DCLK1 inhibitors and therapeutic methods

Abstract

This disclosure relates to DCLK1 inhibitor compounds and therapeutic methods. The inhibitor compounds generally have a structure corresponding to Formula 1, where A is a 5 or 6-membered heterocycle, R1, R2, and Ra are each independently selected from the group consisting of -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl, and alkaryl, or R1 and R2 together with the carbon to which they are bonded to form a condensed 5 or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bonded, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(0)NH-, and J1 and J2 are each independently -CH- or -N-. JPEG2026534530000026.jpg24128

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 587,362, titled DCLK1 INHIBITORS AND METHODS OF TREATMENT, filed on 2 October 2023, which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) The following application includes a sequence listing submitted electronically as a standard ST.26 compliant XML file titled "SequenceListing_61013.xml" created on 2 October 2024, with a size of 6,341 bytes, the contents of which are incorporated herein by reference.

[0003] (Field of Invention) This disclosure relates to doublecortin-like kinase 1 (DCLK1) inhibitor compounds, therapeutic compositions comprising such inhibitor compounds, and methods for treating cancer using the same. [Background technology]

[0004] Colon cancer is the second leading cause of cancer-related death in the United States. Recent advances in early diagnosis and treatment are not curative in patients with advanced disease. Standard treatment involves three different combination regimens of chemotherapy, each of which has significant toxicity. At least 50% of colon cancer patients receiving these treatments develop metastases. Other combination therapies include immunotherapy, but these suffer from the development of resistance and increased side effects. Therefore, there remains a high and unmet medical need to improve the treatment of colon cancer.

[0005] Doublecortin-like kinase 1 (DCLK1) is considered a cancer stem cell (CSC) marker. The DCLK1 signaling pathway involves AKT phosphorylation at Thr308, which leads to Notch1 activation. Notch1 activates the expression of the transcription factor Pregnane X receptor (PXR). PXR induces cancer cell proliferation and metastasis and regulates the expression of multidrug resistance protein 1 and other proteins involved in drug metabolism, including cytochrome P4503A4 (one of six enzymes responsible for metabolizing approximately 90% of all small molecule drugs). [Overview of the Initiative]

[0006] This disclosure relates to a DCLK1 inhibitor compound having a structure corresponding to Formula 1,

[0007] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R1, R2, and R3 are each independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 R1 and R2 are selected from alkyl-substituted C3-C6 aryl groups, or R1 and R2, together with the carbons to which they are bonded, form a 5 or 6-membered ring fused with the adjacent 6-membered ring to which R1 and R2 are bonded, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, and J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 or J2 is -N-.

[0008] In addition, this disclosure relates to a DCLK1 inhibitor compound having a structure corresponding to Formula 1A,

[0009] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R3 is independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 Selected from alkyl-substituted C3-C6 aryls, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, and J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 or J2 is -N-.

[0010] Also disclosed are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of the DCLK1 inhibitor compound described herein.

[0011] This disclosure relates to a method for treating cancer in a patient requiring cancer treatment, comprising administering a DCLK1 inhibitor compound or a pharmaceutical composition described herein in a dose effective for treating cancer. This disclosure also relates to a method for treating various cancers, including but not limited to colon cancer, cholangiocarcinoma, genitourinary cancer, gynecological cancer, gastrointestinal cancer, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain tumor, or glioma.

[0012] Other subjects and characteristics are partially evident and partially noted below. [Brief explanation of the drawing]

[0013] [Figure 1]Figure of an exemplary embodiment using a peptide-based nanosponge carrier for delivery of DCLK1 inhibitors using a trimaleimide-based core structure, which should not be construed as limiting the possible nanosponge structures or other possible carriers contemplated herein. [Figure 2] Shows a graph of the concentration of the DCLK1 inhibitor compound versus the percent proliferation of the colorectal cancer cell line SW480 cells relative to a control containing no DCLK1 inhibitor compound. [Figure 3] Shows a graph of the concentration of the DCLK1 inhibitor compound versus the percent proliferation of the colorectal carcinoma cell line HCT116 cells relative to a control containing no DCLK1 inhibitor compound. [Figure 4] (Top) Shows representative colony formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at the IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay for 48 hours. (Bottom) Quantification of colony number and size. Data are expressed as the mean number and size from three independent experiments, *p<0.05. [Figure 5] (Top) Shows representative colony formation images of HUH28 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at the IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay for 48 hours. (Bottom) Quantification of colony number and size. Data are expressed as the mean number and size from three independent experiments, *p<0.05. [Figure 6] (Left) Shows representative spheroid formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at the IC50, 1 / 2 IC50, and 1 / 4 IC50 doses previously determined by hexosaminidase assay. (Right) Quantification of spheroid number. Data are expressed as the mean from three independent experiments, *p<0.05.

[0014] Throughout the drawings, corresponding reference numerals indicate corresponding parts. Detailed Description of the Invention

[0015] Oral administration of representative small molecule DCLK1 inhibitors demonstrated in vivo antitumor activity in a mouse colon cancer xenograft model. The present inventors were able to demonstrate that inhibition of DCLK1 is associated with an antitumor response, and used initial lead candidates to generate more pharmaceutically tractable compounds. The present inventors further used a delivery vehicle for targeted delivery, which can reduce the effective dose required for treatment, and possibly reduce side effects and off-target effects. Nanosponges linked to small molecule DCLK1 inhibitors can be delivered directly to the tumor site in the colon, making it possible to enhance antitumor activity while "protecting" the small molecule DCLK1 inhibitors from potential metabolism.

[0016] DCLK1 is a cancer stem cell (CSC) marker. CSCs are rare, and isolating and identifying cells within tumor masses is difficult. Multiple methods used to identify CSCs include globular ability, dynamolytic elimination by overexpression of efflux pumps, intracellular enzyme activity, and expression of cell surface markers. In previous reports, DCLK1 was identified in the gut. DCLK1 has been confirmed as a CRC and pancreatic cancer stem cell marker. DCLK1 is a member of the calmodulin-like protein kinase superfamily. The full-length encoded protein contains two N-terminal doublecortin (DCX) domains that bind to microtubules and regulate microtubule polymerization, a C-terminal serine / threonine kinase domain homologous to Ca2+ / calmodulin-dependent protein kinases, and a serine / proline-rich domain between the doublecortin and protein kinase domains. DCLK1 is preferentially transcribed from a second promoter in colon cancer cells. The AKT1 peptide around Thr308 interacts with the kinase domain of the DCLK1 protein, which is a shorter form lacking the N-terminal DCX domain. The DCLK1 knockout mouse model suppresses colon polyps in APCmin / + mice but does not affect the normal gut. These data suggest that DCLK1+ cells are CSCs, not normal stem cells. An important consequence of this finding is that targeting DCLK1 specifically targets CSCs while leaving normal proliferating cells intact.

[0017] DCLK1 has been shown to phosphorylate the microtubule-associated protein MAP7D1. Using in silico modeling, AKT1 was identified around the threonine at position 308 (IKDGATMKTFCGTP, SEQ ID NO: 1). Using AutoDock Vina and the crystal structure of the DCLK1 kinase domain (PDB ID 5JZN), it was observed that the AKT1 peptide around Thr308 interacts with the DCLK1 kinase domain (binding energy of -5.7 kcal / mol). DCLK1 amino acids targeted by AKT1 interaction include ASP398, ASP475, GLU515, and THR552. The inventors confirmed the interaction using magnetic relaxation assays and immunoprecipitation-coupled Western blot assays. In vitro assays using recombinant DCLK1 and AKT peptides also confirmed significant phosphorylation of AKT substrates by DCLK. This phosphorylation was lost when DCLK1 was denatured.

[0018] Lead candidate MRL16 has been demonstrated to inhibit colon cancer growth by inhibiting proliferation during the G2 / M phase of the cell cycle, inducing apoptosis, and suppressing stem cell characteristics by inhibiting spheroid proliferation. MRL16 suppressed DCLK1-mediated phosphorylation of AKT1 at Thr308, resulting in downstream inhibition of Notch signaling and PXR. Subsequently, in vivo mouse xenograft studies using MRL16 demonstrated antitumor activity, providing evidence that DCLK1 is a viable therapeutic target for CRC. By combining the inhibition of this novel drug target with a novel approach for selectively delivering DCLK1 inhibitors to CRC tumors, this disclosure aims to improve therapeutic outcomes by enhancing efficacy and reducing toxicity. This specification describes the synthesis of a novel nanosponge-linked DCLK1 inhibitor and its in vitro evaluation. This novel nanosponge drug delivery platform has not been previously used for the treatment of CRC.

[0019] Researchers at the University of Kansas Medical Center isolated marmelin (1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxyaldehyde, marmelin, MRL) from Aegle marmelos, an Ayurvedic treatment for gastrointestinal cancer.

[0020] [ka]

[0021] Based on this scaffolding and its initial ADMET properties, MRL16 was selected for target validation studies. Despite limited water solubility and moderate in vitro metabolic stability, the compound demonstrated excellent probe potential for proof-of-principle studies to validate DCLK1 as a therapeutic target. MRL16 significantly interacts with the kinase domain with a binding energy of -7.9 kcal / mol, and the key amino acid is VAL468.

[0022] [ka]

[0023] DCLK1 is a protein that indicates quiescent stem cells in colon cancer. Stem cells support spheroid growth. The inventors performed a spheroid assay using HCT116 cells in which DCLK1 was knocked down using specific shRNA. 500 cells were seeded in a serum-deficient medium containing factors essential for spheroid proliferation in a very low-adhesion 12-well dish. A significant reduction in spheroid formation was observed compared to the control. The cells were then treated with MRL16 (0.5 μM), showing a significant reduction in primary and secondary colonosphere growth. Secondary spheroids arise from primary spheroids. No additional drugs were added to secondary spheroids, depending on the number of surviving CSCs when the primary spheroids were treated. MRL16 did not cause a significant reduction in DCLK1-deficient cells, confirming that its analogues inhibit spheroid proliferation by suppressing DCLK1+ CSCs.

[0024] Figure 1 shows one embodiment intended for the use of a novel nanosponge for the delivery of a DCLK1 inhibitor, where the trimareimide structure is a core that binds to a first peptide block of 20 lysine residues and a second peptide block of 10 serine, threonine, aspartate, or glutamate residues. Additionally, two of the blocks are capped with lipid caps (cholesterol), and the other position binds to a signaling sequence that can be either a CD44-targeting or SIRPα-targeting sequence ("Don't Eat Me" sequence). Depending on the peptide used in the second 10-residue block, the DCLK1 inhibitor can be covalently bonded to the hydroxyl group of the serine or threonine residue to form a cleavable carbonate bond, or to the carboxylic acid group of the aspartate or glutamate group to form a cleavable ester bond.

[0025] DCLK1 inhibitors can have a structure corresponding to Formula 1,

[0026] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R1, R2, and R3 are each independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 R1 and R2 are selected from alkyl-substituted C3-C6 aryl groups, or R1 and R2, together with the carbons to which they are bonded, form a 5 or 6-membered ring fused with the adjacent 6-membered ring to which R1 and R2 are bonded, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 and J2 is -N-.

[0027] Additionally, DCLK1 inhibitors may have a structure corresponding to formula 1A.

[0028] [ka] In the formula, A is a 5 or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, preferably a nitrogen-containing heteroaryl group, and R3 is independently -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C) 12 , preferably C1-C6 alkyl, and alkaryl (for example, C1-C 12 Selected from alkyl-substituted C3-C6 aryls, R4 is H or -CH3, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, and J1 and J2 are each independently -CH- or -N-, preferably at least one of J1 or J2 is -N-.

[0029] In one or more embodiments, the DCLK1 inhibitor of formula 1 or formula 1A comprises a nitrogen-containing heterocycle with respect to A, and has the following structure:

[0030] [ka] The formula has one of the following, where R is selected from hydrogen or alkyl (e.g., C1-C6, preferably C1-C3 alkyl).

[0031] In one or more embodiments, the DCLK1 inhibitor also has the following structure

[0032] [ka]

[0033] [ka] It can have.

[0034] Pharmaceutical composition DCLK1 inhibitor compounds can be formulated for various delivery routes and therapeutic modes. Furthermore, pharmaceutical compositions comprising a pharmaceutically acceptable carrier or delivery vehicle and a DCLK1 inhibitor compound (or more compounds) described herein are disclosed herein. In one or more embodiments, the composition may include a mixture of two or more different types of inhibitors.

[0035] A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. Many carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or other solvents or vehicles such as glycols, glycerols, oligosaccharides in water / polyethylene glycol, oils such as olive oil or soybean oil, or injectable organic esters, naturally occurring monosaccharides, disaccharides, and polysaccharides, carbohydrates, sugars, proteins, etc., and any of the following, including mixtures thereof: cellulose such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc. These include their derivatives and their microcrystalline forms, sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, other naturally derived polysaccharides such as dextran, monosaccharides or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, and amylose, dextrins such as α-, β-, or γ-cyclodextrin, dimethyl-β-cyclodextrin, and dextrin, natural starches such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch and their derivatives, and gums such as gum arabic, tragacanth gum, and glucomannan. Preferred carriers may further contain proteins such as serum proteins, casein, and albumin.

[0036] In one or more embodiments, a particularly preferred carrier is a novel peptide-based nanosponge, as illustrated in the examples. Generally, these peptide nanosponges are characterized as a water-soluble sponge-like supramolecular assembly or aggregate comprising a plurality of individual peptide-based building blocks (e.g., 1,000 to 50,000 building blocks), which can dynamically self-assemble, degrade, and reorganize into smaller clusters to facilitate site-selective delivery and uptake of their therapeutic payloads into cells. The peptide nanosponge comprises a plurality of peptide-based building blocks, each building block generally comprising a multi-armed core, two or more linear block copeptide sequences, each having its C-terminus bound to each arm of the core, and its N-terminus typically capped with a lipid moiety. Small molecules (e.g., DCLK1 inhibitors described herein) are covalently bonded to one or more amino acids of the block copeptides via cleavable linkages (e.g., ester bonds). One or more arms of a multi-armed core may contain signaling sequences, which are “Don’t Eat Me” sequences, “Eat Me” sequences, a targeting moiety, and / or a detectable label (e.g., a fluorescent moiety). Details of the nanosponges can be found in concurrently pending international application PCT / US / 2024 / 049592, filed on October 2, 2024, claiming priority to U.S. Provisional Patent Application No. 63 / 542,052, filed on October 2, 2023, entitled “PEPTIDE NANOSPONGES FOR DRUG DELIVERY,” each of which is incorporated herein by reference in whole.

[0037] In one or more embodiments, the pharmaceutically acceptable carrier comprises one or more combinations of the above vehicles and is preferably configured to enhance delivery via a desired pathway.

[0038] This inhibitor can be formulated in various solid or liquid dosage forms. In some cases, if such a pharmaceutical composition is for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion across the epithelial barrier), the aqueous solution is pyrogenic or substantially pyrogenic. Excipients can be selected, for example, to result in delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized products for reconstitution, powders, solutions, syrups, suppositories, and injections. The powder can be used for inhalation, packaged in gel caps, compressed into tablets, etc. Such powders may contain one or more inert carriers or vehicles to promote free flow of the powder and reduce aggregation or clamping. The powder can also be reconstituted into liquid form for various routes of administration, for example, via nebulizers, injections, IVs, oral, ocular, ear, or nasal droplets or sprays, or oral gastric tube feeding. This inhibitor can be formulated as sublingual or buccal tablets, drops, lozenges, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0039] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that acts to stabilize, increase the solubility of, or increase the absorption of a compound, such as the compound of the present invention. Examples of such physiologically acceptable agents include carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may be, for example, a liposome or other polymer matrix in which the compound of the present invention can be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0040] The term "pharmaceutically acceptable" is used herein to mean those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues in proportion to a reasonable benefit-to-risk ratio, without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of sound medical judgment.

[0041] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of substances that can function as a pharmaceutically acceptable carrier include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil (10) Oils such as sorghum oil and soybean oil, glycols such as propylene glycol, (11) Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) Esters such as ethyl oleate and ethyl laurate, (13) Agar, (14) Buffers such as magnesium hydroxide and aluminum hydroxide, (15) Alginic acid, (16) Water free of pyrogens, (17) Isotonic saline, (18) Ringer's solution, (19) Ethyl alcohol, (20) Phosphate buffer, and (21) Other non-toxic and suitable substances used in pharmaceutical formulations. Particularly preferred carriers include sugars in water and oligoethylene glycols in water, dissolved in nonionic micelles.

[0042] Pharmaceutical compositions (formulations) can be administered to a target by any of a variety of routes of administration, including, for example, oral (e.g., aqueous or non-aqueous solutions or suspensions, such as liquid drugs, tablets, capsules including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue), absorption through the oral mucosa (e.g., sublingual, buccal), subcutaneous, transdermal (e.g., as a patch applied to the skin), ocular (e.g., as drops, injections, etc.), intravenous or infusion administration, and topical (e.g., as a cream, ointment, or spray applied to the skin). Compounds may also be formulated for inhalation. In certain embodiments, compounds may be simply dissolved or suspended in sterile water.

[0043] The inhibitor can be administered in one or more doses, and these doses can be administered via the same or different routes to achieve the desired prophylactic or therapeutic effect. In some embodiments, the composition can be provided in unit dosage forms in a suitable container. The term “unit dosage form” refers to a physically separated unit suitable as a unit dose for use in humans or animals. Each unit dosage form may contain a predetermined amount of the inhibitor (and / or other activators) in a carrier, calculated to produce the desired effect. In other embodiments, the inhibitor can be provided separately from the carrier (e.g., in its own vial, ampoule, sachet, or other suitable container) for mixing in situ before administration to a subject. Kits containing the inhibitor are also disclosed herein. The kit further includes instructions for administering the inhibitor to a subject, including reconfiguration as necessary. The inhibitor can be provided as part of a dose unit already dispersed in a pharmaceutically acceptable carrier, or separately from the carrier. The kit may further include instructions for preparing the inhibitor for administration to a subject.

[0044] Treatment method Furthermore, a method for treating cancer in patients requiring cancer treatment is disclosed, comprising administering a DCLK1 inhibitor compound or a pharmaceutical composition described herein in a dose effective for treating cancer.

[0045] Treatment methods are also disclosed, and cancers include colon cancer, bile duct cancer, genitourinary cancer, gynecological cancer, gastrointestinal cancer, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain tumor, or glioma, and in some cases cancer is colon cancer, and in other cases cancer is bile duct cancer.

[0046] The treatment method is suitable for human or veterinary treatment. In one embodiment, the patient is human. In one embodiment, the patient is a non-human animal. In one embodiment, the patient is a companion animal such as a dog, cat, or horse. In one embodiment, the patient is a livestock or production animal such as a cattle, pig, sheep, goat, chicken, or llama.

[0047] Furthermore, for the therapeutic methods disclosed herein, DCLK1 inhibitor compounds or pharmaceutical compositions may be administered orally, ophthalmologically, intravenously, parenterally, sublingually, orally, rectally, cutaneously, intracranially, topically, transdermally, by inhalation, by injection, or in combination thereof.

[0048] The DCKL1 inhibitor compounds described herein may be administered in therapeutically effective doses. As used herein, “therapeutically effective” dose or “therapeutic dose” means the amount or dosage that elicits a biological or medical response in a tissue, system, or subject, as sought by the researcher or clinician, such as to induce some desired therapeutic or prophylactic effect against a disease or condition, depending on the activator delivered. Those skilled in the art will recognize that a dose may be considered therapeutically “effective” even if the disease, condition, or symptom is not completely eradicated or prevented, but it or its symptoms and / or effect may be partially improved or mitigated in the subject (e.g., reduction of tumor volume). The actual effective dose for a particular application depends on the patient (e.g., age, weight), the condition being treated, and the route of administration. Determining an effective dose is well within the capabilities of those skilled in the art, particularly in light of the disclosures herein. Effective doses for human use can be estimated from animal models.

[0049] Unless otherwise stated, the “alkyl” groups described herein, either alone or as part of another group, are optionally substituted linear saturated monovalent hydrocarbon radicals containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, or optionally substituted branched saturated monovalent hydrocarbon radicals containing 3 to 20 carbon atoms, preferably 3 to 8 carbon atoms. Examples of unsubstituted alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, and t-pentyl.

[0050] As used herein, either alone or as part of another group, the term “aryl” refers to an optionally substituted monovalent aromatic hydrocarbon radical, preferably a monovalent monocyclic or bicyclic group, containing 6 to 12 carbon atoms in its ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl. Phenyl and substituted phenyl are more preferred aryl groups. The term “aryl” also includes heteroaryl groups.

[0051] As used herein, either alone or as part of another group, the term "heteroaryl" refers to a monovalent monocyclic or bicyclic aromatic radical substituted with an optional number of 5 to 10 ring atoms in protonated or deprotonated form, where one or more, preferably 1, 2, or 3 ring atoms are heteroatoms independently selected from N, O, and S, and the remaining ring atoms are carbon. Exemplary heteroaryl moieties include benzofuranyl, benzo[d]thiazolyl, benzo[d]thiazolium, isoquinolinyl, isoquinolinium, quinolinyl, quinolinium, thiophenyl, imidazolyl, imidazolium, oxazolyl, oxazolium, furanyl, thiazolyl, thiazolium, pyridinyl, pyridinium, furyl, thienyl, pyridyl, pyrrolyl, pyrrolidinium, indolyl, and indolinium.

[0052] The term "substituted," as in "substituted aryl" or "substituted alkyl," means that in the group in question (i.e., alkyl, aryl, or any other group following the term), at least one hydrogen atom bonded to a carbon atom is replaced by one or more substituents such as hydroxy(-OH), alkylthio, phosphino, amide (-CON(RA)(RB), where RA and RB are independently hydrogen, alkyl, or aryl), amino (-N(RA)(RB), where RA and RB are independently hydrogen, alkyl, or aryl), halo (fluoro, chloro, bromo, or iodo), silyl, nitro (-NO2), ether (-ORA, where RA is alkyl or aryl), ester (-OC(O)RA, where RA is alkyl or aryl), keto (-C(O)RA, where RA is alkyl or aryl), heterocyclo, etc. When the term "substituted" introduces a list of possible substituted groups, the term is intended to apply to all members of that group. In other words, the phrase "arbitrarily substituted alkyl or aryl" should be interpreted as "arbitrarily substituted alkyl or arbitrarily substituted aryl."

[0053] The additional advantages of various embodiments of the present invention will become apparent to those skilled in the art by examining the disclosure herein and the following examples. It will be understood that the various embodiments described herein are not necessarily mutually exclusive unless otherwise stated herein. For example, features described or depicted in one embodiment may, but do not necessarily, be included in other embodiments. Accordingly, the present invention encompasses various combinations and / or integrations of the specific embodiments described herein.

[0054] As used herein, the phrase "and / or" means that, when used in a list of two or more items, any one of the listed items may be used alone, or any combination of two or more of the listed items may be used. For example, if it is stated that a composition contains or excludes components A, B, and / or C, the composition may contain or exclude only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0055] This description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. When numerical ranges are provided, it should be understood that such ranges are to be interpreted as providing literal support to claims that specify only the lower limit of a range, and to claims that specify only the upper limit of a range. For example, the disclosed numerical range of about 10 to about 100 provides literal support to claims that specify "about 10 or more" (no upper limit) and to claims that specify "less than about 100" (no lower limit).

[0056] Having described this disclosure in detail, it will be clear that modifications and variations are possible without departing from the scope of the invention as defined in the attached claims. [Examples]

[0057] The following examples illustrate methods according to the present invention. However, it should be understood that the following non-limiting examples are provided for illustrative purposes only and do not limit the overall scope of the invention.

[0058] Example 1 Synthesis Procedure for DCLK and MRL Compounds Example 1A: Preparation of MRL16

[0059] [ka]

[0060] In a dry round-bottom flask, add 1'-hydroxy-2'-acetonaphthone (1 equivalent) and isonicotinic acid hydrazide (1 equivalent), followed by the addition of 0.5 mL of concentrated HCl and 5 mL of methanol. The reaction mixture is then heated to 60°C and stirred for 4 hours. During this time, a precipitate may form. If no precipitate forms after this time, slowly cool the reaction flask to room temperature, then place it in the freezer to induce recrystallization of the product. After either the reaction time or recrystallization, filter the mixture and wash the solid with DCM. The yellow solid was isolated, and no further purification was required.

[0061] Example 1B: Preparation of MRL17

[0062] [ka]

[0063] In a dry round-bottom flask, add 1'-hydroxy-2'-acetonaphthone (1 equivalent) and nicotinic acid hydrazide (1 equivalent), followed by the addition of 0.5 mL of concentrated HCl and 5 mL of methanol. The reaction mixture is then heated to 60°C and stirred for 4 hours. During this time, a precipitate may form. If no precipitate forms after this time, slowly cool the reaction flask to room temperature, then place it in the freezer to induce recrystallization of the product. After either the reaction time or recrystallization, filter the mixture and wash the solid with DCM. The yellow solid was isolated, and no further purification was required.

[0064] Regarding the synthesis of both MRL compounds, it is important to note that while Z isomers are also formed, they are likely to be present only in trace amounts due to steric hindrance. Therefore, only the E isomer is shown.

[0065] Example 1C: Preparation of DCLK3

[0066] [ka]

[0067] Step 1: Dissolve naphthalene-1-ol (7.2 g, 50 mmol) in EtOH (28 mL) to prepare a mixture of NaOH (14.4 g, 36.0 mmol) and H2O (30 mL), and add it dropwise. Then heat the mixture to 75°C. Once the desired temperature is reached, add chloroform (6.5 mL, 80 mmol) dropwise and heat the reaction mixture for 2-3 hours. Once the reaction is complete (monitored by TLC), acidify the mixture with 50 mL of 1N HCl and extract with DCM. Wash the organic layer with brine, dry over Na2SO4, filter, concentrate, and purify via silica gel flash column chromatography using a hexane:ethyl acetate gradient.

[0068] Step 2: Iodomethane (3 equivalents) was added dropwise to a mixture of DCLK3_Int1 (1 equivalent) and K2CO3 (1 equivalent) in 10 mL of THF:DMF (10:1) under argon at room temperature. The mixture was then stirred overnight. After overnight, water (15 mL) was added to the mixture. The aqueous phase was extracted with ethyl acetate, washed with brine, and dried over sodium sulfate. The organic phase was filtered and concentrated, and the crude product was purified by silica column chromatography using a hexane:DCM gradient.

[0069] Step 3: Add dimethyl(1-diazo-2-oxopropyl)phosphonate (1 equivalent) all at once at room temperature to a mixture of DCLK3_Int2 (1 equivalent) and potassium carbonate (1.4 equivalents) in 5 mL of MeOH under argon. Stir the mixture overnight at room temperature and monitor the reaction by TLC. After overnight, concentrate the reaction mixture and purify it via silica column chromatography using a hexane:DCM gradient.

[0070] Step 4: Add KHCO3 (2 equivalents) to a mixture of DCLK3_Int3 (1 equivalent) and ethyl 2-chloro-2-(hydroxyimino)acetate (2.2 equivalents) dissolved in 5 mL of ethyl acetate. Flash the mixture under argon and stir overnight at room temperature. After overnight, add heptane (5 mL) and stir for a further 5 minutes. Filter the mixture using heptane to aid filtration. Concentrate the filtrate and purify it via silica column chromatography using a hexane:DCM gradient.

[0071] Step 5: 1N NaOH (79 equivalents) was added to a mixture of DCLK3_Int4 (1 equivalent) in MeOH (48 equivalents). The mixture was stirred overnight at room temperature. After overnight, the reaction mixture was further diluted with water (approximately 25 mL), the pH was adjusted to 2-3 using 1M HCl, and the mixture was stirred for another hour. Within 1 hour, a white precipitate formed. The mixture was then filtered and washed with water. The precipitate was collected and dried under reduced pressure at 50°C.

[0072] Step 6: Dissolve DCLK_Int5 (1.0 equivalent), 4-aminopyridine (2.0 equivalents), and DIPEA (5.0 equivalents) in 1 mL of DMF, flush under argon, and place in a dry ice:acetonitrile bath in the dark. Add a solution of T3P in EA (50% by weight, 2.0 equivalents) dropwise while maintaining the internal temperature at -30°C to -10°C. After addition, heat to -10°C and stir for 1 hour. Then, heat the reaction mixture to 0°C and add water. Add ethyl acetate to the resulting suspension. Separate the layers, wash the organic layer with an aqueous K2CO3 solution (5% by weight), dry over magnesium sulfate, then filter and concentrate to obtain the product. This procedure was modified for DCLK3a and DCLK4.

[0073] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and flush under argon. While maintaining 0°C, add 1 M BCl3 in DCM (68 equivalents) dropwise. After addition, keep the reaction mixture in the ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stir for a further 1 hour. Then, further dilute the reaction mixture with water and wash multiple times with DCM. Isolate the organic layer, dry it over Na2SO4, and concentrate it to obtain DCLK3. No further purification was necessary.

[0074] Example 1D: Preparation of DCLK3a

[0075] [ka]

[0076] Steps 1-5 of DCLK3a are the same as steps of DCLK3.

[0077] Step 6: A mixture of DCLK3a_Int5 (1 equivalent), 3-aminopyridine (1.5 equivalents), and triethylamine (3.4 equivalents) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While maintaining 0°C, a solution of T3P in ethyl acetate (50% by weight, 1.7 equivalents) is added dropwise with stirring. After addition, the reaction mixture is stirred at 0°C for a further 1 hour. Then, it is warmed to room temperature and stirred in the dark for 3-4 days. The reaction is monitored by TLC (5:95 methanol:DCM). After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (×2), saturated sodium bicarbonate (×1), and brine (×1). The organic layer is isolated, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by silica column chromatography using a DCM:methanol gradient (99:1 → 90:10).

[0078] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and flush under argon. While maintaining 0°C, add 1 M BCl3 in DCM (68 equivalents) dropwise. After addition, keep the reaction mixture in the ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stir for a further 1 hour. Then, further dilute the reaction mixture with water and wash multiple times with DCM. Isolate the organic layer, dry it over Na2SO4, and concentrate it. The crude residue was purified by silica column chromatography using a DCM:methanol gradient (99:1) to obtain DCLK3a.

[0079] Example 1E: Preparation of DCLK4

[0080] [ka]

[0081] Steps 1-5 are the same as for DCLK3.

[0082] Step 6: A mixture of DCLK4_Int5 (1 equivalent), N,O-dimethylhydroxylamine hydrochloride (1.5 equivalents), and triethylamine (3.4 equivalents) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While maintaining 0°C, a solution of T3P in ethyl acetate (50% by weight, 1.7 equivalents) is added dropwise with stirring. After the addition, the reaction mixture is stirred for a further 1 hour at 0°C. Then, it is warmed to room temperature and stirred in the dark for 3-4 days. The reaction is monitored by TLC (5:95 methanol:DCM). After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (×2), saturated sodium bicarbonate (×1), and brine (×1). The organic layer is isolated, dried over Na2SO4, filtered, and concentrated to obtain the crude product. It is important to note that DCLK4_Int6 is photosensitive and must be kept in the dark during synthesis and storage. If not handled properly, it degrades rapidly. All attempts to purify it have resulted in decomposition. Therefore, DCLK4_Int6 is used as is.

[0083] Step 7: A suspension of 4-pyridylmagnesium bromide in freshly prepared THF was prepared by treating 4-iodopyridine (16.6 mmol, 6.5 equivalents) in 55 mL of THF with 16.6 mL of 1.0 M ethylmagnesium bromide. To this suspension, 1 equivalent of DCLK4_Int6 in 3.5 mL of THF was added dropwise. After 2 hours, the reaction was quenched with 1.0 mL of acetic acid and saturated NH4Cl, and then extracted with 2× ethyl acetate at 0–5°C. The organic layer extract was dried over MgSO4, filtered, and concentrated using a rotary evaporator. The residue was suspended in ethyl acetate, filtered, and the filtration cake was washed with ethyl acetate to obtain a pale yellow solid as the first harvest of the product. The filtrate was concentrated to dryness on silica gel to obtain a free-flowing solid, which was dried and packed onto a heptane-moistened silica gel column. The column was sequentially eluted with ethyl acetate:heptane (1:3, 1:2, and 1:1), and the resulting product fractions were combined and distilled under vacuum at 35°C to obtain a second harvest of the product as a white solid.

[0084] Example 2 Trimaleimide linker (CORE) synthesis

[0085] [ka]

[0086] Step 1: Add maleic anhydride (1 equivalent) to a dry round-bottom flask and dissolve in 5-10 mL of tetrahydrofuran. Add furan (1 equivalent) dropwise to this mixture at room temperature while stirring. After the addition, stir the reaction mixture overnight in RT. A precipitate may form during this time. After overnight, filter the reaction mixture, wash the precipitate with hexane (3 × 5 mL), collect it, and dry it under vacuum. Heat the filtrate, cool it to room temperature, and place it in the freezer to induce recrystallization of the product.

[0087] If no precipitate forms overnight, add 15 mL of hexane to the reaction mixture. The change in polarity may cause the product to precipitate. If a precipitate forms, filter the mixture, collect the precipitate, and dry it under vacuum. If no precipitate forms, recrystallize the mixture as described above. Collect the recrystallized product via vacuum filtration as described above. No further purification is necessary.

[0088] [ka]

[0089] Step 2: Add intermediate A (5.2 equivalents) to a dry round-bottom flask and dissolve in 10 mL of methanol. Place this mixture in an ice bath and stir. Prepare a solution of tris(2-aminoethyl)amine (1 equivalent) in methanol (3 mL) and add it dropwise to the cold mixture. After addition, stir the resulting mixture at 0°C for 5 minutes, then warm to room temperature and stir for a further 30 minutes. After this, heat the reaction mixture under reflux for 4 hours (70°C). Then, slowly cool it to room temperature and leave it in the freezer overnight to induce recrystallization of the product. Then, filter it, wash the solid with cold methanol (3 × 5 mL), isolate it, and dry it under vacuum. No further purification is necessary.

[0090] [ka]

[0091] Step 3: Add 1 equivalent of intermediate B to a dry round-bottom flask and suspend in 10 mL of toluene. Heat the mixture under reflux overnight (115°C). Then concentrate the reaction mixture, isolate the product, and dry it under vacuum overnight. No further purification is required after collecting the product.

[0092] Example 3 Solid-phase peptide synthesis protocol All peptides listed in Table 1 below were synthesized according to the Fmoc(N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis procedure (Wang H., Udukala DN, Samarakoon TN, Basel MT, Kalita M., Abayaweera G., Manawadu H., Malalasekera A., Robinson C., Villanueva D., Maynez P., Bossmann L., Riedy E., Barriga J., Wang N., Li P., Higgins DA, Zhu G., Troyer DL, and Bossmann, SH Nanoplatforms for Highly Sensitive Fluorescence Detection of Cancer-Related Proteases. Photochemistry & Photobiology Sciences. 2014, 13:231-240 and Duro-Castano A., Conejos-Sanchez I., and Vicent M. Peptide-Based Polymer). (Therapeutics.Polymers.2014,6(2):515-551). Briefly, a 2-ClTrt (2-chlorotrityl) resin containing the first amino acid in the peptide sequence (in this case, Gly) was first swollen in dichloromethane (DCM) for 20 minutes, and then washed with N,N-dimethylformamide (DMF) for 1 minute. After washing, a solution containing Fmoc-protecting amino acids (resin:amino acid, 1:3 molar ratio) and O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU) (resin:HBTU, 1:29 molar ratio) as a coupling agent was prepared in a 1:23 diisopropylethylamine (DIEA):DMF solution and added to the resin, and stirred for 30 minutes for the coupling reaction. Each amino acid was added twice to enhance the addition of amino acids to the peptide chain.Prior to moving to the next amino acid, the last Fmoc-protected amino acid was deprotected using a 20% diethylamine solution in DMF, followed by 5 washes with DMF to remove any excess diethylamine present. Each deprotection and addition of amino acids is an iterative cycle during peptide synthesis until all amino acids on the peptide sequence chain are added. After the synthesis was completed, several peptides were labeled with cholesterol, cholic acid, or cleaved unlabeled. For cholesterol and cholic acid addition, they were coupled to the N-terminus of the peptide using 1,1'-carbonyldiimidazole (CDI) as the coupling reagent. Cholesterol was coupled after 5 additions within a 24-hour reaction, while cholic acid was coupled after a total of 7 additions within a 24-hour reaction. Cleavage of the completed sequence (labeled or unlabeled) is performed using trifluoroacetic acid with a 3-hour incubation. After the 3-hour cleavage reaction, the peptide is precipitated in cold ether, collected via centrifugation (4,000×g, 5 minutes), and washed a total of 4 to 6 times with cold ether. The cleaved peptide was then dried and stored at -20°C under argon. S containing the C-terminal linked amino acid (cysteine) and spacer residues (glycine, from resin synthesis). 10 K 20 (SEQ ID NO: 3) and D 10 K 20 (SEQ ID NO: 4) block copolypeptides were prepared.

[0093] [Table 1] k * : D-Lys amino acid

[0094] Example 4 Nanosponge organization and Small molecule coupling protocol The trimaleimide linker used to organize the nanosponges contained three -NH2 groups and acted as an anchor for linking up to three different peptides, which determined the molar ratio used for organization. Nanosponges organized in the presence of the "Don't Eat Me Peptide" sequence consisted of a 1:2:1 ratio of linker:cholesterol block copeptide:"Don't Eat Me Peptide". On the other hand, for nanosponges organized without the "Don't Eat Me Peptide", the ratio was 1:3 linker:cholesterol / cholic acid-block copeptide. In detail, prior to organization, 1× phosphate-buffered saline (PBS, pH 7.2) and anhydrous DMF were degassed with argon. In separate vials, the tris-maleimide linker was dissolved in 10-15 μL of degassed DMF, and the peptide was dissolved together in 1-2 mL of degassed PBS. The two solutions were then combined and stirred at room temperature under an argon atmosphere (deoxygenated) for 24 hours. After 24 hours, to remove unbound components, the nanosponges were transferred to a dialysis membrane tube (3,500 Da MWCO) and dialyzed in water for 1-2 hours. The nanosponges were then freeze-dried overnight and stored at -20°C.

[0095] CDI or HBTU (D) are used as coupling reagents. 10 K 20 Small molecules were coupled to the nanosponge using CG (SEQ ID NO: 6) (only in the case of the nanosponge). Briefly, for the DCLK1 small molecule, 10% (by weight) of each small molecule and CDI were dissolved in anhydrous DMF and stirred at room temperature for 10 minutes. In a separate vial, the nanosponge was dissolved in DMF, the solution was transferred to the small molecule-CDI reaction solution, and the reactants were stirred at room temperature for 24 hours. Dialysis was repeated for 1 hour on the nanosponge-small molecule complex to remove any unbound components, and then freeze-dried overnight. Table 3 summarizes all the compounds organized for the project. Figure 1 shows a diagram of the nanosponge building block with small molecules according to formula 1A coupled to the signal sequence on two arms and a third arm of the trimareimide linker (core).

[0096] [Table 2]

[0097] Example 5 In vitro results Figure 2 shows that DCLK3(C-3) and DCLK4(C-4) inhibit the proliferation of the colon cancer cell line SW480, as evaluated by the hexosaminidase assay described below.

[0098] Figure 3 shows that DCLK3(C-3) and DCLK4(C-4) inhibit the proliferation of the colorectal cancer cell line HCT116, as evaluated by the hexosaminidase assay described below.

[0099] The compounds were screened using a colorimetric hexosaminidase assay. Briefly, cells were seeded in a 96-well plate. The cells were then treated with escalating doses of the compound for 48 hours. The medium was aspirated, and the cells were incubated with a substrate buffer containing p-nitrophenol-N-acetyl-β-D-glucosaminidase. The plates were read at 405 nm absorbance.

[0100] The survival rate was calculated as the percentage of absorbance of the treated organism compared to the control. The obtained IC50 data are shown in Table 5.

[0101] [Table 3]

[0102] Cells were seeded in 6-well plates for colony formation. The cells were then treated with test substances at IC50 and 1 / 2 IC50 doses for 48 hours. The medium was then aspirated and replaced with complete medium free of the drug. Colonies were then grown for 2 weeks, stained, counted, and their size determined using ImageJ software.

[0103] Cells were seeded in 24-well low-adhesion plates for spheroid formation. After 72 hours of spheroid formation, test substances were added at IC50, 1 / 2IC50, and 1 / 4IC50 doses. The spheroids were grown for a further 96 hours, after which imaging and quantification were performed.

[0104] Figure 4 shows representative colony formation images of HUCCT1 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments. * p<0.05.

[0105] Figure 5 shows representative colony formation images of HUH28 cells treated for 48 hours with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50 and 1 / 2 IC50 previously determined by hexosaminidase assay. Quantification of colony number and size. Data are expressed as the average number and size from three independent experiments. * p<0.05.

[0106] Figure 6 shows representative spheroid formation images of HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50, 1 / 2IC50, and 1 / 4IC50 doses previously determined by hexosaminidase assay. Spheroid count quantification. Data are expressed as the mean from three independent experiments. * p<0.05.

[0107] When describing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to indicate that there is one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that there may be additional elements other than those listed.

[0108] Considering the above, it will be evident that several objectives of this disclosure have been achieved and other favorable results have been obtained.

[0109] Because various modifications can be made in the manner described above without departing from the scope of this disclosure, all matters contained in the above description are intended to be interpreted as illustrative and not limited in meaning.

Claims

1. A DCLK1 inhibitor compound having a structure corresponding to Formula 1, 【Chemistry 1】 In the formula, A is a 5- or 6-membered complex ring. R １ , R ２ , and R ３ are each independently selected from the group consisting of -H, -O, -N, -S, -F, -Cl, -OH, -OCH ３ , NO ２ , alkyl, and alkaryl, or R １ and R ２ together with the carbon to which they are bonded form a 5- or 6-membered ring fused to the adjacent 6-membered ring to which R １ and R ２ are bonded, R ４ is H or -CH ３ And, X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-, J １ and J ２ These are DCLK1 inhibitor compounds, each independently being -CH- or -N-.

2. J １ or J ２ The DLC1 inhibitor compound according to claim 1, wherein at least one of them is -N-.

3. R １ and R ２ The DCLK1 inhibitor compound according to claim 1, wherein they combine with the carbon atoms to which they are bonded to form a 5 or 6-membered ring by condensation with an adjacent 6-membered ring.

4. R １ and R ２ However, together with the carbon atoms to which they are bonded, they form a condensed six-membered ring, and the compound is given by formula 1A: 【Chemistry 2】 A DLC1 inhibitor compound according to any one of claims 1 to 3, having a structure corresponding to the above.

5. A is a nitrogen-containing heterocycle, the DCLK1 inhibitor compound according to any one of claims 1 to 4.

6. A, 【Transformation 3】 The DCLK1 inhibitor compound according to claim 5.

7. A DCLK1 inhibitor compound according to any one of claims 1 to 6, wherein X is -C(O)-, -NHC(O)-, or -C(O)NH-.

8. A DCLK1 inhibitor compound according to any one of claims 1 to 6, wherein X is -C(O)- or -C(O)NH-.

9. A DCLK1 inhibitor compound according to any one of claims 1 to 6, wherein X is -C(O)-.

10. A DCLK1 inhibitor compound according to any one of claims 1 to 6, wherein X is -C(O)NH-.

11. J １ If J is -CH-, ２ However, it is either -N- or J １ However, it is -N- and J ２ A DLC1 inhibitor compound according to any one of claims 1 to 10, wherein the compound is -CH-.

12. The above compound has the following structure 【Chemistry 4】 A DLC1 inhibitor compound according to claim 1 or 4, having one of the following.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a DCLK1 inhibitor compound according to any one of claims 1 to 12.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable carrier comprises sugars (e.g., lactose, glucose, or sucrose), starches (e.g., corn starch or potato starch), cellulose or its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, or cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (e.g., cocoa butter or suppository wax), oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, or soybean oil), glycols (e.g., propylene glycol), polyols (e.g., glycerin, sorbitol, mannitol, or polyethylene glycol), esters (e.g., ethyl oleate or ethyl laurate), agar, buffers (e.g., magnesium hydroxide or aluminum hydroxide), alginic acid, water free of pyrogens, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer, or other pharmaceutically acceptable, non-toxic, compatible substances.

15. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable carrier comprises a multi-arm peptide-based nanosponge, and the DLC1 inhibitor compound is bound to the nanosponge via cleavable linkages.

16. A method for treating cancer, the method comprising administering a therapeutically effective amount of a DCLK1 inhibitor compound or a pharmaceutical composition thereof according to any one of claims 1 to 12 to a subject in need thereof.

17. The method according to claim 16, wherein the cancer is colon cancer, bile duct cancer, genitourinary cancer, gynecological cancer, gastrointestinal cancer, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain tumor, or glioma.

18. The method according to claim 17, wherein the cancer is colon cancer or bile duct cancer.

19. A pharmaceutical product for treating cancer, preferably colon cancer or bile duct cancer, in a subject requiring such treatment, wherein the pharmaceutical product contains a therapeutically effective amount of a DCLK1 inhibitor compound according to any one of claims 1 to 12.

20. Use of a DCLK1 inhibitor compound according to any one of claims 1 to 12 for treating cancer in a subject requiring cancer treatment.

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