Pyrrolopyrimidine inhibitors of wild-type and mutant LRRK2

Abstract

The present disclosure relates to compounds, compositions, and methods for treating diseases or disorders that are associated with aberrant activity of LRRK2.

Description

Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO PYRROLOPYRIMIDINE INHIBITORS OF WILD-TYPE AND MUTANT LRRK2 RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 656,726, filed June 6, 2024, which is incorporated herein by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0002] Parkinson's disease (PD) is a movement disorder resulting from progressive loss of dopamine producing neurons. It is the second most common neurodegenerative disease in the world, and affects over 1 million Americans. More than 60,000 patients are newly diagnosed each year (Gandhi et al., J. Neurosci. Res.87:1283−1295 (2009); Daniëls et al., Neurosignals 19:1−15 (2011)). Symptoms associated with Parkinson’s disease include motor impairment, tremor, bradykinesia, instability, and other movement related disorders. There are also non-motor symptoms such as cognitive dysfunction, autonomic dysfunction, and sleep disruption. These symptoms greatly reduce the quality of life of those suffering from PD.

[0003] Recent genetic studies have revealed that genetic variation is estimated to contribute approximately 25% to the overall risk of developing PD, which provides new opportunities for the discovery of molecularly targeted therapeutics that may ameliorate neurodegeneration (Day et al., Genes (Basel) 12(7):1006 (2021)). Some of the genes associated with PD, e.g., SNCA (α- synuclein), LRRK2 (leucine-rich repeat kinase 2), VPS35 (vacuolar protein sorting ortholog 35), PRKN (Parkin), DJ1 (DJ-1), and PINK1 (PTEN-induced kinase 1), are highly expressed in immune cells, and variants in these genes have been found to be involved in the pathogenesis of inflammatory bowel disease (IBD), particularly in Crohn’s disease (CD) (Day et al., Genes (Basel) 12(7):1006 (2021); Liu et al., Cell Res.22:1092-1093 (2012)). Therefore, their roles in immune functions and in neurodegeneration of PD have been studied (Rui et al., Curr. Neuropharmacol. 16(9):1348-1357 (2018)). Research has shown that these genes contribute to PD potentially by altering innate immune response to brain pathology (Ikezu et al., J. Neuroimmune Pharmacol. 15:794-800 (2020)).

[0004] LRRK2 has been identified as the most common genetic cause of PD. It is a large multidomain protein that bears two enzymatic functions: kinase and GTPase, and several protein– 1 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO protein interaction domains (Wojewska et al., Biomolecules 11(8): 1101 (2021)). There are many genomic PD variants of LRRK2, some of them have been repeatedly confirmed as pathogenic, others are considered risk factors, and some are benign (Monfrini et al., Adv. Neurobiol.14: 3-30 (2017)). LRRK2 has also been implicated in various cancers (Yan et al., Genomics 114:316-327 (2022)).

[0005] Currently, few compounds with LRRK2 inhibiting properties are known. Therefore, a need exists for novel therapeutics that inhibit both wild-type and mutant forms of LRRK2 (Hu et al., Eur. J. Med. Chem.225:115475 (2023)). SUMMARY OF THE DISCLOSURE

[0006] A first aspect of the present disclosure is directed to a compound having a structure represented by formula I: I), or a pharmaceutically acceptablewherein: X is CH or N; Y is N(R4)2 or OR5, wherein each R4 is independently H or C1-C3 alkyl, and R5 is H or C1- C3alkyl; R1 is H, C1-C3 alkyl, SO2R6, C(O)R7, or 4-6-membered O-containing heterocyclyl, wherein R6 is C1-C3 alkyl, and R7 is H or C1-C3 alkyl; R2is OR8, wherein R8is C1-C3alkyl optionally substituted with one or more halo group; and R3 is H, halo, CN, or optionally substituted C1-C3 alkyl.

[0007] A second aspect of the present disclosure is directed to a pharmaceutical composition containing the compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 2 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0008] Another aspect of the present disclosure is directed to a method of treating a disease or disorder that is associated with (e.g., characterized or mediated by) aberrant activity of LRRK2, comprising administering to a subject in need thereof the compound of formula I, or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the disease or disorder is Parkinson’s disease (PD). In some embodiments, the disease is cancer. In some embodiments, the disease is inflammatory bowel disease (IBD) such as Crohn’s disease.

[0010] Further aspects of the present disclose are directed to methods of making the compounds.

[0011] Compounds of the present disclosure inhibit the activity of both wild-type and mutant forms of LRRK2. Compounds of the present disclosure may thus provide a therapeutic entrée for diseases involving aberrant LRRK2 function, such as PD, by inhibiting LRRK2.

[0012] Previous reports demonstrate that despite advancement in the development of LRRK2 inhibitors over the past decade, treatment of PD patients with LRRK2 mutations remains a major challenge. Presently disclosed compounds inhibit wild-type LRRK2 as well as mutant forms of the protein, thereby offering a wider spectrum of clinical benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1 is a Western blot showing the amounts of total LRRK2, LRRK2 phosphorylated at Serine 935 (pSer935 (pS935) LRRK2), α-tubulin, phosphorylated Rab 10 (pRab 10) and total Rab 10 in cell lines treated with indicated concentrations of compound I-1 as LRRK2 inhibitors for 90 min. Gels show LRRK2 wild-type (WT) and LRRK2 (G2019S) mutant Mouse Embryonic Fibroblasts (MEFs) treated with various concentrations of compound I-1 in triplicate with α- tubulin as a loading control.

[0014] FIG. 2 is a Western blot showing the amounts of total LRRK2, pSer935 LRRK2, α- tubulin, pRab 10 and total Rab 10 in cell lines treated with indicated concentrations of compound I-5 as LRRK2 inhibitors for 90 min in the same system as FIG.1.

[0015] FIG. 3 is a Western blot showing the amounts of total LRRK2, pSer935 LRRK2, α- tubulin, pRab 10 and total Rab 10 in cell lines treated with indicated concentrations of compound I-10 as LRRK2 inhibitors for 90 min in the same system as FIG.1. 3 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0016] FIG. 4 is a Western blot showing the amounts of total LRRK2, pSer935 LRRK2, α- tubulin, pRab 10 and total Rab 10 in cell lines treated with indicated concentrations of compound I-11 as LRRK2 inhibitors for 90 min in the same system as FIG.1.

[0017] FIG.5 is a series of graphs showing relative pS935 and pRab levels in wide-type LRRK2 and LRRK2(G2019S) mutant treated with indicated concentrations of compound I-1.

[0018] FIG. 6 is a series of graphs showing relative pS935 levels in wide-type LRRK2 and LRRK2(G2019S) mutant treated with indicated concentrations of compound I-5.

[0019] FIG. 7 is a series of graphs showing relative pS935 levels in wide-type LRRK2 and LRRK2(G2019S) mutant treated with indicated concentrations of compound I-10.

[0020] FIG.8 is a series of graphs showing relative pS935 and pRab levels in wide-type LRRK2 and LRRK2(G2019S) mutant treated with indicated concentrations of compound I-11. DETAILED DESCRIPTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated in order to facilitate the understanding of the present disclosure.

[0022] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Therefore, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0023] Unless stated otherwise, the term “about” means within 10% (e.g., within 5%, 2%, or 1%) of the particular value modified by the term “about.”

[0024] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group that that minimum number of heteroatoms. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the 4 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the disclosure.

[0025] With respect to compounds of the present disclosure, and to the extent the following terms are used herein to further describe them, the following definitions apply.

[0026] As used herein, the term “alkyl” refers to a saturated linear or branched-chain monovalent hydrocarbon radical. In some embodiments, and to the extent not disclosed otherwise for any one or more groups of the compounds of formula I, the alkyl radical is a C0-C6, C0-C5, C0-C3, C1-C6, C1-C5, C1-C4 or C1-C3 group (wherein C0 alkyl refers to a bond). In some embodiments, an alkyl group is a C1-C3alkyl group.

[0027] As used herein, the term “O-containing heterocyclyl” refers to a group that used alone or as part of a larger moiety, contains a saturated, partially unsaturated, or aromatic ring system having 3 to 12 carbon atoms, wherein one or more (e.g., 1, 2, 3, 4, or 5) carbon atoms have been replaced with a heteroatom or heteroatom-containing group (e.g., O, N, N(O), S, S(O), or S(O)2) including at least one oxygen atom. The term O-containing heterocyclyl includes mono-, bi-, tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In some embodiments, an O- containing heterocyclyl group includes 3-12 ring atoms and includes monocycles, bicycles, tricycles and spiro ring systems, wherein the ring atoms are carbon, and one to 5 ring atoms is a heteroatom such as nitrogen, sulfur or oxygen, including at least one oxygen atom. In some embodiments, O-containing heterocyclyl includes 3- to 7-membered monocycles having one or more heteroatoms selected from O, N, and S, including at least one oxygen atom. Representative examples of O-containing heterocyclyls include furanyl, dihydrofuranyl, tetrahydropyranyl, morpholinyl, dioxyanyl, pyranyl, dihydropyranyl, tetrahydropyranyl, benzofuranyl, chromenyl, chromanyl.

[0028] As used herein, the term “halogen” (or “halo” or “halide”) refers to fluorine, chlorine, bromine, or iodine.

[0029] Unless stated otherwise, and to the extent not further defined for any particular group(s) in the compounds of formula I, any of the groups described herein may be substituted or unsubstituted. To the extent not disclosed otherwise for any particular group(s), representative examples of substituents may include alkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), substituted alkyl (e.g., substituted C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), alkoxy (e.g., C1-C6, C1- C5, C1-C4, C1-C3, C1-C2, C1), substituted alkoxy (e.g., substituted C1-C6, C1-C5, C1-C4, C1-C3, C1- 5 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO C2, C1), haloalkyl (e.g., CF3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., substituted C2-C6, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g., substituted C2-C6, C2-C5, C2-C4, C2-C3, C2), cyclic (e.g., C3-C12, C5-C6), substituted cyclic (e.g., substituted C3-C12, C5-C6), carbocyclic (e.g., C3-C12, C5-C6), substituted carbocyclic (e.g., substituted C3-C12, C5-C6), heterocyclic (e.g., 3- to 12-membered, 5-to 6- membered), substituted heterocyclic (e.g., substituted 3- to 12-membered, 5-to 6-membered), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or substituted phenyl), heteroaryl (e.g., pyridyl or pyrimidyl), substituted heteroaryl (e.g., substituted pyridyl or substituted pyrimidyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halo, hydroxyl, aryloxy (e.g., C6-C12, C6), substituted aryloxy (e.g., substituted C6-C12, C6), alkylthio (e.g., C1-C6), substituted alkylthio (e.g., substituted C1-C6), arylthio (e.g., C6-C12, C6), substituted arylthio (e.g., substituted C6-C12, C6), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfinamide, substituted sulfinamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid, and peptide groups. Terminal substituents, unless otherwise specified, include unsubstituted C1-C4alkyl, halides, -OH, NH2and -CN.

[0030] In one aspect, compounds of the disclosure are represented by formula I: I), or a pharmaceutically acceptablewherein: X is CH or N; Y is N(R4)2 or OR5, wherein each R4 is independently H or C1-C3 alkyl, and R5 is H or C1- C3 alkyl; R1is H, C1-C3alkyl, SO2R6, C(O)R7, or 4-6-membered O-containing heterocyclyl, wherein R6 is C1-C3 alkyl, and R7 is H or C1-C3 alkyl; 6 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO R2 is OR8, wherein R8 is C1-C3 alkyl optionally substituted with one or more halo group; and R3is H, halo, CN, or optionally substituted C1-C3alkyl.

[0031] In some embodiments, X is CH.

[0032] In some embodiments, X is N.

[0033] In some embodiments, Y is NHR4.

[0034] In some embodiments, R1is H.

[0035] In some embodiments, R1 is C1-C3 alkyl.

[0036] In some embodiments, R1is CH3.

[0037] In some embodiments, R1is SO2R6.

[0038] In some embodiments, R6 is methyl and R1 is SO2CH3.

[0039] In some embodiments, R1 is C(O)R7.

[0040] In some embodiments, R7is CH3and R1is COCH3.

[0041] In some embodiments, R1 is a 4-6-membered O-containing heterocyclyl.

[0042] In some embodiments, R1 is oxetanyl.

[0043] In some embodiments, R8is C1-C3alkyl optionally substituted with one or more fluoro group.

[0044] In some embodiments, R8 is methyl, and R2 is OCH3.

[0045] In some embodiments, R8is CH2CF3, and R2is OCH2CF3.

[0046] In some embodiments, R3is H, F, Cl, CN, or perhalo-substituted C1-C3alkyl.

[0047] In some embodiments, R3 is H.

[0048] In some embodiments, R3is Cl.

[0049] In some embodiments, R3is CN.

[0050] In some embodiments, X is N; Y is NHCH3; R1 is H, CH3, SO2Me or oxetanyl; R2 is OCH3 or OCH2CF3; and R3 is H, Cl or CN.

[0051] The disclosed compounds also embrace combinations of the specific X, Y, R1, R2and R3groups disclosed above.

[0052] In some embodiments, the compounds of the present disclosure are represented by any of the following structures: 7 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO 3), ), ),Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO (I-

[0053] Compounds of the present disclosure may be in the form of a free acid or free base, or a pharmaceutically acceptable salt. A pharmaceutically acceptable salt of the compounds of this disclosure can be formed, for example, by reaction of an appropriate free base of a compound of the disclosure and an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions well known in the art. See, for example, Gould, P. L., “Salt selection for basic drugs,” International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, R. J., et al., “Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,” Organic Process Research and Development, 4:427-435 (2000); and Berge, S. M., et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, 66:1-19 (1977).

[0054] In some embodiments, a compound of the present disclosure is an isotopic derivative in that it has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound includes deuterium or multiple deuterium atoms. As used herein, the term “hydrogen”, i.e., H, refers to all isotopes of hydrogen, including protium (1H) and deuterium (2H). As used herein, the term “compound” embraces isotopic derivatives.

[0055] Compounds of the present disclosure may also be in the form of N-oxides, crystalline forms (also known as polymorphs), co-crystals, active metabolites of the compounds having the same type of activity, prodrugs, tautomers, and unsolvated as well as solvated (e.g., hydrated) forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, of the compounds. As used herein, the term “compound” embraces all these forms. Methods of Synthesis

[0056] In some aspects, the present disclosure is directed to a method for making a compound of formula I, or a pharmaceutically acceptable salt thereof. Broadly, the compounds or 9 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO pharmaceutically acceptable salts thereof, may be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present disclosure will be better understood in connection with the synthetic schemes that are described in various working examples that illustrate non-limiting methods by which the compounds of the disclosure may be prepared. Pharmaceutical Compositions

[0057] Another aspect of the present disclosure is directed to a pharmaceutical composition that includes the compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier,” as known in the art, refers to a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present disclosure to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous alike, and combinations thereof), solids, encapsulating materials, gases, and combinations thereof (e.g., semi-solids), and gases, that function to carry or transport the compound from one organ, or portion of the body, to another organ, or portion of the body. A carrier is “acceptable” in the sense of being physiologically inert to and compatible with the other ingredients of the formulation and not injurious to the subject (also referred to herein as “patient”). Depending on the type of formulation, the composition may also include one or more pharmaceutically acceptable excipients.

[0058] Broadly, compounds of formula I, and their pharmaceutically acceptable salts may be formulated into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20thed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988- 1999, Marcel Dekker, New York), each of which is incorporated herein by reference in its entirety. The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), and intrasternal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, intradermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal). 10 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0059] In general, the most appropriate route of administration will depend upon a variety of factors including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

[0060] The compositions may include an excipient, representative examples of which include preservatives, antioxidants, buffering agents, solubilizing agents, and surfactants. Representative examples of preservatives that may be suitable include alcohols, quaternary amines, organic acids, parabens, and phenols. Representative examples of antioxidants that may be suitable include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents like EDTA and citric acid. Representative examples of buffering agents that may be suitable include citric, hydrochloric, and lactic acid buffers. Representative examples of solubilizing agents that may be suitable include quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.

[0061] In other embodiments, the compositions may be formulated for oral, buccal, sublingual administration (examples of which include tablets, lozenges and gels), inhalation, topical, ophthalmic and rectal administration. Dosage Amounts

[0062] The amount of the compound of formula I administered to a patient may be effective in producing the desired therapeutic response in the patient. Therefore, the amount of the compound of formula I, or a pharmaceutically acceptable salt or thereof, may induce a positive modification in the disease or disorder to be treated, prevent development or progression of the disease or disorder, or alleviate to some extent, one or more of the symptoms of the disease or disorder being treated in a subject, or protect diseased cells from further damage (e.g., dopamine producing neurons), or kills or inhibits the growth of diseased cells (e.g., cancer cells), or reduces the amount of LRRK2 in diseased cells.

[0063] The total daily dosage of the compounds and usage thereof may be decided in accordance with standard medical practice, e.g., by the attending physician using sound medical judgment. The specific dose for any particular subject may depend upon a variety of factors including the disease or disorder being treated and the severity thereof (e.g., its present status); 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 11 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO treatment; drugs used in combination or coincidental with the compound; and like factors well known in the medical arts (see, for example, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10thEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001), which is incorporated herein by reference in its entirety.

[0064] Compounds of formula I, and their pharmaceutically acceptable salts may be effective over a wide dosage range. In some embodiments, the total daily dosage (e.g., for adult humans) may range from about 0.01 to about 1600 mg per day, from 0.05 to about 500 mg per day, from about 0.1 to about 100 mg per day, from about 0.5 to about 100 mg per day, from 1 to about 100 mg per day, from about 1 to about 50 mg per day, and from about 5 to about 40 mg per day, and in yet other embodiments from about 10 to about 30 mg per day. Individual dosages may be formulated to contain the desired dosage amount depending upon the number of times the compound is administered per day. By way of example, unit dosages (such as capsules or tablets) may be formulated with from about 1 to about 200 mg of a compound. In some embodiments, individual dosages may be formulated to contain the desired dosage amount depending upon the number of times the compound is administered per day. Methods of Use

[0065] In some aspects, the present disclosure provides a method of treating a disease or disorder that is associated with (e.g., characterized or mediated by) aberrant activity of LRRK2, comprising administering to a subject in need thereof a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0066] In a related aspect, the present disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in treating a disease or disorder that is associated with (e.g., characterized or mediated by) aberrant activity of LRRK2.

[0067] In another related aspect, the present disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for treating a disease or disorder that is associated with (e.g., characterized or mediated by) aberrant activity of LRRK2.

[0068] In yet another related aspect, the present disclosure provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a disease or disorder that is associated with (e.g., characterized or mediated by) aberrant activity of LRRK2. 12 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0069] As used herein, the term “LRRK2” embraces wild-type LRRK2 and mutant forms thereof such as missense mutations of LRRK2 (e.g., G2019S, R1441G, R1441C, R1441H, Y1699C, I2020T) that are involved in disease (Wojewska et al., Biomolecules 11(8): 1101 (2021)).

[0070] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone to or suffering from the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or a non-human mammal. A subject “in need of” treatment according to the present disclosure may be “suffering from or suspected of suffering from” a specific disease or disorder may have been positively diagnosed or otherwise presents with a sufficient number of risk factors or a sufficient number or combination of signs or symptoms such that a medical professional could diagnose or suspect that the subject was suffering from the disease or disorder. Thus, subjects suffering from, and suspected of suffering from, a specific disease or disorder are not necessarily two distinct groups.

[0071] In some embodiments, the disease is cancer. In some embodiments, the cancer is papillary thyroid carcinoma, colorectal cancer, pancreatic cancer, intrahepatic cholangiocarcinoma, breast cancer, lung cancer or kidney cancer.

[0072] In some embodiments, the disease is Parkinson’s Disease (PD). In some embodiments, the PD is associated with a missense mutation of LRRK2. In some embodiments, the missense mutation is G2019S.

[0073] In some embodiments, the disease is inflammatory bowel disease (IBD). In some embodiments, the IBD is Crohn’s disease (CD).

[0074] Modes of administration and dosage amounts are as disclosed above.

[0075] These and other aspects of the present disclosure will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the disclosure but are not intended to limit its scope, as defined by the claims. 13 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO EXAMPLES General synthetic Scheme

[0076] Example 1: Synthesis of Compounds 2-((2-methoxy-6-(methylsulfonyl)-5,6,7,8- tetrahydro-1,6-naphthyridin-3-yl)amino)-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidine-5- carbonitrile (I-2)nyl)-3-nitro-5,6,7,8-tetrahydro-1,6-naphthyridine (i3)

[0078] A solution of 1-methyl-3,5-dinitro-pyridin-2-one (i1) (5 g, 25.12 mmol) and 1- (methylsulfonyl)piperidin-4-one (i2) (4.9 g, 27.63 mmol) in NH3 / MeOH (7 N, 50 mL) was stirred in a sealed tube at 50 °C overnight. After cooled down to room temperature, the mixture was concentrated, and the residue was partitioned between DCM (100 mL) and saturated aqueous NaHCO3 solution (50 mL). The layers were separated, and the aqueous layer was extracted with DCM (50 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated to provide 6-(methylsulfonyl)-3-nitro-5,6,7,8-tetrahydro-1,6-naphthyridine as red solid (3.87 g, 60% yield).

[0079] LC-MS (ESI) m / z: 258.62 [M+H]+. 14 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO nyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (i5)

[0081] To a solution of 6-(methylsulfonyl)-3-nitro-5,6,7,8-tetrahydro-1,6-naphthyridine (3.87 g, 15.04 mmol) in ethanol (50 mL) and THF (50 mL) was added 10% Pd / C (1.65 g), and the mixture was stirred under H2 (1 atm) for 2 hours. After filtration, the cake was washed with EtOH (5 mL x 2) and the filtrate was concentrated to provide 6-(methylsulfonyl)-5,6,7,8-tetrahydro-1,6- naphthyridin-3-amine as yellow solid (3.42 g, 100% yield).

[0082] LC-MS (ESI) m / z: 228.47 [M+H]+. thylsulfonyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (i6)

[0084] To a solution of 6-(methylsulfonyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (3.42 g, 15.04 mmol) and AcONa (1.23 g, 30.08 mmol) in acetic acid (40 mL) was added bromine (2.40 g, 15.04 mmol) at room temperature and the mixture was stirred at room temperature for 1 hour. The mixture was diluted in DCM (100 mL) and water (30 mL), and slowly adjusted to pH ~9 with NaHCO3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified with flash column chromatography on silica gel (DCM / MeOH = 20 / 1, v / v) to provide 2-bromo-6-(methylsulfonyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine as yellow solid (1.93 g, 42%).

[0085] LC-MS (ESI) m / z: 307.76 [M+H]+.methylsulfonyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (i7)

[0087] To a mixture of 2-bromo-6-(methylsulfonyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3- amine (1.93 g, 6.3 mmol), cesium carbonate (4.1 g, 12.6 mmol) and MeONa (2.05 mL, 9.48 mmol) in MeOH (20 mL) were added CuI (120 mg, 0.63 mmol) and 1,10-phenanthroline monohydrate (227 mg, 1.26 mmol). The mixture was stirred under N2 in microwave conditions at 120 °C for 2 hours. After cooling to room temperature, the residue was purified by flash column 15 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO chromatography on silica gel (DCM / MeOH = 15 / 1, v / v) to give 2-methoxy-6-(methylsulfonyl)- 5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine yellow solid (778 mg, 48% yield).

[0088] LCMS: m / z = 258.62 (M + H)+. ) (100 mg, 0.39 mmol), i8 (138 mg, 0.41mmol) and K2CO3(161 mg,1.17 mmol) in degassed dioxane (5 mL) was added BrettPhos Pd G3 (35 mg, 0.039 mmol). The mixture was stirred at 100°C for 2 h at which time the mixture was cooled to room temperature (rt), filtered through celite rinsing with EtOAc. The organic layer was washed with water, brine, dried over MgSO4and condensed to give a dark brown oil that was dissolved in DCM (10 mL) and TFA (1 mL). The mixture was stirred at rt for 2 h at which point the solvent was removed under reduced pressure. The resulting brown residue was dissolved in THF:H2O 1:1 (10 mL) and saturated aqueous NaHCO3(10 mL) was added. The mixture was stirred for 3h at rt. The reaction was quenched with H2O and extracted with EtOAc (3 × 50mL). The organic layer was washed with water, brine, dried over MgSO4 and condensed to give a dark brown oil that was purified by reversed phase HPLC using a gradient of 2% to 70% ACN in H2O with 0.025% trifluoroacetic acid to give a white solid (28 mg, 17% over 3 steps).

[0090] 1H NMR (500 MHz, DMSO-d6) δ 12.28 (br, 1H), 8.55 (s, 1H), 7.88 (d, J = 4 Hz, 1H), H), -3-Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0093] Compound I-1 was prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid (23% yield over 3 steps).

[0094] 1H NMR (500 MHz, DMSO-d6) δ 12.29 (br, 1H), 8.52 (s, 1H), 7.9 (d, J = 4 Hz, 1H), 7.58 (s, 1H), 6.62 (br, 1H), 4.45 (s, 2H), 4.33-4.27 (m, 2H), 3.97 (s, 3H), 3.77- 3.70 (m, 1H), 3.49-3.40 (m, 1H), 3.02 (d, J = 5 Hz, 3H), 2.98 (s, 3H).

[0095] LCMS: m / z = 365.52 (M + H)+.

[0096] Example 3: Synthesis of 2-((6-methyl-2-(2,2,2-trifluoroethoxy)-5,6,7,8-tetrahydro-1,6- naphthyridin-3-yl)amino)-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (I-3) ) prepared in an analogous manner to compound I-2 in Example 1 andwas isolated as a white solid (33% yield over 3 steps).

[0098] 1H NMR (500 MHz, DMSO-d6) δ 12.28 (br, 1H), 8.55 (s, 1H), 7.91 (d, J = 4 Hz, 1H), 7.58 (s, 1Hz, 2H), 4.51 (s, 2H), 4.36-4.30 (m, 2H), 3.77- 3.70 (m, 1H), 3.51-3.41 (m, 1H), 3.02 (d, J = 5 Hz, 3H), 2.98 (s, 3H).

[0099] LCMS: m / z = 433.71 (M + H)+.

[0100] Example 4: Synthesis of 4-(methylamino)-2-((6-(methylsulfonyl)-2-(2,2,2- trifluoroethoxy)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine- 5-carbonitrile (I-4) )s prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 31% yield over 3 steps. 17 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0102] 1H NMR (500 MHz, DMSO-d6) δ 12.27 (br, 1H), 8.58 (s, 1H), 7.88 (d, J = 4 Hz, 1H), 7.45 (s, 1H), 6.65 (br, 1H), 5.07 (q, J = 8 Hz, 2H), 4.36 (s, 2H), 3.52 (t, J = 7 Hz, 2H), 3.01 (d, J = 5 Hz, 3H), 2.99 (s, 3H), 2.88 (t, J = 7 Hz, 2H).

[0103] LCMS: m / z = 497.59 (M + H)+.

[0104] Example 5: Synthesis of 5-chloro-N2-(2-methoxy-6-(methylsulfonyl)-5,6,7,8- tetrahydro-1,6-naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-5) ) as prepared in an analogous manner to compound I-2 in Example 1 andwas isolated as a white solid 37% yield over 3 steps.

[0106] 1H NMR (500 MHz, DMSO-d6) δ 11.61 (br, 1H), 8.59 (s, 1H), 7.65 (br, 1H), 7.05 (s, 1H), 6.91 (br, 1H), 4.33 (s, 2H), 3.96 (s, 3H), 3.51 (t, J = 7 Hz, 2H), 3.01 (d, J = 5 Hz, 3H), 2.98 (s, 3H), 2.87 (t, J = 7 Hz, 2H).

[0107] LCMS: m / z = 438.82 (M+H)+.

[0108] Example 6: Synthesis of N2-(2-methoxy-6-(methylsulfonyl)-5,6,7,8-tetrahydro-1,6- naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-6) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 32% yield over 3 steps.

[0110] 1H NMR (500 MHz, DMSO-d6) δ 11.89 (br, 1H), 8.85 (s, 1H), 8.56 (br, 1H), 7.04 (s, 1H), 6.62 (br, 1H), 4.36 (s, 2H), 3.99 (s, 3H), 3.53 (t, J = 7 Hz, 2H), 3.07 (m, 3H), 2.99 (s, 3H), 2.90 (t, J = 7 Hz, 2H).

[0111] LCMS: m / z = 404.76 (M+H)+. 18 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0112] Example 7: Synthesis of 5-chloro-N2-(2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6- naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-7) ) as prepared in an analogous manner to compound I-2 in Example 1 andwas isolated as a white solid 29% yield over 3 steps.

[0114] 1H NMR (500 MHz, DMSO-d6) δ 11.55 (br, 1H), 8.57 (br, 1H), 7.61 (br, 1H), 7.04 (br, 1H), 6.86 (br, 1H), 4.46 (s, 2H), 4.34-4.27 (m, 2H), 3.98 (s, 3H), 3.76-3.70 (m, 1H), 3.49-3.41 (m, 1H), 3.02 (d, J = 5 Hz, 3H), 2.98 (s, 3H).

[0115] LCMS m / z = 374.69 (M + H)+.

[0116] Example 8: Synthesis of N2-(2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin- 3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-8) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 23% yield over 3 steps.

[0118] 1H NMR (500 MHz, DMSO-d6) δ 11.7 (br, 1H), 10.31 (br, 1H), 8.60 (br, 1H), 7.61 (br, 1H), 6.98 (br, 1H), 6.56 (br, 1H), 4.48 (s, 2H), 4.36-4.28 (m, 2H), 4.0 (s, 3H), 3.78-3.70 (m, 1H), 3.51-3.41 (m, 1H), 3.03 (m, 2H), 2.99 (s, 3H).

[0119] LCMS m / z = 340.28 (M + H)+.

[0120] Example 9: Synthesis of 5-chloro-N2-(2-methoxy-6-(oxetan-3-yl)-5,6,7,8-tetrahydro- 1,6-naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-9) 19 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO ) as prepared in an analogous manner to compound I-2 in Example 1 andwas isolated as a white solid 34% yield over 3 steps.

[0122] 1H NMR (500 MHz, DMSO-d6) δ 11.61 (br, 1H), 8.57 (br, 1H), 7.69 (br, 1H), 7.06 (br, 1H), 6.91 (br, 1H), 4.87-4.79 (m, 4H), 4.59-4.53 (m, 2H), 4.37-4.14 (m, 3H), 3.98 (s, 3H), 3.61- 3.30 (m, 2H), 3.01 (d, J = 5 Hz, 3H).

[0123] LCMS m / z = 416.73 (M + H)+.

[0124] Example 10: Synthesis of N2-(2-methoxy-6-(oxetan-3-yl)-5,6,7,8-tetrahydro-1,6- naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-10) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 39% yield over 3 steps.

[0126] 1H NMR (500 MHz, DMSO-d6) δ 11.85 (br, 1H), 8.56 (br, 1H), 7.02 (br, 1H), 7.60 (br, 1H), 4.87-4.79 (m, 4H), 4.61-4.50 (m, 2H), 4.40-4.16 (m, 3H), 3.99 (s, 3H), 3.65-3.40 (m, 2H), 3.01 (d, J = 5 Hz, 3H).

[0127] LCMS m / z = 382.59 (M + H)+.

[0128] Example 11: Synthesis of 5-chloro-N4-methyl-N2-(6-methyl-2-(2,2,2-trifluoroethoxy)- 5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-11) 20 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO ) as prepared in an analogous manner to compound I-2 in Example 1and was isolated as a white solid 39% yield over 3 steps.

[0130] 1H NMR (500 MHz, DMSO-d6) δ 11.49 (br, 1H), 8.64 (br, 1H), 7.46 (br, 1H), 7.04 (br, 1H), 6.78 (br, 1H), 5.10 (d, J = 8 Hz, 2H), 4.49 (s, 2H), 4.37-4.09 (m, 2H), 3.77-3.71 (m, 1H), 3.5- 3.4 (m, 1H), 3.02 (d, J = 5 Hz, 2H), 2.99 (s, 3H).

[0131] LCMS m / z = 442.63 (M + H)+.

[0132] Example 12: Synthesis of N4-methyl-N2-(6-methyl-2-(2,2,2-trifluoroethoxy)-5,6,7,8- tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-12) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 34% yield over 3 steps.

[0134] 1H NMR (500 MHz, DMSO-d6) δ 11.57 (br, 1H), 8.85 (br, 1H), 8.62 (s, 1H), 7.05 (s, 1H), 6.97 (s, 1H), 6.55 (br, 1H), 5.10 (d, J = 8 Hz, 2H), 4.57-4.47 (m, 2H), 4.40-4.33 (m, 2H), 3.79-3.72 (m, 1H), 3.52-3.44 (m, 1H), 3.01 (m, 3H), 2.99 (s, 3H).

[0135] LCMS m / z = 408.84 (M + H)+.

[0136] Example 13: Synthesis of 5-chloro-N4-methyl-N2-(6-(oxetan-3-yl)-2-(2,2,2- trifluoroethoxy)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4- diamine (I-13) 21 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO ) as prepared in an analogous manner to compound I-2 in Example 1and was isolated as a white solid 27% yield over 3 steps.

[0138] 1H NMR (500 MHz, DMSO-d6) δ 11.53 (br, 1H), 8.62 (br, 1H), 7.54 (br, 1H), 7.05 (br, 1H), 6.83 (br, 1H), 5.12 (d, J = 8 Hz, 2H), 4.86-4.81 (m, 4H), 4.57-4.50 (m, 2H), 4.40-4.32 (m, 3H), 3.79-3.72 (m, 1H), 3.52-3.41 (m, 1H), 3.0 (d, J = 5 Hz, 3H).

[0139] LCMS m / z = 484.71 (M + H)+.

[0140] Example 14: Synthesis of N4-methyl-N2-(6-(oxetan-3-yl)-2-(2,2,2-trifluoroethoxy)- 5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-14) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 23% yield over 3 steps.

[0142] 1H NMR (500 MHz, DMSO-d6) δ 11.75 (br, 1H), 8.56 (br, 1H), 8.44 (br, 1H), 7.01 (br, 1H), 6.58 (br, 1H), 5.15 (d, J = 8 Hz, 2H), 4.81 (m, 4H), 4.58-4.41 (m, 3H), 4.40-4.32 (m, 2H), 3.77-3.70 (m, 1H), 3.53-3.44 (m, 1H), 3.02 (d, J = 5 Hz, 3H).

[0143] LCMS m / z = 450.35 (M + H)+.

[0144] Example 15: Synthesis of 5-chloro-N2-(2-methoxy-5,6,7,8-tetrahydro-1,6- naphthyridin-3-yl)-N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-15) 22 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO ) as prepared in an analogous manner to compound I-2 in Example 1and was isolated as a white solid 39% yield over 3 steps.

[0146] 1H NMR (500 MHz, DMSO-d6) δ 11.57 (br, 1H), 9.06 (br, 2H), 8.58 (br, 1H), 7.62 (br, 1H), 7.05 (br, 1H), 6.88 (br, 1H), 4.25 (m, 2H), 3.97 (s, 3H), 3.51-3.45 (m, 2H), 3.01 (d, J = 5 Hz, 3H), 2.95 (t, J = 7 Hz, 2H).

[0147] LCMS m / z = 360.53 (M + H)+.

[0148] Example 16: Synthesis of N2-(2-methoxy-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)- N4-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-16) )as prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 27% yield over 3 steps.

[0150] 1H NMR (500 MHz, DMSO-d6) δ 11.73 (br, 1H), 9.15 (br, 2H), 8.61 (br, 1H), 7.01 (br, 1H), 6.58 (br, 1H), 4.27 (m, 2H), 3.99 (s, 3H), 3.52-3.46 (m, 2H), 3.04 (m, 3H), 2.96 (t, J = 7 Hz, 2H).

[0151] LCMS m / z = 326.61 (M + H)+.

[0152] Example 17: Synthesis of 5-chloro-N4-methyl-N2-(2-(2,2,2-trifluoroethoxy)-5,6,7,8- tetrahydro-1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-17) 23Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0153] Compound I-17 was prepared in an analogous manner to compound I-2 in Example 1 and was isolated as a white solid 38% yield over 3 steps.

[0154] 1H NMR (500 MHz, DMSO-d6) δ 11.52 (br, 1H), 9.13 (br, 1H), 8.63 (s, 1H), 7.52 (br, 1H), 7.06 (br, 1H), 6.83 (br, 1H), 5.10 (d, J = 8 Hz, 2H), 4.29 (m, 2H), 3.52-3.46 (m, 2H), 3.0 (d, J = 5 Hz, 2H), 2.95 (t, J = 7 Hz, 2H).

[0155] LCMS m / z = 428.64 (M + H)+.

[0156] Example 18: Synthesis of N4-methyl-N2-(2-(2,2,2-trifluoroethoxy)-5,6,7,8-tetrahydro- 1,6-naphthyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (I-18) ) as prepared in an analogous manner to compound I-2 in Example 1and was isolated as a white solid 26% yield over 3 steps.

[0158] 1H NMR (500 MHz, DMSO-d6) δ 11.64 (br, 1H), 9.16 (br, 1H), 8.64 (s, 1H), 6.98 (br, 1H), 6.56 (br, 1H), 5.13 (d, J = 8 Hz, 2H), 4.31 (m, 2H), 3.54-3.48 (m, 2H), 3.02 (d, J = 5 Hz, 2H), 2.98 (t, J = 7 Hz, 2H).

[0159] LCMS m / z = 394.57 (M + H)+.

[0160] Example 19: Cell line generation, culturing and treatment

[0161] The LRRK2[G2019S] cl.1.6 homozygous knock-in and littermate matched wild-type (WT) Mouse Embryonic fibroblasts (MEFs) cl.1.2 were generated from LRRK2[G2019S] knock- in mice obtained from Taconic-generated mice (model 13940) as described in Steger et al., ELife 5:e12813 (2016).

[0162] Cells were cultured in complete growth medium (Dulbecco's modified Eagle medium (Glutamax and Gibco) supplemented with 10% (v / v) Fetal Bovine Serum; 100 U / ml penicillin and 100 µg / ml streptomycin, 1mM MEM Non-essential Amino Acid Solution (Life Technologies) and 1 mM sodium pyruvate (Life Technologies)). WT MEFs and homozygous MEFs LRRK2-G2019S mutants’ cells were plated at equal density into 6-well plates in a final volume of 3 ml of complete growth medium / well. 24 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO

[0163] Inhibitors were reconstituted in DMSO (Sigma, #D2650) and used at 1:1000 in cells, i.e., 3 µl / 3 ml. The same volume of DMSO was added to the control wells. After reaching over 90% confluence, the cells were treated in triplicate for 90 minutes at 37°C. After incubation, media were aspirated, plates were placed on ice, and cells were washed with DPBS (Dulbecco’s Phosphate Buffered Saline). 120 ^l of an ice-cold lysis buffer containing 50 mM Tris–HCl, pH 7.5, 1% (v / v) Triton X-100, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM NaF, 0.1% (v / v) 2-mercaptoethanol, 10 mM 2-glycerophosphate, 5 mM sodium pyrophosphate, 0.1 µg / ml microcystin-LR (Enzo Life Sciences), 270 mM sucrose and complete EDTA-free protease inhibitor cocktail (Sigma–Aldrich Cat # 11836170001) was added per well. Lysates were centrifuged at 20 817 g (14,000 rpm) for 15 min at 4°C and supernatants were used to determine protein concentration using Bradford assay (Pierce™ Coomassie (Bradford) Protein Assay Kit, Thermo Scientific Cat #23200) and for Western blot analysis. Lysates were snap-frozen in liquid nitrogen and stored at -80 °C until required.

[0164] Example 20: Western blotting

[0165] Cell lysates were mixed with 4x SDS–PAGE sample buffer [50 mM Tris–HCl, pH 6.8, 2% (w / v) SDS, 10% (v / v) glycerol, 0.02% (w / v) Bromophenol Blue and 1% (v / v) 2- mercaptoethanol] to a final total protein concentration of 2 µg / µl and heated at 95°C for 10 min. 30 ^g of samples were loaded onto NuPAGE 4-12% Bis-Tris gradient gels (Life Technologies) along with 3 µl of BIO-RAD protein marker (Precision Plus Protein™ All Blue Prestained Protein Standards #1610373kDa), gel was run at 120 V for 2.5 h with the NuPAGE MOPS SDS running buffer (Life Technologies, Cat# NP0001-02). After electrophoresis, the separated proteins were transferred onto the nitrocellulose membrane (GE Healthcare, Amersham Protran 0.45 µm NC) at 90 V for 90 min. Transferred membranes were briefly stained with Ponceau S stain and divided into 3 strips (Fan et al., Biochem. J.475:23-44 (2018)). Briefly, the upper strip was cut from the top of the membrane to 75 kDa, the middle strip cut was between 75 kDa – 40 kDa, and the bottom strip cut was from 40 kDa- to the bottom of the membrane. Membrane strips were blocked at room temperature with 5% (w / v) dried skimmed milk dissolved in TBS-T [20 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.1% (v / v) Tween 20] for 1 h, washed four times with 10-min intervals in TBS- T and incubated with primary antibodies diluted in 5% (w / v) BSA (bovine serum albumin) in TBS- T overnight at 4°C. Primary antibodies were used as follow: the upper strip was incubated with 1 µg / ml of rabbit anti-LRRK2 pSer935 UDD2 antibody combined with mouse anti-LRRK2 C- 25 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO terminus (Dardarin) total antibody; the middle strip was incubated with rabbit anti-α-tubulin (Cell Signaling Technology #5174). The bottom strip was blotted with rabbit MJFF-pRAB10 monoclonal antibody multiplexed with a mouse MJFF-total Rab10 monoclonal antibody at a final concentration of 0.5 µg / mL for each antibody. Membranes were washed as before and incubated at room temperature for 1 h with anti-rabbit and anti-mouse near-infrared fluorescent IRDye antibodies (LI-COR #925-68070, #925-32211) diluted (1: 30 000 and 1:15000, respectively) in 5% (w / v) dried skimmed milk dissolved in TBS-T. Following incubation in secondary antibodies, membrane strips were washed, and the signal was developed using the Li-COR Odyssey CLx Western Blot imaging system and quantified using the Image Studio software (FIG.1-FIG.8).

[0166] As shown in FIG.1 to FIG.4, levels of LRRK2 phosphorylated at serine 935 (pSer935 LRRK2) is reduced as the compound concentrations are increased from 0 to 1000 nM in both wild- type (WT) and LRRK2 G2019S (mutant) cells. Likewise, levels of phosphorylated Rab 10 (pRab 10) were reduced suggesting that the disclosed compounds inhibit both LRRK2 autophosphorylation and downstream phosphorylation (Kania et al., Cell Death Dis., 14:436 (2023)). These results were quantified in terms of relative phosphorylation in FIG.4 to FIG.8 by quantifying pRab10 / Rab10 total (downstream phosphorylation) and / or pS935 / LRRK2 total (autophosphorylation). The reduction in phosphorylation was plotted against the compound concentration to calculate inhibition constants (IC50) for wild-type (WT) and the G2019S mutant in fibroblast systems. The quantified signal for each inhibitor concentration tested in LRRK2 WT and G2019S mutant cells was averaged across three replicates. The values were then normalized to the DMSO control (0 nM) set at 1. The IC50 values were calculated using nonlinear regression analysis with variable slope using GraphPad Prism (GraphPad Software 10). The results are shown in Table 1. 26 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO Table 1. IC50 of exemplary compounds in cells. Compound Cell Line Relative Protein Levels IC50[nM] logIC50I 1 G2019S R b10 / R b10 t t l 7253 186 [0016

[0168] IC50experiments were performed for the disclosed compounds. The results are shown in Table 2. An exemplary kit to perform this assay includes the Adapta™ Screening Protocol and Assay Conditions by Invitrogen™ (Fisher Scientific™, Catalog No. PV5099).

[0169] All LRRK2 kinase inhibition assays were conducted by using Promega ADP-Glo™ Kinase assay systems (Catalog number; V4475). Inhibitors were tested with 12 concentrations over a 5-fold serial dilution series (50 µM, 10 µM, 2 µM, 400 nM, 80 nM, 16 nM, 3.2 nM, 640 pM, 128 pM, 25.6 pM, 5.12 pM and 1.024 pM) and PF06447475 as positive control. For the LRRK2 wild-type enzyme assay, each inhibitor was mixed with 0.2 µg / µL of substrate (LRRKtide, SignalChem, Richmond, BC, Canada), 10 μmol / L ATP (Invitrogen™, Carlsbad, CA), 24 ng of 27 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO LRRK2 WT enzyme (Thermo Fisher Scientific, PR8604B) in 384-well plate. For LRRK G2019S, each compound was mixed with 0.2 µg / µL of substrate, 25 μmol / L ATP, 16 ng of LRRK2 G2019S enzyme (SignalChem, L10-12GG). All samples were diluted with kinase reaction buffer (40 mmol / L TrisHCl, 10 mmol / L MgCl2, and 0.1 μg / μL BSA (bovine serum albumin)). After 2 h at 25oC, ADP-Glo™ (Promega, Madison, WI) reagent was added and the samples were incubated at rt for 40 min. Finally, Kinase Detection Reagent was added. The resulting mixtures were allowed to react at rt for 10 min. Luminescence signals were detected using Synergy™ Neo2 microplate reader (Bio-Tek). Compound inhibition curve was fitted using Graphpad Prism 8.0 software. Table 2 shows that the compounds of the disclosure are potent inhibitors of both wild-type and G2019S LRRK2 with IC50values generally within a ~3-fold range for a given compound. Furthermore, the compounds demonstrated selectivity for LRRK over TTK. Table 2. IC50 of disclosed compounds. Compound Wild-type IC50[nM] G2019S IC50[nM] TTK IC50[nM]28 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO I-15 8 5 13 I-16 85 32 384 l ofany specific portions thereof that are referenced) are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0171] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. 29 AFSDOCS:301794960.1

Claims

Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO What is claimed is:

1. A compound having a structure represented by formula I: I), or a pharmaceutically acceptablewherein: X is CH or N; Y is N(R4)2or OR5, wherein each R4is independently H or C1-C3alkyl, and R5is H or C1-C3alkyl; R1 is H, C1-C3 alkyl, SO2R6, C(O)R7, or 4-6-membered O-containing heterocyclyl, wherein R6 is C1-C3alkyl, and R7is H or C1-C3alkyl; R2is OR8, wherein R8is C1-C3alkyl optionally substituted with one or more halo group; and R3 is H, halo, CN, or optionally substituted C1-C3 alkyl.

2. The compound of claim 1, wherein X is CH.

3. The compound of claim 1, wherein X is N.

4. The compound of any one of claims 1-3, wherein Y is NHR4.

5. The compound of claim 4, wherein R4 is methyl, and Y is NHCH3.

6. The compound of any one of claims 1-5, wherein R1is H.

7. The compound of any one of claims 1-5, wherein R1 is C1-C3 alkyl.

8. The compound of claim 7, wherein R1is CH3.

9. The compound of any one of claims 1-5, wherein R1 is SO2R6. 30 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO 10. The compound of claim 9, wherein R6 is methyl and R1 is SO2CH3.

11. The compound of any one of claims 1-5, wherein R1is C(O)R7.

12. The compound of claim 11, wherein R7 is CH3 and R1 is COCH3.

13. The compound of any one of claims 1-5, wherein R1is a 4-6-membered O-containing heterocyclyl.

14. The compound of claim 13, wherein R1is oxetanyl.

15. The compound of any one of claims 1-14, wherein R8 is C1-C3 alkyl optionally substituted with one or more fluoro group.

16. The compound of claim 15, wherein R8is methyl, and R2is OCH3.

17. The compound of claim 15, wherein R8is CH2CF3, and R2is OCH2CF3.

18. The compound of any one of claims 1-17, wherein R3 is H, F, Cl, CN, or perhalo- substituted C1-C3 alkyl.

19. The compound of claim 18, wherein R3is H.

20. The compound of claim 18, wherein R3 is Cl.

21. The compound of claim 18, wherein R3is CN.

22. The compound of claim 1, which is: -3),Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO ), 2), 5), 8),. 32 AFSDOCS:301794960.1Date of Deposit: June 5, 2025 Attorney Docket No.046094-789001WO 23. A pharmaceutical composition, comprising the compound or pharmaceutically acceptable salt thereof, of any one of claims 1-22, and a pharmaceutically acceptable carrier.

24. The method of treating a disease or disorder that is associated with aberrant activity of LRRK2, comprising administering to a subject in need thereof the compound or a pharmaceutically acceptable salt thereof, of any one of claims 1-22.

25. The method of claim 24, wherein the disease is cancer.

26. The method of claim 24, wherein the disease is Parkinson’s disease (PD).

27. The method of claim 26, wherein the PD involves aberrant activity of LRRK2 that has a missense mutation.

28. The method of claim 27, wherein the missense mutation is G2019S.

29. The method of claim 24, wherein the disease is inflammatory bowel disease (IBD).

30. The method of claim 29, wherein the IBD is Crohn’s disease. 33 AFSDOCS:301794960.1

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