Fatty acid elongase attenuator compounds targeting KRAS mutant variants and their uses

Abstract

Provided herein are novel inhibitors of ELOVL6. Also provided herein are methods of use of the novel inhibitors to inhibit the growth of KRAS-dependent cells across KRAS mutant variants.

Description

FATTY ACID ELONGASE ATTENUATOR COMPOUNDS TARGETING KRAS MUTANT VARIANTS AND THEIR USESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 658,415 filed June 10, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] KRAS is a commonly mutated oncogene and therapeutic targeting of its mutants is critical for the development of new cancer therapies (1-4). KRAS mutations are frequently observed in highly fatal cancers, including pancreatic ductal adenocarcinoma (86%), colorectal cancer (41%), and non small-cell lung cancer (32%) (2). Notably, the G12 codon accounts for 89% of the mutant KRAS variants (5). Covalent inhibitors have emerged over the last several years that target G12C KRAS via the thiol moiety on cysteine and trap the protein in its GDP bound inactive form (6, 7). While the KRAS G12C inhibitors have shown potent target engagement and encouraging results in the clinic (8-11), using this strategy to selectively inhibit protein function is not applicable to other KRAS mutants. The G12V KRAS variant, prevalent in colorectal (30%), pancreatic (35%), and lung (23%) cancer, represents a significant therapeutic challenge with no G12V KRAS therapeutics yet available (2).[0(103] Therapeutics that impede the activity of oncogenic mutants of KRAS offer significant clinical impact. Despite the recent success in targeting the G12C mutant, targeted therapy for most prevalent KRAS mutant variants - G12V KRAS, G12D KRAS - has lagged far behind. There are no existing alternative options for small-molecule inhibitor molecules based allele- selective targeted degradation of G12V or G12D KRAS mutant proteins. Herein is described the generation of novel chemical inhibitors of ELOVL6 that show high potency and efficacy in cell culture systems. The novel compounds exhibit the potential to be selective cytotoxic agents against KRAS-dependent cancer cells. The compounds may have further applications involving modulation of de novo lipid biogenesis, inner membrane leaflet protein membrane anchoring, and modulation of cellular signaling cascades via ELOVL6 inhibition.SUMMARY[00041 KRAS remains one of the deadly cancers with KRAS mutations accounting for up to 90% of all pancreatic cancers, 45% of colorectal and 35% of lung cancers. Globally there are 9.6 million deaths from cancer every year with an annual economic cost exceeding $1.16 trillion. For the 4.3 million new KRAS mutation diagnoses every year (> 250,000 in the US), current standard of care is chemotherapy or tyrosine and / or MEK kinase inhibitors that are susceptible to adaptive therapeutic resistance, limiting their long-term utility as therapeutics. To date, there are no small-molecule based allele-selective degraders for mutant KRAS protein.

[0005] ELOVL6, a druggable enzyme, can be targeted to control the production of lipids that are exploited by mutated forms of KRAS for function and trigger the targeted degradation of the protein. Here is described the generation of novel chemical inhibitors of ELOVL6 that show high potency and efficacy in cell culture systems. Here Applicant describes the use of small-molecule ELOVL6 inhibitors that leads to the selective degradation of mutant KRAS protein. The ELOVL6 functional activity attenuation based selective clearance of mutant KRAS protein is anticipated to mitigate the incidence of therapeutic resistance, a common occurrence and significant liability of existing anti-KRAS therapies.

[0006] Described herein are compounds which target KRAS mutant variants including G12V, G12D and G13D. Collectively, these variants account for over 90% of the existing KRAS patient population.BRIEF DESCRIPTION OF THE FIGURES(0007[ FIGURES 1A-1E. ELOVL6 genetic validation and anti-KRAS therapeutic potential assessment. (FIG. 1A) Colony formation and cellular proliferation assessment of unmodified wild type KRAS expressing HT-29 and CRISPR-edited polyclonal companion ELOVL6 knock-out cells. Error bars indicate standard deviations across three independent experiments. (FIG. IB) Colony formation and cellular proliferation evaluations of unmodified mutant KRAS G12V variant expressing homozygous KRAS mutant cell line (SW480) and the CRISPR-edited polyclonal companion ELOVL6 knock-out cells. Error bars indicate standard deviations across three independent trials. (FIG. 1C) Flow cytometry basedtotal KRAS protein expression analysis of unmodified parental and CRISPR-edited EL0VL6 knockout companion cells in wild type (HT-29) and heterozygous KRAS G12V mutant cell lines from colorectal (SW403) and lung cancer (NCI-H441) background. (FIG. ID) Flow cytometry based endogenous KRAS protein expression and cell viability analysis of wild type (HT-29) and KRAS mutant (NCI-H441) cells treated with small molecule EL0VL6 inhibitor. Error bars indicate standard deviations across multiple trials. (FIG. IE) Quantitative in vivo anti -turn or activity assessment of EL0VL6 inhibitor using heterozygous KRAS xenografts (NCIH441) in nude mice. The data represents mean tumor volume ±SEM from 10 animals dosed twice daily (100 mg / kg). ELOVL6 inhibitor in FIG. IE has the formula according to Nagase et a / :(2009) J. Med. Chem. 52, 4111-4114. FIG. 1A-1E * indicates p <0.05, ** indicates p<0.01, *** indicates p<0.001, n.s. indicates not significant.

[0008] FIGURES 2A-2C. Development of novel ELOVL6 functional activity attenuators as mutant KRAS selective cytotoxic agents. (FIG. 2A) Schematic depiction of the privileged scaffold and chemical derivatives generated for the SAR analysis to identify novel ELOVL6 functional activity attenuators. (FIG. 2B) Cell viability analysis of wild-type KRAS expressing (control) and mutant KRAS expressing (test) cell lines treated with the indicated ELOVL6 inhibitors. Viability analysis was performed after 3 days of treatment. Each inhibitor was tested at 30pM.. (FIG. 2C) Cell viability analysis of wild-type KRAS expressing (control) and mutant KRAS expressing (test) cell lines treated with the indicated ELOVL6 inhibitor. Viability analysis was performed after 7 days of treatment. Each inhibitor was tested at 30pM. FIG. 2B-2C * indicates p<0.05, ** indicates p<0.01, n.s. indicates not significant.

[0009] FIGURES 3A-3D. Functional evaluation of novel ELOVL6 functional activity attenuators as mutant KRAS selective cytotoxic agents. (FIG. 3A) ELOVL6 inhibitor treatment response in wild type KRAS expressing cell line (HT-29 KRAS wild-type). Each inhibitor was tested at 30 pM. Cells were treated for 7 days before cell viability measurements. Error bars indicate standard deviation. UN: untreated control cells. 1W: reference compound 1W. # indicates p values < 0.05. ** indicates p-values < 0.01. n.s. not significant. (FIG. 3B) ELOVL6 inhibitor treatment response in G12V mutant KRAS expressing cell line (NCI-H727 KRAS G12V). Each inhibitor was tested at 30 pM. Cellswere treated for 7 days before cell viability measurements. Error bars indicate standard deviation. UN: untreated control cells. 1W: reference compound 1W. # or * indicates p values < 0.05. ** indicates p-values < 0.01. *** indicates p-values < 0.001. (FIG. 3C) ELOVL6 inhibitor treatment response in G12D mutant KRAS expressing cell line (HP AC G12D). Each inhibitor was tested at 30 pM. Cells were treated for 7 days before cell viability measurements. Error bars indicate standard deviation. UN: untreated control cells. 1W: reference compound 1W. # or * indicates p-value < 0.05. ** indicates p-values < 0.01. *** indicates p-values < 0.001. (FIG. 3D) Quantitative cell viability assessment of dosedependent novel ELOVL6 inhibitor treatment response in mutant KRAS expressing cell line (HP AC KRAS G12D). Cells were treated for 7 days before cell viability measurements. Error bars indicate standard deviation.

[0010] FIGURES 4A-4C. Functional evaluation of novel ELOVL6 functional activity attenuator as mutant KRAS selective cytotoxic agent. (FIG. 4A) Chemical structure of a top candidate novel chemical entity displaying selective cytotoxicity in mutant KRAS expressing cell lines. (FIG. 4B) Quantitative cell viability assessment of dose-dependent novel ELOVL6 inhibitor treatment response in wild-type KRAS expressing HT-29 control cell line. n.s. not significant. (FIG. 4C) Quantitative cell viability assessment of dosedependent novel ELOVL6 inhibitor treatment response in mutant KRAS expressing HP AC G12D test cell line. *** indicates p-values < 0.001. In FIGS. 4B and 4C cells were treated for 7 days before cell viability measurements.DETAILED DESCRIPTION

[0011] The following documents, each of which is incorporated herein by reference in its entirety, may be useful for understanding this disclosure: (1) WO2011103516; (2) U.S. Provisional Patent Application No. 63 / 658,416 filed June 10, 2024; (3) T. Nagase, J. Med. Chem. 52, 4111-4114 (2009); (4) Takahashi, J Med Chem 2009 52(10):3142-5.

[0012] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0013] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the disclosure. All the various embodiments of the present disclosure will not be described herein. Many modifications and variations of the disclosure can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0014] In practicing the present technologies, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rdedition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4, 683, 195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization;Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology.

[0015] The fatty acid elongase ELOVL6 can diminish mutant KRAS protein levels and aberrant oncogenic signaling. Applicants have seen that ELOVL6 inhibition hindered G12V KRAS localization at the cell membrane. Further, lipidomic analysis has showed alterationsin lipid species linked to G12V KRAS PM anchoring. Applicants observed increases in long- chain PUFA in EL0VL6i treated cells. Without wishing to be bound by any particular theory, the formation of active KRAS nanoclusters at the plasma membrane is critical for the signaling function of the protein (Fuentes et al (2018) Cancer Res. 78, 3899-3912), and ELOVL6 inhibition appears to deplete critical membrane components derived from PUFAs. ELOVL6 is a key enzyme in the elongation of 16-carbon saturated and monounsaturated fatty acids to 18-carbon fatty acids, which is the rate-limiting step for the de novo synthesis of VLCFs (Mantsuzaka and Shimano (2009) J. Mol. Med (Berl). 87, 379-384).

[0016] Inhibition or deletion of ELOVL6 reduces cell activity (e.g. proliferation, KRAS expression, viability) of KRAS mutant variants (FIG. 1). Described herein are novel inhibitors of ELOVL6.Definitions

[0017] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al. , Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0018] As used herein, the term “administering” of an agent to a subject includes any route of introducing or delivering the agent to the subject to perform its intended function. Administration can be carried out by any suitable route, including, but not limited to, orally, intravenously, intramuscularly, intraperitoneally, subcutaneously, transdermal, buccal, ocular, suppository and other suitable routes as described herein. Administration includes selfadministration and the administration by another.

[0019] As used herein, the term “combination therapy” refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents. When used in combination therapy, two or more different agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of the two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations. In another alternative, a first agent can be administered just followed by one or more additional agents. In the separate administration protocol, two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart.

[0020] As used herein, the term “effective amount” or “therapeutically effective amount” refers to a quantity of an agent sufficient to achieve a beneficial or desired clinical result upon treatment. In the context of therapeutic applications, the amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the therapeutic agent administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art.[00211 In certain embodiments, the terms “disease” “disorder” and “condition” are used interchangeably herein, referring to a cancer, a status of being diagnosed with a cancer, or a status of being suspect of having a cancer.

[0022] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and may be used interchangeablywith the term “tumor.” In some embodiments, the cancer is a glioma or glioblastoma. “Cell associated with the cancer” refers to those subject cells that demonstrate abnormal uncontrolled replication.

[0023] “Cancer”, which is also referred to herein as “tumor”, is a known medically as an uncontrolled division of abnormal cells in a part of the body, benign or malignant. In one embodiment, cancer refers to a malignant neoplasm, a broad group of diseases involving unregulated cell division and growth, and invasion to nearby parts of the body. Non-limiting examples of cancers include carcinomas, sarcomas, leukemia and lymphoma, e.g., colon cancer, colorectal cancer, rectal cancer, gastric cancer, esophageal cancer, head and neck cancer, breast cancer, brain cancer, lung cancer, stomach cancer, liver cancer, gall bladder cancer, or pancreatic cancer In one embodiment, the term “cancer” refers to a solid tumor, which is an abnormal mass of tissue that usually does not contain cysts or liquid areas, including but not limited to, sarcomas, carcinomas, and certain lymphomas (such as NonHodgkin's lymphoma). In another embodiment, the term “cancer” refers to a liquid cancer, which is a cancer presenting in body fluids (such as, the blood and bone marrow), for example, leukemias (cancers of the blood) and certain lymphomas.[0024| Additionally or alternatively, a cancer may refer to a local cancer (which is an invasive malignant cancer confined entirely to the organ or tissue where the cancer began), a metastatic cancer (referring to a cancer that spreads from its site of origin to another part of the body), a non-metastatic cancer, a primary cancer (a term used describing an initial cancer a subject experiences), a secondary cancer (referring to a metastasis from primary cancer or second cancer unrelated to the original cancer), an advanced cancer, an unresectable cancer, or a recurrent cancer. As used herein, an advanced cancer refers to a cancer that had progressed after receiving one or more of: the first line therapy, the second line therapy, or the third line therapy.100251 A “solid tumor” is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, gliomas, and lymphomas. The solid tumor can be localized or metastatic.Compounds

[0026] In one aspect, as described herein is a compound according to formula (I):wherein A is N or CRi; wherein Z is selected from the group consisting of:wherein # is an N, O, or S; wherein Q is -(CH2)nPh; wherein Ph is phenyl optionally substituted with R1 and / or R2; wherein n is 0-3; wherein M is H or F; wherein X is selected from the group consisting of -SC>2-(CH2)n-, -C(O)(CH2)n, -CH=CHRi; wherein Y is selected from the group consisting of:andwherein n is 1-4 in Y; wherein [3 is optionally N or CH; wherein Ri is selected from the group consisting of H, D, halogen, -OH, oxo, mercapto, cyano, -CD3, -C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkane Base, -OC1- C6 alkylphenyl, -C1-C6 alkyl-OH, -C1-C6 alkyl-SH, -C1-C6 alkyl-O-Cl -C6 alkyl, -OC1-C6 haloalkyl, -NH2 , -C1-C6 alkyl-NH2, -N(C1-C6 alkyl)2, -NH(C1-C6 alkyl), -N( C1-C6 alkyl) (C1-C6 alkylphenyl), -NH (C1-C6 alkylphenyl), -N (C1-C6 alkyl) (C6-10 aryl), -NH (6 -10-membered aryl), nitro, -C(O)-OH, -C(O)OC1-C6 alkyl, -CONR1R“ , -NHC(O)(C1-C6 alkyl), -NHC(O)(phenyl),-N(Cl-C6 alkyl)C(O)(Cl-C6 alkyl), -N(C1-C6 alkyl)C(O)(phenyl),- C( O)C1-C6 alkyl, -C(O)C1-C6 alkylphenyl, -C(O)C1-C6 haloalkyl, -OC(O) C1-C6 alkyl, - S(O)2-C1-C6 alkyl, -S(O)-C1-C6 alkyl, -S(O)2-phenyl, -S(O)2-Cl - C6 haloalkyl, -S(O)2NH2, -S(O)2NH(C1-C6 alkyl), -S(O)2NH(phenyl), -NHS(O)2(C1-C6 alkyl), -NHS(O)2(phenyl) and -NHS(O)2(C1-C6 haloalkyl); wherein R1and R11are selected from H, lower alkyl, phenyl and benzyl; wherein R2is selected from the group consisting of H, lower alkyl, and lower alkoxy, and wherein R3 is an alicyclic or aromatic ring fused to the pyridine comprised of carbocyclic or heterocyclic groups optionally substituted with Ri.In one aspect, as described herein is a compound according to formula (II)wherein Ri is selected from the group consisting of 2-pyridyl, 2-quinolyl, -CH2-2-pyridyl, 1- naphthyl, 2-pyrimidinyl, 2-pyrazinyl, 2-thiazolyl, and 5-bromo-2-pyridyl; and wherein R2 is selected from the group consisting of 4-NO2PI1, 4-CN Ph, 4-cyclopropyl Ph, 2- napththyl, 4-acetyl Ph, trans 4-CFs cyclohexyl, 4-CFs Ph, 3 -tetrahydrofuranyl (rac), 1- adamantyl, 4-piperidinyl, l-acetyl-4-piperidinyl, 3-pyrazolyl, 3-(l-methylpyrazolyl), 4-(l- methylpiperidinyl), 3 -(1 -acetyl pyrazolyl), and 2-thiophenyl, and wherein Ph is a phenyl.10027] In some aspects, the compound according to formula (II) has an Ri of 2-quinolyl, - CH2-2-pyridyl, 2-pyrimidinyl, 2-pyrazinyl, 1-napthyl, or 5-bromo-2-pyridyl and R2 is 4- CFsphenyl. In some aspects, the compound is selected from the compounds in Table 1.

[0028] In some aspects, the compound according to formula (II) has an Ri of 2-pyridyl, and R2 is 4-acetylphenyl, 2-napthyl, or N-methylpiperidine-4-yl. In some aspects, the compound is selected from the compounds in Table 1.

[0029] In some aspects, the compound according to formula (II) is:Compositions

[0030] Another aspect is a composition comprising one or more of the compounds as described herein. In one aspect the composition further includes a pharmaceutically acceptable carrier.

[0031] In some embodiments, one or more compounds is present in a concentration between 0.0001 pM and 100 pM.

[0032] The compositions may include pharmaceutical solutions comprising carriers, diluents, excipients, preservatives, and surfactants, as known in the art. Further, the compositions mayinclude preservatives (e.g., anti-microbial or anti-bacterial agents such as benzalkonium chloride). The compositions also may include buffering agents (e.g., in order to maintain the pH of the composition between 6.5 and 7.5).

[0033] The pharmaceutical compositions may be administered therapeutically to a patient in an amount sufficient to elicit a therapeutic effect (e.g., a response which cures or at least partially arrests or slows symptoms and / or complications of disease (i.e., a "therapeutically effective dose").

[0034] In some embodiments, compositions are formulated for systemic delivery, such as oral or parenteral delivery. Additional exemplary routes of administration include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, buccal, intrapulmonary, intradermal, topical, dermal, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, and / or intralymphatic. In some embodiments, minimally invasive microneedles and / or iontophoresis may be used to administer the composition. In some embodiments, compositions are formulated for sitespecific administration, such as by injection into a specific tissue or organ, topical administration (e.g., by patch applied to the target tissue or target organ). In some embodiments, the composition is formulated to be delivered through the blood-brain barrier. By way of example, but not by way of limitation, such methods may include ultrasound treatment with or without concomitant administration of microbubbles, convection enhanced drug delivery, biodegradable wafers that release the drug, peptide-drug conjugates, and nanoparticle-drug coupling to enhance drug penetration.

[0035] In some embodiments, the methods include administration of the therapeutic compositions once per day; in some embodiments, the composition may be administered multiple times per day, e.g., at a frequency of one or two times per day, or at a frequency of three or four times per day or more. In some embodiments, the methods include administration of the composition once per week, once per month, or as symptoms dictate.

[0036] In some embodiments, the compositions are used in combination with additional therapeutic agents and methods. In some embodiments, the compositions described herein are applied sequentially or consecutively with additional therapeutic agents.Patient Populations

[0037] As used herein, the terms “patient,” and “subject” are used interchangeably.

[0038] In some embodiments, a subject in need thereof in accordance with the technologies (i.e. methods and compositions) disclosed herein include, but are not limited to, humans and non-human vertebrates. In some embodiments, a subject in need thereof in accordance with the technologies disclosed herein comprise, for example, a mammal. In some embodiments the mammal is a human. In some such embodiments, a mammal includes, for example and without limitation, a companion animal (e.g., a dog, a cat, a rabbit, a ferret, a hamster, etc.), a livestock or farm animal (e.g., a cow, a pig, a sheep, a goat, a chicken or another poultry), a horse, a monkey, a laboratory animal (e.g., a mouse, a rat, a rabbit, etc.) and the like. In a preferred embodiment, the subject in need thereof in accordance with technologies described herein is a human.

[0039] In some aspects, the technologies of the present disclosure can be utilized in a subject that has cancer. A subject that has cancer is a subject that has detectable cancer cells. In some embodiments, a cancer involves one or more tumors.(0040] In some aspects, the subject has a cancer selected from lung cancer, colorectal cancer, and pancreatic cancer.

[0041] In some aspects, the subject has a mutated KRAS gene (KRAS gene mRNA NCBI NM_001369786.1). The KRAS gene is in the Ras family of oncogenes. KRAS encodes the GTPase KRAS protein. In some aspects, the subject has the G12V KRAS or G12D KRAS mutant variant, which are the most common variants. In other aspects, the subject has a different KRAS mutant variant, including but not limited to A146V, D47N, G12C, Q61L, KI 17N, and T50P. The prevalence of KRAS mutant variants in a cohort of 127 patients with surgically treated intrahepatic cholangiocarcinoma is described in Zhou et al (2022) JAMA Surg. 157(l):59-65.

[0042] In some aspects, the subject in need thereof has undergone one or more other cancer therapies (e.g., chemotherapy, radiotherapy, immunotherapy).(0043] In some embodiments, the subject in need thereof is an adult from 18-75 years of age. In some embodiments, the subject in need thereof is a pediatric subject (e.g., a human subject under the age of 12).Methods of Use

[0044] Also provided are methods to inhibit a mutant KRAS expressing cancer cell line by treating the cell lines with the compound or compositions described herein, thereby inhibiting growth of the cell, wherein the contacting is in vivo, in vitro, in situ, or ex vivo.

[0045] In some aspects, the mutant KRAS expressing cancer cell line is HP AC (G12D), H727 (G12V), or AsPCl (G12D). G12D and G12V are common KRAS mutant variants.

[0046] In other aspects, the KRAS expressing cancer cell line are other cell lines such as SW1573, Mia-paca2, Capan-2, Pane 02.03, HCT116, SW480, PSN-1, Pane 03.27, SW403, NCI-H441, GP2D, LS180, COR-L23, LS1034, Capan-1, SK-LU-1, SW1990, NCI-H1944, LS513, Pane 04.03, NCI-H647, NCI-H2444, Calu-6, A427, HUP-T4, CFPAC-1, SK-CO-1, AsPC-1, NCI-H727, and HP AC. The cell lines can have mutant variants including G12V, G12D, G12C, G12R, G13D, Q61X, and A146T.

[0047] In some aspects, the compound or composition can inhibit the growth of at least two of mutant KRAS expressing cancer cell lines. In some aspects, the mutant KRAS expressing cancer cell lines are selected from HP AC (G12D), H727 (G12V), and AsPCl (G12D) (see FIG. 3, Tables 3-5)

[0048] In yet other aspects, the compound or composition can inhibit the growth of three mutant KRAS expressing cancer cell lines. In some aspects, the three mutant KRAS expressing cancer cell lines are HP AC (G12D), H727 (G12V), and AsPCl (G12D) (see Tables 3-5).(0049] Also described herein are methods to treat cancer in a subject in need thereof, comprising administering the compound or composition described herein to the subject inneed. In some aspects, the cancer is selected from liver cancer, head and neck cancer, lung cancer, colorectal cancer, and pancreatic cancer. These cancer types are intended to be exemplary, not limiting. In some aspects, the subject has a KRAS mutation, for example G12V, G12D, G12C, G12R, G13D, G61X, or A146T. The KRAS mutations listed here are intended to be exemplary, and some subjects may have other mutations. Two of the most common KRAS mutations in cancer patients are G12V and G12D.Examples10050] Applicant saw that a fatty acid elongase (ELOVL6), showed substantial selectivity in diminishing mutant KRAS mutant protein levels and aberrant oncogenic signaling. FIG. 1 shows KRAS expression and cell viability in wildtype and KRAS mutants that have the ELOVL6 gene knocked out. KRAS expression and cell viability was significantly reduced in KRAS mutant cells with ELOVL6 knocked out.

[0051] ELOVL6, a druggable enzyme, can be targeted to control the production of lipids that are exploited by mutated forms of KRAS for function and trigger the targeted degradation of the protein. FIG. ID shows reduced cell viability in KRAS mutant cells, as well as diminished tumor growth, in the presence of commercially available ELOVL6 inhibitor ELOVL6-IN-2. (Nagase et al: J. Med. Chem. 2009, 52, 4111-4114). Further, Applicant saw that tumor volume in G12V mutant KRAS xenografts in mice were lower in animals dosed with the commercially available ELOVL6 inhibitor as compared to the negative control (FIG. IE). As described herein, Applicant designed novel compounds to inhibit the growth and / or viability of cell KRAS mutant cell lines. The novel compounds exhibit the potential to be selective cytotoxic agents against KRAS-addicted cancer cells.ELOVL6 targetsUnless otherwise noted, all solvents, chemicals, and reagents were obtained commercially and used without purification. Phenyl arylcarbamates were commercially available or prepared from phenyl chloroformate and corresponding anilines.1H-NMR spectra were obtained at 400MHz on an AVANCE NEO spectrometer from Bruker using RT -PROBES with BBFO with chemical shift (8, ppm) reported relative to TMS as an internal standard. Mass spectra were recorded with electron-spray ionization (ESI) on Agilent 1290 Infinity IIwith 6130 and 6125 MSD. All final compounds had a HPLC purity of >95% with purification methods specified below.Synthetic procedures100521 Bicyclic exo mesylate (1) was prepared following the procedures of Nagase et al: J. Med. Chem. 2009, 52, 4111-4114. Briefly, displacement of the mesylate provided endo sulfides (2) which were oxidized to sulfones (3) using KMnC>4. The Boc groups were removed via hydrolysis and the resulting secondary amines (4) were condensed with phenyl carbamates to provide target molecules (5).

[0053] Synthesis of t-butyl 3-hydroxy-8-azabicyclo[3.2. l]octane-8-carboxylate:

[0054] To a stirred solution of t-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (1 g, 4.44 mmol) in THF (15 ml) was added sodium borohydride (0.420 g, 11.10 mmol) portion wise at 0 °C. Then the reaction mixture was stirred at 25°C for 2 h. The reaction was monitored by TLC. After completion of reaction, cooled to 0°C and added sodium bicarbonate solution and extracted with EtOAc(4 x 50 ml). The combined organic layer was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to yield crude mass which was purified by silica gel (230-400 nm mesh) column chromatography with eluent 0-40% (EtOAcin petroleum ether). The pure fractions was concentrated separately to afford t- butyl 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate (200 mg, 0.875 mmol, 19.70 % yield) as white solid (Non polar spot on TLC identified as endo, undesired) and t-butyl 3- hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate (780 mg, 3.43 mmol, 78 % yield) as white solid (more polar spot on TLC as exo, desired).|0055|1HNMR of exo isomer: (400 MHz, CDCh): 54.26 (s, 2H), 4.17-4.08 (m, 1H), 2.00- 1.95 (m, 4H), 1.64-1.59 (m, 4H), 1.49 (s, 9H). MS (ESI) m / z 128.3 (M+H).Compounds

[0056] Table 1 below shows the final products (i.e. compounds) following the general chemical formula:

[0057] Table 1. Compounds according to the chemical formula (5). Ph = phenyl. The compound in Table 1 are shown in FIG. 2. The identifier (e.g. 5a) indicates a scaffold according to formula (5) above.

[0058] The compounds in Table 1 are characterized below:[0059| 5a: 'HNMR (400 MHz, deDMSO) 5 8.90 (1H, m), 8.80 (d, 1H, J=8 Hz), 8.17-8.10 (m, 2H), 7.78 (d, 2H, J= 2 Hz), 7.70 (m, 1H), 7.63 (d, 2H, J= 2 Hz), 4.47 (bm, 2H), 3.98 (m, 1H), 2.50 (s, 3H), 2.33 (m, 2H), 2.0-1.85 (m, 6H). MS (APCI) m / z 414 (M+H)+.

[0060] 5b: 'HNMR (400 MHz, deDMSO) 5 8.80 (1H, m), 8.78 (s, 1H), 8.18-8.05 (m, 3H), 7.76 (m, 4H), 7.62 (m, 1H), 7.60 (m, 1H), 7.43 (m, 1H), 4.50 (bm, 2H), 3.90 (m, 1H), 2.50 (m, 2H), 1.90 (m, 6H). MS (APCI) m / z 422 (M+H)+.[00611 5c: 'HNMR (400 MHz, deDMSO) 5 8.56 (s, 1H), 8.20 (m, 2H), 8.10 (m, 1H), 7.86 (m, 1H), 7.74 (m, 2H), 7.63 (d, 2H, J=9 Hz), 7.55 (d, 2H, J=9 Hz), 4.43 (m, 2H), 2.51 (m, 1H), 2.31 (m, 2H), 1.90 (m, 6H). MS (APCI) m / z 489 (M+H)+.

[0062] 5d: 'HNMR (400 MHz, deDMSO) 5 8.46 (m, 1H), 8.19 (m, 1H), 8.08 (m, 1H), 7.95 (m, 1H), 7.85-7.73 (m, 3H), 7.57 (bm, 4H), 4.38 (m, 3H), 2.54 (m, 2H), 2.20 (m, 6H). MS (APCI) m / z 490 (M+H)+.[0063[ 5e: 'HNMR (400 MHz, deDMSO) 5 8.45 (m, 1H), 7.76 (m, 1H), 7.54 (m, 4H), 7.27 (m, 2H), 6.57 (s, 1H), 4.40 (m, 2H), 4.35 (m, 2H) 3.40 (m, 1H), 2.57 (m, 2H). 2.05 (m, 6H). MS (APCI) m / z 454 (M+H)+.

[0064] 5f: 'H NMR (400 MHz, deDMSO) 5 8.76 (m, 1H), 8.14 (m, 2H), 7.71 (m, 1H), 7.50 (m, 1H), 4.88 (m, 2H), 4.31 (m, 1H), 3.87 (bm, 1H), 3.32 (m, 1H), 2.91 (m, 2H), 2.31 (s, 3H), 2.45 (m, 2H), 2.12 (m, 2H), 1.98 (bm, 8H), 1.50 (m, 2H). MS (APCI) m / z 393 (M+H)+.

[0065] 5g: 'HNMR (400 MHz, deDMSO) 5 9.03 (d, 2H, J=5 Hz), 7.77 (m, 1H), 7.65 (m, 2H), 7.56 (m, 2H), 4.53 (m, 2H), 4.26 (m, 1H), 2.58 (m, 2H), 2.16 (bm, 6H). MS (APCI) m / z 441 (M+H)+.

[0066] 5h: 'HNMR (400 MHz, deDMSO) 5 9.27 (d, 1H, J=2 Hz), 9.05 (d, 1H, J=2 Hz), 8.90 (m, 2H), 7.71 (d, 2H, J=8 Hz), 7.58 (d, 2H, J=8 Hz), 4.48 (bs, 2H), 3.93 (m, 1H), 2.35 (m, 2H), 1.97 (bm, 6H). MS (APCI) m / z 441 (M+H)+.

[0067] 5i: 'H NMR (400 MHz, deDMSO) 5 9.66 (s, 1H), 8.89 (s, 1H), 8.45 (m,lH), 8.05 (d, 1H, J = 2Hz), 7.71 (d, 2H, J=9 Hz), 7.59 (d, 2H, J=9 Hz), 4.46 (bs, 2H), 3.87 (m, 1H), 2.34 (m, 2H), 1.94 (bm, 6H). MS (APCI) m / z 518 (M+H)+.Analytical Methods

[0068] Multiple analytical methods were used to determine the chemical properties of each compound. Table 2 shows which methods were used for each compound, with further explanation of the methods below.[0069| Table !. General Analytical Methods used to determine the properties of the compounds in Table 1.HPLC:[0070[ Method B: Mobile Phase: A: 0.1% FA in H2O: B: 0.05% FA in ACN; Flow Rate: 0.8 mL / min; Column: BEH Cl 8 (50 x 2.1 mm), 1.7 pM.

[0071] Method C: Method: A-0.1% TFA in H2O, B- ACN, flow rate: 1.2 mL / min; column: Atlantis T3 (150X4.6)mm, 3.0pm

[0072] Method D: Method: A-0.1% TFA in H2O, B-ACN, flow rate: 2.0 mL / min; column: X-Bridge C8 (50 X 4.6) mm, 3.5 pm.

[0073] Method F: 5mM NH4HCO3 in H2O, B- ACN; flow rate: 1.2 mL / min; column: X- Bridge C8 (50 x 4.6 mm, 3.5 pm).LCMS:

[0074] Method B: Mobile Phase: A: 0.1% FA in H2O: B: 0.05% FA in ACN; Flow Rate: 0.8 mL / min; Column: BEH Cl 8 (50 x 2.1 mm), 1.7 pM.

[0075] Method C: Mobile Phase: A: 0.1% TFA in H2O, B: ACN; Flow Rate: 2.0 mL / min; Column: XBridge C8 (50 x 4.6 mm), 3.5 pM.

[0076] Method F: Mobile Phase: A: 5 mM Ammonium Bicarbonate in H2O, B: ACN; Flow Rate: 1.0 mL / min; Column: ACQUITY BEH C8 (50x2.1mm)1.7 gmNormal Phase purification:

[0077] Method A: Crude product was purified by 230-400 silica gel column chromatography with eluent 20 to 50% pet ether and EtOAc to afford pure product. Purification instrument: Biotage Isolera with Flow: 20 mL / min. Run time: 20 to 30 min{0078] Method B: Crude product was purified by 230-400 silica gel column chromatography with eluent 60 to 100% pet ether and EtOAc to afford pure product. Purification instrument: Biotage Isolera with Flow: 20 mL / min. Run time: 20 to 30 minCell Viability Assays

[0079] Compounds from Table 1 were tested in in vitro assays with KRAS control and mutant cell lines. The compounds of the present study were tested against two known ELOVL6 inhibitors, Compound B and Compound Iw. Cell viability was assessed after 3 or 7 days of treatment (FIGS. 2B and 2C). FIGS. 2B and 2C show exemplary data of the viability assays. Full results are in Tables 3-5.

[0080] Compound B and Iw (3-(pyridin-2-ylsulfonyl)-N-(4-(trifluoromethyl)phenyl)-8- azabicyclo[3.2.1]octane-8-carboxamide) were previously characterized in Shimamura et al (2010) Eur J Pharmacol. 630(1 -3): 34-41.

[0081] Tables 3-5 shows the functional evaluation summary of the compounds in Table 1 against the KRAS mutant cell lines.

[0082] Table 3. Cell viability assay in KRAS mutant cell line HP AC G12D. Cells were treated for 7 days before cell viability measurements and comparison with the reference compound responses. Compounds were tested at 30 pM. Compounds with a “+” passed the activity threshold set by applicant. Compounds in green passed the activity threshold and had mutant selectivity. Activity threshold 1 is defined as performance greater than reference compound 1W. Activity threshold 2 is defined as performance greater than reference compound B. Mutant selectivity is defined as compound showing greater impact on cell viability in the mutant KRAS expressing cell lines over the wild-type KRAS expressing cell lines.

[0083] Table 4. Cell viability assay in KRAS mutant cell line AsPCl G12D. Cells were treated for 7 days before cell viability measurements and comparison with the reference compound responses. Compounds were tested at 30 pM. Compounds with a “+” passed the activity threshold set by applicant. Compounds in green passed the activity threshold and had mutant selectivity. Activity threshold 1 is defined as performance greater than reference compound 1W. Activity threshold 2 is defined as performance greater than reference compound B. Mutant selectivity is defined as compound showing greater impact on cell viability in the mutant KRAS expressing cell lines over the wild-type KRAS expressing cell lines.

[0084] Table 5. Cell viability assay in KRAS mutant cell line H727 G12V. Cells were treated for 7 days before cell viability measurements and comparison with the reference compound responses. Compounds were tested at 30 pM. Compounds with a “+” passed the activity threshold set by applicant. Compounds in green passed the activity threshold and had mutant selectivity. Activity threshold 1 is defined as performance greater than reference compound 1W. Activity threshold 2 is defined as performance greater than reference compound B. Mutant selectivity is defined as compound showing greater impact on cell viability in the mutant KRAS expressing cell lines over the wild-type KRAS expressing cell lines.

[0085] When tested in the three KRAS mutant cell lines (HP AC G12D, AsPCl G12D, and H727 G12V), four compounds: 5d, 5e, 5g , and 5h performed better than Compound B and Compound 1W across all the mutant cell lines.

[0086] Two compounds: 5b and 5c performed better than Compound B and Compound 1W in mutant cell lines AsPCl G12D and H727 G12V.

[0087] Two compounds: 5a and 5f performed better than Compound B and Compound 1W in mutant cell line H727 G12V.

[0088] Applicant identified preferred candidates as the compounds effective at inhibiting the growth of the three mutant cell lines (FIGS 3A-3C). Further, FIG. 3D shows that select candidate compounds are effective across a gradient of concentrations. Further, preferred candidates exhibited mutant selectivity.References1. M. Malumbres, Nat. Rev. Cancer. 3, 459-465 (2003).2. A. R. Moore, Nat. Rev. Drug. Discov. 19, 533-552 (2020).3. M. H. Hofmann, Cancer Discov. 12, 924-937 (2022).4. S. R. Punekar, Nat. Rev. Clin. Oncol. 19, 637-655 (2022).5. S. Lu, Sci. Rep. 6, 21949 (2016).6. J. M. Ostrem, Nature 503, 548-551 (2013).7. P. Lito, Science 351, 604-608 (2016).8. J. Canon, Nature 575, 217-223 (2019).9. J. Hallin, Cancer Discov. 10, 54-71 (2020).10. D. S. Hong, N. Engl. J. Med. 383, 1207-1217 (2020).11. F. Skoulidis, N. Engl. J. Med. 384, 2371-2381 (2021).Equivalents

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0090] Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.[00911 The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0092] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0093] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure alsoform part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0094] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0095] All publications, patent applications, patents, GenBank citations, ATCC citations, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the specification, including definitions, will control.

Claims

WHAT IS CLAIMED IS:

1. A compound according to formula (I):wherein A is N or CRi; wherein Z is selected from the group consisting of:wherein # is an N, O, or S; wherein Q is -(CH2)nPh; wherein Ph is phenyl optionally substituted with R1 and / or R2; wherein n is 0-3; wherein M is H or F; wherein X is selected from the group consisting of -SC>2-(CH2)n-, -C(O)(CH2)n, -CH=CHRi; wherein Y is selected from the group consisting of:andwherein n is 1-4 in Y; wherein [3 is optionally N or CH; wherein Ri is selected from the group consisting of H, D, halogen, -OH, oxo, mercapto, cyano, -CD3, -C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkane Base, -OC1- C6 alkylphenyl, -C1-C6 alkyl-OH, -C1-C6 alkyl-SH, -C1-C6 alkyl-O-Cl -C6 alkyl, -OC1-C6 haloalkyl, -NH2 , -C1-C6 alkyl-NH2, -N(C1-C6 alkyl)2, -NH(C1-C6 alkyl), -N( C1-C6 alkyl) (C1-C6 alkylphenyl), -NH (C1-C6 alkylphenyl), -N (C1-C6 alkyl) (C6-10 aryl), -NH (6 -10-membered aryl), nitro, -C(O)-OH, -C(O)OC1-C6 alkyl, -CONR1R“ , -NHC(O)(C1-C6 alkyl), -NHC(O)(phenyl),-N(Cl-C6 alkyl)C(O)(Cl-C6 alkyl), -N(C1-C6 alkyl)C(O)(phenyl),- C( O)C1-C6 alkyl, -C(O)C1-C6 alkylphenyl, -C(O)C1-C6 haloalkyl, -OC(O) C1-C6 alkyl, - S(O)2-C1-C6 alkyl, -S(O)-C1-C6 alkyl, -S(O)2-phenyl, -S(O)2-Cl - C6 haloalkyl, -S(O)2NH2, -S(O)2NH(C1-C6 alkyl), -S(O)2NH(phenyl), -NHS(O)2(C1-C6 alkyl), -NHS(O)2(phenyl) and -NHS(O)2(C1-C6 haloalkyl); and wherein R1and R11are selected from H, lower alkyl, phenyl and benzyl; wherein R2is selected from the group consisting of H, lower alkyl, and lower alkoxy; and wherein R3 is an alicyclic or aromatic ring fused to the pyridine comprised of carbocyclic or heterocyclic groups optionally substituted with Ri.

2. A compound according to formula (II)(II), wherein Ri is selected from the group consisting of 2-pyridyl, 2-quinolyl, -CH2-2-pyridyl, 1- naphthyl, 2-pyrimidinyl, 2-pyrazinyl, 2-thiazolyl, and 5-bromo-2-pyridyl; and wherein R2 is selected from the group consisting of 4-NO2PI1, 4-CN Ph, 4-cyclopropyl Ph, 2- napththyl, 4-acetyl Ph, trans 4-CFs cyclohexyl, 4-CFs Ph, 3 -tetrahydrofuranyl (rac), 1- adamantyl, 4-piperidinyl, l-acetyl-4-piperidinyl, 3-pyrazolyl, 3-(l-methylpyrazolyl), 4-(l- methylpiperidinyl), 3-(l-acetylpyrazolyl), and 2-thiophenyl, and wherein Ph is a phenyl.

3. The compound of claim 2, wherein the compound is selected from:

4. A composition comprising the compound of any one of claims 1-3 and optionally a pharmaceutically acceptable carrier.

5. The composition of any one of claims 1-4, wherein the compound is present in a concentration between 0.001 and lOOuM.

6. A method to inhibit a mutant KRAS expressing cancer cell line comprising contacting the cell lines with the compound of any one of claims 1-3 or the composition of any one of claims 4-5, thereby inhibiting growth of the cell, wherein the contacting is in vivo, in vitro, in situ, or ex vivo.

7. The method of claim 6, wherein the mutant KRAS expressing cancer cell line is HP AC G12D, H727 G12V, or AsPCl G12D.

8. The method of claim 6, wherein the compound of any of claims 1-3 or the composition of claim 4 or 5 inhibits the growth of at least two of the mutant KRAS expressing cancer cell lines HP AC G12D, H727 G12V, and AsPCl G12D.

9. The method of claim 6, wherein the compound of any of claims 1-3 or the composition of claim 4 or 5 inhibits the growth of the mutant KRAS expressing cancer cell lines HP AC G12D, H727 G12V, and AsPCl G12D.

10. A method to treat cancer in a subject in need thereof, comprising administering the compound of any of claims 1-3 or the composition of claim 4 or 5 to the subject.

11. The method of claim 10, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, and pancreatic cancer.

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