Methods of treating KRAS-related cancers

Abstract

The present invention relates to methods of treating KRAS-related cancers. Disclosed is a method of treating a cancer characterized by the expression of at least one KRAS mutation using a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a prodrug thereof: # imgabs0. Also disclosed is a method of altering the level of KRAS or cMet in a cell characterized by the expression of at least a KRAS mutation with a compound of Formula I.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 20, 2020, application number 202080059528.X, and invention name “Method for treating KRAS-related cancers”. Technical Field

[0002] The present disclosure relates to the use of heterocyclic compounds for treating KRAS-related cancers. Background Art

[0003] The RAS family consists of three members: KRAS, NRAS, and HRAS. KRAS is the single most commonly mutated oncogene in human cancer. In the United States, KRAS mutations are prevalent in cancer cells of patients with any of the three most difficult-to-treat cancer types: 95% of pancreatic cancer, 45% of colorectal cancer, and 35% of lung cancer.

[0004] Because KRAS mutations are prevalent in particularly recalcitrant cancers, a large amount of drug discovery effort has been devoted to developing therapeutic strategies that block KRAS function. These efforts include (i) direct targeting approaches, such as disruption of protein-protein (e.g., RAS-Raf) interactions and covalent irreversible KRAS-G12C inhibition, and (ii) indirect targeting approaches, such as reduction of RAS populations at the plasma membrane and targeting of downstream effector signaling proteins (e.g., ERK or mTOR). Despite these intensive efforts, clinically viable cancer therapies that effectively block KRAS function remain elusive.

[0005] Therefore, there is a need to develop new approaches that can effectively block KRAS function to treat KRAS-related cancers. Summary of the invention

[0006] The present disclosure relates to treating KRAS-related cancers with heterocyclic compound DGD1202 or a pharmaceutically acceptable salt thereof or a prodrug thereof. Unexpectedly, DGD1202 exhibits superior anti-tumor efficacy against certain cancers clearly characterized by the expression of mutant KRAS compared to known EGFR inhibitors.

[0007] One aspect of the present disclosure is a method for treating a cancer characterized by expression of a mutant KRAS protein and optionally a mutant EGFR protein. The method comprises administering to a subject in need thereof an amount sufficient to alter a KRAS-related activity (e.g., KRAS or cMet) caused by the KRAS mutation, wherein the therapeutic agent is a compound of Formula I as shown below (interchangeably referred to as "DGD1202"), or a pharmaceutically acceptable salt thereof, or a prodrug thereof:

[0008]

[0009] In some embodiments, the cancer is a KRAS driven cancer. Examples of KRAS driven cancers include, but are not limited to, pancreatic cancer, colorectal cancer, head and neck cancer, and lung cancer.

[0010] In various embodiments, the cancer is characterized by the presence of at least one deleterious KRAS mutation, which refers to a KRAS mutation that contributes to increased cell proliferation when present in cancer cells. The deleterious KRAS mutation can be one of the following mutations: G12D, G12V, and G13D. The cancer can also be characterized by the presence of one or more of the following EGFR mutations: L858R, T790M, C797S, S768I, del Exon 19, or a combination thereof.

[0011] In an exemplary method, the KRAS mutation is G12D or G13D and the EGFR mutation is L858R or T790M.

[0012] In some embodiments, the cancer is resistant to an EGFR inhibitor (eg, cetuximab or osimertinib).

[0013] In some embodiments, the above therapeutic agent can degrade EGFR or block EGFR dimerization. Generally, the amount of the therapeutic agent administered is sufficient to change the activity of KRAS. The therapeutic agent can be administered (e.g., orally) at a dose of 1 to 500 mg / kg (e.g., 10 to 100 mg / kg, 10 to 60 mg / kg, or 20 to 40 mg / kg).

[0014] Compositions comprising a therapeutic agent and a pharmaceutically acceptable carrier are also disclosed. The carrier in the pharmaceutical composition must be "acceptable" in that it is compatible with the therapeutic agent of the composition (and preferably, capable of stabilizing the therapeutic agent) and is harmless to the subject to be treated. One or more solubilizing agents can be used as pharmaceutical excipients for delivering the therapeutic agent. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

[0015] The above therapeutic agents can be administered to a subject orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted cartridge. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0016] The composition containing the above therapeutic agent for oral administration can be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersants and solutions. In the case of tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are usually added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When an aqueous suspension or emulsion is administered orally, the therapeutic agent (i.e. DGD1202) can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If necessary, certain sweeteners, flavorings or coloring agents can be added. Oral solid dosage forms can be prepared by: spray drying technology; hot melt extrusion strategy, micronization and nano grinding technology.

[0017] Nasal aerosol or inhalation compositions can be prepared according to techniques known in the field of pharmaceutical preparations. For example, benzyl alcohol or other suitable preservatives, absorption enhancers, fluorocarbons and / or other solubilizing agents or dispersants known in the art can be used to prepare such compositions as saline solutions. Compositions containing DGD1202 as therapeutic agents can also be used for rectal administration in the form of suppositories.

[0018] The term "treatment" refers to the application or administration of a therapeutic agent to a subject in order to cure, alleviate, relieve, alter, remedy, improve or affect a disease, symptom or constitution. "Effective amount" or "effective amount" refers to the amount of a compound of Formula I required to impart the desired effect to a subject. As will be appreciated by those skilled in the art, the effective amount varies, depending on the route of administration, the use of excipients, and the possibility of co-use with other therapeutic methods, such as the use of other active agents.

[0019] Another aspect of the present disclosure is a method of altering KRAS levels in a cell characterized by expression of at least a KRAS mutation. The method comprises contacting the cell with a compound of Formula I in an amount effective to degrade EGFR activity in the cell.

[0020] Generally, KRAS-associated cells are characterized by expression of KRAS G12D, KRAS G12V, KRAS G13D, EGFR L858R, EGFR T790M, EGFR C797S, EGFR S768I, EGFR del Exon 19, or a combination thereof.

[0021] Also within the scope of the present disclosure is a method of altering cMet levels in a cell characterized by expression of at least a KRAS mutation, the method comprising contacting the cell with an amount of a compound of Formula I effective to degrade EGFR activity in the cell.

[0022] Similarly, cMet-associated cells are often characterized by expression of KRAS G12D, KRAS G12V, KRAS G13D, EGFR L858R, EGFR T790M, EGFR C797S, EGFR S768I, EGFR del Exon 19, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Attached Figure 1 Effects of DGD1202 in a mutant KRAS (and EGFR co-driven) head and neck (UMSCC74B) tumor model are depicted. KRAS-driven UMSCC74B xenograft-bearing nude mice were treated with vehicle, cetuximab (100 mg / kg, Monday) or DGD1202 (30 mg / kg, Monday-Friday) for 2 weeks, and the effects on tumor growth were measured and plotted.

[0024] Figure 2 Effect of DGD1202 KRAS mutant cell lines HCT116 and LoVo are depicted. The effect of DGD1202 (left line in each graph) was assessed in KRAS mutant colorectal cell lines (HCT116, Lovo) compared to cetuximab (right line in each graph) using a clonogenic assay.

[0025] Figure 3 The effects of DGD1202 on EGFR, KRAS, and downstream signaling are depicted. A HCT-116 (colorectal cancer) cells were treated with DGD1202 for 5 or 15 hours, and whole-cell lysates were probed with the listed antibodies. B The effects of DGD1202 on a KRAS G12D-driven pancreatic cell line (Panc1) tumor model were assessed; the effects of treatment on steady-state levels of EGFR and mtKRAS were determined by immunoblotting; and GAPDH expression was assessed as a loading control.

[0026] Figure 4 Shown are the effects of DGD1202 on pancreatic intraepithelial neoplasia (PanIns) in mutant KRAS mice compared to controls.

[0027] Figure 5 The effect of DGD1202 on osimertinib-resistant NCI-H1975 lung cancer xenografts is depicted. A SCID mice bearing NCI-H1975 osimertinib-resistant xenografts were treated with DGD1202 (75 mg / kg, daily, PO). The arrow marks the start of DGD1202 treatment. The changes in tumor volume are plotted. B The effect of DGD1202 treatment on EGFR was assessed in tumors harvested 3 days after DGD1202 treatment.

[0028] Figure 6 Shown are the percentage of lung damage and total lung damage in genetically engineered KRAS-LSL-G12D mice administered DGD1202 compared to controls. DETAILED DESCRIPTION

[0029] Disclosed herein first in detail is a method of using the disclosed therapeutic agents to treat a cancer characterized by expression of at least a KRAS mutation.

[0030] KRAS plays an important role in EGFR-induced signaling pathways. For example, see Knickelbein et al., Genes & Diseases, 2015, 2, 4-12 ("Knickelbein"). As reported by Knickelbein, the activation of EGFR upon ligand binding and its subsequent autophosphorylation creates a docking site for the SOS / GRB2 complex, resulting in nucleotide exchange between SOS and the GTP-bound form of KRAS; subsequently, KRAS promotes cell growth and inhibits apoptosis through RAF / MEK / ERK and PI3K / AKT cascade signals. Therefore, anti-EGFR antibodies including cetuximab and panitumumab bind to EGFR and prevent ligand binding and subsequent KRAS activation, which leads to growth inhibition and cell death due to inhibition of the RAF / MEK / ERK and PI3K / AKT pathways. See Knickelbein, first paragraph, page 6. On the other hand, mutant KRAS can override the effects of anti-EGFR antibodies that lead to cell growth and survival. See Id.

[0031] As noted above, the present disclosure provides a method of treating a cancer characterized by at least a KRAS mutation.

[0032] More specifically, the method comprises administering to a subject in need thereof an amount of a therapeutic agent sufficient to alter KRAS-related activity (e.g., KRAS or cMet) caused by the KRAS mutation, wherein the therapeutic agent is a compound of Formula I as shown below, or a pharmaceutically acceptable salt thereof, or a prodrug thereof:

[0033]

[0034] As described above, cancer is characterized by the presence of at least one deleterious KRAS mutation and optionally one or more EGFR mutations. The term "deleterious KRAS mutation" herein refers to a KRAS mutation that, when present in a cancer cell, contributes to an increase in cell proliferation. Examples of deleterious KRAS mutations include, but are not limited to, G12D, G12V, and G13D. Examples of EGFR mutations include, but are not limited to, L858R, T790M, C797S, S768I, and del Exon 19. In one embodiment, the deleterious KRAS mutation is G12D or G13D, and the EGFR mutation is L858R or T790M.

[0035] Compared with similar structural analogs, the above compounds show significant improvements in pharmacological properties and biological activities. For example, DGD1202 shows better liver microsomal stability compared with the other two similar structural compounds as shown below:

[0036]

[0037] DGD1202 is stable, has a hepatic microsomal half-life of more than 46 minutes, is soluble in water at pH 3.5, is bioavailable by systemic injection and oral administration, and has a submicromolar IC in a clonogenic cell assay 50 Importantly, this compound degrades EGFR rather than inhibiting its activity. It also blocks EGF-induced EGFR dimerization and binds directly to purified EGFR and is selectively active in EGFR-driven osimertinib-resistant cell lines and in xenograft models.

[0038] In order to further study the anti-tumor activity of the compound of formula I (ie, DGD1202), NCI-60 cell line screening was performed. The data from this experiment (see the table in Example 3 below) showed that the compound was active not only against tumor cells driven by EGFR, but also against a variety of cells expressing mutant KRAS and showing resistance to EGFR targeting antibodies (such as cetuximab).

[0039] IC of DGD1202 against these cetuximab-resistant cell lines 50 The values ​​ranged from 0.5 μM to 2.2 μM. In immortalized cells of non-cancer origin, the IC 50 The values ​​were much higher than 20 μM.

[0040] In addition, DGD1202 showed activity against mutant KRAS-positive cell lines and in a transgenic mouse model in which KRAS G12D is expressed in the pancreas leading to the formation of pancreatic intraepithelial neoplasia (PanIns). In addition, it also showed activity in a KRAS-mutant cetuximab-resistant head and neck tumor model (UMSCC74B).

[0041] Cetuximab (Erbitux, an anti-EGFR antibody) has been shown to be beneficial for patients with colorectal cancer (CRC). However, only a subset of WT-EGFR patients show durable clinical responses. Patients with KRAS mutations generally do not respond to treatment with cetuximab to inhibit EGFR kinase activity. Recent studies by different groups using preclinical models have shown that degradation of oncoproteins is more effective than inhibition of kinase activity. For example, see Raina et al., Proc Natl Acad Sci USA., 2016, 113: 7124-7129; and Corcoran et al., Cancer Discovery, 2012, 2: 227-235. This may be because the EGFR protein scaffold may continue to function by interacting with other proteins even when its kinase activity is inhibited. An important advantage of degrading oncoproteins (over-inhibiting their activity) is that it is conceivable that resistance mediated by acquired mutations is expected to be lower. In addition to acquired mutations, even patients who initially respond to EGFR inhibitor treatment may develop resistance due to upregulation of compensatory signals. In CRC, the EGFR-KRAS axis is known to activate MEK-ERK signaling, thereby promoting cancer progression, which in turn leads to primary and secondary resistance to existing kinase inhibitor therapies. In KRAS-mutated CRC tumors, EGFR continues to signal through the ERK pathway even when the ERK pathway is inhibited, leading to resistance to EGFR inhibitor therapy. Therefore, the method of using the compounds of Formula I to treat cancers characterized by at least a KRAS mutation provides unexpected advantages over known treatment methods.

[0042] In order to implement the above-mentioned treatment methods, a pharmaceutically acceptable salt of the compound of formula I can be used. As used herein, the term "pharmaceutically acceptable salt" refers to those salts suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of reasonable medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the above-mentioned compounds include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or organic acids (such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid) or by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts of compounds containing carboxylic acids or other acidic functional groups can be prepared by reaction with a suitable base. Such salts include, but are not limited to, alkali metals, alkaline earth metals, aluminum salts, ammonium, N + (C 1-4The present invention also contemplates the quaternization of any basic nitrogen-containing group of the compounds provided herein. Water-soluble or oil-soluble or dispersible products can be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium and amine cations formed when appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Exemplary pharmaceutically acceptable salts of compounds of Formula I include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, methanesulfonates, ethanesulfonates, isethionates, toluenesulfonates, naphthylsulfonates, benzenesulfonates, acetates, propionates, maleates, benzoates, salicylates, fumarates, glutamates, aspartates, citrates, lactates, succinates, tartrates, glycolates, caproates, caprylates, caprates, oleates, stearates, pamoates and polystyrenesulfonates.

[0043] In some embodiments, prodrugs of the compound of formula I (DGD1202) can also be used. As used herein, the term "prodrug" refers to a drug or compound that is metabolized (i.e., converted in vivo) into a pharmacologically active drug after administration. Therefore, the prodrug of DGD1202 refers to a drug or compound that is converted into DGD1202 in vivo after administration. For technicians, the corresponding prodrug can be used instead to improve the absorption, distribution, metabolism and excretion of the drug, thereby improving the pharmacological effect played in a subject (e.g., a person) in need. Examples of prodrugs that can be used in the present invention include, but are not limited to, amides, carbamates, sulfonamides, formamides, N-oxides, N-acyloxyalkyl derivatives, N-hydroxyalkyl derivatives, and N-(phosphoryloxy)alkyl derivatives.

[0044] A general synthesis of compounds of Formula I is provided below:

[0045]

[0046] Typically, acetamide B (1 equivalent) is added to a solution of 3-(4-bromophenyl)-8-methyl-1,4,8-triazaspiro[4.5]dec-3-ene-2-thione A in an organic solvent (e.g., anhydrous acetonitrile). The reaction mixture is heated to a predetermined temperature (e.g., 40° C.). Next, a base (e.g., 2M aqueous potassium carbonate, 1 equivalent) is added to the reaction mixture. The reaction is maintained at the same temperature until TLC shows loss of starting material and the appearance of new R f Once the reaction was complete by TLC, it was worked up and the resulting crude compound was purified by flash chromatography to provide the desired product: (2-((3-(4-bromophenyl)-8-methyl-1,4,8)-triazaspiro[4.5]dec-1,3-dien-2-yl)thio)-N-(quinolin-3-yl)acetamide).

[0047] The synthesis methods of intermediates A and B used to prepare compounds of formula I are well known in the art. For example, see R. Larock, Comprehensive Organic Transformations (2nd edition, VCH Publishers 1999); PGM Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis (4th edition, John Wiley and Sons 2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2nd edition, John Wiley and Sons 2009); P. Roszkowski, JK Maurin, Z. Czarnocki "Enantioselective synthesis of (R)-(–)-praziquantel (PZQ)" Tetrahedron: Asymmetry 17(2006)1415-1419; and L. Hu, S. Magesh, L. Chen, T. Lewis, B. Munoz, L. Wang "Direct inhibitors of keap1-nrf2 interaction as antioxidantinflammation modulators," WO2013 / 067036.

[0048] The compounds of Formula I described herein or their pharmaceutically acceptable salts (referred to as therapeutic agents) can be administered to a subject in a therapeutically effective amount (e.g., an amount sufficient to prevent or alleviate the symptoms of a disease or condition associated with a KRAS mutation). The therapeutic agent can be administered alone or as part of a pharmaceutically acceptable composition. In addition, the therapeutic agent can be administered all at once, multiple times, or delivered substantially uniformly over a period of time.

[0049] It will be appreciated by the skilled person that the dosage of the therapeutic agent can vary over time. The specific dosing regimen for a particular subject will depend in part on the compound, the amount of the compound administered, the route of administration, and the cause and extent of any side effects. The amount of the compound administered to a subject (e.g., a mammal, such as a human) according to the disclosure should be sufficient to affect the desired reaction within a reasonable time frame. The dosage generally depends on the route, time, and frequency of administration. Therefore, the clinician titrates the dosage and changes the route of administration to obtain the best therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art.

[0050] By way of illustration only, the above-described method of treating cancer includes administering, for example, about 1 mg / kg to about 500 mg / kg of a compound of formula I, depending on the above-mentioned factors. In other embodiments, the dosage range may be from 5 mg / kg to about 100 mg / kg; or from 10 mg / kg to about 100 mg / kg, or from 10 mg / kg to about 60 mg / kg, or from 20 mg / kg to about 40 mg / kg. Some conditions require extended treatment, which may or may not result in multiple administrations followed by administration of a lower dose of the compound. If necessary, the dose of the compound is administered in two, three, four, five, six or more sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. The treatment period will depend on the specific circumstances and may last from one day to several months.

[0051] The suitable administration method of the pharmaceutically acceptable composition comprising the compound of formula I is well known in the art. Although more than one approach can be used to administer the compound, a specific approach can provide a more direct and more effective reaction than another approach. Depending on the circumstances, the pharmaceutical composition comprising the compound is administered or instilled into the body cavity, absorbed by the skin or mucous membrane, ingested, sucked and / or introduced into the circulation. For example, in some cases, it is desirable to deliver the pharmaceutical composition comprising the therapeutic agent orally by injection or in one of the following ways: intravenous, intraperitoneal, intracerebral (in the substance), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, urethral, ​​vaginal or rectal. The compound can be administered by a sustained release system or an implant.

[0052] For the convenience of administration, in various aspects, the therapeutic agent is formulated into a pharmaceutically acceptable composition comprising a carrier (e.g., vehicle, adjuvant or diluent). The specific carrier used is limited by chemical-physical considerations (such as solubility and insufficient reactivity with the compound) and the route of administration. Pharmaceutically acceptable carriers are well known in the art. Illustrative drug forms suitable for injection purposes include sterile aqueous solutions or dispersions and sterile powders for temporary preparation of sterile injection solutions or dispersions (e.g., see U.S. Patent No. 5,466,468). Injectable compositions are further described in the following documents: for example, "Pharmaceutics and Pharmacy Practice", JB Lippincott Publishing Company (JB Lippincott Co.), Pennsylvania. Pa., Banker and Chalmers, pp. 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pp. 622-630 (1986)). On the one hand, a pharmaceutical composition comprising a therapeutic agent is placed in a container together with packaging material that provides instructions for the use of such pharmaceutical composition. Typically, such instructions include a tangible expression describing the concentration of the reagent, and the relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be needed to reconstruct the pharmaceutical composition in certain embodiments.

[0053] Compositions suitable for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstructing as sterile injection solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, suitable fluidity can be maintained.

[0054] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Microbial contamination may be prevented by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. The absorption of injectable pharmaceutical compositions may be prolonged by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0055] Solid dosage forms for oral administration include capsules, tablets, powders and granules. In such solid dosage forms, the therapeutic agent is mixed with: at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, such as starch, lactose, sucrose, mannitol and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrants, such as agar-agar, calcium carbonate, potato tapioca starch, alginic acid, certain complex silicates and sodium carbonate; (e) solution retarders, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin and bentonite; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate or mixtures thereof. In the case of capsules and tablets, the dosage form may also contain buffering agents.Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0056] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the art. Solid dosage forms may also contain opacifiers. In addition, solid dosage forms may be embedded compositions that release the therapeutic agent in a delayed manner in a specific portion of the intestinal tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The compound of formula I may also be in microcapsule form, optionally containing one or more excipients.

[0057] Oral liquid dosage forms include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the therapeutic agent, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (specifically cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, or mixtures of these substances, etc.

[0058] In addition to such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and perfuming agents. In addition to the therapeutic agent, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, or mixtures of these substances, and the like.

[0059] After formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The composition is easily administered in a variety of dosage forms (such as injectable solutions, drug release capsules, etc.). For example, for parenteral administration in aqueous solution, if necessary, the solution should be appropriately buffered and the liquid diluent first made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

[0060] Formula I compounds can modulate EGFR in a unique manner. In some embodiments, the compounds block or inhibit EGFR dimerization. In various embodiments, the compounds induce EGFR degradation.

[0061] Although EGFR has been clearly identified as an important molecular target in oncogenes and cancers, there is still a great need and opportunity for improved methods to regulate the activity of the oncogene. By using cell-penetrating peptides that block dimerization (interferon) or siRNA, it has been shown that EGFR degradation has a profound effect on cell survival, even in TKI-resistant cells. For example, see Raina et al., Proc Natl Acad Sci USA., 2016, 113: 7124-7129. By degrading EGFR protein rather than simply inhibiting its kinase activity, a wide range of activity has been demonstrated in preclinical models, while because the agent only affects the EGFR combined with EGF (compared with normal tissue, it is abundant in tumor cells), thereby improving the ability to target tumor tissue, thereby improving safety and treatment range.

[0062] Approaches that degrade EGFR rather than simply inhibit its kinase activity overcome the resistance to osimertinib that develops in patients with non-small cell lung cancer. See, for example, Corcoran et al., Cancer Discovery, 2012, 2:227-235. Although the application of EGFR degradation has focused on lung cancer, additional important clinical opportunities exist in other cancers driven by EGFR or KRAS, such as head and neck cancer, colorectal cancer, and pancreatic cancer.

[0063] As used herein, the term "treatment" and words related thereto do not necessarily imply 100% or complete treatment. On the contrary, there are different degrees of treatment, one of which is considered by a person of ordinary skill in the art to have a potential benefit or therapeutic effect. In this regard, the method for treating cancer disclosed herein can provide any amount or any level of cancer treatment. In addition, the treatment provided by the method disclosed herein can include treating one or more symptoms or symptoms of the cancer being treated. Moreover, the treatment provided by the method disclosed herein can cover slowing the progression of cancer. For example, the disclosed method can treat cancer by reducing the growth of tumors or cancers, reducing the metastasis of tumor cells, increasing cell death of tumors or cancer cells, etc.

[0064] Cancers treatable by the methods disclosed herein can be any KRAS-associated cancer or KRAS-driven cancer characterized by at least a KRAS mutation.

[0065] In some aspects, the cancer is one selected from pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), ovarian cancer, cervical cancer, gastric cancer, breast cancer, hepatocellular carcinoma, glioblastoma, liver cancer, malignant mesothelioma, melanoma, multiple myeloma, prostate cancer, and kidney cancer. In some embodiments, the cancer is pancreatic cancer, colorectal cancer, head and neck cancer, or lung cancer. In some embodiments, the cancer is cetuximab-resistant cancer or osimertinib-resistant cancer.

[0066] Still within the scope of the present disclosure is a method of altering the level of KRAS or cMet in a cell characterized by expression of at least a KRAS mutation. The method comprises contacting the cell with an amount of a compound of Formula I effective to degrade EGFR activity in the cell.

[0067] Typically, KRAS or cMet-related cells are characterized by the presence of at least one KRAS mutation and optionally one or more EGFR mutations. Examples of KRAS mutations include, but are not limited to, G12D, G12V, and G13D. Examples of EGFR mutations include, but are not limited to, L858R, T790M, C797S, S768I, and del Exon 19. In some embodiments, the KRAS mutation is G12D or G13D, and the EGFR mutation is L858R or T790M.

[0068] In some embodiments, the disclosed methods of administering a compound of Formula I result in degradation of EGFR or blockade of EGFR dimerization, thereby altering the level of KRAS or cMet in the cell.

[0069] Without further elaboration, it is believed that those skilled in the art can fully utilize the present disclosure based on the above description. Therefore, the following specific embodiments (ie, embodiments 1 to 5) should be interpreted as merely illustrative and not limiting the rest of the present disclosure in any way.

[0070] Example

[0071] Example 1 - Synthesis and characterization of DGD1202

[0072]

[0073] To a solution of 3-(4-bromophenyl)-8-methyl-1,4,8-triazaspiro[4.5]dec-3-ene-2-thione A in anhydrous acetonitrile was added acetamide C (1 eq). The reaction mixture was warmed to 40°C. Next, 2M aqueous potassium carbonate solution (1 eq) was added to the reaction mixture. The reaction was maintained at 40°C until TLC showed loss of starting material and the appearance of new R f point (usually 2 to 6 hours). Once the reaction is completed by TLC, it is post-processed. The crude reaction mixture is poured into a separatory funnel, and ethyl acetate and water are added. The organic layer is separated and then washed with brine (1x). The organic layer is then dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to obtain a crude product. The crude product is purified by flash chromatography and eluted in 10% methanol / dichloromethane solution to obtain DGD1202: (2-((3-(4-bromophenyl)-8-methyl-1,4,8-triazaspiro[4.5]dec-1,3-diene-2-yl)thio)-N-(quinolin-3-yl)acetamide). 1 H NMR (400MHz, DMSO-d6) δ10.89(br s,1H),8.95(s,1H),8.67(s,1H),7.96(br d,J=8.33Hz,1H),7.92(br d,J=8.33Hz,1H),7.78-7.87(m,4H),7.52-7.72(m,2H),4.29(s,2H),2.43-2.52(m,4H),2.18(brs,3H),1.55-1.85(m,4H); MS(ESI+m / z 523.05, ESI-m / z 521.00); TLC: (90:10:0.5, DCM:MeOH:NH4OH)R f =0.47.

[0074] Example 2 - Evaluation of DGD1202 in KRAS mutant head and neck cancer

[0075] A study was conducted to evaluate the efficacy and toxicity of DGD1202 in a KRAS G12D-driven, cetuximab-resistant head and neck cancer tumor model (UMSCC74B) Figure 1 ).

[0076] Tumor-bearing mice were treated with DGD1202 at a dose of 30 mg / kg every two weeks for one week by oral gavage. This dose was selected based on its single-dose PK profile. The final effect of DGD1202 on tumor volume was compared with control mice that did not receive the test compound and control mice that received the known EGFR inhibitor cetuximab.

[0077] Unexpectedly, this treatment dose and schedule was safe, and mice treated with DGD1202 showed significant tumor growth inhibition (P < 0.001), in stark contrast to cetuximab which failed to show meaningful efficacy.

[0078] Example 3 - Evaluation of DGD1202 in KRAS mutant colorectal and pancreatic cancer cell lines

[0079] A study was conducted to evaluate the efficacy of DGD1202 in a KRAS G13D-driven cetuximab-resistant colorectal cell line (HCT-116) using a clonogenic survival assay and in a pancreatic cancer cell line (Panc1) containing a KRAS G12D mutation ( Figure 3 B) activity.

[0080] More specifically, cells were plated at clonal density in 60 or 100 mm dishes in triplicate one day before treatment with a range of concentrations (e.g., 0 to 10 μM). Eight to twelve days later, cells were fixed with acetic acid / methanol (1:7, v / v), stained with crystal violet (0.5%, w / v), and counted using a stereomicroscope. Drug cytotoxicity (surviving drug-treated cells) was measured and normalized to the survival of untreated control cells.

[0081] Figure 2 and Figure 3 The data shown in B indicate that DGD1202 is effective in treating mutant KRAS-driven colorectal and pancreatic cancer cells. It is hypothesized that the anti-tumor efficacy is due to the loss of EGFR protein, which blocks the reactivation of ERK / AKT signaling, most likely through EGFR.

[0082] The effects of DGD1202 on EGFR, ERK, and AKT were also evaluated by immunoblotting. Preliminary data of DGD1202 in mutant KRAS-driven HCT-116 (CRC) and Panc1 (pancreatic) cell lines are available in Figure 3 A and Figure 3 Shown in B. It was observed that the compound affected EGFR and mtKRAS (Panc1) levels (detected by KRAS G12D specific antibody in case of Panc1 cell line and by PAN-RAS antibody in case of HCT116), which in turn affected downstream signaling in pAKT and pERK.

[0083] Perform immunoblotting according to the following protocol:

[0084] The cells were plated at 3 × 10 5 The density of cells was seeded in a 60 mm culture dish and cultured overnight or to 70% confluence. Cells were treated with vehicle (DMSO) or DGD1202 and then harvested at different time points. The precipitate was washed twice with ice-cold PBS and resuspended in lysis buffer for 30 minutes. After sonication, the particulate material was removed by centrifugation at 13000 rpm for 10 minutes at 4 ° C. The soluble protein portion was heated to 95 ° C in 5 minutes and then applied to a 4-12% Bis-Tris precast gel (Invitrogen) and transferred to a PVDF membrane. At room temperature, the membrane was incubated in a blocking buffer consisting of 5% BSA and 1% normal goat serum in Tris buffered saline for 1 hour (137mM NaCl, 20mM Tris-HCl (pH 7.6), 0.1% (v / v)). The membrane was then incubated with a primary antibody in a blocking buffer at 4 ° C overnight, washed, and incubated with a secondary antibody coupled to horseradish peroxidase for 1 hour. After washing three more times in Tris-buffered saline, bound antibodies were detected by enhanced chemiluminescence plus reagent. To quantify relative protein levels, immunoblot membranes were scanned and analyzed using Image J 1.32j software. The relative protein levels shown represent comparisons to untreated controls.

[0085] At this point, DGD1202 was tested for activity against 60 different human tumor cell lines at the National Cancer Institute using the standard NCI 60 screening protocol. The table below gives the percentage of growth inhibition for the best performing cell lines. The cell lines with the highest response (e.g., HCT-116) displayed mutations in the KRAS gene (e.g., KRAS G13D), which are frequently mutated in CRC and pancreas and are associated with cetuximab resistance.

[0086] Group Cell lines Growth percentage BRAF KRAS Melanoma SK-MEL-5 -96.2 BRAF V600E Colon cancer HCT-116 -90.7 KRAS G13D Melanoma M14 -83.8 BRAF V600E Kidney cancer 786-0 -81.7 Melanoma UACC-62 -78.9 BRAF V600E Melanoma LOX IMVI -78.5 BRAF V600E Colon cancer COLO 205 -76.2 BRAF V600E Melanoma MALME-3M -75.5 BRAF V600E Melanoma SK-MEL-28 -70.1 BRAF V600E Colon cancer HT29 -67.1 BRAF V600E leukemia K-562 -61.5 Melanoma UACC-257 -61.2 BRAF V600E Colon cancer HCC-2998 -51.3 Breast cancer MDA-MB-468 -43.8 Breast cancer MCF7 -42.7 leukemia HL-60(TB) -40.7 NRAS p.Q61L Breast cancer MDA-MB-231 / ATCC -40.5 BRAF G464V KRAS G13D

[0087] Example 4 - Evaluation of DGD1202 in KRAS mutant pancreatic cancer

[0088] A study was conducted to evaluate the efficacy of DGD1202 in a KRAS G12D-driven pancreatic tumor model.

[0089] More specifically, 6-week-old KC mice were treated with DGD1202 by oral gavage (30 mg / kg body weight per day) for 5 weeks. The final effect on PanIn (pancreatic intraepithelial neoplasia) levels was observed compared to control mice that did not receive DGD1202. All mice were sacrificed at week 11 and the effect of PanIn was scored, as Figure 4 Mice treated with DGD1202 showed a significantly reduced tendency to develop PanIn, a type of pancreatic duct injury.

[0090] These data suggest that DGD1202 exhibits unexpected efficacy in treating pancreatic cancer driven by mutant KRAS.

[0091] Example 5 - Evaluation of DGD1202 in EGFR-mutated lung cancer

[0092] A study was conducted to evaluate the efficacy of DGD1202 in mutant EGFR-driven lung cancer models ( Figure 5 ).

[0093] Briefly, SCID mice bearing locally advanced NCI-H1975-AZR (osimertinib-resistant) xenografts were treated with DGD1202 (75 mg / kg) by oral gavage daily for two weeks. Tumor volume was measured using a caliper at least three times a week. The effect of DGD1202 treatment on EGFR expression was determined by IHC staining 3 days after the start of treatment.

[0094] Figure 5 The data presented in the present study demonstrate that DGD1202 has unexpected efficacy in treating mutant EGFR-driven lung cancer that is resistant to osimertinib.

[0095] Example 6 - Evaluation of DGD1202 in a genetically engineered KRAS-LSL-G12D mouse lung cancer model

[0096] The efficacy of DGD1202 treatment on the development and progression of KRAS mutant lung tumors was evaluated. A genetically engineered KRAS-LSL-G12D mouse lung cancer model was used. The mouse carries a Lox-Stop-Lox (LSL) sequence followed by a KRAS G12D point mutation allele. Tumorigenesis was initiated by intranasal delivery of adenoviral-Cre particles (1.5x10e 7 pfu) that transduced lung epithelial cells to express Cre recombinase, which deleted the LSL cassette and allowed expression of the mutant KRAS oncoprotein. These mice exhibited lung hyperplasia at 6 to 8 weeks of age and adenomas at 12 weeks of age.

[0097] Eight weeks after viral delivery, mice were treated with vehicle or DGD1202 at 30 mg / kg by oral gavage for 4 weeks on a Monday to Friday schedule. When mice were 20 weeks old, lungs were harvested and H&E sections were evaluated morphologically. The histopathological spectrum of lung lesions was recorded, and alveolar or bronchial hyperplasia, hyperplastic pattern, adenoma, focal dysplasia, bronchial dysplasia, adenoma, and atypical adenomatous hyperplasia were recorded in a double-blind fashion. The percentage of lung lesions in each lobe and the total area of ​​lung lesions were scored in each sample for both treatment groups and are shown in Table 1. Figure 6 The paired t-test showed that the difference between the two cohorts tended to be significant (p = 0.0577).

[0098] Other embodiments

[0099] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0100] In addition, through the above description, those skilled in the art can easily determine the essential characteristics of the present disclosure, and without departing from its spirit and scope, various changes and modifications can be made to the present disclosure to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

Claims

1. A method of treating a cancer characterized by expression of at least a KRAS mutation, the method comprising administering to a subject in need thereof an amount of a therapeutic agent sufficient to alter KRAS or cMet activity caused by the KRAS mutation, wherein the therapeutic agent is a compound of the following formula I, or a pharmaceutically acceptable salt thereof, or a prodrug thereof:

2. The method of claim 1, wherein the cancer is a KRAS driven cancer.

3. The method of claim 2, wherein the cancer is selected from the group consisting of pancreatic cancer, colorectal cancer, head and neck cancer, and lung cancer.

4. The method of any one of claims 1 to 3, wherein the cancer is characterized by expression of: at least one KRAS mutation selected from G12D, G12V, and G13D; and optionally an EGFR mutation selected from L858R, T790M, C797S, S768I, del Exon 19, and combinations thereof.

5. The method of claim 4, wherein the KRAS mutation is G12D or G13D.

6. The method of claim 4 or 5, wherein the EGFR mutation is L858R or T790M.

7. The method of any one of claims 1 to 6, wherein the therapeutic agent is administered in an amount sufficient to alter the activity of the KRAS.

8. The method of any one of claims 1 to 7, wherein the cancer is resistant to an EGFR inhibitor.

9. The method of claim 8, wherein the EGFR inhibitor is cetuximab or osimertinib.

10. The method of any one of claims 1 to 9, wherein the therapeutic agent degrades EGFR.

11. The method of any one of claims 1 to 10, wherein the therapeutic agent blocks EGFR dimerization.

12. The method of claim 8, wherein the cancer is a KRAS driven cancer.

13. The method of claim 12, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, head and neck cancer, and lung cancer.

14. The method of claim 8, wherein the cancer is characterized by expression of: at least one KRAS mutation selected from G12D, G12V, and G13D; and optionally an EGFR mutation selected from L858R, T790M, C797S, S768I, delExon 19, and combinations thereof.

15. The method of claim 14, wherein the KRAS mutation is G12D or G13D.

16. The method of claim 14, wherein the EGFR mutation is L858R or T790M.

17. The method of any one of claims 1 to 16, wherein the therapeutic agent is administered at a dose of 1 to 500 mg / kg.

18. The method of claim 17, wherein the therapeutic agent is administered at a dose of 20 to 40 mg / kg.

19. The method of any one of claims 1 to 18, wherein the therapeutic agent is administered orally.

20. The method of claim 1, wherein the cancer is resistant to an EGFR inhibitor and is characterized by expression of: at least one KRAS mutation selected from G12D, G12V, and G13D; and optionally an EGFR mutation selected from L858R, T790M, C797S, and combinations thereof.

21. The method of claim 20, wherein the cancer is a KRAS driven cancer and the therapeutic agent is administered orally at a dose of 1 to 500 mg / kg.

22. The method of claim 21, wherein the cancer is selected from pancreatic cancer, colorectal cancer, head and neck cancer, and lung cancer, and the therapeutic agent is administered orally at a dose of 20 to 40 mg / kg.

23. The method according to any one of claims 1 to 22, wherein the therapeutic agent is a compound of formula (I) or a pharmaceutically acceptable salt thereof.

24. A method of altering KRAS levels in a cell characterized by expression of at least a KRAS mutation, the method comprising contacting the cell with an amount of a compound effective to degrade EGFR activity in the cell, wherein the compound is a compound of Formula I below, or a pharmaceutically acceptable salt thereof:

25. The method of claim 24, wherein the cells are characterized by expression of: at least one KRAS mutation selected from G12D, G12V, and G13D; and optionally an EGFR mutation selected from L858R, T790M, C797S, S768I, delExon19, and combinations thereof.

26. The method of claim 25, wherein the KRAS mutation is G12D or G13D.

27. The method of claim 25 or 26, wherein the EGFR mutation is L858R or T790M.

28. The method of any one of claims 24 to 27, wherein the compound degrades EGFR.

29. The method of any one of claims 24 to 28, wherein the compound blocks EGFR dimerization.

30. A method of altering cMet levels in a cell characterized by expression of at least a KRAS mutation, the method comprising contacting the cell with a compound in an amount effective to degrade EGFR activity in the cell, wherein the compound is a compound of Formula I below, or a pharmaceutically acceptable salt thereof:

31. The method of claim 30, wherein the cells are characterized by expression of: at least one KRAS mutation selected from G12D, G12V, and G13D; and optionally an EGFR mutation selected from L858R, T790M, C797S, S768I, delExon19, and combinations thereof.

32. The method of claim 31 , wherein the KRAS mutation is G12D or G13D.

33. The method of claim 31 or 32, wherein the EGFR mutation is L858R or T790M.

34. The method of any one of claims 30 to 33, wherein the compound degrades EGFR.

35. The method of any one of claims 30 to 34, wherein the compound blocks EGFR dimerization.

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