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WO2025104149A1 - Macrocyclic compounds as kras mutant inhibitors for the treatment of cancer - Google Patents

Macrocyclic compounds as kras mutant inhibitors for the treatment of cancer Download PDF

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Publication number
WO2025104149A1
WO2025104149A1 PCT/EP2024/082296 EP2024082296W WO2025104149A1 WO 2025104149 A1 WO2025104149 A1 WO 2025104149A1 EP 2024082296 W EP2024082296 W EP 2024082296W WO 2025104149 A1 WO2025104149 A1 WO 2025104149A1
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Prior art keywords
compound
fluoro
methyl
methoxyethyl
cancer
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French (fr)
Inventor
Jianguo Chen
Weixing Zhang
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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F Hoffmann La Roche AG
Hoffmann La Roche Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/18—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • 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.
  • 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.
  • G12C, G12D and G12V represent more than half of all K-RAS-driven cancers across colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), lung adenocarcinoma (LU AD).
  • CRC colorectal cancer
  • PDAC pancreatic ductal adenocarcinoma
  • LU AD lung adenocarcinoma
  • 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.
  • MAPK mitogen-activated protein kinase
  • PI3K phosphatidylinositol 3 -kinase
  • AKT protein kinase B
  • KRAS protein switches between an inactive to an active form via binding to GTP and GDP, respectively.
  • GEFs guanine nucleotide exchange factors
  • SOS1 Son Of Sevenless Homolog 1
  • GAPs GTPase- activating proteins
  • 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.
  • RAF RBD RAF RAS binding domains
  • 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.
  • MEK Mitogen- activated protein kinase
  • ERK extracellular signal-regulated kinase
  • BRAF is most frequently mutated and remains the most potent activator of MEK.
  • 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).
  • the above compounds show good KRAS inhibition for G12C.
  • the compounds of current invention had much improved pharmacokinetic properties and surprisingly better performance in anti-tumor activity in difficult KRAS mutated xenograft tumor model.
  • the compounds of this invention showed superior cancer cell inhibition and human hepatocyte stability.
  • the compounds of this invention also show good or improved cytotoxicity and solubility profiles.
  • pharmaceutically acceptable salts denotes salts which are not biologically or otherwise undesirable.
  • Pharmaceutically acceptable salts include both acid and base addition salts.
  • 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,
  • pharmaceutically acceptable base addition salt denotes those pharmaceutically acceptable salts formed with an organic or inorganic base.
  • 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, N-ethylpiperidine, and polyamine resins.
  • substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, trieth
  • 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.
  • 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.
  • 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.
  • pharmaceutically acceptable 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.
  • the present invention relates to (i) a compound which is 1-[4-(dimethylamino)-4-methyl- pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3- pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.1 2,6 .1 10,14 .0 23,27 ]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
  • Another embodiment of present invention is (ii) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-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]carbamoyl]-2-methyl-propyl]-
  • Another embodiment of present invention is (iii) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-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-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]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
  • Another embodiment of present invention is (iv) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2- [(1S)-1-methoxyethyl]-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]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
  • Another embodiment of present invention is (ix) the use of a compound according to any one of (i) to (iv) for inhibiting the propagating oncogenic MAPK and PI3K signaling.
  • Another embodiment of present invention is (x) the use of a compound according to any one of (i) to (iv) 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 (xi) the use of a compound according to any one of (i) to (iv) 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 (xii) a compound or pharmaceutically acceptable salt according to any one of (i) to (iv) 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 (xiii) the use of a compound according to any one of (i) to (iv) 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 (xiv) 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 (iv).
  • 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.
  • 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.
  • 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.
  • a compound of formula (I) is formulated in an acetate buffer, at pH 5.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • An embodiment includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof.
  • 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.
  • composition A 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
  • 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.
  • 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.
  • 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% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).
  • Mass spectra 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.
  • Step 2 Preparation of 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine (Intermediate A) To a solution of 3-bromo-2-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (compound A2, 2.5 g, 7.3 mmol) in ACN (40 mL) was added N- iodosuccinimide (4.1 g, 18.27 mmol). The mixture was stirred at 90 °C for 40 hrs under N 2 protection.
  • 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.
  • Step 3 Preparation of 4-bromo-2-[[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5- dihydropyrazin-2-yl]methyl]thiazole (compound B5)
  • compound B5 To a mixture of (R)-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.
  • Step 4 Preparation of methyl (2S)-2-amino-3-(4-bromothiazol-2-yl)propanoate (compound B6)
  • Step 5 Preparation of methyl (2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoate (compound B7)
  • compound B6 methyl (2S)-2-amino-3-(4-bromothiazol-2-yl)propanoate (compound B6)
  • triethylamine 2.9 g, 29.23 mmol
  • (Boc)2O 3.8 g, 17.54 mmol
  • Step 6 Preparation of (2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)- propanoic acid (compound B8)
  • Step 7 Preparation of methyl (3S)-1-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (Intermediate B) To a solution of (2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)propanoic 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), EDCI (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.
  • 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.
  • Step 1 Preparation of (2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-tert- butoxycarbonylmorpholin-2-yl]propanoic acid (compound C2)
  • Step 2 Preparation of tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-[(3S)-3- methoxycarbonylhexahydropyridazin-1-yl]-3-oxo-propyl]morpholine-4-carboxylate (compound C3)
  • (2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-tert- butoxycarbonylmorpholin-2-yl]propanoic acid compound C2, 20.0 g, 48.97 mmol
  • methyl (3S)-hexahydropyridazine-3-carboxylate;dihydrochloride compound B9, 21.3 g, 97.93 mmol,) in DMF (300 mL) was added DIEA (42.6 mL, 244.83 mmol) and T4P (70.6 g, 97.93 mmol) at
  • Step 3 Preparation of methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)- morpholin-2-yl]propanoyl]hexahydropyridazine-3-carboxylate (intermediate C) To a solution of tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-[(3S)-3- methoxycarbonylhexahydropyridazin-1-yl]-3-oxo-propyl]morpholine-4-carboxylate (compound C3, 5.0 g, 9.35 mmol) in DCM (20 mL) was added TFA (19.8 mL) at 0 °C.
  • Step 2 Preparation of [3-(5-bromo-6-fluoro-1H-indol-3-yl)-2,2-dimethyl-propoxy]- tert-butyl-diphenyl-silane (compound D4)
  • Step 3 Preparation of [3-(5-bromo-6-fluoro-2-iodo-1H-indol-3-yl)-2,2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (intermediate D) To a mixture of [3-(5-bromo-6-fluoro-1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl- diphenyl-silane (compound D4, 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.
  • Step 2 1-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3- pyridyl]-4-(2,2,2-trifluoroethyl)piperazine (compound E3).
  • Step 3 Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl- diphenyl-silane (compound E4).
  • Step 4 Preparation of [3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound E5).
  • Step 5 Preparation of 3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-1-ol (compound E6).
  • Step 6 Preparation of 3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-1-ol (compound E7).
  • Step 7 Preparation of methyl (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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-carboxylate (compound E8).
  • Step 8 Preparation of (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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).
  • Step 9 Preparation of tert-butyl N-[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-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]carbamate (compound E10).
  • Step 10 Preparation of (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-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 E).
  • Step 2 Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-1H- indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F2) To a mixture of 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)pyridine (compound F1, 18.0 g, 30.78 mmol) and [3-(5-bromo-6-fluoro-2-iodo-1H-indol-3- yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate D, 20.5 g, 30.78 mmol) and [3-(5-bromo-6-fluoro-2-
  • Step 3 Preparation of [3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]- 3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F3)
  • compound F2 7 g, 10.39 mmol,
  • reaction mixture was added with a solution of iodoethane (1.1 mL, 13.51 mmol) in toluene (2 mL). After being stirred at 25 °C for 6 hrs, the reaction mixture was filtered and the filtrate was concentrated under vacuum to give a residue.
  • Step 4 Preparation of methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[3- [3-[tert-butyl(diphenyl)silyl]oxy-2,2-dimethyl-propyl]-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1- methoxyethyl]-3-pyridyl]indol-5-yl]morpholin-2-yl]propanoyl]hexahydropyridazine-3- carboxylate (compound F4) To the mixture of [3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-3- pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (
  • Step 6 Preparation of benzyl N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.1 2,6 .1 10,14 .0 23,27 ]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamate (compound F6) To a solution of (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[1-ethyl-6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]
  • Step 7 Preparation of (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.1 2,6 .1 10,14 .0 23,27 ]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F) To a solution of benzyl N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-diox
  • Step 2 Preparation of 4-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-pyridyl]morpholine (compound G2)
  • 4-[5-bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]morpholine compound G1, 21 g, 63.3 mmol
  • bis(pinacolato)diboron (24.0 g, 94.63 mmol)
  • KOAc (13.6 g, 138.79 mmol) in toluene (500 mL) was added Pd(dppf)Cl2 (4.4 g, 6.31 mmol).
  • Step 3 Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound G3)
  • 4-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)-3-pyridyl]morpholine compound G2, 40.6 g, 46.65 mmol
  • [3-(5-bromo-6-fluoro-2-iodo- 1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate C5, 31 g, 46.65 mmol) in
  • reaction mixture was degassed and purged with nitrogen, 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 Na 2 SO 4 , filtered and the filtrate was concentrated in vacuo to give a residue.
  • Step 4 Preparation of [3-[5-bromo-1-ethyl-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound G4)
  • compound G4 To a solution of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3- pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-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,
  • Step 5 Preparation of 3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]- 5-morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propan-1-ol (compound G5) and 3-[5- bromo-1-ethyl-6-fluoro-(2P)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]indol-3- yl]-2,2-dimethyl-propan-1-ol (compound G6) To a solution of [3-[5-bromo-1-ethyl-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino- 3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-buty
  • Step 7 Preparation of methyl (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1- ethyl-6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3- carboxylate (compound G8) To a mixture of 3-[1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3- pyridyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl
  • Step 10 Preparation of (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) To a solution of tert-butyl N-[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-morpholino-3-pyridyl]-17,17
  • Step 1 Preparation of N,N,2-trimethylbut-3-yn-2-amine (compound I2) A mixture of 3-chloro-3-methyl-1-butyne (20.0 g, 195.01 mmol) and dimethylamine (22.0 g, 195.01 mmol) was stirred at 25 °C for 12 hrs. After the reaction completed, the suspension was filtered. The collected solid was washed with ice-water (50 mL, three times) and dissolved in cold HCl (3 N, 10 mL).
  • Step 2 Preparation of 4-(dimethylamino)-4-methyl-pent-2-ynoic acid (intermediate I) To a solution of N,N,2-trimethylbut-3-yn-2-amine (compound I2, 3.2 g, 28.78 mmol) in THF (120 mL) was added dropwise n-BuLi (12.7 mL, 31.66 mmol, 2M in hexane) at -70 °C. After being stirred at -70°C for 5 min, the reaction mixture was warmed to 0 °C and stirred for another 10 min. After being cooled to -70 °C, the reaction mixture was bubbled with carbon dioxide for 30 min.
  • Step 4 Preparation of N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.1 2,6 .1 10,14 .0 23,27 ]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (compound 1d) To a solution of tert-butyl 4-[[(1S)-1-[[(6S,8S,14S)-22-
  • Step 5 Preparation of 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1- [[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18- dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.1 2,6 .1 10,14 .0 23,27 ]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (Example 1) To a mixture of N-[
  • Example 2 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-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]carbamoyl]-2-methyl-propyl]-N-methyl-piperidine-4-carboxamide
  • Example 3 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-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-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]-4-fluoro-N-methyl- piperidine-4-carboxamide
  • the title compound was prepared in analogy to the preparation of Example 1 by using (7S,13S)-7-amino-21-
  • Example 4 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro- (20M)-20-[2-[(1S)-1-methoxyethyl]-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]-4-fluoro-N-methyl-piperidine-4-carboxamide
  • PK pharmacokinetics
  • Example 1 has excellent pharmacokinetic properties in mouse model. Especially Example 1 has almost 3.7 folds of C max , 6 folds of AUC 0-last and significant lower clearance compared with compound RMC-6291, which makes Example 1 more suitable for treating cancers with RAS mutation as an orally therapeutic active ingredient in clinic.
  • Example 6 In vivo xenograft studies The purpose of the study was to assess the anti-tumor activity in difficult KRAS mutated xenograft tumor model, NCI-H2122 which is a non-small cell lung cancer (NSCLC), by examining the tumor suppression post-administration of Example 1 and RMC-6291. Studies were conducted at Wuxi AppTec (Nantong, China). All CDX mouse studies and procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the relevant Institutional Animal Care and Use Committee (IACUC). Mice were maintained under pathogen-free conditions, and food and water was provided ad libitum. Female BALB/c nude mice at 6-8 weeks old from Vital River Co., LTD.
  • IACUC Institutional Animal Care and Use Committee
  • each mouse was inoculated at the right flank with tumor cells in 200 ⁇ L of PBS and 50% matrigel matrix in the right hind flank with 5 ⁇ 10 6 cells.
  • Mouse health was monitored daily, and caliper measurements began when tumors were palpable.
  • Tumor volume measurements were determined utilizing the formula 0.5 ⁇ L ⁇ W 2 in which L refers to length and W refers to width of each tumor. When tumors reached an average tumor volume of approximately 350 mm 3 , mice were randomized into treatment groups.
  • animals were orally administered daily, tumor volumes and body weights were measured 3 times per week. Study day on efficacy plots indicates the day after which treatment was initiated.
  • TGI (tumor growth inhibition)% (Final treated tumor volume- Initial treated tumor volume)/(Vehicle final treated tumor volume-Vehicle initial treated tumor volume) ⁇ 100%
  • Tumor regression (-100%) ⁇ (1 – (Final treated tumor volume)/(Initial treated tumor volume))
  • Tumor growth 100% ⁇ (Final treated tumor volume – Initial treated tumor volume)/(Initial treated tumor volume)
  • TGI mean tumor growth inhibition
  • Figure 2 Table 2.
  • Example G12C IC50 (nM)
  • Example 1 0.42
  • Example 2 0.44
  • Example 3 0.62
  • Example 4 0.41
  • Example 8 KRAS-BRAF with CYPA (500 nM) interaction assay
  • TR-FRET was also used to measure the compound or compound-CYPA dependent disruption of the KRAS G12C-BRAF complex.
  • Compound was present in plate wells as a 16-point 3-fold dilution series starting at a final concentration of 10 ⁇ M 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).
  • NCI-H358 ATCC- CRL5807 cells was grown and maintained using RPMI-1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin/streptomycin.
  • RPMI-1640 medium Thermo Fisher Scientific
  • cells were plated in tissue culture-treated 96 well plates (Corning-3699) at a density of 30,000 cell/well for NCI-H358 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 ⁇ L 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 ⁇ L of chilled methanol for 10 minutes. Non-specific antibody binding to the plates was blocked using 50 ⁇ L 1X 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 ⁇ L aliquoted to each well, and incubated overnight at 4 °C.
  • PBS phosphate buffered saline

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Abstract

The present invention relates to four specific macrocyclic compounds for use as inhibitors of KRAS mutant for the treatment of cancer. Example (1): 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide.

Description

MACROCYCLIC COMPOUNDS AS KRAS MUTANT INHIBITORS FOR THE TREATMENT OF CANCER
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 and 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 (RAFRBD), 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-BRAFRBD 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. SUMMARY OF THE INVENTION The present invention relates to novel compounds: 1. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1- [[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl- 9,15-dioxo-5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa- 1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4- carboxamide, or a pharmaceutically acceptable salt thereof. 2. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. 3. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-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- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. 4. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1- [[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-17,17-dimethyl- 8,14-dioxo-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl- piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. The above compounds show good KRAS inhibition for G12C. In one embodiment, compared with reference compound, the compounds of current invention had much improved pharmacokinetic properties and surprisingly better performance in anti-tumor activity in difficult KRAS mutated xenograft tumor model. In another embodiment, the compounds of this invention showed superior cancer cell inhibition and human hepatocyte stability. In addition, the compounds of this invention also show good or improved cytotoxicity and solubility profiles. BRIEF DESCRIPTION OF THE FIGURE Figure 1. X-ray crystallographic analysis of compound G5. Figure 2. In vivo H2122 xenograft studies of Example 1 and RMC-6291. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS 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, ethanesulfonic 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, N-ethylpiperidine, 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 which is 1-[4-(dimethylamino)-4-methyl- pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3- pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (ii) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-dimethyl-8,14- dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (iii) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-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-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen- 7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (iv) a compound which is 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro-(20M)-20-[2- [(1S)-1-methoxyethyl]-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. Another embodiment of present invention is (v) a compound or pharmaceutically acceptable salt according to any one of (i) to (iv) for use as therapeutically active substance. Another embodiment of present invention is (vi) a pharmaceutical composition comprising a compound in accordance with any one of (i) to (iv) and a pharmaceutically acceptable excipient. Another embodiment of present invention is (vii) the use of a compound according to any one of (i) to (iv) for treating a KRAS G12C protein-related disease. Another embodiment of present invention is (viii) the use of a compound according to any one of (i) to (iv) for inhibiting RAS interaction with downstream effectors, wherein the downstream effectors are RAF and PI3K. Another embodiment of present invention is (ix) the use of a compound according to any one of (i) to (iv) for inhibiting the propagating oncogenic MAPK and PI3K signaling. Another embodiment of present invention is (x) the use of a compound according to any one of (i) to (iv) 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 (xi) the use of a compound according to any one of (i) to (iv) 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 (xii) a compound or pharmaceutically acceptable salt according to any one of (i) to (iv) 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 (xiii) the use of a compound according to any one of (i) to (iv) 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 (xiv) 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 (iv). 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. 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)2O Di-tert-butyldicarbonate (R)-binap (R)-(+)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl CDCl3: deuterated chloroform CMPI 2-Chloro-1-methylpyridinium iodide Cs2CO3 Cesium carbonate DMF: dimethyl formamide DMSO: dimethyl sulfoxide EDCI: N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc or EA: ethyl acetate HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate) hr(s): hour(s) HPLC: high performance liquid chromatography HOBt: N-hydroxybenzotriazole [Ir(OMe)(COD)]2 (1,5-Cyclooctadiene)(methoxy)iridium(I) dimer MS: (ESI): mass spectroscopy (electron spray ionization) min(s) minute(s) NMM: N-Methylmorpholine NMR: nuclear magnetic resonance NPLC: Normal phase liquid chromatography Pd(dppf)Cl2: [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(dtbpf)Cl2: [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) Pd(OH)2: Palladium hydroxide Pd-PESSI-IPent Cl: [1,3-bis[2,6-bis(1-ethylpropyl)phenyl]-4,5-dichloro-imidazol-2- yl]-dichloro-(3-chloro-1-pyridyl)palladium prep-HPLC: preparative high performance liquid chromatography RT or rt: room temperature sat.: saturated SFC: supercritical fluid chromatography TBAF: Tetrabutylammonium fluoride TEA: triethylamine TFA: trifluoroacetic acid THF: tetrahydrofuran T4P: 1,3,5,2,4,6-Trioxatriphosphorinane, 2,4,6-tributyl-, 2,4,6-trioxide 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 Å, particle size: 40-60 µm; 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 XBridgeTM Prep-C18 (5 µm, OBDTM 30 × 100 mm) column, SunFireTM Prep-C18 (5 µm, OBDTM 30 × 100 mm) column, Phenomenex Synergi-C18 (10 µm, 25 × 150 mm) or Phenomenex Gemini-C18 (10 µm, 25 × 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% HCl 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 µm, 30 × 250 mm), AS (10 µm, 30 × 250 mm) or AD (10 µm, 30 × 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 IPA) or CO2 and MeOH (0.1% NH3∙H2O in MeOH), back pressure 100bar, detection UV@ 254 or 220 nm. LC/MS spectra of compounds were obtained using a LC/MS (WatersTM 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% NH3·H2O 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 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine
Figure imgf000015_0001
The title intermediate A was prepared according to the following scheme:
Figure imgf000015_0002
A1 A2 Intermediate A Step 1: Preparation of 3-bromo-2-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (compound A2) To a solution of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (compound A1, 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-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (compound A2, 2.4 g) as yellow oil.1H NMR (400 MHz, CDCl3) δ 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-[(1S)-1-methoxyethyl]pyridine (Intermediate A) To a solution of 3-bromo-2-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (compound A2, 2.5 g, 7.3 mmol) in ACN (40 mL) was added N- iodosuccinimide (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 Na2SO3 (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 (intermediate A, 660 mg) as yellow oil. MS calc’d 342 (MH+), measured 341.8 (MH+). Intermediate B Methyl (3S)-1-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)- propanoyl]hexahydropyridazine-3-carboxylate
Figure imgf000016_0001
Figure imgf000016_0002
The intermediate B was prepared according to the following scheme:
B8 Intermediate B Step 1: Preparation of (4-bromothiazol-2-yl)methanol (compound B2) To a solution of 4-bromothiazole-2-carboxaldehyde (compound B1 ̧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,5R)-5-isopropyl-3,6-dimethoxy-2,5- dihydropyrazin-2-yl]methyl]thiazole (compound B5) To a mixture of (R)-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 hour. The reaction was quenched with saturated solution of NH4Cl (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-[[(2S,5R)-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-[[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-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 NaHCO3 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 (Boc)2O (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 HCl 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 (2S)-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)-1-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (Intermediate B) To a solution of (2S)-3-(4-bromothiazol-2-yl)-2-(tert-butoxycarbonylamino)propanoic 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), EDCI (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)-1-[(2S)-3-(4-bromothiazol-2-yl)-2- (tert-butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (intermediate B, 2.4 g). MS calc’d 477(MH+), measured 476.9 (MH+). Intermediate C Methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-morpholin-2- yl]propanoyl]hexahydropyridazine-3-carboxylate
The compound was prepared according to the following scheme:
Figure imgf000020_0001
intermediate C Step 1: Preparation of (2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-tert- butoxycarbonylmorpholin-2-yl]propanoic acid (compound C2) To a solution of tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-methoxy-3-oxo- propyl]morpholine-4-carboxylate (CAS: 2641824-60-2, 20.0 g, 47.34 mmol) in THF (250 mL)/water (50 mL) was added LiOH·H2O (4.0 g, 94.68 mmol) at 0°C. After being stirred at 25 °C for 1.5 hrs, the reaction mixture was acidified by citric acid aq. until pH=6. The mixture was extracted with EtOAc (200 mL, three times). The combined organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under vacuum to afford (2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-tert- butoxycarbonylmorpholin-2-yl]propanoic acid (compound C2, 20.0 g) as a yellow solid. MS calc’d 409.2 (MH+), measured 409.2 (MH+). Step 2: Preparation of tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-[(3S)-3- methoxycarbonylhexahydropyridazin-1-yl]-3-oxo-propyl]morpholine-4-carboxylate (compound C3) To a mixture of (2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-tert- butoxycarbonylmorpholin-2-yl]propanoic acid (compound C2, 20.0 g, 48.97 mmol) and methyl (3S)-hexahydropyridazine-3-carboxylate;dihydrochloride (compound B9, 21.3 g, 97.93 mmol,) in DMF (300 mL) was added DIEA (42.6 mL, 244.83 mmol) and T4P (70.6 g, 97.93 mmol) at 0 °C. After being stirred at 25 °C for 0.5 h, the reaction mixture was poured into water (300 mL) and extracted with EtOAc (150 mL, three times). The combined organic layer was washed with brine (150 mL, three times), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to get a residue. The residue was purified by silica gel chromatography to tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-[(3S)-3-methoxycarbonylhexahydropyridazin-1-yl]- 3-oxo-propyl]morpholine-4-carboxylate (compound C3, 22.7 g) as yellow oil. MS calc’d 535.3 (MH+), measured 535.3 (MH+). Step 3: Preparation of methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)- morpholin-2-yl]propanoyl]hexahydropyridazine-3-carboxylate (intermediate C) To a solution of tert-butyl (2S)-2-[(2S)-2-(benzyloxycarbonylamino)-3-[(3S)-3- methoxycarbonylhexahydropyridazin-1-yl]-3-oxo-propyl]morpholine-4-carboxylate (compound C3, 5.0 g, 9.35 mmol) in DCM (20 mL) was added TFA (19.8 mL) at 0 °C. The mixture was stirred at 25°C for 1 hour. After the reaction was completed, the reaction mixture was concentrated under vacuum to give a residue. Sat. NaHCO3 aq. (20 mL) was added and the mixture was extracted with EtOAc (30 mL, three times). The combined organic layer was washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-morpholin-2- yl]propanoyl]hexahydropyridazine-3-carboxylate (intermediate C, 4.0 g) as a brown solid. MS calc’d 435.2 (MH+), measured 435.2 (MH+). Intermediate D [3-(5-bromo-6-fluoro-2-iodo-1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl- silane
The compound was prepared according to the following scheme:
Figure imgf000022_0001
Intermediate D Step 1: Preparation of 1-(5-bromo-6-fluoro-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl) oxy)-2,2-dimethylpropan-1-one (compound D3) To a mixture of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoyl chloride (compound D1, 35.0 g, 116.8 mmol) in DCM (400 mL) at 0 °C was added a solution of SnCl4 (97.2 mL, 121.5 mmol) slowly. After the mixture was stirred at - 40 °C for 0.5 hour, 5-bromo-6- fluoro-1H-indole (compound D2, 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.NaHCO3 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 Na2SO4, filtered and concentrated in vacuo. The residue was triturated in a mixed solvent (100 mL, Petroleum ether: Ethyl acetate = 8:1) and filtered. The collected solid was dried in vacuo to afford 1-(5-bromo-6-fluoro-1H-indol-3-yl)-3-((tertbutyldiphenylsilyl)oxy)-2,2- dimethylpropan-1-one (compound D3, 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-1H-indol-3-yl)-2,2-dimethyl-propoxy]- tert-butyl-diphenyl-silane (compound D4) To a mixture of 1-(5-bromo-6-fluoro-1H-indol-3-yl)-3-((tertbutyldiphenylsilyl)oxy)-2,2- dimethylpropan-1-one (compound D3, 50.0 g, 90.49 mmol) in THF (600 mL) was added LiBH4 (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 Na2SO4, 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-1H-indol-3-yl)-2,2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (compound D4, 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-1H-indol-3-yl)-2,2-dimethyl- propoxy]-tert-butyl-diphenyl-silane (intermediate D) To a mixture of [3-(5-bromo-6-fluoro-1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl- diphenyl-silane (compound D4, 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. Na2SO3 aq. (400 mL) and EtOAc (400 mL) and the reaction mixture was filtered. The organic layer was washed with brine (100 mL), dried over Na2SO4, 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-1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate D, 43.0 g) as a yellow solid. MS calc’d 664.0 (MH+), measured 664.1 (MH+). Intermediate E (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- 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
The compound was prepared according to the following scheme:
E10 Intermediate E Step 1: Preparation of 1-[5-bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]-4-(2,2,2- trifluoroethyl)piperazine (compound E2). To a mixture of 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine (intermediate A, 2.03 g, 5.95 mmol) and 1-(2,2,2-trifluoroethyl)piperazine (compound E1, 1.0 g, 5.95 mmol) in toluene (15 mL) were added Cs2CO3 (4.85 g, 14.88 mmol), (R)-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 1-[5- bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]-4-(2,2,2-trifluoroethyl)piperazine (compound E2, 2.0 g) as yellow oil. MS calc’d 382.2 (MH+), measured 382.1 (MH+). Step 2: 1-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3- pyridyl]-4-(2,2,2-trifluoroethyl)piperazine (compound E3). To a solution of 1-[5-bromo-6-[(1S)-1-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 KOAc (2.1 g, 20.93 mmol) in toluene (50 mL) was added Pd(dppf)Cl2 (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 1-[6-[(1S)-1-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-C6H10+H+). Step 3: Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl- diphenyl-silane (compound E4). To a solution of 1-[6-[(1S)-1-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, 4.41 mmol), [3-(5-bromo- 6-fluoro-2-iodo-1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate D, 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)Cl2 (231 mg, 0.37 mmol). The mixture was degassed and purged with nitrogen, then 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-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]- 1H-indol-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-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound E5). To a solution of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1H-indol-3-yl]-2,2-dimethyl-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-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-3- pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-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-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-1-ol (compound E6). To a solution of [3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-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-[(1S)-1-methoxyethyl]-5- [4-(2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-1-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-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2- dimethyl-propan-1-ol (compound E7). To a solution of 3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2-dimethyl-propan- 1-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)Cl2 (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-(2M)-2-[2-[(1S)-1- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol- 3-yl]-2,2-dimethyl-propan-1-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)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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-carboxylate (compound E8). To a mixture of 3-[5-bromo-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2- trifluoroethyl)piperazin-1-yl]-3-pyridyl]-1-(2,2,2-trifluoroethyl)indol-3-yl]-2,2-dimethyl-propan- 1-ol (compound E7, 0.3 g, 0.41 mmol) and methyl (3S)-1-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (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)Cl2 (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 (3S)-1-[(2S)-2-(tert- butoxycarbonylamino)-3-[4-[6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1- 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-carboxylate (compound E8, 200.0 mg) as yellow gum. MS calc’d 1001.4 (MH+), measured 1001.4 (MH+). Step 8: Preparation of (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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). To a mixture of methyl (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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-carboxylate (compound E8, 200.0 mg, 0.2 mmol) in DCE (5 mL) was added Me3SnOH (200.0 mg, 1.11 mmol). The mixture was stirred at 60 °C for 12 hrs, and then 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 (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3- [4-[6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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 of tert-butyl N-[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1- methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-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]carbamate (compound E10). To a mixture of (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[6-fluoro-3-(3-hydroxy- 2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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, 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 Na2SO4, filtered and concentrated under vacuum to give a residue which was purified by column chromatography (EtOAc in PE : 50% - 70%) to afford tert-butyl N-[(7S,13S)- 24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-5-[4-(2,2,2-trifluoroethyl)piperazin-1-yl]-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]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-[(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-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 E). To a solution of tert-butyl N-[(7S,13S)-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-5-[4- (2,2,2-trifluoroethyl)piperazin-1-yl]-3-pyridyl]-17,17-dimethyl-8,14-dioxo-21-(2,2,2- 2,5 9,13 22,26 trifluoroethyl)-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-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. NaHCO3 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-(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-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 E, 98.0 mg) as a yellow solid. MS calc’d 869.4 (MH+), measured 869.2 (MH+). Intermediate F (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]- 18,18-dimethyl-5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa- 1(26),20,23(27),24-tetraene-9,15-dione
Figure imgf000031_0001
The compound was prepared according to the following scheme:
F6 intermediate F Step 1: Preparation of 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)pyridine (compound F1) To a solution of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (compound A1, 46.0 g, 212.88 mmol) and bis(pinacolato)diboron (108.1 g, 425.77 mmol) in 2-methyltetrahydrofuran (1 L) were added potassium carbonate (88.3 g, 638.65 mmol), Pd(dppf)Cl2 (15.6 g, 21.29 mmol) and pivalic acid (10.87 g, 106.44 mmol). Then the mixture was degassed with N2 (this sequence was repeated three times). After being stirred at 80 °C for 3 hrs under N2, the reaction mixture was filtered and the collected solid was washed with EtOAc (100 mL). The filtrate was poured into water (400 mL), extracted with EA (400 mL, three times).The combined organic layer was washed with brine (300 mL, twice), dried over Na2SO4, filtered, and concentrated under vacuum to give the crude product which was purified by silica gel chromatography to afford 2-[(1S)-1- methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (compound F1, 75.0 g) as brown oil. MS calc’d 264.1 (MH+), measured 264.0 (MH+). Step 2: Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-1H- indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F2) To a mixture of 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)pyridine (compound F1, 18.0 g, 30.78 mmol) and [3-(5-bromo-6-fluoro-2-iodo-1H-indol-3- yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate D, 20.5 g, 30.78 mmol) in 1,4-dioxane (200 mL)/water (70 mL)/toluene (70 mL) were added potassium phosphate (19.6 g, 92.35 mmol) and Pd(dppf)Cl2 (2.3 g, 3.08 mmol). The mixture was stirred at 70 °C for 16 hrs under N2. After the reaction completed, the cooling reaction mixture was filtered and the filtrate was concentrated under vacuum to remove the solvent. The resultant mixture was extracted with EtOAc (200 mL, three times). The combined organic layer was washed with brine (100 mL, twice), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography to afford [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]- 1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F2, 22.0 g) as a brown solid. MS calc’d 674.7 (MH+), measured 675.3 (MH+). Step 3: Preparation of [3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]- 3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F3) To a solution of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-1H-indol-3- yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F2, 7 g, 10.39 mmol,) in toluene (20 mL) were added benzyl(triethyl)azanium;bromide (5.6 g, 20.78 mmol), molecular sieve (4Å) (7.5 g, 31.17 mmol) and KOH (1.2 g, 20.78 mmol). The reaction mixture was added with a solution of iodoethane (1.1 mL, 13.51 mmol) in toluene (2 mL). After being stirred at 25 °C for 6 hrs, the reaction mixture was filtered and the filtrate was concentrated under vacuum to give a residue. The residue was purified by NPLC (Column: Welch Ultimate XB-SiOH 250 mm × 100 mm × 10 um, Hexane-EtOH) and concentrated to give [3-[5-bromo-1-ethyl-6-fluoro-(2M)- 2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl- silane (compound F3, 7.0 g) as yellow oil. MS calc’d 702.8 (MH+), measured 703.2 (MH+). Step 4: Preparation of methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[3- [3-[tert-butyl(diphenyl)silyl]oxy-2,2-dimethyl-propyl]-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1- methoxyethyl]-3-pyridyl]indol-5-yl]morpholin-2-yl]propanoyl]hexahydropyridazine-3- carboxylate (compound F4) To the mixture of [3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-3- pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F3, 2.1 g, 2.99 mmol) in 1,4-dioxane (20 mL) was added methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3- [(2S)-morpholin-2-yl]propanoyl]hexahydropyridazine-3-carboxylate (intermediate C, 1.6 g, 3.59 mmol), Pd-PESSI-IPent Cl (251.4 mg, 0.3 mmol) and cesium carbonate (2.9 g, 8.98 mmol). The mixture was stirred at 115 °C for 16 hrs. The cooling reaction mixture was added with EtOAc (40 mL) and water (40 mL) and layers were separated. The aqueous phase was extracted with EtOAc (30 mL, twice). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and purified by column chromatography to afford methyl (3S)-1-[(2S)-2- (benzyloxycarbonylamino)-3-[(2S)-4-[3-[3-[tert-butyl(diphenyl)silyl]oxy-2,2-dimethyl-propyl]- 1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol-5-yl]morpholin-2- yl]propanoyl]hexahydropyridazine-3-carboxylate (compound F4, 1.0 g) as a yellow solid. MS calc’d 1055.5 (MH+), measured 1055.6 (MH+). Step 5: Preparation of (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[1-ethyl-6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol- 5-yl]morpholin-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound F5) A solution of methyl (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[3-[3-[tert- butyl(diphenyl)silyl]oxy-2,2-dimethyl-propyl]-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1- methoxyethyl]-3-pyridyl]indol-5-yl]morpholin-2-yl]propanoyl]hexahydropyridazine-3- carboxylate (compound F4, 1.0 g, 0.95 mmol) in TBAF (7.6 mL, 7.6 mmol, 1 M in THF) was stirred at 40 °C for 12 hrs. After the reaction completed, the mixture was concentrated under vacuum to remove most solvent. The residue was diluted with 1N HCl aqueous solution (15 mL) and extracted with EtOAc (50 mL, three times). The combined organic layer was washed by aq. NH4Cl (30 mL, twice), brine (40 mL, twice), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give a crude product. The crude product was purified by reversed phase chromatography to afford (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[1-ethyl-6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol-5- yl]morpholin-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound F5, 500.0 mg) as a yellow solid. MS calc’d 803.4 (MH+), measured 803.4(MH+). Step 6: Preparation of benzyl N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamate (compound F6) To a solution of (3S)-1-[(2S)-2-(benzyloxycarbonylamino)-3-[(2S)-4-[1-ethyl-6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol-5-yl]morpholin- 2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound F5, 700.0 mg, 0.87 mmol) in DCM (70 mL) were added DIEA (1.1 g, 8.72 mmol), HATU (1.7 g, 4.36 mmol) and 1- methylimidazole (715.8 mg, 8.72 mmol) at 25 °C. After being stirred at rt for 1 h, the mixture was concentrated under vacuum and purified by reversed phase chromatography to give benzyl N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18- dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa- 1(26),20,23(27),24-tetraen-8-yl]carbamate (compound F6, 320.0 mg) as a light yellow solid. MS calc’d 785.5 (MH+), measured 785.4(MH+). Step 7: Preparation of (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F) To a solution of benzyl N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamate (compound F6, 300.0 mg, 0.38 mmol) in THF (3 mL) was added Pd(OH)2 on activated carbon (200 mg). The mixture reaction was stirred at 25 °C for 2 hrs. The mixture was filtered and the filtrate was concentrated under vacuum to afford (6S,8S,14S)-8-amino-22-ethyl-25-fluoro- (21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F, 270.0 mg) as a yellow solid. MS calc’d 651.4 (MH+), measured 651.4 (MH+). Intermediate G (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-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
Figure imgf000035_0001
The compound was prepared according to the following scheme:
Figure imgf000036_0001
G10 intermediate G Step 1: Preparation of 4-[5-bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]morpholine (compound G1) To a mixture of 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine (intermediate A, 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), (R)-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-[(1S)-1-methoxyethyl]-3- pyridyl]morpholine (compound G1, 21 g) as yellow oil. MS calc’d 301.1 (MH+), measured 301.1 (MH+). Step 2: Preparation of 4-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-3-pyridyl]morpholine (compound G2) To a solution of 4-[5-bromo-6-[(1S)-1-methoxyethyl]-3-pyridyl]morpholine (compound G1, 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)Cl2 (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-[(1S)-1-methoxyethyl]- 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridyl]morpholine (compound G2, 45 g) as a yellow gum, which was used in the next step directly. MS calc’d 349.2 (MH+), measured 349.2 (MH+). Step 3: Preparation of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound G3) To a solution of 4-[6-[(1S)-1-methoxyethyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)-3-pyridyl]morpholine (compound G2, 40.6 g, 46.65 mmol), [3-(5-bromo-6-fluoro-2-iodo- 1H-indol-3-yl)-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (intermediate 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)Cl2 (1.7 g, 0.29 mmol). The mixture was degassed and purged with nitrogen, 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 Na2SO4, 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-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]-1H-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-1-ethyl-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound G4) To a solution of [3-[5-bromo-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3- pyridyl]-1H-indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-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 Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by column chromatography to afford [3-[5- bromo-1-ethyl-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-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-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]- 5-morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propan-1-ol (compound G5) and 3-[5- bromo-1-ethyl-6-fluoro-(2P)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]indol-3- yl]-2,2-dimethyl-propan-1-ol (compound G6) To a solution of [3-[5-bromo-1-ethyl-6-fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino- 3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]-tert-butyl-diphenyl-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-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]indol-3-yl]- 2,2-dimethyl-propan-1-ol (compound G5, 6 g, faster eluted) as colorless foam and 3-[5-bromo-1- ethyl-6-fluoro-(2P)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]indol-3-yl]-2,2- dimethyl-propan-1-ol (compound G6, 4.5 g, slower eluted) as colorless foam. Compound G5: MS calc’d 548.2 (MH+), measured 548.2 (MH+).1H NMR (400MHz, Methanol-d4) δ = 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, J = 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-[1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indol-3-yl]-2,2- dimethyl-propan-1-ol (compound G7) To a solution of 3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propan-1-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)Cl2 (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-[1-ethyl-6-fluoro-(2M)- 2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)indol-3-yl]-2,2-dimethyl-propan-1-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)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1- ethyl-6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3- carboxylate (compound G8) To a mixture of 3-[1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3- pyridyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indol-3-yl]-2,2-dimethyl-propan-1-ol (compound G7, 4.5 g, 7.56 mmol) and methyl (3S)-1-[(2S)-3-(4-bromothiazol-2-yl)-2-(tert- butoxycarbonylamino)propanoyl]hexahydropyridazine-3-carboxylate (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)Cl2 (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)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1- ethyl-6-fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-5- morpholino-3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-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)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1-ethyl-6- fluoro-3-(3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino- 3-pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylic acid (compound G9) To a mixture of methyl (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1-ethyl-6-fluoro-3- (3-hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3- pyridyl]indol-5-yl]thiazol-2-yl]propanoyl]hexahydropyridazine-3-carboxylate (compound G8, 3.8 g, 4.39 mmol) in DCE (76 mL) was added Me3SnOH (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)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1-ethyl-6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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 N-[(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- 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- hexaen-7-yl]carbamate (compound G10) To a mixture of (3S)-1-[(2S)-2-(tert-butoxycarbonylamino)-3-[4-[1-ethyl-6-fluoro-3-(3- hydroxy-2,2-dimethyl-propyl)-(2M)-2-[2-[(1S)-1-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 Na2SO4, filtered and concentrated under vacuum to give a residue which was purified by column chromatography to afford tert-butyl N-[(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]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-[(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) To a solution of tert-butyl N-[(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]carbamate (compound G10, 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. NaHCO3 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-[(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, 1 g) as a yellow solid. MS calc’d 734.3 (MH+), measured 734.3 (MH+). Intermediate H (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-17,17- dimethyl-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione
Figure imgf000042_0001
The title compound was prepared in analogy to the preparation of Intermediate G by using [3-[5-bromo-1-ethyl-6-fluoro-(2M)-2-[2-[(1S)-1-methoxyethyl]-3-pyridyl]indol-3-yl]-2,2- dimethyl-propoxy]-tert-butyl-diphenyl-silane (compound F3) instead of [3-[5-bromo-1-ethyl-6- fluoro-2-[2-[(1S)-1-methoxyethyl]-5-morpholino-3-pyridyl]indol-3-yl]-2,2-dimethyl-propoxy]- tert-butyl-diphenyl-silane (compound G4). Intermediate I 4-(dimethylamino)-4-methyl-pent-2-ynoic acid
Figure imgf000043_0001
The compound was prepared according to the following scheme:
Figure imgf000043_0002
Step 1: Preparation of N,N,2-trimethylbut-3-yn-2-amine (compound I2) A mixture of 3-chloro-3-methyl-1-butyne (20.0 g, 195.01 mmol) and dimethylamine (22.0 g, 195.01 mmol) was stirred at 25 °C for 12 hrs. After the reaction completed, the suspension was filtered. The collected solid was washed with ice-water (50 mL, three times) and dissolved in cold HCl (3 N, 10 mL). The acidic solution was extracted with methyl tert-butyl ether (50 mL, twice). The resulting aqueous phase was cooled at 0 °C and made strongly alkaline with cold NH3.H2O. The yellow precipitate was collected, washed with NH3.H2O (50 mL, three times) and H2O (50 mL, three times). The collected solid was concentrated in vacuo to afford N,N,2- trimethylbut-3-yn-2-amine (compound I2, 4.0 g) as a yellow solid. MS calc’d 112.1 (MH+), measured 112.1 (MH+). Step 2: Preparation of 4-(dimethylamino)-4-methyl-pent-2-ynoic acid (intermediate I) To a solution of N,N,2-trimethylbut-3-yn-2-amine (compound I2, 3.2 g, 28.78 mmol) in THF (120 mL) was added dropwise n-BuLi (12.7 mL, 31.66 mmol, 2M in hexane) at -70 °C. After being stirred at -70°C for 5 min, the reaction mixture was warmed to 0 °C and stirred for another 10 min. After being cooled to -70 °C, the reaction mixture was bubbled with carbon dioxide for 30 min. The reaction mixture was stirred at 0°C for 2 hrs. After the reaction completed, the reaction mixture was carefully hydrolyzed at 0°C by water (40 mL). Two phases were separated and the aqueous layer was acidified pH to 1-2 with 6N HCl. The aqueous was concentrated under vacuum to afford 4-(dimethylamino)-4-methyl-pent-2-ynoic acid (intermediate I, 4.3 g) as a white solid, which was used in the next step without purification. MS calc’d 156.1 (MH+), measured 156.1 (MH+).1H NMR (400 MHz, DEUTERIUM OXIDE-D2O) δ = 2.99 - 2.91 (m, 6H), 1.77 - 1.68 (m, 6H). Example 1 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro- (21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa- 2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide
Figure imgf000044_0001
The compound was prepared according to the following scheme:
Figure imgf000044_0002
Step 1: Preparation of tert-butyl N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21- [2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-N-methyl-carbamate (compound 1a) To a mixture of (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F, 270.0 mg, 0.41 mmol) and (2S)-2-[tert-butoxycarbonyl(methyl)amino]-3- methyl-butanoic acid (191.9 mg, 0.83 mmol) in DMF (3 mL) were added DIEA (0.36 mL, 2.07 mmol) and T4P (597.9 mg, 0.83 mmol) at 0 °C. The mixture was stirred at 25 °C for 1h. The mixture was concentrated under vacuum to get a residue, which was purified by reversed phase chromatography to afford tert-butyl N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2- [(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-N-methyl-carbamate (compound 1a, 210.0 mg) as a brown solid. MS calc’d 864.5 (MH+), measured 864.5(MH+). Step 2: Preparation of (2S)-N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]-3-methyl- 2-(methylamino)butanamide (compound 1b) To a solution of tert-butyl N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-N-methyl-carbamate (compound 1a, 210.0 mg, 0.24 mmol) in DCM (2 mL) was added TFA (1.0 mL, 12.98 mmol). The mixture was stirred at 25 °C for 1 h. After the reaction completed, the mixture was concentrated under vacuum to remove most solvent, then poured into sat. NaHCO3 aq. (5 mL) and extracted with EtOAc (30 mL, five times). The combined organic layer was washed by brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford (2S)-N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)- 1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]-3-methyl-2- (methylamino)butanamide (compound 1b, 225.0 mg) as a brown solid. MS calc’d 764.4 (MH+), measured 764.4 (MH+). Step 3: Preparation of tert-butyl 4-[[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-21-[2- [(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-methyl-carbamoyl]-4-fluoro-piperidine-1-carboxylate (compound 1c) To a solution of (2S)-N-[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]-3-methyl-2- (methylamino)butanamide (compound 1b, 200.0 mg, 0.26 mmol) in DMF (2.5 mL) were added DIEA (0.5 mL, 2.62 mmol), 1-tert-butoxycarbonyl-4-fluoro-piperidine-4-carboxylic acid (194.2 mg, 0.79 mmol) and CMPI (200.6 mg, 0.79 mmol) at 0 °C. The mixture was stirred at 25 °C for 1 h. The mixture was poured into water (30 mL) and extracted with EtOAc (30 mL, three times). The combined organic layer was washed by brine (30 mL, twice), dried over Na2SO4, filtered and concentrated under vacuum to give a residue. The residue was purified by reversed phase chromatography to afford tert-butyl 4-[[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-methyl-carbamoyl]-4-fluoro-piperidine-1-carboxylate (compound 1c, 200.0 mg) as yellow oil. MS calc’d 993.6 (MH+), measured 993.6 (MH+). Step 4: Preparation of N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (compound 1d) To a solution of tert-butyl 4-[[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-methyl-carbamoyl]-4-fluoro-piperidine-1-carboxylate (compound 1c, 200.0 mg, 0.2 mmol) in DCM (2 mL) was added TFA (1.1 mL, 14.35 mmol) at 0°C. The mixture was stirred at 25°C for 1 hour. After the reaction completed, the mixture was concentrated under vacuum to remove most solvent, then poured into sat. NaHCO3 aq. (5 mL) and extracted with EtOAc (20 mL, three times). The combined organic layer was washed by brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford N-[(1S)-1- [[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl- 9,15-dioxo-5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa- 1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4- carboxamide (compound 1d, 200.0 mg) as yellow oil. MS calc’d 893.5 (MH+), measured 893.5 (MH+). Step 5: Preparation of 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1- [[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18- dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8- yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (Example 1) To a mixture of N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (compound 1d, 100.0 mg, 0.11 mmol) and 4-(dimethylamino)-4-methyl-pent-2-ynoic acid (intermediate I, 34.7 mg, 0.22 mmol,) in DMF (2 mL) were added DIEA (0.1 mL, 0.56 mmol) and T4P (161.4 mg, 0.22 mmol) at 0 °C. The mixture was stirred at 25 °C for 1h, then purified by Prep-HPLC to afford 1-[4- (dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)-22-ethyl-25-fluoro-(21M)-21- [2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraen-8-yl]carbamoyl]-2- methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide (Example 1, 45.0 mg) as a yellow solid. MS calc’d 1030.6 (MH+), measured 1030.6 (MH+).1H NMR (400 MHz, METHANOL-d4) δ = 8.82 ( s, 1H), 8.25 - 8.13 (m, 1H), 7.75 - 7.65 (m, 1H), 7.33 - 7.23 (m, 2H), 5.74 - 5.64 (m, 1H), 4.65 - 4.60 (d, J = 10.8 Hz, 1H), 4.55 - 4.44 (m, 1H), 4.40 - 4.30 (m, 1H), 4.30 - 4.17 (m, 2H), 4.16 - 4.06 (m, 1H), 3.98 - 3.89 (m, 2H), 3.89 - 3.84 (m, 1H), 3.80 (dd, J = 4.0, 11.2 Hz, 2H), 3.65-3.55 (m, 2H), 3.50 - 3.37 (m, 1H), 3.26 - 3.18 (m, 4H), 3.16 (d, J = 5.2 Hz, 2H), 3.10 - 3.08 (m, 1H), 3.02 ( s, 5H), 2.98 - 2.94 (m, 1H), 2.84 - 2.76 (m, 1H), 2.71 - 2.58 (m, 1H), 2.38 - 2.25 (m, 2H), 2.23 - 2.03 (m, 5H), 1.99 - 1.85 (m, 3H), 1.80 (s, 6H), 1.75 - 1.56 (m, 4H), 1.51 - 1.39 (m, 5H), 1.11-1.01 (m, 3H), 1.00 - 0.89 (m, 6H), 0.85 (d, J = 6.4 Hz, 2H), 0.82 - 0.73 (m, 4H). Example 2 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-piperidine-4-carboxamide
Figure imgf000048_0001
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- 9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaene- 8,14-dione (intermediate E) instead of (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2- [(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F). Example 2 (38.5 mg) was obtained as a yellow solid. MS calc’d 1248.6 (MH+), measured 1248.6 (MH+).1H NMR (400 MHz, METHANOL-d4) δ = 8.69 (dd, J = 2.0, 7.6 Hz, 1H), 8.51 - 8.45 (m, 1H), 8.42 (d, J = 2.0 Hz, 1H), 7.68 - 7.66 (m, 1H), 7.52 - 7.44 (m, 1H), 5.78 - 5.67 (m, 1H), 5.22 - 5.12 (m, 1H), 4.76 (dd, J = 2.4, 11.2 Hz, 1H), 4.47 - 4.35 (m, 2H), 4.27 - 4.17 (m, 3H), 3.82 - 3.75 (m, 1H), 3.73 - 3.59 (m, 2H), 3.51 - 3.43 (m, 1H), 3.42 - 3.34 (m, 7H), 3.28 - 3.19 (m, 5H), 3.18 - 3.10 (m, 3H), 3.01 (d, J = 7.2 Hz, 6H), 2.88 (t, J = 5.2 Hz, 4H), 2.84 - 2.77 (m, 1H), 2.68 - 2.58 (m, 1H), 2.39 - 2.12 (m, 6H), 2.00 - 1.92 (m, 1H), 1.79 (d, J = 4.8 Hz, 7H), 1.71 - 1.58 (m, 1H), 1.45 (d, J = 6.4 Hz, 3H), 1.15 - 1.04 (m, 1H), 1.03 - 0.94 (m, 6H), 0.89 (d, J = 6.8 Hz, 3H), 0.47 (d, J = 6.0 Hz, 3H). Example 3 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-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-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl- piperidine-4-carboxamide
Figure imgf000049_0001
The title compound was prepared in analogy to the preparation of Example 1 by using (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- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (intermediate G) instead of (6S,8S,14S)-8-amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1- methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16-dioxa-2,10,22,28- tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene-9,15-dione (intermediate F). Example 3 (16.5 mg) was obtained as a white solid. MS calc’d 1113.6 (MH+), measured 1113.6 (MH+).1H NMR (400 MHz, METHANOL-d4) δ = 8.66 (dd, J = 7.6 Hz, 3.2 Hz, 1H), 8.42 (d, J = 2.8 Hz, 1H), 7.62 (dd, J= 12.4 Hz, 0.8 Hz, 1H), 7.39 - 7.28 (m, 2H), 5.84 - 5.66 (m, 1H), 4.80 - 4.75 (m, 1H), 4.49 - 4.32 (m, 3H), 4.30 - 4.14 (m, 4H), 3.88 (t, J = 4.4 Hz, 4H), 3.81 - 3.70 (m, 2H), 3.69 - 3.57 (m, 1H), 3.50 - 3.41 (m, 1H), 3.36 - 3.30 (m, 10H), 3.22 (t, J = 6.4 Hz, 3H), 3.10 - 3.01 (m, 1H), 2.89 - 2.76 (m, 1H), 2.70 - 2.58 (m, 1H), 2.39 (s, 3H), 2.36 (s, 3H), 2.33 - 2.26 (m, 2H), 2.25 - 2.13 (m, 3H), 2.01 - 1.92 (m, 1H), 1.87 - 1.75 (m, 1H), 1.71 - 1.57 (m, 1H), 1.50 (s, 5H), 1.45 (d, J =3.0 Hz, 3H), 1.38 - 1.27 (m, 1H), 1.07 - 0.93 (m, 9H), 0.89 (d, J =3.2 Hz, 3H), 0.51 (d, J =4.2 Hz, 3H). Example 4 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(7S,13S)-21-ethyl-24-fluoro- (20M)-20-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia- 9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23- hexaen-7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide
The title compound was prepared in analogy to the preparation of Example 1 by using (7S,13S)-7-amino-21-ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-17,17- dimethyl-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaene-8,14-dione (intermediate H) instead of (6S,8S,14S)-8- amino-22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-5,16- dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24-tetraene- 9,15-dione (intermediate F). Example 4 (44.0 mg) was obtained as a white solid. MS calc’d 1028.5 (MH+), measured 1028.5 (MH+).1H NMR (400 MHz, METHANOL-d4) δ = 8.75 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 8.67 (dd, J = 7.2 Hz, 2.4 Hz, 1H), 7.85 (dd, J = 7.6 Hz, 1.2 Hz, 1H), 7.62 (dd, J = 12.8 Hz, 2.4 Hz, 1H), 7.55 (dd, J = 8.0 Hz, 5.2 Hz, 1H), 7.35 (dd, J = 11.6 Hz, 5.2 Hz, 1H), 5.80 - 5.65 (m, 1H), 4.99 - 4.93 (m, 1H), 4.81 - 4.73 (m, 1H), 4.49 - 4.31 (m, 4H), 4.28 - 4.10 (m, 3H), 3.80 - 3.70 (m, 2H), 3.68 - 3.57 (m, 1H), 3.49 - 3.40 (m, 1H), 3.39 - 3.34 (m, 3H), 3.30 - 3.25 (m, 1H), 3.23 - 3.18 (m, 3H), 3.10 - 3.02 (m, 1H), 2.89 - 2.77 (m, 1H), 2.66 - 2.55 (m, 1H), 2.39 (s, 3H), 2.36 (s, 3H), 2.33 - 2.27 (m, 2H), 2.27 - 2.11 (m, 4H), 2.01 - 1.90 (m, 1H), 1.87 - 1.74 (m, 1H), 1.69 - 1.59 (m, 1H), 1.53 - 1.41 (m, 9H), 1.04 - 0.83 (m, 12H), 0.48 (d, J = 6.8 Hz, 3H). BIOLOGICAL EXAMPLE Compound A647 (page 176 of Table.1, or referred as “RMC-6291”), from WO2021091982 was cited as reference compound for this invention.
Example 5 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 3 mg/kg, iv 30 mg/kg, po Compound CL Cmax AUC0-last (mL/min/kg) (ng/mL) (h×ng/mL) RMC-6291 44.2 2706 4116 Example 1 8.4 9919 24959 From Table 1, it can be seen that Example 1 has excellent pharmacokinetic properties in mouse model. Especially Example 1 has almost 3.7 folds of Cmax, 6 folds of AUC0-last and significant lower clearance compared with compound RMC-6291, which makes Example 1 more suitable for treating cancers with RAS mutation as an orally therapeutic active ingredient in clinic. Example 6 In vivo xenograft studies The purpose of the study was to assess the anti-tumor activity in difficult KRAS mutated xenograft tumor model, NCI-H2122 which is a non-small cell lung cancer (NSCLC), by examining the tumor suppression post-administration of Example 1 and RMC-6291. Studies were conducted at Wuxi AppTec (Nantong, China). All CDX mouse studies and procedures related to animal handling, care and treatment were conducted in compliance with all applicable regulations and guidelines of the relevant Institutional Animal Care and Use Committee (IACUC). Mice were maintained under pathogen-free conditions, and food and water was provided ad libitum. Female BALB/c nude mice at 6-8 weeks old from Vital River Co., LTD. were used for these studies. In order to generate subcutaneous xenograft tumors, each mouse was inoculated at the right flank with tumor cells in 200 μL of PBS and 50% matrigel matrix in the right hind flank with 5×106 cells. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 × L × W2 in which L refers to length and W refers to width of each tumor. When tumors reached an average tumor volume of approximately 350 mm3, , mice were randomized into treatment groups. Mice were treated by oral gavage at 10 mg/kg with either vehicle consisting of DMSO: Solutol HS15 :water=10:10:80 (%v/v/v) or compounds (Example 1 or RMC-6291) in vehicle at indicated doses. For efficacy studies, animals were orally administered daily, tumor volumes and body weights were measured 3 times per week. Study day on efficacy plots indicates the day after which treatment was initiated. TGI (tumor growth inhibition)%=(Final treated tumor volume- Initial treated tumor volume)/(Vehicle final treated tumor volume-Vehicle initial treated tumor volume) ×100% Tumor regression=(-100%)×(1 – (Final treated tumor volume)/(Initial treated tumor volume)) Tumor growth=100%×(Final treated tumor volume – Initial treated tumor volume)/(Initial treated tumor volume) Daily administration of Example 1 at 100 mg/kg was well-tolerated based on body weights and resulted in 108% mean tumor growth inhibition (TGI) or -32% tumor regression following 21 days treatment in comparison to RMC-6291 that leads to only 92% TGI or unwanted 35% tumor growth at same dose regiments. (Figure 2) Table 2. Summary of in vivo efficacy in NCI-H2122 xenograft study Tumor Volume (mm3, Mean/SEM) TGI Tumor regression Tumor growth Day 0 Day 21 (Day 21) (Day 21) (Day 21) Vehicle, QD 348±26 1799±223 RMC-6291 100mpk,QD 348±29 470±49 92% 35% Example 1 100mpk, QD 348±29 237±11 108% -32% Example 7 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 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) 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 G12C IC50 (nM) Example 1 0.42 Example 2 0.44 Example 3 0.62 Example 4 0.41 Example 8 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. 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 MgCl2, 10 µM GMPPNP (Guanosine 5′-[β,γ- imido]triphosphate trisodium salt hydrate, Sigma, G0635), tagless CYPA, GMPPNP loaded 6His-KRAS proteins, and GST-BRAFRBD were mixed in a well of a 384-well assay plate at final concentrations of 50 nM, 6.25 nM and 1nM, respectively. Compound was present in plate wells as a 16-point 3-fold dilution series starting at a final concentration of 10 µM 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 G12C IC50 (nM) Example 1 0.50 Example 2 0.60 Example 3 0.50 Example 4 0.70 Example 9 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. NCI-H358 (ATCC- CRL5807) cells was 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 for NCI-H358 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 µL 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 µL of chilled methanol for 10 minutes. Non- specific antibody binding to the plates was blocked using 50 µL 1X 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 µL aliquoted to each well, and incubated overnight at 4 ℃. 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 µL was added to each well and incubated 1-2 hrs at room temperature. Cells was washed 5 times for 5 minutes with PBST, 100µL TMB ELISA substrate (abcam-ab171523) were added and gently shake for 20 minutes.50µL stop solution (abcam-ab171529) 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 G12C IC50 (nM) Example 1 1.54 Example 2 0.96 Example 3 2.31 Example 4 1.29

Claims

CLAIMS 1. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(6S,8S,14S)- 22-ethyl-25-fluoro-(21M)-21-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-18,18-dimethyl-9,15-dioxo- 5,16-dioxa-2,10,22,28-tetrazapentacyclo[18.5.2.12,6.110,14.023,27]nonacosa-1(26),20,23(27),24- tetraen-8-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof. 2. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-4-fluoro-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-dimethyl-8,14-dioxo-21-(2,2,2-trifluoroethyl)-15-oxa-4-thia-9,21,27,28- tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),
2,5(28),19,22(26),23-hexaen-7- yl]carbamoyl]-2-methyl-propyl]-N-methyl-piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
3. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-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-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl- piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
4. A compound which is 1-[4-(dimethylamino)-4-methyl-pent-2-ynoyl]-N-[(1S)-1-[[(7S,13S)-21- ethyl-24-fluoro-(20M)-20-[2-[(1S)-1-methoxyethyl]-3-pyridyl]-17,17-dimethyl-8,14-dioxo-15- oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa- 1(25),2,5(28),19,22(26),23-hexaen-7-yl]carbamoyl]-2-methyl-propyl]-4-fluoro-N-methyl- piperidine-4-carboxamide, or a pharmaceutically acceptable salt thereof.
5. A compound or pharmaceutically acceptable salt according to any one of claims 1 to 4 for use as therapeutically active substance.
6. A pharmaceutical composition comprising a compound in accordance with any one of claims 1 to 4 and a pharmaceutically acceptable excipient.
7. The use of a compound according to any one of claims 1 to 4 for treating a KRAS G12C protein-related disease.
8. The use of a compound according to any one of claims 1 to 4 for inhibiting RAS interaction with downstream effectors, wherein the downstream effectors are RAF and PI3K.
9. The use of a compound according to any one of claims 1 to 4 for inhibiting the propagating oncogenic MAPK and PI3K signaling.
10. The use of a compound according to any one of claims 1 to 4 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.
11. The use of a compound according to any one of claims 1 to 4 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.
12. A compound or pharmaceutically acceptable salt according to any one of claims 1 to 4 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.
13. The use of a compound according to any one of claims 1 to 4 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.
14. 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 claims 1 to 4.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025255438A1 (en) 2024-06-07 2025-12-11 Revolution Medicines, Inc. Methods of treating a ras protein-related disease or disorder

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021091982A1 (en) 2019-11-04 2021-05-14 Revolution Medicines, Inc. Ras inhibitors
WO2023133543A1 (en) * 2022-01-10 2023-07-13 Revolution Medicines, Inc. Ras inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021091982A1 (en) 2019-11-04 2021-05-14 Revolution Medicines, Inc. Ras inhibitors
WO2023133543A1 (en) * 2022-01-10 2023-07-13 Revolution Medicines, Inc. Ras inhibitors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANSEL, HOWARD C. ET AL.: "Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems.", 2004, LIPPINCOTT, WILLIAMS & WILKINS
GENNARO, ALFONSO R. ET AL.: "Remington: The Science and Practice of Pharmacy.", 2000, LIPPINCOTT, WILLIAMS & WILKINS
ROWE, RAYMOND C.: "Handbook of Pharmaceutical Excipients. Chicago", 2005, PHARMACEUTICAL PRESS

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025255438A1 (en) 2024-06-07 2025-12-11 Revolution Medicines, Inc. Methods of treating a ras protein-related disease or disorder

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