WO2021190502A1 - Composé de pyridopyrimidinone - Google Patents

Composé de pyridopyrimidinone Download PDF

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Publication number
WO2021190502A1
WO2021190502A1 PCT/CN2021/082391 CN2021082391W WO2021190502A1 WO 2021190502 A1 WO2021190502 A1 WO 2021190502A1 CN 2021082391 W CN2021082391 W CN 2021082391W WO 2021190502 A1 WO2021190502 A1 WO 2021190502A1
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Prior art keywords
compound
pharmaceutically acceptable
acceptable salt
mmol
present
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PCT/CN2021/082391
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English (en)
Chinese (zh)
Inventor
吴立方
孙飞
丁照中
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202180023225.7A priority Critical patent/CN115335377B/zh
Publication of WO2021190502A1 publication Critical patent/WO2021190502A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • 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
    • 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

Definitions

  • the present invention relates to the field of medicine, in particular to a new type of benzopyrimidinone compound or a pharmaceutically acceptable salt thereof, a preparation method thereof, and application thereof in the preparation of drugs for treating related diseases.
  • HBV hepatitis B virus
  • Hepatitis B virus is the pathogen that causes hepatitis B (hepatitis B for short) and belongs to the family of hepatotropic DNA viruses. After HBV adheres to the surface of liver cells, it enters the cell through viral endocytosis mediated by sodium ion-taurocholic acid transport polypeptide (NTCP), releases the capsid in the cytoplasm, and enters the nucleus and transforms the rcDNA into a covalently closed loop DNA (cccDNA). All subgenomic RNA (sgRNA) and pregenomic RNA (pgRNA) are formed by cccDNA transcription.
  • sgRNA subgenomic RNA
  • pgRNA pregenomic RNA
  • sgRNA After exiting the nucleus, sgRNA is translated into X protein and three other envelope proteins, and pgRNA is translated into core protein and viral polymerase. pgRNA and core protein self-assemble under the action of polymerase to form RNA that wraps the nucleocapsid. In the nucleocapsid, pgRNA is reverse-transcribed into negative-strand DNA, and the positive strand of DNA is further synthesized from this to form rcDNA.
  • the rcDNA wrapped by nucleocapsid is re-uncoated into the nucleus to further amplify the cccDNA; on the other hand, it recombines with the envelope protein and releases the cell through the endoplasmic reticulum to form a new HBV.
  • cccDNA has a high degree of stability and is a template for HBV to replicate continuously. It exists in the form of minichromosomes in the nucleus of the host liver cell, and it is difficult to completely remove it with current treatment methods. This is also the main reason why hepatitis B is difficult to cure at present.
  • nucleoside (acid) compounds and interferons. Nucleoside (acid) drugs, such as lamivudine, entecavir, tenofovir (ester), etc., can effectively inhibit HBV DNA replication, but these drugs cannot eliminate cccDNA, and the disease often rebounds after stopping the drug.
  • Interferon drugs can partially activate the patient's immune system and inhibit hepatitis B virus through the body's autoimmune effect.
  • these drugs have relatively large side effects and are not tolerated by patients. What is more serious is the response rate of different populations to interferon therapy. Significant difference, but overall the response rate is low (usually less than 30%) (Nat. Rev. Gastro. Hepat. 8 (2011), 275-284).
  • Existing clinical treatment programs have a low functional cure rate, and there is still a large unmet clinical need for hepatitis B treatment.
  • the present invention discloses a compound of formula (II) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from H, F, OH, CN, C 1-3 alkyl, -C 1-3 alkyl- and C 1-3 alkoxy, the C 1-3 alkyl and C 1-3 alkane group are independently optionally substituted with one, two or three R a;
  • R 2 is selected from H, F, Cl, Br, CN and CF 3 ;
  • R 3 , R 4 , R 5 and R 6 are each independently selected from H, F, Cl, Br, CN, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3 ⁇ 6-membered heterocycloalkyl, the C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally selected by 1, 2 Or 3 R b substitutions;
  • L is selected from -O-, -S-, -SO 2 -, -N(R 7 )- and -C(R 7 ) 2 -;
  • L 1 is selected from -C(R 7 ) 2 -;
  • L 2 is selected from -C(R 7 ) 2 -;
  • R 7 is each independently selected from H, F, Cl, Br, I, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl, so The C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally substituted with 1, 2 or 3 R c ;
  • T 1 , T 2 , T 3 and T 4 are independently selected from CR 8 and N respectively;
  • R 8 is each independently selected from H, F, Cl, Br, I, CN, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl ,
  • the C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally substituted with 1, 2 or 3 R d ;
  • m is selected from 1, 2, 3 and 4;
  • R 9 is each independently selected from H, F, Cl, Br, I, and C 1-3 alkyl groups, the C 1-3 alkyl groups are optionally substituted with 1, 2 or 3 R e ;
  • R a, R b, R c , R d and R e are each independently selected from F, Cl, Br, I, OH, CN, NH 2, COOH, CF 3, -CHF 2, -CH 2 F, -OCH 3 , CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -NHCH 3 , -N(CH 3 ) 2 and cyclopropyl;
  • the C 1-6 heteroalkyl group and 3-6 membered heterocycloalkane each independently include 1, 2, 3, or 4 atoms or heteroatom groups independently selected from O, N, S, and NH.
  • R 1 is selected from H, F, OH, CN, CH 3 and OCH 3, OCH 3 and CH 3 said optionally substituted with 1, 2 or 3 R a, the other variables are as Defined by the present invention.
  • R 1 is selected from H and CH 3 , and other variables are as defined in the present invention.
  • R 1 is selected from H, CH 3 , F, OCH 3 and OH, and other variables are as defined in the present invention.
  • R 2 is selected from Cl, and other variables are as defined in the present invention.
  • R 3 , R 4 , R 5 and R 6 are each independently selected from H, and other variables are as defined in the present invention.
  • the above-mentioned L is selected from -O-, and other variables are as defined in the present invention.
  • the above-mentioned R 7 is selected from H, F, Cl, Br, I, -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl, the -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl are optionally substituted with 1, 2 or 3 R c , and other variables are as defined in the present invention.
  • the above-mentioned R 7 is selected from H, F, Cl, Br, I, CF 3 , -CHF 2 , -CH 2 F, -OCH 3 , CH 3 , -CH 2 CH 3 , -CH (CH 3 ) 2 , -NHCH 3 , -N(CH 3 ) 2 and cyclopropyl, and other variables are as defined in the present invention.
  • the above-mentioned L is selected from -NH- and -CH 2 -, and other variables are as defined in the present invention.
  • L 1 and L 2 are independently selected from -CH 2 -, -CHF- and -CF 2 -, and other variables are as defined in the present invention.
  • the above-mentioned R 8 is independently selected from H, F, Cl, Br, I, -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl, said- OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl are optionally substituted with 1, 2 or 3 Rd , and other variables are as defined in the present invention.
  • R 8 is independently selected from H, F and Cl, and other variables are as defined in the present invention.
  • R 9 is independently selected from H, and other variables are as defined in the present invention.
  • R 1 , R 2 , R 8 , L 1 and L 2 are as defined in the present invention.
  • the present invention also discloses the compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from H, F, OH, CN, C 1-3 alkyl group and C 1-3 alkoxy group, said C 1-3 alkyl group and C 1-3 alkoxy group are each independently optionally 1 , 2 or 3 R a substitutions;
  • R 2 is selected from H, F, Cl, Br, CN and CF 3 ;
  • R 3 , R 4 , R 5 and R 6 are each independently selected from H, F, Cl, Br, CN, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3 ⁇ 6-membered heterocycloalkyl, the C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally selected by 1, 2 Or 3 R b substitutions;
  • L is selected from -O-, -S-, -SO 2 -, -N(R 7 )- and -C(R 7 ) 2 -;
  • L 1 is selected from -C(R 7 ) 2 -;
  • L 2 is selected from -C(R 7 ) 2 -;
  • R 7 is each independently selected from H, F, Cl, Br, I, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl, so The C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally substituted with 1, 2 or 3 R c ;
  • T 1 , T 2 , T 3 and T 4 are independently selected from CR 8 and N respectively;
  • R 8 is selected from H, F, Cl, Br, I, CN, C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl, said C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 heteroalkyl and 3-6 membered heterocycloalkyl are each independently optionally substituted with 1, 2 or 3 R d ;
  • n is selected from 1, 2, 3 and 4;
  • R 9 is each independently selected from H, F, Cl, Br, I, and C 1-3 alkyl groups, the C 1-3 alkyl groups are optionally substituted with 1, 2 or 3 R e ;
  • R a, R b, R c , R d and R e are each independently selected from F, Cl, Br, I, OH, CN, NH 2, COOH, CF 3, -CHF 2, -CH 2 F, -OCH 3 , CH 3 , -CH 2 CH 3 , -CH(CH 3 ) 2 , -NHCH 3 , -N(CH 3 ) 2 and cyclopropyl;
  • the C 1-6 heteroalkyl group and 3-6 membered heterocycloalkane each independently include 1, 2, 3, or 4 atoms or heteroatom groups independently selected from O, N, S, and NH.
  • R 1 is selected from H, F, OH, CN, CH 3 and OCH 3, OCH 3 and CH 3 said optionally substituted with 1, 2 or 3 R a, the other variables are as Defined by the present invention.
  • R 1 is selected from H and CH 3 , and other variables are as defined in the present invention.
  • R 2 is selected from Cl, and other variables are as defined in the present invention.
  • R 3 , R 4 , R 5 and R 6 are each independently selected from H, and other variables are as defined in the present invention.
  • the above-mentioned L is selected from -O-, and other variables are as defined in the present invention.
  • the above-mentioned R 7 is selected from H, F, Cl, Br, I, -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl, the -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl are optionally substituted with 1, 2 or 3 R c , and other variables are as defined in the present invention.
  • the above-mentioned R 7 is selected from H, F, Cl, Br, I, CF 3 , -CHF 2 , -CH 2 F, -OCH 3 , CH 3 , -CH 2 CH 3 , -CH (CH 3 ) 2 , -NHCH 3 , -N(CH 3 ) 2 and cyclopropyl, and other variables are as defined in the present invention.
  • the above-mentioned L is selected from -NH- and -CH 2 -, and other variables are as defined in the present invention.
  • L 1 and L 2 are independently selected from -CH 2 -, -CHF- and -CF 2 -, and other variables are as defined in the present invention.
  • the above-mentioned R 8 is selected from H, F, Cl, Br, I, -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl, the -OCH 3 , CH 3 , -CH 2 CH 3 , -NHCH 3 and cyclopropyl are optionally substituted with 1, 2 or 3 R d , and other variables are as defined in the present invention.
  • R 8 is selected from H, F and Cl, and other variables are as defined in the present invention.
  • R 9 is independently selected from H, and other variables are as defined in the present invention.
  • the present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof, the compound is selected from
  • the above-mentioned compound is selected from
  • the present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of a medicament for treating hepatitis B virus.
  • the compound of the present invention exhibits unexpected activity of inhibiting cccDNA (labeled by HBeAg) in HepDES19 cell line, and exhibits unexpected activity of inhibiting hepatitis B surface antigen in human primary hepatocytes, and has an ideal in vivo PK. nature.
  • the compounds of the present invention can be used for diseases caused by HBV infection, such as the treatment of hepatitis B.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror-image relationship.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a three-dimensional center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key Or straight dashed key
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomer also called prototropic tautomer
  • proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers include some recombination of bonding electrons to carry out mutual transformations.
  • keto-enol tautomerization is the tautomerism between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the term “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refers to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • any variable such as R
  • its definition in each case is independent.
  • the group can be optionally substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond, and -C 0 alkyl-A means that the structure is actually -A.
  • substituents When the listed substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom.
  • a pyridyl group can pass through any one of the pyridine ring as a substituent. The carbon atom is attached to the substituted group.
  • the bond of a substituent can be cross-connected to two or more atoms on a ring, the substituent can be bonded with any atom on the ring, for example, a structural unit It means that the substituent R can be substituted at any position on the cyclohexyl or cyclohexadiene.
  • the middle linking group L is -MW-, at this time -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right It can also be formed by connecting ring A and ring B in the opposite direction to the reading order from left to right
  • Combinations of the linking groups, substituents, and/or variants thereof are only permitted if such combinations result in stable compounds.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • the connection method of the chemical bond is not positioned, and there is a H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will correspondingly decrease with the number of chemical bonds connected to become the corresponding valence number ⁇ The group.
  • the chemical bond between the site and other groups can be a straight solid bond Straight dashed key Or wavy line Express.
  • the straight solid bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy line in indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms;
  • the number of atoms in a ring is usually defined as the number of members of the ring.
  • a "5- to 7-membered ring” refers to a “ring” in which 5 to 7 atoms are arranged around.
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl, etc.; it may Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl) and the like.
  • heteroalkyl by itself or in combination with another term means a stable linear or branched alkyl group or a combination thereof composed of a certain number of carbon atoms and at least one heteroatom or heteroatom group.
  • the heteroatoms are selected from B, O, N, and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen heteroatoms are optionally quaternized.
  • the heteroalkyl is a C 1-6 heteroalkyl; In other embodiments, the heteroalkyl is a heteroalkyl C 1- 3.
  • heteroatom or heteroatom group can be located in any internal position of the heteroalkyl group, including the position of attachment of the alkyl group to the rest of the molecule.
  • alkoxy "alkylamino” and “alkylthio” (or thioalkoxy) are customary expressions and refer to those connected to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.
  • Alkyl group is customary expressions and refer to those connected to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively.
  • Up to two heteroatoms can be continuous
  • C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms attached to the rest of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy and the like.
  • Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy) and the like.
  • C 3-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic and bicyclic ring system.
  • the C 3-6 cycloalkyl includes C 3 to 5 , C 4 to 5 and C 5 to 6 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • the term "3- to 6-membered heterocycloalkyl" by itself or in combination with other terms means a saturated cyclic group consisting of 3 to 6 ring atoms, with 1, 2, 3 or 4 ring atoms.
  • a heteroatom may occupy the connection position between the heterocycloalkyl group and the rest of the molecule.
  • the 3- to 6-membered heterocycloalkyl group includes 4- to 6-membered, 5- to 6-membered, 4-, 5-, and 6-membered heterocycloalkyl.
  • 3- to 6-membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophene ⁇ 2-yl and tetrahydrothiophene ⁇ 3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2 ⁇ yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2- Piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), Dioxanyl, dithiazinyl, isoxazolidinyl, iso
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • SXRD single crystal X-ray diffraction
  • the cultured single crystal is collected with the Bruker D8 venture diffractometer to collect the diffraction intensity data
  • the light source is CuK ⁇ radiation
  • the scanning method After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.
  • the solvent used in the present invention is commercially available.
  • DMF stands for N,N-dimethylformamide
  • Na 2 CO 3 stands for sodium carbonate
  • K 2 CO 3 stands for potassium carbonate
  • Bn stands for benzyl
  • Tf stands for trifluoromethanesulfonyl
  • TBS-Cl stands for tert-butylchlorodimethylsilane
  • TBS stands for tert-butyldimethylsilyl
  • DMAP stands for N,N-lutidine-4-amine
  • TMS-CN stands for trimethylsilylcarbonitrile
  • TMS stands for Trimethylsilyl
  • EtOAc stands for ethyl acetate
  • THF stands for tetrahydrofuran
  • MeOH stands for methanol
  • DCM stands for dichloromethane
  • DMSO stands for dimethyl sulfoxide
  • EtOH stands for ethanol
  • CH 3 CN stands for acetonitrile
  • TFA stands for trifluoroacetic acid
  • DIPEA stands
  • the present invention will be described in detail through the following examples, but it is not meant to impose any disadvantageous restriction on the present invention.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.
  • 1 H NMR 400 MHz, CDCl 3
  • the preparation of intermediate B refers to the preparation process of intermediate A, and the compound A-3 in step B is replaced with (cis)-3-hydroxy-1-methylcyclobutyric acid methyl ester (B-1).
  • the intermediate BA is prepared by the following method:
  • BA-1 500 mg, 3.47 mmol was added to toluene (15 mL), followed by A-2 (1.78 g, 6.94 mmol) and N,N-diisopropylethylamine (896.45 mg, 6.94 mmol).
  • the reaction mixture was stirred at 90 degrees Celsius for 12 hours, then diluted with water (30 mL), and extracted with ethyl acetate (40 mL/time, 3 times). After the organic phases were combined, they were dried with anhydrous sodium sulfate.
  • Step A Dissolve potassium hydroxide (12.74 g, 227.00 mmol) in methanol (70 mL) and cool to room temperature. Then, under stirring at 0 degrees Celsius, the solution was added dropwise to a mixed solution of D-1 (5 g, 28.37 mmol) and tribromomethane (57.37 g, 227.00 mmol, 19.85 mL). Remove the ice water bath after adding. The reaction mixture was stirred at 20-25 degrees Celsius for 18 hours, quenched by adding water (100 mL), and then extracted with dichloromethane (80 mL/time, extraction 3 times).
  • the water phase was adjusted to a pH of about 3 with 0.5 mol/L dilute hydrochloric acid, and then extracted with ethyl acetate (100 ml/time, extraction 3 times).
  • the ethyl acetate phases obtained by the extraction were combined, washed with saturated brine (50 ml/time, washed twice), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain D-2, which was directly used in the next reaction.
  • Step B Dissolve D-2 (3.5 g, 14.81 mmol) in dry N,N-dimethylformamide (30 mL), then add iodoethane (3.47 g, 22.22 mmol, 1.78 mL) And potassium carbonate (4.09 g, 29.63 mmol).
  • the reaction mixture was stirred at 20-25 degrees Celsius for 6 hours, then diluted with water (150 mL), and extracted with ethyl acetate (50 mL/time, 3 times). After the organic phases were combined, they were washed once with saturated brine (50 mL), and then dried with anhydrous sodium sulfate.
  • the reaction system was replaced with hydrogen three times, and stirred under a hydrogen pressure of 15 psi and 25 degrees Celsius for 17 hours. After filtering through celite, the filtrate was concentrated under reduced pressure to obtain D-4, which was directly used in the next reaction.
  • Step D Add D-4 (500 mg, 2.87 mmol), A-2 (1.48 g, 5.74 mmol) and N,N diisopropylethylamine (742 mg, 5.74 mmol) to toluene (15 mL) Mole, 1 ml). After the system was replaced with nitrogen three times, the reaction mixture was stirred at 90 degrees Celsius for 48 hours under nitrogen protection. After cooling to room temperature, water (30 mL) and ethyl acetate (120 mL) were added to the reaction mixture, which was stirred and left to separate into layers. The organic phase was separated and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure.
  • Step A Dissolve D-1 (2 g, 11.35 mmol) in dry tetrahydrofuran (15 mL), then add sodium carbonate (601.49 mg, 5.67 mmol) and TMS-CN (1.13 g) at 20 degrees Celsius. , 11.35 mmol, 1.42 ml). The reaction mixture was stirred at 20-25 degrees Celsius for 5 hours under the protection of nitrogen, and then concentrated under reduced pressure to remove the solvent. The residue was diluted with water (50 mL), and extracted with ethyl acetate (30 mL/time, extraction twice). The combined organic phase was washed once with saturated brine (30 mL), and then dried over anhydrous sodium sulfate.
  • Step B At 0 degrees Celsius, add thionyl chloride (5.75 g, 48.36 mmol, 3.51 ml) to a solution of E-2 (7.4 g, 26.87 mmol) in methanol (50 ml) dropwise. After the addition, The reaction mixture was continuously stirred at 55 degrees Celsius for 3 hours. After concentration under reduced pressure to remove methanol, tert-butyl methyl ether (50 mL) was added to the residue, stirred at room temperature for 10 minutes, and filtered.
  • Step C Dissolve E-3 (500 mg, 2.12 mmol) in dry dichloromethane (50 mL), add TBS-Cl (478.45 mg, 3.17 mmol), imidazole (216.11 mg, 3.17 mmol) in sequence ) And DMAP (25.85 mg, 211.63 micromolar).
  • the reaction mixture was stirred at slight reflux (approximately 42 degrees Celsius) for 16 hours.
  • water (50 mL) and dichloromethane (200 mL) were added, and the mixture was allowed to stand and separate into layers after stirring.
  • Step F Add E-5 (150 mg, 576.03 micromole), A-2 (280.98 mg, 1.09 mmol) and N,N diisopropylethylamine (141.28 mg, 1.09 mmol) to toluene (15 mL) Mol, 190.40 microliters). After the system was replaced with nitrogen three times, the reaction mixture was stirred at 90 degrees Celsius for 48 hours under nitrogen protection. After cooling to room temperature, water (30 mL) and ethyl acetate (120 mL) were added to the reaction mixture, which was stirred and left to separate into layers. The organic phase was separated and dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure.
  • F-1 150 mg, 1.06 mmol was added to toluene (5 mL), followed by A-2 (544.90 mg, 2.12 mmol) and N,N-diisopropylethylamine (273.99 mg, 2.12 mmol).
  • the reaction mixture was stirred at 90 degrees Celsius for 48 hours, then diluted with water (20 mL), and extracted with ethyl acetate (20 mL/time, extraction twice). After the organic phases were combined, they were washed once with saturated brine (20 mL), and then dried with anhydrous sodium sulfate.
  • Compound 1 was prepared by the following synthetic route:
  • Step C Mix compound 1-3 (2 g, 7.03 mmol), compound 1-4 (904.37 mg, 7.03 mmol) and p-toluenesulfonic acid (1.21 g, 7.03 mmol) and heat to 150 degrees Celsius.
  • the reaction mixture was stirred at 150 degrees Celsius for 1 hour, and after natural cooling, water (20 mL) was added, and then washed with ethyl acetate (50 mL/time, washing twice).
  • Step D Dissolve compound 1-5 (200 mg, 551.26 micromole) in anhydrous dichloromethane (5 mL), add boron tribromide (1 mol /L, 1.1 mL), after the addition, the reaction mixture was stirred at minus 78 degrees Celsius for 0.5 hours and quenched with methanol (10 mL). Then water (50 mL) was added, and it was extracted with ethyl acetate (50 mL/time, twice extraction). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude product 1-6, which was directly used in the next reaction. MS(ESI) m/z: 273 [M+H + ].
  • Step E Compound 1-6 (150 mg, 550.08 ⁇ mol) and Intermediate A (138.14 mg, 550.08 ⁇ mol) were sequentially added to acetonitrile (2 mL), and then potassium carbonate (152.05 mg, 1.1 mmol) was added And potassium iodide (9.13 mg, 55.01 micromolar). The reaction mixture was stirred at 85 degrees Celsius for 12 hours. Then water (20 mL) was added, and it was extracted with ethyl acetate (50 mL/time, twice extraction). The combined organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude product 1-7, which was directly used in the next reaction. MS (ESI) m/z: 443 [M+H + ].
  • Step F Add compound 1-7 (0.2 g, 451.58 micromolar) to a mixture of tetrahydrofuran (1 ml), methanol (0.5 ml) and water (0.5 ml), and add lithium hydroxide monohydrate (94.75 mg, 2.26 mmol). After the reaction mixture was stirred at 25 degrees Celsius for 0.5 hours, it was diluted by adding water (20 mL), followed by washing with ethyl acetate (50 mL/time, washing twice). The pH of the water phase is adjusted to about 3 to 4 with 1 mol/L hydrochloric acid, and a solid is precipitated.
  • Step A Add 2-1 (1 g, 5.86 mmol) and potassium carbonate (1.22 g, 8.79 mmol) to N,N-dimethylformamide (10 mL), followed by benzyl bromide (1.02 g , 5.98 mmol). The reaction mixture was stirred at 15-25 degrees Celsius for 14 hours, then poured into water (50 mL), and then extracted with ethyl acetate (25 mL/time, extraction twice). The combined organic phase was washed once with saturated brine (30 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 2-2, which was directly used in the next reaction.
  • Step D Dissolve 2-4 (40 mg, 100.69 micromoles) in dichloromethane (3 mL), and then add boron tribromide (50.54 mg, 201.38 micromoles) at minus 20 degrees Celsius.
  • the reaction mixture was stirred at 0 degrees Celsius for 1 hour. After quenching by adding methanol (2 mL), it was concentrated under reduced pressure to remove the solvent. The remaining solid was washed with water (5 mL) and tert-butyl methyl ether (2 mL) successively, and dried under reduced pressure to obtain 2-5, which was directly used in the next reaction.
  • Step E Add 2-5 (20 mg, 53.50 micromole), intermediate A (15.22 mg, 64.20 micromole), potassium carbonate (14.79 mg, 107.00 micromole) and potassium iodide (0.88 mg, 5.35 micromole) in sequence Into acetonitrile (4 mL). The reaction mixture was stirred at 90 degrees Celsius for 14 hours. After being naturally cooled, it was diluted by adding water (20 ml), and then extracted with ethyl acetate (15 ml/time, extraction 2 times). After the organic phases were combined, they were washed once with saturated brine (15 mL), and then dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 2-6, which was directly used in the next reaction. MS(ESI) m/z: 463 [M+H + ].
  • the preparation of compound 3 refers to the preparation process of compound 1, replacing compound 1-1 in step A with 3-chloro-4-hydroxyacetophenone (3-1).
  • Step A Add 4-1 (1 g, 6.49 mmol) and potassium carbonate (1.35 g, 9.74 mmol) to N,N-dimethylformamide (10 mL), followed by benzyl bromide (1.17 g , 6.81 mmol). The reaction mixture was stirred at 20 degrees Celsius for 12 hours, then poured into water (50 mL), and extracted with ethyl acetate (50 mL/time, twice extraction). The combined organic phase was washed once with saturated brine (50 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 4-2, which was directly used in the next reaction.
  • Step B Add sodium hydride (60% purity, 1.30 g, 32.55 mmol) to N,N-dimethylformamide (15 ml), then lower the temperature to minus 5 degrees Celsius, and add 4-2 (1.59 g, 6.51 mmol). After stirring for 30 minutes, additional dimethyl carbonate (2.93 g, 32.55 mmol) was added. The reaction mixture was slowly warmed to 20 degrees Celsius, and stirring was continued for 12 hours. The reaction mixture was poured into water (80 mL), and extracted with ethyl acetate (80 mL/time, extraction 2 times). The organic phases were combined, washed once with saturated brine (50 mL), and dried over anhydrous sodium sulfate.
  • Step C After mixing 4-3 (1.48 g, 4.90 mmol), 2-amino-3-chloropyridine (629.41 mg, 4.90 mmol) and p-toluenesulfonic acid (168.62 mg, 919.17 micromol), the mixture is heated at 150 Stir for 12 hours at °C. After cooling, water (30 mL) was added to wash. After filtration, the filter cake was collected to obtain 4-4.
  • Step D Dissolve 4-4 (200 mg, 525.21 ⁇ mol) in dichloromethane (5 mL), and then add boron tribromide (263.16 mg, 1.05 mmol) at minus 20 degrees Celsius. The reaction mixture was stirred at 20 degrees Celsius for 30 minutes. After quenching by adding methanol (10 mL), adding water (20 mL) for dilution, and then extracting with dichloromethane (30 mL/time, extraction twice). The combined organic phase was washed once with saturated brine (30 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 4-5, which was directly used in the next reaction. MS (ESI) m/z: 291 [M+H + ].
  • Step E Add 4-5 (152 mg, 522.92 ⁇ mol), Intermediate A (130.18 mg, 549.06 ⁇ mol), potassium carbonate (144.54 mg, 1.05 mmol) and potassium iodide (8.68 mg, 52.29 ⁇ mol) in sequence Into acetonitrile (5 mL). The reaction mixture was stirred at 85 degrees Celsius for 12 hours. After being naturally cooled, it was diluted by adding water (50 ml), and then extracted with ethyl acetate (50 ml/time, extraction 2 times). After the organic phases were combined, they were washed once with saturated brine (50 mL), and then dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 4-6, which was directly used in the next reaction. MS(ESI) m/z: 447 [M+H + ].
  • Step F Add 4-6 (233 mg, 521.42 micromoles) to a mixture of methanol (3 mL), tetrahydrofuran (3 mL) and water (3 mL), and then add lithium hydroxide monohydrate (54.70 mg, 1.30 mmol).
  • Compound 5 was prepared by the following synthetic route:
  • Step A Add 1-6 (400 mg, 1.47 mmol), intermediate B (369.14 mg, 1.47 mmol), potassium carbonate (304.75 mg, 2.21 mmol) and potassium iodide (48.80 mg, 294.00 micromol) in sequence Into acetonitrile (25 mL). The reaction mixture was stirred at 85 degrees Celsius for 12 hours under the protection of nitrogen. Acetonitrile was removed by distillation under reduced pressure to obtain 5-1 crude product, which was directly used in the next reaction. MS (ESI) m/z: 443 [M+H+].
  • Step B Add 5-1 crude product (650 mg, 1.47 mmol) to a mixture of methanol (5 mL), tetrahydrofuran (5 mL) and water (5 mL), and then add lithium hydroxide monohydrate (92.38 mg) , 2.20 mmol).
  • the reaction mixture was stirred at 25 degrees Celsius for 2 hours.
  • Compound 5A was prepared by the following method:
  • Step A Dissolve 1-6 (300 mg, 1.19 mmol) in acetonitrile (10 mL), add potassium carbonate (247.66 mg, 1.79 mmol), potassium iodide (59.49 mg, 358.40 micromol) and intermediate BA in sequence (325.77 mg, 1.19 mmol).
  • the reaction mixture was stirred at 85 degrees Celsius for 12 hours.
  • the solvent was distilled off under reduced pressure to obtain the crude product of 5A-1, which was directly used in the next reaction.
  • Step B Add 5A-1 (529 mg, 1.19 mmol) to a mixture of tetrahydrofuran (3 mL), methanol (3 mL) and water (3 mL), and then add lithium hydroxide monohydrate (100.24 mg, 2.39 mmol). The reaction mixture was stirred at 25 degrees Celsius for 2 hours. The solvent was distilled off under reduced pressure, and the residue was separated and purified by preparative HPLC (column: Waters Xbridge 150 ⁇ 25mm ⁇ 5 ⁇ m; mobile phase: phase A: 0.05% ammonia solution; phase B: acetonitrile, 5% to 35%; 10 minutes). Get 5A-2. MS (ESI) m/z: 429 [M+H + ].
  • Step A Dissolve 1-6 (120 mg, 440.07 micromole) in acetonitrile (10 mL), add potassium carbonate (91.23 mg, 660.10 micromole), potassium iodide (36.53 mg, 220.03 micromole) and Intermediate C in sequence (104.34 mg, 440.07 micromolar).
  • the reaction mixture was stirred at 85 degrees Celsius for 48 hours.
  • the solvent was distilled off under reduced pressure to obtain crude product 6-2, which was directly used in the next reaction.
  • Step B Add 6-2 (189 mg, 440.70 micromole) to a mixture of tetrahydrofuran (2 mL), methanol (2 mL) and water (1 mL), and then add lithium hydroxide monohydrate (36.99 mg, 881.40 micromolar). After the reaction mixture was stirred at 20 degrees Celsius for 2 hours, it was neutralized to pH ⁇ 3 with 2 mol/L dilute hydrochloric acid.
  • the preparation of compound 7 refers to the preparation process of compound 1, and the compound intermediate A in step E is replaced with intermediate D.
  • Example 7 was separated and purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 ⁇ 30 mm ⁇ 3 ⁇ m; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: acetonitrile, 30% to 60%; 7 min).
  • Step A Add 1-6 (74.23 mg, 408.33 micromoles) and Intermediate E (100 mg, 272.22 micromoles) to dry acetonitrile (5 mL), and then add potassium carbonate (56.44 mg, 408.33 micromoles) And potassium iodide (9.04 mg, 54.44 micromolar). The reaction mixture was stirred at 85 degrees Celsius for 12 hours. After acetonitrile was removed by distillation under reduced pressure, the residue was 8-2 crude product, which was directly used in the next reaction. MS(ESI)m/z:559[M+H + ]
  • Step B Take the above 8-2 crude product (150 mg) and add it to a mixture of methanol (2 mL), tetrahydrofuran (2 mL) and water (2 mL), and then add lithium hydroxide monohydrate (22.52 mg). The reaction mixture was stirred at 25 degrees Celsius for 2 hours. After adding water (30 mL) to dilute, it was extracted with dichloromethane (40 mL/time, extraction 3 times). After the organic phases were combined, they were washed with saturated brine (50 ml/time, twice), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain 8-3 crude product, which was directly used in the next reaction. MS(ESI)m/z:545[M+H + ]
  • Compound 8 was prepared by the following method:
  • Step A Dissolve 1-6 (164.20 mg, 602.16 micromole) in acetonitrile (5 mL), add potassium carbonate (124.83 mg, 903.25 micromole), potassium iodide (20 mg, 120.43 micromole) and Intermediate G in sequence (150 mg, 602.16 micromolar).
  • Compound 10 was prepared by the following method:
  • Step A Compound 8 (100 mg, 232.11 micromoles) was dissolved in anhydrous methanol (2 mL), and then thionyl chloride (55.23 mg, 464.21 micromoles) was added at 0 degrees Celsius. The reaction mixture was naturally heated and stirred at 20-25 degrees Celsius for 3 hours. The solvent was distilled off under reduced pressure to obtain 10-1, which was directly used in the next reaction.
  • Step B Dissolve diethylaminosulfur trifluoride (76.09 mg, 472.05 micromoles) in dichloromethane (2 mL), and then add 10-1 (105 mg, 236.03 micromoles) of dichloromethane ( 1 ml) solution. After the addition, the reaction mixture was stirred at 25 degrees Celsius for 1 hour. Water (2 mL) was added to quench the reaction, and the liquid was separated to obtain an organic phase. After the organic phase was concentrated under reduced pressure, 10-2 was obtained, which was directly used in the next reaction.
  • Step C Dissolve 10-2 (100 mg, 223.79 micromoles) in a mixture of tetrahydrofuran (1 mL) and water (1 mL), and then add lithium hydroxide monohydrate (18.78 mg, 447.57 micromoles). The reaction mixture was stirred at 25 degrees Celsius for 1 hour. After concentrating under reduced pressure to remove the solvent, the residue was subjected to preparative HPLC (column: Phenomenex Gemini-NX C18 75 ⁇ 30mm ⁇ 3 ⁇ m; mobile phase: phase A: 0.05% ammonia solution; phase B: acetonitrile, 3%-30%; 7 minutes ) Isolation and purification to obtain compound 10.
  • the HepDES19 cell line contains a 1.1-unit-length HBV genome, and the transcription of pgRNA is controlled by tetracycline. In the absence of tetracycline, the transcription of pgRNA is induced, but because the very short leader sequence before the start codon of HBV e antigen (HBeAg) interferes with the promoter, pgRNA cannot produce HBeAg. Only after the formation of cccDNA, the missing leader sequence and promoter mutation can be restored, and then HBeAg can be synthesized. Therefore, HBeAg can be used as a surrogate marker for cccDNA. (Antiviral Res.; 2006,72,116-124; Virol.; 2007,81,12472-12484).
  • the HBeAg content of HepDES19 cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA) to evaluate the compound's inhibitory effect on HBV.
  • ELISA enzyme-linked immunosorbent assay
  • DMEM/F12 medium source: Gibco Cat.11330057
  • 10% fetal bovine serum Fetal Bovine Serum, FBS, source: Clontech
  • 100unis/mL 100 ⁇ g/mL penicillin/streptomycin (Penicillin/streptomycin)
  • Source: Hyclone 2mM GlutaMAX (source: Gibco), 1% non-essential amino acid solution (MEM NEAA, source: Gibco), 0.1mM aminoglycoside antibiotic (Geneticin, source: Gibco), 1 ⁇ g/mL tetracycline hydrochloride (Tetracycline) Hydrochloride, source: Sigma), subcultured and expanded at a ratio of 1/3, then planted HepDES19 into a T150 culture flask at a cell density of 4 ⁇ 10 6 cells, replaced with a tetracycline-free medium and cultured for 8 days, and finally The cells were collected and cryopreserved in liquid nitrogen (1 ⁇ 10 7
  • HepDES19 cells Resuscitate HepDES19 cells, plant HepDES19 cells in a 96-well plate (6 ⁇ 10 4 cells/well), and incubate overnight at 37 degrees Celsius and 5% CO 2.
  • the compound was diluted to a total of 8 concentrations in 3-fold serial dilutions. Compounds of different concentrations were added to the culture wells, and the wells were duplicated. The final concentration of DMSO in the culture broth is 0.5%.
  • the culture medium in the culture well was collected, and the content of hepatitis B virus HBeAg was determined by ELISA. After aspirating the culture medium in the culture wells, add Celltiter-Glo reagent to each well of the 96-well plate, and the microplate reader detects the chemiluminescence value of each well to detect cell viability.
  • hepatitis B virus HBeAg The content of hepatitis B virus HBeAg is determined by ELISA.
  • the specific steps refer to the product manual. The steps are briefly described as follows: Take 50 microliters of sample and standard substance into the reaction plate, and then add 50 microliters of enzyme conjugate to each well, shake and mix well. Incubate at 37 degrees Celsius for 60 minutes, then wash the plate 6 times with washing solution, then add 50 microliters of luminescent substrate to each well, mix, and react at room temperature for 10 minutes in the dark, and finally detect the chemiluminescence intensity with a microplate reader.
  • %Inh. (1-HBeAg value in sample/DMSO control HBeAg value) ⁇ 100.
  • % Cell viability (sample luminescence value-medium control luminescence value) / (DMSO control luminescence value-medium control luminescence value) ⁇ 100%.
  • hepatitis B surface antigen in the culture supernatant of human primary hepatocytes was detected by enzyme-linked immunosorbent assay (ELISA), and the EC 50 value of the compound was used as an indicator to evaluate the inhibitory effect of the compound on HBV; at the same time, the CellTiter- glo detects cell viability to evaluate the toxicity of compounds to cells.
  • ELISA enzyme-linked immunosorbent assay
  • PHH 800GE/cell
  • type D HBV concentrated from HepG2.2.15 cell culture supernatant
  • the compound was diluted to a total of 7 concentrations, with a 3-fold gradient dilution. Add different concentrations of compounds to the culture wells, double-repeat the wells. The final concentration of DMSO in the culture broth is 2%.
  • %Viability value in sample/DMSO control value ⁇ 100.
  • %Inh. (1—value in sample/DMSO control value) ⁇ 100.
  • the compound of the present invention has significant anti-HBV activity in vitro.
  • This experiment aims to evaluate the pharmacokinetic behavior of the compound after a single intravenous injection or intragastric administration in mice.
  • the compound is formulated as a clear solution of 0.2 mg/mL, and the solvent: 5% DMSO/5% dodecyl hydroxystearate (solutol)/90% water;
  • the compound is formulated as 0.3 mg/mL mL of suspension, solvent: 0.5% sodium carboxymethyl cellulose/0.2% Tween 80/99.3% water.
  • the concentration of the compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the retention time of the compound and the internal standard (diclofenac), the collection of chromatograms and the integration of the chromatograms are processed by the software Analyst (Applied Biosystems), and the data statistics are processed by the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems).
  • the unit of the analyte concentration in the sample is ng/mL, with 3 significant digits reserved, and all values expressed in percentage (such as% deviation and% coefficient of variation, etc.) are reserved to one decimal place.
  • Each calibration curve contains at least 6 concentration levels.
  • the preparation of calibration standards requires stock solutions from different sources from the quality control samples.
  • the standard sample should be rejected in the regression analysis.
  • the rejected calibration standards should be less than 25%, and each calibration curve contains at least 6 calibration standards that meet the acceptance criteria. If the lower limit of quantification and upper limit of quantification need to be rejected, the upper and lower limit of quantification of the analysis batch will be increased and lowered accordingly.
  • the non-compartmental model of WinNonlin TM Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process plasma concentration, and the pharmacokinetic parameters were calculated using the linear logarithmic trapezoidal method.
  • the pharmacokinetic parameters to be calculated include but are not limited to (data allowed) T 1/2 , MRT 0-24h , Vd ss , CL, AUC 0-24h in the IV group; C max , T max , AUC 0 in the PO group -24h , oral bioavailability (F%).
  • the following table 3 shows the pharmacokinetic parameters in mice of the embodiment of the present invention at a dose of 1 mg/Kg for intravenous injection and 3 mg/Kg for oral gavage.
  • the compound of the present invention exhibits a longer drug residence time in the body and a higher drug plasma exposure.
  • the introduction of a methyl group at the alpha position of the terminal carboxylic acid significantly prolongs the residence time of the drug, and at the same time significantly increases the exposure in the body, which effectively improves the bioavailability.
  • T0, T15, T30, T60, T90, T0-MC, T90-MC and blank matrix Take out the resuscitation medium and incubation medium in advance, and place them in a 37°C water bath to preheat. Take out the frozen liver cells from the liquid nitrogen tank and immediately immerse them in a 37°C water bath (about 90 seconds). After the frozen parts are melted and loosened, pour them into centrifuge tubes containing 40 mL of resuscitation medium, and gently invert them to resuspend the cells in the resuscitation medium.
  • test compound and control compound working solution 2 ⁇ L
  • mix well 2 ⁇ L
  • incubation plate 2 ⁇ L
  • the incubation conditions are 37°C, saturated humidity, and 5% carbon dioxide.
  • the final concentration of the test substance is 1 ⁇ M
  • the final concentration of the reference substance is 3 ⁇ M
  • the final concentration of hepatocytes is (0.5 ⁇ 10 6 cells/mL)
  • the final concentration of total organic solvents is 0.96%.
  • the final concentration is 0.1%.
  • the incubation plate was taken out, 25 ⁇ L of the mixture of the compound and the control compound and the cells were taken out and added to the sample plate containing 125 ⁇ L of stop solution (acetonitrile solution containing 200 ng/mL tolbutamide and Rabenol).
  • stop solution acetonitrile solution containing 200 ng/mL tolbutamide and Rabenol
  • the compound of the present invention has good metabolic stability in hepatocytes.

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Abstract

L'invention concerne une classe de composés de pyridopyrimidinone, et spécifiquement un composé tel que représenté par la formule (II) et un sel pharmaceutiquement acceptable de celui-ci.
PCT/CN2021/082391 2020-03-23 2021-03-23 Composé de pyridopyrimidinone WO2021190502A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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