors; Figure 35B shows the resulting number of tumors measured ≤1 mm; Figure 35C shows the resulting number of tumors measured 1.5 mm to 2.5 mm; and Figure 35D shows the number of tumors measured ≥3 mm. Similar or significantly improved effects are expected with compounds of the present technology. [Modes for carrying out the invention]

[0006] To provide a substantial understanding of this technology, please understand that certain aspects, styles, embodiments, variations, and features of the method of the present invention will be described below at various levels of detail.

[0007] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this art belongs. As used herein and in the appended claims, singular articles such as “a,” “an,” and “the,” and similar references in relation to the descriptions of elements (particularly in relation to the following claims), should be interpreted as encompassing both singular and plural forms unless otherwise stated herein or unless clearly inconsistent with the context. Unless otherwise stated herein, the enumeration of ranges of values ​​herein serves merely as a way of omitting the individual listing of each distinct value contained within that range, and each distinct value is incorporated herein as if it were listed separately herein. Unless otherwise stated herein or unless clearly inconsistent with the context, all methods described herein may be carried out in any preferred order. The use of any and all examples or illustrative words (e.g., “etc.”) is merely for the purpose of better illustrating these embodiments and, unless otherwise stated, does not limit the claims. Nothing in this specification should be interpreted as indicating that elements not described in the claims are essential. For example, references to "cells" include combinations of two or more cells. In general, the nomenclature used herein, as well as the experimental procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below, are well known and commonly used in the art.

[0008] Where used herein, “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. Where there is a use of a term that is not clear to those skilled in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of a particular term—for example, “about 10% by mass” will be understood to mean “9% to 11% by mass.” Where “about” precedes a term, it should be understood that the term should be interpreted as disclosing both the term modified by “about” and the term as it is not modified by “about”—for example, “about 10% by mass” discloses “9% to 11% by mass” and “10% by mass.”

[0009] As used herein, “administration” of a drug or substance to a subject includes any route through which the compound is introduced or delivered to the subject to perform its intended function. Administration may be carried out by any preferred route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration may include self-administration and administration by another person. As used in this disclosure, the phrase "and / or" is understood to mean any one of the enumerated members individually or any two or more combinations thereof—for example, "A, B, and / or C" means "A, B, C, A and B, A and C, or B and C."

[0010] As used herein, the terms “cancer,” “neoplasm,” and “tumor” are interchangeable and refer to malignantly transformed cells that become pathological to a host organism. Primary cancer cells (i.e., cells obtained near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells as used herein includes not only primary cancer cells but also any cells derived from the ancestors of cancer cells. This includes metastatic cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that typically manifests as a solid tumor, a “clinically detectable” tumor is one that is detectable based on the tumor mass by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or detectable for the expression of one or more cancer-specific antigens in a sample available from the patient.

[0011] As used herein, “control” refers to an alternative sample used in an experiment for comparative purposes. A control may be “positive” or “negative.” For example, if the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that is not treated or is receiving a placebo) are typically used.

[0012] As used herein, the terms “metastasis” or “metastatic” refer to the ability of cancer cells to invade surrounding tissues, enter the circulatory system, and establish malignant growth in new sites. "Non-metastatic" refers to tumors that do not spread beyond their original site of origin, and in particular do not enter the circulatory system or establish malignant growth in new sites. As used herein, “prevention,” “prevention,” or “prevention” of a disease or condition means, in a statistical sample, that reduces the incidence of a disease or condition in a treated sample compared to an untreated control sample, or delays the onset of one or more symptoms of a disease or condition compared to an untreated control sample. As used herein, prevention includes preventing or delaying the onset of symptoms of a disease or condition. As used herein, prevention also includes preventing the recurrence of one or more signs or symptoms of a disease or condition.

[0013] As used herein, the term “sample” refers to a clinical sample obtained from a subject. A biological sample may include tissues, cells, cellular protein or membrane extracts, mucus, sputum, bone marrow, bronchoalveolar lavage (BAL), bronchial lavage (BW), and body fluids (e.g., ascites or cerebrospinal fluid (CSF)) separated from the subject, as well as tissues, cells, and body fluids (e.g., blood, plasma, saliva, urine, serum) present within the subject. As used herein, the term “separate” therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously via different routes. As used herein, the term “sequential” therapeutic use refers to the administration of at least two active ingredients at different times, either via the same or different routes of administration. More specifically, sequential use refers to the total administration of one active ingredient prior to the administration of the other or before the commencement of any other administration. Thus, it is possible to administer one of the active ingredients over several minutes, hours, or days before administering any other or multiple active ingredients. In this case, there is no simultaneous treatment. As used herein, the term “concurrent” therapeutic use refers to the administration of at least two active ingredients simultaneously or substantially simultaneously via the same route. As used herein, the terms “subject,” “individual,” or “patient” are interchangeable and refer to individual organisms, vertebrates, mammals, or humans. In certain embodiments, the individual, patient, or subject is a human.

[0014] As used herein, “to treat,” “to treat,” or “treatment” encompasses treatment of a disease or disorder described herein in an object such as a human, and includes (i) inhibiting the disease or disorder, i.e., preventing its onset; (ii) reducing the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, mitigating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, for example, reduced, cured, or put into remission. The various forms of treatment or prevention of medical diseases and conditions described are intended to mean “substantial,” and it should be understood that this includes both total treatment or prevention and treatments that do not constitute total treatment or prevention but yield some biological or medically relevant results. Treatment may be continuous, long-term treatment for a chronic disease, or a single or multiple administration for the treatment of an acute condition.

[0015] Generally, a reference to a particular element, such as hydrogen or H, implies the inclusion of all isotopes of that element. For example, if the R group is defined as containing hydrogen or H, then deuterium and tritium are also included. Therefore, tritium, 14 C, 32 P and 35 Compounds containing radioactive isotopes such as 360 ions are within the scope of this technology. Procedures for inserting such labels into compounds of this technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0016] Generally, "substituted" refers to an organic group (e.g., an alkyl group) as defined below, in which one or more bonds to a hydrogen atom are replaced by bonds to a non-hydrogen or non-carbon atom. Substituents also include groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds to a heteroatom, including double or triple bonds. Thus, substituents are substituted with one or more substituents unless otherwise specified. In some embodiments, substituents are substituted with one, two, three, four, five, or six substituents. Examples of substituents include halogens (i.e., F, Cl, Br, and I); hydroxyl; alkoxy groups, alkenoxy groups, aryloxy groups, aralkyloxy groups, heterocyclyl groups, heterocyclylalkyl groups, heterocyclyloxy groups, and heterocyclylalkoxy groups; carbonyl (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanil (i.e., SF5), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN); and others.

[0017] Substituted ring groups, such as substituted cycloalkyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups, also include rings and ring systems in which bonds to hydrogen atoms are replaced by bonds to carbon atoms. Therefore, substituted cycloalkyl groups, aryl groups, heterocyclyl groups, and heteroaryl groups can also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups, as defined below.

[0018] Alkyl groups include linear and branched alkyl groups having 1 to 12 carbon atoms, typically 1 to 10 carbon atoms, or in some embodiments 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups can be substituted or unsubstituted. Examples of linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Typical substituted alkyl groups can be substituted once or multiple times with the substituents described above and include, but are not limited to, haloalkyls (e.g., trifluoromethyl), hydroxyalkyls, thioalkyls, aminoalkyls, alkylaminoalkyls, dialkylaminoalkyls, alkoxyalkyls, and carboxyalkyls.

[0019] Cycloalkyl groups include monocyclic, bicyclic, or tricyclic alkyl groups having 3 to 12 carbon atoms in the ring, or in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups can be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups have 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Bicyclic and tricyclic ring systems include, but are not limited to, both cross-linked cycloalkyl groups and fused rings, such as bicyclo[2.1.1]hexane, adamantyl, and dekalinyl. Substituted cycloalkyl groups can be substituted once or multiple times with non-hydrogen and non-carbon groups, as defined above. However, substituted cycloalkyl groups also include rings substituted with the linear or branched alkyl groups defined above. Typical substituted cycloalkyl groups may be monosubstituted or polysubstituted. Examples, though not limited to these, include 2,2-, 2,3-, 2,4-2,5-, or 2,6-disubstituted cyclohexyl groups, which may be substituted with the substituents described above.

[0020] A cycloalkylalkyl group is an alkyl group as defined above, in which the hydrogen or carbon bonds of the alkyl group are replaced by bonds to the cycloalkyl group as defined above. Cycloalkylalkyl groups can be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. A substituted cycloalkylalkyl group can be substituted with the alkyl, cycloalkyl, or both the alkyl and cycloalkyl moieties of the group. Typical substituted cycloalkylalkyl groups may be monosubstituted or polysubstituted, and may, for example, be monosubstituted, disubstituted, or trisubstituted with the substituents described above.

[0021] Alkenyl groups include linear and branched alkyl groups as defined above, except that they have at least one double bond between two carbon atoms. Alkenyl groups can be substituted or unsubstituted. Alkenyl groups have 2 to 12 carbon atoms, and typically 2 to 10 carbon atoms, or in some embodiments 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkenyl groups have one, two, or three carbon-carbon double bonds. Examples include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), and -C(CH2CH3)=CH2. Typical substituted alkenyl groups may be monosubstituted or polysubstituted, and may be monosubstituted, disubstituted, or trisubstituted with the substituents described above, but are not limited to these examples.

[0022] Cycloalkenyl groups include cycloalkyl groups defined above, which have at least one double bond between two carbon atoms. Cycloalkenyl groups can be substituted or unsubstituted. In some embodiments, cycloalkenyl groups may have one, two, or three double bonds but do not contain aromatic compounds. Cycloalkenyl groups have 4 to 14 carbon atoms, or in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentadienyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl. A cycloalkenylalkyl group is an alkyl group as defined above, in which the hydrogen or carbon bond of the alkyl group is replaced by a bond to the cycloalkenyl group as defined above. Cycloalkenylalkyl groups can be substituted or unsubstituted. A substituted cycloalkenylalkyl group can be substituted with the alkyl, cycloalkenyl, or both the alkyl and cycloalkenyl moieties of the group. Typical substituted cycloalkenylalkyl groups can be substituted once or multiple times with substituents as described above.

[0023] Alkynyl groups include linear and branched alkyl groups as defined above, except that they have at least one triple bond between two carbon atoms. Alkynyl groups can be substituted or unsubstituted. Alkynyl groups have 2 to 12 carbon atoms, and typically 2 to 10 carbon atoms, or in some embodiments 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, alkynyl groups have one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to, -C≡CH, -C≡CCH3, -CH2C≡CCH3, and -C≡CCH2CH(CH2CH3)2. Typical substituted alkynyl groups may be monosubstituted or polysubstituted, and may be monosubstituted, disubstituted, or trisubstituted with the substituents described above, but are not limited to these examples.

[0024] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups as used herein include monocyclic, bicyclic, and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Therefore, examples of aryl groups include, but are not limited to, phenyl, azlenyl, heptalenyl, biphenyl, fluorenyl, phenantrenyl, anthracenyl, indenyl, indanyl, pentarenyl, and naphthyl groups. In some embodiments, the aryl group contains 6 to 14 carbon atoms in the ring portion of the group, and in other embodiments, 6 to 12, and even 6 to 10 carbon atoms. In some embodiments, the aryl group is phenyl or naphthyl. The phrase "aryl group" includes groups containing fused rings, such as fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.). Typical substituted aryl groups may be monosubstituted (e.g., tolyl) or polysubstituted. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which can be substituted with the substituents described above.

[0025] An aralkyl group is an alkyl group as defined above, in which the hydrogen or carbon bond of the alkyl group is replaced by a bond to the aryl group defined above. Aralkyl groups can be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. A substituted aralkyl group can be substituted with the alkyl, aryl, or both alkyl and aryl moieties of the group. Typical aralkyl groups include, but are not limited to, the benzyl and phenethyl groups, as well as condensed (cycloalkylaryl) alkyl groups such as 4-indanylethyl. Typical substituted aralkyl groups can be substituted once or multiple times with the substituents described above.

[0026] Heterocyclyl groups include aromatic (also called heteroaryl) and non-aromatic ring compounds containing three or more ring members, one or more of which are heteroatoms such as, but not limited to, N, O, and S. Heterocyclyl groups can be substituted or unsubstituted. In some embodiments, heterocyclyl groups contain one, two, three, or four heteroatoms. In some embodiments, heterocyclyl groups include monocyclic, bicyclic, and tricyclic rings having 3 to 16 ring members, while other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups include aromatic, partially unsaturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl groups. The phrase “heterocyclyl group” includes condensed ring species, such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxynyl, and benzo[1,3]dioxolyl, which contain condensed aromatic and non-aromatic groups. This phrase also includes bridging polycyclic ring systems containing heteroatoms, such as quinuclidyl. This phrase includes heterocyclyl groups, called “substituted heterocyclyl groups,” which have another group, such as an alkyl, oxo, or halo group, bonded to one of the ring members.Examples of heterocyclyl groups include azilidinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranil, dioxolyl, furanil, thiophenyl, pyrrolyl, pyrrolinil, imidazolyl, imidazolinil, pyrazolyl, pyrazolinil, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, and morpholinyl. L, thiomorpholinyl, tetrahydropyranil, tetrahydrothiopyranil, oxatian, dioxyl, dithianil, pyranil, pyridyl, pyrimidinil, pyridazinil, pyrazinil, triazinil, dihydropyridyl, dihydrodithinyl, dihydrodithionyl, homopiperazinil, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolidinil, benzotriazolyl, benzimidazolyl, benzofuranil, benzothiophenyl, Benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithinyl, benzoxathinyl, benzothiadinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolidinyl, quinoxalinyl, quinazolinyl, cinolinyl, phthalazi Examples include, but are not limited to, the yl, naphthilidinyl, pteridinyl, thianafthyl, dihydrobenzothiadiazine, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxynyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Typical substituted heterocyclyl groups may be monosubstituted or polysubstituted, and may include, for example but not limited to, pyridyl or morpholinyl groups that are 2-, 3-, 4-, 5-, or 6-substituted or disubstituted with various substituents as described above.

[0027] A heteroaryl group is an aromatic ring compound containing five or more ring members, one or more of which are heteroatoms such as, but not limited to, N, O, and S. Heteroaryl groups can be substituted or unsubstituted. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanil, benzofuranil, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaftyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds where all rings are aromatic, such as indolyl groups, and fused ring compounds where only one ring is aromatic, such as 2,3-dihydroindolyl groups. Typical substituted heteroaryl groups can be substituted once or multiple times with various substituents as described above.

[0028] A heterocyclylalkyl group is an alkyl group as defined above, in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups can be substituted or unsubstituted. A substituted heterocyclylalkyl group can be substituted with the alkyl, heterocyclyl, or both the alkyl and heterocyclyl moieties of the group. Representative heterocyclylalkyl groups include, but are not limited to, morpholine-4-ylethyl, furan-2-ylmethyl, imidazole-4-ylmethyl, pyridine-3-ylmethyl, tetrahydrofuran-2-ylethyl, and indole-2-ylpropyl. Representative substituted heterocyclylalkyl groups can be substituted once or multiple times with the substituents described above.

[0029] A heteroaralkyl group is an alkyl group as defined above, in which the hydrogen or carbon bond of the alkyl group is replaced by a bond to a heteroaryl group as defined above. Heteroaralkyl groups can be substituted or unsubstituted. A substituted heteroaralkyl group can be substituted with both the alkyl, heteroaryl, or alkyl-partial heteroaryl moiety of the group. Typical substituted heteroaralkyl groups can be substituted once or multiple times with substituents as described above.

[0030] Groups described herein that have two or more bonding sites (i.e., divalent, trivalent, or polyvalent) within the compounds of this technology are indicated by the use of the suffix "ene". For example, a divalent alkyl group is an alkylene group, a divalent aryl group is an arylene group, a divalent heteroaryl group is a divalent heteroarylene group, and so on. Substituents having a single bonding site to the compounds of this technology are not referred to using the "ene" designation. Therefore, for example, chloroethyl is not referred to as chloroethylene herein.

[0031] An alkoxy group is a hydroxyl group (-OH) in which the bond to the hydrogen atom is replaced by a bond to the carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups can be substituted or unsubstituted. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, and hexoxy. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, and isohexoxy. Examples of cycloalkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Typical substituted alkoxy groups can be substituted once or multiple times with the substituents described above. As used herein, the terms “alkanoyl” and “alkanoyloxy” may refer to -C(O)-alkyl groups and -OC(O)-alkyl groups, respectively, that contain 2 to 5 carbon atoms. Similarly, “aryloyl” and “aryloyloxy” may refer to -C(O)-aryl groups and -OC(O)-aryl groups.

[0032] The terms "aryloxy" and "arylalkoxy" refer to substituted or unsubstituted aryl groups bonded to an oxygen atom, and substituted or unsubstituted aralkyl groups bonded to an oxygen atom via alkyl groups, respectively. Examples include, but are not limited to, phenoxy, naphthyloxy, and benzyloxy. Typical substituted aryloxy and arylalkoxy groups can be substituted once or multiple times with the substituents described above. As used herein, the term "carboxylate" refers to the -COOH group. The term "ester" as used herein refers to -COOR 70 And refers to the -C(O)OG group. 70is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to those skilled in the art. A comprehensive list of protecting groups for carboxylate functional groups can be found in Protective Groups in Organic Synthesis, Greene, TW; Wuts, PGM, John Wiley & Sons, New York, NY, (3rd Edition, 1999), which can be added or removed using the procedures described therein and is incorporated herein by reference in its entirety for any purpose as if it were fully described herein.

[0033] The term "amide" (or "amido") refers to the C- and N-amide groups, i.e., -C(O)NR, respectively. 71 R 72 and -NR 71 C(O)R 72 Includes the group R 71 and R 72 This is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Therefore, the amide group may include, but is not limited to, a carbamoyl group (-C(O)NH2) and a formamide group (-NHC(O)H). In some embodiments, the amide is -NR 71 C(O)-(C 1-5 In other embodiments, the amide is -NHC(O)-alkyl, and this group is called "carbonylamino". As used herein, the terms "nitrile" or "cyano" refer to the -CN group.

[0034] The urethane groups include N- and O-urethane groups, i.e., -NR groups, respectively.73 C(O)OR 74 and -OC(O)NR 73 R 74 The base is R. 73 and R 74 R is independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. 73 It can also be H. As used herein, the term "amine" (or "amino") is defined as -NR 75 R 76 It refers to the base, R 75 and R 76 This is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. In some embodiments, the amine is an alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0035] The term "sulfonamide" refers to the S- and N-sulfonamide groups, i.e., -SO2NR, respectively. 78 R 79 and -NR 78 SO2R 79 Includes the group R 78 and R 79 This group is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Therefore, sulfonamide groups include, but are not limited to, sulfamoyl (-SO2NH2) groups. In some embodiments herein, the sulfonamide is -NHSO2-alkyl and is referred to as an "alkylsulfonylamino" group. The term "thiol" refers to the -SH group, while "sulfide" refers to the -SR group. 80 The group is included, and "sulfoxide" is -S(O)R 81 The group is included, and "sulfone" is -SO2R 82 The group is included, and "sulfonyl" is -SO2OR 83 Includes R 80 , R 81 , R 82 , and R 83 Each of these is independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. In some embodiments, the sulfide is an alkylthio group, an -S-alkyl group.

[0036] The term "urea" is -NR 84 -C(O)-NR 85 R 86 It refers to the base. R 84 , R 85 , and R 86 This is independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. The term "amidine" is -C(NR 87 )NR 88 R 89 and -NR 87 C(NR 88 )R 89 It refers to R 87 , R 88 , and R 89 Each of these is independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein. The term "guanidine" is -NR 90 C(NR 91 )NR 92 R 93 It refers to R 90 , R 91 , R92 and R 93 is, independently, hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0037] The term "enamine" refers to -C(R 94 )=C(R 95 )NR 96 R 97 and -NR 94 C(R 95 )=C(R 96 )R 97 where R 94 , R 95 , R 96 , and R 97 is, independently, hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. As used herein, the term "halogen" or "halo" refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0038] As used herein, the term "hydroxyl" can refer to -OH or its ionized form -O - . A "hydroxyalkyl" group is a hydroxyl-substituted alkyl group such as HO-CH2-. The term "imide" refers to -C(O)NR 98 C(O)R 99 where R 98 and R 99 is, independently, hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. The term "imine" refers to -CR 100 (NR 101 ) group and -N(CR100 R 101 ) refers to the base, R 100 and R 101 R 100 and R 101 Each is independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein, provided that neither is simultaneously hydrogen. As used herein, the term "nitro" refers to the -NO2 group. As used herein, the term "trifluoromethyl" refers to -CF3.

[0039] As used herein, the term "trifluoromethoxy" refers to -OCF3. The term "Azid" refers to -N3. The term "trialkylammonium" refers to a -N(alkyl)3 group. Because the trialkylammonium group is positively charged, it usually has an associated anion, such as a halogen anion. The term "isocyano" refers to -NC. The term "isothiocyan" refers to -NCS. The term "pentafluorosulfanil" refers to -SF5.

[0040] As will be understood by those skilled in the art, for all purposes, and especially in terms of providing written explanations, all scopes disclosed herein also encompass any possible subscopes and combinations thereof. It will be readily apparent that each enumerated scope is sufficiently described so that the same scope can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-restrictive example, each scope discussed herein can be readily broken down into lower thirds, middle thirds, upper thirds, etc. As will also be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” includes the enumerated number and refers to a scope that can then be broken down into subscopes as described above. Finally, as will be understood by those skilled in the art, a scope includes individual members. Thus, for example, a group having 1 to 3 atoms refers to a group having 1, 2, or 3 atoms. Similarly, a group having 1 to 5 atoms refers to a group having 1, 2, 3, 4, or 5 atoms, and so on.

[0041] pharmaceutically acceptable salts of the compounds described herein include acid or base addition salts that are within the scope of this art, retain the desired pharmacological activity, and are not biologically undesirable (e.g., the salt is not excessively toxic, allergenic, or irritating, and is biologically usable). If the compounds of this art have a basic group, such as an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (e.g., hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginic acid, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (e.g., aspartic acid and glutamic acid). If the compounds of this art have an acidic group, such as a carboxylic acid group, it can be formed with metals, such as alkali metals and alkaline earth metals (e.g., Na + Li + , K + Ca 2+ Mg2+ Zn 2+ ), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine, and ornithine) can form salts. Such salts can be prepared in situ when isolating or purifying the compound, or by reacting the purified compound in the form of a free base or free acid separately with a suitable acid or base, respectively, and then isolating the salts thus formed.

[0042] Those skilled in the art will understand that the compounds of this technology may exhibit tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism. Since the drawings of the formulas in the specification and claims can only show one of the possible tautomerized forms, conformational isomerized forms, stereochemical isomerized forms, or geometric isomerized forms, it should be understood that this technology encompasses any tautomerized forms, conformational isomerized forms, stereochemical isomerized forms, and / or geometric isomerized forms of one or more useful compounds described herein, as well as mixtures of various different forms thereof. "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The presence and concentration of isomers depend on the environment in which the compound is found, for example, whether the compound is a solid, an organic solution, or an aqueous solution. For example, in aqueous solution, quinazolinone may exhibit the following isomeric forms, which are all called tautomers.

[0043] [ka] As another example, guanidine may exhibit the following isomeric forms in protic organic solutions, which are also called tautomers.

[0044] [ka] Because there are limitations to presenting compounds using structural formulas, please understand that all chemical formulas of the compounds described herein represent all tautomer forms of the compounds and are within the scope of this technology.

[0045] The stereoisomers (also known as optical isomers) of a compound include all chiral, diastereomer, and racemic forms of the structure unless a specific stereochemistry is explicitly indicated. Therefore, the compounds used in this technique, as is evident from the description, include concentrated or divided optical isomers at any or all chiral atoms. Both racemic and diastereomer mixtures, as well as individual optical isomers, can be isolated or synthesized so as to be substantially free of their enantiomer or diastereomer partners, and all such stereoisomers are within the scope of this technique. The compounds of this technology may exist as solvates, particularly hydrates. Hydrates may form during the preparation of the compound or a composition containing the compound, or they may form over time due to the hygroscopic nature of the compound. The compounds of this technology may also exist as organic solvent hydrates, including, in particular, DMF, ethers, and alcohol solvates. The identification and preparation of specific solvates are within the scope of the skills of those skilled in the art of synthetic organic or medicinal chemistry.

[0046] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying citations. Within this disclosure, there are Arabic numerals indicating the referenced citations, the complete bibliographic details of which are provided immediately before the claims. These publications, patents, and published patent specifications are incorporated by reference to more fully illustrate the current art relating to the present invention.

[0047] The technology according to the present invention As described in the Art (for example, in U.S. Patent Application No. 2009 / 022642 and International Publication No. WO2015 / 089495, respectively, which are incorporated herein by reference), the Id protein has been shown to play a crucial role as a regulator of stem cell identity in both colorectal cancer and malignant glioma, and is essential for both the self-renewal and tumor-initiating capabilities of cancer stem cells. Despite the complexity and unclear pathways of the mechanisms involved in cancer stem cell development, anti-Id compounds disrupt stem cell identity and impair stem cell tumor initiation by inactivating the Id protein on a critical basis. Anti-Id compounds with anti-metastatic and anti-angiogenic effects can specifically target tumor stem cell viability, proliferation, tumor-initiating potential, and / or cell fate determination, resulting in a significant reduction in the population of novel tumor-inducing stem cells present in new or established tumors.

[0048] The compounds in this technology target Id1 and Id3-positive "quiescent" stem cells. These cells represent a pool of cancer progenitor cells that are relatively resistant to chemotherapy (based on the non-proliferative state of quiescent cells, such cells evade first-line chemotherapy that targets proliferating cells). Thus, these stem cells frequently escape first-line cancer treatment, after which they can rebound and generate a new population of cancer cells. Further additional evidence presented here indicates that the compounds in this technology also affect their effects, either alone or in combination with conventional chemotherapy cancer treatments, eliminating acquired resistance. For example, quiescent stem cells or cancer cells that evade first-line treatment due to mutations (e.g., cells that acquire resistance to chemotherapy drugs due to mutations) cannot further evade the compounds in this technology or develop resistance to them. While not bound by theory, functionally important, evolutionarily constrained / conserved Id binding interfaces (targeted by the compounds in this technology) cannot be substantially altered to generate "acquired resistance." This is because any structural mutation would produce a biologically non-functional Id protein that is functionally ineffective with respect to the essential purpose for which such proteins serve in cancer cells.

[0049] Therefore, in one embodiment, this disclosure relates to formula I [ka] (I) The present invention provides compounds thereof, or pharmaceutically acceptable salts and / or solvates thereof. (In the formula, R 1 , R 2 , and R 3 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanil, halo, or -N(R) 10 )(R 11 ) and; R 4 , R 5 , R 6 , and R 7 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanil, halo, or -N(R) 12 )(R 13 ) and; R 8 It is either an aryl or heteroaryl; R 9 is H, C1-C3 alkyl, or fluoro; R 10 , R 11 , R 12 , and R 13 (Each of these is independently a C1-C3 alkyl group.)

[0050] In any embodiment of this specification, R 1 , R 2 , and R 3 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(R) 10 )(R 11 ) may be. In any embodiment of this specification, R 1 , R2 , and R 3 Each of these can independently be H, C1-C3 alkyl, C1-C3 alkoxy, halo, or -N(Me)2. In any embodiment of this specification, R 1 , R 2 , and R 3 Each of these can independently be H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. In any embodiment of this specification, R 3 It may be methoxy.

[0051] In any embodiment of this specification, R 4 , R 5 , R 6 , and R 7 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(R) 12 )(R 13 ) may be. In any embodiment of this specification, R 4 , R 5 , R 6 , and R 7 Each of these can independently be H, C1-C3 alkyl, C1-C3 alkoxy, halo, or -N(Me)2. In any embodiment of this specification, R 4 , R 5 , R 6 , and R 7 Each of these can independently be H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. In any embodiment of this specification, R 6 It may be isopropoxy.

[0052] In any embodiment of this specification, the compound of formula I is formula IA [ka] (IA) It may be a compound of, or a pharmaceutically acceptable salt and / or solvate thereof.

[0053] In any embodiment of this specification, the compound of formula I is the compound of formula IB [ka] (IA) It may be a compound of, or a pharmaceutically acceptable salt and / or solvate thereof. (In the formula, R 14 , R 15 , and R 16 (Each of these is independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanyl, halo, aryloxy, arylroyl, hydroxyl, amino, or amide.)

[0054] In any embodiment of this specification, R 14 , R 15 , and R 16 Each of these can independently be H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, aryloxy, aryloyl, or -N(C1-C3 alkyl)2. In any embodiment herein, R 14 , R 15 , and R 16 Each of these can independently be H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(Me)2. In any embodiment herein, R 14 , R 15 , and R 16 Each of these can independently be H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. In any embodiment of this specification, R 14 , R 15 , and R 16 Each of these can independently be H.

[0055] In any embodiment of this specification, the compound of formula I is [ka] JPEG0007880815000008.jpg147170 JPEG0007880815000009.jpg137162 or a pharmaceutically acceptable salt and / or solvate thereof.

[0056] In any embodiment of this specification, the compound of formula I is formula IC [ka] (I C) It may be a compound of, or a pharmaceutically acceptable salt and / or solvate thereof.

[0057] In any embodiment of this specification, the compound of formula I is [ka] JPEG0007880815000012.jpg143170 JPEG0007880815000013.jpg138164 or a pharmaceutically acceptable salt and / or solvate thereof.

[0058] In one embodiment, a composition is provided comprising a compound of formula I in any embodiment disclosed herein (e.g., a compound according to formula I, a compound disclosed above, a pharmaceutically acceptable salt and / or solvate of any compound disclosed above) and a pharmaceutically acceptable carrier or one or more excipients, fillers or pharmaceuticals (collectively referred to as “pharmaceutically acceptable carriers” unless otherwise specified and / or expressed). In related embodiments, a pharmaceutical is provided comprising a compound of formula I in any embodiment disclosed herein. In related embodiments, a pharmaceutical composition is provided comprising (i) an effective amount of a compound of formula I in any embodiment disclosed herein, and (ii) a pharmaceutically acceptable carrier. For ease of reference, the compositions, pharmaceuticals, and pharmaceutical compositions of the Art may be collectively referred to herein as “compositions.” In further related embodiments, the Art provides a method comprising a compound and / or a composition of any embodiment disclosed herein, as well as the use of a compound and / or a composition of any embodiment disclosed herein. Such methods and uses may involve an effective amount of the compound in any embodiment disclosed herein. In any aspect or embodiment disclosed herein (collectively referred to herein as “any embodiment herein,” “any embodiment disclosed herein,” etc.), the effective amount may be an amount that treats pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproproliferative disease in a subject. Where used herein, “subject” or “patient” is typically a mammal such as a cat, dog, rodent, or primate. Typically, the subject is a human, preferably a human who has or is suspected of having pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproproliferative disease. The terms subject and patient may be used interchangeably.

[0059] Accordingly, the technology of the present invention provides pharmaceutical compositions and pharmaceutically acceptable

[0060] Pharmaceutical compositions and pharmaceuticals of the present technology can be prepared by mixing one or more compounds of the present technology, their pharmaceutically acceptable salts, their stereoisomers, their tautomers, or their solvates with pharmaceutically acceptable carriers, excipients, binders, diluents, etc. The compounds and compositions described herein can be used to prepare formulations and pharmaceuticals for the prevention or treatment of pathogenic cell proliferation, angiogenesis, cancer, metastatic diseases, and / or pathogenic angioproliferative diseases. Such compositions may be in the form of granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions, for example. The compositions can be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, or vaginal administration, or via implanted reservoirs. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injections. The following dosage forms are given as examples and should not be construed as limiting the present technology.

[0061] For oral, sublingual, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gel caps, and caplets are acceptable solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the technology of the present invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive, such as starch or other additives. Suitable additives include sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Optionally, the oral dosage form may contain other ingredients that aid administration, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens or sorbic acid, antioxidants such as ascorbic acid, tocopherol or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings or fragrances. Tablets and pills can be further treated with suitable coating materials known in the art.

[0062] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions that may contain an inert diluent such as water. Pharmaceutical formulations and pharmaceuticals can be prepared as liquid suspensions or solutions using sterile liquids, including but not limited to oils, water, alcohols, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents, and emulsifiers may be added for oral or parenteral administration.

[0063] As described above, the suspension may contain oil. Examples of such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. The suspension preparation may also contain esters of fatty acids, such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Examples of alcohols in the suspension formulation include, but are not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. However, ethers such as poly(ethylene glycol), mineral oil, and petroleum hydrocarbons such as petrolatum; and water may also be used in the suspension formulation.

[0064] Injectable dosage forms generally include aqueous or oily suspensions that can be prepared using suitable dispersants or wetting agents and suspending agents. Injectable forms may be in the form of a solution phase or suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic saline. Alternatively, sterile oil can be used as a solvent or suspending agent. Typically, the oil or fatty acid is non-volatile and includes natural or synthetic oils, fatty acids, monoglycerides, diglycerides, or triglycerides. In the case of injection, the pharmaceutical formulation and / or pharmaceutical may be a powder suitable for reconstitution with the appropriate solution as described above. Examples of these include, but are not limited to, lyophilized, tumble-dried or spray-dried powders, amorphous powders, granules, precipitates, or particles. In the case of injection, the formulation may also contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0065] The compounds of this technology can be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation include solutions, sprays, dry powders, or aerosols, which contain a suitable solvent and may also contain other compounds such as stabilizers, antimicrobial agents, antioxidants, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof. Carriers and stabilizers vary depending on the requirements of the specific compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), amino acids such as serum albumin, sorbitan esters, oleic acid, lecithin, and glycine, buffers, salts, sugars, or harmless proteins such as sugar alcohols. Aqueous and non-aqueous (e.g., in fluorocarbon propellants) aerosols are typically used for the delivery of the compounds of this technology by inhalation.

[0066] Dosage forms for topical (including oral and sublingual) or transdermal administration of the compounds of this technology include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. The active ingredients may be mixed with pharmaceutically acceptable carriers or excipients under sterile conditions, and any preservatives or buffers as needed. Powders and sprays can be prepared using excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powders, or mixtures thereof. Ointments, pastes, creams, and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof. Absorption enhancers can also be used to increase the flux of the compounds of this technology across the skin. The rate of such flux can be controlled by providing a rate-controlled membrane (e.g., as part of a transdermal patch) or by dispersing the compounds in a polymer matrix or gel.

[0067] In addition to the representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are therefore included in the art of the present invention. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference. Formulations of this technology can be designed to be short-acting, rapid-release, long-acting, and sustained-release, as shown below. Therefore, pharmaceutical formulations can also be formulated for controlled-release or sustained-release. The compositions of the present invention may also include, for example, micelles or liposomes, or several other encapsulated forms, or may be administered in a sustained-release form to provide long-term storage and / or delivery effects. Thus, pharmaceutical formulations and pharmaceuticals can be compressed into pellets or cylinders and implanted as intramuscular or subcutaneous depot injections, or as implants such as stents. Known inert materials such as silicones and biodegradable polymers may be used for such implants.

[0068] The specific dosage can be adjusted according to the disease state, age, weight, general health status, sex, and the subject's diet, administration interval, route of administration, excretion rate, and drug combination. Any of the above dosage forms containing an effective amount are well within the scope of standard experiments and, therefore, well within the scope of the present invention. Those skilled in the art can easily determine an effective dose by simply administering the compounds of this art to a patient in increasing amounts until the desired result is achieved. The compounds of this art can be administered to patients at dose levels ranging from about 0.1 to about 1,000 mg per day. For a normal adult weighing about 70 kg, a dose ranging from about 0.01 to about 100 mg per kg of body weight per day is sufficient. However, the specific dose used may be modified or adjusted as deemed appropriate by those skilled in the art. For example, the dose may depend on several factors, including the patient's requirements, the severity of the condition, and the pharmacological activity of the specific compound being used. Determining the optimal dose for a particular patient is well known to those skilled in the art.

[0069] Various assays and model systems can be readily used to determine the therapeutic effect of treatment using this technology. The effectiveness of the composition and method of this technology can also be demonstrated by a reduction in symptoms. For each of the conditions described herein, compared to placebo treatment or other suitable control subjects, the test subjects will show a reduction of 10%, 20%, 30%, 50% or more, up to 75–90%, or 95% or more in one or more symptoms caused by or associated with the subject's disorder.

[0070] For example, the efficacy of the compounds, compositions, and methods of this technology against cancer and metastatic diseases can be monitored in terms of clinical success by any of the following methods, for example, by tumor imaging using X-ray or MRI (e.g., to determine whether the size or number of tumors in treated patients has decreased). Efficacy can often be determined by X-ray or MRI observation of tumor size reduction. Effective compounds, compositions, and methods of this technology for treating cancer typically result in a reduction of at least 10%, 25%, 50%, 75%, or 90% or more in tumor size in treated patients, or the average tumor size between groups of treated patients, compared to a qualified equivalent control. The efficacy of the compounds, compositions, and methods of this technology against cancer and metastatic diseases can be further determined by measuring the number of circulating tumor cells in blood samples between a suitable test subject and a control. This can be achieved by any applicable means, including but not limited to immunomagnetic selection, flow cytometry, immunobead capture, fluorescence microscopy, cytomorphological analysis, or cell separation techniques. The effective compounds, compositions, and methods of this technology for treating cancer typically result in a reduction of at least 10%, 25%, 50%, 75%, or 90% in circulating tumor cells in blood samples of treated patients or between groups of treated patients, compared to a qualified equivalent control. The efficacy of the compounds, compositions, and methods of this technology against cancer and metastatic diseases can be further determined by detecting or measuring the occurrence or number of primary tumor cells in secondary tissues or organs, including but not limited to bone, lymph nodes, and lungs. The effective compounds, compositions, and methods of this technology for treating cancer typically result in a reduction of at least 10%, 25%, 50%, 75%, or 90% in the occurrence or number of metastatic primary tumor cells in secondary tissues or organs between treated patients, compared to a qualified equivalent control.

[0071] In any embodiment or aspect of this specification, the anti-angiogenic compounds, compositions, and methods of the Technology may be effective in reducing neovascularization of pathological eyes in mammalian subjects. These methods may employ monotherapy or combination therapy. The compounds, compositions, and methods of the Technology are “anti-angiogenic effective,” for example, to reduce the incidence, size, or number of vascular lesions in the ocular tissue of subjects with age-related macular degeneration (AMD). “Reduction in neovascularization” may correspond to the observation of a decrease in the histopathological or ocular angiographic index of AMD lesion size, e.g., a decrease in the incidence, size, number, or distribution of lesions or “foci” of lesions observed in secondary ocular sites. The effectiveness against anti-angiogenicity can be determined by a positive change in the treatment index of one or more patients that correlates with effective prevention and / or treatment of AMD, e.g., by an increase in the duration of disease-free or disease-stable status in subjects receiving the Compounds / Compositions of the Technology compared to a suitable control subject not receiving the Compounds / Compositions of the Technology.

[0072] The anti-AMD lesion efficacy of the compounds, compositions, and methods of this technology, for example, the efficacy of reducing or stabilizing the proliferation of neovascular lesion complexes, may result in substantial therapeutic benefits and improved treatment outcomes in patients treated for ocular conditions (or other pathogenic conditions) involving harmful neovascularization as part of the underlying disease. For example, patients treated with the compounds, compositions, and methods of this technology may show improved treatment outcomes with no increase or with observed reduction of adverse side effects. These benefits, examples of the compounds, compositions, and methods of this technology may result in an increase of at least 20% of one or more positive clinical therapeutic indices, such as a beneficial change in the AMD lesion index (e.g., a decrease in the occurrence, size, number, or distribution of lesions observed in secondary ocular sites or primary lesion "foci"). The anti-AMD lesion efficacy of the compounds, compositions, and methods of this technology may be indirectly demonstrated by an increase of at least 20% in disease-free or disease-stable states in patients treated with the compounds / compositions of this technology compared to the survival rate determined in suitable control patients (not treated with the compounds / compositions of this technology). The compounds, compositions, and methods of this technology may yield even greater anti-AMD clinical benefits, for example, a 20-50% increase in the positive treatment index, a 50-90% increase, and up to a 75-100% increase, including complete remission of observed primary AMD lesions, for example, lasting 6 months to 1 year, 1-2 years, 2-5 years, more than 5 years, and more than 10 years. In subjects treated with the compounds / compositions of this technology versus untreated or placebo-treated subjects, the compounds, compositions, and methods of this technology may be effective anti-AMD treatments resulting in at least a 20% reduction in lesion size, 20-50%, 50-75%, and up to more than 90%. The effectiveness of anti-AMD treatment may also correlate with the absence or reduction of observed symptoms of AMD, such as increased or decreased vision loss, between patients treated with the compounds / compositions of this technology and positive control subjects.For example, subjects treated with monotherapy via the compounds / compositions of this technology, and subjects treated with combination therapies such as the compounds / compositions of this technology plus anti-VEGF therapy, may not show an increase in Snellen's target score compared to positive control subjects treated with conventional (e.g., anti-VEGF) therapy, but may show an increase of at least 20%, 20-50%, or up to 50-90% or more in Snellen's target score.

[0073] The compounds of this technology may also be administered to a patient together with other conventional therapeutic agents that may be useful in treating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angiogenic disorders. Dosage may include oral, parenteral, or nasal administration. In any of these embodiments, administration may include subcutaneous, intravenous, intraperitoneal, or intramuscular injection. In any of these embodiments, administration may include oral administration. The method of this technology may also include administering, sequentially or in combination with one or more compounds of this technology, an amount of a conventional therapeutic agent that may be potentially or synergistically effective in treating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angiogenic disorders.

[0074] In one embodiment, the compounds of the present technology are administered to a patient in an amount or dosage suitable for therapeutic use. Generally, the unit dose containing the compounds of the present technology varies depending on patient considerations. Such considerations include, for example, age, protocol, condition, sex, severity of disease, contraindications, and concomitant therapies. Exemplary unit doses based on these considerations may also be adjusted or modified by physicians in the art. For example, a unit dose to a patient containing the compounds of the present technology might be 1 × 10⁻⁶. -4 g / kg to 1g / kg, preferably 1 × 10 -3 The dosage may vary from / kg to 1.0g / kg. The dosage of the compound in this technology may also vary from 0.01mg / kg to 100mg / kg, or preferably from 0.1mg / kg to 10mg / kg.

[0075] The compounds of this technology can also be modified by covalent bonding or conjugates of organic moieties to improve pharmacokinetic properties, toxicity, or bioavailability (e.g., by increasing the in vivo half-life). The conjugates may be linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. The polymer groups may include molecular weights that can be adjusted by those skilled in the art to improve pharmacokinetic properties, toxicity, or bioavailability. Exemplary conjugates may include polyalkane glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and fatty acid or fatty acid ester groups, each of which may contain about 8 to about 70 carbon atoms. Conjugates for use with the compounds of this technology can also function, for example, as linkers to any suitable substituents or groups, radiolabels (markers or tags), halogens, proteins, enzymes, polypeptides, other therapeutic agents (e.g., pharmaceuticals or drugs), nucleosides, dyes, oligonucleotides, lipids, phospholipids, and / or liposomes. In one embodiment, the conjugate may include polyethyleneamine (PEI), polyglycine, a hybrid of PEI and polyglycine, polyethylene glycol (PEG), or methoxypolyethylene glycol (mPEG). The conjugate may also be used to link the compounds of the Art to, for example, a label (fluorescent or luminescent) or a marker (radionuclide, radioisotope, and / or isotope) to constitute a probe of the Art. In one embodiment, a conjugate for use with the compounds of the Art can improve the in vivo half-life. Other exemplary conjugates for use with the compounds of the Art, as well as their applications and related technologies, include those generally described by U.S. Patent No. 5,672,662, which is incorporated herein by reference.

[0076] In another embodiment, the Art provides a method for identifying a target of interest, comprising contacting the target of interest with a detectable or imaging-effective amount of the Art's labeled compound. The detectable or imaging-effective amount is the amount of the Art's labeled compound required to be detected by a selected detection method. For example, the detectable amount may be a dose sufficient to enable detection of binding of the labeled compound to a target of interest, including, but not limited to, cells or tissues associated with pathogenic cell proliferation, angiogenesis, cancer, metastatic diseases, and / or pathogenic angioproliferative diseases. Suitable labels are known to those skilled in the art and may include, for example, radioisotopes, radionuclides, isotopes, fluorescent groups, biotin (in combination with streptavidin complex formation), and chemiluminescent groups. Once the labeled compound has bound to the target of interest, the target can be further characterized, for example, by isolation, purification, and determination of its amino acid sequence.

[0077] The terms “associate” and / or “bond” may, for example, refer to a chemical or physical interaction between a compound of the Art and a target of interest. Examples of association or interaction include covalent bonds, ionic bonds, hydrophilic-hydrophilic interactions, hydrophobic-hydrophobic interactions, and complexes. Association may also generally refer to “bonding” or “affinity,” as these terms can be used to describe various chemical or physical interactions. Measuring bonding or affinity is also standard practice for those skilled in the art. For example, a compound of the Art may bond to or interact with a target of interest or its precursors, parts, fragments, and peptides, and / or their precipitates.

[0078] Combination therapy As previously shown in this disclosure, in any embodiment or aspect of this specification, the compounds, compositions, or pharmaceutical compositions of any embodiment of the Art may be combined with one or more additional therapies for the prevention or treatment of pathogenic cell proliferation, angiogenesis, cancer, metastatic diseases, and / or pathogenic angiopuroproliferative diseases. Additional therapeutic agents include, but are not limited to, chemotherapeutic agents, immunotherapeutic agents, surgical procedures, radiotherapy, anti-angiogenic agents, nonsteroidal anti-inflammatory drugs, or any combination thereof.

[0079] Additionally or alternatively, in any of the embodiments disclosed herein, additional therapeutic agents may be selected from the group consisting of alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, antimetabolites, immunotherapeutic agents, mitotic inhibitors, nitrogen mustard, nitrosourea, alkyl sulfonates, platinum agents, taxanes, vinca agents, anti-estrogen agents, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, endocrine / hormone agents, bisphosphonate therapeutic agents, phenformin, anti-angiogenic agents, histone deacetylase inhibitors, and nonsteroidal anti-inflammatory drugs (NSAIDs).

[0080] Additionally or alternatively, in any of the embodiments disclosed herein, additional therapeutic agents include cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxane, paclitaxel, ABRAXANE® (albumin-conjugated paclitaxel), protein-conjugated paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifen, toremifene, and fulvestran. Gemcitabine, irinotecan, ixabepyrone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, avalerix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab Oxaliplatin, melphalan, etoposide, mechloretamine, bleomycin, microtubule toxin, nonacetate acetogenin, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altoretamine, 6-mercaptopurine (6-MP), cytarabine, phloxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, camptothecin, 9-nitrocamptothecin, 9-aminocamptothecin Lubifen, Gimatecan, Diflomotecan, BN80927, DX-8951f, MAG-CPT, Amsacrine, Etoposide Phosphate, Teniposide, Azacitidine (Vidaza), Decitabine, Baccatin III, 10-Deacetyltaxol, 7-Xylosyl-10-Deacetyltaxol, Cephalomannin, 10-Deacetyl-7-Epitaxol, 7-Epitaxol, 10-Deacetylbaccatin III, 10-Deacetylcephalomannin, Streptozotocin, Nimustine, Ranimustine, Bendamustine,The chemotherapeutic agent may be selected from the group consisting of uracil mustard, estramustine, mannosulfan, camptothecin, exatecan, ruthecan, lamelin D 9-aminocamptothecin, amsacrine, ellipticin, oulintricarboxylic acid, HU-331, and mixtures thereof.

[0081] In addition or alternatively, in some embodiments, additional therapeutic agents may be antimetabolites selected from the group consisting of 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, and mixtures thereof. In addition or alternatively, in some embodiments, additional therapeutic agents may be taxanes selected from the group consisting of baccatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannin, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetylcephalomannin, and mixtures thereof. Additionally or alternatively, in some embodiments, additional therapeutic agents may be DNA alkylating agents selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, bendamustine, uracil mustard, estramustine, carmustine, lomustine, nimustine, ranimustine, streptozotocin; busulfan, mannosulfan, and mixtures thereof.

[0082] Additionally or alternatively, in some embodiments, additional therapeutic agents may be topoisomerase I inhibitors selected from the group consisting of SN-38, ARC, NPC, camptothecin, topotecan, 9-nitrocamptothecin, exatecan, ruthecan, lamelin D 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, and mixtures thereof. Additionally or alternatively, in some embodiments, additional therapeutic agents may be topoisomerase II inhibitors selected from the group consisting of amsacrin, etoposide, etoposide phosphate, teniposide, daunorubicin, mitoxantrone, amsacrin, ellipticin, oulintricarboxylic acid, doxorubicin, and HU-331 and combinations thereof. In addition or alternatively, in some embodiments, additional therapeutic agents may be immunotherapies selected from the group consisting of immune checkpoint inhibitors (e.g., antibodies targeting CTLA-4, PD-1, and PD-L1), ipilimumab, 90Y-cribatuzumab tetraxetan, pembrolizumab, nivolumab, trastuzumab, cixutumumab, ganitumab, demcizumab, cetuximab, nimotuzumab, darotuzumab, ciproisel T, CRS-207, and GVAX.

[0083] In addition or alternatively, in some embodiments, additional therapeutic agents may be anti-angiogenic agents selected from the group consisting of bevacizumab, sediranib, axitinib, anginex, sunitinib, sorafenib, pazopanib, batalanib, cabozantinib, ponatinib, lenvatinib, SU6668, everolimus (Afinitor®), lenalidomide (Revlimid®), ramucirumab (Cyramza®), regorafenib (Stivarga®), thalidomide (Synovir, Thalomid®), vandetanib (Caprelsa®), and aflibercept (Zaltrap®).

[0084] In addition or alternatively, in some embodiments, additional therapeutic agents may be histone deacetylase inhibitors selected from the group consisting of trichostatin A (TSA), tubasin, apicidine, depsipeptide, MS275, BML-210, RGFP966, MGCD0103, LBH589, spritomycin, FK228, phenylbutyrate, SAHA, bellinostat, panabiostat, gibinostat, resminostat, abexinostat, quisinostat, rosilinostat, prasinostat, CHR-3996, valproic acid, butyrate, entinostat, tasejinarin, 4SC202, mosetinostat, romidepsin, nicotinamide, siltinol, canbinol, and EX-527.

[0085] In addition or alternatively, in some embodiments, the additional therapeutic agent may be an NSAID selected from the group consisting of indomethacin, fenoprofen, ibuprofen, flufenamic acid, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ketorolac, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sarsalate, sulindac, and tolmetine. Examples of antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, phloxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, and mixtures thereof.

[0086] Examples of taxanes include baccatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannin, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetylcephalomannin, and mixtures thereof. Examples of immunotherapies include immune checkpoint inhibitors (e.g., antibodies targeting CTLA-4, PD-1, and PD-L1), ipilimumab, 90Y-cribatuzumab tetraxetan, pembrolizumab, nivolumab, trastuzumab, cyclostomumab, ganitumab, demcizumab, cetuximab, nimotuzumab, darotuzumab, ciproisel T, CRS-207, and GVAX. Examples of anti-angiogenic agents include bevacizumab, cediranib, axitinib, anginex, sunitinib, sorafenib, pazopanib, batalanib, cabozantinib, ponatinib, lenvatinib, SU6668, everolimus (Afinitor®), lenalidomide (Revlimid®), ramucirumab (Cyramza®), regorafenib (Stivarga®), thalidomide (Synovir, Thalomid®), vandetanib (Caprelsa®), and aflibercept (Zaltrap®).

[0087] Examples of histone deacetylase inhibitors include trichostatin A (TSA), tubasin, apicidine, depsipeptide, MS275, BML-210, RGFP966, MGCD0103, LBH589, spritomycin, FK228, phenylbutyrate, SAHA, bellinostat, panabiostat, gibinostat, resminostat, abexinostat, quisinostat, rosilinostat, prasinostat, CHR-3996, valproic acid, butyrate, entinostat, tasejinarin, 4SC202, mosetinostat, romidepsin, nicotinamide, siltinol, canbinol, and EX-527. Examples of NSAIDs include indomethacin, fenoprofen, ibuprofen, flufenamic acid, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ketorolac, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sarsalate, sulindac, and tolmetine.

[0088] In any case, in any embodiment of this specification, multiple therapeutic agents may be administered in any order or simultaneously. If administered simultaneously, the multiple therapeutic agents may be provided in a single unified form or in multiple forms (for example only, as a single injection, two separate injections, or an injection combined with a pill). One of the therapeutic agents may be administered in multiple doses, or both may be administered in multiple doses. If not administered simultaneously, the timing between multiple doses may vary from more than 0 weeks to less than 4 weeks. In addition, the combination methods, compositions, and formulations should not be limited to the use of only two agents.

[0089] kit This disclosure also provides kits comprising compounds and / or compositions of the Technology, as well as instructions for using them to prevent and / or treat pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angiopurogenic disease. Optionally, the above components of a kit of the Technology may be packaged in a suitable container and labeled for the prevention and / or treatment of pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angiopurogenic disease.

[0090] The above components can be stored in unit-dose or multi-dose containers, e.g., sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous, preferably sterile solutions, or, for reconstitution, as lyophilized, preferably sterile formulations. The kit may further include a second container for holding a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable carriers of the pharmaceutical composition and saline solutions. Furthermore, the kit may include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. Containers can be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (for example, a container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous injection needle). The kit may further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and culture media for one or more suitable hosts. The kit may also include instructions that are customarily included in the commercial packaging of the therapeutic or diagnostic product, such as information regarding indications, usage, dosage, manufacturing, administration, contraindications, and / or warnings relating to the use of such therapeutic or diagnostic product.

[0091] The kit may also include, for example, buffers, preservatives, or stabilizers. The kit may also include control samples or a set of control samples, which can be assayed and compared to the test sample. Each component of the kit may be sealed in a separate container, and all the various containers, along with instructions for interpreting the results of assays performed using the kit, may be included in a single package. The kits of this technology may include products indicated on or inside the kit container. The indicated products describe how to use the reagents included in the kit. In certain embodiments, the use of the reagents may follow the methods of this technology. In any embodiment of this specification, the indicated products may direct the execution of the methods according to any embodiment described herein. [Examples]

[0092] The Art is further illustrated by the following examples, which should not be construed as limiting. The examples herein are provided to illustrate the advantages of the Art and to further assist those skilled in the art in preparing or using the compositions and systems of the Art. The examples should not be construed as limiting the scope of the Art as defined by the appended claims. The examples may include or incorporate any of the above-described variations, aspects, or embodiments of the Art. Each of the above-described variations, aspects, or embodiments may also further include or incorporate any other or all variations, aspects, or embodiments of the Art. The following examples demonstrate the preparation, characterization, and use of exemplary compositions of the Art that inhibit the Id protein.

[0093] (Example 1) Synthesis of AGX51. The desired adduct 3 was obtained in high yield by the reaction of 2-methoxycinnamaldehyde 1 with potassium aryl trifluoroborate 2a or a common arylboronic acid 2b (Scheme 1). Aldehyde 3 was reductively aminated with benzylamine and sodium borohydride to obtain amine 4. Amine 4 was acylated with propionyl chloride to obtain the final product 5 (AGX51) in 75% of the total yield.

[0094] Scheme 1. Synthesis of compound AGX51 (compound 5): [ka]

[0095] (Example 2) Synthesis of AGX-A. Compound 7 was produced in one step by conjugate addition of trifluoroborate 6 to aldehyde 1 in the presence of catalytic amounts of palladium dibenzylideneacetone and triphenylphosphine. Reductive amination of aldehyde 7 yielded secondary amine 8 (AGX-A) in high yield as a viscous oil, which was converted to the corresponding hydrochloride salt 9 and isolated as a white solid.

[0096] Scheme 2. Synthesis of compound 8 (AGX-A) and its corresponding hydrochloride salt. [ka]

[0097] (Example 3) Reductive amination using an amine containing a carbonyl group. The two-step reductive amination of aldehyde 3 (Scheme 3) using amines with miscible groups, such as ketones, which can be reduced by reagents such as NaBH4, was instead carried out by hydrogenation using hydrogen under palladium catalysis on charcoal. Compound 10 was obtained by hydrogenating the intermediate imine using 10% Pd on charcoal under a 1 atmosphere of hydrogen.

[0098] Scheme 3. Synthesis of compound 11. [ka]

[0099] (Example 4) Chiral partitioning of AGX51 As shown in Scheme 4, the racemic AGX51 was split using a chiral AS-H preparative column (Chiral Technologies) to extract compound P1 (peak 1, >99%ee, [α] 21.6 D +22.53 (c 0.8, MeOH) and compound P2 (peak 2, approximately 93% ee, [α] 21.6 A value of D -25.77 (c 0.8, MeOH) was obtained.

[0100] Scheme 4. Chiral partitioning of AGX51 [ka]

[0101] (Example 5) Crystallization and X-ray structure determination. Neither P1 nor P2 provided X-ray quality crystals. However, since the corresponding amine salt is crystalline, the amide may be hydrolyzed under certain conditions, which does not impair the chiral center. Therefore, as shown in Scheme 5, the (+)-enantiomer was exposed to 4.0 N HCl in dioxane-water at 85°C for 24 hours, and the corresponding amine was provided after base post-treatment.

[0102] Scheme 5. Chiral partitioning [ka]

[0103] Next, the (+)-chiral amine thus obtained was crystallized with several acids: (+)-camphorsulfonic acid, D-(-)-tartaric acid, L-(+)-tartaric acid, L-(-)-malic acid, and fumaric acid in ethanol (in a toluene external chamber). The crystals were collected and submitted for X-ray crystal structure analysis. Only the crystals from the fumarate diffracted and clearly provided the conformation of the chiral center as the R-(+)-enantiomer. The X-ray structure of P1, the R-(+) enantiomer (i.e., R-(+)-3-(benzo[d][1,3]dioxol-5-yl)-N-benzyl-3-(2-methoxyphenyl)propane-1-amine)) is shown in Figure 20.

[0104] (Example 6) Deacylation of both enantiomers of AGX51. As shown in Scheme 6, deacylation of both optically pure amides P1 and P2 was further achieved by in-situ activation of an inactive tertiary amide followed by the addition of a phenyl Grignard reagent (Huang et al., Tetrahedron, 71(2015): 4248-4254). NMR analysis of both optical amines is identical to the analysis of amine 4 synthesized in Scheme 1.

[0105] Scheme 6. Deacylation of both enantiomers of AGX51. [ka]

[0106] (Example 7) Synthesis of crosslinker compound 16. Furthermore, as shown in Scheme 7, direct chiral synthesis could be achieved by organocatalysis using a chiral catalyst such as R-(C7F7)2-BINOL (Angew. Chem. Int. Ed. Eng., 54(34): 9931-9935 (2015)). In fact, when aldehyde 1 was reacted with trifluoroborate 2a using molecular sieves in the presence of a catalytic amount of R-(C7F7)2-BINOL in toluene at 95 °C, chiral aldehyde 3 was produced, which was advanced to the corresponding chiral amine 12. Following functional group manipulations to set the trifluoroacetamide and remove the Boc protecting group, reductive amination of the amine obtained using aldehyde 15 gave diazirine 16 showing a specific rotation of [α] 22 D -70.39 (c 1.0, CH2Cl2). The enantiomeric excess was not determined. Aldehyde 15 was the product of Dess-Martin periodinane oxidation of alcohol 14.

[0107] Scheme 7. Synthesis of crosslinker compound 16.

Chemical formula

[0108] (Example 8) Synthesis of AGX-E. As illustrated in Scheme 8, additional analogs were modified by functional group manipulations. The chloride of compound 17 (AGX-C) was substituted with acetate using sodium acetate under microwave irradiation to produce AGX-D (18), which was hydrolyzed to produce AGX-E (19) (Scheme 6).

[0109] Scheme 8. Late-stage modification.

Chemical formula

[0110] (Example 9) Synthesis of more AGX51 analogs. As shown in Scheme 9, compounds 21(AGX-F) and 24(AGX-H) were provided in good yield by amide formation with secondary amine 4 and corresponding acids 20 and 22 (Scheme 7).

[0111] Scheme 9. Synthesis of more AGX51 analogs. [ka]

[0112] (Example 10) Crosslinker AGX51 analog synthesis. As shown in Scheme 10, several crosslinks and fluorescent probes were also synthesized. The AGX51-BODIPY analog can be prepared by coupling amine 4 and PEG spacers 28 and 31 with the BODIPY NHS ester 25 (Scheme 7).

[0113] Scheme 10. Synthesis of cross-linker AGX51 analogs. [ka]

[0114] (Example 11) Experimental Procedure Typical procedure A for the conjugate addition of 2a organic potassium trifluoroborate to 2-methoxycinnamaldehyde. In a flask, under argon, 6.183 g, 27 mmol, 2.5 equivalents of 2a organic potassium trifluoroborate, 1.76 g, 10.85 mmol, 122 mg, 0.54 mmol, 5 mol%, 339 mg, 2.17 mmol, 20 mol%, 10 mL of HOAc, 5.4 mL of THF, and 3.2 mL of H₂O were added. The mixture was stirred and heated at 60°C for 2-3 days. The reaction mixture was cooled to room temperature, filtered, and washed with ethyl acetate. The filtrate was neutralized with saturated NaHCO₃ and then extracted with ethyl acetate (x3). The combined organic layers were washed with saturated NaCl, dried, and concentrated. The residue was purified using an ISCO CombiFlash SiO2 (12g) column (20% ethyl acetate / cyclohexane) to obtain product 3 (2.83g) as a yellow oil in 92% yield.

[0115] Typical procedure B for conjugate addition of arylboronic acid to 2-methoxycinnamaldehyde. Under argon, arylboronic acid 6a (2.82 g, 15.67 mmol, 2.5 equivalents), 2-methoxycinnamaldehyde 1 (1 g, 6.17 mmol), Pd(OAc)2 (69 mg, 0.308 mmol, 5 mol%), bpy (192 mg, 1.22 mmol, 20 mol%), HOAc (6 mL), THF (3 mL), and H2O (1.8 mL) were added to a flask. The mixture was stirred and heated at 60°C for 2-3 days. The reaction mixture was neutralized with saturated NaHCO3 and then extracted with ethyl acetate (×3). The combined organic layer was washed with saturated NaCl, dried, (Na2SO4) and concentrated. The residue was purified using an ISCO CombiFlash SiO2 (24g) column (25% ethyl acetate / cyclohexane) to obtain product 7 (1.46g) as a yellow oil in 80% yield.

[0116] Basic Procedure C Reductive Amination: To a solution of aldehyde 3 (7.66 g, 26.96 mmol) and benzylamine (3.17 g, 29.66 mmol) in dichloromethane (150 mL), anhydrous magnesium sulfate (4.85 g, 40.44 mmol) was added. After stirring under reflux for 1 hour, the reaction mixture was filtered to remove the drying agent, and the solvent was removed under reduced pressure. Next, the crude imine was dissolved in methanol (100 mL), and sodium borohydride (2.04 g, 53.92 mmol) was added while stirring at 0°C. After stirring for another 1 hour under reflux, the reaction mixture was quenched with water, concentrated, and methanol was removed. The residue was diluted with dichloromethane and washed with water. The aqueous layer was extracted with dichloromethane (×3), and the combined organic layers were washed with saturated NaCl, dried, and concentrated in (Na2SO4). The residue was purified using an ISCO CombiFlash SiO2 (120g) column (5% MeOH / ethyl acetate) to obtain product 5 (9.36g) as a yellow oil in 92% yield.

[0117] 3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propanal 3. 1 H NMR (CDCl, 600 MHz) δ 9.69 (t, J =2.2 Hz, 1H), 7.19 (dt, J = 7.5, 1.6 Hz, 1H), 7.07 (dd, J = 7.6, 1.5 Hz, 1H), 6.9 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.73 (m, 3H), 5.91 (s, 2H), 4.95 (t, J = 7.9 Hz, 1H), 3.82 (s, 3H), 3.05 (dd, J = 7.9, 2.2 Hz, 2H); 13 C NMR (CDCl3, 150 MHz) δ 201.75, 156.53, 147.71, 146.06, 136.75, 131.65, 127.88, 127.84, 120.88, 120.71, 110.78, 108.66, 108.14, 100.92, 55.40, 48.63, 37.96.

[0118] N-benzyl-3-(4-isopropoxyphenyl)-3-(2-methoxyphenyl)propan-1-amine 4 1 H NMR (CDCl3, 600 MHz) δ 7.30-7.19 (m, 6H), 7.15 (dt, J = 8.1, 1.4 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.73 (m, 2H), 6.68 (d, J = 8.0 Hz, 1H), 5.86 (s, 2H), 4.41 (t, J = 7.8 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 2H), 2.60 (t, J = 7.1 Hz, 2H), 2.17 (m, 2H); 13 C NMR (CDCl3, 150 MHz) δ 156.99, 147.58, 145.72, 140.67, 138.46, 133.46, 128.51, 128.23, 127.66, 127.32, 127.00, 121.10, 120.82, 110.84, 108.80, 108.10, 100.89, 55.61, 54.03, 47.95, 40.74, 35.45.

[0119] N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzylpropionamide 5 (AGX51) Basic Procedure D: To a solution of amine 8 (6.14 g, 16.37 mmol) and triethylamine (45.6 mL, 32.75 mmol) in anhydrous dichloromethane (100 mL), propionyl chloride (3.57 mL, 40.92 mmol) was added dropwise at 0°C. The reaction mixture was left at room temperature overnight, and the resulting solution was poured into water and separated. The aqueous layer was extracted with dichloromethane (×3). The combined organic layers were washed with saturated NaCl, then dried with (Na2SO4) and concentrated. The residue was purified by ISCO CombiFlash SiO2 (120 g) column (30-40% ethyl acetate / hexane) to obtain product 10 (6.35 g) as a viscous yellow oil in 90% yield. The resulting clear viscous syrup was treated with ethanol (9 mL) and mixed well. After being left in the freezer overnight, a white precipitate formed. The white solid was filtered and washed with cold ethanol to obtain 5.77 g (AGX51) of dry white powder. Mp: 87~88°C. 1 H NMR (DMSO-d6, 600 MHz) δ 7.30-7.18 (m, 4H), 7.15 (m, 1H), 7.08 (m, 2H), 6.92-6.88 (m, 2H), 6.84-6.74 (m, 2H), 6.68 (m, 1H), 5.93 (m, 2H), 4.54-4.40 (m, 2H), 4.18 (t, J = 7.9 Hz, 1H), 3.73, 3.72 (s, s, 3H), 3.16-2.98 (m, 2H), 2.54-2.05 (m, 4 H), 0.95 (t, J = 7.3Hz, 3H); 13C NMR (CDCl3, 150 MHz) δ 173.91, 173.83, 156.68, 147.66, 147.41, 145.91, 145.59, 138.34, 137.96, 137.47, 137.21, 132.79, 132.26, 128.80, 128.45, 128.24, 127.58, 127.44, 127.33, 127.19, 127.16, 126.34, 120.83, 120.81, 120.72, 120.68, 110.72, 110.66, 108.56, 108.48, 108.11, 107.95, 100.87, 100.70, 55.41, 55.37, 51.48, 48.05, 45.99, 45.36, 40.90, 40.50, 33.61, 32.54, 26.55, 26.02, 9.67, 9.48; ; High decomposition energy MS C 27 H 30 NO 24 The calculated value is 432.2175, the measured value is 432.2191 (M+H).

[0120] 3-(4-イソプロポキシフェニル)-3-(2-メトキシフェニル)プロパナール7. 1 H NMR (CDCl3, 600 MHz) δ 9.69 (t, J = 2.3 Hz, 1H), 7.18 (dt, 1H, J = 8.1, 1.7 Hz), 7.14 (m, 2H), 7.05 (dd, J = 7.6, 1.6 Hz, 1H), 6.88 (dt, J = 7.5, 0.9 Hz, 1H), 6.85 (d, J = 8.2, 0.9 Hz, 1H), 6.80 (m, 2H), 4.96 (t, J = 7.9 Hz, 1H), 4.49 (m, 1H), 3.81 (s, 3H), 3.05 (dd, J = 7.9, 2.3 Hz, 2H), 1.31(d, J = 6.0 Hz, 6H); 13C NMR (CDCl3, 150 MHz) δ 202.15, 156.57, 156.43, 134.55, 132.05, 129.03, 128.08, 127.68, 120.67, 115.72, 110.73, 69.79, 55.39, 48.63, 37.53, 22.10.

[0121] N-benzyl-3-(4-isopropoxyphenyl)-3-(2-methoxyphenyl)propan-1-amine 8(AGX-A). 1 H NMR (CDCl3, 600 MHz) δ 7.29-7.18 (m, 6H), 7.16-7.12 (m, 3H), 6.88 (dt, J = 7.5, 0.9 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.75 (m, 2H), 4.48-4.41 (m, 2H), 3.75(s, 3H), 3.71 (s, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.19 (m, 2H), 1.29 (d, J = 6.1 Hz, 6H); 13 C NMR (CDCl3, 150 MHz) δ 156.90, 156.04, 140.54, 136.62, 133.71, 129.02, 128.09, 127.72, 127.00, 126.83, 120.64, 115.53, 110.68, 69.77, 55.47, 53.88, 47.91, 40.09, 35.29, 30.39, 22.18; High resolution MS C 26 H 32 The calculated value for NO2 was 390.2433, and the measured value was 390.2426 (M+H).

[0122] Formation of AGX-A hydrochloride 9: Benzylamine AGX-A 8 (9.92 g, 25.5 mmol) in dichloromethane (40 mL) was treated with HCl (2 N in ether, 14 mL, 28 mmol) in an ice bath. The mixture was stirred for 2 h. After removing the solvent, the sticky syrup was treated twice with hexane and concentrated under reduced pressure. The syrup (5 g) was triturated with ethyl acetate (10 mL) to form a white precipitate. The white solid was filtered and washed with hexane to give AGX-A HCl 9 as a white powder (10.6 g). Mp: 158 - 160 °C. (4-(((3-(Benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)amino)methyl)phenyl)(phenyl)methanone 10. 4-(Aminomethyl)phenyl](phenyl)methanone hydrochloride (66 mg, 0.27 mmol) was pretreated with K2CO3 (5 eq) in dichloromethane (10 mL) and water (2 mL). After stirring at room temperature for 2 h, the aqueous layer was extracted with dichloromethane (×3), the combined organic layers were washed with saturated NaCl, then dried (Na2SO4) and concentrated to give a colorless oil for direct use in the next step.

[0123] The resulting free amine was treated with aldehyde 3 using basic procedure C to give an imine, which was treated with 10% Pd / C (48 mg) in MeOH (4 mL) and dichloromethane (2 mL) using a hydrogen balloon for 2 h. The reaction mixture was filtered through celite and washed with a mixture of MeOH and dichloromethane. After removing the solvent, the residue was purified by ISCO combiflash SiO2 (4 g) column (5% MeOH / dichloromethane) to give product 10 (60 mg) as a foamy solid in 50% yield. 1H NMR (CDCl3, 500 MHz) δ 7.75 (m, 4H), 7.59 (t, J = 7.4 Hz, 1H), 7.53 (d, J = 7.9 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H),7.13 (m, 2H), 6.86 (t, J = 7.4 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.70 (m, 2H), 6.63 (d, J =8.3 Hz, 1H), 5.83 (s, 2H), 4.36 (t, J = 7.5 Hz, 1H), 3.97 (s, 2H), 3.75 (s, 3H), 2.71 (t, J = 7.3 Hz, 2H), 2.41 (m, 2H); 13 C NMR (CDCl3, 125 MHz) δ 195.06, 180.18, 170.20, 155.69, 146.56, 144.86, 136.57, 136.34, 136.29, 131.63, 130.96, 129.52, 129.04, 128.27, 127.37, 126.58, 126.48, 119.85, 119.81, 109.76, 107.51, 107.06, 99.82, 59.43, 54.49, 39.57, 20.08, 13.22.

[0124] N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-(4-benzoylbenzyl)propionamide 13. The above amine 10 was treated with propionyl chloride (29 μL, 0.313 mmol), followed by treatment according to basic procedure D, to obtain compound 11 (66 mg, 98%) as a sticky syrup. 1H NMR (CDCl3, 600 MHz) δ 7.81-7.70 (m, 4H), 7.60 (m, 1H), 7.49 (m, 2H), 7.25-7.12 (m, 4H), 6.91 (m, 1H), 6.85-6.79 (m, 1H), 6.75-6.67 (m, 3H), 5.88-5.84 (m, 2H), 4.69-4.51 (m, 2H), 4.31-4.23(t, t, J = 7.9, 7.9 Hz, 1H), 3.78, 3.75 (s, s, 3H), 3.34, 3.17 (t, t, J = 7.6, 7.6 Hz, 2H), 2.30-2.22 (m, 4H), 1.12 (t, J = 7.4 Hz, 3H); 13 C NMR (CDCl3, 150 MHz) δ 196.35, 196.21, 174.07, 173.93, 156.68, 147.71, 147.48, 145.99, 145.67, 142.84, 142.05, 138.21, 137.64, 137.45, 137.35, 136.91, 136.54, 132.69, 132.56, 132.14,130.68, 130.38, 130.02, 130.00, 128.36, 128.29, 127.87, 127.69, 127.31, 127.13, 126.21, 120.80, 120.78, 120.72, 110.78, 110.72, 108.53, 108.44, 108.16, 108.00, 100.92, 100.75, 60.41, 55.45, 55.42, 51.36, 48.12, 46.14, 45.81, 40.90, 40.47, 33.71, 32.58, 26.60, 26.02, 21.07, 9.66, 9.47.

[0125] Preparation of chiral AGX51 using a chiral column. Racemic AGX515 (1 g) was divided using a Waters chiral AS-H preparative column eluted at 15% MeOH / liquid CO2 3.5 ml / min to obtain AGX51 P1 (99% ee), 460 mg syrup, [α] = 22.53 (c = 0.8 MeOH) and AGX51 P2 (93% ee), 480 mg syrup, [α] = -25.77 (c = 0.8 MeOH).

[0126] Basic procedure for directly converting a chiral tertiary amide to a chiral amine. 1.2 equivalents of Tf2O were added dropwise to a chilled (-78°C) solution of amide AGX51 (P1) and 2,6-di-tert-butyl-4-methylpyridine (DTBMP, 1.2 equivalents) in CH2Cl2 (5 mL). The reaction mixture was warmed to 0°C over 2 hours. A solution of Grignard reagent PhMaBr (1N THF, 1.0 equivalent) was added dropwise to the resulting mixture at -78°C and stirred at the same temperature for 2 hours. The reaction mixture was then quenched with an aqueous solution of NH4Cl. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (×3). The combined organic layers were washed with brine, dried, and concentrated with (Na2SO4). The residue was purified using an ISCO CombiFlash SiO2 column (50-70% ethyl acetate / hexane) to obtain chiral product 4. Amine 4P1, AGX51(P1), [α] 21 It was obtained from D 39.66 (c 1.7, MeOH). Amine 4P2 was converted to AGX51 (P2), [α] 21 It was obtained from D -38.49 (c 1.3, MeOH).

[0127] 3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propanal (3). 0.7 g of powdered 4 Å molecular sieve was added to a flask fitted with a stirring bar, and the flask was flame-dried under reduced pressure. The flask was cooled to room temperature under argon. Aldehyde 1 (100 mg, 0.616 mmol), ligand R-(C7F7)2BINOL (88 mg, 0.12 mol, 0.2 equivalents), and potassium trifluoroborate aryl salt 2a (422 mg, 1.85 mmol, 3 equivalents) were added, followed by anhydrous toluene (12 mL). The reaction mixture was heated at 95°C for 3 days. Next, the reaction mixture was filtered through Celite and washed with ether. After concentration, the residue was purified using a 4g silica gel column with elution using 5-10% acetone / hexane to obtain product 3 (70mg, 40%), and the starting material aldehyde 1 was recovered. 1 H NMR (CDCl3, 600 MHz) δ 9.69 (t, J =2.2 Hz, 1H), 7.19 (dt, J = 7.5, 1.6 hz, 1H), 7.07 (dd, J = 7.6, 1.5 Hz, 1H), 6.9 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.73 (m, 3H), 5.91 (s, 2H), 4.95 (t, J = 7.9 Hz, 1H), 3.82 (s, 3H), 3.05 (dd, J = 7.9, 2.2 Hz, 2H); 13 C NMR (CDCl3, 150 MHz) δ 201.75, 156.53, 147.71, 146.06, 136.75, 131.65, 127.88, 127.84, 120.88, 120.71, 110.78, 108.66, 108.14, 100.92, 55.40, 48.63, 37.96. [α] 22 D -70.39 (c 1.0, CH2Cl2).

[0128] tert-butyl(4-(((3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)amino)methyl)phenyl)carbamate 12 Basic procedure. A mixture of aldehyde 3 (20 mg, 0.07 mmol), tert-butyl (4-(aminomethyl)phenyl) carbamate (18 mg, 0.081 mmol), and acetic acid (5 μL, 0.086 mmol) in ClCH2CH2Cl (1 ml) was stirred at room temperature for 30 minutes, after which sodium triacetoxyborohydride (37 mg, 0.175 mmol) was added. The resulting mixture was stirred at room temperature for 18 hours. The mixture was concentrated, dissolved in MeOH, and purified by 4 g silica gel column to obtain product 12 (25 mg, 60%) as a colorless oil. 1 H NMR (CDCl3, 600 MHz) δ 7.26 (m, 1H), 7.19-7.13 (m, 4H), 6.89 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.71-6.66 (m, 3H), 6.55 (brs, 1H), 6.23 (br, 2H), 5.87 (s, 2H), 4.34 (t, J = 7.9 Hz, 1H), 3.75 (s, 3H), 3.73(s, 2H), 2.61 (m, 2H), 2.23 (m, 2H), 1.51(s, 9H); 13 C NMR (CDCl3, 150 MHz) δ 156.73, 147.49, 145.70, 138.05, 137.87, 132.68, 129.46, 127.75, 120.89, 120.75, 118.56, 110.71, 108.57, 108.00, 100.76, 80.57, 55.45, 51.79, 46.38, 40.57, 33.44, 28.35; [α] 22 D -30.45 (c 1.25, MeOH); High resolution MS C 29 H 35 The calculated value for N2O5 is 491.2546, and the measured value is 491.2534 (M+H).

[0129] N-(4-aminobenzyl)-N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-2,2,2-trifluoroacetamide 13. A solution of amine 12 (25 mg, 0.051 mmol) and triethylamine (21 μl, 0.153 mmol) in CH2Cl2 (1 mL) was treated with anhydrous trifluoroacetic acid (18 μL, 0.127 mmol) at room temperature. After stirring the reaction mixture for 4 hours, the solution was concentrated and purified by sieving on a 4 g silica gel column to obtain the product (18 mg, 60%). 1 H NMR (CDCl3, 600 MHz) δ 7.30 (m, 1H), 7.21-7.15 (m, 1H), 7.10 (t, J = 8.9 Hz, 1H), 6.92 (dd, J = 8.3, 16.7 Hz, 2H), 6.92-6.80 (m, 2H), 6.72-6.67 (m, 3H), 6.48 (d, J = 19.1Hz, 1H), 5.89 (m, 2H), 4.51 (s, 1H), 4.46(s, 1H), 4.22 (m, 1H), 3.78, 3.76 (s, s, 3H), 3.22 (m, 2H), 2.28-2.14 (m, 2H), 1.51 (s, 9H); 19 F decoupling H δ -67.96, -68.96; [α] 22 D -24.02 (c 0.85, MeOH); ES MS C 31 H 33 The calculated value for FN2O6 was 586.23, and the measured value was 609.3 (M+Na).

[0130] The intermediate prepared above (17 mg, 0.029 mmol) was dissolved in dioxane / HCl solution (4N, 0.2 mL) and MeOH (1 mL) and stirred at room temperature for 3 hours. The reaction mixture was concentrated, and the residue was converted to a free base with concentrated NaHCO3 aqueous solution. This material was purified by chromatography with silica eluted by 5% MeOH in CH2Cl2 to obtain product 13 (13 mg, 92%). 1H NMR (CDCl3, 600 MHz) δ 7.19-7.15 (m, 1H), 7.10 (m, 1H), 6.93-6.80 (m, 4 H), 6.72-6.66 (m, 3H), 6.61 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 5.89 (dd, J = 4.9, 12.9 Hz, 2H), 4.46 (s, 1H), 4.39 (s, 1H), 3.78, 3.76 (s, s, 3H), 3,20 (m, 2H), 2.29-2.10 (m, 2H); ); 19 F decoupling H δ -67.82, -68.94; [α] 22 D -29.16 (c 0.55, MeOH); ES MS C 26 H 25 The calculated value for N2O4 was 486.18, and the measured value was 509.3 (M+Na).

[0131] N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-(4-((2-(3-(buto-3-in-1-yl)-3H-diazilin-3-yl)ethyl)amino)benzyl)-2,2,2-trifluoroacetamide 16. Compound 16 can be synthesized using a basic procedure involving the treatment of amine 13 (13 mg, 0.0267 mmol) and aldehyde 15 (5 mg, 0.04 mmol). 1 H NMR (CDCl3, 600 MHz) δ 7.19-7.15 (m, 1H), 7.10 (m, 1H), 6.93-6.80 (m, 4 H), 6.72-6.66 (m, 3H), 6.51 (d, J = 8.5 Hz, 1H), 6.47 (d, J = 8.5 Hz, 1H), 5.89 (m, 2H), 4.46 (s, 1H), 4.22 (s, 1H), 3.78, 3.76 (s, s, 3H), 3.20 (m, 2H), 2.92 (m, 2H), 2.25-2.04 (m, 2H), 2.01 (m, 3H), 1.80 (m, 2H), 1.67 (m, 2H); 19F decoupling H δ -67.79, -68.94; ES MS C 33 H 33 F3N 24 The calculated value for O4 was 606.25, and the measured value was 607.3 (M+H).

[0132] 2-((3,4-dimethoxybenzyl)(3-(4-methoxyphenyl)-3-phenylpropyl)amino)-2-oxoethyl acetate (22, AGX-D). A mixture of GY-AGX-C17 (47 mg, 1 mmol) in benzene (1 mL) in a microwave tube was mixed with NaOAc (52 mg, 6 mmol), followed by tetrabutylammonium bromide (TBABr, 6 mg, 0.02 mmol). The reaction mixture was heated under microwave at 120°C for 30 minutes. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified using a silica column (4 g) eluted with 50% EA / hexane to obtain product 18 (46 mg, 93%). 1 H NMR (CDCl3, 600 MHz) δ 7.28-7.25 (m, 2H), 7.17-7.11 (m, 3H), 7.08 (m, 2H), 6.83-6.63 (m, 5H), 4.69, 4.51 (s, s, 2H), 4.45, 4.27 (s, s, 2H), 3.88-3.74 (m, 9H), 3.30, 3.05 (m, 2H), 2.29 (m, 2H), 2.17 (s, 3H); ES MS C 29 H 33 The calculated value for NO6 was 491.23, and the measured value was 492.3 (M+H).

[0133] N-(3,4-dimethoxybenzyl)-2-hydroxy-N-(3-(4-methoxyphenyl)-3-phenylpropyl)acetamide (19, AGX-E). To the reaction mixture of acetate 18 (42 mg, 0.085 mmol) in MeOH (1 mL), K2CO3 (38 mg, 0.26 mmol) was added and heated at 50°C for 2 hours. The mixture was filtered, washed with DCM, and the solvent was concentrated. The resulting residue was purified using a silica (4 g column) eluted with 50% EA / hexane to obtain product 19 (32 mg, 86%). 1 H NMR (CDCl3, 600 MHz) δ 7.29-7.24 (m, 2H), 7.20-7.16 (m, 3H), 7.14-7.07 (m, 2H), 6.84-6.74 (m, 3H), 6.70-6.50 (m, 2H), 4.55(s, 1H), 4.15 (s, 1H), 3.92 (d, J = 4.1 Hz, 1H), 3.86-3.71 (m, 9H), 3.65, 3.61 (m, 1H), 3.35 (m, 1H), 2.95 (m, 1H), 2.31-2.21 (m, 2H); ES MS C 27 H 31 The calculated value for NO5 was 449.22, and the measured value was 450.3 (M+H).

[0134] A classic amide formation method. Amine 4 (68 mg, 0.18 mmol) and carboxylic acid (1.2 equivalents) in DCM (2 mL) were treated with EDC (1.3 equivalents, 45 mg) and HOBt (1.3 equivalents, 32 mg), followed by treatment with DIEPA (4 equivalents, 126 μL). The reaction mixture was stirred overnight at room temperature, diluted with DCM, and washed with saturated NaHCO3. The organic layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by eluting silica (4 g column) with 5% MeOH / DCM to obtain products 21 (53 mg, 65%) and 23 (75%).

[0135] N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzyl-2-(dimethylamino)acetamide(21,AGX-F). 1H NMR (CDCl3, 600 MHz) δ 7.35-7.03 (m, 7H), 6.94-6.79 (m, 2H), 6.73-6.66 (m, 3H), 5.86 (m, 2H), 4.65-4.35 (m, 2H), 4.19 (m, 1H), 3.94-3.54 (m, 5H), 3.35, 3.05 (m, 2H), 2.91-2.86 (m, 6H), 2.16 (m, 2H); ES MS C 28 H 32 The calculated value for N2O4 is 460.24, and the measured value is 461.2 (M+H).

[0136] 2-((3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)(benzyl)amino)-2-oxoethylacetate(23,AGX-G). 1 H NMR (CDCl3, 600 MHz) δ 7.35-7.24 (m, 3H), 7.20-7.09 (m, 4H), 6.91-6.78 (m, 2H), 6.67 (m, 3H), 5.90 (2H), 4.66-4.55 (m, 2H), ES MS C 28 H 29 The calculated value for NO6 was 475.20, and the measured value was 476.2 (M+H). N-(3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)-N-benzyl-2-hydroxyacetamide(24,AGX-H). Compound 24 can be obtained using the same method as that used to produce compound 19. 1H NMR (CDCl3, 600 MHz) δ 7.31-7.25 (m, 3H), 7.18-7.03 (m, 4H), 6.90 (m, 1H), 6.84 (m, 1), 6.71-6.65 (m, 3H), 5.89-5.85 (m, 2H), 4.63 (s, 1H), 4.30-4.14 (m, 3H), 3.99 (s, 2H), 3.78, 3.75(s, s, 3H), 3.36, 2.96 (m, T, 2H), 2.24-2.14 (m, 2H); 26 H 27 The calculated value for NO5 was 433.19, and the measured value was 434.2 (M+H).

[0137] General coupling reaction of amine 4 and NHS ester 25: To a solution of amine 4 (1 equivalent) and NHS ester 25 (1 equivalent) in DMF (1 mL), DIEPA (3 equivalents) was added. The reaction mixture was stirred overnight at room temperature and then purified. Hydrolysis of the NBoc reaction: AGX-PEG analogs 28 and 31 in MeOH (1 mL) were treated with 4N HCl (10 equivalents in doxane). The reaction mixture was stirred at room temperature for 4 hours, then concentrated and dried under reduced pressure. Each HCl salt was used directly in the next step without purification. HPLC purification of AGX-BODIPY analog: A solution of the crude BODIPY mixture in MeOH and water is injected into an HPLC XBridge™ preparative C18 column (5 μm, 19 × 150 mm), eluted with 60-90% ACN / water (0.05% TFA in each solution) for 10 minutes, and the product is collected in clumps at a flow rate of 20 ml / min.

[0138] tert-butyl(2-(3-((3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)(benzyl)amino)-3-oxopropoxy)ethyl)carbamate 28. 1H NMR (CDCl3, 600 MHz) δ 7.33-7.24 (m, 3H), 7.20-7.08 (m, 4H), 6.92-6.79 (m, 2H), 6.70 (m, 3H), 5.90-5.86 (m, 2H), 5.07, 4.98 (brs, 1H), 4.57, 4.45 (s,s, 2H), 4.26 (m, 1H), 3.81, 3.74 (s, s, 3H), 3.73 (m, 2H), 3.48 (m, 2H), 3.33-3.13 (m, 4H), 2.56, 2.43 (t, t, 2H), 2.20 (m, 2H), 1.43, 1.41 (s, s, 9H); ES MS C 34 H 42 The calculated value for N2O7 is 590.30, and the measured value is 591.3 (M+H).

[0139] tert-butyl(2-(2-(3-((3-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)propyl)(benzyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate 31. 1 H NMR (CDCl3, 600 MHz) δ 7.32-7.22 (m, 3H), 7.21-7.07 (m, 4H), 6.92-6.79 (m, 2H), 6.69 (m, 3H), 5.90-5.86 (m, 2H), 5.05, 4.98 (brs, 1H), 4.56, 4.46 (s,s, 2H), 4.29-4.11 (m, 1H), 3.80 (m, 2H), 3.78, 3.75 (s, s, 3H), 3.58-3.50 (m, 6H), 3.33-3.14 (m, 4H), 2.60, 2.52 (t, t, 2H), 2.15 (m, 2H), 1.42 (s, 9H); ES MS C 36 H 46 The calculated value for N2O8 was 634.33, and the measured value was 635.3 (M+H).

[0140] AGX-BODIPY26. 1H NMR (CDCl3, 600 MHz) δ 10.35 (brs, 1H), 7.28 (m, 2H), 7.17-7.02 (m, 6 H), 6.97 (m, 2H), 6.91-6.58 (m, 7H), 6.36 (m, 1H), 6.21(m, 1H), 5.88(m, 2H), 4.59-4.42 (m, 1H), 4.29-4.15 (m, 1H), 3.75, 3.68 (s, s, 3H), 3.37-3.11 (m, 4H), 2.78-2.63 (m, 2H), 2.23-2.08 (m, 2H); 19 F NMR (CDCl3, 600 MHz) δ -75.67 (TFA), -140.09 (m, BF); ES MS C 40 H 37 The calculated value of BF2N4O4 is 686.29, and the measured value is 687.3 (M+H).

[0141] AGX-PEG1-BODIPY29. 1 H NMR (CDCl3, 600 MHz) δ 10.38 (brs, 1H), 7.21-7.16 (m, 2H), 7.12-7.04 (m, 3H), 7.02-6.92 (m, 5H), 6.90-6.77 (m, 4H), 6.66 (m, 3H), 6.35 (m, 1H), 6.26 (m, 1H), 5.86 (m, 2H), 4.51, 4.37 (s, s, 2H), 4.20 (m, 1H), 3.74 (s, 3H), 3.66 (m, 2H), 3.47-3.40 (m, 4H), 3.32-3.10 (m, 4H), 2.70 (m, 2H), 2.49, 2.40 (t, t, 2H), 2.11 (m, 2H); 19 F NMR (CDCl3, 600 MHz) δ -75.84 (TFA), -140.08 (m, BF); ES MS C 45 H 46 The calculated value of BF2N5O6 is 801.35, and the measured value is 802.3 (M+H). AGX-PEG2-BODIPY32. 1H NMR (CDCl3, 600 MHz) δ 10.38 (brs, 1H), 7.24-6.95 (m, 9H), 6.88-6.78 (m, 3H), 6.67 (m, 3H), 6.56 (m, 1H), 6.35-6.26 (m, 2H), 5.88 (m, 2H), 4.51, 4.37 (s, s, 2H), 4.25 (m, 1H), 3.73 (m, 4H), 3.51 (m, 5H), 3.41 (m, 2H), 3.33-3.08 (m, 4H), 2.66-2.43 (m, 4H), 2.16 (m, 2H); 19 F NMR (CDCl3, 600 MHz) δ -75.76 (TFA), -140.06 (m, BF); ES MS C 47 H 50 The calculated value for BF2N5O7 is 845.38, and the measured value is 846.3 (M+H).

[0142] (Example 12) Details of the experimental model and subject In vivo animal studies. Animal studies were conducted in an open-label manner in accordance with facility regulations (MO16M130 for CNV angiogenesis studies, M016M138 for ROP studies). Pharmacokinetic analysis. To determine the pharmacokinetic parameters of AGX51, 8-week-old male Balb / c mice (Taconic farms) were administered a single dose by intravenous injection (ip) using 30 mg / kg, 50 mg / kg, or 100 mg / kg of AGX51 prepared in 70% DMSO (n = 3 mice per group). Another set of 3 mice was administered 100 mg / kg of AGX51 prepared in 100% DMSO. Blood samples were collected 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours after AGX51 administration, and plasma was analyzed by LC-MS according to a protocol previously validated at the MSKCC Antitumor Assessment Core Facility. After blood collection, the mice were euthanized by CO2 asphyxiation, and the eyes of the 30 mg / kg treatment group were collected and flash-frozen for analysis. Data obtained from LC-MS were analyzed for pharmacokinetic parameters via WinNonLin software (version 8.1).

[0143] Toxicity analysis. To assess toxicity, 6-8 week old female athymoid nude mice (Envigo) were administered either a control vehicle (70% DMSO in water) or AGX51 at a dose of 60 mg / kg twice daily for 14 consecutive days. Mice were euthanized 24 hours after the last test dose. Macroscopic and complete necropsy, as well as clinicopathological analysis, were performed on all mice. Measured clinicochemical parameters included BUN, creatine, ALP, ALT, AST, GGT, bilirubin, total protein, albumin, globulin, phosphorus, glucose, cholesterol, phosphorus, calcium, sodium, potassium, and chloride. Measured hematological parameters included leukocytes (lymphocytes, monocytes, eosinophils, basophils, neutrophils), erythrocytes, hemoglobin, hematocrit, MCV, MCH, MCHC, RDW, and platelets. The organs / tissues analyzed were the lungs, heart, thymus, kidneys, liver, spleen, gallbladder, pancreas, duodenum, jejunum, ileum, cecum, colon, bone marrow, femur, tibia, sternum, brain, eyes, ears, nasal and oral cavities, teeth, mesentery, and tracheal lymph nodes.

[0144] A mouse model of ocular angiogenesis. CNV was induced as described above. Tobe et al., Am. J. Pathol., 153, 1641-1646 (1998). In summary, 4-6 week old female Id1 mice. - / - or Id3 - / - Mouse and littermate control Id1 + / + or Id3 + / + In mice (all in C57BL / 6 background), laser-induced Bruch's membrane rupture was observed at three locations in each eye. After 14 days, the mice were euthanized, the eyes were removed, and choroidal flat mounts were stained with FITC-labeled Griffonia simplicifolialectin (Vector Laboratories, Burlingame, CA), which selectively stains vascular cells (n=12 mice per group). The flat mounts were examined under a fluorescence microscope, and the area of ​​each CNV was measured by image analysis using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) by an observer masked for the experimental group. In other experiments, wild-type 4-6 week old female C57BL / 6 mice underwent Bruch's membrane rupture at three locations in each eye, followed immediately and 7 days later by intravitreal injection of 1-30 μg of AGX51 (racemic, E1 or E2) or vehicle into one eye, or by ip injection of 500 μg of AGX51 or vehicle twice daily for 14 days (n=7-10 mice per group). The area of ​​the CNV was measured 14 days after Bruch's membrane rupture. In aflibercept and AGX-A treatment experiments, after Bruch's membrane rupture, mice were treated with 40 μg of aflibercept, 10 μg of AGX51E2, 1-5 μg of AGX51, 1-5 μg of AGX-A, DMSO, or a combination thereof. The mice were 4-6 week old female C57BL / 6. The mice were euthanized after 14 days, and CNV was measured as described above.

[0145] In the ROP experiment, 26 C57BL / 6 offspring and 36 Id - / - and 22 Id3 - / -The pups were placed in 75% O2 at 7 days postnatal. At 12 days postnatal, the mice were returned to room air, and at 17 days postnatal, the mice were euthanized, and retinal neovascularization was measured as described above. In the AGX51 ROP experiment, C57BL / 6 pups were placed in 75% O2 at 7 days postnatal. At 12 days postnatal, the mice were returned to room air, and 10 μg of AGX51 or DMSO was injected into the eye in FE (N=15 mice / group). At 17 days postnatal, the mice were euthanized, and retinal neovascularization was measured as described above.

[0146] Cell lines and bacterial lines Cell lines. HCT116 (male type), 4T1 (female type), and 293T (female type) cell lines were purchased from ATCC (Manassas, VA, USA) and grown in RPMI (HCT116) or DME (4T1 and 293T) medium supplemented with 10% FBS (fetal bovine serum), 1% penicillin-streptomycin, and 1% L-glutamine. HUVEC cells were purchased from Corning (sex unspecified) (Oneonta, NY, USA) and grown in EGM-2 medium (Lonza, Walkersville, MD, USA). Cells were cultured at 37°C. Bacterial strains. Id1 and Id3 were purified from Rosetta2 (DE3) competent cells (Sigma Millipore).

[0147] (Example 13) Method details Id protein purification. pGEV-PSP-mId1 and mId3 expression constructs were transformed into Rosetta2(DE3) competent cells (Sigma Millipore) for protein expression. To produce recombinant GST-tagged proteins, 50 mL of LB / ampicillin (100 μg / m) + chloramphenicol (25 μg / mL) cultures were grown overnight at 37°C. Early the next morning, the overnight cultures were diluted 1:100 with 6 L LB / ampicillin + chloramphenicol and OD (Oxygen-Dose). 600The cultures were incubated at 37°C for approximately 4 hours until the concentration reached 0.7. The cultures were induced with 1 mM IPTG and incubated at 16°C for 16–18 hours. Cells were collected by centrifugation at 4000 rpm for 15 minutes at 4°C, and the pellet was resuspended in lysis buffer (50 mM Tris pH 8.0; 400 mM NaCl; 0.5 mM TCEP, protease inhibitor cocktail) (25 mL / L culture). To lyse the cells, Triton X-100 and lysozyme were added at 0.1% and 10 μg / mL, respectively, and incubated on ice for 30 minutes, followed by sonication. The lysates were spun at 17,000 rpm for 30 minutes at 4°C, the supernatant was filtered through a 0.45 μM filter, and then incubated with glutathione Sepharose at 4°C for 2 hours. The protein-bound Sepharose was passed through a polypropylene column and washed twice with 50 mL of washing buffer (50 mM Tris pH 8.0; 400 mM NaCl). The protein was then eluted with 10 mL of 50 mM glutathione elution buffer (pH 8.0). The protein was then buffer-changed using a PD10 column (Sigma) with 12 mL of storage buffer (50 mM Tris HCl pH 8.0, 400 mM NaCl, 10 mM EDTA, 1 mM DTT, 10% glycerol).

[0148] The GST tags were cleaved with PreScission protease (0.1 μL of 0.3 μg / μL PreScission protease cleaved 10 μg of GST-mId1 after incubation at 4°C for 4 hours), and the cleaved GST tags were isolated by incubation with glutathione Sepharose. The cleaved mId1 proteins were further washed by incubation with GST antibodies (Abcam#ab9085-200 μL anti-GST rabbit polyclonal; ThermoFisher#MA4-004 anti-GST mouse monoclonal) before Kumma-Siegel analysis. The cleaved proteins were concentrated to the desired intensity using an Amicon 3000MCO Ultra-4 centrifugation column (UFC800308).

[0149] Crystallization. Crystals of mouse Id1(51-104) were grown at 4°C by the hanging-drop vapor diffusion method. Aliquots (1.5 μL) of 2.8 mg / mL of protein in 20 mM Tris buffer (pH 8.0), 0.25 M NaCl, and 5 mM DTT were mixed with 1.5 μL of reservoir buffer containing 0.1 M sodium citrate (pH 6.5), 0.2 M magnesium acetate, and 10% PEG8000. Crystals were collected and transferred stepwise to a solution containing 0.1 M sodium citrate (pH 6.5), 0.2 M magnesium acetate, 11% PEG8000, and 30% ethylene glycol, cryoprotected, and rapidly frozen in liquid nitrogen. Crystals of mouse Id1(58-104) were grown at 4°C by the sitting-drop vapor diffusion method. Aliquotes of 2 mg / mL (2 μL) of protein in 20 mM Tris buffer (pH 8.0), 0.25 M NaCl, and 9% ethanol were mixed with 2 μL of reservoir buffer containing 0.1 M MES (pH 6.5) and 0.2 M sodium acetate. Crystals were collected and transferred to a solution containing 0.1 M MES (pH 6.5), 0.2 M sodium acetate, 10% PEG8000, and 30% ethylene glycol, cryoprotected, and then rapidly frozen in liquid nitrogen.

[0150] Crystals of the mouse Id1(51-104)-human E47(348-399) complex were grown at 22°C by hanging-drop vapor diffusion. Aliquots (1 μL) of 9 mg / mL of protein in 20 mM MES buffer (pH 6.5), 0.3 M NaCl, and 5 mM DTT were mixed with 1 μL of reservoir buffer containing 0.1 M potassium phosphate (pH 6.0), 0.25 M NaCl, and 22.5% PEG8000. The crystals were collected, transferred to a solution containing 0.1 M potassium phosphate (pH 6.0), 0.25 M NaCl, 23% PEG8000, and 16% ethylene glycol, cryoprotected, and rapidly frozen in liquid nitrogen.

[0151] Structural determination. Diffraction data for Id1(51-104) and Id1-E47 were collected from single crystals at beamline BNL-X9A with resolutions of 1.8 and 1.9 Å, respectively. For Id1(58-104), data were collected at CHESS with a resolution of 1.5 Å. Indexing and merging of diffraction data were performed using HKL2000 (Otwinowski and Minor, 1997). The structure of Id1(51-104) was analyzed by molecular substitution using PDB entry 1MDY as the search model. The search model was shortened to match the length of the construct used for crystallization. The structures of the Id1(58-104) and Id1-E47 complexes were analyzed by molecular substitution using the refined structure of Id1(51-104) as the search model. Molecular substitution, model construction, and refinement were performed using Phenix. Figure 16 summarizes the statistics for the collection and refinement of diffraction data.

[0152] In silico screening. The initial docking study was performed on the Id1-E47 X-ray structure (deposit ID: D_1000223931 PDB ID: (6MGN)). A compiled list of commercially available compounds (libraries available from ChemBridge, ChemDiv, Maybridge, and Salor) was screened using a beta release of Autodock 4.0 (The Scripps Research Institute, Molecular Graphics Laboratory, La Jolla, California 92037) with standard settings. For docking, the target was a cleavage adjacent to the loop region of Id1 present in the Id1-E47 heterodimer. The docking study was performed on a Linux-running Sun Microsystems (Menlo Park, CA 94025) workstation. Monte Carlo simulations of the Id1-small molecule complex were performed at 1x10⁻¹⁶ levels. 6The process was performed in steps, and 100 conformations were collected and analyzed. The complex conformations with the highest score and lowest total energy were selected for further analysis. 3000 compounds offering a promising docking score > 6.0 were further filtered by computation by calculated physical properties: ClogP < 5 (1-octanol-water partition coefficient), tPSA > 80 (topological polar surface area), MW < 600, and chemical and biochemical stability. 364 compounds that were hits obtained by computation were purchased from vendors and screened for their ability to inhibit Id1-E47 homodimerization.

[0153] In silico modeling. For all ligand preparation and docking calculations, Schrodinger Suite version 2016-1 was used with default settings unless otherwise specified. Small molecules were prepared for docking from sketched 2D structures using LigPrep. 3D structures were generated using the OPLS3 force field, and the ionization state was determined using Epik at pH 7.0 + / - 2.0. Id1 monomers, residues 58-104, were prepared from crystallographic coordinates using the Protein Preparation Wizard. The protonation state of the protein was assigned to pH 7.0 using PROPKA. The protein was minimized using the OPLS3 force field. Independent of the in silico screening step, the binding pocket within the Id1 monomer was identified using SiteMap with default settings, and this was used to rotate the hydroxyl hydrogens (Tyr66, Ser67, Thr75, and Ser83) using Glide to generate the receptor grid used for docking. Docking was performed using Glide with ultra-high-precision, flexible ligand sampling, and ring conformation and nitrogen inversion sampling. Strain sampling was biased towards amides to penalize non-planar conformations.

[0154] BRET probes derived from AGX51 (AGX51 tracer) were prepared in 4 to 3 steps of advanced intermediate amine by reaction with a commercially available N-Boc-aminoPEG2-NHS ester, removal of the Boc protecting group, and reaction with Bodipy-558 / 568-NHS ester.

[0155] Circular dichroism. Far-ultraviolet circular dichroism (CD) measurements were performed at room temperature using a Jasco J-1500 spectropolarimeter with 0.1 cm cells and 0.1 mg / mL protein solution in the absence of AGX51, AGX51E1, AGX51E2, or AGX-A. The minimum wavelength that could be scanned was limited due to the presence of DMSO in the samples.

[0156] NanoBRET® Target Binding Assay. The N-terminal NanoLuc® luciferase ID1 fusion protein was synthesized into a pUC57 backbone by Genewiz (South Plainfield, NJ, USA) and cloned into a pcDNA3.1 plasmid using EcoRI-HF and XbaI (both enzymes from New England Biolabs, Ipswich, MA, USA). The assay was performed essentially as described in the NanoBRET® TE Intracellular BET BRD Assay Kit Manual (Promega Corporation, Madison, WI, USA). In summary, cells were transfected and plated 24 hours later in a flat-bottom, unbound white polystyrene 96-well plate (Corning Incorporated, Kennebunk, ME, USA). Next, AGX51 tracer (0–4 μM) was added, followed by digitonin (Sigma, St. Louis, MO, USA), and the cells (50 μg / mL) were permeabilized. Next, NanoBRET® Nano-Glo® substrate (Promega) was added, and readings were performed using a GloMax Discover System instrument (Promega). In the competitive assay, cells were treated with 2 μM AGX51 tracer and 0–60 μM AGX-A or AGX51.

[0157] Covalent binding of an AGX51 derivative to Id1. An analog of AGX51 containing a benzophenone photoreactive moiety (AGX51-XL2) was used. 1 μg of purified Id1 (aa59-104) and 19.7 ng of AGX51-XL2 (dissolved in DMSO) were combined in the dark and then exposed to UV light for 20 minutes, including a negative control without UV exposure. The samples were then electrophoresed on a 15% denatured gel in the dark, stained with silver according to the manufacturer's protocol (SilverQuest staining kit, Invitrogen, Grand Island, NY, USA), and the bands were excised for mass spectrometry as described below. To evaluate the ability of AGX51 to compete with AGX51-XL2, and also against AGX51-XL2, another sample containing a 10-fold excess of AGX51 was added, and the above experiment was repeated.

[0158] Intragel digestion for mass spectrometry. Intragel digestion was performed using the method by Shevchenko et al. (Nat Protoc 1(6):2856-60(2006)). In summary, gel bands were cut out, washed with 1:1 (acetonitrile:100 mM ammonium bicarbonate) for 30 minutes, dehydrated with 100% acetonitrile for 10 minutes until the gel section shrunk and excess acetonitrile was removed, and the section was dried in a speed-vacuum for 10 minutes without heating. The gel section was reduced with 5 mM DTT at 56°C for 30 minutes with gentle mixing in a thermostat-controlled mixer, removed, cooled to room temperature, and alkylated with 11 mM IAA in the dark for 30 minutes. The gel section was washed with 100 mM ammonium bicarbonate and 100% acetonitrile for 10 minutes each. Excess acetonitrile was removed, and the section was dried in a speed-vacuum for 10 minutes without heating. Next, the gel sections were rehydrated on ice for 30 minutes with a solution of 25 ng / μL trypsin in 50 mM ammonium bicarbonate. Digestion was carried out overnight at 37°C on a thermostat-controlled heater with gentle mixing. The digested peptides were collected and further extracted from the gel sections with extraction buffer (1:2 vol / vol) (5% formic acid / 50% acetonitrile) by rapid mixing. The extracts were combined and dried using a vacuum centrifuge. The peptides were desalted using a C18 resin-packed stage-tip, freeze-dried, and then reconstituted with 3% acetonitrile / 0.1% formic acid for LC-MS / MS analysis.

[0159] LC-MS / MS analysis. LC-MS / MS was performed using a Waters NanoAcquity LC system consisting of a 180 μm × 2 cm trap column connected to a ThermoQ-Exactive Plus Orbitrap mass spectrometer (using a 100 μm inner diameter × 10 cm length C18 column (1.7 μm BEH130; Waters)). Trapping was performed for 1 minute with 15 μL / min 0.1% formic acid (buffer A). The LC gradient was 0.5% to 50% B (100% acetonitrile, 0.1% formic acid) over 90 minutes at 300 nL / min. MS data were collected in data-dependent acquisition (DDA) mode using top 10 precursor ion sorting for HCD fragmentation. A full MS scan was performed using the following parameters: resolution: 70,000; AGC target: 1e6; maximum IT: 50 ms; scan range: 400–1600 m / z. The DDA parameters were as follows: Resolution: 17,500; AGC target: 5e4; Maximum IT: 50 milliseconds; Isolation window: 1.5 m / z; NCE: 27; Minimum AGC target: 2e3; Intensity threshold: 4e4; Dynamic exclusion: 15 seconds; Charge exclusion: Unassigned, 1, 6-8, >8.

[0160] Crosslinked peptide identification analysis. MS raw files were processed by searching a custom mouse ID1 database using Byonic version 2.5 (Protein Metrics, San Carlos, USA). Search criteria included a 10 ppm mass tolerance for MS spectra, a 40 ppm mass tolerance for MS / MS spectra, up to two acceptable cleavage failures, fixed carbamide methylcysteine ​​modification, variable methionine oxidation, glutamine and asparagine deamidation, N-terminal protein acetylation, and monoisotopic mass of the AGX51-XL2 crosslinked product (419.1885 Da). A Pep2D significance threshold of 0.005 or less was considered significant. Crosslinked peptides were further examined by visual analysis.

[0161] Electrophoretic mobility shift assay. To test the activity of compounds identified by in silico screening, full-length E47 was purified from bacteria and tested in the presence or absence of purified full-length Id1, muscle creatine kinase (MCK) enhancer, BSA, DTT, polydI-dC, salmon sperm, and P derived from HeLa nuclear extract. 32 The labeled E-box sequences were mixed. Various test compounds, at increased concentrations, dissolved in DMSO, or DMSO alone, were added to the reaction mixture for 30 minutes, separated on a 5% non-denaturing polyacrylamide gel, and autoradiography was performed. Electrophoretic mobility shift assay (EMSA) was performed on the total cell lysates from AGX51-treated cells, and EMSA was carried out as previously described (Tournay and Benezra, Mol Cell Biol, 16: 2418-30 (1996)).

[0162] Immunoblotting. For immunoblotting, cells were collected by trypsin treatment, washed with PBS, and lysed in homogenization buffer (0.3 M sucrose, 10 mM Tris (pH 8.0), 400 mM sodium chloride, 3 mM magnesium chloride, 0.5% NP40 / IGEPAL, 100 μg / mL aprotinin + protease inhibitor cocktail (Roche #11 836 153 001)). Proteins were separated by SDS-PAGE, transferred to a membrane (LI-COR), probed with primary antibody overnight at 4°C, and probed with secondary antibody (LI-COR) for 1-2 hours at room temperature. Proteins were visualized using the LI-COR Odyssey infrared imaging detection system. The following primary antibodies were used. Id1, Id2, Id3, and Id4 (195-14, 9-2-8, 17-3, and 82-12, respectively, all from Biocheck), cyclin D1 (2978, Cell Signaling), actin (A2066, Sigma), and tubulin (T4026, Sigma) were used. Western blot quantification was performed using intensity data from channels 700 and 800 of the Odyssey application software version 3.0.30 (LI-COR), after subtracting the blank value and normalizing to tubulin.

[0163] Immunoprecipitation. Cells were lysed in NP40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1.5 mM Na3VO4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerol phosphate, and EDTA-free protease inhibitor cocktail (Roche)) or RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1.5 mM Na3VO4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerol phosphate, and EDTA-free protease inhibitor cocktail (Roche)). The lysates were clarified by centrifugation at 15,000 rpm for 15 minutes at 4°C. For immunoprecipitation, the cell lysates were incubated overnight at 4°C with primary antibodies (FLAG M2 affinity gel, Sigma, F2426; ID1(C-20), Santa Cruz, sc-488; E2A(N-649), Santa Cruz, sc-763) and protein G / A beads (Santa Cruz, sc-2003). The beads were washed four times with lysis buffer and eluted with 2× SDS sample buffer. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBS containing 5% skim milk and 0.1% Tween20, and probed with primary antibodies. The antibodies and concentrations used were as follows: ID11:500 (C-20, sc-488) and E2A 1:1000 (N-649, sc-763), obtained from Santa Cruz Biotechnology; HA 1:1000 (C29F4, #3724), obtained from Cell Signaling Technology; β-actin 1:8000 (A5441), vinculin 1:8000 (V9131), and FLAG M2 1:500 (F1804), obtained from Sigma. Horseradish peroxidase conjugate secondary antibodies were purchased from Pierce, and ECL solution (Amersham) was used for detection.

[0164] Ubiquitination assay. HCT116 cells were transfected with pcDNA3-ID1-Flag and pcDNA3-HA-ubiquitin using Lipofectamine 3000 (ThermoFisher). 36 hours after transfection, cells were treated with 60 μM AGX51 for 2 hours, followed by 20 μM MG132 (EMD Millipore) for a further 6 hours. After washing twice with ice-cold PBS, cells were dissolved in 100 μL of TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 2% SDS and boiled at 100°C for 10 minutes. The lysate was diluted in 900 μL of TBS containing 1% NP40 and an EDTA-free protease inhibitor cocktail (Roche) and clarified by centrifugation at 15,000 rpm at 4°C for 15 minutes. Immunoprecipitation was performed using 1 mg of cell lysates containing FLAG M2 affinity gel (Sigma, F2426). Ubiquitinated proteins were analyzed by immunoblotting using the indicated antibodies.

[0165] qRT-PCR. RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA, USA), and cDNA was generated from 1 μg of RNA using the SuperScript IV First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA). Quantitative PCR was performed using the SYBR Green QuantiTect Primer Assay (Qiagen) on a 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Grand Island, NY, USA) according to the manufacturer's instructions. Primer pairs for individual genes were obtained from a bioinformatics-validated QuantiTect library, and are shown below: ID1 (QT00230650), ID3 (QT01673336), and GAPDH (QT01192646). The multiplicative changes in gene expression were calculated using the delta-delta CT method.

[0166] Cell viability assay. Cell lines were seeded in 96-well plates (5000 cells per well). After incubation overnight, cells were treated with AGX51 and incubated for 24 hours. Then, MTT reagent (5 mg / mL) was added to each well, and the cells were incubated for 4 hours. After incubation, the medium was aspirated and 200 μL of DMSO was added per well. Absorbance was then measured at 570 nm using a plate reader (Synergy2, BioTek). Cell proliferation profiles were determined by seeding 38,000 cells in 24-well plates three times at each time point, and counting cells using trypan blue exclusion for dead cells on days 1, 3, and 5 after seeding.

[0167] Cell cycle analysis. Cells were treated with AGX51 or DMSO, collected by trypsin treatment, washed with 1×PBS, resuspended in 500 μL of 1×PBS, diluted in 6 mL of 70% ethanol, and stored at -20°C until analysis. For cell cycle analysis, cells were centrifuged at 1000 rpm for 5 minutes, washed with 1×PBS, resuspended in 0.5 mL of PI / RNase staining buffer (550825, BD Biosciences), incubated at room temperature for 15 minutes, and analyzed by flow cytometry (LSRII).

[0168] HUVEC cell branching assay. In the branching assay, 350 μL of Matrigel was packed into each well of a 24-well plate on ice, and the plate was incubated at 37°C for 30 minutes to solidify the Matrigel. 80,000 HUVEC cells in 0.5 mL of EGM-2 medium at the indicated AGX concentration were plated onto the solidified Matrigel. After 18–20 hours of incubation, when tube formation peaked, the medium was carefully removed from the wells and fixed with 10% buffered formalin for 15 minutes. Each well was washed with DPBS. The morphology of the capillary-like structures was visualized using an inverted microscope and photographed with a digital camera at 10x magnification. To quantify the tube network, ImageJ was installed with the Angiogenesis Analyzer plugin (a public domain Java-based image processing program; reference: Carpentier G. ImageJ contribution: Angiogenesis Analyzer. ImageJ News. 2012) and the analysis of the number of nodes, junctions, meshes, and the total length of branches was performed according to the instructions. Statistical data analysis was performed using the Wilcoxon test.

[0169] HUVEC scratch assay. HUVEC cells were seeded in 24-well plates coated with 0.1% fibronectin. After 24 hours, when the cells had grown to confluence, they were serum-starved for 4 hours in endothelial basal medium (EBM, Lonza) and scraped with a sterile P200 pipette tip to generate a cell-free zone. The cells were washed with PBS and stimulated for 24 hours in EGM-2 medium at the indicated AGX concentration. Scratched areas at 0 and 24 hours were visualized using an inverted microscope and photographed with a digital camera at 20x magnification.

[0170] Quantification and statistical analysis. Statistical details of the experiments can be found herein. Three copies were generally used for each experimental condition in the in vitro experiments, and each mouse experiment typically used 5 mice per group. Sample size was determined based on the expected large effect size. With three copies per condition, a small effect size of 3 could be detected using a two-sample t-test with 80% power at a two-sided significance level of 0.05. Using 5 mice per group, a small effect size of 2 could be detected using a two-sample t-test with 80% power at a two-sided significance level of 0.05. If greater variability in the data was observed and the data was pooled for analysis, additional experiments could be performed. Generally, Welch's t-test was used to examine the difference between two groups. ANOVA was used to examine the difference between multiple experimental groups. The data may be transformed to ensure that the underlying normality assumption is met. If heteroscedasticity was observed, weighted linear regression analysis was used, with data points for each group weighted, typically by the reciprocal of the standard deviation of the data for each group. For data pooled from multiple experiments, the model included both the experiment and the experiment with the interaction of the treatment group as a covariate to explain potential differences in the experiment. The significance of the linear contrast of interest was assessed based on estimates obtained from weighted least squares. QQ plots of the residuals were examined to confirm that the assumptions of the underlying model were met. A p-value < 0.05 was considered statistically significant.

[0171] (Example 14) Genetic loss of Id1 or Id3 reduces neovascularization in the eye. While not bound by theory, we hypothesized that the Id protein is involved in CNVs occurring in AMD. The mouse model of laser-induced choroidal angiogenesis (CNV) used herein has previously provided results predicting outcomes in clinical trials investigating AMD treatments. Laser-induced rupture of Bruch's membrane is associated with Id1 - / - or Id3 - / - Mouse and littermate control Id1 + / + or Id3 + / +The study was conducted in mice. After 14 days, the mice were euthanized, and choroidal flat mounts were stained with FITC-labeled Griffonia simplicifolialectin, which selectively stains vascular cells, and the area of ​​CNVs was measured. As shown in Figures 1A and 1B, gene deletions of Id1 or Id3 significantly suppressed CNVs compared to the wild type (p<0.03).

[0172] The pathology of retinal neovascularization can also be studied in a mouse model of retinopathy of prematurity (ROP). In this model, mice 7 days postnatally (P) are placed in a 75% O2 chamber, which induces the loss of retinal capillaries. At 12 days postnatally, the mice are returned to room air, which leads to the development of retinal ischemia and proliferative vascular disease in the retinal vascular system. When this model was applied to Id1 or Id3 knockout mice, a significant reduction in retinal neovascularization was observed compared to wild-type mice (p<0.0001) (Figure 1C). These studies, taken together, demonstrate that pharmacologically antagonizing the Id protein may be a useful approach for treating ocular neovascularization. Therefore, the compounds in this technology may be useful in treating ocular neovascularization.

[0173] (Example 15) In silico screening was used to identify AGX51, an Id1 antagonist. To facilitate the search for small molecules that could antagonize the Id protein, we analyzed the crystal structures of two fragments of Id1 containing the HLH domain, residues (51-104) and (59-104) (identical in mouse and human), and the E47-Id1 complex: Id1(59-104)-E47(558-609) (Figures 2A and 16). As shown in Figure 2A, similar to other members of the HLH superfamily, the structure consisted of two α-helices connected by a loop of 10 residues from Id1 and 7 residues from E47. The crystal structure showed that the HLH domain of Id1 is homodimer, and the α-helices from both monomers form a 4-helix bundle. The interface region is 1045 Å. 2This is formed by hydrophobic interactions and a leucine zipper-like region of seven hydrogen bonds (Figure 2A). The Id1-E47 interface is formed by the same Id1 region as the homodimer interface. The E47 residue interacting with Id1 is structurally equivalent to the Id1 interface region, thereby creating a 1131 Å interface. 2 A four-helix bundle similar to the structure of the Id1 homodimer with a filled region is obtained (Figure 2A). In addition to hydrophobic interactions and six hydrogen bonds (Id1-L59:E47-Q590; Id1-Q89:E47-R558; Id1-Q89:E47-V559; Id1-Y94:E47-E600; Id1-L102:E47-R606; Id1-S104:E47-R606), two salt bridges (Id1-R99:E47-E568; E47-E568:E47-R571) are formed (Figure 2A). The crystal structure further shows that the loop region between the two helices of Id1 is flexible, which results in the different conformations and high B factor observed in the three structures. The crystal structure of the HLH region of E47 alone (Ahmadpour et al., PLoS One, 7: e32136 (2012)) and complexes with other proteins and DNA have been previously published: El Omari et al., Cell Rep, 4, 135-47 (2013); Longo et al., Biochemistry, 47, 218-29 (2008). All structures overlapping the E47 structure reported herein have an RMSD of 0.4–0.8 Å between Cα atoms. The dimerization interface in both the E47 homodimer and the heterodimer with T cell acute lymphoblastic leukemia protein 1 and neuronal differentiation factor 1 was found to be the same as that in the Id1-E47 complex. However, these studies showed that the loop region of E47 is more rigid than the loop region of Id1 because the conformation of the loop is similar in all E47 structures.

[0174] As shown in Figure 2B, hydrophobic gap analysis revealed a gap adjacent to the Id1 loop region present in the Id1-E47 heterodimer, indicating that the Id1 region is highly conserved among family members and species and is crucial for maintaining Id activity. Pesce and Benezra, Mol. Cell. Biol., 13, 7874-7880 (1993). In silico screening of 2,234,000 compounds for gap binding was performed, yielding 3,000 hits, which were reduced for drug-like properties. 364 candidates emerged and were tested for their ability to inhibit the Id1's ability to antagonize E47 binding to DNA by EMSA, as previously described (Benezra, 1994). Representative EMSA images of three of the tested compounds are shown in Figure 2C, where compounds B and C showed a dose-dependent increase in E protein binding (lanes 6-8 and 9-16, respectively). Compound A did not show such a recovery. The potent recovery of E protein binding activity compared to E protein alone (comparing lanes 1–16) suggested that compound C antagonized Id1 activity and had little effect on E47-DNA binding. In summary, only two compounds showed strong activity in the EMSA assay, resulting in a hit rate of 2 / 364 or 0.55%. This anti-Id1 activity is most likely due to perturbation of the Id1-E47 interaction, although other explanations are possible, such as the binding of the Id1-E47 complex to DNA in the presence of the compound. Previously, EMSA was performed in the presence of reticulocyte lysates (Benezra et al., Cell, 61: 49–59 (1990)), and here again, HeLa nuclear extract was added to facilitate the observed interaction. Compound C, which was potenter than B, was selected for further analysis as an Id1 antagonist and is called AGX51. The structures of A, B, and C from Figure 2C are shown in Figure 2D.

[0175] AGX51 has a single stereocenter (Figure 2D). As shown in Figure 2E, the binding pocket of Id1 was predicted using SiteMap (Schrodinger release 2016-1: Lig Prep, version 3.7, Schrodinger, LLC, New York, NY), which was the same cleavage identified in Figure 2B, but the AGX51 binding pocket at E47 was not found. Analysis of the AGX51 binding pose (predicted by subsequent high-resolution docking calculations using Glide XP) showed that Lys70 forms a hydrogen bond with AGX51, bringing AGX51 into proximity to 7 residues of the loop domain and 4 residues of helix 1 of Id1 (Figure 2F). Importantly, as shown in Figure 8, the majority of these residues (7 / 11) are highly conserved among the four Id family members, and 9 / 11 generated the consensus amino acid sequence found in the Drosophila Id ortholog.

[0176] To demonstrate the physical interaction between Id1 and AGX51, circular dichroism (CD) measurements were performed. As shown in Figure 2G, analysis of the CD spectrum showed that Id1 interacts with AGX51, resulting in a significant change in the secondary structure of Id1. The CD change saturated at 20 μM, which meant that the dissociation constant was within this range. Importantly, there was no evidence of interaction between AGX51 and E47, nor were any changes observed attributable to DMSO or the buffer used (Figure 2G). DMSO exhibits strong absorbance at the wavelengths used in these assays, causing spikes in the spectrum. Unfortunately, since AGX51 is insoluble in water, and it was necessary to include DMSO and the wavelength range used in these assays, such noise could not be avoided. We were unable to purify a sufficient amount of Id1 with mutations in the pocket region, which likely reflects that they are inactive (Pesce and Benezra, Mol. Cell. Biol., 13, 7874-7880 (1993)) and possibly unstable. CD assays were also performed using purified Id3, and as shown in Figure 9, there was a small but reproducible effect of AGX51 on the secondary structure of Id3, following the trend observed with Id1. While aggregation of AGX51 may affect the CD spectrum of a protein, this possibility is ruled out here, as such an effect is not expected to be more specific to the Id protein than to the more relevant E47 bHLH protein. Co-crystallization of AGX51 and Id1 was also attempted, but these efforts were unsuccessful despite trying over 1000 conditions, including all commonly used sparse matrix screenings and immersion of Id1 crystals. Interestingly, when Id1 crystals were exposed to AGX51, they melted, but when exposed to DMSO alone, they did not melt. The dissolution of Id1 crystals after AGX51 exposure is consistent with the CD data and suggests a conformational change that does not fit lattice formation.

[0177] To demonstrate target binding to cells, a NanoBRET® assay was developed for ID1 and AGX51. This assay is based on the NanoBRET® Target Engagement (TE) Intracellular BET BRD assay (Promega). Constructs expressing NanoLuc® luciferase ID1 fusion protein and AGX51-fluorescent tracer were generated (Figure 10A). Upon target binding, bioluminescence resonance energy transfer (BRET) occurs, with luminescence energy transferred from NanoLuc® luciferase to the fluorescent tracer bound to the target protein portion of the fusion protein. When permeabilized 293T cells transfected with the fusion protein were treated with AGX51 tracer (0-4 μM) (Figure 10B), a dose-dependent increase in the BRET ratio was observed, indicating target binding. Furthermore, as shown in Figure 10C, the AGX51 tracer may compete with AGX51 and may more efficiently compete with AGX-A, which has greater activity, in biological assays (as described herein). The effective compound concentrations identified in these assays differ from those found in biochemical and cell-based assays (see below), because these assays use the AGX51 tracer, which is a different entity from AGX51 and has its own unique properties, and the NanoLuc® luciferase ID1 fusion protein rather than endogenous ID1. In addition, the size of the AGX51 tracer allows the assay to be performed in digitonin-permeable cells, which may alter the endogenous cellular state and affect the binding properties. These results support the idea that ID1 is a direct target of AGX51 in the cellular environment.

[0178] Further verification of physical interaction data was conducted to investigate which Id1 residues are AGX51, the AGX51 analog, and which interact with AGX51-XL2 (Figure 8B), which incorporates a benzophenone moiety that forms covalent bonds with adjacent residues upon UV irradiation. AGX51-XL2 was predicted to bind to the same pocket as AGX51 in Id1 (data not shown). AGX51-XL2 and purified Id1 (59-104) were mixed, exposed to UV light, and the samples were analyzed by mass spectrometry. As shown in Figure 8C, evidence of AGX51-XL2 covalently binding to Id1 was found at six residues (V73, P74, T75, P77, Q78, and R80). These residues overlapped with four helix-1 residues and seven loop residues predicted to be adjacent to AGX51 (Figures 8 and 17). No evidence of covalent bonding was observed in samples not exposed to UV light, nor was there any evidence of AGX51-XL2 binding to E47 (data not shown). To support the concept that AGX51 binds to the same region as AGX51-XL2, an excess amount of AGX51 was added to create competition for the Id1 binding site. Mass spectrometry revealed that after adding a 10-fold excess of AGX51, the binding of AGX51-XL2 to Id1 was significantly reduced (Figure 18). The cell permeability and UV reactivity of AGX51 could not be developed to perform this analysis in living cells. These data support the direct interaction between AGX51 and Id1, which is consistent with the in silico screening and modeling described above.

[0179] (Example 16) The effect of AGX51 on intracellular Id protein The activity of AGX51 was tested in two cell types with different ID1-4 expression profiles: primary human umbilical vein endothelial cells (HUVECs) and the HCT116 colorectal cancer cell line. Unbound Id proteins are short-lived, with half-lives on the order of 10-20 minutes, but are significantly stabilized when complexed with E proteins. As observed in vitro, if the Id-E interaction is disrupted by AGX51 in cultured cells, this is expected to lead to an increase in unbound Id proteins, which are then predicted to be rapidly degraded. HUVECs were treated with increasing concentrations of AGX51 (0-40 μM) for 24 hours, and a significant decrease in ID1 protein levels was observed at 10 μM (Figure 3A). A similar protein loss pattern was observed for ID3 (Figure 3A); ID2 and ID4 proteins were not detected in this cell line (data not shown). The effect of AGX51 on ID3 loss in HUVEC cells was a decrease in the range of 20–40 μM compared to ID1, consistent with weaker disturbances observed in CD spectra. A similar decrease in the effect on Id3 was also observed in 4T1 breast cancer cells (data not shown). In HCT116 cells, AGX51 treatment reduced the levels of ID1, ID2, ID3, and ID4, suggesting that AGX51 antagonized all four members of the protein family (Figure 11A). Paradoxically, while ID protein levels decreased with AGX51 treatment, an increase in ID1 mRNA levels was observed, likely due to activation of the ID1 promoter by free E protein (Figure 11B). These results indicate that the reduction in ID1 steady-state protein levels by AGX51 is strong enough to overcome a significant increase in ID1 mRNA production.

[0180] The Id protein is degraded by the ubiquitin 26S proteasome system. Lasorella et al., Nat Rev Cancer, 14: 77-91 (2014). Next, we investigated whether this degradation pathway mediates the effect of AGX51 on Id protein levels. Because transfecting expression constructs into HUVECs is difficult, HCT116 cells were used for this assay. HCT116 cells were transfected with a construct expressing Flag-ID1 and treated with 60 μM AGX51 for 2–24 hours (Figure 3B). Next, HCT116 and U87 glioma cells were co-transfected with FLAG-ID1 and HA-ubiquitin and treated with either the vehicle or AGX51 for 2 hours to visualize ubiquitination before degradation. After adding the proteasome inhibitor MG132 for an additional 6 hours, immunoprecipitation was performed with an anti-FLAG antibody, and ubiquitinated ID1 was visualized by immunoblotting using an anti-HA antibody. ID1 ubiquitination was observed in lysates from cells treated with MG132 (Figure 3C). Treatment with AGX51 further increased ID1 polyubiquitination in both cell types.

[0181] While not bound by theory, we hypothesized that, assuming minimal compound interference with the E protein itself, the loss of Id protein in response to AGX51 should result in an increase in E protein binding activity. After treating HCT116 cells with AGX51 for 24 hours, as expected, a slight increase in E protein binding in cell lysates was observed compared to controls (Figure 11C). To determine whether the loss of Id activity precedes the loss of Id protein, EMSA was performed using cell lysates from HCT116 cells treated with AGX51 for 1 hour. A similar increase in E protein binding in response to AGX51 was observed when ID1 protein levels did not decrease to a detectable level (Figure 11C), suggesting that the observed increase in E protein binding was due to the disruption of the ID1-E protein heterodimer, rather than a decrease in overall ID protein levels. Similar results were observed in 4T1 breast cancer cells treated with AGX51 (data not shown).

[0182] To confirm that the increased binding of E47 to DNA in the presence of AGX51 was caused by AGX51-induced dissociation of the endogenous E47-ID1 cell complex, immunoprecipitation using ID1 or E47 antibodies and Western blotting of endogenous E47 or ID1 were performed, respectively. In both assays, treatment with AGX51 significantly reduced the level of co-precipitated protein before detectable loss of ID1 protein. Thus, AGX51 can block intracellular ID1-E47 PPI (Figure 3D). Taken together, these results suggest that AGX51 treatment disrupts the ID1-E47 complex, leading to proteasome-mediated degradation of ID1 and the release of the transcription-promoting E protein. However, there is also a formal possibility that a ubiquitination event precedes this, potentially promoting the complete dissociation of the complex and subsequent Id degradation.

[0183] (Example 17) The effect of AGX51 on cell growth AGX51 treatment resulted in decreased cell viability, G0 / G1 growth arrest, and reduced cyclin D1 levels in both HUVEC (Figures 4A-4C) and HCT116 cells (Figures 12A-12C). Changes in other proteins were also observed by whole proteome SILAC analysis after AGX51 treatment (data not shown). These data are consistent with genetic experiments showing that threshold levels of Id protein expression are essential for the growth and / or survival of essentially all cell types examined in culture, but not essential in most adult tissues where these proteins are silenced (see toxicity studies below).

[0184] To characterize the effect of AGX51 on HUVEC vascular bifurcation, the number of nodes, junctions, and meshes, and the length of bifurcation were measured after AGX51 treatment. As shown in Figures 4D–4E, when HUVECs were cultured on Matrigel for 18–20 hours in the presence of AGX51, vascular bifurcation was significantly impaired in a dose-dependent manner across all parameters tested compared to the vehicle control (p<0.05). After monolayers were scratched and then cultured in AGX51-containing medium for 24 hours, HUVEC migration was also significantly impaired by AGX51 (Figure 4F). Therefore, AGX51 treatment impaired the normal proliferative characteristics of human endothelial cells in culture.

[0185] (Example 18) Pharmacokinetics and toxicity of AGX51 after intraperitoneal injection. Next, we investigated and determined the feasibility of systemic administration of AGX51 for the treatment of ocular retinopathy. To determine the half-life of AGX51 in serum, mice were treated with a single intraperitoneal (ip) injection of 30 mg / kg or 50 mg / kg of AGX51 in 70% DMSO, and blood was collected over 24 hours. A time-dependent decrease in serum AGX51 levels was observed with a half-life of approximately 3 hours. The mean maximum serum concentrations of AGX51 achieved after administration of 30 mg / kg or 50 mg / kg were 1.1 and 1.6 μg / mL (2.7 and 4 μM), respectively, and did not increase further when mice were treated with a 100 mg / kg dose. Notably, significant Id loss was observed with 10 μM HUVEC. Higher mean serum concentrations could be achieved with 100% DMSO (approximately 12 μM at 100 mg / kg), but the animals exhibited injection site toxicity with DMSO alone. Therefore, 70% of the formulations were used in all future studies.

[0186] No deaths or morbidities were observed after a 14-day treatment period in mice administered either vehicle or AGX51 at 60 mg / kg twice daily via intravenous injection (IP). Overall, all mice appeared healthy and exhibited normal behavior throughout; no significant weight loss was observed in either group, and all clinical chemistry parameters and hematological parameters were within the normal range (Figure 19). No abnormal findings were detected during macroscopic autopsy or after complete histopathological evaluation of all major organs. Therefore, AGX51 treatment is non-toxic. Consequently, the compounds of this technology are useful in treating proliferative diseases, including neovascular diseases and cancers of the eye.

[0187] (Example 19) The effect of AGX51 treatment on neovascularization in the eye To determine whether AGX51 treatment phenotypicly mimics the effects observed in the Id1 and Id3 loss gene models described above, the AMD mouse models discussed herein were used again. Tobe et al., Am. J. Pathol., 153, 1641-1646 (1998). As shown in Figures 5A-5B, two intravitreal injections of 10 μg of AGX51 at one-week intervals (2 hours and 7 days after laser treatment; analysis performed on day 14) significantly suppressed CNV compared to vehicle alone (p<0.05). Similar results were observed with a single dose of AGX51 2 hours after laser treatment and analysis on day 14 (data not shown). 5 μg of AGX51 was also effective in significantly reducing CNV, but 1 μg was not (p<0.05) (Figure 13A). Twice-daily intravenous injections of AGX51 (approximately 30 mg / kg) also significantly reduced CNVs compared to vehicle-treated mice (p<0.05) (Figures 5C-5D). As shown in Figure 5E (top panel, arrows), the Id1 protein was readily detected in the control eye (head of arrow) in the CNV region and co-localized with lectin-stained endothelial cells. Treatment with AGX51 did not produce Id1-positive cells in areas lacking CNVs (see Figure 5E, middle panel), and Id1 staining was absent in the rare sections where CNVs were observed (Figure 5E, bottom panel).

[0188] The efficacy observed with intravitreal (IP) administration suggested that AGX51 may reach the eye after systemic injection. Therefore, the concentration of AGX51 was measured by mass spectrometry over 24 hours in the eyes of mice administered 30 mg / kg of AGX51 via IP. The maximum concentration of AGX51 at 30 minutes was approximately 4 ng / eye, with a half-life of 3.7 hours. The amount of AGX51 reaching the eye was far less than the amount required for intravitreal injection to demonstrate efficacy, which may be due to incomplete recovery from excision of the excised eye or significant differences in the effective dose range depending on the delivery route. The effects of AGX51 in a ROP mouse model were also evaluated. Mice exposed to hyperxic and then normoxic conditions were treated with AGX51 at postnatal day 12 and euthanized at postnatal day 17, and the degree of angiogenesis was measured. As shown in Figure 5F, intraocular injection of AGX51 significantly reduced retinal angiogenesis (p<0.01), consistent with Id1 and Id3 knockout data. These results are consistent with AGX51 targeting the Id protein for degradation in the CNV region, and the genetic loss in expression studies is phenotypicly mimicked.

[0189] Because AGX51 has one chiral center, the relative activity of two AGX51 enantiomers (called AGX51E1 and E2) was measured. Stereospecific synthesis of the two enantiomers was performed, and X-ray crystallography studies identified AGX51E1 and AGX51E2 as the R and S forms of the molecule, respectively. The effects of ip injection of the racemic mixture, AGX51E1, AGX51E2, and vehicle control were compared using a CNV assay. As shown in Figures 6A and 6B, only the racemic mixture and AGX51E2 significantly reduced the CNV area compared to the vehicle control (p<0.05 and p=0.0014, respectively). In the intravitreal injection assay, dose settings for AGX51E2 showed significant efficacy at doses of 30 and 10 μg (p=0.03) compared to the fellow eye (FE), but not at doses of 3 or 1 μg (Figure 6C). Interestingly, AGX51E2 showed higher activity than AGX51E1 in the CD assay (Figure 14), further supporting the idea that it is a more active enantiomer.

[0190] Current clinically approved treatments for AMD include aflibercept, a VEGF trap that inhibits neovascularization. To determine the relative efficacy of AGX51E2 and aflibercept, direct and combined treatments were performed in a CNV assay. AGX51E2 significantly reduced CNV in this assay compared to FE (p=0.0014), while aflibercept, while showing inhibitory activity, did not reach statistical significance under these conditions. Furthermore, AGX51 + aflibercept combination treatment worked better than aflibercept alone (p<0.05) (Figure 6D). Overall, these results suggest that AGX51 targeting of the Id protein in pathological angiogenesis via systemic or intravitreous administration may be a valuable therapeutic approach. Therefore, AGX51 treatment impaired the normal proliferative characteristics of human endothelial cells in culture.

[0191] (Example 20) Characterization of AGX-A AGX-A (Figure 7A) was identified as exhibiting higher activity than AGX51 in CD (Figure 7B) and NanoBRET assays (Figure 10C). In cell-based assays, AGX-A showed approximately a one-quarter reduction in IC50 value and a decrease in Id protein levels at lower concentrations than AGX51 (Figures 7C-7D). Furthermore, AGX-A performed better than AGX51 in a CNV assay at a dose of 1 μg (Figure 7E).

[0192] In this disclosure, we have shown that genetic loss of the Id protein reduces angiogenesis in two models of ophthalmic vascular disease. AGX51 is a small molecule antagonist of the Id protein family. This molecule was identified by in silico screening of compounds that can interact with the hydrophobic pocket within the highly conserved loop region of the Id HLH dimerization motif. CD data showed that AGX51 interacts with Id1 and Id3, rather than E47, and modifies the Id1 2° structure. The interaction with Id1 occurred in the 20 μM range, which is consistent with EMSA and co-IP data. The concentrations of AGX51 required to confirm the effect on the Id protein were similar to those required for Myc-Max inhibition (approximately 20–50 μM), and the dimer consists of two bHLH proteins with adjacent leucine zippers that interact to form a 4-helix bundle. Importantly, the concentrations of AGX51 used in in vitro and cell-based assays are in the micromolar range, without associated toxicity, and achieved in mouse serum after iP injection, thus suggesting the feasibility of systemic anti-Id therapy. While not bound by theory, it is hypothesized that the absence of effects on bone marrow function and the general lack of apparent toxicity are likely due to the fact that the Id protein is primarily required by adult stem cells when these cells are induced to enter the cycle in response to stress or injury.

[0193] The analyses disclosed herein demonstrate that AGX51 disrupts the intracellular endogenous Id1-E47 PPI, which is consistent with a proposed model in which AGX51 disrupts the ability of Id1 to associate with E proteins by binding to a highly conserved functional loop domain of the Id family. It is noteworthy that in vitro cell lysates are required to observe the disruption of the Id1 / E47 interaction, suggesting that cellular factors (possibly ubiquitination of Id1 upon AGX51 binding) are necessary to promote the destabilization of the PPI. Importantly, immediately after the disruption of this PPI in cell culture, Id1 protein levels steadily decrease, which, at least for Id1, is due to increased ubiquitin-mediated proteolysis. This destabilization of Id proteins is consistent with genetic analyses that have shown a dramatic increase in Id3 stability upon co-expression of E proteins. SILAC analysis (data not shown) revealed that none of the other 13 proteins downregulated in response to AGX51 are known substrates of Id1 deubiquitinase (USP1), making USP1 unlikely to be a drug target. Since AGX51 appears to act as an antagonist and degradation factor for Id protein in cell culture and tissue, levels of Id protein in tissue or circulating Id-expressing cells could potentially serve as a biomarker for AGX51 activity.

[0194] The loss of Id protein in response to AGX51 treatment in both cells and animals clearly indicates that they are drug targets, but we do not subscribe to the theory, although we hypothesize that Id protein is a key target of the AGX51-induced phenotype. If so, compounds are expected to replicate the effects of loss-of-function mutations in Id, and this prediction has been supported by multiple assays: AGX51 inhibits cell proliferation and induces G0 / G1 arrest; inhibits ocular angiogenesis in mouse models of AMD and ROP; and phenotypicly mimics the effects of Id1 and Id3 loss in various cancer models, including ROS production and metastasis suppression (data not shown). Furthermore, partial reduction of Id1 and Id3 by shRNA reduces the IC50 of AGX51 in cells, and significantly attenuates cell death in quiescent cells where Id protein is not detected (data not shown). It is not yet possible to strictly rule out the possibility that other unintended targets may also contribute to the observed phenotype.

[0195] The results of the CD, crosslinking, and NanoBRET® assays presented herein, as well as crystal lattice disruption, support direct target binding between ID1 and AGX51 both in vitro and intracellularly. Furthermore, AGX-A acted at lower concentrations than AGX51, inducing secondary changes in Id1 in the CD assay and competing for tracer binding in the NanoBRET assay; correspondingly, intracellular AGX-A-degraded Id protein at lower concentrations than AGX51 exhibited lower IC50 and higher activity in the CNV assay. Conversely, higher concentrations of AGX8 (compound B in Figure 2) were required to disrupt Id1 in the EMSA assay, degrade intracellular Id protein, and exhibit high IC50 in the cell viability assay (data not shown). Taken together, these data support the direct binding of AGX51 and AGX-A to the target ID protein.

[0196] Intravitreal and / or systemic administration of AGX51 suppressed ocular angiogenesis in two models of neovascular eye disease: AMD and ROP. Importantly, the efficacy in the ROP model may also predict efficacy in diabetic retinopathy. Furthermore, this specification shows that AGX51 functioned similarly to currently available AMD treatments, aflibercept, and combination therapies, and acted better than aflibercept alone. The ability of systemic delivery of AGX51 to inhibit retinal angiogenesis is consistent with intravitreal delivery, suggesting that Id-dependent circulating endothelial progenitor cells may contribute to the phenotype. While molecular signaling that promotes angiogenesis is not necessarily identical across all organs, the involvement of the Id protein in ocular and tumor angiogenesis suggests that AGX51 may have therapeutic potential for other diseases complicated by angiogenesis.

[0197] In conclusion, identified herein is AGX51, a first-in-class Id protein antagonist and degradation factor that phenotypicly mimics Id gene loss in pathological conditions. This suggests that, in addition to being a useful biological tool for studying the Id protein, it could also be developed as a therapeutic agent that could bring clinical benefits to a variety of Id-related human medical conditions, including proliferative disorders such as ocular angiogenesis and cancer.

[0198] (Example 21) A comparative study of the anti-Id compounds AGX51 and AGX-A for their anti-Id efficacy, anticancer efficacy, and for targeting dormant stem cells in cholangiocarcinoma to prevent acquired resistance associated with standard chemotherapy. This example provides a controlled comparative study of AGX51 and AGX-A. These studies demonstrate the broad and flexible range of the compounds of this technology to mediate anti-Id, anti-cancer, and anti-pathogenic angiogenesis, and to support other therapeutic effects in clinical use. The studies herein show that compounds of this technology, such as AGX-A, are potent anti-Id agents and function well as effective and dose-dependent anticancer compounds in accepted animal models of cancer drug efficacy in humans. As will be described in more detail below, AGX-A showed significantly superior efficacy compared to AGX51 in several experiments and showed equivalent or significantly superior efficacy at lower doses.

[0199] AGX-A and AGX51 were compared in their ability to mediate Id knockdown in cell culture according to the methods described herein (see, for example, Examples 12-13). Figure 21 shows the control-comparative effects of AGX51 and AGX-A on Id1 knockdown in TFK1 cell culture according to the examples. Figure 22 shows the control-comparative effects of AGX51 and AGX-A on Id3 knockdown in TFK1 cell culture according to the examples. In particular, the most relevant cell lines do not express Id3 when cultured (e.g., SNU1079, SNU1196), while TFK1 expresses Id3 when cultured. In these experiments, AGX-A showed superior anti-Id1 and anti-Id3 efficacy compared to AGX51, and showed equivalent or superior Id knockdown activity at lower concentrations compared to AGX51.

[0200] The clinical target of cholangiocarcinoma was selected for further comparative studies for several reasons. Preliminary studies have shown that cholangiocarcinoma cells and tumors overexpress Id1 and are also positive for Id3, indicating that these targets are susceptible to anti-Id1 and anti-Id3 interference. Importantly, cholangiocarcinoma is a refractory cancer type that often shows relapse with acquired resistance after first-line standard treatment (SOC) with the potent chemotherapy agent gemcitabine. The following expanded studies show that anti-Id therapy with this technology targets Id1 and Id3-positive "quiescent" stem cells. These cells represent a pool of cancer progenitor cells that are relatively resistant to chemotherapy (based on their non-proliferative state during quiescence, such cells evade first-line chemotherapy that targets proliferating cells). Thus, these stem cells frequently escape first-line cancer treatment, after which they can rebound and generate a new population of cancer cells. Further additional evidence presented herein shows that the compounds of this technology also influence their efficacy and eliminate acquired resistance, either alone or in combination with conventional chemotherapy cancer treatments. For example, dormant stem cells or cancer cells that escape primary treatment through mutation (e.g., cells that acquire resistance to chemotherapy drugs through mutation) cannot further evade the compounds of this technology or develop resistance to them. While not bound by theory, functionally important, evolutionarily constrained / conserved Id binding interfaces (targeted by the compounds of this technology) cannot be altered to generate "acquired resistance." This is because any structural mutation would produce a biologically non-functional Id protein that is functionally ineffective for the essential purpose of such proteins in cancer cells.

[0201] Further additional data in this specification further demonstrate that the anti-Id compounds of this technology potently target dormant stem cells that are resistant to first-line chemotherapy, radiation, and other cancer treatments that target highly proliferative cell populations. Histochemical studies were performed, and the results therein showed that dormant nonproliferative stem cells in two different tumor types, cholangiocarcinoma and triple-negative breast cancer (TNBC), express Id1 mutually and exclusively with Ki67, a marker of cancer cell proliferation. Comparable data showed that nonproliferative stem cells similarly express Id3 mutually and do not express Ki67. Further studies showed a dramatic increase in the ratio of Id1+ / Ki67+ cells in tumors after chemotherapy, which means that the relative population of dormant stem cells that are Id1+ / Ki67- was greatly enriched by chemotherapy. These data demonstrate that Id1 is a resting stem cell marker for both types of cancer, that resting Id+ / Ki67- stem cells are resistant to conventional chemotherapy (and other cancer treatments targeting proliferation), and that the anti-Id compounds of this technology eradicate resting Id+ stem cells associated with cancer recurrence after chemotherapy / remission. To target these resting stem cells, the anti-Id compounds of this technology can be effectively used as a second-line treatment following primary treatment with chemotherapeutic agents or other conventional treatments, in a coordinated treatment protocol with chemotherapy or other conventional cancer treatments, or alone as an effective first-line treatment that specifically and broadly targets both proliferating cancer cells and resting Id+ stem cells.

[0202] A: Comparative effect of AGX51 and AGX-A on cell proliferation In in vitro studies, the anti-Id-mediated effects of AGX51 and AGX-A on various cholangiocarcinoma cell lines, as listed in Table 1 below, were investigated. [Table 1]

[0203] The effects of AGX51 and AGX-A on the survival rate of cholangiocarcinoma cells were evaluated in six different cholangiocarcinoma cell lines. For comparison, the IC50 values ​​(μM) measured in these studies are shown in Table 2 below, along with the ratio of AGX51 IC50 to AGX-A IC50. [Table 2]

[0204] As shown in Table 2, both AGX51 and AGX-A had a negative Id knockdown-related effect on the survival rate of cholangiocarcinoma cells, and the anticancer properties of AGX-A were consistently superior to those of AGX51. In particular, the anticancer properties of AGX-A were significantly superior to those of AGX51 in SNU-1079, EGI-1, and WITT cells. The dose-dependent effects of AGX51 and AGX-A on TFK1 cell viability were evaluated in a 24-hour relevant study, and exemplary data are shown in Figure 23. These data demonstrate that AGX-A has superior anticancer potential compared to AGX51, and that its inhibition of cell viability at low concentrations is equivalent to or significantly superior.

[0205] B: In vivo efficacy of AGX-A with and without gemcitabine in a mouse model of cholangiocarcinoma. Additional studies in animal models of cholangiocarcinoma, which predict the drug efficacy against cholangiocarcinoma in humans, have demonstrated dose-dependent anticancer effects of AGX-A both alone and in combination with the SOC chemotherapy agent gemcitabine. The first round of the study ("Round 1") was planned as outlined in the following Round 1 study protocol, using gemcitabine in saline and AGX-A in DMSO for administration.

[0206] Round 1 Research Protocol - Animal Models and Experimental Design (AGX-A in DMSO): • Mice: Female NSG (Non-Stress Growth) at 6-8 weeks of age • PDX:RomeP_PHCH_X_0008a, SC serial transplantation using Matrigel • Treatment group (at least 6 animals / group): Tumors 100 mm 3 Treatment was initiated when the condition was reached (Day 0 for each animal). Group 1. Physiological saline, IP once a week ("QW") for 3-4 weeks ("×3-4 weeks"). Group 2: Gemcitabine 25 mg / kg ip QW × 3-4 weeks Group 3. AGX-A 10 mg / kg ip once daily for 5 consecutive days ("QDx5") × 3-4 weeks Group 4. Gemcitabine 25 mg / kg ip QW + AGX-A 10 mg / kg ip QD x 5 × 3-4 weeks Tumor volume, body weight, and clinical signs were monitored at least twice a week for each animal in each group throughout the study.

[0207] In Round 1 of the study, the body weight of animals in groups 1 and 2 remained essentially unchanged throughout the study period. However, weight loss was observed in animals treated with AGX-A in DMSO (groups 3 and 4), indicating that DMSO exhibited some degree of toxicity. Therefore, treatment for groups 3 and 4 was discontinued after day 9, and tumor volume, body weight, and clinical signs were continued for each subject in each group at least twice a week. Subsequently, the weight loss in animals in groups 3 and 4 ceased, and instead, body weight began to increase again. Meanwhile, treatment for groups 1 and 2 continued as planned. Due to changes in the planned study, Round 1 was terminated on day 20. The tumor volume data from the Round 1 study are presented in Figure 24. Notably, although treatment in group 3 was discontinued after 9 days, the data show that AGX-A alone performed better than the SOC drug gemcitabine. Furthermore, the data from group 4 show that combination therapy using AGX-A with gemcitabine (again, treatment was discontinued after only 9 days) yielded an even greater anti-cancer effect.

[0208] To overcome the problems posed by DMSO, we conducted research to develop a formulation compatible with AGX-A and suitable for administration. In one study, we investigated the use of sulfobutyl ether-β-cyclodextrin (CAPTISOL) together with the anti-Id compound of this technology, and formulations containing sulfobutyl ether-β-cyclodextrin neutralized AGX-A-induced cell death in cholangiocarcinoma cells. Ultimately, a particularly effective formulation using 2-hydroxypropyl-β-cyclodextrin ("HPBCD") was discovered. HPBCD was found to effectively solubilize AGX-A in aqueous solution while maintaining AGX-A activity against cholangiocarcinoma cells. Representative data are shown in Figures 25A-25B, which provide results from 24-hour cell viability studies of SNU1079 and SNU1196 cells, respectively. These studies and data demonstrate that HPBCD and equivalent solubilizers can be effectively used in pharmaceutical formulations containing anti-Id compounds of this technology.

[0209] Given this finding, we conducted a "Round 2" study as shown below, in which AGX-A was administered with a saline solution containing 12.5% ​​by mass of HPBCD, and gemcitabine was administered with saline (as gemcitabine was administered in the first round of the study). Round 2 Research Protocol - Animal Models and Experimental Design (AGX-A + HPBCD): • Mice: Female NSG (Non-Stress Growth) at 6-8 weeks of age • PDX:RomeP_PHCH_X_0008a, SC serial transplantation using Matrigel • Treatment group (at least 6 animals / group): Tumors 100 mm 3 Treatment was initiated when the condition was reached (Day 0 for each animal). Group 1.1 2.5% by weight of saline solution containing HPBCD ("vehicle"), ip, QD x 5 x 1 week each week Group 2: Gemcitabine 15 mg / kg ip QW x 1 week Group 3. AGX-A 10 mg / kg ip QDx5x 1 week Group 4. AGX-A 15 mg / kg ip QDx5x 1 week Group 5. Gemcitabine 15 mg / kg ip QW + AGX-A 10 mg / kg ip QD x 5 x 1 week Group 6. Gemcitabine 15 mg / kg ip QW + AGX-A 15 mg / kg ip QD x 5 x 1 week

[0210] During the study, tumor volume, body weight, and clinical signs were monitored at least twice a week for each animal in each group. No weight loss was observed in any of the animals in either group during this Round 2 study period, indicating that HPBCD is well-tolerated. The tumor volume data from this Round 2 study are presented in Figure 26. The data from this Round 2 study confirmed the significant efficacy and effectiveness of the anti-Id compound demonstrated in the Round 1 study, both as monotherapy and in some combination therapies.

[0211] (Example 22) Effects of AGX51 and the compounds of this technology on various additional cancer cell lines Four T1 mouse mammary cancer cell lines representing major mammary cancer subtypes (ER+, HER2+, and TNBC) and nine other mammary cancer cell lines, e.g., MDA-MB-157, MDA-MB-436, MDA-MB-231, MDA-MB-453, MDA-MB-361, BT-474, SK-BR-3, MCF-7, and T47-D, are grown in RPMI or DMEM (Dulbecc's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), 1% penicillin-streptomycin, and 1% L-glutamine. Patient-derived xenograft cell lines BR7, BR11, and IBT are established directly from bone metastasis specimens of mammary cancer surgically resected from patients with informed consent. BR7 and BR11 specimens are obtained from ER-positive (HER2-negative and PR-negative) metastatic mammary cancer patients, and IBT specimens are obtained from metastatic triple-negative mammary cancer patients. Fresh tumor tissue is quickly washed with ice-cold PBS and finely chopped into approximately 1-3 mm pieces in MEM medium (FBS-free) using a sterile razor blade. A portion of the finely chopped original tumor tissue is incubated with collagenase / hyaluronidase enzyme mixture (1,000 units, Voden Medical, Lombardia, Italy) in FBS-free MEM medium (5 mL / 250 mg tissue) for 2-4 hours. The dissociated tumor tissue is then filtered through a 70 μm nylon filter, the cells are concentrated by centrifugation at room temperature, seeded, and each is induced into primary cell cultures in MEM medium containing 3% FBS (Sigma). The primary cell cultures are transduced with a fluorescent td tomato- / EGFP-luciferase fusion protein expressing a lentiviral vector for 18-24 hours, and the primary cells are then maintained in MEM medium supplemented with 3% FBS, 1% penicillin-streptomycin, and 1% L-glutamine. Aliquots of primary cell cultures are also cryopreserved after a minimum number (3-4) in vitro passages. Cells are treated with one of the compounds of this technique, such as 100 μM DMSO, AGX51, or AGX-A. Cell morphology and proliferation are monitored daily for one week by microscopic examination for morphological changes or cell death. Cancer cell lines are seeded into 96-well plates (5000 cells per well) to determine IC50.After overnight incubation, cells were treated with AGX51 (0, 5, 10, 20, 40, 60 μM) or the compounds of this technology (0, 5, 10, 20, 40, 60 μM) and incubated for 24, 48, and 72 hours, repeating each condition three times. At each time point, 40 μL of MTT reagent (5 mg / mL) was added per well, and the cells were incubated for 4 hours. After incubation, the medium was aspirated and 200 μL of DMSO was added per well. Absorbance was then measured at 570 nm using a plate reader (Synergy2, BioTek). To support future comparisons with the compounds of this technology, the study was first conducted on AGX51 as described above, providing the data shown in Table 3 below.

[0212] [Table 3]

[0213] The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on 4T1 cells were also evaluated using the Alamer Blue viability assay according to the manufacturer's instructions. In summary, 5000 4T1 cells were seeded in a 96-well plate, treated with 40 μM AGX51 for 24 hours the following day, and then a 1:10 dilution of Alamer Blue cell viability reagent was added to the cells. Absorbance was measured 2, 3, 4, 5, and 6 hours after reagent addition using a plate reader (Synergy2, BioTek). To support future comparisons with compounds of this technology, such assays were performed with AGX51, and the results are provided in Figure 27.

[0214] The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on pancreatic cancer cells and organoid cells will also be investigated. The cell lines to be tested are human pancreatic cancer cell lines Panc1 and A21, mouse pancreatic cancer cell lines 806 (KrasG12D;Ink4a- / -;Smad4- / -), NB44 (KrasG12D;Ink4a- / -), and 4279 (KrasG12D;Ink4a- / -), and mouse pancreatic organoid cell lines T7 and T8. Pancreatic spheroids will be grown in ultra-low adhesion DMEM culture plates (Corning, Oneonta, NY, USA) supplemented with Glutamax (2 mM) and heparin (5 μg / mL). Pancreatic organoids are prepared as described in Boj, SF, et al., Cell 160: 324-338 (2015), incorporated herein by reference, using B-27 (12587-010, Life Technologies, Carlsbad, CA), HEPES (10mM), 50% Wnt / R-spondin / Noggin conditioned medium (ATCC, CRL-3276), Glutamax (2mM, Invitrogen, Carlsbad, Calif.), N-acetylcysteine ​​(1mM, Sigma, St. Louis, MO, USA), nicotinamide (10mM, Sigma, St. Louis, MO, USA), and epidermal growth factor (50ng / mL, Peprotech, Rocky). Cell lines and organoids were embedded in Matrigel using Advanced DMEM / F12 (12634-028, Gibco, Carlsbad, CA) supplemented with gastrin (10 nM, Sigma-Aldrich, St. Louis, MO), fibroblast growth factor-10 (100 ng / mL, Peprotech, Rocky Hill, NJ), and A83-01 (0.5 μM, Tocris, Bristol, United Kingdom). All cell lines and organoids were maintained at 37°C and 5% CO2. Cell viability was measured using Cell Titer-Glo (Promega, Madison, WI) according to the manufacturer's instructions.To support future comparisons with compounds of this technology, the study was first performed using AGX51, and the results are shown in Figure 28 (mouse organoids), Figure 29 (mouse cell lines 806, NB44, and 4279) and Figure 30 (human cell lines Panc1 and A21). The compounds of this technology showed similar or significantly improved effects, and it is expected that this will provide further evidence that the compounds of this technology are useful for treating cancer.

[0215] (Example 23) Effects of AGX51 and the compounds of this technology on xenografts of mouse cancer cell lines. The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on primary tumors will be tested in a xenograft model of primary tumors using MDA-MB-231 cells, with and without the use of paclitaxel. Orthotopic mammary lipomas will be studied in 5 × 10⁻⁶ cells. 6 MDA-MB-231 cells (1:1 PBS:Matrigel) are generated by injecting them into the right caudal mammary fat pad of 8-12 week old female athymoid nu / nu mice. Mice are obtained from Simonsen Laboratories, Gilroy, CA. The tumor is approximately 100 mm. 3The tumors are allowed to grow to a certain size, at which point they are divided into 12 groups of 5 mice, each with approximately the same tumor size, and then treatment is initiated. Group 1 was the vehicle (DMSO, administered q5d), Group 2 received 15 mg / kg of paclitaxel once daily for 5 days, Group 3 received 60 mg / kg of AGX51 twice daily for 19 days, Group 4 received a combination of 15 mg / kg of paclitaxel once daily and 6.7 mg / kg of AGX51 twice daily for 19 days, Group 5 received a combination of 15 mg / kg of paclitaxel once daily and 20 mg / kg of AGX51 twice daily for 19 days, Group 6 received a combination of 15 mg / kg of paclitaxel once daily and 60 mg / kg of AGX51 twice daily for 19 days, and Group 7 received 15 mg / kg of paclitaxel once daily for 19 days, along with 60 mg / kg of AGX51. Group 8 receives AGX51 twice daily for the first 7 days, Group 9 receives 60 mg / kg of the compound of this technology (e.g., AGX-A) twice daily for 19 days, Group 9 receives a combination of 15 mg / kg of paclitaxel once daily and 6.7 mg / kg of AGX-A twice daily for 19 days, Group 10 receives a combination of 15 mg / kg of paclitaxel once daily and 20 mg / kg of AGX-A twice daily for 19 days, Group 11 receives a combination of 15 mg / kg of paclitaxel once daily and 60 mg / kg of AGX-A twice daily for 19 days, and Group 12 receives 15 mg / kg of paclitaxel once daily for 19 days, while simultaneously receiving 60 mg / kg of AGX51 twice daily for the first 7 days. The treatment agents are administered intravenously. Tumor volume was determined throughout the study using digital calipers and the formula: Tumor volume = 1 / 2(length × width²), where the maximum longitudinal diameter is the tumor length and the maximum transverse diameter is the width. At the end of the study, mice were euthanized by cervical dislocation. To support future comparisons with compounds of this technique, the study was first performed with AGX51 (i.e., provided to groups 1-7, except for one mouse in group 2 which died during the study). Exemplary data of the effect of AGX51 on primary tumors with and without paclitaxel are shown in Figure 31.The compounds of this technology are expected to demonstrate similar or significantly improved effects, providing further evidence that these compounds are useful in treating triple-negative breast cancer.

[0216] The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on metastasis will also be investigated using a lung colonization model. Lung metastases will be generated by injecting 50,000 luciferase-labeled 4T1 cells into the tail vein of 6-8 week old female Balb / c mice. 24 hours after tail vein injection, mice will be treated once daily with DMSO, 50 mg / kg AGX51, or twice daily with 50 mg / kg, e.g., AGX-A (at least 5 mice per treatment group) via intravenous injection. The development of lung metastases will be monitored using the IVIS-200 in vivo imaging system. To support future comparisons with compounds of this technology, studies will be performed using MSO and AGX51, and exemplary data on the effect of AGX51 on lung colonization will be presented in Figure 32. The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on established lung metastases will also be investigated. Once evidence of lung metastasis was observable by in vivo imaging, the mice were divided into groups of five, each with approximately the same tumor load. The groups were as follows: Group 1: Vehicle (DMSO) for 5 days; Group 2: 50 mg / kg AGX51 twice daily for 5 days; Group 3: 15 mg / kg paclitaxel once daily for 5 days; Group 4: A combination of 50 mg / kg AGX51 twice daily and 15 mg / kg paclitaxel once daily for 5 days; Group 5: 50 mg / kg, e.g., AGX-A, twice daily for 5 days; Group 6: A combination of 50 mg / kg AGX51 twice daily and 15 mg / kg paclitaxel once daily for 5 days. At the end of the experiment, the mice were euthanized and tissues were collected for further analysis. Lung tumor load was quantified blindly by a pathologist. To support future comparisons with compounds of this technology, studies were conducted using DMSO and AGX51, and exemplary data on the established effect of AGX51 on lung metastases are presented in Figure 33. It is expected that compounds of this technology will show similar or significantly improved effects.

[0217] To evaluate the inhibition of extravasation and early dissemination at secondary sites, or the progression of extravasation of cancer cells into tumors, GFP-labeled 4T1 cells were injected into mice via the tail vein, and the mice were treated for 24 or 48 hours with DMSO, AGX51 (5 mg / kg), or a compound of this technology (e.g., AGX-A; 50 mg / kg). The lungs were then stained for GFP (i.e., tumor cells), and the number of tumor cells was quantified. To support future comparisons with compounds of this technology, the study was first performed using DMSO and AGX51, and exemplary data on the effect of AGX51 on early dissemination of cancer cells at secondary sites are shown in Figure 34A for all cells and in Figure 34B for all tissue area. It is expected that compounds of this technology will show similar or significantly improved effects.

[0218] The effects of DMSO, AGX51, or compounds of this technology (such as AGX-A) on sporadic tumors will be investigated using an azoxymethane (AOM) colon tumor model, a chemically induced spontaneous adenoma model. Spontaneous colon tumors will be induced in 30 four-week-old male A / J mice (Jackson Laboratory) by treating them with AOM (10 mg / kg; Sigma Aldrich) once a week via ip injection for six weeks. During the experiment, the mice will be fed AIN-93G purified diet (Research Diets). After a three-week treatment break, the mice will be treated with DMSO, AGX51 (15 mg / kg), or compounds of this technology (e.g., AGX-A; 15 mg / kg) for three weeks via ip injection. Following the final injection, the mice will be euthanized, and the colon tumors will be formalin-fixed to assess tumor volume. The number and size of tumors will be determined in the entire tissue mount after methylene blue staining. To support future comparisons with compounds of this technology, studies were conducted using DMSO and AGX51, and exemplary data on the effect of AGX51 on sporadic tumors are shown in Figures 35A-35D. In particular, AGX51 treatment resulted in a significant reduction in the number of colon tumors (p=0.008), as shown in Figure 35A. In addition, tumors in the AGX51 group were smaller than those in the DMSO group (Figures 35B-35D), and this difference was significant when comparing tumors measured >3 mm, as shown in Figure 35D (p=0.004). Compounds of this technology are expected to show similar or significantly improved effects.

[0219] (Example 24) Effects of AGX51 and the compounds of this technology on prostate cancer cell lines Prostate cancer cell lines DU145 and PC3 (10% BCS) are cultured in Ham's F12 (Gibco, Carlsbad, Calif.) medium containing 10% BCS (Hyclone, Logan, Utah) and appropriate antibiotics (pen / strep, fungizone, and gentamicin (Invitrogen Inc., Carlsbad, CA)). All cells are cultured at 37°C in a fully humidified atmosphere containing 5% CO2. At 50% confluence, cells are treated with either 100 μM DMSO, AGX51, or a compound of this technology (such as AGX-A). Cell morphology and proliferation are monitored daily for one week by microscopic examination for morphological changes or cell death. Apoptosis is determined by measuring the activity of caspase-3 and caspase-7 using the Promega((Madison, Wis.)) Caspase-Glo3 / 7 assay system.

[0220] AGX51 and the compounds of this technology are expected to show significant effects on DU145 cells: after 3 days, the cells appear very unhealthy and are unable to proliferate compared to the control. Furthermore, after 6 days, treatment of DU145 cells with either AGX51 or the compounds of this technology results in cell death. The compounds of this technology are expected to show similar or significantly improved effects compared to AGX51. The molecular mechanism underlying the effects of the compound in this technology on prostate cancer cells will be evaluated by measuring the activity of caspase 3 / 7, a major mediator of apoptosis. Low concentrations of the compound in this technology will result in a significant increase in caspase 3 / 7 in both DU145 and PC3 cells, which is expected to be higher than the caspase activity in cells treated with staurosporine (10 μm), a known apoptosis inducer.

[0221] (Example 25) Effect of AGX51 and the compound of this technology on anti-angiogenic activity in mouse Matrigel assays Matrigel plugs treated with VEGF-165 and FGF-2 are implanted into C57BL / 6 mice on day 0. Mice are treated with either a vehicle, AGX51, or a compound of this technology (e.g., AGX-A). AGX51 and the compound of this technology are provided either as a plug (25 μg / mg) or as daily ip treatment for 10 days (30 or 100 mg / kg). Plugs are harvested, fixed, and paraffin-embedded on day 10. Three sections (5 μM thick) of each plug are stained with anti-CD31 antibody and counterstained with hematoxylin and eosin. CD31-positive microvessels are counted for one entire section per plug to determine the mean microvascular density ± SD vessels. Student's t-test is used for statistical analysis.

[0222] Compared to vehicle-only control animals, treatment with AGX51 or the compounds of this technology is expected to provide significant protection from new angiogenesis. Typical photographs of Matrigel plug sections are expected to show the presence of complete vascularization in vehicle-only controls. In contrast, the presence of endothelial cells is expected to be significantly reduced by treatment with AGX51 or the compounds of this technology. The compounds of this technology are expected to show similar or significantly improved effects compared to AGX51.

[0223] (Example 26) Effects of AGX51 and the compounds of this technology on transferability activity in LLC mouse models 30 C57BL / 6 mice, 7.5 x 10 5LLC cancer cells / animals are transplanted. Seven days after transplantation, 5M / 5F animals per group are treated daily with an ip treatment for 25 days using either the administration vehicle (DMSO), AGX51, or a compound of this technology (e.g., AGX-A). Fourteen days after transplantation, another group of 5M / 5F animals is treated daily with an ip treatment for 18 days using 50 mg / kg of AGX51, and another group of 5M / 5F animals is treated daily with an ip treatment for 18 days using a compound of this technology (e.g., AGX-A). Tumors are measured three times from day 7 to day 14. Tumors are resected on day 14. Animals are autopsied for the presence of lung metastases on days 32 and 18 after resection. Treatment with AGX51 and the compounds of this technology is expected to significantly reduce lung metastases. Furthermore, the compounds of this technology are expected to show similar or significantly improved effects compared to AGX51.

[0224] Equal parts While certain embodiments are illustrated and described, those skilled in the art, after reading the foregoing specification, can make modifications, substitutions of equivalents, and other types of changes to the compounds of the Art described herein, or their salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures. Each of the above embodiments may also include or incorporate variations or embodiments disclosed in relation to any or all of the other embodiments. This technology is also intended as a single example of individual embodiments of this technology and should not be limited to any particular embodiment described herein. Many modifications and variations of this technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods within the scope of this technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It should be understood that this technology is not limited to any particular method, reagent, compound, composition, labeled compound, or biological system, and may, of course, change. It should also be understood that the terms used herein are intended solely to describe a particular embodiment and are not intended to limit it. Accordingly, this specification is intended to be considered only as an example with respect to the breadth, scope, and spirit of this technology, as indicated only by the appended claims, the definitions therein, and their equivalents.

[0225] The embodiments described herein as exemplary may be suitably carried out even without any elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be read broadly and without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only and not limitation, and in the use of such terms and expressions, it is not intended to exclude equivalents of any or any of the indicated and described features, and it is recognized that various modifications are possible within the scope of the claimed technology. Furthermore, the phrase “essentially consisting of” should be understood to include the specifically enumerated elements and any additional elements that do not substantially affect the fundamental and novel characteristics of the claimed technology. The phrase “consisting of” excludes any elements not explicitly stated.

[0226] In addition, where any feature or aspect of this disclosure is described in terms of the Markush group, those skilled in the art will recognize that this disclosure also describes in terms of any individual member or subgroup of any member of the Markush group. Each of the narrower species and subtribe groups within the scope of the general disclosure also forms part of the invention. This includes the general description of the invention having conditions or negative limitations on removing any subject matter from a genus, whether or not the excised material is specifically enumerated herein.

[0227] As will be understood by those skilled in the art, for all purposes, and especially in terms of providing written descriptions, all scopes disclosed herein also encompass any possible subscopes and combinations thereof. It will be readily apparent that each enumerated scope is sufficiently described so that the same scope can be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-restrictive example, each scope discussed herein can be readily divided into lower thirds, middle thirds, upper thirds, etc. As will also be understood by those skilled in the art, all language such as “up to,” “at least,” “greater than,” and “less than” refers to a scope that includes the enumerated number and can then be divided into subscopes as described above. Finally, as will be understood by those skilled in the art, a scope includes its individual members.

[0228] All publications, patent applications, granted patents, and other documents (e.g., periodicals, articles, and / or textbooks) referenced herein are incorporated by reference as if each individual publication, patent application, granted patent, or other document were specifically and individually indicated to be incorporated by reference in whole. Definitions contained in the incorporated material are excluded insofar as they conflict with the definitions in this disclosure. This technology may include, but is not limited to, the features and combinations of features listed in the following textualized sections, and it should be understood that the following paragraphs should not be construed as limiting the claims attached to this document or as requiring all such features to necessarily be included in such claims. A. Formula I

[0229] [ka] (I) Compounds thereof, or pharmaceutically acceptable salts and / or solvates thereof. (In the formula, R 1 , R 2 , and R 3 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanil, halo, or -N(R) 10 )(R 11 ) and; R 4 , R 5 , R 6 , and R 7 These are independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanil, halo, or -N(R) 12 )(R 13 ) and; R 8 It is either an aryl or heteroaryl; R 9 is H, C1-C3 alkyl, or fluoro; R 10 , R 11 , R 12 , and R 13 (Each of these is independently a C1-C3 alkyl group.) BR 1 , R 2 , and R 3However, each is independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(R) 10 )(R 11 The compound in item A is ). CR 1 , R 2 , and R 3 The compound according to item A or B, wherein each is independently H, C1-C3 alkyl, C1-C3 alkoxy, halo, or -N(Me)2. DR 1 , R 2 , and R 3 A compound from any one of items A to C, wherein each is independently H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. ER 3 However, it is a compound from any one of items A to D, which is methoxy. FR 4 , R 5 , R 6 , and R 7 However, each is independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(R) 12 )(R 13 A compound that is one of the compounds in items A to E. GR 4 , R 5 , R 6 , and R 7 A compound from any one of items A to F, wherein each is independently H, C1-C3 alkyl, C1-C3 alkoxy, halo, or -N(Me)2. HR 4 , R 5 , R 6 , and R 7 A compound from any one of items A to G, wherein each is independently H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. IR 6 However, it is an isopropoxy compound, one of the compounds listed in items A to H. J. Formula IA

[0230] [ka] (IA) A compound of any one of items A to I, or a pharmaceutically acceptable salt and / or solvate thereof. K.Formula IB

[0231] [ka] (IA) A compound of any one of items A to I, or a pharmaceutically acceptable salt and / or solvate thereof. (In the formula, R 14 , R 15 , and R 16 (Each of these is independently H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, trialkylammonium, pentafluorosulfanyl, halo, aryloxy, arylroyl, hydroxyl, amino, or amide.) LR 14 , R 15 , and R 16 The compounds of item K, each independently being H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, aryloxy, arylroyl, or -N(C1-C3 alkyl)2. MR 14 , R 15 , and R 16 The compounds of item K or L, each independently being H, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl, trifluoromethoxy, halo, or -N(Me)2. NR 14 , R 15 , and R 16 A compound of any one of the K to M items, wherein each is independently H, methyl, methoxy, isopropyl, isopropoxy, fluoro, or -N(Me)2. Ure 14 , R 15 , and R16 However, each is independently a hydrogen atom, and the compound is one of the K-N terms. P.

[0232] [ka] JPEG0007880815000031.jpg145170 The compound in any one of sections A to O, or a pharmaceutically acceptable salt and / or solvate thereof, is JPEG0007880815000032.jpg139166. Q. IC

[0233] [ka] (I C) A compound of any one of items A to O, or a pharmaceutically acceptable salt and / or solvate thereof. R.

[0234] [ka] JPEG0007880815000035.jpg143170 The compound in any one of items A to Q, or a pharmaceutically acceptable salt and / or solvate thereof, is JPEG0007880815000036.jpg141159.

[0235] A compound from any one of the SA-R items; and Pharmacologically acceptable carriers A composition containing the following: T. A compound from any one of items A to R in an effective amount for treating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease in the subject; and Pharmacologically acceptable carriers A pharmaceutical composition containing the following: U. A pharmaceutical composition of item T, wherein the pathogenic angioplomatic disorder includes pathogenic angiogenesis associated with a cancerous disease or condition. V. A pharmaceutical composition of item T or U, in which pathogenic angioproliferative disease includes ocular diseases.

[0236] W. A pharmaceutical composition according to any one of items T to V, wherein the pathogenic angioproliferative disorder is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, retinal vein occlusion, central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular macular degeneration, or ocular cancer. X. A pharmaceutical composition according to any one of items T to W, wherein the pathogenic angioproliferative disorder includes age-related macular degeneration (AMD). Y. A pharmaceutical composition according to any one of items T to X, wherein the pathogenic angioproliferative disorder includes wet-type and exudative-type age-related macular degeneration.

[0237] Z. A pharmaceutical composition according to any one of items T to Y, wherein the cancer includes cholangiocarcinoma, triple-negative breast cancer, or colorectal cancer. AA. A pharmaceutical composition according to any one of items T to Z, formulated for parenteral administration, intravenous administration, subcutaneous administration, and / or oral administration. AB. A pharmaceutical composition according to any one of items T to AA, wherein the pharmaceutically acceptable carrier comprises 2-hydroxypropyl-β-cyclodextrin. AC. A pharmaceutical composition according to any one of items T to AB, formulated for use in mammals suffering from neoplasms. AD. A pharmaceutical composition according to any one of items T to AC, formulated for use in mammals that have a neoplasm or present a history of neoplasm.

[0238] AE. A pharmaceutical composition according to any one of the items T to AD, formulated for use in mammals that have a neoplasm or have previously been treated for a neoplasm and achieved clinical remission. AF. A pharmaceutical composition according to any one of the clauses T to AE, formulated for use in mammals suffering from conditions mediated by or contributed to by pathogenic angiogenesis. AG. A method for treating a condition in a subject, comprising administering to the subject an effective amount of a compound from any one of items A to R for treating the condition, wherein the condition includes one or more of the following: pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease. AH. Pathogenic angioproproliferative disorders include methods of the AG clause, including pathogenic angiogenesis associated with cancerous disease or condition. AI. Pathogenic angioproproliferative disorders, including ocular diseases, by the AG or AH method. AJ. A method according to any one of items AG to AI, wherein the pathogenic angioproliferative disorder is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, retinal vein occlusion, central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular macular degeneration, or ocular cancer. AK.X. Pathogenic angioproliferative disorder, including age-related macular degeneration (AMD), as defined in any one of the AG-AJ sections. AL. Pathogenic angioproliferative disease, including wet and exudative age-related macular degeneration, as defined in any one of the AG-AK criteria. AM. A method according to any one of the AG-AL clauses, where the cancer includes cholangiocarcinoma, triple-negative breast cancer, or colorectal cancer. AN. A method according to any one of sections AG to AM, wherein the pharmaceutical composition is formulated for parenteral administration, intravenous administration, subcutaneous administration, and / or oral administration.

[0239] Other embodiments are described in such claims, along with the entire scope of equivalents to which the following claims are given.

Claims

1. Formula IB 【Chemistry 1】 (IB) Compounds thereof, or pharmaceutically acceptable salts and / or solvates thereof. (In the formula, R 1 and R 2 These are H and C, respectively, independently. 1 -C 3 Alkyl, C 1 -C 3 It is an alkoxy or halo; R 3 is C 1 -C 3 alkyl, C 1 -C 3 alkoxy, or halo; R 4 , R 5 , R 6 , and R 7 These are H and C, respectively, independently. 1 -C 3 Alkyl, C 1 -C 3 It is an alkoxy or halo; R 9 H, C 1 -C 3 Alkyl or fluoro; R 14 , R 15 , and R 16 Each of them is independently C 1 -C 3 (It is alkyl or hydroxyl.)

2. R 1 and R 2 However, each is independently H, methyl, methoxy, isopropyl, isopropoxy, or fluoro, and R 3 The compound according to claim 1, wherein the compound is methyl, methoxy, isopropyl, or isopropoxy.

3. R 3 The compound according to claim 1, wherein is methoxy.

4. R 4 , R 5 , R 6 , and R 7 The compound according to claim 1, wherein each is independently H, methyl, methoxy, isopropyl, isopropoxy, or fluoro.

5. R 6 The compound according to claim 1, wherein isopropoxy. 【Request Item 6】 【Chemistry 2】 A compound that is a compound, or a pharmaceutically acceptable salt and / or solvate thereof.

7. Formula IC 【Transformation 3】 (I C) The compound according to claim 1, or a pharmaceutically acceptable salt and / or solvate thereof. 【Request Item 8】 【Chemistry 4】 A compound that is a compound, or a pharmaceutically acceptable salt and / or solvate thereof.

9. The compound according to any one of claims 1 to 8; and Pharmacologically acceptable carriers A composition containing the following:

10. The compound according to any one of claims 1 to 8; and Pharmacologically acceptable carriers A pharmaceutical composition for treating pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease in a subject, including the above.

11. The pharmaceutical composition according to claim 10, wherein the pathogenic angioproliferative disorder includes pathogenic angiogenesis associated with cancer or a disease.

12. The pharmaceutical composition according to claim 10, wherein the pathogenic angioproliferative disease includes ocular diseases.

13. The pharmaceutical composition according to claim 12, wherein the eye disease is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, retinal vein occlusion, central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular macular degeneration, or ocular cancer.

14. The pharmaceutical composition according to claim 10, wherein the pathogenic angioproliferative disorder includes age-related macular degeneration (AMD).

15. The pharmaceutical composition according to claim 10, wherein the pathogenic angioproliferative disorder includes wet-type and exudative-type age-related macular degeneration.

16. The pharmaceutical composition according to claim 10, wherein the cancer includes cholangiocarcinoma, triple-negative breast cancer, or colorectal cancer.

17. The pharmaceutical composition according to claim 10, which is formulated for parenteral administration, intravenous administration, subcutaneous administration, and / or oral administration.

18. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable carrier comprises 2-hydroxypropyl-β-cyclodextrin.

19. The pharmaceutical composition according to claim 10, formulated for use in mammals suffering from neoplasms.

20. The pharmaceutical composition according to claim 10, formulated for use in mammals that are suffering from or have a history of neoplasms.

21. The pharmaceutical composition according to claim 10, formulated for use in mammals that are suffering from a neoplasm or have previously been treated for a neoplasm and achieved clinical remission.

22. The pharmaceutical composition according to claim 10, formulated for use in mammals suffering from a condition mediated by or contributed to by pathogenic angiogenesis.

23. Use of the compound according to any one of claims 1 to 8 in the manufacture of a pharmaceutical product for treating one or more of the following: pathogenic cell proliferation, angiogenesis, cancer, metastatic disease, and / or pathogenic angioproliferative disease.

24. The use according to claim 23, wherein the pathogenic angioproliferative disorder includes pathogenic angiogenesis associated with cancer or a condition.

25. The use according to claim 23, wherein the pathogenic angioproliferative disease includes ocular diseases.

26. The use according to claim 23, wherein the pathogenic vascular proliferative disease is selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, retinal vein occlusion, central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), neovascular macular degeneration, or ocular cancer.

27. The use according to claim 23, wherein the pathogenic angioproliferative disorder includes age-related macular degeneration (AMD).

28. The use according to claim 23, wherein the pathogenic angioproliferative disorder includes wet and exudative age-related macular degeneration.

29. The use according to claim 23, wherein the cancer includes cholangiocarcinoma, triple-negative breast cancer, or colorectal cancer.

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