Treatment of cancer with isoindolinone-based protacs

Abstract

Provided herein are methods of treating particular cancers (pancreatic ductal adenocarcinoma, non-small cell lung cancer, and colorectal cancer) comprising a compound of Formula (I), (I): or a pharmaceutically acceptable salt thereof.

Description

[0001] ARVN0199WO2; ARVN-199-PCT

[0002] TREATMENT OF CANCER WITH ISOINDOLINONE-BASED PROTACS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to and the benefit of U.S. Provisional Application Ser. No. 63 / 714,700, filed October 31 , 2024, the contents of which are hereby incorporated by reference in their entirety.

[0005] BACKGROUND

[0006] The Kirsten rat sarcoma (KRAS) gene is an oncogene encoding the KRAS protein, which is a small GTPase signal transduction protein. Several other highly similar Ras homologs are also in this protein family, including HRAS and NRAS (Signal Transduct. Target Ther. 2023, 8, 212). Ras proteins associate with the plasma membrane and act as switches in the transduction of extracellular signals to intracellular response by cycling between an ON state (bound to GTP) and an OFF state (bound to GDP), thereby regulating cellular processes.

[0007] In its ON state, KRAS binds to multiple effector proteins, including B / CRAF and PI3K, and activates the downstream signaling cascades. In this way, KRAS regulates cellular processes including proliferation, survival, and differentiation. In the case of wild-type (WT) KRAS, the protein exists primarily in the OFF state and is only activated transiently through multiple mechanisms such as receptor tyrosine kinase (RTK) activation. In contrast to the WT protein, multiple point mutations in KRAS lead to the protein being locked in the ON state and signaling through the downstream pathways becomes constitutive, leading to aberrant cell growth (Cold Spring Harb. Perspect. Med. 2018, 8, a031518; Signal Transduct. Target. Ther. 2021 , 6, 386; Cancers 2022, 14, 2837).

[0008] Numerous activating or gain-of-function mutations of the KRAS gene are known, and in fact, KRAS 'is the most frequently mutated gene in cancer. Specifically, KRAS is altered in 20- 25% of all instances of cancer (Cancer Res. 2020, 80, 2969; npj Precis. One. 2022, 6, 91). Mutation hotspots exist at codons 12, 13, and 61 , with the highest prevalence occurring at codon 12 (J. Cell Sci. 2016, 129, 1287). Of all mutations in KRAS, the G12D mutation is the most common, and is found in highest prevalence in pancreatic cancer, colorectal cancer (CRC), and lung cancer (Nat. Commun. 2021 , 12, 1808; npj Precis. One. 2022, 6, 91 ).

[0009] Mutation in the KRAS gene is often the initiating event in the formation of pancreatic cancer (Semin. Oncol. 2021 , 48, 10) and an early event in CRC (N. Engl. J. Med. 2009, 361 , 2449; Cancer Lett. 2024, 585, 216639) and lung cancer (Cancer Sci. 2004, 95, 197; N. Engl. J. ARVN0199WO2; ARVN-199-PCT

[0010] Med. 2017, 376, 2109). Decades of research have shown that mutant KRAS protein drives initiation and progression of these cancers and that targeting it will be a successful treatment strategy for patients whose tumors bear a mutant KRAS allele (J. Biomed. Biotechnol. 2010, 2010, 150960; Cancer Cell 2020, 37, 543; Nat. Rev. Drug Discov. 2020, 19, 533; Signal Transduct. Target. Ther. 2023, 8, 212). This prediction has been, at least, partially confirmed by the clinical efficacy observed with inhibitors of the KRAS G12C mutant, such as sotorasib (N. Engl. J. Med. 2020, 383, 1207; Clin. Cancer Res. 2021 , 28, 1482), adagrasib (N. Engl. J. Med.

[0011] 2022, 387, 120; J. Clin. Oncol. 2022, 40, 2530; N. Engl. J. Med. 2022, 388, 44; J. Clin. Oncol.

[0012] 2023, 41 , 4097), and divarasib (N. Engl. J. Med. 2023, 389, 710).

[0013] Thus, an ongoing need exists in the art for effective cancer therapies that directly target KRAS mutants including G12D.

[0014] SUMMARY

[0015] Provided herein are methods of treating particular cancers by administering PROTAC (Proteolysis-Targeting Chimera) compounds, chimeric small molecules, that bind to both an E3 ligase and a target protein, i.e., a KRAS protein, and function to recruit the KRAS protein or mutated version thereof, to the E3 ubiquitin ligase, i.e., CRBN, for targeted ubiquitination and subsequent proteasomal degradation. These methods are useful in treating particular cancers such as pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), non- squamous non-small cell lung cancer, colorectal cancer (CRC), and non-squamous colorectal cancer.

[0016] In an aspect, provided herein is a method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I:

[0017] I, or a pharmaceutically acceptable salt thereof, ARVN0199WO2; ARVN-199-PCT wherein the cancer is pancreatic ductal adenocarcinoma, non-small cell lung cancer, or colorectal cancer and wherein the variables are defined herein.

[0018] In an embodiment, the compound of Formula I is a compound of Formula la: la, or a pharmaceutically acceptable salt thereof, wherein the variables are defined herein.

[0019] In another embodiment, the compound of Formula I is a compound of Formula lb: lb, or a pharmaceutically acceptable salt thereof, wherein the variables are defined herein.

[0020] In yet another embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is pancreatic ductal adenocarcinoma (PDAC). In still another embodiment, the cancer is non-small cell lung cancer (NSCLC). In another embodiment, the cancer is non-squamous non- small cell lung cancer. In an embodiment, the cancer is colorectal cancer (CRC). In another embodiment the cancer is non-squamous colorectal cancer. ARVN0199WO2; ARVN-199-PCT

[0021] In yet another embodiment, the cancer is pancreatic ductal adenocarcinoma. In still another embodiment, the cancer is non-small cell lung cancer. In an embodiment, the cancer is colorectal cancer.

[0022] In another aspect, provided herein is a method of treating pancreatic cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0023] 1 , or a pharmaceutically acceptable salt thereof, wherein the pancreatic cancer is selected from pancreatic ductal adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic squamous cell carcinoma, pancreatic squamous cell carcinoma, a giant cell tumor of the pancreas, pancreatoblastoma, and an invasive mucinous cystic neoplasm.

[0024] In an embodiment, the pancreatic cancer is pancreatic adenosquamous carcinoma.

[0025] In an embodiment, the pancreatic cancer is pancreatic squamous cell carcinoma.

[0026] In an embodiment, the pancreatic cancer is acinar cell carcinoma.

[0027] In an embodiment, the pancreatic cancer is a giant cell tumor of the pancreas.

[0028] In an embodiment, the pancreatic cancer is pancreatoblastoma.

[0029] In an embodiment, the pancreatic cancer is an invasive intraductal papillary mucinous neoplasm.

[0030] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 : ARVN0199WO2; ARVN-199-PCT

[0031] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0032] 1 , or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating non-squamous colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of Compound 1 : ARVN0199WO2; ARVN-199-PCT

[0033] In an embodiment, the colorectal cancer is colorectal adenocarcinoma.

[0034] In an embodiment, the colorectal cancer is colorectal squamous cell carcinoma.

[0035] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0036] 1 , or a pharmaceutically acceptable salt thereof.

[0037] In still another aspect, provided herein is method of treating non-squamous non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of Compound 1 : ARVN0199WO2; ARVN-199-PCT

[0038] 1 , or a pharmaceutically acceptable salt thereof.

[0039] In another embodiment, the cancer (e.g., pancreatic ductal adenocarcinoma, non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, or non-squamous colorectal cancer) is characterized by a KRAS G12D mutation.

[0040] In yet another embodiment, the subject is identified as having a cancer characterized by a KRAS G12D mutation.

[0041] In still another embodiment, the therapeutically effective amount of Compound 1 is administered intravenously to the subject.

[0042] In still another embodiment, the therapeutically effective amount of Compound 1 is administered subcutaneous to the subject.

[0043] In an embodiment, the therapeutically effective amount of Compound 1 is administered to the subject once per week.

[0044] In an embodiment, the therapeutically effective amount of Compound 1 is administered to the subject once every two weeks.

[0045] In another embodiment, the therapeutically effective amount of Compound 1 is about 10 mg to about 250 mg.

[0046] In another embodiment, the therapeutically effective amount of Compound 1 is about 5 mg to about 125 mg. In yet another embodiment, the therapeutically effective amount of Compound 1 is about 10 mg to about 30 mg.

[0047] In still another embodiment, the method further comprises administering to the subject an effective amount of at least one additional anti-cancer agent. ARVN0199WO2; ARVN-199-PCT

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, help explain the principles of the disclosure. The drawings are only for the purpose of illustrating an embodiment of the disclosure and are not to be construed as limiting the disclosure. Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.

[0050] FIG. 1A Representative western blot of KRAS G12D degradation in GP2d.

[0051] FIG. 1 B Representative degradation curves for endogenous KRAS G12D.

[0052] FIG. 2A Binding in cells.

[0053] FIG. 2B HiBiT degradation.

[0054] FIG. 2C Endogenous degradation.

[0055] FIG. 3A PD is dose-responsive; no degradation with E3-dead.

[0056] FIG. 3B KRAS degradation is sustained for > one week after a single dose.

[0057] FIG. 3C MAPK signaling after single treatment of 3 mpk Compound 1 in GP2d tumors.

[0058] FIG. 3D Apoptosis after single treatment of 3 mpk Compound 1 in GP2d tumors.

[0059] FIG. 4A Panc04.03 Pancreatic cancer model.

[0060] FIG. 4B SW1990 Pancreatic cancer model.

[0061] FIG. 4C HPAC Pancreatic cancer model.

[0062] FIG. 4D AsPC-1 Pancreatic cancer models.

[0063] FIG. 4E GP2d Colorectal cancer model.

[0064] FIG. 5A Ternary complex formation TR-FRET assay.

[0065] FIG. 5B SEC purified ternary complex.

[0066] FIG. 5C In vitro ubiquitination TR-FRET assay.

[0067] FIG. 5D Cryo-EM 2D class averages.

[0068] FIG. 6A Baseline KRAS G12D levels.

[0069] FIG. 6B Baseline Cereblon protein levels.

[0070] FIG. 7A Loading controls.

[0071] FIG. 7B Loading controls from KRAS blots.

[0072] FIG. 7C Loading controls from HRAS / NRAS blots.

[0073] FIG. 8 Antiproliferative activity of Compound 1 .

[0074] FIG. 9A Plasma PK.

[0075] FIG. 9B Tumor PK. ARVN0199WO2; ARVN-199-PCT

[0076] FIG. 10 Response to 3 mpk IV BiW Compound 1 .

[0077] FIG. 11 KRAS G12D levels after single treatment of 3 mpk Compound 1 .

[0078] FIG. 12 KRAS G12D levels after single treatment of 3 mpk Compound 1 in AsPC-1 tumors.

[0079] FIG. 13A Dose dependent TGI SW1990 model.

[0080] FIG. 13B Dose dependent degradation SW1990 model.

[0081] FIG. 14A Dose dependent degradation GP2d model.

[0082] FIG. 14B KRAS G12D levels after single treatment of 3 mpk Compound 1 GP2d model. FIG. 15A MAPK signaling after single treatment of 3 mpk Compound 1 in GP2d tumors.

[0083] FIG. 15B AKT signaling after single treatment of 3 mpk Compound 1 in GP2d tumors.

[0084] FIG. 16A MAPK signaling after single treatment of 3 mpk Compound 1 in HPAC tumors.

[0085] FIG. 16B AKT signaling after single treatment of 3 mpk Compound 1 in HPAC tumors.

[0086] FIG. 17A MAPK signaling after single treatment of 3 mpk Compound 1 in AsPC-1 tumors.

[0087] FIG. 17B AKT signaling after single treatment of 3 mpk Compound 1 in AsPC-1 tumors.

[0088] DETAILED DESCRIPTION

[0089] Provided herein are methods of treating particular cancers by administering PROTAC compounds that bind to both an E3 ligase and a target protein, i.e., a KRAS protein, and function to recruit the KRAS protein or mutated version thereof, to the E3 ubiquitin ligase, i.e., CRBN, for targeted ubiquitination and subsequent proteasomal degradation. These methods are useful in treating particular cancers such as pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), non-squamous non-small cell lung cancer, colorectal cancer (CRC), and non-squamous colorectal cancer.

[0090] Definitions

[0091] Listed below are definitions of various terms used to describe the compounds and compositions disclosed herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0092] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, ARVN0199WO2; ARVN-199-PCT organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0093] Specific compounds of the present invention may be identified in the present specification by chemical name and / or chemical structure. When a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or the structure encompasses one of the possible stereoisomers or geometric isomers free of the others, or a mixture of the encompassed stereoisomers or geometric isomers. R*” and “S*” denote the relative configurations of substituents around one or more chiral carbon atoms, i.e., the compound is a single enantiomer, but the absolute configuration is unknown.

[0094] As used herein, the term “Ubiquitin Ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. For example, cereblon (CRBN) is an E3 Ubiquitin Ligase protein that alone or in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono-ubiquitinated proteins are not targeted to the proteasome for degradation but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.

[0095] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0096] As used herein, the term “about” as part of a quantitative expression such as “about X”, as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” encompasses variations of ±10%, (i.e., 10% higher or lower than X) including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate ARVN0199WO2; ARVN-199-PCT to perform the disclosed methods. The term also includes any numerical value that falls between X-10% and X+10%.

[0097] The term “administration” or the like as used herein refers to providing a therapeutic agent to a subject. The related terms “administering” and “administration of” (and grammatical equivalents) refer both to direct administration, which may be administration to a subject by a medical professional or by self-administration by the subject, and / or to indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and / or provides a patient with a prescription for a drug is administering the drug to the patient. Multiple techniques of administering a therapeutic agent exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0098] As used herein, the term “biweekly” is synonymous with the phrases “once every other week” and “once every two weeks.”

[0099] The term “treat,” “treated,” “treating,” or “treatment” includes the diminishment, alleviation, reversing, delaying the onset of, or inhibiting the progress of a disease or disorder or at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises alleviating the symptoms of cancer.

[0100] As used herein, the term “prevent” or “prevention” means stopping the onset of the symptoms or complications of the disease, condition or disorder.

[0101] As used herein, the terms “subject” and “patient” may be used interchangeably and refer to a human or a non-human mammal in need of treatment. Non-human mammals include, for example, livestock or farm animals (e.g., cows, pigs, horses, sheep, goats and the like) companion animals (e.g., dogs, cats, and the like), laboratory animals (e.g., rats, mice, guinea pigs and the like), vine, bovine, porcine, canine, feline and marine mammals. Typically the subject is a human in need of treatment.

[0102] As used herein, the terms “effective amount,” “pharmaceutically effective amount,” and “therapeutically effective amount” refer to an amount of the free base of a compound, or the equivalent amount of a pharmaceutically acceptable salt of a compound, that is sufficient to treat, ameliorate, or prevent the disease or disorder, or a symptom of the disease or disorder, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The effective amount for a particular subject may depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and whether additional therapeutics are to be administered to the subject. An appropriate therapeutic amount ARVN0199WO2; ARVN-199-PCT in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0103] As used herein, the term “pharmaceutically acceptable salt” refers to a salt form of compound, as well as hydrates of the salt form with one or more water molecules present. Such salt and hydrated forms retain the biological activity of the compound of the disclosure and are not biologically or otherwise undesirable, i.e. , exhibit minimal, if any, toxicological effects. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. The phrase “pharmaceutically acceptable salt” is not limited to a mono, or 1 :1 , salt. For example, “pharmaceutically acceptable salt” also includes bis-salts, such as a bis-hydrochloride salt. Representative "pharmaceutically acceptable salts" include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3- hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1 ,1 -methene-bis-2-hydroxy-3- naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0104] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject. Multiple techniques of administering a compound exist in the art including, but not ARVN0199WO2; ARVN-199-PCT limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0105] As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, non-toxic carrier (examples include but are not limited to, a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material), adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions described herein include, but are not limited to, sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose-based substances, and derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol and polyethylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, wool fat and other non-toxic compatible substances employed in pharmaceutical formulations. Other additional ingredients that may be included in the pharmaceutical compositions are known in the art and described, for example, in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0106] As used herein, the term “alkyl,” when used alone or as part of a larger moiety, such as “haloalkyl ”, “hydroxyalkyl” and the like, means saturated straight chain or branched monovalent hydrocarbon radical having, unless otherwise specified, from 1 to 20 carbon atoms such as C1 - 10, C1 -6, or C1 -4. A C1 -6 alkyl includes e.g. methyl, ethyl, propyl (e.g. , n-propyl, isopropyl), butyl (e.g. n-butyl, isobutyl, tert-butyl, sec-butyl), pentyl (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (e.g. n-hexyl). It will be understood that ARVN0199WO2; ARVN-199-PCT when specified, optional substituents on an alkyl group may be present on any substitutable position.

[0107] The term “haloalkyl” includes mono, poly, and perhaloalkyl groups where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

[0108] The term “hydroxyalkyl” includes mono, poly, and perhydroxy alkyl groups where one or more hydrogen atoms are replaced by OH.

[0109] As used herein, the term “alkoxy” refers to an alkyl radical attached through an oxygen linking atom, represented by — O-alkyl, wherein alkyl is as defined herein. Non-limiting examples include, methoxy, ethoxy, propoxy, 2-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy pentyloxy, and hexyloxy. It will be understood that when specified, optional substituents on an alkoxy group may be present on any substitutable position.

[0110] The term “haloalkoxy” includes mono, poly, and perhaloalkoxy groups where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

[0111] The term “alkynyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. The term “Cn-m alkynyl” refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1 -yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0112] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine.

[0113] Unless otherwise indicated, the term “compound” refers to any hetero-bifunctional compound described herein. In certain aspects, where specified, one or more hydrogen atoms on a disclosed compound may be replaced with deuterium. Such deuterated compounds may have one or more improved pharmacokinetic or pharmacodynamic properties (e.g., longer halflife) compared to the equivalent “un-deuterated” compound.

[0114] One or more of the compounds described herein may exist in various tautomeric forms and are part of the present disclosure. The terms “tautomers” or “tautomeric” refer to two or more interconvertible compounds / substituents resulting from at least one formal migration of a hydrogen atom and at least one change in valency. All such isomeric forms of such compounds are expressly included. Thus, when a compound herein is represented by a structural formula or ARVN0199WO2; ARVN-199-PCT designated by a chemical name herein, all tautomeric forms which may exist for the compound are encompassed by the structural formula.

[0115] Compounds having one or more chiral centers can exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric, enantiomeric, and epimeric forms as well as racemates and mixtures thereof. A “geometric isomer” refers to isomers that differ in the orientation of substituent group in relationship to a carbon-carbon double bond, a cycloalkyl ring, or a bridged bicyclic system. Atoms (other than H) on each side of a carbon-carbon double bond may be in an E (substituents are on opposite sides of the carbon-carbon double bond) or Z (substituents are oriented on the same side) configuration. “Cis” refers to substituents oriented on the same side of the ring, whereas “trans” refers to substituents oriented on opposite sides of the ring.

[0116] When the stereochemical configuration at a chiral center in a compound having one or more chiral centers is depicted by its chemical name (e.g., where the configuration is indicated in the chemical name by “R” or “S”) or structure (e.g., the configuration is indicated by “wedge” bonds), the enrichment of the indicated configuration relative to the opposite configuration is greater than 50%, 60%, 70%, 80%, 90%, 99% or 99.9%. “Enrichment of the indicated configuration relative to the opposite configuration” is a mole percent and is determined by dividing the number of compounds with the indicated stereochemical configuration at the chiral center(s) by the total number of all of the compounds with the same or opposite stereochemical configuration in a mixture.

[0117] When a geometric isomer is depicted by name or structure, the enrichment of the indicated isomer relative to the opposite isomer is greater than 50%, 60%, 70%, 80%, 90%, 99% or 99.9%. “Enrichment of the indicated isomer relative to the opposite isomer” is a mole percent and is determined by dividing the number of compounds with the indicated geometrical configuration by the total number of all of the compounds with the same or opposite geometrical configuration in a mixture.

[0118] The term “independently selected” is used herein to indicate that, for a variable which occurs in more than one location in a genus, the identity of the variable is determined separately in each instance. For example, if Rxappears as a substituent on two different atoms, the two instances of Rxmay be the same moiety, or different moieties. The same is true if a single atom is substituted with more than one instance of Rx. The identity of Rxin each instance is determined independently of the identity of the other(s). ARVN0199WO2; ARVN-199-PCT

[0119] Methods of Treatment

[0120] E3 ubiquitin ligases like Cereblon (CRBN) confer substrate specificity for ubiquitination, and therefore are more attractive therapeutic targets than general proteasome inhibitors due to their specificity for certain protein substrates. Bifunctional compounds, such as those described in U.S. Patent Application Publication Nos. 2015 / 0291562 and 2014 / 0356322 (both incorporated herein by reference), function to recruit endogenous proteins to an E3 ubiquitin ligase for ubiquitination and degradation. In particular, these publications describe bifunctional or Proteolysis-Targeting Chimera (PROTAC) compounds, which find utility as modulators of targeted ubiquitination of a variety of polypeptides and proteins, which are then degraded via the proteasome system. These bifunctional compounds function to recruit endogenous proteins to an E3 ubiquitin ligase for degradation.

[0121] The methods disclosed herein involve treating particular cancers by administering PROTAC compounds that function to recruit a KRAS protein or mutated version thereof, to an E3 ubiquitin ligase, i.e., CRBN, for targeted ubiquitination and subsequent proteasomal degradation. These methods are useful in treating particular cancers such as pancreatic ductal adenocarcinoma, non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, and non-squamous colorectal cancer. In an embodiment, the PROTAC compounds function to selectively recruit KRAS G12D protein. In another embodiment, the cancer is characterized by a KRAS G12D mutation.

[0122] In an aspect, provided herein is a method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I:

[0123] I, or a pharmaceutically acceptable salt thereof, ARVN0199WO2; ARVN-199-PCT wherein the cancer is pancreatic ductal adenocarcinoma, non-small cell lung cancer, non-squamous non-small cell lung cancer, colorectal cancer, or non-squamous colorectal cancer; and wherein:

[0124] Q1, Q2, Q3, Q4, Q5, and Q6are each independently CRLor N;

[0125] Q7is selected from C(R4)2, 0(0), and NR4;

[0126] R1and R2are each independently selected from H, halo, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, and C1-6 haloalkyl;

[0127] R3and R4are each independently selected from H, halo, C1-6 alkyl, C1-6 alkoxy, and C1-6 haloalkyl; each RLis independently H, halo, or ON; and p and q are each independently 1 , 2, 3, 4, 5, or 6.

[0128] In an embodiment,

[0129] Q1, Q2, Q3, Q4, Q5, and Q6are each independently CRLor N;

[0130] Q7is selected from C(R4)2, 0(0), and NR4;

[0131] R1is selected from H, halo, C1-6 alkyl, C1-6 alkoxy, and C1-6 haloalkyl;

[0132] R2is selected from H, C1-6 alkyl, G2-6 alkynyl, C1-6 alkoxy, and C1-6 haloalkyl;

[0133] R3is selected from H, halo, C1-6 alkyl, C1-6 alkoxy, and C1-6 haloalkyl; each R4is independently selected from H, C1-6 alkyl, C1-6 alkoxy, and Ci-s haloalkyl; each RLis independently H, halo, or ON; and p and q are each independently 1 , 2, 3, 4, 5, or 6.

[0134] In another embodiment,

[0135] Q1, Q2, Q3, Q4, Q5, and Q6are each independently CRLor N;

[0136] Q7is selected from C(R4)2, 0(0), and NR4;

[0137] R1is selected from H, halo, C1-6 alkyl, and C1-6 haloalkyl;

[0138] R2is selected from H, C1-6 alkyl, C2-6 alkynyl, and C1-6 alkoxy;

[0139] R3is selected from H, halo, C1-6 alkyl, and C1-6 alkoxy; each R4is independently selected from H, C1-6 alkyl, and C1-6 alkoxy; each RLis independently H, halo, or ON; and p and q are each independently 1 , 2, 3, 4, or 5.

[0140] In yet another embodiment, the compound of Formula I is a compound of Formula la: ARVN0199WO2; ARVN-199-PCT la, or a pharmaceutically acceptable salt thereof.

[0141] In still another embodiment, the compound of Formula I is a compound of Formula lb: lb, or a pharmaceutically acceptable salt thereof.

[0142] In an embodiment, R1and R2are each independently selected from H, halo, C1-6 alkyl, and C2-6 alkynyl. In another embodiment, R1is halo or C1-6 alkyl. In yet another embodiment, R1is halo.

[0143] In still another embodiment, R2is selected from H, C1-6 alkyl, and C=CH. In an embodiment, R2is selected from C1-6 alkyl and C=CH. In another embodiment, R2is C1-6 alkyl. In yet another embodiment, R2is C=CH.

[0144] In still another embodiment, R3is selected from H, halo, and C1-6 alkoxy. In an embodiment, R3is selected from H and halo. In another embodiment, R3is H. In yet another embodiment, R3is halo. ARVN0199WO2; ARVN-199-PCT

[0145] In still another embodiment, each R4is independently H or C1-6 alkyl. In an embodiment, each R4is H.

[0146] In another embodiment, each RLis independently H or halo. In yet another embodiment, Q1, Q2, Q3, Q4, Q5, and Q6are each independently is independently CH or N. In still another embodiment, at least three of Q1, Q2, Q3, Q4, Q5, and Q6are N. In yet another embodiment, three of Q1, Q2, Q3, Q4, Q5, and Q6are N. In still another embodiment, four of Q1, Q2, Q3, Q4, Q5, and Q6are N.

[0147] In an embodiment, Qi is N. In another embodiment, Q2is selected from CH and C-halo. In yet another embodiment, Q2is CH. In still another embodiment, Q2is C-halo. In an embodiment, Q3is selected from CH, C-halo, and N. In another embodiment, Q3is CH. In yet another embodiment, Q3is C-halo. In still another embodiment, Q3is N. In an embodiment, Q4is selected from CH and N. In another embodiment, Q4is CH. In yet another embodiment, Q4is N. In still another embodiment, Q5is selected from CH, C-halo, and N. In another embodiment, Q5is CH. In yet another embodiment, Q5is C-halo. In still another embodiment, Q5is N. In an embodiment, Q6is N.

[0148] In another embodiment, Q7is CH2 or C(O). In yet another embodiment, Q7is CH2. In still another embodiment, Q7is C(O).

[0149] In an embodiment, p and q are each independently 1 , 2, 3, or 4. In another embodiment, p and q are each independently 1 , 2, or 3. In yet another embodiment, p is 1 . In still another embodiment, q is 1 .

[0150] In another embodiment, the compound is a compound of Formula I, la or lb, wherein:

[0151] Q1, Q4, and Q6are N;

[0152] Q2, Q3, and Q5are each independently CRLor N;

[0153] Q7is selected from C(R4)2, C(O), and NR4;

[0154] R1is H or halo;

[0155] R2is H, C1-6 alkyl or C2-6 alkynyl;

[0156] R3is H or halo; each R4is independently H or C1-6 alkyl; each RLis independently H or halo; and p and q are each 1 .

[0157] In another embodiment, the compound is a compound of Formula I, la or lb, wherein:

[0158] Q1, Q4, and Q6are N;

[0159] Q2, Q3, and Q5are each independently CRL;

[0160] Q7is C(R4)2; ARVN0199WO2; ARVN-199-PCT

[0161] R1is halo;

[0162] R2is C26 alkynyl ;

[0163] R3is halo; each R4is H; each RLis H; and p and q are each 1 .

[0164] In another embodiment, the compound is selected from a compound in Table 1 , or a pharmaceutically acceptable salt thereof.

[0165] Table 1. ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT

[0166] ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT ARVN0199WO2; ARVN-199-PCT

[0167] In yet another embodiment, the compound is selected from a compound in Table 2, or a pharmaceutically acceptable salt thereof. ARVN0199WO2; ARVN-199-PCT

[0168] Table 2.

[0169] In another aspect, the disclosure provides a compound selected from a compound in Table 3, or a pharmaceutically acceptable salt thereof.

[0170] Table 3. ARVN0199WO2; ARVN-199-PCT

[0171] It is generally well known in the art that any compound that will be converted in vivo to provide a compound disclosed herein is a prodrug within the scope of the present disclosure.

[0172] Compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the disclosure can be replaced or substituted with isotopes of the atoms in natural or non-natural abundance. In some embodiments, the compound includes at least one deuterium atom. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced or substituted by deuterium. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 deuterium atoms. Synthetic methods for including isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton- Century-Crofts, 1971 ; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, ARVN0199WO2; ARVN-199-PCT

[0173] Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011 ). Isotopically labeled compounds can used in various studies such as NMR spectroscopy, metabolism experiments, and / or assays.

[0174] In yet another embodiment, the cancer to be treated by the compounds disclosed herein is pancreatic cancer. In an embodiment the cancer to be treated by the compounds disclosed herein is pancreatic ductal adenocarcinoma. In still another embodiment, the cancer to be treated by the compounds disclosed herein is non-small cell lung cancer. In an embodiment, the cancer to be treated by the compounds disclosed herein is non-squamous non-small cell lung cancer. In an embodiment, the cancer to be treated by the compounds disclosed herein is colorectal cancer. In an embodiment, the cancer to be treated by the compounds disclosed herein is non-squamous colorectal cancer.

[0175] In another aspect, provided herein is a method of treating pancreatic cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0176] 1 , or a pharmaceutically acceptable salt thereof, wherein the pancreatic cancer is selected from pancreatic ductal adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic squamous cell carcinoma, pancreatic squamous cell carcinoma, a giant cell tumor of the pancreas, pancreatoblastoma, and an invasive mucinous cystic neoplasm.

[0177] In an embodiment, the pancreatic cancer is pancreatic adenosquamous carcinoma.

[0178] In an embodiment, the pancreatic cancer is pancreatic squamous cell carcinoma.

[0179] In an embodiment, the pancreatic cancer is acinar cell carcinoma.

[0180] In an embodiment, the pancreatic cancer is a giant cell tumor of the pancreas. ARVN0199WO2; ARVN-199-PCT

[0181] In an embodiment, the pancreatic cancer is pancreatoblastoma.

[0182] In an embodiment, the pancreatic cancer is an invasive intraductal papillary mucinous neoplasm.

[0183] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0184] 1 , or a pharmaceutically acceptable salt thereof.

[0185] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0186] 1 , or a pharmaceutically acceptable salt thereof. ARVN0199WO2; ARVN-199-PCT

[0187] In yet another aspect, provided herein is a method of treating non-squamous colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of Compound 1 :

[0188] 1 , or a pharmaceutically acceptable salt thereof.

[0189] In an embodiment, the colorectal cancer is colorectal adenocarcinoma.

[0190] In an embodiment, the colorectal cancer is colorectal squamous cell carcinoma.

[0191] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :

[0192] 1 , or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is method of treating non-squamous non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of Compound 1 : ARVN0199WO2; ARVN-199-PCT

[0193] 1 , or a pharmaceutically acceptable salt thereof.

[0194] In an embodiment, the non-small cell lung cancer is squamous non-small cell lung cancer.

[0195] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 2: or a pharmaceutically acceptable salt thereof.

[0196] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 2: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0197] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 2: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 3: ARVN0199WO2; ARVN-199-PCT

[0198] 3, or a pharmaceutically acceptable salt thereof.

[0199] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 3:

[0200] 3, or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 3: ARVN0199WO2; ARVN-199-PCT

[0201] 3, or a pharmaceutically acceptable salt thereof.

[0202] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 4:

[0203] 4, or a pharmaceutically acceptable salt thereof.

[0204] In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 4: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0205] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 4: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 5: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0206] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 5: or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 5: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0207] In an aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 6:

[0208] 6, or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 6:

[0209] ARVN0199WO2; ARVN-199-PCT

[0210] 6, or a pharmaceutically acceptable salt thereof.

[0211] In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 6:

[0212] 6, or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 7:

[0213] ARVN0199WO2; ARVN-199-PCT

[0214] 7, or a pharmaceutically acceptable salt thereof.

[0215] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 7:

[0216] 7, or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 7: ARVN0199WO2; ARVN-199-PCT

[0217] 7, or a pharmaceutically acceptable salt thereof.

[0218] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 8: or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 8: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0219] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 8: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 9: ARVN0199WO2; ARVN-199-PCT

[0220] 9, or a pharmaceutically acceptable salt thereof.

[0221] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 9:

[0222] 9, or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 9: ARVN0199WO2; ARVN-199-PCT

[0223] 9, or a pharmaceutically acceptable salt thereof.

[0224] In an aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 10: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 10: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0225] In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 10: or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 11 : ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0226] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 11 :

[0227] 11 , or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 11 : ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0228] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 12:

[0229] 12, or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 12: ARVN0199WO2; ARVN-199-PCT

[0230] 12, or a pharmaceutically acceptable salt thereof.

[0231] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 12:

[0232] 12, or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 13: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0233] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 13:

[0234] 13, or a pharmaceutically acceptable salt thereof.

[0235] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 13:

[0236] 13, or a pharmaceutically acceptable salt thereof.

[0237] In an aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 14: ARVN0199WO2; ARVN-199-PCT

[0238] 14, or a pharmaceutically acceptable salt thereof.

[0239] In another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 14: or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 14: ARVN0199WO2; ARVN-199-PCT

[0240] 14, or a pharmaceutically acceptable salt thereof.

[0241] In still another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 15: or a pharmaceutically acceptable salt thereof. In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 15: ARVN0199WO2; ARVN-199-PCT

[0242] 15, or a pharmaceutically acceptable salt thereof.

[0243] In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 16: ARVN0199WO2; ARVN-199-PCT

[0244] 16, or a pharmaceutically acceptable salt thereof.

[0245] In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 16:

[0246] 16, or a pharmaceutically acceptable salt thereof. In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 16: ARVN0199WO2; ARVN-199-PCT

[0247] 16, or a pharmaceutically acceptable salt thereof.

[0248] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 17: or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 17: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0249] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 17: or a pharmaceutically acceptable salt thereof.

[0250] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 18: ARVN0199WO2; ARVN-199-PCT

[0251] 18, or a pharmaceutically acceptable salt thereof.

[0252] In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 18:

[0253] 18, or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 18: ARVN0199WO2; ARVN-199-PCT

[0254] 18, or a pharmaceutically acceptable salt thereof.

[0255] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 19: or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 19: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0256] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 19: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 20: ARVN0199WO2; ARVN-199-PCT

[0257] 20, or a pharmaceutically acceptable salt thereof.

[0258] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 20:

[0259] 20, or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 20: ARVN0199WO2; ARVN-199-PCT

[0260] 20, or a pharmaceutically acceptable salt thereof.

[0261] In an aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 21 : or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 21 : ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0262] In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 21 : or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 22: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0263] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 22:

[0264] 22, or a pharmaceutically acceptable salt thereof.

[0265] In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 22: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0266] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 23:

[0267] 23, or a pharmaceutically acceptable salt thereof.

[0268] In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 23: ARVN0199WO2; ARVN-199-PCT

[0269] 23, or a pharmaceutically acceptable salt thereof.

[0270] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 23:

[0271] 23, or a pharmaceutically acceptable salt thereof.

[0272] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 24:

[0273] 24, or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 24: ARVN0199WO2; ARVN-199-PCT

[0274] 24, or a pharmaceutically acceptable salt thereof.

[0275] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 24:

[0276] 24, or a pharmaceutically acceptable salt thereof. In an aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 25:

[0277] 25, or a pharmaceutically acceptable salt thereof. ARVN0199WO2; ARVN-199-PCT

[0278] In another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 25: 25, or a pharmaceutically acceptable salt thereof.

[0279] In yet another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 25:

[0280] 25, or a pharmaceutically acceptable salt thereof.

[0281] In still another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 26: ARVN0199WO2; ARVN-199-PCT

[0282] 26, or a pharmaceutically acceptable salt thereof.

[0283] In an aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 26:

[0284] 26, or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 26: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0285] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 27:

[0286] 27, or a pharmaceutically acceptable salt thereof.

[0287] In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 27:

[0288] 27, or a pharmaceutically acceptable salt thereof.

[0289] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 27: ARVN0199WO2; ARVN-199-PCT

[0290] 27, or a pharmaceutically acceptable salt thereof.

[0291] In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 28:

[0292] 28, or a pharmaceutically acceptable salt thereof. In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 28: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0293] In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 28:

[0294] 28, or a pharmaceutically acceptable salt thereof.

[0295] In yet another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 29: or a pharmaceutically acceptable salt thereof.

[0296] In still another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 29: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0297] In an aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 29: or a pharmaceutically acceptable salt thereof. In another aspect, provided herein is a method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 30: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0298] In yet another aspect, provided herein is a method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 30: or a pharmaceutically acceptable salt thereof. In still another aspect, provided herein is a method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 30: ARVN0199WO2; ARVN-199-PCT or a pharmaceutically acceptable salt thereof.

[0299] In an embodiment, the non-small cell lung cancer is non-squamous non-small cell lung cancer.

[0300] In another aspect, provided herein is a method of treating pancreatic cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of Compounds 1-30, or a pharmaceutically acceptable salt thereof, wherein the pancreatic cancer is selected from pancreatic ductal adenocarcinoma, pancreatic adenosquamous carcinoma, pancreatic squamous cell carcinoma, pancreatic squamous cell carcinoma, a giant cell tumor of the pancreas, pancreatoblastoma, and an invasive mucinous cystic neoplasm.

[0301] In an embodiment, the pancreatic cancer is pancreatic adenosquamous carcinoma.

[0302] In an embodiment, the pancreatic cancer is pancreatic squamous cell carcinoma.

[0303] In an embodiment, the pancreatic cancer is acinar cell carcinoma.

[0304] In an embodiment, the pancreatic cancer is a giant cell tumor of the pancreas.

[0305] In an embodiment, the pancreatic cancer is pancreatoblastoma.

[0306] In an embodiment, the pancreatic cancer is an invasive intraductal papillary mucinous neoplasm.

[0307] In yet another aspect, provided herein is a method of treating non-squamous colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of any of Compounds 1 -30, or a pharmaceutically acceptable salt thereof,

[0308] In an embodiment, the colorectal cancer is colorectal adenocarcinoma.

[0309] In an embodiment, the colorectal cancer is colorectal squamous cell carcinoma.

[0310] In still another aspect, provided herein is method of treating non-squamous non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a ARVN0199WO2; ARVN-199-PCT therapeutically effective amount of any of Compounds 1 -30, or a pharmaceutically acceptable salt thereof,

[0311] In another embodiment, the cancer is characterized by a KRAS G12D mutation. In yet another embodiment, the subject is identified as having a cancer characterized by a KRAS G12D mutation.

[0312] In still another embodiment, the method further comprises administering to the subject an effective amount of at least one additional anti-cancer agent.

[0313] Administration / Dosages / Formulations

[0314] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0315] Injectable preparations such as intravenous preparations and subcutaneous preparations (for example, sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0316] To prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend ARVN0199WO2; ARVN-199-PCT upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0317] Compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax that are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0318] 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 and the like.

[0319] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

[0320] Dosage forms for topical or transdermal administration of a compound of this disclosure include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this disclosure.

[0321] The ointments, pastes, creams, and gels may contain, in addition to an active compound of this disclosure, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0322] Powders and sprays can contain, in addition to the compounds of this disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. ARVN0199WO2; ARVN-199-PCT

[0323] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0324] Compounds of the present disclosure may be administered intratympanically, wherein a long, narrow, bore needle is passed through the ear canal and through the eardrum to administer medications into the middle ear space where they are absorbed by the inner ear.

[0325] According to the methods of treatment of the present disclosure, disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of a compound of the disclosure, in such amounts and for such time as is necessary to achieve the desired result. As is well understood in the medical arts a therapeutically effective amount of a compound of this disclosure will be at a reasonable benefit / risk ratio applicable to any medical treatment.

[0326] In general, compounds of the disclosure will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art.

[0327] In an embodiment, the therapeutically effective amount of compound is administered intravenously to the subject. In another embodiment, the therapeutically effective amount of compound is administered subcutaneously to the subject.

[0328] In another embodiment, the therapeutically effective amount of compound is administered to the subject once per week or once every two weeks. In yet another embodiment, the therapeutically effective amount of compound is administered to the subject once per week. In still another embodiment, the therapeutically effective amount of compound is administered to the subject once every two weeks.

[0329] In still another embodiment, the subject is in a fed state at the time of administration. In an embodiment, the subject is in a fasted state at the time of administration.

[0330] In another embodiment, the therapeutically effective amount of compound is about 10 mg to about 250 mg. In yet another embodiment, the therapeutically effective amount of compound is about 10 mg to about 225 mg. In still another embodiment, the therapeutically effective amount of compound is about 10 mg to about 200 mg. In an embodiment, the therapeutically effective amount of compound is about 10 mg to about 175 mg. In another embodiment, the therapeutically effective amount of compound is about 10 mg to about 150 mg.

[0331] In another embodiment, the therapeutically effective amount of compound is about 5 mg to about 125 mg. In yet another embodiment, the therapeutically effective amount of compound ARVN0199WO2; ARVN-199-PCT is about 10 mg to about 100 mg. In yet another embodiment, the therapeutically effective amount of compound is about 5 mg to about 100 mg. In still another embodiment, the therapeutically effective amount of compound is about 5 mg to about 85 mg. In still another embodiment, the therapeutically effective amount of compound is about 5 mg to about 75 mg. In an embodiment, the therapeutically effective amount of compound is about 5 mg to about 60 mg.

[0332] In an embodiment, the therapeutically effective amount of compound is about 5 mg to about 50 mg. In another embodiment, the therapeutically effective amount of compound is about 5 mg to about 40 mg. In another embodiment, the therapeutically effective amount of compound is about 5 mg to about 30 mg. In yet another embodiment, the therapeutically effective amount of compound is about 10 mg to about 30 mg. In still another embodiment, the therapeutically effective amount of compound is about 10 mg to about 20 mg.

[0333] In an embodiment, the therapeutically effective amount of compound is about 10 mg. In another embodiment, the therapeutically effective amount of compound is about 15 mg. In yet another embodiment, the therapeutically effective amount of compound is about 20 mg. In still another embodiment, the therapeutically effective amount of compound is about 25 mg. In an embodiment, the therapeutically effective amount of compound is about 30 mg.

[0334] In any of the above embodiments, the compound is a compound of Formulae I, la, or lb, or Compound 1 through Compound 30. In another embodiment, the compound is Compound 1 .

[0335] A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors (e.g., the known heterogeneity of the disclosed cancers, or the dependency of the tumor on KRAS signaling). In general, satisfactory results are indicated to be obtained systemically at weekly dosages or biweekly dosages of from about 5 mg to about 250 mg of a compound of the disclosure. In an embodiment, the weekly dose of the compound is about 10 mg to about 30 mg. In another embodiment, the biweekly dose of the compound is about 10 mg to about 250 mg.

[0336] Upon improvement of a subject’s condition, a maintenance dose of a compound or composition of this disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained; when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. ARVN0199WO2; ARVN-199-PCT

[0337] It will be understood, however, that the dosage amount and frequency of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; and like factors well known in the medical arts.

[0338] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate; disodium hydrogen phosphate; potassium hydrogen phosphate; sodium chloride; zinc salts; colloidal silica; magnesium trisilicate; polyvinyl pyrrolidone; polyacrylates; waxes; polyethylenepolyoxypropylene-block polymers; wool fat; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such a propylene glycol or polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; and phosphate buffer solutions. Further, non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The protein kinase inhibitors or pharmaceutical salts thereof may be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions, which comprise an amount of the protein inhibitor effective to treat or prevent a protein kinase- mediated condition and a pharmaceutically acceptable carrier, are other embodiments of the present disclosure. ARVN0199WO2; ARVN-199-PCT

[0339] While various disclosure embodiments have been particularly shown and described in the present disclosure, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the embodiments disclosed herein and set forth in the appended claims.

[0340] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0341] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0342] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0343] EXAMPLES

[0344] The compounds and methods disclosed herein are further illustrated by the following examples, which should not be construed as further limiting. The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic synthesis, cell biology, cell culture, and molecular biology, which are within the skill of the art.

[0345] The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings of the present disclosure as set forth.

[0346] Processes for preparing the compounds disclosed herein can be found, at least, in WO 2024 / 159164 and US 2024 / 0287101 , the contents of which are incorporated in their entirety.

[0347] Abbreviations as used herein have respective meanings as follows:

[0348] Abbreviation Term

[0349] APC Allophycocyanin

[0350] ATCC American Type Culture Collection

[0351] AUG Area under the curve

[0352] BCA Bicinchoninic acid

[0353] BRET Bioluminescence resonance energy transfer ARVN0199WO2; ARVN-199-PCT

[0354] BSA Bovine serum albumin CDX Cell line-derived xenograft CRBN Cereblon E3 ligase CRC Colorectal cancer CRL Cullin-RING ligase DC50 Half-maximal degradation concentration Dmax Maximum degradation, as % of control DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DPBS Dulbecco’s phosphate-buffered saline DTT Dithiothreitol EC50 Half-maximal efficacious concentration EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent assay EMEM Eagle’s Minimum Essential Medium Eu Europium FBS Fetal bovine serum GDP Gaunosine 5'-diphosphate GPPNP Guanosine 5'-[P,y-imido]triphosphate GTP Gaunosine 5'-triphosphate HEPES 2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethanesulfonic acid HPbCD Hydroxypropyl-p-cyclodextrin HRP Horseradish peroxidase IACUC Institutional Animal Care and Use Committee IC50 Half-maximal inhibitory concentration IMAC Immobilized metal affinity chromatography IMID Immunomodulatory imide drug IV Intravenous kDa Kilodalton LDS Lithium dodecyl sulfate MAPK Mitogen-activated protein kinase MgCh Magnesium chloride MOPS 3-morpholinopropane-1 -sulfonic acid NaCI Sodium chloride NanoBRET Nano Bioluminescence Resonance Energy Transfer NSCLC Non-small cell lung cancer PD Pharmacodynamic PDAC Pancreatic ductal adenocarcinoma PDX Patient-derived xenograft PK Pharmacokinetic PROTAC PROteolysis TArgeting Chimera PTM Post translational modification RPMI Roswell Park Memorial Institute media RT Room temperature ARVN0199WO2; ARVN-199-PCT

[0355] RTK Receptor tyrosine kinase

[0356] RIPA Radio-immunoprecipitation assay

[0357] SCID Severe combined immunodeficiency disease

[0358] SD Standard deviation

[0359] SDS Sodium dodecyl sulfate

[0360] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0361] STR Short tandem repeats

[0362] TBS Tris-buffered saline

[0363] TEST T ris-buffered Saline, 0.1 % Tween® 20

[0364] TCEP T ris(2-carboxyethyl)phosphine

[0365] TGI Tumor growth inhibition, expressed as %

[0366] TR-FRET Time-resolved Forster's resonance energy transfer

[0367] TWB Tween wash buffer

[0368] Ub Ubiquitin

[0369] ULA Ultra-low attachment

[0370] UPS Ubiquitin proteasome system

[0371] WT Wild-type

[0372] Example 1 : In Vitro Pharmacology of Compound 1

[0373] 1. Methods and Materials

[0374] 1.1. Cell Culture Each human derived cell line was cultured in the medium recommended by the vendor and maintained at 37°G, 5% CO2. A427, AsPC-1 , H520, H647, HeLa, HPAC, LS180, MiaPacCa-2, Pane 04.03, Pane 08.13, SNU-C2B, SW620, SK-LU-1 , and SW1990 were purchased from the American Tissue Culture Collection (ATCC), GP2d was purchased from Millipore Sigma, and HEK293 cell line stably expressing NanoLuc-CRBN was purchased from Promega. All cell lines (except HeLa cells and HEK293 NanoLuc-CRBN cells) were verified by short tandem repeat (STR) analysis. Additional culturing conditions are below.

[0375] Table 1.1.1. ARVN0199WO2; ARVN-199-PCT

[0376] All cell lines were grown in tissue culture flasks within humidified 37°O, 5% CO2 incubators and detached via trypsin for passaging and expansion within sterile biosafety cabinets. The growth conditions for each cell line are listed below.

[0377] A427 cells (ATCC, USA) were maintained at ~2-3 x 106cells / mL, passaged once per week at a ratio 1 :30 in EMEM (ATCC, USA) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0378] AsPC-1 cells (ATCC, USA) were maintained at ~1-2 x 105cells / mL, passaged once per week at a ratio of 1 :10 in RPMI-1640 (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0379] GP2d cells (Millipore Sigma, USA) were maintained at ~2-3 x 106cells / mL, passaged once per week at a ratio of 1 :12 in DMEM (Gibco) + 10% FBS (Gibco) + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0380] H520 cells (ATCC, USA) were maintained at ~2-3 x 106cells / mL, passaged once per week at a ratio of 1 :15 in RPMI-1640 (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0381] H647 cells (ATCC, USA) were maintained at ~2-3 x 106cells / mL, passaged once per week at a ratio of 1 :15 in RPMI-1640 (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0382] HEK293 NanoLuc-CRBN cells (Promega, USA) were maintained ~2-3 x 106cells / mL, passaged once per week at a ratio of 1 :30 in DMEM (Gibco) + 10% FBS (Gibco) + 1% Geneticin (Gibco) at 37°C, 5% CO2.

[0383] HeLa cells (ATCC, USA) were maintained at ~1 -2 x 106cells / mL, passaged twice per week at a ratio 1 :10 in DMEM (ATCC, USA) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0384] HPAC cells (ATCC, USA) were maintained at ~2-4 x 106cells / mL, passaged once per week at a ratio of 1 :30 in DMEM / F12 (Gibco) + 5% FBS (Gibco) + 1% PenStrep (Gibco) + 1 pg / ml EGF (Gibco) + 40ng / ml hydrocortisone (Millipore Sigma) + ITS supplement (R&D Systems) at 37°C, 5% CO2.

[0385] LS180 cells (ATCC, USA) were maintained at ~2-3 x 106cells / mL, passaged once per week at a ratio 1 :15 in EMEM (ATCC, USA) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2. ARVN0199WO2; ARVN-199-PCT

[0386] MiaPaCa-2 cells (ATCC, USA) were maintained at ~2-4 x 105cells / mL, passaged once per week at a ratio of 1 :30 in DMEM (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0387] Pane 04.03 cells (ATCC, USA) were maintained at ~2-4 x 106cells / mL, passaged once per week at a ratio of 1 :12 in RPMI-1640 (Gibco) + 15% FBS (Gibco) + 20 units / ml human recombinant insulin (Millipore Sigma) at 37°C, 5% CO2.

[0388] Pane 08.13 cells (ATCC, USA) were maintained at ~2-4 x 106cells / mL, passaged once per week at a ratio of 1 :12 in RPMI-1640 (Gibco) + 15% FBS (Gibco) + 20 units / ml human recombinant insulin (Millipore Sigma) at 37°C, 5% CO2.

[0389] SK-LU-1 cells (ATCC, USA) were maintained ~5x106cells / mL, passaged once per week at a ratio of 1 :3 in EMEM (ATCC, USA) +10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0390] SNU-C2B (ATCC, USA) were maintained at ~1-2 x 106cells / mL, passaged once per week at a ratio of 1 :12 in RPMI-1640 (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0391] SW620 cells (ATCC, USA) were maintained at ~2-4 x 106cells / mL, passaged once per week at a ratio of 1 :30 in DMEM (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0392] SW1990 cells (ATCC, USA) were maintained at ~ 1 -2 x 10scells / mL, passaged once per week at a ratio of 1 :10 in DMEM (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37°C, 5% CO2.

[0393] 1.2. NanoBRET Cellular Target Engagement

[0394] RAS:CRAF protein :protein interaction displacement assay:

[0395] HeLa cells were plated in T-182 flasks with 24 mL at 4.0x105cells / mL per flask and transfected to express NanoLuc-tagged Ras and Halo-Tagged CRAF-RBD-CRD. In brief, 1200 pL of Optimem (Gibco, cat #11058-021 ), 24 pg pFdR4Hc CRAF RBD-CRD-HaloTag plasmid, 2.4 pg pFN31 K NanoLuc-Ras plasmids (Promega non-catalog items), and 72 pL of FuGene HD transfection reagent (Promega, cat #E2312) were premixed and added to each T-182 flask of HeLa cells. Following the transfection, the flask was returned to 37°C.

[0396] Twenty-four hours later, the cells were harvested and resuspended in Optimem supplemented with 4% FBS to a density of 2.3x105cell / mL. MLN4924 (Sigma, cat #5054770001 ) was added to the cultures for a final concentration of 1 pM for a one-hour pretreatment. MLN4924 inhibits NEDDylation, a critical post-translational modification for Cullin- RING Ligase (CRL) (CRBN ligase family) activity and blocks CRL-based proteasomal degradation (Nature 2009, 458, 732). The cultures were further prepared according to the ARVN0199WO2; ARVN-199-PCT manufacturer’s protocol (section 1 .3), which includes the addition of the +618 ligand, a small molecule that binds the HaloTag and acts as an energy acceptor in the assay.

[0397] The prepared cells were plated in 384-well, white, TC-treated assay plates (Corning Costar, cat #3570) with 35 pL / well for a final density of 8000 cells / well. The plates were treated at a top concentration of 12.5 pM in 5-fold, 10-point serial dilutions (0.625% DMSO) with one of three compounds: Compound 1 , Compound 31 and Compound 32. After compound treatment, the plates were returned to the 37°C incubator for twenty-four hours and treated with detection reagent according to manufacturer’s protocol (section 1 .3) before being read on an Envision 2105 (Revvity).

[0398] The data were processed by subtracting background signal (averaged signal from DMSO treated cells without +618 ligand) and normalizing to negative control (DMSO treated cells with +618 ligand).

[0399] CRBN probe displacement assay:

[0400] HEK293 cells stably expressing NanoLuc-CRBN (Promega cell line, cat #CS1810C398) were resuspended in Optimem (Gibco, cat #11058-021 ) to a concentration of 3.23x105cells / mL and plated in 384-well, white, non-binding surface assay plates (Corning Costar, cat #3574) for a final density of 10,000 cells / well.

[0401] The assay plates were then treated with a final concentration of 10 pM top, 3-fold, 11- point serial dilution of the compounds or DMSO vehicle alone (0.33% final) and with 500 nM of Nano Bioluminescence Resonance Energy Transfer (NanoBRET) target engagement CRBN tracer or tracer dilution buffer alone (Promega, cat #N2912). After a two-hour incubation at 37°C, 5% CO2, the plates were acclimated to room temperature (RT) for 15 minutes and 20 pL of detection reagent was added before being read on the Synergy Multimode Hybrid plate reader (BioTek). The data was normalized to the positive and negative controls (presence vs absence of the CRBN tracer) on a scale of 0-1 .

[0402] 1.3 NanoBRET Protein-Protein Interaction Protocol

[0403] Table 1.3.1. ARVN0199WO2; ARVN-199-PCT

[0404] Transfection Reagent:

[0405] • 1200 p L of Optimem (Gibco, cat #11058-021 ),

[0406] • 24 pg RBD-CRD plasmid DNA,

[0407] • 2.4 pg NanoLuc-GTPase plasmids for KRAS and NRAS variant, or 1 .2 pg NanoLuc- GTPase plasmids for HRAS

[0408] • 72 pL of FuGene HD transfection reagent

[0409] Cell Transfection:

[0410] • Hela cells were plated in T-182 flasks with 24 mL at 4.0x105cells / mL and allowed to adhere.

[0411] • Transfection reagent was incubated for 10 minutes and added to one T-182 flask of Hela cells.

[0412] • The cells are returned to incubate for 24 hours at 37°C, 5% CO2.

[0413] Plating:

[0414] • Cells were harvested, centrifuged to pellet, and resuspended in Optimem supplemented with 4% PBS to a density of 2.3x105cell / mL.

[0415] • MLN4924 was added for a one-hour pre-treatment at a final concentration of 1 pM to inhibit NEDDylation a PTM required for CRL activity which eliminates KRAS degradation from the analysis.

[0416] • 5 mL of MLN-treated culture was then removed to a different conical tube and treated with 5 pL DMSO.

[0417] • To the remaining culture, 1000X +618 ligand was added 1 :1000 and mixed by swirling.

[0418] • The cells were plated in 384-well, white, TC-treated assay plates with 35 pl / well using a Multidrop Combi with a small dispensing cassette for a final of 8000 cells / well.

[0419] • The plates were treated with compounds at a top concentration of 12.5 pM in 5-fold, 10-point serial dilutions for a final 0.625% DMSO.

[0420] • After compound treatment, the plates were returned to the 37°C incubator for 24 hours.

[0421] Detection:

[0422] • The assay plates were cooled to room temperature. ARVN0199WO2; ARVN-199-PCT

[0423] • NanoGio substrate was diluted 1 :100 in Optimem and vortexed to mix.

[0424] • The resulting detection reagent was added to the plates, with 10 pL per well using a Multidrop Combi with a small dispensing cassette.

[0425] • The plates were sealed with adhesive film (VWR, cat #89024-752).

[0426] • Donor emission (460nm) and acceptor emission (618nm) were measured within

[0427] 10 minutes of substrate addition using a PerkinElmer EnVision® Multilabel Reader.

[0428] • The acceptor emission value (618nm) was divided by the donor luminescence value (460nm) then multiplied by 1000 for each sample to generate raw NanoBRET™ ratio values.

[0429] • Z' and Z factor calculations were calculated to gauge assay consistency.

[0430] Envision Setup:

[0431] • Mirror (643): Luminescence - Slot4

[0432] • Emission filter (704): Chroma Cat.# E600LP - EmSlot4

[0433] • Second emission filter (703): Chroma Cat.# AT460 / 50m - EmSlotl

[0434] • Measurement height (mm): 6.5

[0435] • Measurement time (seconds): 0.1

[0436] Using a protocol described or similarly described herein, the results are shown in Fig. 2A.

[0437] 1.4. Compound 1 Dose Response Assay

[0438] Cells were seeded in 12-well plates (Celltreat, cat #229111 ) with approximately 4-5x105per well and allowed to adhere overnight prior to Compound 1 treatment. Depending on the cell line or experiment, cells were treated with either: a 3000 nM top 3-point 10-fold serial dilution, a 300 nM top 11 -point 3-fold serial dilution, or a 3 nM top 7-point 3-fold serial dilution (0.1% DMSO). For each serial dilution replicate a DMSO alone control was included and used for normalization. Cells were treated for 24 hours and processed immediately for immunoblotting as described in section 1 .5 or the wells were rinsed with ice-cold DPBS and stored at -80°C for future processing.

[0439] 1.5. Immunoblotting

[0440] Cells were lysed by adding ice-cold radio-immunoprecipitation assay (RIPA) buffer to the 12-well plates and mechanically disrupting on an orbital plate shaker. Recovered lysates were clarified by centrifugation and total protein levels were quantified by bicinchoninic acid (BCA) assay according to the manufacturer’s directions (Pierce, cat #23225). Lysates were then normalized and prepared for SDS-PAGE by adding additional RIPA buffer, 4X lithium dodecyl sulfate (LDS) sample buffer, and 10X reducing agent. Samples were denatured by boiling, ARVN0199WO2; ARVN-199-PCT resolved on 4-12% Bis-Tris gels in MOPS / SDS buffer and transferred to nitrocellulose membrane.

[0441] Membranes were cut horizontally to blot for multiple proteins simultaneously and then blocked for 1 hour at RT with one of the following: 3-5% (w / v) BSA in TBST, 3-5% (w / v) non-fat dry milk in TBST, or Intercept Blocking Buffer (LI-COR cat#927-60001 ). Primary antibodies were diluted in 3-5% BSA or milk in TBST and incubated with the membranes overnight at 4°C. After washing, the blots were hybridized with secondary antibodies diluted in 3-5% BSA or milk in TBST for 1 hour at RT followed with additional washes. The secondary antibody signal was then detected either on a LI-COR Odyssey M (fluorescence detection) or treated with SuperSignal™ West Femto (ThermoFisher, cat #34095) for ~2 minutes and imaged on a Bio-Rad ChemiDoc MP (chemiluminescence detection).

[0442] Densitometry analysis was then carried out using Image Studio™ 5.2 software (LI-COR) for fluorescent antibody detection and ImageJ 1 .51 (NIH) or Image Lab 6.1 .0 (Bio-Rad) for chemiluminescent antibody detection. Data was normalized by dividing the KRAS G12D value with the loading control protein value and then normalizing relative to the DMSO alone samples. A list of antibodies and antibody dilutions along with a detailed protocol are provided in section 1 .6.

[0443] 1.6. Immunoblotting Protocol and Antibodies

[0444] The following antibodies at the indicated dilution factor (v / v) were used for immunoblotting in this study:

[0445] Table 1.6.1. ARVN0199WO2; ARVN-199-PCT

[0446] Table 1.6.2.

[0447] Day 1: Harvest samples / lysate preparation / BOA assay: Prepared complete RIPA lysis buffer (with protease inhibitor, phosphatase inhibitor, and EDTA, which are all 100X stock).

[0448] Cells were washed 2X with ice-cold DPBS, while being kept on ice.

[0449] Lysed cells by adding 100 pL of complete RIPA lysis buffer on a plate shaker 4°C for 10 minutes. Lysate was collected and transferred to 1 .5 m L microfuge tubes.

[0450] Cell lysate was spun at full speed for 10 minutes at 4°C in a benchtop microfuge.

[0451] Supernatant was collected into a U-bottom 96 well plate on ice.

[0452] Protein quantification was conducted using the Pierce™ BCA Protein Assay Kit assay according to the manufacturer’s directions. Cell lysates were frozen at -80°C.

[0453] Protein concentrations were calculated based on the BCA assay results. Calculations were made to prepare a 10Opil gel sample at a concentration of 0.5 pg / pL. ARVN0199WO2; ARVN-199-PCT

[0454] Gel samples were prepared using lysate, 4X LDS sample buffer, 10X reducing agent, and additional RIPA buffer.

[0455] Samples were boiled for 5 min in a heat block at 95°C then stored at -20°C or -80°C.

[0456] Day 2: Immunoblotting:

[0457] Samples were boiled for 5 min in a heat block at 95°C.

[0458] 15 pL of samples were loaded into a 4-12% Bis-Tris gel and run at 150V in MOPS / SDS running buffer until the dye front migrated to the bottom of the gel.

[0459] Proteins were transferred to a nitrocellulose membrane using the Trans- Blot® Turbo™ Transfer System with the 25V 1 1 min transfer protocol.

[0460] Membranes were cut horizontally into sections and blocked with LICOR Intercept Blocking buffer for downstream fluorescent antibody detection or with 3% BSA / milk in TEST for chemiluminescent detection buffer at room temperature for 1 hour.

[0461] Primary antibodies were diluted as denoted in Table 1 .6.1 in 3% BSA / milk in TBST and membranes were incubated overnight at 4°C on a platform rocker.

[0462] Day 3: Developing blots:

[0463] Membranes were washed 4 x 5 minutes with TBS-T.

[0464] The appropriate secondary antibodies were diluted in 3% BSA / milk in TBST and hybridized to the blots for 1 hour at room temperature.

[0465] For fluorescent antibody detection:

[0466] 1 . Membranes were washed 3 x 5 minutes with TBS-T, and 1 X 5 minutes with TSB.

[0467] 2. Membranes were developed using the LICOR Odyssey M.

[0468] For chemiluminescent antibody detection:

[0469] 1 . Membranes were washed 4 x 5 minutes with TBS-T.

[0470] 2. Treated membranes with SuperSignalTM West Femto (ThermoFisher cat#34095) for ~2 minutes and imaged on a Bio-Rad ChemiDoc MP.

[0471] Using a protocol described or similarly described herein, the results are shown in Fig. 1 A, 1 B, 2C, 7A, 7B, 7C and Table 1.

[0472] 1.7. Mechanism of Action in AsPC-1 Cells

[0473] KRAS G12D homozygous AsPC-1 cells were pretreated with UPS inhibitors or lenalidomide, a CRBN binder. Excess lenalidomide was added to compete with Compound 1 for CRBN occupancy. The two UPS inhibitors used were MG-132 (Santa Cruz Biotechnology, cat #sc-201270) which directly inhibits the catalytic activity of the proteasome (Cell 1994, 78, 761 ; J. Biol. Chem. 1996, 271 , 27280) and MLN4924 which blocks CRL-based proteasomal degradation (Nature 2009, 458, 732) (MilliporeSigma, cat #5054770001 ).

[0474] Roughly 4x105AsPC-1 cells were seeded in each well of 12-well plates (Celltreat, cat# 229111 ) and incubated overnight prior to Compound 1 treatment. The next day select wells ARVN0199WO2; ARVN-199-PCT were treated with DMSO alone (0.1 or 0.15%) or final concentrations of 10 pM MG-132 (0.1 % DMSO), 1 pM MLN4924 (0.1% DMSO), and 30 pM lenalidomide (0.15% DMSO) and returned to the 37°C incubator. After 60 minutes select wells were treated with DMSO alone or 30 nM Compound 1 (0.1 %) and the plates were further incubated for 4, 6, or 8 hours before being washed with ice-cold DPBS and stored at -80°C for future immunoblot processing (section 1 .5).

[0475] 1.8. Biochemical KRAS :CRBN Ternary Assay

[0476] Equimolar amounts (10 nM final) of recombinant Biotin-Avi tagged-KRAS 1 -169 (WT, G12C, G12D, or G12V) and CRBN 41 -442 / 6His-DDB1 complex were combined with TR-FRET detection reagents: 1 nM final Europium (Eu)-W1024 labeled streptavidin (Revvity, cat #AD0062) and 100 nM final anti-6His antibody conjugated to allophycocyanin (APC) (Revvity, cat #AD0059H) in a buffer promoting nucleotide exchange (20 mM HEPES pH 7.5, 100 mM NaCI, 4 mM EDTA, 2 mM MgCh, 0.02% (v / v) Triton X-100, 1 mM DTT) with an excess (10 pM) of either GDP (MilliporeSigma, cat #G7127-25mg) or GPPNP, a nonhydrolyzable analog of GTP (MilliporeSigma, cat #G0635-25mg). These master mixes were dispensed to a white, 384-well low volume ProxiPlate (Revvity, cat #6008280) and mixed with a 10 pM top 3-fold 12-step serial dilution of Compound 1 , Compound 31 , or Compound 32 in the above buffer with GDP or GPPNP (1 .25% DMSO final) and allowed to incubate overnight at RT before reading on the Envision 2105 (Revvity) by exciting at 320 nM, and capturing emission spectra at 615 nM (Eu) and 665 nM (APC). A ratio of 665 over 615 multiplied by 10,000 was used to represent the TR- FRET data. Recombinant protein production methods are provided in section 1 .9. Using a protocol described or similarly described herein, the results are shown in Fig. 5A.

[0477] 1.9. Recombinant protein preparation

[0478] Gentamicin (Thermo Fisher Scientific, 15710064) Sf-900TM II SFM (Thermo Fisher Scientific, 10902104) 1 M HEPES pH7.5 (TEKNOVA, H1035) 2M Imidazole (TEKNOVA, I6909) NaCI (Sigma, S6546) Glycerol (Sigma, G6279) Protease Inhibitor Tablets (ROCHE, COEDTAF-RO) TCEP (Fisher Scientific, 501532844) Benzonase (Sigma, E1014-25KU) ARVN0199WO2; ARVN-199-PCT

[0479] CRBN (41-442) protein sequence:

[0480] GGGRAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQ TLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAI GRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKW WQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVA ACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVN PHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQGKICASHIGWKFTATKKDMSPQKF WGLTRSALLPTIPDTEDEISPDKVILCL

[0481] DDB1-Thrombin-His protein sequence:

[0482] MSYNYVVTAQKPTAVNGCVTGHFTSAEDLNLLIAKNTRLEIYVVTAEGLRPVKEVGMYG KIAVMELFRPKGESKDLLFILTAKYNACILEYKQSGESIDIITRAHGNVQDRIGRPSETGIIGIIDPE CRMIGLRLYDGLFKVIPLDRDNKELKAFNIRLEELHVIDVKFLYGCQAPTICFVYQDPQGRHVKT YEVSLREKEFNKGPWKQENVEAEASMVIAVPEPFGGAIIIGQESITYHNGDKYLAIAPPIIKQSTIV CHNRVDPNGSRYLLGDMEGRLFMLLLEKEEQMDGTVTLKDLRVELLGETSIAECLTYLDNGVV FVGSRLGDSQLVKLNVDSNEQGSYVVAMETFTNLGPIVDMCVVDLERQGQGQLVTCSGAFKE GSLRIIRNGIGIHEHASIDLPGIKGLWPLRSDPNRETDDTLVLSFVGQTRVLMLNGEEVEETELM GFVDDQQTFFCGNVAHQQLIQITSASVRLVSQEPKALVSEWKEPQAKNISVASCNSSQVVVAV GRALYYLQIHPQELRQISHTEMEHEVACLDITPLGDSNGLSPLCAIGLWTDISARILKLPSFELLH KEMLGGEIIPRSILMTTFESSHYLLCALGDGALFYFGLNIETGLLSDRKKVTLGTQPTVLRTFRSL STTNVFACSDRPTVIYSSNHKLVFSNVNLKEVNYMCPLNSDGYPDSLALANNSTLTIGTIDEIQK LHIRTVPLYESPRKICYQEVSQCFGVLSSRIEVQDTSGGTTALRPSASTQALSSSVSSSKLFSSS TAPHETSFGEEVEVHNLLIIDQHTFEVLHAHQFLQNEYALSLVSCKLGKDPNTYFIVGTAMVYPE EAEPKQGRIVVFQYSDGKLQTVAEKEVKGAVYSMVEFNGKLLASINSTVRLYEWTTEKELRTE CNHYNNIMALYLKTKGDFILVGDLMRSVLLLAYKPMEGNFEEIARDFNPNWMSAVEILDDDNFL GAENAFNLFVCQKDSAATTDEERQHLQEVGLFHLGEFVNVFCHGSLVMQNLGETSTPTQGSV LFGTVNGMIGLVTSLSESWYNLLLDMQNRLNKVIKSVGKIEHSFWRSFHTERKTEPATGFIDGD LIESFLDISRPKMQEVVANLQYDDGSGMKREATADDLIKVVEELTRIHSGSSLVPRGSHHHHHH

[0483] Full-length DDB1 was cloned into pFastBad with a C-terminal thrombin-cleavable 6xHis tag. CRB (41-442) was cloned into pFastBad with an N-terminal TEV-cleavable Strep tag. The two plasmids were used separately to generate recombinant baculovirus using the Bac-to-Bac® method (Invitrogen), stored as baculovirus-infected insect cell (Bl IC) stocks. For large-scale expression 10 L of Sf21 cells were prepared at a viable cell density of 1 ,3x106 / mL in Sf-900TM ARVN0199WO2; ARVN-199-PCT

[0484] II media supplemented with 5 pg / mL Gentamicin. The cells were co-infected with DDB1 and CRB viruses at a multiplicity of infection (MOI) of 1 (assuming 100 pfu / BIIC). The cells were incubated at 27 °C and harvested 72 h post-infection. The cell pellet was lysed by sonication (35% amplitude, 2 sec on, 2 sec off for 3 min) in lysis buffer [50 mM HEPES pH 7.5, 300 mM NaCI, 1 mM TCEP, 10 mM Imidazole, and a protease inhibitor tablet]. The lysate was clarified by high-speed centrifugation (19,000 x g, 20 min) and the supernatant was used in subsequent purification by immobilized metal affinity chromatography (IMAC). Clarified lysate was applied to a HisTrap Crude FF column (5 ml, Cytiva) and washed with wash buffer [50 mM HEPES pH 7.5, 300 mM NaCI, 1 mM TCEP, and 20 mM imidazole]. Protein was eluted over a gradient of wash buffer and elution buffer [50 mM HEPES pH 7.5, 300 mM NaCI, 1 mM TCEP, 500 mM imidazole] over 20 column volumes by FPLC (AKTA, Cytiva). Eluted protein was incubated with TEV protease while dialyzed into dialysis buffer [50 mM HEPES pH 7.5, 300 mM NaCI, 1 mM TCEP, 10 mM Imidazole] overnight at 4°C. The cleaved protein sample was diluted 6-fold with no-salt buffer [25 mM Tris pH 8, 1 mM TCEP] and loaded onto a 5 ml HiTrap Q column (Cytiva). The column was washed with 10 column volumes of buffer A [25 mM Tris pH 8, 50 mM NaCI, 1 mM TCEP], and protein was eluted over a gradient of buffer A and buffer B [50 mM Tris pH 8, 800 mM NaCI, 1 mM TCEP] over 50 column volumes. Fractions containing the CRBN / DDB1 complex were concentrated and applied to and S200 26 / 60 column (Cytiva) pre-equilibrated with storage buffer [50 mM HEPES pH 7.3, 150 mM NaCI, 1 mM TCEP], Fractions containing pure protein were concentrated to 4.3 mg / mL and stored at -80QC

[0485] Expression and purification of Biotinylated Avitag-KRAS 4B (1-169) G12D:

[0486] Unless otherwise noted,

[0487] Reagents:

[0488] HEPES Buffer (Fisher BioReagents, BP299-1) Sodium Chloride (Fisher Scientific, 50-227-4160) TCEP (UBPBio, P1021 -100) Magnesium Chloride (Millipore, 442611 )

[0489] Protein Sequence:

[0490] GLNDIFEAQKIEWHEMTEYKLVVVGADGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDV PMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEK ARVN0199WO2; ARVN-199-PCT

[0491] Gene Sequence:

[0492] ATGCATCATCATCATCATCACAGCAGCGGCAGAGAAAACTTGTATTTCCAGGGCCT GAATGACATCTTTGAAGCGCAGAAGATCGAATGGCACGAGATGACTGAATATAAACTTGTG GTAGTTGGAGCTGATGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATT TTGTGGACGAATATGATCCAACAATAGAGGATTCCTACAGGAAGCAAGTAGTAATTGATGG AGAAACCTGTCTCTTGGATATTCTCGAGACAGCAGGTCAGGAGGAGTACAGTGCAATGAG GGACCAGTACATGAGGAGTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAA TCATTTGAAGATATTCACCATTATAGAGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGT ACCTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAG GCTCAGGACTTAGCAAGAAGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACA GGGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGTGA

[0493] KRAS constructs comprising residues 1 -169 harboring a G12D mutation and an N- terminal His-Avi tag were expressed recombinantly in E. coli BL21 . BirA enzyme was coexpressed for efficient in vivo biotinylation of the Avi-tag. After cell lysis, KRAS was purified from the soluble fraction via IMAC. The biotinylated protein was further enriched using SoftLink Soft Release Avidin resin, and polished by size exclusion chromatography using a HiLoad 2660 Superdex 75 column equilibrated in 20 mM HEPES / NaOH, 150 mM NaCI, 5 mM MgCI2, 1 mM DTT, pH 7.5. KRAS was concentrated to 8-10 mg / mL and stored at -80°C. The protein was thawed and incubated with TEV protease (Biovision 7847-1000) for 3 days at 4eC. The protein was passed through 1 ml of HisPur™ Cobalt Resin (Thermo Scientific, 89965) in reverse IMAC format where the flow-through and wash samples were collected. The column was washed with 5 ml of storage buffer. The combined flow-through and wash fractions were concentrated using an 10K MWCO centrifugal concentrator (Amicon-15, Millipore) and purified by size exclusion chromatography on a Superdex 75 10 / 300 GL column (Cytiva) pre-equilibrated with storage buffer II [50 mM HEPES pH 7.5, 100 mM NaCI, 5 mM MgCh, and 1 mM TCEP]. Fractions containing pure biotinylated Ras protein were pooled and concentrated to 0.82 mg / mL and stored at -80QC.

[0494] 1.10. Cell-free CRBN-based KRAS Ubiquitination Assay

[0495] Biotin-Avi tagged-KRAS 1 -169 G12D was tested in a TR-FRET assay for direct ubiquitination by CRBN using KRAS as the FRET donor and Cy5-labeled ubiquitin (Ub) as the acceptor. The ubiquitination reactions were setup by mixing 4, 2.5 pL master mixes sequentially to a white, 384-well low volume ProxiPlate (Revvity cat#6008280). First, Compound 1 , ARVN0199WO2; ARVN-199-PCT

[0496] Compound 31 , and Compound 32 were serially diluted in reaction buffer (25 mM HEPES pH 7.4, 100 mM NaCI, 10 mM MgCk, 2 mM DTT, 100 pg / mL BSA) and dispensed on an assay plate with a 50 nM top 3-fold, 12-point titration (1 .25% DMSO) final concentration in the assay. Second, Biotin-Avi tagged-KRAS 1-169 G12D diluted in reaction buffer was then added to the wells for a final concentration of 5 nM. The plate was incubated at RT for 90 minutes before adding reaction buffer containing recombinant human polypeptides of 15 nM final concentration of UBE1 / E1 -activating enzyme (R&D Systems, cat #E-305), 400 nM UBE2D1 / E2-conjugating enzyme (R&D Systems, cat #E2-616), and 10 nM Cul4A / Rbx1 / DDB1 / CRBN E3-ligase complex (R&D Systems, cat# E3-650-025). Finally, the detection reagents along with an excess of ATP and unlabeled Ub diluted in reaction buffer was added to the plate; 1 nM Eu-W1024 labeled streptavidin (Revvity, cat # AD0062), 300 nM Cy5-Ub (South Bay Bio, cat #SBB-TR0015), 3 pM Ub (R&D Systems, cat #U-100H), and 2 mM ATP (MilliporeSigma, cat #A2383-1g). The plate was incubated at RT for 5.5 hours before being read on the Envision 2105 (Revvity) as in section 1 .8. Using a protocol described or similarly described herein, the results are shown in Fig. 5C.

[0497] 1.11. Time Course Treatment of AsPC-1 Spheroids

[0498] AsPC-1 cells were grown as spheroids in RPMI, 10% FBS in ULA plates (Corning, cat #4520) at 2x106per well in 200 pL medium and placed in a CO2 incubator overnight to form spheroids. Compound 1 was dosed the next day with 0.1 mM DMSO stocks using a D300E compound dispenser (Tecan) with a top dose of 100 nM, half log steps and normalizing to the highest DMSO concentration (0.1 %) and returned to the incubator for 24, 48 or 72 hours. Spheroids were harvested by filtration using a pre-wet filter plate (Millipore, cat #MSBVN1210) and lysed in MSD Tris Lysis Buffer, 80 pL per well with shaking at 4 -C, 15 minutes. Lysates were collected by centrifugation at 1000xg for 5 minutes in polypropylene 96-well plates and mixed with 5x reducing SDS sample buffer (ProteinSimple) and denatured at 95QC for 5 minutes. Samples were analyzed by capillary electrophoresis on a JESS instrument (ProteinSimple) with a 12-230kDa cartridge and probed for KRAS G12D (CST, cat #14429) and pERK (CST, cat #9101 ). The peak area was normalized to total protein in the capillary.

[0499] 1.12. Apoptosis Assay

[0500] AsPC-1 , GP2d and SK-LU-1 cells were grown as spheroids in RPMI, or DMEM, or EMEM supplemented with 10% FBS, P / S in ULA plates (SBio, cat #MS-9096UZ), at a density of 2000 cells / well in 0.2 mL complete medium and allowed to recover overnight. Compounds were ARVN0199WO2; ARVN-199-PCT added the next day using the D300E dispenser (Tecan) with a top dose of 1 pM and 1 / 3 log steps for 9 points total. Caspase 3 / 7 dye (Satorius, cat #4440) was added to the cultures at 5 pM, and the spheroids were imaged on an Incucyte instrument (Satorius), collecting brightfield, phase and green fluoresce images with a 4x objective every 12 hours for 3 days. The spheroid image analysis algorithm was used to quantify the green fluorescence based on the spheroid area / well. Each data set was normalized to the baseline level in time=0 measurement and then expressed as % of baseline caspase activation. Two independent datasets were collected for each cell line.

[0501] 1.13. 3D Proliferation Assay

[0502] 3D proliferation assays were used to test the activity of Compound 1 in inhibiting proliferation of cancer cells grown as spheroids. 500 cells / well of various KRAS G12D mutant cell lines were added to a 384-well ULA spheroid plate (Corning, cat #3830) and allowed to aggregate into spheroids for 24 hours. The spheroids were then treated with a 10-step 3-fold serial dilution of Compound 1 with a top concentration of 300 nM or a DMSO vehicle control in at least duplicate and then incubated at 37eC, 5% CO2. After 5 days of treatment, cell viability of the spheroids was measured with the CellTiter-Glo 3D reagent (Promega, cat #G9683). A protocol was adapted from manufacturer’s recommendations for this reagent, which can be found in section 1.15. Luminescent signal readout was completed on an Envision 2105 (Revvity) plate reader. Proliferation was also measured by image analysis of spheroid area on the Incucyte (Satorius). Images were collected every 12 hours for 6 days with a 4x objective and the spheroid area was calculated using the spheroid algorithm (Satorius). Data was plotted as a percentage of the vehicle (DMSO) control and the IC50 of proliferation was calculated for each cell line. Data was obtained for two biological replicates per cell line.

[0503] 1.14. 3D Proliferation Assay Protocol

[0504] Materials:

[0505] • Corning 384 well, black with clear round bottom ULA spheroid plates (Corning, #3830)

[0506] • Cell Titer GLO3D reagent (Promega #G9683)

[0507] • Complete media (cell line dependent)

[0508] Day 1 - Plating Cells:

[0509] • Cells were rinsed, trypsinized, and counted. Cells were then diluted to 1 1 ,000 cells / mL and the Multi-drop Combi dispenser was used to plate 45 pL of cell suspension to wells in columns 1 -23. Column 24 received 45 pL of media only.

[0510] • Plates were returned to incubator overnight to allow spheroids to form. ARVN0199WO2; ARVN-199-PCT

[0511] Day 2 - Treating cells and reading baseline plate:

[0512] • A 3-fold, 10-step, 300mM top concentration serial dilution of desired compounds was prepared in DMSO (this gave a 300uM final concentration on cells in 0.1% DMSO).

[0513] • A 1 pL stamp copy of serial dilutions was made into a Grenier (#781280) clear 384 well plate. Compound serial dilutions were horizontal from columns 1 -10 and 11 -20. DMSO (1 pL) was added for vehicle controls in remaining columns (final DMSO will be 0.1 %).

[0514] • The 1 pL stamp copy plate was re-suspended with 99 pL of complete media and was mixed 10x with the Integra384 well Viaflow pipetting station.

[0515] • Compounds (5 pL) were repeat dispensed into the assay plates.

[0516] • Assay plates were returned to incubator for 5 days.

[0517] 5 days post-treatment - Reading assay plates:

[0518] • Cell assay plates were cooled to room temperature and 12 mL aliquots of CTG 3D reagent were thawed.

[0519] • CTG3D reagent (30 pL) was added to all wells

[0520] • Assay plates were incubated for 5 minutes

[0521] • Assay plates were shaken vigorously for 5 seconds with Combi dispenser

[0522] • Assay plates were incubated plate for 15 minutes

[0523] • The Integra384 Viaflow pipetting station was used to mix 12x (to completely disperse / lyse the spheroids)

[0524] • Assay plates were incubated for an additional 15 minutes

[0525] • Plates were sealed with clear seal (VWR#60941 -078) and were read on Envision reader with “Pete 384 LUMI” protocol.

[0526] • MS Excel and GraphPad PRISM were used to analyze data, fit curves, and determine IC5o values

[0527] Using a protocol described or similarly described herein, the results are shown in Fig. 8.

[0528] Degradation data is shown below in Table 1.

[0529] Table 1. ARVN0199WO2; ARVN-199-PCT 2

[0530] Values are mean ± STD with n > 3 biological replicates

[0531] Example 3: Pharmacology of Compound A in Xenograft Models

[0532] 3. Methods and Materials

[0533] 3.1. Cell Culture

[0534] Each human derived cell line used in this study was cultured in the medium recommended by the vendor and maintained at 37 °C, 5% CO2. AsPC-1 and SW1990 were purchased from the American Type Culture Collection (ATCC), GP2d was purchased from Millipore Sigma. All cell lines were verified by short tandem repeat (STR) analysis. Cells were seeded into HYPER flasks (Corning) as per manufacturer’s instructions, grown for eight days, and harvested for implantation into mice at a final concentration of 25 x 10scells / mL in 50% Matrigel (Corning) and 50% phenol red-free media (Gibco DMEM or RPMI-1640, depending on cell line). For detailed culturing conditions see section 3.2.

[0535] 3.2. Reagents for Cell Culture and Xenograft Implantation

[0536] DMEM - Gibco I ThermoFisher # 10566016

[0537] RPMI-1640 - Gibco I ThermoFisher # 61870036

[0538] DMEM (phenol red-free) - Gibco / ThermoFisher #21063029 RPMI-1640 (phenol red-free) - Gibco / ThermoFisher #1 1835030 DPBS - Gibco / ThermoFisher #14190144

[0539] Trypsin / EDTA - Gibco / ThermoFisher #25200056

[0540] FBS - Gibco / ThermoFisher #26140079

[0541] Penicillin-Streptomycin (PenStrep) - Gibco / ThermoFisher #15140122

[0542] EMEM - ATCC #302003 ARVN0199WO2; ARVN-199-PCT

[0543] Matrigel - Corning #354234

[0544] HYPER flask - Corning# 10034

[0545] All cell lines were grown in tissue culture flasks within humidified 37 °C, 5% CO2 incubators and detached via trypsin for passaging and expansion within sterile biosafety cabinets. The growth conditions for each cell line used in this study is listed below: GP2d cells (Millipore Sigma, USA) were maintained at ~2-3 x 105cells / mL, passaged once per week at a ratio of 1 :12 in DM EM (Gibco) + 10% FBS (Gibco) + 1% PenStrep (Gibco) at 37 °C, 5% CO2. This model was implanted into mice in DMEM (phenol red-free, Gibco) + 50% Matrigel (Corning).

[0546] AsPC-1 cells (ATCC, USA) were maintained at ~1-2 x 106cells / mL, passaged once per week at a ratio of 1 :10 in RPMI-1640 (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37 °C, 5% CO2. This model was implanted into mice in RPMI-1640 (phenol red-free, Gibco) + 50% Matrigel (Corning).

[0547] SW1990 cells (ATCC, USA) were maintained at ~1-2 x 106cells / mL, passaged once per week at a ratio of 1 :10 in DMEM (Gibco) + 10% FBS + 1% PenStrep (Gibco) at 37 °C, 5% CO2. This model was implanted into mice in DMEM (phenol red-free, Gibco) + 50% Matrigel (Corning).

[0548] Panc04.03 and HPAC cells were prepared using similar protocol as described herein.

[0549] 3.3. Murine Xenograft Models

[0550] CB17 SCID female mice purchased from Charles River Laboratories were 6-8 weeks old at arrival, acclimatized for 5-7 days and implanted with 200 pL of cell suspension (5 x 106cells mouse) subcutaneously on the left flank under sterile conditions. Studies were initiated when tumors reached an average of approximately 200 mm3. Tumor dimensions were measured with calipers and the tumor volume calculated according to:

[0551] Tumor volume = (width x width x length) / 2 where all measurements are in mm and the tumor volume is in mm3.

[0552] Tumor measurements were performed twice a week throughout efficacy studies and the TGI was calculated using the following formula:

[0553] % TGI = (Vc-Vt) / (Vc-Vo) x 100 where Vc and Vt are the average tumor volume of control and treated groups at the end of the study, and Vo is the average tumor volume of all arms at the beginning of the study. ARVN0199WO2; ARVN-199-PCT

[0554] Mice were dosed IV either once per week (QW), twice per week (BiW), or once every 2 weeks (Q2W). Compounds were formulated by adding to vehicle (30% hydroxypropyl- - cyclodextrin [HPbCD] in 10 mM citrate buffer, pH 4.0) and sonicating until fully dissolved (pH was adjusted as needed). Efficacy studies were run from 14-28 days depending on growth rate (or other ethical considerations) of each tumor model tested. Under anesthesia and upon cessation of heartbeat, blood was collected via cardiac punch or trunk blood collected postdecapitation. Whole blood was collected in Lithium-Heparin 2 mL blood collection tubes and centrifuged within 30 minutes at 4000xg for seven minutes at 4 °C. Plasma was aliquoted into a 96 well plate and stored at -80 °C until analysis. Samples were shipped on dry ice to Drumetix Laboratories, North Carolina for pharmacokinetic (PK) analysis. Approximately 1 mg of dry compound in a 1-dram vial was shipped to use as a standard reference. Tumors harvested at the indicated timepoints post-dose were snap frozen on dry ice and stored at -80 °C until needed. Using a protocol described or similarly described herein, the Results are shown in Fig. 4A, 4B, 4C, 4D, 4E, 10, 13A and 13B.

[0555] For PK / PD time course study, mice were dosed with a single IV administration of 3 mpk of Compound 1 . Tumors were collected at various time points between 6 hours and 168 hours post dose and concentration of Compound 1 was assessed along with levels of KRAS G12D and multiple relevant signal markers. Using a protocol described or similarly described herein, the results are shown in Fig. 3A, 3B, 3C, 3D, 9A, 9B, 11 , 12, 14B, 15A, 15B, 16A, 16B, 17A and 17B.

[0556] 3.4. Tumor Lysate Preparation and Immunoblotting

[0557] See section 3.5 for a detailed materials list. Complete radio-immunoprecipitation assay (RIPA) buffer (containing protease inhibitor, phosphatase inhibitor, and EDTA) was added to frozen tumor pieces at a volume of 1 pL / mg of tumor tissue in a 2 mL tube. A single metal bead (5mm) was added to each tube and tumors were lysed by rapid shaking in a TissueLyser II (Qiagen) for 4 minutes at 24 Hz at 4 °C. Beads were removed from tubes and lysates were centrifuged for 15 minutes at 4 °C at 15000 rpm in a benchtop microcentrifuge. The supernatants (cleared lysates) were collected and used to make 1 :30 dilutions in complete RIPA buffer. The concentrated and diluted tumor lysates were frozen at -80 °C. Total protein concentration was determined with the bicinchoninic acid (BCA) assay, run according to manufacturer’s protocol (ThermoFisher) with provided bovine serum albumin (BSA) standard ARVN0199WO2; ARVN-199-PCT curve (range = 2 mg / mL - 0.1 mg / mL) in 96 well plate format. The previously prepared 1 :30 lysate dilutions were used for both the BCA assay and for preparation of gel samples. Plates were read (OD562) using a BioTek Cytation3 plate reader running Gen5 3.11 software. Protein concentration of tumor lysates was determined with Graph Pad Prism by interpolating sample ODs with BCA assay standard curve. Lysates were normalized and prepared for SDS-PAGE by combining an appropriate volume of lysate, sample buffer, reducing agent, and ultrapure water. Gel samples were prepared to allow equal volume loading of 8 pg of total protein / lane for all tumor samples. Gels were loaded with 8 pg of total protein per well on 26 well, 4-12% Bis-Tris gels (ThermoFisher). Bio-Rad Precision Plus molecular weight markers were loaded into lanes 1 and 26 (4 pL per lane). Gels were electrophoresed using MOPS / SDS running buffer for 65 minutes at 150 volts. Protein was transferred to nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo semi-dry system. Transfer protocol conditions were midi gel format at 25 volts, 2.5 amps, for 7 minutes.

[0558] Membranes were cut into horizontal strips at desired molecular weights and then blocked for 1 hour in 3% BSA / TBS-T. Primary antibodies were diluted as per manufacturer’s instructions in 3% BSA / TBS-T and incubated overnight at 4 °C with gentle rocking. Membranes were then washed 5 x 5 minutes with TBS-T at room temperature. Secondary antibodies were diluted 1 :20000 in 3% BSA / TBS-T and incubated at room temperature for 1 hour. Membranes were washed 5 x 5 minutes with TBS-T. Membranes were developed for 2 minutes with SuperSignal™ West Femto Maximum Sensitivity Substrate and imaged with a Bio-Rad ChemiDoc MP imager running Image Lab software v 5.2.1. Densitometry analysis was done using Imaged software, all samples were normalized to a loading control and graphed as a percent of control as compared to vehicle treated tumors. A one-way ANOVA with Dunnett’s multiple comparisons was used to determine statistical significance of results.

[0559] 3.5. Reagents for Tumor Immunoblot Analysis

[0560] RIPA buffer - ThermoFisher #89900

[0561] HALT protease inhibitor - ThermoFisher #78437 HALT phosphatase inhibitor - Thermofisher #78420 EDTA, 0.5M - Thermofisher #1861275 BCA Protein Assay kit - ThermoFisher #23225 NuPAGE 4x LDS sample buffer - ThermoFisher #NP0007 Bolt 10x sample reducing agent - Thermofisher #B0009 NuPAGE 4-12% Bis-Tris Midi gel, 26 well - ThermoFisher #WG1403BX10 ARVN0199WO2; ARVN-199-PCT

[0562] BioRad Precision Plus Dual Color ladder - BioRad #1610374 Trans-blot midi Nitrocellulose transfer pack - Biorad #1704159 MOPS / SDS running buffer - Thermofisher #NP0001 -02 SuperSignal™ West Femto - ThermoFisher #34095 TBS-T (1X TBS with 0.1% tween-20)

[0563] BSA, bovine serum albumin, fraction V - AmericanBio #AB00440-01000

[0564] 5 mm stainless steel bead - Qiagen #69989

[0565] Ras (G12D mutant specific)(D8H7) Rabbit mAb - Cell Signaling Technology #14429S Vinculin (E1 E9V) XP Rabbit mAb - Cell Signaling Technology #13901 S Anti-rabbit IgG, HRP-linked Antibody - Cell Signaling Technology #7074P2 Phospho-ERK1 / 2 (Thr202 / Tyr204) rabbit antibody - Cell Signaling Technology #9101 c-MYC (Y69) rabbit antibody - Abeam #ab32072

[0566] Anti-mitochondria (Mito.c) mouse monoclonal antibody [113-1] - Abeam #92824

[0567] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0568] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

ARVN0199WO2; ARVN-199-PCTCLAIMS1 . A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I:or a pharmaceutically acceptable salt thereof, wherein the cancer is pancreatic ductal adenocarcinoma, non-small cell lung cancer, or colorectal cancer; and wherein:Q1, Q2, Q3, Q4, Q5, and Q6are each independently CRLor N;Q7is selected from C(R4)2, C(O), and NR4;R1and R2are each independently selected from H, halo, Ci-e alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, and C1-6 haloalkyl;R3and R4are each independently selected from H, halo, C1-6 alkyl, C1-6 alkoxy, and C1-6 haloalkyl; each RLis independently H, halo, or CN; and p and q are each independently 1 , 2, 3, 4, 5, or 6.

2. The method of claim 1 , wherein the compound of Formula I is a compound of Formula la:ARVN0199WO2; ARVN-199-PCTla, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 , wherein the compound of Formula I is a compound of Formula lb:lb, or a pharmaceutically acceptable salt thereof.

4. The method of any one of claims 1-3, wherein R1and R2are each independently selected from H, halo, C1-6 alkyl, and C2-6 alkynyl.

5. The method of any one of claims 1-4, wherein R1is halo or C1-6 alkyl.

6. The method of any one of claims 1-5, wherein R2is selected from H, C1-6 alkyl, and C=CH.ARVN0199WO2; ARVN-199-PCT7. The method of any one of claims 1-6, wherein R3is selected from H, halo, and C1-6 alkoxy.

8. The method of any one of claims 1-7, wherein each R4is independently H or C1-6 alkyl.

9. The method of any one of claims 1-8, wherein each RLis independently H or halo.

10. The method of any one of claims 1-9, wherein Q1, Q2, Q3, Q4, Q5, and Q6are each independently is independently CH or N.1 1. The method of any one of claims 1-10, wherein Q7is CH2 or C(O).

12. The method of any one of claims 1-11 , wherein p and q are each independently 1 , 2, 3, or 4.

13. The method of any one of claims 1-12, wherein p and q are each independently 1 , 2, or 3.

14. The method of any one of claims 1-3, whereinQ1, Q4, and Q6are N;Q2, Q3, and Q5are each independently CRL;Q7is C(R4)2;R1is halo;R2is C2-6 alkynyl;R3is halo; each R4is H; each RLis H; and p and q are each 1 .

15. The method of any one of claims 1-13, wherein the compound is selected from:ARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTARVN0199WO2; ARVN-199-PCTor a pharmaceutically acceptable salt thereof.

16. The method of any one of claims 1-13, wherein the compound is selected from:ARVN0199WO2; ARVN-199-PCTor a pharmaceutically acceptable salt thereof.

17. The method of any one of claims 1-16, wherein the cancer is pancreatic ductal adenocarcinoma.

18. The method of any one of claims 1-16, wherein the cancer is non-small cell lung cancer.

19. The method of any one of claims 1-16 or 18, wherein the cancer is non-squamous non- small cell lung cancer.

20. The method of any one of claims 1-16, wherein the cancer is colorectal cancer.

21. The method of any one of claims 1-16 and 20, wherein the cancer is non-squamous colorectal cancer.

22. A method of treating pancreatic ductal adenocarcinoma in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :ARVN0199WO2; ARVN-199-PCT1 , or a pharmaceutically acceptable salt thereof.

23. A method of treating colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :1 , or a pharmaceutically acceptable salt thereof.

24. A method of treating non-squamous non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :ARVN0199WO2; ARVN-199-PCT1 , or a pharmaceutically acceptable salt thereof.

25. A method of treating non-squamous colorectal cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :1 , or a pharmaceutically acceptable salt thereof.

26. A method of treating non-small cell lung cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound that is Compound 1 :ARVN0199WO2; ARVN-199-PCT1 , or a pharmaceutically acceptable salt thereof.

27. The method of any one of claims 1-26, wherein the therapeutically effective amount of Compound 1 is administered to the subject once per week.

28. The method of any one of claims 1-26, wherein the therapeutically effective amount of Compound 1 is administered to the subject once every two weeks.

29. The method of any one of claims 1-28, wherein the subject is in a fed state at the time of administration.

30. The method of any one of claims 1-28, wherein the subject is in a fasted state at the time of administration.31 . The method of any one of claims 1-30, wherein the therapeutically effective amount of Compound 1 is about 5 mg to about 125 mg.

32. The method of any one of claims 1-31 , wherein the therapeutically effective amount of Compound 1 is about 5 mg to about 100 mg.

33. The method of any one of claims 1-32, wherein the therapeutically effective amount of Compound 1 is about 5 mg to about 75 mg.ARVN0199WO2; ARVN-199-PCT34. The method of any one of claims 1-33, wherein the therapeutically effective amount of Compound 1 is about 5 mg to about 50 mg.

35. The method of any one of claims 1-34, wherein the therapeutically effective amount of Compound 1 is about 5 mg to about 30 mg.

36. The method of any one of claims 1-35, wherein the therapeutically effective amount of Compound 1 is about 10 mg to about 30 mg.

37. The method of any one of claims 1-36, wherein the therapeutically effective amount of Compound 1 is about 10 mg to about 20 mg.

38. The method of any one of claims 1-28, wherein the therapeutically effective amount of Compound 1 is about 10 mg to about 250 mg.

39. The method of any one of claims 1-38, wherein the therapeutically effective amount of Compound 1 is about 10 mg.

40. The method of any one of claims 1-38, wherein the therapeutically effective amount of Compound 1 is about 15 mg.41 . The method of any one of claims 1-38, wherein the therapeutically effective amount of Compound 1 is about 20 mg.

42. The method of any one of claims 1-38, wherein the therapeutically effective amount of Compound 1 is about 25 mg.

43. The method of any one of claims 1-38, wherein the therapeutically effective amount of Compound 1 is about 30 mg.

44. The method of any one of claims 1-43, wherein the method further comprises administering to the subject an effective amount of at least one additional anti-cancer agent.

45. A compound selected from:ARVN0199WO2; ARVN-199-PCTin free or pharmaceutically acceptable salt form.

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