DCLK1 inhibitors and methods of treatment

Abstract

[This disclosure is directed to DCLK1 inhibitor compounds and treatment methods. The inhibitor compounds generally have a structure corresponding to Formula 1. wherein 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, alkyls, and alkaryls, or R1 and R2 together with the carbons they are attached to form a fused 5- or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bound; 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-.

Description

24KU021M-01 61013-PCT DCLK1 INHIBITORS AND METHODS OF TREATMENT CROSS-REFRENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 587,362, filed October 2, 2023, entitled DCLK1 INHIBITORS AND METHODS OF TREATMENT, incorporated by reference in its entirety herein. SEQUENCE LISTING

[0002] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled " SequenceListing_61013.xml," created on October 2, 2024, as 6,341 bytes in size, the contents of which are incorporated herein. TECHNICAL FIELD

[0003] This disclosure is directed to Doublecortin Like Kinase 1 (DCLK1) inhibitor compounds, therapeutic composition comprising the inhibitor compounds, and methods of treating cancer using the same. BACKGROUND

[0004] Colon cancer is the second-leading cause of cancer-related deaths in the United States. Recent advances in early diagnosis and treatment are not curative in patients with advanced disease. The standard of care includes three different combination regimens of chemotherapy, each one associated with significant toxicity. At least 50% of colon cancer patients on these treatments develop metastases. Other combination therapies include immunotherapies, but these suffer from the development of resistance and increased adverse effects. Therefore, there continues to be a high unmet medical need to improve colon cancer treatment.

[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 transcription factor Pregnane X Receptor (PXR) expression. 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 the metabolism of approximately 90% of all small24KU021M-01 61013-PCT molecule drugs). SUMMARY

[0006] The disclosure is directed to a DCLK1 inhibitor compound having a structure corresponding to Formula 1 1 , wherein A is a 5- or 6-containing heterocycle, preferably a nitrogen-containing heteroaryl group; R1 , R2 , and R3 are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyls (e.g., C1-C12, preferably C1-C6 alkyls), and alkaryls (e.g., C1-C12alkyl-substituted C3-C6aryls), or R1and R2together with the carbons they are attached to form a fused 5- or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bound; R4 is H or -CH3; X is -C(O)-, -NHC(O)-, -C(O)NH-, or –NHC(O)NH-; and J1and J2are each independently -CH- or -N-, and preferably with the proviso that at least one of J1or J2is -N-.

[0007] Additionally, the disclosure is directed to a DCLK1 inhibitor compound having a structure corresponding to Formula 1A , wherein A is a 5-containing heterocycle, preferably a nitrogen-containing heteroaryl group; R3is independently selected from -H, -O, -N, - S, -F, -Cl, -OH, -OCH3, NO2, alkyls (e.g., C1-C12, preferably C1-C6alkyls), and alkaryls (e.g., C1-24KU021M-01 61013-PCT C12alkyl-substituted C3-C6aryls); R4is 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 with the proviso that at least one of J1 or J2 is -N-.

[0008] Also disclosed is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the DCLK1 inhibitor compounds described herein.

[0009] The disclosure is directed to a method for treatment of cancer in a patient in need thereof comprising administering the DCLK1 inhibitor compounds described herein or a pharmaceutical composition described herein in a dose effective for treating the cancer. The disclosure also concerns methods of treating various cancers including, but not limited to, colon cancer, cholangiocarcinoma, a genitourinary cancer, a gynecologic cancer, a gastrointestinal cancer, lymphoma, melanoma or skin cancer, a head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain cancer, or a glioma.

[0010] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is an illustration of an exemplary embodiment using a peptide-based nanosponge carrier for delivery of the DCLK1 inhibitors using a trimaleimide-based core structure, which is not to be taken as limiting of possible nanosponge structures or other possible carriers contemplated herein.

[0012] FIG. 2 show graphs of the concentration of the DCLK1 inhibitor compounds versus the percent proliferation of colorectal cancer cell line SW480 cells relative to a control not containing the DCLK1 inhibitor compound.

[0013] FIG. 3 show graphs of the concentration of the DCLK1 inhibitor compounds versus the percent proliferation of colorectal carcinoma cell line HCT116 cells relative to a control not containing the DCLK1 inhibitor compound.

[0014] FIG.4 shows (top) representative colony formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and ½ IC50 as previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p<0.05.

[0015] FIG. 5 shows (top) representative colony formation images of HUH28 cells treated with24KU021M-01 61013-PCT Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and ½ IC50 as previously determined by hexosaminidase assay. (Bottom) Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p<0.05.

[0016] FIG. 6 shows (Left) representative spheroid formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at IC50, ½ IC50 and ¼ IC50 doses, as previously determined by hexosaminidase assay. (Right) Quantification of spheroid number. Data are represented as the mean from 3 independent experiments, * p<0.05.

[0017] Corresponding reference characters indicate corresponding parts throughout the drawings. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0018] Oral administration of representative small molecule DCLK1 inhibitors demonstrated in vivo antitumor activity in a mouse colon cancer xenograft model. We were able to demonstrate that inhibition of DCLK1 was associated with antitumor response, and used the initial lead candidates to generate compounds more pharmaceutically tractable. We further used delivery vehicles for targeted delivery, which can lower the effective dose needed for treatment, possibly reducing side effects and off-target effects. The nanosponge linked to small molecule DCLK1 inhibitors could allow for delivery directly at the tumor site in the colon to enhance antitumor activity while “protecting” small molecule DCLK1 inhibitors from potential metabolism.

[0019] DCLK1 is a cancer stem cell (CSC) marker. CSCs are rare and difficult to isolate and identify cells within the tumor mass. Multiple methods used to identify CSCs include sphere- forming ability, dye exclusion due to overexpression of efflux pumps, intracellular enzyme activity and expression of cell surface markers. Previous reports identified DCLK1 in the intestine. DCLK1 is 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, which bind microtubules and regulate microtubule polymerization, a C-terminal serine / threonine kinase domain that has homology to Ca2+ / calmodulin-dependent protein kinase, and a serine / proline-rich domain in between the doublecortin and the protein kinase domains. DCLK1 is predominantly transcribed from a second promoter in colon cancer cells. AKT1 peptide surrounding Thr308 interacts with kinase domain of DCLK1 protein, a short form that lacks the N-terminal DCX domains. DCLK1 knock out mouse models suppress colon polyps in APCmin / + mice but do not affect normal intestine. These data24KU021M-01 61013-PCT suggest that DCLK1+ cells mark CSCs, not normal stem cells. An important consequence of this finding is that targeting DCLK1 will specifically target CSCs while sparing normal proliferating cells.

[0020] DCLK1 has been shown to phosphorylate the microtubule-associated protein MAP7D1. Using in silico modeling, AKT1 was identified surrounding threonine at position 308 (IKDGATMKTFCGTP, SEQ ID NO:1). Using AutoDock Vina, and the crystal structure of the kinase domain of DCLK1 (PDB ID 5JZN), it was observed that the AKT1 peptide surrounding Thr308 interacts with the DCLK1 kinase domain (binding energy of -5.7 kcal / mol). DCLK1 amino acids targeted for AKT1 interaction include ASP398, ASP475, GLU515, and THR552. We confirmed the interaction using a magnetic relaxation assay and an immunoprecipitation-coupled western blot assay. An in vitro assay using recombinant DCLK1 and the AKT peptide also confirmed significant phosphorylation of the AKT substrate by DCLK. This phosphorylation was lost when DCLK1 was denatured.

[0021] It has been demonstrated that lead candidate MRL16 inhibits colon cancer growth by inhibiting proliferation at the G2 / M phase of the cell cycle, inducing apoptosis, and suppressing stemness by inhibiting spheroid growth. MRL16 suppressed DCLK1-mediated phosphorylation of AKT1 at Thr308, resulting in downstream suppression of Notch signaling and PXR. In vivo mouse xenograft studies with MRL16 subsequently demonstrated antitumor activity providing evidence that DCLK1 is a viable CRC therapeutic target. Combining 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 enhancing efficacy and reducing toxicity. Described herein is the synthesis of novel nanosponge-linked DCLK1 inhibitors and evaluation of the agents in vitro. This novel nanosponge drug delivery platform has not been previously employed for the treatment of CRC.

[0022] Researchers at the University of Kansas Medical Center isolated marmelin (1-hydroxy-5,7- dimethoxy-2-naphthalene-carboxaldehyde, MRL) from Aegle marmelos, an Ayurveda treatment for gastrointestinal cancers:24KU021M-01 61013-PCT

[0023] From this scaffold, MRL16, was selected for target validation studies based on its early ADMET properties. Despite limited aqueous solubility and moderate in vitro metabolic stability, the compounds proved excellent probes 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, with the key amino acid being VAL468. MW 305.33

[0024] DCLK1 is acancer. Stem cells support spheroid growth. We performed spheroid assays with HCT116 cells where DCLK1 is knocked down using specific shRNA. 500 cells were plated in an ultra-low-attachment 12-well dish in a medium devoid of serum but containing essential factors for spheroid growth. A significant reduction in spheroid formation compared to controls was observed. Cells were then treated with MRL16 (0.5 µM) and showed a significant reduction in primary and secondary colonosphere growth. Secondary spheroids are developed from primary spheroids. In secondary spheroids, no additional drug is added, relying on the number of CSCs that survived when primary spheroids were treated. MRL16 did not invoke a significant reduction in cells lacking DCLK1, confirming the analogs suppress DCLK1+ CSCs to inhibit spheroid growth.24KU021M-01 61013-PCT

[0025] FIG. 1 shows one embodiment contemplated for using a novel nanosponge for delivery of the DCLK1 inhibitors wherein the trimaleimide structure is the core that is attached to a first peptide block of 20 lysine residues and a second peptide block of 10 residues of serine, threonine, aspartic acid / aspartate, or glutamic acid / glutamate. Additionally, two of the blocks are capped with a lipid cap (cholesterol) and the other position is attached to a signaling sequence that can be either a sequence targeting CD44 or targeting SIRPα (“don’t eat me” sequence). Depending on the peptides used for the second 10-residue block, the DLCK1 inhibitor can be covalently attached to the hydroxyl groups of serine or threonine residues to form a cleavable carbonate bond or covalently attached to the carboxylic acid groups of aspartic acid or glutamic acid groups to form cleavable ester bonds.

[0026] The DCLK1 inhibitors can have a structure corresponding to Formula 1 , wherein A is a 5-containing heterocycle, preferably a nitrogen-containing heteroaryl group; R1, R2, and R3are each independently selected from -H, -O, -N, -S, -F, -Cl, -OH, -OCH3, NO2, alkyls (e.g., C1-C12, preferably C1-C6 alkyls), and alkaryls (e.g., C1-C12 alkyl-substituted C3-C6 aryls), or R1 and R2 together with the carbons they are attached to form a fused 5- or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bound; 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 with the proviso that at least one of J1and J2is -N-.

[0027] Additionally, the DCLK1 inhibitors can have the structure corresponding to Formula 1A24KU021M-01 61013-PCT , wherein A is a 5- containing heterocycle,preferably a group; from -H, -O, -N, - S, -F, -Cl, -OH, -OCH3, NO2, alkyls (e.g., C1-C12, preferably C1-C6alkyls), and alkaryls (e.g., C1- C12 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- and preferably with the proviso that at least one of J1or J2is -N-.

[0028] In one or more embodiment, the DCLK1 inhibitors of Formula 1 or Formula 1A comprise a nitrogen-containing heterocycle for A, have one of the following structures:24KU021M-01 61013-PCT ,

[0029] In one or more embodiments, the DCLK1 inhibitors can also have the following structures N ,24KU021M-01 61013-PCT ,24KU021M-01 61013-PCT Pharmaceutical Compositions

[0030] The DCLK1 inhibitor compounds can be formulated for various routes of delivery and modalities of treatment. Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier or delivery vehicle and a DCLK1 inhibitor compound (or plurality of compounds) described herein. In one or more embodiments, the compositions can comprise a mixture of two or more different types of inhibitors.

[0031] 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 physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oligo / polyethylene glycols in water, oils such as olive oil, soy oil, or injectable organic esters, , naturally occurring mono, di-, and polysaccharides, carbohydrates, sugars, proteins, and the like, including any of the following including mixtures thereof: celluloses, derivatives thereof, and microcrystalline forms thereof such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and the like; other naturally derived polysaccharides such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, dextran; mono or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, amylose, and the like; dextrins such as α-, β- or γ- cyclodextrin, dimethyl-β-cyclodextrin, dextrin; native starches and their derivatives such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch; as well as gums such as gum arabic, tragacanth gum and glucomannan. Suitable carriers can further include proteins, such as serum proteins, casein, albumin, and the like.

[0032] In one or more embodiments, a particularly preferred carrier is a novel peptide-based nanosponge as exemplified in the working examples. In general, these peptide nanosponges are characterized as water-soluble sponge-like supramolecular assemblies or aggregates comprising a plurality of 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 nanosponge comprises a plurality of peptide-based building block, where each building block will generally comprise a multi-armed core; two or more linear block co-peptide sequences, each having its C- terminal end attached to a respective arm of the core and its N-terminal end typically capped with a lipid moiety. Small molecules (e.g., the DCLK1 inhibitors described herein) are covalently24KU021M-01 61013-PCT attached to one or more amino acids of the block co-peptide via a cleavable linkage (e.g., ester linkage). One or more arms of the multi-armed core can include a signaling sequence, which is a “don’t eat me” sequence, an “eat me” sequence, a targeting moiety, and / or a detectable label (e.g., fluorescent moiety). Details of the nanosponges can be found in co-pending PCT / US / 2024 / 049592 filed October 2, 2024, claiming priority to U.S. Provisional Patent Application Serial No. 63 / 542,052, filed October 2, 2023, entitled PEPTIDE NANOSPONGES FOR DRUG DELIVERY, each of which incorporated by reference in its entirety herein.

[0033] In one or more embodiments, the pharmaceutically-acceptable carrier comprises a combination of one or more of the above-described vehicles, and preferably is configured for enhancing delivery via the desired route.

[0034] The inhibitors can be formulated in a variety of solid or liquid dosage forms. In some cases, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in unit dosage form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection, or the like. Powders can be used for inhalation, packaged in gel caps, compressed into tablets, and the like. Such powders can include one or more inert carriers or vehicles to facilitate free-flowing powder and reduce aggregation or clumping. Powders can also be reconstituted into a liquid form for various routes of administration, e.g., via nebulizer, injection, IV, oral, ocular, otic, or nasal drops or sprays, oral gavage, and the like. The inhibitors can be formulated as sublingual or buccal tablets, drops, lozenges, and the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0035] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The24KU021M-01 61013-PCT choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self- microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0036] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0037] The phrase “pharmaceutically acceptable carrier” as used herein 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 of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve 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; (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 soybean oil; (10) 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) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Particularly preferred carriers include sugars in water, oligoethylene glycol in water, dissolved in non-ionic micelles.

[0038] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (e.g., drenches as in aqueous or24KU021M-01 61013-PCT non-aqueous solutions or suspensions, tablets, capsules, including sprinkle capsules and gelatin capsules, boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually, buccally); subcutaneously; transdermally (e.g., as a patch applied to the skin); ocularly (e.g., as drops, injection, etc.); intravenous or infusion administration; and topically (e.g., as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water.

[0039] The inhibitors may be administered by one or more dosages, which dosages can be administered, by the same or different route, to achieve the desired prophylactic or therapeutic effect. In some embodiments, the composition can be provided in unit dosage form in a suitable container. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of the inhibitor (and / or other active agents) in the carrier calculated to produce the desired effect. In other embodiments, the inhibitor can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject. A kit comprising the inhibitors is also disclosed herein. The kit further comprises instructions for administering the inhibitors to a subject, including reconstituting it if needed. The inhibitors can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable carrier, or it can be provided separately from the carrier. The kit can further comprise instructions for preparing the inhibitors for administration to a subject. Methods of Treatment

[0040] Also disclosed is a method for treatment of cancer in a patient in need thereof comprising administering the DCLK1 inhibitor compound described herein or a pharmaceutical composition described herein in a dose effective for treating the cancer.

[0041] Also disclosed are methods of treatment, wherein the cancer is colon cancer, cholangiocarcinoma, a genitourinary cancer, a gynecologic cancer, a gastrointestinal cancer, lymphoma, melanoma or skin cancer, a head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain cancer, or a glioma; in some cases, the cancer is colon cancer; and in other cases, the cancer is cholangiocarcinoma.24KU021M-01 61013-PCT

[0042] The methods of treatment are suitable for human or veterinary treatment. In one aspect, the patient is a human. In one aspect, the patient is a non-human animal. In one aspect, the patient is a companion animal, such as a canine, feline, equine, etc. In one aspect, the patient is livestock or a producer animal such as cows, pigs, sheep, goats, chickens, llamas, and the like.

[0043] Also, for the methods of treatment disclosed herein the DCLK1 inhibitor compound or pharmaceutical composition can be administered orally, ocularly, intravenously, parenterally, sublingually, buccally, rectally, cutaneously, intracranially, topically, transdermally, via inhalation, via infusion, or a combination thereof.

[0044] The DCKL1 inhibitor compounds described herein can be administered in a therapeutically effective amount. As used herein, a “therapeutically effective” amount or “therapeutic dose” refers to the amount or dosage that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, such as to elicit some desired therapeutic or prophylactic effect as against the disease or condition depending upon the active agent delivered. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the disease, condition, or symptom is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject (e.g., reduction in tumor volume, 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. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the disclosure herein. The effective amount for use in humans can be estimated from animal models.

[0045] Unless otherwise indicated, an "alkyl" group as described herein alone or as part of another group is an optionally substituted linear saturated monovalent hydrocarbon radical containing from one to twenty carbon atoms and preferably one to twelve carbon atoms, or an optionally substituted branched saturated monovalent hydrocarbon radical containing three to twenty carbon atoms, and preferably three to eight 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, t-pentyl, and the like.

[0046] The term "aryl" as used herein alone or as part of another group denotes an optionally substituted monovalent aromatic hydrocarbon radical, preferably a monovalent monocyclic or bicyclic group containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl,24KU021M-01 61013-PCT naphthyl, substituted phenyl, substituted biphenyl, or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl groups. The term "aryl" also includes heteroaryl.

[0047] The term "heteroaryl," as used herein alone or as part of another group, denotes an optionally substituted monovalent monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms in protonated or unprotonated form, where 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]thiazolium, isoquinolinyl, isoquinolinium, quinolinyl, quinolinium, thiophenyl, imidazolyl, imidazolium, oxazolyl, oxazolium, furanyl, thiazolyl, thiazolium, pyridinyl, pyridinium, furyl, thienyl, pyridyl, pyrrolyl, pyrrolidinium, indolyl, indolinium, and the like.

[0048] The term "substituted" as in "substituted aryl," "substituted alkyl," and the like, means that in the group in question (i.e., the alkyl, aryl or other group that follows the term), at least one hydrogen atom bound to a carbon atom is replaced with one or more substituent groups such as hydroxy (-OH), alkylthio, phosphino, amido (-CON(RA)(RB), wherein RA and RB are independently hydrogen, alkyl, or aryl), amino(-N(RA)(RB), wherein RA and RB are independently hydrogen, alkyl, or aryl), halo (fluoro, chloro, bromo, or iodo), silyl, nitro (-NO2), an ether (-ORA wherein RA is alkyl or aryl), an ester (-OC(O)RA wherein RA is alkyl or aryl), keto (-C(O)RA wherein RA is alkyl or aryl), heterocyclo, and the like. When the term "substituted" introduces a list of possible substituted groups, it is intended that the term apply to every member of that group. That is, the phrase "optionally substituted alkyl or aryl" is to be interpreted as "optionally substituted alkyl or optionally substituted aryl."

[0049] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein.

[0050] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or24KU021M-01 61013-PCT excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0051] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0052] Having described the disclosure in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. EXAMPLES

[0053] The following examples set forth methods in accordance with the invention. It is to be understood, however, that following non-limiting examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. EXAMPLE 1 DCLK AND MRL COMPOUND SYNTHETIC PROCEDURES

[0054] Example 1A: Preparation of MRL16 N

[0055] hydrazide (1 eq.) followed by 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 precipitate may form. If precipitate does not form after this time, the reaction flask is allowed to cool slowly to RT and24KU021M-01 61013-PCT then placed in the freezer to induce recrystallization of product. After either the reaction time or recrystallization, the mixture is filtered and the solid is washed with DCM. The yellow solid is isolated, and no further purification was needed.

[0056] Example 1B: Preparation of MRL17 OH O O O Nhydrazide (1 eq.) followed by 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 precipitate may form. If precipitate does not form after this time, the reaction flask is allowed to cool slowly to RT and then placed in the freezer to induce recrystallization of product. After either the reaction time or recrystallization, the mixture is filtered and the solid is washed with DCM. The yellow solid is isolated, and no further purification was needed.

[0058] It is important to note for the synthesis of both MRL compounds, it is likely that the Z isomer also forms but in trace amounts due to steric hindrance of the Z isomer. As such only the E isomer is being shown.

[0059] Example 1C: Preparation of DCLK324KU021M-01 61013-PCT

[0060] Step 1: Naphthalen-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 made and added dropwise. The mixture is then heated to 75°C. Once at the desired temperature, chloroform (6.5 mL, 80 mmol) was added dropwise, and the reaction is heated for 2-3 hours. Upon the completion of the reaction (monitored by TLC), the mixture was acidified with 50mL of 1N HCl, and extracted with DCM. The organic layer is washed with brine and dried over Na2SO4, filtered, concentrated, and purified via silica gel flash column chromatography using hexane: ethyl acetate gradient.

[0061] Step 2: To a mixture of DCLK3_Int1 (1 eq.) and K2CO3 (1 eq.) in 10 mL of THF: DMF (10:1) under argon is iodomethane (3 eq.) added dropwise at RT. the mixture is then stirred for overnight. After overnight, water (15 mL) was added to the mixture. The aqueous phase was extracted with ethyl acetate, washed with brine, dry with sodium sulfate. The organic phase was filtered and concentrated, and the crude product was purified via silica column chromatography using hexane: DCM gradient.

[0062] Step 3: To a mixture of DCLK3_Int2 (1 eq.) and potassium carbonate (1.4 eq.) in 5 mL of MeOH, under argon, is added dimethyl (1-diazo-2-oxopropyl) phosphonate (1 eq.) in one portion at RT. This mixture is stirred at RT for overnight, TLC to monitor reaction. After overnight, the reaction mixture is concentrated and purified via silica column chromatography using hexane: DCM gradient.

[0063] Step 4: To a mixture of DCLK3_Int3 (1 eq.) and ethyl 2-chloro-2-(hydroxyimino) acetate (2.2 eq) dissolved in 5 mL of ethyl acetate is KHCO3 (2 eq.) added. The mixture is flushed under argon and stirred at RT for overnight. After overnight, heptane (5 mL) was added and allowed to stir for an additional 5 minutes. The mixture was filtered using heptane to aid in the filtration. The filtrate is concentrated and purified via silica column chromatography using hexane: DCM gradient.

[0064] Step 5: To a mixture of DCLK3_Int4 (1 eq) in MeOH (48 eq) was added 1N NaOH (79 eq). The mixture was stirred at RT for overnight. After overnight, the reaction mixture was further diluted with water (~25 mL) and adjusted the pH to 2~3 using 1M HCl and stirred for an additional hour. Within the hour, a white precipitate was generated. The mixture was then filtered and washed with water. The precipitate was collected and dried under reduced pressure at 50°C.

[0065] Step 6: DCLK_Int5 (1.0 eq.), 4-aminopyridine (2.0 eq.) and DIPEA (5.0 eq.) were dissolved in 1 mL of DMF, flushed under argon and placed in a dry ice: acetonitrile bath in the24KU021M-01 61013-PCT dark. To this was added a solution of T3P in EA (50 wt%, 2.0 eq.) dropwise while keeping the internal temperature between -30°C and -10 °C. Following the dropwise addition, the resulting mixture was warmed to -10°C and stirred for an hour. After, the reaction mixture was warmed to 0°C, and water was added. Ethyl acetate was added to the resulting suspension. The layers were separated and the organic layer was washed with aqueous K2CO3solution (5 wt %), and dried over magnesium sulfate followed by filtration and concentration to yield product. This procedure was changed for DCLK3a and DCLK4.

[0066] Step 7: DCLK_Int6 (1 eq.) was dissolved in 1 mL of DCM, placed in an ice bath, and flushed under argon. While at 0°C, 1M BCl3 in DCM (68 eq.) is added dropwise. Post dropwise addition, the reaction mixture is kept in the ice bath for overnight. After overnight, the reaction mixture is quenched via the addition of ice water (5 mL) and stirring for an additional hour. The reaction mixture is then further diluted with water and washed multiple times with DCM. The organic layer is isolated, dried over Na2SO4 and concentrated to yield DCLK3. No further purification was needed.

[0067] Example 1D: Preparation of DCLK3a

[0069] Step 6: A mixture of DCLK3a_Int5 (1 eq.), 3-aminopyridine (1.5 eq.), triethylamine (3.4 eq.) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While at 0°C, a solution of T3P in ethyl acetate (50 wt%, 1.7 eq.) is added dropwise while stirring. Post dropwise addition the reaction mixture is allowed to stir at 0°C for an additional hour. This is then allowed to warm to RT and left to stir for 3-4 days in the dark. TLC (5: 95 Methanol: DCM) to monitor the24KU021M-01 61013-PCT reaction. After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (x2), saturated sodium bicarbonate (x1) and brine (x1). The organic layer is isolated, dried over Na2SO4, filtered and concentrated. The crude residue was purified via silica column chromatography using DCM: Methanol gradient (99:1 ^ 90:10).

[0070] Step 7: DCLK_Int6 (1 eq.) was dissolved in 1 mL of DCM, placed in an ice bath, andflushed under argon. While at 0°C, 1M BCl3 in DCM (68 is added dropwise. Post dropwise addition, the reaction mixture is kept in the ice bath for overnight. After overnight, the reaction mixture is quenched via the addition of ice water (5 mL) and stirring for an additional hour. The reaction mixture is then further diluted with water and washed multiple times with DCM. The organic layer is isolated, dried over Na2SO4 and concentrated. The crude residue was purified via silica column chromatography using DCM: Methanol (99:1) to yield DCLK3a.

[0071] Example 1E: Preparation of DCLK4 O OH OH O O O

[0073] Step 6: A mixture of DCLK4_Int5 (1 eq.), N,O-Dimethylhydroxylamine hydrochloride (1.5 eq.), triethylamine (3.4 eq.) in 3 mL of DCM is flushed under argon and placed in an ice bath in the dark. While at 0°C, a solution of T3P in ethyl acetate (50 wt%, 1.7 eq.) is added dropwise while stirring. Post dropwise addition the reaction mixture is allowed to stir at 0°C for an additional hour. This is then allowed to warm to RT and left to stir for 3-4 days in the dark. TLC (5: 95 Methanol: DCM) to monitor the reaction. After the reaction time, the mixture is further diluted with DCM and the organic layer is washed with water (x2), saturated sodium bicarbonate (x1) and brine (x1). The organic layer is isolated, dried over Na2SO4, filtered, and concentrated to yield crude product. It is important to note that DCLK4_Int6 is light sensitive and must be kept in the dark during synthesis and storage. This will quickly degrade if not handled properly. Purification24KU021M-01 61013-PCT efforts of this all resulted in degradation. As such, DCLK4_Int6 is used crude.

[0074] Step 7: To a suspension of 4-pyridylmagnesiurn bromide in THF, which was freshly prepared by treating 4- iodopyridine (16.6 mmol, 6.5 eq) in 55 mL of THF with 16.6 mL of 1.0M ethyl magnesium bromide at room temperature, was added DCLK4_Int6 (1 eq.) in 3.5 mL of THF dropwise. After 2 hours, the reaction was quenched with 1.0 mL of acetic acid and sat. NH4Cl, and then extracted with 2 x ethyl acetate at 0-5 °C. The organic layer extracts were dried over MgSO4, filtered, concentrated by rotary evaporator. The residue was suspended in ethyl acetate, filtered, and washed the filter cake with ethyl acetate, to give a light-yellow solid as the first crop of product. The filtrate was concentrated with silica gel to dryness, providing a free-flowing solid, which was dry loaded onto a column of silica gel wet with heptane. The column was sequentially eluted with ethyl acetate: heptane (1:3, 1:2 and 1:1), the resulting product fractions were combined and distilled under vacuum at 35 °C to give the second crop of product as a white solid. EXAMPLE 2 TRIMALEIMIDE LINKER (CORE) SYNTHESIS O O O A

[0075] Step 1: To a dry round bottom flask is maleic anhydride (1 eq.) added and dissolved in 5- 10 mL of tetrahydrofuran. To this mixture is furan (1 eq.) added dropwise at RT while stirring. Post addition the reaction mixture is stirred overnight at RT. During this time, a precipitate may form. After overnight, the reaction mixture is filtered and the precipitate is washed with hexane (3x5mL), collected, and dried in vacuo. The filtrate is heated, cooled to RT, and placed in a freezer to induce recrystallization of the product.

[0076] If no precipitate forms overnight, 15mL of hexane is added to the reaction mixture. The change in polarity may cause product to precipitate out. If precipitate is formed, filter, collect and dry the precipitate in vacuo. If no precipitate generates, subject the mixture to recrystallization as mentioned. Collect the recrystallized product as mentioned before via vacuum filtration. No further purification is necessary.24KU021M-01 61013-PCT O

[0077] in 10 mL of methanol. This mixture is placed in an ice bath and stirred. A solution of tris(2-aminoethyl) amine (1 eq.) in methanol (3 mL) is prepared and added to cold mixture dropwise. After dropwise addition, the resulting mixture is stirred at 0°C for 5 minutes, and then allowed to warm to RT and stirred for 30 additional minutes. After this time, the reaction is heated to reflux (70°C) for 4 hours. This is then allowed to cool to RT slowly and placed in a freezer overnight to induce recrystallization of product. This is then filtered, the solid washed with cold methanol (3x5mL), isolated and dried in vacuo. No further purification is necessary. O O

[0078] Step 3:suspended in 10 mL of toluene. This mixture is heated to reflux (115°C) for overnight. After, the reaction mixture is concentrated, and the product is isolated and further dried in vacuo overnight. After the product is collected and no further purification is necessary. EXAMPLE 3 SOLID PHASE PEPTIDE SYNTHESIS PROTOCOL

[0079] All peptides mentioned in Table 1 below were synthesized following the Fmoc (N-(9- fluorenyl)methoxycarbonyl) solid phase peptide synthesis procedure (Wang H., Udukala D. N., Samarakoon T. N., Basel M. T., Kalita M., Abayaweera G., Manawadu H., Malalasekera A., Robinson C., Villanueva D., Maynez P., Bossmann L., Riedy E., Barriga J., Wang N., Li P.,24KU021M-01 61013-PCT Higgins D. A., Zhu G., Troyer D. L., and Bossmann, S. H. 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 (Gly in this case), was first swelled in dichloromethane (DCM) for 20 minutes and then washed with N,N-dimethylformamide (DMF) for 1 minute. After washing, a solution containing Fmoc-protected amino acid (resin:amino acid, 1:3 molar ratio) and O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) as a coupling agent (resin:HBTU, 1:29 molar ratio) was prepared in a 1:23 diisopropylethylamine (DIEA):DMF solution and was added to the resin and allowed to swirled for 30 minutes for the coupling reaction. Each amino acid was added two times to enhance addition of amino acid to the peptide chain. Before 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 an amino acid is a repeated cycle during peptide synthesis until all amino acids on the peptide sequence chain have been added. After synthesis was completed, some peptides were labeled with cholesterol, cholic acid, or cleaved unlabeled. For cholesterol and cholic acid additions, these were coupled on the N-terminal end of the peptide using 1,1'-Carbonyldiimidazole (CDI) as the coupling reagent. Cholesterol was coupled following 5 additions within 24-hours reaction, while cholic acid was coupled following a total of 7 additions within 24-hours reaction. Cleaving of completed sequences (labeled or unlabeled) is performed using trifluoroacetic acid incubated for 3 hours. After the 3-hr cleaving reaction, peptide is precipitated in cold ether, collected via centrifugation (4,000 xg for 5 min), and washed with cold ether for a total of 4-6 times. Cleaved peptides were then dried and kept under argon at -20°C. S10K20(SEQ ID NO: 3) and D10K20(SEQ ID NO:4) block co-peptides were prepared that included a C-terminal linking amino acid (cysteine) and spacer residue (glycine, from resin synthesis). Table 1. Peptide sequences including block co-peptides synthesized for DCLK1 nanosponges Calculated molecular weight P tid n / n m24KU021M-01 61013-PCT k*GNYTCEVTELSREGKTVIELKCk*G (SEQ ID NO:2) S10K20CG unlabeled (SEQ ID NO:5) 3612.478EXAMPLE 4 NANOSPONGE ASSEMBLY AND COUPLING OF SMALL MOLECULE PROTOCOLS

[0080] The trimaleimide linker used to assemble nanosponges contains three -NH2 groups, serving as the anchor to link up to 3 different peptides, which determined the molar ratio to use for assembly. Nanosponges assembled in the presence of the “Don’t eat me peptide” sequence consisted in a 1:2:1 ratio of linker : cholesterol-block co-peptide : “Don’t eat me peptide”. On the other hand, for nanosponges assembled without the “Don’t eat me peptide,” the ratio consisted in 1:3 linker : cholesterol / cholic acid-block co-peptide. In detail, prior to assembly, 1X Phosphate Buffered Saline (PBS, pH 7.2) and anhydrous DMF were degassed with argon. In separate vials, tris-maleimide linker was dissolved in 10-15 µL of degassed DMF and peptides were dissolved together in 1-2 mL of degassed PBS. Both solutions were then combined and stirred for 24 hours at room temperature under argon atmosphere (deoxygenated). After 24 hours, nanosponge was transferred to a dialysis membrane tubing (3,500 Da MWCO) to carry dialysis against water for 1- 2 hours in order to remove unbound components. Nanosponge was then lyophilized overnight to dry and stored at -20°C.

[0081] Small molecules were coupled to the nanosponges using CDI or HBTU (only for D10K20CG (SEQ ID NO:6) nanosponges) as the coupling reagents. Briefly, for DCLK1 small molecules, 10% (per weight) of each small molecule and CDI were dissolved in anhydrous DMF and stirred for 10 min at room temperature. In a separate vial, nanosponge was dissolved in DMF and solution was transferred to small molecule-CDI reaction solution; reaction was stirred for 24 hours at room temperature. Dialysis was repeated on the nanosponge-small molecule complex for 1 hour to24KU021M-01 61013-PCT remove any unbound components, followed by lyophilization overnight to dry. Table 3 summarizes all compounds assembled for the project. FIG. 1 shows an illustration for a nanosponge building block with small molecules according to Formula 1A coupled to two arms and a signal sequence on the third arm of the trimaleimide linker (core). Table 3. Nanosponge-small molecule assembled for DCLK1 SEQ Don't eat Cholic Small molecule Compound Nanosponge ID Cholesterol me24KU021M-01 61013-PCT IA-DC-123 D10K20CG 6 X No DCLK3A IA-DC-124 D10K20CG 6 X No - control -IN VITRO RESULTS

[0082] FIG. 2 shows that DCLK3 (C-3) and DCLK4 (C-4) inhibit proliferation of colon cancer cell line SW480 cells as assessed by hexosaminidase assay described below.

[0083] FIG. 3 shows that DCLK3 (C-3) and DCLK4 (C-4) inhibit proliferation of colorectal carcinoma cell line HCT116 cells as assessed by hexosaminidase assay described below.

[0084] Compounds were screened using a colorimetric hexosaminidase assay. Briefly, cells are plated in 96 well plates. Cells are then treated with increasing doses of compound for 48 hours. Media was aspirated and cells were incubated with substrate buffer containing p-nitrophenol-N- acetyl-beta-D-glucosaminidase. Plates were read at an absorbance of 405 nm.

[0085] Viability was calculated as a percentage of absorbance of treatment relative to control. The resulting IC50 data is shown in Table 5. Table 5. Nanosponge IC50 Nanosponge Small molecule coupled HUCCT148hr IC50 HUH2848hr IC5024KU021M-01 61013-PCT IA-DC-113 - control - 52 µM 25 µM IA-DC-114 Gemcitabine 1 M 30 M[ ] or co ony orma on, ce s were p a e n -we p a es. e s were en rea e w test articles at IC50, and ½ IC50 doses for 48h. Media is then aspirated and replaced with complete media without drug. Colonies are then allowed to grow for 2 weeks before staining, counting, and size determination using ImageJ software.

[0087] For spheroid formation, cells were plated in 24-well low attachment plates. Spheroids were allowed to form for 72 hours before the addition of test articles at IC50, ½ IC50, and ¼ IC50 doses. Spheroids were allowed to grow for an additional 96 hours before imaging and quantification.

[0088] FIG. 4 shows representative colony formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and ½ IC50 as previously determined by hexosaminidase assay. Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p<0.05.

[0089] FIG. 5 shows representative colony formation images of HUH28 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 for 48h with IC50 and ½ IC50 as previously determined by hexosaminidase assay. Quantification of colony number and size. Data are represented as mean number and size from 3 independent experiments, * p<0.05.

[0090] FIG. 6 shows representative spheroid formation images of HUCCT1 cells treated with Gemcitabine (Gem), IA-DC-103, DCLK3a, IA-DC-125 at IC50, ½ IC50 and ¼ IC50 doses, as24KU021M-01 61013-PCT previously determined by hexosaminidase assay. Quantification of spheroid number. Data are represented as the mean from 3 independent experiments, * p<0.05.

[0091] When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0092] In view of the above, it will be seen that the several objects of the disclosure are achieved and other advantageous results attained.

[0093] As various changes could be made in the above methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

Claims

24KU021M-01 61013-PCT CLAIMS:

1. A DCLK1 inhibitor compound having a structure corresponding to Formula 1 , wherein A is aR1 , R2 , and R3 are each independently selected from the group consisting of -H, -O, -N, - S, -F, -Cl, -OH, -OCH3, NO2, alkyls, and alkaryls, or R1and R2together with the carbons they are attached to form a fused 5- or 6-membered ring with the adjacent 6-membered ring to which R1 and R2 are bound; R4is 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-.

2. The DCLK1 inhibitor compound of claim 1, wherein at least one of J1or J2is -N-.

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

4. The DCLK1 inhibitor compound of any one of claims 1-3, wherein R1 and R2 together with the carbons they are attached to form a fused 6-membered ring, wherein said compound has a structure corresponding to Formula 1A:24KU021M-01 61013-PCT .

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

6. The DCLK1 inhibitor compound of claim 5, wherein A is:24KU021M-01 61013-PCT . is –C(O)-, -NHC(O)-, or -C(O)NH-.

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

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

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

11. The DCLK1 inhibitor compound of any one of claims 1 to 10, wherein when J1is -CH-, J2is -N-, or wherein J1 is -N- and J2 is -CH-.

12. The DCLK1 inhibitor compound of claim 1 or 4, wherein the compound has one of the following structures ,24KU021M-01 61013-PCT ,13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the DCLK1 inhibitor compound of any one of claims 1 to 12.24KU021M-01 61013-PCT 14. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable carrier comprises a sugar (e.g., lactose, glucose, or sucrose); a starch (e.g., corn starch or potato starch); cellulose or a derivative thereof (e.g., sodium carboxymethyl cellulose, ethyl cellulose or cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (e.g., cocoa butter or suppository wax); an oil (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil or soybean oil); a glycol (e.g., propylene glycol); a polyol (e.g., glycerin, sorbitol, mannitol or polyethylene glycol; an ester (e.g., ethyl oleate or ethyl laurate); agar; a buffering agent (e.g., magnesium hydroxide or aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; 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 said DCLK1 inhibitor compound is attached to said nanosponge via cleavable linkages.

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

17. The method of claim 16, wherein the cancer is colon cancer, cholangiocarcinoma, a genitourinary cancer, a gynecologic cancer, a gastrointestinal cancer, lymphoma, melanoma or skin cancer, a head and neck cancer, lung cancer, intestinal cancer, kidney cancer, pancreatic cancer, breast cancer, stomach cancer, leukemia, bone cancer, thyroid cancer, brain cancer, or a glioma.

18. The method of claim 17, wherein the cancer is colon cancer or a cholangiocarcinoma.

19. A medicament for treating cancer, preferably colon cancer or a cholangiocarcinoma in a subject in need thereof, the medicament comprising a therapeutically-effective amount of a DCLK1 inhibitor compound according to any one of claims 1 to 12.24KU021M-01 61013-PCT 20. Use of a DCLK1 inhibitor compound according to any one of claims 1 to 12 to treat cancer in a subject in need thereof.

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