WO2020052647A1 - 作为lsd1抑制剂的杂螺环类化合物及其应用 - Google Patents
作为lsd1抑制剂的杂螺环类化合物及其应用 Download PDFInfo
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- WO2020052647A1 WO2020052647A1 PCT/CN2019/105680 CN2019105680W WO2020052647A1 WO 2020052647 A1 WO2020052647 A1 WO 2020052647A1 CN 2019105680 W CN2019105680 W CN 2019105680W WO 2020052647 A1 WO2020052647 A1 WO 2020052647A1
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- ZBFSUWRDHTUQLB-MERJSTESSA-N O=C(C(F)(F)F)N([C@H](C1)[C@@H]1c1ccccc1)C(C1)COC11CCNCC1 Chemical compound O=C(C(F)(F)F)N([C@H](C1)[C@@H]1c1ccccc1)C(C1)COC11CCNCC1 ZBFSUWRDHTUQLB-MERJSTESSA-N 0.000 description 2
- QHWSLFXGFFNBEW-GJGLBJJNSA-N CCN(CC)CC(N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)=O Chemical compound CCN(CC)CC(N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)=O QHWSLFXGFFNBEW-GJGLBJJNSA-N 0.000 description 1
- BFMGELUWRKWGSN-JTGIGXABSA-N CCN(CC)CCC(N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)=O Chemical compound CCN(CC)CCC(N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)=O BFMGELUWRKWGSN-JTGIGXABSA-N 0.000 description 1
- KEYBCCDRYHJYGK-UQJFVLDMSA-N NS(CCN(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)(=O)=O Chemical compound NS(CCN(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1)(=O)=O KEYBCCDRYHJYGK-UQJFVLDMSA-N 0.000 description 1
- OGEPASNIDPEOMC-NRRUETGQSA-N O=C(CN(CCC1)C1=O)N(CC1)CCC1(C1)OCC1N([C@H](C1)[C@@H]1c1ccccc1)C(C(F)(F)F)=O Chemical compound O=C(CN(CCC1)C1=O)N(CC1)CCC1(C1)OCC1N([C@H](C1)[C@@H]1c1ccccc1)C(C(F)(F)F)=O OGEPASNIDPEOMC-NRRUETGQSA-N 0.000 description 1
- YORQGGCOJAZVRV-NRRUETGQSA-N O=C(CN(CCC1)C1=O)N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1 Chemical compound O=C(CN(CCC1)C1=O)N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1 YORQGGCOJAZVRV-NRRUETGQSA-N 0.000 description 1
- AGYCLNMYLZFJPI-GJGLBJJNSA-N O=C(CN1CCCC1)N(CC1)CCC1(C1)OCC1N([C@H](C1)[C@@H]1c1ccccc1)C(C(F)(F)F)=O Chemical compound O=C(CN1CCCC1)N(CC1)CCC1(C1)OCC1N([C@H](C1)[C@@H]1c1ccccc1)C(C(F)(F)F)=O AGYCLNMYLZFJPI-GJGLBJJNSA-N 0.000 description 1
- NTDIZMRCITZDNW-GJGLBJJNSA-N O=C(CN1CCCC1)N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1 Chemical compound O=C(CN1CCCC1)N(CC1)CCC1(C1)OCC1N[C@H](C1)[C@@H]1c1ccccc1 NTDIZMRCITZDNW-GJGLBJJNSA-N 0.000 description 1
- JGPIWNNFLKDTSR-UHFFFAOYSA-N OC(CN(CCC1)C1=O)=O Chemical compound OC(CN(CCC1)C1=O)=O JGPIWNNFLKDTSR-UHFFFAOYSA-N 0.000 description 1
- IPXNXMNCBXHYLQ-UHFFFAOYSA-N OC(CN1CCCC1)=O Chemical compound OC(CN1CCCC1)=O IPXNXMNCBXHYLQ-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
-
- 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
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C211/00—Compounds containing amino groups bound to a carbon skeleton
- C07C211/33—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings
- C07C211/34—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings of a saturated carbon skeleton
- C07C211/35—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of rings other than six-membered aromatic rings of a saturated carbon skeleton containing only non-condensed rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
- C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
- C07C217/74—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with rings other than six-membered aromatic rings being part of the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
- C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
- C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/14—Nitrogen atoms not forming part of a nitro radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
- C07D211/10—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms
- C07D211/14—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with radicals containing only carbon and hydrogen atoms attached to ring carbon atoms with hydrocarbon or substituted hydrocarbon radicals attached to the ring nitrogen atom
Definitions
- the present invention relates to a class of heterospirocyclic compounds that are lysine-specific demethylase 1 (LSD1) inhibitors, and their use in the preparation of a medicament for treating diseases associated with LSD1. Specifically, it relates to a compound represented by formula (I), an isomer thereof, and a pharmaceutically acceptable salt thereof.
- LSD1 lysine-specific demethylase 1
- LSD1 Lysine specific demethylase1
- KDM1A is the first reported histone lysine demethylase, which regulates the methylation of histone lysine Status, extensive participation in transcriptional regulation, affecting many physiological processes such as cell proliferation and differentiation, and pluripotency of embryonic stem cells.
- the LSD1 structure includes three main parts: the N-terminal SWIRM domain, the C-terminal aminooxidase domain (AOL), and the central Tower domain. [Ruchi Anand, Ronen Marmorstein, Journal of Biological Chemistry, 2007, 35425–35429].
- the C-terminal amino oxidase domain includes two active pockets, one is a site for FAD binding, and the other is a site for recognition and binding to substrates [Pete Stavropoulos, Günter Blobel, André Hoelz, Nature Structral & Molecular Biology , 2006, 626-632].
- the function of the SWIRM domain has not been clearly defined. It does not directly participate in the binding of FAD or substrates, but mutation or removal of this region will reduce the activity of LSD1. Therefore, it is speculated that this region may affect the active region by adjusting the conformation. effect. [Yong Chen, Yuting Yang, Feng Wang Wang et al., Biochemistry, 2006, 13956–13961].
- the Tower domain is the binding domain of LSD1 to other protein factors.
- LSD1 combines with different protein factors and acts on different substrates, thus playing different roles in regulating histones and gene expression.
- the combination of LSD1 and CoREST will preferentially act on histone H3K4. By demethylation, the related histone markers will be removed and gene transcription will be inhibited.
- LSD1 After binding to androgen receptor protein, recombinant LSD1 will preferentially act In H3K9, androgen receptor-related gene transcription is activated by demethylation [Ruchi Anand, Ronen Marmorstein, Journal of Biological Chemistry, 2007, 35425–35429; Eric Metzger, Melanie Wissmann, Na Yin Yin et al., Nature, 2005 , 436-439.]. In addition, LSD1 also regulates the methylation status of some non-histone substrates, including the tumor suppressor gene p53 and DNA methyltransferase 1 (DNMT1), etc. [Yi Chao Zhao, Jinlian Ma, Zhiru Wang, Medical Research Reviews, 2015, 1032–1071].
- LSD1 is a FAD-dependent amino oxidase, in which proton transfer is considered to be its most probable oxidation mechanism [Zheng Y C, Yu B, Chen Z S, et al. Epigenomics, 2016, 8, 651-666.].
- the N-CH 3 bond of the substrate is converted into an imine bond through proton transfer.
- This imine ion intermediate undergoes a hydrolysis reaction to generate demethylated amines on one side and formaldehyde on the other.
- LSD1 is abnormally expressed in many different types of tumors. LSD1 is highly expressed in acute myeloid leukemia (AML) subtypes and is an important factor in maintaining the potential of leukemia stem cells (LSCs). LSD1 is highly expressed in a variety of solid tumors such as lung cancer, breast cancer, prostate cancer, liver cancer and pancreatic cancer, which is closely related to the poor prognosis of the tumor. LSD1 inhibits the expression of cadherin and is closely related to tumor invasion and epithelial-mesenchymal transition (EMT) [Hosseini A, Minucci S. Epigenomics, 2017, 9, 1123-1142.].
- EMT epithelial-mesenchymal transition
- the present invention provides a compound of formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof,
- n 0, 1 or 2;
- n 0, 1, or 2, and m and n cannot be 0 at the same time;
- r is 0 or 1;
- q is 0 or 1
- g 0, 1, 2, 3, or 4;
- R is selected from F, Cl, Br, I, OH, NH 2 and C 1-3 alkyl
- the 4-7 membered heterocycloalkyl group comprises 1, 2, 3 or 4 heteroatoms or heteroatoms independently selected from -NH-, -O-, -S- and N;
- the carbon atoms with "*" are chiral carbon atoms and exist in the form of (R) or (S) single enantiomer or are rich in one enantiomer;
- Carbon atoms with "#” are chiral carbon atoms and exist in the form of a single enantiomer (R) or (S) or are rich in one enantiomer.
- the R is selected from the group consisting of F, Cl, Br, I, OH, NH 2 , CH 3 and -CH 2 CH 3 , and other variables are as defined in the present invention.
- the R a is selected from the group consisting of F, Cl, Br, I, OH, NH 2 , CN, COOH, Which said It is optionally substituted by 1, 2 or 3 R, other variables are as defined in the present invention.
- the R a is selected from the group consisting of F, Cl, Br, I, OH, NH 2 , CN, COOH, Other variables are as defined in the present invention.
- R 1 is selected from the group consisting of NH 2 , CN, COOH, -CH 3 , -CH 2 -CH 3 , Other variables are as defined in the present invention.
- the present invention provides a compound of formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof,
- n 0, 1 or 2;
- n 0, 1, or 2, and m and n cannot be 0 at the same time;
- r is 0 or 1;
- q is 0 or 1
- g 0, 1, 2, 3, or 4;
- R a is selected from F, Cl, Br, I, OH, NH 2 , CN, COOH, and C 1-3 alkylamino, wherein the C 1-3 alkylamino is optionally substituted with 1, 2 or 3 R;
- R is selected from F, Cl, Br, I, OH, NH 2 and CH 3 ;
- the carbon atoms with "*" are chiral carbon atoms and exist in the form of (R) or (S) single enantiomer or are rich in one enantiomer;
- Carbon atoms with "#” are chiral carbon atoms and exist in the form of a single enantiomer (R) or (S) or are rich in one enantiomer.
- Other variables are as defined in the present invention.
- the R a is selected from the group consisting of F, Cl, Br, I, OH, NH 2 , CN, COOH, and Which said It is optionally substituted by 1, 2 or 3 R, other variables are as defined in the present invention.
- the R a is selected from the group consisting of F, Cl, Br, I, OH, NH 2 , CN, COOH, and Other variables are as defined in the present invention.
- R 1 is selected from the group consisting of NH 2 , CN, COOH, CH 3 , Other variables are as defined in the present invention.
- the present invention provides a compound of formula (I), an isomer thereof, or a pharmaceutically acceptable salt thereof,
- n 0, 1, or 2, and m and n cannot be 0 at the same time;
- r is 0 or 1;
- q is 0 or 1
- g 0, 1, 2, 3, or 4;
- R a is selected from F, Cl, Br, I, OH, NH 2 , CN, and OOH, wherein the phenyl group is optionally substituted with 1, 2 or 3 R;
- R is selected from F, Cl, Br, I, OH and NH 2 ;
- the carbon atoms with "*" are chiral carbon atoms and exist in the form of (R) or (S) single enantiomer or are rich in one enantiomer;
- Carbon atoms with "#” are chiral carbon atoms and exist in the form of a single enantiomer (R) or (S) or are rich in one enantiomer.
- R a is selected from F, Cl, Br, I, OH, NH 2, CN and COOH, other variables are as defined in the present invention.
- R 1 is selected from the group consisting of NH 2 , CN, COOH, CH 3 , Other variables are as defined in the present invention.
- the aforementioned compound, an isomer thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of
- g and R 1 are as defined in the present invention.
- the invention also provides a compound of the formula, an isomer thereof, or a pharmaceutically acceptable salt thereof,
- the aforementioned compound, an isomer thereof, or a pharmaceutically acceptable salt thereof is aforementioned compound, an isomer thereof, or a pharmaceutically acceptable salt thereof,
- the present invention also provides the aforementioned compound, an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from the hydrochloride salt.
- the invention also provides the use of the above-mentioned compound, its isomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease associated with LSD1.
- the compounds of the present invention have significant inhibitory activity on LSD1 and obvious inhibitory activity on NCI-H1417, HL60 and MV-4-11 cell proliferation; meanwhile, they have good pharmacokinetic properties; and CT-26 mouse colon cancer xenograft model combined with PD-L1 monoclonal antibody and MC38 mouse colon cancer xenograft model combined with PD-1 monoclonal antibody have excellent tumor suppressive effect.
- pharmaceutically acceptable refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound 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 a compound of the present invention, prepared from a compound having a specific substituent and a relatively non-toxic acid or base found in the present invention.
- base addition salts can be obtained by contacting a sufficient amount of a base with a neutral form of such compounds in a pure solution or a suitable inert solvent.
- Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
- acid addition salts can be obtained by contacting the neutral form of such compounds 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, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc .; and organic acid salts, such as 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; salts of amino acids such as arginine , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into
- the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by a conventional chemical method. Generally, such salts are prepared by reacting these compounds in the form of a free acid or base with a stoichiometric appropriate base or acid in water or an organic solvent or a mixture of the two.
- the compounds of the invention may exist in specific geometric or stereoisomeric forms.
- This invention contemplates all such compounds, including cis and trans isomers, (-)-and (+)-enantiomers, (R)-and (S) -enantiomers, diastereomers Isomers, (D) -isomers, (L) -isomers, and racemic and other mixtures thereof, such as enantiomeric or diastereomeric enriched mixtures, all of which belong to the present invention Within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All these isomers and their mixtures are included in the scope of the present invention.
- enantiomers or “optical isomers” refer to stereoisomers in mirror image relationship to each other.
- cis-trans isomer or “geometric isomer” are caused by the inability of a double bond or a single bond of a ring-forming carbon atom to rotate freely.
- diastereomer refers to a stereoisomer in which a molecule has two or more centers of chirality and is in a non-mirror relationship between molecules.
- wedge solid line key And wedge dashed keys Represents the absolute configuration of a solid center, using straight solid line keys And straight dashed keys Represents the relative configuration of the solid center, with wavy lines Represents a wedge solid line key Or wedge-shaped dotted key Or with wavy lines Represents a straight solid line key And straight dashed keys
- the following formula (A) indicates that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2) Exists in the form of a mixture;
- the following formula (B) represents that the compound exists as a single isomer of the formula (B-1) or (B-2) or in the form of both (B-1) and (B-2) The isomers exist as a mixture.
- the following formula (C) represents that the compound exists as a single isomer of the formula (C-1) or (C-2) or in the form of the two isomers of the formula (C-1) and the formula (C-2) It exists as a mixture.
- tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be quickly converted to each other. If tautomers are possible (eg in solution), the chemical equilibrium of the tautomers can be reached.
- proton tautomers also known as prototropic tautomers
- proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
- Valence tautomers include recombination of some bonding electrons for mutual conversion.
- a specific example of the keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
- the terms “rich in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enantiomerically enriched” refer to one of the isomers or the The enantiomeric content is less than 100%, and the content of the isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or more 99.9%.
- the terms “isomer excess” or “enantiomeric excess” refer to the difference between the two isomers or the relative percentages of the two enantiomers. For example, if one of the isomers or enantiomers is 90% and the other isomer or enantiomer is 10%, the isomer or enantiomeric 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 an enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
- a diastereomeric salt is formed with a suitable optically active acid or base, and then a conventional method known in the art Diastereomeric resolution is performed and the pure enantiomer is recovered.
- the separation of enantiomers and diastereoisomers is usually accomplished by using chromatography that employs a chiral stationary phase and optionally is combined with chemical derivatization (such as the generation of amino groups from amines) Formate).
- the compounds of the invention may contain atomic isotopes in unnatural proportions on one or more of the atoms constituting the compound.
- compounds such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C) can be labeled with radioisotopes.
- deuterated drugs can be replaced by heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have lower toxicity and increased drug stability , Enhance efficacy, extend the biological half-life of drugs and other advantages. Transformations of all isotopic compositions of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
- “Optional” or “optionally” refers to events or conditions described later that may, but need not, occur, and that the description includes situations in which the events or conditions occur and situations in which the events or conditions do not occur.
- substituted refers to the replacement of any one or more hydrogen atoms on a specific atom with a substituent, and can include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
- O oxygen
- Oxygen substitution does not occur on aromatic groups.
- optionally substituted means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemically achievable.
- any variable such as R
- its definition in each case is independent.
- the group may be optionally substituted with at most two R, and R in each case has independent options.
- combinations of substituents and / or variants 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- , the linking group is a single bond.
- substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X is vacant in AX, it means that the structure is actually A.
- a substituent's bond can be cross-linked to two or more atoms on a ring, the substituent can be bonded to any atom on the ring, for example, a structural unit It means that the substituent R may be substituted at any position on the cyclohexyl or cyclohexadiene. .
- substituents do not indicate which atom is connected to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be passed through any The carbon atom is attached to a substituted group.
- the intermediate linking group L is -MW-.
- -MW- can be connected to ring A and ring B in the same direction as the reading order from left to right. You can also connect ring A and ring B in the opposite direction from the reading order from left to right.
- the number of atoms on a ring is generally defined as the number of rings, for example, a "5-7 member ring” refers to a “ring” arranged around 5-7 atoms.
- 3-12 membered ring means a cycloalkyl, heterocycloalkyl, cycloalkenyl or heterocycloalkenyl group consisting of 3 to 12 ring atoms.
- the ring includes a single ring, and also includes a double ring or a multi-ring system such as a spiro ring, a parallel ring and a bridge ring.
- the ring optionally contains 1, 2, or 3 heteroatoms independently selected from O, S, and N.
- the 3-12 member ring includes 3-10 members, 3-9 members, 3-8 members, 3-7 members, 3-6 members, 3-5 members, 4-10 members, 4-9 members, 4- 8 yuan, 4-7 yuan, 4-6 yuan, 4-5 yuan, 5-10 yuan, 5-9 yuan, 5-8 yuan, 5-7 yuan, 5-6 yuan, 6-10 yuan, 6- 9 yuan, 6-8 yuan and 6-7 yuan ring, etc.
- the term "5- to 7-membered heterocycloalkyl” includes piperidinyl and the like, but does not include phenyl.
- ring also includes ring systems containing at least one ring, each of which "ring” independently meets the above definition.
- 5- to 6-membered ring means a cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aromatic group consisting of 5 to 6 ring atoms. Or heteroaryl.
- the ring includes a single ring, and also includes a double ring system such as a spiro ring, a parallel ring and a bridge ring. Unless otherwise specified, the ring optionally contains 1, 2, or 3 heteroatoms independently selected from O, S, and N.
- the 5-6 membered ring includes a 5-membered, 6-membered ring, and the like.
- 5-6 membered ring includes, for example, phenyl, pyridyl, piperidinyl, and the like; on the other hand, the term “5-6 membered heterocycloalkyl” includes piperidinyl and the like, but does not include phenyl.
- ring also includes ring systems containing at least one ring, each of which "ring” independently meets the above definition.
- C 1-6 alkyl is used to indicate a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms.
- the C 1-6 alkyl 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 polyvalent (such as methine).
- C 1-6 alkyl examples 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 and the like.
- C 1-4 alkyl is used to indicate a straight or branched chain saturated hydrocarbon group consisting of 1 to 4 carbon atoms.
- the C 1-4 alkyl group includes C 1-2 , C 1-3 and C 2-3 alkyl, etc .; it may be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as (Such as methine).
- Examples of C 1-4 alkyl 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) and the like.
- C 1-3 alkyl is used to indicate a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms.
- the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, and the like; it may be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methine).
- Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
- C 1-6 alkoxy refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through one oxygen atom.
- the C 1-6 alkoxy group includes C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 , C 5 , C 4 and C 3 alkoxy, etc. .
- C 1-6 alkoxy examples include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutyl (Oxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy, and the like.
- C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule through one 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 include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
- C1-3 alkylamino refers to those alkyl groups containing 1 to 3 carbon atoms that are attached to the rest of the molecule through an amino group.
- the C 1-3 alkylamino group includes C 1-2 , C 3 and C 2 alkylamino, and the like.
- Examples of C 1-3 alkylamino include, but are not limited to, -NHCH 3 , -N (CH 3 ) 2 , -NHCH 2 CH 3 , -N (CH 3 ) CH 2 CH 3 , -NHCH 2 CH 2 CH 3 ,- NHCH 2 (CH 3 ) 2 and the like.
- C 6-10 aromatic ring and “C 6-10 aryl” in the present invention are used interchangeably, and the terms “C 6-10 aromatic ring” or “C 6-10 aryl” mean A cyclic hydrocarbon group with a conjugated ⁇ -electron system consisting of 6 to 10 carbon atoms, which can be a monocyclic, fused bicyclic or fused tricyclic system, where each ring is aromatic. It may be monovalent, divalent, or polyvalent, and C 6-10 aryl includes C 6-9 , C 9 , C 10, and C 6 aryl, and the like. Examples of C 6-10 aryl include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, etc.).
- the heteroatom may occupy the position of attachment of the heterocycloalkyl group to the rest of the molecule.
- the 4-7 membered heterocycloalkyl includes 5-6 members, 4 members, 5 members, 6 members, 7 members, and the like.
- 4- to 7-membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuryl (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, dithiazyl, isoxazolidinyl, isothiazolyl
- a heteroatom may occupy a connection position between the heterocycloalkyl group and the rest of the molecule.
- the 5- to 6-membered heterocycloalkyl includes 5- and 6-membered heterocycloalkyl.
- Examples of 5- to 6-membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothien-2-yl and tetrahydrothien-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, dithiaalkyl, isoxazolidinyl, isothiazolyl Alkyl, 1,2-oxazinyl, 1,2-thiazinyl
- C n-n + m or C n -C n + m includes any specific case of n to n + m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , and also include any range from n to n + m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.
- n yuan to n + m means that the number of atoms on the ring is n to n + m.
- 3-12-membered rings include 3-, 4-, 5-, 6-, 7-, 8-, and 9-membered rings.
- 10-membered ring, 11-membered ring, and 12-membered ring including any range from n to n + m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring Ring, 5-7 member ring, 6-7 member ring, 6-8 member ring, and 6-10 member ring, etc.
- leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, an affinity substitution reaction).
- representative leaving groups include triflate; chlorine, bromine, and iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, and p-toluenesulfonic acid. Esters, etc .; acyloxy, such as acetoxy, trifluoroacetoxy and the like.
- protecting group includes but is not limited to "amino protecting group", “hydroxy protecting group” or “mercapto protecting group”.
- amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
- Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-methoxyphenyl) methyl; silyl, such as trimethylsilyl (TMS) and tert-butyld
- hydroxy protecting group refers to a protecting group suitable for preventing side reactions of a hydroxyl group.
- Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl, and tert-butyl; acyl groups such as alkanoyl (such as acetyl); aryl methyl groups such as benzyl (Bn), Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and more.
- alkyl groups such as methyl, ethyl, and tert-butyl
- acyl groups such as alkanoyl (such as acetyl)
- aryl methyl groups such as benzyl (Bn), Oxybenzyl (PMB), 9-fluorenylmethyl
- 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 familiar to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
- the solvent used in the present invention is commercially available.
- the present invention uses the following abbreviations: aq stands for water; HATU stands for O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurea hexafluorophosphate ; EDC stands for N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent, equivalent; CDI stands for Carbonyl diimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for diisopropyl azodicarboxylate; DMF stands for N, N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOAc stands for ethyl acetate EtOH for ethanol; MeOH for methanol; CBz for benzy
- the reaction solution was diluted with dichloromethane (80 mL), and then washed with a saturated aqueous sodium hydrogen carbonate solution (100 mL x 3), water (100 mL x 2), and saturated brine (100 mL x 1), and then dried over anhydrous sodium sulfate and filtered.
- the obtained mother liquor was concentrated to obtain compound 1-4.
- reaction solution was stirred at 25 ° C for 11.5 hours.
- the reaction solution was poured into a saturated aqueous sodium hydrogen carbonate solution (10 mL), and extracted with ethyl acetate (20 mL ⁇ 3).
- MS-ESI calculated [M + H] + 568, found 568.
- the purpose of this test is to detect the inhibitory activity of the compound on LSD1 in vitro.
- the enzyme used in this test is human-derived LSD1, and the standard substrate is the histone H3K4me peptide (20 ⁇ M).
- the enzyme-fluorescence coupling method was used to detect the H produced by the reaction of LSD1 by horseradish peroxidase (HPR) and the fluorescent reagent Amplex Red The method of 2 O 2 determines the activity of the compound. 3 fold dilution beginning from 10 ⁇ M, the IC 50 value of 10 concentrations of the test compound.
- test compounds have LSD1 inhibitory activity, and the results are shown in Table 1.
- test compounds have inhibitory activity on NCI-H1417 cell proliferation, and the results are shown in Table 2.
- the compound of the present invention has obvious inhibitory activity on the proliferation of NCI-H1417 cells.
- RPMI-1640 medium, fetal bovine serum, penicillin / streptomycin antibiotics were purchased from Vicente.
- CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
- the HL60 cell line was purchased from Nanjing Kebai Life Technology Co., Ltd. Nivo Multi-Label Analyzer (PerkinElmer).
- HL60 cells were seeded in white 384-well plates, each well containing 40 ⁇ L of cell suspension, which contained 600 HL60 cells.
- Cell plates were cultured overnight in a carbon dioxide incubator. Dilute the test compound 5 times with a row gun to the 10th concentration, that is, dilute from 2mM to 1.024nM, and set up a double well experiment.
- the cell plate was cultured in a carbon dioxide incubator for 6 days.
- test compound has HL60 cell proliferation inhibitory activity, and the results are shown in Table 3.
- Table 3 Test results of HL60 cell proliferation inhibition compounds of the present invention
- the compound of the present invention has obvious inhibitory activity on HL60 cell proliferation.
- IMDM medium fetal calf serum, penicillin / streptomycin antibiotics were purchased from Vicente.
- CellTiter-Glo cell viability chemiluminescence detection reagent
- the MV-4-11 cell line was purchased from Nanjing Kebai Life Technology Co., Ltd. Nivo Multi-Label Analyzer (PerkinElmer).
- MV-4-11 cells were seeded in a white 96-well plate, 80 ⁇ L of cell suspension per well, which contained 6000 MV-4-11 cells. Cell plates were cultured overnight in a carbon dioxide incubator.
- test compound has inhibitory activity on MV-4-11 cell proliferation, and the results are shown in Table 4.
- the compound of the present invention has obvious inhibitory activity on the proliferation of MV-4-11 cells.
- CD-1 mice male, 7-9 weeks old, Shanghai Slark
- Intravenous and oral vehicles are a mixed vehicle of 10% dimethyl sulfoxide and 90% 10% hydroxypropyl ⁇ -cyclodextrin.
- This project uses four male CD-1 mice and two mice for intravenous administration at a dose of 1 mg / kg, collected for 0 h (before administration) and 0.0833, 0.25, 0.5, 1, 2 after administration , 4,8,24h plasma samples, and two other mice were administered by oral gavage at a dose of 2mg / kg, collected 0h (before administration) and 0.25, 0.5, 1, 2, 4 after administration,
- the supernatant was sampled by centrifugation with water, and the plasma drug concentration was quantitatively analyzed by LC-MS / MS analysis method, and the pharmacokinetic parameters were calculated, such as peak concentration (C max ), clearance (CL), and half-life (T 1/2 ), Tissue distribution (Vdss), area under the curve (AUC 0-last ), bioavailability (F), etc.
- the compounds of the present invention have good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life, and clearance rate.
- Age and weight 7 weeks of age, weight 18-23 grams
- CT-26 mouse colon cancer cell
- the culture medium was 1640 medium containing 10% fetal bovine serum, and the culture conditions were 37 ° C and 5% carbon dioxide.
- the passage ratio is 1: 2 to 1: 3, and the passage is performed 2 to 3 times a week.
- 0.1 mL (3 ⁇ 10 5 ) cells were inoculated subcutaneously on the right back of each mouse. Animals were randomly divided into groups based on body weight on the same day.
- the solvent used in the experiment was a 0.5% methylcellulose solution.
- the preparation method was to weigh 5g of methylcellulose, dissolve it in 800mL of ultrapure water, and stir to volume to 1000mL with ultrapure water.
- the test substance was dissolved in a solvent to prepare a uniform solution with a certain concentration and stored at 4 ° C.
- the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
- Tumor diameter was measured twice a week with vernier calipers.
- T / C% T RTV / C RTV ⁇ 100% (T RTV : RTV in the treatment group; C RTV : RTV in the negative control group).
- PD-L1 monoclonal antibody source BioXcell.
- MC38 mouse colon cancer cell
- the culture medium was 1640 medium containing 10% fetal bovine serum, and the culture conditions were 37 ° C and 5% carbon dioxide.
- the passage ratio is 1: 2 to 1: 3, and the passage is performed 2 to 3 times a week.
- 0.1 mL (2 ⁇ 10 5 ) cells were subcutaneously inoculated on the right back of each mouse. Animals were randomly divided into groups based on body weight on the same day.
- the solvent used in the experiment was a 0.5% methylcellulose solution.
- the preparation method was to weigh 5g of methylcellulose, dissolve it in 800mL of ultrapure water, and stir to volume to 1000mL with ultrapure water.
- the test substance was dissolved in a solvent to prepare a uniform solution with a certain concentration and stored at 4 ° C.
- the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
- Tumor diameter was measured twice a week with vernier calipers.
- T / C% T RTV / C RTV ⁇ 100% (T RTV : RTV in the treatment group; C RTV : RTV in the negative control group).
- PD-1 monoclonal antibody source BioXcell.
- CONCLUSION The combination of the compound of the present invention and PD-1 monoclonal antibody has excellent antitumor effect on MC38 mouse colon cancer xenograft model.
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Abstract
涉及一类可作为赖氨酸特异性去甲基化酶1（LSD1）抑制剂的杂螺环类化合物，还涉及其在制备治疗与LSD1相关疾病的药物中的应用。具体涉及一种如式(I)所示的化合物、其异构体及其药学上可接受的盐。
Description
本申请主张如下优先权：
CN201811070310.5，申请日：2018.09.13；
CN201910099370.8，申请日：2019.01.31。
本发明涉及一类作为赖氨酸特异性去甲基化酶1(LSD1)抑制剂的杂螺环类化合物，及其在制备治疗与LSD1相关疾病的药物中的应用。具体涉及式(Ⅰ)所示化合物、其异构体及其药学上可接受的盐。
组蛋白翻译后修饰包括甲基化、乙酰化、磷酸化、泛素化等过程，是表观遗传学的重要调控手段，通过改变染色质结构影响基因表达[Xueshun Wang,Boshi Huang,Takayoshi Suzuki et al.,Epigenomics,2015,1379-1396；]。尽管这些修饰并不改变DNA的基础序列，但这种表观遗传的变化可能通过细胞分裂在整个细胞生命周期或者细胞迭代过程持续存在[Adrian Bird,Nature,2007,396-398]。因此表观遗传学功能异常与各种疾病的病理过程密切相关[James T Lynch,William J Harris&Tim C P Somervaille,Expert Opin.Ther.Targets,2012,1239-1249]，比如各种实体瘤，血液瘤，病毒感染，神经系统异常等疾病。因此，表观遗传学现在成为药物研发领域的研究热点。组蛋白的甲基化状态由组蛋白甲基转移酶和组蛋白去甲基化酶共同调控。赖氨酸特异性去甲基化酶(Lysine specific demethylase 1，LSD1，又名KDM1A)是第一个被报道的组蛋白赖氨酸去甲基化酶，通过调控组蛋白赖氨酸的甲基化状态，广泛参与转录调控，影响细胞增殖和分化、胚胎干细胞多能性等诸多生理过程。[Yujiang Shi,Fei Lan,Caitlin Matson et al.,Cell,2004,941–953][Daniel P.Mould,Alison E.McGonagle,Daniel H.Wiseman et al.,Medicinal Research Reviews,2015,586–618]。LSD1结构包括三个主要部分：N-末端的SWIRM结构域，C-末端的氨基氧化酶结构域(AOL)和中央的Tower域。[Ruchi Anand,Ronen Marmorstein,Journal of Biological Chemistry,2007,35425–35429]。C-末端的氨基氧化酶结构域包括两个活性口袋，一个是FAD结合的位点，另一个是用于识别并与底物结合的位点[Pete Stavropoulos,Günter Blobel,André Hoelz,Nature Structral&Molecular Biology,2006,626-632]。SWIRM结构域的功能还没有明确的结论，它不直接参与FAD或者底物的结合，但是这个区域的突变或者是去除都会降低LSD1的活性，因此推测该区域可能是通过调整构象，影响活性区域的作用。[Yong Chen,Yuting Yang,Feng Wang et al.,Biochemistry,2006,13956–13961]。Tower结构域是LSD1与其他蛋白因子的结合域。LSD1与不同蛋白因子相结合后，作用于不同底物，从而对组蛋白以及基因表达起到不同的调控作用。比如LSD1与CoREST相结合后，会优先作用于组蛋白H3K4，通过去甲基化，去除激活相关的组蛋白标记，抑制基因转录；而与雄激素受体蛋白结合后，重组的LSD1会优先作用于H3K9，通过去甲基化激活雄激素受体相关的基因转录 [Ruchi Anand,Ronen Marmorstein,Journal of Biological Chemistry,2007,35425–35429；Eric Metzger,Melanie Wissmann,Na Yin et al.,Nature,2005,436-439.]。此外，LSD1还调控部分非组蛋白底物的甲基化状态，包括抑癌基因p53和DNA甲基转移酶1(DNA methyltransferase 1，DNMT1)等[Yi Chao Zheng,Jinlian Ma,Zhiru Wang,Medicinal Research Reviews,2015,1032–1071]。
LSD1是FAD依赖的氨基氧化酶，其中质子转移被认为是其最可能的氧化机理[Zheng Y C,Yu B,Chen Z S,et al.Epigenomics,2016,8,651-666.]。首先通过质子转移，将底物的N-CH
3键转化成亚胺键，这个亚胺离子中间体发生水解反应，一边生成去甲基的胺，另一边生成甲醛。在这个催化循环过程中，FAD被还原成FADH2，随后又被一分子的氧气氧化回到FAD，同时生成一分子H2O2[Yujiang Shi,Fei Lan,Caitlin Matson,Cell,2004,941–953]。
LSD1在多种不同类型的肿瘤中异常表达。LSD1在急性髓性白血病(acute myeloid leukemia，AML)亚型中高表达，是维持白血病干细胞(leukemia stem cell，LSC)潜能的重要因素。LSD1在多种实体瘤如肺癌、乳腺癌、前列腺癌、肝癌和胰腺癌中高表达，与肿瘤的预后不良密切相关。LSD1抑制钙粘蛋白的表达，与肿瘤的侵袭和上皮-间质转移(epithelial-mesenchymal transition,EMT)密切相关[Hosseini A,Minucci S.Epigenomics,2017,9,1123-1142.]。
LSD1抑制剂目前没有药物获批上市，已有8个药物处于临床研究阶段，主要用于血液肿瘤、小细胞肺癌和尤文氏肉瘤等疾病的治疗。然而，面对巨大的未满足市场，该领域仍然需要活性更好，药代动力学参数更优的候选化合物推进临床试验，以满足治疗需求。
发明内容
本发明提供了式(Ⅰ)化合物、其异构体或其药学上可接受的盐，
其中，
L
1选自-(CH
2)g-、-C(＝O)-、-S(＝O)
2-、-C(＝O)-O-和-C(＝O)-NH-；
R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、C
1-6烷基、-C(＝O)-C
1-4烷基-苯基和-C
1-4烷基-4-7元杂环烷基，其中所述C
1-6烷基、-C(＝O)-C
1-4烷基-苯基和-C
1-4烷基-4-7元杂环烷基任选被1、2或3个R
a取代；
m为0、1或2；
n为0、1或2，且m和n不能同时为0；
r为0或1；
q为0或1；
g为0、1、2、3或4；
R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH、C
1-3烷氨基和-NH-C(＝O)-C
1-3烷基，其中所述C
1-3烷氨基和-NH-C(＝O)-C
1-3烷基任选被1、2或3个R取代；
R选自F、Cl、Br、I、OH、NH
2和C
1-3烷基；
所述4-7元杂环烷基包含1、2、3或4个独立选自-NH-、-O-、-S-和N的杂原子或杂原子团；
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；
带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。
本发明的一些方案中，上述R选自F、Cl、Br、I、OH、NH
2、CH
3和-CH
2CH
3，其他变量如本发明所定义。
本发明的一些方案中，上述R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH、
其中所述
任选被1、2或3个R取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH、
其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、C
1-4烷基、-C(＝O)-C
1-3烷基-苯基和-C
1-3烷基-5～6杂环烷基，其中所述C
1-4烷基、-C(＝O)-C
1-3烷基-苯基和-C
1-3烷基-5～6杂环烷基任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、-CH
3、-CH
2-CH
3、
其中所述CH
3、-CH
2-CH
3、
任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、
-CH
3、-CH
2-CH
3、
其他变量如本发明所定义。
本发明的一些方案中，上述L
1选自单键、-CH
2-、-(CH
2)
2-、
-C(＝O)-、和-S(＝O)
2-，其他变量如本发明所定义。
本发明提供了式(Ⅰ)化合物、其异构体或其药学上可接受的盐，
其中，
L
1选自-(CH
2)g-、-C(＝O)-、-S(＝O)
2-、-C(＝O)-O-和-C(＝O)-NH-；
R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、C
1-6烷基和-C(＝O)-C
1-4烷基-苯基，其中所述C
1-6烷基和-C(＝O)-C
1-4烷基-苯基任选被1、2或3个R
a取代；
m为0、1或2；
n为0、1或2，且m和n不能同时为0；
r为0或1；
q为0或1；
g为0、1、2、3或4；
R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH和C
1-3烷氨基，其中所述C
1-3烷氨基任选被1、2或3个R取代；
R选自F、Cl、Br、I、OH、NH
2和CH
3；
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；
带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。
本发明的一些方案中，上述L
1选自-(CH
2)g-时，R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、C
1-6烷基和-C(＝O)-C
1-4烷基-苯基，其中所述C
1-6烷基和-C(＝O)-C
1-4烷基-苯基任选被1、2或3个R
a取代，其 他变量如本发明所定义。
本发明的一些方案中，上述L
1选自-C(＝O)-、-S(＝O)
2-、-C(＝O)-O-和-C(＝O)-NH-时，R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2和C
1-6烷基，其中所述C
1-6烷基任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH和
其中所述
任选被1、2或3个R取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
a选自F、Cl、Br、I、OH、NH
2、CN、COOH和
其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、C
1-4烷基和-C
1-3烷基-苯基，其中所述C
1-4烷基和-C
1-3烷基-苯基任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、CH
3、-CH
2-CH
3、
其中所述CH
3、-CH
2-CH
3、
任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、
CH
3、
其他变量如本发明所定义。
本发明的一些方案中，上述L
1选自单键、-CH
2-、-(CH
2)
2-、
-C(＝O)-、和-S(＝O)
2-，其他变量如本发明所定义。
本发明提供了式(Ⅰ)化合物、其异构体或其药学上可接受的盐，
其中，
L
1选自-(CH
2)g-、-C(＝O)-、-S(＝O)
2-、-C(＝O)-O-和-C(＝O)-NH-；
R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2和C
1-6烷基，其中所述C
1-6烷基任选被1、2或3个R
a取代；m为0、1或2；
n为0、1或2，且m和n不能同时为0；
r为0或1；
q为0或1；
g为0、1、2、3或4；
R
a选自F、Cl、Br、I、OH、NH
2、CN和OOH，其中所述苯基任选被1、2或3个R取代；
R选自F、Cl、Br、I、OH和NH
2；
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；
带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。
本发明的一些方案中，上述R
a选自F、Cl、Br、I、OH、NH
2、CN和COOH，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2和C
1-4烷基，其中所述C
1-4烷基任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、-S(＝O)
2-NH
2、CH
3、-CH
2-CH
3、
其中所述CH
3、-CH
2-CH
3、
任选被1、2或3个R
a取代，其他变量如本发明所定义。
本发明的一些方案中，上述R
1选自NH
2、CN、COOH、
CH
3、
其他变量如本发明所定义。
本发明的一些方案中，上述L
1选自单键、-CH
2-、-(CH
2)
2-、
-C(＝O)-、和-S(＝O)
2-，其他变量如本发明所定义。
本发明还有一些方案是由上述各变量任意组合而来。
本发明的一些方案中，上述化合物、其异构体或其药学上可接受的盐，其选自
其中，
g和R
1如本发明所定义。
本发明还提供了下式化合物、其异构体或其药学上可接受的盐，
本发明的一些方案中，上述化合物、其异构体或其药学上可接受的盐，
本发明还提供了上述的化合物、其异构体或其药学上可接受的盐，其中所述的药学上可接受的盐选自盐酸盐。
本发明还提供了上述的化合物、其异构体或其药学上可接受的盐在制备治疗LSD1相关病症的药物上的应用。
技术效果
作为新型的LSD1抑制剂，本发明的化合物对LSD1具有显著的抑制活性，对NCI-H1417、HL60和MV-4-11细胞增殖抑制活性明显；同时，具有良好的药代动力学性质；并且在CT-26小鼠结肠癌移植瘤模型中与PD-L1单抗联用、在MC38小鼠结肠癌移植瘤模型中与PD-1单抗联用具有优异的抑瘤效果。
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的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。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的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。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，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
除非另有说明，用楔形实线键
和楔形虚线键
表示一个立体中心的绝对构型，用直形实线键
和直形虚线键
表示立体中心的相对构型，用波浪线
表示楔形实线键
或楔形虚线键
或用波浪线
表示直形实线键
和直形虚线键
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原 子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线
连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来 制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(
3H)，碘-125(
125I)或C-14(
14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。“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“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。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。
当一个连接基团的数量为0时，比如-(CRR)
0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当一个取代基的键可以交叉连接到一个环上的两一个以上原子时，这种取代基可以与这个环上的任意原子相键合，例如，结构单元
表示其取代基R可在环己基或者环己二烯上的任意一个位置发生取代。。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
中连 接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
所述连接基团、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，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
除非另有规定，“3-12元环”表示由3至12个环原子组成的环烷基、杂环烷基、环烯基或杂环烯基。所述的环包括单环，也包括螺环、并环和桥环等双环或多环体系。除非另有规定，该环任选地包含1、2或3个独立选自O、S和N的杂原子。所述3-12元环包括3-10元、3-9元、3-8元、3-7元、3-6元、3-5元、4-10元、4-9元、4-8元、4-7元、4-6元、4-5元、5-10元、5-9元、5-8元、5-7元、5-6元、6-10元、6-9元、6-8元和6-7元环等。术语“5-7元杂环烷基”包括哌啶基等，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
除非另有规定，“5-6元环”表示由5至6个环原子组成的环烷基、杂环烷基、环烯基、杂环烯基、环炔基、杂环炔基、芳基或杂芳基。所述的环包括单环，也包括螺环、并环和桥环等双环体系。除非另有规定，该环任选地包含1、2或3个独立选自O、S和N的杂原子。所述5-6元环包括5元、6元环等。“5-6元环”包括例如苯基、吡啶基和哌啶基等；另一方面，术语“5-6元杂环烷基”包括哌啶基等，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
除非另有规定，术语“C
1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C
1-6烷基包括C
1-5、C
1-4、C
1-3、C
1-2、C
2-6、C
2-4、C
6和C
5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C
1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C
1-4烷基”用于表示直链或支链的由1至4个碳原子组成的饱和碳氢基团。所述C
1-4烷基包括C
1-2、C
1-3和C
2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C
1-4烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)等。
除非另有规定，术语“C
1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C
1-3烷基包括C
1-2和C
2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C
1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C
1-6烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至6个碳原子的烷基基团。所述C
1-6烷氧基包括C
1-4、C
1-3、C
1-2、C
2-6、C
2-4、C
6、C
5、C
4和C
3烷氧基等。C
1-6烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)、丁氧基(包括n-丁氧基、异丁氧基、s-丁氧基和t-丁氧基)、戊氧基(包括n-戊氧基、异戊氧基和新戊氧基)、己氧基等。
除非另有规定，术语“C
1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C
1-3烷氧基包括C
1-2、C
2-3、C
3和C
2烷氧基等。C
1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，术语“C
1-3烷氨基”表示通过氨基连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C
1-3烷氨基包括C
1-2、C
3和C
2烷氨基等。C
1-3烷氨基的实例包括但不限于-NHCH
3、-N(CH
3)
2、-NHCH
2CH
3、-N(CH
3)CH
2CH
3、-NHCH
2CH
2CH
3、-NHCH
2(CH
3)
2等。
除非另有规定，本发明术语“C
6-10芳环”和“C
6-10芳基”可以互换使用，术语“C
6-10芳环”或“C
6-10芳基”表示由6至10个碳原子组成的具有共轭π电子体系的环状碳氢基团，它可以是单环、稠合双环或稠合三环体系，其中各个环均为芳香性的。其可以是一价、二价或者多价，C
6-10芳基包括C
6-9、C
9、C
10和C
6芳基等。C
6-10芳基的实例包括但不限于苯基、萘基(包括1-萘基和2-萘基等)。
除非另有规定，术语“4-7元杂环烷基”本身或者与其他术语联合分别表示由4至7个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，碳、氮和硫杂原子可任选被氧化(即C(＝O)、NO和S(O)
p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“4-7元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述4-7元杂环烷基包括5-6元、4元、5元、6元和7元杂环烷基等。4-7元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基、六氢哒嗪基、高哌嗪基、高哌啶基或吡咯烷-2-酮基等。
除非另有规定，术语“5-6元杂环烷基”本身或者与其他术语联合分别表示由5至6个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，碳、氮和硫杂原子可任选被氧化(即C(＝O)、NO和S(O)
p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“5-6元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述5-6元杂环烷基包括5元和6元杂环烷基。5-6元杂环烷基的实例包括但不限于吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶 基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基、六氢哒嗪基、高哌嗪基、高哌啶基或吡咯烷-2-酮基等。
除非另有规定，C
n-n+m或C
n-C
n+m包括n至n+m个碳的任何一种具体情况，例如C
1-12包括C
1、C
2、C
3、C
4、C
5、C
6、C
7、C
8、C
9、C
10、C
11、和C
12，也包括n至n+m中的任何一个范围，例如C
1-12包括C
1-3、C
1-6、C
1-9、C
3-6、C
3-9、C
3-12、C
6-9、C
6-12、和C
9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
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。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸盐；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二异丙酯；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基，是一种胺保护基团；BOC代表叔丁氧羰基是一种胺保护基团；HOAc代表乙酸；NaCNBH
3代表氰基硼氢化钠；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc
2O代表二-叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA 代表二异丙基乙基胺；本发明化合物的盐酸盐，加入饱和碳酸氢钠溶液调节pH到中性，经过高效液相色谱法分离(中性,碳酸氢铵体系)得到化合物的游离碱。
化合物依据本领域常规命名原则或者使用
软件命名，市售化合物采用供应商目录名称。
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。
实施例1
合成路线:
第一步
将氢氧化钠(279g,6.99mol)溶于水(3L)中,用冰水浴将反应液降温至10℃,将化合物1-1(997g,3.49mol)分批加入至反应液,在10℃下搅拌反应2小时。向反应液中加入乙酸乙酯(2L x 1)萃取,乙酸乙酯(1.6L x 1)萃取。合并有机相用水(1.5L x 1)洗涤,再用饱和食盐水(1.5L x 1)洗涤,有机相用无水硫酸钠干燥,过滤,减压浓缩除去溶剂后得到化合物1-2。
1H NMR(400MHz,CDCl
3)δ7.18-7.14(m,2H),7.08-7.04(m,1H),6.95-6.92(m,2H),2.48-2.44(m,1H),1.80-1.76(m,1H),0.98-0.87(m,2H)。
第二步
将化合物1-3(1.00g,3.92mmol)和化合物1-2(522mg,3.92mmol)溶于无水二氯甲烷(20mL)中,向反应液中加入冰醋酸(706mg,11.8mmol)。反应液在20℃下搅拌1小时,加入醋酸硼氢化钠(2.49g,11.8mmol),反应液在20℃下继续搅拌10小时。反应液用二氯甲烷(80mL)稀释后依次用饱和碳酸氢钠水溶液(100mL x 3),水(100mL x 2),饱和食盐水(100mL x 1)洗涤,再用无水硫酸钠干燥,过滤,所得母液浓缩得到化合物1-4。
1H NMR(400MHz,CDCl
3)δ7.18-7.16(m,2H),7.11-7.17(m,1H),6.96-6.94(m,2H),3.92-3.89(m,1H),3.61-3.55(m,1H),3.52-3.48(m,3H),3.27-3.23(m,2H),2.24-2.21(m,1H),1.99-1.94(m,1H),1.85-1.79(m,1H),1.55-1.47(m,6H),1.38(s,9H),1.01-0.90(m,2H)。MS-ESI计算值[M+H]
+373,实测值373。
第三步
将化合物1-4(1.10g,2.95mmol)溶于无水二氯甲烷中(20mL),加入三乙胺(448mg,4.43mmol)和三氟乙酸酐(930mg,4.43mmol)。反应液在15℃下搅拌反应12小时。向反应液中加入二氯甲烷(50mL),有机相用盐酸(1M,50mL x 1)和饱和食盐水(50mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩,粗产物经过柱层析法分离(5/1二氯甲烷/甲醇,Rf＝0.38)得到化合物1-5。MS-ESI计算值[M-56+H]
+413,[M-Boc+H]
+369,实测值413,369。
第四步
将化合物1-5(600mg,1.28mmol)溶于无水二氯甲烷(6mL)中,在20℃下加入三氟乙酸(4.62g,40.5mmol)。反应液在20℃下搅拌反应2小时,减压浓缩除去溶剂,剩余物溶于二氯甲烷(6mL)中,再向其中加入三乙胺(250μL)后在室温下搅拌半小时,减压浓缩除去溶剂,得到化合物1-6。MS-ESI计算值[M+H]
+369,实测值369。
第五步
将化合物1-6(200mg,0.543mmol)和三乙胺(164mg,1.63mmol)溶解在乙腈(10mL)中,加入化合物1-7(157mg,0.869mmol),反应液在50℃条件下反应12小时。继续向反应液中加入1-7(49.1mg,0.271mmol)和三乙胺(54.9mg,0.543mmol),反应液在50℃条件下反应24小时。减压浓缩除去溶剂,产物先用二氯甲烷(50mL x 1)溶解,再依次以饱和碳酸氢钠(15mL x 1),水(15mL x 1)和饱和食盐水(15mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩。产物经过薄层层析法分离(10/1二氯甲烷/甲醇,Rf＝0.56)得到化合物1-8。MS-ESI计算值[M+H]
+469,实测值469。
第六步
将化合物1-8(100mg,0.213mmol)溶解在四氢呋喃(2mL)、乙醇(2mL)和水(2mL)的混合溶液中,加入氢氧化钠(25.6mg,0.640mmol)。50℃条件下反应2小时。减压浓缩除去有机相,剩余物用水(10mL)溶解,用盐酸(1mol/L)调节pH值到4,经过高效液相色谱法分离(酸性,盐酸体系)得到化合物1的盐酸盐。
1H NMR(400MHz,D
2O)δ7.36-7.32(m,2H),7.28-7.25(m,1H),7.17-7.16(m,2H), 4.21-4.19(m,1H),4.16-4.08(m,2H),3.49-3.36(m,4H),3.17-3.11(m,2H),2.94-2.93(m,1H),2.84(t,J＝6.8Hz,2H),2.59-2.50(m,1H),2.45-2.39(m,1H),2.08-1.98(m,4H),1.85-1.77(m,1H),1.52-1.50(m,1H),1.44-1.39(m,1H)。MS-ESI计算值[M+H]
+345,实测值345。
实施例2
合成路线:
第一步
参照实施例1第五步得到化合物2-2。MS-ESI计算值[M+H]
+483,实测值483。
第二步
参照实施例1第六步得到化合物2的盐酸盐。
1H NMR(400MHz,D
2O)δ7.34-7.30(m,2H),7.26-7.23(m,1H),7.16-7.14(m,2H),4.19-4.15(m,1H),4.08-4.07(m,2H),3.43(t,J＝13.2Hz,2H),3.09-3.05(m,3H),2.94-2.88(m,1H),2.53-2.49(m,1H),2.43-2.36(m,3H),2.13-1.91(m,7H),1.83-1.75(m,1H),1.53-1.48(m,1H),1.41-1.36(m,1H)。MS-ESI计算值[M+H]
+359,实测值359。
实施例3
合成路线:
第一步
将化合物3-1(200mg,1.01mmol)溶解在无水二氯甲烷(5mL)中,加入三乙胺(306mg,3.03mmol)并冷却至0℃,在0℃氮气氛下加入化合物3-2(231mg,2.02mmol),在25℃条件下反应3小时,减压蒸馏除去有机溶剂,将剩余物和三乙胺(275mg,2.71mmol)溶解在乙腈(5mL)中，继续加入化合物1-6(333mg,0.905μmol),25℃条件下反应12小时。减压浓缩除去溶剂,用二氯甲烷(30mL)溶解剩余物,依次用饱和碳酸氢钠(10mL x 2)溶液,饱和食盐水(10mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩。经过薄层层析法分离(1/1石油醚/乙酸乙酯,Rf＝0.56)得到化合物3-3。MS-ESI计算值[M+H]
+447，实测值447。
第二步
将化合物3-3(120mg,0.269mmol)溶解在四氢呋喃(2mL)、乙醇(2mL)和水(2mL)的混合溶液中,加入氢氧化钠(32.3mg,0.806mmol)。50℃条件下反应2小时。减压浓缩除去有机溶剂,剩余物用水(10mL)溶解,用盐酸(1mol/L)调节pH值到4,经过高效液相色谱法分离(酸性,盐酸体系)得到化合物3的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.30(m,2H),7.25-7.22(m,1H),7.19-7.17(m,2H),4.19-4.02(m,3H),3.46-3.42(m,2H),3.17-3.06(m,2H),3.03-2.99(m,1H),2.83(s,3H),2.52-2.48(m,1H),2.38(m,1H),1.96-1.85(m,4H),1.71-1.63(m,1H),1.53(m,1H),1.43(m,1H)。MS-ESI计算值[M+H]
+351,实测值351。
实施例4
合成路线:
第一步
将化合物1-6(300mg,0.622mmol),化合物4-1(125mg,0.933mmol)和三乙胺(189mg,1.87mmol)溶于乙腈(5mL)中。反应液在50℃下搅拌10小时,减压浓缩除去溶剂,剩余物用二氯甲烷(50mL)溶解,有机相依次用水(50mL x 1)及饱和食盐水(50mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩,粗产物经过薄层层析法分离(1/2石油醚/乙酸乙酯,Rf＝0.24)得到化合物4-2。MS-ESI计算值[M+H]
+422,实测值422。
第二步
将化合物4-2(100mg,0.237mmol)溶于四氢呋喃(2mL)、水(2mL)和乙醇(2mL)中,加入氢氧化钠(28.5mg,0.712mmol)。反应液在50℃下搅拌2小时,减压浓缩除去四氢呋喃和乙醇,剩余物用水(10mL)溶解,用盐酸(1M)调节pH值到4,减压浓缩后剩余物经过高效液相色谱法分离(酸性,盐酸体系)得到化合物4的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.23-7.20(m,2H),7.15-7.10(m,3H),4.15-4.10(m,1H),4.07-4.02(m,2H),3.48-3.36(m,3H),3.32-3.29(m,1H),3.17-3.06(m,3H),2.91-2.90(m,1H),2.73-2.71(m,1H),2.56-2.53(m,1H),2.33-2.30(m,1H),2.08-2.01(m,4H),1.90-1.87(m,1H),1.56-1.52(m,1H),1.31-1.29(m,1H)。MS-ESI计算值[M+H]
+326,实测值326。
实施例5
合成路线:
第一步
将化合物5-1(56.5mg,0.298mmol),O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(155mg,0.407mmol)和N,N-二异丙基乙胺(70.2mg,0.543mmol)溶于二氯甲烷(6mL)中,反应液在30℃下搅拌0.5小时,之后向反应液中加入化合物1-6(100mg,0.271mmol),新的反应液在30℃下继续搅拌10小时,将其用二氯甲烷(10mL)稀释,依次用水(20mL x 1)及饱和食盐水(20mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩,粗产物经过薄层层析法分离(2/1石油醚/乙酸乙酯,Rf＝0.3)得到化合物5-2。MS-ESI计算值[M+H]
+540,实测值540。
第二步
将化合物5-2(70.0mg,0.130mmol)在30℃下溶于盐酸甲醇(4mol/L,5mL)中。反应液在30℃下搅拌反应10小时,减压浓缩除去溶剂,将剩余物溶于四氢呋喃(2mL)、水(2mL)和乙醇(2mL)中,加入氢氧化钠(15.6mg,0.389mmol)。反应液在50℃下搅拌2小时,减压浓缩除去四氢呋喃和乙醇,剩余物用水(10mL)稀释,用盐酸(1mol/L)调节pH值到4,减压浓缩后剩余物经过高效液相色谱法分离(酸性,盐酸体系)得到化合物5的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.48-4.45(m,1H),4.20-3.97(m,4H),3.71-3.49(m,2H),3.37-3.17(m,1H),3.01(t,J＝3.2Hz,1H),2.66-2.63(m,1H),2.43-2.41(m,1H),2.08-1.99(m,1H),1.97-1.80(m,3H),1.72-1.58(m,2H),1.51-1.40(m,4H)。MS-ESI计算值[M+H]
+344,实测值344。
实施例6
合成路线:
第一步
参照实施例5第一步得到化合物6-2。MS-ESI计算值[M+H]
+568,实测值568。
第二步
参照实施例4第二步得到粗品化合物6-3。MS-ESI计算值[M+H]
+472,实测值472。
第三步
将化合物6-3(70.0mg,148μmol)在25℃下溶于盐酸甲醇(4M,10mL)中。反应液在25℃下搅拌反应1小时,减压浓缩除去溶剂,剩余物经过高效液相色谱法分离(酸性,盐酸体系)得到化合物6的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.40-4.35(m,1H),4.20-3.96(m,4H),3.81-3.64(m,1H),3.62-3.43(m,1H),3.48-3.16(m,1H),3.03-3.01(m,1H),2.68-2.62(m,1H),2.47-2.37(m,1H),2.22-2.18(m,1H),2.09-2.05(m,1H),1.97-1.81(m,3H),1.68-1.58(m,2H),1.45-1.40(m,1H),1.14-1.10(m,3H),1.05-1.02(m,3H)。MS-ESI计算值[M+H]
+372,实测值372。
实施例7
合成路线:
第一步
将化合物7-1(80.0mg,0.639mmol)溶于无水四氢呋喃(6mL)中,将其冷却至0℃后向溶液中加入N,N-二异丙基乙胺(165mg,1.28mmol)和甲烷磺酰氯(110mg,0.959mmol),反应液在29℃下搅拌1小时。反应液用乙酸乙酯(30mL)稀释后,依次用饱和碳酸氢钠溶液(30mL x 1)和饱和氯化钠溶液(30mL x 1)洗涤,有机相用无水硫酸钠干燥,过滤,减压浓缩,得到粗品化合物7-2。
1H NMR(400MHz,DMSO-d
6)δ4.56(t,J＝6.4Hz,2H),3.52(t,J＝6.4Hz,2H),3.29(s,3H)。
第二步
参照实施例4第一步得到化合物7-3。MS-ESI计算值[M+H]
+476,实测值476。
第三步
参照实施例4第二步得到化合物7的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.36-7.32(m,2H),7.28-7.24(m,1H),7.22-7.20(m,2H),4.25-4.21(m,1H),4.18-4.12(m,2H),3.67-3.65(m,2H),3.62-3.50(m,4H),3.32-3.26(m,2H),3.06-3.03(m,1H),2.63-2.58(m,1H),2.48-2.43(m,1H),2.27-2.06(m,4H),2.00-1.94(m,1H),1.64-1.59(m,1H),1.47-1.42(m,1H)。MS-ESI计算值[M+H]
+380,实测值380。
实施例8
合成路线:
第一步
参照实施例4第一步得到化合物8-2。MS-ESI计算值[M+H]
+490,实测值490。
第二步
参照实施例4第二步得到化合物8的盐酸盐。
1H NMR(400MHz,D
2O)δ7.38-7.34(m,2H),7.30-7.27(m,1H),7.20-7.18(m,2H),4.26-4.21(m,1H),4.16-4.08(m,2H),3.63-3.46(m,2H),3.33-3.25(m,4H), 3.19-3.07(m,2H),2.98-2.93(m,1H),2.64-2.53(m,1H),2.47-2.41(m,1H),2.28-2.20(m,2H),2.16-1.95(m,4H),1.89-1.79(m,1H),1.56-1.50(m,1H),1.46-1.41(m,1H)。MS-ESI计算值[M+H]
+394,实测值394。
实施例9
合成路线：
第一步
将化合物1-6(150mg,0.407mmol)溶于乙腈(2mL)中,将化合物9-1(221mg,2.04mol)和三乙胺(206mg,2.04mmol)加入至反应液,将体系升至50℃搅拌反应12小时。减压浓缩除去溶剂,粗产物经过薄层层析法(10/1二氯甲烷/甲醇,Rf＝0.4)分离纯化得到化合物9-2。MS-ESI计算值[M+H]
+441,实测值441。
第二步
将化合物9-2(131mg,0.291mmol)溶于四氢呋喃(2mL)和无水乙醇(2mL),将氢氧化钠(33.8mg,0.845mmol)溶于水(2mL)后滴加至溶液中,在50℃下搅拌2小时。将反应液减压浓缩去除溶剂,用盐酸(1mol/L)调pH值到5后减压浓缩,其粗产物经过高效液相色谱(酸性,盐酸体系)分离纯化得到化合物9的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.28(m,2H),7.25-7.19(m,3H),4.22-7.14(m,3H),3.68-3.54(m,2H),3.36-3.25(m,3H),3.17-3.15(m,1H),3.04-3.00(m,1H),2.66-2.60(m,1H),2.55-2.39(m,1H),2.34-2.19(m,1H),2.16-1.96(m,4H),1.67-1.60(m,1H),1.45-1.35(m,7H)。MS-ESI计算值[M+H]
+345,实测值345。
实施例10
合成路线
第一步
将化合物10-1(5.00g,2.87mmol)溶于乙酸乙酯(5mL)和盐酸乙酸乙酯(4mol/L,15mL)中,反应液15℃下搅拌1小时。减压浓缩得到粗品化合物10-2。
1HNMR(400MHz,DMSO-d
6)δ8.51(s,3H),1.38(s,6H)。
第二步
将化合物10-2(200mg,1.43mmol)溶解在甲醇(5ml)中,加入甲醛(465mg,37％水溶液,5.73mmol)和Pd/C(153mg,10％湿钯碳,0.143mmol),反应液在氢气氛(50psi),20℃下反应16小时,反应液过滤浓缩得到化合物10-3。
1HNMR(400MHz,DMSO-d
6)δ2.74(s,6H),1.50(s,6H)。
第三步
将化合物10-3(100mg,0.762mmol)溶解在N,N-二甲基甲酰胺(5ml)中,继续加入化合物1-6(247mg,0.610mmol),三乙胺(386mg,3.81mmol)和O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(386mg,2.29mmol),反应液在30℃条件下搅拌15小时,向反应液中加入乙酸乙酯(20mL)、饱和食盐水(5mL),有机层用饱和食盐水(10mL x 2)洗涤,无水硫酸钠干燥,过滤,滤液减压浓缩,粗产物经过柱层析法分离(20/1二氯甲烷/甲醇,Rf＝0.51)得到化合物10-4。
1H NMR(400MHz,CDCl
3)δ7.33-7.30(m,2H),7.24-7.22(m,1H),7.07-7.05(m,2H),4.72-4.49(m,1H),4.10-3.97(m,2H),3.03-3.01(m,1H),2.96(s,1H),2.88(s,1H),2.80(s,6H),2.41-2.08(m,6H),1.96-1.66(m,4H),1.61-1.45(m,4H),1.26-1.24(m,3H)。MS-ESI计算值[M+H]
+482,实测值482。
第四步
将化合物10-4(143mg,0.173mmol)溶于乙醇(3mL),四氢呋喃(3mL)和水(3mL),加入氢氧化钠(20.8mg),反应液在50℃下搅拌2小时,反应液减压浓缩，粗品经高效液相色谱法分离(酸性,盐酸体系)得到化合物10的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.32-7.28(m,2H),7.24-7.18(m,3H),4.07-3.99(m,5H),3.58-3.33(m,2H),3.02-2.99(m,1H),2.89-2.65(m,6H),2.63-2.58(m,1H),2.43-2.37(m,1H), 2.06-1.99(m,1H),1.93-1.81(m,3H),1.67(s,6H),1.64-1.57(m,2H),1.43-1.38(m,1H)。MS-ESI计算值[M+H]
+386,实测值386。
实施例11
合成路线:
第一步
将化合物11-1(64.9mg,0.298mmol),O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(124mg,0.326mmol)和N,N-二异丙基乙胺(105mg,0.814mmol)溶于N,N-二甲基甲酰胺(2mL)中,反应液在25℃下搅拌1小时,之后向反应液中加入化合物1-6(100mg,0.271mmol),新的反应液在25℃下继续搅拌12小时。将其用水(30mL)稀释,用乙酸乙酯(10mL x 3)萃取,用饱和食盐水(30mL x 1)洗涤,无水硫酸钠干燥,过滤,母液浓缩,粗产物经过薄层层析法分离(1:1石油醚/乙酸乙酯,Rf＝0.8)得到化合物11-2。
1H NMR(400MHz,CDCl
3)δ7.34-7.31(m,2H),7.25-7.23(m,1H),7.11-7.06(m,2H),5.45-5.44(m,1H),4.64-4.58(m,1H),4.10-4.05(m,1H),4.00-3.91(m,3H),3.50-3.32(m,2H),3.24-3.21(m,2H),2.41-2.33(m,1H),2.20-2.07(m,2H),1.76-1.73(m,2H),1.54-1.43(m,11H),1.28-1.25(m,8H)。MS-ESI计算值[M+H]
+568,实测值568。
第二步
将化合物11-2(150mg,0.264mmol)溶于四氢呋喃(2mL)和无水乙醇(2mL),将氢氧化钠(21.1mg,0.528mmol)溶于水(2mL)后滴加至溶液中,在20℃下搅拌2小时。用盐酸(1mol/L)调pH值到5后减压浓缩,得到粗品化合物11-3。MS-ESI计算值[M+H]
+472,实测值472。
第三步
将化合物11-3(150mg,0.318mmol)溶于二氯甲烷(2mL)中,反应液在0℃下加入三氟乙酸(109mg, 0.954mmol),自然升温至25℃后搅拌反应1小时。减压浓缩除去溶剂,剩余物经过高效液相色谱法分离(酸性,盐酸体系)得到化合物11的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.34-7.30(m,2H),7.26-7.19(m,3H),4.22-4.07(m,3H),4.00-3.97(m,2H),3.43-3.40(m,2H),3.05-3.01(m,3H),2.62-2.58(m,1H),2.44-2.37(m,1H),2.04-1.98(m,1H),1.87-1.75(m,3H),1.64-1.55(m,2H),1.45-1.40(m,7H)。MS-ESI计算值[M+H]
+372,实测值372。
实施例12
合成路线:
第一步
参照实施例11第一步得到化合物12-2。
1H NMR(400MHz,CDCl
3)δ7.34-7.31(m,2H),7.25-7.23(m,1H),7.11-7.06(m,2H),5.36-5.32(m,1H),4.64-4.61(m,1H),4.51-4.44(m,1H),4.09-4.04(m,1H),3.99-3.95(m,1H),3.75-3.40(m,2H),3.26-3.17(m,1H),2.40-2.33(m,1H),2.20-2.07(m,2H),1.97-1.83(m,2H),1.76-1.69(m,2H),1.60-1.58(m,2H),1.51-1.43(m,11H),0.99-1.86(m,6H)。MS-ESI计算值[M+H]
+568,实测值568。
第二步
参照实施例11第二步得到化合物12-3。MS-ESI计算值[M+H]
+472,实测值472。
第三步
参照实施例11第三步得到化合物12的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.34-7.30(m,2H),7.26-7.19(m,3H),4.38-4.32(m,1H),4.24-3.97(m,4H),3.79-3.16(m,3H),3.02-3.00(m,1H),2.63-2.57(m,1H),2.46-2.35(m,1H),2.22-2.14(m,1H),2.07-1.98(m,1H),1.91-1.75(m,3H),1.70-1.53(m,2H),1.44-1.39(m,1H),1.12-1.08(m,3H),1.03-1.00(m,3H)。MS-ESI计算值[M+H]
+372,实测值372。
实施例13
合成路线:
第一步
参照实施例11第一步得到化合物13-2。MS-ESI计算值[M+H]
+540,实测值540。
第二步
参照实施例11第二步得到化合物13-3。MS-ESI计算值[M+H]
+444,实测值444。
第三步
参照实施例11第三步得到化合物13的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.29(m,2H),7.25-7.18(m,3H),4.47-4.42(m,1H),4.17-3.98(m,4H),3.69-3.43(m,2H),3.27-3.15(m,1H),3.02-2.99(m,1H),2.61-2.56(m,1H),2.45-2.35(m,1H),2.06-1.96(m,1H),1.93-1.72(m,3H),1.69-1.51(m,2H),1.48-1.39(m,4H)。MS-ESI计算值[M+H]
+344,实测值344。
实施例14
合成路线:
第一步
参照实施例11第一步得到化合物14-2。
1H NMR(400MHz,CDCl
3)δ7.34-7.30(m,2H),7.27-7.23(m,1H),7.10-7.05(m,2H),5.25-5.24(m,1H),4.69-4.57(m,1H),4.19-4.04(m,1H),4.01-3.94(m,1H),3.59-3.37(m,2H),3.29-3.14(m,1H),2.80(s,2H),2.47-2.32(m,3H),2.21-2.04(m,2H),1.76-1.69(m,2H),1.60-1.42(m,3H)1.29-1.24(m,6H)。MS-ESI计算值[M+H]
+469,实测值469。
第二步
参照实施例11第三步得到化合物14的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.29(m,2H),7.25-7.18(m,3H),4.16-4.06(m,4H),3.84-3.77(m,1H),3.51-3.46(m,1H),3.27-3.19(m,1H),3.04-2.97(m,1H),2.63-2.52(m,3H),2.40-2.35(m,1H),1.99-1.96(m,1H),1.86-1.77(m,3H),1.65-1.51(m,2H),1.44-1.39(m,1H),1.28(s,6H)MS-ESI计算值[M+H]
+373,实测值373。
实施例15
合成路线:
第一步
参照实施例11第一步得到化合物15-2。MS-ESI计算值[M+H]
+526,实测值526。
第二步
参照实施例11第二步得到化合物15-3。MS-ESI计算值[M+H]
+430,实测值430。
第三步
参照实施例11第三步得到化合物15的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.29(m,2H),7.24-7.18(m,3H),4.17-3.91(m,6H),3.54-3.41(m,2H),3.30-3.22(m,1H),3.02-3.00(m,1H),2.64-2.60(m,1H),2.42-2.36(m,1H),2.06-1.98(m,1H),1.89-1.73(m,3H),1.69-1.52(m,2H),1.43-1.38(m,1H)。MS-ESI计算值[M+H]
+330,实测值330。
实施例16
合成路线:
第一步
参照实施例11第一步得到化合物16-2。MS-ESI计算值[M+H]
+554,实测值554。
第二步
参照实施例11第二步得到化合物16-3。MS-ESI计算值[M+H]
+458,实测值458。
第三步
参照实施例11第三步得到化合物16的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.29(m,2H),7.24-7.18(m,3H),4.17-4.08(m,3H),4.05-3.85(m,2H),3.52-3.33(m,2H),3.02-3.00(m,1H),2.63-2.56(m,1H),2.43-2.37(m,1H),2.04-1.98(m,1H),1.86-1.80(m,3H),1.68-1.57(m,8H),1.44-1.39(m,1H)。MS-ESI计算值[M+H]
+358,实测值358。
实施例17
合成路线:
第一步
将化合物17-1(109mg,0.503mmol),O-(7-偶氮苯并三氮唑)-N,N,N,N,-四甲基脲六氟磷酸酯(225mg,0.593mmol),N,N-二异丙基乙胺(95.8mg,0.741mmol)溶于N,N-二甲基甲酰胺(3mL),在25℃下搅拌0.5小时。将化合物1-6(200mg,0.494mmol)加入到反应液中。反应液在25℃下搅拌反应11.5小时,反应液倒入饱和碳酸氢钠水溶液(10mL)中,用乙酸乙酯萃取(20mL×3),合并有机相,有机相用饱和氯化钠水溶液洗涤(20mL×1),无水硫酸钠干燥,过滤,粗产物经过柱层析法分离(2/1石油醚/乙酸乙酯,Rf＝0.15)得到化合物17-2。MS-ESI计算值[M+H]
+568,实测值568。
第二步
将化合物17-2(204mg,0.359mmol)和氢氧化钠(43.1mg,0.450mmol)溶于四氢呋喃(1mL),乙醇(1mL)和水(1mL)中,反应液在20℃条件下搅拌反应2小时,减压浓缩后得到化合物17-3。MS-ESI计算值[M+H]
+472,实测值472。
第三步
将化合物17-3(216mg,0.458mmol)溶于盐酸甲醇(5mL)中。反应液在25℃氮气条件下搅拌反应1小时,反应液减压浓缩后剩余物经过高效液相色谱法分离(酸性,盐酸体系)得到化合物17的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.19(m,5H),4.17-4.10(m,4H),3.53-3.49(m,1H),3.48-3.46(m,1H),3.42-3.31(m,1H),3.02-3.00(m,1H),2.82-2.73(m,2H),2.62(s,1H),2.41-2.37(m,1H),2.01-2.03(m,1H),1.88-1.73(m,3H),1.63-1.50(m,2H),1.42(s,7H)。MS-ESI计算值[M+H]
+372,实测值372。
实施例18
合成路线:
第一步
将化合物18-1(56.5mg,0.299mmol)溶于无水N,N-二甲基甲酰胺(2mL)中,向反应液中加入N,N-二异丙基乙胺(70.2mg,0.543mmol)和O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(155mg,0.407mmol)。反应液在30℃下搅拌1小时。再向反应液中加入化合物1-6(100mg,0.271mmol)。反应液在30℃下搅拌4小时。向反应液中加入水(10mL),用乙酸乙酯萃取(10mL x 3),合并有机相,用饱和氯化钠溶液(20mL x 1)洗,有机相用无水硫酸钠干燥,过滤,粗产物经过薄层层析法分离(1/1石油醚/乙酸乙酯,Rf＝0.41)得到化合物18-2。MS-ESI计算值[M+H]
+540,实测值540。
第二步
将化合物18-2(130mg,0.238mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(19.1mg,0.477mmol)。反应液在50℃下搅拌反应3小时,减压浓缩除去溶剂,残渣用水(10mL)稀释,用乙酸乙酯萃取(10mL x 3),合并有机相,用饱和氯化钠(30mL x 1)洗，有机相用无水硫酸钠干燥,过滤,浓缩得到化合物18-3。MS-ESI计算值[M+H]
+444,实测值444。
第三步
将化合物18-3(100mg,0.225mmol)溶于乙酸乙酯(42mL)中,向反应液中加盐酸乙酸乙酯(4mol/L,0.563mL,2.25mmol)。反应液在25℃下搅拌反应2小时,减压浓缩除去溶剂。用高效液相色谱法(酸性,盐酸体系)制备得到化合物18的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.25-7.21(m,3H),4.19-4.11(m,4H),3.68-3.61(m,1H),3.52-3.46(m,1H),3.24-3.21(m,3H),3.04-3.02(m,1H),2.89-2.83(m,2H),2.65-2.64(m,1H),2.42-2.40(m,1H),2.07-2.02(m,1H),1.91-1.73(m,3H),1.72-1.53(m,2H),1.45-1.42(m,1H)。MS-ESI计算值[M+H]
+344,实测值344。
实施例19
合成路线:
第一步
将化合物19-1(429mg,4.12mmol)溶于无水N,N-二甲基甲酰胺(10mL)中,向反应液中加入N,N-二异丙基乙胺(665mg,5.15mmol),1-羟基苯并三氮唑(556mg,4.12mmol)和1-(3-二甲氨基丙基)-3-乙醛盐酸盐(789mg,4.12mmol)。反应液在30℃下搅拌1小时。再向反应液中加入化合物1-6(800mg,2.06mmol)。反应液在30℃下搅拌12小时。向反应液中加入水(20mL),用乙酸乙酯萃取(10mL x 3),合并有机相,用饱和氯化钠(20mL x 1)洗,有机相用无水硫酸钠干燥,过滤,粗产物经过薄层层析法分离(1/1石油醚/乙酸乙酯,Rf＝0.51)得到化合物19-2。MS-ESI计算值[M+H]
+455,实测值455。
第二步
将化合物19-2(700mg,1.54mmol)溶于四氢呋喃(4mL)和水(4mL)中,向反应液中加氢氧化钠(123mg,3.08mmol)。反应液在50℃下搅拌反应3小时,减压浓缩除去溶剂,残渣用水(10mL)稀释,并用盐酸水溶液(1mol/L)调节pH到4左右。用高效液相色谱法(酸性,盐酸体系)制备得到化合物19的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.36-7.32(m,2H),7.28-7.26(m,1H),7.17-7.16(m,2H),4.22-4.20(m,1H),4.16-4.12(m,1H),4.07-4.05(m,2H),3.84-3.56(m,2H),3.49-3.20(m,1H),2.95-2.91(m,1H),2.53-2.49(m,1H),2.46-2.40(m,1H),1.97-1.92(m,1H),1.84-1.69(m,3H),1.65-1.58(m,1H),1.53-1.49(m,1H),1.45-1.41(m,1H),1.39(s,6H)。MS-ESI计算值[M+H]
+359,实测值359。
实施例20
合成路线:
第一步
将化合物20-1(53.8mg,0.407mmol)溶于无水N,N-二甲基甲酰胺(2mL)中,向反应液中加入N,N-二异丙基乙胺(87.7mg,0.679mmol),O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(206mg,0.543mmol)。反应液在30℃下搅拌1小时。再向反应液中加入化合物1-6(100mg,0.271mmol)。反应液在30℃下搅拌4小时。向反应液中加入水(10mL),用乙酸乙酯萃取(10mL x 3),合并有机相,用饱和氯化钠(20mL x 1)洗,有机相用无水硫酸钠干燥,过滤,粗产物经过薄层层析法分离(1/1石油醚/乙酸乙酯,Rf＝0.40)得到化合物20-2。MS-ESI计算值[M+H]
+483,实测值483。
第二步
将化合物20-2(100mg,0.207mmol)溶于四氢呋喃(1mL)和水(1mL)中,向反应液中加氢氧化钠(24.9mg,0.622mmol)。反应液在60℃下搅拌反应3小时,减压浓缩除去溶剂,残渣用水(10mL)稀释,并用盐酸水溶液(1mol/L)调节pH＝7左右。用高效液相色谱法(中性体系)制备得到化合物20。
1H NMR(400MHz,CD
3OD)δ7.27-7.23(m,2H),7.16-7.12(m,1H),7.08-7.06(m,2H),3.96-3.92(m,1H),3.73-3.64(m,3H),3.56-3.50(m,1H),3.30-3.23(m,1H),2.67-2.64(m,2H),2.53-2.50(m,2H),2.36-2.32(m,1H),2.18-2.13(m,1H),1.99-1.92(m,1H),1.81-1.63(m,6H),1.11-1.04(m,2H)。MS-ESI计算值[M+H]
+373,实测值373。
实施例21
合成路线:
第一步
将化合物1-6(100mg,0.271mmol)和化合物21-1(50.0mg,0.299mmol)溶于乙腈(6mL)中,向反应液中加入三乙胺(54.9mg,0.543mmol)。反应液在50℃下搅拌12小时。将反应液直接浓缩,粗产物经过薄层层析法分离(10/1二氯甲烷/甲醇,Rf＝0.50)得到化合物21-2。MS-ESI计算值[M+H]
+455,实测值455。
第二步
将化合物21-2(120mg,0.264mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(31.7mg,0.792mmol)。反应液在60℃下搅拌反应3小时,减压浓缩除去溶剂,残渣用水(10mL)稀释,并用盐酸水溶液(1mol/L)调节pH＝5左右。用高效液相色谱法(酸性,盐酸体系)制备得到化合物21的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.24-4.14(m,3H),3.56-3.49(m,2H),3.33-3.26(m,4H),3.05-3.03(m,1H),2.72-2.67(m,1H),2.46-2.43(m,1H),2.19-2.09(m,4H),1.98-1.94(m,3H),1.71-1.66(m,1H),1.43-1.41(m,1H),1.28(s,6H)。MS-ESI计算值[M+H]
+359,实测值359。
实施例22
合成路线:
第一步
参照实施例11第一步得到化合物22-2。MS-ESI计算值[M+H]
+602,实测值602。
第二步
参照实施例11第二步得到化合物22-3。MS-ESI计算值[M+H]
+506,实测值506。
第三步
参照实施例11第三步得到化合物22的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.60-7.43(m,5H),7.30-7.13(m,5H),5.55-5.51(m,1H),4.21-4.01(m,4H),3.48-3.37(m,2H),3.24-3.05(m,1H),2.98-2.94(m,1H),2.56-2.55(m,1H),2.41-2.17(m,1H),2.08-1.94(m,1H),1.86-1.68(m,2H),1.57-1.35(m,4H)。MS-ESI计算值[M+H]
+406,实测值406。
实施例23
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物23-1(61.1mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N- 二异丙基乙胺(128mg,0.988mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,无水硫酸钠干燥,减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.40)得到化合物23-2。MS-ESI计算值[M+H]
+454,实测值454。
第二步
将化合物23-2(203mg,0.439mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(17.5mg,0.439mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,用盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物23的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.37-4.29(m,2H),4.23-4.08(m,4H),3.50-3.42(m,2H),3.30-3.23(m,1H),3.06-3.02(m,1H),2.99-2.98(m,6H),2.68-2.64(m,1H),2.44-2.39(m,1H),2.08-2.03(m,1H),1.95-1.78(m,3H),1.73-1.59(m,2H),1.46-1.40(m,1H)。MS-ESI计算值[M+H]
+358,实测值358。
实施例24
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物24-1(76.6mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.988mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.42)得到化合物24-2。MS-ESI计算值[M+H]
+480,实测值480。
第二步
将化合物24-2(201mg,0.412mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(16.5mg,0.412mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物24的盐酸盐。
1H NMR(400MHz, CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.48-4.37(m,2H),4.20-4.13(m,4H),3.77-3.76(m,2H),3.51-3.47(m,2H),3.30-3.20(m,3H),3.04-3.02(m,1H),2.68-2.64(m,1H),2.44-2.39(m,1H),2.20-2.17(m,2H),2.10-2.06(m,3H),1.94-1.80(m,3H),1.75-1.59(m,2H),1.45-1.40(m,1H)。MS-ESI计算值[M+H]
+384,实测值384。
实施例25
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物25-1(86.1mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.42)得到化合物25-2。MS-ESI计算值[M+H]
+496,实测值496。
第二步
将化合物25-2(205mg,0.411mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(16.4mg,0.411mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物25的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.46-4.35(m,2H),4.20-4.05(m,6H),3.94-3.88(m,2H),3.61-3.58(m,2H),3.53-3.49(m,2H),3.33-3.22(m,3H),3.04-3.03(m,1H),2.66-2.65(m,1H),2.44-2.39(m,1H),2.09-2.03(m,1H),1.95-1.80(m,3H),1.75-1.59(m,2H),1.45-1.40(m,1H)。MS-ESI计算值[M+H]
+400,实测值400。
实施例26
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物26-1(84.9mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.42)得到化合物26-2。MS-ESI计算值[M+H]
+494,实测值494。
第二步
将化合物26-2(200mg,0.402mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(16.1mg,0.402mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物26的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.36-4.26(m,2H),4.20-4.05(m,4H),3.59-3.43(m,4H),3.30-3.23(m,1H),3.13-3.11(m,2H),3.04-2.99(m,1H),2.71-2.67(m,1H),2.43-2.40(m,1H),2.09-2.05(m,1H),1.93-1.83(m,8H),1.71-1.67(m,2H),1.61-1.56(m,1H),1.42-1.39(m,1H)。MS-ESI计算值[M+H]
+398,实测值398。
实施例27
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物27-1(77.8mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.47)得到化合物27-2。MS-ESI计算值[M+H]
+482,实测值482。
第二步
将化合物27-2(209mg,0.428mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(17.1mg,0.428mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物27的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.35-4.25(m,2H),4.20-4.11(m,4H),3.57-3.46(m,2H),3.32-3.25(m,5H),3.04-3.02(m,1H),2.70-2.62(m,1H),2.44-2.40(m,1H),2.10-2.03(m,1H),1.95-1.81(m,3H),1.72-1.57(m,2H),1.45-1.40(m,1H),1.38-1.33(m,6H)。MS-ESI计算值[M+H]
+386,实测值386。
实施例28
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物28-1(69.4mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.43)得到化合物28-2。MS-ESI计算值[M+H]
+468,实测值468。
第二步
将化合物28-2(206mg,0.437mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(17.5mg,0.437mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物28的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.14-7.10(m,2H),7.05-7.00(m,3H),4.06-3.92(m,5H),3.83-3.81(m,1H),3.43-3.37(m,1H),3.30-.3.21(m,1H),3.10-3.00(m,1H),2.84-2.81(m,1H),2.48-2.44(m,1H),2.22-2.18(m,1H),1.99(s,3H),1.89-1.82(m,1H),1.70-1.56(m,3H),1.49-1.35(m,2H),1.24-1.18(m,1H)。MS-ESI计算值[M+H]
+372,实测值372。
实施例29
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物29-1(86.1mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.52)得到化合物29-2。MS-ESI计算值[M+H]
+496,实测值496。
第二步
将化合物29-2(208mg,0.383mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(15.3mg,0.383mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物29的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.22-4.05(m,6H),3.64-3.60(m,1H),3.49-3.45(m,1H),3.30-3.23(m,1H),3.06-3.02(m,1H),2.67-2.56(m,2H),2.43-2.37(m,1H),2.05-2.02(m,1H),1.89-1.77(m,3H),1.68-1.56(m,2H),1.45-1.40(m,1H),1.18-1.15(d,J＝6.8,6H)。MS-ESI计算值[M+H]
+400,实测值400。
实施例30
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物30-1(84.9mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.43)得到化合物30-2。MS-ESI计算值[M+H]
+494,实测值494。
第二步
将化合物30-2(201mg,401umol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(16.1mg,402umol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物30的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.26-7.21(m,3H),4.27-4.19(m,5H),4.16-4.12(m,1H),3.57-3.53(m,4H),3.25-3.20(m,1H),3.04-3.03(m,1H),2.67-2.66(m,1H),2.52-2.50(m,2H),2.49-2.48(m,1H),2.14-2.05(m,3H),1.78-1.68(m,3H),1.66-1.50(m,2H),1.43-1.41(m,1H)。MS-ESI计算值[M+H]
+398,实测值398。
实施例31
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物31-1(86.1mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.34)得到化合物31-2。MS-ESI计算值[M+H]
+496,实测值496。
第二步
将化合物31-2(209mg,0.352mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(14.1mg,0.352mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物31的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.27-7.21(m,3H),4.21-4.15(m,4H),3.69-3.66(m,1H),3.56-3.48(m,1H),3.45-3.42(m,2H),3.31-3.26(m,4H),3.05-3.02(m,2H),2.99-2.97(m,3H),2.70-2.60(m,1H),2.44-2.38(m,1H),2.13-2.01(m,1H),1.93-1.77(m,3H),1.69-1.62(m,2H),1.38-1.35(m,6H)。MS-ESI计算值[M+H]
+400,实测值400。
实施例32
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物32-1(94.4mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.32)得到化合物32-2。MS-ESI计算值[M+H]
+510,实测值510。
第二步
将化合物32-2(101mg,67.0umol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(2.68mg,67.0umol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物32的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.32(m,2H),7.25-7.20(m,3H),4.20-4.05(m,6H),3.88-3.82(m,2H),3.73-3.66(m,1H),3.59-3.46(m,5H),3.27-3.17(m,3H),3.05-3.00(m,3H),2.62-2.59(m,1H),2.44-2.38(m,1H),2.06-1.97(m,1H),1.94-1.78(m,3H),1.63-1.59(m,2H),1.47-1.41(m,1H)。MS-ESI计算值[M+H]
+414,实测值414。
实施例33
合成路线:
第一步
将化合物1-6(200mg,0.494mmol)和化合物33-1(86.6mg,0.593mmol)溶于N,N-二甲基甲酰胺(4mL)中,向反应液中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟磷盐(282mg,0.741mmol),N,N-二异丙基乙胺(128mg,0.998mmol)。反应液在25℃下搅拌12小时。加水(10mL),用乙酸乙酯(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.34)得到化合物33-2。MS-ESI计算值[M+H]
+497,实测值497。
第二步
将化合物33-2(186mg,0.334mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(13.4mg,0.334mmol)。反应液在50℃下搅拌反应12小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物33的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.35-7.31(m,2H),7.27-7.21(m,3H),4.25-4.20(m,1H),4.18-4.16(m,2H),3.30-3.25(m,4H),3.06-3.04(m,1H),2.67-2.63(m,1H),2.59(s,2H),2.50-2.43(m,1H),2.15-2.02(m,4H),1.90-1.83(m,1H),1.67-1.64(m,1H),1.46-1.42(m,1H),1.28-1.26(m,6H)。MS-ESI计算值[M+H]
+401,实测值401。
实施例34
合成路线:
第一步
将化合物34-1(200mg,0.127mmol)溶于二氯甲烷(2mL)中,在0度下向反应液中加入草酰氯(194mg,0.153mmol)和N,N-二甲基甲酰胺(9.30mg,0.127mol)。反应液在0-15℃下搅拌1小时。将反应液直接浓缩,得到粗产品化合物34-2。
第二步
将化合物34-2(200mg,1.14mmol)和化合物1-6(210mg,0.569mmol)溶于二氯甲烷(5mL)中,向反应液中加入三乙胺(230mg,2.28mmol)。反应液在15℃下搅拌3小时。加水(10mL),用二氯甲烷(10mL x 3)萃取,有机相用饱和氯化钠(20mL x 1)洗,用无水硫酸钠干燥,过滤,滤液减压浓缩。粗产物经过薄层层析法分离(1：1石油醚/乙酸乙酯,Rf＝0.31)得到化合物34-3。MS-ESI计算值[M+H]
+508,实测值508。
第三步
将化合物34-3(144mg,0.282mmol)溶于四氢呋喃(2mL)和水(2mL)中,向反应液中加氢氧化钠(22.6mg,0.564mmol)。反应液在50℃下搅拌反应3小时,减压浓缩除去有机溶剂,加盐酸(1mol/L)调节pH至5。用高效液相色谱法(酸性,盐酸体系)制备得到化合物34的盐酸盐。
1H NMR(400MHz,CD
3OD)δ7.33-7.31(m,2H),7.27-7.21(m,3H),4.23-4.15(m,4H),3.60-3.59(m,1H),3.57-3.56(m,2H),3.42-3.41(m,1H),3.40-3.39(m,2H),3.38-3.37(m,1H),3.05-2.99(m,5H),2.65-2.63(m,1H),2.48-2.47(m,1H),2.02-2.01(m,1H),1.94-1.80(m,8H),1.67-1.57(m,3H),1.43-1.42(m,1H)。MS-ESI计算值[M+H]
+412,实测值412。
生物化学检测：
实验例1：酶活性评价
本试验目的是检测化合物对LSD1的体外抑制活性。本试验采用的酶为人源LSD1，标准底物为组蛋白H3K4me肽(20μM)，采用酶荧光偶联法，通过辣根过氧化酶(HPR)和荧光试剂Amplex Red联合检测LSD1反应后生成的H
2O
2的方法测定化合物的活性。从10μM开始3倍稀释，检测化合物的10个浓度下IC
50值。化合物在加入底物开始反应前，酶和底物共孵化30分钟。荧光检测器：EnVision，激发波长：Ex/Em＝530/590nM。
测试化合物对LSD1抑制活性，结果如表1所示。
表1：本发明化合物体外酶活性筛选试验结果
| 化合物编号 | IC 50(nM) | 化合物编号 | IC 50(nM) |
| 化合物1的盐酸盐 | 116.5 | 化合物18的盐酸盐 | 8.54 |
| 化合物2的盐酸盐 | 2289 | 化合物19的盐酸盐 | 317.8 |
| 化合物3的盐酸盐 | 388.5 | 化合物20的盐酸盐 | 935.2 |
| 化合物4的盐酸盐 | 14.62 | 化合物21的盐酸盐 | 34.06 |
| 化合物5的盐酸盐 | 46.42 | 化合物22的盐酸盐 | 29.29 |
| 化合物6的盐酸盐 | 86.24 | 化合物23的盐酸盐 | 15.72 |
| 化合物7的盐酸盐 | 640 | 化合物24的盐酸盐 | 13.19 |
| 化合物8的盐酸盐 | 12.21 | 化合物25的盐酸盐 | 68.14 |
| 化合物9的盐酸盐 | 60.42 | 化合物26的盐酸盐 | 18.83 |
| 化合物10的盐酸盐 | 36.58 | 化合物27的盐酸盐 | 8.74 |
| 化合物11的盐酸盐 | 11.36 | 化合物28的盐酸盐 | 41.1 |
| 化合物12的盐酸盐 | 37.35 | 化合物29的盐酸盐 | 66.24 |
| 化合物13的盐酸盐 | 7.05 | 化合物30的盐酸盐 | 84.8 |
| 化合物14的盐酸盐 | 44.76 | 化合物31的盐酸盐 | 35.53 |
| 化合物15的盐酸盐 | 12.89 | 化合物32的盐酸盐 | 27.99 |
| 化合物16的盐酸盐 | 3.7 | 化合物34的盐酸盐 | 42.15 |
| 化合物17的盐酸盐 | 12.89 |
结论：本发明化合物对LSD1抑制活性明显。
实验例2：对NCI-H1417细胞增殖抑制活性评价：
实验目的：检测待测化合物对H1417细胞增殖抑制活性。
实验材料：RPMI 1640培养基，胎牛血清，Promega CellTiter-Glo试剂。NCI-H1417细胞系购自ATCC。Envision多标记分析仪(PerkinElmer)。
实验方法：将化合物溶解到10mM,在化合物板里用DMSO 5倍稀释化合物，化合物起始为2mM，用Bravo进行三倍稀释，10个浓度，用Echo转板250nL到空白的384细胞板的上下双复孔，往转了250nL DMSO/化合物里面加入每孔/1000个细胞/50μL的细胞悬液，化合物稀释了200倍，即起始作用浓度是10μM。细胞板置于二氧化碳培养箱中培养10天。向细胞板中加入每孔25μL的Promega CellTiter-Glo试剂，室温振荡10分钟使发光信号稳定。采用PerkinElmer Envision多标记分析仪读数。
数据分析：利用方程式(Max-Ratio)/(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(XLFIT5中205模式得出，iDBS)。
测试化合物对NCI-H1417细胞增殖抑制活性，结果如表2所示。
表2：本发明化合物对NCI-H1417细胞增殖抑制试验结果
| 化合物编号 | IC 50(nM) | 化合物编号 | IC 50(nM) |
| 化合物1的盐酸盐 | 32.49 | 化合物16的盐酸盐 | 0.90 |
| 化合物5的盐酸盐 | 1.28 | 化合物17的盐酸盐 | 3.95 |
| 化合物6的盐酸盐 | 1.74 | 化合物18的盐酸盐 | 2.84 |
| 化合物9的盐酸盐 | 2.28 | 化合物19的盐酸盐 | 10.25 |
| 化合物10的盐酸盐 | 8.17 | 化合物21的盐酸盐 | 3.04 |
| 化合物11的盐酸盐 | 2.79 | 化合物22的盐酸盐 | 2.26 |
| 化合物12的盐酸盐 | 7.20 | 化合物23的盐酸盐 | 6.95 |
| 化合物13的盐酸盐 | 1.83 | 化合物25的盐酸盐 | 22.38 |
| 化合物14的盐酸盐 | 24.05 | 化合物30的盐酸盐 | 19.09 |
| 化合物15的盐酸盐 | 2.18 | 化合物32的盐酸盐 | 10.39 |
结论：本发明化合物对NCI-H1417细胞增殖抑制活性明显。
实验例3：对HL60细胞增殖抑制活性评价：
实验目的：检测待测化合物对HL60细胞增殖抑制活性。
实验材料：RPMI-1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。HL60细胞系购自南京科佰生命科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：将HL60细胞种于白色384孔板中，40μL细胞悬液每孔，其中包含600个HL60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行5倍稀释至第10个浓度，即从2mM稀释至1.024nM，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10μL每孔到细胞板中。细胞板置于二氧化碳培养箱中培养6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。向此细胞板每孔加入20μL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)。
测试化合物对HL60细胞增殖抑制活性，结果如表3所示。
表3：本发明化合物对HL60细胞增殖抑制试验结果
| 化合物编号 | IC 50(nM) | 化合物编号 | IC50(nM) |
| 化合物1的盐酸盐 | 15.29 | 化合物17的盐酸盐 | 0.6 |
| 化合物5的盐酸盐 | 0.73 | 化合物18的盐酸盐 | 1.05 |
| 化合物6的盐酸盐 | 1.9 | 化合物19的盐酸盐 | 3.14 |
| 化合物10的盐酸盐 | 7.97 | 化合物21的盐酸盐 | 4.51 |
| 化合物11的盐酸盐 | 0.46 | 化合物22的盐酸盐 | 0.59 |
| 化合物12的盐酸盐 | <0.005 | 化合物23的盐酸盐 | 3.52 |
| 化合物13的盐酸盐 | 0.79 | 化合物25的盐酸盐 | 14.32 |
| 化合物14的盐酸盐 | 6.77 | 化合物30的盐酸盐 | 7.46 |
| 化合物15的盐酸盐 | 2.30 | 化合物32的盐酸盐 | 2.89 |
| 化合物16的盐酸盐 | 0.32 |
结论：本发明化合物对HL60细胞增殖抑制活性明显。
实验例4：对MV-4-11细胞增殖抑制活性评价：
实验目的：检测待测化合物对MV-4-11细胞增殖抑制活性。
实验材料：IMDM培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。MV-4-11细胞系购自南京科佰生命科技有限公司。Nivo多标记分析仪(PerkinElmer)。
实验方法：将MV-4-11细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含6000个MV-4-11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行5倍稀释至第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。细胞板置于二氧化碳培养箱中培养6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。向此细胞板每孔加入25μL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)。
测试化合物对MV-4-11细胞增殖抑制活性，结果如表4所示。
表4：本发明化合物对MV-4-11细胞增殖抑制试验结果
| 化合物编号 | IC 50(nM) | 化合物编号 | IC 50(nM) |
| 化合物1的盐酸盐 | 26.67 | 化合物17的盐酸盐 | 2.41 |
| 化合物5的盐酸盐 | 0.4 | 化合物18的盐酸盐 | 0.56 |
| 化合物6的盐酸盐 | 1.16 | 化合物19的盐酸盐 | 3.46 |
| 化合物10的盐酸盐 | 2.63 | 化合物21的盐酸盐 | 1.74 |
| 化合物11的盐酸盐 | 1.18 | 化合物22的盐酸盐 | 1.16 |
| 化合物12的盐酸盐 | 0.99 | 化合物23的盐酸盐 | 1.93 |
| 化合物13的盐酸盐 | 1.01 | 化合物25的盐酸盐 | 3.74 |
| 化合物14的盐酸盐 | 7.22 | 化合物30的盐酸盐 | 3.56 |
| 化合物15的盐酸盐 | 1.32 | 化合物32的盐酸盐 | 1.7 |
| 化合物16的盐酸盐 | <0.128 |
结论：本发明化合物对MV-4-11细胞增殖抑制活性明显。
实验例5：化合物药代动力学评价
实验目的：测试化合物在CD-1小鼠体内的药代动力学
实验材料：
CD-1小鼠(雄性,7～9周龄,上海斯莱克)
实验操作：
以标准方案测试化合物静脉注射及口服给药后的啮齿类动物药代特征,实验中候选化合物配成澄清溶液,给予小鼠单次静脉注射及口服给药。静注及口服溶媒为10％二甲基亚砜与90％的10％的羟丙基β环糊精配成的混合溶媒。该项目使用四只雄性CD-1小鼠,两只小鼠进行静脉注射给药,给药剂量为1mg/kg,收集0h(给药前)和给药后0.0833,0.25,0.5,1,2,4,8,24h的血浆样品,另外两只小鼠口服灌胃给药,给药剂量为2mg/kg,收集0h(给药前)和给药后0.25、0.5,1,2,4,8,24h的血浆样品,收集24小时内的全血样品,3000g离心15分钟,分离上清得血浆样品,加入4倍体积含内标的乙腈溶液沉淀蛋白,离心取上清液加入等倍体积的水再离心取上清进样,以LC-MS/MS分析方法定量分析血药浓度,并计算药代参数,如达峰浓度(C
max),清除率(CL),半衰期(T
1/2),组织分布(Vdss),药时曲线下面积(AUC
0-last),生物利用度(F)等。
实验结果如表5所示：
表5药代动力学测试结果
结论：本发明化合物具有良好的药代动力学性质,包括良好的口服生物利用度,口服暴露量,半衰期和清除率等。
实验例6：化合物对CT-26小鼠结肠癌移植瘤模型的体内药效学研究
3.1实验目的：
本实验的目的是研究本发明化合物对CT-26小鼠结肠癌移植瘤模型体内药效进行评估。
3.2实验动物：
种属：小鼠
品系：BALB/c小鼠
周龄及体重：7周龄,体重18-23克
性别：雌性
供应商：上海必凯实验动物有限公司
3.3实验方法与步骤
3.3.1细胞培养
名称：CT-26(小鼠结肠癌细胞)
细胞来源：ATCC
细胞培养：培养液为含有10％胎牛血清的1640培养基,培养条件为37℃,5％二氧化碳。传代比例为1:2～1:3,每周传代2～3次。
3.3.2肿瘤细胞接种
将0.1mL(3×10
5个)细胞皮下接种于每只小鼠的右后背。同日将动物根据体重随机分组。
3.3.3受试物的配制
实验用溶媒为0.5％甲基纤维素溶液,配制方法为称取5g甲基纤维素,溶解于800mL超纯水中,搅拌均匀后用超纯水定容至1000mL。受试物用溶媒溶解,配制成一定浓度均一溶液,于4℃保存。
3.3.4肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b
2,a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T
RTV/C
RTV×100％(T
RTV：治疗组RTV；C
RTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume,RTV),计算公式为RTV＝V
t/V
0,其中V
0是分组给药时(即d
0)测量所得平均肿瘤体积,V
t为某一次测量时的平均肿瘤体积,T
RTV与C
RTV取同一天数据。
3.4实验结果如表6所示：
表6受试化合物对CT-26小鼠结肠癌移植瘤模型的抑瘤药效评价(基于分组后第32天肿瘤体积计算得出)
PD-L1单抗来源：BioXcell。
结论：本发明化合物与PD-L1单抗联用对CT-26小鼠结肠癌移植瘤模型具有优异的抑瘤效果。
实验例7：化合物对MC38小鼠结肠癌移植瘤模型的体内药效学研究
7.1实验目的：
本实验的目的是研究本发明化合物对MC38小鼠结肠癌移植瘤模型体内药效进行评估。
7.2实验动物：
种属：小鼠
品系：C57BL/6小鼠
周龄：6-8周龄
性别：雌性
供应商：上海斯莱克实验动物有限公司
7.3实验方法与步骤
7.3.1细胞培养
名称：MC38(小鼠结肠癌细胞)
细胞来源：ATCC
细胞培养：培养液为含有10％胎牛血清的1640培养基,培养条件为37℃,5％二氧化碳。传代比例为1:2～1:3,每周传代2～3次。
7.3.2肿瘤细胞接种
将0.1mL(2×10
5个)细胞皮下接种于每只小鼠的右后背。同日将动物根据体重随机分组。
7.3.3受试物的配制
实验用溶媒为0.5％甲基纤维素溶液,配制方法为称取5g甲基纤维素,溶解于800mL超纯水中,搅拌均匀后用超纯水定容至1000mL。受试物用溶媒溶解,配制成一定浓度均一溶液,于4℃保存。
7.3.4肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b
2,a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝T
RTV/C
RTV×100％(T
RTV：治疗组RTV；C
RTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume,RTV),计算公式为RTV＝V
t/V
0,其中V
0是分组给药时(即d
0)测量所得平均肿瘤体积,V
t为某一次测量时的平均肿瘤体积,T
RTV与C
RTV取同一天数据。
7.4实验结果如表7所示：
表7受试化合物对MC38小鼠结肠癌移植瘤模型的抑瘤药效评价(基于分组后第27天肿瘤体积计算得出)
PD-1单抗来源：BioXcell。
结论：本发明化合物与PD-1单抗联用对MC38小鼠结肠癌移植瘤模型具有优异的抑瘤效果。
Claims (13)
- 式(Ⅰ)化合物、其异构体或其药学上可接受的盐，
其中，L 1选自-(CH 2)g-、-C(＝O)-、-S(＝O) 2-、-C(＝O)-O-和-C(＝O)-NH-；R 1选自NH 2、CN、COOH、-S(＝O) 2-NH 2、C 1-6烷基、-C(＝O)-C 1-4烷基-苯基和-C 1-4烷基-4-7元杂环烷基，其中所述C 1-6烷基、-C(＝O)-C 1-4烷基-苯基和-C 1-4烷基-4-7元杂环烷基任选被1、2或3个R a取代；m为0、1或2；n为0、1或2，且m和n不能同时为0；r为0或1；q为0或1；g为0、1、2、3或4；R a选自F、Cl、Br、I、OH、NH 2、CN、COOH、C 1-3烷氨基和-NH-C(＝O)-C 1-3烷基，其中所述C 1-3烷氨基和-NH-C(＝O)-C 1-3烷基任选被1、2或3个R取代；R选自F、Cl、Br、I、OH、NH 2和C 1-3烷基；所述4-7元杂环烷基包含1、2、3或4个独立选自-NH-、-O-、-S-和N的杂原子或杂原子团；带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。 - 根据权利要求1所述的化合物、其异构体或其药学上可接受的盐，其中，R选自F、Cl、Br、I、OH、NH 2、CH 3和-CH 2CH 3。
- 根据权利要求1或2所述的化合物、其异构体或其药学上可接受的盐，其中，R a选自F、Cl、Br、I、OH、NH 2、CN、COOH、其中所述
任选被1、2或3个R取代。
- 根据权利要求3所述的化合物、其异构体或其药学上可接受的盐，其中，R a选自F、Cl、Br、I、OH、NH 2、CN、COOH、
- 根据权利要求1所述的化合物、其异构体或其药学上可接受的盐，其中，R 1选自NH 2、CN、COOH、 -S(＝O) 2-NH 2、C 1-4烷基、-C(＝O)-C 1-3烷基-苯基和-C 1-3烷基-5～6杂环烷基，其中所述C 1-4烷基、-C(＝O)-C 1-3烷基-苯基和-C 1-3烷基-5～6杂环烷基任选被1、2或3个R a取代。
- 根据权利要求5所述的化合物、其异构体或其药学上可接受的盐，其中，R 1选自NH 2、CN、COOH、-S(＝O) 2-NH 2、-CH 3、-CH 2-CH 3、
其中所述CH 3、-CH 2-CH 3、
任选被1、2或3个R a取代。
- 根据权利要求6所述的化合物、其异构体或其药学上可接受的盐，其中，R 1选自NH 2、CN、COOH、-CH 3、-CH 2-CH 3、
- 根据权利要求1所述的化合物、其异构体或其药学上可接受的盐，其中，L 1选自单键、-CH 2-、-(CH 2) 2-、-C(＝O)-、和-S(＝O) 2-。
- 根据权利要求1-7任意一项所述的化合物、其异构体或其药学上可接受的盐，其化合物选自
其中，g如权利要求1所定义；R 1如权利要求1-6任意一项所定义。 - 下式化合物、其异构体或其药学上可接受的盐，
- 根据权利要求10所示的化合物、其异构体或其药学上可接受的盐，
- 根据权利要求1-11任意一项所述的化合物、其异构体或其药学上可接受的盐，其中所述的药学上可接受的盐选自盐酸盐。
- 根据权利要求1-12任意一项所述的化合物、其异构体或其药学上可接受的盐在制备治疗LSD1相关病症的药物上的应用。
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| WO2026015801A1 (en) | 2024-07-12 | 2026-01-15 | Revolution Medicines, Inc. | Methods of treating a ras related disease or disorder |
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| WO2022267495A1 (zh) * | 2021-06-22 | 2022-12-29 | 南昌弘益药业有限公司 | 含氮氧杂螺环类化合物及其应用 |
| CN116888122A (zh) * | 2021-06-22 | 2023-10-13 | 南昌弘益药业有限公司 | 含氮氧杂螺环类化合物及其应用 |
| WO2023217758A1 (en) | 2022-05-09 | 2023-11-16 | Oryzon Genomics, S.A. | Methods of treating malignant peripheral nerve sheath tumor (mpnst) using lsd1 inhibitors |
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| WO2025255438A1 (en) | 2024-06-07 | 2025-12-11 | Revolution Medicines, Inc. | Methods of treating a ras protein-related disease or disorder |
| WO2025265060A1 (en) | 2024-06-21 | 2025-12-26 | Revolution Medicines, Inc. | Therapeutic compositions and methods for managing treatment-related effects |
| WO2026006747A1 (en) | 2024-06-28 | 2026-01-02 | Revolution Medicines, Inc. | Ras inhibitors |
| WO2026015796A1 (en) | 2024-07-12 | 2026-01-15 | Revolution Medicines, Inc. | Methods of treating a ras related disease or disorder |
| WO2026015790A1 (en) | 2024-07-12 | 2026-01-15 | Revolution Medicines, Inc. | Methods of treating a ras related disease or disorder |
| WO2026015801A1 (en) | 2024-07-12 | 2026-01-15 | Revolution Medicines, Inc. | Methods of treating a ras related disease or disorder |
| WO2026015825A1 (en) | 2024-07-12 | 2026-01-15 | Revolution Medicines, Inc. | Use of ras inhibitor for treating pancreatic cancer |
| WO2026050446A1 (en) | 2024-08-29 | 2026-03-05 | Revolution Medicines, Inc. | Ras inhibitors |
| WO2026072904A2 (en) | 2024-09-26 | 2026-04-02 | Revolution Medicines, Inc. | Compositions and methods for treating lung cancer |
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| Publication number | Publication date |
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| CN112672994A (zh) | 2021-04-16 |
| CN112672994B (zh) | 2022-09-13 |
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