Chemical inhibitors of Id proteins for the treatment of cancer and other diseases

Abstract

The present invention relates to compounds that inhibit the expression of Id1 and / or Id3 and their use in the treatment of cancer and other diseases and conditions associated with the expression of Id1 and / or Id3.

Description

[Technical field]

[0001] The present invention relates to compounds that inhibit the expression of Id1 and / or Id3 and their use in the treatment of cancer and other diseases and conditions associated with the expression of Id1 and / or Id3. [Background technology]

[0002] Effective treatment of cancer remains a major unmet clinical need. In 2018, approximately 18 million new cases of cancer occurred worldwide, projected to increase to 23.6 million new cases annually by 2030. Currently, approximately one in six total deaths worldwide is attributable to cancer, representing 9.6 million deaths in 2018. Approximately 90% of these deaths are estimated to be caused by the direct and indirect effects of metastasis. Metastatic disease remains essentially incurable, and new therapies are urgently needed. Given the scale of the problem, the global cancer therapeutics market is estimated to be worth $178.863 billion by 2023.

[0003] The cancer stem cell theory states that tumor growth is driven by a subset of cancer stem cells (CSCs) that can self-renew, give rise to heterogeneous progeny, and initiate new tumor growth. In non-CSC subpopulations, tumor cells do not possess these properties and are therefore non-tumorigenic. The presence of CSCs has many predicted important ramifications, for example, it should be possible to effectively treat cancer by targeting tumor cells that possess stem cell properties. As metastasis is caused by the seeding of new tumors in various organs of the body, targeting stemness should also be a means to prevent or treat metastasis, since CSCs are, by definition, the only cells that can initiate new tumor growth. Recent evidence suggests that acquisition of stemness by dormant tumor cells may lead to their growth as overt metastases.

[0004] Tumor initiation in vivo is currently the gold standard for defining CSCs. The majority of published studies use co-injection of tumor cells with Matrigel to determine tumor initiation rates in vivo. In syngeneic animal models of breast cancer and melanoma, we could show that Matrigel, laminin, and collagen all have a strong and significant enhancing effect on tumor incorporation rates. Gene expression profiling and subsequent validation showed that the culture of tumor cells in a 3D microenvironment composed of these ECM components strongly upregulates the expression of the transcriptional regulators Id1 and Id3. This upregulation is explained, at least in part, by autocrine BMP signaling, which is promoted in the 3D matrix by the local accumulation of self-produced BMPs around the cells.

[0005] Id1 and Id3 are genes that have been shown to play a crucial role in the initiation of primary tumors and metastatic growth. They are involved in determining and maintaining the CSC characteristics of several types of tumors, including gliomas and colorectal cancer. Thus, their expression correlates with poor prognosis in many types of cancer. In addition to their role in regulating stem cell characteristics, Id1 and Id3, individually or together, are involved in promoting tumor cell invasiveness and resistance to chemotherapy.

[0006] Cancer entities with elevated Id1 and / or Id3 levels include prostate cancer, B acute lymphoblastic leukemia, non-small cell lung cancer, ovarian tumors, esophageal squamous cell carcinoma, breast cancer, and melanoma. In many cases, Id1 and Id3 are co-expressed in tumor tissues, highlighting the importance of simultaneously inhibiting both Id proteins.

[0007] Id1 and Id3 are also involved in the induction of angiogenesis and lymphangiogenesis, in part through upregulation of VEGF-A and VEGF-C, respectively. Thus, inhibition of these genes may inhibit tumor growth and progression, not only through direct effects on the tumor cells themselves, but also through inhibition of angiogenesis and lymphangiogenesis. Because angiogenesis and lymphangiogenesis are hallmarks of many diseases other than cancer, inhibition of Id1 and / or Id3 may also have therapeutic impact in these settings.

[0008] Both Id1 and Id3 are involved in determining the differentiation state of immune cells and are important drivers of diseases involving immune dysregulation. Id1 expands the myeloid-derived suppressor cell population and mediates dendritic cell differentiation and CD8 + Id3 inhibits T cell proliferation, generating a tumor-promoting, immunosuppressive immune environment. H 17 Inhibits helper T cell differentiation and Foxp3 + T reg Promotes cell development. Foxp3 + T reg Id1 and Id3 suppress T cell immunity, promote tolerance, and inhibit anti-cancer immune responses in the context of cancer. Moreover, Id3 is required for strong TCR signals to promote T cells to adopt a γδ fate and oppose an αβ fate outcome. Neoplastic γδ T cells promote tumor progression by inducing an immunosuppressive tumor microenvironment, stimulating angiogenesis via cytokines they produce, interfering with dendritic cell effector function, and inhibiting anti-tumor T cell immunity via the PD-L1 pathway. Thus, inhibition of Id1 and Id3 may inhibit tumor growth and progression not only by direct effects on tumor cells, but also by suppressing the tumor-promoting immunosuppressive microenvironment. These observations also indicate that inhibitors of Id1 and Id3 may be useful for the treatment of cancer in combination with immune checkpoint inhibitors.

[0009] Id1 is involved in mediating epithelial-to-mesenchymal transition (EMT), which is believed to be a critical process in metastasis. Furthermore, EMT contributes to several fibrotic diseases, such as pulmonary, hepatic, renal, or cardiac fibrosis. Direct evidence suggests an important role for Id1 in liver fibrogenesis. Inhibiting Id1 / Id3 function may impair EMT, thereby preventing metastasis and fibrosis.

[0010] Recently, knockdown of Id3 was shown to render melanoma cells more sensitive to vemurafenib treatment, suggesting a possible role for Id3 in mediating adaptive therapy resistance in melanoma. Interestingly, resistance to paclitaxel treatment in nasopharyngeal carcinoma cells is mediated by activation of the Raf / MEK pathway, which results in increased Id1 levels. Combining BRAF / MEK inhibitors with Id1 / Id3 inhibitors may be useful for improving melanoma treatment.

[0011] The expression and activity of Id1 and Id3 are also implicated in rare diseases. Id1 plays a central role in Castleman disease, a rare lymphoproliferative disorder. Id3 plays a role in the pathogenesis of fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by extraskeletal bone formation by endochondral ossification. Thus, inhibition of Id1 and Id3 may have therapeutic utility in these diseases.

[0012] In normal physiological situations, Id1 inhibits adipogenic differentiation and Id3 may play a similar role, and thus inhibition of Id1 and / or Id3 may be beneficial in the context of modulating adipogenesis in vivo or differentiation of cultured stem cells, particularly iPSCs.

[0013] Furthermore, Id3 has been reported to inhibit implantation of trophoblast into the uterine wall and has been implicated in recurrent miscarriage. Thus, inhibition of Id3 may enhance fertility by promoting implantation and inhibiting miscarriage during natural pregnancy or as part of an in vitro fertilization procedure.

[0014] Several strategies have been reported to target Id protein or gene expression. Peptide-based approaches have been used in vitro and in vivo. Difficulties in using these approaches for therapy include the delivery of the molecules to the target cells and the pharmacological properties of the substances. In addition, small molecule inhibitors have been investigated. However, each approach lacked specificity, since the expression of many other genes besides Id was also affected.

[0015] Furthermore, natural products and substances have been considered, but their specificity for inhibiting Id expression has hardly been explored. Among such substances, cannabidiol (CBD) inhibits the expression of Id1 at the transcriptional level, and some preclinical studies suggest that CBD can inhibit tumor growth and metastasis. CBD-induced inhibition of primary tumor growth and lung metastasis in an orthotopic breast cancer model was associated with reduced Id1 levels in tumor tissue. Cannabidiol has low affinity for CB1 and CB2, which explains why CBD is considered non-psychotropic. CBD inhibited the expression of Id1 in a glioma model, resulting in reduced invasion in vivo and prolonged survival of tumor-bearing mice. However, other genes besides Id1 are also regulated by CBD. Nevertheless, among the current approaches for therapeutic inhibition of Id proteins, CBD has emerged as a benchmark due to its lack of psychotropic effects, its pharmacological properties, and the fact that it is well tolerated. Summary of the Invention [Problem to be solved by the invention]

[0016] In view of the above, the technical problem underlying the present invention is the provision of means for the inhibition of Id1 and / or Id3, and their respective uses for the treatment of diseases and conditions associated with Id1 and / or Id3 expression. [Means for solving the problem]

[0017] The solution to the above technical problem is achieved by the embodiments characterized in the claims.

[0018] In particular, in a first aspect, the present invention provides a compound having a structure according to formula (I): [ka] (In the formula, R' is H or methyl; R a is H, alkyl, or cycloalkyl; R b is H, alkyl, or cycloalkyl; R c is H, alkyl, or cycloalkyl, or R a , R b and R c are linked together to form a 5- or 6-membered alicyclic ring, R d is alkyl, aryl, or -CH2-aryl, the aryl portion of which may be substituted with one or more groups selected from the group consisting of alkyl, alkoxy, hydroxy, amino, monoalkylamino, dialkylamino, halogen, and trifluoromethyl.

[0019] With respect to the compounds of formula (I), "alkyl" refers to a straight or branched C1-C 24 , especially C1~C 10 More specifically, it means a C1 to C6 alkyl group.

[0020] Further, with respect to the compounds of formula (I), "aryl" refers to unsubstituted or substituted C6-C 10It means an aryl group, preferably a phenyl group, having one or more substituents selected from the group consisting of halogen atoms, linear or branched C1-C6 alkyl groups (which may be further substituted by halogen atoms, preferably fluorine atoms, for example -CF3 groups), C1-C6 alkoxy groups (which may be further substituted by halogen-functionalized groups, preferably fluorine groups, for example -OCF3 groups), hydroxy groups, and amino groups, including mono- or di-substituted amino groups, such as dimethylamino.

[0021] The -CH2-aryl group is in particular a benzyl group, a -CH2-mesitylene group, or a -CH2-pyridyl group, with the -CH2 substitution in the ortho, meta, or para position relative to the nitrogen atom of the pyridine moiety.

[0022] In preferred embodiments of the compounds according to formula (I) of the present invention, said compounds have a structure according to any one of formulas (Ia), (Ib), (Ic) or (Id): [ka] (In the formula, R e is -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, benzyl or hydroxybenzyl, n is 0 or 1); [ka] (In the formula, R f is -(CH2)5CH3 or -(CH2)6CH3); [ka] (In the formula, Each group R g is H or both R g are linked together to form a 5-membered alicyclic ring; [ka] (In the formula, Both R h are linked together to form a five-membered alicyclic ring).

[0023] In a particularly preferred embodiment, the compound according to formula (I) of the present invention is selected from the group consisting of the following compounds X6632, X6760, X6779, X6631, X6777, X6768, X6633, X5549 / X1384, X81, X106, X1312, X6945, X6910, X6404, X6770, X6778, X6758, X6944, X7776 and X7401, with compounds X6632, X6760, X81 and X106 being particularly preferred and compound X6632 being even more preferred: [ka]

[0024] In a second aspect, the present invention relates to compounds including compounds having a structure according to formula (II): [ka] (In the formula, X is CH or N; Y is CH or NR, R is H, alkyl, aryl, or -CH-aryl, or Y is absent; Z is CH or NR, R is H, alkyl, aryl, or -CH-aryl, or Z is absent; R 1 is alkyl or -CH-aryl, R 2 is alkyl, aryl or -CH2-aryl, R 3 is H, alkyl, Br, Cl, F, I, CN or CF3).

[0025] With respect to the compounds of formula (II), "alkyl" refers to a straight or branched C1-C 24 , especially C1~C 10 More specifically, it means a C1 to C6 alkyl group.

[0026] Further, with respect to the compounds of formula (II), "aryl" refers to unsubstituted or substituted C6-C 10 It means an aryl group, preferably a phenyl group, having one or more substituents selected from the group consisting of halogen atoms, linear or branched C1-C6 alkyl groups (which may be further substituted by halogen atoms, preferably fluorine atoms, for example -CF3 groups), C1-C6 alkoxy groups (which may be further substituted by halogen-functionalized groups, preferably fluorine groups, for example -OCF3 groups), hydroxy groups, and amino groups, including mono- or di-substituted amino groups, such as dimethylamino.

[0027] The -CH2-aryl group is in particular a benzyl group, a -CH2-mesitylene group, or a -CH2-pyridyl group, with the -CH2 substitution in the ortho, meta, or para position relative to the nitrogen atom of the pyridine moiety.

[0028] In preferred embodiments, the aryl moiety can be substituted with one or more groups selected from the group consisting of alkyl, alkoxy, hydroxy, amino, monoalkylamino, dialkylamino, halogen, and trifluoromethyl.

[0029] In further preferred embodiments of the compound according to formula (II) of the present invention, said compound has a structure according to any one of formulas (IIa), (IIb) or (IIc): [ka] (In the formula, R 1 is alkyl or -CH-aryl, R 4 is H or CF3); [ka] (In the formula, R 1 is alkyl or -CH2-aryl); [ka] (In the formula, R 1 is alkyl or -CH-aryl, R 2 is aryl).

[0030] In a particularly preferred embodiment, the compound according to formula (II) of the present invention is selected from the group consisting of the following compounds X8706, X8765, X8166, X8762, X8702, X8572, X8766, X8571 and X8035, with the compounds X8706, X8166, X8766 and X8035 being particularly preferred and the compound X8166 being even more preferred: [ka]

[0031] In a third aspect, the present invention relates to a compound of the invention for use in medicine.

[0032] In a fourth aspect, the present invention relates to a compound of the invention for use in the prevention or treatment of a condition or disease associated with cells expressing Id1 and / or Id3, an angiogenesis-dependent condition or disease, and / or a lymphangiogenesis-dependent condition or disease.

[0033] The condition or disease associated with cells expressing Id1 and / or Id3 may be selected from the group consisting of initiation of tumor formation, initiation of tumor metastasis, tumor growth, metastatic growth, tumor-promoting immune response, tumor cell dissemination, and cancer, preferably prostate cancer, B acute lymphoblastic leukemia, non-small cell lung cancer, ovarian tumors, esophageal squamous cell carcinoma, breast cancer, and melanoma.

[0034] Further, the angiogenesis-dependent condition or disease can be selected from the group consisting of initiation of tumor formation, initiation of tumor metastasis, tumor growth, metastatic growth, vascular adhesion, angiofibroma, arteriovenous malformation, arthritis, atherosclerotic plaque, corneal graft vascularization, delayed wound healing, diabetic retinopathy, granulation, burn, hemangioma, hemophilic arthropathy, hypertrophic scar, neovascular glaucoma, non-union fracture, Osler-Weber syndrome, psoriasis, pyogenic granuloma, retrolental fibroplasia, scleroderma, cancer, trachoma, and von Hippel-Lindau syndrome.

[0035] Further, the lymphangiogenesis dependent condition or disease may be selected from the group consisting of organ transplantation, cancer, filariasis, Gorham's disease, dry eye disease, pulmonary fibrosis, inflammatory bowel disease, diabetes, chronic inflammatory disease, chronic obstructive pulmonary disease (COPD), inflammatory arthritis, ulcerative colitis, psoriasis, and ocular surface disease.

[0036] In a fifth aspect, the present invention relates to a compound of the invention for use in the prevention or treatment of a condition or disease selected from the group consisting of Castleman's disease, fibrodysplasia ossificans progressiva (FOP), miscarriage, and fibrosis.

[0037] In a sixth aspect, the present invention relates to a compound of the present invention for use in targeting cancer stem cells, inhibiting angiogenesis, enhancing chemosensitivity of tumor cells, inducing tumor cell dormancy, maintaining tumor cell dormancy, inhibiting EMT (epithelial to mesenchymal transition), suppressing VEGF-A (vascular endothelial growth factor A) expression, suppressing VEGF-C (vascular endothelial growth factor C) expression, inducing differentiation of stem cells and iPSCs (induced pluripotent stem cells), improving implantation of trophoblasts into the uterine wall, preventing a TGF-beta (transforming growth factor beta) immunosuppressive phenotype of immune cells, and / or inhibiting myeloid derived suppressor cells.

[0038] In a preferred embodiment of the compound for use according to the invention, said compound is administered in combination with one or more further compounds and / or therapies selected from the group consisting of immune checkpoint inhibitors, BRAF (B-Raf) inhibitors, MEK (MAPK / ERK kinase) inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, hormonal therapy, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapy, immunoconjugates, toxin delivery molecules, small molecule kinase inhibitors, antibody-based therapy, adoptive cell transfer, Bacillus Calmette-Guerin therapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, and oncolytic virus therapy.

[0039] In a seventh aspect, the present invention relates to a method for preventing or treating any of the conditions and / or diseases defined in the above fourth, fifth and sixth aspects of the invention, comprising administering to a subject in need thereof one or more compounds defined in the first, second and third aspects of the invention. In a preferred embodiment, the subject is a human subject.

[0040] The compounds of the present invention advantageously provide dual inhibition of Id1 and Id3, more potent inhibition of Id1 and / or Id3 compared to known compounds, improved pharmaceutical properties compared to known compounds, and reduced cytotoxicity compared to known compounds.

[0041] The drawings show: [Brief description of the drawings]

[0042] [Figure 1]Genetic ablation of Id1 and Id3 by CRISPR / Cas9 significantly impairs tumor growth of melanoma cells in vivo. Mouse melanoma cells (B16-F10 or Ret) were co-injected with Matrigel into a syngeneic mouse model. (A) Single knockdown of Id1 or Id3 impairs tumor growth of Ret cells in vivo. (B) Simultaneous knockdown of Id1 and Id3 in B16-F10 and Ret melanoma cells significantly inhibits tumor growth in vivo. (C) Simultaneous knockdown of Id1 and Id3 in Ret cells is superior to single knockdown in inhibiting tumor growth in vivo. Relative tumor growth is shown by normalization to the mean of the corresponding control group. Error bars = SEM. *p ≤ 0.05; ***p ≤ 0.001. [Diagram 2] Loss of Id1 and Id3 significantly reduces colonies growing in 3D Matrigel culture. B16-F10 (left panel) or Ret (right panel) control or CRISPR / Cas9 Id1 / Id3 cells were plated in 3D Matrigel (10 mg / ml) and grown for 6 days before analysis. The number of colonies in 6 positions in each well of a 48-well plate was counted at each z-level (5-10 z-levels were imaged per position). [Diagram 3] Figure 1 shows an example Western blot from a compound library screen of compounds structurally related to cannabidiol (CBD) for their potential to inhibit BMP4-mediated Id1 and Id3 expression in B16-F10 cells. Of the 33 compounds tested in this subset, 18 reduced Id1 and Id3 protein levels 24 hours after co-treatment with BMP4 (10 ng / ml) and DMSO, CBD (10 μM) or test compound (10 μM). [Figure 4]BOILED-Egg analysis demonstrates superior pharmacological properties of X8166 compared to CBD. The Brain Or Intestinal Estimated permeation method predicts gastrointestinal absorption and brain access by lipophilicity (WLOGP) and polarity (tPSA) of small molecules. [Diagram 5] Figure 1: X6632 and X8166 significantly inhibit melanoma growth and development in vivo. B16-F10 cells (A) or Ret (B) cells were co-injected with Matrigel into syngeneic mice. Mice were treated (intraperitoneally) daily with DMSO, CBD, X6632 or X8166 for the first 14 days. Numbers indicate the number of tumor-bearing animals in each group. Bar graphs show the mean tumor volume in each group on the day the first animal reached the legal tumor size limit. Kaplan-Meier curves show the proportion of tumor-free animals in percent as a measure of tumor development. Error bars = SEM. * / #p=0.05;## / **p=0.01;### / ***p=0.001. *Compared to DMSO, #Compared to CBD. [Figure 6] Figure 1 shows that X8166 is less cytotoxic than CBD and X6632. (A) Loss of expression of Id1 and Id3 does not significantly alter proliferation of B16-F10 and Ret cells in 2D culture. (B) Cannabidiol (CBD) is more toxic to mouse embryonic fibroblasts (MEFs) than X8166. (C) High concentrations of CBD are more cytotoxic in B16-F10 and Ret cells compared to X8166. [Figure 7] Figure 1: X8166 inhibits melanoma cell growth in 3D Matrigel. B16-F10 (A) or Ret (B) control or CRISPR / Cas9 Id1 / Id3 cells were plated in 3D Matrigel (10 mg / ml) and grown for 6 days before analysis. B16-F10 or Ret cells were treated daily with DMSO or X8166 (7.5 μM). The number of colonies in 6 positions in each well of a 48-well plate was counted at each z-level (5-10 z-levels were imaged per position). [Figure 8] Assays to determine the chemical space of indole- and indazole-type compounds required for inhibition of Id1 and Id3. (A) Ret cells were treated with BMP4 and the indicated compounds (3.3 μM) for 24 h before lysis. (B) B16-F10 or Ret cells were treated with BMP4 and the indicated compounds (10 μM) for 24 h before lysis. [Figure 9] Figure 1: Analysis to determine the chemical space of coumarin-type compounds required for inhibition of Id1 and Id3. (A) B16-F10 or Ret cells were treated with BMP4 and the indicated compounds (10 μM) for 24 h before lysis. (B) Several compounds inhibit Id1 and Id3 expression at 3.3 μM, whereas CBD does not inhibit Id1 or Id3 expression at this concentration. [Figure 10] Human umbilical vein endothelial cells (HUVEC) and lymphatic endothelial cells (LEC) were cultured in EBM™-2 endothelial cell growth basal medium (hereafter referred to as endothelial cell growth medium) supplemented with EGM™-2MV Microvascular Endothelial Cell Growth Medium-2 SingleQuots™ Kit (both from Lonza, Cat. Nos. CC-3156 and CC4176, respectively). HUVEC and LEC were cultured in the presence of the indicated concentrations of X6632 or with DMSO as a solvent control. After 24 hours, cells were harvested. Lysates were Western blotted and probed with antibodies against Id1 and Id3. Western blots probed with vinculin antibodies served as loading controls. [Figure 11]HUVECs (A) and LECs (B) were cultured in endothelial cell growth medium. Cells were incubated with the indicated concentrations of X6632. Incubation with DMSO served as a solvent control. After the indicated periods of culture, cells were stained with the CyQUANT™ Cell Proliferation Assay Kit (Thermo Fisher Scientific). CyQUANT™ dye fluoresces after excitation at 485 nm when incorporated into double-stranded DNA, allowing DNA content to be used as a measure of cell number. Plates were incubated at 37°C for 15 minutes to allow incorporation of CyQUANT™ dye into DNA. Fluorescence was measured using a Tecan Infinite M200 reader by exciting at 485 nm and detecting emission (signal intensity) at 530 nm at 25 positions in each well. [Figure 12] HUVECs or LECs were resuspended in endothelial cell growth medium containing 20% ​​methylcellulose at 1.6×104 cells per milliliter. Twenty-five microliters of the cell suspension was pipetted as a droplet onto a non-adherent plastic plate, which was then inverted to form a hanging drop culture. The plate was incubated at 37° C. for 24 hours. Spheroids were harvested and embedded in type I collagen (2 mg / ml) containing 0.5% methylcellulose. After polymerization, gels were cultured with endothelial cell growth medium containing 50 ng / ml hVEGF-A in the presence or absence of X6632 (10 μM) or DMSO as a solvent control. Microscope-based image acquisition was used to assess sprouting after 24 hours of culture. Representative images are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0044] Materials and Methods: Synthetic Routes to Compounds According to Formula IIa (e.g., Compound X6632) a) KHMDS, THF, dibromoalkane, -16°C to room temperature, 16 hours, 78%; b) DIBAL-H, DCM, -78°C, 1 hour, 92%; c) 1) n-propyltriphenylphosphonium bromide, KHMDS, THF, 0°C to 10°C, 30 minutes, 2) starting material, 10°C, 1 hour, 99%; d) Pd / C, H2-atmosphere, ethyl acetate, 24 hours, 97%; e) 1.) TMEDA, diethyl 1) 0 °C, n-BuLi, 2 h, rt, 2.) 0 °C, DMF, 4 h, rt, 91%; f) AlCl3, NaI, ACN, DCM, 1.5 h, rt, 80%; g) hexanoic anhydride, K2CO3, microwave irradiation (180 °C, 65 min, 300 W), 82%; h) BBr3 in CH2Cl2 (5.00 equiv.), CH2Cl2, -78 °C to rt (30 min), 92% (Scheme 1). [ka]

[0045] Formula Ib(R 1 = butyl) (Scheme 2) [ka]

[0046] CRISPR / Cas9 The generation of CRISPR / Cas9 Id1 / Id3 clones used in this study has been reported previously. Briefly, cells were co-transfected with gRNA vector, hCas9 plasmid and sequence-specific single-stranded donor oligonucleotides using Lipofectamine 2000 reagent according to the manufacturer's protocol. Single-cell clones were expanded and screened for alterations in the genomic DNA sequence of Id1 and Id3 genes using sequence-specific primers. Colonies with alterations in the genomic DNA sequence were selected and expression of Id1 / 3 proteins was confirmed by Western blot analysis. Colonies lacking bands specific for Id1 / 3 proteins were selected, plated as single cells in 96-wells and subsequently analyzed for genomic alterations and loss of protein expression of Id1 and Id3. Single-cell clones obtained from the parental cell line were used as controls.

[0047] 3D Matrigel assay cells (2×10 3) was mixed with Matrigel to obtain a 150 μl cell / Matrigel (10 mg / ml, #354262, Corning Inc.) solution, which was plated in wells of a 48-well plate. The gel was allowed to solidify for 30 min at 37°C and then covered with 500 μl of complete growth medium. After 6 days of culture, images were captured using a Leica DMI6000 B microscope at 6 different x / y positions in every well and a minimum of 5 horizontal layers (z levels) at each position to focus all cells for image analysis. Image analysis was performed using Fiji software as follows: first, images were converted to 8-bit files (pixel values ​​ranging from 0 (black) to 255 (white)). The scale was set to distance=0, known=0, pixel=1, and unit=pixel global. The integrated semi-automatic magic wand tool was used to detect the outline of each colony in focus in all images. Once the outline was consistent with the colony morphology, the colony was filled in white (pixel value 255) using the fill function. Thresholding of pixel values ​​(threshold = 255) allowed obtaining black (background) and white (colony) images of the filled colony. The "Analyze Particles" function was applied to measure the area and perimeter of the colony with a minimum pixel size of 1500. The invasion index was calculated as follows: invasion index = (perimeter) 2 / area, resulting in a unitless measure of invasion.

[0048] Tumor growth and development in vivo Cells were harvested using trypsin / EDTA and washed with PBS. The indicated number of live melanoma cells was injected subcutaneously into the flank of syngeneic mice in 100 μl PBS. For co-injection experiments, the indicated number of live cells was resuspended in 100 μl Matrigel HC (10 mg / ml, #354262, Corning Inc.) and injected subcutaneously into the flank. Tumor volumes were calculated using the formula 4 / 3π(d1 / 2×d2 / 2×d3 / 2), where d1–d3 represent the diameter of the tumor in three dimensions. Animals were treated with DMSO, CBD, X6632 or X8166 (30 μl in DMSO intraperitoneally) for 14 days. All animal experiments were approved by the local authorities and performed in accordance with the German legal requirements. Tumor initiating cell (TIC) frequencies and p-values ​​were calculated using ELDA software.

[0049] Compound Screen To screen for compounds with inhibitory effects on the expression of Id1 and Id3, cells were simultaneously stimulated with BMP4 (10 ng / ml, 315-27, Peprotech) and the indicated concentrations of substances for 24 h. DMSO served as the solvent control. The effects on the expression levels of Id1 and Id3 were analyzed by Western blotting.

[0050] Western blot Western blot analysis was performed using standard methods. The following antibodies were used for protein detection: Id1 (195-14, CalBioreagents), Id3 (17-3, CalBioreagents), β-actin (AC-15, Sigma Aldrich), and vinculin (VIN-11-5, Sigma Aldrich). Protein bands were detected using HRP-conjugated secondary antibodies (P0447, P0448, Agilent Technologies) and enhanced chemiluminescence (32106, Thermo Fisher Scientific).

[0051] Example 1: Establishing Id1 and Id3 as targets for cancer therapy To demonstrate the role of Id1 and Id3 in melanoma initiation and growth, we used CRISPR / Cas9 genome editing to permanently turn off these genes in B16-F10 and Ret melanoma cells, then subcutaneously implanted the tumor cells into syngeneic experimental animals and compared melanoma initiation and growth to controls.

[0052] Loss of either Id1 or Id3 strongly and significantly reduced tumor growth of Ret cells in vivo (Figure 1A). Simultaneous loss of Id1 and Id3 significantly inhibited tumor growth of B16-F10 and Ret cells in vivo (Figure 1B), with a more pronounced effect than the single knockout cells (Figure 1C). Although all animals eventually developed tumors, the onset of tumor growth was also significantly delayed (Table 1), consistent with a role for Id1 and Id3 in stem cell properties that endow the cells with enhanced tumorigenic properties. Furthermore, loss of Id1 and Id3 significantly impaired the growth of B16-F10 and Ret cells in 3D Matrigel culture in vitro (Figure 2). In Ret cells, a reduction in invasive behavior was observed when expression of Id1 and Id3 was lost (Figure 2, left panel). These results indicate that targeting Id1 and Id3 should be of therapeutic value.

[0053] [Table 1]

[0054] Five or 50 B16-F10 or Ret control or CRISPR / Cas9 cells were co-injected with Matrigel (10 mg / ml) into syngeneic mice. Each group (control or CRISPR / Cas9 Id1 / Id3) consisted of 24 animals (8 animals for each of the three cell clones tested). 28-30 days after tumor cell injection, 1000 mm 3 Tumor incidence was recorded if a tumor of size > 1. The frequency of tumor initiating cells (TICs) was measured using limiting dilution analysis (ELDA) software.

[0055] Example 2: Identifying novel inhibitors of Id1 and Id3 protein expression by screening unique chemical libraries A unique chemical library containing 168 novel compounds structurally related to CBD was synthesized. B16-F10 and Ret melanoma cells were treated with BMP4 to induce Id1 and Id3 expression and simultaneously treated with each compound individually for 24 hours. The ability of the compounds to inhibit Id1 and Id3 protein expression compared to CBD was assessed using Western blotting analysis. Approximately one-third of the compounds had an inhibitory effect on both Id1 and Id3 protein expression, and 22 compounds reduced Id1 and Id3 expression more potently than CBD (Figure 3, Table 2). The compounds showing the strongest inhibitory activity were X81, X106, X6632, X6760, X8035, X8166, X8706 and X8766. [ka]

[0056] [Table 2]

[0057] Gut absorption and brain access are two important pharmacokinetic parameters. The brain or intestine estimated permeation method (BOILED-Egg) predicts gut absorption and brain access depending on the lipophilicity (WLOGP) and polarity (tPSA) of small molecules. BOILED-Egg analysis of the identified compounds predicts better pharmacokinetic properties of X8166 compared to CBD, while X6632 has similar properties to CBD (Figure 4). Based on these predicted values ​​and in vitro solubility analysis, X6632 and X8166 were further investigated in subsequent experiments.

[0058] Example 3: Novel compound inhibits tumor growth better than CBD To investigate the antitumor properties of selected Id1 / Id3-inhibiting compounds, groups of experimental mice (8 per group) were subcutaneously injected with B16-F10 or Ret melanoma cells. During the first 2 weeks after melanoma cell implantation, mice were injected daily with DMSO as a solvent control, CBD as a reference compound, or test substances X6632 and X8166 (Figure 5). Melanoma initiation and growth were assessed over a 90-day period. Compounds X6632 and X8166 significantly inhibited melanoma growth and development compared to DMSO treatment (Figure 5). X8166 inhibited tumor growth and development more significantly compared to CBD, while X6632 was significantly more effective than CBD in the B16-F10 experiment and showed a trend towards reduced tumor initiation and growth in the Ret experiment. Importantly, none of the animals injected with B16-F10 cells and treated with X8166 had tumors, and only three animals treated with X6632 had tumors, whereas all animals treated with DMSO and seven of eight mice treated with CBD developed tumors. When mice were injected with Ret cells and treated with X6632 or X8166, one mouse in the X6632 group and two mice in the X8166 group also did not develop tumors. These results indicate that X6632, and especially X8166, may prevent tumor development and inhibit melanoma growth. [ka]

[0059] Among the compounds tested, X8166 showed the best toxicity profile (no traces of toxicity were observed with X8166 in treated mice). In contrast, daily intraperitoneal injections of CBD led to the death of two mice in experiments with tumors derived from Ret cells (cause of death unknown). Moreover, when the mice were sacrificed at the end of the experiment, all CBD-treated mice showed toxicity-related damage to the intestinal organs.

[0060] Genetic ablation of Id1 and Id3 expression by CRISPR / Cas9 genome editing inhibited melanoma cell growth and invasiveness in 3D culture (Figure 2), but did not significantly alter melanoma cell proliferation in standard 2D plastic culture (Figure 6A). In 2D culture, X8166 also had only a moderate inhibitory effect on melanoma cell proliferation and non-transformed mouse embryonic fibroblasts (MEFs), mainly at high concentrations (Figure 6B, Figure 6C). Similar to genetic deletion of Id1 and Id3, X8166 also significantly impaired melanoma cell growth in 3D Matrigel (Figure 7). In contrast, CBD and X6632 treatment strongly inhibited the proliferation of both melanoma cells and MEFs in 2D culture, and 3D assays using daily application of CBD and X6632 could not be performed due to cytotoxicity. Taken together, these observations suggest that CBD and X6632 exert additional effects on melanoma cells in addition to inhibiting expression of Id1 and Id3. Importantly, these data also show that X8166 exhibits fewer off-target effects than the other substances tested, phenocopies the effects of genetic deletion of Id1 and Id3, and suggests that X8166 is a much more specific inhibitor of Id1 and Id3 than the other substances.

[0061] In summary, these data identify a novel class of substances that exert superior inhibitory effects on Id1 and Id3 expression compared to CBD. X6632 and X8166 are demonstrated to be potent inhibitors of melanoma growth and development in two independent syngeneic mouse models, and X8166 is shown to be well tolerated by mice and non-transformed cells.

[0062] Example 4: Defining the chemical space required for inhibition of Id1 / Id3 expression Compounds X8166 and X6632 belong to the indole and coumarin compound classes. X8166 was shown to be superior in terms of in vivo studies and cytotoxicity in MEFs, whereas compound X6632 was more potent in inhibiting Id1 / Id3 expression in cells. Derivatives of both compound classes were tested in further in vitro studies to determine their potential compared to the original compounds with in vivo results and to determine the chemical space required to inhibit Id1 and Id3 expression.

[0063] Indazole- and indole-type compounds that demonstrated inhibitory activity against Id1 and Id3 expression are shown in Figure 8 and their chemical structures are described in this application. Three closely related subclasses of compounds with inhibitory activity were identified.

[0064] The coumarin-type compounds that exhibited inhibitory activity against the expression of Id1 and Id3 are shown in FIG. 9 and their chemical structures are described in this application.

[0065] Example 5: Synthesis of compound X6632 1-(3,5-dimethoxyphenyl)cyclohexane-1-carbonitrile [ka] To a solution of 7.50 g of 2-(3,5-dimethoxyphenyl)acetonitrile (42.3 mmol, 1.00 equiv.) in 200 mL of anhydrous tetrahydrofuran was added 25.3 g of KHMDS (127 mmol, 3.00 equiv.) at -16°C under argon counterflow. The mixture was stirred at the same temperature for 3 min, then 6.24 mL of 1,4-dibromopentane (10.6 g, 46.6 mmol, 1.10 equiv.) diluted in 50 mL of anhydrous tetrahydrofuran was added dropwise. The mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched by adding ammonium chloride solution (150 mL) and diluted with 100 mL of diethyl ether. The organic layer was extracted with 3 x 200 mL of diethyl ether and the combined organic layers were dried over sodium sulfate. Removal of volatiles under reduced pressure and purification by flash column chromatography (CH / EtOAc 5:1) gave 8.09 g (78%) of pure product as a colorless oil. Analytical data is consistent with the literature.

[0066] R f (CH / EtOAc 5:1): 0.43. 1 H NMR (300 MHz, CDCl3): δ = 6.63 (d, J = 2.2 Hz, 2H, 2 × H Ar ), 6.40 (t, J = 2.2 Hz, 1H, H Ar ), 3.81 (s, 6H, 2 × OCH3), 2.21 - 2.16 (m, 2H, CH2), 1.93 - 1.65 (m, 6H, CH2), 1.46 - 1.02 (m, 2H, CH2) ppm.

[0067] 1-(3,5-dimethoxyphenyl)cyclohexane-1-carbaldehyde [ka] A solution of 7.97 g of 1-(3,5-dimethoxyphenyl)cyclohexane-1-carbonitrile (32.5 mmol, 1.00 equiv.) in 250 mL of anhydrous dichloromethane was cooled to -78°C under an argon atmosphere and 81.2 mL of DIBAL-H (1 m in dichloromethane, 81.2 mmol, 2.50 equiv.) was added dropwise. The mixture was stirred at the same temperature for an additional hour and then the reaction was quenched by adding dropwise 120 mL of 10% aqueous potassium sodium tartrate solution. After thawing to room temperature, the mixture was stirred for an additional 40 minutes and the aqueous layer was extracted with 3 x 200 mL of ethyl acetate. The combined organic layers were washed with 300 mL of brine and dried over sodium sulfate. Removal of the volatiles under reduced pressure and purification by flash column chromatography (CH / EtOAc 10:1) gave 7.39 g (92%) of pure product as a colorless oil. Analytical data is consistent with the literature. R f (CH / EtOAc 20:1): 0.20. 1 H NMR (300 MHz, CDCl3): δ = 9.34 (s, 1H, CHO), 6.46 (d, J = 2.2 Hz, 2H, 2 × H Ar ), 6.37 (t, J = 2.2 Hz, 1H, H Ar ), 3.78 (s, 6H, 2 × OCH3), 2.27-2.22 (m, 2H, CH2), 1.85-1.78 (m, 2H, CH2), 1.69-1.57 (m, 3H, CH2), 1.52-1.25 (m, 3H, CH2) ppm.

[0068] (Z)-1-(1-(but-1-en-1-yl)cyclohexyl)-3,5-dimethoxybenzene [ka] To a suspension of 36.3 g n-propyltriphenylphosphonium bromide (94.3 mmol, 3.00 equiv.) in 300 mL anhydrous tetrahydrofuran at 0° C., 18.8 g KHMDS (94.3 mmol, 3.00 equiv.) was added under argon counterflow. The mixture was stirred for 30 min at 10° C. and a solution of 7.81 g 1-(3,5-dimethoxyphenyl)cyclohexane-1-carbaldehyde (33.7 mmol, 1.00 equiv.) in 50 mL anhydrous tetrahydrofuran was added dropwise. After stirring for another 60 min, the reaction was quenched by adding 200 mL ammonium chloride solution. The aqueous layer was extracted with 3×200 mL diethyl ether and the combined organic layers were dried over sodium sulfate. Removal of the volatiles under reduced pressure and purification by flash column chromatography (CH / EtOAc 10:1) gave 8.62 g (99%) of the pure product as a colorless oil. The analytical data are consistent with the literature.

[0069] R f (CH / EtOAc 5:1): 0.65. 1 H NMR (300 MHz, CDCl3): δ = 6.58 (d, J = 2.3 Hz, 2H, 2 × H Ar ), 6.28 (t, J = 2.3 Hz, 1H, H Ar ), 5.63 (dt, J = 11.2 Hz, J = 1.7 Hz, 1H, H DB ), 5.34 (dt, J = 11.2 Hz, J = 7.4 Hz, 1H, H DB ), 3.78 (s, 6H, 2 × OCH3), 1.95-1.90 (m, 2H, CH2), 1.72-1.56 (m, 9H, CH2), 1.31-1.24 (m, 1H, CH2), 0.72 (t, J = 7.5 Hz, 3H, CH3) ppm.

[0070] 1-(1-butylcyclohexyl)-3,5-dimethoxybenzene [ka] To a solution of 8.50 g of ((Z)-1-(1-(but-1-en-1-yl)cyclohexyl)-3,5-dimethoxybenzene in ethyl acetate was added 1.73 g of palladium on activated carbon (10% Pd / C). Hydrogen gas was bubbled through the solution for several hours followed by maintaining under a hydrogen atmosphere for 24 hours. Filtration through Celite™, rinsing with ethyl acetate and removal of volatiles gave 8.31 g (97%) of pure product as a colorless oil. Analytical data is consistent with the literature.

[0071] R f (CH / EtOAc 5:1): 0.68. 1 H NMR (300 MHz, CDCl3): δ = 6.48 (d, J = 2.3 Hz, 2H, 2 × H Ar ), 6.3 (t, J = 2.3 Hz, 1H, H Ar ), 3.80 (s, 6H, 2 × OCH3), 2.02-1.98 (m, 2H, CH2), 1.57-1.36 (m, 10H, 5 × CH2), 1.18-1.09 (m, 2H, CH2), 0.96-0.88 (m, 2H, CH2), 0.78 (t, J = 7.3 Hz, 3H, CH3) ppm.

[0072] 4-(1-Butylcyclohexyl)-2,6-dimethoxybenzaldehyde [ka] 8.26 g of 1-(1-butylcyclohexyl)-3,5-dimethoxybenzene (29.9 mmol, 1.00 equiv.) was dissolved in 60 mL of diethyl ether and 6.72 mL of TMEDA (5.21 g, 44.8 mmol, 1.50 equiv.) was added dropwise. The solution was cooled to 0° C. and 17.9 mL of n-butyllithium (2.5 m in n-hexane, 44.8 mmol, 1.50 equiv.) was added slowly. After stirring at room temperature for 4 h, the solution was cooled to 0° C. and 6.89 mL of dimethylformamide (6.551 g, 89.7 mmol, 3.00 equiv.) was added and the mixture was stirred at room temperature for an additional 4 h. The reaction was quenched by adding 60 mL of brine and extracted with 3×15 mL of diethyl ether. The combined organic layers were dried over sodium sulfate, the volatiles were removed under reduced pressure, and the residue was then purified by flash column chromatography (CH / EtOAc 10:1) to give 8.31 g (91%) of the product as a yellow oil that was used directly in the next step. Analytical data is consistent with the literature.

[0073] R f (CH / EtOAc 10:1): 0.19. 1 H NMR (300 MHz, CDCl3): δ = 10.46 (s, 1H, CHO), 6.52 (s, 2H, 2 × H Ar ), 3.89 (s, 6H, 2 × OCH3), 2.07-1.94 (m, 2H, CH2), 1.66 - 1.33 (m, 10H, 5 × CH2), 1.29-1.04 (m, 2H, CH2), 1.01-0.85 (m, 2H, CH2), 0.79 (t, J = 7.3 Hz, 3H, CH3) ppm.

[0074] 4-(1-Butylcyclohexyl)-2-hydroxy-6-methoxybenzaldehyde [ka] 8.00 g of 4-(1-butylcyclohexyl)-2,6-dimethoxybenzaldehyde (1.00 equiv., 26.3 mmol) was dissolved in a mixture of 100 mL of dry acetonitrile and 50 mL of dry dichloromethane, cooled to 0° C., and 8.76 g of aluminum trichloride (65.7 mmol, 2.50 equiv.) and 9.85 g of sodium iodide (65.7 mmol, 2.50 equiv.) were added slowly under argon counterflow. The reaction mixture was stirred at room temperature for 1.5 h, quenched with water, extracted with 3×30 mL of dichloromethane, the combined organic layers were washed with sodium thiosulfate solution, dried over sodium sulfate, and after removal of volatiles the crude product was purified by flash column chromatography (CH / EtOAc 40:1) to give 6.11 g (80%) of a yellow oil. Analytical data are consistent with the literature.

[0075] R f (CH / EtOAc 40:1): 0.26. 1 H NMR (300 MHz, CDCl3): δ = 11.92 (s, 1H, C2-OH), 10.26 (s, 1H, CHO), 6.51 (d, J = 1.3 Hz, 1H, H Ar ), 6.34 (d, J = 1.3 Hz, 1H H Ar ), 3.88 (s, 3H, OCH3), 2.00-1.32 (m, 12H, 6 × CH2), 1.20-1.10 (m, 2H, CH2), 0.96-0.88 (m, 2H, CH2), 0.79 (t, J = 7.3 Hz, 3H, CH3) ppm.

[0076] 7-(1-Butylcyclohexyl)-5-methoxy-3-butyl-2H-chromen-2-one [ka] 200 mg of 4-(1-butylcyclohexyl)-2-hydroxy-6-methoxybenzaldehyde (0.69 mmol, 1.00 equiv), 0.56 mL of hexanoic anhydride (517 mg, 2.41 mmol, 3.50 equiv) and 4.8 mg of potassium carbonate (270 μmol, 0.05 equiv) were placed in a microwave vial and heated at 180° C. for 65 min with 300 W microwave irradiation. The resulting mixture was cooled to room temperature, poured onto crushed ice and the pH was adjusted to about 7 with sodium bicarbonate. The mixture was then extracted with 3×50 mL of ethyl acetate and the combined organic layers were dried over sodium sulfate. Removal of the volatiles under reduced pressure and purification by flash column chromatography (CH / EtOAc 100:1) afforded 210 mg (82%) of an off-white solid.

[0077] R f (CH / EtOAc 50:1): 0.31. -MP: 143.8℃ - 1 H NMR (400 MHz, CDCl3): δ = 7.81 (s, 1H, 4-CH), 6.90-6.86 (m, 1H, H Ar ), 6.66 (d, J = 1.5 Hz, 1H, H Ar ), 3.92 (s, 3H, OCH3), 2.55 (t, J = 7.7 Hz, 2H, CH2), 2.11-1.94 (m, 2H, CH2), 1.70-1.30 (m, 14H, 7 × CH2), 1.13 (p, J = 7.3 Hz, 2H, CH2), 1.00-0.85 (m, 5H, CH2, CH3), 0.77 (t, J = 7.3 Hz, 3H, CH3) ppm. - 13 C NMR (100 MHz, CDCl3): δ = 162.4 (C quart. , COO), 155.4 (C quart. , C Ar ), 154.2 (C quart. , C Ar ), 152.4 (C quart. , C Ar ), 133.5 (+, 4-C Ar H), 127.2(Cquart. , C Ar ), 108.0 (C quart. , C Ar ), 107.9 (+, C Ar H), 103.9 (+, C Ar H), 55.9 (+, OCH3), 43.6(C quart. , C CH ), 42.3 (-, CH2), 36.5 (-, 2 × CH2), 30.8 (-, CH2), 30.5 (-, CH2), 26.6 (-, CH2), 25.8 (-, CH2), 23.4 (-, CH2), 22.6 (-, 2 × CH2), 14.1 (+, CH3), 14.0 (+, CH3) ppm. -IR (KBr): v~ = 2926 (m), 2854 (w), 1714 (m), 1613 (m), 1570 (w), 1495 (w), 1453 (m), 1413 (m), 1376 (w), 1344 (w), 1291 (w), 1245 (m), 1163 (w), 1104 (m), 1073 (w), 1044 (m), 991 (m), 943 (w), 906 (w), 834 (w), 798 (w), 760 (w), 712 (w), 684 (w), 653 (w), 558 (w), 494 (vw), 429 (vw) cm -1 . -MS (70 eV, EI): m / z (%) = 370 (96) [M] + , 328 (7), 327 (8), 315 (10), 314 (45), 313 (100) [M - C4H9] + , 274 (6), 271 (5), 259 (5), 246 (10), 245 (54), 233 (19), 203 (7), 202 (11), 189 (5), 81 (7). -HRMS(C 24 H 34 O3): Calculated 370.2502, Found 370.2502. -Elemental analysis: C 24 H 34 O3: Calculated value: C77.80, H9.25, Actual value: C77.78, H9.43.

[0078] 7-(1-Butylcyclohexyl)-5-hydroxy-3-butyl-2H-chromen-2-one [ka] 116 mg of 7-(1-butylcyclohexyl)-5-methoxy-3-butyl-2H-chromen-2-one (356 μmol, 1.00 equiv.) was dissolved in 5 mL of dry dichloromethane. The solution was cooled to −78° C. and 1.78 mL of boron tribromide (1 m in dichloromethane, 1.78 mmol, 5.00 equiv.) was added dropwise. The mixture was stirred at this temperature for 30 min and then warmed to room temperature. The reaction was quenched after 16 h at 0° C. by the addition of sodium bicarbonate. The aqueous layer was extracted with 3×15 mL of dichloromethane and the combined organic layers were washed with brine, dried over sodium sulfate and the volatiles were removed under reduced pressure. The crude product was then purified by flash column chromatography (CH / EtOAc 5:1) to give the product as 116 mg (92%) of a white solid.

[0079] R f (CH / EtOAc 5:1): 0.43. -MP: 154.0℃ - 1 H NMR (400 MHz, CDCl3): δ = 7.89 (s, 1H, 4-CH), 6.83 (d, J = 1.4 Hz, 1H, H Ar ), 6.73 (d, J = 1.5 Hz, 1H, H Ar ), 6.54 (s, 1H, OH), 2.70-2.48 (m, 2H, CH2), 2.04-1.93 (m, 2H, CH2), 1.68-1.58 (m, 2H, CH2), 1.57-1.27 (m, 12H, 6 × CH2), 1.17-1.05 (m, 2H, CH2), 0.94 (t, J = 7.3 Hz, 3H, CH3), 0.92-0.83 (m, 2H, CH2), 0.75 (t, J = 7.3 Hz, 3H, CH3) ppm. - 13C NMR (100 MHz, CDCl3): δ = 163.3 (C quart. , COO), 154.3 (C quart. , C Ar ), 152.7 (C quart. , C Ar ), 152.2 (C quart. , C Ar ), 134.3 (+, 4-C Ar H), 126.8 (C quart. , C Ar ), 109.1 (+, C Ar H), 107.5 (+, C Ar H), 107.1 (C quart. , C Ar ), 43.8 (-, CH2), 42.0 (C quart. , C CH ), 36.4 (-, 2 × CH2), 30.7 (-, CH2), 30.5 (-, CH2), 26.6 (-, CH2), 25.8 (-, CH2), 23.4 (-, CH2), 22.6 (-, CH2), 22.5 (-, 2 × CH2), 14.1 (+, CH3), 14.0 (+, CH3) ppm。-IR (KBr): v~ = 3171 (w), 2925 (w), 2853 (w), 1670 (m), 1613 (m),1573 (w), 1451 (w), 1421 (m), 1345 (w), 1288 (w), 1253 (w), 1185 (w), 1124 (w), 1101 (w), 1066 (w), 939 (w), 862 (w), 842 (w), 782 (w), 745 (w), 728 (w), 608 (vw), 529 (w), 414 (vw) cm -1 。 - MS (70 eV, EI): m / z (%) = 356 (71) [M] + , 331 (8), 314 (12), 301 (8), 300 (46), 299 (100) [M - C4H9] + , 281 (8), 262 (7), 260 (9)。-HRMS(C23 H 32 O3): Calculated 356.2346, Found 356.2347. -Elemental analysis: C 23 H 32 O3: Calculated values: C77.49, H9.05; measured values: C77.27, H9.09.

[0080] Example 6: Synthesis of compound X8166 [ka] 1-H indole (1.00 g, 8.54 mmol, 1.00 equiv) was dissolved in 50 mL of dry DMF under an inert atmosphere. Sodium hydride (512 mg, 12.80 mmol, 1.50 equiv) and 1-iodobutane (2.36 g, 1.46 mL, 13 mmol, 1.50 equiv) were added at 0° C. and the reaction was stirred at 21° C. overnight. The reaction was worked up by pouring it onto ice and extracting the organic phase with ethyl acetate. The combined organic layers were dried over Na2CO3, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography with cyclohexane:ethyl acetate 50:1. R f =0.73 (cyclohexane / ethyl acetate 4:1).

[0081] 1 H NMR (400 MHz, CDCl3, ppm) δ = 0.98 (t, J = 7.3 Hz, 3H), 1.39 (dq, J = 15.1 Hz, J = 7.4 Hz, 2H), 1.81-1.91 (m, 2H), 4.16 (t, J = 7.1 Hz, 2H), 6.53 (dd, J = 3.1 Hz, J = 0.7 Hz, 1H), 7.11-7.17 (m, 2H), 7.25 (td, J = 7.6 Hz, J = 1.1 Hz, 1H), 7.39 (dd, J = 8.3 Hz, J = 0.7 Hz, 1H), 7.68 (dt, J = 7.8 Hz, J = 0.9 Hz, 1H). 13C NMR (100 MHz, CDCl3, ppm) δ = 14.0, 20.5, 32.6, 46.4, 101.1, 109.7, 119.4, 121.2, 121.6, 128.1, 128.8, 136.2. EI (m / z, 70 eV, 15 ℃): 173 (52), 130 (100). HRMS(C 12 H 15 N): Calculated 173.1199, Measured 173.1199; IR (ATR, v~) = 3050, 2955, 2928, 2870, 1611, 1572, 1509, 1483, 1461, 1399, 1376, 1365, 1351, 1334, 1314, 1256, 1240, 1198, 1153, 1138, 1112, 1085, 1027, 1011, 942, 922, 884, 840, 762, 736, 714, 691, 606, 585, 569, 487, 464, 425, 395, 382 cm -1 .

[0082] [ka] 1-Butylindole (671 mg, 3.88 mmol, 1.00 equiv) was dissolved in 40 mL of anhydrous methylene chloride (N2 atmosphere) and dimethylarmanilium in hexane; chloride (1 M, 4.65 mL, 4.65 mmol, 1.20 equiv) was added at 0 °C. The reaction was stirred at 0 °C for 15 min and 2-phenylacetyl chloride (1.20 g, 1.02 mL, 7.75 mmol, 1.00 equiv) was added. The reaction was allowed to warm to room temperature and stirred at 21 °C for 14 h. The reaction was worked up by washing the reaction mixture with saturated NaHCO3 solution. The organic layer was separated, dried over Na2SO4 and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography with cyclohexane:ethyl acetate (gradient: 10:1 to 4:1) to give the target compound in 85% yield (955 mg, 3.3 mmol). R f =0.41 (cyclohexane / ethyl acetate 4:1).

[0083] 1 H NMR (400 MHz, CDCl3, ppm) δ = 0.91 (t, J = 7.3 Hz, 3H), 1.30 (dq, J = 15.1 Hz, J = 7.4 Hz, 2H), 1.72-1.86 (m, 2H), 4.03-4.16 (m, 4H), 7.13-7.35 (m, 8H), 7.72 (s, 1H), 8.33-8.44 (m, 1H); 13 C NMR (100 MHz, CDCl3, ppm) δ = 13.6, 20.0, 31.8, 46.9, 47.0, 109.8, 116.1, 122.6, 122.8, 123.3, 126.6, 126.7, 127.3, 128.5, 128.5, 129.3, 129.3, 134.9, 135.9, 136.7, 192.7; EI (m / z, 70 eV, 100 ℃): 291 (12), 200 (100), 144 (20). HRMS(C 20 H 21 ON): calculated value 291.1618, measured value 291.1617; IR (ATR, v~) = 3117, 3050, 2958, 2928, 2870, 1624, 1575, 1526, 1492, 1483, 1464, 1433, 1385, 1338, 1284, 1235, 1206, 1184, 1155, 1129, 1104, 1076, 1055, 1031, 1012, 952, 929, 915, 871, 857, 841, 800, 777, 746, 719, 697, 643, 610, 602, 579, 554, 521, 481, 429 cm -1 .

[0084] Example 7: X6632 blocks angiogenesis and neovascularization The effect of X6632 on the expression of Id1 and Id3 in cultured proliferating human umbilical vein endothelial cells (HUVEC) was examined. X6632 completely abolished the protein expression of Id1 and Id3 (Figure 10A). The coumarin derivative 4-methylumbelliferone (4-MU) did not reduce the expression of Id1 or Id3. LDN-193189, an antagonist of the BMP receptor isotypes ALK2 and ALK3, served as a positive control for the inhibition of Id1 and Id3 expression. Further experiments showed that X6632 inhibited the protein expression of Id1 and Id3 in both HUVEC and lymphatic endothelial cells (LEC) (Figure 10B). Consistent with these results, X6632 dose-dependently inhibited the proliferation of both HUVEC and LEC (Figure 11).

[0085] The inhibitory activity of X6632 on the expression of Id1 and Id3 in HUVEC and LEC, as well as their proliferation, suggests that X6632 may affect angiogenesis and lymphangiogenesis. To determine whether this is the case, the effect of X8166 on angiogenic and lymphangiogenic sprouting from endothelial cell spheroids was investigated. For this purpose, HUVEC and LEC were grown as spheroids in hanging drop cultures. The spheroids were then embedded in collagen. Sprouting from the spheroids in the presence and absence of X6632 was monitored using microscopy-based image analysis. As shown in Figure 12, X6632 substantially inhibited sprouting from both HUVEC and LEC spheroids. Taken together, these results suggest that X6632 can inhibit both angiogenesis and lymphangiogenesis.

Claims

1. angiogenesis-dependent conditions or diseases; and / or Lymphangiogenesis-dependent conditions or diseases Used in the prevention or treatment of A composition comprising a compound having a structure according to any one of formulas (Ia), (Ic) or (Id): 【Chemistry 1】 (In the formula, R ｅ is -CH ３ , -CH ２ CH ３ , -(CH ２ ) ２ CH ３ , -(CH ２ ) ３ CH ３ or benzyl, n is 0 or 1; 【Chemistry 2】 (In the formula, Each group R ｇ is H or both R ｇ are linked together to form a 5-membered alicyclic ring; 【Chemistry 3】 (In the formula, Both R ｈ are linked together to form a 5-membered alicyclic ring).

2. The composition for use according to claim 1, selected from the group consisting of the following compounds: X6632, X6760, X6779, X6631, X6777, X6768, X6633, and X6945: 【Chemistry 4】

3. angiogenesis-dependent conditions or diseases; and / or Lymphangiogenesis-dependent conditions or diseases Used in the prevention or treatment of A composition comprising a compound selected from the group consisting of the following compounds: X6910, X6404, X6770, X6778, X6758, X6944, X7776, and X7401: 【Chemistry 5】

4. 4. The composition for use according to any one of claims 1 to 3, wherein the angiogenesis dependent condition or disease is selected from the group consisting of initiation of tumor formation, initiation of tumor metastasis, tumor growth, metastatic growth, cancer, vascular adhesion, angiofibroma, arteriovenous malformation, arthritis, atherosclerotic plaque, corneal graft angiogenesis, delayed wound healing, diabetic retinopathy, granulation, burns, hemangiomas, hemophilic arthropathy, hypertrophic scars, neovascular glaucoma, pseudoarthrosis, Osler-Weber syndrome, psoriasis, pyogenic granuloma, retrolental fibroplasia, scleroderma, trachoma, and von Hippel-Lindau syndrome.

5. 4. The composition for use according to any one of claims 1 to 3, wherein the lymphangiogenesis dependent condition or disease is selected from the group consisting of organ transplantation, cancer, filariasis, Gorham's disease, dry eye disease, pulmonary fibrosis, inflammatory bowel disease, diabetes, chronic inflammatory diseases, chronic obstructive pulmonary disease (COPD), inflammatory arthritis, ulcerative colitis, psoriasis, and ocular surface diseases.

6. 4. The composition of any one of claims 1 to 3 for use in targeting cancer stem cells, inhibiting angiogenesis, enhancing chemosensitivity of tumor cells, inducing tumor cell dormancy, maintaining tumor cell dormancy, inhibiting EMT (epithelial-mesenchymal transition), suppressing VEGF-A (vascular endothelial growth factor A) expression, suppressing VEGF-C (vascular endothelial growth factor C) expression, inducing differentiation of stem cells and iPSCs (induced pluripotent stem cells), improving trophoblast implantation into the uterine wall, preventing a TGF-beta (transforming growth factor beta) immunosuppressive phenotype of immune cells, and / or inhibiting myeloid derived suppressor cells.

7. 7. The composition for use according to any one of claims 1 to 6, wherein the compound is administered in combination with one or more further compounds and / or therapies selected from the group consisting of immune checkpoint inhibitors, BRAF inhibitors, MEK inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, hormonal therapy, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapy, immunoconjugates, toxin delivery molecules, small molecule kinase inhibitors, antibody based therapy, adoptive cell transfer, Bacillus Calmette-Guerin therapy, cancer vaccines, chimeric antigen receptor (CAR) T cell therapy, cytokine therapy, gene therapy, and oncolytic virus therapy.

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