WO2023104018A1 - Substituted bicyclic heteroaryl compound as kras g12d inhibitor - Google Patents
Substituted bicyclic heteroaryl compound as kras g12d inhibitor Download PDFInfo
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- WO2023104018A1 WO2023104018A1 PCT/CN2022/136857 CN2022136857W WO2023104018A1 WO 2023104018 A1 WO2023104018 A1 WO 2023104018A1 CN 2022136857 W CN2022136857 W CN 2022136857W WO 2023104018 A1 WO2023104018 A1 WO 2023104018A1
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- cancer
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- mmol
- compound
- ethyl acetate
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- VPAYJEUHKVESSD-UHFFFAOYSA-N trifluoroiodomethane Chemical compound FC(F)(F)I VPAYJEUHKVESSD-UHFFFAOYSA-N 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- CCRMAATUKBYMPA-UHFFFAOYSA-N trimethyltin Chemical compound C[Sn](C)C.C[Sn](C)C CCRMAATUKBYMPA-UHFFFAOYSA-N 0.000 description 1
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- NHDIQVFFNDKAQU-UHFFFAOYSA-N tripropan-2-yl borate Chemical compound CC(C)OB(OC(C)C)OC(C)C NHDIQVFFNDKAQU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- 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
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- 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
- C07D487/02—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 two hetero rings
- C07D487/08—Bridged systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Definitions
- the invention belongs to the field of medicine, in particular to a substituted bicyclic heteroaryl compound, which can be used as a KRAS G12D inhibitor.
- the human RAS gene family includes three types of RAS genes KRAS, NRAS and HRAS, encoding four different RAS proteins (KRAS-4A, KRAS-4B, NRAS and HRAS).
- RAS protein belongs to the GTPase protein family, it is in an inactive state when it binds to GDP, and it is in an active state after binding to GTP, which can lead to the activation of downstream RAF-MAPK, PI3K-Akt and other signaling pathways, leading to the anti-apoptosis and anti-apoptosis of cells. Proliferation (Cell, 2017; 170(1):17-33; Cell, 2020; 183(4):850-859; Nat Rev Drug Discov, 2020; 19(8):533-552.).
- KRAS Activating mutations in the RAS gene are the most prevalent oncogenic driver genes in human cancers, among which KRAS is the most frequent oncogenic activating mutation.
- KRAS has a mutation rate of 86-96% in pancreatic cancer, 40-54% in colorectal cancer, and 27-39% in lung cancer (PNAS, 2019; 116(32):15823-15829 ; Pathol Res Pract, 2009; 205, 858–862; Nature, 2012; 491, 399–405; Nature, 2014; 511, 543–550).
- Oncogenic driver mutations can occur at multiple sites in the KRAS gene, the most common mutations occur at the G12 site, including G12C, G12D, G12V, etc. These mutations can reduce the GTPase activity of the KRAS protein, resulting in a long-term Active state, leading to malignant transformation of cells and carcinogenesis (Cell, 2017; 170(1): 17-33; Nat Rev Drug Discov, 2020; 19(8): 533-552). In different cancer types, the frequently occurring KRAS mutation types are different.
- KRAS G12D small molecule MRTX1133 for intravenous administration, but it has not yet entered clinical research, and its oral absorption is weak.
- the KRAS G12D small molecule drug of the present invention has excellent activity and higher in vivo exposure, and is expected to Realize oral administration in order to solve clinical unmet needs.
- the invention provides a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof, wherein Said compound is selected from:
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of the present invention, and optionally a pharmaceutically acceptable excipient.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient, which also comprises other therapeutic agents.
- the present invention provides the use of the compound of the present invention in the preparation of a medicament for treating and/or preventing KRAS G12D mutein-mediated diseases.
- the present invention provides a method for treating and/or preventing a KRAS G12D mutein-mediated disease in a subject, comprising administering to the subject a compound of the present invention or a composition of the present invention.
- the present invention provides the compound of the present invention or the composition of the present invention for the treatment and/or prevention of KRAS G12D mutein-mediated diseases.
- the diseases treated by the present invention include cancers selected from the group consisting of acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (e.g., lymphoma and Kaposi's sarcoma), anal cancer , appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, Carcinoid tumors, atypical teratoids, embryonal tumors, germ cell tumors, primary lymphomas, cervical cancers, childhood cancers, chordomas, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia ( CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lympho
- CLL
- KRAS G12D refers to a mutant form of the mammalian KRAS protein that contains an amino acid substitution of aspartic acid for glycine at amino acid position 12.
- the term "pharmaceutically acceptable salt” refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention, which are suitable for use in contact with patient tissues within the scope of sound medical judgment without undue toxicity, Irritation, allergic effects, etc., commensurate with a reasonable benefit/risk ratio, are valid for their intended use, including, where possible, zwitterionic forms of the compounds of the invention.
- Subjects for administration include, but are not limited to: human (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adult, middle-aged adult or older adult)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep , goats, rodents, cats and/or dogs.
- the subject is a human.
- the subject is a non-human animal.
- the terms "human", “patient” and “subject” are used interchangeably herein.
- an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response.
- an effective amount of a compound of the invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the condition of the subject. Age Health conditions and symptoms.
- An effective amount includes a therapeutically effective amount and a prophylactically effective amount.
- Combination and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent.
- the compounds of the invention may be administered with the other therapeutic agent simultaneously or sequentially in separate unit dosage forms, or together with the other therapeutic agent in a single unit dosage form.
- the compound of the present invention refers to the following compounds, their pharmaceutically acceptable salts, enantiomers, diastereoisomers, solvates, hydrates or isotopic variants, and their mixture.
- the invention relates to a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof, wherein Said compound is selected from:
- the compounds of the present invention may include one or more asymmetric centers, and thus may exist in various stereoisomeric forms, eg, enantiomeric and/or diastereomeric forms.
- the compounds of the invention may be individual enantiomers, diastereoisomers or geometric isomers (eg cis and trans isomers), or may be in the form of a mixture of stereoisomers, Racemic mixtures and mixtures enriched in one or more stereoisomers are included.
- Isomers can be separated from mixtures by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be obtained by prepared by asymmetric synthesis.
- HPLC high pressure liquid chromatography
- the compounds of the invention may also exist as tautomers.
- a said compound is not limited to any particular tautomeric form, but is intended to encompass all tautomeric forms.
- organic compounds may form complexes with solvents in which they react or from which they are precipitated or crystallized. These complexes are known as "solvates”. When the solvent is water, the complex is called a "hydrate”. The invention covers all solvates of the compounds of the invention.
- solvate refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
- solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
- Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
- “Solvate” includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
- hydrate refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined.
- a hydrate of a compound can be represented, for example, by the general formula R.x H 2 O, where R is the compound, and x is a number greater than zero.
- a given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)).
- the compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
- polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
- the invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (A), but with one or more atoms represented by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature replaced.
- isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl.
- the compounds of the present invention their prodrugs and pharmaceutically acceptable salts of the compounds or the prodrugs containing the above-mentioned isotopes and/or other isotopes of other atoms all belong to the scope of the present invention.
- Certain isotopically-labeled compounds of the invention eg, those incorporating radioactive isotopes (eg, 3H and14C ), are useful in drug and/or substrate tissue distribution assays. Tritium, ie3H , and carbon-14, ie14C isotopes are particularly preferred because of their ease of preparation and detection.
- isotope-labeled compound of formula (A) of the present invention and its prodrug can generally be prepared in this way, when carrying out the processes disclosed in the following schemes and/or examples and preparation examples, replace the non-isotope with easily available isotope-labeled reagents Labeled reagents.
- prodrugs are also included within the context of the present invention.
- the term "prodrug” as used herein refers to a compound that is converted in vivo to its active form having a medical effect, for example by hydrolysis in blood.
- Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon, and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, per intro This article is for reference.
- the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient.
- the pharmaceutical composition comprises an effective amount of a compound of the invention.
- the pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention.
- the pharmaceutical composition comprises a prophylactically effective amount of a compound of the invention.
- a pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle which does not destroy the pharmacological activity of the compound formulated together.
- Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin Protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- Block polymers, polyethylene glycol
- kits eg, pharmaceutical packs.
- kits can include a compound of the invention, another therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container).
- first and second containers e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container.
- provided kits can also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent.
- a compound of the invention and other therapeutic agent provided in a first container and a second container are combined to form a unit dosage form.
- parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal, intralesional, and intracranial injection or infusion techniques, preferably intravenously.
- an effective amount of a compound provided herein is administered.
- the amount of the compound actually administered can be determined by the physician according to the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
- the compounds provided herein are administered to a subject at risk of developing the condition, typically on the advice and supervision of a physician, at dosage levels as described above.
- Subjects at risk of developing a particular condition generally include those with a family history of the condition, or those determined by genetic testing or screening to be particularly susceptible to developing the condition.
- Chronic administration refers to administering a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, For example, the rest of the subject's life.
- chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within the therapeutic window.
- compositions may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to effective levels.
- the bolus dose depends on the target systemic level of the active ingredient through the body, for example, an intramuscular or subcutaneous bolus dose provides slow release of the active ingredient, while a bolus delivered directly into a vein (e.g., by IV intravenous infusion) ) can be delivered more rapidly, so that the concentration of the active ingredient in the blood rises rapidly to effective levels.
- the pharmaceutical compositions may be administered as a continuous infusion, eg, by IV infusion, to provide a steady state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
- Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More usually, however, the compositions will be presented in unit dosage form for ease of precise dosing.
- unit dosage form refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
- Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions.
- the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful for forming the desired administration form. Carriers or excipients and processing aids.
- a typical regimen is one to five oral dosages per day, especially two to four oral dosages, typically three oral dosages.
- each dose provides from about 0.01 to about 20 mg/kg of the compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially about 1 to about 5 mg/kg.
- the transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
- Injection dosage levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially 24 to 96 hours.
- a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given in order to achieve adequate steady state levels.
- the maximum total dose should not exceed approximately 2 g/day.
- Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering, suspending and dispersing agents, coloring agents, flavoring agents, and the like.
- the solid form may comprise, for example, any of the following components, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or flavoring agents, for example, peppermint, water Methyl sylate or orange flavoring.
- binders such as microcrystalline cellulose, tragacanth, or gelatin
- excipients such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch
- Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art.
- the active compound is typically a minor component, often from about 0.05 to 10% by weight, the remainder being injectable excipients and the like.
- Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient.
- the active ingredients When formulated in an ointment, the active ingredients are typically combined with a paraffinic or a water-miscible ointment base.
- the active ingredients may be formulated in a cream, with, for example, an oil-in-water cream base.
- Such transdermal formulations are well known in the art, and generally include other ingredients for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope of the present invention.
- transdermal administration can be achieved using patches of the reservoir or porous membrane type, or various solid matrices.
- compositions for oral administration, injection or topical administration are representative only. Other materials and processing techniques, etc. are described in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, Part 8, which is incorporated herein by reference.
- the compounds of the invention may also be administered in sustained release form, or from a sustained release delivery system.
- sustained release materials can be found in Remington's Pharmaceutical Sciences.
- the invention also relates to pharmaceutically acceptable formulations of the compounds of the invention.
- the formulation comprises water.
- the formulation comprises a cyclodextrin derivative.
- the most common cyclodextrins are ⁇ -, ⁇ -, and ⁇ -cyclodextrins composed of 6, 7, and 8 ⁇ -1,4-linked glucose units, respectively, optionally including a or multiple substituents including, but not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions.
- the cyclodextrin is a sulfoalkyl ether ⁇ -cyclodextrin, eg, sulfobutyl ether ⁇ -cyclodextrin, also known as Captisol. See, eg, U.S. 5,376,645.
- the formulation includes hexapropyl- ⁇ -cyclodextrin (eg, 10-50% in water).
- the reagents used in the present invention are commercial reagents purchased directly or synthesized by common methods well known in the art.
- Step 1 Dissolve the raw material 2-amino-3-fluoro-4-bromobenzoic acid a1-1 (15g, 64.1mmol) in 100mL DMF, slowly add N-chlorosuccinimide (8.56g, 64.1mmol) , the temperature was raised to 80° C. for 16 hours, and the reaction was stopped. The reaction solution was poured into 500 mL of ice water, filtered with suction to obtain a filter cake, and dried to obtain intermediate a1-2 (13.2 g, 49.2 mmol). Yield: 77%.
- LC-MS: [MH] - 267.
- Step 2 Add urea (35.3g, 588mmol) and intermediate a1-2 (10.5g, 39.2mmol) into a 200mL round bottom flask, and heat up to 200°C for 12 hours. After cooling down to 80°C, 100 mL of water was added to the system, refluxed for 10 minutes, cooled to room temperature, filtered to obtain a filter cake, washed with water, and dried in an oven to obtain intermediate a1-3 (4.0 g, 13.7 mmol). Yield: 35%.
- LC-MS: [M+H] + 294.
- Step 3 Dissolve the intermediate a1-3 (4.0g, 13.7mmol) and N,N-diisopropylethylamine (5.3g, 41.1mmol) in 15mL phosphorus oxychloride, and heat up to 120°C After reacting for 8 hours, stop the reaction. The solvent was evaporated under reduced pressure and separated by column chromatography to obtain intermediate a1 (1.9 g, 5.8 mmol). Yield: 42%.
- LC-MS: [M+H] + 331.
- Step 1 Under ice bath, dissolve 1-(methoxycarbonyl)cyclopropane-1-carboxylic acid a4-2 (3.0g, 20.8mmol) in 60mL of dichloromethane, slowly add oxalyl chloride (10.5g, 83.3 mmol), added 5 drops of anhydrous DMF, and reacted at 0° C. for 20 minutes. The ice bath was removed, the temperature was raised to room temperature and stirring was continued for 1 hour, and the solvent was evaporated under reduced pressure.
- Step 2 At -78°C, the intermediate a4-3 (2.6g, 13.2mmol) was dissolved in 30mL of anhydrous tetrahydrofuran, and lithium aluminum hydride (26.4mL, 1M) was added slowly. The temperature was raised to room temperature for 2 hours, and the reaction was stopped. Slowly pour 80 mL of ice water into the reaction solution, evaporate the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate, and separate by flash column chromatography to obtain intermediate a4 (900 mg, 5.8 mmol). Yield: 44%.
- LC-MS: [M+H] + 156.
- Step 1 Under ice bath, dissolve 6-bromo-2,3-difluorobenzaldehyde a7-1 (25g, 113mmol) in 250mL of methanol, slowly add sodium borohydride (8.54g, 226mmol), and stir for 20 minutes , the temperature was raised to room temperature and the reaction was continued for 1 hour, and the reaction was stopped. The reaction solution was slowly poured into saturated aqueous NH 4 Cl solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid a7-2 (23.9 g, 107 mmol). Yield: 95%.
- Step 2 Dissolve intermediate a7-2 (23.9g, 107mmol) and N,N-diisopropylethylamine (20.7g, 161mmol) in 200mL anhydrous tetrahydrofuran under ice bath, and slowly add methylsulfonate Acid anhydride (20.5 g, 118 mmol), after stirring for 20 minutes, the ice bath was removed, and the reaction was continued at room temperature for 18 hours, and the reaction was stopped. The reaction solution was slowly poured into ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain oil a7-3 (19 g, 63.1 mmol). Yield: 59%.
- Step 3 Dissolve the above intermediate a7-3 (19g, 63.1mmol) in 240mL of a mixed solution of ethanol and water (v/v, 6/1), and add potassium cyanide (4.49g, 69mmol). React under reflux condition for 1 hour, stop the reaction. The organic solvent was evaporated under reduced pressure, the reaction solution was poured into saturated sodium carbonate solution, stirred for 20 minutes, extracted with dichloromethane, concentrated, and separated by column chromatography to obtain intermediate a7-4 (10.4g, 44.8mmol). Yield: 71%.
- LC-MS: [M+H] + 233.
- Step 4 Under ice bath, dissolve the intermediate a7-4 (10.4g, 44.8mmol) in 80mL DMF, slowly add potassium tert-butoxide (5.3g, 47.2mmol), and stir for 20 minutes, the solution turns red , DMF solution (6.2 g, 47.2 mmol, 5 mL) of ethyl isothiocyanatoformate (6.2 g, 47.2 mmol, 5 mL) prepared in advance was slowly added dropwise. After continuing to stir the reaction liquid for 1 hour, the temperature was raised to 100° C. for 30 minutes to react.
- Step 5 The above intermediate a7-5 (6.7g, 19.5mmol) was dissolved in 30mL DMSO, and 30mL aqueous sodium hydroxide solution (5M) was added. React under reflux conditions for 4 hours, stop the reaction. After cooling to room temperature, 100 mL of ice water was slowly added to the reaction solution to quench the reaction, and the filter cake was obtained by suction filtration, washed with water, and dried in a vacuum oven to obtain the crude intermediate a7-6 (3.8 g).
- LC-MS: [M+H] + 272.
- Step 6 Dissolve the above crude product a7-6 (3.8g) and DMAP (122mg, 1mmol) in a mixed solution of 30mL THF/DMF (v/v, 1/1), add Boc anhydride (4.3g, 19.5mmol ). React at room temperature for 12 hours, then stop the reaction. Add 80 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude intermediate a7-7 (3.3 g).
- Step 7 Under the protection of nitrogen, the crude product a7-7 (3.3g) and raw material a7-8 (6.01g, 26.7mmol) were dissolved in 30mL 1,4-dioxane, and potassium acetate (2.62g, 26.7 mmol) and Pd(DPEphos)Cl 2 (643mg, 0.9mmol), the temperature was raised to 100°C for 1 hour, and the reaction was stopped. Cool to room temperature, filter, wash with saturated brine, extract with dichloromethane, dry, concentrate, and separate by flash column chromatography to obtain intermediate a7 (2.5g, 6.2mmol).
- LC-MS: [M+H] + 405.
- Step 1 Dissolve starting material 3-methoxy-2,2-dimethyl-3-oxalonic acid a15-1 (1.0g, 6.8mmol) and starting material a5-1 (850mg, 6.8mmol) in 20mL without Add EDCI (1.56g, 8.2mmol), N,N-diisopropylethylamine (1.77g, 13.6mmol) and HOBt (1.1g, 8.2mmol) to water dichloromethane, react at room temperature for 16h, stop reaction. Add 60 mL of ice water to the reaction solution, extract with dichloromethane, and dry over anhydrous sodium sulfate. The mixture was separated by Flash column chromatography to obtain intermediate a15-2 (1.04g, 4.6mmol) with a yield of 68%.
- LC-MS: [M+H] + 218.
- Step 2 At -78°C, the intermediate a15-2 (1.04g, 4.6mmol) was dissolved in 15mL of anhydrous tetrahydrofuran, and lithium aluminum hydride (350mg, 9.2mmol) was added slowly. The temperature was raised to 0°C for 1 hour, and the reaction was stopped. Slowly add 2 mL of 10% NaOH aqueous solution to the reaction liquid, precipitate flocs, filter with suction, concentrate the filtrate under reduced pressure, and separate by flash column chromatography to obtain intermediate a15 (700 mg, 4.0 mmol). Yield: 87%.
- LC-MS: [M+H] + 176.
- Step 1 Add raw material 3,3-difluorocyclobutane-1-amine a24-1 (1.0g, 9.34mmol) and intermediate a19-2 (3.16g, 9.34mmol) in a 100mL reaction flask, 40mL anhydrous THF dissolves. After stirring for 5 minutes, NaBH(OAc) 3 (2.57g, 12.34mmol) and 10 drops of acetic acid were added to the reaction solution, reacted at room temperature for 10 hours, and the reaction was stopped. The reaction solution was poured into 200mL of ice water and extracted with ethyl acetate.
- Step 2 Add intermediate a24-2 (2.62g, 5.91mmol) and aqueous formaldehyde (0.26mL) into a 100mL reaction flask, and dissolve in 40mL THF. After stirring for 5 minutes, NaBH(OAc) 3 (1.63g, 7.68mmol) and 10 drops of acetic acid were added to the reaction solution, reacted at room temperature for 2 hours, and the reaction was stopped. The reaction solution was poured into 100mL of ice water and extracted with ethyl acetate.
- Step 3 The above intermediate a24-3 (2.0 g, 4.51 mmol) was dissolved in 20 mL of tetrahydrofuran, and tetrabutylammonium fluoride (2.2 g, 9.0 mmol) was added. The reaction was carried out at room temperature for 12 hours, and the reaction was complete as monitored by TLC.
- Step 2 Intermediate a25-2 (1.21g, 4.13mmol) was dissolved in 25mL of anhydrous THF under ice bath, and lithium aluminum hydride (310mg, 8.3mmol) was added slowly. The temperature was raised to room temperature for 2 hours, and the reaction was stopped. Slowly add 2 mL of 10% NaOH aqueous solution to the reaction liquid, precipitate flocs, filter with suction, concentrate the filtrate under reduced pressure, and separate by flash column chromatography to obtain intermediate a25 (180 mg, 0.76 mmol). Yield: 18%.
- LC-MS: [M+H] + 238.
- Step 1 Add oxetane-3,3-diyldimethanol a26-1 (4.1g, 33.9mmol) and triethylamine (4.11g, 40.63mmol) in a 50mL reaction flask, 40mL of anhydrous dichloro Methane dissolves.
- tert-butyldiphenylchlorosilane (7.18g, 33.86mmol) was added to the reaction liquid, reacted at room temperature for 2 hours, stopped the reaction, poured the reaction liquid into 200mL ice water, extracted with dichloromethane, organic The phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 12 g of crude intermediate a26-2.
- Step 2 At -10°C, dissolve the crude intermediate a26-2 (12.0 g) from the previous step in 40 mL of tetrahydrofuran, add pyridine sulfur trioxide (12.8 g, 81.0 mmol), and react under ice cooling for 3 hours to stop the reaction.
- the reaction solution was poured into 100 mL of ice water, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain intermediate a26-3 (8.0 g, 22.7 mmol), two Step yield: 67%.
- Step 1 Dissolve intermediate a1 (3.0g, 9.15mmol) and raw material 4,7-diazaspiro[2.5]octane-4-carboxylate tert-butyl a27-1 (2.12g, 10.1mmol) at room temperature
- N,N-diisopropylethylamine (2.3 g, 17.4 mmol) was added, and reacted at room temperature for 8 hours.
- LC-MS: [M+H] + 505.
- Step 2 Under the protection of nitrogen, the intermediate a27-2 (3.74g, 7.4mmol) was dissolved in 75mL of anhydrous DMF, CsF (3.4g, 22.2mmol) was added, the temperature was raised to 60°C for 8h, and the reaction was stopped. The reaction solution was added to 300 mL of water, the mixture was extracted three times with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate a27 with a yield of 90%.
- LC-MS: [M+H] + 489.
- Step 1 Dissolve 2-fluoro-3-methyl-4-bromopyridine a29-1 (4.5g, 23.9mmol) in 25mL carbon tetrachloride, slowly add N-bromosuccinimide NBS (6.35g , 35.8mmol) and azobisisobutyronitrile AIBN (390mg, 2.3mmol), reacted at room temperature for 3 hours, and stopped the reaction.
- the reaction solution was slowly poured into 150 mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (PE/EtOAc, 5/1) to obtain oil a29-2 (6.1 g, 22.9 mmol). Yield: 94%.
- Step 2 Intermediate a29-2 (5.2g, 3.7mmol) and trimethylcyanosilane TMSCN (2.9g, 5.6mmol) were dissolved in 20mL of acetonitrile, and a solution of tetrahydrofuran (29mL) containing TBAF (5.5mmol) was added , reacted at room temperature for 16 hours, and stopped the reaction. The solvent was evaporated under reduced pressure and separated by column chromatography (PE/EtOAc, 5/1) to obtain oil a29-3 (3.4g, 15.9mmol). Yield: 81%.
- Step 3 Under ice bath, dissolve the above intermediate a29-3 (3.4g, 15.9mmol) in 20mL DMF, slowly add NaH (2.9g, 19.1mmol), stir for 30 minutes, the solution turns red, slowly gradually A DMF solution of ethyl isothiocyanate (1.8 g, 15.9 mmol, 5 mL) prepared in advance was added dropwise. The reaction solution was heated up to 100° C. to react for 1 hour, and then cooled to room temperature.
- Step 4 Dissolve the intermediate a29-4 (1.0 g, 3.1 mmol) in 10 mL of DMSO, and add 10 mL of aqueous sodium hydroxide solution (5M). React under reflux conditions for 4 hours, stop the reaction. After cooling to room temperature, 100 mL of ice water was slowly added to the reaction solution to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was separated by column chromatography (PE/EtOAc, 1/1) to obtain a light yellow solid a29- 5 (450 mg, 1.8 mmol). Yield: 20%.
- LC-MS: [M+H] + 254.
- Step 5 Dissolve the intermediate a29-5 (450mg, 1.8mmol) and DMAP (5mg, 0.04mmol) in 20mL THF/DMF mixed solution (v/v, 1/1), add Boc anhydride (465mg ,2.16mmol). React at room temperature for 12 hours, then stop the reaction. Add 50 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain crude intermediate a29-6 (760 mg).
- Step 1 Under the protection of nitrogen, dissolve the raw material a31-1 (10.0g, 40.5mmol) in 100mL of anhydrous tetrahydrofuran, slowly add LiAlH 4 (2.3g, 60.7mmol), react at room temperature for 2 hours, and monitor the reaction by LC-MS Completely, stop the reaction. Add 300mL of ice water to the system, distill off the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain light yellow oil a31-2 (5.9g, 26.9mmol) , Yield: 67%.
- LC-MS: [M+H]+ 220.
- Step 2 In ice bath, under the protection of nitrogen, dissolve the intermediate a31-2 (5.9g, 26.9mmol) and imidazole (3.66g, 53.8mmol) in 50mL of anhydrous dichloromethane, and slowly add tert-butyl Diphenylchlorosilane TBDPSCl (7.4 g, 26.92 mmol). The reaction solution was stirred at room temperature for 30 minutes, and the reaction was stopped by monitoring the completion of the reaction by LC-MS.
- Step 3 Dissolve the intermediate a31-3 (11.4g, 24.93mmol) in the 1,4-dioxane solution of hydrogen chloride (4M, 30mL), stir at room temperature for 1 hour, stop the reaction, and filter with suction. The filter cake was dried to obtain hydrochloride a31-4 (7.0 g, 17.8 mmol), yield: 71%.
- LC-MS: [M+H] + 358.
- Step 5 Dissolve the intermediate a31-6 (7.78g, 15.2mmol) and tetrabutylammonium fluoride TBAF (7.96g, 30.44mmol) in 80mL tetrahydrofuran, raise the temperature to 50°C for 6 hours, and stop the reaction . Add 200 mL of water to the system, distill off the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography to obtain brown oil a31-7 (2.3 g, 8.42 mmol), yield : 55%.
- LC-MS: [M+H] + 274.
- Step 1 In ice bath, the starting materials 2-nitro-4-bromo-5-hydroxy-benzoic acid methyl ester a32-1 (25g, 90.6mmol) and triethylamine (27.5g, 272mmol) were dissolved in 250mL of dichloromethane In, acetyl chloride (10.66g, 135.9mmol) was slowly added dropwise. After dropping, the reaction was continued for 3 hours, and the reaction was stopped. Add 500 mL of water to the system, extract with dichloromethane, and distill off the solvent under reduced pressure to obtain the crude intermediate a32-2.
- Step 2 Dissolve the crude product a32-2 and reduced iron powder (27.2g, 487.3mmol) in 310mL of glacial acetic acid, raise the temperature to 40°C for 2 hours, and stop the reaction.
- the crude product was filtered through celite, and the filtrate was concentrated under reduced pressure.
- LC-MS: [M+H] + 290.
- Step 3 Mix the crude product a32-3 and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane di(tetrafluoroborate) salt (selectfluor, 30.8g, 87.0 mmol) was dissolved in 170 mL of acetonitrile, reacted at room temperature for 15 hours, and stopped the reaction. Add saturated aqueous sodium bicarbonate solution to the reaction solution to adjust the pH to about 8, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound a32-4 (1.6g, 5.2mmol), three Step yield: 6%.
- LC-MS: [M+H] + 306.
- Step 4 Dissolve the above intermediate a32-4 (1.6g, 5.2mmol) and potassium carbonate (1.44g, 10.5mmol) in 16mL of methanol, react at room temperature for 2 hours, and stop the reaction.
- the pH of the reaction solution was adjusted to about 5 with 1M dilute hydrochloric acid aqueous solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to obtain compound a32-5 (1.2 g, 4.54 mmol), yield 87 %.
- LC-MS: [M+H] + 264.
- Step 5 In ice bath, dissolve the intermediate a32-5 (1.2g, 4.54mmol) and cesium carbonate (2.96g, 9.1mmol) in 12mL DMF, stir for 5 minutes, slowly add methyl iodide (710mg, 5.0mmol), after dropping, the reaction was continued for 2 hours, and the reaction was stopped. 50 mL of ice water was added to the reaction solution, extracted with ethyl acetate, washed with saturated brine, concentrated under reduced pressure, and the crude product was separated by column chromatography to obtain compound a32-6 (1.1 g, 3.96 mmol), with a yield of 87%.
- LC-MS: [M+H] + 278.
- Step 8 Dissolve the intermediate a32-8 (1.1 g, 3.81 mmol) and DIEA (984 mg, 7.62 mmol) in 11 mL of phosphorus oxychloride, raise the temperature to 90°C for 15 hours, and stop the reaction.
- the reaction solution was concentrated under reduced pressure, and the crude product was slowly poured into 100 mL of ice water to precipitate a solid, which was filtered with suction, and the filter cake was washed with water and dried to obtain intermediate 11 (1.2 g, 3.68 mmol). Yield 96.6%.
- LC-MS: [M+H] + 325.
- Step 2 Dissolve the above intermediate b1-2 (1.0g, 4.9mmol), palladium acetate (55mg, 0.24mmol) and NBS (0.87g, 4.87mmol) in 10mL of anhydrous acetic acid, and heat the reaction solution to 80°C The reaction was stopped for 1 hour, cooled to room temperature, the reaction liquid was poured into water, filtered, and the filter cake was dried to obtain a brown solid b1-3 (1.03 g, 3.6 mmol).
- LC-MS: [M+H] + 284.
- Step 3 Dissolve the intermediate b1-3 (12.5g, 43.99mmol) in the above step in a mixed solution of 60mL concentrated hydrochloric acid and 100mL 1,4-dioxane.
- the reaction solution was heated to reflux and stirred for 1 hour, then the reaction was stopped, and concentrated under reduced pressure.
- LC-MS: [M+H] + 255.
- Step 4 Under nitrogen protection, the intermediate b1-4 (7.90g, 30.97mmol) and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoro Borate) salt (Selectfluor, 16.46g, 46.5mmol) was dissolved in 80mL of methanol, and 0.3mL of concentrated sulfuric acid was slowly added dropwise. The reaction solution was heated to 50° C. for 5 hours, then the reaction was stopped, and concentrated under reduced pressure.
- Step 7 In ice bath, under the protection of nitrogen, the intermediate b1-7 (21.0 g, 77.5 mmol) and pyridine (18.38 g, 232.41 mmol) were dissolved in 200 mL of dichloromethane. Trifluoromethanesulfonic anhydride (26.2 g, 92.96 mmol) was slowly added dropwise to the reaction solution, and the mixture was slowly raised to room temperature for 1 hour to stop the reaction, and the solvent was evaporated under reduced pressure.
- LC-MS: [M+H]+ 314.
- Step 1 Dissolve the raw material 4-fluorophenylacetic acid b2-1 (50.0g, 324.4mmol) and cyclo(ethylene)isopropyl malonate b2-2 (51.4g, 356.8mmol) in 500mL of acetonitrile, add 4- Dimethylaminopyridine (DMAP, 3.57 g, 29.2 mmol) and DIEA (88.0 g, 681.2 mmol). After stirring for 5 minutes, pivaloyl chloride (43.0 g, 356.8 mmol) was slowly added dropwise. The reaction solution was heated to 45°C and stirred for 3 hours, then cooled to room temperature.
- DMAP Dimethylaminopyridine
- DIEA 88.0 g, 681.2 mmol
- Step 2 Slowly add intermediate b2-3 (54.0 g, 192.7 mmol) in the above step into trifluoromethanesulfonic acid (228.5 g, 1.5 mol).
- the reaction solution was stirred at room temperature for 2 hours, and the reaction was complete as monitored by LC-MS.
- Step 3 Dissolve the intermediate b2-4 (66.0g, 295.96mmol) in the above step in a mixed solution of 660mL acetonitrile and water (v/1, 1/1), raise the temperature to 80°C for 13 hours, and stop the reaction. The solvent was evaporated under reduced pressure, washed with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain pale yellow compound b2-5 (51.8 g, 291.01 mmol), yield: 97%.
- LC-MS: [M+H] + 179.
- Step 6 Under nitrogen protection, at -40°C, the crude product b2-7 (58.4g, 113.43mmol) and DIEA (51.3g, 397.0mmol) were dissolved in 300mL of dichloromethane, and trifluoromethanesulfonic anhydride (54.4 g, 192.8mmol), after 3 hours of dripping, the stirring was continued for 0.5 hour, and the reaction was stopped. The reaction solution was poured into 500 mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated.
- Step 7 Under nitrogen protection, the intermediate b2-8 (61.6g, 95.2mmol), triethylamine (38.5g, 380.9mmol) and pinacol borane (48.7g, 380.9mmol) were dissolved in 600mL of acetonitrile, After stirring for 5 minutes, the catalyst Pd(dppf) Cl2 (4.2 g, 5.7 mmol) was added. The reaction solution was heated to 80°C and stirred for 4 hours, then cooled to room temperature. The mixture was slowly quenched with MeOH, keeping the temperature below 25 °C, and a solid precipitated out. After suction filtration, the filter cake was washed with MeOH and dried to obtain compound b2 (45.9 g, 73.4 mmol) as a white solid, yield: 77%.
- LC-MS: [M+H] + 625.
- LC-MS: [M+H] + 543.
- Step 2 Dissolve the intermediate b3-2 (7.6g, 14mmol) and DIEA (1.8g, 14.0mmol) in 80mL of acetonitrile, slowly add diethyl phosphite (1.95g, 14.0mmol), and react at room temperature 1 hour, stop the reaction.
- LC-MS: [M+H] + 465.
- LC-MS: [M+H] + 412.
- Step 4 In ice bath, under the protection of nitrogen, dissolve the intermediate b3-4 (3.6g, 8.76mmol) of the previous step in 40mL of anhydrous tetrahydrofuran, add NaH (700mg, 17.5mmol), stir for 10 minutes, and then add dropwise 1,2-Dibromoethane (2.0g, 10.5mmol), after dropping, warmed up to room temperature and reacted for 10 hours, then stopped the reaction.
- NaH 700mg, 17.5mmol
- 1,2-Dibromoethane 2.0g, 10.5mmol
- Step 1 Dissolve the raw material 4-bromo-7-fluoro-1H-indole (3.5g, 16.4mmol) in 35mL DMF, add N-iodosuccinimide NIS (3.69g, 16.4mmol), and heat up to The reaction was carried out at 60° C. for 1 hour, and then the reaction was terminated.
- Step 2 In ice bath, under the protection of nitrogen, dissolve the intermediate 3-iodo-4-bromo-7-fluoro-1H-indole b4-2 (2.0g, 5.9mmol) in 20mL of anhydrous tetrahydrofuran, add NaH (235mg, 8.9mmol) and iodomethane (92mg, 6.5mmol) were heated to room temperature for 2 hours and the reaction was stopped.
- Step 4 Under the protection of nitrogen, the intermediate b4-4 (350mg, 1.4mmol), biboronic acid pinacol ester (706mg, 2.8mmol) and potassium acetate (408mg, 4.2mmol) were dissolved in 10mL of 1,4- Add catalyst Pd(dppf)Cl 2 (100mg, 0.14mmol) to dioxane, heat up to 100°C and react for 8 hours, then cool to room temperature.
- Pd(dppf)Cl 2 100mg, 0.14mmol
- Step 1 Dissolve raw material b7-1 (1.0g, 5.9mmol) in 10mL DMF, add N-iodosuccinimide NIS (1.99g, 8.85mmol), heat up to 70°C for 1 hour, stop the reaction .
- LC-MS: [M+H] + 296.
- Step 2 In ice bath, under the protection of nitrogen, the intermediate b7-2 (800mg, 2.70mmol), anhydrous methanol (430mg, 13.6mmol) and triphenylphosphine (1.07g, 4.1mmol) were dissolved in 20mL DMF , diethyl azodicarboxylate (710 mg, 4.1 mmol) was added to the reaction solution, and the temperature was raised to room temperature for 12 hours to stop the reaction.
- LC-MS: [M+H] + 356.
- LC-MS: [M+H] + 283.
- Step 3 The above intermediate b8-3 (175mg, 0.62mmol), triethylamine (190mg, 1.86mmol), 4-dimethylaminopyridine DMAP (15mg, 0.12mmol) and di-tert-butyl dicarbonate (160mg , 0.74mmol) was dissolved in 20mL of dichloromethane, reacted at room temperature for 4 hours, and stopped the reaction. Add 80 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude intermediate b8-4.
- LC-MS: [M+H] + 383.
- Step 7 Under nitrogen protection, the crude product b8-4 (100mg, 0.26mmol), potassium acetate (77mg, 0.78mmol) and biboronic acid pinacol ester (79mg, 0.31mmol) were dissolved in 7mL 1,4-dioxane
- LC-MS: [M+H] + 431.
- Step 1 The starting material 2-amino-3-cyano-4-bromoindole b9-1 (3.8g, 16.10mmol) and 4-dimethylaminopyridine DMAP (196mg, 1.61mmol) were dissolved in 50mL THF, slowly Add di-tert-butyl dicarbonate (14.1 g, 64.4 mmol), react at room temperature for 10 hours, and stop the reaction. Add 200 mL of ice water to the reaction liquid, extract with ethyl acetate, and concentrate to obtain the crude product b9-2.
- LC-MS: [M+H] + 436.
- Step 1 Under the protection of nitrogen, the compound 4-bromo-benzofuran-2-carboxylic acid b10-1 (5.5g, 23mmol) and triethylamine (6.0mL) were dissolved in 50mL tert-butanol, and diphosphoric acid diazide was added Phenyl ester DPPA (9.4g, 7.4mL) was stirred for 10 minutes and then heated to 95°C for 3 hours to stop the reaction. The solvent was evaporated under reduced pressure, the crude product was dissolved in 50 mL of dichloromethane, di-tert-butyl dicarbonate (5.0 g, 23 mmol) was added, and the reaction was carried out at room temperature for 2 hours to stop the reaction.
- Phenyl ester DPPA 9.4g, 7.4mL
- Step 2 Dissolve the intermediate b10-2 (1.0g, 3.2mmol) in 10mL of anhydrous tetrahydrofuran at -70°C under the protection of nitrogen, add the raw material chlorosulfonic acid isocyanate (0.42mL), and stir for 1 hour. Chlorosulfonic acid isocyanate (0.42 mL) was added again, the reaction was continued for 1 hour, and the temperature was raised to room temperature. 10 mL of anhydrous DMF was added to the reaction solution, and after stirring for 1 hour, the reaction was stopped.
- Step 3 Under the protection of nitrogen, the intermediate b10-3 (0.5g, 1.5mmol), potassium acetate (440mg, 4.5mmol) and biboronic acid pinacol ester (460mg, 1.8mmol) were dissolved in 10mL 1,4 - Add Pd(dppf) 2 Cl 2 (11mg, 0.15mmol) to dioxane, heat up to 100°C and react for 2 hours, stop the reaction, and filter. Add 50 mL of ice water to the reaction liquid, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound b10 (270 mg, 0.7 mmol), with a yield of 46%.
- LCMS: [M+1] + 385.
- Step 2 Dissolve the crude product c1-2 (25g, 77.3mmol) in 300mL DMF, add sodium methylthiolate (6.5g, 92.8mmol) at room temperature, raise the temperature to 90°C and stir for 16 hours to stop the reaction.
- the reaction solution was poured into 500 mL ice water, extracted with ethyl acetate, washed with saturated brine, concentrated, and the crude product was separated by column chromatography to obtain compound c1-3 (10 g, 36.4 mmol), with a yield of 47%.
- LC-MS: [M+H] + 275.
- Step 3 Dissolve the intermediate c1-3 (16.5g, 51.0mmol) in 200mL of anhydrous tetrahydrofuran at -78°C under the protection of nitrogen, slowly add LDA (12.8g, 76.6mmol) dropwise, and continue stirring for 0.5 Hour.
- Step 6 In an ice bath, dissolve the crude product c1-7 (2.1 g, 9.8 mmol) in 20 mL of tetrahydrofuran, add lithium aluminum hydride (750 mg, 19.5 mmol), and continue stirring for 1 hour to stop the reaction. Add 10 mL of methanol to the system to quench, filter, concentrate under reduced pressure, add 50 mL of water to the mixture, extract with dichloromethane, dry over anhydrous sodium sulfate, and concentrate to obtain crude product c1 (directly used in the next reaction).
- LC-MS: [M+H] + 188.
- Step 7 Substitution of intermediate c1-5 by c1-6 yields intermediate c2.
- Step 2 In an ice bath, the intermediate c9-2 (12.0 g, 56.3 mmol) and triethylamine (17.3 g, 170.5 mmol) were dissolved in 120 mL of dichloromethane. After stirring for 5 minutes, a dichloromethane solution (20 mL) of methanesulfonic anhydride (14.9 g, 85.2 mmol) was added dropwise to the reaction liquid, and the stirring was continued for 1 hour after the dropping was completed. Stop the reaction, add 200 mL of ice water to the system, extract with dichloromethane, and concentrate under reduced pressure to obtain the crude product to c9-3.
- Step 3 The crude product c9-3 and potassium thioacetate (9.4 g, 82.4 mmol) were dissolved in 150 mL of DMF, and the temperature was raised to 60° C. for 15 hours to stop the reaction. Add 300mL ice water to the system, extract 3 times with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, the crude product is separated by column chromatography to obtain intermediate c9-4 (15g, 55.4 mmol), two-step yield: 10%.
- LC-MS: [M+H] + 272.
- Step 4 In ice bath, the intermediate c9-4 (15g, 55.4mmol) of the previous step was dissolved in 300mL of anhydrous tetrahydrofuran, and lithium aluminum tetrahydride (5.2g, 138mmol) was added. After stirring for 5 minutes, the ice bath was removed, and the temperature was raised to 60° C. to react for 2 hours. Stop responding. Dilute hydrochloric acid was added to the system to quench the reaction, and the pH was adjusted to about 7. A solid was precipitated, filtered with suction, and the filter cake was washed with ethyl acetate to obtain intermediate c9.
- LC-MS: [M+H] + 174.
- LC-MS: [M+H] + 214.
- Step 1 In an ice bath, in a 250mL reaction flask, dissolve the raw material d1-1 (5.0g, 18.6mmol) in 80mL of pyridine, slowly add 20mL monoethyl oxalyl chloride dropwise to the system, after the drop is complete, stir at room temperature After 1 hour, the temperature was raised to 50° C. for 2 hours to stop the reaction. The reaction solution was slowly poured into 300 mL of ice water, suction filtered, and the filter cake was dried to obtain intermediate d1-2 (5.2 g, 14.8 mmol), with a yield of 80%.
- LC-MS: [M+H] + 350.
- Step 2 Dissolve the above intermediate d1-2 (5.2g, 14.8mmol) in 100mL of absolute ethanol, add 4.0g of ammonium acetate and 1mL of acetic acid. Raise the temperature to reflux for 1 hour, and cool to room temperature. Slowly add 1M dilute hydrochloric acid dropwise to the system to adjust the pH to about 5, a solid precipitates out, is suction filtered, and the filter cake is dried to obtain the crude product d1-3.
- LC-MS: [M+H] + 349.
- Step 3 Dissolve the crude product d1-3 and DIEA (2.2 g, 17.2 mmol) in 20 mL of phosphorus oxychloride, raise the temperature to 85° C. for 2 hours, and stop the reaction. The solvent was evaporated under reduced pressure, the crude product was slowly poured into 100 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain compound d1-4 (2.5 g, 6.8 mmol). rate 46%.
- LC-MS: [M+H] + 367.
- Step 4 In an ice bath, dissolve the intermediate d1-4 (2.5g, 6.8mmol) and DIEA (1.75g, 13.6mmol) in 20mL of anhydrous tetrahydrofuran, and a2-1 (1.44g, 6.8 mmol) THF solution (3.0 mL) was slowly added dropwise to the reaction solution, after the drop was completed, the temperature was raised to room temperature and reacted for 40 minutes, and the reaction was stopped. 100 mL of water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain compound d1 (3.5 g, 6.4 mmol), with a yield of 94%.
- LC-MS: [M+H] + 543.
- Step 3 Dissolve P1-3 (120 mg, 0.15 mmol) in 4 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling.
- the reaction solution was placed under nitrogen protection to continue the reaction for 1 hour, and then the reaction was stopped.
- the solvent was distilled off under reduced pressure, and the residue was purified by preparative HPLC chromatography to obtain the target compound P1 (10.2 mg).
- LC-MS: [M+H] + 607.
- Step 3 Dissolve H1-2 (80 mg, 0.10 mmol) in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 0.5 hours, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative SFC (Xselect CSH C18 OBD) to give target compounds H1a (4.0 mg) and H1b (4.1 mg).
- Step 1 Dissolve intermediate a5 (420 mg, 2.42 mmol) in 10 mL of anhydrous THF in a 50 mL reaction flask, add potassium tert-butoxide (340 mg, 3.64 mmol), and stir at room temperature for 30 minutes to obtain solution S1.
- the intermediate a14 1.0g, 2.0mmol was dissolved in 10mL of anhydrous THF, and the prepared solution S1 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction.
- the reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Step 1 Dissolve intermediate a5 (170 mg, 0.94 mmol) in 10 mL of anhydrous THF in a 50 mL reaction flask, add potassium tert-butoxide (180 mg, 1.56 mmol), and stir at room temperature for 30 minutes to obtain solution S2.
- the intermediate a17 400mg, 0.78mmol was dissolved in 10mL of anhydrous THF, and the prepared solution S2 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction.
- the reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Embodiment 6 is a diagrammatic representation of Embodiment 6
- Step 1 In a 50 mL reaction flask, the intermediate a19 (150 mg, 0.55 mmol) was dissolved in 5 mL of anhydrous THF, potassium tert-butoxide (93 mg, 0.82 mmol) was added, and stirred at room temperature for 30 minutes to obtain solution S3.
- the intermediate a14 In another 50mL reaction flask, the intermediate a14 (280mg, 0.58mmol) was dissolved in 5mL of anhydrous THF, and the prepared solution S3 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Embodiment 7 is a diagrammatic representation of Embodiment 7:
- Embodiment 8 is a diagrammatic representation of Embodiment 8
- Embodiment 9 is a diagrammatic representation of Embodiment 9:
- Step 1 Dissolve intermediate a5 (142mg, 0.82mmol) in 5mL anhydrous THF in a 20mL reaction flask, add potassium tert-butoxide (138mg, 2.34mmol), and stir at room temperature for 30 minutes to obtain solution S4.
- the intermediate a27 400mg, 0.82mmol was dissolved in 5mL of anhydrous THF, and the prepared solution S4 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction.
- the reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Step 2 Under nitrogen protection, 2,2-diethoxyethanol (33.8g, 252mmol) was dissolved in 450mL of anhydrous DMF, NaH (10.08g, 252mmol) was slowly added at 0°C, and after stirring for 1 hour, The intermediate P14-2 (45 g, 210 mmol) of the previous step was added. The ice bath was removed, and the temperature was raised to 50° C. to react for 2 hours. The reaction was stopped, cooled to room temperature, 1 L of water was added to the system, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Step 3 Add 100 mL of toluene to polyphosphoric acid (10.42 g), raise the temperature to 100°C, add the intermediate P14-3 (10.0 g, 30.4 mmol) in the previous step, continue to react at this temperature for 2 hours, and stop the reaction.
- the reaction solution was slowly poured into a large amount of ice water, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether/ethyl acetate, 10/1) to obtain yellow solid P14-4 (1.04 g, 4.4 mmol), yield: 14%.
- LC-MS: [M+H] + 236.
- Step 4 Dissolve the intermediate P14-4 (8.3g, 35.0mmol) in 150mL of ethanol, add KOH aqueous solution (7.94g, 50mL), heat up to 90°C for 4 hours, and stop the reaction.
- the organic solvent was evaporated under reduced pressure, 100 mL of ice water was added to the reaction solution, extracted with dichloromethane, and concentrated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether/dichloromethane, 2/1) to obtain yellow solid P14-5 (4.0 g, 15.7 mmol), yield: 45%.
- LC-MS: [M+H] + 256.
- Step 5 Under the protection of nitrogen, the intermediate P14-5 (3.3g, 13.0mmol) in the previous step was dissolved in 33mL of anhydrous THF, and a tetrahydrofuran solution containing triphosgene (3.6g, 12.4mmol) was slowly added dropwise at 0°C (20mL). The temperature was raised to room temperature for 2 hours, and the reaction was stopped. 100 mL of water was added to the system, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure to obtain crude product P14-6 (2.8 g).
- LC-MS: [M+H] + 282.
- Step 7 At room temperature, intermediate P14-7 (1.0g, 3.15mmol) and raw material 3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl a2-1 (670mg, 3.15mmol) was dissolved in 20mL 1,4-dioxane, N,N-diisopropylethylamine (1.7mL, 9.5mmol) was added, and the reaction was carried out at 50°C for 2 hours. Add 60 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain white solid P14-8 (900 mg, 1.82 mmol). Yield: 58%.
- LC-MS: [M+H] + 495.
- Step 8 Under nitrogen protection, dissolve intermediate P14-8 (520mg, 1.05mmol) and cesium carbonate (684mg, 2.10mmol) in 5mL DMF, add intermediate a5 (363mg, 2.10mmol), and heat up to 140°C React for 2 hours. Cool to room temperature, add 60 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography (petroleum ether/ethyl acetate, 1/1) to obtain a yellow solid P14-9 (155 mg, 0.25 mmol). Yield: 23%.
- LC-MS: [M+H] + 630.
- Step 9 Under the protection of nitrogen, the intermediate P14-9 (155mg, 0.25mmol) and cesium carbonate (200mg, 0.62mmol) were dissolved in 5mL of toluene, and the intermediate a7 (139mg, 0.34mmol) and Pd(DPEPhos)Cl were added 2 (52mg, 0.074mmol), heated to 105°C and reacted for 3 hours.
- Step 10 Dissolve P14-10 (65 mg, 0.077 mmol) in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure.
- Step 1 In ice bath, dissolve intermediate c1 (330mg, 1.76mmol) in 1mL of anhydrous tetrahydrofuran, add NaH (85mg, 3.52mmol), warm to room temperature and stir for 0.5 hours, and the reaction solution is set aside.
- Intermediate a3 350mg, 0.82mmol was dissolved in 1mL of anhydrous tetrahydrofuran, added to the above reaction solution, reacted at room temperature for 1 hour, and stopped the reaction. Concentrated under reduced pressure, the crude product was separated by flash column chromatography to obtain compound P18-1 (240mg, 0.42mmol), with a yield of 24%.
- LC-MS: [M+H] + 579.
- Step 2 Under nitrogen protection, compound P18-1 (240mg, 0.42mmol), intermediate b2 (270mg, 0.44mmol), cesium carbonate (270mg, 0.83mmol) and methanesulfonic acid [n-butylbis(1-adamantine Alkyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium (Pd-G3, 10mg, 0.014mmol) was dissolved in a mixed solution of 2mL 1,4-dioxane and water (v/v, 5/1), the temperature was raised to 95° C. for 1 hour, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound P18-2 (300 mg, 0.29 mmol), with a yield of 70%.
- LC-MS: [M+H] + 1041.
- Step 3 Compound P18-2 (270 mg, 0.26 mmol) and cesium fluoride (80 mg, 0.52 mmol) were dissolved in 1 mL of DMF, heated to 50° C. for 1 hour, and the reaction was stopped. Add 5 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain crude product P18-3.
- LC-MS: [M+H] + 729.
- Step 4 Dissolve the crude product P18-3 from the previous step in 1 mL of hydrogen chloride in 1,4-dioxane solution (4M concentration), react at room temperature for 1 hour, and slowly add saturated aqueous sodium bicarbonate solution to the system to adjust the pH to 8 Left and right, extracted with ethyl acetate, concentrated under reduced pressure, and purified by TLC thin layer chromatography to obtain compound P18 (50 mg).
- LC-MS: [M+H] + 629.
- the compound P29-3 (450mg, 0.91mmol) and the raw material P9-1 (560mg, 1.1mmol) were dissolved in 10mL of 1,4-dioxane and 2mL of water, and the catalyst XPhos Pd G2 (215mg, 0.3 mmol) and cesium carbonate (1.18g, 3.64mmol).
- the reaction solution was reacted at 95° C. for 2 hours, and the reaction was stopped.
- Step 1 Intermediate d2 (1.4g, 1.85mmol) was dissolved in 10mL of tetrahydrofuran, and saturated lithium hydroxide aqueous solution (5.0mL) was slowly added dropwise, reacted at room temperature for 40 minutes, and stopped the reaction. Slowly add 1M dilute hydrochloric acid dropwise to the reaction to adjust the pH to about 5, extract with ethyl acetate, and concentrate to obtain the crude product M1-1, which is directly used in the next reaction.
- LC-MS: [M+H] + 727.
- Step 2 In an ice bath, dissolve the crude product M1-1 from the previous step in a mixed solution of trifluoroacetic acid and dichloromethane (4.0 mL, 1/1), react in an ice bath for 1 hour, and stop the reaction. The solvent was evaporated under reduced pressure, and the crude product was separated by preparative HPLC chromatography to obtain compound M1 (10 mg).
- LC-MS: [M+H] + 527.
- Step 1 In ice bath, compound M1-1 (400mg, 0.55mmol), raw material M2-1 (109mg, 0.83mmol) and DIEA (213mg, 1.65mmol) were dissolved in 10mL DMF, stirred for 5 minutes, then added HATU ( 315mg, 0.83mmol), react at room temperature for 2 hours. The reaction solution was poured into 150 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain a colorless oil M2-2 (393 mg, 0.47 mmol). Yield 85%.
- LC-MS: [M+H] + 837
- Step 2 Dissolve the intermediate M2-2 (393 mg, 0.47 mmol) in 4.0 mL of dichloromethane, add 1.0 mL of trifluoroacetic acid, react at room temperature for 1 hour, and stop the reaction. The solvent was evaporated under reduced pressure, the crude product was dissolved in 10 mL of water, the pH was adjusted to about 8 with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain compound M2 (89 mg , 0.14mmol), yield: 30%.
- LC-MS: [M+H] + 637.
- Step 1 Under nitrogen protection, in a 50 mL reaction flask, dissolve intermediate a31 (300 mg, 1.0 mmol) in 5 mL of anhydrous THF, add potassium tert-butoxide (224 mg, 2.0 mmol), and stir at room temperature for 30 minutes to obtain a solution S5.
- the intermediate a14 (488mg, 1.0mmol) was dissolved in 5mL of anhydrous THF, and the prepared solution S5 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction.
- the reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Step 1 In a 50 mL reaction flask, the intermediate a18 (176 mg, 0.94 mmol) was dissolved in 10 mL of anhydrous THF, potassium tert-butoxide (180 mg, 1.56 mmol) was added, and stirred at room temperature for 30 minutes to obtain solution S6. In another 50mL reaction flask, the intermediate a17 (400mg, 0.78mmol) was dissolved in 10mL of anhydrous THF, and the prepared solution S2 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure.
- Step 1 -25°C, under the protection of nitrogen, the compound H10-1 (1.1g, 1.68mmol) obtained by referring to the route of Example 4 was dissolved in 20mL of anhydrous tetrahydrofuran, and isopropylmagnesium bromide (5.0mL, 5.0 mmol), after dropping, isopropanol pinacol borate (1.56 g, 8.4 mmol) was added to the reaction liquid, reacted at -25°C for 2 hours, and stopped the reaction.
- isopropanol pinacol borate (1.56 g, 8.4 mmol
- LC-MS: [M+H] + 835.
- Step 1 -25°C, under the protection of nitrogen, refer to the compound H10-1 (100mg, 0.15mmol), hexamethylditin (74mg, 0.22mmol), tricyclohexylphosphine (8.4mg, 0.03 mmol) and lithium chloride (19mg, 0.45mmol) were dissolved in 10mL 1,4-dioxane, the catalyst Pd 2 dba 3 (27mg, 0.03mmol) was added, the temperature was raised to 95°C for 12 hours, and the reaction was stopped.
- Step 3 Dissolve compound H32-2 (20 mg, 0.03 mmol) in 1 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) in an ice bath, react at room temperature for 1 hour, and stop the reaction , slowly add saturated sodium bicarbonate solution to the system and adjust the pH to about 8, extract with ethyl acetate, and concentrate under reduced pressure. The residue was separated by flash column chromatography to obtain H32 (2.1 mg) as a yellow solid.
- LC-MS: [M+H] + 650.
- Step 1 -60°C, under the protection of nitrogen, the compound H24-1 (1.0g, 1.34mmol) obtained in the route of Example 18 was dissolved in 20mL of anhydrous tetrahydrofuran, and isopropylmagnesium bromide (1.3mL, 1.3 mmol), after stirring for 30 minutes, anhydrous DMF (196 mg, 2.38 mmol) was added to the reaction solution, and after stirring for 30 minutes again, the reaction was stopped. Add 20 mL of saturated aqueous ammonium chloride solution to the mixture to quench the reaction, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate to obtain crude product H33-1.
- LC-MS: [M+H] + 650.
- LC-MS: [M+H] + 652.
- Step 1 In ice bath, dissolve raw material H40-1 (600mg, 2.54mmol) and potassium tert-butoxide (404mg, 3.6mmol) in 5mL THF, and stir for 10 minutes; pour the above mixture into intermediate a14 (882mg , 1.8mmol) in tetrahydrofuran (10mL). The mixture was continued to react for 1 hour under ice-bath condition, and the reaction was stopped. Add 50 mL of ice water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound H40-2 (500 mg, 0.71 mmol) with a yield of 39%.
- LC-MS: [M+H] + 705.
- Step 2 Under the protection of nitrogen, the above compound H40-2 (100mg, 0.14mmol), cesium carbonate (92mg, 0.28mmol) and intermediate a7 (80mg, 0.20mmol) were dissolved in 5mL of toluene, and the catalyst bis(di Phenylphosphine phenyl ether) palladium dichloride (10mg, 0.014mmol), heated to 105°C for 2 hours, stopped the reaction, and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound H40-3 (90 mg, 0.098 mmol), with a yield of 69%.
- LC-MS: [M+H] + 917.
- LC-MS: [M+H] + 767.
- Step 2 Dissolve the above compound M10-1 (38 mg) in a mixed solution of trifluoroacetic acid and dichloromethane (3.0 mL, 1/1), react in an ice bath for 1 hour, evaporate the solvent under reduced pressure, and the crude product is HPLC preparative chromatography gave compound M10 (8 mg).
- LC-MS: [M+H] + 567.
- Step 1 Under the protection of nitrogen, the raw materials 2-fluoro-5-methylaniline H44-1 (5.0g, 39.96mmol), potassium carbonate (13.8g, 99.90mmol) and potassium iodide (6.63g, 39.96mmol) were dissolved in 50mL N-methylpyrrolidone NMP was stirred for 5 minutes; 4-methoxybenzyl chloride (12.83 g, 81.92 mmol) was slowly added to the above mixture, and the mixture was heated to 65° C. for 1 hour to stop the reaction.
- Step 2 Dissolve 2,2,6,6-tetramethylpiperidine (1.55g, 11.0mmol) in 10mL of anhydrous tetrahydrofuran under nitrogen protection at -5°C, and slowly add n-butyllithium (4.38mL , 2.5M), after stirring for 10 minutes, cool down to -60°C; add compound H44-2 (1.0g, 2.74mmol) from the previous step, and stir for 30 minutes. Triisopropyl borate (620mg, 3.29mmol) was added to the reaction solution, and the reaction was continued for 30 minutes before stopping the reaction.
- Step 4 Dissolve the above compound H44-4 (190mg, 0.25mmol) and N-iodosuccinimide NIS (62mg, 0.28mmol) in 2mL of acetonitrile, add 2 drops of trifluoroacetic acid, and react at room temperature for 0.5 hours , stop responding.
- the reaction solution was poured into 20 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to obtain compound H44-5 (210 mg), yield: 95%.
- LC-MS: [M+H] + 900.
- Step 6 Under nitrogen protection, the intermediate a18 (78 mg, 0.42 mmol) was dissolved in 4 mL of anhydrous THF, potassium tert-butoxide (95 mg, 0.84 mmol) was added, and stirred at room temperature for 30 minutes to obtain a solution S0. In another reaction flask, compound H44-6 (350 mg, 0.42 mmol) was dissolved in 2 mL of anhydrous THF, and the prepared solution S0 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction.
- potassium tert-butoxide 95 mg, 0.84 mmol
- the compound is tested for the inhibition of p-ERK mediated by KRAS G12D (directly reflecting the inhibitory effect of the test compound on the cellular level). details as follows:
- AGS cells cultured in F-12K medium (Gibco, Cat.No.30-2004) containing 10% fetal calf serum and 1% penicillin were seeded on 384-well microplates at 37°C, 5% Incubate overnight under carbon dioxide conditions. 200 microliters of compounds at different concentrations (0.5% dimethyl sulfoxide final concentration) were added to each well and incubated at 37°C for 3 hours. Then, the cells were fixed in 8% fixative solution (Solarbio, Cat. No. P1112) and washed once with phosphate buffered saline (PBS). After washing, a blocking solution (LI-COR, Cat. No. 927-40000) was added to each well to block for 1 hour at room temperature.
- F-12K medium Gibco, Cat.No.30-2004
- Example 28 Inhibitory activity of compounds against GTP-KRAS
- KRAS-G12D were all from the commercially available kit KRAS-G12D/cRAF BINDING ASSAY KITS (Cisbio, Cat. No. 63ADK000CB21PEG); in the detection of KRAS-WT, GTP was purchased from Sigma (Cat. No.V900868), GST-cRAF was prepared by Beijing Pharmaron (Cat.No.20190718), MAb Anti GST-Tb cryptate was purchased from Cisbio (Cat.No.61GSTTLA), and other key reagents were from the commercially available kit KRAS- WT/SOS1 BINDING ASSAY KITS (Cisbio, Cat. No. 63ADK000CB15PEH).
- Relative ratio (relative ratio, RR) (Ratio 665/615 -Ratio background )
- Inhibition percentage [1-(RR compound -RR positive control well average value)/(RR negative control well average value-RR positive control well average value)] ⁇ 100
- IC 50 calculation: Y lower plateau signal+(upper plateau signal-lower plateau signal)/(1+10 ⁇ (LogIC 50 -X) ⁇ Hill slope).
- X logarithmic value of compound concentration; Y: percent inhibition.
- the compound to be tested (1 ⁇ M as the initial concentration, diluted 3 times, a total of 10 concentrations) was co-incubated with the cells for 7 days, and added to each well 3D reagent (Promega, catalog number G9683), read the luminescence value with Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104), the light signal is directly proportional to the ATP amount in the system, and the content of ATP directly characterizes the ATP in the system number of viable cells. Finally, XLFIT software was used to obtain the IC 50 (half inhibitory concentration) of the compound with a nonlinear fitting formula.
- Inhibition rate (%) 100 ⁇ (negative control average value-compound reading value)/(negative control average value-positive control average value)
- Negative control DMSO.
- Positive control culture medium.
- PANC-1 pancreatic cancer cells were cultured in a medium containing 10% fetal bovine serum (Ausgenex, catalog number FBS500-S) and 1% penicillin/streptomycin (Gibco, Cat. No. 15140-122) in DMEM medium (GIBCO).
- the compound to be tested (1 ⁇ M as the initial concentration, diluted 3 times, a total of 10 concentrations) was co-incubated with the cells for 7 days, and added to each well 3D reagent (Promega, catalog number G9683), read the luminescence value with Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104), the light signal is directly proportional to the amount of ATP in the system, and the content of ATP directly characterizes the amount of ATP in the system number of living cells. Finally, XLFIT software was used to obtain the IC 50 (half maximal inhibitory concentration) of the compound with a nonlinear fitting formula.
- Inhibition rate (%) 100 ⁇ (negative control average value-compound reading value)/(negative control average value-positive control average value)
- Negative control DMSO.
- Positive control culture medium.
- the compound to be tested is co-incubated with liver microsomes of different species with or without adding NADPH, the final concentration of the compound to be tested in the test system is 1 ⁇ M, and the final concentration of NADPH 1mM, the final concentration of liver microsomes is 0.5mg/ml. Detect the concentration of the compound in the incubation supernatant at different time points within 60 minutes and calculate the pharmacokinetic parameters (such as clearance rate Clint).
- Some molecules (such as H1b, H10b, H24, H25, H35b-H38b, P20, P24, etc.) have lower clearance rates and slower metabolism in humans than the control MRTX1133.
- Colorectal cancer tumor cell GP2D with KRAS G12D mutation was cultured, and the tumor cells were inoculated into 6-8 week old female BALB/c nude mice (body weight about 20 g), and all mice were inoculated subcutaneously. Mice were raised in an SPF-grade experimental environment, and all mice had free access to commercially certified standard diets.
- Daily intraperitoneal (ip) administration of test compounds began when the average tumor volume of the mice had grown to approximately 150 mm3 .
- the dosage is: blank group vehicle (10% Captisol in 50mM citrate buffer pH 5.0).
- the dosage of the administration group was 10mg/kg, twice a day.
- Tumor volumes were measured with two-dimensional calipers three times a week, and animals were weighed daily. After 10 days of continuous administration, the inhibition rate (TGI/100%) was calculated according to the final tumor volume.
- Qualified healthy ICR mice (aged 6-8 weeks, body weight 18-20g) were selected, 3 in each group, for single intravenous administration respectively.
- a single intravenous administration test was carried out, and the dose was explored from 2mg/kg. If no death was observed, the dose was increased, and if death occurred, the increase would be stopped.
- the intravenous administration solution of MRTX1133 and the compound P20 intravenous administration vehicle are: DMSO/Tween80/Solutol/normal saline (the volume ratio of the four is 5/3/10/82), vortex ultrasonic to make it fully dissolved, and carry out administration disposal .
- MRTX1133 infusion administration solution and compound P20 infusion administration vehicle are: DMSO/Tween80/Solutol/normal saline (the volume ratio of the four is 5/3/10/82), vortex ultrasonic to make it fully dissolved, then administer Drug disposal.
- CD1 female mice were used as test animals, and administered orally/intravenously (the oral administration dose was 20 mg/kg, and the vehicle was: DMSO-Solutol-H 2 O).
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Abstract
Provided in the present invention is a substituted bicyclic heteroaryl compound as a KRAS G12D inhibitor. Also provided in the present invention are a pharmaceutical composition containing the compound, and the use thereof in the treatment of cancers.
Description
本发明属于医药领域,具体涉及取代的双环杂芳基化合物,其可作为KRAS G12D抑制剂。The invention belongs to the field of medicine, in particular to a substituted bicyclic heteroaryl compound, which can be used as a KRAS G12D inhibitor.
人RAS基因家族包含3类RAS基因KRAS、NRAS和HRAS,编码四种不同的RAS蛋白(KRAS-4A,KRAS-4B,NRAS和HRAS)。RAS蛋白属于GTP酶蛋白家族,其结合于GDP时处于非活性状态,而结合于GTP后处于活性状态,可以导致下游RAF-MAPK、PI3K-Akt等信号通路的激活,导致细胞的抗凋亡和增殖(Cell,2017;170(1):17-33;Cell,2020;183(4):850-859;Nat Rev Drug Discov,2020;19(8):533-552.)。RAS基因的激活性突变是人类癌症中最普遍的致癌驱动基因,其中KRAS最经常发生致癌性激活突变。举例来说,KRAS在胰腺癌中的突变率为86-96%,在结肠直肠癌中为40-54%,在肺癌中为27-39%(PNAS,2019;116(32):15823-15829;Pathol Res Pract,2009;205,858–862;Nature,2012;491,399–405;Nature,2014;511,543–550)。The human RAS gene family includes three types of RAS genes KRAS, NRAS and HRAS, encoding four different RAS proteins (KRAS-4A, KRAS-4B, NRAS and HRAS). RAS protein belongs to the GTPase protein family, it is in an inactive state when it binds to GDP, and it is in an active state after binding to GTP, which can lead to the activation of downstream RAF-MAPK, PI3K-Akt and other signaling pathways, leading to the anti-apoptosis and anti-apoptosis of cells. Proliferation (Cell, 2017; 170(1):17-33; Cell, 2020; 183(4):850-859; Nat Rev Drug Discov, 2020; 19(8):533-552.). Activating mutations in the RAS gene are the most prevalent oncogenic driver genes in human cancers, among which KRAS is the most frequent oncogenic activating mutation. For example, KRAS has a mutation rate of 86-96% in pancreatic cancer, 40-54% in colorectal cancer, and 27-39% in lung cancer (PNAS, 2019; 116(32):15823-15829 ; Pathol Res Pract, 2009; 205, 858–862; Nature, 2012; 491, 399–405; Nature, 2014; 511, 543–550).
在KRAS基因中多个位点均可以发生致癌驱动突变,最常见的突变发生在G12位点,包括G12C、G12D、G12V等,这些突变可以降低KRAS蛋白的GTP酶活性,从而导致KRAS蛋白长期处于活性状态,导致了细胞的恶性转化和癌症发生(Cell,2017;170(1):17-33;Nat Rev Drug Discov,2020;19(8):533-552)。在不同癌症类型中,经常出现的KRAS突变类型有所不同,例如大约13%的肺癌患者出现G12C突变(N Engl Med J,2021;384(25):2382-2393);而在胰腺癌中,33.8%的患者出现G12D突变,但仅有1.7%的患者出现G12C突变;在结肠直肠癌中,约10-12%的患者出现G12D突变,而G12C突变的发生率低于3%(Nat Rev Cancer 2018;18(12):767-777)。Oncogenic driver mutations can occur at multiple sites in the KRAS gene, the most common mutations occur at the G12 site, including G12C, G12D, G12V, etc. These mutations can reduce the GTPase activity of the KRAS protein, resulting in a long-term Active state, leading to malignant transformation of cells and carcinogenesis (Cell, 2017; 170(1): 17-33; Nat Rev Drug Discov, 2020; 19(8): 533-552). In different cancer types, the frequently occurring KRAS mutation types are different. For example, about 13% of lung cancer patients have G12C mutations (N Engl Med J, 2021; 384(25):2382-2393); in pancreatic cancer, G12D mutation occurred in 33.8% of patients, but only 1.7% of patients developed G12C mutation; in colorectal cancer, about 10-12% of patients had G12D mutation, while the incidence of G12C mutation was less than 3% (Nat Rev Cancer 2018;18(12):767-777).
不同于ATP依赖性的蛋白激酶(蛋白与ATP的亲和力在微摩尔水平),KRAS蛋白与GDP/GTP的亲和力在皮摩尔水平,化合物分子很难与GDP/GTP产生有效竞争以抑制KRAS信号通路,这严重阻碍了KRAS抑制剂的研发(Cell,2017;170(1):17-33;Cell,2020;183(4):850-859;Nat Rev Drug Discov,2020;19(8):533-552.)。在近些年,在KRAS蛋白上发现了可以与小分子有效结合的非GDP/GTP竞争的“别构”结合腔,这些发现极大促进了靶向KRAS突变驱动肿瘤的靶向药物研发。当前,靶向KRAS G12C的MRTX-849(Adagrasib)和AMG510(Sotorasib)已经在临床研究中展示了优异疗效(N Engl J Med.2020;383(13):1207-1217;Cancer Discov.2020;10(1):54-71),且AMG510已在2021年5月顺利获得FDA批准上市。相对于KRAS G12C药物的研发,靶向KRAS G12D突变的药物研发还处于早期阶段;然而,如上所示,在多种肿瘤中有非常高的KRAS G12D突变率,KRAS G12D抑制剂的研发具有极其重要的意义。Unlike ATP-dependent protein kinases (the affinity between protein and ATP is at the micromolar level), the affinity between KRAS protein and GDP/GTP is at the picomolar level, and it is difficult for compounds to compete effectively with GDP/GTP to inhibit the KRAS signaling pathway. This has seriously hindered the development of KRAS inhibitors (Cell, 2017; 170(1): 17-33; Cell, 2020; 183(4): 850-859; Nat Rev Drug Discov, 2020; 19(8): 533- 552.). In recent years, an "allosteric" binding cavity that can compete with non-GDP/GTP that can effectively bind small molecules has been discovered on the KRAS protein. These discoveries have greatly promoted the development of targeted drugs targeting KRAS mutation-driven tumors. Currently, MRTX-849 (Adagrasib) and AMG510 (Sotorasib) targeting KRAS G12C have demonstrated excellent efficacy in clinical studies (N Engl J Med.2020; 383(13):1207-1217; Cancer Discov.2020; 10 (1):54-71), and AMG510 has successfully obtained FDA approval for marketing in May 2021. Compared with the development of KRAS G12C drugs, the development of drugs targeting KRAS G12D mutations is still at an early stage; however, as shown above, there are very high KRAS G12D mutation rates in various tumors, and the development of KRAS G12D inhibitors is extremely important meaning.
美国Mirati公司报道了静脉给药的KRAS G12D小分子MRTX1133,但尚未进入临床研究,且口服吸收较弱,本发明的靶向KRAS G12D小分子药物具有优异的活性和更高的体内暴露量,有望实现口服给药,以期解决临床未满足需求。Mirati Corporation of the United States reported the KRAS G12D small molecule MRTX1133 for intravenous administration, but it has not yet entered clinical research, and its oral absorption is weak. The KRAS G12D small molecule drug of the present invention has excellent activity and higher in vivo exposure, and is expected to Realize oral administration in order to solve clinical unmet needs.
发明内容Contents of the invention
在一个方面,本发明提供了化合物,或其药学上可接受的盐、同位素变体、互变异构体、立体异构体、前药、多晶型、水合物或溶剂合物,其中所述化合物选自:In one aspect, the invention provides a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof, wherein Said compound is selected from:
在另一个方面,本发明提供了一种药物组合物,所述药物组合物含有本发明化合物,和任选地药学上可接受的赋形剂。In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, and optionally a pharmaceutically acceptable excipient.
在另一个方面,本发明提供了含有本发明化合物和药学上可接受的赋形剂的药物组合物,其还含有其它治疗剂。In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient, which also comprises other therapeutic agents.
在另一个方面,本发明提供了本发明化合物在制备用于治疗和/或预防KRAS G12D突变蛋白介导的疾病的药物中的用途。In another aspect, the present invention provides the use of the compound of the present invention in the preparation of a medicament for treating and/or preventing KRAS G12D mutein-mediated diseases.
在另一个方面,本发明提供了在受试者中治疗和/或预防KRAS G12D突变蛋白介导的疾病的方法,包括向所述受试者给药本发明化合物或本发明组合物。In another aspect, the present invention provides a method for treating and/or preventing a KRAS G12D mutein-mediated disease in a subject, comprising administering to the subject a compound of the present invention or a composition of the present invention.
在另一个方面,本发明提供了本发明化合物或本发明组合物,其用于治疗和/或预防KRAS G12D突变蛋白介导的疾病。In another aspect, the present invention provides the compound of the present invention or the composition of the present invention for the treatment and/or prevention of KRAS G12D mutein-mediated diseases.
在具体实施方案中,本发明治疗的疾病包括选自以下的癌症:急性髓细胞样白血病、青少年癌症、儿童肾上腺皮质癌、AIDS相关的癌症(例如淋巴瘤和卡波西氏肉瘤)、肛门癌、阑尾癌、星形细胞瘤、非典型畸胎样、基底细胞癌、胆管癌、膀胱癌、骨癌、脑干神经胶质瘤、脑瘤、乳腺癌、支气管肿瘤、伯基特淋巴瘤、类癌瘤、非典型畸胎样、胚胎肿瘤、生殖细胞肿瘤、原发性淋巴瘤、宫颈癌、儿童癌症、脊索瘤、心脏肿瘤、慢性淋巴细胞性白血病(CLL)、慢性髓细胞性白血病(CML)、慢性骨髓增殖性病症、结肠癌、结肠直肠癌、颅咽管瘤、皮肤T细胞淋巴瘤、肝外导管原位癌(DCIS)、胚胎肿瘤、CNS癌症、子宫内膜癌、室管膜瘤、食道癌、嗅神经母细胞瘤、尤文氏肉瘤、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、骨骼的纤维组织细胞瘤、胆囊癌、胃癌、胃肠道类癌瘤、胃肠道间质瘤(GIST)、生殖细胞肿瘤、妊娠滋养细胞肿瘤、毛细胞白血病、头颈癌、心脏癌、肝癌、霍奇金氏淋巴瘤、下咽癌、眼内黑色素瘤、胰岛细胞瘤、胰腺神经内分泌瘤、肾癌、喉癌、唇和口腔癌、肝癌、小叶原位癌(LCIS)、肺癌、淋巴瘤、转移性鳞状颈癌伴隐匿原发灶、中线道癌、口腔癌、多发性内分泌瘤综合征、 多发性骨髓瘤/浆细胞瘤、蕈样真菌病、骨髓发育不良综合征、骨髓发育不良/骨髓增殖性瘤、多发性骨髓瘤、梅克尔细胞癌、恶性间皮瘤、骨骼的恶性纤维组织细胞瘤和骨肉瘤、鼻腔和鼻窦癌、鼻咽癌、神经母细胞瘤、非霍奇金氏淋巴瘤、非小细胞肺癌(NSCLC)、口腔癌、唇和口腔癌、口咽癌、卵巢癌、胰腺癌、乳头瘤、副神经节瘤、鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、胸膜肺母细胞瘤、原发性中枢神经系统(CNS)淋巴瘤、前列腺癌、直肠癌、移行性细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、皮肤癌、胃癌、小细胞肺癌、小肠癌、软组织肉瘤、细胞淋巴瘤、睾丸癌、喉癌、胸腺瘤和胸腺癌、甲状腺癌、肾盂和输尿管的移行性细胞癌、滋养细胞肿瘤、儿童罕见的癌症、尿道癌、子宫肉瘤、阴道癌、外阴癌或病毒诱导的癌症,优选胰腺癌、结肠直肠癌或非小细胞肺癌。In specific embodiments, the diseases treated by the present invention include cancers selected from the group consisting of acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (e.g., lymphoma and Kaposi's sarcoma), anal cancer , appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, Carcinoid tumors, atypical teratoids, embryonal tumors, germ cell tumors, primary lymphomas, cervical cancers, childhood cancers, chordomas, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia ( CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancers, endometrial cancer, ventricular duct Thymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor , pancreatic neuroendocrine tumors, renal cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract cancer, oral cancer , multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplasia/myeloproliferative neoplasm, multiple myeloma, Merkel cell carcinoma, malignant mesenchymal Skin tumors, malignant fibrous histiocytoma and osteosarcoma of the bone, nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and mouth cancer carcinoma, oropharyngeal carcinoma, ovarian carcinoma, pancreatic carcinoma, papilloma, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile carcinoma, pharyngeal carcinoma, pleuropulmonary blastoma, primary central nervous system (CNS ) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, gastric cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, cellular lymphoma, testicular cancer, laryngeal cancer , thymoma and thymus carcinoma, thyroid carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors, rare cancers of childhood, urethral carcinoma, uterine sarcoma, vaginal carcinoma, vulvar carcinoma or virus-induced cancer, preferably pancreatic, colon Colorectal cancer or non-small cell lung cancer.
由随后的具体实施方案、实施例和权利要求,本发明的其它目的和优点将对于本领域技术人员显而易见。Other objects and advantages of the present invention will become apparent to those skilled in the art from the ensuing detailed description, examples and claims.
定义definition
术语“KRAS G12D”是指哺乳动物KRAS蛋白的突变形式,其在12位氨基酸处含有天冬氨酸对甘氨酸的氨基酸取代。The term "KRAS G12D" refers to a mutant form of the mammalian KRAS protein that contains an amino acid substitution of aspartic acid for glycine at amino acid position 12.
本文所用的术语“药学上可接受的盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐,它们在可靠的医学判断范围内适用于与患者组织接触,不会产生不恰当的毒性、刺激作用、变态反应等,与合理的益处/风险比相称,就它们的预期应用而言是有效的,包括(可能的话)本发明化合物的两性离子形式。As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention, which are suitable for use in contact with patient tissues within the scope of sound medical judgment without undue toxicity, Irritation, allergic effects, etc., commensurate with a reasonable benefit/risk ratio, are valid for their intended use, including, where possible, zwitterionic forms of the compounds of the invention.
给药的“受试者”包括但不限于:人(即,任何年龄组的男性或女性,例如,儿科受试者(例如,婴儿、儿童、青少年)或成人受试者(例如,年轻的成人、中年的成人或年长的成人))和/或非人的动物,例如,哺乳动物,例如,灵长类(例如,食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中,受试者是人。在一些实施方案中,受试者是非人动物。本文可互换使用术语“人”、“患者”和“受试者”。"Subjects" for administration include, but are not limited to: human (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adult, middle-aged adult or older adult)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep , goats, rodents, cats and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.
“疾病”、“障碍”和“病症”在本文中可互换地使用。"Disease", "disorder" and "condition" are used interchangeably herein.
通常,化合物的“有效量”是指足以引起目标生物反应的数量。正如本领域普通技术人员所理解的那样,本发明化合物的有效量可以根据下列因素而改变:例如,生物学目标、化合物的药代动力学、所治疗的疾病、给药模式以及受试者的年龄健康情况和症状。有效量包括治疗有效量和预防有效量。In general, an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response. As will be appreciated by those of ordinary skill in the art, an effective amount of a compound of the invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the condition of the subject. Age Health conditions and symptoms. An effective amount includes a therapeutically effective amount and a prophylactically effective amount.
“组合”以及相关术语是指同时或依次给药本发明化合物和其它治疗剂。例如,本发明化合物可以与其它治疗剂以分开的单位剂型同时或依次给药,或与其它治疗剂一起在单一单位剂型中同时给药。"Combination" and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent. For example, the compounds of the invention may be administered with the other therapeutic agent simultaneously or sequentially in separate unit dosage forms, or together with the other therapeutic agent in a single unit dosage form.
本文中,“本发明化合物”指的是以下的化合物、其药学上可接受的盐、对映异构体、非对映异构体、溶剂合物、水合物或同位素变体,以及它们的混合物。Herein, "the compound of the present invention" refers to the following compounds, their pharmaceutically acceptable salts, enantiomers, diastereoisomers, solvates, hydrates or isotopic variants, and their mixture.
在一个实施方案中,本发明涉及化合物,或其药学上可接受的盐、同位素变体、互变异构体、立体异构体、前药、多晶型、水合物或溶剂合物,其中所述化合物选自:In one embodiment, the invention relates to a compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, stereoisomer, prodrug, polymorph, hydrate or solvate thereof, wherein Said compound is selected from:
本发明化合物可包括一个或多个不对称中心,且因此可以存在多种立体异构体形式,例如,对映异构体和/或非对映异构体形式。例如,本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体),或者可为立体异构体的混合物的形式,包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离,所述方法包括:手性高压液相色谱法(HPLC)以及手性盐的形成和结晶;或者优选的异构体可通过不对称合成来制备。The compounds of the present invention may include one or more asymmetric centers, and thus may exist in various stereoisomeric forms, eg, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereoisomers or geometric isomers (eg cis and trans isomers), or may be in the form of a mixture of stereoisomers, Racemic mixtures and mixtures enriched in one or more stereoisomers are included. Isomers can be separated from mixtures by methods known to those skilled in the art, including: chiral high pressure liquid chromatography (HPLC) and formation and crystallization of chiral salts; or preferred isomers can be obtained by prepared by asymmetric synthesis.
本发明化合物还可能以互变异构体存在。在不同的互变异构形式存在的化合物,一个所述化合物并不局限于任何特定的互变异构体,而是旨在涵盖所有的互变异构形式。The compounds of the invention may also exist as tautomers. For compounds that exist in different tautomeric forms, a said compound is not limited to any particular tautomeric form, but is intended to encompass all tautomeric forms.
本领域技术人员将理解,有机化合物可以与溶剂形成复合物,其在该溶剂中发生反应或从该溶剂中沉淀或结晶出来。这些复合物称为“溶剂合物”。当溶剂是水时,复合物称为“水合物”。本发明涵盖了本发明化合物的所有溶剂合物。Those skilled in the art will appreciate that organic compounds may form complexes with solvents in which they react or from which they are precipitated or crystallized. These complexes are known as "solvates". When the solvent is water, the complex is called a "hydrate". The invention covers all solvates of the compounds of the invention.
术语“溶剂合物”是指通常由溶剂分解反应形成的与溶剂相结合的化合物或其盐的形式。这个物理缔合可包括氢键键合。常规溶剂包括水、甲醇、乙醇、乙酸、DMSO、THF、乙醚等。本文所述的化合物可制备成,例如,结晶形式,且可被溶剂化。合适的溶剂合物包括药学上可接受的溶剂合物且进一步包括化学计量的溶剂合物和非化学计量的溶剂合物。在一些情况下,所述溶剂合物将能够分离,例如,当一或多个溶剂分子掺入结晶固体的晶格中时。“溶剂合物”包括溶液状态的溶剂合物和可分离的溶剂合物。代表性的溶剂合物包括水合物、乙醇合物和甲醇合物。The term "solvate" refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline forms, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
术语“水合物”是指与水相结合的化合物。通常,包含在化合物的水合物中的水分子数与该水合物中该化合物分子数的比率确定。因此,化合物的水合物可用例如通式R·x H
2O代表,其中R是该化合物,和x是大于0的数。给定化合物可形成超过一种水合物类型,包括,例如,单水合物(x为1)、低级水合物(x是大于0且小于1的数,例如,半水合物(R·0.5H
2O))和多水合物(x为大于1的数,例如,二水合物(R·2H
2O)和六水合物(R·6H
2O))。
The term "hydrate" refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, a hydrate of a compound can be represented, for example, by the general formula R.x H 2 O, where R is the compound, and x is a number greater than zero. A given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R·2H 2 O) and hexahydrate (R·6H 2 O)).
本发明化合物可以是无定形或结晶形式(多晶型)。此外,本发明化合物可以以一种或多种结晶形式存在。因此,本发明在其范围内包括本发明化合物的所有无定形或结晶形式。术语“多晶型物”是指特定晶体堆积排列的化合物的结晶形式(或其盐、水合物或溶剂合物)。所有的多晶型物具有相同的元素组成。不同的结晶形式通常具有不同的X射线衍射图、红外光谱、熔点、密度、硬度、晶体形状、光电性质、稳定性和溶解度。重结晶溶剂、结晶速率、贮存温度和其他因素可导致一种结晶形式占优。化合物的各种多晶型物可在不同的条件下通过结晶制备。The compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
本发明还包括同位素标记的化合物(同位素变体),它们等同于式(A)所述的那些,但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素,分别例如
2H、
3H、
13C、
11C、
14C、
15N、
18O、
17O、
31P、
32P、
35S、
18F和
36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如
3H和
14C)的那些可用于药物和/或底物组织分布测定。氚、即
3H和碳-14、即
14C同位素是特别优选的,因为它们容易制备和检测。进而,被更重的同位素取代,例如氘、即
2H,由于代谢稳定性更高可以提供治疗上的益处,例如延长体内半衰期或减少剂量需求,因而在有些情况下可能是优选的。同位素标记的本发明式(A)化合物及其前体药物一般可以这样制备,在进行下述流程和/或实施例与制备例所公开的工艺时,用容易得到的同位素标记的试剂代替非同位素标记的试剂。
The invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (A), but with one or more atoms represented by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature replaced. Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl. The compounds of the present invention, their prodrugs and pharmaceutically acceptable salts of the compounds or the prodrugs containing the above-mentioned isotopes and/or other isotopes of other atoms all belong to the scope of the present invention. Certain isotopically-labeled compounds of the invention, eg, those incorporating radioactive isotopes (eg, 3H and14C ), are useful in drug and/or substrate tissue distribution assays. Tritium, ie3H , and carbon-14, ie14C isotopes are particularly preferred because of their ease of preparation and detection. Furthermore, substitution with heavier isotopes, such as deuterium, ie2H , may be preferred in some circumstances since greater metabolic stability may afford therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements. The isotope-labeled compound of formula (A) of the present invention and its prodrug can generally be prepared in this way, when carrying out the processes disclosed in the following schemes and/or examples and preparation examples, replace the non-isotope with easily available isotope-labeled reagents Labeled reagents.
此外,前药也包括在本发明的上下文内。本文所用的术语“前药”是指在体内通过例如在血液中水解转变成其具有医学效应的活性形式的化合物。药学上可接受的前药描述于T.Higuchi和V.Stella,Prodrugs as Novel Delivery Systems,A.C.S.Symposium Series的Vol.14,Edward B.Roche,ed.,Bioreversible Carriers in Drug Design,American Pharmaceutical Association and Pergamon Press,1987,以及D.Fleisher、S.Ramon和H.Barbra“Improved oral drug delivery:solubility limitations overcome by the use of prodrugs”,Advanced Drug Delivery Reviews(1996)19(2)115-130,每篇引入本文作为参考。Furthermore, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo to its active form having a medical effect, for example by hydrolysis in blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon, and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, per intro This article is for reference.
药物组合物和试剂盒Pharmaceutical compositions and kits
在另一方面,本发明提供了药物组合物,其包含本发明化合物(还称为“活性组分”)和药学上可接 受的赋形剂。在一些实施方案中,所述药物组合物包含有效量的本发明化合物。在一些实施方案中,所述药物组合物包含治疗有效量的本发明化合物。在一些实施方案中,所述药物组合物包含预防有效量的本发明化合物。In another aspect, the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a compound of the invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of a compound of the invention.
用于本发明的药学上可接受的赋形剂是指不会破坏一起调配的化合物的药理学活性的无毒载剂、佐剂或媒剂。可以用于本发明组合物中的药学上可接受的载剂、佐剂或媒剂包括(但不限于)离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(如人类血清白蛋白)、缓冲物质(如磷酸盐)、甘氨酸、山梨酸、山梨酸钾、饱和植物脂肪酸的偏甘油酯混合物、水、盐或电解质(如硫酸鱼精蛋白)、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、三硅酸镁、聚乙烯吡咯烷酮、基于纤维素的物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧丙烯-嵌段聚合物、聚乙二醇以及羊毛脂。A pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle which does not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin Protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- Block polymers, polyethylene glycols and lanolin.
本发明还包括试剂盒(例如,药物包装)。所提供的试剂盒可以包括本发明化合物、其它治疗剂,以及含有本发明化合物、其它治疗剂的第一和第二容器(例如,小瓶、安瓿瓶、瓶、注射器和/或可分散包装或其它合适的容器)。在一些实施方案中,提供的试剂盒还可以任选包括第三容器,其含有用于稀释或悬浮本发明化合物和/或其它治疗剂的药用赋形剂。在一些实施方案中,提供在第一容器和第二容器中的本发明化合物和其它治疗剂组合形成一个单位剂型。The invention also includes kits (eg, pharmaceutical packs). Provided kits can include a compound of the invention, another therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container). In some embodiments, provided kits can also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent. In some embodiments, a compound of the invention and other therapeutic agent provided in a first container and a second container are combined to form a unit dosage form.
给药medication
本发明提供的药物组合物可以通过许多途径给药,包括但不限于:口服给药、肠胃外给药、吸入给药、局部给药、直肠给药、鼻腔给药、口腔给药、阴道给药、通过植入剂给药或其它给药方式。例如,本文使用的肠胃外给药包括皮下给药、皮内给药、静脉内给药、肌肉内给药、关节内给药、动脉内给药、滑膜腔内给药、胸骨内给药、脑脊髓膜内给药、病灶内给药、和颅内的注射或输液技术,优选通过静脉内给药。The pharmaceutical composition provided by the present invention can be administered by many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal cavity administration, buccal administration, vaginal administration Drugs, by implants, or by other means of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal, intralesional, and intracranial injection or infusion techniques, preferably intravenously.
通常,给予有效量的本文所提供的化合物。按照有关情况,包括所治疗的病症、选择的给药途径、实际给予的化合物、个体患者的年龄、体重和响应、患者症状的严重程度,等等,可以由医生确定实际上给予的化合物的量。Typically, an effective amount of a compound provided herein is administered. The amount of the compound actually administered can be determined by the physician according to the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
当用于预防本发明所述病症时,给予处于形成所述病症危险之中的受试者本文所提供的化合物,典型地基于医生的建议并在医生监督下给药,剂量水平如上所述。处于形成具体病症的危险之中的受试者,通常包括具有所述病症的家族史的受试者,或通过遗传试验或筛选确定尤其对形成所述病症敏感的那些受试者。When used to prevent a condition described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically on the advice and supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition generally include those with a family history of the condition, or those determined by genetic testing or screening to be particularly susceptible to developing the condition.
还可以长期给予本文所提供的药物组合物(“长期给药”)。长期给药是指在长时间内给予化合物或其药物组合物,例如,3个月、6个月、1年、2年、3年、5年等等,或者可无限期地持续给药,例如,受试者的余生。在一些实施方案中,长期给药意欲在长时间内在血液中提供所述化合物的恒定水平,例如,在治疗窗内。Long-term administration of the pharmaceutical compositions provided herein ("chronic administration") can also be used. Long-term administration refers to administering a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, For example, the rest of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within the therapeutic window.
可以使用各种给药方法,进一步递送本发明的药物组合物。例如,在一些实施方案中,可以推注给药药物组合物,例如,为了使化合物在血液中的浓度提高至有效水平。推注剂量取决于通过身体的活性组分的目标全身性水平,例如,肌内或皮下的推注剂量使活性组分缓慢释放,而直接递送至静脉的推注(例如,通过IV静脉滴注)能够更加快速地递送,使得活性组分在血液中的浓度快速升高至有效水平。在其它实施方案中,可以以持续输液形式给予药物组合物,例如,通过IV静脉滴注,从而在受试者身体中提供稳态浓度的活性组分。此外,在其它实施方案中,可以首先给予推注剂量的药物组合物,而后持续输液。Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, pharmaceutical compositions may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to effective levels. The bolus dose depends on the target systemic level of the active ingredient through the body, for example, an intramuscular or subcutaneous bolus dose provides slow release of the active ingredient, while a bolus delivered directly into a vein (e.g., by IV intravenous infusion) ) can be delivered more rapidly, so that the concentration of the active ingredient in the blood rises rapidly to effective levels. In other embodiments, the pharmaceutical compositions may be administered as a continuous infusion, eg, by IV infusion, to provide a steady state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
口服组合物可以采用散装液体溶液或混悬剂或散装粉剂形式。然而,更通常,为了便于精确地剂 量给药,以单位剂量形式提供所述组合物。术语“单位剂型”是指适合作为人类患者及其它哺乳动物的单元剂量的物理离散单位,每个单位包含预定数量的、适于产生所需要的治疗效果的活性物质与合适药学赋形剂。典型的单位剂量形式包括液体组合物的预装填的、预先测量的安瓿或注射器,或者在固体组合物情况下的丸剂、片剂、胶囊剂等。在这种组合物中,所述化合物通常为较少的组分(约0.1至约50重量%,或优选约1至约40重量%),剩余部分为对于形成所需给药形式有用的各种载体或赋形剂以及加工助剂。Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More usually, however, the compositions will be presented in unit dosage form for ease of precise dosing. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful for forming the desired administration form. Carriers or excipients and processing aids.
对于口服剂量,代表性的方案是,每天一个至五个口服剂量,尤其是两个至四个口服剂量,典型地是三个口服剂量。使用这些剂量给药模式,每个剂量提供大约0.01至大约20mg/kg的本发明化合物,优选的剂量各自提供大约0.1至大约10mg/kg,尤其是大约1至大约5mg/kg。For oral dosages, a typical regimen is one to five oral dosages per day, especially two to four oral dosages, typically three oral dosages. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially about 1 to about 5 mg/kg.
为了提供与使用注射剂量类似的血液水平,或比使用注射剂量更低的血液水平,通常选择透皮剂量,数量为大约0.01至大约20%重量,优选大约0.1至大约20%重量,优选大约0.1至大约10%重量,且更优选大约0.5至大约15%重量。In order to provide blood levels similar to, or lower than, the injected dose, the transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
从大约1至大约120小时,尤其是24至96小时,注射剂量水平在大约0.1mg/kg/小时至至少10mg/kg/小时的范围。为了获得足够的稳定状态水平,还可以给予大约0.1mg/kg至大约10mg/kg或更多的预载推注。对于40至80kg的人类患者来说,最大总剂量不能超过大约2g/天。Injection dosage levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially 24 to 96 hours. A preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given in order to achieve adequate steady state levels. For a human patient of 40 to 80 kg, the maximum total dose should not exceed approximately 2 g/day.
适于口服给药的液体形式可包括合适的水性或非水载体以及缓冲剂、悬浮剂和分散剂、着色剂、调味剂,等等。固体形式可包括,例如,任何下列组份,或具有类似性质的化合物:粘合剂,例如,微晶纤维素、黄蓍胶或明胶;赋形剂,例如,淀粉或乳糖,崩解剂,例如,褐藻酸、Primogel或玉米淀粉;润滑剂,例如,硬脂酸镁;助流剂,例如,胶体二氧化硅;甜味剂,例如,蔗糖或糖精;或调味剂,例如,薄荷、水杨酸甲酯或橙味调味剂。Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering, suspending and dispersing agents, coloring agents, flavoring agents, and the like. The solid form may comprise, for example, any of the following components, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or flavoring agents, for example, peppermint, water Methyl sylate or orange flavoring.
可注射的组合物典型地基于可注射用的无菌盐水或磷酸盐缓冲盐水,或本领域中已知的其它可注射的赋形剂。如前所述,在这种组合物中,活性化合物典型地为较少的组分,经常为约0.05至10%重量,剩余部分为可注射的赋形剂等。Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. In such compositions, as previously mentioned, the active compound is typically a minor component, often from about 0.05 to 10% by weight, the remainder being injectable excipients and the like.
典型地将透皮组合物配制为含有活性组分的局部软膏剂或乳膏剂。当配制为软膏剂时,活性组分典型地与石蜡或可与水混溶的软膏基质组合。或者,活性组分可与例如水包油型乳膏基质一起配制为乳膏剂。这种透皮制剂是本领域中公知的,且通常包括用于提升活性组分或制剂的稳定的皮肤渗透的其它组份。所有这种已知的透皮制剂和组份包括在本发明提供的范围内。Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient. When formulated in an ointment, the active ingredients are typically combined with a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream, with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art, and generally include other ingredients for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope of the present invention.
本发明化合物还可通过经皮装置给予。因此,经皮给药可使用贮存器(reservoir)或多孔膜类型、或者多种固体基质的贴剂实现。The compounds of the invention may also be administered by transdermal devices. Thus, transdermal administration can be achieved using patches of the reservoir or porous membrane type, or various solid matrices.
用于口服给予、注射或局部给予的组合物的上述组份仅仅是代表性的。其它材料以及加工技术等阐述于Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania的第8部分中,本文以引用的方式引入该文献。The foregoing components of compositions for oral administration, injection or topical administration are representative only. Other materials and processing techniques, etc. are described in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, Part 8, which is incorporated herein by reference.
本发明化合物还可以以持续释放形式给予,或从持续释放给药系统中给予。代表性的持续释放材料的描述可在Remington's Pharmaceutical Sciences中找到。The compounds of the invention may also be administered in sustained release form, or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.
本发明还涉及本发明化合物的药学上可接受的制剂。在一个实施方案中,所述制剂包含水。在另一个实施方案中,所述制剂包含环糊精衍生物。最常见的环糊精为分别由6、7和8个α-1,4-连接的葡萄糖单元组成的α-、β-和γ-环糊精,其在连接的糖部分上任选包括一个或多个取代基,其包括但不限于:甲基化的、羟基烷基化的、酰化的和磺烷基醚取代。在一些实施方案中,所述环糊精为磺烷基醚β-环糊精,例如,磺丁基醚β-环糊精,也称作Captisol。参见,例如,U.S.5,376,645。在一些实施方案中,所述制剂包括六丙基-β-环糊精(例如,在水中,10-50%)。The invention also relates to pharmaceutically acceptable formulations of the compounds of the invention. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises a cyclodextrin derivative. The most common cyclodextrins are α-, β-, and γ-cyclodextrins composed of 6, 7, and 8 α-1,4-linked glucose units, respectively, optionally including a or multiple substituents including, but not limited to, methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, eg, sulfobutyl ether β-cyclodextrin, also known as Captisol. See, eg, U.S. 5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (eg, 10-50% in water).
实施例Example
本发明所采用的试剂为直接购买的商业化试剂或经本领域熟知的常用方法合成。The reagents used in the present invention are commercial reagents purchased directly or synthesized by common methods well known in the art.
如下示例的具体反应路线或步骤为本发明所用,具体如下:The specific reaction scheme or steps of the following examples are used in the present invention, specifically as follows:
实施例1Example 1
关键中间体a1-a38的制备Preparation of key intermediate a1-a38
中间体a1的合成Synthesis of intermediate a1
步骤1:将原料2-氨基-3-氟-4-溴苯甲酸a1-1(15g,64.1mmol)溶于100mL DMF中,缓慢加入N-氯代琥珀酰亚胺(8.56g,64.1mmol),升温至80℃反应16小时,停止反应。将该反应液倒入500mL冰水中,抽滤得到滤饼,干燥,得到中间体a1-2(13.2g,49.2mmol),收率:77%。LC-MS:[M-H]
-=267。
Step 1: Dissolve the raw material 2-amino-3-fluoro-4-bromobenzoic acid a1-1 (15g, 64.1mmol) in 100mL DMF, slowly add N-chlorosuccinimide (8.56g, 64.1mmol) , the temperature was raised to 80° C. for 16 hours, and the reaction was stopped. The reaction solution was poured into 500 mL of ice water, filtered with suction to obtain a filter cake, and dried to obtain intermediate a1-2 (13.2 g, 49.2 mmol). Yield: 77%. LC-MS: [MH] - = 267.
步骤2:向200mL圆底烧瓶中加入尿素(35.3g,588mmol)和上步中间体a1-2(10.5g,39.2mmol),升温至200℃反应12小时。降温至80℃后,向体系加水100mL,回流10分钟后冷却至室温,过滤得到滤饼,加水洗涤,在烘箱中干燥,得到中间体a1-3(4.0g,13.7mmol)。收率:35%。LC-MS:[M+H]
+=294。
Step 2: Add urea (35.3g, 588mmol) and intermediate a1-2 (10.5g, 39.2mmol) into a 200mL round bottom flask, and heat up to 200°C for 12 hours. After cooling down to 80°C, 100 mL of water was added to the system, refluxed for 10 minutes, cooled to room temperature, filtered to obtain a filter cake, washed with water, and dried in an oven to obtain intermediate a1-3 (4.0 g, 13.7 mmol). Yield: 35%. LC-MS: [M+H] + =294.
步骤3:将上步中间体a1-3(4.0g,13.7mmol)和N,N-二异丙基乙基胺(5.3g,41.1mmol)溶于15mL三氯氧磷中,升温至120℃反应8小时,停止反应。减压蒸除溶剂,柱层析分离,得到中间体a1(1.9g,5.8mmol)。收率:42%。LC-MS:[M+H]
+=331。
Step 3: Dissolve the intermediate a1-3 (4.0g, 13.7mmol) and N,N-diisopropylethylamine (5.3g, 41.1mmol) in 15mL phosphorus oxychloride, and heat up to 120°C After reacting for 8 hours, stop the reaction. The solvent was evaporated under reduced pressure and separated by column chromatography to obtain intermediate a1 (1.9 g, 5.8 mmol). Yield: 42%. LC-MS: [M+H] + = 331.
中间体a2,a3,a16,a30的合成Synthesis of intermediates a2, a3, a16, a30
步骤:室温下,将中间体a1(1.9g,5.8mmol)和原料3,8-二氮杂双环[3.2.1]辛烷-8-羧酸叔丁酯a2-1(1.85g,8.7mmol)溶于20mL二氯甲烷中,加入N,N-二异丙基乙基胺(2.3g,17.4mmol),室温反应8小时。向体系加水60mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离,得到淡黄色固体a2(1.2g,2.4mmol)。收率:41%。LC-MS:[M+H]
+=507。
Step: At room temperature, intermediate a1 (1.9g, 5.8mmol) and raw material 3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl a2-1 (1.85g, 8.7mmol ) was dissolved in 20 mL of dichloromethane, N,N-diisopropylethylamine (2.3 g, 17.4 mmol) was added, and reacted at room temperature for 8 hours. Add 60 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain a light yellow solid a2 (1.2 g, 2.4 mmol). Yield: 41%. LC-MS: [M+H] + = 507.
参照中间体a2的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a2, the following intermediate was synthesized.
中间体a4-a6,a8-a13的合成Synthesis of Intermediates a4-a6, a8-a13
步骤1:冰浴下,将1-(甲氧基羰基)环丙烷-1-羧酸a4-2(3.0g,20.8mmol)溶于60mL二氯甲烷中,缓慢加入草酰氯(10.5g,83.3mmol),加入5滴无水DMF,0℃下反应20分钟后。撤去冰浴,升温至室温并继续搅拌1小时,减压蒸除溶剂。将该混合物溶于40mL二氯甲烷中,加入N,N-二异丙基乙基胺(5.4g,41.2mmol)和四氢吡咯a4-1(1.78g,25.0mmol),室温反应3小时。停止反应,减压蒸除溶剂,flash柱层析分离,得到中间体a4-3(2.6g,13.2mmol)。收率:64%。LC-MS:[M+H]
+=198。
Step 1: Under ice bath, dissolve 1-(methoxycarbonyl)cyclopropane-1-carboxylic acid a4-2 (3.0g, 20.8mmol) in 60mL of dichloromethane, slowly add oxalyl chloride (10.5g, 83.3 mmol), added 5 drops of anhydrous DMF, and reacted at 0° C. for 20 minutes. The ice bath was removed, the temperature was raised to room temperature and stirring was continued for 1 hour, and the solvent was evaporated under reduced pressure. The mixture was dissolved in 40 mL of dichloromethane, N,N-diisopropylethylamine (5.4 g, 41.2 mmol) and tetrahydropyrrole a4-1 (1.78 g, 25.0 mmol) were added, and reacted at room temperature for 3 hours. Stop the reaction, evaporate the solvent under reduced pressure, and separate by flash column chromatography to obtain intermediate a4-3 (2.6 g, 13.2 mmol). Yield: 64%. LC-MS: [M+H] + =198.
步骤2:-78℃下,将中间体a4-3(2.6g,13.2mmol)溶于30mL无水四氢呋喃中,缓慢加入四氢铝锂(26.4mL,1M)。升温至室温反应2小时,停止反应。缓慢向反应液倒入冰水80mL,减压蒸除有机溶剂后,乙酸乙酯萃取,无水硫酸钠干燥,过滤,浓缩,flash柱层析分离,得到中间体a4(900mg,5.8mmol)。收率:44%。LC-MS:[M+H]
+=156。
Step 2: At -78°C, the intermediate a4-3 (2.6g, 13.2mmol) was dissolved in 30mL of anhydrous tetrahydrofuran, and lithium aluminum hydride (26.4mL, 1M) was added slowly. The temperature was raised to room temperature for 2 hours, and the reaction was stopped. Slowly pour 80 mL of ice water into the reaction solution, evaporate the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate, and separate by flash column chromatography to obtain intermediate a4 (900 mg, 5.8 mmol). Yield: 44%. LC-MS: [M+H] + = 156.
参照中间体a4的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a4, the following intermediate was synthesized.
中间体a7的合成Synthesis of intermediate a7
步骤1:冰浴下,将6-溴-2,3-二氟苯甲醛a7-1(25g,113mmol)溶于250mL甲醇中,缓慢加入硼氢化钠(8.54g,226mmol),搅拌20分钟后,升温至室温继续反应1小时,停止反应。将该反应液缓慢倒入饱和NH
4Cl水溶液中,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,得到白色固体a7-2(23.9g,107mmol)。收率:95%。
Step 1: Under ice bath, dissolve 6-bromo-2,3-difluorobenzaldehyde a7-1 (25g, 113mmol) in 250mL of methanol, slowly add sodium borohydride (8.54g, 226mmol), and stir for 20 minutes , the temperature was raised to room temperature and the reaction was continued for 1 hour, and the reaction was stopped. The reaction solution was slowly poured into saturated aqueous NH 4 Cl solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid a7-2 (23.9 g, 107 mmol). Yield: 95%.
步骤2:冰浴下,将中间体a7-2(23.9g,107mmol)和N,N-二异丙基乙基胺(20.7g,161mmol)溶于200mL无水四氢呋喃中,缓慢加入甲基磺酸酐(20.5g,118mmol),搅拌20分钟后,撤去冰浴,室温继续反应18小时,停止反应。将该反应液缓慢倒入冰水中,乙酸乙酯萃取,无水硫酸钠干燥,过滤,浓缩,得到油状物a7-3(19g,63.1mmol)。收率:59%。Step 2: Dissolve intermediate a7-2 (23.9g, 107mmol) and N,N-diisopropylethylamine (20.7g, 161mmol) in 200mL anhydrous tetrahydrofuran under ice bath, and slowly add methylsulfonate Acid anhydride (20.5 g, 118 mmol), after stirring for 20 minutes, the ice bath was removed, and the reaction was continued at room temperature for 18 hours, and the reaction was stopped. The reaction solution was slowly poured into ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain oil a7-3 (19 g, 63.1 mmol). Yield: 59%.
步骤3:将上步中间体a7-3(19g,63.1mmol)溶于240mL乙醇和水的混合溶液(v/v,6/1)中,加入氰化钾(4.49g,69mmol)。回流条件下反应1小时,停止反应。减压蒸除有机溶剂,将反应液倒入饱和碳酸钠溶液中,搅拌20分钟后,二氯甲烷萃取,浓缩,柱层析分离,得到中间体a7-4(10.4g,44.8mmol)。收率:71%。LC-MS:[M+H]
+=233。
Step 3: Dissolve the above intermediate a7-3 (19g, 63.1mmol) in 240mL of a mixed solution of ethanol and water (v/v, 6/1), and add potassium cyanide (4.49g, 69mmol). React under reflux condition for 1 hour, stop the reaction. The organic solvent was evaporated under reduced pressure, the reaction solution was poured into saturated sodium carbonate solution, stirred for 20 minutes, extracted with dichloromethane, concentrated, and separated by column chromatography to obtain intermediate a7-4 (10.4g, 44.8mmol). Yield: 71%. LC-MS: [M+H] + =233.
步骤4:冰浴下,将上步中间体a7-4(10.4g,44.8mmol)溶于80mL DMF中,缓慢加入叔丁醇钾(5.3g,47.2mmol),搅拌20分钟后,溶液变红,缓慢逐滴加入提前配制的异硫氰酰甲酸乙酯的DMF溶液(6.2g,47.2mmol,5mL)。将该反应液继续搅拌1小时后,升温至100℃反应30分钟。冷却至室温,向该反应液缓慢倒入冰水淬灭反应,抽滤得到滤饼,正己烷洗涤,真空干燥箱干燥,得到中间体a7-5(13.7g,40.0mmol)。收率:89%。LC-MS:[M+H]
+=344。
Step 4: Under ice bath, dissolve the intermediate a7-4 (10.4g, 44.8mmol) in 80mL DMF, slowly add potassium tert-butoxide (5.3g, 47.2mmol), and stir for 20 minutes, the solution turns red , DMF solution (6.2 g, 47.2 mmol, 5 mL) of ethyl isothiocyanatoformate (6.2 g, 47.2 mmol, 5 mL) prepared in advance was slowly added dropwise. After continuing to stir the reaction liquid for 1 hour, the temperature was raised to 100° C. for 30 minutes to react. After cooling to room temperature, the reaction solution was slowly poured into ice water to quench the reaction, and the filter cake was obtained by suction filtration, washed with n-hexane, and dried in a vacuum oven to obtain intermediate a7-5 (13.7 g, 40.0 mmol). Yield: 89%. LC-MS: [M+H] + = 344.
步骤5:将上步中间体a7-5(6.7g,19.5mmol)溶于30mL DMSO中,加入30mL氢氧化钠水溶液(5M)。回流条件下反应4小时,停止反应。冷却至室温,向该反应液缓慢加入100mL冰水淬灭反应, 抽滤得到滤饼,水洗,真空干燥箱干燥,得到粗品中间体a7-6(3.8g)。LC-MS:[M+H]
+=272。
Step 5: The above intermediate a7-5 (6.7g, 19.5mmol) was dissolved in 30mL DMSO, and 30mL aqueous sodium hydroxide solution (5M) was added. React under reflux conditions for 4 hours, stop the reaction. After cooling to room temperature, 100 mL of ice water was slowly added to the reaction solution to quench the reaction, and the filter cake was obtained by suction filtration, washed with water, and dried in a vacuum oven to obtain the crude intermediate a7-6 (3.8 g). LC-MS: [M+H] + =272.
步骤6:将上步粗品a7-6(3.8g)和DMAP(122mg,1mmol)溶于30mL THF/DMF的混合溶液(v/v,1/1)中,加入Boc酸酐(4.3g,19.5mmol)。室温反应12小时,停止反应。向体系加水80mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,得到粗品中间体a7-7(3.3g)。Step 6: Dissolve the above crude product a7-6 (3.8g) and DMAP (122mg, 1mmol) in a mixed solution of 30mL THF/DMF (v/v, 1/1), add Boc anhydride (4.3g, 19.5mmol ). React at room temperature for 12 hours, then stop the reaction. Add 80 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude intermediate a7-7 (3.3 g).
步骤7:氮气保护下,将粗品a7-7(3.3g)和原料a7-8(6.01g,26.7mmol)溶于30mL 1,4-二氧六环中,缓慢加入醋酸钾(2.62g,26.7mmol)和Pd(DPEphos)Cl
2(643mg,0.9mmol),升温至100℃反应1小时,停止反应。冷却至室温,过滤,饱和食盐水洗涤,二氯甲烷萃取,干燥,浓缩,flash柱层析分离,得到中间体a7(2.5g,6.2mmol)。LC-MS:[M+H]
+=405。
Step 7: Under the protection of nitrogen, the crude product a7-7 (3.3g) and raw material a7-8 (6.01g, 26.7mmol) were dissolved in 30mL 1,4-dioxane, and potassium acetate (2.62g, 26.7 mmol) and Pd(DPEphos)Cl 2 (643mg, 0.9mmol), the temperature was raised to 100°C for 1 hour, and the reaction was stopped. Cool to room temperature, filter, wash with saturated brine, extract with dichloromethane, dry, concentrate, and separate by flash column chromatography to obtain intermediate a7 (2.5g, 6.2mmol). LC-MS: [M+H] + = 405.
中间体a14,a17的合成Synthesis of intermediates a14, a17
步骤:氮气保护下,将中间体a2(50g,98.77mmol)溶于750mL无水DMF中,加入CsF(45g,29.63mmol),升温至60℃下反应8h,停止反应。将反应液加入到1L水中,混合液用750mL乙酸乙酯分3次萃取,有机相用饱和盐水洗涤,无水硫酸钠干燥,过滤,得到中间体a14,收率90%。LC-MS:[M+H]
+=489。
Step: Under the protection of nitrogen, the intermediate a2 (50g, 98.77mmol) was dissolved in 750mL of anhydrous DMF, CsF (45g, 29.63mmol) was added, the temperature was raised to 60°C for 8h, and the reaction was stopped. The reaction solution was added to 1L of water, and the mixture was extracted three times with 750 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate a14 with a yield of 90%. LC-MS: [M+H] + = 489.
参照中间体a14的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a14, the following intermediate was synthesized.
中间体a15,a18,a33-a37的合成Synthesis of intermediates a15, a18, a33-a37
步骤1:将原料3-甲氧基-2,2-二甲基-3-氧丙二酸a15-1(1.0g,6.8mmol)和原料a5-1(850mg,6.8mmol)溶于20mL无水二氯甲烷中,加入EDCI(1.56g,8.2mmol)、N,N-二异丙基乙基胺(1.77g,13.6mmol)和HOBt(1.1g,8.2mmol),室温下反应16h,停止反应。向反应液中加入60mL冰水,二氯甲烷萃取,无水硫酸钠干燥。混合物经Flash柱层析分离,得到中间体a15-2(1.04g,4.6mmol),收率68%。LC-MS:[M+H]
+=218。
Step 1: Dissolve starting material 3-methoxy-2,2-dimethyl-3-oxalonic acid a15-1 (1.0g, 6.8mmol) and starting material a5-1 (850mg, 6.8mmol) in 20mL without Add EDCI (1.56g, 8.2mmol), N,N-diisopropylethylamine (1.77g, 13.6mmol) and HOBt (1.1g, 8.2mmol) to water dichloromethane, react at room temperature for 16h, stop reaction. Add 60 mL of ice water to the reaction solution, extract with dichloromethane, and dry over anhydrous sodium sulfate. The mixture was separated by Flash column chromatography to obtain intermediate a15-2 (1.04g, 4.6mmol) with a yield of 68%. LC-MS: [M+H] + = 218.
步骤2:-78℃下,将中间体a15-2(1.04g,4.6mmol)溶于15mL无水四氢呋喃中,缓慢加入四氢铝锂(350mg,9.2mmol)。升温至0℃反应1小时,停止反应。缓慢向反应液加入10%NaOH水溶液2mL,析出絮状物,抽滤,滤液减压浓缩,flash柱层析分离,得到中间体a15(700mg,4.0mmol)。收率:87%。LC-MS:[M+H]
+=176。
Step 2: At -78°C, the intermediate a15-2 (1.04g, 4.6mmol) was dissolved in 15mL of anhydrous tetrahydrofuran, and lithium aluminum hydride (350mg, 9.2mmol) was added slowly. The temperature was raised to 0°C for 1 hour, and the reaction was stopped. Slowly add 2 mL of 10% NaOH aqueous solution to the reaction liquid, precipitate flocs, filter with suction, concentrate the filtrate under reduced pressure, and separate by flash column chromatography to obtain intermediate a15 (700 mg, 4.0 mmol). Yield: 87%. LC-MS: [M+H] + =176.
参照中间体a15的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a15, the following intermediate was synthesized.
中间体a19-a23的合成Synthesis of intermediate a19-a23
步骤1:在50mL反应瓶中加入(3S,4R)-4-氟-3-羟基吡咯烷a19-1(300mg,2.86mmol)和中间体a19-2(1.06g,3.14mmol),加入5mL无水THF溶解。搅拌5分钟后,向反应液中加入NaBH(OAc)
3(1.81g,8.58mmol)和5滴醋酸,室温下反应10小时,停止反应,将反应液倒入150mL冰水中,乙酸乙酯萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,得到1.1g无色油状粗品中间体a19-3,LC-MS:[M+H]
+=428。
Step 1: Add (3S,4R)-4-fluoro-3-hydroxypyrrolidine a19-1 (300mg, 2.86mmol) and intermediate a19-2 (1.06g, 3.14mmol) into a 50mL reaction flask, add 5mL without Aqueous THF dissolves. After stirring for 5 minutes, NaBH(OAc) 3 (1.81g, 8.58mmol) and 5 drops of acetic acid were added to the reaction solution, reacted at room temperature for 10 hours, and the reaction was stopped. The reaction solution was poured into 150mL of ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 1.1 g of the crude intermediate a19-3 as a colorless oil, LC-MS: [M+H] + =428.
步骤2:将上步粗品1.1g中间体a19-3(1.1g)溶于15mL无水THF中,加入3,4-二氢-2H-吡喃(390mg)和对甲苯磺酸(200mg),室温下搅拌1小时,TLC显示反应完毕。将反应液倒入80mL冰水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到1.5g粗品中间体a19-4,LC-MS:[M+H]
+=514。
Step 2: Dissolve 1.1g of intermediate a19-3 (1.1g) of the crude product in the previous step in 15mL of anhydrous THF, add 3,4-dihydro-2H-pyran (390mg) and p-toluenesulfonic acid (200mg), After stirring at room temperature for 1 hour, TLC showed that the reaction was complete. The reaction solution was poured into 80 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain 1.5 g of crude intermediate a19-4, LC-MS: [M+H] + =514.
步骤3:将上步中间体a19-4(1.5g)溶于15mL四氢呋喃中,加入四丁基氟化铵(1.5g)。室温下反应3小时,TLC监测反应完全。将反应液溶于50mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离(PE/EA=1/1),得到无色油状物a19(200mg,0.73mmol),收率:35%,LC-MS:[M+H]
+=274。
Step 3: The above intermediate a19-4 (1.5g) was dissolved in 15mL of tetrahydrofuran, and tetrabutylammonium fluoride (1.5g) was added. The reaction was carried out at room temperature for 3 hours, and the reaction was complete as monitored by TLC. The reaction solution was dissolved in 50 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (PE/EA=1/1) to obtain a19 (200 mg, 0.73 mmol) as a colorless oil , Yield: 35%, LC-MS: [M+H] + =274.
参照中间体a19的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a19, the following intermediate was synthesized.
中间体a24的合成Synthesis of intermediate a24
步骤1:在100mL反应瓶中加入原料3,3-二氟环丁烷-1-胺a24-1(1.0g,9.34mmol)和中间体a19-2(3.16g,9.34mmol),40mL无水THF溶解。搅拌5分钟后,向反应液中加入NaBH(OAc)
3(2.57g,12.34mmol)和10滴醋酸,室温下反应10小时,停止反应,将反应液倒入200mL冰水中,乙酸乙酯萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,flash柱层析分离(PE/EA,1/1)得到2.62g无色油状中间体a24-2,LC-MS:[M+H]
+=430。
Step 1: Add raw material 3,3-difluorocyclobutane-1-amine a24-1 (1.0g, 9.34mmol) and intermediate a19-2 (3.16g, 9.34mmol) in a 100mL reaction flask, 40mL anhydrous THF dissolves. After stirring for 5 minutes, NaBH(OAc) 3 (2.57g, 12.34mmol) and 10 drops of acetic acid were added to the reaction solution, reacted at room temperature for 10 hours, and the reaction was stopped. The reaction solution was poured into 200mL of ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by flash column chromatography (PE/EA, 1/1) to obtain 2.62g of a colorless oily intermediate a24-2, LC-MS: [M+H ] + =430.
步骤2:在100mL反应瓶中加入中间体a24-2(2.62g,5.91mmol)和甲醛水溶液(0.26mL),40mL四氢呋喃溶解。搅拌5分钟后,向反应液中加入NaBH(OAc)
3(1.63g,7.68mmol)和10滴醋酸,室温下反应2小时,停止反应,将反应液倒入100mL冰水中,乙酸乙酯萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,flash柱层析分离(PE/EA,10/1)得到2.0g无色油状中间体a24-3,LC-MS:[M+H]
+=444。
Step 2: Add intermediate a24-2 (2.62g, 5.91mmol) and aqueous formaldehyde (0.26mL) into a 100mL reaction flask, and dissolve in 40mL THF. After stirring for 5 minutes, NaBH(OAc) 3 (1.63g, 7.68mmol) and 10 drops of acetic acid were added to the reaction solution, reacted at room temperature for 2 hours, and the reaction was stopped. The reaction solution was poured into 100mL of ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by flash column chromatography (PE/EA, 10/1) to obtain 2.0 g of a colorless oily intermediate a24-3, LC-MS: [M+H ] + = 444.
步骤3:将上步中间体a24-3(2.0g,4.51mmol)溶于20mL四氢呋喃中,加入四丁基氟化铵(2.2g,9.0mmol)。室温下反应12小时,TLC监测反应完全。将反应液溶于50mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离(DCM/MeOH,1/1),得到无色油状物a24(800mg,0.73mmol),收率:87%,LC-MS:[M+H]
+=206。
Step 3: The above intermediate a24-3 (2.0 g, 4.51 mmol) was dissolved in 20 mL of tetrahydrofuran, and tetrabutylammonium fluoride (2.2 g, 9.0 mmol) was added. The reaction was carried out at room temperature for 12 hours, and the reaction was complete as monitored by TLC. The reaction solution was dissolved in 50 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (DCM/MeOH, 1/1) to obtain a24 (800 mg, 0.73 mmol) as a colorless oil , Yield: 87%, LC-MS: [M+H] + =206.
中间体a25的合成Synthesis of intermediate a25
步骤1:将原料a25-1(1.0g,4.5mmol)和原料a5-1(680mg,5.4mmol)溶于50mL无水二氯甲烷中,加入EDCI(1.29g,6.75mmol)、N,N-二异丙基乙基胺(1.74g,13.5mmol)和HOBt(910mg,6.75 mmol),室温下反应4h,停止反应。向反应液加入到80mL冰水,二氯甲烷萃取,无水硫酸钠干燥,浓缩,得到粗品中间体a25-2(1.21g),LC-MS:[M+H]
+=294。
Step 1: Dissolve raw material a25-1 (1.0g, 4.5mmol) and raw material a5-1 (680mg, 5.4mmol) in 50mL of anhydrous dichloromethane, add EDCI (1.29g, 6.75mmol), N,N- Diisopropylethylamine (1.74g, 13.5mmol) and HOBt (910mg, 6.75mmol) were reacted at room temperature for 4h, and the reaction was stopped. Add 80 mL of ice water to the reaction solution, extract with dichloromethane, dry over anhydrous sodium sulfate, and concentrate to obtain the crude intermediate a25-2 (1.21 g), LC-MS: [M+H] + =294.
步骤2:冰浴下,将中间体a25-2(1.21g,4.13mmol)溶于25mL无水四氢呋喃中,缓慢加入四氢铝锂(310mg,8.3mmol)。升温至室温反应2小时,停止反应。缓慢向反应液加入10%NaOH水溶液2mL,析出絮状物,抽滤,滤液减压浓缩,flash柱层析分离,得到中间体a25(180mg,0.76mmol)。收率:18%。LC-MS:[M+H]
+=238。
Step 2: Intermediate a25-2 (1.21g, 4.13mmol) was dissolved in 25mL of anhydrous THF under ice bath, and lithium aluminum hydride (310mg, 8.3mmol) was added slowly. The temperature was raised to room temperature for 2 hours, and the reaction was stopped. Slowly add 2 mL of 10% NaOH aqueous solution to the reaction liquid, precipitate flocs, filter with suction, concentrate the filtrate under reduced pressure, and separate by flash column chromatography to obtain intermediate a25 (180 mg, 0.76 mmol). Yield: 18%. LC-MS: [M+H] + =238.
中间体a26,a38的合成Synthesis of intermediates a26, a38
步骤1:在50mL反应瓶中加入氧杂环丁烷-3,3-二基二甲醇a26-1(4.1g,33.9mmol)和三乙胺(4.11g,40.63mmol),40mL无水二氯甲烷溶解。搅拌5分钟后,向反应液中加入叔丁基二苯基氯硅烷(7.18g,33.86mmol),室温下反应2小时,停止反应,将反应液倒入200mL冰水中,二氯甲烷萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,得到12g粗品中间体a26-2。Step 1: Add oxetane-3,3-diyldimethanol a26-1 (4.1g, 33.9mmol) and triethylamine (4.11g, 40.63mmol) in a 50mL reaction flask, 40mL of anhydrous dichloro Methane dissolves. After stirring for 5 minutes, tert-butyldiphenylchlorosilane (7.18g, 33.86mmol) was added to the reaction liquid, reacted at room temperature for 2 hours, stopped the reaction, poured the reaction liquid into 200mL ice water, extracted with dichloromethane, organic The phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 12 g of crude intermediate a26-2.
步骤2:-10℃下,将上步粗品中间体a26-2(12.0g)溶于40mL四氢呋喃中,加入三氧化硫吡啶(12.8g,81.0mmol),冰浴下反应3小时,停止反应,将反应液倒入100mL冰水中,二氯甲烷萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,柱层析分离,得到中间体a26-3(8.0g,22.7mmol),两步收率:67%。Step 2: At -10°C, dissolve the crude intermediate a26-2 (12.0 g) from the previous step in 40 mL of tetrahydrofuran, add pyridine sulfur trioxide (12.8 g, 81.0 mmol), and react under ice cooling for 3 hours to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain intermediate a26-3 (8.0 g, 22.7 mmol), two Step yield: 67%.
步骤3:在100mL反应瓶中加入上步中间体a26-3(8.0g,22.7mmol)和中间体a5-1(3.4g,27.1mmol),40mL无水THF溶解。搅拌5分钟后,向反应液中加入NaBH(OAc)
3(7.18g,33.86mmol)和10滴醋酸,室温下反应10小时,停止反应,将反应液倒入200mL冰水中,乙酸乙酯萃取,有机相用饱和食盐水洗涤,无水硫酸钠干燥,浓缩,得到5.0g粗品中间体a26-4,LC-MS:[M+H]
+=428。
Step 3: Add intermediate a26-3 (8.0 g, 22.7 mmol) and intermediate a5-1 (3.4 g, 27.1 mmol) of the previous step into a 100 mL reaction flask, and dissolve in 40 mL of anhydrous THF. After stirring for 5 minutes, NaBH(OAc) 3 (7.18g, 33.86mmol) and 10 drops of acetic acid were added to the reaction solution, reacted at room temperature for 10 hours, and the reaction was stopped. The reaction solution was poured into 200mL of ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 5.0 g of crude intermediate a26-4, LC-MS: [M+H] + =428.
步骤4:将上步粗品a26-4(5.0g,11.69mmol)溶于30mL四氢呋喃中,加入四丁基氟化铵(4.58g,17.5mmol)。升温至40℃下反应12小时,TLC监测反应完全。将反应液溶于150mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离(DCM/MeOH,1/1),得到无色油状物a26(1.9g,10.2mmol),收率:88%,LC-MS:[M+H]
+=190。
Step 4: The above crude product a26-4 (5.0 g, 11.69 mmol) was dissolved in 30 mL of tetrahydrofuran, and tetrabutylammonium fluoride (4.58 g, 17.5 mmol) was added. The temperature was raised to 40° C. for 12 hours, and the reaction was complete as monitored by TLC. The reaction solution was dissolved in 150mL water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (DCM/MeOH, 1/1) to obtain a colorless oil a26 (1.9g, 10.2mmol ), yield: 88%, LC-MS: [M+H] + =190.
参照中间体a26的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a26, the following intermediate was synthesized.
中间体a27-a28的合成Synthesis of intermediate a27-a28
步骤1:室温下,将中间体a1(3.0g,9.15mmol)和原料4,7-二氮杂螺[2.5]辛烷-4-甲酸叔丁酯a27-1(2.12g,10.1mmol)溶于30mL四氢呋喃中,加入N,N-二异丙基乙基胺(2.3g,17.4mmol),室温反应8小时。向体系加水100mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离,得到淡黄色固体a27-2(3.74g,7.4mmol)。收率:81%。LC-MS:[M+H]
+=505。
Step 1: Dissolve intermediate a1 (3.0g, 9.15mmol) and raw material 4,7-diazaspiro[2.5]octane-4-carboxylate tert-butyl a27-1 (2.12g, 10.1mmol) at room temperature In 30 mL of tetrahydrofuran, N,N-diisopropylethylamine (2.3 g, 17.4 mmol) was added, and reacted at room temperature for 8 hours. Add 100 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain a light yellow solid a27-2 (3.74 g, 7.4 mmol). Yield: 81%. LC-MS: [M+H] + = 505.
步骤2:氮气保护下,将中间体a27-2(3.74g,7.4mmol)溶于75mL无水DMF中,加入CsF(3.4g,22.2mmol),升温至60℃下反应8h,停止反应。将反应液加入到300mL水中,混合液用乙酸乙酯分3次萃取,有机相用饱和盐水洗涤,无水硫酸钠干燥,过滤,得到中间体a27,收率90%。LC-MS:[M+H]
+=489。
Step 2: Under the protection of nitrogen, the intermediate a27-2 (3.74g, 7.4mmol) was dissolved in 75mL of anhydrous DMF, CsF (3.4g, 22.2mmol) was added, the temperature was raised to 60°C for 8h, and the reaction was stopped. The reaction solution was added to 300 mL of water, the mixture was extracted three times with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to obtain intermediate a27 with a yield of 90%. LC-MS: [M+H] + = 489.
参照中间体a27的合成路线,合成如下中间体。Referring to the synthetic route of intermediate a27, the following intermediate was synthesized.
中间体a29的合成Synthesis of intermediate a29
步骤1:将2-氟-3-甲基-4-溴吡啶a29-1(4.5g,23.9mmol)溶于25mL四氯化碳中,缓慢加入N-溴代琥珀酰亚胺NBS(6.35g,35.8mmol)和偶氮二异丁腈AIBN(390mg,2.3mmol),室温下反应3小时,停止反应。将该反应液缓慢倒入150mL冰水中,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离(PE/EtOAc,5/1),得到油状物a29-2(6.1g,22.9mmol)。收率:94%。Step 1: Dissolve 2-fluoro-3-methyl-4-bromopyridine a29-1 (4.5g, 23.9mmol) in 25mL carbon tetrachloride, slowly add N-bromosuccinimide NBS (6.35g , 35.8mmol) and azobisisobutyronitrile AIBN (390mg, 2.3mmol), reacted at room temperature for 3 hours, and stopped the reaction. The reaction solution was slowly poured into 150 mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (PE/EtOAc, 5/1) to obtain oil a29-2 (6.1 g, 22.9 mmol). Yield: 94%.
步骤2:将中间体a29-2(5.2g,3.7mmol)和三甲基氰基硅烷TMSCN(2.9g,5.6mmol)溶于20mL乙腈中,加入含有TBAF(5.5mmol)的四氢呋喃(29mL)溶液,室温下反应16小时,停止反应。减压蒸除溶剂,柱层析分离(PE/EtOAc,5/1),得到油状物a29-3(3.4g,15.9mmol)。收率:81%。Step 2: Intermediate a29-2 (5.2g, 3.7mmol) and trimethylcyanosilane TMSCN (2.9g, 5.6mmol) were dissolved in 20mL of acetonitrile, and a solution of tetrahydrofuran (29mL) containing TBAF (5.5mmol) was added , reacted at room temperature for 16 hours, and stopped the reaction. The solvent was evaporated under reduced pressure and separated by column chromatography (PE/EtOAc, 5/1) to obtain oil a29-3 (3.4g, 15.9mmol). Yield: 81%.
步骤3:冰浴下,将上步中间体a29-3(3.4g,15.9mmol)溶于20mL DMF中,缓慢加入NaH(2.9g,19.1mmol),搅拌30分钟后,溶液变红,缓慢逐滴加入提前配制的异硫氰酰甲酸乙酯的DMF溶液 (1.8g,15.9mmol,5mL)。将该反应液升温至100℃下反应1小时,冷却至室温。向该反应液缓慢倒入冰水淬灭反应,乙酸乙酯萃取,无水硫酸钠干燥,粗品经柱层析分离(PE/EtOAc,1/1),得到淡黄色固体a29-4(1.05g,3.2mmol)。收率:20%。LC-MS:[M+H]
+=325。
Step 3: Under ice bath, dissolve the above intermediate a29-3 (3.4g, 15.9mmol) in 20mL DMF, slowly add NaH (2.9g, 19.1mmol), stir for 30 minutes, the solution turns red, slowly gradually A DMF solution of ethyl isothiocyanate (1.8 g, 15.9 mmol, 5 mL) prepared in advance was added dropwise. The reaction solution was heated up to 100° C. to react for 1 hour, and then cooled to room temperature. The reaction solution was slowly poured into ice water to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was separated by column chromatography (PE/EtOAc, 1/1) to obtain a light yellow solid a29-4 (1.05g ,3.2mmol). Yield: 20%. LC-MS: [M+H] + = 325.
步骤4:将上步中间体a29-4(1.0g,3.1mmol)溶于10mL DMSO中,加入10mL氢氧化钠水溶液(5M)。回流条件下反应4小时,停止反应。冷却至室温,向该反应液缓慢加入100mL冰水淬灭反应,乙酸乙酯萃取,无水硫酸钠干燥,粗品经柱层析分离(PE/EtOAc,1/1),得到淡黄色固体a29-5(450mg,1.8mmol)。收率:20%。LC-MS:[M+H]
+=254。
Step 4: Dissolve the intermediate a29-4 (1.0 g, 3.1 mmol) in 10 mL of DMSO, and add 10 mL of aqueous sodium hydroxide solution (5M). React under reflux conditions for 4 hours, stop the reaction. After cooling to room temperature, 100 mL of ice water was slowly added to the reaction solution to quench the reaction, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the crude product was separated by column chromatography (PE/EtOAc, 1/1) to obtain a light yellow solid a29- 5 (450 mg, 1.8 mmol). Yield: 20%. LC-MS: [M+H] + = 254.
步骤5:将上步中间体a29-5(450mg,1.8mmol)和DMAP(5mg,0.04mmol)溶于20mL THF/DMF的混合溶液(v/v,1/1)中,加入Boc酸酐(465mg,2.16mmol)。室温反应12小时,停止反应。向体系加水50mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,得到粗品中间体a29-6(760mg)。Step 5: Dissolve the intermediate a29-5 (450mg, 1.8mmol) and DMAP (5mg, 0.04mmol) in 20mL THF/DMF mixed solution (v/v, 1/1), add Boc anhydride (465mg ,2.16mmol). React at room temperature for 12 hours, then stop the reaction. Add 50 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain crude intermediate a29-6 (760 mg).
步骤6:氮气保护下,将粗品a29-6(760mg)和原料a7-8(590mg,2.6mmol)溶于10mL 1,4-二氧六环中,缓慢加入醋酸钾(432mg,4.32mmol)和Pd(DPEphos)Cl
2(46mg,0.065mmol),升温至100℃反应1小时,停止反应。冷却至室温,过滤,饱和食盐水洗涤,二氯甲烷萃取,干燥,浓缩,flash柱层析分离,得到中间体a29(320mg,0.82mmol)。LC-MS:[M+H]
+=388。
Step 6: Under nitrogen protection, the crude product a29-6 (760mg) and raw material a7-8 (590mg, 2.6mmol) were dissolved in 10mL 1,4-dioxane, and potassium acetate (432mg, 4.32mmol) and Pd(DPEphos)Cl 2 (46mg, 0.065mmol), the temperature was raised to 100°C for 1 hour, and the reaction was stopped. Cool to room temperature, filter, wash with saturated brine, extract with dichloromethane, dry, concentrate, and separate by flash column chromatography to obtain intermediate a29 (320mg, 0.82mmol). LC-MS: [M+H] + =388.
中间体a31的合成Synthesis of intermediate a31
步骤1:氮气保护下,将原料a31-1(10.0g,40.5mmol)溶于100mL无水四氢呋喃中,缓慢加入LiAlH
4(2.3g,60.7mmol),室温下反应2小时,LC-MS监测反应完全,停止反应。向体系加入300mL冰水,减压蒸除有机溶剂,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到淡黄色油状物a31-2(5.9g,26.9mmol),收率:67%。LC-MS:[M+H]+=220。
Step 1: Under the protection of nitrogen, dissolve the raw material a31-1 (10.0g, 40.5mmol) in 100mL of anhydrous tetrahydrofuran, slowly add LiAlH 4 (2.3g, 60.7mmol), react at room temperature for 2 hours, and monitor the reaction by LC-MS Completely, stop the reaction. Add 300mL of ice water to the system, distill off the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain light yellow oil a31-2 (5.9g, 26.9mmol) , Yield: 67%. LC-MS: [M+H]+=220.
步骤2:冰浴,氮气保护下,将上步中间体a31-2(5.9g,26.9mmol)和咪唑(3.66g,53.8mmol)溶于50mL无水二氯甲烷中,缓慢滴加叔丁基二苯基氯硅烷TBDPSCl(7.4g,26.92mmol)。反应液在室温下搅拌30分钟,LC-MS监测反应完全,停止反应。向反应液加入100mL冰水,二氯甲烷萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得到淡黄色固体物a31-3(11.4g,24.9mmol),收率:93%。LC-MS:[M+H]
+=458。
Step 2: In ice bath, under the protection of nitrogen, dissolve the intermediate a31-2 (5.9g, 26.9mmol) and imidazole (3.66g, 53.8mmol) in 50mL of anhydrous dichloromethane, and slowly add tert-butyl Diphenylchlorosilane TBDPSCl (7.4 g, 26.92 mmol). The reaction solution was stirred at room temperature for 30 minutes, and the reaction was stopped by monitoring the completion of the reaction by LC-MS. Add 100 mL of ice water to the reaction solution, extract with dichloromethane, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography to obtain light yellow solid a31-3 (11.4 g, 24.9 mmol), yield: 93%. LC-MS: [M+H] + = 458.
步骤3:将上步中间体a31-3(11.4g,24.93mmol)溶于氯化氢的1,4-二氧六环溶液中(4M,30mL),室温下搅拌1小时,停止反应,抽滤,滤饼干燥,得到盐酸盐a31-4(7.0g,17.8mmol),收率:71%。LC-MS:[M+H]
+=358。
Step 3: Dissolve the intermediate a31-3 (11.4g, 24.93mmol) in the 1,4-dioxane solution of hydrogen chloride (4M, 30mL), stir at room temperature for 1 hour, stop the reaction, and filter with suction. The filter cake was dried to obtain hydrochloride a31-4 (7.0 g, 17.8 mmol), yield: 71%. LC-MS: [M+H] + = 358.
步骤4:氮气保护下,将上步中间体a31-4(7.0g,17.8mmol)、原料环丁基-1,1-二羧酸单乙酯a31-5(4.6g,26.7mmol)和DIEA(6.2mL)溶于60mL DMF中,加入HATU(10.2g,26.7mmol),反应液在室温下反应4小时,停止反应。向体系加入200mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得褐色固体物a31-6(7.78g,15.2mmol),收率:85%。LC-MS:[M+H]
+=512。
Step 4: Under the protection of nitrogen, the intermediate a31-4 (7.0g, 17.8mmol) of the previous step, the raw material cyclobutyl-1,1-dicarboxylate monoethyl ester a31-5 (4.6g, 26.7mmol) and DIEA (6.2mL) was dissolved in 60mL DMF, HATU (10.2g, 26.7mmol) was added, the reaction solution was reacted at room temperature for 4 hours, and the reaction was stopped. Add 200mL of ice water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, the crude product is separated by column chromatography to obtain brown solid a31-6 (7.78g, 15.2mmol), yield: 85%. LC-MS: [M+H] + = 512.
步骤5:将上步中间体a31-6(7.78g,15.2mmol)和四丁基氟化铵TBAF(7.96g,30.44mmol)溶于80mL四氢呋喃中,升温至50℃下反应6小时,停止反应。向体系加入200mL水,减压蒸除有机溶剂,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得褐色油状物a31-7(2.3g,8.42 mmol),收率:55%。LC-MS:[M+H]
+=274。
Step 5: Dissolve the intermediate a31-6 (7.78g, 15.2mmol) and tetrabutylammonium fluoride TBAF (7.96g, 30.44mmol) in 80mL tetrahydrofuran, raise the temperature to 50°C for 6 hours, and stop the reaction . Add 200 mL of water to the system, distill off the organic solvent under reduced pressure, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by column chromatography to obtain brown oil a31-7 (2.3 g, 8.42 mmol), yield : 55%. LC-MS: [M+H] + =274.
步骤6:将上步中间体a31-7(2.3g,8.42mmol)、二氢吡喃DHP(2.12g,25.3mmol)和对甲苯磺酸TsOH(145mg,0.84mmol)溶于20mL二氯甲烷中,反应液在室温下搅拌5小时,LC-MS监测反应完全,停止反应。向体系加入80mL冰水,二氯甲烷萃取,减压蒸除溶剂,粗品经柱层析分离,得油状物a31-8(1.5g,4.2mmol),收率:50%。LC-MS:[M+H]
+=358。
Step 6: Dissolve the above intermediate a31-7 (2.3g, 8.42mmol), dihydropyran DHP (2.12g, 25.3mmol) and p-toluenesulfonic acid TsOH (145mg, 0.84mmol) in 20mL of dichloromethane , the reaction solution was stirred at room temperature for 5 hours, and the LC-MS monitored that the reaction was complete, and the reaction was stopped. Add 80 mL of ice water to the system, extract with dichloromethane, evaporate the solvent under reduced pressure, and separate the crude product by column chromatography to obtain oil a31-8 (1.5 g, 4.2 mmol), yield: 50%. LC-MS: [M+H] + = 358.
步骤7:氮气保护下,将上步中间体a31-8(1.5g,4.2mmol)溶于20mL无水四氢呋喃中,缓慢加入LiAlH
4(319mg,8.40mmol),室温下搅拌2小时,LC-MS监测反应完全,向体系加入100mL冰水淬灭反应。混合物用乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得褐色固体a31(760mg,2.5mmol),收率:60%。LC-MS:[M+H]
+=302。
Step 7: Under the protection of nitrogen, the intermediate a31-8 (1.5g, 4.2mmol) in the previous step was dissolved in 20mL of anhydrous tetrahydrofuran, LiAlH 4 (319mg, 8.40mmol) was slowly added, stirred at room temperature for 2 hours, LC-MS After monitoring the completion of the reaction, 100 mL of ice water was added to the system to quench the reaction. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain a brown solid a31 (760 mg, 2.5 mmol), yield: 60%. LC-MS: [M+H] + =302.
中间体a32的合成Synthesis of intermediate a32
步骤1:冰浴,将原料2-硝基-4-溴-5-羟基-苯甲酸甲酯a32-1(25g,90.6mmol)和三乙胺(27.5g,272mmol)溶于250mL二氯甲烷中,缓慢滴加乙酰氯(10.66g,135.9mmol)。滴毕,继续反应3小时,停止反应。向体系加入500mL水,二氯甲烷萃取,减压蒸除溶剂,得到粗品中间体a32-2。Step 1: In ice bath, the starting materials 2-nitro-4-bromo-5-hydroxy-benzoic acid methyl ester a32-1 (25g, 90.6mmol) and triethylamine (27.5g, 272mmol) were dissolved in 250mL of dichloromethane In, acetyl chloride (10.66g, 135.9mmol) was slowly added dropwise. After dropping, the reaction was continued for 3 hours, and the reaction was stopped. Add 500 mL of water to the system, extract with dichloromethane, and distill off the solvent under reduced pressure to obtain the crude intermediate a32-2.
步骤2:将上步粗品a32-2和还原铁粉(27.2g,487.3mmol)溶于310mL冰醋酸中,升温至40℃下反应2小时,停止反应。粗品经硅藻土过滤,滤液减压浓缩。向滤液中加入饱和碳酸氢钠水溶液调节pH至8左右,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到粗品中间体a32-3(16.7g)。LC-MS:[M+H]
+=290。
Step 2: Dissolve the crude product a32-2 and reduced iron powder (27.2g, 487.3mmol) in 310mL of glacial acetic acid, raise the temperature to 40°C for 2 hours, and stop the reaction. The crude product was filtered through celite, and the filtrate was concentrated under reduced pressure. Add saturated aqueous sodium bicarbonate solution to the filtrate to adjust the pH to about 8, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate to obtain the crude intermediate a32-3 (16.7 g). LC-MS: [M+H] + =290.
步骤3:将上步粗品a32-3和1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷二(四氟硼酸)盐(selectfluor,30.8g,87.0mmol)溶于170mL乙腈中,室温下反应15小时,停止反应。向反应液加入饱和碳酸氢钠水溶液调节pH至8左右,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到化合物a32-4(1.6g,5.2mmol),三步收率:6%。LC-MS:[M+H]
+=306。
Step 3: Mix the crude product a32-3 and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane di(tetrafluoroborate) salt (selectfluor, 30.8g, 87.0 mmol) was dissolved in 170 mL of acetonitrile, reacted at room temperature for 15 hours, and stopped the reaction. Add saturated aqueous sodium bicarbonate solution to the reaction solution to adjust the pH to about 8, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound a32-4 (1.6g, 5.2mmol), three Step yield: 6%. LC-MS: [M+H] + = 306.
步骤4:将上步中间体a32-4(1.6g,5.2mmol)和碳酸钾(1.44g,10.5mmol)溶于16mL甲醇中,室温下反应2小时,停止反应。反应液用1M稀盐酸水溶液调节pH至5左右,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到化合物a32-5(1.2g,4.54mmol),收率87%。LC-MS:[M+H]
+=264。
Step 4: Dissolve the above intermediate a32-4 (1.6g, 5.2mmol) and potassium carbonate (1.44g, 10.5mmol) in 16mL of methanol, react at room temperature for 2 hours, and stop the reaction. The pH of the reaction solution was adjusted to about 5 with 1M dilute hydrochloric acid aqueous solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to obtain compound a32-5 (1.2 g, 4.54 mmol), yield 87 %. LC-MS: [M+H] + = 264.
步骤5:冰浴,将上步中间体a32-5(1.2g,4.54mmol)和碳酸铯(2.96g,9.1mmol)溶于12mL DMF中,搅拌5分钟后,缓慢滴加碘甲烷(710mg,5.0mmol),滴毕,继续反应2小时,停止反应。向反应液加入50mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,减压浓缩,粗品经柱层析分离,得到化合物a32-6(1.1g,3.96mmol),收率87%。LC-MS:[M+H]
+=278。
Step 5: In ice bath, dissolve the intermediate a32-5 (1.2g, 4.54mmol) and cesium carbonate (2.96g, 9.1mmol) in 12mL DMF, stir for 5 minutes, slowly add methyl iodide (710mg, 5.0mmol), after dropping, the reaction was continued for 2 hours, and the reaction was stopped. 50 mL of ice water was added to the reaction solution, extracted with ethyl acetate, washed with saturated brine, concentrated under reduced pressure, and the crude product was separated by column chromatography to obtain compound a32-6 (1.1 g, 3.96 mmol), with a yield of 87%. LC-MS: [M+H] + =278.
步骤6/7:将上步中间体a32-6(1.1g,3.96mmol)溶于11mL甲醇中,滴加NaOH水溶液(791mg,19.8mmol),室温下反应6小时,停止反应。向反应液中加入氰酸钾水溶液(963mg,11.88mmol), 升温至50℃下反应2小时,反应过程中用6M浓度稀盐酸调节pH至6.5左右。LC-MS监测反应完成,停止反应,抽滤,得到灰色固体a32-8(1.1g,3.81mmol)。LC-MS:[M+H]
+=291。
Step 6/7: The above intermediate a32-6 (1.1g, 3.96mmol) was dissolved in 11mL of methanol, NaOH aqueous solution (791mg, 19.8mmol) was added dropwise, reacted at room temperature for 6 hours, and the reaction was stopped. Aqueous potassium cyanate solution (963mg, 11.88mmol) was added to the reaction solution, and the temperature was raised to 50°C for 2 hours. During the reaction, the pH was adjusted to about 6.5 with 6M dilute hydrochloric acid. The completion of the reaction was monitored by LC-MS, the reaction was stopped, and suction filtration was performed to obtain a gray solid a32-8 (1.1 g, 3.81 mmol). LC-MS: [M+H] + =291.
步骤8:将上步中间体a32-8(1.1g,3.81mmol)和DIEA(984mg,7.62mmol)溶于11mL三氯氧磷中,升温至90℃下反应15小时,停止反应。将反应液减压浓缩,粗品缓慢倒入100mL冰水中,析出固体,抽滤,滤饼用水洗涤,干燥,得到中间体11(1.2g,3.68mmol)。收率96.6%。LC-MS:[M+H]
+=325。
Step 8: Dissolve the intermediate a32-8 (1.1 g, 3.81 mmol) and DIEA (984 mg, 7.62 mmol) in 11 mL of phosphorus oxychloride, raise the temperature to 90°C for 15 hours, and stop the reaction. The reaction solution was concentrated under reduced pressure, and the crude product was slowly poured into 100 mL of ice water to precipitate a solid, which was filtered with suction, and the filter cake was washed with water and dried to obtain intermediate 11 (1.2 g, 3.68 mmol). Yield 96.6%. LC-MS: [M+H] + = 325.
关键中间体b1-b10制备Preparation of key intermediate b1-b10
中间体b1的合成Synthesis of intermediate b1
步骤1:室温下,将原料b1-1(10.00g,56.8mmol),甲氧基胺盐酸盐(6.73g,83.2mmol)和吡啶(5.69g,68.10mmol)溶于10mL无水乙醇中。反应液于室温下反应2小时,停止反应,减压浓缩。残余物溶于二氯甲烷中,分别用稀盐酸(2N)、饱和碳酸氢钠水溶液、饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,得到粗品无色油状物b1-2(11.30g,55.1mmol)。LC-MS:[M+H]
+=206。
Step 1: Dissolve raw material b1-1 (10.00g, 56.8mmol), methoxylamine hydrochloride (6.73g, 83.2mmol) and pyridine (5.69g, 68.10mmol) in 10mL absolute ethanol at room temperature. The reaction solution was reacted at room temperature for 2 hours, then the reaction was stopped, and concentrated under reduced pressure. The residue was dissolved in dichloromethane, washed with dilute hydrochloric acid (2N), saturated aqueous sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product b1-2 (11.30g ,55.1mmol). LC-MS: [M+H] + = 206.
步骤2:将上步中间体b1-2(1.0g,4.9mmol)、醋酸钯(55mg,0.24mmol)和NBS(0.87g,4.87mmol)溶于10mL无水乙酸中,反应液升温至80℃下反应1小时,停止反应,冷却至室温,将反应液倒入水中,过滤,滤饼干燥,得到褐色固体b1-3(1.03g,3.6mmol)。LC-MS:[M+H]
+=284。
Step 2: Dissolve the above intermediate b1-2 (1.0g, 4.9mmol), palladium acetate (55mg, 0.24mmol) and NBS (0.87g, 4.87mmol) in 10mL of anhydrous acetic acid, and heat the reaction solution to 80°C The reaction was stopped for 1 hour, cooled to room temperature, the reaction liquid was poured into water, filtered, and the filter cake was dried to obtain a brown solid b1-3 (1.03 g, 3.6 mmol). LC-MS: [M+H] + =284.
步骤3:将上步中间体b1-3(12.5g,43.99mmol)溶于60mL浓盐酸和100mL 1,4-二氧六环的混合溶液中。反应液升温至回流下搅拌1小时,停止反应,减压浓缩。残余物溶于乙酸乙酯,经氢氧化钠水溶液(1N)、饱和食盐水洗涤,无水硫酸钠干燥,浓缩,flash柱层析分离(PE/EA=4/1),得到黄色固体b1-4(10.9g,42.7mmol),收率:97%。LC-MS:[M+H]
+=255。
Step 3: Dissolve the intermediate b1-3 (12.5g, 43.99mmol) in the above step in a mixed solution of 60mL concentrated hydrochloric acid and 100mL 1,4-dioxane. The reaction solution was heated to reflux and stirred for 1 hour, then the reaction was stopped, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with aqueous sodium hydroxide solution (1N) and saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by flash column chromatography (PE/EA=4/1) to obtain a yellow solid b1- 4 (10.9 g, 42.7 mmol), yield: 97%. LC-MS: [M+H] + = 255.
步骤4:氮气保护下,将中间体b1-4(7.90g,30.97mmol)和1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷二(四氟硼酸)盐(Selectfluor,16.46g,46.5mmol)溶于80mL甲醇中,缓慢滴加0.3mL浓硫酸。反应液升温至50℃下反应5小时,停止反应,减压浓缩。残余物溶解于乙酸乙酯,饱和食盐水洗涤,无水硫酸钠干燥,过滤,浓缩,flash柱层析分离(PE/EA=10/1),得到红色固体b1-5(6.37g,23.35mmol),收率:75%。LC-MS:[M+H]
+=273。
Step 4: Under nitrogen protection, the intermediate b1-4 (7.90g, 30.97mmol) and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoro Borate) salt (Selectfluor, 16.46g, 46.5mmol) was dissolved in 80mL of methanol, and 0.3mL of concentrated sulfuric acid was slowly added dropwise. The reaction solution was heated to 50° C. for 5 hours, then the reaction was stopped, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by flash column chromatography (PE/EA=10/1) to obtain a red solid b1-5 (6.37g, 23.35mmol ), yield: 75%. LC-MS: [M+H] + =273.
步骤5:氮气保护下,将中间体b1-5(32.0g,117.2mmol)和三溴化吡啶盐(41.22g,128.89mmol)溶于300mL乙腈中,升温至60℃下反应0.5小时,停止反应,减压蒸除溶剂。饱和食盐水洗涤,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离(PE/EA=10/1),得到黄色固体b1-6(36.0g,102.3mmol),收率:87%。LC-MS:[M+H]
+=350。
Step 5: Under the protection of nitrogen, the intermediate b1-5 (32.0g, 117.2mmol) and pyridinium tribromide salt (41.22g, 128.89mmol) were dissolved in 300mL of acetonitrile, and the temperature was raised to 60°C for 0.5 hours to stop the reaction , and distill off the solvent under reduced pressure. Washed with saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (PE/EA=10/1) to obtain a yellow solid b1-6 (36.0g, 102.3mmol). Rate: 87%. LC-MS: [M+H] + =350.
步骤6:氮气保护下,将中间体b1-6(36.0g,102.3mmol)和溴化锂(19.5g,225mmol)溶于100 mL DMF中,升温至100℃下反应0.5小时,停止反应。向体系中加入300mL水,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,过滤,浓缩。粗品经flash柱层析分离(PE/EA=10/1),得到淡黄色固体b1-7(21.0g,77.5mmol),收率:75%。LC-MS:[M+H]
+=271。
Step 6: Under the protection of nitrogen, the intermediate b1-6 (36.0g, 102.3mmol) and lithium bromide (19.5g, 225mmol) were dissolved in 100 mL DMF, heated to 100°C and reacted for 0.5 hours to stop the reaction. Add 300 mL of water to the system, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate. The crude product was separated by flash column chromatography (PE/EA=10/1) to obtain a pale yellow solid b1-7 (21.0 g, 77.5 mmol), yield: 75%. LC-MS: [M+H] + =271.
步骤7:冰浴,氮气保护下,将中间体b1-7(21.0g,77.5mmol)和吡啶(18.38g,232.41mmol)溶于200mL二氯甲烷中。缓慢向反应液滴加三氟甲磺酸酐(26.2g,92.96mmol),缓慢升至室温下反应1小时,停止反应,减压蒸除溶剂。粗品用饱和食盐水洗涤,二氯甲烷萃取,无水硫酸钠干燥,浓缩,经flash柱层析分离(PE/EA=8/1),得到黄色固体b1-8(27.50g,68.2mmol),收率:88%。LC-MS:[M+H]
+=403。
Step 7: In ice bath, under the protection of nitrogen, the intermediate b1-7 (21.0 g, 77.5 mmol) and pyridine (18.38 g, 232.41 mmol) were dissolved in 200 mL of dichloromethane. Trifluoromethanesulfonic anhydride (26.2 g, 92.96 mmol) was slowly added dropwise to the reaction solution, and the mixture was slowly raised to room temperature for 1 hour to stop the reaction, and the solvent was evaporated under reduced pressure. The crude product was washed with saturated brine, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and separated by flash column chromatography (PE/EA=8/1) to obtain a yellow solid b1-8 (27.50g, 68.2mmol), Yield: 88%. LC-MS: [M+H] + =403.
步骤8:氮气保护下,将中间体b1-8(1.0g,2.48mmol),氰化锌(146mg,1.24mmol),Pd
2(dba)
3(114mg,0.12mmol)和1,1'-双(二苯基膦)二茂铁(dppf,137mg,0.25mmol)溶于10mL无水DMF中。反应液升温至70℃下反应3小时,冷却至室温。将粗品倒入50mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩。粗品经flash柱层析色谱分离(PE/EA=5/1),得到白色固体b1-9(270mg,0.96mmol),收率:38%。LC-MS:[M+H]
+=208。
Step 8: Under nitrogen protection, intermediate b1-8 (1.0g, 2.48mmol), zinc cyanide (146mg, 1.24mmol), Pd 2 (dba) 3 (114mg, 0.12mmol) and 1,1'-bis (Diphenylphosphine)ferrocene (dppf, 137 mg, 0.25 mmol) was dissolved in 10 mL of anhydrous DMF. The reaction liquid was heated to 70° C. for 3 hours and then cooled to room temperature. The crude product was poured into 50 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA=5/1) to obtain a white solid b1-9 (270 mg, 0.96 mmol), yield: 38%. LC-MS: [M+H] + =208.
步骤9:-78℃,氮气保护下,将中间体b1-9(50mg,0.18mmol)溶于2mL二氯甲烷中,缓慢滴加0.44mL三溴化硼二氯甲烷溶液(浓度2M)。将体系缓慢升温至0℃下反应16小时,加入10mL甲醇淬灭反应。减压蒸除溶剂,粗品经flash柱层析色谱分离(PE/EA=3/1),得到白色固体b1-10(20mg,0.075mmol),收率:42%。LC-MS:[M+H]
+=266。
Step 9: Dissolve intermediate b1-9 (50mg, 0.18mmol) in 2mL dichloromethane at -78°C under nitrogen protection, and slowly add 0.44mL boron tribromide dichloromethane solution (concentration 2M) dropwise. The system was heated slowly to 0°C for 16 hours, and 10 mL of methanol was added to quench the reaction. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography (PE/EA=3/1) to obtain a white solid b1-10 (20 mg, 0.075 mmol), yield: 42%. LC-MS: [M+H] + = 266.
步骤10:氮气保护下,将中间体b1-10(430mg,26.7mmol),联硼酸频那醇酯(823mg,3.24mmol),乙酸钾(477mg,4.86mmol),Pd
2(dba)
3(74mg,0.081mmol)和三环己基膦(45mg,0.016mmol)溶于8mL 1,4-二氧六环中,升温至105℃下反应10小时,停止反应。冷却至室温,过滤,将体系倒入30mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩。粗品经flash柱层析色谱分离(PE/EA=3/1),得到淡黄色固体b1(350mg,6.2mmol),收率:69%。LC-MS:[M+H]+=314。
Step 10: Under nitrogen protection, intermediate b1-10 (430mg, 26.7mmol), pinacol diboronate (823mg, 3.24mmol), potassium acetate (477mg, 4.86mmol), Pd 2 (dba) 3 (74mg , 0.081mmol) and tricyclohexylphosphine (45mg, 0.016mmol) were dissolved in 8mL 1,4-dioxane, heated to 105°C and reacted for 10 hours to stop the reaction. Cool to room temperature, filter, pour the system into 30 mL of ice water, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate. The crude product was separated by flash column chromatography (PE/EA=3/1) to obtain light yellow solid b1 (350 mg, 6.2 mmol), yield: 69%. LC-MS: [M+H]+=314.
中间体b2的合成Synthesis of intermediate b2
步骤1:将原料4-氟苯乙酸b2-1(50.0g,324.4mmol)和丙二酸环(亚)异丙酯b2-2(51.4g,356.8mmol)溶于500mL乙腈中,加入4-二甲氨基吡啶(DMAP,3.57g,29.2mmol)和DIEA(88.0g,681.2mmol)。搅拌5分钟后,缓慢滴加特戊酰氯(43.0g,356.8mmol)。反应液升温至45℃下搅拌3小时后,冷却至室温。将反应液置于冰浴下,滴加4N盐酸水溶液将pH调节至5左右,继续搅拌1小时后,用水稀释,再次用4N盐酸将反应液pH调节至2左右,有大量固体析出,抽滤,用水洗涤滤饼,干燥,得到白色固体b2-3(104g,371.1mmol),收率定量。LC-MS:[M+H]
+=281。
Step 1: Dissolve the raw material 4-fluorophenylacetic acid b2-1 (50.0g, 324.4mmol) and cyclo(ethylene)isopropyl malonate b2-2 (51.4g, 356.8mmol) in 500mL of acetonitrile, add 4- Dimethylaminopyridine (DMAP, 3.57 g, 29.2 mmol) and DIEA (88.0 g, 681.2 mmol). After stirring for 5 minutes, pivaloyl chloride (43.0 g, 356.8 mmol) was slowly added dropwise. The reaction solution was heated to 45°C and stirred for 3 hours, then cooled to room temperature. Put the reaction solution under an ice bath, add dropwise 4N hydrochloric acid aqueous solution to adjust the pH to about 5, continue to stir for 1 hour, dilute with water, and adjust the pH of the reaction solution to about 2 with 4N hydrochloric acid again, a large amount of solids are precipitated, filter with suction , washed the filter cake with water, dried to obtain a white solid b2-3 (104g, 371.1mmol), and the yield was quantitative. LC-MS: [M+H] + =281.
步骤2:将上步中间体b2-3(54.0g,192.7mmol)缓慢加入到三氟甲磺酸(228.5g,1.5mol)中。反应液在室温下搅拌2小时,LC-MS监测反应完全。将反应液缓慢倒入500mL冰水中,有固体析出,抽滤,用水洗涤滤饼,干燥,得到棕色固体b2-4(66.0g,295.96mmol)。收率:93%,LC-MS:[M+H]
+ =223。
Step 2: Slowly add intermediate b2-3 (54.0 g, 192.7 mmol) in the above step into trifluoromethanesulfonic acid (228.5 g, 1.5 mol). The reaction solution was stirred at room temperature for 2 hours, and the reaction was complete as monitored by LC-MS. The reaction solution was slowly poured into 500 mL of ice water, solids precipitated out, filtered with suction, the filter cake was washed with water, and dried to obtain a brown solid b2-4 (66.0 g, 295.96 mmol). Yield: 93%, LC-MS: [M+H] + =223.
步骤3:将上步中间体b2-4(66.0g,295.96mmol)溶于660mL乙腈和水的混合溶液中(v/1,1/1),升温至80℃下反应13小时,停止反应。减压蒸除溶剂,饱和碳酸氢钠水溶液洗涤,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到淡黄色化合物b2-5(51.8g,291.01mmol),收率:97%。LC-MS:[M+H]
+=179。
Step 3: Dissolve the intermediate b2-4 (66.0g, 295.96mmol) in the above step in a mixed solution of 660mL acetonitrile and water (v/1, 1/1), raise the temperature to 80°C for 13 hours, and stop the reaction. The solvent was evaporated under reduced pressure, washed with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain pale yellow compound b2-5 (51.8 g, 291.01 mmol), yield: 97%. LC-MS: [M+H] + =179.
步骤4:氮气保护下,将粗品b2-5(20.0g,112.3mmol),(2-溴乙炔基)三异丙基硅烷(30.8g,112.3mmol),乙酸钾(22.0g,224.5mmol)和二氯双(4-甲基异丙基苯基)钌(II)(2.06g,3.4mmol)溶于200mL1,4-二氧六环中,升温至100℃下反应4小时,LC-MS监测反应完全,过滤。减压蒸除溶剂,向体系加水100mL,乙酸乙酯萃取,用无水硫酸钠干燥,浓缩。粗品经flash柱层析色谱分离(PE/EA=10/1),得到淡黄色固体b2-6(28.0g,78.2mmol),收率:69%。LC-MS:[M+H]
+=359。
Step 4: Under nitrogen protection, the crude product b2-5 (20.0g, 112.3mmol), (2-bromoethynyl)triisopropylsilane (30.8g, 112.3mmol), potassium acetate (22.0g, 224.5mmol) and Dichlorobis(4-methylisopropylphenyl)ruthenium(II) (2.06g, 3.4mmol) was dissolved in 200mL of 1,4-dioxane, heated to 100°C for 4 hours, monitored by LC-MS The reaction is complete and filtered. The solvent was evaporated under reduced pressure, 100 mL of water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA=10/1) to obtain light yellow solid b2-6 (28.0 g, 78.2 mmol), yield: 69%. LC-MS: [M+H] + = 359.
步骤5:将上步中间体b2-6(28g,78.2mmol)和DIEA(20.19g,156.20mmol)溶于300mL二氯甲烷中,缓慢滴加三异丙基氯硅烷TIPSCl(18.1g,93.7mmol),滴毕,反应液在室温下搅拌1小时,LC-MS监测反应完全。将反应液倒入500mL冰水,二氯甲烷萃取,无水硫酸钠干燥,浓缩,得到粗品红色油状化合物b2-7。LC-MS:[M+H]
+=515。
Step 5: Dissolve the intermediate b2-6 (28g, 78.2mmol) and DIEA (20.19g, 156.20mmol) in 300mL of dichloromethane, and slowly add triisopropylchlorosilane TIPSCl (18.1g, 93.7mmol) ), after dropping, the reaction solution was stirred at room temperature for 1 hour, and the LC-MS monitoring reaction was complete. The reaction solution was poured into 500 mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated to obtain crude magenta oily compound b2-7. LC-MS: [M+H] + = 515.
步骤6:氮气保护,-40℃下,将粗品b2-7(58.4g,113.43mmol)和DIEA(51.3g,397.0mmol)溶于300mL二氯甲烷中,缓慢滴加三氟甲磺酸酐(54.4g,192.8mmol),3小时滴毕,继续搅拌0.5小时,停止反应。将反应液倒入500mL冰水中,二氯甲烷萃取,无水硫酸钠干燥,浓缩。粗品经flash柱层析色谱分离(PE/EA=10/1),得到淡红色油状化合物b2-8(57.7g,89.2mmol),收率:78%。LC-MS:[M+H]
+=647。
Step 6: Under nitrogen protection, at -40°C, the crude product b2-7 (58.4g, 113.43mmol) and DIEA (51.3g, 397.0mmol) were dissolved in 300mL of dichloromethane, and trifluoromethanesulfonic anhydride (54.4 g, 192.8mmol), after 3 hours of dripping, the stirring was continued for 0.5 hour, and the reaction was stopped. The reaction solution was poured into 500 mL of ice water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by flash column chromatography (PE/EA=10/1) to obtain light red oily compound b2-8 (57.7g, 89.2mmol), yield: 78%. LC-MS: [M+H] + = 647.
步骤7:氮气保护下,将中间体b2-8(61.6g,95.2mmol)、三乙胺(38.5g,380.9mmol)和频那醇硼烷(48.7g,380.9mmol)溶于600mL乙腈中,搅拌5分钟后,加入催化剂Pd(dppf)Cl
2(4.2g,5.7mmol)。反应液升温至80℃下搅拌4小时,冷却至室温。将混合物用MeOH缓慢淬灭反应,温度保持在25℃以下,有固体析出。抽滤,MeOH洗涤滤饼,干燥,得到白色固体化合物b2(45.9g,73.4mmol),收率:77%。LC-MS:[M+H]
+=625。
Step 7: Under nitrogen protection, the intermediate b2-8 (61.6g, 95.2mmol), triethylamine (38.5g, 380.9mmol) and pinacol borane (48.7g, 380.9mmol) were dissolved in 600mL of acetonitrile, After stirring for 5 minutes, the catalyst Pd(dppf) Cl2 (4.2 g, 5.7 mmol) was added. The reaction solution was heated to 80°C and stirred for 4 hours, then cooled to room temperature. The mixture was slowly quenched with MeOH, keeping the temperature below 25 °C, and a solid precipitated out. After suction filtration, the filter cake was washed with MeOH and dried to obtain compound b2 (45.9 g, 73.4 mmol) as a white solid, yield: 77%. LC-MS: [M+H] + = 625.
中间体b3的合成Synthesis of intermediate b3
步骤1:将原料b3-1(6.0g,15.5mmol)和AIBN(1.27g,7.75mmol)溶于60mL四氯化碳中,缓慢加入N-溴代琥珀酰亚胺NBS(13.8g,77.5mmol)。升温至60℃反应6小时,停止反应。减压蒸除溶剂,残余物经flash柱层析色谱分离(石油醚/乙酸乙酯=10/1),得到淡黄色固体b3-2(7.6g,14.0mmol),收率:90%。LC-MS:[M+H]
+=543。
Step 1: Dissolve raw material b3-1 (6.0g, 15.5mmol) and AIBN (1.27g, 7.75mmol) in 60mL carbon tetrachloride, slowly add NBS (13.8g, 77.5mmol ). The temperature was raised to 60° C. for 6 hours, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the residue was separated by flash column chromatography (petroleum ether/ethyl acetate=10/1) to obtain a light yellow solid b3-2 (7.6 g, 14.0 mmol), yield: 90%. LC-MS: [M+H] + = 543.
步骤2:将上步中间体b3-2(7.6g,14mmol)和DIEA(1.8g,14.0mmol)溶于80mL乙腈中,缓慢加入亚磷酸二乙酯(1.95g,14.0mmol),室温下反应1小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=10/1),得到淡黄色固体b3-3(4.6g,10.0mmol),收率:72%。LC-MS:[M+H]
+=465。
Step 2: Dissolve the intermediate b3-2 (7.6g, 14mmol) and DIEA (1.8g, 14.0mmol) in 80mL of acetonitrile, slowly add diethyl phosphite (1.95g, 14.0mmol), and react at room temperature 1 hour, stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=10/1) to obtain a light yellow solid b3-3 (4.6g, 10.0mmol), yield: 72%. LC-MS: [M+H] + = 465.
步骤3:将上步中间体b3-3(4.6g,10mmol)、碳酸钾(2.8g,20.0mmol)和三甲基硅基氰基TMSCN(1.5g,15.0mmol)溶于50mL乙腈中,室温下反应4小时,停止反应。将反应液倒入300mL冰水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=3/1),得到淡黄色固体b3-4(3.6g,8.76mmol),收率:87%。LC-MS:[M+H]
+=412。
Step 3: Dissolve the intermediate b3-3 (4.6g, 10mmol), potassium carbonate (2.8g, 20.0mmol) and trimethylsilyl cyano TMSCN (1.5g, 15.0mmol) in 50mL of acetonitrile at room temperature The reaction was carried out for 4 hours, and the reaction was stopped. The reaction solution was poured into 300 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=3/1) to obtain a light yellow solid b3-4 (3.6g, 8.76mmol), yield: 87%. LC-MS: [M+H] + = 412.
步骤4:冰浴,氮气保护下,将上步中间体b3-4(3.6g,8.76mmol)溶于40mL无水四氢呋喃中,加入NaH(700mg,17.5mmol),搅拌10分钟后,逐滴加入1,2-二溴乙烷(2.0g,10.5mmol),滴毕,升温至室温反应10小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=3/1),得到淡黄色固体b3(1.2g,3.56mmol),收率:40%。LC-MS:[M+H]
+=338。
Step 4: In ice bath, under the protection of nitrogen, dissolve the intermediate b3-4 (3.6g, 8.76mmol) of the previous step in 40mL of anhydrous tetrahydrofuran, add NaH (700mg, 17.5mmol), stir for 10 minutes, and then add dropwise 1,2-Dibromoethane (2.0g, 10.5mmol), after dropping, warmed up to room temperature and reacted for 10 hours, then stopped the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=3/1) to obtain a light yellow solid b3 (1.2 g, 3.56 mmol), yield: 40%. LC-MS: [M+H] + =338.
中间体b4-b6的合成Synthesis of intermediates b4-b6
步骤1:将原料4-溴-7-氟-1H-吲哚(3.5g,16.4mmol)溶于35mL DMF中,加入N-碘代琥珀酰亚胺NIS(3.69g,16.4mmol),升温至60℃下反应1小时,停止反应。将反应液倒入150mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=3/1),得到淡黄色固体b4-2(2.0g,5.9mmol),收率:36%。LC-MS:[M+H]
+=340。
Step 1: Dissolve the raw material 4-bromo-7-fluoro-1H-indole (3.5g, 16.4mmol) in 35mL DMF, add N-iodosuccinimide NIS (3.69g, 16.4mmol), and heat up to The reaction was carried out at 60° C. for 1 hour, and then the reaction was terminated. The reaction solution was poured into 150 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=3/1) to obtain light Yellow solid b4-2 (2.0 g, 5.9 mmol), yield: 36%. LC-MS: [M+H] + =340.
步骤2:冰浴,氮气保护下,将上步中间体3-碘-4-溴-7-氟-1H-吲哚b4-2(2.0g,5.9mmol)溶于20mL无水四氢呋喃中,加入NaH(235mg,8.9mmol)和碘甲烷(92mg,6.5mmol),升温至室温反应2小时,停止反应。向反应液加入80mL饱和氯化铵水溶液,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=3/1),得到淡黄色固体b4-3(1.1g,3.12mmol),收率:53%。LC-MS:[M+H]
+=354。
Step 2: In ice bath, under the protection of nitrogen, dissolve the intermediate 3-iodo-4-bromo-7-fluoro-1H-indole b4-2 (2.0g, 5.9mmol) in 20mL of anhydrous tetrahydrofuran, add NaH (235mg, 8.9mmol) and iodomethane (92mg, 6.5mmol) were heated to room temperature for 2 hours and the reaction was stopped. Add 80 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography (petroleum ether/ethyl acetate=3/1) to obtain a light yellow solid b4 -3 (1.1 g, 3.12 mmol), yield: 53%. LC-MS: [M+H] + = 354.
步骤3:氮气保护下,将上步中间体b4-3(1.1g,3.12mmol)和CuCN(1.1g,12.4mmol)溶于20mL DMSO中,升温至100℃下反应3小时,冷却至室温。将反应液倒入到150mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得到淡黄色固体b4-4(0.55g,2.2mmol),收率:70%。LC-MS:[M+H]
+=253。
Step 3: Under the protection of nitrogen, the intermediate b4-3 (1.1g, 3.12mmol) and CuCN (1.1g, 12.4mmol) were dissolved in 20mL DMSO, heated to 100°C for 3 hours, and cooled to room temperature. The reaction solution was poured into 150 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain Pale yellow solid b4-4 (0.55g, 2.2mmol), yield: 70%. LC-MS: [M+H] + = 253.
步骤4:氮气保护下,将上步中间体b4-4(350mg,1.4mmol)、联硼酸频哪醇酯(706mg,2.8mmol)和醋酸钾(408mg,4.2mmol)溶于10mL 1,4-二氧六环中,加入催化剂Pd(dppf)Cl
2(100mg,0.14mmol),升温至100℃下反应8小时,冷却至室温。将反应液倒入到50mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得到淡黄色固体b4(0.27g,0.9mmol),收率:65%。LC-MS:[M+H]
+=301。
Step 4: Under the protection of nitrogen, the intermediate b4-4 (350mg, 1.4mmol), biboronic acid pinacol ester (706mg, 2.8mmol) and potassium acetate (408mg, 4.2mmol) were dissolved in 10mL of 1,4- Add catalyst Pd(dppf)Cl 2 (100mg, 0.14mmol) to dioxane, heat up to 100°C and react for 8 hours, then cool to room temperature. The reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain Pale yellow solid b4 (0.27g, 0.9mmol), yield: 65%. LC-MS: [M+H] + = 301.
参照中间体b4的合成路线,合成如下中间体。Referring to the synthetic route of intermediate b4, the following intermediates were synthesized.
中间体b7的合成Synthesis of intermediate b7
步骤1:将原料b7-1(1.0g,5.9mmol)溶于10mL DMF中,加入N-碘代琥珀酰亚胺NIS(1.99g,8.85mmol),升温至70℃下反应1小时,停止反应。将反应液倒入50mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=9/1),得到黄色固体b7-2(800mg,2.70mmol),收率:46%。LC-MS:[M+H]
+=296。
Step 1: Dissolve raw material b7-1 (1.0g, 5.9mmol) in 10mL DMF, add N-iodosuccinimide NIS (1.99g, 8.85mmol), heat up to 70°C for 1 hour, stop the reaction . The reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=9/1) to obtain a yellow Solid b7-2 (800mg, 2.70mmol), yield: 46%. LC-MS: [M+H] + = 296.
步骤2:冰浴,氮气保护下,将上步中间体b7-2(800mg,2.70mmol)、无水甲醇(430mg,13.6mmol)和三苯基膦(1.07g,4.1mmol)溶于20mL DMF中,向反应液中加入偶氮二甲酸二乙酯(710mg,4.1mmol),升温至室温反应12小时,停止反应。向反应液加入80mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=5/1),得到黄色固体b7-3(530mg,1.72mmol),收率:64%。LC-MS:[M+H]
+=310。
Step 2: In ice bath, under the protection of nitrogen, the intermediate b7-2 (800mg, 2.70mmol), anhydrous methanol (430mg, 13.6mmol) and triphenylphosphine (1.07g, 4.1mmol) were dissolved in 20mL DMF , diethyl azodicarboxylate (710 mg, 4.1 mmol) was added to the reaction solution, and the temperature was raised to room temperature for 12 hours to stop the reaction. 80 mL of ice water was added to the reaction solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=5/1) to obtain a yellow solid b7-3 (530 mg, 1.72 mmol), yield: 64%. LC-MS: [M+H] + =310.
步骤3:氮气保护下,将上步中间体b7-3(530mg,1.71mmol)和CuCN(610mg,5.84mmol)溶于20mL DMSO中,升温至100℃下反应6小时,冷却至室温。将反应液倒入到80mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=3/1),得到黄色固体b7(85mg,0.41mmol),收率:24%。LC-MS:[M+H]
+=209。
Step 3: Under the protection of nitrogen, the intermediate b7-3 (530mg, 1.71mmol) and CuCN (610mg, 5.84mmol) were dissolved in 20mL DMSO, heated to 100°C for 6 hours, and cooled to room temperature. The reaction solution was poured into 80 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=3/1) to obtain Yellow solid b7 (85 mg, 0.41 mmol), yield: 24%. LC-MS: [M+H] + =209.
中间体b8的合成Synthesis of intermediate b8
步骤1:氮气保护下,将化合物b8-1(300mg,1.23mmol)和异硫氰酰甲酸乙酯(240mg,1.84mmol)溶于15mL 1,4-二氧六环中,升温至100℃下反应4小时,停止反应。向该反应液缓慢倒入50mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(PE/EA=5/1),得到化合物b8-2(210mg,0.59mmol),收率:48%。LC-MS:[M+H]
+=356。
Step 1: Under nitrogen protection, compound b8-1 (300mg, 1.23mmol) and ethyl isothiocyanate (240mg, 1.84mmol) were dissolved in 15mL 1,4-dioxane, and the temperature was raised to 100°C After reacting for 4 hours, stop the reaction. The reaction solution was slowly poured into 50 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (PE/EA=5/1) to obtain compound b8-2 (210 mg, 0.59 mmol), yield: 48%. LC-MS: [M+H] + = 356.
步骤2:将上步中间体b8-2(210mg,0.59mmol)溶于8mL DMSO中,加入4mL氢氧化钠水溶液(5N),回流条件下反应4小时,停止反应。冷却至室温,向该反应液缓慢加入50mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离(PE/EA=3/1),得到化合物b8-3,收率定量。LC-MS:[M+H]
+=283。
Step 2: Dissolve the intermediate b8-2 (210mg, 0.59mmol) in 8mL DMSO, add 4mL sodium hydroxide aqueous solution (5N), react under reflux for 4 hours, and stop the reaction. After cooling to room temperature, 50 mL of ice water was slowly added to the reaction solution, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (PE/EA=3/1) to obtain Compound b8-3, quantitative yield. LC-MS: [M+H] + =283.
步骤3:将上步中间体b8-3(175mg,0.62mmol)、三乙胺(190mg,1.86mmol)、4-二甲氨基吡啶DMAP(15mg,0.12mmol)和二碳酸二叔丁酯(160mg,0.74mmol)溶于20mL二氯甲烷中,室温下反应4小时,停止反应。向体系加水80mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,得到 粗品中间体b8-4。LC-MS:[M+H]
+=383。
Step 3: The above intermediate b8-3 (175mg, 0.62mmol), triethylamine (190mg, 1.86mmol), 4-dimethylaminopyridine DMAP (15mg, 0.12mmol) and di-tert-butyl dicarbonate (160mg , 0.74mmol) was dissolved in 20mL of dichloromethane, reacted at room temperature for 4 hours, and stopped the reaction. Add 80 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude intermediate b8-4. LC-MS: [M+H] + =383.
步骤7:氮气保护下,将粗品b8-4(100mg,0.26mmol)、醋酸钾(77mg,0.78mmol)和联硼酸频哪醇酯(79mg,0.31mmol)溶于7mL 1,4-二氧六环中,加入催化剂醋酸钯(12mg,0.052mmol),升温至100℃下反应3小时,停止反应。冷却至室温,过滤,减压蒸除溶剂,粗品经flash柱层析分离(PE/EA=5/1),得到中间体b8(60mg,0.14mmol),收率:54%。LC-MS:[M+H]
+=431。
Step 7: Under nitrogen protection, the crude product b8-4 (100mg, 0.26mmol), potassium acetate (77mg, 0.78mmol) and biboronic acid pinacol ester (79mg, 0.31mmol) were dissolved in 7mL 1,4-dioxane The catalyst palladium acetate (12 mg, 0.052 mmol) was added to the ring, and the temperature was raised to 100° C. for 3 hours to stop the reaction. Cool to room temperature, filter, evaporate the solvent under reduced pressure, and separate the crude product by flash column chromatography (PE/EA=5/1) to obtain intermediate b8 (60mg, 0.14mmol), yield: 54%. LC-MS: [M+H] + = 431.
中间体b9的合成Synthesis of intermediate b9
步骤1:将原料2-氨基-3-氰基-4-溴吲哚b9-1(3.8g,16.10mmol)和4-二甲氨基吡啶DMAP(196mg,1.61mmol)溶于50mL四氢呋喃中,缓慢加入二碳酸二叔丁酯(14.1g,64.4mmol),室温下反应10小时,停止反应。向反应液中加入200mL冰水,乙酸乙酯萃取,浓缩,得到粗品b9-2。LC-MS:[M+H]
+=436。
Step 1: The starting material 2-amino-3-cyano-4-bromoindole b9-1 (3.8g, 16.10mmol) and 4-dimethylaminopyridine DMAP (196mg, 1.61mmol) were dissolved in 50mL THF, slowly Add di-tert-butyl dicarbonate (14.1 g, 64.4 mmol), react at room temperature for 10 hours, and stop the reaction. Add 200 mL of ice water to the reaction liquid, extract with ethyl acetate, and concentrate to obtain the crude product b9-2. LC-MS: [M+H] + = 436.
步骤2:氮气保护下,将上步粗品b9-2(600mg,1.38mmol)、醋酸钾(405mg,4.1mmol)和联硼酸频哪醇酯(489mg,1.93mmol)溶于10mL 1,4-二氧六环中,加入催化剂Pd(dppf)Cl
2(102mg,0.14mmol),升温至100℃下反应2小时,停止反应。过滤,减压蒸除溶剂,粗品经flash柱层析分离,得到中间体b9(190mg,0.39mmol)。LCMS:[M+1]
+=484。
Step 2: Under the protection of nitrogen, the crude product b9-2 (600mg, 1.38mmol), potassium acetate (405mg, 4.1mmol) and pinacol borate (489mg, 1.93mmol) were dissolved in 10mL of 1,4-di Add catalyst Pd(dppf)Cl 2 (102mg, 0.14mmol) to the oxane, heat up to 100°C and react for 2 hours, then stop the reaction. After filtration, the solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain intermediate b9 (190mg, 0.39mmol). LCMS: [M+1] + =484.
中间体b10的合成Synthesis of intermediate b10
步骤1:氮气保护下,将化合物4-溴-苯并呋喃-2-甲酸b10-1(5.5g,23mmol)和三乙胺(6.0mL)溶于50mL叔丁醇中,加入叠氮磷酸二苯酯DPPA(9.4g,7.4mL),搅拌10分钟后升温至95℃下反应3小时,停止反应。减压蒸除溶剂,粗品溶于50mL二氯甲烷中,加入二碳酸二叔丁酯(5.0g,23mmol),室温反应2小时,停止反应。将反应液倒入100mL冰水中,二氯甲烷萃取,减压蒸除溶剂,粗品经flash柱层析分离,得到化合物b10-2(4.0g,12.9mmol),收率:56%。LCMS:[M+1]
+=312。
Step 1: Under the protection of nitrogen, the compound 4-bromo-benzofuran-2-carboxylic acid b10-1 (5.5g, 23mmol) and triethylamine (6.0mL) were dissolved in 50mL tert-butanol, and diphosphoric acid diazide was added Phenyl ester DPPA (9.4g, 7.4mL) was stirred for 10 minutes and then heated to 95°C for 3 hours to stop the reaction. The solvent was evaporated under reduced pressure, the crude product was dissolved in 50 mL of dichloromethane, di-tert-butyl dicarbonate (5.0 g, 23 mmol) was added, and the reaction was carried out at room temperature for 2 hours to stop the reaction. The reaction solution was poured into 100 mL ice water, extracted with dichloromethane, and the solvent was evaporated under reduced pressure. The crude product was separated by flash column chromatography to obtain compound b10-2 (4.0 g, 12.9 mmol), yield: 56%. LCMS: [M+1] + =312.
步骤2:-70℃,氮气保护下,将上步中间体b10-2(1.0g,3.2mmol)溶于10mL无水四氢呋喃中,加入原料氯磺酸异氰酸酯(0.42mL),搅拌1小时后,再次补加氯磺酸异氰酸酯(0.42mL),继续反应1小时,升温至室温。向反应液中加入10mL无水DMF,搅拌1小时后,停止反应。将反应液倒入100mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离得到化合物b10-3(0.5g,1.5mmol),收率46%。LCMS:[M+1]
+=337。
Step 2: Dissolve the intermediate b10-2 (1.0g, 3.2mmol) in 10mL of anhydrous tetrahydrofuran at -70°C under the protection of nitrogen, add the raw material chlorosulfonic acid isocyanate (0.42mL), and stir for 1 hour. Chlorosulfonic acid isocyanate (0.42 mL) was added again, the reaction was continued for 1 hour, and the temperature was raised to room temperature. 10 mL of anhydrous DMF was added to the reaction solution, and after stirring for 1 hour, the reaction was stopped. The reaction solution was poured into 100 mL ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to obtain compound b10-3 (0.5 g, 1.5 mmol), yield 46 %. LCMS: [M+1] + =337.
步骤3:氮气保护下,将上步中间体b10-3(0.5g,1.5mmol)、醋酸钾(440mg,4.5mmol)和联硼酸频哪醇酯(460mg,1.8mmol)溶于10mL 1,4-二氧六环中,加入Pd(dppf)
2Cl
2(11mg,0.15mmol),升温至100℃下反应2小时,停止反应,过滤。向反应液中加入50mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离得到化合物b10(270mg,0.7mmol),收率46%。LCMS:[M+1]
+=385。
Step 3: Under the protection of nitrogen, the intermediate b10-3 (0.5g, 1.5mmol), potassium acetate (440mg, 4.5mmol) and biboronic acid pinacol ester (460mg, 1.8mmol) were dissolved in 10mL 1,4 - Add Pd(dppf) 2 Cl 2 (11mg, 0.15mmol) to dioxane, heat up to 100°C and react for 2 hours, stop the reaction, and filter. Add 50 mL of ice water to the reaction liquid, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound b10 (270 mg, 0.7 mmol), with a yield of 46%. LCMS: [M+1] + =385.
关键中间体c1-c12制备Preparation of key intermediates c1-c12
中间体c1-c8的合成Synthesis of intermediates c1-c8
步骤1:冰浴下,将原料c1-1(20.0g,81.5mmol)和TEA(16.5g,163.1mmol)溶于250mL二氯甲烷中,缓慢向体系加入甲磺酸酐(15.6g,89.7mmol),滴毕,升温至室温反应2小时,停止反应。向体系中加入300mL冰水,二氯甲烷萃取,无水硫酸钠干燥,浓缩,得粗品c1-2,LC-MS:[M+H]
+=324。
Step 1: Dissolve raw material c1-1 (20.0g, 81.5mmol) and TEA (16.5g, 163.1mmol) in 250mL dichloromethane under ice bath, and slowly add methanesulfonic anhydride (15.6g, 89.7mmol) to the system After dropping, the temperature was raised to room temperature for 2 hours, and the reaction was stopped. Add 300 mL of ice water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, and concentrate to obtain crude product c1-2, LC-MS: [M+H] + =324.
步骤2:将粗品c1-2(25g,77.3mmol)溶于300mL DMF中,室温下加入甲硫醇钠(6.5g,92.8mmol),升温至90℃下搅拌16小时,停止反应。将反应液倒入500mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,浓缩,粗品经柱层析分离,得到化合物c1-3(10g,36.4mmol),收率47%。LC-MS:[M+H]
+=275。
Step 2: Dissolve the crude product c1-2 (25g, 77.3mmol) in 300mL DMF, add sodium methylthiolate (6.5g, 92.8mmol) at room temperature, raise the temperature to 90°C and stir for 16 hours to stop the reaction. The reaction solution was poured into 500 mL ice water, extracted with ethyl acetate, washed with saturated brine, concentrated, and the crude product was separated by column chromatography to obtain compound c1-3 (10 g, 36.4 mmol), with a yield of 47%. LC-MS: [M+H] + = 275.
步骤3:-78℃,氮气保护下,将中间体c1-3(16.5g,51.0mmol)溶于200mL无水四氢呋喃中,缓慢滴加LDA(12.8g,76.6mmol),滴毕,继续搅拌0.5小时。缓慢向体系滴加1-溴-3-氯丙烷(40.2g,255.2mmol),滴加完毕,升温至室温继续反应1小时,向反应液中加入100mL冰水淬灭。乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,得到粗品c1-4,LC-MS:[M+H]
+=352。
Step 3: Dissolve the intermediate c1-3 (16.5g, 51.0mmol) in 200mL of anhydrous tetrahydrofuran at -78°C under the protection of nitrogen, slowly add LDA (12.8g, 76.6mmol) dropwise, and continue stirring for 0.5 Hour. 1-Bromo-3-chloropropane (40.2 g, 255.2 mmol) was slowly added dropwise to the system. After the dropwise addition was completed, the temperature was raised to room temperature to continue the reaction for 1 hour, and 100 mL of ice water was added to the reaction solution to quench it. Extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain crude product c1-4, LC-MS: [M+H] + =352.
步骤4:将上步粗品c1-4溶于50mL二氯甲烷中,加入150mL三氟乙酸,室温搅拌1小时,停止反应。减压蒸除溶剂,混合物用饱和碳酸氢钠水溶液调至弱碱性,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析纯化得化合物c1-5(2.5g)与c1-6(1.3g),LC-MS:[M+H]
+=252。
Step 4: Dissolve the crude product c1-4 in the previous step in 50 mL of dichloromethane, add 150 mL of trifluoroacetic acid, stir at room temperature for 1 hour, and stop the reaction. The solvent was evaporated under reduced pressure, the mixture was adjusted to weak alkaline with saturated aqueous sodium bicarbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by column chromatography to obtain compound c1-5 (2.5 g) and c1- 6 (1.3 g), LC-MS: [M+H] + =252.
步骤5:将中间体c1-5(2.5g,10.5mmol)、碘化钾(0.17g,1.1mmol)和碳酸钾(6.9g,21.0mmol)溶于25mL甲醇中,室温下反应16小时,停止反应。向体系加入100mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,得到粗品c1-7,LC-MS:[M+H]
+=216。
Step 5: Dissolve intermediate c1-5 (2.5g, 10.5mmol), potassium iodide (0.17g, 1.1mmol) and potassium carbonate (6.9g, 21.0mmol) in 25mL of methanol, react at room temperature for 16 hours, and stop the reaction. Add 100 mL of ice water to the system, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain the crude product c1-7, LC-MS: [M+H] + =216.
步骤6:冰浴,将粗品c1-7(2.1g,9.8mmol)溶于20mL四氢呋喃中,加入四氢铝锂(750mg,19.5mmol),继续搅拌1小时,停止反应。向体系加入10mL甲醇淬灭,过滤,减压浓缩,向该混合物加水50mL,二氯甲烷萃取,无水硫酸钠干燥,浓缩,得到粗品c1(直接用于下一步反应)。LC-MS:[M+H]
+=188。
Step 6: In an ice bath, dissolve the crude product c1-7 (2.1 g, 9.8 mmol) in 20 mL of tetrahydrofuran, add lithium aluminum hydride (750 mg, 19.5 mmol), and continue stirring for 1 hour to stop the reaction. Add 10 mL of methanol to the system to quench, filter, concentrate under reduced pressure, add 50 mL of water to the mixture, extract with dichloromethane, dry over anhydrous sodium sulfate, and concentrate to obtain crude product c1 (directly used in the next reaction). LC-MS: [M+H] + =188.
步骤7:将中间体c1-5替换为c1-6,得到中间体c2。Step 7: Substitution of intermediate c1-5 by c1-6 yields intermediate c2.
参照中间体c1的合成路线,合成如下中间体。Referring to the synthetic route of intermediate c1, the following intermediates were synthesized.
中间体c9的合成Synthesis of intermediate c9
步骤1:冰浴,将原料c9-1(15.0g,71.0mmol),溶于150mL无水四氢呋喃(150mL)中,缓慢加入硼氢化钠(806mg,21.3mmol),在冰浴下反应3小时,停止反应。减压蒸除溶剂,向混合物中加入50mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩。粗品经柱层析分离,得到中间体c9-2(12.0g,56.3mmol)。LC-MS:[M+H]
+=214。
Step 1: In ice bath, dissolve raw material c9-1 (15.0 g, 71.0 mmol) in 150 mL of anhydrous tetrahydrofuran (150 mL), slowly add sodium borohydride (806 mg, 21.3 mmol), and react under ice bath for 3 hours, Stop responding. The solvent was evaporated under reduced pressure, 50 mL of ice water was added to the mixture, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by column chromatography to obtain intermediate c9-2 (12.0 g, 56.3 mmol). LC-MS: [M+H] + =214.
步骤2:冰浴,将上步中间体c9-2(12.0g,56.3mmol)和三乙胺(17.3g,170.5mmol)溶于120mL二氯甲烷中。搅拌5分钟后,向反应液中滴加甲磺酸酐(14.9g,85.2mmol)的二氯甲烷溶液(20mL),滴毕,继续搅拌1小时。停止反应,向体系加入200mL冰水,二氯甲烷萃取,减压浓缩,得到粗品至c9-3。Step 2: In an ice bath, the intermediate c9-2 (12.0 g, 56.3 mmol) and triethylamine (17.3 g, 170.5 mmol) were dissolved in 120 mL of dichloromethane. After stirring for 5 minutes, a dichloromethane solution (20 mL) of methanesulfonic anhydride (14.9 g, 85.2 mmol) was added dropwise to the reaction liquid, and the stirring was continued for 1 hour after the dropping was completed. Stop the reaction, add 200 mL of ice water to the system, extract with dichloromethane, and concentrate under reduced pressure to obtain the crude product to c9-3.
步骤3:将粗品c9-3和硫代乙酸钾(9.4g,82.4mmol)溶于150mL DMF中,升温至60℃下反应15小时,停止反应。向体系中加入300mL冰水,乙酸乙酯萃取3次,合并有机相,饱和食盐水洗涤,无水硫酸钠干燥,减压浓缩,粗品经柱层析分离得到中间体c9-4(15g,55.4mmol),两步收率:10%。LC-MS:[M+H]
+=272。
Step 3: The crude product c9-3 and potassium thioacetate (9.4 g, 82.4 mmol) were dissolved in 150 mL of DMF, and the temperature was raised to 60° C. for 15 hours to stop the reaction. Add 300mL ice water to the system, extract 3 times with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, the crude product is separated by column chromatography to obtain intermediate c9-4 (15g, 55.4 mmol), two-step yield: 10%. LC-MS: [M+H] + =272.
步骤4:冰浴,将上步中间体c9-4(15g,55.4mmol)溶于300mL无水四氢呋喃中,加入四氢铝锂(5.2g,138mmol)。搅拌5分钟后,撤去冰浴,升温至60℃下反应2小时。停止反应。向体系加入稀盐酸淬灭反应,调节pH至7左右,析出固体,抽滤,滤饼用乙酸乙酯洗涤,得到中间体c9。LC-MS:[M+H]
+=174。
Step 4: In ice bath, the intermediate c9-4 (15g, 55.4mmol) of the previous step was dissolved in 300mL of anhydrous tetrahydrofuran, and lithium aluminum tetrahydride (5.2g, 138mmol) was added. After stirring for 5 minutes, the ice bath was removed, and the temperature was raised to 60° C. to react for 2 hours. Stop responding. Dilute hydrochloric acid was added to the system to quench the reaction, and the pH was adjusted to about 7. A solid was precipitated, filtered with suction, and the filter cake was washed with ethyl acetate to obtain intermediate c9. LC-MS: [M+H] + =174.
中间体c10的合成Synthesis of intermediate c10
步骤:将中间体c9(2.0g,11.5mmol)溶于20mL DMF中,加入NaH(920mg,23mmol)。室温下反应30分钟。降温至-40℃,向体系加入三氟碘甲烷(3.4g,17.3mmol)的DMF溶液(5mL)。缓慢升温至室温并继续搅拌1小时,停止反应。向反应液加入50mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,减压浓缩,flash柱层析分离,得到中间体c10(1.3g,5.4mmol),收率47%。LC-MS:[M+H]
+=242。
Step: Intermediate c9 (2.0 g, 11.5 mmol) was dissolved in 20 mL of DMF, and NaH (920 mg, 23 mmol) was added. React at room temperature for 30 minutes. The temperature was lowered to -40°C, and a DMF solution (5 mL) of trifluoroiodomethane (3.4 g, 17.3 mmol) was added to the system. Slowly warm to room temperature and continue stirring for 1 hour to stop the reaction. 50 mL of ice water was added to the reaction liquid, extracted with ethyl acetate, washed with saturated brine, concentrated under reduced pressure, and separated by flash column chromatography to obtain intermediate c10 (1.3 g, 5.4 mmol) with a yield of 47%. LC-MS: [M+H] + = 242.
中间体c11的合成Synthesis of intermediate c11
步骤:冰浴,将中间体c9(2.2g,12.7mmol)溶于30mL DMSO中,加入氢化钠(1.02g,25.9mmol)。搅拌30分钟后,加入溴代环丙烷(2.3g,19.0mmol),室温下反应1小时,停止反应。向体系中加入100mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,减压浓缩,得到中间体c11。LC-MS:[M+H]
+=214。
Step: In ice bath, the intermediate c9 (2.2g, 12.7mmol) was dissolved in 30mL DMSO, and sodium hydride (1.02g, 25.9mmol) was added. After stirring for 30 minutes, bromocyclopropane (2.3 g, 19.0 mmol) was added and reacted at room temperature for 1 hour to stop the reaction. Add 100 mL of ice water to the system, extract with ethyl acetate, wash with saturated brine, and concentrate under reduced pressure to obtain intermediate c11. LC-MS: [M+H] + =214.
中间体c12的合成Synthesis of intermediate c12
步骤:冰浴,将中间体c9(1.6g,9.2mmol)溶于30mL DMSO中,加入氢化钠(738mg,18.4mmol)。搅拌30分钟后,加入溴代环丁烷(1.87g,13.9mmol),室温下反应1小时,停止反应。室温下反应1小时,停止反应。向体系中加入100mL冰水,乙酸乙酯萃取,饱和食盐水洗涤,减压浓缩,得到中间体c12。LC-MS:[M+H]
+=228。
Step: In ice bath, the intermediate c9 (1.6g, 9.2mmol) was dissolved in 30mL DMSO, and sodium hydride (738mg, 18.4mmol) was added. After stirring for 30 minutes, bromocyclobutane (1.87 g, 13.9 mmol) was added and reacted at room temperature for 1 hour to stop the reaction. After reacting at room temperature for 1 hour, the reaction was stopped. Add 100 mL of ice water to the system, extract with ethyl acetate, wash with saturated brine, and concentrate under reduced pressure to obtain intermediate c12. LC-MS: [M+H] + = 228.
关键中间体d1-d5制备Preparation of key intermediates d1-d5
中间体d1的合成Synthesis of intermediate d1
步骤1:冰浴,在250mL的反应瓶中,将原料d1-1(5.0g,18.6mmol)溶于80mL吡啶中,缓慢向体系中滴加20mL草酰氯单乙酯,滴毕,室温下搅拌1小时后,升温至50℃反应2小时,停止反应。将反应液缓慢倒入300mL冰水中,抽滤,滤饼干燥,得到中间体d1-2(5.2g,14.8mmol),收率80%。LC-MS:[M+H]
+=350。
Step 1: In an ice bath, in a 250mL reaction flask, dissolve the raw material d1-1 (5.0g, 18.6mmol) in 80mL of pyridine, slowly add 20mL monoethyl oxalyl chloride dropwise to the system, after the drop is complete, stir at room temperature After 1 hour, the temperature was raised to 50° C. for 2 hours to stop the reaction. The reaction solution was slowly poured into 300 mL of ice water, suction filtered, and the filter cake was dried to obtain intermediate d1-2 (5.2 g, 14.8 mmol), with a yield of 80%. LC-MS: [M+H] + =350.
步骤2:将上步中间体d1-2(5.2g,14.8mmol)溶于100mL无水乙醇中,加入4.0g醋酸铵和1mL醋酸。升温至回流反应1小时,冷却至室温。向体系缓慢滴加1M浓度稀盐酸调节pH至5左右,析出固体,抽滤,滤饼干燥,得到粗品d1-3。LC-MS:[M+H]
+=349。
Step 2: Dissolve the above intermediate d1-2 (5.2g, 14.8mmol) in 100mL of absolute ethanol, add 4.0g of ammonium acetate and 1mL of acetic acid. Raise the temperature to reflux for 1 hour, and cool to room temperature. Slowly add 1M dilute hydrochloric acid dropwise to the system to adjust the pH to about 5, a solid precipitates out, is suction filtered, and the filter cake is dried to obtain the crude product d1-3. LC-MS: [M+H] + = 349.
步骤3:将上步粗品d1-3和DIEA(2.2g,17.2mmol)溶于20mL三氯氧磷中,升温至85℃下反应2小时,停止反应。减压蒸除溶剂,将粗品缓慢倒入100mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得到化合物d1-4(2.5g,6.8mmol),两步收率46%。LC-MS:[M+H]
+=367。
Step 3: Dissolve the crude product d1-3 and DIEA (2.2 g, 17.2 mmol) in 20 mL of phosphorus oxychloride, raise the temperature to 85° C. for 2 hours, and stop the reaction. The solvent was evaporated under reduced pressure, the crude product was slowly poured into 100 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain compound d1-4 (2.5 g, 6.8 mmol). rate 46%. LC-MS: [M+H] + = 367.
步骤4:冰浴,将上步中间体d1-4(2.5g,6.8mmol)和DIEA(1.75g,13.6mmol)溶于20mL无水四氢呋喃中,将提前配置的a2-1(1.44g,6.8mmol)的THF溶液(3.0mL)缓慢滴加至反应液中,滴毕,升温至室温反应40分钟,停止反应。向体系加入100mL水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到化合物d1(3.5g,6.4mmol),收率94%。LC-MS:[M+H]
+=543。
Step 4: In an ice bath, dissolve the intermediate d1-4 (2.5g, 6.8mmol) and DIEA (1.75g, 13.6mmol) in 20mL of anhydrous tetrahydrofuran, and a2-1 (1.44g, 6.8 mmol) THF solution (3.0 mL) was slowly added dropwise to the reaction solution, after the drop was completed, the temperature was raised to room temperature and reacted for 40 minutes, and the reaction was stopped. 100 mL of water was added to the system, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain compound d1 (3.5 g, 6.4 mmol), with a yield of 94%. LC-MS: [M+H] + = 543.
中间体d2的合成Synthesis of intermediate d2
步骤:氮气保护下,将中间体d1(2.0g,3.7mmol)和中间体a7(2.1g,1.02mmol)溶于50mL甲苯中,依次加入碳酸铯(3.6g,11.1mmol)和催化剂Pd(DPEPhos)Cl
2(600mg,1.1mmol)。反应液升温至105℃下反应4小时,冷却至室温。过滤,滤液减压浓缩,粗品经flash柱层析色谱分离,得到化合物d2(1.4g,1.85mmol),收率:50%。LC-MS:[M+H]
+=755。
Step: under nitrogen protection, intermediate d1 (2.0g, 3.7mmol) and intermediate a7 (2.1g, 1.02mmol) were dissolved in 50mL of toluene, cesium carbonate (3.6g, 11.1mmol) and catalyst Pd(DPEPhos ) Cl 2 (600 mg, 1.1 mmol). The reaction solution was heated up to 105° C. for 4 hours and then cooled to room temperature. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound d2 (1.4 g, 1.85 mmol), yield: 50%. LC-MS: [M+H] + = 755.
中间体d3的合成Synthesis of intermediate d3
步骤1:冰浴,将原料N-Boc-2-氨基-2-甲基-1-丙醇d3-1(500mg,2.64mmol)溶于10mL二氯甲烷中,缓慢加入Dess Martin氧化剂(1.68g,3.96mmol),升温至室温反应2小时,停止反应。将反应液倒入100mL水中,二氯甲烷萃取,减压蒸除溶剂。粗品经flash柱层析色谱分离(PE/EA=4/1),得到油状物d3-2(380mg,2.02mmol),收率:76%。Step 1: In an ice bath, dissolve the raw material N-Boc-2-amino-2-methyl-1-propanol d3-1 (500mg, 2.64mmol) in 10mL of dichloromethane, slowly add Dess Martin oxidant (1.68g , 3.96mmol), the temperature was raised to room temperature for 2 hours, and the reaction was stopped. The reaction solution was poured into 100 mL of water, extracted with dichloromethane, and the solvent was evaporated under reduced pressure. The crude product was separated by flash column chromatography (PE/EA=4/1) to obtain oily substance d3-2 (380 mg, 2.02 mmol), yield: 76%.
步骤2:氮气保护下,将上步中间体d3-2(380mg,2.02mmol)和原料(R)-3-氟-四氢吡咯a5-1(303mg,2.42mmol)溶于10mL无水THF中,缓慢加入三乙酰氧基硼氢化钠(852mg,4.04mmol)。反应液于室温下反应10小时,停止反应。向体系中加入50mL冰水,二氯甲烷萃取,减压蒸除溶剂。粗品经flash柱层析色谱分离(PE/EA=2:/1),得到白色固体d3-3(250mg,0.96mmol),收率:48%。LC-MS:[M+H]
+=261。
Step 2: Under the protection of nitrogen, the intermediate d3-2 (380mg, 2.02mmol) and the starting material (R)-3-fluoro-tetrahydropyrrole a5-1 (303mg, 2.42mmol) were dissolved in 10mL of anhydrous THF , sodium triacetoxyborohydride (852 mg, 4.04 mmol) was added slowly. The reaction solution was reacted at room temperature for 10 hours, and then the reaction was stopped. Add 50 mL of ice water to the system, extract with dichloromethane, and evaporate the solvent under reduced pressure. The crude product was separated by flash column chromatography (PE/EA=2:/1) to obtain white solid d3-3 (250 mg, 0.96 mmol), yield: 48%. LC-MS: [M+H] + = 261.
步骤3:氮气保护下,将上步中间体d3-3(250mg,0.96mmol)溶于8mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。反应液于室温下反应1小时,停止反应,缓慢加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色油状物d3(200mg)。LC-MS:[M+H]
+=161。
Step 3: Under the protection of nitrogen, the intermediate d3-3 (250 mg, 0.96 mmol) in the previous step was dissolved in 8 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1). The reaction solution was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain d3 (200 mg) as a yellow oil. LC-MS: [M+H] + = 161.
参照中间体d3的合成路线,合成如下中间体。Referring to the synthetic route of intermediate d3, the following intermediates were synthesized.
实施例2:Example 2:
步骤1:氮气保护下,将中间体a3(2.4g,5.61mmol)溶于50mL二氧六环中,加入原料P1-1(1.34g,8.42mmol)和N,N-二异丙基乙胺DIEA(1.45g,11.2mmol)。反应液在80℃下搅拌12小时,冷却至室温。向体系加入100mL水,乙酸乙酯萃取,干燥,过滤,减压蒸除溶剂,柱层析分离(石油醚:乙酸乙酯=3:1)纯化,得到化合物P1-2(2.7g,4.9mmol)。收率:87%,LC-MS:[M+H]
+=552。
Step 1: Under the protection of nitrogen, the intermediate a3 (2.4g, 5.61mmol) was dissolved in 50mL of dioxane, and the raw material P1-1 (1.34g, 8.42mmol) and N,N-diisopropylethylamine were added DIEA (1.45 g, 11.2 mmol). The reaction solution was stirred at 80°C for 12 hours and cooled to room temperature. Add 100mL water to the system, extract with ethyl acetate, dry, filter, evaporate the solvent under reduced pressure, and purify by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound P1-2 (2.7g, 4.9mmol ). Yield: 87%, LC-MS: [M+H] + =552.
步骤2:氮气保护下,上步化合物P1-2(300mg,0.54mmol)和磷酸钾(229mg,1.08mmol)混于6mL无水甲苯中,随后依次加入中间体a7(262mg,0.65mmol),Xphos Pd G3(93mg,0.11mmol)和Xphos(53mg,0.11mmol)。在氮气保护下,反应液于100℃下反应1小时。停止反应,冷却至室温,过滤,减压蒸除溶剂。残余物通过TLC色谱分离,得到化合物P1-3(120mg,0.15mmol)。收率:28%,LC-MS:[M+H]
+=807。
Step 2: Under nitrogen protection, compound P1-2 (300mg, 0.54mmol) and potassium phosphate (229mg, 1.08mmol) in the previous step were mixed in 6mL of anhydrous toluene, and then intermediate a7 (262mg, 0.65mmol) was added in turn, Xphos Pd G3 (93 mg, 0.11 mmol) and Xphos (53 mg, 0.11 mmol). Under nitrogen protection, the reaction solution was reacted at 100° C. for 1 hour. The reaction was stopped, cooled to room temperature, filtered, and the solvent was evaporated under reduced pressure. The residue was separated by TLC chromatography to obtain compound P1-3 (120 mg, 0.15 mmol). Yield: 28%, LC-MS: [M+H] + =807.
步骤3:冰浴下,将P1-3(120mg,0.15mmol)溶于4mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。将反应液置于氮气保护下继续反应1小时,停止反应。缓慢向体系加入饱和碳酸氢钠溶液并调节至pH 8左右。乙酸乙酯萃取,减压蒸除溶剂,残留物用制备HPLC色谱纯化,得到目标化合物P1(10.2mg)。LC-MS:[M+H]
+=607。
Step 3: Dissolve P1-3 (120 mg, 0.15 mmol) in 4 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. The reaction solution was placed under nitrogen protection to continue the reaction for 1 hour, and then the reaction was stopped. Slowly add saturated sodium bicarbonate solution to the system and adjust the pH to about 8. After extraction with ethyl acetate, the solvent was distilled off under reduced pressure, and the residue was purified by preparative HPLC chromatography to obtain the target compound P1 (10.2 mg). LC-MS: [M+H] + = 607.
1H NMR(400MHz,DMSO-d
6)δ9.07(s,1H),8.08(s,2H),7.41(dd,J=8.4,5.3Hz,1H),7.14(dd,J=9.5,8.4Hz,1H),5.27(br,J=72Hz,1H),4.42(d,J=12.3Hz,2H),4.13(d,J=10.4Hz,1H),4.03(d,J=10.3Hz,1H),3.71–3.52(m,4H),3.13–3.06(m,2H),3.01(s,1H),2.83(q,J=8.5Hz,1H),2.35–2.27(m,1H),2.17–2.10(m,1H),2.08–1.98(m,2H),1.88–1.74(m,3H),1.65(d,J=6.6Hz,2H),1.56(d,J=7.5Hz,2H).
1 H NMR (400MHz, DMSO-d 6 ) δ9.07(s, 1H), 8.08(s, 2H), 7.41(dd, J=8.4, 5.3Hz, 1H), 7.14(dd, J=9.5, 8.4 Hz, 1H), 5.27(br, J=72Hz, 1H), 4.42(d, J=12.3Hz, 2H), 4.13(d, J=10.4Hz, 1H), 4.03(d, J=10.3Hz, 1H ),3.71–3.52(m,4H),3.13–3.06(m,2H),3.01(s,1H),2.83(q,J=8.5Hz,1H),2.35–2.27(m,1H),2.17– 2.10(m,1H),2.08–1.98(m,2H),1.88–1.74(m,3H),1.65(d,J=6.6Hz,2H),1.56(d,J=7.5Hz,2H).
参照化合物P1的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound P1, using a similar skeleton structure, the following target molecules were synthesized.
实施例3:Example 3:
步骤1:氮气保护下,将中间体a2(500mg,0.99mmol)溶于6mL无水DMF中,接着加入原料P1-1(314mg,2.0mmol)和碳酸铯(966mg,2.96mmol),在氮气保护下,反应液在140℃下反应2小时,停止反应,冷却至室温。向体系加入30mL水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过TLC 色谱分离(石油醚:乙酸乙酯=1:4)纯化,得到淡黄色固体H1-1(110mg,0.18mmol)。收率:18%,LC-MS:[M+H]
+=630。
Step 1: Under the protection of nitrogen, the intermediate a2 (500mg, 0.99mmol) was dissolved in 6mL of anhydrous DMF, then the raw material P1-1 (314mg, 2.0mmol) and cesium carbonate (966mg, 2.96mmol) were added, and the , the reaction solution was reacted at 140° C. for 2 hours, the reaction was stopped, and cooled to room temperature. Add 30 mL of water to the system, extract with ethyl acetate, and evaporate the solvent under reduced pressure. The residue was purified by TLC chromatography (petroleum ether: ethyl acetate = 1:4) to obtain pale yellow solid H1-1 (110 mg, 0.18 mmol). Yield: 18%, LC-MS: [M+H] + =630.
步骤2:氮气保护下,将H1-1(210mg,0.34mmol)溶于5mL无水甲苯中,依次加入中间体a7(189mg,0.47mmol),Pd(DPEPhos)Cl
2(72mg,0.10mmol)和无水碳酸铯(273mg,0.84mmol)。在氮气保护下,反应液于105℃下反应6小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过TLC色谱分离(石油醚:乙酸乙酯=1:2),得到黄色固体H1-2(80mg,0.10mmol)。收率:28%,LC-MS:[M+H]
+=841。
Step 2: Under nitrogen protection, H1-1 (210mg, 0.34mmol) was dissolved in 5mL of anhydrous toluene, and intermediate a7 (189mg, 0.47mmol), Pd(DPEPhos)Cl 2 (72mg, 0.10mmol) and Anhydrous cesium carbonate (273mg, 0.84mmol). Under the protection of nitrogen, the reaction solution was reacted at 105° C. for 6 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by TLC chromatography (petroleum ether: ethyl acetate = 1:2) to obtain yellow solid H1-2 (80 mg, 0.10 mmol). Yield: 28%, LC-MS: [M+H] + =841.
步骤3:冰浴下,将H1-2(80mg,0.10mmol)溶于3mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应0.5小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残留物用制备SFC(Xselect CSH C18 OBD)纯化,得到目标化合物H1a(4.0mg)和H1b(4.1mg)。Step 3: Dissolve H1-2 (80 mg, 0.10 mmol) in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 0.5 hours, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative SFC (Xselect CSH C18 OBD) to give target compounds H1a (4.0 mg) and H1b (4.1 mg).
H1a:
1H NMR(300MHz,DMSO-d
6)δ8.23(s,1H),8.11(s,1H),7.85(s,1H),7.26(dd,J=8.4,5.3Hz,1H),7.15(dd,J=9.5,8.4Hz,1H),5.27(br,J=72Hz,1H),4.29(dd,J=20.9,12.2Hz,3H),4.14–3.91(m,2H),3.67–3.39(m,5H),3.21–2.95(m,3H),2.92–2.70(m,1H),2.14(d,J=6.8Hz,1H),2.09–1.93(m,2H),1.88–1.52(m,6H).
H1a: 1 H NMR (300MHz, DMSO-d 6 )δ8.23(s,1H),8.11(s,1H),7.85(s,1H),7.26(dd,J=8.4,5.3Hz,1H), 7.15(dd, J=9.5, 8.4Hz, 1H), 5.27(br, J=72Hz, 1H), 4.29(dd, J=20.9, 12.2Hz, 3H), 4.14–3.91(m, 2H), 3.67– 3.39(m,5H),3.21–2.95(m,3H),2.92–2.70(m,1H),2.14(d,J=6.8Hz,1H),2.09–1.93(m,2H),1.88–1.52( m,6H).
H1b:
1H NMR(400MHz,DMSO-d
6)δ8.21(s,1H),8.09(s,2H),7.84(s,1H),7.26(dd,J=8.4,5.3Hz,1H),7.18–7.11(m,1H),5.27(br,J=72Hz,1H),4.27(dd,J=19.6,12.0Hz,2H),4.08(d,J=10.3Hz,1H),3.99(d,J=10.3Hz,1H),3.49–3.55(m,3H),3.08(d,J=9.5Hz,3H),3.02(d,J=11.5Hz,2H),2.81(s,1H),2.14–2.12(m,1H),2.02(d,J=18.1Hz,2H),1.91–1.71(m,4H),1.66–1.54(m,4H).
H1b: 1 H NMR (400MHz, DMSO-d 6 )δ8.21(s,1H),8.09(s,2H),7.84(s,1H),7.26(dd,J=8.4,5.3Hz,1H), 7.18–7.11(m,1H),5.27(br,J=72Hz,1H),4.27(dd,J=19.6,12.0Hz,2H),4.08(d,J=10.3Hz,1H),3.99(d, J=10.3Hz, 1H), 3.49–3.55(m, 3H), 3.08(d, J=9.5Hz, 3H), 3.02(d, J=11.5Hz, 2H), 2.81(s, 1H), 2.14– 2.12(m,1H),2.02(d,J=18.1Hz,2H),1.91–1.71(m,4H),1.66–1.54(m,4H).
实施例4:Example 4:
步骤1:在50mL反应瓶中,将中间体a5(420mg,2.42mmol)溶于10mL无水THF,加入叔丁醇钾(340mg,3.64mmol),室温下搅拌30分钟—得溶液S1。在另一50mL反应瓶中将中间体a14(1.0g,2.0mmol)溶于10mL无水THF中,冰浴下,缓慢加入配置好的溶液S1,继续搅拌1小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色 谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体H2-1(916mg,1.42mmol)。收率:71%,LC-MS:[M+H]
+=644。
Step 1: Dissolve intermediate a5 (420 mg, 2.42 mmol) in 10 mL of anhydrous THF in a 50 mL reaction flask, add potassium tert-butoxide (340 mg, 3.64 mmol), and stir at room temperature for 30 minutes to obtain solution S1. In another 50mL reaction flask, the intermediate a14 (1.0g, 2.0mmol) was dissolved in 10mL of anhydrous THF, and the prepared solution S1 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid H2-1 (916 mg, 1.42 mmol). Yield: 71%, LC-MS: [M+H] + =644.
步骤2:氮气保护下,将H2-1(480mg,0.75mmol)溶于10mL无水甲苯中,依次加入中间体a7(414mg,1.02mmol),Pd(DPEPhos)Cl
2(22mg,0.03mmol)和碳酸铯(438mg,1.35mmol)。在氮气保护下,反应液于105℃下反应10小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:2),得到黄色固体H2-2(450mg,0.53mmol)。收率:70%,LC-MS:[M+H]
+=854。
Step 2: Under nitrogen protection, H2-1 (480mg, 0.75mmol) was dissolved in 10mL of anhydrous toluene, and intermediate a7 (414mg, 1.02mmol), Pd(DPEPhos)Cl 2 (22mg, 0.03mmol) and Cesium carbonate (438 mg, 1.35 mmol). Under nitrogen protection, the reaction solution was reacted at 105° C. for 10 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain yellow solid H2-2 (450 mg, 0.53 mmol). Yield: 70%, LC-MS: [M+H] + =854.
步骤3:冰浴下,将H2-2(600mg,0.70mmol)溶于10mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H2(320mg,0.49mmol),LC-MS:[M+H]
+=654。收率:70%。
Step 3: Dissolve H2-2 (600 mg, 0.70 mmol) in 10 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H2 (320 mg, 0.49 mmol), LC-MS: [M+H] + =654. Yield: 70%.
将H2经手性柱:Unichiral CMD-5H纯化,得到目标化合物H2a(peak1)和H2b(peak2)。Purify H2 through a chiral column: Unichiral CMD-5H to obtain the target compounds H2a (peak1) and H2b (peak2).
参照化合物H2a或H2b的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound H2a or H2b, the following target molecules were synthesized using similar skeleton structures.
实施例5:Example 5:
步骤1:在50mL反应瓶中,将中间体a5(170mg,0.94mmol)溶于10mL无水THF,加入叔丁醇钾(180mg,1.56mmol),室温下搅拌30分钟—得溶液S2。在另一50mL反应瓶中将中间体a17(400mg,0.78mmol)溶于10mL无水THF中,冰浴下,缓慢加入配置好的溶液S2,继续搅拌1小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体H11-1(300mg,0.41mmol)。收率:44%,LC-MS:[M+H]
+=734。
Step 1: Dissolve intermediate a5 (170 mg, 0.94 mmol) in 10 mL of anhydrous THF in a 50 mL reaction flask, add potassium tert-butoxide (180 mg, 1.56 mmol), and stir at room temperature for 30 minutes to obtain solution S2. In another 50mL reaction flask, the intermediate a17 (400mg, 0.78mmol) was dissolved in 10mL of anhydrous THF, and the prepared solution S2 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid H11-1 (300 mg, 0.41 mmol). Yield: 44%, LC-MS: [M+H] + =734.
步骤2:氮气保护下,将H11-1(300mg,0.41mmol)和CuCN(150mg,1.64mmol)溶于8mL无水DMF中,反应液于100℃下反应6小时,冷却至室温,停止反应。向反应液中加入50mL水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=2:1),得到黄色固体H11-2(110mg,0.17mmol)。收率:42%,LC-MS:[M+H]
+=633。
Step 2: Under nitrogen protection, H11-1 (300mg, 0.41mmol) and CuCN (150mg, 1.64mmol) were dissolved in 8mL of anhydrous DMF, and the reaction solution was reacted at 100°C for 6 hours, then cooled to room temperature to stop the reaction. 50 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain a yellow solid H11-2 ( 110 mg, 0.17 mmol). Yield: 42%, LC-MS: [M+H] + =633.
步骤3:氮气保护下,将H11-2(110mg,0.17mmol)溶于10mL无水甲苯中,依次加入中间体a7(94mg,0.22mmol),Pd(DPEPhos)Cl
2(22mg,0.03mmol)和碳酸铯(160mg,0.48mmol)。在氮气保护下,反应液于110℃下反应6小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层 析色谱分离(石油醚:乙酸乙酯=1:1),得到黄色固体H11-3(90mg,0.11mmol)。收率:63%,LC-MS:[M+H]
+=845。
Step 3: Under nitrogen protection, H11-2 (110mg, 0.17mmol) was dissolved in 10mL of anhydrous toluene, and intermediate a7 (94mg, 0.22mmol), Pd(DPEPhos)Cl 2 (22mg, 0.03mmol) and Cesium carbonate (160mg, 0.48mmol). Under the protection of nitrogen, the reaction solution was reacted at 110° C. for 6 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain yellow solid H11-3 (90 mg, 0.11 mmol). Yield: 63%, LC-MS: [M+H] + =845.
步骤4:冰浴下,将H11-3(90mg,0.11mmol)溶于8mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H11(30mg,0.05mmol),LC-MS:[M+H]
+=645。收率:43%。
Step 4: Dissolve H11-3 (90 mg, 0.11 mmol) in 8 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H11 (30 mg, 0.05 mmol), LC-MS: [M+H] + =645. Yield: 43%.
1H NMR(400MHz,DMSO-d
6)δ8.45(s,1H),8.26(s,2H),7.35(dd,J=8.4,5.2Hz,1H),7.21(t,J=8.8Hz,1H),4.58(d,J=13.6Hz,1H),4.47(d,J=12.8Hz,1H),4.34(s,2H),4.15(d,J=16.8Hz,2H),4.02(d,J=13.6Hz,2H),3.88(d,J=12.8Hz,2H),3.52-3.16(m,4H),1.96-1.83(m,6H),1.23(s,2H),0.85–0.74(m,4H).
1 H NMR (400MHz, DMSO-d 6 )δ8.45(s,1H),8.26(s,2H),7.35(dd,J=8.4,5.2Hz,1H),7.21(t,J=8.8Hz, 1H), 4.58(d, J=13.6Hz, 1H), 4.47(d, J=12.8Hz, 1H), 4.34(s, 2H), 4.15(d, J=16.8Hz, 2H), 4.02(d, J=13.6Hz, 2H), 3.88(d, J=12.8Hz, 2H), 3.52-3.16(m, 4H), 1.96-1.83(m, 6H), 1.23(s, 2H), 0.85–0.74(m ,4H).
参照化合物H11的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound H11, using a similar skeleton structure, the following target molecules were synthesized.
实施例6:Embodiment 6:
步骤1:在50mL反应瓶中,将中间体a19(150mg,0.55mmol)溶于5mL无水THF,加入叔丁醇钾(93mg,0.82mmol),室温下搅拌30分钟—得溶液S3。在另一50mL反应瓶中将中间体a14(280mg,0.58mmol)溶于5mL无水THF中,冰浴下,缓慢加入配置好的溶液S3,继续搅拌1小时,停止反应。将反应液倒入50mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体H13-1(205mg,0.28mmol)。收率:51%,LC-MS:[M+H]
+=744。
Step 1: In a 50 mL reaction flask, the intermediate a19 (150 mg, 0.55 mmol) was dissolved in 5 mL of anhydrous THF, potassium tert-butoxide (93 mg, 0.82 mmol) was added, and stirred at room temperature for 30 minutes to obtain solution S3. In another 50mL reaction flask, the intermediate a14 (280mg, 0.58mmol) was dissolved in 5mL of anhydrous THF, and the prepared solution S3 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid H13-1 (205 mg, 0.28 mmol). Yield: 51%, LC-MS: [M+H] + =744.
步骤2:氮气保护下,将H13-1(205mg,0.28mmol)溶于6mL无水甲苯中,依次加入中间体a7(200mg,0.50mmol),Pd(DPEPhos)Cl
2(13mg,0.015mmol)和碳酸铯(175mg,0.54mmol)。在氮气保护下,反应液于105℃下反应10小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:2),得到黄色固体H13-2(120mg,0.13mmol)。收率:45%,LC-MS:[M+H]
+=955。
Step 2: Under nitrogen protection, H13-1 (205mg, 0.28mmol) was dissolved in 6mL of anhydrous toluene, and intermediate a7 (200mg, 0.50mmol), Pd(DPEPhos)Cl 2 (13mg, 0.015mmol) and Cesium carbonate (175 mg, 0.54 mmol). Under nitrogen protection, the reaction solution was reacted at 105° C. for 10 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain yellow solid H13-2 (120 mg, 0.13 mmol). Yield: 45%, LC-MS: [M+H] + =955.
步骤3:冰浴下,将H13-2(120mg,0.13mmol)溶于6mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H13(32mg),LC-MS:[M+H]
+=671。
Step 3: Dissolve H13-2 (120 mg, 0.13 mmol) in 6 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H13 (32 mg), LC-MS: [M+H] + =671.
1H NMR(400MHz,DMSO-d
6)δ8.26(s,2H),7.97(d,J=11.2Hz,1H),7.38–7.09(m,2H),5.88–5.43(m,2H),4.70–4.37(m,2H),4.29–4.00(m,6H),3.82-3.64(m,,4H),3.74–3.36(m,4H),1.93(m,4H),1.60-1.50(m,2H),1.40-1.20(m,2H).
1 H NMR (400MHz,DMSO-d 6 )δ8.26(s,2H),7.97(d,J=11.2Hz,1H),7.38–7.09(m,2H),5.88–5.43(m,2H), 4.70–4.37(m,2H),4.29–4.00(m,6H),3.82-3.64(m,,4H),3.74–3.36(m,4H),1.93(m,4H),1.60-1.50(m, 2H),1.40-1.20(m,2H).
参照化合物H13的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound H13, using a similar skeleton structure, the following target molecule was synthesized.
实施例7:Embodiment 7:
步骤1:氮气保护下,将中间体H1-1(1.0g,1.59mmol)溶于12mL甲醇中,接着加入甲醇钠(102mg,4.77mmol),反应液升温至60℃下反应2小时,停止反应,冷却至室温。向体系加入40mL水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离,得到淡黄色固体P5-1(305mg,0.48mmol)。收率:30%,LC-MS:[M+H]
+=640。
Step 1: Under the protection of nitrogen, the intermediate H1-1 (1.0g, 1.59mmol) was dissolved in 12mL of methanol, then sodium methoxide (102mg, 4.77mmol) was added, and the reaction solution was heated to 60°C for 2 hours to stop the reaction , cooled to room temperature. Add 40 mL of water to the system, extract with ethyl acetate, and distill off the solvent under reduced pressure. The residue was separated by flash column chromatography to obtain pale yellow solid P5-1 (305 mg, 0.48 mmol). Yield: 30%, LC-MS: [M+H] + =640.
步骤2:氮气保护下,将P5-1(305mg,0.48mmol)溶于5mL无水甲苯中,依次加入中间体a7(270mg,0.67mmol),Pd(DPEPhos)Cl
2(90mg,0.14mmol)和无水碳酸铯(330mg,0.96mmol)。在氮气保护下,反应液于105℃下反应8小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过柱层析色谱分离(PE/EA,1/2),得到黄色固体P5-2(327mg,0.38mmol)。收率:80%,LC-MS:[M+H]
+=853。
Step 2: Under nitrogen protection, dissolve P5-1 (305mg, 0.48mmol) in 5mL of anhydrous toluene, add intermediate a7 (270mg, 0.67mmol), Pd(DPEPhos)Cl 2 (90mg, 0.14mmol) and Anhydrous cesium carbonate (330mg, 0.96mmol). Under the protection of nitrogen, the reaction solution was reacted at 105° C. for 8 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (PE/EA, 1/2) to obtain yellow solid P5-2 (327 mg, 0.38 mmol). Yield: 80%, LC-MS: [M+H] + =853.
步骤3:冰浴下,将P5-2(327mg,0.38mmol)溶于4mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残留物用HPLC制备色谱纯化,得到目标化合物P5(74mg,0.11mmol)。收率:30%,LC-MS:[M+H]
+=653。
Step 3: Dissolve P5-2 (327 mg, 0.38 mmol) in 4 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative HPLC chromatography to obtain the target compound P5 (74 mg, 0.11 mmol). Yield: 30%, LC-MS: [M+H] + = 653.
1H NMR(400MHz,DMSO-d
6)δ8.16(s,2H),7.87(d,J=0.8Hz,1H),7.29(dd,J=8.4,5.3Hz,1H),7.22–7.11(m,1H),5.68-5.54(m,1H),4.28(dt,J=35.0,17.4Hz,2H),4.06(dd,J=35.9,10.3Hz,2H),3.56(t,J=18.0Hz,4H),3.21(s,3H)3.18–3.08(m,2H),3.03(s,1H),2.90–2.76(m,1H),2.19–1.75(m,6H),1.63(dd,J=20.6,9.9Hz,4H).
1 H NMR (400MHz, DMSO-d 6 ) δ8.16 (s, 2H), 7.87 (d, J = 0.8Hz, 1H), 7.29 (dd, J = 8.4, 5.3Hz, 1H), 7.22–7.11 ( m,1H),5.68-5.54(m,1H),4.28(dt,J=35.0,17.4Hz,2H),4.06(dd,J=35.9,10.3Hz,2H),3.56(t,J=18.0Hz ,4H),3.21(s,3H),3.18–3.08(m,2H),3.03(s,1H),2.90–2.76(m,1H),2.19–1.75(m,6H),1.63(dd,J= 20.6,9.9Hz,4H).
参照化合物P5的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound P5, using a similar skeleton structure, the following target molecules were synthesized.
实施例8:Embodiment 8:
步骤1:冰浴,氮气保护下,将中间体H2-1(500mg,0.78mmol)和三氟乙醇(156mg,1.56mmol)溶于10mL四氢呋喃中,接着加入NaH(94mg,2.34mmol),反应液升温至室温下反应3小时,停止反应,冷却至室温。向体系加入30mL水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离,得到淡黄色固体P7-1(337mg,0.47mmol)。收率:60%,LC-MS:[M+H]
+=722。
Step 1: In ice bath, under the protection of nitrogen, the intermediate H2-1 (500mg, 0.78mmol) and trifluoroethanol (156mg, 1.56mmol) were dissolved in 10mL of tetrahydrofuran, then NaH (94mg, 2.34mmol) was added, and the reaction solution Raise the temperature to room temperature and react for 3 hours, stop the reaction, and cool to room temperature. Add 30 mL of water to the system, extract with ethyl acetate, and evaporate the solvent under reduced pressure. The residue was separated by flash column chromatography to obtain pale yellow solid P7-1 (337mg, 0.47mmol). Yield: 60%, LC-MS: [M+H] + =722.
步骤2:氮气保护下,将P7-1(305mg,0.42mmol)溶于5mL无水甲苯中,依次加入中间体a7(270mg,0.67mmol),Pd(DPEPhos)Cl
2(90mg,0.14mmol)和无水碳酸铯(330mg,0.96mmol)。在氮气保护下,反应液于105℃下反应8小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过柱层析色谱分离(PE/EA,1/2),得到黄色固体P7-2(313mg,0.34mmol)。收率:80%,LC-MS:[M+H]
+=934。
Step 2: Under nitrogen protection, dissolve P7-1 (305mg, 0.42mmol) in 5mL of anhydrous toluene, add intermediate a7 (270mg, 0.67mmol), Pd(DPEPhos)Cl 2 (90mg, 0.14mmol) and Anhydrous cesium carbonate (330mg, 0.96mmol). Under the protection of nitrogen, the reaction solution was reacted at 105° C. for 8 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (PE/EA, 1/2) to obtain yellow solid P7-2 (313 mg, 0.34 mmol). Yield: 80%, LC-MS: [M+H] + =934.
步骤3:冰浴下,将P7-2(313mg,0.34mmol)溶于5mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残留物用HPLC制备色谱纯化,得到目标化合物P7(82mg,0.11mmol)。收率:33%,LC-MS:[M+H]
+=734。
Step 3: Dissolve P7-2 (313 mg, 0.34 mmol) in 5 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative HPLC chromatography to obtain the target compound P7 (82 mg, 0.11 mmol). Yield: 33%, LC-MS: [M+H] + =734.
1H NMR(400MHz,DMSO-d
6)δ8.02(s,2H),7.82(s,1H),7.20(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz,1H),5.24-5.10(m,1H),4.96–4.75(m,2H),4.24(dd,J=10.8,4.4Hz,2H),3.65(d,J=13.2Hz,4H),3.51(d,J=12.1Hz,2H),2.84(dd,J=23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J=15.9,9.7Hz,2H),2.21–1.76(m,2H),1.76–1.60(m,4H),0.66–0.60(m,2H),0.46–0.41(m,2H).
1 H NMR (400MHz, DMSO-d 6 )δ8.02(s,2H),7.82(s,1H),7.20(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz, 1H), 5.24-5.10(m, 1H), 4.96–4.75(m, 2H), 4.24(dd, J=10.8, 4.4Hz, 2H), 3.65(d, J=13.2Hz, 4H), 3.51(d ,J=12.1Hz,2H),2.84(dd,J=23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J=15.9,9.7Hz, 2H),2.21–1.76(m,2H),1.76–1.60(m,4H),0.66–0.60(m,2H),0.46–0.41(m,2H).
实施例9:Embodiment 9:
步骤1:冰浴,氮气保护下,将中间体H2-1(500mg,0.78mmol)和4-甲氧基苄醇(215mg,1.56mmol)溶于10mL四氢呋喃中,接着加入NaH(94mg,2.34mmol),反应液升温至室温下反应3小时, 停止反应,冷却至室温。向体系加入30mL水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离,得到淡黄色固体P8-1(385mg,0.51mmol)。收率:65%,LC-MS:[M+H]
+=760。
Step 1: In ice bath, under nitrogen protection, intermediate H2-1 (500mg, 0.78mmol) and 4-methoxybenzyl alcohol (215mg, 1.56mmol) were dissolved in 10mL THF, then NaH (94mg, 2.34mmol) was added ), the reaction solution was warmed up to room temperature and reacted for 3 hours, the reaction was stopped, and cooled to room temperature. Add 30 mL of water to the system, extract with ethyl acetate, and evaporate the solvent under reduced pressure. The residue was separated by flash column chromatography to obtain pale yellow solid P8-1 (385 mg, 0.51 mmol). Yield: 65%, LC-MS: [M+H] + =760.
步骤2:氮气保护下,将P8-1(319mg,0.42mmol)溶于5mL无水甲苯中,依次加入中间体a7(270mg,0.67mmol),Pd(DPEPhos)Cl
2(90mg,0.14mmol)和无水碳酸铯(330mg,0.96mmol)。在氮气保护下,反应液于105℃下反应8小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过柱层析色谱分离(PE/EA,1/2),得到黄色固体P8-2(122mg,0.13mmol)。收率:30%,LC-MS:[M+H]
+=972。
Step 2: Under nitrogen protection, dissolve P8-1 (319mg, 0.42mmol) in 5mL of anhydrous toluene, add intermediate a7 (270mg, 0.67mmol), Pd(DPEPhos)Cl 2 (90mg, 0.14mmol) and Anhydrous cesium carbonate (330mg, 0.96mmol). Under the protection of nitrogen, the reaction solution was reacted at 105° C. for 8 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (PE/EA, 1/2) to obtain yellow solid P8-2 (122 mg, 0.13 mmol). Yield: 30%, LC-MS: [M+H] + =972.
步骤3:冰浴下,将P8-2(122mg,0.13mmol)溶于5mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残留物用HPLC制备色谱纯化,得到目标化合物P8(19mg,0.03mmol)。收率:23%,LC-MS:[M+H]
+=653。
Step 3: Dissolve P8-2 (122 mg, 0.13 mmol) in 5 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative HPLC chromatography to obtain the target compound P8 (19 mg, 0.03 mmol). Yield: 23%, LC-MS: [M+H] + =653.
1H NMR(400MHz,DMSO-d
6)δ8.02(s,2H),7.82(s,1H),7.20(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz,1H),5.32-5.18(m,1H),4.24(dd,J=10.8,4.4Hz,2H),3.65(d,J=13.2Hz,4H),3.51(d,J=12.1Hz,2H),2.84(dd,J=23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J=15.9,9.7Hz,2H),2.21–1.76(m,2H),1.76–1.60(m,4H),0.68–0.60(m,2H),0.48–0.41(m,2H).
1 H NMR (400MHz, DMSO-d 6 )δ8.02(s,2H),7.82(s,1H),7.20(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz, 1H),5.32-5.18(m,1H),4.24(dd,J=10.8,4.4Hz,2H),3.65(d,J=13.2Hz,4H),3.51(d,J=12.1Hz,2H), 2.84(dd,J=23.1,10.0Hz,2H),2.72–2.58(m,1H),2.48(s,1H),2.36(dt,J=15.9,9.7Hz,2H),2.21–1.76(m, 2H),1.76–1.60(m,4H),0.68–0.60(m,2H),0.48–0.41(m,2H).
实施例10:Example 10:
步骤1:冰浴,氮气保护下,将中间体a3(900mg,2.1mmol)和中间体a5(360mg,2.1mmol)溶于15mL四氢呋喃中,接着加入NaH(100mg,4.2mmol),反应液升温至40℃下反应3小时,停止反应,冷却至室温。向体系加入30mL水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离,得到黄色固体P9-2(490mg,0.81mmol)。收率:38%,LC-MS:[M+H]
+=565。
Step 1: In ice bath, under the protection of nitrogen, intermediate a3 (900mg, 2.1mmol) and intermediate a5 (360mg, 2.1mmol) were dissolved in 15mL of tetrahydrofuran, then NaH (100mg, 4.2mmol) was added, and the reaction solution was heated to React at 40°C for 3 hours, stop the reaction, and cool to room temperature. Add 30 mL of water to the system, extract with ethyl acetate, and evaporate the solvent under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid P9-2 (490 mg, 0.81 mmol). Yield: 38%, LC-MS: [M+H] + =565.
步骤2:氮气保护下,将P9-2(440mg,0.78mmol)溶于6mL 1,4-二氧六环和水的混合溶液中(v/v,5/1),依次加入原料P9-1(480mg,0.94mmol),Xphos Pd G1(180mg,0.23mmol)和无水碳酸铯(1.02g,3.12mmol)。在氮气保护下,反应液于95℃下反应3小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过柱层析色谱分离(PE/EA,1/2),得到黄色固体P9-3(500mg,0.55mmol)。收率:70%,LC-MS:[M+H]
+=915。
Step 2: Under nitrogen protection, dissolve P9-2 (440mg, 0.78mmol) in 6mL of a mixed solution of 1,4-dioxane and water (v/v, 5/1), and add raw materials P9-1 in sequence (480mg, 0.94mmol), Xphos Pd G1 (180mg, 0.23mmol) and anhydrous cesium carbonate (1.02g, 3.12mmol). Under nitrogen protection, the reaction solution was reacted at 95° C. for 3 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by column chromatography (PE/EA, 1/2) to obtain yellow solid P9-3 (500mg, 0.55mmol). Yield: 70%, LC-MS: [M+H] + =915.
步骤3:将上步中间体P9-3(500mg,0.55mmol)溶于20mL四氢呋喃中,加入四丁基氟化铵(220mg,0.9mmol)。室温下反应12小时,TLC监测反应完全。将反应液溶于50mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到粗品P9-4(400mg),LC-MS:[M+H]
+=759。
Step 3: The above intermediate P9-3 (500 mg, 0.55 mmol) was dissolved in 20 mL of tetrahydrofuran, and tetrabutylammonium fluoride (220 mg, 0.9 mmol) was added. The reaction was carried out at room temperature for 12 hours, and the reaction was complete as monitored by TLC. The reaction solution was dissolved in 50 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain crude product P9-4 (400 mg), LC-MS: [M+H] + =759.
步骤4:冰浴下,将粗品P9-4(400mg,0.53mmol)溶于5mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残留物用HPLC制备色谱纯化,得到目 标化合物P9(45mg,0.07mmol)。收率:14%,LC-MS:[M+H]
+=615。
Step 4: The crude product P9-4 (400 mg, 0.53 mmol) was dissolved in 5 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was purified by preparative HPLC chromatography to obtain the target compound P9 (45 mg, 0.07 mmol). Yield: 14%, LC-MS: [M+H] + =615.
1H NMR(400MHz,DMSO-d
6)δ10.30(s,1H),9.10(s,1H),7.98(dd,J=9.2,6.0Hz,1H),7.47(t,J=9.0Hz,1H),7.41(d,J=2.4Hz,2H),7.37(s,1H),7.24(s,1H),7.22(s,2H),7.11(s,1H),4.65(d,J=13.7Hz,1H),4.49(s,1H),4.35(s,3H),4.18(s,4H),4.0-3.90(m,6H),1.97(m,7H).
1 H NMR (400MHz, DMSO-d 6 )δ10.30(s, 1H), 9.10(s, 1H), 7.98(dd, J=9.2, 6.0Hz, 1H), 7.47(t, J=9.0Hz, 1H), 7.41(d, J=2.4Hz, 2H), 7.37(s, 1H), 7.24(s, 1H), 7.22(s, 2H), 7.11(s, 1H), 4.65(d, J=13.7 Hz,1H),4.49(s,1H),4.35(s,3H),4.18(s,4H),4.0-3.90(m,6H),1.97(m,7H).
参照化合物P9的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound P9, using a similar skeleton structure, the following target molecules were synthesized.
实施例11:Example 11:
步骤1:在20mL反应瓶中,将中间体a5(142mg,0.82mmol)溶于5mL无水THF,加入叔丁醇钾(138mg,2.34mmol),室温下搅拌30分钟—得溶液S4。在另一20mL反应瓶中将中间体a27(400mg,0.82mmol)溶于5mL无水THF中,冰浴下,缓慢加入配置好的溶液S4,继续搅拌1小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体P12-1(420mg,0.66mmol)。收率:80%,LC-MS:[M+H]
+=642。
Step 1: Dissolve intermediate a5 (142mg, 0.82mmol) in 5mL anhydrous THF in a 20mL reaction flask, add potassium tert-butoxide (138mg, 2.34mmol), and stir at room temperature for 30 minutes to obtain solution S4. In another 20mL reaction flask, the intermediate a27 (400mg, 0.82mmol) was dissolved in 5mL of anhydrous THF, and the prepared solution S4 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid P12-1 (420 mg, 0.66 mmol). Yield: 80%, LC-MS: [M+H] + =642.
步骤2:氮气保护下,将P12-1(420mg,0.66mmol)溶于5mL无水甲苯中,依次加入中间体a7(352mg,0.87mmol),Pd(DPEPhos)Cl
2(22mg,0.03mmol)和碳酸铯(438mg,1.35mmol)。在氮气保护下,反应液于105℃下反应10小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:2),得到黄色固体P12-2(450mg,0.53mmol)。收率:80%,LC-MS:[M+H]
+=854。
Step 2: Under nitrogen protection, P12-1 (420mg, 0.66mmol) was dissolved in 5mL of anhydrous toluene, and intermediate a7 (352mg, 0.87mmol), Pd(DPEPhos)Cl 2 (22mg, 0.03mmol) and Cesium carbonate (438 mg, 1.35 mmol). Under nitrogen protection, the reaction solution was reacted at 105° C. for 10 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain yellow solid P12-2 (450 mg, 0.53 mmol). Yield: 80%, LC-MS: [M+H] + =854.
步骤3:冰浴下,将P12-2(450mg,0.53mmol)溶于6mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体P12(104mg,0.16mmol),LC-MS:[M+H]
+=654。收率:30%。
Step 3: Dissolve P12-2 (450 mg, 0.53 mmol) in 6 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice-cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid P12 (104 mg, 0.16 mmol), LC-MS: [M+H] + =654. Yield: 30%.
1H NMR(400MHz,DMSO-d
6)δ8.15(s,2H),7.81(s,1H),7.26(dd,J=8.4,5.3Hz,1H),7.15(t,J=8.9Hz,1H),5.24–5.10(m,1H),4.22(dt,J=19.4,10.7Hz,2H),3.86–3.62(m,4H),3.01(s,2H),2.87–2.75(m,2H),2.42-2.33(m,3H),2.20–1.95(m,2H),1.91–1.75(m,1H),0.67–0.38(m,8H).
1 H NMR (400MHz, DMSO-d 6 )δ8.15(s, 2H), 7.81(s, 1H), 7.26(dd, J=8.4, 5.3Hz, 1H), 7.15(t, J=8.9Hz, 1H),5.24–5.10(m,1H),4.22(dt,J=19.4,10.7Hz,2H),3.86–3.62(m,4H),3.01(s,2H),2.87–2.75(m,2H) ,2.42-2.33(m,3H),2.20–1.95(m,2H),1.91–1.75(m,1H),0.67–0.38(m,8H).
参照化合物P12的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound P12, using a similar skeleton structure, the following target molecules were synthesized.
实施例12:Example 12:
步骤1:在高压反应釜中,将原料4-溴-2,6-二氟苯腈a5(100g,459mmol)溶于240mL乙醇中,加入400mL氨水,升温到90℃下反应16小时,冷却至室温,停止反应。减压蒸除溶剂,向反应液加入100mL冰水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚/乙酸乙酯,9/1)纯化,得到黄色固体P14-2(74.9g,352mmol)。收率:77%,LC-MS:[M+H]
+=215。
Step 1: In an autoclave, dissolve the raw material 4-bromo-2,6-difluorobenzonitrile a5 (100g, 459mmol) in 240mL of ethanol, add 400mL of ammonia water, heat up to 90°C for 16 hours, and cool to room temperature, stop the reaction. The solvent was evaporated under reduced pressure, 100 mL of ice water was added to the reaction solution, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate, 9/1) to obtain yellow solid P14-2 (74.9g, 352mmol). Yield: 77%, LC-MS: [M+H] + =215.
步骤2:氮气保护下,将2,2-二乙氧基乙醇(33.8g,252mmol)溶于450mL无水DMF中,在0℃下缓慢加入NaH(10.08g,252mmol),搅拌1小时后,加入上步中间体P14-2(45g,210mmol)。撤去冰浴,升温至50℃下反应2小时,停止反应,冷却至室温,向体系中加入1L水,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚/乙酸乙酯,10/1),得到黄色固体P14-3(25.3g,77.1mmol)。收率:37%,LC-MS:[M+H]
+=329。
Step 2: Under nitrogen protection, 2,2-diethoxyethanol (33.8g, 252mmol) was dissolved in 450mL of anhydrous DMF, NaH (10.08g, 252mmol) was slowly added at 0°C, and after stirring for 1 hour, The intermediate P14-2 (45 g, 210 mmol) of the previous step was added. The ice bath was removed, and the temperature was raised to 50° C. to react for 2 hours. The reaction was stopped, cooled to room temperature, 1 L of water was added to the system, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether/ethyl acetate, 10/1) to obtain yellow solid P14-3 (25.3 g, 77.1 mmol). Yield: 37%, LC-MS: [M+H] + =329.
步骤3:将100mL甲苯加入到多聚磷酸(10.42g)中,升温至100℃下加入上步中间体P14-3(10.0g,30.4mmol),继续在该温度下反应2小时,停止反应。将反应液缓慢倒入大量冰水中,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离(石油醚/乙酸乙酯,10/1),得到黄色固体P14-4(1.04g,4.4mmol),收率:14%。LC-MS:[M+H]
+=236。
Step 3: Add 100 mL of toluene to polyphosphoric acid (10.42 g), raise the temperature to 100°C, add the intermediate P14-3 (10.0 g, 30.4 mmol) in the previous step, continue to react at this temperature for 2 hours, and stop the reaction. The reaction solution was slowly poured into a large amount of ice water, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether/ethyl acetate, 10/1) to obtain yellow solid P14-4 (1.04 g, 4.4 mmol), yield: 14%. LC-MS: [M+H] + = 236.
步骤4:将上步中间体P14-4(8.3g,35.0mmol)溶于150mL乙醇中,加入KOH水溶液(7.94g,50mL),升温至90℃下反应4小时,停止反应。减压蒸除有机溶剂,向反应液加入100mL冰水,二氯甲烷萃取,减压浓缩。残余物通过flash柱层析色谱分离(石油醚/二氯甲烷,2/1),得到黄色固体P14-5(4.0g,15.7mmol),收率:45%。LC-MS:[M+H]
+=256。
Step 4: Dissolve the intermediate P14-4 (8.3g, 35.0mmol) in 150mL of ethanol, add KOH aqueous solution (7.94g, 50mL), heat up to 90°C for 4 hours, and stop the reaction. The organic solvent was evaporated under reduced pressure, 100 mL of ice water was added to the reaction solution, extracted with dichloromethane, and concentrated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether/dichloromethane, 2/1) to obtain yellow solid P14-5 (4.0 g, 15.7 mmol), yield: 45%. LC-MS: [M+H] + = 256.
步骤5:氮气保护下,将上步中间体P14-5(3.3g,13.0mmol)溶于33mL无水THF中,在0℃下缓慢滴加含有三光气(3.6g,12.4mmol)的四氢呋喃溶液(20mL)。升温至室温反应2小时,停止反应,向体系中加入100mL水,乙酸乙酯萃取,减压蒸除溶剂,得到粗品P14-6(2.8g)。LC-MS:[M+H]
+=282。
Step 5: Under the protection of nitrogen, the intermediate P14-5 (3.3g, 13.0mmol) in the previous step was dissolved in 33mL of anhydrous THF, and a tetrahydrofuran solution containing triphosgene (3.6g, 12.4mmol) was slowly added dropwise at 0°C (20mL). The temperature was raised to room temperature for 2 hours, and the reaction was stopped. 100 mL of water was added to the system, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure to obtain crude product P14-6 (2.8 g). LC-MS: [M+H] + =282.
步骤6:氮气保护下,将上步粗品P14-6(2.8g)溶于50mL三氯氧磷中,加入5mLN,N-二异丙基乙胺,升温至100℃下反应2小时。减压蒸除溶剂,向体系中加入100mL水,乙酸乙酯萃取,浓缩,残余物通过flash柱层析色谱分离(石油醚/乙酸乙酯,3/1),得到黄色固体P14-7(1.1g,3.5mmol),两步收率:27%。LC-MS:[M+H]
+=319。
Step 6: Under the protection of nitrogen, dissolve the crude product P14-6 (2.8 g) from the previous step in 50 mL of phosphorus oxychloride, add 5 mL of N,N-diisopropylethylamine, and heat up to 100 ° C for 2 hours. The solvent was evaporated under reduced pressure, 100 mL of water was added to the system, extracted with ethyl acetate, concentrated, and the residue was separated by flash column chromatography (petroleum ether/ethyl acetate, 3/1) to obtain a yellow solid P14-7 (1.1 g, 3.5 mmol), two-step yield: 27%. LC-MS: [M+H] + = 319.
步骤7:室温下,将中间体P14-7(1.0g,3.15mmol)和原料3,8-二氮杂双环[3.2.1]辛烷-8-羧酸叔丁酯a2-1(670mg,3.15mmol)溶于20mL 1,4-二氧六环中,加入N,N-二异丙基乙基胺(1.7mL,9.5mmol),升温至50℃下反应2小时。向体系加水60mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,柱层 析分离,得到白色固体P14-8(900mg,1.82mmol)。收率:58%。LC-MS:[M+H]
+=495。
Step 7: At room temperature, intermediate P14-7 (1.0g, 3.15mmol) and raw material 3,8-diazabicyclo[3.2.1]octane-8-carboxylate tert-butyl a2-1 (670mg, 3.15mmol) was dissolved in 20mL 1,4-dioxane, N,N-diisopropylethylamine (1.7mL, 9.5mmol) was added, and the reaction was carried out at 50°C for 2 hours. Add 60 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain white solid P14-8 (900 mg, 1.82 mmol). Yield: 58%. LC-MS: [M+H] + = 495.
步骤8:氮气保护下,将中间体P14-8(520mg,1.05mmol)和碳酸铯(684mg,2.10mmol)溶于5mL DMF中,加入中间体a5(363mg,2.10mmol),升温至140℃下反应2小时。冷却至室温,向体系加水60mL,乙酸乙酯萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离(石油醚/乙酸乙酯,1/1),得到黄色固体P14-9(155mg,0.25mmol)。收率:23%。LC-MS:[M+H]
+=630。
Step 8: Under nitrogen protection, dissolve intermediate P14-8 (520mg, 1.05mmol) and cesium carbonate (684mg, 2.10mmol) in 5mL DMF, add intermediate a5 (363mg, 2.10mmol), and heat up to 140°C React for 2 hours. Cool to room temperature, add 60 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography (petroleum ether/ethyl acetate, 1/1) to obtain a yellow solid P14-9 (155 mg, 0.25 mmol). Yield: 23%. LC-MS: [M+H] + =630.
步骤9:氮气保护下,将中间体P14-9(155mg,0.25mmol)和碳酸铯(200mg,0.62mmol)溶于5mL甲苯中,加入中间体a7(139mg,0.34mmol)和Pd(DPEPhos)Cl
2(52mg,0.074mmol),升温至105℃下反应3小时。冷却至室温,向体系加水30mL,乙酸乙酯萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离(石油醚/乙酸乙酯,1/4),得到黄色固体P14-10(85mg,0.25mmol)。收率:41%。LC-MS:[M+H]
+=842。
Step 9: Under the protection of nitrogen, the intermediate P14-9 (155mg, 0.25mmol) and cesium carbonate (200mg, 0.62mmol) were dissolved in 5mL of toluene, and the intermediate a7 (139mg, 0.34mmol) and Pd(DPEPhos)Cl were added 2 (52mg, 0.074mmol), heated to 105°C and reacted for 3 hours. Cool to room temperature, add 30 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography (petroleum ether/ethyl acetate, 1/4) to obtain a yellow solid P14-10 (85 mg, 0.25 mmol). Yield: 41%. LC-MS: [M+H] + = 842.
步骤10:冰浴下,将P14-10(65mg,0.077mmol)溶于3mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过HPLC制备色谱分离(X Bridge Shield RP18 OBD柱,19*150mm,5μm;流动相A:水(10mmol/L NH
4HCO
3),流动相B:乙腈;流速:25mL/min),得到白色固体P14(9.2mg),LC-MS:[M+H]
+=642。
Step 10: Dissolve P14-10 (65 mg, 0.077 mmol) in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by preparative HPLC chromatography (X Bridge Shield RP18 OBD column, 19*150mm, 5μm; mobile phase A: water (10mmol/L NH 4 HCO 3 ), mobile phase B: acetonitrile; flow rate: 25mL/min), to obtain White solid P14 (9.2 mg), LC-MS: [M+H] + =642.
1H NMR(400MHz,DMSO-d
6)δ8.11(d,J=4.0Hz,1H),7.97(brs,2H),7.36-7.31(m,2H),7.17-7.11(m,1H),6.57(d,J=4.0Hz,1H),5.17(d,J=56.0Hz,1H),4.31-4.18(m,2H),4.07-4.03(m,1H),3.93-3.89(m,1H),3.49-3.45(m,2H),2.89-2.73(m,3H),2.41-2.27(m,3H),2.17-2.08(m,2H),1.93-1.79(m,3H),1.66-1.62(m,2H),1.32(s,1H),0.64-0.61(m,2H),0.45-0.42(m,2H).
1 H NMR (400MHz, DMSO-d 6 )δ8.11 (d, J=4.0Hz, 1H), 7.97 (brs, 2H), 7.36-7.31 (m, 2H), 7.17-7.11 (m, 1H), 6.57(d,J=4.0Hz,1H),5.17(d,J=56.0Hz,1H),4.31-4.18(m,2H),4.07-4.03(m,1H),3.93-3.89(m,1H) ,3.49-3.45(m,2H),2.89-2.73(m,3H),2.41-2.27(m,3H),2.17-2.08(m,2H),1.93-1.79(m,3H),1.66-1.62( m,2H),1.32(s,1H),0.64-0.61(m,2H),0.45-0.42(m,2H).
实施例13:Example 13:
步骤1:冰浴,将中间体c1(330mg,1.76mmol)溶于1mL无水四氢呋喃中,加入NaH(85mg,3.52mmol),升温至室温搅拌0.5小时,反应液备用。将中间体a3(350mg,0.82mmol)溶于1mL无水四氢呋喃中,加入上述反应液,室温下反应1小时,停止反应。减压浓缩,粗品经flash柱层析分离,得到化合物P18-1(240mg,0.42mmol),收率24%。LC-MS:[M+H]
+=579。
Step 1: In ice bath, dissolve intermediate c1 (330mg, 1.76mmol) in 1mL of anhydrous tetrahydrofuran, add NaH (85mg, 3.52mmol), warm to room temperature and stir for 0.5 hours, and the reaction solution is set aside. Intermediate a3 (350mg, 0.82mmol) was dissolved in 1mL of anhydrous tetrahydrofuran, added to the above reaction solution, reacted at room temperature for 1 hour, and stopped the reaction. Concentrated under reduced pressure, the crude product was separated by flash column chromatography to obtain compound P18-1 (240mg, 0.42mmol), with a yield of 24%. LC-MS: [M+H] + = 579.
步骤2:氮气保护下,将化合物P18-1(240mg,0.42mmol)、中间体b2(270mg,0.44mmol)、碳酸铯(270mg,0.83mmol)和甲磺酸[正丁基二(1-金刚烷基)膦](2-氨基-1,1'-联苯-2-基)钯(Pd-G3,10mg,0.014mmol)溶于2mL 1,4-二氧六环和水的混合溶液中(v/v,5/1),升温至95℃下反应1小时,停止反应。减压蒸除溶剂,粗品经flash柱层析分离,得到化合物P18-2(300mg,0.29mmol),收率70%。LC-MS:[M+H]
+=1041。
Step 2: Under nitrogen protection, compound P18-1 (240mg, 0.42mmol), intermediate b2 (270mg, 0.44mmol), cesium carbonate (270mg, 0.83mmol) and methanesulfonic acid [n-butylbis(1-adamantine Alkyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium (Pd-G3, 10mg, 0.014mmol) was dissolved in a mixed solution of 2mL 1,4-dioxane and water (v/v, 5/1), the temperature was raised to 95° C. for 1 hour, and the reaction was stopped. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound P18-2 (300 mg, 0.29 mmol), with a yield of 70%. LC-MS: [M+H] + = 1041.
步骤3:将化合物P18-2(270mg,0.26mmol)和氟化铯(80mg,0.52mmol)溶于1mL DMF中, 升温至50℃下反应1小时,停止反应。向体系中加入5mL水,乙酸乙酯萃取,无水硫酸钠干燥,减压浓缩,得粗品P18-3。LC-MS:[M+H]
+=729。
Step 3: Compound P18-2 (270 mg, 0.26 mmol) and cesium fluoride (80 mg, 0.52 mmol) were dissolved in 1 mL of DMF, heated to 50° C. for 1 hour, and the reaction was stopped. Add 5 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain crude product P18-3. LC-MS: [M+H] + =729.
步骤4:将上步粗品P18-3溶于1mL氯化氢的1,4-二氧六环溶液中(4M浓度),室温下反应1小时,向体系缓慢加入饱和碳酸氢钠水溶液调至pH至8左右,乙酸乙酯萃取,减压浓缩,经TLC薄层色谱纯化,得到化合物P18(50mg)。LC-MS:[M+H]
+=629。
Step 4: Dissolve the crude product P18-3 from the previous step in 1 mL of hydrogen chloride in 1,4-dioxane solution (4M concentration), react at room temperature for 1 hour, and slowly add saturated aqueous sodium bicarbonate solution to the system to adjust the pH to 8 Left and right, extracted with ethyl acetate, concentrated under reduced pressure, and purified by TLC thin layer chromatography to obtain compound P18 (50 mg). LC-MS: [M+H] + = 629.
1H NMR(400MHz,DMSO-d
6)δ10.17(s,1H),9.06(s,1H),7.99(dd,J=9.2,5.9Hz,1H),7.48(t,J=9.0Hz,1H),7.41(d,J=2.5Hz,1H),7.19(t,J=2.2Hz,1H),4.49(d,J=11.4Hz,1H),4.33(d,J=12.6Hz,1H),4.12(dd,J=10.5,3.4Hz,1H),4.02(dd,J=10.6,2.7Hz,1H),3.95(s,1H),3.69–3.53(m,4H),3.45–3.25(m,2H),2.95–2.85(m,1H),2.72–2.64(m,1H),2.57–2.52(m,1H),2.47–2.32(m,2H),2.09(s,3H),1.95–1.72(m,4H),1.90–1.60(m,4H),1.51(t,J=11.7Hz,1H).
1 H NMR (400MHz, DMSO-d 6 )δ10.17(s, 1H), 9.06(s, 1H), 7.99(dd, J=9.2, 5.9Hz, 1H), 7.48(t, J=9.0Hz, 1H), 7.41(d, J=2.5Hz, 1H), 7.19(t, J=2.2Hz, 1H), 4.49(d, J=11.4Hz, 1H), 4.33(d, J=12.6Hz, 1H) ,4.12(dd,J=10.5,3.4Hz,1H),4.02(dd,J=10.6,2.7Hz,1H),3.95(s,1H),3.69–3.53(m,4H),3.45–3.25(m ,2H),2.95–2.85(m,1H),2.72–2.64(m,1H),2.57–2.52(m,1H),2.47–2.32(m,2H),2.09(s,3H),1.95–1.72 (m,4H),1.90–1.60(m,4H),1.51(t,J=11.7Hz,1H).
参照化合物P18的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound P18, using a similar skeleton structure, the following target molecules were synthesized.
实施例14:Example 14:
冰浴下,将原料P29-1(305mg,2.4mmol)和中间体a3(500mg,1.98mmol)溶于10mL二氯甲烷中,加入三乙胺(301mg,2.97mmol),室温下反应2小时。向体系加水30mL,二氯甲烷萃取,无水硫酸钠干燥,过滤,浓缩,柱层析分离,得到白色固体P29-2(650mg,1.9mmol),收率:95%。LC-MS:[M+H]
+=344。
Under ice-cooling, raw material P29-1 (305 mg, 2.4 mmol) and intermediate a3 (500 mg, 1.98 mmol) were dissolved in 10 mL of dichloromethane, triethylamine (301 mg, 2.97 mmol) was added, and reacted at room temperature for 2 hours. Add 30 mL of water to the system, extract with dichloromethane, dry over anhydrous sodium sulfate, filter, concentrate, and separate by column chromatography to obtain white solid P29-2 (650 mg, 1.9 mmol), yield: 95%. LC-MS: [M+H] + = 344.
冰浴,在50mL反应瓶中,将化合物P29-2(300mg,0.87mmol)溶于6mL无水四氢呋喃中,加入氢化钠(70mg,1.74mmol),搅拌30分钟得溶液S1。向溶液S1中加入中间体a18,升温至70℃下反应16小时,冷却至室温,停止反应。向体系加入30mL冰水淬灭反应,乙酸乙酯萃取,减压浓缩除去溶剂。残余物通过flash柱层析色谱分离(DCM/MeOH=10/1)纯化,得到白色固体P29-3(200mg,0.40mmol),收率:46%。LC-MS:[M+H]
+=495。
In ice bath, in a 50 mL reaction flask, compound P29-2 (300 mg, 0.87 mmol) was dissolved in 6 mL of anhydrous THF, sodium hydride (70 mg, 1.74 mmol) was added, and stirred for 30 minutes to obtain solution S1. The intermediate a18 was added to the solution S1, the temperature was raised to 70°C and the reaction was carried out for 16 hours, and then cooled to room temperature to stop the reaction. Add 30 mL of ice water to the system to quench the reaction, extract with ethyl acetate, and concentrate under reduced pressure to remove the solvent. The residue was purified by flash column chromatography (DCM/MeOH=10/1) to obtain white solid P29-3 (200 mg, 0.40 mmol), yield: 46%. LC-MS: [M+H] + = 495.
氮气保护下,将化合物P29-3(450mg,0.91mmol)和原料P9-1(560mg,1.1mmol)溶于10mL1,4-二氧六环和2mL水中,依次加入催化剂XPhos Pd G2(215mg,0.3mmol)和碳酸铯(1.18g,3.64mmol)。反应液于95℃下反应2小时,停止反应。减压浓缩除去溶剂,残余物通过flash柱层析色谱分离(DCM/MeOH=20/1),得到黄色固体P29-4(500mg,0.59mmol)。收率:65%,LC-MS:[M+H]+=845。Under the protection of nitrogen, the compound P29-3 (450mg, 0.91mmol) and the raw material P9-1 (560mg, 1.1mmol) were dissolved in 10mL of 1,4-dioxane and 2mL of water, and the catalyst XPhos Pd G2 (215mg, 0.3 mmol) and cesium carbonate (1.18g, 3.64mmol). The reaction solution was reacted at 95° C. for 2 hours, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the residue was separated by flash column chromatography (DCM/MeOH=20/1) to obtain a yellow solid P29-4 (500 mg, 0.59 mmol). Yield: 65%, LC-MS: [M+H]+=845.
室温下,将化合物P29-4(200mg,0.24mmol)溶于4mL四氢呋喃中,加入四丁基氟化铵(TBAF,130mg,0.48mmol),室温下反应1小时。向体系加水20mL,乙酸乙酯萃取,无水硫酸钠干燥,过滤,浓缩,得到粗品黄色油状物P29-5(160mg,0.23mmol)。收率:98%。LC-MS:[M+H]
+=689。
Compound P29-4 (200 mg, 0.24 mmol) was dissolved in 4 mL of tetrahydrofuran at room temperature, tetrabutylammonium fluoride (TBAF, 130 mg, 0.48 mmol) was added, and reacted at room temperature for 1 hour. Add 20 mL of water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, and concentrate to obtain crude yellow oil P29-5 (160 mg, 0.23 mmol). Yield: 98%. LC-MS: [M+H] + = 689.
将上步粗品P29-5(163mg,0.24mmol)溶于2mL二氯甲烷中,滴加1mL氯化氢的1,4-二氧六环溶液(浓度4N),室温下反应1小时,停止反应。缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到10mg黄色固体P29。LC-MS:[M+H]
+=645。
Dissolve the crude product P29-5 (163 mg, 0.24 mmol) from the previous step in 2 mL of dichloromethane, add 1 mL of hydrogen chloride in 1,4-dioxane solution (concentration 4 N) dropwise, react at room temperature for 1 hour, and stop the reaction. Slowly add saturated sodium bicarbonate solution to the system to adjust the pH to about 8, extract with ethyl acetate, and concentrate under reduced pressure. The residue was separated by flash column chromatography to obtain 10 mg of yellow solid P29. LC-MS: [M+H] + = 645.
1H NMR(400MHz,DMSO-d
6)δ10.16(s,1H),9.34(s,1H),8.77(s,1H),8.01(dd,J=9.2,5.9Hz,1H),7.49(t,J=9.0Hz,1H),7.42(d,J=2.4Hz,1H),7.19(t,J=3.5Hz,1H),5.30–5.02(m,1H),4.54–4.41(m,2H),4.22-4.05(m,1H),4.00-3.92(m,1H),3.87-3.68(m,1H),2.85–2.60(m,5H),2.42-2.20(m,7H),2.16–1.67(m,14H).
1 H NMR (400MHz, DMSO-d 6 ) δ10.16(s, 1H), 9.34(s, 1H), 8.77(s, 1H), 8.01(dd, J=9.2, 5.9Hz, 1H), 7.49( t,J=9.0Hz,1H),7.42(d,J=2.4Hz,1H),7.19(t,J=3.5Hz,1H),5.30–5.02(m,1H),4.54–4.41(m,2H ),4.22-4.05(m,1H),4.00-3.92(m,1H),3.87-3.68(m,1H),2.85–2.60(m,5H),2.42-2.20(m,7H),2.16–1.67 (m,14H).
实施例15:Example 15:
步骤1:将中间体d2(1.4g,1.85mmol)溶于10mL四氢呋喃中,缓慢滴加饱和氢氧化锂水溶液(5.0mL),室温下反应40分钟,停止反应。缓慢向反应中滴加1M浓度稀盐酸调节pH至5左右,乙酸乙酯萃取,浓缩,得到粗品M1-1,直接用于下一步反应。LC-MS:[M+H]
+=727。
Step 1: Intermediate d2 (1.4g, 1.85mmol) was dissolved in 10mL of tetrahydrofuran, and saturated lithium hydroxide aqueous solution (5.0mL) was slowly added dropwise, reacted at room temperature for 40 minutes, and stopped the reaction. Slowly add 1M dilute hydrochloric acid dropwise to the reaction to adjust the pH to about 5, extract with ethyl acetate, and concentrate to obtain the crude product M1-1, which is directly used in the next reaction. LC-MS: [M+H] + =727.
步骤2:冰浴,将上步粗品M1-1溶解于三氟乙酸和二氯甲烷的混合溶液中(4.0mL,1/1),冰浴下反应1小时,停止反应。减压蒸除溶剂,粗品经HPLC制备色谱分离,得到化合物M1(10mg)。LC-MS:[M+H]
+=527。
Step 2: In an ice bath, dissolve the crude product M1-1 from the previous step in a mixed solution of trifluoroacetic acid and dichloromethane (4.0 mL, 1/1), react in an ice bath for 1 hour, and stop the reaction. The solvent was evaporated under reduced pressure, and the crude product was separated by preparative HPLC chromatography to obtain compound M1 (10 mg). LC-MS: [M+H] + = 527.
1H NMR(400MHz,DMSO-d
6)δ8.10(s,2H),7.81(s,1H),7.32–7.21(m,1H),7.18–7.10(m,1H),4.27(d,J=10.7Hz,2H),3.47(s,4H),1.58(s,4H).
1H NMR (400MHz, DMSO-d 6 )δ8.10(s,2H),7.81(s,1H),7.32–7.21(m,1H),7.18–7.10(m,1H),4.27(d,J= 10.7Hz,2H),3.47(s,4H),1.58(s,4H).
实施例16:Example 16:
步骤1:冰浴,将化合物M1-1(400mg,0.55mmol)、原料M2-1(109mg,0.83mmol)和DIEA(213mg,1.65mmol)溶于10mL DMF中,搅拌5分钟后,加入HATU(315mg,0.83mmol),室温下反应2小时。将反应液倒入150mL冰水中,乙酸乙酯萃取,饱和食盐水洗涤,无水硫酸钠干燥,浓缩,粗品经柱层析色谱分离,得到无色油状物M2-2(393mg,0.47mmol),收率85%。LC-MS:[M+H]
+=837
Step 1: In ice bath, compound M1-1 (400mg, 0.55mmol), raw material M2-1 (109mg, 0.83mmol) and DIEA (213mg, 1.65mmol) were dissolved in 10mL DMF, stirred for 5 minutes, then added HATU ( 315mg, 0.83mmol), react at room temperature for 2 hours. The reaction solution was poured into 150 mL of ice water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain a colorless oil M2-2 (393 mg, 0.47 mmol). Yield 85%. LC-MS: [M+H] + = 837
步骤2:将上步中间体M2-2(393mg,0.47mmol)溶于4.0mL二氯甲烷中,加入1.0mL三氟乙酸,室温反应1小时,停止反应。减压蒸除溶剂,将粗品溶解于10mL水中,用饱和碳酸氢钠水溶液调节pH至8左右,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经柱层析分离,得到化合物M2(89mg,0.14mmol),收率:30%。LC-MS:[M+H]
+=637。
Step 2: Dissolve the intermediate M2-2 (393 mg, 0.47 mmol) in 4.0 mL of dichloromethane, add 1.0 mL of trifluoroacetic acid, react at room temperature for 1 hour, and stop the reaction. The solvent was evaporated under reduced pressure, the crude product was dissolved in 10 mL of water, the pH was adjusted to about 8 with saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to obtain compound M2 (89 mg , 0.14mmol), yield: 30%. LC-MS: [M+H] + = 637.
1H NMR(400MHz,DMSO-d
6)δ8.5(d,J=8.4Hz,1H),8.12(s,2H),7.97(s,1H),7.33–7.25(m,1H),7.17(t,J=8.9Hz,1H),4.49(t,J=11.4Hz,1H),4.37(t,J=10.4Hz,1H),4.17(t,J=7.5Hz,1H),3.68-3.51(m,5H),2.86–2.80(m,1H),2.68–2.66(m,1H),2.34–2.31(m,1H),2.02–1.92(m,1H),1.84–1.75(m,1H),1.71–1.46(m,9H),1.25–1.22(m,2H).
1 H NMR (400MHz, DMSO-d 6 ) δ8.5 (d, J=8.4Hz, 1H), 8.12(s, 2H), 7.97(s, 1H), 7.33–7.25(m, 1H), 7.17( t,J=8.9Hz,1H),4.49(t,J=11.4Hz,1H),4.37(t,J=10.4Hz,1H),4.17(t,J=7.5Hz,1H),3.68-3.51( m,5H),2.86–2.80(m,1H),2.68–2.66(m,1H),2.34–2.31(m,1H),2.02–1.92(m,1H),1.84–1.75(m,1H), 1.71–1.46(m,9H),1.25–1.22(m,2H).
参照化合物M2的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound M2, using a similar skeleton structure, the following target molecules were synthesized.
*表示手性分子,未进行手性拆分。* Indicates chiral molecules without chiral resolution.
实施例17:Example 17:
步骤1:氮气保护下,在50mL反应瓶中,将中间体a31(300mg,1.0mmol)溶于5mL无水THF,加入叔丁醇钾(224mg,2.0mmol),室温下搅拌30分钟—得溶液S5。在另一50mL反应瓶中将中间体a14(488mg,1.0mmol)溶于5mL无水THF中,冰浴下,缓慢加入配置好的溶液S5,继续搅拌1小时,停止反应。将反应液倒入50mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体H23-1(240mg,0.32mmol)。收率:31%,LC-MS:[M+H]
+=757。
Step 1: Under nitrogen protection, in a 50 mL reaction flask, dissolve intermediate a31 (300 mg, 1.0 mmol) in 5 mL of anhydrous THF, add potassium tert-butoxide (224 mg, 2.0 mmol), and stir at room temperature for 30 minutes to obtain a solution S5. In another 50mL reaction flask, the intermediate a14 (488mg, 1.0mmol) was dissolved in 5mL of anhydrous THF, and the prepared solution S5 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 50 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid H23-1 (240 mg, 0.32 mmol). Yield: 31%, LC-MS: [M+H] + =757.
步骤2:氮气保护下,将化合物H23-1(240mg,0.32mmol)溶于6mL无水甲苯中,依次加入中间体a7(188mg,0.45mmol),Pd(DPEPhos)Cl
2(46mg,0.064mmol)和碳酸铯(209mg,0.64mmol)。在氮气保护下,反应液于105℃下反应10小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:2),得到黄色固体H23-2(250mg,0.26mmol)。收率:79%,LC-MS:[M+H]
+=982。
Step 2: Under nitrogen protection, compound H23-1 (240mg, 0.32mmol) was dissolved in 6mL of anhydrous toluene, and intermediate a7 (188mg, 0.45mmol) and Pd(DPEPhos)Cl 2 (46mg, 0.064mmol) were added successively and cesium carbonate (209mg, 0.64mmol). Under nitrogen protection, the reaction solution was reacted at 105° C. for 10 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain yellow solid H23-2 (250 mg, 0.26 mmol). Yield: 79%, LC-MS: [M+H] + =982.
步骤3:冰浴下,将化合物H23-2(250mg,0.26mmol)溶于6mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H23(10mg),LC-MS:[M+H]
+=698。
Step 3: Compound H23-2 (250 mg, 0.26 mmol) was dissolved in 6 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H23 (10 mg), LC-MS: [M+H] + =698.
1H NMR(400MHz,DMSO-d
6)δ8.16(s,2H),7.87(s,1H),7.28(dd,J=8.4,5.3Hz,1H),7.17(t,J=8.9Hz,1H),5.19–5.05(m,1H),4.38–4.29(m,5H),3.68–3.33(m,9H),3.18–3.09(m,1H),3.01(d,J=13.1Hz,1H),2.88-2.85(m,1H),2.67–2.56(m,2H),2.09-1.53(m,12H).
1 H NMR (400MHz, DMSO-d 6 )δ8.16(s,2H),7.87(s,1H),7.28(dd,J=8.4,5.3Hz,1H),7.17(t,J=8.9Hz, 1H),5.19–5.05(m,1H),4.38–4.29(m,5H),3.68–3.33(m,9H),3.18–3.09(m,1H),3.01(d,J=13.1Hz,1H) ,2.88-2.85(m,1H),2.67–2.56(m,2H),2.09-1.53(m,12H).
实施例18:Example 18:
步骤1:在50mL反应瓶中,将中间体a18(176mg,0.94mmol)溶于10mL无水THF,加入叔丁醇钾(180mg,1.56mmol),室温下搅拌30分钟—得溶液S6。在另一50mL反应瓶中将中间体a17(400mg,0.78mmol)溶于10mL无水THF中,冰浴下,缓慢加入配置好的溶液S2,继续搅拌1小时,停止反应。将反应液倒入100mL冰水中,乙酸乙酯萃取,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1)纯化,得到淡黄色固体H24-1(337mg,0.45mmol)。收率:48%,LC-MS:[M+H]
+=749。
Step 1: In a 50 mL reaction flask, the intermediate a18 (176 mg, 0.94 mmol) was dissolved in 10 mL of anhydrous THF, potassium tert-butoxide (180 mg, 1.56 mmol) was added, and stirred at room temperature for 30 minutes to obtain solution S6. In another 50mL reaction flask, the intermediate a17 (400mg, 0.78mmol) was dissolved in 10mL of anhydrous THF, and the prepared solution S2 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 100 mL of ice water, extracted with ethyl acetate, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain pale yellow solid H24-1 (337 mg, 0.45 mmol). Yield: 48%, LC-MS: [M+H] + =749.
步骤2:氮气保护下,将H24-1(1.0g,1.34mmol)、碳酸钾和甲基硼酸(80mg,1.34mmol)溶于10mL无水DMA中,加入Pd(PPh
3)Cl
2(100mg,0.13mmol),反应液于90℃下反应2小时,冷却至室温,停止反应。向反应液中加入50mL水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=3:1),得到黄色固体H24-2(340mg,0.54mmol)。收率:40%,LC-MS:[M+H]
+=636。
Step 2: Under nitrogen protection, H24-1 (1.0g, 1.34mmol), potassium carbonate and methylboronic acid (80mg, 1.34mmol) were dissolved in 10mL of anhydrous DMA, and Pd(PPh 3 )Cl 2 (100mg, 0.13 mmol), the reaction solution was reacted at 90°C for 2 hours, cooled to room temperature, and the reaction was stopped. 50 mL of water was added to the reaction solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain a yellow solid H24-2 ( 340 mg, 0.54 mmol). Yield: 40%, LC-MS: [M+H] + = 636.
步骤3:氮气保护下,将上步中间体H24-2(340mg,0.54mmol)溶于10mL无水甲苯中,依次加入中间体a7(900mg,2.2mmol),Pd(DPEPhos)Cl
2(170mg,0.24mmol)和碳酸铯(1.0g,3.2mmol)。在氮气保护下,反应液于110℃下反应6小时,停止反应,冷却至室温,减压蒸除溶剂。残余物通过flash柱层析色谱分离(石油醚:乙酸乙酯=1:1),得到黄色固体H24-3(300mg,0.35mmol)。收率:65%,LC-MS:[M+H]
+=848。
Step 3: Under nitrogen protection, dissolve the intermediate H24-2 (340mg, 0.54mmol) in 10mL of anhydrous toluene, and then add intermediate a7 (900mg, 2.2mmol), Pd(DPEPhos)Cl 2 (170mg, 0.24mmol) and cesium carbonate (1.0g, 3.2mmol). Under the protection of nitrogen, the reaction solution was reacted at 110° C. for 6 hours, the reaction was stopped, cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was separated by flash column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain yellow solid H24-3 (300 mg, 0.35 mmol). Yield: 65%, LC-MS: [M+H] + =848.
步骤4:冰浴下,将H24-3(300mg,0.35mmol)溶于3mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中。在氮气保护下,将反应液于置于室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过HPLC制备色谱分离,得到黄色固体H24(10mg),LC-MS:[M+H]
+=648。
Step 4: Dissolve H24-3 (300 mg, 0.35 mmol) in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) under ice cooling. Under the protection of nitrogen, the reaction liquid was reacted at room temperature for 1 hour, the reaction was stopped, and saturated sodium bicarbonate solution was slowly added to the system to adjust the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by preparative HPLC chromatography to obtain yellow solid H24 (10 mg), LC-MS: [M+H] + =648.
1H NMR(400MHz,DMSO-d
6)δ8.03(s,2H),7.61(s,1H),7.22–7.08(m,2H),5.24-5.10(m,1H),4.38(s,2H),4.27(dd,J=25.6,12.1Hz,2H),3.53–3.42(m,5H),2.80–2.62(m,5H),2.40–2.30(m,1H),2.07(s,3H),2.01–1.66(m,12H).
1 H NMR (400MHz,DMSO-d 6 )δ8.03(s,2H),7.61(s,1H),7.22–7.08(m,2H),5.24-5.10(m,1H),4.38(s,2H ),4.27(dd,J=25.6,12.1Hz,2H),3.53–3.42(m,5H),2.80–2.62(m,5H),2.40–2.30(m,1H),2.07(s,3H), 2.01–1.66(m,12H).
实施例19:Example 19:
步骤1:-25℃,氮气保护下,参考实施例4路线获得的化合物H10-1(1.1g,1.68mmol)溶于20mL无水四氢呋喃中,缓慢滴加异丙基溴化镁(5.0mL,5.0mmol),滴毕,向反应液加入异丙醇频哪醇硼酸酯(1.56g,8.4mmol),在-25℃下反应2小时,停止反应。向混合物中加入20mL饱和氯化铵水溶液淬灭反应,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得到淡黄色固体H26-1(800mg,1.28mmol),收率:76%。LC-MS:[M+H]
+=622。
Step 1: -25°C, under the protection of nitrogen, the compound H10-1 (1.1g, 1.68mmol) obtained by referring to the route of Example 4 was dissolved in 20mL of anhydrous tetrahydrofuran, and isopropylmagnesium bromide (5.0mL, 5.0 mmol), after dropping, isopropanol pinacol borate (1.56 g, 8.4 mmol) was added to the reaction liquid, reacted at -25°C for 2 hours, and stopped the reaction. The reaction was quenched by adding 20 mL of saturated aqueous ammonium chloride solution to the mixture, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain light Yellow solid H26-1 (800 mg, 1.28 mmol), yield: 76%. LC-MS: [M+H] + = 622.
步骤2:氮气保护下,将化合物H26-1(250mg,0.74mmol)、碳酸铯(480mg,1.48mmol)和中间体b3(690mg,1.11mmol)溶于10mL无水甲苯中,加入催化剂双(二苯基膦苯基醚)二氯化钯(100mg,0.15mmol),反应液于105℃下反应3小时,停止反应,过滤。减压蒸除溶剂,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=1/2),得淡黄色固体H26-2(160mg,0.19mmol),收率:26%。LC-MS:[M+H]
+=835。
Step 2: Under nitrogen protection, compound H26-1 (250mg, 0.74mmol), cesium carbonate (480mg, 1.48mmol) and intermediate b3 (690mg, 1.11mmol) were dissolved in 10mL of anhydrous toluene, and the catalyst bis(di Phenylphosphine phenyl ether) palladium dichloride (100mg, 0.15mmol), the reaction solution was reacted at 105°C for 3 hours, the reaction was stopped, and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=1/2) to obtain light yellow solid H26-2 (160 mg, 0.19 mmol), yield: 26%. LC-MS: [M+H] + = 835.
步骤3:冰浴下,将化合物H26-2(160mg,0.19mmol)溶于5mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中,室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H26(20mg,0.032mmol)。LC-MS:[M+H]
+=635。
Step 3: Dissolve compound H26-2 (160 mg, 0.19 mmol) in 5 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) in an ice bath, react at room temperature for 1 hour, and stop the reaction , slowly add saturated sodium bicarbonate solution to the system and adjust the pH to about 8, extract with ethyl acetate, and concentrate under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H26 (20 mg, 0.032 mmol). LC-MS: [M+H] + = 635.
1HNMR(400MHz,DMSO-d
6)δ7.85(s,1H),7.55(d,J=8.6Hz,1H),6.55(d,J=8.6Hz,1H),6.39–6.38(m,2H),5.11(dt,J=9.4,5.1Hz,1H),4.42(s,2H),4.29(d,J=12.3Hz,2H),3.70–3.62(m,6H),2.81–2.66(m,3H),2.65–2.57(m,3H),2.33(q,J=8.2Hz,1H),2.10–1.80(m,9H),1.75–1.65(m,5H).
1 HNMR (400MHz, DMSO-d 6 ) δ7.85(s, 1H), 7.55(d, J=8.6Hz, 1H), 6.55(d, J=8.6Hz, 1H), 6.39–6.38(m, 2H ),5.11(dt,J=9.4,5.1Hz,1H),4.42(s,2H),4.29(d,J=12.3Hz,2H),3.70–3.62(m,6H),2.81–2.66(m, 3H),2.65–2.57(m,3H),2.33(q,J=8.2Hz,1H),2.10–1.80(m,9H),1.75–1.65(m,5H).
实施例20:Example 20:
步骤1:-25℃,氮气保护下,参考实施例4路线获得的化合物H10-1(100mg,0.15mmol)、六甲 基二锡(74mg,0.22mmol)、三环己基磷(8.4mg,0.03mmol)和氯化锂(19mg,0.45mmol)溶于10mL 1,4-二氧六环中,加入催化剂Pd
2dba
3(27mg,0.03mmol),升温至95℃下反应12小时,停止反应。向混合物中加入40mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得到黄色固体H32-1(80mg,0.11mmol),收率:72%。LC-MS:[M+H]
+=742。
Step 1: -25°C, under the protection of nitrogen, refer to the compound H10-1 (100mg, 0.15mmol), hexamethylditin (74mg, 0.22mmol), tricyclohexylphosphine (8.4mg, 0.03 mmol) and lithium chloride (19mg, 0.45mmol) were dissolved in 10mL 1,4-dioxane, the catalyst Pd 2 dba 3 (27mg, 0.03mmol) was added, the temperature was raised to 95°C for 12 hours, and the reaction was stopped. Add 40 mL of ice water to the mixture, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain yellow solid H32-1 (80 mg , 0.11 mmol), yield: 72%. LC-MS: [M+H] + =742.
步骤2:氮气保护下,将化合物H32-1(80mg,0.11mmol)、碳酸铯(110mg,0.33mmol)和中间体b7(25mg,0.12mmol)溶于10mL 1,4-二氧六环中,加入催化剂双(三苯基膦)二氯化钯(15mg,0.022mmol),反应液于100℃下反应4小时,停止反应,过滤。减压蒸除溶剂,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得淡黄色固体H32-2(20mg,0.03mmol),收率:24%。LC-MS:[M+H]
+=750。
Step 2: Under nitrogen protection, compound H32-1 (80mg, 0.11mmol), cesium carbonate (110mg, 0.33mmol) and intermediate b7 (25mg, 0.12mmol) were dissolved in 10mL 1,4-dioxane, The catalyst bis(triphenylphosphine)palladium dichloride (15mg, 0.022mmol) was added, and the reaction solution was reacted at 100°C for 4 hours, then stopped and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain light yellow solid H32-2 (20 mg, 0.03 mmol), yield: 24%. LC-MS: [M+H] + =750.
步骤3:冰浴下,将化合物H32-2(20mg,0.03mmol)溶于1mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中,室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H32(2.1mg)。LC-MS:[M+H]
+=650。
Step 3: Dissolve compound H32-2 (20 mg, 0.03 mmol) in 1 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1) in an ice bath, react at room temperature for 1 hour, and stop the reaction , slowly add saturated sodium bicarbonate solution to the system and adjust the pH to about 8, extract with ethyl acetate, and concentrate under reduced pressure. The residue was separated by flash column chromatography to obtain H32 (2.1 mg) as a yellow solid. LC-MS: [M+H] + = 650.
1H NMR(400MHz,DMSO-d
6)δ7.98(s,1H),6.67(s,2H),5.19–5.05(m,1H),4.42(s,2H),4.29(d,J=12.3Hz,2H),3.70–3.62(m,6H),2.81–2.66(m,3H),2.65–2.57(m,3H),2.33–1.80(m,9H),1.75–1.65(m,5H).
1 H NMR (400MHz, DMSO-d 6 )δ7.98(s,1H),6.67(s,2H),5.19–5.05(m,1H),4.42(s,2H),4.29(d,J=12.3 Hz,2H),3.70–3.62(m,6H),2.81–2.66(m,3H),2.65–2.57(m,3H),2.33–1.80(m,9H),1.75–1.65(m,5H).
实施例21:Example 21:
步骤1:-60℃,氮气保护下,实施例18路线获得的化合物H24-1(1.0g,1.34mmol)溶于20mL无水四氢呋喃中,缓慢滴加异丙基溴化镁(1.3mL,1.3mmol),搅拌30分钟后,向反应液加入无水DMF(196mg,2.38mmol),再次搅拌30分钟后,停止反应。向混合物中加入20mL饱和氯化铵水溶液淬灭反应,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得到粗品H33-1。LC-MS:[M+H]
+=650。
Step 1: -60°C, under the protection of nitrogen, the compound H24-1 (1.0g, 1.34mmol) obtained in the route of Example 18 was dissolved in 20mL of anhydrous tetrahydrofuran, and isopropylmagnesium bromide (1.3mL, 1.3 mmol), after stirring for 30 minutes, anhydrous DMF (196 mg, 2.38 mmol) was added to the reaction solution, and after stirring for 30 minutes again, the reaction was stopped. Add 20 mL of saturated aqueous ammonium chloride solution to the mixture to quench the reaction, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate to obtain crude product H33-1. LC-MS: [M+H] + = 650.
步骤2:冰浴,将上步粗品H33-1溶于10mL甲醇中,慢慢加入硼氢化钠(102mg,2.68mmol),升温至室温下反应1小时,停止反应。向反应液加入30mL饱和氯化铵水溶液,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离(石油醚/乙酸乙酯=2/1),得到淡黄色固体H33-2(200mg,0.31mmol),两步收率:23%。LC-MS:[M+H]
+=652。
Step 2: In an ice bath, dissolve the crude product H33-1 from the previous step in 10 mL of methanol, slowly add sodium borohydride (102 mg, 2.68 mmol), warm up to room temperature for 1 hour, and stop the reaction. Add 30 mL of saturated ammonium chloride aqueous solution to the reaction solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography (petroleum ether/ethyl acetate=2/1) to obtain light yellow solid H33 -2 (200 mg, 0.31 mmol), two-step yield: 23%. LC-MS: [M+H] + = 652.
步骤3:氮气保护下,将上步中间体H33-2(200mg,0.31mmol)、碳酸铯(201mg,0.62mmol)和中间体a7(150mg,0.50mmol)溶于10mL无水甲苯中,加入催化剂双(二苯基膦苯基醚)二氯化钯(44mg,0.06mmol),反应液升温至105℃下反应3小时,停止反应,过滤,减压蒸除溶剂。粗品经 flash柱层析色谱分离(石油醚/乙酸乙酯=1/2),得淡黄色固体H33-3(150mg,0.17mmol),收率:56%。LC-MS:[M+H]
+=864。
Step 3: Under the protection of nitrogen, the above intermediate H33-2 (200mg, 0.31mmol), cesium carbonate (201mg, 0.62mmol) and intermediate a7 (150mg, 0.50mmol) were dissolved in 10mL of anhydrous toluene, and the catalyst was added Bis(diphenylphosphinephenyl ether)palladium dichloride (44mg, 0.06mmol), the reaction solution was heated to 105°C for 3 hours, the reaction was stopped, filtered, and the solvent was evaporated under reduced pressure. The crude product was separated by flash column chromatography (petroleum ether/ethyl acetate=1/2) to obtain light yellow solid H33-3 (150 mg, 0.17 mmol), yield: 56%. LC-MS: [M+H] + = 864.
步骤4:氮气保护下,将化合物H33-3(150mg,0.17mmol)溶于3mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中,室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过flash柱层析色谱分离,得到黄色固体H33(30mg),LC-MS:[M+H]
+=664。
Step 4: Under nitrogen protection, compound H33-3 (150 mg, 0.17 mmol) was dissolved in 3 mL of a mixed solution of trifluoroacetic acid and dichloromethane (v/v, 1/1), reacted at room temperature for 1 hour, and stopped the reaction , slowly add saturated sodium bicarbonate solution to the system and adjust the pH to about 8, extract with ethyl acetate, and concentrate under reduced pressure. The residue was separated by flash column chromatography to obtain yellow solid H33 (30 mg), LC-MS: [M+H] + =664.
1HNMR(400MHz,DMSO-d
6)δ8.01(s,2H),7.84(s,1H),7.21(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz,1H),5.35–5.25(m,1H),5.13(dt,J=56.1,5.6Hz,1H),4.40(s,2H),4.29–4.13(m,4H),3.52(s,2H),3.48–3.37(m,2H),2.81–2.66(m,3H),2.65–2.61(m,2H),2.36(dd,J=15.3,8.2Hz,1H),2.10–1.80(m,9H),1.73–1.65(m,4H).
1 HNMR (400MHz, DMSO-d 6 )δ8.01(s,2H),7.84(s,1H),7.21(dd,J=8.3,5.4Hz,1H),7.12(t,J=8.9Hz,1H ),5.35–5.25(m,1H),5.13(dt,J=56.1,5.6Hz,1H),4.40(s,2H),4.29–4.13(m,4H),3.52(s,2H),3.48– 3.37(m,2H),2.81–2.66(m,3H),2.65–2.61(m,2H),2.36(dd,J=15.3,8.2Hz,1H),2.10–1.80(m,9H),1.73– 1.65(m,4H).
实施例22:Example 22:
步骤1:冰浴,将原料H40-1(600mg,2.54mmol)和叔丁醇钾(404mg,3.6mmol)溶于5mL四氢呋喃中,搅拌10分钟;将上述混合液倒入含有中间体a14(882mg,1.8mmol)的四氢呋喃中(10mL)。混合物在冰浴条件下继续反应1小时,停止反应。向体系中加入50mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到化合物H40-2(500mg,0.71mmol),收率39%。LC-MS:[M+H]
+=705。
Step 1: In ice bath, dissolve raw material H40-1 (600mg, 2.54mmol) and potassium tert-butoxide (404mg, 3.6mmol) in 5mL THF, and stir for 10 minutes; pour the above mixture into intermediate a14 (882mg , 1.8mmol) in tetrahydrofuran (10mL). The mixture was continued to react for 1 hour under ice-bath condition, and the reaction was stopped. Add 50 mL of ice water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash column chromatography to obtain compound H40-2 (500 mg, 0.71 mmol) with a yield of 39%. LC-MS: [M+H] + =705.
步骤2:氮气保护下,将上步化合物H40-2(100mg,0.14mmol)、碳酸铯(92mg,0.28mmol)和中间体a7(80mg,0.20mmol)溶于5mL甲苯中,加入催化剂双(二苯基膦苯基醚)二氯化钯(10mg,0.014mmol),升温至105℃下反应2小时,停止反应,过滤。减压蒸除溶剂,粗品经flash柱层析分离,得到化合物H40-3(90mg,0.098mmol),收率69%。LC-MS:[M+H]
+=917。
Step 2: Under the protection of nitrogen, the above compound H40-2 (100mg, 0.14mmol), cesium carbonate (92mg, 0.28mmol) and intermediate a7 (80mg, 0.20mmol) were dissolved in 5mL of toluene, and the catalyst bis(di Phenylphosphine phenyl ether) palladium dichloride (10mg, 0.014mmol), heated to 105°C for 2 hours, stopped the reaction, and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound H40-3 (90 mg, 0.098 mmol), with a yield of 69%. LC-MS: [M+H] + =917.
步骤3:将上步化合物H40-3(90mg)溶于4mL三氟乙酸和二氯甲烷(v/v,1/1)的混合溶液中,室温下反应1小时,停止反应,缓慢向体系加入饱和碳酸氢钠溶液并调节pH至8左右,乙酸乙酯萃取,减压浓缩。残余物通过TLC薄层色谱分离,得到黄色固体H40(10mg),LC-MS:[M+H]
+=597。
Step 3: Dissolve the above compound H40-3 (90mg) in a mixed solution of 4mL trifluoroacetic acid and dichloromethane (v/v, 1/1), react at room temperature for 1 hour, stop the reaction, and slowly add Saturated sodium bicarbonate solution and adjusted the pH to about 8, extracted with ethyl acetate, and concentrated under reduced pressure. The residue was separated by TLC to give yellow solid H40 (10 mg), LC-MS: [M+H] + =597.
1H NMR(400MHz,DMSO-d
6)δ8.14(s,2H),7.89(s,1H),7.32(dd,J=8.4,5.2Hz,1H),7.23–7.17(m,1H),4.73(t,J=5.4Hz,1H),4.35–4.26(m,4H),3.64–3.35(m,7H),1.90–1.87(m,5H),1.73–1.60(m,5H).
1 H NMR (400MHz, DMSO-d 6 )δ8.14(s,2H),7.89(s,1H),7.32(dd,J=8.4,5.2Hz,1H),7.23–7.17(m,1H), 4.73(t,J=5.4Hz,1H),4.35–4.26(m,4H),3.64–3.35(m,7H),1.90–1.87(m,5H),1.73–1.60(m,5H).
实施例23:Example 23:
步骤1:将中间体d2(75mg,0.10mmol)溶于三氟乙酸和二氯甲烷的混合溶液中(3.0mL,1/1),冰浴下反应1小时,停止反应。减压蒸除溶剂,粗品经HPLC制备色谱分离,得到化合物M9(30mg)。LC-MS:[M+H]
+=555。
Step 1: Intermediate d2 (75mg, 0.10mmol) was dissolved in a mixed solution of trifluoroacetic acid and dichloromethane (3.0mL, 1/1), reacted under ice bath for 1 hour, and stopped the reaction. The solvent was evaporated under reduced pressure, and the crude product was separated by preparative HPLC chromatography to obtain compound M9 (30 mg). LC-MS: [M+H] + = 555.
1H NMR(400MHz,DMSO-d
6)δ8.18(s,2H),8.07(s,1H),7.28(dd,J=8.4,5.3Hz,1H),7.22–7.15(m,1H),4.64(d,J=13.7Hz,1H),4.44(d,J=13.7Hz,1H),4.38(q,J=7.1Hz,2H),4.18(d,J=27.8Hz,2H),4.03(d,J=13.4Hz,1H),3.84(d,J=13.6Hz,1H),1.95(m,J=12.9Hz,2H),1.88–1.76(m,2H),1.34(m,J=7.1Hz,4H).
1 H NMR (400MHz, DMSO-d 6 )δ8.18(s,2H),8.07(s,1H),7.28(dd,J=8.4,5.3Hz,1H),7.22–7.15(m,1H), 4.64(d, J=13.7Hz, 1H), 4.44(d, J=13.7Hz, 1H), 4.38(q, J=7.1Hz, 2H), 4.18(d, J=27.8Hz, 2H), 4.03( d,J=13.4Hz,1H),3.84(d,J=13.6Hz,1H),1.95(m,J=12.9Hz,2H),1.88–1.76(m,2H),1.34(m,J=7.1 Hz,4H).
实施例24:Example 24:
步骤1:氮气保护下,将化合物M1-1(35mg,0.048mmol)、4-二甲氨基吡啶DMAP(1.2mg,0.009mmol)和环丙醇(5.0mg,0.072mmol)溶于6mL二氯甲烷中,加入N,N'-二环己基碳二亚胺DCC(45mg,0.096mmol),室温下反应12小时,停止反应。向反应液中加入30mL冰水,二氯甲烷萃取,减压蒸除溶剂,粗品经flash柱层析分离(PE/EA=3/1),得到化合物M10-1(38mg),收率定量。LC-MS:[M+H]
+=767。
Step 1: Under nitrogen protection, compound M1-1 (35 mg, 0.048 mmol), 4-dimethylaminopyridine DMAP (1.2 mg, 0.009 mmol) and cyclopropanol (5.0 mg, 0.072 mmol) were dissolved in 6 mL of dichloromethane , N,N'-dicyclohexylcarbodiimide DCC (45mg, 0.096mmol) was added, reacted at room temperature for 12 hours, and stopped the reaction. Add 30 mL of ice water to the reaction liquid, extract with dichloromethane, evaporate the solvent under reduced pressure, and separate the crude product by flash column chromatography (PE/EA=3/1) to obtain compound M10-1 (38 mg) in quantitative yield. LC-MS: [M+H] + =767.
步骤2:将上步化合物M10-1(38mg)溶于三氟乙酸和二氯甲烷的混合溶液中(3.0mL,1/1),冰浴下反应1小时,减压蒸除溶剂,粗品经HPLC制备色谱分离,得到化合物M10(8mg)。LC-MS:[M+H]
+=567。
Step 2: Dissolve the above compound M10-1 (38 mg) in a mixed solution of trifluoroacetic acid and dichloromethane (3.0 mL, 1/1), react in an ice bath for 1 hour, evaporate the solvent under reduced pressure, and the crude product is HPLC preparative chromatography gave compound M10 (8 mg). LC-MS: [M+H] + = 567.
1H NMR(400MHz,DMSO-d
6)δ9.54(s,1H),8.16(s,2H),8.04(s,1H),7.29(dd,J=8.0,4.0Hz,1H),7.18(t,J=8.0Hz,1H),4.53(dd,J=76.0,12Hz,2H),4.38–4.34(m,1H),3.93–3.71(m,4H),3.73(d,J=12Hz,1H),2.67(d,J=20Hz,1H),2.33(d,J=12Hz,1H),2.01–1.82(m,5H).
1 H NMR (400MHz, DMSO-d 6 ) δ9.54(s, 1H), 8.16(s, 2H), 8.04(s, 1H), 7.29(dd, J=8.0, 4.0Hz, 1H), 7.18( t, J=8.0Hz, 1H), 4.53(dd, J=76.0, 12Hz, 2H), 4.38–4.34(m, 1H), 3.93–3.71(m, 4H), 3.73(d, J=12Hz, 1H ),2.67(d,J=20Hz,1H),2.33(d,J=12Hz,1H),2.01–1.82(m,5H).
参照化合物M10的合成路线,采用类似的骨架结构,合成如下目标分子。Referring to the synthetic route of compound M10, using a similar skeleton structure, the following target molecules were synthesized.
*表示手性分子,未进行手性拆分。* Indicates chiral molecules without chiral resolution.
实施例25:Example 25:
步骤1:冰浴,将丙酰胺(1.0g,2.05mmol)溶于10mL无水四氢呋喃中,加入NaH(123mg,3.07mmol),搅拌30分钟后,向反应体系中加入中间体a14(976mg,2.0mmol)。升温至室温反应2小时,LC-MS监测反应完全,向反应液加入50mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到化合物M13-1(663mg,1.23mmol),收率:61%。LC-MS:[M+H]
+=542。
Step 1: In ice bath, dissolve propionamide (1.0g, 2.05mmol) in 10mL of anhydrous tetrahydrofuran, add NaH (123mg, 3.07mmol), stir for 30 minutes, then add intermediate a14 (976mg, 2.0 mmol). The temperature was raised to room temperature and reacted for 2 hours. LC-MS monitored that the reaction was complete. Added 50 mL of ice water to the reaction liquid, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to obtain compound M13-1 (663 mg , 1.23mmol), yield: 61%. LC-MS: [M+H] + = 542.
步骤2:氮气保护下,将上步化合物M13-1(300mg,0.55mmol)、碳酸铯(360mg,1.11mmol)和中间体a7(269mg,0.66mmol)溶于溶剂5mL甲苯中,加入催化剂双(二苯基膦苯基醚)二氯化钯(40mg,0.055mmol)。升温至105℃反应12小时,停止反应,过滤。减压蒸除溶剂,粗品经flash柱层析色谱分离,得到化合物M13-2(85mg,0.11mmol),收率:21%。LC-MS:[M+H]
+=754。
Step 2: Under the protection of nitrogen, the above compound M13-1 (300mg, 0.55mmol), cesium carbonate (360mg, 1.11mmol) and intermediate a7 (269mg, 0.66mmol) were dissolved in the solvent 5mL toluene, and the catalyst bis( Diphenylphosphine phenyl ether) palladium dichloride (40 mg, 0.055 mmol). Raise the temperature to 105°C to react for 12 hours, stop the reaction, and filter. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound M13-2 (85 mg, 0.11 mmol), yield: 21%. LC-MS: [M+H] + =754.
步骤3:上步化合物M13-2(85mg)溶于三氟乙酸和二氯甲烷的混合溶液中(3.0mL,1/1),冰浴下反应1小时,减压蒸除溶剂,粗品经HPLC制备色谱分离,得到化合物M13(15mg)。LC-MS:[M+H]
+=554。
Step 3: Compound M13-2 (85mg) from the previous step was dissolved in a mixed solution of trifluoroacetic acid and dichloromethane (3.0mL, 1/1), and reacted in an ice bath for 1 hour. The solvent was evaporated under reduced pressure, and the crude product was subjected to HPLC Preparative chromatography gave compound M13 (15 mg). LC-MS: [M+H] + = 554.
1H NMR(400MHz,DMSO)δ10.53(s,1H),8.14(s,2H),7.89(s,1H),7.26(dd,J=8.4,5.3Hz,1H),7.15(t,J=8.9Hz,1H),4.51(dd,J=79.5,13.4Hz,2H),4.10(d,J=21.7Hz,2H),3.95(d,J=13.1Hz,1H),3.76(d,J=13.3Hz,1H),2.54(d,J=7.4Hz,2H),2.05–1.76(m,5H),1.05(t,J=7.5Hz,3H).
1 H NMR (400MHz,DMSO)δ10.53(s,1H),8.14(s,2H),7.89(s,1H),7.26(dd,J=8.4,5.3Hz,1H),7.15(t,J =8.9Hz,1H),4.51(dd,J=79.5,13.4Hz,2H),4.10(d,J=21.7Hz,2H),3.95(d,J=13.1Hz,1H),3.76(d,J =13.3Hz, 1H), 2.54(d, J=7.4Hz, 2H), 2.05–1.76(m, 5H), 1.05(t, J=7.5Hz, 3H).
实施例26:Example 26:
步骤1:氮气保护下,将原料2-氟-5-甲基苯胺H44-1(5.0g,39.96mmol)、碳酸钾(13.8g,99.90mmol)和碘化钾(6.63g,39.96mmol)溶于50mL N-甲基吡咯烷酮NMP中,搅拌5分钟;向上述混合液中缓慢加入4-甲氧基苄氯(12.83g,81.92mmol),混合物升温至65℃下反应1小时,停止反应。向体系中加入200mL冰水,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析分离,得到化合物H44-2(14.0g),收率:95%。LC-MS:[M+H]
+=366。
Step 1: Under the protection of nitrogen, the raw materials 2-fluoro-5-methylaniline H44-1 (5.0g, 39.96mmol), potassium carbonate (13.8g, 99.90mmol) and potassium iodide (6.63g, 39.96mmol) were dissolved in 50mL N-methylpyrrolidone NMP was stirred for 5 minutes; 4-methoxybenzyl chloride (12.83 g, 81.92 mmol) was slowly added to the above mixture, and the mixture was heated to 65° C. for 1 hour to stop the reaction. Add 200mL ice water to the system, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, the crude product is separated by flash column chromatography to obtain compound H44-2 (14.0g), yield: 95%. LC-MS: [M+H] + = 366.
步骤2:-5℃,氮气保护下,将2,2,6,6-四甲基哌啶(1.55g,11.0mmol)溶于10mL无水四氢呋喃中,缓慢滴加正丁基锂(4.38mL,2.5M),搅拌10分钟后,降温至-60℃;加入上步化合物H44-2(1.0g,2.74mmol),搅拌30分钟。向反应液中加入硼酸三异丙酯(620mg,3.29mmol),继续反应30分钟后,停止反应。缓慢向反应液中加入50mL饱和氯化铵水溶液,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash反向柱层析分离,得到化合物H44-3(770mg),收率:68%。LC-MS:[M+H]
+=:410。
Step 2: Dissolve 2,2,6,6-tetramethylpiperidine (1.55g, 11.0mmol) in 10mL of anhydrous tetrahydrofuran under nitrogen protection at -5°C, and slowly add n-butyllithium (4.38mL , 2.5M), after stirring for 10 minutes, cool down to -60°C; add compound H44-2 (1.0g, 2.74mmol) from the previous step, and stir for 30 minutes. Triisopropyl borate (620mg, 3.29mmol) was added to the reaction solution, and the reaction was continued for 30 minutes before stopping the reaction. Slowly add 50mL saturated aqueous ammonium chloride solution to the reaction solution, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate, and separate the crude product by flash reverse column chromatography to obtain compound H44-3 (770mg), yield: 68% . LC-MS: [M+H] + =: 410.
步骤3:氮气保护下,将上步化合物H44-3(209mg,0.51mmol)、碳酸铯(499mg,1.53mmol)和中间体a14(250mg,0.51mmol)溶于5mL 1,4-二氧六环中,加入催化剂Pd(dppf)Cl
2(39mg,0.052mmol),升温至105℃下反应16小时,停止反应,过滤。减压蒸除溶剂,粗品经flash柱层析分离,得到化合物H44-4(190mg),收率:48%。LC-MS:[M+H]
+=774。
Step 3: Under the protection of nitrogen, the above compound H44-3 (209mg, 0.51mmol), cesium carbonate (499mg, 1.53mmol) and intermediate a14 (250mg, 0.51mmol) were dissolved in 5mL 1,4-dioxane , the catalyst Pd(dppf)Cl 2 (39mg, 0.052mmol) was added, the temperature was raised to 105°C and the reaction was carried out for 16 hours, then the reaction was stopped and filtered. The solvent was evaporated under reduced pressure, and the crude product was separated by flash column chromatography to obtain compound H44-4 (190 mg), yield: 48%. LC-MS: [M+H] + =774.
步骤4:将上步化合物H44-4(190mg,0.25mmol)和N-碘代琥珀酰亚胺NIS(62mg,0.28mmol)溶于2mL乙腈中,加入2滴三氟乙酸,室温下反应0.5小时,停止反应。将反应液倒入20mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash反向柱层析分离,得到化合物H44-5(210mg),收率:95%。LC-MS:[M+H]
+=900。
Step 4: Dissolve the above compound H44-4 (190mg, 0.25mmol) and N-iodosuccinimide NIS (62mg, 0.28mmol) in 2mL of acetonitrile, add 2 drops of trifluoroacetic acid, and react at room temperature for 0.5 hours , stop responding. The reaction solution was poured into 20 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to obtain compound H44-5 (210 mg), yield: 95%. LC-MS: [M+H] + =900.
步骤5:氮气保护下,将上步化合物H44-5(140mg,0.16mmol)、氟磺酰基二氟乙酸甲酯(308mg, 1.6mmol)和催化剂CuI(152mg,0.80mmol)溶于3mL DMF中,室温下反应16小时,停止反应。将反应液倒入30mL水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash反向柱层析分离,得到化合物H44-6(70mg),收率:53%。LC-MS:[M+H]
+=842。
Step 5: Under nitrogen protection, the above compound H44-5 (140mg, 0.16mmol), methyl fluorosulfonyl difluoroacetate (308mg, 1.6mmol) and catalyst CuI (152mg, 0.80mmol) were dissolved in 3mL DMF, After reacting at room temperature for 16 hours, the reaction was terminated. The reaction solution was poured into 30 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash reverse column chromatography to obtain compound H44-6 (70 mg), yield: 53%. LC-MS: [M+H] + = 842.
步骤6:氮气保护下,将中间体a18(78mg,0.42mmol)溶于4mL无水四氢呋喃中,加入叔丁醇钾(95mg,0.84mmol),室温下搅拌30分钟—得溶液S0。在另一反应瓶中将化合物H44-6(350mg,0.42mmol)溶于2mL无水THF中,冰浴下,缓慢加入配置好的溶液S0,继续搅拌1小时,停止反应。将反应液倒入30mL冰水中,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品经flash柱层析色谱分离,得到淡黄色固体H44-7(300mg)。收率:71%,LC-MS:[M+H]
+=1009。
Step 6: Under nitrogen protection, the intermediate a18 (78 mg, 0.42 mmol) was dissolved in 4 mL of anhydrous THF, potassium tert-butoxide (95 mg, 0.84 mmol) was added, and stirred at room temperature for 30 minutes to obtain a solution S0. In another reaction flask, compound H44-6 (350 mg, 0.42 mmol) was dissolved in 2 mL of anhydrous THF, and the prepared solution S0 was slowly added under ice cooling, and the stirring was continued for 1 hour to stop the reaction. The reaction solution was poured into 30 mL of ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by flash column chromatography to obtain light yellow solid H44-7 (300 mg). Yield: 71%, LC-MS: [M+H] + =1009.
步骤7:将上步化合物H44-7(300mg,0.30mmol)溶于2mL甲苯中,加入2mL三氟乙酸和甲基磺酸的混合溶液(v/v,1/1),升温至50℃下反应16小时,停止反应。减压蒸除溶剂,残余物通过HPLC制备色谱分离,得到白色固体H44(20mg)。收率:9%,LC-MS:[M+H]
+=669。
Step 7: Dissolve the above compound H44-7 (300mg, 0.30mmol) in 2mL of toluene, add 2mL of a mixed solution of trifluoroacetic acid and methanesulfonic acid (v/v, 1/1), and heat up to 50°C After 16 hours of reaction, the reaction was stopped. The solvent was distilled off under reduced pressure, and the residue was separated by preparative HPLC chromatography to obtain white solid H44 (20 mg). Yield: 9%, LC-MS: [M+H] + =669.
1H NMR(400MHz,DMSO-d
6)δ7.89(s,1H),6.84(d,J=8.9Hz,1H),6.03(s,2H),5.11(dt,J=10.9,5.8Hz,1H),4.40(s,2H),4.31–4.23(m,2H),3.64–3.46(m,5H),2.79–2.59(m,5H),2.38–2.32(m,4H),2.05–1.77(m,8H),1.67(s,4H).
1 H NMR (400MHz, DMSO-d 6 )δ7.89(s, 1H), 6.84(d, J=8.9Hz, 1H), 6.03(s, 2H), 5.11(dt, J=10.9, 5.8Hz, 1H),4.40(s,2H),4.31–4.23(m,2H),3.64–3.46(m,5H),2.79–2.59(m,5H),2.38–2.32(m,4H),2.05–1.77( m,8H), 1.67(s,4H).
实施例27:Example 27:
化合物对于KRAS G12D介导的p-ERK的抑制测试(直接反映待测化合物的细胞水平抑制效果)。具体如下:The compound is tested for the inhibition of p-ERK mediated by KRAS G12D (directly reflecting the inhibitory effect of the test compound on the cellular level). details as follows:
将培养在包含10%胎牛血清和1%青链霉素的F-12K培养基(Gibco,Cat.No.30-2004)中的AGS细胞接种在384孔微板上,37℃、5%二氧化碳条件下温育过夜。在各孔中加入200微升不同浓度化合物(二甲基亚砜终浓度为0.5%)并在37℃温育3小时。然后,细胞固定于8%的固定液中(Solarbio,Cat.No.P1112)并使用磷酸缓冲液(PBS)清洗一次。清洗后在各孔中加入阻断液(LI-COR,Cat.No.927-40000)室温阻断1小时。移除阻断液后,各孔中加入phospho-p44/42 MAPK(T202/Y204)Rabbit mAb(CST,Cat.No.97166S)和GAPDH(D4C6R)Mouse mAb(CST,Cat.No.4370S)抗体工作液,4℃孵育过夜。使用包含0.1%吐温-80的PBS溶液(PBST)清洗微孔板三次,加入IRDye 800CW Goat anti-Rabbit IgG(H+L)(LI-COR,Cat.No.926-32211)和IRDye 680RD Goat anti Mouse IgG(H+L)(LI-COR,Cat.No.926-68070)抗体工作液,微孔板在室温避光温育。使用PBST清洗微孔板三次后,在1000rpm离心微孔板1分钟,使用Odyssey CLx(LI-COR)仪器扫描读板并记录信号值。AGS cells cultured in F-12K medium (Gibco, Cat.No.30-2004) containing 10% fetal calf serum and 1% penicillin were seeded on 384-well microplates at 37°C, 5% Incubate overnight under carbon dioxide conditions. 200 microliters of compounds at different concentrations (0.5% dimethyl sulfoxide final concentration) were added to each well and incubated at 37°C for 3 hours. Then, the cells were fixed in 8% fixative solution (Solarbio, Cat. No. P1112) and washed once with phosphate buffered saline (PBS). After washing, a blocking solution (LI-COR, Cat. No. 927-40000) was added to each well to block for 1 hour at room temperature. After removing the blocking solution, add phospho-p44/42 MAPK (T202/Y204) Rabbit mAb (CST, Cat.No.97166S) and GAPDH (D4C6R) Mouse mAb (CST, Cat.No.4370S) antibodies to each well Working solution, incubate overnight at 4°C. Wash the microplate three times with PBS solution (PBST) containing 0.1% Tween-80, add IRDye 800CW Goat anti-Rabbit IgG (H+L) (LI-COR, Cat.No.926-32211) and IRDye 680RD Goat anti Mouse IgG (H+L) (LI-COR, Cat. No. 926-68070) antibody working solution, incubate the microplate at room temperature in the dark. After washing the microplate three times with PBST, the microplate was centrifuged at 1000 rpm for 1 minute, and the plate was scanned and read with an Odyssey CLx (LI-COR) instrument and the signal value was recorded.
IC
50的计算公式
Calculation formula of IC 50
使用非线性回归方程计算化合物IC
50值:Y=下平台信号+(上平台信号-下平台信号)/(1+10^((LogIC
50-X)*希尔斜率));X=化合物浓度对数。
Compound IC50 values were calculated using nonlinear regression equation: Y=lower plateau signal+(upper plateau signal-lower plateau signal)/(1+10^(( LogIC50 -X)*Hill slope)); X=compound concentration logarithm.
表1:化合物对于p-ERK半数有效浓度抑制效果[MRTX1133作为阳性对照]Table 1: Inhibitory effect of compounds on p-ERK half effective concentration [MRTX1133 as positive control]
化合物compound | p-ERK/IC 50/nM p-ERK/IC 50 /nM | 化合物compound | p-ERK/IC 50/nM p-ERK/IC 50 /nM |
H1aH1a | 0.290.29 | P1P1 | 5454 |
H1bH1b | 4.64.6 | P2P2 | 534534 |
H2aH2a | 4242 | P3P3 | 450450 |
H2bH2b | 1.11.1 | P4P4 | 21twenty one |
H3aH3a | 4040 | P5P5 | 169169 |
H3bH3b | 0.330.33 | P6P6 | 117117 |
H4H4 | 2.62.6 | P7P7 | >1000>1000 |
H5aH5a | >1000>1000 | P8P8 | 1313 |
H5bH5b | 4242 | P9P9 | 0.880.88 |
H6H6 | 4.14.1 | P10P10 | 1.91.9 |
H7H7 | 1010 | P11P11 | 2.82.8 |
H8H8 | 4040 | P12P12 | 259259 |
H9H9 | 1010 | P13P13 | 1818 |
H10aH10a | 9898 | P14P14 | 633633 |
H10bH10b | 0.510.51 | P15P15 | 158158 |
H11H11 | 4.04.0 | P16P16 | 9494 |
H12aH12a | 112112 | P17P17 | 7272 |
H12bH12b | 0.940.94 | P18P18 | 7.97.9 |
H13H13 | 780780 | P19P19 | 3.63.6 |
H14H14 | 358358 | P20P20 | 0.770.77 |
H15H15 | 5.55.5 | P21P21 | 1313 |
H16H16 | 24twenty four | P22P22 | 3.03.0 |
H17H17 | 4.04.0 | P23P23 | 1313 |
H18H18 | 21twenty one | P24P24 | 0.420.42 |
H19aH19a | >1000>1000 | P25P25 | 5.25.2 |
H19bH19b | 7.57.5 | P26P26 | 1.41.4 |
H20H20 | 3.23.2 | P27P27 | 1.21.2 |
H21H21 | 5151 | P28P28 | 1.91.9 |
H22H22 | 23twenty three | P29P29 | 105105 |
H23H23 | 2525 | M1M1 | >1000>1000 |
H24H24 | 1.91.9 | M2M2 | 117117 |
H25H25 | 5.55.5 | M3M3 | 112112 |
H26H26 | >1000>1000 | M4M4 | 2828 |
H27H27 | 3.93.9 | M5M5 | 8484 |
H28aH28a | 4242 | M6M6 | 2929 |
H28bH28b | 1.61.6 | M7M7 | 1919 |
H29H29 | >1000>1000 | M8M8 | 204204 |
H30H30 | >1000>1000 | M9M9 | 4848 |
H31H31 | >1000>1000 | M10M10 | 401401 |
H32H32 | >1000>1000 | M11M11 | 9797 |
H33H33 | 3737 | M12M12 | 165165 |
H34H34 | 6969 | M13M13 | 193193 |
H35aH35a | 184184 | the | the |
H35bH35b | 2.82.8 | the | the |
H36aH36a | 261261 | the | the |
H36bH36b | 3.63.6 | the | the |
H37aH37a | 716716 | the | the |
H37bH37b | 2.02.0 | the | the |
H38aH38a | 384384 | the | the |
H38bH38b | 2.02.0 | the | the |
H39H39 | 3131 | the | the |
H40H40 | 239239 | the | the |
H41aH41a | 167167 | the | the |
H41bH41b | 1010 | the | the |
H42aH42a | 613613 | the | the |
H42bH42b | 3.33.3 | the | the |
H43H43 | 3737 | the | the |
H44H44 | 277277 | the | the |
MRTX1133MRTX1133 | 1.01.0 | the | the |
MRTX1133结构:MRTX1133 structure:
实施例28:化合物对GTP-KRAS的抑制活性Example 28: Inhibitory activity of compounds against GTP-KRAS
以4倍浓度梯度稀释待测化合物,使用ECHO(Labcyte)向384孔微板中每孔转移0.1μL不同浓度的待测化合物,每孔中相继加入5μL稀释好的Tag2-KRAS G12D>P或Tag2-KRAS WT>P,1000RPM离心1分钟;然后在各孔中加入5μL稀释好的Tag1-cRAF,1000RPM离心1分钟,25℃温孵15分钟;每孔中加入10μL的anti-Tag1-Tb3和anti-Tag2-XL665混合物,1000RPM离心1分钟,4℃温孵3小时;使用Envision在665/615nm扫描读板并记录信号值。Dilute the test compound with a 4-fold concentration gradient, use ECHO (Labcyte) to transfer 0.1 μL of different concentrations of the test compound to each well of a 384-well microplate, and add 5 μL of diluted Tag2-KRAS G12D>P or Tag2-KRAS to each well WT>P, centrifuge at 1000RPM for 1 minute; then add 5μL of diluted Tag1-cRAF to each well, centrifuge at 1000RPM for 1 minute, incubate at 25°C for 15 minutes; add 10μL of anti-Tag1-Tb3 and anti-Tag2-XL665 to each well The mixture was centrifuged at 1000RPM for 1 minute and incubated at 4°C for 3 hours; the plate was scanned at 665/615nm using Envision and the signal value was recorded.
上述分析中,KRAS-G12D的相关检测试剂均来源于市售试剂盒KRAS-G12D/cRAF BINDING ASSAY KITS(Cisbio,Cat.No.63ADK000CB21PEG);KRAS-WT的检测中,GTP购自Sigma(Cat.No.V900868),GST-cRAF由北京康龙化成制备(Cat.No.20190718),MAb Anti GST-Tb cryptate购自Cisbio(Cat.No.61GSTTLA),其他关键试剂来自于市售试剂盒KRAS-WT/SOS1 BINDING ASSAY KITS(Cisbio,Cat.No.63ADK000CB15PEH)。In the above analysis, the relevant detection reagents of KRAS-G12D were all from the commercially available kit KRAS-G12D/cRAF BINDING ASSAY KITS (Cisbio, Cat. No. 63ADK000CB21PEG); in the detection of KRAS-WT, GTP was purchased from Sigma (Cat. No.V900868), GST-cRAF was prepared by Beijing Pharmaron (Cat.No.20190718), MAb Anti GST-Tb cryptate was purchased from Cisbio (Cat.No.61GSTTLA), and other key reagents were from the commercially available kit KRAS- WT/SOS1 BINDING ASSAY KITS (Cisbio, Cat. No. 63ADK000CB15PEH).
根据以下公式分析计算结果:Analyze the calculation results according to the following formula:
相对比值(relative ratio,RR)=(Ratio
665/615-Ratio
背景)
Relative ratio (relative ratio, RR) = (Ratio 665/615 -Ratio background )
抑制百分率=[1-(RR
化合物-RR
阳性对照孔平均值)/(RR
阴性对照孔平均值-RR
阳性对照孔平均值)]×100
Inhibition percentage=[1-(RR compound -RR positive control well average value)/(RR negative control well average value-RR positive control well average value)]×100
IC
50计算:Y=下平台信号+(上平台信号-下平台信号)/(1+10^(LogIC
50-X)×希尔斜率)。X:化合物浓度对数值;Y:抑制百分率。
IC 50 calculation: Y=lower plateau signal+(upper plateau signal-lower plateau signal)/(1+10^(LogIC 50 -X)×Hill slope). X: logarithmic value of compound concentration; Y: percent inhibition.
表2 化合物对GTP-KRAS的抑制活性效果Table 2 The inhibitory effect of compounds on GTP-KRAS
化合物compound | KRAS G12D-CRAF/IC 50/nM KRAS G12D-CRAF/IC 50 /nM |
H1aH1a | 5.15.1 |
H2bH2b | 9.29.2 |
H4H4 | 7.47.4 |
H10bH10b | 8.58.5 |
H19bH19b | 1010 |
P4P4 | 1616 |
P20P20 | 1111 |
MRTX1133MRTX1133 | 1111 |
该结果表明本发明分子对于KRAS G12D蛋白激活具有优异的抑制效果。This result shows that the molecule of the present invention has an excellent inhibitory effect on KRAS G12D protein activation.
实施例29:Example 29:
化合物对于KRAS G12D突变的胰腺癌AsPC-1细胞系的3D抗增殖效果。具体如下:3D antiproliferative effect of compounds on KRAS G12D mutated pancreatic cancer AsPC-1 cell line. details as follows:
细胞培养:在T75培养瓶(Corning,目录号430641)中,AsPC-1胰腺癌细胞培养于包含10%胎牛血清(Ausgenex,目录号FBS500-S)和1%青/链霉素(Gibco,目录号15140-122)的RPMI 1640培养基(Hyclone,目录号SH3080901B)中。Cell culture: In T75 culture flasks (Corning, catalog number 430641), AsPC-1 pancreatic cancer cells were cultured in a medium containing 10% fetal bovine serum (Ausgenex, catalog number FBS500-S) and 1% penicillin/streptomycin (Gibco, Cat. No. 15140-122) in RPMI 1640 Medium (Hyclone, Cat. No. SH3080901B).
试验过程:利用纳升移液系统(LABCYTE,目录号Echo550)将稀释好的待测化合物加入384孔低吸附细胞培养板(Labcyte,目录号PP-0200),铺入细胞后,将培养板放置于37℃,5%CO
2恒温培养箱培养。待测化合物(1μM作为起始浓度,3倍稀释,共10个浓度)与细胞共孵育7天后,在每孔中加入
3D试剂(Promega,目录号G9683),用Envision多功能酶标仪(Perkin Elmer, 目录号Envision 2104)读取发光值,光信号和体系中ATP量成正比,而ATP的含量直接表征体系中的活细胞数。最后使用XLFIT软件用非线性拟合公式得到化合物的IC
50(半数抑制浓度)。
Experimental process: Add the diluted compound to be tested into a 384-well low-adsorption cell culture plate (Labcyte, catalog number PP-0200) using a nanoliter pipetting system (LABCYTE, catalog number Echo550). After the cells are spread, place the culture plate Incubate at 37°C, 5% CO 2 incubator. The compound to be tested (1μM as the initial concentration, diluted 3 times, a total of 10 concentrations) was co-incubated with the cells for 7 days, and added to each well 3D reagent (Promega, catalog number G9683), read the luminescence value with Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104), the light signal is directly proportional to the ATP amount in the system, and the content of ATP directly characterizes the ATP in the system number of viable cells. Finally, XLFIT software was used to obtain the IC 50 (half inhibitory concentration) of the compound with a nonlinear fitting formula.
抑制率(%)=100×(阴性对照平均值-化合物读值)/(阴性对照平均值-阳性对照平均值)Inhibition rate (%)=100×(negative control average value-compound reading value)/(negative control average value-positive control average value)
阴性对照:DMSO。阳性对照:培养基。Negative control: DMSO. Positive control: culture medium.
表3:化合物对于AsPC-1细胞半数有效浓度抗增殖效果Table 3: Anti-proliferation effect of compounds on AsPC-1 cell half effective concentration
化合物compound | IC 50/nM IC 50 /nM |
H1aH1a | 4.24.2 |
H1bH1b | 4949 |
H2bH2b | 23twenty three |
P4P4 | 851851 |
P11P11 | 5050 |
P20P20 | 4747 |
该结果表明本发明分子对于KRAS G12D突变的肿瘤细胞系具有良好的抗增殖效果。This result shows that the molecule of the present invention has a good anti-proliferation effect on KRAS G12D mutated tumor cell lines.
实施例30:Example 30:
化合物对于KRAS G12D突变的胰腺癌PANC-1细胞系的3D抗增殖效果。具体如下:3D antiproliferative effect of compounds on KRAS G12D mutated pancreatic cancer PANC-1 cell line. details as follows:
细胞培养:在T75培养瓶(Corning,目录号430641)中,PANC-1胰腺癌细胞培养于包含10%胎牛血清(Ausgenex,目录号FBS500-S)和1%青/链霉素(Gibco,目录号15140-122)的DMEM培养基(GIBCO)中。Cell culture: In T75 culture flasks (Corning, catalog number 430641), PANC-1 pancreatic cancer cells were cultured in a medium containing 10% fetal bovine serum (Ausgenex, catalog number FBS500-S) and 1% penicillin/streptomycin (Gibco, Cat. No. 15140-122) in DMEM medium (GIBCO).
试验过程:利用纳升移液系统(LABCYTE,目录号Echo550)将稀释好的待测化合物加入384孔低吸附细胞培养板(Labcyte,目录号PP-0200),铺入细胞后,将培养板放置于37℃,5%CO
2恒温培养箱培养。待测化合物(1μM作为起始浓度,3倍稀释,共10个浓度)与细胞共孵育7天后,在每孔中加入
3D试剂(Promega,目录号G9683),用Envision多功能酶标仪(Perkin Elmer,目录号Envision 2104)读取发光值,光信号和体系中ATP量成正比,而ATP的含量直接表征体系中的活细胞数。最后使用XLFIT软件用非线性拟合公式得到化合物的IC
50(半数抑制浓度)。
Experimental process: Add the diluted compound to be tested into a 384-well low-adsorption cell culture plate (Labcyte, catalog number PP-0200) using a nanoliter pipetting system (LABCYTE, catalog number Echo550). After the cells are spread, place the culture plate Incubate at 37°C, 5% CO 2 incubator. The compound to be tested (1μM as the initial concentration, diluted 3 times, a total of 10 concentrations) was co-incubated with the cells for 7 days, and added to each well 3D reagent (Promega, catalog number G9683), read the luminescence value with Envision multifunctional microplate reader (Perkin Elmer, catalog number Envision 2104), the light signal is directly proportional to the amount of ATP in the system, and the content of ATP directly characterizes the amount of ATP in the system number of living cells. Finally, XLFIT software was used to obtain the IC 50 (half maximal inhibitory concentration) of the compound with a nonlinear fitting formula.
抑制率(%)=100×(阴性对照平均值-化合物读值)/(阴性对照平均值-阳性对照平均值)Inhibition rate (%)=100×(negative control average value-compound reading value)/(negative control average value-positive control average value)
阴性对照:DMSO。阳性对照:培养基。Negative control: DMSO. Positive control: culture medium.
表4:化合物对于PANC-1细胞半数有效浓度抗增殖效果Table 4: Anti-proliferation effect of compounds on PANC-1 cell half effective concentration
化合物compound | IC 50/nM IC 50 /nM |
H2bH2b | 1.31.3 |
H3bH3b | 1.01.0 |
H10bH10b | 1.81.8 |
H12bH12b | 2.42.4 |
H19bH19b | 7.77.7 |
P20P20 | 3.03.0 |
MRTX1133MRTX1133 | 3.63.6 |
该结果表明本发明分子对于KRAS G12D突变的肿瘤细胞系具有良好的抗增殖效果。This result shows that the molecule of the present invention has a good anti-proliferation effect on KRAS G12D mutated tumor cell lines.
实施例31:Example 31:
化合物的肝微粒体稳定性实验。具体如下:Liver microsomal stability assay of compounds. details as follows:
对本发明化合物进行肝微粒体稳定性试验研究,将待测化合物在加入或不加入NADPH情况下与不同种属的肝微粒体进行共孵育,试验体系中待测化合物终浓度为1μM,NADPH终浓度为1mM,肝微粒体终浓度为0.5mg/ml。检测60分钟内不同时间点孵育上清中的化合物浓度并计算药代动力学参数(例如清除率Clint)。Carry out liver microsome stability test research on the compound of the present invention, the compound to be tested is co-incubated with liver microsomes of different species with or without adding NADPH, the final concentration of the compound to be tested in the test system is 1 μM, and the final concentration of NADPH 1mM, the final concentration of liver microsomes is 0.5mg/ml. Detect the concentration of the compound in the incubation supernatant at different time points within 60 minutes and calculate the pharmacokinetic parameters (such as clearance rate Clint).
该结果表明本发明分子具有较好的代谢稳定性(尤其在人体中,具有较好的代谢稳定性)。This result shows that the molecule of the present invention has better metabolic stability (especially in human body, it has better metabolic stability).
部分分子(例如H1b、H10b、H24、H25、H35b-H38b、P20、P24等)相比对照MRTX1133在人体中清除率更低,代谢更慢。Some molecules (such as H1b, H10b, H24, H25, H35b-H38b, P20, P24, etc.) have lower clearance rates and slower metabolism in humans than the control MRTX1133.
化合物compound | Human Clint/(mL/min/kg)Human Clint/(mL/min/kg) | MouseClint/(mL/min/kg)MouseClint/(mL/min/kg) |
H1aH1a | 2727 | 451451 |
H1bH1b | 9.79.7 | 456456 |
H2bH2b | 40.440.4 | 156156 |
H3bH3b | 44.944.9 | 229229 |
H4H4 | 33.733.7 | 211.7211.7 |
H5bH5b | 32.932.9 | 244244 |
H10bH10b | 14.214.2 | 167167 |
H11H11 | 39.639.6 | 298298 |
H12bH12b | 24.124.1 | 182182 |
H19bH19b | 32.932.9 | 177177 |
H24H24 | 19.819.8 | 166166 |
H25H25 | 10.310.3 | N.D.N.D. |
H35bH35b | 17.817.8 | 143143 |
H36bH36b | 12.712.7 | 102102 |
H37bH37b | 14.314.3 | 110110 |
H38bH38b | 20.020.0 | 152152 |
P4P4 | 40.840.8 | 595595 |
P11P11 | 29.729.7 | 547547 |
P20P20 | 16.216.2 | 334334 |
P24P24 | 20.020.0 | 403403 |
MRTX1133MRTX1133 | 28.728.7 | 260260 |
N.D.=未测试N.D. = not tested
实施例32:Example 32:
BALB/c裸鼠体内药效实验。具体如下:Drug efficacy experiment in BALB/c nude mice. details as follows:
培养KRAS G12D突变的结肠直肠癌肿瘤细胞GP2D,将该肿瘤细胞接种到6-8周的雌性BALB/c裸鼠中(体重约20g左右),所有小鼠皮下接种。小鼠培养于SPF级实验环境中,所有小鼠可自由获取商业认证的标准饮食。当小鼠平均肿瘤体积成长到150mm
3左右时,将试验化合物开始每日腹腔(ip)给药。给药剂量为:空白组溶媒(10%Captisol in 50mM柠檬酸盐缓冲液pH 5.0)。给药组剂量为10mg/kg,每日两次。肿瘤体积一周三次用二维卡尺测量,每天动物称重。连续给药10天后,根据最终肿瘤体积计算抑制率(TGI/100%)。体积计算公式为:V=1/2a*b
2,a代表肿瘤长径,b代表肿瘤短径。
Colorectal cancer tumor cell GP2D with KRAS G12D mutation was cultured, and the tumor cells were inoculated into 6-8 week old female BALB/c nude mice (body weight about 20 g), and all mice were inoculated subcutaneously. Mice were raised in an SPF-grade experimental environment, and all mice had free access to commercially certified standard diets. Daily intraperitoneal (ip) administration of test compounds began when the average tumor volume of the mice had grown to approximately 150 mm3 . The dosage is: blank group vehicle (10% Captisol in 50mM citrate buffer pH 5.0). The dosage of the administration group was 10mg/kg, twice a day. Tumor volumes were measured with two-dimensional calipers three times a week, and animals were weighed daily. After 10 days of continuous administration, the inhibition rate (TGI/100%) was calculated according to the final tumor volume. The volume calculation formula is: V=1/2a*b 2 , where a represents the long diameter of the tumor, and b represents the short diameter of the tumor.
受试药物Test drug | 给药剂量Dosage | TGITGI |
空白组blank group | 00 | 0%0% |
MRTX1133MRTX1133 | 10mg/kg,BID10mg/kg, BID | 186%186% |
P20P20 | 10mg/kg,BID10mg/kg, BID | 187%187% |
该结果表明本发明分子具有较好体内药效,能够抑制KRAS G12D突变肿瘤的生长,且效果优于MRTX1133。The results show that the molecule of the present invention has better drug efficacy in vivo, can inhibit the growth of KRAS G12D mutant tumors, and the effect is better than MRTX1133.
实施例33:Example 33:
本发明化合物的动物体内安全性实验。具体如下:In vivo safety experiment of the compound of the present invention in animals. details as follows:
选择合格的健康ICR小鼠(年龄6-8周,体重18-20g),每组3只,分别用于单次静脉给药。先进行单次静脉给药预试,剂量从2mg/kg开始摸索,如果未见死亡,将剂量增加,如果出现死亡,将停止增加。Qualified healthy ICR mice (aged 6-8 weeks, body weight 18-20g) were selected, 3 in each group, for single intravenous administration respectively. First, a single intravenous administration test was carried out, and the dose was explored from 2mg/kg. If no death was observed, the dose was increased, and if death occurred, the increase would be stopped.
MRTX1133静脉给药溶液和化合物P20静脉给药溶媒为:DMSO/Tween80/Solutol/生理盐水(四者体积比为5/3/10/82),涡旋超声使其充分溶解后,进行给药处置。The intravenous administration solution of MRTX1133 and the compound P20 intravenous administration vehicle are: DMSO/Tween80/Solutol/normal saline (the volume ratio of the four is 5/3/10/82), vortex ultrasonic to make it fully dissolved, and carry out administration disposal .
化合物compound | 给药方式Method of administration | 给药剂量Dosage | 死亡率/3只Mortality/3 |
MRTX1133MRTX1133 | 静脉ivvein iv | 2mpk2mpk | 0/30/3 |
MRTX1133MRTX1133 | 静脉ivvein iv | 4mpk4mpk | 1/31/3 |
P20P20 | 静脉ivvein iv | 2mpk2mpk | 0/30/3 |
P20P20 | 静脉ivvein iv | 4mpk4mpk | 0/30/3 |
P20P20 | 静脉ivvein iv | 8mpk8mpk | 0/30/3 |
P20P20 | 静脉ivvein iv | 16mpk16mpk | 2/32/3 |
选择合格的健康ICR小鼠(年龄6-8周,体重18-20g),每组3只,分别用于单次输液给药。以静脉给药的最大剂量为起始,如果未见死亡,将剂量增加,如果出现死亡,将停止增加。MRTX1133输注给药溶液和化合物P20输注给药溶媒为:DMSO/Tween80/Solutol/生理盐水(四者体积比为5/3/10/82),涡旋超声使其充分溶解后,进行给药处置。Qualified healthy ICR mice (age 6-8 weeks, body weight 18-20g) were selected, 3 mice in each group, for single infusion administration respectively. Start with the maximum dose administered intravenously and increase the dose if no deaths are seen and stop if deaths occur. MRTX1133 infusion administration solution and compound P20 infusion administration vehicle are: DMSO/Tween80/Solutol/normal saline (the volume ratio of the four is 5/3/10/82), vortex ultrasonic to make it fully dissolved, then administer Drug disposal.
化合物compound | 给药方式Method of administration | 给药剂量Dosage | 死亡率/3只Mortality/3 |
MRTX1133MRTX1133 | 输注infInfuse inf | 4mpk4mpk | 0/30/3 |
MRTX1133MRTX1133 | 输注infInfuse inf | 8mpk8mpk | 0/30/3 |
MRTX1133MRTX1133 | 输注infInfuse inf | 16mpk16mpk | 0/30/3 |
MRTX1133MRTX1133 | 输注infInfuse inf | 32mpk32mpk | 2/32/3 |
P20P20 | 输注infInfuse inf | 16mpk16mpk | 0/30/3 |
P20P20 | 输注infInfuse inf | 32mpk32mpk | 0/30/3 |
P20P20 | 输注infInfuse inf | 96mpk96mpk | 0/30/3 |
P20P20 | 输注infInfuse inf | 150mpk150mpk | 0/30/3 |
该结果表明本发明分子具有较好体内安全性,不论是静脉给药还是输注给药,安全性均远优于MRTX1133。The results show that the molecule of the present invention has better in vivo safety, and the safety is far superior to MRTX1133 no matter it is administered intravenously or infused.
实施例34:Example 34:
化合物的小鼠体内药代动力学实验。具体如下:In vivo pharmacokinetic experiments of compounds in mice. details as follows:
以CD1雌性小鼠为受试动物,口服/静脉给药(口服给药量为20mg/kg溶媒为:DMSO-Solutol-H
2O)。
CD1 female mice were used as test animals, and administered orally/intravenously (the oral administration dose was 20 mg/kg, and the vehicle was: DMSO-Solutol-H 2 O).
实验方案:口服组每组3只。收集给药前(0h)和给药后(0.25,0.5,1,2,4,8,24h)的血浆样品;用LC/MS/MS法测定小鼠口服给药后的血浆的血液浓度,采集的数据用AB Sciex QTRAP 6500软件计算,实验结果如下:Experimental scheme: oral administration group with 3 rats in each group. Collect plasma samples before administration (0h) and after administration (0.25,0.5,1,2,4,8,24h); use LC/MS/MS method to measure the blood concentration of plasma after oral administration in mice, The collected data is calculated with AB Sciex QTRAP 6500 software, and the experimental results are as follows:
*=50mg/kg*=50mg/kg
该结果表明本发明分子具有较好的小鼠体内吸收,部分分子具有非常高的小鼠体内暴露量,且远高于MRTX1133。临床上有望实现口服给药的可能。The results show that the molecules of the present invention have better absorption in mice, and some molecules have very high exposure in mice, which is much higher than MRTX1133. Clinically, it is expected to realize the possibility of oral administration.
Claims (7)
- 药物组合物,其含有权利要求1中的化合物,或其药学上可接受的盐、对映异构体、非对映异构体、溶剂合物、水合物或同位素变体,和药学上可接受的赋形剂;优选地,其还含有其它治疗剂。A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereoisomer, solvate, hydrate or isotopic variant thereof, and pharmaceutically acceptable Acceptable excipients; preferably, also contain other therapeutic agents.
- 权利要求1的化合物或其药学上可接受的盐、对映异构体、非对映异构体、溶剂合物、水合物或同位素变体在制备用于治疗和/或预防KRAS G12D突变蛋白介导的疾病的药物中的用途。The compound of claim 1 or its pharmaceutically acceptable salt, enantiomer, diastereoisomer, solvate, hydrate or isotope variant is used for the treatment and/or prevention of KRAS G12D mutant protein in the preparation Use in medicines for mediated diseases.
- 一种在受试者中治疗和/或预防KRAS G12D突变蛋白介导的疾病的方法,所述方法包括向所述受试者给药权利要求1的化合物或其药学上可接受的盐、对映异构体、非对映异构体、溶剂合物、水合物或同位素变体或权利要求2的药物组合物。A method for treating and/or preventing KRAS G12D mutant protein-mediated diseases in a subject, the method comprising administering the compound of claim 1 or a pharmaceutically acceptable salt thereof to the subject, Enantiomers, diastereomers, solvates, hydrates or isotopic variants or the pharmaceutical composition of claim 2.
- 权利要求1的化合物或其药学上可接受的盐、对映异构体、非对映异构体、溶剂合物、水合物或同位素变体或权利要求2的药物组合物,其用于治疗和/或预防KRAS G12D突变蛋白介导的疾病。The compound of claim 1 or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, hydrate or isotopic variant thereof or the pharmaceutical composition of claim 2 for use in the treatment of And/or prevent KRAS G12D mutant protein-mediated diseases.
- 权利要求3的用途或权利要求4的方法或权利要求5的化合物或组合物的用途,其中所述KRAS G12D突变蛋白介导的疾病选自急性髓细胞样白血病、青少年癌症、儿童肾上腺皮质癌、AIDS相关的癌症(例如淋巴瘤和卡波西氏肉瘤)、肛门癌、阑尾癌、星形细胞瘤、非典型畸胎样、基底细胞癌、胆管癌、膀胱癌、骨癌、脑干神经胶质瘤、脑瘤、乳腺癌、支气管肿瘤、伯基特淋巴瘤、类癌瘤、非典型畸胎样、胚胎肿瘤、生殖细胞肿瘤、原发性淋巴瘤、宫颈癌、儿童癌症、脊索瘤、心脏肿瘤、慢性淋巴细胞性白血病(CLL)、慢性髓细胞性白血病(CML)、慢性骨髓增殖性病症、结肠癌、结肠直肠癌、颅咽管瘤、皮肤T细胞淋巴瘤、肝外导管原位癌(DCIS)、胚胎肿瘤、CNS癌症、子宫内膜癌、室管膜瘤、食道癌、嗅神经母细胞瘤、尤文氏肉瘤、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、骨骼的纤维组织细胞瘤、胆囊癌、胃癌、胃肠道类癌瘤、胃肠道间质瘤(GIST)、生殖细胞肿瘤、妊娠滋养细胞肿瘤、毛细胞白血病、头颈癌、心脏癌、肝癌、霍奇金氏淋巴瘤、下咽癌、眼内黑色素瘤、胰岛细胞瘤、胰腺神经内分泌瘤、肾癌、喉癌、唇和口腔癌、肝癌、小叶原位癌(LCIS)、肺癌、淋巴瘤、转移性鳞状颈癌伴隐匿原发灶、中线道癌、口腔癌、多发性内分泌瘤综合征、多发性骨髓瘤/浆细胞瘤、蕈样真菌病、骨髓发育不良综合征、骨髓发育不良/骨髓增殖性瘤、多发性骨髓瘤、梅克尔细胞癌、恶性间皮瘤、骨骼的恶性纤维组织细胞瘤和骨肉瘤、鼻腔和鼻窦癌、鼻咽癌、神经母细胞瘤、非霍奇金氏淋巴瘤、非小细胞肺癌(NSCLC)、口腔癌、唇和口腔癌、口咽癌、卵巢癌、胰腺癌、乳头瘤、副神经节瘤、鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、胸膜肺母细胞瘤、原发性中枢神经系统(CNS)淋巴瘤、前列腺癌、直肠癌、移行性细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、皮肤癌、胃癌、小细胞肺癌、小肠癌、软组织肉瘤、细胞淋巴瘤、睾丸癌、喉癌、胸腺瘤和胸腺癌、甲状腺癌、肾盂和输尿管的移行性细胞癌、滋养细胞肿瘤、儿童罕见的癌症、尿道癌、子宫肉瘤、阴道癌、外阴癌或病毒诱导的癌症。The use of claim 3 or the method of claim 4 or the use of the compound or composition of claim 5, wherein the disease mediated by the KRAS G12D mutant protein is selected from acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (eg, lymphoma and Kaposi's sarcoma), anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glia Glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonal tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, Cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic catheter in situ Carcinoma (DCIS), embryonal tumor, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, bone Fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, cholangiocarcinoma Chiggin's lymphoma, hypopharyngeal carcinoma, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, Metastatic squamous neck cancer with occult primary, midline tract cancer, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic syndrome/ Myeloproliferative neoplasms, multiple myeloma, Merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of the bone, nasal and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer (NSCLC), Oral Cancer, Lip and Mouth Cancer, Oropharyngeal Cancer, Ovarian Cancer, Pancreatic Cancer, Papilloma, Paraganglioma, Sinus and Nasal Cancer, Parathyroid Cancer, Penile Cancer , pharyngeal carcinoma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, gastric cancer, small Lung cell lung cancer, small bowel cancer, soft tissue sarcoma, cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymus carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors, rare cancers of childhood, urethral cancer, uterus Sarcomas, vaginal, vulvar, or virus-induced cancers.
- 权利要求3的用途或权利要求4的方法或权利要求5的化合物或组合物的用途,其中所述KRAS G12D突变蛋白介导的疾病选自胰腺癌、结肠直肠癌或非小细胞肺癌。The use of claim 3 or the method of claim 4 or the use of the compound or composition of claim 5, wherein the disease mediated by the KRAS G12D mutant protein is selected from pancreatic cancer, colorectal cancer or non-small cell lung cancer.
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