Novel tetraheterocycle compounds

Abstract

The present invention relates to novel tetraheterocyclic compounds, and more particularly to novel tetraheterocyclic compounds, isomers thereof, useful as KRAS protein inhibitors, and pharmaceutical compositions containing the same for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to novel tetraheterocyclic compounds, and more particularly to novel isomeric tetraheterocyclic compounds useful as KRAS protein inhibitors, and pharmaceutical compositions containing the same for treating cancer. [Background technology]

[0002] RAS genes are involved in signal transduction within the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) pathways and are known to be oncogenes due to frequent mutations. The RAS gene family is classified as KRAS, NRAS, and HRAS, and these three genes encode four proteins: the splice variants K-Ras4A and K-Ras4B, and the N-Ras and H-Ras. K-Ras is the most frequently mutated isoform in Ras-induced cancers (86%), followed by N-Ras (11%) and H-Ras (3%) (Non-Patent Document 1). For example, oncogenic alterations in KRAS are observed in 15.95% of cancers, including pancreatic cancer, lung cancer, colon adenocarcinoma, colon-rectal cancer, and rectal adenocarcinomas (Non-Patent Document 2).

[0003] Gain-of-function missense mutations, mostly located at codons 12, 13, and 61, constitutively activate the RAS protein and are detected in various types of human cancer. 98% of tumor Ras mutations are found at the active site amino acid residues G12, G13, and Q61, and these mutations impair intrinsic and GAP-mediated GTP hydrolysis, resulting in abnormal activation of downstream signaling (Non-Patent Document 3). K-Ras G12 mutations (89%) are predominant in human cancers, followed by G13 mutations (9%) and Q61 mutations (1%). Codon 12 mutations include codon 12 Gly → Asp (G12D) (36%), codon 12 Gly → Val (G12V) (23%), and codon 12 Gly → Cys (G12C) (14%), with G12D being the most common mutation among codon 12 mutations. Mutations at codon 13 Gly → Asp (G13D) (7%) and codon 61 Gln → His (Q61H) (0.6%) were also observed (Non-Patent Document 1).

[0004] The well-known role of KRAS in malignant tumors and reports of KRAS mutations in various tumor types suggest that KRAS may also be an effective target for cancer therapy. The present inventors have developed novel KRAS inhibitors to complete the present invention. [Prior art documents] [Non-patent literature]

[0005] (Non-patent document 1) Scientific Reports 6(1):21949 (Non-patent document 2) J Cancer Metastasis Treat 2021;7:26 (Non-patent document 3) Cancer Biol Ther. 2006 August; 5(8): 928-932 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides novel tetraheterocyclic compounds, isomers, and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing the same for treating cancer, and production methods and intermediates for providing the same. [Means for solving the problem]

[0007] To achieve the above object, one aspect of the present invention provides a compound of Chemical Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof:

[0008] Another aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof.

[0009] Another aspect of the present invention provides processes for producing a compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof, and intermediates used therein. [Effects of the Invention]

[0010] The present invention provides novel tetraheterocyclic compounds useful as KRAS protein inhibitors, or stereoisomers, diastereomers, enantiomers, rotationally hindered isomers, solvates, isotopic variants, tautomers, or pharmaceutically acceptable salts thereof, and pharmaceutical compositions containing the same, which can exhibit KRAS protein inhibitory activity and can be effectively used in cancer treatment. DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art in the field related to the present invention. In addition, although preferred methods and samples are described in the present specification, similar or equivalent methods and samples are also included in the scope of the present invention.

[0012] Tetraheterocycle compounds

[0013] One aspect of the present invention provides a compound of Formula 1 below, or a stereoisomer, diastereomer, enantiomer, hindered rotational isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof: [ka] In the above formula, R 1 is hydroxy, halogen, C 1-3 Haloalkyl, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 1-3 optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; phenyl, pyridinyl, naphthyl, indazolyl, benzothiazolyl, or benzothiophenyl; R 2 is hydrogen or halogen; R 3 is hydrogen, C 1-3 Alkyl, C 3-6 Cycloalkyl, 4-10 membered heterocycle, or -O-(L) m -A1, where A1 is one or more R 7 and unsubstituted C 1-3 Alkyl, C 3-6 is a cycloalkyl, a 4-10 membered heterocycle, a 6-10 membered aryl, a 5-10 membered heteroaryl, or a 4-10 membered fused heteroaryl; L is R7 Substituted or unsubstituted C 1-3 Alkylene or C 3-8 is cycloalkylene; R 7 are each independently halogen, oxo (=0), =CH2, -OCF3, -OCHF2, amino, cyano, -N(C 1-3 alkyl)2, -NH(C 1-3 alkyl), substituted or unsubstituted C 1-3 Alkyl, substituted or unsubstituted C 1-3 Haloalkyl, substituted or unsubstituted C 1-3 Alkoxy, substituted or unsubstituted C 3-4 cycloalkyl, or a substituted or unsubstituted heterocycle; R 4 are each independently hydrogen, hydroxy, halogen, or C 1-3 Haloalkyl or C 1-3 Alkyl, C 1-3 is alkoxy; X is O, CH2 or NR 8 and; R 8 is hydrogen or R 9 C selectively substituted 1-3 times with 1-6 Alkyl, C 3-6 Cycloalkyl or C 3-6 is heterocycloalkyl; R 9 is, in each occurrence, independently oxygen, hydroxy, -C 1-4 Alkyl or -OC 1-4 is alkyl; Y is NH, O, S, SO, or SO; Z is CR 6 or N; R 5 is hydrogen, C 1-3 alkyl, cyano, or amino; R 6 is hydrogen, hydroxy, halogen, C 1-3 is alkyl; n1, n2, n3, and m are each an integer from 1 to 3; Heterocycle, heteroaryl, and fused heteroaryl each contain one or more N, S, or O heteroatoms.

[0014] In one embodiment, R 1 is hydroxy, halogen, C 1-3 Haloalkyl, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 1-3 optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; phenyl, naphthyl, benzothiazolyl, or benzothiophenyl; X is O, CH or NH; Y is also O.

[0015] In one embodiment, R 3 is -O-(L) m -A1, where A1 is one or more R 7 and each may be a substituted or unsubstituted 4-10 membered heterocycle or a 5-10 membered fused heteroaryl.

[0016] In one embodiment, R 1 is hydroxy, halogen, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 It is optionally unsubstituted or substituted with one or more substituents independently selected from cycloalkyl, amino, and cyano.

[0017] In claim 1, R 1 is hydroxy, halogen, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6optionally unsubstituted or substituted with one or more substituents independently selected from cycloalkyl, amino, and cyano; [ka] and; R 3 is -O-(L) m -A1, where A1 is one or more R 7 each of which is a substituted or unsubstituted 4-10 membered heterocycle or 5-10 membered fused heteroaryl; L is C 1-3 Alkylene or C 3-8 is cycloalkylene; R 7 are each independently halogen, oxo (=0), =CH2, -OCF3, -OCHF2, amino, cyano, C 1-3 Alkyl, C 1-3 Haloalkyl or C 1-3 is alkoxy; R 4 is hydrogen; X is O, CH or NH; Z is CR 6 or N; R 5 is hydrogen or C 1-3 is alkyl; R 6 is a halogen; A compound in which n1, n2, n3, and m are each an integer of 1 to 3.

[0018] In one embodiment, where Z is N, R 1 is hydroxy, halogen, C 1-3 Haloalkyl, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 1-3 optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; is phenyl or naphthyl; Z is CR 6 If R 1 is hydroxy, halogen, C 1-3 Haloalkyl, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 1-3 optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; Also benzothiazolyl or benzothiophenyl.

[0019] In one embodiment, R 3 is -O-(L) m -A2, where A2 is [ka] and they are halogens, C 1-3 Alkyl, C 1-3 substituted or unsubstituted with one or more substituents each independently selected from alkoxy, and =CH2; -(L) m - is methylene or [ka] It is also.

[0020] In one embodiment, R 1 is hydroxy, halogen, C 1-3 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 optionally unsubstituted or substituted with one or more substituents independently selected from cycloalkyl, amino, and cyano; [ka] and; R 2 is a halogen; R 3 is -O-(L) m -A1, where A1 is a halogen, C 1-3 Alkyl, C 1-3 substituted or unsubstituted with one or more substituents independently selected from alkoxy and =CH2; [ka] In this case, -(L) m - is methylene or [ka] and; R 4 is hydrogen; X is O, CH or NH; Y is O; Z is CH, C(C 1-3 alkyl), or N; R 5 is hydrogen or C 1-3 It is also alkyl.

[0021] In one embodiment, the compound of Formula 1 is any one selected from the group consisting of the following compounds, or a stereoisomer, diastereomer, enantiomer, hindered rotational isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof: [ka] .

[0022] Another aspect provides a pharmaceutical composition for treating cancer comprising, as an active ingredient, any one of the above-mentioned compounds, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof.

[0023] In one embodiment, the composition also exhibits KRAS protein inhibitory activity.

[0024] definition As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine, unless otherwise specified. The term "alkyl," as used herein, unless otherwise specified, refers to a straight-chain or branched saturated monovalent hydrocarbon radical.

[0025] As used herein, the term "alkylene" refers to (-CH-) n and includes, but is not limited to, methylene, ethylene, propylene, butylene, isobutylene, and the like.

[0026] As used herein, unless otherwise specified, the term "alkenyl" refers to a monovalent hydrocarbon group containing at least one carbon-carbon double bond, each of which may have an E or Z configuration. As used herein, unless otherwise specified, the term "alkynyl" refers to a monovalent hydrocarbon radical containing at least one carbon-carbon triple bond. The term "alkoxy," as used herein, unless otherwise specified, refers to a straight-chain or branched hydrocarbon residue linked through an oxygen.

[0027] The term "aryl," as used herein, unless otherwise specified, refers to a substituted or unsubstituted aromatic group, including monocyclic or bicyclic or higher cyclic substituted or unsubstituted aromatic groups, including unsaturated or partially saturated aryls, such as C 6-15 Aryl includes, but is not limited to, phenyl, biphenyl, naphthyl, toluyl, or naphthalenyl.

[0028] The term "heteroaryl," as used herein, unless otherwise specified, refers to a monocyclic or bicyclic or higher cyclic, substituted or unsubstituted aromatic group containing one or more heteroatoms selected from N, O, and S, and includes unsaturated or partially saturated heteroaryls, such as C 4-15 Heteroaryl may include, but is not limited to, morpholinyl, piperidinyl, pyrrolidinyl, or pyrrolizinyl.

[0029] As used herein, the term "fused heteroaryl" refers to an unsaturated or partially saturated, substituted or unsubstituted ring system in which the heteroaryl group is further connected in a fused manner to another aryl, heteroaryl, or heterocycloalkyl group. For example, C 8-20 Heteroaryl includes, for example, 9-, 10-, 11-, 12-, 13-, 14-, or 15-membered benzofused heteroaryl groups. For example, fused heteroaryl can be a 5+5-, 5+6-, 5+7-, 6+6-, or 6+7-membered fused ring system. Fused heteroaryl also includes, but is not limited to, pyrrolidine, benzothiazole, benzthiazolinyl, benzothiophenyl, benzofuranyl, isobenzofuranyl, benzothionyl, indolyl, isoindolinyl, indazolinyl, benzimidazolinyl, benzoxazolinyl, benzisoxazolinyl, benzthiadiazolyl, benzoxadiazolinyl, benztriazolinyl, quinolinyl, isoquinolinyl, or quinazolinyl.

[0030] As used herein, the term "partially saturated" refers to containing at least one site of saturation, i.e., at least one single bond, within the aryl, heteroaryl, or fused heteroaryl ring as defined above. As used herein, the term "unsaturated" refers to containing no sites of saturation, i.e., no single bond, within the aryl, heteroaryl, or fused heteroaryl ring as defined above.

[0031] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a substituted or unsubstituted, saturated or partially unsaturated, monocyclic, bicyclic, or polycyclic hydrocarbon ring, including bridged cycloalkyls, fused cycloalkyls, and spirocycloalkyls. For example, C 3-10 Cycloalkyl or C 3-6 Cycloalkyl includes, but is not limited to, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. As used herein, the term "cycloalkylene" refers to a divalent radical derived from a cycloalkene.

[0032] As used herein, unless otherwise specified, the term "heterocycle" or "heterocycloalkyl" refers to a substituted or unsubstituted, saturated or partially unsaturated, monocyclic, bicyclic, or polycyclic non-aromatic ring containing one or more heteroatoms selected from N, O, and S, including bridged heterocycles, fused heterocycles, and spiro heterocycles. The term includes monocyclic or polycyclic cyclic alkyls, such as C 4-15 Heterocycloalkyl includes, but is not limited to, piperidinyl, piperazinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydro-2H-pyranyl, imidazolidinyl, pyrrolidin-2-one, pyrrolidinyl, or pyrrolyl. Heterocycles can also be carbon-linked or heteroatom-linked groups. For example, a heterocycle linked to the base molecule through a nitrogen ring atom of the heterocycle can be, but is not limited to, N-morpholinyl, N-piperidinyl, N-pyrrolidinyl, or N-pyrrolyl.

[0033] As used herein, the term "fused heterocycle" or "fused cycloalkyl" refers to a substituted or unsubstituted ring system unless otherwise specified. Depending on the number of rings, fused cycloalkyls can be classified as bicyclic, tricyclic, tetracyclic, or higher polycyclic fused cycloalkyls. Fused cycloalkyls refer to polycyclic cycloalkyls in which each ring shares an adjacent pair of carbon atoms with another ring, and one or more rings may share one or more double bonds, but none of the rings has a completely conjugated π-electron system, e.g., C 3-20 For example, a bicyclic fused cycloalkyl, also referred to as a "bicycloalkyl," can be a 5+6 bicycloalkyl or a 6+6 bicycloalkyl depending on the number of atoms in each of the two fused rings.

[0034] The term "fused heterocycloalkyl," as used herein, unless otherwise stated, refers to a substituted or unsubstituted fused cycloalkyl containing one or more heteroatoms selected from N, O, and S, such as C 8-20 This may include heterofused cycloalkyl. For example, bicyclic fused heterocycloalkyl is also referred to as "heterobicycloalkyl" and may be a 5+6 fused heterobicycloalkyl or a 6+6 fused heterobicycloalkyl depending on the number of atoms in each of the two fused rings. For example, hexahydro-1H-pyrrolizine is also included, but is not limited to these.

[0035] As used herein, the term "stereoisomer" refers to a compound of the present invention or a salt thereof that has the same chemical or molecular formula but is optically or sterically different. As used herein, the term "enantiomeric isomer" refers to the various stereoisomers and geometric isomers that may exist for a compound according to the present invention.

[0036] The compounds described herein may possess asymmetric centers, geometric centers (e.g., double bonds), or both. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.

[0037] The compounds of Formula 1 according to one aspect of the present invention may have asymmetric carbon centers (chiral carbons) and therefore may exist as enantiomeric isomers (R or S isomers), racemates, diastereomers, or any mixtures thereof, and all such isomers and mixtures are included within the scope of the present invention.

[0038] The compounds described herein contain chiral centers, and unless otherwise specified, each chiral center is understood to be independently in the R or S configuration, or a mixture thereof. Accordingly, the compounds described herein include optical isomers enriched or resolved at any or all asymmetric atoms. Racemic mixtures of the R- and S-enantiomers, and enantiomerically enriched stereoisomeric mixtures containing not only the R- and S-enantiomers but also individual optical isomers, isolated or synthesized substantially free of their enantiomers or diastereomeric partners, are within the scope of the present technology.

[0039] Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. Methods for preparing optically active forms, such as by resolution of racemic forms, by synthesis from optically active starting materials, or by use of chiral auxiliaries, are well known in the art.

[0040] As used herein, the term "rotationally hindered isomer" refers to all stereoisomers that are separate from one another. These isomers arise when a single carbon-carbon bond in a compound cannot be freely rotated due to bulky substituents. This refers to stereoisomers formed from an asymmetric axis, and can result from restricted rotation around a single bond where the rotational barrier is high enough to allow differentiation of isomeric species, including the complete isolation of stable, non-interconverting diastereomeric or enantiomeric species. The compound according to one embodiment may have a high probability of occurring as a rotationally hindered isomer.

[0041] Compounds according to certain embodiments may also include "tautomeric" forms. Tautomeric forms arise from the swapping of a single bond with an adjacent double bond and the accompanying migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0042] Compounds according to one embodiment may include "isotopic variations." The present disclosure also includes isotopically labeled compounds that are identical to the compounds described herein except that one or more atoms are replaced by an atom having an atomic mass or mass number that differs from the atomic mass or mass number commonly found in nature (an "isotope"). Compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. Examples of isotopes that may be incorporated into compounds described herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, e.g., 2 H("D"), 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F and 36 For example, the compounds described herein may have one or more H atoms replaced by deuterium. Generally, reference or description of a particular element, such as hydrogen or H, is meant to include all isotopes of that element. For example, if an R group is defined as including hydrogen or H, deuterium and tritium are also included. Deuterated starting materials are readily available and can be adapted to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds. Many deuterated reagents and building blocks are commercially available from chemical supply companies such as Aldrich Chemical Co.

[0043] As used herein, the term "solvate" may include a molecular complex comprising a compound of Formula 1 and one or more pharmaceutically acceptable solvent molecules, such as ethanol or water. A complex in which the solvent molecule is water is also referred to as a "hydrate."

[0044] As used herein, the term "pharmaceutically acceptable salt" includes any salt that has low toxicity to the human body and does not adversely affect the biological activity and physicochemical properties of the parent compound.

[0045] KRAS protein inhibitors Kirsten Rat Sarcoma Oncogene Homolog (KRAS) is a GTPase that integrates extracellular signals into intracellular growth and survival signals. It is a member of the small GTPase family, which includes Ras, Rho, Rab, Arf, and Ran. It transmits signals from multiple receptor tyrosine kinases, particularly EGFR, via the KRAS GTPase cycle and then transmits signals to Raf and PI3K, regulating various processes, including cell proliferation.

[0046] The GTPase cycle is activated by the action of guanine nucleotide exchange factors (GEFs), which bind GTP to effector proteins, and then interacts with GTPase-activating proteins (GAPs) to become inactivated by binding GDP.

[0047] In addition to the KRAS GTP / GDP binding site, switch I / II, which is involved in the protein structural change involved in GTP / GDP binding, has been reported, proving the possibility of inhibiting the GTPase cycle.

[0048] The relationship between KRAS and cancer has been reported, and although there have been various attempts to inhibit KRAS activity, its potential as a direct drug target is low. However, the approval of sotorasib and adagrasib for non-small cell lung cancer confirmed the possibility of drug development.

[0049] The compounds disclosed herein are novel KRAS inhibitor compounds that inhibit KRAS, including, but not limited to, at least one mutation in any one of KRAS codons 12, 13, and 61, such as KRAS G12D, KRAS G12V, KRAS G12C, KRAS G13D, and KRAS Q61H mutations.

[0050] Pharmaceutical Compositions One aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising as an active ingredient a compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof.

[0051] In one embodiment, the composition may comprise a therapeutically effective amount of a compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof.

[0052] In one embodiment, the composition may further comprise another therapeutic agent in addition to the compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof. In one embodiment, the composition may further comprise a pharmaceutically acceptable carrier or excipient.

[0053] In one embodiment, the composition comprises a compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof, or any one of other therapeutic agents, in the range of 0.005% to 100%, with the remainder being a pharmaceutically acceptable carrier or excipient.

[0054] In one embodiment, the compound of Formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, may be used for the treatment of cancer.

[0055] In one embodiment, the compound of formula 1, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, may exhibit KRAS protein inhibitory activity.

[0056] Route of administration and dosage form The compounds and pharmaceutical compositions of the presently disclosed subject matter may be provided in any suitable administration route and dosage form acceptable in the pharmaceutical arts.

[0057] Manufacturing method The compounds of the present disclosure can be synthesized using organic synthesis techniques known to those of ordinary skill in the relevant art. Some compounds and / or intermediates of the present disclosure are commercially available, known in the literature, or can be prepared by those of ordinary skill in the art by selecting an appropriate synthesis method from known organic synthesis techniques. Some compounds of the present disclosure can be synthesized using the reaction schemes, examples, or intermediates described herein. Those of ordinary skill in the art will recognize that the reaction times, number of equivalents of reagents, and / or temperatures can be varied from the synthesis methods described herein, and that different workup and / or purification techniques can be used. The structures of the synthesized compounds may be verified by methods known to those of ordinary skill in the art, such as nuclear magnetic resonance (NMR) spectroscopy and / or mass spectroscopy.

[0058] General information Unless otherwise stated, reagents and solvents obtained from commercial suppliers were used without purification or drying. 1 H NMR was recorded using a Broker 400 MHz and 500 MHz, with TMS used as the internal standard. LCMS analysis was performed on a Waters UPLC equipped with an SQD-2 mass detector (single quadruple), and HPLC analysis was performed on an Acquit UPLC H CLASS (WATERS).

[0059] Numerical values ​​described herein are considered to include the meaning of "about" even if not explicitly stated. As used herein, the term "about" means within 5%, preferably within 1% to 2%, of a given value or range. Numerical ranges expressed herein using the term "to" include ranges that include the numerical values ​​stated before and after the term "to" as the lower and upper limits, respectively. All references, publications, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0060] The present invention will be explained in more detail below with reference to the following synthetic reaction schemes, examples, and test examples, which are intended to aid in understanding the present invention and are not intended to limit the scope of the present invention in any way. Synthesis reaction scheme of Example 1 [ka]

[0061] Example 1 Preparation of 5-ethyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol Step 1) Preparation of Intermediate 2 of Example 1: 2,6-dichloro-3-fluoropyridin-4-amine

[0062] To a stirred solution of 2,6-dichloropyridin-4-amine (Intermediate 1 in Example 1 (25.0 g, 154 mmol) in a solvent mixture of MeOH and water (1:1, 500 mL, 20 vol) was added Selectfluoro (60.1 g, 169 mmol) at room temperature, and the reaction mixture was heated to 45° C. and stirred for 16 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with water and extracted with ethyl acetate. The separated organic layer was dried over anhydrous NaSO, filtered, and evaporated under reduced pressure to give the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography eluting with 2% ethyl acetate in petroleum ether (PET ether) to give 2,6-dichloro-3-fluoropyridin-4-amine (Intermediate 2 in Example 1) (20.0 g, 72% yield) as a white solid. 1 H NMR(400 MHz, CDCl3) δ 4.52(brs, 2H), 6.62(d, J = 5.2 Hz, 1H). MS(LCMS): 180.96 m / z [M+H].

[0063] Step 2) Preparation of Intermediate 3 of Example 1: tert-butyl (tert-butoxycarbonyl) (2,6-dichloro-3-fluoropyridin-4-yl)carbamate

[0064] To a stirred solution of 2,6-dichloro-3-fluoropyridin-4-amine (Intermediate 2 from Example 1) (1.00 g, 2.63 mmol) in THF (10 mL, 10 vol) were added DMAP (0.06 g, 0.26 mmol) and Boc anhydride (1.10 mL, 5.26 mmol) at room temperature, and the reaction mixture was heated to 60° C. and maintained for 6 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate. The separated organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 1% ethyl acetate in petroleum ether to give tert-butyl (tert-butoxycarbonyl) (2,6-dichloro-3-fluoropyridin-4-yl)carbamate (Intermediate 3 of Example 1) (0.50 g, 50% yield). 1 H NMR(400 MHz, CDCl3) δ 1.47(s, 18H), 7.15(d, J = 4.40Hz, 1H). Step 3) Preparation of Intermediate 4 of Example 1: tert-butyl 4-((tert-butoxycarbonyl)amino)-2,6-dichloro-5-fluoronicotinate

[0065] To a stirred solution of tert-butyl (tert-butoxycarbonyl) (2,6-dichloro-3-fluoropyridin-4-yl)carbamate (Intermediate 3 of Example 1) (28.0 g, 73.7 mmol) in THF (280 mL, 10 vol) was added LDA (1 M in THF) (147 mL, 147 mmol) at −78° C. and stirred for 30 minutes, followed by the addition of Boc anhydride (14.4 g, 66.3 mmol). The reaction mixture was stirred at −78° C. for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and extracted with ethyl acetate. The separated organic layer was dried over anhydrous NaSO, filtered, and evaporated under reduced pressure to give the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 5% ethyl acetate in petroleum ether to give tert-butyl 4-((tert-butoxycarbonyl)amino)-2,6-dichloro-5-fluoronicotinate (Intermediate 4 of Example 1) (26.0 g, 92% yield) as an off-white solid. 1H NMR(400 MHz, CDCl3) δ 1.50(s, 9H), 1.62(s, 9H), 7.21(brs, 1H). MS(LC-MS): 381.17 m / z [M+H]. Step 4) Preparation of Intermediate 5 of Example 1: 4-amino-2,6-dichloro-5-fluoronicotinic acid

[0066] To a stirred solution of tert-butyl 4-((tert-butoxycarbonyl)amino)-2,6-dichloro-5-fluoronicotinate (Intermediate 4 in Example 1) (26.0 g, 68.4 mmol) in 1,4-dioxane (130 mL, 5 vol) at 0° C. was added concentrated HCl (130 mL, 5 vol), and the reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, washed with DCM, and filtered to give crude compound 4-amino-2,6-dichloro-5-fluoronicotinic acid (Intermediate 5 in Example 1) (18.0 g, crude compound) as a white solid. The crude compound was used directly in the next step without further purification. 1 H NMR(400 MHz, CDCl3) δ 5.71(brs, 2H), 7.31(brs, 1H). MS(LC-MS): 224.82 m / z [M+H]. Step 5) Preparation of Intermediate 6 of Example 1: 5,7-dichloro-8-fluoro-2-mercaptopyrido[4,3-d]pyrimidin-4(3H)-one

[0067] A solution of 4-amino-2,6-dichloro-5-fluoronicotinic acid (Intermediate 5 in Example 1) (18.0 g, 80.3 mmol) in SOCl (540 mL, 30 vol) was heated to 50 °C and stirred for 8 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in acetone (20 mL), to which NHSCN (1.0 g in 20 mL acetone) was added dropwise at room temperature and stirred for 1 hour. Upon completion, the reaction mixture was diluted with water, filtered, washed with water, and dried under high vacuum to give crude compound 5,7-dichloro-8-fluoro-2-mercaptopyrido[4,3-d]pyrimidin-4(3H)-one (Intermediate 6 in Example 1) (10.0 g, 49% yield) as an off-white solid. The compound was used directly in the next step without further purification. 1H NMR(400 MHz, CDCl3) δ 13.31(brs, 1H), 12.91(s, 1H). MS(LC-MS): 265.94 m / z [M+H]. Step 6) Preparation of Intermediate 7 of Example 1: 5,7-dichloro-8-fluoro-2-(methylthio)pyrido[4,3-d]pyrimidin-4(3H)-one

[0068] To a stirred solution of 5,7-dichloro-8-fluoro-2-mercaptopyrido[4,3-d]pyrimidin-4(3H)-one (Intermediate 6 in Example 1) (10.0 g, 37.7 mmol) in MeOH (1500 mL, 150 vol) was added 0.1 M NaOH (1500 mL, 150 vol) and MeI (2.50 mL, 41.5 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was diluted with water (500 mL) and acidified with concentrated HCl to approximately pH 6 to obtain a solid. The solid was filtered, washed with water, and dried under high vacuum to obtain crude compound 5,7-dichloro-8-fluoro-2-(methylthio)pyrido[4,3-d]pyrimidin-4(3H)-one (Intermediate 7 in Example 1) (8.0 g, crude compound) as a light brown solid. The crude compound was used directly in the next step without further purification. 1 H NMR(400 MHz, CDCl3) δ 2.60(s, 3H), 13.33(s, 1H). MS(LC-MS): 279.95 m / z [M+H].

[0069] Step 7) Preparation of Intermediate 9 of Example 1: tert-butyl (S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate

[0070] To a stirred solution of 5,7-dichloro-8-fluoro-2-(methylthio)pyrido[4,3-d]pyrimidin-4(3H)-one (Intermediate 7 from Example 1) (8.00 g, 28.7 mmol) in THF (80 mL, 10 vol) was added NaH (4.90 g, 129 mmol) at 0°C. After stirring for 30 minutes, tert-butyl (S)-3-(2-hydroxyethyl)piperazine-1-carboxylate (7.90 g, 34.4 mmol) was added at room temperature, and the reaction mixture was stirred at room temperature for 2 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with ice-cold water and extracted with ethyl acetate. The separated organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to give the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 5% methanol in DCM to give tert-butyl (S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 of Example 1) (6.0 g, 44% yield) as a brown solid. 1 H NMR (400 MHz, DMSO-d6): δ 1.42(s, 9H), 2.06(brs, 2H), 2.42(s, 3H), 3.04(d, J = 10.1Hz, 3H), 3.46(brs, 2H), 4.02(brs, 2H), 4.27(brs, 1H), 4.52(d, J = 8.8, 1H), 9.45(brs, 1H). MS(LC-MS): 474.37 m / z [M+H].

[0071] Step 8) Preparation of Intermediate 10 of Example 1: tert-butyl (S)-2-chloro-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0072] To a stirred solution of tert-butyl (S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 from Example 1) (6.00 g, 12.7 mmol) in DCM (60 mL, 10 vol) was added DIPEA (32.9 mL, 190 mmol) at 0° C. and stirred for 10 minutes, followed by the addition of BOP-Cl (11.6 g, 45.6 mmol) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 5 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and extracted with DCM. The separated organic layer was washed with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give the crude compound. The crude compound was purified by preparative HPLC (prep-HPLC) to give tert-butyl (S)-2-chloro-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 10 of Example 1) (0.80 g, 14% yield) as a brown solid. 1 H NMR(400 MHz, DMSO-d6): δ 1.42(s, 9H), 1.97-2.10(m, 2H), 2.54(s, 3H), 3.07-3.22(m, 2H), 3.55(d, J = 5.60 Hz, 1H), 3.78(d, J = 12.8Hz, 1H), 3.86-3.91(m, 1H), 3.97(d, J = 12.8 Hz, 1H), 4.22(t, J = 10.8Hz, 1H), 4.46(dd, J = 12.8, 4.8 Hz, 1H), 4.66-4.76(m, 1H). MS(LC-MS): 456.37 m / z [M+H].

[0073] Step 9) Preparation of Intermediate 11 of Example 1: tert-butyl (S)-2-chloro-1-fluoro-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0074] To a stirred solution of tert-butyl (S)-2-chloro-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 10 from Example 1) (0.40 g, 0.88 mmol) in a mixture of solvent ACN and water (2:1, 48 mL) at 0°C, oxone (0.65 g, 1.05 mmol) was added and the reaction mixture was stirred for 20 min at room temperature. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give crude tert-butyl (S)-2-chloro-1-fluoro-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 11 of Example 1) (0.34 g, crude compound) as an off-white solid. MS (LCMS): 488.32 m / z [M+H].

[0075] Step 10) Preparation of Intermediate 13 of Example 1: tert-butyl (S)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0076] To a stirred solution of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (0.01 g, 0.62 mmol) in THF (2.0 mL, 10 vol) was added NaH (0.031 g, 0.82 mmol) at 0° C. and stirred for 30 minutes, followed by the addition of tert-butyl (S)-2-chloro-1-fluoro-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 11 from Example 1) (0.20 g, 0.41 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was diluted with water and extracted with ethyl acetate. The separated organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 2% methanol in DCM to obtain tert-butyl (S)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 13 of Example 1) (0.02 g, 9% yield) as an off-white solid. MS (LC-MS): 567.44 m / z [M+H].

[0077] Step 11) Preparation of Intermediate 15 of Example 1: tert-butyl (S)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0078] In a mixture of solvent 1,4-dioxane and water (2.0 mL, 10 vol), tert-butyl (S)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8 To a stirred solution of 2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate 13 of Example 1) (0.20 g, 0.35 mmol) and 2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Intermediate 14 of Example 1) (0.19 g, 0.53 mmol) was added CsCO (0.34 g, 1.06 mmol) at room temperature and degassed under N gas for 10 minutes. Pd(dppf)Cl·DCM (0.028 g, 0.03 mmol) was added at room temperature, and the reaction mixture was heated to 80 °C and stirred for 16 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and extracted with ethyl acetate. The separated organic layer was washed with brine, dried over anhydrous NaSO, filtered, and evaporated under reduced pressure to obtain the crude compound. The crude compound was purified by preparative HPLC to give tert-butyl (S)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 15 of Example 1) (0.04 g, 15% yield) as a pale yellow solid. MS (LC-MS): 765.71 m / z [M+H].

[0079] Step 12) Example 1: Preparation of 5-ethyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol

[0080] To a stirred solution of tert-butyl (S)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 15 from Example 1) (0.04 g, 0.05 mmol) in DCM (1.2 mL, 30 vol) was added 4 M HCl in 1,4-dioxane (0.80 mL) at 0° C. and the reaction mixture was allowed to warm to room temperature and stirred for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain a crude compound. The crude compound was purified by preparative HPLC to obtain 5-ethyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol (Example 1) (0.006 g, 19% yield) as an off-white solid.

[0081] 1H NMR(400 MHz, DMSO-d6) δ 0.76(t, J = 7.2 Hz, 2H), 0.85(t, J = 7.2 Hz, 1H), 1.75-1.79(m, 2H), 1.79-1.81(m, 2H), 1.99(brs, 2H), 2.05(brs, 1H), 2.12-2.28(m, 2H), 2.61(d, J = 12.8Hz, 1H), 2.82-2.86(m, 2H), 2.93(brs, 1H), 3.02(s, 1H), 3.09(d, J = 4.12 Hz, 4H), 3.74-3.86(m, 1H), 3.98 (d, J = 10.26 Hz, 1H), 4.02-4.20 (m, 2H), 4.43-4.48 (m, 1H), 5.03 (t, J = 12.8 Hz, 1H), 5.21 & 5.35 (brs, 1H), 6.91 & 7.11 (d, J = 2.50 Hz, 1H), 7.28-7.30 (m, 1H), 7.31 (d, J = 8.06 Hz, 1H), 7.74 (dd, J = 8.88, 5.88 Hz, 1H), 9.92 (brs, 1H). NH and -OH protons were not visible by 1H-NMR. MS (LC-MS): 621.40 m / z [M+H].

[0082] Synthesis reaction scheme of Example 2 [ka]

[0083] Example 2 Preparation of 5-ethynyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol

[0084] Step 1) Preparation of Intermediate 2 of Example 2: tert-butyl (S)-3-(2-((2-(ethylthio-8-fluoro-7-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-4-iodo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate

[0085] In a mixture of solvent 1,4-dioxane and water (4:1, 2.5 mL), tert-butyl (S)-3-(2-((7-chloro-2-(ethylthio-8-fluoro-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 of Example 1) (0.10 g, 0.20 mmol) and ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5, To a stirred solution of 5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane (Intermediate 1 of Example 2) (0.13 g, 0.25 mmol), KPO (0.13 g, 0.62 mmol) was added at room temperature and degassed under N gas for 10 minutes. Ruphos-Pd-G (0.02 g, 0.02 mmol) was added at room temperature, and the reaction mixture was heated to 90 °C. The mixture was stirred for 3 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and extracted with ethyl acetate (twice). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to give the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 5% methanol in DCM to give tert-butyl (S)-3-(2-((2-(ethylthio-8-fluoro-7-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 2 of Example 2) (0.35 g, total yield of 4 batches (4 x 100 mg), crude compound) as a pale yellow solid. MS (LC-MS): 838.54 m / z [M+H].

[0086] Step 2) Preparation of Intermediate 3 of Example 2: tert-butyl (S)-12-(ethylthio-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0087] To a stirred solution of tert-butyl (S)-3-(2-((2-(ethylthio-8-fluoro-7-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 2 from Example 2) (0.15 g, 0.28 mmol) in DCM (3.0 mL, 20 vol) was added BOP-Cl (0.254 g, 1.00 mmol) and DIPEA (0.70 mL, 4.04 mmol) (3.0 mL, 20 vol) at room temperature and the reaction mixture was heated to 50° C. and stirred for 5 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 5% MeOH in DCM to obtain tert-butyl (S)-12-(ethylthio-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 3 of Example 2) (0.13 g, overall yield for two batches, 57% yield) as a brown solid. MS (LCMS): 820.49 m / z [M+H].

[0088] Step 3) Preparation of Intermediate 4 of Example 2: tert-butyl (S)-12-(ethylsulfonyl)-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0089] To a stirred solution of tert-butyl (S)-12-(ethylthio-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 3 from Example 2) (0.13 g, 0.16 mmol) in a mixture of solvents ACN and water (2:1, 15.6 mL) was added oxone (0.24 g, 0.79 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC for 2 hours. After completion of the reaction, the reaction mixture was diluted with water (10 The resulting mixture was diluted with 1 mL of ethyl acetate (2×20 mL) and the combined organic layer was washed with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give crude tert-butyl (S)-12-(ethylsulfonyl)-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 4 of Example 2) (0.135 g, crude compound) as a pale yellow solid. The crude compound was used directly in the next step without further purification. MS (LC-MS): 852.61 m / z [M+H].

[0090] Step 4) Preparation of Intermediate 6 of Example 2: tert-butyl (S)-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0091] To a stirred solution of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (Intermediate 5 of Example 2) (0.031 g, 0.20 mmol) in THF (2.0 mL, 15 vol) was added NaOtBu (0.025 g, 0.26 mmol) at 0° C. and stirred for 10 minutes, followed by tert-butyl(S)-12-(ethylsulfonyl)-1-fluoro-2-methyl-2-pyrrolidin-2-yl. -(7-Fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 4 of Example 2) (0.135 g, 0.16 mmol) was added at 0° C. The reaction mixture was stirred at 0° C. for 20 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with water and extracted with ethyl acetate (twice), and the combined organic layers were washed with brine solution, dried over anhydrous Na2SO4, filtered, and evaporated. The crude compound tert-butyl (S)-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 6 of Example 2) (0.135 g, crude compound) was obtained as a pale yellow solid under reduced pressure. The crude compound was used directly in the next step without further purification. MS (LC-MS): 917.64 m / z [M+H].

[0092] Step 5) Preparation of Intermediate 7 of Example 2: tert-butyl (S)-2-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0093] To a stirred solution of tert-butyl (S)-1-fluoro-2-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 6 from Example 2) (0.135 g, 0.15 mmol) in THF (3.4 mL, 25 vol) was added TBAF (2.0 mL, 15 vol) at 0° C. and the reaction mixture was stirred at 0° C. for 1 hour. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched with aqueous NaHCO (5 mL) and extracted with ethyl acetate (twice). The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and evaporated under reduced pressure to give the crude compound. The crude compound was purified by preparative HPLC to give tert-butyl (S)-2-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 7 of Example 2) (0.03 g, 26% yield) as an off-white solid. MS (LC-MS): 761.55 m / z [M+H].

[0094] Step 6) Example 2: Preparation of 5-ethynyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol

[0095] To a stirred solution of tert-butyl (S)-2-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R),7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (Intermediate 7 of Example 2) (0.12 g, 0.16 mmol) in 1,4-dioxane (1.2 mL) was added 4 M HCl at 0° C. The reaction mixture was stirred at 0° C. for 30 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude compound was purified by preparative HPLC to give 5-ethynyl-6-fluoro-4-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol (Example 2) (0.0061 g, 6% yield) as an off-white solid.

[0096] 1 H NMR(401 MHz, DMSO-d6) δ 1.72-2.23(m, 8H), 2.58-2.63(m, 1H), 2.82-2.87(m, 2H), 2.98-3.16(m, 6H), 3.73(dd, J = 10.88, 1.38 Hz, 1H), 3.94-3.98(m, 1H), 4.06-4.17(m, 3H), 4.42(dd, J = 12.51, 5.50 Hz, 1H), 5.03(d, J = 12.51 Hz, 1H), 5.21-5.34(m, 1H), 7.04-7.24(m, 1H), 7.35-7.36(m, 1H), 7.42-7.48(m, 1H), 7.95(dd, J = 9.13, 5.88 Hz, 1H), exchangeable protons are 1Not observed by H NMR. MS(LC-MS): 617.31 m / z [M+H].

[0097] Synthesis reaction scheme of Example 3 [ka]

[0098] Example 3 Preparation of 4-((14aS)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazolin-10-yl)-7-fluorobenzo[d]thiazol-2-amine Step 1) Preparation of Intermediate 2 of Example 3: 3-Bromo-2,5-difluoroaniline

[0099] To a stirred solution of 1-bromo-2,5-difluoro-3-nitrobenzene (Intermediate 1 of Example 3) (10.0 g, 42.0 mmol) in a mixture of solvents EtOH and HO (4:1, 250 mL) was added Fe (7.03 g, 126 mmol) and NH4Cl (13.5 g, 252 mmol) at room temperature, and the reaction mixture was heated to 80 °C and stirred for 6 h. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was filtered through a Celite pad using ethyl acetate (100 mL) as a washing solvent to obtain a filtrate, which was then washed with water (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude compound. The crude compound was purified by silica gel (100-200 mesh) column chromatography, eluting with 10% ethyl acetate in petroleum ether to give 3-bromo-2,5-difluoroaniline (Intermediate 2 of Example 3) (10.0 g, crude compound) as a pale yellow viscous liquid. 1 H NMR(400 MHz, CDCl3) δ 3.93(brs, 2H), 6.40-6.45(m, 1H), 6.59-6.64(m, 1H). MS(LC-MS): 208.02 m / z [M+H]. Step 2) Preparation of Intermediate 3 of Example 3: (Z)—N-(3-bromo-2,5-difluorophenyl)-2-(hydroxyimino)acetamide

[0100] To a stirred solution of 3-bromo-2,5-difluoroaniline (Intermediate 2 from Example 3) (5.00 g, 24.1 mmol) in HO (100 mL, 20 vol), NHOH.HCl (5.02 g, 72.5 mmol), NaSO (27.4 g, 217 mmol), and 1,2,2-trichloroethane-1,1-diol (5.99 g, 36.2 mmol) were added at 0 °C. Concentrated HCl (3.5 mL) was added and the reaction mixture was heated to 60 °C and stirred for 16 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction mixture was cooled to room temperature to form a solid. The solid was filtered using water (50 mL) as a washing solvent and dried under high vacuum to obtain crude compound (Z)—N-(3-bromo-2,5-difluorophenyl)-2-(hydroxyimino)acetamide (Intermediate 3 of Example 3) (10.0 g, total yield of 2 batches (2×5 g), crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. 1 H NMR(401MHz, DMSO-d6) δ 7.50-7.54(m, 1H), 7.78(s, 1H), 7.82-7.87(m, 1H), 10.11(s, 1H), 12.45(s, 1H). MS(LC-MS): 279.02 m / z [M+H]. Step 3) Preparation of Intermediate 4 of Example 3: 6-Bromo-4,7-difluoroindoline-2,3-dione

[0101] (Z)—N-(3-bromo-2,5-difluorophenyl)-2-(hydroxyimino)acetamide (Intermediate 3 of Example 3) (5.00 g, 17.9 mmol) was added to HSO (50 mL, 10 vol) at 60° C., and the reaction mixture was stirred at 90° C. for 2 hours. The progress of the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was diluted with ice-cold water (50 mL) to obtain a solid, which was filtered and dried under vacuum to obtain crude compound 6-bromo-4,7-difluoroindoline-2,3-dione (Intermediate 4 of Example 3) (4.0 g, crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. MS (LC-MS): 259.88 m / z [M−H]. Step 4) Preparation of Intermediate 5 of Example 3: 2-amino-4-bromo-3,6-difluorobenzoic acid

[0102] To a stirred solution of 6-bromo-4,7-difluoroindoline-2,3-dione (Intermediate 4 in Example 3) (4.00 g, 15.3 mmol) in 2 M NaOH solution (84 mL, 11 vol) was added 30% HO (9.53 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was acidified with concentrated HCl at 0 °C to approximately pH 3 to obtain a solid. The solid was filtered and dried under high vacuum to obtain crude compound 2-amino-4-bromo-3,6-difluorobenzoic acid (Intermediate 5 in Example 3) (4.0 g, total yield for two batches (2 x 4 g), crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. 1 H NMR(400 MHz, DMSO-d6) δ 6.72(dd, J = 10.76, 5.25 Hz, 1H), 6.77-7.00(m, 2H), 13.42(brs, 1H).MS(LCMS): 252.06 m / z [M+H]. Step 5) Preparation of Intermediate 6 of Example 3: 2-amino-4-bromo-5-chloro-3,6-difluorobenzoic acid

[0103] To a stirred solution of 2-amino-4-bromo-3,6-difluorobenzoic acid (Intermediate 5 in Example 3) (3.00 g, 11.9 mmol) in DMF (40 mL, 13 vol) was added NCS (2.48 g, 14.3 mmol) at room temperature, and the reaction mixture was heated to 90° C. and stirred for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (100 mL) to obtain a solid. The solid was filtered and dried under high vacuum to obtain crude compound 2-amino-4-bromo-5-chloro-3,6-difluorobenzoic acid (Intermediate 6 in Example 3) (3.0 g, crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. MS (LCMS): 286.09 m / z [M+H]. Step 6) Preparation of Intermediate 7 of Example 3: 2-amino-4-bromo-5-chloro-3,6-difluorobenzamide

[0104] To a stirred solution of 2-amino-4-bromo-5-chloro-3,6-difluorobenzoic acid (Intermediate 6 in Example 3) (1.50 g, 5.26 mmol) in DMF (15 mL, 10 vol) was added NHCl (0.56 g, 10.5 mmol), HATU (2.20 g, 5.79 mmol), and DIPEA (2.03 g, 15.8 mmol) at 0 °C, and the reaction mixture was allowed to warm to room temperature and stirred for 2 h. The progress of the reaction was monitored by TLC. Upon completion of the reaction, the reaction mixture was diluted with water (20 mL) to obtain a solid, which was filtered and dried under high vacuum to obtain crude compound 2-amino-4-bromo-5-chloro-3,6-difluorobenzamide (Intermediate 7 in Example 3) (0.80 g, crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. MS(LCMS): 285.08 m / z [M+H]. Step 7) Preparation of Intermediate 8 of Example 3: 7-Bromo-2,6-dichloro-5,8-difluoroquinazolin-4(3H)-one

[0105] To a stirred solution of 2-amino-4-bromo-5-chloro-3,6-difluorobenzamide (Intermediate 7 in Example 3) (0.80 g, 2.82 mmol) in 1,4-dioxane (8.0 mL, 10 vol) was added thiophosgene (0.96 g, 8.45 mmol) at 0° C. and stirred at room temperature for 1 hour. The reaction mixture was heated to 105° C. and stirred for 2 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue, which was washed with 20% ethyl acetate in petroleum ether to give a solid. The solid was filtered and dried under high vacuum to give crude compound 7-bromo-2,6-dichloro-5,8-difluoroquinazolin-4(3H)-one (Intermediate 8 in Example 3) (0.70 g, crude compound) as a pale yellow solid. The crude compound was used directly in the next step without further purification. MS(LC-MS): 329.02 m / z [M+H].

[0106] Step 8) Preparation of Intermediate 9 of Example 3: tert-butyl (S)-3-(2-((7-bromo-2,6-dichloro-8-fluoro-4-oxo-3,4-dihydroquinazolin-5-yl)oxy)ethyl)piperazine-1-carboxylate

[0107] To a stirred solution of tert-butyl (S)-3-(2-hydroxyethyl)piperazine-1-carboxylate (1.19 g, 3.65 mmol) in THF (10 mL, 14 vol) was added NaH (0.58 g, 24.3 mmol) at 0° C. and stirred at 0° C. for 30 minutes. To this was added a solution of 7-bromo-2,6-dichloro-5,8-difluoroquinazolin-4(3H)-one (Intermediate 8 from Example 3) (0.70 g, 3.04 mmol) in THF (7.0 mL, 10 vol) at 0° C., and the reaction mixture was warmed to room temperature and stirred for 3 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with saturated NH4Cl solution (20 mL) and extracted with ethyl acetate (2x20 mL), and the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give crude compound tert-butyl (S)-3-(2-((7-bromo-2,6-dichloro-8-fluoro-4-oxo-3,4-dihydroquinazolin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 of Example 3) (0.45 g, crude compound) as a brown solid. The crude compound was used directly in the next step without further purification. 1 H NMR (400 MHz, DMSO-d6) δ 1.16-1.23 (m, 2H), 1.37-1.43 (m, 9H), 1.99-2.10 (m, 2H), 2.81-3.13 (m, 3H), 3.96-4.13 (m, 4H). Exchangeable protons are 1 Not observed by H NMR. MS(LC-MS): 539.16 m / z [M+H].

[0108] Step 9) Preparation of Intermediate 10 of Example 3: tert-butyl (S)-10-bromo-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate

[0109] To a stirred solution of tert-butyl (S)-3-(2-((7-bromo-2,6-dichloro-8-fluoro-4-oxo-3,4-dihydroquinazolin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 of Example 3) (0.45 g, 0.77 mmol) in DCM (5 mL, 11 vol) was added BOP-Cl (0.68 g, 2.69 mmol) and DIPEA (1.48 g, 11.5 mmol) at 0° C., and the reaction mixture was warmed to room temperature and stirred for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with DCM (15 mL) and washed with water (10 mL), and the separated organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified on silica gel (100-200 mesh) eluting with 40% ethyl acetate in petroleum ether to give tert-butyl (S)-10-bromo-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 10 of Example 3) (0.15 g, crude compound) as a brown solid. MS (LC-MS): 521.23 m / z [M+H].

[0110] Step 10) Preparation of Intermediate 11 of Example 3: tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate

[0111] To a stirred solution of tert-butyl (S)-10-bromo-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 10 of Example 3) (0.15 g, 0.28 mmol) and tert-butyl (7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-yl)carbamate (0.095 g, 0.31 mmol) in a mixture of solvents 1,4-dioxane and water (2:1, 3.0 mL), CsCO (0.27 g, 0.84 mmol) was added at room temperature and degassed for 5 min. Dichloro[bis(diphenylphosphinophenyl)ether]palladium(II) (0.02 g, 0.03 mmol) was added thereto at room temperature, and the reaction mixture was heated to 80 °C and stirred for 4 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with ice-cold water (10 mL) and extracted with ethyl acetate (2 × 15 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified on silica gel (100-200 mesh) eluting with 40% ethyl acetate in petroleum ether to give tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 11 of Example 3) (0.07 g, 35% yield) as a pale yellow viscous solid. MS (LCMS): 709.35 m / z [M+H].

[0112] Step 11) Preparation of Intermediate 12 of Example 3: tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1′,2′:5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate

[0113] To a stirred solution of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (0.04 g, 0.25 mmol) in DMF (1.0 mL, 6 vol) was added NaH (0.008 g, 0.32 mmol) at 0° C. and stirred for 1 h. To this solution was added tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-7,11-dichloro-9-fluoro-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 11 from Example 3) (0.15 g, 0.21 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction progress was monitored by TLC. Upon completion of the reaction, the reaction mixture was diluted with saturated NH4Cl solution (10 mL) and extracted with ethyl acetate (2 x 15 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude compound. The crude compound was purified on silica gel (100-200 mesh) eluting with 5% methanol in DCM to give tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 12 of Example 3) (0.05 g, 28% yield) as a brown solid. MS (LCMS): 832.41 m / z [M+H].

[0114] Step 12) Example 3: Preparation of 4-((14aS)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazolin-10-yl)-7-fluorobenzo[d]thiazol-2-amine

[0115] A solution of tert-butyl (14aS)-10-(2-((tert-butoxycarbonyl)amino)-7-fluorobenzo[d]thiazol-4-yl)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazoline-2-carboxylate (Intermediate 12 from Example 3) (0.05 g, 0.06 mmol) in 4 M HCl in dioxane (1.0 mL, 20 vol) was stirred at room temperature for 5 hours. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give the crude compound. The crude compound was purified by preparative HPLC to give 4-((14aS)-11-chloro-9-fluoro-7-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-1,3,4,13,14,14a-hexahydro-2H-pyrazino[1',2':5,6][1,5]oxazocino[4,3,2-de]quinazolin-10-yl)-7-fluorobenzo[d]thiazol-2-amine (Example 3) (0.0075 g, 20% yield) as an off-white solid.

[0116] 1 H NMR(400 MHz, DMSO-d6) δ 1.70-1.91(m, 4H), 1.93-2.16(m, 3H), 2.54-2.56(m, 1H), 2.64-2.70(m, 1H), 2.77-2.82 (m, 3H), 3.00-3.10(m, 4H), 3.19-3.24(m, 1H), 3.64-3.74(m, 1H), 3.94(dd, J = 10.51, 2.25Hz, 1H), 4.06(dd, J = 10.51, 2.75Hz, 1H), 4.24(q, J = 11.6Hz, 1H), 4.43(brs, 1H), 4.55-4.93(m, 1H), 5.20-5.34(m, 1H), 7.01- 7.06(m, 1H), 7.14-7.23(m, 1H), 7.89(d, J = 16.76 Hz, 2H), -NH protons are1 Not observed by H NMR. MS(LCMS): 632.20 m / z [M+H].

[0117] Synthesis reaction scheme of Example 4 [ka]

[0118] Example 4 Preparation of 3-chloro-4-cyclopropyl-5-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)phenol Step 1) Preparation of Intermediate 2 of Example 4: 1-Bromo-3-chloro-2-cyclopropylbenzene

[0119] A mixture of 1-bromo-3-chloro-2-iodobenzene (22 g, 69.65 mmol), cyclopropylboronic acid (15.21 g, 90.54 mmol), Pd(dppf)Cl (2.3 g, 3.16 mmol), and KPO (24.2 g, 113.96 mmol) in dioxane:water (100 mL:30 mL) was stirred at 100 °C for 7 h under N gas. TLC (PE / EA = 1 / 0) showed the consumption of SM1 (R = 0.6) and the formation of a new spot (R = 0.7). The reaction mixture was diluted with water and extracted with ethyl acetate (200 mL × 2). The combined organic layers were washed with 70 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA=1 / 0) to give 1-bromo-3-chloro-2-cyclopropyl-benzene (13 g, 81.25% yield) as a colorless liquid.

[0120] 1H NMR(400 MHz, CDCl3) δ 7.46(dd, J = 8.0, 0.9 Hz, 1H), 7.30(dd, J = 8.0, 0.9Hz, 1H), 6.99(t, J = 8.0 Hz, 1H), 1.81 - 1.71(m, 1H), 1.22 - 1.14(m, 2H), 0.81 - 0.73(m, 2H).

[0121] Step 2) Preparation of Intermediate 3 of Example 4: 2-(3-bromo-5-chloro-4-cyclopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0122] A mixture of 1-bromo-3-chloro-2-cyclopropylbenzene (13 g, 56.52 mmol), [Ir(OMe)(COD)) (3.74 g, 5.65 mmol), 4,4'-di-tert-butyl-2,2'-bipyridine (1.82 g, 6.78 mmol), and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21.70 g, 169.56 mmol) in hexane (130 mL) was stirred at 60 °C for 3 h under N gas. TLC (PE / EA = 50 / 1) showed the consumption of SM1 (Rf = 0.3) and the formation of a new spot (Rf = 0.5). The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (PE:EA=50:1) to give 2-(3-bromo-5-chloro-4-cyclopropyl-phenyl)-4,4,5,5 tetramethyl-1,3,2-dioxaborolane (10.7 g, 53.18% yield) as a colorless oil.

[0123] 1 H NMR(400 MHz, CDCl3) δ 7.87(s, 1H), 7.70(s, 1H), 1.78(tt, J = 8.5, 5.8 Hz, 1H), 1.33(s, 12H), 1.22 - 1.14(m, 2H), 0.77(q, J = 5.8 Hz, 2H).

[0124] Step 3) Preparation of Intermediate 4 of Example 4: 2-(3-bromo-5-chloro-4-cyclopropylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0125] To a solution of 2-(3-bromo-5-chloro-4-cyclopropyl-phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (10.5 g, 29.49 mmol) in THF / HO (100 / 50 mL) was added AcOH (113.4 g, 1888.42 mmol, 107.5 mL) and HO (2.05 g, 4.79 mmol, 15.96 mL, 30% purity) at 0 °C, and the mixture was stirred at 0 °C for 1 h. TLC (PE / EA = 15 / 1) showed the consumption of SM1 (Rf = 0.7) and the formation of a new spot (Rf = 0.4). The reaction mixture was quenched with NaSO (10% aq, 80 mL) at 0 °C. The mixture was extracted with EA (100 mL × 2). The combined organic layers were washed with 100 mL of brine and dried over NaSO. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (PE:EA=10:1) to give 3-bromo-5-chloro-4-cyclopropyl-phenol as a colorless oil (5.7 g, 78.62% yield).

[0126] 1 H NMR(400 MHz, CDCl3) δ 7.00(d, J = 2.6 Hz, 1H), 6.83(d, J = 2.6 Hz, 1H), 3.73(d, J = 47.7 Hz, 1H), 1.66(ddd, J = 14.0, 7.0, 4.2Hz, 1H), 1.15 - 1.08(m, 2H), 0.71(q, J = 5.7Hz, 2H). Step 4) Preparation of Intermediate 5 of Example 4: 1-Bromo-3-chloro-2-cyclopropyl-5-(methoxymethoxy)benzene

[0127] To a solution of 3-bromo-5-chloro-4-cyclopropylphenol (5.7 g, 23.17 mmol) in DCM (57 mL) was added DIEA (4.49 g, 34.76 mmol) and methoxymethyl hypobromite (3.19 g, 25.49 mmol) at 0 °C. The reaction mixture was then stirred at 25 °C for 2 h. The reaction mixture was diluted with water and extracted with DCM (50 mL × 2). The combined organic layers were washed with 70 mL of brine, dried over Na SO , filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 20 / 1) to give 1-bromo-3-chloro-2-cyclopropyl-5-(methoxymethoxy)benzene (4 g, 59.52% yield) as a colorless oil.

[0128] 1 H NMR(400 MHz, CDCl3) δ 7.12(d, J = 2.5 Hz, 1H), 6.96(d, J = 2.5 Hz, 1H), 5.04(s, 2H), 3.38(s, 3H), 1.61(s, 1H), 1.08 - 1.02(m, 2H), 0.65(dd, J = 5.6, 1.1 Hz, 2H).

[0129] Step 5) Preparation of Intermediate 6 of Example 4: 2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0130] To a solution of 1-bromo-3-chloro-2-cyclopropyl-5(methoxymethoxy)benzene (4 g, 13.79 mmol), KOAc (4.06 g, 41.38 mmol), and 4,4,5,5-tetramethyl-2-(4,5,5-trimethyl-4-methyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (7.0 g, 27.59 mmol) in dioxane (80 mL) was added Pd(dppf)Cl (1.0 g, 1.38 mmol). The mixture was stirred at 100 °C for 16 h. The reaction mixture was diluted with water and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with 70 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 10 / 1) to give 2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.4 g, 30.04% yield) as a colorless oil. MS: m / z = 339.1 (M+H + , ESI+)

[0131] Step 6) Preparation of Intermediate 7 of Example 4: tert-butyl (3S)-3-(2-((7-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-8-fluoro-2-(methylthio-4-oxo-4,4a-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate

[0132] tert-Butyl (3S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-4,4a-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (Intermediate 9 of Example 1, 1.4 g, 2.96 mmol), 2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.0 g, 5.92 mmol), Ruphos-Pd-G3 (742 mg, 0.89 mmol), and 1,3,2-dioxaborolane (2.0 g, 5.92 mmol) in dioxane / HO (16 mL / 4 mL). A solution of KPO (1.88 g, 8.88 mmol) was stirred under N gas at 90 °C for 3 h. LCMS showed that the starting material was consumed and the desired mass was formed. The reaction mixture was quenched with water (60 mL) and extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with 60 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 0 / 1) to give tert-butyl (3S)-3-(2-((7-(3-chloro-2-cyclopropyl-5-(methoxymethyl)phenyl)-8-fluoro-2-(methylthio-4-oxo-4,4a-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (700 mg, 36.46% yield) as a yellow solid. MS: m / z = 650.3 (M+H). + , ESI+)

[0133] Step 7) Preparation of Intermediate 8 of Example 4: tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0134] To a solution of tert-butyl (3S)-3-(2-((7-(3-chloro-2-cyclopropyl-5-(methoxymethyl)phenyl)-8-fluoro-2-(methylthio-4-oxo-4,4a-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)ethyl)piperazine-1-carboxylate (700 mg, 1.08 mmol) in DCM (47 mL) was added BoPCl (822 mg, 3.24 mmol) and DIEA (1.25 g, 9.71 mmol). The mixture was then stirred at 25° C. for 16 h. LCMS showed that the starting material had been consumed and the desired mass had formed. The reaction mixture was diluted with water (20 ml). The resulting mixture was extracted with DCM (40 mL × 2). The combined organic layers were washed with 60 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 1 / 1) to give tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (140 mg, 68.06% yield) as a yellow solid. MS: m / z = 632.4 (M+H). + , ESI+)

[0135] Step 8) Preparation of Intermediate 9 of Example 4: tert-butyl (6aS)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylsulfinyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0136] To a solution of tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (110 mg, 0.17 mmol) in ACN / HO (10 mL / 10 mL) was added oxane (536 mg, 0.87 mmol) and stirred at 25 °C for 1 h. LCMS indicated that the starting material had been consumed and the desired mass was detected. The reaction mixture was diluted with water (20 mL) and extracted with DCM (30 mL × 2). The combined organic layers were washed with 20 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give tert-butyl (6aS)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylsulfinyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (120 mg, crude compound) as a yellow solid. MS: m / z = 648.2 (M+H). + , ESI+)

[0137] Step 9) Preparation of Intermediate 10 of Example 4: tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0138] To a solution of tert-butyl (6aS)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(methylsulfinyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (120 mg, 0.18 mmol) and ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (44 mg, 0.28 mmol) in THF (20 mL) was added t-BuONa (36 mg, 0.37 mmol), and the mixture was stirred at 0 °C for 1 h. LCMS indicated that the starting material had been consumed and the desired mass was detected. The reaction mixture was diluted with water (30 mL) and extracted with DCM (30 mL × 2). The combined organic layers were washed with 20 mL of brine, dried over NaSO, filtered, and concentrated in vacuo to give a residue. The residue was purified by pre-TLC (DCM / MeOH = 10 / 1) to give tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (70 mg, 52.24% yield) as a yellow solid. MS: m / z = 743.3(M+H + , ESI+)

[0139] Step 10) Example 4: Preparation of 3-chloro-4-cyclopropyl-5-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)phenol

[0140] To a solution of tert-butyl (S)-2-(3-chloro-2-cyclopropyl-5-(methoxymethoxy)phenyl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (70 mg, 0.09 mmol) in EA (2 mL) was added 3 M HCl (7 mL) and the mixture was stirred at 0 °C for 1 h; LCMS showed that the starting material was consumed and the desired mass was formed. The reaction mixture was concentrated in vacuo to give a residue. The residue was purified by pre-HPLC (FA conditions) to give 3-chloro-4-cyclopropyl-5-((S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)phenol (Example 4) (2.76 mg, 4.9% yield) as a white solid.

[0141] 1H NMR (400 MHz, MeOD) δ 8.35 (brs, 2H, FA), 6.95 (d, J = 2.4 Hz, 1H), 6.77 (s, 1H), 5.52 (d, J = 52.6 Hz, 1H), 5.35 - 5.17 (m, 1H), 4.69 - 4.45 (m, 3H), 4.40 - 4.25 (m, 1H), 4.20 - 4.10 (m, 1H), 4.01 - 3.57 (m, 3H), 3.48 - 3.32 (m, 3H), 3.25 - 3.13 (m, 2H), 3.06 - 2.93 (m, 1H), 2.73 - 2.44 (m, MS: m / z = 599.6(M+H) + , ESI+)

[0142] Synthesis reaction scheme of Example 5 [ka]

[0143] Example 5 Preparation of 5-ethyl-6-fluoro-4-((6aS)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol

[0144] Step 1) Preparation of Intermediate 2 of Example 5: tert-butyl (S)-3-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1-carboxylate

[0145] To a mixture of tert-butyl (S)-3-(2-hydroxyethyl)piperazine-1-carboxylate (8 g, 34.78 mmol), DMAP (848 mg, 6.95 mmol), and imidazole (4.73 g, 69.65 mol) in DCM (80 mL) was added TBDPSCl (19.11 g, 69.65 mmol), and the reaction mixture was stirred at 25 °C for 16 h. TLC (PE / EA = 2 / 1) showed the consumption of SM1 (Rf = 0.2) and the formation of a new spot (Rf = 0.4). The reaction mixture was diluted with HO (100 mL) and extracted with DCM (150 mL × 3). The combined organic layers were washed with brine (150 mL) and dried over NaSO. Filtration and concentration under vacuum gave a residue. The residue was purified by column chromatography (PE:EA=3:1) to give tert-butyl (S)-3-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1-carboxylate (14.55 g, 91% yield) as a colorless oil. MS: m / z = 469.4 (M+H + , ESI+)

[0146] Step 2) Preparation of Intermediate 3 of Example 5: 1-benzyl 4-(tert-butyl)(S)-2-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1,4-dicarboxylate

[0147] To a mixture of tert-butyl (S)-3-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1-carboxylate (14.55 g, 31.08 mmol) and DIEA (6.01 g, 46.62 mmol) in DCM (150 mL) was added CBZCl (13.21 g, 77.7 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 1 h under N gas. The reaction mixture was diluted with HO (100 mL) and extracted with EA (150 mL × 3). The combined organic layers were washed with brine (150 mL) and dried over NaSO. Filtration and concentration under vacuum gave a residue. The residue was purified by column chromatography (PE:EA=50:1) to give 1-benzyl 4-(tert-butyl)(S)-2-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1,4-dicarboxylate (13.806 g, 73% yield) as a colorless oil. MS: m / z = 688.2 (M+H +, ESI+)

[0148] 1 H NMR(400 MHz, CDCl3) δ 7.64(d, J = 6.7 Hz, 4H), 7.35(ddd, J = 40.3, 20.2, 11.1 Hz, 11H), 5.11(d, J = 17.9 Hz, 2H), 4.44 (s, 1H), 4.02 - 3.56 (m, 5H), 2.92 (td, J = 12.8, 2.9 Hz, 3H), 1.89 - 1.70 (m, 2H), 1.40 (s, 9H), 1.03 (s, 9H). Step 3) Preparation of Intermediate 4 of Example 5: 1-benzyl 4-(tert-butyl)(S)-2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate

[0149] To a solution of 1-benzyl 4-(tert-butyl)(S)-2-(2-((tert-butyldiphenylsilyl)oxy)ethyl)piperazine-1,4-dicarboxylate (13.806 g, 22.93 mmol) in THF (150 mL) was added TBAF (91.7 mL, 1 M in THF), and the mixture was stirred at room temperature under N gas for 2 h. LCMS showed that the starting material was consumed and the target compound was detected. The reaction mixture was diluted with H2O (100 mL) and extracted with EA (150 mL × 3). The combined organic layers were washed with brine (150 mL) and dried over Na2SO4. Filtration and concentration in vacuo gave a residue. The residue was purified by column chromatography (PE:EA=5:1) to give 1-benzyl 4-(tert-butyl)(S)-2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate (7.4 g, 92% yield) as a colorless oil. MS: m / z = 387.2 (M+H + , ESI+) Step 4) Preparation of Intermediate 5 of Example 5: 1-benzyl 4-(tert-butyl)(S)-2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate

[0150] To a solution of oxalyl chloride (6.41 g, 50.5 mmol) in DCM (60 mL) was added a solution of dimethyl sulfoxide (5.51 mg, 70.7 mmol) in dichloromethane (50 mL) at −78° C. After stirring at −78° C. for 0.5 h, a solution of (S)-1-benzyl 4-tert-butyl 2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate (7.4 g, 20.2 mmol) in DCM (20 mL) was added to the mixture. After stirring at −78° C. for 3 h, TEA (15.3 g, 151.5 mmol) was added dropwise. The reaction mixture was warmed to 25° C. for an additional 0.5 h. LCMS indicated that the starting material had been consumed and the target compound had been detected. The reaction mixture was diluted with HO (60 mL) and extracted with DCM (60 mL × 3). The combined organic layers were washed with brine (30 mL) and dried over Na2SO4. Filtration and concentration in vacuo gave a residue. The residue was purified by column chromatography (PE:EA=5:1) to give 1-benzyl 4-(tert-butyl)(S)-2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate (5.4 g, 74.5% yield) as a colorless oil. MS: m / z = 363.2 (M+H + , ESI+)

[0151] Step 5) Preparation of Intermediate 6 of Example 5: 1-benzyl 4-(tert-butyl)(2S)-2-(2-hydroxypropyl)piperazine-1,4-dicarboxylate

[0152] To a stirred solution of 1-benzyl 4-(tert-butyl)(S)-2-(2-hydroxyethyl)piperazine-1,4-dicarboxylate (5.4 g, 14.9 mmol) dissolved in dry THF (25 mL) was added 1 M CHMgBr (1 M in THF, 14.9 mL) at 0 °C. The reaction was stirred at 25 °C for 2 h, and LCMS showed that the starting material was consumed and the target compound was detected. The reaction mixture was quenched with saturated NHCl (40 mL) and extracted with DCM (60 mL × 3). The combined organic layers were washed with brine (30 mL) and dried over NaSO. Filtration and concentration in vacuo gave a residue. The residue was purified by column chromatography (PE:EA=5:1) to give 1-benzyl 4-(tert-butyl)(2S)-2-(2-hydroxypropyl)piperazine-1,4-dicarboxylate (2 g, 35.5% yield) as a colorless oil. MS: m / z = 401.2 (M+Na+, ESI+). Step 6) Preparation of Intermediate 7 of Example 5: tert-butyl (3S)-3-(2-hydroxypropyl)piperazine-1-carboxylate

[0153] To a suspension of 1-benzyl 4-(tert-butyl)(2S)-2-(2-hydroxypropyl)piperazine-1,4-dicarboxylate (2 g, 5.29 mmol) in MeOH (20 mL) was added Pd(OH) (200 mg), and the mixture was stirred under a hydrogen atmosphere at 25 °C for 2 h. The mixture was filtered through a Celite pad, and the filtrate was evaporated to give tert-butyl (3S)-3-(2-hydroxypropyl)piperazine-1-carboxylate (1.01 g, 83% yield) as a colorless oil. MS: m / z = 245.2 (M+H + , ESI+)

[0154] Step 7) Preparation of Intermediate 8 of Example 5: tert-butyl (3S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate

[0155] To a mixture of tert-butyl (3S)-3-(2-hydroxypropyl)piperazine-1-carboxylate (1.01 g, 4.14 mmol) in THF (15 mL) was added NaH (553 mg, 13.8 mmol, 60% suspension in mineral oil), and the mixture was stirred at 0 °C for 0.5 h. 5,7-Dichloro-8-fluoro-2-(methylthio)pyrido[4,3-d]pyrimidin-4(3H)-one (1.29 g, 4.61 mmol) was added, and the mixture was stirred at 60 °C for an additional 1 h. TLC (EA / MeOH = 15 / 1) showed that SM1 (Rf = 0.2) was consumed and a new spot (Rf = 0.4) was formed. The reaction mixture was quenched with water (40 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were washed with 60 mL of brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA=0 / 1) to give tert-butyl (3S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate (934 mg, 46.3% yield) as a white solid. MS: m / z = 488.1 (M+H + , ESI+)

[0156] Step 8) Preparation of Intermediate 9 of Example 5: tert-butyl (3S)-3-(2-((7-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate

[0157] A mixture of tert-butyl (3S)-3-(2-((7-chloro-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate (934 mg, 1.92 mmol), 2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (691.2 mg, 1.92 mmol), Ruphos-Pd-G3 (481.5 mg, 0.576 mmol), and K3PO4 (1.22 g, 5.76 mmol) in dioxane / HO (8 mL / 2 mL) was stirred at 90 °C under N2 gas for 3 h. LCM S indicated that the starting material had been consumed and the target compound had been detected. The reaction mixture was diluted with water (30 mL) and extracted with DCM (40 mL × 2). The combined organic layers were washed with 60 mL of brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 0 / 1) to give tert-butyl (3S)-3-(2-((7-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate (235 mg, 20% yield) as a pale yellow solid. MS: m / z = 686.3 (M+H) + , ESI+)

[0158] Step 9) Preparation of Intermediate 10 of Example 5: tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-5-methyl-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0159] To a solution of tert-butyl (3S)-3-(2-((7-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-8-fluoro-2-(methylthio-4-oxo-3,4-dihydropyrido[4,3-d]pyrimidin-5-yl)oxy)propyl)piperazine-1-carboxylate (100 mg, 0.15 mmol) in DCM (4 mL) was added DIEA (169 mg, 1.31 mmol) and BOPCl (114 mg, 0.45 mmol). The mixture was then stirred at 25° C. for 16 h. LCMS showed that the starting material had been consumed and the target compound had been detected. The reaction mixture was diluted with water (20 ml) to give DC 100:107 (4H, 1H, 2H, 3H). The mixture was extracted with M (30 mL × 2). The combined organic layers were washed with 60 mL of brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (PE / EA = 1 / 1) to give tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-5-methyl-12-(methylthio-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (40 mg, 41.2% yield) as a white solid. MS: m / z = 668.3 (M+H). + , ESI+)

[0160] Step 10) Preparation of Intermediate 11 of Example 5: tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-5-methyl-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0161] To a solution of tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-5-methyl-12-(methylthio)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (40 mg, 0.11 mmol) in MeCN / HO (1 mL / 1 mL) was added oxone (190.3 mg, 0.55 mmol) and stirred at 25 °C for 1 h. LCMS indicated that the starting material had been consumed and the desired mass had been detected. The reaction mixture was diluted with water (20 mL) and extracted with DCM (30 mL × 2). The combined organic layers were washed with 20 mL of brine, dried over Na2SO4, filtered, and concentrated in vacuo to give tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl-1-fluoro-5-methyl-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (50 mg, 65% yield crude compound) as a white solid. MS: m / z = 700.2 (M+H+, ESI+).

[0162] Step 10) Preparation of Intermediate 12 of Example 5: tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate

[0163] To a solution of tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-5-methyl-12-(methylsulfonyl)-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (50 mg, 0.071 mmol) and ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (16.9 mg, 0.106 mmol) in dry THF (2 mL) was added t-BuONa (11 mg, 0.14 mmol), and the mixture was stirred at −60 °C under N gas for 1 h. LCMS showed that the starting material was consumed and the desired mass was formed. The reaction mixture was quenched with water (60 mL) and extracted with ethyl acetate (60 mL x 3). The combined organic layers were washed with 60 mL of brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a residue. The residue was purified by pre-TLC (PE / EA=0 / 1) to give tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (20 mg, 36% yield). LC-MS MS: m / z=779.3 (M+H+, ESI+).

[0164] Step 11) Example 5: Preparation of 5-ethyl-6-fluoro-4-((6aS)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-6,6a,7,8,9,10-hexahydro-5H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalen-2-yl)naphthalen-2-ol

[0165] To a solution of tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (20 mg, 0.25 mmol) in EA (1 mL) was added 3 M HCl (2 mL) and the mixture was stirred at 0 °C for 1 min. After 1 h, LCMS indicated that the starting material was consumed and the desired mass had formed. The mixture was concentrated in vacuo to give a residue. The residue was purified by pre-HPLC to give tert-butyl (6aS)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5-methyl-5,6,6a,7,9,10-hexahydro-8H-4-oxa-3,8,10a,11,13-pentaazabenzo[4,5]cycloocta[1,2,3-de]naphthalene-8-carboxylate (2.63 mg, 16.1% yield).

[0166] 1H NMR (400 MHz, MeOD) δ 8.49 (brs, 1H, FA), 7.71 - 7.57 (m, 1H), 7.32 - 6.90 (m, 3H), 5.55 - 5.20 (m, 2H), 4.64 - 4.52 (m, 1H), 4.41 (dd, J = 59.4, 11.3 Hz, 2H), 4.02 (dd, J = 30.6, 12.5 Hz, 1H), 3.66 - 3.40 (m, 3H), 3.26 - 2.92 (m, 5H), 2.91 - 2.65 (m, 2H), 2.60 - 2.34 (m, 3H), 2.28 - 2.08 (m, 4H), 2.05 - 1.92 (m, 1H), 1.88 - 1.74 (m, 1H), 1.47 (d, J = 5.9 Hz, 3H), 0.98 - 0.82 (m, 3H). MS: m / z = 635.4(M+H) + , ESI+)

[0167] Synthesis reaction scheme of Example 6 [ka]

[0168] Example 6 Preparation of 4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-amine Step 1) Preparation of Intermediate 2 of Example 6: 2-amino-4-bromo-5-chloro-3,6-difluorobenzoyl chloride

[0169] A mixture of 2-amino-4-bromo-5-chloro-3,6-difluorobenzoic acid (2 g, 6.99 mmol) in SOCl (20 mL) was stirred at 50° C. for 3 hours. The reaction mixture was concentrated under reduced pressure to give the desired product, 2-amino-4-bromo-5-chloro-3,6-difluorobenzoyl chloride (2.13 g, 100% yield) as a brown oil. The crude product was used in the next step without further purification. Step 2) Preparation of Intermediate 3 of Example 6: 7-Bromo-6-chloro-5,8-difluoro-2-mercaptoquinazolin-4-ol

[0170] To a mixture of NHSCN (1.6 g, 21.02 mmol) dissolved in acetone (20 mL) was added a solution of 2-amino-4-bromo-5-chloro-3,6-difluorobenzoyl chloride (2.13 g, 6.98 mmol) dissolved in acetone (10 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water (200 mL) and the solution was extracted with EtOAc (100 mL × 3). The combined organic phases were washed with brine (50 mL), dried over NaSO(s), filtered, and concentrated to give the desired product, 7-bromo-6-chloro-5,8-difluoro-2-mercaptoquinazolin-4-ol (2.78 g, crude compound) as a yellow solid. MS: m / z = 326.9 (M+H). + , ESI+). The crude product was used in the next step without further purification. Step 3) Preparation of Intermediate 4 of Example 6: 7-Bromo-6-chloro-5,8-difluoro-2-(methylthio)quinazolin-4-ol

[0171] To a mixture of 7-bromo-6-chloro-5,8-difluoro-2-mercaptoquinazolin-4-ol (2.78 g, 8.53 mmol) in MeOH (30 mL) was added NaOH (1 M, 17 mL, 17 mmol) and MeI (1.06 mL, 17.05 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours. After the reaction mixture was adjusted to pH = 7 using 1 M HCl, the mixture was diluted with water (150 mL) and extracted with EtOAc (100 mL × 3). The combined organic phases were washed with brine (50 mL), dried over Na2SO4(s), filtered, and concentrated to give a residue that was purified by silica gel chromatography with PE / EA = 5:1 to give the desired product 7-bromo-6-chloro-5,8-difluoro-2-(methylthio)quinazolin-4-ol (1.85 g, 64% yield) as a yellow solid. MS: m / z = 340.9 (M+H + , ESI+)

[0172] Step 4) Preparation of Intermediate 5 of Example 6: 5-(azepan-2-ylmethoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol

[0173] To a mixture of azepan-2-ylmethanol hydrochloride (387 mg, 2.34 mmol) in THF (5 mL) was added NaH (60% in oil, 280 mg, 11.67 mmol) at 0 °C. The reaction mixture was stirred under N gas at 0 °C for 10 min, and a solution of 7-bromo-6-chloro-5,8-difluoro-2-(methylthio)quinazolin-4-ol (794 mg, 2.33 mmol) in THF (15 mL) was added to the mixture. The reaction mixture was stirred under N gas at 25 °C for 16 h. The reaction mixture was quenched with concentrated NH Cl(aq) (150 mL), and the mixture was extracted with EA (80 mL × 3). The organic phase was washed with brine (50 mL), dried over Na SO (s), filtered, and concentrated to dryness in vacuo to give a yellow solid. The yellow solid was purified by silica gel chromatography eluting with DCM / MeOH=5:1 to give the desired product 5-(azepan-2-ylmethoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol (706 mg, 67.4% yield) as a yellow solid. MS: m / z = 451.9 (M+H + , ESI+)

[0174] Step 5) Preparation of Intermediate 6 of Example 6: 2-Bromo-3-chloro-1-fluoro-13-(methylthio-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0175] To a mixture of 5-(azepan-2-ylmethoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol (706 mg, 1.57 mmol) and DIEA (2.4 mL, 14.08 mmol) in DCM (20 mL) was added BOPCl (1.2 g, 4.71 mmol) at 0 °C under N gas. The reaction mixture was stirred at room temperature under N gas for 2 h. The reaction mixture was diluted with water (200 mL) and the solution was extracted with EtOAc (100 mL × 3). The combined organic phase was washed with brine (80 mL), dried over Na SO (s), filtered, and concentrated to dryness in vacuo to give a yellow solid. The yellow solid was purified by silica gel chromatography eluted with PE / EA = 10:1 to give the desired product, 2-bromo-3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (640 mg, 74.5% yield) as a yellow solid. MS: m / z = 433.9 (M+H) + , ESI+)

[0176] Step 6) Preparation of Intermediate 7 of Example 6: tert-butyl (4-(3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0177] To a mixture of 2-bromo-3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (300 mg, 0.7 mol), KPO (443 mg, 2.1 mmol), and tert-butyl (7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-yl)carbamate (548 mg, 1.39 mmol) in dioxane / HO (9:1, 5 mL) was added Ruphos-Pd-G (174 mg, 0.21 mmol). The solution was purged with N2 three times and stirred under N2 gas at 100 °C for 2 h. The reaction was diluted with water (100 mL) and the solution was extracted with EtOAc (50 mL × 3). The combined organic phases were washed with brine (50 mL), dried over Na2SO4(s), filtered, and concentrated to dryness in vacuo to give a yellow solid. The yellow solid was purified by silica gel chromatography eluted with PE / EA = 10:1 to give the desired product tert-butyl (4-(3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (454 mg, crude compound) as a yellow solid. MS: m / z = 620.3 (M+H + , ESI+)

[0178] Step 7) Preparation of Intermediate 8 of Example 6: tert-butyl (4-(3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0179] To a mixture of tert-butyl (4-(3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3]oxone) in ACN:HO (3:1, 6 mL) was added oxone (2.2 g, 3.58 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 5 h. The reaction was quenched with aq. NaSO (60 mL) and extracted with EtOAc (30 mL × 3). The combined organic phase was washed with aq. NaSO (20 mL × 2) and The residue was washed with HCl and brine (20 mL), dried over NaSO(s), filtered, and concentrated to dryness in vacuo to give the desired product, tert-butyl (4-(3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (434 mg, crude compound) as a yellow solid. MS: m / z = 652.2 (M+H + , ESI+)

[0180] Step 8) Preparation of Intermediate 9 of Example 6: tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0181] To a mixture of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (51 mg, 0.32 mmol) in THF (2 mL) was added NaH (60% in oil, 39 mg, 1.62 mmol) at 0° C. The reaction mixture was stirred under N gas at 0° C. for 10 minutes, and tert-butyl (4-(3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (210 mg, 0.32 mmol) in THF (1 mL) was added to the solution. The reaction mixture was stirred under N2 gas at 25 °C for 1 h. The reaction mixture was quenched with concentrated NH4Cl(aq) (40 mL) and the mixture was extracted with EA (20 mL x 3). The organic phase was washed with brine (20 mL), dried over Na2SO4(s), filtered, and concentrated to dryness in vacuo to give a brown solid. The brown solid was purified by silica gel chromatography eluting with PE / EA=1:2 to give the desired product tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (65 mg, 27.6% yield) as a white solid. m / z = 731.3 (M+H + , ESI+)

[0182] Step 9) Example 6: Preparation of 4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-amine

[0183] To a mixture of tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (65 mg, 0.089 mmol) in DCM (1.5 mL) was added HCl / dioxane (1.5 mL) at room temperature. The reaction mixture was stirred at room temperature for 23 hours. The reaction was concentrated to dryness in vacuo to give a yellow solid. The yellow solid was purified by preparative HPLC (Waters 2767 / Qda, column: SunFire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% FA / HO, B: ACN, flow rate: 20 mL / min, gradient: 20% to 30%) to give the desired product Example 6 (22.82 mg, 40.7% yield) as a white solid.

[0184] 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (s, 0.31H, FA), 7.96 - 7.85 (m, 2H), 7.20 (t, J = 6.6 Hz, 1H), 7.04 (q, J = 8.0 Hz, 1H), 5.27 (d, J = 55.1 Hz, 1H), 4.87 - 4.59 (m, 2H), 4.48 - 4.37 (m, 1H), 4.11 - 3.95 (m, 3H), 3.14 - 2.96 (m, 4H), 2.87 - 2.76 (m, 1H), 2.14 - 1.55 (m, 13H), 1.30 - 1.16 (m, 1H). m / z = 631.4(M+H + , ESI+)

[0185] Synthesis reaction scheme of Example 7 [ka]

[0186] Example 7 Preparation of 4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-amine

[0187] Step 1) Preparation of Intermediate 2 of Example 7: tert-butyl (S)-3-(3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propoxy)propanoate

[0188] To a solution of tert-butyl (S)-(1-(benzyloxy)-3-hydroxypropan-2-yl)carbamate (9 g, 31.99 mmol) and CsCO (10.6 g, 32.53 mmol) in t-BuOH (72 mL) was added tert-butyl acrylate (72 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (250 mL) and the solution was extracted with ethyl acetate (150 mL × 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography with petroleum ether (PET) / ethyl acetate=10:1 to give the desired product tert-butyl (S)-3-(3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propoxy)propanoate (10.05 g, 76.8% yield) as a colorless oil. MS: m / z = 410.2 (M+H + , ESI+) Step 2) Preparation of Intermediate 3 of Example 7: (S)-3-(2-amino-3-(benzyloxy)propoxy)propanoic acid

[0189] A mixture of tert-butyl (S)-3-(3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)propoxy)propanoate (2 g, 4.89 mmol) in HCl / dioxane (20 mL) was stirred at room temperature for 2 hours. The reaction was concentrated in vacuo to give the crude product (S)-3-(2-amino-3-(benzyloxy)propoxy)propanoic acid (1.81 g, HCl salt) as a yellow oil. MS: m / z = 254.0 (M+H +, ESI+). The crude product was used in the next step without further purification. Step 3) Preparation of Intermediate 4 of Example 7: (S)-3-((benzyloxy)methyl)-1,4-oxazepan-5-one

[0190] To a solution of (S)-3-(2-amino-3-(benzyloxy)propoxy)propanoic acid (1.71 g, 6.72 mmol) and TEA (7.5 mL, 53.96 mmol) in DCM (18 mL) was added HATU (3.08 g, 8.1 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with water (150 mL) and the solution was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography with petroleum ether / ethyl acetate = 1:1 to give the desired product (S)-3-((benzyloxy)methyl)-1,4-oxazepan-5-one (1.43 g, 90.5% yield) as a yellow solid. MS: m / z = 236.1 (M+H) + , ESI+) Step 4) Preparation of Intermediate 5 of Example 7: (S)-3-((benzyloxy)methyl)-1,4-oxazepane

[0191] To a mixture of LAH (1 M in THF, 11.5 mL, 11.33 mmol) in THF (5 mL) was added (S)-3-((benzyloxy)methyl)-1,4-oxazepan-5-one (1.33 g, 5.66 mmol) in THF (5 mL) at 0 °C under N gas. The reaction mixture was stirred at 25 °C for 5 h under N gas. The reaction mixture was quenched with HO (1 mL) at 0 °C, and then NaSO was added to the solution. The mixture was stirred at room temperature for 10 min. The mixture was filtered, and the filtrate was collected and concentrated in vacuo. The residue was purified by silica gel chromatography with petroleum ether / ethyl acetate = 1:2 to give the desired product (S)-3-((benzyloxy)methyl)-1,4-oxazepane (1 g, 80% yield) as a yellow oil. MS: m / z = 222.2 (M+H + , ESI+) Step 5) Preparation of Intermediate 6 of Example 7: (R)-(1,4-oxazepan-3-yl)methanol

[0192] To a mixture of (S)-3-((benzyloxy)methyl)-1,4-oxazepane (0.95 g, 4.30 mmol) in MeOH (10 mL) was added Pd / C (0.5 g, 60%) at room temperature. The reaction mixture was stirred under H2 (15 psi) at 25 °C for 40 h. The reaction was filtered through Celite, and the filtrate was collected and concentrated in vacuo to give crude (R)-(1,4-oxazepan-3-yl)methanol (637 mg, crude compound) as a white solid. MS: m / z = 132.1 (M+H + , ESI+). The crude product was used in the next step without further purification.

[0193] Step 6) Preparation of Intermediate 7 of Example 7: (S)-5-((1,4-oxazepan-3-yl)methoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol

[0194] To a mixture of (R)-(1,4-oxazepan-3-yl)methanol (300 mg, 2.29 mmol) in THF (4 mL) was added NaH (60% in oil, 275 mg, 11.46 mmol) at 0 °C. The reaction mixture was stirred under N gas at 0 °C for 10 min, and the solution was added to a solution of 7-bromo-6-chloro-5,8-difluoro-2-(methylthio)quinazolin-4-ol (779 mg, 2.29 mmol) in THF (4 mL). The reaction mixture was stirred under N gas at 25 °C for 16 h. The reaction mixture was quenched with concentrated NH Cl(aq) (150 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (50 mL), dried over anhydrous Na SO , filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with DCM / MeOH=5:1 to give the desired product (S)-5-((1,4-oxazepan-3-yl)methoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol (319 mg, 30.9% yield) as a yellow solid. MS: m / z = 454.0 (M+H + , ESI+)

[0195] Step 7) Preparation of Intermediate 8 of Example 7: (S)-11-bromo-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0196] To a mixture of (S)-5-((1,4-oxazepan-3-yl)methoxy)-7-bromo-6-chloro-8-fluoro-2-(methylthio)quinazolin-4-ol (319 mg, 0.71 mmol) and DIEA (1 mL, 6.05 mmol) in DCM (5 mL) was added BOPCl (540 mg, 2.12 mmol) under N gas at 0 °C. The reaction mixture was stirred at room temperature under N gas for 16 h. The reaction was quenched with water (150 mL) and the solution was extracted with ethyl acetate (70 mL × 3). The combined organic phase was washed with brine (50 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography, eluting with petroleum ether / ethyl acetate = 4:1, to give the desired product (S)-11-bromo-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (142 mg, 46.4% yield) as a yellow solid. MS: m / z = 435.9 (M+H + , ESI+)

[0197] Step 8) Preparation of Intermediate 9 of Example 7: tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0198] To a mixture of (S)-11-bromo-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (142 mg, 0.33 mol), KPO (209 mg, 0.98 mmol), and tert-butyl (7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]thiazol-2-yl)carbamate (259 mg, 0.66 mmol) in dioxane / HO (9:1, 5 mL) was added Ruphos-Pd-G (82 mg, 0.10 mmol). The solution was purged with N2 three times and stirred under N2 gas at 100 °C for 2 h. The reaction mixture was diluted with water (100 mL) and the solution was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate = 1:1 to give the desired product tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (143 mg, 70% yield) as a yellow solid. MS: m / z = 622.2(M+H + , ESI+)

[0199] Step 9) Preparation of Intermediate 10 of Example 7: tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0200] To a mixture of tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (140 mg, 0.23 mmol) in THF:HO (1:1, 4 mL) was added oxone (693 mg, 1.13 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction was quenched with aq. NaSO (80 mL), and the solution was extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with aq. NaSO (20 mL × 2) and brine (20 mL), dried over NaSO, filtered, and concentrated in vacuo to give crude product tert-butyl (4-((14aS)-12-chloro)-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (140 mg, crude compound) as a yellow solid. MS: m / z = 654.2 (M+H). + , ESI+)

[0201] Step 10) Preparation of Intermediate 11 of Example 7: tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate

[0202] To a mixture of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (34 mg, 0.21 mmol) in THF (2 mL) was added NaH (60% in oil, 26 mg, 1.08 mmol) at 0°C. The reaction mixture was stirred under N2 gas at 0 °C for 10 min, and a solution of tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (140 mg, 0.21 mmol) in THF (3 mL) was added to the solution. The reaction mixture was stirred under N2 gas at 25 °C for 16 h. The reaction mixture was quenched with concentrated NH4Cl(aq) (40 mL), and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phase was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by pre-TLC (ethyl acetate, Rf=0.4) to give the desired product tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (30 mg, 19% yield) as a white solid. MS: m / z = 733.3 (M+H + , ESI+)

[0203] Step 11) Example 7: Preparation of 4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-amine

[0204] A mixture of tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[d]thiazol-2-yl)carbamate (30 mg, 0.04 mmol) in HCl / dioxane (2 mL) was stirred at room temperature for 16 hours. The reaction was concentrated in vacuo. The residue was subjected to preparative HPLC (Waters 2767 / Qda, column: SunFire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% FA / HO, B: ACN, flow rate: 20 mL / min; gradient: 20% to 30%) to give the desired product Example 7 (2.47 mg, 9.5% yield) as a white solid.

[0205] 1 H NMR (400 MHz, CD3OD) δ 8.56 (s, 0.72H-FA), 7.24 - 7.14 (m, 1H), 6.97 (t, J = 8.8 Hz, 1H), 5.39 - 5.25 (m, 1H), 5.11 - 5.00 (m, 1H), 4.83 - 4.70 (m, 3H), 4.62 - 4.54 (m, 1H), 4.47 - 4.42 (m, 1H), 4.39 - 4.12 (m, 4H), 4.08 - 3.97 (m, 1H), 3.85 - 3.76 (m, 1H), 3.60 - 3.46 (m, 2H), 3.14 -3.05 (m, 1H), 2.36 - 2.17 (m, 4H), 2.06 - 1.87 (m, 4H). MS: m / z = 633.3(M+H + , ESI+)

[0206] Synthesis reaction scheme of Example 8 [ka]

[0207] Example 8 Preparation of 2-amino-4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0208] Step 1) Preparation of Intermediate 1 of Example 8: (S)-11-bromo-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0209] To a mixture of (S)-11-bromo-12-chloro-10-fluoro-8-(methylthio)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (197 mg, 0.45 mmol) in THF / HO (1:1, 8 mL) was added oxone (1.4 g, 2.28 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. The reaction was quenched with aq. NaSO (80 mL), and the solution was extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with aq. NaSO (20 mL × 2) and brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness in vacuo to give crude (S)-11-bromo-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (227 mg, crude compound) as a yellow solid. MS: m / z = 467.9 (M+H). + , ESI+). The crude product was used in the next step without further purification.

[0210] Step 2) Intermediate 2 of Example 8: (S)-11-bromo-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0211] To a stirred solution of (1-(pyrrolidin-1-ylmethyl)cyclopropyl)methanol (79 mg, 0.51 mmol) in THF (2 mL) was added NaH (60% in mineral oil, 20 mg, 0.51 mmol) under nitrogen gas at 0 °C and stirred for 20 min. (S)-11-Bromo-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (160 mg, 0.34 mmol) was added and stirred at room temperature for an additional 16 h. After completion of the reaction, aqueous ammonium chloride (20 mL) was added to quench the reaction, and the organic layer was extracted with ethyl acetate (3 x 25 mL). The organic layer was washed with brine (60 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue, which was purified by silica gel column chromatography using methanol / DCM = 1:6 as an eluent to give the desired compound (S)-11-bromo-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (50 mg, 26.6% yield).

[0212] Step 3) Intermediate 3 of Example 8: tert-butyl (4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate

[0213] To a stirred solution of (S)-11-bromo-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (50 mg, 0.09 mmol) in 1,4-dioxane (3 mL) was added tert-butyl ether. (3-cyano-4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-7-fluorobenzo[b]thiophen-2-yl)carbamate (73 mg, 0.18 mmol), KPO (57 mg, 0.27 mmol), KF (10 mg, 0.18 mmol), and DPEphos-PdCl (14 mg, 0.02 mmol) were added and stirred at 105 °C for 4 hours under nitrogen gas. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain a residue. The residue was purified by reverse phase chromatography (column: SunFire Sunfire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% HCOOH / HO, B: acetonitrile; flow rate: 20 mL / min; gradient: 35%-50%) to give the desired compound tert-butyl (4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (12 mg, 17.3% yield) as a white solid. MS: m / z = 753.4 (M+H + , ESI+)

[0214] Step 4) Example 8: 2-amino-4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0215] A stirred solution of tert-butyl (4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (12 mg, 0.16 mmol) in a 1,4-dioxane / hydrochloric acid mixture (4 M, 2.5 mL) was allowed to stand at room temperature for 6 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a residue. The resulting residue was purified by prep-HPLC (column: XBridge XBridge C18 19*250 mm, 10 μm; mobile phase A: 0.1% NH4HCO3 / HO, B: acetonitrile; flow rate: 20 mL / min; gradient: 45% to 50%; residence time: 6.4–9 min out of 16 min) to give the desired compound, 2-amino-4-((S)-12-chloro-10-fluoro-8-((1-(pyrrolidin-1-ylmethyl)cyclopropyl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile (0.70 mg, 6.7% yield).

[0216] 1 H NMR (400 MHz, MeOD-d4) δ 8.55 (brs, 2.25H, FA), 7.22 (dd, J = 8.4, 5.1 Hz, 1H), 7.10 - 7.02 (m, 1H), 4.53 - 4.44 (m, 4H), 4.40 - 3.33 (m, 1H), 4.30 - 4.16 (m, 3H), 4.10 - 3.94 (m, 2H), 3.91 - 3.74 (m, 2H), 3.62 - 3.45 (m, 5H), 2.28 - 2.16 (m, 1H), 2.08 - 1.92 (m, 5H), 0.94 - 0.74 (m, 4H).MS: m / z = 653.0 (M+H + , ESI+)

[0217] Synthesis reaction scheme of Example 11 [ka]

[0218] Example 11 Preparation of 2-amino-4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0219] Step 1) Preparation of Intermediate 1 of Example 11: 2-Bromo-3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0220] To a mixture of 2-bromo-3-chloro-1-fluoro-13-(methylthio)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (300 mg, 0.70 mmol) in THF / HO (1:1, 4 mL) was added oxone (2.14 g, 3.48 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with aq. NaSO (80 mL), and the solution was extracted with ethyl acetate (30 mL × 3). The combined organic phase was washed with aq. NaSO (20 mL × 2) and brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give the crude product 2-bromo-3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (365 mg, crude compound) as a yellow solid. MS: m / z = 466.0 (M+H). + , ESI+). The crude product was used in the next step without further purification.

[0221] Step 2) Preparation of Intermediate 2 of Example 11: 2-Bromo-3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0222] To a mixture of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (125 mg, 0.79 mmol) in THF (2 mL) was added NaH (60% in oil, 95 mg, 3.96 mmol) at 0° C. The reaction mixture was stirred under N gas at 0° C. for 10 minutes, and a solution of 2-bromo-3-chloro-1-fluoro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-13-(methylsulfonyl)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazoline (365 mg, 0.79 mmol) in THF (10 mL) was added to the solution. The reaction mixture was stirred under N2 gas at 25 °C for 16 h. The reaction mixture was quenched with concentrated NH4Cl(aq) (100 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate = 1:2 to give the desired product, 2-bromo-3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (319 mg, 74.7% yield) as a yellow solid. MS: m / z = 545.0(M+H + , ESI+)

[0223] Step 3) Preparation of Intermediate 3 of Example 11: tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2′,1′:3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate

[0224] A mixture of 2-bromo-3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazoline (220 mg, 0.41 mol), CsCO (396 mg, 1.22 mmol), and DPEphos-PdCl (73 mg, 0.10 mmol) in 5 mL of toluene was purged with N three times. The solution was stirred under N gas at 80 °C for 10 min. Then, tert-butyl (3-cyano-4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-7-fluorobenzo[b]thiophen-2-yl)carbamate (328 mg, 0.81 mmol) was added to the mixture. The mixture was purged with N2 three times and then stirred at 110 °C for 2 h. The reaction was diluted with water (200 mL) and the solution was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with brine (80 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel chromatography with petroleum ether / ethyl acetate=1:2 to give the desired product tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (176 mg, crude compound) as a yellow oil. MS: m / z = 755.4 (M+H + , ESI+)

[0225] Step 4) Preparation of Intermediate 4 of Example 11: 2-amino-4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0226] A mixture of tert-butyl (4-(3-chloro-1-fluoro-13-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-azepino[2',1':3,4][1,4]oxazepino[5,6,7-de]quinazolin-2-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (166 mg, 0.22 mmol) in HCl / dioxane (5 mL) was stirred at room temperature for 16 hours. The reaction was concentrated in vacuo to dryness. The residue was subjected to preparative HPLC (Waters 2767 / Qda, column: SunFire Sunfire C18, 19*250mm, 10μm; mobile phase A: 0.1% FA / HO, B: ACN, flow rate: 20mL / min; gradient: 20% to 30%) to obtain Example 11a (40mg) as a white solid and Example 11b (17.08mg) as a white solid, as the desired product. HPLC results showed that Example 11a was impure and was purified by preparative HPLC (Waters 2767 / Qda, column: XBridge XBridge C18, 19*250 mm, 10 μm; mobile phase A: 10 mmol NH4HCO3 / H2O, B: ACN, flow rate: 20 mL / min, gradient: 30%-40%, retention time: 7-8 min, 16 min) to give pure Example 11a (15.29 mg) as a white solid. MS: m / z = 655.4 (M+H + , ESI+)

[0227] H NMR (Example 11a): 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 2H), 7.30 - 7.21 (m, 1H), 7.18 - 7.10 (m, 1H),5.27 (d, J = 54.7 Hz, 1H), 4.86 - 4.71 (m, 1H), 4.70 - 4.58 (m, 1H), 4.52 - 4.41 (m,1H), 4.12 - 4.02 (m, 2H), 4.01 - 3.95 (m, 1H), 3.15 - 2.94 (m, 4H), 2.88 - 2.77 (m, 1H), 2.14- 1.45 (m, 14H)

[0228] H NMR (Example 11b): 1 H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 0.40H-FA), 8.08 (s, 2H), 7.29 - 7.21 (m,1H), 7.19 - 7.04 (m, 1H), 5.29 (d, J = 53.8 Hz, 1H), 4.75 - 4.56 (m, 2H), 4.39 - 4.25 (m, 1H), 4.17 - 3.98 (m, 3H), 3.18 - 2.99 (m, 4H), 2.91 - 2.81 (m, 1H), 2.20 - 1.50 (m, 14H)

[0229] Synthesis reaction scheme of Example 12 [ka]

[0230] Example 12 Preparation of 2-amino-4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0231] Step 1) Preparation of Intermediate 1 of Example 12: (S)-11-bromo-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline

[0232] To a mixture of ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (78 mg, 0.49 mmol) in THF (5 mL) was added NaH (60% in oil, 60 mg, 2.5 mmol) at 0°C. The reaction mixture was stirred at 0 °C for 10 min under N gas, and a solution of (S)-11-bromo-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-14-tetrahydro-12-chloro-10-fluoro-8-(methylsulfonyl)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (227 mg, 0.49 mmol) in THF (5 mL) was added to the solution. The reaction mixture was stirred at 25 °C for 16 h under N gas. The reaction mixture was quenched with concentrated NH Cl(aq) (100 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phase was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel chromatography eluting with ethyl acetate to give the desired product (S)-11-bromo-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (122 mg, 46% yield) as a yellow oil. MS: m / z = 547.1 (M+H) + , ESI+)

[0233] Step 2) Preparation of Intermediate 2 of Example 12: tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate

[0234] (S)-11-bromo-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazoline (91 mg, 0.17 mmol), tert-butyl ether, in 4 mL of dioxane. A mixture of t-butyl (3-cyano-4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-7-fluorobenzo[b]thiophen-2-yl)carbamate (135 mg, 0.33 mmol), KPO (107 mg, 0.5 mmol), KF (19 mg, 0.33 mmol), and DPEphos-PdCl (24 mg, 0.034 mmol) was purged with N three times. The solution was then stirred at 105 °C for 5 h. The reaction mixture was allowed to cool to room temperature and diluted with water (80 mL). The solution was extracted with ethyl acetate (40 mL × 3). The organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to dryness in vacuo. The residue was purified by silica gel chromatography eluting with ethyl acetate to give the desired product tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H)-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (40 mg, 31.7% yield) as a yellow oil. MS: m / z = 757.3 (M+H + , ESI+)

[0235] Step 3) Example 12: Preparation of 2-amino-4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-7-fluorobenzo[b]thiophene-3-carbonitrile

[0236] A mixture of tert-butyl (4-((14aS)-12-chloro-10-fluoro-8-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-4,5,14,14a-tetrahydro-1H,3H-[1,4]oxazepino[3',4':3,4][1,4]oxazepino[5,6,7-de]quinazolin-11-yl)-3-cyano-7-fluorobenzo[b]thiophen-2-yl)carbamate (40 mg, 0.05 mmol) in HCl / dioxane (2 mL) was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. The residue was subjected to preparative HPLC (Waters 2767 / Qda, column: SunFire Sunfire C18, 19*250 mm, 10 μm; mobile phase A: 0.1% FA / HO, B: ACN, flow rate: 20 mL / min; gradient: 20% to 30%) to give the desired products Example 12a (1.94 mg, 5.6% yield) and Example 12b (1.32 mg, 3.8% yield) as white solids. MS: m / z = 657.3 (M+H + , ESI+)

[0237] H NMR (Example 12a): 1 H NMR (400 MHz, CD3OD) δ 7.15 (dd, J = 8.4, 5.1 Hz, 1H), 7.02 - 6.92 (m, 1H), 5.59 -5.33 (m, 1H), 5.06 - 4.96 (m, 1H), 4.69 - 4.36 (m, 4H), 4.27 - 4.10 (m, 2H), 4.01 - 3.69 (m,5H), 3.55 - 3.34 (m, 3H), 2.70 - 2.42 (m, 2H), 2.32 - 2.23 (m, 2H), 2.17 - 1.88 (m, 4H)

[0238] H NMR (Example 12b): 1H NMR (400 MHz, CD3OD) δ 8.43 (brs, 0.61H-FA), 7.18 - 7.09 (m, 1H), 7.03 - 6.90(m, 1H), 5.52 - 5.31 (m, 1H), 5.04 - 4.92 (m, 1H), 4.73 - 4.63 (m, 2H), 4.59 - 4.37 (m, 4H), 4.26 - 4.18 (m, 1H), 4.17 - 4.08 (m, 1H), 3.99 - 3.89 (m, 1H), 3.85 - 3.58 (m, 4H), 3.57 - 3.43 (m, 2H), 2.57 - 2.37 (m, 2H), 2.35 - 2.24 (m, 1H), 2.22 - 2.11 (m,3H), 1.99 - 1.89 (m, 2H)

[0239] In a similar manner, the compounds of Examples 1 to 15 in Table 1 below were prepared using the appropriate materials for preparing the compounds described in each Example.

[0240] [Table 1] TIFF2025532125000020.tif252164TIFF2025532125000021.tif253164TIFF20255321250 00022.tif246170TIFF2025532125000023.tif254166TIFF2025532125000024.tif253166 TIFF2025532125000025.tif202170TIFF2025532125000026.tif254166TIFF20255321250 00027.tif239170TIFF2025532125000028.tif254169TIFF2025532125000029.tif142170

[0241] <Experimental Example 1: KRAS Nucleotide Exchange Assay> Test Purpose KRAS WT , KRAS G12D, KRAS G12V Evaluation of the inhibitory effect of compounds on SOS1-mediated nucleotide exchange activity of mutants Test Principle

[0242] This method monitors the SOS1-mediated exchange of unlabeled KRAS-bound GDP and fluorescently labeled GTP. Detection is based on the energy transfer between the two fluorophores when the donor, a Tb cryptate-labeled GST antibody, and the acceptor, DY-647P1 GTP, are in close proximity. Testing conditions and procedures material GST-tagged KRAS WT or mutant protein (amino acids 2-169), SOS1 (amino acids 564-1049), Labeled GTP (GTP-DY-647P1),

[0243] Assay Buffer (20mM HEPES pH 7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT, 0.05% BSA, 0.0025% NP40) Examination Procedures

[0244] 1. KRAS protein was diluted with assay buffer to a final concentration of 1.5 times, and then mixed with Tb cryptate anti-GST antibody. 10 μL of this mixture was added to each assay well (final concentration: KRAS WT , KRAS G12D , KRAS G12V Both 20nM). 2. The compounds were dissolved in DMSO and then diluted to a final concentration 100 times higher.

[0245] 3. Compounds were dispensed into assay wells using an ECHO acoustic dispenser (Beckman), gently mixed with the KRAS / Ab mixture, and incubated for 60 minutes.

[0246] 4. SOS1 and labeled GTP were mixed and diluted with assay buffer to three times the final concentration. 5 μL of the solution was added to the assay well to initiate the reaction (final labeled GTP concentration: 0.15 μM, final SOS1 concentrations: 7.5 nM for WT, 12.5 nM for G12D, and 50 nM for G12V). Blank wells contained only assay buffer and labeled GTP. 5. The reaction was monitored using a Pherastar Plate Reader (BMG) with Ex / Em = (337 / 665; 337 / 620). 6. The HTRF signal was analyzed approximately 25 minutes after the start of the reaction (60 minutes reaction time for G12V).

[0247] 7. Nucleotide exchange activity is expressed as a percentage difference from the DMSO reaction value, and the IC 50 The values ​​were calculated based on a four-parameter logistic equation using GraphPad 4.0 software.

number

[0248] [Table 2]

[0249] <Experimental Example 2: KRAS NanoBRET Assay> Test Purpose NanoBRET target binding assessment of compounds against KRAS (WT, G12D, or G12V) HEK293 cells Testing conditions and procedures Compound production

[0250] Test compounds were dissolved in 10 mM stock solutions, and reference compounds BI-2582 and MRTX1133 (MedChemExpress) were dissolved in DMSO in 10 mM and 1 mM stock solutions, respectively. cell culture

[0251] The NanoBRET KRAS (WT, G12D, or G12V)-NanoLuc fusion vector and tracer K-2 were purchased from Promega, and the HEK293 cell line was purchased from ATCC. HEK293 cells were cultured in EMEM medium supplemented with 10% FBS and 100 μg / mL penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Examination Procedures

[0252] 1. HEK293 cells are transfected with the NanoBRET KRAS (WT, G12D, or G12V)-NanoLuc Fusion Vector.

[0253] 2. The density of cells transfected in Opti-MEM without Phenol red was increased to 2 × 10 5 Adjust the concentration to cells / mL and mix 20xK-2 tracer with the cells. 3. Dispense the cell and tracer mixture into 384 wells and leave in a 37°C, 5% CO2 incubator for 1 hour, then leave at room temperature for 15 minutes. 4. Apply the substrate and test compound solution to the 384 wells containing the cells and tracer, and react for 15 minutes at room temperature.

[0254] 5. Use an Envision 2104 plate reader to measure the donor emission wavelength (460 nm) and the acceptor emission wavelength (600 nm).

[0255] 6. Calculate the BRET ratio by dividing the acceptor emission value (600 nm) by the donor emission value (460 nm), and correct for background by subtracting the BRET ratio that does not contain the tracer. 6. Calculate the BRET response by taking the BRET ratio when treated with DMSO versus the BRET ratio when treated with the compound * 100. 7. IC calculated based on the sigmoidal dose-response equation using the GraphPad Prism 4 program. 50 Calculate the value.

[0256] <Experimental Example 3: Cell proliferation assay> Test Purpose

[0257] Test compounds were tested for cell viability in AsPC-1 pancreatic cancer (KRAS G12D mutation) or SW480 colon cancer (KRAS G12V mutation) cells for 72 hours. Testing conditions and procedures material

[0258] The reference compound, Staurosporine, was purchased from Sigma-Aldrich (Saint Louis, MI), and CellTiter-Glo® 2.0 Luminescent cell viability assay reagent (cat# G9243) was purchased from Promega (Madison, WI). AsPC-1 and SW480 cell lines were purchased from American Type Culture Collection (Manassas, VA). AsPC-1 cells were cultured in RPMI-1640 (ATCC, cat# 30-2001), and SW480 cells were cultured in DMEM (ATCC cat# 30-2002), supplemented with 10% FBS (Sigma-Aldrich, cat# F2442) and 100 μg / mL penicillin-streptomycin (Sigma-Aldrich, cat# P4333). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Examination Procedures

[0259] 1. Test compounds and the reference compound Staurosporine were dissolved in DMSO solution to prepare 20 mM (test compounds) and 10 mM (reference compound Staurosporine) in a source plate, which were then diluted 3-fold, 10 doses, with DMSO.

[0260] 2. Using the Source Plate, 10 volumes of 125 nL of test compound or 25 nL of reference compound were dispensed into wells of a 384-well culture plate (VWR, cat#82050-076) using an Echo 655. 3. 25 μL of culture medium containing 2000 AsPC-1 or SW480 cells was dispensed into a 384-well cell culture plate. 4. The cells were incubated with the compounds at 37°C in 5% CO2 for 72 hours. 5. 25 μL of CellTiter-Glo 2.0 reagent was added to each well of the plate. 6. The contents were mixed on an orbital shaker for 2 minutes, and then the luminescence signal was stabilized at room temperature for 15 minutes.

[0261] 7. Luminescence signals were measured with an Envision 2104 Multilabel Reader (PerkinElmer, Santa Clara, CA), and the number of viable cells was determined through quantification of ATP present in each culture medium. 8. IC calculated based on the sigmoidal dose-response equation using the GraphPad Prism 4 program. 50 values ​​were calculated. The test results are shown in Table 3.

[0262] [Table 3]

[0263] While the present invention has been described above with reference to specific examples, and while the present invention has been specifically illustrated and described based on preferred and various alternative embodiments, those skilled in the relevant art will recognize that various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. A compound of the following formula 1, or a stereoisomer, diastereomer, enantiomer, hindered rotational isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof: 【Chemical 1】 In the above formula, R １ is hydroxy, halogen, C １－３ Haloalkyl, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ Cycloalkyl, C １－３ optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; phenyl, pyridinyl, naphthyl, indazolyl, benzothiazolyl, or benzothiophenyl; R ２ is hydrogen or halogen; R ３ is hydrogen, C １－３ Alkyl, C ３－６ cycloalkyl, 4-10 membered heterocycle, or -O-(L) ｍ -A １ In this case, A １ is one or more R ７ and unsubstituted C １－３ Alkyl, C ３－６ cycloalkyl, 4-10 membered heterocycle, 6-10 membered aryl, 5-10 membered heteroaryl, or 5-10 membered fused heteroaryl; L is R ７ Substituted or unsubstituted C １－３ Alkylene or C ３－８ is cycloalkylene; R ７ are each independently a halogen, oxo (=0), or ═CH ２ , -OCF ３ , -OCHF ２ , amino, cyano, -N(C １－３ alkyl) ２ , —NH(C １－３ alkyl), substituted or unsubstituted C １－３ Alkyl, substituted or unsubstituted C １－３ Haloalkyl, substituted or unsubstituted C １－３ Alkoxy, substituted or unsubstituted C ３－４ cycloalkyl, or a substituted or unsubstituted heterocycle; R ４ are each independently hydrogen, hydroxy, halogen, C １－３ haloalkyl, or C １－３ Alkyl, C １－３ is alkoxy; X is O, CH ２ or NR ８ and R ８ is hydrogen or R ９ C selectively substituted 1-3 times with １－６ Alkyl, C ３－６ cycloalkyl, or C ３－６ is heterocycloalkyl; R ９ is independently in each occurrence oxygen, hydroxy, -C １－４ Alkyl or —O—C １－４ is alkyl; Y is NH, O, S, SO, or SO ２ and Z is CR ６ or N; R ５ is hydrogen, C １－３ alkyl, cyano, or amino; R ６ is hydrogen, hydroxy, halogen, C １－３ is alkyl; n1, n2, n3, and m are each an integer from 1 to 3; Heterocycle, heteroaryl, and fused heteroaryl each contain one or more N, S, or O heteroatoms.

2. R １ is hydroxy, halogen, C １－３ Haloalkyl, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ Cycloalkyl, C １－３ optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; phenyl, naphthyl, benzothiazolyl, or benzothiophenyl; X is O, CH ２ or NH; The compound of claim 1 , wherein Y is O.

3. R ３ is -O-(L) ｍ -A １ In this case, A １ is one or more R ７ 2. The compound of claim 1, wherein each of the following is a substituted or unsubstituted 4-10 membered heterocycle or 5-10 membered fused heteroaryl:

4. R １ is hydroxy, halogen, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ optionally unsubstituted or substituted with one or more substituents independently selected from cycloalkyl, amino, and cyano; 【Chemistry 2】 and R ３ is -O-(L) ｍ -A １ In this case, A １ is one or more R ７ each of which is a substituted or unsubstituted 4-10 membered heterocycle or 5-10 membered fused heteroaryl; L is C １－３ Alkylene or C ３－８ is cycloalkylene; R ７ are each independently a halogen, oxo (=0), or ═CH ２ , -OCF ３ , -OCHF ２ , amino, cyano, C １－３ Alkyl, C １－３ haloalkyl, or C １－３ is alkoxy; R ４ is hydrogen; X is O, CH ２ or NH; Z is CR ６ or N; R ５ is hydrogen or C １－３ is alkyl; R ６ is a halogen; 2. The compound of claim 1, wherein n1, n2, n3, and m are each an integer from 1 to 3.

5. When Z is N, R １ is hydroxy, halogen, C １－３ Haloalkyl, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ Cycloalkyl, C １－３ optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; phenyl or naphthyl; Z is CR ６ If R １ is hydroxy, halogen, C １－３ Haloalkyl, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ Cycloalkyl, C １－３ optionally unsubstituted or substituted with one or more substituents independently selected from alkoxy, amino, and cyano; 10. The compound of claim 1 which is benzothiazolyl or benzothiophenyl.

6. R ３ is -O-(L) ｍ -A ２ In this case, A ２ teeth 【Chemistry 3】 and They are halogens, C １－３ Alkyl, C １－３ Alkoxy, and =CH ２ and - (L) ｍ - is methylene or 【Chemistry 4】 2. The compound of claim 1, wherein:

7. R １ is hydroxy, halogen, C １－３ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－６ optionally unsubstituted or substituted with one or more substituents independently selected from cycloalkyl, amino, and cyano; 【Chemistry 5】 and R ２ is a halogen; R ３ is -O-(L) ｍ -A １ In this case, A １ is a halogen, C １－３ Alkyl, C １－３ Alkoxy, and =CH ２ or unsubstituted with one or more substituents independently selected from 【Chemistry 6】 In this case, -(L) ｍ - is methylene or 【Chemistry 7】 and R ４ is hydrogen; X is O, CH ２ or NH; Y is O; Z is CH, C(C １－３ alkyl), or N; R ５ is hydrogen or C １－３ The compound of claim 1 , wherein the aryl group is alkyl.

8. The compound of formula 1 is any one selected from the group consisting of the following compounds: The compound of claim 1, or a stereoisomer, diastereomer, enantiomer, hindered rotation isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof: 【Chemistry 8】 。

9. A pharmaceutical composition for cancer treatment comprising, as an active ingredient, a compound according to any one of claims 1 to 8, or a stereoisomer, diastereomer, enantiomer, rotationally hindered isomer, solvate, isotopic variant, tautomer, or pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition of claim 9, which exhibits KRAS protein inhibitory activity.

Images