WO2023125540A1 - 吡唑-1(2h)-酞嗪酮类化合物及其应用 - Google Patents

吡唑-1(2h)-酞嗪酮类化合物及其应用 Download PDF

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WO2023125540A1
WO2023125540A1 PCT/CN2022/142362 CN2022142362W WO2023125540A1 WO 2023125540 A1 WO2023125540 A1 WO 2023125540A1 CN 2022142362 W CN2022142362 W CN 2022142362W WO 2023125540 A1 WO2023125540 A1 WO 2023125540A1
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compound
alkynyl
present
pharmaceutically acceptable
cycloalkyl
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PCT/CN2022/142362
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French (fr)
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付志飞
罗妙荣
俞晨曦
周凯
高娜
张杨
黎健
陈曙辉
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南京明德新药研发有限公司
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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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C07D237/32—Phthalazines with oxygen atoms directly attached to carbon atoms of the nitrogen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • 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

Definitions

  • the present invention relates to a series of pyrazole-1(2H)-phthalazinone compounds and applications thereof, in particular to compounds represented by formula (III) and pharmaceutically acceptable salts thereof.
  • Protein arginase methyltransferase 5 (protein arginase methyltransferase 5, PRMT5), an epigenetic target with clinical potential, is a type II arginine methyltransferase that Chin, post-translational modification of histones and other proteins, current studies have shown elevated expression levels of PRMT5 in a variety of tumors.
  • the metabolic enzyme 5-methylthioadenonine phosphorylase (5'-methylthioadenonine phosphorylase, MTAP) gene is very close to the tumor suppressor gene CDKN2A on human chromosome 9p21, so the tumors with CDKN2A gene deletion are often accompanied by MTAP loss, which is the tumor One of the most frequently mutated genes. Loss of MTAP leads to the accumulation of its substrate methylthioadenosine (MTA), which selectively inhibits the activity of PRMT5 methyltransferase and makes it more sensitive to further PRMT5 inhibition.
  • MTA substrate methylthioadenosine
  • PRMT5 inhibitors could be a highly selective treatment for patients with MTAP-deficient or low-expressing tumors.
  • PRMT5 has received special attention as a potential target for tumor therapy.
  • a large number of small molecule inhibitors of PRMT5 have been reported.
  • the compounds occupy the SAM binding site they can be divided into two categories, SAM non-competitive inhibitors and SAM competitive inhibitors, in which SAM non-competitive inhibitors occupy the substrate The binding site competes with the substrate, whereas the SAM competitive inhibitor occupies the SAM binding site.
  • SAM non-competitive inhibitors although it can effectively inhibit the growth of many cell lines in vitro, it lacks the selectivity of tumor cell lines, and its ability to inhibit tumor cells has nothing to do with the state of MTAP in the cells, which may lead to Potential risk of clinical toxicity.
  • MTA accumulates and partially inhibits PRMT5 in cells, making tumor cells sensitive to PRMT5 inhibitors. Therefore, a small molecule is designed to cooperate with MTA to bind to PRMT5 and specifically bind to the PRMT5 ⁇ MTA complex, thereby inhibiting Tumor cell growth is an effective means to improve the selectivity of PRMT5 inhibitors.
  • the present invention provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl, the C 2-5 alkenyl Base, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl are independently optionally substituted by 1, 2 or 3 R a ;
  • R 2 is selected from halogen and C 2-3 alkynyl, the C 2-3 alkynyl is optionally substituted by 1, 2 or 3 halogens;
  • R 3 is selected from CH 3 and CD 3 ;
  • R is selected from H and halogen
  • Each R a is independently selected from halogen and C 1-3 alkyl, and the C 1-3 alkyl is optionally substituted by 1, 2 or 3 halogens;
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 Cycloalkyl, the C 2-5 alkenyl , C 2-5 alkynyl and -OC 2-3 alkynyl are optionally substituted by 1, 2 or 3 R a , the -OC 3-5 cycloalkyl and C 3-5 cycloalkyl is substituted by 1, 2 or 3 R a ;
  • R 4 is selected from halogen.
  • R a are independently selected from F, Cl, CH 3 , CHF 2 and CF 3 , and other variables are as defined in the present invention.
  • R 1 is selected from C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl, and the C 2 -3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl are independently optionally substituted by 1, 2 or 3 R a , other variables are as in this invention defined.
  • R 1 is selected from said are independently optionally substituted by 1, 2 or 3 R a , and other variables are as defined in the present invention.
  • R 2 is selected from F, Cl and said Optionally substituted with 1, 2 or 3 halogens, other variables are as defined herein.
  • R 2 is selected from Cl, Other variables are as defined herein.
  • R 2 is selected from Cl, and R 1 is selected from Other variables are as defined herein.
  • R 2 is selected from Cl
  • R 3 is selected from CD 3
  • other variables are as defined in the present invention.
  • R 3 is selected from CD 3 , and other variables are as defined in the present invention.
  • R 4 is selected from H and F, and other variables are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 2 , R 3 and R a are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 3 and R a are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • R a is as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • R a is as defined in the present invention.
  • the present invention also provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl, the C 2-5 alkenyl Base, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl are independently optionally substituted by 1, 2 or 3 R a ;
  • R 2 is selected from halogen and C 2-3 alkynyl, the C 2-3 alkynyl is optionally substituted by 1, 2 or 3 halogens;
  • R 3 is selected from CH 3 and CD 3 ;
  • R is selected from H and halogen
  • Each R a is independently selected from halogen and C 1-3 alkyl
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 ring Alkyl, the C 2-5 alkenyl, C 2-5 alkynyl and -OC 2-3 alkynyl are optionally substituted by 1, 2 or 3 R a , the -OC 3-5 cycloalkyl and C 3-5 cycloalkyl is substituted by 1, 2 or 3 R ; or
  • R 2 when R 2 is selected from halogen, R 3 is selected from CD 3 ;
  • R 4 is selected from halogen.
  • the R a are independently selected from F, Cl, CH 3 , CHF 2 and CF 3 , and other variables are as defined in the present invention.
  • said R 1 is selected from C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl, said C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl are independently optionally substituted by 1, 2 or 3 R a , other variables such as defined in the present invention.
  • said R 1 is selected from said are independently optionally substituted by 1, 2 or 3 R a , and other variables are as defined in the present invention.
  • said R 1 is selected from Other variables are as defined herein.
  • said R 2 is selected from F, Cl and said Optionally substituted with 1, 2 or 3 halogens, other variables are as defined herein.
  • the R 2 is selected from Cl, Other variables are as defined herein.
  • the R 3 is selected from CD 3 , and other variables are as defined in the present invention.
  • the R 4 is selected from H and F, and other variables are as defined in the present invention.
  • the present invention also provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl, the C 2-5 alkenyl Base, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl are independently optionally substituted by 1, 2 or 3 R a ;
  • R 2 is selected from halogen and C 2-3 alkynyl, the C 2-3 alkynyl is optionally substituted by 1, 2 or 3 halogens;
  • R 3 is selected from CH 3 and CD 3 ;
  • Each R a is independently selected from halogen and C 1-3 alkyl
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 ring Alkyl, the C 2-5 alkenyl, C 2-5 alkynyl and -OC 2-3 alkynyl are optionally substituted by 1, 2 or 3 R a , the -OC 3-5 cycloalkyl and C 3-5 cycloalkyl is substituted by 1, 2 or 3 R ; or
  • R 3 is selected from CD 3 .
  • the R a are independently selected from F, Cl, CH 3 , CHF 2 and CF 3 , and other variables are as defined in the present invention.
  • said R 1 is selected from C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl, said C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl are independently optionally substituted by 1, 2 or 3 R a , other variables such as defined in the present invention.
  • said R 1 is selected from said are independently optionally substituted by 1, 2 or 3 R a , and other variables are as defined in the present invention.
  • said R 1 is selected from Other variables are as defined herein.
  • said R 2 is selected from F, Cl and said Optionally substituted with 1, 2 or 3 halogens, other variables are as defined herein.
  • the R 2 is selected from Cl, Other variables are as defined herein.
  • the present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl, the C 2-5 alkenyl Base, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl are independently optionally substituted by 1, 2 or 3 R a ;
  • R 2 is selected from halogen and C 2-3 alkynyl, the C 2-3 alkynyl is optionally substituted by 1, 2 or 3 halogens;
  • Each R a is independently selected from halogen and C 1-3 alkyl
  • R 1 is selected from C 2-5 alkenyl, C 2-5 alkynyl, -OC 3-5 cycloalkyl, -OC 2-3 alkynyl and C 3-5 cycloalkyl , the C 2-5 alkenyl, C 2-5 alkynyl and -OC 2-3 alkynyl are optionally substituted by 1, 2 or 3 R a , the -OC 3-5 cycloalkyl and C 3 -5 cycloalkyl is substituted by 1, 2 or 3 R a .
  • the R a are independently selected from F, Cl, CH 3 , CHF 2 and CF 3 , and other variables are as defined in the present invention.
  • said R 1 is selected from C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl, said C 2-3 alkenyl, C 2-3 alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl are independently optionally substituted by 1, 2 or 3 R a , other variables such as defined in the present invention.
  • said R 1 is selected from said are independently optionally substituted by 1, 2 or 3 R a , and other variables are as defined in the present invention.
  • said R 1 is selected from Other variables are as defined herein.
  • said R 2 is selected from F, Cl and said Optionally substituted with 1, 2 or 3 halogens, other variables are as defined herein.
  • the R 2 is selected from Cl, Other variables are as defined herein.
  • the present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,
  • the above compound or a pharmaceutically acceptable salt thereof is selected from,
  • the present invention also provides the application of the above compound or a pharmaceutically acceptable salt thereof in PRMT5 inhibitor-related drugs.
  • the present invention also provides the application of the above compound or a pharmaceutically acceptable salt thereof in PRMT5 ⁇ MTA complex inhibitor-related drugs.
  • the present invention also provides following synthetic method:
  • the present invention also provides following test method:
  • Test method 1 PRMT5 enzyme inhibitory activity test
  • the inhibition rates were imported into Excel to calculate the inhibition rates of different concentrations of compounds, and then XLfit software was used to draw inhibition curves and calculate related parameters, including minimum inhibition rate, maximum inhibition rate and IC 50 .
  • Test method 2 pharmacokinetic evaluation in mice
  • mice Male C57BL/6 mice were used as the experimental animals, and the plasma drug concentration at different times after intravenous and oral administration of the compound was determined by LC/MS/MS method. Study the pharmacokinetic behavior of compounds in mice and evaluate their pharmacokinetic characteristics.
  • the compound of the present invention has excellent binding effect on PRMT5, can effectively inhibit PRMT5, and has significant inhibitory effect on PRMT5 ⁇ MTA complex; has obvious inhibitory activity on MTAP-deficient tumor cells, and has weak inhibitory effect on wild-type tumor cells. Shows good selectivity; has good liver microsomal stability and good in vivo pharmacokinetic properties, and the species difference is small; the tumor inhibitory effect is significant on the human large cell lung cancer LU99 subcutaneous xenograft tumor model, and the administration The body weight of the mice was maintained well.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
  • Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
  • such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the invention may exist in particular 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 and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
  • Certain compounds of the invention may exist as atropisomers, which are conformational isomers that occur when rotation about a single bond in the molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule.
  • the compounds disclosed in the present invention include all atropisomers, which may be pure individual atropisomers, or be enriched in one of the atropisomers, or be respective non-specific mixtures. If the rotational potential around a single bond is high enough, and the interconversion between conformations is slow enough, it can allow the separation of isomers.
  • )and (or ) is a pair of atropisomers, wherein, on the phenyl Indicates that the stereoscopic orientation of this side is outward, Indicates that the stereoscopic orientation of this side is inward.
  • any variable eg, R
  • its definition is independent at each occurrence.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
  • any one or more sites of the group can be linked to other groups through chemical bonds.
  • connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
  • the chemical bonds that the site is connected with other groups can use straight solid line bonds Straight dotted key or tilde express.
  • the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dotted line bond indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups;
  • halogen or halogen by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.
  • C2-5 alkenyl is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 5 carbon atoms containing at least one carbon-carbon double bond, a carbon-carbon double bond can be located anywhere in the group.
  • the C 2-5 alkenyl includes C 2-3 , C 5 , C 4 , C 3 and C 2 alkenyl, etc.; the C 2-5 alkenyl can be monovalent, divalent or multivalent.
  • Examples of C alkenyl include, but are not limited to, ethenyl, propenyl, butenyl, butadienyl, and the like.
  • C2-3 alkenyl is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, a carbon-carbon double bond can be located anywhere in the group.
  • the C 2-3 alkenyl includes C 3 and C 2 alkenyl; the C 2-3 alkenyl can be monovalent, divalent or multivalent. Examples of C 2-3 alkenyl include, but are not limited to, ethenyl, propenyl, and the like.
  • C 2-5 alkynyl is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 5 carbon atoms containing at least one carbon-carbon triple bond, a carbon-carbon triple bond can be located anywhere in the group.
  • the C 2-4 alkynyl includes C 2-3 , C 5 , C 4 , C 3 and C 2 alkynyl and the like. It may be monovalent, divalent or polyvalent. Examples of C alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, and the like.
  • C 2-3 alkynyl is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon triple bond, a carbon-carbon triple bond can be located anywhere in the group. It may be monovalent, divalent or polyvalent.
  • the C 2-3 alkynyl includes C 3 and C 2 alkynyl. Examples of C alkynyl include, but are not limited to, ethynyl, propynyl, and the like.
  • C 3-5 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 5 carbon atoms, which is a monocyclic ring system, and the C 3-5 cycloalkyl includes C 3 -4 and C 4-5 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-5 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and the like.
  • 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 the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
  • the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
  • SXRD single crystal X-ray diffraction
  • solutol represents polyethylene glycol-15 hydroxystearate
  • MC (4000cps) represents methylcellulose (viscosity 4000)
  • tolbutamide represents tolbutamide
  • labetalol represents labetalol
  • the solvent used in the present invention is commercially available.
  • the molecular docking process was carried out by using Maestro ( Induced Fit Docking [1-2] default option in Version 2021-2).
  • the crystal structure of PRMT5 in the PDB database (PDB ID: 5FA5) was selected as the docking template.
  • PDB ID: 5FA5 The crystal structure of PRMT5 in the PDB database
  • hydrogen atoms were added using the Protein Preparation Wizard module of Maestro [2] and energy minimization was performed using the OPLS4 force field.
  • the 3D structure of the molecule was generated using LigPrep [3] and energy minimized using the OPLS4 force field.
  • Trp579 of 5FA5 as the center, boxes are automatically generated to place example compounds during molecular docking.
  • the prediction diagram of the binding mode of compound A and PRMT5 is shown in Figure 1
  • the prediction diagram of the binding mode of compound B and PRMT5 is shown in Figures 2-5
  • the prediction diagram of the binding mode of compound C and PRMT5 is shown in Figures
  • the prediction diagram of the binding mode is shown in Figure 10
  • the prediction diagram of the binding mode of compound F and PRMT5 is shown in Figure 11.
  • the compound of the present invention has excellent binding effect with PRMT5.
  • compound M2-1 (30g, 154.61mmol) was dissolved in acetonitrile (300mL), then cesium carbonate (50.37g, 154.61mmol) and deuteroiodomethane (22.38g, 157.70mmol) were added, and reacted at 20°C for 24 hours .
  • the reaction system was filtered, the filter cake was washed with acetonitrile (300 mL), and the filtrate was concentrated under reduced pressure.
  • the crude product was diluted with 300 mL of water, extracted with ethyl acetate (3x300 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, eluent ethyl acetate/petroleum ether, acetic acid Ethyl ester ratio: 0-20%) was purified to obtain compound M4.
  • Compound 1 was separated by SFC (chromatographic column: DAICEL CHIRAL PAKIC (250mm*30mm, 10 ⁇ m); mobile phase: phase A (CO 2 ), phase B (ethanol containing 0.1% ammonia water); gradient (B%): 50% -50%) yielded Compound 1A and Compound 1B.
  • SFC chromatographic column: DAICEL CHIRAL PAKIC (250mm*30mm, 10 ⁇ m); mobile phase: phase A (CO 2 ), phase B (ethanol containing 0.1% ammonia water); gradient (B%): 50% -50%) yielded Compound 1A and Compound 1B.
  • Compound 2 was separated by SFC (chromatographic column: DAICEL CHIRALPAK IC (250mm*30mm, 10 ⁇ m); mobile phase: [A: CO 2 , B: isopropanol containing 0.1% ammonia water]; B%: 65%-65% ) to obtain compound 2A and compound 2B.
  • SFC chromatographic column: DAICEL CHIRALPAK IC (250mm*30mm, 10 ⁇ m)
  • mobile phase [A: CO 2 , B: isopropanol containing 0.1% ammonia water]; B%: 65%-65%
  • the aqueous phase was extracted with ethyl acetate (10 mL*2).
  • the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • McCoy's 5A medium penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • HCT116 WT cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • HCT116 MTAP KO cells were constructed from Horizon Corporation. Envision Multilabel Analyzer (PerkinElmer).
  • HCT116 WT/MTAP KO cells were planted in ultra-low adsorption 96-well U-shaped plates, 80 ⁇ L of cell suspension per well, which contained 1000 HCT116 cells. Cell plates were cultured overnight in a carbon dioxide incubator.
  • the compound to be tested was diluted 5 times to 8 concentrations with a row gun, that is, diluted from 2mM to 25.6nM, and a double-well experiment was set up.
  • Compound concentrations transferred to the cell plate ranged from 10 [mu]M to 0.128 nM.
  • Cell plates were cultured in a carbon dioxide incubator for 10 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • Example-Min Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of IC50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on the proliferation of HCT116WT/MTAP KO cells.
  • the compound of the present invention has obvious inhibitory activity on MTAP-deficient HCT116 cells, and has weak inhibitory effect on wild-type tumor cells, showing good selectivity.
  • Buffer preparation take 10 ml as an example.
  • 10mM MTA Add 10mg MTA into 3296 ⁇ l DMSO, dissolve, aliquot and store at -80°C. On the day of the experiment, configure it for immediate use. See Table 2 and Table 3 for the configuration scheme.
  • Element storage concentration Final concentration Amount added Bicine, pH 7.6 1000mM 20mM 200 ⁇ L NaCl 5000mM 25mM 50 ⁇ L DTT 1000mM 2mM 20 ⁇ L Pig skin collagen (gelatin) 0.08% 0.005% 625 ⁇ L MTA 10mM 2.6 ⁇ M 2.6 ⁇ L Tween-20 1% 0.01% 100 ⁇ L deionization - - 9002.4 ⁇ L
  • Element storage concentration Final concentration Amount added Bicine, pH 7.6 1000mM 20mM 200 ⁇ L NaCl 5000mM 25mM 50 ⁇ L DTT 1000mM 2mM 20 ⁇ L Pig skin collagen (gelatin) 0.08% 0.005% 625 ⁇ L Tween-20 1% 0.01% 100 ⁇ L deionized - - 9005.0 ⁇ L
  • the compound was dissolved in DMSO to obtain a mother solution of the compound with a concentration of 10 mM. Perform serial dilution in the compound dilution plate to obtain four compound wells, the concentrations of which are: 1 mM, 37.037 ⁇ M, 1.3717 ⁇ M, 0.0508 ⁇ M. Transfer these four concentrations of compounds into a compound transfer plate in a transfer volume of 8 ⁇ L for each concentration. Additional DMSO was added to the empty wells on the compound transfer plate for later use. Use the serial dilution function of the micro liquid transfer device Echo550 to transfer the liquid to the experimental plate, and the experimental plate is obtained after the liquid transfer is completed.
  • a mixed solution of the enzyme solution and the substrate was prepared using a buffer, the concentration of PRMT5 was 7.6 nM, the concentration of polypeptide H4 (1-21) was 0.32 ⁇ M, and the concentration of SAM was 2.6 ⁇ M.
  • This experiment adopts PE company's time-resolved fluorescence resonance energy transfer technology ( Ultra) for detection.
  • Ultra time-resolved fluorescence resonance energy transfer technology
  • two antibodies were added, of which Super europium anti-methyl histone H4 arginine 3 (H4R3me) antibody as an energy donor can specifically bind to the methylation site on polypeptide H4 (1-21), while Ulight as an energy acceptor can bind to polypeptide H4 ( 1-21) specifically binds to the biotin tag carried on it. If excited by a laser with a certain wavelength (the wavelength of the excitation light in this experiment is 340nm), the energy donor can emit emission light with a wavelength of 615nm.
  • energy transfer can occur between the energy donor and the energy acceptor, so that the energy acceptor emits emission light with a wavelength of 665nm.
  • Use LANCE buffer to prepare antibody mixture solution The concentration of Ultra Europium-anti-methyl-Histone H4 Arginine 3 (H4R3me) Antibody is 4nM, and the concentration of Ulight is 53.3nM.
  • the detection solution was added to the positive control, negative control and compound wells of the experimental plate at a volume of 10 ⁇ L per well using an electric multichannel pipette, centrifuged and incubated at room temperature for one hour, and read using a plate reader Envision 2104.
  • Use the interpolation method to calculate the inhibition rate of the compound use the four-parameter Logis equation curve and XLfit software to make the compound inhibition curve and calculate the relevant parameters, including the minimum inhibition rate, maximum inhibition rate and IC 50 .
  • the compound of the present invention has significant inhibitory effect on PRMT5 ⁇ MTA complex.
  • Liver microsomes Human and animal microsomes were purchased from Corning or Xenotech and stored in a -80°C refrigerator;
  • NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate
  • Control compounds testosterone, diclofenac, propafenone.
  • T60 incubation plate Prepare two 96-well incubation plates, named T60 incubation plate and NCF60 incubation plate respectively.
  • microsomal working solution hepatic microsomal protein concentration: 0.56 mg/mL
  • stop solution acetonitrile solution containing 200 ng/mL tolbutamide and 200 ng/mL labetalol
  • CD-1 mice female, 15-30g, 6-9 weeks, Beijing Weitong Lihua
  • the pharmacokinetic characteristics of the rodents after intravenous injection and oral administration of the compound were tested according to the standard protocol.
  • the candidate compound was formulated into a clear solution and administered to mice for a single intravenous injection and a homogeneous suspension was administered to mice for a single oral administration.
  • the dosage for intravenous injection is 3mg/kg, and the vehicle is 5% DMSO/10% Solutol/85% water; the oral dose is 30mg/kg, and the vehicle is 0.5% MC (4000cps)/0.2% Tween 80.
  • the compound of the present invention has good oral absorption bioavailability and plasma exposure in mice, and has good pharmacokinetic properties.
  • the pharmacokinetic characteristics of the compound were tested in dogs after intravenous injection and oral administration according to the standard protocol.
  • the test compound was made into a clear solution and administered to the dog for a single intravenous and a single oral administration.
  • the dosage for intravenous injection is 2mg/kg, and the vehicle is 5% DMSO/10% Solutol/85% water; the oral dose is 10mg/kg, and the vehicle is 5% DMSO/10% Solutol/85% water.
  • the compound of the present invention has good oral absorption bioavailability and plasma exposure in Beagle dogs, and has good pharmacokinetic properties.
  • mice Female BALB/c nude mice were subcutaneously inoculated with human large cell lung cancer LU99 cell line, and randomly divided into groups according to body weight and tumor volume after inoculation, with 6 animals in each group, and the administration was performed as described below.
  • Group 1 (vehicle group): After inoculation, when the tumor volume was 207 ⁇ 9mm, the drug was administered, and the vehicle (5% DMSO/10% Solutol/85% double distilled water) was intragastrically administered once a day according to the dose of 0.1mL/10g .
  • the 2nd group (treatment group): start administration when tumor volume is 207 ⁇ 10mm after inoculation, once a day, dose 12.5mg/kg, administration test compound (compound is dissolved in 5%DMSO/10%Solutol /85% double distilled water).
  • the 3rd group (treatment group): start administration when tumor volume is 207 ⁇ 10mm after inoculation, once a day, dosage 25mg/kg, oral administration test compound (compound is dissolved in 5%DMSO/10%Solutol/ 85% double distilled water).
  • the 4th group (treatment group): start administration when tumor volume is 207 ⁇ 14mm after inoculation, once a day, dose 50mg/kg, administration test compound (compound is dissolved in 5%DMSO/10%Solutol/ 85% double distilled water).
  • mice were weighed twice a week, and the tumor diameter was measured with a vernier caliper, the tumor volume was calculated, and then the tumor growth inhibition rate (TGI) was calculated. Mice were euthanized and sampled on day 29 after experimental dosing. The calculation formula is:
  • Tumor volume 0.5 long diameter ⁇ wide diameter 2 ;
  • TGI(%) [1-(T i -T 0 )/(C i -C 0 )] ⁇ 100%, where T i is the average tumor volume of a certain administration group on a certain day, and T 0 is the administration group The average tumor volume at the beginning of drug administration; C i is the average tumor volume of the control group on a certain day (the same day as T i ), and C 0 is the average tumor volume of the control group at the beginning of drug administration.
  • the compound of the present invention has a significant antitumor effect in the subcutaneous xenograft tumor model of human large cell lung cancer LU99 cells, and the body weight of the mice is maintained well after administration.

Abstract

提供一种吡唑-1(2H)-酞嗪酮类化合物及其应用，作为PRMT5·MTA复合物抑制剂，用于制备治疗相关疾病的药物。

Description

吡唑-1(2H)-酞嗪酮类化合物及其应用
本发明主张如下优先权：
CN202111616977.2，申请日2021年12月27日；
CN202210062133.6，申请日2022年01月19日；
CN202210142771.9，申请日2022年02月16日；
CN202211213451.4，申请日2022年09月29日。
技术领域
本发明涉及一系列吡唑-1(2H)-酞嗪酮类化合物及其应用，具体涉及了式(Ⅲ)所示化合物及其药学上可接受的盐。
背景技术
蛋白质精氨酸酶甲基转移酶5(protein arginase methyltransferase 5,PRMT5)是一种具有临床潜力的表观遗传靶点，它是一种II型精氨酸甲基转移酶，通过对称二甲基化，翻译后修饰组蛋白和其他蛋白质，目前研究表明在多种肿瘤中PRMT5表达水平升高。
代谢酶5-甲硫腺苷磷酸化酶(5'-methylthioadenonine phosphorylase，MTAP)基因与人类染色体9p21上的抑癌基因CDKN2A非常接近，因此在CDKN2A基因缺失的肿瘤中经常伴随MTAP缺失，是肿瘤中突变频率最高的基因之一。MTAP缺失使其底物甲硫腺苷(methylthioadenosine，MTA)积累，MTA能选择性抑制PRMT5甲基转移酶的活性，并使其对进一步的PRMT5抑制更为敏感。PRMT5抑制剂可以成为MTAP缺失或低表达肿瘤患者的一种高度选择性的治疗方法。
近年来，PRMT5作为肿瘤治疗的潜在靶点受到特别关注。目前已有大量PRMT5小分子抑制剂报道，根据化合物是否占据SAM结合位点，可将其分为两类，SAM非竞争性抑制剂和SAM竞争性抑制剂，其中SAM非竞争抑制剂占据底物结合位点与底物竞争，而SAM竞争抑制剂则是占据SAM的结合位点。
对于SAM非竞争性抑制剂，虽然其能够有效地抑制许多细胞株的体外生长，但缺乏肿瘤细胞株选择性，并且它对肿瘤细胞的抑制能力与细胞中MTAP的状态无关，这就有可能导致潜在的临床毒性风险。在MTAP缺乏的细胞中，MTA累积并部分抑制细胞中的PRMT5，使肿瘤细胞对PRMT5抑制剂敏感，因此设计一种小分子协同MTA与PRMT5结合，并特异性结合PRMT5·MTA复合物，从而抑制肿瘤细胞生长，是一种有效提高PRMT5抑制剂选择性的手段。
发明内容
本发明提供了式(Ⅲ)所示化合物或其药学上可接受的盐，
Figure PCTCN2022142362-appb-000001
其中，
R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基、-O-C 3- 5环烷基、-O-C 2-3炔基和C 3-5环烷基分别独立地任选被1、2或3个R a取代；
R 2选自卤素和C 2-3炔基，所述C 2-3炔基任选被1、2或3个卤素取代；
R 3选自CH 3和CD 3；
R 4选自H和卤素；
各R a分别独立地选自卤素和C 1-3烷基，所述C 1-3烷基任选被1、2或3个卤素取代；
且至少满足以下一个条件：
(1)当R 2选自卤素时，R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2- 5烯基、C 2-5炔基和-O-C 2-3炔基任选被1、2或3个R a取代，所述-O-C 3-5环烷基和C 3-5环烷基被1、2或3个R a取代；
(2)当R 2选自卤素时，R 3选自CD 3；
(3)当R 2选自卤素时，R 4选自卤素。
在本发明的一些方案中，上述R a分别独立地选自选自F、Cl、CH 3、CHF 2和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1选自C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基，所述C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1选自
Figure PCTCN2022142362-appb-000002
所述
Figure PCTCN2022142362-appb-000003
Figure PCTCN2022142362-appb-000004
分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1选自
Figure PCTCN2022142362-appb-000005
Figure PCTCN2022142362-appb-000006
其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自F、Cl和
Figure PCTCN2022142362-appb-000007
所述
Figure PCTCN2022142362-appb-000008
任选被1、2或3个卤素取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自Cl、
Figure PCTCN2022142362-appb-000009
其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自Cl，R 1选自
Figure PCTCN2022142362-appb-000010
Figure PCTCN2022142362-appb-000011
其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自Cl，R 3选自CD 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 3选自CD 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自H和F，其他变量如本发明所定义。
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，
Figure PCTCN2022142362-appb-000012
其中，R 2、R 3和R a如本发明所定义。
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，
Figure PCTCN2022142362-appb-000013
其中，R 3和R a如本发明所定义。
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，
Figure PCTCN2022142362-appb-000014
其中，R a如本发明所定义。
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，
Figure PCTCN2022142362-appb-000015
其中，R a如本发明所定义。
本发明还提供了式(Ⅲ)所示化合物或其药学上可接受的盐，
Figure PCTCN2022142362-appb-000016
其中，
R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基、-O-C 3- 5环烷基、-O-C 2-3炔基和C 3-5环烷基分别独立地任选被1、2或3个R a取代；
R 2选自卤素和C 2-3炔基，所述C 2-3炔基任选被1、2或3个卤素取代；
R 3选自CH 3和CD 3；
R 4选自H和卤素；
各R a分别独立地选自卤素和C 1-3烷基；
且至少满足以下一个条件，
1)当R 2选自卤素时，R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基和-O-C 2-3炔基任选被1、2或3个R a取代，所述-O-C 3-5环烷基和C 3-5环烷基被1、2或3个R a取代；或者
2)当R 2选自卤素时，R 3选自CD 3；
3)当R 2选自卤素时，R 4选自卤素。
在本发明的一些方案中，所述R a分别独立地选自选自F、Cl、CH 3、CHF 2和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基，所述C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000017
所述
Figure PCTCN2022142362-appb-000018
Figure PCTCN2022142362-appb-000019
分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000020
Figure PCTCN2022142362-appb-000021
其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自F、Cl和
Figure PCTCN2022142362-appb-000022
所述
Figure PCTCN2022142362-appb-000023
任选被1、2或3个卤素取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自Cl、
Figure PCTCN2022142362-appb-000024
其他变量如本发明所定义。
在本发明的一些方案中，所述R 3选自CD 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 4选自H和F，其他变量如本发明所定义。
本发明还提供了式(Ⅱ)所示化合物或其药学上可接受的盐
Figure PCTCN2022142362-appb-000025
其中，
R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基、-O-C 3- 5环烷基、-O-C 2-3炔基和C 3-5环烷基分别独立地任选被1、2或3个R a取代；
R 2选自卤素和C 2-3炔基，所述C 2-3炔基任选被1、2或3个卤素取代；
R 3选自CH 3和CD 3；
各R a分别独立地选自卤素和C 1-3烷基；
条件是，
1)当R 2选自卤素时，R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基和-O-C 2-3炔基任选被1、2或3个R a取代，所述-O-C 3-5环烷基和C 3-5环烷基被1、2或3个R a取代；或者
2)当R 2选自卤素时，R 3选自CD 3。
在本发明的一些方案中，所述R a分别独立地选自选自F、Cl、CH 3、CHF 2和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基，所述C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000026
所述
Figure PCTCN2022142362-appb-000027
Figure PCTCN2022142362-appb-000028
分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000029
Figure PCTCN2022142362-appb-000030
其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自F、Cl和
Figure PCTCN2022142362-appb-000031
所述
Figure PCTCN2022142362-appb-000032
任选被1、2或3个卤素取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自Cl、
Figure PCTCN2022142362-appb-000033
其他变量如本发明所定义。
本发明还提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2022142362-appb-000034
其中，
R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基、-O-C 3- 5环烷基、-O-C 2-3炔基和C 3-5环烷基分别独立地任选被1、2或3个R a取代；
R 2选自卤素和C 2-3炔基，所述C 2-3炔基任选被1、2或3个卤素取代；
各R a分别独立地选自卤素和C 1-3烷基；
条件是，
当R 2选自卤素时，R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基和-O-C 2-3炔基任选被1、2或3个R a取代，所述-O-C 3-5环烷基和C 3-5环烷基被1、2或3个R a取代。
在本发明的一些方案中，所述R a分别独立地选自选自F、Cl、CH 3、CHF 2和CF 3，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基，所述C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000035
所述
Figure PCTCN2022142362-appb-000036
Figure PCTCN2022142362-appb-000037
分别独立地任选被1、2或3个R a取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 1选自
Figure PCTCN2022142362-appb-000038
Figure PCTCN2022142362-appb-000039
其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自F、Cl和
Figure PCTCN2022142362-appb-000040
所述
Figure PCTCN2022142362-appb-000041
任选被1、2或3个卤素取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R 2选自Cl、
Figure PCTCN2022142362-appb-000042
其他变量如本发明所定义。
本发明还有一些方案由上述变量任意组合而来。
本发明还提供了下式所示化合物或其药学上可接受的盐，
Figure PCTCN2022142362-appb-000043
在本发明的一些方案中，上述化合物或其药学上可接受的盐选自，
Figure PCTCN2022142362-appb-000044
Figure PCTCN2022142362-appb-000045
Figure PCTCN2022142362-appb-000046
Figure PCTCN2022142362-appb-000047
本发明还提供了上述化合物或其药学上可接受的盐在PRMT5抑制剂相关药物中的应用。
本发明还提供了上述化合物或其药学上可接受的盐在PRMT5·MTA复合物抑制剂相关药物中的应用。
本发明还提供了下列合成方法：
方法1
Figure PCTCN2022142362-appb-000048
Figure PCTCN2022142362-appb-000049
方法2
Figure PCTCN2022142362-appb-000050
方法3
Figure PCTCN2022142362-appb-000051
方法4
Figure PCTCN2022142362-appb-000052
方法5
Figure PCTCN2022142362-appb-000053
方法6
Figure PCTCN2022142362-appb-000054
本发明还提供了下列测试方法：
测试方法1：PRMT5酶抑制活性试验
实验目的：测试化合物对PRMT5酶活性的抑制作用
实验材料：PRMT5酶
实验操作：使用LABCYTE Echo 550将测试化合物以一定浓度加入白色透明底384孔板中，加入PRMT5，设置溶媒对照(加入DMSO，不含化合物)和空白对照(加入DMSO，不含PRMT5)。在25℃孵育30分钟，然后加入底物，在25℃反应90分钟。90分钟后采用PerkinElmer LANCE Ultra TR-FRET标准方法，将检测试剂加入384孔板中，使其与酶-底物反应产物相结合，在25℃下反应1小时后，在PerkinElmer EnVision 2105 Multimode Plate Reader读板器上检测信号。原始数据用下列公式来计算检测化合物的抑制率：
Figure PCTCN2022142362-appb-000055
将抑制率分别导入Excel中计算不同浓度化合物的抑制率，然后用XLfit软件作抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率及IC 50。
测试方法2：小鼠药代动力学评价
实验目的：以雄性C57BL/6小鼠为受试动物，应用LC/MS/MS法测定化合物经静脉、口服给药后不同时刻血浆药物浓度。研究化合物在小鼠体内的药代动力学行为，评价其药动学特征。
实验操作：称取适量样品，用无菌生理盐水配成澄清溶液。取健康雄性C57BL/6小鼠2只，购买自北京维通利华实验动物有限公司，正常饮食。动物给药后，在0.083、0.25、0.5、1、2、4、8、12及24小时分别采血25μL，置于预先加有EDTA-K2的商品化抗凝管中。将试管离心10分钟分离血浆，并于-60℃保存。用LC/MS/MS法测定血浆样品中相应化合物的含量。
技术效果
本发明化合物对PRMT5具有优异的结合作用，可有效抑制PRMT5，对PRMT5·MTA复合物具有显著的抑制作用；对MTAP缺失的肿瘤细胞具有明显的抑制活性，对野生型肿瘤细胞抑制作用较弱，表现出良好的选择性；具有良好的肝微粒体稳定性和良好的体内药代动力学性质，且种属差异小；在人大细胞肺癌LU99皮下异种移植肿瘤模型上抑瘤效果显著，且给药后小鼠体重维持良好。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠 的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，用楔形实线键
Figure PCTCN2022142362-appb-000056
和楔形虚线键
Figure PCTCN2022142362-appb-000057
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2022142362-appb-000058
和直形虚线键
Figure PCTCN2022142362-appb-000059
表示立体中心的相对构型，用波浪线
Figure PCTCN2022142362-appb-000060
表示楔形实线键
Figure PCTCN2022142362-appb-000061
或楔形虚线键
Figure PCTCN2022142362-appb-000062
或用波浪线
Figure PCTCN2022142362-appb-000063
表示直形实线键
Figure PCTCN2022142362-appb-000064
或直形虚线键
Figure PCTCN2022142362-appb-000065
本发明的某些化合物可以以阻转异构体存在，其是构象异构体，当由于与分子的其它部分的空间相互作用而阻止或大大减缓绕分子中单键的旋转时出现。本发明公开的化合物包括所有的阻转异构体，可以是纯的单独的阻转异构体、或者是富含其中一种阻转异构体、或者是各自的非特异性混合物。如果围绕单键的旋转势能足够高，并且构象之间的相互转化足够慢，则可以允许分离异构体。例如，
Figure PCTCN2022142362-appb-000066
)与
Figure PCTCN2022142362-appb-000067
(或
Figure PCTCN2022142362-appb-000068
)为一对阻转异构体，其中，苯基上的
Figure PCTCN2022142362-appb-000069
表示该侧立体朝向为向外，
Figure PCTCN2022142362-appb-000070
Figure PCTCN2022142362-appb-000071
表示该侧立体朝向为向内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2022142362-appb-000072
直形虚线键
Figure PCTCN2022142362-appb-000073
或波浪线
Figure PCTCN2022142362-appb-000074
表示。例如-OCH 3中的直形 实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2022142362-appb-000075
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2022142362-appb-000076
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；
Figure PCTCN2022142362-appb-000077
表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括
Figure PCTCN2022142362-appb-000078
Figure PCTCN2022142362-appb-000079
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2022142362-appb-000080
仍包括
Figure PCTCN2022142362-appb-000081
这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。
除非另有规定，“C 2-5烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至5个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C 2-5烯基包括C 2-3、C 5、C 4、C 3和C 2烯基等；所述C 2-5烯基可以是一价、二价或者多价。C 2-4烯基的实例包括但不限于乙烯基、丙烯基、丁烯基、丁间二烯基等。
除非另有规定，“C 2-3烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至3个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C 2-3烯基包括C 3和C 2烯基；所述C 2-3烯基可以是一价、二价或者多价。C 2-3烯基的实例包括但不限于乙烯基、丙烯基等。
除非另有规定，“C 2-5炔基”用于表示直链或支链的包含至少一个碳-碳三键的由2至5个碳原子组成的碳氢基团，碳-碳三键可以位于该基团的任何位置上。所述C 2-4炔基包括C 2-3、C 5、C 4、C 3和C 2炔基等。其可以是一价、二价或者多价。C 2-5炔基的实例包括但不限于乙炔基、丙炔基、丁炔基等。
除非另有规定，“C 2-3炔基”用于表示直链或支链的包含至少一个碳-碳三键的由2至3个碳原子组成的碳氢基团，碳-碳三键可以位于该基团的任何位置上。其可以是一价、二价或者多价。所述C 2-3炔基包括C 3和C 2炔基。C 2-3炔基的实例包括但不限于乙炔基、丙炔基等。
除非另有规定，“C 3-5环烷基”表示由3至5个碳原子组成的饱和环状碳氢基团，其为单环体系，所述C 3-5环烷基包括C 3-4和C 4-5环烷基等；其可以是一价、二价或者多价。C 3-5环烷基的实例包括，但不限于，环丙基、环丁基、环戊基等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2022142362-appb-000082
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本发明的缩略词：solutol表示聚乙二醇-15羟基硬脂酸酯；MC(4000cps)表示甲基纤维素(粘度4000)；tolbutamide表示甲苯磺丁尿；labetalol表示拉贝洛尔；
本发明所使用的溶剂可经市售获得。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2022142362-appb-000083
软件命名，市售化合物采用供应商目录名称。
附图说明
图1.化合物A与PRMT5结合模式预测图。
图2.化合物B(构象1)与PRMT5结合模式预测图。
图3.化合物B(构象2)与PRMT5结合模式预测图。
图4.化合物B(构象3)与PRMT5结合模式预测图。
图5.化合物B(构象4)与PRMT5结合模式预测图。
图6.化合物C(构象1)与PRMT5结合模式预测图。
图7.化合物C(构象2)与PRMT5结合模式预测图。
图8.化合物C(构象3)与PRMT5结合模式预测图。
图9.化合物C(构象4)与PRMT5结合模式预测图。
图10.化合物D与PRMT5结合模式预测图。
图11.化合物F与PRMT5结合模式预测图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
计算例1
Figure PCTCN2022142362-appb-000084
分子对接过程是通过使用Maestro(
Figure PCTCN2022142362-appb-000085
版本2021-2)中的Induced Fit Docking [1-2]默认选项进行的。选取PDB数据库中PRMT5的晶体结构(PDB ID:5FA5)作为对接模板。为了准备蛋白质，使用Maestro [2]的蛋白质准备向导模块添加氢原子，并使用OPLS4力场进行能量最小化。对于配体的准备，使用LigPrep [3]生成了分子的三维结构，并使用OPLS4力场进行能量最小化。以5FA5的Trp579作为中心，自动生成盒子，在分子对接过程中放置实例化合物。分析蛋白质与实例化合物的相互作用，然后根据计算得到的IFD score以及结合模式选择并保存了合理对接构象。化合物A与PRMT5结合模式预测图如图1所示，化合物B与PRMT5结合模式预测图如图2～5所示，化合物C与PRMT5结合模式预测图如图6～9所示，化合物D与PRMT5结合模式预测图如图10所示，化合物F与PRMT5结合模式预测图如图11所示。
[1]Induced Fit Docking protocol；Glide,
Figure PCTCN2022142362-appb-000086
LLC,New York,NY,2021.
[2]Prime,
Figure PCTCN2022142362-appb-000087
LLC,New York,NY,2021.
[3]Maestro,
Figure PCTCN2022142362-appb-000088
LLC,New York,NY,2021.
[4]LigPrep,
Figure PCTCN2022142362-appb-000089
LLC,New York,NY,2021.
结论：本发明化合物与PRMT5具有优异的结合作用。
参考例1：化合物M1
Figure PCTCN2022142362-appb-000090
步骤1
将化合物M1-1(4.2g,48.90mmol)和化合物M1-2(9.99g,48.90mmol)溶于无水二氯甲烷(80mL)中，加入4.2g
Figure PCTCN2022142362-appb-000091
分子筛，氮气置换三次，25℃下搅拌16小时。反应液过滤，滤饼用30mL二氯甲烷洗涤。滤液减压浓缩，得到化合物M1-3，无需纯化直接投下一步。 1H NMR(400MHz,CDCl 3)δ：6.62-6.78(m,1H),5.57(s,2H),5.41-5.50(m,1H),3.21(s,6H),2.98(s,6H),1.81-1.89(m,3H)。
步骤2
在预先干燥的三口瓶中，加入无水二氯甲烷(120mL)，氮气置换三次，加入二乙基锌(1M,140.50mL)，冷却至-65℃，滴加二碘甲烷(56.45g,210.75mmol)，搅拌20分钟。-65℃下滴加化合物M1-3(11.9g,46.83mmol)的无水二氯甲烷(120mL)溶液。滴加完毕，-65℃下搅拌2小时。-65℃下向反应液中缓慢滴加150mL饱和氯化铵水溶液，缓慢升至室温。分液，水相用二氯甲烷萃取(30mL*3)。合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物M1-4，无需纯化直接投下一步。 1H NMR(400MHz,CDCl 3)δ:5.49(s,2H),3.17(s,6H),2.97(s,6H),0.95-1.12(m,4H),0.68-0.78(m,1H),0.38-0.46(m,1H),0.46-0.34(m,1H)。
步骤3
将化合物M1-4(4.95g,18.46mmol)溶于甲基叔丁基醚(75mL)和水(75mL)中，加入四水合高硼酸钠(28.41g,184.62mmol)，25℃下搅拌12小时。分液，水相用甲基叔丁基醚萃取(15mL*3)。合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物M1，无需纯化直接用于下一步。 1H NMR(400MHz,CDCl 3)δ:3.32-3.43(m,1H),2.19(br s,1H),1.08-1.19(m,4H),0.83-0.96(m,1H),0.48(q,J＝5.6Hz,1H)。
参考例2：化合物M2A和M2B
Figure PCTCN2022142362-appb-000092
氮气保护，将化合物M2-1(30g,154.61mmol)溶于乙腈(300mL)，然后加入碳酸铯(50.37g,154.61mmol)和氘代碘甲烷(22.38g,157.70mmol)，20℃反应24小时。反应体系过滤，滤饼使用乙腈(300mL)清洗，滤液减压浓缩。然后加入甲基叔丁基醚(400mL)，过滤，滤饼使用甲基叔丁基醚(300mL)洗涤，滤液减压浓缩，粗品经SFC分离纯化(色谱柱：DAICEL CHIRALCEL OD(250mm*50mm,10μm)；流动相：A相(CO 2)，B相(异丙醇，含0.1％氨水)；梯度：B％＝11％-11％)得到化合物M2A。 1H NMR(400MHz,CDCl 3)δ：7.47(d,J＝1.6Hz,1H),6.70(d,J＝1.6Hz,1H),1.33(s,12H)。
SFC分析方法：色谱柱:Chiralcel OD-3,150×4.6mm I.D.,3μm；流动相：A相(CO 2)，B相(含0.1％异丙胺的乙醇)；梯度：B％＝10％-50％)化合物M2A的保留时间为0.895min，其异构体M2B的保留时间为1.398min。
参考例3：化合物M3
Figure PCTCN2022142362-appb-000093
步骤1
将化合物M3-1(50g,234.71mmol)溶于N,N-二甲基甲酰胺二甲基缩醛(180.12g,1.51mol)，加入叔丁醇钾(2.63g,23.47mmol)，氮气置换三次，加热至110℃，搅拌20小时。反应液减压浓缩。粗品加入100mL石油醚，16℃搅拌0.5小时。过滤，收集固体，加入200mL乙酸乙酯，加热至80℃，搅拌5小时。过滤，收集固体得到化合物M3-2。 1H NMR(400MHz,DMSO-d6)δ：7.98(d,J＝0.8Hz,1H),7.60(d,J＝8.4Hz,1H),7.29(dd,J＝8.4,1.6Hz,1H),7.01(s,1H),3.11(s,6H)。
步骤2
将化合物M3-2(29.1g,108.54mmol)溶于无水乙醇(450mL)，加入98％水合肼(11.85g,236.71mmol)，氮气置换三次，16℃搅拌0.5小时，然后加热至70℃，搅拌12小时。反应液冷却至室温，过滤，滤饼用30mL乙醇洗涤，收集滤饼，减压浓缩除去残留溶剂，得到化合物M3-3。 1H NMR(400MHz,DMSO-d6)δ：12.66(s,1H),8.34(d,J＝1.6Hz,1H),8.13(d,J＝8.4Hz,1H),7.93-8.04(m,1H),3.61(s,2H),2.19(s,6H)。
步骤3
将化合物M3-3(20.7g,73.37mmol)溶于无水四氢呋喃(300mL)，氮气置换三次。冷却至0℃，滴加氯甲酸异丁酯(12.02g,88.04mmol)。加料完毕，加热至25℃，搅拌12小时。反应液冷却至0℃，滴加300mL0.5M的盐酸水溶液，搅拌0.5小时。反应液过滤，滤饼用四氢呋喃洗涤(30mL*3)。收集固体，减压干燥除去溶剂，得到化合物M3-4。 1H NMR(400MHz,DMSO-d6)δ：12.92(s,1H),7.90-8.52(m,3H),5.07(s,2H)。
步骤4
将化合物M3-4(15.6g,57.04mmol)溶于N，N-二甲基甲酰胺(170mL)，加入邻苯二甲酰亚胺钾盐(11.62g,62.74mmol)，氮气置换三次，加热至25℃，搅拌4小时。向反应液中滴加200mL 0.5M的稀盐酸水溶液，过滤，滤饼依次用100mL饱和碳酸氢钠水溶液和100mL水洗涤。滤饼再加入50mL乙醇，70℃搅拌1小时。过滤，收集固体，固体减压干燥得到化合物M3-5。 1H NMR(400MHz,DMSO-d6)δ：12.60(s,1H),8.43(d,J＝1.6Hz,1H),8.03-8.23(m,2H),7.83-8.00(m,4H),5.19(s,2H)。
步骤5
将化合物M3-5(2.5g,6.51mmol),双联嚬哪醇硼酸酯(2.48g,9.76mmol),醋酸钾(1.92g,19.52mmol)溶于无水二氧六环(25mL)，氮气置换三次，加入1,1-双(二苯基膦)二茂铁氯化钯(476.14mg,650.73μmol)，氮气置换三次，加热至80℃，搅拌2小时。反应液减压浓缩。粗品用20mL乙腈搅拌0.5小时，过滤，收集滤饼得到化合物M3。 1H NMR(400MHz,DMSO-d6)δ：12.40(s,1H),8.01-8.35(m,3H),7.84-7.98(m, 4H),5.17(s,2H),1.24(s,12H)。
参考例4：化合物M4
Figure PCTCN2022142362-appb-000094
将化合物1-2(20g,79.23mmol)，化合物1-8(32.97g,158.45mmol)，碳酸钠(25.19g,237.68mmol)和[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(2.58g,3.96mmol)溶于600毫升N,N-二甲基甲酰胺中，氮气置换3次，使混合物在80℃下搅拌48小时。反应完成后，将反应液浓缩。粗品用300毫升水稀释，用乙酸乙酯(3x300mL)萃取，有机相无水硫酸钠干燥，过滤，减压浓缩，粗品用快速色谱柱纯化(硅胶，淋洗液乙酸乙酯/石油醚，乙酸乙酯比例：0～20％)纯化得到化合物M4。 1H NMR(400MHz,CDCl 3)δ＝7.67(d,J＝2.0Hz,1H),7.47(dd,J＝5.6,7.6Hz,1H),6.57(d,J＝2.0Hz,1H),3.86(d,J＝1.2Hz,3H)。
参考例5：化合物M5
Figure PCTCN2022142362-appb-000095
第一步
将化合物M3-5(6g,15.62mmol)和水合肼(3.68g,62.47mmol,85％纯度)溶于100毫升乙醇中，使混合物在80℃搅拌15小时。反应完成后，将反应液浓缩，得到化合物M5-1。MS-ESI计算值[M+1] +254.0，256.0，实测值253.8，255.8。
第二步
将化合物M5-1(3.9g,15.35mmol)，Boc酸酐(6.70g,30.70mmol)和三乙胺(4.66g,46.05mmol)溶于100毫升二氯甲烷中，使混合物在25℃下搅拌24小时。反应完成后，过滤，收集滤饼，干燥，得到化合物M5-2。 1H NMR(400MHz,DMSO-d 6)δ＝12.67(br s,1H),8.27(s,1H),8.17(d,J＝8.3Hz,1H),8.02(d,J＝8.5Hz,1H),4.41(s,2H),1.41(s,9H)。
第三步
将化合物M5-2(4.1g,11.58mmol)，双联嚬哪醇硼酸酯(4.41g,17.36mmol)，1,1-双(二苯基膦)二茂铁氯化钯(846.98mg,1.16mmol)和乙酸钾(3.41g,34.73mmol)加入到100毫升无水1,4-二氧六环中，氮气置换3次，使混合物在100℃下搅拌15小时。反应完成后，冷却至室温，放置2小时，过滤，将滤液浓缩，加入20毫升石油醚/乙酸乙酯(体积比10:1)的混合溶剂，在室温下打浆搅拌1小时，过滤，收集滤饼得到化合物M5。MS-ESI计算值[M+1] +402.2，实测值402.2。
实施例1
Figure PCTCN2022142362-appb-000096
步骤1
向预先干燥的单口瓶中加入化合物1(5g,28.81mmol)，N-溴代丁二酰亚胺(10.26g,57.62mmol)，对甲苯磺酸(2.48g,14.41mmol)，1,2-二氯乙烷(50mL)，醋酸钯(646.82mg,2.88mmol)，充分氮气置换后，75℃搅拌20小时。将反应液过滤，滤饼使用二氯甲烷(20mL*2)洗涤，滤液减压浓缩，粗品用硅胶柱分离纯化(乙酸乙酯/石油醚，乙酸乙酯比例为0～10％)，得到化合物1-2。 1H NMR(400MHz,CDCl 3)δppm 7.28(d,J＝5.50Hz,1H)。
步骤2
将化合物1-7(500mg,6.93mmol)溶于无水四氢呋喃(50mL)，氮气置换三次，冷却至0℃，加入钠氢(416.06mg,10.40mmol，60％纯度)，搅拌10分钟。0℃加入化合物1-2(1.75g,6.93mmol)，缓慢升至16℃，搅拌2小时。反应液倒入100mL饱和氯化铵水溶液中，乙酸乙酯萃取(10mL*3)。合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝100:1至10:1)，得化合物1-3。
步骤3
将化合物1-3(1.2g,3.94mmol)，化合物1-8(2.46g,11.82mmol)和碳酸钠(1.67g,15.76mmol)溶于二氧六环(20mL)和水(5mL)中，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(256.81mg,394.03μmol)，升至80℃，搅拌16小时。反应倒入100mL饱和食盐水中，乙酸乙酯萃取(30mL*3)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)得化合物1-4。 1H NMR(400MHz,CDCl 3)δppm 7.61(d,J＝2.0Hz,1H),7.42(d,J＝6.0Hz,1H),6.50(d,J＝2.0Hz,1H),3.81(d,J＝1.2Hz,3H),1.63(s,3H),1.09-1.16(m,2H),0.82-0.90(m,2H)。
步骤4
将化合物1-4(760mg,2.49mmol)溶于乙腈(40mL)，氮气置换三次，加入N-溴代丁二酰亚胺(884.87mg,4.97mmol)，升至40℃，搅拌2小时。减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)，得到化合物1-5。 1H NMR(400MHz,CDCl 3)δppm 7.61(s,1H),7.48(d,J＝6.0Hz,1H),3.80(s,3H),1.65(s,3H),1.10-1.19(m,2H),0.83-0.94(m,2H)。
步骤5
将化合物1-5(400mg,1.04mmol)，化合物M3(538.18mg,1.25mmol)和无水磷酸钾(662.24mg,3.12mmol,3eq)溶于二氧六环(6mL)和水(1.5mL)，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(67.78mg,104.00μmol)，升至80℃，搅拌12小时。反应倒入30mL饱和食盐水中，乙酸乙酯萃取(10mL*3)。合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩。粗品经硅胶柱纯化(二氯甲烷：甲醇＝100:1至30:1)，得到化合物1-6。
步骤6
将化合物1-6(330mg,541.87μmol)溶于无水乙醇(7mL)，加入98％水合肼(310mg,6.19mmol)，升至 80℃，搅拌2小时。反应液减压浓缩，粗品加入4mL的DMF，过滤，收集滤液。粗品经prep-HPLC分离(色谱柱:Phenomenex C18 75*30mm*3μm；流动相A:水(0.1％HCl)，流动相B:乙腈；运行梯度:B％:10％-45％)得到化合物1。
步骤7
化合物1再经SFC分离(色谱柱:DAICEL CHIRAL PAKIC(250mm*30mm,10μm)；流动相:A相(CO 2)，B相(含0.1％氨水的乙醇)；梯度(B％):50％-50％)得到化合物1A和化合物1B。
化合物1A：SFC分析方法：色谱柱：Chiralpak IC-3,150×4.6mm I.D.,3μm；流动相：[A(CO 2)和B(异丙醇，含0.1％二乙胺)]；梯度：B％＝50％，保留时间为4.221min，ee＝98.74％； 1HNMR(400MHz,CD 3OD)δppm 8.27(d,J＝8.53Hz,1H),7.76-7.82(m,2H),7.61(d,J＝8.03Hz,1H),7.25(s,1H),4.05-4.18(m,2H),3.85(s,3H),1.64(s,3H),1.07-1.13(m,2H),0.89-0.94(m,2H)。MS m/z:479.0[M+H] +。
化合物1B：SFC分析方法：色谱柱：Chiralpak IC-3,150×4.6mm I.D.,3μm；流动相：流动相：[A(CO 2)和B(异丙醇，含0.1％二乙胺)]；梯度：B％＝50％，保留时间为6.128min，ee＝99.01％； 1HNMR(400MHz,CD 3OD)δppm 8.24-8.31(m,1H),7.74-7.84(m,2H),7.61(br d,J＝9.79Hz,1H),7.17-7.29(m,1H),4.03-4.17(m,2H),3.85(s,3H),1.64(s,3H),1.06-1.13(m,2H),0.88-0.94(m,2H)。MS m/z:479.0[M+H] +。
实施例2
Figure PCTCN2022142362-appb-000097
步骤1
向预先干燥的三口瓶中加入化合物M1(270mg,3.74mmol)，四氢呋喃(4mL)，0℃加入氢化钠(299.53mg,7.49mmol，60％纯度)，0℃搅拌10分钟，加入化合物1-2(945.28mg,3.74mmol)的四氢呋喃(2mL)溶液，20℃搅拌2小时。将反应水溶液(10mL)淬灭，加入饱和食盐水(10mL)，乙酸乙酯(10mL*3)萃取，收集合并有机相加入无水硫酸钠干燥过滤，滤液减压浓缩，粗品用硅胶柱分离纯化(乙酸乙酯：石油醚＝1：10)得到化合物2-1。
步骤2
向预先干燥的单口瓶中加入化合物2-1(2.1g,6.90mmol)，化合物1-8(2.87g,13.80mmol)，1,4-二氧六环 (40mL)，水(10mL)，磷酸钾(4.39g,20.70mmol)，充分氮气置换加入1,1-二(叔丁基膦)二茂铁氯化钯(539.30mg,828.00μmol)，80℃搅拌20小时。将反应液减压浓缩，加入饱和食盐水(15mL)，乙酸乙酯(20mL*3)萃取，收集合并有机相加入无水硫酸钠干燥过滤，滤液减压浓缩，粗品用硅胶柱分离纯化(乙酸乙酯：石油醚＝1：10)得到化合物2-2。MS m/z:305.9[M+H] +。
步骤3
向预先干燥的单口瓶中加入化合物2-2(320mg,1.05mmol)，N-溴代丁二酰亚胺(372.58mg,2.09mmol)，乙腈(5mL)，40℃搅拌2小时。减压浓缩。粗品用硅胶柱分离纯化(乙酸乙酯：石油醚＝1：10)得到化合物2-3。MS m/z:383.9[M+H] +,385.9[M+H] +。
步骤4
向预先干燥的单口瓶中加入化合物2-3(500mg,1.30mmol)，化合物M3(689.54mg,1.60mmol)，磷酸钾(551.87mg,2.60mmol)，1,4-二氧六环(10mL)，水(2.5mL)充分氮气置换后加入1,1-二(叔丁基膦)二茂铁氯化钯(101.67mg,155.99μmol)，85℃搅拌3小时，加入饱和食盐水(50mL)，乙酸乙酯(15mL*3)萃取，收集合并有机相减压浓缩。粗品用硅胶柱分离纯化(乙酸乙酯：石油醚＝1：10)得到化合物2-4。MS m/z:609.1[M+H] +。
步骤5
向预先干燥的单口瓶中加入化合物2-4(100mg,164.20μmol)，乙醇(2mL)，98％水合肼(16.78mg,328.40μmol)，80℃搅拌2小时。将反应液减压浓缩，先经prep-HPLC制备分离(色谱柱:Phenomenex luna C18 80*40mm*3μm；流动相:[水(盐酸)-乙腈]；梯度(乙腈％):1％-35％)得到化合物2。
步骤6
化合物2再经SFC分离(色谱柱:DAICEL CHIRALPAK IC(250mm*30mm,10μm)；流动相:[A:CO 2,B:含0.1％氨水的异丙醇]；B％:65％-65％)得到化合物2A和化合物2B。
化合物2A：SFC分析方法：色谱柱：Chiralpak IC-3,50×4.6mm I.D.,3μm；流动相:[A:CO 2,B:含0.1异丙胺的异丙醇)],A:B＝50:50，保留时间为1.352min，ee＝96.56％。 1H NMR(400MHz,CDCl 3)δppm 9.50-10.07(m,1H)8.24(br dd,J＝3.94,2.31Hz,1H)7.83(s,1H)7.56(s,1H)7.40-7.46(m,1H)7.38(br d,J＝5.75Hz,1H)3.96(br d,J＝1.88Hz,2H)3.76(s,3H)3.40-3.49(d,1H)1.14-1.21(m,1H)1.04-1.12(m,3H)0.90-1.02(m,1H)0.71–0.73(m,1H)。MS m/z:479.2[M+H] +。
化合物2B在SFC(色谱柱：Chiralpak IC-3,50×4.6mm I.D.,3μm；流动相：[A:CO 2,B:含0.1异丙胺的异丙醇,A:B＝50:50，保留时间为2.269min，ee＝97.23％。 1H NMR(400MHz,CDCl 3)δppm 10.07-10.67(s,1H)8.26(d,J＝7.75Hz,1H)7.84(s,1H)7.58(s,1H)7.33-7.49(m,2H)3.98(d,2H)3.76(s,3H)3.45(d,J＝3.00Hz,1H)1.18(m,J＝1.75Hz,1H)1.05-1.14(m,3H)0.85-0.99(m,1H)0.52-0.72(m,1H)。MS m/z:479.2[M+H] +。
实施例3
Figure PCTCN2022142362-appb-000098
步骤1
将化合物1-3(1g,3.28mmol)，化合物M2A(1.73g,8.21mmol)，无水磷酸钾(2.79g,13.13mmol)溶于二氧六环(16mL)和水(4mL)，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(214.01mg,328.36μmol)，氮气置换三次，加热至80℃，搅拌2小时。反应液减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)得到化合物3-1。 1H NMR(400MHz,CDCl 3)δ:7.61(d,J＝2.0Hz,1H),7.42(d,J＝6.0Hz,1H),6.50(d,J＝2.0Hz,1H),1.63(s,3H),1.07-1.19(m,2H),0.81-0.89(m,2H)。
步骤2
将化合物3-1(480mg,1.55mmol),N-溴代丁二酰亚胺(553.40mg,3.11mmol)溶于乙腈(10mL)，氮气置换三次，加热至40℃，搅拌2小时。反应液减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)得到化合物3-2。 1H NMR(400MHz,CDCl 3)δ:7.61(s,1H),7.48(d,J＝6.0Hz,1H),1.65(s,3H),1.09-1.20(m,2H),0.82-0.91(m,2H)。
步骤3
将化合物3-2(500mg,1.29mmol),化合物M3(834.35mg,1.93mmol)和无水磷酸钾(821.37mg,3.87mmol)溶于二氧六环(8mL)和水(2mL)，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(84.06mg,128.98μmol)，加热至80℃，搅拌2小时。反应液减压浓缩。粗品经硅胶柱纯化(二氯甲烷：甲醇＝100:1至30:1)得到化合物3-3。
步骤4
将化合物3-3(600mg,980.35μmol)溶于无水乙醇(12mL)，加入98％水合肼(260mg,4.15mmol)，升至80℃，搅拌2小时。反应液减压浓缩。粗品先经高效液相制备色谱分离纯化(色谱柱:Phenomenex Luna 75*30mm*3μm；流动相A:水(0.1％HCl)，流动相B:乙腈；运行梯度:B％:20％-50％)得到化合物3。
步骤5
化合物3经SFC分离纯化(柱子：DAICEL CHIRALPAK IC(250mm*30mm,10μm)；流动相：A(CO 2)和B(乙腈/异丙醇＝1:1，含0.1％氨水)；梯度：B％＝60％-60％)，含产物的分离洗脱液分别进行后处理：减压浓缩，剩余物加乙腈/水溶解，再冷冻干燥，得到化合物3A以及化合物3B。
化合物3A：SFC分析方法(柱子：Chiralpak IC-3,50×4.6mm I.D.,3μm；流动相：A(CO 2)和B(异丙醇，含0.1％异丙胺)；梯度：B％＝50％)条件下的保留时间为2.221min，ee＝96.70％； 1H NMR(400MHz,CD 3OD)δppm 8.24(d,J＝8.4Hz,1H),8.14(s,1H),7.66-7.82(m,2H),7.52-7.66(m,1H),3.89-4.13(m,2H),1.62(s,3H),1.02-1.15(m,2H),0.79-0.98(m,2H)。MS m/z:482.2[M+H] +。
化合物3B：SFC分析方法(柱子：Chiralpak IC-3,50×4.6mm I.D.,3μm；流动相：A(CO 2)和B(异丙醇，含 0.1％异丙胺)；梯度：B％＝50％)条件下的保留时间为1.312min，ee＝95.56％。 1H NMR(400MHz,CD 3OD)δ:8.22(t,J＝9.2Hz,1H),8.13(d,J＝2.0Hz,1H),7.77(d,J＝6.0Hz,1H),7.70(d,J＝4.4Hz,1H),7.59(t,J＝7.0Hz,1H),3.97(s,2H),1.61(s,3H),0.99-1.17(m,2H),0.81-0.97(m,2H)。MS m/z:482.1[M+H] +。
实施例4
Figure PCTCN2022142362-appb-000099
步骤1
将化合物2-1(1.04g,3.41mmol)，化合物M2A(2.16g,10.24mmol)，无水磷酸钾(2.90g,13.66mmol)溶于二氧六环(20mL)和水(5mL)中，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(222.57mg,341.50μmol)，氮气置换三次，加热至80℃，搅拌12小时。反应液减压浓缩,加入20mL水和10mL乙酸乙酯，过滤，分液。水相用乙酸乙酯萃取(10mL*2)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)得到化合物4-1。 1H NMR(400MHz,CDCl 3)δ:7.61(d,J＝2.0Hz,1H),7.38(d,J＝5.6Hz,1H),6.50(d,J＝2.0Hz,1H),3.50-3.59(m,1H),1.23-1.34(m,1H),1.16-1.21(m,3H),1.01-1.11(m,1H),0.68-0.77(m,1H)。
步骤2
将化合物4-1(440.00mg,1.43mmol)溶于乙腈(2mL)，氮气置换三次，加入N-溴代丁二酰亚胺(507.29mg,2.85mmol)，加热至40℃，搅拌8小时。反应液减压浓缩。粗品经硅胶柱纯化(石油醚:乙酸乙酯＝10:1至3:1)得到化合物4-2。 1H NMR(400MHz,CDCl 3)δ:7.61(s,1H),7.45(d,J＝6.0Hz,1H),3.50-3.60(m,1H),1.25-1.38(m,1H),1.20(d,J＝6.4Hz,3H),1.02-1.14(m,1H),0.68-0.80(m,1H)。
步骤3
将化合物4-2(500mg,1.29mmol)，化合物M3(834.35mg,1.93mmol)和无水磷酸钾(821.37mg,3.87mmol)溶于二氧六环(10mL)和水(2mL)，氮气置换三次，加入[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(84.06mg,128.98μmol)，加热至80℃，搅拌12小时。反应液减压浓缩。粗品经硅胶柱纯化(二氯甲烷：甲醇＝100:1,50:1,30:1)，得到化合物4-3。 1H NMR(400MHz,DMSO-d6)δ:12.44(s,1H),8.33(s,1H),8.12-8.20(m,1H),7.87-8.01(m,5H),7.82-7.87(m,1H),7.52-7.65(m,1H),5.00-5.15(m,2H),3.89-3.98(m,1H),1.14-1.23(m,1H),1.04-1.12(m,3H),0.89-0.96(m,1H),0.68-0.76(m,1H)。
步骤4
将化合物4-3溶于无水乙醇(12mL)，加入水合肼(420mg,8.22mmol)，加热至80℃，搅拌2小时。反应液减压浓缩。粗品经高效液相制备色谱分离纯化(色谱柱:Phenomenex Luna 80*30mm*3um；流动相A:水(0.1％HCl)，流动相B:乙腈；运行梯度:B％:1％-30％)得到化合物4。
步骤5
化合物4经过SFC分离纯化(柱子：DAICEL CHIRALPAK IC(250mm*30mm,10μm)；流动相：A(CO 2)和B(乙醇，含0.1％氨水)；梯度：B％＝60％-60％)得到化合物4A以及化合物4B。
化合物4A：SFC分析(柱子：Chiralpak IC-3,50×4.6mm I.D.,3um；流动相：A(CO 2)和B(EtOH，含0.1％异丙胺)；梯度：B％＝40％)中的保留时间2.056min，ee＝100％。 1H NMR(400MHz,CD 3OD)δ:8.21(d,J＝8.0Hz,1H),8.13(s,1H),7.66-7.79(m,2H),7.59(d,J＝8.0Hz,1H),3.98(s,2H),3.73-3.84(m,1H),1.12-1.32(m,4H),0.90-1.02(m,1H),0.73(J＝6.0Hz,1H)。MS m/z:482.1[M+H] +。
化合物4B：SFC分析(柱子：Chiralpak IC-3,50×4.6mm I.D.,3um；流动相：A(CO 2)和B(EtOH，含0.1％异丙胺)；梯度：B％＝40％)中的保留时间1.479min，ee＝98.83％。 1H NMR(400MHz,CD 3OD)δ:8.23(d,J＝8.4Hz,1H),8.13(s,1H),7.76(d,J＝6.0Hz,1H),7.71(s,1H),7.60(dd,J＝8.4,1.2Hz,1H),3.97(d,J＝3.2Hz,2H),3.73-3.82(m,1H),1.19-1.26(m,1H),1.13-1.17(m,3H),0.92-1.02(m,1H),0.73(q,J＝6.4Hz,1H)。MS m/z:482.1[M+H] +。
实施例5
Figure PCTCN2022142362-appb-000100
步骤1
将2-(甲基磺酰基)乙醇(587.42mg)加入到N,N-二甲基甲酰胺(6mL)中，反应体系氮气保护。0℃下分批加入钠氢(189.23mg,60％纯度)，搅拌0.5小时。0℃下滴加化合物M4(600mg)的N,N-二甲基甲酰胺(6mL)溶液，然后在20℃下搅拌1小时。加入水稀释(30mL)，用乙酸乙酯萃取(10mL×2)。水相用1M盐酸调至pH＝2～3，然后用乙酸乙酯萃取(15mL×3)，有机相合并，用饱和食盐水洗(10mL)，无水硫酸钠干燥，过滤，滤液减压浓缩得化合物5-1。MS m/z:252.0[M+H] +。
步骤2
将化合物5-1(500mg)加入到二氯甲烷(10mL)中，然后加入吡啶(628.67mg)。0℃下加入三氟甲磺酸酐(840.89mg)，反应液在20℃下搅拌1小时。加入水(20mL)，搅拌5分钟，分液。水相用二氯甲烷萃取(10mL)，有机相合并，用饱和食盐水洗(10mL)，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品用快速硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例为0～20％)得化合物5-2。MS m/z:383.9[M+H] +。
步骤3
将化合物5-2(600mg)加入到N,N-二甲基甲酰胺(9mL)中，然后加入三甲基硅乙炔(168.94mg)，二氯双(三苯基膦)钯(II)(109.76mg)，碘化亚铜(14.89mg)和三乙胺(237.34mg)，反应液在20℃下搅拌3小时。加入水(30mL)，加入乙酸乙酯(20mL)，搅拌5分钟，过滤。滤液分液，水相用乙酸乙酯萃取(10mL×2)。有机相合并，用饱和食盐水洗，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品用快速硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例为0～20％)得化合物5-3。MS m/z:332.1[M+H] +。
步骤4
将化合物5-3(170mg)加入到醋酸(3mL)中，然后加入N-碘琥珀酰亚胺(230.51mg)，反应液在20℃下搅拌12小时。反应液减压浓缩，加入饱和碳酸氢钠溶液(10mL)，加入乙酸乙酯(10mL)，搅拌5分钟，分液。水相用乙酸乙酯萃取(5mL)，有机相合并，用饱和食盐水洗(5mL)，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品用快速硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例为0～20％)得化合物5-4。MS m/z:457.9[M+H] +。
步骤5
将化合物5-4(50mg)加入到1,4-二氧六环(2mL)中，加入水(0.2mL)，然后加入化合物M5(65.75mg)，[1,1-双(二叔丁基膦)二茂铁]二氯化钯(II)(3.56mg)和磷酸钾(46.37mg)，氮气保护，反应液在80℃下搅拌12小时。反应液冷却到室温，加入水(5mL)，加入乙酸乙酯(5mL)，搅拌5分钟，过滤。滤液分液，水相用乙酸乙酯萃取(5mL)。有机相合并，用饱和食盐水洗(5mL)，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品用制备薄层色谱(石油醚:乙酸乙酯＝1:1)纯化得化合物5-5。MS m/z:533.2[M+H] +。
步骤6
将化合物5-5(5mg)加入到二氯甲烷(0.6mL)中，然后加入三氟乙酸(0.2mL)，反应液在20℃下搅拌0.5小时。应液减压浓缩得粗品。粗品经制备高效液相色谱纯化(柱子:Welch Xtimate C18 100*40mm*3μm；流动相:[水(三氟乙酸)-乙腈]；梯度(乙腈％):10％-40％)得化合物5的三氟乙酸盐。
MS m/z:433.1[M+H] +。 1H NMR(400MHz,CD 3OD)δ8.28(d,J＝8.03Hz,1H),8.18(s,1H),8.14(d,J＝7.03Hz,1H),7.85(s,1H),7.50(d,J＝10.04Hz,1H),4.51(br d,J＝5.02Hz,2H),4.28(s,1H),3.86(s,3H)。
生物测试数据
实验例1：体外HCT116 WT/MTAP KO细胞增殖实验
实验材料：
McCoy's 5A培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。3D CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。HCT116 WT细胞系购自南京科佰生物科技有限公司。HCT116 MTAP KO细胞构自Horizon公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将HCT116 WT/MTAP KO细胞种于超低吸附96孔U型板中，80μL细胞悬液每孔，其中包含1000个HCT116细胞。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进5倍稀释8个浓度，即从2mM稀释至25.6nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.128nM。细胞板置于二氧化碳培养箱中培养10天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。
向细胞板中加入每100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC 50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表1提供了本发明化合物对HCT116WT/MTAP KO细胞增殖的抑制活性。
表1：本发明化合物体外筛选试验结果
化合物编号 HCT116 MTAP KO IC 50(nM) HCT116 WT IC 50(μM)
1A 8.00 >10
2A 3.99 >10
3A 7.43 4.7
结论：本发明化合物对MTAP缺失的HCT116细胞具有明显的抑制活性，对野生型肿瘤细胞抑制作用较弱，表现出良好的选择性。
实验例2：酶抑制活性试验
实验目的：测试本发明化合物对PRMT5·MTA复合物的抑制作用
实验材料：PRMT5/MEP50，多肽H4(1-21)，
Figure PCTCN2022142362-appb-000101
超铕抗甲基组蛋白H4精氨酸3(H4R3me)抗体，Ulight链霉亲和素，LANCE检测缓冲溶液，SAM(腺苷甲硫氨酸)，猪皮胶原蛋白，MTA(甲硫腺苷)实验方法：
(1)缓冲液配制：以10毫升为例。10mM MTA：将10毫克MTA加入3296微升DMSO中，溶解后分装并置于-80℃保存。实验当天先配现用，配置方案见表2和表3。
表2添加MTA的缓冲液配制方案
成分 储存浓度 终浓度 添加量
Bicine,pH 7.6 1000mM 20mM 200μL
NaCl 5000mM 25mM 50μL
DTT 1000mM 2mM 20μL
猪皮胶原蛋白(gelatin) 0.08％ 0.005％ 625μL
MTA 10mM 2.6μM 2.6μL
Tween-20 1％ 0.01％ 100μL
去离子 - - 9002.4μL
表3不添加MTA的缓冲液配制方案
成分 储存浓度 终浓度 添加量
Bicine,pH 7.6 1000mM 20mM 200μL
NaCl 5000mM 25mM 50μL
DTT 1000mM 2mM 20μL
猪皮胶原蛋白(gelatin) 0.08％ 0.005％ 625μL
Tween-20 1％ 0.01％ 100μL
去离子 - - 9005.0μL
(2)化合物准备
将化合物溶解于DMSO得到化合物母液，浓度为10mM。在化合物稀释板中进行梯度稀释，得到四个化合物孔，浓度依次为：1mM，37.037μM，1.3717μM，0.0508μM。将这四个浓度的化合物转移到化合物转移板中，转移体积为每个浓度8μL。另将DMSO加入至化合物转移板上空的孔中备用。使用微量液体转移器Echo550的连续稀释功能转移液体至实验板中，液体转移完成后就得到实验板。
(3)反应
使用缓冲液配制酶溶液与底物混合溶液，PRMT5浓度为7.6nM，多肽H4(1-21)浓度为0.32μM，SAM浓度为2.6μM。
使用电动多通道移液器以每孔5μL的体积将PRMT5溶液添加至实验板的化合物孔以及阴性对照孔中，在阳性对照孔中添加同等体积的缓冲液。使用离心机以1000转每分钟的转速离心一分钟，随后将实验板置于恒温孵育箱中以25℃孵育30分钟。之后用同样的方法将底物混合溶液添加至实验板的阳性对照，阴性对照以及化合物孔中，离心并以25℃孵育90分钟。
(4)检测和结果计算
原理：本实验采用PE公司的时间分辨荧光共振能量转移技术(
Figure PCTCN2022142362-appb-000102
Ultra)进行检测。反应过程中，当PRMT将底物多肽H4(1-21)甲基化以后加入两种抗体，其中
Figure PCTCN2022142362-appb-000103
超铕抗甲基组蛋白H4精氨酸3(H4R3me)抗体作为能量供体能与多肽H4(1-21)上的甲基化位点特异性结合，而Ulight作为能量受体能与多肽H4(1-21)上携带的生物素标签特异性结合。若使用一定波长的激光(本实验的激发光波长为340nm)激发，能量供体能发射出615nm波长的发射光，同时当能量供体与能量受体的空间距离足够接近时(即两个抗体同时连接在多肽H4(1-21)上)，能量供体与能量受体之间能发生能量转移，使得能量受体发射出665nm波长的发射光。使用读板机对两个发射光进行检测并求出665nm与615nm两种信号的比值，通过作图和计算即可求出待测样品的相关参数。
使用LANCE缓冲液配制抗体混合溶液，
Figure PCTCN2022142362-appb-000104
Ultra Europium-anti-methyl-Histone H4 Arginine 3(H4R3me)Antibody浓度为4nM，Ulight浓度为53.3nM。使用电动多通道移液器以每孔10μL的体积将检测溶液添加至实验板的阳性对照，阴性对照以及化合物孔中，离心并在室温孵育一小时，使用读板机Envision 2104读数。使用内插法计算化合物抑制率，将抑制率使用四参数罗吉斯方程曲线和XLfit软件作出化合物抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率及IC 50。
结论：本发明化合物对PRMT5·MTA复合物具有显著的抑制作用。
实验例3：化合物体外微粒体稳定性实验
实验材料
肝微粒体：人和动物微粒体购买于Corning或Xenotech，储存于-80℃冰箱；
还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，供应商：Chem-impex international，货号：00616；
对照化合物：睾酮，双氯芬酸，普罗帕酮。
实验步骤
2.1工作液的配制
储备液：10mM DMSO溶液
工作浓度配制：100％乙腈稀释到100μM(有机相含量：99％乙腈，1％DMSO)
2.2实验步骤
准备2块96孔孵育板，分别命名为T60孵育板和NCF60孵育板。
在T60孵育板和NCF60孵育板上分别加入445μL微粒体工作液(肝微粒体蛋白浓度为0.56mg/mL)，然后将上述孵育板放置于37℃水浴锅中预孵育大约10分钟。
预孵育结束后，在T60孵育板和NCF60孵育板上分别加入5μL供试品或对照化合物工作液，混匀。在NCF60孵育板上每孔添加50μL磷酸钾盐缓冲液启动反应；在T0终止板中加入180μL的终止液(含200ng/mL tolbutamide(甲苯磺丁尿)和200ng/mL labetalol(拉贝洛尔)的乙腈溶液)和6μL的NADPH再生体系工作液，从T60孵育板中取出54μL样品至T0终止板(T0样品产生)。在T60孵育板上每孔添加44μL NADPH再生体系工作液启动反应。在空白板中只添加54μL微粒体工作液、6uL的NADPH再生体系工 作液和180μL的终止液。因此，在供试品或对照化合物的样品中，化合物、睾酮、双氯芬酸和普罗帕酮的反应终浓度为1μM，肝微粒体的浓度为0.5mg/mL，DMSO和乙腈在反应体系中的终浓度分别为0.01％(v/v)和0.99％(v/v)。
孵育适当时间(如5、15、30、45和60分钟)后，分别在每个终止板的样品孔中加入180μL的终止液(含200ng/mL tolbutamide和200ng/mL labetalol的乙腈溶液)，之后从T60孵育板中取出60μL样品以终止反应。
所有样品板摇匀并在3220×g离心20分钟，然后每孔取80μL上清液稀释到240μL纯水中用于液相色谱串联质谱分析。实验结果见表4。
表4：本发明化合物肝微粒体稳定性试验结果
Figure PCTCN2022142362-appb-000105
结论：本发明化合物在肝微粒体测试中表现出中速代谢。
实验例4：小鼠体内药代动力学实验
实验目的：测试本发明化合物在CD-1小鼠体内的药代动力学性质
实验材料：CD-1小鼠(雌性，15-30g，6-9周，北京维通利华)
实验操作：
以标准方案测试化合物静脉注射及口服给药后啮齿类动物的药代特征，实验中候选化合物配成澄清溶液给予小鼠单次静脉及均一混悬液给予小鼠单次口服给药。静脉注射的剂量为3mg/kg，溶媒为5％DMSO/10％Solutol/85％水；口服剂量为30mg/kg，溶媒为0.5％MC(4000cps)/0.2％吐温80。收集24小时内的全血样品，在4℃下3000g离心15分钟，分离上清得血浆样品，加入20倍体积含内标的乙腈溶液沉淀蛋白，涡旋10分钟后，在4℃下3220g离心15分钟，转移上清到96孔板，在4℃下3220g离心5分钟，取上清进样，以LC-MS/MS分析方法定量分析血药浓度，并计算药代参数。
实验结果见表5：
表5:CD-1小鼠药代动力学测试结果
Figure PCTCN2022142362-appb-000106
结论：本发明的化合物在小鼠体内具有良好的口服吸收生物利用度和血浆暴露量，药代动力学性质良好。
实验例5：比格犬体内药代动力学实验
实验目的：测试本发明化合物在比格犬体内的药代动力学性质
实验材料：比格犬(雄性，≥6月龄，北京玛斯)
实验操作：
以标准方案测试化合物静脉注射及口服给药后犬的药代特征，实验中测试化合物配成澄清溶液给予犬单次静脉及单次口服给药。静脉注射的剂量为2mg/kg，溶媒为5％DMSO/10％Solutol/85％水；口服剂量为10mg/kg，溶媒为5％DMSO/10％Solutol/85％水。收集48小时内的全血样品，在4℃下3000g离心15分钟，分离上清得血浆样品，加入20倍体积含内标的乙腈溶液沉淀蛋白，涡旋10分钟后，在4℃下3220g离心15分钟，转移上清到96孔板，在4℃下3220g离心5分钟，取上清进样，以LC-MS/MS分析方法定量分析血药浓度，并计算药代参数。
实验结果：实验结果见表6。
表6:比格犬药代动力学测试结果
Figure PCTCN2022142362-appb-000107
结论：本发明的化合物在比格犬体内具有良好的口服吸收生物利用度和血浆暴露量，药代动力学性质良好。
实验例6：体内药效测试
实验目的：
在人大细胞肺癌LU99皮下异种移植肿瘤模型上考察本发明化合物的抑瘤效果
实验方法：
在雌性BALB/c裸小鼠皮下接种人大细胞肺癌LU99细胞株，接种后按照体重和肿瘤体积随机分组，每组6只动物，并且按照下列描述进行给药处理。
第1组(溶媒组)：接种后当肿瘤体积为207±9mm 3时开始给药，每天一次按照0.1mL/10g的剂量灌胃溶媒(5％DMSO/10％Solutol/85％双蒸水)。
第2组(治疗组)：接种后当肿瘤体积为207±10mm 3时开始给药，每天一次，剂量12.5mg/kg，灌胃给药待测化合物(化合物溶解于5％DMSO/10％Solutol/85％双蒸水)。
第3组(治疗组)：接种后当肿瘤体积为207±10mm 3时开始给药，每天一次，剂量25mg/kg，灌胃给药待测化合物(化合物溶解于5％DMSO/10％Solutol/85％双蒸水)。
第4组(治疗组)：接种后当肿瘤体积为207±14mm 3时开始给药，每天一次，剂量50mg/kg，灌胃给药待测化合物(化合物溶解于5％DMSO/10％Solutol/85％双蒸水)。
实验分组后，每周二次称量小鼠体重，并用游标卡尺测量肿瘤直径，计算肿瘤体积，再计算肿瘤生长抑制率(TGI)。实验给药后第29天安乐死小鼠并采样。计算公式为：
肿瘤体积＝0.5长径×宽径 2；
TGI(％)＝[1-(T i-T 0)/(C i-C 0)]×100％,其中T i为某一天某给药组的平均肿瘤体积，T 0为此给药组在开始给药时的平均肿瘤体积；C i为某一天(与T i同一天)对照组的平均肿瘤体积，C 0为对照组在给开始药时的平均肿瘤体积。
实验结果：实验结果表7。
表7本发明化合物对人大细胞肺癌LU99皮下异种移植肿瘤模型的抑瘤药效评价
Figure PCTCN2022142362-appb-000108
*：基于给药后第29天肿瘤体积计算
结论：本发明化合物在人大细胞肺癌LU99细胞皮下异种移植肿瘤模型中的抑瘤效果显著，且给药后小鼠体重维持良好。

Claims (15)

  1. 式(Ⅲ)所示化合物或其药学上可接受的盐
    Figure PCTCN2022142362-appb-100001
    其中，
    R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2-5烯基、C 2-5炔基、-O-C 3- 5环烷基、-O-C 2-3炔基和C 3-5环烷基分别独立地任选被1、2或3个R a取代；
    R 2选自卤素和C 2-3炔基，所述C 2-3炔基任选被1、2或3个卤素取代；
    R 3选自CH 3和CD 3；
    R 4选自H和卤素；
    各R a分别独立地选自卤素和C 1-3烷基，所述C 1-3烷基任选被1、2或3个卤素取代；
    且至少满足以下一个条件：
    (1)当R 2选自卤素时，R 1选自C 2-5烯基、C 2-5炔基、-O-C 3-5环烷基、-O-C 2-3炔基和C 3-5环烷基，所述C 2- 5烯基、C 2-5炔基和-O-C 2-3炔基任选被1、2或3个R a取代，所述-O-C 3-5环烷基和C 3-5环烷基被1、2或3个R a取代；
    (2)当R 2选自卤素时，R 3选自CD 3；
    (3)当R 2选自卤素时，R 4选自卤素。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，各R a分别独立地选自选自F、Cl、CH 3、CHF 2和CF 3。
  3. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 1选自C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基，所述C 2-3烯基、C 2-3炔基、-O-环丙基、-O-C 2-3炔基和环丙基分别独立地任选被1、2或3个R a取代；或者，R 1选自
    Figure PCTCN2022142362-appb-100002
    所述
    Figure PCTCN2022142362-appb-100003
    Figure PCTCN2022142362-appb-100004
    分别独立地任选被1、2或3个R a取代。
  4. 根据权利要求3所述化合物或其药学上可接受的盐，其中，R 1选自
    Figure PCTCN2022142362-appb-100005
    Figure PCTCN2022142362-appb-100006
  5. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 2选自F、Cl和
    Figure PCTCN2022142362-appb-100007
    所述
    Figure PCTCN2022142362-appb-100008
    任选被1、2或3个卤素取代；或者，R 2选自Cl、
    Figure PCTCN2022142362-appb-100009
  6. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 2选自Cl，R 1选自
    Figure PCTCN2022142362-appb-100010
    Figure PCTCN2022142362-appb-100011
  7. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 2选自Cl，R 3选自CD 3。
  8. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 4选自H和F。
  9. 根据权利要求1～8任意一项所述化合物或其药学上可接受的盐，其选自，
    Figure PCTCN2022142362-appb-100012
    其中，R 2、R 3和R a如权利要求1～8任意一项所定义。
  10. 根据权利要求9所述化合物或其药学上可接受的盐，其选自，
    Figure PCTCN2022142362-appb-100013
    其中，R 3和R a如权利要求9所定义。
  11. 根据权利要求9所述化合物或其药学上可接受的盐，其选自，
    Figure PCTCN2022142362-appb-100014
    其中，R a如权利要求9所定义。
  12. 根据权利要求11所述化合物或其药学上可接受的盐，其选自，
    Figure PCTCN2022142362-appb-100015
    其中，R a如权利要求11所定义。
  13. 下列所示化合物或其药学上可接受的盐，
    Figure PCTCN2022142362-appb-100016
  14. 根据权利要求13所述化合物或其药学上可接受的盐，其选自，
    Figure PCTCN2022142362-appb-100017
    Figure PCTCN2022142362-appb-100018
    Figure PCTCN2022142362-appb-100019
    Figure PCTCN2022142362-appb-100020
  15. 权利要求1～14任意一项所述化合物或其药学上可接受的盐在PRMT5·MTA复合物抑制剂相关药物中的应用。
PCT/CN2022/142362 2021-12-27 2022-12-27 吡唑-1(2h)-酞嗪酮类化合物及其应用 WO2023125540A1 (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024008176A1 (zh) * 2022-07-07 2024-01-11 西藏海思科制药有限公司 一种可抑制prmt5·mta的杂环化合物及其用途
WO2024022186A1 (zh) * 2022-07-29 2024-02-01 四川科伦博泰生物医药股份有限公司 甲基吡唑化合物、包含其的药物组合物及其制备方法和用途
WO2024027703A1 (zh) * 2022-08-02 2024-02-08 上海艾力斯医药科技股份有限公司 一种prmt5抑制剂、其制备方法及应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101085759A (zh) * 2001-08-07 2007-12-12 辉瑞意大利有限公司 作为激酶抑制剂的氨基－2,3－二氮杂萘酮衍生物,它们的制备方法和含有它们的药物组合物
WO2021050915A1 (en) * 2019-09-12 2021-03-18 Mirati Therapeutics, Inc. Mta-cooperative prmt5 inhibitors
WO2022192745A1 (en) * 2021-03-11 2022-09-15 Mirati Therapeutics, Inc. Mta-cooperative prmt5 inhibitors
WO2022216645A1 (en) * 2021-04-08 2022-10-13 Mirati Therapeutics, Inc. Combination therapies using prmt5 inhibitors for the treatment of cancer
WO2022216648A1 (en) * 2021-04-08 2022-10-13 Mirati Therapeutics, Inc. Combination therapies using prmt5 inhibitors for the treatment of cancer
WO2022256806A1 (en) * 2021-06-02 2022-12-08 Ideaya Biosciences, Inc. Combination therapy comprising a mat2a inhibitor and a type ii prmt inhibitor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101085759A (zh) * 2001-08-07 2007-12-12 辉瑞意大利有限公司 作为激酶抑制剂的氨基－2,3－二氮杂萘酮衍生物,它们的制备方法和含有它们的药物组合物
WO2021050915A1 (en) * 2019-09-12 2021-03-18 Mirati Therapeutics, Inc. Mta-cooperative prmt5 inhibitors
WO2022192745A1 (en) * 2021-03-11 2022-09-15 Mirati Therapeutics, Inc. Mta-cooperative prmt5 inhibitors
WO2022216645A1 (en) * 2021-04-08 2022-10-13 Mirati Therapeutics, Inc. Combination therapies using prmt5 inhibitors for the treatment of cancer
WO2022216648A1 (en) * 2021-04-08 2022-10-13 Mirati Therapeutics, Inc. Combination therapies using prmt5 inhibitors for the treatment of cancer
WO2022256806A1 (en) * 2021-06-02 2022-12-08 Ideaya Biosciences, Inc. Combination therapy comprising a mat2a inhibitor and a type ii prmt inhibitor

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024008176A1 (zh) * 2022-07-07 2024-01-11 西藏海思科制药有限公司 一种可抑制prmt5·mta的杂环化合物及其用途
WO2024022186A1 (zh) * 2022-07-29 2024-02-01 四川科伦博泰生物医药股份有限公司 甲基吡唑化合物、包含其的药物组合物及其制备方法和用途
WO2024027703A1 (zh) * 2022-08-02 2024-02-08 上海艾力斯医药科技股份有限公司 一种prmt5抑制剂、其制备方法及应用

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