CN107602446A - 1,4 disubstituted 1,2,3,6 tetrahydropyridines, its preparation method, pharmaceutical composition and its applications - Google Patents
1,4 disubstituted 1,2,3,6 tetrahydropyridines, its preparation method, pharmaceutical composition and its applications Download PDFInfo
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Abstract
The invention provides 1,4 shown in a kind of formula I disubstituted 1,2,3,6 tetrahydropyridines, its preparation method, pharmaceutical composition and its application.Especially, compound of the present invention is a kind of micromolecular inhibitor at the targeting menin MLL interactions interface of brand new, the inhibitor is used for the interference and treatment of kinds of tumors, it is highly preferred that the tumour is leukaemia, liver cancer, brain tumor, myeloma, cancer of pancreas, breast cancer, colon cancer, prostate cancer, carcinoma of urinary bladder or Multiple Endocrine gland cancer.
Description
Technical field
The present invention relates to pharmaceutical chemistry and pharmacotherapeutics field, relates generally to a kind of Isosorbide-5-Nitrae-disubstituted -1,2,3,6-
Tetrahydropyridines, its preparation method, pharmaceutical composition and its application.Especially, compound of the present invention is one kind
The micromolecular inhibitor at the targeting menin-MLL interactions interface of brand new, the interaction of menin and MLL albumen can
To be considered a kind of potential effective target of molecular therapy, therefore the selectively targeting activated interface, it is advantageously used for phase therewith
The new drug development of pass.
Background technology
Multiple Endocrine cancer protein (menin albumen) is by the type of Multiple Endocrine gland cancer 1 (MEN1) gene code, MEN1
Gene plays tumor suppressor gene in endocrine organ.Menin albumen has interaction with multiple protein, forms multiple
Miscellaneous interactive network.Such as menin and wild type MLL1 and the N-terminal direct interaction of pattern of fusion MLL1 albumen, and simultaneously
With reference to chromatin-associated protein LEDGF (epidermal growth factor in crystalline lens source).LEDGF most at last the composite belt to corresponding
Target gene at, activated gene expression.
Mll gene family includes five members of MLL1-5, and the occurrence and development and deterioration, transfer with kinds of tumors are closely related
(Grembecka etc., Future Medicinal Chemistry, 2014,6 (4), 447-462).MLL1 albumen is as the family
The epigenetic regulation enzyme reported earliest, the mediation histone H 3 K4 modification that methylates, regulates and controls the isogenic transcriptions of HOX, no
There is critical function only in the growth course of normal hematopoiesis system, and fusion protein caused by its gene dystopy is considered as to make
The root fallen ill and deteriorated into MLL leukaemia.Grembecka etc. is based on the boundary that interacted to menin genes and menin-MLL
The further investigation in face, the micromolecular inhibitor MI-2 at menin-MLL interactions interface is reported first, it is follow-up to be proposed one again
The active stronger inhibitor of series structure optimization.The inhibitor series are first in MLL pattern of fusion leukaemia and mouse
Shown on model good therapeutic effect (Grembecka etc., Nature Chemical Biology, 2012,8,227-
284).In addition, MLL1 is considered as in close relations with liver cancer and brain tumor.Newest research shows, in liver cancer cells, MLL1-
Menin participates in the malignant proliferation of regulation and control liver cancer cells, cuts down menin expression effectively suppression by transcriptional upregulation Yap1 gene expressions
Oncogenicity and malignant proliferation of the nude mice processed into liver cancer cells on knurl model.MLL1 albumen is by activating isogenic turn of HOXA10
Record, it is vital for maintaining self-renewing, growth and the oncogenicity of brain tumor stem cell.MLL change cause GATA4 and
The horizontal rise of EST1 H3K4me3 and then the high expression of albumen, be considered as it is relevant with carcinoma of urinary bladder (Song etc., Oncotarget,
2016,7(3),2629-2645).In addition, MLL2 is reported in up-regulated expression in cancer of pancreas, breast cancer, colon cancer, cut down MLL2
Gene expression, suppress tumour cell cycle process, inducing cell apoptosis, it is seen that the occurrence and development and deterioration of MLL2 albumen and tumour
It is closely related.
Research shows menin with MLL1, MLL2 albumen direct interaction for the histone methylated modification of compound
(H3K4) enzyme activity, it is indispensable (Yali etc., Nature to target gene transcriptional control and its corresponding function
Structure＆Molecular Biology, 2006,13,713-719).In view of MLL1, MLL2 albumen are pernicious to kinds of tumors
Propagation is necessary, and does not have enzyme activity during MLL albumen individualisms substantially, it is presumed that it promotes the function of malignancy of tumor propagation
It is likely to and is completed in a big epigenetic regulation compound.In fact, at present in prostate cancer and breast cancer,
Wild type MLL function is also proved to be to rely on itself and the interaction of menin albumen.Before Castration drug resistances
In row gland cancer, MLL epigenetic regulation compounds are accredited as androgen receptor AR synetion, and menin-MLL inhibitor leads to
Cross and disintegrate MLL compounds and destroy its function, can effectively suppress the signal path in AR downstreams, suppress prostate cancer on nude mice model
Growth (Malik etc., Nature Medicine, 2015,21 (4), 344-352).In breast cancer, TP53 gain-of-function
Transmethylase MLL1, MLL2, transacetylase MOZ that type mutant passes through transcriptional upregulation epigenetic regulation expression, make
Help histone methylated and acetylation modification in genome range it is abnormal (Zhu etc., Nature, 2015,525,206-
211).In this kind of tumour, the inhibitor MI2-2 for targetting menin-MLL destroys MLL1 transmethylases compound and suppresses it
Enzyme activity, significantly inhibit tumour cell and grown in nude mice into the knurl body on knurl model.
In addition, menin take part in a variety of important transcriptional control compounds as a kind of nucleoprotein for organizing wide spectrum expression
Formed, a variety of important biological functions are shown as in body.Except participating in, to form MLL1, MLL2 epigenetic regulation compound
Thing, menin albumen are reported and a variety of transcription factors, including JunD, NFKB, SMAD3 interaction, regulate and control the transcription of target gene
Activation suppresses (Agarwal etc., Cell, 1999,96 (1), 143-152；Christina etc., Oncogene, 2001,20
(36), 4917-4925；Kaji, 2001,98 (7), 3837-3842).Menin by with ASK (activator of S-phase
Kinase) interaction, participation cell division propagation and cell cycle regulating (Schnepp etc., Cancer Research,
2004,64 (18), 6791-6796)；Menin influences genome by DNA integrity scanning protein A FANCD2 protein-interactings
Stability (Jin etc., Cancer Research, 2003,63 (14), 4204-4210).Menin and BMP-2 signal path eggs
Bletilla Runx2 interacts, and is considered as (Katalin etc., TRENDS in Endocrinology and relevant with skeleton development
Metabolism, 2006,17 (9), 357-364), regulation and control menin perhaps can influence bone and form or control patients with malignant myeloma
Disease development.It is interesting that the result of study of structure shows, the larger central pocket of menin albumen possesseds is to be situated between
Lead the major interfaces with other protein-interactings.Menin albumen is individually crystallized, menin-MLL compounds cocrystallization,
The crystallographic structural analysis result of menin-JunD cocrystallization shows, menin and MLL and JunD protein bindings, on combination interface
Menin bag structure is consistent, and as the bag structure of the menin albumen of individualism.Thus, we release,
The pocket, not only mediated menin and MLL1, MLL2 albumen interaction, also mediated its with JunD etc. other albumen
Interaction.Thus, the little molecules in inhibiting or its different types of structural derivative for targetting Menin-MLL are accounted for by emulative
According to menin pocket, it would be possible to the interaction of selective destruction menin Yu other different type transcription factors, regulation and control
Corresponding transcriptional events and related biological function, so as to further expand the application of menin-MLL inhibitor.
As can be seen here, it is effective to may be considered a kind of potential molecular therapy for the interaction of menin and MLL fusion proteins
Target, menin-MLL inhibitor are with a wide range of applications, and the selectively targeting activated interface, are advantageously used for phase therewith
The new drug development of pass.
The content of the invention
A kind of disubstituted -1,2,3,6- tetrahydropyridines the classes of the 1,4- as shown in formula I for being related to brand new of the present invention
Compound and preparation method thereof, and binding activity evaluation and related biological experiment, it was demonstrated that such compound can be with efficient targeting
Menin-MLL interactions interface, can be used for opening with the treatment of menin-MLL protein-interacting relevant diseases and new drug
Hair.
It is an object of the present invention to provide the disubstituted -1,2,3,6- tetrahydropyridines classes of a kind of 1,4- shown in formula I
Compound or its pharmaceutically acceptable salt.
It is a further object to provide the disubstituted -1,2,3,6- tetrahydropyridines class chemical combination of the 1,4- shown in formula I
The preparation method of thing.
A further object of the present invention is to provide the disubstituted -1,2,3,6- tetrahydropyridines class chemical combination of 1,4- shown in formula I
Thing or its pharmaceutically acceptable salt are preparing the micromolecular inhibitor as targeting menin-MLL protein-interactings interface
Purposes in medicine, interference and treatment available for the kinds of tumors related to menin-MLL interactions interface.The tumour
For leukaemia, liver cancer, brain tumor, myeloma, cancer of pancreas, breast cancer, colon cancer, prostate cancer, carcinoma of urinary bladder or Multiple Endocrine
Gland cancer.
According to an aspect of the invention, there is provided compound or its pharmaceutically acceptable salt shown in formula I：
Wherein, n=1-4 integer, preferably 1,2,3 or 4；
R1、R2It is identical or different, and be separately：Hydrogen atom, hydroxyl, halogen, C1-C10The alkyl of straight or branched,
C1-C10Alkoxy, the C of straight or branched2-C10Alkenyl, the C of straight or branched3-C10Cycloalkyl, C1-C6Alkyl-carbonyl, C1-
C6Alkoxy carbonyl, amino carbonyl C1-C6Alkyl, C1-C6Alkylamidoalkyl or C5-C20Aryl；More preferably：Hydrogen is former
Son, hydroxyl, halogen, C1-C6Alkyl, the C of straight or branched1-C6Alkoxy, the C of straight or branched2-C6The chain of straight or branched
Alkenyl, C3-C8Cycloalkyl, C1-C6Alkyl-carbonyl, C1-C6Alkoxy carbonyl, amino carbonyl C1-C3Alkyl, C1-C3Alkylamidoalkyl
Or C5-C10Aryl；More preferably H, Br, Cl, methyl, ethyl, propyl group, isopropyl, butyl, the tert-butyl group, methoxyl group, ethoxy
Base, propoxyl group, isopropoxy, butoxy, tert-butoxy, vinyl, acrylic, cyclobutenyl, pentenyl, hexenyl, cyclopropane
Base, cyclobutane base, pentamethylene base, cyclohexyl, cycloheptyl alkyl, methyl carbonyl, ethylcarbonyl group, propyl group carbonyl, Isopropylcarbonyl,
Butyl carbonyl, tert-butyl carbonyl, methoxycarbonyl, ethoxy carbonyl, propoxycarbonyl, isopropoxy carbonyl, butoxy carbonyl,
Tert-butoxycarbonyl or methyl nitrosourea, ethyl acylamino, amino carbonyl methyl, aminocarbonylethyl, phenyl or naphthyl；
R3And R4It is identical or different, and be separately：Hydrogen atom, C1-C10Alkyl, the C of straight or branched2-C10Straight chain
Or alkenyl, the C of side chain3-C10Cycloalkyl, substituted or unsubstituted C5-C20Aryl or substituted or unsubstituted five yuan or six
Unit's heteroaryl, wherein the C substituted5-C20Substituent in aryl or substituted five yuan or six membered heteroaryl be selected from halogen, hydroxyl,
Amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, the Herbicidal sulphonylamino of acyl group substitution
Base, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6Straight chain or branch
Alkyl sulphonyl, the C of chain1-C6Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Straight chain
Or alkoxy carbonyl, the C of side chain1-C6The alkyl or C of straight or branched1-C6One in the alkoxy of straight or branched or
It is multiple；Preferably hydrogen atom, C1-C6Alkyl, the C of straight or branched2-C6Alkenyl, the C of straight or branched3-C8Cycloalkyl, take
Generation or unsubstituted C5-C12Aryl or substituted or unsubstituted five yuan or six membered heteroaryl, wherein the C substituted5-C12Aryl
Substitution five yuan or six membered heteroaryl in substituent be selected from halogen, hydroxyl, amino, by one or two C1-C6Straight chain or branch
Alkyl-substituted amino, the C of chain1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6The alkyl sulphonyl of straight or branched
Amino, C5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6Straight or branched
Alkyl-carbonyl, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Alkoxy carbonyl, the C of straight or branched1-C6Straight chain or
The alkyl or C of side chain1-C6One or more of alkoxy of straight or branched；More preferably methyl, ethyl, third
Base, isopropyl, butyl, the tert-butyl group, methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy, tert-butoxy, vinyl, third
Alkenyl, cyclobutenyl, pentenyl, hexenyl, cyclopropane base, cyclobutane base, pentamethylene base, cyclohexyl, cycloheptyl alkyl, substitution or
Unsubstituted phenyl or substituted or unsubstituted pyridine radicals, wherein, taking in the substituted phenyl or substituted pyridine radicals
Dai Ji be selected from halogen, hydroxyl, amino, trifluoromethyl, by one or two C1-C6The alkyl-substituted amino of straight or branched,
C1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl,
Sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6Alkyl-carbonyl, the C of straight or branched1-C6Straight or branched
Alkyl-carbonyl oxygen, C1-C6Alkoxy carbonyl, the C of straight or branched1-C6The alkyl or C of straight or branched1-C6Straight chain or branch
One or more of alkoxy of chain；
R5For substituted or unsubstituted C5-C12Aryl, substituted or unsubstituted five yuan or six membered heteroaryl, substitution do not take
The benzo five-membered or six membered heteroaryl in generation or substituted or unsubstituted dibenzothiophenes base, wherein, the substituted C5-C12
Aryl, five yuan of substitution or six membered heteroaryl, the benzo five-membered or six membered heteroaryl of substitution or substituted dibenzothiophenes base
In substituent be selected from halogen, hydroxyl, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6
Amino, sulfuryl amino, the C of acyl group substitution1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, fluoroform
Base, sulfonyl, C1-C6Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Straight or branched
Alkoxy carbonyl, amino-sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6The alkyl sulfonyl of straight or branched
Amido, amino C1-C6The alkyl of straight or branched, the phenylSulphon amido of halogen substitution, phenyl, the C of halogen substitution1-C6Straight chain
Or the alkyl or C of side chain1-C6One or more of alkoxy of straight or branched；Preferably, R5For substitution or unsubstituted
Phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted furyl, substitution or not
Substituted pyridine radicals, substituted or unsubstituted pyrimidine radicals, substituted or unsubstituted pyrrole radicals, substituted or unsubstituted imidazole radicals or
In Qu generations, do not substitute oxazolyls, substituted or unsubstituted indazolyl, substituted or unsubstituted indyl, substituted or unsubstituted
Quinolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted benzothiophene
Base, substituted or unsubstituted benzothiazolyl, or substituted or unsubstituted dibenzothiophenes base, wherein, substituent is selected from halogen
Element, hydroxyl, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6The amino of acyl group substitution,
Sulfuryl amino, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6
Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6The alkoxy carbonyl of straight or branched,
Amino-sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6The alkylsulfonamido of straight or branched, amino C1-C6
The alkyl of straight or branched, the phenylSulphon amido of halogen substitution, phenyl, the C of halogen substitution1-C6The alkyl of straight or branched,
Or C1-C6One or more of alkoxy of straight or branched；Most preferably, R5To be unsubstituted or by selected from halogen, trifluoro
Methyl, C1-C6Alkoxy, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Acyl group substitutes
Amino, sulfuryl amino, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, sulfonyl, C1-C6Directly
The alkyl-carbonyl of chain or side chain, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Alkoxy carbonyl, the ammonia of straight or branched
Base C1-C6Alkyl, halogenophenyl, halogenophenyl sulfoamido, amino-sulfonyl or C1-C6At least one in alkylsulfonamido
The phenyl of individual substituent substitution；It is unsubstituted or by selected from halogen, trifluoromethyl, C1-C6Alkoxy, amino, by one or two
C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6Straight or branched
Alkyl sulfonyl-amino, C5-C12Aryl carbonyl, sulfonyl, C1-C6Alkyl-carbonyl, the C of straight or branched1-C6Straight chain or branch
Alkyl-carbonyl oxygen, the C of chain1-C6Alkoxy carbonyl, the amino C of straight or branched1-C6Alkyl, halogenophenyl, halogenophenyl sulphonyl
Amido or C1-C6The pyridine radicals of at least one substituent substitution in alkylsulfonamido；It is unsubstituted or by selected from halogen, trifluoro
Methyl, C1-C6Alkoxy, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Acyl group substitutes
Amino, sulfuryl amino, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, sulfonyl, C1-C6Directly
The alkyl-carbonyl of chain or side chain, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Alkoxy carbonyl, the ammonia of straight or branched
Base C1-C6Alkyl, halogenophenyl, halogenophenyl sulfoamido or C1-C6At least one substituent substitution in alkylsulfonamido
Indyl；It is unsubstituted or by selected from halogen, trifluoromethyl, C1-C6Alkoxy, amino, by one or two C1-C6Straight chain or
Alkyl-substituted amino, the C of side chain1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6The alkyl sulfonyl of straight or branched
Base amino, C5-C12Aryl carbonyl, sulfonyl, C1-C6Alkyl-carbonyl, the C of straight or branched1-C6The alkyl oxycarbonyl of straight or branched
Base oxygen, C1-C6Alkoxy carbonyl, the amino C of straight or branched1-C6Alkyl, halogenophenyl, halogenophenyl sulfoamido or C1-C6
The quinolyl of at least one substituent substitution in alkylsulfonamido；It is unsubstituted or by selected from halogen, trifluoromethyl, C1-C6Alkane
Epoxide, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, the sulphonyl of acyl group substitution
Base amino, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, sulfonyl, C1-C6Straight or branched
Alkyl-carbonyl, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Alkoxy carbonyl, the amino C of straight or branched1-C6Alkyl,
Halogenophenyl, halogenophenyl sulfoamido or C1-C6The thianaphthenyl of at least one substituent substitution in alkylsulfonamido.
Preferably, R3And R4In it is at least one be substituted or unsubstituted phenyl, the substituent in the substituted phenyl
Selected from halogen, hydroxyl, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Acyl group substitution
Amino, sulfuryl amino, C1-C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, trifluoromethyl, sulfonyl,
C1-C6Alkyl sulphonyl, the C of straight or branched1-C6Alkyl-carbonyl, the C of straight or branched1-C6The alkyl-carbonyl of straight or branched
Oxygen, C1-C6Alkoxy carbonyl, the C of straight or branched1-C6The alkyl or C of straight or branched1-C6The alcoxyl of straight or branched
One or more of base.
It is further preferred that compound of the present invention has the structure shown in formula II：
Wherein, R1、R4And R5It is as defined above,
R6For hydrogen atom, halogen, C1-C6Alkyl, trifluoromethyl, hydroxyl, amino, by one or two C1-C6Straight chain or branch
Alkyl-substituted amino, the C of chain1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6The alkyl sulphonyl of straight or branched
Amino, C5-C12Aryl carbonyl, sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6The alkyl oxycarbonyl of straight or branched
Base, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6The alkoxy carbonyl or C of straight or branched1-C6Straight or branched
Alkoxy.
Unless otherwise noted, in the present invention, term is defined as follows：
Halogen represents fluorine, chlorine, bromine or iodine；
Five yuan or six membered heteroaryl represent there is at least one heteroatomic five yuan or six in N, O or S in ring
First aryl, the example include, but not limited to thienyl, furyl, pyridine radicals, pyrimidine radicals, pyrrole radicals, imidazole radicals Huo oxazolyls；
Benzo five-membered or six-membered Hetero-aromatic group represents benzene with having at least one miscellaneous original in N, O or S in ring
Son five yuan or hexa-atomic aryl-condensed formation ring, the example include, but not limited to indazolyl, indyl, quinolyl, naphthyl,
Isoquinolyl, benzofuranyl, benzothienyl or benzothiazolyl.
More specifically, the compound represented by formula I can be any one in following compound：
Table 1
The pharmaceutically acceptable salt bag of the disubstituted -1,2,3,6- tetrahydropyridines of 1,4- of the present invention
Include：The salt formed with inorganic acid, such as hydrochloride, hydrobromate, sulfate, phosphate etc.；And the salt formed with organic acid,
Such as maleate, mesylate, tosilate, trifluoroacetate, acetate, tartrate, malonate etc..
According to another aspect of the present invention, there is provided Isosorbide-5-Nitrae of the present invention-disubstituted -1,2,3,6- tetrahydropyridine classes
The preparation method of compound, it comprises the following steps：
Wherein, n, R1、R2、R3、R4And R5Definition it is same as described above,
A) compound 1 obtains compound 3 with the generation substitution reaction of compound 2；
B) generation of compound 3 reduction reaction obtains compound 4；
C) substitution reaction that compound 4 occurs on amino N atom obtains compound 5, including methylated on N, be acetylating,
Methyl formate, alkylation and arylation.
Preferably, step a) reaction condition be in the presence of alkali, bromo-derivative 1 and 4- aryl -1,2,3,6- tetrahydrochysene pyrroles
Pyridine class compound 2, substitution reaction occurs；The alkali is sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide etc., preferably carbonic acid
Sodium, solvent for use are acetonitrile, Isosorbide-5-Nitrae-dioxane, tetrahydrofuran, 2- methyl-tetrahydros furans, toluene etc., preferably acetonitrile, are reacted
Temperature is the reflux temperature of solvent for use, and preferably acetonitrile reflux temperature is 80 DEG C, hour in reaction time 3-8, preferably 4 hours；
Step b) reaction condition is in the presence of go back original reagent, is amino by cyano reduction, the go back original reagent is
Lithium Aluminium Hydride, diisobutyl aluminium hydride (DIBAL-H), borine and borine/dimethylsulfid complex, additive are anhydrous tri-chlorination
Aluminium, preferably Lithium Aluminium Hydride-aluminum trichloride (anhydrous) combination (mol ratio 1:1), the solvent include ether, tetrahydrofuran, 2- methyl-
Tetrahydrofuran, Isosorbide-5-Nitrae-dioxane etc., preferably tetrahydrofuran；
Step c) reaction condition is reacted to determine by specific, usually condition well known in the art.According to the present invention
Another further aspect, it provides a kind of pharmaceutical composition, and it includes one or more of the present invention the 1 of therapeutically effective amount,
Disubstituted -1,2,3,6- the tetrahydropyridines of 4- or its pharmaceutically acceptable salt and pharmaceutically acceptable auxiliary material.
According to another aspect of the invention, it provides Isosorbide-5-Nitrae-disubstituted -1,2,3,6- tetrahydropyridine class shown in formula I
Compound or its pharmaceutically acceptable salt are preparing the little molecules in inhibiting as targeting menin-MLL protein-interactings interface
Purposes in the medicine of agent.The inhibitor can be used for kinds of tumors interference and treatment, the tumour be leukaemia, liver cancer,
Brain tumor, myeloma, cancer of pancreas, breast cancer, colon cancer, prostate cancer, carcinoma of urinary bladder or Multiple Endocrine gland cancer.
According to another aspect of the invention, it provides a kind of method for treating tumour, and it includes applying to patient
Disubstituted -1,2,3,6- the tetrahydropyridines of 1,4- of the present invention of effective dose or its pharmaceutically acceptable salt.
The beneficial effects of the present invention are：The invention provides a series of disubstituted -1,2,3,6- of the brand-new 1,4- of structures
Tetrahydropyridines and preparation method thereof, such compound useful effect can be used as target in the mutual interfaces of menin-MLL
To the new drug development of the micromolecular inhibitor at menin-MLL protein-interactings interface.Its conventional method can synthesize, through molecule
Horizontal and cellular level test evaluation, series compound activity are obvious.
Brief description of the drawings
Fig. 1 confirms compound D for the thermophoresis experiment of display albumen28With the protein bound charts of menin；
Fig. 2 is that the experiment of display surface plasma resonance confirms compound D28With the protein bound charts of menin；
Fig. 3 confirms compound D to show that competitive nuclear magnetic resonance saturation transfer difference spectrum is tested28It is protein bound with menin
Chart；
Fig. 4 is that co-immunoprecipitation experiment confirms compound D28The mutual of menin albumen and MLL albumen is destroyed in cellular level
The figure of effect；
Fig. 5 is compound D28And D37With the schematic diagram of menin protein binding pattern analyses；
Fig. 6 is compound D28The chart of selective depression MLL pattern of fusion Leukemia Cell Proliferations；
Fig. 7 is compound D28By leukaemia MV4；11 are arrested in the figure of G0/G1 phases；
Fig. 8 is compound D28Inducing leukemia cell MV4；The figure of 11 differentiation；
Fig. 9 is compound D28The figure of inducing leukemia cell KOPN-8 differentiation；
Figure 10 is compound D28Lower the figure of the expression of HOXA9, HOXA10, MEIS1 and DLX2 gene in leukaemia.
Embodiment
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
Embodiment 1：4- aryl -2 ' in the present invention, have in 2 '-two fragrant (alkane) base -1- tetrahydro pyridyl amine derivants
Representational compound prepares as follows：Compound D1Synthesis
1) raw material A is added in reaction bulb1-20(257mg, 0.86mmol, commercial goods, it is purchased from the limited public affairs of lark prestige science and technology
Department), compound B1(174mg, 0.9mmol, commercial goods, being purchased from the resistance to Jilin Chemical (Energy Chemical) of peace), sodium carbonate
(339mg, 3.2mmol), acetonitrile (15mL), react 4 hours under the conditions of 80 DEG C, be extracted with ethyl acetate after the completion of reaction, it is organic
Layer obtains compound C after drying concentration column chromatography1(283mg, 80%).
2) THF solution (2.4M, 2.76mL) of tetrahydrochysene lithium aluminium is added in anhydrous response bottle, three are slowly added dropwise under the conditions of 0 DEG C
The THF solution (10mL) of aluminium chloride (153mg), compound C is then added dropwise1The THF solution (10mL) of (95mg, 0.23mmol),
After moving to room temperature reaction 1 hour, it is heated at reflux 7 hours, is cooled to after room temperature plus water quenching is gone out, it is molten with ether and saturation sodium potassium tartrate tetrahydrate
Liquid extracts, and organic layer dries concentration column chromatography and obtains compound D1(43mg, 45%).
Spectral data：1H NMR(300MHz,CDCl3)δ7.32-7.18(m,14H),6.00(s,1H),3.35(s,2H),
3.11-3.03 (m, 2H), 2.61 (t, J=5.4Hz, 2H), 2.53-2.38 (m, 4H), 2.23-2.14 (m, 2H)；13C NMR
(125MHz,CDCl3)δ146.3,139.2,133.9,132.6,128.4,128.2,128.2,126.2,122.3,53.9,
53.4,50.9,50.6,49.1,33.5,28.1；HRMS (EI) calculated values C27H29ClN2[M]+, 416.2019. measured values,
416.2025.
Embodiment 2：Compound D2-D20Synthesis, it is as follows to prepare formula
Preparation method is similar to embodiment 1, but raw materials used is B2-B20(by substitution phenyl boric acid and 3,6- dihydros -4-
[[(trifluoromethyl) sulphonyl] epoxide] -1 (2H)-pyridine carboxylic acid tert-butyl ester is prepared by Suzuki coupling reactions, commercial goods, purchase
In lark prestige Science and Technology Ltd.), obtain target compound D2-D20, shown in table specific as follows, table 2：
Embodiment 3：Compound D21-D27Synthesis, it is as follows to prepare formula
Preparation method is similar to embodiment 2, but raw materials used is A21-A27, obtain target compound D21-D27, it is specific as follows
Shown in table, table 3：
Embodiment 4：Compound D28Synthesis
Compound A is added in reaction bulb28(397mg), compound B1(229mg), sodium carbonate (318mg) acetonitrile (5mL), 80
React 4 hours under the conditions of DEG C, be extracted with ethyl acetate after the completion of reaction, organic layer obtains compound C after drying concentration column chromatography28
(301mg, 72%).1H NMR(300MHz,CDCl3) δ 7.43-7.33 (m, 9H), 6.00 (s, 1H), 3.45 (t, J=6.3Hz,
2H),3.00(s,2H),2.59-2.50(m,2H),2.41-2.29(m,4H),2.06-1.93(m,4H),1.83-1.71(m,
3H),1.51-1.35(m,4H).
The THF solution (2.4M, 0.15mL) of tetrahydrochysene lithium aluminium is added in anhydrous response bottle, trichlorine is slowly added dropwise under the conditions of 0 DEG C
Change the THF solution (5mL) of aluminium (49mg), compound C is then added dropwise28The THF solution (5mL) of (52mg), it is small to move to room temperature reaction 1
Shi Hou, it is heated at reflux 7 hours, is cooled to after room temperature plus water quenching is gone out, extracted with ether and saturation potassium sodium tartrate solution, organic layer is done
Dry concentration column chromatography obtains compound D28(22mg, 42%).
Spectral data：1H NMR(300MHz,CDCl3)δ7.40-7.20(m,9H),6.02(s,1H),3.60-3.44(m,
2H),3.20-3.00(m,2H),2.90-2.60(m,8H),2.56-2.28(m,4H),1.93-1.75(m,2H),1.68-1.40
(m,4H),1.16-0.90(m,3H)；13C NMR(125MHz,CDCl3)δ141.9,138.8,133.5,132.2,127.9,
127.7,127.4,125.7,125.2,121.9,61.9,58.7,53.6,49.9,46.5,46.2,42.9,32.3,31.2,
27.6,26.6,25.9,23.9,22.5,21.3；HRMS(EI)calcd.for C27H35ClN2[M]+,422.2489.Found,
422.2487.
Embodiment 5：Compound D29-D41Synthesis, it is as follows to prepare formula
Preparation method is similar to embodiment 2, but raw materials used is A29-A35, or B3, B7, B15-B17, and B36, obtain
Target compound D29-D39, shown in table specific as follows, table 4：
Embodiment 6：Compound D42Synthesis
Compound D is added in reaction bulb28(70mg), after being dissolved with acetonitrile (5mL), formalin (37%, 26mg) is added,
Sodium cyanoborohydride (32mg), 1 drop acetic acid, after being stirred at room temperature 40 minutes, is extracted with ethyl acetate and saturated sodium bicarbonate solution,
Organic layer obtains compound D after drying concentration column chromatography42(75mg, 86%).Spectral data：1H NMR(300MHz,CDCl3)δ
7.43-7.15(m,9H),6.06(s,1H),3.26(s,2H),2.88-2.72(m,4H),2.65-2.47(m,4H),2.32-
2.12(m,8H),1.74-1.56(m,2H),1.49-1.31(m,5H),1.17-1.03(m,2H)；13C NMR(125MHz,
CDCl3)δ138.9,134.2,132.8,128.5,128.0,127.6,126.2,125.8,65.3,53.7,53.3,50.4,
48.1,46.4,31.5,27.8,27.4,27.0,24.7,24.5；HRMS(EI)calcd.for C28H37ClN2[M]+,
436.2645.Found,436.2649.
Embodiment 7：Compound D43Synthesis
Sodium hydrogen (20mg) is added in reaction bulb with after DMF (2mL) dissolvings, is stirred at room temperature 30 minutes, adds compound D28
(70mg), monovalent iodomethane is added, after 1h is stirred at room temperature, extracted with ethyl acetate and saturated sodium bicarbonate solution, organic layer
Compound D is obtained after drying concentration column chromatography43(75mg, 41%).Spectral data：1H NMR(300MHz,CDCl3)δ7.26-
7.13(m,9H),6.02(s,1H),3.31(m,2H),2.88(m,4H),2.45(m,4H),2.26-2.12(m,7H),1.67-
1.58(m,2H),1.31(m,3H),1.17-1.02(m,2H)；HRMS(EI)calcd.for C27H35ClN2[M]+,
422.2489.Found,422.2482.
Embodiment 8：Compound D44Synthesis
Compound D is added in reaction bulb28(82mg), after being dissolved with dioxane (5mL), add aceticanhydride (106mg), 4- bis-
Methylamino pyridine DMAP (3.2mg), after being stirred at room temperature 4 hours, extracted with ethyl acetate and saturated sodium bicarbonate solution, organic layer
Compound D is obtained after drying concentration column chromatography43(116mg, 77%).
Spectral data：1H NMR(300MHz,CDCl3)δ7.48-7.20(m,9H),6.11(s,1H),3.32(s,2H),
2.77-2.65(m,4H),2.56-2.41(m,4H),2.34(s,3H),2.29-2.12(m,8H),1.78-1.62(m,2H),
1.51-1.35(m,5H),1.21-1.08(m,2H)；HRMS(EI)calcd.for C28H35ClN2O[M]+,
436.2645.Found,436.2649.
Embodiment 9：Compound D45Synthesis
Compound D is added in reaction bulb28(81mg), after being dissolved with dichloromethane (5mL), add triethylamine and chloro-carbonic acid first
Ester (38mg), after temperature stirs 2 hours, extracted with ethyl acetate and saturated sodium bicarbonate solution, organic layer dries concentration column chromatography
After obtain compound D45(52mg, 34%).Spectral data：1H NMR(300MHz,CDCl3)δ7.81-7.22(m,9H),6.06
(s,1H),3.78(s,3H),3.61(s,2H),3.21(s,2H),2.88-2.59(m,4H),2.57(m,4H),2.43(m,
7H),1.99-1.62(m,2H),1.23(m,5H),1.18-1.06(m,2H)；HRMS(EI)calcd.For C28H35ClN2O2
[M]+,466.2387.Found,466.2388.
Embodiment 10：Compound D46Synthesis
Compound D is added in reaction bulb28(81mg), after being dissolved with dichloromethane (5mL), chloroacetamide (38mg) is added,
Then after 2-3 hours are stirred at room temperature in addition 1M KOH (2mL) solution, are extracted, had with ethyl acetate and saturated sodium bicarbonate solution
Machine layer obtains compound D after drying concentration column chromatography44(52mg, 56%).Spectral data：1H NMR(300MHz,CDCl3)δ
7.93-7.18(m,9H),6.08(s,1H),3.61(s,2H),3.21(s,2H),2.97-2.68(m,4H),2.56-2.45(m,
4H),2.41-2.33(m,8H),1.99-1.62(m,2H),1.59-1.23(m,5H),1.18-1.06(m,2H)；HRMS(EI)
calcd.For C28H36ClN3O[M]+,465.2547.Found,465.2542.
Embodiment 11：Compound D47Synthesis
Compound D is added in reaction bulb28(1mmol), after being dissolved with DMSO (5mL), add cuprous iodide 10%, L- dried meat ammonia
Acid 20%, the equivalent of potassium carbonate 1.5, the equivalent of iodobenzene 1.2, react overnight, with ethyl acetate and saturated sodium bicarbonate solution at 80 DEG C
Extraction, organic layer obtain compound D after drying concentration column chromatography47(37%).Spectral data：1H NMR(300MHz,CDCl3)δ
7.44(s,5H),7.38(m,6H),7.31–7.07(m,8H),7.07–7.02(m,1H),6.69(s,1H),6.58(s,4H),
6.45(s,2H),6.08(s,1H),3.61(s,2H),3.21(s,2H),2.97-2.68(m,4H),2.56-2.45(m,4H),
2.41-2.33(m,8H),1.99-1.62(m,2H),1.59-1.23(m,5H),1.18-1.06(m,2H)；HRMS(EI)
calcd.For C32H37ClN2[M]+,484.2645.Found,484.2641.
Embodiment 12：Compound D48Synthesis
Compound A is added in reaction bulb48(380mg), compound B1(229mg), sodium carbonate (320mg) acetonitrile (5mL), 80
React 4 hours under the conditions of DEG C, be extracted with ethyl acetate after the completion of reaction, organic layer obtains compound after drying concentration column chromatography
C48。
The THF solution (2.4M, 0.15mL) of tetrahydrochysene lithium aluminium is added in anhydrous response bottle, trichlorine is slowly added dropwise under the conditions of 0 DEG C
Change the THF solution (5mL) of aluminium (49mg), compound C is then added dropwise48The THF solution (5mL) of (52mg), it is small to move to room temperature reaction 1
Shi Hou, it is heated at reflux 7 hours, is cooled to after room temperature plus water quenching is gone out, extracted with ether and saturation potassium sodium tartrate solution, organic layer is done
Dry concentration column chromatography obtains compound D48(22mg, 27%).
Product spectral data：1H NMR(400MHz,CDCl3) δ 8.41 (m, 4H), 7.44 (m, 5H), 7.17 (d, J=
20.0Hz,5H),6.17(s,1H),3.53(s,2H),3.24–2.60(m,7H),2.58(s,1H),2.55(s,1H),2.44
(d, J=10.5Hz, 6H), 2.16-1.81 (m, 11H), 1.69 (dd, J=34.9,20.0Hz, 12H)；HRMS(EI)
calcd.for C25H32ClN3[M]+,409.2285.Found,409.2282.
Embodiment 13：Fluorescence polarization experiment (Fluorescence Polarization, FP) measure 1,4- it is disubstituted-
The activity of 1,2,3,6- tetrahydropyridines
In fluorescence polarization experiment, we use 600nM menin albumen and 30nM marked by fluorescein isothiocyanate
MBM1 polypeptides (FITC-MBM1, Suzhou Chinapeptides Co., Ltd.) mix in FP buffer solutions, while add specified dense eventually
The compound of degree, 2h is incubated 4 DEG C of dark places.Negative and sun is used as using the DMSO of same volume, unlabelled MBM1 polypeptides respectively
Property control.Each sample well after being incubated using the Envision multiple labeling micropores board detector measure of PerkinElmer companies
Fluorescence intensity, fluorescence polarization value, try to achieve the inhibiting rate of compound, IC50Value and Ki values are fitted through GraphPad Prism5.0 softwares
Fluorescence polarization value is tried to achieve.
Pass through the activity of the disubstituted -1,2,3,6- tetrahydropyridines of FP measurings 1,4-.NA- is inactive,
IC50<10 μM of overstrikings expressions,
Table 5
Embodiment 14：Albumen thermophoresis experiment confirm the disubstituted -1,2,3,6- tetrahydropyridines of 1,4- with
Menin albumen directly in conjunction with
Albumen thermophoresis experiment is carried out in 20 μ l reaction system, 2.5 μM of menin albumen and compound, 5 × SYPRO
Orange dyestuffs (Invitrogen) mix in buffer solution, then Quant Studio6Flex in real time-PCR instrument (ABI)
On, with 1 DEG C/min slope, 95 DEG C are heated to from 25 DEG C.In order to reduce evaporation, with heat-sealing film (Thermo sciencific))
Hole is sealed.All samples all contain three repetitions.Fluorescence intensity is recorded, and with albumen thermophoresis software Pro tein
(ABI) versions of Thermal Shift Software Version 1.1 are fitted the Tm values of menin albumen.Using DMSO groups as pair
According to the change of calculating compound group menin albumen Tm values.Measurement result is shown in Table 6, and wherein compound concentration is 20 μM.
The albumen thermophoresis of table 6 experiment confirms the combination of multiple compounds and menin albumen
As a result show, multiple Isosorbide-5-Nitraes-disubstituted -1,2,3,6- tetrahydropyridines can make in 20 μM of concentration
The thaw temperature rise of menin albumen, shows that it can make albumen more stable directly in conjunction with menin albumen.Fig. 1 shows chemical combination
Thing D28The thaw temperature of the raising menin albumen of concentration dependent.
Embodiment 15：Surface plasma resonance (Surface Plasmon Resonance, SPR) experiment confirms compound
D28With menin protein bindings
SPR is tested on Biacore T200 instruments, and (GE Healthcare) is carried out in 25 DEG C.Menin albumen acetic acid
Sodium is covalently coupled on CM5 chips.Compound HBS-EP+Buffer solution dilutes a series of concentration, carries out dynamic experiment.Chemical combination
The equilibrium dissociation constant K of thing and menin albumenDValue is calculated through Biacore T200 DAS and tried to achieve.Such as Fig. 2, display
Compound D28Can be directly in conjunction with its equilibrium dissociation constant K with menin albumenD=3.07 μM.
Embodiment 16：Competitive saturation transfer difference spectrum (STD) nuclear magnetic resonance experiment confirms compound D28With menin albumen knots
The region of conjunction is MBM1 peptide fragment binding pockets
In order to illustrate that the calmodulin binding domain CaM of the disubstituted -1,2,3,6- tetrahydropyridines of 1,4- and menin albumen is true
Actually binding pocket of the MBM1 peptide fragments in menin, we used saturation transfer difference spectrum (STD) nuclear magnetic resonance experiment method to survey
Determine representative compound D28With MBM1 competitive relation.It is 5 μM in the concentration containing menin albumen, D28For 200 μM (5%
DMSO-D6 the MBM1 of various concentrations, the combination of competing compound and menin albumen are separately added into system).Experiment exists
Carried out on Bruker Avance III-600MHz NMRs, temperature is 25 DEG C.Such as Fig. 3, by menin albumen and compound
D28Mixing, STD spectrograms are gathered, the compound spectral peak of higher signal value can be obtained.With competitive polypeptide MBM1 addition, chemical combination
Thing peak value gradually reduces, and concentration dependant sexual intercourse is presented.Show the disubstituted -1,2,3,6- tetrahydropyridines of 1,4-
With MBM1 polypeptides competitive binding menin albumen same pocket.
Embodiment 17：Co-immunoprecipitation experiment proves compound D28It is horizontal in the cell to destroy menin albumen and MLL albumen
Between interaction
Expression Flag-MLL will be successfully constructedNPlasmid with PEI reagents (sigma) transfect into 293T cells, transfect 48h
Afterwards cell is handled with certain density compound or isometric DMSO.Cell lysis (Cell Signaling after administration 12h
Technology), it is incubated with ANTI-FLAG M-2 (magnetic bead) in 4 DEG C, 2h and lysate, is enriched with Flag-MLLNAlbumen.Enrichment
After sample afterwards removes foreign protein with phosphate buffer, it is separated by electrophoresis through PAGE gel, immune-blotting method menin eggs
White and Flag-MLLNAlbumen.Compare the menin that DMSO treatment groups sample obtains with co-immunoprecipitation in compound treatment group sample
Protein content.If the interaction that compound can be destroyed between menin albumen and MLL albumen, its menin protein content will be less than
DMSO groups.Such as Fig. 4, compound D28Cell is handled, the menin protein contents of co-immunoprecipitation are considerably less than at 40 μM, 20 μM
DMSO treatment groups, also there is faint reduction at 10 μM.Illustrate compound D28Really can cellular level destroy menin albumen and
Interaction between MLL albumen.
So far, with compound D28For represent, we are tested by fluorescence polarization, albumen thermophoresis experiment, surface plasma
Resonance laboratory, competitive saturation deficit spectrum nuclear magnetic resonance experiment and co-immunoprecipitation experiment, it was demonstrated that Isosorbide-5-Nitrae-disubstituted -1,2,
3,6- tetrahydropyridines also can be in the horizontal phase interaction for destroying menin and MLL albumen of intracellular in can testing in vitro
With the micromolecular inhibitor at the interface that can be interacted as menin-MLL.
Embodiment 18：The binding pattern point of the disubstituted -1,2,3,6- tetrahydropyridines of 1,4- and menin albumen
Analysis
It is mutual in order to be best understood from the disubstituted -1,2,3,6- tetrahydropyridines of 1,4- and menin albumen pockets
The detailed information of effect, present inventor use GLIDE softwares, select wherein representative compound D28And D37To itself and menin eggs
It is white to carry out molecular docking, analyze its binding pattern.Analysis result (Fig. 5) display, compound D28And D37Occupy menin with
The F9 (the amino acids phenylalanine of MLL albumen the 9th) and P10 (the amino acids proline of MLL albumen the 10th) of MBM1 binding pockets
Region, MBM1 and menin albumen combination are simulated well.In addition, compound D37The sulfanilamide (SN) group of afterbody may be with
Menin albumen M322, E326, W341, the pocket that E363 is formed have hydrogen bond and hydrophobic interaction, therefore compared to D28,
Compound D37More excellent inhibitory activity is shown in fluorescence polarization experiment.
Embodiment 19：Disubstituted -1,2,3,6- the tetrahydropyridines of cell survival measuring 1,4- melt to MLL
The increment inhibitory action of mould assembly leukaemia
More ripe MLL pattern of fusion leukaemia's models have been studied in the present inventor's selection, to Isosorbide-5-Nitrae-disubstituted -1,
The cellular level function of 2,3,6- tetrahydropyridines is tested.
We select the leukemia cell line MV4 for having MLL pattern of fusion albumen；11(MLL-AF4),KOPN-8(MLL-ENL),
And the HL60 and Jurkat cell of non-MLL pattern of fusion, multiple emphasis compound processing cells are selected, carry out cell inhibitory effect
Detection.Cell is with 1 × 105mL-1Density be incubated in 96 hole transparent panels, with the DMSO processing of compound or same volume
Cell, after processing 7 days, add AlamarBlue and be incubated, each hole is determined with empty miniature board detector more than PHERAstar BMG
Fluorescence intensity, indicator cells vigor.Half Proliferation Ability concentration GI50Value is tried to achieve through the fitting of the softwares of GraphPad Prism 5.0.Knot
Fruit shows, compound D28The suppression MLL pattern of fusion leukaemias MV4 of energy selectivity；11 and KOPN-8 propagation is specific to suppress
Rate and GI50Value, referring to Fig. 6 and table 7.In addition, multiple compounds are also in MV4；Preferable Proliferation Ability is shown on 11 cells
Effect, it is shown in Table 7.
The experiment of the cell survival rate of table 7 confirms that multiple compounds suppress MLL pattern of fusion leukaemias MV4；11 propagation
| Compound | GI50(μM) |
| D28 | 1.89 |
| D31 | 3.34 |
| D32 | 3.14 |
| D37 | 2.96 |
| D42 | 6.39 |
Embodiment 20：Cell cycle detection confirms compound D28By MLL pattern of fusion leukaemia's Cycle Arrests in G0/G1
Phase
Tested for the cell cycle, 2 × 105/ hole cell kind plate in 12 orifice plates, with the compound of respective concentration or
0.2%DMSO handles 48h.4 DEG C, 800g is collected by centrifugation 5 × 104Individual cell is simultaneously resuspended in the PBS of 300 μ l precoolings, oscillating drop
Add the absolute ethyl alcohol of 700 μ l precoolings, 4 DEG C of fixations are overnight.It is then centrifuged for removing ethanol, and with PBS, is eventually adding 300 μ l
PI/RNase solution (BD, article No.：550825) it is resuspended, room temperature lucifuge is incubated 15min, is then examined on flow cytometer
Survey.As a result Fig. 7 is seen, compared with the cell sample of DMSO treatment groups, compound D28At 15 μM, the cell mass institute in the G0/G1 phases
The percentage accounted for increases, and the percentage shared by the cell mass in S phases and G2 phases is then reduced.Therefore, compound D28Passing through will
MLL pattern of fusion leukaemias are arrested in the G0/G1 phases, so as to suppress the propagation of cell.
Embodiment 21：The experiment of flow cytometer detection CD11b and Rui Shi Giemsa staining confirms compound D28Induce MLL pattern of fusion white
Blood disease cell differentiation
3×105/ hole MV4；11 or KOPN-8 cell kind plates are in 12 orifice plates, with the compound of respective concentration or 0.2%
DMSO is handled 7 or 10 days.To detect cell surface differentiation mark CD11b, drug-treated collects cell after 7 days, with containing
1%BSA PBS removes culture medium and cell is resuspended.Add PE mouse anti-human CD11b Ab (BD
Phamingen), 4 DEG C of lucifuges are incubated 30min, carry out flow cytometer detection.Tested for Rui Shi Giemsa stainings, drug-treated 10 days
Afterwards, with cell centrifuge smearing machine Shandon cytospin3 (Thermo Scientific Cytospin) by cell centrifuge to
On slide.Cell smear is dyed with Rui Shi Giemsa stains (Baso) after airing.
Fig. 8 shows, compound D28Handle MV4；After 11 cells, cell ratio positive cell surface differentiation mark CD11b
Example rises, and Rui Shi Giemsa stainings picture shows that drug-treated can reduce the nucleocytoplasmic ratio of cell, and medicine is presented in variation effect
The concentration dependent of thing.Fig. 9 shows compound D28Also there is the effect of induction differentiation to KOPN-8 cells.Therefore, the implementation
Example explanation compound D28Can be by inducing MLL pattern of fusion leukaemia to break up, so as to suppress the propagation of cell.
Embodiment 22：Real-time quantitative PCR experiment confirms compound D28The high table in MLL pattern of fusion leukaemias can be lowered
The hox gene group reached and gene M EIS1, DLX2
Select MV4；11 cells, compound or 0.2%DMSO processing with respective concentration, collected at specified time point thin
Born of the same parents, total serum IgE is extracted with Trizol kits (Invitrogen), and use RevertAid First Strand cDNA
Synthesis Kit (Thermo Scientific) reverse transcription synthesizes cDNA.SYBR Green are added in the sample
Realtime PCR Master Mix (TOYOBO), and it is enterprising in Quant Studio 6Flex real-time PCRs (ABI)
Row real-time quantitative PCR is reacted, and corresponding target genes expand quantitatively.Figure 10 shown compared with DMSO groups, compound D28Place
Manage MV4；11 cells can lower the hox gene such as HOXA9, HOXA10 and gene of the high expression in MLL pattern of fusion leukaemias
MEIS1、DLX2。
Claims (10)
1. a kind of compound shown in formula I or its pharmaceutically acceptable salt：
Wherein, n=1-4 integer；
R1、R2It is identical or different, and be separately：Hydrogen atom, hydroxyl, halogen, C1-C10Alkyl, the C of straight or branched1-
C10Alkoxy, the C of straight or branched2-C10Alkenyl, the C of straight or branched3-C10Cycloalkyl, C1-C6Alkyl-carbonyl, C1-C6Alkane
Epoxide carbonyl, amino carbonyl C1-C6Alkyl, C1-C6Alkylamidoalkyl or C5-C20Aryl；
R3And R4It is identical or different, and be separately：Hydrogen atom, C1-C10Alkyl, the C of straight or branched2-C10Straight chain or branch
Alkenyl, the C of chain3-C10Cycloalkyl, substituted or unsubstituted C5-C20Aryl or substituted or unsubstituted five yuan or hexa-atomic miscellaneous
Aryl, wherein the C substituted5-C20Substituent in aryl or substituted five yuan or six membered heteroaryl be selected from halogen, hydroxyl, amino,
By one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, sulfuryl amino, the C of acyl group substitution1-
C6Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6The alkane of straight or branched
Base sulfonyl, C1-C6Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Straight or branched
Alkoxy carbonyl, C1-C6The alkyl or C of straight or branched1-C6One or more of alkoxy of straight or branched；
R5For substituted or unsubstituted C5-C12It is aryl, substituted or unsubstituted five yuan or six membered heteroaryl, substituted or unsubstituted
Benzo five-membered or six membered heteroaryl or substituted or unsubstituted dibenzothiophenes base, wherein, the substituted C5-C12Aryl,
Substitution five yuan or six membered heteroaryl, substitution benzo five-membered or six membered heteroaryl or substituted dibenzothiophenes base in
Substituent be selected from halogen, hydroxyl, amino, by one or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Acyl group
Substituted amino, sulfuryl amino, C1-C6Alkyl sulfonyl-amino, trifluoromethyl, sulfonyl, the C of straight or branched1-C6Directly
The alkyl-carbonyl of chain or side chain, C5-C12Aryl carbonyl, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Straight or branched
Alkoxy carbonyl, amino-sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6The alkyl sulfonamide of straight or branched
Base, amino C1-C6The alkyl of straight or branched, the phenylSulphon amido of halogen substitution, phenyl, the C of halogen substitution1-C6Straight chain or
The alkyl or C of side chain1-C6One or more of alkoxy of straight or branched.
2. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, wherein, R1、R2Phase
It is same or different, and be separately：Hydrogen atom, hydroxyl, halogen, C1-C6Alkyl, the C of straight or branched1-C6Straight or branched
Alkoxy, C2-C6Alkenyl, the C of straight or branched3-C8Cycloalkyl, C1-C6Alkyl-carbonyl, C1-C6Alkoxy carbonyl, amino
Carbonyl C1-C3Alkyl, C1-C3Alkylamidoalkyl or C5-C10Aryl.
3. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, wherein, R3And R4Phase
It is same or different, and be separately：Hydrogen atom, C1-C6Alkyl, the C of straight or branched2-C6The alkenyl of straight or branched,
C3-C8Cycloalkyl, substituted or unsubstituted C5-C12Aryl or substituted or unsubstituted five yuan or six membered heteroaryl, wherein taking
The C in generation5-C12Substituent in aryl or substituted five yuan or six membered heteroaryl be selected from halogen, hydroxyl, amino, by one or two
Individual C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6Straight chain or branch
Alkyl sulfonyl-amino, the C of chain5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6The alkyl sulphonyl of straight or branched,
C1-C6Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6The alkoxy carbonyl of straight or branched
Base, C1-C6The alkyl or C of straight or branched1-C6One or more of alkoxy of straight or branched.
4. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, wherein, R5For substitution
Or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted furyl,
It is substituted or unsubstituted pyridine radicals, substituted or unsubstituted pyrimidine radicals, substituted or unsubstituted pyrrole radicals, substituted or unsubstituted
Imidazole radicals or Qu generation or do not substitute oxazolyls, substituted or unsubstituted indazolyl, substituted or unsubstituted indyl, substitution or
It is unsubstituted quinolines base, substituted or unsubstituted isoquinolyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted
Benzothienyl, substituted or unsubstituted benzothiazolyl, or substituted or unsubstituted dibenzothiophenes base, wherein, it is above-mentioned
Substituent in group be selected from halogen, hydroxyl, amino, by one or two C1-C6The alkyl-substituted amino of straight or branched,
C1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6Alkyl sulfonyl-amino, trifluoromethyl, the sulphonyl of straight or branched
Base, C1-C6Alkyl-carbonyl, the C of straight or branched5-C12Aryl carbonyl, C1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Directly
The alkoxy carbonyl of chain or side chain, amino-sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6Straight or branched
Alkylsulfonamido, amino C1-C6The alkyl of straight or branched, the phenylSulphon amido of halogen substitution, halogen substitution phenyl,
C1-C6The alkyl or C of straight or branched1-C6One or more of alkoxy of straight or branched.
5. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, wherein, R3And R4In
It is at least one be substituted or unsubstituted phenyl, the substituent in the substituted phenyl is selected from halogen, hydroxyl, amino, quilt
One or two C1-C6Alkyl-substituted amino, the C of straight or branched1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6
Alkyl sulfonyl-amino, the C of straight or branched5-C12Aryl carbonyl, trifluoromethyl, sulfonyl, C1-C6The alkyl of straight or branched
Sulfonyl, C1-C6Alkyl-carbonyl, the C of straight or branched1-C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6Straight or branched
Alkoxy carbonyl, C1-C6The alkyl or C of straight or branched1-C6One or more of alkoxy of straight or branched.
6. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, the formula I institutes
The compound shown has the structure shown in formula II：
Wherein, R1、R4And R5Be as defined above it is identical in claim 1,
R6For hydrogen atom, halogen, C1-C6Alkyl, trifluoromethyl, hydroxyl, amino, by one or two C1-C6Straight or branched
Alkyl-substituted amino, C1-C6Amino, sulfuryl amino, the C of acyl group substitution1-C6The alkyl sulfonyl-amino of straight or branched,
C5-C12Aryl carbonyl, sulfonyl, C1-C6Alkyl sulphonyl, the C of straight or branched1-C6Alkyl-carbonyl, the C of straight or branched1-
C6Alkyl-carbonyl oxygen, the C of straight or branched1-C6The alkoxy carbonyl or C of straight or branched1-C6The alcoxyl of straight or branched
Base.
7. compound shown in formula I according to claim 1 or its pharmaceutically acceptable salt, it has following knot
Structure：
8. a kind of method for preparing the compound described in claim 1 shown in formula I, it comprises the following steps：
Wherein, n, R1、R2、R3、R4And R5Definition it is identical with the definition in claim 1,
A) compound 1 obtains compound 3 with the generation substitution reaction of compound 2；
B) generation of compound 3 reduction reaction obtains compound 4；
C) substitution reaction that compound 4 occurs on amino N atom obtains the compound shown in formula I.
9. a kind of pharmaceutical composition, its include compound shown in formula I according to any one of claim 1 to 7 or
Its pharmaceutically acceptable salt, and pharmaceutically acceptable auxiliary material.
10. the compound or its pharmaceutically acceptable salt shown in formula I according to any one of claim 1 to 7 exist
Prepare the purposes in the medicine as the micromolecular inhibitor at targeting menin-MLL protein-interactings interface, it is preferable that described
Inhibitor is used as the medicine of interference and the treatment of kinds of tumors, it is highly preferred that the tumour is leukaemia, liver cancer, brain tumor, marrow
Knurl, cancer of pancreas, breast cancer, colon cancer, prostate cancer, carcinoma of urinary bladder or Multiple Endocrine gland cancer.
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| CN110974827A (en) * | 2019-01-28 | 2020-04-10 | 中南大学湘雅医院 | Application of N-aryl-3, 3-difluoro-5-p-toluenesulfonyl-1, 2,3, 6-tetrahydropyridine-4-alcohol |
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| CN110974827A (en) * | 2019-01-28 | 2020-04-10 | 中南大学湘雅医院 | Application of N-aryl-3, 3-difluoro-5-p-toluenesulfonyl-1, 2,3, 6-tetrahydropyridine-4-alcohol |
| CN110974827B (en) * | 2019-01-28 | 2021-08-20 | 中南大学湘雅医院 | Application of N-aryl-3, 3-difluoro-5-p-toluenesulfonyl-1, 2,3, 6-tetrahydropyridine-4-alcohol |
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