The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to inhibition of KRAS mutant useful for treating cancers.

FIELD OF THE INVENTION

RAS is one of the most well-known proto-oncogenes. Approximately 30% of human cancers contain mutations in three most notable members, KRAS, HRAS, and NRAS, making them the most prevalent oncogenic drivers. KRAS mutations are generally associated with poor prognosis especially in colorectal cancer, pancreatic cancer, lung cancers. As the most frequently mutated RAS isoform, KRAS has been intensively studied in the past years. Among the most commonly occurring KRAS alleles (including G12D, G12V, G12C, G13D, G12R, G12A, G12S, Q61H, etc), G12C, G12D, G12V represent more than half of all K-RAS-driven cancers across colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), lung adenocarcinoma (LU AD). Of note, KRAS wild-type amplifications are also found in around 7% of all KRAS- altered cancers (ovarian, esophagogastric, uterine), ranking among the top alterations.

All RAS proteins belong to a protein family of small GTPases that hydrolyze GTP to GDP. KRAS is structurally divided into an effector binding lobe followed by the allosteric lobe and a carboxy-terminal region that is responsible for membrane anchoring. The effector lobe comprises the P-loop, switch I, and switch II regions. The switch I/II loops play a critical role in KRAS downstream signaling through mediating protein-protein interactions with effector proteins that include RAF in the mitogen-activated protein kinase (MAPK) pathway or PI3K in the phosphatidylinositol 3 -kinase (PI3K)/protein kinase B (AKT) pathway.

KRAS protein switches between an inactive to an active form via binding to GTP and GDP, respectively. Under physiological conditions, the transition between these two states is regulated by guanine nucleotide exchange factors (GEFs), such as Son Of Sevenless Homolog 1 (SOS1), or GTPase- activating proteins (GAPs) that involve catalyzing the exchange of GDP for GTP, potentiating intrinsic GTPase activity or accelerating RAS-mediated GTP hydrolysis. In response to extracellular stimuli, the inactive RAS -GDP is converted to active RAS-GTP which directly binds to RAF RAS binding domains (RAF ^RBD ), recruiting RAF kinase family from cytoplasm to membranes, where they dimerize and become active. The activated RAF subsequently carries out a chain of phosphorylation reactions to its downstream Mitogen- activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK), and propagates the growth signal. Of the RAF family of protein kinases (three known isoforms ARAF, BRAF, CRAF/RAF1), BRAF is most frequently mutated and remains the most potent activator of MEK. Despite that individual RAS and RAF family members revealed distinct binding preferences, all RAFs possess the conserved RBD for forward transmission of MAPK singnaling, frequently used for characterize KRAS inhibition (e.g. KRAS-BRAF ^RBD herein). For KRAS, mutations at positions 12, 13, 61, and 146 lead to a shift toward the active KRAS form through impairing nucleotide hydrolysis or activating nucleotide exchange, leading to hyper-activation of the MAPK pathway that results in tumorigenesis.

Despite its well-recognized importance in cancer malignancy, continuous efforts in the past failed to develop approved therapies for KRAS mutant cancer until recently, the first selective drug AMG510 has fast approval as second line treatment in KRAS G12C driven non-small cell lung cancer (NSCLC). Nevertheless, the clinical acquired resistance to KRAS G12C inhibitors emerge rigorously with disease progresses after around 6 month of treatment. All of the mutations converge to reactivate RAS-MAPK signaling, with secondary RAS mutants at oncogenic hotspots (e.g. G12/G13/Q61) and within the switch II pocket (e.g. H95, R68, and Y96) have been observed; moreover, over 85% of all KRAS-mutated or wild-type amplified driven cancers still lack novel agents. Altogether, both the myriad of escape mechanism and various oncogenic alleles, highlight the urgent medical need for additional KRAS therapies. As such, we invented oral compounds that target and inhibit KRAS alleles for the treatment of KRAS mutant driven cancers.

First generation KRAS G12C inhibitors like Sotorosib, Adagrasib targeting on ‘GDP bound off form (RASOFF) of KRAS G12C mutation have demonstrated promising efficacy. While this treatment has benefited many patients with activating KRAS mutations, almost all who initially benefited will eventually acquire resistance via various mechanism. Increasing cases of KRAS G12C second mutations have been identified either from patients’ samples such as Y96D, R68S, H95D, H95Q, H95R, V8L (Tanaka et al., Cancer Discovery (2021), Awad et al., NEJM (2021), Ho et al., EJC (2021), Zhao et al., Nature (2021), Tsai et al., JCI (2022)), or discovered from saturation mutagenesis (Siyu et al, PNAS (2022)) and ENU mutagenesis (Takamasa et al, J Thorac Oncol (2021)) that demonstrated resistance to KRAS(OFF) G12C inhibitors. Therefore, there are unmet needs to prevent the acquisition of one or more mutations in RAS that confer resistance to the RAS(OFF) inhibitor.

SUMMARY OF THE INVENTION

The present invention relates to novel compounds of formula (I) ,

, wherein R ¹ is , 3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c][1,4]oxazinyl or (C1- 6alkyl)oxoimidazolidinyl; wherein R ⁸ is C _1-6 alkyl; R ⁹ is ((C1-6alkyl)2amino)azetidinyl, C1-6alkylpiperazinyl, haloazetidinyl, haloC1-6alkylamino, haloC1-6alkylaminoazetidinyl, haloC1- 6alkylpiperazinyl, hydroxy(C _1-6 alkyl)piperidinyl or morpholinyl; R ² is C1-6alkyl; R ³ is H or halogen; R ⁴ is H or halogen; R ⁵ is C _1-6 alkyl or haloC _1-6 alkyl; R ⁶ is C1-6alkoxyC1-6alkyl; R ⁷ is morpholinyl, (haloC1-6alkyl)piperazinyl or C1-6alkylpiperazinyl; A ¹ is thiazolylene; A ² is C1-6alkylene; with the proviso that R ³ and R ⁴ are not H simultaneously; or a pharmaceutically acceptable salt thereof. The invention also relates to their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula (I) or (Ia) thereof as inhibitor of KRAS. The compounds of formula (I) or (Ia) show good KRAS inhibition for G12C, G12D and G12V. In another embodiment, the compounds of this invention showed superior cancer cell inhibition and human hepatocyte stability. In addition, the compounds of formula (I) or (Ia) also show good or improved cytotoxicity and solubility profiles. Furthermore, the compound of current invention had excellent pharmacokinetic properties comparing with the reference compounds. BRIEF DESCRIPTION OF THE FIGURE Figure 1. X-ray crystallographic analysis of compound G5. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS The term “C _1-6 alkyl” denotes a saturated, linear or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like. Particular “C1-6alkyl” groups are methyl, ethyl and n-propyl. The term “C _1-6 alkoxy” denotes C _1-6 alkyl-O-. The term “C1-6alkylene” denotes a linear or branched saturated divalent hydrocarbon group of 1 to 6 carbon atoms or a divalent branched saturated divalent hydrocarbon group of 3 to 6 ^{carbon atoms. Examples of C} 1-6 ^{alkylene groups include methylene, ethylene, propylene, 2-} methylpropylene, butylene, 2-ethylbutylene, pentylene, hexylene. The term “halogen” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo. The term “haloC _1-6 alkyl” denotes a C _1-6 alkyl group wherein at least one of the hydrogen atoms of the C1-6alkyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of haloalkyl include monofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, or trifluoromethyl. The term “C3-7cycloalkyl” denotes a monovalent saturated monocyclic or bicyclic hydrocarbon group of 3 to 7 ring carbon atoms. Bicyclic means consisting of two saturated carbocycles having one or more carbon atoms in common. Examples for monocyclic cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Examples for bicyclic cycloalkyl are bicyclo[1.1.0]butyl, bicyclo[2.2.1]heptanyl, bicyclo[1.1.1]pentanyl, or bicyclo[2.2.2]octanyl. The term “thiazolylene” denotes a divalent thiazolyl group. The term “oxo” denotes a divalent oxygen atom =0.

The term “oxoimidazolidinyl” denotes

The term “haloazetidinyl” denotes an azetidinyl group wherein at least one of the hydrogen atoms of the azetidinyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of haloazetidinyl include fluoro azetidinyl and difluoro azetidinyl.

The term “dimethylmethylene” denotes

The term “protecting group” denotes the group which selectively blocks a reactive site in a multifunctional compound such that a chemical reaction can be carried out selectively at another unprotected reactive site in the meaning conventionally associated with it in synthetic chemistry. Protecting groups can be removed at the appropriate point. Exemplary protecting groups are amino -protecting groups, carboxy-protecting groups or hydroxy-protecting groups.

The skilled of the art would understand that the following structures of compounds of formula (la) and (la’) are equal especially for the chiral centers:

The term “pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.

The term “pharmaceutically acceptable acid addition salt” denotes those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, and salicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, /V-cthylpipcridinc, and polyamine resins.

The term “A pharmaceutically active metabolite” denotes a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. After entry into the body, most drugs are substrates for chemical reactions that may change their physical properties and biologic effects. These metabolic conversions, which usually affect the polarity of the compounds of the invention, alter the way in which drugs are distributed in and excreted from the body. However, in some cases, metabolism of a drug is required for therapeutic effect.

The term “therapeutically effective amount” denotes an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein. The therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors. The term “pharmaceutical composition” denotes a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable excipients to be administered to a mammal, e.g., a human in need thereof.

The terms “pharmaceutically acceptable excipient”, “pharmaceutically acceptable carrier” and “therapeutically inert excipient” can be used interchangeably and denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents or lubricants used in formulating pharmaceutical products.

INHIBITOR OF KRAS

The present invention relates to (i) a compound of formula (I), wherein

R ¹ is , 3-oxo-5,6,8,8a-tetrahydro-lH-imidazo[5,l-c][l,4]oxazinyl or (Ci-

6alkyl)oxoimidazolidinyl; wherein R ⁸ is Cnealkyl;

R ⁹ is ((Ci-6alkyl)2amino)azetidinyl, Ci -ealky Ipiperazinyl, haloazetidinyl, haloCi-ealkylamino, haloCnealkylaminoazetidinyl, haloCn ealkylpiperazinyl, hydroxy(Ci -ealky l)piperidinyl or morpholinyl; R ² is C1-6alkyl; R ³ is H or halogen; R ⁴ is H or halogen; R ⁵ is C _1-6 alkyl or haloC _1-6 alkyl; R ⁶ is C _1-6 alkoxyC _1-6 alkyl; R ⁷ is morpholinyl, (haloC1-6alkyl)piperazinyl or C1-6alkylpiperazinyl; A ¹ is thiazolylene; A ² is C _1-6 alkylene; with the proviso that R ³ and R ⁴ are not H simultaneously; or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (ii) a compound of formula (Ia), wherein R ¹ is , 3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c][1,4]oxazinyl or (C1- 6alkyl)oxoimidazolidinyl; wherein R ⁸ is C1-6alkyl; R ⁹ is ((C1-6alkyl)2amino)azetidinyl, C1-6alkylpiperazinyl, haloazetidinyl, haloC _1-6 alkylamino, haloC _1-6 alkylaminoazetidinyl, haloC _{1- 6} alkylpiperazinyl, hydroxy(C _1-6 alkyl)piperidinyl or morpholinyl; R ² is C1-6alkyl; R ³ is H or halogen; R ⁴ is H or halogen; R ⁵ is C _1-6 alkyl or haloC _1-6 alkyl; R ⁶ is C1-6alkoxyC1-6alkyl; R ⁷ is morpholinyl, (haloC _1-6 alkyl)piperazinyl or C _1-6 alkylpiperazinyl; A ¹ is thiazolylene; A ² is C1-6alkylene; with the proviso that R ³ and R ⁴ are not H simultaneously; or a pharmaceutically acceptable salt thereof. A further embodiment of present invention is (iii) a compound of formula (I) or (Ia) according to (i) or (ii), or a pharmaceutically acceptable salt thereof, wherein R ¹ is ; wherein R ⁸ is C1-6alkyl; R ⁹ is C1-6alkylpiperazinyl, haloC1-6alkylpiperazinyl or morpholinyl. A further embodiment of present invention is (iv) a compound of formula (I) or (Ia), according to any one of (i) to (iii), or a pharmaceutically acceptable salt thereof, wherein R ¹ is ; wherein R ⁸ is methyl; R ⁹ is 4-methylpiperazin-1-yl, 4-(2,2,2- trifluoroethyl)piperazin-1-yl or morpholinyl. A further embodiment of present invention is (v) a compound of formula (I) or (Ia) according to any one of (i) to (iv), wherein R ¹ is methyl-(4-methylpiperazine-1-carbonyl)amino, methyl-[4-(2,2,2-trifluoroethyl)piperazine-1-carbonyl]amino or methyl(morpholine-4- carbonyl)amino. A further embodiment of present invention is (vi) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (v), wherein R ² is isopropyl. A further embodiment of present invention is (vii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vi), wherein R ³ is H or fluoro. A further embodiment of present invention is (viii) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vii), wherein R ³ is fluoro.

A further embodiment of present invention is (ix) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (viii), wherein R ⁴ is H or fluoro.

A further embodiment of present invention is (x) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (ix), wherein R ⁴ is H.

A further embodiment of present invention is (xi) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (x), wherein R ⁵ is haloCn ealkyl.

A further embodiment of present invention is (xii) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xi), wherein R ⁵ is 2,2,2- trifluoroethyl.

A further embodiment of present invention is (xiii) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xii), wherein R ⁶ is 1- methoxyethyl.

A further embodiment of present invention is (xiv) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xiii), wherein R ⁷ is (haloCi-6alkyl)piperazinyl.

A further embodiment of present invention is (xv) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xiv), wherein R ⁷ is 4- (2,2,2-trifluoroethyl)piperazin- 1 -yl.

A further embodiment of present invention is (xvi) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xv), wherein A ¹ is

, wherein bond “a” connects to indole ring.

A further embodiment of present invention is (xvii) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xvi), wherein A ² is dimethylmethylene .

Another embodiment of present invention is (xviii) a compound of formula (I) or (la), according to (i) or (ii), wherein ¹ R is ; wherein R ⁸ is C1-6alkyl; R ⁹ is C1-6alkylpiperazinyl, haloC1- 6alkylpiperazinyl or morpholinyl; R ² is C _1-6 alkyl; R ³ is halogen; R ⁴ is H; R ⁵ is haloC _1-6 alkyl; R ⁶ is C1-6alkoxyC1-6alkyl; R ⁷ is (haloC1-6alkyl)piperazinyl; A ¹ is , wherein bond “a” connects to indole ring; A ² is C _1-6 alkylene; or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (xix) a compound of formula (I) or (Ia), according to (xviii), wherein R ¹ is methyl-(4-methylpiperazine-1-carbonyl)amino, methyl-[4-(2,2,2- trifluoroethyl)piperazine-1-carbonyl]amino or methyl(morpholine-4-carbonyl)amino; R ² is isopropyl; R ³ is fluoro; R ⁴ is H; R ⁵ is 2,2,2-trifluoroethyl; R ⁶ is (1S)-1-methoxyethyl; R ⁷ is 4-(2,2,2-trifluoroethyl)piperazin-1-yl; , wherein bond “a” connects to indole ring; A ² is dimethylmethylene; or a pharmaceutically acceptable salt thereof. The present invention relates to (i’) a compound of formula (I),

, wherein R ¹ is or (C1-6alkyl)oxoimidazolidinyl; wherein R ⁸ is C _1-6 alkyl; R ⁹ is ((C _1-6 alkyl) ₂ amino)azetidinyl, haloazetidinyl or morpholinyl; R ² is C1-6alkyl; R ³ is H or halogen; R ⁴ is H or halogen; R ⁵ is C1-6alkyl or haloC1-6alkyl; R ⁶ is C1-6alkoxyC1-6alkyl; R ⁷ is morpholinyl, (haloC _1-6 alkyl)piperazinyl or C _1-6 alkylpiperazinyl; A ¹ is thiazolylene; A ² is C1-6alkylene; with the proviso that R ³ and R ⁴ are not H simultaneously; or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (ii’) a compound of formula (Ia),

wherein R ¹ is or (C1-6alkyl)oxoimidazolidinyl; wherein R ⁸ is C _1-6 alkyl; R ⁹ is ((C _1-6 alkyl) ₂ amino)azetidinyl, haloazetidinyl or morpholinyl; R ² is C1-6alkyl; R ³ is H or halogen; R ⁴ is H or halogen; R ⁵ is C1-6alkyl or haloC1-6alkyl; R ⁶ is C1-6alkoxyC1-6alkyl; R ⁷ is morpholinyl, (haloC _1-6 alkyl)piperazinyl or C _1-6 alkylpiperazinyl; A ¹ is thiazolylene; A ² is C1-6alkylene; with the proviso that R ³ and R ⁴ are not H simultaneously; or a pharmaceutically acceptable salt thereof. A further embodiment of present invention is (iii’) a compound of formula (I) or (la) according to (i’) or (ii’), or a pharmaceutically acceptable salt thereof, wherein R ¹ is

; wherein R ⁸ is Ci-ealkyl; R ⁹ is haloazetidinyl or morpholinyl.

A further embodiment of present invention is (iv’) a compound of formula (I) or (la), according to any one of (i’) to (iii’), or a pharmaceutically acceptable salt thereof, wherein R ¹ is

; wherein R ⁸ is methyl; R ⁹ is 3,3-difluoroazetidin-l-yl or morpholinyl.

A further embodiment of present invention is (v’) a compound of formula (I) or (la) according to any one of (i’) to (iv’), wherein R ¹ is (3,3-difluoroazetidine-l-carbonyl)-methyl- amino or methyl(morpholine-4-carbonyl) amino.

A further embodiment of present invention is (vi’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (v’), wherein R ² is isopropyl.

A further embodiment of present invention is (vii’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (vi’), wherein R ³ is H or fluoro.

A further embodiment of present invention is (viii’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (vii’), wherein R ³ is fluoro.

A further embodiment of present invention is (ix’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (viii’), wherein R ⁴ is H or fluoro.

A further embodiment of present invention is (x’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (ix’), wherein R ⁴ is H.

A further embodiment of present invention is (xi’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (x’), wherein R ⁵ is ethyl or 2,2,2-trifluoroethyl. A further embodiment of present invention is (xii’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (xi’), wherein R ⁶ is 1- methoxyethyl.

A further embodiment of present invention is (xiii’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (xii’), wherein R ⁷ is 4- (2,2,2-trifluoroethyl)piperazin-l-yl or morpholinyl.

A further embodiment of present invention is (xiv’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (xiii’), wherein A ¹ is

, wherein bond “a” connects to indole ring.

A further embodiment of present invention is (xv’) a compound of formula (I) or (la), or a pharmaceutically acceptable salt thereof, according to any one of (i’) to (xiv’), wherein A ² is dimethylmethylene .

Another embodiment of present invention is (xvi’) a compound of formula (I) or (la), according to (i’) or (ii’), wherein

R ¹ is ; wherein R ⁸ is Ci-ealkyl; R ⁹ is haloazetidinyl or morpholinyl;

R ² is Ci-ealkyl;

R ³ is halogen;

R ⁴ is H;

R ⁵ is Ci-ealkyl or haloCi-ealkyl;

R ⁶ is Ci-ealkoxyCnealkyl;

R ⁷ is morpholinyl or (haloCi-6alkyl)piperazinyl;

A ¹ is , wherein bond “a” connects to indole ring;

A ² is Ci-ealkylene; or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (xvii’) a compound of formula (I) or (la), according to (xvi’), wherein

R ¹ is (3,3-difluoroazetidine-l-carbonyl)-methyl-amino or methyl(morpholine-4- carbonyl)amino;

R ² is isopropyl;

R ³ is fluoro;

R ⁴ is H;

R ⁵ is ethyl or 2,2,2-trifluoroethyl;

R ⁶ is (1S)-1 -methoxyethyl;

R ⁷ is 4-(2,2,2-trifluoroethyl)piperazin-l-yl or morpholinyl; wherein bond “a” connects to indole ring;

A ² is dimethylmethylene; or a pharmaceutically acceptable salt thereof.

Another embodiment of present invention is (xx) a compound of formula (I) or (la) selected from the following:

3-(dimethylamino)-A-[(lS)-l-[[(7S,13S)-24-fluoro-(20Af)-2 0-[2-[(lS)-l-methoxyethyl]-5- (4-methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-oxa- 4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl] carbamoyl] -2-methyl-propyl] - N- methy 1-azet idine- 1 -carboxamide;

3-(dimethylamino)-A-[(lS)-l-[[(7S,13S)-25-fluoro-(20Af)-2 0-[2-[(lS)-l-methoxyethyl]-5- (4-methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-oxa- 4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl] carbamoyl] -2-methyl-propyl] -/V- methy l-azct idine- 1 -carboxamide;

A-[( IS)- 1-[ [(7S,13S)-24-fluoro-(20M)-20-[2-[( IS)- 1 -methoxyethyl]-5-(4-methylpiperazin- l-yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluo roethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl] -2-methyl-propyl] - N- methy 1- morphol ine-4-carboxam ide ;

3-(dimethylamino)-A-[(lS)-l-[[(7S,13S)-24-fluoro-(20Af)-2 0-[2-[(lS)-l-methoxyethyl]-5- morpholino-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-oxa-4-thia- 9,21 ,27,28-tctrazapcntacyclo| 17.5.2. 1 ² ' ⁵ . | ^{9 l 3} .0 ²² ' ²⁶ |octacosa- 1 (25),2,5(28), l 9,22(26),23-hcxacn- 7 -yl] carbamoyl] -2-methyl-propyl] - N- methy 1-azet idine- 1 -carboxamide;

7V-[(15)-l-[[(75,135)-21-ethyl-24-fluoro-(20M)-20-[2-[(15 )-l-methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl] -2-methyl-propyl] -N- methy l-morpholine-4-carboxamide ;

7V-[(15)-l-[[(75,135)-24-fluoro-(20M)-20-[2-[(15)-l-metho xyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- l-yl]-3-pyridyl]- 17, 17 -dimethyl- 8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-

2 5 9 13 22 26 oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ’ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl] carbamoyl] -2-methyl-propyl] -N- methyl- morpholine-4- carboxamide;

7V-[(15)-l-[[(75,135)-21-ethyl-24-fluoro-(20M)-20-[2-[(15 )-l-methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-3,3-difluoro-2V-methyl-azetid ine-l-carboxamide;

(2S)-AH(7S, 13S)-21-ethyl- 24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5-morpho lino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-(3-methyl-2-oxo-imidazolidin-l-yl)butanamide;

7V-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS )-l-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-dimethyl-8,1 4-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl] -2-methyl-propyl] -N- methy l-morpholine-4-carboxamide ;

7V-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS )-l-methoxyethyl]-5-(4- methylpiperazin-l-yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-1 5-oxa-4-thia-9,21,27,28- tetrazapentacyclo [17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl] -2-methyl-propyl] -N- methy l-morpholine-4-carboxamide ;

A^-[(lS)-l-[[(7S,13S)-24-fluoro-20-(20Af)-[2-[(lS)-l-meth oxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl) -15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-Af-methyl-morpholine-4-carbox amide; 3-(dimethylamino)-A^-[(lS)-l-[[(7S,13S)-24-fluoro-(20Af)-20- [2-[(lS)-l-methoxyethyl]-5- [4-(2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-di methyl-8,14-dioxo-21-(2,2,2-

2 5 9 13 22 26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ’ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl]carbamo yl]-2-methyl-propyl]-2V- methyl- azetidine- 1- carboxamide;

(2S)-AH(7S, 13S)-21-ethyl- 24-fluoro-(20A0-20-[2-[( IS)- l-methoxyethyl]-5-morpho lino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-[methyl(2,2,2-trifluoroethylcarbamoyl)amino]butanam ide;

^-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS) -l-methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-Af-methyl-3-(trifluoromethyl) azetidine-l-carboxamide;

(2S)-A^-[(7S,13S)-21-ethyl-24-fluoro-(20Af)-20-[2-[(lS)-l -methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-(3-oxo-5,6,8,8a-tetrahydro-177-imidazo[5,l-c][l,4]o xazin-2-yl)butanamide;

(37?)-A^-[(lS)-l-[[(7S,13S)-24-fluoro-(20Af)-20-[2-[(lS)- l-methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl) -15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-3-hydroxy-2V,3-dimethyl-piper idine-l-carboxamide;

^-[(lS)-l-[[(7S,13S)-25-fluoro-(20M)-20-[2-[(lS)-l-methox yethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 17, 17 -dimethyl- 8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-

2 5 9 13 22 26 oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ’ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl] carbamoyl] -2-methyl-propyl]-2V- methyl- morpholine-4- carboxamide;

^-[(lS)-l-[[(7S,13S)-24-fluoro-(20M)-20-[2-[(lS)-l-methox yethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1-yl] -3-pyridyl] - 17, 17 -dimethyl- 8, 14-dioxo-21-(2, 2, 2-trifluoroethyl)- 15-

2 5 9 13 22 2fa oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ’ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl]carbamo yl] -2-methyl-propyl] -A^,4-dimethyl-piperazine- 1 -carboxamide; and A-[(15)-l-[[(75,135)-24-fluoro-(20M)-20-[2-[(15)-l-methoxyet hyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 17, 17 -dimethyl- 8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-

2 5 9 13 22 26 oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ’ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl] carbamoyl] -2-methyl-propyl] -A- methyl-4-(2, 2,2- trifluoroethyl)piperazine- 1 -carboxamide ; or a pharmaceutically acceptable salt thereof.

Another embodiment of present invention is related to (xxi) a process for the preparation of a compound according to any one of (i) to (xix) or (i’) to (xvii’) comprising the following step: a) coupling reaction between compound of formula (II), the presence of a coupling reagent and a base to form the compound of formula (I); wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , A ¹ and A ² are defined as in any one of (i) to (xix); the coupling reagent is T3P, HATU, PyBOP or EDCI/HOBt; the base is TEA, DIEPA or DMAP.

Another embodiment of present invention is (xxii) a compound or pharmaceutically acceptable salt according to any one of (i) to (xx) or (i’) to (xvii’) for use as therapeutically active substance.

Another embodiment of present invention is (xxiii) a pharmaceutical composition comprising a compound in accordance with any one of (i) to (xx) or (i’) to (xvii’) and a pharmaceutically acceptable excipient.

Another embodiment of present invention is (xxiv) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for treating a KRAS G12C protein-related disease.

Another embodiment of present invention is (xxv) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for treating a KRAS G12C, G12D and G12V protein-related disease. Another embodiment of present invention is (xxvi) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for inhibiting RAS interaction with downstream effectors, wherein the downstream effectors are RAF and PI3K.

Another embodiment of present invention is (xxvii) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for inhibiting the propagating oncogenic MAPK and PI3K signaling.

Another embodiment of present invention is (xxviii) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer is selected from pancreatic cancer, colorectal cancer, lung cancer, esophageal cancer, gallbladder cancer, melanoma ovarian cancer and endometrial cancer.

Another embodiment of present invention is (xxix) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer is selected from pancreatic adenocarcinoma, colorectal cancer and non- small cell lung cancer.

Another embodiment of present invention is (xxx) a compound or pharmaceutically acceptable salt according to any one of (i) to (xx) or (i’) to (xvii’) for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer is selected from pancreatic adenocarcinoma, colorectal cancer and non-small cell lung cancer.

Another embodiment of present invention is (xxxi) a compound or pharmaceutically acceptable salt according to any one of (i) to (xx) or (i’) to (xvii’) for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer comprises a first mutation that is G12C, and a second mutation at a position selected from V8A, V9Y, S17E, T58I, A59T, S65W, R68S, D69P, M72I, D92R, H95N, Y96D, Q99F, Q99W, Y96H, and F156L.

Another embodiment of present invention is (xxxii) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for the preparation of a medicament for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer is selected from pancreatic adenocarcinoma, colorectal cancer and non-small cell lung cancer.

Another embodiment of present invention is (xxxiii) the use of a compound according to any one of (i) to (xx) or (i’) to (xvii’) for the preparation of a medicament for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer comprises a first mutation that is G12C, and a second mutation at a position selected from V8A, V9Y, SUE, T58I, A59T, S65W, R68S, D69P, M72I, D92R, H95N, Y96D, Q99F, Q99W, Y96H, and F156L.

Another embodiment of present invention is (xxxiv) a method for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer is selected from pancreatic adenocarcinoma, colorectal cancer and non-small cell lung cancer, which method comprises administering a therapeutically effective amount of a compound as defined in any one of (i) to (xx) or (i’) to (xvii’).

Another embodiment of present invention is (xxxv) a method for the treatment or prophylaxis of KRAS mutation driven cancers, wherein the cancer comprises a first mutation that is G12C, and a second mutation at a position selected from V8A, V9Y, S17E, T58I, A59T, S65W, R68S, D69P, M72I, D92R, H95N, Y96D, Q99F, Q99W, Y96H, and F156L.

Another embodiment of present invention is (xxxvi) a compound or pharmaceutically acceptable salt according to any one of (i) to (xx) or (i’) to (xvii’), when manufactured according to a process of (xxi).

PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer, at pH 5. In another embodiment, the compounds of formula (I) are sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.

Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “effective amount” of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit mutant RAS (e.g. KRAS G12C) interaction with RAF, blocking the oncogenic MAPK signaling. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.

In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.1 to 1000 mg/kg, alternatively about 0.1 to 1000 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 1 to about 1000 mg of the compound of the invention.

The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.

A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).

An example of a suitable oral dosage form is a tablet containing about 1 to 1000 mg of the compound of the invention compounded with about 1 to 1000 mg anhydrous lactose, about 1 to 1000 mg sodium croscarmellose, about 1 to 1000 mg polyvinylpyrrolidone (PVP) K30, and about 1 to 1000 mg magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of the PVP. The resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound, for example 5 to 400mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.

An embodiment, therefore, includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.

Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment of mutant KRAS-driven cancers. Another embodiment includes a pharmaceutical composition comprising a compound of Formula (I) for use in the treatment of mutant KRAS-driven cancers.

The following composition A and B illustrate typical compositions of the present invention, but serve merely as representative thereof.

Composition A

A compound of the present invention can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:

Per tablet

Active ingredient 200 mg

Microcrystalline cellulose 155 mg

Corn starch 25 mg

Talc 25 mg

Hydroxypropylmethylcellulose 20 mg

425 mg

Composition B A compound of the present invention can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:

Per capsule

Active ingredient 100.0 mg

Corn starch 20.0 mg

Lactose 95.0 mg

Talc 4.5 mg

Magnesium stearate 0,5 mg

220.0 mg

INDICATIONS AND METHODS OF TREATMENT

The compounds of the invention induce a new binding pocket in KRAS by driving formation of a high affinity tri-complex between KRAS protein and the widely expressed cyclophilin A (CYPA), which inhibit KRAS interaction with downstream effectors, such as RAF and PI3K. Accordingly, the compounds of the invention are useful for inhibiting the propagating oncogenic MAPK and PI3K signaling, reducing cell proliferation, in particular cancer cells. Compounds of the invention are useful for termination of RAS signaling in cells that express RAS mutant, e.g. KRAS mutation driven pancreatic cancer, colorectal cancer, lung cancer, esophageal cancer, gallbladder cancer, melanoma ovarian cancer, endometrial cancer, etc. Alternatively, compounds of the invention are useful for termination of RAS signaling in malignant solid tumor where the oncogenic role of KRAS mutation is reinforced by dysregulation or mutation of effector pathways as MAPK, PI3K-AKT-mTOR (Mammalian target of rapamycin) driven signaling, for targeted therapy in pancreatic adenocarcinoma, colorectal cancer, non- small cell lung cancer, etc.

Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of formula (I), a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

SYNTHESIS

The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R ¹ to R ⁷ , A ¹ and A ² are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.

General synthetic routes for preparing the compound of formula (I) are shown below.

Scheme 1

Compound of formula II was synthesized according to the procedure described in Intermediate A to J. Compound of formula (I) can be obtained by a coupling reaction between acid (III) and compound of formula (II) with coupling reagent(s), such as T3P, HATU, PyBOP and EDCI/HOBt, in the presence of a base, such as TEA, DIEPA and DMAP.

Compounds of this invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, e.g. (chiral) HPLC or SFC. In another embodiment, compound of formula (I) can be obtained according to above scheme by using corresponding chiral starting materials.

This invention also relates to a process for the preparation of a compound of formula (I) comprising following step: a) coupling reaction between compound of formula (II),

in the presence of a coupling reagent and a base to form the compound of formula (I); wherein in step a) the coupling reagent can be, for example, T3P, HATU, PyBOP or EDCI/HOBt; the base can be, for example, TEA, DIEPA or DMAP.

A compound of formula (I) or (la) when manufactured according to the above process is also an object of the invention.

EXAMPLES

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

ABBREVIATIONS

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

Abbreviations used herein are as follows:

ACN acetonitrile aq. Aqueous

BOC-L- valine (.S')-2-((/c/7-butoxycarbonyl)amino)-3-mcthylbutanoic acid

Boc-V-Mc-Val-OH V-(tert-Butoxycarbonyl)-V-methyl-L- valine

(BOC) ₂ O Di-tert-butyldicarbonate

(/?)-binap (7?)-(+)-2,2'-Bis(diphenylphosphino)-l,T-binaphthyl

CDCI3: deuterated chloroform

CDI l,l'-Carbonyldiimidazole

CD3OD: deuterated methanol COMU (l-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino- morpholino -carbenium hexafluoropho sphate

DIEPA: N, A-diethylpropylamine

DIBAL-H Diisobutylaluminium hydride

DMAP: 4-Dimethylaminopyridine

DMF: dimethyl formamide

DMP 1,1,1 -Tris(acetyloxy)- 1 , 1 -dihydro- 1 ,2-benziodoxol-3-( 177)-one

DMSO: dimethyl sulfoxide

EDCI: N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride

EtOAc or EA: ethyl acetate

FRET fluorescence resonance energy transfer

HATU: (l-[Bis(dimethylamino)methylene]-l//-l,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluoropho sphate) hr(s): hour(s)

HPLC: high performance liquid chromatography

HOBt: A-hydroxybenzotriazole

H-VAL-OTBU HC1 (S')-tert-Butyl 2-amino-3-methylbutanoate hydrochloride [Ir(OMe)(COD)] ₂ ( 1 ,5-Cyclooctadiene)(methoxy)iridium(I) dimer LDA Lithium diisopropylamide

MS: (ESI): mass spectroscopy (electron spray ionization) min(s) minute(s) MTBE Methyl tert-butyl ether NMM N- Methylmorpholine NaBH(OAc) ₃ Sodium triaceto xyborohydride NMR: nuclear magnetic resonance NMO 4-Methylmorpholine A-oxide obsd. Observed Pd(dppf)Cl ₂ [1,1 '-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(dtbpf)Cl ₂ [l,r-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium( II) prep-HPLC preparative high performance liquid chromatography PyBOP: benzo triazo 1- 1 - ylo xytripyrro lidinopho sphonium hexafluoropho sphate

RT or rt: room temperature sat. saturated

SFC supercritical fluid chromatography

TEA: triethylamine

TFA: trifluoro acetic acid

THF: tetrahydro furan

TEA: trimethylamine

TMEDA Tetramethylethylenediamine

TMSCF3 Trifluoromethyltrimethylsilane

T3P: propylphospho nic anhydride

GENERAL EXPERIMENTAL CONDITIONS

Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and the Quad 12/25 Cartridge module, ii) ISCO combi-flash chromatography instrument. Silica gel brand and pore size: i) KP-SIL 60 A, particle size: 40-60 pm; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.

Intermediates and final compounds were purified by preparative HPLC on reversed phase column using XBridge™ Prep-C18 (5 pm, OBDTM 30 x 100 mm) column, SunFire™ Prep-C18 (5 pm, OBD™ 30 x 100 mm) column, Phenomenex Synergi-C18 (10 pm, 25 x 150 mm) or Phenomenex Gemini-C18 (10 pm, 25 x 150 mm). Waters AutoP purification System (Sample Manager 2767, Pump 2525, Detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water; acetonitrile and 0.1% FA in water or acetonitrile and 0.1% TFA in water). Or Gilson-281 purification System (Pump 322, Detector: UV 156, solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HC1 in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).

For SFC chiral separation, intermediates were separated by chiral column (Daicel chiralpak IC, 5 pm, 30 x 250 mm), AS (10 pm, 30 x 250 mm) or AD (10 pm, 30 x 250 mm) using Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar 80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IP A) or CO2 and MeOH (0.1% NEL-EhO in MeOH), back pressure lOObar, detection UV @ 254 or 220 nm. LC/MS spectra of compounds were obtained using a LC/MS (Waters™ Alliance 2795- Micromass ZQ, Shimadzu Alliance 2020-Micromass ZQ or Agilent Alliance 6110-Micromass ZQ), LC/MS conditions were as follows (running time 3 or 1.5 mins):

Acidic condition I: A: 0.1% TFA in H2O; B: 0.1% TFA in acetonitrile;

Acidic condition II: A: 0.0375% TFA in H2O; B: 0.01875% TFA in acetonitrile;

Basic condition I: A: 0.1% NH3 H2O in H2O; B: acetonitrile;

Basic condition II: A: 0.025% NFL-FhO in H2O; B: acetonitrile;

Neutral condition: A: H2O; B: acetonitrile.

Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (MH) ⁺ .

NMR Spectra were obtained using Bruker Avance 400 MHz or 500MHz.

The microwave assisted reactions were carried out in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were performed under an argon or nitrogen atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted.

PREPARATIVE EXAMPLES

The following examples are intended to illustrate the meaning of the present invention but should by no means represent a limitation within the meaning of the present invention:

Preparation of Intermediate

Intermediate A l-[6-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2-diox aborolan-2-yl)-3-pyridyl]-4- methyl-piperazine

Cbz

The title intermediate A was prepared according to the following scheme:

A5 Intermediate A

Step 1: Preparation of 3-bromo-2-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2 - dioxaborolan-2-yl)pyridine (compound A2)

To a solution of 3-bromo-2-[(lS)-l-methoxyethyl]pyridine (compound Al, 2.0 g, 9.26 mmol) and bis(pinacolato)diboron (3.5 g, 13.9 mmol) in THF (30 mL) were added 4,4'-di-tert- butyl-2,2'-bipyridin (372.7 mg, 1.39 mmol) and [Ir(OMe)(COD)]2 (306.3 mg, 0.460 mmol). The mixture was stirred at 75 °C for 16 hours under N2 protection. The mixture was filtrated and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (EA/PE: 0-20%) to afford 3-bromo-2-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2 - dioxaborolan-2-yl)pyridine (compound A2, 2.4 g) as yellow oil. ^J H NMR (400 MHz, CDC13) 5 ppm 8.91 (d, J = 1.4 Hz, 1 H), 8.21 (d, J = 1.4 Hz, 1 H), 4.95 (q, J = 6.5 Hz, 1 H), 3.30 (s, 3 H), 1.49 (d, J = 6.5 Hz, 3 H), 1.35 (s, 12 H).

Step 2: Preparation of 3-bromo-5-iodo-2-[(lS)-l-methoxyethyl]pyridine (compound A3)

To a solution of 3-bromo-2-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2 - dioxaborolan-2-yl)pyridine (compound A2, 2.5 g, 7.3 mmol) in ACN (40 mL) was added N- iodo succinimide (4.1 g, 18.27 mmol). The mixture was stirred at 90 °C for 40 hrs under N2 protection. The reaction was quenched with saturated solution of Na2SOa (40 mL) and the reaction mixture was extracted with EtOAc (30 mL, twice). The combined organic layer was washed with brine (50 mL), filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (EA/PE: 0-20%) to afford 3-bromo-5-iodo-2-[(1S)-1- methoxyethyl]pyridine (compound A3, 660 mg) as yellow oil. MS calc’d 342 (MH ⁺ ), measured 341.8 (MH ⁺ ). Step 3: Preparation of benzyl 4-[5-bromo-6-[(1S)-1-methoxyethyl]-3- pyridyl]piperazine-1-carboxylate (compound A5) To a solution of 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine (compound A3, 660 mg, 1.9 mmol) and 1-Cbz-piperazine (compound A4, 425.1 mg, 1.9 mmol) in toluene (10 mL) were added cesium carbonate (1.6 g, 4.83 mmol), (R)-BINAP (60.1 mg, 0.1 mmol) and palladium (II) acetate (43.3 mg, 0.19 mmol). The mixture was stirred at 100 °C for 12 hours under N2 protection. The mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel chromatography (EA/PE: 0-50%) to afford benzyl 4-[5-bromo-6-[(1S)- 1-methoxyethyl]-3-pyridyl]piperazine-1-carboxylate (compound A5, 740 mg) as a yellow solid. MS calc’d 434.1 (MH ⁺ ), measured 434.1 (MH ⁺ ). Step 4: Preparation of 1-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-pyridyl]-4-methyl-piperazine (Intermediate A) To a solution of benzyl 4-[5-bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]piperazine-1- carboxylate (compound A5, 740 mg, 1.7 mmol) and bis(pinacolato)diboron (519.2 mg, 2.04 mmol) in toluene (12 mL) were added KOAc (418.0 mg, 4.26 mmol) and Pd(dppf)Cl ₂ (124.7 mg, 0.170 mmol). The reaction mixture was stirred at 90 °C for 12 hrs under N2 protection. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column to afford 1-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-pyridyl]-4-methyl-piperazine (Intermediate A, 470 mg) as a brown solid. MS calc’d 482.3 (MH ⁺ ), measured 482.2 (MH ⁺ ). Intermediate B Methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonyla mino)- propanoyl]hexahydropyridazine-3-carboxylate

The intermediate B was prepared according to the following scheme:

Intermediate B Step 1: Preparation of (4-bromothiazol-2-yl)methanol (compound B2)

To a solution of 4-bromothiazole-2-carboxaldehyde (compound Bl ₄ 6.0 g, 31.25 mmol) in methanol (70 mL) was added sodium borohydride (1.7 g, 46.87 mmol) at 0 °C. The mixture was stirred at 25 °C for 1 hour. The reaction was quenched with water (300 mL) at 0 °C and the reaction mixture was extracted by ethyl acetate (200 mL, three times). The combined organic phase was washed with brine (150 mL, twice), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum to afford (4-bromothiazol-2-yl)methanol (compound B2, 6g) as colorless oil.

Step 2: Preparation of 4-bromo-2-(bromomethyl)thiazole (compound B3)

To a solution of (4-bromothiazol-2-yl)methanol (compound B2, 6.0 g, 30.92 mmol) in DCM (80 mL) was added CBr4 (15.4 g, 46.38 mmol) and triphenylphosphine (12.1 g, 46.38 mmol) at 0 °C. After being stirred at 25 °C for 1 hour, the mixture was filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column, eluted with ethyl acetate in petroleum ether (0-10%) to afford (4-bromothiazol-2-yl)methanol (compound B3, 6.0 g) as yellow oil. MS calc’d 255.9 (MH ⁺ ), measured 255.9 (MH ⁺ ).

Step 3: Preparation of 4-bromo-2-[[(2S,5/?)-5-isopropyl-3,6-dimethoxy-2,5- dihydropyrazin-2-yl]methyl]thiazole (compound B5)

To a mixture of (7?)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (compound B4, 4.3 g, 23.45 mmol) in THF (60 mL) was added n-butyllithium (10 mL, 25.22 mmol, 2.5 M) at -78 °C slowly. After addition, the mixture was stirred for 0.5 hour at -78 °C. 4-bromo-2- (bromomethyl)thiazole (compound B3, 5.4 g, 21.02 mmol) was added into above mixture at - 78 °C which was stirred for another 1 hour. The reaction was quenched with saturated solution of NH4CI (100 mL) and the reaction mixture was extracted with EtOAc (100 mL, twice). The combined organic layer was washed with brine (150 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum. The residue was purified by reversed - phase chromatography to afford 4-bromo-2-[[(25',57?)-5-isopropyl-3,6-dimethoxy-2,5- dihydropyrazin-2-yl]methyl]thiazole (compound B5, 3.6 g) as yellow oil. MS calc’d 360 (MH ⁺ ), measured 359.9 (MH ⁺ ).

Step 4: Preparation of methyl (2S)-2-amino-3-(4-bromothiazol-2-yl)propanoate (compound B6)

To a solution of 4-bromo-2-[[(25',57?)-5-isopropyl-3,6-dimethoxy-2,5-dihydrop yrazin-2- yl] methyl] thiazole (compound B5, 3.6 g, 10 mmol) in ACN (20 mL) was added hydrochloric acid (66.6 mL, 0.3 M). The mixture was stirred at 25 °C for 2 hours. The mixture was basified by saturated solution of NaHCOs until pH=8. The mixture was extracted with EtOAc (80 mL, six times). The combined organic layer was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum to afford methyl (2S)-2-amino-3-(4-bromothiazol-2- yl)propanoate (compound B6, 3.1 g) as yellow oil. MS calc’d 264.9 (MH ⁺ ), measured 264.9 (MH ⁺ ).

Step 5: Preparation of methyl (2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoate (compound B7)

To a solution of methyl (2S)-2-amino-3-(4-bromothiazol-2-yl)propanoate (compound B6, 3.1 g, 11.69 mmol) in DCM (40 mL) were added triethylamine (2.9 g, 29.23 mmol) and (BochO (3.8 g, 17.54 mmol). After being stirred at 30 °C for 12 hours, the mixture was concentrated under vacuum. The residue was purified by silica gel column, eluted with ethyl acetate in petroleum ether (0-30%) to afford methyl (2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoate (compound B7, 3.2 g) as yellow oil. MS calc’d 387(MNa ⁺ ), measured 386.9 (MNa ⁺ ).

Step 6: Preparation of (2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)- propanoic acid (compound B8)

To a solution of methyl (2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoate (compound B7, 3.2 g, 8.76 mmol) in THF (30 mL), methanol (2 mL) and water (10 mL) was added lithium hydroxide (0.4 mL, 43.81 mmol). After being stirred at 25 °C for 1 hour, the reaction mixture was acidified by 1 M solution of HC1 until pH=5. The mixture was extracted with EtOAc (40 mL, twice). The combined organic layer was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum to afford (25')-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoic acid (compound B8, 3.1 g) as yellow oil. MS calc’d 373(MNa ⁺ ), measured 372.9 (MNa ⁺ ).

Step 7: Preparation of methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2-(terz- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxyl ate (Intermediate B)

To a solution of (2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)pro panoic acid (compound B8, 3.1 g, 8.83 mmol) in DCM (50 mL) was added methyl (3S)- hexahydropyridazine-3-carboxylate;hydrochloride (compound B9, 2.4 g, 13.24 mmol), EDO (3.4 g, 17.65 mmol), 1 -Hydroxybenzotriazole (238.5 mg, 1.77 mmol) and NMM (9.92 mL, 88.26 mmol) at 0 °C. After being stirred at 25 °C for 1 hour, the reaction mixture was diluted with water (60 mL) and extracted with EtOAc (60 mL, three times). The combined organic layer was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum. The residue was purified by silica gel column and eluted with ethyl acetate in petroleum ether (10-30%) to afford methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2- (tert-butoxycarbonylamino)propanoyl]hexahydropyridazine-3-ca rboxylate (intermediate B, 2.4 g). MS calc’d 477(MH ⁺ ), measured 476.9 (MH ⁺ ).

Intermediate C

(75,135)-7-amino-24-fluoro-(20A/)-20-[2-[(15)-l-methoxyet hyl]-5-(4-methylpiperazin-l-yl)- 3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4 -thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione

The title intermediate C was prepared according to the following scheme:

C13 intermediate C

Step 1: Preparation of l-(5-bromo-6-fluoro-lH-indol-3-yl)-3-((tert-butyldiphenylsil yl) oxy)-2,2-dimethylpropan-l-one (compound C3) To a mixture of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoyl chloride (compound Cl, 35.0 g, 116.8 mmol) in DCM (400 mL) at 0 °C was added a solution of SnCU (97.2 mL, 121.5 mmol) slowly. After the mixture was stirred at - 40 °C for 0.5 hour, 5-bromo-6- fluoro-177-indole (compound C2, 25.0 g, 116.8 mmol) in DCM (200 mL) was added dropwise and the mixture was stirred at - 40 °C for another 15 min. After the reaction was completed, it was quenched with sat.NaHCOs aq. (800 mL), and the reaction mixture was extracted with EtOAc (900 mL, twice). The combined organic layer was washed with brine (700 mL), dried over NaiSCU, filtered and concentrated in vacuo. The residue was triturated with the solution (100 mL, Petroleum ether: Ethyl acetate = 8:1) and filtered. The filter cake was dried in vacuo to afford l-(5-bromo-6-fluoro-177-indol-3-yl)-3-((tertbutyldiphenylsil yl)oxy)-2,2-dimethylpropan- 1-one (compound C3, 50.0 g) as a yellow solid. MS calc’d 552.1 (MH ⁺ ), measured 552.1 (MH ⁺ ).

Step 2: Preparation of [3-(5-bromo-6-fluoro- lH-indol-3-yl)-2,2-dimethyl-propoxy ]- tert-butyl-diphenyl-silane (compound C4)

To a mixture of l-(5-bromo-6-fluoro-177-indol-3-yl)-3-((tertbutyldiphenylsil yl)oxy)-2,2- dimethylpropan-l-one (compound C3, 50.0 g, 90.49 mmol) in THF (600 mL) was added LiBPL (48.4 mL, 193.49 mmol, 4 M in THF) dropwise at 0 °C. The mixture was stirred at 70 °C for 24 hrs under nitrogen atmosphere. After the reaction was completed, it was quenched by addition of water (600 mL) at 0 °C slowly and the reaction mixture was extracted with EtOAc (600 mL, twice). The combined organic layer was washed with brine (600 mL), dried over NaiSO4, filtered and concentrated in vacuo. The residue was purified by silica column chromatography (EtOAc in PE = 20% ~ 33%) to afford [3-(5-bromo-6-fluoro-177-indol-3-yl)-2,2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (compound C4, 46.0 g) as a white solid. MS calc’d 538.1 (MH ⁺ ), measured 538.2 (MH ⁺ ).

Step 3: Preparation of [3-(5-bromo-6-fluoro-2-iodo-lH-indol-3-yl)-2,2-dimethyl- propoxy |-/c/7-butyl-diphenyl-silane (compound C5)

To a mixture of [3-(5-bromo-6-fluoro-177-indol-3-yl)-2,2-dimethyl-propoxy]-t ert-butyl- diphenyl- silane (compound C4, 35.4 g, 65.73 mmol) and iodine (18.4 g, 72.3 mmol) in THF (400 mL) was added silver trifluoromethanesulfonate (20.3 g, 78.88 mmol) at 0 °C. The mixture was stirred at 0 °C for 10 min. After the reaction was completed, it was quenched by sat. NaiSCb aq. (400 mL) and EtOAc (400 mL) and the reaction mixture was filtered. The organic layer was washed with brine (100 mL), dried over NaiSO4, filtered and concentrated in vacuo. The residue was purified by silica column chromatography (EtOAc in PE = 0% ~ 2.5%) to afford [3-(5- bromo-6-fluoro-2-iodo-177-indol-3-yl)-2,2-dimethyl-propoxy]- tert-butyl-diphenyl-silane (compound C5, 43.0 g) as a yellow solid. MS calc’d 664.0 (MH ⁺ ), measured 664.1 (MH ⁺ ).

Step 4: Preparation of benzyl 4-[5-[5-bromo-3-[3-[ter/-butyl(diphenyl)silyl]oxy-2,2- dimethyl-propyl]-6-fhioro- lH-indol-2-yl]-6-[( IS)- 1 -methoxyethyl ]-3-pyridyl ]piperazine- 1- carboxylate (compound C6)

To a mixture of [3-(5-bromo-6-fluoro-2-iodo-177-indol-3-yl)-2,2-dimethyl-pro poxy]-tert- butyl-diphenyl- silane (compound C5, 16.7 g, 25.13 mmol) and benzyl 4-[6-[(lS)-l- methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl )-3-pyridyl]piperazine-l- carboxylate (Intermediate A, 16.7 g, 34.69 mmol) in a mixed solution of 1,4-dioxane (270 mL)/toluene (90 mL) /water (90 mL) were added potassium phosphate (15.7 g, 73.92 mmol) and Pd(dppf)Ch (920 mg, 1.26 mmol). The mixture was stirred at 70 °C for 12 hrs under nitrogen atmosphere. After the reaction was completed, the mixture was filtered and concentrated in vacuo. The residue was purified by silica column chromatography (EtOAc in PE = 20% ~ 50%) to afford 4-[5-[5-bromo-3-[3-[tert-butyl(diphenyl)silyl]oxy-2,2-dimeth yl-propyl]-6-fluoro- 177- indol-2-yl]-6-[(lS)-l-methoxyethyl]-3-pyridyl]piperazine-l-c arboxylate (compound C6, 19.5 g) as a white solid. MS calc’d 891.3 (MH ⁺ ), measured 891.3 (MH ⁺ ).

Step 5: Preparation of benzyl 4-[(5M)-5-[5-bromo-3-[3-[tert-butyl(diphenyl)silyl]oxy- 2,2-dimethyl-propyl]-6-fhioro-l-(2,2,2-trifhioroethyl)indol- 2-yl]-6-[(lS)-l-methoxyethyl]-3- pyridyl]piperazine-l-carboxylate(compound C7)

To a solution of 4-[5-[5-bromo-3-[3-[tert-butyl(diphenyl)silyl]oxy-2,2-dimeth yl-propyl]-6- fluoro- 177-indo 1-2-yl] -6- [( IS)- 1 -methoxyethyl] -3-pyridyl]piperazine-l -carboxylate (compound C6, 14.5 g, 16.26 mmol) and CS2CO3 (15. 9 g, 48.77 mmol) in DMF (200 mL) was added 2,2,2- trifluoroethyl trifluoromethanesulfonate (37.7 g, 162.56 mmol) dropwise at 0 °C, and the mixture was stirred at 20 °C for 12 hrs. After the reaction was completed, EtOAc (70 mL) and water (100 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (70 mL, twice). Combined organic layer was washed with brine (100 mL, four times), dried over Na2SO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by silica column chromatography to afford benzyl 4-[(5Af)-5-[5-bromo-3-[3-[tert- butyl(diphenyl)silyl]oxy-2,2-dimethyl-propyl]-6-fluoro-l-(2, 2,2-trifluoroethyl)indol-2-yl]-6- [(lS)-l-methoxyethyl]-3-pyridyl]piperazine-l-carboxylate (compound C7, 8.0 g, faster eluted) as yellow oil. MS calc’d 973.3 (MH ⁺ ), measured 973.2 (MH ⁺ ). Step 6: Preparation of benzyl 4-[(5M)-5-[5-bromo-6-fluoro-3-(3-hydroxy-2,2- dimethyl-propyl)-l-(2,2,2-trifluoroethyl)indol-2-yl]-6-[(15) -l-methoxyethyl]-3- pyridyl]piperazine-l-carboxylate (compound C8)

To a solution of benzyl 4-[(5Af)-5-[5-bromo-3-[3-[terLbutyl(diphenyl)silyl]oxy-2,2- dimethyl-propyl]-6-fhioro-l-(2,2,2-trifhioroethyl)indol-2-yl ]-6-[(15')-l-methoxyethyl]-3- pyridyl]piperazine-l -carboxylate (compound C7, 10.5 g, 10.78 mmol) in DMF (130 mL) was added cesium fluoride (8.2 g, 53.9 mmol) and the mixture was stirred at 60 °C for 24 hrs. After the reaction was completed, EtOAc (100 mL) and water (100 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (100 mL, twice). The combined organic layer was washed with brine (80 mL, three times), dried over NaiSO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by silica column chromatography (EtOAc in PE = 25% ~ 66%) to afford benzyl 4-[(5M)-5-[5-bromo-6-fluoro-3- (3-hydroxy-2,2-dimethyl-propyl)-l-(2,2,2-trifluoroethyl)indo l-2-yl]-6-[(15')-l-methoxyethyl]-3- pyridyl]piperazine-l -carboxylate (compound C8, 6.5 g) as a yellow solid. MS calc’d 735.2 (MH ⁺ ), measured 735.1 (MH ⁺ ).

Step 7: Preparation of benzyl 4-[(5M)-5-[6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)- 5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l-(2,2,2-tri fluoroethyl)indol-2-yl]-6-[(lS)-l- methoxyethyl]-3-pyridyl]piperazine-l-carboxylate (compound C9)

To a solution of benzyl 4-[(5M)-5-[5-bromo-6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl )- 1 -(2,2, 2-trifhioroethyl)indo 1-2-yl] -6- [( 1 S)- 1-methoxyethyl] -3-pyridyl]piperazine- 1 -carboxylate (compound C8, 5.4 g) , bis(pinacolato)diboron (2.8 g, 11.01 mmol) and potassium acetate (1.2 mL, 18.35 mmol) in toluene (70 mL) was added Pd(dppf)Ch (537.1 mg, 0.73 mmol). The mixture was degassed and purged with nitrogen atmosphere for three times and the mixture was stirred at 90 °C for 12 hrs. After the reaction was completed, the mixture was cooled to room temperature. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by silica column chromatography (EtOAc in PE = 25% ~ 66%) to afford benzyl 4-[(5M)-5-[6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-5-(4,4 ,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-l-(2,2,2-trifluoroethyl )indol-2-yl]-6-[(15')-l- methoxyethyl]-3-pyridyl]piperazine-l -carboxylate (compound C9, 5.2 g) as yellow oil. MS calc’d 783.3 (MH ⁺ ), measured 783.3 (MH ⁺ ).

Step 8: Preparation of methyl (3S)-l-[(2S)-3-[4-[(2M)-2-[5-(4- benzyloxycarbonylpiperazin-l-yl)-2-[(lS)-l-methoxyethyl]-3-p yridyl]-6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-l-(2,2,2-trifluoroethyl)indol-5 -yl]thiazol-2-yl]-2-(tert- butoxycarbonylamino)-propanoyl]hexahydropyridazine-3-carboxy late (compound CIO)

To a mixture of methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxyl ate (intermediate B, 2.7 g, 5.69 mmol), benzyl 4-[(5M)-5-[6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-5-(4,4 ,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)-l-(2,2,2-trifluoroethyl )indol-2-yl]-6-[(15')-l- methoxyethyl]-3-pyridyl]piperazine-l -carboxylate (compound C9, 4.9 g, 6.32 mmol) in toluene (60 mL)/l,4-dioxane (20 mL) / water (20 mL) were added K3PO4 (3.4 g, 15.81 mmol) and Pd(dtbpf)C12 (412.2 mg, 0.63 mmol) under nitrogen atmosphere. The mixture was stirred at 70 °C for 12 hrs. After the reaction was completed, the mixture was concentrated in vacuo to give a residue. The residue was purified by silica column (EtOAc in PE = 10% ~ 75%) to afford methyl (3S)-l-[(2S)-3-[4-[(2M)-2-[5-(4-benzyloxycarbonylpiperazin-l -yl)-2-[(lS')-l-methoxyethyl]-3- pyridyl]-6-fhioro-3-(3-hydroxy-2,2-dimethyl-propyl)-l-(2,2,2 -trifluoroethyl)indol-5-yl]thiazol-2- yl]-2-(tert-butoxycarbonylamino)-propanoyl]hexahydropyridazi ne-3-carboxylate (compound CIO, 3.6 g) as a brown solid. MS calc’d 1053.4 (MH ⁺ ), measured 1053.3 (MH ⁺ ).

Step 9: Preparation of (3S)-l-[(2S)-3-[4-[(2M)-2-[5-(4-benzyloxycarbonylpiperazin-l - yl)-2-[(lS)-l-methoxyethyl]-3-pyridyl]-6-fluoro-3-(3-hydroxy -2,2-dimethyl-propyl)-l-(2,2,2- trifluoroethyl)indol-5-yl]thiazol-2-yl]-2-(tert-butoxycarbon ylamino)propanoyl]hexahy- dropyridazine-3-carboxylic acid (compound Cll)

To a solution of methyl (35')-l-[(25')-3-[4-[(2Af)-2-[5-(4-benzyloxycarbonylpiperazi n-l- yl)-2-[(lS)-l-methoxyethyl]-3-pyridyl]-6-fluoro-3-(3-hydroxy -2,2-dimethyl-propyl)-l-(2,2,2- trifluoroethyl)indol-5-yl]thiazol-2-yl]-2-(tert-butoxycarbon ylamino)-propanoyl]- hexahydropyridazine-3-carboxylate (compound CIO, 3.6 g, 3.42 mmol) in DCE (50 mL) was added trimethylstannanol (2.4 g, 13.67 mmol) and the mixture was stirred at 60 °C for 12 hrs. After the reaction was completed, EtOAc (80 mL) and water (60 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (80 mL, twice). The combined organic layer was washed with brine (100 mL), dried over NaiSO4, filtered, and concentrated under vacuum to give (3S)-l-[(25')-3-[4-[(2Af)-2-[5-(4-benzyloxycarbonylpiperazin -l-yl)-2- [(lS)-l-methoxyethyl]-3-pyridyl]-6-fluoro-3-(3-hydroxy-2,2-d imethyl-propyl)-l-(2,2,2- trifluoroethyl)indol-5-yl]thiazol-2-yl]-2-(tert-butoxycarbon ylamino)propanoyl]hexahy- dropyridazine-3-carboxylic acid (compound Cll, 4.3 g) as a brown solid. MS calc’d 1039.4 (MH ⁺ ), measured 1039.2 (MH ⁺ ). Step 10: Preparation of benzyl 4-[5-[(7S,13S)-7-(tert-butoxycarbonylamino)-24- fluoro-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-1 5-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-(20M)- 20-yl]-6-[(15)-l-methoxyethyl]-3-pyridyl]piperazine-l-carbox ylate (compound C12)

To a mixture of (3S)-l-[(2S)-3-[4-[(2Af)-2-[5-(4-benzyloxycarbonylpiperazin- l-yl)-2- [(lS)-l-methoxyethyl]-3-pyridyl]-6-fluoro-3-(3-hydroxy-2,2-d imethyl-propyl)-l-(2,2,2- trifluoroethyl)indol-5-yl]thiazol-2-yl]-2-(tert-butoxycarbon ylamino)propanoyl]hexahy- dropyridazine-3-carboxylic acid (compound Cll, 4.3 g, 4.14 mmol) in DCM (430 mL) was added DIEA (14.4 mL, 82.76 mmol), EDCI (11.9 g, 62.07 mmol) and 1 -hydroxybenzotriazole (1.4 g, 10.35 mmol) at 0 °C. The mixture was stirred at 15 °C for 12 hrs. After the reaction was completed, the mixture was concentrated in vacuo, then diluted with water (80 mL), extracted with EtOAc (80 mL, twice). The combined organic layer was washed with brine (80 mL), dried over NaiSCU, filtered and concentrated in vacuo. The residue was purified by silica column chromatography (EtOAc in PE = 25% ~ 66%) to afford benzyl 4-[5-[(7S,13S)-7-(tert- butoxycarbonylamino)-24-fluoro-17,17-dimethyl-8,14-dioxo-21- (2,2,2-trifluoroethyl)-15-oxa-4- thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hcxacn-(20M)-20-yl|-6-|( IS)- 1 -mcthoxycthyl | -3-pyridyl | piperazine- 1 -carboxylate (compound C12, 3.1 g) as yellow gum. MS calc’d 1021.4 (MH ⁺ ), measured 1021.2 (MH ⁺ ).

Step 11: Preparation of/er/-butvl \-|(7S,13S)-24-nuoro-(20V/)-20-|2-|( IS)- 1- methoxyethyl]-5-(4-methylpiperazin-l-yl)-3-pyridyl]-17,17-di methyl-8,14-dioxo-21-(2,2,2- 25 9 13 2226 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 ’ .1 ’ .0 ’ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl]carbamate (compound C13)

To a mixture of benzyl 4-[5-[(7S,13S)-7-(tert-butoxycarbonylamino)-24-fluoro-17,17- dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia- 9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-(20Af)-20- yl]-6-[(lS)-l-methoxyethyl]-3-pyridyl]piperazine-l-carboxyla te (compound C12, 3.1 g, 3.04 mmol) and formaldehyde aqueous (775.0 mg, 9.55 mmol) in methanol (150 mL) was added Pd(OH)2 on activated carbon (2.79 g, 3.97 mmol). The mixture was degassed and purged with H2 three times. The mixture was hydrogenated at 30 °C for 18 hrs. After the reaction was completed, the mixture was filtered and the filtrate was concentrated in vacuo to afford tert-butyl N- [(7S, 13S)-24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5-(4-methylpiperazin- l-yl)-3-pyridyl]- 17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4 -thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamate (compound C13, 2.6 g) as a brown solid. MS calc’d 901.3 (MH ⁺ ), measured 901.3 (MH ⁺ ).

Step 12: Preparation of (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-(4-methylpiperazin-l-yl)-3-pyridyl]-17,17-di methyl-21-(2,2,2- 25 9 13 2226 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 ’ .1 ’ .0 ’ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14-dione (intermediate C)

To a mixture of tert-butyl A-[(7S,13S)-24-fluoro-(20Af)-20-[2-[( IS)- l-methoxyethyl]-5-(4- methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo-21-(2,2,2-trifluoroethyl)- 15-oxa-4- thia-9, 21,27, 28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23- hexaen-7-yl] carbamate (compound C13, 2.6 g, 2.89 mmol) in DCM (18 mL) was added TFA (14.0 mL, 181.72 mmol). The mixture was stirred at 15 °C for 0.5 h. After the reaction was completed, the mixture was concentrated in vacuo and diluted with sat. NaHCOs (30 mL), extracted with EtOAc (30 mL, three times). The combined organic layer was washed with brine (50 mL), dried over NaiSCU, filtered and concentrated in vacuo to afford (7S,13S)-7-amino-24- fluoro-(20Af)-20-[2-[(lS)-l-methoxyethyl]-5-(4-methylpiperaz in-l-yl)-3-pyridyl]-17,17- dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e (intermediate C, 2.0 g) as a yellow solid, which was used directly in the next step. MS calc’d 801.3 (MH ⁺ ), measured 801.2 (MH ⁺ )

Intermediate D

(75,135)-7-amino-21-ethyl-24-fluoro-(20A/)-20-[2-[(15)-l- methoxyethyl]-5-(4- methylpiperazin- l-yl)-3-pyridyl] - 17,17-dimethyl- 15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ^2,5 .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione

The title compound was prepared in analogy to the preparation of Intermediate C by using iodoethane instead of 2,2,2-trifluoroethyl trifluoromethane sulfonate.

Intermediate E

(75,135)-7-amino-24-fluoro-(20M)-20-[2-[(15)-l-methoxyeth yl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1-yl] -3-pyridyl] - 17,17-dimethyl-21-(2,2,2-trifluoroethyl) -15-oxa-4- thia-9, 21,27, 28-tetrazapentacyclo[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23- hexaene-8, 14-dione

The compound was prepared according to the following scheme:

E10 Intermediate E

Step 1: Preparation of l-[5-bromo-6-[(lS)-l-methoxyethyl]-3-pyridyl]-4-(2,2,2- trifluoroethyl)piperazine (compound E2).

To a mixture of 3-bromo-5-iodo-2-[(lS)-l-methoxyethyl]pyridine (compound A3, 2.03 g, 5.95 mmol) and l-(2,2,2-trifluoroethyl)piperazine (compound El, 1.0 g, 5.95 mmol) in toluene (15 mL) were added CS2CO3 (4.85 g, 14.88 mmol), (/?)-binap (92.6 mg, 0.15 mmol) and Pd(OAc)2 (66.8 mg, 0.3 mmol). The reaction mixture was degassed and purged with nitrogen for 3 times and the mixture was stirred at 100 °C for 12 hrs under nitrogen atmosphere. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography to afford l-[5- bromo-6-[(15')-l-methoxyethyl]-3-pyridyl]-4-(2,2,2-trifluoro ethyl)piperazine (compound E2, 2.0 g) as yellow oil. MS calc’d 382.2 (MH ⁺ ), measured 382.1 (MH ⁺ ).

Step 2: l-[6-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2-diox aborolan-2-yl)-3- pyridyl]-4-(2,2,2-trifluoroethyl)piperazine (compound E3). To a solution of l-[5-bromo-6-[(lS)-l-methoxyethyl]-3-pyridyl]-4-(2,2,2- trifluoroethyl)piperazine (compound E2, 3.2 g, 8.37 mmol), bis(pinacolato)diboron (3.19 g, 12.56 mmol) and KO Ac (2.1 g, 20.93 mmol) in toluene (50 mL) was added Pd(dppf)C12 (306.3 mg, 0.42 mmol). The mixture was degassed and purged with nitrogen for 3 times and the mixture was stirred at 90 °C for 12 hrs under nitrogen atmosphere. After being cooled to the room temperature, the reaction mixture was filtered, the filtrate was concentrated in vacuo to give a residue, which was purified by reversed phase column to afford l-[6-[(lS)-l-methoxyethyl]-5- (4,4,5,5-tetramethyl- 1 ,3,2-dioxaborolan-2-yl)-3-pyridyl] -4-(2,2,2-trifluoroethyl)piperazine (compound E3, 1.9 g) as a yellow gum. MS calc’d 430.2 (MH ⁺ ), measured 348.4 (M-C6HIO+H ⁺ ).

Step 3: Preparation of [3-[5-bromo-6-fluoro-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- l-yl]-3-pyridyl]- 1 //-indol-3-y 1| -2.2-dimethyl- propoxy |-/(T/-butyl- diphenyl-silane (compound E4).

To a solution of l-[6-[(15')-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2-dio xaborolan-2- yl)-3-pyridyl]-4-(2,2,2-trifluoroethyl)piperazine (compound E3, 1.9 g, 4.41 mmol), [3-(5-bromo- 6-fluoro-2-iodo-177-indol-3-yl)-2,2-dimethyl-propoxy]-tert-b utyl-diphenyl- silane (compound C5, 2.1 g, 3.15 mmol) in 1,4-dioxane (24 mL), water (8 mL) and toluene (8 mL) was added K3PO4 (2.1 g, 9.5 mmol) and Pd(dppf)C12 (231 mg, 0.37 mmol). The mixture was degassed by bubbling nitrogen for 2 min, and the reaction mixture was stirred at 70 °C for 12 hrs. After being cooled to room temperature, the reaction mixture was filtered. The filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography (EtOAc in PE : 30% - 60%) to afford [3-[5-bromo-6-fhioro-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- l -yl|-3-pyridyl|- l /7-indo 1-3-yl] -2, 2-dimethyl-propoxy]-tert-butyl-diphenyl- silane (compound E4, 960.0 mg) as a yellow gum. MS calc’d 839.3 (MH ⁺ ), measured 839.3 (MH ⁺ ).

Step 4: Preparation of [3-[5-bromo-6-fluoro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2-tr ifluoroethyl)indol-3-yl]-2,2- dimethyl-propoxy |-/c/7-butyl-diphenyl-silane (compound E5).

To a solution of [3-[5-bromo-6-fluoro-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 177- indo 1-3-yl] -2, 2-dimethy 1-propoxy] -tert-butyl- diphenyl- silane (compound E4, 1 g, 1.14 mmol) in DMF (35 mL) was added CS2CO3 (1.1 g, 3.44 mmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (2.7 g, 11.63 mmol) at 0 °C. After being stirred at 20 °C for 15 hrs, the reaction mixture was poured into water (100 mL), and extracted with EtOAc (50 mL, three times). The combined organic was washed with brine (50 mL, three times), dried over Na2SO4, filtered and concentrated under vacuum to give a residue which was purified by column chromatography (EtOAc in PE: 30% - 40%) to afford [3-[5- bromo-6-fhioro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2,2,2-t rifluoroethyl)piperazin-l-yl]-3- pyridyl] - 1 -(2,2,2-trifluoroethyl)indol-3-yl] -2,2-dimethyl-propoxy] -/c/7-but yl-diphcny I- silane (compound E5, 640.0 mg, 0.69 mmol, faster eluted) as a white solid. MS calc’d 921.3 (MH ⁺ ), measured 921.4 (MH ⁺ ).

Step 5: Preparation of 3-[5-bromo-6-fluoro-(2Af)-2-[2-[(lS)-l-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2-tr ifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-l-ol (compound E6).

To a solution of [3-[5-bromo-6-fhioro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2 ,2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indo 1-3-yl] -2, 2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (compound E5, 640.0 mg, 0.69 mmol) in DMF (7 mL) was added cesium fluoride (421.8 mg, 2.78 mmol). The mixture was stirred at 60 °C for 16 hrs. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography (EtOAc in PE : 30% - 60%) to afford 3-[5-bromo-6-fluoro-(2M)-2-[2-[(15')-l-methoxyethyl]-5- [4-(2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2 -trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-l-ol (compound E6, 360.0 mg) as yellow oil. MS calc’d 683.2 (MH ⁺ ), measured 683.1 (MH ⁺ ).

Step 6: Preparation of 3-[5-bromo-6-fluoro-(2Af)-2-[2-[(lS)-l-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2-tr ifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-l-ol (compound E7).

To a solution of 3-[5-bromo-6-fhioro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2, 2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indo 1-3-yl] -2, 2-dimethyl-propan- l-ol (compound E6, 360.0 mg, 0.53 mmol), bis(pinacolato)diboron (200.6 mg, 0.79 mmol) in toluene (6 mL) was added potassium acetate (129 mg, 1.32 mmol) and Pd(dppf)C12 (40 mg, 0.1 mmol). The reaction mixture was degassed by bubbling nitrogen for 5 min then stirred at 80 °C for 15 hrs. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography (EtOAc in PE : 30% - 50%) to afford 3-[5-bromo-6-fluoro-(2Af)-2-[2-[(15')-l- methoxyethyl] -5- [4-(2, 2, 2-trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indol- 3-yl]-2,2-dimethyl-propan-l-ol (compound E7, 300.0 mg) as yellow gum. MS calc’d 731.4 (MH ⁺ ), measured 731.4 (MH ⁺ ).

Step 7: Preparation of methyl (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6- fhioro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[( lS)-l-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2-trifluoro ethyl)indol-5-yl]thiazol-2- yl]propanoyl]hexahydropyridazine-3-carboxylate (compound E8).

To a mixture of 3-[5-bromo-6-fhioro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-[4-(2, 2,2- trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indo 1-3-yl] -2, 2-dimethyl-propan- l-ol (compound E7, 0.3 g, 0.41 mmol) and methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxyl ate (intermediate B, 196.7 mg, 0.41 mmol) in toluene (3 mL), 1,4-dioxane (1 mL) and water (1 mL) were added K3PO4 (221.3 mg, 1.04 mmol) and Pd(dtbpf)C12 (27.05 mg, 0.04 mmol). The mixture was stirred at 70 °C for 12 hrs under nitrogen atmosphere. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography (EtOAc in PE : 60% - 80%) to afford methyl (35)-l-[(25)-2-(tcrt- butoxycarbonylamino)-3-[4-[6-fluoro-3-(3-hydroxy-2,2-dimethy l-propyl)-(2Af)-2-[2-[(15')-l- methoxyethyl] -5- [4-(2, 2, 2-trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indol- 5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylat e (compound E8, 200.0 mg) as yellow gum. MS calc’d 1001.4 (MH ⁺ ), measured 1001.4 (MH ⁺ ).

Step 8: Preparation of (3S)-l-[(2S)-2-(fcrt-butoxycarbonylamino)-3-[4-[6-fluoro-3-( 3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(lS)-l-methoxyethyl] -5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-l-(2,2,2-trifluoro ethyl)indol-5-yl]thiazol-2- yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound E9).

To a mixture of methyl (3S)-l-[(2S)-2-(ter^butoxycarbonylamino)-3-[4-[6-fluoro-3-(3 - hydroxy-2,2-dimethyl-propyl)-(2Af)-2-[2-[(15')-l-methoxyethy l]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1-yl] -3-pyridyl] -l-(2, 2, 2-trifluoroethyl)indol-5-yl]thiazo 1-2- yl]propanoyl]hexahydropyridazine-3-carboxylate (compound E8, 200.0 mg, 0.2 mmol) in DCE (5 mL) was added MeaSnOH (200.0 mg, 1.11 mmol). The mixture was stirred at 60 °C for 12 hrs. The reaction mixture was concentrated under vacuum to give a residue. EtOAc (10 mL) and water (10 mL) were added to the residue and the layers were separated. The aqueous phase was extracted with EtOAc (15 mL, twice). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under vacuum to afford (3.S')- l-|(2.S')-2-(/c/7- butoxycarbonylamino)-3-[4-[6-fluoro-3-(3-hydroxy-2,2-dimethy l-propyl)-(2Af)-2-[2-[(15')-l- methoxyethyl] -5- [4-(2, 2, 2-trifluoroethyl)piperazin- 1 -yl] -3-pyridyl] - 1 -(2,2, 2-trifluoroethyl)indol- 5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound E9, 188.0 mg) as a brown solid. MS calc’d 987.4 (MH ⁺ ), measured 987.4 (MH ⁺ ). Step 9: Preparation ol'/c/7-butyL\-|(7.S.LTS)-24-fhioro-(20V/)-20-|2-|( LS)-l- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-l-yl]-3-p yridyl]-17,17-dimethyl-8,14- dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamate (compound E10).

To a mixture of (3S)-l-[(2S)-2-(ter^butoxycarbonylamino)-3-[4-[6-fluoro-3-(3 -hydroxy- 2, 2-dimethyl-propyl)-(2Af)-2-[2-[( IS)- 1 -methoxyethyl] -5-[4-(2, 2, 2-trifluoroethyl)piperazin-l- yl]-3-pyridyl]-l-(2,2,2-trifluoroethyl)indol-5-yl]thiazol-2- yl]propanoyl]hexahydropyridazine-3- carboxylic acid (compound E9, 188.0 mg, 0.19 mmol) in DCM (20 mL) were added DIEA (0.7 mL, 3.81 mmol), EDCI (550.0 mg, 2.87 mmol) and HOBt (65.0 mg, 0.48 mmol) at 0 °C. After being stirred at 20 °C for 12 hrs, the reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL, three times). The combined organic layer was washed with brine (30 mL), dried over NaiSCU, filtered and concentrated under vacuum to give a residue which was purified by column chromatography (EtOAc in PE : 50% - 70%) to afford tert-butyl A-[(7S,13S)- 24-fluoro-(20M)-20-[2-[(lS)-l-methoxyethyl]-5-[4-(2,2,2-trif luoroethyl)piperazin-l-yl]-3- pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl) -15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamate (compound E10, 110.0 mg) as a yellow solid. MS calc’d 969.4 (MH ⁺ ), measured 969.5 (MH ⁺ ).

Step 10: Preparation of (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-l-yl]-3-p yridyl]-17,17-dimethyl-21- (2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione (Intermediate E).

To a solution of tert-butyl A-[(7S,13S)-24-fluoro-(20Af)-20-[2-[(lS)-l-methoxyethyl]-5-[ 4- (2,2,2-trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-dimet hyl-8,14-dioxo-21-(2,2,2-

25 913 2226 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ’ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl] carbamate (compound E10, 110.0 mg, 0.11 mmol) in DCM (1 mL) was added TFA (1.0 mL, 12.98 mmol). The mixture was stirred at 20 °C for 1 h. After the reaction was completed, the reaction mixture was concentrated under vacuum to give a residue. Sat. NaHCOs aq. (20 mL) was added and the mixture was extracted with EtOAc (15 mL, three times). The combined organic layer was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford (7S,13S)-7-amino-24-fluoro-(20Af)- 20-[2-[(15')-l-methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)pipe razin-l-yl]-3-pyridyl]-17,17- dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e

(Intermediate E, 98.0 mg) as a yellow solid. MS calc’d 869.4 (MH ⁺ ), measured 869.2 (MH ⁺ ).

Intermediate F

(75,135)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(15)-l-m ethoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin- 1-yl] -3-pyridyl] - 17,17-dimethyl- 15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione

The title compound was prepared in analogy to the preparation of Intermediate E by using iodoethane instead of 2,2,2-trifluoroethyl trifluoromethane sulfonate.

Intermediate G

(75,135)-7-amino-21-ethyl-24-fluoro-(20A/)-20-[2-[(15)-l- methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-15-oxa-4-thia-9,21,27,28-tetrazapent acyclo-

[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14-dione

G10 intermediate G

Step 1: Preparation of 4-[5-bromo-6-[(lS)-l-methoxyethyl]-3-pyridyl]morpholine (compound Gl) To a mixture of 3-bromo-5-iodo-2-[(lS)-l-methoxyethyl]pyridine (compound A3, 30 g, 87.73 mmol) and morpholine (7.6 g, 87.73 mmol) in toluene (450 mL) were added CS2CO3 (57.2 g, 175.45 mmol), (/?)-binap (2.7 g, 4.39 mmol) and Pd(OAc)2 (0.98 g, 4.39 mmol). The reaction mixture was degassed and purged with nitrogen for 3 times and the mixture was stirred at 90 °C for 12 hrs under nitrogen atmosphere. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography to afford 4-[5-bromo-6-[(15')-l-methoxyethyl]-3- pyridyl] morpholine (compound Gl, 21 g) as yellow oil. MS calc’d 301.1 (MH ⁺ ), measured 301.1 (MH ⁺ ).

Step 2: Preparation of 4-[6-[(lS)-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-3-pyridyl]morpholine (compound G2)

To a solution of 4-[5-bromo-6-[(lS)-l-methoxyethyl]-3-pyridyl]morpholine (compound Gl, 21 g, 63.3 mmol), bis(pinacolato)diboron (24.0 g, 94.63 mmol) and KOAc (13.6 g, 138.79 mmol) in toluene (500 mL) was added Pd(dppf)C12 (4.4 g, 6.31 mmol). The mixture was degassed and purged with nitrogen for 3 times and the mixture was stirred at 90 °C for 12 hrs under nitrogen atmosphere. After being cooled to the room temperature, the reaction mixture was filtered, the filtrate was concentrated in vacuo to give crude product 4-[6-[(lS)-l-methoxyethyl]- 5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-3-pyridyl]mo rpholine (compound G2, 45 g) as a yellow gum, which was used to the next step. MS calc’d 349.2 (MH ⁺ ), measured 349.2 (MH ⁺ ).

Step 3: Preparation of [3-[5-bromo-6-fluoro-2-[2-[(lS)-l-methoxyethyl]-5- morpholino-3-pyridyl]- l//-indol-3-yl| -2.2-dimethyl- propoxy |-/c/7-butyl-diphenyl-silane (compound G3)

To a solution of 4-[6-[(15')-l-methoxyethyl]-5-(4,4,5,5-tetramethyl-l,3,2-dio xaborolan-2- yl)-3-pyridyl] morpholine (compound G2, 40.6 g, 46.65 mmol), [3-(5-bromo-6-fluoro-2-iodo- 177-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-si lane (compound C5, 31 g, 46.65 mmol) in 1,4-dioxane (420 mL) and water (80 mL) was added K3PO4 (29.7 g, 2.33 mmol) and Pd(dppf)C12 (1.7 g, 0.29 mmol). The mixture was degassed by bubbling nitrogen for 2 min, and the reaction mixture was stirred at 90 °C for 18 hrs. After being cooled to room temperature, the reaction mixture was extracted with EA (200 mL, three times). The combined organic layer was washed with brine (200 mL), dried over Na2SC>4, filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography to afford [3-[5- bromo-6-fhioro-2-[2-[(lS)-l-methoxyethyl]-5-morpholino-3-pyr idyl]-177-indol-3-yl]-2,2- dimethyl-propoxy]-tert-butyl-diphenyl- silane (compound G3, 17.2 g) as yellow oil. MS calc’d 758.3 (MH ⁺ ), measured 758.3 (MH ⁺ ). Step 4: Preparation of [3-[5-bromo-l-ethyl-6-fluoro-2-[2-[(lS)-l-methoxyethyl]-5- morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-terf- butyl-diphenyl-silane (compound G4)

To a solution of [3-[5-bromo-6-fluoro-2-[2-[(lS)-l-methoxyethyl]-5-morpholino -3- pyridyl]-177-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-di phenyl-silane (compound G3, 15 g, 19.77 mmol) in DMF (300 mL) was added CS2CO3 (19.3 g, 59.3 mmol) and iodoethane (6.16 g, 39.53 mmol) at 0 °C. After being stirred at 20 °C for 16 hrs, the reaction mixture was poured into water (200 mL), and extracted with EtOAc (200 mL, three times). The combined organic layer was washed with brine (10 mL, three times), dried over NaiSCU, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography to afford [3-[5- bromo-l-ethyl-6-fhioro-2-[2-[(lS)-l-methoxyethyl]-5-morpholi no-3-pyridyl]indol-3-yl]-2,2- dimethyl-propoxy]-tert-butyl-diphenyl- silane (compound G4, 14.7 g) as yellow oil. MS calc’d 786.3 (MH ⁺ ), measured 786.4 (MH ⁺ ).

Step 5: Preparation of 3-[5-bromo-l-ethyl-6-fluoro-(2M)-2-[2-[(lS)-l-methoxyethyl]- 5-morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propan-l-ol (compound G5) and 3-[5- bromo-l-ethyl-6-fluoro-(2P)-2-[2-[(lS)-l-methoxyethyl]-5-mor pholino-3-pyridyl]indol-3- yl]-2,2-dimethyl-propan-l-ol (compound G6)

To a solution of [3-[5-bromo-l-ethyl-6-fhioro-2-[2-[(lS)-l-methoxyethyl]-5-mo rpholino- 3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphe nyl-silane (compound G4, 14.7 g, 18.68 mmol) in DMF (160 mL) was added cesium fluoride (14.2 g, 93.41 mmol). The mixture was stirred at 60 °C for 48 hrs. After being cooled to room temperature, the reaction mixture were added with EtOAc (300 mL) and water (300 mL) and the layers were separated. The aqueous phase was extracted with EtOAc (200 mL, three times). The combined organic layer was washed with brine (200 mL, four times), dried over Na2SO4, filtered, and concentrated under vacuum to give a residue. The residue was purified by column chromatography to afford 3-[5- bromo- l-ethyl-6-fluoro-(2Af)-2-[2-[( IS)- l-methoxyethyl]-5-morpholino-3-pyridyl]indo 1-3-yl]- 2,2-dimethyl-propan-l-ol (compound G5, 6 g, faster eluted) as colorless foam and 3-[5-bromo-l- ethyl-6-fluoro-(2P)-2-[2-[(lS)-l-methoxyethyl]-5-morpholino- 3-pyridyl]indol-3-yl]-2,2- dimethyl-propan-l-ol (compound G6, 4.5 g, slower eluted) as colorless foam. Compound G5: MS calc’d 548.2 (MH ⁺ ), measured 548.2 (MH ⁺ ). ^J H NMR (400MHz, Methanol-^) d = 8.41 (d, J = 2.4 Hz, 1H), 7.92 (d, J = 6.8 Hz, 1H), 7.37 - 7.33 (m, 2H), 4.58 (s, 1H), 4.05 - 3.98 (m, 2H), 3.87-3.82 (m, 5H), 3.27 - 3.23 (m, 4H), 3.15 - 3.13 (m, 1H), 3.00 (s, 3H), 2.75-2.71 (m, 1H), 2.24 - 2.22 (m, 1H), 1.42 (d, 7 = 6.4 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H), 0.76 (s, 3H), 0.76 (s, 3H). X-ray crystallographic analysis of compound G5

Absolute configuration structure of compound G5 was confirmed by X-ray crystallographic analysis of its single crystal. (Figure 1).

Step 6: Preparation of 3-[l-ethyl-6-fluoro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5- morpholino-3-pyridyl]-5-(4,4,5,5-tetramethyl-l,3,2-dioxaboro lan-2-yl)indol-3-yl]-2,2- dimethyl-propan-l-ol (compound G7)

To a solution of 3-[5-bromo-l-ethyl-6-fluoro-(2M)-2-[2-[(15')-l-methoxyethyl] -5- morpholino-3-pyridyl] indo 1-3-yl] -2, 2-dimethyl-propan-l-ol (compound G5, 6 g, 10.94 mmol), bis(pinacolato)diboron (4.2 g, 16.41 mmol) in toluene (60 mL) was added potassium acetate (2.7 g, 27.35 mmol) and Pd(dppf)Ch (0.8 g, 1.09 mmol). The reaction mixture was degassed by bubbling nitrogen for 5 min then stirred at 90 °C for 15 hrs. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography to afford 3-[l-ethyl-6-fluoro-(2M)- 2-[2-[(lS)-l-methoxyethyl]-5-morpholino-3-pyridyl]-5-(4,4,5, 5-tetramethyl-l,3,2-dioxaborolan- 2-yl)indo 1-3-yl] -2, 2-dimethyl-propan-l-ol (compound G7, 4.5 g) as colorless gum. MS calc’d 596.4 (MH ⁺ ), measured 596.4 (MH ⁺ ).

Step 7: Preparation of methyl (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[l- ethyl-6-fhioro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[ (lS)-l-methoxyethyl]-5- morpholino-3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexah ydropyridazine-3- carboxylate (compound G8)

To a mixture of 3-[l-ethyl-6-fhioro-(2M)-2-[2-[(lS)-l-methoxyethyl]-5-morpho lino-3- p yridyl]-5-(4, 4, 5, 5-tetramethyl-l, 3, 2-dioxaborolan-2-yl)indol-3-yl]-2, 2-dimethyl-propan-l-ol (compound G7, 4.5 g, 7.56 mmol) and methyl (3S)-l-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxyl ate (intermediate B, 3.6 g, 7.56 mmol) in toluene (45 mL), 1,4-dioxane (15 mL) and water (15 mL) were added K3PO4 (4.0 g, 18.89 mmol) and Pd(dtbpf)C12 (492.5 mg, 0.75 mmol). The mixture was stirred at 70 °C for 12 hrs under nitrogen atmosphere. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo to give a residue. The residue was purified by column chromatography to afford methyl (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[l- ethyl-6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2Af)-2-[2- [(15')-l-methoxyethyl]-5- morpholino-3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexah ydropyridazine-3-carboxylate (compound G8, 3.8 g) as colorless gum. MS calc’d 866.4 (MH ⁺ ), measured 866.4 (MH ⁺ ). Step 8: Preparation of (3S)-l-[(2S)-2-(tcrt-butoxycarbonylamino)-3-[4-[l-ethyl-6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(lS)-l-m ethoxyethyl]-5-morpholino- 3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridaz ine-3-carboxylic acid (compound G9)

To a mixture of methyl (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[l-ethyl-6-fl uoro-3- (3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(lS)-l-methoxyeth yl]-5-morpholino-3- pyridyl] indo l-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxyl ate (compound G8, 3.8 g, 4.39 mmol) in DCE (76 mL) was added MeaSnOH (3.2 g, 17.55 mmol). The mixture was stirred at 60 °C for 48 hrs. The reaction mixture was concentrated under vacuum to give a residue. EtOAc (200 mL) and water (100 mL) were added to the residue and the layers were separated. The aqueous phase was extracted with EtOAc (150 mL, twice). The combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under vacuum to afford (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[l-ethyl-6-fl uoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(lS)-l-methoxyethyl] -5-morpholino-3-pyridyl]indol- 5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound G9, 3.7 g) as a brown solid. MS calc’d 852.4 (MH ⁺ ), measured 852.4 (MH ⁺ ).

Step 9: Preparation of tert-butyl 2V-[(7S, 13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-8,14-di oxo-15-oxa-4-thia- 9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl]carbamate (compound G10)

To a mixture of (3S)-l-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[l-ethyl-6-fl uoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(lS)-l-methoxyethyl] -5-morpholino-3-pyridyl]indol- 5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound G9, 2.5 g, 2.93 mmol) in DCM (250 mL) were added DIEA (7.58 mL, 58.68 mmol), EDCI (8.4 g, 44.01 mmol) and HOBt (991.2 mg, 0.91 mmol) at 0 °C. After being stirred at 20 °C for 12 hrs, the reaction mixture was poured into water (100 mL) and extracted with EtOAc (100 mL, three times). The combined organic layer was washed with brine (30 mL), dried over NaiSO4, filtered and concentrated under vacuum to give a residue which was purified by column chromatography to afford tert-butyl A-[(7S,13S)-21-ethyl-24-fhioro-(20M)-20-[2-[(lS')-l-methoxye thyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamate (compound G10, 1.2 g) as a yellow oil. MS calc’d 834.4 (MH ⁺ ), measured 834.4 (MH ⁺ ). Step 10: Preparation of (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-15-oxa- 4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione (Intermediate G)

To a solution of tert-butyl A-[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-8,14-di oxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .I ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamate (compound GIO, 1.2 g, 1.44 mmol) in DCM (12 mL) was added TFA (6.0 mL). The mixture was stirred at 20 °C for 3 hrs. After the reaction was completed, the reaction mixture was concentrated under vacuum to give a residue. Sat. NaHCOs aq. (60 mL) was added and the mixture was extracted with EtOAc (80 mL, three times). The combined organic layer was washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford (7S, 13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-15-oxa-4-thia-9,21,27,2 8- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e (Intermediate G, 1 g) as a yellow solid. MS calc’d 734.3 (MH ⁺ ), measured 734.3 (MH ⁺ ).

Intermediate H

(7S,13S)-7-amino-25-fluoro-(20Af)-20-[2-[(lS)-l-methoxyet hyl]-5-(4-methylpiperazin-l-yl)- 3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4 -thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione

The title compound was prepared in analogy to the preparation of Intermediate C by using 5-bromo-4-fluoro-177-indole instead of 5-bromo-6-fluoro-177-indole (compound C2). Intermediate I

(75,135)-7-amino-24-fluoro-(20A/)-20-[2-[(15)-l-methoxyet hyl]-5-morpholino-3-pyridyl]-

17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9, 21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14- dione

The title compound was prepared in analogy to the preparation of Intermediate E by using morpholine instead of l-(2,2,2-trifluoroethyl)piperazine (compound El).

Intermediate J (75,135)-7-amino-25-fluoro-(20A/)-20-[2-[(15)-l-methoxyethyl ]-5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-dimethyl-21- (2,2,2-trifluoroethyl)-15-oxa-4- thia-9, 21,27, 28-tetrazapentacyclo[17.5.2.1 ^2,5 .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25), 2, 5(28), 19, 22(26), 23- hexaene-8, 14-dione The title compound was prepared in analogy to the preparation of Intermediate E by using

[3-(5-bromo-4-fluoro-2-iodo-177-indol-3-yl)-2,2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (compound JI) instead of [3-(5-bromo-6-fluoro-2-iodo-177-indol-3-yl)-2,2-dimethyl-pro poxy]- /<?/7-butyl-di phenyl- silane (compound C5).

The compound JI was prepared in analogy to the preparation of compound C5 by using 5- bromo-4-fluoro-lH- indole instead of 5-bromo-6-fluoro-177-indole (compound C2). Example 1

3-(dimethylamino)-A^-[(15)-l-[[(75,135)-24-fluoro-(20A/)- 20-[2-[(15)-l-methoxyethyl]-5-(4- methylpiperazin-l-yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-2 1-(2,2,2-trifluoroethyl)-15- oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl|carbamoyl|-2-methyl-propyl|-\-methyl-azetidin e- 1- carboxamide

The compound was prepared according to the following scheme:

Step 1: Preparation of benzyl (2S)-2-[tert-butoxycarbonyl(methyl)amino]-3-methyl- butanoate (compound IB)

To a mixture of BOC-A-ME-VAL-OH (5.0 g, 21.62 mmol) and potassium carbonate (4.5 g, 32.43 mmol) in DMF (70 mL) was added benzyl bromide (4.1 g, 23.78 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. After the reaction was completed, the reaction mixture was diluted with water (70 mL), extracted with EtOAc (70 mL, twice). The combined organic layer was washed with brine (200 mL), dried over NaiSCU, filtered and concentrated in vacuo. The residue was purified by reversed phase column to afford benzyl (2S)-2-[tert- butoxycarbonyl(methyl)amino]-3-methyl-butanoate (compound IB, 6.7 g) as yellow oil. ^J H NMR (400 MHz, CHLOROFORM-d) 5 = 7.34 (br s, 5H), 5.21 - 5.10 (m, 2H), 2.88 - 2.74 (m, 3H), 2.31 - 2.12 (m, 1H), 1.68 - 1.64 (m, 1H), 1.44 (br d, J= 15.0 Hz, 9H), 0.96 (d, 7 = 6.6 Hz, 3H), 0.90 (br d, J= 3.8 Hz, 3H). MS calc’d 322.2 (MH ⁺ ), measured 344.2 (MNa ⁺ ).

Step 2: Preparation of benzyl (2S)-3-methyl-2-(methylamino)butanoate (compound 1C)

A mixture of benzyl (25')-2-[tert-butoxycarbonyl(methyl)amino]-3-methyl-butanoat e (compound IB, 6.7 g, 21.47 mmol) in HCl/l,4-dioxane (50.0 mL, 2 M) was stirred at 25 °C for 1 h. After the reaction was completed, the reaction mixture was concentrated in vacuo to get a residue. The residue was diluted with sat. NaHCOs (40 mL), extracted with EtOAc (50 mL, three times). The combined organic layer was washed with brine (100 mL), dried over NaiSCU, filtered and concentrated in vacuo to afford benzyl (2S)-3-methyl-2-(methylamino)butanoate (compound 1C, 4.7 g) as colorless oil. MS calc’d 222.1 (MH ⁺ ), measured 222.2 (MH ⁺ ).

Step 3: Preparation of benzyl (2S)-2-[[3-(dimethylamino)azetidine-l-carbonyl]- methyl- amino] -3-methyl-butanoat (compound ID)

To a mixture of benzyl (2S)-3-methyl-2-(methylamino)butanoate (compound 1C, 1.0 g, 4.52 mmol) and DIEA (0.9 mL, 4.97 mmol) in DCM (15 mL) was added triphosgene (1.3 g, 4.52 mmol) at 0 °C. After being stirred for 0.5 h, the reaction was added with a solution of N,N- dimethylazetidin-3-amine dihydrochloride (compound ID, 782.1 mg, 4.52 mmol) and DIEA (1.97 mL, 11.3 mmol) in DCM (4 mL). The reaction mixture was stirred at 20 °C for another 1 h. After the reaction was completed, the reaction mixture was quenched with water (40 mL), extracted with EtOAc (40 mL, twice). The combined organic layer was washed with brine (60 mL), dried over NaiSO4, filtered and concentrated in vacuo to get a residue. The residue was purified by prep-HPLC to afford benzyl (2S)-2-[[3-(dimethylamino)azetidine- 1 -carbonyl] - methyl-amino]-3-methyl-butanoat (compound IE, 1.0 g) as yellow oil. MS calc’d 348.2 (MH ⁺ ), measured 348.2 (MH ⁺ ).

Step 4: Preparation of (2S)-2-[[3-(dimethylamino)azetidine-l-carbonyl]-methyl- amino]-3-methyl-butanoic acid (compound IF)

To a mixture of benzyl (2S)-2-[[3-(dimethylamino)azetidine-l-carbonyl]-methyl-amino ]-3- methyl-butanoat (compound IE, 500.0 mg, 1.44 mmol) in methanol (15 mL) was added Pd(OH)2 on activated carbon (400.0 mg, 0.57 mmol). The reaction mixture was degassed and purged into hydrogen for three times and stirred at 20 °C for 12 hrs under hydrogen atmosphere (15 psi). After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated in vacuo to afford (2S)-2-[[3-(dimethylamino)azetidine-l-carbonyl]-methyl- amino]-3-methyl- butanoic acid (compound IF, 370.0 mg) as yellow oil. MS calc’d 258.1 (MH ⁺ ), measured 258.3 (MH ⁺ ).

Step 5: Preparation of 3-(dimethylamino)-A^-[(lS)-l-[[(7S,13S)-24-fluoro-(20M)-20-[ 2- [(lS)-l-methoxyethyl]-5-(4-methylpiperazin-l-yl)-3-pyridyl]- 17,17-dimethyl-8,14-dioxo-21- (2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl|carbamoyl|-2-methyl-propyl|-\-methyl-azetidimM -carboxamide (Example 1)

To a solution of (2S)-2-[[3-(dimethylamino)azetidine-l -carbonyl] -methyl- amino] -3- methyl-butanoic acid (compound IF, 32.13 mg, 0.12 mmol) in DMF (2 mL) were added DIEA (0.1 mL, 0.25 mmol), HATU (47.47 mg, 0.12 mmol) and (7S,13S)-7-amino-24-fluoro-17,17- dimethyl-20-[5-(4-methylpiperazin-l-yl)-2-[rac-(lS)-l-methox yethyl]-3-pyridyl]-21-(2,2,2- 25 913 2226 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ’ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14-dione (intermediate C, 50.0 mg, 0.06 mmol) at 0 °C. After being stirred at 20 °C for 1 h, the reaction mixture was diluted with water (15 mL), extracted with EtOAc (15 mL, three times). The combined organic layer was washed with brine (20 mL), dried over NaiSCU, filtered and concentrated in vacuo to get a residue. The residue was purified by prep-HPLC to afford Example 1 (35.1 mg) as a white solid. MS calc’d 1040.5 (MH ⁺ ), measured 1040.8 (MH ⁺ ), ^J H NMR (400 MHz, Methanol-^) 6 = 8.68 (d, 7 = 7.6 Hz, 1H), 8.51 (d, J = 2.8 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.57 - 7.44 (m, 2H), 5.79 - 5.69 (m, 1H), 5.25 - 5.11 (m, 1H), 4.97 - 4.89 (m, 2H), 4.51 - 4.39 (m, 2H), 4.31 - 4.12 (m, 7H), 4.12 - 3.88 (m, 2H), 3.87 - 3.67 (m, 3H), 3.67 - 3.52 (m, 2H), 3.47 (d, J = 14.8 Hz, 2H), 3.35 (s, 3H), 3.29 - 3.22 (m, 2H), 3.22 - 3.10 (m, 2H), 2.99 (s, 3H), 2.92 (s, 8H), 2.87 - 2.77 (m, 1H), 2.58 (d, J= 14.4 Hz, 1H), 2.30 - 2.16 (m, 2H), 2.03 - 1.90 (m, 1H), 1.88 - 1.74 (m, 1H), 1.72 - 1.58 (m, 1H), 1.45 (d, J = 6.0 Hz, 3H), 1.03 - 0.89 (m, 9H), 0.45 (s, 3H).

Example 2 3-(dimethylamino)-^-[(lS)-l-[[(7S,13S)-25-fluoro-(20M)-20-[2 -[(lS)-l-methoxyethyl]-5-(4- methylpiperazin-l-yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-2 1-(2,2,2-trifluoroethyl)-15- oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl|carbamoyl|-2-methyl-propyl|-\-methyl-azetidin e- 1- carboxamide

The title compound was prepared in analogy to the preparation of Example 1 by using (7S,13S)-7-amino-25-fhioro-(20M)-20-[2-[(lS)-l-methoxyethyl] -5-(4-methylpiperazin-l-yl)-3- pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-t hia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e (intermediate H) instead of (7S,13S)-7-amino-24-fhioro-(20M)-20-[2-[(lS')-l-methoxyethyl ]-5- (4-methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-21-(2,2,2-trifluoroethyl)- 15-oxa-4-thia- 9,21,27 ,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .I ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25),2,5(28), 19, 22(26), 23-hexaene- 8, 14-dione (Intermediate C). Example 2 (50.0 mg) was obtained as a yellow solid. MS calc’d 1040.5 (MH ⁺ ), measured 1040.8 (MH ⁺ ). ^J H NMR (400MHz, Methanol-^) d = 8.55 - 8.49 (m, 1H), 7.63 - 7.56 (m, 1H), 7.52 - 7.39 (m, 3H), 6.03 - 5.81 (m, 1H), 5.20 - 5.01 (m, 1H), 4.70 - 4.61 (m, 1H), 4.45 - 4.36 (m, 2H), 4.30 - 4.23 (m, 2H), 4.17 - 3.93 (m, 7H), 3.77 - 3.63 (m, 3H), 3.56 - 3.48 (m, 2H), 3.42 - 3.34 (m, 2H), 3.29 - 3.21 (m, 3H), 3.20 - 3.11 (m, 3H), 3.00 - 2.98 (m, 3H), 2.94 - 2.87 (m, 10H), 2.68 - 2.51 (m, 2H), 2.23 - 2.14 (m, 1H), 1.68 - 1.53 (m, 1H), 1.47 - 1.41 (m, 3H), 1.35 - 1.15 (m, 3H), 0.95 - 0.88 (m, 6H), 0.81 (s, 3H), 0.72 - 0.58 (m, 3H).

Example 3 ^-[(15)-l-[[(75,135)-24-fluoro-(20M)-20-[2-[(15)-l-methoxyet hyl]-5-(4-methylpiperazin-l- yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoro ethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-A^-methyl-morpholine-4-carbox amide

The title compound was prepared in analogy to the preparation of Example 1 by using morpholine instead of MAMimcthylazctidin-3-aminc dihydrochloride (compound ID). Example 3 (25 mg) was obtained as an off-white solid. MS calc’d 1027.4 (MEE), measured 1027.7 (MH ⁺ ), ^J H NMR (400 MHz, Methanol-^) d = 8.71 (d, 7 = 7.6 Hz, 1H), 8.49 (d, 7= 2.8 Hz, 1H), 7.72 - 7.67 (m, 2H), 7.48 (d, 7 = 12.8 Hz, 1H), 5.76 (d, 7= 8.8 Hz, 1H), 5.26 - 5.13 (m, 1H), 4.84 - 4.75 (m, 1H), 4.43 (d, 7 = 12 Hz, 1H), 4.30 - 4.24 (m, 1H), 4.20 (dd, 7 = 12, 2.8 Hz, 1H), 4.17 - 4.03 (m, 2H), 4.00 (s, 1H), 3.85 - 3.62 (m, 8H), 3.55 - 3.44 (m, 2H), 3.42 - 3.34 (m, 6H), 3.30 - 3.18 (m, 5H), 3.15 - 3.09 (m, 1H), 2.99 (s, 3H), 2.91 (s, 3H), 2.87 - 2.77 (m, 1H), 2.71 - 2.58 (m, 1H), 2.33 - 2.14 (m, 2H), 2.01 - 1.90 (m, 1H), 1.87 - 1.74 (m, 1H), 170 - 1.57 (m, 1H), 1.46 (d, 7 = 6.0 Hz, 3H), 1.01 - 0.87 (m, 9H), 0.50 (s, 3H).

Example 4

3-(dimethylamino)-A^-[(15)-l-[[(75,135)-24-fluoro-(20A/)- 20-[2-[(15)-l-methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-tr ifluoroethyl)-15-oxa-4-thia- 9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl]carbamoyl]-2-methyl-propyl]-2V-methyl-azetidine- l-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using (7S,13S)-7-amino-24-fluoro-(20Af)-20-[2-[(lS)-l-methoxyethyl ]-5-morpholino-3-pyridyl]- 17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21, 27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e (Intermediate I) instead of (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(lS)-l-methoxyethyl] -5-(4- methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-21-(2,2,2-trifluoroethyl)- 15-oxa-4-thia- 9,21,27 ,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .I ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25),2,5(28), 19, 22(26), 23-hexaene- 8, 14-dione (Intermediate C). Example 4 (12.9 mg) was obtained as a yellow solid. MS calc’d 1027.5 (MH ⁺ ), measured 1027.5 (MH ⁺ ). ^J H NMR (400MHz, Methanol-^) d = 8.69 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 2.9 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.48 (d, J = 12.7 Hz, 1H), 5.74 (br d, J= 9.3 Hz, 1H), 5.18 (br dd, J = 8.3, 16.6 Hz, 1H), 4.87 - 4.79 (m, 2H), 4.49 - 4.38 (m, 2H), 4.33 - 4.11 (m, 7H), 3.87 (t, 7= 4.6 Hz, 4H), 3.78 (br d, J = 11.2 Hz, 1H), 3.72 - 3.66 (m, 1H), 3.48 (br d, J = 14.7 Hz, 1H), 3.39 - 3.33 (m, 6H), 3.26 - 3.21 (m, 1H), 3.15 - 3.10 (m, 1H), 2.97 - 2.90 (m, 8H), 2.89 - 2.76 (m, 2H), 2.66 - 2.60 (m, 1H), 2.26 - 2.17 (m, 2H), 2.01 - 1.91 (m, 1H), 1.87 - 1.78 (m, 1H), 1.70 - 1.60 (m, 1H), 1.49 - 1.41 (m, 3H), 1.01 - 0.84 (m, 9H), 0.48 (s, 3H).

Example 5 ^-[(15)-l-[[(75,135)-21-ethyl-24-fluoro-(20M)-20-[2-[(15)-l- methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-2V-methyl-morpholine-4-carbox amide

The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (IS, 13S)-7-amino-2 l-ethyl-24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5- morpholino-3-pyridyl]- 17, 17-dimethyl-15-oxa-4-thia-9, 21, 27, 28-tetr azapentacyclo-

[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaene-8, 14-dione (Intermediate G) instead of MAMimcthylazctidin-3-aminc dihydrochloride (compound ID) and (75,135)-7- amino-24-fluoro-(20Af)-20-[2-[(15')-l-methoxyethyl]-5-(4-met hylpiperazin-l-yl)-3-pyridyl]- 17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21, 27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaene-8,14-dion e (Intermediate C). Example 5 (131.3 mg) was obtained as a yellow solid. MS calc’d 960.5 (MH ⁺ ), measured 960.3 (MH ⁺ ). ^J H NMR (400 MHz, CHLOROFORM -d) 3 = 8.97 (br d, J= 1.6 Hz, 1H), 8.69 (d, J = 7.5 Hz, 1H), 8.14 - 7.98 (m, 1H), 7.61 (d, J= 1.7 Hz, 1H), 7.38 (d, J= 1.5 Hz, 1H), 7.13 (d, J = 12.5 Hz, 1H), 5.81 (br d, J= 17.7 Hz, 1H), 4.68 - 4.57 (m, 1H), 4.41 (d, J = 6.5 Hz, 1H), 4.29 - 4.22 (m, 1H), 4.19 - 4.07 (m, 2H), 4.00 - 3.88 (m, 6H), 3.84 - 3.78 (m, 2H), 3.77 - 3.70 (m, 3H), 3.49 - 3.42 (m, 6H), 3.39 - 3.31 (m, 6H), 3.23 - 3.14 (m, 2H), 2.91 (s, 3H), 2.77 - 2.68 (m, 1H), 2.49 - 2.42 (m, 1H), 2.38 - 2.28 (m, 2H), 2.02 - 1.96 (m, 2H), 1.82 (br d, J= 12.7 Hz, 1H), 1.69 - 1.60 (m, 1H), 1.57 (d, J = 6.2 Hz, 3H), 1.03 - 0.96 (m, 9H), 0.91 (d, J= 6.6 Hz, 3H), 0.55 - 0.43 (m, 3H).

Example 6

^-[(15)-l-[[(75,135)-24-fluoro-(20M)-20-[2-[(15)-l-methox yethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-dimethyl-8,1 4-dioxo-21-(2,2,2-

25 9 13 2226 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 ’ .1 ’ .0 ’ ]octacosa-

1(25), 2, 5(28), 19, 22(26), 23-hexaen-7-yl]carbamoyl]-2-methyl-propyl]-2V-methyl-morphol ine-

4-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (75',135')-7-amino-24-fluoro-(20M)-20-[2-[(15')-l-methoxyeth yl]-5-[4-(2,2,2- trifluoroethyl)piperazin-l-yl]-3-pyridyl]-17,17-dimethyl-21- (2,2,2-trifluoroethyl)-15-oxa-4-thia- 9,21,27 ,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ J ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25),2,5(28), 19, 22(26), 23-hexaene- 8, 14-dione (Intermediate E) instead of MMdimcthylazctidin-3-aminc dihydrochloride (compound ID) and (7S,13S)-7-ammo-24-fhioro-(20A0-20-[2-[(15')-l-methoxyethyl] -5-(4- methylpiperazin- l-yl)-3-pyridyl]- 17, 17-dimethyl-21-(2,2,2-trifluoroethyl)- 15-oxa-4-thia- 9,21,27 ,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ J ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25),2,5(28), 19, 22(26), 23-hexaene- 8, 14-dione (Intermediate C). Example 6 (8.8 mg) was obtained as an off-white solid. MS calc’d 1095.5 (MH ⁺ ), measured 1095.5 (MH ⁺ ). ^J H NMR (400 MHz, Methanol-^) 8 = 8.71 (d, J = 7.2 Hz, 1H), 8.45 - 8.38 (m, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.63 - 7.56 (m, 1H), 7.48 (d, J = 12.8 Hz, 1H), 5.87 - 5.69 (m, 1H), 5.24 - 5.11 (m, 1H), 4.49 - 4.37 (m, 1H), 4.29 - 4.16 (m, 2H), 4.01 (d, J = 11.2 Hz, 1H), 3.83 - 3.57 (m, 7H), 3.50 - 3.36 (m, 10H), 3.26 - 3.06 (m, 5H), 2.93 - 2.77 (m, 8H), 2.71 - 2.60 (m, 1H), 2.31 - 2.16 (m, 2H), 2.00 - 1.91 (m, 1H), 1.87 - 1.75 (m, 1H), 1.70 - 1.58 (m, 1H), 1.46 (d, J = 6.0 Hz, 3H), 1.34 - 1.26 (m, 1H), 1.00 - 0.88 (m, 9H), 0.50 (s, 3H).

Example 7 ^-[(15)-l-[[(75,135)-21-ethyl-24-fluoro-(20M)-20-[2-[(15)-l- methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- y l|carbamoyl | -2-methyl- propyl | -3.3-difhioro-\- met hy 1-azetidine- 1 -carboxamide

The compound was prepared according to the following scheme:

Step 1: tert-butyl A-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-8,14-di oxo-15-oxa-4-thia- 9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl]carbamoyl]-2-methyl-propyl]-2V-methyl-carbamate (compound 7A)

To a solution of BOC-A-ME-VAL-OH (compound 1A, 66.2 mg, 0.29 mmol) in DMF (1 mL) were added DIEA (0.3 mL, 1.90 mmol) and HATU (67.3 mg, 0.29 mmol) at 0 °C. After being stirred at 0 °C for 15 min, the reaction mixture was added with (7S,13S)-7-amino-21-ethyl- 24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5-morpholino-3-pyridyl]- 17, 17-dimethyl- 15-oxa- 4-thia-9,21,27,28-tetrazapentacyclo-[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaene- 8, 14-dione (intermediate G, 70.0 mg, 0.09 mmol). After the reaction was stirred at 20 °C for 0.5 h, the reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL, three times). The combined organic layer was washed with brine (10 mL), and dried over NaiSCU, filtered and concentrated under vacuum to give a residue. The residue was purified by prep-TLC to afford tert-butyl A-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20Af)-20-[2-[(lS)-l - methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-8,14-di oxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-A-methyl-carbamate (compound 7A, 65.1 mg) as a light yellow solid. MS calc’d 947.5 (MH ⁺ ), measured 947.4 (MH ⁺ ).

Step 2: [(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l-me thoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-methyl-ammonium;2,2,2-trifluo roacetate (compound 7B)

To the mixture solution of tert-butyl A ^f -|( I S)- l -||(7S. I 3S)-2 l-cthyl-24-nuoro-(20A7)-20-|2- [( IS)- l-methoxyethyl]-5-morpholino-3-pyridyl] - 17, 17-dimethyl-8, 14-dioxo- 15-oxa-4-thia- 9,21,27 ,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen- 7-yl]carbamoyl]-2-methyl-propyl]-A-methyl-carbamate (compound 7A, 40.0 mg, 0.04 mmol) in DCM (1 mL) was added TFA (0.2 mL, 2.7 mmol) at 20 °C. After being stirred at 20 °C for 1 h, the reaction mixture was concentrated in vacuo to get a residue. The residue was purified prep- HPLC to afford [( IS)- 1-[[(7S, 13S)-21-ethyl-24-fluoro-(20M)-20-[2-[( IS)- l-methoxyethyl]-5- morpholino-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo- 15-oxa-4-thia-9, 21, 27, 28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-methyl-ammonium;2,2,2-trifluo roacetate (compound 7B, 20.0 mg) as a white solid. MS calc’d 847.5 (MH ⁺ ), measured 847.4 (MH ⁺ ). ^J H NMR (400 MHz, Methanol-eU) 6 = 8.68 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 2.8 Hz, 1H), 7.65 (d, J= 2.4 Hz, 1H), 7.56 (br s, 1H), 7.35 (d, J = 12.6 Hz, 1H), 5.95 - 5.92 (m, 1H), 5.04 - 4.96 (m, 1H), 4.94 - 4.91 (m, 3H), 4.45 - 4.40 (m, 1H), 4.35 - 4.05 (m, 4H), 3.88 - 3.83 (m, 4H), 3.80 - 3.69 (m, 3H), 3.56 - 3.50 (d, J = 14.5 Hz, 1H), 3.35 - 3.31 (m, 4H), 3.07 - 2.97 (m, 1H), 2.90 - 2.80 (m, 1H), 2.75 - 2.65 (m, 4H), 2.30 - 2.15 (m, 2H), 2.03 - 1.91 (m, 1H),1.89 - 1.75 (m, 1H), 1.73 - 1.56 (m, 1H), 1.45 - 1.43 (m, 3H), 1.15 - 1.09 (m, 3H), 1.09 - 1.07 (m, 3H), 1.03 - 1.01 (m, 3H), 0.95 (s, 3H), 0.56 (s, 3H).

Step 3: A-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5- morpholino-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thi a-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7- y l|carbamoyl | -2-methyl- propyl | -3.3-difhioro- \- met hy 1-azetidine- 1 -carboxamide (Example 7)

To a solution of [(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l-me thoxyethyl]- 5-morpholino-3-pyridyl]- 17, 17-dimethyl-8, 14-dioxo- 15-oxa-4-thia-9, 21, 27, 28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .1 ⁹ ’ ¹³ .0 ²² ’ ²⁶ ]octacosa- 1(25), 2, 5(28), 19, 22(26), 23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-methyl-ammonium;2,2,2-trifluo roacetate (compound 7B, 50.0 mg, 0.06 mmol) and DIEA (45.8 mg, 0.35 mmol) in DCM (1 rnL) was added triphosgene (6.1 mg, 0.02 mmol) at 0 °C. After being stirred at 20 °C for 1 h, the reaction mixture was added with 3,3-difluoroazetidine hydrochloride (compound 7C, 7.6 mg, 0.06 mmol) at 0 °C and stirred at 20 °C for 11 hrs. After the reaction was completed, the reaction mixture was poured into H2O (10 mL), extracted with EtOAc (10 rnL, three times). The organic layer was washed with brine (10 mL, twice), dried over NaiSCU, filtered and concentrated under vacuum to get a residue. The residue was purified by prep-HPLC to afford Example 7 (13.0 mg) as a light yellow solid. MS calc’d 966.5 (MH ⁺ ), measured 966.4 (MH ⁺ ). ^J H NMR (400 MHz, Methanol-^) 6 = 8.70 - 8.64 (m, 1H), 8.49 - 8.35 (m, 1H), 7.62 (d, J = 2.2 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 12.8 Hz, 1H), 5.78 (br t, J= 8.2 Hz, 1H), 5.02 - 4.96 (m, 1H), 4.68 - 4.52 (m, 1H), 4.46 - 4.43 (m, 1H), 4.42 - 4.32 (m, 4H), 4.22 - 4.05 (m, 3H), 3.93 - 3.82 (m, 4H), 3.81 - 3.66 (m, 2H), 3.50 - 3.42 (m, 1H), 3.35 - 3.31 (m, 6H), 3.28 - 3.21 (m, 1H), 3.09 - 2.96 (m, 1H), 2.88 (s, 3H), 2.84 - 2.77 (m, 1H), 2.26 - 2.14 (m, 2H), 1.95 - 1.90 (m, 1H), 1.87 - 1.74 (m, 1H), 1.69 - 1.59 (m, 1H), 1.43 (d, J = 6.2 Hz, 3H), 1.36 - 1.27 (m, 2H), 1.03 - 0.89 (m, 12H), 0.53 (s, 3H).

Example 8 (25)-^-[(75,135)-21-ethyl-24-fluoro-(20M)-20-[2-[(15)-l-meth oxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ^{9 13} .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-(3-methyl-2-oxo-imidazolidin-l-yl)butanamide

The title compound was prepared in analogy to the preparation of Example 1 by using (2S)-3-methyl-2-(3-methy 1-2-oxo-imidazo lidin- 1-yl) butanoic acid (compound 8H) and (75, 13.8')- 7-amino-21 -ethyl- 24-fluoro-(20A7)-20- [2- [( 1 S)- 1 -methoxyethyl] -5-morpholino-3-pyridyl] - 17, 17- 25 913 2226 dimethyl-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo-[17.5.2. 1 .1 .0 ’ ]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (intermediate G) instead of (2S)-2-[[3- (dimethylamino)azetidine-1-carbonyl]-methyl-amino]-3-methyl- butanoic acid (compound 1F) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1- yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-o xa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 8 (50.4 mg) was obtained as a yellow solid. MS calc’d 916.5 (MH ⁺ ), measured 916.3 (MH ⁺ ). ¹ H NMR (400 MHz, CHLOROFORM-d) δ = 8.84 (br s, 1H), 8.66 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.10 (d, J = 12.3 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 5.83 (t, J = 9.0 Hz, 1H), 4.59 (br d, J = 11.9 Hz, 1H), 4.39 - 4.31 (m, 1H), 4.27 - 4.14 (m, 2H), 4.11 - 4.03 (m, 1H), 3.95 (s, 1H), 3.93 - 3.89 (m, 4H), 3.84 (br d, J = 11.5 Hz, 1H), 3.71 (d, J = 11.0 Hz, 1H), 3.52 - 3.46 (m, 1H), 3.42 - 3.31 (m, 11H), 3.21 - 3.07 (m, 3H), 2.89 (s, 3H), 2.76 - 2.59 (m, 2H), 2.46 (br d, J = 14.4 Hz, 1H), 2.29 - 2.18 (m, 2H), 2.00 - 1.91 (m, 1H), 1.80 (br d, J = 12.2 Hz, 1H), 1.66 - 1.59 (m, 1H), 1.54 - 1.50 (m, 3H), 1.00 - 0.90 (m, 12H), 0.49 (s, 3H). The compound 8H was prepared according to the following scheme: 8H Step 1: Preparation of benzyl (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoate (compound 8B) To a mixture of BOC-L-valine (compound 8A, 1.0 g, 4.6 mmol) and potassium carbonate (763.4 mg, 5.52 mmol) in DMF (10 mL) was added benzyl bromide (0.6 mL, 5.06 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 hrs. After the reaction was completed, the reaction mixture was diluted with water (70 mL), extracted with EtOAc (70 mL, three times). The combined organic layer was washed with brine (50 mL) and dried over Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography to afford benzyl (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoate (compound 8B, 1.0 g) as colorless oil. MS calc’d 307.4 (MH ⁺ ), measured 208.0 (M-Boc+H ⁺ ). Step 2: Preparation of benzyl (2S)-2-amino-3-methyl-butanoate (compound 8C) A mixture of benzyl (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoate (compound 8B, 1.0 g, 3.11 mmol) in THF (5 mL) was added with HCl/dioxane (20.0 mL, 80.0 mmol, 4 N), at 0 °C. After the reaction being stirred at 25 °C for 1 h, the reaction mixture was concentrated under vacuum to give a yellow gum. The gum was diluted with aq.NaHCO3 (40 mL), extracted with EtOAc (50 mL, three times). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under vacuum to afford benzyl (2S)-2-amino-3- methyl-butanoate (compound 8C, 600.0 mg) as colorless oil, which was used to the next step without purification. Step 3: Preparation of benzyl (2S)-2-[2-[tert-butoxycarbonyl(methyl)amino]- ethylamino]-3-methyl-butanoate(compound 8E) To a solution of benzyl (2S)-2-amino-3-methyl-butanoate (compound 8C, 600.0 mg, 2.89 mmol) and N-BOC-(methylamino)acetaldehyde (compound 8D, 601.7 mg, 3.47 mmol) in Methanol (20 mL) was added MgSO4 (2.1 g, 14.47 mmol). After being stirred at 20 °C for 1 h, the reaction mixture was added with NaBH(OAc)3 (1.2 g, 5.79 mmol) and stirred at 20 °C for another 1 h. After the reaction was completed, the reaction mixture was filtered and the filtrate was concentrated under vacuum to give colorless oil. The oil was purified by column chromatography to afford benzyl (2S)-2-[2-[tert-butoxycarbonyl(methyl)amino]-ethylamino]-3- methyl-butanoate(compound 8E, 700.0 mg) as a colorless gum. MS calc’d 365.2 (MH ⁺ ), measured 365.1 (MH ⁺ ). Step 4: Preparation of benzyl (2S)-3-methyl-2-[2- (methylamino)ethylamino]butanoate;hydrochloride (compound 8F) To a solution of benzyl (2S)-2-[2-[tert-butoxycarbonyl(methyl)amino]-ethylamino]-3- methyl-butanoate(compound 8E, 700.0 mg, 1.92 mmol) in THF (5 mL) at 0 °C was added HCl/dioxane (15.0 mL, 60.0 mmol, 4N). After being stirred at 25 °C for 2 hrs, the reaction mixture was concentrated under vacuum to give a white solid. The residual solvent was removed via azeotrope distillation with THF twice to afford benzyl (2S)-3-methyl-2-[2- (methylamino)ethylamino]butanoate;hydrochloride (compound 8F, 600.0 mg) as a white solid. It was used directly to next step without further purification. MS calc’d 265. 2 (MH ⁺ ), measured

265.2 (MH ⁺ ).

Step 5: Preparation of benzyl (2S)-3-methyl-2-(3-methyl-2-oxo-imidazolidin-l- yl)butanoate (compound 8G)

To a solution of benzyl (2S)-3-methyl-2-[2- (methylamino)ethylamino]butanoate;hydrochloride (compound 8F, 600.0 mg, 1.99 mmol) and DIEA (772.5 mg, 5.98 mmol) in ACN (50 mL) was added CDI (646.4 mg, 3.99 mmol). After being stirred at 0 °C for 2 hrs, the reaction mixture was added with water (10 mL), extracted with EA (40 mL, twice). The combined organic layer was washed with brine (20 mL), dried (NaiSCU) and concentrated under vacuum to give a yellow gum. The gum was purified by reversed-phase column to afford benzyl (2S)-3-methyl-2-(3-methyl-2-oxo-imidazo lidin- 1- yl)butanoate (compound 8G, 300.0 mg) as a colorless gum. MS calc’d 291.2 (MH ⁺ ), measured

291.2 (MH ⁺ ).

Step 6: Preparation of (2S)-3-methyl-2-(3-methyl-2-oxo-imidazolidin-l-yl)butanoic acid (compound 8H)

To a solution of benzyl (2S)-3-methyl-2-(3-methyl-2-oxo-imidazo lidin- l-yl)butanoate (compound 8G, 280.0 mg, 0.96 mmol) in Methanol (10 mL) was added Pd(OH)2 on activated carbon (0.2 g, 10% purity). The reaction mixture was hydrogenated at rt for 5 hrs. The reaction mixture was filtered and the filtrate was concentrated under vacuum to afford (2S)-3-methyl-2- (3-methy 1-2-oxo-imidazo lidin- 1-yl) butanoic acid (compound 8H, 150.0 mg) as a colorless gum. MS calc’d 201.1 (MH ⁺ ), measured 201.0 (MH ⁺ ).

Example 9 ^-[(lS)-l-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(lS)-l- methoxyethyl]-5-[4-(2,2,2- trifhioroethyl)piperazin- 1-yl] -3-pyridyl] - 17,17-dimethyl-8,14-dioxo-15-oxa-4-thia- 9,21,27,28-tetrazapentacyclo[17.5.2.1 ² ’ ⁵ .l ⁹¹³ .0 ²² ’ ²⁶ ]octacosa-l(25),2,5(28),19,22(26),23- hexaen-7-yl]carbamoyl]-2-methyl-propyl]-A-methyl-morpholine- 4-carboxamide

The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimet hyl-15-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate F) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1-yl)-3- pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-t hia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 9 (26.8 mg) was obtained as an off-white solid. MS calc’d 1041.5 (MH ⁺ ), measured 1041.7 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ = 8.71 (d, J = 7.6 Hz, 1H), 8.38 (d, J = 2.8 Hz, 1H), 7.87 (d, J = 2.8 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.36 (d, J = 12.8 Hz, 1H), 5.86 (d, J = 8.4 Hz, 1H), 4.48 - 4.32 (m, 2H), 4.30 - 4.19 (m, 1H), 4.18 - 4.11 (m , 1H), 4.08 - 3.97 (m, 2H), 3.80 - 3.67 (m, 6H), 3.51 - 3.47 (m, 4H), 3.39 - 3.33 (m, 5H), 3.29 - 3.08 (m, 6H), 3.03 - 2.96 (m, 1H), 2.93 - 2.84 (m, 7H), 2.84 - 2.70 (m, 2H), 2.28 - 2.14 (m, 2H), 1.99 - 1.92 (m, 1H), 1.86 - 1.74 (m, 1H), 1.70 - 1.58 (m, 1H), 1.48 - 1.43 (d, J = 6.0 Hz, 3H) 1.03 (t, J = 6.8 Hz, 3H), 0.96 - 0.89 (m, 9H), 0.61 (s, 3H). Example 10 N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-8,14-dioxo-1 5-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-morpholine-4-carboxa mide

The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]-5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-15-oxa-4-thi a-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate D) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1-yl)-3- pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-t hia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 10 (10.3 mg) was obtained as a yellow solid. MS calc’d 973.5 (MH ⁺ ), measured 973.5 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ = 8.67 (d, J = 7.6 Hz, 1H), 8.49 (d, J = 2.8 Hz, 1H), 7.70 - 7.60 (m, 2H), 7.34 (d, J = 12.4 Hz, 1H), 5.80 (d, J = 8.8 Hz, 1H), 4.43 (d, J = 10.8 Hz, 1H), 4.36 - 4.28 (m, 1H), 4.27 - 4.05 (m, 4H), 4.01 (d, J = 10.8 Hz, 1H), 3.81 - 3.62 (m, 8H), 3.49 - 3.43 (m, 2H), 3.42 - 3.33 (m, 7H), 3.29 - 3.17 (m, 4H), 3.07 - 3.02 (m, 1H), 2.99 (s, 3H), 2.89 (s, 3H), 2.86 - 2.79 (m, 1H), 2.67 - 2.63 (m, 1H), 2.30 - 2.15 (m, 2H), 2.01 - 1.91 (m, 1H), 1.86 - 1.74 (m, 1H), 1.70 - 1.57 m, 1H), 1.44 (d, J = 6.0 Hz, 3H), 1.03 - 0.88 (m, 13H), 0.53 (s, 3H). Example 11 N-[(1S)-1-[[(7S,13S)-24-fluoro-20-(20M)-[2-[(1S)-1-methoxyet hyl]-5-morpholino-3-pyridyl]- 17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4 -thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-morpholine-4-carboxa mide

The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-morpholino- 3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4 -thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate I) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1-yl)-3- pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-oxa-4-t hia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 11 (26.3 mg) was obtained as a white solid. MS calc’d 1013.5 (MH ⁺ ), measured 1013.8 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ = 8.71 (d, J = 7.6 Hz, 1H), 8.49 - 8.35 (d, J = 2.8, 1H), 7.73 - 7.69 (d, J = 2.4, 1H), 7.66 - 7.59 (d, J = 2.4, 1H), 7.53 - 7.43 (d, J = 12.4, 1H), 5.88 - 5.70 (d, J = 8.8, 1H), 5.25 - 5.13 (m, 1H), 4.53 - 4.38 (m, 1H), 4.31 - 4.15 (m, 2H), 4.07 - 3.98 (d, J = 10.8, 1H), 3.91 - 3.84 (t, J = 4.8, 4H), 3.83 - 3.67 (m, 6H), 3.52 - 3.45 (m, 1H), 3.43 - 3.34 (m, 10H), 3.30 - 3.18 (m, 3H), 3.11 (m, 1H), 2.99 - 2.89 (s, 3H), 2.88 - 2.78 (m, 1H), 2.72 - 2.60 (d, J = 14.8, 1H), 2.34 - 2.15 (m, 2H), 2.01 - 1.91 (m, 1H), 1.90 - 1.74 (m, 1H), 1.72 - 1.57 (m, 1H), 1.52 - 1.42 (d, J = 6.0, 3H), 1.05 - 0.88 (m, 9H), 0.57 - 0.45 (s, 3H). Example 12 3-(dimethylamino)-N-[(1S)-1-[[(7S,13S)-24-fluoro-(20M)-20-[2 -[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimet hyl-8,14-dioxo-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-p ropyl]-N-methyl-azetidine-1- carboxamide

The title compound was prepared in analogy to the preparation of Example 1 by using (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-21- (2,2,2-trifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate E) instead of (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-(4-methylpiperazin-1-yl)-3-pyridyl]-17,17-di methyl-21-(2,2,2-trifluoroethyl)- 2,5 9,13 22,26 15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 12 (45.3 mg) was obtained as a white solid. MS calc’d 1108.5 (MH ⁺ ), measured 1108.5 (MH ⁺ ). ¹ H NMR (400MHz, Methanol-d4) δ = 8.68 (d, J = 7.6 Hz, 1H), 8.42 (d, J = 2.8 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 12.8 Hz, 1H), 7.32 -7.27 (m, 1H), 5.74 - 5.68 (m, 1H), 5.18 - 5.06 (m, 2H), 4.93 - 5.00 (m, 2H), 4.58 - 4.56(m, 5H), 4.32 - 4.23 (m, 1H), 4.22 - 4.14 (m, 2H), 4.14 - 4.07 (m, 1H), 4.00 - 3.89 (m, 1H), 3.87 - 3.67 (m, 2H), 3.53 - 3.39 (m, 1H), 3.34 - 3.32 (m, 4H), 3.19 - 3.09 (m, 4H), 2.90 - 2.79 (m, 8H), 2.63 - 2.54 (m, 1H), 2.26 - 2.15 (m, 8H), 1.57 - 1.98 (m, 3H), 1.43 (d, J = 6.0 Hz, 3H), 0.99 - 0.89 (m, 9H), 0.44 (s, 3H). Example 13 (2S)-N-[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-[methyl(2,2,2-trifluoroethylcarbamoyl)amino]butanam ide

The title compound was prepared in analogy to the preparation of Example 1 by using 2,2,2-Trifluoroethylamine and (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-15-oxa- 4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo-[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14- dione (Intermediate G) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1- yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-o xa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 13 (13.4 mg) was obtained as a white solid. MS calc’d 972.4 (MH ⁺ ), measured 972.4 (MH ⁺ ). ¹ H NMR (400 MHz, CHLOROFORM-d) δ = 8.64 (d, J = 7.6 Hz, 1H), 8.47 (d, J = 2.8 Hz, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.10 (s, 1H), 7.08 - 7.03 (m, 2H), 5.84 (t, J = 9.1 Hz, 1H), 5.21 (br s, 1H), 4.58 (br d, J = 12.1 Hz, 1H), 4.32 - 4.21 (m, 3H), 4.08 - 4.02 (m, 2H), 3.92 - 3.88 (m, 4H), 3.84 (br d, J = 11.0 Hz, 1H), 3.73 - 3.70 (m, 1H), 3.38 (s, 3H), 3.27 - 3.21 (m, 4H), 3.17 - 3.04 (m, 2H), 2.97 (s, 3H), 2.74 - 2.65 (m, 1H), 2.46 (br d, J = 14.4 Hz, 1H), 2.31 - 2.21 (m, 5H), 1.96 (br d, J = 13.7 Hz, 1H), 1.85 - 1.72 (m, 1H), 1.66 - 1.54 (m, 1H), 1.44 (d, J = 6.1 Hz, 3H), 1.25 (t, J = 7.0 Hz, 1H), 0.99 - 0.86 (m, 12H), 0.45 (s, 3H). Example 14 N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-3-(trifluoromethyl)a zetidine-1-carboxamide

The title compound was prepared in analogy to the preparation of Example 1 by using 3- (Trifluoromethyl)azetidine and (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-15-oxa- 4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo-[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14- dione (Intermediate G) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4-methylpiperazin-1- yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-trifluoroethyl)-15-o xa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 14 (3.1 mg) was obtained as a white solid. MS calc’d 998.5 (MH ⁺ ), measured 998.5 (MH ⁺ ). ¹ H NMR (400 MHz, CHLOROFORM-d) δ = 8.64 (d, J = 7.5 Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H), 7.58 (s, 1H), 7.32 (br d, J = 9.6 Hz, 1H), 7.11 - 7.04 (m, 2H), 5.81 (br t, J = 8.8 Hz, 1H), 4.59 (br d, J = 9.8 Hz, 1H), 4.35 (br t, J = 8.7 Hz, 1H), 4.30 - 4.21 (m, 4H), 4.13 (br dd, J = 7.9, 15.0 Hz, 3H), 4.08 - 3.99 (m, 3H), 3.90 (br s, 4H), 3.84 (br d, J = 11.3 Hz, 1H), 3.43 (br d, J = 14.6 Hz, 1H), 3.37 (s, 3H), 3.24 (br d, J = 4.3 Hz, 4H), 3.18 - 3.13 (m, 1H), 2.84 (s, 3H), 2.75 - 2.62 (m, 1H), 2.47 (br d, J = 14.4 Hz, 1H), 2.31 - 2.17 (m, 2H), 2.05 (s, 3H), 1.94 (br d, J = 1.8 Hz, 1H), 1.51 (br s, 2H), 1.46 - 1.46 (m, 1H), 1.44 - 1.44 (m, 1H), 1.15 (td, J = 6.4, 13.0 Hz, 12H), 0.46 (s, 3H). Example 15 (2S)-N-[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7-yl]-3- methyl-2-(3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c][1,4]ox azin-2-yl)butanamide

The title compound was prepared in analogy to the preparation of Example 1 by using (2S)-3-methyl-2-(3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c] [1,4]oxazin-2-yl)butanoic acid (compound 15g) and (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]- 5-morpholino-3-pyridyl]-17,17-dimethyl-15-oxa-4-thia-9,21,27 ,28-tetrazapentacyclo- 2,5 9,13 22,26 [17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (intermediate G) instead of (2S)-2-[[3-(dimethylamino)azetidine-1-carbonyl]-methyl-amino ]-3-methyl-butanoic acid (compound 1F) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-tr ifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate C). Example 15 (37.5 mg) was obtained as a white solid. MS calc’d 958.5 (MH ⁺ ), measured 958.5 (MH ⁺ ). ¹ H NMR (400 MHz, METHANOL-d4) δ = 8.70 - 8.61 (m, 1H), 8.53 - 8.30 (m, 1H), 7.64 - 7.59 (m, 1H), 7.38 - 7.27 (m, 2H), 5.86 - 5.72 (m, 1H), 5.03 - 4.92 (m, 1H), 4.82 - 4.78 (m, 1H), 4.67 - 4.53 (m, 3H), 4.47 - 4.36 (d, J=12.8 Hz,1H), 4.29 - 4.12 (m, 4H), 4.10 - 4.01 (d, J=10.8 Hz,1H), 3.89 - 3.84 (m, 4H), 3.79 - 3.68 (m, 4H), 3.65 - 3.54 (m, 1H), 3.50 - 3.36 (m, 4H), 3.28 (m, 4H), 3.20 - 3.10 (m, 2H), 3.03 - 2.95 (m, 1H), 2.88 - 2.75 (m, 1H), 2.69 - 2.58 (m, 1H), 2.24 - 2.07 (m, 2H), 2.01 - 1.89 (m, 1H), 1.87 - 1.71 (m, 1H), 1.70 - 1.56 (m, 1H), 1.51 - 1.37 (m, 3H), 1.03 - 0.87 (m, 12H), 0.62 - 0.42 (m, 3H). The compound 15g was prepared according to the following scheme: Step1: tert-butyl 3-[[(1S)-1-benzyloxycarbonyl-2-methyl-propyl]carbamoyl]- morpholine-4-carboxylate (compound 15c) The solution of 4-tert-butoxycarbonylmorpholine-3-carboxylic acid (compound 15b, 2.8 g, 12.31 mmol) and L-valine benzyl ester hydrochloride (compound 15a, 3.0 g, 12.31 mmol) in DMF (30 mL) were added with DIEA (4.29 mL, 24.62 mmol) and HATU (5.1 g, 13.54 mmol). After being stirred at 25 °C for 1 h, the reaction mixture were added with EtOAc (40 mL) and water (40 mL). The layers were separated and the aqueous phase was extracted with EtOAc (30 mL, twice). The combined organic layer was washed with brine (60 mL), dried over Na2SO4, filtered, and concentrated under vacuum to give tert-butyl 3-[[(1S)-1-benzyloxycarbonyl-2- methyl-propyl]carbamoyl]morpholine-4-carboxylate (compound 15c, 7.5 g) as white oil. MS calc’d 420.23(MH ⁺ ), measured 321.2 (M-Boc+H ⁺ ). Step2: benzyl (2S)-3-methyl-2-(morpholine-3-carbonylamino)-butanoate; hydrochloride (compound 15d) To a solution of tert-butyl 3-[[(1S)-1-benzyloxycarbonyl-2-methyl- propyl]carbamoyl]morpholine-4-carboxylate (compound 15c, 7.2 g, 17.16 mmol) in 1,4-dioxane (10 mL) was added 1,4-dioxane/HCl (4M, 15.0 mL, 60.0 mmol). The mixture was stirred at 25 °C for 2 hrs. The mixture was concentrated under vacuum to afford benzyl (2S)-3-methyl-2- (morpholine-3-carbonylamino)-butanoate; hydrochloride (compound 15d, 6.7 g) as white oil. MS calc’d 321.2 (MH ⁺ ), measured 321.2 (MH ⁺ ). Step3: benzyl (2S)-2-(1,3-dioxo-5,6,8,8a-tetrahydroimidazo[5,1-c][1,4]oxaz in-2-yl)-3- methyl-butanoate (compound 15e) The solution of benzyl (2S)-3-methyl-2-(morpholine-3-carbonylamino)butanoate (compound 15d, 6.5 g, 20.29 mmol) in THF (1 mL) was added TEA (8.5 mL, 60.87 mmol) and N.N'-carbonyldiimidazole (3.9 g, 24.35 mmol). After being stirred at 25 °C for 2 hrs, the reaction mixture was concentrated under vacuum to give a residue. The residue was purified by reversed- phase chromatography column to afford benzyl (2S)-2-(1,3-dioxo-5,6,8,8a- tetrahydroimidazo[5,1-c][1,4]oxazin-2-yl)-3-methyl-butanoate (compound 15e, 2.36 g) as white oil. MS calc’d 346.4 (MH ⁺ ), measured 369.4 (MNa ⁺ ). Step4: benzyl (2S)-3-methyl-2-(1-oxo-5,6,8,8a-tetrahydro-3H-imidazo[5,1- c][1,4]oxazin-2-yl)butanoate (compound 15e) To a solution of benzyl (2S)-2-(1,3-dioxo-5,6,8,8a-tetrahydroimidazo[5,1-c][1,4]oxaz in- 2-yl)-3-methyl-butanoate (compound 15e, 1 g, 2.89 mmol) in THF (5 mL) were added boron trifluoride diethyl etherate (1M, 1.2 mL, 1.2 mmol) and borane-THF (5.77 mL, 5.77 mmol) at 0°C. After being stirred at 20 °C for 18 hrs, the reaction was quenched with water (40 mL). The aqueous phase was extracted with EtOAc (30 mL, twice). The combined organic layer was washed with brine (60 mL), dried over Mg ₂ SO ₄ , filtered, and concentrated under vacuum to give a residue. The residue was purified by reversed phase column to give benzyl (2S)-3-methyl-2-(1- oxo-5,6,8,8a-tetrahydro-3H-imidazo[5,1-c][1,4]oxazin-2-yl)bu tanoate (compound 15f, 220.0 mg) as colorless oil. MS calc’d 333.2 (MH ⁺ ), measured 333.2 (MH ⁺ ). Step5: (2S)-3-methyl-2-(3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c] [1,4]oxazin-2- yl)butanoic acid (compound 15f) To a solution of benzyl (2S)-3-methyl-2-(1-oxo-5,6,8,8a-tetrahydro-3H-imidazo[5,1- c][1,4]oxazin-2-yl)butanoate (compound 15e, 130.0 mg, 0.39 mmol) in THF (1 mL) was added Pd/C (40.0 mg, 0.78 mmol). The reaction mixture was stirred at 25 °C for 1 h under hydrogen atmosphere. The mixture was filtered, and the filtrate was concentrated under vacuum to give (2S)-3-methyl-2-(3-oxo-5,6,8,8a-tetrahydro-1H-imidazo[5,1-c] [1,4]oxazin-2-yl)butanoic acid (compound 15g, 62.0 mg) as colorless oil. MS calc’d 243.1 (MH ⁺ ), measured 243.2 (MH ⁺ ). Example 16 (3R)-N-[(1S)-1-[[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1-meth oxyethyl]-5-morpholino-3- pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl) -15-oxa-4-thia-9,21,27,28- 2,5 9,13 22,26 tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-3-hydroxy-N,3-dimethyl-piperi dine-1-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using (3R)-3-methylpiperidin-3-ol;hydrochloride and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)- 1-methoxyethyl]-5-morpholino-3-pyridyl]-17,17-dimethyl-21-(2 ,2,2-trifluoroethyl)-15-oxa-4- 2,5 9,13 22,26 thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23- hexaene-8,14-dione (Intermediate I) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-tr ifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate C). Example 16 (13.9 mg) was obtained as a white solid. MS calc’d 1042.5 (MH ⁺ ), measured 1042.5 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ = 8.73 - 8.69 (d, J = 7.2 Hz, 1H), 8.44 - 8.39 (d, J = 2.8 Hz, 1H), 7.70 - 7.67 (d, J = 2.8 Hz, 1H), 7.67 - 7.64 (d, J = 2.4 Hz, 1H), 7.51 - 7.45 (d, J = 12.4 Hz, 1H), 5.80 - 5.74 (d, J = 8.4 Hz, 1H), 5.23 - 5.13 (m, 1H), 4.83 - 4.76 (m, 1H), 4.47 - 4.40 (m, 1H), 4.29 - 4.23 (q, J = 6.4 Hz, J = 12.0 Hz, 1H), 4.23 - 4.17 (dd, J = 2.4, 11.6 Hz, 1H), 4.05 - 3.99 (d, J = 10.8 Hz, 1H), 3.89 - 3.85 (t, J = 4.4 Hz, 4H), 3.82 - 3.78 (d, J = 11.2 Hz, 1H), 3.74 - 3.69 (d, J = 11.2 Hz, 1H), 3.56 - 3.45 (m, 2H), 3.39 - 3.36 (m, 6H), 3.29 - 3.19 (m, 2H), 3.14 - 3.06 (m, 1H), 3.03 - 2.96 (d, J = 12.8 Hz, 2H), 2.91 - 2.89 (s, 3H), 2.87 - 2.78 (m, 1H), 2.71 - 2.63 (m, 1H), 2.29 - 2.16 (m, 2H), 2.00 - 1.90 (m, 3H), 1.88 - 1.76 (m, 1H), 1.74 - 1.68 (m, 1H), 1.67 - 1.53 (m, 3H), 1.49 - 1.45 (d, J = 6.4 Hz, 3H), 1.34 - 1.27 (m, 1H), 1.25 - 1.21 (s, 3H), 0.99 - 0.93 (dd, J = 6.0, 12.0 Hz, 9H), 0.56 - 0.49 (s, 3H). Example 17 N-[(1S)-1-[[(7S,13S)-25-fluoro-(20M)-20-[2-[(1S)-1-methoxyet hyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-8,1 4-dioxo-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-p ropyl]-N-methyl-morpholine- 4-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using morpholine and (7S,13S)-7-amino-25-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-21- (2,2,2-trifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate J) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-tr ifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate C). Example 17 (12.9 mg) was obtained as a yellow solid. MS calc’d 1094.5 (MH ⁺ ), measured 1094.5 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d4) δ = 8.49 - 8.39 (m, 1H), 7.74 (br s, 1H), 7.53 - 7.44 (m, 3H), 5.97 (br d, J = 5.9 Hz, 1H), 5.18 - 5.11 (m, 1H), 4.67 - 4.58 (m, 1H), 4.42 - 4.33 (m, 1H), 4.10 - 3.97 (m, 1H), 3.94 - 3.80 (m, 2H), 3.76 - 3.63 (m, 5H), 3.58 - 3.42 (m, 7H), 3.26 - 3.14 (m, 8H), 3.13 - 2.99 (m, 2H), 2.93 (br d, J = 14.4 Hz, 8H), 2.62 - 2.53 (m, 1H), 2.30 - 2.14 (m, 1H), 1.61 - 1.49 (m, 1H), 1.43 (br d, J = 6.1 Hz, 3H), 1.34 - 1.15 (m, 3H), 0.92 (br t, J = 7.3 Hz, 6H), 0.85 - 0.69 (m, 6H). Example 18 N-[(1S)-1-[[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyet hyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-8,1 4-dioxo-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-p ropyl]-N,4-dimethyl- piperazine-1-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using 1- methylpiperazine and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimet hyl-21-(2,2,2-trifluoroethyl)-15-oxa- 2,5 9,13 22,26 4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23- hexaene-8,14-dione (Intermediate E) instead of N,N-dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl] -5-(4- methylpiperazin-1-yl)-3-pyridyl]-17,17-dimethyl-21-(2,2,2-tr ifluoroethyl)-15-oxa-4-thia- 2,5 9,13 22,26 9,21,27,28-tetrazapentacyclo[17.5.2.1 .1 .0 ]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (Intermediate C). Example 18 (10.8 mg) was obtained as a white solid. MS calc’d 1108.5 (MH ⁺ ), measured 1108.5 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d4) δ = 8.70 - 8.65 (d, J = 7.6 Hz, 1H), 8.45 - 8.40 (d, J = 2.8 Hz, 1H), 7.70 - 7.65 (d, J = 2.4 Hz, 1H), 7.50 - 7.43 (d, J = 12.8 Hz, 1H), 7.33 - 7.28 (d, J = 2.8 Hz, 1H), 5.78 - 5.70 (d, J = 9.2 Hz, 1H), 5.20 - 5.05 (m, 1H), 4.62 - 4.61 (s, 1H), 4.46 - 4.40 (m, 1H), 4.28 - 4.20 (m, 1H), 4.19 - 4.13 (m, 1H), 4.06 - 3.99 (d, J = 11.2 Hz, 1H), 3.83 - 3.65 (m, 3H), 3.53 - 3.40 (m, 4H), 3.27 - 3.07 (m, 6H), 2.94 - 2.81 (m, 9H), 2.63 - 2.45 (m, 6H), 2.37-2.34 (s, 3H), 2.29 - 2.17 (m, 3H), 2.00 - 1.93 (m, 1H), 1.90 - 1.75 (m, 2H), 1.71 - 1.56 (m, 1H), 1.46 - 1.41 (d, J = 6.4 Hz, 3H), 1.33 - 1.15 (m, 1H), 0.98 - 0.91 (m, 9H), 0.47 - 0.42 (s, 3H). Example 19 N-[(1S)-1-[[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyet hyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-8,1 4-dioxo-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-p ropyl]-N-methyl-4-(2,2,2- trifluoroethyl)piperazine-1-carboxamide The title compound was prepared in analogy to the preparation of Example 1 by using 1- (2,2,2-trifluoroethyl)piperazine and (7S,13S)-7-amino-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-3-p yridyl]-17,17-dimethyl-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[1 7.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate E) instead of N,N- dimethylazetidin-3-amine dihydrochloride (compound 1D) and (7S,13S)-7-amino-24-fluoro- (20M)-20-[2-[(1S)-1-methoxyethyl]-5-(4-methylpiperazin-1-yl) -3-pyridyl]-17,17-dimethyl-21- 2,5 9,13 22,26 (2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28-tetrazapenta cyclo[17.5.2.1 .1 .0 ]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (Intermediate C). Example 19 (12.1 mg) was obtained as a white solid. MS calc’d 1176.5 (MH ⁺ ), measured 1176.5 (MH ⁺ ). ¹ H NMR (400 MHz, Methanol-d ₄ ) δ = 8.82 - 8.63 (d, J = 7.2 Hz, 1H), 8.48 - 8.38 (d, J = 2.8 Hz, 1H), 7.75 - 7.67 (d, J = 2.0 Hz, 1H), 7.54 - 7.43 (d, J = 12.4 Hz, 1H), 7.35 - 7.28 (d, J = 2.4 Hz, 1H), 5.86 - 5.72 (d, J = 8.8 Hz, 1H), 5.26 - 5.03 (m, 1H), 4.98 - 4.90 (m, 3H), 4.66 - 4.56 (m, 1H), 4.51 - 4.41 (m, 1H), 4.32 - 4.13 (m, 2H), 4.07 - 4.03 (d, J = 11.3 Hz, 1H), 3.86 - 3.77 (d, J = 11.6 Hz, 1H), 3.76 - 3.69 (d, J = 11.2 Hz, 1H), 3.57 - 3.48 (m, 1H), 3.48 - 3.41 (m, 2H), 3.39 - 3.35 (d, J = 3.1 Hz, 4H), 3.29 - 3.23 (m, 1H), 3.22 - 3.10 (m, 6H), 2.96 - 2.92 (s, 3H), 2.91 - 2.86 (t, J = 4.8 Hz, 4H), 2.85 - 2.76 (m, 3H), 2.76 - 2.69 (m, 2H), 2.67 - 2.57 (d, J = 14.4 Hz, 1H), 2.33 - 2.19 (m, 2H), 2.03 - 1.94 (m, 1H), 1.92 - 1.79 (m, 1H), 1.73 - 1.59 (m, 1H), 1.50-1.41 (d, J = 6.0 Hz, 3H), 1.35 - 1.30 (m, 1H), 1.00 - 0.92 (m, 9H), 0.54 - 0.41 (s, 3H). BIOLOGICAL EXAMPLE Compound A555 (page 158 of Table.1) from WO2021091956 was cited as reference compound for this invention. Example 20 Single dose pharmacokinetics (PK) study in female BALB/c mice The purpose of this study was to determine the pharmacokinetics of selected compounds following single intravenous bolus or oral gavage administration in female BALB/c mice. Briefly, two groups of female BALB/c mice (available from Shanghai Lingchang Biotechnology Co., Ltd) (N=3/group) were treated with a single dose of compound intravenously at 3 mg/kg (IV) or orally at 30 mg/kg (PO). Blood samples were collected at 5 min (only for IV), 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h post-dose. Blood samples were placed on ice until centrifugation to obtain plasma samples. The concentration of compound in plasma samples was determined using LC-MS/MS method. The pharmacokinetic parameters were calculated by non- compartmental analysis. Table 1. Results of SDPK From Table 1, it can be seen that Example 6 has excellent pharmacokinetic properties in mouse model, including almost 7 folds of C _max , 15 folds of AUCo-iast and improved bio availability and significant lower clearance compared with compound A555.

Example 21

Human hepatocyte stability Assay

The hepatocyte stability assay measures the rate of disappearance of a compound from incubations with cryopreserved suspension hepatocytes from human. Positive controls, including Midazolam, Raloxifene and Dextromethorphan, are included in every experiment. Incubations consist of 1 μM tested compound and suspension of human hepatocytes (1 x 10 ⁶ cells/mL) in supplemented Williams’ E Medium with 10% FBS and 0.5% Penicillinstreptomycin. The hepatocyte suspension was incubated with intermittent shaking 900 rpm at 37°C, in a 5% CO2 incubator. The reaction was stopped by adding methanol containing internal standard (2 μM Tolbutamide) at 2, 10, 20, 40, 60 and 120 minutes after compound addition, depletion of the parent compound was monitored by LC-MS/MS analysis.

Table 2. Human hepatocytes stability of Examples and Compounds of present invention

Above result clearly shows that reference compounds (A555) had much higher clearance while Example 6 maintained the low clearance in human hepatocytes stability assay. Achieving low clearance is advantageous to improve in vivo performance of the compound, such as dose reduction, exposure enhancement, and half-life prolongation.

Example 22

Cell viability assay

The purpose of this cellular assay was to determine the effects of test compounds on the proliferation of human cancer cell lines NCI-H358 (ATCC-CRL5807) cells, AGS (ATCC-CRL- 1739) cells, SW620 (ATCC-CCL-227) over a 3-day treatment period by quantifying the amount of NADPH present at endpoint using Cell Counting Kit-8. Cells were seeded at 5,000 cells/well (NCI-H358), 2,000 cells/well (AGS) 2,000 cells/well (SW620) in 96-well assay plates (Corning-3699) and incubated overnight. On the day of the assay, diluted compounds were then added in a final concentration of 0.5% DMSO. After 72 hrs incubation, a tenth of the volume of cell counting kit 8(Dnjindo-CK04) was added into each well. Read the signal (OD450 minus OD650) using EnVision after 2 hrs incubation. IC50 was determined by fitting a 4-parameter sigmoidal concentration response model.

Table 3. Activity of Examples and Compounds of present invention in KRAS Cell viability assay

Example 23 KRAS-BRAF with CYPA (500 nM) interaction assay

In this example, TR-FRET was also used to measure the compound or compound-CYPA dependent disruption of the KRAS G12C-BRAF complex. This protocol was also used to measure disruption of KRAS G12D or KRAS G12V binding to BRAF by a compound of the invention, respectively. In assay buffer containing 25mM HEPES PH=7.4 (4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid, Thermo, 15630080), 0.002% Tween20, 0.1% BSA, 100mM NaCl, 5mM MgCF, 10 pM GMPPNP (Guanosine 5'-[P,y-imido]triphosphate trisodium salt hydrate, Sigma, G0635), tagless CYPA, GMPPNP loaded 6His-KRAS proteins, and GST- BRAF ^RBD were mixed in a well of a 384-well assay plate at final concentrations of 50 nM, 6.25 nM and InM, respectively. Compound was present in plate wells as a 16-point 3-fold dilution series starting at a final concentration of 10 pM and incubated for 3 hours. A mixture of MAb Anti-6His-XL665 (Cisbio, 61HISXLB) and Mab anti-GST-TB cryptate (Cisbio, 61GSTTLB)was then added at a final concentration of 6.67 nM and 0.21 nM, respectively, and the plate was incubated for an additional 1.5 hours. TR-FRET signal was read on a PHERstar FSX microplate reader (Ex320 nm, Em 665/615 nm). Compounds that facilitate disruption of the KRAS-BRAF complex were identified as those eliciting a decrease in the TR-FRET ratio relative to DMSO control wells.

Table 4. Activity of Examples and Compounds of present invention in KRAS-BRAF with CYPA (500 nM) interaction assay

Example 24 pERK inhibition assay

This assay is to measure the ability of test compounds in inhibiting the phosphorylation of ERK, the downstream signaling of KRAS G12C in NCI-H358 cells, KRAS G12D in AGS cells, and KRAS G12V in SW620. NCI-H358 (ATCC-CRL5807) cells, AGS (ATCC-CRL-1739) cells, SW620 (ATCC-CCL-227) cells were all grown and maintained using RPMI-1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin/streptomycin. On the day prior to compound addition, cells were plated in tissue culture -treated 96 well plates (Corning-3699) at a density of 30,000 cell/well, 20,000 cell/well, 30,000 cell/well for NCI-H358, AGS and SW620 respectively, and allowed for attachment overnight. Diluted compounds were then added in a final concentration of 0.5% DMSO. After 4 hours of incubation, the medium was removed, 100 pL of 4% formaldehyde was added, and the assay plates were incubated at room temperature for 20 minutes. The plates were then washed once with phosphate buffered saline (PBS), and permeabilized with 100 pL of chilled methanol for 10 minutes. Non-specific antibody binding to the plates was blocked using 50 pL IX BSA blocking buffer (Thermo -37520, 10-fold dilution by Phosphate-Buffered Saline Tween (PBST) for at least 1 hour at room temperature.

The amount of phosphor- ERK was determined using an antibody specific for phosphorylated form of ERK. Primary antibody (pERK, CST-4370, Cell Signaling Technology) was diluted 1:300 in blocking buffer, with 50 pL aliquoted to each well, and incubated overnight at 4 °C. Cells was washed five times for 5 minutes with PBST. Secondary antibody (HRP-linked anti-rabbit IgG, CST-7074, Cell Signaling Technology) was diluted 1:1000 in blocking buffer, and 50 pL was added to each well and incubated 1-2 hrs at room temperature. Cells was washed 5 times for 5 minutes with PBST, lOOpL TMB ELISA substrate (abcam-abl71523) were added and gently shake for 20 minutes. 50pL stop solution (abcam-abl71529) were added, and then read the signal (OD450) by EnVision.

IC50 was determined by fitting a 4-parameter sigmoidal concentration response model. Table 5. Activity of Examples and Compounds of present invention in KRAS pERK inhibition assay

Example 25 Stable KRAS mutant cell lines and cell viability assay.

The aim of the study was to determine the potency and efficacy of compounds for cell proliferation using CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (Promega Corp., Madison, WI) . We cloned 14 KRAS ^G12C variant sequences with secondary mutations (V8A, V9Y, S17E, T58I, A59T, S65W, R68S, D69P, M72I, D92R, H95N, Y96D, Q99F, Q99W, Y96H, and F156L) into the Miapaca-2. Totally 14 stable Miapaca-2 mutant cell lines were established through lentivirus infection. For the cell viability assay, cells were dosed with compounds in a 9- point dose-response using a 4 fold dilution series at a top dose of lOpM. KRAS mutant cells were maintained in DMEM+10%FBS+2.5%HI Horse serum+l%PS+ Ipg/mL Puromycin and seeded into 96- well plates at 800-1,500 cells per well 24 h before compound addition and then incubated with compound for 3 d before assaying viability (CellTiter-Glo, Promega). Assays were performed in biological duplicates. Nonlinear regression curves were fitted using Xfit.

IC50 (absolute IC50) is the dose at which the estimated viability is 50% relative to untreated wells. Inhibition rate of the compound is calculated according to the formula below: %inhibition=100-100x(Luminescence value-HPE) / (ZPE-HPE).

HPE: Luminescence value from the wells with only medium

ZPE: Luminescence value from the wells with DMSO

Table 8. cell viability in mutant cells with KRAS G12C and other mutations