CN109748943A

CN109748943A - 2 '-C- methyl substituted nucleosides class compounds and its preparation and purposes

Info

Publication number: CN109748943A
Application number: CN201711071739.1A
Authority: CN
Inventors: 沈竞康; 李佳; 陈越磊; 周宇波; 熊兵; 刘同超; 任焕明; 谢吴辰; 苏明波; 胡小蓓; 汪玉洁; 徐威
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2017-11-03
Filing date: 2017-11-03
Publication date: 2019-05-14

Abstract

The invention discloses 2 '-C- methyl substituted nucleosides class compounds and its pharmaceutically acceptable salt shown in formula 2, and preparation method thereof.Such compound has DOT1L inhibitor activity, can apply in the drug that preparation treats or prevents cancer.

Description

2' -C-methyl substituted nucleoside compound and preparation and application thereof

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to 2' -C-methyl substituted nucleoside compounds shown as a formula 2, pharmaceutically acceptable salts thereof and a preparation method thereof. The compound has DOT1L enzyme inhibitor activity, and can be applied to the preparation of medicaments for treating or preventing cancers.

Background

There are several references [ 1) Olhava, E.J.methods of synthesizing substistuted purpurinucrosides, WO2014152566A2, 2014; 2) klaus, c.; raimondi, m.a.; daigle, s.r.; pollock, R.M. combination of hormone removal enzymes DOTIL inhibitors and anticancerogen for a treatment of cancer. WO2014153001A1, 2014; 3) yu, w.; smil, d.; li, F.; tempel, w.; fedorov, o.; nguyen, k.t.; bolshan, y.; Al-Awar, R.; knapp, s.; arrowsmith, C.H.; vedadi, m.; brown, p.j.; schapira, M., Bromo-deaza-SAH A patent and selectiveDOT1L inhibitor bioorganic & Medicinal Chemistry2013,21(7), 1787-; 4) anglin, j.l.; deng, l.; yao, y.; cai, g.; liu, z.; jiang, h.; cheng, g.; chen, p.; dong, s.; song, Y., Synthesis and Structure-Activity Relationship Investigation of Adenosine-containment Inhibitors of tissue metabolism factor DOT1L. journal of medical chemistry2012,55(18),8066 and 8074; 5) yu, w.; chord, e.j.; wernimont, a.k.; tempel, w.; scopton, a.; federation, A.; marineau, j.j.; qi, j.; Barsyte-Lovejoy, d.; yi, j.; marcellus, r.; iacobs, r.e.; engen, j.r.; griffin, c.; aman, a.; wienholds, e.; li, F.; pineda, j.; estiu, g.; shatseva, t.; hajian, t.; al-aware, R.; dick, j.e.; vedadi, m.; brown, p.j.; arrowsmith, C.H.; bradner, j.e.; schapira, M., Catalytic laboratory modifying soft he DOT1L methyl transfer enzyme by selective inhibitors Nat Commun2012,3,1288 ] describes nucleoside DOT1L inhibitors. DOT1L inhibitors generally have the structure shown in 1.

Disclosure of Invention

The invention aims to provide 2' -C-methyl substituted nucleoside compounds shown in formula 2 or pharmaceutically acceptable salts thereof, and the compounds have good DOT1L enzyme inhibitor activity and lower toxicity.

Another object of the present invention is to provide a process for producing the above 2' -C-methyl-substituted nucleoside compounds.

It is another object of the present invention to provide a pharmaceutical composition comprising the above 2' -C-methyl substituted nucleoside compounds and/or pharmaceutically acceptable salts thereof.

Still another object of the present invention is to provide the use of the above 2' -C-methyl substituted nucleoside compounds and/or pharmaceutically acceptable salts thereof for the preparation of a medicament for treating or preventing cancer.

The invention provides a 2' -C-methyl substituted nucleoside compound shown as the following formula 2:

wherein,

x and Y are each independently selected from carbon or nitrogen, Z is selected from carbon, nitrogen, CR⁰Wherein R is⁰Is halogen or cyano; preferably, X and Z are simultaneously nitrogen, YIs carbon; x and Z are both carbon, Y is nitrogen; y and Z are both nitrogen, and X is carbon; or, X is nitrogen, Y is carbon and Z is carbon;

m is

Ar is selected from unsubstituted or substituted by C₁-C₆Alkyl or halogen substituted C₆-C₁₂An aryl group, a heteroaryl group,

ring A being unsubstituted or substituted by C₁-C₆Alkyl or halogen substituted C₆-C₁₂An aryl group, a heteroaryl group,

l is selected from substituted or unsubstituted C₂-C₆Straight or branched alkylene, substituted or unsubstituted C₂-C₆Straight or branched alkenylene, substituted or unsubstituted C₂-C₆Straight or branched alkynylene, substituted or unsubstituted C₃-C₆A cycloalkylene group, or a substituted or unsubstituted C₂-C₆Alkylene radical C₃-C₆A cycloalkylene group, the substituted substituent being selected from halogen, C₁-C₆Alkoxy radical, said C₃-C₆The cycloalkylene group is preferably a cycloalkylene group

R⁴Is selected from C₁-C₆Acyl or H;

m is preferably

Preferably, the compound represented by formula 2 is a 2' -C-methyl substituted nucleoside compound represented by one of the following general formulae:

wherein M is as defined in formula 2.

In a preferred embodiment of the present invention, the 2' -C-methyl substituted nucleoside compound represented by the general formula 2 is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

The 2' -C-methyl substituted nucleosides of the present invention can contain one or more chiral centers and can therefore produce enantiomers, diastereomers and other stereoisomeric forms that can be defined as (R) -or (S) -forms according to absolute stereochemistry. Unless otherwise indicated, the present invention is intended to include all such possible isomers, including all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds.

Suitable methods for determining the amount of an enantiomer are well known to those skilled in the art. Such as HPLC on a chiral stationary base and NMR spectroscopic studies using chiral shift reagents.

Among the terms used in the present invention, C-nucleoside analogs can be classified into alpha-C-nucleoside analogs and beta-C-nucleoside analogs according to the relative positions of the 1-and 5-position substituents of ribose on both sides of ribose, and specifically, when the substituents at the 1-and 5-positions of ribose are on opposite sides of ribose, the configuration of nucleoside is defined as alpha-type; when the substituents at the 1-and 5-positions of the ribose are on the same side of the ribose, the configuration of the nucleoside is defined as beta-type, e.g., nucleoside analog 2;

in the present invention, the term "C₁-C₆Alkyl "means a straight or branched chain alkyl group having 1 to 6 carbon atoms in the main chain, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like; ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl are preferred.

In the present invention, the term "C₃-C₆Cyclic alkyl "refers to cyclic alkyl groups having 3 to 6 carbon atoms in the ring, including, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

In the present invention, the term "C₆-C₁₂The "aryl" refers to a hetero atom-free aromatic ring group having 6 to 12 carbon atoms, for example, phenyl or naphthyl.

In the context of the present invention, the term "acyl" includes groups having an alkyl, alkenyl, alkynyl, cycloalkyl, aryl or arylalkyl group attached at one of the two available valence positions to the carbon atom of a carbonyl group, for example, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl. "C₁-C₆Acyl is C₁-C₆Alkylcarbonyl, and the like.

In the present invention, the term "isomers" refers to non-distinct compounds having the same molecular formula, but differing in atomic arrangement and conformation. Furthermore, the term "stereoisomer" as used herein refers to any of the various stereoisomeric configurations that may exist for a given compound of the invention and includes geometric isomers.

In a second aspect of the present invention, there is also provided a method for preparing the 2' -C-methyl substituted nucleoside compound 2, the method comprising one or more of the following steps:

(a) carrying out protection reaction on the amino group of the heterocyclic compound 3 and RCl or ROR to obtain a compound 4;

(a3) carrying out hydroxyl protection reaction on hydroxyl of a sugar compound 5 and PgBr or PgCl to obtain a lactone compound 5-1;

(b) removing proton from the amino protected compound 4, performing Br-metal exchange, performing addition reaction with a lactone compound 5-1, and reducing to obtain a nucleoside compound 6;

(c) removing a protecting group Pg of a hydroxyl group from the compound 6 to obtain a compound 11;

(d) carrying out condensation reaction on a base compound 8 and an acyl protected sugar compound 9 to obtain a nucleoside compound 10;

(e) removing O-acyl protection from the compound 10 to obtain a nucleoside compound 11;

(f) then protecting the nucleoside compound 11 with a protective reagent to obtain a compound 7;

(g) compound 7 with acylating agent LgCl or Lg₂Carrying out acylation reaction on O to obtain a compound 12;

(h) and (3) carrying out azide substitution on the compound 7 or the compound 12 to obtain a compound 13, and then carrying out reduction and deprotection to obtain a compound 14.

(i) Reacting compound 7 or 12 with Pg¹NH is substituted by amino to obtain the compound 15 or 15-1, and Pg of the compound 15 or 15-1¹Deprotection to compound 14;

(j) carrying out reductive amination reaction on the compound 14 to obtain a compound 16;

(k) substituting the amino group of the compound 12 to obtain a compound 16;

(l) Carrying out reductive amination reaction on the compound 16 and M ═ O to obtain a compound 17;

(M) reductive amination of compound 14 with M ═ O to give compound 16-1;

(n) subjecting compound 16-1 to reductive amination reaction to obtain compound 17;

(o) removing acetal protection of dihydroxy from compound 17 to obtain compound 2;

wherein,

x and Y are each independently selected from carbon or nitrogen, Z is selected from carbon, nitrogen, CR⁰Wherein R is⁰Is halogen or cyano; preferably, X and Z are both nitrogen and Y is carbon; x and Z are both carbon, Y is nitrogen; y and Z are both nitrogen, and X is carbon; or, X is nitrogen, Y is carbon and Z is carbon;

r is selected from tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), acetyl, isobutyryl, tert-butyryl (Piv), benzoyl, substituted by halogen or C₁-C₆Alkyl-substituted benzoyl, preferably said R is tert-butoxycarbonyl;

R¹selected from H, tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), acetyl, isobutyryl, tert-butyryl (Piv), benzoyl, substituted by halogen or C₁-C₆Alkyl-substituted benzoyl, preferably, said R¹Is H or tert-butoxycarbonyl;

lg is selected from p-methyl benzenesulfonyl, methylsulfonyl, phenylsulfonyl, imidazolesulfonyl or trifluoromethanesulfonyl;

pg is selected from 2-naphthylmethyl, 1-naphthylmethyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, triethylsilyl, benzyl, p-methoxybenzyl, substituted by halogen or C₁-C₆Alkyl-substituted benzyl, preferably 2-naphthylmethyl, p-methoxybenzyl;

Pg¹selected from phthaloyl;

acyl is selected from C₁-C₁₆Acyl, preferably selected from benzoyl, by halogen or C₁-C₆Alkyl-substituted benzoyl, acetyl, further preferably benzoyl and acetyl;

m is

Wherein,

ring A being unsubstituted or substituted by halogen or C₁-C₆Alkyl substituted C₆-C₁₂An aryl group, a heteroaryl group,

l is selected from substituted or unsubstituted C₂-C₆Straight or branched alkylene, substituted or unsubstituted C₂-C₆Straight or branched alkenylene, substituted or unsubstituted C₂-C₆Straight or branched alkynylene, substituted or unsubstituted C₃-C₆A cycloalkylene group, or a substituted or unsubstituted C₂-C₆Alkylene radical C₃-C₆A cycloalkylene group, the substituted substituent being selected from halogen, C₁-C₆An alkoxy group,

said C is₃-C₆The cycloalkylene group is preferably a cycloalkylene group

R⁴Is selected from C₁-C₆Acyl or H;

m is preferably

Preferably, the first and second electrodes are formed of a metal,

in the step (a), the first step of the method,

the amino protection reaction is carried out in two steps of a1 and a 2;

step a 1: heterocyclic compound 3 is taken as a raw material, and is reacted with amino protective reagent a1RCl or ROR under the existence of solvent a1, alkali a1 and catalyst to obtain intermediate a 1;

the solvent a1 can be a mixed solvent of one or more of ethers, toluene, hexane and dichloromethane; the ethers are selected from one or more of diethyl ether, tert-butyl methyl ether (TBME), isopropyl ether, dioxane, 2-methyltetrahydrofuran and Tetrahydrofuran (THF); the solvent a1 is preferably tetrahydrofuran and dichloromethane;

the amino protecting reagent a1RCl or ROR is selected from di-tert-butyl dicarbonate (Boc)₂O), tert-butyl chloroformate (Boc-Cl), benzyloxyformic anhydride (Cbz)₂O), benzyl chloroformate (CbzCl), pivaloyl chloride, acetic anhydride, acetyl chloride, isobutyric anhydride, isobutyryl chloride, benzoyl chloride, benzoic anhydride, halogen or C₁-C₆Alkyl-substituted benzoyl, halogen or C₁-C₆Alkyl substituted benzoic anhydride, said amino protecting reagent preferably being Boc₂O；

The base a1 is selected from pyridine, triethylamine, diisopropylethylamine and dimethylaminopyridine; the base a1 is preferably triethylamine;

the catalyst is dimethylamino pyridine;

the reaction temperature in the step a1 is between-30 ℃ and +60 ℃, and the reaction time is between 0.5 and 24 hours;

step a 2: reacting the intermediate a1 prepared in the step a1 with a base a2 in the presence of a2 solvent;

the solvent a2 can be a mixed solvent of one or more of ethers, alcohols and water; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, dioxane, 2-methyltetrahydrofuran and tetrahydrofuran; the alcohol is selected from one or more of methanol, ethanol and isopropanol; the solvent a2 is preferably water;

the alkali a2 is selected from sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, triethylamine, diisopropylethylamine and calcium hydroxide;

after the alkali a2 and the solvent a2 are mixed, the concentration of the alkali is selected from 1M to 20M, preferably 2M to 4M;

the reaction temperature in the step a2 is between 0 ℃ and +100 ℃, and the reaction time is between 0.5 hours and 24 hours.

In the step (a3),

the hydroxyl protection reaction takes a sugar compound 5 as a raw material to react with a hydroxyl protection reagent PgBr or PgCl in the presence of a solvent a3 and an alkali a3 to obtain a compound 5-1;

the solvent a3 can be a mixed solvent of one or more of ethers and amide solvents; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the amide is selected from N, N-Dimethylformamide (DMF), N, N-Dimethylacetamide (DMF), and formamide; the solvent is preferably tetrahydrofuran and N, N-dimethylformamide;

the hydroxyl protecting reagent PgBr or PgCl is selected from a bromization reagent PgBr and a chlorination reagent PgCl; the hydroxyl protecting reagent is preferably 2-naphthylmethyl bromide and p-methoxybenzyl chloride;

the base a3 is selected from pyridine, triethylamine, diisopropylethylamine, dimethylaminopyridine, sodium hydride NaH and sodium hexamethyldisilazide, and the base is preferably NaH;

the temperature of the hydroxyl protection reaction is between-30 ℃ and +150 ℃, and the reaction time is between 0.5 and 12 hours.

In the step (b), the step (c),

pg in the substrate 5-1 of the reaction is selected from tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, benzyl, p-methoxybenzyl, halogen-substituted benzyl, 1-naphthylmethyl, 2-naphthylmethyl, preferably 2-naphthylmethyl and p-methoxybenzyl;

the deprotonation reaction is carried out by taking a compound 4 as a raw material and a metal organic reagent in the presence of a solvent b 1;

the solvent b1 can be a mixed solvent of one or more of ethers, toluene, hexane and dichloromethane; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the solvent b1 is preferably tetrahydrofuran;

the metal organic reagent is selected from one or more of isopropyl magnesium chloride, methyl magnesium bromide, isopropyl magnesium chloride-LiCl mixture (Turbo-Grignard reagent), isobutyl magnesium chloride-LiCl mixture, lithium dichloride (2,2,6, 6-tetramethylpiperidine), lithium diisopropylamide, hexamethyldisilazane lithium, n-butyl lithium, isobutyl lithium, tert-butyl lithium, methyl lithium and phenyl lithium; preferably isopropyl magnesium chloride, lithium diisopropylamide;

the temperature of the deprotonation reaction is between-78 ℃ and +30 ℃, and the reaction time is between 0.5 and 12 hours.

The Br-metal exchange reaction is carried out with a metal organic reagent b2 in the presence of a solvent b 2;

the solvent b2 can be a mixed solvent of one or more of ethers, toluene, hexane and dichloromethane; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the solvent is preferably tetrahydrofuran.

The metal organic reagent b2 is selected from one or more of isopropyl magnesium chloride, methyl magnesium bromide, isopropyl magnesium chloride-LiCl mixture (Turbo-Grignard reagent), isobutyl magnesium chloride-LiCl mixture, lithium dichloride (2,2,6, 6-tetramethylpiperidine) salt, lithium diisopropylamide, hexamethyldisilazane alkyl lithium, n-butyl lithium, isobutyl lithium, tert-butyl lithium, methyl lithium and phenyl lithium; preferably an isopropyl magnesium chloride-LiCl mixture (Turbo-Grignard reagent), n-butyllithium;

the temperature of the Br-metal exchange reaction is between-100 ℃ and-40 ℃, and the reaction time is between 0.5 and 12 hours.

The addition reaction is carried out in the presence of a solvent b 3;

the solvent b3 can be a mixed solvent of one or more of ethers, toluene, hexane and dichloromethane; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the solvent b3 is preferably tetrahydrofuran.

Preferably, the addition reaction is carried out in the presence of a lewis acid catalyst selected from CuI, cuprous triflate (CuOTf), CuCN, LiCl, CuBr-dimethylsulfide complex, boron trifluoride etherate, TMSCl, and the like.

The temperature of the addition reaction is between-100 ℃ and-10 ℃, and the reaction time is between 0.5 and 12 hours.

Preferably, the deprotonation reaction, the Br-metal exchange reaction, and the addition reaction are preferably performed in a "one-pot" manner.

The reduction reaction is carried out in the presence of a solvent b4, a reducing agent b4 and Lewis acid b4 by taking the crude product of the deprotonation reaction, the Br-metal exchange reaction and the addition reaction which are boiled in one pot as a raw material to obtain a nucleoside compound 6;

the reducing agent b4 is selected from silicon hydride derivatives, preferably triethylsilane and polymethylhydrosiloxane;

the reductive reaction is carried out in the presence of Lewis acid catalyst b4, the Lewis acid catalyst b4 is selected from boron trifluoride diethyl etherate or trimethylsilyl trifluoromethanesulfonate (TMSOTf) and the like; the lewis acid catalyst b4 is preferably trimethylsilyl trifluoromethanesulfonate (TMSOTf).

The solvent b4 can be a mixed solvent of one or more of ethers, toluene, hexane and halogenated alkanes; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the halogenated alkane is one or more of dichloromethane, 1, 2-dichloroethane, 1, 1-dichloroethane and chloroform. The solvent is preferably dichloromethane.

The temperature of the reductive reaction is between-100 ℃ and-40 ℃, and the reaction time is between 0.5 and 12 hours;

in the step (c),

the reaction for removing the protecting group Pg is carried out in the presence of a fluorine-containing reagent c1, by using a compound 6 as a raw material, in the presence of an oxidant c2 and a solvent c2 or in the presence of a catalyst c3 containing Pd, Pt or Ni, a hydrogen donor c3 and a solvent c3 to obtain a compound 11;

when the reaction for removing the protecting group Pg is carried out in the presence of fluorine-containing reagent c1, the fluorine-containing reagent c1 is selected from tetrabutyl fluorideAmmonium Trihydrate (TBAF), potassium hydrogen fluoride (KHF)₂) Or potassium fluoride (KF), etc.;

when the reaction for removing the protecting group Pg is carried out in the presence of an oxidizing agent c2 and a solvent c2, the oxidizing agent c2 is selected from dichlorodicyanoquinone (DDQ), etc.;

the solvent c2 is one or more of dichloromethane, trichloromethane, methanol, ethanol and water; the solvent c2 is preferably a mixture of dichloromethane and methanol;

the reaction for removing the protecting group Pg is carried out in the presence of a catalyst c3 containing Pd, Pt or Ni and a hydrogen donor c3 in a solvent c 3;

the Pd, Pt or Ni containing catalyst c3 includes, but is not limited to, palladium on carbon, palladium black, palladium oxide, palladium acetate, platinum on carbon, platinum oxide, raney nickel, etc.;

the hydrogen donor c3 comprises hydrogen, transfer hydrogenation hydrogen donor or a combination of active metal and proton solvent as hydrogen donor; the transfer hydrogenation hydrogen donor is selected from ammonium formate, cyclohexene, tetrahydronaphthalene, cyclohexadiene, methylcyclohexadiene, or diethyl 2, 6-dimethyl-1, 4-dihydro-3, 5-pyridinedicarboxylate (Hantzsch ester), etc.; the method adopts the combination of active metal and proton solvent as a hydrogen donor, wherein the active metal is selected from zinc, zinc amalgam, sodium amalgam or magnesium, and the proton solvent is selected from methanol, ethanol, isopropanol or water;

the solvent c3 is selected from one or more solvent mixture of methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, acetone, water, TBME, DMF and THF;

the reaction temperature of the deprotection reaction c is between-20 ℃ and +70 ℃, and the reaction time is between 0.5 and 24 hours;

preferably, the deprotection reaction is such that after one deprotection reaction is completed, the product is reacted again in a reaction selected from the group consisting of the presence of a fluorine-containing reagent, the presence of an oxidizing agent and a solvent c2, or the presence of a catalyst containing Pd, Pt or Ni, a hydrogen donor, and a solvent c 3.

In the step (d), the step (c),

the condensation reaction is carried out by taking a compound 8 and a compound 9 as raw materials and a silanization reagent d in the presence of a solvent d and Lewis acid d to obtain a nucleoside compound 10;

the Acyl of 9 in the substrate of the condensation reaction is Acyl selected from benzoyl, substituted benzoyl and acetyl, and is preferably benzoyl and acetyl.

The solvent d can be a mixed solvent of one or more of ethers, toluene, hexane, acetonitrile and halogenated alkane; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the halogenated alkane is selected from one or more of dichloromethane, 1, 2-dichloroethane, 1, 1-dichloroethane and trichloromethane; the solvent d is preferably acetonitrile.

The silylating agent is N, O-bis (trimethylsilyl) acetamide (BSA).

The Lewis acid d is selected from tin tetrachloride, titanium tetrabromide, aluminum trichloride and trimethylsilyl trifluoromethanesulfonate. The lewis acid is preferably trimethylsilyl trifluoromethanesulfonate.

The condensation reaction temperature is between 0 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours.

In the step (e), the O-acyl deprotection reaction is selected from the condition e1 or the condition e2

Condition e 1: the O-acyl removing protection is carried out by taking a compound 10 as a raw material in the presence of an alkali e1 and a solvent e1 to obtain a nucleoside compound 11;

the base e1 is selected from sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, triethylamine, diisopropylethylamine and calcium hydroxide;

after the alkali e1 and the solvent e1 are mixed, the concentration of the alkali is selected from 1M to 20M, preferably 2M to 4M;

the solvent e1 can be a mixed solvent of one or more of ethers, toluene, hexane, acetonitrile and halogenated alkanes; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the solvent is preferably tetrahydrofuran;

the reaction temperature is between-20 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours;

r in the deacylation-protected Compound 10 and Compound 11¹Selected from benzoyl, pivaloyl and acetyl.

Condition e 2: the de-O-acyl protection is carried out in a solvent e2 in the presence of a base e 2;

the alkali is selected from ammonia water, ammonia gas, sodium methoxide, potassium carbonate and triethylamine, and preferably ammonia gas;

the solvent e2 is selected from methanol, ethanol and isopropanol, and is preferably methanol;

r in the compound 10 for removing O-acyl protection reaction¹R is selected from benzoyl, pivaloyl, acetyl, compound 11¹Selected from benzoyl;

in the step (f),

the protection reaction is to react a compound 11 serving as a raw material with a protection reagent in the presence of a solvent f1 and an acid catalyst to obtain a nucleoside compound 7;

the solvent f1 is selected from one or more of acetone, methanol, ethanol, dichloromethane, chloroform, acetonitrile, tert-butyl methyl ether (TBME), N-Dimethylformamide (DMF), water and Tetrahydrofuran (THF) mixture;

the protective reagent is selected from acetone, 2, 2-dimethoxypropane (CAS ═ 77-76-9);

the acid catalyst is selected from p-toluenesulfonic acid, benzenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, hydrochloric acid, sulfuric acid, camphorsulfonic acid;

the temperature of the protection reaction is between-20 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours.

In the step (g), the step (c),

the acylation reaction is carried out by taking a compound 7 as a raw material and acylating agent LgCl or Lg₂O, in the presence of a solvent g1 and an acid-binding agent, reacting to obtain a nucleoside compound 12;

the acylating agent LgCl or Lg₂O is selected from p-toluenesulfonyl chloride, methanesulfonyl chloride, benzenesulfonyl chloride, trifluoromethanesulfonic anhydride or the like;

the acid-binding agent is selected from triethylamine, N-Diisopropylethylamine (DIPEA) or diazabicyclo (DBU, CAS: 6674-22-2), etc.;

the solvent g1 can be a mixed solvent of one or more of ethers, toluene, hexane and halogenated alkanes; the ethers are selected from one or more of diethyl ether, TBME, isopropyl ether, 2-methyl tetrahydrofuran and tetrahydrofuran; the halogenated alkane is selected from one or more of dichloromethane, 1, 2-dichloroethane, 1, 1-dichloroethane and trichloromethane; the solvent g1 is preferably dichloromethane.

The temperature of the acylation reaction is between-100 ℃ and +100 ℃, and the reaction time is 1 hour to 24 hours.

In the step (h), the step (c),

when the compound 7 is used as a raw material,

the azide substitution reaction is carried out on a compound 7 and azido diphenyl phosphate in the presence of a solvent h1 and an alkali h1 to obtain a nucleoside compound 13;

or, the azide substitution reaction can also be carried out in two steps of h2-1 and h2-2 to obtain a nucleoside compound 13;

the solvent h1 is selected from dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, toluene, xylene, chlorobenzene, preferably toluene and dioxane;

said base h1 is selected from Diazabicyclo (DBU), diisopropylethylamine, preferably Diazabicyclo (DBU); the temperature of the azide substitution reaction is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours.

The step h2-1 is carried out with azido diphenyl phosphate in the presence of a solvent h2-1 and an alkali h2-1 by taking a compound 7 as a raw material;

the solvent h2-1 is selected from dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, toluene, xylene, chlorobenzene, preferably toluene and dioxane;

the base h2-1 is selected from Diazabicyclo (DBU), diisopropylethylamine, preferably Diazabicyclo (DBU);

the reaction temperature of the h2-1 is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours.

The step h2-2 is to take the product of the step h2-1 as a raw material, react with sodium azide and alkali h2-2 in the presence of a phase transfer catalyst h2-2 in a solvent h2-2 to obtain a nucleoside compound 13;

the solvent h2-2 is selected from dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, toluene, xylene and chlorobenzene, and is preferably dioxane;

the base h2-2 is selected from Diazabicyclo (DBU), diisopropylethylamine, preferably Diazabicyclo (DBU).

The phase transfer catalyst h2-2 is selected from tetra-n-butylammonium bromide, tetra-n-butylammonium chloride, tetra-n-butylammonium iodide, and crown ether; the phase transfer catalyst is preferably 15-crown-5;

the reaction temperature is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours;

preferably, the azide substitution reaction is carried out in two steps of h2-1 and h 2-2;

when the compound 12 is used as a raw material, the azide substitution reaction is carried out with sodium azide in a solvent h3 to obtain a nucleoside compound 13;

the solvent h3 is selected from dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, and preferably is dimethylformamide.

The temperature of the azide substitution reaction is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours.

The reduction reaction of the compound 13 can be carried out in the presence of a phosphine reagent h4 or a hydride h4 in a solvent h4 to obtain a nucleoside compound 14,

or,

the reduction reaction of the compound 13 can also be carried out in the presence of a catalyst h5, a hydrogen donor h5 and a solvent h5 to obtain a nucleoside compound 14;

when the reduction reaction of the compound 13 is carried out in the presence of the phosphine reagent h4, or hydride h4, in the solvent h4,

the phosphine reagent h4 is selected from triphenylphosphine, trimethylphosphine or tri-n-butylphosphine;

the hydride h4 is selected from sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride and the like;

the solvent h4 is selected from one or more solvent mixture of water, dichloromethane, trichloromethane, acetonitrile, TBME, DMF and THF;

the temperature of the reduction reaction is between-20 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours.

When the reduction reaction h5 of the compound 13 is carried out in the presence of a solvent h5, a catalyst h5 and a hydrogen donor h5,

the solvent h5 is selected from one or more solvent mixture of methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, acetone, water, TBME, DMF and THF;

the catalyst h5 is a catalyst containing Pd, Pt or Ni, including but not limited to palladium carbon, palladium black, palladium oxide, palladium acetate, platinum carbon, platinum oxide, Raney nickel, etc.;

the hydrogen donor h5 comprises hydrogen, transfer hydrogenation hydrogen donor or a combination of active metal and proton solvent as the hydrogen donor; the transfer hydrogenation hydrogen donor is selected from ammonium formate, cyclohexene, tetrahydronaphthalene, cyclohexadiene, methylcyclohexadiene, or diethyl 2, 6-dimethyl-1, 4-dihydro-3, 5-pyridinedicarboxylate (Hantzsch ester), etc.; the method adopts the combination of active metal and proton solvent as a hydrogen donor, wherein the active metal is selected from zinc, zinc amalgam, sodium amalgam or magnesium, and the proton solvent is selected from methanol, ethanol, isopropanol or water.

The temperature of the reduction reaction is between-100 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours.

In the step (i),

when the compound 7 is used as a raw material,

the amido substitution reaction is a compound 7 and an amination reagent Pg¹NH i1, and triphenylphosphine, and condensing agent i1, in solvent i1 to give nucleoside compound 15;

the amination reagent Pg¹NH i1 is phthalimide;

the condensing agent i1 is selected from diethyl azodicarboxylate and diisopropyl azodicarboxylate;

the solvent i1 is selected from one or more of dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethanol, methanol, water, dichloromethane, trichloromethane, acetonitrile, tert-butyl methyl ether (TBME) and a mixture of solvents, wherein the solvent is preferably THF;

the temperature of the amino substitution reaction is between-20 ℃ and +100 ℃, and the reaction time is between 1 hour and 24 hours;

when the compound 12 is used as a raw material,

the amino substitution reaction is carried out on a compound 12 and an amination reagent i2 in a solvent i2 to obtain a nucleoside compound 15 or 15-1;

the amination reagent i2 is selected from phthalimide and phthalimide potassium salt;

the solvent i2 is one or more solvent mixtures selected from dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethanol, methanol, water, dichloromethane, trichloromethane, acetonitrile and TBME;

the temperature of the amino substitution reaction is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours.

The deprotection reaction is carried out on a compound 15 or 15-1 and hydrazine hydrate in a solvent i3 to obtain a nucleoside compound 14;

the solvent i3 is selected from one or a mixture of more solvents of dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethanol, methanol, water, dichloromethane, trichloromethane, acetonitrile and TBME; the solvent i3 is preferably ethanol;

the deprotection reaction temperature is between 0 ℃ and +150 ℃, and the reaction time is between 1 hour and 24 hours.

In the step (j),

the reductive amination reaction j1 is carried out in an alkylating reagent j1, a reducing agent j1, an acid catalyst j1 and a solvent j1 by taking a compound 14 as a raw material to obtain a nucleoside compound 16;

the alkylating agent j1 is selected from acetone, dimethoxyacetone ketal, preferably acetone;

the reducing agent j1 is selected from sodium cyanoborohydride or sodium triacetoxyborohydride, preferably sodium triacetoxyborohydride;

the pH of the reaction solution is controlled to be between 2 and 10 by the acid catalyst j1, and the acid catalyst is selected from trifluoroacetic acid, hydrochloric acid, formic acid or acetic acid, and is preferably acetic acid;

the solvent j1, wherein the solvent j1 is a mixture of one or more solvents selected from dichloromethane, trichloromethane, acetonitrile, TBME, methanol, ethanol, acetone, water and THF, and is preferably methanol;

the temperature of the reductive amination reaction j1 is between-20 ℃ and +100 ℃, and the reaction time is 1 hour to 24 hours.

In the step (k), the step (c),

performing an amino substitution reaction of the compound 12 to obtain a compound 12 and an amination reagent in a solvent k1 to obtain a nucleoside compound 16;

the amination reagent is selected from isopropylamine and potassium isopropylamine;

the solvent k1 is selected from one or a mixture of more solvents of isopropylamine, dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethanol, methanol, water, dichloromethane, chloroform, acetonitrile and TBME; the solvent k1 is preferably ethanol or isopropylamine;

In the step (l), the step (a),

the reductive amination reaction is carried out in an alkylating reagent M ═ O, a reducing agent, an acid catalyst and a solvent l1 by using a compound 16 as a raw material to obtain a nucleoside compound 17;

the alkylating reagent M ═ O is selected from ketone reagentsOr aldehyde reagentsPreferably, it is

The reducing agent is selected from sodium cyanoborohydride or sodium triacetoxyborohydride; preferably sodium triacetoxyborohydride;

controlling the pH of the reaction solution to be between 2 and 10 in the reductive amination reaction process;

the acid catalyst is selected from trifluoroacetic acid, hydrochloric acid, formic acid or acetic acid; preferably acetic acid;

the solvent l1 is selected from one or more of dichloromethane, trichloromethane, acetonitrile, TBME, methanol, ethanol, water and THF, preferably acetonitrile;

the temperature of the reductive amination reaction is between 0 ℃ and +100 ℃, and the reaction time is between 12 hours and 36 hours.

In the step (m), the step (c),

the reductive amination reaction is carried out in an alkylating reagent M ═ O, a reducing agent, an acid catalyst and a solvent M1 by taking a compound 14 as a raw material to obtain a nucleoside compound 16-1;

the acid catalyst is selected from trifluoroacetic acid, hydrochloric acid, formic acid or acetic acid, preferably acetic acid;

the solvent m1 is selected from one or more of dichloromethane, trichloromethane, acetonitrile, TBME, methanol, ethanol, water and THF mixture, preferably methanol or acetonitrile;

In the step (n),

the reductive amination reaction is carried out in an alkylating reagent, a reducing agent, an acid catalyst and a solvent n1 by taking a compound 16-1 as a raw material to obtain a nucleoside compound 17;

the alkylating agent is selected from acetone, 2, 2-dimethoxypropane, preferably acetone;

the solvent n1 is selected from one or more of dichloromethane, trichloromethane, acetonitrile, TBME, methanol, ethanol, water and THF mixture, preferably methanol or acetonitrile;

In the step (o), the step (c),

the deprotection reaction is carried out in a solvent o1 in the presence of an acidic substance by using a compound 17 as a raw material to obtain a nucleoside compound 2;

the acidic substance is selected from p-toluenesulfonic acid, camphorsulfonic acid, hydrogen chloride, acetic acid, sulfuric acid or trifluoroacetic acid and the like; the acidic substances are preferably hydrogen chloride and trifluoroacetic acid;

the solvent o1 is selected from one or more solvent mixtures of dichloromethane, trichloromethane, acetonitrile, TBME, methanol, ethanol, water and THF; the solvent o1 is preferably dichloromethane or water;

the deprotection reaction temperature is between-20 ℃ and +100 ℃, and the reaction time is 1 hour to 24 hours.

The term "one-pot" as used in the present invention means that the reaction product of the respective stages of the reaction is carried out in one step without isolation and purification.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the group consisting of 2' -C-methyl-substituted nucleosides according to formula 2 of the present invention and/or pharmaceutically acceptable salts thereof as an active ingredient, and optionally pharmaceutically acceptable carriers and/or excipients (e.g., diluents, etc.).

The fourth aspect of the present invention is to provide the use of the 2' -C-methyl substituted nucleoside compound of formula 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof in the preparation of an anticancer drug. The compound or the pharmaceutically acceptable salt thereof can be independently administered or administered in combination with other pharmaceutically acceptable chemotherapeutic agents, or kinase inhibitors, epigenetic target drugs and antibody antitumor drugs, particularly in combination with other antitumor drugs. Such chemotherapeutic agents include, but are not limited to: oxaliplatin (Oxaliplatin), vinblastine (Vinorebine), Paclitaxel (Paclitaxel), Docetaxel (Docetaxel), Gemcitabine (Gemcitabine), Capecitabine (Capecitabine), Rituximab (Rituximab), hydroxycamptothecin (Hydroxycampothecin), Pirarubicin (Pirarubicin), Epirubicin (Epirubicin), Azacitidine (Azacitidine), Decitabine (Decitabine), Cytarabine (Cytarabine); such kinase inhibitors include, but are not limited to: MEK (mitogen-activated protein kinase) inhibitors including, but not limited to, Trametinib (Trametinib), cobitinib (Cobimetinib); EGFR inhibitors (EGFR inhibitors) including, but not limited to, iressa (gefitinib), erlotinib (erlotinib), afatinib (afatinib), brigitinib, icotinib (icotinib), osetinib (ositinib); the epigenetic target drugs include, but are not limited to, Histone Deacetylase (HDAC) inhibitors including, but not limited to, Vorinostat (Vorinostat), Panobinostat (Panobinostat), Belinostat (Belinostat); the antibody antineoplastic drugs include, but are not limited to, bevacizumab (bevacizumab), cetuximab (cetuximab), Yiprimumab (ipilimumab), nivolumab (nivolumab), Pembrolizumab (Pembrolizumab). The ingredients to be combined may be administered simultaneously or sequentially, in a single formulation or in different formulations. The combinations include not only combinations of a compound of the invention and one other active agent, but also combinations of a compound of the invention and two or more other active agents.

Accordingly, in a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the group consisting of the 2' -C-methyl substituted nucleoside compounds according to the present invention and pharmaceutically acceptable salts thereof as an active ingredient and other pharmaceutically acceptable therapeutic agents, particularly other anticancer drugs. The pharmaceutical composition optionally may further comprise a pharmaceutically acceptable carrier and/or excipient (e.g., diluent, etc.).

In a sixth aspect of the present invention, there is provided a method for treating or preventing cancer, which comprises administering a therapeutically effective amount of one or more selected from the group consisting of the 2 '-C-methyl-substituted nucleoside compounds according to the present invention and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to the present invention comprising as an active ingredient a therapeutically effective amount of one or more selected from the group consisting of the 2' -C-methyl-substituted nucleoside compounds according to the present invention and pharmaceutically acceptable salts thereof, to a patient in need of such treatment.

In the present invention, the cancer is a non-solid tumor, including but not limited to a hematological tumor, including: lymphoma, acute leukemia, and chronic leukemia.

Advantageous effects

The invention provides a nucleoside compound 2 with 2-C-methyl substitution and a preparation method thereof. Compound 2 has comparable activity as a DOT1L enzyme inhibitor, comparable potency in vivo, and lower toxicity relative to known nucleoside DOT1L inhibitors characterized by structure 1.

Drawings

FIG. 1 shows the body weight and tumor suppression of cis-2e administered subcutaneously in an MV (4,11) transplantable tumor animal model.

Detailed Description

The invention further refers to the following examples describing in detail the preparation and use of the salts and crystalline forms of the invention. It will be apparent to those skilled in the art that many changes, both to materials and methods, may be practiced without departing from the scope of the invention.

In the following preparation examples, the following examples were conducted,¹H-NMR was measured with a Varian Mercury AMX300 type apparatus, a Varian Mercury-300High Performance Digital FT-NMR type apparatus, a Bruker Ultrashield 500NMR type apparatus, a Varian Mercury-400High Performance Digital FT-NMR type apparatus, an Agilent 1260Prospekt2Bruker Assicend 600NMR type apparatus, and deuterated chloroform (CDCl)₃) Deuterated methanol (MeOD-d)₄) Tetramethylsilane (TMS) was used as an internal standard. Mass spectra were measured on a Thermo Finnigan MAT-95 type instrument, a Waters Q-TofUltima Globalspectrometer type instrument. Melting points were determined on an SGW X-4 melting point apparatus.

In the following preparation examples, reagents such as petroleum ether, ethanol, ethyl acetate and the like with a boiling range of 60 to 90 ℃ are analytically pure and provided by chemical reagents of the national drug group, ltd, and the used reagents and solvents are not specially treated except for special specifications. All solvents were redistilled before use and the anhydrous solvents used were dried according to standard procedures. All reactions were carried out under nitrogen and followed by TLC, all work-up was carried out with a saturated saline wash and anhydrous magnesium sulfate drying, except as indicated. The purification of the product was performed by column chromatography using silica gel (200-300 mesh) including 200-300 mesh, GF₂₅₄Produced by Qingdao oceanic plant or tobacco terrace edge Bo silica gel company.

Common reagents and solvents are available from Shanghai Biao medical science and technology, Inc., Shanghai book sub-medical science and technology, Inc., Shanghai Tatanke technology, Inc., Annagi chemical or national drug group chemical reagents, Inc., unless otherwise specified.

The optical rotation (+/-) was measured by an OR-2090 chiral detector (Hg-Xe lamp, 150W) manufactured by Japan chemical Co., Ltd. (JASCO).

High Performance Liquid Chromatography (HPLC) assay conditions: agilent 1260 analytical high performance liquid chromatography system (Agilent Inc.) and LC3000 preparative high performance liquid chromatography system (Beijing Innovation technology, Inc.).

Chiral OD or OJ columns were purchased from xylonite drug chiral technology (shanghai) ltd, column size 2cm Φ X25 cm.

Analytical high performance liquid chromatography conditions: c18 column (5 μm,4.6X 250mm), ultraviolet detection band 214 and 280nm, elution conditions 0-90% acetonitrile (containing 0.1% V/VTFA) gradient wash for 30 minutes.

Preparing high performance liquid chromatography conditions: c18 column (5 μm,19X 250mm), ultraviolet detection band 214 and 280nm, elution conditions 0-90% acetonitrile (containing 0.1% V/VTFA) gradient wash for 30 minutes.

In the above discussion and in the examples below, the following abbreviations have the following meanings. An abbreviation has a generally accepted meaning if it is not defined.

TLC is thin layer chromatography;

DMF is N, N-dimethylformamide;

EtOAc is ethyl acetate;

DMAP is 4-dimethylaminopyridine;

THF is tetrahydrofuran;

DMSO is dimethyl sulfoxide;

DCM is dichloromethane;

Et₃n is triethylamine;

TBME is tert-butyl methyl ether;

Boc₂o is di-tert-butyl dicarbonate;

Cbz₂o is benzyloxy formic anhydride;

CbzCl is benzyl chloroformate;

TMSOTf is trimethylsilyl trifluoromethanesulfonate;

DDQ is dichloro dicyano benzoquinone;

BSA is N, O-bis (trimethylsilyl) acetamide;

STAB is sodium triacetoxyborohydride.

Example 1

Preparation of Compound 5-1 a:

dissolving raw material 5(5.0g,30.84mmol) [ prepared according to the Tetrahedron: Asymmetry 2007,18,500-512 method ] in anhydrous DMF (200mL), cooling to-10 ℃, slowly adding 60% NaH (1.6g, 40.09mmol), stirring at-10 ℃ for reaction for 1 hour, slowly adding 2-bromomethylnaphthalene (10.2g, 46.26mmol), stirring for 30 minutes, then adding 60% NaH (1.6g, 40.09mmol) and 2-bromomethylnaphthalene (10.2g, 46.26mmol), repeating twice, stirring at-10 ℃ for reaction for 48 hours, quenching the reaction liquid in 500mL ice water, extracting with ethyl acetate (100mLx 4), combining organic phases, washing with 200mL saturated brine organic layer, drying over anhydrous sodium sulfate, concentrating, separating (10:1, V/V, petroleum ether: 5-12 g) to obtain colorless product (12.a), 21.59mmol), yield: 70 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₉H₃₅O₅ ⁺Theoretical value is 583.2479, found 583.2471.

Example 2

Preparation of compound 4 a:

compound 3a (5.0g, 23.36mmol) was suspended in THF (100mL), and triethylamine (16.3mL,0.12mol) was added) And dimethylaminopyridine (287mg,2.35mmol) were added portion wise slowly to Boc₂O (15.4g,70.42mmol), stirring at room temperature overnight, after the reaction is completed, adding 3M aqueous sodium hydroxide (100mL), heating to 60 ℃, stirring and reacting for 6 hours, returning to room temperature, separating an organic layer, extracting an aqueous layer with ethyl acetate (20mLx 3), combining the organic layers, washing the organic layer with saturated saline (100mL), drying with anhydrous sodium sulfate, concentrating, separating with silica gel column (10:1, V/V, petroleum ether: EtOAc), to obtain a white solid product 4a (6.3g, 20.09mmol), yield: 86 percent.

M.p. 160.5-163.8 ℃ (petroleum ether-EtOAc); r_f0.29(5:1, V/V, petroleum ether: EtOAc); HPLCt_R＝2.72min；¹H-NMR(400MHz,CDCl₃)δ8.62(s,1H,ArH),8.58(s,1H,NH),7.70(s,1H,ArH),1.57(s,9H,C(CH ₃)₃)；¹³C-NMR(126MHz,CDCl₃)δ149.51(C＝O),149.41,148.68,133.48,129.16,103.44,83.87(C(CH₃)₃),28.14(C(CH₃)₃) (ii) a High resolution mass spectrometry (ESI)⁺)：C₁₀H₁₂BrN₅NaO₂ ⁺Theoretical value is 336.0067, found 336.0058.

Example 3

Preparation of compound 4 b:

compound 3b (5.0g,23.47mmol) was suspended in THF (100mL), triethylamine (16.3mL,0.12mol) and dimethylaminopyridine (287mg,2.35mmol) were added and Boc was added slowly in portions₂O (15.4g,70.42mmol), reacting overnight under stirring at room temperature, adding 3M aqueous sodium hydroxide (100mL) after reaction, heating to 60 deg.C, reacting under stirring for 6 hr, recovering to room temperature, separating organic layer, extracting water layer with ethyl acetate (20mLx 3), combining organic phases, washing organic phase with saturated saline (100mL)Dried over anhydrous sodium sulfate, concentrated and isolated by silica gel column (10:1, V/V, petroleum ether: EtOAc) to give product 4b (6.6g, 21.12mmol) as a white solid in yield: 90 percent.

M.p.180.0 deg.C (decomposition), R_f0.21(10:1, V/V, petroleum ether: EtOAc); HPLC t_R＝2.79min；¹H-NMR(400MHz,CDCl₃)δ8.28(s,1H,ArH),8.24(s,1H,NH),7.39(d,J＝4.8Hz,1H,ArH),6.90(d,J＝4.8Hz,1H,ArH),1.58(s,9H,C(CH ₃)₃)；¹³C-NMR(126MHz,CDCl₃)δ151.52(C＝O),150.60,147.00,116.57,115.34,108.71,103.35,83.13(C(CH₃)₃),28.29(C(CH₃)₃) (ii) a High resolution mass spectrometry (ESI)⁺)：C₁₁H₁₃BrN₄NaO₂ ⁺Theoretical value is 335.0114, found 335.0119.

Example 4

Preparation of compound 6 a:

cooling THF (20mL) solution of 4b (1.0g, 3.19mmol) to-30 deg.C under anhydrous and oxygen-free conditions, adding diisopropylamino lithium THF solution (2.1mL, 4.15mmol, 2M tetrahydrofuran solution) dropwise, controlling the temperature at-30 deg.C, stirring for reaction for 50min, further cooling to-78 deg.C, adding n-BuLi (5.0mL, 7.98mmol, 1.6M hexane solution) dropwise, stirring for reaction for 5min, adding THF (20mL) solution of 5-1a (5.6g, 9.58mmol) dropwise, maintaining the temperature at-78 deg.C, reacting for 2 hr, detecting by TLC, adding saturated NH₄The reaction was quenched with aqueous Cl (50mL), brought to room temperature, extracted 4 times with ethyl acetate (40 mL. times.4), the organic layers were combined, washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, concentrated, and the residue was dissolved in 20mL of dry CH₂Cl₂In (b), cooled to-78 ℃, Et is added₃SiH(2.0mL,12.77mmol), stirring for 5min, adding TMSOTf (1.2mL,6.39mmol) dropwise, reacting at-78 deg.C for 2h, after TLC detection, adding saturated aqueous sodium bicarbonate solution (40mL) to quench the reaction, and adding CH to the aqueous layer₂Cl₂Extraction (15 mL. times.3) was performed 3 times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column separation (4:1, V/V, petroleum ether: EtOAc) to give product 6a (1.9g,2.40mmol) as a white amorphous solid in yield: 75 percent.

¹H-NMR(400MHz,CDCl₃) beta-configuration characteristic peak δ 6.0(s,1H, H-1); high resolution mass spectrometry (ESI)⁺)：C₅₀H₄₉N₄O₆ ⁺Theoretical value is 801.3647, found 801.3643.

Example 5

Preparation of compound 6 b:

cooling THF (10mL) solution of 4a (0.5g, 1.59mmol) to-30 deg.C under anhydrous and oxygen-free conditions, adding diisopropylamino lithium THF solution (1.03mL, 2.07mmol, 2M tetrahydrofuran solution) dropwise, controlling the temperature at-30 deg.C, stirring for reaction for 50min, further cooling to-78 deg.C, adding n-BuLi (2.5mL, 3.98mmol, 1.6M hexane solution) dropwise, stirring for reaction for 5min, adding THF (20mL) solution of 5-1a (2.8g, 4.77mmol) dropwise, maintaining the temperature at-78 deg.C, reacting for 2 hr, detecting by TLC, adding saturated NH₄Quenching the reaction with a Cl aqueous solution (40mL), returning to room temperature, extracting with ethyl acetate (20mL × 4) for 4 times, combining the organic layers, drying with anhydrous sodium sulfate, and concentrating to obtain a crude intermediate product; dissolving the crude product in dry MeCN under nitrogen protection, cooling to-40 deg.C, adding Et₃SiH (1.02mL,6.37mmol), stirred for 5min, and then BF was added dropwise₃·Et₂O (402 mu L,3.18mmol), naturally heating to room temperature, reacting for 2h, after TLC detection reaction,the reaction was quenched by addition of saturated aqueous sodium bicarbonate (10mL) and the aqueous layer was CH-washed₂Cl₂Extraction (15 mL. times.3) was performed 3 times, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column separation (25:1, V/V, dichloromethane: methanol) to obtain white foamy solid 6b (726mg,1.03mmol), yield: 65 percent.

¹H-NMR(400MHz,CDCl₃) beta-configuration characteristic peak δ 5.8(s,1H, H-1); high resolution mass spectrometry (ESI)⁺)：C₄₄H₄₀N₅O₄ ⁺Theoretical value is 702.3075, found 702.3070.

Example 6

Preparation of compound 7 a:

intermediate 6a (1.5g,1.87mmol) was dissolved in 40mL of dichloromethane/methanol (4:1) at room temperature, 10 drops of water were added, DDQ (2.6g, 11.24mmol) was added, the reaction was stirred at room temperature for 72 hours, after the reaction was completed, 50mL of saturated sodium bicarbonate solution was added to quench the reaction, the organic layer was separated, dried over anhydrous sodium sulfate and concentrated to give a slightly reddish brown amorphous solid 11 a. High resolution mass spectrometry (ESI)⁺)：C₁₇H₂₅N₄O₆ ⁺Theoretical value is 381.1769, found 381.1777.

Dissolving the intermediate 11a in 40mL of acetone, adding 2, 2-dimethoxypropane (20mL, V/V ═ 2), dropwise adding concentrated HCl to adjust the pH to about 3, stirring at room temperature overnight for reaction, after the reaction is finished, adding a saturated sodium bicarbonate solution to adjust the pH to be neutral, spin-drying the reaction solution, and separating and purifying with a silica gel column (15:1, dichloromethane: methanol) to obtain a white amorphous solid 7a (432mg, 1.35mmol), wherein the yield: 72 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₅H₂₁N₄O₄ ⁺Theoretical value is 321.1557, found 321.1565.

Example 7

Preparation of compound 7 b:

at room temperature, dissolving intermediate 6b (1.0g,1.42mmol) in 40mL dichloromethane/methanol (4:1), adding 10 drops of water, adding DDQ (1.9g, 8.55mmol), stirring at room temperature for reaction for 72 hours, adding 50mL saturated sodium bicarbonate solution after the reaction is finished to quench the reaction, separating an organic layer, drying over anhydrous sodium sulfate, and concentrating to obtain a slightly reddish brown amorphous solid 11 b. High resolution mass spectrometry (ESI)⁺)：C₁₁H₁₆N₅O₄ ⁺Theoretical value is 282.1197, found 282.1203.

Dissolving the intermediate 11b in 40mL of acetone, adding 2, 2-dimethoxypropane (20mL, V/V ═ 2), dropwise adding concentrated HCl to adjust the pH to about 3, stirring at room temperature overnight for reaction, after the reaction is finished, adding a saturated sodium bicarbonate solution to adjust the pH to be neutral, spin-drying the reaction solution, and separating and purifying with a silica gel column (15:1, dichloromethane: methanol) to obtain a white amorphous solid 7b (321mg, 1.00mmol), wherein the yield: 70 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₄H₂₀N₅O₄ ⁺Theoretical value is 322.1510, found 322.1517.

Example 8

Preparation of compound 12 a:

dissolving the intermediate 7a (1.0g, 3.12mmol) in 20mL of dry DCM, cooling to 0 ℃, adding triethylamine (2.2mL, 15.61mmol), slowly dropping MsCl (0.3mL, 3.75mmol), stirring at 0 ℃ for reaction for 30min, after the reaction is finished, adding 2mL of methanol to quench the reaction, stirring for 10min, washing the reaction solution with 20mL of saturated bicarbonate solution, separating an organic layer, drying over anhydrous sodium sulfate, concentrating, separating a silica gel column, purifying (1:1, V/V, petroleum ether: EtOAc) to obtain a white amorphous solid 12a (1.0g, 2.56mmol), yield: 82 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₆H₂₃N₄O₆S⁺Theoretical value is 399.1333, found 399.1339.

Example 9

Preparation of compound 13 a:

dissolving the compound 7a (1.0g, 3.12mmol) in 20mL of anhydrous THF, cooling to 0 ℃ under the protection of nitrogen, adding triphenylphosphine (983mg, 3.75mmol), dropwise adding diisopropyl azodicarboxylate (0.7mL, 3.75mmol), keeping the temperature at 0 ℃ and continuing stirring for 10 minutes, adding diphenyl azidophosphate (0.8mL, 3.75mmol), heating to room temperature for reaction for 5 hours, after the reaction is finished, concentrating the reaction solution, separating and purifying with silica gel column (25:1, V/V, dichloromethane: methanol) to obtain white amorphous solid 13a (927mg, 2.68mmol), and obtaining the yield: 86 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₅H₂₀N₇O₃ ⁺Theoretical value is 346.1622, found 346.1628.

Example 10

Preparation of compound 12 b:

dissolving intermediate 7b (4.3g, 13.38mmol) in 150mL dry DCM, cooling to 0 deg.C, adding triethylamine (9.2mL, 66.20mmol), slowly dropping MsCl (1.23mL, 15.89mmol), stirring at 0 deg.C for 30min, after the reaction is finished, adding 2mL methanol to quench the reaction, stirring for 10min, washing the reaction solution with 100mL saturated bicarbonate solution, separating the organic layer, drying over anhydrous sodium sulfate, concentrating, separating and purifying with silica gel column (30:1, V/V, DCM: MeOH) to obtain white solid 12b (4.0g, 10.01mmol), yield: 75 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₅H₂₂N₅O₆S⁺Theoretical value is 400.1285, found 400.1277.

Example 11

Preparation of compound 16 a:

compound 12a (1.0g, 2.51mmol) was dissolved in 10mL of isopropylamine, reacted for 48 hours in a pressure resistant reaction tube heated to 50 ℃ and after completion of the reaction, isopropylamine was evaporated to dryness and the residue was purified by silica gel column separation (15:1, V/V, DCM: MeOH) to give light brown amorphous solid 16a (844mg, 2.33mmol) with yield: 93 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₈H₂₈N₅O₃ ⁺Theoretical value is 362.2187, found 362.2181.

Example 12

Preparation of compound 16 b:

compound 12b (4.0g, 10.01mmol) was dissolved in 50mL of isopropylamine, heated to 50 ℃ in a pressure-resistant reaction tube and reacted for 48 hours, after the reaction was completed, isopropylamine was evaporated to dryness, and the residue was purified by silica gel column separation (15:1, V/V, DCM: MeOH) to give light brown amorphous solid 16b (3.4g, 9.51mmol), yield: 95 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₇H₂₇N₆O₃ ⁺Theoretical value is 363.2139, found 363.2135.

Example 13

Preparation of compound 14 a:

compound 12a (1.0g, 2.51mmol) was dissolved in anhydrous DMF (10mL) and NaN was added₃(326mg,5.02mmol), stirring to form a suspension, heating to 80 ℃, stirring for reaction for 2 hours, cooling the reaction liquid to room temperature after the raw materials are completely removed, diluting with DCM (50mL), washing with water (20mL) and saturated NaCl solution (20mL) in sequence, drying with anhydrous sodium sulfate, and concentrating to obtain a crude compound 13 a; high resolution mass spectrometry (ESI)⁺)：C₁₅H₂₀N₇O₃ ⁺Theoretical value is 346.1622, found 346.1628. Crude 13a was dissolved in THF (20mL), and PPh was added to the solution in order₃(790mg,3.01mmol) and water (2.0mL) were heated to 40 ℃ and stirred for reaction overnight, after completion of the reaction, the reaction mixture was concentrated and purified by column chromatography to obtain compound 14a (601mg, 1.88mmol), yield: 75 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₅H₂₂N₅O₃ ⁺Theoretical value320.1717, found 320.1723.

Example 14

Preparation of compound 14 b:

compound 12b (5.0g, 12.52mmol) was dissolved in anhydrous DMF (50mL) and NaN was added₃(1.6g,25.04mmol), stirring to form a suspension, heating to 80 ℃, stirring for reaction for 2 hours, cooling the reaction liquid to room temperature after the raw materials are completely reacted, diluting with DCM (150mL), washing with water (100mL) and saturated NaCl solution (100mL) in sequence, drying with anhydrous sodium sulfate, and concentrating to obtain a crude compound 13 b; high resolution mass spectrometry (ESI)⁺)：C₁₄H₁₉N₈O₃ ⁺Theoretical value is 347.1575, found 347.1570. Crude 13b was dissolved in THF (50mL), and PPh was added to the solution in order₃(306g,13.77mmol) and water (5.0mL), heating to 40 ℃, stirring for reaction overnight, after completion of the reaction, concentrating the reaction solution, and purifying by column chromatography to obtain compound 14b (2.8g, 8.76mmol), yield: 70 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₄H₂₁N₆O₃ ⁺Theoretical value is 321.1670, found 321.1661.

Example 15

Preparation of compound 16 a:

under the protection of nitrogen, compound 14a (1.0g, 3.13mmol) was dissolved in 20mL of anhydrous methanol, acetone (2.4mL, V/m ═ 2.4) and AcOH (0.4mL, 6.26mmol) were added, the mixture was stirred at room temperature for 10 minutes, and after slow addition of STAB (1.3g, 6.26mmol), the reaction was continued for 2 hours, after completion of TLC detection reaction, dichloromethane (40mL) was added to dilute the reaction mixture, the reaction mixture was washed with saturated sodium bicarbonate solution (40mL), the reaction mixture was washed with saturated saline (50mL), dried over anhydrous sodium sulfate and concentrated, and the mixture was separated and purified by silica gel column to obtain amorphous white solid 16a (996mg, 2.76mmol), yield: 88 percent.

Example 16

Preparation of compound 16 b:

under nitrogen protection, compound 14b (2.5g, 7.80mmol) was dissolved in 50mL of anhydrous methanol, acetone (6mL, V/m ═ 2.4), AcOH (0.9mL, 15.61mmol) was added, after stirring at room temperature for 10 minutes, STAB (3.0g, 14.30mmol) was slowly added, the reaction was continued for 2 hours, after completion of TLC detection reaction, DCM and MeOH (50mL, 10:1, V/V) were added to dilute the reaction, the reaction was washed with saturated sodium bicarbonate solution (50mL), washed with saturated brine (50mL), dried over anhydrous sodium sulfate, concentrated, isolated and purified by silica gel column to give amorphous white solid 16b (2.4g, 6.63mmol), yield: 85 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₇H₂₇N₆O₃ ⁺Theoretical value is 363.2139, found 363.2132.

Example 17

Preparation of compound 10 a:

weighing compound 8a (20.0g, 83.60mmol) and suspending in 300mL acetonitrile, adding N, O-bis (trimethylsilyl) acetamide (53.4mL, 217.36mmol), stirring at room temperature until the reaction solution is clear, adding 9a (53.4g, 91.96mmol), adding 200mL acetonitrile, stirring until all acetonitrile is dissolved, adding trimethylsilyl trifluoromethanesulfonate (53.0mL, 292.51mmol), heating to 80 ℃, stirring for 8 hours, after the reaction is finished, adding saturated sodium bicarbonate solution to adjust pH to 7, distilling off acetonitrile, adding dichloromethane 500mL to dissolve residues, separating an organic layer, washing once with saturated saline (200mL), drying with anhydrous sodium sulfate, concentrating, separating and purifying with silica gel column (1:1, V/V, petroleum ether: EtOAc) to obtain an amorphous white solid product 10a (47.2g, 67.72mmol), yield: 81 percent.

¹H-NMR(400MHz,CDCl₃)δ9.17(s,1H),8.91(s,1H),8.29(s,1H),8.19–7.95(m,8H),7.68–7.32(m,12H),6.84(s,1H),6.24(d,J＝6.0Hz,1H),5.01–4.89(m,2H),4.75(td,J＝6.0,3.7Hz,1H),1.79(d,J＝0.7Hz,3H)。

Example 18

Preparation of compound 11 a:

compound 10a (40.0g, 57.33mmol) was dissolved in ethanol/tetrahydrofuran (600mL, 1:2), 3mol/L aqueous sodium hydroxide solution 100mL was added, the reaction was stirred at room temperature for 2 hours, the reaction mixture was neutralized with 1mol/L aqueous hydrochloric acid solution, the organic solvent was evaporated, the residue was dissolved in a mixed solvent of dichloromethane and methanol (1000mL, 10:1, V/V), 200mL saturated saline was added, the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to give Compound 11 a.

High resolution mass spectrometry (ESI)⁺)：C₁₈H₂₀N₅O₅ ⁺Theoretical value is 386.1459, found 386.1451.

Example 19

Preparation of compound 7 c:

the crude compound 11a obtained in example 17 was suspended in 200mL of acetone, 2-methoxypropane (200mL) was added, p-toluenesulfonic acid (39.5g, 229.33mmol) was added, the reaction was stirred at room temperature for 10 hours, after completion of the reaction, sodium bicarbonate (19.3g, 229.33mmol) was added, the reaction was stirred for 1 hour, the organic solvent was evaporated, the residue was diluted with 600mL of ethyl acetate, washed with 600mL of water, the organic phase was separated, the aqueous layer was extracted with ethyl acetate (400mLx 3), the organic layers were combined, washed with saturated brine (500mL), dried over anhydrous sodium sulfate, and isolated and purified by silica gel column (40:1, V/V, DCM: MeOH) to obtain a white solid product 7c (15.6g, 36.69mmol) in two steps: and 64 percent.

¹H-NMR(400MHz,MeOD-d₄) δ 11.24(s,1H),8.77(s,1H),8.70(s,1H),8.05(dd, J ═ 8.4,1.4Hz,2H), 7.69-7.61 (m,1H),7.55(ddd, J ═ 8.3,6.6,1.3Hz,2H),6.38(s,1H),4.64(d, J ═ 2.1Hz,1H),4.30(td, J ═ 3.9,2.0Hz,1H),3.75(qd, J ═ 12.2,3.9Hz,2H),1.60(s,3H),1.39(s,3H),1.16(s, 3H). High resolution mass spectrometry (ESI)⁺)：C₂₁H₂₄N₅O₅ ⁺Theoretical value is 426.1772, found 426.1765.

Example 20

Preparation of compound 12 c:

dissolving compound 7c (15.6g, 36.69mmol) in anhydrous dichloromethane (150mL), cooling to 0 ℃ in ice bath, adding N, N-diisopropylethylamine (12.1mL, 73.33mmol), slowly dropping methanesulfonyl chloride (3.4mL, 44.00mmol), after dropping, stirring at room temperature for 1 hour, adding 10mL methanol to quench the reaction, washing with saturated sodium bicarbonate solution (200mL), separating the organic phase, washing with saturated brine (200mL), drying with anhydrous sodium sulfate, concentrating, separating and purifying by silica gel column to obtain amorphous white solid product 12c (15.9g, 31.53mmol), yield: 86 percent.

High resolution mass spectrometry (ESI)⁺)：C₂₂H₂₆N₅O₇S⁺Theoretical value is 504.1547, found 504.1540.

Example 21

Preparation of compound 13 c:

compound 12c (15.9g, 31.53mmol) was dissolved in 100mL anhydrous DMF and NaN was added₃(6.2g, 94.73mmol), heating to 80 deg.C, reacting overnight, distilling off DMF under reduced pressure, and separating and purifying the residue on silica gel column to obtain white solid product 13c (12.1g, 26.84mmol), yield: 85 percent.

¹H-NMR(400MHz,DMSO-d₆)δ11.25(s,1H),8.80(s,1H),8.60(s,1H),8.06(d,J＝7.4Hz,2H),7.66(t,J＝7.3Hz,1H),7.56(t,J＝7.6Hz,2H),6.42(s,1H),4.60(d,J＝2.7Hz,1H),4.41(dt,J＝7.1,3.9Hz,1H),3.94(dd,J＝13.1,7.2Hz,1H),3.72(dd,J＝13.1,4.3Hz,1H),1.62(s,3H),1.40(s,3H),1.25(s,3H)。

Example 22

Preparation of compound 15 a:

suspending compound 7c (500mg, 1.18mmol) in 10mL tetrahydrofuran, adding phthalimide (346mg, 2.35mmol) and triphenylphosphine (617mg, 2.35mmol), cooling to 0 ℃ in an ice bath, slowly dropping DIAD (466 μ L, 2.35mmol), returning to room temperature after dropping, stirring to react overnight, evaporating the solvent, isolating and purifying with silica gel column to obtain compound 15a (521mg, 0.94mmol), yield: 80 percent.

High resolution mass spectrometry (ESI)⁺)：C₂₉H₂₇N₆O₆ ⁺Theoretical value is 555.1987, found 555.1993.

Example 23

Preparation of Compound 15-1 a:

dissolving a compound 12c (500mg, 0.99mmol) in 10mL of absolute ethanol, adding phthalimide (730mg, 4.96mmol), sealing a tube, heating to 100 ℃ for overnight reaction, concentrating the reaction solution after the reaction is finished, and separating and purifying by a silica gel column to obtain a compound 15-1a (358mg, 0.79mmol), wherein the yield is: 80 percent.

High resolution mass spectrometry (ESI)⁺)：C₂₂H₂₃N₆O₅ ⁺Theoretical value is 451.1724, found 451.1728.

Example 24

Preparation of compound 14 c:

dissolving the compound 15a (500mg, 0.90mmol) in 10mL ethanol, adding hydrazine monohydrate (168. mu.L, 5.41mmol), refluxing for 2 hours, evaporating the reaction solution, and separating and purifying the residue with silica gel column to obtain the compound 14c (257mg, 0.80mmol) in yield: 89 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₄H₂₁N₆O₃ ⁺Theoretical value is 321.1670, found 321.1676.

Example 25

Preparation of compound 14 c:

dissolving the compound 15-1a (500mg, 1.11mmol) in 10mL ethanol, adding hydrazine monohydrate (207 μ L, 6.65mmol), refluxing for 2 hours, evaporating the reaction solution, and separating and purifying the residue with silica gel column to obtain the compound 14c (326mg, 1.02mmol) with yield: 92 percent.

High resolution mass spectrometry (ESI)⁺)：C₁₄H₂₁N₆O₃ ⁺Theoretical value is 321.1670, found 321.1675.

Example 26

Preparation of compound 14 c:

compound 13c (12.0g, 26.64mmol) was dissolved in 120mL tetrahydrofuran, triphenylphosphine (9.1g, 34.63mmol) and water (2.4mL, 133.20mmol) were added, the mixture was heated to 40 ℃ and stirred overnight for reaction, after completion of the reaction, the solvent was evaporated off, the residue was dissolved in 7mol/L methanolic ammonia (100mL), stirred at room temperature for 5 hours, and the solvent was evaporated off to give crude 14 c.

High resolution mass spectrometry (ESI)⁺)：C₁₄H₂₁N₆O₃ ⁺Theoretical value is 321.1670, found 321.1665.

Example 27

Preparation of compound 16 c:

dissolving the crude product of 14c in 100mL of anhydrous methanol under the protection of nitrogen, adding acetone (20.5mL) and acetic acid (3.1mL, 53.28mmol) while stirring, stirring at room temperature for 10 minutes, adding sodium triacetoxyborohydride (11.3g, 53.28mmol) in portions, continuing to react at room temperature for 5 hours, adjusting the pH to 7 with saturated aqueous sodium bicarbonate after the reaction is finished, extracting the reaction solution (200mL x 3) with a mixed solvent (10:1, V/V) of dichloromethane and methanol, combining organic phases, washing with saturated brine (200mL), drying with anhydrous sodium sulfate, concentrating, separating and purifying with a silica gel column (15:1, V/V, DCM: MeOH) to obtain an amorphous white solid 16c (6.2g, 17.05mmol), wherein the yield: and 64 percent.

¹H-NMR(400MHz,MeOD-d₄)δ8.29(s,1H),8.21(s,1H),6.27(s,1H),4.54–4.51(m,1H),4.42–4.34(m,1H),3.08(dd,J＝12.5,8.4Hz,1H),3.00(dd,J＝12.5,4.5Hz,1H),2.93(p,J＝6.4Hz,1H),1.65(s,3H),1.43(s,3H),1.21(s,3H),1.12(d,J＝6.3Hz,6H),NH₂From the beginning to the end. High resolution mass spectrometry (ESI)⁺)：C₁₇H₂₇N₆O₃ ⁺Theoretical value is 363.2139, found 363.2133.

Example 28

Preparation of compound 16 c:

compound 12c (5.0g, 9.93mmol) was dissolved in 50mL of isopropylamine, reacted for 48 hours in a pressure resistant reaction tube heated to 50 ℃, after completion of the reaction, isopropylamine was evaporated to dryness, and the residue was purified by silica gel column separation (20:1, DCM: MeOH) to give light brown amorphous solid 16c (3.2g, 8.94mmol), yield: 90 percent.

¹H-NMR(400MHz,MeOD-d₄)δ8.29(s,1H),8.21(s,1H),6.27(s,1H),4.54–4.51(m,1H),4.42–4.34(m,1H),3.08(dd,J＝12.5,8.4Hz,1H),3.00(dd,J＝12.5,4.5Hz,1H),2.93(p,J＝6.4Hz,1H),1.65(s,3H),1.43(s,3H),1.21(s,3H),1.12(d,J＝6.3Hz,6H),NH₂From the beginning to the end. High resolution mass spectrometry (ESI)⁺)：C₁₇H₂₇N₆O₃ ⁺Theoretical value is 363.2139, found 363.2134.

Example 29

Preparation of compound 17 b:

dissolving compound 16b (0.2g, 0.55mmol) in anhydrous acetonitrile (20mL) under nitrogen, adding AcOH (0.06mL, 1.10mmol) and side chain 1 (according to W.Yu, E.J.Chory, A.K.Wernimont, W.Tempel, A.Scopton, A.Federation, J.J.Marineau, J.Qi, D.Barsyte-Lovejoy, J.Yi, R.Marcellus, R.E.Iacob, J.R.Engen, C.Griffin, A.Aman, E.Wienholes, F.Li, J.Pineda, G.Estimu, T.Shatseva, T.Hajian, R.Al-Diwar, J.E.E.Vedak.P.P.Pinedi, G.Eszewiu, T.Shaweva, T.Hajian, R.Al-Schwann, R.Al-Wawar, J.E.E.Vedak.P.P.P.Piroda, G.Piroda, G.G.E.S.S.S.P.P.P.H, H.P.N, H.P.H.N, H.P.H.P.H.H.N, after adjusting the pH to 10mmol, stirring to 10 M.V.V.S. under stirring to 10 M.H, stirring to a, adding saturated aqueous solution, stirring to react at room temperature, stirring to obtain a solution, stirring, keeping the reaction solution, adding saturated aqueous solution, stirring to react at 0.N, stirring to obtain a solution, stirring, adding sodium hydroxide solution, stirring to obtain a solution, stirring, the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, concentrated, and isolated and purified on silica gel to give a white amorphous solid 17b (246mg, 0.41mmol), yield: 75 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₁H₄₇N₈O₄ ⁺Theoretical value is 595.3715, found 595.3709.

Example 30

Preparation of compound 2 b:

compound 17b (50.0mg, 0.08mmol) was dissolved in trifluoroacetic acid/water (9:1, V/V), reacted at room temperature for 2.5 hours with stirring, after completion of the reaction, trifluoroacetic acid was distilled off, 15mL of a mixed solvent of dichloromethane and methanol (4:1, V/V) was added to dissolve the residue, made alkaline with saturated aqueous sodium bicarbonate solution, the organic layer was separated, washed with saturated brine (10mL), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give product 2b (42.0mg, 0.076mmol) as a white solid, a yield: 90 percent.

High resolution mass spectrometry (ESI)⁺)：C₂₈H₄₃N₈O₄ ⁺Theoretical value is 555.3402, found 555.3394.

Example 31

Preparation of compound 17 c:

dissolving compound 16c (0.2g, 0.55mmol) in anhydrous acetonitrile (20mL) under nitrogen, adding AcOH (0.06mL, 1.10mmol) and side chain 1 (according to W.Yu, E.J.Chory, A.K.Wernimont, W.Tempel, A.Scopton, A.Federation, J.J.Marineau, J.Qi, D.Barsyte-Lovejoy, J.Yi, R.Marcellus, R.E.Iacob, J.R.Engen, C.Griffin, A.Aman, E.Wienholes, F.Li, J.Pineda, G.Estimu, T.Shatseva, T.Hajian, R.Al-Diwar, J.E.E.Vedak.P.P.Pinedi, G.Eszewiu, T.Shaweva, T.Hajian, R.Al-Schwann, R.Al-Wawar, J.E.E.Vedak.P.P.P.Piroda, G.Piroda, G.G.S.S.S.P.P.P.H, G.S.S.S.S. Purpu, S. N, S. K.N, after stirring to 10 M.H.H.N, adding saturated sodium hydroxide solution, stirring to react at room temperature, stirring to a, stirring to react at room temperature, stirring to neutrality, adding 0.26, stirring to 10 M.V, stirring to obtain a solution, stirring to obtain a solution, adding saturated solution, stirring to obtain a solution, stirring to, the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, concentrated, and isolated and purified on silica gel to give amorphous white amorphous solid 17c (230mg, 0.39mmol), yield: 70 percent.

Example 32

Preparation of compound 2 c:

dissolving compound 17c (50.0mg, 0.08mmol) in trifluoroacetic acid/water (9:1, V/V), stirring at room temperature for reaction for 2.5 hours, after the reaction is completed, evaporating trifluoroacetic acid, adding 15mL of a mixed solvent of dichloromethane and methanol (4:1, V/V) to dissolve the residue, adjusting to be alkaline with saturated aqueous sodium bicarbonate solution, separating an organic layer, washing with saturated saline (10mL), drying with anhydrous sodium sulfate, concentrating, and purifying by column chromatography to obtain an amorphous white solid product 2c (42.9mg, 0.077mmol), yield: 92 percent.

R_f＝0.53(8:1:0.01，V/V/V，DCM:MeOH:NH₄OH)；[α]_D ²⁰＝17.00(c＝0.100,CH₃OH)；HPLC t_R＝2.47min；¹H-NMR(500MHz,MeOD-d₄) Δ 8.23(s,1H, ArH),8.21(s,1H, ArH), 7.26-7.23 (m,2H, ArH), 7.21-7.17 (m,2H, ArH),6.04(s,1H, H-1),4.31-4.28(m,2H, H-3, H-4), 3.61-3.37 (m,2H, H-5, isopropyl-C)H),3.30–3.17(m,3H,H-5’,-C ₂H-),3.09–2.83(m,2H,-C ₂H-),1.84(p,J＝6.6Hz,2H,-C ₂H-),1.28(s,9H,C(CH ₃)₃) 1.23(d, J ═ 5.9Hz,3H, isopropyl-C ₃H) 1.19(d, J ═ 6.5Hz,3H, isopropyl-C) ₃H),0.92(s,3H,2-C ₃H),NH,NH₂OH is absent;¹³C-NMR(126MHz,MeOD-d₄) Delta 158.76(ArC),157.43(ArC),153.88(ArC),150.00(ArC),146.57(ArC),141.41(ArC),137.93(ArC),126.54(2 xAlC), 120.69(ArC),120.31(2 xAlC), 94.29(C-1),80.64(C-4),79.73(C-2),77.32(C-3),54.83 (isopropyl-CH),53.01(C-5),50.26(-CH₂-),38.66(-CH₂-),35.00(C(CH₃)₃),31.85(C(CH₃)₃),28.10(-CH₂-),20.14(2-CH₃) 17.42 (isopropyl-CH₃) 17.39 (isopropyl-CH₃) (ii) a High resolution mass spectrometry (ESI)⁺)：C₂₈H₄₃N₈O₄ ⁺Theoretical value is 555.3402, found 555.3390.

Example 33

Preparation of compound 17 d:

under nitrogen, compound 16b (0.2g, 0.55mmol) was dissolved in anhydrous acetonitrile (20mL), AcOH (0.06mL, 1.10mmol) and side chain 2 (prepared according to the method in e.j.olhava, US 20160024134a 1; 224mg, 0.83mmol) were added, after stirring for 10 minutes, warmed to 60 ℃, STAB (234mg, 1.10mmol) was added in portions, after stirring at 60 ℃ for 48 hours, the reaction was placed in an ice bath, a saturated sodium bicarbonate solution was added to adjust to neutrality, A3M sodium hydroxide solution was added to adjust pH to 10, the reaction was extracted with a mixed solution of DCM and MeOH (20mL x3, 8:1, V/V), the organic phases were combined, a saturated brine (50mL) was washed, anhydrous sodium sulfate was dried, concentrated, and isolated and purified by silica gel column to give an amorphous white amorphous solid 17d (279mg, 0.45mmol), yield: 82 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₄H₄₉N₈O₃ ⁺Theoretical value is 617.3922, found 617.3920.

Example 34

Preparation of compound 17 d:

dissolving compound 14b (0.2g, 0.62mmol) in anhydrous acetonitrile (20mL) under nitrogen, adding AcOH (0.07mL, 1.25mmol) and side chain 2 (prepared according to the method in E.J. Olhova, US 20160024134A 1; 251mg, 0.93mmol), stirring for 10 minutes, adding sodium triacetoxyborohydride (265mg, 1.25mmol) in portions, stirring at room temperature for reaction for 48 hours, placing the reaction solution in an ice bath, adding saturated sodium bicarbonate solution to adjust to neutral, adding 3M sodium hydroxide solution to adjust pH to 10, extracting the reaction solution with a mixed solution of DCM and MeOH (20mLx 3, 8:1, V/V), combining the organic phases, washing with saturated brine (50mL), drying with anhydrous sodium sulfate, concentrating, and purifying with a column to obtain 16-1 a; dissolving 16-1a in anhydrous dichloroethane (20mL), adding AcOH (0.07mL, 1.25mmol) and anhydrous acetone (0.6mL, V/M ═ 3) under the protection of nitrogen, stirring for 30 minutes, adding sodium triacetoxyborohydride (265mg, 1.25mmol) in portions, stirring at room temperature for reaction for 72 hours, placing the reaction solution in an ice bath, adding saturated sodium bicarbonate solution to adjust to neutrality, adding 3M sodium hydroxide solution to adjust the pH to 10, extracting the reaction solution with a mixed solution of DCM and MeOH (20mL of x3, 8:1, V/V), combining organic phases, washing with saturated saline (50mL), drying over anhydrous sodium sulfate, concentrating, separating and purifying to obtain an amorphous white solid 17d (208mg, 0.34mmol) by silica gel column, wherein the yield: 54 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₄H₄₉N₈O₃ ⁺Theoretical value is 617.3922, found 617.3929.

Example 35

Preparation of compound 2 d:

dissolving compound 17d (50.0mg, 0.08mmol) in 2mL of methanol, adding 6N HCl solution (0.4mL, V/V ═ 5) at room temperature, heating to 60 ℃, reacting overnight, after the reaction is finished, placing in ice bath, adding saturated sodium bicarbonate solution to adjust to neutral, adding 3M sodium hydroxide solution to adjust pH to 10, extracting the reaction solution with a mixed solution of DCM and MeOH (10mL x4, 4:1, V/V), combining organic phases, washing with saturated brine (20mL), drying with anhydrous sodium sulfate, concentrating, separating and purifying with silica gel column to obtain amorphous white solid 2d (42mg, 0.07mmol), yield: 90 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₁H₄₅N₈O₃ ⁺Theoretical value is 577.3609, found 577.3602.

Example 36

Preparation of the Compounds cis-2d and trans-2 d:

compound 2d prepared in example 30 (36.0mg, 0.06mmol) was isolated by column chromatography to give cis-2d (23.5mg, 0.04mmol) and trans-2d (7.5mg, 0.01 mmol).

Compound cis-2 d: high resolution mass spectrometry (ESI)⁺)：C₃₁H₄₅N₈O₃ ⁺Theoretical value is 577.3609, found 577.3601.

The compound trans-2 d: high resolution mass spectrometry (ESI)⁺)：C₃₁H₄₅N₈O₃ ⁺Theoretical value is 577.3609, found 577.3598.

Example 37

Preparation of compound 17 e:

under nitrogen, compound 16c (0.2g, 0.55mmol) was dissolved in anhydrous acetonitrile (20mL), AcOH (0.06mL, 1.10mmol) and side chain 2 (prepared according to the method in e.j.olhava, US 20160024134a 1; 224mg, 0.83mmol) were added, after stirring for 10 minutes, warmed to 60 ℃, STAB (234mg, 1.10mmol) was added in portions, after stirring at 60 ℃ for 48 hours, the reaction was placed in an ice bath, a saturated sodium bicarbonate solution was added to adjust to neutrality, A3M sodium hydroxide solution was added to adjust pH to 10, the reaction was extracted with a mixed solution of DCM and MeOH (20mL x3, 8:1, V/V), the organic phases were combined, a saturated brine (20mL) was washed, anhydrous sodium sulfate was dried, concentrated, and isolated and purified by silica gel column to give amorphous white solid 17e (259mg, 0.42mmol), yield: 76 percent.

High resolution mass spectrometry (ESI)⁺)：C₃₄H₄₉N₈O₃ ⁺Theoretical value is 617.3922, found 617.3915.

Example 38

Preparation of compound 2 e:

dissolving compound 17e (0.5g, 0.81mmol) in 5mL of methanol, adding 6N HCl solution (0.25mL) at room temperature, heating to 60 ℃, reacting overnight, after the reaction is finished, placing in ice bath, adding saturated sodium bicarbonate solution to adjust to neutral, adding 3M sodium hydroxide solution to adjust pH to 10, extracting the reaction solution with a mixed solution of DCM and MeOH (10mL x4, 4:1, V/V), combining organic phases, washing with saturated brine (10mL), drying with anhydrous sodium sulfate, concentrating, separating and purifying with silica gel column to obtain amorphous white solid 2e (0.4g, 0.71mmol), yield: 87 percent.

Example 39

Preparation of the Compounds cis-2e and trans-2 e:

compound 2e prepared in example 33 (400mg, 0.69mmol) was isolated by column chromatography to give cis-2e (282mg, 0.49mmol) and trans-2e (68mg, 0.12 mmol).

Compound cis-2e [ α ]]_D ²⁰＝26.43(c＝0.140,CH₃OH)；¹H-NMR(500MHz,MeOD-d₄) δ 8.42(s,1H, purine-H), 8.22(s,1H, purine-H), 7.47(s,1H, ArH),7.37(d, J ═ 8.4Hz,1H, ArH),7.26(dd, J ═ 8.5,1.8Hz,1H, ArH),6.03(s,1H, H-1), 4.10-4.01 (m,2H, H-4, H-3),3.25(p, J ═ 9.1Hz,1H, N-cyclobutyl-C, H, C, g, CH-, 3.07(p, J ═ 6.6Hz,1H, isopropyl-CH),3.02–2.90(m,2H,H-5,H-5’),2.76(p,J＝7.6Hz,2H,-C ₂H-), 2.25-2.14 (m,2H, cyclobutyl-C ₂H-),1.86(t,J＝5.7Hz,3H，-C ₂H-, cyclobutyl-CH-, 1.64(p, J ═ 9.1Hz,2H, cyclobutyl-C ₂H-),1.36(s,9H,C(CH ₃)₃) 1.07(d, J ═ 6.7Hz,3H, isopropyl-C ₃H) 1.00(d, J ═ 6.6Hz,3H, isopropyl-C ₃H),0.92(s,3H,2-C ₃H),NH,NH₂OH is absent;¹³C-NMR(126MHz,MeOD-d₄) Delta 157.37 (purine-C), 156.48(ArC),153.90 (purine-C), 150.29 (purine-C), 146.63(ArC),146.63(ArC),146.62(ArC),146.61(ArC),141.34 (purine-C), 121.10(ArC),121.10(ArC),120.41 (purine-C), 93.39(C-1),82.68(C-4),79.79(C-2),77.13(C-3),53.48 (N-cyclobutyl-CH-),52.30 (isopropyl-CH) 49.72(C-5),36.29 (cyclobutyl-CH₂-),36.26(-CH₂-, 36.20 (cyclobutyl-CH₂-),35.58(C(CH₃)₃),32.29(C(CH₃)₃) 29.58 (cyclobutyl-CH-),27.73(-CH₂-),20.27(2-CH₃) 19.08 (isopropyl-CH₃) 18.57 (isopropyl-CH₃) (ii) a High resolution mass spectrometry (ESI)⁺)：C₃₁H₄₅N₈O₃ ⁺Theoretical value is 577.3609, found 577.3594.

Compound trans-2e [ α ]]_D ²⁰＝20.87(c＝0.115,CH₃OH)；¹H-NMR(600MHz,MeOD-d₄) δ 8.45(s,1H, purine-H), 8.22(s,1H, purine-H), 7.47(s,1H, ArH),7.38(d, J ═ 8.2Hz,1H, ArH),7.27(dd, J ═ 8.5,1.8Hz,1H, ArH),6.05(s,1H, H-1),4.08(ddd, J ═ 8.9,6.9,2.2Hz,1H, H-4),4.04(d, J ═ 9.0Hz,1H, H-3), 3.68-3.61 (m,1H, N-cyclobutyl-C, 1H), CH-, 3.07(p, J ═ 6.6Hz,1H, isopropyl-CH),3.02(dd,J＝14.9,2.0Hz,1H,H-5),2.96(dd,J＝14.9,6.7Hz,1H,H-5’),2.82–2.76(m,2H,-C ₂H-), 2.28-2.19 (m,2H, cyclobutyl-C ₂H-),2.01(dq,J＝14.3,7.2Hz,3H,-C ₂H-, cyclobutyl-CH-, 1.81(t, J ═ 9.4Hz,2H, cyclobutyl-C ₂H-),1.36(s,9H,C(CH ₃)₃) 1.07(d, J ═ 6.7Hz,3H, isopropyl-C ₃H) 1.00(d, J ═ 6.6Hz,3H, isopropyl-C ₃H),0.93(s,3H,2-C ₃H),NH,NH₂OH is absent;¹³C-NMR(151MHz,MeOD-d₄) Delta 157.36 (purine-C), 156.51(ArC),153.89 (purine-C), 150.28 (purine-C), 146.68(ArC),146.66(ArC),146.63(ArC),146.61(ArC),141.30 (purine-C), 121.12(2 xAlC), 120.36 (purine-C), 93.31(C-1),82.83(C-4),79.80(C-2),77.03(C-3),54.42 (N-cyclobutyl-CH-),52.42 (isopropyl-CH),49.35(C-5),35.57(C(CH₃)₃),35.33(-CH₂-) 33.90 (cyclobutyl-CH₂-, 33.77 (cyclobutyl-CH₂-),32.29(C(CH₃)₃) 29.12 (cyclobutyl-CH-),28.25(-CH₂-),20.27(2-CH₃) 19.33 (isopropyl-CH₃) 18.56 (isopropyl-CH₃) (ii) a High resolution mass spectrometry (ESI)⁺)：C₃₁H₄₅N₈O₃ ⁺Theoretical value is 577.3609, found 577.3594.

Example 40

Compounds were tested for DOT1L inhibitory activity.

Multifunctional microplate reader Envision (PerkinElmer, USA). Dot1L was purchased from BPS Bioscience; the detection kit is AlphaLISA DOT1L Histone H3 Lysine-N-methytranferase Assay (Perkin Elmer). The detection method comprises the following steps: activity was measured using AlphaLISA DOT1L Histone H3Lysine-N-methyl ransferase assay (Perkin Elmer).

Dot1L activity detection method:

the human recombinant Dot1L protein is purchased from BPS Bioscience, the histone methyltransferase activity is detected by a Dot1LHistone H3Lysine-N-methyl ransferase assay kit of Perkins Elmer, the nucleosome is taken as a substrate, SAM provides methyl, and after the 79 th Lysine on the histone H3 on the nucleosome is methylated under the catalytic action of Dot1L, a fluorescent signal with the emission wavelength of 615nm can be generated under the excitation light with the wavelength of 680nm by using the method of AlphaLISA. The viability of Dot1L is reflected by changes in the fluorescence signal.

mu.L of enzyme, 2. mu.L of substrate and 1. mu.L of test compound at different concentrations were added to 384 reaction plates (ProxiPlate. TM. -384Plus, Perkinelmer), respectively. After incubation for 3 hours at room temperature, 2.5. mu.L of stop solution was added to stop the reaction, and then the enzyme activity was detected by AlphaLISA kit. And simultaneously setting a solvent control group, an EPZ5676 positive control group and a blank control group which replace the compound to be detected by DMSO, and setting 3 multiple wells for each concentration of each sample. The final volume of the reaction was 12.5. mu.L, and the specific reaction system was 2% DMSO, 5nM Dot1L, 0.15ng/mL nucleosome, 20. mu.M SAM. The data processing is carried out by plotting the logarithm value of the concentration to the activity percentage, then calculating a fitting curve by adopting nonlinear regression, and calculating by utilizing a software GraphPadprism 5 formula log (inhibitor) vs₅₀The value is obtained.

In structure 2, the DOT1L inhibitory activity of representative compounds is as follows:

table 1 bioactivity assay data for compound 2

***IC₅₀Between 0.1-100 nM. IC₅₀Between 100 and 200 nM. IC₅₀Above 200 nM.

EXAMPLE 41

Female nude mice (purchased from beijing weitonglihua laboratory animals ltd., china), 17-23 g, the experiments met the requirements of the animal regulatory committee. Inoculating MV (4,11) cell strain to the right axilla of nude mice, inoculating 100 μ L of the strain per mouse, and inoculating 5 × 10 cells⁶A/only. After the tumor formation, the diameter of the transplanted tumor is measured by a vernier caliper, and the tumor volume TV is 0.5 multiplied by the length multiplied by the width²The tumor grows to 100-300mm³Animals were divided into three groups by body weight and tumor volume, i.e., a negative control group, EPZ5676 group and cis-2e group, with 6 animals per group. Mice in the EPZ5676 group and the cis-2e group were given a subcutaneous injection of 40mg/kg of EPZ5676 and cis-2e, respectively, 3 times a day, 5 days per week, 2 days at rest, 2 weeks. The negative control group was administered 3 times daily with an equal amount of the blank solvent, and during the course of the experiment, the drug was administered for 5 days, and the test was continued for 2 days. During the experiment, the diameter of the transplanted tumor was measured 2 times per week and the body weight of the mice was weighed, during which the change in body weight and T/C% of the animals were observed.

The curative effect of the compound is comprehensively evaluated in two aspects of anti-tumor activity index T/C (%) and body weight. T/C (%) ═ (RTV)_T/RTV_C) X 100%, wherein RTV refers to the relative tumor volume (ratio of tumor volume after treatment to initial tumor volume), footmark "_T"refers to EPZ5676 group or cis-2e group"_C"refers to a negative control group. Body weight changes indicate compound toxicity.

As shown in fig. 1A, cis-2e had less effect on animal body weight than the positive drug EPZ-5676 within 2 weeks of administration, but exhibited comparable antitumor activity to the positive (fig. 1B): T/C of compound cis-2e ═ 49.1; the T/C of the positive compound EPZ-5676 in the same batch was 45.6.

Claims

1. A 2' -C-methyl substituted nucleoside compound represented by formula 2 or a pharmaceutically acceptable salt thereof:

wherein,

x and Y are each independently selected from carbon or nitrogen, Z is selected from carbon, nitrogen, CR⁰Wherein R is⁰Is halogen or cyano;

m is

R⁴is selected from C₁-C₆Acyl or H.

2. The 2' -C-methyl substituted nucleoside compound according to claim 1, or a pharmaceutically acceptable salt thereof,

x and Z are both nitrogen and Y is carbon; x and Z are both carbon, Y is nitrogen; y and Z are both nitrogen, and X is carbon; or, X is nitrogen, Y is carbon and Z is carbon;

said C is₃-C₆Cycloalkylene is

Preferably, the first and second electrodes are formed of a metal,

m is

3. The 2 '-C-methyl-substituted nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula 2 is a 2' -C-methyl-substituted nucleoside compound represented by one of the following general formulae:

wherein M is as defined in claim 1.

4. The 2 '-C-methyl-substituted nucleoside compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the 2' -C-methyl-substituted nucleoside compound represented by formula 2 is selected from the group consisting of:

5. a process for preparing a 2' -C-methyl substituted nucleoside compound or pharmaceutically acceptable salt thereof according to claim 1, comprising one or more of the following steps:

(h) carrying out azide substitution on the compound 7 or the compound 12 to obtain a compound 13, and then carrying out reduction and deprotection to obtain a compound 14;

(k) substituting the amino group of the compound 12 to obtain a compound 16;

(M) reductive amination of compound 14 with M ═ O to give compound 16-1;

wherein,

x, Y, Z is as defined in claim 1,

r is selected from tert-butyloxycarbonyl, benzyloxycarbonyl, acetyl, isobutyryl, tert-butyryl, benzoyl, substituted by halogen or C₁-C₆Alkyl-substituted benzoyl, preferably said R is tert-butoxycarbonyl;

R¹selected from H, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, isobutyryl, tert-butyryl, benzoyl, substituted by halogen or C₁-C₆Alkyl-substituted benzoyl, preferably, said R¹Is H, tert-butyloxycarbonyl;

pg is selected from 2-naphthylmethyl, 1-naphthylmethyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, triethylsilyl, benzyl, p-methoxybenzyl, substituted by halogen or C₁-C₆Alkyl-substituted benzyl, preferably 2-naphthylmethyl, benzyl or p-methoxybenzyl;

Pg¹selected from phthaloyl;

acyl is selected from C₁-C₁₆Acyl, preferably selected from benzoyl, by halogen or C₁-C₆An alkyl-substituted benzoyl group, an acetyl group, and more preferably a benzoyl group or an acetyl group;

m is

Wherein,

ring A being unsubstituted or substituted by halogen or C₁-C₆Alkyl substitutionC of (A)₆-C₁₂An aryl group, a heteroaryl group,

said C is₃-C₆The cycloalkylene group is preferably a cycloalkylene group

R⁴Is selected from C₁-C₆Acyl or H.

6. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the group consisting of 2' -C-methyl substituted nucleosides of formula 2 according to any one of claims 1-4 and/or pharmaceutically acceptable salts thereof as an active ingredient, and optionally pharmaceutically acceptable carriers and/or excipients.

7. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the group consisting of 2' -C-methyl substituted nucleosides of formula 2 according to any one of claims 1 to 4 and/or pharmaceutically acceptable salts thereof as an active ingredient and other pharmaceutically acceptable therapeutic agents.

8. Use of a 2' -C-methyl substituted nucleoside compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 6 or 7 for the preparation of a medicament for the treatment of cancer.

9. The use of claim 8, wherein the cancer is a non-solid tumor.