WO2021252339A1 - Substituted purine-2,6-dione compounds as kras inhibitors - Google Patents

Abstract

The present disclosure relates to novel compounds that inhibits KRAS G12C, pharmaceutical compositions containing such compounds, and their use in prevention and treatment of cancer and related diseases and conditions.

Description

SUBSTITUTED PURINE-2, 6-DIONE COMPOUNDS AS KRAS INHIBITORS Cross Reference to Related Application

[1] This application claims the benefit of priority to United States Provisional Patent Application No. 63/035,896, filed June 8, 2020, which is hereby incorporated by reference in its entirety.

Field of the Disclosure

[2] The present invention relates to compounds that inhibit KRas G12C. In particular, the present invention relates to compounds that irreversibly inhibit the activity of KRas G12C, pharmaceutical compositions comprising the compounds and methods of use therefor.

Background of the Disclosure

[3] The KRAS, NRAS and HRAS genes encode a set of closely related small GTPase proteins KRas, NRas and HRas, collectively referred to herein as the Ras proteins or Ras, that share 82-90% overall sequence identity. The Ras proteins are critical components of signalling pathways transmitting signals from cell-surface receptors to regulate cellular proliferation, survival and differentiation. Ras functions as a molecular switch cycling between an inactive GDP-bound state and an active GTP-bound state. The GDP/GTP cycle of Ras is tightly regulated in cells by guanine nucleotide exchange factors (GEFs) such as Sosl and Sos2, which promote the exchange of GDP for GTP, and GTPase activating proteins (GAPs) such as NF-1 and pl20RasGAP which stimulate the intrinsic GTPase activity of Ras hydrolysing GTP to GDP.

[4] The Ras proteins are 188-189 amino acids in length and have a highly conserved N-terminal G- domain containing the p-loop region, which binds nucleotide, and the switch I and switch II regions which are important for regulatory and effector protein interactions. The C-terminal region of the Ras proteins are more divergent and contain elements which regulate the association of Ras with the membrane including the conserved carboxyl terminal CAXX box motif which is necessary for post- translational prenylation modifications. On binding to GTP the switch I and switch II regions of Ras undergo a conformational change which enables its interaction and activation of effector proteins to regulate down-stream signalling pathways. The best characterized effector of Ras is the serine/threonine kinase Raf which regulates the activity of the mitogen-activate protein kinase (MAPK) pathway. The PI3K pathway is another important effector pathway down-stream of Ras with the pi 10 catalytic subunit of the class I phosphoinositide 3-kinases interacting with Ras. Other effectors of Ras including RalGDS, TiamF, PFC-e and Rassfl have been have also been described (See, Cox et al. Nature Reviews Drug Discovery, 13:828-851 (2014)).

[5] RAS mutations are frequently found in cancer and approximately 30% of all human cancers have a mutation in KRAS, NRAS or HRAS genes. Oncogenic Ras is typically, but not exclusively, associated with mutations at glycine 12, glycine 13 or glutamine 61 of Ras. These residues are located at the active site of Ras and mutations impair intrinsic and/or GAP-catalysed GTPase activity favouring the formation of GTP bound Ras and aberrant activation of down-stream effector pathways. KRAS is the most frequently mutated RAS gene in cancer followed by NRAS and then HRAS. [6] Glycine to cysteine mutation at residue 12 of Ras (the G12C mutation) is generated from a G.C to T.A base transversion at codon 12, a mutation commonly found in RAS genes that accounts for 14% of all KRAS, 2% of all NRAS and 2% of all HRAS mutations across cancer types. The G12C mutation is particularly enriched in KRAS mutant non-small cell lung cancer with approximately half carrying this mutation, which has been associated with the DNA adducts formed by tobacco smoke. The G12C mutation is not exclusively associated with lung cancer and is found in other RAS mutant cancer types including 8% of all KRAS mutant colorectal cancer.

[7] There are several tumour types that exhibit a high frequency of activating mutations in KRAS including pancreatic (-90% prevalence), colorectal (-40% prevalence) and non-small cell lung cancer (-30% prevalence). KRAS mutations are also found in other cancer types including multiple myeloma, uterine cancer, bile duct cancer, stomach cancer, bladder cancer, diffuse large B cell lymphoma, rhabdomyosarcoma, cutaneous squamous cell carcinoma, cervical cancer, testicular germ cell cancer and others.

[8] There remains an unmet medical need for new medical treatments for patients with pancreatic cancer, lung adenocarcinoma, or colorectal cancer, especially those who have been diagnosed to have such cancers characterized by a KRAS mutation, and including those who having cancer that progressed after chemotherapy.

Summary of The Disclosure

[9] In some embodiments, provided herein are compounds of Formula (I), or a pharmaceutically acceptable salt:

wherein:

X₁ is CR₁ or N;

X₂ is CR₂ or N;

X₃ is CR₃ or N;

X₄ is CR₄ or N;

X₅ is CR₅ or N each of R₁, R₂, R₃, R₄, and R₅ is independently selected from hydrogen, halogen, hydroxyl group, -CN, C₁-C₅alkyl, C₁-C₅alkoxy, and C₁-C₅haloalkyl;

L is a linker of 1 to 16 carbon atoms in length, wherein one or more carbon atoms are optionally and independently replaced by a group selected from C(=O), O, N(R₉), S, C₂-alkenyl, C2-alkynyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the R₉ C2-alkenyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 0, 1, 2, or 3 Rio; R7 is independently selected from hydrogen, C₁-C₅alkyl, and C₁-C₅haloalkyl, each of which is substituted with 0, 1, 2, or 3 R_10;

X is selected from -CH₂-, -(CH₂)₂-, and -(CH₂)₃-, each of which is optionally substituted at one or more hydrogens with Ri 1;

A is selected from cycloalkyl, aryl, heterocycle, and heteroaryl, each of which is substituted with 1, 2, or 3 R₈;

R₈ is independently selected from a,b-unsaturated carbonyl derivative, carboxamide, C₁-C₅alkyl, C₁-C₅alkoxy, C₁-C₅haloalkyl, each of which is optionally substituted with 1, 2, or 3 substituents selected from halogen, hydroxyl, C₁-C₃alkyl, C₁-C₃alkoxy, C₁-C₃haloalkyl, -NHCH3, -N(CH₃)2, and -CN; each R9 is independently selected from hydrogen, C₁-C₃alkyl, -C(=O)-(C₁-C₃alkyl), -C(=O)-O- (C₁-C₃alkyl), and -C(=O)-NH-(C₁-C₃alkyl), each of which is substituted with 0, 1, 2, or 3 R₁₀; each R₁₀ is independently selected from halogen, hydroxyl, C₁-C₃alkyl, C₁-C₃alkoxy, Ci- C₃haloalkyl, -N(R₉)₂, and -CN; and each Rn is independently selected from C₁-C₃alkyl, C₁-C₃alkoxy, Ci-Cshaloalkyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the C₁-C₃alkyl, C₁-C₃alkoxy, Ci-Cihaloalkyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 1, 2, or 3 substituents selected from halogen, -NO2, hydroxyl, -NH₂, -NHCH₃, -N(CH₃)₂, -CN, C₁-C₃alkyl, C₁-C₃alkoxy, C₁-C₃haloalkyl, C₃- C₆cycloalkyl, C₃-C₆heterocyclyl, C6-Ci2aryl, and C₆-C ₁₂ heteroaryl; wherein each hydrogen atom is independently and optionally replaced by a deuterium atom.

[10] In some embodiments, the compound of Formula (I) is a compound of Formula (1A):

[11] In some embodiments, the compound of Formula (I) is a compound of Formula (IB):

[12] Also provided herein is a method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of a compound disclosed herein. In some embodiments, the cancer is selected from breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gall bladder cancer, thyroid cancer, bile duct cancer, ovarian cancer, endometrial cancer, prostate cancer, and esophageal cancer.

BRIEF DESCRIPTION OF THE FIGURES

[13] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, the attached drawings illustrate some, but not all, alternative embodiments. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities shown. These figures, which are incorporated into and constitute part of the specification, assist in explaining the principles of the disclosures.

[14] Figures 1 A illustrates molecular weight shift of KRAS-G12C mutant and wild type proteins on SDS-PAGE after 18 hours of coincubation at 25 °C with exemplary Compounds 4 and 5.

[15] Figures IB illustrates molecular weight shift of KRAS-G12C mutant and wild type proteins on SDS-PAGE after 18 hours of coincubation at 25 °C with exemplary Compounds 20 and 21.

[16] Figure 2 A depicts the crystal structure of Compound 5 complex with KRAS (G12C).

[17] Figure 2B depicts the crystal structure of Compound 21 complex with KRAS (G12C).

DETAILED DESCRIPTION

Definitions

[18] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -CN is attached through the carbon atom.

[19] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C₁-C₆ alkyl” is intended to encompass C₁, C₂, C₃, C₄, C₅, C₆, C_1-6, C_1-5, C_1-4, C_1-3, C₁- ₂, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_5-6 alkyl.

[20] The term “acyl” as used herein refers to R-C(O)- groups such as, but not limited to, (alkyl)-C(O)-, (alkenyl)-C(O)-, (alkynyl)-C(O)-, (aryl)-C(O)-, (cycloalkyl)-C(O)-, (heteroaryl)-C(O)-, and (heterocyclyl)-C(O)-, wherein the group is attached to the parent molecular structure through the carbonyl functionality. In some embodiments, it is a Ci-10 acyl radical which refers to the total number of chain or ring atoms of the, for example, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or heteroaryl, portion plus the carbonyl carbon of acyl. For example, a C4-acyl has three other ring or chain atoms plus carbonyl.

[21] The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-8 carbon atoms, referred to herein as (C₂-C₈)alkenyl. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-butene)-pentenyl.

[22] The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-8 carbon atoms, referred to herein as C_M alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl- 1 -propyl, 2-methyl-2-propyl, 2- methyl-1 -butyl, 3 -methyl- 1 -butyl, 2-methyl-3-butyl, 2,2-dimethyl- 1 -propyl, 2-methyl- 1 -pentyl, 3-methyl- 1 -pentyl, 4-methyl- 1 -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- 1- butyl, 3,3-dimethyl- 1 -butyl, 2-ethyl- 1 -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. In some embodiments, “alkyl” is a straight-chain hydrocarbon. In some embodiments, “alkyl” is a branched hydrocarbon.

[23] The term “alkoxy” means a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms containing a terminal “O” in the chain, e.g., -O(alkyl). Examples of alkoxy groups include, without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[24] The term “alkylene” as used herein referes to a divalent alkyl radical. Representative examples of Ci-io alkylene include, but are not limited to, methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3- methylhexylene, 2,2-dimethylpentylene, 2,3-dimethylpentylene, n-heptylene, n-octylene, n-nonylene and n-decylene.

[25] The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-8 carbon atoms, referred to herein as (C2-C8)alkynyl. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl- 1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.

[26] The term “aryl” as used herein refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system with 5 to 14 ring atoms. The aryl group can optionally be fused to one or more rings selected from aryls, cycloalkyls, heteroaryls, and heterocyclyls. The aryl groups of this present disclosure can be substituted with groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5, 6,7,8- tetrahydronaphthyl. Exemplary aryl groups also include but are not limited to a monocyclic aromatic ring system, wherein the ring comprises 6 carbon atoms, referred to herein as “C₆-aryl.”

[27] The term “cyano” as used herein refers to -CN.

[28] The term “cycloalkyl” as used herein refers to a saturated or unsaturated cyclic, bicyclic, or bridged bicyclic hydrocarbon group of 3-16 carbons, or 3-8 carbons, referred to herein as “(C3- C₈)cycloalkyl,” derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclohexenes, cyclopentanes, and cyclopentenes. Cycloalkyl groups may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Cycloalkyl groups can be fused to other cycloalkyl (saturated or partially unsaturated), aryl, or heterocyclyl groups, to form a bicycle, tetracycle, etc. The term “cycloalkyl” also includes bridged and spiro-fused cyclic structures which may or may not contain heteroatoms.

[29] The terms “halo” or “halogen” as used herein refer to -F, -Cl, -Br, and/or -I. [30] “Haloalkyl” means an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, etc.

[31] The term “heteroaryl” as used herein refers to a mono-, bi-, or multi-cyclic, aromatic ring system containing one or more heteroatoms, for example 1-3 heteroatoms, such as nitrogen, oxygen, and sulfur. Heteroaryls can be substituted with one or more substituents including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Heteroaryls can also be fused to non-aromatic rings.

Exemplary heteroaryl groups include, but are not limited to, a monocyclic aromatic ring, wherein the ring comprises 2-5 carbon atoms and 1-3 heteroatoms, referred to herein as "(C2-C5)heteroaryl.” Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)- and (l,2,4)-triazolyl, pyrazinyl, pyrimidilyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl, phenyl, isoxazolyl, and oxazolyl. Exemplary heteroaryl groups also include, but are not limited to, a bicyclic aromatic ring, wherein the ring comprises 5-14 carbon atoms and 1-3 heteroatoms, referred to herein as "(CVCi i jhctcroaryl.” Representative examples of heteroaryl include, but not limited to, indazolyl, indolyl, azaindolyl, indolinyl, benzotriazolyl, benzoxadiazolyl, imidazolyl, cinnolinyl, imidazopyridyl, pyrazolopyridyl, pyrrolopyridyl, quinolinyl, isoquinolinyl, quinazolinyl, quinazolinonyl, indolinonyl, isoindolinonyl, tetrahydronaphthyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[32] The terms “heterocycle,” “heterocyclyl,” or “heterocyclic” as used herein each refer to a saturated or unsaturated 3- to 18-membered ring containing one, two, three, or four heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. Heterocycles can be aromatic (heteroaryls) or non-aromatic. Heterocycles can be substituted with one or more substituents including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Heterocycles also include bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from aryls, cycloalkyls, and heterocycles. Exemplary heterocycles include acridinyl, benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, furyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl, isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl, oxazolyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazinyl, pyrazolyl, pyrazolinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, pyrrolyl, quinolinyl, quinoxaloyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, thiomorpholinyl, thiopyranyl, and triazolyl.

[33] The terms “hydroxy” and “hydroxyl” as used herein refer to -OH. [34] “Isomers” means compounds having the same number and kind of atoms, and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms in space.

[35] “Stereoisomer” or “optical isomer” mean a stable isomer that has at least one chiral atom or restricted rotation giving rise to perpendicular dissymmetric planes (e.g., certain biphenyls, allenes, and spiro compounds) and can rotate plane-polarized light. Because asymmetric centers and other chemical structure exist in the compounds of the disclosure which may give rise to stereoisomerism, the disclosure contemplates stereoisomers and mixtures thereof. The compounds of the disclosure and their salts include asymmetric carbon atoms and may therefore exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. Typically, such compounds will be prepared as a racemic mixture. If desired, however, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. As discussed in more detail below, individual stereoisomers of compounds are prepared by synthesis from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, use of chiral resolving agents, or direct separation of the enantiomers on chiral chromatographic columns. Starting compounds of particular stereochemistry are either commercially available or are made by the methods described below and resolved by techniques well-known in the art.

[36] It is well-known in the art that the biological and pharmacological activity of a compound is sensitive to the stereochemistry of the compound. Thus, for example, enantiomers often exhibit strikingly different biological activity including differences in pharmacokinetic properties, including metabolism, protein binding, and the like, and pharmacological properties, including the type of activity displayed, the degree of activity, toxicity, and the like. Thus, one skilled in the art will appreciate that one enantiomer may be more active or may exhibit beneficial effects when enriched relative to the other enantiomer or when separated from the other enantiomer. Additionally, one skilled in the art would know how to separate, enrich, or selectively prepare the enantiomers of the compounds of the disclosure from this disclosure and the knowledge of the prior art.

[37] Thus, although the racemic form of drug may be used, it is often less effective than administering an equal amount of enantiomerically pure drug; indeed, in some cases, one enantiomer may be pharmacologically inactive and would merely serve as a simple diluent. For example, although ibuprofen had been previously administered as a racemate, it has been shown that only the S -isomer of ibuprofen is effective as an anti-inflammatory agent (in the case of ibuprofen, however, although the R-isomer is inactive, it is converted in vivo to the S-isomer, thus, the rapidity of action of the racemic form of the drug is less than that of the pure S-isomer). Furthermore, the pharmacological activities of enantiomers may have distinct biological activity. For example, S -penicillamine is a therapeutic agent for chronic arthritis, while R-penicill amine is toxic. Indeed, some purified enantiomers have advantages over the racemates, as it has been reported that purified individual isomers have faster transdermal penetration rates compared to the racemic mixture. See U.S. Pat. Nos. 5,114,946 and 4,818,541. [38] In some embodiments, the compound is a racemic mixture of (S)- and (R)-isomers. In other embodiments, provided herein is a mixture of compounds wherein individual compounds of the mixture exist predominately in an (S)- or (R)-isomeric configuration. For example, the compound mixture has an (S)-enantiomeric excess of greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or more. In other embodiments, the compound mixture has an (S)-enantiomeric excess of greater than about 55% to about 99.5%, greater than about 60% to about 99.5%, greater than about 65% to about 99.5%, greater than about 70% to about 99.5%, greater than about 75% to about 99.5%, greater than about 80% to about 99.5%, greater than about 85% to about 99.5%, greater than about 90% to about 99.5%, greater than about 95% to about 99.5%, greater than about 96% to about 99.5%, greater than about 97% to about 99.5%, greater than about 98% to greater than about 99.5%, greater than about 99% to about 99.5%, or more. In other embodiments, the compound mixture has an (R)-enantiomeric purity of greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or more. In some other embodiments, the compound mixture has an (R)-enantiomeric excess of greater than about 55% to about 99.5%, greater than about 60% to about 99.5%, greater than about 65% to about 99.5%, greater than about 70% to about 99.5%, greater than about 75% to about 99.5%, greater than about 80% to about 99.5%, greater than about 85% to about 99.5%, greater than about 90% to about 99.5%, greater than about 95% to about 99.5%, greater than about 96% to about 99.5%, greater than about 97% to about 99.5%, greater than about 98% to greater than about 99.5%, greater than about 99% to about 99.5% or more.

[39] Individual stereoisomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by: (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary; (2) salt formation employing an optically active resolving agent; or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically- pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[40] Thus, if one enantiomer is pharmacologically more active, less toxic, or has a preferred disposition in the body than the other enantiomer, it would be therapeutically more beneficial to administer that enantiomer preferentially. In this way, the patient undergoing treatment would be exposed to a lower total dose of the drug and to a lower dose of an enantiomer that is possibly toxic or an inhibitor of the other enantiomer. [41] The term “pharmaceutically acceptable carrier” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

[42] The term “pharmaceutically acceptable composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[43] The term “pharmaceutically acceptable prodrugs” as used herein represents those prodrugs of the compounds of the present disclosure that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present disclosure. A discussion is provided in Higuchi et al, “Prodrugs as Novel Delivery Systems,” ACS Symposium Series, Vol. 14, and in Roche, E.B., ed. Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.

[44] The term “pharmaceutically acceptable salt(s)” refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, matate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-tolucncsul fonatc and pamoate (i.e., l,T-methylene-bis-(2-hydroxy- 3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

[45] Chemical names were generated using PerkinElmer ChemDraw^® Professional, version 17.

[46] The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbol “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. In some embodiments, an enantiomer or stereoisomer may be provided substantially free of the corresponding enantiomer.

[47] As used herein, “cancer” refers to diseases, disorders, and conditions that involve abnormal cell growth with the potential to invade or spread to other parts of the body. Exemplary cancers include, but are not limited to, breast cancer, lung cancer, ovarian cancer, endometrial cancer, prostate cancer, and esophageal cancer.

[48] As used herein, the term “subject” refers to an animal. Typically, the animal is a mammal. A subject also refers to for example, primates (e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.

[49] As used herein, the term “inhibit,” “inhibition,” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[50] As used herein, the term “treat,” “treating,” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically (e.g., through stabilization of a discernible symptom), physiologically, (e.g., through stabilization of a physical parameter), or both. In yet another embodiment, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.

[51] As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[52] Additionally, unless otherwise stated, structures described herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium (²H) or tritium (¾), or the replacement of a carbon by a ¹³C- or ¹⁴C-carbon atom are within the scope of this disclosure. Such compounds may be useful as, for example, analytical tools, probes in biological assays, or therapeutic agents.

Compounds

[53] In some embodiments, provided herein are compounds of Formula (I), or a pharmaceutically acceptable salt:

wherein

X₁ is CR₁ or N;

X₂ is CR₂ or N;

X₃ is CR₃ or N;

X₄ is CR₄ or N;

X₅ is CR₅ or N each of R₁, R₂, R3, R₄, and R5 is independently selected from hydrogen, halogen, hydroxyl group, -CN, C₁-C₅alkyl, C₁-C₅alkoxy, and C₁-C₅haloalkyl;

L is a linker of 1 to 3 carbon atoms in length, wherein one or more carbon atoms are optionally and independently replaced by a group selected from C(=O), O, N(R₉), S, CValkcnyl, C₂-alkynyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the R₉, CValkcnyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 0, 1, 2, or 3 R₁₀;

R₇ is independently selected from hydrogen, C₁-C₅alkyl, and C₁-C₅haloalkyl, each of which is substituted with 0, 1, 2, or 3 R_10;

X is selected from -CH₂-, -(CH₂)₂, and -(CHV-, each of which is optionally substituted at one or more hydrogens with R₁₁;

A is selected from cycloalkyl, aryl, heterocycle, and heteroaryl, each of which is substituted with 1, 2, or 3 R₈;

R₈ is independently selected from a,b-unsaturated carbonyl derivative, carboxamide, C₁-C₅alkyl, C₁-C₅alkoxy, C₁-C₅haloalkyl, each of which is optionally substituted with 1, 2, or 3 substituents selected from halogen, hydroxyl, C₁-C₃alkyl, Ci-C₃alkoxy, C ₁-C₃haloal ky I , -NHCH₃, -N(CH₃ )₂, and -CN; each R₉ is independently selected from hydrogen, C₁-C₃alkyl, -C(=O)-(C₁-C₃alkyl), -C(=O)-O- (C₁-C₃alkyl), and -C(=O)-NH-(C₁-C₃alkyl), each of which is substituted with 0, 1, 2, or 3 R₁₀; each R₁₀ is independently selected from halogen, hydroxyl, C₁-C₃alkyl, C₁-C₃alkoxy, C₁- C₃haloalkyl, -N(R₉)₂, and -CN; and each R₁₁ is independently selected from C₁-C₃alkyl, C₁-C₃alkoxy, C₁-C₃haloalkyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the C₁-C₃alkyl, C₁-C₃alkoxy, C₁-C₃haloalkyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 1, 2, or 3 substituents selected from halogen, -NO₂, hydroxyl, -NH₂, -NHCH₃, -N(CH₃)₂, -CN, C₁-C₃alkyl, C₁-C₃alkoxy, C₁-C₃haloalkyl, C₃- C₆cycloalkyl, C₃-C₆heterocyclyl, C₆-C₁₂aryl, and C₆-C₁₂ heteroaryl; wherein each hydrogen atom is independently and optionally replaced by a deuterium atom.

[54] In some embodiments, X₁ is CR₁, X₂ is CR₂, X₃ is CR₃, X₄ is CR₄, and X5 is CR_5. In a further embodiment, R₁, R₂, R₃, R₄, and R₅ are each independently selected from hydrogen, halogen, hydroxyl group, haloalkyl and -CN. In a further embodiment, R₁, R₂, R₃, and R₅ are each independently selected from H, Cl, F, -CF3, OH, and -CN. In some embodiments, R₁ is F or Cl. In some embodiments, R₁ is - CF₃. In some embodiments, R₁ is -CN. In some enbodiments, R1 is -OH. In some embodiments, R₂ is F. In some embodiments, R₃ is Cl. In some embodiments, R₅ is F. In some embodiments, Ri and R₃ are each Cl. In some embodiments, R₂, R₃, R₄ and R₅ are each H. In some embodiments, R₂, Rn and R₅ are each

H.

[55] In some embodiments, R₇ is selected from hydrogen and C₁-C₃alkyl. In a further embodiment, R₇ is selected from methyl and ethyl. In a further embodiment, R₇ is methyl.

[56] In some embodiments, X is selected from -CH₂- and -(CH₂)₂-, each of which is optionally substituted at one or more hydrogens with R₁₁. In some embodiments, X is -CH₂-. In some embodiments, X is -(CH₂)2-.

[57] In some embodiments, R₁₁ is an optionally substituted C₁-C₃alkyl. In a further embodiment, the C₁-C₃alkyl is substituted with 1, 2, or 3 substituents selected from halogen, -NH₂, -NHCH₃, -N(CH₃)₂, - CN, -NO₂, hydroxyl, C₁-C₃alkyl, C1-C₃alkoxy, C₁ -C₃haloalkyl, C₃-C₆cycloalkyl, C₃-C₆heterocyclyl, C₆- C₁₂aryl, and C₆-C₁₂ heteroaryl. In a further embodiment, the C₁-C₃alkyl is optionally substituted with 1 substituent selected from C₄-C₅heterocyclyl and -N(CH₃)₂· In a further embodiment, R₁₁ is selected from -

. In some embodiments, R₁₁ is selected from -CH₃. In some embodiments, R₁₁ is selected from

. In some embodiments, R₁₁ is selected from

[58] In some embodiments, A is selected from 4- to 6-member cycloalkyl, 5- to 6-member aryl, 4- to 6-member heterocycle, and 5- to 6-member heteroaryl, each of which is substituted with 1, 2, or 3 groups independently selected from a,b-unsaturated carbonyl derivative, carboxamide, C₁-C₅alkyl, C₁- C₅haloalkyl, and optionally substituted C₁-C₅alkoxy. In some embodiments, A is a substituted 6-member aryl. In some further embodiments, the a,b-unsaturated carbonyl derivative is selected from acryloyl, acrylamide, and alkylacrylamide. In some embodiments, the carboxamide is selected from

In some embodiments, the 4- member cycloalkyl is selected from

in some embodiments, the 4- member heterocycle is azetidine, wherein the azetidine is substituted with an a,b-unsaturated carbonyl derivative. In some embodiments, the 6- member heterocycle is piperidine, wherein the piperidine is substituted with an a,b-unsaturated carbonyl derivative. In a further embodiment, the piperidine is selected from

. In some embodiments, the 5-member heteroaryl is a substituted pyrazole.

[59] In some embodiments, R₈ is a substituted a,b-unsaturated carbonyl derivative. In a further embodiment, the a,b-unsaturated carbonyl derivative is selected from acryloyl, acrylamide, and alkylacrylamide.

[60] In some embodiments, R₈ is a carboxamide. In a further embodiment, the carboxamide is an acetamide.

[61] In some embodiments, R₈ is an optionally substituted C₁-C₃ alkyl. In a further embodiment, the C₁-C₃ alky l is selected from methyl and ethyl. In a further embodiment, the C1-C₃alkyl is methyl.

[62] In some embodiments, R₈ is an optionally substituted Ci-C alkoxy. In a further embodiment, the substituted C₁-C₃ alkoxy is

In some embodiments, R₈ is selected from:

, and

. In a further embodiment, R₈ is selected from:

[64] In some embodiments, the

group is selected from

[65] In some embodiments, L is a linker of 1 to 3 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by C(=O), O, N(R₉), S, C₂-alkenyl, C₂-alkynyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the R₉ C2-alkenyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 0, 1, 2, or 3 R₁₀.

[66] In some embodiments, L is selected from -CH₂ -CH₂- CH₂-, -CH₂-CH₂CH₂-.

[67] In some embodiments, provided herein is a compound, or pharmaceutically acceptable salt thereof, chosen from the compounds listed in Table 1.

Table 1. Exemplary Compound of the Present Disclosure

[68] In some embodiments, the compound is a compound of Formula (1 A)

[69] In some embodiments, the compound is a compound of Formula (IB)

Pharmaceutical Compositions

[70] Pharmaceutical compositions of the present disclosure comprise at least one compound of Formula (I), or tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal and parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) administration. The most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used.

[71] Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present disclosure as powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. As indicated, such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association at least one compound of the present disclosure as the active compound and a carrier or excipient (which may constitute one or more accessory ingredients). The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and must not be deleterious to the recipient. The carrier may be a solid or a liquid, or both, and may be formulated with at least one compound described herein as the active compound in a unit-dose formulation, for example, a tablet, which may contain from about 0.05% to about 95% by weight of the at least one active compound. Other pharmacologically active substances may also be present including other compounds. The formulations of the present disclosure may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components.

[72] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmacologically administrable compositions can, for example, be prepared by, for example, dissolving or dispersing, at least one active compound of the present disclosure as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. In general, suitable formulations may be prepared by uniformLy and intimately admixing the at least one active compound of the present disclosure with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, a tablet may be prepared by compressing or molding a powder or granules of at least one compound of the present disclosure, which may be optionally combined with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, at least one compound of the present disclosure in a free-flowing form, such as a powder or granules, which may be optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, where the powdered form of at least one compound of the present disclosure is moistened with an inert liquid diluent.

[73] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising at least one compound of the present disclosure in a flavored base, usually sucrose and acacia or tragacanth, and pastilles comprising the at least one compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[74] Formulations of the present disclosure suitable for parenteral administration comprise sterile aqueous preparations of at least one compound of Formula (I), or tautomers, stereoisomers, pharmaceutically acceptable salts, and hydrates thereof, which are approximately isotonic with the blood of the intended recipient. These preparations are administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing at least one compound described herein with water and rendering the resulting solution sterile and isotonic with the blood. Injectable compositions according to the present disclosure may contain from about 0.1 to about 5% w/w of the active compound. [75] Formulations suitable for rectal administration are presented as unit-dose suppositories. These may be prepared by admixing at least one compound as described herein with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[76] Formulations suitable for topical application to the skin may take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers and excipients which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combinations of two or more thereof. The active compound (i.e., at least one compound of Formula (I), or tautomers, stereoisomers, pharmaceutically acceptable salts, and hydrates thereof) is generally present at a concentration of from about 0.1% to about 15% w/w of the composition, for example, from about 0.5 to about 2%.

[77] The amount of active compound administered may be dependent on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician. For example, a dosing schedule may involve the daily or semi-daily administration of the encapsulated compound at a perceived dosage of about 1 pg to about 1000 mg. In another embodiment, intermittent administration, such as on a monthly or yearly basis, of a dose of the encapsulated compound may be employed. Encapsulation facilitates access to the site of action and allows the administration of the active ingredients simultaneously, in theory producing a synergistic effect. In accordance with standard dosing regimens, physicians will readily determine optimum dosages and will be able to readily modify administration to achieve such dosages.

[78] A therapeutically effective amount of a compound or composition disclosed herein can be measured by the therapeutic effectiveness of the compound. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being used. In one embodiment, the therapeutically effective amount of a disclosed compound is sufficient to establish a maximal plasma concentration. Preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.

[79] Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferable.

[80] Data obtained from the cell culture assays or animal studies can be used in formulating a range of dosage for use in humans. Therapeutically effective dosages achieved in one animal model may be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et ah, Cancer Chemother. Reports 50(4):219-244 (1966) and the following Table for Equivalent Surface Area Dosage Factors). Table 2. Equivalent Surface Area Dosage Factors.

[81] The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Generally, a therapeutically effective amount may vary with the subject's age, condition, and gender, as well as the severity of the medical condition in the subject. The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

Methods of Treatment

[82] In some embodiments, a compound of Formula (I), or a tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof, is administered to treat cancer in a subject in need thereof.

[83] In some embodiments, the compound or the pharmaceutically acceptable salt is an irreversible K- Ras inhibitor. In some embodiments, the compound or the pharmaceutically acceptable salt thereof selectively binds to K-Ras(G12C) without any effect on the K-Ras wild type. In some embodiments, the compound or the pharmaceutically acceptable salt thereof selectively binds to K-Ras(G12C) without any effect on the K-Ras wild type.

[84] In some embodiments, a compound of Formula (I), or a tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof, is administered as a pharmaceutical composition. In some embodiments, the compound or pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.

[85] In some embodiments, the cancer is associated with K-Ras wild-type or mutations. In some embodiments, the cancer is associated with K-Ras(G12C).

[86] In some embodiments, the cancer is chosen from breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gall bladder cancer, thyroid cancer, bile duct cancer, ovarian cancer, endometrial cancer, prostate cancer, and esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is gall bladder cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is bile duct cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is esophageal cancer. [87] In some embodiments, the invention provides for methods for modulating an activity of a K-Ras protein, comprising contacting a K-Ras protein with an effective amount of a compound of Formula (I) or pharmaceutically acceptable salt thereof. In one embodiment, the K-Ras protein is K-Ras(G12C).

[88] In some embodiments, the invention provides for methods for inhibiting KRas G12C activity in a cell, comprising contacting the cell in which inhibition of KRas G12C activity is desired with an effective amount of a compound of Formula (I), pharmaceutically acceptable salts thereof or pharmaceutical compositions containing the compound or pharmaceutically acceptable salt thereof. In one embodiment, the contacting is in vitro. In one embodiment, the contacting is in vivo.

[89] As used herein, the term "contacting" refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, "contacting" a KRas G12C with a compound provided herein includes the administration of a compound provided herein to an individual or patient, such as a human, having KRas G12C, as well as, for example, introducing a compound provided herein into a sample containing a cellular or purified preparation containing the KRas G12C.

[90] In one embodiment, a cell in which inhibition of KRas G12C activity is desired is contacted with an effective amount of a compound of Formula (I) to negatively modulate the activity of KRas G12C. In other embodiments, a therapeutically effective amount of pharmaceutically acceptable salt or pharmaceutical compositions containing the compound of Formula (I) may be used.

[91] By negatively modulating the activity of KRas G12C, the methods described herein are designed to inhibit undesired cellular proliferation resulting from enhanced KRas G12C activity within the cell.

The cells may be contacted in a single dose or multiple doses in accordance with a particular treatment regimen to effect the desired negative modulation of KRas G12C.

[92] The concentration and route of administration to the patient will vary depending on the cancer to be treated.

[93] In one embodiment, a compound of Formula (I), or a tautomer, stereoisomer, pharmaceutically acceptable salt or hydrate thereof, is administered in combination with another therapeutic agent, e.g., chemotherapy, or used in combination with other treatments, such as radiation or surgical intervention, either as an adjuvant prior to surgery or post-operatively.

[94] Also provided herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as defined herein for use in therapy.

[95] Also provided herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof as defined herein for use in the treatment of cancer.

[96] Also provided herein is the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as defined herein in the manufacture of a medicament for the treatment of cancer.

[97] Also provided herein is the use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as defined herein, in the manufacture of a medicament for the treatment of a cancer associated with K-Ras wild-type or mutations. [98] One skilled in the art will recognize that, both in vivo and in vitro trials using suitable, known and generally accepted cell and/or animal models are predictive of the ability of a test compound to treat or prevent a given disorder.

[99] One skilled in the art will further recognize that human clinical trials including first-in-human, dose ranging and efficacy trials, in healthy patients and/or those suffering from a given disorder, may be completed according to methods well known in the clinical and medical arts.

Examples

[100] The examples and preparations provided below further illustrate and exemplify the compounds as disclosed herein and methods of preparing such compounds. It is to be understood that the scope of the present disclosure is not limited in any way by the scope of the following examples and preparations.

[101] The chemical entities described herein can be synthesized according to one or more illustrative schemes herein and/or techniques well known in the art. Unless specified to the contrary, the reactions described herein take place at atmospheric pressure, generally within a temperature range from about -10° C to about 200° C. Further, except as otherwise specified, reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about -10° C to about 200° C over a period that can be, for example, about 1 to about 24 hours; reactions left to run overnight in some embodiments can average a period of about 16 hours.

[102] Isolation and purification of the chemical entities and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. See, e.g., Carey et al. Advanced Organic Chemistry, 3^rd Ed., 1990 New York: Plenum Press; Mundy et al., Name Reaction and Reagents in Organic Synthesis, 2^nd Ed., 2005 Hoboken, NJ: J. Wiley & Sons. Specific illustrations of suitable separation and isolation procedures are given by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can also be used.

[103] In all of the methods, it is well understood that protecting groups for sensitive or reactive groups may be employed where necessary, in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T.W. Greene and P.G.M. Wuts (1999) Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons). These groups may be removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art.

[104] When desired, the (R)- and (S)-isomers of the nonlimiting exemplary compounds, if present, can be resolved by methods known to those skilled in the art, for example, by formation of diastereoisomeric salts or complexes which can be separated, e.g., by crystallization; via formation of diastereoisomeric derivatives which can be separated, e.g., by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, e.g., enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, e.g., on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent. Alternatively, a specific enantiomer can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

[105] The compounds described herein can be optionally contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salts. Also, the compounds described herein can be optionally contacted with a pharmaceutically acceptable base to form the corresponding basic addition salts.

[106] In some embodiments, disclosed compounds can generally be synthesized by an appropriate combination of generally well-known synthetic methods. Techniques useful in synthesizing these chemical entities are both readily apparent and accessible to those of skill in the relevant art, based on the instant disclosure. Many of the optionally substituted starting compounds and other reactants are commercially available, e.g., from Millipore Sigma or can be readily prepared by those skilled in the art using commonly employed synthetic methodology.

[107] The discussion below is offered to illustrate certain of the diverse methods available for use in making the disclosed compounds and is not intended to limit the scope of reactions or reaction sequences that can be used in preparing the compounds provided herein. The skilled artisan will understand that standard atom valencies apply to all compounds disclosed herein in genus or named compound for unless otherwise specified.

The following abbreviations have the definitions set forth below:

ACN Acetonitrile

DCE Dichloroethane

DCM Dichloromethane

DIEA Diisopropylethylamine

DMA N, N-di methyl acetamide

DMF Dimethylformamide

EA Ethyl acetate

HPLC High pressure liquid chromatography

LC/MS Liquid chromatography/Mass spectroscopy

MsCl Methanesulfonyl chloride

NMP N-mcthyl-2-pyrrolidonc

NMR Nuclear magnetic resonance

PE Petroleum ether

SEMC1 2-(Trimethylsilyl)ethoxymethyl chloride

TEA Triethylamine

TFA Trifluoroacetic acid

THF Tetrahydrfuran

TLC Thin layer chromatography

TR-FRET Time-resolved fluorescence energy transfer General Synthetic Schemes

[108] HPLC spectra for all compounds were acquired using an Agilent 1200 Series system with DAD detector. Chromatography was performed on a 2.1x150 mm Zorbax 300SB-C18 5 pm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 mL /min. The gradient program was as follows: 1% B (0-1 min), 1-99% B (1-4 min), and 99% B (4-8 min). High-resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G 1969 A API-TOF with an electrospray ionization (ESI) source. Nuclear Magnetic Resonance (NMR) spectra were acquired on a Bruker spectrometer with 600 MHz or 400 MHz for proton ('H NMR) and 150 MHz for carbon (¹³C NMR); chemical shifts are reported in (d). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector set to 254 nm and 220 nm. Samples were injected onto a Phenomenex Luna 75 x 30 mm, 5 pm, C₁₈ column at room temperature. The flow rate was 40 mL/min. A linear gradient was used with 10% (or 50%) of MeOH in H₂O (with 0.1 % TLA) to 100% of MeOH. HPLC was used to establish the purity of target compounds. All final compounds were determined to be > 95% purity when analyzed according to the HPLC methods described above.

[109] Compounds of Lormula (I) can be synthesized according to schemes 1 - 3. Starting from 3- methyl-8-bromoxanthine A, tandem alkylation with dibromoalkane such as 1,2-dibromoethane, 1,3- dibromopropane or 1,4-dibromobutane, followed by alkyation with substituted benzylbromide to give intermediate B. Treatment of intermediate B with amino-substituted cyclic amine with a suitable protecting group under forcing conditions such as microwave irradiation can provide tricyclic intermediate C. Deprotection of nitrogen protecting group followed by acylation of the amino group can provide desired compound E.

[110] Alternatively, 3-methyl-8-bromoxanthine A can be alkylated with substituted allybromide followed by alkylation with substituted benzylbromide to give intermediate G. The alkene group can undergo hydroboration-oxidation to give primary alcohol intermediate H, which can be converted to alkyl or aryl sulfonate ester I. Final compounds L can be prepared from intermediate I with the same three-step sequence as described in Scheme 1.

Scheme 2

[111] Alternatively, imidazole nitrogen of 3-methyl-8-bromoxanthine A can be selectively protected followed by alkylation with substituted benzylbromide. After deprotection, imidazole nitrogen can be alkylated with l,3-dichloro-2-methylenepropane to form intermediate P. Treatment of intermediate P with amino-substituted cyclic amine with a suitable protecting group under forcing conditions such as microwave irradiation can provide tricyclic intermediate Q. The alkene intermediate Q can be converted to primary alcohol followed by activation as alkyl or aryl sulfonate ester and displaced by acyclic or cyclic secondary amines to give intermediate S. Deprotection of nitrogen protecting group followed by acylation give the desired final compound T.

Examples

Example 1: Preparation of N-(4-(3-(2-chloro-6-fluorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 1)

[112] A mixture solution of 8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (10 g, 40.8 mmol), 3- bromoprop-l-ene (4.9 g, 40.8 mmol) and K2CO3 (8.46 g, 61.2 mmol) in DMF (150 mL) was stirred at rt for 4h. The mixture was diluted with water (150 mL) and extracted with EA (150 mL x 2). The combined organic layers were washed with brine (150 mL x 3), dried, filtered and concentrated. The crude product was washed with EA to obtain the title product 7-allyl-8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6- dione 1A (7.7 g, yield 66%) as a white solid. LCMS: (ES+): 285.0 m/z [M]⁺

[113] A mixture solution of 7-allyl-8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (3.0 g, 10.5 mmol) , 2-(bromomethyl)-l-chloro-3-fluorobenzene (2.59 g, 11.6 mmol) and K2CO3 (2.18 g, 15.8 mmol) in DML (100 mL) was stirred at 60 °C for 3h. The mixture was diluted with water (80 mL) and extracted with EA (100 mL x 2). The combined organic layers were washed with brine (150 mL x 3), dried, filtered and concentrated. The crude product was purified by silica column chromatography (eluent: EA/PE=1:1) to obtain the title product 7-allyl-8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-3,7-dihydro-lH-purine- 2,6-dione IB (3.84g, yield 85%) as a white solid. LCMS: (ES+): 429.0 m/z [M]⁺.

[114] To a solution of cyclohexene (3.68 g, 44.9 mmol) in THE (40 mL) was added BH3-THL (22.4 mL, 22.4 mmol) at 0 °C, then the resulting solution was stirred at 0°C for lh. After that, the solution of 7- allyl-8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-3, 7-dihydro- lH-purine-2,6-dione (3.84 g, 8.98 mmol) in THE (20 mL) was added dropwise to the solution and the resulting solution was stirred at room temperature for overnight. Then the 37% NaOH solution (5 mL), H2O2 (5 mL) was added to the solution at 0 °C successively. And the resulting solution was stirred at room temperature for 3 h. The solution was quenched with saturated Na₂S₂O₃ solution (30 mL) and extracted with ethyl acetate (100 mL x 2). The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by silica column chromatography (eluent: EA/PE=1:2) to obtain the title product 8- bromo-l-(2-chloro-6-fluorobenzyl)-7-(3-hydroxypropyl)-3-methyl-3, 7-dihydro- lH-purine-2,6-dione 1C (2.7 g, yield 67.5%) as a white solid. LCMS: (ES+): 447.0 m/z [M]⁺.

[115] To a solution of 8-bromo-l-(2-chloro-6-fluorobenzyl)-7-(3-hydroxypropyl)-3-methyl-3,7- dihydro-lH-purine-2,6-dione (1.2 g, 2.69 mmol) in DCM (40 mL) was added TEA (0.75 mL, 5.38 mmol), MsCl (0.25 mL, 3.23 mmol) dropwise at 0 °C successively. The reaction mixture was stired at 0 °C for 3 h. Then the reaction was quenched by water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated. The crude product 3-(8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7- yl)propyl methanesulfonate ID (1.21 g, yield 88%) which was used without further purification. LCMS: (ES+): 525.0 m/z [M]⁺.

[116] In a microwave tube was placed 3-(8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-2,6-dioxo- l,2,3,6-tetrahydro-7H-purin-7-yl)propyl methanesulfonate (350 mg, 0.67 mmol), tert-butyl (4- aminophenyl)carbamate (279 mg, 1.34 mmol), NMP (3 mL), then the mixture was irradiated in the microwave at 150 °C for 2 h. Water (20 mL) was added and extracted with ethyl acetate (20 mL x 2) .

The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by Pre-TLC (eluent: EA) to obtain the title product 9-(4-aminophenyl)-3-(2-chloro- 6-fluorobenzyl)-l-methyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione IE as a white solid (114 mg, yield 37.5%). LCMS: (ES+): 455.1 m/z [M]⁺.

[117] To a solution of 9-(4-aminophenyl)-3-(2-chloro-6-fluorobenzyl)-l-methyl-6,7,8,9-tetrahydro pyrimido[2,l-f]purine-2,4(lH,3H)-dione (114 mg, 0.25 mmol) in DCM (10 mL) was added TEA (0.07 mL, 0.5 mmol), acryloyl chloride (0.26 mL, 0.325 mmol) dropwise at 0 °C successively. The reaction mixture was stirred at 0 °C for 3 h. Then the reaction was quenched by water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated. The crude product was purified further by Prep-HPLC to obtain the title product N-(4-(3-(2- chloro-6-fluorobenzyl)-l -methyl-2, 4-dioxo- 1,2, 3,4,7, 8-hexahydropyrimido[2, l-f]purin-9(6H)- yl)phenyl)acrylamide 1 (31.2 mg, yield 24.6%) as white solid. LCMS: (ES+): 509.2 m/z [M]⁺. 1H NMR (400 Hz, DMSO): d 10.18 (s,lH), 7.69-7.67 (d, J= 8.8 Hz, 2H), 7.47-7.45 (d, J= 8.8 Hz, 2H), 7.32-7.27 (m, 2H), 7.15-7.10 (t, J= 8.4 Hz, 1H), 6.47-6.40 (dd, J= 10 Hz, 1H), 6.28-6.24 (d, J= 15.6 Hz, 1H), 5.77-5.74 (d, J= 11.6 Hz, 1H), 5.19 (s, 2H), 4.19 (m, 2H), 3.80-3.78 (m, 2H), 3.26 (s,3H), 2.22 (m, 2H)

Example 2: Preparation of N-(3-(3-(2-chloro-6-fluorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 2)

[118] In a microwave tube was placed 3-(8-bromo-l-(2-chloro-6-fhiorobenzyl)-3-methyl-2,6-dioxo- l,2,3,6-tetrahydro-7H-purin-7-yl)propyl methanesulfonate ID (400 mg, 0.76 mmol), tert-butyl (3- aminophenyl) carbamate (318 mg, 1.52 mmol), DMA (3 mL), then the mixture was irradiated in the microwave at 150 °C for 2 h. Water (20 mL) was added and extracted with EA (20 mL x 2) . The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by Prep-TLC (eluent: EA) to obtain the title product 9-(3-aminophenyl)-3-(2-chloro- 6-fluorobenzyl)-l-methyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 2A as a white solid (120 mg, yield 34.5%). LCMS: (ES+): 455.1 m/z [M]⁺. [119] To a solution of 9-(3-aminophenyl)-3-(2-chloro-6-fluorobenzyl)-l-methyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (120 mg, 0.26 mmol) in DCM (10 mL) was added TEA (0.07 mL, 0.52 mmol), acryloyl chloride (0.026 mL, 0.34 mmol) dropwise at 0 °C successively. The reaction mixture was stired at 0 °C for 1 h. Then the reaction was quenched by water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated. The crude product was purified further by Prep-HPLC to obtain the title product N-(3-(3-(2-chloro-6-fluorobenzyl)- 1 -methyl-2, 4-dioxo- 1,2, 3,4,7, 8-hexahydropyrimido[2, 1- f]purin-9(6H)-yl)phenyl) acrylamide 2 (36.7 mg, yield 27.4%) as white solid. LCMS: (ES+): 509.1 m/z [M]⁺ 1HNMR (400 Hz, DMSO): d 10.19 (s,lH), 7.91 (s, 1H), 7.46-7.44 (d, J= 8.0 Hz, 1H), 7.35-7.24 (m, 4H), 7.15-7.12 (m,lH), 6.45-6.41 (m, 1H), 6.28-6.24 (d, J= 15.2 Hz, 1H), 5.78-5.75 (d, J = 10 Hz, 1H), 5.19 (s, 2H), 4.21 (m, 2H), 3.83-3.80 (m, 2H), 3.28 (s,3H), 2.24 (m, 2H)

Example 3: Preparation of 3-(2-chloro-6-fluorobenzyl)-l-methyl-9-(m-tolyl)-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 3)

[120] In a microwave tube was placed 3-(8-bromo-l-(2-chloro-6-fluorobenzyl)-3- methyl-2, 6-dioxo- l,2,3,6-tetrahydro-7H-purin-7-yl)propyl methanesulfonate ID (150 mg, 0.286 mmol), m-toluidine (61 mg, 0.572 mmol), DMA (1 mL), then the mixture was irradiated in the microwave at 150 °C for 2 h. Water (20 mL) was added and extracted with EA (20 mL x 2) . The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by Pre-TLC (eluent: EA) and then purified further by Prep-HPLC to obtain the title product 3-(2-chloro-6- fluorobenzyl)-l-methyl-9-(m-tolyl)-6,7,8,9-tetrahydropyrimido[2, l-f]purine-2,4(lH,3H)-dione 3 as a white solid (44 mg, yield 33.8%). LCMS: (ES+): 454.2 m/z [M]⁺. ¹H NMR (400 Hz, CDCL₃): d 7.30 (br, 3H), 7.16-7.28 (m, 2H), 7.11 (s, 1H), 6.90-6.95 (t, J= 9.6 Hz, 1H), 5.38 (s, 2H), 4.36 (t, J= 6.0 Hz, 2H), 3.84 (t, J= 5.2 Hz, 2H), 3.43 (s, 3H), 2.37 (s, 3H), 2.29 (t, J= 5.2 Hz, 2H). Example 4: Preparation of 3-(2-chlorobenzyl)-9-(3-methoxyphenyl)-l, 7-dimethyl-6, 7,8,9-

[121] A solution of 8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (980 mg, 4 mmol), 3-bromo-2- methylprop-l-ene (540 mg, 4 mmol) and K2CO3 (1.1 g, 8 mmol) in N,N-dimethylformamide (15 mL) was stirred at 90 °C for 2 h. l-(bromomethyl)-2-chlorobenzene (820 mg, 4 mmol) was added and the reaction was stirred for another 2 h. LCMS showed reaction was completed. The mixture was diluted with EA and extracted with water. The organic phase was dried and concentrated. The crude product was purified by silica column to give 8-bromo-3-methyl-7-(2-methylallyl)-3,7-dihydro-lH-purine-2,6-dione 4B (840 mg, 50%) as a white solid. LCMS: (ES+): m/z = 425.0 [M+l]⁺.

[122] A dry three-neck flask was charged with 8-bromo-l-(2-chlorobenzyl)-3-methyl-7-(2- methylallyl)-3,7-dihydro-lH-purine-2,6-dione (424 mg, 1 mmol) and dry THF (10 mL) and cooled to

0 °C, then borane-THF complex (1.3 mL) was added via a syringe dropwise. The resulting mixture was stirred for 2 h at 0 °C. Sodium hydroxide (4 M, 1 mL) and hydrogen peroxide (1 mL) were added in sequence. The mixture was stirred for 2 h. The reaction mixture was diluted with water, then extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulfate and filtered. After evaporation of solvent, the residue was purified with silica gel chromatography to give 8- bromo-l-(2-chlorobenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-3, 7-dihydro- lH-purine-2,6-dione 4C (300 mg, 67.9%). LCMS: (ES+): m/z = 443.0 [M]⁺.

[123] To a solution of 8-bromo-l-(2-chlorobenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-3,7- dihydro-lH-purine-2,6-dione (220 mg, 0.5 mmol) and TEA (151 mg, 1.5 mmol) in DCM (5 mL) was added MsCl (86 mg, 1.5 mmol) at 0 °C. The reaction mixture was stired at 0 °C for 2 h. LCMS showed small amount of SM remained. The reaction was quenched by water. The organic phase was dried and concentrated in vacuo to give 3-(8-bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H- purin-7-yl)-2-methylpropyl methanesulfonate 4D (230 mg) as a yellow solid which was directly used in the next step. LCMS: (ES+): m z = 520.9 [M+l]⁺, t_R =3.21 min.

[ 124] 3-(8-bromo- 1 -(2-chlorobenzyl)-3-methyl-2,6-dioxo- 1 ,2, 3,6-tetrahydro-7H-purin-7 -yl)-2- methylpropyl methanesulfonate (230 mg) and 3-methoxyaniline(123 mg, 1 mmol) were dissolved in NMP (3 mL). The reaction mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with EA. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give 3-(2-chlorobenzyl)-9-(3-methoxyphenyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 4 as a white solid (50 mg, 21.5%). LCMS: (ES+): m/z = 466.1 [M]+. 1H NMR (400 MHz, CDCl₃-d): d 7.38-7.28 (m, 2H), 7.15-7.12 (m, 3H), 7.03-7.01 (m, 2H), 6.76-6.74 (m, 1H), 5.31(s, 2H), 4.57-4.52 (m, 1H), 3.84-3.78 (m, 5H), 3.56-3.49 (m, 4H), 2.51-2.48 (m, 1H), 1.18 (d, / = 7.4 Hz, 3H).

Example 5: Preparation of N-(3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 5)

[125] To a solution of 8-bromo-l-(2-chlorobenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-3,7- dihydro-lH-purine-2,6-dione (250 mg, 0.566 mmol) in DCM (10 mL) was added TEA (114.5 mg, 1.132 mmol) and MsCI (97 mg, 0.849 mmol) dropwise at 0 °C sequentially. The reaction mixture was stired at 0 °C for 2 h. Then the reaction was quenched by water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated. The crude product 3-(8-bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate 5A (294 mg) was used without further purification.

[126] In a microwave tube was added 3-(8-bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6- tetrahydro-7H-purin-7-yl)-2-methylpropyl methanesulfonate (294 mg), tert-butyl (3-aminophenyl) carbamate (236 mg, 1.132 mmol) and NMP (4 mL). The mixture was irradiated in the microwave at 150 °C for 2 h. Water (20 mL) was added and extracted with ethyl acetate (20 mL x 2). The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by Pre-TLC (eluent: EA/MeOH=10:l) to obtain 9-(3-aminophenyl)-3-(2-chlorobenzyl)-l,7-dimethyl- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 5B as a white solid (160 mg, yield 62.7%). LCMS: (ES+): 451.2 m/z [M]⁺.

[127] To a solution of 9-(3-aminophenyl)-3-(2-chlorobenzyl)-l, 7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (160 mg, 0.35 mmol) in DCM (10 mL) was added TEA (70.8 mg, 0.7 mmol), acryloyl chloride (97 mg, 0.849 mmol) dropwise at 0 °C sequentially. The reaction mixture was stired at 0 °C for 3 h. Then the reaction was quenched with water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated. The crude product was purified further by Prep-HPLC to obtain N-(3-(3-(2- chlorobenzyl)-l,7-dimethyl-2,4-dioxo- 1,2, 3,4,7, 8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide 5 (53.6 mg, yield 29.8%) LCMS: (ES+): 505.1 m/z [M]⁺. 1H NMR (400 Hz, MeOD): d 7.98 (s,lH), 7.37-7.41 (m, 3H), 7.30 (m, 1H), 7.20-7.21 (m, 2H), 6.94 (s, 1H), 6.35-6.44 (m, 2H), 5.78 (d, J= 10 Hz, 1H), 5.24 (s, 2H), 4.43-4.48 (m, 1H), 3.80-3.88 (m, 2H), 3.62 (t, J= 6.0. Hz, 1H), 3.41 (s, 3H), 2.52 (br, 1H), 1.20 (d, J = 6.8 Hz, 3H). Example 6: Preparation of N-(3-(3-(2-cyanobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 6)

[128] A solution of 8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (980 mg, 4 mmol), 3-bromo-2- methylprop-l-ene (540 mg, 4 mmol) and K2CO3 (1.1 g, 8 mmol) i n N,N-di methylformamide (15 mL) was heated at 90 °C for 2 h, 2-(bromomethyl)benzonitrile (784 mg, 4 mmol) was added and the reaction was stirred for another 2 h. The mixture was diluted with ethyl acetate and washed by water. The organic phase was dried and concentrated. The crude product was purified by silica column to give 2-((8-bromo- 3-methyl-7-(2-methylallyl)-2,6-dioxo-2,3,6,7-tetrahydro-lH-purin-l-yl)methyl)benzonitrile 6A (828 mg, yield 50%) as a white solid.

[129] A dry three-neck flask was charged with 2-((8-bromo-3-methyl-7-(2-methylallyl)-2,6-dioxo- 2,3,6,7-tetrahydro-lH-purin-l-yl)methyl)benzonitrile (414 mg, 1 mmol) and dry THF (10 mL) and cooled to 0°C, borane-THF complex (1.3 mL) was added via a syringe dropwise. The resulting mixture was stirred for 2 h at 0°C. Sodium hydroxide (4 M, 1 mL) and hydrogen peroxide (1 mL) were added in sequence. The mixture was stirred for 2h. The reaction mixture was diluted with water, then extracted with ethyl acetate, washed with water and brine, dried over sodium sulfate and filtered. After evaporation of solvent, the residue was purified with silica gel chromatography to give 2-((8-bromo-7-(3-hydroxy-2- methylpropyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-lH-purin-l-yl)methyl)benzonitrile 6B (300 mg, 69.4%). LCMS: (ES+): m/z = 434.0 [M]⁺.

[130] To a solution of 2-((8-bromo-7-(3-hydroxy-2-methylpropyl)-3-methyl-2,6-dioxo-2,3,6,7- tetrahydro-lH-purin-l-yl)methyl)benzonitrile (216 mg, 0.5 mmol), TEA (151 mg, 1.5 mmol) in DCM (5 mL) was added MsCl (86 mg, 1.5 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. LCMS showed small amount of SM remained. The reaction was quenched by water. The organic phase was dried and concentrated in vacuo to give 3-(8-bromo-l-(2-cyanobenzyl)-3-methyl-2,6-dioxo-l,2,3,6- tetrahydro-7H-purin-7-yl)-2-methylpropyl methanesulfonate 6C (230 mg, crude) as a yellow solid which was used in the next step without further purification. LCMS: (ES+): m/z = 510.0 [M+l]⁺.

[131] 3-(8-bromo-l-(2-cyanobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (230 mg, crude), tert-butyl (3-aminophenyl)carbamate (208 mg, 1 mmol) were dissolved in NMP (3 mL). The mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with ethyl acetate. The organic layer was concentrated and purified by Pre-TLC using DCM/MeOH to give 2-((9-(3-aminophenyl)-l,7-dimethyl-2,4-dioxo-l,4,6,7,8,9- hexahydropyrimido[2,l-f]purin-3(2H)-yl)methyl)benzonitrile 6D as a white solid (70 mg, yield 31.7%). LCMS: (ES+): m/z = 442.2 [M+l]⁺. [132] To the mixture of 2-((9-(3-aminophenyl)-l,7-dimethyl-2,4-dioxo-l,4,6,7,8,9- hexahydropyrimido[2,l-f]purin-3(2H)-yl)methyl)benzonitrile (66 mg, 0.15 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added the solution of acryloyl chloride (13.6 mg, 0.15 mmol) in DCM (1 mL) at -70 °C. After reaction completed as monitored by TLC, the reaction was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na₂SO₄, filtered and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain N-(3-(3-(2-cyanobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide 6 (42 mg, yield 56.6%) as a white solid. LCMS: (ES+): m/z = 496.2 [M]⁺. ¹H NMR (400 MHz, CDCl₃-d): d 8.04 (s, 1H), 7.64 (d, 1H), 7.47-7.50 (m, 2H), 7.27-7.40 (m, 4H), 7.15 (d, 1H), 6.44 (d, J = 16.8 Hz, 1H), 6.22-6.28 (m, 1H), 5.80 (d, J = 11.2 Hz, 1H), 5.41(s, 2H), 4.52-4.56 (m, 1H), 3.78-3.83 (m, 2H), 3.54 (m, 1H), 3.48 (s, 3H), 2.51 (br, 1H), E18 (d, /= 6.4 Hz, 3H).

Example 7: Preparation of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7- hexahydro-8H-imidazo[2,l-f]purin-8-yl)cyclobutyl)acetamide (Compound 7)

[133] A solution of 8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (980 mg, 4 mmol), 1,2- dibromoethane (752 mg, 4 mmol) and K2CO3 (1.1 g, 8 mmol) in N,N-dimethylformamide (15 mL) was stirred at 90 °C for 2 h. l-(Bromomethyl)-2-chlorobenzene (820 mg, 4 mmol) was added and the reaction was stirred for additional 2 hr. LCMS showed the reaction completed. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried and concentrated. The crude product was purified by silica column to give 8-bromo-7-(2-bromoethyl)-3-methyl-3,7-dihydro-lH-purine-2,6-dione 7B (400 mg, 21.1% for two steps) as a white solid. LCMS: (ES+): m/z = 476.9 [M]⁺.

[134] 8-Bromo-7-(2-bromoethyl)-3-methyl-3,7-dihydro-lH-purine-2,6-dione (400 mg, 0.84 mmol), tert-butyl ((lR,3R)-3-aminocyclobutyl)carbamate (314 mg, 1.68 mmol) were dissolved in NMP (5 mL). The mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with ethyl acetate. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give tert-butyl (lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7-hexahydro- 8H-imidazo[2,l-f]purin-8-yl)cyclobutyl)carbamate 7C as a white solid (100 mg, 23.8%). LCMS: (ES+): m/z = 501.2 [M+l]⁺.

[135] To tert-butyl (lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7-hexahydro-8H- imidazo[2,l-f]purin-8-yl)cyclobutyl)carbamate (100 mg, 0.2 mmol) in 50 mL flask was added HCl/dioxane (5.0 mL), the reaction was stirred for 2.0 h at room temperature, then concentrated by rotary evaporation to obtain 8-((lR,3R)-3-aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-7,8-dihydro-lH- imidazo[2,l-f]purine-2,4(3H,6H)-dione 7D (80 mg crude). LCMS: (ES+): m/z = 401.2 [M+l]⁺.

[136] To the mixture of 8-((lR,3R)-3-aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-7,8-dihydro-lH- imidazo[2,l-f]purine-2,4(3H,6H)-dione (40 mg, 0.1 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added slowly the solution of acetyl chloride (7.8 mg, 0.1 mmol) in DCM (1 mL) at -70°C. After the reaction completed as monitored by TLC, the mixture solution was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain N-((1R, 3R)-3-(3-(2-chlorobenzyl)-l-methyl-2, 4-dioxo- 1,2, 3,4,6, 7-hexahydro- 8H-imidazo[2,l-f]purin-8-yl)cyclobutyl)acetamide 7 (30 mg, 67.7%) as a white solid. LCMS: (ES+): m/z = 443.1 [M]⁺.

Ή NMR (400 MHz, DMSO-ifc): d 8.30 (d, 7 = 7.2 H_z,IH), 7.46 (d, 7 = 7.2 Hz, 1H), 7.23-7.27 (m, 2H), 6.91 (d, 7 = 7.2 Hz, 1H), 5.06 (s, 2H), 4.35-4.39 (m, 1H), 4.11-4.29 (m, 3H), 3.94-3.98 (m, 2H), 3.38 (s, 3H), 2.63-2.70 (m, 2H), 2.11-2.17 (m, 2H), 1.81 (s, 3H).

Example 8: Preparation of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7- hexahydro-8H-imidazo[2,l-f]purin-8-yl)cyclobutyl)acrylamide (Compound 8)

[137] To the mixture of 8-((lR,3R)-3-aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-7,8-dihydro-lH- imidazo[2,l-f]purine-2,4(3H,6H)-dione 7D (40 mg, 0.1 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added a solution of acryloyl chloride (9.1 mg, 0.1 mmol) in DCM (1 mL) at -70 °C. With the reaction completed as monitored by TLC, the reaction mixture was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The product was purified by column chromatography to obtain N-((1R, 3R)-3-(3-(2-chlorobenzyl)-l -methyl-2, 4-dioxo- 1,2, 3,4,6, 7-hexahydro-8H-imidazo[2, 1- f]purin-8-yl)cyclobutyl)acrylamide 8 (26 mg, 57.1%) as a white solid. LCMS: (ES+): m/z = 455.1 [M]⁺. Ή NMR (400 MHz, DMSO-ifc): d 8.56 (d, 7= 7.2 Hz, 1H), 7.47 (d, 7= 8.0 Hz, 1H), 7.21-7.28 (m, 2H), 6.91 (d, 7= 7.6 Hz, 1H), 6.18-6.25 (m, 1H), 6.11 (dd, J = 2.0, 14.8 Hz, 1H); 5.61 (dd, J = 2.4, 10.0 Hz,

1H ), 5.07 (s, 2H), 4.41 (m, 1H), 4.29 (m, 1H), 4.12-4.16 (m, 2H), 3.96-4.02 (m, 2H), 3.38 (s, 3H), 2.67- 2.75 (m, 2H), 2.16-2.22 (m, 2H).

Example 9: Preparation of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2, 4-dioxo- 1,2, 3, 4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)cyclobutyl)acetamide (Compound 9)

[138] To a mixture of 8-bromo-l-(2-chlorobenzyl)-7-(3-hydroxypropyl)-3-methyl-3,7-dihydro-lH- purine-2,6-dione (400.0 mg, 0.93 mmol), TEA (190 mg, 1.86 mmol) in DCM (5.0 mL) was added MsCl (130 mg, 1.11 mmol) at 0 °C. After stirring at r.t. for 2 h, EA was added. The organic phase was washed with brine, dried and concentrated to give of 3-(8-bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6- tetrahydro-7H-purin-7-yl)propyl methanesulfonate 9A (467 mg) which was used in the next step without further purification.

[139] A solution of 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7- yl)propyl methanesulfonate (467 mg, 0.92 mmol) and tert-butyl ((lR,3R)-3-aminocyclobutyl)carbamate (348 mg, 1.86) in NMP was heated at 150 °C with microwave for 2 h. After cooling to r.t, the solid was collected by filtration and washed by acetonitrile to give 275 mg of tert-butyl ((lR,3R)-3-(3-(2- chlorobenzyl)-l -methyl-2, 4-dioxo- 1,2, 3,4,7, 8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)cyclobutyl)carbamate 9B which was used directly in the next step without purification. LC-MS: (ES⁺): m/z = 515.2 [M+H] ⁺.

[140] tert-Butyl ((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)cyclobutyl)carbamate (275 mg) was stirred in DCM/TFA (2:1) at r.t. for 1 h. The reaction mixture was concentrated under vacuum to give 9-((lR,3R)-3- aminocyclobutyl)-3-(2-chlorobenzyl)-l -methyl-6, 7, 8, 9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)- dione 9C (220 mg) which was used directly in the next step without purification. LC-MS: (ES⁺): m/z 415.1 [M+H] ⁺.

[141] To a solution of 9-((lR, 3R)-3-Aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (110 mg, 0.27 mmol) and TEA (55 mg, 0.54 mmol) in DCM at 0°C was acetyl chloride (25 mg, 0.32 mmol) dropwise and stirred at r.t. for 2 hours. Water was added and extracted with DCM. The organic phase was dried and concentrated followed by prep-HPLC purification to give of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8 hexahydropyrimido[2,l-f]purin-9(6H)-yl)cyclobutyl)acetamide 9 (50 mg) as a TFA salt. LC-MS: (ES⁺): m/z = 457.2 [M+H] ⁺.

Ή NMR (400 MHz, DMSO): d 8.35 (d, = 6.J4 Hz, 1H), 7.45 (d, = 7.2 HJz, 1H), 7.22 -7.26 (m, 2H), 6.88 (d, J= 6.8 Hz, 1H), 5.06 (br s, 3H), 4.06 (m, 3H), 3.43 (t, J= 8.0 Hz, 2H), 3.36 (s, 3H), 2.59-2.63 (m, 2H), 2.07-2.14 (m, 4H), 1.83 (s, 3H).

Example 10: Preparation of N-((1R, 3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)cyclobutyl)acrylamide (Compound 10)

[142] To a solution of 9-((lR, 3R)-3-Aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (110 mg, 0.27 mmol) and TEA (55 mg, 0.54 mmol) in DCM at 0°C was added acryloyl chloride (30 mg, 0.32 mmol) dropwise, then stirred at r.t. for 2 h. Water was added and extracted with DCM. The organic phase was dried, concentrated and purified by prep- HPLC to give of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)cyclobutyl)acrylamide 10 (40 mg) as a TFA salt. LC-MS:

(ES⁺): m/z = 469.1 [M+H] ⁺.

Ή NMR (400 MHz, DMSO): d 8.61 (d, 7=6.0 Hz, 1H), 7.45 (d, J= 7.6 Hz, 1H), 7.22-7.26 (m, 2H), 6.88 (d, J = 6.8 Hz, 1H), 6.24-6.28 (m, 1H), 6.09 (d, J = 14.8 Hz, 1H), 5.59 (d, J = 8.0 Hz, 1H), 5.05 (br s,

3H), 4.17 (m, 1H), 4.06 (t, J= 5.6 Hz, 2H), 3.45 (t, J = 4.8 Hz, 2H), 3.38 (s, 3H), 2.68-2.71 (m, 2H), 2.16 (t, J = 4.8 Hz, 2H), 2.08 (m, 2H).

Example 11: Preparation of N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7,8,9- octahydro-10H-[l,3]diazepino[2,l-f]purin-10-yl)cyclobutyl)acrylamide (Compound 11)

[143] A solution of 8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (1 g,4.08 mmol), 1,4- dibromobutane (0.88 g, 4.08 mmol) and TEA (0.91 g, 8.98 mmol) in DMF (30 mL) were stirred for 16h. Ethyl acetate was added and the organic layer was sequentially washed with water and brine. After separation, the organic phase was dried over sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:l to 4:1) to give 8-bromo-7-(4-bromobutyl)-3-methyl-3,7-dihydro-lH-purine-2,6- dione 11A (0.64 g, 53%). LCMS: (ES+): m/z = 380.9 [M]⁺.

[144] A solution of 8-bromo-7-(4-bromobutyl)-3-methyl-3,7-dihydro-lH-purine-2,6-dione (0.64 g, 1.68 mmol), l-(bromomethyl)-2-chlorobenzene (0.381 g,1.85 mmol) and K2CO3 (0.512 mg, 3.705 mmol) in DMF (10 mL) was stirred at 80 °C for 3 h. Ethyl acetate was added and the organic layer was sequentially washed with water and brine and dried over sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=20:l to 3:1) to give 8-bromo-7-(4-bromobutyl)-l-(2-chlorobenzyl)-3- methyl-3, 7-dihydro- lH-purine-2,6-dione 11B (0.316 g, 40%). LCMS: (ES+): m/z = 502.9 [M]⁺.

[145] 8-Bromo-7-(4-bromobutyl)-l-(2-chlorobenzyl)-3-methyl-3, 7-dihydro- lH-purine-2,6-dione (315 mg, 0.6242mmol) and tert-butyl ((lR,3R)-3-aminocyclobutyl)carbamate (139.53 mg, 0.7491 mmol) were dissolved in NMP (15 mL) and irradiated at 150 °C with microwave for 2 hours. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography using DCM/PE to give tert-butyl((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo- l,2,3,4,6,7,8,9-octahydro-10H-[l,3]diazepino[2,l-f]purin-10-yl)cyclobutyl)carbamate 11C as a colorless oil (130 mg). LCMS: (ES+): m/z = 529.2 [M+l]⁺.

[146] A solution of tert-butyl((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7,8,9- octahydro-10H-[l,3]diazepino[2,l-f]purin-10-yl)cyclobutyl)carbamate (130 mg, crude) in TFA/dioxane (2.0 mL) was stirred for 2.0 h at room temperature. The mixture was concentrated by rotary evaporation to give 10-((lR,3R)-3-aminocyclobutyl)-3-(2-chlorobenzyl)-l -methyl-7, 8, 9, 10-tetrahydro-lH- [l,3]diazepino[2,l-f]purine-2,4(3H,6H)-dione 11D (130 mg, 100%) which was directly used in the next step without further purification. LCMS: (ES+): m z = 429.1 [M+l]⁺.

[147] To the mixture of 10-((lR,3R)-3-aminocyclobutyl)-3-(2-chlorobenzyl)-l-methyl-7,8,9,10- tetrahydro-lH-[l,3]diazepino[2,l-f]purine-2,4(3H,6H)-dione (130 mg, 0.246 mmol) and TEA (49.78 mg, 0.492 mmol) in DCM (2 mL) was slowly added the solution of acryloyl chloride (22.26 mg, 0.246 mmol) in DCM (1 mL) at -70°C. After the reaction completed as monitored by TLC, the mixture solution was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2S04, filtered and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl- 2,4-dioxo-l,2,3,4,6,7,8,9-octahydro-10H-[l,3]diazepino[2,l-f]purin-10-yl)cyclobutyl)acrylamide 11 (10.8 mg, 10%) as a white solid. LCMS: (ES+): m/z = 483.1 [M]⁺.

Ή NMR (400 MHz, CDCfe-d): d 7.34-7.37 (m, 1H), 7.13-7.15 (m, 2H), 6.99-7.01 (m, 1H), 6.30 (d, = J 17.2 H_z,IH), 6.11-6.14 (m, 1H), 5.82 (br, 1H), 5.69 (d, /= 11.2 H_z,IH), 5.31 (s, 2H), 4.36-4.40 (m, 4H), 3.52 (s, 3H), 3.01-3.04 (m, 2H), 2.39-2.47 (m, 4H), 1.83-1.89 (m, 4H).

Example 12: Preparation of 3-(2-Chlorobenzyl)-9-(3-(2-(dimethylamino)ethoxy)phenyl)-l-methyl- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 12)

[148] A mixture solution of 7-allyl-8-bromo-3-methyl-3,7-dihydro-lH-purine-2,6- dione (3.0 g, 10.5 mmol) , l-(bromomethyl)-2-chlorobenzene (2.38 g, 11.6 mmol) and K2CO3 (2.18 g, 15.8 mmol) in DMF (100 mL) was stirred at 60 °C for 3 h. The mixture was diluted with water (80 mL) and extracted with ethyl acetate (100 mL x 2). The combined organic layers were washed with brine (150 mL x 3), dried, filtered and concentrated. The crude product was purified by silica column chromatography (EA/PE=1: 10 to 1:5 to 1:3) to obtain the title product 7-allyl-8-bromo-l-(2-chlorobenzyl)-3-methyl-3,7-dihydro-lH- purine-2,6-dione 12A (4.0 g, yield 93%) as a white solid.

[149] To a solution of cyclohexene (4.0 g, 48.82 mmol) in THF (40 mL) was added BH₃-THF (24.4 mL, 24.4 mmol) at 0 °C, then the resulting solution was stirred at 0 °C for 1 h. After that, the solution of 12A (4.0 g, 9.76 mmol) in THF (20 mL) was added dropwise to the solution and the resulting solution was stirred at room temperature for overnight. Then 37% NaOH solution (5 mL), H2O2 (5 mL) was added to the solution at 0 °C sequentially, and the resulting solution was stirred at room temperature for 3 h. The solution was quenched with saturated Na₂S₂C>₃ solution (30 mL) and extracted with ethyl acetate (100 mL x 2). The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by silica column chromatography (EA/PE=1:2) to obtain the title product 8- bromo-l-(2-chlorobenzyl)-7-(3-hydroxypropyl)-3-methyl-3, 7-dihydro- lH-purine-2,6-dione 12B (1.9 g, yield 45%) as a white solid.

[150] To a solution of 8-bromo-l-(2-chlorobenzyl)-7-(3-hydroxypropyl)-3-methyl-3,7- dihydro-lH- purine-2,6-dione (300 mg, 0.7 mmol) in DCM (5 mL) was added TEA (142 mg, 1.4 mmol) and MsCl (96 mg, 0.84 mmol) dropwise at 0 °C. The reaction mixture was stired at 0 °C for 3 h. The reaction was quenched with water (20 mL) and extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated to give 3-(8-bromo-l- (2-chlorobenzyl)-3- methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)propyl methanesulfonate 12C (355 mg) which was used without further purification.

[151] A solution of 3-(8-bromo-l-(2-chlorobenzyl)-3-methyl-2,6- dioxo-l,2,3,6-tetrahydro-7H-purin-7- yl)propyl methanesulfonate (355 mg), 3-(2-(dimethylamino)ethoxy)aniline (253 mg, 1.4 mmol) in DMA (4 mL) was irradiated with microwave at 150 °C for 2 h. Water (20 mL) was added and extracted with ethyl acetate (20 mL x 2) . The combined organic layers were washed with brine (50 mL), dried, filtered and concentrated. The crude product was purified by Prep-TLC first (eluent: EA) and further purified by Prep-HPLC to obtain 3-(2-chlorobenzyl)-9-(3-(2-(dimethylamino)ethoxy)phenyl)-l-methyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 12 as a white solid of a TFA salt (45 mg, yield 12.5%). LCMS: (ES+): 509.2 m/z [M]⁺.

Ή NMR (400 Hz, MeOD): d 7.36-7.41 (m, 2H),7.28 (s, 1H), 7.16-7.24 (m, 3H), 6.90-6.94 (m, 2H), 5.23 (s, 2H), 4.37-4.40 (t, /= 4.8 Hz, 2H), 4.29-4.32 (t, /= 6.0 Hz, 2H), 3.88-3.90 (t, /= 5.2 Hz, 2H), 3.60- 3.63 (t, /= 4.8 Hz, 2H), 3.43 (s,3H), 3.00 (s, 6H), 2.31-2.34 (m, 2H)

Example 13: Preparation of 9-(l-Acryloylpiperidin-4-yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 13)

[152] 8-Bromo-3-methyl-3,7-dihydro-lH-purine-2,6-dione (980 mg, 4 mmol), 3-bromo-2-methylprop- 1-ene (540 mg, 4 mmol) and K2CO3 (1.1 g, 8 mmol) was suspended in N,N-dimethylformamide (15 mL). After stirring at 90 °C for 2 h, 2-(bromomethyl)-l-chloro-3-fluorobenzene (890 mg, 4 mmol) was added and the reaction was stirred for another 2 h. LCMS showed completion of the reaction. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried and concentrated. The crude product was purified by silica column to give 8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-7-(2- methylallyl)-3,7-dihydro-lH-purine-2,6-dione 13B (880 mg, 50%) as a white solid. LCMS: (ES+): m/z = 442.9 [M]⁺.

[153] A dry three-neck flask was charged with 8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-7-(2- methylallyl)-3,7-dihydro-lH-purine-2,6-dione (440 mg, 1 mmol) and dry THE (10 mL) and cooled to

0 °C. Borane-THF complex (1.3 mL) was added via a syringe dropwise. The resulting mixture was stirred for 2 h at 0 °C. Sodium hydroxide (4 M, 1 mL) and hydrogen peroxide (1 mL) were added sequentially. The mixture was stirred for 2 h. The reaction mixture was diluted with water, then extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulfate. After filtration, organic solvent was evaporated under vacuum and the residue was purified with silica gel chromatography to give 8-bromo- 1 -(2-chloro-6-fluorobenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-

3.7-dihydro- 1 H-purine-2,6-dione 13C (300 mg, 65.5%). LCMS: (ES+): m/z = 461.0 [M]⁺.

[154] To a solution of 8-bromo-l-(2-chloro-6-fluorobenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-

3.7-dihydro-lH-purine-2,6-dione (230 mg, 0.5 mmol) and TEA (151 mg, 1.5 mmol) in DCM (5 mL) at 0 °C was added MsCl (86 mg, 1.5 mmol). The reaction mixture was stirred at 0 °C for 2 h. LCMS showed small amount of SM remained. The reaction was quenched with water and the organic phase was dried and concentrated in vacuo to give 3-(8-bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6- tetrahydro-7H-purin-7-yl)-2-methylpropyl methanesulfonate 13D (250 mg) as a yellow solid which was directly used in the next step. LCMS: (ES+): m/z = 539.0 [M+l]⁺. [155] 3-(8-Bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)- 2-methylpropyl methanesulfonate (250 mg) and tert-butyl 4-aminopiperidine-l-carboxylate (200 mg, 1 mmol) were dissolved in NMP (4 mL). The mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with EA. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give tert-butyl 4-(3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl- 2,4-dioxo-l,2,3,4,7,8-hexa hydropyrimido[2,l-f]purin-9(6H)-yl)piperidine-l-carboxylate 13E as a white solid (60 mg, 21.4%). LCMS: (ES+): m/z = 561.3 [M]⁺.

[156] A solution of tert-butyl 4-(3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)piperidine-l-carboxylate (60 mg, 0.11 mmol) in HCl/dioxane (3.0 mL) was stirred at room temperature for 2 h. The mixture was concentrated to give 3-(2-chloro-6- fluorobenzyl)-l,7-dimethyl-9-(piperidin-4-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 13F (55 mg, 100%) which was used in the next step without purification.

[157] To the mixture of 3-(2-chloro-6-fluorobenzyl)-l, 7-dimethyl-9-(piperidin-4-yl)-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (55 mg, 0.11 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (10 mg, 0.11 mmol) in DCM (1 mL) at - 70°C. After the reaction completed as monitored by TLC, ice water was added and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The product was purified by column chromatography to obtain 9-(l-acryloylpiperidin-4-yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 13 (31 mg, 54.7%) as a white solid. LCMS: (ES+): m/z = 515.2 [M]⁺.

Ή NMR (400 MHz, CDCL-i/): d 7.10-7.17 (m, 2H), 6.89-6.93 (m, 1H), 6.57-6.63 (dd, /= 10.4, 16.8 H_z,IH), 6.32 (d, /= 16.8 H_z,IH), 5.72 (d, /= 12.4 H_z,IH), 5.37 (s, 2H), 4.82-4.86 (m, 1H), 4.48-4.54 (m, 1H), 4.37-4.41 (m, 1H), 4.11-4.13 (m, 1H), 3.63-3.67 (m, 1H), 3.48 (s, 3H), 3.24 (m, 2H), 2.74-2.87 (m, 2H), 2.23 (m, 1H), 1.59-1.90 (m, 4H), 1.09 (d, /= 6.8 Hz, 3H).

Example 14: Preparation of 9-(l-acryloylpiperidin-4-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9- tetrahydro pyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 14)

[158] 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (260 mg, crude) and tert-butyl 4-aminopiperidine-l-carboxylate (200 mg, 1 mmol) were dissolved in NMP (4 mL). The mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with EA. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give tert-butyl 4-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4- dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)piperidine-l-carboxylate 14A as a white solid (60 mg, 22%). LCMS: (ES+): m/z = 543.2 [M]⁺. [159] tert-Butyl 4-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l- f]purin-9(6H)-yl)piperidine-l-carboxylate (60 mg, 0.11 mmol) was stirred for 2.0 h at room temperature in HCl/dioxane (3.0 mL). The mixture was concentrated by rotary evaporation to give 3-(2- chlorobenzyl)-l,7-dimethyl-9-(piperidin-4-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 14B (55 mg, 100%) which was used in the next step without further purification. LCMS: (ES+): m/z = 443.2 [M]⁺.

[160] To the mixture of 3-(2-chlorobenzyl)-l, 7-dimethyl-9-(piperidin-4-yl)-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (55 mg, 0.11 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (10 mg, 0.11 mmol) in DCM (1 mL) at - 70°C. After the reaction was completed as monitored by TLC, the mixture solution was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by evaporation. The crude product was purified by column chromatography to afford 9-(l-acryloylpiperidin-4-yl)-3-(2-chlorobenzyl)-l, 7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 14 (31 mg, 56.7%) as a white solid. LCMS: (ES+): m/z = 497.2 [M]⁺.

Ή NMR (400 MHz, CDCL-i/): d 7.35 (br s, 1H), 7.13 (br s, 2H), 7.01 (br s, 1H), 6.58-6.65 (dd, /= 10.4, 16.8 H_z,IH), 6.29-6.33 (d, =J 16.4 H_z,IH), 5.71-5.74 (d, /= 9.6 H_z,IH), 5.29 (s, 2H), 4.85 (br, 1H),

4.54 (br, 1H), 4.40 (br, 1H), 4.15 (br, 1H), 3.68 (t, J = 10.8 Hz, 1H), 3.53 (s, 3H), 3.27 (br, 2H), 2.88-2.76 (br, 2H), 2.25 (br, 1H), 1.85 (br, 2H), 1.761 (br, 2H), 1.09 (d, J = 6.0 Hz, 3H).

Example 15: Preparation of 9-(l-acryloylazetidin-3-yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 15)

[161] 3-(8-Bromo-l-(2-chloro-6-fhiorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)- 2-methylpropyl methanesulfonate (260 mg) and tert-butyl 3-aminoazetidine-l-carboxylate (172 mg, 1 mmol) were dissolved in NMP (4 mL). The mixture was irradiated in the microwave at 150 °C for 2 h. Water was added and the mixture was extracted with EA.The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give tert-butyl 3-(3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-2,4- dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)azetidine-l-carboxylate 15A as a white solid (60 mg, yield: 23%). LCMS: m/z = 533.2 [M]⁺.

[162] A solution of tert-butyl 3-(3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)azetidine-l-carboxylate (60 mg, 0.11 mmol) in HCl/dioxane (3.0 mL) was stirred for 2.0 h at room temperature. The mixture was Concentrated to give 9-(azetidin-3- yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 15B (55 mg) which was used in the next step without further purification. [163] To the mixture of 9-(azetidin-3-yl)-3-(2-chloro-6-fluorobenzyl)-l, 7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (55 mg, 0.11 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (10 mg, 0.11 mmol) in DCM (1 mL) at - 70°C. After the reaction completed as monitored by TLC, the mixture solution was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain 9-(l-acryloylazetidin-3-yl)-3-(2-chloro-6-fluorobenzyl)-l,7- dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 15 (24 mg, yield: 44.8%) as a white solid. LCMS: (ES+): m/z = 487.2 [M]⁺.

Ή NMR (400 MHz, CDCL-i/): d 7.13-7.173 (m, 2H), 6.89-6.94 (m, 1H), 6.38 (d, = 16.8 Hz,J 1H), 6.20 (dd, J = 10.4, 16.8 Hz, 1H), 5.72 (d, J = 10.4 Hz, 1H), 5.36 (s, 2H), 5.06-5.14 (m, 1H), 4.30-4.54 (m, 5H), 3.69 (t, /= 12.0 H_z,IH), 3.45 (br S, 4H), 3.02-3.07 (m, 1H), 2.36 (br, 1H), 1.15 (d, J = 6.8 Hz, 3H).

Example 16: Preparation of 9-(l-acryloylazetidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9- tetrahydro pyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 16)

[164] 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (260 mg) and tert-butyl 3-aminoazetidine-l-carboxylate (172 mg, 1 mmol) were dissolved in NMP (4 mL). The mixture was irradiated with microwave at 150 °C for 2 h. Water was added and the mixture was extracted with ethyl acetate. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give tert-butyl 3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4- dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)azetidine-l-carboxylate 16A as a white solid (60 mg, 23%). LCMS: (ES+): m/z = 515.2 [M]⁺.

[165] A solution of tert-butyl 3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)azetidine-l-carboxylate (60 mg, 0.11 mmol) in HCl/dioxane (3.0 mL) was stirred for 2.0 h at room temperature. The mixture was concentrated to give 9-(azetidin-3- yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 16B (55 mg) which was used in the next step without further purification.

[166] To the mixture of 9-(azetidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (55 mg, 0.11 mmol) and DIEA (65 mg, 0.5 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (10 mg, 0.11 mmol) in DCM (1 mL) at - 70°C. After the reaction completed as monitored by TLC, the reaction was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The crude product was purified by Biotage to provide 9-(l-acryloylazetidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 16 (17 mg, 33.1%) as a white solid. LCMS: (ES+): m z = 469.1 [M]⁺.

Ή NMR (400 MHz, CDCL-i/): d 7.36 (d, /= 8.8 Hz, 1H), 7.13-7.16 (m, 2H), 6.98-7.00 (m, 1H), 6.39 (d, /= 16.8 Hz, 1H), 6.24 (dd, /= 10.4, 16.8 Hz, 1H), 5.74(d, /= 10.0 Hz, 1H), 5.29 (s, 2H), 5.10-5.11 (m, 1H), 4.25-4.53 (m, 5H), 3.72 (t, /= 6.2 Hz, 1H), 3.49 (br s, 4H), 3.09 (m, 1H), 2.39(br, 1H) , 1.16 (d, = J 6.4 Hz, 3H).

Example 17: Preparation of 9-((R)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl- 6,7,8,9-tetra hydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 17)

[167] 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (220 mg, 0.42 mmol) and tert-butyl (R)-3-aminopiperidine-l-carboxylate (93.24mg, 0.47 mmol) were dissolved in NMP (3 mL) and irradiated with microwave atl50 °C for 2 hours. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography using DCM/PE to give tert-butyl (3R)-3-(3-(2- chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)piperidine-l- carboxylate 17A as a white solid (65 mg). LCMS: (ES+): m/z = 543.3 [M+l]⁺.

[168] A solution of tert-butyl (3R)-3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido [2, l-f]purin-9(6H)-yl)piperidine-l -carboxylate (65 mg) was in HCl/dioxane (2.0 mL) was stirred for 2.0 h at room temperature. Concentration by rotary evaporation gave 3-(2-chlorobenzyl)- l,7-dimethyl-9-((R)-piperidin-3-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 17B (65 mg, 100%) which was directly used in the next step without further purification. LCMS: (ES+): m/z = 443.2 [M+l]⁺.

[169] To the mixture of 3-(2-chlorobenzyl)-l,7-dimethyl-9-((R)-piperidin-3-yl)-6,7,8,9-tetrahydro pyrimido[2,l-f]purine-2,4(lH,3H)-dione (65 mg) and DIEA (74.54 mg, 0.5869 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (10.62 mg, 0.117 mmol) in DCM (1 mL) at -70°C. After reaction completed as monitored by TLC, the reaction was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The cmde product was purified by column chromatography to afford 9-((R)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 17 (30.5 mg, 41.91%) as a white solid. LCMS: (ES+): m z = 497.2 [M]⁺.

Ή NMR (400 MHz_,DMSO-d6): d 7.46 (d, /= 7.2 Hz, 1H), 7.22-7.29 (m, 2H), 6.90 (d, /= 7.2 Hz, 1H), 6.83 (m, 1H), 6.14 (d, =J 16.8 H_z,IH), 5.70 (d, J = 9.2 H_z,IH), 5.07 (s, 2H), 4.44 (br, 1H), 4.19 (m, 1H), 4.03-4.10 (m, 2H), 3.50-3.64 (m, 1H), 3.48 (d, = 11.J6 Hz, 1H), 3.38 (s, 3H), 3.19-3.28 (m, 1H), 2.85- 3.12 (m, 2H), 2.51 (m, 1H), 2.23 (br, 1H), 1.81-1.99 (m, 3H), 1.05 (d, J = 6.4 Hz, 3H). Example 18: Preparation of 9-((S)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl- 6,7,8,9-tetra hydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 18)

[170] 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (174 mg, 0.33 mmol) and tert-butyl (S)-3-aminopiperidine-l-carboxylate (80.45 mg, 0.40 mmol) were dissolved in NMP (3 mL) and irradiated with microwave at 150 °C for 2 hours. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography using DCM/PE to give tert-butyl (3S)-3-(3-(2- chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)piperidine-l- carboxylate 18A as a white solid (110 mg). LCMS: (ES+): m/z = 543.3 [M+l]⁺.

[171] A solution of tert-butyl (3S)-3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f] purin-9(6H)-yl)piperidine-l-carboxylate (110 mg) in HCl/dioxane (2.0 mL) was stirred for 2.0 h at room temperature. Evaporation of the mixture gave 3-(2-chlorobenzyl)-l,7- dimethyl-9-((S)-piperidin-3-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4-(lH,3H)-dione 18B (100 mg, 100%) which was directly used in the next step without further purification. LCMS: (ES+): m/z = 443.2 [M+l]⁺.

[172] To the mixture of 3-(2-chlorobenzyl)-l,7-dimethyl-9-((S)-piperidin-3-yl)-6, 7,8,9- tetrahydropyrimido [2,l-f]purine-2,4(lH,3H)-dione (100 mg) and DIEA (124.23 mg, 0.9782 mmol) in DCM (2 mL) was added slowly a solution of acryloyl chloride (17.7 mg, 0.195 mmol) in DCM (1 mL) at -70°C. After the reaction completed as monitored by TLC, the mixture solution was quenched with ice water and extracted with DCM (10 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The crude product was purified by HPLC to give 9-((S)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l, 7-dimethyl-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (50 mg, 45%) as a TLA salt of a white solide. LCMS: (ES+): m/z = 497.2 [M]⁺.

Ή NMR (400 MHz, MeOH -d): d 7.39 (d, /= 8.8 Hz, 1H), 7.18 -7.22 (m, 2H), 6.91 (d, J = 8.2 Hz, 1H), 6.70-6.90 (m, 1H), 6.25 (m, 1H), 5.75 (d, /= 10.8 H_z,IH), 5.23 (s, 2H), 4.50-4.70 (m, 1H), 4.25-4.35 (m, 1H), 4.10-4.22 (m, 2H), 3.66-3.69 (m, 1H), 3.50-3.60 (m, 4H), 2.95-3.20 (m, 3H), 2.32 (br, 1H), 1.91- 2.06 (m, 3H), 1.58-1.70 (br, 1H), 1.13 (d, /= 6.8 Hz, 3H).

Example 19: Preparation of 3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-9-(l-methyl-lH-pyrazol-4-yl)- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 19)

[173] 3-(8-Bromo-l-(2-chloro-6-fluorobenzyl)-3-methyl-2,6-dioxo-l,2,3,6-tetrahydro-7H-purin-7-yl)- 2-methylpropyl methanesulfonate (260 mg), 1 -methyl- lH-pyrazol-4-amine (65.02 mg, 0.6695 mmol) were dissolved in NMP (4 mL). The mixture was irradiated in the microwave at 150 °C for 2 h. Water was added and the mixture was extracted with EA. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give 3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-9-(l-methyl-lH-pyrazol- 4-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 19 as a white solid(60 mg, 26%). LCMS: (ES+): m/z = 458.2 [M]⁺.

Ή NMR (400 MHz, CDCl -7): d 7.85 (s, 1H), 7.57 (s, 1H), 7.13-7.18 (m, 2H), 6.90-6.94 (m, 1H), 5.39 (s, 2H), 4.53-4.58 (m, 1H), 3.92 (s, 3H), 3.71-3.77 (m, 2H), 3.53 (s, 3H), 3.35-3.41 (m, 1H), 2.49 (br,

1H), 1.18 (d, 7= 6.4 Hz, 3H).

Example 20: Preparation of 3-(2-chlorobenzyl)-l,7-dimethyl-9-(l-methyl-lH-pyrazol-4-yl)-6,7,8,9- tetra hydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (Compound 20)

[174] 3-(8-Bromo-l-(2-chlorobenzyl)-3-methyl-2,6-dioxo- 1,2,3, 6-tetrahydro-7H-purin-7-yl)-2- methylpropyl methanesulfonate (174 mg, 0.3347 mmol) and 1 -methyl- lH-pyrazol-4-amine (65.02 mg, 0.6695 mmol) were dissolved in NMP (2.5 mL) and irradiated at 150 °C with microwave for 1 hr. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography with DCM/PE to give 3-(2-chlorobenzyl)-l,7-dimethyl-9-(l-methyl- lH-pyrazol-4-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione 20 as a white solid (40 mg, 34%). LCMS: (ES+): m/z = 440.2 [M]⁺.

Ή NMR (400 MHz, CDCE-T): d 7.87 (s, 1H), 7.59 (s, 1H), 7.35-7.37 (m, 1H), 7.14-7.17 (m, 2H), 6.99- 7.02 (m, 1H), 5.30 (br, 2H), 4.52-4.57 (dd, 7 = 3.6, 12.4 Hz, 1H), 3.93 (s, 3H), 3.73-3.77 (m, 2H), 3.57 (s, 3H), 3.37-3.42 (dd, 7= 10.0, 11.6 Hz, 1H), 2.48 (br, 1H), 1.20 (d, J = 6.8 Hz, 3H). Example 21: Preparation of N-(3-(3-(2-chlorobenzyl)-7-((dimethylamino)methyl)-l-methyl-2,4- dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 21)

[175] 8-Bromo-3-methyl-lH-purine-2,6 (3H,7H)-dione (5 g, 20.41 mmol) was dissolved in DMF (50 mL). SEMCI (3.4 g, 20.41 mmol) and K2CO3 (4.23 g, 30.61 mmol) were added. The reaction mixture was stirred at room temperature overnight. LCMS showed small amount of SM remained. The reaction was quenched with water and extracted with ethyl acetate (100 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation to give 8- bromo-3-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-lH-purine-2,6 (3H,7H)-dione 21A (6.5 g, yield: 85%).

[176] 8-Bromo-3-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-lH-purine-2,6 (3H,7H)-dione (6.5 g, 17.32 mmol) was dissolved in DMF (50 mL). l-(Bromomethyl)-2-chlorobenzene (3.91 g, 19.05 mmol) and K2CO3 (3.6 g, 25.98 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. LCMS showed small amount of SM remained. The reaction was quenched with water and extracted with EA (100 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The product was purified by column chromatography on silica gel (PE/EA= 5/1) to afford 8-bromo-l-(2-chlorobenzyl)-3-methyl-7-((2- (trimethylsilyl)ethoxy)methyl)-lH-purine-2,6 (3H,7H)-dione 21B (6.8 g, yield: 78%).

[177] To a solution of 8-bromo-l-(2-chlorobenzyl)-3-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-lH- purine-2,6 (3H,7H)-dione (6.8 g, 13.6 mmol) in 60 mLof DCM was added trifluoroacetic acid (15 mL). The mixture was stirred for 2.0 h at room temperature, then concentrated by rotary evaporation to provide 8-bromo-l-(2-chlorobenzyl)-3-methyl-lH-purine-2,6 (3H,7H)-dione 21C (4.8 g, yield: 95%). LCMS: (ES+): m/z = 371 [M]⁺.

[178] 8-Bromo-l-(2-chlorobenzyl)-3-methyl-lH-purine-2,6 (3H,7H)-dione (1 g, 2.71 mmol) was dissolved in DMF (10 mL). 3-Chloro-2-(chloromethyl)prop-l-ene (372 mg, 2.98 mmol) and K2CO3 (3.56 g, 3.56 mmol) were added. The reaction mixture was stirred at room temperature overnight. LCMS showed only small amount of SM remained. The reaction was quenched with water and extracted with ethyl acetate (100 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The product was purified by column chromatography on silica gel (PE/EA= 5/1) to afford 8-bromo-l-(2-chlorobenzyl)-7-(2- (chloromethyl)allyl)-3-methyl-lH-purine-2,6 (3H,7H)-dione 21D (700 mg, yield: 56%). LCMS: (ES+): m/z 459.0 = [M+l]⁺.

[179] 8-Bromo-l-(2-chlorobenzyl)-7-(2-(chloromethyl)allyl)-3-methyl-lH-purine-2,6 (3H,7H)-dione (600 mg, 1.31 mmol) was dissolved in NMP (10 mL). Then tert-butyl (3-aminophenyl)carbamate (546 mg, 2.62 mmol) was added and irradiated with microwave at 150°C for 1 h. The reaction was quenched with water and extracted with ethyl acetate (100 mL x 3), the organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation. The product was purified by column chromatography on silica gel (DCM/MeOH= 20/1) to give 9-(3-aminophenyl)-3-(2- chlorobenzyl)- 1 -methyl-7 -methylene-6,7, 8, 9-tetrahydropyrimido [2, 1 -f]purine-2,4( 1 H,3H)-dione 21E (700 mg). LCMS: (ES+): m/z = 449.1 [M+l]⁺.

[180] A suspension containing 9-(3-aminophenyl)-3-(2-chlorobenzyl)-l-methyl-7-methylene-6, 7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (500 mg, 1.11 mmol), B0C2O (365 mg, 1.67 mmol) and K2CO3 (462 mg, 3.34 mmol) in DCM (10 mL) and H2O (2 mL) were stirred overnight. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography using PE/EA to give (3-(3-(2-chlorobenzyl)-l-methyl-7-methylene- 2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)carbamate 21F (150 mg, yield: 24% for two steps). LCMS: (ES+): m/z = 549.2 [M]⁺.

[181] tert-Butyl (3-(3-(2-chlorobenzyl)-l -methyl-7 -methylene-2, 4-dioxo- 1,2, 3, 4, 7, 8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)carbamate (150 mg, 0.27 mmol) was dissolved in THE (4 mL) at 0 °C, and 0.6 mL of a solution of 1 M B¾ in THE was added. The reaction solution was stirred at 30 °C overnight followed by the addition of 4N NaOH solution (1 mL) and 30% I¾q₂(1 mL). The reaction solution was warmed up to room temperature and stirred for 5 hours. The reaction was quenched with water and extracted with ethyl acetate (100 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by rotary evaporation to provide tert-butyl (3-(3-(2-chlorobenzyl)-7-(hydroxymethyl)- 1 -methyl-2, 4-dioxo- 1,2, 3,4,7, 8-hexahydropyri mido[2,l- f]purin-9(6H)-yl)phenyl)carbamate 21G (120 mg, yield: 77%). LCMS: (ES+): m/z = 567.2 [M]⁺.

[182] To a solution of tert-butyl (3-(3-(2-chlorobenzyl)-7-(hydroxymethyl)-l-methyl-2,4-dioxo- l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)carbamate (120 mg, 0.21 mmol) in DCM (3.0 mL) was added TEA (43 mg, 0.42 mmol) and MsCl (37 mg, 0.31 mmol) at 0°C. The reaction was stirred at room temperature for 5 hrs. Water was added and the product was extracted with dichloromethane (20 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by evaporation. The product was purified by column chromatography on silica gel (DCM/MeOH= 20/1) to afford (9-(3-((tert-butoxycarbonyl)amino)phenyl)- 3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-l,2,3,4,6,7,8,9-octahydropyrimido[2,l-f]purin-7-yl)methyl methanesulfonate 21H (100 mg). LCMS: (ES+): m/z = 646.1 [M+l]⁺.

[183] (9-(3-((tert-Butoxycarbonyl)amino)phenyl)-3-(2-chlorobenzyl)-l -methyl-2, 4-dioxo- l,2,3,4,6,7,8,9-octahydropyrimido[2,l-f]purin-7-yl)methyl methanesulfonate (100 mg, 0.15 mmol) was a dissolved in (CI¾)2NH in THE (5 mL). The reaction was stirred at 60 °C for 48 hrs and the mixture was evaporated to afford tert-butyl (3-(3-(2-chlorobenzyl)-7-((dimethylamino)methyl)-l-methyl-2,4-dioxo- l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)carbamate 211 (90 mg) which was directly used in the next step without further purification. LCMS: (ES+): m/z = 594.3 [M]⁺.

[184] tert-Butyl (3-(3-(2-chlorobenzyl)-7-((dimethylamino)methyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[2,l-f]purin-9(6H)-yl)phenyl)carbamate (90 mg) was dissolved in DCM (2 mL). HCl/dioxane (0.5 mL) was added and the mixture reaction was stirred for 1 h at room temperature. Water was added and the mixture was extracted with DCM. The organic layer was concentrated and purified by silica gel column chromatography using DCM/MeOH to give 9-(3-aminophenyl)-3-(2-chlorobenzyl)-7- ((dimethylamino)methyl)-l-methyl-6,7,8,9-tetrahydropyrimido[2, l-f]purine-2,4(lH,3H)-dione 21J as a colorless oil (32 mg, yield: 43% for three steps). LCMS: (ES+): m/z = 494.2 [M]⁺.

[185] To the mixture of 9-(3-aminophenyl)-3-(2-chlorobenzyl)-7-((dimethylamino)methyl)-l-methyl- 6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione (32 mg, 0.06 mmol) and DIEA (64.6 mg, 0.50 mmol) in DCM (3 mL) was added slowly a solution of acryloyl chloride (6 mg, 0.06 mmol) in DCM (1 mL) at -20 °C. the reaction mixture was stirred at -20 °C for 1 h. LCMS showed only small amount of SM remained. The mixture solution was quenched with ice water and extracted with DCM (10 mLx3). The organic layer was washed with water and brine, dried over Na2S04, filtered and the solvent was removed by evaporation. The product was purified by HPLC to provide N-(3-(3-(2-chlorobenzyl)-7- ((dimethylamino)methyl)-l-methyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide 21 (24 mg, yield: 67%) as a white solid of a TLA salt. LCMS: (ES+): m z = 548.2 m z [M]⁺.

Ή NMR (400 MHz, MeOD) d 8.12 (s, 1H), 7.40 (t, = 7.7 J Hz, 2H), 7.31 (d, /= 7.4 Hz, 2H), 7.21 (dt, / = 14.9, 7.4 Hz, 2H), 6.94 (d, /= 6.6 Hz, 1H), 6.41 (qd, /= 16.9, 5.8 Hz, 2H), 5.80 (dd, /= 9.6, 2.1 Hz, 1H), 5.24 (s, 2H), 4.53 - 4.59 (m, 1H), 4.03 - 4.20 (m, 2H), 3.81 (dd, /= 12.1, 8.2 Hz, 1H), 3.45 (s, 3H), 3.43 - 3.36 (m, 2H), 2.99 (s, 7H).

Example 22: Preparation of N-(3-(3-(2-chloro-4-hydroxybenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[l,2-f]purin-9(6H)-yl)phenyl)acrylamide (Compound 31)

To a solution of 8-bromo-3-methyl-7-(2-methylallyl)-lH-purine-2,6(3H,7H)-dione (3 g, 0.01 mol) in DMF (30 mL) was added K2CO3 (2.08 g, 0.015 mol). After stirring at room temperature for 10 min, 2-chloro-l-(chloromethyl)-4-methoxybenzene (3.83 g, 0.02 mol) was added. The mixture was stirred for 2 hr at 60 °C and then filtered to remove the K2CO3. The filtrate was concentrated to dryness under vacuum. The crude product was purified by silica gel column to give 8-bromo-l-(2-chloro-4- methoxybenzyl)-3-methyl-7-(2-methylallyl)-lH-purine-2,6(3H,7H)-dione 31A (2.92 g, 64.6% yield). LCMS: (ES+): m/z 453 [M]⁺

To a solution containing 8-bromo-l-(2-chloro-4-methoxybenzyl)-3-methyl-7-(2-methylallyl)-lH- purine-2,6(3H,7H)-dione (1.2 g, 2.65 mmol) and dry THF (20 mL) cooled to 0 °C was added borane-THF complex (4 mL) via a syringe dropwise. The resulting mixture was stirred at 0°C for 4 hr. Sodium hydroxide (4.0 M, 3 mL) and hydrogen peroxide (3 mL) were added sequentially. The mixture was stirred for 12 hr. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with water and brine, dried over sodium sulfate and filtered. After evaporation of the solvent under vacuum, the residue was purified with silica gel chromatography to give 8-bromo-l-(2-chloro-4- methoxybenzyl)-7-(3-hydroxy-2-methylpropyl)-3-methyl-lH-purine-2,6(3H,7H)-dione 31B (743 mg, 59.5% yield). LCMS: (ES+): m/z 471 [M]⁺

To a solution containing 8-bromo-l-(2-chloro-4-methoxybenzyl)-7-(3-hydroxy-2-methylpropyl)- 3-methyl-lH-purine-2,6(3H,7H)-dione (443 mg, 0.94 mmol), TEA (0.4 mL, 2.82 mmol) in DCM (10 mL) was added MsCl (323 mg, 1.5 mmol) at 0 ^°C. The reaction mixture was stirred at 0 ^°C for 2 hr, and then quenched with water. After extraction the organic phase was dried and concentrated in vacuo to give 3-(8- bromo-l-(2-chloro-4-methoxybenzyl)-3-methyl-2,6-dioxo-2, 3-dihydro- lH-purin-7(6H)-yl)-2- methylpropyl methanesulfonate 31C (468 mg) as a yellow solid which was used in the next step without further purification.

A solution of 3-(8-bromo-l-(2-chloro-4-methoxybenzyl)-3-methyl-2,6-dioxo-2,3-dihydro-lH- purin-7(6H)-yl)-2-methylpropyl methanesulfonate (468 mg), tert-butyl 3-aminophenylcarbamate (355 mg, 1.7 mmol) in NMP (5 mL) was irradiated in the microwave at 180 ^°C for 2 hr. Water was added and the mixture was extracted with EA. The organic layer was concentrated and purified by Prep-TLC using DCM/MeOH to give 9-(3-aminophenyl)-3-(2-chloro-4-methoxybenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[l,2-f]purine-2,4(lH,3H)-dione 31D (260 mg, 63.5% yield) as a white solid.

To a solution of 9-(3-aminophenyl)-3-(2-chloro-4-methoxybenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydro pyrimido[l,2-f]purine-2,4(lH,3H)-dione (160 mg, 0.33 mmol) in DCM (10 mL) was added BBr3 dropwise (0.99 mL, 0.99 mmol, 1M in CH2CI2) at -30 ^°C. The reaction mixture was stirred at -30 ^°C for 15 min. The reaction mixture was quenched with saturated aqueous NaHCCL solution and extracted with EtOAc three times. The combined organic phases were washed with brine and dried over anhydrous sodium sulfate, concentrated and purified by silica gel chromatography (2-6% MeOH gradient in CH2CI2) to afford of 9-(3-aminophenyl)-3-(2-chloro-4-hydroxybenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydropyrimido[l,2-f]purine-2,4(lH,3H)-dione 31E (31 mg, 20.0% yield).

To the mixture of 9-(3-aminophenyl)-3-(2-chloro-4-hydroxybenzyl)-l,7-dimethyl-6, 7,8,9- tetrahydro pyrimido[l,2-f]purine-2,4(lH,3H)-dione (42 mg, 0.14 mmol) and TEA (23 mg, 0.42 mmol) in DCM (5 mL) was added slowly a solution of acrylic anhydride (12 uL, 0.11 mmol) in DCM (3 mL) at -60 ^°C. After the reaction completed as monitored by TLC, the reaction solution was quenched with ice water and extracted with DCM (20 mL x 3). The organic layer was washed with water and brine, dried over Na2SC>4, filtered and the solvent was removed by evaporation. The crude product was purified by Prep- TLC plate to obtain N-(3-(3-(2-chloro-4-hydroxybenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8- hexahydropyrimido[l,2-f]purin-9(6H)-yl)phenyl)acrylamide (11.3 mg, 78.7%) as a white solid (Compound 31). LCMS: (ES+): m/z 521.1 [M]⁺.

Ή NMR (400 MHz, CDC1 ) d 8.08 (s, 1H), 7.35-7.31 (m, 3H), 7.09 (m, 1H), 6.89 (d, 7= 8.4 Hz, 1H), 6.80 (s, 1H), 6.58 (d, 7 = 8.0 Hz, 1H), 6.46 (J = 16.4 Hz, 1H), 6.30-6.27 (m, 1H), 5.80 (d, 7 = 10.4 Hz, 1H), 5.23 (s, 2H), 4.58-4.53 (m, 1H), 3.84-3.81 (m, 2H), 3.55 (m, 1H), 3.50 (s, 3H), 2,51 (br, 1H), 1.18 (d, 7 = 6.8 Hz, 3H).

Example 23: Gel shift assay to determine the activity of the exemplary compounds in modifying mutant KRAS (KRAS G12C) and wild type KRAS

[186] 200 mM compounds were incubated with 1 mM GDP-bound untagged KRAS (G12C or wild type) proteins at 25 °C for 18 hours in 40 pi reaction buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM MgC12, 1 mM DTT). The reactions were quenched with 10 pi SDS-PAGE sample loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol and 50 mM DTT) and then analyzed via SDS-PAGE using 4%-20% gradient polyacrylamide gel followed by Coomassie blue stain.

[187] Figures 1A and IB illustrate molecular weight shift of KRAS-G12C mutant and wild type proteins on SDS-PAGE after 18 hours of coincubation at 25 °C with exemplary Compounds 4, 5 (1A), 20 and 21 (IB) of the present disclosure respectively, indicative of covalent cysteine conjugation.

[188] Figures 2A and 2B depict the co-crystal structures of KRAS(G12C) in complex with exemplary Compounds 5 (A) and 21 (B) of the present disclosure, respectively.

[189] Table 3 illustrates molecular weight shift of KRAS-G12C mutant and wild type proteins on SDS- PAGE after 18 hours of coincubation at 25 °C with exemplary compounds of the present disclosure, indicative of covalent cysteine conjugation.

Table 3 Molecular weight shift of KRAS-G12C and wild type

[190] The many features and advantages of the present disclosure are apparent from the detailed specification, and thus it is intended by the appended claims to cover all such features and advantages of the present disclosure that fall within the true spirit and scope of the present disclosure. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the present disclosure to the exact construction and operation illustrated and described and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the present disclosure.

[191] Moreover, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be used as a basis for designing other structures, methods, and systems for carrying out the several purposes of the present disclosure. Accordingly, the claims are not to be considered as limited by the foregoing description or examples.

Claims

WHAT IS CLAIMED IS:

1 A compound of Formula (1) or a pharmaceutically acceptable salt thereof:

wherein:

Xi is CRi or N;

X₂ is CR₂ or N;

X₃ is CR₃ or N;

X₄ is CR or N;

Xs is CRs or N each of Ri, R₂, R₃, R_t, and Rs is independently selected from hydrogen, halogen, hydroxyl group, -CN, C₁-C₅alkyl, Ci-C₅alkoxy, and C₁-C₅haloalkyl;

L is a linker of 1 to 3 carbon atoms in length, wherein one or more carbon atoms are optionally and independently replaced by a group selected from C(=0), O, N(R₉), S, CValkcnyl, C alkynyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the R_¾ C₂-alkcnyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 0, 1, 2, or 3 Rio;

R₇ is independently selected from hydrogen, C₁-C₅alkyl, and C₁-C₅haloalkyl, each of which is substituted with 0, 1, 2, or 3 Rio_;

X is selected from -CH₂-, -(CH₂)₂-, and -(CH₂)₃-, each of which is optionally substituted at one or more hydrogens with Ri ₁;

A is selected from cycloalkyl, aryl, heterocycle, and heteroaryl, each of which is substituted with 1, 2, or 3 Rg;

Rs is independently selected from a,b-unsaturated carbonyl derivative, carboxamide, C₁-C₅alkyl, C₁-C₅alkoxy, C₁-C₅haloalkyl, each of which is optionally substituted with 1, 2, or 3 substituents selected from halogen, hydroxyl, Ci-C₃alkyl, Ci-C₃alkoxy, Ci-C₃haloalkyl, -NHCH₃, -N(CH₃)₂, and -CN; each R₉ is independently selected from hydrogen, Ci-C₃alkyl, -C(=0)-(Ci-C₃alkyl), -C(=0)-0- (Ci-C₃alkyl), and -C(=0)-NH-(Ci-C₃alkyl), each of which is substituted with 0, 1, 2, or 3 Rio; each Rio is independently selected from halogen, hydroxyl, Ci-C₃alkyl, Ci-C₃alkoxy, Ci- C₃haloalkyl, -N(R₉)₂, and -CN; and each R is independently selected from Ci-C₃alkyl, Ci-C₃alkoxy, Ci-C₃haloalkyl, cycloalkyl, aryl, heterocycle, or heteroaryl, wherein the Ci-C₃alkyl, Ci-C₃alkoxy, Ci-C₃haloalkyl, cycloalkyl, aryl, heterocycle, and heteroaryl are each independently substituted with 1, 2, or 3 substituents selected from halogen, -NO2, hydroxyl, -NH₂, -NHCH₃, -N(CH₃)₂, -CN, Ci-C alkyl, Ci-C alkoxy, Ci-C haloalkyl, C - C₆cycloalkyl, C3-C6heterocyclyl, C6-Ci2aryl, and C₆-C 12 heteroaryl; wherein each hydrogen atom is independently and optionally replaced by a deuterium atom.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein Xi is CRi, X₂ is CR₂, X₃ is CR , X₄ is CR₄, and X₅ is CR₅.

3. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein Ri, R₂, R₃, R₄, and R₅ are each independently selected from hydrogen, halogen, hydroxyl group, haloalkyl and - CN.

4. The compound or pharmaceutically acceptable salt thereof according to claim 3, wherein Ri, R₂, R₃, and R₅ are each independently selected from H, Cl, F, -CF₃, -OH, and -CN.

5. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein Ri is F or Cl.

6. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein Ri is - CN.

7. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein Ri is -

CF₃.

8. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein Ri is -OH.

9. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R2 is F.

10. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R3 is Cl.

11. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R5 is F.

12. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein Ri and

R3 are each Cl.

13. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R₂, R₃, R₄, and R₅ are each H.

14. The compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R₂, R₄, and R5 are each H.

15. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R7 is selected from hydrogen and C₁-C₃alkyl.

16. The compound or pharmaceutically acceptable salt thereof according to claim 15, wherein R7 is selected from methyl and ethyl.

17. The compound or pharmaceutically acceptable salt thereof according to claim 16, wherein R7 is methyl.

18. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein X is selected from -CH2- and -(ϋ¾)2-, each of which is optionally substituted at one or more hydrogens with R1 1.

19. The compound or pharmaceutically acceptable salt thereof according to claim 18, wherein X is - CH2-.

20. The compound or pharmaceutically acceptable salt thereof according to claim 18, wherein X is - (CH₂)2-.

21. The compound or pharmaceutically acceptable salt thereof according to claim 18, wherein Rn is an optionally substituted C₁-C₃alkyl.

22. The compound or pharmaceutically acceptable salt thereof according to claim 21, wherein the Ci- Csalkyl is substituted with 1, 2, or 3 substituents selected from halogen, -NH2, -NHCH3, -N(CH3)2, -CN, - NO2, hydroxyl, C₁-C₃alkyl, C₁-C₃alkoxy, Ci-Cihaloalkyl, C3-C6cycloalkyl, C3-C6heterocyclyl, C6-Ci2aryl, and C6-C12 heteroaryl.

23. The compound or pharmaceutically acceptable salt thereof according to claim 21, wherein the Ci- Csalkyl is optionally substituted with 1 substituent selected from C^Csheterocyclyl and -N(CH3)2·

24. The compound or pharmaceutically acceptable salt thereof according to claim 23, wherein Rn is

25. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein Rn is - CH₃.

26. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein Rn is

27. The compound or pharmaceutically acceptable salt thereof according to claim 24, wherein Rn is

28. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein A is selected from 4- to 6-member cycloalkyl, 5- to 6-member aryl, 4- to 6-member heterocycle, and 5- to 6-member heteroaryl, each of which is substituted with 1, 2, or 3 groups independently selected from a,b-unsaturated carbonyl derivative, carboxamide, C₁-C₅alkyl, C₁-C₅haloalkyl, and optionally substituted C₁-C₅alkoxy.

29. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein A is a substituted 6-member aryl.

30. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the a,b-unsaturated carbonyl derivative is selected from acryloyl, acrylamide, and alkylacrylamide.

31. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the carboxamide is selected from

.

32. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the 4- member cycloalkyl is selected from

33. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the 4- member heterocycle is azetidine, wherein the azetidine is substituted with an a,b- unsaturated carbonyl derivative.

34. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the 6-member heterocycle is piperidine, wherein the piperidine is substituted with an a,b- unsaturated carbonyl derivative.

35. The compound or pharmaceutically acceptable salt thereof according to claim 34, wherein the piperidine is selected from

, and

36. The compound or pharmaceutically acceptable salt thereof according to claim 28, wherein the 5- member heteroaryl is a substituted pyrazole.

37. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R₈ is a substituted a,b-unsaturated carbonyl derivative.

38. The compound or pharmaceutically acceptable salt thereof according to claim 37, wherein the a,b-unsaturated carbonyl derivative is selected from acryloyl, acrylamide, and alkylacrylamide.

39. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R₈ is a carboxamide.

40. The compound or pharmaceutically acceptable salt thereof according to claim 39, wherein the carboxamide is an acetamide.

41. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R₈ is an optionally substituted C₁- C₃ alkyl.

42. The compound or pharmaceutically acceptable salt thereof according to claim 41, wherein the C₁- C₃ alkyl is selected from methyl and ethyl.

43. The compound or pharmaceutically acceptable salt thereof according to claim 42, wherein the C₁- C₃ alkyl is methyl.

44. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R₈ is an optionally substituted C₁- C₃ alkoxy.

45. The compound or pharmaceutically acceptable salt thereof according to claim 44, wherein the substituted C₁- C₃ alkoxy is

46. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R₈ is selected from:

47. The compound or pharmaceutically acceptable salt thereof according to claim 46, wherein R₈ is selected from:

48. The compound or pharmaceutically acceptable salt thereof according to claim 47, wherein R₈ is

49. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the

group is selected from

50. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the

51. The compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein L is selected from -CH₂-, -CH₂-CH₂-, or -CH₂-CH₂-CH₂-.

52. The compound according to claim 1, wherein the compound is chosen from: N-(4-(3-(2-chloro-6-fluorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide;

N-(3-(3-(2-chloro-6-fluorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide;

3-(2-chloro-6-fluorobenzyl)-1-methyl-9-(m-tolyl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)- dione;

3-(2-chlorobenzyl)-9-(3-methoxyphenyl)-1,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-

2,4(lH,3H)-dione;

N-(3-(3-(2-chlorobenzyl)-1,7-dimethyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide;

N-(3-(3-(2-cyanobenzyl)-1,7-dimethyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide;

N-((lR,3R)-3-(3-(2-chlorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,6,7-hexahydro-8H-imidazo[2,l-f]purin-8- yl)cyclobutyl)acetamide;

N-((lR,3R)-3-(3-(2-chlorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,6,7-hexahydro-8H-imidazo[2,l-f]purin-8- yl)cyclobutyl)acrylamide;

N-((lR,3R)-3-(3-(2-chlorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)cyclobutyl)acetamide;

N-((lR,3R)-3-(3-(2-chlorobenzyl)-1-methyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)cyclobutyl)acrylamide; N-((lR,3R)-3-(3-(2-chlorobenzyl)-l-methyl-2,4-dioxo-1,2,3,4,6,7,8,9-octahydro-10H-[l,3]diazepino[2,l- f] purin- 10-y l)cy clobutyl) acrylamide;

3-(2-chlorobenzyl)-9-(3-(2-(dimethylamino)ethoxy)phenyl)-l-methyl-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

9-(l-acryloylpiperidin-4-yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

9-(l-acryloylpiperidin-4-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-

2,4(lH,3H)-dione;

9-(l-acryloylazetidin-3-yl)-3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

9-(l-acryloylazetidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l-f]purine-

2,4(lH,3H)-dione;

9-((R)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

9-((S)-l-acryloylpiperidin-3-yl)-3-(2-chlorobenzyl)-l,7-dimethyl-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

3-(2-chloro-6-fluorobenzyl)-l,7-dimethyl-9-(l-methyl-lH-pyrazol-4-yl)-6,7,8,9-tetrahydropyrimido[2,l- f]purine-2,4( 1 H,3H)-dione;

3-(2-chlorobenzyl)-l,7-dimethyl-9-(l-methyl-lH-pyrazol-4-yl)-6,7,8,9-tetrahydropyrimido[2,l-f]purine-

2,4(lH,3H)-dione;

N-(3-(3-(2-chlorobenzyl)-7-((dimethylamino)methyl)-l -methyl-2, 4-dioxo- 1,2, 3, 4, 7, 8- hexahydropyrimido [2, 1 -f]purin-9(6H)-yl)phenyl)acrylamide;

N-(3-(3-(2-chloro-3-fluorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-

9(6H)-yl)phenyl)acrylamide;

N-(3-(l,7-dimethyl-2,4-dioxo-3-(2-(trifluoromethyl)benzyl)-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-

9(6H)-yl)phenyl)acrylamide;

N-(3-(3-(2-chlorobenzyl)-l -methyl-2, 4-dioxo-7-(pyrrolidin-l-ylmethyl)- 1,2, 3, 4,7,8- hexahydropyrimido [2, 1 -f]purin-9(6H)-yl)phenyl)acrylamide;

N-(3-(3-(2,4-dichlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)acrylamide;

N-(3-(3-(2,4-dichlorobenzyl)-7-((dimethylamino)methyl)-l -methyl-2, 4-dioxo- 1,2, 3, 4,7,8- hexahydropyrimido [2, 1 -f]purin-9(6H)-yl)phenyl)acrylamide;

N-(2-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)benzyl)acrylamide;

N-(3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)benzyl)acrylamide;

N-(3-(3-(2-chlorobenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,l-f]purin-9(6H)- yl)phenyl)-N-methylacrylamide; 3-(2,4-dichloro-5-methylbenzyl)-1-methyl-9-(l-methyl-lH-pyrazol-4-yl)-7-methylene-6,7,8,9- tetrahydropyrimido[2,l-f]purine-2,4(lH,3H)-dione; and

N-(3-(3-(2-chloro-4-hydroxybenzyl)-l,7-dimethyl-2,4-dioxo-l,2,3,4,7,8-hexahydropyrimido[2,1-f]purin-

9(6H)-yl)phenyl)acrylamide; or a pharmaceutically acceptable salt thereof, wherein each hydrogen is independently and optionally replaced by a deuterium.

53. The compound of Formula (1) according to claim 1, wherein the compound is a compound of Formula (1A)

54. The compound of Formula (1) according to claim 1, wherein the compound is a compound of Formula (IB)

55. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is an irreversible K-Ras inhibitor.

56. A pharmaceutical composition comprising at least one compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 55 and a pharmaceutically acceptable carrier.

57. The pharmaceutical composition according to claim 56, wherein the compound or pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.

58. A method of treating cancer in a subject in need thereof, comprising administering to said subject a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 55 or a pharmaceutical composition according to claim 56 or 57.

59. The method according to claim 58, wherein the cancer is associated with K-Ras wild-type or mutations.

60. The method according to claim 59, wherein the cancer is associated with K-Ras(G12C).

61. The method according to claim 58, wherein the cancer is selected from breast cancer, lung cancer, pancreatic cancer, colorectal cancer, gall bladder cancer, thyroid cancer, bile duct cancer, ovarian cancer, endometrial cancer, prostate cancer, and esophageal cancer.

62. The method according to claim 59, wherein the compound or the pharmaceutically acceptable salt thereof selectively binds to K-Ras(G12C) without any effect on the K-Ras wild type.

63. A method of modulating an activity of a K-Ras protein, comprising contacting a K-Ras protein with an effective amount of a compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 55.

64. The method according to claim 63, wherein the K-Ras protein is K-Ras(G12C).

65. A use of a compound or pharmaceutically acceptable salt thereof according to any one of claims

1 to 55 or a pharmaceutical composition according to claim 56 or 57 in the manufacture of a medicament.

66. The use according to claim 65, wherein the medicament is for the treatment of cancer, wherein the cancer is associated with K-Ras wild-type or mutations.