Dclk1 inhibitors and methods of treatment

Abstract

The present disclosure relates to DCLK1 inhibitor compounds and methods of treatment. The inhibitor compounds generally have a structure (I) corresponding to Formula 1, wherein A is a 5 or 6 membered heterocycle; R1, R2, and R3 are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl, and alkylaryl, or R1 and R2 together with the carbon to which they are attached and the adjacent 6 membered ring to which R1 and R2 are bound form a fused 5 or 6 membered ring; 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-.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 587,362, filed October 2, 2023, entitled DCLK1 INHIBITORS AND METHODS OFTREATMENT, which is incorporated herein by reference in its entirety. sequence list

[0002] The following application contains a sequence list submitted electronically as an XML file conforming to the ST.26 standard, entitled “SequenceListing_61013.xml”, created on October 2, 2024, and measuring 6,341 bytes, the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to doublecortin-like kinase 1 (DCLK1) inhibitor compounds, therapeutic compositions comprising the inhibitor compounds, and methods of using the same to treat cancer. Background Technology

[0004] Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Recent advances in early diagnosis and treatment are ineffective for patients with advanced disease. Standards of care include three different combination chemotherapy regimens, each associated with significant toxicity. At least 50% of colorectal cancer patients receiving these treatments develop metastases. Other combination therapies include immunotherapy, but these have issues with developing resistance and increasing side effects. Therefore, a high unmet medical need remains for improving colorectal cancer treatment.

[0005] Biscortin-like kinase 1 (DCLK1) is considered a biomarker for cancer stem cells (CSCs). 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 P450 3A4 (one of the six enzymes responsible for the metabolism of approximately 90% of all small molecule drugs). Summary of the Invention

[0006] This disclosure relates to DCLK1 inhibitor compounds having a structure corresponding to Formula 1. 1 , Wherein A is a 5- or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, and more preferably a nitrogen-containing heteroaryl group; R1, R2, and R3 are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C...). 12 Preferably C1-C6 alkyl) and alkylaryl (e.g., C1-C6 alkyl) 12 The alkyl-substituted C3-C6 aryl group, or R1 and R2 together with their attached carbons form a fused 5 or 6-membered ring with the adjacent 6-membered rings of R1 and R2; 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-, and preferably at least one of J1 or J2 is -N-.

[0007] Furthermore, this disclosure relates to DCLK1 inhibitor compounds having a structure corresponding to Formula 1A. 1A , Wherein A is a 5- or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, and more preferably a nitrogen-containing heteroaryl group; R3 is independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C...). 12 Preferably C1-C6 alkyl) and alkylaryl (e.g., C1-C6 alkyl) 12 (alkyl-substituted C3-C6 aryl); 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-, and preferably at least one of J1 or J2 is -N-.

[0008] Pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of the DCLK1 inhibitor compound described herein are also disclosed.

[0009] This disclosure relates to methods of treating cancer in patients with a corresponding need, the methods comprising administering a DCLK1 inhibitor compound or a pharmaceutical composition described herein at a dose effective in treating cancer. This disclosure also relates to methods of treating a variety of cancers, including but not limited to colon cancer, bile duct cancer, genitourinary cancers, gynecological cancers, gastrointestinal cancers, lymphoma, melanoma or skin cancer, head and neck cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, breast cancer, gastric cancer, leukemia, bone cancer, thyroid cancer, brain cancer, or glioma.

[0010] Other purposes and features are obvious and are partly indicated below. Brief description of the attached diagram

[0011] Figure 1This is an illustration of an exemplary embodiment of delivering a DCLK1 inhibitor using a peptide-based nanosponge carrier with a trimaleimide-based core structure, which should not be considered as a limitation on possible nanosponge structures or other possible carriers contemplated herein.

[0012] Figure 2 The graph shows the percentage of DCLK1 inhibitor concentration versus proliferation of colorectal cancer cell line SW480, relative to a control without the DCLK1 inhibitor compound.

[0013] Figure 3 The graph shows the percentage of proliferation of colorectal cancer cell line HCT116 cells at the concentration of the DCLK1 inhibitor compound relative to a control without the DCLK1 inhibitor compound.

[0014] Figure 4 The top panel shows representative colony formation images of HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 for 48 h, with IC50 and ½ IC50 as previously determined by hexosaminease assay. The bottom panel shows quantification of colony number and size. Data are presented as mean number and size from three independent experiments, *p<0.05.

[0015] Figure 5 The top panel shows representative colony formation images of HUH28 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 for 48 h, with IC50 and ½ IC50 as previously determined by hexosaminease assay. The bottom panel shows quantification of colony number and size. Data are presented as mean number and size from three independent experiments, *p<0.05.

[0016] Figure 6 The left panel shows representative images of globule formation in HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50, ½ IC50, and ¼ IC50 doses as previously determined by hexosaminease assay. The right panel shows the quantification of globule number. Data are presented as mean from three independent experiments, *p<0.05.

[0017] In all the accompanying drawings, the corresponding reference numerals indicate the corresponding parts. Detailed Description of Implementation Plans - Preferred Implementation Plans

[0018] Oral administration of a representative small-molecule DCLK1 inhibitor demonstrated in vivo antitumor activity in a mouse xenograft model of colon cancer. We were able to demonstrate that DCLK1 inhibition is associated with an antitumor response and generate a more pharmaceutically tractable compound using the initial lead candidate. We further employed a delivery vehicle for targeted delivery, which reduces the effective dose required for treatment and may decrease side effects and off-target effects. Nanosponges linked to small-molecule DCLK1 inhibitors allow for direct delivery to tumor sites in the colon to enhance antitumor activity while simultaneously “protecting” the small-molecule DCLK1 inhibitors from potential metabolism.

[0019] DCLK1 is a biomarker for cancer stem cells (CSCs). CSCs are rare and difficult to isolate and identify within tumor masses. Various methods have been used to identify CSCs, including spheroidization ability, dye rejection due to efflux pump overexpression, intracellular enzyme activity, and expression of cell surface markers. Previous reports have identified DCLK1 in the gut. DCLK1 has been confirmed as a biomarker for CRC and pancreatic cancer stem cells. DCLK1 is a member of the calmodulin-like protein kinase superfamily. The full-length encoded protein contains two N-terminal dicortin (DCX) domains that bind 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 located between the dicortin and kinase domains. DCLK1 is primarily transcribed by a second promoter in colon cancer cells. The AKT1 peptide around Thr308 interacts with the kinase domain of the DCLK1 protein (a short form lacking the N-terminal DCX domain). DCLK1 knockout mouse models suppressed colonic polyps in APCmin / + mice but did not affect the normal intestine. These data indicate that DCLK1+ cells label CSCs but not normal stem cells. An important implication of this finding is that targeting DCLK1 specifically targets CSCs without affecting normally proliferating cells.

[0020] DCLK1 has been shown to phosphorylate the microtubule-associated protein MAP7D1. Using computer modeling, AKT1 (IKDGATMKTFCGTP, SEQ ID NO:1) was identified around a threonine residue at position 308. Using the AutoDock Vina and the crystal structure of the DCLK1 kinase domain (PDB ID 5JZN), the interaction between the AKT1 peptide around Thr308 and the DCLK1 kinase domain was observed (binding energy -5.7 kcal / mol). DCLK1 amino acids targeted for AKT1 interaction include ASP398, ASP475, GLU515, and THR552. This interaction was confirmed using magnetic relaxation assays and immunoprecipitation-coupled protein blot assays. In vitro assays using recombinant DCLK1 and the AKT peptide also confirmed significant phosphorylation of the AKT substrate by DCLK. This phosphorylation was lost upon DCLK1 denaturation.

[0021] The lead candidate MRL16 has been demonstrated to inhibit colon cancer growth by suppressing cell cycle proliferation during the G2 / M phase, inducing apoptosis, and inhibiting stemness by suppressing globular growth. MRL16 inhibits DCLK1-mediated phosphorylation of AKT1 at Thr308, leading to downstream inhibition of Notch signaling and PXR. In vivo mouse xenograft studies using MRL16 subsequently 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 to selectively deliver DCLK1 inhibitors to CRC tumors, this disclosure aims to improve treatment outcomes by enhancing efficacy and reducing toxicity. This article describes the synthesis and in vitro pharmaceutical evaluation of a novel nanosponge-linked DCLK1 inhibitor. This novel nanosponge drug delivery platform has not been previously used for the treatment of CRC.

[0022] Researchers at the University of Kansas Medical Center obtained information from the wood orange ( Aegle marmelos marmelin (1-hydroxy-5,7-dimethoxy-2-naphthaldehyde, MRL) was isolated from (an Ayurvedic treatment material for gastrointestinal cancer): Marmelin (MRL), MW 232.23 Based on its early ADMET properties, MRL16 was selected from this scaffold for target validation studies. Despite limited water solubility and moderate in vitro metabolic stability, these compounds have proven to be excellent probes for validating DCLK1 as a therapeutic target in proof-of-principle studies. MRL16 interacts significantly with the kinase domain, with a binding energy of -7.9 kcal / mol, and the key amino acid is VAL468.

[0023] MW 305.33 DCLK1 is a protein that marks quiescent stem cells in colorectal cancer. Stem cells support colonosphere growth. We performed colonosphere assays using HCT116 cells, in which DCLK1 was knocked down using a specific shRNA. 500 cells were seeded in ultra-low adhesion 12-well culture dishes containing serum-free medium but essential for colonosphere growth. A significant reduction in colonosphere formation was observed compared to controls. Cells were then treated with MRL16 (0.5 µM), showing a significant reduction in both primary and secondary colonosphere growth. Secondary colonospheres developed from primary colonospheres. In secondary colonospheres, without the addition of additional drugs, growth depended on the number of surviving CSCs at the time of primary colonosphere treatment. MRL16 did not induce a significant reduction in DCLK1-deficient cells, confirming that the analogue inhibits DCLK1+ CSCs to suppress colonosphere growth.

[0024] Figure 1 An embodiment for the intended delivery of a DCLK1 inhibitor using a novel nanosponge is shown, wherein a trimaleimide structure is the core, which is attached to a first peptide block of 20 lysine residues and a second peptide block of 10 serine, threonine, aspartic acid / aspartate, or glutamic acid / glutamate residues. Furthermore, both blocks are capped with lipid caps (cholesterol), and a signal transduction sequence is attached to another site, which may be a CD44-targeting sequence or a SIRPα-targeting sequence (“Don’t eat me” sequence). Depending on the peptide used for the second 10-residue block, the DLCK1 inhibitor may be covalently attached to the hydroxyl group of a serine or threonine residue to form a cleavable carbonate bond, or covalently attached to the carboxylic acid group of an aspartic or glutamate group to form a cleavable ester bond.

[0025] DCLK1 inhibitors may have a structure corresponding to Formula 1. 1 , Wherein A is a 5- or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, and more preferably a nitrogen-containing heteroaryl group; R1, R2, and R3 are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C...). 12 Preferably C1-C6 alkyl) and alkylaryl (e.g., C1-C6 alkyl) 12The alkyl-substituted C3-C6 aryl group, or R1 and R2 together with their attached carbons form a fused 5 or 6-membered ring with the adjacent 6-membered rings of R1 and R2; 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-, and preferably at least one of J1 and J2 is -N-.

[0026] Furthermore, DCLK1 inhibitors may have a structure corresponding to Formula 1A. 1A , Wherein A is a 5- or 6-membered heterocycle, preferably a nitrogen-containing heterocycle, and more preferably a nitrogen-containing heteroaryl group; R3 is independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl (e.g., C1-C...). 12 Preferably C1-C6 alkyl) and alkylaryl (e.g., C1-C6 alkyl) 12 (alkyl-substituted C3-C6 aryl); 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-, and preferably at least one of J1 or J2 is -N-.

[0027] In one or more embodiments, the DCLK1 inhibitor of Formula 1 or Formula 1A comprises a nitrogen-containing heterocycle as A, having one of the following structures: , , , , , , , , , , , or , R is selected from hydrogen or alkyl (e.g., C1-C6, preferably C1-C3 alkyl).

[0028] In one or more embodiments, the DCLK1 inhibitor may also have the following structure , , , , , , , , , , or .

[0029] Pharmaceutical Composition DCLK1 inhibitor compounds can be formulated for various delivery routes and modes of treatment. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier or delivery medium and one or more DCLK1 inhibitor compounds described herein are also disclosed. In one or more embodiments, the composition may comprise a mixture of two or more different types of inhibitors.

[0030] Pharmaceutically acceptable carriers are any carriers suitable for in vivo administration. Many carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or media such as glycols, glycerol, oligomeric glycols / polyethylene glycol in water, oils such as olive oil, soybean oil, or injectable organic esters, naturally occurring monosaccharides, disaccharides, and polysaccharides, carbohydrates, sugars, proteins, etc., including any of the following substances (including mixtures thereof): cellulose, its derivatives, and their microcrystalline forms, such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, etc. Methylcellulose, sodium carboxymethylcellulose, etc.; other naturally derived polysaccharides such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, and dextran; monosaccharides or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, and amylose; dextrins such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, dimethyl-β-cyclodextrin, and dextrin; natural starches and their derivatives such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch; and gums such as gum arabic, tragacanth, and glucomannan. Suitable carriers may further include proteins such as serum albumin, casein, and albumin.

[0031] In one or more embodiments, a particularly preferred carrier is the novel peptide-based nanosponges exemplified in the working examples. Typically, these peptide nanosponges are characterized as water-soluble sponge-like supramolecular assemblies or aggregates comprising multiple individual peptide-based building blocks (e.g., 1,000-50,000 building blocks) that can dynamically self-assemble, disassemble, and reassemble into small clusters to facilitate site-selective delivery and uptake of their therapeutic payload into cells. The peptide nanosponges comprise multiple peptide-based building blocks, each typically comprising a multi-armed core; two or more linear block conpeptide sequences, each sequence having its C-terminus attached to a corresponding arm of the core, and each sequence typically having its N-terminus capped with a lipid moiety. Small molecules (e.g., the DCLK1 inhibitor described herein) are covalently attached to one or more amino acids of the block conpeptide via a cleavable linkage (e.g., ester bond). One or more arms of the multi-armed core may include signal transduction sequences, such as “don’t eat me” sequences, “eat me” sequences, targeting portions, and / or detectable markers (e.g., fluorescent portions). Details of the nanosponges can be found in co-pending PCT / US / 2024 / 049592, filed October 2, 2024, which claims priority to U.S. Provisional Patent Application Serial No. 63 / 542,052, filed October 2, 2023, entitled “PEPTIDE NANOSPONGES FORDRUG DELIVERY,” each of which is incorporated herein by reference in its entirety.

[0032] In one or more embodiments, the pharmaceutically acceptable carrier comprises a combination of one or more of the aforementioned mediators and is preferably configured to enhance delivery via the desired route.

[0033] Inhibitors can be formulated into various solid or liquid dosage forms. In some cases, when such pharmaceutical compositions are intended 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 pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of the drug or selective targeting of one or more cells, tissues, or organs. Pharmaceutical compositions can be in unit dosage forms, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized formulations for reconstitution, powders, solutions, syrups, suppositories, injections, etc. Powders can be used for inhalation, packaged in gel capsules, compressed into tablets, etc. Such powders can contain one or more inert carriers or mediators to facilitate free flow of the powder and reduce aggregation or clumping. Powders can also be reconstituted into liquid forms for use in various routes of administration, such as via nebulizer, injection, IV, oral, ophthalmic, ocular, or nasal drops or sprays, oral tube feeding, etc. Inhibitors can be formulated as sublingual or buccal tablets, drops, lozenges, etc. The composition can also be present in transdermal delivery systems (e.g., skin patches). The composition can also be present in solutions suitable for topical application, such as lotions, creams, or ointments.

[0034] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that, for example, stabilize the compound (e.g., the compound of the present invention), increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, 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 choice of pharmaceutically acceptable carrier (including physiologically acceptable agents) 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 also be a liposome or other polymer matrix in which compounds of the present invention may be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers, and their preparation and administration are relatively simple.

[0035] The phrase “pharmaceutically acceptable” as used herein means a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0036] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium, 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 other components of the formulation and harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and sesame oil. Oils and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, 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) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations. Particularly preferred carriers include sugars in water, oligomeric polyethylene glycols in water, and those dissolved in nonionic micelles.

[0037] The pharmaceutical composition (formulation) may be administered to a subject via any of many routes of administration, including, for example, oral (e.g., as an aqueous or non-aqueous solution or suspension, a drench, tablet, capsule (including oral capsules and gelatin capsules), pills, powder, granules, or paste for application to the tongue); absorption through the oral mucosa (e.g., sublingual, buccal); subcutaneous; transdermal (e.g., as a patch for application to the skin); ocular (e.g., as drops, injections, etc.); intravenous or infusion administration; and topical (e.g., as a cream, ointment, or spray for application to the skin). The compound may also be formulated for inhalation. In some embodiments, the compound may simply be dissolved or suspended in sterile water.

[0038] Inhibitors can be administered in one or more doses, which can be administered via the same or different routes to achieve the desired preventive or therapeutic effect. In some embodiments, the composition can be provided in a unit dosage form in a suitable container. The term "unit dosage form" refers to a physically discrete unit suitable for use as a single dose in humans or animals. Each unit dosage form may contain a predetermined amount of inhibitor (and / or other active agent) calculated to produce the desired effect in a carrier. In other embodiments, the inhibitor may be provided separately from the carrier (e.g., in its own vial, ampoule, sachet, or other suitable container) for on-site mixing prior to administration to a subject. Kits containing inhibitors are also disclosed herein. The kit further includes instructions for administering the inhibitor to a subject, including reconfiguring it (if necessary). The inhibitor may be provided as part of a dosage unit already dispersed in a pharmaceutically acceptable carrier, or it may be provided separately from the carrier. The kit may further include instructions for preparing the inhibitor for administration to a subject.

[0039] Treatment Methods for treating cancer in patients with appropriate need are also disclosed, the methods comprising administering the DCLK1 inhibitor compound or the pharmaceutical composition described herein at a dose effective for treating cancer.

[0040] Treatment methods were also disclosed, including cancers such as 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 cancer or glioma; in some cases, the cancer is colon cancer; and in others, the cancer is bile duct cancer.

[0041] Treatment methods are applicable to both humans and veterinary patients. In one case, the patient is a human. In another case, the patient is a non-human animal. In one case, the patient is a companion animal, such as a canine, feline, or equine. In another case, the patient is livestock or producer animal, such as a cow, pig, sheep, goat, chicken, or llama.

[0042] Furthermore, for the treatment methods disclosed herein, DCLK1 inhibitor compounds or pharmaceutical compositions may be administered orally, ocularly, intravenously, parenterally, sublingually, buccally, rectally, dermally, intracranially, topically, percutaneously, by inhalation, by infusion, or in combination thereof.

[0043] The DCKL1 inhibitor compounds described herein can be administered in therapeutically effective amounts. As used herein, a “therapeutically effective” amount or “therapeutic dose” means an amount or dose that will elicit a biological or medical response in an investigator’s or clinician’s desired tissue, system, or subject, such as an amount or dose that will elicit some desired therapeutic or preventative effect against a disease or condition, depending on the active agent delivered. Those skilled in the art will recognize that an amount may be considered therapeutically “effective” even if the disease, condition, or symptom is not completely eradicated or prevented, but its or its symptoms and / or effects are partially improved or alleviated in a subject (e.g., tumor volume reduction, etc.). The actual amount effective for a particular application will depend on the patient (e.g., age, weight, etc.), the condition being treated, and the route of administration. Determining an effective amount is entirely within the capabilities of those skilled in the art (especially in light of the disclosure herein). Effective amounts for human use can be estimated using animal models.

[0044] Unless otherwise stated, "alkyl" as described herein, whether alone or as part of another group, is an optionally substituted straight-chain saturated monovalent hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, or an optionally substituted branched saturated monovalent hydrocarbon group containing 3 to 20 carbon atoms, preferably 3 to 8 carbon atoms. Examples of unsubstituted alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, tert-pentyl, etc.

[0045] As used herein, the term "aryl" alone or as part of another group means an optionally substituted monovalent aromatic group, preferably a monovalent monocyclic or bicyclic group comprising 6 to 12 carbons in the ring moiety, 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.

[0046] As used herein, the term "heteroaryl," whether alone or as part of another group, refers to a monovalent monocyclic or bicyclic aromatic group of 5 to 10 ring atoms, either protonated or unprotonated, wherein one or more, preferably one, two, or three 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]thiazolyl, isoquinolinyl, isoquinolinlyl, quinolinyl, quinolinlyl, thiophene, imidazolyl, imidazolyl, oxazolyl, oxazollyl, furanyl, thiazolyl, thiazolyl, pyridinyl, pyridinyl, furanyl, thiophene, pyridyl, pyrrolidinium, indole, indole, etc.

[0047] The term "substituted" (as in "substituted aryl", "substituted alkyl", etc.) means that in the group under discussion (i.e., the alkyl, aryl, or other group following the term), at least one hydrogen atom bonded to a carbon atom is replaced by one or more substituents such as: hydroxyl (-OH); alkylthio; phosphine; amino (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; halogen ( Fluorine, chlorine, bromine, or iodine); silyl; nitro (-NO2); ether (-ORA, where RA is alkyl or aryl); ester (-OC(O)RA, where RA is alkyl or aryl); ketone (-C(O)RA, where RA is alkyl or aryl), heterocyclic, etc. When the term “substituted” introduces a list of possible substituted groups, it is intended that the term apply to each member of said group. In other words, the phrase “optionally substituted alkyl or aryl” should be interpreted as “optionally substituted alkyl or optionally substituted aryl.”

[0048] Other advantages of the various embodiments of the invention will become apparent to those skilled in the art upon reading the disclosure herein and the working examples below. It should be understood that, unless otherwise stated herein, the various embodiments described herein are not necessarily mutually exclusive. For example, a feature described or depicted in one embodiment may be included in, but is not necessarily included in, other embodiments. Therefore, the invention covers various combinations and / or integrations of the specific embodiments described herein.

[0049] As used herein, when used in a list of two or more items, the phrase “and / or” means that 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 a composition is described as containing or excluding components A, B, and / or C, the composition may contain or exclude A alone; B alone; C alone; 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.

[0050] This specification also uses numerical ranges to quantify certain parameters related to various embodiments of the invention. It should be understood that when numerical ranges are provided, these ranges should be interpreted as providing textual support for claims that only state the lower limit of the range and claims that only state the upper limit of the range. For example, the disclosed numerical range of about 10 to about 100 provides textual support for claims stating "greater than about 10" (no upper limit) and claims stating "less than about 100" (no lower limit).

[0051] Having described this disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims.

[0052] Example The following embodiments illustrate the method according to the present invention. However, it should be understood that the following non-limiting embodiments are provided in an illustrative manner, and nothing therein should be considered as limiting the overall scope of the invention.

[0053] Example 1 Synthetic procedures for DCLK and MRL compounds Example 1A: Preparation of MRL16 Add 1′-hydroxy-2′-naphthyl ethylone (1 equivalent) and isonicotinamide (1 equivalent) to a dry round-bottom flask, followed by 0.5 mL of concentrated hydrochloric acid and 5 mL of methanol. Heat the reaction mixture to 60 °C and stir for 4 hours. A precipitate may form during this time. If no precipitate forms after this period, allow the reaction flask to cool slowly to RT and then place it in a refrigerator to induce recrystallization of the product. After the stated reaction time or recrystallization, filter the mixture and wash the solid with DCM. Separate the yellow solid; no further purification is required.

[0054] Example 1B: Preparation of MRL17 Add 1′-hydroxy-2′-naphthyl ethylone (1 equivalent) and nicotinamide (1 equivalent) to a dry round-bottom flask, followed by 0.5 mL of concentrated hydrochloric acid and 5 mL of methanol. Heat the reaction mixture to 60 °C and stir for 4 hours. A precipitate may form during this time. If no precipitate forms after this period, allow the reaction flask to cool slowly to RT and then place it in a refrigerator to induce recrystallization of the product. After the reaction time or recrystallization, filter the mixture and wash the solid with DCM. Separate the yellow solid; no further purification is required.

[0055] It is important to note that the synthesis of the two MRL compounds may also have resulted in the formation of... Z Isomers, but due to Z The steric hindrance of the isomer is trace. Therefore, only a small amount is shown. E Isomers.

[0056] Example 1C: Preparation of DCLK3 Step 1: Naphthalene-1-ol (7.2 g, 50 mmol) was dissolved in EtOH (28 mL), and a mixture of NaOH (14.4 g, 36.0 mmol) and H₂O (30 mL) was prepared and added dropwise. The mixture was then heated to 75 °C. Once the desired temperature was reached, chloroform (6.5 mL, 80 mmol) was added dropwise, and the reaction mixture was heated for 2–3 hours. After the reaction was complete (monitored by TLC), the mixture was acidified with 50 mL of 1N HCl and extracted with DCM. The organic layer was washed with brine, dried over Na₂SO₄, filtered, concentrated, and purified by silica gel rapid column chromatography using a hexane:ethyl acetate gradient.

[0057] Step 2: Iodomethane (3 equivalents) was added dropwise to a mixture of DCLK3_Int1 (1 equivalent) and K2CO3 (1 equivalent) in 10 mL THF:DMF (10:1) under an argon atmosphere. The mixture was then stirred overnight. After overnight incubation, 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 purified by silica gel column chromatography using a hexane:DCM gradient.

[0058] Step 3: To a mixture of DCLK3_Int2 (1 equivalent) and potassium carbonate (1.4 equivalent) in 5 mL of MeOH under an argon atmosphere, dimethyl (1 equivalent) phosphonate was added in a single step at RT. The mixture was stirred at RT overnight, and the reaction was monitored by TLC. After overnight incubation, the reaction mixture was concentrated and purified by silica gel column chromatography using a hexane:DCM gradient.

[0059] Step 4: Add KHCO3 (2 equivalents) to a mixture of DCLK3_Int3 (1 equivalent) and 2-chloro-2-(hydroxyimino)ethyl acetate (2.2 equivalents) dissolved in 5 mL of ethyl acetate. Wash the mixture under an argon atmosphere and stir overnight at RT. After overnight, add heptane (5 mL) and stir for another 5 minutes. Filter the mixture using heptane to aid filtration. Concentrate the filtrate and purify it by silica gel column chromatography using a hexane:DCM gradient.

[0060] Step 5: Add 1N NaOH (79 equivalents) to the mixture of DCLK3_Int4 (1 equivalent) and MeOH (48 equivalents). Stir the mixture at RT overnight. After overnight, further dilute the reaction mixture with water (about 25 mL) and adjust the pH to 2-3 with 1M HCl, then stir for another hour. A white precipitate forms within one hour. Filter the mixture and wash with water. Collect the precipitate and dry under reduced pressure at 50°C.

[0061] Step 6: Dissolve DCLK_Int5 (1.0 equivalent), 4-aminopyridine (2.0 equivalent), and DIPEA (5.0 equivalent) in 1 mL of DMF, rinse under an argon atmosphere, and place in a dry ice:acetonitrile bath in the dark. Add dropwise a solution of T3P in EA (50 wt%, 2.0 equivalent) while maintaining an internal temperature of -30°C to -10°C. After dropwise addition, warm the resulting mixture to -10°C and stir for 1 hour. Then, warm 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 solution of K₂CO₃ (5 wt%), dry it over magnesium sulfate, filter, and concentrate to obtain the product. This procedure has been modified for DCLK3a and DCLK4.

[0062] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and wash under an argon atmosphere. Meanwhile, add 1 M BCl3 (68 equivalents) dropwise to the DCM at 0°C. After the dropwise addition, keep the reaction mixture in an ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stirring for another 1 hour. Then, further dilute the reaction mixture with water and wash several times with DCM. Separate the organic layer, dry with Na2SO4, and concentrate to obtain DCLK3. No further purification is required.

[0063] Example 1D: Preparation of DCLK3a Steps 1-5 of DCLK3a are the same as steps 1-5 of DCLK3.

[0064] Step 6: Under an argon atmosphere, wash the mixture of DCLK3a_Int5 (1 equivalent), 3-aminopyridine (1.5 equivalent), and triethylamine (3.4 equivalent) in 3 mL of DCM and place it in an ice bath in the dark. Meanwhile, at 0°C, add dropwise a solution of T3P in ethyl acetate (50 wt%, 1.7 equivalent) while stirring. After the dropwise addition, stir the reaction mixture at 0°C for another 1 hour. Then warm it to RT and stir in the dark for 3–4 days. Monitor the reaction by TLC (5:95 methanol:DCM). After the reaction time, further dilute the mixture with DCM, and wash the organic layer with water (x2), saturated sodium bicarbonate (x1), and brine (x1). Separate the organic layer, dry it with Na2SO4, filter, and concentrate. Use a DCM:methanol gradient (99:1) The crude residue was purified by silica gel column chromatography (90:10).

[0065] Step 7: Dissolve DCLK_Int6 (1 equivalent) in 1 mL of DCM, place in an ice bath, and wash under an argon atmosphere. Meanwhile, add 1 M BCl3 (68 equivalents) dropwise to the DCM at 0°C. After the dropwise addition, keep the reaction mixture in an ice bath overnight. After overnight, quench the reaction mixture by adding ice water (5 mL) and stirring for another 1 hour. Then, further dilute the reaction mixture with water and wash several times with DCM. Separate the organic layer, dry over Na2SO4, and concentrate. Purify the crude residue by silica gel column chromatography using DCM:methanol (99:1) to obtain DCLK3a.

[0066] Example 1E: Preparation of DCLK4 Steps 1-5 are the same as DCLK3.

[0067] Step 6: Under an argon atmosphere, wash a mixture of DCLK4_Int5 (1 equivalent), N,O-dimethylhydroxylamine hydrochloride (1.5 equivalent), and triethylamine (3.4 equivalent) in 3 mL of DCM and place it in an ice bath in the dark. Meanwhile, at 0°C, add dropwise a solution of T3P in ethyl acetate (50 wt%, 1.7 equivalent) while stirring. After the dropwise addition, stir the reaction mixture at 0°C for another 1 hour. Then warm it to RT and stir in the dark for 3–4 days. Monitor the reaction by TLC (5:95 methanol:DCM). After the reaction time, further dilute the mixture with DCM, and wash the organic layer with water (x2), saturated sodium bicarbonate (x1), and brine (x1). Separate the organic layer, dry it with Na2SO4, filter, and concentrate to obtain the crude product. It is important to note that DCLK4_Int6 is photosensitive and must be stored in the dark during synthesis and storage. It will degrade rapidly if not handled properly. All attempts to purify it resulted in degradation. Therefore, a crude version of DCLK4_Int6 was used.

[0068] Step 7: 3.5 mL of DCLK4_Int6 (1 equivalent) in THF was added dropwise to a suspension of 4-pyridylmagnesium bromide in THF, freshly prepared by treating 55 mL of 4-iodopyridine (16.6 mmol, 6.5 equivalent) in THF with 16.6 mL of 1.0 M ethyl magnesium bromide at room temperature. After 2 hours, the reaction mixture was quenched with 1.0 mL of acetic acid and saturated NH4Cl, and then extracted with 2x ethyl acetate at 0–5 °C. The organic layer extract was dried over MgSO4, filtered, and concentrated by a rotary evaporator. The residue was resuspended in ethyl acetate, filtered, and the filter cake was washed with ethyl acetate to give a pale yellow solid as the first product. The filtrate was concentrated to dryness with silica gel to provide a free-flowing solid, which was then loaded onto a silica gel column moistened with heptane. The column was eluted sequentially with ethyl acetate:heptane (1:3, 1:2 and 1:1), the resulting product fractions were combined, and the product was vacuum distilled at 35°C to obtain a second batch of product as a white solid.

[0069] Example 2 Trimaleimide Linker (Core) Synthesis Step 1: Add maleic anhydride (1 equivalent) to a dry round-bottom flask and dissolve it in 5–10 mL of tetrahydrofuran. Add furan (1 equivalent) dropwise to the mixture at RT while stirring. After addition, stir the reaction mixture at RT overnight. During this time, a precipitate may form. After overnight, filter the reaction mixture, wash the precipitate with hexane (3 x 5 mL), collect the precipitate, and dry it under vacuum. Heat the filtrate, cool it to RT, and place it in a refrigerator to induce recrystallization of the product.

[0070] If no precipitate forms overnight, add 15 mL of hexane to the reaction mixture. A change in polarity may cause the product to precipitate. If a precipitate forms, filter, collect, and vacuum dry the precipitate. If no precipitate forms, recrystallize the mixture as described above. Collect the recrystallized product via vacuum filtration as previously described. No further purification is required. Step 2: Add intermediate A (5.2 equivalents) to a dry round-bottom flask and dissolve it in 10 mL of methanol. Place the 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 dropwise addition, stir the resulting mixture at 0 °C for 5 min, then warm to RT and stir for another 30 min. Afterward, heat the reaction mixture under reflux (70 °C) for 4 h. Then allow it to cool slowly to RT and place it in a refrigerator overnight to induce recrystallization of the product. Filter the solid, wash it with cold methanol (3 x 5 mL), separate it, and dry it under vacuum. No further purification is required. Step 3: Add intermediate B (1 equivalent) to a dry round-bottom flask and suspend it in 10 mL of toluene. Heat the mixture under reflux (115°C) overnight. Then, concentrate the reaction mixture, separate the product, and further dry it under vacuum overnight. The product is then collected without further purification.

[0071] Example 3 Solid-phase peptide synthesis scheme All peptides mentioned in Table 1 below are in accordance with Fmoc ( N Synthesis of (9-fluorenyl)methoxycarbonyl) solid-phase peptides using a synthetic program (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). In short, 2-ClTrt (2-chlorotriphenylmethyl) resin containing the first amino acid in the peptide sequence (Gly in this case) was first swollen in dichloromethane (DCM) for 20 minutes, and then... N,N Wash with dimethylformamide (DMF) for 1 minute. After washing, prepare a solution containing Fmoc-protected amino acids (resin:amino acid, 1:3 molar ratio) and o-benzotriazole-N,N,N',N'-tetramethyl-ureonium hexafluorophosphate (HBTU) as a coupling agent in a 1:23 diisopropylethylamine (DIEA):DMF solution, and add it to the resin, vortexing for 30 minutes to carry out the coupling reaction. Each amino acid is added twice to enhance the addition of amino acids in the peptide chain. Before moving to the next amino acid, the last Fmoc-protected amino acid is deprotected with a 20% diethylamine solution in DMF, and then washed 5 times with DMF to remove any excess diethylamine. During peptide synthesis, each deprotection and amino acid addition is repeated in a cycle until all amino acids in the peptide sequence chain have been added. After synthesis, some peptides are labeled with cholesterol, bile acids, or unlabeled peptides are cleaved. For the addition of cholesterol and bile acids, 1,1'-carbonyldiimidazole (CDI) was used as the coupling agent to couple them to the N-terminus of the peptide. Cholesterol was coupled after 5 additions within a 24-hour reaction period, while bile acids were coupled after a total of 7 additions within a 24-hour reaction period. The completed sequences (labeled or unlabeled) were lysed by incubation with trifluoroacetic acid for 3 hours. After the 3-hour lysis reaction, the peptides were precipitated in cold diethyl ether, collected by centrifugation (4,000 x g for 5 min), and washed with cold diethyl ether a total of 4–6 times. The lysed peptides were then dried and stored at -20°C under an argon atmosphere. 10 K 20 (SEQ IDNO: 3) and D 10 K 20 (SEQ ID NO:4) The block peptide was prepared to include a C-terminal linked amino acid (cysteine) and a spacer residue (glycine, derived from resin synthesis).

[0072] Table 1. Synthesized peptide sequences containing block conopeptides for DCLK1 nanosponges Example 4 Nanosponge assembly and small molecule coupling scheme The trimaleimide linker used to assemble the nanosponges contains three -NH2 groups, serving as anchors for linking up to three different peptides, which determines the molar ratio used in assembly. Nanosponges assembled in the presence of the "Don't Eat Me Peptide" sequence have a 1:2:1 linker:cholesterol block conopeptide: "Don't Eat Me Peptide" ratio. Conversely, for nanosponges assembled in the absence of "Don't Eat Me Peptide," a 1:3 linker:cholesterol / cholesterol acid block conopeptide ratio is present. Specifically, prior to assembly, 1X phosphate-buffered saline (PBS, pH 7.2) and anhydrous DMF were degassed with argon. In separate vials, the trimaleimide linker was dissolved in 10–15 µL of degassed DMF, and the peptides were dissolved together in 1–2 mL of degassed PBS. The two solutions were then combined and stirred at room temperature for 24 hours under an argon atmosphere (deoxygenation). After 24 hours, the nanosponges were transferred to dialysis membrane tubing (3,500 Da MWCO) for dialysis against water for 1–2 hours to remove unbound components. The nanosponges were then lyophilized overnight and stored at -20°C.

[0073] Use CDI or HBTU (for D only) 10 K 20 CG (SEQ ID NO:6) nanosponge was used as a coupling agent to couple small molecules to 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 min. In separate vials, the nanosponge was dissolved in DMF, and the solution was transferred to the small molecule-CDI reaction solution; the reactants were stirred at room temperature for 24 h. The nanosponge-small molecule complex was repeatedly dialyzed for 1 h to remove any unbound components, and then lyophilized overnight to dryness. Table 3 summarizes all compounds assembled for the project. Figure 1 A diagram of the nanosponge building blocks is shown, in which a small molecule according to Formula 1A is coupled to two arms and the signal sequence is on the third arm of the trimaleimide linker (core).

[0074] Table 3. Assembled nanosponges for DCLK1 - small molecules Example 5 In vitro results Figure 2 DCLK3 (C-3) and DCLK4 (C-4) inhibited the proliferation of the colon cancer cell line SW480, as assessed by the hexosaminease assay described below.

[0075] Figure 3DCLK3 (C-3) and DCLK4 (C-4) inhibited the proliferation of colorectal cancer cell line HCT116, as assessed by the hexosaminease assay described below.

[0076] Compounds were screened using a colorimetric hexosaminease assay. Briefly, cells were seeded in 96-well plates. Cells were then treated with escalating doses of the compound for 48 hours. The culture medium was aspirated, and the cells were incubated with a substrate buffer containing p-nitrophenol-N-acetyl-β-D-glucosidase. The absorbance of the plates was read at 405 nm.

[0077] Viability was calculated as a percentage of absorbance of the treatment relative to the control. The obtained IC50 data are shown in Table 5.

[0078] Table 5. IC50 of Nano Sponges For colony formation, cells were seeded in 6-well plates. Cells were then treated with test items at IC50 and ½ IC50 doses for 48 h. The culture medium was then aspirated and replaced with complete, drug-free medium. Colonies were then allowed to grow for 2 weeks, followed by staining, counting, and size determination using ImageJ software.

[0079] For globule formation, cells were seeded onto 24-well low-attachment plates. Globule formation was allowed to occur for 72 hours, followed by the addition of test items at IC50, ½ IC50, and ¼ IC50 doses. Globule regrowth was then allowed for 96 hours before imaging and quantification.

[0080] Figure 4 Representative colony formation images of HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 for 48 h are shown. IC50 and ½ IC50 were determined as previously by hexosaminease assay. Quantification of colony number and size is presented. Data are expressed as mean number and size from three independent experiments. *p<0.05.

[0081] Figure 5 Representative colony formation images of HUH28 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 for 48 h are shown. IC50 and ½ IC50 were determined as previously by hexosaminease assay. Quantification of colony number and size is presented. Data are expressed as mean number and size from three independent experiments, *p<0.05.

[0082] Figure 6Images of representative spheroid formation in HUCCT1 cells treated with gemcitabine (Gem), IA-DC-103, DCLK3a, and IA-DC-125 at IC50, ½ IC50, and ¼ IC50 doses as previously determined by hexosaminease assay are shown. Quantification of spheroid number is also presented. Data are expressed as the mean of three independent experiments, *p<0.05.

[0083] When describing elements of this disclosure or its preferred embodiments, the articles “a,” “an,” “the,” and “said” are intended to indicate the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and indicate that additional elements may exist in addition to those listed.

[0084] As will be seen from the foregoing, several objectives of this disclosure have been achieved and other favorable results have been obtained.

[0085] Various modifications can be made to the above methods without departing from the scope of this disclosure, and all content contained in the above description should be interpreted as illustrative rather than restrictive.

Claims

1. A DCLK1 inhibitor compound having a structure corresponding to Formula 1 1 ， Where A is a 5- or 6-membered heterocyclic ring; R1, R2 and R3 are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyl and alkylaryl, or R1 and R2 together with the carbons attached to them form a fused 5 or 6-membered ring with the adjacent 6-membered rings bonded to R1 and R2. R4 is H or -CH3; X is -C(O)-, -NHC(O)-, -C(O)NH-, or -NHC(O)NH-; and J1 and J2 are independently -CH- or -N-.

2. The DCLK1 inhibitor compound according to claim 1, wherein at least one of J1 or J2 is -N-.

3. The DCLK1 inhibitor compound according to claim 1, wherein R1 and R2, together with the carbons to which they are attached, form a fused 5- or 6-membered ring with the adjacent 6-membered ring.

4. The DCLK1 inhibitor compound according to any one of claims 1-3, wherein R1 and R2 together with the carbons to which they are attached form a fused 6-membered ring, wherein the compound has a structure corresponding to formula 1A: 1A 。 5. The DCLK1 inhibitor compound according to any one of claims 1 to 4, wherein A is a nitrogen-containing heterocycle.

6. The DCLK1 inhibitor compound according to claim 5, wherein A is: 、 、 、 、 、 、 、 、 、 , , or .

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

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

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

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

11. The DCLK1 inhibitor compound according to any one of claims 1 to 10, wherein J2 is -N- when J1 is -CH-, or wherein J1 is -N- and J2 is -CH-.

12. The DCLK1 inhibitor compound according to claim 1 or 4, wherein the compound has one of the following structures 、 、 , , , , , , or .

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 of 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 derivatives thereof (e.g., sodium carboxymethyl cellulose, ethyl cellulose, or cellulose acetate); powdered tragacanth gum; 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., glycerol, sorbitol, mannitol, or polyethylene glycol); esters (e.g., ethyl oleate or ethyl laurate); agar; buffers (e.g., magnesium hydroxide or aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; or other pharmaceutically acceptable non-toxic compatible substances.

15. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable carrier comprises a multi-arm peptide-based nanosponge, wherein the DCLK1 inhibitor compound is attached to the nanosponge via a cleavable linker structure.

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

17. The method of 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 cancer or glioma.

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

19. A medicament for treating cancer in a subject with a corresponding need, said medicament comprising a therapeutically effective amount of a DCLK1 inhibitor compound according to any one of claims 1 to 12, said cancer preferably colon cancer or bile duct cancer.

20. Use of the DCLK1 inhibitor compound according to any one of claims 1 to 12 for the treatment of cancer in a subject with appropriate need.

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